96. Jahrestagung der Deutschen Gesellschaft für Pathologie e. V ...

Inhalt Der Pathologe · Supplement 1 · Mai 2012
96. Jahrestagung
der Deutschen Gesellschaft
für Pathologie e.V.
Schwerpunkt der Jahrestagung
F Gastrointestinale Pathologie
F Translationale Forschung in der Pathologie
Vorsitzender der Gesellschaft
Prof . Dr . med . Manfred Dietel
Tagungspräsident
Prof . Dr . med . Gustavo Baretton
Herausgeber
im Auftrag der Gesellschaft
Holger Moch, Zürich
Kongressorganisation und Industrieausstellung
Elisabeth Jacob
Project Manager
MCI Berlin Office
Elisabeth .Jacob@mci-group .com
Titelbild: © W .M .
96. Jahrestagung der Dt. Ges. f. Pathologie e.V. Berlin, 31. Mai–3. Juni 2012
Editorial
Gustavo B . Baretton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Eingeladene Referate und Keynote Lectures
Kolorektales Karzinom 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-001 – VO-003 . . . . . . . . . . .6
Kolorektales Karzinom 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-005 . . . . . . . . . . . . . . . . . . . .7
Keynote Lecture –
Deep Sequencing - new frontiers in GI-tumor pathology
N. Papadopoulos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-007 . . . . . . . . . . . . . . . . . . . .7
Primäre Entzündungen im GI-Trakt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-008 – VO-013 . . . . . . . . . . .7
Keynote Lecture –
Genetic determinants for cancer progression and
individual therapy selection
A. Ullrich . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-014 . . . . . . . . . . . . . . . . . . . . .9
Translationale Forschung und Diagnostik –
Lunge, Sarkome, GIST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-015 – VO-017 . . . . . . . . . . .9
Translationale Forschung und Diagnostik –
Niere, abl . Harnwege, Prostata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-018 – VO-020 . . . . . . . . . .10
Gastric cancer – English . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-024 – VO-026 . . . . . . . . . .11
Keynote Lecture –
Mechanisms of androgen resistance in prostate cancer
D.J. Tindall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-027 . . . . . . . . . . . . . . . . . . . .12
Pankreaskarzinom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-029 – VO-031 . . . . . . . . . .12
Keynote Lecture
The epithelial-mesenchymal transition and cancer stem cells
R.A. Weinberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .VO-032 . . . . . . . . . . . . . . . . . . .13
Mechanisms of Progression and
Therapy resistance of Cancer I – English . . . . . . . . . . . . . . . . . . . . . . . . . .VO-033 – VO-035 . . . . . . . . . .13
Mechanisms of Progression and
Therapy Resistance of Cancer II – English . . . . . . . . . . . . . . . . . . . . . . . .VO-036 – VO-038 . . . . . . . . . .14
Translationale Forschung und
Diagnostik – Mamma/Schilddrüse/Melanom . . . . . . . . . . . . . . . . . . . .VO-039 – VO-040 . . . . . . . . . .15
Der Pathologe · Supplement 1 · 2012 |
1

Inhalt Der Pathologe · Supplement 1 · Mai 2012
2 | Der Pathologe · Supplement 1 · 2012
Ausgewählte Vorträge aus den Einsendungen (Hauptprogramm
und Arbeitsgemeinschaften)
AG Gastroenterologische Pathologie I – Leber . . . . . . . . . . . . . . . . . . .DO-001 – DO-007 . . . . . . . . .15
AG Gastroenterologische Pathologie IV – Oberer GI-Trakt . . . . . . . .DO-015 – DO-018 . . . . . . . . .18
AG Gastroenterologische Pathologie VI – Unterer GI-Trakt . . . . . . . .DO-020 – DO-024 . . . . . . . . .19
AG Pneumopathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-025 – DO-029 . . . . . . . . .21
AG Pneumopathologie III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-032 – DO-036 . . . . . . . . .23
AG Hämatopathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-043 – DO-054 . . . . . . . . .24
AG Hämatopathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-055 – DO-066 . . . . . . . . .28
AG Orthopädische Pathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-079 – DO-086 . . . . . . . . .32
AG Oralpathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-087 – DO-098 . . . . . . . . .35
AG Herz- und Gefäßpathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-100 – DO-110 . . . . . . . . . .38
AG Molekularpathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .DO-111 – DO-125 . . . . . . . . . .42
Workshop Informatik – Strukturierte Befunde . . . . . . . . . . . . . . . . . . .DO-001 – DO-002 . . . . . . . . .46
AG Dermatopathologie und AG Zytopathologie I –
Endokrine Themen I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-001 – FR-008 . . . . . . . . . . .47
AG Dermatopathologie und AG Zytopathologie II –
Endokrine Themen II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-009 – FR-015 . . . . . . . . . . .49
Aktuelle Habilitationen I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-016 – FR-020 . . . . . . . . . . .51
Aktuelle Entwicklungen in der Forschung mit
Vortrag des Promotionspreisträgers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-021 – FR-024 . . . . . . . . . . .53
Promotionspreis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-026 . . . . . . . . . . . . . . . . . . . .54
Translationale Forschung und AG Molekularpathologie . . . . . . . . . .FR-027 – FR-035 . . . . . . . . . . .55
Aktuelle Habilitationen II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-001 – SA-004 . . . . . . . . . .58
Aktuelle Entwicklungen in der Forschung II –
Gastrointestinaltrakt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-001 – SO-009 . . . . . . . . . .59
Aktuelle Entwicklungen in der Forschung III –
Translationale Forschung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-010 – SO-018 . . . . . . . . . .62
AG Gynäkopathologie und Mammapathologie I . . . . . . . . . . . . . . . . .SO-019 – SO-027 . . . . . . . . . .65
AG Gynäkopathologie und Mammapathologie II . . . . . . . . . . . . . . . . .SO-029 – SO-037 . . . . . . . . . .69
AG Paidopathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-038 – SO-045 . . . . . . . . . .72
AG Paidopathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-046 – SO-058 . . . . . . . . . .75
AG Urologische Pathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-061 – SO-69 . . . . . . . . . . .78
AG Urologische Pathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SO-071 – SO-074 . . . . . . . . . .81

Inhalt Der Pathologe · Supplement 1 · Mai 2012
Deutsch-Chinesisches Symposium
Colorectal Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SG-129 – SG-136 . . . . . . . . . . .83
Pancreatic Adenocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SG-137– SG-140 . . . . . . . . . . .85
Mammary Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SG-141 – SG-145 . . . . . . . . . . .86
Malignant Lymphoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SG-146 – SG-147 . . . . . . . . . . .87
Poster
Poster: Deutsch-Chinesisches Symposium . . . . . . . . . . . . . . . . . . . . . . .SG-P-112 – SG-P-133 . . . . . . .88
Poster: GI-Trakt: Ösophagus, Magen . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-036 – FR-P-055 . . . . . . .94
Poster: GI-Trakt: GIST, Dünndarm, Kolorektum . . . . . . . . . . . . . . . . . . .FR-P-056 – FR-P-074 . . . . . .101
Poster: GI-Trakt: Kolorektum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-075 – FR-P-093 . . . . . .107
Poster: GI-Trakt: Hepatobiliäres System und Pankreas . . . . . . . . . . . .FR-P-094 – FR-P-111 . . . . . .113
Poster: Herz- und Gefäßpathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-112 – FR-P-129 . . . . . . .119
Poster: Pneumopathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-130 – FR-P-146 . . . . . 125
Poster: Hämatopathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-147 . . . . . . . . . . . . . . . . .130
Poster: Autopsie/Fallstudien/Sonstiges . . . . . . . . . . . . . . . . . . . . . . . . . .FR-P-161 – FR-P-180 . . . . . 134
Poster: Gynäkopathologie und Mammapathologie I . . . . . . . . . . . . .SA-P-005 – SA-P-019 . . . . .141
Poster: Gynäkopathologie und Mammapathologie II . . . . . . . . . . . . .SA-P-020 . . . . . . . . . . . . . . . . .145
Poster: Molekularpathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-035 – SA-P-053 . . . . .150
Poster: Molekularpathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-054 – SA-P-068 . . . . .156
Poster: Paidopathologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-069 – SA-P-077 . . . . 160
Poster: Uropathologie I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-078 – SA-P-089 . . . . 163
Poster: Uropathologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-090 – SA-P-100 . . . . 166
Poster: Informatik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SA-P-101 – SA-P-111 . . . . . .170
Brahestraße 13 • 04347 Leipzig
Tel.: 0341 / 2 33 44 05 Fax. 2 33 44 06
Internet: http://www.hollborn.de
E-mail: Medizinchemie@hollborn.de
Seit1880
Reagenz- und Farbstofflösungen
• für die Mikroskopie und Zelldiagnostik • für naturwissenschaftliche Bereiche
Auch Sonderanfertigungen

Herausgeber Der Pathologe · Supplement 1 · April 2012
Organ der Deutschen Gesellschaft für Pathologie
Organ der Deutschen Abteilung der Internationalen Akademie für Pathologie
Organ der Österreichischen Gesellschaft für Pathologie
Organ der Schweizerischen Gesellschaft für Pathologie
Organ des Bundesverbandes Deutscher Pathologen
Federführende Schriftleitung / Editor-in-Chief
Univ.-Prof. Dr. K.W. Schmid, Institut für Pathologie und Neuropathologie,
Universitätsklinikum Essen
In Zusammenarbeit mit / In cooperation with
Prof. Dr. G.B. Baretton, Institut für Pathologie, Universitätsklinikum
„Carl Gustav Carus“, TU Dresden
Prof. Dr. R. Büttner, Institut für Pathologie, Universitätsklinikum Köln
Prof. Dr. H.H. Kreipe, Institut für Pathologie, Medizinische Hochschule Hannover
Prof. Dr. H. Moch, Institut für Klinische Pathologie, UniversitätsSpital Zürich,
Schweiz
Prof. Dr. P. Schirmacher, Pathologisches Institut, Universität Heidelberg
Eigentümer & Copyright © Springer-Verlag 2012, Springer Medizin c/o Springer-Verlag
GmbH, Tiergartenstr . 17, 69121 Heidelberg, Tel. +49 6221/487-0, www .springer .de .
Springer Medizin ist Teil der Fachverlagsgruppe Springer Science+Business Media
Geschäftsführung Springer Medizin: Harm van Maanen (Executive Vice President),
Stephan Kröck, Dr . Esther Wieland, Matthias Wissel
Leitung Fachzeitschriften: Dr . Paul Herrmann (v .i .S .d .P .)
Stellv.: Monika Kretz
Chef vom Dienst/Redaktion: Dr . Frank Sommerauer
Redaktion „Der Pathologe“: Elisabeth Althaus, elisabeth .althaus@springer .com
Redaktionsleitung: Dr . Bettina Koch, Tel . -8491, Fax -68491, bettina .koch@springer .com
Eingangsredaktion: Elisabeth Althaus, elisabeth .althaus@springer .com
Leitung Copy-Editing/Lektorat: Sabine Hofmann,
Tel . -8468, sabine .hofmann@springer .com
Technische Redaktion: Rita Kieser, Tel . -8429, Fax -68429, rita .kieser@springer .com
Leitung Herstellung: Alison Hepper, alison .hepper@springer .com
Chef vom Dienst/Herstellung: Jutta Daum, jutta .daum@springer .com
Gesamtleitung Sales & Marketing: Stephan Kröck
Anzeigen: Jens Dessin (Leitung Sales & Advertising);
Silvia Ziemann (Anzeigenleitung, verantwortlich), silvia .ziemann@springer .com,
Tel . +49 30/82787-5713, www .mediadaten .springermedizin .de
Gesamtleitung Corporate Publishing: Ulrike Hafner
Druck: Stürtz GmbH, Alfred-Nobel-Str . 33, 97080 Würzburg . Printed in Germany
Erscheinungsweise: zweimonatlich
Papierausgabe: ISSN 0172-8113, gedruckt auf säurefreiem Papier.
Elektr. Ausgabe: ISSN 1432-1963
Die elektronische Version finden Sie unter www .DerPathologe .de . Die Formulierungen
der Beitragsinhalte können zwischen Online- und Druck ausgabe geringfügig voneinander
abweichen .
springerlink@springer .com, Tel . +49 6221/345-4303, Fax -4229
4 | Der Pathologe · Supplement 1 · 2012
Schriftleitung / Editors
Prof. Dr. L. Bubendorf, Institut für Pathologie, Universitätsspital Basel, Schweiz
Prof. Dr. W. Feiden, MVZ für Histologie, Zytologie und Molekulare
Diagnostik, Trier
Prof. Dr. C. Kuhnen, Institut für Pathologie am Clemenshospital
Münster
Univ.-Prof. Dr. S. Lax, Institut für Pathologie, LKH Graz West,
Österrreich
Prof. Dr. T. Mentzel, Dermatopathologische Gemeinschaftspraxis,
Friedrichshafen
Prof. Dr. W. Saeger, Institut für Pathologie des Marienkrankenhauses Hamburg
Prof. Dr. D. Schmidt, Institut für Pathologie, Referenzzentrum für
Gynäkopathologie, Mannheim
Prof. Dr. Annette Schmitt-Gräff, Abt. Allgemeine Pathologie und
Pathologische Anatomie, Institut für Pathologie, Universitätsklinikum Freiburg
PD Dr. M. Vieth, Institut für Pathologie, Klinikum Bayreuth GmbH
PD Dr. M. Werner, Institut für Pathologie, HELIOS Klinikum Emil von Behring,
Berlin
Rubrikherausgeber / Section editors
Pitfalls / Pitfalls
Prof. Dr. C. Kuhnen, Münster
Bezugspreise: Vorzugspreis für persönliche Abonnenten inkl. Online-Basis-Lizenz 2012:
EUR 340,– (unverb . Preis empfehlung inkl . 7% deutscher MwSt .) zzgl . Versand kosten .
Vorzugspreis für Ärzte in Aus- und Weiterbildung und Studenten inkl. Online-Basis-
Lizenz 2012: EUR 204,00 (unverb . Preis em pfehlung inkl . 7% deut scher MwSt .)
zzgl . Ver sandkosten .
Institutspreis inkl. Online-Basis-Lizenz 2012: EUR 588,50 (unverb . Preis empfehlung
inkl . 7% deutscher MwSt .) zzgl . Versandkosten (Deutsch land: EUR 22,-, Ausland: EUR 39,-) .
Einzelheftpreis 2012: EUR 66,– (unverb . Preis empfehlung inkl . 7% deutscher MwSt .)
zzgl . Versandkosten . Der Bezugspreis ist im Voraus zu zahlen . Das Abonnement kann
bis 30 Tage vor Ende des Bezugszeitraums gekündigt werden .
Bestellungen oder Rückfragen nimmt jede Buchhandlung oder der Verlag entgegen .
Springer Customer Service Center GmbH, Haberstr . 7, 69126 Heidelberg,
Tel. +49 62 21-345-4303, Fax +49 62 21-345-4229, Leserservice@springer .com
(Mo .-Fr . 8 .00 Uhr bis 18 .00 Uhr)
Copyright & allgemeine Hinweise: Mit der Annahme eines Beitrags zur Veröffentlichung
erwirbt der Verlag vom Autor alle Nutzungsrechte, insbe sondere das Recht der weiteren
Vervielfältigung und Verbreitung zu gewerblichen Zwecken mit Hilfe fotomechanischer oder
anderer Verfahren . Die Zeit schrift sowie alle in ihr enthaltenen einzelnen Bei träge und
Abbildun gen sind urheberrechtlich geschützt . Jede Verwer tung, die nicht aus drücklich vom
Urheberrechtsgesetz zugelassen ist, bedarf der vor heri gen schriftlichen Zustimmung des
Verlags . Das gilt insbesondere für Vervielfältigungen, Bearbeitungen, Übersetzungen,
Mikro verfil mun gen und die Einspeicherung und Verarbeitung in elek tronischen Systemen .
Autoren können unter bestimmten Voraussetzungen an der Ausschüttung der Bibliotheks-
und Fotokopietantiemen teilnehmen . Einzelheiten bei VG WORT, Abt . Wissenschaft,
Goethestr . 49, 80336 München .
Angaben über Dosierungsanweisungen und Applikationsformen sind anhand anderer
Literaturstellen oder der Packungsbeilage auf ihre Richtigkeit zu überprüfen . Der Verlag
übernimmt keine Gewähr .
Indexed in Current Contents (Science Edition of the Journal Citation Report) and Medline .

Editorial
Liebe Kolleginnen und Kollegen,
sehr geehrte Damen und Herren,
ich freue mich, Sie bei der 96. Jahrestagung
der Deutschen Gesellschaft für Pathologie e.V.
in Berlin begrüßen zu dürfen, die zum vierten
Mal gemeinsam mit der Tagung des Bundesverbandes
Deutscher Pathologen e.V. und mit
Beteiligung der Internationalen Akademie
für Pathologie, Deutsche Abteilung e.V., als
„Berliner Woche der Pathologie“ stattfindet.
Mit den diesjährigen Hauptthemen
„Gastro intestinale Pathologie“ und „Translationale
Forschung in der Pathologie/Prädiktive
Diagnostik“ werden hoch relevante
und aktuelle Aspekte der gewebsbasierten
Forschung und Diagnostik in den Fokus
der Tagung gestellt. Für beide Themenbereiche
ist es gelungen, national und international
renommierte Experten aus dem
Fach Pathologie und aus anderen Feldern
der Biomedizin und Grundlagenforschung
zu gewinnen. Die Beiträge der eingeladenen
Referenten sollen einen umfassenden Überblick
über den aktuellen Stand der Forschung
bieten und aufzeigen, in welche Richtung
sich das Fach Pathologie weiterentwickelt.
Mit der Verbindung von wissenschaftlichen
Keynote­Vorträgen und der Präsentation
von wichtigen diagnostischen Themen (teilweise
im Dialog mit Klinikern) soll sowohl
für junge Pathologinnen und Pathologen als
auch für erfahrene Kolleginnen und Kollegen
ein interessanter Bogen geschlagen werden.
Die Sitzungen der Arbeitsgemeinschaften,
welche das Hauptprogramm umrahmen,
runden das Programm ab und bieten Einblicke
in neueste wissenschaftliche Erkenntnisse
in den verschiedenen Organsystemen.
Gerade der Nachwuchsförderung soll auch
auf der 96. Jahrestagung wieder große Beachtung
geschenkt werden. Dem erfolgreichen
96. Jahrestagung
der Deutschen Gesellschaft
für Pathologie e. V.
Berlin, 31. Mai – 3. Juni 2012
Format der im letzten Jahr in Leipzig erstmals
durchgeführten Nachwuchslounge wird
diesmal noch mehr Zeit eingeräumt. Es soll
damit ein möglichst großer Kreis von interessierten
jungen Nachwuchswissenschaftlern
angesprochen und motiviert werden, sich
weiter für die Forschung in der Pathologie
zu begeistern und Kontakte zu knüpfen.
Erfreulich ist außerdem das große Interesse
der Industrie an unserem Fach, was durch
die große Zahl der Aussteller und zahlreiche
Satelliten­Symposien zum Ausdruck kommt.
Dabei weitet sich das Spektrum von den
Anbietern labortechnischer und analytischapparativer
Verfahren zunehmend auch auf
die pharmazeutische Industrie aus. Dahinter
steht die Intention der Hersteller, eine
flächendeckende qualitätsgesicherte prädiktive
Diagnostik für die zielgerichteten Therapeutika
sicherzustellen, die eine zunehmende
Bedeutung in der Onkologie spielen.
Die Bündelung der Veranstaltungen der
96. Jahrestagung der Deutschen Gesellschaft
für Pathologie e.V., des Bundesverbandes
Deutscher Pathologen e.V. und der Internationalen
Akademie für Pathologie, Deutsche
Abteilung e.V., als „4. Berliner Woche
der Pathologie“ soll nachdrücklich die
Gewebekompetenz der Pathologie als Alleinstellungsmerkmal
unseres Fachs dokumentieren.
Es soll gezeigt werden, dass die
Grenzen zwischen akademischer Pathologie
und praktischer pathodiagnostischer Tätigkeit
offen sind und offen bleiben müssen;
denn gerade die zeitnahe Integration translationaler
Forschungsergebnisse in die Praxis
bringt – wie in der Vergangenheit immer
wieder eindrucksvoll dokumentiert – unser
Fach weiter voran und sichert seine Zukunft.
Die erfolgreiche Zusammenarbeit mit der
Internationalen Akademie für Pathologie/
IAP wird in bewährter Weise fortgesetzt,
indem die IAP dankenswerterweise wieder
4 Tutorials im Rahmen der Tagung anbietet.
Eine Besonderheit der diesjährigen
Tagung stellt das angeschlossene „Chinesischdeutsche
Symposium“ dar, welches mit Unterstützung
durch die Deutsche Forschungsgemeinschaft/DFG
die Zusammenarbeit in
der wissenschaftlichen Pathologie zwischen
der deutschen und der chinesischen Fachgesellschaft
weiter voranbringen soll. Insbesondere
junge Nachwuchswissenschaftler/innen
aus beiden Ländern sollen so die
Möglichkeit erhalten, persönlich in Kontakt
zu kommen und bilaterale Kooperationen in
der Forschung einzugehen.
Ich wünsche Ihnen eine interessante
Tagung mit vielen neuen Erkenntnissen
sowie der Möglichkeit zum kollegialen Dialog
und regen wissenschaftlichen Austausch.
Prof. Dr. Gustavo B. Baretton
Tagungspräsident der 96. Jahrestagung
der DGP e.V.
Korrespondenzadresse
Prof. Dr. G. B. Baretton
Institut für Pathologie
Universitätsklinikum Dresden
Fetscherstr . 74
01307 Dresden
gustavo .baretton@uniklinikum-dresden .de
Der Pathologe · Supplement 1 · 2012 |
5

Abstracts
Pathologe 2012 · 33:6–176
DOI 10 .1007/s00292-012-1584-x
© Springer-Verlag 2012
Eingeladene Referate und Keynote Lectures
Kolorektales Karzinom 1
VO-001
The natural history of colorectal adenomas and early colorectal
cancer
M . Risio 1
1 Institute for Cancer Research and Treatment (IRCC), Candiolo – Torino, Italy
It is well known that adenomas represent the morphologically categorized
precursor of the vast majority of colorectal cancers. In accordance
with the stochastic modeling of colorectal tumor progression, each step
of the adenoma-carcinoma sequence, from single-crypt dysplasia to cancerised
adenoma, can be probabilistically profiled in terms of evolutive
pathways: although every adenoma has the capacity of malignant evolution,
most adenomas stabilize their progression or even regress. Easily
identifiable pathological features (size, type, architectural growth, grade,
and gross organization of dysplasia) are predictive of the natural history
of adenomas in terms of potential and times of cancerisation. Interestingly,
the link between size and adenoma malignancy is greater than
the one linking dysplasia and malignancy, suggesting the need to perfect
the histological criteria now used for grading dysplasia in order to improve
the detection rates of faster-progressing precursors. Regression of
both micro- and gross adenomas is histologically well established, but it
is thought to be a dynamic process, with cycling fluctuation of phases of
regression and growth of adenomatous tissue.
Colorectal carcinoma invading the submucosa but not the muscular layer
(pT1, early invasive cancer) represents the earliest form of clinically
relevant colorectal cancer. Neoplastic invasion of the submucosa, in fact,
opens the way to metastasis and the choice between surveillance and
major surgery will turn on its metastatic potential. Grade of differentiation
of carcinoma, lymphovascular invasion and state of the resection
margin predict the risk of metastasis and the different clinical outcomes:
a positive resection margin is predictive of local disease, vascular
invasion of lymph node metastasis, poorly differentiated carcinoma of
hematogenous metastasis and cancer-related mortality. Microstaging of
invasive cancer, namely the width and the depth of submucosal invasion,
together with tumor budding at the advancing edge allow the metastatic
risk to be further stratified in minimal, low, and high. There evidence
exists that cancerised adenomas represent the end point of two different,
6 | Der Pathologe · Supplement 1 · 2012
96. Jahrestagung
der Deutschen Gesellschaft
für Pathologie e. V.
Berlin, 31. Mai – 3. Juni 2012
although morphologically undistinguishable, tumorigenic pathways,
the former blocking the growth of early cancer, the latter allowing its
fast progression towards advanced cancer: new biomarkers are needed to
distinguish progressive from non-progressive pT1 neoplasia.
VO-003
The mismatch repair deficient crypt focus – Der mismatch repair
defiziente Kryptenfokus
H . Bläker1 , M . Kloor2 1Institut für Pathologie, Campus Charite Mitte, Universitätsmedizin Berlin,
Institute of Pathology, Campus Mitte, Berlin, 2Heidelberg, Institute of
Pathology
Keimbahnmutationen in einem der Mismatch-Repair(MMR)-Gene,
zumeist MLH1 und MSH2, sind ursächlich für das Lynch-Syndrom,
häufig auch als „hereditary non-polyposis colorectal cancer“-Syndrom
(HNPCC) bezeichnet. Anders als bei den Polyposis-Syndromen, insbesondere
der familiären Adenomatosis polyposis coli, fehlt bei HNPCC
die deutlich erhöhte Zahl der adenomatösen Karzinom-Vorläufer.
Der MMR-defiziente Kryptenfokus, der hier vorgestellt wird, ist eine
neu entdeckte, nichtpolypöse Läsion, die in hoher Zahl, etwa einmal pro
cm2, in der nichttumorös veränderten Dünn- und Dickdarmschleimhaut
von HNPCC-Patienten auftritt. Der MMR-defiziente Kryptenfokus
zeigt die typischen molekularen Veränderungen HNPCC-assoziierter
Tumoren. Während kleine, nur eine bis wenige Krypten umfassende
Läsionen histologisch unauffällig sind, finden sich mit zunehmender
Größe architekturelle und zytologische Veränderungen, die sich keiner
Adenomform zuweisen lassen.
Das Transformationspotential der MMR-defizienten Kyrptenfoci muss
in Anbetracht der erheblichen Diskrepanz zwischen Menge an MMRdefizienten
Foci und Anzahl von Karzinom bei HNPCC ausgesprochen
gering sein. Möglicherweise sind diese Läsionen selbstlimitierend, wobei
die Mechanismen der Elimination noch offen sind.
Zusammenfassend erklärt die Assoziation des HNPCC-Syndroms mit
dem MMR-defizienten Kryptenfokus das Fehlen einer Polyposis bei
diesem Syndrom. Die weitere Charakterisierung der MMR-defizienten
Kryptenfoci und ihr Verlauf versprechen neue Erkenntnisse über initialisierende
und limitierende Mechanismen des autonomen Wachstums.

Kolorektales Karzinom 2
VO-005
Translating biology of colorectal cancer into clinical applications
G .A . Meijer 1
1VU University Medical Center, VU University Medical Center, Amsterdam,
Netherlands
Colorectal cancer (CRC) is one of the most common malignancies and
represents a substantial burden for society, in terms of patient suffering
as well as economically. As cancer is an evolutionary process in which
genotype drives phenotype, knowledge of the biological mechanisms
underlying CRC can help to improve patient outcome. Translational research
in CRC mainly focuses on unmet clinical needs in three stages of
the disease, i.e. early detection or screening, prognostication in primary
CRC and prediction of response to drug therapy in metastatic CRC.
As CRC develops over a number of years from a detectable precursor
(adenoma), there is a window of opportunity for early detection and curative
intervention. Our increased understanding of CRC biology, and in
particular adenoma to carcinoma progression, has yielded a number of
markers based on e.g. DNA or proteins that hold great promise for the
next generation CRC screening tests.
In primary CRC, standard UICC staging is still the most important
method for stratifying patients by risk of recurrence. Yet, the substantial
number of stage II patients that do develop recurrences, and the still
small proportion of stage III patients that actually benefit from adjuvant
therapy, underline the need for additional diagnostic arsenal. Since CRC
biologically is a heterogeneous disease, it does not come as a surprise
that there is a large number of markers for which some level of evidence
exists that they could have additional prognostic value. The main challenge
here is to make the next step to get these markers validated and
implemented in routine diagnostic practice.
In a similar way, much translational research has successfully translated
biology of CRC into diagnostic tests that more rapidly have been implemented,
like KRAS testing for anti-EGFR therapy. The fact that these
predictive markers have entered the field much more rapidly than the
prognostic markers most likely is associated with more efficient business
development, stimulated by the companion diagnostic concept. Yet, also
here challenges remain, as because of the complexity of CRC biology it
is much more likely that we will have complex decision trees rather than
single parameter tests to stratify patients for drug therapy. The exciting
developments that will bring whole cancer genome sequencing within
the reach of pathologists will eliminate current barriers for the full exploration
of tumor biology for the purpose of diagnostic pathology.
Keynote Lecture
VO-007
Deep Sequencing - new frontiers in GI-tumor pathology
N . Papadopoulos
Johns Hopkins University, Baltimore, USA
Inflammatory bowel diseases represent a chronic disorder accompanying
mostly young people throughout their life. Thus therapeutic strategies
allowing for a normal life are mandatory. Although the incidence is
increasing and the research of the last decades provides a detailed view
of the pathogenesis, the available therapeutic strategies are still symptomatic.
The primary aim is to induce a stable remission. Depending on
disease localization as well as associated complications such as fistulas,
abscesses and malnutrition, the therapeutic strategies range from local
applications up to systemic immunosuppressive medications. Despite
the availability of highly potent agents including azathioprine, calci-
neurin inhibitors as well as anti-TNF antibodies, there is still a subset
of patients that remains with active disease. Thus it is crucial to predict
the disease course early on, in order to decide whether early aggressive
therapy is required or a less aggressive treatment is sufficient. Some factors
helping with this decision have already been identified. Novel data
suggest that the expression profile of CD8+ cells may help in predicting
the disease course. However, these data will have to be confirmed in
prospective studies. Equally important is the question when immunosuppressive
therapies can be paused with low risk. Do we need clinical,
endoscopic or even histological remission? On a long-term view mucosal
healing gains impact since it reduces the risk of developing colorectal
cancer. The discussed points emphasize that therapeutic strategies in
inflammatory bowel diseases represent an increasingly individualized
therapy in order to allow for a high life quality of our patients.
Primäre Entzündungen im GI-Trakt
VO-008
Clinical view and novel therapeutic strategies in inflammatory
bowel diseases
B . Siegmund1 1Charité – Universitätsmedizin Berlin, Klinik für Gastroenterologie, Infektiologie
und Rheumatologie der Charite Campus Benjamin Franklin, Berlin
Inflammatory bowel diseases represent a chronic disorder accompanying
mostly young people throughout their life. Thus therapeutic strategies
allowing for a normal life are mandatory. Although the incidence is
increasing and the research of the last decades provides a detailed view
of the pathogenesis, the available therapeutic strategies are still symptomatic.
The primary aim is to induce a stable remission. Depending on
disease localization as well as associated complications such as fistulas,
abscesses and malnutrition, the therapeutic strategies range from local
applications up to systemic immunosuppressive medications. Despite
the availability of highly potent agents including azathioprine, calcineurin
inhibitors as well as anti-TNF antibodies, there is still a subset
of patients that remains with active disease. Thus it is crucial to predict
the disease course early on, in order to decide whether early aggressive
therapy is required or a less aggressive treatment is sufficient. Some factors
helping with this decision have already been identified. Novel data
suggest that the expression profile of CD8+ cells may help in predicting
the disease course. However, these data will have to be confirmed in
prospective studies. Equally important is the question when immunosuppressive
therapies can be paused with low risk. Do we need clinical,
endoscopic or even histological remission? On a long-term view mucosal
healing gains impact since it reduces the risk of developing colorectal
cancer. The discussed points emphasize that therapeutic strategies in
inflammatory bowel diseases represent an increasingly individualized
therapy in order to allow for a high life quality of our patients.
VO-010
Microscopic colitis: clinical appearance and therapy
S . Miehlke1 1Magen-Darm-Zentrum, Hamburg
Microscopic colitis (MC) is a chronic inflammatory bowel disease which
is increasingly recognized as a common cause of chronic, non-bloody
diarrhoea. Besides watery diarrhea, many patients also suffer from abdominal
pain, weight loss and fecal incontinence which severely deteriorate
their quality of life. There is a female predominance with an average
age at diagnosis around 60 years. Smoking appears to be a relevant risk
factor. Epidemiological studies have shown a rising incidence in the last
Der Pathologe · Supplement 1 · 2012 |
7

Abstracts
decade and meanwhile it appears that MC is almost as common as classic
IBD, i.e. Crohn‘s disease and ulcerative colitis.
The endoscopic appearance of the colon is usually normal or may show
only subtile alterations. The diagnosis can be made only by histology and
the specific findings reveal also the subtypes of MC, lymphocytic (LC)
or collagenous colitis (CC). The key histological feature is a thickened
subepithelial collagenous band >10 µm in CC, and an increase number of
surface intraepithelial lymphocytes >20 IEL/100 epithelial cells) in LC.
Patients with chronic diarrhea not completely fulfilling the histological
CC/LC criteria may have incomplete MC.
The primary aim of medical therapy is to achieve and maintain clinical
remission and to improve patient‘s quality of life. The strongest evidence
is currently available for budesonide, a locally acting corticosteroid
with an extensive first-pass metabolism in the liver. Three randomized
controlled trials in CC and two in LC have proven budesonide 9 mg per
day effective for induction of clinical remission with a pooled response
rate of 81%, and a NNT of 2 patients. The majority of patients response
rapidly and experience a substantial improvement in their quality of life.
After cessation of budesonide, symptomatic relapse may occur in 60–
80% of patients. Two randomized controlled trials have now shown that
clinical remission and histological response can be maintained in the
majority of patients with budesonide 6 mg per day for 6 months with a
pooled response rate of 83% and a NNT of 2 patients. Other drugs such as
mesalazine, bismuth or loperamide are occasionally used; however, the
benefit is unclear due to lack of adequate clinical trials.
There is currently also no evidence to recommend immunosuppressives.
However, recent case reports suggest that azathioprin or anti-TNF natibodies
might be an option in individual refractory cases.
VO-011
Histopathology of microscopic colitis
D . Aust1 1Institute for Pathology TU Dresden, Dresden
Microscopic colitis (MC) is recognized to be a common cause of chronic,
non-bloody diarrhea. Numerous epidemiological studies, mainly in the
USA and Sweden, have shown a rising incidence in the last decade. The
diagnosis can only be made by histology and the specific histological findings
define the subtypes of MC, lymphocytic (LC) or collagenous colitis
(CC). As LC and CC share clinical similarities and histopathological features,
and many patients with CC also fulfil the histological criteria for
LC, it has been discussed whether the two are in fact different stages of
disease development. Conversion of LC to CC or vice versa is infrequent,
and at present LC and CC is considered two separate but related entities.
In MC, the lamina propria shows increased numbers of plasma cells and
lymphocytes with loss of the normal gradient, even eosinophilic and
neutrophilic granulocytes may be present. But these histological features
do not warrant the diagnosis of MC even though they may be responsible
for the clinical symptoms.
The key histological feature of LC is an increased number of surface
intraepithelial lymphocytes (IEL). Usually >20 IELs/100 epithelial cells
are requested to warrant the diagnosis of LC. IELs are mostly cytotoxic
CD8+ T-lymphocytes. The epithelium itself can show regressive changes
with focal or diffuse flattening of the columnar cells, loss of mucin,
decreased goblet cells and signs of degeneration such as cytoplasmic vacuoles
and pycnotic nuclei.
The key histological criterion for CC is a continuous subepithelial fibrous
band underneath the surface epithelium (>10 µm). Other hallmarks of
CC are chronic mucosal inflammation, the collagen band contains entrapped
capillaries, red blood cells and inflammatory cells. Damaged
epithelial cells appear flattened, mucin depleted and irregularly oriented.
Focally, small strips of surface epithelium may lift off from their basement
membrane.
The terms MC not otherwise specified (MCnos) or MCi (microscopic colitis
incomplete) was suggested for a subgroup of patients with diarrhea
8 | Der Pathologe · Supplement 1 · 2012
and an increase in cellular infiltrate in the colonic lamina propria and
either an abnormal collagenous layer and/or intraepithelial lymphocytes
coming short of fulfilling the criteria for CC and LC. The histological
features of MC, diagnostic algorithms and possible differential diagnoses
of MC will be discussed in this talk.
VO-012
Eosinophilic esophagitis: role of the gastroenterologist
A . Schöpfer1 1University of Lausanne, Department of Gastroenterology and Hepatology,
CHUV, Lausanne, Switzerland
Eosinophilic esophagitis (EoE), first described in the early 1990’s, has rapidly
evolved as distinctive chronic inflammatory oesophageal disease
with increasing incidence and prevalence in the westernized countries
(prevalence of about 1/2000). Currently, EoE represents the main cause
of dysphagia in adult patients. EoE is defined as chronic, immune/antigen-mediated
esophageal disease characterized clinically by symptoms
related to esophageal dysfunction and histologically by eosinophil-predominant
inflammation. The presence of at least 15 eosinphils per high
power field is considered a minimum threshold for EoE diagnosis. The
disease is isolated to the esophagus, and other causes of esophageal eosinophilia
should be excluded. Other diseases associated with esophageal
eosinophilia are e.g. gastroesophageal reflux disease (GERD), eosinophilic
gastrointestinal disorders, celiac disease, Crohn’s disease, invasive
parasites, achalasia, or drug hypersensitivity. EoE is more prevalent in
males and is frequently associated with allergies. It is currently under
discussion to what extent and by which methods allergic testing should
be performed. Therapeutic strategies for EoE can be summarized by
the “3 D’s”: drugs, diet, dilation. Topical corticosteroids lead to a rapid
improvement of active EoE clinically and histologically. Especially in
children, elimination diets can have similar efficacy as topical corticosteroids.
Oesophageal dilation of EoE-induced oesophageal strictures can
also be effective in improving symptoms, but this therapy has no effect
on the underlying inflammation. Neither the diagnostic nor long-term
therapeutic strategies are yet defined.
VO-013
The role of the pathologist in the diagnosis of eosinophilic
esophagitis (EoE)
C . Bussmann1 1Pathology Cantonal Hospital Lucerne, Luzern, Switzerland
EoE is a chronic, immune-mediated esophageal disease with clinical
symptoms and eosinophil-predominant inflammation. The diagnosis of
EoE is therefore complex. It is not reflux or infectious or drug-induced.
Histological cases with a high number of eosinophils are not a diagnostic
problem. However, limits are of necessity in borderline cases. These must
be based on the high power field (HPF) size and the percentage of squamous
epithelium covering an HPF, which may greatly vary. Also, it must
be considered that EoE is patchy in nature. In order to establish a reliable
diagnosis a minimal number of biopsies are essential.
Alongside the number of eosinophils further histological features associated
with EoE, such as abscesses of eosinophils or basal layer enlargement,
may be of diagnostic help in borderline cases.

Keynote Lecture
VO-014
Genetic determinants for cancer progression and individual
therapy selection
A . Ullrich 1
1Department of Molecular Biology, Max Planck Institute of Biochemistry,
Martinsried
For the past years we have investigated various aspects of signaling systems
in tumor cells in order to identify critical switch points in the patho-physiological
process that results in malignancy. These efforts aim
at the selective blockade of abnormal, disease-promoting signaling mechanisms
by monoclonal antibodies, or small molecule kinase inhibitors.
This strategic approach began with the cloning of the EGF receptor
cDNA and the related receptor HER-2/neu and translated the animal
oncogene concept into target-directed personalized therapy of human
cancer. This work yielded the first specific oncogene target-based, FDAapproved
(1998) therapeutic agent, “Herceptin”, for the treatment of metastatic
breast cancer. Earlier and subsequent “target-driven drug development”
efforts that employed various genomic analysis strategies led
to the cancer therapies that are based on EGFR, HER3, FGFR4, Axl/Ufo
and Flk-1/VEGFR2 as critical signaling elements in tumor progression.
The latter served, in cooperation with SUGEN Inc./Pharmacia/Pfizer, as
basis for the development of SU11248. The drug discovery process that
led to SU11248 represents a prototypical example for the adaptation of
cancer therapeutics from highly specific to multi-targeted drugs.
While all novel cancer therapies target genetic alterations in tumor tissues
innovative strategies are aimed at investigating the contribution of
germ line determinants of the patient to disease progression and therapy
response. One example is the common polymorphism at codon position
388 in the human FGFR4 gene of which the Arg388 allele represents a
target for the development of individual genotype-dependent cancer
therapy development. Current findings and their consequences for patient-specific
cancer therapy will be discussed.
PSI Grünewald
Neu: EXAKT Pathosäge, Arbeitssicherheit,
Diamant-Trennband, Edelstahlausführung
• Abstrichinstrumente
• Objektträger-Aufbewahrung
• Färbeautomaten
• Laborabzugsgeräte
• Sonderlösungen/-entwicklungen
PSI Grünewald
Gottlieb-Daimler-Straße 1, 69514 Laudenbach
Tel.: 06201/71343, Fax: 06201/45542
psi-gruenewald@t-online.de, www.psi-gruenewald.de
Translationale Forschung und Diagnostik –
Lunge, Sarkome, GIST
VO-015
Translational lung cancer research
S . Perner1 1University Hospital of Bonn, Institute of Pathology, Bonn
Lung cancer is the most common malignant disease leading to death
worldwide. Histologically, it is broadly subcategorized into small cell
lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with the
latter mainly consisting of the three major entities – adenocarcinoma,
squamous cell carcinomas and large cell carcinomas. In the recent past,
surgical resection and chemotherapy were the only therapeutic options
available. However, genetic profiling of various lung cancer entities have
revealed major genetic differences within distinct histological tumor
entities, enabling specific diagnosis, individual prognosis and rational
treatment for the disease. Mutation of the Epidermal Growth Factor
Receptor (EGFR) in lung cancer of non-smoking patients was the first
major discovery leading to novel therapeutic strategies, which included
treatment with tyrosin kinase inhibitors (TKI) gefitinib and erlotinib.
EGFR mutated cases are more sensitive to TKI treatment, whereas cases
harboring KRAS mutations are associated with a resistance to TKI.
Moreover, the occurrence of KRAS and EFGR mutations are mutually
exclusive and are correlated with poor prognosis.
In an effort to further subclassify lung cancer at the molecular level, large
lung cancer cohorts were characterized using high-throughput technologies.
The lineage survival oncogene TTF1 is found to be the most
common amplification occurring in pulmonary adenocarcinomas. In
squamous cell lung cancer, SOX2 was identified as the most frequently
amplified lineage survival oncogene. Amplification of either gene proved
to be associated with better overall survival rates. In 2010, Weiss et al.
described Fibroblast Growth Factor Receptor 1 (FGFR1) amplification in
20% of squamous cell lung cancer. FGFR1 amplified tumors were shown
to be sensitive to FGFR1 small molecule inhibitors in cell lines and murine
xenograft models. This finding paved the way to the first rational
therapy in a significant subset of molecularly defined squamous cell lung
cancers. Moreover, our discovery of FGFR1 amplification in squamous
cell cancers of the head and neck area might broaden the therapeutic
spectrum of the FGFR1 inhibitors.
These findings, among others, can only estimate the genetic complexity
of lung tumors. Large-scale molecular profiling has the potential to
identify novel diagnostic, prognostic and predictive markers as well as
therapeutic targets.

Abstracts
VO-017
Translational research and diagnostics – GIST
E . Wardelmann 1
1 University of Cologne, Institute of Pathology, Köln
Gastrointestinal stromal tumors (GIST) are the most common mesenchymal
tumors in the digestive tract. In up to 90% of cases, they are
characterized by activating mutations in the KIT or the PDGFRA gene.
GIST turn out to be a paradigm for successful targeted treatment with
tyrosine kinase inhibitors (TKI). Since the approval of the TKI imatinib
in 2002 the survival of patients with metastatic GIST has been tripled.
The next logical step was the concept to use imatinib in an adjuvant
approach which recently showed to increase the overall survival significantly.
In both settings, the mutational status has high predictive implications.
In detail, GIST with KIT exon 11 mutations show the best response
rates with partial remission rates of up to 80%. In KIT exon 9 mutations,
a doubled daily dose of 800 mg imatinib is now the standard. The
PDGFRA exon 18 mutation D842V has been shown to lead to a primary
resistance. Conclusively, the treatment strategy in GIST is driven by their
molecular characterisation.
Further research has increased the knowledge about resistance mechanisms
in solid tumors against TKI. The number of patients with secondary
resistance due to acquired KIT mutations is increasing with treatment
duration. Strategies to address this situation are the introduction of
novel pathway inhibitors targeting different levels of signal transduction
pathways such as the mTOR/Akt pathway, the RAS/RAF pathway, histone
deacetylation, and others.
Among the GIST without mutations in the common hot spot regions
of KIT and PDGFRA, so-called wild type GIST, further genomic subgroups
have been identified. One such subgroup carries inactivating
germline mutations in the genes encoding succinate dehydrogenase B,
C, or D. They are associated with the occurrence of paragangliomas, the
so-called Carney-Stratakis syndrome. Most frequently, GIST are located
in the stomach and show an epithelioid phenotype and a multinodular
growth pattern. They preferentially occur in young females and often
show lymph node metastases. In Carney’s triad additional pulmonary
chondromas are observed. Another small subgroup of sporadic GIST
present with BRAF mutations as an alternative genomic event. Finally,
very rare kindreds with germline mutations in either KIT or PDGFRA
have been described.
In summary, the molecular characterisation of GIST has revolutionized
their treatment because of increasing knowledge about the high relevance
of predictive molecular typing in solid tumors.
Translationale Forschung und Diagnostik –
Niere, abl. Harnwege, Prostata
VO-018
Biomarker for diagnosis, prognosis and prediction in renal cancer
H . Moch1 1University Zurich, Institute for Pathology, Zürich, Switzerland
Biomarkers are frequently used in aiding diagnosis, and to predict prognosis
or response to therapy in neoplasms. In renal cancer, immunohistochemistry
is commonly used in the routine for the classification of
renal tumors. Conventional karyotyping, fluorescence in situ hybridization
and molecular cytogenetics are less commonly used. Expression
profiling, and mutational analysis are currently performed to identify
specific molecular pathways in renal cancer, mainly for research purposes.
In this presentation, we document results of a Working Group Meeting
conducted by the International Society of Urological Pathology (ISUP) in
10 | Der Pathologe · Supplement 1 · 2012
Vancouver 2012. The Working Group discussed the use of immunohistochemical
markers as well as the use of cytogenetics or other molecular
technologies in the characterization of renal neoplasms. In this presentation,
the results of the Working Group Meeting are summarized. In
detail, the value of immunohistochemistry for the differential diagnosis
in different diagnostic situations, e.g. in renal tumors with clear or granular
cytoplasm, diagnosis of unclassified renal cancer is commented.
Further, the view of the Working Group regarding use of predictive or
prognostic biomarkers in routine pathology is provided.
VO-019
Translational research and diagnostics: urinary tract
R . Knüchel-Clarke1 , N .T . Gaisa2 , M . Rose2 , C . Henkel3 , E . Dahl2 1 2 Universitätsklinikum Aachen, Institut für Pathologie, Aachen, University
Hospital, RWTH Aachen, Institut für Pathologie, Aachen, 3Ruhr-University Bochum, Medical Proteomics Center, Bochum
In urothelial cancer as a frequent tumor entity two main fields of the
clinical situation deserve special attention and need support from basic
research.
1. Amongst the most frequent non-invasive papillary low grade tumors
morphology alone is insufficient to predict recurrence rate or even more
importantly progression.
2. Amongst the already muscle invasive tumors which mostly undergo
cystectomy, neoadjuvant and adjuvant chemotherapy concepts still have
a limited supportive role, and more effective individualized treatment
concepts are desirable.
Ad 1. While the WHO classification of tumors has integrated data from
genetic analysis of tumors to diagnose genetically stable (low grade) and
unstable (high grade) tumors, FGFR3 mutations and additional molecular
alterations may help to define the non-progressing, i.e. nearly benign
tumors within the group of low grade tumors. This could support therapy
decision favoring avoidance of intravesical chemotherapy and allowing
less follow up. A multiparametric approach will be necessary and
becomes increasingly feasible due to e.g. epigenetic or proteomic marker
sets. Ideally these marker sets are not only valid for tissue analysis but
also sufficiently sensitive to predict benign disease course in cytology
specimens.
Ad 2. Within the group of genetically instable tumors it seems necessary
to define tumors different from mere urothelial differentiation as
squamous and glandular as well as variants of urothelial carcinoma as
micropapillary or small cell carcinoma in a first step. All these tumor
variants are found in other organs as well. Consequently the multicentric
collection of cases and the molecular analysis of pathways of growth
signalling is an apt approach for target identification. Data should be
compared to already established data in other cancers as colon and lung
cancer and will allow the inclusion of cases in prospective studies using
e.g. small molecule- like tyrosine kinase inhibitors. Own research efforts
and recent data from the literature that intend to improve the two clinical
settings in urothelial cancer will be presented.
With support of a START grant RWTH Aachen (MR, NTG and ED) and
a DFG grant GA 1384/2-1 (NTG).
VO-020
Translational research and diagnosis of prostate cancer
G . Kristiansen1 1Institut für Pathologie der Universitätsklinik Bonn, Bonn
Prostate cancer is the most common non-cutaneous malignant tumour
in men and is a major research focus of pathologists, urologists and urooncologists
alike. Due to PSA-screening and risen patient awareness, the
practising pathologist is confronted with a steadily increasing number of
prostate biopsies, necessitating ancillary tests in morphologically challenging
cases. Next to basal cell markers, additional positive markers

(AMACR, FASN, GOLM1, GSP-pi, ERG) that aid in the differential diagnosis
are presented and discussed here.
The clinical decision of urologists or radiooncologists, whom and how
to treat these men, still rests predominantly on histological parameters.
This urges us to increase standardization of the way we handle and diagnose
our specimens. Processing and diagnosing of prostatectomy specimens
has been the topic of the 2009 consensus conference of the International
Society of Urologic Pathology (ISUP) and the most important
recommendations of this conference will be discussed in comparison to
the current S3-guidelines.
As Gleason Score is one of the strongest prognostic parameters in prostate
cancer, standardization is particularly important. The long overdue
update on Gleason scoring by the ISUP 2005 recommendations has generally
increased awareness, but has also led to some confusion among
pathologists and clinicians and has possibly even induced a systematic
over-grading of biopsies. A recent study conducted by the European Network
of Urinary Pathologists focussed on particularly challenging cases
to discriminate problematic areas in the differentiation of Gleason patterns
3 and 4 in order to establish helpful diagnostic guidelines.
Despite the clinical need to identify insignificant and lethal prostate cancer
at the biopsy stage, this estimation is still left exclusively to conventional
clinical and histological parameters and no molecular biomarker has
entered clinical practice yet. A critical overview of recent developments
of prognostic biomarkers in prostate cancer is given.
Finally, a brief outline of the PREFERE study, a prospective therapy study
beginning in late 2012 in Germany, that aims to compare the outcomes
of 7600 patients over a period of nearly 20 years, will be presented.
Gastric Cancer – English
VO-024
Hereditary gastric cancer
F . Carneiro1 1Institute of Molecular Pathology and Immunology of the University of
Porto (IPATIMUP) and Medical Faculty of Porto/Centro Hospitalar S . João,
Porto, Portugal, Porto, Portugal
Familial aggregation of gastric cancer (GC), both of the diffuse and of
the intestinal type, occurs in a variable proportion of cases, pointing to
genetic predisposition in these settings.
In 1998, Guilford et al identified the first inherited gastric cancer syndrome,
designated as Hereditary Diffuse Gastric Cancer (HDGC), caused
by germline alterations at the CDH1 (E-cadherin) gene. In 1999, the
International Gastric Cancer Linkage Consortium (IGCLC) defined the
criteria for the different types of familial gastric cancer syndromes: 1)
two GC cases in a family, one confirmed DGC 80% at the age of 80
in both genders, and lobular breast cancer is 60% in women by age 80.
About one third of families fulfilling the criteria for HDGC carry germline
alterations of the CDH1 gene. To date, about 100 different germline
CDH1 alterations have been identified in HDGC families, mainly point
mutations and large deletions. In 2010, the IGCLC updated the recommendations
for CDH1 testing, including: 1) histological confirmation of
DGC only required for one family member; 2) DGC

Abstracts
second only to lung cancer. In recent decades we witnessed major advancements
in the understanding of the epidemiology, pathology and
pathogenesis of gastric cancer. Infection with H. pylori or Epstein-Barr
virus, dietary and lifestyle factors contribute to the risk of developing
gastric cancer. With regard to pathogenesis, at least three distinct types
of gastric cancer exist, i.e. (1) proximal, (2) distal diffuse, and (3) distal
non-diffuse type. Genetic and epigenetic alterations are related to oncogene
mutations and tumor suppressor gene inactivations, e.g. by loss
of heterozygosity or methylation. Canonical oncogenic pathways such
as E2F, KRAS, p53, and WNT/β-catenin signaling are de-regulated in
gastric cancer. Microsatellite instability is observed in approximately
10–15% of the cases. Hereditary and familial type gastric cancers are currently
linked to 122 different CDH1-mutations (25–30% of the cases with
Hereditary Diffuse Gastric Cancer) and various gene polymorphisms
determining disease susceptibility. Molecular subtypes of gastric cancer
were identified, which separate diffuse from intestinal type gastric cancer
and are not entirely congruent with the histopathological phenotype
according to Laurén, but may influence chemosensitivity. Putative cancer
stem cell markers of gastric cancer were found (e.g. ADAM17, CD133,
FZD7, LGR5), and correlate with patient prognosis. Perioperative chemotherapy
has improved patient survival and targeted therapy is applied
in patients overexpressing Her2/neu. With regard to patient prognosis,
complete surgical resection is still the most important predictor of patient
outcome, followed by tumor stage, lymph node ratio, and mucin
phenotype (Muc2). Among the diverse anxillary biomarkers that have
been sought and identified, including class I histone deacetylases, none
has reached a broader clinical application. Thus, molecular phenotyping
of gastric cancer is still in its infancies and the search continues for novel
diagnostic, prognostic and predictive biomarkers.
Keynote Lecture
VO-027
Mechanisms of androgen resistance in prostate cancer
D .J . Tindall1 1Department of Urology, Mayo Clinic Foundation, Rochester, United States
The androgen receptor (AR) signaling axis plays a critical role in the development,
function and homeostasis of the prostate. The classical action
of AR is to regulate gene transcriptional processes via AR nuclear
translocation, binding to androgen response elements on target genes
and recruitment of, or crosstalk with, transcription factors. Prostate cancer
initiation and progression is also uniquely dependent on AR. Androgen
deprivation therapy remains the standard of care for treatment of
advanced prostate cancer. Despite an initial favorable response, almost
all patients invariably progress to a more aggressive, castrate-resistant
phenotype. Considerable evidence now supports the concept that development
of castrate-resistant prostate cancer (CRPC) is causally related
to continued transactivation of AR. Understanding the critical events
and complexities of AR signaling in the progression to CRPC is essential
in developing successful future therapies. This talk provides a synopsis
of AR structure and signaling in prostate cancer in progression, with a
special focus on recent findings on the role of AR in CRPC. Clinical implications
of these findings and potential directions for future research
are also outlined.
12 | Der Pathologe · Supplement 1 · 2012
Pankreaskarzinom
VO-029
Molecular alterations in pancreatic ductal adenocarcinoma
B . Sipos1 1University of Tübingen, Department of Pathology, Tübingen
In the last decade significant results have been achieved in understanding
the molecular pathogenesis of pancreatic ductal adenocarcinoma
(PDAC). Holistic approaches searching for genetic abnormalities indicate
that PDACs harbor one or more genetic alterations in the majority
of core pathways. These pathways include apoptosis, DNA damage and
G1-S phase transition control; homophilic cell adhesion and integrin
signaling; c-Jun, K-ras and other small GTPases associated pathways;
TGF-beta, hedgehog and Wnt/notch signalling and finally the regulation
of invasion. In the initiation of human PDACs K-ras, p53, DPC4/
Smad4 and p16/CDKN2a play a pivotal role, which is demonstrated by
the increasing rate of alterations of these genes during progression of
pancreatic intraepithelial neoplasia.
Beyond these descriptive data from human studies on pancreatic intraepithelial
neoplasia, experiments using genetically engineered mouse
models (GEMM) provide functional evidence that K-ras, p53, p16/
CDKN2a alterations are key factors in emerging PDACs. GEMM that
express mutant oncogenes and/or tumor suppressor genes under the
control of pancreas specific promoters such as elastase, Pdx-1 and p48/
Ptf1a recapitulate the progression of pancreatic intraepithelial neoplasia
to PDAC, following a characteristic acinar-ductal metaplasia in the pancreatic
parenchyma. These GEMM give rise to PDAC, but also to other
types of cancer. Targeting K-ras and p53 in GEMM results in well-reproducible
tumor development, which allows reliable pre-clinical in vivo
experiments in conjunction with sophisticated small animal imaging.
High stroma content and paucity of intratumoral vessels are hallmarks
of human PDAC. The stroma contains stellate cells, immune cells and
large amounts of extracellular matrix, which all contribute to the malignant
traits of PDACs and may even exert a selective pressure on tumor
cells. Desmoplasia probably contributes to the innate chemoresistance of
PDACs by hindering drug delivery.
To date, there is no efficient targeted therapeutics for human PDAC. Targeted
treatment may fail due to numerous affected pathways that facilitate
evasion of tumor cells from the pressure of selective blocking agents.
In the next decade the big challenge is to find the Achilles’ heel of PDAC,
probably using intelligent combination of smart molecules and targeting
of the stroma.
VO-030
The CRM concept of pancreatic cancer – a proposal for the new
S3-Guideline
A . Tannapfel1 1Institut für Pathologie, Bochum
Pancreatic ductal adenocarcinoma is diagnosed in about 13,000 patients
each year in Germany being the fourth leading cause of cancer mortality.
The 5-year survival rate remains less than 5% because of metastatic disease
at time of initial diagnosis. It becomes evident, that ductal pancreatic
cancer develops in a sequential process from lesions, named as Pancreatic
Intraepithelial Neoplasia (PanIn). The curative removal of the tumor
(R0 resection) may improve survival, but survival remains poor even in
optimally resected patients. Loco regional and metastatic recurrence is
frequent. The rate of microscopic margin involvement (R1) varies markedly
in the current literature (from 5 to 85%). The rate of R1 resections
is frequently underreported. One possible reason is the lack of the uniform
use of R classification, followed by the lack of quality assessment
for pathological examination of pancreaticoduodenectomy specimens.
A standardized protocol for pathological examination should be used

to assess the correct rate of R1 resected patients and also the correlation
between R1 resections and clinical outcome. The optimal standardized
protocol for Whipple specimens, involving multicolor margin staining,
axial slicing and extensive tissue sampling, has to be applied. The R classification
is defined for any given tumor as R0, no residual tumor; R1, microscopic
residual tumor; R2, macroscopic residual tumor. Since these
definitions are internationally accepted, the concept of „circumferential
resection margins“ is introduced for pancreatic cancer, describing the
relation of the tumor edge to the circumferential margin (CRM). The
pathologist has to measure the exact distance of the tumor to the definite
resection margin. Tumors with a minimal distance from the CRM of
< or=1 mm are categorized as CRM-positive, tumor with a distance of
>1 mm are CRM-negative – in both cases a curative resection occurred.
Only in the case of tumor cells visible directly at the resection margin, a
R1 resection is diagnosed. The different use of both definitions of resection
margin involvement improves valid comparisons between reports
on treatment results. The CRM concept of pancreatic carcinoma will be
introduced in the revised version of the S3 guideline.
VO-031
Molecular pathology of pancreatic adenocarcinoma
P . Michl1 1Klinik für Gastroenterologie, Endokrinologie und Stoffwechsel, Philipps-
Universität, Marburg
Pancreatic cancer carries the most dismal prognosis of all solid tumors
and is associated with a 5-year survival rate of less than 5%. Identification
of novel, urgently required therapeutic modalities depends on detailed
knowledge of the underlying molecular pathology. During recent years,
progress has been made in deciphering the complex network of signaling
cascades involved in cancerogenesis and tumor progression in pancreatic
cancer. Furthermore, genetically engineered mouse models have been
developed, including a model with conditionally activating mutation of
K-Ras and inactivating mutation of p53, both of which are frequently
found in the human disease. The murine tumors closely recapitulate histological
and clinical presentation of human pancreatic tumor development
and progression and are suitable for studying the impact of genetic
manipulation of specific genes as well as for testing novel pharmacological
inhibitors. In addition, accumulating evidence suggests a dominant
role for the desmoplastic stromal reaction that comprises up to 90% of
the tumor volume in mediating tumor progression. Signaling events within
the tumor stroma such as activation of the hedgehog pathway have
been shown to influence tumor growth, angiogenesis and resistance to
chemotherapy. Moreover, infiltrating inflammatory cells within the
stromal compartment have been shown to contribute to the resistant
phenotype of this tumor entity. Numerous preclinical and clinical trials
targeting tumor cell autonomous and non-autonomous stromal signaling
cascades are currently underway to overcome the resistance to chemotherapy
and to improve the appalling prognosis of pancreatic cancer.
Keynote Lecture
VO-032
The epithelial-mesenchymal transition and cancer stem cells
R .A . Weinberg1 1Whitehead Institute, Massachusetts Institute of Technology, Cambridge,
United States
The molecular and the cellular mechanism of tumor progression have
been elusive until recently. In particular, the mechanisms of invasion
and metastasis have proven difficult to elucidate. These last steps of malignant
progression involve a succession of steps often termed the “in-
vasion-metastasis cascade”, which includes local invasion by primary
tumor cells, intravasation, passage through the systemic circulation, extravasation,
formation of micrometastatic colonies, and the outgrowth
of the latter in macroscopic growths, often termed colonization.
In recent years, a cell-biological program termed the epithelial-mesenchymal
transition (EMT) has been studied in great depth because it
holds the promise of explaining many of the steps of this complex cascade.
Thus, by passing through this program, a carcinoma cell acquires the
attributes required to complete most of the steps of the invasion-metastasis
cascade, quite possibly all of them except for the last step, colonization.
This represents an enormous simplification of our conceptualization
of this process, as it may represent nothing more than the activation
of an otherwise-latent cell-biological program that is normally operative
during embryogenesis and, in adults, wound-healing.
Over the past several years, an additional aspect of the EMT program has
come to light, as it has been found to be intertwined with the epithelial
stem cell program. Thus, cells that have passed through the EMT program
approach the stem-cell state. This holds important implications for
both normal epithelial and neoplastic epithelial cells, as they both exploit
the same program to organize themselves. In the context of metastasis,
this implies that a cell that has pass through an EMT also acquires the
self-renewal capability needed to seed a new colony of metastatic cells, an
important prerequisite to successful colonization
Mechanisms of Progression and Therapy
Resistance of Cancer I – English
VO-033
Programmed necrosis
W . Roth1 1Institute of Pathology, University Hospital Heidelberg, and German Cancer
Research Center, Heidelberg
The research on cell death regulation has become one of the most important
areas in tumor biology. Due to the central role of cell death regulation
in tumor development and therapy resistance, a detailed knowledge
of the molecular mechanisms of cell death opens up the possibility to
develop novel rational therapeutic approaches for cancer. During the last
decades the research on cell death was dominated by an oversimplified
dual concept of apoptosis versus necrosis: Apoptosis was defined as an
active, molecularly determined signaling cascade leading to “programmed
cell death”, whereas necrosis was conceived as a passive process resulting
from unspecific cellular damage. However, in the last few years
this dogmatic conception has dramatically changed. Several studies clearly
demonstrate that certain forms of necrotic cell death are executed
in a strictly regulated and ordered fashion. The best known type of programmed
necrosis is “necroptosis” which describes a signaling cascade
mediated by TNF receptor 1 and RIP1 leading to cell death. Necroptosis
plays an important role in the embryonic development, but also in the
pathophysiology of certain diseases such as chronic inflammatory bowel
disease. Yet another necrosis-like type of cell death can be induced by
the HMGB1 protein. The HMGB1-induced cell death is morphologically
characterized by the formation of giant mitochondria and metabolically
by a severe deregulation of mitochondrial oxidative phosphorylation.
The elucidation of the molecular mechanisms of necrotic cell death is
an important step to develop novel strategies to overcome the classical
resistance to apoptotic cell death which is one of the main reasons for
therapy resistance in many types of cancer.
Der Pathologe · Supplement 1 · 2012 |
13

Abstracts
VO-034
Interplay of cadherins in breast cancer progression
M . Rezaei 1 , K . Friedrich 1 , A . Kettelhake 1 , B . Wielockx 1 , G . Baretton 1 , G . Breier 1
1 University Hospital Carl Gustav Carus, TU Dresden, Institute of Pathology,
Dresden
Introduction. Deregulation of cadherin expression, such as the loss of
epithelial (E-)cadherin and gain of neural (N-)cadherin, has been implicated
in carcinoma progression. We have previously shown that vascular
endothelial (VE-)cadherin can be expressed on human breast cancer
cells, in addition to tumor endothelial cells. This aberrant expression
pattern was recapitulated in a mouse mammary carcinoma model, and
functional studies showed that VE-cadherin promotes experimental tumor
growth by stimulating transforming growth factor (TGF)-beta signaling
in cancer cells. Here, we have investigated the functional interplay
between N-cadherin and VE-cadherin in breast cancer.
Methods. The expression of N-cadherin and VE-cadherin was evaluated
by immunohistochemistry in a tissue micro-array with 84 invasive
human breast carcinomas. VE-cadherin and N-cadherin expression in
mouse breast cancer cells was manipulated by RNA-interference or overexpression
and analysed by immunofluorescence, reverse transcriptasepolymerase
chain reaction, and western blot. Experimental tumors were
generated by transplantation of the modified mouse breast cancer cells
into immunocompetent mice. Tumor growth was monitored, and tumor
tissue was subjected to histological analysis.
Results. VE-cadherin and N-cadherin were largely co-expressed in invasive
human breast cancers. Silencing of N-cadherin in mouse mammary
carcinoma cells led to decreased VE-cadherin expression and induced
changes indicative of mesenchymal-epithelial reverting transition
(MET), as indicated by re-induction of E-cadherin, localisation of β-catenin
at the cell membrane, decreased expression of vimentin and SIP1,
and gain of epithelial morphology. Suppression of N-cadherin expression
in mammary carcinoma cells inhibited tumor growth in vivo even
with forced expression of VE-cadherin.
Conclusions. The results underline the critical role of N-cadherin in
breast cancer progression and show that N-cadherin is involved in the
maintenance of the malignant fibroblastoid tumor cell phenotype. Ncadherin
prevents the re-expression of E-cadherin and the localisation
of β-catenin at the plasma membrane; consequently, β-catenin can exert
its known protumorigenic activity in the cell nucleus. N-cadherin is also
required to maintain the expression and protumorigenic activity of VEcadherin
in malignant tumor cells but not vice versa. Thus, N-cadherin
acts in concert with VE-cadherin to promote tumor growth.
VO-035
Cancer stem cells: targets and potential biomarkers for radiotherapy
M . Krause1 1Dept . of Radiation Oncology, OncoRay Center for Radiation Research in
Oncology
Radiotherapy has a curative potential in solid human tumours. Even in
locally advanced, inoperable tumours, many patients can still be cured
by radiotherapy or radiochemotherapy, e.g. up to 40% in advanced head
and neck cancer.
The current understanding of cancer stem cells (CSC) defines a CSC as a
tumour cell that has the unique potential to self-renew and to regenerate
a complete tumour with all its sublines of tumour cells. This definition
implies that all CSC need to be inactivated to reach a permanent local
tumour control, or, that a single surviving CSC after treatment will cause
a recurrence. Thus, CSC should be ideal biomarkers and targets for
radiotherapy.
Today, there some evidence for a higher radioresistance of CSC measured
by surface markers that are higher expressed in CSC versus non-
CSC. If such biological differences hold true, CSC need to be included
14 | Der Pathologe · Supplement 1 · 2012
into the development of new predictive biomarkers. Recently, for the
first time a systematic clinical study has shown a predictive value for the
expression of the surface marker CD44 for local tumour control after
primary radiotherapy of early laryngeal cancer. However, it has to be
expected that in other tumour entities, the heterogeneity will be larger
due to confounding factors of radiation resistance, e.g. tumour size or
tumour micromilieu.
The talk will give an overview on the current knowledge of the potential
value of CSC for prediction of tumour control after radiotherapy. First
attempts of specific targeting approaches will be discussed.
Mechanisms of Progression and Therapy
Resistance of Cancer II – English
VO-036
The role of HIF-prolyl hydroxylase-2 (PHD2) during physiological
and pathological processes in mice
B . Wielockx 1
1TUDresden – Pathology, Dresden
Hypoxia is a prominent feature during development and physiological
as well as pathological conditions in adults. An oxygen-sensing machinery
is therefore very important to help the cells adapt instantaneously
to any unacceptable O2 level. Such a system relies on the oxygen dependent
HIF-prolyl hydroxylases (PHD1–3), enzymes that can inactivate
the alpha subunit of the hypoxia inducible transcription factor (HIF).
HIF1α is ubiquitously expressed in all tissues, whereas HIF2α is restricted
to certain cell types. In case of low oxygen availability, PHDs lose
their functionality and allow the HIF complex, composed of HIFα and
a constitutive HIFβ subunit, to promote biochemical and physiological
changes including anaerobic glycolysis, angiogenesis and hematopoiesis.
We produced a mouse line that lacks HIF prolyl hydroxylase2 (PHD2)
in different cell types (e.g. hematopoietic cells, epithelial cells). Moreover,
these conditional PHD2-deficient mice display strongly elevated
hematocrit levels (up to 85%) together with high EPO concentrations in
the blood produced by kidney and brain. Remarkably, these mice show
no premature lethality. In addition, we observed an enlargement of the
spleen which we showed to be the major organ responsible for the enormous
overproduction of RBCs. Double cKO mice revealed that the erythrocytosis
phenotype is exclusively driven by HIF2 α whereas HIF1 α
is responsible for the survival of cKO mice.
Next, we found that the hematopoietic stem cell (HSC) compartment in
the bone marrow was significantly altered. Detailed FACS analyses demonstrated
that cKO mice contain much more proliferating multipotent
progenitors (MPPs) under steady state conditions; an effect induced by
HIF1 α . On the other hand, severe stress situations pushed quiescent
cKO CD34neg HSCs to self-renewal.
In addition, we subjected these cKO mice to different in vivo models
highlighting the central role of PHD2 during inflammatory related disorders.
VO-037
Role of autophagy in cancer
K . Datta1 1Department of Biochemistry, University of Nebraska Medical Center,
Nebraska, United States
Autophagy is a regulated catabolic pathway that promotes lysosomal
degradation of damaged proteins, cellular organelles, and other macromolecules.
This self-digestion process, which facilitates the recycling of
bioenergetic components, is activated by a number of stimuli, including
the presence of reactive oxygen species, deprivation of growth factors,

DNA damage, and cytotoxic drugs. Autophagy dysregulation is associated
with a number of disease states, including cancer. Autophagy plays
different roles during the initiation and progression of cancer. While
autophagy acts as a tumor suppressor during the initiation phase of cancer,
it promotes tumor progression and metastasis in established cancers.
Metastatic cancer cells that usually grow in a nutrient-poor microenvironment
utilize autophagy to fulfil their high metabolic demand. Autophagy
can facilitate survival during anchorage-independent growth
or anoikis, and promotes therapeutic resistance. Furthermore, recent
studies indicated that genetic or pharmacologic inhibition of autophagy
sensitized tumor cells to anti-cancer treatment. It is therefore important
to study the role of autophagy and its regulations in cancer cells, which
will help defining optimal strategies to modulate autophagy for therapeutic
advantage.
VO-038
Markers of autophagy in cancer
M . Muders1 1University Hospital Carl Gustav Carus at the University of Dresden,
Institute of Pathology, Dresden
Autophagy has been implicated in cancer progression and therapy resistance.
Accordingly, different methods to identify and quantify autophagy
in tissue samples and cell culture models will be applied more frequently.
The traditional way to visualize autophagy at the ultrastructural
level is electron microscopy. Electron microscopy has the ability to detect
important structures that are involved during lysosomal degradation of
organelles like autophagosomes. Easier and more cost effective is the immunohistochemical
detection of autophagy substrates like LC3 or p62.
In addition, proteins involved in autophagy like Beclin-1 could serve as
an indicator of autophagy. In cell culture models, functional studies like
detection of autophagic flux as well as long-lived protein degredation are
used to monitor autophagic activity. However, all methods have their limitations
and should be applied only after careful consideration of their
strengths and weaknesses.
Translationale Forschung und Diagnostik –
Mamma/Schilddrüse/Melanom
VO-039
Translationale Forschung und Diagnostik: Karzinome der Schilddrüse
A . Perren1 , A .M . Schmitt 1 , M . Dettmer2 1 2 Institut für Pathologie, Bern, Switzerland, University of Pittsburgh, Department
of Pathology and Laboratory Medicine, Pittsburgh, United States
Histopathology and treatment of thyroid carcinomas poses several challenges:
On a diagnostic level there is a group of tumors difficult (or impossible)
to classify as benign or malignant, more importantly, the 10%
of patients that cannot be cured by surgery and radio-iodine treatment
is difficult to predict. The grey zone of follicular thyroid tumors of unknown
malignant potential and morphological criteria of an adverse
outcome (poorly differentiated thyroid carcinomas and tall-cell papillary
thyroid carcinomas) will be discussed.
On a molecular level, well differentiated thyroid carcinomas are well
classified and the genetic basis is well known, however genetic events
during carcinoma progression are less well understood. The most important
genetic changes helping diagnosis, determination of prognosis
will be discussed. Their role in guiding future targeted therapy will have
to be shown in clinical trials.
VO-040
Translational research and diagnostics: Melanoma
J . Rüschoff and Panel Members of DGP/BDP BRAF Testing Ring Study 1
1Pathologie Nordhessen
Most recently the first molecularly defined targeted therapy in metastatic
and/or irresectable melanoma has been approved by EMA (20.2.2012).
Vemurafinib (Zelburaf) is a small molecule that selectively inhibits BRAF
kinase in its mutated form. About 50% of metastatic melanoma exhibit
mutations within the BRAF oncogene almost exclusively at codon V600
activating the RAS-RAF-MEK-ERK signal transduction pathway. About
90% of mutations lead to an exchange of valin and glutamate (V600E).
Within a large phase III clinical trial (BRIM3) median survival of Vemurafinib
treated patients was 5.3 months instead of 1.6 months after chemotherapy
with response rates of 48.4% versus 5.5%, respectively (Chapman
PB et al. NEJM 2011; Sosman JA et al. NEJM 2012).
In light of these data mutation testing of BRAF is becoming standard
of care in malignant melanoma. This raises the question about testing
methods and quality assurance. A working group of the DGP and BDP
has addressed these aspects by performing a ring study where 9 Institutes
of Pathology together with their clinical colleagues participated.
Recommendations of testing and evaluation have been determined and
a QUIP-based approach of quality assurance will be available in 04/2012
headed by the Universities of Heidelberg and Berlin [1]. Sensitivity and
specificity of testing platforms (Sanger-, Pyro-, 454-Sequencing, real-time-PCR-based
cobas® 4800) will be discussed together with the need of
a strict tissue based approach making use of tumor cell enrichment, e.g.
by microdissection.
In addition to BRAF testing in melanomas further mutation analyses
will be needed, e.g. of NRAS and CKIT, where targeted drugs are either
available (CKIT) or under development (NRAS). Potential impact of
new targeted drugs on testing probably in specific tumor subtypes such
as acral or mucosal melanoma will be discussed.
References
1 . Panel Members: M . Dietel (Berlin), A . Enk (Heidelberg), A . Lehmann (Berlin),
J .N . Bauer (Tübingen), C . Garbe (Tübingen), U . Kellner (Minden), T . Kirchner
(München), A . Jung (München), H . Kreipe (Hannover), S . Merkelbach-Bruse
(Köln), R . Büttner (Köln), W . Schlake (Gelsenkirchen), P . Schirmacher (Heidelberg),
R . Stadler (Minden) als Verfasser entsprechender Ankündigungspublikationen in
JDDG und Der Pathologe, eingereicht 2012 .
Ausgewählte Vorträge aus den Einsendungen
(Hauptprogramm und Arbeitsgemeinschaften)
AG Gastroenteropathologie I – Leber
DO-001a
miR-101 is involved in steatosis and steatohepatitis of Non-
Alcoholic Fatty Liver Disease (NAFLD)
K .S . Ommer1 , N . Elfimova1 , A . Noetel1 , H .-P . Dienes1 , M . Odenthal1 ,
N . Winkler 2 , M . Quasdorff2 , I . Strack1 , J . Riemer1 1 2 University Hospital of Cologne, Institute for Pathology, Köln, University
Hospital of Cologne, Department of Gastroenterology and Hepatology, Köln
Aims. The Non-Alcoholic Fatty Liver Disease (NAFLD) is a rising widespread
disease. Frequently, steatosis results in steatohepatitis (NASH),
however the factors, responsible for inflammatory progression, are yet
unknown. Since previous profiling studies have pointed to miR-101 as
a candidate involved in steatohepatitis, we have studied the role of miR-
101.
Der Pathologe · Supplement 1 · 2012 |
15

Abstracts
Methods. LDL-receptor knockout mice were fed with a fatty diet. Subsequently,
the miR-101 level was detected by Real-Time PCR. Primary
hepatocytes of mice as well as human hepatoma cells were stimulated
with free fatty acids or with proinflammatory factors TNF-a or IL-6.
Following, the cellular and extra-cellular miR-101 levels were analyzed
by PCR. Additionally, miR-101 was quantified in histological characterized
biopsies (NASH-score 1–8) and serum samples of 55 patients with
NAFLD (Ntissue, Nserum=55) and correlated to clinical data as well as
liver apoptosis determined by M30/M65 measurement.
Results. The expression of miR-101 was increased in fatty livers of LDLreceptor
mouse model as well as in human patients with steatosis. In
contrast, the miRNA level was diminished in livers with inflammatory
changes. Both, the miR-101 enhancement in fatty liver as well as repression
of miR-101 upon inflammation could be mimicked in vitro in
a hepatoma cells stimulated with free fatty acids or proinflammatory
mediators, respectively. Interestingly, miR-101 was also detected in human
serum. These circulating miR-101 levels were associated with the
M30 apoptosis values and with grades of steatosis and inflammation. In
addition, circulating miR-101 levels inversely correlated to the expression
of miR-101 in liver tissues.
Conclusions. In NAFLD, the expression of miR-101 in liver tissues is contrarily
influenced by free fatty acids and proinflammatory mediators.
Alterations of miR-101 serum levels are suggested to indicate NAFLD
progression into steatohepatitis.
DO-002a
Regulation and function of the nuclear transport factor CAS in
hepatocarcinogenesis
J . Winkler1 , J . Samarin1 , V . Ehemann1 , K . Breuhahn1 , P . Schirmacher1 , S . Singer1 1Institute of Pathology/University Hospital Heidelberg, Heidelberg
Aims. There is rising evidence that deregulation of nuclear transport
factors contributes to cancer formation. An important member of the
nucleocytoplasmic transport machinery is the RanGTPase dependent
exporter CAS (Cellular Apoptosis Susceptibility) that recycles importinalpha
from the nucleus to the cytoplasm. CAS was also shown to bind to
p53 target gene promoters (e.g. PIG-3) and to be involved in apoptosome
formation. Considering these pro-apoptotic properties high expression
levels of CAS observed in different malignant tumors suggest additional
protumorigenic functions. Here, we analyzed the expression, function,
and regulation of CAS in hepatocarcinogenesis.
Methods. CAS expression analyses in 188 HCCs, 9 dysplastic nodules
and 20 normal liver samples were performed by using immunhistochemistry
and in a subset of samples by semiquantitative real-time PCR
(qRT-PCR). The impact of siRNA mediated CAS knockdown on cell viability,
cell cycle, and apoptosis was analyzed in different HCC cell lines
by using MTT-assays and FACS. The role of p53 and mTOR (the mammalian
target of rapamycin) in regulating CAS expression in HCC cell lines
was investigated by Westernblot (WB) and qRT-PCR upon treatment
with appropriate compounds.
Results. CAS was overexpressed on mRNA and protein level in up to
~70% of the HCCs analyzed and its expression was positively correlated
with tumor dedifferentiation, proliferation (Ki-67) and nuclear accumulation
of p53. A significantly decreased cell viability and increased
apoptosis was observed upon CAS knockdown. Induction of wild-type
p53 by Nutlin-3 led to reduced CAS protein and mRNA expression levels.
Lowered levels of CAS protein were also observed after Rapamycin
(mTOR inhibitor) treatment.
Conclusions. Our data suggest a protumorigenic role of CAS in hepatocarcinogenesis
apparently linked to a pro-survival function. We also
conclude that CAS is a target of p53 mediated repression and identified
mTOR as a positive regulator of CAS expression. Future studies in vitro
and in vivo are required to gain further mechanistic insights into CAS
dependent functions and to examine if CAS is a potential therapeutic
target in HCC.
16 | Der Pathologe · Supplement 1 · 2012
DO-003
Molecular and functional analysis of long non-coding RNAs in
hepatocellular carcinoma
M . Hämmerle1 , T . Gutschner1 , M . Polycarpou-Schwarz 1 , C . Hildenbrand1 ,
K . Breuhahn2 , T . Longerich2 , P . Schirmacher2 , S . Diederichs1 1Institute of Pathology, University Hospital & German Cancer Research
Center (DKFZ), Heidelberg, 2Institute of Pathology, University Hospital,
Heidelberg
Aims. The vast majority of the human genome is represented by nonprotein-coding
RNAs (ncRNAs), which are ribonucleic acids of different
lengths without an open reading frame. Recently, different functions
have been attributed to the few well-characterized ncRNAs, e.g. in epigenetics
and cancer. However, the function of most of the newly discovered
long ncRNAs is still unknown and a detailed analysis is lacking.
Since cancer research has focused on protein-coding genes for the last
decades, the potential of involvement of ncRNAs in the pathogenesis and
prognosis of hepatocellular carcinoma (HCC) is not known so far. Therefore,
our study aimed at identifying differentially expressed ncRNAs
in HCC compared to control liver samples and at elucidating their role
on the cellular and molecular level in order to draw conclusions about
their contribution to the development of HCC.
Methods. We screened for the expression of 17,000 ncRNAs in 32 cases of
HCC and 7 control tissue samples. After identifying tumor-specific candidates,
their expression was validated in HepG2 and Huh7 cells. Their
impact on cell viability was uncovered after siRNA-mediated ncRNA
knockdown in liver cancer cell lines. By using RNA affinity purification
(RNA-AP), protein interaction partners were identified.
Results. Statistical analysis unravelled 187 upregulated and 278 downregulated
ncRNAs in HCC. One ncRNA, LOHC (Long non-coding RNA
Overexpressed in Hepatocellular Carcinoma), was highly expressed in
liver cancer compared to normal liver patient samples. Knockdown of
LOHC expression significantly reduced cell viability and influenced
cell cycle progression of HepG2 and Huh7 cells. Using RNA-AP, IGF2
mRNA binding proteins (IMPs) were identified as LOHC interaction
partners. Moreover, interaction of LOHC and IMPs was largely different
in diverse stages of cell cycle, which additionally influenced LOHC expression
and stability over time.
Conclusions. LOHC is an important ncRNA in HCC, which regulates cell
viability and cell cycle. It was found to be an interaction partner of IMPs,
which can regulate LOHC stability and have a major role in the pathogenesis
of liver cancer. These data show that besides protein-coding genes,
the expression of ncRNAs could be highly and specifically regulated in
HCC, which will allow conclusions about the use of ncRNAs as potential
diagnostic and prognostic markers. Most importantly, ncRNA expression
profiling in cancer has identified functionally important players in
liver tumorigenesis.
DO-004
miR-125b regulates the lin28/IGF-II axis during hepatocellular
carcinogenesis
N . Elfimova1 , K .S . Ommer1 , N . Winkler2 , M . Quasdorff2 , I . Strack1 , J . Riemer1 ,
A . Noetel1 , H .-P . Dienes1 , M . Odenthal1 1 2 University Hospital of Cologne, Institute for Pathology, Köln, University
Hospital of Cologne, Department of Gastroenterology and Hepatology, Köln
Aims. MicroRNA (miRNA), involved in posttranscriptional regulation
of gene expression, play an important role in cell proliferation and differentiation.
miR-125b expression was shown to be divergently expressed
in liver carcinogenesis. Here, we focused on the role of miR-125b in development
of hepatocellular carcinoma (HCC).
Methods. A Cre-expressing adenoviral vector was applied to Alb-SV40
T-Ag transgenic mice in order to induce liver carcinogenesis. Expression
levels of miR-125b were determined at different time points of tumorgenesis
and in human hepatoma cell lines. Additionally, from 52 human

HCV-positive formalin-fixed and paraffin-embedded biopsies, sections
were prepared and HCC were macrodissected. Subsequently, miR-125b
was analyzed by real-time PCR. Putative miR-125b binding sites were fused
to the luciferase reporter and reporter assays were carried out with
miR-125b treated hepatoma cells.
Results. During development of mouse HCC, the expression of miR-125b
progressively decreased. In agreement miR-125b was reduced in human
hepatoma cells in comparison to normal liver. Furthermore, miR-125b
decrease depending on the progression of hepatocarcinogenesis was
confirmed in human samples, showing significant lower levels in HCC
high grades than in dysplastic foci or cirrhosis. Overexpression of miR-
125b in Hep3B and Pop10 cells resulted in a pronounced reduction of cell
growth. Screening of putative miR-125b target transcripts by various
algorithm calculations identified various pathways involved in proliferation
and apoptosis. Reporter assays of 3’-UTR-regions of the putative
targets identified miR-125b binding sites in lin-28 mRNA. Since lin28 is
known to effect synthesis of the mitogen IGF-II, the miR-125b/lin28 axis
is suggested to be involved in HCC pathogenesis by IGF-II mediated regulation
of cell growth.
Conclusions. Expression of miR-125b is down-regulated during progression
of hepatocarcinogenesis leading to up-regulation of lin-28 that in
turn triggers enhanced cell growth and proliferation.
DO-005
The PI3K-AKT-mTOR axis contributes to the functional inactivation
of p53 through stabilization of MDM4 in human hepatocellular
carcinoma
R . Pellegrino1 , O . Neumann1 , P . Schirmacher1 , T . Longerich1 1University Hospital Heidelberg/Institute of Pathology, Heidelberg
Aims. Mutational inactivation of p53 gene is rare in Western hepatocellular
carcinoma (HCC). MDM4, one of the main p53-regulating factors,
is frequently upregulated in human HCC. This overexpression can be
in part explained by chromosomal gains at 1q34.1. Here we investigated
the role of the PI3K-AKT axis in the stabilization of the MDM4 protein.
Methods. All experiments were performed in human HCC cell lines
with different p53 gene status. PI3K and mTOR were specifically inhibited
using chemical compounds in vitro; specific siRNAs were transiently
transfected to target AKT1 and ATK2 and to validate the results from
drug treatment. Realtime RT-PCR analysis was used to check for restored
p53 transcriptional activity. In addition, combined cycloheximide
and siRNAs treatment was performed to study the protein stability of
MDM4 under these experimental conditions.
Results. Using a specific PI3K inhibitor or siRNAs targeting AKT1/2 in
HCC cell lines, we observed a strong decrease of MDM4 protein levels,
which resulted in the activation of p53 target genes (e.g. PUMA, BAX and
p21) indicating restored p53 gene function in these lines. Cycloheximide
treatment combined with siRNA-mediated AKT inhibition indicated
that MDM4 is phosphorylated by AKT2 and that this phosphorylation
is responsible for the stabilization of the protein via the protection from
proteasomal degradation. This effect was independent from both MDM2
and p53 gene status. Furthermore, we showed that the Eukaryotic translational
Elongation Factor 1 alpha 2 (EEF1A2), which we reported upregulated
in human HCC, is involved in the activation of the PI3K-AKT
axis. Specific siRNA inhibition of EEF1A2 resulted in decreased pAKT
and MDM4 protein levels in HCC cell lines. Moreover, treatment with
the mTOR inhibitors, Rapamycin and PI-103, decreased MDM4 protein
levels indicating that the PI3K-AKT-mTOR axis is involved in the
MDM4 regulation in vitro.
Conclusions. Our data demonstrate that the EEF1A2-PI3K-AKT-mTOR
axis is involved in maintaining protumorigenic MDM4 levels in human
HCC cell lines, which in turn promotes functional inactivation of
p53. Moreover, we showed that the AKT-mediated phosphorylation of
MDM4 is the crucial mechanism to prevent its proteasomal degradation.
DO-006
HSF1 is a downstream effector of Ras and AKT protooncogenes
and contributes to hepatocellular carcinoma development and
progression
D .F . Calvisi1 , S . Mattu1 , S . Delogu1 , V . De Murtas1 , G . Gasparetti1 , G . Destefanis1 ,
X . Chen2 , F . Dombrowski1 , M . Evert1 1University Medicine Greifswald, Institute for Pathology, Greifswald,
2University of San Francisco, Liver Center, San Francisco, United States
Aims. Recent evidence suggests an oncogenic role of heat shock transcription
factor 1 (HSF1) in cancer, but its functional relevance in hepatocellular
carcinoma (HCC) remains poorly delineated.
Methods. We have investigated HSF1 function both via in vitro and in
vivo approaches as well as in a collection of human HCC.
Results. In human liver specimens, we found that HSF1 was progressively
induced from non-tumorous surrounding livers to HCC, reaching the
highest levels in tumors with a poorer outcome (as defined by the length
of patient’s survival). In HCC cell lines, overexpression of HSF1 resulted
in increased activity of MAPK and AKT/mTOR pathways and suppression
of JNK cascade, leading to augmented proliferation and angiogenesis
and reduced apoptosis in vitro. Conversely, suppression of HSF1 in
HCC cell lines decreased MAPK and AKT/mTOR activity, and induced
JNK-dependent apoptosis. Forced overexpression of either Ras or AKT
protooncogenes triggered upregulation of HSF1 in HCC cell lines via the
small RalA GTPase. Of note, HSF1-overexpressing cells were specifically
sensitive to growth inhibition and induction of apoptosis following the
treatment with either AMPK activators or hexokinase inhibitors. Finally,
overexpression of a HSF1 dominant negative form by hydrodynamic
gene delivery strongly reduced the oncogenic potential of activated Ras
and AKT in a mouse model of aggressive liver cancer.
Conclusions. Altogether, the present data indicate that activation of HSF1
plays a major role in hepatocarcinogenesis by enhancing the activity of
Ras and AKT, and might represent a valuable candidate for innovative
targeted therapies against human HCC.
DO-007
Perturbation of hepatocytes metabolism by AKT contributes to
growth in insulin-induced hepatocarcinogenesis and is reverted
by the PI3K/mTOR dual inhibitor NVP-BEZ235
M . Evert1 , D .F . Calvisi1 , K . Evert1 , V . De Murtas1 , G . Gasparetti1 , S . Mattu1 ,
G . Destefanis1 , S . Thiel1 , A . Thiele1 , S . Ribback1 , F . Dombrowski1 1University Medicine Greifswald, Institute for Pathology, Greifswald
Aims. Mounting evidence supports a role of insulin signaling deregulation
and diabetes mellitus in human hepatocarcinogenesis. To study the
oncogenic effect of chronically elevated secretion of insulin on hepatocytes
in the presence of mild hyperglycemia, we developed a model of
pancreatic islet transplantation into the liver.
Methods. In this model, islets of a donor rat are transplanted into the
liver of a recipient diabetic rat, with resulting local hyperinsulinism that
leads to the development of preneoplastic lesions and hepatocellular carcinoma
(HCC). Here, we investigated the metabolic and growth properties
of the AKT pathway in this model of insulin-induced hepatocarcinogenesis.
These findings were recapitulated in HCC cell lines in vitro.
Results. We found that activation of insulin signaling triggers a strong
induction of the AKT cascade that is paralleled by increased synthesis
of fatty acids, cholesterol, and triglycerides, induction of glycolysis and
decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic
and neoplastic liver lesions when compared with normal liver. AKT-dependent
metabolic effects of insulin on hepatocytes were recapitulated in
vitro using human HCC cell lines. In these cells, suppression of lipogenesis,
glycolysis, and the pentose phosphate pathway triggered a strong
growth restraint despite insulin administration. Of note, metabolic abnormalities
and proliferation driven by insulin were effectively reverted
Der Pathologe · Supplement 1 · 2012 |
17

Abstracts
using the dual PI3K/mTOR inhibitor, NVP-BEZ235, both in vitro and
in vivo.
Conclusions. Thus, AKT activation by unconstrained insulin signaling
induces a defined module of metabolic alterations in hepatocytes contributing
to aberrant cell growth. The inhibition of AKT and related
metabolic changes might represent a novel preventive and therapeutic
approach to effectively inhibit insulin-induced hepatocarcinogenesis.
AG Gastroenteropathologie IV – Oberer GI-Trakt
DO-015
STAT3 activation and Mcl-1 and MMP9 target gene expression is
preferentially seen in esophageal squamous cell carcinomas, but
not Barrett‘s adenocarcinomas
S . Timme1 , K . Atanasov1 , C .D . Fichter 1 , A . Schoepflin1 , L . Bogatyreva2 ,
D . Hauschke2 , L . Tang 3 , H . Geddert4 , G . Faller 4 , D . Klimstra 3 , O . Opitz5 ,
M . Werner1 , S . Lassmann1 1 2 Institute of Pathology, University Medical Center, Freiburg, Institute of
Medical Biometry and Medical Informatics, University Medical Center,
Freiburg, 3Dept . of Pathology, Memorial Sloan Kettering Cancer Center, New
York, United States, 4Institute of Pathology, St-Vincentius-Kliniken, Karlsruhe,
5Tumorzentrum Ludwig Heilmeyer – CCCF, Freiburg
Aims. Active (phosphorylated) signal transducer and activator of transcription
3 (P-STAT3) is translocated to the nucleus. By this, (P-)STAT3
suppresses apoptosis or induces cell migration via Mcl-1, Bcl-xl and Survivin
or matrix metalloproteinases (e.g. MMP9) expression, respectively.
This STAT3 activity can be triggered by an active EGFR. To complement
our data on P-STAT3 expression in esophageal squamous cell carcinomas
(ESCC) and Barrett’s adenocarcinomas (BAC), we investigated
EGF-mediated STAT3 activity in ESCC and BAC cell lines as well as inactive
STAT3 expression in ESCCs and BACs.
Methods. Serial sections of 105 esophageal carcinomas (n=60 BAC; n=45
ESCC) were evaluated for STAT3 expression by semi-quantitative immunohistochemistry.
Data of nuclear P-STAT3 expression was already
available. Statistical analysis was performed using Mann-Whitney-U
tests at p

well as MET activation were examined by Western blot analysis, flow
cytometry and immunofluorescence staining. Cells were treated with
varying concentrations of cetuximab and cisplatin and 5-fluorouracil in
tumor relevant concentrations. The biological end point was cell viability,
which was measured by XTT cell proliferation assay. Response to
treatment was evaluated using statistical methods.
Results. We assessed the activity of cetuximab in five gastric cancer cell
lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two
cell lines, MKN1 and MKN28, was significantly reduced by cetuximab
treatment. High EGFR expression and low levels of receptor activation
were associated with cetuximab responsiveness. MET activation as well
as mutations of KRAS and E-cadherin were associated with cetuximab
resistance.
Conclusions. These data indicate that our examinations may be clinically
relevant and the candidate markers should therefore be tested in clinical
studies.
DO-018
Notch2 expression and chemoresistance in neoadjuvant treated
gastric cancer
L . Bauer1 , R . Langer1 , M . Mandl1 , K . Becker1 , J . Slotta-Huspenina1 , A . Novotny2 ,
A . Hapfelmeier3 , H . Höfler1 , G . Keller1 1Technische Universität München, Department of Pathology, München,
2Technische Universität München, Department of Surgery, München,
3Technische Universität München, Department of Medical Statistics and
Epidemiology, München
Aims. In a recent study analyzing the prognostic significance of the expression
of cancer stem cell (CSC) related genes in residual gastric tumor
cells after neoadjuvant chemotherapy, Wnt and Notch signaling genes,
among others, showed a prominent association with survival. The aim of
this study was to assess selected genes for differential expression between
pretherapeutic biopsies and resected specimens. In vitro we investigated
the impact of Notch activity on chemosensitivity in gastric cancer cell
lines.
Methods. Expression of 12 genes was compared between corresponding
biopsies and resected specimens from patients treated with neoadjuvant
chemotherapy (CTx) demonstrating partial (n=22) or minimal/
no tumor regression (n=22). mRNA was isolated from macrodissected
FFPE tissues and gene expression was quantified by real time PCR using
TaqMan® low density arrays. Immunohistochemical staining (IHC) for
Notch2 was performed on biopsy/resected-specimen pairs from patients
with sub-total, partial and minimal/no tumor regression (n=22, each)
and from patients not treated by CTx. (n=16) and evaluated by a semiquantitative
scoring system. Chemosensitivity of three gastric cancer
cell lines to the gamma-secretase inhibitor DAPT alone or in combination
with cisplatin was determind by XTT or colony formation assays.
Results. Differential expression analysis revealed an increase of Notch2
and POU5F1 from biopsies to resected tumors in tumors with partial
response (p=0.002 and 0.028) and minimal/non responding tumors
(p=0.062 and 0.002). In contrast a decrease in expression was observed
in both tumor groups for Notch1 (p=0.072 and 0.001). Immunhistochemical
analysis of Notch2 revealed that cytoplasmic staining intensities of
tumor cells decreased significantly in all groups of CTx-treated patients
(p=0.016, .001 and 0.017) but not in non-CTx patients. IHC analysis of
Notch1 is in progress. Treatment of gastric cancer cell lines with 10 µM
DAPT and 2 µM cisplatin led to a synergistic reduction of metabolic activity
in comparison to the single drugs.
Conclusions. The comparison of mRNA expression between corresponding
biopsies and resected specimens revealed alterations consistent with
an enrichment of chemotherapy resistant residual tumor cells. Results of
Notch2 IHC are in line with the mRNA data. The synergistic effect of
cisplatin and the gamma-secretase inhibitor DAPT in vitro suggests that
Notch signaling might be involved in chemoresistance of gastric cancers.
AG Gastroenteropathologie VI – Unterer GI-Trakt
DO-020
Aurora-A protein expression is associated with multipolar mitoses
independent of molecular class of colorectal carcinomas*
D . Batarello 1 , A . Schoepflin1 , D . Hauschke2 , M . Werner3 , S . Lassmann1 1Institute of Pathology, University Medical Center Freiburg, Freiburg,
2Institute of Medical Biometry and Medical Informatics, University Medical
Center Freiburg, Freiburg, 3Institute of Pathology, University Medical Center
Freiburg
Aims. Aurora-A overexpression may induce supernumerary centrosomes,
respective multipolar mitoses, and aneuploidy in model systems.
Here, we examined the occurrence of Aurora-A positive multipolar mitoses
in aneuploid (microsatellite-stable, CIN-type) versus near-diploid
(microsatellite-instable, MIN-type) colorectal carcinomas (CRC).
Methods. Three-dimensional immunofluorescence (3D-IF) of Aurora-A
was performed on 8µm thick FFPE tissue sections of 18 previously characterized
colorectal carcinomas. Stained sections were screened by a
x63/1.3 oil objective at 0.7µm image stacks (one x63 field = high power
field/HPF; total of 374 HPFs, range 10–46 HPF per case). Total numbers
of mitoses (n=476, range 11–57 per case) and numbers of bipolar (2 Aurora-A
positive centrosomes/spindle poles) and aberrant multipolar (>2
Aurora-A positive centrosomes/spindle poles) mitoses were counted.
For differences of mitotic counts and frequencies between CIN-type and
MIN-type CRCs, the Wilcoxon Test (exact, two-sided; with p

Abstracts
cosa. MiR-214 expression was stably reconstituted in the colorectal cancer
cell lines RKO (CIMP+; MSI) and SW480 (CIMP−; MSS) via retroviral
gene transfer. Its impact on tumor growth and response to oxaliplatin
and 5-FU treatment was analyzed by MTT assays, Annexin V staining
and cell cycle analysis by flow cytometry.
Results. Up-regulation of miR-214 in CIMP+ colorectal adenocarcinomas
could be demonstrated in more than 45% of the tumor specimens
analyzed. MiR-214 overexpressing cells showed an increased proliferation
regardless of the CIMP background in vitro. Further, we observed
that only the CIMP+ colorectal adenocarcinoma cell line RKO overexpressing
miR-214 was more resistant to oxaliplatin as well as 5-FU treatment.
Conclusions. Up-regulation of miR-214 expression is frequently observed
in CIMP+ tumors. Its increased expression is linked to a higher growth
rate. Additionally, we were able to show that the overexpression of miR-
214 in a cell line with a CIMP+ phenotype leads to a 5-FU and oxaliplatin
chemoresistance.
DO-022
EWSR1: Identification and functional characterization of a novel
target gene locus in Lynch syndrome
S . Piscuoglio1 , S . Kishore2 , V . Mele3 , F . Trapani3 , M . Zavolan2 , M . Kovac1 ,
L . Terracciano 3 , K . Heinimann1 1 2 University of Basel, Department of Biomedicine, Basel, Switzerland, University
of Basel, Biozentrum and Swiss Institute of Bioinformatics, Basel,
Switzerland, 3University of Basel, Institute of Pathology, Basel, Switzerland
Aims. Lynch syndrome represents the most common, autosomal dominantly
inherited cancer predisposition worldwide and accounts for 3-5%
of the total colorectal cancer (CRC) burden. It is caused by germline
mutations in DNA mismatch repair (MMR) genes. MMR deficiency results
in microsatellite instability (MSI). MSI, used as a diagnostic tool to
identify HNPCC-related CRCs, can also affect repeat tracts within or
immediately adjacent to the coding sequence of so-called target genes
which are thought to fuel carcinogenesis in HNPCC. In search for novel
target gene loci we identified a large poly-T tract, (T)16, in the 3’ untranslated
region of the Ewing sarcoma breakpoint region 1 (EWSR1) gene.
Our aims are: to determine 1) type and frequency of instability at the
EWSR1 (T)16 in 78 HNPCC and 123 sporadic colorectal cancers and 2) its
possible effect on EWS expression.
Methods. We determined the length of the EWSR1 3’UTR tract motif,
(T)16, by PCR amplification and fragment analysis of 78 CRCs from
HNPCC patients with identified germline mutation, 123 sporadic microsatellite-stable
CRCs as well as 5 CRC cell lines (2 MMR proficient,
3 MMR deficient). EWS protein expression was assessed by immunohistochemistry
(IHC) on a tissue microarray and immunoreactivity scored
semi-quantitatively. Statistical comparisons were performed using
Chi-square or Student’s t test where appropriated (two-tailed p-values,
considering p6 (11.92%). Similar observations
were made for the MMR-deficient cell lines whereas the MMR-proficient
ones were stable. RNA secondary structure prediction suggested
gross structural alterations for deletions >4 Ts. IHC showed significant
downregulation of EWS expression in sporadic CRCs (p

or BRAF mutation, c-MYC and SIRT1 expression was not found increased.
In addition, within the group of serrated lesions with wild type
KRAS and BRAF, a subgroup was characterized by elevated c-MYC and
SIRT1 expression. In these lesions with mostly high grade intraepithelial
neoplasia and carcinomas, nuclear localization of β-catenin suggested
that activation the wnt signalling pathway may mediate the induction of
c-MYC at the transcriptional level.
Conclusions. In summary, we established a link of oncogenic K-Ras and
B-Raf as well as wnt signalling to activation of the c-MYC oncogene and
SIRT1 in the serrated route to colorectal cancer. The elevated expression
levels observed within higher grades of malignancy point to a crucial
function of c-MYC and SIRT1 in the malignant transformation.
AG Pneumopathologie I
DO-025
HOPE-technique improves diagnostics of Bronchoalveolar
Lavage (BAL)
E . Vollmer 1 , S . Marwitz1 , M . Abdullah1 , C . Vock1 , J .S . Fine2 , S . Visvanathan2 ,
K . Gaede1 , H .-P . Hauber1 , P . Zabel1 , T . Goldmann1 1 2 Research Center Borstel, Borstel, Roche, Inflammation Discovery,
United States
Aims. Besides its application in pulmonary routine diagnostics BAL is
a useful tool for scientific investigations. Because of its limitations in
long term storage we explored in this study the utility of a novel fixative
(HOPE, Hepes glutamic acid buffer mediated Organic solvent Protection
Effect) for retrospective and standardized cell analysis also in regard of
immunological and molecular techniques. This study has been performed
in accordance with the 1964 Declaration of Helsinki and its later
amendments.
Methods. BAL samples, obtained by flexible bronchoscopy from patients
with different diseases, were diluted to a standard cell number of
one million cells and incubated in HOPE-fixative as well as in neutral
buffered 4% formalin with a subsequent paraffin-bloc-embedding. In
addition, for addressing RNA preservation, fresh frozen samples were
included. For enhanced/expanded high-throughput analyses of multiple
BAL samples tissue microarrays of paraffin embedded HOPE-BAL were
also produced.
Results. We have shown that HOPE-BAL cells have an excellent morphology;
besides that this technique allows archiving of BAL cells. By
preservation of proteins and nucleic acids it allows the application of immunocytochemistry
as well as a plethora of molecular techniques like in
situ hybridization, quantitative PCR, transcription microarray analysis,
2-D-Gel-Electrophoresis etc. We showed by targeting some exemplary
molecules the power of screening and validating HOPE-BAL for new
biomarkers.
Conclusions. The HOPE-BAL-technique allows long term storage of BAL
cells and is a unique and novel tool for various molecular based applications
in pulmonary medicine. It combines easy handling in the form of
paraffin blocks with almost no limitations in readout techniques thus
being a step forward into enhanced molecular diagnostics and biobanking.
DO-026
Tissue sparing application of the newly proposed IASLC/ATS/
ERS classification of non-small cell lung cancer shows practical
diagnostic and prognostic impact
W . Sterlacci1 , S . Savic2 , T . Schmid3 , M . Fiegl4 , A . Tzankov 2
1 2 Academic Teaching Hospital Feldkirch, Feldkirch, Austria, University Hospital
Basel, Switzerland, 3Medical University Innsbruck, Center of Operative
Medicine, Austria, 4Medical University Innsbruck, Department of Internal
Medicine, Austria
Aims. The histologic subtype of non-small cell lung cancer (NSCLC) determines
treatment strategies and the need for genetic analyses. Since
most NSCLC are diagnosed on small biopsies or cytology specimens, an
accurate but also tissue sparing approach is necessary. To date, consensus
for a general diagnostic algorithm is lacking.
Methods. To test the diagnostic and clinical relevance of the recently published
multidisciplinary guidelines by the International Association for
the Study of Lung Cancer, American Thoracic Society and European Respiratory
Society, we examined 371 surgically resected NSCLC brought
into tissue microarray format as a surrogate for small biopsies. Adenocarcinomas
(ACA) were graded according to architecture considering
lepidic as well, acinary and papillary as moderately and micropapillary
and solid as poorly differentiated.
Results. The antibody panel TTF-1, p63, CK5/6 and CK7 proved diagnostic
for most cases. The positive predictive value (PPV) of p63 and CK5/6
for squamous cell carcinoma (SCC), and of CK7 and TTF1 for ACA was
88.9%, 84.9%, 88.4% and 97.7%, respectively. The negative predictive value
(NPV) of p63 and CK5/6 for SCC, and of CK7 and TTF1 for ACA was
99.5%, 99.5%, 93.6% and 76.9%, respectively. Faint/focal staining for CK7
is negligible for classificatory purposes and focal expression of TTF-1
with variable staining intensity is a feature compatible with SCC (approximately
3% of cases). Overall survival in months for ACA according to
architecture-based tumor grade was 72.5 for well, 71.0 for moderate and
35.7 for poor differentiation (p=0.039).
Conclusions. We propose double stains combining an above mentioned
nuclear and membranous marker, which is highly diagnostic for NSCLC
on small biopsies while conserving tumor tissue for subsequent analyses.
No recommendations using less than 2 sections exist, however a panel
consisting of TTF-1 in combination with CK5/6 may be feasible, since
TTF-1 appears to be the most specific discriminating marker between
ACA and SCC (best PPV for ACA) and the only unequivocally evaluable
staining combination is with cytoplasmic staining for CK5/6, which
also achieved the best NPV for ACA. When grading ACA, the histologic
tumor architecture should be the determining factor. This approach primarily
has prognostic implications but will also result in easier comparisons
of future studies.
DO-027
Multi-immunassay with concurrent staining of 6 antibodies
allows tissue-sparing diagnosis on small tissue samples on nonsmall
cell lung carcinomas with high diagnostic accuracy
G . Kayser1 , A . Csanadi 1 , C . Otto1 , S . Dango2 , B . Passlick2 , M . Werner1 1 2 Institute of Pathology, University Hospital Freiburg, Freiburg, Department
of Thoracic Surgery, University Hospital Freiburg, Freiburg
Aims. Today the histological differentiation of non-small cell lung carcinomas
NSCLC into adenocarcinomas (LAC), squamous cell carcinomas
(SCC), and large cell neuroendocrine carcinomas (LCNEC) is not only of
prognostic relevance but far more of predictive value for different therapeutic
regimes. As these decisions are of utmost relevance in advanced
cancer stages, pathologists are asked to perform a highly accurate diagnosis
on small tissue samples. We here investigated the possibility of simultaneous
staining of widely agreed upon markers for the histological
classification of NSCLC.
Der Pathologe · Supplement 1 · 2012 |
21

Abstracts
Methods. Of 261 NSCLC patients tissue multi arrays (TMA) with a
core diameter of 2 mm were composed which served as a simulation
model for endobronchial biopsies. The TMAs were stained with TTF1
(8G7G3/1) and Vimentin (VIM3B4) visualized by amino-ethylcarbazol
first. Second staining was performed with p63 (4A4) and a neuroendocrine
(NE) cocktail (CD56 (NCL-L-CD56-1B6), synaptophysin (SPII),
chromogranin (DAK-A3)) visualized by diaminobenzidine. For histological
classification hematoxilin-eosin (HE) stained TMA-slides were
evaluated. Independently from HE-classification immunohistochemical
(IHC) classification was performed by a stepwise decision tree: 1) morphologically
clear adenoid or squamous growth patterns: LAC or SCC; 2)
TTF1 positive: LAC; 3) TTF1 negative and p63 positive: SCC; 4) TTF1 and
p63 negative: large cell carcinoma; 5) TTF1 negative, p63 negative, NE
positive: LCNEC. Statistical analyses included kappa-values and Kaplan
Meier survival curves.
Results. Evaluation of specific staining of the different antibodies was
easy to perform and compared to a set of carcinomas which were stained
with the multi-IHC protocol and each antibody separate did not show
any different staining patterns. Analyzing the inter-core variability, IHC
classification was superior to HE-diagnosis. Furthermore, a better separation
of the Kaplan-Meier survival curves could be achieved by IHC
classification as compared to HE classification alone.
Conclusions. Multi-immune assays for classification of NSCLC are feasible
and deliver more accurate results than HE-diagnosis alone. IHC
classification shows higher intra-tumor homogeneity. As different entities
are most probable to show different biological behavior IHC classification
delivers the best separation of survival curves and should thus be
applied to all lung cancer specimens for accurate pathological classification
on small biopsies.
DO-028
The novel IASLC/ATS/ERS classification is a stage-independent
predictor of survival and correlates with the response to adjuvant
therapies
A . Warth1 , T . Muley2 , M . Meister3 , A . Stenzinger1 , J . Cortis1 , M . Thomas4 ,
P . Schirmacher1 , P .A . Schnabel1 , J . Budczies5 , H . Hoffmann6 , W . Weichert1 1 2 University Hospital Heidelberg, Institute for Pathology, Heidelberg, Thoraxklinik
Heidelberg, Translational Research Unit, 3Heidelberg University
Hospital, Translational Research Unit, 4Thoraxklinik Heidelberg, Oncology,
5 6 Charité University Hospital Berlin, Institute for Pathology, Thoraxklinik
Heidelberg, Thoracic Surgery
Aims. Our aim was to analyze and to validate the prognostic impact of
the novel IASLC/ATS/ERS proposal for an architectural classification of
invasive pulmonary adenocarcinomas (ADC) across all tumor stages.
Methods. The architectural pattern of a large cohort of 500 resected ADC
(stages I–IV) was retrospectively analyzed in 5% increments and classified
according to their predominant architecture (lepidic, acinar, solid,
papillary, micropapillary), as proposed by the IASLC/ATS/ERS. Subsequently,
histomorphological data were correlated with clinical data, adjuvant
therapy and patient outcome.
Results. Overall survival differed significantly between lepidic
(78.5 months), acinar (67.3 months), solid (58.1 months), papillary
(48.9 months), and micropapillary (44.9 months) predominant ADC
(p=0.007). When patterns were lumped into groups this resulted in even
more pronounced differences in survival (pattern group 1: 78.5 months,
group 2: 67.3 months, group 3: 57.2 months, p=0.001). Comparable differences
were observed for overall, disease specific and disease free survival.
Pattern and pattern groups were stage- and therapy-independent
prognosticators for all three survival parameters. Survival differences
according to patterns were influenced by adjuvant radiochemotherapy,
especially solid predominant tumors had an improved prognosis under
adjuvant radiotherapy. The predominant pattern was tightly linked to
the risk of developing nodal metastases (p

AG Pneumopathologie III
DO-032
Remodelling-related molecular profiles in interstitial pulmonary
fibrosis
D . Jonigk 1 , J . Rische 1 , L . Maegel 1 , H . Golpon 2 , N . Izykowski 1 , C . Bockmeyer 1 , T .
Welte 2 , S . Janciauskiene 3 , J . Gottlieb 2 , G . Warnecke 4 , A . Haverich 5 , H . Kreipe 1 ,
F . Laenger 1
1 Hannover Medical School (MHH), Institute of Pathology, Hannover,
2 Hannover Medical School (MHH), Department of Pneumology, Hannover,
3 Hannover Medical School (MHH), Department of Respiratory Medicine,
Hannover, 4 Hannover Medical School (MHH), Department of Throracic Surgery,
Hannover, 5 Hannover Medical School (MHH), Department of Thoracic
Surgery, Hannover
Aims. Idiopathic pulmonary fibrosis (IPF) is the most important representative
of the idiopathic interstitial pneumonia group (IIP) and is a disease
characterized by an overall poor prognosis and unresponsiveness
to currently available therapies. Thus elucidation of molecular pathways
to gain better insight into the pathogenesis and identify potential therapeutic
targets is warranted.
Methods. We performed compartment-specific analyses using laser
microdissection, RT-PCR based microarray techniques and immunohistochemistry
in lung samples from well defined patients with UIP,
nonspecific interstitial pneumonia (NSIP), organizing pneumonia (OP)
patterns and controls (n=5 of each group).
Results. Notably, we identified cardinal regulatory genes that were differentially
up-regulated in UIP (BMP 4 and MMP13), NSIP (BMP6 and
CXCR4) and OP (BMP1, IL-13 and TGFB3), respectively. In UIP, remodelled
areas showed a prominent up-regulation of fibrosis-associated
genes like BMP7, MMP2 and TIMP2, while non-remodelled zones were
characterized by a significantly higher expression of BMP6 and pro-inflammatory
mediators IL-8 and IL-17A.
Conclusions. Our findings show that distinct, morphologically defined
IPF subgroups show specific cytokine expression patterns. Moreover,
BMPR2 and MMP13 up-regulation correlates significantly with the absence
of interstitial scarring in UIP pattern lungs. These results are promising
regarding their potential as diagnostic adjunct and therapeutic
targets.
DO-033
MALAT1 is essential for lung cancer metastasis in a novel human
knockout model
T . Gutschner1 , M . Eißmann2 , M . Hämmerle1 , M . Baas1 , C . Hildenbrand1 ,
M . Groß1 , M . Zörnig2 , S . Diederichs1 1Heidelberg University Hospital & German Cancer Research Center (DKFZ),
Institute of Pathology, Heidelberg, 2Georg-Speyer-Haus, Frankfurt
Aims. The highly conserved long non-coding RNA MALAT-1 (Metastasis-Associated
in Lung Adenocarcinoma Transcript 1) had been discovered
as a prognostic marker associated with poor survival and development
of distant metastasis in lung adenocarcinoma. Since then, it
has been found to be deregulated in numerous tumor entities and has
been linked to splicing. However, its functional relevance in tumor cells
remains to be elucidated. Knockdown models for MALAT1 have been
described but suffer from insufficient silencing efficiency of the highly
abundant, nuclear non-coding RNA (ncRNA).
Methods. In this project, we have developed a novel strategy to create
ncRNA knockouts in human cancer cell lines.
Results. We have successfully used a synthetic Zinc Finger Nuclease
engineered to target the 5’-region of MALAT1 to stably and biallelically
integrate RNA-destabilizing elements into the genome of human lung
cancer cells (A549). This approach resulted in a specific and more than
1000-fold silencing of MALAT1 in individual clones compared to a less
than 5-fold silencing using siRNAs. Thus, this approach can be used to
create functional knockouts of coding as well as non-coding genes also
in human tumor cell lines allowing loss-of-function studies also of nonconserved
ncRNAs in the future. Phenotypically, the MALAT1-Knockout
cells (KO) greatly differ from their parental cell line and wild type
clones (WT): Next to morphological changes, the migration of the KO
cells is largely impaired as shown in scratch assays. In xenograft assays
after i.v. injection, the KO cells form significantly fewer and smaller lung
metastases than their WT counterparts. Since no large difference was
observed after subcutaneous injection of the WT and the KO cells, this
indicates a specific, active and essential function of MALAT1 in metastasis.
Conclusions. Taken together, we have developed a novel, highly effective
approach for the knockout of genes that can be used for non-coding as
well as coding RNAs in human tumor cells. Knockout of MALAT1 in
human lung cancer cells revealed its essential function in migration and
metastasis.
DO-034
Rationale for treatment of metastatic squamous cell carcinoma of
the lung using FGFR inhibitors
A . Franzen 1 , F . Göke1 , R . Menon1 , D . Goltz1 , R . Kirsten1 , D . Böhm1 , W . Vogel 1 ,
A . Göke1 , V . Scheble2 , J . Ellinger3 , U . Gerigk4 , F . Fend5 , P . Wagner6 , A . Schröck7 ,
S . Perner1 1 2 University Hospital of Bonn, Institute of Pathology, Bonn, University of
Tübingen, Department of Hematology and Oncology, Tübingen, 3University Hospital of Bonn, Department of Urology, Bonn, 4Malteser Hospital Bonn,
Department of Thorax Surgery, Bonn, 5University Hospital of Tübingen,
Institute of Pathology, Tübingen, 6University of Pittsburgh Medical Center,
Division of Surgical Oncology, Pittsburgh, United States, 7University Hospital
of Bonn, Department of Head and Neck Surgery, Bonn
Aims. We previously identified amplification of the fibroblast growth factor
receptor 1 (FGFR1) gene as a potential therapeutic target for a small
molecule inhibitor therapy in squamous cell lung cancer (L-SCC). Currently,
clinical phase 1 trials are underway to examine whether patients
with FGFR1-amplified L-SCC benefit from a targeted therapy approach
using small molecule inhibitors of FGFR. As most lung cancer patients
present with metastatic disease, we investigated whether lymph node
metastases in L-SCC share the FGFR1 amplification status of their corresponding
primary tumor.
Methods. Our study cohort consisted of 72 patients with L-SCC, 39 of
whom presented with regional lymph node metastases. Tissue microarrays
were constructed from formalin-fixed, paraffin-embedded tissue
of the primary tumors and, where present, of the corresponding lymph
node metastasis. A biotin-labeled target probe spanning the FGFR1 locus
(8p11.22-23) was used to determine the FGFR1 amplification status by
fluorescence in-situ hybridization (FISH).
Results. FGFR1 amplification was detected in 16% (12/72) of all primary
lung SCC. Among patients with metastatic L-SCC, 18% (7/39) of the
lymph node metastases displayed a FGFR1 amplification, and an exact
correlation between the FGFR1 amplification status was observed between
the primary tumor and metastatic tissue.
Conclusions. FGFR1 amplification is a common genetic event in squamous
cell carcinomas of the lung. Moreover, lymph node metastases derived
from FGFR1-amplified L-SCCs also exhibit FGFR1 amplification.
Therefore, we suggest that the FGFR1 amplification is a clonal event in
tumor progression. Beyond this biologically relevant observation, our
findings carry therapeutic implications, in that small-molecule inhibitors
may be applicable to the treatment of squamous cell carcinomas of
metastatic squamous cell carcinoma of the lung.
Der Pathologe · Supplement 1 · 2012 |
23

Abstracts
DO-035
FISH assays for the detection of FGFR1 amplifications and EML4-
ALK translocations in NSCLC
H .-U . Schildhaus 1 , K . Schmitz 1 , L . Heukamp 1 , S . Merkelbach-Bruse 1 ,
R . Büttner 1
1 University of Cologne, Institute of Pathology, Köln
Aims. Easy, reliable and standardized tests for predictive diagnoses and
targeted therapies are needed. A small subset of pulmonary adenocarcinomas
(AC) harbor therapeutically relevant EML4-ALK translocations.
The incidence of squamous cell carcinomas (SC) of the lung increases
dramatically, but targeted therapeutic options are not well established
for this subgroup of NSCLC. Recently, Fibroblast Growth Factor Receptor
1 (FGFR1) amplification in SC was described to be associated with
tumor growth and survival, suggesting that FGFR inhibitors may be a
viable therapeutic option in this cohort of patients.
Methods. A total of 400 NSCLC samples were included in this study. For
detection of EML4-ALK translocations a triple color FISH assay was
used: two probes labeled orange and green flank the breakpoint region of
ALK in telomeric and centromeric direction, and a third probe, labeled
in blue, spans the entire EML4 gene. A dual color FGFR1/CEN8 probe
was used to evaluate the prevalence of FGFR1 amplification in SC and to
develop an evaluation strategy for FGFR1 FISH assays.
Results. Using the triple color EML4-ALK probe specific signal patterns
were found for different types of ALK rearrangements. An inversion of
chromosome 2p results in (1) split (separated) orange and green signals,
(2) a split (doubled) blue signal and (3) a colocalisation (fusion) of the
separated orange and green signals with blue signals. Additional specific
signals patterns were seen in cases with inversions/deletions and
interstitial deletions alone. Investigating a subset of 254 SC with a two
colour FISH assay we found FGFR1 amplifications in 20% of SCC but
not in AC. Low amplification levels (as defined by ≥5 FGFR1 signals in
≥50% of tumor cells) were rare with a frequency of 6.3% while high level
amplifications (as defined by a FGFR1/CEN8 ≥2.0, or average number
of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor
cells containing ≥15 FGFR1signals or large clusters ≥10%) occurred in 15%
of all SC.
Conclusions. The novel triple color split/fusion approach decreases the
number of questionable or borderline cases at high sensitivity and specificity
and allows the diagnosis of specific types of ALK translocations.
FGFR1 amplification is one of the most frequent therapeutically tractable
genetic lesion in NSCLC. Standardized reporting of FGFR1 amplification
in SCC will become increasingly important to correlate therapeutic
responses to FGFR1 inhibitors in clinical studies.
DO-036
Mutation analysis from the REASON study, a Registry for the
Epidemiologic and Scientific evaluation of EGFR mutation status
in newly diagnosed NSCLC patients stage IIIB/IV
P . Schirmacher1 , W . Schütte2 , M . Thomas3 , W . Eberhardt4 , J .-M . Graf von der
Schulenburg5 , S . Zaun6 , M . Dietel7 1 2 University of Heidelberg, Institute of Pathology, Heidelberg, Städtisches
Krankenhaus Martha Maria, Halle-Dölau, 3Heidelberg University Hospital,
Thoraxklinik, Heidelberg, 4Universitätsklinikum der GSH Essen, Essen,
5 6 Leipniz University of Hannover, Hannover, AstraZeneca GmbH, Wedel,
7Humbold University Berlin, Institute of Pathology, Berlin
Aims. Somatic mutations in the EGFR gene predict for sensitivity to
EGFR tyrosine kinase inhibitors (TKI) in patients with advanced
NSCLC, however, little is known about the prevalence of such mutations
in the German population. The REASON study aims to generate
key data on the EGFR mutation status and its association with major
clinicopathological parameters derived from a sufficiently large sample
of stage IIIB/IV NSCLC patients with a predominantly Caucasian ethnic
background in Germany.
24 | Der Pathologe · Supplement 1 · 2012
Methods. REASON is an AstraZeneca sponsored German registry. 4255
subjects with stage IIIB/IV NSCLC for whom EGFR mutation testing
was planned were enrolled at 151 participating sites throughout Germany
(129 hospital-based, 22 office-based). EGFR mutation testing was done at
56 QuIP certified and 11 additional pathology institutions. While analysis
of exons 19 and 21 was obligatory, analysis of exons 18 and 20 was
not routinely done at all labs. The primary aim was to collect epidemiological
data on EGFR mutation status (M+, M−) in the German population
and to correlate EGFR mutation status with clinicopathological
characteristics (e.g. smoking status, gender, histology, etc.). As secondary
objectives, real-life clinical outcome data of all EGFR M+ patients (PFS,
OS,DCR), clinical management and pharmaco-economic data (resource
use) associated with diagnosis and treatment of EGFR M+ patients will
be collected.
Results. Interim analysis data was available for 3612 patients (Caucasian
patients only, 99.4% of all patients). 62% of patients are male and 38%
female; with 81% being ever-smokers vs. 19% non-smokers. Adenocarcinoma
histology is most frequent (68%), followed by squamous epithelial
carcinoma subtype (20%). 358 EGFR mutations were found, the rate of
EGFR mutations predicting TKI sensitivity was 9.5%, with 0.4% resistant
mutations. Most common mutations were exon 19 deletions (49.6%; predominantly
Del E746-A750) followed by exon 21 mutations [38%, mostly
L858R (93 or 26.8% of all mutations)]. Exon 18 and 20 mutations (incl. 3
T790M) were found in 6.3% and 9.2% of patients, respectively. The differentiated
panel of identified mutations will be shown.
Conclusions. REASON provides the largest data base to date on EGFR
mutations status of patients with newly diagnosed stage IIIB/IV NSCLC
in Germany. In addition, data on baseline epidemiological and further
clinicopathological characteristics will enable us to better understand
the association of these factors with different mutations and clinical outcomes.
AG Hämatopathologie I
DO-043
ID2 and ID3 protein expression differs between hematopoietic
cell lineages and acute leukemias of distinct clinicopathological
entities
A .M . May1 , A .-V . Pfister1 , L . Bogatyreva2 , M . Benkisser3 , D . Hauschke2 , M . Lübbert4
, M . Werner1 , J . Hasskarl4 , S . Lassmann1 1 2 University Freiburg Medical Center, Institute of Pathology, Freiburg, University
Freiburg Medical Center, Institute of Medical Biometry und Medical
Informatics, Freiburg, 3University Freiburg, Freiburg, 4University Freiburg
Medical Center, Department of Hematology and Oncology, Freiburg
Aims. Inhibitors of DNA binding (ID) proteins regulate cellular differentiation
and proliferation through formation of heterodimers with
basic helix-loop-helix transcription factors. To elucidate the role of ID
proteins in hematopoiesis and in acute leukemia, we analysed ID2 and
ID3 protein expression in hematopoietic cells and leukemic blasts.
Methods. Primary bone marrow biopsies (BMB) of patients with AML
with myelodysplasia related changes (AML-MD) (n=19), de novo AML
(n=20), cALL (n=23) and T-ALL (n=19) as well as BMB of healthy bone
marrow stem cell donors (n=19) were stained for ID2 and ID3 protein
expression by immunohistochemistry (IHC). In healthy BMB, each
200 cells/hematopoietic lineage were evaluated, differentiating between
mature and immature granulopoiesis. In acute leukemias, the staining
pattern and intensity of 200 blast cells/BMB were assessed. Statistical
analyses were done by the nonparametric Kruskal-Wallis test (two-sided,
significance level 0.05), and in case of a significant result by an additional
pairwise Wilcoxon test.
Results. In normal BMB hematopoietic cells and maturation stages displayed
significant differences in ID2/3 protein expression. While the

immature granulopoiesis showed a strong ID3 protein expression, the
more mature granulocytes only displayed a minimal reactivity. In contrast,
erythropoiesis remained negative for ID2/3 (p

Abstracts
ray-based expression profiling in 468 tissue samples from 25 healthy organs
from more than 210 patients.
Results. We found that CD317 protein was expressed to varying degrees
in all organs tested and detected in a number of specialized cell types,
including hepatocytes, pneumocytes, ducts of major salivary glands,
pancreas and kidney, Paneth cells, epithelia, Leydig cells, plasma cells,
bone marrow stromal cells, monocytes, and vascular endothelium. Although
many of these cell types are in vivo targets for pathogenic viruses,
restriction by CD317 or virus-encoded antagonists has been documented
in only some of them. Limited cell type-dependent co-expression
of CD317 with the IFN biomarker MxA in vivo and lack of responsive
stimulation in organ explants suggest that interferons may only partially
regulate CD317.
Conclusions. This in vivo expression profiling sheds light on the biology
and species-specificity of CD317, identifies multiple thus far unknown
interaction sites of viruses with this restriction factor, and refutes the
concept of its restricted constitutive expression and primary IFN inducibility.
CD317’s widespread expression calls into question its suitability as
a target for immunotherapy.
DO-047
Frequent detection of the Merkel cell polyomavirus in B-lymphocytes:
implications for non-Hodgkin lymphomagenesis?
D . Rennspiess1 , A . Haugg1 , J . Beckervordersandforth1 , A .K . Kurz2 , R . Plusquin1 ,
G . Cathomas3 , C . Wendtner4 , E .-J . Speel1 , H . Kvasnicka5 , A . zur Hausen1 1Maastricht University Medical Center, Department of Pathology, Maastricht,
Netherlands, 2University Hospital Aachen, Department of Internal
Medicine IV, Aachen, 3Kantonsspital Liestal, Institute of Pathology, Switzerland,
4University Hospital Cologne, Department I of Internal Medicine, Köln,
5University Hospital Frankfurt, Institute of Pathology, Frankfurt
Aims. The recent discovery of the Merkel cell polyomavirus (MCPyV)
in Merkel cell carcinomas (MCC) also had an important impact on the
well established epidemiological association of CLL/SLL with MCC. We
have recently demonstrated the presence of MCPyV DNA in highly purified
CD5+/CD19+ CLL cells. Meanwhile, the presence of MCPyV DNA
in CLL/SLL cells has been independently demonstrated by two other
groups reporting MCPyV-DNA in approx. 21–33%. In addition, we were
able to demonstrate MCPyV integration by fluorescence in situ hybridisation
(FISH). Here we extended our analyses to different types of non
Hodgkin lymphomas (NHL) as well as to non neoplastic reactive follicular
lymph nodes for the presence of MCPyV by FISH and/or MCPyV
DNA PCR.
Methods. DNA PCR was carried out on 8 formalin-fixed and paraffin
embedded SLL cases and 1 DLBCL case and on 39 reactive lymph nodes
as previously described. On these and on a tissue microarray (TMA) containing
CLL/SLL (n=43), MZL (n=44), FL (n=40), MALT (n=47), DLBCL
(n=32), and T cell lymphomas (n=19) MCPyV FISH was performed.
Results. MCPyV DNA was detected by PCR in 6 of the 8 SLL cases and in
13 of the 39 reactive lymph nodes. By MCPyV FISH sharply punctate dots
– compatible with viral integration – were identified in 29% (n=15/51) of
CLLs, in 9% (n=4/45) of MZL, in 35% (n=14/40) of FL, in 15% (n=7/47)
of MALT, and in 18% (n=6/32) of DLBCL. All T cell lymphomas were
MCPyV negative. Of interest, small clusters of MCPyV DNA positive B
cells were detected in the follicle centres of the reactive lymph nodes.
These hybridization signals were punctate and multiple, and some of
them revealed a diffuse hybridization pattern.
Conclusions. MCPyV FISH confirms our previous data on the integrated
presence of MCPyV in CLL. Other B-cell NHL might be associated with
the presence of integrated MCPyV. The finding of small clusters of follicular
B-cells harbouring integrated MCPyV DNA is suggestive for an
early and rather common MCPyV infection of premature B-cells which
normally – during the process of follicular B-cell maturation – are eliminated.
MCPyV-positive follicular B-cells which are not eliminated,
thus not recognised as “foreign”, are likely to be the reservoir of cells
26 | Der Pathologe · Supplement 1 · 2012
which during a long-term transformation process by an accumulation of
oncogenic mutations or deletions turn into a NHL cell. Functional studies
concerning MCPyV and lymphomagenesis are ongoing in order to
elucidate a possibly underlying etiopathogenic role for MCPyV in NHL
lymphomagenesis.
DO-048
The majority of immunohistochemically BCL2 negative FL grade I/
II carry a t(14;18) with mutations in exon 1 of the BCL2 gene and
can be identified with the BCL2 E17 antibody
I . Bonzheim1 , R . Baumann1 , P . Adam1 , F . Fend1 , L . Quintanilla-Martinez 1
1University Hospital Tübingen, Institute of Pathology and Neuropathology,
Tübingen
Aims. Follicular lymphoma (FL) is characterized by the translocation
t(14;18)(q32;q21) resulting in constitutional overexpression of the antiapoptotic
protein BCL2. However, 10–15% of FL grade I/II remain negative
in the immunohistochemical (IHC) staining for BCL2. The aims of
this study were: 1) to investigate the incidence of IHC BCL2 negative FL
grade I/II diagnosed at our institution, 2) to analyze BCL2 IHC negative
FL with the alternative BCL2 antibody (clone E17) and perform FISH
analysis for the t(14;18), and 3) to elucidate the molecular mechanism of
immunohistochemical BCL2 negativity.
Methods. FL grade I/II diagnosed between 01/2005 and 08/2011 at the
Institute of Pathology of the University of Tübingen were included in
the study. All cases were stained with the standard BCL2 antibody (clone
100D5; DCS). BCL2 negative cases were subsequently stained with the
BCL2 antibody, clone E17 (Zytomed) and analyzed by FISH using a BCL2
break-apart probe (LSI BCL2 BAP, Vysis). Exon 1 of the BCL2 gene, where
the epitope of the standard BCL2 antibody resides, was amplified and
sequenced.
Results. 23 (9.6%) of the 240 identified cases of FL grade I/II were negative
with the standard BCL2 antibody. Of these, 13 cases (57%) were
positive for the E17 antibody and 10 cases (43%) remained negative. All
E17 positive cases showed a BCL2 break by FISH analysis, indicative of
a t(14;18), whereas the E17 negative cases lacked BCL2 alterations. Two
of the E17 negative cases carried a BCL6/IGH translocation. Sequencing
of BCL2 exon 1 revealed missense point mutations resulting in amino
acid substitutions in all 9 analyzable E17-positive cases, with a hot spot
around codon 144.
Conclusions. The incidence of immunohistochemically BCL2-negative
FL grade I/II in our series is comparable to published data. The E17 antibody
reveals the presence of BCL2 protein undetectable with the standard
antibody due to exon 1 missense mutations in the majority of BCL2
“negative” FL grade I/II and correlates with the presence of the t(14;18).
The molecular pathogenesis of the BCL2 (E17)- and t(14;18) negative FL
grade I/II remains to be determined.
DO-049
Follicular lymphoma with prominent mantle zones – a FICTION
analysis
P . Kosmidis1 , P . Adam1 , I . Bonzheim1 , L . Quintanilla-Fend1 , P . Bauer2 ,
M . Scharpf1 , T . Henopp1 , F . Fend1 1Eberhard-Karls-University, Institute of Pathology and Neuropathology, Tübingen,
2Eberhard-Karls-University, Institute of Human Genetics, Tübingen
Aims. Follicular lymphoma (FL) is characterized by the recurrent translocation
t(14;18), resulting in BCL2 protein overexpression. Most cases
show an indolent clinical course, which in part may be a result of the
influence of the complex tumor microenvironment of FL. Involvement
of different lymph node compartments may indicate a biologically more
advanced lymphoma, whereas restriction of neoplastic cells to germinal
centers might represent earlier disease stages. We have therefore exami-

ned, if well-defined mantle zones, which can be identified in a subset of
FL including FL “in situ”, are part of the malignant clone.
Methods. FL cases with morphologically detectable mantle zone structures
and a case of follicular lymphoma “in situ” were selected from a series
of 240 FL grade 1/2. Fluorescence in situ hybridization (FISH) for the
detection of the chromosomal translocation t(14;18)(q32;q21) was combined
with a simultaneous immunofluorescence staining for IgD for the
identification of mantle zone cells (FICTION).
Results. 10 of 17 (59%) FL cases with morphological detectable mantle
zone structures lacked a t(14;18) by FISH. In the remaining 7 t(14;18) positive
FL cases and the FLIS case, the IgD+ mantle zone cells did not show
a chromosomal break in the BCL2 gene locus.
Conclusions. 1) A significant part of FL cases with prominent mantle zone
structures are t(14;18) negative. Further investigations are necessary to
elucidate the molecular background of this subgroup and to define the
border with nodal marginal zone lymphoma with follicular colonization.
2) The mantle zone cells in t(14;18)+ FL with prominent mantle zones
in their majority are not part of the malignant clone and therefore most
likely represent either pre-existent or newly recruited reactive cells.
DO-050
Reactive tumor infiltrating T-cells predict survival in mantle cell
lymphoma: an immunohistochemical study of 81 patients
C . Schrader1 , Ö . Akalthun1 , P . Meusers2 , G . Brittinger2 , J . Claasen1 , W . Klapper3 1 2 2 University Hospital of Kiel, nd Department of Medicine, Kiel, University
Essen, Department of Hämatology, 3UKSH, Campus Kiel, Department of
Pathology
Aims. The role of tumor infiltrating T-Cells in malignant B-Cell lymphomas
is discussed controversial. There are only limited data on CD 8 and
FOXP3 positive cells in mantle cell lymphoma.
Methods. 81 biopsy specimens of patients (64 men and 17 women) with
mantle cell lymphoma and a median age of 64 years (range: 41 to 86 years)
were included in this study. The slides were stained immunohistochemically
with CD3, CD8 and FOXP3. Positive T-cells of 10 High power
fields (HPF) were counted and the average value was calculated.
Results. The CD 8 staining showed a range of 0 to 138 positive cells per
HPF with a mean value of 19.4/HPF. A high account of CD 8 positive
cells was associated with a significantly longer overall survival time
(42 months) compared to MCL with a low account of CD 8 positive cells
(28.8 months, p=0.029). FOXP3 staining had a range of 0 to 104/HPF
with a mean value of 28. Patients with MCL and a high number (>25/
HPF) of FOXP3 positive cells had a median survival time of 38.2 months
compared with the group with low account (

Abstracts
DO-053
Account of tumor infiltrating macrophages is a prognostic factor
for patients with mantle cell lymphoma
C . Schrader 1 , F . Sirin 1 , P . Meusers 2 , G . Brittinger 2 , J . Claasen 1 , W . Klapper 3
1 University Hospital of Kiel, 2 nd Department of Medicine, Kiel, 2 University
Essen, Department of Hämatology, 3 UKSH, Campus Kiel, Department of
Pathology
Aims. Mantle cell lymphoma (MCL) is a malignant lymphoma associated
with a relatively aggressive clinical course and a median overall survival
time of 3–4 years. Only limited data about tumor associated macrophages
and their influence on survival in MCL exists.
Methods. We analyzed the amount of CD68 macrophages in relation to
the clinical outcome in patients with MCL. Lymph node biopsies of 77
untreated patients (17 women and 60 men) enrolled in two multicenter
trials (1975–1985) with a median age of 66 years (range 41–86 years) were
included in this study. Biopsy specimens were investigated immunohistochemically
with monoclonal antibodies against CD68 (Ki-M1P).
10 High power fields (HPF) were evaluated by random.
Results. Patients with low account (less than 10/HPF) of CD 68 positive
macrophages had a median overall survival time of 38.2 months, compared
to 24.2 months for patients with high (more 10/HPF) CD 68 positive
macrophages. The Kaplan-Meier analysis showed a significant difference
in the overall survival time (p=0.0027).
Conclusions. Patients with mantle cell lymphoma and a low number of
CD 68 positive macrophages have a better prognosis and can predict
outcome.
DO-054
Transformation of gastritis to gastric marginal zone lymphoma is
associated with deregulated expression of microRNAs
C . Thorns1 , J . Kuba1 , A .C . Feller1 , V . Bernard 1 , A . Senft2 , S . Szymczak2 ,
H .-W . Bernd1 1 2 UKSH, Campus Lübeck, Pathology, University Lübeck, Institute for medical
bioinformatics and statistics
Aims. Gastric extranodal marginal zone lymphoma (MALT lymphoma)
generally evolve from a chronic Helicobacter pylori-positive gastritis.
The mechanisms that promote the malignant transformation from gastritis
to lymphoma are not well understood. This study aims to identify
microRNAs that might be involved in the process of neoplastic transformation.
Methods. Gastric biopsies were scored as 0 (normal), 1 (gastritis), 2 (follicular
gastritis), 3 (suspicious, probably reactive), 4 (suspicious, probably
lymphoma) and 5 (MALT lymphoma) (Wotherspoon scores). Groups 3,
4, and 5 were further evaluated for monoclonality by immunohistochemistry
for immunoglobulin light chains and/or by polymerase-chainreaction
for the immunoglobulin heavy chain locus (IgH). MicroRNAsignatures
of 68 cases were generated by RT-PCR for 376 miRNAs.
Results. MicroRNA signatures revealed a total of 41 miRNAs that were
significantly upregulated (n=33) or down regulated (n=8) in succession
from normal mucosa to gastritis and to MALT-lymphoma. Some of these
reflect the normal expression in lymhocytes (e.g. miR-566 and -212),
while others are known to be the effect of Helicobacter pylori infection
(e.g. miR-155 and let7f). A group of five miRNAs (miR-150, -550, -124a,
-518b and -539) were differentially expressed in gastritis (Wotherspoon
scores 1, 2 and polyclonal 3 and 4) and lymphomas (monoclonal scores 3,
4, and 5). These are likely to be involved in the malignant transformation
of gastritis into MALT lymphoma.
Conclusions. The development of gastric MALT-lymphoma out of chronic
gastritis is paralleled by the deregulation of distinct microRNAs
which might thus be centrally involved in the process of malignant
transformation.
28 | Der Pathologe · Supplement 1 · 2012
AG Hämatopathologie II
DO-055
Bcl6 expression is not limited to germinal center B-cells and is
associated with progression of extranodal marginal zone B-cell
lymphomas of the gastrointestinal tract
U . Boruschek1 , L . Floßbach1 , M . Buck1 , S . Brüderlein1 , P . Möller1 , T .F .E . Barth1 1Ulm University, Pathology, Ulm
Aims. We have previously shown that progression in gastrointestinal
B-cell lymphomas is associated with changes in the BCL6 locus and
with an increased expression of Bcl6 [Flossbach et al. Int J Cancer 2011;
129(1):70–7]. Bcl6 is a well known germinal center marker; the extranodal
marginal zone B-cell lymphomas (MALT lymphomas) are supposed to
stem from cells of the extrafollicular space, therefore the expression of
Bcl6 in these lymphomas during progression seems conflicting. We have
analyzed the detailed expression of Bcl6 in lymph nodes with toxoplasmosis
since one of the cells discussed to be the potential progenitor of
MALT lymphomas is the monocytoid B-cell.
Methods. We studied lymph node paraffin sections of four patients with
toxoplasmosis by double immunofluorescence staining using a broad
panel of antibodies. For this purpose we first established a new technique
based on sequential heating of the samples that now even allows the
application of antibodies from the same species or the simultaneous use
of monoclonal and polyclonal antibodies. Further we performed stimulation
experiments with lymphoblastoid B-cells.
Results. We found a strong expression of Bcl6 in the germinal center. However,
we also detected some scattered Bcl6 positive cells in the extrafollicular
space. These extrafollicular Bcl6-positive cells were characterized
as: CD19+,CD75+, AID+, IgA+, IgG+/−, CD30−, CD10−, CD38−, Bcl2−,
ZAP70−, cRel−, IgM−, IgD−, CD11c−. Monocytoid B-cells expressed
Bcl6 in a subfraction of less than 1%. The profile of the Bcl6+ extrafollicular
B-cells corresponds to an activated post germinal center B-cell differing
from monocytoid B-cells. Expression of Bcl6 was partially inducible
in lymphoblastoid EBV transformed B-cells by TNFα, TPA, and LPS.
Conclusions. We conclude that Bcl6 expression is not limited to germinal
B-cells but is also found in extrafollicular B-cells. We suggest that the
blastic variant of MALT lymphoma may have preserved the potential of
up-regulating Bcl6 as an antiapoptotic mechanism.
DO-056
Characterization of gastrointestinal marginal zone B-cell lymphoma
and large cell variants using high-resolution SNP-arrays
L . Floßbach1 , K . Holzmann2 , T . Mattfeldt 1 , P . Möller1 , T .F .E . Barth1 1 2 Ulm University, Pathology, Ulm, Ulm University, IZKF, Ulm
Aims. Gastrointestinal marginal zone B-cell lymphomas (MALT Lymphomas)
are a model for tumor progression since we and others have
shown that the frequently coexisting more aggressive large cell component
is clonally related to the small cell lymphoma. We used SNP analysis
to further characterize these lymphomas.
Methods. We extracted genomic DNA from frozen tissue samples of 28
gastrointestinal marginal zone B-cell lymphomas (n=7) and large cell
variants (n=21). We performed SNP analysis using the Affymetrix HGW
SNP array 6.0 platform. Results were correlated with FISH and IHC analyses.
Results. While small cell lymphomas have on the average 8 aberrations
longer than 0.2MB each case, large cell variants have more than 14. Both
small and large cell lymphomas have losses in regions 1p13 and 6q15 as
well as gains on 1p36 and 17q21. Gains on 9q12i-j (2/7) are restricted to
small cell lymphomas. Losses on 6q24 (5/21) and gains on 11q23 (8/21)
are restricted to the large cell lymphomas. Most frequent losses or deletions
concern region 6q14.1a-c containing HTR1B, IRAK1BP1, PHIP,
HMGN3, LCA5 and SH3BGRL2 (5/21 large cell and 1/7 small cell lym-

phomas). Most prominent gains or amplifications (6/21 large cell lymphomas)
concern region 2p16.1a-15d containing PAPOLG, REL, PUS10
and PEX13. Amplification of REL was confirmed by FISH in these cases.
In all these lymphomas, immunohistochemical staining for c-Rel was
positive in at least 30% of the tumor cells. Regions with putative acquired
uniparental disomies (aUPD) are more present in the large cell lymphomas:
on the average 40 regions vs. 9 regions in the small cell lymphomas.
Comparing the SNP profiles of two areas of the same tumor both with
a t(11;18) Api2/Malt1 but with slightly different morphology, the analysis
revealed additional gains in the more blastic part. Investigating two
lymphoma samples from the same patient with an interval of two years,
FISH analysis showed a signal pattern pointing to a large deletion in the
IGH locus exclusively in the later sample. SNP analysis confirmed the
FISH result and revealed about ten additional aberrations illustrating increasing
genomic complexity during lymphoma progression.
Conclusions. Small and large cell variants of gastrointestinal marginal
zone B-cell lymphomas have distinct patterns of genomic aberrations
but share some overlapping features. REL is frequently amplified in the
large cell variants. In general, during lymphoma progression, the SNP
data correlate with a more complex pattern of aberrations.
DO-057
Comparative analysis of gene expression profiles defines large
cell variants of gastric marginal zone B-cell lymphoma as a distinct
subgroup
L . Floßbach1 , J . Kraus2 , K . Holzmann3 , P . Möller1 , H .A . Kestler2 , T .F .E . Barth1 1 2 Ulm University, Pathology, Ulm, Ulm University, Neural Information Processing,
Ulm, 3Ulm University, IZKF, Ulm
Aims. Gastrointestinal marginal zone B-cell lymphomas (MALT Lymphomas)
often collocalize with a more aggressive, large cell component.
The WHO classifies these lymphomas as “extranodal DLBCL with or
without residing MALT component”. We have shown that the small and
the large cell components of these composite lymphomas (ComL) are
mostly clonal and, in addition to that, that the gene expression profiles
of small cell MALT lymphomas and lymphoma components are similar
to those of the large cell components or lymphomas. This suggests
that most of the gastrointestinal DLBCL are indeed blastic variants of
marginal zone B-cell lymphomas (MZBL) [Barth et al., J Pathol 2007;
211(3):305; Flossbach et al. Int J Cancer 2011 129(1):70]. To further distinguish
these large cell variants of MZBL from nodal and extranodal
DLBCL we performed a comparative analysis of gene expression profiles
from B-cell lymphomas.
Methods. We extracted RNA from frozen tissue samples of 28 gastrointestinal
marginal zone B-cell lymphomas (n=7), large cell components
of ComL (n=8) and large cell variants (n=13). Gene expression profiling
was performed using the Affymetrix U 133 plus 2.0 array. Additional datasets
created with the same chip from DLBCL (n=119), PMBL (n=20),
BL (n=33), FL (n=38), MCL (n=7), pulmonary MALT lymphomas (n=35)
and normal B-cells (n=20) were obtained from the Gene Expression
Omnibus (GEO) database. After normalization and based on a subset of
NF-κB target genes [Compagno et al., Nature, 459, 717, 2009], we performed
hierarchical clustering analysis. Additionally, we performed cluster
robustness analysis using the k-means algorithm.
Results. Cluster number estimation was robust for k=8. These eight
clusters consisted mostly of: 1. naïve B-cells and memory B-cells, 2. centroblasts
and centrocytes, 3. Burkitt’s lymphoma, 4. pulmonary MALT
lymphoma, 5. follicular lymphoma, mantle cell lymphoma, and gastrointestinal
MALT lymphoma, 6. PMBL and DLBCL, 7. DLBCL, 8. blastic
MZBL and DLBCL. The dendrogramm, generated by the hierarchical
average linkage clustering process, was consistent with this classification.
In comparison to all DLBCLs and PMBLs, the blastic MZBLs had
relatively underexpressed PTPN3 and relatively overexpressed BANK,
CD44, CD63, and FAS.
Conclusions. These data confirm our view of blastic marginal zone B-cell
lymphomas as a distinct group of extranodal diffuse large B-cell lymphomas.
DO-058
Prognostic phenotypic and genotypic in situ biomarkers in diffuse
large B-cell lymphomas: preliminary translational report of the
prospective SAKK 38/07 trial
A . Tzankov1 , N . Leu1 , S . Muenst1 , D . Klingbiel2 , C . Mamot3 , S . Dirnhofer1 1 2 University Hospital Basel, Pathology, Basel, Switzerland, SAKK Coordinating
Center, Swiss Group for Clinical Cancer Research, Bern, Switzerland,
3Cantonal Hospital Aarau, Aarau, Switzerland
Aims. Diffuse large-B cell lymphoma (DLBCL) exhibits variable outcomes
and risk assessment is based on the international prognostic index
(IPI), which takes into account primarily patient-related parameters. The
prognostic role of tumor-related parameters is a matter of controversy.
Methods. We prospectively analyzed the prognostic value of phenotypic
and genotypic profiles suggested to play a role in DLBCL on a clinical trial
collective of 124 DLBCL patients homogenously treated with six cycles
of rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone,
followed by 2 cycles rituximab (R-CHOP). Evaluation of the role of positron
emission tomography was a main objective, and was performed
before, after 2 cycles of therapy and at the end of treatment. Immunohistochemical
(BCL2, BCL6, CD5, CD10, CD20, CD95, CD168, Cyclin
E, FOXP1, GCET, Ki-67, MUM1p, pSTAT3) and in situ hybridization
analyses [BCL2 break apart probe (BAP), C-MYC BAP and C-MYC/
IgH double-fusion probe (DFP) and Epstein-Barr virus probe (EBER)]
were performed and correlated with clinicopathological parameters and
outcome.
Results. The median patients’ age was 59 years; 68 were males, 56 females.
BCL2 gene breaks were observed in 11% of cases, and those cases also expressed
BCL2 in a mean of 95% of tumor cells, compared to 42% in nonrearranged
instances; 85% of the rearranged cases were of the germinal
center (GC) phenotype according to the Tally algorithm. 3% of cases (all
of the non-GC phenotype) showed BCL2 amplifications. C-MYC breaks
were observed in 10% of cases; 66% were of the GC phenotype. Of the C-
MYC rearranged cases only a third displayed C-MYC/IgH fusions corresponding
to t(8;14), the others being assumed to have alternative C-MYC
rearrangement partners. Cases with rearranged C-MYC showed as high
Ki-67 labeling as non-rearranged. Cases with both BCL2 and C-MYC
rearrangements were not observed. A complete response (CR) defined
by Cheson’s criteria was achieved in 90 out of 117 patients, for 7 there
were no data. Factors that were linked to failure to achieve CR were CD5
positivity (11% compared to 2%, p=0.051), EBER positivity (4% of cases
compared to 0% of those with CR, p=0.072) and presence of either BCL2
or C-MYC gene rearrangements (46% compared to 18%, p=0.132), but not
IPI or Tally phenotype.
Conclusions. Phenotypic and genotypic studies with carefully selected
biomarkers like CD5, EBER and BCL2- as well C-MYC BAP might be of
prognostic value in DLBCL patients treated by R-CHOP.
DO-059
Phenotype of primary testicular diffuse large B-cell lymphomas
T . Menter1 , M . Ernst1 , S . Dirnhofer1 , A . Braghorn2 , P . Went1 , A . Tzankov1 1University of Basel, Institute of Pathology, Basel, Switzerland,
2Medica Zürich, Switzerland
Aims. Primary testicular diffuse large B-cell lymphomas (tDLBCL) are
rare neoplasms with few comprehensive studies conducted so far. We
therefore aimed to systematically analyze the morphology and phenotype
of tDLBCL.
Der Pathologe · Supplement 1 · 2012 |
29

Abstracts
Methods. Forty-three patients from 3 different Swiss hospitals were included
in the study. The tumors were diagnosed between 1972 and 2009.
The protein expression profile was assessed by immunohistochemistry.
Results. 39 of the tumors showed centroblastic and 1 immunoblastic morphology,
three were not classifiable. All cases were positive for CD79a,
followed by CD20 and PAX5 (95% of cases) and CD19 (93%). Most cases
(68%) expressed the post-germinal center (GC) marker FOXP1 and
21% expressed MUM1, while the GC markers CD10, LMO2, BCL6 and
GCET1 were expressed in 27, 16, 8 and 6%, respectively (cut-off levels for
the respective markers were determined by ROC). BCL2 was expressed
on >50% of the tumor cells in 69% of cases. 83% of cases were phenotypically
classifiable as non-GC DLBCL according to the Tally algorithm.
There was no evidence for EBV- or HHV8-association. 70% of the tumors
showed active STAT signaling by expression of either pSTAT1 or pSTAT3,
but not pSTAT5. p53 was expressed in 12% of cases, but p21 staining (p21/
p53) did not suggest presence of TP53 mutations.. Mean mitotic index
was 18/mm2, median MIB1 labeling index was 40% (±25%). Tumors with
lymphoepithelial lesions in seminiferious tubules showed a lower mitotic
activity, although the association was weak. Interestingly, one tumor was
positive for OCT4. All 43 cases were negative for NUT1 and PLAP. Only
limited clinical data were available: mean age at diagnosis was 69 years
(range: 43–87 years, n=41). There was no side predilection of the tumors.
One tumor was bilateral at diagnosis, one tumor presented simultaneously
in the testis and the CNS. Of ten tumors, five did not relapse
(mean follow up time 48 months). Five tDLBCL relapsed, thereof two in
the contralateral testis, two in the CNS and one in the skin.
Conclusions. We conclude that tDLBCL are predominantly centroblastic
and of non-GC phenotype. Since occasionally tDLBCL can express germ
cell markers or be CD20-negative, multimarker phenotyping is important
for lineage determination. There was no shift of morphology or protein
expression profile over time. tDLBCL have active STAT signalling
mediated through pSTAT1 and pSTAT3. tDLBCL are of non-/post-GC
origin and not hyper-proliferative. TP53 mutations are unlikely.
DO-060
C-MYC aberrations characterize a subset of patients with diffuse
large B-cell lymphoma with poor outcome when treated with
rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone
A . Tzankov1 , M . Gerhard1 , S . Dirnhofer1 , C . Visco2 , K . Young3 1 2 University Hospital Basel, Pathology, Basel, Switzerland, San Bortolo Hospital,
Internal Medicine, Vicenza, Italy, 3The University of Texas MD Anderson
Cancer Center, Pathology, Houston, United States
Aims. Diffuse large-B cell lymphoma (DLBCL) exhibits variable outcomes
and risk assessment is based on the international prognostic index
(IPI), which takes into account primarily patient-related parameters.
Gene expression profiling (GEP) can stratify patients with different
prognoses into germinal center B-cell (GCB) and activated B-cell subtype
(ABC). These groups remain of prognostic importance with the addition
of rituximab (R) to chemotherapy. The prognostic role of C-MYC
gene status in the era of modern treatment is still debatable.
Methods. To address this question, we analyzed C-MYC gene abnormalities
by interphase fluorescence in situ hybridization (FISH) utilizing
break-apart- (BAP) and a IgH/C-MYC double-fusion (DFP) probes in
601 patients with de novo DLBCL treated with R, cyclophosphamide,
doxorubicin, vincristine, prednisone (R-CHOP) and 332 patients treated
with CHOP; all patients had clinical follow-up and GEP data.
Results. C-MYC gene abnormalities were detected in 67 of 672 evaluable
cases (10%), including 7 amplifications (2 ABC, 5 GCB), 4 rearrangements,
detectable only with the DFP (2 ABC, 2 GCB), 23 rearrangements,
detectable only with the BAP (5 ABC, 18 GCB) and 33 rearrangements,
detectable with both the BAP and DFP (12 ABC, 21 GCB; p=0.032 for
the differences between ABC and GCB). In multivariable models the last
two C-MYC aberration types represented IPI- and GEP-independent
30 | Der Pathologe · Supplement 1 · 2012
prognostic factors for event-free survival in R-CHOP treated patients
(p=0.003; relative risk 1.46) and in patients with GCB (p=0.014; relative
risk 1.29), but (though being of prognostic significance in univariable
models) not in CHOP treated ones and in ABC, where IPI was the sole
prognosticator.
Conclusions. C-MYC aberrations are observed in 10% of DLBCL, more
commonly in GCB cases. Alternative C-MYC rearrangements with non-
IgH partners, which are detectable only with BAP, account for a third of
all C-MYC structural genetic abnormalities. C-MYC aberrations detected
by BAP add IPI independent prognostic information for individual
DLBCL risk estimation in R-CHOP treated cases.
DO-061
MYC-binding sites in Burkitt lymphoma identified by deep
sequencing
V . Seitz1 , P . Butzhammer2 , B . Hirsch1 , J . Hecht3 , I . Gütgemann4 , A . Ehlers1 ,
D . Lenze1 , E . Oker1 , A . Sommerfeld1 , E . von der Wall1 , C . König5 , C . Zinser6 ,
R . Spang2 , M . Hummel1 1 2 Institute of Pathology, Charité – University Medicine, Berlin, University
of Regensburg, Institute for Functional Genomics, Regensburg, 3Charité –
University Medicine, Berlin-Brandenburg Center for Regenerative Therapies
(BCRT), Berlin, 4University Hospital of Bonn, Department of Pathology, Bonn,
5 6 imaGenes GmbH, Source BioScience, Berlin, Genomatix Software GmbH,
Genomatix Personalized Medicine, München
Aims. MYC is a key transcription factor involved in central cellular processes
such as regulation of the cell cycle, histone acetylation and ribosomal
biogenesis. It is overexpressed in the majority of human tumors
including aggressive B-cell lymphoma. Especially Burkitt lymphoma
(BL) is a highlight example for MYC overexpression due to a chromosomal
translocation involving the c-MYC gene. However, so far genomewide
analysis of MYC-binding sites by chromatin immunoprecipitation
(ChIP) followed by next generation sequencing (ChIP-Seq) has not been
conducted in BL. Therefore, our objective was the precise and representative
consideration of the MYC landscape in BL due to a much higher
coverage of MYC-binding sites employing ChIP-Seq.
Methods. We carried out ChIP in 5 human BL cell lines, employing a
MYC-specific antibody followed by next generation sequencing of the
precipitated DNA fragments. To assess the functional consequences of
MYC binding, the ChIP-Seq data were supplemented with siRNA-mediated
knock-downs of MYC in BL cell lines followed by gene expression
profiling.
Results. Our ChIP-Seq analysis gave rise to 7,054 MYC-binding sites after
bioinformatics analysis of a total of approx. 19 million sequence reads. In
line with previous findings, binding sites accumulate in genes known to
be involved in the cell cycle, ribosomal biogenesis, histone acetylation
and DNA-methylation demonstrating a regulatory role of MYC in these
processes. Unexpectedly, MYC-binding sites also accumulate in many
B-cell relevant genes and expression analysis after knock-down of MYC
by siRNA identified genes involved in the B-cell function which are upregulated
in response to MYC silencing.
Conclusions. The 7,054 MYC-binding sites identified by our ChIP-Seq approach
greatly extend the knowledge regarding MYC binding in BL and
shed further light on the enormous complexity of the MYC regulatory
network. Especially our observations that (i) many B-cell relevant genes
are targeted by MYC and (ii) that MYC down-regulation leads to an upregulation
of B-cell genes highlight an interesting aspect of BL biology.

DO-062
Tumor-associated macrophages in pediatric classical Hodgkin
lymphoma: associations with Epstein-Barr virus, lymphocyte
subsets and prognostic impact
M . Barros 1 , R . Hassan 2 , G . Niedobitek 1
1 Unfallkrankenhaus Berlin, Institut für Pathologie, Berlin, 2 Brazilian National
Cancer Institute, Bone Marrow Transplantation Center, Rio de Janeiro, Brazil
Aims. The number and type of tumor-associated macrophages are implicated
in the biology of classical Hodgkin lymphoma (cHL) in adults.
Due to the immunological differences between children and adults, and
to the cross-talk between innate and adaptive immune system, it is hypothesized
that number, function and prognostic impact of macrophages
in pediatric cHL may be different from adult cases.
Methods. We analyzed the number of macrophages and dendritic cells
(DCs) in the tumor microenvironment of pediatric cHL by immunohistochemistry
(IHC) and image analysis system. Epstein-Barr virus (EBV)
status was determined by EBER-specific in situ hybridization and IHC.
Results were analyzed in the context of age group, histological characteristics
and clinical follow-up.
Results. The younger ages were characterized by a more intense CD14+
and CD163+ cell infiltrate (p=0.01 and p=0.025, respectively). In relation
to nodular sclerosis, mixed cellularity subtype was characterized by high
number of CD14+, (p=0.003) and CD163+ cells (p=0.027). EBV+ cases
exhibited higher numbers of CD14+ (p

Abstracts
Methods. Three ALK + ALCL cell lines were transduced with C/EBPβ
shRNA by lentiviral gene transfer. The C/EBPβ knockdown was quantified
by RT-qPCR and Western Blot. MiRNA expression in ALK+ ALCL
cell lines with and without C/EBPβ knockdown, ALK − ALCL cells and
T-cells were analyzed by deep-sequencing, to determine differentially
regulated and expressed miRNAs in ALK+ ALCL. The influence of C/
EBPβ on the expression of miRNA candidates and the differential expression
in ALK+ ALCL was validated by RT-qPCR in cell lines and in
primary tumors.
Results. Next-generation sequencing analysis resulted in 80 significantly
regulated miRNAs after C/EBPβ knockdown in the three ALK+ ALCL
cell lines. Three of these miRNAs (miR-181a*, miR-146b-5p, miR-203)
were significantly regulated in all three cell lines. The C/EBPβ dependent
regulation of these miRNAs was confirmed in two cell lines by RTqPCR.
Comparison of the results of the ALK+ ALCL cell line SUDHL-1
and T-cells or SUDHL-1 and the ALK− ALCL cell line revealed 379 or
301 significantly regulated miRNAs respectively. ALK− ALCL cells and
T-cells showed a significant difference in the expression levels of 366
miRNAs. A hundredfold change in expression levels was observed for
a few interesting miRNAs of the different signatures. The differential
expression of some of the most remarkable miRNAs in ALK+ ALCL
was validated in primary human ALK+ and ALK− ALCLs by RT-qPCR.
Several of these miRNAs play important roles in diverse cancers with
tumor-suppressing or oncogenic functions.
Conclusions. Three miRNAs were found to be regulated by C/EBPβ in
two ALK+ ALCL cell lines. Numerous miRNAs are differentially expressed
in ALK+ or ALK− ALCL cells and T-cells. Several miRNAs which are
significant differentially expressed in ALK+ and ALK− ALCLs separate
ALCLs depending on their ALK status. We identified a miRNA profile
specific to ALK+ ALCLs.
DO-066
The role of C/EBPβ in the phenotype of ALK+ anaplastic large cell
lymphoma
J .-A . Schmidt 1 , I . Bonzheim1 , S . Schäfer1 , F . Fend1 , L . Quintanilla-Martinez1 1University Hospital Tübingen, Institute of Pathology and Neuropathology,
Tübingen
Aims. ALK+ anaplastic large cell lymphoma is characterized by the loss
of pan T-cell antigens and the unusual expression of myelomonocytic
surface markers. The forced overexpression of the transcription factor C/
EBPβ in B and T-cells has been shown to induce transdifferentiation into
macrophages. Since C/EBPβ has been shown to play a central role in the
pathogenesis of ALK+ ALCL, the aim of the study was to investigate its
influence on the unusual phenotype of ALK+ ALCL.
Methods. The expression of 242 surface antigens was investigated in
ALK+ ALCL cell lines before and after the specific knockdown of C/
EBPβ using the BD Lyoplate Human Cell Surface Marker Screening Panel
by flow cytometry. A highly specific C/EBPβ-shRNA was transduced
by lentiviral infection. The expression of significant surface markers was
validated by immunohistochemistry and Western blot in primary ALK+
and ALK− ALCL cases.
Results. The surface marker screening confirmed the loss of T-cell specific
markers, such as TCR chains and CD3, and overexpression of EMA
and activation markers CD25 and CD30 but did not support the unusual
expression of myeloid markers as CD13 or CD33, as previously described.
Activation surface markers, including CD25, CD30, CD97 and CD98,
showed downregulation after C/EBPβ knockdown indicating a role for
C/EBPβ in the activation of ALK + ALCL cells. CD147 (EMMPRIN) was
strongly expressed in ALK + ALCL cells, and was downregulated after C/
EBPβ knockdown. Interestingly, CD147 was differentially expressed in
ALK + ALCL when compared to ALK − ALCL primary cases.
Conclusions. Surface marker screening in ALK+ ALCL revealed an influence
of C/EBPβ on the activation phenotype of the neoplastic T-cells and
expression of CD147, an inductor of matrix metalloproteinases implica-
32 | Der Pathologe · Supplement 1 · 2012
ted in tumor progression and metastasis in solid tumors. The differential
expression of CD147 in ALK + ALCL suggests its involvement in the pathogenesis
of ALK + ALCL.
AG Orthopädische Pathologie
DO-079
Histomorphological and clinical characterisation of Epstein-Barr
virus-associated post-transplant smooth muscle tumours
L . Maegel1 , J . Rische1 , C . Tiede1 , J . Salem1 , F . Laenger1 , H . Kreipe1 , K . Hussein1 ,
D . Jonigk1 1Hannover Medical School, Institute of Pathology, Hannover
Aims. Histomorphological and clinical characterisation of Epstein-Barr
virus-associated post-transplant smooth muscle tumours.
Methods. Own cohort: Five PTSMT were examined by histology,
immunohistochemistry and molecular methods [mean age of patients
10 years; PTSMT developed after a mean interval of 44 months following
liver (n=2), heart (n=2) or bone marrow (n=1) transplantation]. Metadata
analysis: Data of 64 PTSMT cases (including our cohort) of the last
20 years were re-evaluated by applying survival analysis. Molecular analyses:
All specimen of our cohort underwent compartment-specific laser
microdissection and processed for further quantitative real-time PCR
analysis. Gene expression of transcripts for a set of 20 EBV-associated
endogenous human genes were analysed by qPCR: transcription factors
MYC, TP53, NFKB1; apoptosis factors such as BAX; JAK3/STAT signal
factors; cytokines such as VEGF; miR-155 and miR-146a. To evaluate the
origin of PTSMT – either from the recipient or from the donor – short
tandem repeat (STR)-PCR was performed (“molecular fingerprinting”).
Results. Histomorphology and immunoprofile (total cohort, n=64):
– Spindle shaped, leiomyogenous cells (actin+/desmin+/EBER+), local
invasion.
– Most PTSMT show no marked cellular atypia, no tumour necrosis,

DO-080
DOG1: an immunohistochemical marker for neoplastic chondroblasts
in chondroblastoma
H . Akpalo 1 , C . Lange 2 , J . Zustin 1
1 University Medical Center Hamburg-Eppendorf, Institute of Pathology,
Hamburg, 2 University Medical Center Hamburg-Eppendorf, Clinic for Stem
Cell Transplantation, Hamburg
Aims. Chondroblastoma represents less than 1% of all primary bone tumors
and typically presents in the epiphysis of long bones of young patients.
The tumor is composed of cartilaginous and osseous matrix along
with cellular portions with polygonal chondroblasts with indented nuclei
and scattered osteoclast-type multinucleated cells. In the current study,
we wished to investigate the expression of several established immunohistochemical
markers in the cellular portions of chondroblastomas.
Methods. Nine chondroblastomas, seven chondromyxoid fibromas, five
giant cell tumors of bone and tissues from five fetal femoral bone endings
were analyzed using immunohistochemical antibodies (CD34, SMA,
DOG1, CD117, and CD163).
Results. The cellular areas of chondroblastoma cases contained nests of
DOG1+ SMA+ CD117− CD34− chondroblasts, a phenotype that was not
detected in chondromyxoid fibroma cases or in giant cell tumors. The
remaining chondroblasts showed only low expression of DOG1 or negative
reaction. Furthermore, numerous CD163+ macrophages were detected
in all tumors which were analyzed in our study: chondroblastomas,
chondromyxoid fibromas and giant cell tumors.
Conclusions. We described membranous DOG1+ chondroblasts located
within cellular portions of chondroblastoma along with CD163+ macrophages
and multinucleated osteoclastic giant cells. Therefore, chondroblastoma
can be added to the tumors that are usually positive for DOG1,
alongside GIST, rare solid-pseudopapillary neoplasms of the pancreas as
well as exceptional mesenchymal tumors including peritoneal leiomyomatosis,
uterine type retroperitneal leiomyoma, and synovial sarcoma.
DO-081
Bone erosion and inflammation – polymorphonuclear neutrophils
promote generation of osteoclasts in osteomyelitis patients
M .M . Gaida1 , B . Mayer2 , S . Stegmaier2 , P . Schirmacher1 , C . Wagner3 ,
G .M . Hänsch2 1 2 University of Heidelberg, Institute of Pathology, Heidelberg, University
of Heidelberg, Institute of Immunology, Heidelberg, 3BG Trauma Center
Ludwigshafen, Ludwigshafen
Aims. Chronic and persistent inflammatory processes in the proximity of
bones may lead to severe bone erosion, requiring the amputation of the
respective limb. Aim of the present study was to elucidate the process of
bone erosion in the context of inflammation.
Methods. To explore the relationship between inflammation and bone
erosion, biopsies of patients with osteomyelitis due to arterial occlusive
disease or to diabetes mellitus were examined (n=31) and the inflammatory
infiltrate, bone erosion and infiltration of osteoclasts were quantified.
In parallel, interleukin (IL)-8-induced differentiation of CD14+
monocytes derived from the peripheral blood of healthy individuals
to osteoclasts was tested in vitro. The cells were cultivated with monocyte
colony stimulating factor (M-CSF) and IL-8, and for comparison
with the well established osteoclast-inducing receptor activator of Nf κ
B ligand (RANKL). To verify the activity of newly generated osteoclasts
the ability to degrade ivory slices was tested. The classical pathway of
the osteoclastogenesis (NFATc1 and c-fos) was explored by Western blot
analysis of isolated cytoplasmic and nuclear proteins after stimulating
the isolated monocytes with IL-8.
Results. In tissue sections of osteomyelitis patients, in areas of bone destruction,
the number of osteoclasts correlated significantly with the
extent of the leukocytic infiltrate, particularly with the number of polymorphonuclear
neutrophils (PMN). PMN recovered from the infec-
ted sites showed characteristics of activation and produced interleukin
(IL)-8. CD14 + monocytes derived from the peripheral blood of healthy
individuals were cultivated with (M-CSF) and IL-8, and within 3 days,
a translocation of the transcription factor NFATc1 into the nucleus was
seen, as begin of the differentiation into osteoclasts. By 10 to 20 days,
multinucleated cells with the typical osteoclast morphology appeared
which expressed tartrate-resistant acid phosphatase (TRAP) and cathepsin
K. Moreover, these cells were able to resorb bone.
Conclusions. In patients with persistent inflammatory disease and loss of
bone, the abundance of PMN in areas of bone resorption correlated with
the number of osteoclasts. Since activated PMN are known to produce
IL-8, which is able to induce osteoclast formation, we propose that PMN
promote bone destruction by local generation of osteoclasts and thus
provide a link between the inflammation and bone erosion.
DO-082
High IGF2 and FGFR3 are associated with tumor progression in
pleomorphic undifferentiated sarcomas, but EGFR and FGFR3
mutations are a rare event
K . Rüping1 , D . Katenkamp1 , Y . Chen1 , A . Altendorf Hofmann2 , U . Settmacher2 ,
I . Petersen1 , T . Knösel1 1 2 Friedrich-Schiller University, Institute of Pathology, Jena, Friedrich-Schiller
University, Department of General, Visceral und Vascular Surgery, Jena
Aims. Pleomorphic undifferentiated sarcoma (formerly known as malignant
fibrous histiocytoma, MFH) is meanwhile recognized as a morphological
growth pattern shared by a wide variety of poorly differentiated
malignant neoplasms, which include specific subtypes of pleomorphic
sarcomas. Nevertheless prognostic and therapeutic options in these tumors
are urgently needed.
Methods. 327 fibroblastic/myofibroblastic differentiated tumors consisted
of 203 pleomorphic undifferentiated sarcomas, 42 low grade sarcomas
(10 low grade fibromyxoid sarcoma, 32 low grade myofibroblastic
sarcomas) and 82 pseudosarcomatous tumors of the fasciitis family were
analyzed immunohistochemically and correlated with clinicopathological
parameters. Additionally mutational analysis was performed on high
expressed specimens of EGFR and FGFR3.
Results. High expression was found in PDGFRA (45%), PDGFRB (35%),
EGFR (3.4%), TFE (30%), KDR (1.5%), IGF2 (68%), FGFR1 (6.5%) and
FGFR3 (52%). High expression of IGF2 and FGFR3 was significantly
correlated with higher tumor grading (low versus high, p5 cm, p

Abstracts
study was to evaluate the expression and mutational status of potential
molecular therapeutic targets in synovial sarcomas.
Methods. 38 well characterized and molecularly confirmed cases of synovial
sarcomas were included in this study. Immunohistochemical stainings
of tyrosine kinase receptors of the EGF-R family (EGF-R, HER2/
neu, HER3, HER4), the hepatocyte growth factor c-met, and signaling
molecules implicated in the mTOR pathway (AKT, mTOR, PTEN), as
well as E-Cadherin and snail was performed. In addition, cases were
screened for mutations in the EGFR, PIK3C, B-RAF, K-RAS and N-RAS
genes.
Results. Members oft the EGF-receptor family of kinases as well as E-
Cadherin and snail are important for defining the tumor phenotype by
determining epithelial-mesenchymal transition of synovial sarcomas.
Activation of c-met and signaling molecules oft the mTOR pathway are
seen in a significant number of cases. Mutations of the genes studied
(EGFR, PIK3C, B-RAF, K-RAS, N-RAS) are an overall rare event in synovial
sarcomas.
Conclusions. EGF-R expression is found in many synovial sarcomas, however,
activating mutations in the tyrosine kinase domain or downstream
signaling molecules appear to be a rare event or are absent. Activation
of c-met and molecules oft the mTOR pathway is frequently seen in
synovial sarcomas. The benefit of targeted therapy against these genes in
synovial sarcomas remains to be determined.
DO-084
Allergy to metal implants: Immunological und histological analysis
of patients with intolerance reaction after knee arthroplasty
J . Schneider1 , M . Flaig1 , B . Summer1 , C . von der Helm1 , C . Schopf1 , V . Krenn2 ,
M . Thomsen3 , L . Frommelt4 , P . Thomas1 1 2 Ludwig-Maximilians-University, Munich, Dermatology, München, Zentrum
für Histologie, Zytologie und Molekulare Diagnostik Trier, 3Orthopädische- DRK-Klinik, Baden-Baden, 4Endoklinik, Hamburg
Aims. Allergic reaction to metal implants as a reason for implant loosening
or other complications is controversially discussed. Therefore we
analysed 10 patients with metal allergy but no infection or mechanical
problems in knee arthroplasty who needed revision surgery.
Methods. In periprosthetic tissue of the 10 patients with metal hypersensitivity
(patch testing and/or lymphocyte transformation test (LTT)
positive) and CoCrMo based arthroplasty, histological classification (according
to consensus classification of periprosthetic interface membranes)
and molecular cytokine analysis (Realtime-PCR) was performed.
The results were compared with a control group of 5 patients without
metal hypersensitivity. After implant replacement to titanium or oxinium
based arthroplasty the symptoms were monitored with the WO-
MAC-Score.
Results. Patch testing: 4/10 reactivity to nickel, 3/10 to cobalt, 1/10 to chromium.
Enhanced LTT reactivity: 8/10 to nickel, 1/10 to cobalt. Cytokine
expression: IFNy 4/10 vs. 0/5; TGFβ 8/10 vs. 5/5; IL-8 8/10 vs. 0/5; IL-6 6/10
vs. 1/5, IL10 7/10 vs. 5/5. In histopathology primarily the indeterminate
type of periprosthetic tissue (type IV) or arthrofibrosis and a varying
degree of lymphocytic infiltration were detected. The WOMAC-Score
increased from 40.4±20.58 to 55.59±20.14.
Conclusions. The combination of allergological, immunological and histopathological
diagnostic steps helps to identify patients with implant
intolerance reaction and may support the decision to a replacement with
alternative material, such as titanium based arthroplasty.
34 | Der Pathologe · Supplement 1 · 2012
DO-085
Expression patterns of microRNA in SFT – prediction of malignancy?
C . Poremba1 , C . Altmann1 , N . Arens1 , J . Kriegsmann1 , P . Knöß2 1Research Park Trier, Center of Histopathology, Cytology and Molecular
Diagnostics (CHCMD) Trier, Trier, 2Center of Histopathology, Cytology and
Molecular Diagnostics Trier
Aims. The clinical and biologic behavior of solitary fibrous tumor (SFT)
has been a problematic issue, mainly because of inconclusional criteria.
SFT harboring “malignant” features such as high cellularity, >4 mitotic
figures/10HPF and necrosis/hemorrhage are at risk for metastasis/recurrence.
However, in biopsies those criteria may not be evident. In our
study, we analyze if expression patterns of microRNA, a class of posttranscriptional
regulators, differs between localized, relapsed and metastatic
SFT, and may help to predict clinical and biologic behavior in this
tumor entity.
Methods. In this pilot study, tumor tissues from 6 patients suffering from
SFT (3 localized without recurrence/metastasis, 3 with recurrence/metastasis)
are analyzed by microRNA assay (Applied Biosystems, Carlsbad,
CA USA). Clinical follow-up is available up to 10 years after initial diagnosis.
Expression patterns of microRNAs are compared between localized
SFT versus relapsed/metastatic SFT and within this group between
the initial tumor (areas of different cellularity) and its respective recurrence/metastasis
after microdissection.
Results. In ongoing analyses, expression patterns of microRNAs are
compared between localized vs. relapsed/metastatic SFT and are correlated
to clinical outcome.
Conclusions. We investigate if different expression patterns of microR-
NAs between localized vs. metastatic/relapsed SFT may help to identify
patients with higher risk for unfavourable clinical outcome based on the
initial biopsy of SFT. Statistical analyses are ongoing.
DO-086
Defining arthrofibrosis in histopathological specimens:
an evolving concept
P . Knöß1 , C . Dierkes1 , M . Ruppert2 , T . Gehrke3 , D . Kendoff3 , C . Theiß4 , V . Krenn1 1Medical health center for histology, cytology and molecular diagnostics
Trier, 2Brothers of mercy hospital Trier, 3ENDO Clinic Hamburg, 4Ruhr University
Bochum
Aims. Arthrofibrosis is the most severe complication in endoprothetic
surgery leading to a complete loss of joint function. In the past we reported
a proposal for a histopathological grading system for arthrofibrosis
(grade 1–3). This time we wanted to verify our results immunohistochemically
using β-catenin, a marker known for his association with fibromatosis.
Methods. 262 specimens of patients with clinical evidence of arthrofibrosis
were graded semiquantitatively for fibroblast density using our
proposed grading system, stained with antibodies against β-Catenin and
compared with a reference group of 29 neosynovialitis type 4 specimens.
Results. In 85.1% of the specimens the histopathologic diagnosis was arthrofibrosis.
The distribution for the fibroblast density was 28.8% grade 1,
47.7% grade 2 and 23.4% grade 3. The cellularity was significantly higher
in every arthrofibrosis grade than in the reference group (p

AG Oralpathologie
DO-087
Detection of human papillomavirus infection in head and neck
squamous cell carcinoma
J . Dreyer 1 , M . Barros 1 , G . Niedobitek 1
1 Unfallkrankenhaus Berlin, Institute for Pathology, Berlin
Aims. A sub-group of head and neck squamous cell carcinoma (HNSCC)
is associated with HPV infection, mainly with HPV16. The incidence of
this subset has increased and there is evidence to suggest that HPV-positive
HNSCC show a more favourable prognosis. There is, however, controversy
regarding the most suitable method for the detection of HPV
infection in this setting. We have compared HPV DNA in situ hybridisation
(ISH) with p16 immunohistochemistry (IHC).
Methods. 141 cases of HNSCC diagnosed between 1997 and 2010 were
identified. 119 patients (84.4%) were male and 22 (15.6%) were female.
Primary site and pTNM stage as well as all other relevant clinicopathological
data were extracted from the clinical and pathological files. Primary
sites were oropharynx in 65 cases, hypopharynx in 21 cases, floor
of mouth in 31 cases, tongue in 20 cases and other sites in the oral cavity
in 4 cases. Tissue-micro-arrays (TMA) with three 2 mm cores per case
were constructed. IHC for p16 (mtm laboratories) and ISH for high-risk
and low-risk HPV-DNA (Ventana) were carried out.
Results. 23 cases (16.3%) were positive for p16. Of these, 17 cases (12.1%
overall) also showed a nuclear signal for high-risk HPV-DNA by ISH.
No p16- cases were positive by ISH and no infection with low-risk HPV
types was detected. Thus, there was a statistically significant association
of p16 expression and high-risk HPV infection in our series (p

Abstracts
the nasopharyngeal tonsil might relate to the anatomical restriction of
respiratory surface epithelium to this type of tonsil and might thereby
give a clue of the primary site of malignant transformation by EBV.
DO-090
Analysis of human papilloma virus (HPV) and Epstein-Barr virus
(EBV) in salivary gland adenocarcinomas
E . Senft1 , H . Kreipe1 , K . Hussein1 1Hannover Medical School, Institute of Pathology, Hannover
Aims. Viruses are known to be associated with neoplastic proliferation,
e.g. epitheliotropic human papilloma virus (HPV) can be detected in
squamous neoplasms such as cervix carcinoma and oropharynx carcinoma
while Epstein-Barr virus (EBV) infects B cells and can induce malignant
lymphomas. Furthermore, EBV persists in the ductal epithelial
cells of salivary glands and can be associated with solid neoplasms such
as benign Warthin tumour. Systematic analyses have been performed
in squamous carcinomas of the head and neck but not in salivary gland
adenocarcinomas.
Methods. Histological re-evaluation and selection of samples: adenoidcyctic
carcinomas (n=20), adenocarcinomas, NOS (n=17), mucoepidermoid
carcinomas (n=11), pleomorphic adenoma (n=4), carcinoma
ex pleomorphic adenoma (n=3), non-neoplastic salivary glands (n=65).
Analysis of multi-blocks with a total of 120 formalin-fixed and paraffinembedded
(FFPE) tissue samples by HPV immunhistochemistry and
EBER in situ hybridisation. DNA extraction from FFPE tumour samples
and evaluation of HPV by multiplex PCR.
Results. HPV and EBV were not detectable in salivary gland carcinomas.
Conclusions. This is the first systematic analysis which demonstrates that
the two human pathogenic viruses HPV and EBV are not involved in the
pathobiology of salivary gland adenocarcinomas.
DO-091
SOX2 amplification is a common event in sinunasal squamous cell
and undifferentiated carcinomas
F . Göke1 , A . Franzen2 , R . Menon2 , S . Huss3 , D . Boehm2 , W . Vogel2 , F . Bootz4 ,
S . Ihrler5 , A . Schroeck4 , S . Perner1 1 2 3 University Hospital Bonn, Pathology, University Hospital Bonn, University
Hospital Cologne, Institute of Pathology, 4University Hospital Bonn,
Head and neck department, 5Laboratory for Dermatohistology and Oral
Pathology
Aims. Although carcinomas of the nasal cavities are known to differ significantly
from other cancers of the head and neck, regarding causing
noxa, clinical behavior, and treatment, they share histological appearance.
SOX2, a transcription factor-coding gene located at 3q26.33, is known
to be recurrently amplified in squamous cell carcinomas (SCCs) of the
lung, esophagus, skin, penis, cervix uteri and oral cavity. The aim of our
study was to assess if SOX2 amplifications also occur in different tumor
entities of the paranasal sinuses.
Methods. Using fluorescence in-situ hybridization, we assessed for SOX2
amplification status in a cohort consisting of sinonasal SCCs (n=65), sinonasal
undifferentiated carcinomas (SNUC, n=18), adenocarcinomas
(n=25), and adenoid cystic carcinomas (n=18). Furthermore, we performed
SOX2 immunohistochemical staining to quantify protein expression.
Results. We detected SOX2 amplifications in 36% of sinunasal SCCs, 35%
of SNUCs, 9% of adenocarcinomas, but none of the adenoid cystic carcinomas.
Moreover, we found that the SOX2 amplification is associated
with a SOX2 protein overexpression in SCCs and SNUCs.
Conclusions. SOX2 amplification is not an organ site specific event, but
is a frequent genetic alteration occurring in SCCs of various organs. Since
SNUCs also harbor SOX2 amplifications in similar frequencies, we
36 | Der Pathologe · Supplement 1 · 2012
hypothesize that SNUCs may be undifferentiated SCCs of the sinunasal
cavity.
DO-092
Expression of differentiation factor caspase 14 in oral squamous
carcinomas
C . Scharenberg1 , H . Kreipe1 , K . Hussein1 1Hannover Medical School, Institute of Pathology, Hannover
Aims. Caspase 14 is not involved in apoptosis (in contrast to all other
caspase family members) but in differentiation of squamous epithelia.
Caspase 14 is expressed mainly in the suprabasal layers, particularly the
Str. intermedium/spinosum. Systematic analyses have been performed
in cervix carcinomas and skin cancer but not oral cavity squamous carcinomas.
Methods. Histological re-evaluation and selection of samples: squamous
carcinomas of the oral cavity (n=30) and oral leukoplakia (n=10). Caspase
14 expression analysis by immunhistochemical evaluation of formalin
fixed and paraffin embedded (FFPE) tissue samples.
Results. Nuclear and cytoplasmatic caspase 14 expression is evident in
non-neoplastic epithelial cells of the Str. intermedium but absent or weak
in the basal and superficial layers. In leukoplakia the protein expression
is increased in cells with keratinisation. In invasive lesions, caspase 14 is
mainly expressed in the cells with keratinisation but absent or weak in
neoplastic cells without keratinisation.
Conclusions. This is the first experimental evidence that differentiation
factor caspase 14 is expressed in oral cavity squamous carcinomas.
Similar to leukoplakia expression of caspase 14 is increased in carcinoma
cells with keratinisation.
DO-093
FGFR1 amplification in metastatic squamous cell carcinoma of the
head and neck – a potential target for a rational therapy?
F . Göke1 , A . Franzen2 , R . Menon2 , R . Kirsten2 , D . Boehm2 , W . Vogel2 , F . Bootz3 ,
A . Schroeck3 , S . Perner1 1 2 3 University Hospital Bonn, Pathology, University Hospital Bonn, University
Hospital Bonn, Head and neck department
Aims. Currently, patients with FGFR1 amplified squamous cell lung cancers
(L-SCC) are treated in phase I clinical trials using small molecule
inhibitors. Of interest, SCC of the lung share common molecular alterations
with squamous cell head and neck cancers (HN-SCC). Aim of our
study is to assess if HN-SCCs also harbor FGFR1 amplifications. Furthermore,
we aim to identify a HN-SCC cell line harbouring FGFR1 amplification
and inhibit cell proliferation using a small molecule inhibitor.
Methods. We put together a cohort of 227 patients suffering from HN-
SCC, with 97 of these suffering from metastatic disease. Primary tumors
and, where available, metastatic tumors were assessed for FGFR1 copy
number status using fluorescence in-situ hybridization (FISH). We tested
different cell lines for FGFR1 amplification status and inhibited these
with small molecule inhibitors.
Results. 20.3% of primary HN-SCC displayed FGFR1 amplifications. Of
interest, almost all metastatic tumor samples revealed a FGFR1 amplification
if the corresponding primary tumor harbored the amplification.
The cell lines HN and SCC-25 harboured FGFR1 amplifications. HN cell
proliferation was inhibitable with small molecule inhibitors.
Conclusions. FGFR1 amplification frequently occurs in primary and metastatic
HN-SCC and proves as a potential target for small molecule therapy
in non-operable or metastatic disease. Furthermore, cell growth of
FGFR1 amplified cell lines is inhibitable with small molecule inhibitors.
Additional functional studies and subsequent clinical trials are needed
for further validation of our findings.

DO-094
Do activated fibroblasts influence epidermal growth factor receptor
(EGFR) inhibitor sensitivity in oral squamous cell carcinoma
cells (OSCC)?
P . Richter 1 , N . Neumann 2 , J . Schulte 3 , K . Schult 1 , S . Weisheit 4 , M . Franz 5 ,
O . Guntinas-Lichius 6 , C . Liebmann 4 , I . Petersen 1 , A . Berndt 1
1 Jena University Hospital, Institute of Pathology, Jena, 2 University Hospital
Zurich, Institute of Surgical Pathology, Zurich, Switzerland, 3 Ludwig-Maximilian-University,
Munich, Großhadern Medical Center/Department of Cardiac
Surgery, München, 4 Friedrich Schiller University Jena, Institute of Biochemistry
and Biophysics, Jena, 5 Jena University Hospital, Clinic for Internal
Medicine I, Jena, 6 Jena University Hospital, Department of Otorhinolaryngology,
Jena
Aims. Although EGFR is involved in development of OSCC and EGFRinhibitor
sensitivity could be shown in OSCC cell lines, a therapeutic
benefit of an EGFR-inhibitor therapy can be observed only in a minority
of patients. It was suggested that the carcinoma microenvironment have
modulating effects on inhibitor sensitivity in vivo. One possible hypothesis
is that carcinoma associated fibroblasts induce phenotype changes
such as epithelial-mesenchymal transition (EMT) which are accompanied
by alterations in EGFR signalling. Thus, the study was aimed at investigating
the influence of growth factor activated fibroblasts on EGFRinhibitor
sensitivity of OSCC in vitro applying Gefitinib.
Methods. “Activated” fibroblasts were generated by stimulation of
hTERT-BJ1 fibroblasts with TGFbeta1, PDGFAB, aFGF, and TGFbeta1/
aFGF, respectively. They were characterized with regard to proliferation,
expression of fibroblast markers, activation of EGFR signalling, and
the capability to induce OSCC cell invasion as well as their Gefitinib
sensitivity using immunohistochemistry, western blotting, rtRT-PCR,
MTT test and Boyden chamber assay. Furthermore, Gefitinib sensitivity
was evaluated in different OSCC cell lines by MTT test. The impact of
TGFbeta1 and TGFbeta1/aFGF activated fibroblasts on EGFR inhibitor
sensitivity was assessed by pre-culturing of OSCC cells in fibroblast conditioned
media.
Results. In vitro activated fibroblasts showed a strong upregulation of
ASMA and fibronectin due to stimulation with TGFbeta1. PDGFAB stimulation
induced proliferation with up-regulation of EGFR and pAKT.
aFGF and TGFbeta/aFGF stimulation resulted in an intermediate phenotype.
TGFbeta1 stimulated fibroblasts exhibited the highest capability
to induce invasion in the OSCC cell line PE/CA-PJ15 with upregulation of
N-cadherin. Interestingly, activated fibroblasts differed in their Gefitinib
sensitivity (TGFbeta-stim.=”low” and PDGFAB stim.=”high”). OSCC
cell lines tested so far show a different Gefitinib sensitivity. Furthermore,
pre-culturing in fibroblast conditioned medium leads to a partial reversibility
of the Gefitinib effect.
Conclusions. Results indicate that: 1) EGFR inhibition by Gefitinib
affects OSCC cells as well as activated stromal fibroblasts, 2) the different
Gefitinib sensitivity of fibroblast phenotypes may lead to a selective
accumulation of myofibroblasts during treatment, and 3) OSCC cell sensitivity
is modulated by stromal fibroblasts.
DO-095
Evaluation of post-transplant lymphoproliferative diseases
(PTLD) with manifestation in the oral cavity
C . Tiede1 , B . Maecker-Kolhoff 2 , H . Kreipe1 , K . Hussein1 1 2 Hannover Medical School, Institute of Pathology, Hannover, Hannover
Medical School, Hannover
Aims. Inspection of the oral cavity can be easily performed in transplanted
patients with an oral tumour mass and suspected Epstein-Barr virus
(EBV)-associated post-transplant lymphoproliferative disease (PTLD).
We aimed to evaluate the main sites of manifestation and the morphological
subtypes of PTLD in the oral cavity.
Methods. Evaluation of histomorphology and clinical data on early lesion,
polymorphic, and monomorphic PTLD. Total cohort of 212 patients
(median age 9 years, range 0.5–70 years, 57% males/43%females, 75.5%
children/24.5% adults).
Results. Oral cavity PTLD manifestation was found in 61/212 patients
(29%): 42/61 early lesion PTLD (20%), 12/61 polymorphic PTLD (6%)
and 7/61 monomorphic PTLD (3%). Tonsils were the most frequent site
of manifestation (n=57/61, 93%) including early lesion PTLD (n=42/57,
74%), polymorphic PTLD (n=12/57, 21%) and monomorphic B cell PTLD
(n=3/57, 5%). Other localisations were gingiva (n=2/61, 3%; EBV+ plasmocytomas),
maxillary bone (n=1/61, 1.5%; EBV+ plasmocytomas) and
larynx (n=1/61, 1.5%; EBV-T-cell PTLD).
Conclusions. Tonsils are the main site of PTLD manifestation in the oral
cavity and comprise mainly benign early lesion PTLD and polymorphic
PTLD. Oral cavity monomorphic PTLD is rare and is located outside of
the tonsils in a considerable proportion (n=4/7, 57%) of cases.
DO-096
Non-sebaceous lymphadenoma of salivary glands: proposed
development from intraparotid lymph nodes and risk of misdiagnosis
C . Weiler1 , A . Agaimy2 , P . Zengel3 , J . Zenk4 , T . Kirchner1 , S . Ihrler5 1 2 Ludwig Maximilian University, Institute of Pathology, München, University
Hospital Erlangen, Institute of Pathology, Erlangen, 3Ludwig Maximilian
University, Head and Neck Surgery, München, 4University Hospital Erlangen,
Head and Neck Surgery, 5Laboratory for Dermatohistology and Oral Pathology,
München
Aims. Non-sebaceous lymphadenoma (NSLA) is a rare benign salivary
gland tumour composed of lymphoid and epithelial components. Definitionally,
the epithelial component lacks sebaceous differentiation and,
instead, displays a wide range of histological differentiation. In this study,
we have collected 9 cases of NSLA to characterize their histological
and immunohistochemical profile.
Methods. The samples were histologically reviewed, and immunohistochemical
stains for CK5/6, CK7, CK14, CK18, p63, and Ki67 performed.
Patients were 6 males and 3 females (mean age, 50 years).
Results. All tumours were located in the parotid gland and showed
intimate intermingling of lymphoid tissue with islands or strands of
epithelium with a wide spectrum of histological differentiation. The
immunohistochemical profiles mirrored the epithelial differentiation;
hence, areas with basaloid or lymphoepithelial differentiation strongly
expressed CK5/6, CK14, and p63, while areas with ductal differentiation
showed strong positivity for CK18/CK7 and CK5/6/CK14/p63 in luminal
and basal cell layers, respectively. A hilus structure with salivary inclusions
or D2-40 (podoplanin) positive marginal sinus were identifiable in 4
and 9 of the cases, respectively, confirming origin within intra-/periparotid
lymph nodes. Six cases were initially misdiagnosed as other benign
(n=4) or malignant tumours (n=2).
Conclusions. Our study on the largest series of NSLA reported to date
provides strong evidence that NSLA belongs to the group of salivary
gland tumours that pathogenetically develop from embryonic salivary
gland inclusions in intra-/periparotid lymph nodes. Knowledge of the
wide histological spectrum of this rare and presumably underreported
tumour is important in order to avoid misdiagnosis, particularly as malignant
tumour.
Der Pathologe · Supplement 1 · 2012 |
37

Abstracts
DO-097
Cytokeratin-positive epithelioid angiosarcoma presenting in the
tonsil: a diagnostic challenge
A . Agaimy 1 , H . Kirsche 2 , S . Semrau 3 , H . Iro 2 , A . Hartmann 1
1 Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen,
2 Friedrich-Alexander University of Erlangen, Department of Otorhinolaryngology,
Head and Neck Surgery, Erlangen, 3 Friedrich-Alexander University of
Erlangen, Department of Radiation oncology, Erlangen
Aims. The majority of malignant neoplasms of the head and neck represent
squamous cell carcinomas while sarcomas are rare in this anatomic
region. Primary oral cavity sarcomas are exceedingly rare and may pose
a great diagnostic challenge.
Methods. A 71-year-old woman without previous history of malignancy
or radiation to the head and neck presented with an antibiotic-refractory
diffuse painful swelling of the right tonsil necessitating tonsillectomy.
Within months the patient underwent surgical resection of multiple
bleeding intraoral and gastrointestinal metastases. She is currently alive
with disease 9 months from diagnosis.
Results. Histological evaluation revealed subtotal replacement of the
right tonsil by a high-grade epithelioid neoplasm displaying extensive
ulceration, necrosis and primitive vasoformation. Immunohistochemistry
showed strong/diffuse expression of pancytokeratin (CK) antibodies
KL-1 and Lu5, CK8, CK18, CK19, vimentin, CD31, ERG and FLI-1. High
molecular weight cytokeratins (CK5, 34ß12), CK7, CK13 and CK20 were
not expressed.
Conclusions. To our knowledge, this case represents the first well documented
primary epithelioid angiosarcoma of the tonsil. The strong cytokeratin
expression in epithelioid angiosarcomas represents a diagnostic
pitfall. Thus, awareness of this rare and highly aggressive neoplasm is necessary
for distinguishing it from poorly differentiated and acantholytic
squamous cell carcinoma and diffuse large cell lymphoma.
DO-098
Lipomatous neoplasms of the salivary glands. A series of 23 cases
with emphasis on oncocytic lipoadenoma and sebaceous differentiation
A . Agaimy1 , B . Märkl2 , H . Arnholdt2 , J . Zenk3 , V . Bonkowsky4 , M . Michal5 ,
A . Skalova5 , A . Hartmann1 , S . Ihrler6 1Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen,
2Augsburg Clinic Center, Augsburg, 3Friedrich-Alexander University of
Erlangen, Department of Otorhinolaryngology, Head and Neck Surgery, Erlangen,
4Nürnberg Clinic Center, Department of Otorhinolaryngology, Head
and Neck Surgery, Nürnberg, 5Charles University, Medical Faculty Hospital,
Department of Pathology, Pilsen, Czech Republic, 6Ludwig Maximilian University,
Munich, Department of Pathology, München
Aims. Lipomatous tumors of salivary glands are rare and have been the
subject of rare case reports in the literature. Accordingly, their morphologic
spectrum and clinicopathological features from a consecutive case
series have not been studied.
Methods. We collected 23 fatty tumors from consecutive surgical pathology
files at four large hospitals (n=17) and from consultation files of two
centers (n=6). Fat-containing pleomorphic adenoma and lipomatous
myoepitheliomas were excluded.
Results. There were 15 males and 8 females aged 18–89 yrs (mean, 55 yrs).
Most tumors (n=21) originated in the parotid gland. Two affected the
submandibular gland. Histologically, the tumors could be categorized
into three groups: ordinary lipoma (n=16; 70%), oncocytic lipoadenoma
(n=4) and non-oncocytic adenolipoma/sialolipoma (n=2). Ordinary lipomas
were either completely intraglandular or they have been submitted
as a lipomatous nodule containing minor foci of residual atrophic
serous acini at the periphery of the lipoma beneath the capsule. None
of the lipomas contained sebaceous elements or oncocytic cells. The less
38 | Der Pathologe · Supplement 1 · 2012
common oncocytic lipoadenoma (synonym: oncocytic sialolipoma) had
a fatty component ranging from 10% to 95% of the lesion that was usually
interspersed between the oncocytic acini. Sebaceous islands were found
in three of the four cases. The oncocytes showed either diffuse solid
sheets with a lobular architecture interrupted by scattered adipocytes
or were scattered between plentiful fatty tissues. The two non-oncocytic
adenolipoma (synonym: non-oncocytic sialolipoma) were predominantly
fatty (70–80%) and prominently lobulated. They displayed a biphasic
pattern with serous tissue diffusely distributed between the fatty components.
One lesion showed foci of sebaceous metaplasia.
Conclusions. Lipomatous tumors of the salivary glands are rare and most
represent intraglandular ordinary lipomas that are otherwise similar
to their soft tissue and cutaneous counterparts. While fairly absent in
ordinary salivary lipomas, sebaceous differentiation seems to be a common
feature of oncocytic lipoadenoma and non-oncocytic sialolipoma.
Lipomatous lesions of the salivary glands do not seem to be association
with other significant salivary gland pathology or to carry a risk of malignant
degeneration. Although lipomatous components in these lesions
might derive from intraglandular adipose tissue, the pathogenesis of the
oncocytic and sebaceous elements (metaplastic vs. neoplastic) remains
unclear.
AG Herz- und Gefäßpathologie
DO-100
microRNA-143 is essential in arteriogenesis
K . Troidl1 , G . Jung1 , C . Troidl2 , W . Schaper 1 , T . Schmitz-Rixen3 1Max-Planck-Institute for Heart and Lung Research, Bad Nauheim,
2 3 Kerckhoff Heart Centre, Bad Nauheim, Goethe University, Frankfurt
Aims. Arteriogenesis – the growth of pre-existing collateral arterioles to
functional arteries – is triggered by increased fluid shear stress (FSS).
MicroRNAs (miRNA) are implicated in post-transcriptional regulation
of gene expression. A FSS-induced signature pattern of miRNAs during
collateral growth might influence signal transduction of the physical
stimulus into a cellular response. We investigated the involvement of
miRNAs in arteriogenesis in a rat model of chronically elevated FSS in
collateral arteries.
Methods. 6 sprague dawley rats were subjected to femoral artery ligature
(FAL). A side-to-side anastomosis distal to the ligature was created
between the femoral artery and the accompanying vein, which leads to
chronically elevated FSS inside the collaterals. Following dissection of
collateral tissue 7 d after surgery, miRNA was isolated and an expression
profile was generated by microarray analysis. Differential expression was
confirmed by qRT-PCR. Cellular localization of selected miRNAs was
assessed by in situ hybridisation combined with immunostaining. By local
blockage of specific miRNAs in mouse collaterals we analyzed their
functional involvement in arteriogenesis.
Results. Growing collaterals showed a significant up-regulation of 6
miRNAs when compared to sham operated controls. miR-143, miR-195,
and miR-24 were localized in the media of growing collaterals. miR-24 is
also expressed in the FSS-stimulated endothelium. Blockage of miR-143
led to severe impairment of arteriogenesis.
Conclusions. These data indicate that miRNas are involved in arteriogenesis.
miR-143 belongs to a cluster which has been assigned to the phenotypic
switch of smooth muscle cells. We identified a functional implication
during vascular remodeling. Targeted modulation of this miRNA
in vivo suggests new treatment options in improvement of collateral
growth.

DO-101
Histopathological analysis of proteases in abdominal aortic
aneurysm wall
J . Pelisek 1 , M . Rudelius 2 , C . Reeps 1 , F . Lohoefer 1 , C . Lipp 1 , H .-H . Eckstein 1
1 Klinikum rechts der Isar der TU München, Clinic of Vascular Surgery,
München, 2 Klinikum rechts der Isar der TU München, Institute of Pathology,
München
Aims. Abdominal aortic aneurysm (AAA) wall is characterised by degradation
of extracellular matrix through plethora of proteases. In contrast
to the already well explored matrix metalloproteinases (MMPs), little is
known about other groups, such as ADAM family of metalloproteases
(a disintegrin and metalloprotease) or cathepsins. The aim of the study
was therefore a detailed analysis of expression of selected ADAMs
and cathepsins with known proteolytic activity of relevant extracellular
components of the vessel wall and their inhibitors in specimens of patients
with AAA.
Methods. Tissue samples of vessel wall of 35 AAA patients and 10 organ
donors were analysed by immunohistochemistry for expression of
MMP-1, -2, -3, -7, -8, -9, -12, -13, ADAM 8, 9, 10, 12, 15, 17, and cathepsin B,
D, K, L, S in all cells located within AAA. In addition, the known inhibitors
of these proteases TIMP-1, -3, and cystatin C were analysed.
Results. Endothelial cells (ECs) were positive for MMP-1, -3, -9, neovessels
expressed all MMPs tested except for MMP-13. Aortic medial
smooth muscle cells (SMCs) expressed MMP-1,-2,-3,-9. Inflammatory
infiltrates expressed all MMPs tested except for MMP-2, macrophages
expressed all MMPs. ADAMs were expressed in both AAA and control
aorta without any significant differences between the groups. SMCs,
neovessels, and macrophages were positive for all ADAMs tested. ECs of
AAA were positive for cathepsin D and partially for cathepsin B, K, and
S. Macrophages, neovessels and SMCs were positive for all cathepsins
tested. Inflammatory infiltrates expressed all cathepsins in the following
manner: D>B=S>K

Abstracts
Conclusions. The AIM2 expression and induction patterns suggest a role
in vascular pathogenesis. AIM2 might act as a danger signal in vascular
EC, SMC and infiltrating inflammatory cells.
DO-104
Carbamylated EPO-fusion protein and recombinant human EPO
during porcine kidney I/R injury
F . Simon1 , M . Gröger2 , O . McCook2 , E . Calcia2 , P . Radermacher2 , H . Schelzig1 1 2 University of Düsseldorf, University of Ulm, Ulm
Aims. A newly carbamylated erythropoietin-fusion protein (cEPO-FC)
and recombinant human EPO (rhEPO) equally protected against spinal
cord I/R injury in young, healthy swine [1]. In a recent clinical trial,
however, rh-EPO did not affect acute kidney injury in ICU patients [2].
Since patients often present with vascular disease and consecutive organ
dysfunction, we compared cEPO-FC and rh-EPO in swine with ubiquitous
atherosclerosis [3].
Methods. Pigs randomly received either of cEPO-FC (50 μg/kg), rh-EPO
(5000 IU/kg) or vehicle twice over 30 min before and during the first
4 h of reperfusion after 120 min of aortic occlusion using inflatable balloons.
We assessed creatinine-clearance, fractional Na+ excretion, blood
NGAL, cytokine and NO levels together with tissue histology and immune-histochemistry
and -blotting for iNOS, HO-1, HIF-1α , NF-κB,
and markers of apoptosis.
Results. All pigs presented with reduced glomerular filtration (creatinine-clearance
74±24 vs. 90–140 mL/min normal value) and pre-existing
histological organ damage. Neither cEPO-FC nor rh-EPO beneficially
influenced the I/R-induced kidney dysfunction, histological organ damage
nor tissue inflammation and apoptosis.
Conclusions. Pre-existing atherosclerosis-induced kidney dysfunction
and tissue damage may reduce the efficacy of cEPO-FC and rh-EPO to
prevent I/R-induced kidney damage.
References
1 . Simon F et al (2011) . Intensive Care Med, in press
2 . Thim T et al (2010) . EuroIntervention 6:261–8
3 . Endre Z et al (2010) . Kidney Int 77:1020–30
DO-106
Antibody-mediated rejection in cardiac transplant recipients is a
seasonal disease
K . Wassilew1 , N .E . Hiemann1 , D . Kemper1 , R . Hetzer1 1Deutsches Herzzentrum Berlin, Berlin
Aims. Diagnosis of antibody-mediated rejection (AMR) is still a matter of
controversial discussion. We suggested that staining for immunoglobulins
might improve the diagnostic spectrum of AMR because of seasonal
effects in complement deposition.
Methods. We studied prospectively all endomyocardial biopsies harvested
since 01/2011 (n=205) for acute cellular rejection, activated endothelial-cells
and deposition of C4d, C3d and IgA/M/G in interstitial
capillaries by paraffin immunohistochemistry. Histologic and immunohistochemical
parameters of AMR were classified according to the
ISHLT and studied for seasonal effects in an ordinal (Jan–Mar vs. Apr–
Jun vs. Jul–Sept vs. Oct–Dec) and nominal model (Oct–Mar vs. Apr–
Sept).
Results. Overall, 16% of biopsies showed signs of acute cellular rejection
of any grade and 46% of samples showed evidence of endothelial-cell
swelling. In the ordinal model (Jan–Mar vs. Apr–Jun vs. Jul–Sep vs.
Oct–Dec), seasonal effects were found for endothelial cell swelling (83%
vs. 14% vs. 49% vs. 40%; p

DO-108
Identification of potential criteria for a successful Rituximab
salvage therapy in kidney allograft rejection
M . Dämmrich 1 , C . Blume 2 , C . Bockmeyer 1 , S . Immenschuh 3 , A . Schwarz 2 ,
D . Agustian 1 , V . Broecker 1 , H . Kreipe 1 , J . Becker 1
1 Hannover Medical School, Institute for Pathology, Hannover,
2 Hannover Medical School, Centre for Internal Medicine, Hannover,
3 Hannover Medical School, Institute for Transfusion Medicine, Hannover
Aims. Rituximab (anti-CD-20 antibody) is often used in kidney transplantation
to treat rejection refractory to standard treatment. Rituximab
therapy carries a risk for serious infectious complications and is not
effective in all cases. Therefore clinica, serological or histopathological
criteria to predict Rituximab response are most desirable. As a first step
for the identification of useful criteria we did a retrospective exploratory
analysis to identify criteria that were different between Rituximab responders
and non-responders.
Methods. 18 renal transplant recipients who received Rituximab (375 g/
m2 body surface, 1–2 courses after steroid bolus therapy, 15× combined
with up to 5 courses of plasmapheresis) for standard-therapy resistant
rejection were included in the study with their last biopsy before therapy.
10 were identified by terminal loss of transplant function as nonresponders,
8 as responders. Clinical, serological and histopathological
parameters were compared between both cohorts by Wilcoxon- or χ2
tests. P-values were regarded as descriptive in this retrospective analysis.
Results. At time of biopsy more of the Rituximab non-responders had
panel-reactive antibodies (>85%), and their serum creatinine before therapy
was higher. Among the histopathological criteria tubulitis was less
severe in responders. No significant differences were found for any of the
other clinical, serological or histopathological criteria.
Conclusions. In this retrospective exploratory study we identified lower
serum creatinine before therapy, the presence of panel- reactive antibodies
in more than 85% at time of biopsy, and a less severe Banff t-score as
possible criteria to predict responsiveness to Rituximab therapy. These
criteria need validation in future prospective randomized studies.
DO-109
Splenectomy and postconditioning with the sphingosine-1-phosphate
agonist FTY720 protect the myocardium against ischemia
reperfusion injury
D . Goltz1 , S . Huss2 , E . Ramadori1 , L . Diehl3 , R . Büttner2 , R . Meyer4 1 2 University of Bonn, Dept . of Pathology, Bonn, University of Cologne,
Dept . of Pathology, Köln, 3University of Bonn, Institute of Molecular Medicine,
Bonn, 4University of Bonn, Physiology II, Bonn
Aims. The pathogenesis of myocardial ischemia reperfusion injury
(MI/R) involves the inflammatory response of the innate immune system.
A modulation of this response could be a potential future target in
the management of the acute coronary syndrome. Recently, the spleen
has been proved an important origin of a Ly6-Cpos monocyte subset
that readily invades the injured myocardium upon ischemic damage.
We operated a murine myocardial ischemia reperfusion model in splenectomised
animals to reduce the invasion of phagocytic active immune
cells. In a second pharmacologic approach we applied the sphinosine-1phosphate
analogue FTY720 with reperfusion to interfere with the maturation
of monocytic derived macrophages. The aim of this study was
to evaluate the short and long term outcome of MI/R after modulation
the immune response.
Methods. In a murine closed-chest ischemia-reperfusion model, myocardial
infarct volume was assessed by TTC staining after 24 h of reperfusion
and by planimetric quantification of fibrosis on the Masson
stained specimen after 21 days of reperfusion in control animals, splenectomised
animals and FTY720 treated mice. Within the same interval,
cardiac function was evaluated by catheterisation using a Millar cathe-
ter. The immune response was characterised by FACS analysis in whole
heart specimens after 24 h.
Results. After 24 h of reperfusion, splenectomised animals and FTY720
treated animals revealed a significant reduction of infarct volume. The
invasion of monocytes, especially of Ly6-Cpos monocytes was significantly
reduced in splenectomised animals compared to the control
group. Moreover, the ratio of inflammatory macrophages to resident
macrophages was significantly smaller in the splenectomised group.
FTY720 treated animals showed an almost exclusive invasion by monocytes
within the remote myocardium. Functional data show a significantly
improved systolic cardiac function in both, the splenectomised
and the FTY720 treated groups compared to the control animal after
21 days of reperfusion. Quantification of the area of fibrosis, however,
revealed a trend towards reduced myocardial scarring in both target
groups, but the decline failed to reach a level of significance.
Conclusions. Both, splenectomy and postconditioning with FTY720 are
cardioprotective within 3 weeks after MI/R. In splenectomised animals,
this effect is due to a reduction of early invading monocytes. The mechanism
of FTY720 efficiency still warrants further investigation.
DO-110
Nestin expression in fetal and adult lungs vessels as well as vascular
tumors
S . Gerlach1 , G . Kristiansen1 , A .M . Müller2 1University Bonn Medical Center, Institute of Pathology, Bonn,
2University Bonn, Department of Pediatric Pathology, Bonn
Aims. The neural stem/progenitor cell marker Nestin is a class VI intermediate
filament protein. It is not only expressed by undifferentiated
central nervous system (CNS) cells during development and adult CNS
cells but various tumour cells as well as in injured and regenerating tissues,
indicating nestin as a marker for activated, migrating, proliferating
cells. Recently it has been described in proliferating endothelial cells
(EC). We were interested in any differences Nestin expression in fetal
and adult vessels as well as vascular tumors.
Methods. Endothelial Nestin and D2-40 expression was studied by immunolocalisation
in chorionic tissue from early abortions (n=5), fetal
(n=21) and adult (n=3) lungs, infantile hemangiomas (n=5) and angiosarcomas
(n=5).
Results. Although Nestin was expressed by EC of all blood vessels but not
lymphatic vessels there were differences concerning expression intensity.
In the first trimester, EC in chorionic and villous tissue as well as EC of
pulmonary veins, arteries and capillaries showed the same strong Nestin-positivity.
Starting in the second half of the second trimester Nestin
expression in venous EC began to fade, while arterial EC still showed a
strong staining. In lungs of neonates and adults the Nestin expression
was even weaker, although in arteries it was still stronger expressed than
in veins. In angiosarcomas Nestin was strongly expressed by the tumour
cells. EC of all infantile hemangiomas were clearly but in comparison to
angiosarcomas weaker stained.
Conclusions. The intermediate filament Nestin can be regarded as endothelial
marker expressed by vascular EC already during the first weeks
of life. As it is not expressed by lymphatic EC it can help discriminating
blood vessel endothelium from lymphatic endothelium. In accordance
with other endothelial markers like Angiotensin I converting enzyme it
is weaker expressed in adult pulmonary veins than adult pulmonary arteries.
The strong Nestin expression in fetal vessels – when compared to
EC of mature vessels or hemangiomas – corresponds to a strong Nestinpositivity
in malignant endothelial tumor cells. At present we analyse
the role of Nestin in colorectal cancer resp. its tumour vessels concerning
expression patterns in different stages of cancer.
Der Pathologe · Supplement 1 · 2012 |
41

Abstracts
AG Molekularpathologie
DO-111
Improved method of detecting the ERG gene rearrangement in
prostate cancer using combined dual-color chromogenic and
silver in-situ hybridization
M . Braun 1 , J . Stomper 1 , D . Böhm 1 , W . Vogel 1 , V . Scheble 2 , N . Wernert 1 ,
Z . Shaikhibrahim 1 , F . Fend 3 , G . Kristiansen 1 , S . Perner 1
1 University Hospital Bonn, Institute of Pathology, Bonn, 2 University Hospital
Tübingen, Division of Hematology and Oncology, Tübingen, 3 University
Hospital Tübingen, Institute of Pathology, Tübingen
Aims. The recently detected TMPRSS2-ERG fusion revealed as a recurrent
and prevalent prostate cancer (PCa) specific event, which potentially
qualifies it for clinical utilizations. To detect this alteration, fluorescence
in-situ hybridization (FISH) is the method of choice. However, FISH
harbors some disadvantages for widespread adoption in clinical practice.
Subsequently, the chromogenic in-situ hybridization (CISH), that uses
organic chromogens, and the enzymatic metallography silver in-situ hybridization
(SISH) emerged as promising bright-field alternatives. Compared
to CISH, SISH signals are very distinct and superior with regard to
signal clarity and resolution, but rule out a multi-color protocol. However,
the precise localization of genomic targets using a dual-color approach
is indispensable for gene break-apart and fusion assays. In order to
bridge this gap, we aimed to develop a dual-colour combined CISH and
SISH (CS-ISH) gene break-apart assay on the example of the ERG gene
commonly rearranged in PCa.
Methods. On the basis of the ERG break-apart FISH assay, we established
a dual-colour ERG break-apart CS-ISH assay and compared these results
with those obtained by FISH. We assessed 178 PCa and 10 benign specimens
for their ERG rearrangement status applying a dual-colour FISH
and CS-ISH ERG break-apart assay on consecutive sections.
Results. We observed a highly significant concordance (97.7%) between
FISH-based and CS-ISH-based results (Pearson’s correlation coefficient
0.955, p

Conclusions. In conclusion, 454 parallel sequencing is a very useful and
economic approach for molecular pathology due to sample multiplexing
and simultaneous target analyses. Both, FFPE extracted DNA and DNA
from cell preparations may be applied to the approach. If the mutation
rate is higher than 10% in the FFPE sample, the mutation is easily detected.
DO-114
IMDA: a methodical approach enhancing molecular diagnostic of
microcarcinomas and small biopsies
F . Mairinger1 , K . Worm1 , W . Grüning2 , T . Mairinger3 , K .W . Schmid1 1University Hospital Essen, Department of Pathology und Neuropathology,
Essen, 2Helios Klinikum Emil von Behring, Department of Pneumology,
Berlin, 3Helios Klinikum Emil von Behring, Department of Pathology, Berlin
Aims. The isothermal multiple displacement amplification (IMDA)
would be a powerful tool in molecular routine diagnostics for preamplification
of extraordinary small tumor samples (biopsies containing
small amount of tumor, microcarcinomas) but is not banked in pathological
laboratories. We designed a study to check the feasibility and convenience
of these methods for routine diagnostics on LCM microdissected
FFPE samples.
Methods. For validation of the IMDA assay, different benign FFPE tissue
samples were microdissected using LCM technology (areas ca. 50×50 µm
up to 150×100 µm) and afterwards preamplified by an commercial IM-
DA-kit (Qiagen REPLI-g FFPE assay). Chromosomal representation was
tested using qPCR of genes spanning regions on different chromosomes.
Afterwards, patient samples from the Helios Klinikum Emil von Behring
with a too small amount of material for conventional molecular
analysis were pre-amplified by IMDA and further processed with the
“normal” routine samples for EGFR analysis.
Results. With an amplification time duration of 3h and a starting material
of 50×50 µm extension a yield of total 250 µg DNA (concentration
of 5 µg/µl) could be generated. Preliminary results show an acceptable
relative chromosomal representation, the presence of diagnosis relevant
genes in clinical samples could be proven. Mutational analysis of clinical
samples was accomplishable and shows concordance with earlier diagnostically
findings.
Conclusions. We could proof the diagnostic feasibility and convenience
of IMDA for routine diagnostics. Also small amount samples, until now
not analyzable with molecular methods, will be sufficient for all-embracing
molecular routine diagnostics.
DO-115
miRNA 26b stabilizes the pro-apoptotic DAP Kinase by inhibiting
its E3 ubiquitin ligase DIP1
S . Knaup1 , S . Wach2 , A . Agaimy1 , J . Schulze-Luehrmann 1 , M . Hugele1 ,
S . Chakilam1 , R . Atreya3 , R . Wirtz4 , T .T . Rau1 , R . Schneider-Stock 1
1University of Erlangen-Nuremberg, Institute of Pathology, Erlangen,
2University of Erlangen-Nuremberg, Institute of Urology, Erlangen,
3University of Erlangen-Nuremberg, Department of Medicine I, Erlangen,
4STRATIFYER Molecular Pathology GmbH, Cologne
Aims. Tumor necrosis factor α (TNF) is a pro-inflammatory cytokine
involved in the inflammatory reaction of the intestinal mucosa, but
also mediates tumor eliminating effect. We have shown recently that
treatment of HCT116 colorectal tumor cells with TNF led to a higher
expression of death-associated protein kinase (DAPK) and induced caspase-dependent
apoptosis. It is also known, that DAPK can be found
in a complex together with DAPK-inter-acting protein (DIP1) that antagonizes
the pro-apoptotic function of DAPK by ubiquitination. Upon
TNF treatment a decrease of the DIP1 protein level could be detected,
while there were no matching changes in the DIP1 mRNA levels. This
raised the question of a potential involvement of a miRNA binding to
the 3‘UTR of DIP1 regulating its degradation or translational inhibition.
A miRNA microarray analysis of TNF-treated HCT116 cells revealed a
significant up-regulation of miRNA 26b.
Methods. The potential of miRNA 26b to target DIP1 and thereby influencing
apoptosis through regulation of DAPK had to be verified. This
was achieved by a luciferase reporter assay and overexpression of miRNA
26b as well as knock-down of the miRNA and DIP1, followed by westernblot
and Real-Time PCR analysis. To assess the in vitro findings in vivo,
in situ hybridization was performed on tissue microarrays of normal, inflamed
(ulcerative colitis) and inflammation-associated colorectal tumor
samples to check the localization and abundance of miRNA 26b.
Results. In the luciferase reporter assay, miRNA 26b binds to DIP1,
confirming it as a miRNA 26b target. This was further verified by overexpression,
as well as a knock-down of miRNA 26b. More miRNA led
to a decrease of DIP1 protein levels and in return to a stabilization and
increase of DAPK protein levels. Vice versa, reduction of miRNA 26b
resulted in higher DIP1 protein levels and less DAPK protein. Knockdown
of DIP1 by siRNA showed an increase of apoptosis via caspase 3
cleavage. In human tissues of ulcerative colitis patients, in situ hybridization
verified a higher level of miRNA 26b in the inflamed colon crypts,
consistent with the grade of inflammation.
Conclusions. miRNA 26b promotes apoptosis in human colon cancer
cells by targeting the E3 ubiquitin ligase DIP1 and thereby stabilizing the
pro-apoptotic DAPK. miRNA 26b is strongly expressed in the inflamed
tissue of ulcerative colitis patients, suggesting a possible role in the regulation
of the inflammatory process.
DO-116
MRNA and microRNA stability in surgical tissue: an issue for biobanking
and biomarker identification
C . Schuster1 , W .E . Thasler2 , K .-F . Becker3 , T . Kirchner1 , F . Hlubek1 1Ludwig-Maximilians-University München, Institute of Pathology, München,
2Ludwig-Maximilians-University München, Department of Surgery,
Grosshadern Hospital, München, 3Technical University Munich, Institute of
Pathology
Aims. Human frozen tissues are one of the best sources for molecular
analyses such as microarrays, qPCR or Next-Generation-Sequencing. In
addition, frozen tissues are particularly valuable for biomarker identification.
Several biobanks comprising non-fixed frozen tissues have been
established alongside with corresponding clinical data repositories to
facilitate biomarker studies relevant for clinical diagnostics. Apart from
proteomic approaches, mRNA- and microRNA-expression profiles have
shown to be highly valuable for biomarker studies. Since RNA is generally
a fragile molecule, RNA integrity in tissue specimens has tremendous
impact on gene expression analyses, requiring a rigorous quality assessment
of biobank tissue samples.
Methods. To address this issue, we established a tissue quality test system
based on RNA integrity and differential gene expression. We used normal
and cancerous surgical tissue that was stored under various conditions
and for different time periods of ischemia prior to being snap frozen.
Results. The RNA was isolated and the quality was assessed in four steps:
First, the RNA was quantitated by spectrophotometry (NanoDrop) and
total RNA quality was determined by on-chip electrophoresis (Experion).
Second, the degree of RNA degradation and the maximum length of
RNA molecules available for downstream applications were determined
by amplicon length analysis of housekeeping genes using PCR amplification.
Third, the RNA expression level of selected genes were determined
and correlated to the time and condition of ischemia. The genes
analysed comprised highly regulated genes, signaling pathway genes and
genes induced by hypoxia or apoptosis. Fourth, the expression of selected
miRNAs representing different expression levels was determined by
quantitative PCR.
Der Pathologe · Supplement 1 · 2012 |
43

Abstracts
Conclusions. Taken together we analysed mRNA and miRNA quality in
correlation to the time and condition of warm and cold ischemia of the
same tissue samples. The results enabled us to establish a RNA-based
quality assessment procedure of tissue specimen for frozen tissue biobanks.
This study may facilitate and optimise the logistics of biobanking
processes.
DO-117
Quantitative genome-wide methylation profiling of human
breast cancer reveals subtype-specific patterns of epigenetic
instability
U . Lehmann1 , J . Rößler1 , O . Ammerpohl2 , J . Gutwein2 , R . Geffers3 , W . Hofmann4
, F . Länger1 , H . Kreipe1 1 2 Medical School Hannover, Institute of Pathology, Hannover, University
Hospital Schleswig-Holstein, Institute for Human Genetics, Kiel, 3Helm holtz Centre for Infection Research, Genom analysis/Gen Regulation and
Differentation, Braunschweig, 4Medical School Hannover, Institute of Cell
and Molecular Pathology
Aims. This project addresses the question whether a subgroup of human
breast cancer is characterized by widespread epigenetic instability, in
contrast to genetic instability which is typical for e.g., familial breast
cancer.
Methods. High molecular weight DNA was isolated from 28 histologically
examined fresh-frozen human breast cancer specimens and 4 normal
mammary epithelial fractions using standard procedures. DNA methylation
patterns were analyzed using the newly developed 450k methylation
array from Illumina as well as methyl binding domain (MBD)-based
affinity enrichment and subsequent hybridization to a CpG-island
and promotor array from Agilent. For data analysis software provided
by the manufacturers of the arrays were employed. These analyses were
complemented and extended by employing commercially as well as freely
available software packages (Omics Explorer from Qlucore and the R
package IMA). The results for individual loci were validated using conventional
pyrosequencing and independent breast cancer specimens.
Results. In comparison to normal mammary epithelial cell fractions all
breast cancer specimens display several thousand statistically significant
aberrations in DNA methylation (number of CpG sites for pA), and tamoxifen-resistant MCF7 cells (T-
MCF7) were treated with the allosteric mTOR complex 1 (mTORC1)
inhibitor Everolimus and the active-site mTORC1/mTORC2 kinase inhibitor
PP242. In this setting, the effects of insulin receptor signalling on
cell growth, motility and viability were investigated by stimulation with
insulin or IGF1 and in the presence of siRNA inhibition of the insulin
receptor (IR) and insulin like growth factor 1 receptor (IGF-1R).
Results. T-MCF7 showed elevated level of IR/IGFR expression as well
as an activated (phosphorylated) ERK1/2 in contrast to the untreated
MCF7. The addition of insulin resulted in an increased signal transduction
via AKT and ERK1/2. Simultaneous inhibition of mTORC1/2 through
PP242 abolished AKT-phosphorylation and led to a complete cell cycle
arrest in G0/G1 as well as a substantial decrease of cell viability in MCF7
and T-MCF7. However, mTORC1-inhibition alone using Everolimus resulted
only in a partial G0/G1-arrest which could be reversed by addition
of insulin. siRNA inhibition of IR demonstrated an effective reduction of
MAPK-signalling in both MCF7 and T-MCF7 while siRNAs against IR
or IGF1R resulted in an additional decrease of cell viability.
Conclusions. Inhibition of mTOR-signalling reduced cell viability and
proliferation in PIK3CA-mutated breast cancer cells independent of an
acquired Tamoxifen resistance. However, our data indicate that IR and
IGF1R-conferred cell growth may reduce the effects of isolated mTOR
inhibition in Tamoxifen-resistant breast cancer cells and that additional
targeting of the insulin receptor pathway may prove useful in this
setting.
DO-120
Strong negative feedback from Erk to Raf confers robustness to
MAPK signaling
R . Fritsche1 , F . Witzel2 , A . Sieber1 , R . Herr3 , N . Schmidt1 , S . Braun3 , T . Brummer3 ,
C . Sers1 , N . Blüthgen1 1 2 Charité University Hospital Berlin, Pathology, Berlin, Charité University
Hospital Berlin, Pathology, 3ZBSA, Albert Ludwigs-University, Freiburg
Aims. Protein levels within signal transduction pathways vary strongly
from cell to cell. For example, it has been reported that concentrations
of the last kinase within the MAPK signalling module, Erk, varies about
4-fold between clonal cells under the same conditions. In the present study,
we analysed how signalling pathways can still process information
quantitatively despite strong heterogeneity in protein levels.
Methods. Mathematical analysis of isolated de- and phosphorylation
cycles predicts that phosphorylation of a signalling molecule is proportional
to the protein concentration. We combined mathematical modelling
and experimental analysis and systematically perturbed the protein
levels of Erk by siRNA. Our experiments also included the analysis of
Erk phosphorylation under Mek overexpression, measuring transcript
levels of negative feedback regulators, and the application of generic inhibitors.
Results. We found that the steady-state phosphorylation of Erk is very robust
against perturbations of Erk protein level, suggesting that there are
mechanisms that provide robustness to the pathway against protein fluctuations.
Using mathematical modelling, we identified three potential
mechanisms that may provide robustness: 1. kinetic effects, 2. transcriptional
negative feedbacks, 3. negative feedbacks on the post-translational
level. By experimental analysis of the systems we could exclude kinetic
effects and transcriptional negative feedback as mechanisms of robustness.
By analysing a panel of cell lines we found that cells are robust as
long as the signal passes through Raf-1. In contrast, cells where the pathway
is activated by a mutation in B-Raf loose robustness. Therefore,
once the feedback is broken, the system loses robustness and can be readily
modulated by low concentrations of targeted inhibitors. In contrast,
if the feedback is intact, inhibition of the pathway is inefficient.
Conclusions. This finding explains why Mek inhibition has shown little
success in the past in cancer treatment. However, it also shows that a

subgroup of patients with B-Raf mutation will likely benefit, and that
due to the robustness of the healthy cells that have no B-Raf mutation
side effects might be minimal. We believe that analysing robustness of
other signalling pathways in a similar way will be the key to devise efficient
targeted interventions for these, and will unveil which mutations in
the pathway will break robustness and thereby open the door for efficient
intervention.
DO-121
The nuclear localization and transcriptional activation of
β-catenin are independent of each other
S . Ormanns1 , T . Kirchner1 , A . Jung1 1Ludwig-Maximilians-University, Institute of Pathology, München
Aims. Mutations in components of the Wnt signaling pathway are the
drivers of carcinogenesis in the majority of colorectal cancers. They
result in the accumulation of the transcription factor β-catenin which
exerts its function by the induction of the hallmarks of cancer like epithelio-mesenchymal
transition, stemness, chemoresistance, proliferation,
invasion or apoptosis besides others. The transcriptional activity
of β-catenin depends on its nuclear localization and posttranslational
modifications. As it is unknown how both processes are regulated, we
asked if the nuclear accumulation of β-catenin and its activation induced
via the PI3K-AKT signaling pathway were independent of each other.
Methods. To discriminate between the nuclear localization and additional
activation steps of β-catenin an experimental cell culture system
was designed that allowed the forced nuclear translocation of β-catenin
independent of additional activation steps. The subcellular localization
of β-catenin was assessed by immunofluorescence. The transcriptional
activity of β-catenin was determined by TOP-flash luciferase reporter
gene assays. The activity of PI3K-AKT was interfered by the specific inhibitor
LY294.002.
Results. Inhibiting PI3K/AKT lead to a significant dose-dependent reduction
of endogenous β-catenin activity in the cell lines SW480 and
RWP-1 harbouring inactivating mutations in the APC gene. Conversely,
stimulating the Wnt-signaling pathway or adding degradation resistant
β-catenin both resulted in the activation of β-catenin in the cell lines
293T, CHO or HeLa. Here, the nuclear translocation of β-catenin also
resulted in an activation of its transcriptional activity which could be
blocked by inhibiting PI3K. In contrast the nuclear translocation of β-catenin
did not result in transcriptional activity in A431 cells.
Conclusions. We provide experimental evidence that the nuclear transport
and the transcriptional transactivation of β-catenin are independent
processes. Thus, β-catenin signaling depends at least on active PI3K/
AKT signaling. Taken together, the transcriptional activity of β-catenin
is regulated at least by two signals which might open the opportunity
for clinically interfering with the hallmark of CRC, the activation of the
β-catenin pathway.
DO-122
Activation of the EGFR-MAPK signaling pathway is dependent on
FAM125 proteins
S . Müller1 , G . Baretton2 , G . Fitze1 , M . Haase3 1 2 TU Dresden, Pediatric Surgery, Dresden, TU Dresden, Pathology, Dresden,
3TU Dresden, Pediatric Surgery and Pathology, Dresden
Aims. Ionizing radiation leads to complex changes in tissues such as changes
in cell survival, cell differentiation and loss of function. In order to
find proteins that are overexpressed in irradiated tissue, we constructed
a differential cDNA library. Among other proteins, we found FAM125A
(family 125A), a protein component of transport vesicles that has been
reported to play a role in the internalization of epidermal growth factor
receptor (EGFR). The aim of the study was to get further insights into the
function of FAM125 proteins.
Methods. A differential cDNA library was constructed from cDNA obtained
from radiated lung tissue of the rat. mRNA expression analysis
was done by quantitative RT-PCR. Protein expression was quantified
by western blot analysis. Tissue distribution was analyzed by immunohistochemistry
on tissue microarrays (TMAs). Down-regulation of
mRNA/proteins was achieved by stable transfection of sh-RNA vectors
into HELA cells. Protein extracts from these cells were prepared from
the membrane, cytoplasmic and nuclear fractions.
Results. FAM125A protein is expressed in most cell types. A very high
expression is seen in tissues with very active membrane transport processes
such as renal tubule cells and glandular cells. FAM125A mRNA
and protein are overexpressed in irradiated tissue. Down-regulation of
FAM125A and B leads to a decrease of total EGFR and phosphorylated
EGFR (Y1045 and Y1068) in the membrane fraction. In addition, it leads
to an accumulation of phosphorylated Akt (S473) and c-Src (T416)
in the membrane fraction whereas phosphorylation of p42/44 MAPK
(T202-Y204) is decreased.
Conclusions. FAM125 proteins seem to play a role in transport processes
of molecules including signaling proteins. Down-regulation of EGFRactivity
correlates with decreased activity of the p42/44 MAPK pathway
whereas Akt and c-Src activity are not affected. This suggests that the
EGFR-MAPK pathway is dependent on FAM proteins whereas the Akt
and c-Src pathways act independently. Further research should clarify
the association of various signaling molecules to transport vesicles and
should provide an insight into signaling processes in radiation-damaged
cells.
DO-123
DUSP4 expression increases cell proliferation in colorectal cancer
(CRC) cells and is associated with microsatellite instability in CRC
B . Gröschl1 , M . Bettstetter1 , W . Dietmaier1 1University of Regensburg, Institute of Pathology, Regensburg
Aims. DUSP4, a member of the mitogen-activated protein kinase phosphatase
(MKP) family and a potential tumor suppressor, negatively regulates
the MAPKs (Mitogen-activated protein kinases) ERK, p38 and
JNK which play a crucial role in cancer development and progression.
Our aim was to investigate DUSP4 expression in high frequent microsatellite
unstable (MSI-H) and microsatellite stable (MSS) colorectal
cancers (CRC) as well as its influence on potential MAPK downstream
targets and its effect on the proliferation in CRC cells.
Methods. We studied DUSP4 mRNA levels in 19 MSI-H and 19 MSS CRC
compared to matched normal tissue as well as in CRC cell lines by RTqPCR.
Promotor methylation of the DUSP4 gene was analyzed using
Methy-QESD (Quantification of Endonuclease-Resistent DNA) and
coding regions were assessed for mutations through Sanger sequencing.
We overexpressed DUSP4 in CRC cell lines and analyzed expression of
potential downstream target genes as well as cell growth by Real-Time
Cell Analysis (RTCA).
Results. DUSP4 mRNA was elevated in all 19 MSI-H tumors and in
14 MSS tumors. Median expression levels in MSI-H tumors were significantly
higher than in MSS-tumors (p

Abstracts
DO-124
Gastrointestinal stromal tumors of the stomach rarely harbour
KIT exon 9 mutations and are mostly associated with a low or no
malignant potential
H . Löser 1 , S . Huss 1 , W . Jeske 1 , M . Fielenbach 1 , P . Hohenberger 2 , P . Reichardt 3 ,
H .-U . Schildhaus 1 , R . Büttner 1 , E . Wardelmann 1
1 University of Cologne, Institute of Pathology, Köln, 2 University of Heidelberg,
Department of Surgery, Mannheim, 3 Helios Klinikum, Hematology/
Oncology, Bad Saarow
Aims. Gastrointestinal stromal tumors (GISTs) are the most common
mesenchymal tumors in the gastrointestinal (GI) tract. Up to 90% of
them carry an activating mutation in the KIT or the PDGFRA (platelet-derived
growth factor alpha) gene both encoding type III receptor
tyrosine kinases. In both genes, hot spot regions have been identified,
i.e. exons 9, 11, 13, and 17 in KIT and exons 12, 14 and 18 in PDGFRA.
The distribution among these different exons is not balanced. More than
60% of cases carry KIT exon 11 mutations followed by 10 to 15% of tumors
carrying either a KIT exon 9 or a PDGFRA exon 18 mutation. All other
locations are very rare (less than 2% for each exon). The different mutational
subtypes in KIT and PDGFRA are found in variable frequences in
different parts of the GI tract. In detail, KIT exon 9 mutations are nearly
always found in the small bowel and rectum and but only rarely in gastral
GISTs. In contrast, PDGFRA mutations are nearly always restricted
to gastric GISTs. We were interested to know how often stomach tumors
carry KIT exon 9 mutations and whether there is an association with an
aggressive behavior as demonstrated for GISTs in a non-gastric location.
Methods. We evaluated more than 2000 cases in our GIST and Sarcoma
Registry Cologne/Bonn (GSRCB) for gastric GISTs with KIT exon 9
mutations. Sequences were analysed by direct Sanger Sequencing. We
evaluated pathomorphological and clinical data and compared them to
GISTs in other primary locations.
Results. We could identify 19 gastric cases carrying a KIT exon 9 mutation.
The average tumor diameter was 4.1 cm. According to the AFIP
classification (Miettinen 2006), 15 tumors belonged to the groups of no or
low aggressive behavior. Three GISTs were classified as high risk lesions
with a mitotic count of 13, 25 and 132/50 HPFs, resp. one other belonged to
the intermediate risk group. 18 tumors carried the classical 6 base pairs
insertion in KIT exon 9 (p.A502_Y503dup). One low-risk tumor showed
a novel 12 bp deletion in KIT exon 9 (p.K484_G487del) which has not
been described before.
Conclusions. KIT exon 9 mutations (typically a 6 bp insertion; p.A502_
Y503dup) occur preferentially in a non-gastric location of GISTs. In these,
the mutational subtype frequently implicates an aggressive behavior.
In contrast, gastric tumors with exon 9 mutation often are associated
with a low or no malignant potential. Conclusively, in the vast majority
of these lesions there is no implication for an adjuvant treatment with
imatinib.
DO-125
Functional phosphoproteomics for therapy response prediction
in malignant thymomas and thymic carcinomas
S . Küffer1 , A .-L . Bohlender1 , C . Sauer1 , D . Belharazem1 , A . Marx1 , P . Ströbel1 1University Medical Centre Mannheim of the University of Heidelberg/Institute
of Pathology, Mannheim
Aims. Thymomas (TH) and thymic carcinomas (TC) are rare mediastinal
tumor with a high tendency for local therapy failures. Relapsed
tumors require first or second line adjuvant treatments, which are not
well established. Our group has recently reported clinical response to
the multikinase inhibitor sunitinib in a small series of patients with metastatic
TC. In an attempt to better understand the underlying molecular
conditions and to eventually predict sunitinib response, we investigated
TH and TC by different phosphoproteomic approaches.
46 | Der Pathologe · Supplement 1 · 2012
Methods. Functional kinomics were carried out by spiking sunitinib into
a tumor lysate from one patient with clinical response to sunitinib and
from one patient with a resistant tumor and subsequent measurement
of 144 receptor tyrosine kinase (RTK) substrates. Snap-frozen tumour
tissues of 63 TH and TC samples were analyzed using Phospho-Protein-
Arrays to detect activation of RTKs and MAPKs. Subsequently, primary
cells from 10 tumor samples with known RTK/MAPK activation status
were tested with sunitinib, an Akt inhibitor and a JNK inhibitor.
Results. Comparing the clinical responder and the non-responder,
44/144 peptide substrates were found a) significantly inhibited by sunitinib
and b) significantly different between the two samples. Pathway analysis
revealed a prominent role of the EGFR, and to some extent, VEGFR
signalling pathways, with involvement of PI3K and ras/raf as downstream
targets. Analysis of a large number of TH and TC revealed activation
of the EGFR alone or in combination with other RTKs in 40/63 cases
(63%). Analysis of MAPK revealed a dichotomic pattern with actvation
of the PI3K/AKT pathway in 46% and activation of JNK kinases in 54%
of cases. Preliminary data with ex vivo cell cultures from TH and TC
treated with AKT and JNK inhibitors suggested better responses to AKT
inhibitors in the “AKT group” and vice versa.
Conclusions. Our results suggest that the EGFR – the single most frequently
activated RTK in TH and TC – as well as its downstream effectors
PI3K/AKT and the ERK pathway may be a prominent sunitinib
target in these tumors. Our findings are surprising, since small clinical
trials using targeted EGFR inhibition (e.g. through gefitinib) were disappointing.
Given the possible involvement of the VEGFR, inhibition of
multiple kinases may be a preferable therapeutic approach. Our results
also indicate that inhibition of specific pathways (such as the AKT) in
highly selected patients may further improve therapeutic response rates.
Workshop Informatik – Strukturierte Befunde
DO-001b
Structured reports in pathology – current status and activities
T . Schrader1 , F . Oemig2 , J . Thümmler 3 , U . Altmann4 , G . Haroske5 1University of Applied Sciences Brandenburg, FB Informatics & Media,
Brandenburg, 2Agfa Healthcare, Standards and Interoperability, 3Vivantes GmbH, Berlin, Ressort IT/TK, 4Justus-Liebig-Universität Gießen, Institut für
Medizinische Informatik, 5Krankenhaus Dresden-Friedrichstadt, Institut für
Pathologie
Aims. The application of structured reports (SR) is a pestering request of
clinicians, tumor centers, tumor registers and pathologists. In various
countries (especially France and Spain) SR’s were developed under the
auspices of IHE (Integrating the Healthcare Enterprises) which influences
the current discussion of standards development. In Germany new
efforts were done to promote the adoption of SR in Pathology and to govern
the National and International activities.
Methods. The current situation in application and development of SR’s
were analyzed. Together with the Federal Society of German Pathologist
and with experts from the German Society of Tumor Centers HL7
Germany has identified information blocks of a SR which are then restructured
into new templates. They are then compared with current IHE
Anatomic Pathology Structured Report (APSR).
Results. HL7 Germany coordinates together with the German Society of
Tumor Centers a comprehensive balloting process in order to establish
Pathology reports. As result of this balloting, different templates of Pathology
reports will be approved and could be implemented by vendors
of Pathology Laboratory Information Systems.
Conclusions. A German implementation guide for CDA-based pathology
reports based on APSR is in development including a German description
on how to use diagnostic terms and classifications, esp. ICD-10 and
TNM.

DO-002b
Impact of terminologies in tumor pathology structured reports
G . Haroske 1 , T . Schrader 2
1 Dresden-Friedrichstadt General Hospital, Institute of Pathology, Dresden,
2 University of Applied Sciences Brandenburg, Department Informatics and
Media, Brandenburg
Aims. For information exchange and data mining structured reports in
tumor pathology have to be based on controlled vocabulary as to get a
model of meaning. So far there is no universal terminology for the wide
variety of concepts in tumor pathology. SNOMED CT will probably become
a global health terminology standard. National and international
initiatives are necessary to reach a growing agreement on particular
aspects and needs towards it. Interface terminologies are a tool for drawing
existing separate terminology systems to a finally global standard.
Methods. Controlled vocabularies in guidelines of German pathologists
for a series of tumors, in the basic tumor documentation of cancer registries,
and in the HL7 Germany have been mapped to PathLex, an interface
terminology of IHE.
Results. On average a pathology guideline describes 50 terms which have
to be registered as to fulfill the minimum documentation requirements.
PathLex provides between 30 to 40 terms per tumor entity, only 80% of
them are identical with the German guideline vocabulary. The coincidence
of PathLex with HL7 Germany vocabulary or the basic data set
of cancer registries is still lower. In contrast to PathLex there is no separation
between general and organ-specific information in the German
guideline vocabulary.
Conclusions. Although based on internationally agreed understanding,
sharing the same concepts of tumor pathology, the terminology differences
among the different sources are quite obvious. They have to be
overcome as to ascertain a reliable information exchange between different
actors in the care of tumor patients. Terminology mapping is one
solution, but not the optimal one. A closer collaboration with international
terminology bodies as well as a sharpened realization of the impact
of terminology in home made guidelines would contribute to a better
standing of German pathology. A SNOMED membership of Germany
would be very helpful.
AG Dermatopathologie und AG Zytopathologie I –
Endokrine Themen I
FR-001
Unusual HBME1-expression in a hyalinizing trabecular tumor of
the thyroid gland: a case report
D . Lenggenhager1 , E . Marques Maggio1 , B . Bösch2 , A . Elisa2 , M . Rössle1 1UniversityHospital Zurich, Institute of Clinical Pathology, Zürich, Switzerland,
2Stadtspital Triemli, Institute of Pathology, Zürich, Switzerland
Aims. Hyalinizing trabecular tumour (HTT) is a rare thyroid neoplasm
of follicular cell origin with a trabecular growth pattern, marked intratrabecular
hyalinization and nuclear features, which are typically found
in papillary thyroid carcinoma (PTC). The role of HBME1 in the diagnostic
process of PTC has been demonstrated in several studies. We
present a case of HTT with patchy, but abundant, hitherto not reported
membranous and intrabecular HBME1-positivity.
Methods. A 70-year-old woman underwent total thyroidectomy because
of cytological diagnosis of PTC. Pathomorphological investigation of the
resected specimen was performed.
Results. Histologically, the typical trabecular architecture of HTT with
elongated tumor cells, markedly hyalinized intratrabecular stroma and
oval shaped nuclei with grooves and inclusions was seen. Immunohistochemically,
tumour cells were diffusely and strongly positive for thyreoglobulin
and TTF1, focally and weekly positive for Galektin3 and
HBME1, but negative for calcitonin, ki67 and CK19. The intertrabecular
hyalinized material was positive for diastase-resistant PAS, Collagen IV,
and HBME1, exhibiting a filiform and stellate staining pattern. Mutational
analysis showed a BRAF wild type.
Conclusions. This case shows that HBME1-positivity may occur in HTT,
and therefore should be interpreted with caution in differentiating HTT
from PTC.
FR-002
Secondary tumours to the thyroid an uncommon but potentially
challenging entity: the experience of a single general hospital
C . Cacchi1 , H . Jähnig1 , G . Schenkirsch2 , M . Füller 3 , H . Arnholdt1 , B . Märkl1 1 2 Klinikum Ausburg Insitute for Pathology, Augsburg, Klinikum Augsburg,
3Klinikum Augsburg, Oncology and Hematology Unit
Aims. Despite its rich vascular supply, thyroid is a very uncommon location
of metastasis. It has been reported that secondary malignancies
representing less of 2% of thyroid tumours; the aim of these study is to
present experience of a single institution focusing on the differential histological
diagnosis.
Methods. A total of 13 (8 male and 5 female patients) cases with metastatic
disease to the thyroid have been retrieved from the archive of our
tumour-registry between 1985 and 2011. All patients have documented
histology for both primary and secondary tumour. Patient age, sex, survival,
outcome were reordered.
Results. The median age of the patients is 69.8 years, actually 4 patients
are still alive. Among the other patients only four died as consequence
of progression of the primary tumour. The operable cases (9) received
in four cases a simple lobectomy, in five cases a total thyroidectomy. A
primary tumour was identified in 11 cases: of clear cell renal carcinoma
in 6 cases, squamous cell carcinoma in 4 cases (two lung , one larynx
and one oesophagus respectively) and one small cell carcinoma of the
lung . In two cases the patients have had other malignancies (melanoma,
colorectal carcinomas and bile-pancreatic adenocarcinoma). The time
between the diagnosis of primary tumours and metastasis is interestingly
longer in case of renal carcinoma (144 months) respect to an average
value for all cases (77.5 months). Five cases have been studied pre-operative
with a fine needle aspiration (FNA), in two cases this procedure
were diagnostic.
Conclusions. Patients with metastases to the thyroid gland are an uncommon
but clinically pathologically relevant issue, particularly when
the tumor mass is evident as a single nodule (in our experience in 3 of
13 cases); for example a clear cell renal carcinoma could close mimic a
clear cell thyroid adenoma or follicular carcinoma. Moreover, neuroendocrine
tumor can metastasise to the thyroid gland simulating a primary
thyroid tumour (particularly a medullary carcinoma): a rare but
treacherous possibility. To avoid misdiagnosis and to ensure an optimal
follow-up for these patients clinical data and a comprehensive immunohistochemical
panel are mandatory.
Der Pathologe · Supplement 1 · 2012 |
47

Abstracts
FR-003
Glucagon cell adenomatosis: a novel multifocal neuroendocrine
neoplasia restricted to the dorsal pancreas anlage
M . Anlauf 1 , P . Gerlach 1 , S . Schinner 2 , M . Schott 2 , M . Krausch 3 , K . Cupisti 3 , R .S .
Lanzmann 4 , C . Antke 5 , S . Schulz 6 , G . Klöppel 7 , H .E . Gabbert 1 , W .T . Knoefel 3 ,
W .A . Scherbaum 2
1 Institute of Pathology, University of Düsseldorf, 2 Department of Endocrinology,
Diabetes and Rheumatology, University of Düsseldorf, 3 Department of
General-, Visceral- and Pediatric Surgery, University of Düsseldorf, 4 Institute
of Diagnostic and Interventional Radiology, University of Düsseldorf,
5 Department of Nuclear Medicine, University of Düsseldorf, 6 Institute of
Pharmacology and Toxicology, University of Jena, 7 Institute of Pathology,
Technical University of München
Aims. Based on observations in four patients, glucagon cell adenomatosis
(GCA) has recently been proposed as a novel multifocal neuroendocrine
tumor disease of the pancreas.
Methods. We report on a 58-year-old patient treated by duodenopancreatectomy
who showed distinct features of GCA. In order to study the
tumor development and distribution the entire pancreas was embedded
and systematically analyzed.
Results. The patient presented with recent onset non-insulin-requiring
diabetes and abdominal discomfort. On CT the pancreas showed multiple
tumors and diffuse nodular enlargement corresponding to the
SPECT scintigraphy. The imaging findings were more pronounced in the
pancreatic body and tail than in the head. Pathology analysis revealed
12 neuroendocrine macrotumors and more than 10,000 microadenomas
mainly composed of glucagon cells. Metastases were not detected. The
nontumorous parenchyma revealed a ubiquitous glucagon cell hyperplasia
at the expense of the other endocrine islet cell types. These findings
were restricted to the body, tail and the upper portions of the pancreatic
head. The lower portion of the head of the pancreas that corresponds to
the ventral pancreas anlage had a normal appearance.
Conclusions. GCA is a novel multifocal neuroendocrine neoplastic disease
that is restricted to the dorsal pancreas anlage.
FR-004
Genetic alterations in glucagon cell adenomatosis
T . Henopp1 , M . Anlauf2 , S . Biskup3 , G . Klöppel4 , B . Sipos1 1University Hospital Tübingen, Institute for Pathology and Neuropathology,
Tübingen, 2University Hospital Düsseldorf, Institute for Pathology, Düsseldorf,
3Center for Genomics and Transcriptomics, Tübingen, 4Technische Universität München, Institute of Pathology, München
Aims. Glucagon cell adenomatosis (GCA) was recently recognized by us
as a multifocal neoplastic disease of the endocrine pancreas unrelated to
MEN1. Multiple micro- and a few macrotumors are found on the background
of a hyperplasia of glucagon cells. The disease may cause unspecific
abdominal symptoms and only rarely a glucagonoma syndrome.
Recently a mutation in the glucagon receptor (GCGR) gene was described
in one GCA patient. The aim is to investigate GCGR gene changes in
five patients with GCA.
Methods. Paraffin embedded and formalin fixed pancreatic tissues from
five patients showing multiple microadenomas and in three cases also
macroadenomas of glucagon cells were macro- or microdissected. The
extracted DNA was sequenced and the GCGR gene analysed for mutations.
Results. Sequencing of the GCGR gene revealed germline mutations in
three out of five patients. One patient shows two different heterozygous
point mutations in the hyperplastic alpha cells as well as in the non-tumorous
tissue leading to two premature stop codons. One patient harbors
a homozygous stop mutation. The third patient shows two homozygous
missense mutations of the GCGR gene that most likely also led to a
dysfunction of the GCGR. In the two other patients no germ line mutati-
48 | Der Pathologe · Supplement 1 · 2012
ons of the GCGR gene were detected. These variants were not identified
in healthy subjects.
Conclusions. The finding of germ line and somatic “loss of function” mutations
of the GCGR gene in three of five patients with GCA suggests that
a change in the signalling function of the GCGR may cause glucagon cell
adenomatosis via glucagon cell hyperplasia.
FR-005
Proliferative activity is not associated with tumor aggressiveness
in ileal neuroendocrine tumors
T . Henopp1 , J . Sperveslage1 , M . Anlauf2 , G . Klöppel3 , B . Neumayer1 ,
C .P . Gerlach2 , P . Rexin4 , T .M . Gress5 , R . Moll4 , B . Sipos1 1University Hospital Tübingen, Institute for Pathology and Neuropathology,
Tübingen, 2University Hospital Düsseldorf, Institute for Pathology, Düsseldorf,
3Technische Universität München, Institute of Pathology, München,
4University Hospital Giessen and Marburg, Institute for Pathology, Marburg,
5University Hospital Giessen and Marburg, Department of Internal Medicine,
Marburg
Aims. The accuracy of the new WHO grading system for neuroendocrine
neoplasms, which is based on proliferative activity, has not yet been
validated for ileal neuroendocrine tumors (iNETs). Aim of this study is
to analyse the proliferation rates in primary iNETS, lymph node and distant
metastases in order to determine the prognostic power of grade 1
and grade 2 categories in the WHO 2010 classification.
Methods. 64 primary iNETs, 35 matched node metastases and 20 distant
metastases were analysed for proliferation rate (Mib1, Phospho H3) using
automated image analysis assessing the maximum (hot spot) and overall
proliferative activity. Proliferation rates were compared in different
prognostic relevant cohorts (N0M0, N1M0 and N1M1).
Results. The maximum Mib1 proliferative rates were: 0.6% (range 0.27–
2.78) for N0M0, 0.77% (range 0.03–2.94) for N1M0 and 1.02% (range
0.2–12.17) for N1M1 primary iNETs. Corresponding node metastases
(0.66%; range 0.02–3.38) and distant metastases (0.83%, range 0.01–10.94)
showed comparable proliferative rates like primary iNETs. The number
of grade 1 and grade 2 iNETs was not different in the three cohorts.
Primary iNETs of N1M0 (2 cm; range 1–4.8) and N1M1 (1.95 cm; range
0.4–5) cohorts were significantly larger than N0M0 tumors (0.2 cm; range
0.2–1.6).
Conclusions. Grade 1 and grade 2 categories in the WHO classification
2010 do not have discriminatory power regarding prognosis for iNETs.
In fact, tumor spread is independent of proliferative activity in iNETs.
FR-006
Comprehensive assessment of Merkel cell polyomavirus in Merkel
cell carcinomas: fluorescence in situ hybridization versus qPCR?
A . Haugg1 , D . Rennspiess1 , A . zur Hausen1 , E .-J . Speel1 , G . Cathomas2 ,
J . Becker3 , D . Schrama3 1Maastricht University Medical Center, Department of Pathology, Maastricht,
Netherlands, 2Kantonsspital Liestal, Institute of Pathology, Switzerland,
3Medical University of Graz, Division of General Dermatology Department of
Dermatology, Graz, Austria
Aims. Merkel cell polyoma virus (MCPyV) is detected in 80% of Merkel
cell carcinomas (MCC). The clonal integration and tumor specific mutations
in the large T Antigen (LTAg) gene identify MCPyV as a novel
human tumor virus. To date the relationship between the viral presence
and cancer induction, development or clinical prognosis is discussed
controversially. Yet almost all studies are based on quantitative virus
detection, i.e. PCR or qPCR. Here we aimed to gain information about
the quality of the viral presence on the single cell level in the histomorphological
context.

Methods. We performed MCPyV-FISH of formalin fixed and paraffin
embedded (FFPE) MCCs (n=62) on tissue microarrays (TMAs), determined
the hybridization patterns and correlated these with qPCR data
on the basis of a determined cut-off. MCPyV-FISH was established using
the MKL-1 cell line which harbors integrated copies of MCPyV DNA.
For MCPyV-qPCR a LT primer pair (Becker et al., 2009) was used on
whole tissue sections.
Results. MCPyV-FISH on FFPE MKL-1 cells revealed punctate signals
compatible with viral integration. The MCPyV-FISH positive MCC cores
(76%) mainly revealed two different signal patterns: a punctate pattern
(85%) which correlated with a moderate relative viral abundance
and in some areas the punctate pattern was combined with a diffuse pattern
(15%) indicating the episomal presence of the virus which is linked
to viral replication. A mixed hybridization pattern was associated with
very high qPCR values. Comparing MCPyV-FISH and qPCR data the results
highly correlated (83%) with the MCPyV positive evaluated group,
whereas the negative group showed a concordance of 93%. The mean of
the qPCR values of all MCPyV positive cores differed significantly from
the negative cores (p=0.0076). In some tumor areas of one and the same
patients the FISH signals were heterogeneous in intensity, pattern and
nuclear localization.
Conclusions. A strong correlation between MCPyV FISH and the relative
MCPyV abundance by qPCR was detected. Thus, while presence
of MCPyV can be verified by qPCR, the quality of the presence can be
visualized by MCPyV specific FISH analysis. In this regard, MCPyV
qPCR and MCPyV FISH are important complementary tools to gain
maximum biological information of the presence of MCPyV in MCC
and thus to further elucidate MCPyV related carcinogenesis.
FR-007
A novel BRAF mutation in a patient with metastatic melanoma
R . Schneider-Stock 1 , L . Heinzerling2 , E . Kämpgen2 , M . Erdmann2 , P . Keikavoussi2
, A . Agaimy1 , A . Hartmann1 , G . Schuler2 1University of Erlangen-Nuremberg, Institute of Pathology, Erlangen,
2University of Erlangen-Nuremberg, Department of Dermatology, Erlangen
Aims. BRAF mutations in melanoma have been identified as a new target
for therapy. The V600E mutation is found in 50–68% of malignant
melanomas and can be specifically treated with e.g., the BRAF inhibitor
PLX4032 now registered as Vemurafenib. We describe a patient with a
metastasized nodular melanoma on the right lumbar region which was
excised two years before but had already spread to the inguinal lymph
nodes. Subsequently, the patient developed multiple metastases of the
brain, metastases of the mediastinal, cervical, retroperitioneal, retrocrural,
mesenterial, paraaortal and interaortocaval lymph nodes, in the
liver, the stomach and the intestines. Previous therapy included lymphadenectomy,
radiation therapy, hyperthermia, radiochemotherapy
with temozolomide, sorafenib, fotemustine and paclitaxel/carboplatin.
Despite these attempts he showed progressive disease and inclusion into
a BRAF inhibitor study was considered. Thus the tumor was sent for mutation
analysis.
Methods. Formalin-fixed and paraffin-embedded tumor sample was
used for the extraction of genomic DNA. Mutational analysis was carried
out by Pyrosequencing and for confirmation by ABI capillary sequencing
of exon 15 of BRAF.
Results. In this melanoma patient a new complex mutation was found
in BRAF with base substitution of a valine residue at position 600 for
glutamic acid (GTG GAA) and deletion of codon 601 (p.V600EK601del;
c.1799_1801del3). With this mutation the patient did not qualify for the
BRAF inhibitor study. The clinical course of the patient was rapidly progressive
with various acute complications (renal insufficiency, ileus) and
the patient deceased only 2 months after analysis of the mutation.
Conclusions. The V600E mutation constitutively activates RAF/MEK
signaling, a major driver of carcinogenesis in various malignancies.
Other rather rare mutations like V600K, V600R or V600D might also
cause this constitutive activation and are discussed to be correlated with
a yet more aggressive behaviour. It is unclear whether the new mutation
described in this case can be considered for targeted BRAF inhibitor therapy.
FR-008
The aid of immunohistochemistry in differential diagnosis between
benign and malignant phenotype of difficult melanocytic
lesions
T . Papadopoulos1 1Klinikum Nürnberg, Institute of Pathology, Nürnberg
Aims. A panel of biological markers differentially expressed in common
nevi, dysplastic nevi, Spitz nevi and malignant melanomas are introduced
providing a potential tool to differentiate benign from malignant
phenotype in difficult melanocytic lesions. The panel includes Cyclin
D1, p16, p21 and p53.
Methods. Cyclin D1 is markedly expressed in radial growth phase malignant
melanomas but is negative in common nevi and in the deepest
part of vertical growth phase melanomas as well. The cell cycle inhibitor
p16 is positive in benign nevi and radial growth phase melanomas
but becomes often negative as malignant melanoma progresses to the
vertical growth phase. The cell cycle inhibitor p21 is positive in radial
growth phase melanomas and in thin vertical growth phase melanomas
but becomes negative in thick malignant melanomas. p21 is negative in
common melanocytic nevi. Both cell cycle inhibitors p16 and p21 are
upregulated in Spitz nevi and Spitzoid nevi. Finally, p53 is negative or
positive at a very low level in melanocytic nevi. Its expression increases
with tumor progression from dysplastic nevus to radial growth phase
malignant melanoma and vertical growth phase malignant melanomas.
Conclusions. This presentation demonstrates characteristic expression
patterns of the above mentioned panel that may enable pathologists to
differentiate benign from malignant melanocytic lesions even in cases
when morphology by itself may not allow a clear classification of the melanocytic
lesion.
Notiz an die Gutachter des Abstracts: Der Abstract wurde nach Rücksprache
und auf Wunsch von Prof . Meister (München) eingereicht . Vorgesehen ist ein
etwa 15- bis 20-minütiger Vortrag, quasi als Review für die Immunhistochemie
unklarer melanozytärer Läsionen .
AG Dermatopathologie und AG Zytopathologie II –
Endokrine Themen
FR-009
Epidermal growth factor receptor mutation analysis in pleural
effusions of advanced non-small cell lung cancer patients:
When only cells are available
A . Zimpfer1 , B . Schneider1 , A . Polak1 , A . Bier2 , J . Kölbel1 , J .C . Virchow2 ,
A . Erbersdobler1 1 2 University of Rostock, Institute of Pathology, Rostock, University of Rostock,
Rostock
Aims. Epidermal growth factor receptor (EGFR) mutations are associated
with an improved clinical outcome due to response to tyrosine kinase
inhibitors in patients with non-small cell lung cancer (NSCLC). The
overwhelming majority of bronchial cancer is diagnosed on often very
limited tumor material which is also requested for secondary mutational
analysis. This study assessed the feasibility of using PCR and gene-sequencing
to screen for (EGFR) mutations in pleural effusions of advanced
NSCLC patients.
Der Pathologe · Supplement 1 · 2012 |
49

Abstracts
Methods. EGFR mutation analysis was performed in 16 cases of malignant
pleural effusions in lung cancer patients. 6 patients were male (range
48–77 years, median 60.5 years) and 8 were female (range 66–86 years,
median 77.5 years). In 1 case 3 pleural effusion specimens of 1 male patient
were examined. Precipitations of fibrin in pleural effusions were subjected
to cell block technique. Tumor cell enrichment was performed in
2/16 cases. The DNA was extracted and exons 18–21 of EGFR amplified.
Sequence analysis of the PCR products was performed using an Applied
Biosystems 3500 system.
Results. Molecular analysis of all EGFR target sequences was achieved
in 14 of 16 (87.5%) cases. EGFR mutations were identified in 3/14 (21.4%)
of fully evaluated cases (1 pleomorphic carcinoma and 2 NSCLC). Two
cases carried a deletion in exon 19 (K745-A750) and the third case a point
mutation in exon 20 (V769M, G779F).
Conclusions. EGFR mutations in pleural effusions from advanced
NSCLC patients can be detected and characterised by PCR and gene sequencing.
Thus, further and costly procedures for tissue retrieval would
be unnecessary in some cases. The availability of EGFR mutation testing
of cytological specimen is a valuably tool in the concerted cytologic/histologic
diagnosis of NSCLC and help to spare precious tissue material for
additional tests.
FR-010
Significant increased detection for cervical dysplasia using the
ThinPrep Integrated Imager
G . Richter1 , U . Hahlbohm1 , D . Teschner1 1Institute of Pathology Dr . Richter, Hameln
Aims. The liquid based cytology using the ThinPrep PAP test is a standardised
method of smear testing and cell processing that was developed
in the USA. The ThinPrep Integrated Imager has been available for computer
assisted liquid based cytology since autumn 2009. This presentation
compares the results of the ThinPrep Integrated Imager of 2010 with
the so called normally produced liquid based cytology in 2007.
Methods. As a DIN/ISO17020 accredited Institute we have been carrying
out the conventional PAP smear test and since 2000 also the liquid based
cytology using the ThinPrep PAP test. Since November 2009 the liquid
based cytology is carried out computer assisted.
Results. In 2007 we manually evaluated 3582 ThinPrep PAP tests of which
98% were of an inconspicous PAP group I or II; 1.6% of PAP group IIW;
0.03% of PAP group III; 0.5% of PAP group IIID; 0.14% of PAP group IVa;
0.03% of PAP group IVb. In 2010 we evaluated 4408 ThinPrep PAP tests
using the Integrated Imager of which 94% were of an inconspicuous PAP
group I or II; 3.4% of PAP group IIW, 0.05% of PAP group III; 2.6% of
PAP group IIID; 0.2% of PAP group IVa and 0.02% of PAP group IVb
(p

FR-013
HPV-genotype distribution in cytological screening, histology
and impact of vaccination
M . de Jonge 1 , G . Busecke 2 , A . Heinecke 3 , O . Bettendorf 4
1 Institute for Pathology and Cytology Schüttorf/Leer, 2 Medical office
of General Medicine, Weener, 3 Department of Medical Informatics and
Biomathematics, University of Münster, 4 Insitute of Pathology and Cytology
Schüttorf/Leer, Schüttorf
Aims. The objective of the research was to investigate the HPV-type distribution
in our screening population as well as its correlation to cytological
diagnosis and histological outcome, and to calculate the impact of
primary HPV vaccination.
Methods. HPV genotyping results of 3381 women who attended to the
German cervical screening program were retrospectively analysed. The
PapilloCheck®-Test was used for HPV genotyping. The distribution of
HPV-types with corresponding cytological diagnosis, and – if available
– histological outcome, was statistically analysed (SPSS 17.0, SAS9.3,
Pearson’s χ2 test). To estimate the possible impact of HPV vaccination on
our cohort we calculated the theoretically minimum impact as well as
the maximum impact in the prevention of HPV-positive lesions.
Results. The HPV-type-distribution showed marked differences among
cervical lesions and age. Although HPV-51 was common in all cervical
lesions, it was rarely detected as single-type HPV infection in CIN3-lesions.
HPV-16 and/or HPV-18 were found in only 58% of the CIN3-lesions,
which was still significantly more than the 22% in CIN2-lesions
(p

Abstracts
detect alterations in histopathological inconspicuous urothelium. Gene
expression profiling, TMA technology, promoter methylation analysis,
qRT-PCR, aCGH and in vitro studies were performed for the identification
and functional characterization of progression-related candidate
genes in BC. A comprehensive molecular, immunohistochemical and
clinicopathological analysis of a large collection of plasmacytoid BC
samples was performed.
Results. Deletions on chromosomes 9 and 8p were detected in approx.
10% of normal urothelial samples from BC patients. The most common
deleted region was on chromosome 9q33.3. Deletions of chromosome 8p
were identified as progression markers for papillary BC. Loss of sFRP1
expression (located on 8p11.21) was found in 35% of BC cases and was
caused by gene copy loss and/or promoter methylation. Functional studies
revealed an influence of sFRP1 on proliferation, cell viability and
migration in papillary BC. Patients with papillary BC and sFRP1 loss
showed a significant decreased overall survival which was not relevant
in patients with solid tumors. Plasmacytoid BC displayed molecular alterations
of advanced, aggressive BC. A complete loss of membranous
E-Cadherin underlined the high malignant and metastatic potential of
this BC variant.
Conclusions. BC is not affecting the complete urothelium. Molecular alterations
in the normal urothelium could be origins of multifocal tumor
growth and argue for the “field cancerization” theory. Deletions on chromosome
8p and expression loss of sFRP1 might act as entity-specific progression
markers for papillary BC. The profile of molecular alterations in
plasmacytoid BC might help to find the suitable therapeutic intervention
for patients with this highly aggressive BC variant.
FR-017
Novel approaches to progressive renal diseases
P . Boor1 1RWTH Aachen University, Aachen
Aims. Essentially all chronic renal diseases but also repeated or serious
acute insults inevitably lead to chronic kidney disease and renal fibrosis.
We currently lack effective treatment options for renal fibrosis; the
development of an effective therapy would be invaluable virtually for all
renal patients.
Methods. We have used various in vivo and in vitro models of renal fibrosis.
Results. We have identified PDGF-CC, PDGF-DD, C5a and PPAR-α as
novel treatment targets in renal fibrosis. By identifying these targets as
crucial components of renal tubulointerstitial fibrosis we also uncovered
the mechanisms relevant for their actions, including mitogenic effect
of PDGF-DD and proinflammatory effects of PDGF-CC on interstitial
fibroblasts and effects of C5a/C5aR and PPAR-α on tubular epithelial
cells resulting in reduced profibrotic paracrine signaling to interstitial
fibroblasts. We verified all of the targets using tools used in, tested for or
developed for clinical use. These studies lay an important experimental
basis for translating these targets to clinical practice. In this regard, we
also showed that serum PDGF-DD is specifically increased in patients
with mesangioproliferative but not other types of glomerulonephritis. In
further studies on renal fibrosis we showed that irrelevant IgG has antifibrotic
effects in a model of progressive mesangioproliferative glomerulonephritis
resulting in improved survival. We also documented that the
renal profibrotic effects of cyclosporine A are mediated by Y-box binding
protein-1 (YB-1) and that mesenchymal stem cells might maldifferentiate
and induce local fibrotic response in progressive glomerulonephritis in
rats. We also pointed out that simple non-pharmacological approaches
in diabetic rats, e.g. regular moderate exercise, reduced renal fibrosis in a
very early stage when no functional changes were yet observed. The mechanism
seemed to be via improving metabolism and interfering with an
important pathogenic pathway in diabetes, the advanced glycation. We
have also identified environmental factors, which led to renal but also
cardiac fibrosis in healthy rats, i.e. passive smoking and industrial dust
52 | Der Pathologe · Supplement 1 · 2012
particles amozite. These environmental risk factors are external, modifiable
and could lead to better monitoring of patients with such (occupational)
risk.
Conclusions. Still much is to be learned about renal fibrosis. We hope to
have uncovered some pieces of this immense puzzle and surely aim to
continue to put it together in the future.
FR-018
Using genome-wide molecular screening for the identification of
new marker and target genes of human hepatocellular carcinoma
T . Longerich1 1University Hospital Heidelberg/Institute of Pathology, Heidelberg
Aims. To identify new diagnostic and prognostic markers that may be
used as therapeutic targets and to develop an oncogenetic progression
model of human hepatocellular carcinoma (HCC).
Methods. A well-characterized human HCC collection was used for
systematic genome-wide screening. Identified potential candidate genes
were validated via expression analysis and selected candidates were further
characterized in vitro using suitable HCC cell lines.
Results. Using a meta-analysis of classical comparative genomic hybridisation
(CGH) analysis (24 dysplastic nodules, 871 HCCs) a genomic
progression model of tumour dedifferentiation (1q – 8q – 4q – 16q – 13q)
was generated. Array-based CGH analysis revealed recurrent chromosomal
gains at 1q, 6p, 8q, 17q, 19p, and 20q, while genomic losses were
observed at 1p, 4q, 8p, 13q, 16q, and 17p. The mouse double minute homolog
4 (MDM4) that functions as a negative p53 regulator could be identified
as a target gene of 1q gains in human HCC. Aetiology-dependent
copy number gains and MYC overexpression was detected in viral and
alcohol-related HCCs. In contrast, cryptogenic HCCs showed neither
8q24 gains, nor MYC overexpression, nor target gene activation. The
role of Polo-like kinase family (PLK) members was analyzed in human
HCC. PLK1 levels were upregulated in human HCC, reaching the highest
expression in tumours with poorer outcome. PLK1 overexpression
resulted from activation of the Ha-Ras/FOXM1/PLK1-axis. In contrast
PLK2, PLK3, and PLK4 expression were downregulated in HCC, with
the lowest levels being detected in HCC with shorter survival. PLK2-4
down-regulation was paralleled by promoter hypermethylation and/or
loss of heterozygosity. Immunohistological analysis revealed that a diffuse
sinusoidal expression of Annexin A2 has diagnostic value for the
biopsy diagnosis of highly-differentiated HCC and may increase the accuracy
of the established diagnostic marker panel (GPC3, GS, HSP70)
for HCC detection.
Conclusions. Five genomic aberrations allow the generation of a robust
progression model of human hepatocarcinogenesis. MDM4 upregulation
may result in functional p53-inactivation in HCCs that may allow
p53-reactivation as a therapeutic strategy. Differential oncogenic and tumour
suppressive roles of Polo-like kinases could be identified in human
HCC. Systematic genome-wide screening analysis were used to identify
new diagnostic marker and potential oncogenic and tumorsuppressive
factors that may be promising future therapeutic targets.
FR-019
In situ hybridization in clinical pathology
T . Gaiser1 1Philipps-University Marburg, Institute of Pathology, Marburg
Aims. Over the last decade in situ hybridization (ISH) has emerged as a
powerful clinical and research tool for the assessment of target DNA dosages
within interphase and metaphase nuclei. HER2 detection is the widest
application for ISH in routine pathology but EGFR ISH or the newly
introduced melanoma FISH are further possible applications, which can
be additionally improved by computer based analysis systems.

Methods. Three different techniques FISH, CISH and SISH for in situ
hybridization were evaluated regarding their use in routine pathology.
Furthermore, a multi-colour probe in malignant melanoma samples was
evaluated as test tool in histological borderline melanoma cases and a
computer based algorithm was implemented for simplifying and standardization.
Results. EGFR SISH amplification studies in archival paraffin-embedded
glioblastoma samples showed that SISH can accelerate the diagnostic
process in a cost-effective way. Melanoma FISH probes failed to detect
a sufficient amount of chromosomal changes necessary for a clinically
useful diagnostic tool. Computer based algorithms helped to standardize
ISH evaluation.
Conclusions. Specific genetic alterations will define new disease entities,
requiring ISH as prerequisite to establish the diagnosis. ISH can help to
sub-classify morphologically similar neoplasia in terms of therapeutical
response and will help to define genetic subgroups within distinct diagnostic
groups for treatment purposes.
FR-020
Epithelial-mesenchymal transition of non-small cell lung cancer
A . Soltermann1 , V . Tischler1 , L . Morra1 , W . Weder2 , H . Moch1 1University of Zurich, Institute of Surgical Pathology, Zurich, Switzerland,
2University Hospital Zurich, Division of Thoracic Surgery, Zurich, Switzerland
Aims. Non-small cell lung cancer (NSCLC) is a highly fibrotic malignancy,
elaborating a prominent desmoplastic stroma reaction or tumor microenvironment,
respectively. Four main modes of carcinoma invasion
into its own newly formed stroma are generally recognized: Epithelialmesenchymal
transition (EMT), amoeboid, infiltration in cohorts and
in collective sheets. We aimed for characterization of the matricellular
N-glycoprotein periostin, a major EMT indicator, in the tumor microenvironment
of NSCLC.
Methods. Malignant pleural effusions from lung adenocarcinoma were
screened for N-glycoproteins by shotgun proteomics using liquid chromatography
following tandem mass spectrometry (LC-MS/MS). The
identified EMT protein periostin was validated on both a tissue microarray
of surgically resected patients with NSCLC (n total=532) and on tumor
whole sections (n=30) by immunohistochemistry. Isoform-specific
PCR following sequencing was performed in frozen specimens.
Results. In the pleural effusions, 170 non-redundant N-glycoproteins
were identified with high protein probability >0.9, belonging mainly to
serum factors and extracellular matrix (ECM) constituents. Periostin
was the most robustly identified ECM protein in malignant effusions.
On tissue microarrays, strong protein upregulation was predominantly
observed at the invasive front in both tumor epithelia and the surrounding
extracellular matrix, the so-called matricellular space. In comparison
to structural ECM proteins such as collagen, elastin, vimentin and
versican, high periostin was found to be most closely associated with
clinicopathologic parameters of tumor progression such as higher stage,
higher pT and larger size; as well as the squamous cell histotype (all
p-values

Abstracts
ted genes will in the near future allow for providing helpful information
on individual cancer-genomes for individualized tumor therapy. Also,
NGS will become applicable to rather small specimen, including biopsies,
circulating tumor cells and circulating free DNA in plasma. Even so
NGS is already used in the clinical setting, for instance for the detection
of disease gene mutations of familial breast and ovarian cancer and for
genetic diseases, data analysis and interpretation using biostatistics, bioinformatics,
systems biological and mathematical approaches including
mathematical modeling is still rather complex. Robust strategies for data
analysis are being needed and are being developed.
Solving the key problems in cancer biology will therefore only be accomplished
by orchestrating a manifold collaboration between different
disciplines (life sciences, mathematics, and informatics). Platform comparison
will be provided for expression data comparing RNA seq and
array based methods. We will glimpse into the future and discuss third
generation sequencing and single-molecule sequencing technologies.
Important goals are to improve our knowledge on the regulation and
function of genes and proteins in single cells, tissue and organs.
While NGS is just on its way to be established as a routine diagnostics
tool, the next next-gen sequencing techniques are already emerging.
Whereas current NGS techniques depend on optics and thus require
elaborate signal detection, the third and fourth generation techniques
simply measure changes of electric current, either when DNA passes
through nanopores or bends nanowires in a sequence dependent manner.
Being electronic devices like computer processors, the third and
fourth generation sequencing machines will be very fast, very small and
rather cheap.
FR-023
Prognostoc biomarkers of gastric cancer
V . Warneke 1 , H .-M . Behrens 1 , C . Röcken1 , J . Haag1 , K . Balschun1 , C . Böger1 ,
C . Böger1 , T . Becker2 , J . Hartmann3 , M . Ebert4 , C . Röcken5 1 2 Christian-Albrechts-University, Department of Pathology, Kiel, Christian-
Albrechts-University, Department of Surgery, Kiel, 3Christian-Albrechts-Uni versity, Department of Internal Medicine II, Kiel, 4University of Mannheim,
Dept . of Medicine II, 5University Hospital Schleswig-Holstein, Campus Kiel,
Department of Pathology, Kiel
Aims. Chemotherapy for the treatment of gastric cancer (GC) is evolving
rapidly and continues to improve patient survival. We studied phenotypic
and genotypic biomarkers for GC, in order to test whether these
biomarkers are independent prognosticators of patient survival and
whether any of these biomarkers should be considered to tailor patient
treatment in the future.
Methods. 485 patients (299 men, 186 women; median age 68 years) with
GC had undergone either total or partial gastrectomy for adenocarcinomas
of the stomach or oesophagogastric junction. Survival data and
date of death were available for 469 patients. The pTNM-stage was based
on surgical pathological examination. The Laurén and mucin phenotype
was assessed. H. pylori- and Epstein-Barr virus infections were documented.
The following markers were studied: BRAF-, KRAS-, NRAS-
and PIK3CA (exon 9 and 20)-genotype, microsatellite instability, Her2/
neu-status, E-cadherin, β-catenin and EpCAM-expression.
Results. An intestinal type GC was found in 184 patients, a diffuse type
in 224. A persistent H. pylori-infection was found in 64 (15.5%) patients,
an EBV-infection in 15 (4.0%). Seventeen (3.6%) GCs showed a KRAS-,
12 (2.5%) a PIK3CA (exon 9)- and 9 (1.8%) a PIK3CA (exon 20)-mutation.
No NRAS- and BRAF-mutation was found in our series. 424 (95.1%)
GCs were EpCAM- and 46 (10.2%) Her2/neu-positive. 33 (7.3%) GCs
were highly microsatellite unstable, 31 (93.9%) of which showed loss of
expression of MLH1 and PMS2. Patient survival correlated with Laurén
phenotype, MSI-H and BerEP4-expression. Patient age, stage grouping
according to the Kiel-proposal, lymph node ratio and Mucin 2 were independent
prognosticators of patient survival.
54 | Der Pathologe · Supplement 1 · 2012
Conclusions. A thorough staging and surgical pathological examination
is the most important tool to assess patient prognosis. KRAS, PIK3CA
(exon 9 and 20), BerEP4-, Her2/neu- and MSI-status may be considered
to tailor patient treatment in the future.
FR-024
Deep-sequencing: Speed-up diagnostics of colorectal carcinoma
M . Rechsteiner1 , A . Bohnert 1 , A . von Teichmann 1 , S . Schmid-Brun1 ,
P . Schraml1 , H . Moch1 1University Hospital Zurich, Surgical Pathology, Zürich, Switzerland
Aims. In colorectal carcinoma, KRAS mutations have emerged as a major
predictor of resistance to anti-EGFR antibody treatment. Although the
role of BRAF mutations, in predicting the response to anti-EGFR drugs
still remains controversial, patients with a mutated BRAF gene exhibit
a significant shorter survival than patients without a mutation. In this
project we aimed to establish a high-troughput ultra-deep sequencing
platform to cope with the increased demand for sequence information
at medical institutions. With this platform we intend to unravel low frequency
mutations below the detection limit of Sanger sequencing and to
elucidate throughput power for diagnostics.
Methods. A cohort of 120 patients, diagnosed with colorectal carcinoma,
was established. The cohort consisted of 45 patients with KRAS mutations
in exons 2 or 3 and 75 patients without a KRAS mutation. This was
assessed by Sanger sequencing. The 75 patients with a wild-type KRAS
gene were further analysed for BRAF mutations in exon 15 by Sanger
sequencing.
Results. Fifty ng of genomic DNA isolated from FFPE tissue blocks were
found to give reproducible results as input material of the PCR to generate
amplicons used for deep-sequencing. The target amplicons were
KRAS exons 2 and 3 and BRAF exon 15. The exons 5 to 8 of the p53 gene
were also analysed due to high mutation rates in colorectal carcinoma.
The amplicons of each patient were labelled with multiplex identifiers
(MIDs), and these were shown to be highly specific in the data analysis
after deep-sequencing. Seven amplicons of 9 patients were pooled in
one single 454 Junior Sequencing run. On average, each amplicon was
covered 1000-fold which allowed us to identify mutations at a 4% frequency.
Integrated computational down-stream analyses enabled us to
speed up the detection and classification of mutations. Results from the
first 17 patients yielded 14 mutations located in the p53 gene (82%) and 1
in the BRAF gene (6%). The BRAF mutation was identified as the well
known activating mutation V600E. We also included a patient with a
known KRAS mutation as control which was successfully verified by
deep-sequencing.
Conclusions. This newly established method allowed us to analyse 7 amplicons
of 9 patients (63 amplicons in total) in one deep-sequencing run
within 1 week. The capacity limit of a Junior 454 is 4 runs per week which
would allow us to analyse the mutation status for the KRAS, BRAF, and
p53 genes of 36 patients with colorectal carcinoma
Promotionspreis
FR-026
Colocalization algorithms for conversion of traditional immunohistochemistry
into virtual multicolor stains
A .-S .K . Meyer1 , P . Möller1 , J .K . Lennerz1 1University Ulm, Institute of Pathology, Ulm
Aims. Diagnostic immunophenotyping is performed via “mentally”
combining single immunohistochemistry (IHC) stains. The discrepancy
to research settings, were co-visualization predominates, is due to technical-
(i.e. species identity, detection systems) and/or practical hurdles

(i.e. investment in equipment and expertise). Here we present simple pixel-algorithms
that allow electronic merging and color-range conversion
of traditional IHC stains.
Methods. Single-label IHC-stains, performed on subsequent sections,
are digitized and used as input data. After manual positioning, a merge
algorithm compares the input images and selects pixels with higher
values. A re-coloring algorithm reassigns color ranges. Algorithms are
achieved via a customized link between software platforms (Aperio ImageScope10.0;
Adobe PhotoshopCS3; ImageJ Version10.2) using AutoIT
(v3.2.12.0), a freeware scripting language for automating the Microsoft
Windows GUI.
Results. The merge algorithm emulates double staining, which includes
preservation of the traditional IHC-views (operative comfort zone). To
allow distinction of similarly labeled stains in the merge, a re-coloring
algorithm converts the color range of stained elements from each image
(e.g. brown vs. red). As a specific re-coloring mode, stained elements
can be extracted and converted into pseudo-immunofluorescence (IF),
which includes all downstream assessments of co-localized elements
(virtual IF-stain). The algorithms solve the common problem of species
identity of primary antibodies because they allow co-visualization of
markers without compromising staining specificity, while at the same
time employing individual stains that remain available for traditional
evaluation. When compared to investments in IF-equipment or validation
of physical double-stains, the effort to learn and perform the conversion
algorithms is negligible.
Conclusions. The presented imaging tools emulate the principal advantages
of multi-color fluorescence microscopy as well as multi-color IHC.
These techniques represent a powerful expansion of one of the most versatile
molecular tools in diagnostic pathology. Thereby, the combination
of established methods (IHC) and imaging algorithms also exemplifies
the potential of digital pathology.
Translationale Forschung und
AG Molekularpathologie
FR-027
Delay to preservation does not induce a systematic phosphoprotein
response during tissue processing
S . Gündisch1 , C . Schott1 , K . Grundner-Culemann2 , M . Machatti2 , D . Groelz3 ,
C . Schaab2 , A . Tebbe 2 , K .-F . Becker1 1Technische Universität München, Institute of Pathology, München,
2 3 Evotec AG, Martinsried, PreAnalytiX GmbH, Hombrechtikon, Switzerland
Aims. The quality of tissue samples can have a significant impact on
analytical data sets for biomarker research. In particular, posttranslational
modifications such as phosphorylation need to be systematically
investigated in that phosphorylated protein levels indicate the activation
status of signal transduction pathways controlled by kinases. However,
little is known about the impact of pre-analytical factors on phosphoprotein
stability. The aim of this study was to characterize the potential
effects of delayed preservation and different preservation methods on the
stability of phosphoproteins using targeted and non-targeted proteomic
approaches.
Methods. Murine and rat liver samples were exposed to different ischemic
conditions before preservation and either cryopreserved, formalinfixed
or fixed with the PAXgene Tissue System, a new non-crosslinking
formalin-free fixative. The phosphoproteome was analyzed using quantitative
tandem mass spectrometry (LC-MS/MS) and reverse phase protein
array (RPPA) technology.
Results. The phosphoproteomic analysis of ischemic mouse liver tissue
samples by LC-MS/MS indicated no significant global alterations of
more than 5000 phosphosite ratios analysed during 60 minutes of delayed
cryopreservation. The analysis of ischemic rat liver tissue samples
by RPPA revealed similar results as investigated phosphoproteins, including
phospho-Akt, phospho-p38 MAPK or phospho-p44/42 MAPK,
showed very stable profiles during the time-course experiment, independent
of the preservation method applied.
Conclusions. Since we could not detect significant global changes of the
phosphoprotein profiles, neither with a targeted nor a non-targeted approach,
we conclude that the phosphoproteome seems to be more stable
than expected with regard to delayed preservation. This allows accurate
quantitative measurements of the activation state of signalling pathways
of tissue samples which had not been immediately preserved. This result
is essential for the development of new targeted therapies involving kinase
inhibitors which have recently been a focus in the field of personalized
medicine. Studies are ongoing to validate our results in human tissue
samples as inter-patient variability may occur which is absent in our well
controlled model systems.
This work has received funding from the Munich Biotech Cluster m4 (www .
m4 .de) and the European project SPIDIA (www .spidia .eu) .
FR-028
Validation of a novel DNA methylation based 2-gene biomarker
panel for early detection of bladder cancer using urine samples
M . Rose1 , D . Fiedler1 , N .T . Gaisa1 , C . Schubert 1 , P . Antony1 , R . Davtalab1 ,
D . Pfister2 , A . Heidenreich2 , R . Knüchel1 , E . Dahl1 1RWTH Aachen University/Institute of Pathology, Aachen,
2RWTH Aachen University/Clinic of Urology, Aachen
Aims. The early detection of bladder cancer and its recurrent tumours is
currently based on cystoscopy, which is highly sensitive and specific, but
also invasive and expensive. Alternative methods still lack suitable sensitivity
especially with regard to non-invasive bladder tumours. In the line
with accumulating evidence suggesting that DNA methylation pattern
could serve as sensitive and specific biomarkers, we aimed to identify
novel DNA methylation loci potentially useful for early cancer detection
and treatment stratification of bladder cancer patients, respectively.
Methods. In order to discover potential biomarkers we analyzed the methylation
levels of array-based candidate genes by using MSP in bladder
cell lines (n=5), non-cancerous (n=20) and cancerous bladder tissues
(n=60). Subsequently, we determined the methylation status of selected
genes performing MSP assays on a so called “screening cohort” containing
DNA extracted from urine sediments of bladder cancer patients
(n=60). Age-matched non-urological-cancer patients (n=30) as well as
prostate cancer patients (n=15) were included as controls to ensure specificity.
The biomarker potential of the most frequently methylated gene
loci were then discriminated by using quantitative pyrosequencing in a
“validation urine sample cohort” (currently; n=30) of bladder cancer patients
in comparison to controls. The performance of a 2-gene-panel was
optimised using receiver operator characteristics (ROC) curve analysis.
Results. MSP assays performed on bladder cells lines and bladder cancer
tissue revealed novel candidate genes exhibiting frequently cancer
specific promoter hypermethylation. In the urine screening samples of
bladder cancer patients, these gene loci showed frequent methylation
with an overall-panel sensitivity of 81.5%, i.e. 81.5% of samples presented
promoter methylation in at least one of the two genes. Importantly, none
of the age-matched controls exhibited methylation signals excluding rare
and weak age-depended side effects. By using quantitative pyrosequencing
technique we were able to increase the sensitivity of the 2-gene panel
up to 83.3% (p≤0.001, AUC=0.940) with 100% specificity.
Conclusions. We have identified a 2-gene putative biomarker panel based
on detection of DNA promoter methylation. Applying this panel in urine
sediments using pyrosequencing may provide a highly sensitive and
specific, non-invasive approach for early detection of primary bladder
tumours.
Der Pathologe · Supplement 1 · 2012 |
55

Abstracts
FR-029
Whole exome sequencing identifies potential therapeutic
targets for castration resistant prostate cancer
R . Menon 1 , M . Deng 1 , D . Boehm 1 , F . Fend 2 , D . Boehm 3 , S . Biskup 3 , S . Perner 1
1 Institute of Prostate Cancer Research, Institute of Pathology, University Hospital
of Bonn, Bonn, 2 Institute of Pathology, University Hospital Tübingen,
Tübingen, 3 Center for Genomics and Transcriptomics, Tübingen
Aims. Castration resistant prostate cancer (CR-PCa) is the most aggressive
form of prostate cancer (PCa) having a poor prognosis, and is a
significant therapeutic challenge. The key to the development of novel
therapeutic targets for CR-PCa is to decipher the molecular alterations
underlying this lethal disease. The aim of our study was to perform whole
exome sequencing and gene copy number analysis on 5 CR-PCa/normal
paired formalin fixed paraffin embedded (FFPE) samples using the
SOLiD4 next generation sequencing platform.
Methods. Genomic DNA was extracted from 5 CR-PCa/normal paired
FFPE samples. The DNA was subjected to targeted exon capture using
the Agilent Sure Select kit. The captured DNA was sequenced using the
SOLiD4 next generation sequencing platform. The sequencing output
was mapped, sorted, filtered and annotated using well-known human
genome databases. The results were further analyzed for SNPs and copy
number variations. A set of amplified/deleted genes were validated using
fluorescence in-situ hybridization (FISH) assays with a PCa progression
cohort. The cohort consisted of 138 cases for localized cancer, 105 patients
with primary PCa and corresponding LN metastasis, and 39 samples for
castration resistant tumors.
Results. Whole exome sequencing analysis identified focal regions of deletion,
which included well-known tumor suppressors such as NKX3.1
and PTEN. Focal regions of amplification included well-known genes
such as cmYC and AR that are known to play a role in PCa. Furthermore,
we identified several amplified genes as druggable targets e.g. HDAC6,
NTRK1, PLD1, SPHK1, and SIRT7. NTRK1 is a kinase that plays an active
role in cell proliferation. HDAC6, PLD1, SPHK1 and SIRT7 regulate numerous
complex cellular processes including signal transduction, transcription
and apoptosis.
Conclusions. This is the first study to use whole exome sequencing approaches
on FFPE CR-PCa material to identity novel therapeutic targets.
Validation studies would further shed light into the biological understanding
of the disease and its plausible treatment options.
FR-030
Cut-offFinder – a web application for cut-off optimization for
molecular markers
J . Budczies1 , F . Klauschen1 , W . Schmitt1 , C . Denkert1 1Charité Hospital, Institute of Pathology, Berlin
Aims. In order to translate a continuous diagnostic variable into a clinical
decision, it is necessary to determine a cut-off point and to stratify
patients into two groups, each of which requires a different kind of treatment.
Methods. Cut-offFinder is implemented as Java Server Pages (JSPs) that
connect to the statistical engine R. Using a web browser, the user can
upload a molecular data set, assign biomarker and outcome variables
and determine an optimal cut-off point for the biomarker. The web application
offers three different methods for cut-off determination: The
first method fits a mixture model of two Gaussian distribution to the
distribution of the variable. The optimal cut-off is determined as the value
where both probability density functions coincide. For the two other
methods, all possible cut-off points are scanned. The second method
correlates the dichotomized biomarker with a binary outcome variable
using logistic regression. The optimal cut-off is defined as the point with
the most significant (Fisher’s exact test) split. The third method fits Cox
proportional hazard models to the dichotomized variable and the sur-
56 | Der Pathologe · Supplement 1 · 2012
vival variable. Then, the optimal cut-off is defined as the point with the
most significant (log rank test) split.
Results. As example, we have analyzed gene expression data of estrogen
receptor (ESR1) and progesterone receptor (PGR) from a publicly available
microarray data set of 286 breast cancer samples (GSE2034 at www.
ncbi.nlm.nih.gov/geo) using Cut-offFinder. Histograms of the distribution
of ESR1 and PGR showed a clear bimodal shape. Distribution derived
cut-offs were located at 10.6 (ESR1) and 5.0 (PGR). The dependence
on the cut-off of the odds ratio (OR) for correlation ERS1 expression with
ER status (determined by immunohistochemistry) was analyzed and
visualized. The optimal cut-off was determined as 10.1 with OR=67.8
(30.2–152.1). Using ERS1 expression measured by the microarray, determination
of ER status was feasible with a sensitivity of 85.7% and a specificity
of 91.9%. The dependence on the cut-off of the hazard ratio (HR)
for correlation of PGR expression with distance-metastasis-free survival
was analyzed and visualized. The optimal cut-off was determined as 2.5
with HR=0.46 (0.30–0.71). Kaplan Meier analysis showed a significantly
better outcome for patients with high PGR expression (p=0.00028).
Conclusions. In summary, Cut-offFinder is a comprehensive and easyto-use
web application for cut-off determination for molecular markers.
FR-031
BRAF-testing with pyrosequencing: a reliable alternative for the
analysis of highly pigmented and degraded FFPE material
A . Lehmann1 , C . Schewe1 , K . Jöhrens1 , C . Denkert1 , J . Budczies1 , M . Dietel1 1Charité Universitätsmedizin Berlin, Institute of Pathology, Berlin
Aims. The approval of new BRAF inhibitors for the treatment of metastasized
melanoma has led to a great demand for BRAF testing in molecular
pathology laboratories. However, molecular analysis of formalin-fixed,
paraffin-embedded (FFPE) melanoma tissue is challenging. Sanger sequencing
often fails due to high amplification lengths and the influence
of melanin. In this study we tested the applicability of a new pyrosequencing
assay for BRAF analysis on 118 FFPE melanoma tissues and compared
the results with those of Sanger sequencing.
Methods. The study comprised 118 formalin-fixed, paraffin-embedded
tissues of malignant melanoma which were referred to our department
of Molecular Pathology for routine BRAF testing. DNA was extracted
(QIAamp® DNA Mini Kit, Qiagen) and samples were subjected to both,
Sanger sequencing (in-house method) and pyrosequencing (therascreen
BRAF Pyro Kit®, Qiagen) of BRAF exon 15, codons 599 and 600.
Results. BRAF sequences of 102 of 118 samples (86.4%) were evaluable
by both methods pyrosequencing and Sanger sequencing. Mutational
status of these samples was consistent in 98.0% (Cohen’s kappa coefficient
=0.96, p=0.000). Sanger sequencing failed for 15 samples, which
were mostly highly pigmented. Interestingly, 11 of these samples were
still evaluable with pyrosequencing. With a success rate of 95.8% (CI95
[90.5–98.2%]), significantly more cases could be evaluated by pyrosequencing
than by Sanger sequencing [success rate Sanger =87.3%, CI95
(80.1–92.1%); p=0.035].
Conclusions. Pyrosequencing requires comparably short target sequences
for amplification which makes this method feasible even for problematic
material for which standard methods like Sanger sequencing
often fail. Particularly with regard to the high impact of BRAF-testing
for therapy decision, pyrosequencing is a fast and reliable alternative for
BRAF-testing.

FR-032
COLD-PCR: a powerful tool in routine-diagnostic for cost-neutral
detection of minor clones using real-time PCR or pyrosequencing
quoted by the example of EGFR mutation analysis
F . Mairinger 1 , A . Streubel 2 , A . Roth 2 , O . Landt 3 , W . Grüning 4 , J . Kohlmeier 4 ,
T . Mairinger 2
1 University Hospital Essen, Department of Pathology und Neuropathology,
Essen, 2 Helios Klinikum Emil von Behring, Department of Pathology, Berlin,
3 TIB MOlBIOL Gmbh, Berlin, 4 Helios Klinikum Emil von Behring, Department
of Pneumology, Berlin
Aims. COLD-PCR (co-amplification at lower denaturation temperature-PCR)
is a novel method to enrich minority alleles from mixtures of
wild-type (wt) and mutation containing (mt) sequences, irrespective of
localization and property within the analyzed sequence. The heteroduplexes
generated after an initial denaturation step will be preferentially
melted at the critical denaturation temperature resulting in a radical
enrichment of minor variants, enabling their detection with conventional
methods which are intended to analyze normal bi-allelic variations.
Molecular testing of tissues is faced with samples containing mixtures
of tissues frequently containing only small fractions of mutations. A
study was designed to overcome this problem of the too low sensitivity
of nowadays routinely used molecular methods. For this, the effect of
COLD-PCR in probe-based real-time PCR analysis or as initiating step
for pyrosequencing to the sensitivity was tested.
Methods. Different dilution steps of artificial EGFR T790M mutated and
wild-type EGFR exon20 DNA were analyzed by a LightCycler assay or
amplified by full-COLD-PCR using different protocols and afterwards
sequenced by pyrosequencing to determine the detection-limit of these
methods.
Results. For the LightCycler-assay, the best results are rendered with a
combination of 10 cycles conventional PCR followed by 45 cycles COLD-
PCR using 84°C as denaturation temperature. A dilution of down to
0.125% mt-DNA/total-DNA is still detectable. With COLD-PCR amplified
DNA, a dilution of 0.125% mt-DNA/total-DNA is still detectable by
pyrosequencing in reproducible results.
Conclusions. Our results show the exceeding potential of COLD-PCR in
enrichment of DNA of underrepresented clones in clinical samples. Because
of this, problems like dilution of potentially mutated tumor cells
(showing for example EGFR-resistance mutation T790M) with non-resistant
tumor cells or benign cells debt to macrodissection or tumor heterogeneity
resulting in failing to detect clinically relevant minor clones
could be overcome.
FR-033
Fast and reliable detection of mutations in exon 9 of the KIT gene
by high resolution melting analysis
H . Künstlinger1 , M . Kleine1 , J . Fassunke1 , E . Wardelmann1 , R . Büttner1 ,
S . Merkelbach-Bruse1 , H .-U . Schildhaus1 1University Hospital Cologne, Institute of Pathology, Köln
Aims. Gastrointestinal stromal tumours (GISTs) are the most common
mesenchymal tumours of the gastrointestinal tract. They harbour activating
mutations in the KIT or platelet-derived growth factor (PDGF)
receptor. Imatinib mesylate is a potent inhibitor of KIT signalling and
is therefore widely used as targeted therapy for GISTs. The mutational
status of KIT exon 9 is of special importance for the therapy with Imatinib,
because cases with exon 9 mutations need a higher dose of Imatinib.
Therefore, in metastasized or high-risk GISTs as well as in primary notoperable
tumours it is necessary to obtain the mutational result as fast
as possible after diagnosis. At present, mutational analysis of KIT and
PDGFR is routinely carried out by Sanger sequencing. This method has
certainly its lasting relevance for DNA sections with high variability of
mutations (e.g. KIT exon 11), but is relatively expensive and time consuming.
Therefore alternative methods need to be established for other
exons (like KIT exon 9) or other certain mutational types to enable a fast
and cost efficient detection.
Methods. High Resolution Melting (HRM) is a post-PCR mutation scanning
tool that detects the change in fluorescence caused by the progressive
release of a saturating intercalating dye from DNA duplexes while
they are denatured by slight increases in temperature. HRM assays were
developed using specifically designed primers and genomic DNA isolated
from formalin-fixed paraffin-embedded GIST samples. Melting
curve analyses were performed on the LightCycler 480 platform (Roche)
and mutation analyses were additionally confirmed by Sanger Sequencing.
Results. Conditions for High Resolution Melting analysis of KIT exon 9
could be established using more than 60 GIST samples with known mutational
status of the KIT gene. Sensitivity was determined as a minimal
proportion of 12.5% mutated alleles. A prospective screening of more
than 100 additional GIST samples showed a complete concordance between
HRM assay and traditional Sanger sequencing.
Conclusions. The established high resolution melting assay represents a
highly reliable method for the detection of mutations in exon 9 of the
KIT gene. It allows a faster and more cost-effective mutational analysis of
KIT exon 9 in the future, which is especially important for dose finding
of Imatinib in GIST therapy. The determined sensitivity is comparable to
the sensitivity of currently performed Sanger sequencing.
FR-034
Morphological and clinical characterization of a novel mouse
model for mutation-activated JAK1
S . Wagner1 , E . Janas2 , B . Lorenz-Depiereux3 , J . Calzada-Wack 2 , A . Benet-Pagès3
, S . Eck3 , J .A . Aguilar Pimentel4 , B . Rathkolb4 , V . Gailus-Durner4 ,
H . Fuchs4 , H . Höfler2 , M . Hrabé de Angelis1 , T . Strom3 , F . Neff2 1Helmholtz Zentrum München, Institute of Experimental Genetics, Neuherberg,
2Helmholtz Zentrum München, Institute of Pathology, Neuherberg,
3Helmholtz Zentrum München, Institute of Human Genetics, Neuherberg,
4Helmholtz Zentrum München, German Mouse Clinic, Neuherberg
Aims. The members of the Janus kinase family (JAK1, JAK2, JAK3) play
important roles in signalling downstream of cytokine receptor activation
and are implicated in various physiological processes including the
hematopoietic, immune and neuronal systems. However, the lack of
successful mouse models for mutation-activated JAK1-induced diseases
hampers the understanding of disease pathology. Here, we have produced
a novel mutant mouse line leading to an amino acid substitution in
the pseudokinase domain of JAK1 using N-ethyl-N-nitrosourea (ENU)
mutagenesis. This mutation corresponds to a JAK1-activating mutation
(Ser646Phe) described in humans and associated with acute lymphoblastic
leukemia (Mullighan et al. 2009).
Methods. The ENU mutagenesis was generated in C3HeB/FeJ genetic
background. Mutation screening was performed after linkage analysis
using single nucleotide polymorphisms (SNP) and chromosome sorting
by next generation sequencing. A total of 64 mice at the age of 18 to 31
weeks were analyzed for clinical and immunological parameters as well
as histology. Thirty organs were examined by H&E staining and immunohistochemistry.
Results. All mutant mice showed a loss of ear cartilage starting with
4 months of age without signs of inflammation, a significant loss in body
weight due to an alternation in body composition. In serum, a significant
increase of auto-antibodies was observed. Most strikingly, histopathological
analysis revealed a nodular regenerative hyperplasia of the liver
with a remarkable increased neovascularisation. This vascularisation
was also observed in the skin of ears indicating a systemic vasculitis.
Conclusions. A significantly increase in auto-antibodies together with
the pathological changes observed implicates that the introduced mutation
in the pseudokinase domain of JAK1 induces a systemic autoimmune
disease.
Der Pathologe · Supplement 1 · 2012 |
57

Abstracts
FR-035
MAP3K7 deletions have prognostic relevance in prostate cancer
M . Kluth 1 , A . Krohn 1 , J . Hesse 1 , R . Simon 1 , T . Schlomm 2 , H . Huland 2 , G . Sauter 1 ,
S . Minner 1
1 University Medical Center Hamburg-Eppendorf, Hamburg, 2 Martini-Clinic,
Hamburg
Aims. MAP3K7 (mitogen-activated protein kinase kinase kinase 7) gene
is a component of the MAPK-pathway and plays a central role in cell
growth, differentiation and apoptosis. The MAP3K7 gene is located at
6q15, a region, which is commonly deleted in prostate cancer. The aim
of this study was to examine the potential relevance of MAP3K7 (6q15)
deletions in prostate cancer with respect to tumor phenotype and clinical
outcome.
Methods. A prostate tissue microarray (TMA) with clinical follow-up
data consisting of over 4500 prostate cancer samples was analyzed for
MAP3K7 deletions by fluorescence in situ hybridization (FISH). Results
were correlated with tumor phenotype, clinical outcome and ERG
expression. In addition, 15 tumors with known MAP3K7 deletion and
14 tumors without MAP3K7 deletions were examined for the presence of
MAP3K7 mutations (Exon 1–17).
Results. Heterozygous MAP3K7 deletions were found in 18.5% (423/2289)
of all analyzable prostate cancers. MAP3K7 deletions were associated
with advanced tumor stage (p

dissection (MBLND) technique to improve the LN staging in gastrointestinal
cancers. Moreover, we combined this technique with an ex-vivo
sentinel mapping method to further improve the staging.
Methods. The technique has been established in pilot studies and than
confirmed in prospective and randomized studies. The injections were
performed in fresh state. Subserosal or submucosal injection of black ink
was performed for the sentinel mapping. Methylene blue (MB) solution
was injected afterwards into the main arteries.
Results. LN harvest was highly significant improved in the MB groups
in comparison to the control groups (colon cancer: 35±18 vs. 17±10; rectal
cancer: 30±14 vs. 17±11). MB technique ensured sufficient LN harvest in
almost all cases including cases of neoadjuvantly treated rectal cancer.
The evSLN detection rate, sensitivity and accuracy in gastric cancer were
87%, 81% and 93%, respectively. Isolated tumor cells were detected after
immunohistochemical staining in 3 of 17 cases (18%). The chance to detect
a metastasis in an evSLN was 2 times higher in comparison to conventional
LNs. In 8% of all cases only evSLN were affected by metastases.
Conclusions. MBLND is a highly effective method of improving the LN
harvest in gastric cancer. Further application of evSLN mapping is feasible
and has the potential to heighten the sensitivity of metastasis detection.
SA-004
EBV-assoziierte Tumoren in Interaktion mit dem Immunsystem
M . Büttner1 1University Hospital Erlangen, Institute of Pathology, Erlangen
Aims. EBV-associated tumours are characterized by a prominent inflammatory
infiltrate, which, however, is not able to eliminate the tumour
cells. Viral proteins like latent membrane protein 1 (LMP1) can interact
with intracellular signalling pathways and thereby modulate the immune
reaction. In order to better understand immunological processes in
EBV-associated tumours different mechanisms were investigated.
Methods. Paraffin embedded material of classical Hodgkin lymphomas
(cHL), nasopharyngeal carcinomas (NPC) and gastric carcinomas was
investigated by immunohistochemistry and in situ hybridization with
regard to the expression of EBV-encoded and cellular genes, which are
involved in the regulation of the immune reaction. Cell culture experiments
were performed for a better understanding of the mechanisms
involved on cellular basis.
Results. Hodgkin-Reed-Sternberg (HRS) cells of cHL are B cell derivates
with a phenotype which is neither typical for a germinal center B-cell
nor of a plasma cell, but rather reminds of an abortive plasma cell phenotype.
STAT3 a transcription factor with a central role in the linkage of
inflammation and tumorigenesis is expressed in cHL and NPC. LMP1
can induce the chemokines RANTES and MCP-1 in an at least partially
NFkappaB- dependent manner. However, those chemokines are predominantly
expressed in the inflammatory infiltrate rather than the tumour
cells. In EBV-associated cardiac carcinoma an increased number
of cytotoxic T cells is found, which might be a sign of an on-going antiviral
response.
Conclusions. In EBV-associated tumours a complex interaction of cellular
and viral factors takes place, which can modulate the intratumoural
immune reaction. A better understanding of those processes could help
to find potential immune evasive mechanisms applied by the tumours,
which could interfere with an effective immunotherapy.
Aktuelle Entwicklungen in der Forschung II –
Gastrointestinaltrakt
SO-001
TNF induces STAT3-mediated inflammation in normal colon
epithelial cells
S . Chakilam1 , A . Agaimy1 , R . Atreya 2 , M . Gandesiri1 , J . Ivanovska1 , M . Rave-
Fränk 3 , H . Christiansen3 , T .T . Rau1 , R . Schneider-Stock 1
1University of Erlangen-Nuremberg, Institute of Pathology, Erlangen,
2University of Erlangen-Nuremberg, Department of Medicine I, Erlangen,
3University Göttingen, Department of Radiation Oncology
Aims. Tumor necrosis factor α (TNF) is a pleiotropic cytokine that participates
in a wide range of biological activities, including inflammation,
growth, differentiation, and apoptosis. TNF is a key molecule in the cytokine
network, capable of regulating the expression of other cytokines,
such as IL-6 which is known to be a major mediator of inflammation
through the activation of the STAT-3 pathway. To simulate the effects of
TNF to normal intestine we treated immortalized human colon epithelial
cells (HCEC) with 0.66 ng/ml TNF and analyzed the possible TNFmediated
functions and signal transduction.
Methods. HCEC cells were stimulated for various time points (1, 6, 24,
48 and 72 h). Activation of transcription factors (NF-kB, ATF-2, and
STAT3) was assessed by western blotting. Inflammation was detected by
real-time RT-PCR and cytokine ELISA. Transcriptional transactivation
was measured by EMSA and chromatin immunoprecipitation. Apoptosis
was analyzed by Annexin V binding assay and M30 staining.
Results. TNF-induced a biphasic pattern of activation of transcription
factors NF-kB, ATF-2, and STAT3 with NF-κB and ATF-2 phosphorylation
at 1 and 6 h, whereas STAT3 activation (pSTAT3Tyr705) and transcriptional
activity was induced later at 48 h. TNF also induced secretion
of the pro-inflammatory cytokines IL-6 and IL-8. We assumed that
the released IL-6 at early time points causes STAT3 activation. Indeed,
IL-6 neutralisation significantly attenuated TNF-induced STAT3 phosphorylation.
Moreover, AG490 (JAK inhibitor) pre-treatment decreased
TNF-induced STAT3 activation and significantly decreased TNF-augmented
IL-6 secretion. As expected, IL-6 stimulation alone also increased
pSTAT3Tyr705 levels. In parallel, there was no remarkable apoptosis
induction despite an increase in DNA-damage measured by pH2AX foci
formation. STAT3 signaling was verified in human tissues from ulcerative
colitis patients.
Conclusions. These data indicate that HCEC cells seem to develop an
inflammatory phenotype upon TNF stress. The TNF-induced inflammation
is regulated by IL-6 and STAT3. We hypothesize that the normal
mucosa is also actively participating in the development and maintenance
of inflammatory conditions in the gut.
SO-002
Establishment of a cell culture model to study inflammationassociated
oxidative stress as initiator of colorectal neoplasias
A . Poehlmann1 , K . Reissig1 , P . Schoenfeld2 , P . Steinberg3 , T . Guenther1 ,
A . Roessner1 1Otto-von-Guericke University Magdeburg, Department of Pathology,
Magdeburg, 2Otto-von-Guericke University Magdeburg, Department of
Biochemistry and Cell Biology, Magdeburg, 3Institut of Food Toxicology and
Analytical Chemistry, Hannover
Aims. Accumulating evidence shows that oxidative stress displayed by
reactive oxygen species (ROS) is a major contributor to inflammationassociated
cancer. Whether oxidative stress in inflammatory bowel disease
(IBD)-associated carcinogenesis is only a promoting factor or linked
to tumor initiation is still an open question. To find evidence for the
transforming capacity and tumor-initiation effects of ROS, we generated
Der Pathologe · Supplement 1 · 2012 |
59

Abstracts
a cell culture model. We mimicked ROS exposure of the epithelium in
human IBD using the non-tumor human colon epithelial cell line HCEC
and H2O2 as ROS.
Methods. The clinical course of IBD with multiple recurrences was simulated
by repeated H2O2 exposure cycles of the HCEC cell cultures. We
generated ten cell lines and confirmed neoplastic transformation by analyzing
them for clonal growth, enhanced proliferation, Colony Formation,
ROS-release, and β-galactosidase activity. The p53, Kras, and APC
mutation status, as well as microsatellite instability (MSI) were assigned.
Results. After three treatment cycles with H2O2, HCEC lost cell-cell
contact and started pilling up. Surviving HCEC showed uncontrolled
proliferation. Anchorage-independent growth in soft agar is often predictive
of tumorigenicity in vivo and is considered the most stringent
assay for malignant cell transformation in vitro. As ROS-injured HCEC
form colonies in soft agar, we concluded HCEC transformation. By
checking further hallmarks of cancer, we detected (i) self-sufficiency in
growth signals presumably via ROS-induced ROS-release, (ii) a limitless
replicative potential by overcoming cellular senescence, and (iii) evading
apoptosis. There were no alterations in p53, Kras, and APC gene, or MSI.
Conclusions. Our HCEC cell model provides novel insights into tumorinitiating
molecular events in IBD-associated carcinogenesis as these
cells are more likely to represent the potential target for tumor initiation
in vivo. Oxidative stress alone is sufficient to initiate HCEC transformation.
Transformed HCEC do not show well-known genetic alterations.
We therefore suppose early epigenetic changes to play an important role
in the initiation of the IBD-carcinoma pathway. This would further provide
the possibility of unravelling novel subsequent genetic alterations
that account for tumor initiation.
SO-003
Acetone compression and its impact on tumor staging of colorectal
cancer
J . Kitz1 , A . Gehoff2 , L .-C . Conradi3 , T . Sprenger3 , O . Basten4 , T . Liersch3 ,
P . Middel2 , J . Rüschoff2 1University Medical Center Göttingen, Department of Pathology, Göttingen,
2 3 Institute of Pathology Nordhessen, Kassel, University Medical Center Göttingen,
Department of General and Visceral Surgery, Göttingen, 4Institute of
Pathology Marburg, Marburg
Aims. Acetone compression (AC; Basten et al. Pathologe 2010) is a method
that allows for complete lymph node evaluation in colorectal cancer
(CRC), which is particularly important in rectal carcinomas pretreated
neoadjuvantly (Gehoff et al. Am J Surg Pathol 2011, accepted). In addition,
AC also facilitates detection of any further metastases in mesenteric
adipose tissue, e.g. perineural infiltration, which has its own prognostic
significance (Poeschl et al. JCO 2010). The present study deals with
the significance of AC for tumor staging of colorectal cancer. Not only
lymph node status but particularly also the significance of tumor cell
deposits and perineural infiltration are discussed.
Methods. A total of 245 specimens with CRC were analysed prospectively
via AC, half of which were rectal carcinomas. In consecutive cases (10–30
tissue samples each) the following parameters were evaluated: a. AC effect
on lymph node size and distribution of metastases in comparison
to preceding manual dissection on the same sample; b. The incidence of
metastatic spread to lymph nodes and as a tumor deposit or perineural
invasion in relation to the tumor location (distal, proximal and at the
level of the tumor).
Results. In the 245 tested specimens with colorectal cancer, a mean of
30 lymph nodes were assessed. There was no significant difference observed
between neoadjuvantly treated or untreated rectal carcinomas. The
main effect of AC was seen in lymph node size and metastatic status: In
the 10 manually dissected samples, the number of lymph nodes which
were

SO-005
Prevalence of mutations in signaling pathway components
downstream of the epidermal growth-factor receptor (EGFR) in
colorectal cancer
J . Neumann 1 , L . Wehweck 1 , S . Maatz 1 , F . Mourched 1 , A . Hendrowarsito 1 ,
J . Engel 2 , T . Kirchner 1 , A . Jung 1
1 Ludwig-Maximilians-Universität München, Department of Pathology, München,
2 Ludwig-Maximilians-Universität München, Institut für medizinische
Informationsverarbeitung, Biometrie und Epidemiologie, München
Aims. In metastatic colorectal cancer (mCRC) KRAS-mutations are associated
with clinical resistance to treatment with monoclonal antibodies
targeting the epidermal growth factor receptor (EGFR). However,
up to 50% of these patients do not respond to this therapy. To identify
novel biomarkers, downstream effectors of the EGFR-pathway are currently
validated according their predictive potential. Aim of this study
was to determine the frequency and overlap of key-mutations in the
EGFR-pathway. Furthermore, the concordance between primary tumor
and distant metastasis was analyzed.
Methods. Key mutations of KRAS, BRAF, AKT and PIK3CA were analyzed
by pyrosequencing in a collection of 63 mCRC patients in primary
tumor samples and corresponding metastases, respectively. Furthermore
PTEN- and EGFR-Immunohistochemistry were applied.
Results. Nine of the investigated 63 tumors (14.3%) had mutations in more
than one gene of the EGFR-pathway. KRAS- and BRAF-mutations were
detected in 50.8% and 7.9%, respectively, and were mutually exclusive.
Mutations in the PIK3CA-gene (15.9%) showed huge overlap with KRASmutations
(8 of 10 cases). Only one case with a mutation in the AKT-gene
(1.6%) could be detected showing a simultaneous BRAF-mutation. Mutation
analysis for KRAS, BRAF and AKT revealed a 100% concordance,
whereas PIK3CA exhibited one discordant case showing a mutation in
the primary tumor which could not be detected in the matched distant
metastasis (Κ=0.9). Immunohistochemistry revealed in 49.2% high levels
of EGFR-expression and in 42.9% loss of PTEN-expression, both showing
huge overlap with KRAS-, BRAF-, AKT and exon 9 PIK3CA-mutations
but not with exon 20 PIK3CA-mutations. Furthermore, high rates
of discordant cases were found.
Conclusions. We could demonstrate that KRAS- and PIK3CA-mutations
as well as BRAF- and AKT-mutations can co-occur within a single
tumor. However, the predictive value of these individual tumor profiles
for anti-EGFR targeted therapies has to be validated in further clinical
studies. Additionally, our data indicate that for molecular analysis of
KRAS, BRAF and AKT either the primary tumor or the distant metastasis
is suitable. Due to the lower concordance rates of PIK3CA-mutations
the primary tumor site should be selected. Analysis of PTEN and EGFR
protein expression are afflicted with a huge amount of discordant cases.
Therefore, before these markers can be used in pathological routine diagnostics,
standardized protocols are needed.
SO-006
Synergistic cytotoxicity of recombinant HMGB1 and pro-apoptotic
drugs in colon carcinomas
G . Gdynia1 , C . Zhang1 , M . Keith1 , J . Kopitz2 , P . Schirmacher2 , W . Roth1 1Institute of Pathology, University Hospital Heidelberg, and German Cancer
Research Center, Heidelberg, 2Institute of Pathology, University Hospital
Heidelberg, Heidelberg
Aims. Colon carcinoma cells are highly resistant to chemotherapeutic
drugs. We recently described a new form of necrosis-like cell death in
cancer cells induced by the HMGB1 protein. The aim of this study was to
investigate whether the co-activation of cell death pathways by apoptosis
inducers and HMGB1 can overcome the intrinsic resistance of colon carcinoma
cells to pro-apoptotic therapeutics.
Methods. ATP luciferase assay, LDH-linked lactate accumulation measurement,
OXPHOS flux, FACS, western blot, immunoprecipitation,
electron microscopy, subcellular fractionation, liposome transfection,
generation of stably Flag-/Myc-tagged-HMGB1 and stably Bcl-2 overexpressing
cells, crystal violet cytotoxicity assay, oxygen consumption
measurements.
Results. Colon carcinoma cell lines were differentially sensitive to recombinant
HMGB1. HMGB1 treatment resulted in the formation of giant
mitochondria and a steady decline of ATP. Overexpressed HMGB1
specifically bound to cytochrome c oxidase (COX IV) thus impairing
mitochondrial respiration measured by a marked decrease of mitochondrial
oxygen consumption. Colon carcinoma cells with depleted
mitochondrial DNA (rho zero cells) were less susceptible to the cytotoxic
effects of HMGB1. Combined treatment of colon cancer cells with
subtoxic concentrations of HMGB1 and the death ligand TRAIL or the
Bcl-2 inhibitor ABT-737 resulted in a strongly synergistic induction of
cytotoxicity. The cell death was accompanied by an early activation of
caspase-3 and a continuous decline of ATP. However, no cytochrome c
release was measured in the co-treated cells, and the overexpression of
the mitochondrial outer membrane associated anti-apoptotic Bcl-2 protein
only moderately inhibited the HMGB1-mediated cytotoxicity.
Conclusions. HMGB1 inhibits oxidative respiration of colon carcinoma
cells thus depleting intracellular ATP levels. The administration of
HMGB1 and pro-apoptotic compounds greatly increases their cytotoxic
effects on colon carcinoma cells by activating both the necrotic and
apoptotic cell death machinery.
SO-007
Opposite effects of tissue inhibitor of metalloproteinases- 1
(TIMP-1) overexpression and knockdown on colorectal liver
metastases
O .R . Bandapalli1 , E . Paul1 , P . Schirmacher1 , K . Brand1 1Institute of Pathology, Heidelberg
Aims. Tissue inhibitors of metalloproteinases (TIMPs) and the corresponding
metalloproteinases are integral parts of the protease network
and have been shown to be involved in cancer development and metastasis.
Paradoxically, for TIMP-1, tumor promoting as well as tumor inhibitory
effects have been observed.
Methods. To address this paradox, we utilized the BALB/c/CT26 mouse
model that reliably leads to liver metastasis after splenic tumor cell
injection and variegated the type of target cells for therapeutic intervention
and the modalities of gene transfer. Since we have observed before
that overexpression of TIMP-1 in liver host cells leads to efficient tumor
growth inhibition in this model, we now examined whether targeting
the tumor cells themselves will have a similar effect.
Results. In concordance with the earlier results, TIMP-1 overexpression
in tumor cells led to a dramatic reduction of tumor growth as well. To
evaluate any influence of treatment modality, we further examined
whether TIMP-1 knockdown in the same animal model would have the
opposite effect on tumor growth than TIMP-1 overexpression. Indeed,
TIMP-1 knockdown led to a marked increase in tumor burden.
Conclusions. These data indicate that in the BALB/c/CT26 model, the
modification of TIMP-1 has concordant effects irrespective of the type
of target cell or the technique of modulation of TIMP-1 activity, and that
TIMP-1 is unequivocally tumor inhibitory in this model.
Der Pathologe · Supplement 1 · 2012 |
61

Abstracts
SO-008
KRAS mutational status is a prognostic biomarker in pancreatic
ductal adenocarcinoma that is not influenced by p53 protein
expression
B .V . Sinn 1 , J .K . Striefler 2 , A . Lehmann 1 , M .A . Rudl 1 , M . Sinn 2 , A . Stenzinger 3 ,
M . Bahra 4 , W . Weichert 3 , H . Riess 2 , M . Dietel 1 , C . Denkert 1
1 Charité Universitätsmedizin Berlin, Institut für Pathologie, Berlin, 2 Charité
Universitätsmedizin Berlin, Medizinische Klinik mit Schwerpunkt Hämatologie,
Onkologie und Tumorimmunologie, Berlin, 3 Ruprecht-Karls-Universität
Heidelberg, Institut für Pathologie, Heidelberg, 4 Charité Universitätsmedizin
Berlin, Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Berlin
Aims. Mutations in the KRAS and p53 genes belong to the most frequently
observed genetic alterations in pancreatic ductal adenocarcinoma
(PDAC). Whereas p53 protein expression is of no prognostic value in
most studies, the prognostic role of KRAS mutational status remains
controversial. The aim of this study was to examine the frequency and
prognostic impact of KRAS mutations in patients with PDAC (study
group; n=153). In addition, we attempted to define molecular subgroups
with distinct biological behaviour by combined analysis of KRAS sequencing
data with p53 protein expression data.
Methods. DNA was extracted from formalin-fixed and paraffin-embedded
tissue cores using a fully automated extraction method. Codons 12
and 13 of the KRAS gene were sequenced using sanger sequencing technology
(n=153). P53 immunostaining was performed on tissue microarrays
from the same paraffin blocks. The impact of KRAS mutational
status and nuclear p53 expression on patient outcome was evaluated.
Results. KRAS mutations in codon 12 or 13 were found in 68% of cases.
Nuclear positivity for p53 in at least 60% of tumor cells was observed in
47% of cases. We found no statistically significant association between
KRAS mutational status and nuclear p53 positivity. KRAS mutational
status, but not p53 expression, was an independent prognostic marker in
the study group (p=0.011). Subgrouping of patients in four groups according
to KRAS status and p53 expression failed to define subgroups with
distinct biological behaviour and could not stratify patients beyond the
impact of KRAS mutational status.
Conclusions. In line with in vitro and in vivo data, we could demonstrate
that the KRAS mutational status plays a crucial role in pancreatic cancer
biology. KRAS may serve as a prognostic marker and potentially as
a predictive marker for targeted therapies. P53 could not contribute to
stratification of patients according to survival.
SO-009
Collagen type V affects the tumour-stroma interaction in pancreatic
cancer
S . Berchtold1 , I . Esposito1 1Technische Universität München, Institute of Pathology, München
Aims. Pancreatic cancer (PDAC) is characterized by a dense stroma sustaining
the cancer cells. The aim of this study is to understand the role
that collagen V (Col V) plays in the interaction between stromal and
epithelial cells during the progression of PDAC.
Methods. The expression of Col V was analysed in human PDAC, precursor
lesions and PDAC cells by immunohistochemistry and immunoblotting,
respectively. To study the influence of Col V on PDAC cells,
in vitro assays (adhesion, migration, invasion, chemoresistance) were
performed. Col V-dependent signalling pathways were investigated by
immunoblotting and immunofluorescence. The impact of Col V on angiogenesis
was verified by tube formation assay in Col V knocked-down
HUVEC cells.
Results. A progressively increasing stromal expression of Col V could be
shown during cancer progression. Moreover, Col V significantly affected
adhesion, migration, invasion and promoted chemoresistance of PDAC
cells. Tube formation was impaired in Col V-deficient cells; however, no
62 | Der Pathologe · Supplement 1 · 2012
significant correlation between Col V expression and neoangiogenesis
was found.
Conclusions. The malignant phenotype of pancreatic cancer cells is enhanced
by stromal Col V, potentially through activation of the integrin
signalling pathway.
Aktuelle Entwicklungen in der Forschung III –
Translationale Forschung
SO-010
The expression CDK4 and MDM2 in lipomas may point to a progression
to GI liposarcomas
M . Haab1 , M . Buck1 , L . Flossbach1 , S . Brüderlein1 , A . von Baer2 , M . Schult heiss2 ,
R . Mayer-Steinacker3 , M . Wittau4 , P . Möller1 , T .F . Barth1 1 2 Ulm University, Institute of Pathology, Ulm, Ulm University, University
Hospital, Department for Orthopaedic Trauma, Hand- and Reconstructive
Surgery, Ulm, 3Ulm University, University Hospital, Internal Medicine III, Ulm,
4Ulm University, University Hospital, Department of General, Visceral and
Transplantation Surgery, Ulm
Aims. Lipomatous tumors have the potential to progress from benign
lesions to liposarcomas. Our goal was to specify changes during progression
to distinguish progressing neoplasms from entirely benign lesions.
Methods. We analyzed 31 lipomas of different regions including subcutaneous
and deeply localized lesions, 42 liposarcomas GI and 8 hibernomas
by morphology, immunohistochemistry with antibodies for
MDM2, CDK4 and FISH with probes for the MDM2- and CDK4-region.
Included were one lipoma with a recurrence, two ipomas that progressed
to a GI liposarcoma after 8 years and 6 years respectively as well as one
retroperitoneal lipomatous tumor with a lipoma and GI and GIII liposarcoma
components. Furthermore, we included two own liposarcoma
cell lines (LISA1 and LISA2) derived from dedifferentiated liposarcomas.
Results. Of 31 lipomas 13 were CDK4+ and 8 were MDM2+ in various
combinations. 19/19 showed no CDK4 aberration while two were amplified
for MDM2. In GI liposarcomas 32/42 were CDK4+ and 31/42 were
MDM2+. 9/13 GI sarcomas were amplified for CDK4 and 16/20 showed
an amplification of MDM2. One/8 hibernoma each expressed MDM2
and CDK4 while no genetic aberrations of CDK4 or MDM2 genomic
regions were detected. One lipoma with progression to GI liposarcoma
showed a neoexpression of CDK4 and acquired an amplification of
CDK4 and MDM2 in the GI sarcoma; in the other one an additional amplification
of MDM2 was found. In one retroperitoneal lipomatous tumor
we detected a neoexpression of CDK4/MDM2 in the sarcoma with
an increase in copy numbers for CDK4 and MDM2. The cell lines LISA1
and LISA2 showed a heterogeneous expression of CDK4 and MDM2.
Conclusions. The different patterns of CDK4 and MDM2 expression and
gene amplifications in lipomas point to a progression to GI liposarcoma
in morphologically unsuspicious tumors. Since hibernomas are generally
negative for these markers they have different biology with no progression.
LISA1 and LISA2 are in vitro models to functionally test the
impact of CDK4 and MDM2 for the malignant phenotype. Immunohistochemistry
and FISH-analysis for CDK4 and MDM2 may be crucial in
lipomas for risk estimation.

SO-011
Methylation profiling and integrated genomic and transcriptional
analysis reveal new tumor suppressor genes of human
hepatocarcinogenesis
O . Neumann 1 , M . Kesselmeier 2 , B . Radlwimmer 3 , P . Schemmer 4 , P . Lichter 3 ,
P . Schirmacher 1 , J . Lorenzo Bermejo 2 , T . Longerich 1
1 University Hospital Heidelberg/Institute of Pathology, Heidelberg, 2 Institute
of Medical Biometry and Informatics, University Heidelberg, Heidelberg,
3 Division of Molecular Genetics, German Cancer Research Centre,
Heidelberg, 4 Department of General, Visceral and Transplantation Surgery,
University Hospital Heidelberg, Heidelberg
Aims. We aimed at the identification of new tumor suppressor gene candidates
of human hepatocarcinogenesis by vertical integration of genome-wide
array-based CGH, methylation, and expression data from a
cohort of well-characterized human hepatocellular carcinomas (HCC).
Methods. Bisulfite converted DNA from 64 HCCs and 10 normal control
livers was analyzed for the methylation status of about 14,000 genes
using the Illumina Infinium 27k Methylation array. After determining
the differentially methylated genes between HCC and normal liver, we
integrated their genomic alterations as previously determined by array-CGH-data.
The gene set that contained the genes with both hypermethylation
and regional genomic losses was than correlated with gene
expression data to select genes potentially silenced by promoter hypermethylation.
Aberrant methylation of selected candidates was verified
by pyrosequencing and methylation-dependent gene silencing was validated
after treatment of suitable HCC cell lines with 5’-Azacytidine.
Results. Methylation profiling revealed 2239 CpG-sites differentially
methylated between normal control liver and HCCs. 540 CpG-sites of
these were hypermethylated, whereas 1699 showed promoter hypomethylation.
The hypermethylated group was enriched for genes known to
be inactivated by the polycomb repressor complex 2 (PRC2), while the
group of hypomethyated genes was enriched for imprinted genes showing
loss of imprinting in the HCC samples. We identified 18 candidate
genes matching both criteria hypermethylation and regional genomic
loss. After integrating the expression data three candidates finally remained.
Functional characterization of one of these promising candidates
after re-expression in vitro was performed and the data will be presented
during the meeting.
Conclusions. Our data shows that vertical integration of methylation data
with high resolution genomic and transcriptomic data is suitable for the
identification of new tumors suppressor genes in human HCCs.
SO-012
AKT and N-Ras co-activation in the mouse liver promotes rapid
development of hepatocellular carcinomas that are sensitive to
Rapamycin treatment
D .F . Calvisi1 , C . Ho2 , C . Wang2 , S . Mattu1 , G . Destefanis1 , S . Delogu1 ,
J . Armbruster1 , X . Chen2 , F . Dombrowski1 , M . Evert1 1University Medicine Greifswald, Institute for Pathology, Greifswald,
2University of San Francisco, Liver Center, San Francisco, United States
Aims. Activation of AKT and Ras pathways is often implicated in carcinogenesis.
As yet unknown, the aim of this study is to unravel the
mechanisms, underlying the oncogenic cooperation between these two
cascades in relationship to hepatocellular carcinoma (HCC).
Methods. Therefore, we generated a mouse model characterized by combined
overexpression of activated forms of AKT and N-Ras protooncogenes
in the liver via hydrodynamic transfection. Hepatocarcinogenesis
and the anti-tumorigenic effect of Rapamycin treatment was investigated
by morphological and molecular methods and these findings were
verified in vitro, using HCC cell lines.
Results. Co-expression of AKT and N-Ras resulted in a dramatic acceleration
of liver tumor development when compared with mice overexpressing
AKT alone, whereas N-Ras alone did not lead to tumor for-
mation. Accelerated hepatocarcinogenesis driven by AKT and N-Ras
resulted from a strong activation of mammalian target of rapamycin
complex 1 (mTORC1). Furthermore, elevated expression of FOXM1/
SKP2 and c-Myc also contributed to rapid tumor growth in AKT/Ras
mice, yet via mTORC1-independent mechanisms. Of note, treatment of
AKT/Ras mice with the mTORC1 inhibitor Rapamycin was able both to
strongly constrain the growth of AKT/Ras preneoplastic lesions and to
impede malignant transformation. However, liver tumors rapidly emerged
in AKT/Ras mice following Rapamycin withdrawal. This was associated
with induction of the MAPK/ERK cascade. The biological effects
of co-activation of AKT and N-Ras can be recapitulated in vitro using
HCC cell lines which supported the functional significance of mTORC1,
FOXM1/SKP2 and c-Myc signaling cascades in mediating AKT- and N-
Ras-induced liver tumor development.
Conclusions. Thus, our data demonstrate the in vivo crosstalk between
the AKT and Ras pathways in promoting liver tumor development, and
the pivotal role of mTORC1-dependent and independent pathways in
mediating AKT- and Ras-induced hepatocarcinogenesis.
SO-013
Contribution of keratin-18 in SH- and SH-induced HCC development
A .K . Mehta1 , K . Bettermann1 , E . Lederer1 , C . Diwoky2 , A . Thüringer1 ,
C . Stumptner1 , H . Mueller1 , T . Kolbe3 , T .M . Magin4 , C . Lackner1 , H . Denk1 ,
K . Zatloukal1 , J . Haybaeck1 1 2 Medical University of Graz, Graz, Austria, Graz University of Technology,
Austria, 3University of Veterinary Medicine Vienna, Biomodels Austria (Biat),
Vienna, Austria, 4University of Leipzig, Translational Centre for Regenerative
Medicine Leipzig, Leipzig
Aims. Steatohepatitis (SH) is a liver disease morphologically characterized
by steatosis, hepatocyte ballooning and occurrence of cytoplasmic
protein aggregates termed Mallory-Denk bodies (MDBs). SH affects about
20% of alcoholics and up to 50% of obese type II diabetics. MDBs
mainly consist of misfolded keratin (K) 8, 18 and in part p62 and ubiquitin.
Keratin aggregates are known to be essential for MDB formation
and thereby link keratins to SH which is a major precondition for the
development of liver cirrhosis and hepatocellular carcinoma (HCC). In
this study we aimed at elucidating the pathophysiological and molecular
mechanisms of loss of K18 on hepatocarcinogenesis in mice and its functional
implication in human NASH-induced liver cancer.
Methods. 17 month-old krt18-deficient (krt18-/-) mice (129P2/Ola background)
were investigated for the occurrence of SH- and SH-induced
liver tumors by radiology, histology, immunohistochemistry, gene expression
analysis and immunoblotting.
Results. Aged krt18-/- mice developed the entire morphological spectrum
of SH whereas aged wild-type mice displayed simple steatosis.
Aminotransaminase levels were also elevated in aged krt18-/- mice. Interestingly,
91% of male and 46% of 17 months-old krt18-/- female mice
developed liver tumors revealing morphological and genetic features of
HCC. Moreover, 75% of male and 36% of female age-matched krt18+/-
mice developed HCC.
Conclusions. Aged krt18-/- mice represent a new spontaneous SH- and
SH-driven HCC model. Alterations of hepatocellular K18 therefore seem
to determine the susceptibility towards SH and SH-induced HCC.
Der Pathologe · Supplement 1 · 2012 |
63

Abstracts
SO-014
Genome-wide mRNA expression analysis reveals massive
transcriptional deregulation of cell proliferation and mitosis at
multiple steps as a key factor for tumor progression in gastrointestinal
stromal tumors (GISTs)
F . Haller 1 , D .J . Zhang 2 , I .-M . Schaefer 3 , S . Cameron 4 , B .M . Ghadimi 4 ,
A . Agaimy 5 , S . Wiemann 6 , Ö . Sahin 2
1 Albert Ludwigs University, Institute of Pathology, Freiburg, 2 German
Cancer Research Center, Heidelberg, 3 Georg August University, Institute
of Pathology, Göttingen, 4 Georg August University, Göttingen, 5 Friedrich
Alexander University, Institute of Pathology, Erlangen, 6 German Cancer
Research Center
Aims. Gastrointestinal stromal tumors (GISTs) carry mutations in the
receptor tyrosine kinases KIT and PDGFRA, leading to ligand-independent
autophosphorylation with constitutive activation of downstream
intracellular signalling cascades and accelerated cell proliferation. Next
to genotype, anatomical localisation and mitotic counts are two clinicopathological
parameters with significant impact on clinical behavior
in GISTs. The aim of the current study was to determine the effect of
genotype, anatomical localisation and mitotic counts on global mRNA
expression.
Methods. Genome-wide mRNA expression analyses were performed
using Sentrix HumanWG-6 arrays (Illumina, San Diego, CA) in a series
of 20 GISTs with either KIT or PDGFRA mutation from different anatomical
localisations, and with low and high mitotic counts.
Results. Using two-dimensional principal component analysis, tumors
were clearly separated according to genotype and anatomical localisation,
with clustering of tumors from the stomach, duodenum, jejunum/
ileum and rectum, respectively. Moreover, tumors with high mitotic
counts were separated from tumors with low mitotic counts. Group-wise
comparison of gene expression levels revealed significant upregulation
of 269 genes in GISTs with high mitotic counts, compared to 88 genes
that were downregulated. Further functional enrichment analysis using
this signature revealed a significant enrichment of genes allocated to 38
GO terms associated with cell proliferation and mitosis.
Conclusions. Deregulation of several key players of cell cycle regulation at
different steps of the cell cycle contributes to increased cell proliferation
and tumor progression in GISTs, and determination of their expression
may improve prognostication of clinical behavior.
SO-015
SRC signalling is crucial in the growth of synovial sarcoma cells
S . Michels1 , E . Sievers1 , M . Trautmann1 , D . Kindler1 , S . Huss1 , M . Renner2 ,
R . Penzel2 , O . Larsson3 , A . Kawai4 , S . Tanaka5 , P . Schirmacher2 , G . Mechtersheimer2
, E . Wardelmann1 , R . Büttner1 , W . Hartmann1 1 2 University Hospital Cologne, Institute of Pathology, Köln, University
Hospital Heidelberg, Institute of Pathology, Heidelberg, 3The Karolinska
Institute, Department of Oncology & Pathology, Stockholm, Sweden, 4Natio nal Cancer Center Hospital, Division of Orthopaedic Surgery, Tokyo, Japan,
5Hokkaido University Graduate School of Medicine, Laboratory of Molecular
& Cellular Pathology, Sapporo, Japan
Aims. Synovial sarcoma is a malignant soft tissue tumor, which affects
mainly adolescents and young adults. It is molecularly characterized
by a reciprocal balanced t(X;18) translocation, resulting in a chimeric
transcriptional modifier. Several receptor tyrosine kinases including
the IGF-IR and the EGFR have been shown to be expressed in synovial
sarcomas, leading to an activation of common intracellular kinase signalling
cascades. The SRC tyrosine kinase is an important interaction
partner for different growth factor receptors and effectors of intracellular
kinase signalling pathways and has been shown to be of particular
importance in a variety of tumors. This study was performed to examine
64 | Der Pathologe · Supplement 1 · 2012
the functional relevance SRC in synovial sarcomas and to evaluate if it
might represent a target for innovative therapeutic approaches.
Methods. Immunohistochemical analyses of differentially phosphorylated
SRC and the SRC regulators CSK and PTP1B were performed in a set
of 30 synovial sarcomas. Functional aspects of SRC signals in synovial
sarcomas and dependence of SRC activation on the SS18/SSX fusion proteins
were subsequently analyzed in vitro.
Results. Activated p-(Tyr416)-SRC was detected in the majority of synovial
sarcomas; deregulation of CSK and PTP1B could be excluded to be
the reason for the activation of the kinase. In a T-Rex293-based in vitro
model, expression of the SS18/SSX fusion proteins was associated with
increased p-(Tyr416)-SRC levels, at least partially due to an induction of
the Insulin-like growth factor signalling pathway. Four human synovial
sarcoma cell lines treated with the SRC inhibitor dasatinib displayed a
significant and dose-dependent inhibition of cellular growth in MTT assays,
which was accompanied by decreased phosphorylation of the SRC
targets FAK, STAT3, IGF-IR and AKT. In flow cytometric analyses, the
growth effects exerted by the inhibitor were shown to be due to a reduction
of cellular proliferation combined with an increase of apoptosis.
Concurrent exposure of synovial sarcoma cells to dasatinib and conventional
chemotherapeutic agents resulted in positive interactions. Finally,
synovial sarcoma cell migration and invasion was found to be dependent
on signals transmitted by SRC.
Conclusions. In summary, our data show that the SRC kinase might represent
a promising therapeutic target in synovial sarcomas.
SO-016
Targeting endometrial stromal sarcoma: histone deacetylase and
PI3K/Akt/mTOR signaling
P . Quan1 , E . Lederer1 , I . halbwedl1 , H . Denk1 , K . Zatloukal1 , J . Haybaeck1 1Med . Uni . Graz/Department of Pathology, Graz, Austria
Aims. Endometrial stromal sarcoma (ESS), derived from mesenchymal
cells, is a rare gynecological malignancy with an unclear molecular pathogenesis
and thus few therapeutic options. Previously, histone deacetylase
(HDAC) 2 expression was shown to be upregulated in human ESS
specimens. The HDAC inhibitor SAHA reduced in vitro growth of the
ESS cell line ESS-1 through the inhibition of mTOR signaling. The PI3K/
Akt/mTOR signaling, central to translational regulation and vital to
the growth and survival of cancer cells is an important target in cancer
therapy. This study aims at investigating (1) if HDAC and the PI3K/Akt/
mTOR signaling are involved in ESS pathogenesis and (2) how altered
HDAC levels regulate PI3K/Akt/mTOR signaling and its downstream
translation regulators in ESS.
Methods. The expression levels of HDAC1 and 2 in ESS were examined by
using a tissue microarray. The mRNA and protein levels of HDAC1 and 2
were determined by Q-PCR and Western blotting in ESS cell lines (ESS-1
and MESSA) and the appropriate control endometrial stromal cell line
HESC. The role of HDAC in regulating the PI3K signaling was checked
in cells treated with SAHA.
Results. 1.) HDAC1 and 2 are overexpressed in ESS tissues, compared to
normal endometrium. 2.) Higher levels of HDAC1 and 2, increased cell
growth, upregulated Akt and mTOR activation were detected in ESS cell
lines, relative to HESC. 3.) SAHA inhibited cell growth of three cell lines.
However, HESC is less sensitive to SAHA with a higher IC50 value than
other cells. 4.) SAHA reduced phosphorylated 4EBP1 and BCL-2 protein
levels in all cell lines. 5.) SAHA dose-dependently inhibited activation of
Akt and p70S6k in ESS-1, but not in MESSA and HESC cells.
Conclusions. HDACs and the PI3K signaling are involved in the ESS
pathogenesis. Despite of different drug sensitivity and response rates
among all cell lines, SAHA reduced cell growth via the PI3K/Akt/mTOR
signaling and its downstream effectors 4EBP1 and p70S6K, indicating
an integration of HDACs with the PI3K signaling and translation regulation.
This connection offers a promise for a combination therapy, i.e.
SAHA with various inhibitors for the PI3K signaling, which might be

more effective than SAHA alone and possible for an individualized ESS
therapy.
SO-017
High-resolution 3D visualization and semi-automated characterization
of tumor angiogenesis
J . Ehling1 , B . Theek2 , F . Gremse2 , S . Baetke2 , R . Knüchel3 , F . Kiessling2 ,
T . Lammers2 1RWTH Aachen, Institute of Pathology & Institute of Biomedical Engineering,
Aachen, 2RWTH Aachen, Institute of Biomedical Engineering, Aachen,
3RWTH Aachen, Institute of Pathology, Aachen
Aims. The visualization and quantification of functional tumor blood
vessels is essential for assessing treatment responses to anti-angiogenic
therapies. In experiments using tumor xenografts, anti-angiogenic
effects are generally evaluated by immunohistochemistry (IHC). This
method has several limitations: for example, the 3D architecture and the
vessel functionality are not fully considered. To overcome these shortcomings,
we established a protocol based on ex vivo high-resolution microcomputed
tomography (µCT) for 3D visualization and characterization
of tumor angiogenesis.
Methods. Six different tumor models (A431, A549, Calu-6, DU145, MDA-
MB-231, MLS), differing in blood vessel density and maturation, were
analysed. After tumors reached a diameter of ~6 mm, mice were intracardially
perfused with the radiopaque contrast agent Microfil, which
polymerises intravascularly. Subsequently, the tumor was harvested and
scanned in a high-resolution µCT scanner (SkyScan, Belgium) with a
maximal spatial resolution of 3 µM. Tumor microvessels were visualized,
and vessel density, vessel size, number and order of branches, were analyzed
using a 3D rendering software (MeVisLab). Histological validation
was performed by CD31 and SMA staining.
Results. Vascular casting and high-resolution µCT analysis of the vasculature
in tumor models with large and highly mature vessels (CD31-positive
and SMA-positive), such as A549 or MLS, enabled the detection
of vessel branches up to the 7th order. Conversely, tumors primarily
containing small and immature vessels (CD31-positive and SMA-negative),
such as A431 or Calu-6, presented many randomly arranged small
vessels, without proper hierarchy and architecture. The rising order of
branches, the total number of branches and the distribution of branches
correlated very well with the SMA levels. In addition, a highly significant
correlation was observed between the vessel diameters measured by
high-resolution µCT and by histology.
Conclusions. An imaging protocol based on vascular casting and highresolution
µCT has been developed for the 3D visualization of the micromorphology
of tumor blood vessels. In addition, evidence is provided
showing that high-resolution 3D µCT imaging of vascular casts allows
for a semi-automated analysis of the micro-architecture of tumor vessels,
thereby making it an exquisite and highly useful tool for supplementing
IHC in translational focusing on tumor angiogenesis and antiangiogenic
treatment responses.
SO-018
Eyetracking experiments identify “fast and frugal” heuristics
during cancer grading
D . Bombari1 , B . Mora2 , S . Schaefer3 , F . Mast1 , H .-A . Lehr4 1 2 University of Berne, Cognitive Psychology, Bern, Switzerland, CHUV, Lausanne,
Institute of Pathology, Lausanne, Switzerland, 3Inselspital, Institute
of Pathology, Bern, Switzerland, 4CHUV, Lausanne, Institute of Pathology,
Lausanne, Switzerland
Aims. In prior studies on prostate carcinomas we found that during nuclear
grading, pathologists are heavily biased by the architectural growth
patter of the carcinomas (Fandel et al., J Pathol. 2008).
Methods. We asked 20 pathologists to assign nuclear grades to images
of high power fields of 40 prostate carcinomas projected on a computer
screen with an inbuilt eyetracking device. We wondered if pathologists
fixate on different nuclei when the same circular high power fields
(albeit turned and flipped to avoid recognition) were displayed before
background images of well-differentiated, tubule-rich or poorly differentiated,
solid carcinomas. Using Photoshop-based image analysis, we
analyzed nuclear size, chromasia, and roundness of those nuclei that the
pathologists fixated, and compared these morphometric data to a random
sample of nuclei contained within each high power field.
Results. (i) The selection of fixated nuclei largely followed the confirmation
bias induced by the tumor architecture. (ii) The selection of “matching”
nuclei explained only about one tenth of the manipulation of
nuclear grades induced by the tumor architecture, suggesting that it represents
nothing but an unconscious effort of our minds to vindicate the
gravitation of nuclear grades towards the tumor architecture. (iii) The
majority of pathologists based their subjective nuclear grade on a single
morphometric criterion (mostly nuclear size) and only few pathologists
considered more than one nuclear criterion during nuclear grading. This
observation has in the meantime been confirmed in a study on 44 pathologist
asked to grade nuclei in breast carcinomas.
Conclusions. (i) Counter the general expectation that pathologists rely in
their diagnosis solely (or mostly) on what they see through the microscope,
our experiments suggest that unconscious, poorly recognized biases
influence not only the interpretation of the histological image but also
the way we view the image (unwitting fixation on selected parts/aspects
of the image). (ii) While we eloquently teach medical students that nuclei
of aggressive tumors are enlarged, angulated, and hyper- as well as heterochromatic,
we personally ignore most of these different morphometric
criteria during our daily diagnostic routine and rather base nuclear
grades on a single criterion (mostly size). (iii) It may appear judicious to
recognize cognitive biases and heuristics as part of a consensuous error
culture in diagnostic anatomical pathology and in translational research
and to develop mechanisms to counteract/prevent ensuing blurs and/or
outright diagnostic errors.
This project was funded by a grant from the Fonds National Suisse (Proj .-N°
32000-120417) .
AG Gynäkopathologie und Mammapathologie I
SO-019
p16/Ki-67 dual-stained cytology using ThinPrep® liquid-based
cytology – sub-analyses of more than 9,000 PALMS participants
H . Griesser1 , F . Alameda2 , C . Bergeron3 , M . Labadie 4 , V . Maccalini5 , M . Sideri6 ,
R . Dachez7 , R . Ridder8 1 2 Center for Pathology and Cytodiagnostics, Koeln, Hospital del Mar, Barcelona,
Spain, 3Laboratoire Cerba, Cergy Pontoise, France, 4Laboratoire GRC,
Limonest, France, 5Ospedale Atri, Unita Gestionale Screening Regionale,
Atri, Italy, 6European Institute of Oncology, Milano, Italy, 7Institute Alfred
Fournier, Paris, France, 8mtm laboratories, Heidelberg
Aims. The PALMS trial (Primary ASC-US LSIL Marker Study) evaluated
the diagnostic performance of the novel p16/Ki-67 dual-stained cytology
testing in cervical cancer screening as well as in the triage of ASC-US or
LSIL Pap cytology results. The outcomes were compared to Pap cytology
and HPV testing in the screening setting, and to HPV testing in the triage
of ASC-US or LSIL. For both Pap cytology and dual-stained cytology
testing, liquid-based cytology and conventional cytology methods were
included in the PALMS trial.
Methods. We performed an analysis of the diagnostic performance of
dual-stained cytology, Pap cytology and HPV testing limited to the subcohort
of 9,231 women enrolled to the PALMS trial who had ThinPrep®
(Hologic) liquid-based cytology testing for both Pap and dual-stain.
Der Pathologe · Supplement 1 · 2012 |
65

Abstracts
Results. Test positivity rates for dual-stained cytology, Pap, and HPV
testing over all ages were 5.7%, 5.6%, and 11.2%, respectively. Sensitivity
of dual-stained cytology for CIN2+ (n=67 cases) was found at 95.6%
(95% CI 87.1–98.6%), significantly higher than Pap cytology (80.2%;
95% CI 68.5–88.3%). Specificity levels were identical for both methods
(95.0% for dual-stained cytology vs. 95.1% for Pap testing). In women
aged 30 and older, sensitivity (specificity) of dual-stained cytology for
CIN2+ was 91.2% (96.0%), compared to 77.9% (95.9%) for Pap testing and
96.1% (92.1%) for HPV testing. For the triage of abnormal Pap cytology
results, dual-stained cytology showed identical (ASC-US triage: 100%
for both tests) or similar (LSIL triage: 94.5 vs. 100%) sensitivity as HPV
testing for the detection of underlying CIN2+, at significantly higher specificity
rates.
Conclusions. p16/Ki-67 dual-stained cytology performed on ThinPrep®
liquid-based cytology may achieve a sensitivity level as high as 95% for
underlying CIN2+ in primary screening of women of all ages, combined
with a 95% specificity. Furthermore, dual-stained cytology was shown to
be a highly efficient tool for the triage of abnormal Pap cytology results
when performed as a reflex test out of ThinPrep liquid-based cytology
specimens.
SO-020
Frequency of BRAF-p.V600E mutation in serous ovarian borderline
tumors and its peritoneal implants
A .K . Höhn1 , U . Siebolts1 , J . Einenkel2 , L .-C . Horn1 1 2 University of Leipzig, Institute of Pathology, Leipzig, University of Leipzig,
Department of Obstetrics and Gynecology (Institute of Trier), Leipzig
Aims. Genes of the RAF family, which mediate cellular responses to
growth signals, encode kinases that are regulated by RAS and participate
in the RAS/RAF/MEK/ERK/MAP-kinase pathway. Activating mutations
in BRAF have been identified to play a major role in the pathogenesis
of low-grade serous ovarian carcinomas (LG-OCA) via serous borderline
tumors (s-BLT; Sieben et al. 2004, Mayr et al. 2006; Vang et al. 2009).
But, limited information exists about a possible clonal relation comparing
s-BLT and its peritoneal implants which we want to illuminate by
BRAF p.V600E analysis.
Methods. Thirteen cases of s-BLT with peritoneal implants (invasive and
non-invasive) were identified from our files with subsequent macro- or
microdissection followed by DNA extraction of the adequate tissue. To
reveal the activating mutation of BRAF p.V600E we performed pyrosequencing
of 48 samples with a sensitivity of at least 5% mutated alleles.
Molecular analysis was performed from the ovarian tumor as well as
within one to 6 peritoneal implants from different sites.
Results. Five s-BLT (38.5%) showed BRAF-p.V600E mutation within the
ovarian tumor. In three of those cases BRAF-p.V600E mutation was also
identified within the peritoneal implants suggesting a clonal origin in
terms of abdominal tumor spread.
Conclusions. The frequency of BRAF-p.V600E mutation in s-BLT is concordant
with the reported frequency within LG-OCA. In case of abdominal
spread, peritoneal implants represent clonal origin of the primary
tumor in about two thirds of the informative cases. Further studies, examining
additional members of the RAS/RAF/MEK/ERK/MAP-kinase
pathway and using laser-capture microdissection in cases of rare tumor
epithelium by atrophy are required.
66 | Der Pathologe · Supplement 1 · 2012
SO-021
The relevance of steroid hormone receptors (estrogene alpha and
beta, progesterone), luteinizing hormone and follicle-stimulation
hormone receptor as well as aromatase activity in granulosa cell
tumors (GCTs) of the ovary
M .C . Jarrin Franco1 , T . Kirchner1 , J . Engel2 , S . Lauf3 , A . Mayerhofer4 , D . Mayr1 1 2 University of Munich, Institute of Pathology, München, University of
Munich, Munich Tumor Registry, München, 3University of Munich, Institute
of Cell biology, München, 4University of Munich, Institute of Anatomy,
München
Aims. Granulosa cell tumors of the ovary (GCTs) are rare neoplasms
from sex-cord stromal cells with a general trend toward late relapse and
metastasis. In contrast to ovarian carcinomas, tumor stage is the only
prognostic factor. The pathophysiology of ovarian tumors and relationship
to hormonal control are not yet fully understood, but the ovary is
the principal source of estrogens and one of their target organs. Some
aromatase inhibitors are relatively well-tolerated oral drugs commonly
used in breast cancer treatment. The aim of this study was to investigate
the tumorigenesis of GCTs, regarding steroid hormone receptors, luteinizing
hormone and follicle-stimulation hormone receptor and aromatase
activity with correlation to clinical data and survival.
Methods. 43 cases of GC-tumors of 36 patients from the archive of the
Department of Pathology, LMU Munich were selected. Immunohistochemical
assays (IHC) and RT-PCR were performed by standard methods.
The IHC for ER alpha, ER beta and progesterone was evaluated
by using the IRS-score. For aromatase activity, FSH and LH a 4-tired
scoring system was developed. The results were correlated to each other
and to clinical data (e. m. TNM-stage, Ki-67 proliferation index, progression)
and survival.
Results. Positive results for IHC: ER alpha in 79.1%, ER beta in 86%, PR
in 97.7%, FSHR in 14%, LHR in 25.6% and aromatase in 51.2%. Positive results
for RT-PCR: ER alpha in 88.4%, ER beta in 100%, PR in 86%, FSHR
in 55.8%, LHR in 76.7% and aromatase in 74.4%. No statistical correlation
between IHC and RT-PCR could be demonstrated. A significant correlation
between T-stage and IHC for ER alpha (p=0.026), ER beta (p=0.01),
progesterone (p=0.042) and Ki67 (p=0.046) was observed. Only for ER
beta-IHC a high statistical significance (p=0.019) in correlation to progression
could be demonstrated. No statistical significance between Tstage
and RT-PCR results could be demonstrated in any case. Regarding
the progression and survival, the only statistical significance could be
demonstrated for RT-PCR of LHR (p=0.030).
Conclusions. At the present time tumor stage and in some studies Ki-67
proliferation are the only established prognostic factors for GCTs. A long
follow-up is the only way of validation the success of treatment. Regarding
our results, ER beta and RT-PCR of LHR could be new prognostic
signs and beyond that a basis for a new therapeutic strategy. Analyses of
a larger number of cases, as well as studies of cell culture are already in
progress.

SO-022
Specialized pathology review in patients with ovarian cancer:
highly recommended to assure adequate treatment.
Results from a prospective study
S . Kommoss 1 , J . Pfisterer 2 , A . Reuss 3 , A . du Bois 4 , J . Diebold 5 , S . Hautmann 6 , D .
Schmidt 7 , F . Kommoss 7 , for the AGO study group 8
1 University of British Columbia, Department of Pathology, Vancouver, Canada,
2 Klinikum Solingen, Dept of Gynecology, Solingen, 3 Philipps-Universität
Marburg, Koordinierungszentrum für klinische Studien, Marburg, 4 Kliniken
Essen Mitte (KEM), Dept Gynecology & Gyn .Oncology, 5 Luzerner Kantonsspital,
Institute of Pathology, Luzern, Switzerland, 6 Institute of Pathology,
Allgäu-Oberschwaben, Wangen i . A ., 7 Institute of Pathology, A 2 , 2 , Mannheim,
8 AGO study group, Wiesbaden
Aims. In view of retrospective findings on second opinion pathology in
ovarian cancer it seems certain, that a considerable number of ovarian
borderline tumors (BOTs) or metastatic non-ovarian primaries are being
erroneously diagnosed as ovarian carcinomas. If BOTs are misdiagnosed
as cancer, patients may not only suffer from non-beneficial morbidity
at unnecessary high cost but may have to cope with an incorrect diagnosis
of cancer for the rest of their lives. In cases of metastatic disease
mistaken for an ovarian primary, more adequate therapeutic modalities
may be withheld from some patients. Finally, clinical trials may be biased
through disregarding of histological inclusion criteria. We hypothesized
that 5% of all patients in clinical trials of ovarian carcinomas have lesions
other than epithelial ovarian cancer. This is the first such study with a
prospective approach.
Methods. Patients who were enrolled into a chemotherapy trial of ovarian
carcinoma were asked to consent to a translational subprotocol. Contributing
pathologists were asked to submit all original slides as well as
paraffin material. Specialized central pathology review of all cases was
performed by two experienced gynecopathologists. In cases of clinically
relevant diagnostic discrepancies, the contributing pathologist was contacted.
If a given discrepancy could not be resolved, a panel of experts
was available for clarification.
Results. 454 patients with an outside diagnosis of ovarian epithelial
cancer were recruited. In 6.8% (n=31), a major diagnostic discrepancy
of potential clinical relevance was found. Most frequently (n=15), serous
BOTs had been misdiagnosed as invasive cancer. Ovarian metastases
constituted the second most frequent misdiagnosis (n=12). As minor discrepancies,
a divergent histological typing of ovarian carcinomas was
found in 28.2% (n=128).
Conclusions. This study clearly shows that central pathology review by
experienced gynecopathologists is highly recommendable if overtreatment
with chemotherapy of patients with BOTs and inadequate treatment
of patients with ovarian metastases is to be avoided in the future.
Specialized pathology review should become standard procedure in study
protocols prior to randomization. In order to further optimize the
quality of care, a high throughput infrastructure for specialized pathology
review will have to be established. The authors propose a internetbased
ovarian cancer network, capable of providing specialized second
opinion pathology within 10 working days.
SO-023
Development of a consortial database with pathological and
clinical data for fresh frozen breast cancer specimen
P . Bronsert1 , E . Stickeler2 , S . Schmid1 , K . Aumann1 , F . Haller3 , C . Röcken4 ,
N . Arnold5 , C . Mundhenke5 , F . Fend6 , A . Stäbler6 , T . Fehm7 , U . Vogel6 ,
M . Werner1 , O . Opitz8 1Albert-Ludwigs-University Freiburg, Institute of Pathology, Freiburg,
2Albert-Ludwigs-University Freiburg, Department of Obstetrics and
Gynecology, Freiburg, 3Friedrich-Alexander-University Erlangen-Nürnberg,
Institute of Pathology, Erlangen, 4Christian-Albrechts-University, Institute of
Pathology, Kiel, 5Christian-Albrechts-University, Department of Gynecology
and Obstetrics, Kiel, 6Eberhard Karls University Tübingen, Institute of
Pathology, Tübingen, 7Eberhard Karls University Tübingen, Department of
Gynecology and Obstetrics, Tübingen, 8Albert-Ludwigs-University Freiburg,
Tumour Center Ludwig Heilmeyer, Freiburg
Aims. Over the past two decades biomedical research technology in
tumor banking has enabled significant advances in the molecular characterization
of cancers, especially in research projects. Essential for the
functioning of a high quality tumor bank is a standardized implementation
of clinical and pathological information of tumor tissue. Each specific
specimen cohort reflects certain quality characteristics of a tumor
bank database and has to be handled in a certain manner. By combining
three independent breast cancer biobanks with two different disciplines
per facility together, it has to be assured, that each attendant is working
with synchronized and harmonized standard operating procedures
(SOP) and datasets.
Methods. At all three attended facilities rules of internal procedure were
defined, a Shared Resources Advisory was established, SOPs were partially
new elaborated, harmonized and synchronized. Minimal obligate
and facultative breast-cancer-specific, datasets for pathological and clinical
diagnoses were established. A periodically updated, secured database
with an automated combination of all consortial data was developed. A
web page for public presentation and research access was designed. To
attest effective consortial operation, tissue micro arrays (TMA) of a well
specified, facility research related, patient cohort were established, sectioned
and send to each facility where immunohistochemical staining
was performed. The results will be published in a consortial publication.
Results. From 2001 to 2011, the consortial breast cancer tissue bank contains
3400 fresh frozen, prospectively collected and immunohistochemically
classified breast cancer samples from all three facilities. Nearly
half of the patients were diagnosed with a ductal carcinoma in situ. The
median age was 61 years. The common pT category was pT1c (n=1063),
the most frequently pN category was pN0 (1693) followed by pN2 (n=197),
pN1 (194) and pN3 (82). 1113 patients were ER and/or PR and/or HER2/
neu positive. After written request, access for researchers to the consortial
internet accessible breast cancer database can be granted.
Conclusions. A well planed clinicopathological, IT linked infrastructure
is the fundament of a consortial database and the basic principle for multicentric
translational research.
Der Pathologe · Supplement 1 · 2012 |
67

Abstracts
SO-024
Evaluation of tumor proliferation and hormone receptor status in
breast cancer. Comparison of quantitative real time PCR,
image analysis of IHC, and visual scoring
H .-P . Sinn 1 , M . Keller 1 , N . Waldburger 2 , A . Schneeweiss 3 , R . Wirtz 4
1 University of Heidelberg, Institute for Pathology, Heidelberg, 2 University
of Heidelberg, Dept . of Pathology, Heidelberg, 3 University of Heidelberg,
National Center for Tumor Diseases, Heidelberg, 4 St . Elisabeth Krankenhaus,
Dept . of Pathology, Köln
Aims. The exact determination of hormone receptors, HER2 and proliferation
is of outmost importance for clinical decision making in breast
cancer. According to the 12th St Gallen Guidelines systemic therapy recommendations
follow the subtypes classification originally defined by
genetic array testing. These molecular subtypes can be approximated by
clinicopathological parameters combining immunohistochemistry assessments
of ER, PR, HER2 and Ki67.
Methods. Matched fresh and fixed pretreatment biopsy samples were
available from 90 patients participating in neoadjuvant clinical trial. RTqPCR
data were available after extracting RNA using Qiagen kits. RNA
was isolated from fixed tissue samples by using coated magnetic particles.
Multiplex RT-qPCR was performed by TaqMan® based primer probe
sets for ESR1, PGR, Ki67, as well as CALM2 as reference gene. Single Step
RT-qPCR was performed by using Invitrogen reagents on a Stratagene
MX3005p. RNA results were then reported as 40-CT values, which correlate
proportionally to the mRNA expression level of the target gene. All
cases were also used for quantitative immunohistology of nuclear antigens
(ER, PR, Ki67) by automated image analysis on a virtual microscopy
system (Aperio Technologies, Vista, CA, USA).
Results. We observed a significant correlation of mRNA data from independent,
matched fresh and fixed biopsy samples by using RT-qPCR
(ER1 r=0,91; PR r=0,79, Ki67 r=0,72). When comparing automated image
analysis results with conventional histological evaluation of IHC markers,
there were only 3 cases misclassified for ER positivity or negativity,
and 4 cases misclassified for PR positivity or negativity. Correlation of
RT-qPCR data for ER and PR with quantitative immunohistology was
highly significant (p

SO-027
Ki-67 in mitotic score groups of the Nottingham Grading System
for breast cancer
C . Focke 1 , D . Gläser 1 , K . Finsterbusch 1 , T . Decker 1
1 Dietrich-Bonhoeffer-Klinikum Neubrandenburg, Department of Pathology,
Neubrandenburg
Aims. The 2011 St. Gallen Consensus suggested the addition of Ki-67 for
defining proliferation and thus the difference between luminal A and
B clinicopathological subtypes. Whereas a Ki-67 cut-off of 14% is cited
from literature it became obvious from the discussion that no standardized
methodology or cut-off definition for Ki-67 is available so far. To
estimate the capability of immunohistochemically quantified Ki-67 rates
to discriminate the Nottingham Grading System (NGS) mitotic score
groups and to determine respective cut-offs in breast cancer (BC).
Methods. We retrospectively analyzed routinely H&E stained slides of 50
invasive BC (9 G1, 23 G2, and 18 G3). Immunohistochemistry for Ki-67
was done prospectively according to an in house protocol, evaluated in a
national interlaboratory trial for quality assurance in immunohistochemistry.
To rate Ki-67, 100 tumor cells within an area of the “hot spot “
were evaluated by counting all stained nuclei regardless of intensity. We
used the mitotic activity index (MAI) per mm2 as gold standard for proliferation
measurement. By assessing the MAI ranges within the mitotic
score subgroups of the Nottingham Grading System (NGS) we determined
respective MAI cut-offs. Using these MAI cut-offs we adjusted the
observed Ki-67 rates.
Results. Whereas the cut-offs of MAI for NGS mitotic scores 1, 2, and 3
were 0–32, 33–70, and 71–582, respectively, the resulting ranges of Ki-67
were 7–30%, 13–54%, and 33–98%. The median Ki-67 rates for NGS scores
1–3 came out with 21 (SD±7.5), 41 (SD±11), and 59 (±18.8), respectively.
Conclusions. Whereas there is obviously a trend of higher Ki-67 rates in
NGS score groups with higher MAI, our data indicate that 1) it was not
possible to discriminate the NGS mitotic score groups by using Ki-67
rates, 2) the cited cut-off of

Abstracts
SO-031
Decentral gene expression analysis to predict outcome of ER+/
Her2− breast cancer – results of a proficiency testing program for
the EndoPredict assay
C . Denkert 1 , R . Kronenwett 2 , W . Schlake 3 , K . Bohmann 2 , R . Penzel 4 ,
K .E . Weber 2 , H . Höfler 5 , U . Lehmann 6 , P . Schirmacher 4 , K . Specht 5 , M . Rudas 7 ,
H . Kreipe 6 , P . Schraml 8 , G . Schlake 3 , Z . Bago-Horvath 7 , F . Tiecke 3 , Z . Varga 8 ,
H . Moch 8 , M . Schmidt 9 , J . Prinzler 1 , D . Kerjaschki 7 , B .V . Sinn 1 , B .M . Müller 1 ,
M . Filipits 10 , C . Petry 2 , M . Dietel 1
1 Charité University Hospital, Institute of Pathology, Berlin, 2 Sividon Dignostics
GmbH, Köln, 3 Institute of Pathology, Gelsenkirchen, 4 University of Heidelberg,
Institute of Pathology, Heidelberg, 5 Technical University Munich,
Institute of Pathology and Pathological Anatomy, München, 6 Medizinische
Hochschule Hannover, Institute of Pathology, Hannover, 7 Medical University
of Vienna, Institute of Pathology, Austria, 8 University Hospital Zurich,
Institute of Surgical Pathology, Zürich, Switzerland, 9 University of Mainz,
Department of Gynecology and Obstetrics, 10 Medical University of Vienna,
Institute of Cancer Research, Austria
Aims. Gene expression profiles provide important information about the
biology of breast tumors and can be used to develop predictive tests. However,
the implementation of quantitative RNA-based testing in routine
molecular pathology has not been accomplished, so far. The EndoPredict
assay has recently been described as a quantitative RT-PCR-based multigene
expression test to identify a subgroup of hormone-receptor positive
tumors that have an excellent prognosis with endocrine therapy only. To
transfer this test from bench to bedside it is essential to evaluate the testperformance
in a multicenter setting in different molecular pathology
laboratories. In this study, we have evaluated the EndoPredict assay in
seven different molecular pathology laboratories in Germany, Austria
and Switzerland.
Methods. A set of ten formalin-fixed paraffin-embedded tumors from
patients with breast cancer was tested in the different labs. The Endo-
Predict score (EP score) ranging from 0 to 15 was calculated and patients
were classified into low or high risk for the occurrence of distant recurrence
using a validated cut-off. The variance and accuracy of the Endo-
Predict assays were determined using predefined reference values.
Results. Extraction of a sufficient amount of RNA and generation of a
valid EP score was possible for all 70 study samples (100%). The EP scores
measured by the individual participants showed an excellent correlation
with the reference values, respectively, as reflected by Pearson correlation
coefficients ranging from 0.987 to 0.999. The Pearson correlation coefficient
of all values compared to the reference value was 0.994. All laboratories
determined EP scores for all samples differing not more than 1.0
score units from the pre-defined references. All samples were assigned
to the correct EP risk group, resulting in a sensitivity and specificity of
100%, a concordance of 100%, and a kappa of 1.0.
Conclusions. Taken together, the EndoPredict test could be successfully
implemented in all seven participating laboratories and is feasible for
reliable decentralized assessment of prognosis in luminal breast cancer.
70 | Der Pathologe · Supplement 1 · 2012
SO-032
Cyclin D1 gene amplification is rarely heterogeneous in breast
cancer
E . Burandt1 , M . Grünert1 , A . Lebeau1 , M . Choschzick1 , V . Müller2 , C . Bokemeyer3
, R . Simon1 , G . Sauter1 , F . Jänicke2 , W . Wilczak1 1University Medical Center Hamburg-Eppendorf, Department of Pathology,
Hamburg, 2University Medical Center Hamburg-Eppendorf, Department of
Gynecology, Hamburg, 3University Medical Center Hamburg-Eppendorf,
Department of Internal Medicine II, Oncology Center, Hamburg
Aims. Amplification of Cyclin D1 (CCND1) occurs in about 10–20% of
breast cancers and has been suggested to predict resistance to anti-hormonal
therapy. As the diagnostic accuracy of predictive biomarkers can
be substantially limited by regional expression differences within tumors,
heterogeneity of CCND1 amplification was assessed in this study.
To assess heterogeneity, a novel tissue microarray based analysis platform
was developed.
Methods. To comprehensively asses the three-dimensional molecular
composition of breast cancers, a “heterogeneity TMA” was constructed
containing 8 different tissue cylinders from as many different cancer
containing tumor blocks as possible (at least 4) from 147 primary breast
cancers. Additional tissue samples were taken from 1–4 corresponding
nodal metastases from 35 of these patients. CCND1 amplification was
assessed by dual labeling fluorescence in situ hybridization (FISH).
Results. The analysis revealed amplification in 28 of 133 (21.05%) patients
with informative FISH results. CCND1 amplification was significantly
associated with high tumor grade (p=0.042). The preference of ER positive
tumors (p=0.061) was not statistically significant. No association was
found between CCND1 amplification and tumor type (p=0.307), stage
(p=0.540) and PR expression (p=0.871). A discordant Cyclin D1 amplification
status was initially detected in 6 out of 28 (21.43%) amplified tumors
by TMA analysis. Re-testing on large sections confirmed CCND1
heterogeneity in only 3 of them. Discordant results of the other cases
were due to variable interpretation of the TMA cores within the borderline
range (CCND1/centromer 11 ratios between 1.7 and 2.3). Overall
CCND1 genetic heterogeneity was observed in 3 out of 133 informative
tumors (2.3%). No discrepancies were detected between 22 primary tumors
and their matched lymph node metastases.
Conclusions. The high degree of homogeneity seen for CCND1 amplification
suggests that this alteration represents an early event in breast
cancer development in a small subset of breast cancers. Thus, CCND1
status determined in a core biopsy is highly representative of the entire
tumor and appropriate for predicting treatment outcome if applicable.
SO-033
Prognostic relevance of AIB1 (NCoA3) amplification and overexpression
in breast cancer
E . Burandt1 , G . Jens1 , F . Holst1 , F . Jänicke2 , V . Müller2 , C . Bokemeyer3 , W . Wilczak1
, L . Terracciano 4 , R . Simon1 , G . Sauter1 , A . Lebeau1 1University Medical Center Hamburg-Eppendorf, Department of Pathology,
Hamburg, 2University Medical Center Hamburg-Eppendorf, Department of
Gynecology, Hamburg, 3University Medical Center Hamburg-Eppendorf,
Department of Internal Medicine II, Oncology Center, Hamburg, 4University of Basel, Department of Pathology, Basel, Switzerland
Aims. AIB1 is an estrogen receptor co-activator, know to be amplified in
a fraction of breast cancers. Aim of this study was to test the potential
clinical significance of AIB1 expression levels and its relationship with
ER alpha expression, tumor phenotype and prognosis.
Methods. To analyze AIB1 expression and amplification immunohistochemistry
and Fluorescence in situ hybridization (FISH) was performed
on a pre-existing breast cancer tissue microarray (TMA) containing tumor
samples of 2197 breast cancer patients.
Results. AIB1 expression was found in 60% of our tumors including 29%
weak, 7% moderate and 24% strong expressors. AIB1 expression was sig-

nificantly associated with advanced tumor stage (p=0.003), high BRE
grade (p

Abstracts
is known about the functionality of SFRP1. In this project we have analyzed
the functional consequences of a SFRP1 re-expression in human breast
cancer cell lines. We also conducted a systematic expression analysis
to determine possible links to other pathways after SFRP1 re-expression.
Methods. Using standardized methods SFRP1 overexpressing clones of
two human breast cancer cell lines, BT20 (basal-like) and SKBR3 (luminal-like),
were generated and analyzed in cell-culture based assays. The
ability of SFRP1 expressing BT20 clones to grow in nude mice will be
also tested. Using DNA array expression profiling, we searched for genes
activated or repressed after forced re-expression of SFRP1 in these two
tumour cell lines (“SFRP1 target genes”). Validation of differential gene
expression was performed by real-time PCR comparing stable SFRP1
and control clones.
Results. SFRP1 re-expression in stable clones of two breast cancer cell
lines (BT20 and SKBR3) was validated on mRNA and protein level. We
found a correlation between SFRP1 expression and the growth behaviour
of both breast cancer cell lines in functional assays. SFRP1 re-expression
resulted in a significant reduced proliferation (p

liver did not correlate with the mutation status. However, the number
of CD34, MPO, GlycophorinA and CD61 positive hematopoietic cells
varied considerably from case to case and the number of mutated cases
was rather small. No mutation could be found in the lung specimens of
each autopsy.
Conclusions. GATA1 exon 2 mutations are acquired mutations and may
occur already in fetal hematopoiesis as early as 13th week of gestation. We
could not demonstrate a phenotypic correlation with quantitative fetal
liver hematopoiesis.
SO-040
Fetal acute myeloid leukemia in Down syndrome causing intrauterine
fetal death
H . Löser1 , M . Engels1 , S . Huss1 , R . Büttner1 , A . Müller2 , J . Fries1 1 2 University of Cologne, Institute of Pathology, Köln, University of Bonn,
Institute for Pathology
Aims. Down syndrome (DS) is associated with a 10- to 20-fold higher
risk for developing acute leukemia compared to non-DS children. About
10% of DS newborns are diagnosed with a “transient myeloproliferative
disorder” (TMD) and up to 30% of these children sustain a progression
to an acute myeloid leukemia (AML) within the following 3 years.
Methods. We report of a fetal, intrauterine AML in trisomy 21. While
no further fetal malformation was recognised in the pervious prenatal
screening of the 42-years old woman the fetus died in utero in the 24th
week of gestation without any detectable clinical cause. After informed
consent was obtained by the mother an autopsy was performed at the
Institute of Pathology, University of Cologne.
Results. We saw a phenotypically female fetus with an almost gestational
age-appropriate development without any apparent anomalies. The
internal organs regular, the membranous part of the ventricular septum
already closed. Histologically we found extensive tissue and vascular infiltrations
with megakaryoblasts in all organs including thymus gland,
heart, lungs, liver, pancreas, spleen, adrenal glands, kidneys, intestine,
and organs of the lesser pelvis. Immunohistologically positive for CD61,
this finding was consistent with an AML FAB-subtype M7. The placenta
showed pronounced infiltrations of leukemic cells in the villous vessels,
the larger villi and in the umbilical vein. The literature describes mutations
of the exon 2 and 3 of the GATA1 gene, an erythroid transcription
factor particularly in children with AML and DS.
Conclusions. Until now, no data has been published about prenatal GA-
TA1-mutations, although this is there most likely point in time of occurrence
when AML develops in early childhood. In the present case, we are
in the process of analysing whether GATA-1 mutations can be detected
in the fetal tissue by sequencing. Considering that the heart was also affected
by the massive intra- and extravascular dissemination of AML, it
is very likely that this is the crucial reason for the intrauterine fetal demise.
In accordance with published literature this is the first report of fetal
AML in DS. It extends the spectrum of possible differential diagnosis of
intrauterine fetal death in DS.
SO-041
On the correlation of coronal clefts and chromosomal aberrations
E . Doberenz1 , U . Gembruch2 , R . Schumacher3 , A .M . Müller4 1 2 University Bonn Medical Center, Institute of Pathology, Bonn, University
Bonn Medical Center, Dept . of Prenatal Medicine and Obstetrics, Bonn, 3Uni versity Clinic Freiburg, Department of Pediatrics, Freiburg, 4University Bonn,
Department of Pediatric Pathology, Bonn
Aims. Coronal vertebral clefts, radiologically defined as vertical radiolucent
bands in the middle of the vertebral body, are to our observation
associated with chromosomal aberrations. Published studies concerning
this are missing.
Methods. Hence 443 aborted fetuses were studied radiologically concerning
the incidence of coronal clefts and their association with proven
chromosomal aberrations. Furthermore they were studies histologically
concerning remnants of the notochord which are still discussed as cause
of coronal clefts.
Results. In 44 cases coronal clefts were visualized radiologically. In 93.2%
of these fetuses chromosomal aberrations were proven. On the other
hand, of all fetuses with trisomy only 30% showed this diagnostic finding.
Histologically coronal clefts displayed a missing ossification at the
center of the vertebral body. Remnants of the notochord could be excluded.
Conclusions. Summing up, coronal clefts represent a retarded ossification
of vertebral bodies in fetal development, nearly solely found in fetus
with chromosomal aberrations and malformations. On the other hand,
the genetic diagnosis of chromosomal malformation, especially trisomy,
does not automatically implicate coronal clefts.
SO-042
Molecular-genetic classification of glomerulocystic kidney
disease
J .K . Lennerz1 , H . Liapis2 1 2 University Ulm, Institute of Pathology, Ulm, Washington University,
Department of Pathology and Immunology, St . Louis, United States
Aims. Glomerular cysts (GCs) are defined as Bowman space dilatation
>2–3× normal that occupy >5% of glomeruli. GCs occur in pediatric and
adult kidneys. GCs are associated with a plethora of genetic- and nongenetic
diseases and present significant diagnostic challenges. We aim to
develop a molecular-genetic classification scheme to facilitate diagnosis.
Methods. We reviewed our biopsy and nephrectomy material and the
medical literature of >100 years (20+230 cases). Additionally, we performed
in silico experiments mapping gene-protein networks (Ingenuity-
Systems; MetaCoreV4.5).
Results. We identified 5 categories: Type I represents GCK in polycystic
kidney disease (PKD). Type II represents molecularly-recognized
(UMOD/TCF2) and inherited subtypes of GCK (other than PKD).
Type III represents syndromic-GCK, associated with malformations/
syndromes. Type IV includes obstructive-GCK (±dysplasia). Type V encompasses
sporadic-GCK, including ischemic- and drug-induced types,
which lack an inheritance pattern, syndromic features or obstruction/
dysplasia. In this scheme, types I/II can be regarded primary-GCK whereas
III–V represent secondary-GCK and emerge (not necessarily from
constitutional mutations but) from a loss-of-function of ‘maintenance
kidney genes’ involved in injury repair.
Conclusions. The clinical relevance of the various associations forms the
basis for this molecular-genetic classification scheme that provides a
framework for a structured differential diagnosis, suggests screening for
probable mutations, and opens new avenues in understanding common
kidney injuries.
SO-043
RSV- and hMPV-infections in BALB/c mice
M . Neumann1 , J . Lüsebrink2 , V . Ditt2 , O . Schildgen2 , A .M . Müller3 1 2 University Bonn Medical Center, Institute of Pathology, Bonn, University
Bonn Medical Center, Institute of Virology, Bonn, 3University Bonn, Department
of Pediatric Pathology, Bonn
Aims. hRSV (human respiratory syncytial virus) and hMPV (human
metapneumovirus) cause respiratory infections, leading to death in
approximately 200,000 toddlers and old patients each year. Numerous
aspects of the pathomechanisms and resulting pathomorphologic changes
of infection are sparsely studied. Murine models differ concerning
methodical parameters as well as inoculation methods resulting in a
limited comparability of published studies. By using a mild inoculati-
Der Pathologe · Supplement 1 · 2012 |
73

Abstracts
on method avoiding cough reflex, we studied, whether an infectiously
sufficient viral load can be achieved, if it causes significant pulmonary
pathomorphological findings and if those findings differ for each virus
and can be correlated by age.
Methods. 43 mice (group I: 4 weeks of age; group II: 16 months of age)
were either mock infected, inoculated with hRSV, hMPV or half the
quantity of both viruses. Five days after inoculation the lungs were analysed
by RTq-PCR for containing viral loads and by light microscopy and
immunohistochemistry concerning pathomorphological findings.
Results. Only lungs of infected mice showed a significantly raised viral
load. In young hMPV-infected mice pathomorphological findings
(broadened septae, focal poor ventilation) were far more prominent than
in older animals. RSV-infection and co-infection caused increased severity
of pathomorphology in older animals, while only displaying focal
poor ventilation in young mice. By immunohistochemistry, a more
proximal hRSV-infestation of the bronchi was found for co-infections
than for solitary hRSV-infections. Old infected animals displayed virus
proteins within macrophages and an enhanced BALT-activation.
Conclusions. The raised viral loads affirm the effectivity of the inoculation
method under inhalative short time anaesthesia, suppressing cough
reflex without putting a strain on the animals concerning side-effects
of anesthesia, e.g. vomitus. In all, pathomorphological changes were
mild. Nevertheless, viral- and age-specific differences were found which
might be related to age-dependent immune responses. An explanation
for the more proximal bronchial distribution of hRSV during co-infection
could be a result of competing receptors for attachment (as they are
in large parts identical with hMPV) or a mutual inhibition during the
intracellular replication process.
SO-044
Genetic analysis of relapsed childhood germ cell tumors by CGH
L . Wulf1 , C . Vokuhl1 , D . Schneider2 , G . Calaminus3 , I . Leuschner1 1 2 University of Kiel, Department of Pediatric Pathology, Kiel, Municipal Clinics
Dortmund, Department of Pediatrics and Adolescent Medicin, 3Univer sity Hospital Münster, Department of Pediatric Oncology and Hematology
Aims. Germ cell tumours are rare heterogeneous malignancies in childhood.
Pure teratomas in childhood normally don’t show genetic changes
in terms of aberrations and they are associated with a relapse-free prognosis.
Higher grade of immaturity in teratomas increases probability of
york sac tumour presence which concurrently decreases prognosis for
relapse-free survival, especially if the tumour can not be completely excised.
It is meaningful whether the rare cases of relapsing teratomas are
genetic changes that are likely to predict recurrence of these tumours.
This assumption disposed us to use chromosomal genomic hybridisation
for primary tumours with relapse and comparing them to teratomas
without relapse.
Methods. Formalin-fixed, paraffin-embedded tissue blocks were retrieved
from the files of the German Pediatric Tumor Registry. Sufficient
DNA from 9 patients included in the Malignant Germ Cell Study Group
(MAKEI) could be extracted, among them 9 primary tumors and 8 relapses.
Tumor DNA was labeled with spectrum-green dUTPs, normal
reference DNA with spectrum-red dUTPs. After co-hybridisation on
normal male human metaphase spreads, CGH was analysed using ISIS
CGH software (Metasystems).
Results. All of the primaries were teratomas, beneath the relapses there
were 4 teratomas and 4 tumors with at least a microfocus of YST. All but
two of the primary teratomas had chromosomal aberrations detectable
by CGH. The average number of chromosome arm aberrations per tumor
was 1.9 (range: 0–5). Copy number changes were gain of chromosome
17, 7, 1q, 3 and 12p and loss of chromosome 13q, 5q and 5p. When
comparing the two groups (primary tumor and relapse) most of the
chromosomal imbalances detected in the primary tumor could also be
found in the relapse. Furthermore some of the tumor relapses had additional
changes (e.g. amplification of chromosome 8q).
74 | Der Pathologe · Supplement 1 · 2012
Conclusions. Pediatric germ cell tumors are a heterogeneous group with
generally good relapse-free prognosis. Regardless most of the children
are cured, some tumors relapse. In this study we wanted to search for
differences between primary and relapsed tumors to find possible markers
which could predict tumor relapse. In contrast to most teratomas
which generally show a normal karyotype, the teratomas with relapse in
our study showed chromosomal aberrations in 78% (7/9) of cases. Taken
together the detection of chromosomal aberrations in teratoms could be
a risk factor for tumor relapse. This assumption has to be evaluated on a
bigger cohort of patients.
SO-045
Genetic and immunhistochemical analysis of embryonal rhabdomyosarcoma
with good and poor prognosis
T . Heilmann1 , C . Vokuhl1 , T . Dantonello2 , E . Koscielniak2 , I . Leuschner1 1 2 University of Kiel, Department of Pediatric Pathology, Kiel, Olgaspital,
Klinikum Stuttgart, Pediatric 5
Aims. Embryonal rhabdomyosarcoma (ERMS) is the most common soft
tissue sarcoma in children, typically affecting children younger than
5 years of age. Contrary to alveolar rhabdomyosarcoma, ERMS don’t
show specific translocations or specific genetic changes. Prognosis depends
on the tumor size, localization and the age of the patient. Even if
the overall survival with approximately 70% is generally good, there are
some cases of ERMS with unfavourable prognosis. In our study we used
comparative genomic hybridization (CGH) and immunohistochemistry
(IHC) to analyse the tumours with good prognosis comparing to tumors
with unfavourable prognosis.
Methods. Formalin-fixed, paraffin-embedded tissue blocks were retrieved
from the files of the German Pediatric Tumor Registry. Sufficient
DNA from 28 patients for the CGH included in the Cooperative Weichteilsarkom
(Soft Tissue Sarcoma) Study Group (CWS) could be extracted.
For the CGH tumour DNA was labelled with spectrum-green
dUTPs, normal reference DNA with spectrum-red dUTPs. After co-hybridization
on normal male human metaphase spreads, CGH was analysed
using ISIS CGH software (Metasystems). Furthermore we analysed
the expression of the cell-cycle-proteins p16ink4, pRb and cyclin D1 as
well as p53 in 40 cases.
Results. The most common chromosome arm copy number changes
were loss of chromosome 4q (39%), 6q (32%) and 5q (29%). Frequent gains
were on chromosome 20q (54%), 22q (50%), 8p (46%), 8q (43%) and 11q
(39%). When comparing the two groups we found gain on chromosome
6p (2/14), 14q (4/14) and 18p (3/14) and loss on chromosome 3p (3/14) only
in cases with unfavourable prognosis. Only in the group of good outcome
we found gains on chromosome 1q (2/14), 2q (2/14) and 11p (2/14). The
results from the IHC showed abnormal expression of p53 5/40, Cyclin
D1 10/40, pRb 6/40 and p16 24/40. Comparing the two groups the 5 cases
with abnormal p53 expression were only in the group with poor prognosis.
Conclusions. Although embryonal rhabdomyosarcoma generally has a
good prognosis there are cases with less favourable prognosis. In this
study we were able to confirm the frequent genetic changes in embryonal
rhabdomyosarcoma. Furthermore we found some genetic changes
(3p, 6p, 14q and 18p) and abnormal expression of p53 only in the tumour
group with less favourable prognosis. To find better tools for risk stratification
in embryonal rhabdomyosarcomas, future studies should be
concentrated on possible genes within the chromosomal regions we have
found in our study.

AG Paidopathologie II
SO-046
Overexpression of co stimulatory ICAM1 enhances killing of RMS
cell lines by NKT and chimeric T cells
K . Simon-Keller 1 , A .-L . Bohlender 1 , K . Mößinger 1 , S . Küffer 1 , P . Ströbel 1 ,
A . Marx 1
1 University Medical Center Mannheim, Institute of Pathology, Mannheim
Aims. Rhabdomyosarcomas (RMS) are the most common soft tissue sarcoma
of childhood and adolescence. Recent efforts to enhance overall
survival of patients with clinically advanced RMS have failed. Different
types of immunotherapies have been suggested as new perspective.
However, little is known about immune escape mechanisms in RMS.
Killing of RMS cells with specific chimeric T (cTCs) and NKT cells was
markedly attenuated in comparison to killing of CEA-expressing colon
carcinoma cells by respective cTCs. Therefore, we wondered whether resistance
to killing might be due to lack of co-stimulatory molecules and
if so, whether it is possible to improve killing efficiency by overexpression
of defective co-receptors.
Methods. We compared four alveolar RMS cell lines and two embryonal
RMS cell lines with 16 embryonal and 12 alveolar RMS biopsies. Expression
status of different surface molecules was checked by FACS analysis,
Western blot, and qRT-PCR to characterize RMS cell lines. Biopsies were
analyzed by IHC, Western blot and qRT-PCR. Cell lines were transfected
with CD54 by electroporation and checked by FACS for CD54 surface expression.
Cell survival after co-cultivation of RMS and chimeric T cells
was checked by MTT test and FACS analysis.
Results. Studying expression levels of MHC class I, MHC class II, CD80,
CD86, ICAM-1/CD54 in RMS cell lines and biopsies we found low to
absent expression of crucial co-receptors, e.g. ICAM1 and CD86 both in
vitro and in vivo. To functionally prove the influence of ICAM-1 expression
on killing efficiency, we overexpressed ICAM1 in six RMS cell lines.
On co-incubation with cTC and NKT cells, all RMS cell lines showed
substantially increased susceptibility to cTC and NKT cell mediated killing
after overexpression of ICAM1.
Conclusions. The results imply that RMS cell lines lack expression of crucial
co-receptors for cytotoxic T cell responses. Up-regulation of ICAM
appears as promising strategy to enhance the cytotoxic effect of the immune
system against RMS cells, e.g. following RMS-directed vaccination
or adoptive transfer strategies.
SO-047
The role of homeobox genes in the development of nephroblastomas
K . Koller1 , M . Pichler2 , K . Koch1 , M . Zandl1 , I . Leuschner1 , G . Höfler1 , B . Gürtl1 1 2 Medical University of Graz, Institute of Pathology, Graz, Austria, Medical
University of Graz, Department of Internal Medicine, Graz, Austria
Aims. Different homeobox genes and some of their binding partners feature
already known tumorigenic properties, but their role in the pathogenesis
of nephroblastomas has hardly been investigated. In our study
we therefore focused on the expression of HOXA9 and its binding partners
in different subtypes of nephroblastomas.
Methods. The mRNA expression levels were investigated by quantitative
REALtime PCR, protein expression levels by immunohistochemistry.
Results. All nephroblastomas investigated so far had a significant upregulation
of MEIS1 mRNA expression levels. In parallel in most of these
cases a nuclear positivity with antibodies against MEIS1 was identified.
The majority of nephroblastomas investigated so far also showed an
overexpression of PBX2 mRNA, some of them additionally a distinct
positive nuclear staining in the immunohistochemical investigation. The
mRNA expression levels of HOXA9 were significantly higher in most of
the tumors investigated.
Conclusions. Our results show that the upregulation of homoebox genes
and their binding partners might play a role in the development of nephroblastomas.
SO-048
Gain of chromosome 8 and IGF1R in pleuropulmonary blastoma
C . Vokuhl1 , L . de Leon1 , S . Kirsch2 , E . Koscielniak2 , I . Leuschner1 1 2 University of Kiel, Department of Pediatric Pathology, Kiel, Olgaspital,
Klinikum Stuttgart, Pediatric 5
Aims. Pleuropulmonary blastoma (PPB) is a very rare, aggressive primary
intrathoracic tumor of early childhood. This tumor is composed
of a malignant mesenchymal component, namley a rhabdomyomatous,
chondroid or fibrosarcomatous, and an epithelial component regarded
as entrapped elements. Histopathologically, three different types are described:
Type 1 PPB is composed exclusively of cysts lined by a benign
epithelium. The cyst walls consist of small, malignant mesenchymal
cells, type 2 is composed of cystic and solid areas and type 3 is an entirely
solid tumor. The outcome is poor, with 5-years survival rates of 45-49%
and 66%, respectively. Because of the rarity of this tumor type, the genetics
are poorly understood.
Methods. In this study we want to analyse the PPB cases of the Kiel Pediatric
Tumor Registry. Sufficient DNA from 16 patients could be extracted.
We analysed these cases by comparative genomic hybridisation and
confirmed tumors with gain of chromosome 8 and of IGF1R by FISH.
Results. The most frequently recurring change was gain of chromosome
8 and the short arm of chromosome 8, respectively (9/17). Six pleuropulmonary
blastomas show gain of chromosome 20 (pq and p). Genomic
amplification was observed in 5 cases, four related to 15q25qter, and one
to 1p. We were able to perform Cen8 FISH in all but one of the cases with
chromosome 8 gain. Two tumors show trisomy of chromosome 8, 4 tumors
a polysomy (in average 4–5.8 signals per nucleus). The remaining
one showed normal signal pattern. FISH could confirm three low-level
amplifications and one high-level amplification of the IFG1R gene.
Conclusions. Pleuropulmonary blastoma is a very rare pediatric tumor,
therefore only case reports or small series of genetic studies were published.
In our series of 18 PPBs we could show a gain of chromosome 8 in
51% (9/17). In addition we found five amplifications, four of which are in
the 15q21qter region where the insulin-like growth factor type 1 (IGF1R)
gene (15q26) lies. All of these were pleuropulmonary blastomas type III,
indicating that it is a later event in tumor progression. In future we have
to correlate these results with prognosis and these findings suggest the
possibility of use of targeted agents in the therapy of a subset of these
rare tumors.
SO-049
Expression of cancer testis (CT) antigens in fetal thymus
A . Jungbluth1 , D . Frosina1 , M . Holz1 , G . Spagnoli 2 , S . Gnjatic1 , A .M . Müller3 1Ludwig Institute for Cancer Research, Memorial Sloan-Kettering Cancer
Center, New York, United States, 2University Hospital Basel, Department of
Biomedicine, Basel, Switzerland, 3University Bonn, Department of Pediatric
Pathology, Bonn
Aims. CT antigens such as NY-ESO-1, MAGE, GAGE, and CT7 are named
after their characteristic pattern of expression, since they are found
in various types of cancer and in normal adult tissues are restricted to
testicular germ cells. They are also present in fetal ovarian germ cells
and occasionally in placenta. In cancer patients, some CT antigens are
highly immunogenic and due to their limited expression in normal tissues,
are used as vaccine targets for the immunotherapy of cancer. They
also serve as markers of malignancy. Little is known about the biology
of CT antigens and especially their role in the normal immune system.
Der Pathologe · Supplement 1 · 2012 |
75

Abstracts
Consequently, here was analyzed the presence of CT antigens in a series
of fetal thymic tissues.
Methods. Archival thymus tissue from 40 cases (week 15–42) were available
for analysis. Immunohistochemistry employing the following mAbs
(to the following CT antigens) were used: MA454 (MAGE-A1), 57B
(MAGE-A4), E978 (NY-ESO-1), #26 (GAGE), CT7-33 (CT7), and CT10#5
(CT10).
Results. Expression of the tested CT antigens was highly variable. NY-
ESO-1 remained completely negative. MAGE-A1 was solely found in
single cells of three thymi. CT7 and CT10 were present in 10 and 11 thymi
respectively, both showing solely focal staining. GAGE and MAGE-A4
were most abundantly expressed: GAGE was present in 22/40 and MA-
GE-A4 in 27/40 tissues; both antigens displaying larger groups of positive
cells. For all tested antigens, immunopositive cells were restricted to the
medulla and were exclusively epithelial cells. There was not predilection
of any gestational age for any of the tested antigens.
Conclusions. The present study shows that -irrespective of fetal development/gestational
age- several CT antigens are consistently present in fetal
thymus, albeit to a variable extent. Expression is restricted to thymus
epithelial cells and ranges from a few cells to larger groups of cells; GAGE
and MAGE-A4 are most abundantly present. Interestingly, there was no
NY-ESO-1 expression in any of the tested thymi. These data complement
serological data in cancer patients which show rare immune responses
to those antigens, which were highly expressed in our series of thymus
tissues. NY-ESO-1, however, is the most immunogenic antigen in cancer
patients. The lack of NY-ESO-1 expression in fetal thymus could be the
cause of lacking pre-existing immunotolerance to NY-ESO-1 rendering
cancer patients more sensitive to the presence of NY-ESO-1 in cancer tissue
and/or vaccine applications.
SO-050
Hepatobiliary rhabdomyosarcoma in a 2-year-old: a case report
K . Wieczorek1 , G . Fitze2 , R . Knöfler3 , G . Hahn4 , G . Baretton1 1University Hospital “Carl Gustav Carus”, TU Dresden, Department of
Pathology, Dresden, 2University Hospital “Carl Gustav Carus”, TU Dresden,
Department of Pediatric Surgery, Dresden, 3University Hospital “Carl Gustav
Carus”, TU Dresden, Department of Pediatric Oncology, Dresden, 4Univer sity Hospital “Carl Gustav Carus”, TU Dresden, Department of Radiology,
Dresden
Aims. Although representing the most common sarcoma in the pediatric
patient, rhabdomyosarcomas of the liver account for only 0.8% of all
rhabdomyosarcomas, and 1% of all liver tumors. Unless they present in
the characteristic setting of a botryoid mass in the biliary tree, they are
difficult to diagnose, as they share many histomorphologic similarities
with undifferentiated embryonal sarcomas of the liver. This case report
summarizes clinical data and histomorphologic and immunohistochemical
features of a hepatobiliary rhabdomyosarcoma in a 2-year-old boy.
Methods. A 2-year-old boy presented to an external hospital with severe
jaundice and hepatomegaly. Initially, an infectious process was suspected,
and the boy was referred to the pediatrics department of the Dresden
university hospital. MR imaging revealed a very large liver tumor and
multiple metastases in the lung and bone. The gallbladder and the biliary
tree were initially not visible due to the size and partially cystic appearance
of the tumor. A biopsy of the liver was taken.
Results. Liver biopsy showed a malignant mesenchymal neoplasia consisting
of primitive round to spindled cells with multiple mitoses. A
zonal architecture was noticed around small bile ducts. Very few (

Results. Histomorphology of the ovary displayed nodules of luteinized
cells and multiple luteinized ovarian cysts within an edematous stroma,
typical histological findings of a pregnancy luteoma. Placental examination
revealed signs of insufficiency.
Conclusions. Pregnancy luteoma is a rare ovarian lesion which can macroscopically
be misinterpreted as malignancy. Awareness of this entity
can avoid unnecessary adnexectomy in young patients as pregnancy
luteomas regress spontaneously. Although hyperandrogenism can be
associated with IURG it is hardly probable that in the present case fetal
hypotrophy was due to pregnancy luteoma because the level of secreted
androgens was not high enough to cause manifest hyperandrogenism.
Instead IUGR in our case is more likely attributable to placental insufficiency
and/or beta thalassemia major.
SO-053
Infantile digital fibromatosis (IDF) – A case report and review of
the literature
U . Titze1 , R . Rödl2 , G . Köhler1 1University Hospital Münster, Gerhard-Domagk-Institute for Pathology,
Münster, 2University Hospital Münster, Department of General and Tumor
Orthopedics, Münster
Aims. We report on a 7 months old male infant who received excisionbiopsy
of a rapidly growing dermal tumor from the 2nd toe of the right
side.
Methods. Histological, immunohistological and ultrastructural examination
revealed typical findings of an infantile digital fibromatosis
(WHO: inclusion body fibromatosis).
Results. Infantile digital fibromatosis (IDF) is a rare, distinctive benign
fibroblastic/myofibroblastic tumor of infancy typically arising in the digits
of the hands or feet. Characteristic morphological findings in the
proliferating spindled cells are characteristic rounded eosinophilic paranuclear
inclusions.
Conclusions. Etiology of IDF remains uncertain. Despite of its benign
behavior, local recurrence was seen in up to 60% of cases after surgical
therapy. Local installations of corticosteroids do not reduce the size of
these lesions. Current management of IDF recommends avoiding surgical
intervention, as spontaneous involution is the rule.
SO-054
Male fetus with ectrodactyly ectodermal dysplasia clefting (EEC)
syndrome
F . Fronhoffs1 , S . Detering2 , S . Gerlach1 , C . Berg3 , M . Born4 , A .M . Müller5 1 2 University Bonn Medical Center, Institute of Pathology, Bonn, University
Bonn Medical Center, Institute of Pathology, Bonn, 3University Clinic of Cologne,
Department of Prenatal Medicine and Ultrasound, Köln, 4University Bonn Medical Center, Institute of Radiology, Bonn, 5University Bonn, Department
of Pediatric Pathology, Bonn
Aims. Characteristics of the ectrodactyly ectodermal dysplasia clefting
(EEC) syndrome, first described by Rüdiger et al in 1970, are ectrodactyly,
dysplasia of skin, its adnexal structures and/or teeth as well as orofacial
clefts. Its exact prevalence is unknown. Until now, about 300 cases
have been reported in literature. In more than 90% of all cases, a missense
mutation in the gene TP63 can be detected.
Methods. 33-year-old mother, gravida 1, para 1. Proof of bilateral complete
cleft of lip and palate and ectrodactyly of both feet and hands and
tentative sonographic diagnosis of complex cloacal persistence and malformation.
Confirmation of EEC-syndrome by genetic testing. Feticide
in 24th+4 week of gestation.
Results. Male fetus, appropriate for gestational age, with bilateral complete
cleft of lip and palate accompanied by deformation of the nasal apex.
Ectrodactyly of both feet and hands. Right hand with five metacarpals (I,
III–V regular, II shortened) and agenesis of phalanges II and III. At the
left hand only a rudimentary anlage of digitus II but regularly formed
digitus I and III–V. Right foot with five metacarpals but shortened metacarpal
II. Left foot with five regularly shaped metacarpal bones, but only
four phalanges (I and III–V), i.e. missing second toe. Time-adequate development
of the nails. Histologically, in the skin biopsy only very few
perspiratory and sebaceous glands. Very scarce scalp hair. Additionally,
a megacystis with pseudodiverticulum and dilatated and sidled ureters.
Conclusions. This fetus presented classic findings of the EEC syndrome.
Because of the additional urogenital anomalies the diagnosis was expanded
to ectrodactyly ectodermal dysplasia syndrome with urinary tract
pathology (EECUT).
SO-055
Femur fibula ulna (FFU) complex
A .M . Müller1 , S . Detering2 , U . Gembruch3 1 2 University Bonn, Department of Pediatric Pathology, Bonn, University
Bonn Medical Center, Institute of Pathology, Bonn, 3University Bonn Medical
Center, Dept . of Prenatal Medicine and Obstetrics, Bonn
Aims. Femur fibula ulna (FFU) complex is a sporadic, non-lethal malformation
characterized by typical unilateral combination of defects of the
femur and fibula and contralateral defect of the ulna.
Methods. We present a fetus of 23 weeks of gestation of consanguine parents.
Results. Macroscopically the fetus showed a short collar, hypertelorism,
slightly down sloping palpebral fissures, short, flat nose, small lips and
high arched palate and dysmorphic ear concha. In comparison to the
right side, left sided upper and lower leg were significantly shortened, the
foot appeared as clubfoot. Furthermore, in comparison to the left side,
the right forearm was shortened, the right hand displaying four fingers
and an aplasia of the thumb.
Conclusions. Etiology of the sporadically occurring FFU complex is unknown.
Up to now there are no signs a paternal age effect or an association
with consanguinity. Neither could chromosomal studies reveal any
abnormalities. Furthermore there is no evidence for an infectious or teratogenic
cause. Children show normal mental development and normal
life expectancy. As – dependent on the number of involved malformed
limbs – the FFU complex is grouped in four groups (I–IV) this case can
be assigned to type II.
SO-056
Fetal manifestation of the Peters’ plus syndrome associated with
lenticular ectopia and occipital meningocele in one of the cases
K . Schoner1 , J . Kohlhase2 , J . Steinhard3 , R . Bald4 , A . Schwan5 , P . Wieacker6 ,
H . Rehder1 1 2 Philipps University Marburg, Institute of Pathology, Marburg, Praxis of
Human Genetics, Freiburg, 3Department of Obstetrics, Münster, 4Clinic of
Gynecology, Leverkusen, 5Division of Human Genetics, Dortmund, 6Institute of Human Genetics, Münster
Aims. Fetal pathology aims to recognize syndrome specific patterns of
malformations and dysmorphic features for goal directed mutation analyses,
genetic counselling of the parents and early prenatal molecular
diagnoses in consecutive pregnancies. Here we report on four fetuses
with Peters’ plus syndrome from two distinct families.
Methods. We performed fetal autopsies after prenatal ultrasound diagnoses
of malformations and termination of pregnancy and carried out
molecular genetic investigations on fetal and parental DNA.
Results. Four fetuses of 16 to 22 gestational weeks presented with multiple
malformations and dysmorphic signs in addition to Peters’ anomaly of
the eyes. The latter comprised central sclerocornea, absence of the posterior
corneal stroma, and a variable degree of iris and lenticular attachments
to the central posterior cornea in association with microphthal-
Der Pathologe · Supplement 1 · 2012 |
77

Abstracts
mia and lenticular ectopia. Additional features concerned hydrocephaly,
a characteristic round face with cleft lip and palate, hypertelorism and
prominent front, short stature, brachydactyly, and also cardiac, renal,
genital and cerebral malformations including occipital meningocele. Peters’
plus syndrome was confirmed by sequence analysis of the B3GALTL
gene revealing homozygosity for the common 660+1G>A donor splice
site mutation in intron 8 in all four cases and heterozygosity for this mutation
in the Caucasian, non-consanguineous parents.
Conclusions. The four affected fetuses show a characteristic facial aspect
that in association with the accompanying malformations should enable
the diagnosis of a Peters’ plus syndrome. Peters’ anomaly of the eyes,
representing an evolutive feature, is already evident at 18 weeks of gestation.
However, manifestation of the disorder is variable. Occipital meningocele
is a novel finding in Peters’ plus syndrome.
SO-057
Massive ovarian edema (MOE)
V . Sailer1 , S . Huss1 , F . Fronhoffs1 , E . Wardelmann1 , A .M . Müller2 1University Clinic of Bonn, Institute of Paidopathology and Institute of Pathology,
Bonn, 2University Bonn, Department of Pediatric Pathology, Bonn
Aims. Massive ovarian edema (MOE) is a very rare benign tumor-like
condition found in young women resulting from accumulation of fluid
mostly due to partial or intermittent torsion of the ovary or secondary to
a pre-existing ovarian lesion.
Methods. We report a case of a 13-year-old girl that presented with an
ovarian mass measuring 16 cm in diameter. Ultrasound and CT-scan
revealed a multilobulated cystic mass. CA-12-5 levels were increased.
Concerns regarding underlying malignancy lead to unilateral salpingooophorectomy.
Results. Pathological evaluation revealed a MOE and multiple thromboses
of ovarian veins.
Conclusions. Differentiation MOE from malignant tumor is crucial to
prevent unnecessary surgery potentially resulting in hormonal dysfunction
and infertility. Conservative treatment is possible and may be more
appropriate in cases when histology on frozen section supports a benign
lesion.
SO-058
Infantile myofibroma of the thyroid gland
A . Agaimy1 , D . Schmidt 2 , P . Klein3 , R . Carbon4 , W . Holter5 1Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen,
2Friedrich-Alexander University of Erlangen, Department of Nuclear Medicine,
Erlangen, 3Friedrich-Alexander University of Erlangen, Department of
Surgery, Erlangen, 4Friedrich-Alexander University of Erlangen, Department
of Surgery, Erlangen, 5Friedrich-Alexander University of Erlangen, University
Children‘s Hospital, Erlangen, Erlangen
Aims. Spindle cell lesions of the thyroid gland are rare and may thus be
diagnostically challenging. They encompass a heterogeneous group of
reactive mesenchymal lesions, and a variety of benign and malignant
neoplasms of epithelial and mesenchymal origin.
Methods. A 5-year-old girl presented with a rapidly growing firm nodular
cervical mass localized to the right thyroid lobe associated with bilateral
lymphadenopathy. Because of symptoms and concern about malignancy,
an open surgical biopsy was performed followed by resection
of the right lobe and biopsy of the cervical nodes. The patient is alive with
no evidence of recurrence 18 months after surgery.
Results. The specimen contained a 3.8 cm firm tan circumscribed nodular
mass surrounded by a thin rim of thyroid tissue. Histological examination
displayed a moderately cellular lesion composed of alternating
fascicles of eosinophilic myoid spindled cells and primitive looking
small rounded cells with hemangiopericytoma-like vascular pattern and
a prominent myointimal proliferation at the periphery of the lesion. The
78 | Der Pathologe · Supplement 1 · 2012
myoid cells expressed strongly alpha-smooth muscle actin but were negative
for desmin, h-caldesmon, epithelial membrane antigen, pankeratin,
CK7, thyroglobulin, TTF-1, protein S100, TLE1, ALK-1, beta-catenin,
CD31, CD34 and CD99. The lymph nodes showed reactive florid hyperplasia
without evidence of tumor.
Conclusions. To our knowledge, this case represents the first report of
solitary myofibroma presenting as a thyroid mass. Awareness of this differential
diagnosis is necessary to avoid misinterpretation as a sarcoma
with the sequelae of unnecessary over-treatment.
AG Urologische Pathologie I
SO-061
Mechanisms of VEGF-C and Neuropilin-2 induced therapy resistance
in prostate cancer
M . Muders1 , S . Haberlau 1 , M . Stanton 2 , H . Zhang3 , S . Dutta2 , M . Krause4 ,
K . Datta2 , G .B . Baretton1 1University Hospital Carl Gustav Carus at the University of Dresden, Institute
of Pathology, Dresden, 2University of Nebraska Medical Center, Omaha, NE,
United States, 3Mayo Clinic, Department of Urology, Rochester, MN, United
States, 4University Hospital Carl Gustav Carus at the University of Dresden,
Department of Radiation Oncology, Dresden
Aims. Resistance to treatment is a major contributor to prostate cancer
mortality in advanced stages. Therefore, in order to develop new treatment
modalities and improve the efficacy of current ones, it is important
to understand the molecular mechanisms that promote resistance to
therapy in prostate cancer cells.
Methods. To investigate the signaling pathways involved in induction
of therapy resistance by VEGF-C and Neuropilin-2 we knocked down
VEGF-C or Neuropilin 2 by RNA interference in standard prostate cancer
cells lines and treated these cell lines with ionizing irradiation or Docetaxel.
During or after treatment autophagic pathways were evaluated
by studying autophagic flux.
Results. VEGF-C and Neuropilin-2 are important molecules of radiation
and chemotherapy resistance in prostate cancer. Furthermore, we
have found that the VEGF-C/NRP-2 axis is involved in the activation
of autophagy, which maintains cancer cell survival following treatment.
Blocking autophagy also limits the ability of Neuropilin2 and VEGF-C
to induce therapy resistance in prostate cancer cell lines.
Conclusions. Together, these data suggest a link between the VEGF-C/
NRP-2 axis in prostate cancer cell survival in the presence of therapy-induced
stress by activating autophagy. Effective targeting of this pathway
may lead to the development of new cancer therapies.
SO-062
The ETS family of transcription factors and prostate cancer: The
role of the family prototype ETS-1
Z . Shaikhibrahim1 , N . Wernert1 1University Hospital Bonn, Institute of Pathology, Bonn
Aims. The ETS family of transcription factors plays important roles in
both normal and neoplastic cells for various biological processes. In
prostate cancer (PCa), recurrent gene fusions occurring between the
androgen-regulated prostate-specific serine protease TMPRSS2 gene,
and several ETS family members, most commonly ERG, are frequently
reported. ETS-1, the prototype of the family is reported to be overexpressed
in latent and clinically manifest PCas. The ETS-1 gene encodes three
distinct proteins, ETS-1 p51 encoded by a full-length mRNA, ETS-1 p42
and ETS-1 p27 encoded by an alternatively spliced mRNA lacking exon
VII and exons III–VI, respectively. Even though ETS-1 p42 and p27 have
been investigated in functional terms, the presence and roles of ETS-1

p42 and p27 have not yet been investigated in PCa. Therefore, we aimed
in this study at investigating the role of ETS-1 in PCa cell lines, and whether
the ETS-1 splice variants p42 and p27 are expressed in PCa cell lines.
Methods. We first examined the expression of all 27 ETS family members
using quantitative RT-PCR in androgen-sensitive and insensitive PCa
cell lines. As ETS-1 was found to be highly expressed in the androgeninsensitive
PCa cell lines PC3 and DU-145, we investigated the effect of
blocking ETS-1 in PC3 cells on genes involved in the metastatic cascade
using comprehensive gene expression microarrays, and correlated these
findings with PCa tissues. The expression of the ETS-1 splice variants p42
and p27 was assessed using an anti-ETS-1 antibody directed against the
DNA-binding domain (DBD), and RT-PCR.
Results. Assessment of ETS-1 blockade yielded many genes which are
known to be implicated in PCa. Correlating these genes with findings
from PCa tissues identified 16 genes that are up or down regulated in
healthy compared to tumorous PCa glands. Bioinformatical analysis revealed
that 13/16 of these genes have potential ETS-1 binding sites within
their promoter regions, and 4 were reported to be regulated by members
of the ETS family. Furthermore, our results show for the first time the
novel identification of the ETS-1 splice variants p42 and p27 in both the
androgen-dependent and the androgen-independent PCa cell lines.
Conclusions. Future studies will address the roles of the ETS-1 splice variants
in PCa cell lines and tissues. These findings provide in vitro and
in vivo evidence for the importance of ETS-1 in development and progression
of PCa
SO-063
Serum and prostate cancer tissue signatures of ERG rearrangement
derived from quantitative analysis of the PTEN conditional
knockout mouse proteome
N .J . Rupp1 , I . Cima2 , R . Schiess3 , P .J . Schüffler4 , T . Fuchs5 , N . Fankhauser2 , M .
Kälin6 , S . Gillessen6 , R . Aebersold3 , W . Krek2 , M .A . Rubin7 , H . Moch1 , P .J . Wild1 1University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland,
2ETH Zurich, Institute of Cell Biology, Zürich, Switzerland, 3ETH Zurich,
Institute of Molecular Systems Biology, Zürich, Switzerland, 4ETH Zurich,
Department of Computer Science, Zürich, Switzerland, 5California Institute
of Technology, Pasadena, CA, United States, 6Cantonal Hospital St . Gallen,
Department of Oncology, St . Gallen, Switzerland, 7Weill Cornell Medical
College, Department of Pathology and Laboratory Medicine, New York, NY,
United States
Aims. Applying a systems biology approach to assess serum and tissue
signatures of ERG rearrangement in patients with prostate cancer with
the goal of determining downstream molecular targets for gene fusion
cancers.
Methods. Prostate tissue from a conditional PTEN knockout mouse
model of prostate cancer was investigated, using selective enrichment of
N-glycopeptides and mass spectrometry-based label-free quantification.
Mouse tissue signatures were validated in sera and tissue of mice (n=12)
and humans (n=105) by selected reaction monitoring (SRM), ELISA, and
immunohistochemistry. ERG rearrangement status was assessed using
fluorescence in situ hybridization (FISH) on two independent tissue microarray
based prostatectomy cohorts (n=41, n=348). A Random forest
model was trained and validated to identify serum and tissue signatures
of ERG rearrangement.
Results. A comprehensive PTEN dependent protein catalogue representing
over 700 glycoproteins was established. TMPRSS2-ERG gene fusions
occurred in 41% (17/41) of analyzable prostate cancers of the training
cohort. ERG dependent serum signatures could be found. Predicted serum
signatures were systematically tested on two prostate cancer tissue
microarrays (training and test cohort) using immunohistochemistry for
15 candidate proteins and members of the PI3K/AKT/mTOR pathway.
Conclusions. This is the first study to demonstrate a proteomic signature
of ERG rearrangement prostate cancer. Given the challenges of directly
targeting ETS transcription factors, this study has potential clinical im-
plications providing important insights into future targetable downstream
pathways.
SO-064
Landscape of chromosome number changes during prostate
cancer progression
J . Stomper1 , M . Braun1 , W . Vogel1 , D . Böhm1 , V . Scheble2 , F . Fend3 , S . Perner1 1 2 University Hospital of Bonn, Institute of Pathology, Bonn, University
Hospital of Tübingen, Division of Oncology, Tübingen, 3University Hospital
of Tübingen, Institute of Pathology, Tübingen
Aims. Genetic instability resulting in both aneuploidy and polyploidy is
discussed to be involved in prostate cancer (PCa) development and progression.
However, a complete survey of numerical chromosomal changes
in PCa is lacking so far. The aim of this study was to comprehensively
characterize the ploidy level in PCa with regard to disease progression
via fluorescence in situ hybridization (FISH). Since aneuploidy and aggressive
disease are often associated with increased tumor cell proliferation,
we also assessed the expression of two common mitosis markers
within the same PCa cohort.
Methods. We studied a cohort comprising 186 localized PCa, 75 PCa with
125 corresponding lymph node metastases, and 42 hormone-refractory
distant metastases. Using dual-color FISH, we assessed the cohort for
losses and gains of all 24 chromosomes. Conducting immunohistochemistry
studies with the markers pHH3 and Ki67, we quantified the proliferation
rate within the same cohort.
Results. We observed a sharp and significant increase in aneuploidy with
advancing tumor stage (p

Abstracts
SO-065
Mutation analysis of squamous bladder tumours
N .T . Gaisa 1 , J . Korb 1 , N . Reimer 1 , S . Denzinger 2 , S . Koufou 2 , E . Eltze 3 , M . Toma 4 ,
S . Siegert 5 , A . Hartmann 6 , R . Stöhr 6 , R . Knüchel 1
1 RWTH Aachen University, Institute of Pathology, Aachen, 2 University
Hospital Regensburg, Department of Urology, Regensburg, 3 Institute of
Pathology Saarbrücken-Rastpfuhl, Saarbrücken, 4 University Hospital Dresden,
Institute of Pathology, Dresden, 5 LMU Munich, Institute of Pathology,
München, 6 University Hospital Erlangen, Institute of Pathology, Erlangen
Aims. The identity and impact of genetic changes in non-Schistosoma
associated squamous carcinoma of the bladder and urothelial carcinoma
with squamous differentiation are still unknown. Therefore, in this
study squamous tumours have been analyzed for frequent somatic mutations
in urothelial cancer.
Methods. Pure squamous carcinoma (n=34) and mixed urothelial cancers
with additional squamous differentiation (n=42) as well as their
precursor lesions have been screened for mutations in TP53, FGFR3
and PIK3CA. Sanger Sequencing was performed for TP53; FGFR3 and
PIK3CA were analyzed by SnapShot-method.
Results. 47% of pure squamous carcinoma (16/34) and 62% of urothelial
cancer with squamous differentiation (26/42) showed TP53 mutations.
The most frequent mutation was p.R175H (5 times, 3 in pure squamous
carcinoma, 2 in mixed carcinoma). FGFR3 mutations (exclusively
S249C) were detected in 9% (3/34) and 12% (5/42) respectively, PIK3CA
mutations (E542K and E545K) in 18% (6/34) and 17% (7/42). Both FGFR3
and PIK3CA mutations were found in 2 patients (pure squamous carcinoma)
only. TP53 and FGFR3/PIK3CA mutations were not mutually
exclusive, and TP53 mutations associated with either FGFR3 or PIK3CA
mutations were found in 9 cases (n=4 pure squamous carcinoma, n=5
mixed carcinoma). FGFR3 mutations were not related to any particular
morphological phenotype.
Conclusions. TP53 mutations occurred with slightly higher frequency in
squamous parts of mixed carcinoma, but the overall incidence of TP53
mutations was similar to reports of pure urothelial carcinoma in the
literature. TP53 mutations may play a critical role in the development
of squamous bladder tumours, whereas FGFR3 and PIK3CA mutations
seem to be less relevant.
SO-066
The impact of bladder cancer histology on overall survival of
patients treated by cystectomy and adjuvant cisplatin-based
chemotherapy
B . Keck1 , R . Stöhr2 , S . Wach1 , H . Taubert1 , F . Kunath1 , S . Bertz2 , J . Lehmann3 ,
M . Stöckle4 , B . Wullich1 , A . Hartmann2 1 2 University of Erlangen, Department of Urology, Erlangen, University Erlangen,
Department of Pathology, Erlangen, 3Urology Practice Prüner Gang,
Kiel, 4Saarland University, Department of Urology
Aims. To evaluate the impact of conventional urothelial (UC), plasmacytoid
(PUC) and micropappilary (MPC) histology on survival of bladder
cancer patients treated by cystectomy and adjuvant cisplatin-based chemotherapy
within the prospective and randomized trial AUO-AB05/95.
Methods. Tumor samples of 221 patients with a majority treated within
the randomized AUO-AB05/95 trial by radical cystectomy and adjuvant
cisplatin-based chemotherapy were reviewed for identifying histologic
subtypes of locally advanced bladder cancer. 191 UC, 20 PUC and 10 MPC
of the bladder were identified. For the definition of PUC and MPC, at
least 50% of the tumor had the specific histologic pattern. Kaplan Meier
analysis and multivariate Cox’s proportional hazards regression analysis
were performed to compare overall survival (OS) of UC, MPC and PUC.
Of these 221 patients, 50 patients were treated with gemcitabine/cisplatin,
83 patients with M-VEC and 88 patients with cm.
Results. Median OS of PUC was 29.9months and significantly worse compared
to patients suffering from UC (62.8 months) or MPC (64.2 months;
80 | Der Pathologe · Supplement 1 · 2012
p=0.04). Multivariate Cox’s proportional hazards regression analysis adjusted
to chemotherapy showed a hazard ratio of 1.9 (p=0.034) for UC
(n=191) and 2.7 (p=0.083) for PUC (n=20) in contrast to patients suffering
from MPC (n=10).
Conclusions. Bladder cancer histology gives important information on
the prognosis of patients suffering from locally advanced bladder cancer.
As OS of UC, PUC and MPC differs in patients treated by radical cystectomy
and adjuvant cisplatin-based chemotherapy further prospective
studies comparing histologic variants of bladder cancer are necessary in
order to tailor therapeutic strategies in the future.
SO-067
Prognostic role of androgen receptor in bladder cancer
R . Wirtz1 , S . Bertz2 , B . Keck3 , S . Claas2 , L . Dyrskjøt4 , T . Orntoft5 , B . Wullich3 ,
R . Hake6 , S . Eidt6 , A . Hartmann1 1 2 University Cancer Center Erlangen, Institute of Pathology, Erlangen, University
Cancer Center Erlangen, Institute of Pathology, 3University Cancer
Center Erlangen, Urology University Clinic, 4Aarhus University Hospital, Denmark,
5Aarhus University Hospital, Molecular Medicine, Denmark, 6Institute of Pathology at the St-Elisabeth-Hospital Cologne
Aims. Hormone receptors are the prototype predictive marker in breast
and prostate cancer. Hormone receptor positive cancers have a better
prognosis, increased tropism to metastasize into the bones and respond
to endocrine treatment options. The prognostic value of hormone receptor
in urothelial carcinoma of the bladder (UCB) is less established.
This may in part result from technical limitations of immunhistochemical
detection methods. Interestingly, female gender has recently been
identified as strong adverse factor in advanced UCB (May et al. 2011).
By analyzing whole genome expression data from non-muscle invasive
bladder cancer patients, we have evaluated the potential of top candidate
genes (ESR1, PGR, AR, CYP19, HER2, RACGAP1) commonly used to
stratify breast cancer patients to predict bladder cancer progression. In
view of the gender specific effects, we have focused on the prognostic role
of androgen receptor expression on tumor invasion, disease progression
and survival.
Methods. Affymetrix microarray data from 41 non-metastatic bladder
cancer patients undergoing curative surgery were analyzed. Prognostic
value of androgen receptor mRNA expression was analyzed by unsupervised
Cluster analysis, partitioning tests, Mann Whitney tests and Kaplan
Meier estimates of cancer specific survival.
Results. Cluster analysis in the microarray date of the superficial UCB
cohort identified a hormone receptor positive subtype and a proliferation
dominated subtype of equal size. Androgen receptor expression
was negatively associated with cancer specific death (r=−0.42; p=0.005),
while proliferation correlated with increased risk of cancer specific death
(r=0.46; p=0.003). In addition, low androgen receptor expression was
associated with higher tumor stage (pTa vs pT1-4; p=0.017). In Kaplan
Meier analysis, the cancer specific survival was significantly better in tumors
exhibiting high androgen receptor levels (80% vs. 20%; p

SO-068
Recent advances in understanding the genetic and epigenetic
networks of germ cell tumors
D . Nettersheim 1 , B . Westernstroer 2 , S . Schlatt 2 , H . Schorle 1
1 University of Bonn Medical School, Institute of Pathology, Bonn,
2 University of Münster, CERA, Münster
Aims. In the past decades the incidence of germ cell tumors (GCT) has
been rising constantly. It is believed, that GCT arise from an arrested
progenitor to form an IGCNU (CIS). With puberty, CIS eventually progress
into seminoma and nonseminoma. The genetics of this progression
is poorly understood, so knowledge of genetic and epigenetic markers
is mandatory. Further, the establishment of a murine model system for
CIS/seminoma and embryonal carcinoma would further help in addressing
functional questions relating to GCT initiation, progression and
treatment.
Methods. We used cell lines derived from seminoma (TCam-2) and embryonal
carcinoma (2102EP) as well as paraffin sections from human fetal
material and germ cell tumors to elucidate the specificity of the markers
for primordial germ cells BLIMP1, PRMT5 and TFAP2C. Furthermore,
we generated knock down cell lines as well as knockout-models for
TCFAP2C in order to analyze the requirement of these factors. Finally,
we transplanted various GTC cell lines to nude mice and analyzed tumor
initiation and development.
Results. In our hands, the markers BLIMP1 and TFAP2C are highly
specific for CIS and seminoma. They are downregulated in embryonal
carcinoma, forced loss of these factors leads to upregulation of somatic
marker genes indicative for differentiation. A lack of Tfap2c in the murine
model results in sterility. Transplantation of the TCam2 seminoma
cell line revealed, that this cells grew as CIS/seminoma or embryonal
carcinoma depending on the microenvironment.
Conclusions. We demonstrate that TFAP2C and BLIMP1 might be suitable
for the diagnostics of GCTs. We show that these proteins are required
for the maintenance of the undifferentiated status of CIS and seminoma.
Finally, the establishment of a murine model for GCT enables for in vivo
studies relating to germ cell tumor biology.
SO-069
Progressive kidney lesions in mutant mouse models for uromodulin-associated
kidney disease
E . Kemter1 , S . Sklenak 1 , P . Prueckl1 , B . Rathkolb 1 , F . Habermann2 , M . Hrabé de
Angelis3 , E . Wolf1 , B . Aigner1 , R . Wanke 4
1LMU Munich, Chair for Molecular Animal Breeding and Biotechnology,
München, 2LMU Munich, Chair for Veterinary Anatomy, Histology, and
Embryology, Department of Veterinary Sciences, München, 3Helmholtz Zentrum München, Institute of Experimental Genetics, Neuherberg, 4LMU Munich, Institute of Veterinary Pathology, Center for Clinical Veterinary
Medicine, München
Aims. Uromodulin-associated kidney disease (UAKD) is a heritable renal
disease in humans caused by mutations in the uromodulin (UMOD)
gene. Impaired maturation of mutant uromodulin with retention of
uromodulin in the hyperplastic endoplamic reticulum is considered to
be causative for thick ascending limb (TALH) cell dysfunction. Besides
clinical symptoms like hyperuricemia, gout, and alteration of urine concentrating
ability, histological kidney alterations like tubulointerstitial
nephritis, cysts, and interstitial fibrosis are heterogeneously present in
affected humans. Progression of disease leading to end-stage-kidney
disease is a feature of UAKD, which pathophysiology is unknown. The
objective of this study was to analyze the age-associated development of
kidney alterations of two recently described mutant mouse lines carrying
two different Umod mutations.
Methods. Histopathology, immunohistochemistry, confocal laser scanning
microscopy, quantitative stereology, and transmission electron microscopy.
Results. Both the kind of uromodulin mutation and the allelic status
influence onset, severity, and progression of renal dysfunction, as demonstrated
by a continuous age-related increase of plasma urea concentrations
in mutant mice. Interstitial fibrosis and tubular atrophy (IFTA)
as well as interstitial infiltrates of inflammatory cells were constantly
present in kidneys of aged mutant mice of both lines. An association of
different Umod mutations and allelic status with differences in onset,
degree, and progression of these nephropathological alterations was detected.
Further, glomerulocystic and tubulocystic changes were occasionally
observed in kidneys of aged mutant mice.
Conclusions. Both Umod mutant mouse lines represent valuable models
for human UAKD and enable insights into the pathogenesis of this disease.
Intracellular accumulation and release of mutated uromodulin
might trigger an inflammatory response leading to interstitial fibrosis.
Intracellular accumulation of the mutated misfolded protein in the hyperplastic
ER might lead to TALH cell death leading to tubular atrophy.
Supported by the German Research Foundation (KE1673/1-1) .
AG Urologische Pathologie II
SO-071
Renal cell carcinoma – Implications for cooperation of pathologists
and urologists
T . Steiner 1
1HELIOS hospital Erfurt, Erfurt
Aims. Over the last ten years various new options have been developed
for the management of patients with renal cell carcinoma (RCC).
Methods. Due to improved imaging techniques small renal masses
(SRM) are detected more frequently. About 20% of renoparenchymal
tumors with less then 3 cm in diameter have benign histology. Despite
RCC histology a significant amount of the remaining tumors show
only minimal growth over time. Therefore, in elderly patients or those
with significant comorbidities active surveillance strategies should be
considered. Renal mass biopsy enhanced by molecular profiling holds
promise for assessing aggressive potential in this scenarium. Histologic
subtypes and genetic aberrations of RCC predict different probability of
development of local recurrence, lymph node and distant metastases and
should be considered in follow up after local surgery for RCC.
Results. The development of targeted therapy revolutionized the management
of metastatic RCC. It was based on the detection of VHL mutations
with consecutive expression of growth factors in clear cell RCC.
For other histologic RCC subtypes VEGF targeted therapy seems to be
less effective. But even in clear cell RCC primary and secondary resistance
occurs. In the future predictive molecular markers have to be determined
to give patients a chance of really individualized medicine.
Conclusions. In conclusion, cooperation between urologists and pathologists
is of increasing meaning for optimal management of RCC.
Der Pathologe · Supplement 1 · 2012 |
81

Abstracts
SO-072
Therapeutic and prognostic implications of an isolated Banff II
acute cellular rejection without tubulointerstitial component in
renal transplants
V . Broecker 1 , M . Hirzallah 2 , C .L . Bockmeyer 1 , P .A . Agustian 1 , M .E . Dämmrich 3 ,
A . Schwarz 4 , J .U . Becker 3
1 Hannover Medical School, Institute of Pathology, Hannover, 2 Klinikum
Region Hannover Krankenhaus, Oststadt-Heidehaus Medizinische Klinik
I, Hannover, 3 MHH, Institute of Pathology, Hannover, 4 Hannover Medical
School, Clinic for Nephrology and Hypertension, Hannover
Aims. In order to avoid unnecessary therapy it is debated intensely, whether
acute cellular rejection with Banff components v≥1, i0, t0 (v_only)
has the same therapeutic and prognostic implications as acute cellular
rejections with Banff components v≥1, i≥1, t≥1 (v_plus). We examined
this question retrospectively in renal transplant biopsies from a single
center.
Methods. All 23 renal transplant biopsies (from 23 patients) from our biopsy
archive with v_only were compared to 23 biopsies from 23 patients
with v_plus. All patients, v_only and v_plus, received therapy. They were
followed for 135 weeks ±106 (v_only) or 201 weeks ±151 (v_plus).
Results. No significant difference was found between v_only and v_plus
regarding donor sex, donor age, immunosuppressive regimen, recipient
age, time between transplantation and biopsy, number of glomeruli in
the biopsy, Banff components v, g, mm, ah, cg, cv, ci, ptc, ratio of of preglomerular
vessels with endothelialits to sum of preglomerular vessels,
C4d-positivity of preglomerular endothelium, of peritubular capillary
endothelium (Banff C4d), of glomerular endothelium, eGFR at biopsy,
kind of anti-rejection therapy, eGFR one week after initiation of anti-rejection
therapy, eGFR slope per week between biopsy and end of followup.
v_only had significantly more HLA-mismatches, a higher Banff ct
component and less cortical tubular interstitial edema.
Conclusions. The present data indicate marginal histological differences
between v_only and v_plus (with the exception of the defining Banff
components i and t). We could not find a difference regarding response
to anti-rejection therapy, transplant function or prognosis between
v_plus and v_only. The higher number of HLA-mismatches in v_only
might suggest that this rare lesion could be associated with donor specific
antibodies. We will examine this association further.
SO-073
Renal cell carcinoma and senescence: p400 as a novel prognostic
marker
S . Macher-Göppinger 1 , J . Lorenzo Bermejo2 , M . Hohenfellner3 , N . Wagener3 ,
P . Schirmacher1 , W . Roth1 1 2 University of Heidelberg, Institute of Pathology, Heidelberg, University of
Heidelberg, Institute of Medical Biometry and Informatics, Heidelberg, 3Uni versity Heidelberg, Department of Urology, Heidelberg
Aims. Mutations of the von Hippel-Lindau (VHL) tumor suppressor
gene cause hereditary and sporadic renal cell carcinomas (RCCs). The
best characterized function of VHL protein is suppression of the alpha
subunit of hypoxia inducible factor (HIF). Additional VHL functions
have been reported, including induction of senescence mediated by loss
of chromatin remodelling factor p400. Induction of senescence either by
oncogene activation or inactivation of tumor suppressors is considered
as a critical feature of mammalian cells to suppress tumorigenesis. Here,
we studied the expression of p400 in a large and well documented series
of RCCs with long term follow-up information.
Methods. Expression of p400 was examined by immunohistochemistry
using a tissue micro array containing RCC tumor tissue samples and
corresponding normal tissue samples from 932 patients. Regression
models were used to investigate the possible relationship between p400
expression and Ki-67 proliferative index, clinical and pathologic parameters
and disease-specific survival.
82 | Der Pathologe · Supplement 1 · 2012
Results. Loss of p400 expression was detected in 64% of all tumor specimens
and was associated with advanced tumor stage, higher grade
of malignancy and regional lymph node metastasis. Among well differentiated
RCCs, high proliferation (Ki-67 index 10+) was found in 10%
of carcinomas with a positive p400 expression, compared to 3% in p400
negative RCCs. Importantly, multivariate Cox regression indicated that
patients with high-proliferative tumors, those with p400-positive RCCs
have a 159% increased cancer-specific mortality risk compared to p400negative
RCCs.
Conclusions. Loss of p400 expression is common in RCCs and the proportion
of carcinomas with loss of p400 increases alongside advancing
tumor stage and decline of differentiation. In contrast, a subgroup of
tumors defined by high proliferation and expression of p400 shows significantly
shorter cancer-specific survival in multivariate analyses than
the subgroup of tumors with high Ki-67 labeling index without co-expression
of p400. Our data suggest that the highly proliferative, p400positive
subgroup of RCC represent tumors that are characterized by a
loss of the tumor-suppressive mechanism of senescence.
SO-074
Infiltration by tumor associated macrophages and CCR2/CCL7
expression correlates with brain metastases from clear cell renal
cell carcinoma
L .G . Wyler – von Ballmoos1 , C . Urrejola2 , P .H . Schraml1 , H . Moch1 1Institute of Surgical Pathology, University Hospital Zurich, Zürich, Switzerland,
2Institut of Pathology, University Hospital Basel, Basel, Switzerland
Aims. Renal cancer patients with brain metastasis have a poor prognosis.
The mechanisms, by which renal cancer metastasize to the brain is not
entirely understood. The Goal of this study was to find out more about
pathways of metastases from clear cell renal cell carcinoma (ccRCC) and
whether the expression of CCL2, CCL7, CCR2 and CXCR4 by tumor
cells and tumor associated macrophages (TAMs) correlates with brain
metastasis and/or outcome.
Methods. Among 40’021 routine autopsies, the reports from those 636
with metastatic renal cancer were rewied and the metastatic sites were
analyzed. For imunohistochemical staining tissue microarrays were
constructed from biopsy samples of 333 primary RCCs and 51 brain metastases
from ccRCCs. Stainings for CCL2, CCL7, CCR2, CXCR4 and
CD68 were analyzed and compared.
Results. Hematogenous metastases were present in 39% of the autopsy
cases, with most frequent involvement being lung (75%), liver and bone
(40% each), and soft tissue (34%). Brain metastases were observed in 15%.
The rate of brain metastases was higher in patients with lung metastases,
but was also observed in patients without thoracic metastases and
as isolated brain metastases. A strong expression of CXCR4 was seen in
31% of primary ccRCCs and 45% of brain metastases however a moderate
expression was seen in over 80% in both. CCR2 and CCL7 showed a
significantly higher expression in brain metastases of ccRCCs compared
to primary ccRCCs (p40) macrophage infiltrate. Within these CCR2 expressing TAMs correlated
with higher Fuhrman grade (p=0.003) and were more frequent in
brain metastases (p

SO-075
A novel, dual role of CCN3 in experimental glomerulonephritis:
pro-angiogenic and anti-mesangioproliferative effects
P . Boor 1 , C . van Roeyen 1 , E . Borkham-Kamphorst 2 , S . Rong 1 , U . Kunter 1 ,
I .V . Martin 1 , A . Kaitovic 1 , S . Fleckenstein 1 , B . Perbal 3 , R . Weiskirchen 2 , T . Ostendorf
1 , J . Floege 1
1 RWTH Aachen University, Aachen, 2 RWTH Aachen University, Inst . of
Clinical Chemistry and Pathobiochemistry, Aachen, 3 R&D L’Oréal, Clark, NJ,
United States
Aims. In contrast to factors promoting mesangial cell proliferation, little
is known about their endogenous inhibitors. During experimental mesangioproliferative
nephritis, glomerular CCN3 (also known as NOV or
nephroblastoma overexpressed gene) expression is reduced prior to the
proliferative phase and overexpressed in glomeruli and serum when mesangial
cell proliferation subsides.
Methods. To further elucidate its role in mesangioproliferative glomerulonephritis,
CCN3 was systemically overexpressed by muscle electroporation
in healthy or nephritic rats. This increased CCN3 serum concentrations
more than 3-fold for up to 56 days.
Results. At day 5 after disease induction, CCN3-transfected rats exhibited
an increase in glomerular endothelial area and in glomerular mRNA
levels of the pro-angiogenic factors VEGF and PDGF-C. In the mesangioproliferative
phase (day 7), CCN3 overexpression decreased mesangial
cell proliferation including expression of α-smooth muscle actin and
matrix accumulation of fibronectin and type IV collagen. In progressive
nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria,
glomerulosclerosis and reduced cortical collagen type I accumulation.
In healthy rat kidneys, overexpression of CCN3 induced no
morphological changes but regulated glomerular gene transcripts (reduced
transcription of PDGF-B, PDGF-D, PDGFR-β and fibronectin and
increased PDGFR-β and PDGF-C mRNA).
Conclusions. The above data identify a dual role of CCN3 in experimental
glomerulonephritis with pro-angiogenic and anti-mesangioproliferative
effects. Manipulation of CCN3 may represent a novel approach to help
repair glomerular endothelial damage and mesangioproliferative changes.
Deutsch-Chinesisches Symposium
Colorectal Carcinoma
SG-129
ACSL5, a modifier of WNT activity
C . Klaus1 , U . Schneider1 , R . Knuechel1 , N . Gaßler1 1RWTH Aachen University, Institute of Pathology, Aachen
Aims. The mitochondrial localized Acyl-CoA synthetase 5 (ACSL5) converts
free long-chain fatty acids into fatty acyl-CoA esters, and thereby
plays a key role in lipid biosynthesis and fatty acid degradation. In particular,
ACSL5 has been recently identified to be involved in apoptotic
cell death of senescent enterocytes along the intestinal crypt-villus axis
and to interact with mitochondrial proteins. The aim of this study was
to investigate ACSL5-dependent effects on intestinal signalling pathways
that coordinate proliferation and/or differentiation of enterocytes, particularly
the Wnt pathway.
Methods. Wnt signalling was analysed in an ACSL5 overexpressing cell
culture model using luciferase assays, immunohistochemistry, qRT-PCR
and Western blot. The findings were substantiated with expression studies
in human colon carcinomas and in a Wnt-associated mouse model.
Results. ACSL5 transgenic intestinal-derived cells displayed a strong
susceptibility to pro-apoptotic stimuli. This phenomenon was accom-
panied by caspase-3 activation and significant down-regulation of Wnt
signalling activity. In particular, Wnt pathway-associated mitochondrial
localized molecules were identified as interaction partners of ACSL5 activity.
Conclusions. Molecular mechanisms underlying ACSL5-dependent
apoptosis susceptibility of enterocytes are probably bivalent including
pro-apoptotic and anti-proliferative activities.
SG-130
Regulation of differential WNT activity in colorectal cancer
D . Horst1 , J . Chen2 , T . Kirchner1 , R . Shivdasani2 1Ludwig-Maximilians-Universität München, Pathologisches Institut,
München, 2Harvard Medical School, Dana-Farber Cancer Institute, Boston,
United States
Aims. Most colorectal cancers (CRCs) express the WNT-effector protein
β-catenin in a heterogeneous pattern. Strong nuclear expression is
often confined to a small fraction of tumor cells at the tumor’s leading
edge. Recent data suggest a role for Mitogen Activated Protein Kinase
(MAPK) signaling in nuclear accumulation of beta-catenin. We therefore
investigated if MAPK activity regulates overtly differential WNT
activity in CRC cell subpopulations.
Methods. We used gene expression profiling, and immunohistochemistry
to assess interdependence of MAPK and WNT pathway activity in
CRC. Lentivirus- and drug-based pathway modification in CRC xenograft
tumors of primary human colon cancers and colon cancer cell lines
was used to study the effect of MAPK activation or repression on differential
WNT activity.
Results. CRC cells with high WNT activity showed coincident overexpression
of MAPK target genes and high levels of phospho-ERK, indicating
active MAPK signaling. Forced MAPK activation by lentiviral
expression of constitutively active KRAS enhanced WNT pathway activity
in vivo in CRC xenograft tumors, whereas drug based inhibition of
EGFR signaling attenuated it.
Conclusions. Although CRC is characterized by mutational activation of
the WNT pathway, MAPK signaling influences intratumoral β-catenin
heterogeneity, revealing a mechanism for external stimuli to modulate
pathway activity. Because MAPK signaling does not merely coincide
with nuclear β-catenin but also regulates it, this may account for the high
frequency of KRAS mutations in CRC.
SG-132
IGFBP7 and WNT signaling pathway in tumor stroma interactions
C . Rao1 , J . Xu1 , M . Liu1 , H . Deng1 1Department of Pathology, School of Medicine, Zhejiang, China
Aims. To find out the mechanism of the up-regulation of IGFBP7and the
biological changes in fibroblasts during the interactions with colorectal
cancer cells.
Fibroblasts (HELFs) were cultured in colorectal cancer cells conditioned
media (SW620-CM), treated by TGF-β 1, TGF-β1 receptor antagonist
(SB431542), TGF-β1 specific antibody (AF), Wnt signaling pathway
agonist (LiCl) and inhibitor (DKK-1) respectively. Q-PCR, Western Blot,
ELISA, Immunofluorescence microscopy and flow cytometry were used
to detect the expression of related targeted genes and proteins of TGF-β
and Wnt signaling pathways.
HELFs cultured in SW620-CM were activated with abundant expression
of α-SMA and showed strong proliferation and weak apoptosis and senescence.
The expression of IGFBP7 of HELFs was up-regulated in timedependent
and dose-dependent manners when cultured with SW620-
CM, while TGF-β signaling were activated as Smad2, P-Smad2 and
TGF-βRΠ were up-regulated in HELFs. These effects could be strengthened
by TGF-β1 and inhibited by SB431542 or AF. During the interactions,
the downstream genes of Wnt signaling pathway such as c-myc, cyclinD1
Der Pathologe · Supplement 1 · 2012 |
83

Abstracts
were up-regulated and the proteins of Wnt signaling pathway such as
β-catenin, Dvl3, Dvl2 and Naked2 were obviously up-regulated which
suggested the canonical Wnt signaling pathway was also activated.
TGF-β and Wnt signaling pathways were activated during colorectal
cancer cells-fibroblasts interactions. Upregulating of IGFBP7 in HELFs
was mainly through TGF-β1/ALK5/ Smad-2 signaling pathway. Wnt signaling
pathway may also play an important role in this process.
SG-134
FMNL2 is regulated by MIR-137 and promotes actin assembly and
cell invasion
Y . Zeng1 , Y . Qiao1 , W . Zhao1 , X . Zhu1 , J . Wang2 , X . Zhang2 , X . Bian3 , Y . Ding2 ,
L . Liang2 1Department of Pathology, School of Basic Medical Sciences, Guangzhou,
China, 2Guangdong Province Key Laboratory of Molecular Tumor Pathology,
Guangzhou, China, 3Institute of Pathology and Southern Cancer Center,
Southwest hospital, Chongqing, China
Aims. FMNL2 is a member of diaphanous-related formins which act as
effectors of Rho family GTPases and control the actin-dependent processes
such as cell motility or invasion. We previously validated FMNL2
as a positive regulator of cell motility and metastasis in colorectal carcinoma
(CRC) but the mechanisms of FMNL2 in CRC remain unclear.
The aim was to investigate the association of FMNL2 with Rho signaling
pathway in the invasion of CRC.
Methods. Rho family GTPase activity was tested by Rho pull-down assay.
In vitro invasive ability of cells was detected by Boyden Chamber
assay. And luciferase activities of MAL/SRF were detected using dualluciferase
reporter assay. In addition, Immunofluorescent analyses were
performed to examine F-actin stained by phalloidin and co-localization
of FMNL2 and LARG or p115RhoGEF. Co-immunoprecipitation was
used to determine the direct binding of FMNL2 and LARG. Finally,
GST pull-down assay was used to detect the binding of LARG-CT with
FMNL2 in the absence or presence of active RhoAV14.
Results. In this study, we showed that FMNL2 activated Rho/ROCK pathway,
and required ROCK to promote cell invasion. Moreover, FMNL2
promoted the formation of stress fiber and filopodia, and activated the
SRF transcription factor in the Rho-dependent manner. We also demonstrated
that FMNL2 was necessary for LPA-induced invasion, Rho/
ROCK activation, actin polymerization and SRF activation. FMNL2 is
an essential component of LPA signal transduction toward Rho by directly
interacting with LARG. Finally, we found FMNL2, LARG and
RhoA constituted a positive feedback loop. LARG silencing inhibited
Rho/ROCK pathway and cell invasion.
Conclusions. Our findings provide evidence for the positive feedback
between FMNL2 and RhoA, which promotes actin assembly and cell
invasion of CRC.
SG-135
The p53 target gene desmocolin 3 acts as a novel tumor suppressor
through inhibiting AKT signaling pathoway in human colon
cancer
T . Cui1 , Y . Chen1 , L . Yang1 , T . Knösel1 , K . Zöller1 , O . Huber2 , I . Petersen1 1 2 institute of pathology, Jena, Institute of Biochemistry II
Aims. Desmocollin 3 (DSC3), a member of the cadherin superfamily and
integral component of desmosomes, is involved in carcinogenesis. However,
the role of DSC3 in human colorectal cancer (CRC) has not yet
been established. Our aim of the study was to explore the role of DSC3 in
human colorectal cancer.
Methods. DSC3 expression in CRC cell lines was analyzed by RT-PCR
and western blotting. Methylation status of DSC3 was examined by demethylation
tests, methylation-specific PCR, and bisulfite sequencing
(BS). The regulatory role of p53 was investigated by transfection.
84 | Der Pathologe · Supplement 1 · 2012
Results. DSC3 was downregulated in CRC cells at both mRNA and protein
levels. DSC3 expression was restored in five out of seven cell lines
after 5-aza-2’-deoxycytidine (DAC) treatment. A heterogeneous methylation
pattern was detected by BS in promoter region and exon 1 of
DSC3. Methylation of DSC3 genomic sequences was found in 41% (41 out
of 99) of primary CRC, being associated with poor prognosis (p=0.002).
Transfection of p53 alone or in combination of DAC increased the DSC3
expression. Similarly, treatment with p53 inducer adriamycin alone or in
combination with DAC enhanced DSC3 expression.
Conclusions. DNA methylation contributes to downregulation of DSC3
in CRC cell lines. Methylation status of DSC3 DNA is a prognostic marker
for CRC. P53 appears to play an important role in regulating DSC3
expression.
SG-136
Connexin 43 reverses malignant phenotypes of glioma stem cells
by modulating E-cadherin
X .-W . Bian1 , S .-c . Yu1 , H .-l . Xiao1 , X .-f . Jiang1 , Q .-l . Wang1 , Y . Li1 , X .-j . Yang1 ,
Y .-f . Ping1 , J .-j . Duan1 , X . Zhang1 1Third Military Medical University, Institute of Pathology and Southwest
Cancer Center, Chongqing, China
Aims. The purpose of this study was to investigate the role of gap junction
related proteins such as connexins for sustaining the malignant behavior
of cancer stem cells (CSCs) in glioma.
Methods. Tumorspheres from U87 cells and primary specimens were
isolated and characterized as previously described. CD133-positive cells
were sorted by flow sorting cytometry and termed as glioma stem cells
(GSCs). Electron microscopy was used for observation of ultrastructure
of GSCs. Laser confocal microscopy was used for fluorescence recovery
after photobleaching (FRAP) on GSCs. Cells and tissues were immunocyto(histo)chemically
stained by using stemness and differentiation
markers. Real time RT-PCR and western blotting were used for detection
of mRNA and protein levels of the stem cell markers. Cx43 or E-cadherin
pulldown assays were performed with anti-Cx43 antibody or anti-E-cadherin
antibody using the Co-immunoprecipitation. In addition, analyses
for promoter methylation of GJA1 gene, overexpression and knock-down
of Cx43, gene expression array and gene set enrichment analysis (GSEA)
were performed. Proliferation assay Matrigel invasion assay were carried
out in vitro and in vivo experiments were performed in nude mice.
Results. We obtained tumorspheres formed by glioma stem cells (GSCs)
and adherent GSCs, and then examined their GJIC. All GSCs showed
reduced GJIC, and differentiated glioma cells had more gap junction-like
structures than GSCs. GSCs expressed very low level of connexins, Cx43
in particular, which are key components of gap junction. We observed
hypermethylation in the promoter of gap junction protein ***1 (GJA1),
which encodes Cx43 in GSCs. Reconstitution of Cx43 in GSCs inhibited
their capacity of self-renewal, invasiveness and tumorigenicity via influencing
E-cadherin and its coding protein, which leads to changes of the
expression of Wnt/***-catenin targeting genes.
Conclusions. In this study, we provide evidence for the first time that
dysfunction of GJIC is an important feature of GSCs. Reconstitution of
Cx43, the key component to maintain the functional GJIC, does not alter
functional status of GJIC in GSCs but ablates their self-renewal, invasiveness
and tumorigenicity though the interaction with E-cadherin and
its coding protein to downregulate the express of those Wnt/β-catenin
targeting genes. Our results identify a potential role of Cx43 in maintaining
the malignant phenotype of GSCs.

Pancreatic Adenocarcinoma
SG-137
Serum MiR-192 and MiR-194 as tumor biomarker for pancreatic
ductal adenocarcinoma
J . Zhang 1 , C .-y . Zhao 1 , D .-h . Yu 1 , Y . Chen 1 , Q .-h . Liu 1 , M . Shi 1 , J .-t . Zhang 1 , G . Jin 1 ,
P . Cheng 1 , X .-g . Hu 1 , C .-r . Ni 1 , M .-h . Zhu 1
1 Department of Pathology, Changhai Hospital, Shanghai, China
Aims. To assess the validity of using serum miRNA signatures of PDAC
as biomarkers for diagnosis.
Methods. MiRNA microarray was used to detect the differences between
PDAC samples and normal pancreatic tissues. MiR-192 and miR-194
were found in the tissues of human PDAC and in the explants in tumorbearing
SCID mice by locked nucleic acid-based in situ hybridization
(LNA-ISH). Serum levels of miR-192 and miR-194 in PDAC patients,
duodenal adenocarcinoma patients and healthy controls, as well as six
pancreatic cancer cell lines and their culture supernatants were determined
by real-time PCR.
Results. Eight miRNAs were found overexpressed and eight were lowly
expressed in PDAC tissues compared with those in the normal pancreatic
tissues. MiR-192 and miR-194 were found overexpressed in the tissues
of human PDAC and in the explants in tumor-bearing SCID mice.
The levels of miR-192 and miR-194 in the supernatants of six pancreatic
cancer cell lines were positively correlated with their cellular expressions
(r=0.849, p

Abstracts
SG-140
Sialinomycin induces autophagic and apoptotic cell death in
pancreatic carcinoma cell lines
M . Vogt 1 , B . Verdoodt 1 , S .-T . Liffers 1 , A . Tannapfel 1 , A . Mirmohammadsadegh 1
1 Ruhr-University of Bochum, Institute of Pathology, Bochum
Aims. Salinomycin a polyether antibiotic, is produced by a strain of
streptomyces albus and has anti-microbial and anti-coccodial activities.
Recently, a number of studies described anti-tumor properties of salinomycin,
in particular its effect on chemoresistant tumor initiating cells.
In the present study, we investigated the impact of salinomycin mediated
activation of MEK/ERK signalling pathway on autophagy and apoptotic
cell death in pancreatic cancer cell lines.
Methods. Two pancreatic cell lines MIAPaCa-2 and PaTu8902 were used
to analyze the effect of salinomycin on cell viability, autophagy and
apoptosis. The effect of salinomycin on autophgay was investigated by
transmission electron microscopy (TEM) for detection of autophagic
vesicles and processing of LC3B, microtubule-associated protein 1 light
chain 3 isoform B (LC3B). Towards investigating the role of salinomycin
on apoptotic cell death we used caspase3/7, FACS, Western blot analysis
and immunofluorescence staining.
Results. Salinomycin treatment inhibits cell viability and colony forming
in MIA PaCa-2 and PaTu8902 in a time and concentration depending
manner. In both cell lines salinomycin was able to induce autophagic cell
death, detected by LC3 processing and formation of autophagic vacoules.
Salinomycin was able to induce autophagic and apoptotic cell death
in MIAPaCa-2 cell lines. In MIAPaCa-2 cells the salinomycin induced
autophagic cell death was dependent on ERK1/2 pathway. In contrast,
salinomycin induced autophagic cell death in PaTu8902 was independent
of ERK1/2.
Conclusions. Salinomycin, a novel anti-tumor drug is able to induce autophagic
and apoptotic cell death depending on cellular background.
Mammary Cancer
SG-141
Kindlin2 up-regulation promotes tumor progression
W . Fang1 , T . Zhao1 , H .-q . Zhang2 1Department of Pathology, Key Laboratory of Carcinogenesis and Translational
Research, Beijing, China, 2Peking University Health Science Center,
Beijing, China
Aims. Kindlin-2 has been confirmed as an essential element of bidirectional
integrin signaling. In recent years, the relationship between Kindlin-2
expression and cancers has been a focus of interest. Our previous
studies have shown that Kindlin-2 expression was up-regulated in several
types of human cancers, and a strong correlation between Kindlin-2
expression and clinical outcome of breast cancer patients was found.
However, the functional role of Kindlin-2 in breast cancer has not been
studied. This study was designed to investigate the role of Kindlin-2 in
the progression of human breast cancer cells.
Methods. Firstly, Kindlin-2 expression at protein level was detected by
Western blot in several breast cancer cell lines. Two luminal-like breast
cancer cell lines, MCF-7 and T47D, expressed low level of Kindlin-2.
Two basal-like breast cancer cell lines, MDA-MB-231 and HS578T, expressed
moderate levels of the protein. Then, Kindlin-2 gene was overexpressed
by transfected into MCF-7 cells. In comparison, short hairpin
RNA (ShRNA)-mediated knockdown of Kindlin-2 was performed in
HS578T cells. Vector controls were also done in the same cell lines. Ki67
Li, FCM cell cycle, anchorage-independent colony formation (in vitro
tumorigenesis), and in vivo tumorigenesis in NOD/SCID mice were observed.
Apoptotic cells were labeled by fluorescent annexin V assay and
86 | Der Pathologe · Supplement 1 · 2012
quantified by FACS. Array CGH analysis and spectral karyotyping were
performed to detect the genomic instability of these cells.
Results. The growth rate of Kindlin-2-transfected MCF-7 cells was much
quicker than that of the controls. The proportion of G2-M phase cells,
clone formation and tumorigenicity were significantly higher than these
of the controls. The change of Kindlin-2-ShRNA transfected cells was
just the reverse. Moreover, Up-regulation of Kindlin-2 can also reduce
the rate of apoptosis induced by the chemotherapy drugs, and these cells
showed much more genomic instability compared with the controls.
Conclusions. These findings suggested that up-regulation of Kindlin-2
promotes the progression of human breast cancer cells by increasing
their proliferation, drug resistance, genomic instability, and tumorigenesis.
SG-142
ECRG4 is frequently downregulated by CpG island hypermethylation
in human breast cancer
W . Zhang1 1Zhejiang University, Institute of Pathology, Hangzhou, China
Aims. Esophageal cancer related gene4 (ECRG4) is a recently reported
candidate tumor suppressor gene frequently hypermethylated in several
human tumor types, including esophageal squamous cell carcinoma,
colorectal carcinoma, glioma and prostatic carcinoma. This study is to
investigate if ECRG4 is transcriptionally silenced by promoter CpG island
methylation in breast cancer.
Methods. We analyzed several breast cell lines and 12 pairs of fresh samples
from breast cancer patients for ECRG4 promoter CpG island methylation
by COBRA and bisulfite sequencing. ECRG4 mRNA and protein
expression analysis was carried out by semi-quantitative RT-PCR, realtime
PCR, Western Blot and immunohistochemistry.
Results. We found that all of three immortalized normal breast cell lines
and three normal breast tissues expressed ECRG4 proteins, whereas
ECRG4 expression were reduced or absent in most breast cancer cell lines
and primary breast cancer samples. We also identified that ECRG4
promoter was frequently methylated in breast cancer samples. An inverse
correlation between mRNA expression and methylation status of
the ECRG4 promoter CpG island was observed in primary breast cancer
samples.
Conclusions. The expression of ECRG4 is frequently decreased due to
promoter CpG island hypermethylation in breast cancer.
SG-143
Novel biomarkers in breast carcinoma: DKK3, ITIH5, SFRP-1
V . Kloten1 , B . Becker1 , M .G . Schrauder 2 , P .A . Fasching 2 , A . Hartmann3 ,
J . Veeck1 , R . Knüchel1 , E . Dahl1 1University Hospital of the RWTH Aachen, Institute of Pathology, Aachen,
2University Hospital Erlangen, University Breast Center, Erlangen,
3University Hospital Erlangen, Erlangen
Aims. For early detection of breast cancer the development of robust
blood-based biomarkers that accurately reflect the host tumor is mandatory
and thus a growing field of research. The most common alterations
in human cancers including breast cancer are changes in the status of
DNA methylation, which are therefore quickly emerging as a new pool
of potential biomarkers. Thus, we investigated the feasibility of detecting
aberrant tumor suppressor gene methylation in cancer cell-derived free
circulating DNA in the bloodstream of patients.
Methods. Using qualitative MSP, we examined the methylation status of
six biologically significant putative tumor suppressor genes, i.e. ITIH5,
DKK3, WIF1, SFRP1, SFRP2 and SFRP5 in DNA extracted from serum.
Clinical performance was determined in a large training study on 150
serum samples (120 breast cancers, 30 healthy controls). 20 benign di-

sease and 30 colon cancer serum samples were included for additional
specificity testing.
Results. Based on the training study we could evaluate the top candidate
biomarkers with the best values for sensitivity and specificity. A marker
panel of DKK3 and ITIH5 detected breast cancer with a sensitivity of 46%
(55/120). Specificity of the panel was sufficient with 83%, 100% and 93% in
colon cancer samples, benign and healthy control samples, respectively.
Control samples revealed unacceptable high methylation rates of SFRP1
and SFRP5 in DNA extracted from colon cancer sera, whereas SFRP2
and WIF1 showed a considerable methylation frequency in sera from
healthy controls.
Conclusions. The current study suggests that cancer-specific methylation
of ITIH5 and DKK3 in serum-derived tumor-borne DNA might be
valuable biomarkers for the early detection of breast cancer. In the second
phase of this project we are currently validating ITIH5 and DKK3
as reliable methylation biodiagnostic markers in an independent test set
consisting of 160 breast cancer serum samples and 160 control samples.
SG-145
Slug and SOX9 in aggressive breast carcinoma
V . Tischler1 , A . Noske1 , U . Zürrer-Härdi1 , F . Ingold1 , J .-P . Theurillat1 , H . Moch1 , Z .
Varga1 , W . Guo2 1University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland,
2Albert Einstein College of Medicine, Ruth L . and David S . Gottesman
Institute for Stem Cell Biology and Regenerative Medicine, New York, United
States
Aims. Co-expression of the transcription factors Slug and Sox9 was recently
used to determine mammary stem cell state. Our intention was
to study the expression of Slug and Sox9 in primary breast cancer and
to correlate it with clinicopathological parameters including overall survival.
Methods. Formalin-fixed paraffin embedded tumor tissue of 306 patients
with primary breast cancer [pT1 132 (43.1%), pT2 134 (43.8%), pT3 21
(6.9%), pT4 19 (6.2%); pN0 92 (34.1%), pN1 136 (50.4%), pN2 22 (8.1%), pN3
20 (7.4%); G1 41 (13.4%), G2 144 (47.1%), G3 121 (39.5%)] were assembled
on a tissue microarray (TMA). Mean follow-up was 40 months (range
4–324 months). Immunohistochemical Slug and Sox9 expression was semi-quantitatively
evaluated. The median value was used as cut-off point
for dichotomization into “low Slug”/”high Slug” and “low Sox9”/”high
Sox9” expressing groups.
Results. Slug and Sox9 expression was identified in the tumor cell nuclei.
High Slug expression was found in 41% of the cases (126/306) and high
Sox9 expression in 74% (226/306). High expression of both Slug and Sox9
was found in 92/306 (30.1%). Co-expression of Slug and Sox9 significantly
correlated with higher tumor grade (p

Abstracts
our study to identify overlapping molecular characteristics in a variety
of histologically defined lymphoma entities. To achieve this goal, comprehensive
expression profiles of non-coding and coding transcripts derived
from a broad spectrum of lymphomas were established. Our data
show that overlapping molecular features can be identified beyond the
current lymphoma classification.
Methods. RNA was extracted from frozen tissue blocks of distinct human
B- and T-cell lymphoma entities as well as tonsils (n=116). The small
(micro) RNA fraction was sequenced by next generation technology
(SOLiD) and total RNA was subjected to Affymetrix Exon 1.0 ST array
hybridisation. In addition immunohistochemical and clinical data was
acquired. Bioinformatic analyses were then applied for subgroup detection.
Results. Unsupervised clustering based on the mature micro RNA expression
derived from deep sequencing demonstrated the presence of
two distinct large clusters within the case collection. One cluster contained
predominantly the so-called indolent lymphoma types, but also a
considerable number of aggressive B-cell lymphoma cases. In the second
cluster the majority of cases were aggressive B-cell lymphoma but interestingly
intermingled with the T-cell lymphoma cases. Differentially
expressed micro RNAs between the two clusters were determined and
logistic regression identified a micro RNA classificator able to distinguish
the two groups. The micro RNA expression data was combined with
the coding RNA data in order to identify the involved pathways.
Conclusions. Micro RNA expression profiles obtained by deep sequencing
were able to identify two groups within a lymphoma collection that
separated the cases beyond the histopathological classification system.
Discriminative micro RNAs and associated pathways were identified.
These findings give new insights into lymphoma biology and point to
alternative treatment options.
*These authors contributed equally to the study .
Poster
Poster: Deutsch-Chinesisches Symposium
SG-P-112
Mitochondrial mortalin (HSPA9) expression in enterocytes is
regulated by ACSL5
N . Gaßler 1 , C . Klaus 1 , E . Kämmerer 2 , M . Adolf 1 , A . Reinartz 1
1 RWTH Aachen University, Institute of Pathology, Aachen, 2 RWTH Aachen
University, Klinik für Kinder- und Jugendmedizin, Aachen
Aims. Mitochondrial acyl-CoA synthetase 5 (ACSL5), a long-chain fatty
acid activating enzyme, is preferentially found in small intestinal enterocytes.
ACSL5 activities are associated with complex changes in the
mitochondrial lipid metabolism and are important for epithelial differentiation
and cell death. The aim of the present study was to identify
and characterize ACSL5 related regulation of mitochondrial proteins.
Methods. Mitochondria isolated from ACSL5 transfected CaCo2 cells
and controls were homogenized and further separated with 2D-gel
electrophoresis. Spots were analyzed using special proteomics software.
Protein spots differentially expressed were further identified with MAL-
DI-TOF. Additional techniques were used for target validation. Laser microdissected
enterocytes from normal human small intestinal mucosa
were investigated to substantiate the in vitro findings.
Results. 14 mitochondrial protein species, probably regulated by ACSL5,
were identified. ACSL5 dependent expression and synthesis of the candidate
molecule mortalin (HSPA9) was confirmed using qRT-PCR,
Western blotting, and siRNA ACSL5 knockdown. Using this approach,
a positive correlation of ACSL5 and mortalin expression was verified.
The positive correlation of ACSL5 and mortalin expression was additio-
88 | Der Pathologe · Supplement 1 · 2012
nally found in human normal small intestinal mucosa. Strong mortalin
expression was seen in ACSL5-rich enterocytes isolated from intestinal
villi, whereas mortalin was diminished in low ACSL5 expressing enterocytes
isolated from intestinal crypts.
Conclusions. In conclusion, ACSL5 activities are associated with changes
in lipid metabolism as well as expression of mitochondrial proteins.
ACSL5-dependent expression and synthesis of mitochondrial mortalin
is probably a phenomenon of stress-response.
SG-P-113
ACSL5 as putative modifier of Wnt activity in intestinal cells
C . Klaus1 , U . Schneider1 , R . Knuechel1 , N . Gaßler1 1RWTH Aachen University, Institute of Pathology, Aachen
Aims. The mitochondrial localized Acyl-CoA synthetase 5 (ACSL5) converts
free long-chain fatty acids into fatty acyl-CoA esters, and thereby
plays a key role in lipid biosynthesis and fatty acid degradation. In particular,
ACSL5 has been recently identified to be involved in apoptotic
cell death of senescent enterocytes along the intestinal crypt-villus axis
and to interact with mitochondrial proteins. The aim of this study was
to investigate ACSL5-dependent effects on intestinal signalling pathways
that coordinate proliferation and/or differentiation of enterocytes, particularly
the Wnt pathway.
Methods. Wnt signalling was analysed in an ACSL5 overexpressing cell
culture model using luciferase assays, immunohistochemistry, qRT-PCR
and Western blot. The findings were substantiated with expression studies
in human colon carcinomas and in a Wnt-associated mouse model.
Results. ACSL5 transgenic intestinal-derived cells displayed a strong
susceptibility to pro-apoptotic stimuli. This phenomenon was accompanied
by caspase-3 activation and significant down-regulation of Wnt
signalling activity. In particular, Wnt pathway associated mitochondrial
localized molecules were identified as interaction partners of ACSL5 activity.
Conclusions. Molecular mechanisms underlying ACSL5-dependent
apoptosis susceptibility of enterocytes are probably bivalent including
pro-apoptotic and anti-proliferative activities.
SG-P-114
MAPK signaling regulates differential WNT activity in colorectal
cancer
D . Horst1 , J . Chen2 , T . Kirchner1 , R . Shivdasani2 1Ludwig-Maximilians-Universität München, Pathologisches Instiut,
München, 2Harvard Medical School, Dana-Farber Cancer Institute, Boston,
United States
Aims. Most colorectal cancers (CRCs) express the WNT effector protein
β-catenin in a heterogeneous pattern. Strong nuclear expression is
often confined to a small fraction of tumor cells at the tumor’s leading
edge. Recent data suggest a role for Mitogen Activated Protein Kinase
(MAPK) signaling in nuclear accumulation of β-catenin. We therefore
investigated if MAPK activity regulates overtly differential WNT activity
in CRC cell subpopulations.
Methods. We used gene expression profiling, and immunohistochemistry
to assess interdependence of MAPK and WNT pathway activity in
CRC. Lentivirus- and drug-based pathway modification in CRC xenograft
tumors of primary human colon cancers and colon cancer cell lines
was used to study the effect of MAPK activation or repression on differential
WNT activity.
Results. CRC cells with high WNT activity showed coincident overexpression
of MAPK target genes and high levels of phospho-ERK, indicating
active MAPK signaling. Forced MAPK activation by lentiviral
expression of constitutively active KRAS enhanced WNT pathway activity
in vivo in CRC xenograft tumors, whereas drug based inhibition of
EGFR signaling attenuated it.

Conclusions. Although CRC is characterized by mutational activation of
the WNT pathway, MAPK signaling influences intratumoral β-catenin
heterogeneity, revealing a mechanism for external stimuli to modulate
pathway activity. Because MAPK signaling does not merely coincide
with nuclear β-catenin but also regulates it, this may account for the high
frequency of KRAS mutations in CRC.
SG-P-115
Influence of an anti-TLR2-Intrabody on rheumatoid arthritis
B . Küttner1 , V . Seiffert1 , S . Laggies1 , G . Gross1 , T . Böldicke1 , A . Dellmann2 ,
K . Donhuijsen2 1 2 Helmholtz-Centre, Infection Research, Braunschweig, academical hospital
Braunschweig, department of pathology, Braunschweig
Aims. Rheumatoid arthritis (RA) is a chronic, systemic inflammatory
disease. Recent results showed that toll-like receptors (TLRs) particularly
TLR2 and its signaling pathway is involved in the pathogenesis of
rheumatic arthritis and might be a “key target” in prevention of RA. An
intracellular antibody (intrabody) fused with an endoplasmatic reticulum
retention sequence has been developed that inhibits the function of
TLR2 by preventing the translocation of TLR2 from the ER to the cell
surface.
Methods. To test if the intrabody has the potential to inhibit inflammation
during rheumatoid arthritis two mouse models were established: a
collagen-induced arthritis model and a model based on the injection of
inflammatory synovial fibroblasts into joints. Investigations by immunohistochemistry,
confocal microscopy, flow cytometry and paw swelling
showed the course of the disease. At first we analyzed the efficiency
of the anti-TLR2 intrabody in the collagen arthritis mouse model.
Results. A strong inflammatory response was seen in both rheumatoid
mouse models in the absence of the intrabody. Anti-TLR2 intrabody inhibited
significantly the paw swelling after adenoviral anti-TLR2 intrabody
gene transfer into the joints. Joints with an inflammatory response
showed migration of cells (HE staining) into the lamina synovialis and
the fat-tissue, in fact macrophages (CD11b+) and fibroblasts (CD40+).
Conclusions. Analysis of TLR2 surface representation and intracellular
intrabody expression of cells prepared from joints of rheumatoid mice
treated with intrabody-adenovirus and eGFP adenovirus (control) is
currently under investigation. In the rheumatoid arthritis model using
synovial cells with inflammation, we plan to analyze the influence of
cellular anti-TLR2-intrabody expression on the manifestation of the disease.
SG-P-116
TGF-β and Wnt signaling pathways regulate the expression of
IGFBP7 of fibroblasts in the tumor-stroma interactions
C . Rao1 , J . Xu1 , M . Liu1 , H . Deng1 1Department of Pathology, School of Medicine, Zhejiang, China
Aims. To find out the mechanism of the up-regulation of IGFBP7and the
biological changes in fibroblasts during the interactions with colorectal
cancer cells.
Fibroblasts (HELFs) were cultured in colorectal cancer cells conditioned
media (SW620-CM), treated by TGF-β 1, TGF-β1 receptor antagonist
(SB431542), TGF-β1 specific antibody (AF), Wnt signaling pathway
agonist (LiCl) and inhibitor (DKK-1) respectively. Q-PCR, Western Blot,
ELISA, Immunofluorescence microscopy and flow cytometry were used
to detect the expression of related targeted genes and proteins of TGF-β
and Wnt signaling pathways.
HELFs cultured in SW620-CM were activated with abundant expression
of α-SMA and showed strong proliferation and weak apoptosis and senescence.
The expression of IGFBP7 of HELFs was up-regulated in timedependent
and dose-dependent manners when cultured with SW620-
CM, while TGF-β signaling were activated as Smad2, P-Smad2 and
TGF-βRΠ were up-regulated in HELFs. These effects could be strengthened
by TGF-β1 and inhibited by SB431542 or AF. During the interactions,
the downstream genes of Wnt signaling pathway such as c-myc, cyclinD1
were up-regulated and the proteins of Wnt signaling pathway such as
β-catenin, Dvl3, Dvl2 and Naked2 were obviously up-regulated which
suggested the canonical Wnt signaling pathway was also activated.
TGF-β and Wnt signaling pathways were activated during colorectal
cancer cells-fibroblasts interactions. Upregulating of IGFBP7 in HELFs
was mainly through TGF-β1/ALK5/ Smad-2 signaling pathway. Wnt signaling
pathway may also play an important role in this process.
SG-P-117
MicroRNA-137, a HMGA1 target, suppresses cell invasion and
metastasis of colorectal carcinoma by directly targeting FMNL2
X . Li1 , X . Zhang1 , W . Zhao1 , J . Wang1 , X .W . Biang2 , L . Liang3 , Y . Ding3 1Department of Pathology, School of Basic Medical Sciences, Guangzhou,
China, 2Institute of Pathology and Southern Cancer Center, Southwest Hospital,
Chongqing, China, 3Guangdong Province Key Laboratory of Molecular
Tumor Pathology, Guangzhou, China
Aims. FMNL2, a member of diaphanous-related formins, has been
strongly associated with tumor progression but the post-transcriptional
regulatory mechanism of FMNL2 remains unknown. The aim of this
study was to investigate whether increased FMNL2 expression is mediated
by microRNAs in colorectal carcinoma (CRC).
Methods. Real-time PCR or Western blot was performed to detect the expression
level of miR-137 or FMNL2 in CRC cells and tissues. The in vitro
and in vivo functional effect of miR-137 was examined further. A luciferase
reporter assay was conducted to confirm the associations between
miR-137 and FMNL2 3’UTR, HMGA1 and miR-137 promoter. CHIP was
used to assess the direct binding of HMGA1 to miR-137 promoter.
Results. We initially chose miR-137 and miR-142-3p as potential miRNAs
targeting FMNL2 which were predicted by bioinformatics. Only miR-137
showed significant inverse correlation with FMNL2 protein level in CRC
cell lines and tissues. We validated that FMNL2 was a target gene of miR-
137, and miR-137 could inhibit cell proliferation, invasion in vitro, hepatic
or intestinal metastasis in vivo by targeting FMNL2. We further show
that HMGA1 enhances miR-137 transcription by binding its promoter,
and then negatively downregulates FMNL2 expression. Finally, miR-137
inhibited the activation of p-MAPK and p-Akt, followed by the suppression
of MMP2, MMP9 and VEGF.
Conclusions. Our findings demonstrate a novel mechanism of post-transcriptional
regulation of FMNL2 expression and identified miR-137,
induced by its upstream transcription factor HMGA1, can suppress its
direct target FMNL2 and lead to tumor invasion and metastasis, at least
in part through inhibiting PI3K/Akt and MAPK/ERK pathways.
SG-P-118
The positive feedback between FMNL2 and RhoA, promotes actin
assembly and cell invasion of colorectal carcinoma
Y . Zeng1 , Y . Qiao1 , W . Zhao1 , X . Zhu1 , J . Wang2 , X . Zhang2 , X . Bian3 , Y . Ding2 ,
L . Liang2 1Department of Pathology, School of Basic Medical Sciences, Guangzhou,
China, 2Guangdong Province Key Laboratory of Molecular Tumor Pathology,
Guangzhou, China, 3Institute of Pathology and Southern Cancer Center,
Southwest hospital, Chongqing, China
Aims. FMNL2 is a member of diaphanous-related formins which act as
effectors of Rho family GTPases and control the actin-dependent processes
such as cell motility or invasion. We previously validated FMNL2
as a positive regulator of cell motility and metastasis in colorectal carcinoma
(CRC) but the mechanisms of FMNL2 in CRC remain unclear.
Der Pathologe · Supplement 1 · 2012 |
89

Abstracts
The aim was to investigate the association of FMNL2 with Rho signaling
pathway in the invasion of CRC.
Methods. Rho family GTPase activity was tested by Rho pull-down assay.
In vitro invasive ability of cells was detected by Boyden Chamber
assay. And luciferase activities of MAL/SRF were detected using dualluciferase
reporter assay. In addition, Immunofluorescent analyses were
performed to examine F-actin stained by phalloidin and co-localization
of FMNL2 and LARG or p115RhoGEF. Co-immunoprecipitation was
used to determine the direct binding of FMNL2 and LARG. Finally,
GST pull-down assay was used to detect the binding of LARG-CT with
FMNL2 in the absence or presence of active RhoAV14.
Results. In this study, we showed that FMNL2 activated Rho/ROCK pathway,
and required ROCK to promote cell invasion. Moreover, FMNL2
promoted the formation of stress fiber and filopodia, and activated the
SRF transcription factor in the Rho-dependent manner. We also demonstrated
that FMNL2 was necessary for LPA-induced invasion, Rho/
ROCK activation, actin polymerization and SRF activation. FMNL2 is
an essential component of LPA signal transduction toward Rho by directly
interacting with LARG. Finally, we found FMNL2, LARG and
RhoA constituted a positive feedback loop. LARG silencing inhibited
Rho/ROCK pathway and cell invasion.
Conclusions. Our findings provide evidence for the positive feedback
between FMNL2 and RhoA, which promotes actin assembly and cell
invasion of CRC.
SG-P-119
DSC3 expression is regulated by p53, and methylation of DSC3
DNA is a prognostic marker in human colorectal cancer
T . Cui1 , Y . Chen1 , L . Yang1 , T . Knösel1 , K . Zöller1 , O . Huber2 , I . Petersen1 1 2 Institute of Pathology, Jena, Institute of Biochemistry II
Aims. Desmocollin 3 (DSC3), a member of the cadherin superfamily and
integral component of desmosomes, is involved in carcinogenesis. However,
the role of DSC3 in human colorectal cancer (CRC) has not yet
been established. Our aim of the study was to explore the role of DSC3 in
human colorectal cancer.
Methods. DSC3 expression in CRC cell lines was analyzed by RT-PCR
and western blotting. Methylation status of DSC3 was examined by demethylation
tests, methylation-specific PCR, and bisulfite sequencing
(BS). The regulatory role of p53 was investigated by transfection.
Results. DSC3 was downregulated in CRC cells at both mRNA and protein
levels. DSC3 expression was restored in five out of seven cell lines
after 5-aza-2’-deoxycytidine (DAC) treatment. A heterogeneous methylation
pattern was detected by BS in promoter region and exon 1 of
DSC3. Methylation of DSC3 genomic sequences was found in 41% (41 out
of 99) of primary CRC, being associated with poor prognosis (p=0.002).
Transfection of p53 alone or in combination of DAC increased the DSC3
expression. Similarly, treatment with p53 inducer adriamycin alone or in
combination with DAC enhanced DSC3 expression.
Conclusions. DNA methylation contributes to downregulation of DSC3
in CRC cell lines. Methylation status of DSC3 DNA is a prognostic marker
for CRC. P53 appears to play an important role in regulating DSC3
expression.
SG-P-120
ECRG4 is frequently downregulated by promoter CpG island
hypermethylation in human breast cancer
W . Zhang1 1Zhejiang University, Institute of Pathology, Hangzhou, China
Aims. Esophageal cancer related gene4 (ECRG4) is a recently reported
candidate tumor suppressor gene frequently hypermethylated in several
human tumor types, including esophageal squamous cell carcinoma,
colorectal carcinoma, glioma and prostatic carcinoma. This study is to
90 | Der Pathologe · Supplement 1 · 2012
investigate if ECRG4 is transcriptionally silenced by promoter CpG island
methylation in breast cancer.
Methods. We analyzed several breast cell lines and 12 pairs of fresh samples
from breast cancer patients for ECRG4 promoter CpG island methylation
by COBRA and bisulfite sequencing. ECRG4 mRNA and protein
expression analysis was carried out by semi-quantitative RT-PCR, realtime
PCR, Western Blot and immunohistochemistry.
Results. We found that all of three immortalized normal breast cell lines
and three normal breast tissues expressed ECRG4 proteins, whereas
ECRG4 expression were reduced or absent in most breast cancer cell lines
and primary breast cancer samples. We also identified that ECRG4
promoter was frequently methylated in breast cancer samples. An inverse
correlation between mRNA expression and methylation status of
the ECRG4 promoter CpG island was observed in primary breast cancer
samples.
Conclusions. The expression of ECRG4 is frequently decreased due to
promoter CpG island hypermethylation in breast cancer.
SG-P-121
Clonality analysis of neuroendocrine cells in gastric adenocarcinoma
L . Wang1 , G . Yao1 , Z . Zhao2 , X . Wei1 , R . Xu3 1Institute of Pathology and Forensic Medicine, Zhejiang University,
Hangzhou, China, 2Zhejiang Provincial People’s Hospital, Hangzhou, China,
3Hangzhou First People’s Hospital, Hangzhou, China
Aims. Gastric cancer remains one of the most common cancers and the
leading causes of cancer death worldwide. Neuroendocrine differentiation
(NED) is a common phenomenon in adenocarcinomas, but there
have been only a few studies of NED in gastric adenocarcinoma. However,
it remains unclear whether the glandular and endocrine cells expand
from two distinct precursors, or arise from a single progenitor cell. Therefore,
we studied the clonality of neuroendocrine (NE) cells in gastric
adenocarcinoma.
Methods. In this study, 120 cases of gastric adenocarcinoma and corresponding
non-neoplastic gastric mucosal tissues were obtained, immunohistochemistry
was carried out using the primary antibody against
NE marker (chromogranin A). Frozen section immunohistochemistry
samples were selected with a large quantity of NE cells. Then laser-capture
microdissection (LCM) was performed to obtain NE cells from gastric
adenocarcinoma, ready for DNA extraction and subsequent genetic
analysis. DNA extraction from the captured cells and whole genome
amplification (WGA) were performed using DNA Micro-kit and DNA
Repli-g Midi kit to obtain a large quantity of DNA. Then we chose 26
microsatellite markers with genome-wide scope, and exon 7 and exon 8
of p53, designed primers, and performed PCR. Genome-wide microsatellite
abnormalities (MSI and LOH) and p53 mutation were detected by
PCR-SSCP silver staining and PCR sequencing to identify the clonality
of NE cells. Statistical analyses were performed using SPSS for Windows
version 15.0.
Results. Thirty samples from a total of 120 that contained a large number
of NE cells were chosen for LCM. Through LCM, about 500 NE cells
were precisely captured from each sample. The total incidence rate of
MSI was 27.4%, and LOH rate was 17.9%. The rates for adenocarcinoma
and NE cells were similar. There was no significant relationship between
the incidence rate of MSI or LOH and clinicopathological characteristics.
According to the coincidence of microsatellite changes, cases 2, 3, 5,
6, 11, 12, 18, 24, 27 and 30 had highest the concordance for the two types
of cells. The other samples had similar microsatellite changes, except for
cases 7 and 10. Most p53 mutations were detected in exons 7 and 8. Concordant
mutations were observed in samples 4, 14, 21 and 27, and there
were different mutations in the two types of cells in case 7. In case 17, mutation
was seen only in adenocarcinoma cells. p53 mutation occurred six
times in adenocarcinoma cells (20.0%) and five times in NE cells (16.7%).
Clinicopathological data showed that mutations were present in poorly

differentiated and TNM stage III or IV tumours. p53 mutation was closely
related with the degree of differentiation, TNM stage, vessel invasion
and lymph node metastasis. By combining the results with microsatellite
changes and p53 mutation, NE and adenocarcinoma cells showed the
same MSI, LOH or p53 mutation in most cases (27/30). In the other three
cases, different MSI, LOH or p53 mutation occurred.
Conclusion. we suggest that, in 27 of 30 cases, NE and adenocarcinoma
cells were generated from the same stem cells. The multipotent stem cells
differentiate to NE and adenocarcinoma cells, initiated by hormonal
change, microenvironmental change, and genomic instability. NE cells
act as parenchyma of carcinoma, and excrete hormones to promote carcinoma.
The remaining three cases might have had different ancestral
cells, and this needs further study.
SG-P-122
IGFBP7 high expression in the colorectal cancer cells is related
to Wnt signaling pathway inhibition during the interactions
between colorectal cancer cells and fibroblasts
J . Xu1 , H . Deng* 1 , C . Rao1 , S . Lin1 1Zhejiang University, School of Medicine, Hangzhou, China
Aims. To study the Wnt signaling pathway in interactions between the
colorectal cancer cells and fibroblasts and its impact on the expression of
IGFBP7 (Insulin-like growth factor binding protein 7) in the colorectal
cancer cells.
Methods. Colorectal cancer cells SW480 or SW620 were cultured in
HELF cells or activated HELF cells conditional media, treated by Wnt
signaling pathway agonist (LiCl) or Wnt signaling pathway inhibitor
(DKK-1). RT-PCR, Real-time PCR, Western blot and immunofluorescence
microscopy were used to detect the related protein and target genes
of Wnt signaling pathway and the expression of IGFBP7.
Results. IGFBP7 expression was increased with times in the colorectal
cancer cells treated by the activated HELF conditional media. And activation
of Wnt signaling pathway reduced IGFBP7 expression, inhibition
of Wnt signaling pathway induced IGFBP7 expression.
Conclusions. The interactions between tumor cells and fibroblasts could
inhibit Wnt signaling pathway in the tumor cells, and induce the expression
of IGFBP7.
SG-P-123
TGF-beta and Wnt signaling pathways regulate the expression of
IGFBP7 of fibroblasts in the tumor-stroma interactions
C . Rao1 , J . Xu1 , M . Liu1 , H . Deng* 1
1Zhejiang University, School of Medicine, Hangzhou, China
Aims. To find out the mechanism of the up-regulation of IGFBP7and the
biological changes in fibroblasts during the interactions with colorectal
cancer cells.
Methods. Fibroblasts (HELFs) were cultured in colorectal cancer cells
conditioned media (SW620-CM), treated by TGF-beta1, TGF-beta1 receptor
antagonist (SB431542), TGF-beta1 specific antibody (AF), Wnt
signaling pathway agonist (LiCl) and inhibitor (DKK-1) respectively. Q-
PCR, Western Blot, ELISA, Immunofluorescence microscopy and flow
cytometry were used to detect the expression of related targeted genes
and proteins of TGF-beta and Wnt signaling pathways.
Results. HELFs cultured in SW620-CM were activated with abundant
expression of alfa-SMA and showed strong proliferation and weak apoptosis
and senescence. The expression of IGFBP7 of HELFs was up-regulated
in time-dependent and dose-dependent manners when cultured
with SW620-CM, while TGF-beta signaling were activated as Smad2,
P-Smad2 and TGF-beta R2 were up-regulated in HELFs. These effects
could be strengthened by TGF-beta1 and inhibited by SB431542 or AF.
During the interactions, the downstream genes of Wnt signaling pathway
such as c-myc, cyclinD1 were up-regulated and the proteins of
Wnt signaling pathway such as beta-catenin, Dvl3, Dvl2 and Naked2
were obviously up-regulated which suggested the canonical Wnt signaling
pathway was also activated.
Conclusions. TGF-beta and Wnt signaling pathways were activated during
colorectal cancer cells-fibroblasts interactions. Upregulating of
IGFBP7 in HELFs was mainly through TGF-beta1/ALK5/ Smad-2 signaling
pathway. Wnt signaling pathway may also play an important role
in this process.
SG-P-124
Serum MiR-192 and MiR-194 as tumor biomarker for pancreatic
ductal adenocarcinoma
J . Zhang1 , C .-y . Zhao1 , D .-h . Yu1 , Y . Chen1 , Q .-h . Liu1 , M . Shi1 , J .-t . Zhang1 , G . Jin1 ,
P . Cheng1 , X .-g . Hu1 , C .-r . Ni1 , M .-h . Zhu1 1Department of Pathology, Changhai Hospital, Shanghai, China
Aims. To assess the validity of using serum miRNA signatures of PDAC
as biomarkers for diagnosis.
Methods. MiRNA microarray was used to detect the differences between
PDAC samples and normal pancreatic tissues. MiR-192 and miR-194
were found in the tissues of human PDAC and in the explants in tumorbearing
SCID mice by locked nucleic acid-based in situ hybridization
(LNA-ISH). Serum levels of miR-192 and miR-194 in PDAC patients,
duodenal adenocarcinoma patients and healthy controls, as well as six
pancreatic cancer cell lines and their culture supernatants were determined
by real-time PCR.
Results. Eight miRNAs were found overexpressed and eight were lowly
expressed in PDAC tissues compared with those in the normal pancreatic
tissues. MiR-192 and miR-194 were found overexpressed in the tissues
of human PDAC and in the explants in tumor-bearing SCID mice.
The levels of miR-192 and miR-194 in the supernatants of six pancreatic
cancer cell lines were positively correlated with their cellular expressions
(r=0.849, p

Abstracts
the pLenti6.3 to generate pLenti6.3-RC-U. Lentiviruses were packaged in
HEK 293T cells. The KRas mRNA and protein expression after transfecting
the pRC-U expression plasmid into human pancreatic cancer
cells or HEK293T cells were detected by real-time reverse transcription
polymerase chain reaction (real-time RT-PCR), Western blotting and
immunofluorescence. After pRC-U transfection, with mg-132 or cycloheximide
treatments, the KRas protein expression was tested by Western
blotting. The interaction between RC-U and KRas, whether KRas could
be ubiquitinated by RC-U or not were both investigated by immunoprecipitation.
The expression of HRas, NRas, phosphorylated extracellular
signal-regulated protein kinases (pERK) and extracellular signal-regulated
protein kinases (ERK) in pancreatic cancer cells was also examined
by Western blotting after pRC-U transfection. The effects of RC-U
on pancreatic cancer cell growth in vitro were detected by CCK-8 and
soft agar colony formation assays after lentivirus infection. In vivo, with
RC-U treatment, the volumes of the subcutaneous xenografts in nude
mice were measured.
Results. RC-U dramatically decreased the protein level of KRas compared
to the controls. No significant change of KRas mRNA expression
within different groups was observed. After pRC-U transfection, the
stability of KRas was significantly increased in the presence of specific
proteasome inhibitor mg-132. HEK293T cells were transfected by mutant
KRas construct together with either pRC-U or empty vector and then
incubated with cycloheximide to inhibit novel protein synthesis. The
exogeneous mutant KRas oncoprotein in pRC-U transfected cells was
degraded more quickly than that in controls. RC-U can bind with KRas.
KRas can be ubiquitinated by RC-U. It was shown that RC-U also degradate
Kras as well as HRas and NRas. The protein expression of pERK was
also decreased, but there was no significant change in total ERK expression.
When the pancreatic cancer cells infected with lentivirus expressing
RC-U, the ability of the cell growth in vitro was decreased. In vivo,
the reduced volumes of the subcutaneous xenografts in nude mice with
RC-U treatment group versus the control group were observed.
Conclusions. Our findings for the first time, implicate the chimeric ubiquitin
ligase RC-U can decrease KRas protein expression. Destruction
of KRas by RC-U through a ubiquitin-dependent, proteasome-mediated
degradation pathway. RC-U degradates HRas and NRas simultaneously.
RC-U inhibited pancreatic cancer cell growth in vitro and in vivo. This
may represent an effective alternative strategy for the targeted therapy of
KRas in pancreatic cancers.
SG-P-126
Neuropilin-2 in progression and therapy resistance of pancreatic
cancer
M . Muders1 , M .J . Stanton2 , S . Dutta2 , P . Hönscheid1 , D . Aust1 , K . Datta2 ,
G .B . Baretton1 1Institute of Pathology, University Hospital “Carl-Gustav-Carus”, Dresden,
2Department of Biochemistry, University of Nebraska Medical Center,
Omaha, United States
Aims. Exocrine pancreatic adenocarcinoma is a deadly disease with
5-year survival rates of 25 to 30 percent in patients without and 10 percent
in patients with lymph node metastasis. Surgical resection is the only
curative treatment option. Unfortunately only 15 to 20 percent of pancreatic
cancer patients are eligible for curative surgical therapy due to
advanced disease at presentation. Therefore, new treatment options are
urgently needed.
Methods. To investigate the role of Neuropilin-2 in therapy resistance
of pancreatic cancer we knocked down Neuropilin-2 and its ligand
VEGF-C by RNA interference in standard pancreatic cancer cells lines
and treated these cell lines with Gemcitabine. After treatment the cell
death was measured using a YO-PRO/PI apoptosis assay. The role of autophagy
in the treatment resistance was tested by a standard autophagic
flux assay.
92 | Der Pathologe · Supplement 1 · 2012
Results. Neuropilin-2 and its ligand VEGF-C are important molecules
of chemotherapy resistance in pancreatic cancer. Furthermore, we have
found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy,
which maintains cancer cell survival following treatment.
Conclusions. Together, these data suggest a link between the VEGF-C/
NRP-2 axis in pancreatic cancer cell progression and therapy resistance.
Effective targeting of this pathway may lead to the development of new
cancer therapies.
SG-P-127
Salinomycin induces autophagic and apoptotic cell death in pancreatic
carcinoma cell lines
M . Vogt1 , B . Verdoodt1 , S .-T . Liffers1 , A . Tannapfel1 , A . Mirmohammadsadegh1 1Ruhr-University of Bochum, Institute of Pathology, Bochum
Aims. Salinomycin a polyether antibiotic, is produced by a strain of
streptomyces albus and has anti-microbial and anti-coccodial activities.
Recently, a number of studies described anti-tumor properties of salinomycin,
in particular its effect on chemoresistant tumor initiating cells.
In the present study, we investigated the impact of salinomycin mediated
activation of MEK/ERK signalling pathway on autophagy and apoptotic
cell death in pancreatic cancer cell lines
Methods. Two pancreatic cell lines MIAPaCa-2 and PaTu8902 were used
to analyze the effect of salinomycin on cell viability, autophagy and
apoptosis. The effect of salinomycin on autophgay was investigated by
transmission electron microscopy (TEM) for detection of autophagic
vesicles and processing of LC3B, microtubule-associated protein 1 light
chain 3 isoform B (LC3B). Towards investigating the role of salinomycin
on apoptotic cell death we used caspase3/7, FACS, Western blot analysis
and immunofluorescence staining.
Results. Salinomycin treatment inhibits cell viability and colony forming
in MIA PaCa-2 and PaTu8902 in a time and concentration depending
manner. In both cell lines salinomycin was able to induce autophagic cell
death, detected by LC3 processing and formation of autophagic vacoules.
Salinomycin was able to induce autophagic and apoptotic cell death
in MIAPaCa-2 cell lines. In MIAPaCa-2 cells the salinomycin induced
autophagic cell death was dependent on ERK1/2 pathway. In contrast,
salinomycin induced autophagic cell death in PaTu8902 was independent
of ERK1/2.
Conclusions. Salinomycin, a novel anti-tumor drug is able to induce autophagic
and apoptotic cell death depending on cellular background.
SG-P-128
Up-regulation of Kindlin-2 promotes progression of human
breast cancer cells by increasing their proliferation, drug resistance,
genomic instability, and tumorigenesis
W . Fang1 , T . Zhao1 , H .-q . Zhang2 1Department of Pathology, Key Laboratory of Carcinogenesis and Translational
Research, Beijing, China, 2Peking University Health Science Center,
Beijing, China
Aims. Kindlin-2 has been confirmed as an essential element of bidirectional
integrin signaling. In recent years, the relationship between Kindlin-2
expression and cancers has been a focus of interest. Our previous
studies have shown that Kindlin-2 expression was up-regulated in several
types of human cancers, and a strong correlation between Kindlin-2
expression and clinical outcome of breast cancer patients was found.
However, the functional role of Kindlin-2 in breast cancer has not been
studied. This study was designed to investigate the role of Kindlin-2 in
the progression of human breast cancer cells.
Methods. Firstly, Kindlin-2 expression at protein level was detected by
Western blot in several breast cancer cell lines. Two luminal-like breast
cancer cell lines, MCF-7 and T47D, expressed low level of Kindlin-2.
Two basal-like breast cancer cell lines, MDA-MB-231 and HS578T, ex-

pressed moderate levels of the protein. Then, Kindlin-2 gene was overexpressed
by transfected into MCF-7 cells. In comparison, short hairpin
RNA (ShRNA)-mediated knockdown of Kindlin-2 was performed in
HS578T cells. Vector controls were also done in the same cell lines. Ki67
Li, FCM cell cycle, anchorage-independent colony formation (in vitro
tumorigenesis), and in vivo tumorigenesis in NOD/SCID mice were observed.
Apoptotic cells were labeled by fluorescent annexin V assay and
quantified by FACS. Array CGH analysis and spectral karyotyping were
performed to detect the genomic instability of these cells.
Results. The growth rate of Kindlin-2-transfected MCF-7 cells was much
quicker than that of the controls. The proportion of G2-M phase cells,
clone formation and tumorigenicity were significantly higher than these
of the controls. The change of Kindlin-2-ShRNA transfected cells was
just the reverse. Moreover, Up-regulation of Kindlin-2 can also reduce
the rate of apoptosis induced by the chemotherapy drugs, and these cells
showed much more genomic instability compared with the controls.
Conclusions. These findings suggested that up-regulation of Kindlin-2
promotes the progression of human breast cancer cells by increasing
their proliferation, drug resistance, genomic instability, and tumorigenesis.
SG-P-129
DKK3 and ITIH5 gene methylation as novel biomarkers for bloodbased
breast cancer screening: Improving early detection of
breast cancer
V . Kloten1 , B . Becker1 , M .G . Schrauder2 , P .A . Fasching2 , A . Hartmann3 ,
J . Veeck1 , R . Knüchel1 , E . Dahl1 1University Hospital of the RWTH Aachen, Institute of Pathology, Aachen,
2 3 University Hospital Erlangen, University Breast Center, Erlangen, University
Hospital Erlangen, Erlangen
Aims. For early detection of breast cancer the development of robust
blood-based biomarkers that accurately reflect the host tumor is mandatory
and thus a growing field of research. The most common alterations
in human cancers including breast cancer are changes in the status of
DNA methylation, which are therefore quickly emerging as a new pool
of potential biomarkers. Thus, we investigated the feasibility of detecting
aberrant tumor suppressor gene methylation in cancer cell-derived free
circulating DNA in the bloodstream of patients.
Methods. Using qualitative MSP, we examined the methylation status of
six biologically significant putative tumor suppressor genes, i.e. ITIH5,
DKK3, WIF1, SFRP1, SFRP2 and SFRP5 in DNA extracted from serum.
Clinical performance was determined in a large training study on 150
serum samples (120 breast cancers, 30 healthy controls). 20 benign disease
and 30 colon cancer serum samples were included for additional
specificity testing.
Results. Based on the training study we could evaluate the top candidate
biomarkers with the best values for sensitivity and specificity. A marker
panel of DKK3 and ITIH5 detected breast cancer with a sensitivity of 46%
(55/120). Specificity of the panel was sufficient with 83%, 100% and 93% in
colon cancer samples, benign and healthy control samples, respectively.
Control samples revealed unacceptable high methylation rates of SFRP1
and SFRP5 in DNA extracted from colon cancer sera, whereas SFRP2
and WIF1 showed a considerable methylation frequency in sera from
healthy controls.
Conclusions. The current study suggests that cancer-specific methylation
of ITIH5 and DKK3 in serum-derived tumor-borne DNA might be
valuable biomarkers for the early detection of breast cancer. In the second
phase of this project we are currently validating ITIH5 and DKK3
as reliable methylation biodiagnostic markers in an independent test set
consisting of 160 breast cancer serum samples and 160 control samples.
SG-P-130
Forced expression of ITIH5 in a luminal-type breast cancer model
confers growth suppressive properties
S . Huth1 , R . Knüchel1 , E . Dahl1 1University Hospital of the RWTH Aachen, Institute of Pathology, Aachen
Aims. Inter-α-trypsin inhibitors are protease inhibitors which consist of
one light chain (bikunin) and different heavy chains (ITIHs). We recently
characterized ITIH5 as a novel member of the ITIH gene family and
showed that its loss in human breast cancer is associated with parameters
of tumour invasion and distant metastasis. Thus, we aimed to analyze
the biological function of ITIH5 in an in vitro model of breast cancer,
the luminal-type breast cancer cell line T47-D.
Methods. T47-D clones expressing ITIH5 and corresponding mock
(empty vector) clones were generated using standard methods and subsequently
validated on DNA, mRNA and protein level. Functional analyses
of ITIH5 clones were performed by standardized assays including
XTT, colony formation, cell-matrix-adhesion and Apo-ONE® Homogeneous
Caspase-3/7 Assay.
Results. Forced ITIH5 expression in T47-D transfectants was successfully
achieved validating integration and expression of ITIH5 on the DNA,
mRNA and protein level, respectively. Using functional assays we observed
significantly reduced proliferation (p=0.01) as well as significantly
increased cell-matrix-adhesion (p

Abstracts
SG-P-132
SiRNA inhibition of Shp-2 reduces the GC phenotype of Burkitt’s
lymphoma cell lines
X . Jiang 1 , R . Zhou 1 , L . He 1 , C . He 1 , J . Wang 1
1 Zhejiang University, School of Medicine, Hangzhou, China
Aims. To investigate the potential role of Shp2 in the GC phenotype regulation
of BL cell lines.
Methods. We used either scrambled siRNA or Shp2 siRNA pools to
transfect human BL cell lines (Daudi, Raji, Ramos). We also expressed
a PTP-inactive Shp2 mutant (Shp2CS) that had the catalytic Cys-459 replaced
with Ser in Raji cells to verify whether the Shp2 PTP activity is required
in BL cells growth. And we analyzed the effects of Src inhibitor-1
(Sigma) and Mek inhibitors (U0126) in BL cells.
Results. Shp2 had been effectively silenced by the Shp-2 siRNA. Shp2
knockdown led to a decrease in Erk1/2 phosphorylation. Concomitantly,
we observed a decrease in the phosphorylation of the tyrosine residue
Y416 in the activation loop of Src, whereas Stat3 and Akt phosphorylations
were not affected. Similar to Shp2 knockdown in BL cell lines, the
C/S protein in Raji cells inhibited ERK1/2 and Src phosphorylation. The
abrogation of Shp2 in both cell lines significantly impaired cellular proliferation
over time compared with cells grown to the control cell lines.
Cell cycle analysis revealed that Shp2 knock down led to a significant
increase in the percentage of BL cells in the G1 phase and a corresponding
decrease in the S and G2-M phases, without changes in the apoptotic
fraction as indicated by the sub-G0-G1 populations. Thus knockdown
of Shp2 significantly inhibited BL tumor growth. We also found
that shp2 inhibition led to a decrease in CD77 positivity in BL cell lines.
Concomitantly, the protein levels of Bcl-6, E2A, AID and Pax-5 were
substantially lower in Shp2-siRNA BL cells than in their Shp2-positive
counterparts. Biochemical analysis also showed that Shp2 knockdown
resulted in down-regulation of c-Myc in BL cells. And Src inhibitor-1 and
U0126 caused decreases of c-Myc and GC factors as CD77, Bcl-6, AID,
E2A and Pax-5 expression level in BL cells. These results indicate that
Src and Erk1/2 activities are necessary for a constitutive GC phenotype
in BL cells.
Conclusions. In this study, we showed that Shp2 regulated BL cells proliferation
in cell culture. Reduction of Shp2 expression led to a decrease
of c-Myc and GC factors (Bcl-6, E2A, AID and Pax-5) in BL cell lines
through Src and Erk1/2 pathway, which suggested Shp2 play a key role in
the series of regulative factors of the GC phenotype in BL cells.
Keywords. Shp2, Burkitt’s lymphoma, GC phenotype
Grants. the NSF of Zhejiang Province (No.Y2090167) and Research Foundation
in Zhejiang Scientific and Technical Bureau (No.2009C33039).
SG-P-133
Identification of lymphoma subtype independent micro RNA
expression profiles
D . Lenze1 *, M .R . Schweiger2 *, M . Piechotta 3 , K . Zimmermann4 , A . Fischer2 ,
S . Boerno2 , C . Dieterich3 , U . Leser4 , M . Hummel1 1 2 Charité – Universitätsmedizin Berlin, Institute of Pathology, Berlin, Max
Planck Institute for Molecular Genetics, Berlin, 3Max-Delbrueck-Center for
Molecular Medicine, Institute for Medical Systems Biology, Berlin, 4Hum boldt-University, Institute for Informatics, Berlin
Aims. The molecular analyses of human cancers revealed that great heterogeneity
on the molecular level exists even within the same tumor
entity which might explain different therapeutic outcome to the same
type of treatment. This holds also true for lymphoma. Therefore it is justified
to assume that lymphomas carrying the same molecular features
might benefit from the same targeted treatment modalities irrespective
of their histological or immunophenotypical features. It was the goal of
our study to identify overlapping molecular characteristics in a variety
of histologically defined lymphoma entities. To achieve this goal, comprehensive
expression profiles of non-coding and coding transcripts de-
94 | Der Pathologe · Supplement 1 · 2012
rived from a broad spectrum of lymphomas were established. Our data
show that overlapping molecular features can be identified beyond the
current lymphoma classification.
Methods. RNA was extracted from frozen tissue blocks of distinct human
B- and T-cell lymphoma entities as well as tonsils (n=116). The small
(micro) RNA fraction was sequenced by next generation technology
(SOLiD) and total RNA was subjected to Affymetrix Exon 1.0 ST array
hybridisation. In addition immunohistochemical and clinical data was
acquired. Bioinformatic analyses were then applied for subgroup detection.
Results. Unsupervised clustering based on the mature micro RNA expression
derived from deep sequencing demonstrated the presence of
two distinct large clusters within the case collection. One cluster contained
predominantly the so-called indolent lymphoma types, but also a
considerable number of aggressive B-cell lymphoma cases. In the second
cluster the majority of cases were aggressive B-cell lymphoma but interestingly
intermingled with the T-cell lymphoma cases. Differentially
expressed micro RNAs between the two clusters were determined and
logistic regression identified a micro RNA classificator able to distinguish
the two groups. The micro RNA expression data was combined with
the coding RNA data in order to identify the involved pathways.
Conclusions. Micro RNA expression profiles obtained by deep sequencing
were able to identify two groups within a lymphoma collection that
separated the cases beyond the histopathological classification system.
Discriminative micro RNAs and associated pathways were identified.
These findings give new insights into lymphoma biology and point to
alternative treatment options.
*These authors contributed equally to the study .
Poster: GI-Trakt: Ösophagus, Magen
FR-P-036
Acanthosis nigricans: a paraneoplastic condition of the esophagus
D . Karimi1 , J . Siveke2 , M . Ringelhahn2 , M . Bettstetter3 , M . Sarbia1 1 2 Pathology München-Nord, Department of Gastroenterology Technical
University München, 3Institute of Molecular Pathology Südbayern
Aims. Presentation of endoskopic and histologic findings of esophageal
acanthosis nigricans.
Methods. The case of a 69-year-old man with dysphagia and weight loss
will be presented.
Results. Endoscopic examination revealed an adenocarcinoma of the
esophagogastric junction as well as multiple small polyps in the middle
and the lower thirds of the esophagus. Histological examination showed
papilloma-like proliferations without atypia, which were diagnosed as
acanthosis nigricans of the esophagus. After completion of the staging
investigation regarding the cardiac carcinoma, combination chemotherapy
was started because of the presence of liver metastases. Subsequently,
partial regression of the carcinoma as well as of the esophageal lesions
was noted.
Conclusions. Acanthosis nigricans is a rare paraneoplastic disease of the
esophagus. As an indicator lesion, its detection should prompt a search
for a malignant tumor in the gastrointestinal tract.

FR-P-037
MALDI imaging reveals COX7A2, TAGLN2 and S100-A10 as novel
prognostic markers in Barrett’s Cancer
M . Elsner 1 , S . Rauser 1 , S . Maier 1 , C . Schöne 1 , B . Balluff 1 , S . Meding 1 , G . Jung 1 , M .
Nipp 1 , H . Sarioglu 1 , G . Maccarone 2 , U . Jütting 1 , A . Feuchtinger 1 , R . Langer 3 , M .
Feith 3 , B . Küster 4 , M . Ueffing 1 , H . Zitzelsberger 1 , H . Höfler 3 , A . Walch 1
1 Helmholtz-Zentrum München, Neuherberg, 2 Max Planck Institute of
Psychiatry, München, 3 Technische Universität München, Neuherberg, 4 Technische
Universität München, Chair of Proteomics and Bioanalytics, München
Aims. In order to characterize proteomic changes found in Barrett’s cancer
and its premalignant stages, the proteomic profiles of histologically
proposed precursor and invasive carcinoma lesions were analyzed by
MALDI imaging mass spectrometry (MALDI imaging).
Methods. For a primary proteomic screening, a discovery cohort of 38
fresh frozen Barrett’s Cancer samples was used, with the goal to find intestinal
metaplasia and Barrett’s cancer specific proteins that might be
used as markers for cancer development as well as for lymph node metastasis
and disease outcome. Based on this cohort we studied 33 areas of
Barrett’s Cancer and 11 areas of intestinal metaplasia. Protein identification
was done by mass spectrometry. To validate the identified proteins,
immunohistochemistry was performed on an independent validation
set of 102 Barrett’s Cancer cases and the results were correlated to clinical
data.
Results. We detected 60 m/z species that are significantly differentially
expressed in intestinal metaplasia and invasive carcinoma (p50 IEL/HPF) and mild (

Abstracts
FR-P-040
MicroRNA expression profiling for prediction of resistance to
neoadjuvant radiochemotherapy in squamous cell carcinoma of
the oesophagus
J . Slotta-Huspenina 1 , E . Drecoll 1 , M . Feith 2 , C . Wagner 3 , H . Höfler 1 , 4 ,
K . Becker 1 , R . Langer 1
1 Technical University of Munich, Institute of Pathology, München, 2 Technical
University of Munich, Department of Surgery, 3 IMGM Laboratories GmbH,
Martiensried, 4 Institute of Pathology, Helmholtz-Zentrum München, Oberschleissheim
Aims. MicroRNA (miRNA) expression has been shown to play an important
role in biology of malignant tumours, including sensitivity to
chemotherapy and radiation. Neoadjuvant radiochemotherapy (RCTX)
followed by surgery is a standard treatment strategy for locally advanced
oesophageal squamous cell carcinoma (ESCC). However, a subset
of patients does not respond to RCTX. In the present study we evaluated
whether miRNA profiles can predict resistance to RCTX in ESCC.
Methods. 31 patients with locally advanced ESCC (cT3-4, cN1-3, M0-1)
underwent preoperative radiochemotherapy with cisplatin, 5-fluorouracil
and 30-45 Gy, followed by resection of the oesophagus. Tumour response
was evaluated by histopathological tumour regression. MiRNA
profiling was done in pre-therapeutic formalin-fixed and paraffin embedded
(FFPE) biopsies using the Agilent Human Microarray platform
(Release 16.0), encompassing 1205 human miRNAs. Differential expression
was identified in responders (n=15) and non-responders (n=16) by
applying appropriate biostatistics to the data and validated by real-time
quantitative PCR (qRT-PCR).
Results. Even if the miRNA expression profiles of pre-therapeutic ESCC
biopsies within and between non-responders (n=16) and responders
(n=15) were highly similar (average correlation coefficients r=0.96, 0.94
and 0.95), ten differentially expressed miRNAs could be identified by microarray
analysis in non-responders and responders of ESCC (p

in the context of therapy response before. Immunohistochemistry of
the mitochondrial protein COX7A2 in the validation set confirmed the
MALDI Imaging results and revealed the predictive impact of COX7A2
(p=0.0015). By electron microscopy we found, that the loss of these proteins
is strongly associated with a severe mitochondrial damage.
Conclusions. MALDI Imaging is able to detect novel proteomic patterns
distinguishing responders from non-responders. These protein patterns
provide new insights in mechanisms of response. For the first time we
could show that mitochondrial dysfunction is a predisposition for response
to neoadjuvant chemotherapy in Barrett’s cancer.
FR-P-043
Heterogeneity of TP53 mutations in gastric adenocarcinomas as
well as their corresponding lymph node metastases
D . Mones1 , G . Cadeddu1 , K .-L . Schäfer1 , H .E . Gabbert1 , S .E . Baldus1 1University of Düsseldorf, Institute of Pathology, Düsseldorf
Aims. The frequency of TP53 mutations in gastric adenocarcinomas as
well as the prognostic and predictive value of this biomarker is still controversially
discussed. In order to elucidate if genetic mosaicism may
explain at least in part the conflicting results obtained in previous studies,
we investigated the intratumoral heterogeneity of TP53 mutations
analysing tissue specimens from tumor center, invasion front and lymphonodal
metastases.
Methods. We studied a series of 75 gastric adenocarcinomas comprising
25 diffuse type, 24 intestinal type and 26 mixed type carcinomas according
to Laurén. DNA of the paraffin-embedded tissue samples from tumor
center and invasion front (n=75) as well as corresponding lymph
node metastases (n=47) was obtained by microscopically controlled manual
microdissection. DNA was purified and subjected to PCR amplification
of TP53 exon 5–8 followed by direct sequencing.
Results. TP53 mutations were observed in 34 of the 75 primary tumors
(45%). Intratumoral heterogeneity of TP53 mutations was found in 13
primary tumors (17%): Two samples showed the mutation only in the
invasion front, ten cases only in the tumor center and one case showed
different mutations in invasion front and tumor center. There was no
significant difference between the mutation rates in diffuse type (40%)
compared to intestinal type adenocarcinomas (50%), while mixed type
carcinomas showed a mutation in 46% of the cases. In addition, there
was no difference between pT1/pT2 tumors (46%) and pT3/pT4 tumors
(45%). Mutations in the lymph node metastases were found in 19 of 47
specimens (40%). Genetic heterogeneity between primary tumor and
lymph node metastases was observed in 12 of the 47 cases (26%) comprising
six cases showing the TP53 mutation only in the primary tumor,
but not in the lymph node metastasis as well as four cases exhibiting a
mutation in the lymph node metastasis, but not in the primary tumor.
In two cases different mutations in primary tumor and corresponding
lymph node metastasis were observed.
Conclusions. Intratumoral heterogeneity of TP53 mutations is present in
17% of gastric adenocarcinomas and may therefore contribute to the controversial
results regarding the prognostic value of the TP53 status. In
addition, in 26% of the cases heterogeneity between TP53 mutations in
primary tumors and lymph node metastases was observed. This should
be considered in future studies on the predictive and prognostic value of
TP53 mutation analysis in gastric adenocarcinomas.
FR-P-044
Gastric Polyvinylpyrrolidon (PVP) Athrozytosis in chronic hemodialysed
patients
S .A . Thies1 , B . Kaduk2 , R . Ott3 , S . Turi4 , M . Sarbia5 1University of Zurich, Institute of Surgical Pathology, Zürich, Switzerland,
2 3 Kaduk Medical Service (KMS) GmbH, Baar, Switzerland, Gastroenterologie
Bogenhausen, Gemeinschaftspraxis Dr . Schatke, Munich, 4Medizentrum Erlangen, Gemeinschaftspraxis Internist/Gastroenterologie, Erlangen,
5Pathologie München Nord, Munich
Aims. The originally as plasma expander developed and applied high
molecular Polyvinylpyrrolidone (PVP) – a polymer of the monomer
N-Vinylpyrrolidon cannot be eliminated by diuresis nor metabolised
by liver. These PVP polymers were phagocytosed by macrophages and
permanently arrested. According to the type and topos of application of
PVP – intradermal, subcutaneous, intracavitary, intraperitoneal, interpleural
or intravenous – the phenotype of this storage disease differs and
is predictive. The only non-predictive manifestation and severity of the
accumulation and conditio sine qua non is due to the entrance of the
high molecular PVP polymers into the blood circulation. This induces a
high risk situation for the patients. For that reason high molecular PVP
is not yet used as plasma expander. But nevertheless there is an ubiquitary
(generalised) PVP-thesaurismosis, e.g. using PVP-coating fibre filters
in human hemodialysis.
Methods. We examined gastric biopsies of two patients with chronic hemodialysis
in history without defined clinical question. The main histological
topic was aggregates of isolated or grouped large macrophages
with a certain but non-characteristic staining pattern (indirect indicators
for PVP; i.e. advice):
– H&E: intracytoplasmic blue-gray globules
– Sirius red: brightly red deposits on a pale yellow background
– Argentic impregnation: markedly coloured dark black deposits
– PAS: negative
– v. Kossa: negative
– Immunocytochemically: CD68+++
Results. The definitive morphological typing of the macrophages as high
molecular PVP-containing macrophages could only approved by the
investigation of the ultrastructure in transmission electron microscopy
(direct indicator for PVP; i.e. evidence): intracytoplasmic globules of different
size and periodic lamellate internal structure.
Conclusions. The results of this study are essential for the manufacture
of dialyse membranes by advancement the adhesion of PVP and for the
patients to prohibit the generalized PVP-thesaurismosis. Also the findings
are helpful for the clinicians, notably for nephrologists in the surveillance
of chronic hemodialysed patients, for gastroenterologists and
for pathologists to receive an algorithm of the diagnostic relevant panel.
FR-P-045
Interaction of cathepsin X with galectin-2 in H. pylori dependent
gastric carcinogenesis
A . Teller1 , D . Kuester1 , A . Roessner1 , S . Krueger1 1Otto-v .-Guericke University, Department of Pathology, Magdeburg
Aims. Our previous studies have shown an association between Helicobacter
pylori infection, the strong up-regulation of cathepsin X (CTSX),
and the development of gastric cancer. The physiological function of
CTSX still needs to be clarified. In a yeast two-hybrid screen we could
identify galectin-2 as a novel interaction partner of cathepsin X. Now we
suppose an interactive role of CTSX and galectin-2 in modulation of the
immune system in response to H. pylori-infected gastric epithelial cells.
Methods. Background for further experiments was the screening of the
yeast two-hybrid system, where we could identify galectin-2 as a novel
interaction partner of CTSX. Using Western blots and immunohistochemistry
we want to analyze galectin-2 levels in biopsy specimens of
H. pylori-infected and non-infected patients as well as in gastric cancer
Der Pathologe · Supplement 1 · 2012 |
97

Abstracts
samples which showed increased CTSX levels in disease progression.
Functional consequences of the interaction of CTSX and galectin-2 were
tested on siRNA treated primary human epithelial and immune cells in
confrontation and migration experiments with and without infection
with H. pylori.
Results. Interaction of CTSX and galectin-2 was clearly characterized
by immunoprecipitation, double immunofluorescence and pull-down
assays. Immunohistochemistry and western blots on tissue samples
indicated an inversely expression of CTSX and galectin-2. H. pylori-negative
samples showed low expression of CTSX but high expression of
galectin-2, whereas galectin-2 expression decreased and CTSX increased
with progression of disease. Macrophages with high expression of CTSX
rapidly migrate into epithelial cell monolayers and down regulate whereby
their galectin-2 levels.
Conclusions. The interaction of CTSX and galectin-2 seems to be a major
regulatory element to regulate the activity of the immune system against
H. pylori colonization and cancer development. As both enzymes known
to be effecting T-cell function and spreading our further experiments
will focus on possible mechanisms to induce an efficient anti-tumoral
immune response by influencing CTSX/galectin-2 signalling.
FR-P-046
The impact of HER2 amplification in the dysplasia-carcinomasequence
in the stomach
T . Vlajnic1 , S . Eppenberger1 , S . Schneider1 , L . Terracciano 1 , L . Tornillo1 ,
G . Cathomas2 1 2 Institute of Pathology, University Hospital Basel, Switzerland, Cantonal
Institute of Pathology, Liestal, Switzerland
Aims. HER2 amplification and overexpression was demonstrated in
gastric carcinoma soon after its discovery in breast carcinoma. There is
growing evidence that HER2 has an important role in tumorigenesis in
gastric cancer with a reported prevalence of amplification/overexpression
in 7–34%. However, the role of HER2 in the progression of dysplasia
to gastric carcinoma has not yet been investigated. The aim of this study
was to determine the HER2 status in precursor lesions of gastric carcinoma,
i.e. in gastric dysplasia and early cancer.
Methods. A tissue microarray consisting of gastric carcinomas (n=370)
was evaluated immunohistochemically and by FISH and SISH analysis
for the HER2 status. Whole tissue sections with gastric cancer (n=206)
were then re-evaluated for gastric dysplasia and in case of presence of
dysplasia next to carcinoma an immunohistochemical analysis was performed.
Additionally, immunohistochemistry and SISH analysis was
performed on gastric biopsies with dysplasia (n=62) without coexisting
carcinoma.
Results. HER2 amplification was found in 7.9% of gastric carcinomas.
50 cases showed gastric dysplasia next to carcinoma and the HER2 status
in the dysplasia was the same as in the respective invasive carcinoma.
However, the prevalence of HER2 amplification in the cases of dysplasia
alone was only 3.2%.
Conclusions. Interestingly, our data indicate that HER2 amplification/
overexpression may be an early event and may induce a rapid progression
from dysplasia to invasive carcinoma in the stomach. Further studies
are needed to elucidate the potential role for the anti-HER2 targeted
therapy in patients with gastric dysplasia.
98 | Der Pathologe · Supplement 1 · 2012
FR-P-047
Epstein-Barr virus (EBV) in the development of gastric cancer
M . Cathomas1 , V . Genitsch1 , L .M . Terracciano 2 , L . Tornillo2 , A . Lugli2 ,
A .H . Marx3 , G . Sauter3 , F . Carneiro4 , F . Hofstädter5 , N . Willi1 , G . Cathomas1 1 2 Institute of Pathology, Liestal, Switzerland, Institute of Pathology, University
Basel, Switzerland, 3Institute of Pathology, University Medical Center
Hamburg-Eppendorf, Hamburg, 4Institute of Molecular Pathology of the
University of Porto (IPATIMUP), Portugal, Portugal, 5Institute of Pathology,
University Regensburg, Regensburg
Aims. Epstein-Barr virus (EBV) infections are associated with a number
of tumours, including lymphoproliferative diseases and nasopharyngeal
carcinomas. In addition, a subset of gastric tumours has been associated
with EBV, ranging from 1.3% to 20.2% of all gastric cancers. The role
of EBV in the development and progression of gastric cancer, however,
remains to be elucidated. Aim of the study was the assessment of the prevalence
of EBV associated gastric cancers and the presence of the virus
in the development of individual tumours.
Methods. Based on tissue micro arrays (TMA), the presence of EBV was
evaluated in gastric cancers of 5 institutions within Europe (Liestal and
Basel, Switzerland; Hamburg and Regensburg; Porto, Portugal) by using
a commercial in situ hybridization assay for EBER. In a second step, nontumorous
and tumorous tissue including dysplasia, intramucosal and
invasive carcinomas as well as metastasis were analyzed for the presence
of EBV.
Results. A total of 610 (91.6%) of 666 tumours on TMAs were available
for analysis. Overall, 4.9% of gastric cancers were positive for EBER, ranging
form 2.2% to 9.1% within the different institutions. No age difference
was observed within EBV positive and negative patients [68.2 vs.
66.2 (n=23/515)], but EBV positive tumours showed a male predominance
[87.0% vs. 60.6%; p

on primary cells of wild-type and cathepsin X knockout epithelial cells
with infection by H. pylori were performed to confirm the descriptive
data on tissue samples.
Results. Galectin-2 is constitutively expressed in gastric mucosa of wildtype
and cathepsin-X knockout mice. Galectin-2 expression is decreased
in gastric inflammation and spasmolytic polypeptide-expressing metaplasia
(SPEM). H. pylori infection leads to lower mRNA- and protein-levels
of galectin-2 in wild-type and cathepsin-X knockout mice. However,
levels of galectin-2 were significantly higher in cathepsin-X knockout
mice compared to wild-type mice independent of H. pylori infection.
Infection of primary cells revealed comparable results.
Conclusions. Because galectin-2 expression is decreased in gastric inflammation
and cathepsin-X is increased we propose an inverse regulation
of these molecules in gastric inflammatory disease. Since there is an
intense and smooth expression of galectin-2 in healthy gastric mucosa,
treatment of patients with galectin-2 could help to prevent epithelial damage
and thus decelerate gastric carcinogenesis.
FR-P-049
Krukenberg tumor with partial yolk sac differentiation or collision
tumor? A case report
A . Weber1 , A . Schmieder1 , K .-H . Berghaeuser1 , T . Friedrich1 1Institut of Pathology, Saalfeld
Aims. A 28-year-old woman suffered from a diffuse infiltrating gastric
carcinoma. After chemotherapy with ECF, subtotal gastrectomy with
Roux-Y reconstruction was performed before.
Methods. In microscopic examination of the tumor diffuse carcinoma
without any other differentiation was diagnosed. Tumor regression was
estimated to 40% at time of surgery staging was ypT2b ypN0 cm1 (PER).
Despite of adjuvant chemotherapy six months later malignant ascites
and peritoneal dissemination of the tumor with the same differentiation
occurred. Two years later two metastases were removed from the
peritoneum with the same histological appearance. At that time both
enlarged ovaries were removed.
Results. Histologically, in both ovaries metastases of the diffuse carcinoma
were found. In addition in the left side was a definite area with large
clear cells according to yolk sac differentiation. Immunhistologically
this area with cells reacted positively with AFP and CK 7.
Conclusions. Adenocarcinomas of stomach with yolk sac differentiation
are extremely rare. It remains to be clarified if in this case there is one of
this rare adenocarcinomas or a collision tumor combining metastases of
signet ring cell carcinoma of the stomach with a primary yolk sac tumor
of the ovary.
FR-P-050
FoxO6 expression in gastric cancer
J . Haag1 , B . Ingold-Heppner2 , H .-M . Behrens1 , T . Aigner3 , C . Röcken1 1 2 Christian-Albrechts-University Kiel, Institute of Pathology, Kiel, Charité
Campus Mitte, Institute of Pathology, Berlin, 3Klinikum Coburg, Institute of
Pathology
Aims. The expression of the transcription factor FoxO6 in gastric cancer
and the correlation of FoxO6 expression levels to clinicopathological tumor
characteristics were evaluated.
Methods. Study population: 485 cases with cancer of the stomach or oesophagogastric
junction were characterized for type of surgery, age at
diagnosis, gender, tumor localization and tumor size, tumor type, tumor
grade, depth of invasion, number of lymph nodes resected, and number
of lymph nodes containing metastases. Tumors were classified according
to the Laurén classification and the mucin phenotype. pTNM-stage of all
study patients was determined according to the 7th edition of the UICC
guidelines and our recent proposal (“Kiel-stage”). Mismatch DNA repair
capacity and microsatellite instability was analyzed by immuno-
histochemistry and a pentaplex PCR assay. Analysis of FoxO6 expression:
polyclonal rabbit antisera were generated against human FoxO6. A
FoxO6 specific peptide (NH2-)CAQDAYGPRARAGTP(-CONH2) was
synthesized, coupled to a carrier molecule and injected into two rabbit
hosts. Antisera were obtained at day 84 of immunization and purified
by peptide affinity chromatography using a FoxO6 specific peptide affinity
column (Innovagen, Lund, Sweden). Tissue micro arrays (TMA)
were prepared to study the expression of FoxO6 in the study population.
FoxO6 immunostaining of the TMAs was evaluated by applying
an immunoreactivity scoring system [0 (no immunostaining), 1 (weak),
2 (moderate), and 3 (strong)]. Statistics: Statistical analyses were done
with SPSS 18.0. With respect to continuous variables, cases were divided
into two groups by splitting at the median value. Significance of correlation
between clinicopathological parameters and FoxO6 expression was
tested using Fisher’s exact test. For parameters with ordinal scale (T, N,
stage) we applied Kendall’s tau test instead. Generally, p-values less than
0.05 were considered statistically significant.
Results. FoxO6 staining was found exclusively in the cytoplasm, no nuclear
staining was detected. Expression levels were heterogeneous when
different cases were compared. Statistically significant correlation to the
FoxO6 expression levels were found for the Lauren phenotype, the Tcategory,
the mucin phenotype and the microsatellite instability status.
Conclusions. The correlation of FoxO6 expression levels with major clinicopathological
tumor characteristics warrants further analysis of the
biological role of FoxO6 in gastric cancer.
FR-P-051
Differential expression of LGR4, LGR6, GPR34, GPR160 and
GPR171 in gastric carcinoma
J .S . Steffen1 , E . Simon1 , K . Balschun1 , C . Böger1 , C . Röcken1 1Christian-Albrechts-University, Institute of Pathology, Kiel
Aims. Gastric cancer (GC) is one of the most common causes of cancerrelated
deaths worldwide. Due to its poor prognosis, the identification of
novel therapeutic targets is of high priority. G-protein-coupled receptors
(GPCRs) are prime candidates for novel cancer prevention and treatment-strategies,
since they play an important role in regulating physiological
and pathophysiological processes. These receptors form the largest
family of human membrane-bound proteins and represent a target
for more than 40% of all drugs. We aimed to elucidate the differential
expression and putative tumor biological significance of LGR4, LGR6,
GPR34, GPR160 and GPR171, in GC.
Methods. Based on our previous microarray analysis we identified five
candidate genes in human gastric cancer samples. Real time RT-PCR
was carried out to validate their expression in malignant and non-malignant
tissue on an independent collective comprising 32 GC patients with
and without lymph node metastases. Selected protein targets LGR4 and
LGR6 were further validated on paraffin-embedded sections of 10 intestinal
and 10 diffuse type gastric carcinomas and their corresponding
non-malignant tissue using immunohistochemistry. Additionally, the
putative tumour biological significance of LGR4 and LGR6 was studied
using tissue micro arrays obtained from a cohort of 488 GCs.
Results. GPR34, GPR160 and GPR171 did not show any differential expression
in real-time RT-PCR analyses. LGR4 and LGR6 were markedly
up-regulated on transcriptional (real-time RT-PCR) and translational
(immunohistochemistry) level in GC. Furthermore, in tissue micro
array analysis LGR6 expression was significantly associated with local
tumor growth (T-category) and correlated with patient survival. LGR4
expression was significantly correlated with the lymph node metastases
(N-category).
Conclusions. LGR6 has recently been identified as stem-cell marker in
the skin whereas LGR4 is of known tumor biological relevance in colon
carcinoma and other malignancies. Our systematic analysis indicates
that these two genes also play a role in gastric cancer biology. Future stu-
Der Pathologe · Supplement 1 · 2012 |
99

Abstracts
dies will have to demonstrate, whether these are also putative diagnostic,
prognostic and/or therapeutic targets for GC.
FR-P-052
Differential expression and abnormal cytoplasmic distribution of
E-cadherin and the desmosomal cadherin desmoglein 2 in gastric
carcinomas: Prognostic relevance of desmoglein 2-plaques
E .C . Scharbatke1 , A . Gamboa-Dominguez2 , F . Fend3 , A . Brunner4 ,
A . Schmidt1 , R . Moll1 1 2 Philipps University of Marburg, Institute of Pathology, Marburg, Instituto
Nacional de Sciencias Medicas y Nutricion Salvador Zubiran, Institute of
Pathology, Mexico City, Mexico, 3University Hospital Tübingen, Institute
of Pathology, Tübingen, 4University of Würzburg, Lehrstuhl für Volkswirtschaftslehre,
insbesondere Wirtschaftsordnung und Sozialpolitik, Würzburg
Aims. In gastric carcinomas, expression of E-cadherin has been shown
to be important in pathogenetic and prognostic terms. We compared expression
and subcellular distribution of E-cadherin and of another cellcell
adhesion molecule of the cadherin family, the desmosomal cadherin
desmoglein 2 (Dsg2), in a series of gastric adenocarcinomas of Mexican
patients.
Methods. Specimens of 63 cases of sporadic gastric adenocarcinomas
(26 of intestinal type, 37 of diffuse type; three cases showing CHD1
mutations) were analyzed immunohistochemically for E-cadherin and
Dsg2, using monoclonal antibodies AEC (clone 36; BD Transduction
Laboratories) and G129 (Progen, Heidelberg), respectively, on paraffin
sections after heat-induced antigen retrieval. Expression and subcellular
distribution (membrane staining, intracytoplasmic plaques ≤5 µm and
>5 µM) of these proteins were statistically correlated with clinicopathological
parameters and patient survival.
Results. 53 cases (84%) were positive for E-cadherin [19 cases (30%) with
plaques] and 62 cases (98%) were positive for Dsg2 [54 cases (84%) with
plaques]. Large intracytoplasmic plaques (>5 µM) were found for E-cadherin
in 5 cases (8%) and for Dsg2 in 9 cases (14%; three of intestinal and
six of diffuse type). The presence of E-cadherin or Dsg2 plaques of any
size and of large E-cadherin plaques did not show prognostic relevance.
However, the 9 patients with large Dsg2 plaques exhibited significantly
shorter survival (p=0.020) in multivariate regression analysis. This correlation
was independent from other parameters such as tumor stage.
Conclusions. Both cadherins studied are expressed differentially in gastric
carcinomas. Abnormal subcellular distribution of Dsg2 in the form of
large intracytoplasmatic plaques appears to be an independent predictor
of poor prognosis in gastric carcinomas. Prospective studies on larger
cohorts are required to confirm these results.
FR-P-053
Unexpected role of caspases in the survival of human colon epithelial
cells upon oxidative stress
A . Just1 , K . Reissig1 , R . Hartig2 , P . Steinberg3 , T . Guenther1 , A . Roessner1 ,
A . Poehlmann1 1Otto-von-Guericke University Magdeburg, Department of Pathology,
Magdeburg, 2Otto-von-Guericke University Magdeburg, Department of Molecular
and Clinical Immunology, Magdeburg, 3Institute of Food Toxicology
and Analytical Chemistry, Hannover
Aims. Current evidence suggests an increasing role for inflammation-associated
oxidative stress in colorectal cancer initiation. We found that
human colon epithelial cells (HCEC) survive oxidative stress through
cell cycle arrest. Moreover, caspases were found not to be involved in
apoptosis but in mediating the survival of HCEC. Thus, we aimed to unravel
their unexpected role.
Methods. We simulated acute inflammation by treating HCEC with a
pathophysiologic concentration of H2O2. We performed inhibition stu-
100 | Der Pathologe · Supplement 1 · 2012
dies using a pan-caspase inhibitor. The cells were further analyzed by
immunoblotting and FACS.
Results. H2O2 induces DNA damage in HCEC. As a consequence, HCEC
underwent apoptosis or cell cycle arrest to repair DNA damage. In addition,
a proportion of cells may progress through the cell cycle irrespective
of DNA damage (cellular survival). Surprisingly, caspase 3, 8 and 9
expression was found to be up-regulated, but did not precede cells into
apoptosis. To unravel the role of caspases in cell survival, we performed
inhibition studies using a pan-caspase inhibitor. Immunoblotting showed
(i) p21 and (ii) y-H2AX up-regulation following caspase inhibition.
Cell cycle analysis revealed the accumulation of cells in the G1 phase of
the cell cycle as the first response and increased apoptosis following caspase
inhibition as the second response. Because of these findings, we
suggest caspase-mediated inactivation of the tumor suppressor p21 and
the DNA damage sensor y-H2AX. Among others, this led us to suggest
that caspases rather play a role in survival than in apoptosis.
Conclusions. HCEC provide a model to analyze the molecular relationship
between inflammation-associated oxidative stress and colorectal
cancer initiation by mimicking the in vivo conditions in vitro. We suppose
that caspases promote cell survival upon acute colonic inflammation
through inactivation of p21 and y-H2AX. We presume (i) increased
proliferation due to p21 down-regulation and (ii) accumulation of DNA
damage because of y-H2AX down-regulation. Consequently, caspases
seem to mediate the progression of cells through the cell cycle irrespective
of DNA damage, and this might cause the cell to turn on a one way
street to malignant transformation.
FR-P-054
Uncommon coincidence of B-cell chronic lymphatic leukemia with
rare transversal-colon intussusception on older age flanked by a
simultaneous carcinoma of the right and left flexure – successful
management (only slight complication) with curative intent
M . Petersen1 , R . Kube2 , S . Dalicho1 , D . Wolleschak3 , H . Lippert1 , A . Roessner4 ,
F . Meyer1 1 2 University Hospital, Dept . of Surgery, Magdeburg, Municipal Hospital,
Dept . of Surgery, Cottbus, 3University Hospital, Dept . Hematology & Oncology,
Magdeburg, 4University Hospital, Institute of Pathology, Magdeburg
Aims. By means of an extraordinary case report, the rare hematological
case with a chronic lymphatic leucemia (CLL) and an associated malignoma
of the GI tract with the patient-related clinical finding and treatment
characteristics incl. “outcome” out of the daily clinical practice
with currently intermediary, interdisciplinary therapeutic success is
described.
Methods. Patient and diagnostic finding: approximately 4 weeks after
puncture of the bone marrow resulting in the diagnosis B-cell leukemia
(stage IIa according to the classification by BINET and RAI; initially:
Leucocytosis, hypochromic microcytic anemia), the patient (accompanying
diseases: Coronary heart disease, former acute myocardial infarction,
arterial hypertension, erosive gastritis) underwent surgical intervention
because of a simultaneously diagnosed double carcinoma of the
colon. Endoscopy revealed carcinoma (confirmed by histopathological
investigation of a specimen) of the descending colon and a suspicious
lesion of the ascending colon. CT scan excluded distal metastases and
described an intussusception of the transversal colon, infiltrationg tumor
growth through the whole colonic wall (1) and thickening of the
wall of the right colonic flexure (2).
Results. Treatment and course: intraoperatively, simultaneous double
colonic carcinoma of the right flexure and aborally of the left flexure
(no detection of an intussusception) was found prompting to a subtotal
colectomy with a 2-row ileosigmoideostomy to reconstruct intestinal
tract (1: pT2G2; 2: pT3N1[1/71]M0L0V0G2). There was only a summative
anemia through the postoperative course and following need of blood
transfusion. According to the oncological overall concept, an adjuvant

chemotherapy was recommended and continuing the follow-up observation
of the B-cell CLL with no current need for a specific treatment.
Conclusions. Despite a challenging combination of simultaneous neoplasias
of different and/or the same entit(ies)y, in addition with complicating
factors (2 primary tumor manifestations of the same entity with
the need of an extended standard surgery due to tumor sites, subileus,
perioperatively persisting, initially diagnosed hemoblastosis), the treatment
was successful with curative intention and preserved mid- to longterm
curative potential by abdominal surgery with a low complication
rate. This confirms the indicated primary surgical care of the GI cancers,
also because of the coincidence of a colonic carcinoma at 2 sites. The rare
transversal-colon intussusception in this age, flanked by a carcinoma of
the right and left flexure, is the first report of this type in the literature.
FR-P-055
Seltenes Langerhans-Zell-Sarkom der Milz mit ungewöhnlicher
klinischer Manifestation
J . Arend1 , D . Küster2 , H . Lippert1 , F . Meyer1 1 2 University Hospital, Dept . of Surgery, Magdeburg, University Hospital,
Institute of Pathology, Magdeburg
Aims. Abklärung eines pathohistolischen Zufallsbefundes nach Splenektomie
im Rahmen einer Operation bei Leberparenchymblutung (primär
V. a. akute Cholangitis bei Choledochocystolithiasis).
Methods. Zunächst therapeutische ERCP mit Stentimplantation im
D. hepatocholedochus nach Papillotomie unter antibiotische Abschirmung.
In der Folge-CT multiple Leberherde, die zur histologischen
Abklärung sonographiegestützt bioptiert wurden. Komplikation: intra-
und perihepat. Hämatom, welches bei symptomatischer Größenzunahme
und drohendem sept. KH-Bild (Ursache nur teils durch Staphylokokkenkolonisation
eines i.v.-Portsystems zur Chemotherapie bei
Mamma-Ca erklärt) eine notfallmäßige expl. Laparotomie zur sept.
Fokussanierung erforderte: i) Intraabdominal kein sept. Fokus; ii) Entlastung
des Leberkapselhämatoms und lokale Thermokoagulation/
Hämostyptikaapplikation; iii) intraop. multiple, unklare, teils massiv
blutende fokale Milzläsionen (vulnerable Parenchymoberfläche), die zur
Blutungsbeherrschung und pathohistologische Abklärung die Splenektomie
erforderten; i.v.) Explantation des infizierten i.v.-Ports.
Results. Bei Staphylokokkensepsis (zusätzl. Bakteriämie durch Streptokokkus
hominis) und multiplen chologenen Leberabszessen zunächst
kalkulierte und später resistenzgerechte Antibiotika (keine klein. Befundbesserung).
Im Leberbiopsiepräparat multiple kleine Abszesse mit
schaumzellig-histozytärer, resorptiver Entzündungskomponente. Das
Splenektomiepräparat war diffus mit zytologisch malignen Zellen bei
aufgehobener Milzarchitektur durchsetzt mit einer Zellproliferationsfraktion
(Ki-67-Ag) von ca. 50% (pos. Immunhistologie für S-100, CD1a
und teils CD68; Lysozym, CD3, Cd20, CD30, Alk 1, Myeloperoxidase und
Chlorazetatesterase hingegen negativ). Trotz techn. Operationserfolgs
tolerierte die Patientin die Intervention nur mäßig, durch zunehmende
AZ-Verschlechterung keine weiterführend ggf. diagnosespezifisch indizierte
Therapie möglich. Trotz max. int.-therapeut. Maßnahmen erlag
die Patientin 13 Tage nach stationärer Aufnahme dem foudroyanten Verlauf
im MOV.
Conclusions. Die zur Aufnahme nicht bekannte, seltene Erkrankung
eines Langerhans-Zell-Sarkoms trug wesentlich zum fulminanten Cholangitis-Verlauf
bei. Zusätzliche Komplikationen wie Portinfektion und
Blutung nach Leberpunktion verschlechterten die Prognose weiter. Offen
bleibt, ob klinischer Verdacht oder Sarkomfrühdiagnose den KH-
Verlauf suffizient beeinflusst hätte. Die Seltenheit der Erkrankung und
damit fehlende Erfahrungen in Diagnostik und Therapie erschweren
eine individuelle Therapie massiv.
Poster: GI-Trakt: GIST, Dünndarm, Kolorektum
FR-P-056
Value of epithelioid histomorphology and PDGFRA immunostaining
patterns for prediction of PDGFRA mutated genotype
in GISTs
A . Agaimy1 , C . Otto2 , H . Geddert 3 , I .-M . Schaefer4 , A . Braun2 , R . Schneider-
Stock1 , F . Haller2 1 2 Friedrich Alexander University, Institute of Pathology, Erlangen, Albert
Ludwigs University, Institute of Pathology, Freiburg, 3St . Vincentius Hospital,
Institute of Pathology, Karlsruhe, 4Georg August University, Institute of
Pathology, Göttingen
Aims. A majority of gastrointestinal stromal tumors (GISTs) carry mutations
in the receptor tyrosine kinases KIT or PDGFRA. In a recent
study, we demonstrated upregulated expression of KIT in KIT mutated
GISTs, in contrast to upregulated PDGFRA expression in PDGFRA mutated
GISTs, on mRNA (qRT-PCR) and protein (Western BloT) level.
However, most routinely processed GISTs are formalin-fixed and paraffin-embedded;
thus, these methods are not applicable in daily pathology
routine. Reliable determination of PDGFRA expression by immunohistochemistry
might help to identify GISTs with PDGFRA mutation,
without the necessity for complete genotyping of KIT and PDGFRA by
mutational analysis. The aim of the current study was to evaluate the
predictive value of a combination of histomorphology and PDGFRA immunohistochemistry
in comparison to mutational analyses.
Methods. In order to conduct a tissue microarray, 109 surgically resected
GISTs with known mutation status of KIT (74%) and PDGFRA (16%)
were used. The histomorphological phenotype (spindled, epithelioid,
mixed growth pattern) was determined on H&E sections without knowledge
of the genotype. The staining intensity (negative, 1–25%, 26–50%,
>50%) and the staining pattern (paranuclear, cytoplasmic, membranous)
of PDGFRA were determined without knowledge of the genotype.
Results. PDGFRA-mutated GISTs were significantly more often of epithelioid
phenotype and had a significantly higher expression of PDGFRA
protein, compared to KIT-mutated GISTs. The paranuclear stainig pattern
was almost exclusively observed in PDGFRA mutated GISTs. A
combination of histomorphology, staining intensity and staining pattern
of PDGFRA was a reliable predictor for PDGFRA genotype.
Conclusions. A combination of histomorphology and PDGFRA immunostaining
is a reliable predictor of PDGFRA genotype. The use of this
PDGFRA genotype predictor may help to reduce costs and shorten processing
time of GIST genotyping by excluding KIT mutational analysis
in PDGFRA-overexpressing GISTs. This might be even more important
in less developed countries with restricted health budgets.
FR-P-057
Expression of CD34 in GIST is site-dependent and genotypeassociated
A . Braun1 , C . Otto1 , H . Geddert2 , D .J . Zhang3 , A . Agaimy4 , Ö . Sahin3 , F . Haller1 1 2 Albert Ludwigs University, Institute of Pathology, Freiburg, St . Vincentius
Hospital, Institute of Pathology, Karlsruhe, 3German Cancer Research Center,
Heidelberg, 4Friedrich Alexander University, Institute of Pathology, Erlangen
Aims. Gastrointestinal stromal tumors (GISTs) are the most common
mesenchymal tumors of the gastrointestinal tract. In addition to immunopositivity
for KIT (CD117), 60–70% of cases are positive for CD34.
CD34 is a cell surface glycoprotein, which is expressed especially in early
hematopoietic stem and progenitor cells. Its function is still not yet
sufficiently clarified. Our aim was to gain a better understanding of the
regulation of CD34 expression in GISTs.
Methods. From the archives of our institutes, 109 surgically resected
GISTs were compiled. Mutation analyses of KIT (exon 9, 11, 13 and 17),
Der Pathologe · Supplement 1 · 2012 |
101

Abstracts
PDGFRA (exons 10, 12, 14 and 18), and BRAF (exon 15) were performed.
The expression of CD34 was assessed semi-quantitatively using immunohistochemical
staining on tissue microarrays. Methylation analyses
were performed using pyrosequencing of two CpG loci in the promoter
binding region of the CD34 gene (CD34_P339_R and CD34_P780_R).
Furthermore, GIST cell lines 882 and 48B were treated with different
concentrations of the demethylating agent 5-azacitidine (5-azaC), and
the methylation status as well as the mRNA and protein expression of
CD34 were determined.
Results. KIT-mutated tumors of the stomach and rectum were at both
CpG loci of the CD34 gene significantly less methylated compared to
KIT-mutated small intestinal GISTs (p

aims are twofold: 1) to verify the immunohistochemical expression of
ETV1 in a large series of mesenchymal tumors of the gastrointestinal
tract; 2) to verify its possible relationship with clinicopathologic parameters
(risk classification, survival, mutational status).
Methods. 424 GISTs (381 primary and 43 metastases), 30 leiomyomas,
8 leiomyosarcomas and 1 schwannoma in TMA format. Mean age was
66±1.4 ys. For the risk classification was used the schema of Miettinen
in 5 groups (probably benign,18%, very-low risk, 17%, low-risk, 14%, moderate
risk, 12% high-risk, 10%) adding overtly malignant tumors, 20%
and metastases, 9%. Immunohistochemical staining for ETV1 (AbCam®,
dilution 1:100) was performed. Immunoreactivity was scored by evaluating
the number of positive nuclei over the total number of tumor cells.
For survival analysis the series was dycotomised, using 0 as cut-off.
Results. ETV1 was expressed in the majority of primary GISTs (76% of
cases) and of metastases (86%), but also in leiomyosarcomas (50%) and
in leiomyomas (38%). The maximum score was observed in metastases
(mean 68%) and in primary GISTs (mean 48%). Leiomyosarcomas (mean
37%) and leiomyomas (mean 14%) had a lower score. The differences were
statistically significant (0.0001T) being the most frequent, as well as 8 deletions, and 5 deletion-insertions.
CGH revealed -14q, -1p, and -22q as most the common
aberrations (39, 15, and 10 cases, respectively). The mean number of clonal
net changes was 2.68 (0.6 gains, 2.1 losses), and 10 tumors revealed no
chromosomal imbalances.
Der Pathologe · Supplement 1 · 2012 |
103

Abstracts
Conclusions. PDGFRA-mutated GISTs are rather large tumors of gastric
site with a very low/low risk of progression. D842V is the most common
mutation. Overall, GISTs with PDGFRA mutations exhibit a low degree
of chromosomal instability, compared to KIT-mutated GISTs. Although
PDGFRA-mutated GISTs principally display the same chromosomal
aberrations as KIT-mutated GISTs, the observed low degree of chromosomal
instability might contribute to their generally low malignant phenotype.
FR-P-064
Rare inflammatory pseudotumor with primary (intrapulmonal,
mediastinal, subpleural) manifestation and subsequent occurrence
within mesenteric fatty tissue of the small intestine – secondary
tumor lesion or metastasis?
M . Petersen1 , H . Lippert1 , K . Schütte2 , A . Roessner3 , F . Meyer1 1 2 University Hospital, Dept . of Surgery, Magdeburg, University Hospital,
Dept . of Gastroenterology, Magdeburg, 3University Hospital, Institute of
Pathology, Magdeburg
Aims. The rarely occurring inflammatory pseudotumor (IPT) is considered
a relevant differential diagnosis in regard to frequency, identifiability
and adequate management in diagnostic and therapy of intestinal tumor
lesions.
Methods. By means of an extraordinary case report, this tumor finding is
characterized with occurrence, finding the correct diagnosis, therapeutic
measures and outcome.
Results. In a 39-year-old man, a multifocal recurrent tumor growth within
the thorax (intrapulmonary, mediastinal, subpleural lesions – predominating
within the right upper segment) and a singular abdominal
tumor lesion were diagnosed 12 years after a former successfully resected
pulmonary first manifestation of an IPT. An in-toto resection of the
middle jejunal segment was achieved because of a manifest inflammatory
tumor conglomerate consisting of mesenteric fatty tissue and attached
jejunal loops including a jejunojejunostomy with an uneventful
postoperative course. A surgical re-intervention for the pulmonary/thoracic
tumor lesions was not favored by the thoracic surgeons because of a
superior vena-cava-neighbored tumor site with mediastinal infiltration.
Initially, the patient did not agree to an externally recommended immunosuppressive
treatment with cyclophosphamide and steroids because of
a wish for a baby despite repeatingly expressed demands. Finally, the patient
underwent a 5-month systematic steroid therapy and subsequently,
a periodic repeat of such therapeutic cycles but, however, with no CT
follow-up as recommended. A slight tumor progression was diagnosed.
Histopathological investigation revealed myofibroblastic cell proliferation
and inflammatory infiltrates; in addition, the immunohistochemical
test marker profile (CD117-, Alk1-, chromogranine-negative; vimentin-
and partially SM-actin-positive) led to the diagnosis IPT.
Conclusions. After appropriate literature search, the presented patient is
one of the only few cases with an IPT of the described unusual tumor
site within the jejunal mesenteric tissue, a rare multifocal recurrent tumor
growth after former surgical resection through a long-term course
of 12 years.
104 | Der Pathologe · Supplement 1 · 2012
FR-P-065
Tissue damage through MRI histopatology of small intestine in
swine model due to high frequency radiation of different intensity
and duration
S . Griff1 , T . Mairinger1 , G . Stoltenburg-Didinger 2
1 2 HELIOS Klinikum Emil v . Behring, Berlin, Institute for Cellbiology and
Neurobiology Charité Berlin
Aims. Magnetic resonance imaging (MRI) is an important diagnostic
tool in daily clinical praxis. Regarding the physical aspects, the majority
of radiofrequency (RF) power transmitted for imaging is transformed
into heat within the patient’s body. The thresholds of exposure (specific
absorption rate, SAR) have been defined in the early 1980s without investigation
of changes of internal organs. The changes of organs due to
the impact of high frequency radiation have not been investigated since.
Based on these facts we evaluated the morphological effects in small intestine
in swine in correlation to SAR and core temperature.
Methods. By autopsy, small intestine tissue from 14 pigs was examined by
histology and immunohistochemistry. The animals have been exposed
to long-time whole-body SAR. There were four groups of animals with
different SARs and duration of exposure. The control group (group 0)
was not exposed. The highest exposure was beyond the SAR thresholds.
Results. All exposed animals irrespective of dose and duration of exposure
showed marked histological changes. These were:
– Loss and/or necrosis of surface epithelium
– Hyperaemia
– Focal fibrin thrombi
– Retraction of villi
– Lymphocytic and eosinophilic infiltration of lamina propria
Conclusions. The results of the present study question the so far valid
SAR thresholds. The casuistic reports of complications were considered
as handling error or patient’s erratic behaviour. Such cases have to be
re-evaluated and newly discussed, as there are no histological studies of
internal organs in these situations.
FR-P-066
Her2/neu in gastric cancer: comparison of tissue micro arrays with
corresponding whole tissue sections
C . Böger1 , V . Warneke1 , H .-M . Behrens1 , C . Röcken1 1Christian-Albrechts-University, Kiel, Institute of Pathology, Kiel
Aims. Gastric cancer (GC) is one of the most common causes of cancerrelated
deaths worldwide. The introduction of trastuzumab for Her2/
neu-positive gastric cancer is a recent advancement, with Her2/neu currently
being the only predictive biomarker for gastric cancer. Her2/neu
status is currently assessed by biopsy or resection material, but it is not
validated which influence tissue sampling has on the assessment of the
Her2/neu status.
Methods. 485 patients (299 men, 186 women; median age 68 years) with
GC had undergone either total or partial gastrectomy for adenocarcinomas
of the stomach or oesophago-gastric junction. Formalin-fixed and
paraffin-embedded tissue samples were used to generate tissue micro
arrays (TMA). Briefly, five representative regions of the paraffin “donor”
blocks were chosen. Tissue cylinders of 1.5 mm diameter were punched
from these areas and arrayed into a new “recipient” paraffin block. Afterwards,
4 µM sections of the resulting tumor TMA-block were cut for
further analysis. The Her2/neu-status was assessed using the monoclonal
anti-Her2/neu-antibody (clone 4B5) and HER2-SISH double labelling in
situ hybridization-system, according to the gastric cancer scoring system
by Rüschoff et al. (Virch Arch. 2010 Sep; 457(3):299–307) separately for
resection specimens (whole tissue sections) and for biopsies (TMAs) in
order to test which influence tissue sampling has on the Her2/neu-status.
Statistical analyses: For measuring the inter-rater agreement of Her2/neu
scores between two observers, Cohen’s kappa coefficient was calculated.

Results. Her2/neu status in TMAs and corresponding whole tissue sections
were investigated from the same GCs by two surgical pathologists. 37
(8.2%) and 38 (8.5%) GCs were classified as Her2/neu-positive by the two
observers using whole tissue sections. Using the gastric cancer scoring
system for biopsies, Her2/neu was positive in 26 (5.9%) and 27 (6.1%) GCs.
The interobserver agreement was high (98.5% for whole tissue sections
and 98.0% for TMAs; p

Abstracts
cessary accuracy in the differentiation of CU associated IEN and could
therefore only be used with care in this setting.
FR-P-070
Inter-observer variability in the diagnosis of colorectal polyps under
special consideration of serrated lesions – a European study
T .T . Rau1 , A . Agaimy1 , A . Gehoff2 , C . Geppert1 , K . Jung3 , K . Knobloch1 ,
C . Langner4 , A . Lugli5 , I . Nagtegaal6 , J . Rüschoff2 , X . Saegert7 , M . Sarbia8 ,
M . Vieth9 , A . Hartmann1 1 2 University Hospital Erlangen, Institute of Pathology, Erlangen, Institute
of Pathology Nordhessen, Kassel, 3Department of medical statistics, Göttingen,
4Medical University Graz, Institute of Pathology, Graz, Austria, 5Uni versity Bern, Institute of Pathology, Bern, Switzerland, 6UDC St . Radboud,
Institute of Pathology, Nijmegen, Netherlands, 7Catholic University Leuven,
Institute of Pathology, Leuwen, Belgium, 8Institute for Pathology and Cytology,
München, 9Institute of Pathology, Bayreuth
Aims. A huge number of different criteria in the diagnosis of serrated
lesions of the colon are known from the literature. In 2010, a German
consensus conference redefined and simplified criteria for the diagnosis
of SSA, hyperplastic polyp, traditional serrated adenoma and mixed
polyps. As a scientific amendment we aimed to reflect their appliance in
daily practice and to prove their ability to achieve inter-observer concordance.
Methods. Consecutive colorectal polyps of one month (n=1926) were
analysed within a dedicated GI pathology institute (9). All consecutively
diagnosed serrated adenomas such as TSA, SSAs and mixed polyps were
included. To complete the diagnostic spectrum of colorectal lesions a
consecutive number of hyperplastic polyps and classical adenomas were
added and completed with a few probes of normal colonic mucosa to
reach a study set of 200 lesions. Virtual microscopy was enabled with
a Zeiss Mirax Scanner. Hard drives were sent to 10 pathologists with
GI experience in 5 European countries in three rounds. The first round
blanked, the second providing clinical data, the third round after a conference
meeting agreeing upon the recently proposed German consensus
guidelines (Virchow’s Archive, 2010 Sep;457(3):291–7).
Results. Distribution of gender, age and localization highlighted predominance
for certain lesions, but no exclusiveness. Kappa-statistics revealed
a basically moderate average agreement (κ=0.56) in the first round.
Providing clinical data a slight increase could be achieved (κ=0.63),
which was nearly equal to the values under the use of German consensus
criteria (κ=0.61). Single criteria of SSA and TSA diagnosis showed a divergence
in inter-observer reliability (from κ=0.06 to κ=0.82).
Conclusions. Using the simplified German consensus guidelines for serrated
lesions the inter-observer certainty of diagnosing serrated lesions
is maintained. Using the degree of concordance the single criteria of SSA
and TSA diagnosis could be assembled in a hierarchical algorithm. Training
in the diagnosis of TSA and mixed polyp seems to be most promising
to increase quality in GI pathology.
FR-P-071
Role of VEGF-B in the progression of colorectal cancer
C . Jayasinghe1 , N . Simiantonaki1 , C .J . Kirkpatrick2 1Gummersbach Hospital, Institute of Pathology, Gummersbach,
2University Medical Center, Institute of Pathology, Mainz
Aims. The relevance of VEGF-B, a VEGF (vascular endothelial growth
factor) family member, in the progression of colorectal cancer is unclear.
Methods. Therefore, using immunohistochemistry we have investigated
the expression of this VEGFR (VEGF receptor) -1 ligand in 91 non-metastatic
(n=38), lymphogenously metastatic (n=26) and haematogenously
metastatic (n=27) colorectal carcinomas.
106 | Der Pathologe · Supplement 1 · 2012
Results. Independent of the metastatic status,VEGF-B was expressed in
endothelial as well as epithelial cells in colorectal carcinomas. In 89% of
the cases with or without distant metastasis a vascular expression was
found. In contrast, only 62% of the nodal-positive tumors had a VEGF-B
positive vasculature. In relation to the non-metastatic (p=0.01) and haematogenously
metastatic (p=0.027) cases this difference was statistically
significant. Concerning the topological staining distribution, in 2/3 of
the cases a homogenous pattern was seen without differences in immunostaining
between the intratumoral vessels and the vascular fraction
throughout the invasive tumor margin. Using the general endothelial
marker, CD31, and the lymphatic endothelium-specific marker, D2-40,
all VEGF-B positive vessels were of blood but not of lymphatic vascular
origin. Interestingly, distal of the tumor a vascular VEGF-B expression
was not demonstrable. Positive endothelial VEGF-B expression was not
correlated with the metastatic status. In 46% of the investigated cases an
epithelial VEGF-B expression was also seen, in 30%, 46% and 67% of the
tumors without, with lymph node and with distant metastasis, respectively.
In this context positive VEGF-B immunoreactivity was correlated
with haematogenous metastasis (p=0.006). The epithelial VEGF-B immunostaining
positivity was independent of the grade of the tumor cell
dissociation and localization of tumor necrosis.
Conclusions. These morphological observations provide evidence for a
probable pathobiological significance of VEGF-B in the tumor progression
of colorectal cancer, especially in the case of haematogenous metastasis.
FR-P-072
Is there a rationale to record lymphatic invasion in node-positive
colorectal cancer?
N . Schneider1 , J . Betge1 , M .J . Pollheimer1 , R .A . Lindtner1 , P . Kornprat2 ,
P . Rehak3 , C . Langner1 1 2 Medical University of Graz, Institute of Pathology, Graz, Austria, Medical
University of Graz, Department of Surgery, Graz, Austria, 3Medical University
of Graz, Research Unit for Biomedical Engineering & Computing, Graz,
Austria
Aims. The invasion of tumour cells into lymphatic channels represents
a crucial step in the metastatic process. The detection of lymphatic invasion
in histological slides has been associated with decreased survival
and higher recurrence rates. Our study aimed to evaluate prognostic significance
of lymphatic invasion in colorectal cancer when lymph node
metastasis is already present.
Methods. 168 patients with node-positive colorectal cancer (colon, n=98;
rectum, n=70) were retrospectively evaluated. Lymphatic invasion was
assessed on H&E stained slides. Presence of lymphatic invasion was correlated
with different pathological variables applying the chi square test.
Progression-free and cancer-specific survivals were assessed using the
Kaplan-Meier method.
Results. Lymphatic invasion was detected in 95 (57%) cases. There was
a significant association of lymphatic invasion with the number of examined
tissue blocks: 1–3 blocks (n=50): 46% presence of L1, 4–5 blocks
(n=68): 53% presence of L1, and 6 or more blocks (n=50): 72% presence
of L1 (p=0.02). Furthermore, L1 was associated with poor tumour differentiation
(p=0.009) and the number of involved lymph nodes (p

prognostic impact was found in rectal cancer patients. The association
of lymphatic invasion with the number of examined tissue blocks is an
important finding that should be considered establishing practice guidelines
for pathologic work-up of cancer specimens.
FR-P-073
Inflammatory bowel diseases (IBD) are associated with decreased
colonic expression of PEPT1 in humans and mice
T . Wuensch 1 , S . Schulz2 , N . Schaltenberg1 , N . Lill1 , D . Haller1 , H . Daniel1 1Technische Universität München, ZIEL – Research Center for Nutrition and
Food Science, Freising-Weihenstephan, 2Charité – Universitätsmedizin
Berlin, Pathology, Berlin
Aims. The intestinal peptide transporter PEPT1 is found in the brush border
membrane of enterocytes in the small intestine where it contributes
to amino acid absorption from luminal protein digestion. Beside that,
PEPT1 is proposed to be aberrantly expressed during colonic inflammation.
Because of its capability to transport bacteria-derived chemotacticacting
agents such as fMLP (formyl-methionyl-leucine-phenylalanine),
MDP (muramyl dipeptide) or L-Ala-D-Glu-meso-DAP, PEPT1 might act
as an amplifier of inflammation. However, a systematic analysis of PEPT1
expression along the healthy colon has never been described. Here we
provide information on PEPT1 mRNA and protein expression along the
healthy colon of different species and during intestinal inflammation.
Methods. PEPT1 mRNA and protein expression levels were determined
by qRT-PCR, immunofluorescence and Western blotting, respectively,
in intestinal samples from healthy mice, rats and humans. Furthermore,
germ-free mice and three mouse models, resembling Crohn’s-like ileitis
(TNFdeltaARE/WT) and colitis (IL-10-/-, IL- 10XTLR2-/-) as well as intestinal
tissue samples from IBD patients were analyzed by immunohistochemistry.
PEPT1 function in colon was determined by transport studies
using [14C]-Glycyl-sarcosine (Gly-Sar). Moreover, the susceptibility
of PEPT1-deficient (Pept1-/-) mice to dextran sodium sulfate (DSS)-induced
colitis was investigated.
Results. Colonic PEPT1 expression showed a distinct pattern of distribution
with marked differences between proximal and distal segments.
No PEPT1 expression was detectable in the proximal colon but prominent
expression was found from mid colon to rectum of mice rats and
humans. Functional Gly-Sar uptake rates were highest in jejunum and
distal colon (19.76 nmol/20 min/mg protein vs. 1.71–0.42 nmol/20 min/
mg protein) and significantly lower (p

Abstracts
Statistical analyses included Spearman and Mann-Whitney Rank Sum
Tests (significance at p50% Ki-67 expression had a
median overall survival time that was undefined, compared to with
43 months for patients with 50%) and low (

provided information on survival times and utilities. Costs were assessed
from the Swiss health care system perspective.
Results. Remaining life-time costs ranged from EUR 3’983 (no cetuximab)
to EUR 38’662 (no testing). Gains of 0.491 QALYs compared to the
reference strategy were achieved by KRAS/BRAF testing. Compared
to KRAS/BRAF, the KRAS testing strategy yielded an additional gain
of 0.002 QALYs. Testing with KRAS/BRAF was the most cost-effective
approach (incremental cost-effectiveness ratio; EUR 62’653/QALY) compared
to the reference strategy. In Switzerland, KRAS and BRAF testing
could lead to annual savings of EUR 591’170 and a loss of 1.89 QALYs
compared with KRAS alone (1,003 new mCRC patients annually). Compared
with no cetuximab, the usage of KRAS and BRAF testing would
require an annual net investment of about EUR 30.9 million to acquire
a gain of 493 QALYs.
Conclusions. While new predictive test are introduced in pathology, the
health economic implications of these tests remain mainly unknown.
This study has shown that testing for KRAS and BRAF mutations in
mCRC patients prior to cetuximab treatment would be favourable in a
Swiss health care context. This model could also be used for comparable
decision problems arising with other predictive tests, which are of highest
relevance for oncologists, pathologists, and health policy makers.
References
1 . Blank PR, Moch H et al (2011) . Clin Cancer Res
FR-P-079
HER-2/neu expression in locally advanced rectal cancer (cUICC II/
III) and its correlation with long-term prognosis
L .-C . Conradi1 , H . Stycen1 , T . Sprenger1 , J . Gaedcke1 , K . Homayounfar1 ,
H .A . Wolff1 , H . Becker1 , B .M . Ghadimi1 , J . Rüschoff2 , P . Middel1 , T . Beissbarth1 ,
T . Liersch1 1 2 University Medical Center Göttingen, Göttingen, Pathologie Nordhessen,
Kassel
Aims. With the implementation of multimodal treatment strategies including
neoadjuvant radiochemotherapy (RCT), the prognosis of locally
advanced rectal cancer has been improved over the last two decades.
Most actual clinical trials aim to further develop therapy by intensification
of agents administered. We examined Her2/neu in rectal cancer
patients to evaluate its expression as a potential drug target as well as its
capacity as predictive and prognostic biomarker.
Methods. Two-hundred-sixty-four patients (192 male, 72 female; median
age 64 years) with locally advanced rectal cancer (cUICC-II/III) were included
in this study. Preoperative RCT (50.4 Gy and concomitant either
5-FU or 5-FU and oxaliplatin) was administered in 217 patients followed
by surgical resection with total mesorectal excision (TME). Another
75 patients received postoperative RCT. All patients were treated within
phase-II/III trials of the German Rectal Cancer Study Group or analogous
to these standardized protocols. Her2-/neu expression from pretreatment
biopsies and corresponding resection specimens were assessed
by immunohistochemical staining and SISH-analysis
Results. A positive Her-2/neu expression was found in 12.4% of all pretreatment
tumor biopsy samples and in 29.3% of the resection specimens.
The correlation of the pre-treatment Her-2/neu status did not show a
significant correlation with the grade of tumor regression. However,
patients with a higher Her-2/neu protein expression as measured in the
resection specimen showed a significant survival benefit (p=0.045) and a
prolonged disease free survival (p=0.05) compared with patients having
low intratumoral Her-2/neu expression during the follow-up (median
41 months). The 5-year survival rate was 96.5% (Her-2/neu positive) versus
79.9% (Her-2/neu negative).
Conclusions. Her-2/neu might be a promising drug target in a significant
proportion of rectal cancer patients and should be further assessed within
prospective clinical trials. Furthermore the Her-2/neu status seems
to be a valuable prognostic marker within the multimodal treatment of
advanced rectal cancer.
FR-P-080
Functional characterization of necroptosis in colorectal cancer
M . Metzig1 , D . Fuchs2 , S . Macher-Goeppinger 1 , P . Schirmacher3 , W . Roth1 1Institute of Pathology, University of Heidelberg; German Cancer Research
Center (DKFZ), Heidelberg, Heidelberg, 2German Cancer Research Center
(DKFZ), Heidelberg, 3Institute of Pathology, University of Heidelberg, Heidelberg
Aims. Necroptosis is a recently discovered, RIP1-dependent form of programmed
necrosis. Recent evidence indicates that this type of cell death
plays an important role in both physiologic and pathologic cellular processes.
While different stimuli, e.g. members of the tumor necrosis factor
(TNF) family, have been identified as inducers of necroptosis, only little
is known about the underlying signaling cascades and effector molecules
of necroptosis. The aim of our project is to further clarify the mechanism
of programmed necrosis and to analyze its biological significance in colorectal
carcinoma.
Methods. Cytotoxicity and apoptosis assay, FACS analysis, immunohistochemistry,
RNA isolation, quantitative real-time PCR, cloning of expression
vectors, co-immunoprecipitations.
Results. We screened multiple cytotoxic stimuli for the induction of necroptosis
in various colorectal carcinoma cell lines. We found a differential
increase in sensitivity to cell death induced by specific drugs when
co-treated with a pancaspase-inhibitor. Since a RIP1-specific inhibitor
(necrostatin 1) diminished this effect we concluded that cells were dying
due to necroptosis. Drug-induced necroptosis was further dependent
on TNF receptor 1 (TNFR1) signaling. Overexpression of RIP3 has been
shown to reconstitute the ability to undergo necroptosis in response to
different stimuli in so far insensitive colon carcinoma cell lines. To assess
the biological significance of RIP3 in colon cancer we analyzed protein
and mRNA expression levels in human colorectal carcinoma samples.
RIP3 expression was significantly reduced in colon carcinoma specimens
compared to normal tissue.
Conclusions. Chemotherapeutic drugs induce necroptosis in colon carcinoma
cells in a TNFR1 and RIP3 dependent manner. RIP3 expression
is reduced in colon carcinoma suggesting a physiologic function of necroptosis
in tissue homeostasis. The possibility to re-sensitize carcinoma
cells to necroptosis-inducing stimuli points towards novel therapeutic
options in cancer therapy.
FR-P-081
High HSP70 and HSP27 expression levels are independent adverse
prognostic factors in primary resected colon cancer
K . Bauer1 , U . Nitsche2 , J . Slotta-Huspenina1 , E . Drecoll1 , C . Hann von Weyhern1,3
, R . Rosenberg2 , H . Höfler1,4 , R . Langer1 1 2 Technische Universität München, Institute of Pathology, München, Technische
Universität München, Klinikum rechts der Isar, München, 3Städtisches Klinikum München, Klinikum Harlaching, München, 4Institute of Pathology,
Helmholtz-Zentrum München, Oberschleissheim
Aims. The expression of Heat Shock Proteins (HSPs) is increased in various
cancers and has been shown to correlate with biological tumor behaviour.
We aimed to assess the impact of HSP70, HSP60 and HSP27
expression patterns with pathological features of colon carcinomas and
patients survival in a large collection of colon cancer.
Methods. HSP expression was determined by immunohistochemistry on
a tissue microarray containing cancer tissue of 355 patients with primary
resected colon carcinomas. Expression patterns were correlated with pathologic
features (UICC pTNM category, tumor grading) and survival.
Expression of HSP27, HSP60 and HSP70 was assessed in cancer tissue
ranging from negative to high.
Results. Expression of HSP27, HSP60 and HSP70 can be detected in colon
carcinomas. High HSP70 expression was associated with worse overall
survival (p

Abstracts
tioned above. For patients without lymph node or distant metastases
(UICC stages I/II) and with complete tumor excision, HSP70 expression
was the only independent prognostic factor for survival (p=0.001) and
superior to UICC pT category. In left sided UICC stage I/II carcinomas,
high HSP27 expression also had adverse prognostic impact and was an
independent prognostic factor (p=0.016) besides HSP70 (p=0.002). There
was no correlation between HSP27, HSP60 and HSP70 expression
among each other and with UICC pT category, presence of lymph node,
distant metastases or tumor grading.
Conclusions. High HSP70 and HSP27 expression is associated with worse
clinical outcome. Determination of tumoral HSP70 and HSP27 may be
used as additional biomarker for risk stratification in colon cancer patients
especially in UICC stage I/II patients.
FR-P-082
p.G13D mutations in the KRAS oncogene are associated with
sensitivity of colorectal carcinoma cells to anti-EGFR antibody
treatment
I . Messner1 , S .E . Baldus1 , H .E . Gabbert1 , K .-L . Schäfer1 1Heinrich-Heine-University Düsseldorf, Inst . of Pathology, Düsseldorf
Aims. Targeted therapies with the anti-EGFR antibodies panitumumab
(Pmab) or cetuximab (Cmab) have shown significant benefit for patients
suffering from metastatic colorectal carcinoma (mCRC). According to
the approval by the American Food and Drug Administration and the
European Medicines Agency, application of these antibodies is restricted
to tumors without mutation in the KRAS oncogene. However, a recent
metaanalysis of 579 CRC patients treated with cmab indicated that
patients with p.G13D-mutated tumors (exchange of glycine at codon 13
to aspartic acid) showed a longer overall survival compared to patients
with tumors mutated in KRAS codon 12. In order to study the functional
impact of the subtype of KRAS mutation on the efficiency of EGFR-targeted
therapies in CRC, we have determined the KRAS mutation status
of colorectal carcinoma cell lines and correlated these data to the in vitro
sensitivity of these cells to cmab and Pmab.
Methods. Using a set of 15 colorectal cancer cell lines, the KRAS mutation
status was evaluated by Sanger sequencing. Mutations in the potential
predictive biomarkers BRAF and PIK3CA as well as protein expression
of EGFR and PTEN were also determined. In vitro sensitivity of tumor
cells to anti-EGFR antibodies was measured by standard MTT assays.
Results. Seven of 15 cell lines showed a KRAS mutation including four
cell lines exhibiting the p.G13D mutation. If these cell lines were treated
using panitumumab or cetuximab, a significant growth inhibition was
observed, while cell lines showing a KRAS mutation at codon 12 or 61
were not sensitive to anti-EGFR treatment. Only three of eight cell lines
showing KRAS wild type status were sensitive to Pmab and cmab. Noteworthy,
all of the five resistant KRAS wild type cell lines were either
characterized by BRAF mutation or by absence of EGFR or PTEN protein
expression, respectively. No correlation between PIK3CA mutation
status and sensitivity to anti-EGFR treatment could be demonstrated.
Conclusions. Our in vitro data from colon carcinoma cell lines indicate
that tumor cells showing the KRAS p.G13D mutation may respond to
EGFR-targeted therapy. Therefore, subtype analysis of KRAS mutation
should be included in prospective clinical trials analyzing the responsiveness
of CRC to cmab or Pmab. In KRAS wild type tumor cells, additional
aberrations like BRAF mutation and loss of EGFR or PTEN expression
may lead to resistance to EGFR-targeted therapy and should be
considered as additional negative predictive biomarkers.
110 | Der Pathologe · Supplement 1 · 2012
FR-P-083
UBCH10 overexpression in human colorectal cancers
S . Piscuoglio1 , P . Pallante2 , L . Tornillo3 , A . Fusco2 , L . Terracciano 3
1 2 University of Basel, Department of Biomedicine, basel, Switzerland, University
of Naples, Istituto di Endocrinologia ed Oncologia Sperimentale “G .
Salvatore” (IEOS-CNR) c/o Dipartimento di Biologia e Patologia Cellulare e
Molecolare, naples, Italy, 3University of Basel, Institute of Pathology, Basel,
Switzerland
Aims. Colorectal cancer is the result of the accumulation of different
genetic modifications including critical genes involved in the control of
cell proliferation. In a large number of carcinomas with worst prognosis,
lesions are not diagnosed until the disease is at an advanced stage. To
diagnose cancer at an early stage and to establish more effective therapies
featuring of key molecules in carcinogenesis is a critical step. UBCH10
(also known as E2C or UBE2C) is a cell cycle-related protein involved in
mitosis completion. UbcH10 participates in proper metaphase to anaphase
transition, and abrogation of UbcH10 results in the premature
separation of sister chromatids. Thus, UbcH10 is essential for cell cycle
progression, and his expression is cell-cycle dependent and related to
proliferation. The aim of this study is to investigate the association of
UbcH10 gene expression with clinicopathological features and tumor
progression of colorectal cancer.
Methods. We investigated the expression of the UBCH10 genes in a tissue
microarray platform consisting of normal and neoplastic colorectal
cancers (CRC) tissues by immunohistochemistry. UBCH10 was detectable
in 889 patients. Immunoreactivity was scored semi-quantitatively
by evaluating the number of positive tumor cells over the total number
of tumor cells. Scores were assigned using 5% intervals and ranged from
0% to 100%. Median protein expression levels were used as cut-off scores
to define protein marker positivity and the findings were associated with
clinical-pathological parameters.
Results. Our results demonstrated that UBCH10 is overexpressed in
CRCs tissues compared to normal colon (p

Methods. Biopsy specimens from 430 untreated patients were investigated
immunohistochemically with monoclonal antibodies against topo
IIα (Ki-S4).
Results. Patients with low (50%) topo IIα expression. The Kaplan-Meier analysis showed
a significant difference in the overall survival time related to the percentage
of topo IIα (p=0.0012) positive tumor cells.
Conclusions. Patients with colorectal cancer and high expression level of
topo IIα have a superior clinical outcome compared to patients with low
expression levels.
FR-P-085
Stem cell marker cancer testis antigen (CT 45) expression in colorectal
cancer: an immunhistochemical study of 704 patients
C . Schrader1 , F . Gieseler2 , J .H . Bräsen3 , B . Sipos4 , C . Röcken3 , W . Klapper3 ,
H . Kalthoff5 , W . v . Schönfels5 , R . Lucius6 , C . Pflüger1 , S . Hinz5 , H . Held7 , T . Raff1 ,
G . Klöppel8 , J . Claasen1 , S . Nazzal1 , C . Dreyer1 , C . Schafmayer5 1 2 2 University Hospital of Kiel, nd Department of Medicine, Kiel, UKSH, Campus
Lübeck, I . Department of Medicine, 3UKSH, Campus Kiel, Department
of Pathology, 4University of Tübingen, Department of Pathology, 5UKSH, Campus Kiel, Department of Surgery, 6UKSH, Campus Kiel, Department of
Anatomy, 7Hospital Neumünster, Department of Medicine, Kiel, 8University Munich, Department of Pathology
Aims. A subset of colorectal cancer derived from stem cells. Cancer testis
antigen (CT45) is expressed in immature gonocytes, spermatogonia and
also in germ cell tumors. There are only limited data about CT 45 expression
in colorectal cancer and no data if the expression in tumor cells has
an influence on the clinical course of the disease.
Methods. We investigated immunohistochemically embedded tissue
from 704 patients with a monoclonal antibody against CT 45 (Ki-A10).
The percentage of CT45 expressing cells was quantified according to the
following classification: negative, 0–25%, 25–50%, 50–75% and more than
75% positive tumor cells.
Results. The majority of cases were negative (n=633). 71 tumor specimens
were CT45 positive. The Kaplan Meier analysis revealed no significant
difference (p=0.11) in the clinical outcome of CT 45 positive and negative
cases
Conclusions. Cancer testis antigen (CT 45) is expressed in a subset of
colorectal cancer. The CT 45 expression is no prognostic factor for the
clinical outcome of the disease, but it might have clinical implication for
therapy decision of stem cell derived cancer.
FR-P-086
SOX9 promotes cell migration and anchorage-independent
growth of colorectal cancer cell lines
C . Hui1 , X . Enping1 1Zhejiang University, School of Medicine, Hangzhou, China
Aims. SOX9 is a high-mobility group (HMG) domain transcription factor
that plays a critical role in multiple aspects of development and differentiation.
There is a close connection between normal development
and oncogenesis. Recent studies have revealed that SOX9 is implicated
in several types of cancer progression, but SOX9 does not have consistent
function roles in different cancers. Importantly, the function and
role of SOX9 in colorectal cancer (CRC) exactly remains unclear. In our
previous study we identified that SOX9 overexpression in CRC as an independent
predictor of an unfavorable outcome for CRC in Chinese population.
This study is to show the role of SOX9 in CRC cell lines.
Methods. Western blotting, RT-PCR and Q-PCR were performed to
detect the expression of SOX9 in CRC cell lines. Stably SOX9-overexpression
RKO cell was constructed. Subsequently, Cell Counting Kit-8
assay and Soft agar colony formation assay, Annexin v-PE/7-AAD Double
Stain Apoptosis Detection assay, xCELLigence system, cell migration
and 5-Fu cell toxicity test were performed to detect the roles of SOX9 in
vitro.
Results. After performing gain-of-function studies, it was clarified that
the SOX9-overexpression RKO cell had no significant effects on cell
proliferation, cell apoptosis and 5-Fu cell toxicity; In migration assay, it
is revealed that SOX9 could promote cell migration by transwell chamber
and xCELLigence system. Also it was assessed that the SOX9-overexpression
RKO cell had impact on anchorage-independent growth by
using soft agar colony formation assay.
Conclusions. The expression of SOX9 can promote cell migration and
anchorage-independent growth, but it has no effect on cell proliferation.
FR-P-087
RegIV promotes migration and invasion of CRC cell lines
Z . Jie1 , W . Jingyu1 , L . Maode1 1Zhejiang University, School of Medicine, Hangzhou, China
Aims. The regenerating gene IV (RegIV) plays an important role in colorectal
tumorigenesis. However, its biological functions have not been
well elucidated.
Methods. RT-PCR and western blotting analysis were performed to detect
the expression of RegIV in colorectal carcinoma (CRC) cell lines
HT29, RKO and LOVO, then the stably RegIV overexpressed and Reg4
shRNA-silenced cell lines were constructed in the RegIV negative-expressed
and positive-expressed cell lines respectively. Subsequently, Cell
counting Kit-8 (cck-8) assay, EdU assay and xCELLigence system assay
were performed to detect cell proliferation. Transwell chamber and matrigel
assays were used to detect cell migration and invasion.
Results. RT-PCR and western blotting showed that RKO and HT29 cell
lines do not expressed RegIV, while LOVO cell lines expressed RegIV.
After performing gain-of-function and loss-of-function studies, it has
been shown that both RegIV overexpressed and RegIV shRNA-silenced
cell lines have no significant effects on cell proliferation. In contrast to
control, migration and invasion abilities are significantly increased on
RegIV overexpressed cell line and are significantly decreased on RegIV
shRNA- silenced cell lines.
Conclusions. The expression of RegIV can promote CRC cell lines to migrate
and invade, but it has no effect on proliferation.
FR-P-088
Minichromosome maintenance protein 6 (MCM 6) expression in
colorectal cancer: a proliferation marker and a prognostic factor
for clinical outcome
C . Schrader1 , F . Gieseler2 , J .H . Bräsen3 , B . Sipos4 , C . Röcken3 , W . Klapper3 ,
H . Kalthoff5 , W . v . Schönfels5 , R . Lucius6 , C . Pflüger1 , S . Hinz5 , H . Held7 , T . Raff1 ,
G . Klöppel8 , J . Claasen1 , S . Nazzal1 , C . Schafmayer5 1 2 2 University Hospital of Kiel, nd Department of Medicine, Kiel, UKSH, Campus
Lübeck, I . Department of Medicine, 3UKSH, Campus Kiel, Department
of Pathology, 4University of Tübingen, Department of Pathology, 5UKSH, Campus Kiel, Department of Surgery, 6UKSH, Campus Kiel, Department of
Anatomy, 7Hospital Neumünster, Department of Medicine, Kiel, 8University Munich, Department of Pathology
Aims. MCM6 is one of six proteins of the MCM family which are involved
in the initiation of DNA replication and is a marker of proliferating
cells. Proliferation markers and their expression levels as prognostic factors
are discussed controversial in colorectal cancer.
Methods. We investigated paraffin embedded tissue from 570 patients
with colorectal cancer with stage 1 to 4 immunohistochemically with
a monoclonal antibody against MCM6. 500 tumor cells were counted
and the percentage of positive cells was calculated. Overall survival time
Der Pathologe · Supplement 1 · 2012 |
111

Abstracts
was calculated from the date of diagnosis until death or loss to follow-up
evaluation. Univariate survival analysis was computed by means of the
Kaplan-Meier method and significance levels were assessed by means of
the log-rank test.
Results. The percentage of MCM6 expressing tumor cells ranged from
27.6% to 97.0%, with a mean of 82.8%. A high MCM6 expression level
of more than 80% positive cells was associated with a significantly longer
overall survival time (130.5 months) compared to patients with a low
MCM6 expression level of less than 80% (58.7 months, p=0.0016).
Conclusions. Immunohistochemical detection of MCM6 seems to be a
promising marker for predicting the outcome in patients with colorectal
cancer.
FR-P-089
Lymphangiogenesis in regional lymph nodes is an independent
prognostic marker in rectal cancer patients after neoadjuvant
treatment
M . Muders1 , C . Jakob1 , D . Aust1 , G . Folprecht2 , K . Datta3 , G .B . Baretton1 1University Hospital Carl Gustav Carus at the University of Dresden, Institute
of Pathology, Dresden, 2University Hospital Carl Gustav Carus at the University
of Dresden, Department of Internal Medicine, Dresden, 3University of
Nebraska Medical Center, Omaha, NE, United States
Aims. One of the major prognostic factors in rectal cancer is lymph node
metastasis. The formation of lymph node metastases is dependent on
the existence of a premetastatic niche. An important factor preceding
metastasis are lymph vessels which are located in the lymph node. Accordingly,
the occurrence of intranodal lymphangiogenesis is thought to
indicate distant metastasis and worse prognosis. In this study we evaluate
the significance of lymph node lymphangiogenesis in a cohort of rectal
cancer patients who are treated with neoadjuvant radiochemotherapy.
Methods. We studied formalin fixed, paraffin embedded adenocarcinomas
and regional lymph nodes of 203 rectal cancer patients who were
treated with neoadjuvant radiochemotherapy and consecutive curative
surgery with cancer free surgical margins (R0). Regional lymph nodes
were detected by immunohistochemistry for podoplanin (D2-40). These
results were then correlated with disease free survival and stage of disease
using standard statistical methods.
Results. The presence of lymphatic vessels in regional lymph nodes significantly
affects the disease-free survival in univariate and multivariate
analyses. In contrast, there was no correlation between peritumoral or
intratumoral lymph vessel density and prognosis.
Conclusions. Our study demonstrates the importance of lymphangiogenesis
in regional lymph nodes after neoadjuvant radiochemotherapy
and consecutive surgery as an independent prognostic marker. Staining
for intranodal lymphangiogenesis and methods of intravital imaging of
lymphangiogenesis and lymphatic flow may be a useful strategy to predict
long-term outcome in rectal cancer patients. Furthermore, addition
of VEGF-blocking agents to standardized neoadjuvant treatment schemes
might be indicated in advanced rectal cancer.
FR-P-090
FGFR1 amplification in primary and lymph node metastatic
colorectal cancer
A . Göke1 , F . Goeke1 , D . Boehm1 , R . Menon1 , W . Weichert2 , R . Buettner3 ,
S . Perner1 1Universitätsklinikum Bonn/Institut für Pathologie, Institute of Prostate
Cancer Research, 2University Hospital of Heidelberg/Institute of Pathology,
3University Hospital of cologne/Institute of Pathology
Aims. Previous studies showed that up to 20% of squamous cell lung cancers
display an amplification of the fibroblast growth factor receptor 1
(FGFR1) gene and that these cases are potentially sensitive to treatment
112 | Der Pathologe · Supplement 1 · 2012
with a specific inhibitor. By exploring the largest publicly available database
of somatic copy number changes in tumors (www.broadinstitute/tumorscape.org),
we found evidence that also a subset of colorectal
cancers (CRC) might be characterized by FGFR1 amplification. Consequently,
we evaluated the frequency of FGFR1 amplifications in both
primary and lymph node metastatic CRC by applying in-situ detection.
Moreover, our objective was to determine whether FGFR1 can be used
as a potential therapeutic drug target in primary and progressed CRC.
Methods. We assessed 450 paraffin embedded primary CRC samples embedded
in a tissue microarray format. Of these, 94 cases had corresponding
lymph node metastases. A target probe located on the FGFR1 locus
spanning 8p11.23 to 8p11.22 and a centromeric reference probe were used
to determine the FGFR1 amplification status using fluorescence in situ
hybridization (FISH). Furthermore, CRC cell lines are currently being
tested for their FGFR1 amplification status. FGFR1 amplified cell lines
will then be treated with a FGFR specific inhibitor.
Results. 4.2% (19/450) of primary CRC displayed FGFR1 amplification. Of
primary CRCs with corresponding lymph node metastasis 4.2% (4/94)
harbored FGFR1 amplification in both, and 2.1% (2/94) cases showed
FGFR1 amplification in only the primary tumor, but not in the metastasis.
Conclusions. FGFR1 amplification is a recurrent event found in CRC.
Furthermore, we suggest that FGFR1 amplification is a clonal event in
tumor progression since the FGFR1 amplification status transferred to
the corresponding lymph nodes in most of our cases. Our study may indicate
novel therapeutic possibilities for patients suffering from primary
and metastatic CRC.
FR-P-091
Neoadjuvant treated liver metastasis of colorectal cancer:
Is the histopathological regression grade a useful predictor for
survival?
P . Bronsert1 , S . Ruhm2 , H . Neef3 , S . Timme1 , M . Werner1 , G . Illerhaus2 ,
F . Makowiec3 1Albert-Ludwigs-University Freiburg, Institute of Pathology, Freiburg,
2Albert-Ludwigs-University Freiburg, Department of Haemato-Oncology,
Freiburg, 3Albert-Ludwigs-University Freiburg, Department of General and
Visceral Surgery, Freiburg
Aims. Surgical treatment of resectable liver metastasis of colorectal cancer
(CRC-LM) after neoadjuvant therapy (NACT) is a common method
worldwide. For several tumour entities (e.g. esophageal, gastric and
breast cancer) histopathological tumor regression grade (TRG) is well
known as a significant predictive marker. In terms of CRC-LM less is
known about the predictive potential of TRG in relation to overall survival
(OS). In this study we determined the TRG of neoadjuvant treated
CRC-LM and correlated those data with OS and other clinicopathological
parameters.
Methods. The collective contains 89 patients, 68 (76%) of which were
treated with an Oxaliplatin or Irinotecan containing NACT and 21 (27%)
of which were treated with a “targeted therapy” using antibodies. Median
NACT period time was 6 month (1–24 months) and the average
number of liver metastasis per patient was 2 (1–11 metastasis). Complete
hepatical resection (R0) was observed in 80 patients (89%), a complete
overall resection (R0, cm0) in 69 patients (77%). From formalin fixed paraffin
embedded tumor tissue of the resection specimen haematoxylin
and eosin staining was performed. We determined the TRG according
to a previously published score from Rubbia-Brandt et al. – a quantitative
scoring system ranging from score 1 (complete regression) to score 5 (no
cytopathic effect). After scoring, the regression was correlated to clinicopathological
parameters including patient survival.
Results. Most patients (62/89; 69%) showed a poor TRG (score 4/5) and
24/89 (27%) showed a moderate response (score 2/3). A mere 3/89 patients
(4%) had a complete regression (score 1). The 5-year survival rate of all
89 patients was 41%. OS of patients with complete regression was 100%;

the OS rate was 53% for patients with a moderate and 33% for patients
with a poor regression (p=0.08). Other factors such as gender, total tumour
count and size, nodal status, localisation of the primaries, as well
as duration and mode of NACT did not correlate in uni- or multivariate
analysis with OS. Multivariate analysis showed an associated correlation
between overall-resection (p

Abstracts
Results. Amplification of the FGFR1 region was rare, but could be found
in three of 128 cases of pancreatic ductal adenocarcinoma (3/128; 2.3%) in
our collective. Slightly more protein expression of FGFR1 was observed
with commercially available FGFR1 antibodies by standard immunohistochemistry.
So far, none of the pancreatic carcinoma cell lines showed
FGFR1 amplification.
Conclusions. FGFR1 amplification can be identified in a small subset of
pancreatic adenocarcinomas. FGFR1 is a tyrosine kinase receptor protein
which is able to promote tumor progression by altering angiogenesis, tumor
cell proliferation, migration and cell survival. In the future, it will
be necessary to characterize the biological role of FGFR1 amplification
in pancreatic adenocarcinoma. Further studies are needed to determine,
whether the patients with FGFR1 amplification and/or overexpression
need to be pre-selected prior to TKI treatment.
FR-P-095
Paraduodenal pancreatitis: mini-series with regard to vessel
obliteration
T . Hansen1 , F . Vitali2 , R . Kießlich3 , S . Heinrich4 , P . Mildenberger5 , A . Kumar5 ,
L . Frulloni2 , C .J . Kirkpatrick1 1 2 University of Mainz, Institute of Pathology, Mainz, University of Verona,
Department of Medicine, Verona, Italy, 3University of Mainz, Department of
Internal Medicine, Mainz, 4University of Mainz, Department of General and
Abdominal Surgery, Mainz, 5University of Mainz, Department of Radiology,
Mainz
Aims. Paraduodenal pancreatitis comprises a form of chronic pancreatitis
involving the duodenal wall close to the minor papilla within the surrounding
parenchymal tissue and common bile duct (also called groove
area). This disorder most commonly affects male patients in the 5th decade
with a history of alcohol and/or nicotine abuse. Concerning the pathogenesis,
it is believed that alcohol or smoking leads to a resistance of
the pancreatic juice flow and ischemia of the paraduodenal tissue, giving
relapsing episodes of pancreatitis. We report on a series of patients with
paraduodenal pancreatitis with emphasis on vascular changes.
Methods. We describe ten patients (all male, mean age 45.4 yrs). Most of
them presented with abdominal pain (n=8), and weight loss was additionally
found in eight patients as well. All patients were smokers; history
of alcohol abuse could be confirmed in seven cases. In all patients, a
pancreatico-duodenectomy was performed. For histological evaluation,
tissue specimens were routinely processed.
Results. The following characteristic histological phenomena of paraduodenal
pancreatitis were observed: Brunner’s gland hyperplasia occurred
in all cases, while cystic changes of the duodenal wall and adenomyomatosis
of the duodenal wall were found in 9/10 patients. Variable
numbers of a mixed inflammatory infiltrate were present in all patients
analyzed. In 50%, we found foreign body giant cell reaction in the neighbourhood
of some pseudocysts. However, most interestingly obliteration
of segmental arteries was present in 6/10 cases.
Conclusions. This histological study confirms the common morphological
changes in paraduodenal pancreatitis. Interestingly, we found vessel
obliteration in several cases, which has not been described for this subtype
of chronic pancreatitis so far. It remains to be investigated whether
this specific finding might reflect a particular subgroup of paraduodenal
pancreatitis.
114 | Der Pathologe · Supplement 1 · 2012
FR-P-096
Diagnostic value of immunohistochemical IMP3 expression in
core needle biopsies of pancreatic ductal adenocarcinoma
D .L . Wachter1 , A . Schlabrakowski2 , J . Hoegel3 , G . Kristiansen4 , A . Hartmann1 ,
M .-O . Riener1 1University Hospital Erlangen-Nuremberg, Institute of Pathology, Erlangen,
2 3 Nuremberg Clinic Center, Insitute of Pathology, Institute of Human Genetics,
University Hospital Ulm, 4University Hospital Zurich, Zürich, Switzerland
Aims. The oncofetal protein, insulin-like growth factor-II messenger
ribonucleic acid-binding protein 3 (IMP3), has been analyzed in many
different tumors. Various studies have found that IMP3 is a marker for
malignancy and is correlated with increased tumor aggressiveness and
reduced overall survival. The diagnosis of pancreatic ductal adenocarcinoma
(PDAC) in core needle biopsies can be challenging, and immunohistochemical
markers are needed.
Methods. We studied IMP3 expression in 177 core needle biopsies of the
pancreas, including 112 PDACs, 55 cases with chronic sclerosing pancreatitis,
and 10 biopsies with tumor-free pancreatic tissue without inflammation.
An additional 18 biopsies of PDAC metastases (16 liver biopsies
and 2 lymph node biopsies) were analyzed. To study IMP3 expression in
large tissue sections, 45 pancreatic resection specimens (26 with PDAC
and 19 with chronic sclerosing pancreatitis) were investigated.
Results. In contrast to normal or inflamed pancreatic tissue, which was
negative in 47 of 65 (72.3%) cases and weakly positive in 15 of 65 (23.1%)
cases, strong IMP3 expression was found in 99 of 112 (88.4%) PDACs.
Therefore, sensitivity and specificity of IMP3 expression in the differential
diagnosis of PDAC and chronic sclerosing pancreatitis using core
needle biopsies were found to be 88.4% and 94.6%, respectively. These
results were confirmed in the pancreas resection specimens. Furthermore,
strong IMP3 expression was found in 17 of 18 (94.4%) of the PDAC
metastases that were analyzed.
Conclusions. Our study shows that IMP3 is an easy to use and potentially
new immunohistochemical marker for the diagnosis of PDAC in core
needle biopsies.
FR-P-097
Molecular analysis of putative therapeutic targets in pancreatic
acinar cell carcinomas
F . Bergmann1 , H . Bläker2 , A . Harjung1 , P . Mayer1 , B . Sipos3 , G . Klöppel4 ,
P . Schirmacher1 1 2 University of Heidelberg/ Institute of Pathology, Heidelberg, Charité Berlin,
Institute of Pathology, 3University of Tübingen, Institute of Pathology, 4Tech nical University Munich, Institute of Pathology
Aims. Pancreatic acinar cell carcinomas represent aggressive tumors who
frequently display metastases at the time of diagnosis. Because they are
rare, established therapeutic concepts other than surgery practically do
not exist. The aim of the present study was to evaluate established and innovative
therapeutic markers in these to date poorly understood tumors.
Methods. Putative therapeutic targets were investigated by means of direct
sequencing, immunohistochemistry, and/or Western blot analyses
in a series of 60 pancreatic acinar cell carcinomas.
Results. 4% of the tumors displayed k-ras mutations. While 40% of the
acinar cell carcinomas showed immunohistochemical expression of
EGFR, no EGFR mutations were detected. Heat shock protein 90, heat
shock protein 70, L1CAM, and Her2/neu were expressed in 100%, 90%,
65%, and 0% of the tumors, each. mgMT deficiency was found in 28% of
the tumors.
Conclusions. EGFR, heat shock proteins 90 and 70, L1CAM, and mgMT
represent promising targets and predictive markers for the therapy of
inoperable or progressive pancreatic acinar cell carcinomas.

FR-P-098
Immunohistological staining with the monoclonal antibody
Em2G11 is highly specific and sensitive for Echinococcus multilocularis
larvae in human tissue
T .S . Herrmann 1 , D . Tappe 2 , L . Stark 1 , B . Grüner 3 , K . Buttenschön 4 , D . Henne-
Bruns 5 , P . Kern 6 , P . Möller 1 , P . Kern 3 , P . Deplazes 7 , T .F .E . Barth 1
1 University of Ulm, Institute of Pathology, Ulm, 2 University of Würzburg,
3 University Hospital and Medical Center Ulm, Division of Infectious Diseases,
Ulm, 4 University of Alberta, Department of Surgery, Edmonton, Canada,
5 University Hospital of Ulm, Department of General, Visceral, and Transplantation
Surgery, Ulm, 6 University of Ulm, Institute of Epidemiology
and Medical Biometry, Ulm, 7 University of Zurich, Institute of Parasitology,
Zürich, Switzerland
Aims. Alveolar echinococcosis (AE) and cystic echinococcosis (CE) are
two parasitic diseases in humans caused by the metacestode stages of
Echinococcus multilocularis and Echinococcus granulosus, respectively.
Differential diagnosis is fundamental for the choice of specific therapy
strategies and prognosis.
Methods. We have analyzed 96 archived formalin-fixed, paraffin-embedded
tissue samples, including 5 cutting needle biopsies and 3 fine
needle aspirates from patients with AE or CE with the monoclonal antibody
MAbG11, specific for the antigen Em2G11 in the laminated layer of
the metacestode of E. multilocularis.
Results. We show that, in human tissue, staining with MAbG11 is highly
specific for the laminated layer and the calcareous corpuscles of the
E. multilocularis metacestode while no staining was observed in the metacestode
stage of E. granulosus. Furthermore, the antibody marks small
particles of E. multilocularis (Spems) of less than 1 µM in the necrotic
tissue surrounding the main lesion. Spems are also detected in the liver
sinusoids and lymphatic tissue outside the main lesion, most probably
due to shedding from the laminated layer. In 6 of 96 samples, the conventional
histological diagnosis based on haematoxylin and eosin and PAS
stainings alone had to be adjusted when revised by immunohistology
with MAbG11. The specific staining was proved on bioptic material fixed
for more than 60 years in formalin.
Conclusions. We conclude that the MAbG11-antibody is a highly specific
tool in the diagnostic algorithm of AE also on long-time archived formalin-fixed
or paraffin-embedded tissue. The staining modality with small
particles outside the main lesion of E. multilocularis sheds new light on
the interaction of the parasite with the human host and suggests a systemic
effect.
FR-P-099
Chronic HEV hepatitis after liver transplantation: a clinicopathological
follow up over 3 years with implications for the differential
diagnosis of chronic hepatitis
J . Friemel1 , F . Böhm1 , M . Bawohl1 , E . Marques-Maggio1 , J . Seebach2 , P . Dutkowski3
, B . Müllhaupt4 , U . Protzer2 , A . Weber1 1 2 University of Zurich, Pathology, Zürich, Switzerland, Technical University
(TU) Munich, Institute of Virology, München, 3University of Zurich, Visceraland
Transplant Surgery, Zürich, Switzerland, 4University of Zurich, Hepatology
and Gastroenterology, Zürich, Switzerland
Aims. Hepatitis E virus (HEV) infection usually manifests as an acute
and self-limiting hepatitis. Recently, cases of chronic HEV infection following
(solid) organ transplantation have been reported. Here, we present
the case of a 57-year-old man who de novo developed a chronic HEV
infection following liver transplantation (LTX).
Methods. The formalin-fixed and paraffin-embedded (FFPE) tissues
of the explant liver and six liver biopsies (taken 1 day to >3 years after
LTX) were routinely processed for histology. In addition, immunostains
(CMV and ADV) and in situ hybridisation (EBV) were performed as
well as RT-PCR testing on FFPE material for HEV, targeting the HEV
ORF2 gene region. Liver enzymes and viral infections were serologically
monitored.
Results. The patient developed elevated liver enzymes eight months after
LTX. Liver biopsy by that time showed spotty eosinophilic necrosis,
but no inflammation. After 8 month, HEV-RNA was detected serologically
and in the liver biopsy. Follow-up biopsies revealed a chronic
hepatitis with mild portal and lobular inflammation, ductular reaction
and portal fibrosis. No other viruses (hepatitis viruses A, B, C and D;
EBV, ADV, cmV) were detectable. Whereas retrospective testing of the
explanted liver and day 1 biopsy were negative, follow-up biopsies were
HEV-positive for now more than 3 years.
Conclusions. The case presented illustrates that HEV infection is an upcoming
cause of chronic hepatitis in particular in immunosuppressed
patients. The histologic pattern is similar to the chronic hepatitis C infection.
Histologic diagnosis is challenging in cases of unusual histology,
e.g. with little inflammation. Molecular testing is helpful in such cases.
FR-P-100
eIF3a as a putative gall bladder cancer target
A .K . Mehta1 , R . Spilka2 , C . Lackner1 , H . Müller1 , S . Lax3 , P . Obrist2 , J . Haybaeck1 1 2 Institute of Pathology, Medical University of Graz, Austria, Pathologylab
Dr . Obrist & Dr . Brunhuber OG, Zams, Austria, 3Department of Pathology,
General Hospital Graz West, Graz, Austria
Aims. Gallbladder carcinoma (GBC) is the most common type of biliary
tree carcinomas. GBC is an aggressive and often lethal cancer which is
usually diagnosed at advanced stage with a 5-year survival rate

Abstracts
FR-P-101
Metachronic coincidence of a neuroendocrine carcinoma of the
gall bladder after former endometrioid ovarian adenocarcinoma
M . Petersen 1 , T . Kalinski 2 , H . Lippert 1 , J . Bischoff 3 , U .R . Bohr 4 , F . Meyer 1
1 University Hospital, Dept . of Surgery, Magdeburg, 2 University Hospital, Institute
of Pathology, Magdeburg, 3 University Hospital, Dept . of Gynecology
& Obstetrics, Magdeburg, 4 University Hospital, Dept . of Gastroenterology,
Hepatology & Infectious Diseases, Magdeburg
Aims. Along the demographic development, there is an increasing agedependent
multimorbidity. In particular, tumor diseases and second
malignancies will have an impact on the future profile of medical care.
Simultaneous or subsequent (within a short-term interval) occurrence
of two tumor manifestations is always a clinical challenge, especially
with regard to finding the correct diagnosis clarifying malignancy of the
same or different entity.
Methods. By means of an extraordinary and exemplary case report, the
challenge is demonstrated to precisely diagnose a tumor lesion based on
available results of investigations and to differentiate between recurrent
tumor growth and a metastasized tumor manifestation of a second (different)
tumor lesion.
Results. Medical history and clinical finding: In a 73-year-old woman,
histopathological investigation of a specimen after cholecystectomy revealed
a slightly differentiated adenocarcinoma including a lymph node
metastasis. To exclude a metastasis of a known endometrioid ovarian
adenocarcinoma, comparing immunhistological investigations resulted
in: i) CK7-/CA125-negative, CK20-/chromogranin-A-/synaptophysinpositive
(counting for gall bladder and lymph node metastasis); ii) CK7-/
CA125-positive, CK20-/Chromogranin-A-/synaptophysin-negative
(counting for ovar). Therefore, tumor lesion of the gall bladder and the
lymph node was rather associated with a neuroendocrine carcinoma of
the gall bladder than with a metastasis of an ovarian carcinoma. Based
on the diagnosis, re-laparotomy was performed including resection of
the cystic stump and atypical resection of hepatic segment V (“liver bed”
of the gall bladder) as well as lymph node dissection of the hepatoduodenal
ligament. Clinical course: After 14 months, a further metastasis
of a neuroendocrine carcinoma within an additional lymph node was
diagnosed. There had been no recurrent tumor growth of the left-sided
endometrioid ovarian adenocarcinoma since 8 years.
Conclusions. Due to the increased incidence of tumor diseases and second
neoplasias, histopathological and immunohistochemical analyses
are very important in the diagnostic process. According to the available
literature, the presented case is the first description of a metachronous
coincidence of an endometrioid ovarian adenocarcinoma and a neuroendocrine
carcinoma of the gall bladder.
FR-P-102
No crush artifacts: statistical analysis of the histological changes
in the donor bile ducts with special regard to ischemic-type
biliary lesion (ITBL)
T . Hansen1 , D . Hollemann1 , M . Heise2 , M . Hoppe-Lotichius2 , C .J . Kirkpatrick1 ,
G . Otto2 1 2 University of Mainz, Institute of Pathology, Mainz, University of Mainz,
Department of Transplantation/Hepatobiliopancreatic Surgery, Mainz
Aims. Ischemic-type biliary lesion (ITBL) is a feared complication after
liver transplantation as it often leads to graft loss. Recently, we presented
the histological changes of the donor bile ducts taken during liver
transplantation. We now performed a scoring system and statistically
analysed the results. With special reference to ITBL, significant findings
are now demonstrated.
Methods. The histomorphology of the donor bile ducts was studied
84 patients (recipients: 68 male, 16 female, median age 58 yrs; donors:
40 male, 44 female; median age 52.5 yrs). For the characteristic histological
findings, a histological scoring system was established. The single
116 | Der Pathologe · Supplement 1 · 2012
phenomena and the sum score were then statistically reviewed with regard
to the development of ITBL. χ2-test was used to compare the different
score values; p0.001). Correlation of DMBT1 expression with age identified
significant higher expression in older patients (p>0.001).
Conclusions. Our data suggest that DMBT1 seems to play a diverse role
in chronic inflammation of bile ducts and in the development of BTC
dependent on spatiotemporal expression. DMBT1 expression might be of
importance in the progression of BTC and ultimately have an influence
on the outcome of BTC-patients.

FR-P-104
Histopathological evaluation of the iron content in liver biopsies
J . Kelterborn 1 , K . Zöller 1 , J . Biet 1 , Y . Chen 1 , A . Herrmann 2 , M . Kiehntopf 3 ,
A . Stallmach 4 , I . Petersen 1
1 Friedrich-Schiller-University of Jena, Institute of Pathology, Jena, 2 Friedrich-
Schiller-University of Jena, Institute of Internal Medicine, Jena, 3 Friedrich-
Schiller-University of Jena, Institute of Clinical Chemistry, Jena, 4 Friedrich-
Schiller-University of Jena, Institute for Internal Medicine, Jena
Aims. Hereditary hemochromatosis is the most common autosomal recessive
genetic disease in Northern Europe. Iron overload leads to serious
damage of various organ systems. Early diagnosis is desirable due
to the possibility of prevention of severe organ dysfunction. However,
the majority of patients are diagnosed in adulthood in the context of an
uncertain liver pathology. Liver biopsy is an essential tool of analysis. In
the present work two new iron scores were tested for their validity in the
diagnosis of hemochromatosis.
Methods. At the Institute for Pathology of the University Hospital of Jena
all liver samples of a 2-year period were retrieved from the pathology files
and reviewed. In total, 2326 cases were assessed of which 60 were selected
for an in-depth study on iron content. The mean age of the patients was
59 years, 72% originated from male patients. In all 60 samples a mutation
analysis of the two most common gene loci in hemochromatosis were
performed, i.e. HFE282 and HFE63. Moreover, using the clinical diagnosis
documentation system, laboratory values of liver diseases and iron
storage diseases were analyzed. The assessment of the iron content of the
liver biopsies were done by the Prussian blue stain applying two scores
with comprised a published 21-tier and a newly developed 5-tier grading
system.
Results. It was shown that both iron scores were highly correlated with
each other (p

Abstracts
FR-P-107
Comprehensive histological characterization of murine hepatocellular
tumors – towards a classification of murine and human
hepatocellular carcinomas
L . Frick 1 , F . Böhm 2 , Y . Boege 1 , J . Friemel 2 , M . Heikenwaelder 3 , A . Weber 2
1 University Zurich, Department of Pathology, Zürich, Switzerland, 2 University
Zurich, Institute of Surgical Pathology, Zürich, Switzerland, 3 Munich,
Institute for Virology and Helmholtz Zentrum, München
Aims. The aim of our study is to characterise and compare different mouse
models of hepatocellular carcinoma (HCC) which may reflect different
ways of hepatocarcinogenesis in humans (including viral and toxic)
in order to test their applicability to human HCC.
Methods. We have implemented high-throughput, high-quality scanning
technology to make digital copies of all our histological slides. A
viewing software is used to display sequential sections side-by-side on a
computer screen, and to measure tumours and annotate them for later
reference. This enables us to determine the morphological and immunohistochemical
characteristics of each individual lesion efficiently and
comprehensively, even if there are many tumours and dysplastic lesions
per slide, and thus discover correlations that may otherwise have been
missed.
Results. We have found clear differences among the mouse models, not
only in the underlying liver pathology, but also in the spectrum of liver
tumours that arises. In addition, certain regularities and correlations of
tumour morphology and immunophenotype suggest a possible classification
of liver tumours in mice. Using tissue microarrays of human
HCC, we have discovered that the classification of murine tumours has
cross-species applicability to human tumours.
Conclusions. The results of our morphological and immunophenotypic
studies, together with the data from molecular analyses, provide
the basis for a classification of liver tumours that applies to both mice
and humans. We also intend to show which mouse models recapitulate
which aspects of hepatocarcinogenesis in humans, and may thus provide
suitable models for testing therapeutic interventions.
FR-P-108
LGR5 is differentially expressed in hepato-gastrointestinal
tumors
E . Simon1 , D . Petke1 , C . Böger1 , V . Warneke1 , H .-M . Behrens1 , C . Röcken1 1Christian-Albrechts-University, Institute of Pathology, Kiel
Aims. Carcinomas of the hepato-gastrointestinal tract are still leading
cause of cancer related deaths worldwide. The orphan G-protein-coupled
receptor (GPCR) and Wnt-target protein LGR5 was recently identified
as a stem cell marker for cells with intestinal differentiation. In
this study we generated a polyclonal anti-LGR5 antibody to investigate
protein expression in various hepato-gastrointestinal carcinomas and its
correlation with clinicopathological patient characteristics.
Methods. Differential expression of LGR5 was studied on transcriptional
(real time-polymerase chain reaction) and translational level (immunohistochemistry)
in carcinomas and corresponding normal mucosal specimens
comprising seven different primary tumor sites, i.e. oesophagus,
pancreas, stomach, liver, colon and rectum. The putative clinicopathological
relevance of LGR5 expression in terms of patient survival and the
histoanatomical distribution of the protein was studied in 100 patients
with gastric carcinoma.
Results. We succeeded to establish and characterize a highly specific antibody
that recognizes the C-terminal tail of LGR5. LGR5 was differentially
expressed on transcriptional and translational level in adenocarcinomas
of the oesophagus, pancreas, stomach, colon, rectum, hepatocellular
and cholangiocellular carcinoma of the liver compared with the adjacent
non-neoplastic tissue. However, in intestinal type gastric cancer the localization
and number of LGR5+ cells changed during tumorigenesis.
118 | Der Pathologe · Supplement 1 · 2012
Conclusions. Our results substantiate the significance of LGR5 on the biology
of hepatogastrointestinal carcinomas and provide evidence for its
function as potential stem cell marker and candidate therapeutic target
in the stomach. The strikingly changed histoanatomical distribution of
LGR5+ cells during tumorigenesis could be an important observation in
understanding tumor formation and progression. With our anti-LGR5
antibody we now have a useful tool for detection and further analyzes of
LGR5 biological function.
FR-P-109
Gene expression analysis of the Mcl-1delHEP mouse model of
hepatocarcinogenesis reveals overlapping expression profiles
with human hepatocellular carcinoma
F . Böhm1 , Y . Böge2 , R . Maire2 , J . Friemel1 , M . Heikenwälder3 , A . Weber1 1University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland,
2University Hospital Zurich, Institute of Neuropathology, Zürich,
Switzerland, 3Institute of Virology, Helmholtz Center, Munich
Aims. Apoptosis is regulated by a counterbalancing network of proapoptotic
and pro-survival proteins. Mcl-1 is a crucial pro-survival factor,
preventing cells to undergo apoptosis. Many chronic liver diseases
are characterized by a constant loss of hepatocytes. Recently, we have
shown that a mouse model with hepatocyte-specific deletion of Mcl-1
(Mcl-1delhep) reveals increased apoptosis, regeneration, and development
of hepatocellular carcinomas (HCC). To better characterize apoptosis-driving
tumorigenesis, we analyse gene expression patterns in the
Mcl-1delhep model, and compare these to gene expression patterns in
human liver tissues including HCC.
Methods. RNA from livers of Mcl-1delhep mice, several other genetic
mouse models and human HCCs of various etiologies were isolated,
analysed and compared by quantitative real-time PCR.
Results. RNA microarray of livers at 2 months of age uncovered significantly
up- and downregulated genes in Mcl-1delhep mice compared to
wild-type mice. Expression of Top 5 genes was also found to be significantly
upregulated in 12 months old Mcl-1delhep mice (non-tumor and
tumor tissue) and HCCs of various genetic mouse models for hepatocarcinogenesis
as well as in human liver RNA from HCCs with different
etiology.
Conclusions. The Mcl-1delhep mouse model shows that increased apoptosis
might serve as a main trigger of tumorigenesis, and thus recapitulates
the human pathogenesis of chronic liver diseases such as liver
tissue destruction and subsequent cancer formation. Overlapping gene
expression patterns in the Mcl-1delhep mouse model, human tissues of
chronic liver diseases and HCC confirms that the Mcl-1delhep mouse
model is a valuable tool for studying human hepatocarcinogenesis, and
thus also might be suitable for the identification of new molecular targets
and interventional studies.
FR-P-110
Clear cell foci of altered hepatocytes in human liver show an overexpression
of the AKT/mTOR and Ras/Raf1 pathways as well as the
lipogenic phenotype (similar to hepatocarcinogenesis in the rat
and to human hepatocellular carcinoma)
S . Ribback1 , D .F . Calvisi1 , C .-D . Heidecke2 , M . Birth3 , F . Dombrowski1 1 2 Universitätsmedizin Greifswald, Institut für Pathologie, Greifswald, Universitätsmedizin
Greifswald, Klinik und Poliklinik für Chirurgie, Greifswald,
3Hanse-Klinikum Stralsund, Klinik für Allgemein-, Viszeral-, Thorax- und
Gefäßchirurgie, Stralsund
Aims. AKT/mTOR and Ras/Raf1 pathways as well as the lipogenic phenotype
have been shown to be activated in a model of hepatocarcinogenesis
in the rat induced by hyperinsulinemia and in human hepatocellular
carcinomas. In the rat model the activation of these pathways starts

within the earliest morphologic detectable alterations, i.e. clear cell foci
(CCF) of altered hepatocytes. CCF are described in humans indeed, but
it is unclear whether these proto-oncogenic pathways are already activated
within these foci.
Methods. 241 liver resections were examined by using electron microscopy,
histology, enzyme- and immunohistochemistry.
Results. CCF are present within 35% of the extrafocal tissues of non
cirrhotic livers. Electron microscopy illustrates massive glycogen storage
within CCF, largely due to reduced activity of the glycogenolytic
enzyme glucose-6-phosphatase. Hepatocytes of the CCF overexpress
the insulin receptor and the glucose transporter proteins GLUT1 and 4.
Insulin induced protooncogenic pathways AKT/mTOR (AKT, chREBP,
activated mTOR, inactivated AMPKalpha, activated RPS6, inactivated
4EBP1) and Ras/Raf1 (IRS1, Ras, Raf1, MEK-1, ERK1/2, MKP-3, RALA),
enzymes of glycolysis (Glucokinase, PFK, Pyruvate kinase), de novo lipogenesis
(ACLY, ACAC, FASN, USP2, AKR1B10, SCD1), beta-oxidation
(ACADM) and cholesterol synthesis (HMGCoAR, SQS) are upregulated
in the CCF compared to normal liver tissue. Due to activation of these
proto-oncogenic factors the proliferation activity in the CCF is 2-fold
higher than in extrafocal tissue (Ki67-Index 0.75% vs. 0.36%; p=0.002).
Conclusions. CCF reveal a phenotype which is known caused by hyperinsulinism
in experimental hepatocarcinogenesis models as well as in
human hepatocellular carcinomas. These metabolic changes and activation
of proto-oncogenic pathways have not been observed in human
CCF so far. Our results indicate that CCF represent precursor lesions of
hepatocellular carcinoma also in humans.
FR-P-111
Adjuvant fluorescent in situ hybridization in equivocal biliary and
pancreatic duct cytology
M . Schramm1 , A . Thieme1 , N . Pomjanski1 , H . Neuhaus 2 , A . Böcking1 ,
S . Biesterfeld1 1Heinrich Heine University, Institute of Pathology, Düsseldorf,
2Evangelical Hospital, Düsseldorf
Aims. The information whether a stricture or compression of the biliary
and pancreatic ducts is of benign or malignant nature is important
for therapy planning. Brushing of the suspect lesion in the context of
an endoscopic retrograde cholangiopancreaticography is an established
diagnostic procedure with moderate sensitivity and high specificity of
conventional cytological diagnosis. Equivocal cytology due to artefacts
or insufficient sampling is frequent. The detection of aneusomy by multicolour
fluorescent in situ hybridization (FISH) is strongly associated
with malignancy. The concern of our study was to determine if the addition
of FISH subsequent to the cytological investigation could substantially
decrease the number of equivocal cytology diagnoses.
Methods. A cohort of 235 biliary/pancreatic duct brushings sent to the
Department of Cytopathology during January 2005 and December
2007 was enrolled in this study to determine the diagnostic accuracy of
cytology. In a second series of 143 brushings, consisting of all positive
specimens (positive control) and all equivocal specimens in the above
mentioned period and negative specimens in the period from January
to December 2007 (negative control), aneusomy was analysed with the
UroVysion FISH multiprobe. A histological and/or clinical follow up
according to a reference standard, defined in advance had been available
for 219 brushings.
Results. In the whole cohort, cytology achieved a sensitivity and specificity
of 75.3% and 93.1%, respectively. The reduced specificity, compared
with the literature was attributable to the use of cytology as a screening
test; all equivocal specimens were assigned as positive for statistics. FISH
identified 29 out of 38 (76.3%) evaluable cytologically equivocal specimens
as true positive and 9 out of 9 (100%) as true negative, compared to
the follow up. All cytological negative (January to December 2007) and
28 out of 29 positive specimens were confirmed by FISH. Aneusomy was
not observed in benign lesions, according to follow up data.
Conclusions. Adjuvant analysis of aneusomy with multicolour FISH in
biliary/pancreatic duct brushings reduces equivocal cytology rate to a
great extent and is 100% specific.
Poster: Herz- und Gefäßpathologie
FR-P-112
The impact of genetic inactivation of the LIM-ony-protein FHL2
on cardiac remodelling
D . Goltz1 , E . Ramadori1 , S . Huss2 , M . Besmens3 , R . Meyer4 , R . Büttner2 1 2 University of Bonn, Dept . of Pathology, Bonn, University of Cologne, Dept .
of Pathology, Köln, 3University of Bonn, Physiology II, Bonn, 4University of
Bonn, Physiology II, Bonn
Aims. LIM-domain containing proteins play a decisive role during ontogenesis
and cellular differentiation. The LIM-only protein Fhl2 associates
with cytoplasmatic scaffolding proteins and acts intranuclearly as a
co-activator and co-repressor of transcription factors. Fhl2 is specifically
expressed within the cardiovascular system during ontogenesis and high
expression levels continue throughout lifetime. This suggests that Fhl2 is
also of importance during cardiovascular remodelling. Fhl2 deficiency
is known to induce an exacerbated cardiac hypertrophy upon chronic
beta- adrenergic stimulation. This study investigates cardiac remodelling
in the Fhl2 deficient organism caused by an increase in cardiac afterload.
Methods. Experimental transverse aortic constriction (TAC) was induced
in control animals and Fhl2-/- mice. After 14 days, morphometric
and hemodynamic evaluations using a Millar catheter were performed.
Vascular contraction and relaxation was examined using a Mulvany
myograph and renal renin-mRNA-expression was analysed by quantitative
real time PCR. Cardiac fibrosis was addressed by histologic techniques.
Results. TAC induced a rise in pre-stenotic diastolic arterial blood pressure
and a significantly more severe increase in pre-stenotic systolic arterial
blood pressure in the Fhl2-/- animal compared to the control animal.
The transstenotic pressure gradient, however, was identical in the Fhl2-
/- animal and the control animal. In vitro vascular contraction measurements
proved that aortic rings of Fhl2 deficient mice featured combined
contractile dysfunction and disturbed relaxation that was independent
of receptor mediation. As a consequence, renal renin-mRNA-expression
was significantly suppressed in Fhl2-/- mice. Cardiac hypertrophy, however,
was less distinct in Fhl2 deficient animals than in wild type animals
as was cardiac fibrosis.
Conclusions. Fhl2 deficiency encompasses cardiac protection against
chronic increases in cardiac afterload, in this context due to a suppression
of the renin angiotensin axis.
FR-P-113
The impact of Crp2 deficiency on cardiovascular adaptation and
remodelling
D . Goltz1 , E . Ramadori1 , S . Huss2 , R . Meyer3 , R . Büttner2 1 2 University of Bonn, Dept . of Pathology, Bonn, University of Cologne, Dept .
of Pathology, Köln, 3University of Bonn, Physiology II, Bonn
Aims. Lim domain containing proteins among them Crp2 play a central
role in organogenesis and cellular differentiation. Crp2 is specifically
expressed in cardiovascular tissue. Its up-regulation in cardiomyocytes
coincides with vascular smooth muscle differentiation in vitro. We investigated
the vascular phenotype of Crp2 deficient mice in vitro and
addressed the issue of cardiac remodelling under chronic elevation of left
ventricular afterload in vivo.
Der Pathologe · Supplement 1 · 2012 |
119

Abstracts
Methods. Phenylephrine and potassium induced vascular contraction
and endothelially mediated relaxation was investigated in Crp2 deficient
mice and control animals by using the Mulvany Myograph. Transverse
aortic stenosis (TAC) was induced in Crp2-/- mice and wild type animals.
After 14 days, cardiac function was evaluated by cardiac catheterisation
using a Millar catheter. Cardiac hypertrophy and fibrosis was
addressed my morphometric analysis.
Results. Aortic rings of Crp2 deficient animals displayed an isolated contractile
disorder. Vascular contraction was enhanced both under stimulation
with phenylephrine and under increased extracellular potassium
concentrations. Endothelially mediated relaxation was unaffected by
Crp2 deficiency. 14 days after TAC Crp2-/- animals developed exacerbated
cardiac hypertrophy and moderate myocardial fibrosis compared to
the control group. We observed a significantly more severe elevation of
blood pressure levels in Crp2-/- animals compared to the control group.
At the same time the pressure gradient across the stenosis was unchanged
in wild type and Crp2-/- animals. Functional analysis of cardiac contraction
revealed an acute diastolic dysfunction and reduced contractile
reserve in Crp2 deficient mice.
Conclusions. Crp2 deficiency leads to an aggravated vascular contraction
upon both receptor mediated and receptor independent stimuli in vitro.
In vivo, TAC induces an exacerbated cardiac hypertrophy in Crp2-/- animals.
Crp2 plays a central role in cardiac and vascular homoeostasis, its
loss leads to disturbed cardiac remodelling.
FR-P-114
Functional dichotomy of myofibroblasts in chronic cardiac diseases
M . Franz1 , A . Renner2 , M . Ditze3 , K . Grün4 , D . Neri5 , H . Kosmehl6 , H .R . Figulla1 , I .
Petersen3 , A . Berndt3 1University Hospital Jena, Department of Internal Medicine I, Jena,
2Ruhr- University of Bochum, Heart Center North Rhine-Westphalia,, Bad
Oeynhausen, 3University Hospital Jena, Institute of Pathology, Jena, 4Uni versity Hospital Jena, Department of Cardiothoracic Surgery, Jena, 5Swiss Federal Institute of Technology, Institute of Pharmaceutical Sciences, Zürich,
Switzerland, 6HELIOS-Klinikum Erfurt, Institute of Pathology, Erfurt
Aims. Chronic rejection following heart transplantation is accompanied
by allograft vasculopathy (CAV) and fibrosis (CIF) and represents a valuable
model for the cardiac remodelling during ischemia. Processes are
accompanied by extracellular matrix remodelling and the recruitment
of myofibroblasts (MyoFb). ED-A+ fibronectin was recently suggested as
a co-regulator of MyoFb development. The functional role of MyoFb in
the context of hypoxic damage is not fully understood up to now. Aim
of the study was to analyse the relation of MyoFb to the grade of chronic
rejection and to ED-A+ fibronectin deposition in a rat heart transplantation
model. Furthermore, the influence of the MyoFb secretom on cardiac
myocytes under hypoxic conditions was investigated in vitro.
Methods. A model of chronic rejection after rat heart transplantation
(rHTX) was used. Allografts, recipient and control hearts were subjected
to histological assessment of rejection grade and to immunofluorescence
co-localization analysis of ED-A+ Fn and alpha-SMA. For in vitro investigations,
HL-1 cells were used as a model for cardiomyocytes and the
hTERT-BJ1 fibroblasts cell line was as a model for fibroblasts. alpha-SMA
positive myofibroblasts were generated by stimulation with transforming
growth factor-beta 1 (TGF-beta 1. Hypoxia in HL-1 cells was simulated by
cultivation in presence of deoxyglucose. HL-1 cells were incubated with
unconditioned medium and Fb or MyoFb supernatant. LDH as well as
Troponin-I levels were measured in the supernatant by ELISA.
Results. In the rHTX model the grade of chronic rejection showed a clear
association to the amount of alpha-SMA positive cells. Extensive codepositions
of ED-A+ Fn and alpha-SMA occurred in CAV and also in
fibrosis. TGF-beta 1 treated hTERT-BJ1 fibroblasts showed a substantial
increase in alpha-SMA and were therefore designated as MyoFbs. Pre-
120 | Der Pathologe · Supplement 1 · 2012
culturing of HL-1 in MyoFb-conditioned medium resulted in a protection
against deoxyclucose caused cell damage.
Conclusions. The process of chronic rejection accompanied by tissue hypoxia
entails MyoFb activation and associated ED-A+ fibronectin deposition
speaking well for a perpetuating process. alpha-SMA is suggested
as a valuable marker to detect and quantify tissue remodelling in CAV
and fibrosis. A protective effect of MyoFb on myocytes under hypoxic
stress could be evidenced in vitro. With respect to this result, MyoFb
based diagnostic and therapeutic approaches for ischaemic heart failure
should consider this dichotomy.
FR-P-115
Tumours of the heart, great vessels and mediastinum: a retrospective
study
H . Al-Mohamed1 , R . Hetzer1 , K . Wassilew1 1Deutsches Herzzentrum Berlin
Aims. Primary cardiac and pericardial tumours are rare, with a prevalence
ranging from 0.001–0.3%. A retrospective study was conducted with
the focus on epidemiology and pathological features of cardial, pericardial
and mediastinal tumours. Additionally, the results were compared
to those of already existing similar studies.
Methods. In a review of all pathological records of our institution from
2000 to 2010 268 cardial, pericardial and mediastinal tumours were
identified.
Results. The majority of cases (n=209, 78%) were benign neoplasms, myxomas
being the most frequent histological type (n=105, 50.24%). Among
the primary malignant tumours (n=59, 22.01%), sarcomas (n=18), carcinomas
(n=10) and tumours of the haematopoietic system (n=9) were the
most frequent.
Conclusions. This study gives an overview of primary and secondary
neoplasms in the heart, great vessels and mediastinum, with their preferential
localizations and their epidemiological distributions. As found by
other studies, myxoma is the most common tumour.
FR-P-116
Pitfalls treating tumors close to carotid artery bifurcation –
a case report
H .P . Kuhne1 , M . Hegenscheid1 , E . Fietze2 , A . Lieber1 1 2 Hospital of the Armed Forces Germany, Berlin, Clinic for Pathology Berlin,
Berlin
Aims. A 55-year-old patient presented with neuralgia pain in area of right
face for further diagnostic and therapy. He reported about an operation
in the region of lateral neck in his youth with no further information.
There was no tumor palpable, no local pain. Beside this patient is deaf
since birth and suffers of a gynecomastia with hyperprolactinemia treated
with Cabergolin at the moment. An MRI of brain and skull was without
pathological findings.
Methods. An MRI in the region of right bifurcation of carotid artery showed
a tumor with diameter 1.8×2.7×3.6 cm suspected to be a so called
glomus tumor. In a color duplex sonography there was seen a normal
flow, also a normal MRI of brain and supraaortic vessels was done. During
operation we could identify the tumor beginning at the vagus nerve.
We did a complete resection in toto macroscopical, an instantaneous
section showed no signs of malignancy.
Results. Histological result showed a ganglioneuroma with regressive
changes. Tumorspread was into a cleft transection. Most cells showed in
an immunohistology coloration strong expression of S-100 proteine and
very few strongly regressive changed gangliocytes, furthermore with
grained expression of Synaptophysin and marginal CD-56. After inconspicuous
healing process patient was presented in a interdisciplinary tumor
board where no further need of surgery was decided.

Conclusions. In primary imaging a glomus tumor was number one diagnosis
as most frequent entity of tumors in region of bifurcation of carotid
artery. During operation we found a close relationship to vagus nerve
which was confirmed in histology. For differential diagnosis there has
to be considered a hemangioma, myoepithelioma, peripheral paragangliomas
(f. e. M. von Recklinghausen), schwannoma, clotted aneurysms
of carotid artery interne/externe or tumors out of the members with
multiple endocrine neoplasias. Because most of these tumors are found
accidentally by blood pressure disorders, dizziness attacks or syncopes
a fast imaging is being done which detects the majority of these tumors.
Most authors in literature recommend a surgical resection to salve clinical
symptoms and to exclude a malignant tumor. It should be made
clear, that a apparent clear imaging not always can be confirmed in a
histological examination so a definite diagnosis should always be forced.
FR-P-117
Antibody-mediated rejection is associated with microvasculopathy
after heart transplantation
N .E . Hiemann1 , E . Wellnhofer1 , S . Kretschmer1 , C . Christan1 , H . Lehmkuhl1 ,
C . Knosalla1 , R . Hetzer1 , R . Meyer1 1Deutsches Herzzentrum Berlin, Berlin
Aims. Antibody-mediated rejection (AMR) and microvasculopathy are
associated with poor survival after heart transplantation (HTx). Following
the new guidelines of the ISHLT we tested the effect of AMR on the
development of microvasculopathy (MVP) in biopsy.
Methods. We prospectively studied 134 pts (117 men, mean age 50 yrs)
who underwent endomyocardial biopsy at 4 weeks (n=134), 1 yr (n=107)
and 3 yrs (n=61) after HTx. Acute cellular rejection (ACR; ISHLT), MVP
(ratio of luminal radius to diameter of vessel wall) and endothelial swelling
were evaluated in H&E stainings. AMR was assessed by immunohistochemistry
(CD31-positive capillaries to CD68, IgG, IgA, IgM, C1q
and C3c; all x200).
Results. At 4 weeks, 1 yr and 3 yrs, MVP affected 36%, 48% and 43% of
pts, and AMR was present in 37%, 8% and 10% of pts, respectively. Pts
with AMR more frequently presented with MVP at 4 weeks (47% vs. 22%;
p=0.010), 1 yr (74% vs. 46%; p=0.006) and 3 yrs after HTx (81% vs. 45%;
p=0.013). AMR was significantly correlated to ACR, e.g. at 4 weeks 43%
(p

Abstracts
FR-P-120
How effective are heart valve donation from old organ donors?
K . Große 1 , R . Meyer 2 , C . Wesslau 3 , D . Bösebeck 1 , G . Kirste 4 , R . Hetzer 2
1 Deutsche Stiftung Organtransplantation, Northeast Region, Berlin, 2 Deutsches
Herzzentrum Berlin, Berlin, 3 Foundation of European Tissue Banks,
Berlin, 4 Deutsche Stiftung Organtransplantation, Frankfurt am Main
Aims. There is no upper age limit for organ donation and a quarter of all
organ donors are 65 years old and older. The Northeast Region of the
Deutsche Stiftung Organtransplantation and the Cardiovascular Tissue
Bank of the Deutsches Herzzentrum Berlin examined 1.) 1999–2004
whether heart valves from organ donors over the former age limit of
65 years for heart valve donation were morphologically suitable as grafts
and 2.) 2005–2009 after raising the age limit to 70 years the clinical acceptance
of heart valve grafts from organ donors of 65 to 70 years of age.
Methods. 1.) 1999–2004 the heart valves of 100 old organ donors (female/male:
55/45, median age: 71.5) were examined in accordance with the
standards of the Bio Implant Services (BIS). To compare the valve grafts
above and below the age limit of 65 years, we used data on the aortic and
pulmonary valves of 380 organ donors below the age limit in the same
time period. 2.) 2005–2009 we harvested 49 hearts from organ donors 65
to 70 years of age for processing heart valve grafts in accordance with the
BIS-standards. One aortic and 21 pulmonary valves (25%) of these organ
donors (female/male: 11/10, median age: 67) were suitable as grafts. One
year after valve replacement we send the recipients’ physicans a form to
gather information about valve failure, success of transplantation and
current morphological and functional state of the valve graft.
Results. 1.) Half of all heart valves above and below the former age limit
would have fulfilled the morphological standards as grafts. The great
majority (85%) of old pulmonary valves fulfilled the acceptance criteria,
48% even showing good tissue quality. 2.) 2005–2009 the aortic and the
21 pulmonary valve grafts have been allocated to recipients (female/male:
6/15, median age: 32) suffering from late sequelae of congenital diseases
with defects of the either native valves or former grafts. Successful treatment
without morphological and functional alterations of the grafts
1 year after replacement was reported from the aortic and 13 pulmonary
valve grafts. Five further valve grafts were transplanted but follow-up
1 year later is unknown. One recipient of a pulmonary valve graft died
postoperatively of left ventricular failure. In 2 cases accepted valve grafts
were not transplanted because the surgeons decided intra-operatively on
another procedure.
Conclusions. The data clearly demonstrate, that heart valves grafts from
donors 65 years of age and older can safely be used with good long-term
success.
FR-P-121
Change of external surface roughness of vascular implants improves
implant tissue integration
M . Otto1 , J . Kriegsmann1 , S . Bertz2 1Supra-regional Joint Practice of Histology, Cytology and Molecular Diagnostics
Trier – Düren – Düsseldorf, Medical Health Center for Histology,
Cytology and Molecular Diagnostics, Trier, 2University of Erlangen, Institute
of Pathology, Erlangen
Aims. Today, one of the most important problems in implant medicine is
an optimal designed biointegration of implants. A controlled biointegration
of vascular implants is essential for an optimal biofunction as well as
for biosafety. The fast fixation of the vascular prosthesis reduces the risk
of infection as well as thrombosis, two major events which lead to rapid
functional loss. Implants with a silver coating, used to inhibit implant infection
were modified at the outer surface to accelerate tissue integration.
Methods. Silver coated vascular implant material based on expanded
polytetrafluorethylen (ePTFE, expanded Teflon) was modified by application
of a microwave procedure. Two different protocols of microwave
application induce remodeling of the implant surface. At the first step,
122 | Der Pathologe · Supplement 1 · 2012
the material is implanted subcutaneously in a defined sheep model. The
tissue integration of the implant was analyzed after 6 weeks by interposition
of the implants at the Arteria carotis in a sheep model. We used
8 cm long routinely used but surface modified implants with a diameter
of 6 mm. We used two implants, one with highly and one with slightly
modified surface roughness. The implants were histomorphologically
evaluated using qualitative parameters as well as semiquantitative parameters
of vascularization, inflammation, peri-implant fibrous reaction
and giant cell induction. The tissue reaction was compared to the standardized,
clinically used and FDA accredited SilverGraft prosthesis.
Results. After an implantation period of 6 weeks the thrombogenicity
of implants was not significantly changed. Formation of fibrotic neointima
as well the endothelialization of the inner implant surface was
changed. Other histological parameters – lymphocytic infiltration, granulocytic
infiltration and giant cell density – did not show any alteration
by implant surface modification. The slightly modified implants show
a stronger vascularization and mildly increased thickness of peri-implant
fibrosis. The highly modified implants show no significant change
in vascularization but a minimal increase of thickness of peri-implant
membrane.
Conclusions. The change of implant surface roughness effectively improves
the integration of new developed vascular implant devices by
boosting integration into the peri-implant connective tissue without any
change of typical parameters correlating with biosafety of the vascular
implant material. The highest biofunctionality is found in those implants
with slightly increased surface roughness.
FR-P-122
Change of inner surface of ePTFE vascular implants by immobilization
of heparin and heparan sulfate
M . Otto1 , J . Kriegsmann1 , S . Bertz2 1Supra-regional Joint Practice of Histology, Cytology and Molecular Diagnostics
Trier – Düren – Düsseldorf, Medical Health Center for Histology,
Cytology and Molecular Diagnostics, Trier, 2University of Erlangen, Institute
of Pathology, Erlangen
Aims. Today, one of the most important problems in vascular implant
medicine is thrombosis. Complete or incomplete occlusion of vascular
implants by thrombotic masses is the most unfavorable event in vascular
endoprosthesis and results in insufficient biofunctionality of the
implants. During the last decade several implant coatings were used to
reduce thrombotic events.
Methods. The inner implant surface of ePTFE vascular implants (ePTFE,
expanded Teflon) was modified by a coating of polyurethane with covalent
bounded heparin as well as heparin sulfate. To analyze the thrombogenicity
of these modified materials vascular implants were applied
in a sheep model by interposition into the A. carotis. Routinely clinically
used but surface-modified implants with a diameter of 6mm and a
length of 8 cm were applied. Morphology of the implants was analyzed
after 6 weeks of implantation by morphologic evaluation of qualitative
parameters, especially thrombotic changes at the material surface. Further
parameters were semiquantitative analysis of inflammation, periimplant
reaction and giant cell induction. The tissue reaction was compared
to the standardized, routinely used and FDA accredited ePTFE- as
well as GoreTex-prosthesis.
Results. After an implantation period of 6 weeks implants with heparin
and heparin sulfate coating show macroscopically more and stronger
thrombotic events compared to the uncoated ePTFE and GoreTex-controls.
The morphological analysis of the thrombotic material shows in
the heparin group an early thrombosis of the implants, often with luminal
fibrotic organization. On the other hand, the heparan sulfate group
shows late thrombosis without any organization effects. The rate of
morphologically demonstrable thrombotic events in the heparin group
(37.5%) and the heparin sulfate group (75%) exceed the control rate of 10%
by far. The heparin sulfate group shows a considerable reduced rate of

fibrotic neointma development as well as a reduced endothelialization
whereas in the heparin group a significant difference is not demonstrable.
All other estimated parameters did not show any differences between
controls and modified implants.
Conclusions. In the current setting the covalent binding of heparin and
heparin sulfate induce an increased rate of thrombotic events, which
counteract the original purpose of the surface modification. The morphological
changes may be interpreted as an effect of polyurethane, used
as a partner for the covalent binding of the antithrombotic materials.
FR-P-123
Individual risk assessment in AAA – the value of biomarkers and
their correlation to statin treatment
B . Mühling1 , T . Barth2 , K .-H . Orend1 1University of Ulm, Department of cardiothoracic and vascular Surgery, Ulm,
2University of Ulm, Institute of pathology, Ulm
Aims. In patients with infrarenal aortic aneurysm the aneurysm diameter
determines the indication for operative repair. An individual marker
would be interesting in order to assess the individual rupture risk or in
order to control medical treatment, e.g with statins. Hence the activity
of known metalloproteinases (MMP2/9), inflammatory cytokines (Osteoprotegerin,
Interleukin 6/10) and resistin, an adipokin, and C reactive
protein (CRP) in patients with AAA were measured and correlated to
aneurysm diameter and statin therapy.
Methods. In 63 patients with AAA from 4–9 cm with and without statin
therapy serum activity of MMP 2 and 9, Osteoprotegerin (OPG), IL-6
and IL-10, resistin and CRP levels were measured prior to aneurysm repair.
The expression pattern of resistin was also analyzed using immunohistochemistry
in tissue specimen of the aortic wall. As for age, gender
history of coronary artery disease, hypertension and smoking patient
groups were similar. The results obtained were correlated to aneurysm
diameter and statin therapy.
Results. As for CRP and IL 10 levels we found a significant correlation
to aneurysm diameter (r=0.38 and. r=0.42). IL-6, MMP 2 and 9, OPG
and resistin were not correlated. Patients under regular statin therapy
showed significantly lower levels of resistin and CRP (7.73 vs. 11.04 ng/
ml, p=0.005 resp. 1.8 vs. 6.7 mg/ml, p=0.007). Immunohistochemistry
of aneurismal tissue showed in part close co-localization of resistin and
CD 68 positive cells.
Conclusions. The investigated markers are not able to serve as biomarker
for individual risk assessment in AAA patients; however they underscore
the inflammatory nature of AAA pathogenesis. CD 68 positive cells
may mediate this inflammation. Statins have the potential to slow down
this inflammation and should be prescribed in the conservative medical
management of the disease.
FR-P-124
Risc factors for prosthetic vascular graft infection
L . Höller1 , M .K . Schilling1 , M .R . Moussavian1 1Saarland University Medical Center and Saarland University Faculty of
Medicine, Department of General, Visceral and Vascular Surgery, Homburg
Aims. Vascular prosthetic graft infections (VGI) are rare, but are associated
with a high risk of limb loss, re-infection as well as a high mortality.
Furthermore they lead to high economic costs. In this retrospective analysis
predictive factors for VGI were analyzed.
Methods. Out of a prospective SAP based database with 270 datasets,
206 patients/data sets were studied. All 206 patients were operated between
2001 and 2010 and had a re-operation within the same hospital stay.
This cohort was divided into 3 groups: A: Aortal operations B. Arteriovenous
fistulas for dialyses and C. Femoropopliteal bypasses. Patients were
studied for the primary end point infectious complications with and
without prosthetic graft infection. Infection was verified microbiologi-
cally of bacterial growth at the graft. Beside demographic data a clinical
follow-up examination photo documentation of the affected limb was
performed in all patients.
Results. In group A, neither groin incision nor drainage insertion increased
the risk for infection. However, a preoperative low hemoglobin
(12.9±0.9 vs. 10.2±0.4 95% CI 9.4–11.0 vs. 11.4–14.6; p

Abstracts
FR-P-126
Mild hypothermia induced by surface cooling reduced cerebral
cortex lesions after prolonged cardiac arrest in a pig model
S . Högler 1 , F . Sterz 2 , A . Janata 2 , W . Weihs 2 , P . Schmidt 1
1 University of Veterinary Medicine Vienna, Department for Pathobiology,
Wien, Austria, 2 Medical University of Vienna, Department of Emergency
Medicine, Wien, Austria
Aims. A surface cooling system to induce mild therapeutic hypothermia
was tested in a pig model for prolonged cardiac arrest and type and extent
of cerebral cortex lesions were assessed in comparison to a control
group without cooling.
Methods. Experimental ventricular fibrillation cardiac arrest for 10 min
was induced in 22 large white pigs (35–45 kg). After 3 min of basic life
support and 5 min of advanced life support the animals were defibrillated
and randomized into the hypothermia or the control group. Swine
in the hypothermia group were cooled to a core temperature of 33°C by
the LRS ThermoSuit System, which is pumping a thin layer of ice water
over most of the skin surface, and were kept at that temperature for 14 h.
Rewarming was started 16 h post arrest. Control animals were kept at a
constant core temperature of 38.5°C. At day 9 post arrest animals were
deeply anesthetized and the brain was perfused with 4 L of saline and
1 L of paraformaldehyde. Coronary brain sections were embedded in
paraffin and stained with HE. Frontal, parietal, temporal, occipital and
insular cortices were examined by a semi-quantitative method. Type and
extent of lesions were evaluated in each region and assessed. For group
comparisons the Mann-Whitney-U-test was used.
Results. Restoration of spontaneous circulation and subsequent randomization
was achieved in 16 animals. The target temperature in the
cooling group was reached after 9 (5.3–11.9) min. All animals survived
until the endpoint 9 days post arrest. In frontal, parietal, temporal and
occipital cortex statistically highly significant differences (p

FR-P-129
Alpha-1-antitrypsin-PiZ-antibody ATZ11 recognizes von Willebrand
factor (vWF): diagnostic and pathogenetic aspects
K . Hiththetiya 1 , H . Zhou 1 , S . Steiner 1 , H .J . Hertfelder 2 , H .-P . Fischer 1
1 University Hospital Bonn, Institute of Pathology, Bonn, 2 University Hospital
Bonn, Institute of Experimental Hematology and Transfusion Medicine,
Bonn
Aims. Antibody ATZ11 which is directed specifically against the mutated
Alpha-1-antitrypsin PiZ will be tested on further specific binding
sites independent of PiZ. The identity of the underlying ATZ11-binding
proteins will be analysed. Diagnostic und pathogenetic relevance of this
specific cross-reaction will be discussed.
Methods. Comparative immunhistochemical analysis, imunoelectronmicroscopic
analysis, Western-blots of cultivated human vitelin cord
endothelial cells and thrombocyte aggregates.
Results. ATZ11-specific epitope is found immunohistochemically on
megakaryocytes, platelets, endothelial cells of arteries, veins and some
capillaries of normal human tissues. More extensive staining is found
in portal vessels of end-stage liver cirrhoses. Neoexpression is found in
sinusoidal endothelial cells of actively fibrosing liver diseases, especially
in strongly progressive alcoholic steatohepatitis. Microvascular bed of
hepatocellular carcinomas and hepatocellular adenomas remains unstained.
The staining reactivity corresponds to that of von Willebrand
factor (VWF) and P-Selectin. It is independent from the presence of hepatocellular
AAT deposits of PiZ type and AAT genotype. The epitope
can be detected in Weibel-Palade bodies (WPB) of human vitellin cord
endothelial cells (HUVEC) and in the cytoplasm of patelets by immunoelectronmicroscopy.
ATZ11-Western blotting of HUVEC and patelets
visualizes a 240 kD molecule which is in the spectrum of the molecular
weights of multimeric VWF. VWF deficient serum samples lack this
ATZ11-binding molecule.
Conclusions. Apparently ATZ11 binds to a conformation-dependent epitope
of VWF which might reflect a special functional state of this protein.
ATZ11 expression in sinusoids of actively fibrosing steatohepatitis
highlights the possible role of VWF in sinusoidal occlusion by locally
activated coagulation.
Poster: Pneumopathologie
FR-P-130
MAdL- a new specific marker for adenocarcinomas of the lung
H . Schultz1 , S . Marwitz1 , B . Baron-Lühr1 , G . Zissel2 , C . Kugler3 , K .-F . Rabe3 ,
P . Zabel1 , E . Vollmer 1 , J . Gerdes1 , T . Goldmann1 1 2 Research Center Borstel, Borstel, University of Freiburg, Department for
Pneumology, Freiburg, 3Hospital Großhansdorf, Großhansdorf
Aims. Although the common immunohistochemical markers are well
suitable for sub-differentiation a fraction of indistinct cases of NSCLC
still remains, demanding upgrades of the panel by new markers.
Methods. Here we report the generation and evaluation of a new monoclonal
antibody denoted by “Marker of adenocarcinomas of the lung”
(MAdL), which was raised against the cytoplasmatic fraction of primary
isolated human alveolar epithelial cells type II.
Results. Upon screening, one clone was identified as a marker for alveolar
epithelial cells type II, alveolar macrophages and particularly adenocarcinomas
of the lung. With an optimized staining procedure for formalin
fixed tissues this antibody was evaluated together with the established
markers TTF-1, SP-A, pro SP-B and Napsin A in a large-scale study on
a series of 362 lung cancer specimens. MAdL displays a high specificity
for adenocarcinomas of the lung together with a sensitivity of 76.5% and
is able to provide independent additional diagnostic information to the
established markers.
Conclusions. We conclude that MAdL is a new lung specific marker for
adenocarcinomas, which expands sub-differentiation in a notable portion
of non-small cell lung cancers.
FR-P-131
Pulmonary haptoglobin (pHp) can be used as a new specific marker
for adenocarcinomas of the lung
M . Abdullah1 , S . Marwitz1 , D . Kähler1 , H . Schultz1 , C . Kugler2 , P . Zabel3 ,
E . Vollmer 1 , T . Goldmann1 1 2 Research Center Borstel, Clin . & Exp . Pathology, Borstel, Hospital Großhansdorf,
3Research Center Borstel, Borstel
Aims. There are novel chemo-therapeutic approaches which recently have
been developed for NSCLC, known as a largely chemo-resistant tumour.
Substantial differences have been shown between adenocarcinomas and
squamous cell carcinomas with regard to the adequate therapeutic regimens.
Therefore, sub-differentiation of NSCLC is currently getting more
into focus and increasingly turning out to be a central element within
therapeutic decisions. In this study we analyzed the expression of pulmonary
haptoglobin (pHp) in human lung cancer tissues and compare it
to common markers of adenocarcinomas.
Methods. Immunohistochemistry and heat-induced antigen-retrieval
were conducted. For detection, a one-step polymer-system was applied.
119 formalin-fixed, paraffin-embedded tumour tissues were immunohistochemically
analyzed for expression of php, TTF-1, SP-A, SP-B and
Napsin.
Results. Pulmonary haptoglobin was expressed in 35 of 72 (48.6%) adenocarcinomas
of the lung, [TTF-1 (79.1%), SP-A (51.3%), SP-B (48.6%) and
Napsin (84.1%)]. Expression of pHp in squamous cell carcinomas (N=47)
was absent. A proportion of cases exclusively express either pHp and
Napsin (pHp+/Napsin+: 4.7%) or pHp and TTF-1 (pHp+/TTF-1+: 3.1%).
7.8–14% of the samples would have caused difficulties if the expression of
pHp would not have been addressed.
Conclusions. SP-A and SP-B are specific markers for adenocarcinomas
with a limited sensitivity compared to TTF1; the same holds true for
pHp. Therefore, the inclusion of pHp as an additional marker in the panel
has serious impact on diagnostics and therapy.
FR-P-132
The diagnostic value of cytokeratin 5/6, 14, 17, and 18 expression
in human non-small cell lung cancer
Y . Chen1 , T . Cui1 , L . Yang 1 , M . Mireskandari1 , T . Knösel1 , Q . Zhang1 , M . Pacyna-
Gengelbach2 , I . Petersen 1
1 2 University Hospital Jena, Jena, University Hospital Charité, Berlin
Aims. The constitution and expression patterns of cytokeratin filaments
in human epithelial neoplasms are complex and distinctive. The aims
of this study were analysis of the expression of cytokeratins and evaluation
of their diagnostic application in human non-small cell lung cancer
(NSCLC).
Methods. mRNA expression of CK5, CK6, CK14, CK15, CK17, and CK19
was analyzed by Northern blotting. Protein expression of CK5/6, CK7,
CK14, CK17, and CK18 was evaluated by immunohistochemistry on tissue
microarrays.
Results. Northern blotting showed that CKs were highly expressed in
human bronchial epithelial cells and/or small airway epithelial cells.
In NSCLC cell lines, the expression pattern of CKs was heterogeneous.
In the survey of protein expression of CKs in 95 primary lung tumors,
we found that CK5/6, CK14, and CK17 proteins were highly expressed
in squamous cell carcinoma compared to adenocarcinoma (p=0.001,
p=0.030, and p=0.001, respectively) and higher expression is significantly
linked to lower grading (p=0.006, p=0.002, and p=0.001, respectively),
while increased expression of CK7 and CK18 was observed in adenocarcinoma
(p=0.001, respectively).
Der Pathologe · Supplement 1 · 2012 |
125

Abstracts
Conclusions. Our data suggest that CK5/6, CK7, CK14, CK17, and CK18
have diagnostic value in subclassification of NSCLC.
FR-P-133
Malic enzyme expression in non-small cell lung cancer correlates
with acetyl citrate expression and is associated with mediastinal
lymph node metastasis
A . Csanadi 1 , C . Otto 1 , N . Hörter 1 , A . Oser 1 , M . Donauer 1 , T . Plönes 2 , B . Passlick 2 ,
M . Werner 1 , G . Kayser 1
1 Institute of Pathology, University Hospital Freiburg, Freiburg, 2 Department
of Thoracic Surgery, University Hospital Freiburg, Freiburg
Aims. In contrast to normal human tissues malignant tumors utilize glucose
mainly for the production of reductive equivalents and basic modules
of nucleic acid, protein- and cell membrane synthesis, i.e. ribose,
aminoacids and fatty acids. This metabolic shift results in pronounced
production of lactate, known as the Warburg effect. Malic enzyme (ME)
and acetyl citrate lyase (ACL) are two key enzymes involved in glucose
metabolism and its linkage to fatty acid synthesis. We here analyzed the
expression of these two enzymes in non-small cell lung cancer (NSCLC)
and their association with local and systemic tumor disease.
Methods. Protein expression in tissue multi arrays with a core diameter
of 2 mm of 258 NSCLC patients was evaluated by immunohistochemical
staining with ME (clone 3H5) and ACL (Cell Signaling 4331S). An H-score
calculated by multiplication of the percentage of positive tumor cells
with the predominant staining intensity (score 0 to 3) was used for nonparametric
statistical analyses.
Results. Expression of ME and ACL showed highly significant positive
correlation (p

FR-P-136
The expression of central cell cycle regulators in non-small cell
lung cancer (NSCLC) has therapy dependent prognostic impact
J . Cortis 1 , A . Warth 1 , M . Meister 2 , T . Muley 2 , H . Hoffmann 3 , A . Stenzinger 1 ,
P .A . Schnabel 1 , P . Schirmacher 1 , W . Weichert 1
1 University Hospital Heidelberg, Institute of Pathology, Heidelberg,
2 Thoracic Hospital Heidelberg, Heidelberg, 3 Thoracic Hospital Heidelberg,
Department of Thoracic Surgery, Heidelberg
Aims. The relevance of function and expression of central cell cycle regulators
in NSCLC has been discussed for years with controversial and
non-conclusive results. To address this topic, we evaluated expression
patterns of central cell cycle regulators in a very large NSCLC cohort
with the goal to ultimately clarify their clinical-prognostic role.
Methods. Expression of the G1-phase cell cycle regulators CDK4, cyclin
D1 and p16 were analysed by immunhistochemical staining and quantitative
evaluation on tissue microarrays comprising 1045 completely
resected NSCLC. The data were correlated with clinical-pathological
factors, patients’ survival and response to therapy.
Results. Loss of expression of CDK4 (p=0.017), cyclin D1 (p=0.001) and
p16 (p=0.039) were associated with higher UICC stages. In general, high
expression of all cell cycle regulators was associated with better clinical
outcome in a slightly variable way, depending on protein and prognostic
parameter investigated [CDK4, overall survival (OS): p=0.457, disease
specific survival (DSS): p=0.110, disease free survival (DFS): p=0.011;
cyclin D1, OS: p=0.037, DSS: p=0.009, DFS: p=0.111; p16, OS: p=0.204,
DSS: p=0.484, DFS: p=0.004]. These effects were prominent in adenocarcinomas,
adenosquamous carcinomas and in pleomorphic carcinomas,
but less pronounced in squamous cell carcinomas. Interestingly, after
stratification for therapy, the positive association of protein overexpression
with survival was only seen in adenocarcinomas without adjuvant
radio- and chemotherapy whereas the association in tumors with adjuvant
irradiation and chemotherapy was switched in the opposite direction.
Conclusions. The expression of CDK4, cyclin D1 and p16 in NSCLC has
prognostic impact depending on therapy. High expression of all three
proteins was associated with improved clinical outcome in completely
resected NSCLC without adjuvant therapy.
FR-P-137
The FUSE-binding proteins (FBPs) represent essential regulators
responsible for tumor cell proliferation, migration and invasion in
non-small cell lung cancer
M . Malz1 , M . Bovet1 , J . Samarin1 , E . Herpel1 , A . Warth1 , S . Singer1 , T . Muley2 ,
M . Meister2 , H . Hoffmann2 , P . Schnabel1 , P . Schirmacher1 , K . Breuhahn1 1University Hospital Heidelberg, Institute of Pathology, Heidelberg,
2University Hospital Heidelberg, Thoracic Hospital, Heidelberg
Aims. The single-strand nucleic acid binding far upstream element (FU-
SE)-binding proteins (FBP)-1, FBP-2, and FBP-3 represent a multifunctional
protein family, regulating transcriptional and post-transcriptional
processes as well as microRNA biogenesis. Elevated expression and
pro-tumorigenic functions of all FBPs have been described for human
liver cancer. Moreover first data indicated that FBP-1 affects microtubule
dynamics through regulation of MT-destabilizing factors in non-small
cell lung cancer (NSCLC). Therefore we aimed to analyze expression and
functional relevance of FBPs in NSCLC.
Methods. The expression of FBPs was analyzed at the transcript (qPCR)
and protein level (Tissue Microarrays [TMA], Western Blotting) in primary
human NSCLC tissue samples. Using gene-specific siRNAs the
expression of FBPs was inhibited in different NSCLC cell lines (Calu-1,
Calu-6, and A549). Functional consequences of reduced protein expression
on viability (MTT-Assay), proliferation (BrdU-Assay), apoptosis
(FACS-Assay; PARP-cleavage), migration (two-dimensional scratch assay),
and invasion (sprouting assay) were analyzed.
Results. The expression of FBP-1 and FBP-2 was significantly elevated in
human NSCLCs (>60%) in comparison to non-tumorous specimens. In
vitro, transient inhibition of FBP-1 in NSCLC cells (Calu-6) was associated
with decreased tumor cell viability (−76%), proliferation (−83%),
and increased apoptosis (2.8-fold). In contrast, transient inhibition of
FBP-2 predominantly reduced tumor cell migration (−62%) and tumor
cell invasion (−81%), suggesting that both FBP isoforms facilitate distinct
tumor-supporting effects. In addition, FBP-2 inhibition increased FBP-1
expression at the transcript and protein level in A549 cells, demonstrating
that FBP-1 may compensate the loss of FBP-2. Accordingly, the FBP-
1/-2 double-knockdown led to a significant reduction of cell viability
(−69%).
Conclusions. In summary, this study provides evidence that overexpression
of FBP-1 and FBP-2 is frequently detectable in NSCLC tissues and
that both proteins are essential factors for tumor growth and NSCLC
cell dissemination. Furthermore FBP-2 negatively regulates FBP-1 expression,
indicating a functional compensation.
FR-P-138
Interobserver variability in the application of the novel IASLC/
ATS/ERS classification of lung adenocarcinomas
A . Warth1 , A . Stenzinger1 , A .-C . von Brünneck2 , B . Goeppert1 , J . Cortis1 ,
I . Petersen3 , P .A . Schnabel1 , W . Weichert1 1University Hospital Heidelberg, Institute for Pathology, Heidelberg,
2Charité University Hospital Berlin, Institute for Pathology,
3University Hospital Jena, Institute for Pathology
Aims. Recently, an international consensus classification for adenocarcinomas
(ADC) of the lung has been published. The cornerstone of this
new classification is the quantification of different growth patterns. However,
data on the reproducibility performance of this classification in
the routine diagnostic setting are lacking.
Methods. We selected 100 constitutive cases of conventional lung ADC
resection specimens from our archives. All tumor slides were classified
independently by five experienced pulmonary pathologists from three
institutions and by two pathologists in training according to the recommendations
of the IASLC/ATS/ERS.
Results. The most frequent predominant pattern in our cohort was solid
(37%), followed by acinar (35%), lepidic (20%), papillary (5%) and micropapillary
(3%). kappa values for the denomination of a predominant
pattern revealed a substantial agreement for pulmonary pathologists
(0.44—0.72) but only fair agreement for pathologists in training (0.38–
0.47). Interobserver variability was significantly higher in cases with higher
slide numbers (p=0.028) and was considerably reduced in a second
evaluation round after the initiation of a training session. Intraobserver
variability was low (kappa=0.79–0.87). Papillary and micropapillary patterns
were the most complicated patterns to evaluate, while evaluation of
lepidic and solid tumor growth was straightforward. The acinar pattern
ranged in between.
Conclusions. Our data imply that the novel classification of pulmonary
ADC is applicable with adequate interobserver variability if performed
by specifically trained pathologists. However, additional efforts are needed
to harmonize the application of this novel and clinically important
classification scheme of pulmonary ADC.
Der Pathologe · Supplement 1 · 2012 |
127

Abstracts
FR-P-139
Intraoperative subtyping of pulmonary adenocarcinomas according
to the IASLC/ATS/ERS classification: a feasibility study
P .A . Schnabel 1 , H . Hoffmann 2 , E . Herpel 1 , B . Goeppert 1 , H . Dienemann 2 ,
P . Schirmacher 1 , W . Weichert 1 , A . Warth 1
1 University Clinics Heidelberg, Institute of Pathology, Heidelberg,
2 University Clinics Heidelberg, Thoracic Clinic Heidelberg, Heidelberg
Aims. The IASLC/ATS/ERS proposal for a new classification of pulmonary
adenocarcinomas has important impact on prognosis. It may provide
the basis for surgical decisions in operative therapy of multifocal,
multiple, or recurrent adenocarcinomas as well as of adenocarcinomas
in patients with restricted lung function.
Methods. Intraoperative frozen sections from 25 adenocarcinomas were
analyzed covering the complete central lamella of the tumor (one to four,
mean two frozen sections per patient). The results obtained by two independent
investigators from the frozen sections were compared to those
after paraffin embedding of the whole tumor.
Results. In the frozen sections, the 25 tumors showed the following predominant
patterns: acinar 8/25, solid 7/25, micropapillary 4/25, lepidic
4/25, and papillary 2/25. After paraffin embedding two predominant
patterns were different: one changed from solid to acinar, and one from
acinar to lepidic. In both cases these patterns had been reported as the
second frequent occurring pattern. Two patterns were reported, if occurring
at all: the solid pattern in 10/25, and the micropapillary pattern
in 7/25.
Conclusions. Subtyping of pulmonary adenocarcinomas according to the
IASLC/ATS/ERS proposal for a new adenocarcinoma classification can
be reliably applied in frozen sections. These results are encouraging and
indicate that the classification system may be translated into the intraoperative
setting. This may have implications for surgical strategies and
may eventually allow tissue sparing resections, e.g. of predominant lepidic
adenocarcinomas. The study will be continued. As in 2/25 tumors
the second frequent pattern found in cryosections turned out to be the
predominant pattern after paraffin embedding, all patterns should be
reported semi-quantitatively.
FR-P-140
Clonality of multifocal non-small cell lung cancer:
implications for staging and therapy
A . Warth1 , S . Macher-Göppinger 1 , J . Cortis1 , T . Muley2 , M . Thomas3 , H . Hoffmann4
, P .A . Schnabel1 , R . Penzel1 , P . Schirmacher1 , S . Aulmann1 1 2 University Hospital Heidelberg, Institute for Pathology, Heidelberg, Thoraxklinik
Heidelberg, Translational Research Unit, 3Thoraxklinik Heidelberg,
Oncology, 4Thoraxklinik Heidelberg, Thoracic Surgery
Aims. Non-small cell lung cancers (NSCLC) display a variety of morphological
and molecular features. Accurate subtyping of NSCLC has been
shown to predict patient survival as well as response rates and toxicities
of specific drugs. Assessment of multifocal lung tumors and the distinction
of synchronous primary tumors from intrapulmonary metastases
represent an important problem as this decision significantly influences
tumor staging and subsequent treatment strategies.
Methods. In order to provide a basis for evidence-based treatment decisions
in those patients, we analyzed the clonal relationship of multifocal
NSCLC with indistinguishable histomorphology in a series of 78 patients
by allelotyping (using polymorphic short tandem repeat markers) as
well as KRAS and EGFR mutation testing.
Results. Our data demonstrate a common clonal origin indicative of intrapulmonary
metastases in almost two thirds (~62%) of the cases, while
~36% of multifocal NSCLC displayed unique molecular profiles suggesting
separate primary tumors. Divergent KRAS and/or EGFR mutations
were observed in approximately 8% of all cases.
Conclusions. With the increased availability of EGFR-targeted therapy
options, non-resectable, multifocal NSCLC with diverging KRAS and/
128 | Der Pathologe · Supplement 1 · 2012
or EGFR mutations are likely to show different treatment responses underlining
the need to separately analyze multifocal tumors. Obviously,
this also holds true for further, novel molecular predictors of targeted
therapies.
FR-P-141
Localized nodular amyloidosis and adenocarcinoma of the lung: a
rare association
R . Kurth1 , M . Scharpf1 , F . Fend1 1University of Tübingen, Institute of Pathology, Tübingen
Aims. The lung is frequently involved in generalized amyloidosis, whereas
amyloid localized to the respiratory tract is an uncommon finding.
Nodular amyloidosis of the lung is a rare condition with formation of
tumours containing eosinophilic amyloid deposits and a lymphoplasmacytic
infiltrate and usually presents as incidental radiologic finding
in asymptomatic patients.
Methods. Histology, immunohistochemistry, molecular analysis.
Results. We describe the case of a 74-year-old man with a history of smoking
(30PY). Due to a history of cough, a chest X-ray was performed and
revealed an intrapulmonary tumor mass in the left inferior lobe 4.1 cm in
diameter. Bronchoscopy and mediastinoscopy were performed, but the
biopsies of lung and lymph nodes were non-diagnostic. Subsequently a
video-assisted thoracoscopic resection of the lung lesion was performed
and revealed adenocarcioma of the lung in intraoperative frozen section.
Thus, a lobectomy and lymph node dissection was performed. Histological
examination of the tumor revealed extensive nodular amyloidosis
of the lung with accompanying lymphofollicular infiltrates, giant cell
reaction as well as calcifications and ossifications. Within the amyloid
deposits, a moderately differentiated papillary, non mucinous adenocarcinoma
of the lung with a size of 1.1 cm was found. Immunohistochemical
and molecular examination of the lymphoid component failed to
demonstrate evidence for a B-cell neoplasm or clonality, respectively. All
lymph nodes were free of tumor.
Conclusions. To our knowledge, the association of nodular amyloidosis
and pulmonary epithelial malignancies of the lung is a very rare condition.
It is not clear whether this represents a pure chance co-occurrence,
or whether there is a causal relationship between the two lesions as the
result of a chronic inflammatory process.
FR-P-142
CD34+ fibrocytes in neoplasia of the lung and pleura
F . Wötzel1 , P .J . Barth1 1University Hospital Münster, Institute of Pathology, Münster
Aims. CD34+ fibrocytes are a cell population that is found abundantly in
the human connective tissue. Not only do they play a decisive role for the
matrix synthesis but also may appear as antigen-presenting cells. They
are particularly important concerning chronic inflammatory diseases of
the lung as well as systemic and localized fibrosis. Invasive carcinomas
induce a change in the phenotype from CD34+ fibrocytes to SMA+ myofibroblasts.
The significance of CD34+ fibrocytes in chronic inflammatory
diseases of the lung has been analysed thoroughly. However, there
is still a lack of data about CD34+ fibrocytes located in the stroma of
primary bronchial carcinomas, metastasis of the lung and pleural mesotheliomas.
Methods. 30 primary bronchial carcinomas (10 adenocarcinomas,
10 squamous cell carcinomas, 10 small-cell carcinomas), 10 metastasis
and 10 pleural mesotheliomas were analysed immunohistochemically
aiming at the expression of CD34, SMA (smooth muscle actin) and SMM
(smooth muscle myosin). Each case was compared to pulmonary tissue
free of tumor.
Results. Pulmonary tissue free of tumor displays CD34+ fibrocytes with
slender bipolar cytoplasmic projections especially in the bronchial mu-

cosa and in the periarterial tissue. These cells do not display an expression
of SMA or SMM. All analysed malignant tumors demonstrate a loss
of stromal CD34 expression and a phenotype change from CD34+SMA-
SMM- fibrocytes to CD34-SMA+SMM+ myofibroblasts.
Conclusions. Primary and secondary malignant tumors located in the
lung and pleura display a constant stromal phenotype change from
CD34+SMA-SMM- fibrocytes to CD34-SMA+SMM+ myofibroblasts.
This phenomenon is stereotypical for all organs (i.e. pancreas, mamma,
cervix) analysed this far. Furthermore the comparison with in situ carcinomas
indicates the major role of the loss of stromal CD34 expression
in local tumor invasion.
FR-P-143
Cytology-based diagnosis of malignant mesothelioma on behalf
of the German Social Accident Insurance Institution
S . Biesterfeld1 1Heinrich Heine University, Department of Cytopathology, Düsseldorf
Aims. Histology usually represents the gold standard for the diagnosis
of malignant mesothelioma. However, sometimes cytological material,
mainly as pleural fluid, is available solely. Here, we discuss those four
cases which were investigated in our institution in 2010 during the estimation
procedure on the reduction in earning capacity according to the
occupational diseases ordinance (No. 4105: “asbestos-induced malignant
mesothelioma”), ordered by the German Social Accident Insurance Institution.
Methods. In addition to routine cytology, we applied immunocytochemistry
(calretinin, berEP4, HEA125, TTF-1), DNA image cytometry, Ag-
NOR-analysis and FisH (at the region 9p21) and interpreted the results
considering the clinical and occupational history.
Results. In one of the four cases, a malignant effusion due to metastatic
lung cancer was diagnosed. In two cases, the diagnosis of an epitheloid
malignant mesothelioma was made. The fourth case in our opinion revealed
reactive changes only, and manifest tumor cells of a malignant
mesothelioma could not be detected. In all four cases, our interpretation
has been agreed by the Statutory Accident Insurance Funds. The first
three cases were accepted as asbestos-associated occupational diseases,
while the forth case was rejected initially. However, this patient meanwhile
has died, and the autopsy findings, taken in another institution,
led to the acceptance of asbestos-associated malignant mesothelioma.
Conclusions. Cytology-based tumor diagnosis is a useful and reliable
approach in those cases of asbestos-associated tumors in which no histology
can be obtained. The acceptance of cytological diagnoses by the
German Social Accident Insurance Institution enables to come to a conclusive
during the remaining lifetime of those patients by shortening the
procedure to estimate the reduction in earning capacity.
FR-P-144
Transcriptome analyses and validation of the targets reveal
impact of the TGF-β pseudoreceptor BAMBI for COPD
S . Marwitz1 , D . Drömann2 , J . Rupp3 , K . Rohmann3 , S . Osbahr3 , A .-J . Ulmer1 ,
K . Röschmann1 , M . Abdullah1 , H . Schultz1 , E . Vollmer1 , P . Zabel2 , K . Dalhoff2 , T .
Goldmann1 1Research Center Borstel, Leibniz Center for Medicine and Biosciences,
Borstel, 2University Clinic Schleswig-Holstein Campus Lübeck, Medical Clinic
III, Lübeck, 3University Clinic Schleswig-Holstein Campus Lübeck, Institute of
Medical Microbiology and Hygiene, Lübeck
Aims. Transforming Growth Factor beta (TGF-β) signaling events control
a variety of different cellular reactions and are involved on both,
physiologic and pathologic processes. Epithelial as well as cells of the
immune system respond to TGF-β signals throughout the body. The influence
of TGF-β signals in non-malignant lung diseases are up to date
critically discussed and infection-triggered tissue inflammation as well
as remodeling seems to inhabit a central role in the exacerbation and
pathogenesis of chronic obstructive pulmonary diseases (COPD). Infection-triggered
tissue inflammation and remodeling requires tight regulatory
events in the interplay of epithelial and immune cells and TGF-β
is one of the major cytokines involved.
Methods. Ex vivo infected human lung tissues with Non-Typeable Haemophilus
influenzae (NTHI) were subjected to transcriptome analysis.
Infection of lung tissue was verified by RNA in situ hybridization targeting
NTHI mRNA. Pathway analysis of different TGF-β members was
conducted on RNA and protein level and compared to COPD tissues.
Expression and secretion of pro-inflammatory molecules (IL-8, TNF-α)
were analyzed by means of western blotting and ELISA.
Results. 38% of COPD patient samples showed positivity for NTHI on
RNA level in contrast to 0% of controls. Transcriptome based pathway
analysis showed no significant changes of TGF-β receptors as well as cytokines
in contrast to a strong up regulation of Bambi in ex vivo infected
lung tissues as well as in COPD tissues. Bambi was found to be expressed
on alveolar macrophages as well as alveolar epithelial cells.
Conclusions. Here we present the TGF-β pseudoreceptor BMP and Activin
Membrane-Bound Inhibitor (Bambi) as a new modulator of TGF-β
signaling which might play a potent role in controlling the tissue homeostasis
and involvement in infection triggered pathogenesis.
FR-P-145
The contribution of p120-catenin modulated NF-kappa B activation
in airway inflammatory responses
X . Wang1 1Department of Pathology, Tongji Medical College Huazhong University of
Science and Technology, Wuhan, China
Aims. The purpose of this study is to investigate the role of p120-catenin
(p120) modulated nuclear factor-kappa B (NF-kappa B) activation
in airway inflammatory responses, and further to explore the molecular
mechanisms.
Methods. In this study, human bronchial epithelial cells (BECs) were
treated with LPS to establish an airway inflammation model in vitro.
Using confocal immunofluorescence imaging, Western blot, isolation of
cytoplasmic and nuclear proteins, we examined the localizations and expressions
of p120, NF-kappa B, IkappaB alpha and RhoA. Immumoprecipitation
was used to confirm the direct interaction of p120 and RhoA.
The RhoA activity was examined by G-LISA method. Then we detected
the expressions of interleukin-8 (IL-8) by fluorescence quantitative PCR
and enzyme-linked immunosorbent assay. Luciferase reporter analysis
was used to detect the activity of NF-kappa B. Finally, transient transfection
and small interfering RNA (siRNA) were used for p120 overexpression
or knock-down, and then the effects of p120 on NF-kappa B
signaling pathway were detected.
Results. In the present study, we first confirmed that p120 expression was
significantly reduced after LPS stimulation in BECs, the nuclear translocation
of NF-kappa B p65 subunit was promoted, IkappaB alpha was
phosphorylated and degraded, and NF-kappa B activity was rapidly induced.
After LPS stimulation, although the total RhoA and p120-binded
RhoA were unchanged, the RhoA activity is increased. Moreover, the
expression level of IL-8 increased after LPS treatment. Overexpression
of p120 attenuated LPS-stimulated NF-kappa B reporter gene expression
and IL-8 mRNA expression and protein synthesis. On the contrary,
transfection with p120 siRNA significantly elevated LPS-stimulated
NF-kappa B transcriptional activity, p65 nuclear translocation and IL-8
expression.
Conclusions. Collectively, these results indicate an anti-inflammatory effect
of p120 in BECs, through its modulation of NF-kappa B signaling in
a RhoA dependent manner.
Der Pathologe · Supplement 1 · 2012 |
129

Abstracts
FR-P-146
Pneumocystis jirovecii: value of a standardized diagnostic procedure
with molecular pathology combined with cytochemistry
T . Zeiske 1 , A . Schad 1 , T . Wehler 2 , E . Springer 1 , A .J . Ullmann 2 , C .J . Kirkpatrick 1 ,
T . Hansen 1
1 University of Mainz, Institute of Pathology, Mainz, 2 University of Mainz,
Third Department of Internal Medicine, Mainz
Aims. Pneumocystis jirovecii (PJ) is a frequent cause of pulmonary infection
in patients with the acquired immunodeficiency syndrome and
other immunosuppressive conditions. Often PJ pneumonia represents
a life-threatening disorder requiring a sensitive and fast diagnosis. We
report on our experience on a combined diagnostic procedure for the
detection of PJ.
Methods. A total number of 602 cytological specimens were studied between
2008 and 2010 at the University Medical Center Mainz. As a standard
approach, all specimens have been routinely analyzed by Grocott’s
silver stain and nested-polymerase chain reaction (PCR) simultaneously.
The frequency of positive PJ results revealed by both methods was then
compared with respect to two different sampling procedures [bronchoalveolar
lavage (BAL) and sputum specimen]. For a subgroup of 18 patients,
we analyzed the clinical course over a one-year period.
Results. Our cohort included 441 BAL (73.3%) and 161 sputum specimens
(23.7%). In BAL, we found 22.45% of cases positive for PJ (n=99), with
24.2% of these diagnosed by both cytochemistry and PCR, whilst 75.8%
were detected by PCR alone. In sputum specimens, 33 PJ-positive cases
could be found (20.5%), most of them (28/33) being detected by PCR
alone. In about 24% of all specimens evaluated, cytochemistry revealed
various types of fungi such as candida and aspergillus. In the subpopulation
examined for the clinical course, we found 14/18 patients requiring
ventilation by respirator. In that PJ group, the large majority (i.e. 11/14)
was detected solely by PCR.
Conclusions. For the diagnosis of clinically relevant cases with PJ, the
combination of PCR with cytology/cytochemistry is mandatory. It remains
to be investigated, whether additional detection of fungi (by cytochemistry)
influences the clinical outcome of PJ patients.
Poster: Hämatopathologie
FR-P-147
Bone marrow biopsies of patients with haematopoietic and lymphoid
disorders – epidemiology, chromosomal aberrations and
molecular pathology
S . Hehne1 , S .M . Schulze1 , P . Richter1 , C . Geier1 , Y . Chen1 , A . von Deimling 2 ,
I . Petersen1 1 2 Jena University Hospital, Heidelberg University Hospital, Institute of
Neuropathology
Aims. Bone marrow biopsy of the iliac crest is the first and most important
step in the diagnostics of haematopoietic disorders.
Methods. The biopsies of the years 2006 and 2007 from the institute of
pathology of the Jena university hospital were retrospectively analyzed
for clinicopathological parameters. In addition, the Mitelman database
was retrieved for chromosomal aberrations.
Results. The analysis of 2820 reports from 1185 patients revealed that lymphomas,
plasmocytoma and acute leukaemia were most frequent. Males
predominated in myeloproliferative neoplasms and lymphoma subtypes,
particularly CLL, except for plasmocytoma and acute leukaemia. A
peak incidence was seen between 61 and 70 years of age with a varying
pattern for single entities. The database search revealed that ALL, AML,
CLL and cmL were mainly diploid while Hodgkin lymphoma, mature
B-cell lymphoma and multiple myeloma mostly carried hyperdiploid
chromosome numbers. Numerical aberrations like chromosome 8 gains
130 | Der Pathologe · Supplement 1 · 2012
in hyperdiploid cmL were prominent in specific subgroups. Molecular
testing is exemplified in cmL, plasma cell myeloma and hairy cell leukaemia.
Conclusions. The study highlights typical clinicopathological characteristics
and new genetic findings in haematopoietic and lymphoid neoplasms
with relevance for the new WHO classification and beyond. We
hope that it may help in the differential diagnosis of bone marrow biopsies.
FR-P-148
Clonally related nodular lymphocyte-predominant Hodgkin lymphoma
and classical Hodgkin lymphoma occurring as a collision
lymphoma
M . Szczepanowski1 , N . Masqué-Soler1 , I . Oschlies1 , W . Schmidt 2 , A . Lück3 ,
W . Klapper1 1University Hospital Campus Kiel/Institute of Pathology, Section Hematopathology,
House 14 , Kiel, 2Pathology Practice, Rostock, 3Practice for Internal
Medicine, Hematology and Oncology, Rostock
Aims. Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL)
with the typical lymphocyte-predominant (LP) cells, and classical Hodgkin
lymphoma (cHL), characterized by Hodgkin and Reed-Sternberg
(HRS) cells, are considered two distinct diseases whose co-occurrence in
one patient is extremely rare. We report on clonal relatedness in a case of
concurrent NLPHL and cHL, residing in one lymph node in a 48-yearold
male patient.
Methods. We diagnosed synchronous NLPHL (CD20+, CD30−, LMP1−)
and cHL (CD30+, CD20−, LMP1−) in formalin-fixed paraffin-embedded
(FFPE) tissue sections of one lymph node in a 48-year-old male patient.
CD20 or CD30 labeled neoplastic cells were separately collected by laserassisted
microdissection (LCM). Immunoglobulin heavy chain (IGH)
gene rearrangements were pre-amplified by standard multiplex polymerase
chain reaction (PCR) and subjected to fragment analyses and
Sanger sequencing.
Results. In the frame work (FR) 3 multiplex PCR, fragments of 120/121
and 128 base pairs (bp) in LP cells and 120/121 bp in HRS cells were obtained.
The 120/121 bp fragment shared by both LP and HRS cells was
re-amplified by VH1 and JHrev primers as a clear 121 bp fragment.
Sequencing analyses of the shared 121 bp product revealed an identical
rearrangement consisting of IGHV1-2*3/IGHD4-17*01/IGHJ4*02
with identical VH-DH junction (5’-GCCAT-3’) and DH-JH junction
(5’-CCTCTCTTTTGACTG-3’) sequences and a mutation rate of 5.6%
in both entities. Consequently, PCR with allele-specific oligonucleotides
(ASO) yielded identically sized fragments in both LP and HRS cells.
Conclusions. We conclude that both approaches, sequencing of VH1/
JHrev PCR products and amplification of identically sized ASO products,
are highly indicative for a clonal relationship of the concurrent
NLPHL and cHL. The findings point to a common precursor B-cell of
germinal center origin for both, the cHL and the NLPHL, in analogy
to well documented clonal relations in cases of concurrent or sequential
Hodgkin lymphoma (HL) and non-HL.

FR-P-149
Combination of Castleman disease and Langerhans cell histiocytosis
in a supraclavicular lymph node
H . Geddert 1 , A . Dimmler 1 , U . Götschin 2 , M . Schatz 3 , G . Faller 1
1 St . Vincent Hospital, Institute of Pathology, Karlsruhe, 2 St . Vincent Hospital,
Department of General Surgery, Karlsruhe, 3 St . Vincent Hospital, Department
of Hematology and Oncology, Karlsruhe
Aims. A 51-year-old female patient presented to our hospital with a 5 cm
measuring mass in the right supraclavicular fossa. Because of known
nodular goiter an ectopic thyroid nodule was suspected. The patient was
in good clinical condition without B symptoms. To exclude malignant
lymphoma the whole mass was excised.
Methods. During intraoperative frozen section suspicion of Castleman
disease was raised, although a second cellular component remained unclear.
Results. After routine tissue processing diagnosis of Castleman’s disease
of hyaline-vascular subtype in a lymph node was confirmed. The second
component emerged as Langerhans cell histiocytosis. No signs of multicentric
Castleman disease or its associated conditions were found. Systemic
Langerhans cell histiocytosis was excluded clinically.
Conclusions. One year after diagnosis the patient is still in good condition
without any sign of relapse. To our knowledge, this is the first report of a
combination of Castleman disease and Langerhans cell histiocytosis in
a single lymph node.
FR-P-150
Notch2, possible a crucial gene in pathogenesis in lymphoma
X .-x . Zhang1 , Q . Lai1 , R . Zhou1 1Zhejiang University, School of Medicine, Hangzhou, China
Aims. In mammals, there are four kinds of Notch transmembrane receptors
(Notch1 to 4); and there are many kinds of Notch target genes
include Hes family, nuclear factor kappaB (NF-κB), cyclin D1, c-myc,
p21, p27, etc. In recent, researches show that Notch2 signaling may be an
important regulator of B cell lymphocytes activation and effect cell differentiation.
However, the functions of Notch2 in lymphoma remain poorly
understood. In our study, we try to investigate the role which Notch2
plays in the pathogenesis of lymphoma, especially on B cell lymphoma.
Methods. First, Notch2 was detected in 95 B cell lymphoma samples
through PCR, and DNA sequencing was used to see if there were mutations
or not. Then the pEYFP-n1, which contains the Notch2 gene or not,
was transfected into the lymphoma cell lines (include raji, ramos, daudi,
jurkat and molt4) and 293T cell by electro-transfection. And then total
RNA and protein were extracted from the transfected cells, and then
the expression levels of targeted genes, such as c-myc, cyclinD1, p21, p27,
bcl6, p50 and p65, were detected by real-time PCR or Western blot. Statistical
analysis was done by the Student’s t-test between the test groups
and the control group. A P-value of

Abstracts
nal lymph nodes and splenomegaly. Investigation for TCR gamma gene
rearrangement revealed the presence of a dominant amplificate. After
8 cycles of standard cytotoxic chemotherapy according to the CHOP regimen
a complete remission was achieved. Eleven years later, the patient
presented with cervical and axillary lymphadenopathy and splenomegaly.
Histological and immunohistological examination of an excised
lymph node revealed a T-cell lymphoma with the characteristic features
of AITL. Clonality analysis for TCR gamma chain genes revealed a clonal
population with a different amplificate size that those from the initial
manifestation.
Conclusions. To our knowledge, this is the longest recurrence free period
in AITL reported so far. Most interestingly the patient exhibited a novel
T-cell clone at relapse not identifiable even among the minor clones at
initial presentation.
FR-P-153
Severe central nervous system (CNS) graft versus host disease
(GVHD) in a patient without any other GvHD symptoms after
allogeneic stem cell transplantation
C . Schrader1 , R . Stingele2 , W . Brück3 , I . Metz3 , C . Riedel4 , N . Schub1 , A . Humpe1 ,
T . Valerius1 , G . Deuschel2 , M . Gramatzki1 , A . Günther1 1 2 2 University Hospital of Kiel, nd Department of Medicine, Kiel, UKSH, Campus
Kiel, Department of Neurology, 3University of Göttingen, Department of
Pathology, 4UKSH, Campus Kiel, Department of Neuroradiology
Aims. Although graft versus horst disease (GvHD) is the most relevant
complication of allogeneic stem cell transplantation (SCT), it rarely affects
the central nervous system. Recently, a consensus conference proposed
criteria of diagnosis for cerebral GvHD including the obligatory
manifestation of chronic GvHD at other organs [Grauer et al., Brain, 133:
2852, 2010]. We observed a 41-year-old women, who developed spastic
paralysis and fell into coma 2.5 years after having an allogeneic peripheral
blood stem cell stem cell transplantation (PBSCT) for acute myeloblastic
leukemia from an unrelated HLA 9/10-matched donor. The patient
presented with a history of several month of headache supposed to
be caused by migraine. She had a history of acute GvHD stage III (skin
and intestinal) but no signs of chronic GvHD. In addition she had no
history of an independent autoimmunopathy or migraine prior to SCT.
Methods. MRI scan was performed, cerebrospinal fluid was analyzed to
exclude CNS relapse and infectious agents, and finally CNS biopsy was
obtained by open brain surgery.
Results. MRI scan showed disseminated severe leucencephalopathy without
established sign of CNS relapse, lymphoma or typical infection. The
cerebrospinal fluid analysis was normal. Toxoplasmosis and viral infection
such as HSV, VZV, ADV, EBV, cmV, HHV-6 and HIV was excluded
by PCR. The blood lymphocyte subset showed lymphopenia, however
with normal CD4/CD8 ratio. Finally CNS biopsy revealed T-cells close
to blood vessels – a pattern typical for cerebral GvHD. Immunosuppressive
treatment was started with high dose steroids in combination with
mycophenolatmofetil (MMF). She recovered rapidly and became completely
awake within one week. After 9 months of immunosuppression
the patient is competent in activities of daily living.
Conclusions. GVHD of the central nervous system (CNS) is a rare disease
after allogeneic stem cell transplantation. The absence of lymphocytes in
the cerebrospinal fluid and normal CD4/CD8 ratio in peripheral blood
does not exclude GvHD of the CNS. CNS biopsy should be considered
to confirm the diagnosis. This case demonstrates that CNS involvement
can be the only manifestation of chronic GvHD. Immunosuppressive
therapy may provide an impressive benefit in these patients.
132 | Der Pathologe · Supplement 1 · 2012
FR-P-154
Tuberculous lymphadenitis and mycobacterium-negative granulomatous
disease in patients receiving Imatinib mesylate (Glivec)
treatment for gastrointestinal stromal tumors (GIST)
A . Agaimy1 , A . Stegmann2 , A . Mackensen3 , N . Meidenbauer3 1Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen,
2Friedrich-Alexander University of Erlangen, Department of otorhinolaryngology,
head and neck surgery, Erlangen, 3Friedrich-Alexander University of
Erlangen, Medical Clinic 5 , Erlangen
Aims. Imatinib mesylate (IM) represents the standard treatment for patients
with BCR-ABL-positive chronic myelogenous leukemia (CML) and
is the first line adjuvant and palliative treatment modality for those with
disseminated or inoperable gastrointestinal stromal tumor (GIST). IM is
not known to be associated with an elevated risk for tuberculosis, since
only five patients have been reported to date who developed tuberculosis
during or after IM treatment for cmL (n=4) and GIST (n=1).
Methods. We describe our experience with 3 patients (45–79 yrs of age)
who developed necrotizing (caseating) granulomatous disease during
IM treatment for GIST. Mean duration of treatment with Imatinib was
9.5 (range: 9–48) months.
Results. In 3 patients, enlarged lymph nodes with increased metabolism
in Fludoxyglucose-Positron emission tomography (FDG-PET)-computer
tomography-examinations were detected and resected. Affected sites
were subcarinal/mediastinal (1), mediastinal/supraclavicular (1) and periparotideal
cervical (1) lymph nodes. Histologic examination revealed
necrotizing granulomatous disease suggestive of infection with M. tuberculosis
or sarcoidosis. Sputum examination was negative for acid fast
bacilli in all patients and DNA was negative for M. tuberculosis and other
mycobacteria in two cases. However, M. tuberculosis was detected by
PCR in the lymph node of one patient who was then successfully treated
by antituberculous agents. All other patients received no anti-tuberculous
therapy and were on complete response or stable neoplastic disease
without evidence of progressive lymphadenopathy or lung lesions suggestive
of active tuberculosis. Leucocyte and lymphocyte counts have
been within normal limits throughout treatment with IM.
Conclusions. Our observations underline the necessity to sample enlarged
or metabolic active lymph nodes developing during IM treatment
for timely diagnosis and appropriate treatment of these rare complications.
Follow up without treatment is safe for lesions without detection of
M. tuberculosis by PCR. More studies are needed to clarify the potential
causal relationship to IM treatment and to sufficiently explore the pathogenesis
of lesions that were negative for M. tuberculosis.
FR-P-155
Therapeutic Polo-like kinase 1 inhibition results in mitotic arrest
and subsequent cell death of leukemic cells in acute myeloid
leukemia
C . Münch1 , A .M . May1 , A .V . Pfister1 , K . Thurig1 , M . Lübbert2 , R . Wäsch2 ,
T . Taube3 , S . Lassmann1 , M . Werner1 1 2 University Freiburg Medical Center, Institute of Pathology, Freiburg, University
Freiburg Medical Center, Department of Hematology and Oncology,
Freiburg, 3Boehringer Ingelheim Pharma GmbH & Co . KG, Clinical Research
Aims. The mitotic kinase Polo-like kinase 1 (Plk1) is an important cell
cycle regulator which is frequently overexpressed in acute myeloid leukaemia
(AML). Our previous examination of AML blasts in pre- and posttreatment
bone marrow biopsies of AML patients treated with the small
molecule PLK1 inhibitor BI2536 revealed an increase of aberrant mitotic
figures and apoptotic cells 24 hours after administration. The aim of
this study was to extend this examination by investigating the effects of
therapeutic PLK1 inhibition on AML cell lines and lymphoblastoid cell
lines (LCLs) from healthy controls.
Methods. Five AML cell lines (HL-60, KG1, OCIM2, NB4 and THP1)
and two LCLs (LCL1 and LCL2) were cultured for 24 and 48 hours with

or without different concentrations of BI2536 (10 nM, 50 nM, 100 nM,
200 nM and 500 nM). Cell cycle analysis was performed after 24 and
48 hours of culture using FACS (PI-staining). Western blot analysis
was conducted after 24 (H3Ser10ph) and 48 hours (cleaved caspase-3) of
BI2536 treatment. Immunofluorescence staining of cells was done using
antibodies detecting Plk1 (24 h, 100 nM BI2536), cleaved caspase-3 (48 h,
200 nM BI2536) and alpha-tubulin (24 and 48 h).
Results. Cell cycle analyses of AML cells after 24 h of treatment revealed
an accumulation of mitotic cells (4N) and a decrease of cells in G0/
G1 phase (2N) of the cell cycle. Furthermore, elevated protein levels of
the mitosis specific phosphorylated histone 3 (Ser10) were detected by
western blot after 24 h. Immunofluorescence staining of PLK1 and alpha-tubulin
showed an increase of mitotic figures exhibiting monopolar
spindles without Plk1 localization at centrosomes or kinetochores
of prometaphase chromosomes. A strong increase of cleaved caspase-3,
which was used for the detection of apoptotic cells, was visible after 48 h
by western blot and immunofluorescence analyses in inhibitor treated
cells. Furthermore, the less proliferative LCLs arrested in mitosis to a
lower extent and showed fewer apoptotic cells after 48 h compared to the
analyzed AML cells.
Conclusions. Our data confirm the sensitivity of leukemic cells to Plk1
inhibition by BI2536. Treatment with variable doses of BI2536 results in
prometaphase arrest and a pronounced cell death after 48 h in AML cell
lines. In addition, our experiments with lymphoblastoid cell lines indicate
that less proliferative hematopoietic non-leukemic cells show a weaker
response to therapeutic Plk1 inhibition by BI2536.
FR-P-156
Analysis of BCL6 and MYC coexpression in diffuse large B-cell
lymphoma
L . Culemeyer1 , C . Stuhlmann-Laeisz1 , W . Klapper1 1UKSH Campus Kiel, Institute of Pathology, Kiel
Aims. The transcription factor BCL6 is the master regulator of the germinal
centre reaction. cmYC is a transcription factor, which is often overexpressed
in diffuse large B-cell lymphomas (DLBCL), but is not expressed
in the germinal centre under physiological conditions. Herein we aimed
to quantify the unphysiological co-expression of BCL6 and cmYC in
DLBCL. Furthermore, functional effects of this unphysiological coexpression
of BCL6 and cmYC will be estimated by evaluating BCL6 target
gene expression.
Methods. Fluorescence multi-stainings using the combination of BCL6
and cmYC were quantified by digital image analysis (Tissuequest©).
Results. Unphysiological co-expression of BCL6 and cmYC in lymphoma
cells was detected in DLBCL. This co-expression occurred in DLBCL
with and without BCL6 or cmYC translocations and influences BCL6
target gene expression.
Conclusions. The unphysiological co-expression of transcription factors
of the B-cell differentiation can be detected in DLBCL without genetic
alterations affecting these genes. We suggest that this co-expression interferes
with the activation or inhibition of the respective target genes.
We consider this mechanism to be a reason for the oncogenic potential
of transcription factors, which are also expressed under physiological
conditions.
FR-P-157
Conditional PHD2 deficiency leads to non-lethal erythrocytosis
and alters the hematopoietic stem cell fate
B . Wielockx1 1TU Dresden – Pathology, Dresden
Aims. Hypoxia is a prominent feature during development and physiological
as well as pathological conditions in adults. An oxygen-sensing
machinery is therefore very important to help the cells adapt instantaneously
to any inacceptable O2 level. Such a system relies on the oxygen
dependent HIF-prolyl hydroxylases (PHD1-3), enzymes that can inactivate
the alpha subunit of the hypoxia inducible transcription factor
(HIF). In case of low oxygen availability, PHDs lose their functionality
and allow the HIF complex to promote biochemical and physiological
changes including anaerobic glycolysis, angiogenesis and hematopoiesis.
Results. Our research unit produced a mouse line that lacks PHD2 in
a broad spectrum of cell types (e.g. hematopoietic cells, epithelial cells)
which resulted in an unexpected hematologic phenotype. The mice display
strongly elevated hematocrit levels together with high EPO concentrations
in the blood although mice don’t show any premature lethality.
Moreover, we found that the hematopoietic stem cell (HSC) compartment
in the bone marrow was significantly altered compared to WT
mice.
Conclusions. Indeed, detailed FACS analyses demonstrate that cKO mice
contain much more multipotent progenitors (MPPs). Moreover, under
stress conditions in vivo, cKO HSCs are pushed towards self-renewal.
Double deficient cKO mice with one of the two HIFα revealed that the
erythrocytosis phenotype is exclusively driven by HIF2α, whilst HIF1–α
is responsible for the HSC/MPP phenotype.
FR-P-158
Tumor infiltrating T-cells in high risk chronic lymphocytic leukemia
(B-CLL): a clinicopathological study of CD3, CD8 and FOXP3
expression
C . Schrader1 , C . Pflüger1 , S . Stilgenbauer2 , H . Döhner2 , M . Ritgen1 , J . Claasen1 ,
P . Dreger3 , W . Klapper4 1 2 2 University Hospital of Kiel, nd Department of Medicine, Kiel, University of
Ulm, Department of Hematology, 3University of Heidelberg, Department of
Hematology, 4UKSH, Campus Kiel, Department of Pathology
Aims. The prognostic value of the mutation status of the immunoglobulin
heavy chain variable region (IgVH) and cytogenetic abnormalities in
chronic lymphatic leukemia is well known. We investigated the tumour
associated reactive T-cell infiltrates in bone marrow and lymph nodes
biopsies of patient with high risk B-CLL in correlation to other clinical,
biological and genetic markers including age, sex, binet stage, bone marrow
involvement (infiltration pattern and grade), β2 microglobulin level,
leucocytes account, lymphocytes double time, thymidinkinase level, cytogenetic
aberrations (e.g. 11q deletion), IgVH mutations status and ZAP
70 expression
Methods. Bone marrow (n=51) and lymph node biopsies (n=8) from 59
untreated patients (43 men and 16 women) were investigated immunohistochemically
with monoclonal antibodies against CD20, CD5, CD23,
CD3, CD8, FOXP3 and ZAP70. Cells with clear positive staining were
counted and the percentage was calculated. Molecular analysis of IgVH
mutation and FISH analysis was done from fresh peripheral blood tumor
cells.
Results. In 58 biopsies the CD 3 staining, 57 cases of the CD 8 and in
all 59 cases the FOXP staining was evaluable. The CD3 staining had a
range of 0.4% to 35.2% with a median of 9.3% and a mean of 11.2%. In
lymph node tissue a significant higher number of CD3 cells than in bone
marrow biopsies (p=0.0054) was found. Similar results were found in the
CD8 (p=0.0052) and FOXP3 (p=0.0087) stainings with higher T-cells account.
In the analysis of the bone marrow biopsies the nodular pattern/
involvement showed a significant higher number of CD3 cells (p=0.013)
Der Pathologe · Supplement 1 · 2012 |
133

Abstracts
than the diffuse pattern. Similar results could be found concerning
the FOXP3 cells and the infiltration pattern. The nodular pattern had
significant more reactive FOXP3 positive cells than the diffuse pattern
(p=0.018). High number of CD8 cells were found in patients with lower
leucocytes counts (p=0.049) and higher lymphocytes double time (more
than 12 months, p=0.048). All other correlation between clinical, biological
and genetic correlation with T-cells accounts shows no significant
differences.
Conclusions. Lymph node biopsies with B-CLL had a significant higher
account of T-cells than bone marrow biopsies. Patients with B-CLL and
a nodular bone marrow involvement had a significant higher number of
CD3+ and FOXP+ T-Cells. High numbers of CD8+ T-cells cells might
have a positive influence on the lymphocytes double time and leukocytes
level at the time of diagnosis.
FR-P-159
Collision lymphomas in the bone marrow – a diagnostic pitfall
A .M . May1 , L . Morawietz2 , W . Dietrich3 , A . Lindemann4 , G . Faller 5 , P . Fisch1 ,
M . Werner1 1University Freiburg Medical Center, Institute of Pathology, Freiburg,
2 3 Klinikum Stuttgart, Institute of Pathology, Stuttgart, Klinikum Stuttgart,
Stuttgart, 4Oncologic Practice, Ettlingen, 5St . Vincentius-Kliniken, Institute of
Pathology, Karlsruhe
Aims. The simultaneous co-existence of two distinct lymphoma entities
in one bone marrow trephine biopsy (BMB) is rare and can be easily missed,
especially in cases with a high density of infiltration. We present the
cases of two patients with a compact infiltrate in the BMB, which turned
out be composed of two different kinds of lymphoma.
Methods. The distinct lymphoma entities in these two patients’ BMB
were extensively analyzed, using immunohistochemistry and a PCR-based
clonality analysis of immunoglobulin heavy chain gene rearrangements.
Results. One patient’s BMB was infiltrated by hairy cell leukemia (infiltration
density: 75%). In a lymph node biopsy that had been examined separately,
due to a conspicuous abdominal lymphadenopathy, mantle cell
lymphoma was diagnosed. Further studies provided a minimal presence
of mantle cell lymphoma cells in the BMB as well as a discrete population
of hairy cells in the lymph node biopsy. Clonality analysis of the immunoglobulin
heavy chain gene identified two distinct clonal B-cell populations.
In the other patient’s BMB an unusual gigantocellular variant of
B-lymphoblastic lymphoma was diagnosed. The immunohistochemical
analysis indicated a diagnosis of cALL. Flow cytometric analyses found
two separate, but only discrete B-cell populations in the peripheral blood
and bone marrow. One of these populations was immature; the other
population was suspicious of mantle cell lymphoma. Subsequent immunohistochemistry
showed a minute additional presence of mantle cell
lymphoma in the BMB.
Conclusions. In case of clinically unusual presentation, additional examinations,
such as more extensive immunohistochemistry, molecular
methods and flow cytometric analyses, need to be included into the
diagnostic spectrum of lymphomas, to be able to identify rare cases of
collision lymphomas.
134 | Der Pathologe · Supplement 1 · 2012
FR-P-160
Primary gastric ALK-negative anaplastic large cell lymphoma:
a clinicopathologic analysis of four cases
C . Liu1 , Y . Lai1 , X . Wang1 , X . Huang1 , M . Li1 , Z . Gao1 1Peking University Health Science Center, Beijing, China
Aims. To evaluate clinicopathological and immunophenotypic features
of primary gastric ALK-negative anaplastic large cell lymphomas
(ALCLs).
Methods. Formalin-fixed, paraffin embedded tissue blocks of 4 patients
diagnosed with primary gastric ALK-negative ALCL were obtained. Hematoxylin
and Eosin-stained slides were used to evaluate histological
changes and the immunophenotypic features were detected by immunohistochemistry.
In addition, the abnormality of ALK gene was determined
by interphase fluorescence in situ hybridization (FISH).
Results. The cases were comprised of three men and one woman, with
a median age of 58.5 years. All the four cases presented with epigastric
discomfort with or without gastrorrhagia. Endoscopic examination
revealed solitary gastric ulcer in all cases. Endoscopic biopsy from one
patient and surgical specimens from three patients were available. Morphologically,
the normal architecture of gastric wall was effaced by the
diffuse infiltration of tumor cells, in which the characteristic hallmark
cells were easily identified in all cases. The tumor cells of all cases showed
a consistently strong expression of CD30 but lack of the expression
of ALK1. Moreover, the tumor cells demonstrated varying expression of
T cell markers such as CD3 (3/4) and CD43 (1/1), and negative for B-cell
markers CD20 and PAX5. Chromosomal rearrangement involving ALK
gene was not detected by FISH. Three of the four cases underwent total
or partial gastrectomy followed by chemotherapy and till the last followup;
none of them developed a relapse or progression. The rest one patient
refused surgery and chemotherapy, and died 20 months after diagnosis.
Conclusions. Here we reported four ALK-negative ALCLs occurred in
the stomach. Although it’s a rare condition, we should keep in mind
to avoid misdiagnosis especially of the gastric carcinoma. The current
results suggested that ALK-negative ALCL may have a good prognosis
with surgery combined with chemotherapy.
Poster: Autopsie/Fallstudien/Sonstiges
FR-P-161
Postmortem CT in clinical autopsy
S . Westphal1 , J . Apitzsch2 , T . Penzkofer2 , A . Perez-Bouza 1 , B . Sellhaus3 ,
A . Mahnken2 , R . Knüchel-Clarke1 1 2 RWTH Aachen University, Institute of Pathology, Aachen, RWTH Aachen
University, Department of Interventional and Diagnostic Radiology, Aachen,
3RWTH Aachen University, Institute of Neuropathology, Aachen
Aims. While autopsy rates in clinical pathology are declining for the past
decades, and forensic sciences are using virtual autopsy techniques increasingly
in daily routine, pathologists are not yet tapping the potential
of modern imaging techniques. To assess the value of virtual autopsy
techniques in clinical pathology, post- mortem imaging was performed
before autopsy.
Methods. In 29 autopsy cases, a full-body high-definition pmCT (postmortem
computed tomography) scan was performed prior to autopsy.
Images were analyzed by experienced radiologists. Autopsy was performed
following a standard protocol, taking special care of macroscopical
findings, detected in the CT scan previously. Digital macroscopical
pictures of the organs and of their histology were taken. We compared
pmCT and classical autopsy findings regarding cause of death and death-related
diagnoses, reconstruction of the key pathomechanism leading
to death and side diagnoses.

Results. In 18 cases (79%) the cause of death (e.g. hemorrhage), was diagnosed
correctly by pmCT. In 11 cases (38%) the key pathomechanism
of death (e.g. anastomotic leakage from aortic surgical site) was found
by pmCT. Side diagnoses found by pmCT improved and complemented
those found by traditional autopsy. Especially in the documentation and
visualization of bone lesions, CT-imaging was superior to macroscopic
examination because of the simplicity and elegance of the method compared
to classical bone pathology.
Conclusions. pmCT is an elegant and suitable method to complement the
macroscopic and microscopic examination of classical autopsy. Our first
results are a good basis to continue by application of contrast enhancement
or collection of biopsies during pmCT.
FR-P-162
Speed – all that matters?
F . Fronhoffs1 , B . Roick1 , G . Kristiansen1 1University Bonn Medical Center, Institute of Pathology, Bonn
Aims. After change of the head of Pathology Department of the University
Hospital Bonn in spring 2011 we conducted a survey among all our
senders in order to evaluate their expectations concerning diagnostics
and service.
Methods. We sent a questionnaire including 27 topics by mail to all our
senders. They could answer using a scale from “0”(“not important at all”)
to “10” (“very important”).
Results. We sent 2172 questionnaires and received 91 answers (gynaecology
n=17, internal medicine n=16, pathology n=9, surgery n=6, general
practitioners n=6, radiology n=5, haematology n=5, others n=27). Most
important for our senders were “speed of communication of the diagnosis”
(906 of 910 possible points), “personal availability of the responsible
pathologist by phone” (889/910), and “friendliness of contact” (829/910).
Less important were “performing autopsies” (422/910), “24-hours on-call
duty”(322/910) and “service on Saturday” (321/910).
Conclusions. The survey results illustrate the increasing importance of
pathology as an interdisciplinary and cross-linking clinical specialization.
In addition to speed and efficiency, a professional and amiable colleague
is demanded as pathologists play a key role in tumour boards and
personalized therapies. The decreasing importance of autopsies, last but
not least a tool of quality control, is regrettable. However, this general
development is reflected in decreasing numbers of performed autopsies
per annum in our department.
FR-P-163
Retrospective autopsy study of the University of Mainz,
1970–2010
T . Hansen1 , M . Dusolt1 , S . Höring1 , C . Kempe1 , F . Rosendahl1 , M . Kavciakova1 ,
M . Hechtner2 , A . Spriestersbach2 , C .J . Kirkpatrick1 1 2 University of Mainz, Institute of Pathology, Mainz, University of Mainz,
Institute of Medical Biostatistics, Epidemiology and Informatics, Mainz
Aims. In the past, numerous analyses studied several aspects of autopsy,
in particular with regard to the decline of frequency. Especially in the
last years long-term studies comprising more than one decade are sparsely
published.
Methods. We analysed the archival data of the Institute of Pathology of
the University of Mainz for autopsies performed between 1970 and 2010.
We focused on patients at least 14 years old (n=14724) who died in the
University hospital. We compared the number of autopsies with the total
number of deceased patients and studied several epidemiological aspects
with special relevance for the cause of death (COD).
Results. In 1970, the autopsy frequency was 62% and fell to 49.1% in 1980.
In the following decade, there was a steady state (frequency 53.3% in 1985,
and 43.2% in 1990), followed by a remarkable decline between 1995 (30.6%)
and 2000 (9.7%), and finally 2010 (5.6%). The overall mean age increased
during the observation period (59.6 yrs in 1970, 67.5 yrs in 2008). Among
the COD groups, cardiovascular diseases were predominantly recorded
(between 35% in the 1970s and 39% in 1995–2010), followed by infectious
diseases (between 20 and 25%). Malignancies represented the third most
common COD group, with an increase of frequency from about 10.5% in
the 1970s to 17% observed in the last decade. Among the single specific
CODs, pulmonary embolism was most often encountered in the 1970s
(about 11.5%), while in the following decades, myocardial infarction predominated
(up to 15.8% between 1995 and 2010). In the overall period,
lung cancer was the single most common malignancy of the CODs (between
2.5 and 3.9%). In addition, a total number of 57 patients were described
as suffering from infection by Human Immunodeficiency Virus
(HIV). Of this cohort, 31.6% died due to the HIV infection. Concerning
tuberculosis, the frequency fell from 7.9% in 1970 to about 3% in the late
1970s and increased again to 6–7% in the subsequent decades, followed
by a decrease in 1995–2010 to about 2%.
Conclusions. In this study, we were able to analyse autopsy data over a
long-term period. Most of our results are in line with previous reports. In
particular, these data confirm studies showing that in Germany the autopsy
frequency began to remarkably decline in the 1990s (by contrast to
several Anglo-American reports on decrease in the 1980s). With the exception
of some specific findings, we could not observe a general switch
in the COD groups in spite of the dramatically changed autopsy number.
FR-P-164
Introduction of a structured report to quantify additional diagnostic
information by autopsies
G . Ates1 , J . Friemann2 1Department of Internal Medicine II, Klinikum Lüdenscheid, Lüdenscheid,
2Institute of Pathology, Klinikum Lüdenscheid, Lüdenscheid
Aims. Autopsy proves quality of diagnostic procedures as well as success
and adequacy of therapeutic decisions near the end of life. Our investigations
were aimed finding a way to quantitatively describe the gain of
diagnostic information by autopsy and whether this can be made available
as an indicator of quality for inter-institutional analysis.
Methods. From 2004 to 2010, pathological-anatomical diagnoses of 322
autopsies in the Institute of Pathology, Märkische Kliniken GmbH, were
grouped into 4 different categories on a structured report – regarding
to their role in the cause of death and compared to life-time diagnoses
(1. clinically known major disease, 2. revealed unknown major disease,
3. important findings, 4. secondary findings). Further, in every autopsy
the extent of congruency of clinically suspected and pathological-anatomical
assured cause of death was classified (totally congruent, partially
congruent, not congruent).
Results. In the evaluated period 322 autopsies (~5% of the deceased in
hospital) were performed. Predominantly (95%) clinically known major
diseases leading to death were confirmed by autopsy. Additionally in 71%
of the cases autopsy revealed unknown major diseases missed during the
clinical course but significantly contributing to the lethal course. Only in
42% there was total congruency of clinical suspected and pathologicalanatomical
assured immediate cause of death.
Conclusions. This proposal of grouping pathological-anatomical diagnoses
into different categories on a structured report may help to quantify
the gain of additional diagnostic information by autopsy and to introduce
these categories into the quality reports of German hospitals as an
instrument for inter-institutional quality control.
Der Pathologe · Supplement 1 · 2012 |
135

Abstracts
FR-P-165
Potential complications of aortic valve implantation via a transfemoral
artery catheter: an autopsy perspective
H . Löser 1 , H .P . Dienes 1 , J . Fries 1
1 University of Cologne, Institute of Pathology, Köln
Aims. Degenerative or post-endocarditic destruction of aortic valves
with secondary left ventricular hypertension and subsequent cardiac insufficiency
is seen more frequently in patients with increasing age. The
instable health of many of these patients does not permit a removal of
the diseased valve in an open surgical procedure. Instead, an aortic valve
implantation is achieved via either a transapical access or a transfemoral
artery catheter. In Cologne, the catheter carrying a ballon-expandable
stent with a valve of bovine pericardium (Edwards-SAPIEN System) has
been used for the last 3 years. While in the majority of cases this procedure
was completed successfully, we encountered several autopsy cases,
in which unforeseen complications occurred directly related to this type
of valve replacement.
Methods. All patients were in the age range of 65 to 80 years. Preclinical
evaluation had indicated that a conventional surgical approach either by
transsternal access or by intercostal/apical access would not be tolerated
by the patient and an aortic valve implantation via transfemoral catheter
using the Edwards-SAPIEN Systems was performed. Patients had died
within hours of valve implantation and a full body autopsy was performed
in each case once appropriate consent was given.
Results. The observed complications had been: 1. irreversible compression
of implanted valve due to cardiac resuscitation, 2. implantation of a
small diameter valve not properly anchored due to excessive calcification
with paravalvular leak, 3. loss of valve being dislodged before the aortic
isthmus, 4. tilted implantation of valve due to calcifications of the aortic
ring with pressure necrosis of aortic wall and paraaortic bleed, 5. transmural
aortic rupture due to a calcified ring of the aortic valve after balloon
dilatation with intramyocardial/pericardial bleeding leading to coronary
compression with secondary hemorrhagic infarction.
Conclusions. In all cases the preoperative lack of information of the degree
of calcification of the aortic valve leaflets was a common denominator
for postoperative complications. Future improvements of three
dimensional imaging appear necessary to increase the chance of preventing
such complications. Until then, autopsy analysis of complications
may be the only way to detect potential weaknesses of an otherwise lifesaving,
but high risk procedure.
FR-P-166
Situs inversus totalis of twins: an autopsy case report
C . Tóth1 , M . Jäckel2 , K . Bartók2 1University Hospital Heidelberg, Institute of Pathology, Heidelberg,
2Military Hospital, Budapest, Hungary
Aims. Situs inversus totalis is a rare congenital abnormality resulting in
visceral malrotation of the internal organs leading to left-right asymmetry.
In certain cases it is associated with clinically defined syndromes e.g.
Kartagener’s syndrome. In the case presented after 9 attempts of in vitro
fertilization and reduction (from three embryos to two embryos) in the
20th gestation week cesarean section was performed because of acute
purulent chorioamnitis with spontaneous rupture of membranes.
Methods. The medical history of the mother included primary sterility
with 9-times in vitro fertilization with F II 20210 heterozigosity and
MFHFR polymorphism with homozigosity. Further, factor Xa thrombophily
was also in her family diagnosed. After the operation the placenta
and two fetus were macroscopically and histologically examined.
Results. Placenta: 390 g with intact two amnoitic sacs with two umbilical
cords with central origin and three blood vessels (2 arteries and 1 vein).
The cut surface of the placenta shows some grayish areals, the rest is normal.
Fetus A: 304 g, 22 cm long male fetus. Ventricular and ependymal
bleeding in the 4th ventricle. In the thorax the heart apex lies on the
136 | Der Pathologe · Supplement 1 · 2012
right side, the left lung consists of 3 lobes, the right one consists of 2.
The cut heart and the great blood vessels run according to a total situs
inversus situation. In the abdomen after removing a blood clot normal
developed organs with total malrotation can be found. The pelvis organs
macroscopically show no abnormality. Fetus B is a 297 g, 23 cm female
fetus with some petechial skin bleedings. The central nervous system has
no abnormalities. The thoracal and abdominal organs show the same
malrotational changes as described above at fetus A. The changes were
photographically documented.
Conclusions. The cause of spontaneous abortion was an acute purulent
chorioamnitis due to a probably ascending infection which caused the
spontaneous rupture of the membrane. The autopsy cannot explore the
cause of infection. Both of the autopsied fetus showed the rare malrotation
changes, the situs inversus totalis without any other developmental
disturbances. In the daily autopsy practice we need to know about these
rare changes which are important not only in fetopathology but in anatomical
pathology as well.
FR-P-167
Analysis and appraisal of the clinical autopsy results of a surgically
oriented cardiac center in terms of quality assurance
J . Grüning1 , R . Meyer1 , M . Dietel2 , R . Hetzer1 1 2 Deutsches Herzzentrum Berlin, Berlin, Charité, Humboldt-University in
Berlin/ Institute for Anatomic Pathology, Berlin
Aims. We aimed to analyze the quality of clinicopathologic documentation
and communication. Concentrating on the following two questions:
1.) How valuable are the medical documentation of the inspection of the
corpse (post-mortem/clinical autopsy) and the content of the autopsy
requests and are they adequate for a qualified-autopsy? 2.) Are the autopsy
findings and examinations in line with the quality requirements
of a cardiac center?
Methods. Between 2000 and 2009 an average autopsy rate of 37% of all
decedents (range, 28% to 45%) was reached at our institute. During that
time 1063 decedents underwent autopsy. In accordance with the above
objectives the following were analyzed and assessed: documentation of
the inspection of the corpse (causal chain of the causes of death; personal
data; formalities), content of the autopsy requests (clinical procedures),
autopsy reports (date of autopsy; date of compilation of the autopsy report
and granting of access for the clinician have been recorded). The clinical
and autopsied underlying diseases and causes of death were coded
in accordance with ICD 10.
Results. It was evaluated as positive that documentation of the inspection
of the corpse, content of the autopsy requests and the autopsy reports
themselves are in correct form. Also positive is, that the recorded autopsy
results are discussed during weekly medical meetings at the institution.
Regrettably, however, the pathologists do not participate in these
meetings. The fact that the majority of autopsy reports do not reach the
clinician until after 30 days is a serious problem precluding their prompt
evaluation and discussion. The clinicians and pathologists need to change
for the better communication in relation to analyzing and assessing
the autopsies. There have been discrepancies between the clinically determined
causes of death and the autopsy causes of death. In particular
the clinically determined cause of death of “sepsis” needs to be discussed.
The preparation of the autopsy reports has no real influence on the
hospital’s cost accounting (DRG).
Conclusions. It becomes apparent that the autopsy report constitutes an
effective tool for quality assurance. The weekly medical meeting is a suitable
forum to look at quality assurance issues. The quantity and quality
of the communication between clinician and pathologist leave room for
improvement and strengthening.

FR-P-168
Autopsy findings in a 2-year-old boy with EHEC/HUS in the 2011
German outbreak
M . Kuhlmann 1 *, C . Baier 1 *, J .U . Becker 1 , C . Hartmann 2 , F .-C . Bange 3 , T . Ahlenstiel
4 , H . Kreipe 1
1 MH Hannover, Institute of Pathology, Hannover, 2 Ruprecht-Karls University
Heidelberg, Institute of Pathology, Department Neuropathology, 3 MH
Hannover, Department of Medical Microbiology and Hospital Epidemiology,
4 MH Hannover, Department of Pediatric Nephrology
Aims. Demonstration of histopathological findings in a 2-year-old boy
with EHEC/HUS in the 2011 German outbreak.
Methods. Routine autopsy procedure including gross and microscopic
examination with histochemical and immunhistochemical stainings
(MSB, CD 61, GFAP, CD 68), and review of clinical data.
Results. A previously healthy 2-year-old boy was diagnosed with EHEC/
HUS (serotype O104:H4, Shiga-toxin 2 positive) and developed in the
clinical course severe acute renal and heart failure as well as neurological
complications. Our main histopathological findings: Heart: heart weight
was increased above the 90 percentile. Histology revealed rather fresh
focal necrosis of heart muscle cells and thrombocyte rich thrombi in cardial
capillaries. Kidney: on cross sections the kidneys appeared darkly
red and no distinction between renal cortex and medulla was possible.
Histologic examination revealed luminal thrombocyte rich thrombi in
glomeruli and in afferent arterioles, with negligible amounts of stainable
fibrin. Moreover, glomeruli exhibited severe endothelial swelling and
severe mesangiolysis with microaneurysms. Brain: supra- and infratentorial
multiple hemorrhagic infarctions (stage I and II) with severe brain
edema as well as a laminar necrosis of the cortex (stage I) were detectable.
Furthermore there was a severe gliosis with multiple reactive astrocytes
and activated microglia in the white matter. Within the complete central
nervous system no thrombi indicating TMA were found.
Conclusions. In the kidneys endothelial swelling was dominant reflecting
endothelial damage caused by Shiga-toxin 2. Endothelial damage induced
formation of thrombocyte microthrombi without stainable fibrin
content in the heart and in the kidneys with resultant ischemia. While
in previously published cases the thrombi contained fibrin depositions
besides thrombocytes, in this case we only found minimal or no fibrin
with MSB stain. Another key feature of our renal histology was a severe
mesangiolysis, which is described as a rare finding in typical D+ HUS
whereas it is more often seen in D- negative HUS cases. Regarding the
cerebral findings the severe gliosis of the white matter without similar
changes in the cortex could indicate a prior non-ischemic, possibly toxic
damage to the white matter, as is discussed in the literature. Our case
with cerebral, myocardial and renal involvement causing failure of all
three organs argues against an overly rigid clinical separation of HUS
and thrombotic-thrombocytopenic purpura.
*These authors contributed equally to the study .
FR-P-169
Synchronous presentation of gastrointestinal stromal tumor of
the stomach, ganglioneuroma of the adrenal gland and adenomas
of the colon
N . Pawlaczyk1 , K . Neumann1 , J . Knolle1 , H . Zühlke2 1 2 Institute of Pathology, Dessau-Rosslau, Department of General,
Visceral and Vascular Surgery, Lutherstadt Wittenberg
Aims. Gastrointestinal stromal tumor (GIST) is the most common mesenchymal
tumor of the gastrointestinal tract. Its carcinogenesis is driven
by activating mutations of the KIT or PDGFRA gene but a small
subset of GISTs has a negative mutation status. These wild type-GISTs
may develop as part of a multi-neoplastic disease. We present a case with
concurrent presentation of GIST of the stomach, Ganglioneuroma (GN)
of the adrenal gland and adenomas of the colon. To our knowledge, the
synchronous existence of these neoplasias has not yet been reported.
Methods. We describe an 85-year-old male patient with initially acute upper
gastrointestinal bleeding. Explorative laparotomy and subsequently
pathologic analysis of resected specimen revealed three distinct neoplasias:
GIST of the stomach (5,5×3,7×3,8 cm, T,
Gly12Cys). GNs are rarely seen in patients with neurofibromatosis (NF)
type 1. An association between the development of GIST and type 1 NF
has also been established. Our patient showed no further clinical features
of NF. GISTs arising in the setting of type 1 NF are usually KIT- and
PDGFRA-wild type and the tumor suppressor gene Neurofibromin is
inactivated. Testing for a potential silencing of Neurofibromin is underway.
Conclusions. The synchronous manifestation of GIST and other neoplasms
is a common observation. In our case, the co-occurence of GIST,
GN and adenomas of the colon could represent a syndromal setting. Molecular
analysis revealed no amino acid changing mutations of the KIT
and PDGFRA genes what differs from sporadic GISTs. The role of alternative
oncogenes or pathways in the carcinogenesis of wild type-GISTs
as well as in their presentation in a multi-neoplastic context requires
further examination.
FR-P-170
Pleural malacoplakia caused by Rhodoccocus equi infection in a
patient after stem cell transplantation because of a T-PLL
C .L . Behnes1 , S . Neumann2 , S . Schweyer1 , H .-J . Radzun1 1 2 University of Göttingen/Institute of Pathology, University of Göttingen/
Institute of Onkology
Aims. Malakoplakia is a disease especially of the urinary tract with typical
plaques most frequently observed in the bladder’s mucosa, consisting
of accumulated macrophages. The reason for this disease is an impaired
lysosomal degradation of bacteria, especially E. coli. In the context of immunosuppression
malakoplakia can also occur in other organs such as
the prostate, kidney and lung. To our knowledge, affection of the pleura
by malacoplakia has not yet been documented.
Methods. Case report: a 60-year-old man was admitted to the hospital
because of a generalized lymphadenopathy, hepatosplenomegaly and
suspicion of pneumonia. Based on a lymph node biopsy the diagnosis
of a T-cell-prolymphocytic-leukemia was confirmed and treated with
an allogenic stem cell transplantation. A half year later the patient was
admitted to the hospital with retrosternal pain and a reduced state of
condition. The clinical examinations revealed a 12 cm in diameter and
well circumscribed focus within the right upper pleura.
Results. The macroscopical examination of the upper lobe of the right
lung showed a 12 cm in diameter tumor adherent to the pleura, displacing
the lung and showing a gray-white cut surface with central necrotic,
disintegrated areas. The microscopical examinations revealed a tumor
consisting of a monomorphic cell population, which was predominantly
composed of macrophages. Granulomas, multinucleated giant cells, significant
cellular atypia or increased proliferation could not be observed.
The immunohistochemical examinations revealed numerous Ki-M1P/
CD68 positive macrophages with intracellular PAS positive deposits,
which could be identified as calcifications by Kossa staining (Michaelis-
Gutmann-Bodies) being pathognomonic for Malacoplakia. A microbio-
Der Pathologe · Supplement 1 · 2012 |
137

Abstracts
logical examination of tumor tissue during surgery could demonstrate
Rhodococus Equi.
Conclusions. To our knowledge this case represents the first pleural malacoplakia
associated with a Rhodococus equi infection. Extravesikal malacoplakia
is an important differential diagnosis in immunosuppressed
patient especially in case of a proved Rhodococus equi infection.
FR-P-171
Testicular primitive neuroectodermal tumor – molecularpathological
analysis and discussion of developement
S . Brandt1 , B . Lohe2 , A . Vogetseder1 , T . Rüdiger2 , H . Moch1 , P . Bode1 1 2 Universitiy Hospital Zurich, Zürich, Switzerland, Städtisches Klinikum Karlsruhe,
Pathologisches Institut, Karlsruhe
Aims. The occurrence of a testicular primitive neuroectodermal tumor
(PNET) is a rare event in malignant transformation of a teratomatous
component in testicular germ cell tumors. Based on morphological, immunohistochemical
and molecularpathological findings these tumors
resemble central PNETs, as otherwise only seen in children and do not
show a rearrangement of the EWS gene on chromosome 22. We describe
a case of a PNET occurring in a testicular germ cell tumor.
Methods. Routine immunohistochemistry (AE1/AE3-Cytokeratin, S100,
Synaptophysin, CD99, GFAP, Oct-3/4 and CD30) was performed as well
as Fluorescence in situ Hybridization (FISH) and RT-PCR.
Results. Immunohistochemistry showed focal expression of AE1/AE3-
Zytokeratin, S100, Synaptophysin, GFAP and CD99 and no expression
of Oct-3/4 and CD30 in the tumor cells. No translocation of t(11;22)
(EWSR1/FLI1) and t(21;22) (EWSR1/ERG) could be shown by FISH and
RT-PCR.
Conclusions. The occurrence of testicular primitive neuroectodermal
tumor is a rare event. It is believed to originate from malignant transformation
of a teratomatous component in testicular germ cell tumors.
Identification of a PNET component in a testicular germ cell tumor is
of clinical relevance since studies have shown that these tumors do not
respond to conventional cisplatin based chemotherapy in comparison to
usual germ cell tumors. Some authors recommend PNET specific chemotherapy,
as well as retroperitoneal lymph node dissection.
FR-P-172
Complicated Malaria tropica – two typical disease patterns
I . Klempert1 , P . Lohneis1 , W .D . Schmitt1 , M . Dietel1 1Charité University hospital, Institute of Pathology, Berlin
Aims. Malaria – the second most common infectious disease of the world
is rare in Germany. Pathologists are seldom tasked with diagnostics, but
if demanded it is generally presented by fulminant development and
with impressive findings.
Methods. On the basis of two autopsies, which had a fulminant, following
the lethal course of the disease the typical histological pattern and
the correlative medical findings will be demonstrated. The microscopic
slides were prepared with the Hematoxylin-eosin-stain and additionally
examined by the polarizing microscope to differentiate between the
double refracting malaria pigment and stain dependent artefacts.
Results. Anamnestic: Case 1: The patient had a fever, diarrhea and vomiting,
with kidney failure as symptoms of Malaria (tropica) during a
residence in Sierra Leone. Due to the limited therapeutic possibilities
locally, the patient tried to return to his home. The flight back home was
interrupted by a forced landing, because the patient collapsed. He was
brought to the emergency room of the Charité Berlin. The plasmodiumdensity
was very high (>30%). The next day an emergency Splenectomie
by laparotomie was necessary, followed by a secondary haemorrhage.
The cardiovascular system was stable, the plasmodium-density came
down to >1%. Six days later the patient died, caused by a multi organ
failure with a leading liver insufficiency. Case 2: The patient came to his
138 | Der Pathologe · Supplement 1 · 2012
family doctor, because he felt ill and had slurred speech. A few days before,
he had returned from a journey to Cameroon. Within the next few
hours he drifted off more and more and showed increasing intracranial
pressure. He died the next day, caused by a central cardio-vascular system
failure. The density of the plasmodia was 10%. Histologic: Case 1: Expanded
intra- and pericapillary deposits of hemozoin (iron free malaria
pigment) inside the liver, myocardium, kidney and the alveolus of the
lung. Agglutinate erythrocytes were found inside the hepatic sinus and
some vital, adipose hepatocytes were present. Case 2 shows analogous
to case 1 extensive manifestation at the organs. Additionally a massive
edematous brain with plasmodia and hemozoin inside the erythrocytes
of the intracerebral capillaries was found.
Conclusions. The rare disease pattern of a malaria infection appears with
extensive histopathological findings that can be demonstrated by simple
histological methods in correlation with the clinical development.
FR-P-173
Orbital epithelioid sarcoma: a case report
T . Berg1 , J . Knolle1 , S . Knipping2 , C . Kneifel3 , K . Stock4 , I .F . Ciernik5 , T . Mentzel6 1 2 Dessau City Hospital, Institute of Pathology, Dessau, Dessau City Hospital,
Department of Otorhinolaryngology, Dessau, 3Dessau City Hospital, Department
of Ophthalmology, Dessau, 4Dessau City Hospital, Department of Radiology,
Dessau, 5Dessau City Hospital, Department of Radiation Oncology,
Dessau, 6Dermatopathologie Bodensee, Friedrichshafen
Aims. Epithelioid sarcoma (ES) is a rare and aggressive soft tissue neoplasm
most prevalent in the distal extremities of young male adults. The
proximal type of ES is thought to be the morphological progression with
predominance for somewhat older patients and a higher propensity for
metastasis than classic ES.
Methods. We report on a 30-year-old patient who presented with proptosis
of the right eye. He complained of metamorphopsy and visual impairment.
Diagnostic imaging was suspicious for sarcoma. Preoperative
biopsy was performed followed by histological and immunohistochemical
analysis. Antibodies against Cytokeratins (clone MNF116, CK 5/6, CK
8/18, CK 7, CK 20), Vimentin, S100-protein, CD34, CD31, CD10, Desmin,
Aktin, p63, PHH3 and TTF-1 were used. Wide local tumor excision with
curative intent followed.
Results. Histological examination of the biopsy revealed a nodular
growth pattern of atypical eosinophilic epitheloid and spindle cells with
25 mitoses per 10 high-power-fields. By immunohistochemistry, the tumor
was positive for vimentin, cytokeratins and EMA while there was
no reaction for CD34 and INI-1. The diagnosis of proximal-type ES was
established. The patient underwent orbital exenteration with negative
surgical margins. Largest tumor diameter was 27 mm. Eight months
after diagnosis metastatic progression with multiple metastases of the
vertebral column and the base of the skull was noted.
Conclusions. ES (proximal variant) develops predominantely in the pelvis,
perineal region, trunk, mediastinum and genital tract. It is exceedingly
rare in the orbital fossa. To our knowledge only three cases have
been reported. Differential diagnoses include carcinoma, amelanotic
malignant melanoma, epithelioid malignant peripheral nerve sheath tumor
(MPNST), epithelioid angiosarcoma and myoepithelioma. ES has
been associated with an unfavourable prognosis, early and frequent metastasis
and requires adequate surgical treatment at an early stage with
proper assessment of surgical margins. Postoperative radiation oncology
and/or adjuvant systematic treatment might be considered according to
the presentation.

FR-P-174
MPGN-like glomerulonephritis with intracapillary IgM thrombi in
Waldenström’s macroglobulinemia
D . Kratochvil 1 , K . Amann 2 , H . Bruck 1 , M . Büttner 2
1 University Hospital Essen, Internal Medicine, Essen, 2 University Hospital
Erlangen, Institute of Pathology, Erlangen
Aims. Lymphoproliferative disorders causing paraproteinemia can be
associated with various kidney injuries. In the context of a case report
earlier reports in literature and differential diagnoses of IgM-associated
glomerulonephritides (GN) are discussed.
Methods. Case report including the results of clinical, light microscopical,
immunohistochemical and electron microscopical investigations.
Literature search and discussion of earlier descriptions of glomerular
monoclonal IgM deposits in lymphoproliferative diseases.
Results. A 73-year-old female patient with a history of rheumathoid arthritis
and Waldenström’s disease was admitted to hospital for acute renal
failure with a mild proteinuria and hematuria. An MPGN-like GN
with intracapillary IgM thrombi was diagnosed. The patient reported
episodes of palpable purpura reminiscent of cryoglobulinemia. Despite
repeated analyses, however, no cryoglobulines could be detected. The renal
function recovered before the beginning of the immuno-modulatory
therapy.
Conclusions. In contrast to M. Waldenström-associated GN described
by Maroger-Morel in the present case a prominent glomerular hypercellularity
was found. A strict distinction between cryoglobulinemic and
non-cryoglobulinemic GN appears difficult taking previous reports in
literature into account.
FR-P-175
c-Met in undifferentiated pleomorphic sarcomas and fibroblastic/
myofibroblastic tumors
C . Wölfel1 , T . Knösel1 , T . Liehr2 , S . Hauke3 , A . Altendorf Hofmann4 , D . Katenkamp1
, I . Petersen1 1 2 Jena University Hospital, Institute of Pathology, Jena, Jena University Hospital,
Institute of Human Genetics, Jena, 3ZytoVision GmbH, Bremerhaven,
4Jena University Hospital, Department of General Surgery, Jena
Aims. Undifferentiated, pleomorphic sarcomas (UPS), formerly known
as malignant, fibrous histiocytoma (MFH) are defined as a group of
high-grade sarcomas in which any attempt to disclose their line of differentiation
has failed. These undifferentiated, pleomorphic sarcomas tend
to occur in the extremities of elderly patients as a deep-seated, enlarging
mass. The tumors frequently show an aggressive, rapid growth which
contrast with the limited therapeutic options consisting mainly of surgery
and radiotherapy while chemotherapy is usually not effective. c-MET
inhibition is recently evolving as a promising new target in cancer therapy.
C-Met is a proto-oncogene that encodes the hepatocyte growth factor
receptor which possesses tyrosine-kinase activity. An abnormal c-Met
activation in cancer triggers tumor growth, angiogenesis and encourage
metastasis leading to a poor prognosis for the patient. We aimed to analyze
the gene in soft tissue tumors.
Methods. The tumor collective consisted of 327 fibroblastic/myofibroblastic
tumors including 203 undifferentiated, pleomorphic sarcomas, 42 low
grade sarcomas and 82 pseudosarcomatous tumors of the fasciitis family.
It was analysed for c-Met expression by immunohistochemistry and c-
Met amplification by FISH. This was done on TMA sections (3 µm). One
TMA of fasciitis nodularis tissues was used as control. For immunhistochemistry
we applied a polyclonal rabbit anti-c-Met antibody with the
dilution 1:50 at pH 6.1. Furthermore, we used FISH probes located at the
c-Met region at chromosome 7q31.3 and as reference centromere probes
for chromosome 7. The slides were evaluated by fluorescence microscopy.
Results. Immunohistochemically, we found 53 sarcomas (16%) with a
high c-Met expression. In contrast, the fasciitis cases revealed no or only
low expression in all cases. For FISH analyses, 105 samples could so far
be investigated. They showed chromosomal aberrations in 37 cases (39%).
Many cases carried a polysomy of chromosome 7. In addition, 9 cases
(8.6%) revealed a selective amplification of the c-Met locus which, however,
was only rarely associated with a clear-cut c-met overexpression.
Conclusions. Conclusion: c-MET may represent an interesting therapeutic
target in a subset of undifferentiated pleomorphic sarcoma which
needs further evaluation.
FR-P-176
Cement spacers with MicroSilver (Bio-Gate) show decreasing
inflammation without hints for detrimental effects in histological
study of periprosthetic membranes
S . Söder1 , T . Bechert2 , P . Steinrücke2 , R . Ascherl3 , A . Hartmann1 1 2 Erlangen-Nürnberg, Institute of Pathology, Erlangen, Bio-Gate AG, Nürnberg,
3Klinik für Wechselendoprothetik, Chemnitz
Aims. Following hip replacement an infection rate of 2% was observed in
this procedure. So far Gentamycin-spacers are commonly used to control
periprosthetic infections. However especially in the presence of multiresistant
bacteria like MRSA they are often only of limited effect. Silver
has already proven to be effective in vitro. In a randomized prospective
clinical study by Bio-Gate AG, Gentamycin-spacers with additional
Microsilver component were compared with conventional Gentamycinspacers.
In addition to a comprehensive panel of clinical and microbiological
tests the periprosthetic membranes were studied histologically.
Methods. We received blinded samples from 17 different patients with
hip implant infections. Each patient received 2-stage revisions with
2 different cement spacers (Gentamycin and Gentamycin with added
Microsilver) following a randomization. Specimens were formalin fixed,
paraffin embedded, sectioned and hematoxylin-eosin stained.
Results. No silver particles could be identified in any sample, even
though occasionally wear particles were found in both groups. There was
no indication of allergic or toxic effects in the histological examination.
Typically a decline in inflammation was found in the course of the treatment.
Periprosthetic membranes from spacers with added Microsilver
showed typically a stronger or at least equal reduction in inflammation
than spacers with Gentamycin alone.
Conclusions. Histological examination gave no hints for adverse effects of
adding Microsilver to conventional spacers which is in accordance with
preliminary in vitro and animal studies. Furthermore a stronger effect
on inflammation activity was observed.
FR-P-177
GNAS1 mutations in tumorigenesis
B . Walther1 , I . Walther2 , Y . Chen1 , I . Petersen1 1 2 Jena University Hospital, Jena University Hospital, Institute of Pathology
Aims. The GNAS-1 gene is located on chromosome 20 and encodes for
the alpha-subunit of ubiquitary existing stimulatory G-proteins. The two
mutation hotspots R201H and R201C were evaluated which consists of
the replacement of arginine by histidine or cysteine, respectively. These
mutations have been previously reported in intramuscular myxomas representing
benign soft tissue tumors that are often found in near-trunk
muscle tissue of women. Furthermore, the McCune Albright syndrome
(MAS) being characterized by Café-au-lait spots, precocious puberty,
polyostotic fibrous dysplasia (FD) and other endocrine abnormalities is
related to GNAS-1 mutations. In MAS, thyroid adenomas and carcinomas,
adrenocortical hyperplasia and adenomas and pituitary tumors do
occur. Moreover mutations were detected in isolated fibrous dysplasia
(FD) or in combination with intramuscular myxomas in the context of
the so called Mazabraud’s syndrome. The aim of the study was to evaluate
the prevalence of these mutations in intramuscular myxomas and to
verify the usefulness of mutations analysis in the differential diagnosis of
soft tissue and bone lesions.
Der Pathologe · Supplement 1 · 2012 |
139

Abstracts
Methods. Tumor-DNA was extracted from 4–10 µM thick specimen slides
of formalin-fixed and paraffin-embedded tissue after micro dissection.
These probes were amplified with conventional PCR and checked
with agarose-gel-electrophoresis. The mutation status was assessed by
direct DNA sequencing.
Results. 61 intramuscular myxomas were examined and a mutation rate
of 36.07% (22 cases) was detected which correlate with data from published
studies. 75.41% of these were found in women. Both hotspots were
equally affected. Furthermore 26 other tumor entities (angiomyxomas/fibromas,
fibromyxoid sarcomas, chondrosarcomas, liposarcomas, FD,
lipomas and neurothekeomas) were analyzed. In 5 out of 8 FDs (62.5%),
mutations in codon 201 were discovered. In all other entities including
7 atrial myxomas and 31 gastroenteropancreatic-neuroendocrine tumors
(GEP-NET’s) no GNAS-1 mutations were detected.
Conclusions. Our results indicate that the GNAS-1 mutation analysis can
be helpful to differentiate between FD and unspecific bone lesions (e.g.
cystic or inflammatory conditions). It may be particular useful in the
differential diagnosis of myxoid tumors. GNAS-1 mutations were never
detected in any sarcomatous lesions while carrying a considerable prevalence
in intramuscular myxoma. Mutation-positive patient might be
screened for bone lesions compatible with FD to exclude Mazabraud’s
syndrome.
FR-P-178
Valproic acid stimulation induces downregulation of IRAK-1 protein
in a progressive thyroid carcinoma cell line
S . Schwertheim1 , S .-Y . Sheu-Grabellus1 , K . Worm1 , K .W . Schmid1 1University Hospital of Essen, University of Duisburg-Essen, Institute of
Pathology and Neuropathology, Essen
Aims. Valproic acid (VPA) is a drug in clinical phase 2 for the therapy
of advanced/poorly differentiated thyroid cancer with poor prognosis.
miRNA-146a/b has been proved to be deregulated in thyroid carcinoma.
To clarify if miRNA-146a/b plays a role in cells influenced by VPA
we treated the highly progressive thyroid cell line BHT-101 with various
doses of VPA (0, 1.0, 1.5 and 3.0 mM) and analyzed the expression levels
of these miRNAs. As it is documented that miRNA-146/b is associated
with regulation of NF-κB activity we also examined VPA-treated cells on
mRNAs and proteins modulated by NF-κB.
Methods. Cells were seeded in 6-well plates at 60–80% confluence and
incubated with VPA for 48 h; conditioned medium was used for treatment
containing 0.2% FCS, 1% Penicillin and 0.1% Amphotericin B.
miRNA and mRNA expression levels were detected by RT-PCR using
Taq Man miRNA- and Gene Expression-Assays (Applied Biosystems).
Protein analysis was performed by Western blotting.
Results. miRNA-146a/b was upregulated at a concentration of 1 mm
(foldchange 2.35) and 1.5 mM (foldchange 3.43) VPA and decreased at
3.0 mM (foldchange 2.26). VPA significantly and dose-dependently impaired
NF-κB activity, reducing expressions of IRAK-1 (miRNA-146a/b
target gene), phospho-IκBα and p50 protein. Remarkably, 1 mm VPA
treatment induced upregulation of IL-6 and IL-8 mRNA levels, following
reduced expressions at 3.0 mM; examination of IL-8 protein levels
confirmed this.
Conclusions. Our results suggest that miRNA-146a/b and IRAK-1 levels
play a crucial role in VPA’s mechanisms of action and might be promising
tools to regulate the therapy of advanced thyroid cancer.
140 | Der Pathologe · Supplement 1 · 2012
FR-P-179
Dysregulation of miRNA expression in normal thyroid tissue
adjacent to tumor cells
S . Schwertheim1 , S .-Y . Sheu-Grabellus1 , K . Worm1 , K .W . Schmid1 1University Hospital of Essen, University of Duisburg-Essen, Institute of
Pathology and Neuropathology, Essen
Aims. The miRNAs 146a, -146b -181b, -21, -221, -222, 30d, -125b, -26a, -30a-
5p, and -let7c have been proved to be deregulated in thyroid carcinoma.
To clarify if miRNAs can be used to evaluate tumor progression we analyzed
the expression of these miRNAs in 5 normal thyroid tissues adjacent
to highly progressive thyroid carcinomas (4 poorly differentiated, 1
anaplastic thyroid carcinoma) and compared the results with expression
levels in 4 normal thyroid tissues of individuals without any clinical thyroid
disease.
Methods. Paraffin embedded tissues were laser microdissected (PALM
Laser-Micro Beam System, P.A.L.M., Bernried) for RNA analysis. miR-
NA expression levels were detected by RT-PCR using Taq Man miRNA
assays and relative quantification of miRNA expression was calculated
with the 2-ΔΔCt method.
Results. We found significantly (p

Poster: Gynäkopathologie und Mammapathologie I
SA-P-005
HER2 status in breast cancer remains stable with FISH (fluorescence
in situ hybridisation) but is highly variable with IHC
(immunohistochemistry) methodology in view of 10 years
experience
Z . Varga 1 , C . Ramach 2 , B . Padberg 3 , H . Moch 1 , A . Noske 1
1 University Hospital Zurich, Institute of Surgical Pathology, Zürich,
Switzerland, 2 County Hospital St . Gallen, Institute of Pathology, St . Gallen,
Switzerland, 3 Institute of Pathology, County Hospital Graubünden, Chur,
Switzerland
Aims. Gold standard methodology in HER2 status determination in breast
cancer is still a debated issue. Advantage of IHC analysis over timeconsuming
and experience-requiring FISH methodology can be strongly
influenced by pre-analytical differences and interpretational-issues.
Methods. We analysed 6000 consecutive HER2-FISH tests in breast cancer
in 10 years (2001 to 2011) and compared stability of amplification-rate
with immunohistochemical 3+ positivity. Four years long (2001–2004)
FISH tests were performed in all 3+ and 2+ cases and in some of the 1+
and negative (0) cases. For 6 years (2005–2010) HER2 status was determined
with “only FISH” testing. For one year (2011) all cases were tested
with both IHC and FISH.
Results. Between 2001 and 2004, 61% of 3+ IHC was amplified with
FISH (amplification-rate varied from 50%, 67%, 53% and 77%). In 2011,
86% of 3+ IHC cases were amplified with FISH. FISH amplification rate
varied between 15–17% in 2005–2008 and 11–13% in 2009–2011 (due to
modified ASCO criteria of HER2/CEP17 ratio from

Abstracts
SA-P-008
Correlation of anti-PHH3 positive mitoses to pathological response
in neoadjuvantly treated breast cancer
S . Timme 1 , M . Becker 2 , E . Stickeler 2 , P . Bronsert 1 , L . Reischuck 2 , L . Bogatyreva 3 ,
D . Hauschke 3 , A . zur Hausen 4 , M . Werner 1
1 Institute of Pathology, University Medical Center, Freiburg, 2 Department of
Obstetrics and Gynecology, University Medical Center Freiburg, Freiburg,
3 Institute of Medical Biometry and Medical Informatics, University Medical
Center, Freiburg, 4 Department of Pathology, GROW, School for Oncology
and Developmental Biology, Maastricht University Medical Center, Maastricht,
Netherlands
Aims. We evaluated breast cancer (BC) core biopsies taken before neoadjuvant
chemotherapy (NACT) by immunohistochemistry using anti-PhosphohistoneH3
antibody (PHH3) to determine the mitotic count.
The data were correlated to clinicopathological parameters including
intrinsic subtypes as well as the histopathological regression of resected
tumour specimens after NACT with Epirubicin/Cyclophosphamide
(EC) and Taxotere.
Methods. 72 patients with either triple negative (21/72) or luminal type
(51/72) BC obtained NACT with EC and Taxotere. Thereafter, tumour
regression was analyzed in resection specimens using a semiquantitative
score from 0 (no effect) to 4 (no cancer cells) according to Sinn et al.
(1994). Pathological complete response (pCR) was defined as no residual
invasive carcinoma (score 3+4). In 16/72 cases (22.2%) pCR occurred; 9/16
(56.25%) were TNBC and 7/16 were luminal type BC (ER and/or PrR+/
HER2−). In the pre-treatment biopsies immunohistochemical stainings
with PHH3 were performed and mitotic figures were evaluated in 10 high
power fields (HPF). The number of mitoses detected by PHH3 was correlated
to different clinicopathological parameters determined before treatment
(intrinsic subtype, WHO-type, grading, tumour size and nodal
stage) and to regressive changes/pCR after therapy by univariate statistical
analyzes (using SPSS v 18).
Results. The number of PHH3-detected mitoses correlated significantly
with the tumour grading (p=0.001), but there was no correlation with
WHO-type, tumour size or nodal stage. PHH3/10 HPF differs significantly
between the two intrinsic subtypes (p=0.003). PHH3 expression
alone was no predictor for pCR (p=0.399). But tumours with 11 or
more mitoses/10 HPF achieved significantly more often pCR than those
with under 11 mitoses/10 HPF (p=0.031). Furthermore, luminal type
BC with 11 or more mitoses/10 HPF and also TNBC had significantly
more frequent pCR than luminal type BC with under 11 mitoses/10 HPF
(p=0.016).
Conclusions. Strong proliferating BC (11 or more mitoses/10 HPF) have
significantly more pCR compared to low proliferating tumours (under
11 mitoses/10 HPF) after NACT with EC and Taxotere. Furthermore,
mitoses detected by PHH3 are a discriminator for luminal type BC to
predict pCR, because luminal type BC under 11 mitoses/10 HPF rarely
reach pCR. Thus, the determination of mitoses by PHH3 may be recommended
especially for luminal type BC before NACT with EC and
Taxotere.
SA-P-009
Transitions between flat epithelial atypia and low-grade ductal
carcinoma in situ of the breast
S . Aulmann1 , F . Mietzsch1 , R . Penzel1 , P . Schirmacher1 , H .P . Sinn1 1University of Heidelberg, Institute of Pathology, Heidelberg
Aims. Flat epithelial atypia (FEA) of the breast typically is a localised alteration
involving only few, neighbouring terminal ducto-lobular units
(TDLUs). However, occasionally there are cases with extensive FEA and
morphological evidence of direct transitions between FEA and classical
ductal carcinoma in situ (DCIS).
Methods. To investigate the relationship of FEA and DCIS in these cases,
we microdissected multiple foci of the respective lesions in a series
142 | Der Pathologe · Supplement 1 · 2012
of 10 cases and performed comparative allelotyping using a panel of 14
LOH markers. In addition, phylogenetic tree models were calculated on
the basis of mitochondrial DNA sequencing to visualise the clonal relationship
of the different lesions.
Results. FEA and lg-DCIS shared the majority of chromosomal imbalances,
loss of diverging alleles was not detected in any of the 10 cases.
mtDNA sequencing and phylogenetic tree clustering revealed direct
transitions between FEA and lg-DCIS in all 10 cases. However, in three
patients, additional foci of FEA were present which were not directly related
to the rest of the FEA and the lg-DCIS.
Conclusions. In conclusion, our data demonstrate the presence of direct
transitions between FEA and lg-DCIS and support the interpretation of
foci of FEA as part of the lg-DCIS in those unusual cases in which multiple
areas of FEA are interspersed with areas of classical lg-DCIS and
direct transitions of both lesions are apparent.
SA-P-010
Different cytoplasmic and nuclear PARP expression patterns in
hereditary and non-hereditary breast cancer
M .-L . Klauke1 , N . Hoogerbrugge1 , J . Budczies2 , P . Bult1 , J .H .J . van Krieken1 ,
C . Denkert2 , B .M . Müller2 1Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands,
2Charité Hospital, Institute of Pathology, Berlin
Aims. High cytoplasmic poly (adenosine diphosphate-ribose) polymerase
(PARP) expression in non-hereditary breast cancer correlates with an
aggressive tumor pattern and a poor long-term prognosis. Our study was
designed to compare cytoplasmic and nuclear PARP expression between
hereditary and non-hereditary breast cancer.
Methods. Tissue micro arrays containing 39 familiar and 39 non-hereditary
formalin-fixed, paraffin embedded breast cancer tumor samples
were immunohistochemically analyzed for cytoplasmic (cPARP) and
nuclear (nPARP) PARP expression. Stained slides were digitized and
evaluated using the VM Slide Explorer. The intensity and percentage
of positive tumor cells were used to calculate an immunoreactive score
(IRS), which was divided into three subgroups defined as low (IRS 0–2),
intermediate (IRS 3–4) and high (IRS 6–12) expression. The mean age at
diagnosis in patients with familiar breast cancer was about 45±11 years
and in patients with non-hereditary breast cancer about 65±12.1 years.
Results. cPARP and nPARP expression patterns were significantly different
in hereditary and non-hereditary breast cancer. cPARP expression
in hereditary breast cancer was low in 33.3%, intermediate in 25.6% and
high in 41.0% of cases. In contrast, cPARP in non-hereditary breast cancer
was low in 59.0%, intermediate in 25.6% and high in 15.4% of cases.
Statistical analysis showed that cPARP expression was significantly higher
in hereditary compared to non-hereditary breast cancer (p=0.008,
χ2-test for trends). Hereditary breast cancer showed a significant lower
intermediate nPARP expression (23.1%) than non-hereditary breast cancer
(59.0%; p=0.005, χ2-test).
Conclusions. PARP expression in hereditary and non-hereditary breast
cancer differs significantly, which might be related to the higher frequency
of BRCA1 or 2 mutations in hereditary breast cancer and is compatible
with a role of PARP-1 in DNA repair and genomic stability. Because patients
with hereditary breast cancer have a poor long-term prognosis and
show very often an aggressive tumor pattern the overall higher cPARP
expression found in this subgroup suggests that high cPARP expression
might be correlated with an aggressive tumor pattern and a poor longterm
prognosis.

SA-P-011
Genetic aberrations of predictive factors are rare in triple-negative
breast cancer
T . Grob 1 , M . Choschzick 1 , G . Sauter 1 , A . Lebeau 1
1 University Medical Center Hamburg-Eppendorf, Institute of Pathology,
Hamburg
Aims. In the absence of estrogen as well as progesterone receptors and the
lack of HER2 amplification, women with triple-negative breast cancer
TNBC do not benefit from endocrine therapy or trastuzumab and are
left with chemotherapy as their only option. To reduce the elevated risk
of disease progression in these patients, better treatment options are needed
that are less toxic and are more targeted to this patient population.
Therefore, we performed a comprehensive analysis of potential targetable
genetic aberrations affecting the EGFR/HER2/MAPK pathway that
are observed at higher frequencies in adenocarcinomas of other organs.
Methods. 65 consecutive TNBCs were studied by sequence analysis for
HER2, EGFR, KRAS, BRAF mutations. TP53 sequence analysis was included
to control DNA quality and tumor cell content. A tissue microarray
(TMA) representing two samples of each tumor was constructed to
search for EGFR gene copy gain and EML4-ALK fusion by FISH. Triple
negative status was confirmed by immunohistochemistry (IHC) and
FISH on TMA sections. EGFR and CK5/6 IHC were added for identification
of the basal-like phenotype.
Results. Sequence analysis revealed HER2 gene mutation in one patient
(heterozygous missense mutation in exon 19: p.L755S). No mutations
were found in EGFR, KRAS and BRAF. High polysomy of EGFR was
detected in 5 of 62 informative cases by FISH. True EGFR gene amplification
accompanied by strong membraneous EGFR protein expression
was observed in only case. No rearrangement of the ALK gene was detected.
Basal-like phenotype was identified in 38 of the 65 TNBCs (58.5%).
TP53 mutations were detected in 36 of the 63 (57.1%) informative tumors.
Conclusions. Targetable genetic aberrations in the EGFR/HER2/MAPK
pathway occur rarely in TNBC. Nonetheless some patients might benefit
from HER2/EGFR targeted therapy.
SA-P-012
Luminal B breast cancers are not the end stage of a stepwise
dedifferentiation of luminal breast cancers
H . Bürger1 , B . Schymik2 , W . Meinerz2 , E . Korsching3 1University of Münster/Utrecht, Institute of Pathology, Paderborn, Paderborn,
2St .Vinzenz Hospital, Clinics of Gynecology, Paderborn, 3University of
Münster, Medical Faculty, Institute of Bioinformatics, Münster
Aims. Recent molecular data pointed towards the possibility of a stepwise
dedifferentiation in a subgroup of invasive breast cancer (BC) cases. In
detail, it was hypothesized, that estrogen receptor positive (ER+) grade
3 ductal invasive BC’s are the end stage of a dedifferentiation process of
luminal BC. A progression of luminal A towards luminal B breast cancers,
associated with a “progression through grade” seemed the obvious
explanation.
Methods. In order to verify this hypothesis on a morphological and
immunohistochemical level we investigated 865 invasive breast cancer
cases. All cases were reviewed for the presence of intratumoural heterogeneity
in the invasive cancer and the presence of associated ductal
carcinoma in situ (DCIS). With the use of tissue microarrays the molecular
subtype was determined and correlated with clinicopathological
features.
Results. We were able to show that regarding all breast cancer cases the
frequency of ER-positivity decreased with gain of tumour size. In detail,
the frequency of luminal A breast cancer decreased, whereas the number
of luminal B breast cancers remained constant. A constant increase of
the frequency of basal, HER2-driven and triple negative breast cancers
could be seen. Only in 1 out of 865 breast cancer cases a grade 1 and a
grade 3 invasive cancer component within the same breast cancer was
detectable. In 2 cases a ductal invasive grade 1 carcinoma was associated
with a poorly-differentiated DCIS. The frequency of cylinder cell lesions
was evenly distributed between ductal invasive grade 3 carcinomas, irrespectively
of the ER-status.
Conclusions. Our results show that a morphological recognizable “progression
through grade” is a very rare event in the natural course of invasive
breast cancer, including luminal breast cancer. If a progression
through grade occurs, this step is more likely located on the stage of ductal
carcinoma in situ or other suspected precursor lesions.
SA-P-013
Identification of a tissue-based protein signature associated
with triple negative breast cancer by imaging mass spectrometry
(MALDI Imaging)
C . Schöne1 , S . Rauser1 , S . Englert1 , S . Artmeier2 , K . Schulenburg2 , B . Balluff1 , M .
Elsner1 , S . Maier1 , S . Meding1 , M . Schmitt2 , H . Höfler3 , A . Walch1 1 2 Helmholtz Zentrum Munich, Institute of Pathology, Neuherberg, Klinikum
rechts der Isar of the Technische Universität München, Klinische Forschergruppe
of the Frauenklinik, Munich, 3Technische Universität München,
Institute of Pathology, Munich
Aims. The goal of this study is the identification of a tissue-based protein
signature associated with triple negative breast cancer for the purpose of
reliable classification and to identify potential new biomarkers.
Methods. A collective of 25 frozen triple negative and 44 non-triple negative
breast cancer samples was analysed in this study. Protein signatures
of the tissue samples were generated using MALDI Imaging with
a lateral resolution of 70 µM and a mass range of 2,500 to 25,000 Da.
Afterwards the cases were separated into a discovery set, containing 15
triple negative and 15 non-triple negative breast cancer cases, and a validation
set composed of the other cases. The mass spectra of the discovery
set were compared to each other to identify significantly differentially
expressed proteins. The resulting protein signature was then tested on
the validation set using different classification algorithms (divisive hierarchical
clustering, random forest or support vector machine).
Results. We were able to identify a signature composed of 10 different
proteins that could differentiate between triple negative and other breast
cancer samples with an accuracy of over 80%. Three of these proteins
were already encountered in a study dealing with HER2-overexpressing
breast cancer and are of special interest for identification.
Conclusions. MALDI Imaging made it possible to identify protein signatures
associated with triple negative breast cancer. The protein signature
may have potential for classification approaches and contains proteins
that may be of interest as potential biomarkers.
SA-P-014
Expression of RAD23B in invasive breast cancer.
Immunohistochemical study using tissue microarrays
K . Friedrich1 , A . Linge2 , F . Goerl1 , G . Baretton1 1University Hospital “Carl Gustav Carus” Dresden, Institute of Pathology,
Dresden, 2National Institute for Cellular Biotechnology, Dublin, Ireland
Aims. RAD23B is part of the nucleotide excision repair complex (NER)
and involved in repair of DNA damage caused by UV light exposure and
the chemotherapeutic drug cisplatin. The role of RAD23B expression in
invasive breast cancer is still unclear. Thus, the purpose of the study was
to analyse the RAD23B expression in correlation to clinicopathological
characteristics, proliferation and outcome of patients.
Methods. The expression of RAD23B, Ki67, HER-2/neu, estrogen and
progesterone receptor was analysed in 164 formalin fixed, paraffinembedded
specimens of invasive breast carcinoma using tissue microarrays.
All staining results were scored semiquantitatively. The mitotic
count was performed on H&E sections as was histopathological grading
Der Pathologe · Supplement 1 · 2012 |
143

Abstracts
according to Elston & Ellis. The statistical analysis was done by Chisquared
test, t-Test according to student, Kaplan-Meier estimation and
multivariate discriminant analysis.
Results. Nuclear expression of RAD23B was observed in all analysed
cases, ranging from 5–90% of tumor cells with different intensities.
RAD23B expression was correlated with age, histopathological grade
and mitotic activity in univariate analyses. Patients with a disease manifestation
after the age of fifty showed a lower RAD23B expression than
younger patients. A histopathological grade 1 or 2 was associated with a
higher RAD23B expression. The mitotic activity was lower in cases with
high RAD23B expression. The mitotic activity was lower in cases with
high RAD23B expression than in cases with low RAD23B expression.
The multivariate analysis revealed mitotic activity and Ki67 expression
as significant markers. There was no correlation between RAD23B expression
and the other clinicopathological markers or the outcome of
disease.
Conclusions. The correlation of RAD23B expression to proliferation,
especially to mitotic activity may be associated with the function of
RAD23B and the binding of RAD23, XPC and Centrin 2 in DNA damage
recognition complex. Its role in repair of cisplatin-damaged DNA
suggests that RAD23B may be used as a predictive marker for response
to cisplatin based chemotherapy.
SA-P-015
Up-regulation of Kindlin-2 promotes progression of human
breast cancer cells by increasing their proliferation, drug resistance,
genomic instability, and tumorigenesis
W .-g . Fang1 , T . Zhao1 , H .-q . Zhang1 1Peking University, Health Science Center, Beijing, China
Aims. Kindlin-2 has been confirmed as an essential element of bidirectional
integrin signaling. In recent years, the relationship between Kindlin-2
expression and cancers has been a focus of interest. Our previous
studies have shown that Kindlin-2 expression was up-regulated in several
types of human cancers, and a strong correlation between Kindlin-2
expression and clinical outcome of breast cancer patients was found.
However, the functional role of Kindlin-2 in breast cancer has not been
studied. This study was designed to investigate the role of Kindlin-2 in
the progression of human breast cancer cells.
Methods. Firstly, Kindlin-2 expression at protein level was detected by
Western blot in several breast cancer cell lines. Two luminal-like breast
cancer cell lines, MCF-7 and T47D, expressed low level of Kindlin-2.
Two basal-like breast cancer cell lines, MDA-MB-231 and HS578T, expressed
moderate levels of the protein. Then, Kindlin-2 gene was overexpressed
by transfected into MCF-7 cells. In comparison, short hairpin
RNA (ShRNA)-mediated knockdown of Kindlin-2 was performed in
HS578T cells. Vector controls were also done in the same cell lines. Ki67
Li, FCM cell cycle, anchorage-independent colony formation (in vitro
tumorigenesis), and in vivo tumorigenesis in NOD/SCID mice were observed.
Apoptotic cells were labeled by fluorescent annexin V assay and
quantified by FACS. Array CGH analysis and spectral karyotyping were
performed to detect the genomic instability of these cells.
Results. The growth rate of Kindlin-2-transfected MCF-7 cells was much
quicker than that of the controls. The proportion of G2-M phase cells,
clone formation and tumorigenicity were significantly higher than these
of the controls. The change of Kindlin-2-ShRNA transfected cells was
just the reverse. Moreover, Up-regulation of Kindlin-2 can also reduce
the rate of apoptosis induced by the chemotherapy drugs, and these cells
showed much more genomic instability compared with the controls.
Conclusions. These findings suggested that up-regulation of Kindlin-2
promotes the progression of human breast cancer cells by increasing
their proliferation, drug resistance, genomic instability, and tumorigenesis.
144 | Der Pathologe · Supplement 1 · 2012
SA-P-016
GPR30: a predictive marker for Tamoxifen resistance in breast
cancer
T . Kalinski1 , A . Roessner2 , S .-D . Costa2 , A . Ignatov2 1Otto-von-Guericke-University/Department of Pathology, Magdeburg,
2 Magdeburg
Aims. Tamoxifen is the gold standard in the therapy of hormone-dependent
breast cancer. However, the development of Tamoxifen resistance is
a frequent problem in Tamoxifen-responsive tumors during treatment.
The aim was to investigate the role of the new estrogen receptor GPR30
in the development of Tamoxifen resistance.
Methods. Mechanisms of Tamoxifen resistance were investigated in Tamoxifen-resistant
breast cancer cells and wild type cells. The expression
of GPR30 was further analyzed in breast cancer specimens and correlated
with clinical data.
Results. The results proved the important role of GPR30 in the development
of Tamoxifen resistance in breast cancer. GPR30 expression in
breast cancer specimens was associated with Tamoxifen resistance and
negatively correlated with relapse free survival in patients treated with
Tamoxifen.
Conclusions. GPR30 plays an important role in the development of Tamoxifen
resistance in breast cancer cells. GPR30 expression is a predictive
marker for the development of Tamoxifen resistance in breast cancer
specimens.
SA-P-017
CD34+ fibrocytes in the stroma of ductal carcinoma in situ (DCIS)
of the breast
P .J . Barth1 , F . Wötzel1 1University Hospital Münster, Institute of Pathology, Münster
Aims. Im Stroma normalen Brustdrüsengewebes finden sich überwiegend
CD34+-Fibrozyten, die eine große Rolle in der Matrixsynthese und
bei der Aufrechterhaltung der Integrität des mammären Stromas spielen.
Zudem spielen CD34+ Fibrozyten eine Rolle als Antigen präsentierende
Zellen. Das Stroma invasiver duktaler Karzinome zeigt einen kompletten
Verlust der stromalen CD34-Expression und einen Phänotypwechsel
der Stromazellen von CD34+SMA−Fibrozyten zu CD34−SMA+-Myofibroblasten.
Bisher wurden keine Studien zur CD34 Expression im Stroma
von DCIS durchgeführt.
Methods. Wir untersuchten DCIS unterschiedlichen Kernmalignitätsgrades
immunhistochemisch bezüglich der stromalen Expression von
CD34, SMA (Glattmuskel-Aktin) und SMM (Glattmuskel-Myosin). Es
wurden nur DCIS untersucht, die nicht mit einem invasiven Karzinom
assoziiert waren. In jedem Fall lag tumorfreies Brustdrüsengewebe zum
Vergleich vor.
Results. Tumorfreies Brustdrüsengewebe zeigte periduktal und periazinär
dicht gelagerte CD34+ Fibrozyten mit bipolaren zarten Zytoplasmafortsätzen.
SMA+ Stromazellen wurden in tumorfreiem, normalem
Brustdrüsengewebe nicht beobachtet. DCIS niedrigen und mittleren
Kernmalignitätsgrades zeigten eine erhaltene Population von CD34+
Fibrozyten, SMA+ Zellen wurden im Stroma nicht beobachtet. DCIS
hohen Kernmalignitätsgrades zeigten einen Verlust periduktaler CD34+
Fibrozyten; an ihrer Stelle zeigten sich CD34-SMA+ Myofibroblasten,
die konzentrisch um die vom DCIS befallenen Ductus angeordnet waren.
Conclusions. Diese Untersuchung zeigt, dass es bei DCIS hohen Kernmalignitätsgrades
zu Veränderungen des Stromas kommt, die auch bei
invasiven Karzinomen gefunden wurden. Der Verlust der CD34+ Fibrozyten
führt zu einer Störung der Integrität des Stromas, die Voraussetzung
für die Entstehung eines invasiven Karzinoms sein kann. Zudem
kann der Verlust der CD34+ Fibrozyten, bei denen es sich um Antigen
präsentierende Zellen handelt, zu einer Störung der gegen Tumorzellen
gerichteten Immunkontrolle des Organismus führen. Beides sind

Mechanismen, die eine Progression des DCIS zum invasiven duktalen
Mammakarzinom begünstigen.
SA-P-018
Elevated expression of LSD1 (Lysin-specific demethylase 1) during
tumour progression from pre-invasive to invasive ductal carcinoma
of the breast
N . Bektas Serce1 , A . Gnatzy1 , S . Steiner 1 , J . Kirfel1 , R . Büttner2 1 2 University of Bonn, Institute of Pathology, Bonn, University of Cologne,
Institute of Pathology, Köln
Aims. Lysin-specific demethylase 1 (LSD1) is a nuclear protein which
belongs to the aminooxidase enzymes playing an important role in
controlling gene expression. It has also been found highly expressed in
several human malignancies including breast carcinoma. Our aim was
to detect LSD1 expression also in pre-invasive neoplasias of the breast.
In the current study we therefore analyzed LSD1 protein expression in
ductal carcinoma in situ (DCIS) in comparison to invasive ductal breast
cancer (IDC).
Methods. Using immunhistochemistry we systematically analyzed LSD1
expression in low grade DCIS (n=27), intermediate grade DCIS (n=30),
high grade DCIS (n=31) and in invasive ductal breast carcinoma (n=32).
SPSS version 18.0 was used for statistical analysis.
Results. LSD1 was differentially expressed in DCIS and invasive ductal
breast cancer. Interestingly, LSD1 was significantly overexpressed in
high grade DCIS versus low grade DCIS. Differences in LSD1 expression
levels were also statistically significant between low/intermediate DCIS
and invasive ductal breast carcinoma.
Conclusions. LSD1 is also expressed in pre-invasive neoplasias of the breast.
Additionally, there is a gradual increase of LSD1 expression within
tumour progression from pre-invasive DCIS to invasive ductal breast
carcinoma. Therefore upregulation of LSD1 may be an early tumour promoting
event.
SA-P-019
Female adnexal tumor of probable Wolffian origin – case report
and review of literature for epidemiology, course of disease and
differential diagnosis
K . Friedrich1 , M . Toma 1 , J . Wienold2 , G . Baretton1 1University Hospital “Carl Gustav Carus” Dresden, Institute of Pathology,
Dresden, 2Hospital Weißeritztal Kliniken Freital Dippoldiswalde, Deparment
of Gynecology and Obstetrics, Freital
Aims. Clinical history: 35-year-old female with lower abdominal pain on
laparoscopy showed a hematosalpinx and a tumor close to the right fallopian
tube, which was removed laparoscopically. The following laparoscopic
staging did not revealed any further tumors.
Methods. Pathology: Gross examination showed a tumor (7 cm diameter)
with close contact to the fallopian tube, partially covered by serosa
with nodular and focally light gray, glistening pale yellow sectioned surface.
Microscopically, the tumor showed variable histological architecture
with solid, tubular and sieve-like pattern formed by low cuboidal,
attenuated or spindle-shaped cells without atypia and only few mitoses.
The tumor cells expressed cytokeratin 8/18 and 19, CD10, CD99, vimentin
and – at least focally – inhibin and calretinin. EMA, estrogen-, progesterone
receptor, cytokeratin 7 and 20, CD34 and S100 were not detectable.
Based on the histomorphology and the immunohistochemical
expression profile, a female adnexal tumor of probable Wolffian origin
(FATWO) was diagnosed.
Results. Epidemiology, course of disease and differential diagnosis: FAT-
WO are very rare tumors of female adnexal region. A total of 72 cases
have been thus far documented in the literature. The age of reported
patients ranged from 15 to 83 years, most patients are between 40 and
45 years old. Remnants of the Wolffian duct, especially the rete ovarii,
are thought to be the origin of this tumor. The broad ligament is the most
frequent location, but the tumor may also occur in the serosa of the fallopian
tube, the ovary, the mesoalpinx, retroperitoneum and peritoneum.
Conclusions. Most cases are benign, but ten of the reported patients developed
local recurrences, lung or liver metastases. Three patients died
of their tumor. Atypia and a high proliferation activity may predict an
aggressive behaviour. But tumors without these criteria showed recurrences,
too. The differential diagnoses are sex-cord stroma tumors (Sertoli-Leydig
cell tumors, granulosa cell tumors), endometroid adenocarcinomas
(of the fallopian tube), adenomatoid tumors, mesotheliomas
and metastases.
Poster: Gynäkopathologie und Mammapathologie II
SA-P-020
T lymphocytic infiltration in serous ovarian carcinoma:
expression and survival analysis
S . Scheil-Bertram1 , H .-C . Bösmüller2 , A . du Bois3 , F . Heitz3 , P . Harter3 ,
M . Oppitz1 , N . Ewald-Riegler4 , R . Hils4 , A . Fisseler-Eckhoff 1
1 2 Institute of Pathology &Cytology, Wiesbaden, Institute of Pathology, Linz,
Austria, 3Dept Gynecology & Gyn . Oncology, Essen, 4Clinic for Gynecology &
Gyn . Oncology, Wiesbaden
Aims. The lymphocytic infiltration of carcinomas should be a prognostic
marker of antitumoral immunoreactivity and be associated with prolonged
overall survival in ovarian carcinomas. Using CD3 and CD8 antibodies,
we analysed immunreactive T-lymphocyts in a cohort of 78 serous
ovarian carcinoma (OC), and their correlation with patients’ survival.
Methods. We used the CD3 (clone SP7) and CD8 (clone C8/144B) antibodies
(both NeoMarkers). Immunohistochemistry was performed on
multi tissue microarrays (78 patients; median age at diagnosis 64 years).
The stromal and intraepithelial T-cell (CD3 and CD8) density was
quantified, counting the average number of cells per high power field
(HPF=400x) and reviewing a total of 10 HPF for each microarray. The
immunoreactivity was analyzed in a double blind fashion by two pathologists.
Results. The survival of patients with a CD3/CD8 ratio above 1 or 2 respectively
was significantly shorter than with a CD3/CD8 ratio below 1
(17.3 or 15.1 month versus 22.2 month; p=0.0154; hazard ratio:

Abstracts
SA-P-021
The G-protein coupled estrogen receptor 1 (GPER) is differentially
expressed in healthy human ovaries, accompanies progression
into non malignant ovarian diseases, and predicts prognosis in
ovarian cancer patients
S . Heublein 1 , M . Lenhard 2 , J . Schöpfer 3 , C . Kuhn 1 , K . Friese 1 , A . Makrigiannakis
4 , U . Jeschke 1 , D . Mayr 5
1 Ludwig-Maximilians-University of Munich – Campus Innenstadt, Department
of Gynecology and Obstetrics, Munich, 2 Ludwig-Maximilians-University
of Munich, Department of Gynecology and Obstetrics – Campus Grosshadern,
Munich, 3 Ludwig-Maximilians-University of Munich, Department of
Legal Medicine, Munich, 4 University of Crete, Department of Obstetrics and
Gynaecology, Heraklion, Greece, 5 Ludwig-Maximilians-University of Munich,
Department of Pathology, Munich
Aims. In the ovary deregulation of estrogen signalling is tightly linked
to impaired fertility as well as to benign and malignant ovarian diseases.
However several of these estrogen mediated effects are clearly independent
of the classical DNA binding estrogen receptors. Thus to understand
which role the G-protein coupled estrogen receptor 1 (GPER)
plays within these processes might define implications for estrogen or
anti-estrogen based therapies. Therefore we examined GPER in healthy
human ovaries, human folliculogenesis, benign ovarian diseases as well
as in epithelial ovarian cancer (EOC) specimens in a large patient cohort
(n=282).
Methods. Immunohistochemistry, double immune fluorescence and
TaqMan® real time PCR were employed to determine GPER expression.
Ovaries of 32 pre- and 10 postmenopausal women were compared to
84 women affected by follicle cysts (n=14), serous (n=16) and mucinous
(n=18) cystadenofibroma, endometriosis (n=26) or reactive inflammatory
diseases (n=10). We further evaluated how GPER correlates with grading,
FIGO stage and prognosis in 156 EOC patients. Finally different
ovarian cell lines were used to test these interactions functionally.
Results. In healthy follicles GPER is abundantly present in oocytes and
theca cells followed by granulosa cells. In contrast to controls a pronounced
stroma expression of GPER was observed in endometriosis and inflamed
tissue which stresses its immune responsiveness to glucocorticoids
and galectin found in vitro. GPER is highly expressed in the ovarian
outer surface epithelium and down regulated in some entities of benign
neoplasias. In EOC low tumour grade and advanced prognosis were
predicted by elevated GPER. Furthermore here GPER showed positive
correlation to gonadotropine receptors, Galectin-3 and downregulation
of p53.
Conclusions. GPER is detectable in highly specialized cells throughout
human folliculogenesis and thus might be functionally involved in follicle
maturation. Progression into benign and malignant ovarian diseases
is accompanied by GPER, which we found is regulated by immune modulators
in different cell lines. Thus we conclude that GPER is a valuable
tool to further investigate human ovarian (patho-)physiology and that it
might be interesting for therapeutic interventions in EOC patients.
146 | Der Pathologe · Supplement 1 · 2012
SA-P-022
PDGF-C expression in ovarian cancer
A .-K . Zimmermann1 , A . Noske1 , H . Li2 , U . Ericsson2 , A .M . Neville3 , R . Caduff1 , H .
Moch1 , D . Fink4 , G . Kristiansen5 1University Hospital Zurich, Department of Surgical Pathology, Zürich,
Switzerland, 2Ludwig Institute for Cancer Research, Stockholm Branch,
Sweden, 3Ludwig Institute for Cancer Research, University of Oxford Branch,
United Kingdom, 4University Hospital Zurich, Department of Obstetrics
and Gynecology, Zürich, Switzerland, 5University Hospital Bonn, Institute of
Surgical Pathology
Aims. Platelet-derived growth factors (PDGF) and its receptors (PDGFR)
promote tumor growth and angiogenesis and are therefore interesting
targets for anticancer therapies. So far, little is known about the expression
and prognostic role of PDGF-C in ovarian cancer.
Methods. We investigated a cohort of 131 invasive ovarian carcinomas
and 31 borderline tumors for PDGF-C expression by immunohistochemistry
(using a specific antibody against this ligand) and compared expression
data with clinicopathological findings and patient overall survival.
Results. We observed an epithelial and stromal expression of PDGF-C
in 64 (49%) and 62 (48%), respectively of the ovarian carcinomas but did
not find any association with clinicopathological features or overall survival.
In a subgroup analysis of serous carcinomas, we observed a trend
of high PDGF-C levels with higher tumor grade (p=0.047). An epithelial
and stromal expression was also observed in borderline tumors in 65%
(22/34) and 26% (8/31), respectively.
Conclusions. This study shows that PDGF-C is overexpressed in a subset
of ovarian carcinomas but not associated with traditional prognostic
pathological factors or patient survival. However, PDGF-C has potential
as a therapeutic target and in context with the literature it might have a
predictive function in anti-VEGF-treatment.
SA-P-023
AGO VISION-1: Improved utilisation of resources and optimization
of quality of care through internet-based second opinion pathology
– standardized in an optimized ovarian cancer network
S . Kommoss1 , J . Pfisterer2 , J . Diebold3 , S . Lax4 , D . Schmidt5 , A . Staebler6 ,
A . du Bois7 , F . Kommoss5 1University of British Columbia, Department of Pathology, Vancouver,
Canada, 2Klinikum Solingen, Dept of Gynecology, Solingen, 3Luzerner Kantonsspital, Institute of Pathology, Luzern, Switzerland, 4LKH Graz West,
Institute of Pathology, Graz, Austria, 5Institute of Pathology, A2 , 2 , Mannheim,
6 7 University of Tübingen, Institute of Pathology, Tübingen, Kliniken Essen
Mitte (KEM), Dept Gynecology & Gyn . Oncology
Aims. Based on the literature as well as on own results from a prospective
study one may assume that a considerable number of patients in clinical
trials of ovarian carcinoma have diagnoses in conflict with inclusion
criteria. Subsequently clinical trials may be biased through unintended
disregarding of histological inclusion criteria. To avoid the latter limitation,
specialized pathology review should become standard procedure
in study protocols prior to randomization. To facilitate specialized second
opinion pathology prior to randomization and/or treatment decisions
the process of pathological case review has to be completed within
a few working days. We hypothesize that our new, internet-based high
throughput infrastructure will be capable of providing specialized second
opinion pathology within 10 working days.
Methods. Patients scheduled for the AGO OVAR17 trial have to be registered
for a central pathology review, which has to be performed through
the translational subproject termed “AGO VISION-1”. Having provided
written informed consent, the patients will be registered for pathology
review at a central office. Original slides will then be requested from the
outside pathologists in order to be scanned and uploaded to a secured

internet server. A network of internationally recognized gynecological
pathologists is connected to the server through a custom-designed software
platform. If necessary, immunohistochemistry is available through
a collaborating pathology lab.
Results. The new internet-based high throughput infrastructure has
been set up successfully. Five gynecopathologists from Austria, Switzerland
and Germany will provide specialized review of all cases scheduled
for inclusion in the AGO OVAR17 trial for all study centers of the AGO
study group. A centralized office plays a key role in the logistics of the
complex course of action in central pathology review.
Conclusions. Preliminary data suggest that the use of a new internet-based
infrastructure may allow for specialized case review prior to patient
randomization in the AGO OVAR17 trial. This approach might not only
help to avoid disregarding of clinicopathologic inclusion criteria but also
to further improve the quality of patient care through minimization of
overtreatment with chemotherapy of patients with ovarian borderline
tumors and inadequate treatment of patients with ovarian metastases.
SA-P-024
Metastatic sebaceous ovarian cancer
H . Geddert1 , A . Dimmler1 , M . Rauchholz2 , O . Tomé2 , G . Faller1 1 2 St . Vincent Hospital, Institute of Pathology, Karlsruhe, St . Vincent Hospital,
Department of Gynecology and Obstetrics, Karlsruhe
Aims. A 47-year-old patient complained of abdominal tenderness and
pain.
Methods. Ultrasound as well as thoracal, abdominal and pelvic computer
tomography demonstrated a heterogenic tumor of the left ovary measuring
14 cm. A singular lesion suspected of metastasis was identified as
well in the liver and the lung. A core biopsy from the pulmonary lesion
was collected preoperatively. Laparotomy with total abdominal hysterectomy,
bilateral salpingo-oophorectomy, infragastric omentectomy
and liver lesion biopsy was performed. During operation, no macroscopic
sign of peritoneal carcinosis was described.
Results. Histological examination of the ovarian tumor proved a sebaceous
carcinoma. No features of a teratoma were identified in the
completely sampled mass. Hepatic and pulmonary metastases were diagnosed
in the core biopsies. Unfortunately, the patient showed a rapid
progress postoperatively with increasing number and size of liver and
lung metastasis. Therefore, the patient was given chemotherapy with
Carboplatin and Paclitaxel.
Conclusions. To our best knowledge, this is the first reported case of a
sebaceous ovarian carcinoma with synchronous hepatic and lung metastasis.
SA-P-025
Histone-deacetylase-1 expression and intratumoral lymphocyte
density are prognostic parameters for predicting overall survival
in serous ovarian carcinoma
H . Bösmüller1 , J . Peper2 , B . Gückel3 , T . Fehm3 , C . Bachmann3 , D . Wallwiener3 , S .
Stefanovic2 , D . Pham4 , F . Fend4 , A . Staebler4 1Krankenhaus Barmherzige Schwestern, Dept . of Pathology, Linz, Austria,
2 3 University of Tübingen, Dept . of Immunology, Tübingen, University of
Tübingen, Dept . of Gynecology, Tübingen, 4University of Tübingen, Dept . of
Pathology, Tübingen
Aims. High level expression of histone deacetylases 1, 2 and 3 (HDAC) are
associated with progressive disease and poor prognosis in high-grade serous
ovarian carcinoma (SOC) and also are markers for potential treatment
with histone deacetylase inhibitors. Intratumoral lymphocyte density
is a parameter of antitumoral immunoreactivity and is associated
with prolonged overall survival. Since we recently identified HDAC1/2
by HLA ligandome analysis as HLA class I associated antigen in ovarian
cancer, we investigated HDAC 1 expression and T-cell density in a large
series of high-grade serous ovarian carcinomas.
Methods. Primary tumors of 141 patients with SOC in FIGO Stage II–IV
were studied by immunohistochemistry on tissue microarrays containing
6 cores per case. HDAC1 expression was assessed by applying the
semiquantative IRS score. The stromal and intraepithelial T-cell (CD3
and CD8) density was quantified, counting the average number of cells
per high power field (HPF=400x) and reviewing a total of 10 HPF for
each core.
Results. Strong nuclear HDAC1 expression (score 8, 9, 12) was associated
with poor overall survival (OS, median 25 vs. 40 months, p=0.008), but
not disease free survival (DFS median 23 vs. 27 months, p=0.378). Increased
intraepithelial CD3+ lymphocytes (p=0.012) and stromal CD8+ lymphocytes
(p=0.026) were associated with favourable OS, whereas DFS
was only significantly associated with stromal CD8 density (p=0.019).
Strong HDAC 1 expression correlated with increased cytotoxic lymphocyte
density (p=0.021, Pearson-correlation).
Conclusions. Our study of a large collective of advanced SOC confirms
the prognostic relevance of intratumoral lymphocyte density as well
as high HDAC1 expression. The observed correlation between HDAC1
overexpression and intraepithelial CD8+ T-cell density points to a
potential role of HDAC1 as a tumor specific antigen in SOC.
SA-P-026
Effects of MDM2 and MDMX splice variants on p53 pathway in
ovarian carcinomas
S . Hammer1 , S . Pelka1 , A . Wolf1 , A . Böhnke1 , S . Mahner2 , G . Ott3 , S . Hauptmann4
, F . Bartel1 1 2 University of Halle-Wittenberg, Institute of Pathology, Halle/Saale, University
Medical Center Hamburg-Eppendorf, Department of Gynecology,
Hamburg, 3Robert-Bosch-Hospital, Institute of Clinical Pathology, Stuttgart,
4Insitute of Pathology Allgäu-Oberschwaben, Wangen
Aims. Many human tumors show inactivation of p53 pathway e.g. due
to overexpression of the negative regulators MDM2 and MDMX. Both,
MDM2 and MDMX, are spliced alternatively and aberrantly in many tumor
cell lines. So far there are no studies about the expression and effects
of MDM2/X splice variants in ovarian carcinomas.
Methods. Therefore, we screened 33 frozen ovarian carcinoma samples
for the occurrence of MDM2/X splice variants. We detected beside the
known variants MDM2-B, MDMX-S and MDMX-211 new variants such
as MDM2-p53NLS, MDM2-ARZ, MDMX-A, MDMX-Z and MDMX-
ZR containing different domains. Subsequently, the ovarian tumor cell
lines OAW-42 and ES-2 were transfected with these variants and treated
with both cisplatin and taxol in order to analyze their effects on the p53
pathway after DNA damage.
Results. Overexpression of MDM2-p53 and MDM2-p53NLS resulted in
stabilization of p53 but not in induction of p21 expression. In contrast
MDM2-AZR and-B did not change p53 level but slightly increased p21
expression after cisplatin. Both MDMX-A and MDMX-211 stabilize
MDMX which caused a transcriptional inactivation of p53 after cisplatin
treatment.
Conclusions. Our results clearly show, that MDM2 and MDMX splice variants
affect p53 pathway which may had an impact on tumor formation
and/or chemoresistance.
Der Pathologe · Supplement 1 · 2012 |
147

Abstracts
SA-P-027
Primary serous peritoneal carcinoma (PPC) with and without
serous tubal in-situ carcinoma (STIC)
L .-C . Horn 1 , K . Leonhardt 2 , K . Kellner 1 , R . Scherling 2 , J . Einenkel 2
1 University of Leipzig, Institute of Pathology, Leipzig, 2 University of Leipzig,
Department of Obstetrics and Gynecology (Institute of Trier), Leipzig
Aims. Evaluating the frequency of serous tubal in situ carcinoma (STIC)
in cases of primary peritoneal carcinoma.
Methods. The present study evaluates immunohistochemically (Ki-67
and p53-staining) the presence of STIC in completely embedded Fallopian
tubes of 35 consecutive cases meeting the clinicopathologic criteria of
primary high-grade serous carcinoma.
Results. p53 signature was seen in four cases (11%) and STIC in seven patients
(20%). All STIC occurred at the fimbriated end of the Fallopian tube
and in one case a bilateral involvement was seen. These precursor lesions
were missed during the initial routine screening. In two cases repeated
staining for p53 was negative.
Conclusions. STIC and p53-signature as precursor lesions of pelvic serous
cancer are seen in some but not all cases of primary serous peritoneal
cancer. Further studies are required to identify the source of serous cancer
in cases without STIC-lesions.
SA-P-028
KPNA2 protein expression is an independent adverse predictor of
survival in patients with endometrial carcinomas
K . Ikenberg1 , A . Noske1 , R . Caduff1 , A . Dellas2 , H . Moch1 , P .J . Wild1 1University Hospital Zurich, Institute of Surgical Pathology, Zürich, Switzerland,
2University of Basel, Institute of Pathology, Basel, Switzerland
Aims. To analyze rates of expression of karyopherin alpha 2 (KPNA2),
an important mediator of nucleocytoplasmic transport, in different endometrial
cancer subtypes, and to evaluate its prognostic properties for
patients with primary endometrial cancer.
Methods. Tissue microarrays (TMA) contained 527 formalin-fixed, paraffin-embedded
endometrial cancer tissue cores from two different
institutes of pathology. TMAs were stained immunohistochemically for
KPNA2 and p53.
Results. KPNA2 expression was significantly upregulated in carcinomas
of the endometrium and was significantly associated with higher tumor
grade, higher FIGO stage, and overexpression of p53. KPNA2 expression
was not associated with different subtypes of endometrial carcinomas.
Positive nuclear KPNA2 immunoreactivity in at least 10% of nuclei was
identified as a novel predictor of survival, and was independent of the
well-established prognostic factors age, grade, FIGO stage, histological
type, and p53 immunoreactivity in Cox regression analyses (hazard ratio=1.7,
95% CI 1.13–2.56, p=0.01).
Conclusions. KPNA2 is a novel independent prognostic marker for survival
after hysterectomy. This may allow identifying patients who need
more aggressive treatment.
SA-P-029
Loss of ARID1A/BAF250a expression in endometriosis – a new
molecular mechanism for transformation into cancer?
A . Noske1 , N . Samartzis2 , M . Rechsteiner1 , H . Moch1 , P . Imesch2 1University Hospital Zurich, Institute of Pathology, Zürich, Switzerland,
2University Hospital Zurich, Dept . of Gynecology, Zürich, Switzerland
Aims. Mutations of the tumor suppressor gene ARID1A are frequently
found in endometriosis-associated ovarian carcinomas, and correlate
with expression loss of the coding protein BAF250a. We hypothesize that
deficient ARID1A/BAF250a expression may contribute in transformation
of endometriosis into cancer.
148 | Der Pathologe · Supplement 1 · 2012
Methods. We evaluated ARID1A/BAF250a protein expression in 74 endometriosis,
and 30 eutopic endometrium samples by immunohistochemistry.
Further, an analysis of ARID1A/BAF250a and p53 expression in
a cohort of 129 primary ovarian carcinomas was performed. To classify
the ovarian carcinomas in type I and type II, the mutational status of p53,
KRAS, and BRAF will be determined by deep-sequencing technology.
Results. There was a loss of ARID1A/BAF250a expression in three endometriomas
and one deep-infiltrating endometriosis, but in none of
the peritoneal endometriosis and eutopic endometrium samples. Lack
of expression was found in 22.5% of the ovarian carcinomas and was
significantly associated with the endometrioid histological subtype and
wild-type p53 protein.
Conclusions. The deficiency of ARID1A/BAF250a expression in few endometriosis
lesions may indicate an early event in malignant transformation
of endometriosis. In context with the literature, the data support
an association of ARID1A and p53 in ovarian cancer.
SA-P-030
Detection of micrometastasis in paraaortic lymph nodes in patients
with carcinoma of the uterine cervix after negative frozen
section analysis
L .-C . Horn1 , K . Kellner1 , R . Scherling2 , M . Höckel2 , J . Einenkel2 1 2 University of Leipzig, Institute of Pathology, Leipzig, University of Leipzig,
Department of Obstetrics and Gynecology (Institute of Trier), Leipzig
Aims. Previous studies considered the presence of micrometastases
(MM) in pelvic lymph nodes as clinically relevant prognostic indicator
and reported a 2.4 to 3.2 greater risk for recurrent disease and dead of
the disease when compared to nodal negative patients. There is limited
information about the value of (immunohistochemical) ultrastaging in
negative para-aortic lymph nodes (PAN).
Methods. Frozen section analysis was performed in all cases with PA-
LNE because of clinical requirements. From all FS-blocks, routinely
three step sections were performed. After FS-examination nodes were
examined by one H&E-stained slide. All nodes without metastatic disease
after frozen section and permanent section examination were subject
of the present study. 43 patients and 418 PAN were enrolled and immunohistochemical
staining using two cytokeratin-cocktail antibodies (AE
1/AE 3 and KL-1) was performed. MM were defined as foci of tumor cells
ranging from 0.2 mm to no more than 2 mm in size; isolated tumor cells
(ITC) are defined as single tumor cells or small clusters of cells less than
0.2 mm in size within the subcapsular sinuses of the lymph node.
Results. In one case, one single node showed micrometastasis, representing
an incidence of 2.3% of the studied cases and 0.23% of the examined
lymph nodes. In three cases benign endosalpingiosis was seen. The
patient with MM is alive without evidence of disease 96 months after
surgery. ITC were not observed.
Conclusions. The frequency of MM in PAN is very low. There are only limited
data regarding their prognostic impact within the literature. After
careful examination of all removed PAN using H&E-staining (and step
sectioning), immunohistochemical ultrastaging cannot be recommend
for routine use.
SA-P-031
Mismatch repair protein expression of cervical adenocarcinoma
B . Helmchen1 , R . Brand2 , M . Kurrer3 , P . Komminoth1 , H .-P . Sinn2 1Community Hospital Triemli, Dept . of Pathology, Zürich, Switzerland,
2University of Heidelberg, Institute for Pathology, Heidelberg,
3Enge Institute of Pathology, Zürich, Switzerland
Aims. Charakterisierung des immunhistochemischen Expressionsprofils
der Mismatch-Repair-Proteine (MMRP) MLH1, MSH2, MSH6 und
PMS2 an primären zervikalen Adenokarzinomen (ZAK) in Abgrenzung
zu Karzinomen des unteren Uterinsegmentes (UUSK).

Methods. HE-gefärbte Schnittpräparate und pathologische Befunde von
42 archivierten Hysterektomie-Präparaten von Tumorfällen mit der
Diagnose eines ZAK, wurden bezüglich zervikaler Vorläuferläsionen
(ZVL) und Befall des unteren Uterinsegments (UUS) reevaluiert. An
ZVL−/UUS+ Fällen wurde eine PCR-Analyse für HPV-DNA durchgeführt.
Fälle mit ZVL+/− und UUS-, und UUS+/ZVL+ Fälle, sowie
UUS+/ZVL−/HPV+Fälle wurden als primäre ZAK klassifiziert. UUS+/
ZVL−/HPV-Fälle wurden als UUSK reklassifiziert. Repräsentative Tumorschnitte
wurden mittels standardisierter, automatisierter Methoden
für MLH1, MSH2, PMS2 und MSH6 gefärbt. Vollständig fehlende Expression
eines MMRP in Tumorzellen bei positiver interner Kontrolle
im Normalgewebe wurde als Verlust der Proteinexpression gewertet.
Expressionsprofile primärer ZAK und UUSK wurden verglichen und
korreliert (Fisher-Exakt-Test). Zusätzlich wurden alle Tumoren für ER,
Vimentin, CEA und p16 gefärbt.
Results. 37 Fälle wurden als primäre ZAK klassifiziert (37/42; 88.1%),
davon waren 27 UUS−, 8 UUS+/ZVL+ und zwei UUS+/ZVL−/HPV+-
Fälle. Fünf UUS+/ZVL−/HPV−- Fälle (5/42; 11.9%) wurden als UUSK
reklassifiziert. Die MMRP Expression war in sämtlichen primären ZAK
erhalten (37/37; 100%). In vier von fünf UUSK (4/5; 80%) zeigte sich ein
Expressionsverlust von mindestens einem MMRP (p

Abstracts
Methods. Detailed clinical and histopathologic review of a clinical case
and review of the literature using PUBMED.
Results. We report on a 70-year-old woman with FIGO Stage 1A Grade II
(pT1a pN0 L0 V0 R0) endometrial adenocarcinoma who presented 2 years
following total abdominal hysterectomy, bilateral salpingo-oophorectomy
and lymphonodectomy with left radius bone metastasis. A left forearm
amputation was performed. The tumor measured 7.1×6.9×6.5 cm.
The histological diagnosis was a poorly differentiated (G3), endometrioid
endometrial carcinoma. Both carcinomas were oestrogen and progesterone
receptor negative, vimentin, OSCAR and AE1/AE3 positive. Immunohistochemical
stains for S-100, desmin, a-smooth muscle actin and
CD 34 were negative.
Conclusions. This case highlights the rare and unusual presentation of
endometrial cancer. For this reason a review of the literature is also provided
for all cases with evidence of bone metastasis at presentation of the
disease and as a recurrence. Its diagnosis requires immunohistochemistry
and awareness of its possible existence.
Poster: Molekularpathologie I
SA-P-035
Identification of uncommon PIK3CA-mutations in lung cancer by
using pyrosequencing
V . Schildgen1 , J . Lüsebrink 1 , J .D . Appel1 , C . Wübben1 , W . Engel-Riedel1 ,
E . Stoelben1 , O . Schildgen1 , M . Brockmann1 1University Witten/Herdecke, Kliniken der Stadt Köln gGmbH, Köln
Aims. Phospatidylinositol-3-kinases (PI3K) play an important role in various
cell processes. Oncogenic mutations in the PIK3CA gene which codes
for the catalytic subunit have been identified in various malignancies
and activate the PI3K/AKT/mTOR pathway which is a critical driver of
tumorigenesis.
Methods. We tested 41 tumor samples with known KRAS, BRAF, and
EGFR mutation status for the occurrence of mutations in the PIK3CA
gene using a new commercial pyrosequencing kit.
Results. Pyrosequencing revealed 2 mutations (4.9%) in the PIK3CA
gene, one in exon 9 and one in exon 20. Both mutations have not yet been
identified in lung tumor tissue.
Conclusions. The screening of our small patient cohort by pyrosequencing
identified two mutations (4.8%) in PIK3CA, on in exon 9 (Q546H)
and on in exon 20 (M1043T). Both mutations have not yet been described
in lung tumours and seem to be rather uncommon mutations. Future
screening of large patient cohorts with pyrosequencing may contribute
to the detection of more mutations in lung cancer due to the low limit of
detections of this method and may contribute to a better understanding
of the interaction of mutations and tumorigenesis.
SA-P-036
Detection of EML4-ALK fusion oncogenes in NSCLC using a commercial
three-color FISH assay (ZytoLight SPEC TriCheck)
B . Schmitt 1 , H . Schultz1 , F . Stellmacher1 , E . Vollmer 1 , T . Goldmann1 1Borstel Research Center, Clinical & Experimental Pathology, Borstel
Aims. About 5% of non-small cell lung cancers (NSCLC) harbor an inversion
of the short arm of chromosome 2 that causes a rearrangement of
the ALK and EML4 genes. The resulting fusion oncoprotein comprises a
constitutively active tyrosine kinase. Those patients will benefit substantially
from treatment with crizotinib, a Met-/tyrosine kinase inhibitor,
highlighting the need for appropriate tests allowing one to reliably detect
such fusions in biopsy specimens. Existing techniques such as immunohistochemistry,
multiplex PCR, or 2-color FISH using break apart-probes
each possess specific strengths and weaknesses. By design, a novel
150 | Der Pathologe · Supplement 1 · 2012
commercial 3-color FISH assay offers several theoretical advantages.
However, its actual performance in research and diagnostic settings has
not been tested.
Methods. We analyzed formalin-fixed and paraffin embedded (FFPE)
tissue sections (pulmonary adenocarcinomas) and tissue microarrays
(TMA; assembled from adenocarcinomas, squamous and large cell carcinomas)
for EML4-ALK rearrangements by 3-color FISH assay using
ZytoLight SPEC TriCheck probes (Zytovision). Stained slides were evaluated
multiply by pathologists and molecular biologists according to
standardized criteria. Cancers exhibiting a characteristic fluorescence
pattern in at least 15% of tumor cells were taken as EML4-ALK fusion
positive.
Results. High quality signals were obtained in FFPE tissues, in individual
sections as well as in TMAs. Interobserver variability was negligible
for wild type identification. Discrimination between FISH signals from
wild type vs. EML4-ALK fusions in cancer tissue was clear and binary
owing to probe design, and without the problematic intermediate results
observed with break apart-probes in case of a small split. Evaluation was
more laborious and less reproducible, however, when more non-tumor
tissue was interspersed and tumor cells were harder to identify. Assay
hands-on time and total turnover time were less than for multiplex PCR.
Conclusions. Detecting EML4-ALK fusions based on a 3-color FISH assay
using the “ZytoLight SPEC TriCheck”-probes requires specialized
equipment, familiarization of the observer and reliable distinction of
tumor cells from non-tumour cells. The approach does not require assessment
by multiple observers, unlike assays using break-apart probes.
Overall, this approach appears particularly suited for clinical studies and
basic research in that it will also identify rare and putative novel fusion
variants, as well as deletions and amplifications.
SA-P-037
Systematic quantitative cross-validation and content analysis of
the 450k methylation array from Illumina
J . Rößler1 , O . Ammerpohl2 , J . Gutwein2 , U . Lehmann1 1 2 Medical School Hannover, Institute of Pathology, Hannover, University
Hospital Schleswig-Holstein, Institute for Human Genetics, Kiel
Aims. The relationship between the beta-values provided by the newly
released 450k methylation array from Illumina for individual CpG dinucleotides
was compared with quantitative methylation levels obtained
by conventional pyrosequencing. In addition, the representation on the
Illumina array of two groups of genes, which are important in tumour
biology and display extensive aberrations in DNA methylation in cancer
(microRNAs and imprinted loci), was assessed in detail.
Methods. High molecular weight DNA was isolated from histologically
examined 18 fresh-frozen human breast cancer specimens, 4 normal
mammary epithelial fractions and 4 human breast cancer cell lines
(IPH-926, HCC1937, MDA-MB-134, PCM42) using standard procedures.
DNA was bisulfite treated and analyzed on 450k arrays following
the manufacturer’s protocol. For primary data processing and analysis
the software provided by Illumina was employed. These analyses were
complemented and extended by employing the Omics Explorer from
Qlucore and the R package IMA. The beta-values for individual loci were
cross-validated using conventional pyrosequencing of the same DNA
samples independently treated with sodium bisulfite.
Results. The newly released 450k array methylation array from Illumina
shows a high concordance with quantitative pyrosequencing if identical
CpG sites are analysed by the two different methods in cell lines (Spearman
r=0.88, p

Conclusions. The newly released 450k methylation array from Illumina
provides a genome-wide representation of DNA methylation aberrations
in a convenient format with direct quantification of methylation levels
at each individual CpG site. However, the representation of microRNA
genes and imprinted loci is quite uneven and has to be taken into account
during data evaluation and derivation of any conclusion concerning these
two important classes of genes.
SA-P-038
Luminescent conjugated oligothiophenes: novel optical probes
that detect cross beta-sheet conformation of inclusion bodies “in
situ”
V . Mahajan1 , T . Klingstedt2 , R . Simon2 , P . Nilsson2 , A . Thueringer1 , K . Kashofer1 ,
H . Denk1 , K . Zatloukal1 , J . Haybaeck1 1 2 Medical University of Graz, Institute of Pathology, Graz, Austria, Linkoping
University, Sweden
Aims. Mallory-Denk bodies (MDBs) are cellular hallmarks of protein
aggregation diseases such as alcoholic and non-alcoholic steatohepatitis
(ASH and NASH). MDBs are also seen in other liver diseases such as
hepatocellular carcinoma (HCC) and alpha-1-antitrypsin deficiency. Additionally
presence of Intracellular Hyaline bodies (IHBs) is also a common
observation in HCC. Despite of advances in technologies highlighting
protein conformation, structural characterisation of inclusion
bodies remains unresolved. Therefore investigating molecular structure
of the major MDB constituents keratin 8 (K8) and 18 (K18), p62 and ubiquitin
may provide deeper mechanistic insights in MDB/IHB formation.
Methods. Luminescent conjugated oligothiophenes (LCOs), h-HTAA,
p-FTAA and PTAA contain swivelling thiophene backbones whose geometry
modulates fluorescence properties. LCOs were demonstrated to
specifically bind proteins with cross beta sheet conformation. In addition
to the older ones the new generation of LCOs were used for in situ
investigation of conformational changes in MDBs in human and murine
livers.
Results. LCOs demonstrated constant presence of cross beta-sheet conformation
in human MDBs but not in IHBs, alpha-1-antitrypsin and
ground glass inclusions. The spectral signatures collected from MDBs in
ASH, NASH and HCC indicated a similar molecular structure of MDBs
under the various disease conditions. MDBs induced by 3, 5-diethoxycarbonyl-1,
4-dihydrocollidine-feeding of mice revealed h-HTAA and
p-FTAA binding to MDBs in all experimental stages. CHO-K1 cells
transfected with various combinations of SQSTM1/p62, ubi and Krt8/
Krt18 showed that K8 was more prone than K18 to lead to generation
of cross beta-sheets. Aggregates of p62 alone were reproducibly negative
for LCOs. Circular dichroism analysis elucidated intrinsically different
conformational nature of purified K8 and K18 polypeptides.
Conclusions. The comparative higher tendency of K8 to undergo conformational
changes from predominantly Alpha-helical to cross β-sheet
explains its essential role in MDB formation. This elucidates why the absence
of K8 prevents MDB formation whereas its excess facilitates MDB
formation. The nature of keratins to acquire cross beta sheet conformation
appears to be dependent on intrinsic factors but may not be the consequence
of pathological conditions. With PTAA, LCOs may serve as a
multicolour novel diagnostic “in situ” technology that can be applied on
formalin-fixed and paraffin-embedded and fresh frozen tissues.
SA-P-039
Comparison of cobas and HC2 HPV testing in a German routine
laboratory
H . Ikenberg1 , C . Börsch1 , B . Pittel1 , A . Xhaja1 , F . Britz2 , T . Iftner3 1 2 Cytomol, MVZ for Cytology and Molecular Biology, Frankfurt, Roche
Diagnostics, Mannheim, 3University of Tübingen, Institute for Virology,
Tübingen
Aims. Up to now the Digene HC2 test (Qiagen, Hilden) is regarded the
gold standard for human papillomavirus (HPV) DNA testing. Meanwhile
several new test systems for HPV are available, among them the
cobas HPV assay (Roche Diagnostics, Mannheim, Roche) which allows
simultaneous genotyping for HPV 16 and 18. We compared the performance
of the cobas HPV with the HC2 assay in cervical samples collected
with the PreservCyt collection medium (Hologic, Frankfurt).
Methods. 1781 anonymized routine specimens pretested with the HC2
test were available for analysis with cobas HPV. Cases with discrepancies
between the two tests were retested with the Linear Array HPV genotyping
test (LA; Roche). Partially, histologic diagnoses were available.
Results. In 1566 (87.9%) of the cases HPV results were concordant. Of the
215 cases (12.1%) with discrepancies LA results are available in 214 cases.
94 cases were LA-negative: 13 of 105 cobas-pos/HC2-neg and 81 of 99 cobas-neg/HC2-pos
cases. 110 cases were LA-positive: 92 of 105 cobas-pos/
HC2-neg and 18 of 99 cobas-neg/HC2-pos cases. 325 cases with histologically
confirmed CIN2+ were included. In 261 out of 293 cases (89.1%)
where a HC2 result was available this was positive, while 298 out of 318 of
the cases (93.7%) tested with cobas HPV were positive. The rate of HPV
positivity in cytologically normal cases and in HSIL was slightly higher
with cobas HPV, while it was in the same range in ASC-US and in LSIL
cases. With increasing severity of the cytologic findings the rate of HPV
16- and 18 positivity increased proportionally.
Conclusions. In routine specimens from a German commercial laboratory
the cobas HPV test showed similar performance compared to the
HC2 test. Preliminary data point to a potentially higher sensitivity and
specificity with cobas HPV while adding information by HPV 16 and 18
genotyping.
SA-P-040
Comparison of Abbott RealTime and HC2-HPV testing in a routine
laboratory
H . Ikenberg1 , C . Noppen2 , M . Faber1 , A . Xhaja1 , S . Böhm3 1 2 Cytomol, MVZ for Cytology and Molecular Biology, Frankfurt, Viollier AG,
Basel, Switzerland, 3University Hospital Leipzig, Dep . for Gastroenterology
and Rheumatology, Leipzig
Aims. The Digene Hybrid Capture2 test (HC2, Qiagen, Hilden) is the
standard in routine human papillomavirus (HPV) DNA testing. Several
new test systems have become commercially available in the past years,
among them the automated Abbott RealTime-High-Risk-HPV assay
(RealTime-HPV, Abbott Molecular, Wiesbaden). It detects 14 HR-HPV
types and simultaneously differentiates between HPV16, HPV18 and 12
non-HPV16/18 HR types in a single test, while HC2 targets the same HR
types, except HPV66, without typing. RealTime-HPV amplifies human
β-globin in the same reaction. We evaluated RealTime-HPV versus HC2
with cervical specimens from a large German routine laboratory sampled
with the PreservCyt collection medium (Hologic, Frankfurt).
Methods. 505 anonymized routine specimens referred for cytology and
pretested with HC2 were run with RealTime-HPV on the m2000 System
(Abbott). Samples with discordant results between both assays were genotyped
by Linear Array (Roche, Mannheim), targeting 37 HPV genotypes.
Results were correlated with routine histology available for 280 cases
(16.8%

Abstracts
were LA-positive for RealTime-HPV/HC2 targeted HPV types: 27 of 33
RealTime-HPV-pos/HC2-neg and 11 of 40 RealTime-HPV-neg/HC2pos
cases. 35 cases were LA-negative: 6 of 33 RealTime-HPV-pos/HC2neg
and 29 of 40 RealTime-HPV-neg/HC2-pos cases. In 280 histology
confirmed cases overall-agreement between RealTime-HPV and HC2
was 82.5%. Detection rates for CIN2+ and CIN3+ were 90.6% and 90.7%
with RealTime-HPV and 89.3% and 88.3% with HC2, respectively. A high
correlation of HPV16/18 positivity with RealTime-HPV and increasing
histological severity was found.
Conclusions. Clinical performance of RealTime-HPV in routine specimens
was comparable to that of HC2. Discrimination of HPV 16/18 from
other HR HPV types provides additional information for risk stratification.
SA-P-041
FISH analysis does neither reveal genomic ETV1 amplification nor
break-apart in gastrointestinal stromal tumors
E . Wardelmann1 , S . Huss1 , R . Menon2 , F . Göke2 , G . Kristiansen2 , R . Buettner1 , S .
Perner2 1 2 University Hospital Cologne, Institute of Pathology, Köln, University Hospital
Bonn, Institute of Pathology, Bonn
Aims. Gastrointestinal stromal tumors (GISTs) are the most common
mesenchymal tumors of the gastrointestinal tract. Recently the transcription
factor ETV1 was shown to be highly expressed in GISTs both
at transcript and protein levels. ETV1 is a member of ETS family of transcription
factors. The ETS family members share an evolutionary highly
conserved 80-amino-DNA binding domain and individual proteins of
this family are capable of regulating gene promoter activity. Chi et al.
reported that ETV1 is highly expressed in those ICCs, from which GISTs
arise. Taken together, they propose that GISTs originate from ICCs with
high endogenous level of ETV1 when an oncogenic activation via KIT or
PDGFRA mutation occurs. However, they could not detect any genomic
alteration leading to the high ETV1 expression performing FISH analysis
on four GIST samples and two GIST cell lines. The present study was
accomplished to evaluate in a larger cohort whether an ETV1 amplification
or break apart might represent the genomic alteration causing the
reported ETV1 expression.
Methods. For the ETV1 amplification assay, we used the commercially
available Chromosome 7 reference probe CEP 7 (D7Z1), spanning the
region 7p11.1–q11.1. The target probe was located on the ETV1 locus at
7p21.2. The target probe was labelled with biotin to produce a red signal
using CTD-2220I3 BAC clone. For the ETV1 break-apart assay, we
used BAC clones CTD-222013 for centromeric labelling with biotin and
RP-11769K2 for telomeric labelling with digoxigenin. BAC clones were
obtained from Invitrogen (Invitrogen, CA, USA).
Results. We performed FISH analysis on 140 GIST patients using tissue
micro arrays. Neither ETV1 amplification nor break apart was detectable.
Conclusions. The present study ensures that neither a genomic ETV1 amplification
nor a break apart is found in GISTs. We conclude that not
genetic aberrations but rather upregulation of ETV1 due to high KIT
receptor levels are responsible for the high ETV1 expression in GISTs.
152 | Der Pathologe · Supplement 1 · 2012
SA-P-042
A new whole genome amplification method for studying clonal
evolution patterns in malignant colorectal polyps
D . Hirsch1 , J . Camps1 , S . Varma2 , R . Kemmerling3 , T . Ried1 , T . Gaiser1 1Section of Cancer Genomics, Genetics Branch, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Bethesda, United
States, 2HiThru Analytics, Bethesda, United States, 3University Hospital
Salzburg of the Paracelsus Private Medical University, Institute of Pathology,
Salzburg, Austria
Aims. The transition of normal colonic epithelium to adenoma and invasive
carcinoma is defined by chromosomal aberrations. To identify the
genetic drivers involved in this progression in samples from individual
patients, we applied array comparative genomic hybridization (aCGH) to
13 formalin-fixed paraffin-embedded (FFPE) samples of early, localized
human colon adenocarcinomas arising in high-grade adenomas (so called
“malignant polyps”). These lesions are small and hence the amount of
DNA limited. Additionally, the quality of the DNA is compromised due
to the fragmentation as a consequence of formalin fixation. To overcome
these problems, we optimized a newly developed isothermal whole genome
amplification system (NuGEN Ovation® WGA FFPE System).
Methods. DNA was isolated from the FFPE blocks of 13 malignant polyps,
each consisting of areas of adenoma and carcinoma. Starting with
100 ng of FFPE DNA, the amplification system produced 4.01±0.29 µg
(mean ± standard deviation) of DNA. The samples were then analyzed
using Agilent SurePrint G3 Human CGH Microarrays 4×180K.
Results. Genomic imbalances were conserved in this procedure as assessed
by comparing amplified and unamplified FFPE DNA using aCGH.
The excellent quality of amplified DNA was further indicated by a high
signal-to-noise ratio and a low derivative log2 ratio spread. Both, the
amount of amplified DNA and aCGH performance were independent
from the age of the FFPE block and the associated degradation of the
extracted FFPE DNA. We observed losses of chromosome arms 5q and
18q in the malignant polyp samples, while the embedded early carcinomas
revealed losses of 8p, 17p, and 18, and gains of 7, 8q, 13, and 20.
Aberrations detected in the adenoma were invariably maintained in the
embedded carcinomas.
Conclusions. In conclusion, this approach demonstrates that using isothermally
whole genome amplified FFPE DNA is technically suitable for
aCGH. Besides demonstrating clonal origin of the adenoma and carcinoma
part within a malignant polyp, the gain of chromosome arm 20q
was an indicator for progression from adenoma to carcinoma in malignant
polyps.
SA-P-043
Dissimilarities of colorectal (CRAIT) and sinonasal adenocarcinoma
(SNAIT) of intestinal type
K . Donhuijsen1 , I . Kollecker1 , H . Hannig1 , H .-G . Schroeder2 1Academical Hospital Braunschweig, Department of Pathology, Braunschweig,
2Academical Hospital Braunschweig, Department of Otorhinolaryngology,
Braunschweig
Aims. SNAIT and CRAIT are considered to be nearly identically whereas
an uninformed pathologist would diagnose an endonasal metastasis of a
CRAIT instead of a primary of the inner nose. However, in a closer look
are there discrepancies to detect by morphology, immunohistology and
molecular results?
Methods. Fifty consecutive cases of SNAIT and CRAIT were compared
with regard to morphological criteria as subtype, histological inhomogeneity,
mucin production, desmoplastic reaction and vascular invasion.
Further the expression of CDX2, CK7, CK20, Synaptophysin, p53, Ki67,
were compared and also the molecular status (30 cases) by PCR for K-
RAS, b-raf, EGFR and p53.
Results. SNAIT and CRAIT are histologically quite similar but not identical:
adenomatous precursor lesions are absent. Tumor inhomogeneity

and desmoplastic reaction are different. SNAIT revealed more cohesiveness
and lower vascular spreading than CRAIT. CXDX2 and CK20
expressions are identically. CK7 and Synaptophysin demonstrated no reliable
differences, even though SNAIT are more often positive as CRAIT.
Mutations analyses exhibited for 30 SNAIT negative results for EGFR.
One case (1/30) only was positive for K-RAS Mutation (Mut g/g 12 Asp.)
and only one case for b-rafV600E, whereas about 60% of cases revealed a
p53 expression mostly in Exon 5, thrice in Exon 8/9.
Conclusions. Subtile studies detect dissimilarities of CRAIT and SNAIT.
However, if in a given case the discrimination is problematic, the molecular
status can be helpful. Molecular results reflect the different pathway
of these similar tumors. Hence it follows, that a morphologic mimicry
does not imply biologic relatedness!
SA-P-044
The G protein coupled-receptor GPR81 is upregulated in colorectal
carcinomas with KRAS wild type genotype
A .-K . Wenke1 , K . Balschun1 , C . Röcken1 1Christian-Albrechts-University Kiel, Department of Pathology, Kiel
Aims. The KRAS genotype is essential for the response to a targeted therapy
in advanced colorectal carcinoma (CRC). Several studies revealed
that only patients with wild type KRAS (KRASwt) benefit from a therapy
with monoclonal antibodies like cetuximab (Erbitux®) and panitumumab
(Vectibix®) which block the intracellular cascade following
activation of the epidermal-growth-factor-receptor (EGFR). Therefore,
the identification of new therapeutic targets is very important. G protein
coupled-receptors (GPCRs) are interesting candidates, since they
are known to transactivate the EGFR signaling pathway. The aim of this
study was the identification and functional analysis of GPCRs which are
differentially expressed in colorectal carcinoma dependent on KRAS genotype.
Methods. mRNA expression of KRASwt tumor and non-tumor tissue of
eight patients was compared to eight cases with KRASmut tumor and
non-tumor tissue using Affymetrix GeneChip® Human Gene 1.1 ST Arrays.
Differential gene expression of interesting candidate genes was validated
by quantitative RT-PCR and immunohistochemical staining of a
validation series of 46 colorectal carcinomas (28 KRASwt, 18 KRASmut
tumors).
Results. We found 32 differentially regulated GPCRs comparing gene
expression of normal and tumor colon tissue. An increased expression
of GPR81, NIACR1, NIACR2, GPR143, HTR1D and LGR5 in carcinoma
tissue could be confirmed. However, GPR81 is the only receptor being
significantly differentially regulated according to the KRAS genotype of
the tumor.
Conclusions. Our expression analyses demonstrate a correlation of
GPR81 expression with the KRAS genotype in CRC. The functional role
of GPR81 in colorectal carcinogenesis has to be analyzed in further studies.
Additionally, a comprehensive expression analysis on an enlarged
patient series of 2000 cases will show if GPR81 could serve as a proper
therapeutic target or a potential prognostic marker for colorectal carcinoma.
SA-P-045
Polysomy and amplification status of EGFR in primary colorectal
cancer and in matched metastases
C . Giedl1 , A . Kiesl1 , F . Hofstädter1 , W . Dietmaier1 1University of Regensburg, Institute of Pathology, Regensburg
Aims. There is a need for predictive biomarkers that identify patients
most likely to respond to targeted treatment. EGFR is an important target
for therapies using monoclonal antibodies and tyrosine kinase inhibitors
(TKIs). We analyzed the polysomy and amplification status of
EGFR in primary colorectal cancers, matched metastases, and looked if
differences in the polysomy and amplification status exist which could
influence targeted therapies.
Methods. 54 primary colorectal cancers and 94 metastases of primary
colorectal cancers were analyzed. The EGFR gene copy number was evaluated
by fluorescence in situ hybridization (FISH). Specimens that have
>40% of cells displaying ≥4 copies of the EGFR signal and specimens that
showed an EGFR to CEP7 ratio ≥2 over all scored nuclei were counted
as EGFR FISH-positive. Also specimens with gene clusters (≥4 spots) in
≥10% of tumor cells and specimens with at least 15 copies of the EGFR
signals in ≥10% of tumor cells were counted as EGFR FISH-positive.
Results. A high EGFR polysomy was found in 18 of the 54 primary colorectal
cancers (33.3%) and in 30 of the 94 evaluated syn-or metachronous
metastases in lymph nodes, liver, lung, ovary, peritoneum, skin, brain,
urinary bladder and bone (31.9%). In contrast no real gene clusters and
real EGFR amplifications were found. 18 of 22 matched metastases from
EGFR FISH-positive primary colorectal cancers were also EGFR FISHpositive
(81.8%) showing a high accordance but no complete congruence.
In addition, 1 case showed an EGFR FISH-negative primary colorectal
cancer and a EGFR FISH-positive metastasis.
Conclusions. The discrepancy of EGFR polysomy and amplification
status found in primary colorectal cancers and matched metastases demonstrate
a tumor-metastases diversity, which should be considered in
patients’ treatment regime.
SA-P-046
Detection of KRAS and BRAF mutation in Chinese colorectal carcinoma
patients by pyrosequencing
M . Ye1 , J . Chen1 , M . Lai1 1Zhejiang University, School of Medicine, Hangzhou, China
Aims. KRAS and BRAF mutation status were reported to be a possible
biomarker which response to EGFR antibody chemotherapy in colorectal
carcinoma. Recently, pyrosequencing is proved as a powerful and
convenient method for KRAS and BRAF mutation detection by a lot of
groups in the U. S. and Europe. In China, there are rare labs or groups
to setting up pyrosequencing method for molecular clinical diagnosis.
Methods. 180 FFPE CRC tissue samples were microdissected to obtain
the tumor cells and remove the stroma contents, then the genomic DNA
was extracted from these samples by routine method. A pyrosequencing
assay based on PCR was designed to characterize KRAS codon 12, 13 and
BRAF codon 600 mutation status by Pyromark Q24. The SW620 and
HT29 CRC cell lines were used as controls in pyrosequencing.
Results. Mutation status of KRAS codon 12 was identified in 65/180
(36.11%) samples, among them,1 sample was 34G>A mutation(0.56%), 2
samples were 34G>C mutation(1.11%) , 2 samples were 34G>T mutation
(1.11%), 29 samples were 35G>A mutation (16.11%), 1 was 35G>C mutation
(0.56%) and 30 were 35G>T mutation (16.67%). 20 samples were mutant
for KRAS codon 13 (11.11%)with the nucleotide change 38G>A for 19 samples
(10.56%) and 1 for 38G>A (0.56%). 16/180 tumors harbored a mutation
of 1799T>A in BRAF codon 600 mutation (8.89%).
Conclusions. The KRAS and BRAF mutant frequencies of the 180 FFPE
tissue samples are generally consistent with those recently reported. Although
the amount of samples was limited, more CRC patients are needed
to be detected in the year future.
Der Pathologe · Supplement 1 · 2012 |
153

Abstracts
SA-P-047
Alternative splicing of Daxx-beta is reduced in renal cell and
colorectal carcinoma
S . Funke 1 , N . Wethkamp 1 , S . Heikaus 1 , K .L . Schäfer 1 , H .E . Gabbert 1 , C . Mahotka 1
1 University Hospital Düsseldorf, Düsseldorf
Aims. Death associated protein (Daxx) is an important transcriptional
co-repressor for a large number of genes, mostly related to apoptosis.
Daxx interacts with p53 and represses its transcriptional activity. Alternative
splicing of the Daxx results in the generation of a c-terminally
truncated and modified isoform termed Daxx-beta. As we shown before,
the new C-terminus leads to a markedly reduced affinity of Daxx-beta to
PML and p53. Consequently, Daxx-beta is unable to repress transcriptional
activity of p53. Because deregulation of p53 is closely related to carcinogenesis,
alternative splicing of Daxx may also participate in tumour
development and progression. Here, we examined the in vivo splicing
pattern of Daxx-beta during renal and colon carcinoma progression.
Methods. Semi-quantitative real-time PCR were performed with flash
frozen resected tissue of 43 renal cell carcinomas (RCCs) of the clear cell
type and 40 colorectal carcinomas (CCs) from different tumour stages as
well as 49 and 38 samples from the corresponding non-neoplastic kidney
and colon epithelia, respectively.
Results. Statistical calculation revealed a significant reduction of Daxxbeta
isoform in both tumour entities compared to the corresponding
non-neoplastic tissue. These alterations were detected already at early
tumour stages (pT1+pT2) and did not further change in late stages
(pT3+pT4).
Conclusions. Our results indicate for the first time that splicing of Daxxbeta
is reduced in all tumour stages of renal cell and colorectal carcinoma,
and that Daxx-beta could be a potential candidate for a new biomarker
on transcript level.
SA-P-048
Modulation of Wnt and Hedgehog signaling pathways is linked to
retinoic acid-induced amelioration of chronic fibrosing inflammation
P .J . Nelson1 , S . Porubsky2 , C . von Toerne1 , J . Bedke2 , S . Safi2 , N . Gretz2 ,
H .-J . Gröne2 1 2 University of Munich, München, German Cancer Research Center, DKFZ,
Heidelberg
Aims. Chronic renal allograft damage (CAD) is manifested by a smoldering
inflammatory process that leads to transplant glomerulopathy,
diffuse interstitial fibrosis and tubular atrophy with loss of tubular structures.
Methods. Gene expression pathway analysis was used to detect new inducers
of fibrosis. Using a Fischer 344 (RT1lvl) to Lewis (RT1l) rat renal
allograft model, transcriptomic profiling and pathway mapping, we have
previously shown that dynamic dysregulation of the Wnt signaling pathways
may underlie progressive CAD. Retinoic acid, an important regulator
of differentiation during vertebrate embryogenesis, can moderate
the damage observed in this experimental model of CAD.
Results. We show here that subsets of the Hedgehog (Hh) and canonical
Wnt signaling pathways are linked to the pathophysiology of progressive
fibrosis, loss of cilia in epithelia and chronic dysfunction. Oral treatment
with 13cis retinoic acid (13cRA) was found to selectively ameliorate the
dysregulation of the Hh and canonical Wnt pathways associated with
CAD, and lead to a general preservation of cilial structures.
Conclusions. Interplay between these pathways helps explain the therapeutic
effects of retinoic acid treatment in fibrosing inflammation, and
suggests future targets for moderating chronic fibrosing organ damage.
154 | Der Pathologe · Supplement 1 · 2012
SA-P-049
Multiciplicity in sporadic inflammatory fibroid polyps and GISTs
implicating a field effect
S . Huss1 , H . Löser1 , E . Kleimann2 , S . Eidt3 , R . Budde4 , W . Weichert5 , A . Szöke6 , C .
Röcken7 , A . Hölscher8 , W . Hartmann1 , S . Merkelbach-Bruse1 , H .-U . Schildhaus1 ,
R . Buettner1 , E . Wardelmann1 1 2 University Hospital Cologne, Institute of Pathology, Köln, St . Franziskus
Hospital, Cologne, Department of Surgery, 3St . Elisabeth-Krankenhaus Köln
Hohenlind, Institute of Pathology, Köln, 4Institute of Pathology in Cologne –
St . Vinzenz-Hospital, 5University Hospital Heidelberg, Institute of Pathology,
6 7 Institute of Pathology, Cologne – Deuz, Christian-Albrechts-University,
Kiel, Institute of Pathology, 8University Hospital Cologne, Department of
Surgery
Aims. Inflammatory fibroid polyps (IFPs) are rare benign and mesenchymal
tumors. Activating PDGFRA mutations play a central role in the
pathogenesis of IFPs and have been shown to be a key player in a subset
of gastrointestinal stromal tumors (GISTs). Whereas in GIST PDGFRA
mutations are considered to transform one of the subtypes of precursor
cells of interstitial cells of Cajal, in IFP an hitherto not further identified
mesenchymal progenitor cell type of the submucosa is mutated and develops
towards a tumour.
Methods. The vast majority of IFPs occur as solitary tumors. Here, we
report on molecular and clinicopathologic features of three patients presenting
with multiple IPFs and syn- or metachronous GISTs.
Results. The first case had multiple IFPs in a duodenal segment and developed
a metachronous gastric GIST. Both tumors (IFPs and GIST) revealed
exactly the same somatic PDGFRA exon 12 mutation (p.Y555C). The
same observation of an identical PDGFRA exon 18 mutation (p.D842V)
in both a gastric GIST and multiple IFPs in the duodenum was made in
a second case. In the third case, multiple IFPs were found in a jejunal
segment, all carrying the same somatic substitution mutation in exon 18
of PDGFRA (p.[R841K(+)D842I]).
Conclusions. The coincidence of exactly the same sporadic PDGFRA mutation
in multiple IPFs and syn- or metachronous GISTs points at closely
interrelated precursor cells driving both neoplastic processes. With respect
to sporadic multiciplicity, our data suggest a field effect contributing
to the pathogenesis of such IFPs and GISTs.
SA-P-050
Quantitative PCR analysis for detection of cmV infection in patients
suffering from ulcerative colitis using FFPE material
N . Wethkamp1 , C . Bersch1 , H .-W . Kohlmann2 , V . Meister3 , M . Respondek1 1 2 Institute of pathology, Dr . Respondek, Vechta, Praxis f . Pathologie,
Respondek, Vechta, 3St . Marien-Hospital, Gastroenterology, Vechta
Aims. Several lines of evidence indicate that cmV infection can be substantially
associated with the onset of ulcerative colitis, especially in
immunocompromised patients. In order to estimate the impact of cmV
infection and monitoring efficacy of antiviral treatment a real-time PCR
Assay was developed to quantify cmV viral load in gastric tissue samples.
Methods. DNA-samples derived from FFPE material of patients with
ulcerative colitis were quantitatively analysed for cmV infection by Taq-
Man technology. Via two independent PCR reactions cmV DNA and human
GAPDH copy numbers were quantified, using a plasmid coding for
both target sequences as an external standard. Finally, viral load in the
analysed tissue was expressed as cmV copy numbers per 100,000 cells (as
calculated by the obtained GAPDH copy numbers).
Results. A total of 89 samples from 22 patients were evaluated for cmV
infection while varying section numbers (ranging from 1–12) were analysed
per patient. In 29 samples (32%) referring to 50% of the patients cmV
DNA could be detected. Here, three patients (27%) showed a viral load of

one patient revealed more than 9 million cmV/10E5 cells. Histological
tissue assessment of the latter patient also revealed the existence of inclusion
bodies which are typical for a severe cmV infection. Interestingly,
samples with viral loads higher than 8000 cmV/10E5 cells were also
positive for cmV detection using immunohistochemistry. By analysing
different tissue sections of individual patients major differences in viral
loads were notable indicating that cmV infection in ulcerative colitis
could be locally quite heterogeneous. From three patients consecutive
samples were analysed after initiation of antiviral therapy. In every case
a significant reduction in cmV viral load could be detected indicating a
positive response to the therapy.
Conclusions. Quantitative real-time PCR is useful for detection of cmV
infection in tissue samples of patients suffering from ulcerative colitis.
Moreover, the assay seems to be capable for follow up monitoring of therapy
response during antiviral treatment.
SA-P-051
Ion semiconductor sequencing: a novel technology for analysis
of somatic mutations in formalin-fixed and paraffin-embedded
tumor biopsies
K . König1 , U . Koitzsch1 , J . Altmüller2 , S . Merkelbach-Bruse1 , J . Fassunke1 ,
C . Vollbrecht1 , L . Heukamp1 , P . Nürnberg2 , R . Büttner1 , M . Odenthal1 1 2 University Hospital Cologne, Institute for Pathology, Köln, Unversity
Cologne, Cologne Center of Genomics
Aims. Somatic mutations in a panel of genes encoding signal transducers
that are involved in cell growth, proliferation and differentiation take
center stage in molecular pathology due to their impact on tumor prognosis
and therapy response. Recently, an ion semiconductor sequencing
system, based on the semiconductor determination of proton release
after each nucleotide coupling to the DNA strand, was described (Rothberg
et al.; Nature 2011). In the present study, we aimed to evaluate this
novel sequencing technology for mutation detection in formalin-fixed
paraffin-embedded (FFPE) tumor biopsies.
Methods. DNA was purified from FFPE biopsies or from macrodissected
tumor materials by the M48 platform (Qiagen). PCR amplification
of a wide panel of target genes including EGFR exon 18, 19, 20, 21, Kras
codon 12, 13 locus, the Braf codon 600 locus, and Pik3Ca was performed
according to the routine processing in molecular pathology. Up to
109 molecules of the target amplicons of each patient were pooled, sheared,
and adapter and multiple identifier were ligated following the Xpress
protocol (ABI life Technologies). Multiplexed libraries were then used
for clonal amplification by means of emulsion PCR and applied to ion
semiconductor sequencing using the Ion Torrent platform.
Results. Amplicons from EGFR exon 18, 19, 20, 21, Kras codon 12, 13 locus,
the Braf codon 600 locus, and PikCa exons were successfully sequenced
and somatic mutations were detected after bioinformatic analyses.
Therefore, the ion semiconductor sequencing technology combined with
the Xpress adapter ligation approach revealed that amplicons of well established
PCR assays such as assays that are already established in the
routine diagnostics, are suitable templates for parallel sequencing.
Conclusions. In conclusion, parallel ion semiconductor sequencing is a
promising and efficient technology for multiplexed mutation analyses
that may be easily linked to existing PCR approaches of molecular routine
diagnostics.
SA-P-052
Inactivation of JNK as pathogenic factor in colorectal carcinogenesis
upon inflammation
K . Reissig1 , T . Guenther1 , A . Silver2 , R . Hartig3 , P . Steinberg4 , A . Roessner1 ,
A . Poehlmann1 1Otto-von-Guericke University Magdeburg, Department of Pathology,
Magdeburg, 2Queen Mary University London, Colorectal Cancer Genetics,
London, United Kingdom, 3Otto-von-Guericke University Magdeburg,
Department of Molecular and Clinical Immunology, Magdeburg, 4Institut of
Food Toxicology and Analytical Chemistry, Hannover
Aims. We have recently developed a cell culture model using human colon
epithelial cells (HCEC) and H2O2 to study tumor-initiating molecular
events in inflammation-associated colorectal cancer. As we have supposed
epigenetic changes that may account for HCEC transformation,
we aim to find epigenetic signaling events. As DNA damage checkpoints
are important in cancer cell proliferation, their role in HCEC transformation
was analyzed.
Methods. We simulated acute inflammation by treating HCEC with a pathophysiologic
concentration of H2O2 as ROS (reactive oxygen species).
Furthermore, we developed transformed HCEC by repeated treatment
cycles to mimic chronic inflammation. In order to analyze DNA damage
checkpoints, we performed cell cycle analysis using FACS. The function
of JNK was investigated using the JNK inhibitor SP600125. The cells were
further analyzed by immunoblotting.
Results. To prove whether single H2O2 treatment leads to activation of
DNA damage checkpoints, cell cycle analysis was performed. Acute ROS
activated the G1/S and G2/M DNA damage checkpoints. At the same
time, there was prominent activation of JNK signaling. This coincided
with periods when cell cycle arrest was observed. As the JNK inhibitor
was able to rescue S and G2/M arrest, the observed cell cycle arrest is
JNK-dependent. Immunoblotting analysis after JNK inhibition identified
p21, an inhibitor of cell cycle progression, as cellular JNK target. Importantly,
in line with uncontrolled proliferation of transformed HCEC,
we observed altered JNK activation. This down-regulation of activated
JNK caused down-regulation of p21 in transformed HCEC.
Conclusions. JNK inactivation plays an important role in HCEC transformation
and seems to be a pathogenic factor. Subsequent down-stream
p21 down-regulation occurs early, seems to be as efficient as p53 mutation,
and fits very well with the suggestive role of p21 as a potential tumor
suppressor in the colon. We speculate that both inactivation of JNK and
p21 down-regulation might cause the cell to turn on a one way street to
uncontrolled proliferation as one defining feature of the initiation of the
inflammation-carcinoma pathway in the colon.
SA-P-053
Validation of primary antibodies for immunohistochemistry
H .J . Grote1 1Merck KGaA, Histopathology – Biomarker Technologies, Darmstadt
Aims. Tissue biomarkers are gaining increasing importance in establishing
targeted therapies. In oncology tissue biomarkers are used for target
validation and indication seeking, for patient stratification and for pharmacodynamic
assays. Immunohistochemistry on formalin-fixed paraffin-embedded
(FFPE) tissue (IHC-P) is a key technology in biomarker
discovery and development. However, there is increasing evidence that
IHC-P is frequently impacted by limited specificity of primary antibodies.
This report presents strategies, technologies and examples for improved
primary antibody validation.
Methods. Antibody validation is a multi-step process. It starts with gathering
information on target expression and identification of positive
and negative expression controls. Standard laboratory procedures like
Western blot, FACS or qRT-PCR may be useful to corroborate or complement
published expression data in cancer cell lines. A Western blot
may also provide first information about selectivity of a candidate pri-
Der Pathologe · Supplement 1 · 2012 |
155

Abstracts
mary antibody which is intended to be used for IHC-P. Subsequently,
antibody specificity can be tested on multiple cell lines using a FFPE cell
line microarray (CMA). Correlating cmA IHC-P profiles with mRNA
gene expression profiling data may be helpful. If the target is ubiquitously
expressed, evaluation of antibody specificity may require more sophisticated
technologies like siRNA knock down of target in cancer cell lines.
The siRNA knock down or a transfection of cell lines with wild type or
mutated target generates gene expression modified cell lines which may
be assembled to a gene expression modified cmA. Thereby IHC-P can be
linked to functional assays.
Results. In-depth antibody validation discloses that primary antibodies
may be not selective in IHC-P, although the antibody datasheet or published
data suggest the IHC-P application. This observation applies not
only to polyclonal antibodies but also to monoclonals. Using antibodies
that demonstrate a specific staining, IHC-P may correlate remarkably
with alternative protein detection methods like Luminex’s multiplex bead-based
immunoassay that is limited to fresh samples.
Conclusions. The data underline the need for in-house validation of primary
antibodies prior to their use as analytic tool in IHC-P. It is likely
that the unsatisfactory validation status of published antibodies represents
one of the causes for contradictory IHC-P data in the literature.
Concerted activities should be considered to improve the current situation.
Poster: Molekularpathologie II
SA-P-054
The homeobox gene HOPX is methylated in human lung cancer,
and the methylation status of HOPX is a diagnostic marker for
patients with lung adenocarcinoma
Y . Chen1 , T . Cui1 , L . Yang 1 , N . Posorski 2 , I . Petersen 1
1 2 University Hospital Jena, Institute of Pathology, Jena, University Hospital
Jena, Institute of Human genetics, Jena
Aims. The homeobox gene HOPX has been considered as a tumor suppressor
in various cancers. Our previous studies showed that HOPX is
downregulated in lung cancer cell lines and primary lung tumors, and
forced expression of HOPX by stable transfection could inhibit tumor
cell growth. However, the regulation of HOPX in lung cancer has not yet
been well elucidated. Therefore the aim of the study is to investigate the
epigenetic regulation, analyse the potential target genes of HOPX, and
evaluate the clinical relevance of HOPX in lung cancer.
Methods. HOPX protein expression was analysed by western blotting.
Demethylation test by 5-aza-2-deoxycytidine (DAC), bisulfite sequencing
(BS), and methylation-specific-PCR (MSP) were carried out to analyse
the methylation status of HOPX in lung cancer cells and primary
lung tumor samples. cDNA microarray analysis was performed to identify
target genes of HOPX.
Results. In line with decreased expression of HOPX mRNA in lung
cancer cell lines, western blotting showed that HOPX protein was downregulated
compared to normal HBEC cells. Ten out of 12 lung cancer
cell lines restored HOPX expression after treatment with DAC , and the
methylation status of HOPX in the promoter region and exon 1 was confirmed
by bisulfite sequencing. MSP showed that HOPX was methylated
in 64 out of 88 (72.7%) primary lung tumor samples, and methylation of
HOPX was significantly associated with lung adenocarcinoma (p200 patients.
Methods. We performed quantitative real-time PCR to analyse the DNA
methylation of the SHOX2 gene compared with beta-actin using the Epi-
ProLung Test (Epigenomics) in >200 patient undergoing routine diagnostics
for lung cancer at the Charité in 2011. A dichotomized methylation
status was determined by using a ddCT cut-off value of 9.5.
Results. From the 239 cases, 173 were SHOX2-, 44 SHOX2+ and 17 undetermined.
The cytological analysis yielded 200 cases unsuspicious of
cancer (or with only mild atypia) and 34 cases moderately to highly suspicious
of cancer. This comparison between SHOX2 and cytology displays
a sensitivity of 73% and a specificity of 78.4% (AUC 75.1%, SHOX2
testing benchmarked with cytology).
Conclusions. The results demonstrate the utility of SHOX2 methylation
testing for routine diagnostics of lung cancer in direct comparison with
cytology in lavage samples. Clinical, radiological and histological data
will be included upon availability to determine the absolute performance
of SHOX2 testing.
SA-P-056
Detection of a heterogeneous BRAFV600E mutation status in a
patient with metastasized malignant melanoma
R . Marienfeld1 , J . Heinrich2 , S . Jung2 , P . Möller1 , K . Scharffetter-Kochanek3 ,
M . Huber3 , T . Menzel4 , L .-A . Schneider3 , T . Barth2 1 2 University of Ulm, Institute of Pathology, Ulm, University of Ulm, Institute
of Pathology, 3University of Ulm Medical Center, Department of Dermatology
and Allergology, 4Dermatophathology Friedrichshafen
Aims. Targeted tumor therapy is expected to lead to a breakthrough in
the treatment of advanced stage melanoma. By end of this year Vermurafenib®
(Roche PLX4032) is expected to be licensed as the first BRAF
pathway inhibiting substance for metastasised melanoma carrying a
V600E BRAF mutation. Up to 50% of all melanoma patients are estimated
to carry a mutation in the codon 600 of the BRAF gene. It is therefore
in the focus of scientists and clinicians since targeted therapy is supposedly
less harmful and more effective than conventional chemotherapy.
Mutational analysis of the codon 600 of the BRAF gene is strictly required
to determine the eligibility of the patient for a targeted therapy
with Vermurafenib®. The goal our study is to determine the reliability of a
BRAF mutational analysis on the basis of a single tissue specimen. Here,
we present the BRAF mutational analysis of four different specimens of a
patient with metastasized melanoma.
Methods. Microdissection of FFPE tumor tissue. DNA isolation and PCR
amplification of the BRAF gene segment. Pyrosequencing of codon 600.
Results. The patient is a 38-year-old woman with a pTxpN3pM1 metastasized
acrolentigeneous melanoma. We analysed the BRAF mutation
status in the primary tumor as well as in three different biopsies from a
lung axillary lymph node and brain obtained with a time difference of
six years. BRAF mutation has been shown to be an early event in melanoma
and thus should be present in the first biopsy. However, whereas
the analysis of the first biopsy from lymph node revealed a wild type

situation, the V600E mutation in the BRAF locus was observed in the
primary tumor and the later biopsies from the lung and brain implying
that different metastases may resemble different tumor subclones which
need to be analysed separately.
Conclusions. In conclusion, our observations point to a heterogeneous
BRAF mutation status i) during the time course of the disease, and ii) in
a very small subset of tumor cells in a given sample. These results highlight
the need for a critical therapy evaluation in which the this mutational
analysis of the BRAF gene target needs to be embedded in the overall
clinical setting rather then simply rely on a yes-or-no result of the mutational
test of a single biopsy. Thus, the whole setting has to be taken into
account by the pathologist, molecular biologist and clinician.
SA-P-057
Integrin expression in primary tumor and their brain metastasis
A . Vogetseder1 , S . Thies1 , M . Weller2 , P . Schraml1 , H . Moch1 1UnversitätsSpital Zürich, Clinical Pathology, Zürich, Switzerland,
2UnversitätsSpital Zürich, Neurology, Zürich, Switzerland
Aims. To determine the expression of avb3, avb5 and avb8 integrins in
breast, lung and kidney cancer as well as in malignant melanoma and
their brain metastases.
Methods. Novel subtype specific rabbit monoclonal antibodies for avb3,
avb5 and avb8 integrins. Immunohistochemistry on tissue microarrays.
Results. Integrin avb5 is predominantly expressed in all primary tumours
and their metastases. Negative or weak integrin avb3 and avb8
expression in primary lung, renal and breast cancers. Significant increase
of avb3 and avb8 expression in brain metastases. Primary malignant
melanoma and their brain metastases show varying amounts of avb3 and
avb8 expression.
Conclusions. Rabbit monoclonal antibodies allow the analysis of specific
integrin isoforms. The avb3 and avb5 inhibitor cilengitide could also, like
in glioblastoma treatment, be effective in patients with brain metastases
of melanomas as well as lung, breast and kidney carcinomas.
SA-P-058
Activating PDGFRA mutations in inflammatory fibroid polyps
occur in exons 12, 14 and 18 and are associated with tumour
localization
S . Huss1 , E . Wardelmann1 , D . Goltz2 , W . Hartmann1 , E . Binot1 , H . Löser1 ,
S . Merkelbach-Bruse1 , R . Buettner1 , H .-U . Schildhaus1 1 2 University Hospital Cologne, Institute of Pathology, Köln, University Hospital
Bonn, Institute of Pathology, Köln
Aims. Inflammatory fibroid polyps (IFP) are mesenchymal tumours of
the gastrointestinal tract. This study was performed to broaden the base
of evidence of the pathogenic role of PDGFR mutations in IFP.
Methods. A total of 38 IFP, extracted from our consultation files, were
included in this study. Clinicopathological features were collected and
mutational analysis was carried out.
Results. Activating mutations in three different exons of PDGFRA were
found in 25 IFP. For the first time we report two cases with PDGFRAexon
14 mutations (p.N659K; p.[N659K(+)T665A]). The results of our
study and cases reported earlier in the literature clearly indicate that
there is a localization specific pattern: exon 12 mutations predominate
in the small intestine, while exon 18 mutations frequently occur in the
stomach (p

Abstracts
plification identifies a separate subgroup. The identification of distinct
molecular subgroups of AS forms a basis for the identification of novel
pathways that may help to better understand the biology of AS and may
eventually lead to the development of more specific treatments.
SA-P-061
Molecular analysis of COL1A1/PDGFB translocation and PDGFB
amplification in patients with Dermatofibrosarcoma protuberans
K . Walluks1 , S . Hauk2 , C . Wölfel1 , Y . Chen1 , T . Cui1 , L . Yang1 1 2 Universitätsklinikum Jena, Institute of Pathology, Jena, Zytovision, Bremen
Aims. Dermatofibrosarcoma protuberans (DFSP) is a dermal and subcutaneous
tumor of intermediate malignancy. The most remarkable
cytogenetic feature of DFSP is the chromosomal translocation t(17;22)
(q22;q13), causing a fusion of the platelet-derived growth factor beta
chain (PDGFB) gene on 22q13 and the collagen type 1 alpha 1 (COL1A1)
on 17q22. The aim of the study was to analyze the molecular characteristics
of DFSP
Methods. We performed Fluorescence in situ hybridization (FISH) and
multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to
detect chromosomal translocation and fusion gene transcripts in 16 formalin-fixed,
paraffin-embedded DFSP samples. Additionally, we analysed
the gene copy number of PDGFB in the 16 samples by real-time PCR.
Results. Eleven out of 16 samples (68.8%) showed fusion transcripts by
multiplex RT-PCR analysis. Various exons of the COL1A1 gene were fused
with PDGFB gene, among them, exon 25 was found to be more frequently
involved. It turned out that, PDGFB copy numbers in the DFSP
samples was slightly higher than in normal skin tissues (p=0.007).
Conclusions. Our results suggest that the COL1A1/PDGFB fusion transcripts
and amplification of PDGFB may be diagnostic markers for patients
with DFSP, and PDGFB could be a potential target for treatment
of patients with DFSP.
SA-P-062
PHD3 silencing leads to increased tumor growth accompanied by
enlarged tumor vessels
A . Kettelhake1 , M . Rezaei2 , A . Kuzmanov1 , D . Poitz3 , B . Wielockx1 , G . Breier1 1 2 Technical University of Dresden, Department of Pathology, Dresden, Technical
University of Dresden, Department of Pathology, Dresden, 3Technical University of Dresden, Department of Internal Medicine and Cardiology,
Dresden
Aims. The fast growth of solid tumors causes the development of hypoxic
areas that are very often marked by the upregulation of the hypoxia-inducible
factor (HIF). HIF enables the tumor cells to survive under hypoxic
conditions for example by increasing the production of angiogenic
factors. HIF is regulated by prolyl hydroxylase domains proteins (PHD1,
2, 3, 4). However, the function of PHD3 during tumor progression and
angiogenesis has not been investigated in detail so far. It is our aim to
address these open questions.
Methods. We stably silenced PHD3 in a murine osteosarcoma cell line
(LM8) using a lentiviral transduction system and analyzed the cell clones
by quantitative real-time PCR and Western blotting. To investigate the
behavior of these clones in vivo we injected them subcutaneously on the
back of C3H mice and measured the tumor size every 2 days for a period
of 14 days. Perfusion of tumor tissue, immunohistochemical staining as
well as immunofluorescent staining was performed to analyze tumors.
Results. Surprisingly, downregulation of PHD3 does neither affect the
protein level of HIF nor the expression levels of known HIF-target genes.
The other PHD isoforms (PHD1, PHD2, PHD4) as well as factor inhibiting
HIF-1 (FIH) show equal mRNA levels in sh-PHD3 clones compared
to controls. However, transforming growth factor α (TGF-α) and platelet-derived
growth factor c (PDGF-C) are markedly upregulated, whereas
the angiopoietin 2 (Ang2) mRNA level is decreased in the sh-PHD3
158 | Der Pathologe · Supplement 1 · 2012
clones. In vivo experiments show the development of significantly larger
tumors compared to control tumors. Interestingly, the density of tumor
vessels is decreased but the vessels are enlarged as evidenced by PECAM
staining. α-smooth muscle actin (αSMA) immunofluorescence staining
reveals that more vessels in the PHD3 silenced tumors are covered with
αSMA-positive cells. We found no difference in vessel perfusion or leakiness
between shPHD3-tumors versus control tumors.
Conclusions. Our data suggest that PHD3 plays an important role as tumor
suppressor because knocking it down leads to accelerated tumor
growth. Unexpectedly, this function seems to be HIF-independent. The
structure of the shPHD3-tumor vasculature is completely altered indicating
that PHD3 is involved in tumor angiogenesis as well. At the moment
we are determining which factors are responsible for the observed
phenotype. For this, we are using clones simultaneously knocking down
PHD3 and one of the differentially expressed growth factors.
SA-P-063
Ubiquitin, SH2 and UBA domains of p62 regulate interaction of
p62 with K8/18 and aggregation properties
V . Mahajan1 , C . Stumptner1 , A . Thueringer1 , T . Klingstedt2 , P . Nilsson2 ,
K . Metchler3 , K . Kashofer1 , H . Denk1 , K . Zatloukal1 , J . Haybaeck1 1 2 Medical University of Graz, Institute of Pathology, Graz, Austria, Linkoping
University, Sweden, 3Medical University of Vienna, Institute of Molecular
Pathology, Vienna, Austria
Aims. Steatohepatitis and other liver diseases are characterized by presence
of cytoplasmic protein aggregates termed Mallory-Denk bodies
(MDBs) and Intracellular Hyaline Bodies (IHBs. Sequestosome 1/p62,
ubiquitin, Keratin 8 (K8) and Keratin 18 (K18) are the major constituents
of MDBs. We investigated whether interaction of p62 with K8/18
depends on a) ubiquitination, b) phosphorylation, c) conformational alterations,
or d) structural domains of p62.
Methods. The SH2/PB1, ZIP/PB1 and UBA domains of p62 were deleted.
Specific phosphorylation sites (S24 and S152) of p62 were mutated. The
phosphorylation and deletion constructs were co-transfected along with
K8 and K18 in presence or absence of ubiquitin. We used Luminescent
conjugated oligothiophenes (LCOs) to investigate conformational changes
of p62 deletion and phosphorylation mutants.
Results. Deletion of the SH2 domain or partially of the PB1 domain leads
to loss of the filamentous ultrastructure of p62 but resembles an IHBlike
aggregation pattern. Deletion of the ZIP domain or the remaining
PB1 domain lead to irregularly shaped intracytoplasmic aggregates
whereas UBA deleted p62 displayed a diffuse distribution pattern but
only a partial loss of filaments ultrastructure and did not interact with
K8/18 anymore. CHO-K1 cells transfected with various combinations of
SQSTM1/p62, ubi and Krt8/Krt18 demonstrated that SH2 domain deleted
p62 co-localizes with K8 in the absence of ubiquitin. The phosphorylation
sites S24 and S152 do not seem to regulate the interaction of p62
with ubiquitinated keratins but mutation at S24 created a diffuse distribution
pattern of p62. LCO analysis demonstrated the presence of cross
beta-sheet conformation in SH2 domain deleted p62 and K8. Additional
oxidative stress may interfere with MDB components but not with their
interaction.
Conclusions. These findings explain the observation that SH2 and UBA
domains govern the aggregation property of p62 and influence the interaction
patterns with K8/K18. Thus it is clear that filamentous assemblies
of type I MDBs are predominantly due to p62 while type II MDBs may
need p62 and ubiquitin. Alternatively the amorphous granular nature
of type III MDBs results from insoluble K8/K18 aggregates. K8 and SH2
deleted p62 can undergo conformational changes from predominantly
Alpha-helical to cross β-sheet structures which allow their interaction
even in the absence of additional ubiquitin. Therefore the SH2 domain
might regulate the interaction between K8 and p62wt.

SA-P-064
Evolution of molecular pathology at the Institute of Basel
M .P . Bihl 1 , S . Hoeller 1 , A . Foerster 1 , R . Chaffard 1 , S . Schneider 1 , A . Rufle 1 ,
L . Terracciano 1 , L . Tornillo 1
1 University of Basel, Institute of Pathology, Basel, Switzerland
Aims. Molecular genetics in pathology is a very young field. It began with
analysis of haematological diseases or inherited disorders, but in the last
decade, also many of solid tumors have been found to be related to specific
somatic mutations. These mutations can give a hint to drug sensitivity
in a given tumor and therefore are urgently required in modern
oncology. Here we show how this field has developed in the recent years
using the example of the activity of the Institute of Pathology during the
last 6 years.
Methods. The number of PCR based analysis increased from 206 (in
2006) to over 800 analyses (in 2011).
Results. In the beginning only CKIT/PDGFRA mutation and EGFR
mutation analysis and clonality analysis were performed. However, the
overall percentage of analysed sites remained stable with 50% from the
lung, 10–20% from the blood or lymph node and 10–25% from colorectal
or gastrointestinal sites. Recently, new mutations are also routinely
tested like (IDH 1 and 2, BRAF and CTNNB1) and therefore new organs
were included like brain (glioma), skin (melanoma) and liver (adenomas).
From three available assays in 2006 the spectrum of our analysis
expanded to 20 different assays today. The number of performed FISH
analysis stayed stable, while the number of HER2 hybridisations dropped,
but new assays were introduced into the routine panel like 1p19q and
ALK translocation analysis for glioma and adenocarcinoma of the lung,
respectively. Therefore, the dynamic changes in molecular pathology are
due to new tumor classification systems, the development of new tumor
specific drugs and the increased knowledge of drug sensitivity in cancer
patients harboring specific somatic mutations.
Conclusions. In the upcoming years molecular based prognostic markers
and mutation specific therapies will even more expand the spectrum of
molecular testing in pathology. Its role is crucial to improve and optimize
the diagnosis and therapy of tumors and is essential in modern oncology.
Adaptation of the increasing knowledge of pathogenetic pathways
will be a major issue for molecular laboratories in the future.
SA-P-065
The amyloid precursor protein (APP) is a novel biomarker for
transformed human pluripotent stem cells
V . Venkataramani1 , K . Thiele2 , C .-L . Behnes2 , G .G . Wulf1 , P . Thelen3 , L . Opitz4 ,
G . Salinas-Riester4 , O . Wirths5 , T .A . Bayer5 , H .-J . Radzun2 , S . Schweyer2 1University Medicine Göttingen, Department of Hematology and Oncology,
Göttingen, 2University Medicine Göttingen, Department of Pathology, Göttingen,
3University Medicine Göttingen, Department of Urology, Göttingen,
4 5 University Medicine Göttingen, DNA Microarray Facility, Göttingen, University
Medicine Göttingen, Division of Molecular Psychiatry, Göttingen
Aims. There is no doubt that the amyloid precursor protein (APP) and its
proteolytically derived Aβ species significantly contribute to the pathogenesis
of Alzheimer disease. However, the normal physiological role of
this ubiquitously expressed protein has remained largely unknown. In
the current study, we characterized APP expression in a panel of human
testicular germ cell tumors (TGCT) of different histological origin. Furthermore,
we analysed whether histone deacetylase (HDAC) inhibitors
effectively induce cell differentiation and impact stem cell signature and
APP protein levels in embryonal carcinoma (EC) cell lines. These analyses
were also performed in a physiologically relevant in vivo setting
using an established xenograft mouse model.
Methods. Paraffin-embedded tissue blocks from orchiectomy specimens
were used for tissue microarray construction consisting of 173 cases of
pure and mixed TGCTs as well as eight randomly selected normal testicular
tissues. Following TGCT cell lines were used: NCCIT, NTera-2 (EC
cell lines) and TCam-2 (seminoma cell line). Cellular differentiation was
analysed by cell proliferation and cytotoxicity assays, cell morphology
via fluorescence microscopy and expression analyses of stem cell genes
and lineage-specific differentiation markers were determined using microarray
analysis, qRT-PCR and Western blot analysis. APP expression
was selectively down-regulated using target-specific siRNA duplexes.
Xenografts inoculated with NTera-2 were orally treated with the HDAC
inhibitor VPA and tumor growth as well as APP protein levels were compared
to vehicle treated animals.
Results. APP is exclusively expressed in pluripotent germ cell cancer
subtypes (EC and seminoma). Differentiated TGCTs (e.g. teratoma) only
presented low or lack of APP expression. APP knock-down induced the
expression of lineage-specific differentiation markers. HDAC inhibitor
treatment induced cell differentiation, accompanied by down-regulated
APP protein levels and stem cell genes. Moreover, GRP78 could be
identified as a key factor that specifically triggers proteasomal degradation
of APP. Oral administration of VPA significantly suppressed tumor
growth and depleted APP protein levels in vivo.
Conclusions. Our results indicate that APP behaves as a reliable biomarker
for transformed human pluripotent stem cells and also shed light
on the significance of APP as a novel molecular target and furthermore
broaden the therapeutic potential of HDAC inhibitors in the clinical treatment
of TGCT.
SA-P-066
Thymoquinone lowers toxicity and increases efficacy of
5-fluorouracil
C . El-Baba1 , S . Morgenthal2 , M . Ocker3 , H . Gali-Muhtasib4 , R . Schneider-Stock 1
1 2 University of Erlangen-Nuremberg, Institute of Pathology, Erlangen, Christian-Albrechts-University,
Institute of Pathology, Köln, 3Phillips-University of Marburg, Institute of Surgical Research, Marburg, 4American University of
Beirut, Department of Biology, Beirut, Lebanon
Aims. Colorectal cancer is a non-negligible cause of mortality worldwide.
5-fluorouracyl (5-FU) has proved to be one of the most effective chemotherapeutics
for colorectal cancer but an inactivated p53 status leads to a
resistance to 5-FU. We have shown that thymoquinone (TQ), a bioactive
compound extracted from Nigella sativa (black seed) exerts promising
anti-apoptotic effects independent of the p53 status of tumor cells. Our
aim is to investigate if TQ is able to sensitize colorectal cancer cells to
5-FU to minimize its cytotoxic side-effects in the clinical setting.
Methods. Human colon cancer cells HT29 (mutant p53), HCT116 (p53+/+)
and normal intestinal cells HCEC were treated with TQ and/or 5-FU.
Cell viability was measured via crystal violet, mitochondrial activity and
colony formation assays. Cell cycle distribution and apoptosis were assessed
by flow cytometry via propidium iodide (PI), Annexin-V staining,
and Western blotting. A mouse xenograft study was conducted for a better
assessment of potential in vivo effects.
Results. HCT116wt cells were more sensitive to TQ than HT29 cells.
HCEC cells were highly resistant to TQ treatment, which indicates that
TQ has an effect mainly on cancerous cells. In HT29 cells, the combination
TQ/5-FU was equally efficient as the treatment with ten times
higher concentration of 5-FU alone. Annexin-V, propidium iodide-cell
cycle analysis, as well as Caspase 3 and Caspase 9 cleavage along with the
increase of Bax to Bcl2 ratio, revealed a higher apoptosis induction when
the cells are treated with both drugs in comparison to either TQ or 5-FU.
In vivo study showed that the relative tumor size of mice co-treated with
TQ and 5-FU was significantly reduced in comparison with the tumors
of control mice or mice treated with either drug alone.
Conclusions. TQ when combined with the antineoplastic agent 5-FU
was increasing apoptosis in colon cancer cells. The combination therapy
could overcome the resistance to 5-FU in the p53 mutant tumor cells without
showing dramatic cytotoxicity on normal colon cells. Combined
with further clinical studies this approach might be promising for the
improvement of colorectal cancer treatment.
Der Pathologe · Supplement 1 · 2012 |
159

Abstracts
SA-P-067
Overexpression of anti-apoptotic CIAP2 is typical of thymic
squamous cell carcinoma but not thymomas – hints to functional
relevance
D . Belharazem 1 , B . Huang 2 , L . LI 3 , P . Stroebel 1 , A . Marx 1
1 University Medical Center Mannheim; University of Heidelberg, 2 Institut
of Pathology; University of Würzburg, 3 Medical Research Center, Medical
Faculty Mannheim
Aims. Thymomas and thymic carcinomas (TCs) are epithelial tumors of
the thymus. Their molecular oncogenesis is poorly understood. Recent
extensive sequencing of a broad spectrum of receptor and non-receptor
kinase genes in thymomas and TCs (Girard N et al, Clin Cancer Res
15:6790, 2009) failed to identify recurrent mutations except for known
activating KIT mutations in

nic markers such as myf4 and MyoD1 are a helpful and sometimes indispensable
diagnostic tool for the correct diagnosis of rhabdomyosarcoma.
SA-P-070
Angiofibromyxoma of the umbilical cord
A .M . Müller 1 , U . Gembruch 2 , M . Vogel 3
1 University Bonn, Department of Pediatric Pathology, Bonn, 2 University
Bonn Medical Center, Dept . of Obstetrics and Prenatal Therapy, Bonn, 3 University
of Leipzig, Institute of Pathology
Aims. Tumours of the umbilical cord are extremely rare. Differential diagnosis
includes hemangiomas, teratomas, abdominal wall defects and
vascular lesions like hematoma and thrombosis. Hemangiomas are by
far the most common umbilical cord tumours.
Methods. In a 35-year-old woman in the 25th week of gestation a tumour
of the umbilical cord was ultrasonographically diagnosed. Ultrasound
displayed a significantly broadened umbilical cord with hyperechogenic
areas and increased whartons’ jelly and arterio-venous fistulas. Apart
from a slight cardiomegaly fetal heart was regular. In week 38 a healthy
girl was born by cesarean.
Results. Histologically the placenta showed several chorangiomas. The
umbilical cord displayed an angiofibromyxom with – taking into consideration
ultrasound findings – intratumoral arterio-venous malformations.
Conclusions. Angiofibromyxoma with intratumoral arteriovenous malformations
is a very rare subtype of hemangiomatous lesion of the umbilical
cord. Umbilical cord is formed between the sixth and eighth week
of gestation by the approximation of the omphalomesenteric duct resp
yolk sac and the allantoic duct within the body stalk. The two arteries
and one vein derive from the allantoic vessels. Hence cord hemangiomas
are recognized as arising from omphalomesenteric or allantoic vessels.
As they often occur together with the more common chorangiomas –
like in our case – an underlying congenital predisposition to vascular
neoplasms has to be discussed.
SA-P-071
Pregnancy luteoma – an uncommon ovarian tumor: association
with intrauterine growth retardation (IUGR)?
C . Jayasinghe1 , T . Ameziane2 , C . Auerbach2 , A .M . Müller3 1Gummersbach Hospital, Institute of Pathology, Gummersbach,
2St . Elisabeth Hospital Bonn, Department of Obstetrics and Gynecology,
Bonn, 3University Bonn, Department of Pediatric Pathology, Bonn
Aims. Pregnancy luteomas are rare benign lesions of the ovary induced
by hormonal alterations during pregnancy. They present as unilateral
or bilateral tumorous masses and are usually incidental findings during
imaging or caesarean section. Most of the patients are asymptomatic.
However, virilization of the mother occurs in one third of cases and virilization
of the female fetus is found with two thirds of virilized mothers.
Pregnancy luteomas regress postpartum spontaneously, therefore
conservative treatment is sufficient. Nevertheless pregnancy luteomas
are challenging, particularly for clinicians, as they can mimic malignant
ovarian tumors.
Methods. We present the case of a 30-year-old primigravida with beta
thalassemia major who underwent caesarean section at week 37 because
of pathological CTG. A hypotrophic girl was delivered with normal
Apgar score. Both mother and daughter showed no endocrine abnormalities.
Intraoperative both ovaries were enlarged and multicystic. One
ovary was resected for histopathologic examination.
Results. Histomorphology of the ovary displayed nodules of luteinized
cells and muliple luteinized ovarian cysts within an edematous stroma,
typical histological findings of a pregnancy luteoma. Placental examination
revealed signs of insufficiency.
Conclusions. Pregnancy luteoma is a rare ovarian lesion which can macroscopically
be misinterpreted as malignancy. Awareness of this entity
can avoid unnecessary adnexectomy in young patients as pregnancy
luteomas regress spontaneously. Although hyperandrogenism can be
associated with IURG it is hardly probable that in the present case fetal
hypotrophy was due to pregnancy luteoma because the level of secreted
androgens was not high enough to cause manifest hyperandrogenism.
Instead IUGR in our case is more likely attributable to placental insufficiency
and/or beta thalassemia major.
SA-P-072
Infantile digital fibromatosis (IDF) – A case report and review of
the literature
U . Titze1 , R . Rödl2 , G . Köhler1 1University Hospital Münster, Gerhard-Domagk-Institute for Pathology,
Münster, 2University Hospital Münster, Department of General and Tumor
Orthopedics, Münster
Aims. We report on a 7 months old male infant who received excisionbiopsy
of a rapidly growing dermal tumor from the 2nd toe of the right
side.
Methods. Histological, immunohistological and ultrastructural examination
revealed typical findings of an infantile digital fibromatosis
(WHO: inclusion body fibromatosis).
Results. Infantile digital fibromatosis (IDF) is a rare, distinctive benign
fibroblastic/myofibroblastic tumor of infancy typically arising in the digits
of the hands or feet. Characteristic morphological findings in the
proliferating spindled cells are characteristic rounded eosinophilic paranuclear
inclusions.
Conclusions. Etiology of IDF remains uncertain. Despite of its benign
behavior, local recurrence was seen in up to 60% of cases after surgical
therapy. Local installations of corticosteroids do not reduce the size of
these lesions. Current management of IDF recommends avoiding surgical
intervention, as spontaneous involution is the rule.
SA-P-073
Male fetus with ectrodactyly ectodermal dysplasia clefting (EEC)
syndrome
F . Fronhoffs1 , S . Detering2 , S . Gerlach1 , C . Berg3 , M . Born4 , A .M . Müller1 1 2 University Bonn Medical Center, Institute of Pathology, Bonn, University
Bonn Medical Center, Institute of Pathology, Bonn, 3University Clinic of Cologne,
Department of Prenatal Medicine and Ultrasound, Köln, 4University Bonn Medical Center, Institute of Radiology, Bonn
Aims. Characteristics of the ectrodactyly ectodermal dysplasia clefting
(EEC) syndrome, first described by Rüdiger et al in 1970, are ectrodactyly,
dysplasia of skin, its adnexal structures and/or teeth as well as orofacial
clefts. Its exact prevalence is unknown. Until now, about 300 cases
have been reported in literature. In more than 90% of all cases, a missense
mutation in the gene TP63 can be detected.
Methods. 33-year-old mother, gravida 1, para 1. Proof of bilateral complete
cleft of lip and palate and ectrodactyly of both feet and hands and
tentative sonographic diagnosis of complex cloacal persistence and malformation.
Confirmation of EEC-syndrome by genetic testing. Feticide
in 24th+4 week of gestation.
Results. Male fetus, appropriate for gestational age, with bilateral complete
cleft of lip and palate accompanied by deformation of the nasal apex.
Ectrodactyly of both feet and hands. Right hand with five metacarpals (I,
III–V regular, II shortened) and agenesis of phalanges II and III. At the
left hand only a rudimentary anlage of digitus II but regularly formed
digitus I and III–V. Right foot with five metacarpals but shortened metacarpal
II. Left foot with five regularly shaped metacarpal bones, but only
four phalanges (I and III–V), i.e. missing second toe. Time-adequate development
of the nails. Histologically, in the skin biopsy only very few
Der Pathologe · Supplement 1 · 2012 |
161

Abstracts
perspiratory and sebaceous glands. Very scarce scalp hair. Additionally,
a megacystis with pseudodiverticulum and dilatated and sidled ureters.
Conclusions. This fetus presented classic findings of the EEC syndrome.
Because of the additional urogenital anomalies the diagnosis was expanded
to ectrodactyly ectodermal dysplasia syndrome with urinary tract
pathology (EECUT).
SA-P-074
Femur fibula ulna (FFU) complex
A .M . Müller1 , S . Detering2 , U . Gembruch3 1 2 University Bonn Medical Center, Institute of Pathology, Bonn, University
Bonn Medical Center, Institute of Pathology, Bonn, 3University Bonn Medical
Center, Dept . of Prenatal Medicine and Obstetrics, Bonn
Aims. Femur fibula ulna (FFU) complex is a sporadic, non-lethal malformation
characterized by typical unilateral combination of defects of the
femur and fibula and contralateral defect of the ulna.
Methods. We present a fetus of 23 weeks of gestation of consanguine parents.
Results. Macroscopically the fetus showed a short collar, hypertelorism,
slightly down sloping palpebral fissures, short, flat nose, small lips and
high arched palate and dysmorphic ear concha. In comparison to the
right side, left sided upper and lower leg were significantly shortened, the
foot appeared as clubfoot. Furthermore, in comparison to the left side,
the right forearm was shortened, the right hand displaying four fingers
and an aplasia of the thumb.
Conclusions. Etiology of the sporadically occurring FFU complex is unknown.
Up to now there are no signs a paternal age effect or an association
with consanguinity. Neither could chromosomal studies reveal any
abnormalities. Furthermore there is no evidence for an infectious or teratogenic
cause. Children show normal mental development and normal
life expectancy. As – dependent on the number of involved malformed
limbs – the FFU complex is grouped in four groups (I–IV) this case can
be assigned to type II.
SA-P-075
Fetal manifestation of the Peters’ plus syndrome associated with
lenticular ectopia and occipital meningocele in one of the cases
K . Schoner1 , J . Kohlhase2 , J . Steinhard3 , R . Bald4 , A . Schwan5 , P . Wieacker6 ,
H . Rehder1 1 2 Philipps University Marburg, Institute of Pathology, Marburg, Praxis of
Human Genetics, Freiburg, 3Department of Obstetrics, Münster, 4Clinic of
Gynecology, Leverkusen, 5Division of Human Genetics, Dortmund, 6Institute of Human Genetics, Münster
Aims. Fetal pathology aims to recognize syndrome specific patterns of
malformations and dysmorphic features for goal directed mutation analyses,
genetic counselling of the parents and early prenatal molecular
diagnoses in consecutive pregnancies. Here we report on four fetuses
with Peters’ plus syndrome from two distinct families.
Methods. We performed fetal autopsies after prenatal ultrasound diagnoses
of malformations and termination of pregnancy and carried out
molecular genetic investigations on fetal and parental DNA.
Results. Four fetuses of 16 to 22 gestational weeks presented with multiple
malformations and dysmorphic signs in addition to Peters’ anomaly of
the eyes. The latter comprised central sclerocornea, absence of the posterior
corneal stroma, and a variable degree of iris and lenticular attachments
to the central posterior cornea in association with microphthalmia
and lenticular ectopia. Additional features concerned hydrocephaly,
a characteristic round face with cleft lip and palate, hypertelorism and
prominent front, short stature, brachydactyly, and also cardiac, renal,
genital and cerebral malformations including occipital meningocele. Peters’
plus syndrome was confirmed by sequence analysis of the B3GALTL
gene revealing homozygosity for the common 660+1G>A donor splice
162 | Der Pathologe · Supplement 1 · 2012
site mutation in intron 8 in all four cases and heterozygosity for this mutation
in the Caucasian, non-consanguineous parents.
Conclusions. The four affected fetuses show a characteristic facial aspect
that in association with the accompanying malformations should enable
the diagnosis of a Peters’ plus syndrome. Peters’ anomaly of the eyes,
representing an evolutive feature, is already evident at 18 weeks of gestation.
However, manifestation of the disorder is variable. Occipital meningocele
is a novel finding in Peters’ plus syndrome.
SA-P-076
Massive ovarian edema (MOE)
V . Sailer1 , S . Huss1 , F . Fronhoffs1 , E . Wardelmann1 , A .M . Müller1 1University Clinic of Bonn, Institute of Paidopathology and Institute of
Pathology, Bonn
Aims. Massive ovarian edema (MOE) is a very rare benign tumor-like
condition found in young women resulting from accumulation of fluid
mostly due to partial or intermittent torsion of the ovary or secondary to
a pre-existing ovarian lesion.
Methods. We report a case of a 13-year-old girl that presented with an
ovarian mass measuring 16 cm in diameter. Ultrasound and CT-scan
revealed a multilobulated cystic mass. CA-12-5 levels were increased.
Concerns regarding underlying malignancy lead to unilateral salpingooophorectomy.
Results. Pathological evaluation revealed a MOE and multiple thromboses
of ovarian veins.
Conclusions. Differentiation MOE from malignant tumor is crucial to
prevent unnecessary surgery potentially resulting in hormonal dysfunction
and infertility. Conservative treatment is possible and may be more
appropriate in cases when histology on frozen section supports a benign
lesion.
SA-P-077
Infantile myofibroma of the thyroid gland
A . Agaimy1 , D . Schmidt2 , P . Klein3 , R . Carbon4 , W . Holter5 1Friedrich-Alexander University of Erlangen, Institute of Pathology, Erlangen,
2Friedrich-Alexander University of Erlangen, Department of Nuclear Medicine,
Erlangen, 3Friedrich-Alexander University of Erlangen, Department of
Surgery, Erlangen, 4Friedrich-Alexander University of Erlangen, Department
of Surgery, Erlangen, 5Friedrich-Alexander University of Erlangen, University
Children‘s Hospital, Erlangen, Erlangen
Aims. Spindle cell lesions of the thyroid gland are rare and may thus be
diagnostically challenging. They encompass a heterogeneous group of
reactive mesenchymal lesions, and a variety of benign and malignant
neoplasms of epithelial and mesenchymal origin.
Methods. A 5-year-old girl presented with a rapidly growing firm nodular
cervical mass localized to the right thyroid lobe associated with bilateral
lymphadenopathy. Because of symptoms and concern about malignancy,
an open surgical biopsy was performed followed by resection
of the right lobe and biopsy of the cervical nodes. The patient is alive with
no evidence of recurrence 18 months after surgery.
Results. The specimen contained a 3.8 cm firm tan circumscribed nodular
mass surrounded by a thin rim of thyroid tissue. Histological examination
displayed a moderately cellular lesion composed of alternating
fascicles of eosinophilic myoid spindled cells and primitive looking
small rounded cells with hemangiopericytoma-like vascular pattern and
a prominent myointimal proliferation at the periphery of the lesion. The
myoid cells expressed strongly alpha-smooth muscle actin but were negative
for desmin, h-caldesmon, epithelial membrane antigen, pankeratin,
CK7, thyroglobulin, TTF-1, protein S100, TLE1, ALK-1, beta-catenin,
CD31, CD34 and CD99. The lymph nodes showed reactive florid hyperplasia
without evidence of tumor.

Conclusions. To our knowledge, this case represents the first report of
solitary myofibroma presenting as a thyroid mass. Awareness of this differential
diagnosis is necessary to avoid misinterpretation as a sarcoma
with the sequelae of unnecessary over-treatment.
Poster: Uropathologie I
SA-P-078
Rearrangement of the ETS genes ETV-1, ETV-4, ETV-5 and ELK-1 is
a clonal event during prostate cancer progression
M . Braun1 , Z . Shaikhibrahim1 , P . Nikolov1 , D . Böhm1 , V . Scheble2 , R . Menon1 ,
F . Fend3 , G . Kristiansen1 , N . Wernert1 , S . Perner1 1 2 University Hospital Bonn, Institute of Pathology, Bonn, University Hospital
Tübingen, Division of Hematology and Oncology, Tübingen, 3University Hospital Tübingen, Institute of Pathology, Tübingen
Aims. ETS gene rearrangements are frequently found in prostate cancer
(PCa). Several studies have assessed the rearrangement status of the most
commonly found ETS gene, ERG, and the less frequent genes, ETV-1,
ETV-4, ETV-5 and ELK-4 in primary PCa. However, the frequency in
metastatic disease is still not well investigated. Recently, we have assessed
the ERG rearrangement status in both primary PCa and the corresponding
lymph node metastases, and observed that ERG rearrangement in
primary PCa transfers into lymph node metastases, suggesting it to be a
clonal expansion event during PCa progression. As a continuation, we
investigated in this study whether this observation is valid also for the
less frequent ETS rearranged genes ETV-1, ETV-4, ETV-5 and ELK-4.
Methods. Using dual color break-apart FISH assays, we evaluated the status
of all the less frequent ETS gene rearrangements for the first time on
tissue microarrays (TMAs) constructed from a large cohort comprised
of primary PCa and the corresponding lymph node metastases. Additionally,
we evaluated the rearrangement status of all these ETS genes in a
second cohort comprised of distant metastases.
Results. ETV-1, ETV-4, ETV-5 and ELK-4 rearrangements were found in
8/81 (10%), 5/85 (6%), 1/85 (1%) and 2/86 (2%) of the primary PCa, respectively,
and in 6/73 (8%), 5/85 (6%), 4/72 (6%), 1/75 (1%) of the corresponding
lymph node metastases, respectively. Rearrangements of ETV-1 and
ETV-5 were not found in any of the distant metastases cases, whereas
ETV-4 and ELK-4 rearrangements were found in 1/25 (4%) and 1/24 (4%)
of the distant metastases, respectively.
Conclusions. Our results suggest that rearrangement of the less frequent
ETS genes is a clonal event during prostate cancer progression. Our findings
provide insights into potential clonal expansion events during PCa
progression and may have significant implications in understanding the
molecular basis of the metastatic cascade of PCa.
SA-P-079
ERG protein expression and genomic rearrangement status in
primary and metastatic prostate cancer – a comparative study of
two monoclonal antibodies
M . Braun1 , D . Goltz1 , Z . Shaikhibrahim1 , W . Vogel 1 , D . Böhm1 , V . Scheble2 ,
K . Sotlar3 , F . Fend4 , A . Dobi5 , G . Kristiansen1 , N . Wernert1 , S . Perner1 1 2 University Hospital Bonn, Institute of Pathology, Bonn, University Hospital
Tübingen, Division of Hematology and Oncology, Tübingen, 3University Hospital Munich, Institute of Pathology, München, 4University Hospital
Tübingen, Institute of Pathology, Tübingen, 5Uniformed Services University
of the Health Sciences, Center for Prostate Disease Research, United States
Aims. Overexpression of the ERG protein is highly prevalent in prostate
cancer (PCa) and most commonly results from gene fusions involving
the ERG gene. Recently, an N-terminal epitope targeted mouse and a
C-terminal epitope targeted rabbit monoclonal anti-ERG antibody have
been introduced for the detection of the ERG protein. Independent studies
reported that immunohistochemical (IHC) stains with both monoclonal
anti-ERG antibodies (ERG-MAbs) highly correlate with the
underlying ERG gene rearrangement status. However, a comparative
study of both antibodies has not been provided so far. Here, we are the
first to compare the mouse ERG-MAb to the rabbit ERG-MAb for their
concordance on the same PCa cohort. Furthermore, we assessed if the
ERG protein expression is conserved in lymph node and distant PCa metastases,
of which a subset underwent decalcification.
Methods. We evaluated tissue microarrays of 278 specimens containing
265 localized PCa, 29 lymph node, 30 distant metastases, and 13 normal
prostatic tissues. We correlated the ERG protein expression with the
ERG rearrangement status using an ERG break-apart fluorescence insitu
hybridization (FISH) assay and IHC of both ERG antibodies.
Results. ERG protein expression and ERG rearrangement status were
highly concordant regardless of whether the mouse or rabbit ERG-MAb
was used (97.8% versus 98.6%, respectively). Of interest, both ERG antibodies
reliably detected the ERG expression in lymph node and distant
PCa metastases, of which a subset underwent decalcification. Lymphocytes
revealed immunoreactivity using the rabbit ERG-MAb, but not
using the mouse ERG-MAb. If an ERG protein expression was present
in localized PCa, we observed the same pattern in the corresponding
lymph node metastases.
Conclusions. This is the first study to comprehensively compare the two
available ERG-MAbs. By demonstrating a broad applicability of IHC to
study ERG protein expression using either antibody, this study adds an
important step towards a facilitated routine clinical application. Further,
we demonstrate that the clonal nature of the ERG rearrangement is not
restricted the genomic level, but proceeds in the proteome. Together, our
results simplify future efforts to further elucidate the biological role of
ERG in PCa.
SA-P-080
MicroRNA miR-205 is down-regulated in prostate cancer depending
on Gleason score and tumour size
B . Verdoodt1 , M . Vogt1 , V . Kuhn1 , A . Tannapfel1 , A . Mirmohammadsadegh 1 , M .
Neid1 1Ruhr-University Bochum, Institute for Pathology, Bochum
Aims. miR-205 plays a role in the repression of the epithelial to mesenchymal
transition in different epithelial tumours, and targets anti-apoptotic
and cell cycle regulating genes. We studied the expression of miR-
205 and its relation to clinical parameters in archival samples of prostate
cancer with different Gleason scores.
Methods. miRNA was isolated by micro-dissection from 84 formalin
fixed/paraffin embedded prostatectomy specimens with prostate cancer
of Gleason score 3+3 (n=12), 3+4 (n=32), 4+3 (n=30), and 4+4 (n=10),
and from corresponding benign prostate tissue. miR-205 levels were determined
by quantitative real time PCR in comparison to RNU6B (U6
small nuclear RNA 2) as reference gene. Data was correlated with Gleason
score, size, and extraprostatic tumour extension.
Results. Expression of miR-205 was lower in tumour tissue than in benign
tissue from the same patient in 76 of 84 cases (90.5%, median expression
18%). It decreased depending on Gleason score, with median
0.303 in tumours with Gleason score 3+4, median 0.150 in tumours with
Gleason score 4+3, and median 0.087 in tumours of Gleason score 4+4
(p

Abstracts
SA-P-081
Significance of Gleason Grading in a clinical setting that considers
active surveillance as a therapeutic option of prostatic low grade
cancer
B . Helpap 1 , G . Kristiansen 2 , J . Köllermann 3 , U . Oehler 1 , C . Fellbaum 1
1 HBH-Hospital, Dept . Pathology, Singen, 2 University of Bonn, Dept . Pathology,
Bonn, 3 HKS-Wiesbaden, Dept . Pathology, Wiesbaden
Aims. Active surveillance has become an increasingly accepted clinical
strategy to handle patients with presumably insignificant carcinomas
by observant waiting, without endangering the curative intent. Aim of
this analysis was to evaluate the diagnostic value of nuclear features in
addition to Gleason grade in the prediction of non-aggressive disease in
a large and representative prostate cancer cohort.
Methods. A cohort of 968 prostatectomy specimens with matching preceding
biopsies (12 cores) was morphologically analysed. Architectural
features according to Gleason and cytological grading were recorded
and compared.
Results. The combination of architectural and cytoplogical features as
incorporated in the Helpap Grading increase the rate of agreement of
grading between the biopsy and the prostatectomy specimens especially
GS 6 up to 90%. The parallel use of Gleason and Helpap grading allows
for a better prediction of organ confined disease (pT2) following prostatectomy.
Conclusions. By adding cytologic features to Gleason grading, an increased
diagnostic accuracy in the identification of low grade carcinomas,
which may be treated by active surveillance, can be achieved.
SA-P-082
The Microtubule-associated Protein 2 (MAP2) is frequently expressed
in prostate cancer and its precursor lesions
M . Majores1 , E . Krappe1 , N . Wernert1 , J . Ellinger2 , G . Kristiansen1 1 2 University of Bonn, Department of Pathology, Bonn, University of Bonn,
Department of Urology, Bonn
Aims. The microtubule-associated protein 2 (MAP2) is involved in microtubule
assembly and plays a crucial role for nucleation and stabilization
of microtubules. MAP2 is frequently expressed in mature neurons
and tissue with neuroendocrine differentiation.
Methods. We incidentally revealed that MAP2 is expressed in prostate
cancer (PCA) and have evaluated the immunohistochemical characteristics
of MAP2 expression in 107 PCA specimens in comparison to adjacent
normal and dysplastic tissue.
Results. MAP2 expression was strikingly focused on high-grade PIN lesions
and invasive tumour glands: moderate or strong immunolabelling
was found in 92% of high-grade PIN (n=61/68) and in 58% (n=62/107) of
low-grade PIN lesions. In contrast, normal glands or hyperplastic epithelia
of the periurethral zone stained weakly. Invasive carcinoma was
MAP2-positive in 86% of Gleason pattern (GP) 3 glands (n=89/103), in
78% of GP 4 (n=28/36) and in 75% of GP 5 areas (n=6/8). In several cases,
MAP2 was expressed in high-grade PINs with continuous transition to
invasive carcinomas.
Conclusions. Our preliminary findings support MAP2 as a promising
new immunomarker for PCA and PIN lesions and moreover point to
MAP2 as an “interface marker” in the progression from in-situ lesions to
invasive carcinomas. We currently conduct correlations between MAP2
expression and clinicopathological features including patient survival
times.
164 | Der Pathologe · Supplement 1 · 2012
SA-P-083
CyclinD1 expression indicates possible lymph node metastasis in
invasive bladder cancer
J . Rokahr1 , E . Herrmann2 , M . Bögemann2 , S . Bierer2 , E . Eltze3 1 2 University of Muenster, Institute of Pathology, University of Muenster,
Department of Urology, 3Institute of Pathology Saarbrücken Rastpfuhl,
Saarbrücken
Aims. Overexpression of proteins involved in antiapoptosis or proliferation
is often associated with tumour progression and treatment resistance.
Cyclin D1 and BCL2 play a potential role in progression of bladder cancer
like Ki67 and TP53.
Methods. Immunhistochemical stainings were performed for Cyclin D1,
BCL2, TP53 and Ki67 on TMA containing paraffin embedded tissues of
219 invasive bladder cancer patients who had undergone radical cystectomy
between 1987 and 2004. The results were correlated with clinicopathological
parameters and overall and recurrence-free survival.
Results. Expression of the BCL2 was found in only 19 tumours, and only
moderately in 6 of these. Overexpression of BCL2 correlated with a low
proliferation rate (Ki67< 10%, p=0.067) and a low grade (p=0.004). No
correlation could be found to pTstage or TP53 expression or survival
data. Cyclin D1 expression of correlated significantly with pN1 (p=0.031),
whereas no correlations to tumour stage, grade, TP53 expression or proliferation
rate could be detected. Kaplan Meier analysis showed a significant
shorter overall survival for patients with Cyclin D1 expression
tumours (p=0.022).
Conclusions. The correlation between Cyclin D1 expression and lymph
node metastasis and a poor prognosis in invasive bladder cancer after
cystectomy indicates a role in mediating invasion and metastasis of cancer
cells.
SA-P-084
High IMP3 protein expression is a negative prognostic factor in
advanced urothelial carcinoma of the bladder
H . Reis1 , 2 , F . vom Dorp3 , C . Niedworok3 , C . Niedworok4 , A . Melchior-Becker4 ,
J .W . Fischer4 , D . Gödde1 , B .B . Singer5 , A . Bankfalvi2 , I . Romics6 , K .W . Schmid2 ,
S . Störkel1 , S . Ergün5 , H . Rübben3 , T . Szarvas3 , 7
1 2 University of Witten/Herdecke, Institute of Pathology, Wuppertal, University
of Duisburg-Essen, Institute of Pathology and Neuropathology, Essen,
3 4 University of Duisburg-Essen, Department of Urology, Essen, University
of Duisburg-Essen, Institute of Pharmacology and Clinical Pharmacology,
Essen, 5University of Duisburg-Essen, Institute of Anatomy, Essen, 6Semmel weis University Budapest, Department of Urology, Budapest, Hungary,
7Medical University of Vienna, Department of Urology, Wien, Austria
Aims. The identification of the prognostic influence and interactions of
the Insulin-like growth factor mRNA-binding protein 3 (IMP3) in advanced
urothelial carcinoma of the bladder (UCB).
Methods. A total of 224 urothelial bladder carcinoma cases were studied
regarding IMP3 expression by immunohistochemistry, real-time PCR
and Western blot analyses. The molecular targets of IMP3 – CD44, IGF2
and its receptor IGF1-R – were also investigated. Expression levels were
correlated with clinical follow-up data in univariate and multivariate
analyses.
Results. In high-stage and high-grade UCB both IMP3 protein and
mRNA levels were significantly elevated. In muscle-invasive cancer
IMP3 protein but not gene expression proved to be an independent
predictor of disease-specific (HR=2.58, 95%CI 1.28–4.56, p=0.004) and
overall survival (HR=2.07, 95%CI 1.12–3.82, p=0.020). IGF2 and CD44
expression showed no correlation with that of IMP3.
Conclusions. Identification of high IMP3 protein levels in UCB might aid
in the selection of patients at high risk for disease progression and in the
stratification to groups with more intensive therapy or strict follow-up.
No tumor promoting effect of IMP3 in its regulatory action on IGF2 and
CD44 expression was observable.

SA-P-085
Expression of the eukaryotic translation initiation factor 3a in
urinary bladder cancer
R . Spilka 1 , A .K . Mehta 2 , J . Haybaeck 2 , P . Obrist 1
1 Pathologylab Dr . Obrist & Dr . Brunhuber OG, Zams, Austria,
2 Institute of Pathology, Medical University of Graz, Austria
Aims. Urinary bladder cancer (UBC) is a frequent and aggressive urinary
tract cancer, of transitional cell type, with high mortality rates. One
obstacle in defining novel treatment approaches is that the cancer’s aetiology
and genetics are not yet completely understood. eIF3a, the largest
subunit of eukaryotic initiation complex eIF3, is up-regulated in many
cancers including breast, cervix, colon, esophagus, lung and stomach
and its suppression leads to inhibition of tumour cell proliferation in
vitro. We are therefore aiming at evaluating the translation initiation
factor eIF3a in UBC to gain insight in the pathogenesis of these tumors
and to further determine the role of eIF3a in cancer development and
progression.
Methods. eIF3a expression was determined by immunohistochemistry
on paraffin embedded tissues from 178 UBC patients. Protein expression
levels of eIF3a were analysed in five UBC cell lines by western blotting.
Furthermore by manipulating eIF3a levels in tumor cell lines, HT1197
and RT4-31, we want to explore whether eIF3a expression levels can directly
influence global as well as specific translation and proliferation.
We are therefore generating an inducible shRNA mediated eIF3a-knockdown
construct for lentiviral transfection.
Results. eIF3a is upregulated in UBC when compared to normal tissues,
similar to the cancer entities where eIF3a was described to be overexpressed
before. We have successfully tested two lentiviral shRNA constructs
for the inducible knockdown of eIF3a. The knockdown construct
generated proves efficient in all tested cell lines and first results show an
association of eIF3a knockdown with growth retardation of tumor cells.
Conclusions. Overexpression of eIF3a seems to be tumor-associated over
a wide range of tumor entities, with a potential as prognostic marker.
The knockdown of eIF3a leads to reduced proliferation rates, indicative
of subcellular changes arising probably not exclusively from altered
translational profiles.
SA-P-086
A case of clear cell renal carcinoma arising in a renal angiomyolipoma
A . Dellmann1 , A . Vandromme2 , P . Hammerer2 , K . Donhuijsen1 1Academical Hospital Braunschweig, Department of Pathology, Braunschweig,
2Academical Hospital Braunschweig, Department of Urology,
Braunschweig
Aims. Renal angiomyolipoma (AML) is a mesenchymal tumor of the
kidney that usually shows a benign course. MRI usually enables reliable
detection of fat, which is typical for angiomyolipoma, and allows the
differentiation to a renal cell carcinoma. We present a rare case of a renal
cell carcinoma appearing in AML, thus showing that clear cut diagnosis
of AML in MRT in not always possible.
Methods. We report about a case of a renal cell carcinoma appearing in
AML in a 77-year-old male. Pretherapeutic radiologic imaging of the tumour
was fitting for AML. Histological examination including immunohistochemistry
was performed.
Results. In this case of an AML appearing in the right kidney the pretherapeutic
imaging was typical. Histologic examination showed a combined
tumour with features of an AML and of renal cell carcinoma within.
Immunohistochemical studies showed typical results for AML on the
one hand and for RCC on the other. Chromosomal aberrations will be
examined by FISH.
Conclusions. Angiomyolipoma is a combined mesenchymal tumour of
the kidney with a usually benign course. Tumour can be associated with
tuberous sclerosis. Although MRI usually allows the differential diagno-
sis to a renal cell carcinoma, in our case a RCC was found within the
AML. The possibility of RCC appearing in AML has to be kept in mind.
SA-P-087
Proteomic analysis of renal cell carcinoma and adjacent normal
fresh, snap-frozen tissue by MALDI Imaging mass spectrometry
B . Häupl1 , C . Recktenwald1 , D . Berger2 , H .-J . Holzhausen2 , F . Bartel2 ,
B . Seliger1 1University of Halle-Wittenberg, Institute of Medical Immunology, Halle/
Saale, 2University of Halle-Wittenberg, Institute of Pathology, Halle/Saale
Aims. Renal cell carcinoma (RCC) is the most common renal malignancy
among the tumors that are highly resistant to systemic therapy. However,
specific biomarkers for this tumor entity are not well defined. In order
to understand the biology of the tumor and to detect features which
allow the distinction between tumor and adjacent renal tissue and thus
may serve as specific diagnostic biomarkers, in situ-proteome profiling
of matched tumor and normal kidney tissue sections was carried out.
Therefore, respective biopsy samples were dissected and analyzed via
MALDI Imaging mass spectrometry (MALDI-IMS).
Methods. Cryosections (thickness 8 μm) of 28 fresh frozen tumor samples
and the respective adjacent kidney tissue were mounted onto conductive
glass slides and coated with sinapinic acid using an automated
spraying device (Bruker ImagePrepTM) after the removal of salts and
lipids by several washing steps in graded ethanol solutions. Subsequently
the samples were subjected to mass spectrometric measurement with a
MALDI-TOF MS device (Bruker ultrafleXtremeTM) in linear positive
detection mode operating at the following parameters: i) lateral resolution
of 100 μm, ii) mass range between 2 to 20 kDa and iii) 500 laser
shots per measurement position. MS data was further processed using a
software application specialized for MALDI-IMS analysis (Bruker flexImaging)
and the Bruker Clinprotools software package.
Results. Data analysis led to the generation of specific average spectra for
tumor and normal renal tissue. Although the spectra show a quite similar
peak pattern, tumor and normal specific features could be detected.
The significance of these features, which subsequently will be subjected
to mass spectrometric identification, is currently analyzed by different
algorithms such as Support Vector Machine, Genetic Algorithm or
Quick Classifier.
Conclusions. Tissue proteome profiling via MALDI-IMS is able to detect
differentially expressed features and thus allows the identification of biomarkers
for improved diagnosis that may be further used as targets for
immunotherapy of RCC.
SA-P-088
Is the mucinous tubular and spindle cell carcinoma of the kidney
a distinct entity or not?
I . Kollecker1 , A . Dellmann1 , P . Hammerer2 , K . Donhuijsen1 1Academical Hospital Braunschweig, Department of Pathology, Braunschweig,
2Academical Hospital Braunschweig, Department of Urology,
Braunschweig
Aims. Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare,
low grade renal epithelial neoplasm included into the WHO since 2004
as a distinct entity. However, with rising numbers of such cases the histologic
pattern is more and more expanding. The discrimination from
papillary renal cell carcinoma (PRCC) on the one hand and the undifferentiated
renal cell carcinoma on the other can be problematic. The
question arises whether it is really a distinct tumor entity or an new
hotchpotch of renal cell carcinoma.
Methods. We report about a little series of cases with tubular and mucinous
pattern suspect for the diagnosis of MTSCC. The cases were selected
from urologic specimen with 108 non-clear cell carcinoma out of
the last five years. The tumors were analysed on paraffin slides stained
Der Pathologe · Supplement 1 · 2012 |
165

Abstracts
by H&E, Pas-Alcian and immunohistochemical staining by CK7, CK 19,
EMA, AMACR, Vimentin, NSE, Chromogranin, Synaptophysen, CD 10,
CD 15, CD 57 and Ki-67. A FISH-analysis was done for chromosom 7 and
17.
Results. Classic cases of MTSCC mainly show a relative homogeneous
tubular architecture with low grade morphology and typical mucinous
stroma. The latter react pale for Pas and contains in Acian-blue-reaction
acid mucopolysaccids. In most cases more or less areas of papillary,
plexiforme or glomeruloid growth pattern exists. In parts with very close
packed tubuli the cells become a spindle shape character because of
compression of the tubulus but with predominant low grade atypia. Immunohistochemistry
react positive for CK 7, Ck 19, EMA, AMACR and
Vimentin. Neuroendocrine markers show different pattern. CD 10 reacts
negative. FISH-analysis brought different results for PRCC and MTSCC.
Conclusions. The main part of the MTSCC represents the described tubular
architecture with low grade atypia and typical mucinous extracellular
stroma component in variable dimension. Despite the classic
pattern different extent of other differentiation pattern like papillary,
spindle shaped, plexiforme or glomeruloid occur in the cases. Only in
exceptions tubular growth pattern was displaced by last named differentiations.
Especially papillary growth pattern make it difficult to distinguish
MTSCC from other renal cell carcinomas especially PRCC. The
demarcation to the other types is important because of prognostic differences.
Immunohistochemistry could help with CD 10 negativity but
this reaction is however non-specific. Against this background molecularpathologic
methods are due to help, but also with limitations.
SA-P-089
Solitary fibrous tumor of the kidney
M . Gajda1 , S . Harz1 , H . Wunderlich2 , K . Junker2 , M . Grimm2 , D . Katenkamp 1 ,
I . Petersen1 1 2 Institute of Pathology, Jena University Hospital, Department of Urology,
Jena University Hospital
Aims. Solitary fibrous tumors (SFT) are rare spindle cell neoplasms usually
arising in the pleura. They have, however, also been reported at extrapleural
locations. Urogenital localization is rare and to our knowledge,
only 40 cases of SFT of the kidney have been described.
Methods. Detailed clinical and histopathological review of a clinical case
and review of the literature.
Results. We report the case of a typical SFT of the right kidney in a 71-year-old
woman. As part of a CT investigation, a large mass of the right
kidney with suspicion thrombembolic compression and occlusion of the
inferior vena cava was discovered. She underwent radical nephrectomy
and lymphadenoctomy, adrenalectomy and interaortocaval lymph node
dissection. The gross specimen included the kidney (12.8×6.6×7.5 cm),
ureter and adrenal gland. Cut section showed a circumscribed pale
brown mass measuring 9.6×7.4×5.2 cm tumor. The tumors consisted of
bland-looking spindle cells arranged in short, ill-defined fascicles and
storiform pattern with characteristic hemangiopericytoma-like blood
vessels. Immunohistochemistry showed reactivity for vimentin, CD 34,
BCL-2 protein and CD99. Immunohistochemical stains for cytokeratin,
S-100, desmin, a-smooth muscle actin, RCC, EMA and CD 10 were negative.
Ki67 labelling index exceeded 10%.
Conclusions. The possibility of SFT should be considered in the differential
diagnosis of a renal mass which consists of benign-looking spindle
cells and hemangiopericytomatous blood vessels. Its diagnosis requires
immunohistochemistry and awareness of its possible existence.
166 | Der Pathologe · Supplement 1 · 2012
Poster: Uropathologie II
SA-P-090
Topotecan sensitizes renal cell carcinomas towards ABT-263
induced apoptosis
S . Heikaus1 , V . Nitsche1 , S . Funke1 , H .E . Gabbert1 , C . Mahotka1 1Heinrich-Heine University Hospital, Institute of Pathology, Düsseldorf
Aims. Renal cell carcinomas (RCCs) exhibit a marked resistance towards
conventional chemotherapeutic regiments, thus making new therapeutic
approaches necessary. So-called “targeted therapies” could be one strategy
to overcome this broad resistance. Whereas “targeted therapies” with
Kinase-Inhibitors like Sunitinib or Sorafenib are established in RCCs,
therapies directly targeting apoptotic pathways are not validated. In this
context, the BCL-2 inhibitor ABT-263 might be a promising new agent,
aiming at the mitochondrial pathway of apoptosis, which is impaired in
RCCs. We therefore examined the apoptotic effects of ABT-263 alone
and in combination with Topotecan on the apoptosis of RCC cell lines.
Methods. Cell culture, cell count, quantitative real-time detection PCR,
Western Blot, Fluorescent Immunohistochemistry, Ribonuclease Protection
Assay.
Results. 1.) Expression of pro- and antiapoptotic BCL-2 family members
was analysed in 7 RCC cell lines. Surprisingly, all cell lines expressed
not only multiple antiapoptotic but also important proapoptotic BCL-
2 family members of all functional groups on the mRNA and protein
level. 2.) ABT-263 and Topotecan induced apoptosis but not autophagy
in RCCs. 3.) Sensitivity towards ABT-263 induced cell death inversely
correlated with the expression of MCL-1. 4.) Topotecan downregulated
MCL-1 protein expression in RCCs; in contrast, ABT-263 upregulated
MCL-1 protein expression, whereas the expression of most of the other
BCL-2 family members remained unchanged. 5.) Topotecan pretreatment
weakly sensitized RCC cell lines towards ABT-263 induced apoptosis
by synergistically activating the mitochondrial pathway of apoptosis.
Conclusions. Inhibition of antiapoptotic BCL-2 family members by BCL-
2 inhibitors like ABT-263 might be a promising new approach in terms
of a “targeted therapy” in RCCs. Furthermore, a combination with other
chemotherapeutic agents can obviously enhance their proapoptotic effects.
SA-P-091
Cadherin expression in different histological types of papillary
renal cell carcinoma: a diagnostic tool
C .L . Behnes1 , B . Hemmerlein1 , A . Strauss2 , H .-J . Radzun1 , F . Bremmer1 1 2 University of Göttingen, Institute of Pathology, University of Göttingen,
Institute of Urology
Aims. Cadherins constitute a family of transmembrane glycoproteins
and include a variety of subtypes, of which E-cadherin has been widely
studied. Functionally, the cadherins belong to the adhesion molecules
and form cell-cell contacts. Furthermore they also play a role in the development
of different organs and in tumorigenesis. The papillary renal
cell carcinoma (RCC) is a rare tumor and is divided, based on histological
criteria, into two subtypes, from which the type II papillary RCC
do have a worse prognosis. There are no immunohistochemical markers
for the distinction of these subtypes. Therefore we have examined
the expression of four different cadherins in both subtypes of papillary
RCC in order to find any diagnostically relevant differences.
Methods. The expression of N-, P-, E- und KSP-Cadherin was deternined
in 22 papillary RCC of histological type I and 18 papillary RCC of
histological type II (n=40). The intensity of membranous and cytoplasmic
immunhistochemical staining was semiquantitativly evaluated by
a score from 0 to 3. To compare the data for significant differences we
used the Wilcoxon-Test.

Results. The papillary RCC type II were all positive for membranous
N-Cadherin staining, whereas type I did not show any membranous
positivity for N-Cadherin. The E-Cadherin staining showed a stronger
membranous as well as cytoplasmic expression in type II than in type I
RCC. A relevant expression of KSP- and P-Cadherin could not be demonstrated.
Conclusions. Our data show that all papillary RCC type II are characterized
by a membranous N-Cadherin expression. In contrast the papillary
RCC type I do not express N-Cadherin membranous at all. Thus
N-Cadherin represents the first immunhistochemical marker for a clear
differentiation between papillary RCC type I and type II.
SA-P-092
Myoglobin expression in renal cell carcinoma is regulated by
hypoxia
C .L . Behnes1 , J . Bedke2 , A . Strauss3 , F . Bremmer1 , H .-J . Radzun1 1 2 University of Göttingen, Institute of Pathology, University of Tübingen,
Institute of Urology, 3University of Göttingen, Institute of Urology
Aims. Myoglobin (Mb) is a member of the hemoprotein superfamily, including
in addition hemoglobin, neuroglobin and cytoglobin. The functions
of the cytoplasmic localized Mb are the prevention of hypoxia and
radical scavenging, well known from the myocytes of skletal and myocardic
muscles. In case of hypoxia Mb acts as an oxygen reservoir by
binding O2 and improving the diffusion of oxygen into the cell. It could
be shown that some cancers do express Mb preventing hypoxia. In this
study we investigated whether Mb also plays a role in renal cell carcinoma
(RCC) and a potential influence of hypoxia on the expression.
Methods. Four different RCC cell lines were cultivated under hypoxic
conditions and the expression of Mb was evaluated by real-time PCR.
For the correlatin between microvessel density and Mb expression tissue
microarrys (TMAs) with 42 different RCCs were immunhistochemically
stained with a myoglobin- as well as CD31-antibody.
Results. We could show that RCC do express Mb. Immunhistochemical
examinations of RCC tissues show a reverse relationship between Mbexpression
and capillary density. Especially in clear cell RCC a significant
relationship between decrease in capillary density and increased
expression of Mb could be shown. Furthermore, the expression of Mb
in all analyzed RCC cell lines increased under hypoxia up to 60-fold.
Conclusions. Mb especially known as a marker for myogenic differentiation
is expressed in RCC and RCC cell lines under hypoxia. A significant
reverse correlation between capillary density and Mb expression
exist in clear cell RCC. Thus, Mb might be a marker for hypovascularized
tumor entities/tumor areas.
SA-P-093
Impact of early ultrastructural damage after ABO-incompatible
and ABO-compatible kidney transplantation
V . Broecker1 , A . Pfaffenbach1 , C . Bockmeyer1 , M . Dämmrich1 , S . Immenschuh2
, A . Schwarz3 , H . Kreipe1 , F . Heinemann4 , J .U . Becker1 1 2 Hannover Medical School, Institute for Pathology, Hannover, Hannover
Medical School, Institute for Transfusion Medicine, Hannover, 3Hannover Medical School, Department of Nephrology, Hannover, 4Essen University,
Institute for Transfusion Medicine, Essen
Aims. Following ABO-incompatible kidney transplantation (iABO),
c4d in peritubular capillaries (ptc) is frequently positive without further
indicators of humoral rejection. Using electron microscopy (EM), we
investigated the presence and prognostic relevance of early endothelial
damage to glomerular and ptc in transplant kidneys with c4d positivity
under different circumstances.
Methods. In 20 iABO patients (57 biopsies), 16 ABO-compatible (cABO)
patients (26 biopsies with c4d-positivity of ptc) and 14 transplant patients
(14 biopsies) without c4d and signs of humoral rejection (controls)
were included. 10 different EM parameters were semiquantitatively evaluated:
loss of foot processes (FF), lamina rara interna widening (LRI),
swelling of glomerular and ptc endothelial cells (GES and ptcES), inflammatory
cell adhesion to glomerular and ptc endothelial cells (GIS
and ptcIS), glomerular basement membrane double contours (DK), glomerular
and ptc basement membrane lamellation (LG and Lptc), loss of
glomerular endothelial fenestration (FE). Status of donor specific antibodies
(DSA) was available for 35/50 patients. Kidney function (GFR) at
biopsy and follow up (51.8±31.74 months) was available for all patients.
Results. 3/10 EM parameters were significantly less severe in iABO versus
cABO (FF, FE, ptcES); 1/10 significantly more severe in cABO versus
controls (FE). No differences were seen between iABO and controls.
GFR at biopsy and follow up was significantly lower in cABO versus
iABO, although the decline (GFR/time) was not different. GFR at biopsy
and follow up correlated with 2/10 EM parameters in iABO (LRI, FE),
2/10 in cABO (GIS, FF). None of the EM parameters correlated with
GFR decline. Patients with positive DSA (6/35) had significantly lower
GFR at biopsy and follow up, although, GFR decline was not different
between DSA positive and negative patients. The presence of DSA correlated
with 6/10 EM parameters (FF, LRI, GES, ptcES, DK, FE).
Conclusions. Long term outcome in iABO patients is favourable despite
c4d positivity of ptc. In this context the value of EM in the detection of
early endothelial damage, although more pronounced in cABO patients
with signs of humoral rejection, is limited and lacks prognostic relevance.
In contrast, the presence of DSA correlates with ultrastructural
signs of endothelial alteration and is associated with impaired kidney
function.
SA-P-094
Large scale in vivo isolation of glomerular podocytes by cationic
colloidal silica-coated ferromagnetic nanoparticles
A . Blutke1 , R . Wanke1 1Ludwig-Maximilians-University Munich, Institute of Veterinary Pathology
at the Centre for Clinical Veterinary Medicine, München
Aims. Podocyte homeostasis plays a crucial role for maintenance of the
physiological glomerular function and podocyte injury is regarded as a
major determinant of development and progression of various renal diseases.
Since cultured podocytes cannot completely reflect the complex
properties of podocytes in the glomerular environment in vivo, analyses
of the molecular processes occurring during podocyte development
and injury require appropriate methods for preparation of fresh podocyte
isolates.
Methods. A novel, fast, antibody-free and most cost-efficient method
for reproducible large scale isolation of fresh podocytes from mouse
kidney glomeruli is described. Briefly, cationic silica-coated colloidal
ferromagnetic nanoparticles were utilized to bind to negatively charged
cell surfaces of podocytes in preparations of isolated glomeruli. After
enzymatic and mechanical dissociation of the glomerular cells, nanoparticle-coated
podocytes were isolated in a magnetic field.
Results. Podocytes isolated with this method displayed characteristic
phenotypical and ultrastructural features. Protein and mRNA expression
abundances of marker-molecules of podocytes, endothelial or mesangial
glomerular cells indicated a significant enrichment of podocytes
in the generated isolates.
Conclusions. The described method offers a great potential for different
downstream transcript- and protein profiling analysis technologies,
which might contribute to an improved understanding of podocyte
biology and of the molecular mechanisms involved in podocyte injury
in vivo.
Der Pathologe · Supplement 1 · 2012 |
167

Abstracts
SA-P-095
Can fibrils induce a humoral immune reaction? A case report and
review of literature
S . Porubsky 1 , C . Boldt 2 , V . Kliem 2 , H .-J . Gröne 1
1 DKFZ Heidelberg, Heidelberg, 2 Nephrologisches Zentrum Niedersachsen,
Hann . Münden
Aims. Fibrillary glomerulonephritis is a rare disease of later adulthood
and is characterized by randomly arranged Congo-red negative fibrillary
deposits with a diameter of 10–20 nm in glomeruli. Due to sclerosis
of glomeruli and tubulointerstitium the disease leads usually to chronic
kidney failure. We describe a 50-year-old female patient whose medical
history was negative with exception of smoking and a febrile bronchitis
a few days before admission. The patient presented with proteinuria
(3.8 g/d), active urine sediment, hypertension and acute kidney failure.
The laboratory findings revealed solely an ANA-titer of 1:160. ANCA-
and ASLO-titers as well as hantavirus serology were negative. Complement
levels were normal.
Methods. Light-microscopical, immunhistochemical and ultrastructural
investigations of the kidney biopsy and correlation of the findings
with the current literature.
Results. The biopsy showed 15 glomeruli of which 5 were characterized
by a segmental necrosis and extracapillary proliferation. 2 glomeruli
showed a global and 4 a segmental sclerosis. There was moderately severe
chronic interstitial damage. Immunohistochemically IgG, IgA, IgM
C1q and C3 depositions could be detected in mesangium and along the
basement membrane of all glomeruli. Congo-red stain was repeatedly
negative. In mesangium and within the glomerular basement membrane,
electron microscopy visualized randomly arranged fibrils with a
diameter of 10–20 nm. The diagnosis of a fibrillary glomerulonephritis
was made.
Conclusions. Although seldom crescents have been observed in idiopathic
fibrillary glomerulonephritis, a necrotizing form of this disease
has not been described in detail. The simultaneous occurrence – with
a “full house” pattern – of immunoglobulin, complement factors and
fibrils is usually seen in fibrillary glomerulonephritis. Necrosis points to
the potential of fibrillary deposits to induce a pronounced complement
activation and to cause a necrotizing lesion. This is contradictory to the
current paradigm that fibrils are immunologically inert. Furthermore it
demonstrates that glomerulopathies with organized deposits are to be
considered upon a diagnosis of necrotizing glomerulonephritis.
SA-P-096
Pathology of resolving polyomavirus nephropathy
T . Menter1 , M . Mayr2 , H .H . Hirsch3 , M .J . Mihatsch1 , S . Schaub4 , H . Hopfer1 1 2 Institute of Pathology, Basel, Switzerland, Medical Outpatient Department,
Basel, Switzerland, 3Institute of Medical Microbiology, Basel, Switzerland,
4Clinic for Transplantation Immunology and Nephrology, Basel,
Switzerland
Aims. Polyoma virus nephropathy (PVN) is a common complication after
renal transplantation, affecting up to 20% of patients. The standard
therapy is reduction of immunosuppression, which leads to an effective
virus control in more than 90% of cases. So far, the morphology of resolving
PVN has not been investigated. We compared the morphological
findings in protocol biopsies of PVN patients prior to viremia, during
increasing and decreasing viremia as well as after virus clearance.
Methods. The study included 101 transplant biopsies of 34 patients transplanted
between 2005 and 2010 and diagnosed with PVN which were
treated by reduction of immunosuppression only. Biopsies were scored
according to Banff-criteria, the extent of inflammation and interstitial
fibrosis was estimated as% of renal cortex, and the number of tubular
cross sections with SV40+ cells per mm of biopsy length was counted.
Biopsy findings were correlated with virus load in the serum and cli-
168 | Der Pathologe · Supplement 1 · 2012
nical follow-up data, and grouped as pre-, increasing, decreasing, and
post-viremia.
Results. We found a significant increase in interstitial inflammation
(median decreasing viremia 10% vs. increasing viremia 0.3%, p

SA-P-098
Male infertility: assessment of juvenile testes dysfunction and
risk for malignancy in cryptorchic boys based on resin semithinsection
evaluation
J . Schröder 1 , W . Rösch 2 , F . Hofstädter 1
1 University Regensburg, Pathology Dept ., Regensburg,
2 University Regensburg, Clinic St . Hedwig, Regensburg
Aims. Infertility may become more a man’s than a woman’s problem:
new data shows both were level pegging – 40% of cases are linked to women,
40 to men, and 20 to joined problems. Failure in congenital testes
descends (cryptorchidism) is the most frequent genital malformation
affecting approx. 1% of 1-year-old mature birth boys. Untreated maldescensus
testis impairs the germ cell development and reduce significantly
the fertility capacity in adults. Additionally there is an increased risk
for testicular cancer. We report how the pathologic biopsy examination
of juvenile cryptorchic testes can assess infertility and malignancy risk.
Methods. Biopsies of ectopic or cryptorchic testes were immersed in
Karnovsky-fixative, postfixed in osmium-tetroxide, dehydrated in graded
ethanols and routinely embedded in epon resin (EmBed812, LYNXautomated
EM-tissue processor). Semithin sections (1 µM) were prepared
by ultramicrotomy using a diamond knife, mounted on a glass slide
and double stained with toluidine blue/basic fuchsine as well as triple
stained according to Laczko [1975] for intracellular glycogen detection.
Results. A semithin resin section provides an excellent structure preservation
of the testicular tissue and is a proofed basis for light microscopic
spermatogenesis assessment in the tubuli contorti. The evaluation
includes the recognition and counting of different development stages
of the germinal cells and detection of specific germinal glycogen-rich
TIN-cells (testicular intraepithelial neoplasia) which represents a preneoplastic
lesion. The light microscopic biopsy examination allows the
cornerstone parameter estimation for the adult fertility, which are the:
(1) tubular index of total germinal cell number; (2) tubular index of
adult dark (A-dark Spermatogonia) spermatogonia – the stem cell pool
of all future spermatozoa [Hadziselimovic, 1983, 1990]; (3) tubular index
of primary spermatocytes.
Conclusions. The demonstrated assessment of spermatogenesis dysfunction
and malignancy risk in juvenile cryptorchic testes in semithin resin
section is a crucial step for the right therapy. Today there is consent,
that an intrauterine hormonal dysfunction of the hypothalamo-pituitary-gonadal
axis is involved in most testicular maldescended cases. In
general, late diagnosis of undescended testis will have a poor prognosis
for future fertility and increased risk for cancer. In our opinion, an electron
microscopy lab is predisposed for processing testicular biopsies for
fertility assessment.
SA-P-099
Rete testis invasion by malignant germ cell tumors of the testis
A .K . Höhn1 , J .-U . Stolzenburg2 , L .-C . Horn1 1University of Leipzig, Institute of Pathology, Leipzig,
2University of Leipzig, Clinic of Urology, Leipzig
Aims. Rete testis is a communicating network of seminal channels in the
hilum of the testis. Its invasion is described as a risk factor in German
guidelines for the management of malignant germ cell tumors. Tumor
size and rete testis invasion (RTI) were considered as prognostic factors
for recurrent disease within stage I seminomas (Warde et al. 2002) and
was suggested to represent an independent prognostic factor (Vogt et
al. 2010). The present study evaluates the documentation of RTI within
routine workup and the pattern of involvement.
Methods. 100 cases with testicular germ cell tumors were re-evaluated
regarding the presence of rete testis within the examined tissue, the
documentation of rete testis status and, if present, the pattern of RTI
(direct infiltration versus pagetoid) as well as which tumor component
was seen in RTI.
Results. The mean age was 38 years (15–75 years). Mean tumor size was
3.45 cm (0.8–14.0 cm). In the originally reports presence of RTI was recognised
in 27 and its absence in 25 cases. In 48 cases rete testis status
was not reported. After the re-evaluation 51 cases showed RTI. Among
these 31 (61%) represented direct invasion and 3 cases (6%) a pagetoid
pattern. In 17 cases (33%) a mixed pattern of invasion was found. 34 of
51 cases (67%) showed an invasion by a pure seminoma, 16 cases (31%)
showed an invasion by the seminomatous component of a combined
germ cell tumor and 1 case (%) showed a pagetoid pattern of invasion
in a non-seminomatous germ cell tumor. 42 cases showed no evidence
of RTI. In 7 cases the rete testis was not available within the examined,
paraffine-embedded tissue of the specimen.
Conclusions. In 48% the RTI-status was not documented during routine
examination. In case of RTI, the majority of cases represented direct
or mixed type pattern on involvement. The status of RTI should be
mentioned within the pathology report. In case of missing rete testis
recutting with embedding of additional tissue is recommended. Further
analyses of the correlation between the tumor size and RTI and the
prognostic impact of RTI are required.
SA-P-100
N-cadherin expression in malignant germ cell tumors of the
testis
F . Bremmer1 , S . Schweyer1 , B . Hemmerlein1 , A . Strauß2 , H .J . Radzun1 ,
C .L . Behnes1 1University of Göttingen, Department of Pathology, Göttingen,
2University of Göttingen, Department of Urology, Göttingen
Aims. Testicular germ cell tumors (TGCTs) are the most common malignancy
in young men aged 18–35 years. They are clinically and histologically
subdivided into seminomas and non-seminomas. Cadherins
are calcium-dependent transmembrane proteins of the group of adhesion
proteins. They form cell junctions in various tissues including
desmosomes and adherens junction. Furthermore, cadherins play a
role in the stabilization of cell-cell contacts, the embryonic morphogenesis,
in the maintenance of cell polarity and signal transduction. Ncadherin
(CDH1), the neuronal cadherin, stimulates cell-cell contacts
during migration and invasion of cells and is able to suppress tumor cell
growth. The aim of this study was to determine whether N-cadherin is
expressed in TGCT and potentially differentiates between the tumorentities
of TGCT.
Methods. We investigated 74 TGCT (seminoma n=40, embryonic carcinoma
n=14, teratoma n=14, chorioncarcinoma n=3, yolk sac tumor n=5)
by immunohistochemistry for the expression of N-cadherin. Furthermore,
the tumor cell lines NCCIT and NTERA were analyzed by PCR,
Western blot analysis and immunocytochemistry.
Results. The studied TGCT showed a distinct membrane-bound N-cadherin
expression for seminoma, teratoma, yolk sac tumor and chorioncarcinoma.
All examined embryonic carcinoma did not show expression
of N-cadherin. In the investigated mixed tumors each of the
embryonic carcinoma-components was negative for N-cadherin, whereas
the other tumor components were positive for N-cadherin. Both
investigated cell lines expressed N-cadherin mRNA, but only NCCIT
showed N-cadherin protein expression.
Conclusions. In contrast to embryonic carcinomas seminomas, teratomas,
yolk sac tumors and shorioncarcinomas express N-cadherin. Ncadherin
is able to differentiate embryonic carcinomas from other tumor
entities of TGCT also in mixed tumors. Thus, N-cadherin may play
a role in tumor progression and in the pathogenesis of TGCT.
Der Pathologe · Supplement 1 · 2012 |
169

Abstracts
Poster: Informatik
SA-P-101
How virtual slides save pathologist’s time and inspire students
I . Klempert 1 , K . Schlüns 1 , P . Hufnagl 1 , M . Dietel 1
1 Charité University Hospital, Institute of Pathology, Berlin
Aims. Due to the changing curriculum of the Charité Universitätsmedizin
Berlin we have to manage larger groups of students in shorter lessons
with a more important focus on the relation to the clinical aspects
of the program. Less than a third of the lessons of the institute of pathology
are classical histological lessons. We decided to introduce virtual
microscopy to overcome these problems and to offer the students unlimited
access to comprehensive, exciting and clinical related pathohistology.
To realize such an offer, we arranged the slides in cases (as small
exercises).
Methods. We already use the E-learning system “Blackboard”, but histology
was only presentable as still pictures. Additionally, we established
the “Slidebox” System to increase availability of virtual slides. This
software allows the use of each picture ratio of our slide scanners. The
software is password-protected and can be used from every internetaccess
point. The number of well documented cases (of diagnostics) is
increasing and serves as a basis for case-based learning.
Results. The anatomy-histology room allows up to 80 students to work
by microscope and/or PC. The computers are connected by a 2.33 GHZ
XEON Server via 1 GB LAN. This technical foundation is sufficient for
students to work during the lessons. The virtual slides can be annotated
and the system allows them to be viewed down to the single cell
level. The students are able to use the virtual slides during the lessons
(under supervision) or at home. The software works fast and is reliable,
the handling aspects are easy to understand and intuitive. The students
accepted the improvements to the system very quickly, with positive
feedback and good results.
Conclusions. Prepared virtual slides or documents are logged within the
system for repeated use, allowing them to be used with constant quality
or for enhancement. The educator saves time in preparation. The
students are well prepared; are capable of comparing the solutions or
starting discussions. The use as a part of the academic education is just
one possibility. The use in further education of physicians in one location,
or between different institutes or universities is also conceivable.
SA-P-102
Integrative, multimedia-based learning with the aid of a virtual
microscope
C . Brochhausen1 , H . Winther1 , V .H . Schmitt1 , J . Horstmeyer2 , C .J . Kirkpatrick1 1 2 University Medical Centre, Institute of Pathology, Mainz, Johann Wolfgang
Goethe-University, Academy for Educational Research and Teacher
Education, Frankfurt am Main
Aims. Virtual microscopy is already established in teaching curricula at
many universities. However, the technique is the subject of continuous
further development. A variety of features exist in which the individual
applications differ. However, which of the numerous features are considered
useful, necessary or redundant is unknown. Therefore, we have
investigated the needs of the students with a questionnaire and developed
a new application for virtual microscopy on the basis of these data.
Methods. In cooperation with the Center for Quality Assurance and
Development Mainz a questionnaire with a feature-set was created. 216
students assessed the importance of functions such as annotations, additional
teaching texts and a broad scope of target devices. Based on this
evaluation a new application was constructed.
Results. The analysis of the questionnaire revealed that 96.2% of the
students consider virtual microscopy as a meaningful learning opportunity
and desirable for test preparation. The features annotations
170 | Der Pathologe · Supplement 1 · 2012
(87.3%) and additional teaching texts (52.3%) have also been evaluated
positively. Furthermore, many students requested a quiz mode in the
comments section. In contrast, the establishment of a discussion forum
appeared less important to the students (4.2%). These results provided
the basis for the development of a novel application, in which technologies
were used (including HTML1.1, AJAX) both to satisfy the needs
of the students and also to ensure the expansibility (OpenLayers) and
sustainability of the application. Hence, proprietary technologies like
Flash were abandoned.
Conclusions. The survey revealed the requirements of the users, which
formed the basis for the development of a new application. In future innovations
the applications should be able to run on all modern browsers
without proprietary extension. The course of further developments, e.g.
detailed depth portrayal or adventure experience microscopy should be
determined in further questionnaires.
SA-P-103
A web-based virtual microscopy platform as supplement for
histopathology lectures
S . Friedl1 , G . Oehmke2 , T . Wittenberg1 , F . Paulsen3 , A . Hartmann2 , C . Geppert2 1 2 Fraunhofer Institute for Integrated Circuits, Erlangen, University Hospital
Erlangen, Institute of Pathology, Erlangen, 3University of Erlangen-Nürnberg,
Department of Anatomy Chair II, Erlangen
Aims. In academic education of microscopic histology and pathology,
an independent training in the recognition of specimen is an important
supplement to core lectures. To limit the effort regarding hardware,
rooms, and personnel for such an open microscopy, innovative internet
technologies can help to provide flexible and continuous opportunities.
A web-based virtual microscopy platform can offer training possibilities
at any time without the need of constant administration. Such a
platform has been developed and introduced, and the usage and the
feedback of the students have been evaluated during the last two academic
terms.
Methods. To provide specimens for the web-based platform, microscopic
slides have been digitalized using a slide scanner at 40-fold magnification.
Resulting in image data with up to 5 GB per slide, a conversion
of the images is necessary to optimize the transfer over the internet. A
tiled pyramid structure can provide selected tiles in defined resolutions
directly without additional computation. The user can zoom to any preferred
magnification level and pan the slide freely while only the visualized
parts and details are transferred to the client. The digital slides
are categorized by anatomy and findings, and enhanced by additional
descriptions and iconic annotations. Currently approx. 200 specimens
are digitalized and provided to the students.
Results. With 146 5th-semester students using the platform, an average
login count of 180 logins per week was observed. As expected, one week
before the exams the login rate went up near to 1000 per week. According
to a survey of 6th-semester students, 79% indicated, that lack of
time for microscopic examination during the course could be well compensated
by the web-based platform. In the evaluation, students favored
that this environment offers more possibilities to provide additional information
like e.g. attendant macroscopic pictures.
Conclusions. A web-based virtual microscopy platform is an innovative
and valuable opportunity to enable students an independent and flexible
training in the recognition of different specimen. Furthermore,
nowadays students are used to multimedia and internet usage and can
learn and examine the slides any time at any place. The introduced platform
is in use for the second academic term with appropriate access
rates and runs stable with a low amount of administration. The internal
solution offers possibilities to enhance the education with further features
and information according the given lectures.

SA-P-104
Process oriented scientific data management in the Open European
Nephrology Science Center
T . Schrader 1 , S . Niepage 2 , C . Hahn 2 , S . Hanß 3
1 University of Applied Sciences Brandenburg, FB Informatics & Media,
Brandenburg, 2 Charité – Universitätsmedizin Berlin, Institut für Pathologie,
3 Charité – Universitätsmedizin Berlin, Institut für Medizinische Informatik
Aims. The Open European Nephrology Science Center (OpEN.SC) is a
center for research related clinical data supported by the German Research
Foundation. The reuse of clinical for translational medicine is
a cornerstone for the further scientific development. Various projects
try to collect data from different resources for scientific purpose. The
OpEN.SC platform consists of process oriented tools for data import,
management, analysis and presentation and solved the two main problems
in scientific data management: storage of data taking to consideration
the legal issues (secure patient data) and save the intellectual
properties of the diagnostic and therapeutic process by the physicians.
Methods. The platform based on a Service Oriented Architecture and
consists of various web services as modules. The orchestration of these
web services is realized by business process models. At the backend an
Apache Geronimo Application Server ensures the availability of the web
services. The Open Source database engine PostgreSQL is used to store
the data from three different resources. The user interface is realized
by OpenSource Liferay Portal. Different tools for retrieval and image
analysis are available. The processes were modeled using the standard
BPMN (Business Process Modelling Notation by OMG).
Results. The Open European Nephrology Science Center is a flexible
platform for scientific data management. Flexibility means that data
from different resources, with different structures and various file types
can be stored at this server and managed related to the resources.
Each resource has a complete control and transparency for its own data.
Due to the Service Oriented Architecture the platform is scalable. The
process oriented modelling offers the opportunity to adapt the system
for each specific use case and any type of data management model. The
process models can be used for business simulation to evaluate the impact
of changes very early.
Conclusions. Scientific data management should cover different aspects
of data handling: data import, storage, retrieval, access and presentation
concerning all legal issues and changing as well as increasing requirements
to storage, retrieval and analysis. Flexibility is a cornerstone
for management data from different resources and can be realized by
application of business process modeling, executing, analysis and simulation.
SA-P-105
CognitionMaster: an open source biomedical image analysis
development framework
S . Wienert1 , D . Heim1 , K . Saeger2 , C . Denkert1 , P . Hufnagl1 , F . Klauschen1 1 2 Charité University Hospital Berlin, Institute of Pathology, Berlin, VMscope
GmbH, Berlin
Aims. Automated microscopic image analysis has been a research focus
in medical informatics since many years. The topic has also attracted
attention among pathologists who face an increasing demand for a
standardized and quantitative evaluation of (immuno-)histological parameters
in patient specimens. Here, computerized image analysis may
assist pathologists and can also help to more efficiently test hypothesis
on the relevance of certain tissue properties. One limitation in this field
is the difficulty on one side for computer scientists to efficiently develop
and test image analysis algorithms with realistic histological data,
and on the other side for (quantitatively-inclined) pathologists to test
and optimize the potentially useful analysis software: While developers
would usually favour sophisticated software environments that are usually
inaccessible to non-computer scientists and do not facilitate easy
testing of realistic image data, pathologists are normally confronted
with ready-to-use software applications with no or limited flexibility.
Our aim was to design an open and flexible software platform that may
support collaboration between pathologists and computer scientists.
Methods. The software was implemented in C# for Microsoft .NET.
SharpDevelop was used for the integrated editing of C# code. Icons
from the Tango! project were use for the graphical user interface.
Results. We present an open-source software platform that may be used
for a broad spectrum of tasks in the field of medical image analysis:
Ranging from algorithm development with an integrated C# compiler
to one-click analysis provided through a powerful plug-in interface. A
novel object layer structure was designed to handle image objects and
their properties and therefore allow high-level formulations of image
analysis algorithms. The tool provides flexible and interactive functionality
with a variety of image analysis algorithms that may be combined
in process chains, an object model editor, and plotting/statistics functions.
Conclusions. The platform presented here may help both computer
scientists and pathologists to efficiently design and test novel image
analysis approaches and quickly obtain a “first guess” on their practical
utility. It may therefore foster collaboration in the field of quantitative
virtual microscopy and accelerate the integration of novel image analysis
approaches into diagnostic applications.
SA-P-106
Computer-assisted histology for the diagnosis of Barrett’s
Esophagus – a pilot study
C . Dach1 , C . Geppert2 , S . Friedl1 , M . Benz1 , C . Münzenmayer1 , A . Hartmann2 ,
M . Vieth3 , T . Wittenberg1 1 2 Fraunhofer IIS, Erlangen, University Erlangen, Institute for Pathology,
Erlangen, 3Klinikum Bayreuth, Institute for Pathology, Bayreuth
Aims. Gastroesophageal reflux disease (GERD) is one of the most common
and in the frequency increasing diseases in the western world. Intestinal
metaplasia, or “Barrett’s Esophagus”, is a precancerous condition
and complication of GERD. A large interobserver variation is known
in histopathology of Barrett’s Esophagus. Hence, it makes sense to support
pathologists with an automated pre-analysis of the images. Goal of
this study is the evaluation of possibilities to differentiate three types of
tissue automatically, namely Barrett’s Esophagus (BE), normal cardiac
mucosa (CA) and normal squamous epithelium of the esophagus (EP).
Methods. Histological slides of 86 randomly selected patients with Barrett’s
Esophagus have been digitized with a high-resolution whole-slide
scanner (3DHistech). 26 data sets were selected in which equally all 3 types
of tissue (BE, CA, EP) are depicted. In these data sets 50 rectangular
regions for each of the 3 classes were manually labeled. It was evaluated,
which type of image-based features, namely color enhanced 2nd order
texture statistics and statistical-geometrical features, can be used for a
best differentiation of the 3 tissue classes. Furthermore, various parameters
for the texture features were evaluated. For the classification step
a nearest-neighbor classifier with various parameters was applied. For
each experiment all classification rates as well as the confusion tables
were computed.
Results. Using an n-fold cross validation and a combination of 2nd orders
statistical features, a maximum diagnostic classification rate of
90% (BE 88%, CA 84%, EP 100%) could be achieved, denoting the possibility
of a correct differentiation of the diagnostic classes with respect to
the annotated ground truth. The highest possible classification rate for
the discrimination of Barrett’s Esophagus was achieved with 90% on a
basis of color co-occurrence matrices and correlates to a total classification
rate of 89% over all 3 classes (CA 76%, EP 100%).
Conclusions. The results show, that the evaluated classes (BE, CA, EP)
can be differentiated quite well on this image data base by applying color-extended
texture features, whereas the detection and elimination
of EP tissue is possible with a high sensitivity. Hence, the basic criteria
Der Pathologe · Supplement 1 · 2012 |
171

Abstracts
have been defined, on which experiments can be made in order to differentiate
and pre-sort various types of tissue of the esophagus in histological
recordings using image analysis methods and possibly detecting
conspicuous tissue automatically.
SA-P-107
Computer-based morphological analysis of endomyocardial
structure
M . Schmauder1 , J . Zoschke2 , N .E . Hiemann2 , R . Hetzer2 , R . Meyer2 1 2 Realtime Imaging, Berlin, German Heart Institute Berlin
Aims. Histopathological and immunohistological examinations provide
important information to characterize myocardial tissue. The aim
of computer-based analysis of microscopic biopsy images is to detect
and quantify relevant structures of the myocardium by a standardized
procedure. The results provide the basis for correlative assessment with
clinical results.
Methods. The assessment of interstitial changes is done with Sirius redstained
tissue sections. Fibrous parts are extracted and automatically
measured using an online adjustable, adaptive colour segmentation
method. Immunohistochemical CD31 staining is used to determine the
size and distribution of microvessels. Here, the image analysis consists
of optimized colour segmentation and morphological classification followed
by automated evaluation of area ratios and numerical densities
of capillaries per normalized area. For the analysis of myocardial cells
based on haematoxylin eosin stained samples, a user-guided interactive
process was implemented.
Results. In an interdisciplinary project, we developed a new system for
quantitative morphological image analysis. The system includes three
measurement procedures for the analysis of myocardial structure. The
results are summarized in a combined record. Mean assessment time
is 30–60 min. To date, our preliminary experience has been obtained
in endomyocardial biopsy samples from cardiac transplant recipients.
Conclusions. Our investigations are a step towards a standardized computer-based
analysis of myocardial structure.
SA-P-108
The BMBF Initiative to build up centralized biomaterial banks in
Germany: the BioMaterialBank Heidelberg
E . Herpel1 , R . Kirsten2 , J . Berger2 , C . Döllinger2 , E . Frei3 , C . Ulrich3 ,
P . Schirmacher1 1Institute of Pathology, University Hospital Heidelberg, Heidelberg,
2BioMaterialBank Heidelberg, University Hospital Heidelberg, Heidelberg,
3National Center for Tumor disease
Aims. The BMBF Initiative aims to foster the assembly of centralized
structures for biomaterial banks in Germany, based on site-related
strategic concepts. The BioMaterialBank Heidelberg (BMBH) as one of
5 granted centres will merge all on-site high-quality biomaterial collections
(comprising both tissue and liquid samples) on an administrative
level and integrate them into a consistent project and quality management,
with respect to ethical and legal aspects. The overall aim is to provide
biomaterials or biomaterial collectives in a comprehensive way for
research purpose for researchers in Heidelberg and their cooperation
partners.
Methods. Core of the project is the tissue bank of the National Center
for Tumor Diseases (NCT) Heidelberg, where the essential structures,
regulations and procedures are realized and therefore can serve as a
template. Moreover, other tissue and liquid banks with focus on special,
non-tumorous diseases will be integrated, applying the following
measures:
– Setting up central BMBH administration, including IT/data and
quality management
172 | Der Pathologe · Supplement 1 · 2012
– Further developing and integrating liquid biobanking, thereby adapting
the existing structural concept of the NCT tissue bank
– Further development of a QM assessment program for biomaterials
– Setting up a uniform IT solution for all BMBH modules with optimized
interfaces to material administration
– Completing the biobank technology platform with specific emphasis
on improving the generation of derivatives at BMBH
– Expanding the NCT tissue bank outreach and training Program
Results. The BMBH was initiated in May and started to work in July
2011. The following steps of milestone planning were realized so far:
– Assessment of the current state of all processes, regularities and
structures
– Formal integration of all tissue bank modules into BMBH
– Implementation of a consistent IT-structure (STARLIMS)
Conclusions. In the following years, standardization of processes will be
continued, with a special emphasis on IT- and quality management, and
expanded to all other on-site modules. Thereby, we will lay our focus on
the implementation of a conform quality management for tissue banking,
aiming to accredit all tissue bank modules in year 3 of the grant.
Until the end of the grant period (year 5) all processes, regularities and
structures will be continued, to finally become a highly professional and
sustainable biomaterial bank.
SA-P-109
The BMBF Initiative to build up centralized biomaterial banks in
Germany: the Interdisciplinary Bank of Biomaterials and Data
Würzburg (IBDW)
M . Neumann1 , S . Störk2 , R . Lohmüller3 , S . Kircher4 , A . Rosenwald4 , R . Jahns3 1University of Würzburg, Institute of Clinical Biochemistry and Pathobiochemistry,
Würzburg, 2University of Würzburg, Comprehensive Heart
Failure Center, Würzburg, 3University of Würzburg, Interdisciplinary Bank
of Biomaterials and Data Würzburg, Würzburg, 4University of Würzburg,
Institute of Pathology, Würzburg
Aims. The Interdisciplinary Bank of Biomaterials and Data Würzburg
(IBDW) aims to systematically collect liquid (blood/DNA/urine) and
solid biomaterials (BM) from patients and study participants of the Medical
Campus. One key challenge is to integrate acquisition of BM for
research purposes into clinical routine. Further, BM-collection must
comply with the current legal framework and storage conditions with
current OECD recommendations. BM are linked with corresponding
clinical datasets in accordance with current data protection regulations
and ethical principles securing donor’s privacy.
Methods. The Medical Faculty holds full responsibility for the IBDW
governed by its own steering committee. The IBDW is composed of
3 central units (liquid BM/solid BM/database) and a limited number of
decentralized subunits. All units adhere to IBDW standards and rules.
The build-up process is split into four partly overlapping phases: (1) build
up organisation including patient consent and standard operating
procedures (SOP), (2) build up liquid biobank, (3) build up tissue biobank
(4), implement unified IT infrastructure and interface to the clinical
database. All data within the IBDW will be stored pseudonymized
and are protected by an independent gatekeeper.
Results. Together with the local ethics committee a patient and a proband
consent has been developed. The individual subject donates BM to
the IBDW for future research for an unlimited time period. The consent
allows collecting specified amounts of BM from an individual subject
once within a pre-specified time period. Processing of liquid BM samples
is highly automated to ensure a high quality pre-analytic phase.
All BM is managed by a Biobank Management System which tracks all
handling and processing steps of a sample starting with its acquisition
until storage of the sample or its aliquots in the cryo-repository. Quality
control algorithms are currently implemented. Several existing BM
collections within the University Hospital have been identified to be integrated
into the IBDW.

Conclusions. The IBDW offers a unique and professional service to both
clinicians and researchers bringing the “two worlds together”. The
highly standardized and centralized way of collecting BM and corresponding
data secure top quality BM and corresponding clinical data.
This will serve as a basis for high quality research within the local Medical
Campus and for national and international networking.
SA-P-110
A modular and virtual microscope platform based on Eclipse
Technologies
M . Flügge1 , N . Zerbe1 , P . Hufnagl1 1Charité – Universitätsmedizin Berlin, Institute of Pathology, Berlin
Aims. Virtual microscopy is a fast growing field in histology based medical
research. The base of every virtual microscopy system is digital
microscope software, thus several implementations, free and commercial,
are available. The main challenge of most implementations is
the lack of customizability. Often the microscopes are closed in there
functionality and not design to be extended by a third party. But his
in turn is often required especially in research. We demonstrate a new
framework that allows extending the behavior of the virtual microscope
with new features in an easy and flexible way. The system is rather a base
for a new virtual microscope system than a pure microscope with limit
functionality. Thus it is designed with the strong focus on extensibility.
Methods. To build up this extensible platform we are making an extensive
use of Eclipse based technologies expressively the Rich Client Platform
(RCP) architecture and the Eclipse Modeling Framework (EMF).
Using the plug-in concept allows to provide a simple core which can be
extended to more sophisticated applications by simply plug-in in additional
functionality. As the platform in written in Java, it can be easily
combined with other Java-based technologies or frameworks.
Results. We demonstrate an extensible platform which allows adapting
new contexts easily to the scope of virtual microscopy. Leveraging the
advantages of modularizable OSGI/RCP based applications the components
of the platform can be plugged together to provide highly specialized
microscopes for different fields of application. This allows building
up clean and non-overloaded user interface to simplify the work of
pathologists. An extendible layer concept in combination with a fine
grained request/notification mechanism ensures that additional functionality
(e.g false color visualization) can be easily adopted leveraging
automatically build-in support for zooming, panning, rotation and other
editor operations. This is available for standard sized images as well
as whole slide images (WSI). Image access is hidden behind an open service
provider interface which allows to plug-in own implementations.
Conclusions. The introduced architecture allows easy and fast integration
of new functionality to the basic microscope which allows creating
highly customized solutions for special field of applications in term of
virtual microscopy. This reduces the costs and increases the speed of
implementing such applications.
SA-P-111
The BMBF Initiative to build up centralized biomaterial banks in
Germany: the popgen 2.0-Network
U . Nöthlings1 , A . Wolf1 , A . Rüther1 , M . Krawczak1 1Christian-Albrechts-University Kiel, Kiel
Aims. Funded by the Federal Ministry of Education as one of the five
facilities supported within the National Biobank Initiative, the popgen
2.0-Network (P2N) will merge seven data and probe collections currently
existing at the Medical Faculty of the Christian Albrechts-University
(CAU) Kiel. This network will aim towards a uniform and centralised
research infrastructure which will enhance scientific usability
by means of fast and easy access to vast quantities of quality-controlled
data and probes that have been collected in compliance with legal and
ethical regulations.
Methods. Under coordination of popgen, a well established populationbased
biobank, P2N will comprise the data and probes of seven collections,
namely of popgen itself, the Cancer Centre North, the Department
of Neurosurgery, the Institutes of Pathology and Pharmacology,
the University Lung Centre North and the Centre of Family Medicine.
In order to harmonise, standardise and ease access to these manifold
probe types together with the both comprehensive and sensible data
attached to them, P2N has performed a survey of the participating biobanks
by evaluating infrastructure, workflows, probe and data types,
recruitment and consent policies. In addition, P2N has analysed the
portfolios of the leading laboratory information management system
(LIMS) suppliers.
Results. A shared network of peripheral biobanks with centralised access
is merely as useful as the consistent and uniform quality of the probes
and data collected and provided by the network partners. Due to the
broad heterogeneity of the participating probe and data collections the
usage of one common efficient albeit flexible LIMS is a crucial ingredient
for a properly functioning biobank network. Equally important is
a standardised handling of the probes itself and the accompanying data.
Ideally, these routines must adequately be supported by the LIMS and a
uniform set of SOPs.
Conclusions. Within the next 5 years P2N will have set up one of Germany’s
largest probe and data collections, comprising approximately
probes of more than 800,000 individuals. Standardised IT and quality
control infrastructures will facilitate an efficient and data safety-conform
access.
Der Pathologe · Supplement 1 · 2012 |
173

Autorenregister
Autorenregister
A
Abdullah M. FR-P-131
Adam P. DO-051
Agaimy A. DO-097, DO-098,
FR-P-056, FR-P-059, FR-P-154,
SA-P-077, SO-058
Akpalo H. DO-080
Al-Mohamed H. FR-P-115
Andreozzi M. FR-P-128
Anlauf M. FR-003
Arend J. FR-P-055
Ates G. FR-P-164
Aulmann S. SA-P-009
Aumann K. DO-076
Aust D. VO-011
B
Baldensperger M. FR-P-048
Bandapalli OR. SO-007
Barros M. DO-062
Barth PJ. SA-P-017
Barth TFE. FR-P-074
Batarello D. DO-020
Bauer K. FR-P-081
Bauer L. DO-018
Beckervordersandforth J. FR-014
Behnes CL. FR-P-170, SA-P-091,
SA-P-092
Bektas Serce N. SA-P-018
Belharazem D. SA-P-067,
SA-P-068
Benedix F. FR-P-076
Berchtold S. SO-009
Berezowska S. DO-016
Berg T. FR-P-173
Bergmann F. FR-P-097
Bian X-W. SG-136
Biesterfeld S. FR-P-143
Bihl MP. FR-P-060, SA-P-064
Bläker H. VO-003
Blank P. FR-P-078
Blutke A. SA-P-094
Bockmayr M. DO-078
Bockmeyer C. DO-107
Böger C. FR-P-066
Böhm F. FR-P-109
Bombari D. SO-018
Bonzheim I. DO-048
Boor P. FR-017, SO-075
Boruschek U. DO-055
Bösmüller H. SA-P-025
Brachtel E. SO-025
Brandt S. FR-P-171
Braun A. FR-P-057
Braun M. DO-111, SA-P-078,
SA-P-079
Bremmer F. SA-P-100
Brochhausen C. FR-P-093,
SA-P-102
Brockmoeller S. FR-011
Broecker V. SA-P-093, SO-072
Bronsert P. FR-P-091, SO-023
174 | Der Pathologe · Supplement 1 · 2012
Budczies J. FR-030
Burandt E. SO-032, SO-033
Bürger H. SA-P-006, SA-P-012
Bussmann C. VO-013
Büttner M. SA-004
C
Cacchi C. FR-002
Calvisi DF. DO-006, SO-012
Carneiro F. VO-024
Cathomas M. FR-P-047
Chakilam S. SO-001
Chen Y. FR-P-132, SA-P-054
Clauditz TS. SA-P-032
Conradi L-C. FR-P-079
Cortis J. FR-P-136
Csanadi A. FR-P-133, FR-P-134
Cui T. SG-P-119, SG-135
Culemeyer L. FR-P-156
D
Dach C. SA-P-106
Dämmrich M. DO-108
Datta K. VO-037
de Jonge M. FR-013
Decker T. SO-029
Dellmann A. SA-P-086
Denkert C. SO-031
Dihlmann S. DO-103
Doberenz E. SO-041
Donhuijsen K. SA-P-043
Dreyer J. DO-087
E
Ehling J. SO-017
El-Baba C. SA-P-066
Elfimova N. DO-004
Elsner M. FR-P-037, FR-P-042
Erber R. SO-035
Evert M. DO-007
F
Fang W-g. SA-P-015, SG-141
Fichter CD. FR-P-041
Floßbach L. DO-056, DO-057
Flügge M. DO-073, SA-P-110
Focke C. SO-027
Franken L. SO-036
Franz M. FR-P-114
Franzen A. DO-034
Frick L. FR-P-107
Friedl S. SA-P-103
Friedrich K. SA-P-014, SA-P-019
Friemel J. FR-P-099
Fritsche R. DO-120
Fronhoffs F. FR-P-162, SA-P-073,
SO-054
Fuchs D. FR-P-092
Funke S. SA-P-047
G
Gaida MM. DO-081
Gaisa NT. SO-065
Gaiser T. DO-023, FR-019
Gajda M. SA-P-034, SA-P-089
Gaßler N. SG-P-112, SG-128
Gdynia G. SO-006
Geddert H. FR-P-058, FR-P-075,
FR-P-149, SA-P-024
Gerlach S. DO-110
Giedl C. SA-P-045
Goeppert B. FR-P-103
Göke A. FR-P-090
Göke F. DO-091, DO-093
Goltz D. DO-109, FR-P-112,
FR-P-113
Gradhand E. FR-P-068
Griesser H. SO-019
Griff S. FR-P-065
Grob T. SA-P-011
Gröschl B. DO-123
Große K. FR-P-120
Grote HJ. SA-P-053
Grüning J. FR-P-167
Gündisch S. FR-027
Gutschner T. DO-033
H
Haab M. SO-010
Haag J. FR-P-050
Hahn C. DO-069
Haller F. SO-014
Hammer S. SA-P-026
Hämmerle M. DO-003
Hansen T. FR-P-095, FR-P-102,
FR-P-163, SA-P-033
Haroske G. DO-002b
Haugg A. FR-006
Häupl B. SA-P-087
Hehne S. FR-P-147
Heikaus S. SA-P-090
Heilmann T. SO-045
Heindl S. DO-017
Helmchen B. SA-P-031
Helpap B. SA-P-081
Henopp T. FR-004, FR-005,
FR-P-180
Herbach N. SA-001
Herpel E. SA-P-108
Herrmann TS. FR-P-098
Heublein S. SA-P-021
Hiemann NE. FR-P-117, FR-P-118,
FR-P-119
Hirsch D. SA-P-042
Hiththetiya K. FR-P-129
Högler S. FR-P-126
Höhn AK. SA-P-099, SO-020
Höller L. FR-P-124
Höller S. SO-039
Horn L-C. SA-P-027, SA-P-030
Horst D. SG-P-114, SG-130
Hui C. FR-P-086
Huss S. SA-P-049, SA-P-058
Huth S. SG-P-130, SG-144
I
Ikenberg H. FR-012, SA-P-039,
SA-P-040
Ikenberg K. SA-P-028
J
Jakubzik A. SA-P-097
Jarrin Franco MC. SO-021
Jayasinghe C. FR-P-071,
SA-P-071 SO-052
Jiang X. SG-P-132, SG-146
Jie Z. FR-P-087
Jöhrens K. DO-063
Jonigk D. DO-032
Jungbluth A. SO-049
Just A. FR-P-053
K
Kalinski T. SA-P-016
Karimi D. FR-P-036
Kayser G. DO-027, DO-068
Keck B. SO-066
Kelterborn J. FR-P-104
Kemter E. SO-069
Kettelhake A. SA-P-062
Kitz J. SO-003
Klauke M-L. SA-P-010
Klaus C. SG-P-113, SG-129
Klauschen F. SA-P-055
Kleine M. DO-112
Klempert I. FR-P-172, SA-P-101
Kloten V. SG-P-129, SG-143
Kluth M. FR-035
Knaup S. DO-115
Knöß P. DO-086
Knüchel-Clarke R. VO-019
Koitzsch U. DO-113
Kollecker I. SA-P-088
Koller K. SO-047
Kommoss S. SA-P-023, SO-022
König K. SA-P-051
Korsching E. DO-077
Kosmidis P. DO-049
Kratochvil D. FR-P-174
Krause M. VO-035
Kriegl L. DO-024
Kristiansen G. VO-020
Krüger U. FR-P-105
Küffer S. DO-125
Kuhlmann M. FR-P-168
Kuhne HP. FR-P-116
Künstlinger H. FR-033
Kurth R. FR-P-141
Küttner B. SG-P-115

L
Lasitschka F. DO-046
Lehmann A. FR-031
Lehmann U. DO-117
Lehnen NC. FR-P-094
Leisten I. DO-045
Lenggenhager D. FR-001
Lennerz JK. FR-P-151, SO-042
Lenze D. SG-P-133, SG-147
Li XZ. SG-P-117, SG-133
Linge A. SO-037
Liu C. FR-P-160
Lohneis P. FR-P-152
Longerich T. FR-018
Löser H. DO-124, FR-P-165,
SO-040
M
Ma Y. SG-P-125, SG-138
Macher-Göppinger S. SO-073
Maegel L. DO-079
Mahajan V. SA-P-038, SA-P-063
Mairinger F. DO-114, FR-032
Majores M. SA-P-082
Malz M. FR-P-137
Marienfeld R. SA-P-056
Märkl B. SA-003
Marwitz S. FR-P-144
Marx AH. FR-P-039
May AM. DO-043, FR-P-159
Mehta AK. FR-P-100, SO-013
Meijer GA. VO-005
Menon R. FR-029
Menter T. DO-059, SA-P-096
Messner I. FR-P-082
Metzig M. FR-P-080
Meyer A-S. FR-026
Michels S. SO-015
Michl P. VO-031
Miehlke S. VO-010
Mietzsch F. DO-118
Mihic-Probst D. SA-002
Moch H. VO-018
Mogler C. FR-P-106
Mollenhauer M. DO-089
Mones D. FR-P-043
Morawietz L. DO-102
Mößinger K. SA-P-060
Muders M. SG-P-126, FR-P-089,
SO-061, SG-139, VO-038
Mühling B. FR-P-123
Müller AM. SA-P-070, SA-P-074,
SO-51, SO-055
Müller BM. SO-030
Müller S. DO-122
Münch C. FR-P-155
Munding J. DO-021
N
Nelson PJ. SA-P-048
Nettersheim D. SO-068
Neumann J. SO-005
Neumann M. SO-043
Neumann M. SA-P-109
Neumann O. SO-011
Niendorf E. DO-064
Nitta H. FR-021
Noske A. SA-P-029
Nöthlings U. SA-P-111
O
Ommer KS. DO-001a
Omran H. ABS-888, SO-038
Ormanns S. DO-121
Otto M. FR-P121, FR-P-122
P
Papadoupoulus N. VO-007
Papadopoulos T. FR-008
Pawlaczyk N. FR-P-169
Pelisek J. DO-101
Pellegrino R. DO-005
Perner S. VO-015
Perren A. VO-039
Petersen M. FR-P-054, FR-P-064,
FR-P-101
Pfaltz K. SO-034
Pfister F. FR-P-125
Piscuoglio S. DO-022, FR-P-083
Poehlmann A. SO-002
Poremba C. DO-085
Porubsky S. SA-P-095
Q
Quan P. SO-016
R
Rao C. SG-P-123
Rao C. SG-P-116, SG-132
Rau TT. FR-P-069, FR-P-070
Rechsteiner M. FR-024
Reis H. SA-P-084
Reissig K. SA-P-052
Rennspiess D. DO-047
Rezaei M. VO-034
Ribback S. FR-P-110
Richter G. FR-010, SA-P-007
Richter P. DO-094
Riedl E. DO-044
Risio M. VO-001
Röcken C. VO-026
Rokahr J. SA-P-083
Rose M. FR-028
Rößler J. SA-P-037
Roßner M. DO-070
Roth W. VO-033
Rüping K. DO-082
Rupp NJ. SO-063
Rüschoff J. VO-040
S
Sailer V. SA-P-076, SO-057
Schaefer I-M. FR-P-061,
FR-P-063
Scharbatke EC. FR-P-052
Scharenberg C. DO-092
Scheil-Bertram S. SA-P-020
Schierle K. FR-P-062
Schildgen V. SA-P-035
Schildhaus H-U. DO-035
Schirmacher P. DO-036
Schmauder M. SA-P-107
Schmidt J-A. DO-066
Schmidt R. DO-075
Schmitt B. SA-P-036
Schmitz-Rixen T. DO-099
Schnabel PA. FR-P-139
Schneider N. FR-P-072
Schneider J. DO-084
Schneider-Stock R. FR-007
Schöne C. SA-P-013
Schoner K. SA-P-075, SO-056
Schöpfer A. VO-012
Schrader T. DO-001b, DO-074,
SA-P-104
Schrader C. DO-050, DO-053,
FR-P-077, FR-P-084, FR-P-085,
FR-P-088, FR-P-153, FR-P-158
Schramm M. FR-P-111
Schröck E. FR-022
Schröder J. SA-P-098
Schultz H. FR-P-130
Schuster C. DO-116
Schwertheim S. FR-P-178,
FR-P-179
Seitz V. DO-061
Senft E. DO-090
Shaikhibrahim Z. SO-062
Siegmund B. VO-008
Simon E. FR-P-108
Simon F. DO-104
Simon-Keller K. SO-046
Sinn BV. SO-008
Sinn H-P. SO-024
Sipos B. VO-029
Slotta-Huspenina J. FR-P-040
Söder S. FR-P-176
Soltermann A. FR-020
Spilka R. SA-P-085
Steffen JS. FR-P-051
Steiner T. SO-071
Steinhilber J. DO-065
Stenzinger A. DO-088
Sterlacci W. DO-026
Stöhr R. FR-016
Stomper J. SO-064
Straub M. DO-083
Szczepanowski M. FR-P-148
T
Tannapfel A. VO-030
Teller A. FR-P-045
Thies SA. FR-P-044
Thorns C. DO-054
Tiede C. DO-095
Timme S. DO-015, SA-P-008
Tindall DJ. VO-027
Tischler V. SG-P-131, SG-145
Titze U. SA-P-072, SO-053
Tóth C. FR-P-166
Trapani F. SO-004
Troidl K. DO-100
Tzankov A. DO-058, DO-060
U
Ullrich A. VO-014
V
Varga Z. SA-P-005, SO-026
Veits L. FR-P-038
Venkataramani V. SA-P-065
Verdoodt B. SA-P-080
Vlajnic T. FR-P-046
Vogel UF. FR-P-127
Vogetseder A. SA-P-057
Vogt M. SG-P-127, SG-140
Vogt N. DO-052
Vokuhl C. SO-048
Vollmer E. DO-025
von Laffert M. FR-015
von Winterfeld M. FR-P-067
W
Wachter DL. FR-P-096
Wagner S. FR-034
Walluks K. SA-P-061
Walther B. FR-P-177
Wang L. SG-P-121
Wang X. FR-P-145
Wardelmann E. SA-P-041,
VO-017
Warneke V. FR-023
Warth A. DO-028, DO-029,
FR-P-135, FR-P138, FR-P-140
Wassilew K. DO-106
Weber A. FR-P-049
Weiler C. DO-096
Weinberg RA. VO-032
Wenke A-K. SA-P-044
Westphal S. FR-P-161
Wethkamp N. SA-P-050
Wieczorek K. SA-P-069, SO-050
Wielockx B. FR-P-157, VO-036
Wienert S. SA-P-105
Winkler J. DO-002a
Wirtz R. SO-067
Wölfel C. FR-P-175
Wötzel F. FR-P-142
Wuensch T. FR-P-073
Wulf L. SO-044
Wyler-von Ballmoos L-G. SO-074
X
Xu J. SA-P-059
Xu J. SG-P-122
Y
Yasui W. VO-025
Ye M. SA-P-046
Z
Zeiske T. FR-P-146
Zeng Y. SG-P-118, SG-134
Zerbe N. DO-067, DO-071,
DO-072
Zhang J. SG-P-124, SG-137
Zhang W. SG-P-120, SG-142
Zhang X-x. FR-P-150
Zimmermann A-K. SA-P-022
Zimpfer A. FR-009
Der Pathologe · Supplement 1 · 2012 |
175

Rudolf-Virchow-Preis
Ausschreibung 2013
Der Preis wird laut Satzung der Rudolf-Virchow-Stiftung und der Deutschen
Gesellschaft für Pathologie einem Pathologen unter 40 Jahren für eine
noch nicht veröffentlichte oder eine nicht länger als ein Jahr vor der Bewerbung
publizierte wissenschaftliche Arbeit verliehen . Die Verleihung des
Preises erfolgt auf der 97 . Jahrestagung der Deutschen Gesellschaft für
Pathologie e . V . 2013 .
Zusammen mit einem Lebenslauf und einer Publikationsliste
reichen Bewerber ihre Arbeit ein . (Bitte alle Unterlagen in doppelter
Ausfertigung sowie elektronisch einreichen!)
Abgabetermin: bis zum 31. Dezember 2012
Einzureichen bei:
Prof . Dr . med . Holger Moch
Geschäftsführendes Vorstandsmitglied
der Deutschen Gesellschaft für Pathologie e .V .
UniversitätsSpital Zürich
Institut für Pathologie
Schmelzbergstrasse 12
CH – 8091 Zürich
Deutsche Gesellschaft für Pathologie e . V .
– Vorstand – Frühjahr 2012
© neumann-meding