Haematologica 2003 - Supplements

145
STUDY OF BONE MARROW ANGIOGENESIS IN
WALDENSTROM’S MACROGLOBULINEMIA.
PRELIMINARY RESULTS.
E. Hatjiharissi1, M. Papaioannou1, C. Hatjileontis2, V.
Bakaloudi1, N.Eleftheriadis1, D. Mihou1, V. Kaloutsi2, M.A.
Dimopoulos3, J. Christakis1, K. Zervas1.
1Department of Hematogy-Oncology, Theagenio Cancer Hospital,
Thessaloniki, Greece; 2Department of Pathology,Aristotle
University of Thessaloniki, Thessaloniki, Greece; 3Department of
Clinical Therapeutics and Internal Medicine, University of Athens,
School of Medicine, Athens.
Background: Waldenstrom’s Macroglobulinemia (WM) is a rare
clinical syndrome, which is usually associated with an underlying
lymphoplasmacytic lymphoma. It is characterized by infiltration
of bone marrow (BM) with lymphoplasmacytoid cells and
increased number of mast cells. The latter are considered strong
mediators of angiogenesis. It has been reported that in non
Hodgkin’s lymphomas, mast cell density is correlated with the
extent of BM angiogenesis. However, there are limiting data
regarding the presence, extent, and the prognostic significance of
angiogenesis in WM.
Aim of the study. In this study we evaluated bone marrow
angiogenesis (BMA) in conjunction with the presence of mast
cells and the percentage of infiltration by lymphoplasmacytoid
cells in patients with WM at diagnosis and after treatment with
Rituximab (monoclonal Anti-CD20 Ab).
Methods: Eight patients (5 males, 3 females) with median age 66
years (range 47-80) with symptomatic WM were treated with
Rituximab 375mg/m2 iv for 4 consecutive weeks. Treatment was
repeated for additional 4 courses in patients without evidence of
progressive disease, three months after the completion of first
course. Bone marrow biopsies were obtained at diagnosis and
after the completion of treatment. Bone marrow angiogenesis was
estimated by microvessel density (MVD). Microvessels were
identified using the standard immunohistochemical staining for
CD34 moAb and counted in whole cellular bone marrow at 400x
magnification. MVD was expressed as number of vessels per
mm2 Angiogenesis was also studied in a control group of 10
normal bone marrow biopsies. Mast cells were detected by
positive immunostaining of BM specimens for tryptase and c-kit
(CD 117 moAb). Bone marrow specimens were also evaluated
for % of lymphoplasmacytoid infiltration and the expression of
CD 20 by malignant cells.
Results: Five of eight patients responded, according to the usual
criteria. Bone marrow biopsies were repeated in all patients after
a median time of 7 months (range 6-8) since the initiation of
treatment. The pretreatment MVD in patients with WM was
increased in comparison to the control group (median …., range
15.16-26.76, and median 3,06, range 2,3-3,8 vessels/mm3
respectively). Postreatment median MVD was …. (range 15,09-
26.76). In all responders MVD decreased in parallel with the
bone marrow infiltration by neoplastic cells and the number of
mast cells. Decrease in MVD was also observed in the 3 non
responders, according to the strict response criteria used in this
study.
Conclusions. Although the number of patients studied is too
small, we observed that BMA is increased in patients with WM at
diagnosis. This seems to be related to the degree of BM
infiltration by lymphoplasmacytoid cells as well as with the
number of mast cells. This observation is of considerable
biological interest and further studies are warranted to elucidate
the role of angiogenesis in WM.
146
THE PROTEASOME INHIBITOR PS-341 MODULATES
VEGF SECRETION AND ACTIVITY IN MULTIPLE
MYELOMA.
Nicholas Mitsiades1,2, Constantine S. Mitsiades1,2,
Vassiliki Poulaki3, Galinos Fanourakis1,2, Dharminder
Chauhan1,2, Nikhil C. Munshi1,2, Teru Hideshima1,2,
Kenneth C. Anderson1,2.
1. Jerome Lipper Multiple Myeloma Center, Department of Medical
Oncology, Dana-Farber Cancer Institute, Boston MA, USA; 2.
Department of Medicine, Harvard Medical School, Boston, MA,
USA; 3. Massachusetts Eye and Ear Infirmary, Harvard Medical
School, Boston, MA, USA
The proteasome inhibitor PS-341 inhibits IêB degradation,
thereby preventing activation of NF-êB, and induces apoptosis in
several types of cancer cells, including chemoresistant multiple
myeloma (MM) cells. Importantly, it has marked clinical activity
even in the setting of relapsed refractory MM. We have recently
reported that PS-341 in a dose-dependent fashion inhibits tumor
growth, inhibits associated angiogenesis, and prolongs host
survival in a murine plasmacytoma model. We now investigated
the mechanism of PS341-induced inhibition of angiogenesis, by
evaluating its effect on i) human microvascular endothelial cell
(HMVEC) viability and ii) the production of VEGF by the MM
cell line MM.1S in vitro. We found that PS-341 decreased the
viability of human microvascular endothelial cells in a dosedependent
manner. PS-341 also lowered their proliferative
response to stimulation with VEGF and bFGF. Surprisingly, PS-
341 stimulated VEGF mRNA expression and protein release by
MM.1S cells in vitro. To characterize the mechanism of PS341-
induced VEGF upregulation, we transiently transfected MM.1S
cells with a reporter plasmid carrying the 1.7 kb sequence of the
VEGF promoter (VEGFpr), as well as deletion mutants with
respectively 1.0-kb, 0.8-kb, 0.5-kb and 0.1-kb from the VEGFpr,
upstream of a luciferase gene. A promoterless vector served as
control. We found that PS-341 stimulated reporter gene
expression in cells transfected with the full-length (1.7kb)
VEGFpr, but not in cells transfected with the 1.0 kb, 0.8 kb, 0.5
kb, and 0.1 kb promoter fragments, or the control vector. This
step-by-step deletional mapping identified a 0.7 kb region
(between 1.0 kb to 1.7kb upstream of the initiation site) to be
responsible for the PS-341-induced effect on VEGF expression.
Because this region contains HIF-1á response elements, we
investigated the role of the transcription factor HIF-1á in our
model. We found that transfection of HIF-1a anti-sense
oligonucleotides suppressed the constitutive VEGFpr activity and
completely abrogated its stimulation by PS-341. We conclude
that HIF-1á, a well-known substrate of proteasomal degradation,
stimulates VEGF synthesis after proteasome inhibition in MM
cells. However, the direct anti-proliferative effect of PS-341 on
endothelial cells overcomes the increased production of VEGF
and results in net inhibition of angiogenesis.
147
Mean vessel density, endothelial cell proliferation and
carbonic anhydrase IX expression in haematologic
malignancies, MGUS and bone marrow metastases
H. De Raeve, *K. Vanderkerken, P. Vermeulen, **A. Harris
and E. Van Marck
Department of Pathology, University of Antwerp, *Department of
Haematology and Immunology, Free University Brussels (VUB),
**Weatherall Institute of Molecular Medicine, Headington, Oxford, UK
Introduction: Hypoxia inducible factor 1 (HIF 1), which plays a
critical role in oxygen homeostasis, transactivates a large number
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