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

is the gene for the erythropoietin receptor (EpoR). So far, it is not known,
which effects EpoR overexpression has on the t(12;21) positive leukemia,
nor which signaling pathways, potentially different from those used in
erythroblasts, are activated. Aim. The aim of this study was to evaluate
the effects of EpoR signaling on survival and proliferation in t(12;21)
positive leukemias and to investigate the apoptosis-modulatory potential
of Epo. We further planned to explore relevant signaling pathways,
linked to the respective effects and thereby elucidating mechanisms that
might be essential for cell survival. Material and Methods. As model system
for TEL/AML1 positive ALL the only available human BCP ALL cell
line REH was used. Controls included the t(12;21) negative leukemic cell
lines Nalm6, K562 and the Epo-dependent cell line UT7. Proliferation
was measured by 3H-Thymidine incorporation assays. Cell viability and
apoptosis rates were evaluated by MTT assay and Annexin V/ propidium
iodide staining, respectively. Activated members of signaling pathways
were detected by Western blotting using appropriate anti-phospho-antibodies
either after immunoprecipitation or from whole cell
lysates. Results. REH cells exhibited a dose dependent increase in proliferation
when cultured with Epo (10, 50, 100U/mL) for 72 hours. Upon
addition of blocking anti-EpoR antibodies these effects were negated.
Epo-induced proliferation was also abolished when cells were co-cultured
with AG490, a JAK2 inhibitor. We could, however, not detect an
Epo-induced phosphorylation of proteins involved in the
EpoR/JAK2/STAT5 cascade. This observation has been already described
in several non-hematopoietic cancers and suggests that other members
of the JAK/STAT pathway compensate for this specific lack of activation.
REH cells further showed an impaired glucocorticoid (GC)-induced
apoptosis in the presence of Epo. We thus evaluated whether signaling
pathways implicated in survival of tumor cells exposed to apoptotic
stimuli play a role in this rescue mechanism. Preliminary data indicate
that the NF-kB as well as the PI3K/Akt pathway is triggered by Epo.
Since cell lines may have intrinsic changes which could affect their signaling
pathways, we are currently evaluating whether the observed
results can also be reproduced in primary leukemic cells. First results
suggest that also primary t(12;21) positive ALL may exhibit a superior
survival and reduced apoptosis rate to GC in the presence of Epo. Summary
and Conclusions. Our data indicate that in t(12;21) positive leukemias
binding of Epo to its receptor leads to enhanced survival in vitro and negatively
affects the sensitivity to GCs. andrea.inthal@ccri.at
This work was supported in part by the Jubiläumsfonds ÖNB10720, FWF
P17551-B14 and GENAU-CHILD Projekt GZ200.136/1 ' VI/1/2005 to
RPG.
0018
FLOW CYTOMETRY ANALYSIS OF THE CO-EXPRESSION OF T- AND B-CELL MARKERS IN
BLASTS FROM PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA
G. Shubinsky, T. Yermiahu, M. Van den Akker, I. Dulman, I. Levy,
J. Kapelushnik
Soroka University Medical Center, BEER SHEVA, Israel
Analysis of genes encoding Ig- and T-cell receptors (TCR) in acute
lymphoblastic leukemia (ALL) demonstrated frequent lineage infidelity
of blast cells. Rearranged Ig-genes were found in 50-95% cases of T-cell
ALL, and rearranged T-cell receptor genes were found in 70-82% cases
of B-cell ALL. It remains, however, unclear whether the infidelity of lymphoblasts
may be defined at the study of antigenic properties of blasts.
We used multiparameter flow cytometry to test the expression of
CD3/TCR ε'chain and B-cell receptor α-chain (CD79a) in the cytoplasm
of electronically separated (gated) blasts from 51 pediatric and 16 adult
patients with ALL. T-ALL was diagnosed in 16 pediatric and 5 adult
patients, and B-ALL in 35 pediatric and 11 adult patients. Two arbitrary
cutoff thresholds, above the 10% and above the 20% of positive cells
within gated lymphoblasts, were used to define the co-expression of Tand
B-cell markers in blast cells. Statistical analysis of data was performed
using the Fisher exact probability test. At 10% cutoff threshold,
the expression of CD79a was determined in 7 of 21 cases (33%) of T-
ALL with CD3ε+ blasts. The expression of CD3ε was found in 13 of 46
cases (28%) of B-ALL with CD79a+ blasts. Totally the blasts with coexpression
of T- and B-cell markers were found in 30% patients with
ALL. Thirteen from 20 patients (65%) with CD3ε+/CD79a+ doublepositive
blasts had either pre-B or late cortical subtype of ALL. At 20%
cutoff threshold for positive cell count, double-positive blasts were identified
in 8 patients (12%) with ALL. Two of these patients had pre-B, and
5 had late cortical subtype of ALL, indicating more frequent finding of
biphenotypic blasts in patients with these immunologic subtypes of ALL
than in patients with other subtypes of ALL (p=0.01). The proportion of
cases with biphenotypic blasts was higher in patients with T-ALL than
12 th Congress of the European Hematology Association
in patients with B-ALL (p grade III occurred in 9
(36%) and 12 (48%) patients respectively. ANC and platelet nadirs
occurred day 21 (range 5-23) and 14 (range 10-27) respectively. Nonhematologic
side effects were constipation grade I, n=7, liver toxicity
grade I, n=7, nephrotoxicity grade I, n=2, parasthesia grade I, n=1, fever
grade II, n=1. Hospitalisation because of grade IV liver toxicity, arthritis
grade III, and pneumonia were required in three patients. After a median
follow-up of 15 (range 3-78) weeks, 18 (72%) patients responded
[CR, n=7 (39%), PR, n=2 (11%), hematologic improvement, n=4 (22%),
stable disease, n=5 (28%)]. Two (22%) complete cytogenetic remissions
were achieved. Responses occurred after a median of 6 (range 2-16)
weeks after treatment. Median response duration amounts to 36 (range
3-not reached) weeks. Median survival time in responding patients is 57
(range 9-not reached) weeks compared to 16 (range 4-29) weeks in refractory
patients (p