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

ment details and thrombotic histories were evaluated by chart review.
JAK2 V617F mutation was detected qualitatively by allele-specific polymerase
chain reaction method on whole blood DNA. Informed consents
were obtained from patients. Results. Ninety-five patients were included
and all were ethnic Chinese. Median age was 62 (range 21-89). There
were 48 males and 47 females. The median follow-up was 68.4 months
(range 5.9-227). The JAK2 V617F mutation was identified in 60 (63%) of
our ET population, which is slightly higher than those reported in the
literature. Univariate analysis showed that patients with the mutation
were older and had significantly lower platelet counts at diagnosis. They
required lower dose of hydroxyurea to control platelet count but had
higher frequency of thrombotic events (32% vs 9%, p= 0.003). The effect
of JAK2 mutation on rate of thrombotic events was maintained in multivariate
analysis. Conclusions. The frequency of the JAK2 V617F mutation
in our group of Chinese patients with ET was slightly higher than
those reported in the literature. Our results suggested that the presence
of JAK2 V617F mutation in this group of ET patients defines a subset of
patients with a thrombotic tendency. Further prospective studies to evaluate
the role of JAK2 V617F mutation in risk stratification for therapy are
needed.
Table 1. Clinical features of ET patients.
1031
INVOLVEMENT OF TNF α IN LEUKAEMIA ONSET AND BONE MARROW TURNOVER
A.S. Cachaço
Portuguese Institute of Oncology, LISBON, Portugal
Leukaemia is characterized by abnormal ratios of bone marrow cells
from several hematopoietic lineages, meaning that normal tissue
turnover is impaired in this situation. Bone marrow diseases have also
been associated with an increase in bone marrow angiogenesis. It is
known that changes in bone marrow microenvironment may lead to a
loss of homeostasis; important players in this process may be cytokines
and extracellular matrix (ECM) molecules present in bone marrow stroma.
One factor possibly involved in bone marrow cell turnover, in normalcy
and in disease, is TNF-α; this study focused on the functions of
bone marrow TNFα, in regulating cell apoptosis within the bone marrow
microenvironment and also in regulating extracellular matrix (ECM)
turnover. Whilst studying bone marrow turnover following sublethal
irradiation, we observed that TNF-α undergoes significant variations,
acutely increasing following irradiation, and decreasing to normal values
5 days after the stimulus. To understand the possible role of TNF-α in
leukaemia development, we irradiated TNF-α knock-out (KO) and wildtype
(wt) mice thrice (each irradiation was separated 1 month apart), a
model that has been shown to result in leukaemia induction. From the
five mice we irradiated from each genotype, four wt mice died 6-7
months after the last irradiation as a result of leukaemia, while in the KO
group only one mouse succumbed to a possible bone marrow deficiency
(although not overt leukaemia). FACS analysis of peripheral blood (PB)
and bone marrows from the different mice, revealed that WT mice had
increased endothelial progenitors and increased endothelial cells, suggesting
the vascular lineage was increased in these mice, following the
irradiation schedules. In addition, the bone marrows of TNF-α KO mice
12 th Congress of the European Hematology Association
were smaller in diameter, but after irradiation, while wt bone marrows
undergo a notorious reduction, the KO bone marrows remain with more
or less the same size, meaning that probably, the altered mice are more
resistant to irradiation. KO bone marrow (irradiated and control) also
exhibited increased megakaryocyte numbers, accompanied by a
decrease in fibronectin and laminin levels, in irradiated KO mice. Another
important observation is that irradiated wt mice, which developed a
bone marrow disease phenotype, have increased bone marrow angiogenesis,
which consisted of dilated blood vessels. Taken together, these
results suggest that TNF-α plays a role in bone marrow homeostasis,
which is more clearly linked with angiogenesis and ECM turnover. In KO
mice, selective apoptosis may contribute to select leukaemia clones; in
parallel, TNF-α is also crucial in modulating MMP activity, and as a result
its absence may contribute towards a global reduction in bone marrow
MMP activity. Reduced MMPs may in turn lead to a reduced availability
of ECM-bound VEGF, thus regulating bone marrow angiogenesis.
Globally, our data point out for a crucial role of TNF-α in modulating
bone marrow turnover and angiogenesis, which may contribute to
leukaemia onset.
1032
PHOSPHO TYROSINE KINASE PROFILLING IN PRIMARY CULTURES FROM PATIENTS WITH
CHRONIC MYELOID LEUKEMIA
V. Ullmannova, H. Klamova, C. Haskovec, J. Moravcova
Institute of Hematology and Blood Transf, PRAGUE 2, Czech Republic
Background. Chronic myeloid leukemia (CML) is a hematopoietic disorder
characterized by the malignant expansion of bone marrow stem
cells. Pathogenesis of CML is associated with a single genetic defectt(9;22)
reciprocal chromosomal translocation which results in constitutive
activity of BCR-ABL tyrosine kinase. Imatinib mesylate (STI571,
Glivec, Gleevec) is a tyrosine kinase inhibitor specific for ABL kinase as
well as for other kinases such as c-Kit, PDGF-R or ARG. Recently, Imatinib
has been used successfully as the first line therapy for CML patients.
Nevertheless additional alteration of BCR-ABL or other kinase can
appear and lead to the treatment resistance. Investigation of active kinase
pattern can disclose primary resistance prior to treatment as well as suitable
targets for subsequent therapy. Expression of Wilm’s tumor (WT1)
transcription factor is elevated in several types of acute and chronic
leukemia including CML. The positive response or upfront Imatinib
resistance can be predicted on the base of WT1 expression after shortterm
cultivation of primary cells. Aims. The aim of this study was to
establish a profiling pattern of tyrosine kinase activity in primary cells
from CML patients and clarify changes of kinase activity after imatinib
exposure. Methods. The effect of tyrosine kinase inhibitors (Imatinib,
Herbimycin A, PP2 and JAK1) on primary culture was characterized by
the expression of WT-1, proliferation marker Ki-67 and analysis of apoptosis.
Gene expression was performed by quantitative real-time RT-PCR.
Phosphotyrosine profiling array was used for determination of tyrosine
kinase activity profile after Imatinib exposure. Results. Primary cells from
patient in chronic phase of CML were evaluated as Imatinib sensitive by
decrease of WT-1 mRNA expression after short-term exposure in culture.
Response to Imatinib was accompanied by an increase of apoptosis and
inhibition of proliferation. Imatinib inhibited seven (Abl1, Fyn, P85A,
PTPN11, SHC1, SHC2, Src), out of forty, tyrosine kinase domains. On
the other hand, four tyrosine kinase domains (EAT2, GRB14, HCK, and
MATK) exhibited higher activity after short-term exposure to Imatinib.
Untreated cultured cells served as a control. Effect of src kinase inhibitors
(Herbimycin A and PP2) but not Jak kinases inhibitor (JAK1) correlated
with the effect of Imatinib in some patients. Conclusions. Creating a
kinase pattern disclose active signaling pathways, enables to clarify
mechanism of response or resistance to distinct treatment and thus the
therapy can be targeted more efficiently.
Support: VZ MZCR 023736.
1033
LOSS OF HETEROZYGOSITY AND MICROSATELLITE INSTABILITY IN PATIENTS WITH
MULTIPLE MYELOMA
A. Timuragaoglu, 1 E. Kiris, 1 S. Demircin, 1 S. Dizlek, 1 N. Uysalgil, 1
G. Alanoglu, 2 L. Undar1 1 Akdeniz University, School of Medicine, ANTALYA, Turkey; 2 S.Demirel University,
School of Medicine, ISPARTA, Turkey
Background. Chromosome 14 abnormalities -mostly translocations- are
nearly seen 50 percent of multiple myeloma (MM) patients and these
abnormalities are important in the pathogenesis of MM. Genomic insta-
haematologica/the hematology journal | 2007; 92(s1) | 381