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THE LUDWIG INSTITUTE FOR CANCER RESEARCH<br />

ANNUAL RESEARCH REPORT<br />

<strong>2002</strong>/2003<br />

©LUDWIG INSTITUTE FOR CANCER RESEARCH, 2003<br />

ALL RIGHTS RESERVED.<br />

No part of this publication may be reproduced by any means, or transmitted, or translated into any<br />

language without the written permission of the <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong>.<br />

Publication date: June, 2003


TABLE OF CONTENTS<br />

Table of Contents 2<br />

Introduction 4<br />

<strong>LICR</strong> <strong>Research</strong> Programs 5<br />

Angiogenesis Program 5<br />

Signaling Program 5<br />

Genomics Program 6<br />

Bioinformatics Program 6<br />

Cancer Antigen Discovery Program 7<br />

Cancer Vaccine Program 7<br />

Antibody Targeting Program 8<br />

Board of Directors 9<br />

Scientific Committee 10<br />

<strong>Institute</strong> Administration 11<br />

Administration – New York 12<br />

Administration – London 13<br />

Administration – Zurich 14<br />

Administration – Lausanne 15<br />

Branch Addresses<br />

Branch <strong>Report</strong>s<br />

16<br />

Brussels Branch of Human Cancer Cell Genetics 19<br />

Lausanne Branch of Immunology 31<br />

London St Mary’s Branch of Virology and Cell Biology 45<br />

London University College Branch of Cell and Molecular Biology 53<br />

Melbourne Branch of Tumour Biology 67<br />

New York Branch of Human Cancer Immunology 94<br />

San Diego Branch of Cancer Genetics 105<br />

Sao Paulo Branch of Cancer Biology and Epidemiology 117<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 2


TABLE OF CONTENTS<br />

Stockholm Branch of Molecular and Cell Biology 131<br />

Uppsala Branch of Growth Regulation<br />

Affiliate <strong>Research</strong> <strong>Report</strong>s<br />

139<br />

K. Alitalo (Helsinki) 155<br />

V. P. Collins (Cambridge) 159<br />

S. Lakhani (London) 162<br />

K. Miyazono (Tokyo) 164<br />

M. Pfreundschuh & C. Renner (Homburg) 167<br />

E. Pure (Pennsylvania) 169<br />

R. R. Schmidt (Konstanz) 172<br />

P. ten Dijke (Amsterdam) 174<br />

S. Yla-Herttuala<br />

James R. Kerr Program<br />

177<br />

James R. Kerr Program Investigators 180<br />

<strong>Research</strong> <strong>Report</strong> 181<br />

Publications<br />

Central Clinical Program<br />

184<br />

Director’s <strong>Report</strong> (Office of Clinical Trials Management)<br />

Affiliate <strong>Report</strong>s<br />

186<br />

V. Cerundolo (Oxford) 188<br />

A. Knuth, E. Jager, & D. Jager (Frankfurt) 191<br />

E. Oosterwijk (Nijmegen)<br />

Clinical Trials<br />

194<br />

Cancer Vaccines 195<br />

Targeted Antibodies 205<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 3


INTRODUCTION<br />

The <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> (<strong>LICR</strong>) is the largest international academic institute dedicated<br />

to understanding and controlling cancer. With ten Branches in seven countries across Australasia, Europe,<br />

and North and South America, and numerous Affiliates in many other countries, the scientific network<br />

that is the <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> quite literally covers the globe. The <strong>Institute</strong> is not only<br />

associated with established and world-renowned academic institutions, but has also, through its James R.<br />

Kerr Program, expanded its support into countries such as China, Russia, and Turkey, from which<br />

advanced cancer research is beginning to emerge.<br />

The cumulative effect of the <strong>Institute</strong>’s global research model, according to the most comprehensive and<br />

recent study of research organizations† has positioned the <strong>LICR</strong> as one of only twenty international<br />

organizations that produce research articles of extremely high impact. Organizations ranked alongside the<br />

<strong>LICR</strong> include the World Bank, the Red Cross, the World Health Organization, and only one other cancer<br />

institute, the International Agency for <strong>Research</strong> on Cancer, which is part of the World Health<br />

Organization.<br />

The uniqueness of the <strong>LICR</strong> lies not only in its size and scale, but also in its philosophy and ability to<br />

drive its results from the laboratory into the clinic. The <strong>Institute</strong> has developed an impressive portfolio of<br />

reagents, knowledge, expertise, and intellectual property, and, unusually for an academic institution, has<br />

also actively assembled the personnel, facilities, and practices necessary to patent, clinically evaluate,<br />

license, and thus translate the most promising aspects of its own research portfolio into cancer therapies.<br />

The <strong>Institute</strong> believes that the same science-driven investigative rigor that yielded the fundamental<br />

discoveries should, indeed must, be applied in the clinic to fully develop the therapeutic potential of any<br />

discovery.<br />

The goal of the <strong>LICR</strong> is to make a real contribution to the clinical management of cancer. This report<br />

summarizes the progress that <strong>LICR</strong> investigators made to that end in <strong>2002</strong>.<br />

† Da Pozzo, F., Maye, I., Perriard, A.R., and von Ins, M. (2001) “List of the Worldwide Champions League of<br />

<strong>Research</strong> Institutions 1994 – 1999.” Center for Science and Technology Studies, Bern, Switzerland<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 4


ANGIOGENESIS PROGRAM<br />

<strong>LICR</strong> RESEARCH PROGRAMS<br />

Angiogenesis is the process of forming new blood vessels (from endothelial cells). Although part of the<br />

body’s natural processes (in wound healing and reproduction), angiogenesis is also stimulated by the<br />

release of angiogenic growth factors from tumor cells. Without the blood and nutrients that the newlygenerated<br />

blood vessels supply, it is thought that no tumor would grow to be more than a few millimeters<br />

in size.<br />

The <strong>LICR</strong> Angiogenesis Program is a multi-disciplinary research effort that involves collaborations<br />

between the <strong>LICR</strong> Branches in Stockholm, Melbourne, Uppsala, and London (University College)<br />

Branches, and the laboratories of several <strong>LICR</strong> Affiliates. Prior to <strong>2002</strong>, the Program had discovered four<br />

new types of angiogenic growth factor, and two related receptors. The year <strong>2002</strong> saw the addition of a<br />

fifth angiogenic growth factor, and further characterization of the modes of action of these factors and<br />

their receptors.<br />

In particular, one factor has been found by <strong>LICR</strong> scientists to be important for the development of normal<br />

heart. This work was extended in <strong>2002</strong> by <strong>LICR</strong> Affiliates to show that, the factor accelerates the<br />

development of an alternative ("collateral") circulation following experimental occlusion of a coronary<br />

artery, thereby limiting the extent of ischemic damage to the heart. Additionally, angiogenic factors have<br />

recently been found to play a role in mobilizing, from the bone marrow, multipotential stem cells that are<br />

capable of developing into cells that line blood vessels, and thus “feed” tumors. The putative roles of<br />

angiogenic factors in a) limiting ischemic heart damage, and b) causing stem cells to develop into tumor<br />

blood vessels, and the processes through which these occur, are presenting both exciting new research<br />

projects for the Angiogenesis Program, and prospective targets for therapy. Work by <strong>LICR</strong> Affiliates in<br />

<strong>2002</strong>, showed that antibodies that block angiogenic receptors in an animal model prevented<br />

lymphangiogenesis (formation of new lymphatic vessels) and cancer metastasis to particular parts of the<br />

body. The prevention of lymphangiogenesis using antibodies, and gene therapy delivery of a mutant form<br />

of one of the angiogenic factors discovered by the work of <strong>LICR</strong> scientists, are just two of the ongoing<br />

therapy projects within the Angiogenesis Program.<br />

SIGNALING PROGRAM<br />

Cells interact with their environment by sending and receiving signals that initiate and terminate cellular<br />

processes such as cell division, growth, differentiation, migration and survival. Signaling, or signal<br />

transduction, also occurs within the cell, with external signals being passed along a chain of molecules<br />

from the cell surface receptor to, ultimately, the machinery that produces proteins from genes. Cancer<br />

cells have abnormal signal generation and reception, which allow them to grow out of control, invade<br />

other tissues, and escape programmed cell death. Signal reception occurs through the physical<br />

interaction between a receptor and the molecule which activates it, which is also called a ligand. Most of<br />

these interactions are specific, but there are an increasing number of receptor and ligand families that<br />

show complex patterns of interaction. Growth factor receptors and their ligands are prime candidates for<br />

therapeutic intervention, but before one can devise drugs that modulate signaling, it is essential to<br />

understand the complexity of a particular growth factor system.<br />

<strong>LICR</strong> investigators have made substantial contributions to many areas in the field of cell signaling. Work<br />

at the Uppsala and Stockholm Branches has focused on the family of platelet-derived growth factors<br />

(PDGF) and their PDGF receptors (PDGFR). PDGF signaling regulates several aspects of connective<br />

tissue biology, and abnormal activation of the pathway occurs in cancer as well as other diseases,<br />

including arteriosclerosis. During <strong>2002</strong>, <strong>LICR</strong> scientists published the discovery of a fourth PDGF family<br />

member, PDGF-D (the discovery of the third, PDGF-C was published by <strong>LICR</strong> scientists in 2001), and<br />

these findings have added a further dimension to PDGF signaling complexity. Whilst the characterization<br />

of the PDGF-C and -D molecules is ongoing, recent experimental data from <strong>LICR</strong> investigators indicate<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 5


<strong>LICR</strong> RESEARCH PROGRAMS<br />

that these new ligands have properties that are additional to the original two PDGF ligands, and may play<br />

a role in angiogenesis (refer Angiogenesis Program).<br />

<strong>Research</strong> into signaling molecules such as the epidermal growth factor receptor (EGFR) at the Melbourne<br />

and San Diego Branches (see Antibody Targeting Program), cytokines at the Brussels Branch, and<br />

phosphatidylinositol 3-kinases (PI3Ks) at the London University College Branch is ongoing. In particular<br />

the study of mouse models with a disrupted or altered PI3K has shown that at least one of these enzymes<br />

plays a fundamental role in immunology as well as cancer, suggesting that the PI3K inhibitors currently<br />

being developed by the <strong>LICR</strong> may also have an application as an anti-inflammatory agent.<br />

GENOMICS PROGRAM<br />

Cancer arises originally from genetic alterations in normal cells. Key genes that are involved in<br />

oncogenesis fall into two major groups. Tumor suppressor genes protect against cancer, and thus need to<br />

be inactivated or eliminated for cancer to occur. Oncogenes, on the other hand, promote tumor<br />

development and are often found to be hyperactive in cancer cells. Although many single genes have been<br />

identified as oncogenic (i.e. cancer causing), their numbers are relatively few, and it appears that specific<br />

interactions between and among many genes may be responsible for the number and variety of human<br />

cancers.<br />

<strong>LICR</strong> studies on the effects of single gene alterations on normal and tumor cell growth are now being<br />

complemented by use of comprehensive high through-put genomics and proteomics-based strategies that<br />

investigate literally thousands of gene and protein changes that occur as a cell progresses from a normal<br />

to a cancerous state. Using the combination of 'traditional' (single gene) and comprehensive approaches,<br />

the program seeks to understand the causes and effects of genetic changes in cancer. Examples of this are<br />

the studies performed at the São Paulo, London St Mary’s, and London University College Branches that<br />

have identified potential therapeutic, diagnostic and prognostic genetic markers for patients with acute<br />

lymphoblastic leukemia, and aerodigestive tract, breast, bladder, head and neck, prostate, and vulval<br />

cancers.<br />

There are concerted efforts in most <strong>LICR</strong> Branches to identify and characterize particular tumor<br />

suppressor genes and oncogenes. Defining, and determining the function/s of genes implicated in<br />

oncogenesis and cancer progression have allowed <strong>LICR</strong> investigators to unravel many of the cellular<br />

signaling pathways, and identify potential therapeutic targets. Just a few examples of the success of this<br />

approach are: studies of the activation of ‘cancer/testis’ (CT) genes that are expressed in cancer cells and<br />

in normal germ-line cells, and which are being analyzed to complement the Cancer Vaccine Program<br />

(Brussels Branch); the characterization of the ASPP family of proteins that are abnormally expressed in<br />

80% of breast cancers (London St Mary’s Branch); and characterization of the targets, and functions of<br />

transcription factors (San Diego and Uppsala Branches, respectively).<br />

BIOINFORMATICS PROGRAM<br />

As biological datasets grow in size (refer the Genomics Program section), bioinformatics - the<br />

application of informatics and computational tools to biology - is becoming increasingly important to all<br />

aspects of biomedical research. Whether it is identifying genes in DNA sequences, annotating the genome<br />

(i.e. appending information and commentary to the raw DNA sequence) analyzing the volume of data<br />

generated by microarray and proteomic approaches, or collating and compiling transcripts from<br />

ORESTES and ESTs, bioinformatics plays an integral role in advanced cancer research.<br />

The <strong>Institute</strong>'s Office of Information Technology (OIT) in Lausanne has taken on the task of making<br />

bioinformatics tools and relevant databases accessible to all <strong>LICR</strong> scientists. Working with the São Paulo<br />

Branch, which has sequenced over a million new pieces of the transcribed portions of human genes, the<br />

OIT has embarked on a large-scale effort to understand the detailed structure of the human<br />

“transcriptome”. The effort has combined the transcript data generated by the HCGP group, by the US<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 6


<strong>LICR</strong> RESEARCH PROGRAMS<br />

National Cancer <strong>Institute</strong>, and by others, with the draft human genome sequence, and produced a very<br />

detailed map of the regions of the genome that are expressed, i.e. used to make messenger RNA which is<br />

the template from which proteins are made. This knowledge enables the reconstitution of multiple<br />

messages that are produced from each individual gene and to track from which type of tissue each variant<br />

originated. It also makes it possible to predict which proteins are likely to be produced in which tissues,<br />

including cancers. This work is useful to many of the <strong>Institute</strong>'s scientific programs, particularly the<br />

transcriptomic and proteomics efforts of the Genomics Program.<br />

CANCER ANTIGEN DISCOVERY PROGRAM<br />

The path from a normal to a tumor cell is accompanied by changes in the cell’s gene expression and<br />

protein production, and some of these changes are reflected in the number and type of molecules that are<br />

displayed on a cancer cell’s surface, but not a normal cell’s surface. The immune system can recognize<br />

cancer cells, and distinguish them from normal cells, by monitoring unique molecules (or antigens)<br />

displayed on the cancer cells. Antigens that are specific for cancer cells can serve as specific targets for<br />

antibody-based therapies (see Antibody-Targeting Program), or as potential cancer vaccines (see Cancer<br />

Vaccine Program).<br />

<strong>LICR</strong> scientists in Brussels, Lausanne, Melbourne, and New York, as well as <strong>LICR</strong> Affiliates in<br />

Frankfurt, and Homburg, are acknowledged world-leaders in the identification and analysis of cancer<br />

antigens. The effort to identify cancer antigens has grown into a collaborative international Program led<br />

by the <strong>LICR</strong>, with the goal of mapping the entire repertoire of the immune response in cancer patients.<br />

This will define what is becoming known as the 'cancer immunome', that is, all the components of tumor<br />

cells that can elicit an immune response.<br />

The Cancer Antigen Discovery Program also takes advantage of genome- and transcriptome-derived<br />

information in public databases (and data generated within the Genomics Program). For example, in<br />

<strong>2002</strong>, investigators at the <strong>LICR</strong> New York Branch identified over 1000 potential CT antigens (see<br />

Genomics, and Cancer Vaccine Program sections), using bioinformatics techniques to assess the publicly<br />

available EST data. Also in <strong>2002</strong>, the OIT released their newly developed “Cancer Immunome Database”<br />

(CID), which integrates all the information on tumor antigens discovered thus far and the immune<br />

response which each elicits.<br />

CANCER VACCINE PROGRAM<br />

The discovery of specific cancer antigens raises the prospect of stimulating an immune response against<br />

cancer cells via cancer vaccines. This immunotherapy represents a novel alternative in the arsenal of<br />

cancer therapies. The development of a sufficiently potent cancer vaccine requires the systematic study of<br />

candidate antigens and vaccine delivery systems, as well as careful and precise monitoring of the immune<br />

response that is evoked. The ideal cancer vaccine needs to induce the immune system to recognize only<br />

cancer cells as abnormal, and evoke an immune response sufficient to contain tumor growth. Once the<br />

mechanisms for inducing appropriate, consistent and robust immune responses are understood the effects<br />

of such responses on human tumors and, hence, the potential of cancer vaccines may be rationally<br />

evaluated.<br />

Understanding the role of the immune system in cancer development, and the development of cancer<br />

therapies based on immunologic principles continue to be major objectives of the <strong>Institute</strong>. Indeed the<br />

<strong>LICR</strong> has become, arguably, the largest center in the world for cancer immunology studies. Laboratory<br />

research into what constitutes a strong anti-tumor immune response, and how it can be monitored, is<br />

being conducted at the Branches in New York, Lausanne, Brussels, and Melbourne; and by a number of<br />

<strong>LICR</strong> Affiliates.<br />

Understanding how to induce antigen specific immune responses - by comparing various antigen<br />

compositions, adjuvants, cytokines, immunostimulatory molecules and modes of delivery - provides a<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 7


<strong>LICR</strong> RESEARCH PROGRAMS<br />

model for cancer vaccine development. Almost two thirds of ongoing <strong>LICR</strong> clinical trials in the Clinical<br />

Trials Program examine vaccine strategies. The ability to monitor an immune response is critical for the<br />

development of maximally immunogenic vaccines. A prime objective of the program is, therefore, to<br />

develop precise and standardized methodologies capable of monitoring T cell and antibody responses to<br />

cancer antigens following immunization. During <strong>2002</strong>, a Tetramer Facility was established at the<br />

Lausanne Branch to generate the key reagents required, by the <strong>LICR</strong> Clinical Trials Program, for T cell<br />

monitoring.<br />

ANTIBODY TARGETING PROGRAM<br />

Antibodies are naturally occurring proteins that travel through the blood and lymphatic systems. They<br />

seek out and bind to particular antigens with high specificity. Antibody-based cancer therapies selectively<br />

exploit that specificity to target cancer cells. Once an antibody has bound its antigen, it can elicit a local<br />

immune attack against the antigen, or against cells that display the antigen. In addition, antibodies can<br />

be engineered to deliver specific toxins to the tumor. As most therapeutic antibodies are generated in<br />

mice, the antibodies must be genetically engineered to be 'humanized' for long-term therapeutic use.<br />

Over the past 17 years, <strong>LICR</strong> scientists at the New York and Melbourne Branches have accumulated a<br />

portfolio of mouse antibodies and their humanized derivatives, which specifically target a number of<br />

tumors. They have collected a wealth of preclinical and early clinical data on many of these. Recent<br />

efforts have also involved scientists at the San Diego Branch, as well as a number of <strong>LICR</strong> Affiliates and<br />

industrial collaborators.<br />

A particularly exciting project within the Antibody Targeting Program has been the inter-Branch<br />

collaborative venture to characterize the monoclonal antibody (MAb) MAb 806, which was developed by<br />

the New York Branch specifically for the purpose of investigating and targeting the EGFR variant. MAb<br />

806, which was patented in <strong>2002</strong> as part of the <strong>Institute</strong>’s intellectual property portfolio, binds a truncated<br />

version of the EGFR, and also normal EGFR that is over-expressed on many tumor cells, but not normal<br />

cells. Thus MAb 806 may offer significant and very specific tumor (versus normal tissue) targeting over<br />

other closely related antibodies being developed by other researchers for clinical study. Work during <strong>2002</strong><br />

established the biodistribution of the MAb in mice, and found that it shows a very specific tissue targeting<br />

profile that is ideal for clinical use. MAb 806 treatment was also shown to inhibit cellular targets of<br />

EGFR signaling, and was able to reduce the growth of transplanted human brain tumors in mice. The<br />

MAb 806 has now been chimerized (made human-like), and <strong>LICR</strong>-sponsored Phase I trials are planned<br />

for 2003/2004.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 8


MR.R.PALMER BAKER,JR.<br />

Chairman<br />

MR.GEORGES-ANDRE CUENDET<br />

MR.OLIVER DUNANT<br />

DR.ADOLF E. KAMMERER<br />

MR.PIERRE LANGUETIN<br />

MR.EDWARD A. MCDERMOTT,JR.<br />

DR.LLOYD J. OLD<br />

SIR DEREK ROBERTS<br />

MS.SUSY BRÜSCHWEILER<br />

MR.RICHARD D.J. WALKER<br />

Secretary to the Board<br />

BOARD OF DIRECTORS<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 9


DR.LLOYD J. OLD<br />

Chairman<br />

SCIENTIFIC COMMITTEE<br />

SIR WALTER BODMER Weatherall <strong>Institute</strong> of Molecular Medicine, Oxford, England<br />

DR.DOUGLAS T. FEARON University of Cambridge, Cambridge, England<br />

DR.SAMUEL HELLMAN University of Chicago, Chicago, U.S.A.<br />

DR.GEORGE KLEIN Karolinska <strong>Institute</strong>, Stockholm, Sweden<br />

DR.IRA MELLMAN Yale University, New Haven, U.S.A.<br />

DR.ARTHUR B. PARDEE Dana-Farber Cancer <strong>Institute</strong>, Boston, U.S.A.<br />

DR.ELLEN PURE Wistar <strong>Institute</strong>, Philadelphia, U.S.A.<br />

DR.LUCILLE SHAPIRO Stanford University, Stanford, U.S.A.<br />

DR.PHILLIP SHARP Massachusetts <strong>Institute</strong> of Technology, Cambridge, U.S.A.<br />

DR.HARALD ZUR HAUSEN Deutsches Krebschforschungszentrum, Heidelberg, Germany<br />

DR.A.MUNRO NEVILLE<br />

Secretary to the Committee<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 10


INSTITUTE ADMINISTRATION<br />

LUDWIG INSTITUT FÜR KREBSFORSCHUNG REGISTERED ADDRESS<br />

STADELHOFERSTRASSE 22<br />

8001 ZURICH,SWITZERLAND<br />

POSTFACH 8024 ZURICH,SWITZERLAND POSTAL ADDRESS<br />

TELEPHONE: [41] (0)1-267 6262<br />

FAX: [41] (0)1-267 6200<br />

MR.R.PALMER BAKER JR.<br />

Chairman<br />

DR.LLOYD J. OLD<br />

<strong>Institute</strong> Director, and Chief Executive Officer<br />

MR.EDWARD A. MCDERMOTT,JR.<br />

President<br />

DR.A.MUNRO NEVILLE<br />

<strong>Institute</strong> Associate Director<br />

MR.RICHARD D.J. WALKER<br />

Chief Financial Officer<br />

MILBANK,TWEED,HADLEY &MCCLOY,NEW YORK Counsels<br />

NIEDERER,KRAFT &FREY,ZURICH<br />

KPMG FIDES,PEAT,ZURICH Auditors<br />

CREDIT SUISSE,ZURICH Principal Bankers<br />

NATIONAL WESTMINSTER BANK PLC, LONDON<br />

HSBC BANK USA, NEW YORK<br />

AUSTRALIA: A.R.B.N. 001 379 344 Company<br />

BELGIUM:415.420 Registration<br />

SWITZERLAND: CH-020.3.916.330-2 Numbers<br />

UNITED KINGDOM: FC 007198<br />

UNITED STATES OF AMERICA: EIN 23-712-1131<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 11


ADMINISTRATION –NEW YORK<br />

LUDWIG INSTITUTE FOR CANCER RESEARCH ADDRESS<br />

605 THIRD AVENUE /33 RD FLOOR<br />

NEW YORK, NY 10158 U.S.A.<br />

TELEPHONE: [1] 212 450 1500<br />

FAX: [1] 212 450 1555<br />

MR.R.PALMER BAKER,JR. Chairman<br />

MS.KAREN A. HYLAND Executive Assistant<br />

FAX: [1] 212 450 1545<br />

DR LLOYD J. OLD <strong>Institute</strong> Director and CEO<br />

MS NANCY LEYRER Assistant to <strong>Institute</strong> Director and CEO<br />

MS CHARLOTTE DAUB Executive Assistant (until April)<br />

FAX: [1] 212 450 1515<br />

MR.EDWARD A. MCDERMOTT,JR. President<br />

MS.KATHY MEAGHAN Assistant to President<br />

MS.NADETTE BULGIN Deputy Assistant to President<br />

Fax: [1] 212 450 1555<br />

DR.ANDREW J.G. SIMPSON Director, James R. Kerr Program (from October)<br />

Fax: [1] 212 450 1555<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 12


ADMINISTRATION –NEW YORK<br />

DR.ELLEN PURÉ Director OFFICE OF ACADEMIC<br />

MS.TERESA PAPPAS Assistant to Director REVIEW<br />

FAX: [1] 212 450 1525<br />

DR.ERIC W. HOFFMAN Director OFFICE OF CLINICAL<br />

DR.GERD RITTER Assistant Director TRIALS MANAGEMENT<br />

MS.LISA PUGLIESE Manager FAX: [1] 212 450 1535<br />

DR.RALPH VENHAUS Head of Clinical Affairs<br />

DR.MARYJANE RAFII Head of Regulatory Affairs and Safety<br />

MS.JULIE ANDREW Clinical <strong>Research</strong> Associate Manager<br />

MR.LAWRENCE T. PERSAUD IT Portfolio<br />

MR.JOHN C-K. TANG Clinical Data Management (from November)<br />

MR.GARY O’DONNELL Manager Drug Safety (from August)<br />

DR.LINDA PAN Clinical Programs Associate (from August)<br />

MS.JUNE SWIRZ Assistant to Director<br />

MS.ELISA RUIZ Executive Secretary<br />

MR.ERIC SABO External Relations Officer OFFICE OF<br />

MR.JOSHUA CARR Electronic Communications Assistant COMMUNICATIONS<br />

DR.JONATHAN C. SKIPPER Manager OFFICE OF<br />

DR.PÄR OLSSON Scientific Administrative Officer INTELLECTUAL<br />

DR.MONA JHAVERI Scientific Administrative Officer (from September) PROPERTY<br />

DR.JONATHAN C. SKIPPER Director OFFICE OF PROGRAM<br />

DEVELOPMENT<br />

DR.HERBERT F. OETTGEN Chairman OFFICE OF THE<br />

MS.NANCY LEYRER Secretary PROTOCOL REVIEW<br />

COMMITTEE<br />

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ADMINISTRATION -LONDON<br />

LUDWIG INSTITUTE FOR CANCER RESEARCH ADDRESS<br />

HORATIO HOUSE<br />

77-85 FULHAM PALACE ROAD /5 TH FLOOR SOUTH<br />

LONDON, W6 8JC, UNITED KINGDOM<br />

TELEPHONE: [44] (0)20 8735 9240<br />

FAX /MAIN: [44] (0)20 8741 4990<br />

FAX /SECOND: [44] (0)20 8741 5884<br />

DR.A.MUNRO NEVILLE Director OFFICE OF<br />

(Associate Director of <strong>Institute</strong>) INTELLECTUAL<br />

PROPERTY<br />

DR.DEBORAH CARTER Associate Director<br />

DR.PAULINE STASIAK Senior Scientific Administrative Officer<br />

DR.AMY (LAM)JEFFS Scientific Administrative Officer<br />

DR.KATE HUDSON Scientific Administrative Officer (until April)<br />

MR.BEN WILLIAMS Scientific Administrative Assistant (from September)<br />

MS.LINDA A. LENTHALL Assistant to Director<br />

MS.ANITA GANEA Secretary<br />

DR.ROBERT BURNS Director (from May) OFFICE OF<br />

MS.BRENDA GOULDER Secretary (from September) TECHNOLOGY<br />

LICENSING<br />

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ADMINISTRATION -ZÜRICH<br />

LUDWIG INSTITUT FÜR KREBSFORSCHUNG ADDRESS<br />

STADELHOFERSTRASSE 22<br />

8001 ZÜRICH,SWITZERLAND<br />

POSTFACH: 8024 ZÜRICH,SWITZERLAND POSTAL ADDRESS<br />

TELEPHONE: [41] (0)1 267 62 62<br />

FAX /MAIN: [41] (0)1 267 62 00<br />

MR.RICHARD D.J. WALKER Chief Financial Officer OFFICE OF BUDGET<br />

MS.ANDREA KELLER Assistant to Chief Financial Officer AND FINANCE<br />

MR.ODD AARDALSBAKKE Deputy Chief Financial Officer<br />

MR.MARTIN BORN Financial Controller<br />

MS.PATRICIA MCCORMICK Assistant Controller IP<br />

MR.DARREN SHEARER Assistant Controller IP (from May)<br />

MS.HARUKO HARTMANN Assistant IP<br />

MS.PAOLA MAIO Accountant<br />

MR.RETO SCHLÄFLE Accountant (from May)<br />

MR.STEVE BIRNBAUM Manager IT Finance<br />

MR.RICHARD MCCLEARY Budget and Planning Manager/Internal Auditor<br />

MS.KARIN GENHART Secretary<br />

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ADMINISTRATION -LAUSANNE<br />

LUDWIG INSTITUTE FOR CANCER RESEARCH ADDRESS<br />

CHEMIN DES BOVERESSES 155<br />

1066 EPALINGES<br />

SWITZERLAND<br />

TELEPHONE: [41] (0)21 692 59 91<br />

FAX /MAIN: [41] (0)21 692 59 45<br />

DR.C.VICTOR JONGENEEL Director OFFICE OF<br />

INFORMATION<br />

MS.JOCELYNE MULLER Secretary/Administrator TECHNOLOGY<br />

DR.CHRISTIAN ISELI Systems Manager<br />

DR.ABDELALLI KADDOUR Database Programmer (until April)<br />

DR.DMITRI KUZNETSOV Biocomputing Specialist<br />

DR.BRIAN STEVENSON Bioinformatics Specialist<br />

DR.YVES STAUFFER Postdoctoral Fellow<br />

MR.GREGORY THEILER Graduate Student<br />

DR.MONIQUE ZAHN Editorial Assistant “CANCER IMMUNITY”<br />

JOURNAL<br />

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BRUSSELS BRANCH OF HUMAN CANCER CELL GENETICS<br />

AVENUE HIPPOCRATE 74, UCL 7459<br />

1200 BRUSSELS,BELGIUM<br />

TEL: [32] (0)2 764 74 59<br />

FAX: [32] (0)2 762 94 05<br />

DR.THIERRY BOON Branch Director<br />

LAUSANNE BRANCH OF IMMUNOLOGY<br />

CHEMIN DES BOVERESSES 155<br />

1066 EPALINGES S/LAUSANNE,SWITZERLAND<br />

TEL: [41] (0)21 692 59 66<br />

FAX: [41] (0)21 653 44 74<br />

DR.JEAN-CHARLES CEROTTINI Branch Director<br />

LONDON/ST MARY’S BRANCH OF VIROLOGY AND CELL BIOLOGY<br />

IMPERIAL COLLEGE OF SCIENCE TECHNOLOGY AND MEDICINE/ST MARY’S CAMPUS<br />

NORFOLK PLACE,W21PG,LONDON,UK<br />

TEL: [44] (0)20 7724 55 22<br />

FAX: [44] (0)20 7724 85 86<br />

DR.PAUL J. FARRELL Branch Director<br />

LONDON/UNIVERSITY COLLEGE BRANCH OF CELL AND MOLECULAR BIOLOGY<br />

COURTAULD BUILDING,91RIDING HOUSE STREET<br />

W1W 7BS, LONDON UK<br />

TEL: [44] (0)20 7878 4000<br />

FAX: [44] (0)20 7878 4040<br />

DR.MICHAEL D. WATERFIELD Branch Director<br />

MELBOURNE BRANCH OF TUMOUR BIOLOGY<br />

PO BOX 2008 ROYAL MELBOURNE HOSPITAL<br />

PARKVILLE,VICTORIA 3050, AUSTRALIA<br />

TEL: [61] (0)3 9341 31 55<br />

FAX: [61] (0)3 9341 31 04<br />

DR ANTONY W. BURGESS Branch Director<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003<br />

BRANCH ADDRESSES<br />

12


NEW YORK BRANCH OF HUMAN CANCER IMMUNOLOGY<br />

MEMORIAL SLOAN-KETTERING CANCER CENTER<br />

1275 YORK AVENUE,BOX 32<br />

NEW YORK, NY 10021-6007 USA<br />

TEL: [1] 212 639 8600<br />

FAX: [1] 212 717 3100<br />

DR LLOYD J. OLD Branch Director<br />

SAN DIEGO BRANCH OF CANCER GENETICS<br />

UNIVERSITY OF CALIFORNIA SAN DIEGO<br />

9500 GILMAN DRIVE<br />

LA JOLLA, CA 92093-0660 USA<br />

TEL: [1] 858 534 7802<br />

FAX: [1] 619 534 7750<br />

DR WEBSTER K. CAVENEE Branch Director<br />

SÃO PAULO BRANCH OF CANCER GENETICS<br />

RUA PROF.ANTONIO PRUDENTE<br />

109-4 ANDAR,LIBERDADE 01509-010<br />

SÃO PAOLO, SP, BRAZIL<br />

TEL: [55] (0XX)1133883200<br />

FAX:[55](0XX)1132077001<br />

DR RICARDO R. BRENTANI Branch Director<br />

STOCKHOLM BRANCH OF MOLECULAR AND CELL BIOLOGY<br />

KAROLINSKA INSTITUTE,BOX 240<br />

S-17177 STOCKHOLM,SWEDEN<br />

TEL: [46] (0)8 728 71 00<br />

FAX: [46] (0)8 33 28 12<br />

DR RALF E. PETTERSSON Branch Director<br />

UPPSALA BRANCH OF GROWTH REGULATION<br />

BIOMEDICAL CENTER,BOX 595<br />

S-751 24 UPPSALA,SWEDEN<br />

TEL: [46] (0)18 16 04 00<br />

FAX: [46] (0)18 16 04 20<br />

DR.CARL-HENRIK HELDIN Branch Director<br />

BRANCH ADDRESSES<br />

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BRUSSELS BRANCH<br />

OF HUMAN CANCER CELL GENETICS<br />

BRUSSELS,BELGIUM<br />

Staff List: Page 20<br />

Branch Director’s <strong>Report</strong>: Page 24<br />

<strong>Research</strong> <strong>Report</strong>: Page 25<br />

Publications: Page 29<br />

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STAFF LIST<br />

BOON THIERRY, Director, Member*<br />

ANIMAL GROUP<br />

WARNIER,GUY, Associate Investigator<br />

MATTHEWS,JEAN-PIERRE, Technical Assistant<br />

SIRICHANTO,PHONE PRASEUTH, Technical Assistant<br />

SOUVANNARAJ,KEOPRASITH, Technical Assistant<br />

SOUVANNARAJ,KEOTA, Technical Assistant<br />

CYTOKINE GROUP<br />

RENAULD,JEAN-CHRISTOPHE, Member<br />

VAN SNICK,JACQUES, Member<br />

DUMOUTIER,LAURE, Postdoctoral Fellow<br />

LOUAHED,JAMILA, Postdoctoral Fellow<br />

DARCAIGNE,DAVID, PhD Student (from November)<br />

KNOOPS,LAURENT, PhD Student<br />

LEJEUNE,DIANE, PhD Student<br />

RICHARD,MÉLISANDE, PhD Student (until October)<br />

STEENWINCKEL,VALÉRIE, PhD Student (from October)<br />

CORMONT,FRANÇOISE, Technician<br />

DE LESTRE,BRIGITTE, Technician<br />

STEVENS,MONIQUE, Technician<br />

VAN ROOST,EMIEL, Technician<br />

TUMOR GENETICS GROUP<br />

DE PLAEN,ETIENNE, Assistant Member<br />

DE SMET,CHARLES, Assistant Member<br />

LADURON,SANDRA, PhD Student (until September)<br />

BOUCQUEY,MARIE, PhD Student<br />

LORIOT,AXELLE, PhD Student<br />

BLONDIAUX,CLAUDINE, Technician<br />

* Clinical Trials Program participant<br />

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BRUSSELS


HUMAN TUMOR ANTIGEN GROUP*<br />

VAN DER BRUGGEN,PIERRE, Member<br />

WILDMANN,CLAUDE, <strong>Research</strong> Associate<br />

ZHANG,YI, Visiting Scientist<br />

LONCHAY,CHRISTOPHE, PhD Student (until October)<br />

NICOLAY,HUGUES, PhD Student (from November)<br />

PANICHELLI,CHRISTOPHE, PhD Student (until October)<br />

DEMOTTE,NATHALIE, Technician<br />

LOEUILLE,SEBASTIEN, Technician<br />

OTTAVIANI,SABRINA, Technician<br />

TOLLET,BÉNÉDICTE, Technician<br />

TUMOR IMMUNOLOGY GROUP<br />

VAN DEN EYNDE,BENOÎT, Member<br />

CHAPIRO,JACQUES, Postdoctoral Fellow<br />

MA,WENBIN, Postdoctoral Fellow<br />

THÉATE,IVAN, Postdoctoral Fellow<br />

GUILLAUME,BENOÎT, PhD Student (from October)<br />

HUIJBERS,IVO, PhD Student<br />

VIGNERON,NATHALIE, PhD Student<br />

PILOTTE,LUC, <strong>Research</strong> Associate<br />

PIETTE,FANNY, Technician<br />

IMMUNOGENETICS DIAGNOSIS GROUP*<br />

BRASSEUR,FRANCIS, Associate Investigator<br />

CARRASCO,JAVIER, PhD Student<br />

SWINARSKA,MAGDALENA, Technician<br />

* Clinical Trials Program participant<br />

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BRUSSELS


IMMUNOGENETICS MONITORING GROUP*<br />

VAN PEL,ALINE, Associate Member<br />

COLAU,DIDIER, Associate Investigator<br />

LETHÉ,BERNARD, Associate Investigator<br />

LURQUIN,CHRISTOPHE, Associate Investigator<br />

GODELAINE,DANIÈLE, Investigator<br />

HA THI,VINH, Technician<br />

LETELLIER,MARIE-CLAIRE, Technician<br />

PANAGIOTAKOPOULOS,MARIA, Technician<br />

VAN VOOREN,ALICE, Technician (from April)<br />

MOUSE ANTITUMORAL IMMUNIZATION GROUP<br />

UYTTENHOVE,CATHERINE, Senior Investigator<br />

DONCKERS,DOMINIQUE, Technician<br />

SIGNAL TRANSDUCTION GROUP<br />

CONSTANTINESCU,STEFAN, Assistant Member<br />

KUBATZKY,KATHARINA, Postdoctoral Fellow (from June)<br />

MOUCADEL,VIRGINIE, Postdoctoral Fellow<br />

EL NAJJAR,NICOLE, PhD Student (from November)<br />

ROYER,JOHAN, PhD Student<br />

STÄRK,JUDITH, PhD Student<br />

MENU,CATHERINE, Technician<br />

THERAPEUTIC VACCINATION GROUP*<br />

VAN BAREN,NICOLAS, Clinical Investigator<br />

MARCHAND,MARIE, Senior Clinical Investigator<br />

RAIMUNDO,ANA, Postdoctoral Fellow (until October)<br />

BOUSMANS,NATHALIE, Data Manager<br />

DAUBRESSE,PIERRE, Data Manager (from October)<br />

DEGUELDRE,JÉRÔME,Y,Data Manager<br />

GUEUR,MARIE-CHRISTINE, Secretary<br />

* Clinical Trials Program participant<br />

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BRUSSELS


LABORATORY SUPPORT SERVICES<br />

WAUTERS,PAUL, Laboratory Manager<br />

TONON,ANDRÉ, FACS operator<br />

STROOBANT,VINCENT, Chemist (Immunochemistry)<br />

AUTHOM,ANNE, Technician (Immunochemistry) (until July)<br />

LEJEUNE,JOSEPH, Computer Analyst<br />

CALLEJA,MIGUEL, Office Clerk (from December)<br />

VANDENBORRE,JULIEN, Office Clerk (until July)<br />

VERHELST,CEDRIC, Office Clerk (until October)<br />

GERMIS,JEAN-LUC, Technician<br />

ORVIZ,MANUEL, Technical Assistant<br />

VAN GOETSENHOVEN,ANTOINETTE, Technical Assistant<br />

JEBARI,SAMIRA, Laboratory helper<br />

HAMED,MUSTAFA, Laboratory helper<br />

MAHIEU-GAUDY,PASCALE, Laboratory helper<br />

MARIANINI,ENCARNACION, Laboratory helper<br />

REYES,RAFAELA, Cleaner<br />

ADMINISTRATION AND SECRETARIAT<br />

FLOREAN,DARIO, Administrator<br />

VERFAILLE,CHRISTIAN, Clinical Trials Coordinator*<br />

BIEN,ALINE, Administrative Assistant<br />

LEMOINE,CARINE, Administrative Assistant<br />

STAVAUX,PIERRE, Accounting Clerk<br />

HOPPE,SYLVIE, Scientific Secretary<br />

BROUET,JENNY, Secretary (until October)<br />

KRACK,NATHALIE, Secretary<br />

NICULESCU,LILIANA, Secretary (until July)<br />

SCHOONHEYDT,GENEVIÈVE, Secretary<br />

* Clinical Trials Program participant<br />

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BRUSSELS


BRANCH DIRECTOR’S REPORT<br />

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BRUSSELS<br />

The Brussels Branch specializes in cancer immunology and cancer genetics. The notion that the immune<br />

system might be enlisted to rid the body of cancer draws on past work at the Branch which revealed that<br />

most human tumors bear antigens that can be recognized by cytotoxic T lymphocytes (CTLs). Some of<br />

these antigens are highly tumor-specific, whilst others are expressed on certain normal cells. A number of<br />

antigens have been found on many different types of tumors, suggesting that a therapeutic strategy<br />

targeting such antigens could be used to treat a wide range of cancers. The Brussels Branch continues the<br />

search for tumor antigens, and evaluates their therapeutic potential in vaccine trials of cancer patients.<br />

T. Boon


RESEARCH REPORT<br />

CYTOKINE GROUP<br />

* Clinical Trials Program participant<br />

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BRUSSELS<br />

Led by Dr. Jean-Christophe Renauld, the group studies the biology of interleukin (IL) -9 and IL-22, two<br />

cytokines discovered at the <strong>LICR</strong> Brussels Branch. IL-9 is a TH2 cytokine that plays a role in immune<br />

responses against intestinal parasites, and asthma. IL-22, originally identified as a gene induced by IL-9 in<br />

T lymphocytes, upregulates the production of acute phase reagents in liver. Its activity in inflammatory<br />

responses is modulated by a specific antagonist; the IL-22 binding protein. The role of IL-9 and IL-22 in<br />

inflammation is currently investigated using transgenic and gene-targeted mice for these cytokines and<br />

their receptors.<br />

First, IL-9 transgenic mice, which have a high level of this cytokine in all tissues, are characterized by a<br />

high susceptibility to the development of T cell lymphomas. Another major aspect of IL-9 biology is its<br />

effect on the growth and differentiation of mast cells. IL-9 transgenic mice show increased numbers of<br />

mast cells in the gut and airways. Finally, a puzzling activity of IL-9 is a selective increase in the<br />

peritoneal B1b cell subpopulation. Although the specificity of these cells is far from clear, they might be<br />

related to some auto-immune processes. In line with the oncogenic activity of IL-9 in transgenic mice,<br />

this cytokine was shown to be a potent anti-apoptotic factor for T cell lymphomas. The anti-apoptotic<br />

effect of IL-9 does not involve MAP-kinases but is mediated by the Janus tyrosine kinase (JAK)/Signal<br />

transducers and activators of transcription (STAT) pathway. Therefore, the group is now focusing on the<br />

characterization of genes whose expression is regulated by IL-9 through the activation of STAT<br />

transcription factors.<br />

HUMAN TUMOR ANTIGEN GROUP*<br />

The group led by Dr. Pierre van der Bruggen is defining antigenic peptides encoded by genes like those of<br />

the MAGE family, which are expressed by cells from many different cancer types. Therapeutic<br />

vaccination of cancer patients with MAGE peptides is now in progress. The identification of additional<br />

antigenic peptides is important to increase the number and type of patients eligible for therapy, and to<br />

provide tools for a reliable monitoring of the immune response. Stimulation of CD8 + T lymphocytes with<br />

antigen-presenting dendritic cells infected with viruses carrying MAGE genes has led to the identification<br />

of a large number of new antigenic peptides presented by HLA class I molecules. Similarly, stimulation<br />

of CD4 + T cells with dendritic cells pulsed with a MAGE protein has revealed new antigenic peptides<br />

presented by HLA class II molecules. The finding that almost every cancer patient whose tumor expresses<br />

a MAGE gene has at least one HLA molecule presenting a MAGE antigenic peptide suggests numerous<br />

possibilities for therapeutic vaccination. Reliable methods to monitor the CD4 + T cell response to cancer<br />

vaccines are in development.<br />

The group is also involved in the study of functional defects of T cells. We have observed that human<br />

cytotoxic T lymphocytes (CTL) clones lose their specific cytolytic activity and cytokine production under<br />

certain stimulation conditions. These inactive CTL simultaneously lose their labeling by an HLA-peptide<br />

tetramer, even though the amount of TCR-CD3 at their surface is not reduced. Our results suggest the<br />

existence of a new type of functional defect of CTL.


IMMUNOGENETICS MONITORING GROUP*<br />

* Clinical Trials Program participant<br />

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BRUSSELS<br />

Dr. Aline Van Pel’s group collaborates with the group of Dr. Pierre Coulie at the <strong>Institute</strong> for Cellular<br />

Pathology (Catholic University of Louvain, Brussels) to elucidate whether or not a correlation between<br />

the clinical responses and the anti-vaccine T cell responses exists in the minority of patients who display<br />

tumor regression after MAGE vaccination. A very sensitive method of CTL detection was set up last year,<br />

involving in vitro stimulation of blood lymphocytes, tetramer analysis, obtention of CTL clones, and<br />

analysis of their TCR sequences. This approach was used for 12 patients who showed tumor regression<br />

after vaccination with a MAGE-3 peptide presented by HLA-A1. The vaccines contained either peptides<br />

alone, or a recombinant ALVAC canarypox virus, produced by Aventis. An anti-MAGE-3.A1 CTL<br />

response was found in seven patients. All the CTL responses were monoclonal. Among 16 vaccinated<br />

patients who did not display tumor regression, only two had an anti-MAGE-3.A1 CTL response. In<br />

collaboration with the group of Dr. G. Schuler (Erlangen, Germany), CTL responses were analyzed in<br />

melanoma patients vaccinated with mature dendritic cells pulsed with the MAGE-3.A1 peptide. CTL<br />

responses were found in three patients showing tumor regression. In each of these responses, several CTL<br />

clones were amplified, a polyclonality that contrasts with the monoclonal responses observed in patients<br />

vaccinated with peptide or ALVAC. One patient who did not respond clinically to vaccination with<br />

dendritic cells was analyzed, and no MAGE-3.A1 CTL could be found. These results suggest that the<br />

occurrence of a detectable CTL response shows some degree of correlation with tumor regression,<br />

opening the possibility that a limiting factor for the anti-tumor effect of MAGE vaccination is the<br />

intensity of the CTL response to the vaccine antigen.<br />

TUMOR GENETICS GROUP<br />

Human tumors express specific antigens arising from the activation of genes, such as MAGE, BAGE,<br />

GAGE and LAGE/NY-ESO1, which are expressed only in tumors and normal germ cells, and are known<br />

as “cancer/testis” (CT) antigens. As germ cells are not subject to scrutiny by the immune system, CT<br />

antigens encoded by these genes are strictly tumor-specific. The group of Drs. Etienne De Plaen and<br />

Charles De Smet is trying to identify new genes that are specifically expressed in tumors and germ cells.<br />

Screening procedures based on differential expression profiling have isolated several genes with cancer<br />

and germline specific expression. Most of these genes are normally expressed in spermatogonia, the premeiotic<br />

stage of sperm development, and are located on the X chromosome. Efforts are underway to<br />

determining the function of these "cancer-testis" (CT) genes. To analyze the functions of a MAGE<br />

protein, MAGE-A1, Dr. Etienne De Plaen and his group used yeast two-hybrid screening, and found an<br />

interaction between MAGE-A1 and the transcriptional regulator SKIP. SKIP is an adaptor protein that<br />

connects DNA-binding proteins to proteins that activate or repress transcription. The results suggest that<br />

by binding to SKIP and recruiting histone deacetylase 1, MAGE-A1 protein represses transcription. The<br />

group is now trying to identify the genes that are regulated by MAGE-1 by using an inducible transfected<br />

MAGE-A1 gene and microarray technology.<br />

Finally, the group is also studying the mechanisms leading to the activation of CT genes in tumors. The<br />

group has previously shown that DNA methylation is an essential component of their repression in<br />

normal somatic tissues. The promoters of these genes contain a high density of CpG islands, but unlike<br />

classical CpG-rich promoters they are heavily methylated in all somatic tissues. In contrast, the promoters<br />

are unmethylated in germ cells, and in tumors that express these genes. Demethylation and therefore<br />

activation of CT genes in tumors was found to be coincident with overall genome demethylation, a<br />

process known to occur in many cancers. The group is currently studying the mechanisms of<br />

demethylation of these genes in tumors. Activation of CT genes in tumors appears to rely on a historical


* Clinical Trials Program participant<br />

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BRUSSELS<br />

event of demethylation, and on the presence of specific transcription factors that maintain the promoter<br />

region in an unmethylated state.<br />

TUMOR IMMUNOLOGY GROUP<br />

The central research theme of Dr. Benoît Van den Eynde’s group is the study of tumor antigens<br />

recognized by T lymphocytes for cancer vaccines in human patients. As well as a continued effort to<br />

identify additional antigens of interest, the group also addresses a number of fundamental or mechanistic<br />

issues that have a direct impact on the utilization of such antigens. The antigens consist of peptides,<br />

presented by MHC molecules at the cell surface, which derive from intracellular proteins degraded by the<br />

proteasome. The intracellular pathway leading from the antigenic protein to the peptide/MHC complex is<br />

known as “antigen processing”. The group is currently studying the processing of several human tumor<br />

antigens by the proteasome, and is particularly interested in the processing differences observed between<br />

the standard proteasome, which is present in most cells, and the immunoproteasome which is found in<br />

some dendritic cells and in cells exposed to interferon-gamma.<br />

The group is also studying a mouse preclinical model of cancer immunotherapy, to define the optimal<br />

conditions to induce effective anti-tumor responses by various vaccination approaches against particular<br />

antigens. This has uncovered a powerful mechanism of tumor resistance which is based on tryptophan<br />

catabolism by indoleamine-2,3 dioxygenase, an enzyme frequently expressed in tumors. The resulting<br />

local tryptophan shortage appears to prevent the proliferation of lymphocytes at the tumor site. Inhibitors<br />

of indoleamine-2,3 dioxygenase are currently being tested in vivo for their ability to counteract this tumor<br />

resistance mechanism. To obtain the most relevant information from such preclinical models, the group is<br />

attempting to generate a new mouse melanoma model in which tumors expressing a given antigen can be<br />

induced using a transgenic system based on Cre-lox recombination. This should recapitulate the long-term<br />

host/tumor relationship that occurs in humans when a tumor slowly develops within a normal tissue.<br />

THERAPEUTIC VACCINATION GROUP*<br />

Because of their tumor specificity, MAGE antigens are promising candidates for cancer vaccine<br />

development. The group led by Drs. Marie Marchand and Nicolas Van Baren, designs and carries out<br />

such clinical trials. Trials investigating either MAGE peptides or recombinant MAGE proteins, and a<br />

recombinant ALVAC virus encoding a MAGE minigene have been completed or are ongoing. All of<br />

these cancer vaccines are very well tolerated by the patients. Tumor regressions have been observed in<br />

approximately 20% of the vaccinated patients with metastatic melanoma, with complete or partial clinical<br />

responses being observed in approximately 10% of patients.<br />

While it is possible to make CTL recognize and kill autologous tumor cells in vitro, the precise way to<br />

induce an effective CTL response against a MAGE antigen in cancer patients is not yet known. Clinical<br />

vaccination trials have two main objectives. First, the effectiveness of various vaccination modalities can<br />

be assessed by following the clinical evolution of the tumor, by analyzing whether a specific CTL<br />

response to the vaccine antigen occurred, and by determining whether immunological and clinical<br />

responses are correlated. Secondly, T lymphocytes and tumor samples collected at different time points<br />

during vaccination can be analyzed in detail, improving our understanding of what happens in patients<br />

who experience regression of metastatic lesions, and perhaps explaining why this does not happen in the<br />

majority of patients with overall disease progression. This knowledge can then be used to design new<br />

vaccination modalities.


SIGNAL TRANSDUCTION GROUP<br />

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BRUSSELS<br />

The group of Dr. Stefan Constantinescu joined the <strong>LICR</strong> in 2001, and reinforced and extended the focus<br />

on signal transduction mechanisms involving cytokine receptors. Cytokines and their receptors are critical<br />

for the formation of mature blood cells and for the function of the immune system. Signaling by cytokine<br />

receptors is triggered by ligand-induced changes in receptor dimerization/oligomerization, which induces<br />

the activation of cytosolic JAK proteins. Activated JAK proteins phosphorylate downstream STAT<br />

proteins, which dimerize in the cytosol and are translocated to the nucleus where they bind to specific<br />

promoter sequences and regulate transcription. The group studies the signal transduction mechanisms and<br />

biological functions of cytokine receptors such as the receptors for erythropoietin, thrombopoietin, IL-2<br />

and IL-9. The assembly of cell-surface receptor complexes, the structure and orientation of the<br />

transmembrane and cytosolic juxtamembrane domains, and the regulation by JAK kinases of receptor<br />

traffic are major focuses. The group also studies the mechanisms by which STAT proteins become<br />

constitutively activated and how they function in transformed hematopoietic or patient-derived leukemia<br />

cells.


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

* Clinical Trials Program participant<br />

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BRUSSELS<br />

1. BELLADONNA ML, RENAULD JC, BIANCHI R, VACCA C, FALLARINO F, ORABONA C, FIORETTI<br />

MC, GROHMANN U, PUCCETTI P. IL-23 and IL-12 have overlapping, but distinct, effects on<br />

murine dendritic cells. Journal of Immunology (<strong>2002</strong>) Jun 1;168(11):5448-54.<br />

2. BILSBOROUGH J, PANICHELLI C, DUFFOUR MT, WARNIER G, LURQUIN C, SCHULTZ ES,<br />

THIELEMANS K, CORTHALS J, BOON T, VAN DER BRUGGEN P. A MAGE-3 peptide presented by<br />

HLA-B44 is also recognized by cytolytic T lymphocytes on HLA-B18. Tissue Antigens (<strong>2002</strong>)<br />

Jul;60(1):16-24.<br />

3. BILSBOROUGH J, UYTTENHOVE C, COLAU D, BOUSSO P, LIBERT C, WEYNAND B, BOON T, VAN<br />

DEN EYNDE BJ. TNF-mediated toxicity after massive induction of specific CD8+ T cells<br />

following immunization of mice with a tumor-specific peptide. Journal of Immunology. (<strong>2002</strong>)<br />

Sep 15;169(6):3053-60.<br />

4. COULIE PG, KARANIKAS V, LURQUIN C, COLAU D, CONNEROTTE T, HANAGIRI T, VAN PEL A,<br />

LUCAS S, GODELAINE D, LONCHAY C, MARCHAND M, VAN BAREN N, BOON T. Cytolytic T-cell<br />

responses of cancer patients vaccinated with a MAGE antigen. Immunological Reviews (<strong>2002</strong>)<br />

Oct;188(1):33-42.<br />

5. DEMOTTE N, COLAU D, OTTAVIANI S, GODELAINE D, VAN PEL A, BOON T, VAN DER BRUGGEN<br />

P. A reversible functional defect of CD8+ T lymphocytes involving loss of tetramer labeling.<br />

European Journal of Immunology (<strong>2002</strong>) Jun;32(6):1688-97.<br />

6. FLEISCHER A, AYLLON V, DUMOUTIER L, RENAULD JC, REBOLLO A. Proapoptotic activity of<br />

ITM2B(s), a BH3-only protein induced upon IL-2-deprivation which interacts with Bcl-2.<br />

Oncogene (<strong>2002</strong>) May 9;21(20):3181-9.<br />

7. HUAUX F, ARRAS M, TOMASI D, BARBARIN V, DELOS M, COUTELIER JP, VINK A, PHAN SH,<br />

RENAULD JC, LISON D. A profibrotic function of IL-12p40 in experimental pulmonary fibrosis.<br />

Journal of Immunology (<strong>2002</strong>) Sep 1;169(5):2653-61.<br />

8. LEJEUNE D, DUMOUTIER L, CONSTANTINESCU S, KRUIJER W, SCHURINGA JJ, RENAULD JC.<br />

Interleukin-22 (IL-22) activates the JAK/STAT, ERK, JNK, and p38 MAP kinase pathways in a<br />

rat hepatoma cell line. Pathways that are shared with and distinct from IL-10. Journal of<br />

Biological Chemistry (<strong>2002</strong>) Sep 13;277(37):33676-82.<br />

9. LEVY F, BURRI L, MOREL S, PEITREQUIN AL, LEVY N, BACHI A, HELLMAN U, VAN DEN EYNDE<br />

BJ, SERVIS C. The final N-terminal trimming of a subaminoterminal proline-containing HLA<br />

class I-restricted antigenic peptide in the cytosol is mediated by two peptidases. Journal of<br />

Immunology (<strong>2002</strong>) Oct 15;169(8):4161-71.<br />

10. NAGEM RA, LUCCHESI KW, COLAU D, DUMOUTIER L, RENAULD JC, POLIKARPOV I.<br />

Crystallization and synchrotron X-ray diffraction studies of human interleukin-22. ACTA<br />

Crystallographica. Section D: Biological Crystallography (<strong>2002</strong>) Mar;58(Pt 3):529-30.<br />

11. NAGEM RA, COLAU D, DUMOUTIER L, RENAULD JC, OGATA C, POLIKARPOV I. Crystal structure<br />

of recombinant human interleukin-22. Structure (Camb). (<strong>2002</strong>) Aug;10(8):1051-62.


* Clinical Trials Program participant<br />

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12. PALMOWSKI MJ, CHOI EM, HERMANS IF, GILBERT SC, CHEN JL, GILEADI U, SALIO M, VAN PEL<br />

A, MAN S, BONIN E, LILJESTROM P, DUNBAR PR, CERUNDOLO V. Competition between CTL<br />

narrows the immune response induced by prime-boost vaccination protocols. Journal of<br />

Immunology (<strong>2002</strong>) May 1;168(9):4391-8.<br />

13. PANDEY A, IBARROLA N, KRATCHMAROVA I, FERNANDEZ MM, CONSTANTINESCU SN, OHARA<br />

O, SAWASDIKOSOL S, LODISH HF, MANN M. A novel Src homology 2 domain-containing<br />

molecule, Src-like adapter protein-2 (SLAP-2), which negatively regulates T cell receptor<br />

signaling. Journal of Biological Chemistry (<strong>2002</strong>) May 24;277(21):19131-8.<br />

14. PILETTE C, OUADRHIRI Y, VAN SNICK J, RENAULD JC, STAQUET P, VAERMAN JP, SIBILLE Y. IL-<br />

9 inhibits oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human<br />

monocytes through TGF-beta. Journal of Immunology (<strong>2002</strong>) Apr 15;168(8):4103-11.<br />

15. PILETTE C, OUADRHIRI Y, VAN SNICK J, RENAULD JC, STAQUET P, VAERMAN JP, SIBILLE Y.<br />

Oxidative burst in lipopolysaccharide-activated human alveolar macrophages is inhibited by<br />

interleukin-9. European Respiriratory Journal (<strong>2002</strong>) Nov;20(5):1198-205.<br />

16. RUAULT M, VAN DER BRUGGEN P, BRUN ME, BOYLE S, ROIZES G, SARIO AD. New BAGE (B<br />

melanoma antigen) genes mapping to the juxtacentromeric regions of human chromosomes 13<br />

and 21 have a cancer/testis expression profile. European Journal of Human Genetics (<strong>2002</strong>)<br />

Dec;10(12):833-40.<br />

17. SAUVONNET N, LAMBERMONT I, VAN DER BRUGGEN P, CORNELIS GR. YopH prevents monocyte<br />

chemoattractant protein 1 expression in macrophages and T-cell proliferation through inactivation<br />

of the phosphatidylinositol 3-kinase pathway. Molecular Microbiology (<strong>2002</strong>) Aug;45(3):805-15.<br />

18. SCHULTZ ES, CHAPIRO J, LURQUIN C, CLAVEROL S, BURLET-SCHILTZ O, WARNIER G, RUSSO V,<br />

MOREL S, LEVY F, BOON T, VAN DEN EYNDE BJ, VAN DER BRUGGEN P. The production of a new<br />

MAGE-3 peptide presented to cytolytic T lymphocytes by HLA-B40 requires the<br />

immunoproteasome. Journal of Experimental Medicine (<strong>2002</strong>) Feb 18;195(4):391-9.<br />

19. VAN DER BRUGGEN P, ZHANG Y, CHAUX P, STROOBANT V, PANICHELLI C, SCHULTZ ES,<br />

CHAPIRO J, VAN DEN EYNDE BJ, BRASSEUR F, BOON T. Tumor-specific shared antigenic peptides<br />

recognized by human T cells. Immunological Reviews (<strong>2002</strong>) Oct;188(1):51-64.<br />

20. VIGNERON N, OOMS A, MOREL S, DEGIOVANNI G, VAN DEN EYNDE BJ. Identification of a new<br />

peptide recognized by autologous cytolytic T lymphocytes on a human melanoma. Cancer<br />

Immunity (<strong>2002</strong>); 2:9-18.<br />

21. ZAMMATTEO N, LOCKMAN L, BRASSEUR F, DE PLAEN E, LURQUIN C, LOBERT PE, HAMELS S,<br />

BOON T, REMACLE J. DNA microarray to monitor the expression of MAGE-A genes. Clinical<br />

Chemistry (<strong>2002</strong>) Jan;48(1):25-34.<br />

22. ZHANG Y, STROOBANT V, RUSSO V, BOON T, VAN DER BRUGGEN P. A MAGE-A4 peptide<br />

presented by HLA-B37 is recognized on human tumors by cytolytic T lymphocytes. Tissue<br />

Antigens (<strong>2002</strong>) Nov;60(5):365-71.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. DUMOUTIER L, RENAULD JC. Viral and cellular interleukin-10 (IL-10)-related cytokines: from<br />

structures to functions. European Cytokine Network (<strong>2002</strong>) Jan-Mar;13(1):5-15.<br />

2. MACHIELS JP, VAN BAREN N, MARCHAND M. Peptide-based cancer vaccines. Semininars in<br />

Oncology (<strong>2002</strong>) Oct;29(5):494-502.


* Clinical Trials Program participant<br />

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BRUSSELS<br />

3. RENAULD JC, VAN SNICK J. Measurement of mouse and human interleukin-9. In: Current<br />

Protocols in Immunology (Coligan JE, Kruisbeeck AM, Margulies DH, Shevach EM, Strober W,<br />

eds), John Wiley and Sons, New York, (<strong>2002</strong>); 6.13.1-6.13.10.


LAUSANNE BRANCH<br />

OF IMMUNOLOGY<br />

LAUSANNE,SWITZERLAND<br />

Staff List: Page 33<br />

Branch Director’s <strong>Report</strong>: Page 36<br />

<strong>Research</strong> <strong>Report</strong>: Page 37<br />

Publications: Page 41<br />

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STAFF LIST<br />

CEROTTINI JEAN-CHARLES, Director, Member*<br />

ANTIGEN PROCESSING GROUP<br />

LÉVY,FRÉDÉRIC, Assistant Member<br />

LINDHOLM,CECILIA, Postdoctoral Fellow (from October)<br />

CHAPATTE,LAURENCE, PhD Student<br />

VIATTE,SÉBASTIEN, PhD Student<br />

LÉVY,NICOLE, Senior Technician<br />

PEITREQUIN,ANNE-LISE, Senior Technician<br />

CELL FATE DETERMINATION GROUP<br />

RADTKE,FREDDY, Assistant Member<br />

MANCINI,STÉPHANE, Postdoctoral Fellow<br />

COBAS,MONICA, PhD Student (from May)<br />

VAUCLAIR,SOPHIE, PhD Student (from June)<br />

BESSEYRIAS,VALÉRIE, Technician<br />

CLINICAL TUMOR IMMUNOLOGY GROUP*<br />

ROMERO,PEDRO, Member<br />

SPEISER,DANIEL, Assistant Member<br />

VALMORI,DANILA, Assistant Member<br />

GUILLAUME,PHILIPPE N, Assistant Investigator (Tetramer Facility)<br />

AYYOUB,MAHA, Postdoctoral Fellow<br />

PITTET,MIKAËL, Postdoctoral Fellow<br />

PETERSON,DANIEL, Postdoctoral Fellow (until May)<br />

ZIPPELIUS,ALFRED, Postdoctoral Fellow<br />

MINAIDIS,DANIELLE, Clinical Support Staff<br />

GELDHOF,CHRISTINE, Clinical Support Staff<br />

BIOLEY,GILLES, PhD Student (from October)<br />

* Clinical Trials Program participant<br />

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LAUSANNE


BRICARD,GABRIEL, PhD Student<br />

MERALDI RUBIO-GODOY,VERENA, PhD Student (until November)<br />

DUTOIT,VALÉRIE, PhD Student<br />

MONTANDON,NICOLE, Senior Technician<br />

BAROFFIO,CÉLINE, Technician<br />

CORNAZ,ROLF N, Technician (Tetramer Facility)<br />

DEVELOPMENTAL IMMUNOLOGY GROUP<br />

MACDONALD,HUGH ROBSON, Associate Director, Member<br />

WILSON,ANNE, Associate Investigator<br />

FERRERO,ISABEL, <strong>Research</strong> Associate<br />

LEES,ROSEMARY, <strong>Research</strong> Associate<br />

ERNST,BETTINA, Postdoctoral Fellow<br />

ESSLINGER,CHRISTOPH, Postdoctoral Fellow<br />

SCHUMANN,JENS, Postdoctoral Fellow<br />

VOYLE,ROGER, Postdoctoral Fellow<br />

AGOST-MARÉCHAL,CÉLINE, Technician (until August)<br />

MOLECULAR IMMUNOLOGY GROUP*<br />

LUESCHER,IMMANUEL, Member<br />

UMBRICHT,MARIE-SOPHIE, <strong>Research</strong> Associate (June to December)<br />

CEBECAUER,MAREK, Postdoctoral Fellow<br />

GOFFIN,LAURENCE, Postdoctoral Fellow<br />

MARK,SILKE, Postdoctoral Fellow (from December)<br />

BOUCHERON,NICOLE, PhD Student (until May)<br />

BAUMGÄRTNER MEIER,PETRA, <strong>Research</strong> Associate<br />

MOLECULAR TUMOR IMMUNOLOGY GROUP*<br />

CEROTTINI,JEAN-CHARLES, Director, Member<br />

RIMOLDI,DONATA, Associate Investigator<br />

MICHIELIN,OLIVIER, Postdoctoral Fellow<br />

FAGERBERG,THERES, PhD Student<br />

MUEHLETHALER,KATJA, Senior Technician<br />

SALVI,SUZANNE, Senior Technician<br />

* Clinical Trials Program participant<br />

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INNATE IMMUNITY GROUP<br />

HELD,WERNER, Associate Member<br />

COUDERT,JÉRÔME, Postdoctoral Fellow (from May)<br />

ZIMMER,JACQUES, Postdoctoral Fellow<br />

GOUX,DELPHINE, PhD Student<br />

SCARPELLINO,LEONARDO, Senior Technician<br />

VIRAL IMMUNOLOGY GROUP<br />

ACHA-ORBEA,HANS, Associate Member<br />

RYBTSOV,STANISLAV, Visiting Scientist<br />

GÄRDBY,EVA, Postdoctoral Fellow<br />

GENTON,CÉLINE, PhD Student (until August)<br />

BOCHATAY,MARIA, Technician<br />

LAVANCHY,CHRISTINE, Technician (until August)<br />

GENERAL SUPPORT SERVICES<br />

SERVIS,CATHERINE, <strong>Research</strong> Associate<br />

ZAECH,PIERRE, Other Support Staff<br />

BATARD,PASCAL, Other Support Staff<br />

ADMINISTRATION AND SECRETARIAT<br />

MÉAN,MARIE-MADELEINE, Administrator<br />

SERAVALLI,CAPUCINE, Chief Accountant (from October)<br />

MAROUANI,IRIS, Accounting Assistant<br />

NOGARE,NICOLE, Accounting Assistant<br />

LOPEZ,CELIA, Secretary to Director<br />

PONCELET,AUDREY, Secretary<br />

ERISMANN ZOPPI,ANNA, Secretary<br />

VAN OVERLOOP,MARTINE, Secretary<br />

* Clinical Trials Program participant<br />

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BRANCH DIRECTOR’S REPORT<br />

* Clinical Trials Program participant<br />

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The activities at the Lausanne Branch are focused on the basic mechanisms that regulate immunity<br />

mediated by T cells, natural killer (NK) cells and NKT cells, and include a strong patient-oriented<br />

research program devoted to the development of therapeutic cancer vaccines.<br />

The Lausanne Branch is also an active participant in the National Center for Competence in <strong>Research</strong><br />

program entitled “Molecular oncology: from basic research to therapeutic approaches” that is carried out<br />

in partnership with the Swiss <strong>Institute</strong> for Experimental Cancer <strong>Research</strong> (ISREC).<br />

The Branch consists of eight research groups, which have close collaborative links with scientists from<br />

the ISREC, the <strong>Institute</strong> of Biochemistry (University of Lausanne) and the Swiss <strong>Institute</strong> for<br />

Bioinformatics within the Center for Biomedical <strong>Research</strong> on the Epalinges campus, as well as clinicians<br />

from the Multidisciplinary Oncology Center (CePO) on the University Hospital campus.<br />

J.-C. Cerottini


RESEARCH REPORT<br />

ANTIGEN PROCESSING GROUP<br />

* Clinical Trials Program participant<br />

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LAUSANNE<br />

Dr. Frédéric Lévy’s Antigen Processing Group is interested in identifying the intracellular events that<br />

contribute to the production of CD8 + T cell-defined peptide tumor antigens, with special emphasis on the<br />

role of the proteasome and other peptidases. To this end, the group has developed an in vitro proteasomal<br />

degradation assay that is now used routinely to identify potential peptide tumor antigens among peptides<br />

displaying appropriate MHC class I binding motifs. In addition, the group recently identified two<br />

peptidases that act on antigenic peptides downstream of the proteasome. These peptidases play an<br />

essential role in the final trimming of proteasome-generated peptides that are too long to bind efficiently<br />

to MHC class I molecules. Another project deals with the identification of peptide antigens derived from<br />

colon carcinoma-specific proteins. Based on the recently developed large-scale analysis of the human<br />

genome transcriptome, it is now estimated that up to 60% of human genes have at least two splice<br />

variants. In collaboration with Dr. Victor Jongeneel from the <strong>LICR</strong> Office of Information Technology, the<br />

group has developed an in silico approach that aims to identify tumor-associated splice variants<br />

characterized by alternative exon usage. The peptide sequences derived from the alternative exons will<br />

serve as a starting point for the identification of new potential peptide tumor antigens.<br />

CELL FATE DETERMINATION GROUP<br />

Dr. Freddy Radtke’s Cell Fate Determination Group is interested in the molecular mechanisms controlling<br />

lineage commitment and differentiation in the hematopoietic system and the skin. Current attention is<br />

focused on the Notch signaling pathway, which is responsible for numerous binary cell fate decisions in<br />

diverse organisms. Previous studies by the group using a conditional gene targeting strategy have<br />

established an essential role for Notch 1 in specifying T cell fate in a bipotential T/B lymphoid precursor<br />

cell. More recently an additional function of Notch 1 has been uncovered at a later stage of T cell<br />

development, where it appears that Notch 1 is involved in the control of VDJ rearrangement at the T cell<br />

receptor (TCR) beta locus. In other experiments, a novel and unexpected function of Notch 1 as a putative<br />

tumor suppressor gene in the skin has been discovered.<br />

CLINICAL TUMOR IMMUNOLOGY GROUP*<br />

The Clinical Tumor Immunology Group led by Dr. Pedro Romero focuses on the design of molecularly<br />

defined therapeutic cancer vaccines as part of the <strong>LICR</strong> Cancer Vaccine Program. In collaboration with<br />

surgical oncologists at the Multidisciplinary Oncology Center (CePO), a series of closely related phase I<br />

clinical trials with vaccine preparations based on HLA-A2 binding Melan-A peptides were completed in<br />

<strong>2002</strong>. Forty nine patients with Melan-A + high risk stage III and IV melanoma were vaccinated with<br />

Melan-A peptide � adjuvant ± low dose recombinant IL-2. The peptide-specific T cell response in blood<br />

was measured directly ex vivo by both multiparameter flow cytometry combining HLA-A2/Melan-A<br />

peptide tetramers and cell surface markers associated with discrete stages of CD8 T cell differentiation,<br />

and direct IFN gamma ELISPOT. These studies indicate that repeated vaccination with Melan-A peptide<br />

and Montanide (Incomplete Freund’s adjuvant) frequently leads to sustained ex vivo detectable CD8 T<br />

cell responses. The group has already obtained approval to initiate three new, but related, phase I clinical<br />

trials which will test: (i) the immunogenicity of the same peptide + Montanide vaccine combined with<br />

synthetic oligodeoxynucleotides containing bacterial DNA CpG motifs, a new adjuvant that activates<br />

plasmacytoid dendritic cells and B cells via Toll-like receptor-9; (ii) the immunogenicity of Melan-A<br />

peptide admixed with the outer surface membrane protein of Gram negative bacteria OMPA-40, a new


* Clinical Trials Program participant<br />

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LAUSANNE<br />

adjuvant that activates dendritic cells via Toll-like receptor-2; and (iii) a multipeptide vaccine including<br />

peptides derived from Melan-A and the cancer/testis (CT) antigens MAGE-A10 and NY-ESO-1 together<br />

with Montanide. These studies will investigate new combinations of antigens and immunostimulatory<br />

molecules that have the potential to induce protective T cell responses. This approach allows stepwise<br />

progress towards the optimization of Melan-A peptide based cancer vaccines.<br />

The group is also involved in the analysis of naturally acquired CTL responses directed against well<br />

defined tumor antigens. In particular, it has extensively studied Melan-A specific CD8 T cell responses in<br />

both healthy individuals and melanoma patients. In the latter, the group has extended its analyses to the<br />

lymphocytes that can be recovered from the tumor sites. It was found that while the repertoire of<br />

functionally naive Melan-A specific T cells available in healthy individuals is at least 1,000-fold greater<br />

than the usual size of the naive repertoires for common antigens, the relative frequency of Melan-A<br />

antigen-specific CD8 + T cells is markedly increased at the tumor sites. These cells display an antigenexperienced<br />

phenotype. Their low T cell recognition excision circle (TREC) content and shortened<br />

telomeres indicate extensive in vivo expansion. Functionally, although these cells recognize antigen with<br />

higher avidity than their naive counterparts, they are characterized by an antigen-specific defect in their<br />

ability to secrete IFN gamma. This deficit is reversible once the cells are placed in culture together with<br />

cytokines such as IL-2 and IL-7. In collaboration with the Torrey Pines <strong>Institute</strong> for Molecular Sciences<br />

(Torrey Pines, USA), the group has also extensively probed both the specificity and degeneracy of Melan-<br />

A antigen recognition by clonal populations of T cells using synthetic combinatorial peptide libraries in<br />

positional scanning format. A relatively high number of heteroclitic peptides were identified. In a separate<br />

project, the group collaborates with the groups of Drs. H. R. MacDonald and W. Held to elucidate the role<br />

of human NKT cell responses to CD1/alpha galactosylceramide in tumor immunity. Altogether, the<br />

results are of great help to investigate and understand spontaneous and vaccine-induced T cell responses<br />

in cancer patients.<br />

DEVELOPMENTAL IMMUNOLOGY GROUP<br />

The Developmental Immunology Group led by Dr. Hugh Macdonald continues to be interested in the<br />

early stages of T cell development in the thymus. Current projects are aimed at understanding the<br />

relationship between different intrathymic lymphoid lineages including alpha beta T cells, gamma delta T<br />

cells and so-called “lymphoid dendritic cells”. Using chimeric mice reconstituted with hematopoietic<br />

precursor cells deficient in either Notch 1 or Tcf-1 the group has been able to show that the recently<br />

identified plasmacytoid dendritic cell lineage can develop independently of T cells in the thymus.<br />

Another major interest of the group is the intrathymic development and selection of NKT cells. In<br />

contrast to conventional T cells, NKT cells express markers normally associated with NK cells (such as<br />

Ly-49 receptors) as well as a semi-invariant TCR that recognizes glycolipids presented by the<br />

monomorphic CD1d molecule. Using novel mice transgenic for both activating and inhibitory Ly-49<br />

receptors the group has recently shown that forced ligation of Ly-49 molecules can modulate NKT cell<br />

development, presumably by altering the signaling thresholds perceived by the semi-invariant TCR.


INNATE IMMUNITY GROUP<br />

* Clinical Trials Program participant<br />

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LAUSANNE<br />

The Innate Immunity Group led by Dr. Werner Held studies the development and function of NK cells.<br />

NK cell-mediated lysis of target cells is regulated by a dual receptor system, which integrates signals<br />

from activating receptors and inhibitory receptors specific for MHC class I. Target cell lysis can occur<br />

upon the loss of MHC class I molecules from target cells, which results in a derepression of NK cells<br />

(missing-self recognition). Alternatively, NK cell inhibition can be overruled by the expression of novel<br />

ligands that activate NK cell receptors (induced self-recognition). In contrast to NK cell inhibition, NK<br />

cell activation is still relatively poorly understood. The group has shown that NK cell activation by a<br />

subset of target cells depends on the src family kinase Fyn. This subset of target cells is defined by the<br />

absence of ligands for the potent activating NK cell receptor NKG2D, and the lack of MHC class I<br />

molecules. Therefore, Fyn seems be important for missing self-recognition but not for induced-self<br />

recognition.<br />

MOLECULAR IMMUNOLOGY GROUP*<br />

Dr. Immanual Luescher’s Molecular Immunology Goup has continued its molecular analysis of the role<br />

played by the coreceptor CD8 during the process of T cell recognition of, and activation by, peptide-MHC<br />

(p-MHC) complexes on the surface of antigen-presenting cells. CD8 is an alpha-beta heterodimer, which<br />

interacts with the Src-family kinase lck through the cytoplasmic tail of its alpha chain and with the TCR –<br />

CD3 complex through its beta chain. Previous studies from the group have shown that the CD8 beta chain<br />

is palmitoylated, thus mediating the partitioning of CD8 in the membrane microdomains known as lipid<br />

rafts. The TCR-associated CD3 complex consists of four distinct chains that contain, in their cytoplasmic<br />

tails, the so-called immunoreceptor tyrosine-based activation motifs. The group has now determined that<br />

the association between the TCR and CD8 is mediated by the delta chain of the CD3 complex. Moreover,<br />

functional studies demonstrated that the CD3 delta chain-mediated coupling of TCR-CD3 with raftassociated<br />

CD8 is crucial for efficient triggering and activation of CD8 + T cells. The laboratory is also the<br />

host of the <strong>LICR</strong> Tetramer Production Facility, which is in charge of producing the various peptide-MHC<br />

class I tetramers that are used in the monitoring of tumor-specific CD8 + T cell responses during tumor<br />

growth and following vaccination. After careful biochemical and biological characterization, these<br />

reagents are distributed to all the investigators involved in the <strong>LICR</strong> Cancer Vaccine Program.<br />

VIRAL IMMUNOLOGY GROUP<br />

Mouse mammary tumor virus (MMTV) infection establishes chronic infection, long-lasting germinal<br />

centers in the draining lymph node, and a life-long neutralizing antibody response. The Viral Immunology<br />

Group led by Dr. Hans Acha-Orbea has analyzed the role of the lymph node draining the site of MMTV<br />

injection in the maintenance of a chronic neutralizing antibody response. The surgical removal of the<br />

draining lymph node, after establishment of the germinal center reaction, led to complete loss of<br />

neutralizing antibodies despite comparable systemic spread of MMTV-infected lymphocytes.<br />

Importantly, in the absence of neutralization, only the exocrine organs (mammary gland, salivary gland,<br />

pancreas, and skin) showed strikingly increased infection resulting in accelerated mammary tumor<br />

development. Induction of stronger neutralization did not influence chronic infection levels of peripheral<br />

lymphoid organs but strongly inhibited mammary gland infection, and virus transmission to the next<br />

generation. Thus a tight equilibrium in virus neutralization allows limited infection of exocrine organs,<br />

and controls cancer development in susceptible mouse strains. Therefore the antigen deposits in the<br />

draining lymph node, and the chronic immune response in this site, appear to be crucial for the<br />

maintenance of a strong neutralizing immune response.


MOLECULAR TUMOR IMMUNOLOGY GROUP*<br />

* Clinical Trials Program participant<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 40<br />

LAUSANNE<br />

The current research efforts of the Molecular Tumor Immunology Group led by Dr. Jean-Charles<br />

Cerottini are devoted to the characterization of human tumor antigens that are recognized by CD8 + T cells<br />

from melanoma patients. Recent studies of the melanocyte lineage-specific protein Melan-A, a small<br />

transmembrane protein of unknown function that is the target of cancer vaccine trials in the Lausanne<br />

Branch, have revealed that its subcellular localization is distinct from typical melanosomal proteins, such<br />

as tyrosinase. Quantitative immunoelectron microscopy showed that the highest proportion of the cellular<br />

content of Melan-A is found in small vesicles and tubules throughout the cell, whereas the concentration<br />

is maximal in the Golgi region, particularly the trans-Golgi network. Biochemical analyses indicated that<br />

Melan-A is post-translationally acylated and undergoes a rapid turnover. In an attempt to understand the<br />

role of Melan-A in melanocytic cells, in vitro knock-down cells are being generated using the siRNA<br />

technology.<br />

A distinct project, carried out in collaboration with the <strong>LICR</strong> Office of Information Technology, concerns<br />

the development of tools to construct 3D models of T cell receptor-peptide-MHC (TCR-p-MHC)<br />

complexes, combining homology modeling and ab initio predictions. These methods, which have first<br />

been validated in a mouse model system, are now applied to the diverse TCR expressed by CD8 + T cells<br />

directed against the well-defined Melan-A peptide presented by HLA-A2. In addition to these structural<br />

data, functional approaches based on free energy simulation methods have been developed to understand<br />

how peptide mutations alter p-MHC binding as well as TCR-p-MHC binding.


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 41<br />

LAUSANNE<br />

1. ASEFFA A, GUMY A, LAUNOIS P, MACDONALD HR, LOUIS JA AND TACCHINI-COTTIER F. The<br />

early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering<br />

progressive disease in BALB/c mice are subject to the control of regulatory CD4 + CD25 + T cells.<br />

Journal of Immunology (<strong>2002</strong>) 169:3232-3241.<br />

2. AYYOUB M, STEVANOVIC S, SAHIN U, GUILLAUME P, SERVIS C, RIMOLDI D, VALMORI D,<br />

ROMERO P, CEROTTINI J-C, RAMMENSEE H-G, PFREUNDSCHUH M, SPEISER D AND LÉVY F.<br />

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL<br />

infiltrating metastatic melanoma. Journal of Immunology (<strong>2002</strong>) 168:1717-1722.<br />

3. BALMELLI C, DEMOTZ S, ACHA-ORBEA H AND NARDELLI-HAEFLIGER D. Trachea, lung and<br />

tracheobronchial lymph nodes are the major inductive sites after nasal vaccination of mice with<br />

human papillomavirus type 16 virus-like particles. Journal of Virology (<strong>2002</strong>) 76:12596-12602.<br />

4. BATARD P, SZOLLOSI J, LUESCHER I, CEROTTINI J-C, MACDONALD HR AND ROMERO P. Use of<br />

phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow<br />

cytometry: advantages and limitations. Cytometry (<strong>2002</strong>) 48:97-105.<br />

5. BULLANI RR, WEHRLI P, VIARD-LEVEUGLE I, RIMOLDI D, CEROTTINI J-C, SAURAT J-H,<br />

TSCHOPP J AND FRENCH LE. Frequent downregulation of Fas (CD95) expression and function in<br />

melanoma. Melanoma <strong>Research</strong> (<strong>2002</strong>) 12:263-270.<br />

6. BURRI L, SERVIS C, CHAPATTE L AND LÉVY F. A recyclable assay to analyze the NH2-terminal<br />

trimming of antigenic peptide precursors. Protein Expression and Purification (<strong>2002</strong>) 26:19-27.<br />

7. CHAMBERS CA, KANG J, WU Y, HELD W, RAULET DH AND ALLISON JP. The lymphoproliferative<br />

defect in CTLA-4-deficient mice is ameliorated by an inhibitory NK cell receptor. Blood (<strong>2002</strong>)<br />

99:4509-4516.<br />

8. CUNNINGHAM AF, FALLON PG, KHAN M, VACHERON S, ACHA-ORBEA H, MACLENNAN ICM,<br />

MCKENZIE A AND TOELLNER K-M. Th2 activities induced during virgin T cell priming in the<br />

absence of IL-4, IL-13, and B cells. Journal of Immunology (<strong>2002</strong>) 169:2900-2906.<br />

9. DE MAZIÈRE AM, MUEHLETHALER K, VAN DONSELAAR E, SALVI S, DAVOUST J, CEROTTINI J-C,<br />

LÉVY F, SLOT JW AND RIMOLDI D. The melanocytic protein Melan-A/MART-1 has a subcellular<br />

localization distinct from typical melanosomal proteins. Traffic (<strong>2002</strong>) 3:678-693.<br />

10. DUTOIT V, RUBIO-GODOY V, DOUCEY M-A, BATARD P, LIÉNARD D, RIMOLDI D, SPEISER D,<br />

GUILLAUME P, CEROTTINI J-C, ROMERO P AND VALMORI D. Functional avidity of tumor antigenspecific<br />

CTL recognition directly correlates with the stability of MHC/peptide multimer binding<br />

to TCR. Journal of Immunology (<strong>2002</strong>) 168:1167-1171.<br />

11. DUTOIT V, GUILLAUME P, ROMERO P, CEROTTINI J-C, AND VALMORI D. Functional analysis of<br />

HLA-A*0201/Melan-A peptide multimer + CD8 + T cells isolated from an HLA-A*0201- donor:<br />

exploring tumor antigen allorestricted recognition. Cancer Immunity (<strong>2002</strong>) 2:7.<br />

12. DUTOIT V, RUBIO-GODOY V, PITTET MJ, ZIPPELIUS A, DIETRICH P-Y, LEGAL FA, GUILLAUME P,<br />

ROMERO P, CEROTTINI J-C, HOUGHTEN RA, PINILLA C AND VALMORI D. Degeneracy of antigen<br />

recognition as the molecular basis for the high frequency of naive A2/Melan-A peptide multimer +<br />

CD8 + T-cells in humans. Journal of Experimental Medicine (<strong>2002</strong>) 196:207-216.<br />

13. DUTOIT V, GUILLAUME P, CEROTTINI J-C, ROMERO P AND VALMORI D. Dissecting T cell<br />

receptor-MHC/peptide complex interactions with A2/peptide multimers incorporating tumor


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 42<br />

LAUSANNE<br />

antigen peptide variants: crucial role of interaction kinetics on functional outcomes. European<br />

Journal of Immunology (<strong>2002</strong>) 32:3285-3293.<br />

14. DUTOIT V, TAUB RN, PAPADOPOULOS KP, TALBOT S, KEOHAN M-L, BREHM M, GNJATIC S,<br />

HARRIS PE, BISIKIRSKA B, GUILLAUME P, CEROTTINI J-C, HESDORFFER CS, OLD LJ AND<br />

VALMORI D. Multiepitope CD8 + T cell response to an NY-ESO-1 peptide vaccine results in<br />

imprecise tumor targeting. Journal of Clinical Investigation (<strong>2002</strong>) 110:1813-1822.<br />

15. ESSLINGER C, ROMERO P AND MACDONALD HR. Efficient transduction of dendritic cells and<br />

induction of a T-cell response by third-generation lentivectors. Human Gene Therapy (<strong>2002</strong>)<br />

13:1091-1100.<br />

16. FERRERO I, HELD W, WILSON A, TACCHINI-COTTIER F, RADTKE F AND MACDONALD HR.<br />

Mouse CD11c + B220 + Gr1 + plasmacytoid dendritic cells develop independently of the T cell<br />

lineage. Blood (<strong>2002</strong>) 100:2852-2857.<br />

17. FINKE D, ACHA-ORBEA H, MATTIS A, LIPP M AND KRAEHENBUHL J-P. CD4 + CD3- cells induce<br />

Peyer's patch development: Role of �4�1 integrin activation by CXCR5. Immunity (<strong>2002</strong>)<br />

17:363-373.<br />

18. FIORE E, FUSCO C, ROMERO P AND STAMENKOVIC I. Matrix metalloproteinase 9 (MMP-<br />

9/gelatinase B) proteolytically cleaves ICAM-1 and participates in tumor cell resistance to natural<br />

killer cell-mediated cytotoxicity. Oncogene (<strong>2002</strong>) 21:5213-5223.<br />

19. GEERTSEN R, BÖNI R, BLASCZYK R, ROMERO P, BETTS D, RIMOLDI D, HONG X, LAINE E,<br />

WILLERS J AND DUMMER R. Loss of single HLA class I allospecificities in melanoma cells due to<br />

selective genomic abbreviations. International Journal of Cancer (<strong>2002</strong>) 99:82-87.<br />

20. GENOUD S, LAPPE-SIEFKE C, GOEBBELS S, RADTKE F, AGUET M, SCHERER SS, SUTER U, NAVE<br />

K-A AND MANTEI N. Notch1 control of oligodendrocyte differentiation in the spinal cord.<br />

Journal of Cellular Biology (<strong>2002</strong>) 158:709-718.<br />

21. HUNT L, BATARD P, JORDAN M AND WURM FM. Fluorescent proteins in animal cells for process<br />

development: optimization of sodium butyrate treatment as an example. Biotechnology and<br />

Bioengineering (<strong>2002</strong>) 77:528-537.<br />

22. KERRE TCC, DE SMET G, DE SMEDT M, ZIPPELIUS A, PITTET MJ, LANGERAK AW, DE<br />

BOSSCHER J, OFFNER F, VANDEKERCKHOVE B AND PLUM J. Adapted NOD/SCID model supports<br />

development of phenotypically and functionally mature T cells from human umbilical cord blood<br />

CD34 + cells. Blood (<strong>2002</strong>) 99:1620-1626.<br />

23. LEISHMAN AJ, GAPIN L, CAPONE M, PALMER E, MACDONALD HR, KRONENBERG M AND<br />

CHEROUTRE H. Precursors of functional MHC class I- or class II-restricted CD8�� + T cells are<br />

positively selected in the thymus by agonist self-peptides. Immunity (<strong>2002</strong>) 16:355-364.<br />

24. LENS SMA, KATAOKA T, FORTNER KA, TINEL A, FERRERO I, MACDONALD HR, HAHNE M,<br />

BEERMANN F, ATTINGER A, ACHA-ORBEA H, BUDD RC AND TSCHOPP J. The caspase 8 inhibitor<br />

c-FLIPL modulates T-cell receptor-induced proliferation but not activation-induced cell death of<br />

lymphocytes. Molecular and Cellular Biology (<strong>2002</strong>) 22:5419-5433.<br />

25. LÉVY F, BURRI L, MOREL S, PEITREQUIN A-L, LÉVY N, BACHI A, HELLMANN U, VAN DEN<br />

EYNDE BJ AND SERVIS C. The final N-terminal trimming of a subaminoterminal prolinecontaining<br />

HLA class 1-restricted antigenic peptide in the cytosol is mediated by two peptidases.<br />

Journal of Immunology (<strong>2002</strong>) 169:4161-4171.


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 43<br />

LAUSANNE<br />

26. LOWIN-KROPF B, KUNZ B, SCHNEIDER P AND HELD W. A role for the src family kinase Fyn in<br />

NK cell activation and the formation of the repertoire of Ly49 receptors. European Journal of<br />

Immunology (<strong>2002</strong>) 32:773-782.<br />

27. MICHIELIN O AND KARPLUS M. Binding free energy differences in a TCR-peptide-MHC complex<br />

induced by a peptide mutation: A simulation analysis. Journal of Molecular Biology (<strong>2002</strong>)<br />

324:547-569.<br />

28. MICONNET I, KOENIG S, SPEISER D, KRIEG A, GUILLAUME P, CEROTTINI J-C AND ROMERO P.<br />

CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide.<br />

Journal of Immunology (<strong>2002</strong>) 168:1212-1218.<br />

29. NAEHER D, LUESCHER IF AND PALMER E. A role for the �-chain connecting peptide motif in<br />

mediating TCR/CD8 co-operation. Journal of Immunology (<strong>2002</strong>) 169:2964-2970.<br />

30. NGUYEN LT, ELFORD AR, MURAKAMI K, GARZA KM, SCHOENBERGER SP, ODERMATT B,<br />

SPEISER DE AND OHASHI PS. Tumor growth enhances cross-presentation leading to limited T cell<br />

activation without tolerance. Journal of Experimental Medicine (<strong>2002</strong>) 195:423-435.<br />

31. RADTKE F, WILSON A, ERNST B AND MACDONALD HR. The role of Notch signaling<br />

hematopoietic lineage commitment. Immunological Review (<strong>2002</strong>) 187:65-74.<br />

32. RENNO T, WILSON A, DUNKEL C, COSTE I, MAISNIER-PATIN K, BENOIT DE COIGNAC A, AUBRY<br />

J-P, LEES RK, BONNEFOY J-Y, MACDONALD HR AND GAUCHAT J-F. A role for CD147 in thymic<br />

development. Journal of Immunology (<strong>2002</strong>) 168:4946-4950.<br />

33. ROMERO P, VALMORI D, PITTET MJ, ZIPPELIUS A, RIMOLDI D, LÉVY F, DUTOIT V, RUBIO-<br />

GODOY V, MICHIELIN O, GUILLAUME P, BATARD P, LUESCHER IF, LEJEUNE F, LIÉNARD D,<br />

RUFER N, DIETRICH P-Y, SPEISER DE AND CEROTTINI J-C. Antigenicity and immunogenicity of<br />

Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma.<br />

Immunological Review (<strong>2002</strong>) 188:81-96.<br />

34. RUBIO-GODOY V, PINILLA C, DUTOIT V, BORRAS E, SIMON R, ZHAO Y, CEROTTINI J-C, ROMERO<br />

P, HOUGHTEN R AND VALMORI D. Toward synthetic combinatorial peptide libraries in positional<br />

scanning format (PS-SCL)-based identification of CD8 + tumor-reactive T-cell ligands: a<br />

comparative analysis of PS-SCL recognition by a single tumor-reactive CD8 + cytolytic Tlymphocyte<br />

clone. Cancer <strong>Research</strong> (<strong>2002</strong>) 62:2058-2063.<br />

35. RUBIO-GODOY V, AYYOUB M, DUTOIT V, SERVIS C, SCHINK A, RIMOLDI D, ROMERO P,<br />

CEROTTINI J-C, SPEISER D, SIMON R, ZHAO Y, HOUGHTEN RA, PINILLA C AND VALMORI D.<br />

Combinatorial peptide library based identification of peptide ligands for a tumor-reactive CD8 + T<br />

cell clone of unknown specificity. European Journal of Immunology (<strong>2002</strong>) 32:2292-2299.<br />

36. RUBIO-GODOY V, DUTOIT D, ZHAO Y, SIMON R, GUILLAUME P, HOUGHTEN R, ROMERO P,<br />

CEROTTINI J-C, PINILLA C AND VALMORI D. Positional scanning-synthetic peptide library-based<br />

analysis of self-and pathogen-derived peptides cross-reactivity with tumor-reactive Melan-Aspecific<br />

CTL. Journal of Immunology (<strong>2002</strong>) 169:5696-5707.<br />

37. SCHULTZ ES, CHAPIRO J, LURQUIN C, CLAVEROL S, BURLET-SCHILTZ O, WARNIER G, RUSSO V,<br />

MOREL S, LÉVY F, BOON T, VAN DEN EYNDE BJ AND VAN DER BRUGGEN P. The production of a<br />

new MAGE-3 peptide presented to cytolytic T lymphocytes by HLA-B40 requires the<br />

immunoproteasome. Journal of Experimental Medicine (<strong>2002</strong>) 195:391-399.<br />

38. SPEISER DE, LIÉNARD D, PITTET MJ, BATARD P, RIMOLDI D, GUILLAUME P, CEROTTINI J-C AND<br />

ROMERO P. In vivo activation of melanoma-specific CD8 + T cells by endogenous tumor antigen<br />

and peptide vaccines. A comparison to virus-specific T cells. European Journal of Immunology<br />

(<strong>2002</strong>) 32:731-741.


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 44<br />

LAUSANNE<br />

39. SPEISER DE, CEROTTINI J-C AND ROMERO P. Can hTERT peptide540-548-specific CD8 T cells<br />

recognize and kill tumor cells? Cancer Immunity (<strong>2002</strong>) 2:14.<br />

40. VACHERON S, LUTHER SA AND ACHA-ORBEA H. Preferential infection of immature dendritic<br />

cells and B cells by mouse mammary tumor virus. Journal of Immunology (<strong>2002</strong>) 168:3470-3476.<br />

41. VALMORI D, SCHEIBENBOGEN C, DUTOIT V, NAGORSEN D, ASEMISSEN AM, RUBIO-GODOY V,<br />

RIMOLDI D, GUILLAUME P, ROMERO P, SCHADENDORF D, LIPP M, DIETRICH P-Y, THIEL E,<br />

CEROTTINI J-C, LIÉNARD D AND KEILHOLZ U. Circulating tumor-reactive CD8 + T cells in<br />

melanoma patients contain a CD45RA + CCR7 - effector subset exerting ex-vivo tumor-specific<br />

cytolytic activity. Cancer <strong>Research</strong> (<strong>2002</strong>) 62:1743-1750.<br />

42. VALMORI D, DUTOIT V, SCHNURIGER V, QUIQUEREZ A-L, PITTET MJ, GUILLAUME P, RUBIO-<br />

GODOY V, WALKER PR, RIMOLDI D, LIÉNARD D, CEROTTINI J-C, ROMERO P AND DIETRICH P-Y.<br />

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased<br />

functional avidity and tumor reactivity. Journal of Immunology (<strong>2002</strong>) 168:4231-4240.<br />

43. WIRTH S, BILLE F, KOENIG S, WEHRLI N, MICONNET I, LÉVY F, DIGGELMANN H, ROMERO P<br />

AND ACHA-ORBEA H. Testing mouse mammary tumor virus superantigen as adjuvant in cytotoxic<br />

T-lymphocyte responses against a melanoma tumor antigen. International Journal of Cancer<br />

(<strong>2002</strong>) 99:201-206.<br />

44. WIRTH S, VESSAZ A, KRUMMENACHER C, BARIBAUD F, ACHA-ORBEA H AND DIGGELMANN H.<br />

Regions of the mouse mammary tumor virus superantigen involved in the interaction with the<br />

MHC class II I-A molecule. Journal of Virology (<strong>2002</strong>) 76:11172-11175.<br />

45. WOLFER A, WILSON A, NEMIR M, MACDONALD HR AND RADTKE F. Inactivation of Notch1<br />

impairs VDJ� rearrangement and allows pre-TCR-independent survival of early �� lineage<br />

thymocytes. Immunity (<strong>2002</strong>) 16:869-879.<br />

46. ZIPPELIUS A, PITTET MJ, BATARD P, RUFER N, DE SMEDT M, GUILLAUME P, ELLEFSEN K,<br />

VALMORI D, LIÉNARD D, PLUM J, MACDONALD HR, SPEISER DE, CEROTTINI J-C AND ROMERO<br />

P. Thymic selection generates a large T cell pool recognizing a self-peptide in humans. Journal of<br />

Experimental Medicine (<strong>2002</strong>) 195:485-494.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. BOON T AND CEROTTINI J-C. Tumour antigens. In: Oxford Textbook of Oncology. 2nd Edition.<br />

R.L. Souhami, I. Tannock, P. Hohenberger and J.-C. Horiot, eds. (<strong>2002</strong>) Oxford University Press,<br />

New York. pp. 115-126.<br />

2. GUGGISBERG D, CEROTTINI J-P, KRISCHER J, BRAUN R, DIETRICH P-Y AND LIENARD D.<br />

Recommandations pour la prise en charge du mélanome. Revue médicale Suisse romande (<strong>2002</strong>)<br />

122:5-8.<br />

3. MACDONALD HR. Development and selection of NKT cells. Current Opinion in Immunology<br />

(<strong>2002</strong>) 14:250-254.<br />

4. MACDONALD HR. T before NK. Science. 296:481-482, (<strong>2002</strong>).<br />

5. PITTET MJ, ZIPPELIUS A, VALMORI D, SPEISER DE, CEROTTINI J-C AND ROMERO P. Melan-<br />

A/MART-1-specific CD8 T cells: from thymus to tumor. Trends in Immunology (<strong>2002</strong>) 23:325-<br />

328.<br />

6. OTTEN LA, FINKE D AND ACHA-ORBEA H. Can MMTV exploit TLR4? Trends in Microbiology<br />

(<strong>2002</strong>) 10:303-305.


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 45<br />

LAUSANNE<br />

7. Romero P, Pittet MJ, Dutoit V, Zippelius A, Liénard D, Lejeune F, Guillaume P, Rimoldi D,<br />

Valmori D, Speiser DE and Cerottini J-C. Therapeutic cancer vaccines based on molecularly<br />

defined human tumor antigens. Vaccine (<strong>2002</strong>) 20:A2-A7.<br />

8. ROMERO P, PITTET MJ, ZIPPELIUS A, LIENARD D, LEJEUNE FJ, VALMORI D, SPEISER DE AND<br />

CEROTTINI J-C. T cells in tumor immunity. In: Cancer Immune Therapy. G. Stuhler and P.<br />

Walden, (<strong>2002</strong>) eds. Wiley-VCH Verlag GmbH & Co, Weinheim. pp. 40-55.<br />

9. SPEISER DE AND FERRONE S. The Immunobiology of malignant melanoma. In: Immunology for<br />

Surgeons. A.P. Zbar, P.J. Guillou, K.I. Bland and K.N. Syrigos, (<strong>2002</strong>) eds. Springer-Verlag,<br />

Heidelberg. pp. 343-363.<br />

10. ZIPPELIUS A, PITTET MJ AND ROMERO P. Dissecting tumor antigen-specific CD8 T cell responses<br />

in cancer patients. In: American Society for Histocompatibility and Immunogenetics. (<strong>2002</strong>) eds.<br />

ASHI Quaterly, 26:8-12.


LONDON ST.MARY’S BRANCH<br />

OF VIROLOGY AND CELL BIOLOGY<br />

LONDON,ENGLAND<br />

Staff List: Page 46<br />

Branch Director’s <strong>Report</strong>: Page 48<br />

<strong>Research</strong> <strong>Report</strong>: Page 49<br />

Publications: Page 51<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003


STAFF LIST<br />

FARRELL,PAUL J., Director, Member<br />

BLYMPHOCYTE AND EPSTEIN-BARR GROUP<br />

ALLDAY,MARTIN, Professor, Imperial College (Wellcome Trust)<br />

HEANEY,JUDE, Postdoctoral Fellow<br />

SOURISSEAU,TONY, Postdoctoral Fellow<br />

SPYER,MOIRA, Postdoctoral Fellow<br />

HICKABOTTOM,MARK, PhD Student<br />

LEAO,MARIA, PhD Student<br />

LEVI,EVA, PhD Student (until May)<br />

O’NIONS,JENNY, PhD Student (until December)<br />

PARKER,GILLIAN, <strong>Research</strong> Assistant<br />

EPSTEIN-BARR VIRUS AND CELL GROWTH CONTROL GROUP<br />

FARRELL,PAUL J., Member, Director<br />

CROOK,TIMOTHY, Associate Clinical Investigator<br />

SPENDER,LINDSAY, Assistant Investigator<br />

BRYANT,HELEN, Postdoctoral Fellow<br />

REELFS,OLIVIER, Postdoctoral Fellow (until August)<br />

AL-MOZAINI,MAHA, PhD Student (from May)<br />

AMON,WOLFGANG, PhD Student<br />

BINNÉ,ULRICH, PhD Student (until January)<br />

DE JESUS,ORLANDO, PhD Student (until May)<br />

PATTLE,SIMON, PhD Student (from October)<br />

WHITEMAN,HANNAH, PhD Student<br />

SULLIVAN,ALEX, Assistant Laboratory Manager/<strong>Research</strong> Assistant<br />

ATTARD,MARLENE, Technician<br />

ELGUETA,CLAUDIO, Technician<br />

SHEEHAN,JOHN, Technician (July to September)<br />

SYED,NELOFER, Technician (from December)<br />

LONDON ST MARY’S<br />

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ONCOGENE GROUP<br />

WATSON,ROGER, Reader, Imperial College (BBSRC, CRUK)<br />

JOAQUIN,MANEL, Postdoctoral Fellow (until December)<br />

TAVNER,FIONA, Postdoctoral Fellow<br />

ZEGARAC,TEA, PhD Student<br />

CATCHPOLE,STEVEN, Technician<br />

TUMOR SUPPRESSOR GENE GROUP<br />

LU,XIN, Member, Professor of Cancer Biology<br />

BERGAMASCHI,DANIELE, Postdoctoral Fellow<br />

LLANOS,SUSANA, Postdoctoral Fellow (from January)<br />

TRIGIANTE,GUISEPPE, Postdoctoral Fellow (until December)<br />

VIVES,VIRGINIE, Postdoctoral Fellow (from October)<br />

FOGAL,VALENTINA, PhD Student<br />

GILLOTIN,SEBASTIAN, PhD Student (from October)<br />

GODIN,NADIA, PhD Student<br />

SAMUELS-LEV,YARDENA, PhD Student<br />

SU,JIAN, PhD Student<br />

ZHONG,SHAN, Senior Technician<br />

ALI,SOFIA, Technician<br />

RATNAYAKA,INDIRAH, Technician (from April)<br />

SLEE,ELIZABETH, Technician<br />

TECHNICAL SUPPORT<br />

COULSON,LEE, Laboratory Aide<br />

DAVIS,STEVE, Laboratory Aide (until May)<br />

ZLOBECKI,MARTIN, Laboratory Aide (from May)<br />

ADMINISTRATION AND SECRETARIAT<br />

CURTIS,JULIE, Administrator<br />

DE FELICE,JOHN, Laboratory Manager<br />

ZALESKA,ANNA, Secretary (until December)<br />

LONDON ST MARY’S<br />

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BRANCH DIRECTOR’S REPORT<br />

LONDON ST MARY’S<br />

The London St Mary’s Branch studies the molecular mechanisms that cause cancer, and the role of<br />

viruses, particularly Epstein-Barr virus (EBV), in human cancer. We aim to use our discoveries to<br />

develop new diagnostic procedures or cancer therapy strategies. These novel approaches currently include<br />

the ASPP proteins as targets for cancer drugs, p53 polymorphism as a prognostic indicator that may guide<br />

cancer treatment and therapy targeted at EBV present in certain types of cancer. The cell biology groups<br />

investigate the regulation and functions of key proteins involved in control of the cell cycle and apoptosis.<br />

The virology program at the St Mary’s Branch seeks to understand the mechanisms by which EBV causes<br />

cell proliferation and the role of the virus in cancer.<br />

In addition to its core research, this Branch plays a leading role in two <strong>LICR</strong> programs that interact with<br />

many groups across the <strong>Institute</strong> world-wide. A program to create new monoclonal antibodies of value in<br />

diagnosis or treatment of human cancer has been developed in collaboration with the 4 th Military Medical<br />

School in Xian, China and is now delivering cell lines producing antibodies to participating laboratories.<br />

In addition, the human and mouse gene Microarray Program (a collaboration with Cancer <strong>Research</strong> UK<br />

and the Wellcome Trust Sanger <strong>Institute</strong>, UK) provides advanced microarray technology and training to<br />

the <strong>LICR</strong>.<br />

The <strong>Ludwig</strong> <strong>Institute</strong> Branch at St Mary’s operates in close association with the Wright-Fleming <strong>Institute</strong><br />

and Department of Virology of Imperial College Faculty of Medicine (London UK). We continue to<br />

benefit from the interactions that this brings, the steady improvement in our research environment and<br />

access to the new imaging centre that opened in <strong>2002</strong> adjacent to our labs. We are also grateful for our<br />

clinical links with several of the Imperial College group hospitals and other collaborating centres in the<br />

UK and Italy that have provided patient biopsy material essential for our research.<br />

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P. J. Farrell


RESEARCH REPORT<br />

BLYMPHOCYTE BIOLOGY AND EPSTEIN-BARR VIRUS GROUP<br />

LONDON ST MARY’S<br />

The group headed by Dr. Martin Allday is studying EBNA3 proteins and their role in the immortalization<br />

of B cells and the biology of EBV. The group’s work has extended to include EBNA3A that, like<br />

EBNA3C, can co-operate with activated ras in the immortalization and transformation of primary rodent<br />

fibroblasts. EBNA3A also binds to the highly conserved repressor of transcription, CtBP. This<br />

interaction, which is through a novel interaction domain we identified in EBNA3A, is important for the<br />

repression of transcription by EBNA3A, and for its ability to rescue primary rodent cells from the<br />

premature senescence induced by oncogenic ras. In order to explore further our hypothesis that EBV<br />

disrupts cell cycle checkpoints, we have compared checkpoints in EBV-infected B cells and an isogenic<br />

population of normal human B cells stimulated with CD40-ligand/IL4. Preliminary studies have revealed<br />

that EBV can disrupt the G1 checkpoint associated with genotoxic stress by interfering with p21 WAF1 and<br />

CDK2 function.<br />

EPSTEIN-BARR VIRUS AND CELL GROWTH CONTROL GROUP<br />

Epstein-Barr virus (EBV) causes cell proliferation and is involved in several types of human cancer. The<br />

group led by Dr. Paul Farrell is identifying genes and cellular processes that are regulated by EBV. The<br />

viral EBNA2 protein is essential for B cell proliferation induced by EBV, and several targets for<br />

regulation by EBNA2, including cytokines and cell cycle proteins, have been found. The group has<br />

focussed on one of these, RUNX3, and is investigating the role of RUNX proteins in lymphoid cells<br />

infected with EBV. The group is also continuing to search for additional genes within EBV that might<br />

explain its role in those human cancers that lack expression of the viral genes conventionally associated<br />

with B cell immortalization. In this context the group has further characterized the promoter for the<br />

BART RNAs, which are expressed from EBV in nasopharyngeal carcinoma. The extensive analysis of the<br />

mechanism of reactivation of the EBV lytic cycle from the latent state found in tumours has largely<br />

defined the mechanism by which this occurs in Burkitt’s lymphoma cell lines in response to activation of<br />

the B cell receptor. The assay systems used for these studies have been developed into a screen for<br />

compounds that might be able to reactivate the viral lytic cycle in latently infected tumour cells, as a<br />

novel therapeutic strategy specific for EBV associated cancers.<br />

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LONDON ST MARY’S<br />

Molecular phenotyping of tumour suppressor genes in cancers links basic science to disease and helps to<br />

focus on the genes that are most important in human cancer. Inactivation of gene expression is an<br />

important mechanism of removing the functions of some of these genes in cancer, and the patterns of<br />

inactivation guide our interpretations of the functions of the proteins in vivo. Molecular genetic factors<br />

predictive of clinical outcome would be of great value in oncology. The group has focussed on the<br />

involvement of the p53, p73, ASPP, Chk2 and 14-3-3� genes in breast, vulva, skin, and head and neck<br />

cancer. In a relationship between a p53 codon 72 polymorphism and susceptibility to skin cancer has been<br />

found. The group has now shown that p73 is a determinant of cellular response to a range of anti-cancer<br />

drugs, and that polymorphism in p53 influences response in head and neck cancer, at least in part, via its<br />

influence on the inhibition of p73-dependent apoptosis by mutant p53. These results have established a<br />

link between polymorphism in p53 and individual response to cancer therapy, which can potentially be<br />

used in clinical practice.<br />

ONCOGENE GROUP<br />

The Oncogene Group led by Dr. Roger Watson has continued to study the regulation of the mammalian<br />

B-Myb gene, which is transcriptionally repressed, during the G0/early G1 phases of the cell cycle, by<br />

E2F-4 transcription factor complexes containing the p130 and p107 retinoblastoma-related proteins. Our<br />

recent findings show that in vivo occupation of the B-Myb promoter E2F site by repressive p130- and<br />

p107-E2F complexes is regulated by an adjacent co-repressor site, the DRS. Transcriptional repression of<br />

B-Myb in G0 is enforced by recruitment of histone deacetylase (HDAC1) and the Sin3B repressor<br />

protein, which acts as a scaffold for assembly of HDAC1 and chromatin remodelling SWI/SNF proteins.<br />

The B-Myb transcription factor is essential for embryonic development, and the group is currently<br />

establishing transgenic models to study this function. The group has shown that in addition to its direct<br />

role in regulating gene expression, B-Myb also functions to counteract inhibitory regulators of the cell<br />

cycle. Thus, B-Myb binds to an N-terminal domain of p107, containing a cyclin/cdk2 binding domain,<br />

which is in part responsible for the ability of p107 to cause a G1 arrest. The results indicate that B-Myb<br />

prevents the sequestration of cyclin/cdk2 by p107, thereby releasing active kinase that is required at the<br />

G1/S transition for cells to enter S phase.<br />

TUMOUR SUPPRESSOR GENE GROUP<br />

The tumour suppressor function of p53 is lost in approximately 50% of human tumours, and one of the<br />

major objectives of the Tumour Suppressor Gene Group led by Dr. Xin Lu is to develop new strategies to<br />

reactivate p53 function. The group has shown that two members of the ASPP family of proteins, ASPP1<br />

and ASPP2, specifically stimulate the apoptotic function of p53, while a third member of the ASPP<br />

family, iASPP, specifically inhibits the apoptotic function of p53. Importantly, iASPP is the most<br />

conserved member of the ASPP family, and the C. elegans orthologue of iASPP is capable of substituting<br />

for human iASPP in human cells. Approximately 80% of human breast carcinomas containing wild type<br />

p53 have abnormal expression of ASPP family members, i.e. reduced expression of ASPP1 and ASPP2,<br />

or increased expression of iASPP. Moreover, reduced expression of ASPP1 and ASPP2 has also been<br />

observed in some human breast tumours expressing mutant p53. This may due to the fact that ASPP1 and<br />

ASPP2 are common activators of p53 family members. Co-expression of ASPP1 and ASPP2 can also<br />

stimulate the transactivation and apoptotic function of p63 and p73. Since the activity of p53 family<br />

members is critical in controlling cellular response to stress, it is likely that the status of ASPP will play a<br />

pivotal role in regulating cellular sensitivity to stress caused by cancer therapy.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 51


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

LONDON ST MARY’S<br />

1. ATALAY A, CROOK T, OZTURK M AND YULUG IG. Identification of genes induced by BRCA1 in<br />

breast cancer cells. Biochemichal and Biophysical <strong>Research</strong> Communications (<strong>2002</strong>) 299: 839-<br />

846.<br />

2. BARNOUIN K, DUBUISSON ML, CHILD ES, DE MATTOS SF, GLASSFORD J, MEDEMA RH, MANN<br />

DJ AND LAM EWF. Hydrogen peroxide induces a transient multiphase cell cycle arrest in mouse<br />

fibroblasts through modulating cyclin D and p21Cip1 expression. Journal of Biological<br />

Chemistry (<strong>2002</strong>) 277: 13761-13770.<br />

3. BINNÉ UK, AMON W AND FARRELL PJ. Promoter sequences required for reactivation of Epstein-<br />

Barr virus from latency. Journal of Virology (<strong>2002</strong>) 76: 10282-10289.<br />

4. BROOKS LA, SULLIVAN A, O'NIONS H, BELL A, DUNNE B, TIDY JA, EVANS DJ, OSIN P,<br />

VOUSDEN KH, GUSTERSON B, FARRELL PJ, STOREY A, GASCO M, SAKAI T AND CROOK T. E7<br />

proteins from oncogenic human papillomavirus types transactivate p73. British Journal of Cancer<br />

(<strong>2002</strong>) 86: 263-268.<br />

5. BRYANT H AND FARRELL PJ. Signal transduction and transcription factor modification during<br />

reactivation of Epstein-Barr virus from latency. Journal of Virology (<strong>2002</strong>) 76: 10290-10298.<br />

6. CATCHPOLE S, TAVNER F, LE CAM L, SARDET C, WATSON RJ. A B-myb promoter corepressor<br />

site facilitates in vivo occupation of the adjacent E2F site by p107 x E2F and p130 x E2F<br />

complexes. Journal of Biological Chemistry (<strong>2002</strong>) 277: 39015-39024.<br />

7. GASCO M, BELL AK, HEATH V, SULLIVAN A, SMITH P, HILLER L, YULUG I, NUMICO G,<br />

MERLANO M, FARRELL PJ, TAVASSOLI M, GUSTERSON B AND CROOK T. Epigenetic inactivation<br />

of 14-3-3 sigma in oral carcinoma: association with p16INK4a silencing and HPV negativity.<br />

Cancer <strong>Research</strong> (<strong>2002</strong>) 62: 2072-2076.<br />

8. GASCO M, SHAMI S AND CROOK T. The p53 pathway in breast cancer. Breast Cancer <strong>Research</strong><br />

(<strong>2002</strong>) 4: 70-76.<br />

9. GASCO M, SULLIVAN A, REPELLIN C, BROOKS L, FARRELL PJ, TIDY JA, DUNNE B, GUSTERSON<br />

B, EVANS DJ AND CROOK T. Co-incident inactivation of 14-3-3sigma and p16INK4a is an early<br />

event in vulval squamous neoplasia. Oncogene (<strong>2002</strong>) 21: 1876-1881.<br />

10. HICKABOTTOM M, PARKER GA, FREEMONT P, CROOK T, AND ALLDAY MJ. Two non-consensus<br />

sites in the Epstein-Barr virus oncoprotein EBNA3A co-operate to bind the co-repressor CtBP.<br />

Journal of Biological Chemistry (<strong>2002</strong>) 277: 47197-47204.<br />

11. HSIEH J-K, YAP D, O'CONNOR DJ, FOGAL V, FALLIS L, CHAN F, ZHONG S AND LU X. Novel<br />

function of the Cyclin A binding site of E2F in regulating p53-induced apoptosis in response to<br />

DNA damage. Molecular and Cellular Biology (<strong>2002</strong>) 22: 78-93.<br />

12. JOAQUIN M, BESSA M, SAVILLE MK, WATSON RJ. B-Myb overcomes a p107-mediated cell<br />

proliferation block by interacting with an N-terminal domain of p107. Oncogene (<strong>2002</strong>) 21: 7923-<br />

7932.<br />

13. KOPS GJ, MEDEMA RH, GLASSFORD J, ESSERS MA, DIJKERS PF, COFFER PJ, LAM EW,<br />

BURGERING BM. Control of cell cycle exit and entry by protein kinase B-regulated forkhead<br />

transcription factors. Molecular and Cellular Biology (<strong>2002</strong>) 7: 2025-2036.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 52


LONDON ST MARY’S<br />

14. MCGREGOR JM, HARWOOD CA, BROOKS L, FISHER SA, KELLY DA, O'NIONS J, YOUNG AR,<br />

SURENTHERAN T, BREUER J, MILLARD TP, LEWIS CM, LEIGH IM, STOREY A AND CROOK T.<br />

Relationship between p53 codon 72 polymorphism and susceptibility to skin cancer. Journal of<br />

Investigative Dermatology (<strong>2002</strong>) 119: 84-90.<br />

15. RAYMAN JB, TAKAHASHI Y, INDJEIAN VB, DANNENBERG JH, CATCHPOLE S, WATSON RJ, TE<br />

RIELE H, DYNLACHT BD. E2F mediates cell cycle-dependent transcriptional repression in vivo by<br />

recruitment of an HDAC1/mSin3B corepressor complex. Genes and Development (<strong>2002</strong>) 16:<br />

933-947.<br />

16. REDDY A, YUILLE M, SULLIVAN A, REPELLIN C, BELL A, TIDY JA, EVANS DJ, FARRELL PJ,<br />

BARRY GUSTERSON B, GASCO M AND CROOK T. Analysis of CHK2 in vulval neoplasia. British<br />

Journal of Cancer (<strong>2002</strong>) 86: 756-760.<br />

17. SPENDER L, CORNISH GH, SULLIVAN A AND FARRELL PJ. Expression of the transcription factor<br />

AML-2 (RUNX3, CBFa-3) is induced by Epstein-Barr Virus EBNA-2 and correlates with the B<br />

cell activation phenotype. Journal of Virology (<strong>2002</strong>) 76: 4919-4927.<br />

18. SULLIVAN A, YUILLE M, REPELLIN C, REDDY A, REELFS O, BELL A, DUNNE B, GUSTERSON BA,<br />

OSIN P, FARRELL PJ, YULUG I, EVANS A, OZCELIK T, GASCO M AND CROOK T. Concomitant<br />

inactivation of p53 and Chk2 in breast cancer. Oncogene (<strong>2002</strong>) 21: 1316-1324.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. FARRELL PJ. Cell switching and kissing. Nature Medicine (<strong>2002</strong>) 8: 559-560.<br />

2. FARRELL PJ. Tumour viruses - could they be an Achilles heel of cancer? European Journal of<br />

Cancer (<strong>2002</strong>) 38: 1815-1816.<br />

3. O'CONNOR DJ & JONES B. Crashcourse: Pathology, 2nd Edition. Harcourt publishers, 384 pages.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 53


LONDON UNIVERSITY COLLEGE BRANCH<br />

OF CELL AND MOLECULAR BIOLOGY<br />

LONDON,ENGLAND<br />

Staff List: Page 55<br />

Branch Director’s <strong>Report</strong>: Page 60<br />

<strong>Research</strong> <strong>Report</strong>: Page 61<br />

Publications: Page 64<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003


STAFF LIST<br />

WATERFIELD,MICHAEL D., Director, Member<br />

BIOANALYTICAL CHEMISTRY LABORATORY<br />

CRAMER,RAINER, Assistant Member<br />

BURLINGAME,AL, Honorary Member (until February)<br />

THOMPSON,ANDREW, Postdoctoral Fellow<br />

GERRITS,BERTRAN, Graduate Student<br />

HART,SARAH, Graduate Student<br />

RODRIGUEZ-CUTILLAS,PEDRO, Graduate Student<br />

CALNAN,DENIS, Higher Scientific Officer<br />

CORLESS,STEVE, Higher Scientific Officer<br />

SAXTON,MALCOLM, Scientific Officer<br />

BIOINFORMATICS<br />

ZVELEBIL,MARKETA, Associate Investigator<br />

LYKOSTRATIS,KONSTANTINOS, Graduate Student (from October)<br />

PATEL,KETAN, Graduate Student (until September)<br />

HURWITZ,NAAMA, Higher Scientific Officer<br />

YANG,ALICE, Scientific Officer<br />

BREAST CANCER LABORATORY<br />

O'HARE,MIKE, Group Leader<br />

FULFORD,LAURA, Clinical Fellow (from August)<br />

KIM,JONG-BIN, Graduate Student<br />

BURT,AVRIL, Tissue Collector<br />

MACKAY,ALAN, Higher Scientific Officer<br />

DAVIES,SUSAN, Scientific Officer (until October)<br />

GRIGORIADIS,ANITA, Scientific Officer<br />

CANCER BIOCHEMISTRY GROUP<br />

WATERFIELD,MICHAEL, Director, Member<br />

BARNOUIN,KARIN, Postdoctoral Fellow<br />

GAFFNEY,PIERS, Postdoctoral Fellow (until October)<br />

LONDON UNIVERSITY COLLEGE<br />

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TIMMS,JOHN, Postdoctoral Fellow<br />

WHITE,SARAH, Postdoctoral Fellow (until September)<br />

BERTANI,MARIANA, Graduate Student<br />

CHAN,HONG-LIN, Graduate Student<br />

GHARBI,SEVERINE, Graduate Student<br />

FOSBERG,SOFI, Visiting Student (from August)<br />

PAXTON,CAMILLE, Visiting Student (May to August)<br />

MISTRY,KAY, Higher Scientific Officer<br />

CELL REGULATION LABORATORY<br />

GOUT,IVAN, Assistant Member<br />

VERDIER,FREDERIQUE, Postdoctoral Fellow (to July)<br />

ZHYVOLOUP,ALEXANDER, Postdoctoral Fellow<br />

VALOVKA,TARAS, Graduate Student<br />

FENTON,TIMOTHY, Graduate Student<br />

REBHOLZ,HEIKE, Graduate Student<br />

WANG,MONG-LIEN, Graduate Student<br />

NEMEZANYI,IVAN, Visiting Student (June to September)<br />

PANASYUK,GANNA, Visiting Student (July to September)<br />

FOXON,RICHARD, Higher Scientific Officer (from March)<br />

CELL SHAPE AND POLARITY GROUP<br />

BAUM,BENJAMIN, Postdoctoral Fellow<br />

CRAIG,GAVIN, Postdoctoral Fellow<br />

DUCHEK,PETER, Postdoctoral Fellow (from August)<br />

KUNDA,PATRICIA, Postdoctoral Fellow (from May)<br />

SIMS,DAVID, Graduate Student (from October)<br />

DOMINGUEZ,VERONICA, <strong>Research</strong> Assistant<br />

CELL SIGNALING LABORATORY<br />

VANHAESEBROECK,BART, Assistant Member<br />

BILANCIO,ANTONIO, Postdoctoral Fellow<br />

BOX (SAWYER), CAROL, Postdoctoral Fellow<br />

FARJOT,GERALDINE, Postdoctoral Fellow (until June)<br />

FOUKAS,LAZAROS, Postdoctoral Fellow (from October)<br />

LONDON UNIVERSITY COLLEGE<br />

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OKKENHAUG,KLAUS, Postdoctoral Fellow (until December)<br />

SALPEKAR,ASHREENA, Postdoctoral Fellow<br />

ALI,KHALED, Graduate Student<br />

FOUKAS,LAZAROS, Graduate Student (from October)<br />

GEERING,BARBARA, Graduate Student (from October)<br />

WOODS,LIZZIE, Graduate Student<br />

PAXTON,CAMILLE, Visiting Student (May to August)<br />

VAN LEUKEN,RENSKE, Visiting Student (until May)<br />

PEARCE,WAYNE, Scientific Officer<br />

PESKETT,EMMA, Scientific Officer<br />

CELLULAR AND MOLECULAR BIOLOGY LABORATORY<br />

RIDLEY,ANNE, Associate Member<br />

DATTA,ARPITA, <strong>Research</strong> Associate (from September)<br />

ENTWISTLE,ALAN, <strong>Research</strong> Associate<br />

CORY,GILES, Postdoctoral Fellow<br />

DE VILLALONGA SMITH,PRIAM, Postdoctoral Fellow<br />

IVETIC,ALEKSANDAR, Postdoctoral Fellow<br />

KANAGUSUNDARAM,VARUNI, Postdoctoral Fellow (April to October)<br />

LEVERRIER,YANN, Postdoctoral Fellow (until June)<br />

MILLAN MARTINEZ,JAIME, Postdoctoral Fellow<br />

RIENTO,KIRSI, Postdoctoral Fellow<br />

WELLS,CLAIRE, Postdoctoral Fellow<br />

WOJCIAK-STOTHARD,BEATA, Postdoctoral Fellow<br />

FRANZ,CLEMENS, Graduate Student<br />

LYKOSTRATIS,KONSTANTINOS, Graduate Student (from October)<br />

MCKENZIE,JENNY, Graduate Student<br />

SMITH,STEVEN, Graduate Student (from October)<br />

WHEELER,ANN, Graduate Student<br />

EVANS,IWAN, Visiting Student (June to September)<br />

GARG,RITU, Higher Scientific Officer<br />

CLINICAL STUDIES LABORATORY<br />

STEIN,ROBERT, Clinical FellowAssistant Member (until March)<br />

FOXON,RICHARD, Higher Scientific Officer (until March)<br />

LONDON UNIVERSITY COLLEGE<br />

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DROSOPHILA MORPHOGENESIS LABORATORY<br />

BARRETT,KATHERINE, Postdoctoral Fellow<br />

LOH,SAMANTHA HUI YONG, Postdoctoral Fellow<br />

NIKOLAIDOU,KELLY [KYRIAKI], Postdoctoral Fellow<br />

ESCOTT,GARETH, Graduate Student<br />

HILEY,CHARLOTTE, Graduate Student<br />

KITSOU,EFSTATHIA, Graduate Student (from November)<br />

VERBEKE,TOM, Visiting Student (February to June)<br />

BATCHELOR,EMILY, Scientific Officer<br />

DEGUFFROY,BENEDICTE, Scientific Officer (January to June)<br />

PROTEOMICS LABORATORY<br />

NAABY-HANSEN,SOREN, Assistant Member<br />

WARNASURIYA,GAYATHRI, Postdoctoral Fellow<br />

HASTIE,CLAIRE, Visiting Graduate Student<br />

AKPAN,AKUNNA, Higher Scientific Officer<br />

WOOD,CHRIS, Assistant Scientific Officer (from October)<br />

STRUCTURAL BIOLOGY LABORATORY<br />

DRISCOLL,PAUL, Postdoctoral Fellow<br />

VINES,DAVID, Postdoctoral Fellow<br />

SANKAR,ANDREW, Higher Scientific Officer<br />

TRANSFORMATION STUDIES LABORATORY<br />

JAT,PARMJIT, Associate Member<br />

HARDY,KRISTINE, Postdoctoral Fellow<br />

RELPH (BOAST), KATE, Postdoctoral Fellow<br />

TARUNINA,MARINA, <strong>Research</strong> Associate<br />

BENVENUTI,SILVIA, Graduate Student (until October)<br />

MARTIN,ROWENA, Graduate Student<br />

MANSFIELD,LOUISE, Graduate Student (from October)<br />

ARORA,PARINEETA, Scientific Officer<br />

NIKITOPOULOU,ATHENA, Scientific Officer<br />

LONDON UNIVERSITY COLLEGE<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 58


GENERAL SUPPORT SERVICES<br />

SCRACE,GEOFF, <strong>Research</strong> Associate<br />

WILLIAMS,SIMON, Assistant Scientific Officer<br />

GARADY,ADAM, Security<br />

MCCREADIE,JAMES, Security<br />

ADMINISTRATION AND SECRETARIAT<br />

ANNING,RICHARD, Branch Administrator<br />

TARGETT,REBECCA, Administrative Assistant<br />

WARD,ALEX, Administrative Assistant<br />

STONE,DEBORAH, Director's Secretary<br />

AHMADI,KHATEREH, Scientific Administrator<br />

BARNSLEY,SARAH, Assistant Scientific Administrator<br />

LONDON UNIVERSITY COLLEGE<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 59


BRANCH DIRECTOR’S REPORT<br />

LONDON UNIVERSITY COLLEGE<br />

The research program of the London University College Branch is designed to clarify the mechanisms of<br />

signal transduction that controls normal cell differentiation, growth, proliferation, migration and death,<br />

and the ways that cancer cells subvert these processes. The translational objective of this research is to<br />

find ways to detect and reverse the altered signal pathways in cancer cells and has been focused on<br />

collaborative drug development using targets discovered and characterised in the Branch.<br />

The practical approach taken by the Branch program has been based on three fundamental concepts. The<br />

first is that the parallel use of genetically alterable animal model systems, alongside human cultured<br />

cellular models provides a fast track route to deciphering cancer biology. The second is that analytical<br />

protein chemistry and cutting edge cell biology are vital technologies in making progress in cancer<br />

research. The third is that this research can be best translated into patient benefit through industrial<br />

partnership – which is, of course, our ultimate aim.<br />

In the first area we have continued to use Drosophila as a tractable model system to find, establish, and<br />

monitor the genes involved in cell growth and differentiation (see the groups of K Barrettt and B Baum).<br />

This approach proved critical in the case of the phosphatidylinositol 3-kinase (PI3K) pathway and we<br />

believe the system will reap many more benefits in linking post-genomic technologies to molecular<br />

genetics. The second model we use involves targeted knockouts in animals, which is proving essential to<br />

understand the family of PI3K and their signals (see group of B Vanhaesebroeck). The role of cutting<br />

edge cell biology (see group of A Ridley) and analytical protein chemistry (see group of R Cramer) can<br />

be seen in the need to define and visualise alterations in protein expression and interactions, and link these<br />

to cell physiology. The need for partnership to bring discoveries to patients is obvious and during the last<br />

decade the Branch has been enormously successful in developing both basic and applied work on the lipid<br />

kinase mediated signal pathways that were deciphered by the coordinated activities of groups in the<br />

branch. It has now become clear, from the work of many other labs around the world, that loss of control<br />

over PI3Ks, caused by the inability to remove the second messenger (PIP3), which the PI3Ks generate in<br />

response to growth signals, is the second most common defect in cancer cells. The branch scientists have<br />

been working with the pharmaceutical industry for some five years to make drugs that block the PI3Ks,<br />

and this effort has generated new lead compounds with exquisite specificity for the target enzymes. These<br />

compounds will soon move on to be development candidates through more focused industrial<br />

collaboration. This effort has been a major collaboration between the laboratories of Professors M<br />

Waterfield at London University College Branch, and those of P Parker and P Workman of Cancer<br />

<strong>Research</strong> UK (CRUK).<br />

The Cell Signalling, Regulation, Cell and Molecular Biology, and Transformation Groups are actively<br />

continuing the task of defining the importance of signal transduction pathways in cancer.<br />

The Bioinformatics, Bioanalytical Chemistry, Proteomics and Cancer Proteomics Groups are defining<br />

interactions in biological systems that will establish the protein networks linking the subverted pathways<br />

in cancer cells. A more detailed description of up to date research activity by each group can be found on<br />

the Branch’s website.<br />

M. D. Waterfield<br />

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RESEARCH REPORT<br />

BIOANALYTICAL CHEMISTRY GROUP<br />

LONDON UNIVERSITY COLLEGE<br />

The group, under the leadership of Dr. Rainer Cramer, has developed cutting edge mass spectrometry<br />

methods that are being applied to protein identification, and the definition of post-translational<br />

modifications on proteins through more than 30 collaborative projects. The group has established<br />

expertise in ultraviolet and infrared matrix-assisted laser desorption/ionization (MALDI) and high<br />

performance liquid chromatography (HPLC)-linked electrospray instrumentation, and is using an<br />

automated processing robot to assist in sample processing from 2D gels.<br />

BIOINFORMATICS GROUP<br />

The group, led by Dr. Marketa Zvelebil, has worked closely with the Proteomics labs to gather and<br />

interpret protein expression data from 2D-gels, and correlate these with patient and cell data. The group is<br />

moving into new areas by collaborating with the groups of Drs. A Ridley and B Baum to model signal<br />

pathways. Meanwhile they continue their essential modelling of protein structure in the PI3K field<br />

through exploitation of their experience in computational approaches that deciphering interactions<br />

between ligands and proteins.<br />

CANCER PROTEOMICS GROUP<br />

This group, newly formed from Dr. Mike Waterfield’s laboratory, and led by Dr. John Timms, has been<br />

using the unique fluorescent 2D DIGE (Amersham) techniques with advanced laser gel scanners in<br />

special clean rooms to analyse differential cellular protein expression analysis. Linked studies on<br />

transcriptional profiling using the <strong>LICR</strong> Microarray Program have been applied to Erb-B2 dependent<br />

breast cancer cell models developed by Dr. Mike O’Hare.<br />

CELL REGULATION GROUP<br />

The group led by Dr. Ivan Gout is working on the downstream part of the PI3K signal pathway that<br />

controls cell growth and proliferation through a kinase cascade that activates the enzymes S6 kinase and<br />

Tor, and which in turn regulate aspects of translation control. Both enzymes are, or will be, drug targets in<br />

cancer drug development because they, or their regulation, have been altered in cancer cells. The cell<br />

regulation group, discovered the S6 kinase2, and has used several techniques to identify the important<br />

points where regulation of this enzyme occurs. New partners of the enzyme have been discovered through<br />

mutational analysis, the use of yeast two-hybrid searches, and affinity purification techniques,. These<br />

include the enzyme CoA synthase that provides a link to metabolic control and specific phosphorylation<br />

of S6K2 by PKCs, which affects the nucleocytoplasmic movements of this enzyme.<br />

CELL SHAPE AND POLARITY GROUP<br />

Dr. Benjamin (Buzz) Baum’s group joined the Branch in 2001, and is now very active, with five new<br />

members in the team. The group plans to use the technique of RNAi, together with Drosophila molecular<br />

genetics and new cell lines, to study cell morphogenesis. The investment in new cutting edge technology<br />

will be translatable across the Branch programs.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 61


CELL SIGNALLING GROUP<br />

LONDON UNIVERSITY COLLEGE<br />

The continued need to understand the basic science of signal transduction in the lipid kinase area has been<br />

the research focus of Dr. Bart Vanhaesebroeck’s group, which has been successful in developing mouse<br />

models of PI3K defective function. These models will be of great use to both academic and applied<br />

scientists. The PI3K family of enzymes is made up of 4 distinct groups, each of which has one to three<br />

members. This diversity must relate to the need to closely control the output signal – the generation of a<br />

major second messenger, which can deliver the signal for a cell to grow, proliferate or die.<br />

It is very difficult to decipher the function of individual members of such multigene families without<br />

specific drugs and although our work with industry has helped us get some new drugs that target certain<br />

family members, the ability to manipulate the PI3Ks in animal models in vivo has been crucial in our<br />

understanding of the role of the PI3K subtypes. The Cell Signalling Group has this year developed a<br />

p110� knockout mouse model, which has shown that this class IA PI3K enzyme specifically controls<br />

aspects of antigen receptor signaling, and thus could be a novel anti-inflammatory target. The results from<br />

this study are significant as they show that PI3K subtypes have non-redundant functions in the cell.<br />

Models for the other defective PI3K isotypes are being made and will allow the laboratory to examine<br />

predictions about the role of the enzymes in human cancer.<br />

CELLULAR MOLECULAR BIOLOGY GROUP<br />

Dr. Anne Ridley’s group seeks to understand the basis of the process of cell migration, which underlies<br />

the invasion and metastasis process of tumour cells, and is also vital for angiogenesis, and leukocyte<br />

recruitment to tumours. The group has great expertise in cell biology and cell visualisation as well as in<br />

the biochemistry of the small GTP binding proteins that mediate cell movement. The protein kinase<br />

PAK4, which has been implicated in mediating signalling downstream of the growth factor HGF receptor<br />

is one focus of the group. PAK4 triggers the PI3K second messenger system, and controls epithelial<br />

cancer cell invasion. The long-term interest in small GTP binding proteins in the laboratory has<br />

continued, and indeed has made the group world experts in this area. It has been possible to prove that<br />

RhoA and RhoE, whose levels have been shown to be altered in epithelial tumours, control the positive<br />

and negative aspects of migration mediated by the ROCK kinase. The group has also deciphered the 3D<br />

structure of Rho in collaboration with Dr N Keep at Birkbeck College (London, UK). This<br />

multidimensional approach of linking structural and functional studies may prove to be the way to make<br />

this pathway a drug target. Work on leukocyte trans-epithelial migration and macrophage movements has<br />

continued to stress the importance of the small GTP binding proteins and the upstream role of PI3Ks. The<br />

group has been able to extract added value from our cell biology and biochemistry expertise because the<br />

targets have been mapped within complex overlapping networks.<br />

DROSOPHILA MORPHOGENESIS GROUP<br />

The group led by Dr. Kathy Barrett, has focused on research into the control of cell shape and motility –<br />

studies that are linked to those of the Cellular Molecular Biology Group. Using advanced real-time<br />

microscopy, cell physiology events have been visualized, in order to elucidate the role of Rho GTPases.<br />

Through in vivo visual imagery and the powerful genetics of the Drosophila model, several new genes<br />

that are linked to Rho GTPase signalling, and that are required for cell shape change, are being studied.<br />

The relevance of such findings to human cancer is under assessment.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 62


PROTEOMICS GROUP<br />

LONDON UNIVERSITY COLLEGE<br />

The Proteomics Group, headed by Dr. Søren Naaby-Hansen, uses a variety of proteomic methods to<br />

decipher defective signalling pathways in cancer cells, with a focus on prostate cancer. Global proteomic<br />

studies of prostate cells have revealed that stimulation by combinations of cytokines and/or growth factors<br />

have distinct and often opposing effects when compared with cells stimulated with single cytokines or<br />

growth factors. For example, simultaneous exposure to type I and II interferons up-regulates more cell<br />

surface proteins on prostate cancer cells when compared with treatment by a single cytokine. This result<br />

may have implications for the immunotherapy of this disease.<br />

STRUCTURAL BIOLOGY GROUP<br />

This group, led by Dr. Paul Driscoll, which works in the University College London (UCL) Biochemistry<br />

Laboratory, is following a program that involves the development and application of NMR methodology<br />

for the elucidation of structural, dynamic, and functional properties of proteins. Amongst the highlights of<br />

this year’s work are the solution of the structures of the c-terminus of Ku80, a protein involved in<br />

monitoring DNA damage, and a more detailed analysis of the FADD domains that mediate decisions<br />

about cell viability. The group is making progress on developing new methods for optimising protein<br />

expression that could open up ways to solve more important structures.<br />

TRANSFORMATION STUDIES GROUP<br />

The group led by Dr. Parmjit Jat, seeks to find out how the capacity for unlimited growth potential, one of<br />

the critical steps in tumourigenesis, arises from disruption of genes that control the finite lifespan of<br />

normal cells. Through the generation of new cell models where a temperature-sensitive SV40 T antigen<br />

and a telomerase have been introduced, the group Jat has found clues to the origin of aneuploidy. Using<br />

yeast 2-hybrid systems the Bub1 kinase, which is involved in mitotic spindle assembly and is mutated in<br />

some cancers, has also been linked to T antigen and chromosome aberrations.<br />

Post genomic technologies such as proteomic 2D-gel and transcriptional monitoring, through the <strong>LICR</strong><br />

Microarray Program, have proved very useful in these studies linking genes with known roles in cancer<br />

and replicative senescence. Identified genes and proteins include a member of the p63 family of p53related<br />

proteins and transcription targets of p53, both of which are differentially expressed in<br />

conditionally immortalised cells.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 63


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

LONDON UNIVERSITY COLLEGE<br />

1. ARCARO A, KHANZADA UK, VANHAESEBROECK B, TETLEY TD, WATERFIELD MD, AND SECKL<br />

MJ. Two distinct phosphoinositide 3-kinases mediate polypeptide growth factor-stimulated PKB<br />

activation. EMBO Journal (<strong>2002</strong>) 21(19):5097-108<br />

2. BARNOUIN K, DUBUISSON ML, CHILD ES, FERNANDEZ DE MATTOS S, GLASSFORD J, MEDEMA<br />

RH, MANN DJ, AND LAM EW. H2O2 induces a transient multi-phase cell cycle arrest in mouse<br />

fibroblasts through modulating cyclin D and p21Cip1 expression. Journal of Biological<br />

Chemistry (<strong>2002</strong>) 277(16):13761-70.<br />

3. BAUM B. Winging it--actin on the fly. Developmental Cell (<strong>2002</strong>) 2(2):125-6.<br />

4. BAUM B. Drosophila oogenesis: generating an axis of polarity. Current Biology (<strong>2002</strong>)<br />

12(24):835-7.<br />

5. BENVENUTI S, CRAMER R, QUINN CC, BRUCE J, ZVELEBIL M, CORLESS S, BOND J, YANG A,<br />

HOCKFIELD S, BURLINGAME AL, WATERFIELD MD, AND JAT PS. Differential proteome analysis<br />

of replicative senescence in rat embryo fibroblasts. Molecular and Cellular Proteomics (<strong>2002</strong>)<br />

1(4):280-92.<br />

6. BENVENUTI S, CRAMER R, BRUCE J, WATERFIELD MD, AND JAT PS. Identification of novel<br />

candidates for replicative senescence by functional proteomics. Oncogene (<strong>2002</strong>) 21(28):4403-<br />

13.<br />

7. BERRIDGE G, CRAMER R, GALIONE A, AND PATEL S. Metabolism of the novel Ca2+-mobilizing<br />

messenger nicotinic acid-adenine dinucleotide phosphate via a 2'-specific Ca2+-dependent<br />

phosphatase. Biochemical Journal (<strong>2002</strong>) 365(Pt 1):295-301.<br />

8. BUCHMAN VL, LUKE C, BORTHWICK EB, GOUT I, AND NINKINA N. Organization of the mouse<br />

Ruk locus and expression of isoforms in mouse tissues. Gene (<strong>2002</strong>) 295(1):13-17.<br />

9. CHEN HA, PFUHL M, AND DRISCOLL PC. The pH dependence of CD2 domain 1 self-association<br />

and 15N chemical exchange broadening is correlated with the anomalous pKa of Glu41.<br />

Biochemistry (<strong>2002</strong>) 41(50):14680-8.<br />

10. CORY GO, GARG R, CRAMER R, AND RIDLEY AJ. Phosphorylation of tyrosine 291 enhances the<br />

ability of WASp to stimulate actin polymerization and filopodium formation. Journal of<br />

Biological Chemistry (<strong>2002</strong>) 277(47):45115-21.<br />

11. DJELLOUL S, TARUNINA M, BARNOUIN K, MACKAY A, AND JAT PS. Differential protein<br />

expression, DNA binding and interaction with SV40 large tumour antigen implicate the p63family<br />

of proteins in replicative senescence. Oncogene (<strong>2002</strong>) 21(7):981-9.<br />

12. ESPOSITO D, PETROVIC A, HARRIS R, ONO S, ECCLESTON JF, MBABAALI A, HAQ I, HIGGINS CF,<br />

HINTON JC, DRISCOLL PC, AND LADBURY JE. H-NS oligomerization domain structure reveals the<br />

mechanism for high order self-association of the intact protein. Journal of Molecular Biology<br />

(<strong>2002</strong>) 324(4):841-50.<br />

13. FRANZ CM, JONES GE, AND RIDLEY AJ. Cell migration in development and disease.<br />

Developmental Cell (<strong>2002</strong>) 2(2):153-8.<br />

14. GARAVINI H, RIENTO K, PHELAN JP, MCALISTER MS, RIDLEY AJ, AND KEEP NH. Crystal<br />

structure of the core domain of RhoE/Rnd3: a constitutively activated small G protein.<br />

Biochemistry (<strong>2002</strong>) 41(20):6303-10.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 64


LONDON UNIVERSITY COLLEGE<br />

15. GEORGE RR, HARRIS R, NUNN CM, CRAMER R, AND DJORDJEVIC S. Chaperonin assisted<br />

overexpression, purification, and characterisation of human PP2A methyltransferase. Protein<br />

Expression and Purification (<strong>2002</strong>) 26 (2):266-74.<br />

16. GHARBI S, GAFFNEY P, YANG A, ZVELEBIL MJ, CRAMER R, WATERFIELD MD, AND TIMMS JF.<br />

Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis<br />

of a model breast cancer cell system. Molecular and Cellular Proteomics (<strong>2002</strong>) 1(2):91-8.<br />

17. GILMORE AP, VALENTIJN AJ, WANG P, RANGER AM, BUNDRED N, O'HARE MJ, WAKELING A,<br />

KORSMEYER SJ, AND STREULI CH. Activation of BAD by therapeutic inhibition of epidermal<br />

growth factor receptor and transactivation by insulin-like growth factor receptor. Journal of<br />

Biological Chemistry (<strong>2002</strong>) 277(31):27643-50.<br />

18. HARRIS R, MAMAN JD, HINKS JA, SANKAR A, PEARL LH, AND DRISCOLL PC. Backbone 1H,<br />

13C, and 15N resonance assignments for the C-terminal region of Ku86 (Ku86CTR). Journal of<br />

Biomolecular NMR (<strong>2002</strong>) 22(4):373-4.<br />

19. HARRIS RA, YANG A, STEIN RC, LUCY K, BRUSTEN L, HERATH A, PAREKH R, WATERFIELD<br />

MD, O'HARE MJ, NEVILLE MA, PAGE MJ, AND ZVELEBIL MJ. Cluster analysis of an extensive<br />

human breast cancer cell line protein expression map database. Proteomics (<strong>2002</strong>) 2(2):212-23.<br />

20. HART SR, WATERFIELD MD, BURLINGAME AL, AND CRAMER R. Factors governing the<br />

solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by<br />

MALDI TOFMS. Journal of the American Society of Mass Spectrometry (<strong>2002</strong>) 13(9):1042-51.<br />

21. HUTTUNEN HJ, FAGES C, KUJA-PANULA J, RIDLEY AJ, AND RAUVALA H. Receptor for advanced<br />

glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration<br />

and metastasis. Cancer <strong>Research</strong> (<strong>2002</strong>) 62(16):4805-11.<br />

22. IVETIC A, DEKA J, RIDLEY A, AND AGER A. The cytoplasmic tail of L-selectin interacts with<br />

members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent<br />

binding of Moesin but not Ezrin. Journal of Biological Chemistry (<strong>2002</strong>) 277(3):2321-9.<br />

23. KNIGHT D, HARRIS R, MCALISTER MS, PHELAN JP, GEDDES S, MOSS SJ, DRISCOLL PC, AND<br />

KEEP NH. The X-ray crystal structure and putative ligand-derived peptide binding properties of<br />

gamma-aminobutyric acid receptor type A receptor-associated protein. Journal of Biological<br />

Chemistry (<strong>2002</strong>) 277(7):5556-61.<br />

24. LEERKES MR, CABALLERO OL, MACKAY A, TORLONI H, O'HARE MJ, SIMPSON AJ, AND DE<br />

SOUZA SJ. In silico comparison of the transcriptome derived from purified normal breast cells<br />

and breast tumor cell lines reveals candidate upregulated genes in breast tumor cells. Genomics<br />

(<strong>2002</strong>) 79(2):257-65.<br />

25. LIDINGTON EA, RAO RM, MARELLI-BERG FM, JAT PS, HASKARD DO, AND MASON JC.<br />

Conditional immortalization of growth factor-responsive cardiac endothelial cells from H-2K(b)tsA58<br />

mice. American Journal of Physiology Cell Physiology (<strong>2002</strong>) 282(1):67-74.<br />

26. MUSTAPA MFM, HARRIS R, MOULD J, CHUBB NAL, SCHULTZ D, DRISCOLL PC, AND TABOR AB.<br />

Synthesis of cyclic peptides containing nor-lanthionine bridges via a triply-orthogonal protecting<br />

group strategy. Tetrahedron Letters (<strong>2002</strong>) 43(46):8363-8366.<br />

27. MUSTAPA MFM, HARRIS R, MOULD J, CHUBB NAL, SCHULTZ D, DRISCOLL PC, AND TABOR AB.<br />

Synthesis of orthogonally protected lanthionines: a reassessment of the use of alanyl [beta]-cation<br />

equivalents. Tetrahedron Letters (<strong>2002</strong>) 43(46):8359-8362.<br />

28. NAABY-HANSEN S, MANDAL A, WOLKOWICZ MJ, SEN B, WESTBROOK VA, SHETTY J, COONROD<br />

SA, KLOTZ KL, KIM YH, BUSH LA, FLICKINGER CJ, AND HERR JC. CABYR, a novel calcium-<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 65


LONDON UNIVERSITY COLLEGE<br />

binding tyrosine phosphorylation-regulated fibrous sheath protein involved in capacitation.<br />

Developmental Biology (<strong>2002</strong>) 242(2):236-54.<br />

29. OKKENHAUG K, BILANCIO A, FARJOT G, PRIDDLE H, SANCHO S, PESKETT E, PEARCE W, MEEK<br />

SE, SALPEKAR A, WATERFIELD MD, SMITH AJ, AND VANHAESEBROECK B. Impaired B and T<br />

cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science (<strong>2002</strong>)<br />

297(5583):1031-4.<br />

30. PANARETOU B, SILIGARDI G, MEYER P, MALONEY A, SULLIVAN JK, SINGH S, MILLSON SH,<br />

CLARKE PA, NAABY-HANSEN S, STEIN R, CRAMER R, MOLLAPOUR M, WORKMAN P, PIPER PW,<br />

PEARL LH, AND PRODROMOU C. Activation of the ATPase activity of hsp90 by the stressregulated<br />

cochaperone aha1. Molecular Cell (<strong>2002</strong>) 10(6):1307-18.<br />

31. PATEL K, STEIN R, BENVENUTI S, AND ZVELEBIL MJ. Combinatorial use of mRNA and twodimensional<br />

electrophoresis expression data to choose relevant features for mass spectrometric<br />

identification. Proteomics (<strong>2002</strong>) 2(10):1464-73.<br />

32. SALEEM MA, O'HARE MJ, REISER J, COWARD RJ, INWARD CD, FARREN T, XING CY, NI L,<br />

MATHIESON PW, AND MUNDEL P. A conditionally immortalized human podocyte cell line<br />

demonstrating nephrin and podocin expression. Journal of the American Society of Nephrology<br />

(<strong>2002</strong>) 13(3):630-8.<br />

33. SHIBAHARA H, SATO I, SHETTY J, NAABY-HANSEN S, HERR JC, WAKIMOTO E, AND KOYAMA K.<br />

Two-dimensional electrophoretic analysis of sperm antigens recognized by sperm immobilizing<br />

antibodies detected in infertile women. Journal of Reproductive Immunology (<strong>2002</strong>) 53(1-2):1-<br />

12.<br />

34. STENMARK H, AASLAND R, AND DRISCOLL PC. The phosphatidylinositol 3-phosphate-binding<br />

FYVE finger. FEBS Letters (<strong>2002</strong>) 513(1):77-84.<br />

35. SUZUKI RN, ENTWISTLE A, ATHERTON AJ, CLARKE C, LAKHANI SR, AND O'HARE MJ. The<br />

expression patterns of integrin subunits on human breast tissues obtained during pregnancy. Cell<br />

Biology International (<strong>2002</strong>) 26(7):593-8.<br />

36. SWETMAN CA, LEVERRIER Y, GARG R, GAN CH, RIDLEY AJ, KATZ DR, AND CHAIN BM.<br />

Extension, retraction and contraction in the formation of a dendritic cell dendrite: distinct roles<br />

for Rho GTPases. European Journal of Immunology (<strong>2002</strong>) 32(7):2074-83.<br />

37. THOMPSON PW, RANDI AM, AND RIDLEY AJ. Intercellular adhesion molecule (ICAM)-1, but not<br />

ICAM-2, activates RhoA and stimulates c-fos and rhoA transcription in endothelial cells. Journal<br />

of Immunology (<strong>2002</strong>) 169(2):1007-13.<br />

38. TIMMS JF, WHITE SL, O'HARE MJ, AND WATERFIELD MD. Effects of ErbB-2 overexpression on<br />

mitogenic signalling and cell cycle progression in human breast luminal epithelial cells.<br />

Oncogene (<strong>2002</strong>) 21(43):6573-86.<br />

39. VERDIER F, VALOVKA T, ZHYVOLOUP A, DROBOT LB, BUCHMAN V, WATERFIELD M, AND<br />

GOUT I. Ruk is ubiquitinated but not degraded by the proteasome. European Journal of<br />

Biochemistry (<strong>2002</strong>) 269(14):3402-8.<br />

40. VIGONTINA O, EFIMENKO O, YAKOVENKO L, KIYAMOVA R, FILONENKO V, GOUT IT, ROS N,<br />

KOSEY N, TATARCHUK T, SIDORIK L, AND MATSUKA G. Chaperon HSP-60 as autoantigen in the<br />

development of dyshormonal breast diseases. Experimental Oncology (<strong>2002</strong>) 24(1):112-115.<br />

41. WELLS CM, ABO A, AND RIDLEY AJ. PAK4 is activated via PI3K in HGF-stimulated epithelial<br />

cells. Journal of Cell Science (<strong>2002</strong>) 115(Pt 20):3947-56.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 66


LONDON UNIVERSITY COLLEGE<br />

42. WILLS Z, EMERSON M, RUSCH J, BIKOFF J, BAUM B, PERRIMON N, AND VAN VACTOR D. A<br />

Drosophila homolog of cyclase-associated proteins collaborates with the Abl tyrosine kinase to<br />

control midline axon pathfinding. Neuron (<strong>2002</strong>) 36(4):611-22.<br />

43. WOLKOWICZ MJ, NAABY-HANSEN S, GAMBLE AR, REDDI PP, FLICKINGER CJ, AND HERR JC.<br />

Tektin B1 demonstrates flagellar localization in human sperm. Biology of Reproduction (<strong>2002</strong>)<br />

66(1):241-50.<br />

44. ZHANG J, VANHAESEBROECK B, AND RITTENHOUSE SE. Human platelets contain p110delta<br />

phosphoinositide 3-kinase. Biochemical and Biophysical <strong>Research</strong> Communications (<strong>2002</strong>)<br />

296(1):178-81.<br />

45. ZHYVOLOUP A, NEMAZANYY I, BABICH A, PANASYUK G, POBIGAILO N, VUDMASKA M,<br />

NAIDENOV V, KUKHARENKO O, PALCHEVSKII S, SAVINSKA L, OVCHARENKO G, VERDIER F,<br />

VALOVKA T, FENTON T, REBHOLZ H, WANG ML, SHEPHERD P, MATSUKA G, FILONENKO V, AND<br />

GOUT IT. Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis. Journal of<br />

Biological Chemistry (<strong>2002</strong>) 277(25):22107-10.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. CORY GO AND RIDLEY AJ. Cell motility: braking WAVEs. Nature (<strong>2002</strong>) 418(6899):732-3.<br />

2. DJORDJEVIC S AND DRISCOLL PC. Structural insight into substrate specificity and regulatory<br />

mechanisms of phosphoinositide 3-kinases. Trends in Biochemical Science (<strong>2002</strong>) 27(8):426-32.<br />

3. MELLMAN I AND RIDLEY A. Regulation and functional insights in cellular polarity, Madrid, Spain<br />

June 3-5, <strong>2002</strong>. Journal of Cell Biology (<strong>2002</strong>) 158(1):12-6.<br />

4. TIMMS JF. Differential protein analysis using fluorscence two-dimensional difference gel<br />

electrophoresis. (<strong>2002</strong>) In: Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2 nd<br />

Edition.<br />

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MELBOURNE BRANCH<br />

OF TUMOUR BIOLOGY<br />

MELBOURNE,AUSTRALIA<br />

Staff List: Page 69<br />

Branch Director’s <strong>Report</strong>: Page 79<br />

<strong>Research</strong> <strong>Report</strong>: Page 80<br />

Publications: Page 89<br />

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STAFF LIST<br />

BURGESS ANTONY W, Director, Member*<br />

DUNN,ASHLEY, Associate Director<br />

SCOTT,ANDREW, Associate Director – Clinical*<br />

ANGIOGENESIS LABORATORY<br />

ACHEN,MARC, Assistant Member, Joint Head<br />

STACKER,STEVEN, Assistant Member, Joint Head<br />

BALDWIN,MEGAN, Postdoctoral Fellow<br />

HALFORD,MICHAEL, Postdoctoral Fellow<br />

HEINZERLING,LUCIE, Visiting Postdoctoral Fellow (until February)<br />

KUGATHASAN,KUMI, PhD Student<br />

MCCOLL,BRAD, PhD Student<br />

CAESAR,CAROL, Senior <strong>Research</strong> Officer<br />

CHAFFER,CHRISTINE, <strong>Research</strong> Officer<br />

INDER,RACHAEL, <strong>Research</strong> Officer<br />

LAI,PATRICIA, <strong>Research</strong> Officer<br />

MCDONALD,NARELLE, <strong>Research</strong> Officer<br />

ROUFAIL,SALLY, <strong>Research</strong> Assistant<br />

BIOINFORMATICS<br />

HALL,NATHAN, Bioinformatics Officer<br />

MCDONALD,JOSHUA, UROP Student (from February)<br />

COLON MOLECULAR &CELL BIOLOGY LABORATORY<br />

ERNST,MATTHIAS, Assistant Member, Joint Head<br />

HEATH,JOAN, Assistant Member, Joint Head<br />

ABUD,HELEN, Assistant Investigator<br />

JENKINS,BRENDAN, Postdoctoral Fellow<br />

NG,ANNIE, Postdoctoral Fellow<br />

BATEMAN,TRUDIE, PhD Student<br />

DE JONG,TANYA, PhD Student<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

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SAMUEL,MICHAEL, PhD Student<br />

TOMASKOVIC -CROOK,EVA, PhD Student<br />

GRAIL,DIANNE, Chief <strong>Research</strong> Officer<br />

LIU,YONG-SHU, Chief <strong>Research</strong> Officer (from August)<br />

CODY,STEPHEN, Senior <strong>Research</strong> Fellow<br />

CLAY,FIONA, Senior <strong>Research</strong> Officer<br />

INGLESE,MELISSA, Senior <strong>Research</strong> Officer<br />

WHITE,SARA, Senior <strong>Research</strong> Officer<br />

JONES,BENJAMIN, <strong>Research</strong> Officer (April to November)<br />

RICHARDSON,ELSBETH, <strong>Research</strong> Assistant<br />

CYTOKINE BIOLOGY LABORATORY<br />

LIESCHKE,GRAHAM, Assistant Member, Head<br />

LAYTON,JUDY, Senior Investigator<br />

CROWHURST,MEREDITH, PhD Student<br />

HOGAN,BENJAMIN, PhD Student<br />

MCPHEE,DORA, Senior <strong>Research</strong> Officer<br />

VARMA,SONY, Senior <strong>Research</strong> Officer<br />

LONGLAND,RYAN, <strong>Research</strong> Officer<br />

CONNELL,FIONA, <strong>Research</strong> Officer<br />

HAYMAN,JOHN, <strong>Research</strong> Officer (from August)<br />

EPITHELIAL BIOCHEMISTRY LABORATORY<br />

BURGESS,ANTONY, Director, Member*, Head<br />

NICE,EDOUARD, Associate Member<br />

WALKER,FRANCESCA, Associate Investigator<br />

CATIMEL,BRUNO, Assistant Investigator<br />

LACKMANN,MARTIN, <strong>Research</strong> Associate<br />

CLAYTON,ANDREW, Postdoctoral Fellow (from June)<br />

D’ABACO,GIOVANNA, Postdoctoral Fellow (March to August)<br />

FAUX,MAREE, Postdoctoral Fellow<br />

JORISSEN,ROBERT, Postdoctoral Fellow<br />

STENVERS,KAYE, Postdoctoral Fellow<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

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ZHU,HONG-JIAN, Postdoctoral Fellow<br />

LIN,FENG, Visiting Scientist (from May)<br />

KLEIKAMP,SABINE, PhD Student<br />

VEARING,CHRISTOPHER, PhD Student (from September)<br />

WANG,BO, PhD Student<br />

ZAMURS,LAURA, PhD student<br />

KIU,JESSIE-HIU, Honours Student (from August)<br />

PAPAGEORGIOU,IRENE, Honours Student (February to December)<br />

PATANI,NEILL, Visiting Student (from August)<br />

CARTLEDGE,KELLIE, Senior <strong>Research</strong> Officer<br />

CONDRON,MELANIE, Senior <strong>Research</strong> Officer<br />

IARIA,JOSIE, Senior <strong>Research</strong> Officer<br />

KRAVETS,LUCY, Senior <strong>Research</strong> Officer<br />

NERRIE,MAUREEN, Senior <strong>Research</strong> Officer<br />

ORCHARD,SUZANNE, Senior <strong>Research</strong> Officer<br />

ROTHACKER,JULIE, Senior <strong>Research</strong> Officer<br />

WEINSTOCK,JANET, Senior <strong>Research</strong> Officer<br />

ZHANG,HUI-HUA, Senior <strong>Research</strong> Officer<br />

MEEKER,CLARE, <strong>Research</strong> Officer (until February)<br />

GRAS,EMMA, <strong>Research</strong> Officer (from June)<br />

ROSS,JANINE, <strong>Research</strong> Officer<br />

TURSKY,MELINDA, <strong>Research</strong> Officer<br />

WU,FENQIANG, <strong>Research</strong> Officer (from July)<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

JOINT PROTEOMICSLABORATORY (FORMERLY JOINT PROTEIN STRUCTURE LABORATORY)<br />

(A Joint Venture of the <strong>LICR</strong> and the Walter and Eliza Hall <strong>Institute</strong> of Medical <strong>Research</strong>)<br />

SIMPSON,RICHARD, Member, Head<br />

REID,GAVIN, Assistant Member (from April)<br />

JI,HONG, Assistant Investigator<br />

TU,GUO-FEN, Assistant Investigator<br />

LAYTON,MEREDITH, Postdoctoral Fellow<br />

MORITZ,ROBERT, Proteomics Facility Manager<br />

KAPP,EUGENE, Chief <strong>Research</strong> Officer<br />

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CHURCH,NICOLE, <strong>Research</strong> Officer<br />

CONNELLY,LISA, Proteomics Project Service Manager<br />

EDDES,JAMES, Senior <strong>Research</strong> Officer<br />

FRECKLINGTON,DAVID, Senior <strong>Research</strong> Officer<br />

CHURCH,NICOLE, <strong>Research</strong> Officer<br />

HORDEJUK,KATARZYNA, <strong>Research</strong> Officer<br />

TRUBIANO,JASON ANTHONY, <strong>Research</strong> Assistant (from March)<br />

LIM,JUSTIN, PhD Student (from May)<br />

COLE,ADAM, PhD Student (until June)<br />

LIM,KOOK-JIN, Visiting PhD Student (from August)<br />

GOODE,ROBERT, Honours Student<br />

LO,HEO BING, Honours Student (until June)<br />

NUNEZ-IGLESIAS,JUAN, Honours Student (February to November)<br />

HULETT,JOANNE, Summer Student (from November)<br />

O'DAY,JAMES, UROP Student (from July)<br />

ROSEN,JEREMY, Visiting Student (from December)<br />

SOMMER,MICHAEL, Visiting Student (from September)<br />

STRAHM,YVAN, Visiting Student (July to October)<br />

SIGNAL TRANSDUCTION LABORATORY<br />

HIBBS,MARGARET, Assistant Member, Head<br />

HARDNER,KEN, Postdoctoral Fellow<br />

BEAVITT,SARAH -JANE, PhD Student<br />

NAIK,EDWINA, UROP Student<br />

SCHWARZER,WIBKE, Visiting Student (from December)<br />

CASAGRANDA,FRANCA, Chief <strong>Research</strong> Officer<br />

QUILICI,CATHY, Chief <strong>Research</strong> Officer (from January)<br />

KOUNTOURI,NICOLE, Senior <strong>Research</strong> Officer<br />

ZLATIC,KRISTINA, Senior <strong>Research</strong> Officer<br />

COLLINGS,BRIGETTE, <strong>Research</strong> Officer<br />

TURNER,AMANDA, <strong>Research</strong> Officer (from May)<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

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TUMOR SUPPRESSOR LABORATORY<br />

MARUTA,HIROSHI, Associate Member, Head<br />

HE,HONG, <strong>Research</strong> Associate<br />

NHEU,THAO VUONG, Senior <strong>Research</strong> Officer<br />

HIROKAWA,YUMIKO, <strong>Research</strong> Officer<br />

HUYNH,JOHN, Summer Student (from December)<br />

INFORMATION TECHNOLOGY CENTRE (PARKVILLE CAMPUS)<br />

THEGE,GREG, Information Technology Manager<br />

RUNTING,ANDREW, Senior Information Technology Officer<br />

LAY,SAMI, Information Technology Officer<br />

CONTARINO,ALFIO, Information Technology Assistant (September to October)<br />

LABORATORY SUPPORT (PARKVILLE CAMPUS)<br />

DAWS,DOUGLAS, Laboratory Manager<br />

VREMEC,MELINDA, Laboratory Officer<br />

PLENTER,ROBERT, Laboratory Officer<br />

BURGESS,ROSIE, Laboratory Assistant (until November)<br />

COLE,ROBIN, Equipment Officer Maintenance<br />

BRADSHAW,DEREK, Equipment Officer Maintenance<br />

FEAKES,VALERIE, Senior <strong>Research</strong> Officer (Histology)<br />

MEDDINGS,PENELOPE, Media Technician<br />

HELMAN,TRACEY, Animal Facility, Manager<br />

ARNOLD,MELISSA, Technical Officer, Animal Facility<br />

SCOTT BERTRAM, Technical Assistant, Animal Facility (until June)<br />

COHEN,JACQUELINE, Technical Assistant, Animal Facility<br />

DIVISEKERA,UDANA, Technical Assistant, Animal Facility (until June)<br />

DIVISEKERA,UPULIE, Technical Assistant, Animal Facility (until June)<br />

HELMAN,ADAM, Technical Assistant, Animal Facility (from March)<br />

HELMAN,LEONIE, Technical Assistant, Animal Facility<br />

LEE,RUTH, Technical Assistant, Animal Facility (until June)<br />

MCKIERNAN,SARAH, Technical Assistant, Animal Facility (from September)<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

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THORNE,TELEAH, Technical Assistant, Animal Facility<br />

WATSON,PIETA, Technical Assistant, Animal Facility<br />

WEBSTER,GAYE, Technical Assistant, Animal Facility<br />

GINNS,CAROL, Veterinary Assistant, Animal Ethics<br />

DO,CUONG, <strong>Research</strong> Assistant, Aquarium<br />

LONEL,AMBER, Technical Assistant, Washup (until June)<br />

MANEY,WAYNE, Technical Assistant, Washup<br />

TOPHAM,HELEN, Technical Assistant, Washup<br />

ZAMMIT,CARMEN, Technical Assistant, Washup<br />

GIRKE,THOMAS, Information Officer/Librarian<br />

MATHEWS,PETER, Information Officer/Librarian (from May)<br />

MELBOURNE BRANCH CLINICAL PROGRAM (AUSTIN CAMPUS)<br />

TUMOUR TARGETTING PROGRAM*<br />

CLINICAL PROGRAM (PARKVILLE, AND THE WESTERN HOSPITALS)*<br />

(A joint venture of the <strong>LICR</strong>, The Royal Melbourne Hospital, and The Western Hospital)<br />

GIBBS,PETER, Senior Clinical <strong>Research</strong> Fellow<br />

TUMOUR TARGETING LABORATORY*<br />

SCOTT,ANDREW, Associate Member, Head, Associate Director-Clinical<br />

LEE, F.T., Associate Investigator<br />

JOHNS,TERRANCE, Assistant Investigator<br />

LUI,ZHANQI, Assistant Investigator<br />

RAYZMAN,VERONIKA, Postdoctoral Fellow<br />

PANOUSIS,CON, Postdoctoral Fellow (until February)<br />

SPIRKOSKA,VIOLETTA, Chief <strong>Research</strong> Officer (from May)<br />

BUTLER,CLAIRE, Senior <strong>Research</strong> Officer (until March)<br />

HALL,CATHRINE, Senior <strong>Research</strong> Officer<br />

MOUNTAIN,ANGELA, Senior <strong>Research</strong> Officer<br />

RIGOPOULOS,ANGELA, Senior <strong>Research</strong> Officer<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

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VITALI,ANGELA, Senior <strong>Research</strong> Officer<br />

TAVERNESI,MERI, <strong>Research</strong> Officer<br />

WANG,LIPING, <strong>Research</strong> Officer (until February)<br />

LEGGE,SUE, PhD Student<br />

LUWOR,RODNEY, PhD Student (until December)<br />

MARINIC,ANNA, PhD Student<br />

PERERA,RUSHIKA, PhD Student<br />

TAHTIS,KIKI, PhD Student (until January)<br />

HARRIS,MARION, MD Stdent (until January)<br />

KELLY,MARCUS, Honours Student (February to December)<br />

POTASZ,NICOLE, Clinical <strong>Research</strong> Fellow (from February)<br />

SCHLEYER,PAUL, Computer Programmer<br />

BIOLOGICAL PRODUCTION FACILITY*<br />

RUBIRA,MICHAEL, Biological Production Facility Manager<br />

GELEICK DETLEF, Associate Investigator (until August)<br />

MURPHY,ROGER, Associate Investigator<br />

CARTWRIGHT,GLEN, Chief <strong>Research</strong> Officer<br />

JOIS,JENNIFER, Senior <strong>Research</strong> Officer<br />

KYPRIDIS,ANNA, Senior <strong>Research</strong> Officer<br />

WIRTH,VERONIKA, Senior <strong>Research</strong> Officer<br />

TAY,MAUREEN, Quality Compliance Officer (until October)<br />

CANCER CLINICAL TRIALS CENTRE*<br />

HOPKINS,WENDIE, Senior <strong>Research</strong> Nurse<br />

GIBBS,SHAREN, <strong>Research</strong> Nurse<br />

GOLDIE,HEATHER, <strong>Research</strong> Nurse<br />

LEE,SUNANTA, Data Systems Developer<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

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JOINT AUSTIN LUDWIG MEDICAL ONCOLOGY UNIT*<br />

CEBON,JONATHAN, Associate Member, Head<br />

DAVIS,IAN, Assistant Member<br />

MITCHELL,PAUL, Director Cancer Services, <strong>LICR</strong> Associate<br />

PARENTE,PHILLIP, Clinical <strong>Research</strong> Fellow<br />

TEBBUTT,NIALL, Clinical Postdoctoral Fellow<br />

RUDOPH,ANNE, Personal Assistant<br />

Cebon, Michael, Data Entry Operator (until June)<br />

CANCER VACCINE PROGRAM*<br />

CANCER VACCINE LABORATORY*<br />

CEBON,JONATHAN, Associate Member, Head<br />

MARASKOVSKY,EUGENE, Assistant Member, Head, <strong>LICR</strong> Associate (until January)<br />

SCHNURR,MAX, Postdoctoral Fellow (from March)<br />

VAUGHAN,HILARY, Postdoctoral Fellow (until April)<br />

SHIN,AMANDA, <strong>Research</strong> Officer<br />

ST CLAIR,FIONA, <strong>Research</strong> Officer (until April)<br />

RYAN,MARY, <strong>Research</strong> Assistant (August)<br />

JEFFORD,MICHAEL, PhD Student (until August)<br />

JUNAIDI,HAIROL, PhD Student (from September)<br />

CLINICAL DENDRITIC CELL FACILITY*<br />

DAVIS,IAN, Assistant Member, Head<br />

MORRIS,LEONE, Senior <strong>Research</strong> Officer<br />

QUIRK,JULIET, Senior <strong>Research</strong> Officer<br />

STANLEY,MAUREEN, Senior <strong>Research</strong> Officer (until August)<br />

ADELAIDE,CHRISTOPHER, <strong>Research</strong> Officer (from December)<br />

TAVARNESI,MERI, <strong>Research</strong> Officer (until January)<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

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T-CELL LABORATORY*<br />

CHEN,WEISAN, Assistant Member, Head<br />

QIYUAN,CHEN, <strong>Research</strong> Associate<br />

JACKSON,HEATHER, Manager-Clinical Trials Monitoring<br />

DIMOPOULOS,NEKTARIA, Senior <strong>Research</strong> Officer (from June)<br />

TAI,TSIN YEE, Senior <strong>Research</strong> Officer<br />

MASTERMAN,KELLY-ANNE, <strong>Research</strong> Officer<br />

RIZKALLA,MARK, <strong>Research</strong> Officer (until February)<br />

TOY,TRACEY, <strong>Research</strong> Officer<br />

ZHU,HE-CHENG, Visiting Scientist (until December)<br />

BETHUNE,BARBARA, Technician<br />

LABORATORY SERVICES (AUSTIN CAMPUS)<br />

FOX,SIMON, Assistant Laboratory Manager<br />

LEONARD,JEANETTE, Technician, Animal Facility (September to December)<br />

KOUL,USHA, Technical Assistant, Washup<br />

WAPSHOTT,YVONNE, Technical Assistant, Washup (from August)<br />

ADMINISTRATION AND SECRETARIAT (PARKVILLE CAMPUS)<br />

TSAI,KIM, Branch Administrator<br />

FLETCHER,CHELSEA, Assistant Accountant<br />

LANCUBA,SOFIA, Accounts Officer<br />

FORMOSA,NICOLE, Payroll Officer (until January)<br />

GREEN,CASSEY, Payroll Officer/Human Resources Officer (from April)<br />

CLARK,PAULINE, Human Resources Manager<br />

MASKELL,CLAIRE, Human Resources Manager (until February)<br />

GREENWOOD,JANE, Human Resources Officer (until February)<br />

THUMWOOD,CASSANDRA, Scientific Administrator<br />

JONES,PAMELA, Personal Assistant to Branch Director<br />

PAKIN,SIMONE, Personal Assistant to Head of Joint Proteomics Laboratory<br />

O’GORMAN,JANITA, Receptionist (until January)<br />

STEVENS,WENDY, Receptionist (from August)<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

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MAKRIS,POLY, Administration Assistant/Receptionist (from August)<br />

NEAR,CHRISTINE, Ethics Officer<br />

ADMINISTRATION AND SECRETARIAT (AUSTIN CAMPUS)<br />

GROVES,VALDA, Business Manager (until February)<br />

CATANZARITI,NADIA, Financial Accountant (from May)<br />

ARNSTRONG,ADELINA, Administrative Officer (until March)<br />

HUMPHREY,SUSAN, Accounts Officer<br />

LAZAROVSKA,VANESSA, Receptionist/Secretary<br />

COX,SHAYNE, PC/Network Support Officer<br />

SMYTH,FIONA, Scientific Administrative Officer, Clinical Program*<br />

LEWIS,KATH, Personal Assistant to Associate Director, Clinical Program*<br />

<strong>LICR</strong> CLINICAL ASSOCIATES*<br />

* Clinical Trials Program participant<br />

MELBOURNE<br />

ROSENTHAL,MARK, Centre for Developmental Cancer Therapeutics<br />

WARING,PAUL, Peter MacCallum Cancer Centre<br />

WHEATLEY,JENNIFER, Surgeon, Austin Hospital<br />

MACGREGOR,DUNCAN, Department of Pathology, Austin Hospital<br />

ROWE,CHRIS, Department Nuclear Medicine and Centre for PET, Austin Hospital<br />

HANNAH,ANTHONY, Department Nuclear Medicine and Centre for PET, Austin Hospital<br />

BERLANGIERI,SAM, Department Nuclear Medicine and Centre for PET, Austin Hospital<br />

TOCHON-DANGUY,HENRI, Department Nuclear Medicine and Centre for PET, Austin Hospital<br />

O’KEEFE,GRAEME, Department Nuclear Medicine and Centre for PET, Austin Hospital<br />

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BRANCH DIRECTOR’S REPORT<br />

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MELBOURNE<br />

The Melbourne Branch has had a busy year in both the laboratory and clinic. Our research has combined<br />

knowledge of growth factor biology and immunology to improve our understanding of tumor<br />

development, progression, and treatment. All of our laboratories have made fascinating discoveries<br />

including contributions to: lymphangiogenesis; lyn kinase signaling; structures of the IL-6, ephA3 and<br />

epidermal growth factor receptor (EGFR) family structures; aberrant EGFR signaling; TGF-� knockout<br />

biology; zebrafish myelopoiesis and intestinal cell biology; gp130 and gastric mucosa adenomas; and<br />

dendritic cell biology. When these discoveries are combined with our cancer trials using antibodies for<br />

colon and renal cancer, melanoma and the NY-ESO-1 protein vaccine trial in melanoma patients, it is<br />

clear that we have a successful discovery program contributing to the improvement of cancer therapy.<br />

This year also saw some landmark changes at the Branch, with the departure of the Branch’s Associate<br />

Director Dr. Ashley Dunn. Dr. Dunn has provided leadership, scientific excellence, and many world firsts<br />

in the area of hemopoiesis for the past twenty years. Just a few of the highlights from Dr. Dunn’s<br />

laboratory are:<br />

� the first to clone granulocyte-macrophage colony stimulating factor (GM-CSF), which provided<br />

the clinical opportunity (headed at that time by Dr. George Morstyn) to set up the first clinical<br />

trials for GM-CSF and G-CSF to improve cancer treatment;<br />

� the discovery of the cytokine induced release of haemopoietic stem cells and a revolution in stem<br />

cell transplantation for haemopoietic support (resulting in part from the clinical trials with GM-<br />

CSF and G-CSF;<br />

� the production of some of the first transgenic mice in Australia, and the generous sharing of the<br />

art of this technology with many colleagues.<br />

� the discovery of the link between leukemia inhibitory factor (LIF) and embryonal stem cells;<br />

� the introduction of gene knockout technology as an important contribution to both Australian<br />

research and myelopoiesis.<br />

In short, we have been immensely proud of Dr. Dunn’s achievements for both the <strong>LICR</strong> and world<br />

science overall. His election to the Australia Academy of Sciences in 1997 was a most appropriate<br />

accolade to his innovations and discoveries in biology. On a personal note, I have been fortunate to have<br />

Dr. Dunn’s collegial support as our Associate Director since he joined the Branch. He has been a calming<br />

and positive force in all of our successes and challenges. When Dr. Dunn made the decision to close his<br />

own laboratory at the end of 2001, he continued on throughout <strong>2002</strong> in his mentoring capacity and<br />

remains associated with the Branch by his continued involvement in the Neutrophil Promoting Activity<br />

project and chairing our Branch Appointments and Promotions Committee. Dr. Dunn continues to<br />

promote science in Australia by participating in both local and national Committees, both activities<br />

benefit from his immense experience and ability.<br />

A. W. Burgess


RESEARCH REPORT<br />

PARKVILLE CAMPUS<br />

ANGIOGENESIS LABORATORY<br />

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Since the discovery of another member of the vascular endothelial growth factor family (VEGF-D) in the<br />

Angiogenesis Laboratory headed by Drs. Marc Achen and Steven Stacker, there has been a major revival<br />

of research in the area of lymphangiogenesis. The group’s research in this area has led to the development<br />

of a model to study the growth and development of tumor lymph vessels. It is clear that VEGF-D is<br />

produced as an inactive pre-cursor, which upon cleavage at the N- and C-termini, is activated to stimulate<br />

lymphangiogenesis and angiogenesis. Indeed tumors that secrete the activated form of VEGF-D grow<br />

more quickly, and are able to spread more readily from the primary tumor site. Results from the<br />

Angiogenesis Laboratory have highlighted the importance of lymphatic vessels in tumor metastasis, and<br />

the study of the relative roles of the lymphatic and blood vessels in the growth and spread of tumor cells<br />

is a very active research area in the group. If it were possible to inhibit the processing of the VEGF-D precursor,<br />

it might be possible to inhibit the development of lymphatic vessels associated with tumors and, as<br />

a consequence, reduce the spread of the tumor to other sites. The group has identified some of the<br />

enzymes involved in the processing of the VEGF-D pre-cursor and is now searching for inhibitors of<br />

these enzymes. Angiogenesis is also influenced by many other receptor kinases, in particular the Eph<br />

receptors, which have been associated with angiogenesis. The Angiogenesis Laboratory is also continuing<br />

its interest in the RYK “orphan receptor” and has discovered an interaction with EphB3 and EphB3<br />

receptors. The biological significance of this interaction is being evaluated.<br />

COLON MOLECULAR AND CELL BIOLOGY LABORATORY<br />

Several projects at the Branch are aimed at improving our understanding of the signaling systems<br />

regulating the production of intestinal cells. The Colon Molecular and Cell Biology Laboratory, under the<br />

direction of Drs. Matthias Ernst and Joan Heath, is employing the zebrafish system to analyze several<br />

mutants exhibiting gut phenotypes. Positional cloning is well underway to allow identification of the<br />

genes involved, and a genetic screen specifically designed to identify additional gut, liver and pancreatic<br />

mutants is continuing in association with Dr. Didier Stainier (University of California San Francisco,<br />

USA). An in vitro electroporation system has been developed, which allows gene function analyses<br />

without the need to generate transgenic animals. Through collaboration, co-administration of tea and<br />

sulindac to Apc (min) mice, or the A33-�N-�-catenin mouse strain, generated by the group to express an<br />

oncogenic version of �-catenin, was found to reduce histological changes associated with the progression<br />

of colon cancer.<br />

The laboratory has also produced several strains of mice expressing either tissue-specific oncogenes or<br />

“knock-ins” of mutant forms of the gp130 receptor, which is involved in cytokine signaling. Interestingly,<br />

when the interaction site between gp130 and the src-homology tyrosine phosphatase SHP2 is ablated, the<br />

mice develop hyperplastic gastric adenomas within three months. An analysis of these gp130 Y757F mice<br />

has pointed to a critical contribution of gp130-dependent activation of the ras/Erk-pathway in suppressing<br />

gastric hyperplasia. The gp130 receptor also activates the JAK/STAT signaling system. When the<br />

interaction site between gp130 and the STAT1/3 system is deleted, trefoil production is reduced and the<br />

mice fail to repair wounds to the gastro-intestinal mucosa. Clearly, gp130 is involved in many signaling<br />

systems. Results from the work undertaken from the group indicate that the ligand dependent (IL-6 or IL-<br />

11) activation of the SHP2�MAPK pathway is important for cell production, whereas the activation of


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the STAT1/3 system controls the expression of mucosal differentiation and repair. Finally, the src family<br />

kinase, hck, has been known to be involved in signaling from the gp130 receptor, but its precise role in<br />

other cellular systems has been difficult to determine. By generating a gain-of-function hck mouse, it has<br />

been revealed that hck is involved in regulating pulmonary eosinophilia and in stimulating innate<br />

immunity.<br />

CYTOKINE BIOLOGY LABORATORY<br />

Dr. Graham Lieschke heads the Cytokine Biology Laboratory, and he and his colleagues seek to<br />

understand the regulation of white blood cell (leukocyte) development and how this is genetically<br />

dysregulated in leukemia. Many of the genes critical to the developing myeloid hematopoietic system<br />

have also been implicated in the genesis of leukemia, and the group is using its knowledge to try to<br />

identify new targets and mechanisms that interfere with the development of leukemia. The major thrust of<br />

this effort has been to undertake a genetic screen in zebrafish to isolate new mutants with myeloid failure.<br />

To date, the gynogenetic haploid screening protocol using three markers has screened 260 genomes<br />

resulting in 11 putative mutants. Of particular interest, the ES11.36 mutant has been shown to have a<br />

hemapoietic failure and the ES12.1 mutant exhibits a selective myeloid defect with preserved erythroid<br />

development. The challenge now is to identify the genes responsible for these mutants, and three have so<br />

far been positioned on the zebrafish genetic map. In addition, the laboratory is using a transgenic strain of<br />

zebrafish in which the spi1 promotor drives the expression of green fluorescent protein restricted to the<br />

myeloid compartment. This will allow tracking of early myeloid cells in genetic crosses with mutants.<br />

Forward genetic techniques in zebrafish are also being exploited, with over-expression achieved by<br />

mRNA micro-injection or transgene expression, and morpholino antisense oligonucliotides being used to<br />

knock-down the expression specific genes of interest. Finally, a long-term mouse study has demonstrated<br />

that G-CSF is involved in a transgene-driven murine leukemia model and raises novel possibilities for<br />

anti-leukemic therapy.<br />

EPITHELIAL BIOCHEMISTRY LABORATORY<br />

One aspect of the Epithelial Biochemistry Laboratory studies, under the direction of Dr. Antony Burgess,<br />

has been the structure/function studies of signaling proteins. In <strong>2002</strong> successful collaborations produced<br />

3D-structures for: the IL-6 receptor � chain; a fragment of the epidermal growth factor receptor (EGFR)<br />

extracellular domain bound to one of its ligands, TGF-�� and EphB2/ephrinB2 association. The first two<br />

projects involved long-standing collaborations with the Commonwealth Scientific and Industrial <strong>Research</strong><br />

Organisation (CSIRO) and the Biomolecular <strong>Research</strong> <strong>Institute</strong> (Melbourne, Australia) and more recently<br />

the Walter and Eliza Hall <strong>Institute</strong> of Medical <strong>Research</strong> (WEHI, Melbourne, Australia). The EphB2ephrinB2<br />

complex was solved as a result of a vigorous collaboration with the Memorial Sloan-Kettering<br />

Cancer Center (New York, USA). Each of these three signaling systems has been implicated in specific<br />

cancers and the structural data that is now available has opened up new opportunities for identifying<br />

antagonists with potential anti-cancer activity.<br />

Several years ago a truncated form of the soluble EGFR called EGFR501 was discovered through a<br />

collaboration (the Cooperative <strong>Research</strong> Center (CRC) for Cellular Growth Factors) with colleagues from<br />

the Division of Health and Nutritional Sciences of CSIRO and the WEHI. Since this form of the receptor<br />

can act as a ligand trap, its potential as an anti-cancer therapeutic is under investigation. The mechanism<br />

of binding of EGF ligands to the receptor is being analyzed, and a detailed understanding of the activation<br />

determinants has developed as a result. The group believes that it is feasible to develop ligand analogues<br />

that will compete successfully with the natural ligands, but that will prevent receptor activation. Preclinical<br />

studies with the EGFR kinase inhibitor AG1478 continue to progress through the Melbourne<br />

Branch in a joint study between the Epithelial Biochemistry, and Tumour Targeting Laboratories. A


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successful formulation, which has an acceptable acute toxicology profile, is due to undergo final chronic<br />

toxicology testing in rats shortly. Opportunities for combining signaling antagonists are arising from the<br />

group’s experimental work, with structure-function studies with laminin-10 aimed at testing therapeutic<br />

schemes in which both the EGFR and integrin pathways are blocked. Similar experiments in the TGF-�<br />

system have analyzed the effect of knocking out TGFR�III and its role in liver development. A soluble<br />

form of TGF�RIII has been reported in the literature to act as the ligand trap and, since TGF-� has been<br />

implicated in hepatocellular carcinoma, targeting this biological signaling system enables an evalutaion of<br />

the utility of TGF�RIII-related molecules as the basis for a therapy approach.<br />

The Epithelial Biochemistry Laboratory’s interest in tumor suppressor systems has increased in the last<br />

few years. Recent work from the Epithelial Biochemistry Laboratory has revealed that oncogenic ras<br />

down-regulates PTEN, which has been found to be down regulated in many colon cancers, and that this<br />

down regulation is, in part, responsible for the tumor phenotype. When the EGFR system is activated,<br />

PTEN is also down regulated; the group is currently investigating whether EGFR antagonists and/or<br />

inhibitors can induce the re-expression of PTEN in colon cancer cells. Apart from PTEN, there is an<br />

active program on determining the function of the apc tumor suppressor protein in colon cancer. The apc<br />

protein is defective or absent in more than 80% of advanced colon cancers. Expression systems for apc,<br />

and reagents, such as mAbs and peptides, suitable for purifying both native and recombinant forms of the<br />

molecule, have been developed. Using an apc-defective colonic carcinoma cell line, SW480, scientists in<br />

the laboratory have developed an assay for functional apc. This assay allows the analysis of the effects of<br />

full-length apc on �-catenin and E-cadherin distribution, as well as effects on cell adhesion, motility, and<br />

proliferation. Members of the group are convinced that apc has a major regulatory role in cell adhesion<br />

and that it is this activity that is critical for much of its tumor suppressor action. The sub-cellular<br />

distribution of apc has been the subject of much debate. Analyses of the localization of green-fluorescent<br />

protein (GFP)-labelled apc in SW480 cells indicates an unusual microtubule associated distribution (with<br />

clear accumulation of some apc-GFP in focal adhesion pads). The dynamics of apc interaction with the<br />

microtubule system and its role in protein transport are the areas of current investigations within the<br />

group.<br />

JOINT PROTEOMICSLABORATORY<br />

The Joint ProteomicS Laboratory (a joint initiative with the WEHI) moved into its newly renovated<br />

laboratories during the year, and is headed by Dr. Richard Simpson. The facility has dedicated areas for<br />

mass spectrometry, HPLC, electrophoretic gel analysis and bio-informatics. The <strong>LICR</strong> is most grateful to<br />

the Royal Melbourne Hospital, the Victorian State Government, and Atlantic Philanthropy for their<br />

enthusiastic support of this project, and the new premises helps the group to continue at the forefront of<br />

proteomics research. The structural characterization of IL-6 and its receptor provides an opportunity for<br />

identifying ligand antagonists of the IL-6/IL-6R�/gp130 system and continues to be a major project<br />

interest within the group. An IL-6 dimeric antagonist has been characterized, and an in silico screening<br />

project to identify drugs that inhibit IL-6 signaling is underway. As well as projects in the areas of IL-6<br />

receptor, colon cancer protein profiling, and the development of proteomics, the JPSL collaborates with<br />

many biologists at both <strong>LICR</strong> and WEHI to identify proteins in cellular scaffolds and complexes. The<br />

identification of a link between the suppressor of cytokine signaling (SOCS) regulatory system and the<br />

insulin receptor substrate-4 has created considerable interest. The laboratory has continued to identify<br />

proteins regulating several of the feedback loops in the apoptosis regulatory pathways. The progress in<br />

throughput from peptide and protein mass spectrometry is creating new challenges for the rapid<br />

identification of proteins in complex structures. Peptide identification on the basis of single masses, no<br />

matter how accurate, is fraught with danger, but the group has been developing bioinformatic tools such<br />

as CHOMPER, which will allow rapid validation of tandem mass spectrometry search results associated<br />

with high-throughput proteomic strategies. In conjunction with a DNA sequencing scientist at the WEHI


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and the Australian Genome <strong>Research</strong> Facility, a new mass spectrometric detection system has been<br />

developed to sequence small oligonucleotide fragments. This techniques should prove invaluable for both<br />

nucleotide sequencing and eventually the complete analysis of all components of translational or<br />

transcription complexes.<br />

SIGNAL TRANSDUCTION LABORATORY<br />

The src family of kinases is associated with many tumor types, such as leukemia, colon cancer, and<br />

sarcomas. Dr. Margaret Hibbs leads the activities of the Signal Transduction Laboratory, the major focus<br />

of which is the lyn kinase, a member of the src family of intracellular tyrosine kinases. In particular, both<br />

lyn knock-out and lyn gain-of-function mice have been generated by the group, and have enabled a<br />

detailed dissection of this kinase’s complex biological and biochemical activities. Although gain-offunction<br />

lyn mutant mice do not have an increased propensity for tumor formation, B lymphocyte<br />

development is impaired in these mice and they eventually go on to develop a profound auto-immunity.<br />

The lyn knockout mice also exhibit a B cell defect and develop a B lymphoid hyperplasia, eventually<br />

leading on to macrophage-like tumors in several organs. Clearly, the lyn kinase regulates a number of<br />

signaling pathways associated with B cell development and myelopoiesis. Lyn has an indispensable role<br />

in regulating inhibitor pathways which normally restrain and direct lymphoid cell production and the<br />

group is continuing to analyze the intricacies of lyn kinase signaling.<br />

TUMOUR SUPPRESSOR LABORATORY<br />

The ras gene is the most frequent target for oncogenic mutations associated with human cancers, and is<br />

the prime research area for Dr. Hiroshi Maruta and his Tumour Suppressor Laboratory. Activation of the<br />

ras oncogene leads to changes in several signaling pathways, including those that alter cell proliferation,<br />

cell survival, and motility. It has been difficult to identify changes induced by oncogenic ras that are<br />

essential for its transforming action, but the group found an excellent correlation between the ability of<br />

oncogenic ras to activate the molecule PAK-1, and its ability to transform cells. Direct inhibitors of<br />

oncogenic ras have been difficult to identify, but it is possible to inhibit PAK1 either directly, with<br />

staurosporine analogues, or indirectly, using tyrosine kinase inhibitors, which act on the enzymes in the<br />

pathway between oncogenic ras and PAK1. Derivatives of staurosporine, such as K252a, are potent<br />

inhibitors of PAK1, but lack specificity and inhibit many other kinases. In conjunction with Yale<br />

University (New Haven, USA), the group has prepared non-symmetric analogues of K252a that are<br />

predicted to be more specific inhibitors of PAK1. These analogues could be potential compounds for<br />

treating both ras-cancers and neurofibromatosis. The Tumour Suppressor Laboratory has been<br />

collaborating with the Hebrew University (Jerusalem, Israel) to test the anti-tumour activity of a series of<br />

tyrosine kinase inhibitors (tyrphostins). One of these inhibitors, AG879, prevents oncogenic ras from<br />

activating PAK-1. Although the target for AG879 is unknown, the group’s investigations indicate that it is<br />

a potent inhibitor of an enzyme between the kinases ETK and PAK1. Specific antagonist strategies that<br />

lead to PAK1 inhibition are being pursued, and will be tested for efficacy against ras associated tumors in<br />

mouse xenograft experiments.


AUSTIN HOSPITAL CAMPUS<br />

CLINICAL PROGRAM OVERVIEW<br />

* Clinical Trials Program participant<br />

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The Clinical Program of the Melbourne Branch is part of the Clinical Trials Program of the <strong>LICR</strong>, and<br />

consists of the Tumour Targeting Program and the Cancer Vaccine Program. The aims of the activities<br />

are to extend laboratory discoveries from <strong>LICR</strong> research into clinical trials in cancer patients in the areas<br />

of immunology and growth factor signaling. The laboratories and clinical facilities are principally located<br />

at the Austin Hospital (Melbourne, Australia) and the research programs undertaken there work closely<br />

with a number of Austin Hospital’s Departments, namely Oncology, Anatomical Pathology, and Nuclear<br />

Medicine and Positron Emission Tomography (PET). There is close integration of scientists, clinicians,<br />

and students within multiple clinical disciplines including Oncology, Surgery, Nuclear Medicine and<br />

Pathology, and also with the laboratories of the Melbourne Branch at the Parkville Campus. In <strong>2002</strong>, Dr.<br />

Andrew Scott was appointed as Associate Director - Clinical Program. Through clinical trials and the<br />

establishment of a Tissue Bank, the Clinical Program has, in the last 12 months, also extended its<br />

activities at the Royal Melbourne, and the Western Hospitals (Melbourne, Australia) with the help of Dr.<br />

Peter Gibbs.<br />

The <strong>LICR</strong> and the Austin Hospital have continued their association through the Joint Austin <strong>Ludwig</strong><br />

Oncology Unit, which is headed by Dr. Jonathan Cebon. This unit provides medical oncology services<br />

and undertakes clinical research into more effective methods of treating cancer. As one of the busiest<br />

Medical Oncology units in Australia, it has an active commitment to training and teaching, and in recent<br />

years has ranked as the State’s most popular site for trainees wishing to undertake specialist training in<br />

Cancer Medicine.<br />

TUMOUR TARGETING PROGRAM*<br />

The Tumour Targeting Program is directed by Dr. Andrew Scott and has several humanized and radioiodinated<br />

monoclonal antibodies (mAbs) in laboratory, pre-clinical, and clinical development. The<br />

program involves several closely integrated groups (the Tumour Targeting Laboratory, the Biological<br />

Production Facility (BPF), and the <strong>LICR</strong> Clinical Trials Program) and works collaboratively with the<br />

Departments of Nuclear Medicine, and Anatomical Pathology, and the Centre for PET.<br />

TUMOUR TARGETING LABORATORY*<br />

The Tumour Targeting Laboratory focuses on the development and preclinical characterization of<br />

strategies that target tumors with recombinant antibody-derived molecules and signaling inhibitors, for<br />

diagnostic and therapeutic applications. The laboratory is developing two epidermal growth factor<br />

receptor (EGFR) targeted therapeutics: the monoclonal antibody (mAb) 806, which binds to the<br />

extracellular domain of the receptor; and the small molecule inhibitor of the receptor’s enzymatic activity,<br />

AG1478. The mAb 806 is of particular interest to the <strong>LICR</strong>, and is being developed in conjunction with<br />

colleagues in the Epithelial Biochemistry Laboratory at the Melbourne Branch, and groups at the New<br />

York and San Diego Branches. The mAb 806 is unique as it recognizes both a tumor specific variant of<br />

the EGFR (de2-7 EGFR) and a subset of receptors expressed by cells which over-express the wild type<br />

EGFR, but mAb 806 does not bind to normal tissue expressing the non-amplified wild type receptor. To<br />

further understand the EGFR system, the group has continued studying the mechanisms important for the<br />

anti-tumor effect of the EGFR signaling inhibitors through analyses of de2-7 EGFR activation, and<br />

downstream signaling pathways. Furthermore, the group has shown for the first time that the de2-7 EGFR


* Clinical Trials Program participant<br />

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can dimerize and activate the wt EGFR in a ligand independent fashion. A chimeric 806 antibody has<br />

been generated and is the subject of preclinical development and characterization.<br />

Pre-clinical studies in animal xenograft models have explored dual combinations of immunotherapy,<br />

radioimmunotherapy, signaling inhibitors, or conventional cytotoxic therapies using sub-therapeutic dose<br />

regimens. Studies conducted by the group have shown enhanced anti-tumor activity of: 90 Y-CHX-A"-<br />

DTPA-hu3S193 when combined with Taxol in a model of breast cancer; synergistic anti-tumor activity<br />

when mAb 806 is combined with other EGFR therapeutics or more conventional cytoxics. In addition, the<br />

role of complement activation in the anti-tumor activity of cancer specific antibodies is being analyzed.<br />

Using a novel nude rat model, complement activating antibodies have been shown to induce significant<br />

tumor regression if the cell surface complement inhibiting molecules (CD59) are blocked. The<br />

mechanistic effects of radiation delivery and the role of cellular trafficking of antibodies are also being<br />

explored. Recent work by the group has evaluated the murine anti-CD19 antibody, CLB-CD19, in an<br />

animal model of acute lymphocytic leukemia, and demonstrated tumor uptake, in vivo internalization, and<br />

suppressive effects on circulating leukemia cells. These observations indicate a therapeutic potential of<br />

anti-CD19 mAbs suited to the tumor delivery of radioisotopes, including �-emitters. Investigations into<br />

the mechanisms of antibody-antigen binding, and intracellular movement through cancer cells have also<br />

provided insight into receptor function, and suitable radioisotopes for conjugating to tumor targeting<br />

monoclonal antibodies. Finally, several anti-idiotype antibodies suitable for monitoring immune<br />

responses to, and pharmacokinetics of, clinically administered mAbs have been developed within the<br />

program. The generation of novel mAbs targeted to antigens of interest to the <strong>LICR</strong> also continues. The in<br />

vitro evaluation of the binding characteristics and biological activity of a chimeric anti-human GM-CSF<br />

antibody has been completed, and research into stromal targets for antibody based therapeutics is<br />

ongoing.<br />

CLINICAL TRIALS*<br />

As part of the <strong>LICR</strong>’s Clinical Trials Program, Phase I trials in colon, breast, lung, and renal cancers are<br />

being performed to exame the safety, biodistribution and pharmacokinetics of recombinant antibodies.<br />

Trials of a stromal targeting mAb, Sibrotuzumab, in patients with colon and lung cancer have now been<br />

completed. A Phase I trial of 131 I-huA33 in metastatic colorectal cancer patients has also been concluded,<br />

and demonstrated high uptake in colorectal metastases, which has led to the planning of a trial combining<br />

131 I-huA33 with capecitabine. A first-in-man Phase I trial of the Le y targeting mAb, hu3S193, has<br />

continued, and shows impressive results with targeting and immune function. Based on preclinical studies<br />

investigating the enhancement of an antibody’s immune effector function, a pilot study of cG250 and low<br />

doses of subcutaneous IL-2 in patients with advanced renal cell carcinoma has commenced. The clinical<br />

laboratory supports the Program’s trials through the preparation of radiolabelled reagents for<br />

administration to patients, and the analyses of sera samples for pharmacokinetic, immune effector<br />

activity, and immunological response data.<br />

BIOLOGICAL PRODUCTION FACILITY*<br />

The <strong>LICR</strong> is unique in having established a Biological Production Facility (BPF) to manufacture study<br />

drugs, under good manufacturing practice (GMP) conditions, for <strong>LICR</strong>-sponsored cancer vaccine and<br />

mAb clinical trials conducted in Melbourne and internationally. During the last 12 months, formal<br />

certification for GMP was received by the BPF from the Australian Therapeutic Goods Administration. A<br />

vaccine for an early phase clinical trial with the HER-2 protein, to be conducted by Professor Shiku at the<br />

Mie University School of Medicine (Mie, Japan), has been successfully prepared and tested. Further<br />

progress was also achieved with the mAb 806, including media optimisation, a bioreactor production run,


* Clinical Trials Program participant<br />

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MELBOURNE<br />

evaluation of alternate fermenter-type production systems, purification method development, and<br />

establishment of production cell banks, with GMP production planned for 2003. A range of peptides for<br />

clinical trial use, including the NY-ESO-1-DP4 peptide, were formulated and tested during <strong>2002</strong> for the<br />

<strong>LICR</strong>’s Cancer Vaccine Program. In addition, a batch of the tyrosine kinase inhibitor, AG1478, was<br />

formulated and vialed, satisfying all analytical and quality control testing.<br />

CENTRE FOR POSITRON EMISSION TOMOGRAPHY<br />

<strong>Research</strong> in tumor metabolism and molecular imaging continued in the Centre for PET in association with<br />

the Tumour Targeting Program. Clinical studies in glioma, melanoma, lymphoma, renal, lung, and colon<br />

cancer patients were conducted to demonstrate the impact on patient management of 18 F-FDG PET.<br />

<strong>Research</strong> studies in tumor hypoxia imaging in glioma, lung, and renal cancer patients demonstrated<br />

unique data on this important component of tumor metabolism. Radiochemistry research was advanced<br />

with the application of a novel method of 11 C labeling signaling inhibitor compounds.<br />

TISSUE BANK /IMMUNOHISTOCHEMISTRY*<br />

Investigations into cell surface antigens expressed on human tumours, including FAP� and EphA3R, are<br />

continuing in collaboration with Dr. Duncan MacGregor, from the Department of Anatomical Pathology,<br />

Austin Hospital. The group is screening patient archival tumour tissue for the expression of specific<br />

antigens, analysing clinical trial patient biopsy tissue for immunological responses, and evaluating the<br />

relationship between hypoxia, angiogenesis, cell proliferation, and glucose transport in non small cell<br />

lung, brain, and renal cell cancers. The mab 806 is also under investigation in a number of tumour types.<br />

CANCER VACCINE PROGRAM*<br />

The Cancer Vaccine Program is coordinated by Dr. Jonathan Cebon and is comprised of a clinical trials<br />

unit, and three laboratories, the Cancer Vaccine Laboratory, the Clinical Dendritic Cell Facility, and the T<br />

Cell Laboratory. These groups work together to develop a deep understanding of cancer and immunity,<br />

and to apply these findings to clinical trial design and analysis. The trials are conducted at the Austin<br />

Hospital in collaboration with colleagues in the Melbourne-based Centre for Developmental Cancer<br />

Therapeutics (CDCT), a cooperative clinical trials group which focuses on the clinical evaluation of new<br />

cancer treatments. The CDCT also includes the Peter MacCallum Cancer Centre, WEHI, Austin Health,<br />

and the Royal Melbourne, and Western Hospitals. Successful vaccine-based immunotherapy of cancer<br />

requires the induction of an appropriate immune response, for immune effector cells to localize to cancer<br />

sites, and for cancer cells to be susceptible to immune–mediated killing. The laboratories and clinical<br />

group undertake research into each of these facets of cancer immunology and the anti-cancer immune<br />

response. For example, the groups have evaluated the immune responses, in both the laboratory and the<br />

clinic, of 46 patients vaccinated with the CT antigen NY-ESO-1, and the adjuvant ISCOM®, in a clinical<br />

trial performed in collaboration with the Commonwealth Serum Laboratories (CSL) Limited and the<br />

CDCT.


CANCER VACCINE LABORATORY*<br />

* Clinical Trials Program participant<br />

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MELBOURNE<br />

Dr. Jonathan Cebon directs the Cancer Vaccine Laboratory in a continued collaboration with Dr. Eugene<br />

Maraskovsky. CT antigens continue to be attractive targets for cancer immunotherapy since they are<br />

expressed on a variety of different cancer types. The group’s experience with NY-ESO-1 has shown that<br />

it induces broad-based multifaceted (CD4 + , CD8 + , and antibody) immune responses. In addition, more<br />

preliminary work suggests that spontaneous responses to NY-ESO-1 may be associated with improved<br />

clinical outcomes. To understand the biology of these antigens further, members of the group have<br />

collaborated with the Division of Laboratory Medicine, Austin Hospital, to characterize antigen<br />

expression in clinical tumor samples. In other collaborations with Professors Marilyn Monk at the<br />

<strong>Institute</strong> of Child Health (London, UK), and Weifeng Chen at the Peking Medical Health Science Center<br />

(Peking, China) the group has evaluated potential cancer antigens isolated from pre-implantation<br />

embryos, and CT antigens in hepatocellular carcinoma, respectively. These studies will hopefully open<br />

the way for new clinical opportunities in the future.<br />

The group has also been studying subsets of dendritic cells (DCs) and the molecules that regulate the<br />

various functions of DC’s: activation, migration, cytokine secretion, and antigen presentation. These<br />

studies have provided fundamental insights into the roles of key regulators, such as prostaglandins, ATP,<br />

CD40 Ligand, interleukin-1�� and interferon-�. The genetic programs that characterize DC maturation<br />

and activation are being analyzed at the molecular level using microarrays to study subsets, and<br />

differential expression, of genes under different conditions of stimulation. In order to evaluate and<br />

identify the most efficacious DC for clinical application, the group has undertaken detailed comparisons<br />

of peripheral blood DCs and the commonly used monocyte-derived DCs. These studies have<br />

demonstrated fundamental differences in patterns of activation and migration, thereby assisting with the<br />

design of ex vivo strategies for DC therapy.<br />

THE CLINICAL DENDRITIC CELL FACILITY*<br />

The Clinical Dendritic Cell Facility headed by Dr. Ian Davis, is part of the <strong>LICR</strong>’s Clinical Trials<br />

Program, and was established in the context of the wider Cancer Vaccine Program. This program has<br />

arisen from the work of the Cancer Vaccine Laboratory in which basic research into the biology of human<br />

DCs was linked with the observations and experience gained from the group’s involvement in clinical<br />

trials. Previous clinical trials used a variety of cancer antigens delivered using various different adjuvants<br />

and routes of administration, in an attempt to optimize the vaccination process. On the basis of these<br />

studies, the DC team has developed a novel strategy for DC vaccine therapy. This strategy uses DCs<br />

which are mobilized into peripheral blood by the hemopoietic growth factor, Flt 3 ligand, and which are<br />

then harvested by leukopheresis, activated with CD40 Ligand, and exposed to the peptide antigens of<br />

interest, NY-ESO-1, MAGE-4, MAGE-10, and also with control peptides derived from hepatitis B core<br />

antigen and influenza matrix peptides. The DC group has undertaken extensive developmental studies to<br />

optimize the protocol in order to obtain high quality DCs for clinical use. The first trial with the cells was<br />

recently completed, with the primary endpoints of this study being feasibility, safety and immune effects.<br />

The FL-mobilized DCs were well tolerated, and strong immune responses (as determined by delayed type<br />

hypersensitivity responses to peptide-pulsed DCs) were elicited. Results from the T cell assays are still<br />

pending. Further trials, incorporating new information generated from our Vaccine Program are being<br />

planned.


TCELL LABORATORY*<br />

* Clinical Trials Program participant<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 88<br />

MELBOURNE<br />

The T Cell Laboratory has been focusing on the discovery of antigenic epitopes associated with the CT<br />

antigen, NY-ESO-1, under the leadership of Dr. Weisan Chen. Several novel CD4 + and CD8 + epitopes<br />

have been identified during a recent clinical trial of NY-ESO-1 and the adjuvant, ISCOM. The search for<br />

these epitopes in patients is supported by an active mouse-based program using influenza virus to study<br />

the mechanism of immuno-dominance hierarchies and immunological memory. The importance of<br />

various cytokines, and antigen dose and timing, on memory induction is also being assessed in this<br />

process. Using a transgenic system, the group is undertaking microarray analysis of gene expression in T<br />

cell subsets. Cross priming has been demonstrated to result in similar immuno-dominance hierarchy when<br />

compared with conventional viral infection, and the group is continuing to investigate whether antigen<br />

presenting cell competition plays a role in immuno-dominance. In another transgenic model, it is planned<br />

to study how the balance between tumor burden and host immune surveillance is disrupted, which might<br />

help in the understanding of how tumor-related immunological tolerance is established. It is hoped that, as<br />

knowledge of tumor immunology and host responses increases, research conducted by the group will lead<br />

to the design of more effective tumor vaccination regimes.<br />

JOINT AUSTIN LUDWIG ONCOLOGY UNIT*<br />

The Joint Austin <strong>Ludwig</strong> Oncology Unit is headed by Dr. Jonathan Cebon and was established by the<br />

<strong>LICR</strong> and the Austin Hospital a number of years ago. The Unit provides medical oncology services and<br />

engages in clinical research to contribute to the improvement in treating cancer. It is one of the busiest<br />

Medical Oncology units in Australia and has a strong commitment to education in oncology training,<br />

which is reflected in its popularity as a training site for the Cancer Medicine specialty. During <strong>2002</strong>, Dr.<br />

Niall Tebbutt, who recently returned Australia from the Royal Marsden Hospital (London, UK), joined<br />

the unit. His work will develop and expand treatment and research programs in gastrointestinal cancers.<br />

Other highlights include a successful pioneering trial of a clinical vaccine using NY-ESO-1 formulated in<br />

ISCOM® in collaboration with CSL Ltd, commencement of a trial using CpG DNA for the treatment of<br />

renal cancer in collaboration with Coley Pharmaceuticals Ltd, and the completion of the first national trial<br />

for hepatocellular carcinoma with the Australasian Gastrointestinal Trials Group (AGITG), which was<br />

chaired from the Unit. These trials reflect the Unit’s ongoing research focus on the biological therapy of<br />

cancer. The Unit now conducts over 50 clinical trials with much of this effort being directed towards<br />

<strong>LICR</strong> studies, focusing on the cancer immunotherapies of vaccines and mAbs. In addition, trials relevant<br />

to the management of most cancer types, using novel chemotherapy agents, cytokines, and other<br />

biological agents, different chemotherapy regimens, or combination treatments, such as chemoradiotherapy<br />

are conducted. These latter trials are mainly collaborative studies under the auspices of<br />

national or international cooperative groups.


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 89<br />

MELBOURNE<br />

1. ACHEN M.G., WILLIAMS R.A., BALDWIN M.E., LAI P., ROUFAIL S., ALITALO K. AND STACKER<br />

S.A. The angiogenic and lymphangiogenic factor vascular endothelial growth factor-D exhibits a<br />

paracrine mode of action in cancer. Growth Factors (<strong>2002</strong>) 20: 99-107.<br />

2. AKBARZADEH S., JI H., FRECKLINGTON D.F., MARMY-CONUS N., MOK Y-F., BOWES L.,<br />

DEVEREUX L., LINSENMEYER M., SIMPSON R.J. AND DOROW D.S. Mixed lineage kinase 2<br />

interacts with clathrin and influences clathrin-coated vesicle trafficking. Journal of Biological<br />

Chemistry (<strong>2002</strong>) 277: 36280-7.<br />

3. AKBARZADEH S., WARD A.C., MCPHEE D.O., ALEXANDER W.S., LIESCHKE G.J. AND LAYTON<br />

J.E.Tyrosine residues of the granulocyte colony-stimulating factor receptor transmit proliferation<br />

and differentiation signals in murine bone marrow cells. Blood (<strong>2002</strong>) 99: 879-87.<br />

4. ARRETT T.P.J., MCKERN N.M., LOU M.Z., ELLEMAN T.C., ADAMS T.E., LOVRECZ G.O., ZHU H-<br />

J., WALKER F., FRENKEL M.J., HOYNE P.A., JORISSEN R.N., NICE E.C., BURGESS A.W. AND<br />

WARD C.W.Crystal structure of a truncated epidermal growth factor receptor extracellular<br />

domain bound to transforming growth factor alpha. Cell (<strong>2002</strong>) 110: 763-73.<br />

5. BASU S., HODGSON G., KATZ M. AND DUNN A.R. Evaluation of role of G-CSF in the production,<br />

survival, and release of neutrophils from bone marrow into circulation. Blood (<strong>2002</strong>) 100: 854-61.<br />

6. BUTZKUEVEN H., ZHANG J-G., SOILU-HANNINEN M., HOCHREIN H., PETRATOS S., ERNST M.,<br />

BARTLETT P.F. AND KILPATRICK T.J. LIF receptor signaling limits immune-mediated<br />

7.<br />

demyelination by enhancing oligodendrocyte survival. Nature Medicine (<strong>2002</strong>) 8: 613-9.<br />

BYZOVA T.V., GOLDMAN C.K., JANKAU J., CHEN J., CABRERA G., ACHEN M.G., STACKER S.A.,<br />

CARNEVALE K.A., SIEMIONOW M., DICORLETO S.R. AND DICORLETO P.E Adenovirus encoding<br />

vascular endothelial growth factor-D induces tissue-specific vascular patterns in vivo. Blood<br />

(<strong>2002</strong>) 99: 4434-4442.<br />

8. CROWHURST M.O., LAYTON J.E. AND LIESCHKE G.J. Developmental biology of zebrafish<br />

myeloid cells. International Journal of Develomental Biology (<strong>2002</strong>) 46: 483-92.<br />

9. EDDES J.S., KAPP E.A., FRECKLINGTON D.F., CONNOLLY L.M., LAYTON M.J., MORITZ R.L. AND<br />

SIMPSON R.J. CHOMPER: a bioinformatic tool for rapid validation of tandem mass spectrometry<br />

search results associated with high-throughput proteomic strategies. Proteomics (<strong>2002</strong>) 2: 1097-<br />

103.<br />

10. ELSO C., TOOHEY B., REID G.E., POETTER K., SIMPSON R.J. AND FOOTE S.J. Mutation detection<br />

using mass spectrometric separation of tiny oligonucleotide fragments. Genome Resesearch<br />

(<strong>2002</strong>) 12: 1428-33.<br />

11. ERNST M., INGLESE M., SCHOLZ G., HARDER K.W., CLAY F.J., BOZINOVSKI S., WARING P.M.,<br />

DARWICHE R., KAY T., SLY T., COLLINS R., TURNER D.R., HIBBS M.L., ANDERSON G.P. AND<br />

DUNN A.R. Constitutive activation of the SRC family kinase Hck results in spontaneous<br />

pulmonary inflammation and an enhanced innate immune response. Journal of Experimental<br />

Medicine (<strong>2002</strong>) 196: 589-604.<br />

12. GNJATIC S., JAGER E., CHEN W., MATSUO M., LEE S-Y., CHEN Q., NAGATA Y., ATANACKOVIC<br />

D., CHEN Y.T., RITTER G., CEBON J., KNUTH A. AND OLD L.J. CD8(+) T cell responses against a<br />

dominant cryptic HLA-A2 epitope after NY-ESO-1 peptide immunization of cancer patients.<br />

Proceedings of the National Academy of Science U S A (<strong>2002</strong>) 99: 11813-8.


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 90<br />

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13. GRANT S.L., HAMMACHER A., DOUGLAS A.M., GOSS G., MANSFIELD R.K., HEATH J.K. AND<br />

BEGLEY C.G. An unexpected biochemical and functional interaction between gp130 and the EGF<br />

receptor family in breast cancer cells. Oncogene (<strong>2002</strong>) 21: 460-74.<br />

14. GRANT S.L., HAMMACHER A., DOUGLAS A.M., GOSS G.A., MANSFIELD R.K., HEATH J.K. AND<br />

BEGLEY C.G. An unexpected biochemical and functional interaction between gp130 and the EGF<br />

receptor family in breast cancer cells. Oncogene (<strong>2002</strong>) 21: 460-74.<br />

15. HANNAH A., SCOTT A.M., TOCHON-DANGUY H.J., CHAN J.G., AKHURST T., BERLANGIERI S.,<br />

PRICE D., SMITH G.J., SCHELLEMAN T., MCKAY W.J. AND SIZELAND A. Evaluation of 18 Ffluorodeoxyglucose<br />

positron emission tomography and computed tomography with<br />

histopathologic correlation in the initial staging of head and neck cancer. Annals of Surgery<br />

(<strong>2002</strong>) 236: 208-17.<br />

16. HARRIS M., ANGUS P. AND DAVIS I.D. Choledochal cyst and squamous-cell carcinoma of the<br />

biliary tract. Internal Medicine Journal (<strong>2002</strong>) 32: 491.<br />

17. HIBBS M., HARDER K.W., ARMES J., KOUNTOURI N., QUILICI C., CASAGRANDA F., DUNN A.R.<br />

AND TARLINTON D.M. Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity.<br />

Journal of Experimental Medicine (<strong>2002</strong>) 196: 1593-604.<br />

18. JEFFORD M., FEIGEN M., SOMERS G., ANGUS D., READ A. AND DAVIS I.D. Squamous cell<br />

carcinoma of the seminal vesicles - the first case report and review of the related literature.<br />

Urooncology (<strong>2002</strong>) 2: 19-22.<br />

19. JENKINS B.J., QUILICI C., ROBERTS A.W., GRAIL D., DUNN A.R. AND ERNST M. Hematopoietic<br />

abnormalities in mice deficient in gp130-mediated STAT signaling. Experimental Hematology<br />

(<strong>2002</strong>) 30: 1248-56.<br />

20. JOHNS T.G., STOCKERT E., RITTER G., RITTER G., JUNGBLUTH A., HUANG H.J.S., CAVENEE<br />

W.K., SMYTH F.E., HALL C, WATSON N.K., NICE E.C., GULLICK W.J., OLD L.J., BURGESS A.W.<br />

AND SCOTT A.M. Novel monoclonal antibody specific for the de2-7 epidermal growth factor<br />

receptor (EGFR) that also recognizes the EGFR expressed in cells containing amplification of the<br />

EGFR gene. International Journal of Cancer (<strong>2002</strong>) 98: 398-408.<br />

21. JOHNSTONE C.N., WHITE S.J., TEBBUTT N.C., CLAY F.J., ERNST M., BIGGS W.H., VIARS C.S.,<br />

CZEKAY S., ARDEN K.C. AND HEATH J.K. Analysis of the regulation of the A33 antigen gene<br />

reveals intestine-specific mechanisms of gene expression. Journal of Biological Chemistry (<strong>2002</strong>)<br />

277: 34531-9.<br />

22. JORISSEN R.N., TREUTLEIN H.R., EPA V.C. AND BURGESS A.W. Modeling the epidermal growth<br />

factor -- epidermal growth factor receptor l2 domain interaction: implications for the ligand<br />

binding process. Journal of Biomolecular Structure and Dynamics (<strong>2002</strong>) 19: 961-72.<br />

23. KANELLIS J., PAIZIS K., COX A.J., GILBERT R.E., COOPER M.E., STACKER S.A. AND POWER D.A.<br />

Renal ischema-reperfusion increases endothelial VEGFR-2 without increasing VEGF or VEGFR-<br />

2 expression. Kidney International (<strong>2002</strong>) 61: 1696-706.<br />

24. KREBS D.L., UREN R.T., METCALF D., RAKAR S., ZHANG J-G., STARR R., DE SOUZA D.P.,<br />

HANZINIKOLAS K., EYLES J., CONNOLLY L.M., SIMPSON R.J., NICOLA N.A., NICHOLSON S.E.,<br />

BACA M., HILTON D.J. AND ALEXANDER W.S. SOCS-6 binds to insulin receptor substrate 4, and<br />

mice lacking the SOCS-6 gene exhibit mild growth retardation. Molecular Cell Biology (<strong>2002</strong>)<br />

22: 4567-78.<br />

25. LEI G., XUE S., CHERY N., KWAN C.L., FU Y.P., LU Y.M., LIU M., HARDER K.W. AND YU X.M.<br />

Gain control of N-methyl-D-aspartate receptor activity by receptor-like protein tyrosine<br />

phosphatase alpha. EMBO Journal (<strong>2002</strong>) 21: 2977-89.


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 91<br />

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26. LI J.H., ZHU H-J., HUANG X.R., LAI K.N. AND JOHNSON R.J. Smad7 inhibits fibrotic effect of<br />

TGF-� on renal tubular epithelial cells by blocking Smad2 activation. Journal of the American<br />

Society of Nephrology (<strong>2002</strong>) 13: 1464-72.<br />

27. LIESCHKE G.J., OATES A.C., PAW B.H., THOMPSON M.A., HALL N.E., WARD A.C., HO R.K., ZON<br />

L.I. AND LAYTON J.E. Zebrafish SPI-1 (PU1) marks a site of myeloid development independent<br />

of primitive erythropoiesis: implications for axial patterning. Developmental Biology (<strong>2002</strong>) 246:<br />

274-295.<br />

28. LIU Z., SMYTH F.E., RENNER C., LEE F-T., OOSTERWIJK E. AND SCOTT A.M. Anti-renal cell<br />

carcinoma chimeric antibody G250: cytokine enhancement of in vitro antibody-dependent<br />

cellular cytotoxicity. Cancer Immunology and Immunotherapy (<strong>2002</strong>) 51: 171-7.<br />

29. LUFT T., JEFFORD M., LUETJENS P., HOCHREIN H., MASTERMAN K-A., MALISZEWSKI C.R.,<br />

SHORTMAN K., CEBON J.S. AND MARASKOVSKY E. IL-1� enhances CD40 ligand-mediated<br />

cytokine secretion by human dendritic cells (DC): a mechanism for T cell-independent DC<br />

activation. Journal of Immunology (<strong>2002</strong>) 168: 713-22.<br />

30. LUFT T., JEFFORD M., LUETJENS P., HOCHREIN H., MASTERMAN K-A., MALISZEWSKI C.R.,<br />

SHORTMAN K., CEBON J.S. AND MARASKOVSKY E. IL-1� enhances CD40 ligand-mediated<br />

cytokine secretion by human dendritic cells (DC): a mechanism for T cell-independent DC<br />

activation. Journal of Immunology (<strong>2002</strong>) 168: 713-22.<br />

31. LUFT T., JEFFORD M., LUETJENS P., TOY T, HOCHREIN H., MASTERMAN K-A., MALISZEWSKI<br />

C.R., SHORTMAN K., CEBON J. AND MARASKOVSKY E. Functionally distinct dendritic cell (DC)<br />

populations induced by physiologic stimuli: prostaglandin E2 regulates the migratory capacity of<br />

specific DC subsets. Blood (<strong>2002</strong>) 100: 1362-72.<br />

32. LUFT T., LUETJENS P., HOCHREIN H., TOY T, MASTERMAN K-A., RIZKALLA B., MALISZEWSKI<br />

C.R., SHORTMAN K., CEBON J. AND MARASKOVSKY E. IFN-alpha enhances CD40 ligandmediated<br />

activation of immature monocyte-derived dendritic cells. International Immunology<br />

(<strong>2002</strong>) 14: 367-80.<br />

33. MARKUS R., DONNAN G.A., KAZUI S., READ S.J., HIRANO T., SCOTT A.M., O'KEEFE G.J.,<br />

TOCHON-DANGUY H.J., SACHINIDIS J.I. AND REUTENS D.C. Statistical parametric mapping of<br />

hypoxic tissue identified by [(18)F]fluoromisonidazole and positron emission tomography<br />

following acute ischemic stroke. Neuroimage. 16: 425-33, (<strong>2002</strong>).<br />

34. MATHABA L.T., POPE C.H., LENZO J, HARTOFILLIS M, PEAKE H, MORITZ R.L., SIMPSON R.J.,<br />

BUBERT A., THOMPSON P.J. AND STEWART G.A. Isolation and characterisation of a 138-kDa<br />

bacteriolytic enzyme from house dust mite extracts: homology with prokaryotic proteins suggests<br />

that the enzyme could be bacterially derived. FEMS Immunology and Medical Microbiology<br />

(<strong>2002</strong>) 33: 77-88.<br />

35. MIZUTANI K., MIKI H., HE H., MARUTA H. AND TAKENAWA T. Essential role of neural Wiskott-<br />

Aldrich syndrome protein in podosome formation and degradation of extracellular matrix in srctransformed<br />

fibroblasts. Cancer <strong>Research</strong> (<strong>2002</strong>) 62: 669-74.<br />

36. NHEU T.V., HE H., HIROKAWA Y., TAMAKI K., FLORIN L., SCHMITZ M.L., SUZUKI-TAKAHASHI<br />

I., JORISSEN R.N., BURGESS A.W., NISHIMURA S., WOOD J. AND MARUTA H. The K252a<br />

derivatives, inhibitors for the PAK/MLK kinase family selectively block the growth of RAS<br />

transformants. Cancer Journal (<strong>2002</strong>) 8: 328-36.<br />

37. NICE E.C., DOMAGALA T., FABRI L., SETH A., WALKER F., JORISSEN R.N., BURGESS A.W., CUI<br />

D.F. AND ZHANG Y.S. Rapid microscale enzymic semisynthesis of epidermal growth factor<br />

(EGF) analogues. Growth Factors (<strong>2002</strong>) 20: 71-80.


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38. ORNER G.A., DASHWOOD R.H., LI Q., AL-FAGEEH M., AL-FAGEEH M., TEBBUTT N.C., HEATH<br />

J.K., ERNST M., DASHWOOD W-M. AND DASHWOOD R.H. Cellular effects of tea antioxidants in<br />

vitro and in mouse models of colorectal cancer: modulation of Wnt signalling. Free Radical<br />

<strong>Research</strong> (<strong>2002</strong>) 36: 16-20.<br />

39. ORNER G.A., DASHWOOD W-M, BLUM C.A., DIAZ G.D., LI Q., AL-FAGEEH M., TEBBUTT N.C.,<br />

HEATH J.K., ERNST M. AND DASHWOOD R.H. Response of Apc(min) and A33 (delta N beta-cat)<br />

mutant mice to treatment with tea, sulindac, and 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine<br />

(PhIP). Mutation <strong>Research</strong> (<strong>2002</strong>) 506-507: 121-7.<br />

40. SIDEROV J., WENDEL N. AND DAVIS I.D. Non-sedating antihistamines for premedication in<br />

ambulatory oncology patients. Journal of Pharmacy Practice and <strong>Research</strong> (<strong>2002</strong>) 32: 108-9.<br />

41. SIGALOTTI L., CORAL S., ALTOMONTE M., NATALI L., GAUDINO G., CACCIOTTI P., LIBENER R.,<br />

COLIZZI F., VIANALE G., MARTINI F., TOGNON M., JUNGBLUTH A., CEBON J., MARASKOVSKY E.,<br />

MUTTI L. AND MAIO M. Cancer testis antigens expression in mesothelioma: role of DNA<br />

methylation and bioimmunotherapeutic implications. British Journal of Cancer (<strong>2002</strong>) 86: 979-<br />

82.<br />

42. STACKER S.A., BALDWIN M.E. AND ACHEN M.G. The role of tumor lymphangiogenesis in<br />

metastatic spread. FASEB Journal (<strong>2002</strong>) 16: 922-934.<br />

43. TAVERNER T., HALL N.E., OHAIR R.A.J. AND SIMPSON R.J. Characterization of an antagonist<br />

interleukin-6 dimer by stable isotope labeling, cross-linking, and mass spectrometry. Journal of<br />

Biological Chemistry (<strong>2002</strong>) 277: 46487-92.<br />

44. TAVERNER T., SIGALOTTI C.S., ENGELSBERG A., COLIZZI F., CATTAROSSI I., MARASKOVSKY E.,<br />

JAGER E., SELIGER B. AND BROWER M. 5-aza-2'-deoxycytidine-induced expression of functional<br />

cancer testis antigens in human renal cell carcinoma: immunotherapeutic implications. Clinical<br />

Cancer <strong>Research</strong> (<strong>2002</strong>) 8: 2690-5.<br />

45. TEBBUTT N.C., GIRAUD A.S., INGLESE M., JENKINS B.J., WARING P.M., CLAY F.J., MALKI S.,<br />

GRAIL D., HOLLANDE F., HEATH J.K. AND ERNST M. Reciprocal regulation of gastrointestinal<br />

homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice. Nature<br />

Medicine (<strong>2002</strong>) 8: 1089-97.<br />

46. TOCHON-DANGUY H.J., CHAN F., CHAN J.G., HALL C., CHER L., STYLLI S.S., HILL J., KAYE A.<br />

AND SCOTT A.M. Imaging and quantitation of the hypoxic cell fraction of viable tumor in an<br />

animal model of intracerebral high grade glioma using [18F]fluoromisonidazole (FMISO).<br />

Nuclear Medicine and Biology (<strong>2002</strong>) 29: 191-7.<br />

47. TRAN T.T., ZENG J., TREUTLEIN H. AND BURGESS A.W. Effects of thioamide substitutions on the<br />

conformation and stability of alpha- and 3(10)-helices. Journal of the American Chemical Society<br />

(<strong>2002</strong>) 124: 5222-30.<br />

48. VARGHESE J.N., MORITZ R.L., LOU M.Z., VAN DONKELAAR A., JI H., IVANCIC N., BRANSON<br />

K.M., HALL N.E. AND SIMPSON R.J. Structure of the extracellular domains of the human<br />

interleukin-6 receptor alpha -chain. Proceedings of the National Academy of Science U S A<br />

(<strong>2002</strong>) 99: 15959-64.<br />

49. VERHAGEN A.M., SILKE J., EKERT P.G., PAKUSCH M., KAUFMAN H., CONNOLLY L.M., DAY<br />

C.L., TIKOO A., BURKE R., WROBEL C., MORITZ R.L., SIMPSON R.J. AND VAUX D.L. HtrA2<br />

promotes cell death through its serine protease activity and its ability to antagonize inhibitor of<br />

apoptosis proteins. Journal of Biological Chemistry (<strong>2002</strong>) 277: 445-54.


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50. WANG Y., HAN K-J., PANG X-W., VAUGHAN H.A., QU WEI, DONGX-Y.,PENG J-R., ZHAO H.,<br />

RUI J., LENG X., CEBON J., BURGESS A.W. AND CHEN W. Large scale identification of human<br />

hepatocellular carcinoma-associated antigens by autoantibodies. Journal of Immunology (<strong>2002</strong>)<br />

169: 1102-9.<br />

51. WILLIAMS R.T., SENIOR P.V., VAN STEKELENBURG L., LAYTON J.E., SMITH P.J. AND DZIADEK<br />

M.A. Stromal interaction molecule 1 (STIM1), a transmembrane protein with growth suppressor<br />

activity, contains an extracellular SAM domain modified by N-linked glycosylation. Biochimica<br />

et Biophysica Acta (<strong>2002</strong>) 1596: 131-7.<br />

52. XU Y., BEAVITT S-J. E., HARDER K.W., HIBBS M.L. AND TARLINTON D.M. The activation and<br />

subsequent regulatory roles of Lyn and CD19 after B cell receptor ligation are independent.<br />

Journal of Immunology (<strong>2002</strong>) 169: 6910-8.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. BALDWIN M.E., STACKER S.A. AND ACHEN M.G. Molecular control of lymphangiogenesis.<br />

BioEssays (<strong>2002</strong>) 24: 1030-1040.<br />

2. BURGESS A.W. Tyr kinase inhibitors as potential anticancer agents: EGF receptor and ABL<br />

kinases. In, Tumor-suppressing viruses, genes, and drugs: innovative cancer therapy approaches.<br />

pp. 341-359, <strong>2002</strong>, San Diego. Publisher: Academic Press, Editor: Maruta, H.<br />

3. CATIMEL B., ROTHACKER J. AND NICE E.C. Biosensors and proteomics. Today's Life Science<br />

(<strong>2002</strong>) 14: 25-30.<br />

4. DAVIS I.D. Immunotherapy of melanoma. Cancer Forum (<strong>2002</strong>) 26: 115-7.<br />

5. JOHNSTONE C.N., CHANG L. AND ERNST M. AACR special meeting in cancer research: colon<br />

cancer--genetics to prevention. Cancer <strong>Research</strong> <strong>2002</strong> 62: 6779-83.<br />

6. KUZUMAKI M. AND MARUTA H. Cytoskeletal tumor supressor genes. In, Tumor-suppressing<br />

viruses, genes, and drugs: innovative cancer therapy approaches. pp. 177-197, <strong>2002</strong>. San Diego,<br />

Publisher: Academic Press, Editor: Maruta, H.<br />

7. LAYTON J. Colony-stimulating factors and their receptors. Wiley Encyclopedia of Molecular<br />

Medicine (<strong>2002</strong>) 5: 874-878. Publisher: John Wiley & Sons Inc. Editor: Creighton, T.E.<br />

8. LAYTON J. Granulocyte colony-stimulating factor (G-CSF). Wiley Encyclopedia of Molecular<br />

Medicine (<strong>2002</strong>) 5: 1509-1510. Publisher: John Wiley & Sons Inc. Editor: Creighton, T.E.<br />

9. LAYTON J. Granulocyte macrophage colony-stimulating factor. Wiley Encyclopedia of Molecular<br />

Medicine (<strong>2002</strong>) 5: 1510-1512. Publisher: John Wiley & Sons Inc. Editor: Creighton, T.E.<br />

10. LAYTON J. Macrophage colony-stimulating factor (M-CSF). Wiley Encyclopedia of Molecular<br />

Medicine (<strong>2002</strong>) 5: 1987-1988. Publisher: John Wiley & Sons Inc. Editor: Creighton, T.E.<br />

11. LIESCHKE G.J. AND WARD A.C. The zebrafish as a model system for human disease. Frontiers in<br />

Biosciences (<strong>2002</strong>) 7: d827-33.<br />

12. MARUTA H. NF1 and other RAS-binding peptides. In, Tumor-suppressing viruses, genes, and<br />

drug: innovative cancer therapy approaches. pp. 169-175 (<strong>2002</strong>) San Diego. Publisher:<br />

Academic Press, Editor: Maruta, H.<br />

13. MARUTA H. Actin-binding drugs: MKT-077 and Chaetoglobosin K (CK). In, Tumor-suppressing<br />

viruses, genes, and drugs: innovative cancer therapy approaches. pp. 329-339 (<strong>2002</strong>) San Diego.<br />

Publisher: Academic Press, Editor: Maruta, H.


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MELBOURNE<br />

14. MARUTA H., HE H. AND NHEU T. Antagonists of Rho family GTPases : blocking PAKs, ACKs,<br />

and Rock. In, Tumor-suppressing viruses, genes, and drugs : innovative cancer therapy<br />

approaches. pp. 361-377 (<strong>2002</strong>) San Diego. Publisher: Academic Press, Editor: Maruta, H.<br />

15. STACKER S.A. AND ACHEN M.G. Inhibitors of angiogenesis. In, Tumor-suppressing viruses,<br />

genes, and drugs: innovative cancer therapy approaches. Pp. 261-292 (<strong>2002</strong>) San Diego.<br />

Publisher: Academic Press, Editor: Maruta, H.<br />

16. STACKER S.A., ACHEN M.G., BALDWIN M.E., JUSSILA L. AND ALITALO K. Lymphangiogenesis<br />

and cancer metastasis. Nature Reviews Cancer (<strong>2002</strong>) 2: 573-583.<br />

17. SUN X. AND LIESCHKE G.J. Abnormal protein tyrosine kinases associated with human<br />

haematological malignancies. Chinese Journal of Cancer <strong>Research</strong> (<strong>2002</strong>) 14: 79-83.<br />

18. WARD A.C., DUNN A.R. AND BASU S. G-CSF: function and modes of action. International<br />

Journal of Molecular Medicine (<strong>2002</strong>) 10: 3-10.


NEW YORK BRANCH<br />

OF HUMAN CANCER IMMUNOLOGY<br />

NEW YORK CITY, USA<br />

Staff List: Page 96<br />

Branch Director’s <strong>Report</strong>: Page 98<br />

<strong>Research</strong> <strong>Report</strong>: Page 99<br />

Publications: Page 102<br />

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STAFF LIST*<br />

OLD,LLOYD J., Director, Member<br />

RITTER,GERD, Associate Director, Associate Member<br />

RICHARDS,ELIZABETH, Assistant Director (until March)<br />

WELT,SYDNEY, Associate Member<br />

STOCKERT,ELISABETH, Associate Member (until September) §<br />

CHEN,YAO-T., Affiliate, Associate Professor Weill Medical College at Cornell University<br />

GNJATIC,SACHA, Assistant Member<br />

GURE,ALI, Assistant Member<br />

JUNGBLUTH,ACHIM, Assistant Member<br />

SCANLAN,MATTHEW, Assistant Member<br />

LEE,SANG-YULL, Assistant Investigator<br />

MARINO,MICHAEL, Assistant Member (until October)<br />

SHARMA,PADMANEE, Clinical Fellow<br />

ATANACKOVIC,DJORDJE, Postdoctoral Fellow<br />

MATSUO,MITSUTOSHI, Postdoctoral Fellow<br />

SATO,EIICHI, Postdoctoral Fellow<br />

COHEN,LEONARD, <strong>Research</strong> Assistant<br />

FORTUNATO,SHEILA, <strong>Research</strong> Assistant<br />

WILLIAMS,CLARENCE, <strong>Research</strong> Assistant<br />

WILLIAMSON,BARBARA, <strong>Research</strong> Assistant<br />

YIN,BEATRICE, <strong>Research</strong> Assistant (from May)<br />

COPLAN,KEREN, Senior <strong>Research</strong> Technician<br />

GORDON,CLAUDIA, Senior <strong>Research</strong> Technician (until April)<br />

IVERSEN,KRISTIN, Senior <strong>Research</strong> Technician<br />

RITTER,ERIKA, Senior <strong>Research</strong> Technician<br />

KELLY,DAVID, <strong>Research</strong> Technician (from April)<br />

KOLB,DENISE, <strong>Research</strong> Technician<br />

§ Deceased<br />

* Clinical Trials Program participant<br />

NEW YORK<br />

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MENDOZA,THERESA, <strong>Research</strong> Technician (from November)<br />

MURRAY,ANNE, <strong>Research</strong> Technician (from November)<br />

SANTIAGO,DARREN, <strong>Research</strong> Technician<br />

TUBALLES,KEVIN, <strong>Research</strong> Technician<br />

WEI,ISAAC, <strong>Research</strong> Technician (until April)<br />

ADMINISTRATION AND SECRETARIAT<br />

PRASSAS,ADRIENNE, <strong>Research</strong> Administrator (until October)<br />

RIOS,JANNETTE, <strong>Research</strong> Secretary<br />

* Clinical Trials Program participant<br />

NEW YORK<br />

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BRANCH DIRECTOR’S REPORT<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003<br />

NEW YORK<br />

The major objective of the New York Branch is the identification and characterization of suitable<br />

antigenic targets for antibody-based and vaccine-based immunotherapies of human cancer. The<br />

New York Branch, the smallest of the ten <strong>LICR</strong> Branches (an average of 26 staff members)<br />

utilizes a fully integrated and multidisciplinary approach, with the individual research groups -<br />

Antigen Discovery, Biochemistry, Pathology, T Cell Immunology and Serological Typing, and<br />

Clinical Trials Groups - operating in close collaboration with each other. The highly coordinated<br />

activities of the research groups, and their interactions with selected external academic and<br />

industrial research partners, are designed such that new discoveries are rapidly transferred into<br />

early phase clinical evaluation in cancer patients. The New York Branch is an integral part of the<br />

<strong>Institute</strong>’s Clinical Trials Program for immunotherapy of cancer with monoclonal antibodies<br />

(mAbs) and vaccines.<br />

As part of <strong>LICR</strong>’s Targeted Antibody Program a series of cell surface antigens have been<br />

identified and characterized over the past decade by the New York Branch. Six antigens have<br />

been selected for early phase clinical evaluation, and clinical trials with mAbs against those<br />

antigens are ongoing, or are soon to commence, within the <strong>LICR</strong> Clinical Trials Program.<br />

Currently, a major focus is on the generation and development of non-immunogenic human or<br />

chimeric antibody constructs for clinical evaluation. The protocols developed for the clinical<br />

study of the newer generation of antibody constructs are designed to provide the following<br />

information: pharmacokinetic and quantitative biodistribution properties, tumor localization and<br />

imaging characteristics, immunogenicity, and therapeutic activity of antibody alone and in<br />

combination with, or as a carrier of, cytotoxic agents.<br />

As part of <strong>LICR</strong>’s Cancer Vaccine Program, the discovery and characterization of new targets is a<br />

continuing objective of the Branch with particular emphasis on gene products having<br />

characteristics of cancer /testis (CT) antigens. In addition, the Branch’s research groups provide<br />

critical laboratory support to clinical investigators within the Branch or affiliated with the<br />

<strong>Institute</strong>’s Clinical Trials Program in order to monitor, optimize, and characterize the immune<br />

responses to cancer vaccines. The Antigen Discovery and Pathology Groups perform the antigen<br />

typing of tumors to determine the potential eligibility of a patient to participate in clinical trials,<br />

the Biochemistry Group generates monitoring agents, and the T Cell Immunology and<br />

Serological Typing Group monitors humoral and cellular immune responses of patients enrolled<br />

in the cancer vaccine trials.<br />

The Branch is involved in the immunological monitoring for nearly all clinical trials performed<br />

under the auspices of the Cancer Vaccine Collaborative (CVC) which was established in<br />

partnership with the Cancer <strong>Research</strong> <strong>Institute</strong> (New York, USA) in <strong>2002</strong>. Furthermore, the<br />

Branch serves as immunological reference laboratory for the <strong>LICR</strong>’s Clinical Trials Program, as<br />

well as a training laboratory for immunological monitoring and molecular typing techniques<br />

required for the <strong>LICR</strong>’s Cancer Vaccine, and Targeted Antibody Programs.<br />

L.J. Old and G. Ritter<br />

98


RESEARCH REPORT<br />

ANTIBODY DEVELOPMENT AND ANTIBODY-BASED IMMUNOTHERAPY*<br />

* Clinical Trials Program participant<br />

NEW YORK<br />

Over the past decade, a series of cell surface antigens have been identified by the New York<br />

Branch. These antigens are being explored as antigenic targets on cancer cells for antibody-based<br />

immunotherapy within the <strong>LICR</strong> Clinical Trials Program. They include: A33, a 43 K<br />

glycoprotein with selective expression in normal and malignant epithelium of the gastrointestinal<br />

tract (recognized by huAb A33); G250, a glycoprotein expressed by a high percentage of renal<br />

cancers (recognized by chAb G250); Le y , an oligosaccharide epitope expressed on glycolipids<br />

and glycoproteins by a wide range of epithelial cancers (recognized by huAb 3S193); GD3, a<br />

ganglioside with high expression in melanoma and other neuroectodermal tumors (recognized by<br />

chAb KM871); FAP-�, a 95 K glycoprotein strongly expressed in the stromal fibroblasts of<br />

epithelial cancers (recognized by mAb F19 and huAbBIBH1); and a novel aberrant form of the<br />

epidermal growth factor receptor (EGFR), recognized by mAb 806, which is expressed by a<br />

proportion of brain cancers and other tumor types.<br />

Mouse mAb 806 was initially raised, as part of a collaboration with the San Diego Branch, to<br />

recognize a truncated (de2-7 deletion) form of EGFR found predominantly in glioblastoma. The<br />

de2-7 deletion has also been reported to be found in non-CNS malignancies such as breast and<br />

lung carcinomas, and prostate cancers, but the group’s findings do not support the presence of the<br />

de2-7 deletion mutation in tumors other than glioblastoma. Analysis of mAb 806 gave rise to the<br />

surprising finding that mAb 806 showed specificity for overexpressed/amplified EGFR. In a<br />

panel of normal tissues mAb 806 showed little or no reactivity with tissues known to express<br />

wildtype levels of EGFR, including liver and skin. In contrast, tumors known to overexpress<br />

EGFR, e.g. glioblastoma, squamous cell carcinoma of the lung, and head and neck, urinary, and<br />

bladder cancers showed strong reactivity by immunohistochemistry. mAb 806 is now undergoing<br />

further preclinical characterization at the Melbourne Branch, and a chimeric version of this<br />

antibody generated at the <strong>Institute</strong>’s Biological Production Facility in Melbourne is expected to<br />

then enter early phase clinical trials, as part of the <strong>LICR</strong> Clinical Trials Program, in 2004.<br />

Based on evidence that a radiolabeled mouse A33 antibody sensitized patients’ tumors to followup<br />

chemotherapy, a phase I study of escalating doses of humanized A33 + BOF-Strep<br />

chemotherapy was recently completed. Patients with advanced colorectal cancer resistant to at<br />

least two chemotherapeutic regimens were entered into four doses levels (5, 10, 25, and 40<br />

mg/m2). The only adverse events, such as neutropenia, and thrombocytopenia, were related to the<br />

chemotherapy. No significant toxicities specifically related to the combination of<br />

immunochemotherapy were identified. Of the 11 patients completing one treatment cycle, three<br />

had radiographic evidence of partial responses, one had a mixed response, and one patient had<br />

stable disease for nine months. Still, seven of twelve patients developed anti-human A33 activity<br />

(HAHA), according to a novel biosensor-based analysis, specifically developed to allow<br />

unambiguous measurement of human anti-human antibodies (HAHA). In collaboration with<br />

<strong>LICR</strong> Affiliate Dr. Christoph Renner (Homburg, Germany), several new human or chimeric IgG1<br />

A33 antibodies have been generated to overcome the high frequency of HAHA induction of the<br />

first generation antibody, which accelerated antibody serum clearance, blocked tumor targeting<br />

and consequently abrogated tumor response. The genetically engineered antibodies are currently<br />

being produced in mammalian expression systems in collaboration with <strong>LICR</strong> Affiliate Dr.<br />

Florian Wurm (Lausanne, Switzerland).<br />

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* Clinical Trials Program participant<br />

NEW YORK<br />

A new Positron Emission Tomography (PET) Program has been established, as part of the<br />

Targeted Antibody Program, at the Branch’s host institution, the Memorial Sloan- Kettering<br />

Cancer Center (MSKCC) with <strong>LICR</strong> Affiliate Dr. Chaitanya Divgi, and Drs. Steve Larsen and<br />

Ron Finn from MSKCC. This program will allow quantitative assessment of radiolabelled<br />

antibody tumor targeting in real time. The ultimate goal is to replace invasive biopsies to measure<br />

tumor antibody uptake. The initial focus is on huAbA33, hu3S193, and chG250.<br />

ANTIGEN DISCOVERY AND CANCER VACCINES<br />

The Antigen Discovery Group has identified a large array of distinct antigens using serological<br />

screening of cDNA expression cloning (SEREX) (for details see the <strong>LICR</strong> Cancer Immunome<br />

Database). These antigens fall into different categories including differentiation antigens, splice<br />

variants, mutational antigens, overexpressed/amplified antigens, and CT antigens. A new<br />

technique based on SEREX, SADA (serum antibody detection array), has been developed for<br />

sero-epidemiological analyses, and was used to investigate the serological responses of normal<br />

blood donors, and colon or breast cancer patients, to a panel of 278 tumor antigens. Such a<br />

method is not only useful for target identification, but may also be of diagnostic and prognostic<br />

significance. The antigens recognized by sera from patients with breast or colon cancer included<br />

several members of the CT antigen family and other tissue-restricted gene products. One CT<br />

antigen in particular, NY-CO-58, was highly overexpressed in 9/9 cases of colon cancer. Another<br />

technique to identify potential antigens, RAYS (recombinant antigen expression on yeast surface)<br />

is being developed in collaboration with <strong>LICR</strong> Affiliate Dr. Christoph Renner (Homburg,<br />

Germany).<br />

A bioinformatic approach has also been used to identify new members of the CT antigen family.<br />

Over 1300 different CT-associated Unigene clusters were identified and these are now being<br />

investigated further. The SSX gene family of CT antigens has also received detailed analyses<br />

resulting in the identification of nine complete SSX genes and ten pseudogenes or gene fragments<br />

in the human genome. Finally, to further understand the functions and regulation of CT antigens,<br />

the murine homologs of the gene families of SSX and NY-ESO-1 have been cloned. Despite all<br />

human SSX genes having murine homologs, the only NY-ESO-1 family member to have a<br />

homolog was the ubiquitously expressed gene NY-ESO-3.<br />

For an antigen to serve as a potential target for immunotherapy, it is critical to obtain information<br />

on its tissue expression not only on the RNA level, but also on the protein level. Novel mAbs are<br />

being prepared for newly discovered antigens to use in comprehensive immunohistochemical<br />

expression analyses performed by the Pathology Group. The mAbs are prepared by the Branch’s<br />

Biochemistry Group, or in collaboration with Dr. Boquan Jin (Xi’an, China) and/or Dr. Ivan Gout<br />

(Kiev, Ukraine) through the James R. Kerr Program. Extensive immunohistochemical analyses of<br />

a range of CT antigens, including MAGE-A1, MAGE-A3, NY-ESO-1, and CT7 has been carried<br />

out in a large panel of normal and malignant tissues. Normal tissue expression was restricted to<br />

cells undergoing early phases of spermatogenesis. A proportion (20-40%) of a wide range of<br />

different tumor types express CT antigens in a heterogeneous/homogeneous fashion. Synovial<br />

cell sarcoma showed an unusual high frequency (>80%) of expression with NY-ESO-1 expressed<br />

highly homogeneously.<br />

NY-ESO-1, which was discovered in the Branch using SEREX, represents a particularly<br />

promising new target antigen for vaccine development, because of its capacity to induce both<br />

humoral and cellular immune responses in a proportion of cancer patients having NY-ESO-1-<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 100


* Clinical Trials Program participant<br />

NEW YORK<br />

expressing tumors. A major proportion of the Branch’s research activities on cancer vaccines is<br />

focused on this particular antigen, and several clinical vaccine trials utilizing NY-ESO-1 have<br />

now been initiated. Besides safety, the main objective of those trials is to assess NY-ESO-1specific<br />

immunity following vaccination with NY-ESO-1. For this purpose, novel types of assays<br />

have been developed, allowing sensitive and specific monitoring of humoral and both CD4 and<br />

CD8 cellular immune responses to NY-ESO-1. In collaboration with <strong>LICR</strong> Affiliates Drs.<br />

Alexander Knuth, Elke Jäger, and Dirk Jäger (Frankfurt, Germany), the T cell Group has worked<br />

on the detection of NY-ESO-1-antibodies in urine, thus providing a less invasive means of<br />

attaining patient samples for immunological monitoring.<br />

The mechanism of cross-presentation of exogenous antigens by dendritic cells (DCs) is also being<br />

studied by the T cell Group in collaboration with <strong>LICR</strong> Affiliate Dr. Ira Mellman (New Haven,<br />

USA). Anti-NY-ESO-1 antibody immune complexes have been shown to be efficiently taken up<br />

and processed by monocyte-derived dendritic cells (DC), but not by CD34 + -derived DC, before<br />

being presented to CD8 + T cells. In contrast, multiple NY-ESO-1 peptides of 30 amino acids were<br />

found to be efficiently taken up and processed by a wide variety of non-professional antigen<br />

presenting cells. This novel finding has given rise to the concept of using overlapping peptides of<br />

NY-ESO-1 instead of the whole protein as immunogen in cancer vaccines.<br />

Presentation of non-peptide antigens, such as glycolipids like GM2, by non-polymorphic major<br />

histocompatibility complex class I-like glycoproteins is also being investigated in collaboration<br />

with the groups of <strong>LICR</strong> Affiliates Drs. Richard Schmidt (Konstanz, Germany) and Vincenzo<br />

Cerundolo (Oxford, UK). The crystal structure of CD1b complexed with ganglioside GM2 or<br />

phosphatidylinositol has been prepared and analyzed. The structure revealed that in addition to dialkyl<br />

chain lipids, mono-alkyl and triple-alkyl chain lipids can be accommodated in the binding<br />

groove with the sugar portion extruding from the groove allowing recognition by the receptors on<br />

immune cells.<br />

In collaboration with Dr. Nasser Altorki at Weill Medical College at Cornell University, two<br />

<strong>LICR</strong>-sponsored clinical vaccine trials are ongoing in patients with lung cancer. In one of these<br />

trials, cancer patients were vaccinated with HLA-A2-restricted peptides 157-165, and 157-167<br />

from NY-ESO-1. Analysis of CD8 + T cell responses elicited by the vaccine showed that an<br />

unexpected, cryptic epitope had a dominant reactivity, potentially skewing T cells away from<br />

tumor recognition, prompting the removal of peptide 157-167 from clinical studies.<br />

In preparation for a vaccine trial in patients with bladder cancer, the frequency of expression of<br />

NY-ESO-1, and a closely related CT antigen, LAGE, have been studied in over 180 patients with<br />

transitional cell carcinomas (TCC). Over 30% of tumors expressed these antigens. Based on these<br />

findings, a new vaccine trial has been initiated at MSKCC to assess whether immunization with<br />

NY-ESO-1 protein plus adjuvant BCG can elicit or augment humoral and cellular immune<br />

responses in patients at high risk of relapse after cystectomy. In addition, two other new clinical<br />

vaccine trials have been initiated at MSKCC in patients with ovarian cancer and sarcoma under<br />

the auspice of the Cancer Vaccine Collaborative.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 101


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 102<br />

NEW YORK<br />

1. ALPEN B, GURE AO, SCANLAN MJ, OLD LJ, CHEN Y-T. A new member of the NY-ESO-<br />

1 gene family is universally expressed in somatic tissues and evolutionarily conserved.<br />

Gene (<strong>2002</strong>) 297:141-149.<br />

2. ANTONESCU CR, BUSAM KJ, IVERSEN K, KOLB D, COPLAN K, SPAGNOLI GC, LADANYI<br />

M, OLD LJ, JUNGBLUTH AA. MAGE antigen expression in monophasic and biphasic<br />

synovial sarcoma. Human Pathology (<strong>2002</strong>) 33:225-229.<br />

3. ANTONESCU CR, TSCHERNYAVSKY SJ, WOODRUFF JM, JUNGBLUTH AA, BRENNAN MF,<br />

LADANYI M. Molecular diagnosis of clear cell sarcoma: detection of EWS-ATF1 and<br />

MITF-M transcripts and histopathological and ultrastructural analysis of 12 cases.<br />

Journal of Molecular Diagnosis (<strong>2002</strong>) 4:44-52.<br />

4. DHODAPKAR, KM., KRASOVSKY, J., WILLIAMSON, B., AND DHODAPKAR, MV. Antitumor<br />

monoclonal antibodies enhance cross-presentation ofcCellular antigens and the<br />

generation of myeloma-specific killer T cells by dendritic cells. Journal of Experimental<br />

Medicine (<strong>2002</strong>) 195:125-33.<br />

5. DUTOIT, V.,R.N.TAUB, K.P.PAPADOPOULOS, S.TALBOT, M.L.KEOHAN, M.BREHM,<br />

S. GNJATIC, P. E. HARRIS, B.BISIKIRSKA, P.GUILLAUME, J.C.CEROTTINI, C.S.<br />

HESDORFFER, L.J.OLD, AND D. VALMORI. Multiepitope CD8(+) T cell response to a<br />

NY-ESO-1 peptide vaccine results in imprecise tumor targeting. Journal of Clinical<br />

Investigation (<strong>2002</strong>) 110:1813-22.<br />

6. FORTI S, SCANLAN MJ, INVERNIZZI A, CASTIGLIONI F, PUPA S, AGRESTI R, FONTANELLI<br />

R, MORELLI D, OLD LJ, PUPA SM, MENARD S. Identification of breast cancer-restricted<br />

antigens by antibody screening of SKBR3 cDNA library using a preselected patient's<br />

serum. Breast Cancer <strong>Research</strong> and Treatment (<strong>2002</strong>) 73:245-256.<br />

7. GADOLA, SD., ZACCAI, N.,HARLOS, K.,SHEPHERD, D.,CASTRO-PALOMINO, J.,RITTER,<br />

G., SCHMIDT,RS,JONES YE, AND CERUNDOLO, V. Structure of human CD1b with bound<br />

ligands at 2.3A, a maze for alkyl chains. Nature Immunology (<strong>2002</strong>) 3, 721-726.<br />

8. GNJATIC S, JAGER E, CHEN W, ALTORKI NK, MATSUO M, LEE SY, CHEN Q, NAGATA Y,<br />

ATANACKOVIC D, CHEN YT, RITTER G, CEBON J, KNUTH A, OLD LJ. CD8(+) T cell<br />

responses against a dominant cryptic HLA-A2 epitope after NY-ESO-1 peptide<br />

immunization of cancer patients. Proceedings of the National Academy of Sciences USA<br />

(<strong>2002</strong>) 99:11813-11818.<br />

9. GURE AO, WEI IJ, OLD LJ, CHEN YT. The SSX gene family: characterization of 9<br />

complete genes. International Journal of Cancer (<strong>2002</strong>) 101:448-453.<br />

10. JÄGER D, UNKELBACH M, FREI C, BERT F, SCANLAN MJ, JÄGER E, OLD LJ, CHEN Y-T,<br />

KNUTH, A. Identification of tumor-restricted antigens NY-BR-1, SCP-1, and a new<br />

cancer/testis-like antigen NW-BR-3 by serological screening of a testicular library with<br />

breast cancer serum. Cancer Immunity (<strong>2002</strong>) 2:5.<br />

11. JÄGER, D,STOCKERT, E, KARBACH, J,HERRLINGER, K,ATMACA, A,ARAND, M,CHEN,<br />

YT, GNJATIC, S,OLD, LJ,KNUTH, A, AND JÄGER, E. Urine antibody against human<br />

cancer antigen NY-ESO-1. Cancer Immunity (<strong>2002</strong>) 2:10.


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 103<br />

NEW YORK<br />

12. JÄGER, E,KARBACH, J,GNJATIC, S,JÄGER, D,MAEURER, M,ATMACA, A,ARAND, M,<br />

SKIPPER, J,STOCKERT, E,CHEN, YT,OLD, LJ, AND KNUTH, A. Identification of a<br />

naturally processed NY-ESO-1 peptide recognized by CD8+ T cells in the context of<br />

HLA-B51. Cancer Immunity (<strong>2002</strong>) 2:12.<br />

13. JOHNS, TG.,STOCKERT, E.,RITTER, G.,JUNGBLUTH, AA., SU HUANG, H-J., CAVENEE,<br />

WK., SMYTH, FE., HALL, CM., WATSON, N., NICE, EC., GULLICK WJ, OLD, LJ.,<br />

BURGESS, AW., AND SCOTT, AM. A novel monoclonal antibody specific for the de2-7<br />

epidermal growth factor receptor (EGFR) that also recognizes the EGFR expressed in<br />

cells containing amplification of the EGFR gene. International Journal of Cancer (<strong>2002</strong>)<br />

98:398-408.<br />

14. JUNGBLUTH AA, CHEN YT, BUSAM KJ, COPLAN K, KOLB D, IVERSEN K, WILLIAMSON<br />

B, VAN LANDEGHEM FK, STOCKERT E, OLD LJ. CT7 (MAGE-C1) antigen expression in<br />

normal and neoplastic tissues. International Journal of Cancer (<strong>2002</strong>) 99:839-845.<br />

15. LOHMANN CM, IVERSEN K, JUNGBLUTH AA, BERWICK M, BUSAM KJ. Expression of<br />

melanocyte differentiation antigens and ki-67 in nodal nevi and comparison of ki-67<br />

expression with metastatic melanoma. American Journal of Surgical Pathology (<strong>2002</strong>)<br />

26:1351-1357.<br />

16. LIU Z, LEE FT, HANAI N, SMYTH FE, BURGESS AW, OLD LJ, SCOTT AM. Cytokine<br />

enhancement of in vitro antibody-dependent cellular cytotoxicity mediated by chimeric<br />

anti-GD3 monoclonal antibody KM871. Cancer Immunity (<strong>2002</strong>) 2:13.<br />

17. LUO, G., HUANG, S., XIE, X., STOCKERT, E., CHEN, Y.-T., KUBUSCHOK, B. AND<br />

PFREUNDSCHUH M. Expression of cancer-testis genes in human hepatocellular<br />

carcinomas. Cancer Immunity (<strong>2002</strong>) 2:11.<br />

18. NAGATA, Y.,ONO, S.,MATSUO, M.,GNJATIC, S.,VALMORI, D.,RITTER, G.,GARRETT,<br />

W., OLD, LJ, AND MELLMAN, I. Differential expression of a soluble exogenous tumor<br />

antigen, NY-ESO-1, by distinct human dendritic cell populations. Proceedings of the<br />

National Academy of Sciences USA (<strong>2002</strong>) 99:10629-10634.<br />

19. O'DONOGHUE JA, BAIDOO N, DELAND D, WELT S, DIVGI CR, SGOUROS G. Hematologic<br />

toxicity in radioimmunotherapy: dose-response relationships for I-131 labeled antibody<br />

therapy. Cancer Biotherapy and Radiopharmaceuticals (<strong>2002</strong>) 17:435-443.<br />

20. SIGALOTTI, L,CORAL, S,ALTOMONTE, M,NATALI, L, GAUDINO, G,CACCIOTTI, P,<br />

LIBENER, R,COLIZZI, F,VIANALE, G, MARTINI, F, TOGNON, M,JUNGBLUTH, AA,<br />

21.<br />

CEBON, J, MARASKOVSKY, E,MUTTI, L,MAIO, M. Cancer testis antigens expression in<br />

mesothelioma: role of DNA methylation and bioimmunotherapeutic implications. British<br />

Journal of Cancer (<strong>2002</strong>) 86:979-982.<br />

SCANLAN, JM., GORDON, CM.,WILLIAMSON, B., LEE, S-Y., JAEGER, D.,CHEN, Y-T.,<br />

STOCKERT, E.,JUNGBLUTH, A.,RITTER, G.,SOUZA, SJ, AND OLD, LJ. Identification of<br />

cancer/testis genes by database mining and mRNA expression analysis. International<br />

Journal of Cancer (<strong>2002</strong>) 98:458-492.<br />

22. SCANLAN, MJ, WELT, S., HUMM, CG., CHEN, YT., GURE, AO., STOCKERT, E.,<br />

JUNGBLUTH, AA., RITTER, G.,JAEGER, D.,JAEGER, E.,KNUTH, A., AND OLD. LJ.<br />

Cancer-related Serological Recognition of Human Colon Cancer: Identification of<br />

Potential Diagnostic and Immunotherapeutic Targets. Cancer <strong>Research</strong> (<strong>2002</strong>) 62: 4041-<br />

4047.


REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

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NEW YORK<br />

1. IKEDA H, OLD LJ, SCHREIBER RD. The roles of IFN gamma in protection against tumor<br />

development and cancer immunoediting. Cytokine Growth Factor Reviews (<strong>2002</strong>) 13:95-<br />

109<br />

2. DUNN GP, BRUCE AT, IKEDA H, OLD LJ, SCHREIBER RD. Cancer immunoediting: from<br />

immunosurveillance to tumor escape. Nature Immunology (<strong>2002</strong>) 3:991-998<br />

3. SCANLAN MJ, GURE AO, JUNGBLUTH AA, OLD LJ, CHEN, YT. Cancer/Testis antigens:<br />

an expanding family of targets for cancer immunotherapy. Immunological Reviews<br />

(<strong>2002</strong>) 188:22-32


SAN DIEGO BRANCH<br />

OF CANCER GENETICS<br />

SAN DIEGO, USA<br />

Staff List: Page 106<br />

Branch Director’s <strong>Report</strong>: Page 110<br />

<strong>Research</strong> <strong>Report</strong>: Page 111<br />

Publications: Page 113<br />

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STAFF LIST<br />

CAVENEE,WEBSTER K., Director, Member<br />

LABORATORY OF TUMOR BIOLOGY<br />

CAVENEE,WEBSTER, Director, Member<br />

SECTION OF HUMAN CARCINOGENESIS<br />

FURNARI,FRANK, Assistant Investigator<br />

AL-KHOURI,ANNA-MARIA, Postdoctoral Fellow<br />

CHEN,YEN-LIANG, Postdoctoral Fellow<br />

MUKASA,AKITAKE, Postdoctoral Fellow (from December)<br />

Narita, Yoshitaka, Postdoctoral Fellow (until November)<br />

OKUMURA,KOICHI, Visiting <strong>Research</strong> Scholar I<br />

Mendoza, Michelle, PhD Student (until December)<br />

STEELE,JULIANNE, Associated <strong>Research</strong> Staff I<br />

SECTION OF MOLECULAR CYTOGENETICS<br />

ARDEN,KAREN, Associate Investigator<br />

BISHOP,DAVID, Associated <strong>Research</strong> Staff I (from July)<br />

MARC,SUSANNA, Postdoctoral Fellow (until May)<br />

HOSAKA,TAISUKE, Postdoctoral Fellow<br />

LU,WEN, Postdoctoral Fellow<br />

SMITH,WENDY, PhD Student<br />

TIEU,DAVID, Medical Student<br />

BOYER,ANTONIA, Associated <strong>Research</strong> Staff I<br />

LABORATORY OF CANCER GENETICS<br />

KOLODNER,RICHARD, Member<br />

FLORES-ROZAS,HERNAN, Postdoctoral Fellow (until May)<br />

GAZZOLI,ISABELLA, Postdoctoral <strong>Research</strong>er<br />

HESS,MARTIN, Postdoctoral Fellow<br />

HUANG,MENG ER, Postdoctoral Fellow (from September)<br />

MAZUR,DANIEL, Postdoctoral Fellow<br />

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MYUNG,KYUNGJAE, Postdoctoral Fellow (until August)<br />

PENNANEACH,VINCENT, Postdoctoral Fellow<br />

PUTNAM,CHRISTOPHER, Postdoctoral Fellow<br />

SASAKI,NOBUYA, Postdoctoral Fellow<br />

SCHMIDT,KRISTINA, Postdoctoral Fellow<br />

SHIMODAIRA,HIDEKI, Postdoctoral Fellow (until May)<br />

KATS,ELLEN, PhD Student<br />

LAU,PATRICK, PhD Student<br />

MENDILLO,MARC, PhD Student<br />

NARDIN,STEPHANIE, PhD Student (from September)<br />

SHELL,SCARLET, PhD Student (from July)<br />

CASSEL,DENA, Associated <strong>Research</strong> Staff<br />

DERRY,KATRINA, Associated <strong>Research</strong> Staff<br />

MARTINEZ-BARRAGAN,SANDRA, Associated <strong>Research</strong> Staff II<br />

MUELLER,JAMES, Associated <strong>Research</strong> Staff I<br />

NGUYEN,ANN THU, Laboratory Support Staff (from October)<br />

LABORATORY OF CELL BIOLOGY<br />

CLEVELAND,DON, Member<br />

ABRIEU,ARIANE, Postdoctoral Fellow (until May)<br />

BLACK,BEN, Postdoctoral Fellow (from June)<br />

BOILLEE,SEVERINE, Postdoctoral Fellow (from March)<br />

BONDAY,ZAHID, Postgraduate <strong>Research</strong>er<br />

CLEMENT,ALBRECHT, Postdoctoral Fellow (until August)<br />

FOLTZ,DAN, Postdoctoral Fellow (from May)<br />

FUJIMOTO,JIRO, Postdoctoral Fellow (from June)<br />

GARCIA,MICHAEL, Postdoctoral Fellow<br />

KOPS,GEERT, Postdoctoral Fellow<br />

LIU,JIAN, Postdoctoral Fellow (until December)<br />

LOBSIGER,CHRISTIAN, Postdoctoral Fellow<br />

MAO,YINGHUI, Postdoctoral Fellow<br />

SHAH,JAGESH, Postdoctoral Fellow<br />

VAN DE VELDE,CHRISTINE, Postdoctoral Fellow<br />

YAMANAKA,KOJI, Postdoctoral Fellow<br />

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SILK,ALAIN, PhD Student (from May)<br />

TAFARI,ASANTEWA TSAHAI, PhD Student (until November)<br />

WEAVER,BETH, PhD Student<br />

ROY,BERNALI, Undergraduate Intern (from September)<br />

ZHAO,YING, Senior Associated <strong>Research</strong> Staff I (from August)<br />

WANG,YIN, Associated <strong>Research</strong> Staff II (from April)<br />

WARD,CHRISTOPHER, Associated <strong>Research</strong> Staff II<br />

YOUNG,DARREN, Associated <strong>Research</strong> Staff (until August)<br />

LABORATORY OF CHROMOSOME BIOLOGY<br />

DESAI,ARSHAD, Assistant Member (from November)<br />

LABORATORY OF GENE REGULATION<br />

REN,BING, Assistant Member<br />

KIM,TAE HOON, Postdoctoral Fellow (from October)<br />

LI,ZIRONG, Post Graduate <strong>Research</strong>er<br />

HEGLER,JOSEPH, PhD Student (from July)<br />

HEINTZMAN,NATHANIEL, PhD Student (from September)<br />

MAYNARD,NATHANIEL, PhD Student (from August)<br />

QU,CHUNXU, Associated <strong>Research</strong> Staff III (from June)<br />

VAN CALCAR,SARA, Associated <strong>Research</strong> Staff I<br />

LABORATORY OF MITOTIC MECHANISMS<br />

OEGEMA,KAREN, Assistant Member (from November)<br />

LABORATORY OF PROTEOMIC BIOLOGY<br />

ZHOU,HUILIN, Assistant Member (from July)<br />

ZHOU,WEIDONG, Postdoctoral Fellow (from October)<br />

RYAN,JENNIFER, Senior Associated <strong>Research</strong> Staff I (from July)<br />

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GENERAL SERVICES<br />

REIGLE,BRIAN, Technical Support LAN Services<br />

WEGER,JOHN, <strong>Research</strong> Associate (Core Sequencing)<br />

NESS,STEFANIE, Associated <strong>Research</strong> Staff I (Core Sequencing) (from December)<br />

TRESIERIAS,M,Technician (Core Sequencing) (until September)<br />

ADMINISTRATION AND SECRETARIAT<br />

PEACOCK,DIANNE, Branch Administrator (from April)<br />

MACK,ALMA, Branch Grants Officer (from February)<br />

INGLESE,JANE, Payroll Specialist (from August)<br />

CALLAHAN,KIMBERLY, Branch Accountant (from July)<br />

CARDIEL,BLANCA A., Grants Accountant (from June)<br />

GABOR,HERMAN, Financial Officer (until June)<br />

GRAY,BARBARA, Accounts Payable Specialist<br />

KERN,ERIC, Administrative Assistant III (from June)<br />

PIANDES,GARY, Facilities Coordinator (from May)<br />

ROBERTS,R.LEE, Resources Officer (until August)<br />

VAN ROEKEL,ALEXIS, Administrative Assistant<br />

BIRK,HEATHER, Administrative Assistant<br />

MONTGOMERY,ANN, Administrative Assistant<br />

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BRANCH DIRECTOR’S REPORT<br />

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The past year has been quite productive for the Branch in several areas. We continue to make important<br />

discoveries in each of our complementary areas of inquiry; some of these are summarized in the text of<br />

this report. Many of the Branch staff members, of all levels, have roles of significance in scientific<br />

societies, andh hadve journal editorial and program review responsibilities. In <strong>2002</strong>, several international<br />

awards and honors were bestowed on Branch staff. Many postdoctoral fellows and graduate students have<br />

moved on to independent positions in academia or industry after having finished their training with us.<br />

The challenges that Federal grants management has placed on the Branch have been met, and a new and<br />

highly competent Branch administration has been built. Thus, the Branch continues as a vibrant and<br />

challenging environment for training and frontline research.<br />

Over the past year, we have concluded the first phase of the planned Branch expansion. In <strong>2002</strong> we were<br />

fortunate to recruit three outstanding new independent investigators. These were: Dr. Huilin Zhou from<br />

the <strong>Institute</strong> for Systems Biology (Seattle, USA) to head our Laboratory of Proteomic Biology; Dr. Karen<br />

Oegema from the Max Planck <strong>Institute</strong> for Cell Biology (Dresden, Germany) to head our Laboratory of<br />

Mitotic Mechanisms; and Dr. Arshad Desai also from the Max Planck <strong>Institute</strong> (Dresden, Germany) to<br />

head our Laboratory of Chromosome Biology. Their presence, enthusiasm, new ideas, and expertise place<br />

the Branch in an enviable position for future discovery. This has been recognized by the Dean of our<br />

Medical School, Dr. Ed Holmes, as well as the Chairs of their academic departments, who have<br />

enthusiastically welcomed them to the scientific community of the University of California, San Diego.<br />

As well, each of them is competing successfully at the highest levels for national and international honors<br />

and awards. These new faculty promise the sustained excellence of the Branch in future years, a bright<br />

future indeed.<br />

We continue to marvel at the attractiveness and quality of our magnificent physical and intellectual<br />

environment. We are grateful to the University and the <strong>Institute</strong> for such a wonderful opportunity and<br />

look forward to the many outstanding discoveries that will be made here.<br />

W. K. Cavenee


RESEARCH REPORT<br />

LABORATORY OF TUMOR BIOLOGY<br />

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SAN DIEGO<br />

The Laboratory is headed by Dr. Webster Cavenee and comprises two Sections with complementary<br />

interests. The Section of Molecular Cytogenetics is led by Dr. Karen Arden and is focused on three<br />

general areas: the etiologic role of the alveolar rhabdomyosarcoma-associated PAX3-FKHR fusion gene;<br />

the normal function of the FKHR sub-family of genes, each of which has been associated with cancer;<br />

and, the role of DNA methylation in prostate cancer development and progression. The Section of<br />

Human Carcinogenesis, led by Dr. Frank Furnari, is investigating the genetic lesions involved in<br />

advanced stage glioma. Specifically, the focus of this group is on the most prevalent of these lesions;<br />

amplification and truncation of the epidermal growth factor receptor gene (EGFR) and losses of<br />

heterozygosity for chromosome 10, for which the phosphatase and tensin (PTEN) homology gene, located<br />

at 10q23.3 is one target. Deciphering how mutations of these two genes alter their normal functions is an<br />

important step in understanding the transition from grade III glioma to glioblastoma.<br />

LABORATORY OF CANCER GENETICS<br />

The laboratory, headed by Dr. Richard D. Kolodner, is focused on three general areas: 1) the use of the<br />

yeast Saccharomyces cerevisiae as a model organism for identification of genes that function to suppress<br />

the accumulation of mutations and other types of genome rearrangements in DNA; 2) the development of<br />

mice containing mutations in genes that suppress the accumulation of mutations and other types of<br />

genome rearrangements in DNA and the analysis of the role of these genes in suppressing cancer<br />

susceptibility; and 3) gaining an understanding of the role of defects in these genes in inherited and<br />

sporadic cancers in humans.<br />

LABORATORY OF CELL BIOLOGY<br />

The laboratory, headed by Dr. Don Cleveland, is focused on two general areas: how chromosomes are<br />

faithfully moved into each daughter cell at cell division; and the molecular genetics of axonal growth and<br />

mechanisms of human motor neuron disease. Of particular interest is the disease, familiarly known as Lou<br />

Gehrig's disease, or amyotrophic lateral sclerosis (ALS), the most prominent motor neuron disease. One<br />

key interest is deciphering the principles involved in moving chromosomes during mitosis and identifying<br />

components of mammalian centromeres, the structures to which microtubules attach and that contain the<br />

motor molecules for translocation of chromosomes along spindle microtubules. Essentially all human<br />

motor neuron disorders are characterized by the maldistribution of neurofilaments. This is particularly<br />

true in ALS, which is characterized by selective death of motor neurons. The cause of only 1.5% of<br />

human ALS is known and this is point mutations in the enzyme superoxide dismutase. By expression of<br />

these mutations in mice, we have proven that disease arises from a toxic property of the mutant proteins.<br />

LABORATORY OF CHROMOSOME BIOLOGY<br />

The laboratory, headed by Dr. Arshad Desai, is focused on understanding the mechanisms that segregate<br />

chromosomes during cell division. Specifically, the group is analyzing kinetochores, supramolecular<br />

assemblies that form on chromosomes to connect them to spindle microtubules during cell division. The<br />

kinetochore-microtubule connection is central to accurate segregation of chromosomes and prevention of<br />

aneuploidy. This interface is also a key target of anti-mitotic chemotherapeutic drugs, such as taxol. The<br />

group is seeking to define the functionally important components of this interface and to study them in<br />

vivo as well as in vitro. Novel components of this interface are likely to be promising targets for


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 112<br />

SAN DIEGO<br />

chemotherapy and possibly more specific for cell division than tubulin, the cellular target of currently<br />

used anti-mitotics such as taxol.<br />

LABORATORY OF MITOTIC MECHANISMS<br />

The laboratory, headed by Dr. Karen Oegema, is focused on understanding the morphogenetic<br />

transformations required for cell division. Specifically, the group is using the early embryo of the soil<br />

nematode Caenorhabditis elegans as a model system because of the advantages it offers for the molecular<br />

analysis of mitosis. In other metazoans, it is difficult to examine the sub-cellular phenotypes resulting<br />

from the depletion of essential gene products. In contrast, RNA-mediated interference (RNAi) in C.<br />

elegans makes it feasible to analyze the first mitotic division of an embryo depleted of any targeted gene<br />

product. We are combining RNAi-based functional genomics with single-cell high resolution microscopy<br />

assays and biochemical characterization of native protein complexes to study three aspects of cell<br />

division: centrosome duplication and maturation; specification and assembly of kinetochores (a<br />

collaboration with the laboratory of Dr. Arshad Desai, also at the Branch); and, assembly of the cleavage<br />

furrow during cytokinesis.<br />

LABORATORY OF GENE REGULATION<br />

The laboratory, headed by Dr. Bing Ren, is interested in understanding how the complex gene regulatory<br />

networks in mammalian cells control cellular proliferation and differentiation. The research is directed at<br />

development of genomics and bioinformatics tools that allow genome wide identification of regulatory<br />

targets for transcription factors. Specifically, the lab has developed so called genome wide location<br />

analysis to detect in vivo protein-DNA interactions in mammalian cells. These tools are being applied to<br />

elucidating the actions of transcription factors that play critical roles in tumorigenesis. The main research<br />

focus has been on investigating the genomic binding sites of oncogenic transcription factors in normal<br />

and cancerous cells, and understanding the molecular mechanisms whereby their abnormal functions<br />

contribute to tumorigenesis.<br />

LABORATORY OF PROTEOMIC BIOLOGY<br />

The laboratory, headed by Dr. Huilin Zhou, is focused on proteomic analysis of regulation of proteins by<br />

post-translational modifications including protein phosphorylation, and ubiquitination. Aberrant protein<br />

phosphorylation and ubiquitination are well known to be involved many types of diseases. The group is<br />

focused on cell cycle control and receptor signaling in yeast and mammalian cells. In the past year, a new<br />

approach for quantitative analysis of protein phosphorylation by mass spectrometry has been developed<br />

and is being used to study phosphorylation states of the Anaphase Promoting Complex in yeast during<br />

different stages of cell cycle. Work towards a quantitative profiling of protein tyrosine phosphorylation<br />

during cell growth and differentiation in mammalian cells in response to various stimuli has now been<br />

initiated.


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 113<br />

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1. DE SMET C, NISHIMORI H, FURNARI FB, BÖGLER O, HUANG H-J S AND CAVENEE WK. A Novel<br />

Seven Transmembrane Receptor Induced During the Early Steps of Astrocyte Differentiation<br />

Identified by Differential Expression. Journal of Neurochemistry (<strong>2002</strong>) 81: 1-14.<br />

2. GAZZOLI I, LODA M, GARBER JE, SYNGAL S AND KOLODNER RD. An HNPCC Case Associated<br />

with Hypermethylation of the MLH1 Gene in Normal Tissue and Loss of Heterozygosity of the<br />

Unmethylated Allele in the Resulting MSI-H Tumor. Cancer <strong>Research</strong> (<strong>2002</strong>) 62: 3925-3928.<br />

3. HANNAK E, OEGEMA K, KIRKHAM M, GONCZY P, HABERMANN B AND HYMAN AA. The<br />

Kinetically Dominant Assembly Pathway for Centrosomal Asters in Caenorhabditis elegans is �-<br />

Tubulin Dependent. Journal of Cell Biology (<strong>2002</strong>) 157: 591-602.<br />

4. HARDING MA, ARDEN KC, GILDEA JW, GILDEA JJ, PERLMAN, EJ, VIARS C AND THEODORESCU<br />

D. Functional Genomic Comparison of Lineage Related Human Bladder Cancer Cell Lines with<br />

Differing Tumorigenic and Metastatic Potentials by SKY, CGH and a Novel Method of<br />

Positional Expression Profiling. Cancer <strong>Research</strong> (<strong>2002</strong>) 62: 6981-6989.<br />

5. HESS M, DASGUPTA R AND KOLODNER RD. Dominant Saccharomyces cerevisiae msh6<br />

Mutations Cause Increased Mispair Binding and Decreased Dissociation from Mispairs by<br />

MSH2-MSH6 in the Presence of ATP. Journal of Biological Chemistry (<strong>2002</strong>) 277:25545-25553.<br />

6. HOWLAND DS, LIU J, SHE Y, GOAD B, MARGAKIS NJ, KIM B, ERICKSON J, KULIK J, DEVITO L,<br />

PSALTIS G, DEGENNARO LJ, CLEVELAND DW AND ROTHSTEIN JD. Focal Loss of the Glutamate<br />

Transporter EAAT2 in a Transgenic Rat Model of SOD1 Mutant-Mediate Amyotrophic Lateral<br />

Sclerosis (ALS). Proceedings of the National Academy of Sciences USA (<strong>2002</strong>) 99: 1604-1609.<br />

7. JOHNS TG, STOCKERT E, RITTER G, JUNGBLUTH AA, HUANG H-JS, CAVENEE WK, SMYTH FE,<br />

HALL CM, WATSON N, NICE EC, GULLICK WJ, OLD LJ, BURGESS AW AND SCOTT AM. Novel<br />

Monoclonal Antibody Specific for the de2-7 Epidermal Growth Factor Receptor (EGFR) that<br />

Also Recognizes the EGFR Expressed in Cells Containing Amplification of the EGFR Gene.<br />

International Journal of Cancer (<strong>2002</strong>) 98: 398-408.<br />

8. JOHNSTONE CN, WHITE SJ, TEBBUTT NC, CZEKAY S, ARDEN KC AND HEATH JK. Analysis of the<br />

Regulation of the A33 Antigen Gene Reveals Intestine-Specific Mechanisms of Gene Expression.<br />

Journal of Biological Chemistry (<strong>2002</strong>) 277: 34531-34539.<br />

9. KAWAI K, VIARS C, ARDEN K, TARIN D, URQUIDI V AND GOODISON S. Comprehensive<br />

Karyotyping of the HT-29 Colon Adenocarcinoma Cell Line. Genes, Chromosomes and Cancer<br />

(<strong>2002</strong>) 34: 1-8.<br />

10. KITAMURA T, NAKE J, KITAMURA Y, KIDO Y, BIGGS III WH, WRIGHT CVE, WHITE MF, ARDEN<br />

KC AND ACCLI D. The Forkhead Transcription Factor Foxo1 Links Insulin Signaling to Pdx1<br />

Regulation of Pancreatic � Cell Growth. Journal of Clinical Investigation (<strong>2002</strong>) 110: 1839-1847.<br />

11. KUCHERLAPATI M, YANG K, KURAGUCHI M, ZHAO J, LIA M, HEYER J, KANE M, FAN K,<br />

RUSSELL R, BROWN AMC, KNEITZ B, EDELMANN W, KOLODNER RD, LIPKIN M AND<br />

KUCHERLAPATI R. Haplo-insufficiency of Flap Endonuclease (Fen1) Leads to Rapid Tumor<br />

Progression. Proceedings of the National Academy of Sciences USA (<strong>2002</strong>) 99: 9924-9929.<br />

12. LAU PJ, FLORES-ROZAS H AND KOLODNER RD. Isolation and Characterization of New<br />

Proliferating Cell Nuclear Antigen (POL30) Mutator Mutants that are Defective in DNA<br />

Mismatch Repair. Molecular and Cellular Biology (<strong>2002</strong>) 22: 6669-6680.


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SAN DIEGO<br />

13. LEE TI, RINALDI NJ, ROBERT F, ODOM DT, BAR-JOSEPH Z, GERBER GK, HANNETT NM,<br />

HARBISON CT, THOMPSON CM, SIMON I, ZEITLINGER J, JENNINGS EG, MURRAY HL, GORDON<br />

DB, REN B, WYRICK JJ, TAGNE J-B, VOLKERT TL, FRAENKEL E, GIFFORD DK AND YOUNG RA.<br />

Transcriptional Regulatory Networks in Saccharomyces cerevisiae. (<strong>2002</strong>) Science 298: 799-804.<br />

14. MADDOX P, DESAI A, OEGEMA K, MITCHISON TJ AND SALMON ED. Poleward Microtubule Flux<br />

is a Major Component of Spindle Dynamics and Anaphase in Mitotic Drosophila Embryos.<br />

Current Biology (<strong>2002</strong>) 12: 1670-1674.<br />

15. MYUNG KJ AND KOLODNER RD. Suppression of Genome Instability by Redundant S-phase<br />

Checkpoint Pathways in Saccharomyces cerevisiae. Proceedings of the National Academy of<br />

Sciences USA (<strong>2002</strong>) 99: 4500-4507.<br />

16. NAKAE J, BIGGS III WH, KITAMURA T, CAVENEE WK, WRIGHT CVE, ARDEN KC AND ACCILI D.<br />

Regulation of Insulin Action and Pancreatic �-cell Function by Mutated Alleles of the Gene<br />

Encoding Forkhead Transcription Factor Foxo1. Nature Genetics (<strong>2002</strong>) 32: 245-153.<br />

17. NAKAGAWA T AND KOLODNER RD. Saccharomyces cerevisiae MER3 is a DNA Helicase<br />

Involved in Meiotic Crossing Over. Molecular and Cellular Biology (<strong>2002</strong>) 22: 3281-3291.<br />

18. NAKAGAWA T AND KOLODNER RD. The MER3 DNA Helicase Catalyzes the Unwinding of<br />

Holliday Junctions. Journal of Biological Chemistry (<strong>2002</strong>) 277: 28019-28024.<br />

19. NARITA Y, NAGANE M, MISHIMA K, HUANG H-JS, FURNARI FB AND CAVENEE WK. Mutant<br />

Epidermal Growth Factor Receptor Signaling Down-regulates p27 through Activation of the<br />

Phosphatidylinositol 3-kinase/Akt Pathway in Glioblastomas. Cancer <strong>Research</strong> (<strong>2002</strong>) 62:6764-<br />

6769.<br />

20. PUTKEY FR, CRAMER T, MAYHEW MK, SILK AD, JOHNSON RS, MCINTOSH JR AND CLEVELAND<br />

DW. Unstable Kinetochore-Microtubule Capture and Chromosomal Instability Following<br />

Deletion of CENP-E. Developmental Cell (<strong>2002</strong>) 3: 351-365.<br />

21. RAO MV, GARCIA M, MIYAZAKI Y, GOTOW T, YUAN A, CRAWFORD TO, MATTINA S, WARD<br />

CM, UCHIYAMA Y, NIXON RA AND CLEVELAND DW. Gene Replacement in Mice Reveals that<br />

the Heavily Phosphorylated Tail of Neurofilament Heavy Subunit (NF-H) Does Not Affect<br />

Axonal Caliber or the Transit of Cargoes in Slow Axonal Transport. Journal of Cell Biology<br />

(<strong>2002</strong>) 158: 681-693.<br />

22. RAOUL C, ESTEVEZ AG, NISHIMUNE H, CLEVELAND DW, DELAPEYRIERE O, HENDERSON CE,<br />

HASSE G AND PETTMANN B. Motoneurons from Mice with ALS-linked SOD1 Mutations Show<br />

Increased Sensitivity to Fas-induced Death. Neuron (<strong>2002</strong>) 35: 1067-1083.<br />

23. REN B, CAM H, TAKAHASHI Y, VOLKERT T, TERRAGNI J, YOUNG RA AND DYNLACHT BD. E2F<br />

Integrates Cell Cycle Progression with DNA Repair, Replication, and G2/M Checkpoints. Genes<br />

and Development (<strong>2002</strong>) 16: 245-256.<br />

24. SCHMIDT KH, DERRY KL AND KOLODNER RD. Saccharomyces cerevisiae RRM3, a 5’ to 3’ DNA<br />

Helicase, Physically Interacts with Proliferating Cell Nuclear Antigen. Journal of Biological<br />

Chemistry (<strong>2002</strong>) 277: 45331-45337.<br />

25. SMOLKA M, ZHOU H AND AEBERSOLD R. Quantitative Protein Profiling Using Two-Dimensional<br />

Gel Electrophoresis, Isotope-coded Affinity Tag Labeling, and Mass Spectrometry. Molecular<br />

and Cellular Proteomics (<strong>2002</strong>) 1: 19-29.<br />

26. SOUSTELLE C, VEDEL M, KOLODNER RD AND NICOLAS A. Replication Protein A is Required for<br />

the Recombinational Repair of Meiotic Double-strand Breaks in Saccharomyces cerevisiae.<br />

Genetics (<strong>2002</strong>) 161: 535-547.


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SAN DIEGO<br />

27. STEINBACH JP, SUPRA P, HUANG H-JS, CAVENEE WK AND WELLER M. CD95-Mediated<br />

Apoptosis of Human Glioma Cells: Modulation by Epidermal Growth Factor Receptor Activity.<br />

Brain Pathology (<strong>2002</strong>) 12: 12-20.<br />

28. SUBRAMANIAM JR, LYONS WE, LIU J, BARTNIKAS TB, ROTHSTEIN JD, PRICE DL, CLEVELAND<br />

DW, GITLIN JD AND WONG PC. Mutant SOD1 Causes Motor Neuron Disease Independent of<br />

Copper. Nature Neuroscience (<strong>2002</strong>) 5: 301-307.<br />

29. WAHLBERG SS, SCHMEITS J, THOMAS G, LODA M, GARBER JE, SYNGAL S, KOLODNER RD AND<br />

FOX EA. Evaluation of Microsatellite Instability and Immunohistochemistry for the Prediction of<br />

Germ-line MSH2 and MLH1 Mutations in Hereditary Nonpolyposis Colon Cancer Families.<br />

Cancer <strong>Research</strong> (<strong>2002</strong>) 62: 3485-3492.<br />

30. WALCZAK CE, GAN EC, DESAI A, MITCHISON TJ AND KLINE-SMITH S. The Microtubuledestabilizing<br />

Kinesin XKCM1 is Required for Chromosome Positioning During Spindle<br />

Assembly. Current Biology (<strong>2002</strong>) 12: 1885-1889.<br />

31. WATAYA T, NUNOMURA A, SMITH MA, SIEDLAK SL, HARRIS PL, SHIMOHAMA S, SZWEDA LI,<br />

KAMINSKI MA, AVILA J, PRICE DL, CLEVELAND DW, SAYRE LM AND PERRY G. High Molecular<br />

Weight Neurofilament Proteins are Physiological Substrates of Adduction by the Lipid<br />

Peroxidation Product Hydroxynonenal. Journal of Biological Chemistry (<strong>2002</strong>) 277: 4644-4648.<br />

32. ZHOU H, RANISH J, WATTS J AND AEBERSOLD R. Quantitative Proteome Analysis by Solid-phase<br />

Isotope Tagging and Mass Spectrometry. Nature Biotechnology (<strong>2002</strong>) 20: 512-515.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. BIGGS III WH, ARDEN KC. Regulation of the FoxO Family of Transcription Factors by<br />

Phosphatidylinositol-3 Kinase Activated Signaling. Archives of Biochemistry and Biophysics<br />

(<strong>2002</strong>) 403: 292-298.<br />

2. CAVENEE WK, HADJISTILIANOU T, BÖGLER O AND NEWSHAM IF. Retinoblastoma Syndrome. In<br />

Pathology and Genetics of Tumours of Soft Tissue and Bone. C.D.M. Fletcher, K. Krishnan Unni<br />

and F. Mertens, (<strong>2002</strong>) eds. IARC Press, Lyon, France, pp. 363-364.<br />

3. CAVENEE WK. Genetics and New Approaches to Cancer Therapy. Carcinogenesis (<strong>2002</strong>)<br />

23:683-686.<br />

4. CAVENEE WK. Muscling in on Rhabdomyosarcoma. Nature Medicine (<strong>2002</strong>) 8: 1200-1201.<br />

5. DESAI A. The (theoretical) Yin and Yang of Spindle Mechanics. Developmental Cell (<strong>2002</strong>)<br />

3:465-467.<br />

6. KLEIHUES P, LOUIS DN, SCHEITHAUER BW, RORKE LB, REIFENBERGER G, BURGER PC AND<br />

CAVENEE WK. The WHO Classification of Tumors of the Nervous System. Journal of<br />

Neuropathology and Experimental Neurology (<strong>2002</strong>) 61: 215-225.<br />

7. KOLODNER, RD, AND FLORES-ROZAS, H. Mismatch Repair: Biochemistry and Genetics.<br />

Encyclopedia of Cancer, 2nd Edition. (<strong>2002</strong>) Ed., Bertino, J. Academic Press, Elsevier Science,<br />

USA, San Diego 3, 179-185.<br />

8. KOLODNER, RD,PUTNAM, CD,MYUNG, K. Maintenance of Genome Stability in Saccharomyces<br />

cerevisiae. Science (<strong>2002</strong>) 297 552-557.<br />

9. NEWSHAM I, Hadjistilianou D, and Cavenee WK. Retinoblastoma. In The Genetic Basis of<br />

Human Cancer, Second Edition. B. Vogelstein and K.W. Kinzler, (<strong>2002</strong>) eds. McGraw-Hill, New<br />

York, pp. 357-386.


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SAN DIEGO<br />

10. SHAH,JV AND CLEVELAND, DW. Slow Axonal Transport: Fast Motors in the Slow Lane. Current<br />

Opinions in Cell Biology (<strong>2002</strong>) 14, 58-62.


SÃO PAULO BRANCH<br />

OF CANCER BIOLOGY AND EPIDEMIOLOGY<br />

SÃO PAULO,BRAZIL<br />

Staff List: Page 118<br />

Branch Director’s <strong>Report</strong>: Page 123<br />

<strong>Research</strong> <strong>Report</strong>: Page 124<br />

Publications: Page 128<br />

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STAFF LIST<br />

BRENTANI,RICARDO R., Member, Director<br />

CELLULAR AND MOLECULAR BIOLOGY GROUP<br />

BRENTANI,RICARDO R., Member, Director<br />

TORLONI,HUMBERTO, Associate Member<br />

MARTINS,VILMA R., Assistant Member<br />

CASTRO,ROSA MARIA R. P. S., PhD Student<br />

COSTA,FABRICIO FALCONI, PhD Student<br />

FREITAS,ADRIANA R.O., PhD Student<br />

HAJJ,GLAUCIA NOELI MAROSO, PhD Student<br />

LEE,KIL SUN, PhD Student<br />

LIMA,FLÁVIA REGINA SOUZA, PhD Student (from July)<br />

LOPES,MARILENE HOHMUTH, PhD Student<br />

MURAS,ANGELITA GONZALEZ, PhD Student<br />

SILVA,RICARDO LUIS ALVES, PhD Student<br />

KINDLMANN,KATHLEEN, M.Sc.Student<br />

MOTTA,WANDA, M.Sc. Student (until October)<br />

FONSECA,ROGÉRIO DA SILVA, Under Graduate Student (from April)<br />

LANDEMBERGER,MICHELE CHRISTINE, Under Graduate Student<br />

NASCIMENTO,CARLOS F., Technician<br />

NOMIZO,REGINA, Technician<br />

NONOGAKI,SUELY, Technician<br />

DA SILVA,MIYUKI FUKUDA, Technician<br />

COMPUTATIONAL BIOLOGY GROUP<br />

SOUZA,SANDRO JOSÉ, Associate Member<br />

KUVA,SILVIA MARIA, PhD Student<br />

LEERKES,MAARTEN, PhD Student<br />

VIBRANOVSKI,MARIA DULCETTI, PhD Student (from September)<br />

SAKABE,NOBORU JO, M.Sc.Student (from April)<br />

SLAGER,NATANJA SARA KIRSCHBAUM, M.Sc.Student (from November)<br />

GALANTE,PEDRO A. F., Under Graduate Student<br />

LOPES,GRAZIELA MIE P., Under Graduate Student<br />

SÃO PAULO<br />

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SCATOLINI JR., ODAIR, Under Graduate Student (from April)<br />

FABRI,ARTUR PENHA, Technician<br />

DE SOUZA,JORGE STEPHANO SANTANA, Technician<br />

ZAIATS,ANDRE, Technician (until October)<br />

OSORIO,ELISSON, Technician (until October)<br />

LABORATORY OF CANCER GENETICS<br />

SIMPSON,ANDREW J., Member (until October)<br />

CABALLERO,OTÁVIA L. S., Assistant Member<br />

BULGARELLI,ADRIANA, PhD Student<br />

PICOLLI,FABIO DE SIMONI, PhD Student (from May)<br />

SANTOS,MILENA J. S FLÓRIA LIMA, PhD Student<br />

SREDNI,SIMONE TREIGER, PhD Student<br />

VERBISCK,NEWTON VALÉRIO, PhD Student (from November)<br />

DE MOURA,RICARDO PEREIRA, M.Sc.Student<br />

DE OLIVEIRA,MARIANA BRAIT RODRIGUES, M.Sc.Student<br />

GRANATO,MARIANA FACCHINI, Under Graduate Student<br />

DA CUNHA,DANIELLE RENZONI, Technician<br />

PAIXÃO,VALERIA APARECIDA, Technician<br />

SEIXAS,ANDREA QUINTELLA DE ANDRADE, Technician<br />

DE OLIVEIRA,ANDRE LUIZ VETTORE, Senior Investigator<br />

DELLAMANO,MARCIA, M.Sc.Student<br />

LABORATORY OF MOLECULAR BIOLOGY AND GENOMICS<br />

CAMARGO,ANAMARIA ARANHA, Assistant Member<br />

BETTONI,FABIANA, M.Sc.Student<br />

PARMIGIANI,RAPHAEL BESSA, M.Sc.Student<br />

MONTEIRO,ELISANGELA, Technician<br />

SALIM,ANA CHRISTINA DE M., Technician<br />

SILVA,ANA PAULA MEDEIROS, M.Sc.Student<br />

DA ROCHA,JOSE C. M.Sc. Student (until October)<br />

IERARDI,DANIELA FILIPPINI, M.Sc.Student (from February)<br />

DE CARVALHO,FABRÍCIO, Technician (from March)<br />

KAIANO,JANE HARUKO L., Technician<br />

CARRARO,DIRCE MARIA, Senior Investigator<br />

SÃO PAULO<br />

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PASSETTI,FABIO, Technician (from November)<br />

PIRES,LILIAN CAMPOS, Technician<br />

MOLECULAR IMMUNOLOGY GROUP<br />

REIS,LUIZ FERNANDO LIMA, Assistant Member<br />

DE CARVALHO,ALEX FIORINI, Senior Investigator (Microarray)<br />

ABRANTES,EDUARDO FERNANDES, PhD Student<br />

DIAS,ADRIANA ABALEN M., PhD Student<br />

DINIZ,SUZANA NOGUEIRA, PhD Student<br />

MEIRELES,SIBELE INACIO, PhD Student<br />

GOMES,LUCIANA INÁCIA, PhD Student<br />

STOLF,BEATRIZ SIMONSEN, PhD Student<br />

TEMINI,LARA, PhD Student<br />

COLÓ,ANNA ESTELA LUIZA, M.Sc.Student (from August)<br />

ESTEVES,GUSTAVO HENRIQUE, M.Sc.Student<br />

PAGOTTO,ANA HELENA, M.Sc.Student (from November)<br />

NEVES FILHO,EMERSON, Under Graduate Student (from November)<br />

MARTINS,WALESKA KERLLEN, Technician (Microarray)<br />

MENDONÇA NETO,CHAMBERLEIN E., Technician (Microarray)<br />

VIROLOGY GROUP<br />

VILLA,LUISA LINA, Member<br />

DE SOUZA,PATRICIA SAVIO, PhD Student<br />

GONZALEZ,LAURA C. S., PhD Student<br />

JUNES,KATIANA DE S., PhD Student<br />

PIERULIVO,ENRIQUE MARIO BOCCARDO, PhD Student<br />

THOMMAN,PATRÍCIA, PhD Student<br />

TREVISAN,ANDREA, PhD Student<br />

RABACHINI,TATIANA, M.Sc.Student<br />

SILVESTRE,RODRIGO VELLASCO D., M.Sc.Student<br />

DE PAULA,LENICE GALAN, Nurse<br />

PINTO,MARIA LUIZA B. G., Nurse<br />

COSTA,MARIA CECÍLIA, Technician<br />

FERREIRA,SILVANEIDE A., Technician<br />

MACIAG,PAULO, Technician (until July)<br />

SÃO PAULO<br />

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MIYAMURA,ROMULO A., Technician<br />

PEREIRA SOBRINHO,JOÃO S., Technician<br />

PRADO,JOSE CARLOS MANN, Technician<br />

ROSSI,ADRIANA CRISTINA MACHADO, Technician (from October)<br />

SANTOS,MONICA I. R., Technician<br />

MENDONÇA,ROSELI DE SOUZA, Laboratory Worker<br />

LABORATORY SUPPORT -GENERAL<br />

CARNEIRO,IZABEL COSTA, Laboratory Worker<br />

SAMPAIO,SIRLEIA MIRANDA, Laboratory Worker<br />

SOUSA,LEONEL C. B., Photographic Artist<br />

DA CRUZ,JOÃO AQUILAU, Animal Keeper<br />

GONÇALVES,WANDERLEY LOURENÇO, Assistant Animal Keeper (from July)<br />

SILVA,DOMINGOS S. M., Assistant Animal Keeper<br />

SILVA,EUDES, Maintenance Supervisor<br />

MONTEIRO,LUIZ ANTONIO, Maintenance Assistant<br />

SILVA,FRANCISCO SAMPAIO, Maintenance Assistant<br />

ADMINISTRATION AND SECRETARIAT<br />

CAMPOS,PAULO ROBERTO, Manager Accountant<br />

WALTER,ANTJE LUISE, Assistant Administrator<br />

COELHO,VIVIANI AP.MARTINS, Accountant (from May)<br />

SOUZA,MARIA CONCEIÇÃO, General Services<br />

ROCHA,ROSELI Z., Buyer - Importations<br />

DA ROCHA,RUBENS ANTONIO, Storekeeper<br />

MONTEIRO,JAQUELINE, Executive Secretary<br />

COSTA,GISELY DA, <strong>Research</strong> Secretary (Microarray)<br />

HERING,MARIA STELLA LEME, <strong>Research</strong> Secretary (Virology)<br />

MOCHO,CAMILA, <strong>Research</strong> Secretary (Human Cancer Genome) (until July)<br />

PEREIRA,ANA CLÁUDIA, <strong>Research</strong> Secretary (Computational Biology)<br />

RIBEIRO,ELIANE LOPES, <strong>Research</strong> Secretary (Cancer Genetics)<br />

PEREIRA,CARLA REGINA F., Secretary<br />

DA SILVA,RAQUEL HESSEL, Project Clerk (Virology)<br />

CAPELUCCI,RENATA JULIANA SOUZA, Office Clerk<br />

DE SOUSA,ANDRÉA RAMOS, Office Clerk (from April)<br />

SÃO PAULO<br />

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DE SOUZA,RAQUEL RODRIGUES, Office Clerk<br />

PARAGUASSÚ,ERICA, Office Clerk<br />

ROCHA,JULIANA FREITAS, Office Clerk<br />

CRISTINO JÚNIOR,ELIAS LOPES, Office Boy<br />

FIETZEK,BIRGIT, Receptionist<br />

SÃO PAULO<br />

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BRANCH DIRECTOR’S REPORT<br />

SÃO PAULO<br />

The challenge to cancer research is to identify all genes related to cancer, and to characterize their<br />

genomic changes in order to improve detection, diagnosis, intervention and ultimately prevention.<br />

The São Paulo Branch has a strong background in genomics, and has participated over the last<br />

few years in several bacterial genome projects carried out in Brazil. Currently, the Transcript<br />

Finishing Initiative (TFI), a collaborative project funded by FAPESP and the <strong>LICR</strong>, and<br />

involving more than 30 laboratories from the State of São Paulo is identifying new human<br />

transcripts, and defining the structure of approximately 200 human transcripts presently only<br />

partially represented by publicly-available EST data.<br />

There is emerging evidence that the polymorphic frequencies in, and methylation profiles of,<br />

individual cancer types might have important prognostic implications for clinical monitoring, risk<br />

assessment, and even therapeutic considerations. Recently the Laboratory of Molecular Biology<br />

and Genomics, and the Computational Biology Group have focused on exploiting the enormous<br />

amount of publicly-available human genome data in order to identify new genes related to cancer,<br />

and to understand the basic genetic mechanisms underlying tumor development. The Laboratories<br />

of Molecular Biology and Genomics and Cancer Genetics have analyzed the frequencies of<br />

polymorphisms in certain genes, and abnormal sequence methylation to establish population, and<br />

treatment risks, respectively. Most women become infected by human papilloma virus (HPV)<br />

during some part of their lives, and whilst some of these infections progress to malignancy, most<br />

remain self-limiting, subclinical, or benign. The Virology Group is providing valuable<br />

information about the natural history of these infections, including polymorphic variation<br />

between affected and non-affected women, and the risk of cervical disease onset.<br />

The Cell and Molecular Biology Group has now fully characterized the interaction between the<br />

cellular prion protein (PrP c ) and the stress inducible protein (STI1). Additionally the cellular<br />

trafficking of PrP c and its interactions with cellular matrix proteins has been analysed.<br />

The Laboratory of Inflammation has continued its analyses of tumor necrosis factor (TNF), by<br />

generating transgenic mice to use as a model for inflammatory response. Additionally, the<br />

laboratory has continued to optimize microarray technologies, data collection, and analyses.<br />

R. R. Brentani<br />

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RESEARCH REPORT<br />

CELLULAR AND MOLECULAR BIOLOGY GROUP<br />

SÃO PAULO<br />

The Cellular and Molecular Biology Group, led by Dr. Ricardo Brentani, is working on the<br />

physiological properties of the cellular prion protein. The group has fully characterized the<br />

interaction between PrP c and STI1 and showed that this interaction is responsible for cellular<br />

protection against apoptosis through cAMP/PKA pathways. The cellular traffic of PrP c was also<br />

unraveled using a fusion GFP-PrP c protein and live-cell analyses by confocal microscopy. During<br />

the last year the group also showed that PrP c association to laminin in vivo promotes short and<br />

long-term memory formation through cAMP and PKA signaling. PrP c was defined as a cellular<br />

ligand for vitronectin, although it does not bind to fibronectin or type IV collagen. The binding<br />

site to both laminin and vitronectin was mapped and showed that, in vitronectin, the interacting<br />

domain does not include the RGD sequence, which is the major cell attachment site. Therefore,<br />

PrP c binding to this molecule may be in a region where any other cellular receptors, in particular<br />

integrins, might interact. PrP c -vitronectin promotes axonal growth in dorsal-root glangia from<br />

12.5 day embryos, indicating that this molecule induces specific pathways on neuronal<br />

differentiation through distinct protein interactions. Since PrP c is expressed in most cell types we<br />

are presently investigating its physiological functions as a receptor for cellular matrix proteins in<br />

other tissues besides the central or peripheral nervous systems.<br />

COMPUTATIONAL BIOLOGY GROUP<br />

The Computational Biology Group, led by Dr. Sandro Souza, has continued its efforts to decipher<br />

the complexity of the human transcriptome in normal and tumor cells. In collaboration with Drs.<br />

Greg Riggins from Duke University Medical Center (USA) and Bob Strausberg from the<br />

National Cancer <strong>Institute</strong> (USA) the group developed a new strategy to explore the large amount<br />

of SAGE data that are publicly available, and thus easily identify transcripts, redundant tags, and<br />

events of internal priming. To enhance the utility of the data, the group launched SAGE Genie, a<br />

web site for the analysis and presentation of the SAGE data (http://cgap.nci.nih.gov/SAGE).<br />

SAGE Genie provides an automatic link between gene names and SAGE transcript levels,<br />

accounting for many alternative transcripts and many potential errors. The informatics advances<br />

presented in SAGE Genie provides a rapid and intuitive view of transcript expression in the<br />

human body, displayed on the SAGE Genie Anatomic Viewer. Other bioinformatic tools that<br />

have generated for the scientific community include modifications in the ExInt database of<br />

Genbank entries containing annotated introns, and the development of software called pp-Blast<br />

that uses the publicly available Blast package and PVM (parallel virtual machine) to partition a<br />

multi-sequence query across a set of nodes with replicated or shared databases. Benchmark tests<br />

showed that pp-Blast running in a cluster of 14 PCs outperformed conventional Blast running in<br />

large servers, so that we were able to map all human cDNAs onto the draft of the human genes in<br />

less than six days.<br />

The group also continues to use EST data for tumor profiling and identification of transcripts that<br />

are differentially expressed between normal and tumor tissues. One focus has been the<br />

development of a bioinformatics pipeline to identify genes candidates overexpressed in breast<br />

tumors. More than 100 candidates have been identified, and a literature search has shown that for<br />

28 of these candidates, there was evidence of overexpression in breast or other tumors.<br />

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LABORATORY OF CANCER GENETICS<br />

SÃO PAULO<br />

The main goal of the Laboratory of Cancer Genetics, which is headed by Dr. Andy Simpson, is to<br />

identify and evaluate molecular markers for their diagnostic or prognostic value in clinical<br />

oncology. In one project, the group analyzed the promoters of a group of genes frequently<br />

silenced by aberrant methylation in many different tumor types, in formaline-fixed or paraffinembedded<br />

bladder cancer samples, and also in urine samples. The methylation data was then<br />

correlated with clinicopathological parameters. The results suggested that the methylation profile<br />

may be useful as a potential bladder cancer diagnostic and prognostic biomarker.<br />

The group also tested thiopurine methyl transferase (TPMT) polymorphisms as markers of<br />

therapeutic response in patient cohorts with childhood acute lymphoblastic leukemia (ALL).<br />

Patients who have inherited very low levels of TPMT activity are at greatly increased risk for<br />

thiopurine-induced toxicity when treated with standard drug doses, but TPMT-deficient patients<br />

can be successfully treated with a 10- to 15-fold lower dosage of these medications. This was the<br />

first analysis of TPMT polymorphism frequencies in the Brazilian population, and the frequencies<br />

were found to be comparable to other studies. Polymorphisms in the gene for alcohol<br />

dehydrogenase (ADH) 3 were assessed for prognostic markers of aerodigestive tract cancers<br />

(UADT) which have tobacco and alcohol consumption as the main risk factors. ADH3 activity<br />

can produce variability in alcohol metabolic capacity between individuals. The results suggested<br />

that there is a particular genotype that may be a risk factor in Brazilians for small quantities of<br />

ethanol and tobacco. The group extended this study by investigating the relationship between<br />

cyclin D1 (CCND1) polymorphisms and susceptibility for UADT cancers, with the results<br />

suggesting that CCND1 polymorphisms may add to the risk factor for UADT cancers, especially<br />

in individuals without a history of drinking.<br />

LABORATORY OF MOLECULAR BIOLOGY AND GENOMICS<br />

The laboratory, headed by Dr. Anamaria Camargo, has been using an alternative strategy for the<br />

identification of new human genes using publicly available sequencing data. In one study,<br />

alignments between expressed sequences and the human genome have been used to identify<br />

additional human transcripts, including novel disease gene candidates, located at the Hereditary<br />

Prostate Cancer Region (HPC1) on chromosome 1q25. In a second study, involving<br />

collaborations with Dr. S. Antonarakis from the University of Geneva (Switzerland) and Dr.<br />

Victor Jongeneel from the <strong>LICR</strong> Office of Information and Technology, a reliable algorithm to<br />

extract, and stringently map sequences that contain bona fide 3’ transcript ends (3’-tags) to the<br />

genome was developed. The algorithm incorporates features such as CpG islands, repeats and<br />

gene predictions, and identified 19 new transcripts on chromosome 21.<br />

The group is also identifying genes that have differentially methylated promoters in tumors. One<br />

such gene is the adhesion molecule ADAM23 (A Desintegrin And Metalloprotease domain 23),<br />

which exhibits 5’ hypermethylation in two-thirds of tumor cell lines and primary tumors.<br />

Promoter hypermethylation was strongly associated with reductions in both mRNA and protein<br />

expression. Primary breast tumors with a more advanced grade showed a higher degree of<br />

methylation, suggesting that ADAM23 inactivation may play a crucial role in the malignancy of<br />

breast tumors.<br />

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VIROLOGY GROUP<br />

SÃO PAULO<br />

Human papillomavirus-16 (HPV-16) and many related genital HPV types are carcinogenic, and<br />

persistent HPV infection, with the high-risk types, is the primary cause of cervical cancer. Dr.<br />

Luisa Villa’s Virology Group is conducting a study that is one of the largest, and has one of the<br />

longest follow-ups of about 2,000 women from a high- risk area for cervical cancer in Brazil. The<br />

information stemming from this large longitudinal study of the natural history of HPV infections<br />

and risk of cervical cancer is providing seminal information for the development of new<br />

prevention measures to control cervical cancer, including prophylactic HPV vaccines that are<br />

presently in phase III trials to determine their efficacy.<br />

Some of the information that the group is accumulating about the risk of HPV infection, and<br />

ultimately the risk for cervical cancer, includes: that measurement of viral load can identify<br />

women at risk of developing cervical cancer precursors; that both episomes and integrated<br />

genomes of HPV-16 are found in different cervical lesion grades; that intratypic variation of<br />

HPV-16 is an important predictor of progression to clinically relevant cervical lesions; that<br />

enhanced promoter activity could account for differential oncogenic potential associated<br />

epidemiologically with certain variants of HPV-16; and that some alleles and haplotypes of MHC<br />

Class II genes are related to HPV infection prevalence and persistence.<br />

MOLECULAR IMMUNOLOGY GROUP<br />

The Molecular Immunology Group, led by Dr. Luis Reis, continues to concentrate efforts in two<br />

major components: The Laboratory of Inflammation, and the Gene Expression and Microarray<br />

Facility.<br />

The Laboratory of Inflammation<br />

The work related to inflammation is focussed on the structural and functional characterization of<br />

genes that are expressed during the inflammatory response, especially those that are induced by<br />

tumor necrosis factor (TNF), a pleiotropic, pro-inflammatory cytokine that plays an important<br />

role in host defense and also, in the patho-physiology of some malignancies. In order to<br />

contribute to the understanding of the molecular mode of action of TNF, the group has<br />

concentrated its studies on the structural and functional characterization of two TNF-stimulated<br />

genes, TSG-14 / PTX-3 and TSG5. The TSG-14 gene encodes a 42 kDa secreted glycoprotein<br />

that belongs to the pentraxin family of the acute phase response. Transgenic mice over expressing<br />

the murine TSG-14 gene, under the control of its own promoter, have been used as a model for<br />

inflammatory response. TSG-14 transgenic mice are more resistant to the systemic administration<br />

of lipopolysaccharide (LPS), as well as to the sepsis caused by cecal ligation and puncture (CLP).<br />

In contrast, these mice are highly sensitive to ischemia/reperfusion, with higher lethality than the<br />

control group. This pro-inflammatory action of TSG-14 appears to be mediated via increased<br />

production of TNF and some chemokines. More recently, the group has also provided evidence<br />

that macrophages from these mice have a higher capacity to phagocytes and increase fungicidal<br />

activity with increased expression of TLR-2 and Dectini-1.<br />

Gene Expression and Microarray Facility<br />

In relation to the global analysis of gene expression, the group has continued to improve the São<br />

Paulo Branch’s Microarray Facility. Optimization of probe selection, probe preparation,<br />

hybridization, and data acquisition have resulted in data sets of better quality. Additionally, a<br />

protocol for RNA amplification has been established, which allows the utilization of biopsyderived<br />

samples. An important achievement has been the consolidation of a collaborative effort<br />

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SÃO PAULO<br />

with two groups at the Mathematics and Statistics Department at the University of São Paulo. In<br />

collaboration with the group led by Dr. Junior Barreira, new image acquisition software that is<br />

based on image segmentation, has been validated, which confers major advantages in accuracy<br />

and reproducibility. In collaboration with the group led by Drs. Eduardo Neves and Roberto<br />

Hirata, a project-specific set of mathematical and statistical tools for data analyses has been<br />

developed, which imposes stringent criteria for the identification of expression patterns.<br />

The Facility’s research effort is concentrated on the identification of molecular markers that can<br />

contribute to improved, molecular-based, diagnosis and prognosis for human cancer. Main<br />

projects are related to the identification of markers for disease classification in gastric, and head<br />

and neck tumors. The group has also initiated efforts in investigating gene expression profiles in<br />

esophageal cancers, and melanomas.<br />

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PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

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SÃO PAULO<br />

1. BOON, K., OSORIO, E.C., GREENHUT, S.F., SCHAEFER, C.F., SHOEMAKER, J., POLYAK, K.,<br />

MORIN, P.J., BUETOW, K.,STRAUSBERG, R.L., DE SOUZA, S.J., RIGGINS, G.J. An anatomy of<br />

normal and malignant gene expression. Proceedings of the National Academies of Science USA<br />

<strong>2002</strong> 99:11287-11292.<br />

2. BRITO C.F.A, OLIVEIRA G.C., OLIVEIRA S.C., STREET M., RIENGROJPITAK S., WILSON R.A.,<br />

SIMPSON A.J.G., CORREA-OLIVEIRA R. Sm14 gene expression in different stages of the<br />

Schistosoma mansoni life cycle and immunolocalization of the Sm14 protein within the adult<br />

worm. Brazilian Journal of Medical and Biological <strong>Research</strong> <strong>2002</strong> 35:377-381.<br />

3. CABALLERO OTAVIA L., VICENTE A. RESTO, MEERA PATTURAJAN, MING ZHOU GUO, JAMES<br />

ENGLES,ROBERT YOCHEM,EDWARD RATOVITSKI,MICHAEL MATUNIS,DAVID SIDRANSKY, AND<br />

JIN JEN. Interaction and colocalization of an ubiquitin hydrolase, PGP9.5 with p38 JAB1 and p27 KIP1<br />

Oncogene <strong>2002</strong> May 2;21(19):3003-10.<br />

4. CABRAL A.L., LEE KIL S., MARTINS V.R. Regulation pf PrPc gene expression depends on<br />

chromatin conformation Journal of Biological Chemistry <strong>2002</strong> 277 (7):5675,5682.<br />

5. CAMARGO A.A., DE SOUZA S.J., BRENTANI R.R., SIMPSON A.J.G. Human gene discovery through<br />

experimental definition of transcribed regions of the human genome. Current Opinion in<br />

Chemical Biology <strong>2002</strong> 6(1):13-16.<br />

6. Chiarini L.B., Freitas A.R.O., Zanata S.M., Brentani R.R., Martins V.R., Linden R. Cellular prion<br />

protein transduces neuroprotetive signals. EMBO Journal <strong>2002</strong> (21)13:3317-3326.<br />

7. COITINHO, A.S., DIETRICH, M.O, HOFFMANN, A., DAL-IGNA, O.P., SOUZA, D.O.,<br />

MARTINS, V.R., BRENTANI, R.R., IZQUIERDO, I., LARA, D. Decreased hyperlocomotion<br />

induced by MK-801, but not amphetamine and caffeine in mice lacking cellular prion protein.<br />

Molecular Brain <strong>Research</strong> <strong>2002</strong> 107: 190-194,<br />

8. DALLA TORRE C.A., MACIEL R.M.B., PINHEIRO N.A., ANDRADE J.A.D., TOLEDO S.R.C., VILLA<br />

L.L., CERUTTI J.M. TRAP – silver staining, a higly sensitive assay for measuring telomerase<br />

activity in tumor tissue and cell lines. Brazilian Journal of Medical and Biological <strong>Research</strong> <strong>2002</strong><br />

35:65-68.<br />

9. ENDO T., FEDOROV, A., DE SOUZA, S.J. AND GILBERT, W. Do introns favor or avoid regions of<br />

amino acid conservation? Molecular Biology and Evolution <strong>2002</strong> 19: 521-522.<br />

10. FERREIRA L.R.P., ABRANTES E.F., RODRIGUES C.V., CAETANO B., SALIN A.C., REIS L.F.L.,<br />

GAZZINELLI R.T. Identification and characterization of a novel mouse gene encoding a RAS<br />

associated Guanine Nucleotide Exchange Factor: Expression in macrophages and myiocarditis<br />

elicited by Trypanosoma cruzi parasites. Journal of Leukocyte Biology <strong>2002</strong> 72(6):1215-27.<br />

11. ISELI C., STEVENSON B.J., DE SOUZA S.J., SAMAIA H.B., CAMARGO A.A, BUETOW K.H.,<br />

STRAUSBERG R.L., SIMPSON A.J.G., BUCHER P., JONGENEEL V.C. Long-range heterogeneity at<br />

the 3’ ends of human mRNAs. Genome <strong>Research</strong> <strong>2002</strong> 12:1068-1074.<br />

12. LEERKES M.R., CABALLERO O., MACKAY A., TORLONI H., O’HARE M.J.., SIMPSON A.J.G., DE<br />

SOUZA S.J., “In silico” comparison of the transcriptome derived from purified normal breast cells<br />

and breast tumor cell lines reveals candidate up-regulated genes in breast tumor cells, Genomics<br />

<strong>2002</strong> 79 (2):257-265.


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13. LOPES A., BEZERRA A.L.R., PINTO C.A.L., SERRANO S.V., MELLO C.A., VILLA L.L. p53 as a new<br />

prognostic factor for lymph node methasis in penile carcinoma: analysis of 82 patients treated<br />

with amputation and bilateral lymphadenectomy, The Journal of Urology <strong>2002</strong> 168:81-86.<br />

14. MACIAG P.C., SCHLECHT N.F., SOUZA P.S.A., FRANCO E.L., ROHAN T.E., VILLA L.L.<br />

Polymorphisms of the human leukocyte antigen DRB1 and DQB1 genes and the natural history<br />

of human papillomavirus infection. Journal of Infectious Diseases <strong>2002</strong> 186:164-72.<br />

15. MAGALHÃES A.C., SILVA J. A., LEE K.S., MARTINS V.R., PRADO V.F., FERGUSON S.S.G., GOMES<br />

M.V., BRENTANI R.R., PRADO M.A. Endocytic intermediates involved with the intracellular<br />

trafficking of a fluorescent cellular prion protein. Journal of Biological Chemistry <strong>2002</strong><br />

277(36):33311-33318.<br />

16. MARTINS V.R., LINDEN R., PRADO M.A., WALZ R., SAKAMOTO A.C., IZQUIERDO I., BRENTANI<br />

R.R. Cellular prion protein: on the road for functions. FEBS Letters <strong>2002</strong> 512: 25-28.<br />

17. MARTINS, V.R. & BRENTANI, R.R. The biology of the cellular prion protein. Neurochemistry<br />

International <strong>2002</strong> 41:353-355.<br />

18. MINDRESCU C, A. A. M. DIAS,R.J.OLSZEWSKI,M.J.KLEIN,LUIZ F. L. REIS,H-G.WISNIEWSKI.<br />

Reduced susceptibility to Collagen-induced arthritis in DBA/1J mice expressing the TSG-6<br />

transgene. Arthritis & Rhemautism <strong>2002</strong> 46(9):2453-64.<br />

19. NONNENMACHER B., BREITENBACH V., VILLA L.L., PROLLA J.C., BOZZETTI M.C. Genital human<br />

papillomavirus infection identification by molecular biology among asymptomatic women<br />

Revista Saúde Pública <strong>2002</strong> 36(1):95-100.<br />

20. PICCONI M.A., GRONDA J., ALONIO L.V., VILLA L.L., SICHERO L., MIRANDA S, BÁRCENA M.,<br />

TEYSSIE A. Human Papilloma virus in Quechua women from Jujuy with high frequency of<br />

cervical cancer: viral types and HPV-16 variants. Medicina <strong>2002</strong> 62(3):209-220.<br />

21. REYMOND A., CAMARGO A.A., DEUTSCH S., STEVENSON B.J., PARMIGIANI R.B., UCLA C.,<br />

BETTONI F., ROSSIER C., LYLE R., GUIPONNI M., DE SOUZA S.J., ISELI C., JONGENEEL V.,<br />

BUCHER P., SIMPSON A.J.G., ANTONARAKIS S.E. The dynamics process of gene discovery:<br />

characterization of 19 novel transcripts from human chromosome 21. Genomics <strong>2002</strong> 79(6):824-<br />

32.<br />

22. ROPERT C., FERREIRA L.R.P., CAMPOS M.A.S., PROCÓPIO D.O., TRAVASSOS L.R., FERGUSON<br />

M.A.J., REIS L.F.L., TEIXEIRA M.M., ALMEIDA I.C., GAZZINELLI R.T. Macrophage signaling by<br />

glycosylphosphatidyl-inositol-anchored mucin-like glycoproteins derived from Trypanosoma<br />

cruzi trypomastigotes. Microbes Infection <strong>2002</strong> 4:1015-1025.<br />

23. ROSSI B.M., LOPES A., FERREIRA F.O., NAKAGAWA W.T., FERREIRA C.C.N., ROCHA J.C.C.,<br />

SIMPSON C., SIMPSON A.J.G. hMLH1 and hMSH2 Gene Mutation in Brazilian families with<br />

suspected hereditary nonpolyposis colorectal cancer. Annals of Surgical Oncology <strong>2002</strong> 9(6):555-<br />

561.<br />

24. ROULET E., BUSSO S., CAMARGO A.A., SIMPSON A.J.G., MERMOD N., BUCHER P. Highthroughput<br />

SELEX-SAGE method for quantitative modeling of transcription-factor binding sites.<br />

Nature Biotechnology <strong>2002</strong> 20:831-835.<br />

25. SAKHARKAR M., PASSETTI F., DE SOUZA J.E., LONG M., DE SOUZA S.J. ExInt: An Exon Intron<br />

Database. Nucleic Acids <strong>Research</strong> <strong>2002</strong> 30, 1, 191-194,<br />

26. SILVA N.M., RODRIGUES C.V., SANTORO M.M., REIS L.F.L.R., ALVAREZ-LEITE J.I., GAZINELLI<br />

R.T. Expression of indoleamine 2,3-dyoxigenase, tryptophan degradation and kynurenine


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SÃO PAULO<br />

formation during in vivo infection with Toxoplasma gondii: induction by endogenous IFN-� and<br />

requirement of IRF-1. Infection and Immunity <strong>2002</strong> 70(2):859-868.<br />

27. SIMPSON A.J.G. Sequence-based genomics Genome Biology <strong>2002</strong> 3(9): reports 4029.1-4929.2.<br />

28. SOUZA D.G., SOARES A.C., PINHO V., TORLONI H., REIS L.F.L., MARTINS M.T., DIAS A.A.<br />

Increased mortality and inflammation in TSG-14 transgenic mice after ischemia and reperfusion<br />

injury Increased mortality and inflammation in TSG-14 transgenic mice after ischemia and<br />

reperfusion injury. American Journal of Pathology <strong>2002</strong> 160(5):1755-1765,<br />

29. STRAUSBERG RL, CAMARGO AA, RIGGINS GJ, SCHAEFER CF, DE SOUZA SJ, GROUSE LH, LAL<br />

A, BUETOW KH, BOON K, GREENHUT SF, SIMPSON A.J.G. An international database and<br />

integrated analysis tools for the study of cancer gene expression. Pharmacogenomics Journal<br />

<strong>2002</strong> 2(3):156-64.<br />

30. VAN SLUYS M.A., VITORELLO-MNTEIRO C.B., CAMARGO L.E.A., MENCK C.F.M., DA SILVA<br />

A.C.R., FERRO J.A., OLIVEIRA M.C., SETÚBAL J.C., KITAJIMA J.P., SIMPSON A.J.G. Comparative<br />

genomic analysis of plant-associated bacteria. <strong>Annual</strong> Reviews of Phytopathology <strong>2002</strong> 40:169-<br />

189.<br />

31. VILLA LL, BERNARD HU, KAST M, HILDESHEIM A., AMESTOY G., FRANCO E.L. Past, present and<br />

future of HPV research: highlights from the 19 th International Papillomavirus Conference-<br />

HPV2001. Virus <strong>Research</strong> <strong>2002</strong> 89(2):163-173.<br />

32. Villa-Verde D.M., Monteiro E.S., Jasiulionis M.G., Oliveira D.A.F., Brentani R.R., Savino W.,<br />

Chammas R. – Galectin-3 Modulates carbohydrate-dependent thymocyte interactions with the<br />

tymic microenviroment. European Journal Immunology <strong>2002</strong> 32(5):1434-44,<br />

33. WALZ, R., CASTRO, R.M.R.P.S., VELASCO, T.R., CARLOTTI JR C.C., SAKAMOTO, A.C.,<br />

BRENTANI, R.R., MARTINS, V.R. Cellular prion protein: implications in seizures and epilepsy.<br />

Cellular and Molecular Neurobiology <strong>2002</strong> 22(3): 249-257.<br />

34. ZANATA S.M., HOVATTA I., ROHM B. PUSCHEL A.W. Antagonistic effects of Rnd1 and RhoD<br />

GTPases Regulate Receptor activity in Semaphorin 3A- Induced Cytoskeletal Collapse. Journal<br />

of Neuroscience <strong>2002</strong> 22(2):471-477.<br />

35. ZANATA S.M., LOPES M.H., MERCADANTE A. F., HAJJ G., FREITAS A.O., CHIARINI L.B., CABRAL<br />

A.L.B., NOMIZO R., LEE K.S., JULIANO M.A., OLIVEIRA E., JACHIERI S.G., BURLINGAME A.,<br />

HUANG L., LINDEN R., BRENTANI R.R., MARTINS V.M. Stress-inducible protein 1 is a cell surface<br />

ligand for a cellular prion that triggers neuroprotection, EMBO Journal <strong>2002</strong> (21) 13:3307-3316.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. VILLA LL, VIRUS E CANCER: IN: RECURSOS GENETICOS E MELHORAMENTOS Microorganismos.<br />

Eds: Itamar Soares de Melo, Maria Clélia Valadares-Inglis, Luciano Lourenço Nass e Afonso<br />

Celso Candeira Valois. Editora Embrapa Meio Ambiente, <strong>2002</strong> 743p-pp 620-635.


STOCKHOLM BRANCH<br />

OF MOLECULAR AND CELL BIOLOGY<br />

STOCKHOLM,SWEDEN<br />

Staff List: Page 132<br />

Branch Director’s <strong>Report</strong>: Page 134<br />

<strong>Research</strong> <strong>Report</strong>: Page 135<br />

Publications: Page 137<br />

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STAFF LIST<br />

PETTERSSON,RALF F., Director, Member<br />

DEVELOPMENTAL BIOLOGY GROUP<br />

ERIKSSON,ULF, Head, Member<br />

LI,XURI, Assistant Investigator<br />

FIEBER,CHRISTINA, Postdoctoral Fellow<br />

FARNEBO,FILIP, PhD Student (until October)<br />

FOLESTAD,ERIKA, PhD Student<br />

FREDRIKSSON,LINDA, PhD Student<br />

LI,HONG, PhD Student<br />

LIDÉN,MARTIN, PhD Student<br />

PONTÉN,ANNICA, PhD Student<br />

SOHL,MARCUS, PhD Student<br />

TRYGGVASON,KRISTIAN, PhD Student<br />

ÅKERBLOM,BARBARA, Senior Technician<br />

GENE EXPRESSION GROUP<br />

PERLMANN,THOMAS, Head, Associate Member<br />

BENOIT,GERARD, Postdoctoral Fellow<br />

BORGIUS,LOTTA, Postdoctoral Fellow (from May)<br />

CASTANEDA,PATRICIA, Postdoctoral Fellow (until November)<br />

JOSEPH,BETRAND, Postdoctoral Fellow (until November)<br />

MATA DE URQUIZA,ALEXANDER, Postdoctoral Fellow (from April)<br />

MUHR,JONAS, Postdoctoral Fellow (from April)<br />

PETERSSON,SUSANNA, Postdoctoral Fellow (from October)<br />

WERME,MARTIN, Postdoctoral Fellow<br />

ARVIDSSON,MARIETTE, PhD Student<br />

BYLUND,MAGDALENA, PhD Student (from August)<br />

FRILING,STINA, PhD Student (from October)<br />

HERMANSON,ELISABETH, PhD Student<br />

LENGQUIST,JOHAN, PhD Student<br />

WALLÉN,ÅSA, PhD Student (until October)<br />

JOODMARDI,ELIZA, Technician<br />

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STOCKHOLM


PROTEIN TRANSPORT GROUP<br />

PETTERSSON,RALF, Director, Member<br />

FUXÉ,JONAS, Postdoctoral Fellow (part-time)<br />

NEVE,ETIENNE, Postdoctoral Fellow<br />

SOLLERBRANT,KERSTIN, Postdoctoral Fellow<br />

MIRZA,MOMINA, PhD Student<br />

RASCHPERGER,ELISABETH, PhD Student<br />

BERGSTRÖM,ANITA, Senior Technician<br />

SVENSSON,KERSTIN, Senior Technician<br />

YEAST CELL AND MOLECULAR BIOLOGY GROUP<br />

LJUNGDAHL,PER O., Head, Associate Member<br />

MARTINEZ RODRIGUEZ,PAULA, Postdoctoral Fellow<br />

ZARGARI,AREZOU, Postdoctoral Fellow<br />

ANDRÉASSON,CLAES, PhD Student<br />

BOBAN,MIRTA, PhD Student (from August)<br />

KOTA,JHANSI, PhD Student<br />

TECHNICAL SUPPORT<br />

TOLLMAN,INGER, Laboratory Manager<br />

ENRIONE,ERIKA, Laboratory Support<br />

ANDERLING,MATS, System Administrator<br />

ADMINISTRATION AND SECRETARIAT<br />

LINDERHOLM,CHARLOTTA, Administrator<br />

FREIDENFELDT,BIRGITTA, Secretary<br />

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STOCKHOLM


BRANCH DIRECTOR’S REPORT<br />

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STOCKHOLM<br />

The research program of the four groups at the Stockholm Branch spans quite broad areas in the general<br />

fields of cell and molecular biology. <strong>Research</strong> interests during the past year have centered on the<br />

following topics: regulation of export of membrane proteins from the endoplasmic reticulum (ER);<br />

function of the adenovirus receptor; mechanisms of cellular nutrient assessments, and metabolic<br />

compartmentalization in yeast; cell biology of retinoids (vitamin A compounds); mechanisms of action of<br />

nuclear receptors, including the retinoid receptors and some orphan receptors; and characterization of<br />

novel growth factors and their receptors that regulate the formation of new blood vessels (angiogenesis).<br />

R.F. Pettersson


RESEARCH REPORT<br />

DEVELOPMENTAL BIOLOGY GROUP<br />

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STOCKHOLM<br />

The research program of the group led by Dr. Ulf Eriksson, has two distinct focuses: the cell biology of<br />

retinoid processing enzymes, and the biology of members of the vascular endothelial growth<br />

factor/platelet-derived growth factor (VEGF/PDGF) family. Retinoids are known to have important<br />

functions in adult physiology and embryonic development. Retinoic acid (RA, 9-cis, and all-trans) and<br />

11-cis retinal are the physiologically active retinoids in higher animals. The RA isoforms regulate<br />

transcription via the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Little<br />

is known about the mechanisms that control the biosynthetic pathways that generate the two classes of<br />

active retinoids, the RAs and 11-cis retinal. Using several known enzymes in this pathway, an in vivo<br />

reconstituted system in transfected cells has been developed that allows for the generation of RA from<br />

retinol. Using this experimental system, we are now studying structure-function aspects of the involved<br />

enzymes.<br />

VEGFs and their receptors (VEGFRs) have important roles in normal and pathological conditions of the<br />

vasculature. VEGF-B, which was discovered by the Developmental Biology Group, and shown to<br />

selectively bind to VEGFR-1, is one focus of study. VEGF-B-deficient mice develop normally and<br />

display no obvious gross morphological defects, thus the group has not yet been able to define the exact<br />

molecular function of this growth factor. The potential roles of VEGF-B in different pathological<br />

conditions of angiogenesis and tissue repair are now being investigated. The PDGFs are important in<br />

connective tissue and vascular biology, and play critical roles in arteriosclerosis, malignancies and fibroproliferative<br />

diseases. PDGFs bind to two receptors (PDGFRs) on target cells, which are mainly of<br />

mesenchymal origin. The family of ligands has now been expanded with two novel PDGFs, PDGF-C and<br />

PDGF-D, being identified by the group. Both PDGF-C and PDGF-D are latent growth factors requiring<br />

proteolytic processing for receptor binding and biological activity. Overexpression of PDGF-C in the<br />

heart of transgenic mice results in severe hypertrophy and fibrosis, while over-expression in NIH/3T3<br />

cells results in cellular transformation. Characterization of activating enzymes, and elucidation of<br />

structure-function relationships of both factors in vitro and in vivo are presently being carried out.<br />

GENE EXPRESSION GROUP<br />

Cellular diversity in the central nervous system (CNS) depends on strictly regulated signaling events that<br />

resulte in the coordinated expression of region and cell type specific transcription factors. Understanding<br />

how these transcription factors generate cell diversity in the developing CNS and how they influence<br />

neuronal functions in the adult is attracting considerable interest. The group led by Dr. Thomas Perlmann<br />

is interested in cell signaling via nuclear receptors expressed in the CNS. Nuclear receptors function as<br />

ligand-regulated transcription factors and bind small lipophilic ligands such as retinoids, steroids, and<br />

fatty acids. Many nuclear receptors lack identified ligands and are therefore referred to as orphan<br />

receptors. Our aim is to unravel the roles of a selected number of orphan nuclear receptors and retinoid<br />

receptors.<br />

The orphan receptor Nurr1 has been extensively characterized and shown to be essential for the<br />

development of dopamine (DA) neurons. These are the cells that degenerate in Parkinson’s disease. Nurr1<br />

provides a handle that allows upstream and downstream events in the DA differentiation process to be<br />

identified, and the group is characterizing additional regulatory mechanisms essential for the generation<br />

of these cells. In the past year, several target genes of Nurr1 have been identified that, together with other<br />

data, underscores a role of this transcription factor in the adult brain also. Moreover, the group is<br />

continuing to characterize the function exerted by Nurr1 when in a complex with the RXR. The


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 136<br />

STOCKHOLM<br />

characterization of nuclear receptor ligand identity and distribution in vivo has remained challenging due<br />

to the properties of this class of signaling molecules. Novel transgenic mouse strategies, in vitro detection<br />

methods, and novel biochemical approaches have been used to identify a previously unknown CNSderived<br />

endogenous ligand for RXR. An important project is to unravel the biological significance of this<br />

novel signaling pathway.<br />

PROTEIN TRANSPORT GROUP<br />

Dr. Ralf Pettersson’s group is focusing on the involvement of specific membrane-bound receptors<br />

required for the efficient export of secretory proteins out of the endoplasmic reticulum (ER). One such<br />

receptor is p58/ERGIC-53, a lectin-like membrane protein receptor that recycles between the ER, the ER-<br />

Golgi-intermediate compartment (ERGIC), and the Golgi complex. The crystal structures of the<br />

carbohydrate recognition domain (CRD) of p58 has been solved at 1.46Å resolution, revealing a highly<br />

conserved fold, which has been utilized in many leguminous plant lectins, and also, for example, the ER<br />

chaperone calnexin. A new member of the lectin family, VIPL (VIP36-Like), has been cloned and<br />

characterized. The group is presently attempting to identify glycoprotein ligands, whose export from the<br />

ER is dependent on VIPL.<br />

The group has also initiated a new program focusing on the normal cellular function of the coxsackie- and<br />

adenovirus receptor (CAR). Infection with these viruses is dependent on CAR expression, a fact that has<br />

relevance for the use of adenovirus vectors in cancer gene therapy. The level of CAR expression in<br />

malignant astrocytomas was found to vary substantially both between tumors of different malignancy<br />

grades, and among tumors of the same grade. CAR has been found to be localized to the tight junction of<br />

polarized epithelial cells where it seems to serve as a cell-cell adhesion molecule. One aim is to<br />

understand how CAR transduces signals from the cell surface to the nucleus. The group has shown that<br />

the cytoplasmic tail of CAR binds to a PDZ-protein called LNX. The functional implications of this are<br />

being analyzed.<br />

YEAST CELL AND MOLECULAR BIOLOGY GROUP<br />

The regulation of amino acid uptake in yeast offers an attractive system to investigate mechanisms of<br />

signal transduction in eukaryotic cells. Apart from being metabolites for protein synthesis, amino acids<br />

have an important role in nitrogen homeostasis, and yeast possess sophisticated systems that efficiently<br />

import amino acids from the external environment. Dr. Per Ljungdahl’s group has found that yeast<br />

possess a plasma membrane sensor of external amino acids that functions as a ligand-activated receptor.<br />

This multimeric sensor, dubbed the SPS sensor, initiates signals that regulate the expression of genes<br />

required for proper amino acid uptake. A major breakthrough was made during the past year when it was<br />

found that Stp1p and Stp2p, transcription factors that bind to specific sequences within the promoters of<br />

SPS sensor regulated genes, are synthesized as latent cytoplasmic precursors. In response to extracellular<br />

amino acids, the SPS sensor induces the rapid endoproteolytic processing of Stp1p and Stp2p. These<br />

activated shorter forms, which lack N-terminal inhibitory domains, are targeted to the nucleus where they<br />

transactivate SPS sensor target genes. These results define a novel mode of transducing environmental<br />

signals from the plasma membrane to the nucleus in yeast without the apparent involvement of<br />

previously-characterized signal transducing components, such as kinase cascades, ubiquitins, and<br />

proteasomes. The simple mechanism of SPS sensor signaling suggests that yeast, like metazoan cells,<br />

possess a far broader repertoire of signaling mechanisms than represented by phosphorylation-dependent<br />

processes.


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 137<br />

STOCKHOLM<br />

1. AARNISALO P, CHAE-HEE K, LEE JW AND PERLMANN T. Defining requirements for<br />

2.<br />

heterodimerization between the retinoid X receptor and the orphan nuclear receptor Nurr1.<br />

Journal of Biological Chemistry (<strong>2002</strong>) 38:35118-35123.<br />

AASE K, KARLSSON L, ABRAMSSON A, BETSHOLTZ C AND ERIKSSON U. Expression of PDGF-C<br />

in adult and embryonic mouse tissues. Mechanisms of Development (<strong>2002</strong>) 110:187-191.<br />

3. ANDRÉASSON C AND LJUNGDAHL PO. Receptor-mediated endoproteolytic activation of two<br />

transcription factors at the plasma membrane in yeast. Genes & Development (<strong>2002</strong>) 16:3158-<br />

3172.<br />

4. CAO R, BRÅKENHIELM E, LI X, PIETRAS C, WIDENFALK J, ÖSTMAN A, TSANG M, ERIKSSON U<br />

AND CAO Y. PDGF-CC: A new member in the PDGF family stimulates angiogenesis in vivo.<br />

FASEB Journal (<strong>2002</strong>) 12:1575-83.<br />

5. EITNER F, OSTENDORF T, VAN ROEYEN C, KITAHARA M, LI X, AASE K, GRÖNE H-J, ERIKSSON<br />

U AND FLOEGE J. PDGF-C expression in the developing and normal adult human kidney and in<br />

glomerular diseases. Journal of the American Society of Nephrology (<strong>2002</strong>) 13:910-917.<br />

6. FLICK R, ELGH F, HOBOM G AND PETTERSSON RF. Mutational analysis of the Uukuniemi virus<br />

(Bunyaviridae) promotor reveals two elements of functional importance. Journal of Virology<br />

(<strong>2002</strong>) 76:10849-10860.<br />

7. JIN S-B, ZHAO J, BJÖRK P, SCHMEKEL K, LJUNGDAHL PO AND WIESLANDER L. Mrd1p is<br />

required for processing of pre-rRNA and for maintenance of steady-state levels of 40 S ribosomal<br />

subunits in yeast. Journal of Biological Chemistry (<strong>2002</strong>) 21:18431-18439.<br />

8. LENGQVIST J, GRIFFITHS WJ, PERLMANN T AND SJÖVALL J. Detection of a receptor-ligand noncovalent<br />

complex using a triple quadrupole mass spectrometer.Rapid Communications in<br />

Masspectroscopy (<strong>2002</strong>) 16:2003-2006.<br />

9. PULKKANEN KJ, LAUKKANEN JM, FUXE J, KETTUNEN,MI,REHN M, KANNASTO JM, PARKKINEN<br />

JJ, KAUPPINEN RA, PETTERSSON RF AND YLÄ-HERTTUALA S. The combination of HSV-tk and<br />

endostatingene therapy for the treatment of human renal cell carcinoma in an orthotopic nude<br />

mouse model. Cancer Gene Therapy (<strong>2002</strong>) 9:908-916.<br />

10. TÖRNQVIST N, HERMANSON E, PERLMANN T AND STRÖMBERG I. Generation of tyrosine<br />

hydroxylase-immunoreactive neurons in ventral mesencephalic tissue of Nurr1 deficient mice.<br />

Developmental Brain <strong>Research</strong> (<strong>2002</strong>) 133:37-47.<br />

11. VELLOSO LM, SVENSSON K, SCHNEIDER G, PETTERSSON RF AND LINDQVIST Y. Crystal structure<br />

of the carbohydrate recognition domain of p58/ERGIC-53, a protein involved in glycoprotein<br />

export from the endoplasmic reticulum. Journal of Biological Chemistry (<strong>2002</strong>) 277:15979-<br />

15984.<br />

12. VELLOSO LM, SVENSSON K, LAHTINEN U, SCHNEIDER G, PETTERSSON RF AND LINDQVIST Y.<br />

Expression, purification, refolding and crystallization of the carbohydrate recognition domain of<br />

p58/ERGIC-53, an animal C-type lectin involved in the export of glycoproteins from the<br />

endoplasmic reticulum. Acta Crystallographica D Biologcial Crystallography (<strong>2002</strong>) 58:536-<br />

538.


REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 138<br />

STOCKHOLM<br />

1. ERIKSSON U AND ALITALO K. VEGF receptor 1 stimulates stem-cell recruitment and new hopes<br />

for angiogenesis therapies. Nature Medicine (<strong>2002</strong>) 8:775-777.<br />

2. HELDIN CH, ERIKSSON U AND ÖSTMAN A. New members of the platelet-derived growth factor<br />

family of mitogens. Archives of Biochemistry and Biophysics (<strong>2002</strong>) 15:284-290.<br />

3. PERLMANN T. Retinoid metabolism: a balancing act. Nature Genetics (<strong>2002</strong>) 31:7-8.


UPPSALA BRANCH<br />

OF GROWTH REGULATION<br />

UPPSALA,SWEDEN<br />

Staff List: Page 140<br />

Branch Director’s <strong>Report</strong>: Page 144<br />

<strong>Research</strong> <strong>Report</strong>: Page 145<br />

Publications: Page 148<br />

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STAFF LIST<br />

HELDIN,CARL-HENRIK, Member, Director<br />

APOPTOTIC SIGNALING GROUP<br />

LANDSTRÖM,MARÉNE, Assistant Member, Group Head<br />

KOZAKAI,TAKAHARU, Postdoctoral Fellow<br />

SCHUSTER,NORBERT, Postdoctoral Fellow (until September)<br />

BENDER,HERDIS, PhD Student (from August to December)<br />

GROSS,JULIANE, PhD Student (until January)<br />

GRIMSBY,SUSANNE, Senior Technical Assistant (from September)<br />

CYTOSKELETAL REGULATION GROUP<br />

ASPENSTRÖM,PONTUS, Assistant Member, Group Head<br />

GIZATULLINA,ZEMFIRA, Visiting Investigator (until August)<br />

HÁJKOVÁ,LUCIE, Postdoctoral Fellow (joint with Molecular Signaling Group)<br />

SARAS,JAN, Postdoctoral Fellow<br />

EDLUND,SOFIA, PhD Student<br />

FRANSSON,ÅSA, PhD Student<br />

Johansson, Ann-sofi, PhD Student<br />

Richnau, Ninna, PhD Student<br />

RUUSALA,AINO, Senior Technical Assistant<br />

GENE EXPRESSION GROUP<br />

ERICSSON,JOHAN, Assistant Member, Group Head<br />

BENGOECHEA,MARIA TERESA, Postdoctoral Fellow (from May)<br />

GIANDOMENICO,VALERIA, Postdoctoral Fellow (until October)<br />

GRÖNROOS,EVA, Postdoctoral Fellow<br />

SUNDQVIST,ANDERS, Postdoctoral Fellow (from February)<br />

SIMONSSON,MARIA, PhD Student<br />

GENE TARGETING GROUP<br />

HEUCHEL,RAINER, Assistant Member, Group Head<br />

BRODIN,GREGOR, PhD Student (until June)<br />

ÅHGREN,AIVE, Senior Technical Assistant<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003©<br />

UPPSALA<br />

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GROWTH REGULATION GROUP<br />

ÖSTMAN,ARNE, Associate Member, Group Head<br />

FURUHASHI,MASAO, Visiting Scientist<br />

HELLBERG,CARINA, Postdoctoral Fellow<br />

MICKE,PATRIK, Postdoctoral Fellow (from February)<br />

PIETRAS,KRISTIAN, Postdoctoral Fellow<br />

SJÖBLOM,TOBIAS, Postdoctoral Fellow<br />

HÄGERSTRAND,DANIEL, Student<br />

PERSSON,CAMILLA, Student<br />

BÄCKSTRÖM,GUDRUN, Senior Technical Assistant<br />

SANDSTRÖM LEPPÄNEN,JILL, Technical Assistant<br />

INTEGRATED SIGNALING GROUP<br />

SOUCHELNITSKI,SERHIY, Assistant Member, Group Head<br />

EICHNER,ANNEGRET, Postdoctoral Fellow (until April)<br />

FILYAK,YEVHEN, Postdoctoral Fellow (August to November)<br />

IWAHANA,HIROYUKI, Postdoctoral Fellow (from April)<br />

KANAMOTO,TAKAHASHI, Postdoctoral Fellow (until March)<br />

LOMNYTSKA,MARTA, Postdoctoral Fellow<br />

STASYK,TARAS, Postdoctoral Fellow<br />

YAKYMOVYCH,IHOR, Postdoctoral Fellow<br />

MATRIX BIOLOGY GROUP<br />

HELDIN,PARASKEVI, Associate Investigator, Group Head<br />

ASTERIOU,TRIAS, Postdoctoral Fellow (from February)<br />

TAKAHASHI,YOSHINORI, Postdoctoral Fellow (from April)<br />

LI,LINGLI, PhD Student (from February)<br />

MOLECULAR SIGNALING GROUP<br />

DIKIC,IVAN, Assistant Member, Group Head<br />

HÁJKOVÁ,LUCIE, Postdoctoral Fellow (joint with Molecular Signaling Group)<br />

SHIMOKAWA,NORIAKI, Postdoctoral Fellow (until August)<br />

SOUBEYRAN,PHILIPPE, Postdoctoral Fellow<br />

HAGLUND,KAISA, PhD Student<br />

HUSNJAK,KORALJKA, PhD Student<br />

IVANKOVIC-DIKIC,INGA, PhD Student<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003©<br />

UPPSALA<br />

141


KOWANETZ,KATARZYNA, PhD Student<br />

SZYMKIEWICZ,IWONA, PhD Student<br />

PROTEIN STRUCTURE GROUP<br />

HELLMAN,ULF, Member, Group Head<br />

ENGSTRÖM,ULLA, Senior Technical Assistant<br />

WERNSTEDT,CHRISTER, Senior Technical Assistant<br />

SIGNAL TRANSDUCTION GROUP<br />

RÖNNSTRAND,LARS, Associate Member, Group Head (until July)<br />

CHIARA,FEDERICA, Postdoctoral Fellow (from February)<br />

DEMOULIN,JEAN-BAPTISTE, Postdoctoral Fellow<br />

PALUMBO,ROBERTA, Postdoctoral Fellow (until February)<br />

VOYTYUK,OLEXANDR, Postdoctoral Fellow (until August)<br />

HÄGG,MARIA, PhD Student (until July)<br />

KALLIN,ANDERS, PhD Student<br />

LENARTSSON,JOHAN, PhD Student (until August)<br />

RORSMAN,CHARLOTTE, Senior Technical Assistant<br />

TGF-� SIGNALING GROUP<br />

MOUSTAKAS,ARISTIDIS, Assistant Member, Group Head<br />

GAAL,ANNAMARIA, Postdoctoral Fellow (until February)<br />

KURISAKI,AKIRA, Postdoctoral Fellow (until May)<br />

VALCOURT,ULRICH, Postdoctoral Fellow (from February)<br />

KOWANETZ,MARCIN, PhD Student<br />

PARDALI,EKATERINA, PhD Student<br />

KURISAKI (NEE SHIRAISHI), KEIKO, PhD Student (until May)<br />

MORÉN,ANITA, Senior Technical Assistant<br />

TECHNICAL SUPPORT<br />

EJDESJÖ,BENGT, Purchasing Officer<br />

HEDBERG,ULF, Information Technology Support<br />

HERMANSSON,LARS ERIK, Service Engineer<br />

SCHÖNQUIST,INGER, Laboratory Assistant<br />

PETTERSSON,GULLBRITT, Laboratory Assistant<br />

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ADMINISTRATION AND SECRETARIAT<br />

HALLIN,EVA, Administrator<br />

SCHILLER,INGEGÄRD, Secretary<br />

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UPPSALA<br />

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BRANCH DIRECTOR’S REPORT<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003©<br />

UPPSALA<br />

Malignant cells are characterized by perturbations in signaling pathways that regulate cell growth,<br />

survival, and migration. The aim of the work at the Uppsala Branch is to elucidate the signaling<br />

mechanisms induced by factors that stimulate or inhibit cell growth.<br />

The Branch consists of eleven groups which cover different aspects of this research on growth regulatory<br />

factors and signal transduction. Of special importance are the studies of platelet-derived growth factor<br />

(PDGF), an important mitogen for connective tissue cells, and transforming growth factor-� (TGF-�),<br />

which inhibits the growth of most cell types, but other growth regulatory factors are also studied.<br />

The research activities at our Branch span from basic science to animal models for malignant disease, and<br />

our aim is to use the knowledge we obtain on the molecular mechanisms of signal transduction to devise<br />

improved treatment regimens for cancer patients. In this context, it is satisfying that our work on PDGF<br />

antagonists now has led to a clinical trial in which we will explore the possibility that treatment of<br />

patients that have solid tumors with a PDGF antagonist will cause a lowering of the tumor interstitial fluid<br />

pressure, and an increased uptake of chemotherapeutic drug. Among the other highlights from the year<br />

were the findings in the regulation of endocytosis and degradation of tyrosine kinase receptors through<br />

ubiquitination, and that the stability of the transcription factors Smad and SREBP are regulated by<br />

acetylation.<br />

During <strong>2002</strong>, the Group Leader for the Signal Transduction Group, Dr. Lars Rönnstrand, left to take up a<br />

position as Professor in Molecular Medicine in Malmö. We wish Lars and his associates the best success<br />

in their future work, and we look forward to future interactions with them. The work on stem cell factor<br />

receptors in the Signal Transduction Group will now be continued by Lars in Malmö, but some aspects of<br />

his work on PDGF signaling will be continued at our Branch.<br />

C-H. Heldin<br />

144


RESEARCH REPORT<br />

APOPTOTIC SIGNALING GROUP<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003©<br />

UPPSALA<br />

The Apoptosis Signaling Group, headed by Dr. Maréne Landström, studies the molecular mechanisms by<br />

which TGF-� induces apoptosis in epithelial cells. We have found that Smad7 is required for TGF-�1induced<br />

apoptosis, and that TGF-� activates a MAP kinase pathway consisting of TGF-� activating<br />

kinase 1 (TAK1), mitogen activated protein kinase kinase 3 (MKK3) and p38 mitogen activated protein<br />

(MAP) kinase pathway, resulting in apoptosis. It appears that Smad7, which upon TGF-� stimulation is<br />

exported from the nucleus to the activated receptor-complex, acts as a scaffolding protein for TAK1,<br />

MKK3 and p38, thereby facilitating the activation of p38. We are currently investigating the precise<br />

molecular mechanisms for how TGF-� and in particular Smad7, can activate the TAK1 - MKK3 - p38<br />

MAP kinase pathways.<br />

CYTOSKELETAL REGULATION GROUP<br />

The work in the Cytoskeletal Regulation Group, headed by Dr. Pontus Aspenström, is focused on studies<br />

on the molecular mechanisms that control cell migration and cell growth under normal physiological<br />

conditions, as well as during disease. Signaling pathways involving the Rho family of small GTPases,<br />

including Rho, Rac, and Cdc42, have been found to be of particular importance for the organization of the<br />

actin cytoskeleton, and thereby the control of cell morphogenesis and cell migration. Our studies aim to<br />

identify signaling pathways that lead to the activation of the Rho GTPases. In addition, we aim to identify<br />

effectors downstream of the Rho GTPases, which influence biological processes such as cell migration,<br />

cell proliferation and cell survival.<br />

GENE EXPRESSION GROUP<br />

Dr. Johan Ericsson’s Gene Expression Group studies how the levels and activities of certain transcription<br />

factors are regulated in response to extracellular and intracellular cues. The function of transcription<br />

factors may be affected in a variety of ways, including post-translational modification (e.g.<br />

phosphorylation or acetylation), ligand binding, and interactions with other proteins. Many transcription<br />

factors, particularly those involved in the control of cell growth, are unstable proteins targeted for<br />

degradation by the ubiquitin-proteasome system. The group has found that competition between<br />

ubiquitination and acetylation of overlapping lysine residues constitutes a novel mechanism to regulate<br />

the stability of proteins, including transcription factors. The group is currently trying to elucidate how<br />

these pathways are regulated in response to growth factors and metabolic signals, and to understand how<br />

this affects the activity of various transcription factors.<br />

GENE TARGETING GROUP<br />

This group, headed by Dr. Rainer Heuchel, uses gene targeting in the mouse in an effort to explore the in<br />

vivo importance of specific signaling pathways initiated by PDGF and TGF-�. In an effort to elucidate the<br />

in vivo importance of specific signaling pathways downstream of the PDGF �-receptor, the group mutated<br />

the receptor's binding sites for the signaling molecules phosphatidylinositol-3 kinase (PI3K), and<br />

phospholipase C�.��<br />

Activating point mutations have also been introduced into the kinase domain of the<br />

PDGF �-receptor in order to investigate its possible role in carcinogenesis. In order to shed more light on<br />

the in vivo function of Smad7, a component in the TGF-� signaling pathway, the group has generated<br />

mice with a null mutation in the Smad7 gene in collaboration with Dr. Tony Pawson's laboratory in<br />

Toronto (Canada). The phenotype of the Smad7 knockout animals is currently under investigation.<br />

145


GROWTH REGULATION GROUP<br />

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UPPSALA<br />

The Growth Regulation Group, headed by Dr. Arne Östman, explores PDGF receptors as cancer drug<br />

targets, studies the regulation and function of protein-tyrosine phosphatases (PTPs), and also investigates<br />

the biology of tumor fibroblasts. The animal studies on PDGF receptors as drug targets have shown that<br />

inhibition of PDGF receptors in tumor stroma leads to increased tumor uptake of cytotoxic drugs and a<br />

concomitant increase in therapeutic response. The group has developed a novel assay to determine PTP<br />

activity in cultured cells, which improves the possibilities for analyzing alterations of PTP activity, for<br />

example in the context of cell transformation. TC-PTP has also been identified as a negative regulator of<br />

the PDGF �-receptor. Finally, with a procedure that combines laser-capture microdissection and cDNA<br />

microarray analyses, the group has characterized tumor fibroblasts with the long-term goal to exploit this<br />

cell type as a therapeutic target.<br />

INTEGRATED SIGNALING GROUP<br />

The work in the Integrated Signaling Group, headed by Dr. Serhiy Souchelnytskyi, explores TGF�<br />

signaling in normal and malignant cells. The group’s efforts are focused on the unraveling of mechanisms<br />

of receptor activation, as well as Smad-dependent and Smad-independent signaling down-stream of the<br />

receptors. An important aspect of the work is to elucidate the role of TGF� signaling in carcinogenesis.<br />

Proteomics techniques are important tools used to understand the complexity of integrated intracellular<br />

signaling. Global proteome profiling is performed using two-dimensional gel electrophoresis and protein<br />

identification by mass spectrometry. The effect of TGF-� stimulation on the protein expression and<br />

turnover, as well as on the phosphoproteome and glycoproteome of human breast epithelial and<br />

endothelial cells is being studied. More than 20 novel Smad3- and bone morphogenetic protein (BMP)<br />

receptor-interacting proteins have so far been identified using proteomics techniques. Additionally,<br />

interactions between TGF�/Smads and products of breast cancer susceptibility genes BRCA2 and<br />

BRCA1 have been identified. The group has also investigated six classes of low molecular weight<br />

compounds for inhibition of kinase of the type I TGF� receptor.<br />

MATRIX BIOLOGY GROUP<br />

The Matrix Biology Group, headed by Dr. Paraskevi Heldin, studies the importance of hyaluronan and its<br />

cell surface receptor CD44 with respect to the malignant properties of tumor cells, and tumor cell<br />

invasion. Recent studies have revealed that hyaluronan synthesizing and degrading enzymes affect<br />

tumorigenesis differentially; overproduction of the hyaluronan synthase Has2 by colon carcinoma cells in<br />

vivo promotes tumorigenicity, whereas overexpression of the hyaluronan degrading enzyme Hyal1<br />

suppresses tumor development. Furthermore, the group has demonstrated that hyaluronan fragments, as<br />

well as hyaluronidase treatment, promote differentiation of endothelial cells grown in a 3D collagen gel,<br />

in a CD44-dependent manner. The group is now focusing on the elucidation of the signaling pathways<br />

involved in stimulation of hyaluronan biosynthesis by PDGF-BB and TGF�� and the importance of<br />

hyaluronan-CD44 interactions for cell migration and proliferation.<br />

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MOLECULAR SIGNALING GROUP<br />

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UPPSALA<br />

Dr. Ivan Dikic’s group is dedicated to studying the molecular mechanisms by which signaling pathways<br />

control cell growth and differentiation, and the reasons why they are deregulated in diseases such as<br />

cancer. The group is primarily interested in signal transduction mediated by growth factor activated<br />

receptor tyrosine kinases (RTKs). More recently, the group has become interested in mechanisms<br />

underlying the endocytosis of RTKs, and protein degradation through actions of the scaffold protein CIN<br />

85 and the ubiquitin ligase Cbl. Genetically altered mice, and cells derived from such animals, are<br />

currently being utilized to understand how components of these processes regulate cell functions in vivo.<br />

PROTEIN STRUCTURE GROUP<br />

The Protein Structure Group, headed by Dr. Ulf Hellman, has three major activities. The first is<br />

automated peptide synthesis in which synthetic peptides are made using Fmoc chemistry in an Applied<br />

Biosystems 433A instrument. The peptides are used as antigens for raising anti-peptide antibodies, as<br />

substrates, or as inhibitors, and the group is also able to synthesize peptides modified by phosphorylation<br />

or acetylation. The second major activity is the use of two Bruker mass spectrometers, one Autoflex<br />

MALDI and one Esquire 3000 ESI Ion Trap, for classical proteomics work. Samples are mainly prepared<br />

using 1D- or 2D-gel electrophoresis and, following in-gel tryptic digestion, are analyzed for identity, and<br />

for posttranslational modifications. The third activity is the use of radiolabeled Edman sequencing for the<br />

analysis of phosphorylation sites.<br />

SIGNAL TRANSDUCTION GROUP<br />

The work in the Signal Transduction Group, until June headed by Dr. Lars Rönnstrand and thereafter by<br />

Dr. Jean-Baptiste Demoulin, is focused on the mechanisms of signal transduction via the tyrosine kinase<br />

receptors for PDGF and stem cell factor. Autophosphorylation sites in the receptors are being identified,<br />

and their roles as docking sites for signaling molecules containing SH2 domains are being determined.<br />

The aim is to elucidate the importance of individual signaling pathways for the cellular effects of the<br />

growth factors, i.e. cell proliferation, survival, migration, and actin reorganization. Moreover, microarray<br />

techniques are used to determine the effect of PDGF isoforms on the induction/repression of specific<br />

genes in human fibroblasts.<br />

TGF-� SIGNALING GROUP<br />

This group, headed by Dr. Aristidis Moustakas, investigates TGF-� signaling pathways that regulate cell<br />

growth, differentiation, and tumorigenesis. The group has described new mechanisms of Smad3 protein<br />

nucleocytoplasmic shuttling, and has also found that Smad4 mono-ubiquitination regulates Smad protein<br />

oligomerization. Screens for E3 ubiquitin ligases that regulate this process are now underway. Another<br />

major finding is that nuclear protein YY1 interacts with Smad4, leading to repression of cell<br />

differentiation, without affecting the growth inhibitory response to TGF-�. Unique structural determinants<br />

of the Smad N-terminus that specify interactions with transcription factors and nuclear carriers have been<br />

identified, and thus the group is now attempting to understand signaling specificity at the transcriptional<br />

level. Microarrays have been used to identify new target genes that regulate cell growth, or differentiation<br />

leading to epithelial-to-mesenchymal transition, a prerequisite for carcinoma metastasis. Finally, the<br />

group is also focussing on the role of TGF-� as tumor suppressor and pro-metastatic factor.<br />

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PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

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UPPSALA<br />

1. ASPENSTRÖM P. The WASP-binding protein WIRE has a role in the regulation of the actin<br />

filament system downstream of the platelet-derived growth factor receptor. Experimental Cell<br />

<strong>Research</strong> (<strong>2002</strong>) 279: 21-33.<br />

2. BERGER K, SIVARS U, SÖRHEDE WINZELL M, JOHANSSON P, HELLMAN U, RIPPE C AND<br />

ERLANSON-ALBERTSSON C. Mitochondrial ATP synthase - a possible target protein in the<br />

regulation of energy metabolism in vitro and in vivo. Nutritional Neuroscience (<strong>2002</strong>) 5: 201-210.<br />

3. BJÖRK P, BAURÉN G, JIN S, TONG Y-G, BÜRGLIN TR, HELLMAN U AND WIESLANDER L. A novel<br />

conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis. Molecular<br />

Biology of the Cell (<strong>2002</strong>) 13: 3683-3695.<br />

4. BONDESTAM J, KAIVO-OJA N, KALLIO J, GROOME N, HYDÉN-GRANSKOG C, FUJII M,<br />

5.<br />

MOUSTAKAS A, JALANKO A, TEN DIJKE P AND RITVOS O. Engagement of activin and bone<br />

morphogenetic protein signaling pathway Smad proteins in the induction of inhibin B production<br />

in ovarian granulosa cells. Molecular and Cellular Endocrinology (<strong>2002</strong>) 195: 79-88.<br />

BU S, BLAUKAT A, FU X, HELDIN N-E AND LANDSTRÖM M. Mechanisms for 2-methoxyestradiolinduced<br />

apoptosis of prostate cancer cells. FEBS Letters (<strong>2002</strong>) 531: 141-151.<br />

6. CAO R, BRÅKENHIELM E, LI X, PIETRAS K, WIDENFALK J, ÖSTMAN A, ERIKSSON U AND CAO Y.<br />

Angiogenesis stimulated by PDGF-CC, a novel member in the PDGF family, involves activation<br />

of PDGFR-�� and -�� receptors. FASEB Journal (<strong>2002</strong>) 16: 1575-1583.<br />

7. EDLUND S, LANDSTRÖM M, HELDIN C-H AND ASPENSTRÖM P. Transforming growth factor-�induced<br />

mobilization of actin cytoskeleton requires signaling by small GTPases Cdc42 and<br />

RhoA. Molecular Biology of the Cell (<strong>2002</strong>) 13: 902-914.<br />

8. EICHNER A, BROCK J, HELDIN C-H AND SOUCHELNYTSKYI S. Bone morphogenetic protein-7<br />

(OP1) and transforming growth factor-�1 modulate 1,25(OH)2-vitamin D3-induced<br />

9.<br />

differentiation of human osteoblasts. Experimental Cell <strong>Research</strong> (<strong>2002</strong>) 275: 132-142.<br />

EKMAN S, KALLIN A, ENGSTRÖM U, HELDIN C-H AND RÖNNSTRAND L. SHP-2 is involved in<br />

heterodimer specific loss of phosphorylation of Tyr771 in the PDGF �-receptor. Oncogene<br />

(<strong>2002</strong>) 21: 1870-1875.<br />

10. FREDRIKSSON S, GULLBERG M, JARVIUS J, OLSSON C, PIETRAS K, GUSTAFSDOTTIR SM,<br />

ÖSTMAN A AND LANDEGREN U. Protein detection using proximity-dependent DNA ligation<br />

assays. Nature Biotechnology (<strong>2002</strong>) 20: 473-477.<br />

11. GRÖNROOS E, HELLMAN U, HELDIN C-H AND ERICSSON J. Control of Smad7 stability by<br />

competition between acetylation and ubiquitination. Molecular Cell (<strong>2002</strong>) 10: 483-493.<br />

12. GÖRANSSON O, RESJÖ S, RÖNNSTRAND L, MANGANIELLO V AND DEGERMAN E. Ser-474 is the<br />

major target of insulin-mediated phosphorylation of protein kinase B � in primary rat adipocytes.<br />

Cellular Signalling (<strong>2002</strong>) 14: 175-182.<br />

13. HAGLUND K, SHIMOKAWA N, SZYMKIEWICZ I AND DIKIC I. Cbl-directed monoubiquitination of<br />

CIN85 is involved in regulation of ligand-induced degradation of EGF receptors. Proceedings of<br />

the National Academy of Sciences USA (<strong>2002</strong>) 99: 12191-12196.<br />

14. HELLMAN U AND BHIKHABHAI R. Easy amino acid sequencing of sulfonated peptides using postsource<br />

decay on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer<br />

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UPPSALA<br />

equipped with a variable voltage reflector. Rapid Communications in Mass Spectrometry (<strong>2002</strong>)<br />

16: 1851-1859.<br />

15. HÄGG M, LILJEGREN Å, CARLSSON J, RÖNNSTRAND L AND LENNARTSSON J. EGF and dextranconjugated<br />

EGF induces differential phosphorylation of the EGF receptor. International Journal<br />

of Molecular Medicine (<strong>2002</strong>) 10: 655-659.<br />

16. HÖRNSTEN L, SU C, OSBOURN AE, HELLMAN U AND OLIW EH. Cloning of the manganese<br />

lipoxygenase gene reveals homology with the lipoxygenase gene family. European Journal of<br />

Biochemistry (<strong>2002</strong>) 269: 2690-2697.<br />

17. JACOBSON A, RAHMANIAN M, RUBIN K AND HELDIN P. Expression of hyaluronan synthase 2 or<br />

hyaluronidase 1 differentially affect the growth rate of transplantable colon carcinoma cell<br />

tumors. International Journal of Cancer (<strong>2002</strong>) 102: 212-219.<br />

18. KANAMOTO T, HELLMAN U, HELDIN C-H AND SOUCHELNYTSKYI S. Functional proteomics of<br />

transforming growth factor-�1-stimulated Mv1Lu epithelial cells: Rad51 as a target of TGF�1dependent<br />

regulation of DNA repair. EMBO Journal (<strong>2002</strong>) 21: 1219-1230.<br />

19. KINTSCHER U, LYON C, WAKINO S, BRUEMMER D, FENG X, GOETZE S, GRAF K, MOUSTAKAS A,<br />

STAELS B, FLECK E, HSUEH WA AND LAW RE. PPAR� inhibits TGF-�-induced �5-integrin<br />

transcription in vascular smooth muscle cells by interacting with Smad4. Circulation <strong>Research</strong><br />

(<strong>2002</strong>) 91: e35-44.<br />

20. KOUTSODONTIS G, MOUSTAKAS A AND KARDASSIS D. The role of Sp1 family members, the<br />

proximal GC-rich motifs, and the upstream enhancer region in the regulation of the human cell<br />

cycle inhibitor p21WAF-1/Cip1 gene promoter. Biochemistry (<strong>2002</strong>) 41: 12771-12784.<br />

21. LÉVY F, BURRI L, MOREL S, PEITREQUIN A-L, LÉVY N, BACHI A, HELLMAN U, VAN DEN EYNDE<br />

BJ AND SERVIS C. The final N-terminal trimming of a subaminoterminal proline-containing HLA<br />

class I-restricted antigenic peptide in the cytosol is mediated by two peptidases. Journal of<br />

Immunology (<strong>2002</strong>) 169: 4161-4171.<br />

22. PALUMBO R, GAETANO C, ANTONINI A, POMPILIO G, BRACCO E, RÖNNSTRAND L, HELDIN C-H<br />

AND CAPOGROSSI MC. Different effects of high and low shear stress on platelet-derived growth<br />

factor isoform release by endothelial cells: consequences for smooth muscle cell migration.<br />

Artherosclerosis, Thrombosis and Vascular Biology (<strong>2002</strong>) 22: 405-411.<br />

23. PAPANIKOLAOU V, VROCHIDES D, GAKIS D, PATSIAOURA K, MARGARI P, DOGRAMATZI F,<br />

ANTONIADES A AND HELDIN P. Improvement of rat liver graft function after storage in University<br />

of Wisconsin solution containing testicular hyaluronidase. Liver Transplantation (<strong>2002</strong>) 8: 1028-<br />

1035.<br />

24. PERSSON C, ENGSTRÖM U, MOWBRAY SL AND ÖSTMAN A. Primary sequence determinants<br />

responsible for site-selective dephosphorylation of the PDGF �-receptor by the receptor-like<br />

protein tyrosine phosphatase DEP-1. FEBS Letters (<strong>2002</strong>) 517: 27-31.<br />

25. PICASCIA A, STANZIONE R, CHIEFFI P, KISSLINGER A, DIKIC I AND TRAMONTANO D. Proline-rich<br />

tyrosine kinase 2 regulates proliferation and differentiation of prostate cells. Molecular and<br />

Cellular Endocrinology (<strong>2002</strong>) 186: 81-87.<br />

26. PIETRAS K, RUBIN K, SJÖBLOM T, BUCHDUNGER E, SJÖQUIST M, HELDIN C-H AND ÖSTMAN A.<br />

Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of<br />

chemotherapy. Cancer <strong>Research</strong> (<strong>2002</strong>) 62: 5476-5484.<br />

27. PIIPER A, DIKIC I, LUTZ MP, LESER J, KRONENBERGER B, ELEZ R, CRAMER H, MÜLLER-ESTERL<br />

W AND ZEUZEM S. Cyclic AMP induces transactivation of the receptors for epidermal growth<br />

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UPPSALA<br />

factor and nerve growth factor, thereby modulating activation of MAP kinase, Akt, and neurite<br />

outgrowth in PC12 cells. Journal of Biological Chemistry (<strong>2002</strong>) 277: 43623-43630.<br />

28. PIRPIGNANI ML, RIVERA E, HELLMAN U AND BISCOGLIO DE JIMÉNEZ BONINO M. Structural and<br />

immunological aspects of Polybia scutellaris Antigen 5. Archives of Biochemistry and Biophysics<br />

(<strong>2002</strong>) 407: 224-230.<br />

29. PREOBRAZHENSKA O, YAKYMOVYCH M, KANAMOTO T, YAKYMOVYCH I, STOIKA R, HELDIN C-<br />

H AND SOUCHELNYTSKYI S. BRCA2 and Smad3 synergize in regulation of gene transcription.<br />

Oncogene (<strong>2002</strong>) 21: 5660-5664.<br />

30. RAHMANIAN M AND HELDIN P. Testicular hyaluronidase induces tubular structures of endothelial<br />

cells grown in three-dimensional collagen gel through a CD44-mediated mechanism.<br />

International Journal of Cancer (<strong>2002</strong>) 97: 601-607.<br />

31. ROSENDAHL A, PARDALI E, SPELETAS M, TEN DIJKE P, HELDIN C-H AND SIDERAS P. Activation<br />

of bone morphogenetic protein/Smad signaling in bronchial epithelial cells during airway<br />

inflammation. American Journal of Respiratory Cell and Molecular Biology (<strong>2002</strong>) 27: 160-169.<br />

32. SABRI N, ÖSTLUND FARRANTS A-K, HELLMAN U AND VISA N. Evidence for a posttranscriptional<br />

role of a TFIIIC�-like protein in Chironomus tentans. Molecular Biology of the Cell (<strong>2002</strong>) 13:<br />

1765-1777.<br />

33. SAJAN MP, BANDYOPADHYAY G, KANOH Y, STANDAERT ML, QUON MJ, REED BC, DIKIC I AND<br />

FARESE RV. Sorbitol activates atypical protein kinase C and GLUT4 glucose transporter<br />

translocation/glucose transport through proline-rich tyrosine kinase-2, the extracellular signalregulated<br />

kinase pathway and phospholipase D. The Biochemical Journal (<strong>2002</strong>) 362: 665-674.<br />

34. SALOVAARA R, ROTH S, LOUKOLA A, LAUNONEN V, SISTONEN P, AVIZIENYTE E, KRISTO P,<br />

JÄRVINEN H, SOUCHELNYTSKYI S, SARLOMO-RIKALA M AND AALTONEN LA. Frequent loss of<br />

SMAD4/DPC4 protein in colorectal cancers. Gut (<strong>2002</strong>) 51: 56-59.<br />

35. SHIMOKAWA N, OKADA J, HAGLUND K, DIKIC I, KOIBUCHI N AND MIURA M. Past-A, a novel<br />

proton-associated sugar transporter, regulates glucose homeostasis in the brain. Journal of<br />

Neuroscience (<strong>2002</strong>) 22: 9160-9165.<br />

36. SOUBEYRAN P, KOWANETZ K, SZYMKIEWICZ I, LANGDON WY AND DIKIC I. Cbl-CIN85endophilin<br />

complex mediates ligand-induced downregulation of EGF receptors. Nature (<strong>2002</strong>)<br />

416: 183-187.<br />

37. SZYMKIEWICZ I, KOWANETZ K, SOUBEYRAN P, DINARINA A, LIPKOWITZ S AND DIKIC I. CIN85<br />

participates in Cbl-b-mediated down-regulation of receptor tyrosine kinases. Journal of<br />

Biological Chemistry (<strong>2002</strong>) 277: 39666-39672.<br />

38. VALCOURT U, GOUTTENOIRE J, MOUSTAKAS A, HERBAGE D AND MALLEIN-GERIN F. Functions<br />

of transforming growth factor-� family type I receptors and Smad proteins in the hypertrophic<br />

maturation and osteoblastic differentiation of chondrocytes. Journal of Biological Chemistry<br />

(<strong>2002</strong>) 277: 33545-33558.<br />

39. WESTERMARK GT, FALKMER S, STEINER DF, CHAN SJ, ENGSTRÖM U AND WESTERMARK P. Islet<br />

amyloid polypeptide is expressed in the pancreatic islet parenchyma of the teleostean fish,<br />

Myoxocephalus (cottus) scorpius. Comparative Biochemistry and Physiology (<strong>2002</strong>) 133B: 119-<br />

125.<br />

40. WILLIAMS S, SOUCHELNYTSKYI S AND DANIK M. TGF�2 mediates rapid inhibition of calcium<br />

influx in identified cholinergic basal forebrain neurons. Biochemical and Biophysical <strong>Research</strong><br />

Communications (<strong>2002</strong>) 290: 1321-1327.<br />

150


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UPPSALA<br />

41. YAKYMOVYCH I, ENGSTRÖM U, GRIMSBY S, HELDIN C-H AND SOUCHELNYTSKYI S. Inhibition of<br />

transforming growth factor-� signaling by low molecular weight compounds interfering with<br />

ATP- or substrate-binding sites of the TGF� type I receptor kinase. Biochemistry (<strong>2002</strong>) 41:<br />

11000-11007.<br />

42. ZIELINSKI R, PILECKI M, KUBINSKI K, ZIEN P, HELLMAN U AND SZYSZKA R. Inhibition of yeast<br />

ribosomal stalk phosphorylation by Cu-Zn superoxide dismutase. Biochemical and Biophysical<br />

<strong>Research</strong> Communications (<strong>2002</strong>) 296: 1310-1316.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. BRODIN G. Smad7 in TGF-� signalling. Acta Universitatis Upsaliensis. Comprehensive<br />

2.<br />

Summaries of Uppsala Dissertations from the Faculty of Medicine. (<strong>2002</strong>) Eklundshofs Grafiska,<br />

Uppsala, 1135: pps. 1-57.<br />

DIKIC I. CIN85/CMS family of adaptor molecules. FEBS Letters (<strong>2002</strong>) 529: 110-115.<br />

3. DIKIC I, HAGLUND K AND IVANKOVIC-DIKIC I. Regulation of cell morphology by a proline-rich<br />

tyrosine kinase Pyk2. Periodicum Biologorum (<strong>2002</strong>) 104: 11-18.<br />

4. DIKIC I. Signal transduction by proline-rich tyrosine kinase Pyk2. Acta Universitatis Upsaliensis.<br />

Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (<strong>2002</strong>)<br />

Eklundshofs Grafiska, Uppsala, 1154: pps. 1-66.<br />

5. HELDIN C-H. Perturbation of stimulatory and inhibitory pathways in malignancies. Pezcoller<br />

Foundation Journal (<strong>2002</strong>) 19: 2-13.<br />

6. HELDIN C-H. Platelet-derived growth factor. In: Wiley Encyclopedia of Molecular Medicine,<br />

Creighton T, ed. John Wiley and Sons, Inc., New York, 5: pps. 2520-2522, (<strong>2002</strong>).<br />

7. HELDIN C-H, ERIKSSON U AND ÖSTMAN A. New members of the platelet-derived growth factor<br />

family of mitogens. Archives of Biochemistry and Biophysics (<strong>2002</strong>) 398: 284-290.<br />

8. LENNARTSSON J. Stem cell factor induced signal transduction. Acta Universitatis Upsaliensis.<br />

Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (<strong>2002</strong>)<br />

Eklundshofs Grafiska, Uppsala, 1142: pps. 1-59.<br />

9. MOUSTAKAS A. Smad signalling network. Journal of Cell Science (<strong>2002</strong>) 115: 3355-3356.<br />

10. MOUSTAKAS A AND HELDIN C-H. From mono- to oligo-Smads: The heart of the matter in TGF-�<br />

signal transduction. Genes & Development (<strong>2002</strong>) 16: 1867-1871.<br />

11. MOUSTAKAS A, PARDALI K, GAL A AND HELDIN C-H. Mechanisms of TGF-� signaling in<br />

regulation of cell growth and differentiation. Immunology Letters (<strong>2002</strong>) 82: 85-91.<br />

12. PIETRAS K. Inhibition of PDGF receptor signaling in tumor stroma. Effects on interstitial<br />

hypertension, drug uptake and therapeutic response. Acta Universitatis Upsaliensis.<br />

Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (<strong>2002</strong>)<br />

Eklundshofs Grafiska, Uppsala, 1181: pps. 1-65.<br />

13. SJÖBLOM T. Paracrine and autocrine functions of PDGF in malignant disease. Acta Universitatis<br />

Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine.<br />

(<strong>2002</strong>) Eklundshofs Grafiska, Uppsala, 1190: pps. 1-62.<br />

14. SOUCHELNYTSKYI S. Transforming growth factor-� signaling and its role in cancer. Experimental<br />

Oncology (<strong>2002</strong>) 24: 3-12.<br />

151


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15. SOUCHELNYTSKYI S, MOUSTAKAS A AND HELDIN C-H. TGF-� signaling from a threedimensional<br />

perspective: insight into selection of partners. Trends in Cell Biology (<strong>2002</strong>) 12: 304-<br />

307.<br />

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AFFILIATE RESEARCH REPORTS<br />

K. Alitalo: Page 154<br />

V. P. Collins : Page 158<br />

S. Lakhani: Page 161<br />

K. Miyazono : Page 163<br />

M. Pfreundschuh & C. Renner: Page 166<br />

E. Puré: Page 168<br />

R. R. Schmidt: Page 171<br />

P. ten Dijke: Page 173<br />

S. Ylä-Herttuala: Page 176<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003


KARI ALITALO,PH.D.<br />

MOLECULAR/CANCER BIOLOGY LABORATORY<br />

BIOMEDICUM HELSINKI<br />

UNIVERSITY OF HELSINKI, FINLAND<br />

RESEARCH REPORT<br />

LYMPHANGIOGENIC GROWTH FACTORS AND TUMOR METASTASIS<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 154<br />

ALITALO<br />

The major roles of the lymphatic system include the collection of extravasated fluid, macromolecules and<br />

white blood cells from tissues, and their transportation via the collecting lymphatic vessels and thoracic<br />

duct back into the blood circulation. Studies over the last few years have provided significant insights into<br />

the molecular mechanisms underlying the development of lymphatic vessels, and the roles of<br />

lymphangiogenesis in both physiological and pathological conditions. Two specific lymphangiogenic<br />

growth factors have so far been identified, named vascular endothelial cell growth factor C (VEGF-C)<br />

and VEGF-D. Both VEGF-C and VEGF-D have been shown to induce lymphangiogenesis in transgenic<br />

mice and other in vivo models. Both ligands signal via vascular endothelial cell growth factor receptor<br />

(VEGFR) -3. Although produced as pre-pro-polypeptides, stepwise proteolytic processing increases the<br />

affinities of VEGF-C and VEGF-D for VEGFR-3, and the fully processed mature forms can also bind to<br />

and activate VEGFR-2. Therefore both factors can also exert angiogenic activity via VEGFR-2.<br />

Accumulating data from clinicopathological studies suggest that cancer cell spread to regional lymph<br />

nodes is an early event for many solid tumors, and lymphatic vessels serve as the primary route for this<br />

event. Recent studies using animal models have provided convincing evidence for the role of<br />

lymphangiogenesis in lymphatic metastasis. Human breast cancer cell lines genetically modified to overexpress<br />

VEGF-C, showed enhanced rates of tumor spread to regional lymph nodes by inducing periand/or<br />

intra-tumoral lymphatic vessel growth when implanted orthotopically into the fat pads of<br />

mammary glands. In a genetic model of pancreatic tumorigenesis, over-expression of VEGF-C by �-cells<br />

of the endocrine pancreas increased lymphangiogenesis surrounding the primary tumor and enhanced<br />

tumor spread to the regional lymph nodes.<br />

Our recent study provided further evidence for validating the importance of lymphangiogenesis in<br />

lymphatic metastasis. Over-expression of VEGFR-3-Ig by stably transfecting LNM35 cells, a highly<br />

metastatic human lung cancer cell line expressing high levels of endogenous VEGF-C, or by systemic<br />

adenoviral delivery, showed inhibition of tumor lymphangiogenesis and lymph node metastasis when<br />

tumor cells were implanted into immuno-deficient mice. However, pre-existing skin lymphatics were not<br />

affected by VEGFR-3-Ig treatment. This suggests that newly formed lymphatic vessels are necessary for<br />

lymphatic metastasis.<br />

In spite of the significant advances in the study of lymphatic metastasis over the last few years, there are<br />

still many questions to be addressed. One prominent question is how tumor-associated lymphangiogenesis<br />

is regulated. Tumor-associated lymphatics could be formed by direct co-option of, or sprouting/splitting<br />

from, pre-existing lymphatic vessels in the surrounding tissues, or by recruitment of lymphatic endothelial<br />

cell (LEC) progenitors from bone marrow. The existence of LEC progenitors has been shown in avian<br />

embryogenesis. VEGFR-3 + /CD34 + endothelial precursors have recently been identified in human fetal<br />

liver and blood, and upon culture they were shown to express both vascular and lymphatic EC markers. It<br />

remains to be seen which mechanism is the main contributor to tumor lymphangiogenesis. So far only a<br />

few types of cancer have been shown to contain lymphatic vessels, although expression of VEGF-C<br />

mRNA and/or protein has been shown in a variety of human cancers. Also lymphatics are not


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ALITALO<br />

homogeneously distributed in tumors, and it is rare to detect lymphatic vessels within an expanding tumor<br />

mass. This suggests that lymphatic vessel growth is regulated by other micro-environmental factors in<br />

addition to lymphatic growth factors. Furthermore although newly formed lymphatic vessels are<br />

necessary components for lymphatic metastasis, lymphangiogenesis alone cannot cause lymph node<br />

metastasis. For example, over-expression of VEGF-C by lung carcinoma cells that do not metastatize well<br />

was shown to induce intra- and peritumoral lymphatic vessels, but no increase of lymph node metastases<br />

were detected. This suggests that additional factors are involved in the control of lymphatic metastases.<br />

Switching of tumor cells from a low to a high metastatic rate is a complex process and may involve the<br />

alteration of expression of many genes. Studies using high-throughput approaches such as microarray<br />

analysis will provide significant insights into this phenomenon.<br />

PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

1. SHAH RN, IBBITT JC, ALITALO K, HURST HC. FGFR4 overexpression in pancreatic cancer is<br />

mediated by an intronic enhancer activated by HNF1alpha. Oncogene (<strong>2002</strong>) Nov<br />

28;21(54):8251-61.<br />

2. ILJIN K, PETROVA TV, VEIKKOLA T, KUMAR V, POUTANEN M, ALITALO K. A fluorescent Tie1<br />

reporter allows monitoring of vascular development and endothelial cell isolation from transgenic<br />

mouse embryos. FASEB Journal (<strong>2002</strong>) Nov;16(13):1764-74.<br />

3. SZUBA A, SKOBE M, KARKKAINEN MJ, SHIN WS, BEYNET DP, ROCKSON NB, DAKHIL N,<br />

SPILMAN S, GORIS ML, STRAUSS HW, QUERTERMOUS T, ALITALO K, ROCKSON SG. Therapeutic<br />

lymphangiogenesis with human recombinant VEGF-C. FASEB Journal (<strong>2002</strong>) Dec;16(14):1985-<br />

7.<br />

4. YUAN L, MOYON D, PARDANAUD L, BREANT C, KARKKAINEN MJ, ALITALO K, EICHMANN A.<br />

Abnormal lymphatic vessel development in neuropilin 2 mutant mice. Development (<strong>2002</strong>)<br />

Oct;129(20):4797-806.<br />

5. VEIKKOLA T, ALITALO K. Dual role of Ang2 in postnatal angiogenesis and lymphangiogenesis.<br />

Developmental Cell (<strong>2002</strong>) Sep;3(3):302-4.<br />

6. WICKSTROM SA, ALITALO K, KESKI-OJA J. Endostatin associates with integrin alpha5beta1 and<br />

caveolin-1, and activates Src via a tyrosyl phosphatase-dependent pathway in human endothelial<br />

cells. Cancer <strong>Research</strong> (<strong>2002</strong>) Oct 1;62(19):5580-9.<br />

7. SAARISTO A, VEIKKOLA T, TAMMELA T, ENHOLM B, KARKKAINEN MJ, PAJUSOLA K, BUELER H,<br />

YLA-HERTTUALA S, ALITALO K. Lymphangiogenic gene therapy with minimal blood vascular<br />

side effects. Journal of Experimental Medicine (<strong>2002</strong>) Sep 16;196(6):719-30.<br />

8. SCHOPPMANN SF, BIRNER P, STOCKL J, KALT R, ULLRICH R, CAUCIG C, KRIEHUBER E, NAGY K,<br />

ALITALO K, KERJASCHKI D. Tumor-associated macrophages express lymphatic endothelial<br />

growth factors and are related to peritumoral lymphangiogenesis. American Journal of Pathology<br />

(<strong>2002</strong>) Sep;161(3):947-56.<br />

9. PETROVA TV, MAKINEN T, MAKELA TP, SAARELA J, VIRTANEN I, FERRELL RE, FINEGOLD DN,<br />

KERJASCHKI D, YLA-HERTTUALA S, ALITALO K. Lymphatic endothelial reprogramming of<br />

vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO Journal (<strong>2002</strong>)<br />

Sep 2;21(17):4593-9.


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ALITALO<br />

10. ERIKSSON U, ALITALO K. VEGF receptor 1 stimulates stem-cell recruitment and new hope for<br />

angiogenesis therapies. Nature Medicine (<strong>2002</strong>) Aug;8(8):775-7.<br />

11. ACHEN MG, WILLIAMS RA, BALDWIN ME, LAI P, ROUFAIL S, ALITALO K, STACKER SA. The<br />

angiogenic and lymphangiogenic factor vascular endothelial growth factor-D exhibits a paracrine<br />

mode of action in cancer. Growth Factors (<strong>2002</strong>) Jun;20(2):99-107.<br />

12. CURSIEFEN C, SCHLOTZER-SCHREHARDT U, KUCHLE M, SOROKIN L, BREITENEDER-GELEFF S,<br />

ALITALO K, JACKSON D. Lymphatic vessels in vascularized human corneas:<br />

immunohistochemical investigation using LYVE-1 and podoplanin. Investigative Ophthalmology<br />

and Visual Science (<strong>2002</strong>) Jul;43(7):2127-35.<br />

13. SAARISTO A, VEIKKOLA T, ENHOLM B, HYTONEN M, AROLA J, PAJUSOLA K, TURUNEN P,<br />

JELTSCH M, KARKKAINEN MJ, KERJASCHKI D, BUELER H, YLA-HERTTUALA S, ALITALO K.<br />

Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness<br />

but no sprouting angiogenesis in the skin or mucous membranes. FASEB Journal (<strong>2002</strong>)<br />

Jul;16(9):1041-9.<br />

14. KUBO H, CAO R, BRAKENHIELM E, MAKINEN T, CAO Y, ALITALO K. Blockade of vascular<br />

endothelial growth factor receptor-3 signaling inhibits fibroblast growth factor-2-induced<br />

lymphangiogenesis in mouse cornea. Proceedings of the National Academy of Science U S A<br />

(<strong>2002</strong>) Jun 25;99(13):8868-73.<br />

15. WILTING J, PAPOUTSI M, CHRIST B, NICOLAIDES KH, VON KAISENBERG CS, BORGES J, STARK<br />

GB, ALITALO K, TOMAREV SI, NIEMEYER C, ROSSLER J. The transcription factor Prox1 is a<br />

marker for lymphatic endothelial cells in normal and diseased human tissues. FASEB Journal<br />

(<strong>2002</strong>) Aug;16(10):1271-3.<br />

16. HE Y, KOZAKI K, KARPANEN T, KOSHIKAWA K, YLA-HERTTUALA S, TAKAHASHI T, ALITALO K.<br />

Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular<br />

endothelial growth factor receptor 3 signaling. Journal of the National Cancer <strong>Institute</strong> (<strong>2002</strong>)<br />

Jun 5;94(11):819-25.<br />

17. LEMSTROM KB, KREBS R, NYKANEN AI, TIKKANEN JM, SIHVOLA RK, AALTOLA EM, HAYRY<br />

PJ, WOOD J, ALITALO K, YLA-HERTTUALA S, KOSKINEN PK. Vascular endothelial growth factor<br />

enhances cardiac allograft arteriosclerosis. Circulation (<strong>2002</strong>) May 28;105(21):2524-30.<br />

18. MARCHETTI S, GIMOND C, ILJIN K, BOURCIER C, ALITALO K, POUYSSEGUR J, PAGES G.<br />

Endothelial cells genetically selected from differentiating mouse embryonic stem cells<br />

incorporate at sites of neovascularization in vivo. Journal of Cell Science (<strong>2002</strong>) May 15;115(Pt<br />

10):2075-85.<br />

19. MATTILA MM, RUOHOLA JK, KARPANEN T, JACKSON DG, ALITALO K, HARKONEN PL. VEGF-C<br />

induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7<br />

tumors. International Journal of Cancer (<strong>2002</strong>) Apr 20;98(6):946-51.<br />

20. ZHOU Y, MCMASTER M, WOO K, JANATPOUR M, PERRY J, KARPANEN T, ALITALO K, DAMSKY<br />

C, FISHER SJ. Vascular endothelial growth factor ligands and receptors that regulate human<br />

cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver<br />

enzymes, and low platelets syndrome. American Journal of Pathology (<strong>2002</strong>) Apr;160(4):1405-<br />

23.<br />

21. DIAS S, CHOY M, ALITALO K, RAFII S. Vascular endothelial growth factor (VEGF)-C signaling<br />

through FLT-4 (VEGFR-3) mediates leukemic cell proliferation, survival, and resistance to<br />

chemotherapy. Blood (<strong>2002</strong>) Mar 15;99(6):2179-84.


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ALITALO<br />

22. WITMER AN, BLAAUWGEERS HG, WEICH HA, ALITALO K, VRENSEN GF, SCHLINGEMANN RO.<br />

Altered expression patterns of VEGF receptors in human diabetic retina and in experimental<br />

VEGF-induced retinopathy in monkey. Investigative Ophthalmology and Visual Science (<strong>2002</strong>)<br />

Mar;43(3):849-57.<br />

23. PAAVONEN K, MANDELIN J, PARTANEN T, JUSSILA L, LI TF, RISTIMAKI A, ALITALO K,<br />

KONTTINEN YT. Vascular endothelial growth factors C and D and their VEGFR-2 and 3<br />

receptors in blood and lymphatic vessels in healthy and arthritic synovium. Journal of<br />

Rheumatology (<strong>2002</strong>) Jan;29(1):39-45.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. SAARISTO A, KARKKAINEN MJ, ALITALO K. Insights into the molecular pathogenesis and<br />

targeted treatment of lymphedema. Annals of the New York Academy of Sciences (<strong>2002</strong>)<br />

Dec;979:94-110.<br />

2. STACKER SA, ACHEN MG, JUSSILA L, BALDWIN ME, ALITALO K. Lymphangiogenesis and<br />

cancer metastasis. Nature Reviews Cancer (<strong>2002</strong>) Aug;2(8):573-83.<br />

3. JUSSILA L, ALITALO K. Vascular growth factors and lymphangiogenesis. Physiological Reviews<br />

(<strong>2002</strong>) Jul;82(3):673-700.<br />

4. ALITALO K, CARMELIET P. Molecular mechanisms of lymphangiogenesis in health and disease.<br />

Cancer Cell (<strong>2002</strong>) Apr;1(3):219-27.<br />

5. ALITALO K. Growth factors controlling angiogenesis and lymphangiogenesis. Ugeskr Laeger<br />

(<strong>2002</strong>) Jun 10;164(24):3170-2.<br />

6. KARKKAINEN MJ, ALITALO K. Lymphatic endothelial regulation, lymphoedema, and lymph node<br />

metastasis. Seminars in Cell Biology (<strong>2002</strong>) Feb;13(1):9-18.


V. PETER COLLINS,PH.D.<br />

DIVISION OF MOLECULAR HISTOPATHOLOGY<br />

DEPARTMENT OF PATHOLOGY<br />

UNIVERSITY OF CAMBRIDGE<br />

CAMBRIDGE,UK<br />

RESEARCH REPORT<br />

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COLLINS<br />

The research aims of the group are to define the molecular mechanisms involved in the oncogenesis and<br />

progression of primary human brain tumors. These data will then form the basis for the identification of<br />

prognostic indicators, indicators of sensitivity and resistance to therapy, and the development of logical,<br />

innovative and specific treatment modalities. The research focuses on characterizing the genomic<br />

abnormalities and transcriptosome in a large panel of histologically well-characterized tumors from<br />

patients with careful follow up. Limited proteomic studies on the clinical material are also being done to<br />

correlate and support the genomic and mRNA expression studies. During the past 18 months the<br />

following advances were made.<br />

EPIDERMAL GROWTH FACTOR RECEPTOR FAMILY AND LIGANDS<br />

We have previously reported that approximately 8% of glioblastomas have amplification of the region<br />

adjacent to, but not encompassing, the epidermal growth factor receptor (EGFR) gene. The function of the<br />

gene(s) targeted by the amplification event in these cases is/are unknown, and we are currently cloning<br />

them. In collaboration with the <strong>LICR</strong> New York Branch, a report on a number of monoclonal antibodies<br />

to the mutated receptor has been submitted for publication. In addition a study on the cellular uptake and<br />

retention of an EGF-dextran molecule in a model system has been reported. As the four members of the<br />

EGFR family interact we have extended our interest to all members, and the majority of their known<br />

ligands. We are currently completing a study on the expression of all members of the family, their splice<br />

variants as well as 15 of the known ligands in oligodendroglial tumours (n=99). The expression of the<br />

receptor splice variants and their ligands is proving complex. Correlations of the findings with clinical<br />

data and an analysis of any association between EGFR amplification, and rearrangement and prognosis /<br />

response to treatment in glioblastomas are also underway.<br />

CELL CYCLE CONTROL MECHANISMS IN BRAIN TUMOURS<br />

We have previously reported that deregulation of the Rb1 pathway, accompanied by inactivation of the<br />

p53 pathway, are almost prerequisites for the development of glioblastoma. In our previous studies<br />

amplification of the CDK6 and MDM4 genes, which could, if accompanied by overexpression, disrupt the<br />

Rb1 and p53 pathways, were not studied. An investigation of amplification of these genes identified four<br />

tumours with amplification and overexpression of CDK6 but no cases with amplification of MDM4. The<br />

findings indicate that CDK6 amplification and overexpression is relatively uncommon; occurring in<br />

approximately 3% of glioblastomas. An analysis of aberrations of the p53 and Rb1 pathways as<br />

prognostic indicators in glioblastomas has been completed, and indicates that disruption of the Rb1<br />

pathway is a significant negative prognostic indicator.


CHROMOSOMES 1, 6 AND 22 TILED ARRAYS AND A 10 MB GENOMIC ARRAY<br />

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COLLINS<br />

Tiled arrays for chromosome 1 have been produced and validated in a collaborative project with the<br />

former Chromosome 1 Group at the Wellcome Trust Sanger <strong>Institute</strong> (Hinxton, UK), in order to map<br />

deletions in our series of oligodendroglial, and astrocytic tumours. A number of small homozygous<br />

deletions have been identified in the astrocytic tumours indicating the presence of at least two new tumour<br />

suppressor genes. These homozygous deletions lie within regions that commonly show loss of one allele.<br />

Mutation analysis of genes in these two regions in tumours with hemizygous deletions is currently being<br />

carried out to specifically identify the genes involved. Approximately 1800 PAC/BAC clones forming a<br />

tiling path of chromosome 6, which were used for the human genome sequencing project at the Sanger<br />

<strong>Institute</strong>, have been purified, amplified, arrayed, and printed in-house. Evaluation, and validation of the<br />

arrays has been completed and they are being used to map in detail the commonly deleted regions of<br />

chromosome 6 previously identified in the glioma series (using microsatellite technology), and in<br />

collaborative studies of other cancers. A detailed mapping of a homozygous deletion in a glioblastoma on<br />

chromosome 22 has previously been reported, and a tiled CGH-microarray covering 28 Mb of<br />

chromosome 22 (about 85% of its long arm) has also been constructed to refine commonly deleted<br />

regions detected in previous studies. The homozygous deletion identified is a 200-kb region<br />

encompassing three genes, 14-3-3 eta (YWHAH), and two novel genes, AL050256 and KIAA0645.<br />

Mutation analysis is ongoing on these genes in our series of brain tumours. The rationale was to map and<br />

size 22q deletions around the NF2 gene in sporadic schwannomas using a reliable method with maximal<br />

resolution. We detected heterozygous deletions in 21 tumors (45%), with interesting hemizygous<br />

interstitial deletions in four of those cases. The NF2 locus was not affected in two of those tumours.<br />

TRANSCRIPTOSOME<br />

Ready-made cDNA arrays as well as oligonucleotide array of cDNAs representing 19600 genes have<br />

been developed, and validated in collaboration with Dr. Tom Freeman’s Group at The Human Genome<br />

Mapping Project Resource Centre at the Hinxton Genome Campus (Hinxton, UK). We have explored the<br />

cycle-dependent amplification characteristics of Template-Switching PCR and validated its use for<br />

microarray target labelling. TS-PCR is able to identify at least 80% of the differentially expressed genes<br />

identified by direct labelling using 50ng (1,000 fold less) input RNA. We have shown the sensitivity of<br />

microarray experiments is increased, and we have also validated the fidelity of amplification and show<br />

that the amplified material faithfully represents the starting mRNA population. This method permits at<br />

least three repeat hybridisations from 1�g total RNA. The oligonucleotide arrays are now providing the<br />

first data on genome wide expression patterns in our tumour series.<br />

MENINGIOMAS<br />

A detailed analysis of chromosome 17 in a series of atypical and anaplastic meningiomas was carried out<br />

demonstrating marked variations in copy number at many loci along the long arm in the anaplastic series.<br />

The pattern of allele increase was complex and pointed to a number of regions.<br />

OTHER PROJECTS<br />

In collaboration with the <strong>LICR</strong> Stockholm Branch a study of the expression of the coxsackie and<br />

adenovirus receptor in human astrocytic tumors, xenografts and cell lines was commenced. The human<br />

homologue of the mouse WIG-1/PAG608 (wild type TP53 induced gene) was cloned, sequenced and its<br />

chromosomal localization identified. Evidence for its amplification and increased expression in primary<br />

squamous cell carcinoma of the lung was presented. A case study of tuberous sclerosis presenting in late<br />

adult life was also published.


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

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COLLINS<br />

1. BUCKLEY PG, MANTRIPRAGADA KK, BENETKIEWICZ M, TAPIA-PAEZ I, DIAZ DE STAHL T,<br />

ROSENQUIST M, ET AL. A full-coverage, high-resolution human chromosome 22 genomic<br />

microarray for clinical and research applications. Human Molecular Genetics (<strong>2002</strong>)<br />

11(25):3221-9.<br />

2. ZAREI M, COLLINS VP, CHANDRAN S, VALLER D, HIGGINS JN, COMPSTON DA, ET AL. Tuberous<br />

sclerosis presenting in late adult life. Journal of Neurology, Neurosurgery and Psychiatry (<strong>2002</strong>)<br />

73(4):436-8.<br />

3. BUSCHGES R, ICHIMURA K, WEBER RG, REIFENBERGER G, COLLINS VP. Allelic gain and<br />

amplification on the long arm of chromosome 17 in anaplastic meningiomas. Brain Pathology<br />

(<strong>2002</strong>) 12(2):145-53.<br />

4. OLSSON P, COLLINS VP, LIU L, GEDDA L, LILJEGREN A, CARLSSON J. Internalisation and<br />

retention of EGF-dextran associated radioactivity in transfected Chinese hamster ovary cells<br />

expressing the human EGF-receptor. International Journal of Oncology (<strong>2002</strong>) 20(5):1057-63.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. V. P. COLLINS. Cellular mechanisms targeted during astrocytoma progression. Cancer Letters<br />

(<strong>2002</strong>) 188, 1-7.<br />

2. V. P. COLLINS. Diagnosis of Common Brain Neoplasia. CPD Bulletin, Cellular Pathology (<strong>2002</strong>)<br />

4(2), 92-102.


SUNIL LAKHANI, M.D., F.R.C.PATH.<br />

THE BREAKTHROUGH TOBY ROBINS BREAST CANCER RESEARCH CENTRE<br />

CHESTER BEATTY LABS<br />

INSTITUTE OF CANCER RESEARCH (ICR)<br />

LONDON,UK<br />

RESEARCH REPORT<br />

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LAKHANI<br />

The team based at the Breakthrough Breast Cancer Centre, ICR, London has three major programs of<br />

research in the laboratory, all exploiting modern (loss of heterozygosity, LOH; comparative genome<br />

hybridization, CGH; microarray) and classical (morphology, immunohistochemistry) pathological<br />

methods<br />

The programs include:<br />

� Genetic alterations in normal breast tissues<br />

� The study of myoepithelial tumours of the breast<br />

� Molecular pathology of familial breast cancer<br />

The aim of the first study is to characterise the distribution, extent and, in the longer term, significance of<br />

genetic alterations in normal breast tissue. Using an in vitro cell-cloning technique, Dr. M. O’Hare (<strong>LICR</strong><br />

London University College Branch) and I have demonstrated LOH in luminal as well as myoepithelial<br />

cells of the breast. The frequency and distribution and significance of these genetic changes within the<br />

breast are at present unknown.<br />

During the last year, cells have been cultured from defined areas of nine breast samples, and 1059 clones<br />

have been harvested from two of these. LOH is being studied in one of these samples at seven<br />

microsatellites that were altered in the tumour from that patient. The data thus far show that genetic<br />

alterations appear more frequent in distinct areas, rather than being randomly distributed. The data<br />

suggest a very low level of alteration in the normal breast.<br />

The second program is investigating the molecular profile of ‘myoepithelial’ & ‘basaloid’ tumours with a<br />

view to delineating genetic changes that may play an important role in breast carcinogenesis.<br />

Myoepithelial cells are normally located between the continuous luminal epithelial cells and the basal<br />

lamina of acini and ducts of the breast. Tumours with a myoepithelial cell component have been<br />

recognized for some time within the salivary gland as well as the breast. However myoepithelial cell<br />

differentiation in the neoplastic population of “ordinary” ductal and lobular carcinomas of the breast is<br />

rare. The first part of the study investigating CGH profiles of 43 basaloid tumours and 43 non-basaloid<br />

tumours has been completed. The data demonstrate a different profile of changes between the two sets.<br />

The correlation of these samples with clinical follow-up data has been undertaken and demonstrates<br />

differing prognosis related to the differencing genetic profiles. Dr Laura Fulford (<strong>LICR</strong> London<br />

University College Branch) and Dr Jorge Reis-Filho, both Pathology trainees, have been appointed to<br />

study for PhD degrees, and will extend the work on myoepithelial tumours. Dr Fulford will study the<br />

comparative pathology of myoepithelial lesions in salivary gland, breast, skin and lung, and Dr Reis-Filho<br />

will study metaplastic carcinomas of the breast.<br />

The studies on familial cancers are attempting to delineate the morphological, immunophenotypical and<br />

molecular characteristics of familial cancers compared to sporadic breast cancers. Eight years ago, The<br />

Breast Cancer Linkage Consortium (BCLC) members agreed to submit material to assess the


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LAKHANI<br />

histopathology of tumours associated with BRCA1/2 and BRCAX mutations. This large international<br />

collaborative effort has resulted in a unique dataset comprising morphology and immunohistochemistry<br />

data in familial breast cancer. The studies demonstrated that the morphological features of tumours from<br />

patients harbouring mutations in BRCA1 and BRCA2 gene differ from each other and from sporadic breast<br />

cancers. The morphological analysis has implications for clinical management of patients. CGH arrays<br />

are being developed, and the BCLC set of familial cancers will be studied using this approach in the near<br />

future.<br />

PATHOLOGY FACILITY:BREAKTHROUGH/ICR & <strong>LICR</strong> COLLABORATION<br />

The programmes outlined above are being underpinned by a core pathology facility comprising tissue<br />

cutting and staining, evaluation of novel proteins using immunohistochemistry, and the creation of a<br />

tissue array facility for high throughput screening. A number of tissue microarrays have been developed<br />

including a 600-breast cancer array, for which clinical follow up has been performed.<br />

The Pathology core facility has in this last year cut 28000 sections and 4800 blocks, carried out 10000<br />

immunocytochemical procedures, and has worked up approximately 75 antibodies.<br />

PUBLICATIONS:<br />

PRIMARY RESEARCH ARTICLES<br />

1. LAKHANI, S.R., VAN DE VIJVER, M.J., JACQUEMIER, J., ANDERSON, T.J., OSIN, P.P., MCGUFFOG,<br />

L., AND EASTON, D.F. The pathology of familial breast cancer: predictive value of<br />

immunohistochemical markers estrogen receptor, progesterone receptor, HER-2, and p53 in<br />

patients with mutations in BRCA1 and BRCA2. Journal of Clinical Oncology (<strong>2002</strong>) 20(9): p.<br />

2310-8.<br />

2. SAWYER, E.J., HANBY, A.M., ROWAN, A.J., GILLETT, C.E., THOMAS, R.E., POULSOM, R.,<br />

LAKHANI, S.R., ELLIS, I.O., ELLIS,P., AND TOMLINSON, I.P. The Wnt pathway, epithelial-stromal<br />

interactions, and malignant progression in phyllodes tumours. Journal of Pathology (<strong>2002</strong>)<br />

196(4): p. 437-44.<br />

3. SUZUKI, R.N., ENTWISTLE, A.,ATHERTON, A.J., CLARKE, C., LAKHANI, S.R., AND O'HARE, M.J.<br />

The expression patterns of integrin subunits on human breast tissues obtained during pregnancy.<br />

Cell Biology International (<strong>2002</strong>) 26(7): p. 593-8<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. CLARKE,C. AND LAKHANI, S.R. The significance of early breast lesions. BCO. (<strong>2002</strong>) Vol5: p. 1-<br />

5.<br />

2. FULFORD, L.,RYAN, D.A., AND LAKHANI, S.R., Pathology of Breast Cancer, in International<br />

Handbook of Breast Cancer, Johnstone, S., Editor. (<strong>2002</strong>) Euromed Communications Ltd.<br />

3. LAKHANI, S.R. AND FLANAGAN, A.M., Pathology of the breast and ovary in mutation carriers, in<br />

Familial Breast and Ovarian Cancer, Morrison, P., Hodgson, S., and Haites, N., Editors. (<strong>2002</strong>)<br />

Cambridge University Press.<br />

4. LAKHANI, S.R. AND DOGAN, A., Wound Healing, in Clinical Surgery in General, Kirk, R.M.,<br />

Editor. (<strong>2002</strong>) Churchill Livingstone: London.


KOHEI MIYAZONO,PH.D.<br />

DEPARTMENT OF BIOCHEMISTRY<br />

THE CANCER INSTITUTE<br />

JAPANESE FOUNDATION FOR CANCER RESEARCH (JFCR)<br />

TOKYO,JAPAN<br />

RESEARCH REPORT<br />

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MIYAZONO<br />

At the Department of Biochemistry, in The Cancer <strong>Institute</strong> of the JFCR, we are working on signal<br />

transduction, by the TGF-� superfamily proteins, through Smad proteins. We focus on how TGF-�<br />

regulates the growth of various cells, and how tumor cells become resistant to the growth inhibitory<br />

activity of TGF-�. We are also elucidating how TGF-� signals are positively and negatively regulated by<br />

various molecules. In 2000, Dr. Kohei Miyazono was appointed a Professor in the Department of<br />

Molecular Pathology in the Graduate School of Medicine, at the University of Tokyo (Tokyo, Japan), and<br />

since then, Dr. Miyazono’s two groups (in the Departments of Biochemistry, and Molecular Pathology)<br />

have worked in close collaboration, exchanging information, reagents, and research expertise.<br />

CIS-COMPOUND DISRUPTION OF MURINE SMAD2 GENE IN APC KNOCKOUT MICE<br />

Many tumor cells are resistant to the growth inhibitory activity of TGF-�. Smad2 is a receptor-regulated<br />

Smad (R-Smad) that is activated specifically by TGF-� and activin signaling. To examine the role of<br />

Smad2 inactivation in the process of carcinogenesis, we generated compound heterozygous mice, which<br />

carry both Apc and Smad2 mutations on the same chromosome in the cis-configuration. Compound<br />

inactivation of Smad2 in heterozygous Apc mutant mice increased sudden death from intestinal<br />

obstruction caused by extremely large tumors. Histological examination revealed that Apc/Smad2 ciscompound<br />

heterozygotes developed multiple invasive cancers that had not been observed in Apc single<br />

heterozygotes. Thus it appears that loss of Smad2 alone does not initiate tumorigenesis, but accelerates<br />

malignant progression of tumors to invasive cancer in the late stages of carcinogenesis.<br />

C-MYC AS A DOWNSTREAM TARGET OF TGF-� IN GROWTH INHIBITION<br />

Down-regulation of c-Myc has been suggested to be one of the most critical events for growth inhibition<br />

induced by TGF-�, and we have previously identified a Smad-responsive element in the c-myc promoter.<br />

In addition, we have shown that Smads regulate c-myc transcription through disrupting the interaction<br />

between �-catenin and TCF-4. �-catenin activated the transcriptional activity of the c-myc promoter by<br />

binding to a novel LEF/TCF-responsive element. When TCF-4 was bound to this element, TGF-�<br />

dissociated �-catenin and repressed the transcriptional activity of the c-myc promoter. However, TGF-�<br />

neither dissociated �-catenin nor repressed c-myc transcription when LEF-1, a member of the TCF-4<br />

family, was bound to the element. Since LEF-1 is highly expressed in some colon cancer cells, this<br />

finding will be important to understand how certain tumor cells acquire resistance to the growth inhibition<br />

by TGF-�.


SMURF1 IN REGULATION OF THE FUNCTION OF INHIBITORY SMADS (I-SMADS)<br />

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MIYAZONO<br />

We have previously reported that a HECT type E3 ubiquitin ligase, Smurf1, physically interacts with<br />

Smad7. Smurf1 induced nuclear export of Smad7 to the cytoplasm, and associated with TGF-� type I<br />

receptor (T�R-I) via Smad7, with the subsequent enhancement of degradation of T�R-I and Smad7. We<br />

demonstrated that Smurf1 also inhibits bone morphogenetic protein (BMP) signaling together with the I-<br />

Smads, Smad6/7. Smurf1 bound to BMP type I receptors via I-Smads, and induced ubiquitination of these<br />

receptors. Moreover, Smurf1 associated with R-Smads, Smad1/5, indirectly through I-Smads, and<br />

induced their degradation. Smurf1 thus controls BMP signaling through multiple mechanisms.<br />

INTERACTION OF SMADS WITH TRANSCRIPTIONAL CO-REPRESSORS, C-SKI AND SNON<br />

c-Ski was originally identified as the cellular homologue of the v-ski oncogene product, present in the<br />

avian Sloan-Kettering retroviruses. c-Ski has been shown to repress transcription by recruiting histone<br />

deacetylase. We have previously reported that Smad2/3 interacts with c-Ski in a TGF-�-dependent<br />

manner. Using a three-dimensional modeling system, we found that c-Ski and SnoN, which is structurally<br />

related to c-Ski, bind to the “SE” sequence in the MH2 domain of Smad3, which is exposed on the Nterminal<br />

upper side of the doughnut-like structure of the MH2 oligomer. The “QPSMT” sequence, located<br />

in the vicinity of SE, supports the interaction with c-Ski and SnoN. Our findings demonstrated the<br />

stoichiometry of how multiple molecules can associate with the Smad oligomers, and how the Smadinteracting<br />

proteins functionally interact with each other.<br />

PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

1. YAGI, K.,FURUHASHI, M.,AOKI, H.,GOTO, D.,KUWANO, H.,SUGAMURA, K.,MIYAZONO, K.,<br />

AND KATO, M. c-myc is a downstream target of Smad pathway. Journal of Biological Chemistry<br />

(<strong>2002</strong>) 277, 854-861.<br />

2. SUZAWA, M., TAMURA, Y., FUKUMOTO, S., MIYAZONO, K., FUJITA, T., KATO, S., AND<br />

TAKEUCHI, Y. Stimulation of Smad1 transcriptional activity by Ras-extracellular signal-regulated<br />

kinase pathway: a possible mechanism for collagen-dependent osteoblastic differentiation.<br />

Journal of Bone and Mineral <strong>Research</strong> (<strong>2002</strong>) 17, 240-248.<br />

3. RING, C., OGATA, S.,MEEK, L.,SONG, J., OHTA, T.,MIYAZONO, K., AND CHO, K.W.Y. The role<br />

of a Williams-Beuren syndrome-associated helix-loop-helix domain-containing transcription<br />

factor in activin/nodal signaling. Genes & Development (<strong>2002</strong>) 16, 820-835.<br />

4. SAKUMA, R., OHNISHI, Y.,MENO, C., FUJII, H.,JUAN, H.,TAKEUCHI, J., OGURA, T.,LI, E.,<br />

MIYAZONO, K., AND HAMADA, H. Inhibition of Nodal signaling by Lefty mediated through<br />

interaction with common receptors and efficient diffusion. Genes to Cells (<strong>2002</strong>) 7, 401-412.<br />

5. DENDA, K.,SHIMOMURA, T.,KAWAGUCHI, T.,MIYAZAWA, K., AND KITAMURA, N. Functional<br />

characterization of Kunitz domains in hepatocyte growth factor activator inhibitor type 1. Journal<br />

of Biological Chemistry (<strong>2002</strong>) 277, 14053-14059.<br />

6. SHEN, Z.-J., NAKAMOTO, T.,TSUJI, K.,NIFUJI, A.,MIYAZONO, K.,KOMORI, T.,HIRAI, H., AND<br />

NODA, M. Negative regulation of BMP/Smad signaling by Cas-interacting zinc finger protein in<br />

osteoblasts. Journal of Biological Chemistry (<strong>2002</strong>) 277, 29840-29846.<br />

7. NISHIHARA, A.,WATABE, T.,IMAMURA, T., AND MIYAZONO, K. Functional heterogeneity of<br />

bone morphogenetic protein receptor-II mutants found in patients with primary pulmonary<br />

hypertension. Molecular Biology of the Cell (<strong>2002</strong>) 13, 3055-3063.


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MIYAZONO<br />

8. SUZUKI, C., MURAKAMI, G.,FUKUCHI, M.,SHIMANUKI, T.,SHIKAUCHI, Y.,IMAMURA, T., AND<br />

MIYAZONO, K. Smurf1 regulates the inhibitory activity of Smad7 by targeting Smad7 to the<br />

plasma membrane. Journal of Biological Chemistry (<strong>2002</strong>) 277, 39919-39925.<br />

9. OTA,T.,FUJII,M.,SUGIZAKI,T.,ISHII,M.,MIYAZAWA,K.,ABURATANI,H., AND MIYAZONO,K.<br />

Targets of transcriptional regulation by two distinct type I receptors for transforming growth<br />

factor-� in human umbilical vein endothelial cells. Journal of Cellular Physiology (<strong>2002</strong>) 199,<br />

299-318.<br />

10. HAMAMOTO, T.,BEPPU, H.,OKADA, H.,KAWABATA, M.,KITAMURA, T.,MIYAZONO, K., AND<br />

KATO, M. Compound disruption of Smad2 accelerates malignant progression of intestinal tumors<br />

in Apc knockout mice. Cancer <strong>Research</strong> (<strong>2002</strong>) 62, 5955-5961.<br />

11. KAIBORI, M.,INOUE, T.,ODA, M.,NAKA, D.,KAWAGUCHI, T.,KITAMURA, N.,MIYAZAWA, K.,<br />

KWON, A.H.,KAMIYAMA, Y., AND OKUMURA T. Exogenously administered HGF activator<br />

augments liver regeneration through the production of biologically active HGF. Biochemical and<br />

Biophysical <strong>Research</strong> Communications (<strong>2002</strong>) 290, 475-481.<br />

12. KAIBORI,M.,INOUE,T.,SAKAKURA,Y.,ODA,M.,NAGAHAMA,T.,KWON,A.H.,KAMIYAMA,Y.,<br />

MIYAZAWA, K., AND OKUMURA, T. Impairment of activation of hepatocyte growth factor<br />

precursor into its mature form in rats with liver cirrhosis. Journal of Surgical <strong>Research</strong> (<strong>2002</strong>)<br />

106, 108-114.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. MIYAZONO K. A new partner for inhibitory Smads. Cytokine & Growth Factor Reviews (<strong>2002</strong>)<br />

13, 7-9.<br />

2. MIYAZONO, K. Bone morphogenetic protein receptors and actions. In: Principles of Bone<br />

Biology. Eds. Bilezikian, J.P., Raisz, L.G., and Rodan, G.A. Second ed., Vol. 2. (<strong>2002</strong>) Academic<br />

Press, San Diego, pp. 929-942.<br />

3. MIYAZONO,K., AND MIYAZAWA, K. Id: A target of BMP signaling. Science's STKE (<strong>2002</strong>) PE40.<br />

4. MIYAZAWA, K.,SHINOZAKI, M.,HARA, T.,FURUYA, T., AND MIYAZONO, K. Two major Smad<br />

pathways in TGF-� superfamily signaling, Genes to Cells (<strong>2002</strong>) 7, 1191-1204.<br />

5. MIYAZONO, K. Regulation of transforming growth factor-� signaling and vascular diseases.<br />

Cornea (<strong>2002</strong>) 21 (7 Suppl), S48-53.


MICHAEL PFREUNDSCHUH,M.D.<br />

&CHRISTOPH RENNER,PH.D.<br />

UNIVERSITY OF SAARLAND<br />

HOMBURG,GERMANY<br />

RESEARCH REPORT<br />

PFREUNDSCHUH &RENNER<br />

Our group in Homburg is actively involved in two <strong>LICR</strong> programs, the identification of new tumor<br />

antigens using the SEREX methodology, and the generation of new antibody constructs for the treatment<br />

of cancer.<br />

The further characterisation of tumor antigens identified by SEREX has confirmed previous observations<br />

that most identified antigens are intracellular proteins. Two proteins studied recently include restin and<br />

CT-8/HOM-TES-85, both of which have a physiological role in intracellular trafficking of vesicles or<br />

transcriptional control. The aberrant expression of these proteins in human neoplasms might therefore be<br />

involved in cancer associated alterations of transcriptional or post-transcriptional processes. The<br />

expression analysis of cancer/testis (CT) antigens in human tumors with the focus on lymphoma has<br />

revealed once again that the currently available repertoire of tumor antigens is insufficient to cover a<br />

broad spectrum of patients. Therefore, new tumor antigens with a wider expression pattern are needed.<br />

We have established an eukaryotic cDNA expression system in yeast to improve on the current SEREX<br />

methodology. Recombinant antigen expression on the yeast surface (RAYS) allows for the identification<br />

of antigens that are recognised by the immune system in their native non-linear format. This might open<br />

the field for the detection of cell surface expressed tumor antigens, as their specific recognition often<br />

relies on antibody binding to conformational epitopes or post-translational modifications. Further<br />

experiments will tell if this technical improvement will provide us with a different picture of tumor<br />

related antigens recognised by the immune system.<br />

Within the <strong>LICR</strong> Antibody Program we have continued the generation and expression of recombinant<br />

antibodies that specifically target either tumor-associated antigens, or antigens expressed by the<br />

connective tissue that surrounds tumors, for example, the fibroblast activation protein (FAP). Our primary<br />

focus was set on the generation of so called immunocytokines which are fusion products of an antibody<br />

with a human cytokine. Tumor necrosis factor (TNF) and interleukin-8 (IL-8) were chosen since these<br />

cytokines are very capable of recruiting polymorphic nucleated cells including macrophages which cause<br />

intensive local activation. The TNF-immunocytokine (anti-FAP-TNF) we generated carries a TNF dimer<br />

that binds with moderate affinity to TNF-receptor I (TNF-RI), but very efficiently activates TNF-RII<br />

when binding, via the antibody domain, to tumor cells. The preferential activation of TNF-RII, with low<br />

affinity to TNF-RI, should result in reduced systemic TNF-related side-effects, but result in strong TNF<br />

activity at the tumor site. This could be demonstrated in an animal model where high doses of our<br />

construct could be administered with almost no side effects, and resulted in significant reductions of<br />

tumor size. The immunohistochemical analysis of tumors and surrounding tissue after treatment<br />

demonstrated an intense macrophage infiltration with preferential localisation at the tumor rim. Currently,<br />

combinations of cytokines are being studied to increase the efficacy of the approach and, hopefully, allow<br />

for the transition of the system into the clinic.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 166


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

PFREUNDSCHUH &RENNER<br />

1. PREUSS KD, ZWICK C, BORMANN C, NEUMANN F, PFREUNDSCHUH M. Analysis of the B-cell<br />

repertoire against antigens expressed by human neoplasms. Immunological Reviews (<strong>2002</strong>)<br />

188(1):43-50<br />

2. HUANG S, PREUSS KD, XIE X, REGITZ E, PFREUNDSCHUH M. Analysis of the antibody repertoire<br />

of lymphoma patients. Cancer Immunology and Immunotherapy (<strong>2002</strong>) 51(11-12):655-62<br />

3. SAHIN U, NEUMANN F, TURECI O, SCHMITS R, PEREZ F, PFREUNDSCHUH M. Hodgkin and Reed-<br />

Sternberg cell-associated autoantigen CLIP-170/restin is a marker for dendritic cells and is<br />

involved in the trafficking of macropinosomes to the cytoskeleton, supporting a function-based<br />

concept of Hodgkin and Reed-Sternberg cells. Blood (<strong>2002</strong>) 100(12):4139-45<br />

4. TURECI O, SAHIN U, KOSLOWSKI M, BUSS B, BELL C, BALLWEBER P, ZWICK C, EBERLE T,<br />

ZUBER M, VILLENA-HEINSEN C, SEITZ G, PFREUNDSCHUH M. A novel tumour associated leucine<br />

zipper protein targeting to sites of gene transcription and splicing. Oncogene (<strong>2002</strong>) 21(24):3879-<br />

88<br />

5. SCHMITS R, COCHLOVIUS B, TREITZ G, REGITZ E, KETTER R, PREUSS KD, ROMEIKE BF,<br />

PFREUNDSCHUH M. Analysis of the antibody repertoire of astrocytoma patients against antigens<br />

expressed by gliomas. International Journal of Cancer (<strong>2002</strong>) 98(1):73-7<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 167


ELLEN PURÉ,PH.D.<br />

THE WISTAR INSTITUTE,DEPARTMENT OF MICROBIOLOGY<br />

UNIVERSITY OF PENNSYLVANIA<br />

PHILADELPHIA,USA<br />

RESEARCH REPORT<br />

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PURÉ<br />

Inflammation is typically a self-limiting response to infection or injury. Although inflammation is largely<br />

a reparative response, severe acute inflammation or persistent inflammation can lead to tissue injury. In<br />

fact, inflammation is now recognized to be a critical component of a wide variety of diseases ranging<br />

from acute septic shock to chronic inflammatory diseases to some types of cancer. Our goal is to gain<br />

insights into the molecular and cellular basis of inflammation, particularly in relation to how<br />

inflammation impacts on tumor cell growth and metastasis. Our current studies relate to several important<br />

questions in cancer biology. We are studying the B cell antigen receptor (BCR)-mediated signaling<br />

pathways responsible for the reactivation of Epstein Barr virus (EBV) and EBV-associated<br />

lymphoproliferative disorders and tumors. Furthermore, we are studying the mechanisms that mediate<br />

leukocyte trafficking, and leukocyte and parenchymal cell growth and differentiation, and how these<br />

mechanisms regulate tumor cell growth and metastasis.<br />

SIGNAL TRANSDUCTION IN BLYMPHOCYTES<br />

Engagement of antigen receptors on hematopoietic cells is coupled to non-receptor protein tyrosine<br />

kinases (PTKs). BCR activation of PTKs results in the activation of multiple downstream effector<br />

molecules that regulate cytoskeletal reorganization, protein translation, and gene transcription, thus<br />

driving a variety of biological responses that determine whether B lymphocytes undergo apoptosis,<br />

proliferate, or differentiate to antibody secreting cells or memory cells. We have, for the past year, been<br />

focusing on: 1) BCR-mediated activation of the lytic cycle of Epstein-Barr virus in latently infected B<br />

cells, and 2) BCR-mediated signal transduction and BCR internalization.<br />

Epstein-Barr virus (EBV) is a ubiquitous virus that is carried by over 90% of the world’s population and<br />

has been implicated in the pathogenesis of several cancers including Burkitt’s Lymphoma, and Hodgkins<br />

disease. In immunocompetent individuals the immune response to EBV controls the infection but does<br />

not eradicate it, and the virus persistently resides in a latent state in B lymphocytes. Thus EBV can be<br />

reactivated, particularly in immunosuppressed individuals. The switch between EBV latency and<br />

reactivation is induced, through BCR-coupled signal transduction pathways, by initiating transcriptional<br />

activation of the viral immediate-early gene BZLF1 through either of two promoters, Zp or Rp. The early<br />

signaling events involved in BCR-mediated induction of BZLF1 and EBV reactivation remain, in large<br />

part, to be elucidated. In collaboration with Dr. Paul Lieberman (Wistar <strong>Institute</strong>) we have defined the<br />

requirements for BCR proximal tyrosine kinases in the transcriptional activation of BZLF1.<br />

Receptor signaling and endocytosis are linked to cytoskeletal reorganization, but the underlying<br />

mechanisms remain poorly defined. BCR-mediated signaling induces phosphorylation of Cbl, which we<br />

have demonstrated plays an important role in BCR endocytosis, and Cbl is also required for the<br />

phosphorylation of CrkII. Furthermore, we demonstrated that ligation of BCR induces the interaction<br />

between Cbl and CrkII, Cbl-dependent activation of Rac, and reorganization of the actin cytoskeleton.<br />

Thus, our results indicate that interaction between Cbl, which is coupled to receptor signaling complexes<br />

at the plasma membrane, and CrkII, which is associated with the cytoskeleton, represents a critical link


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PURÉ<br />

between BCR engagement, and reorganization of the actin cytoskeleton, thus providing a mechanism for<br />

efficient receptor endocytosis.<br />

INFLAMMATION,FIBROSIS AND TUMOR GROWTH AND METASTASIS<br />

Inflammation and cancer are characterized by activation of local parenchymal cells, leukocyte infiltration<br />

and, turnover and remodeling of extracellular matrix (ECM). A major focus of our laboratory is on<br />

understanding the cell-cell and cell-matrix interactions that regulate the inflammatory response, and<br />

tumor cell growth and metastasis. Our studies are aimed at testing the following hypotheses: 1)<br />

Modification of ECM plays a critical role in the initiation and progression of inflammatory responses, and<br />

tumor cell growth and metastasis. And 2) Activation and differentiation of parenchymal cells contribute<br />

significantly to the progression of inflammation, and tumor cell growth and metastasis.<br />

One focus of these studies has been on the role of the adhesion receptor CD44 and its ligands. CD44 is a<br />

member of a widely expressed family of adhesion receptors that represent alternatively spliced and<br />

posttranslationally modified products of a single gene. CD44 is a major receptor for hyaluronan (HA), a<br />

glycosaminoglycan that is ubiquitously distributed in ECM, and can, in addition, bind other matrix<br />

components such as fibronectin and collagen, a matrix metalloproteinase (MMP-9), growth factors, and<br />

cytokines and chemokines such as osteopontin and MIP-1�. CD44 was once thought to be confined to<br />

being a simple transmembrane adhesion molecule that also played a critical role in the metabolism of HA.<br />

Intense investigation of CD44 over the past twenty years has led to the definition of additional functions<br />

of CD44 including its capacity to mediate signal transduction, inflammatory cell function, and tumor<br />

growth and metastasis. Importantly, CD44 does not appear to play a critical role in homeostasis making<br />

CD44 particularly attractive as a potential target for novel therapeutic interventions in inflammation and<br />

cancer.<br />

We have been investigating the role of CD44 in several murine models of inflammatory diseases and<br />

cancer by comparing pathology in wild-type and CD44-deficient mice. We are comparing the<br />

inflammatory response and pathology associated with bleomycin-induced fibrotic lung injury (in<br />

collaboration with Dr. Paul Noble, Yale University); infection with toxoplasma gondii (in collaboration<br />

with Dr. Chris Hunter, University of Pennsylvania), allergen-induced airway inflammation and<br />

hyperreactivity (with Dr. Reynold Panettieri Jr., University of Pennsylvania), atherosclerosis (in<br />

collaboration with Dr. Daniel Rader, University of Pennsylvania), and cerebral ischemic injury with<br />

collaborators at Dupont. In each case we found that a deficiency in CD44 had a profound impact on the<br />

initiation and or progression of disease in mice and in each case have defined multiple potential<br />

mechanisms by which CD44 contributes to pathology involving leukocytes, and parenchymal, endothelial<br />

and epithelial cells.<br />

In addition to being expressed as a transmembrane receptor on many cell types, a soluble form of CD44<br />

(sCD44) is found in serum and lymph. Interestingly, immunodeficiency correlates with low plasma levels<br />

of sCD44 whereas malignant diseases, and immune activation and inflammation are often associated with<br />

increased plasma levels of CD44. We have investigated the mechanisms by which soluble CD44 is<br />

generated, and have demonstrated that epithelial and parenchymal cells spontaneously release sCD44.<br />

Exposure to leukocyte- and bacterial-derived proteinases markedly increases the release of sCD44.<br />

Interestingly, we found that the spontaneously released sCD44 was incorporated into high molecular<br />

weight complexes derived from matrix. Consistent with the homology of CD44 to proteoglycan core and<br />

link proteins, our data indicate that CD44 released from cells accumulates as an integral component of the<br />

ECM, and may play a role in the organization of matrices and in anchoring growth factors and<br />

chemokines to the ECM. Increases in plasma CD44 during immune activation and tumor progression are<br />

therefore likely to be, in part, a manifestation of the matrix remodeling that occurs in the face of the


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 170<br />

PURÉ<br />

enhanced proteolytic activity associated with infection, inflammation and tumor metastasis, leading to<br />

alterations in cell-matrix interactions. Thus, CD44 serves as a model for understanding posttranslational<br />

processing and its emerging role as a general mechanism for regulating cell behavior.<br />

Our current studies are focused on defining the role of oxidative stress in modifying components of the<br />

ECM, including HA, and testing the hypothesis that modified components of matrix play a critical part in<br />

inflammation, and tumor cell growth and metastasis. In addition, we are establishing a panel of markers<br />

that will allow us to distinguish parenchymal cells at various stages of activation and differentiation. In<br />

addition, we are investigating the role of cell surface proteinases expressed specifically on activated<br />

parencymal cells in inflammation and surrounding tumors. Furthermore, we are investigating the role of<br />

matrix in maintaining parechymal cells and dormant tumor cells in a quiescent state.<br />

Finally, over the past several years we have identified several novel mechanisms by which the production<br />

of the key proinflammatory mediator interleukin (IL) -12 is regulated. Specifically, we demonstrated that<br />

in addition to its proinflammatory activities, TNF is also a potent inhibitor of macrophage IL-12<br />

production. Furthermore, we discovered that 12/15-lipoxygenase is required for the production of IL-12<br />

by macrophages in vitro and in atherosclerotic lesions in mice. We are pursuing the molecular basis for<br />

these novel mechanisms of regulation of IL-12 gene expression during an inflammatory response.<br />

PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

1. TEDER, P.,W.VANDIVIER, D.JIANG, J.LIANG, L.COGN, E. PURÉ, P.M.HENSON AND P.W.<br />

NOBLE. Resolution of lung inflammation by CD44. Science <strong>2002</strong> 296:155-158.<br />

2. KAWASHIRI,M.,Y.ZHANG,E.PURÉ AND D.J. RADER. Combined effects of cholesterol reduction<br />

and apolipoprotein A-I expression on atherosclerosis in LDL receptor deficient mice.<br />

Atherosclerosis (<strong>2002</strong>) 165:15-22.<br />

3. LAZAAR, A.L., M. PLOTNICK, I., U. KUCICH, I.CRICHTON, S.LOTFI, S.K.P.DAS, S.KANE, J.<br />

ROSENBLOOM, R.A. PANETTIERI JR., N.M. SCHECTER, AND E. PURÉ. Mast cell chymase modifies<br />

cell-matrix interactions and inhibits mitogen-induced proliferation of human airway smooth<br />

muscle cells. Journal of Immunology (<strong>2002</strong>) 169:1014-1020.<br />

4. ZHAO, L.,C.CUFF, E.MOSS, U.WILLE, T.CYRUS, E.A. KLEIN, S.SONG, D.PRATICO, D.J.<br />

RADER, C.A. HUNTER, E.PURÉ AND C.D. FUNK. Selective interleukin-12 synthesis defect in<br />

12/15-lipoxygenase deficient macrophages associated with reduced atherosclerosis in a model of<br />

familial hypercholesterolemia. Journal of Biological Chemistry (<strong>2002</strong>) 277:35350-35356.<br />

5. CICHY, J., R. BALS, J.POTEMPA, A.MANI AND E. PURÉ. Proteinase-mediated release of CD44<br />

from epithelial cells: Extracellular CD44 complexes with components of cellular matrices.<br />

Journal of Biological Chemistry (<strong>2002</strong>) 277:44440-44447.<br />

6. WANG, X.L.XU, H.HONG, Y.ZHAN, E.PURÉ AND G.Z. FEUERSTEIN. CD44 deficiency in mice<br />

protects brain from cerebral ischemic injury. Journal of Neurochemistry (<strong>2002</strong>) 83:1172-9.


RICHARD R. SCHMIDT,PH.D.<br />

FACHBEREICH CHEMIE<br />

UNIVERSITÄT KONSTANZ<br />

KONSTANZ,GERMANY<br />

RESEARCH REPORT<br />

GANGLIOSIDE GM2 AND INVESTIGATION OF DERIVED PROJECTS<br />

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SCHMIDT<br />

In <strong>2002</strong> the group in Konstanz worked in four different areas which were partly supported by the <strong>Ludwig</strong><br />

<strong>Institute</strong> for Cancer <strong>Research</strong>.<br />

SYNTHESIS OF �-GALACTOPYRANOSYL CERAMIDE AND STRUCTURAL VARIANTS<br />

The study of the presentation of ganglioside GM2 by human CD 1b for specific recognition by T<br />

lymphocytes led to the identification of a structurally related class of compounds, which has recently<br />

gained great importance, namely the �-galactosyl ceramides. Some �-galactosyl ceramides, with<br />

phytosphingosine as one of the lipid moieties, have been found to have strong anti-tumour activities in<br />

vivo. This kind of structure is not usually detected in normal mammalian tissues. A particularly interesting<br />

compound is �-D-galactopyranosyl ceramide 1 which was found to have immunostimulatory activity, and<br />

is thus is increasing demand for immunological studies. Based on previous synthesis of this compound in<br />

the Konstanz group, we were able to efficiently synthesize this target molecule, and also a structural<br />

variant having different lipid chains. Both compounds are now available to the <strong>LICR</strong> for biological<br />

studies. This work is being conducted in collaboration with Drs. Gerd Ritter at the <strong>LICR</strong> New York<br />

Branch, and <strong>LICR</strong> Affiliates Vincenzo Cerundolo (Oxford, UK), and Robert Schreiber (St. Louis, USA).<br />

SYNTHESIS OF SAPONINS RELATED TO QS 21<br />

Saponins are complex amphiphilic glycolipid conjugates which are mainly of plant origin, and many of<br />

them exhibit immunostimulatory properties. A particularly interesting class of saponins is obtained from<br />

the bark of Quillaja Saponaria Molina, a tree which grows mainly in South America. The most famous<br />

saponin from this source is QS 21, a bisdesmosidic triterpenoid glycoside with a very complex structure;<br />

this compound is the most potent saponin immune adjuvant known to date. Because of the complexity of<br />

this structure, several structurally simpler derivatives were synthesized by the Konstanz group for<br />

biological studies, with part of the work being patented.<br />

SYNTHESIS OF CARBOHYDRATE BASED VEGF PEPTIDE MIMETICS AS ANGIOGENESIS INHIBITORS<br />

Angiogenesis plays an important role in the pathogenesis of a number of diseases, in particular in<br />

promoting the growth and metastatic spread of solid tumours. There is considerable evidence that<br />

members of the vascular endothelial growth factor (VEGF) family of proteins are key mediators of<br />

angiogenesis. The biological effects of the VEGFs are mediated through receptors (VEGFRs). Recently<br />

VEGF-D has been shown to be expressed in human tumours, and to be angiogenic and lymphangiogenic.<br />

Therefore, VEGF-D may be of particular importance for the malignancy of tumours, as metastases can<br />

spread via either blood vessels or lymphatic vessels. The goal of our work, performed in collaboration<br />

with Drs. Mark Achen and Steven Stacker at the <strong>LICR</strong> Melbourne Branch, and Dr. R. Hughes (University<br />

of Melbourne, Melbourne, Australia), is to use a partial peptide structure of VEGF-D as a lead for the


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SCHMIDT<br />

design of VEGFR-2/-3 inhibitors, which could be potential anti-tumour agents. In order to reduce<br />

biological degradation, sugar residues have been included in the synthesis design. In <strong>2002</strong> some<br />

compounds were provided to the Melbourne group for biological studies.<br />

SYNTHESIS OF SOME BIFUNCTIONAL CHELATING AGENTS FOR RADIOIMMUNOTHERAPY<br />

Tumor-targeting with radioisotope-labelled antibodies is being used for (i) cancer radio immunotherapy,<br />

(ii) cancer diagnosis, and (iii) in vivo magnetic resonance imaging. Central to the success of such<br />

approaches is the development of bifunctional chelating agents containing both a reactive functionality<br />

for covalent attachment to the antibody, and a strong cation-binding group which is capable of rapidly and<br />

selectively forming an inert complex with radionuclides. We have investigated the synthesis of some<br />

novel bifunctional chelating agents which possess a partial carbohydrate backbone. Some target<br />

molecules have been obtained, and have been made available for further evaluation in collaboration with<br />

Drs. Chaitan Divgi, Peter Smith-Jones, and Gerd Ritter at the <strong>LICR</strong> New York Branch, and Drs. Andrew<br />

Scott and FT Lee at the <strong>LICR</strong> Melbourne Branch.<br />

PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

1. S. D. GADOLA,N.R.ZACCAI,K.HARLOS,D.SHEPHERD,J.C.CASTRO-PALOMINO,G.RITTER,R.<br />

R. SCHMIDT,E.Y.JONES,V.CERUNDOLO. Nature Immunology (<strong>2002</strong>) 3, 721-726.<br />

PATENT APPLICATIONS<br />

1. M. C. COSTA,L.GREEN,R.R.SCHMIDT,S.LEY,M.I.ELEUTÉRIO,C.RUBALCAVA,F.CARDOSO,<br />

J. SCHIMMEL,G.RITTER. New Immunomodulator agents, methods for their preparation and their<br />

use in vaccines, international application No. PCT/PT02/0016, 7th November <strong>2002</strong>.


PETER TEN DIJKE,PH.D.<br />

TGF-� RESEARCH GROUP<br />

DIVISION OF CELLULAR BIOCHEMISTRY<br />

NETHERLANDS CANCER INSTITUTE<br />

AMSTERDAM,THE NETHERLANDS<br />

RESEARCH REPORT<br />

TGF-� SIGNAL TRANSDUCTION<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 173<br />

TEN DIJKE<br />

Transforming growth factor-� (TGF-�) family members, which include TGF-�s, activins and bone<br />

morphogenetic proteins (BMPs), regulate a broad spectrum of biological responses in a large variety of<br />

cell types. They signal via specific serine/threonine kinase transmembrane receptors and nuclear effector<br />

proteins, termed Smads. Our goals are to elucidate the molecular mechanisms by which TGF-� family<br />

members elicit their cellular effects, and to generate animal models for human diseases that are caused by<br />

perturbed TGF-� family signaling.<br />

TGF-���SMAD (IN)DEPENDENT SIGNAL TRANSDUCTION<br />

To advance our understanding of Smad function and regulation we have screened for Smad interacting<br />

proteins. In collaboration with Dr. C Ibanez (Karolinska <strong>Institute</strong>, Sweden), we identified GATA3, a key<br />

regulator of T helper cell development as a tissue-restricted partner of Smad3. We found that TGF-�<br />

induces a specific and direct interaction between Smad3 and GATA3 in T-cells, thereby recruiting Smad3<br />

to GATA DNA binding sites and allowing TGF-� regulation of GATA-3 target promoters.<br />

Smad anchor for receptor activation (SARA) regulates the subcellular localization of R-Smads, and<br />

presents R-Smads to the receptor complex. We have found that SARA is localized in early endosomes,<br />

and that its FYVE domain is sufficient for this localization. Moreover, we have demonstrated that the<br />

FYVE domain preferentially binds to PtdIns(3)P in vitro. Consistent with this finding, wortmannin, an<br />

inhibitor of PI3 kinase, induces a mislocalization of SARA thus inhibiting TGF-�/Smad signaling. Our<br />

results raise the possibility that the activated TGF-� receptor complex has to undergo internalization to<br />

induce the phosphorylation of SARA-bound R-Smads.<br />

To obtain more insight into TGF-� receptor-initiated intracellular responses that are distinct from the<br />

Smad pathway, we analysed the activation of small GTP-binding proteins and MAP kinases by TGF-� in<br />

collaboration with Drs. Carl-Henrik Heldin and Aris Moustakas at the <strong>LICR</strong> Uppsala Branch. We<br />

observed that TGF-� can activate Rac, and ERK, JNK and p38 MAP kinases in certain cell types. The<br />

rapid kinetics and the TGF-�-induced activation of these components in cells deficient of Smad4 support<br />

the notion that these effects occur in a Smad-independent manner. In addition, we generated a mutant<br />

TGF-� type I receptor (T�R-I) defective in Smad activation and characterized its signaling properties.<br />

The T�R-I-induced extracellular matrix induction, growth inhibition, and epithelial to mesenchymal<br />

trans-differentiation were found to be dependent on the Smad pathway, but JNK MAP kinase activation<br />

did not require the Smad pathway.


TGF-� AND ANGIOGENESIS<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 174<br />

TEN DIJKE<br />

In most cell types, TGF-� signals via T�R-I/ALK5. However, in collaboration with Dr. Kohei Miyazono,<br />

we found that in endothelial cells TGF-� can bind and transduce signals via two distinct type I receptors,<br />

ALK1 and ALK5. ALK5 is widely expressed and induces the phosphorylation of Smad2 and Smad3,<br />

while ALK1, which is predominantly expressed in endothelial cells, stimulates Smad1 and Smad5<br />

phosphorylation. Interestingly, the TGF-�/ALK5 and TGF-�/ALK1 were found to have opposing effects<br />

on endothelial behaviour; ALK5 inhibits EC migration and proliferation while ALK1 stimulates both<br />

processes. We identified genes that are induced specifically by TGF-�-mediated ALK1 versus ALK5<br />

activation, and found that the activation state of the endothelium may be dependent on the level of TGF-<br />

�-induced ALK1 or ALK5 activation.<br />

CONTROLLING MESENCHYMAL CELL FATE BY BMPS<br />

Depending on their extracellular stimuli, mesenchymal cells can differentiate into muscle, fat, bone, or<br />

cartilage. BMPs have important roles in directing mesenchymal cell fate choices. Id1 was identified as a<br />

direct target of BMPs, but not TGF-�. We identified three distinct sequence elements in the Id1 promoter;<br />

Smad binding elements (SBEs), and GGCG and CAGC sequence motifs, each with a pivotal role in Id1<br />

activation by BMPs. Smads were found to be part of transcription factor complexes that specifically bind<br />

to SBEs and CCGCCC palindromic sequence in response to BMP but not TGF-�. Multimerization of all<br />

three distinct sequence motifs is needed to generate a highly sensitive and BMP/Smad dependent specific<br />

enhancer. We found that clusters of all three sequence elements are also important for BMP-induced<br />

activation of other BMP target genes.<br />

PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

6. KLOEN P, DOTY SB, GORDON E, RUBEL IF, GOUMANS M-J, HELFET DL. Expression and<br />

Activation of the BMP-Signaling Components in Human Fracture Nonunions. Journal of Bone<br />

and Joint Surgery, American Volume (<strong>2002</strong>) 84:1909-18.<br />

7. CHANG W, TEN DIJKE P, WU DK. BMP pathways are involved in otic capsule formation and<br />

epithelial-mesenchymal signaling in the developing chicken inner ear. Developmental Biology<br />

(<strong>2002</strong>) 251:380-94<br />

8. MUEHLEMAN C, KUETTNER KE, RUEGER DC, TEN DIJKE P, CHUBINSKAYA S.<br />

Immunohistochemical localization of osteogenetic protein (OP-1) and its receptors in rabbit<br />

articular cartilage. Journal of Histochemistry and Cytochemistry (<strong>2002</strong>) 50:1341-50<br />

9. BONDESTAM J, KAIVO-OJA N, KALLIO J, GROOME N, HYDEN-GRANSKOG C, FUJII M,<br />

MOUSTAKAS A, JALANKO A, TEN DIJKE P, RITVOS O. Engagement of activin and bone<br />

morphogenetic protein signaling pathway Smad proteins in the induction of inhibin B production<br />

in ovarian granulosa cells. Molecular and Cellular Endocrinology (<strong>2002</strong>) 195:79<br />

10. BONDESTAM J, HUOTARI MA, MOREN A, USTINOV J, KAIVO-OJA N, KALLIO J, HORELLI-<br />

KUITUNEN N, AALTONEN J, FUJII M, MOUSTAKAS A, TEN DIJKE P, OTONKOSKI T, RITVOS O.<br />

cDNA cloning, expression studies and chromosome mapping of human type I serine/threonine<br />

kinase receptor ALK7 (ACVR1C). Cytogenetics and Cell Genetics (<strong>2002</strong>) 95:157-62.<br />

11. HE W, LI AG, WANG D, HAN S, ZHENG B, GOUMANS M-J, TEN DIJKE P, WANG XJ. (<strong>2002</strong>)<br />

Overexpression of Smad7 results in severe pathological alterations in multiple epithelial tissues.<br />

EMBO Journal (<strong>2002</strong>) 21:2580-2590.


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 175<br />

TEN DIJKE<br />

12. GOUMANS M-J, VALDIMARSDOTTIR G, ITOH S, ROSENDAHL A, SIDERAS P, TEN DIJKE P. (<strong>2002</strong>)<br />

Balancing the activation state of the endothelium via two distinct TGF-� type I receptors. EMBO<br />

Journal (<strong>2002</strong>) 21:1743-53.<br />

13. DONG C, ZHU S, WANG T, YOON W, LI Z, ALVAREZ RJ, TEN DIJKE P, WHITE B, WIGLEY FM,<br />

GOLDSCHMIDT-CLERMONT PJ. Deficient Smad7 expression: a putative molecular defect in<br />

scleroderma. Proceedings of the National Academy of Science USA (<strong>2002</strong>) 99:3908-13.<br />

14. OHKAWARA B, IEMURA S, TEN DIJKE P, UENO N. Action range of BMP is defined by its Nterminal<br />

basic amino acid core. Current Biology (<strong>2002</strong>) 12:205-9.<br />

15. KORCHYNSKYI O, TEN DIJKE P. Identification and functional characterization of distinct critically<br />

important bone morphogenetic protein-specific response elements in the Id1 promoter. Journal of<br />

Biological Chemistry (<strong>2002</strong>) 277:4883-91.<br />

16. BLOKZIJL A, TEN DIJKE P, IBANEZ C. GATA3-mediated recruitment of Smad3 to target promoters<br />

allows TGF-� regulation of cytokine expression. Current Biology (<strong>2002</strong>) 12:35-45.<br />

17. FAN X, VALDIMARSDOTTIR G, BRUN A, MAGNUSSON M, TEN DIJKE P, KARLSSON S. Transient<br />

disruprtion of autocrine transforming growth factor-� signaling leads to enhanced survival and<br />

proliferation in single primitive human hematopoietic progenitor cells. Journal of Immunology<br />

(<strong>2002</strong>) 168:755-62.<br />

18. ITOH F, DIVECHA N, BROCKS L, OOMEN L, JANSSEN H, CALAFAT J, ITOH S TEN DIJKE P. The<br />

FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of<br />

SARA in early endosomes and regulates TGF-�/Smad signalling. Genes to Cells (<strong>2002</strong>) 7:321-<br />

31.<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS ~PUBLISHED<br />

1. TEN DIJKE P, GOUMANS M-J, ITOH F, ITOH S. Regulation of cell proliferation by Smad proteins.<br />

Journal of Cell Physiology (<strong>2002</strong>) 191:1-16.<br />

2. DENNLER S, GOUMANS M-J, TEN DIJKE P.� Transforming growth factor beta signal transduction.<br />

Journal of Leukocyte Biology (<strong>2002</strong>) 71:731-40.<br />

3. KORCHYNSKY,O, TEN DIJKE P. Bone morphogenetic protein receptors and their nuclear effectors<br />

in bone formation. In “Bone morphogenetic proteins: from discovery to clinical application.<br />

(<strong>2002</strong>) Ed. S. Vukicevic. (Birkhauser Verlag AG, Basel), pp. 31-60.


SEPPO YLÄ-HERTTUALA, M.D., PH.D,<br />

A.I.VIRTANEN INSTITUTE<br />

UNIVERSITY OF KUOPIO<br />

KUOPIO,FINLAND<br />

RESEARCH REPORT<br />

YLÄ-HERTTUALA<br />

We have tested the efficacy of vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, VEGF-<br />

C156S, VEGF-D and VEGF-D�N�C in an ischemic rabbit hindlimb model. VEGFs were delivered using<br />

adenoviral vectors which have been produced in Kuopio. Adenoviral vectors were given as intra-muscular<br />

(i.m.) injections, and the condition of the ischemic hindlimb was analyzed at six days, and at four weeks<br />

after the gene delivery using histology, flow measurements, and MRI. It was established that VEGF-A<br />

and VEGF-D�N�C were the most efficient growth factors to induce angiogenesis in hindlimbs, whereas<br />

VEGF-C and VEGF-D were the most efficient lymphangiogenic factors. Angiogenic effects were mostly<br />

due to enlargement of capillaries which was NO-mediated whereas lymphangiogenesis was not NOdependent.<br />

MRI and flow measurements confirmed the findings (Rissanen et al, VEGF-D is the strongest<br />

angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses<br />

(Circulation <strong>Research</strong> 2003, in press).<br />

We have also studied the efficacy of VEGF-C adenovirus given together with the platelet-derived growth<br />

factor receptor (PDGFR) tyrosine kinase inhibitor GLEEVEC in a rabbit balloon denudation restenosis<br />

model. It was established that while VEGF-C adenoviral delivery was efficient in reducing restenosis up<br />

to three weeks, the effect was lost due to catch-up growth at the six week timepoint. However, when<br />

VEGF-C adenovirus was combined with PDGFR inhibition, the treatment effect persisted for six weeks.<br />

Gleevec treatment alone did not produce any long-lasting treatment effect. VEGF-C adenovirus therapy<br />

was also associated with faster endothelial regrowth, and reduced monocyte-macrophage infiltration into<br />

the vessel wall. Combination of VEGF-C gene transfer and treatment with the PDGFR inhibitor STI571<br />

leads to persistent reduction in neointima formation in balloon-denuded rabbit aorta.<br />

The efficacy of VEGF-D�N�C adenovirus for the reduction of restenosis has been evaluated in the rabbit<br />

balloon denudation model. VEGF-D�N�C adenovirus reduced intimal thickening both 14 days, and 28 days<br />

after the gene transfer. The treated arteries had thinner intimas after the therapy, and reduced monocytemacrophage<br />

infiltration was detected in the treated arteries. Expression of VEGF-D in human<br />

atherosclerotic lesions has also been evaluated.<br />

The A.I.Virtanen <strong>Institute</strong> Virus Vector Laboratory has produced several adenoviral vectors encoding the<br />

members of the VEGF family, decoy VEGF receptors, PDGF-C and PDGF-D. These studies have been<br />

done in collaboration with Drs. Marc Achen and Steven Stacker at the <strong>LICR</strong> Melbourne Branch, and Drs.<br />

Ulf Eriksson, Arne Östman and Carl-Henrik Heldin at the <strong>LICR</strong> Uppsala Branch, and Dr. Kari Alitalo at<br />

the University of Helsinki (Helsinki, Finland), and have resulted in several joint publications.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 176


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

YLÄ-HERTTUALA<br />

1. LEMSTRÖM, K.B., KREBS, R., NYKÄNEN, A.I., TIKKANEN, J.M., SIHVOLA, R.K., AALTOLA, E.M.,<br />

HÄYRY, P.J., WOOD, J., ALITALO,K.,YLÄ-HERTTUALA,S.,KOSKINEN, P.K. Vascular endothelial<br />

growth factor enhances cardiac allograft arteriosclerosis. Circulation (<strong>2002</strong>) 105: 2524-2530.<br />

2. SAARISTO, A.,VEIKKOLA, T.,ENHOLM, B., HYTÖNEN, M.,AROLA, J., PAJUSOLA, K.,JELTSCH,<br />

M., KÄRKKÄINEN, M.,YLÄ-HERTTUALA, S.,ALITALO, K. Adenoviral VEGF-C overexpression<br />

induces blood vessel enlargement, tortuosity and leakiness, but no sprouting angiogenesis in the<br />

skin or mucous membranes. FASEB Journal (<strong>2002</strong>) 16: 1041-1049.<br />

3. PETROVA, T.V., MÄKINEN, T., MÄKELÄ, T.P., SAARELA, J., VIRTANEN, I., FERRELL R.E.,<br />

FINEGOLD, D.N., KERJASCHKI, D.,YLÄ-HERTTUALA, S.,ALITALO, K. Lymphatic endothelial<br />

reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO<br />

Journal (<strong>2002</strong>) 21: 4593-4599.<br />

4. SAARISTO, A.,VEIKKOLA, T.,TAMMELA, T.,ENHOLM, B., KARKKAINEN, M.J., PAJUSOLA, K.,<br />

BUELER, H.,YLÄ-HERTTUALA, S.,ALITALO, K. Lymphangiogenic gene therapy with minimal<br />

blood vascular side effects. Journal of Experimental Medicine (<strong>2002</strong>) 196: 719-730.<br />

5. HE, Y.,KOZAKI, K.,KÄRPÄNEN, T.,KOSHIKAWA, K.,YLÄ-HERTTUALA, S.,TAKAHASHI, T.,<br />

ALITALO, K. Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking<br />

vascular endothelial growth factor receptor 3 signalling. Journal of the National Cancer <strong>Institute</strong><br />

(<strong>2002</strong>) 92: 819-825.<br />

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JAMES R. KERR PROGRAM<br />

James R. Kerr Program Investigators: Page 179<br />

<strong>Research</strong> <strong>Report</strong>: Page 180<br />

Publications: Page 183<br />

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JAMES R. KERR PROGRAM INVESTIGATORS<br />

CHINA<br />

JAMES R. KERR PROGRAM<br />

PROF.WEI-FENG CHEN Peking University, Beijing<br />

PROF.BOQUAN JIN Fourth Military Medical University, Xi’an<br />

RUSSIA<br />

DR.VADIM AGOL Belozersky <strong>Institute</strong> of Physico-Chemistry and Biology,<br />

Moscow State University, Moscow<br />

DR.ANDREI ALEXEEVSKI Belozersky <strong>Institute</strong> of Physico-Chemistry and Biology,<br />

Moscow State University, Moscow<br />

PROF.PAVEL P. PHILIPPOV Belozersky <strong>Institute</strong> of Physico-Chemistry and Biology,<br />

Moscow State University, Moscow<br />

DR.ANDREY B. VARTAPETIAN Belozersky <strong>Institute</strong> of Physico-Chemical Biology,<br />

Moscow State University, Moscow<br />

PROF.SERGEI NEDOSPASOV Engelhardt Insititute of Molecular Biology, Moscow<br />

PROF.VLADIMIR P. SKULACHEV School of Bioengineering and Bioinformatics, Moscow<br />

State University, Moscow<br />

DR.MIKHAIL GELFAND State Scientific Center GOXNII Genetika, Moscow<br />

TURKEY<br />

DR.UGUR OZBEK <strong>Institute</strong> for Oncology, Istanbul University, Istanbul<br />

UKRAINE<br />

DR.VALERIE FILONENKO <strong>Institute</strong> of Molecular Biology and Genetics, Kyiv<br />

DR.IVAN GOUT <strong>Institute</strong> of Molecular Biology and Genetics, Kyiv<br />

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RESEARCH REPORT<br />

JAMES R. KERR PROGRAM<br />

The James R. Kerr Program integrates the research activities of laboratories affiliated with the <strong>Institute</strong> in<br />

China, Russia, Turkey, and the Ukraine. The goal of the Program is to simultaneously stimulate cancer<br />

research on a broad international basis, and utilize the very high level of expertise that already exists in<br />

the countries where the Program is becoming established, thus strengthening the <strong>LICR</strong>’s research<br />

programs.<br />

A strong component of the James R. Kerr Program lies in the realm of bioinformatics. In this regard, the<br />

laboratory of Dr. Mikhail Gelfand, at State Scientific Center GOSNII Genetika in Moscow, has<br />

concentrated its contribution in the area of alternative transcript splicing. The group has found that<br />

alternative splicing tends to insert or delete complete protein domains more frequently than expected by<br />

chance, whereas disruption of domains and other structural modules is less frequent. However, when<br />

domain regions are disrupted, the functional effect, as predicted from 3D structure, is frequently<br />

equivalent to removal of the entire domain. Interestingly, the short alternative splicing events within<br />

domains that might preserve folded structure, target functional residues more frequently than expected.<br />

Thus positive selection appears to have had a major role in the evolution of alternative splicing. In order<br />

to further investigate the evolution of transcript splicing specificity, the group has also initiated an<br />

analysis of alternative splicing patterns between human and mouse genomes through the comparison of<br />

the alternative splicing isoforms of 166 pairs of orthologous human and mouse genes. It has been found<br />

that about half of the analyzed genes have species-specific splicing isoforms, and about one fourth of<br />

elementary alternatives are not conserved between the human and mouse genomes. Thus splicing<br />

represents an apparently important source of genetic differentiation between species. In order to<br />

disseminate splicing data as widely as possible within the scientific community, the group has also started<br />

developing an automatically generated database of tissue- and stage-specific alternative splicing using<br />

EST data, and a curated database of published alternative splicing isoforms and alternative splicing<br />

regulation in collaboration with the Lawrence Berkeley Laboratory in the U.S.A.<br />

At the Belozersky <strong>Institute</strong> in Moscow, the group headed by Dr. Vadim Agol focuses bioinformatic<br />

capacity on the study of protein and gene families. The group has been developing new methodologies for<br />

defining families of homologous sequences, based on the computation of high-quality multiple<br />

alignments, and is applying these, together with other existing bioinformatics tools, to annotate protein<br />

and nucleotide sequence families with excellent results. A number of new profiles and annotations for<br />

protein families have been developed, covering major subfamilies of homeobox trascriptional factors,<br />

viral RNA-dependent RNA polymerases and novel, putative transposase helix-turn-helix domains. A<br />

method for high-quality automated annotation of protein sequences has also been developed and<br />

subjected to mass testing. This methodology is now provided as a public service. Another new<br />

methodology is a profile developer that provides a working environment for experts to create profiles<br />

from unannotated automatic multiple alignments. Finally, in the course of studies of long terminal repeats<br />

(LTRs) in the human genome, which are of special interest because of their possible role in<br />

tumorigenesis, a Human Endogenous RetroElement databank (HEREB) has been developed (in<br />

collaboration with the <strong>Institute</strong> of Bioorganic Chemistry, Moscow), and is populated with the data for<br />

LTR5 retroelements.<br />

A further aspect of the work of the James R. Kerr Program involves the biochemical analysis of apoptotic<br />

signaling in the laboratories of Professor V.P. Skulachev at the School of Bioengineering and<br />

Bioinformatics, Moscow State University, and Dr. A.B. Vartapetian at the Belozersky <strong>Institute</strong>. The focus<br />

of the work in Professor Skulachev’s laboratory concerns the mechanism of the mitochondrial<br />

amplification of the apoptotic signal of TNF and some other apoptogenic agents. The group has<br />

established that neither respiratory chain nor mitochondrial membrane potential is required for the TNF-<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 180


JAMES R. KERR PROGRAM<br />

or staurosporine-induced apoptosis. However, oligomycin, which is selectively cytotoxic for cancer cells<br />

and is a specific inhibitor of the membranous portion of mitochondrial H + -ATP-synthase, has now been<br />

found to arrest TNF-induced, but not staurosporine-induced, apoptosis. Oligomycin was found to operate<br />

upstream of mitochondrial swelling and cytochrome c release, and to function in the presence of a<br />

protonophorous uncoupler. Aurovertin, an inhibitor of the extramembranous portion of H + -ATP-synthase,<br />

failed to reproduce the action of oligomycin in preventing TNF-induced apoptosis. TNF-induced<br />

apoptosis was shown to induce conversion of mitochondria from a long to a spherical form (the “threadgrain”<br />

transition), which was accompanied by swelling of the mitochondrial matrix. Thereafter, there was<br />

observed to be a pronounced shrinkage of the organelle and formation of a “supercondensed” form that is<br />

unable to respire, and that is eventually consumed by autophagosomes. As an extension of this study, the<br />

group also demonstrated that mitochondria undergoing apoptosis can induce a bystander effect that is<br />

inhibited by catalase, suggesting that H2O2 is the signaling molecule. Finally, a collaboration between the<br />

groups of Professor Skulachev and Dr. Vartapetian, has demonstrated that the truncated oncoprotein<br />

prothymosin-� forms a complex with cytochrome c, which then becomes autoxidable, thus explaining the<br />

anti-apoptotic effect of truncated prothymosin-�. In other studies with this molecule, Dr. Vartapetian’s<br />

group has demonstrated that caspase-3 is the principle executor of prothymosin-� fragmentation in vivo<br />

and that caspase cleavage at the carboxy terminal disrupts the nuclear localization signal. This leads to a<br />

profound alteration in subcellular localization of prothymosin-�, including nuclear escape and exposure<br />

on the cell surface.<br />

The balance of the work undertaken thus far in the James R. Kerr Program has been focused on cancer<br />

immunology. The laboratories of Professor W-F. Chen at Peking University in Beijing, Professor S.<br />

Nedospasov at the Engelhardt Insititute of Molecular Biology in Moscow, and Dr. V. Filonenko and Dr. I.<br />

Gout at the <strong>Institute</strong> of Molecular Biology and Genetics in Kyiv have each been undertaking serological<br />

analyses of immune responses in cancer patients, leading to the identification of novel cancer antigens.<br />

These laboratories have all used the SEREX technology, which involves antibody screening of<br />

recombinant proteins produced from cloned human cDNAs. In Beijing, working with hepatocellular<br />

carcinoma (HCC), the group has identified both novel and known antigenic proteins including CAGE,<br />

galectin 4, galactin 8 and kenectin. The latter was found to antigenic in up to 40% of patients. In Moscow,<br />

the work encompasses colon, breast, ovarian and renal cancers and, in addition to the confirmation of<br />

previously described antigens, novel antigens that include BC-203 and a new homologue of telomereassociated<br />

enzyme, tankyrase, called TNKL or tankyrase-2 have been identified. In addition, using the<br />

recombinant proteins generated in the course of SEREX analysis, this group has also been building<br />

miniarrays for the diagnosis and monitoring of cancer patients based on their antibody responses. The<br />

laboratory in Kyiv has concentrated on melanoma, thyroid, and colon cancers and has identified TOB<br />

(Transducer of ErbB2), FKBP4 (FK506 binding protein 4), pleiotropic regulator 1 (PRL1) and testes<br />

specific A2 homolog (TSGA2) as antigens worthy of follow-up study.<br />

In addition to SEREX analysis, the Beijing Laboratory has also been identifying antigens using a<br />

bioinformatics based approach, by identifying genes for which transcripts are found only in tumors and<br />

normal testis; thus defining the gene products as potential cancer-testis antigens. On this basis, two genes<br />

were selected for further analyses, and subsequent confirmation as antigens. The renamed antigens,<br />

FATE/BJ-HCC-2 and TPTE/BJ-HCC-5, were detected in 66% and 39% of HCC cases respectively.<br />

FATE was also found in 21% of colon cancers, and TPTE was found in 36% of lung cancers. In addition,<br />

antibodies against FATE and TPTE were detected in 7.3% and 25% of HCC patients, respectively. The<br />

molecular biological work of the group has also led to the identification of a gene, HCC-C11 that spans<br />

approximately 1.6 kb on chromosome 8q24.2, has an ORF of 234 nucleotides encoding 77 amino acids,<br />

and has no homology to known proteins. HCC-C11 is expressed only in testis and liver, and its expression<br />

is down regulated in HCC tumors. Comparison of HCC-C11 expression in individual tumors indicated<br />

that loss of expression correlates with tumor progression.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 181


JAMES R. KERR PROGRAM<br />

In the course of antigen discovery, the critical steps are the expression of a recombinant protein and the<br />

subsequent generation of monoclonal antibodies to determine tissue and cellular distribution. Two of the<br />

groups within the James R. Kerr Program have become responsible for these two steps within the Antigen<br />

Discovery Program of the <strong>LICR</strong> as a whole. The laboratory of Dr. V. Filonenko and Dr. I. Gout in the<br />

Ukraine has undertaken the heterologous expression, in bacterial systems, of more than 20 tumorassociated<br />

proteins, providing milligram quantitites for biochemical characterization and the production<br />

of antibodies. The subsequent production of antibodies occurs in the laboratory of Professor B. Jin at the<br />

Fourth Military Medical University in Xi’an, China. The laboratory has the potential to generate<br />

monoclonal antibodies against more than 300 antigens annually. Thus far, the laboratory has successfully<br />

generated 224 mouse monoclonal antibodies against 28 different proteins supplied by <strong>LICR</strong> investigators.<br />

Following initial characterization, these antibodies have been shipped to investigators throughout the<br />

<strong>LICR</strong>.<br />

In addition to the identification of novel antigens, analysis of immune responses to tumor associated<br />

antigens has also been undertaken in the laboratory of Professor W-F. Chen in Beijing. The antigen NY-<br />

ESO-1 was found to be expressed in 44.4% of HCC tumors, and 45% of HLA-A2 + patients with such<br />

tumors were found to have a CD8 + T cell response to the NY-ESO-1b epitope (SLLMWITQC), with late<br />

stage tumors having a better response. Based on these findings a clinical trial evaluating NY-ESO-1b for<br />

immunotherapy in HLA-A2 + HCC patients bearing NY-ESO-1 mRNA-positive cancers is being planned<br />

in Beijing.<br />

One of the most exttensely studied tumor antigens within the <strong>LICR</strong> is A33, which is associated with the<br />

surface membrane of colon epithelial cells. The Kyiv laboratory has been participating in the study of this<br />

antigen by contributing to functional analyses through the identification of potential protein binding<br />

partners in collaboration with the University College London Branch. Two proteins were identified as<br />

interacting with A33: calnexin, a cellular chaperone that functions in assisting protein assembly and<br />

facilitating the quality control apparatus of the endoplasmic reticulum (ER) through the retention of<br />

incorrectly folded proteins; and filamin C, a high molecular mass cytoskeletal protein that organize<br />

filamentous actin in networks and stress fibers. It is thought that these partners serve to correctly<br />

synthesize and position A33 on the surface membrane, although the exact functional significance of the<br />

findings remains to be established.<br />

The remaining two projects concern the very strong antibody responses to some tumor components in<br />

patients with lung cancer. In the laboratory of Prof. P.P. Philippov in Moscow, analysis has focused on<br />

the antigenic protein recoverin. It has been found that antibodies against this protein are present in the<br />

sera of some patients with small cell lung carcinoma (SCLC), and non-small cell lung carcinoma<br />

(NSCLC). However, whilst 70-80% of lung tumors expressed recoverin, less than 20% of patients have<br />

detectable antibodies against recoverin, demonstrating a significant variation in the ability to mount a<br />

detectable immune response to this antigen. In the laboratory of Dr. U. Ozbek at the Istanbul University,<br />

and in collaboration with the New York Branch, the seroreactivities of the neuro-embryonic SOX Group<br />

B (1, 2 and 3) and ZIC2 proteins have been studied in SCLC. A total of 39% of SCLC patients were<br />

found to have detectable antibody to at least one of these proteins. Strikingly, seroreactivity to all<br />

antigens, but especially SOX3 and ZIC2, correlated with better survival. Patients who had antibodies to<br />

none in this set of antigens had a mean survival of 12 months following initial presentation, whereas those<br />

with antibodies to SOX3 had less than 50% mortality at 26 months. Patients with antibodies to ZIC2<br />

exhibited a mean survival of more than 35 months. These results have important implications for patient<br />

prognosis, and the field of cancer immunology.<br />

A.J.G. Simpson<br />

James R. Kerr Program Director<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 182


PUBLICATIONS<br />

PRIMARY RESEARCH PUBLICATIONS<br />

JAMES R. KERR PROGRAM<br />

1. Fisher T., Senin I.I., Philippov P.P. and Koch K.-W. Application of different lipid surfaces to<br />

monitor protein-membrane interactions by surface plasmon resonance spectroscopy.<br />

2.<br />

Spectroscopy (<strong>2002</strong>) 16: 271-279.<br />

Koroleva E.P., Lagarkova M.A., Mesheryakov A.A., Scanlan M.J., Old L.J., Nedospasov S.A.<br />

and Kuprash D.V. Serological identification of antigens associated with renal cell carcinoma.<br />

Russian Journal of Immunology (<strong>2002</strong>) 7: 229-38.<br />

3. Leontovich M., Brodsky L. I., Drachev V. A and Nikolaev V. K. Adaptive Algorithm of<br />

Automated Annotation, Bioinformatics (<strong>2002</strong>) 18: 838-844.<br />

4. Mou D-C, Cai S-L, Peng J-R, Wang Y., Chen H-S, Pang X-W, Leng X-S and Chen W-F.<br />

Evaluation of MAGE-1 and MAGE-3 as tumor-specific markers to detect blood dissemination of<br />

hepatocellular carcinoma cells. British Journal of Cancer (<strong>2002</strong>) 86:110-116.<br />

5. Senin I.I., Fisher T., Komolov K.E., Zinchenko D.V., Philippov P.P. and Koch K.-W. Ca 2+ -<br />

myristoyl switch in the neuronal calcium sensor recoverin requires different functions of Ca 2+ -<br />

binding sites. Journal of Biological Chemistry (<strong>2002</strong>) 52: 50365-50372.<br />

6. Senin I.I., Koch K.-W., Akhtar M. and Philippov P.P. Ca 2+ -Dependent Control of Rhodopsin<br />

Phosphorylation: Recoverin and Rhodopsin Kinase. Advances in Experimental Medical Biology<br />

(<strong>2002</strong>) 514: 69-99.<br />

7. Shchepina L.A.,. Pletjushkina O.Y., Avetisyan A.V., Bakeeva L.E., Fetisova E.K., Izyumov D.S.,<br />

Saprunova V.B., Vyssokikh Y.M, Chernyak B.V. and. Skulachev V.P. Oligomycin, inhibitor of<br />

F0 part of H + -ATP-synthase, suppresses the TNF-induced apoptosis. Oncogene (<strong>2002</strong>) 21: 8149-<br />

8157.<br />

8. Sukhacheva E.A., Evstafieva A.G., Fateeva T.V., Shakulov V.R., Efimova N.A., Karapetian<br />

R.N., Rubstov, Y.P. and Varapetian A.B. Sensing prothymosin alpha origin, mutations an<br />

conformation with monoclonal antibodies. Journal of Immunological Methods (<strong>2002</strong>) 266: 185-<br />

196.<br />

9. Wang Y, Han K.J., Pang X.W., Vaughan H.A., Qu W., Dong X.Y., Peng .JR., Zhao H.T., Rui<br />

J.A., Leng X.S., Cebon J., Burgess A.W. and Chen W.F. Large Scale Identification of Human<br />

Hepatocellular Carcinoma-Associated Antigens by Autoantibodies. Journal of Immunology<br />

(<strong>2002</strong>) 169:1102-1109.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 183


CLINICAL TRIALS PROGRAM<br />

(OFFICE OF CLINICAL TRIALS MANAGEMENT)<br />

Director's <strong>Report</strong>: Page 185<br />

Publications: Page 186<br />

Affiliate <strong>Report</strong>s<br />

V. Cerundolo: Page 187<br />

A. Knuth, E. Jäger, & D. Jäger: Page 190<br />

E. Oosterwijk: Page 193<br />

Clinical Trials<br />

Cancer Vaccines Page 195<br />

Targeted Antibodies Page 205


DIRECTOR'S REPORT<br />

CLINICAL TRIALS PROGRAM<br />

The Clinical Trials Program (CTP) of the <strong>LICR</strong> is comprised of five principal clinical trial centers in<br />

Brussels (Belgium), Lausanne (Switzerland), Melbourne (Australia), and New York (U.S.A), which are<br />

associated with the <strong>LICR</strong> Branches in those cities, and in Frankfurt (Germany). Affiliated clinical trial<br />

centers are in Okayama, Gunma, and Mie (Japan), Oxford (U.K.), Nijmegen (The Netherlands), and<br />

Homburg (Germany). The CTP is managed by the Office of Clinical Trials Management (OCTM), based<br />

in the New York Office. Since its inception in 1996, the OCTM has made significant achievements in its<br />

effort to establish an efficient and productive infrastructure for the CTP, whilst ensuring that all safety,<br />

regulatory, legal, and commercial requirements are met.<br />

The <strong>LICR</strong> currently sponsors over 30 early phase immunotherapy clinical trials which are actively<br />

accruing patients. Twenty-seven (27) clinical protocols are in development and, as in past years, the<br />

OCTM evaluated approximately 20 clinical trial concepts in <strong>2002</strong>.<br />

The <strong>LICR</strong> currently holds nine Investigational New Drug (IND) applications, with three more being<br />

prepared. All clinical trials in the U.S.A, most clinical trials in Australia, and several trials in Frankfurt<br />

are conducted under U.S.A IND regulations. Additional trials in other countries have been submitted to<br />

the appropriate local regulatory authorities. The latest country to implement a review and approval<br />

process for the conduct of clinical trials is Switzerland.<br />

In <strong>2002</strong>, the Biological Production Facility (BPF) located at the Austin Hospital in Melbourne was<br />

certified as a cGMP facility, further validating the high quality of investigational reagents produced at this<br />

facility. The Australian BPF produces most of the chimeric and humanized monoclonal antibodies used in<br />

<strong>LICR</strong> clinical trials. A second BPF for the production of recombinant proteins from bacterial systems was<br />

dedicated in November <strong>2002</strong> at Cornell University, Ithaca, New York (U.S.A.). This BPF is a<br />

collaborative venture between the <strong>LICR</strong> and Cornell University. The unit is modeled after the Australian<br />

facility in that it has both a process development laboratory and a GMP production area.<br />

Also in <strong>2002</strong>, the <strong>LICR</strong> signed an agreement with a commercial software company to develop an <strong>LICR</strong>validated<br />

internet-based clinical trial database. The database will allow remote data entry from our<br />

international sites, and will compile and record all clinical trial data (including patient data, safety<br />

reporting, and clinical trial management processes) in legally-required formats, and to standards set by<br />

international regulatory authorities. The system will be, when fully operational, on the cutting edge of<br />

integrated clinical data management systems.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 185<br />

E.W. Hoffman


PUBLICATIONS<br />

Refer to Affiliate and Branch <strong>Report</strong>s for Primary <strong>Research</strong> Articles<br />

CLINICAL ABSTRACTS<br />

CLINICAL TRIALS PROGRAM<br />

1. Jaeger E, Karbach J, Neumann A, Jager D, Herrlinger K, Gnjatic S, Hoffman E, Old L, and<br />

Knuth A. LUD00-009: phase I study of intensive course immunization with NY-ESO-1 peptides<br />

in HLA-A2+ patients with NY-ESO-1+ cancer. (<strong>2002</strong>) ASCO American Society of Clinical<br />

Oncology Tumor Vaccines Category:1859<br />

2. Gnjatic S, Altorki N, Stockert E, Jungbluth A, Williamson B, Port J, Ferrara CA, Chen Y, Mason<br />

J, Ritter G, Hoffman E, and Old L. NY-ESO-1 peptide immunization of patients with non-small<br />

cell lung cancer expressing NY-ESO-1 antigen. (<strong>2002</strong>) ASCO American Society of Clinical<br />

Oncology Tumor Vaccines Category:1926<br />

3. Cebon JS, Davis ID, Parente P, Shackleton M, Hopkins W, Goldie H, Maraskovsky E, Chen W,<br />

Chen Q, Jackson H, Murphy R, Scott A, McArthur G, MacGregor D, Sturrock S, Green S,<br />

Cuthbertson A, Maher D, Ryan D, McNamara M, Drane D, Miloradovic L, Ritter G, Stockert E,<br />

Chen YT, Gnjatic S, Hoffman E, and Old L. A phase I study of NY-ESO-1 ISCOMS in patients<br />

with NY-ESO-1 positive cancers and minimal residual disease. (<strong>2002</strong>) ASCO American Society<br />

of Clinical Oncology Tumor Vaccines Category:86<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 186


VINCENZO CERUNDOLO, M.A., M.D., PH.D., F.R.C.PATH., F.MED.SCI.<br />

INSTITUTE OF MOLECULAR MEDICINE<br />

JOHN RADCLIFFE HOSPITAL<br />

UNIVERSITY OF OXFORD<br />

OXFORD,UK<br />

RESEARCH REPORT<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 187<br />

CERUNDOLO<br />

VACCINATION STRATEGIES TO EXPAND MULTIPLE TUMOUR-SPECIFIC CYTOTOXIC TLYMPHOCYTES IN<br />

VIVO<br />

Preclinical Studies<br />

In order to identify vaccination strategies capable of generating a broad and long lasting tumour specific T<br />

cell immunity, we compared vaccines based on the use of different non-cross reactive vectors encoding a<br />

string of melanoma cytotoxic T lymphocyte (CTL) epitopes. We engineered several HLA-A2 and HLA-<br />

A1-restricted melanoma epitopes, which were cloned into naked plasmid DNA, Vaccinia virus, Modified<br />

Vaccinia Ankara virus (MVA) and Semliki Forest Virus (SFV). Generation of several vectors encoding<br />

the same string of epitopes provided an opportunity to compare the ability of sequential injections of<br />

different vectors to induce effective CTL responses.<br />

To investigate the strength and diversity of HLA-A2-restricted CTL responses to the poly-epitope<br />

vaccines, HLA-A2 (A2) transgenic mice were vaccinated with different combinations of the poly-epitope<br />

constructs. In order to simultaneously monitor CTL responses directed against several A2 epitopes in A2<br />

transgenic mice, we engineered a chimeric A2/K b class I molecule containing the mouse K b �3 domain,<br />

and demonstrated that A2/K b tetramers are superior to A2 tetramers for staining PBL of A2/K b transgenic<br />

mice. The results of our studies demonstrated that pre-existing immune responses specific to a viral vector<br />

protein are able to suppress CTL responses to recombinant epitopes encoded by the same vector. This<br />

observation may explain the finding that sequential injections of non cross-reactive vectors (i.e.<br />

heterologous prime-boost) lead to greater numbers of specific CTL, than sequential injections of the same<br />

vector. Ex vivo tetramer staining of peripheral blood lymphocytes (PBL) from A2 mice immunized with<br />

prime-boost protocols revealed that the use of poly-epitope vaccines leads to preferential expansion of<br />

CTL with a single immunodominant specificity. Our results demonstrated that competition for antigen<br />

recognition at the surface of APC may significantly skew the immune response, and strongly suggested<br />

that this competition needs to be taken into account for the development of vaccination strategies that<br />

optimally induce poly-valent CTL responses.<br />

Phase I Trial with Poly-epitope Vaccines.<br />

Defective pox-viruses are attractive agents in the design of cancer and infectious disease vaccines, but it<br />

is unclear whether pre-existing anti-pox-viral immune responses may impair the generation of T cells<br />

specific to recombinant gene products. We have carried out a Phase I clinical trial in melanoma patients<br />

vaccinated either with plasmid DNA followed by recombinant MVA (Group A), or with recombinant<br />

MVA only (Group B) encoding a string of melanoma CTL epitopes. Expansion of Melan-A 26-35 -specific<br />

CD8 + T cells was observed after MVA administration in 2/6 patients in Group A (range: 0.19-0.074 %<br />

CD8 + T cells) and in 4/7 patients in Group B (range: 0.060-0.0295 % CD8+ T cells), while no responses<br />

specific to the other epitopes were observed. All patients developed high levels of anti-vaccinia antibodies<br />

following MVA administration. Analysis of the data demonstrates that previous smallpox vaccination<br />

does not affect the ability of poxvirus-based therapeutic vaccines to induce effective Melan-A 26-35 -specific


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 188<br />

CERUNDOLO<br />

CD8 + T cell responses. The lack of responses against the other epitopes is consistent with the notion that<br />

competition between CTL narrows the immune response induced by poly-epitope vaccines, and suggest<br />

that future trials should take this issue into account to ensure a broader CTL response.<br />

We have reported that blood plasmacytoid dendritic cells (PDC) efficiently prime naïve CD8 +<br />

lymphocytes specific for the Melan-A26-35 epitope to become IFN-� producing cells in vitro. In addition,<br />

we found that CD40L-stimulated PDC induce expression on primed melan-A-specific T cells of<br />

cutaneous lymphocyte antigen (CLA) and L-selectin (CD62L), homing receptors that allow the migration<br />

of effector cells to the inflamed skin. Finally, we show that PDC can be found in the peri-tumoral area of<br />

most primary cutaneous melanomas in vivo, and that type I-IFN-containing supernatants derived from<br />

PDC increase melanoma cell surface expression of CD95 and MHC class I and class II molecules in vitro.<br />

Our results demonstrate a new immunomodulatory role for tissue infiltrating PDC, which may prime<br />

tumor-specific T cell responses and affect tumor growth via soluble factors.<br />

ANALYSIS OF GLYCOLIPID-SPECIFIC IMMUNE RESPONSES.<br />

Human CD1 molecules segregate into two groups according to sequence homology. CD1a, CD1b and<br />

CD1c have all been shown to present mycobacterial phospho- and glycolipids as well as self glycolipids,<br />

such as gangliosides, to specific T cells. Analysis of the role of CD1 has been hampered by the lack of<br />

reliable and sensitive techniques to isolate glycolipid-specific T lymphocytes ex-vivo. Over the last three<br />

years we have developed novel protocols for the generation of CD1d/glycolipid tetramers, which allow<br />

sensitive and highly specific isolation and characterisation of CD1 restricted T lymphocytes from<br />

peripheral blood and lymphocytes. More recently, we have extended these results to all the members of<br />

the CD1 family by developing additional protocols to refold denatured CD1 molecules in the presence of<br />

defined lipid ligands and short mono-alkyl detergent molecules. Application of these protocols has led to<br />

the solution of the three dimensional structure of CD1b/ß2m-proteins loaded either with<br />

phosphatidylinositol (PI) or GM2. The analysis of CD1b crystal structure demonstrated for the first time,<br />

in atomic detail, how CD1 molecules present lipid ligands to the T cell receptor. These structures clarified<br />

the ligand-binding mechanisms of CD1b which occur via a network of four interlinked hydrophobic<br />

channels, providing a unique adaptable system required to present a broad range ligands. To date, lipids<br />

containing two alkyl chains are thought to be the only ligands capable of binding to CD1b. The current<br />

structures unexpectedly revealed that monoalkyl and tri-alkyl chain ligands can specifically bind to the<br />

CD1b groove. These observations greatly extend the spectrum of CD1b antigens, and also provide a new<br />

model for in vivo refolding of CD1b molecules.<br />

PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

1. PALMOWSKI, M.,CHOI, E.,HERMANS, I.,GILBERT, S.,CHEN, J-L., GILEADI, U.,SALIO, M.,VAN<br />

PEL, A.,MAN, S.,BONIN, E.,LILJESTROM P., DUNBAR, P.R., CERUNDOLO, V. Competition<br />

between cytotoxic T lymphocytes narrows the immune response induced by prime-boost<br />

vaccination protocols. Journal of Immunology (<strong>2002</strong>) 168 (9): 4391-4398.<br />

2. CHOI , E.M., PALMOWSKI, M.,CHEN J., AND CERUNDOLO, V. The use of chimeric A2K b<br />

tetramers to monitor HLA A2 immune responses in HLA A2 transgenic mice. Journal of<br />

Immunological Methods (<strong>2002</strong>) Oct 1;268(1):35.<br />

3. PALMOWSKI M, SALIO M, DUNBAR RP, CERUNDOLO V. The use of HLA class I tetramers to<br />

design a vaccination strategy for melanoma patients. Immunological Reviews (<strong>2002</strong>)<br />

Oct;188(1):155-63.


© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 189<br />

CERUNDOLO<br />

4. GADOLA, S.D. DULPHY, N.,SALIO, M., AND CERUNDOLO, V., V�24-J�Q-independent CD1drestricted<br />

Recognition of �-Galactosylceramide by Human CD4 + and CD8�� + T Lymphocyte.<br />

Journal of Immunology (<strong>2002</strong>) 168(11):5514-20.<br />

5. GADOLA,S.D., ZACCAI, N. R., HARLOS,K., SHEPHERD,D., RITTER,G.,SCHMIDT, R.R., JONES,<br />

Y., CERUNDOLO, V. Structure of human CD1b with bound ligands at 2.3Å, a maze for alkyl<br />

chains. Nature Immunology (<strong>2002</strong>) 3:721.


ALEXANDER KNUTH, M.D., ELKE JÄGER, M.D., PH.D., & DIRK JÄGER,M.D.<br />

II. MEDIZINISCHE KLINIK,HÄMATOLOGIE -ONKOLOGIE<br />

KRANKENHAUS NORDWEST<br />

FRANKFURT,GERMANY<br />

RESEARCH REPORT<br />

IDENTIFICATION OF NEW NY-ESO-1 T CELL EPITOPES<br />

KNUTH,JÄGER,&JÄGER<br />

We searched for NY-ESO-1 peptide epitopes that are recognized by CD8 + T cells in the context of MHC<br />

class I alleles frequently found in Caucasians (HLA-A1, -A2, -A3, -B35, -B51, -Cw02, -Cw04). Strong<br />

CD8 + T cell responses were detected against NY-ESO-1 p94-102 restricted by the HLA-B51 allele in<br />

NY-ESO-1 antibody positive patients. NY-ESO-1 p94-102 specific CD8 + T cell clones were obtained by<br />

either repetitive stimulation with the HLA-B51 + NY-ESO-1 + melanoma cell line NW-MEL-1539, or with<br />

the NY-ESO-1 p94-102 peptide. These T cell clones also recognize naturally processed NY-ESO-1 in the<br />

context of HLA-B51, as demonstrated in transfection experiments with COS-7 cells, NY-ESO-1, and<br />

different MHC class I allleles. Additional epitopes that may be recognized by CD8 + T cells, in the context<br />

of HLA-A3, -B35, and -B44, are being analyzed. The NY-ESO-1 region p85-120 seems to contain<br />

several epitopes serving as T cell targets.<br />

SPONTANEOUS AND VACCINE-INDUCED CD8 + TCELL RESPONSES AGAINST NY-ESO-1<br />

The frequency of NY-ESO-1 specific CD8 + T cell responses has been assessed in multiple patients with a<br />

spontaneous NY-ESO-1 antibody-positive serum, and in 28 patients following vaccination against NY-<br />

ESO-1. ELISPOT and tetramer assays were compared with respect to sensitivity, and correlated with<br />

functional cytotoxicity assays against NY-ESO-1 peptides and NY-ESO-1 + cancer cell lines. We could<br />

confirm that detectable CD8 + T cell responses were associated with strong serum antibody reactivity in all<br />

cases tested. In collaboration with Dr. Sacha Gnjatic at the <strong>LICR</strong> New York Branch, we showed that the<br />

pattern of recognition against different HLA-A2 restricted NY-ESO-1 epitopes differed significantly in<br />

patients with spontaneous versus vaccine-induced NY-ESO-1 immunity. The epitopes p157-165, p157-<br />

167, and p159-167 were recognized by CD8 + T cells obtained from patients after vaccination with NY-<br />

ESO-1 p157-167 and p157-165. It was demonstrated that the strong reactivity against p157-167 and<br />

p159-167 was not associated with a respective reactivity against NY-ESO-1 + cancer cell lines, whereas<br />

CD8 + T cell responses against p157-165 correlated with a strong tumor reactivity. The fact that patients<br />

with spontaneous NY-ESO-1 immunity do not show T cell reactivity against NY-ESO-1 p159-167<br />

indicates that this epitope is not naturally processed and presented on cancer cells. Therefore, vaccination<br />

with epitopes generating reactivity with p159-167 is not considered as useful for the induction of tumor<br />

reactive CD8 + T cell responses in vivo. The association between CD8 + T cell reactivity induced by<br />

vaccination and the appearance of NY-ESO-1 serum antibody has been studied in two patients to date.<br />

The first patient showed a conversion towards a detectable NY-ESO-1 serum antibody after three months<br />

of NY-ESO-1 peptide vaccination (weekly injections). The second patient showed seroconversion after<br />

three immunizations with recombinant NY-ESO-1 vaccinia constructs. Both patients had a strong NY-<br />

ESO-1 specific CD8 + T cell reactivity at the time of seroconversion.<br />

Comparing different strategies of vaccination against NY-ESO-1 (intradermal injection of NY-ESO-1<br />

peptides, at weekly intervals versus an intensive course over a five-day schedule) showed that peptide<br />

specific CD8 + T cell responses may be induced significantly earlier during the course of vaccination (by<br />

day 15) and at a higher magnitude using the intensive course protocol. This observation may lead to a<br />

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KNUTH,JÄGER,&JÄGER<br />

reconsideration of vaccination protocols towards intensive course loading schedules, possibly followed by<br />

maintenance immunization on a weekly or monthly basis.<br />

NY-ESO-1 SPECIFIC CD4 + TCELL RESPONSES<br />

NY-ESO-1 specific CD4 + T cell responses restricted by HLA-DP4 have been extensively evaluated in<br />

NY-ESO-1 seropositive patients. We could not confirm a previous finding that detectable NY-ESO-1<br />

serum antibody was associated with the presence of the HLA-DP4 allele. Of 34 patients with NY-ESO-1 +<br />

cancers, 17 were antibody positive, and 17 were antibody negative. There were 11 and 10 HLA-DP4 +<br />

patients in the antibody positive and negative patients, respectively.<br />

CD4 + T cells obtained from five different HLA-DP4 + NY-ESO-1 seropositive patients were analyzed by<br />

ELISPOT for their fine-specificity against different NY-ESO-1 peptides, all of which included the DP4<br />

binding sequence p157-170. We demonstrated that the efficacy of recognition is dependent on the presensitization<br />

of effector cells (stimulation with adeno-ESO, or different NY-ESO-1 peptides, or pulsed on<br />

CD4-depleted peripheral blood mononuclear cells (PBMC), or dendritic cells), and the type of antigenpresenting<br />

cell used in the ELISPOT (HLA-DP4-matched allogenic Epstein Barr Virus B cells,<br />

autologous peanut haemagglutinin blasts, or autologous dendritic cells). Using adeno-ESO constructs for<br />

primary pre-sensitization, we were able to clone HLA-DP4-restricted NY-ESO-1 specific CD4 + T cells.<br />

Some of the HLA-DP4-restricted CD4 + T cell clones showed a strong cytotoxic potential against peptidepulsed<br />

and NY-ESO-1 transfected antigen-presenting cells.<br />

IDENTIFICATION OF NY-BR-1 T CELL EPITOPES<br />

Two NY-BR-1 epitopes, p158-167 and p960-968, that bind to HLA-A2, and that are recognized by CD8 +<br />

T cells of breast cancer patient NW1100 have been identified in ELISPOT and cytotoxicity assays.<br />

Peptide specific CD8 + T cell clones were generated by repetitive stimulation with the respective epitopes.<br />

HLA-A2 tetramers, produced by Dr. Hans-Georg Rammensee (University of Tübingen, Germany),<br />

confirmed the specificity of NW1100 CD8 + T cells and clones against these epitopes. The analysis of 15<br />

breast and five prostate cancer patients has led to the identification of one additional prostate cancer<br />

patient with a strong CD8 + T cell reactivity against NY-BR-1 p960-968. In conclusion, CD8 + T cell<br />

reactivity against NY-BR-1 seems to be rare, but further studies on the relationship between spontaneous<br />

NY-BR-1 immunity, and NY-BR-1 expression in primary tumors and metastases are warranted.<br />

IDENTIFICATION AND CHARACTERIZATION OF NEW SEREX-DEFINED ANTIGENS IN BREAST, PROSTATE,<br />

AND COLON CANCERS<br />

Different SEREX screenings performed with sera obtained from patients with metastatic prostate, breast<br />

and colon cancer and favourable clinical courses, have led to the identification of the novel genes, DAZ<br />

(deleted in azoospermia), human GKAP42 (germ cell specific cGMP-dependent protein kinase-anchoring<br />

protein), and SATB1. Extensive analyses are ongoing to study the expression patterns of these genes in<br />

malignant and normal tissues, and evaluate the potential of these new genes to serve as targets for<br />

antigen-specific vaccination against cancer.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 191


PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

KNUTH,JÄGER,&JÄGER<br />

1. CORAL S, SIGALOTTI L, ALTOMONTE M, ENGELSBERG A, COLIZZI F, CATTAROSSI I,<br />

MARASKOVSKY E, JAGER E, SELIGER B AND MAIO M, 5-aza-2'-Deoxycytidine-induced<br />

2.<br />

Expression of Functional Cancer Testis Antigens in Human Renal Cell Carcinoma:<br />

Immunotherapeutic Implications. Clinical Cancer <strong>Research</strong> (<strong>2002</strong>) 8(8): 2690-5.<br />

JAGER E, HOHN H, NECKER A, FORSTER R, KARBACH J, FREITAG K, NEUKIRCH C, CASTELLI C,<br />

SALTER RD, KNUTH A AND MAEURER MJ, Peptide-specific CD8+ T-cell evolution in vivo:<br />

response to peptide vaccination with Melan-A/MART-1. International Journal of Cancer (<strong>2002</strong>)<br />

98(3): 376-88.<br />

3. JAGER E, SALTER R, CASTELLI C, HOHN H, FREITAG K, KARBACH J, NEUKIRCH C, NECKER A,<br />

KNUTH A AND MAEURER MJ, Impact of antigen presentation on TCR modulation and cytokine<br />

release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2<br />

wild-type or HLA-A2 mutant tetrameric complexes. Journal of Immunology (<strong>2002</strong>) 168(6): 2766-<br />

72.<br />

4. MAEURER MJ, NECKER A, SALTER RD, CASTELLI C, HOHN H, KARBACH J, FREITAG K,<br />

NEUKIRCH C, KNUTH A AND JAGER E, Improved detection of melanoma antigen-specific T cells<br />

expressing low or high levels of CD8 by HLA-A2 tetramers presenting a Melan-A/Mart-1 peptide<br />

analogue. International Journal of Cancer (<strong>2002</strong>) 97(1): 64-71.<br />

5. SCANLAN MJ, WELT S, GORDON CM, CHEN YT, GURE AO, STOCKERT E, JUNGBLUTH AA,<br />

RITTER G, JAGER D, JAGER E, KNUTH A AND OLD LJ, Cancer-related serological recognition of<br />

human colon cancer: identification of potential diagnostic and immunotherapeutic targets. Cancer<br />

<strong>Research</strong> (<strong>2002</strong>) 62(14): 4041-7.<br />

6. JÄGER D, STOCKERT E, KARBACH J, HERRLINGER K, ATMACA A, ARAND M, CHEN Y-T,<br />

GNJATIC S, OLD LJ, KNUTH A AND JÄGER E, Urine antibody against human cancer antigen NY-<br />

ESO-1. Cancer Immunity (<strong>2002</strong>) 2(10).<br />

7. JÄGER E, KARBACH J, GNJATIC S, JÄGER D, MAEURER M, ATMACA A, ARAND M, SKIPPER J,<br />

STOCKERT E, CHEN Y-T, OLD LJ AND KNTUH A, Identification of a naturally processed NY-<br />

ESO-1 peptide recognized by CD8+ T cells in the context of HLA-B51. Cancer Immunity (<strong>2002</strong>)<br />

2(12).<br />

REVIEWS/COMMENTARIES/BOOK CHAPTERS<br />

1. JAGER E, JAGER D AND KNUTH A, Clinical cancer vaccine trials. Current Opinions in<br />

Immunology (<strong>2002</strong>) 14(2): 178-82.<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 192


EGBERT OOSTERWIJK,PH.D.<br />

SECTION IMMUNO- AND GENE THERAPY<br />

EXPERIMENTAL UROLOGY<br />

UNIVERSITY MEDICAL CENTER NIJMEGEN<br />

NIJMEGEN,THE NETHERLANDS<br />

RESEARCH REPORT<br />

CLINICAL STUDIES<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 193<br />

OOSTERWIJK<br />

In <strong>2002</strong> we finalized several clinical studies with chimeric monoclonal antibody (mAb) G250 (cG250)<br />

recognizing a renal cell carcinoma (RCC)-associated antigen, which is most abundant on RCC of the clear<br />

cell type. In various clinical trials with cG250 in patients with RCC of clear cell morphology the excellent<br />

imaging capacity of cG250 has been demonstrated, and in general all metastatic sites > 2 cm diameter<br />

have been visualized. Thus, cG250 might be of value for diagnostic imaging. A clinical trial was designed<br />

to compare 131 I-cG250 with 18 F-FDG-PET imaging in metastatic RCC patients, and surprisingly, 18 F-<br />

FDG-PET detected substantially more RCC metastases than 131 I-cG250. One caveat of this study was the<br />

lack of histological confirmation of the metastatic RCC sites. Although in many cases the primary tumor<br />

had been diagnosed as “RCC with clear cell component” it is conceivable that the metastases consisted of<br />

non-clear cell components. Additionally, higher cG250 internalization rates may have obscured the true<br />

cG250 uptake, since metabolized 131 I-cG250 is readily excreted and thus will not be visualized.<br />

In a separate clinical trial we studied the effect of different radiolabels on cG250 tumor accumulation. We<br />

had convincing evidence from animal studies that much higher tumor accumulation can occur when<br />

residualizing radiolabels (radiolabels that are retained in the cell after internalization) are used. Based on<br />

these observations we carried out a study in patients with recurrent or metastasized RCC to investigate<br />

whether, and to what extent, the radiation dose to the tumor could be enhanced when the cG250 antibody<br />

was labeled with a residualizing radionuclide compared to radioiodine. Therefore, an intra-patient<br />

comparison of the tumor targeting of RCC lesions of 131 I-labeled cG250 and 111 In-labeled cG250 was<br />

performed. The study demonstrated that the uptake of 111 In-labelled cG250 in RCC metastases was<br />

significantly higher than the uptake of 131 I-labelled cG250 in most RCC lesions. This difference is most<br />

likely to be due to different intracellular handling of the radiolabeled catabolites of the antibody following<br />

internalization. The higher uptake obtained with 111 In-labelled cG250 implies that potentially higher<br />

radiation doses could be guided to RCC metastases in radioimmunotherapy (RIT) trials when mAb G250<br />

is labeled with a residualizing radionuclide such as 177 Lu.<br />

The excellent targeting of the cG250 to RCC tumor lesions in most RCC patients suggested that the<br />

therapeutic efficacy of an approach might be enhanced when more rapidly clearing F(ab’)2 fragments<br />

were used. To investigate the tumor targeting potential of cG250 F(ab’)2 fragments in RCC patients a<br />

clinical batch of cG250 F(ab’)2 was produced. The immunoreactive fraction (IRF: 96%) and the affinity<br />

(Ka=2.6 x 10 9 M -1 ) of I-131-labeled cG250 F(ab’)2 were similar to those of the radioiodinated intact<br />

cG250 antibody. Five patients with primary RCC received 6 mCi 131 I-labeled cG250 F(ab’)2 fragments<br />

intravenously and images were acquired 0, 1, 2 and 3 days after injection. Pharmacokinetic analysis<br />

indicated that the cG250 F(ab’)2 fragments rapidly cleared from the blood, but uptake in the tumors was<br />

modest and tumor-to-blood ratios clearly were not superior to those obtained with the intact cG250<br />

antibody. From these studies, it was concluded that the intact form of G250 is most suitable for RIT.


PRECLINICAL STUDIES<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 194<br />

OOSTERWIJK<br />

In view of our desire to use a residualizing radionuclide for RIT, cG250 IgG1 was conjugated with the<br />

cyclic anhydride of DTPA (cDTPA), isothiocyanatobenzyl-DTPA (ITC-DTPA) and<br />

isothiocyanatobenzyl-DOTA (ITC-DOTA), and the immunoconjugates (cG250-cDTPA, cG250-ITC-<br />

DTPA and cG250-ITC-DOTA, respectively) were labeled with 177 Lu. 177 Lu is a bone-seeking element and<br />

its release from the antibody not only results in decreased radionuclide uptake in the tumor, but also<br />

enhanced radionuclide uptake in bone. 177 Lu incorporation in the bone potentially enhances the radiation<br />

dose to the bone marrow, the dose limiting organ in RIT, thus reducing the maximum tolerated dose that<br />

can be administered. The stability and immunoreactivity of the three cG250 immunoconjugates labeled<br />

with 177 Lu was studied in vitro and in vivo. In vitro analyses showed that there is virtually no release of<br />

the 177 Lu label from either the cG250-ITC-DTPA or the cG250-ITC-DOTA preparation. Biodistribution<br />

studies in a nude mouse/RCC tumor model showed excellent targeting of the tumor with both<br />

radioimmuno-conjugates, but femur accumulation was lowest with 177 Lu-ITC-DOTA-cG250, suggesting<br />

that this compound has superior radioimmuno-conjugate stability. We also compared tumor targeting of<br />

cG250 labeled with 177 Lu or 131 I; the tumor uptake of cG250 labeled with the residualizing 177 Lu was<br />

more than five times higher than the uptake of the radioiodinated antibody at three days, and at seven<br />

days post-injection (p.i.). At all time points, blood levels were not significantly different. The therapeutic<br />

efficacy of cG250 labeled with 131 I, 177 Lu, 90 Y, and 186 Re was compared in the mouse model; tumor<br />

growth was delayed >175 days with 77 Lu-cG250, whereas tumor growth was delayed for approximately<br />

60 days with 131 I-cG250.<br />

Finally, we investigated the transcription of G250, which is expressed in RCC, but not normal kidney, nor<br />

most other normal tissues. In order to identify the promoter elements that are responsible for the RCCspecific<br />

expression of the G250 gene, we first isolated and characterized its promoter. Sequence analysis of<br />

the G250 5’-flanking region was performed, and several promoter-reporter constructs were prepared and<br />

transiently transfected into G250-positive and -negative cell lines. We found that most G250 promoter<br />

constructs displayed strong promoter activity in G250-positive cells, but no activity in G250-negative<br />

cells. The methylation status of the G250 promoter was not correlated with G250 gene expression.<br />

Mutations in the Sp1- or HIF-binding transcription factor elements completely abolished G250 promoter<br />

activity, indicating that these are critically important for the transcription of G250. DNase-I footprint and<br />

band-shift analyses confirmed that Sp1 and HIF-alpha proteins in nuclear extracts of RCC cells bind to<br />

these elements in the G250 promoter. Cotransfection experiments using E1A and E1A mutants that are<br />

unable to bind the CBP/p300 transcription coactivator indicated that CBP/p300 participates in the<br />

regulation of G250 transcription. Thus it appears that a transcription factor complex consisting of at least<br />

HIF-1, Sp1 and CBP/p300 regulates the expression of G250 in RCC. The minimal promoter region was<br />

required for tumor-specific expression of G250 and is therefore of interest for gene therapy approaches. In<br />

preliminary experiments we have shown that this promoter can be used to drive gene-expression in a<br />

tissue-specific manner.<br />

PUBLICATIONS<br />

PRIMARY RESEARCH ARTICLES<br />

1. GRABMAIER K, DE WEIJERT M, UEMURA H, SCHALKEN J, OOSTERWIJK E. Renal cell carcinomaassociated<br />

G250 methylation and expression: in vivo and in vitro studies. Urology (<strong>2002</strong>) 60:357-<br />

62.<br />

2. BROUWERS AH, DORR U, LANG O, BOERMAN OC, OYEN WJ, STEFFENS MG, OOSTERWIJK E,<br />

MERGENTHALER HG, BIHL H, CORSTENS FH. 131 I-cG250 monoclonal antibody<br />

immunoscintigraphy versus [18 F]FDG-PET imaging in patients with metastatic renal cell<br />

carcinoma: a comparative study. Nuclear Medicine Communications (<strong>2002</strong>) 23:229-36.


CANCER VACCINES<br />

CLINICAL TRIALS –CANCER VACCINES<br />

Active and Closed Clinical Trials – January 1 st <strong>2002</strong> to December 31 st <strong>2002</strong><br />

MAGE<br />

TRIAL: LUD 1995004<br />

PRINCIPAL SITE: Brussels, Belgium<br />

PRINCIPAL INVESTIGATOR: Dr. M. Marchand<br />

TITLE: Pilot Study of Immunization with the MAGE-3.A2 Peptide in Patients with Malignant<br />

Melanoma, Non Small Cell Lung Cancer, Head and Neck Squamous Cell Carcinoma, Esophageal<br />

Squamous Cell Carcinoma or Bladder Carcinoma<br />

CANCER: Melanoma, and others<br />

TRIAL: LUD1996007<br />

PRINCIPAL SITE: Brussels, Belgium<br />

PRINCIPAL INVESTIGATOR: Dr. M. Marchand<br />

TITLE: Pilot Study of Immunization with the MAGE-1.A1 and MAGE-3.A1 Peptides in Patients<br />

with Malignant Melanoma, Non-Small Cell Lung Carcinoma, Head and Neck Squamous Cell<br />

Carcinoma, Esophageal Squamous Cell Carcinoma or Bladder Carcinoma<br />

CANCER: Melanoma, and others<br />

TRIAL: LUD1997004<br />

PRINCIPAL SITE: Brussels, Belgium<br />

PRINCIPAL INVESTIGATOR: Dr. M. Marchand<br />

TITLE: Phase I/II Study of High-Frequency and Prolonged Immunization with the MAGE-3.A<br />

Peptide in Stage III/IV Tumor-Bearing Patients with Malignant Melanoma<br />

CANCER: Melanoma, and others<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 195


TRIAL: LUD1997005<br />

PRINCIPAL SITE: Brussels, Belgium<br />

PRINCIPAL INVESTIGATOR: Dr. M. Marchand<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: Pilot Study of Immunization with Recombinant Canarypox Virus vCP1469A Expressing<br />

the MAGE-1.A1 and MAGE-3.A1 Cytolytic T Lymphocytes Epitopes in Patients with Malignant<br />

Melanoma, Non-Small Cell Lung Carcinoma, Head and Neck Squamous Cell Carcinoma,<br />

Esophageal Squamous Cell Carcinoma or Bladder Carcinoma<br />

CANCER: Melanoma, and others<br />

TRIAL: LUD1999010<br />

PRINCIPAL SITE: New York, USA<br />

PRINCIPAL INVESTIGATOR: Dr. N. Altorki<br />

TITLE: Mage-3 Protein in Patients with Non-small Cell Lung Cancer<br />

CANCER: Non-small Cell Lung Cancer<br />

TRIAL: LUD1999014<br />

PRINCIPAL SITE: Brussels, Belgium<br />

PRINCIPAL INVESTIGATOR: Dr. N. VanBaren<br />

TITLE: Phase 1/2 study of immunization with MAGE-1 and MAGE-3 recombinant proteins in<br />

patients with multiple myeloma<br />

CANCER: Myeloma<br />

TRIAL: LUD2001006<br />

PRINCIPAL SITE: Brussels, Belgium<br />

PRINCIPAL INVESTIGATOR: Dr. N. VanBaren<br />

TITLE: Determination of MAGE in peptide-induced immune responses in patients with resected<br />

primary or regional melanoma at high risk of relapse<br />

CANCER: Melanoma<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 196


TRIAL: LUD2001013<br />

PRINCIPAL SITE: Brussels, Belgium<br />

PRINCIPAL INVESTIGATOR: Dr. M. Marchand<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: A phase 1 study of immunization with MAGE-1 or MAGE-3 encoded proteins and<br />

peptides given before radical cystectomy in patients with invasive transitional cell bladder<br />

carcinoma<br />

CANCER: Bladder<br />

TRIAL: LUD<strong>2002</strong>002<br />

PRINCIPAL SITE: Brussels, Belgium<br />

PRINCIPAL INVESTIGATOR: Dr. M. Marchand<br />

TITLE: Phase 1/2 study of intramuscular immunization with recombinant MAGE-3 protein and<br />

peptides mixed with an immunological adjuvant containing CpG/QS21/MPL (SB AS-15) in<br />

patients with MAGE-3 positive, measurable metastatic cutaneous melanoma<br />

CANCER: Metastatic melanoma<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 197


MELAN-A<br />

TRIAL: LUD1996010<br />

PRINCIPAL SITE: Lausanne, Switzerland<br />

PRINCIPAL INVESTIGATORS: Drs. Lienard, and M. Marchand<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: Phase I Study of Immunization with Melan-A Peptide, Influenza Matrix Peptide and<br />

Adjuvant SB AS-2 in HLA-A2 + Patients with High Risk Stage III or Stage IV Malignant<br />

Melanoma<br />

CANCER: Melanoma<br />

TRIAL: LUD1998001<br />

PRINCIPAL SITE: Frankfurt, Germany<br />

PRINCIPAL INVESTIGATOR: Dr. A. Knuth<br />

TITLE: Phase I Study of Immunization with Melanoma Associates Peptides Alone and combined<br />

with GM-CSF in HLA A2+ Patients with High Risk Stage III and Stage IV Malignant Melanoma<br />

CANCER: Melanoma<br />

TRIAL: LUD1998009<br />

PRINCIPAL SITE: Lausanne, Switzerland<br />

PRINCIPAL INVESTIGATORS: Drs. Lienard and D. Speiser<br />

TITLE: Specific Immunotherapy of Skin Melanoma Patients with Melan-A/MART-1 and<br />

Influenza Peptides and Immunological Analysis of the Vaccine Site Draining Lymph Node<br />

(Phase I)<br />

CANCER: Melanoma<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 198


TRIAL: LUD1999013<br />

PRINCIPAL SITE: Oxford, UK<br />

PRINCIPAL INVESTIGATORS: Drs. V. Cerundolo, and A. Harris<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: Phase 1 study of melanoma poly-epitope DNA (DNA.Mel3) and melanoma poly-epitope<br />

modified vaccinia Ankara (MVA.Mel3) in patients with melanoma<br />

CANCER: Melanoma<br />

TRIAL: 2000005<br />

PRINCIPAL SITE: Melbourne, Australia<br />

PRINCIPAL INVESTIGATORS: Drs. J. Cebon and I. Davis<br />

TITLE: A Phase I Study of Immunization with melanoma antigen in conjunction with GM-CSF in<br />

patients with fully resected stage 2, 3 or 4 malignant melanom<br />

CANCER: Resected Melanoma<br />

TRIAL: LUD2000018<br />

PRINCIPAL SITE: Lausanne, Switzerland<br />

PRINCIPAL INVESTIGATOR: Dr. D. Speiser<br />

TITLE: Immunotherapy of HLA-A2 positive stage III / IV melanoma patients with CpG and<br />

Melan-A peptide (phase I study)<br />

CANCER: Melanoma<br />

TRIAL: LUD2000025<br />

PRINCIPAL SITE: New York, USA<br />

PRINCIPAL INVESTIGATOR: Dr. K. Papadopolous<br />

TITLE: A phase 1 study of peptide-based vaccine therapy in patients with high-risk or metastatic<br />

melanoma<br />

CANCER: Melanoma<br />

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NY-ESO-1<br />

TRIAL: LUD1999008<br />

PRINCIPAL SITE: Melbourne, Australia<br />

PRINCIPAL INVESTIGATOR: Dr. J. Cebon<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: A Phase 1/2 Study of NY-ESO-1 ISCOMs in patients with Advanced NY-ESO-1 Positive<br />

Cancers<br />

CANCER: NY-ESO-1 positive cancers<br />

TRIAL: LUD2000014<br />

PRINCIPAL SITE: Frankfurt, Germany<br />

PRINCIPAL INVESTIGATOR: Dr. A. Knuth<br />

TITLE: Phase 1 study of recombinant vaccinia NY-ESO-1 (rv-NY-ESO-1) and recombinant<br />

fowlpox-NY-ESO-1 (rf-NY-ESO-1) in patients with NY-ESO-1 positive cancers.<br />

CANCER: NY-ESO-1 positive cancers<br />

TRIAL: LUD2000009<br />

PRINCIPAL SITE: Frankfurt, Germany<br />

PRINCIPAL INVESTIGATOR: Dr. A. Knuth<br />

TITLE: Phase 1 Study of Intensive Course Immunization with NY-ESO-1 Derived Peptides<br />

Presented by MHC Class 1 Molecules Alone or Combined with GM-CSF in patients with<br />

Advanced NY-ESO-1 Expressing Cancers<br />

CANCER: NY-ESO-1 positive cancers<br />

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TRIAL: LUD2000019<br />

PRINCIPAL SITE: New York, USA<br />

PRINCIPAL INVESTIGATOR: Dr. N. Altorki<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: NY-ESO-1 peptide immunization of patients with NSCLC expresssing NY-ESO-1 antigen<br />

CANCER: Non-small cell lung cancer (NSCLC)<br />

TRIAL: LUD2000023<br />

PRINCIPAL SITE: Boston, USA<br />

PRINCIPAL INVESTIGATOR: Dr. G. DeMetri<br />

TITLE: Study of NY-ESO-1 protein in blood of patients with synovial sarcoma<br />

CANCER: Synovial sarcoma<br />

TRIAL: LUD2000024<br />

PRINCIPAL SITE: New York, USA<br />

PRINCIPAL INVESTIGATOR: Dr. K. Papadopolous<br />

TITLE: NY-ESO-1 peptide immunization of patients with cancer expressing NY-ESO-1 antigen<br />

CANCER: NY-ESO-1 positive cancers<br />

TRIAL: LUD2000026<br />

PRINCIPAL SITE: Frankfurt, Germany<br />

PRINCIPAL INVESTIGATOR: Dr. A. Knuth<br />

TITLE: Phase 1 study of immuniation NY-ESO-1 derived peptides presented by MHC class I<br />

molecules administered alone or combined with polyarginine in patients with advanced NY-ESO-<br />

1 or LAGE-expressing tumours<br />

CANCER: NY-ESO-1 positive cancers<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 201


TRIAL: LUD2001017<br />

PRINCIPAL SITE: Melbourne, Australia<br />

PRINCIPAL INVESTIGATORS: Drs. J. Cebon and I. Davis<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: Evaluation of NY-ESO-1 immunity in patients who have previously been vaccinated with<br />

NY-ESO-1 protein.<br />

CANCER: NY-ESO-1 positive melanoma<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 202


MULTI-ANTIGEN VACCINES<br />

TRIAL: LUD2001019<br />

PRINCIPAL SITE: Brussels, Belgium<br />

PRINCIPAL INVESTIGATOR: Dr. N. VanBaren<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: Phase 1/2 study of immunization with a combination of MAGE-32.A1/B35, NA17.A2,<br />

tyrosinase.A2 and melan-A.A2 peptides in melanoma patients with measurable metastatic<br />

disease.<br />

CANCER: Metastatic melanoma<br />

TRIAL: LUD2000021<br />

PRINCIPAL SITE: Melbourne, Australia<br />

PRINCIPAL INVESTIGATORS: Drs. I. Davis and J. Cebon<br />

TITLE: A phase I/II trial of FLT-3L-mobilized dendritic cells pulsed with peptides derived from<br />

cancer-testis antigens in HLA-A2 positive patients with treated cancer and minimal residual<br />

disease at high risk of relapse<br />

CANCER: Melanoma and others<br />

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CPGOLIGODEOXYNUCLEOTIDES<br />

TRIAL: LUD2000002<br />

PRINCIPAL SITE: Melbourne, Australia<br />

PRINCIPAL INVESTIGATOR: Dr. I. Davis<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: A Phase I/II Study of CpG Oligodeoxynucleotides 7909 in the Treatment of Patients with<br />

Advanced Renal Cell Carcinoma<br />

CANCER: Renal cell carcinoma<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 204


HER-2<br />

TRIAL: LUD2001016<br />

PRINCIPAL SITE: Mie, Japan<br />

PRINCIPAL INVESTIGATOR: Dr. Shiku<br />

CLINICAL TRIALS –CANCER VACCINES<br />

TITLE: Immunization with cholesterol hydrophobized polysaccharide (CHP)-HER2 complex in<br />

HLA-A2402 positive patients with HER2 overexpressing cancers<br />

CANCER: HER-2 positive cancers<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 205


TARGETED ANTIBODIES<br />

CLINICAL TRIALS –TARGETED ANTIBODIES<br />

Active and Closed Clinical Trials – January 1 st <strong>2002</strong> to December 31 st <strong>2002</strong><br />

A33<br />

TRIAL: LUD1998015<br />

PRINCIPAL SITE: Melbourne, Australia<br />

PRINCIPAL INVESTIGATOR: Dr. A. Scott<br />

TITLE: Phase 1 Trial of 131I-huA33 in Patients with Advanced Colorectal Carcinoma<br />

CANCER: Colorectal<br />

TRIAL: LUD1998016<br />

PRINCIPAL SITE: Gunma, Japan<br />

PRINCIPAL INVESTIGATOR: Dr. Sakamoto<br />

TITLE: Radioimmunolocalization and safety study of huA33 in patients with gastric carcinoma<br />

CANCER: Gastric<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 206


G250<br />

TRIAL: LUD1999005<br />

PRINCIPAL SITE: Nijmegen, The Netherlands<br />

PRINCIPAL INVESTIGATOR: Dr. E. Oosterwijk<br />

CLINICAL TRIALS –TARGETED ANTIBODIES<br />

TITLE: Extended Phase I Radioimmunotherapy Trial With 131I-labeled Chimeric Monoclonal<br />

Antibody G250 in Patients with Metastatic Renal Carcinoma: Radioimmunotherapy with Two<br />

Sequential High-dose 131I-cG250 Treatments<br />

CANCER: Renal Cell<br />

TRIAL: LUD2000010<br />

PRINCIPAL SITE: Melbourne, Australia<br />

PRINCIPAL INVESTIGATOR: Dr. A. Scott<br />

TITLE: A Pilot Study of cG250 and Low Dose Subcutaneous IL-2 in Patients with Advanced<br />

Renal Cell Carcinoma<br />

CANCER: Renal cell carcinoma<br />

TRIAL: LUD2000015<br />

PRINCIPAL SITE: Nijmegen, The Netherlands<br />

PRINCIPAL INVESTIGATORS: Drs. Corstens, Brouwers, and E. Oosterwijk<br />

TITLE: Interferon- alpha to enhance the targeting of 131I-cG250 monoclonal antibody to tumor<br />

lesions in patients with renal cell carcinoma<br />

CANCER: Renal cell carcinoma<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 207


TRIAL: LUD2001014<br />

PRINCIPAL SITE: Frankfurt, Germany<br />

PRINCIPAL INVESTIGATOR: Dr. A. Knuth<br />

CLINICAL TRIALS –TARGETED ANTIBODIES<br />

TITLE: Phase 1 study of chimeric monoclonal antibody of cG250 in combination with vinblastine<br />

administered weekly by intravenous infusion to advanced renal cell carcinoma<br />

CANCER: Renal cell carcinoma<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 208


3S193<br />

TRIAL: LUD1997010<br />

PRINCIPAL SITE: Melbourne, Australia<br />

PRINCIPAL INVESTIGATOR: Dr. A. Scott<br />

CLINICAL TRIALS –TARGETED ANTIBODIES<br />

TITLE: Phase I Trial of hu3S193 in Patients with advanced epithelial carcinoma expressing Lewis<br />

Y antigen<br />

CANCER: Epithelial-origin cancers<br />

© <strong>Ludwig</strong> <strong>Institute</strong> for Cancer <strong>Research</strong> – <strong>Annual</strong> <strong>Research</strong> <strong>Report</strong> <strong>2002</strong>/2003 209

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