12th Congress of the European Hematology ... - Haematologica

12 th Congress of the European Hematology Association
0274
NEOPLASTIC PLASMA CELLS ARE DEMONSTRABLE AT BONE MARROW SITES DISTANT
TO SOLITARY PLASMACYTOMA OF BONE AND PREDICT FOR PROGRESSION TO
MULTIPLE MYELOMA
Q. Hill, 1 A.C. Rawstron, 2 J.A. Child, 2 R.M. De Tute, 2 R.G. Owen, 2
1 2 Leeds General Infirmary, LEEDS, United Kingdom, HMDS, LEEDS, United
Kingdom
Background. Solitary plasmacytoma of bone (SPB) typically present as
single destructive lesions within the spinal column or long bones. Local
radiotherapy is the treatment of choice but approximately 50% of
patient’s progress to myeloma. Many patients have a monoclonal protein
detectable in their serum and/or urine at diagnosis and persistence
of this for >1 year after radiotherapy predicts for progression to myeloma.
Identification of high risk patients at the time of diagnosis is clearly
desirable as it would enable risk stratification and more careful monitoring
of patients. Although prophylactic adjunctive chemotherapy has
not definitively been shown to benefit unselected patients with SPB,
future therapeutic advances might be targeted on patients with a high
risk of progression. Interestingly it has recently been shown that patients
positive for serum free light chains are at greater risk of progression to
myeloma. We and others have previously demonstrated that neoplastic
bone marrow plasma cells are distinguishable from their normal counterparts
by virtue of their lack of CD19 expression and/or their aberrant
expression of CD56. We have developed a multiparameter flow cytometry
assay which predicts outcome following autologous transplantation
in myeloma patients and risk of progression in patients with MGUS.
Aims. In this study we have applied this assay to assess the staging bone
marrow specimens from patients with biopsy proven SPB for the presence
of occult disease at sites distant to the primary lesion. Methods. 52
patients were included in this analysis (31 male, 21 female, median age
65) and in each case the staging bone marrow aspirate and trephine
biopsy (obtained from a site distant from the SPB, typically the right iliac
crest) was not indicative of myeloma (30% CD19- and/or CD56 + as per convention) were demonstrable
35/52 (67%). Neoplastic plasma cells when present comprised a
median of 70% (35-100%) of bone marrow plasma cells. 21 patients
(40%) developed myeloma with a median time to progression of 476
days (range 18-1632). Progression occurred in 18 of the 35 (51%) patients
with neoplastic plasma cells in their staging marrows and in 3/17 (18%)
patients with a normal phenotypic profile. The difference was significant
using Chi-square analysis with Yates’ correction for continuity
(p=0.04). The overall risk of progression was similar in patients with a
'myeloma pattern- in which >90% of bone marrow plasma cells had a
neoplastic phenotype (5/12, 42%) and those with an MGUS or mixed
pattern in which distinct populations of both normal and neoplastic cells
are demonstrable (13/23, 57%). Conclusions. Neoplastic plasma cells are
frequently found at bone marrow sites distant to SPB and their presence
predicts for progression to multiple myeloma. Trials of adjuvant systemic
therapy are warranted in this group.
0275
INHIBITION OF CELL PROLIFERATION BY LENALIDOMIDE IS ASSOCIATED WITH STIMU-
LATION OF EGR1 TRANSCRIPTIONAL ACTIVITY IN A CHROMOSOME 5 DELETED
BURKITTS LYMPHOMA AND MULTIPLE MYELOMA CELL LINE
A. Gandhi, J. Kang, D. Stirling, P. Schafer
Celgene Corporation, SUMMIT, USA
Background. The mechanism by which lenalidomide exerts its antiproliferative
effects in deletion 5q MDS clones or MM cells is not yet fully
elucidated. Early growth response (Egr1) gene is a tumor suppressor
gene located on chromosome 5q31.1 that encodes a transcription factor
involved in the regulation of cell proliferation and apoptosis. Aims. The
present study examines the hypothesis that lenalidomide may act by
enhancing the expression or activity of Egr1 in sensitive hematopoietic
tumor cells, especially those with only a single copy of the Egr1 gene.
Methods. Luciferase assay. Cells were transfected with Egr1-luciferase
(Stratagene) using Lipofectamine 2000 (Invitrogen) reagent according to
manufacturer’s instructions. Six hours post-transfection, cells were treated
with 1 ÌM lenalidomide or DMSO for 16 hours. Luciferase activity
was assayed using luciferase substrate (Promega) and measured using a
luminometer (Turner Designs). qRT-PCR and siRNA silencing. Cells
were transfected with Egr1 or non-targeting siRNA (Dharmacon) using
Dharmafect2 (Dharmacon) reagent according to manufacturer’s instruc-
100 | haematologica/the hematology journal | 2007; 92(s1)
tions. Gene expression analysis used gene expression assays (Applied
Biosystems) and expression was measured on a real-time PCR System
7500 (Applied Biosystems). Relative quantifications were calculated with
SDS v.1.3.1 software. Cell proliferation assay. Cells were incubated in
96-well cell culture plates with compounds for 72 hours and assayed by
3H-thymidine incorporation. IC50s were calculated by nonlinear regression
analyses with GraphPad Prism. Results. Lenalidomide stimulated
the transcriptional activity of Egr1 in the lenalidomide-sensitive chromosome
5 deleted Burkitt’s lymphoma Namalwa CSN.70 and in the MM
cell line LP-1. Egr1 siRNA Namalwa cells proliferated more than mock
controls, indicating that Egr1 functions as a tumor suppressor in Namalwa
cells. Lenalidomide had no effect on expression of Egr1, but augmented
Egr1 nuclear transport in a dose-dependent manner. Lenalidomide
did not affect expression of the Egr1 downstream effector genes
ATF3, fibronectin, p53, PTEN, and TGF-β1, while p21 levels increased.
However, lenalidomide-induced p21 expression was not affected in Egr1
siRNA Namalwa cells. Interestingly, lenalidomide’s anti-proliferative
potency was greater in Egr1 siRNA Namalwa but not in Egr1 siRNA LP-
1 cells. Conclusions. Lenalidomide induces nuclear transport and transcriptional
activation of the tumor suppressor Egr1, which may contribute
to lenalidomide’s anti-proliferative activity. This activity may be
related to the levels of Egr1 expression, explaining why del 5q31
myelodysplastic clones are especially sensitive to the cytotoxic effects
of lenalidomide.
0276
DEPLETION OF BLOOD DENDRITIC CELLS (BDC) AND ITS PROGNOSTIC SIGNIFICANCE
IN PATIENTS WITH MULTIPLE MYELOMA
S.P. Smolewski, B. Cebula, A. Branicka, O. Grzybowska-Izydorczyk,
H. Urbanska-Rys, T. Robak
Medical University of Lodz, LODZ, Poland
Background. The number and function of dendritic cells (DC), which
are an important part of anti-tumor response, may be crucial for development
and natural course of malignancy. There are several types of DC,
including those circulating in the peripheral blood (blood DC; BDC), as
was recently distinguished by the panel of monoclonal antibodies
against BDC antigens (named BDCA-1, -2 and -3). Aims. Since these
BDC subtypes have not been assessed in multiple myeloma (MM) we
aimed to investigate their frequency and absolute numbers in this disease.
Material and Methods. The study was performed in 41 untreated
MM patients. Circulating BDC were detected in blood (MM, control)
and bone marrow (MM) samples by the flow cytometry. Three BDC
subsets were determined: plasmacytoid (PDC), BDCA-2+/CD123+/
HLA-DR+, myeloid 1 (MDC1), BDCA-1+/CD11c+/CD14-/HLA-DR+,
and myeloid 2 (MDC2), characterized by BDCA-3+/CD32-/CD19-
/HLA-DR+ immunophenotype. The rates and absolute numbers of all
DC (total DC; tDC) as well as their particular subsets were calculated.
Results obtained in MM patients were compared with age- and sexmatched
healthy controls. Moreover, they were correlated with the outcome.
The study endpoints were response to first line treatment, progression
free survival (PFS) and overall survival (OS) time. Results. Objective
response to first line treatment was achieved in 27 (66%) patients,
including 9 (22%) complete and 18 (44%) partial responses. The remaining
14 (34%) patients were either resistant or even progressive upon
chemotherapy. The median follow up was 18 months (5-28). The median
PFS of responders was 13.5 months (8-21). In MM patients total frequency
and absolute numbers of BDC were significantly lower than in
healthy subjects (0.69±0.37% vs. 1.24±0.40%; p=0.002, and 16.2±13.5
cells/µL vs. 31.7±13.3 cells/µL; p=0.005, respectively). Significantly lower
rates and counts in MM patients concerned all BDC subtypes, including
MDC1 (p=0.035 and 0.029, respectively), MDC2 (p=0.033 and
p=0.009, respectively) and PDC (p=0.007 and p=0.0005, respectively).
Moreover, all those BDC subtypes were also found in the bone marrow
of MM patients. Importantly, a frequency of blood DC in the bone marrow
was even higher than in the peripheral blood (for tDC - p=0.017,
for MDC2 - p=0.015, and for PDC - p=0.021, respectively). Total DC
rates and counts were significantly higher in responders than in resistant
patients (0.90±0.40% vs. 0.41±0.29; p=0.009, and 25.3±14.7 cells/µL
vs. 9.3±6.0 cells/µL; p=0.032, respectively). The difference was due to
prevalence of blood MDC1 in responders (responders vs. non-responders
- p=0.011 for rates and p=0.008 for numbers, respectively). Lower
(less than median) MDC1 number at the diagnosis correlated with shorter
PFS of patients (log rank test: p=0.027) and showed a trend toward
longer OS (log rank test: p=0.073). Conclusions. This is the first study
demonstrating a profound deficiency of all subtypes of BDC in patients
with MM. It may reflect either an impairment of immune system, facil-