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

1110
CONSTITUTIVE ACTIVATION OF STAT1 AND STAT3 PROTEINS WITHOUT INTERFERON
INDUCIBILITY: RELEVANCE TO THERAPY IN IGVH-UNMUTATED CLL PATIENTS
J. Chumchalova, J. Navratilova, D. Klimesova, H. Francova,
V. Kuhrova, Y. Brychtova, M. Doubek, D. Dvorakova, J. Mayer
University Hospital Brno, BRNO, Czech Republic
Background. STAT proteins play an important role in regulation of proliferation,
differentiation, apoptosis, and immune response. STAT proteins
are constitutively activated in hematopoetic cells, which are transformed
by oncogenic tyrosine kinases and are also connected to pathology
of variety leukemias and lymphomas. Tyrosine phosphorylation of
STAT proteins allows formation of dimers, translocation to the nucleus
and binding to specific DNA sequences within promoters of target genes,
which leads to activation of transcription. Serine phosphorylation is not
necessary to activate STATs, but it modulates gene activation induced by
tyrosine phosphorylation. Constitutive phosphorylation of serine but
not tyrosine in both STAT proteins was found in B-CLL cells. Aims. The
aim of our study was to examine defects in STAT1 and STAT3 pathways
in our CLL patients. Methods. We analyzed defects in STAT1 and STAT3
pathways after stimulation of cells with interferon γ (IFNγ) (10 ng/mL)
and interferon α (IFNα) (5000 IU/mL). We evaluated 59 primary cultures
derived from CLL patients with prevalency of B-CLL cells. Presence of
STAT1 and STAT3 protein isoforms was detected by Western-blotting
analysis using specific polyclonal and monoclonal antibodies. Results.
Constitutive phosphorylation of serine and tyrosine residues of both
STAT proteins was found in majority of CLL patients. STAT1 protein
showed higher ratio of defect in phosphorylation of serine (69,6%) and
tyrosine (43,5%) after induction by interferons compared to phosphorylation
of serine (15,8%) and tyrosine (50%) of STAT3 protein. IFNα is
more effective inductor of both phosphorylations in STAT1 and STAT3
proteins. Only tyrosine phosphorylation of STAT1 protein was markedly
induced by IFNγ. Analysis of relationship STAT proteins and IgVHmutation
status was performed. We did not found significant differences
in phoshporylation of STAT1 and STAT3 proteins. IFNα induced only
tyrosine phosphorylation of STAT1 protein in IgVH-unmutated patients
compared to IgVH-mutated patients where the phosphorylation was
induced by both IFNs. Analysis of IgVH-unmutated patients who
required therapy showed higher proportion of constitutive phosphorylation
without IFN inducibility in both STAT proteins compared to
untreated patients. Consequently, the significant difference was found
among IgVH-unmutated patients with or without therapy. Patients with
therapy showed markedly higher proportion of constitutive phosphorylation
without IFN inducibility of serine and tyrosine residues in STAT1
and serine residue in STAT3 proteins. On the contrary, these patients
manifested a decrease of constitutive phosphorylation without IFN
inducibility on tyrosine residue in STAT3 protein. Conclusions. Our results
show that majority of CLL patients have constitutive phosphorylation
of both STAT proteins. IFNα was shown to be a more effective inductor
in comparison with IFNγ. Presence of constitutive phosphorylation
without IFN inducibility of STAT1 and STAT3 proteins is increased in
IgVH-unmutated CLL patients who required therapy.
Supported by grant NR8443-3/2005 provided by IGA MZCR
1111
CD28 AND ICOS GENE POLYMORPHISMS ARE ASSOCIATED WITH BCELL CHRONIC
LYMPHOCYTIC LEUKEMIA (B-CLL)
I. Frydecka, 1 K. Suwalska, 2 A. Tomkiewicz, 2 E. Pawlak, 2 L. Karabon, 2
T. Dobosz, 1 A. Jonkisz, 1 A. Lebioda, 1 K. Kapelko-Slowik, 1
K. Kuliczkowski, 1 D. Wolowiec1 1 2 Medical University, WROCLAW, Poland; Institute of Immunology and Experimental,
WROCLAW, Poland
Background. There are strong evidences that altered immunological
function entails an increased risk of B- CLL. T cell-specific surface receptors
CD28 and inducible co-stimulator (ICOS) are main costimulatory molecules
expressed on cell surfaces and provide regulatory signals for T-cell
activation. CD28 potently enhances T-cell functions essential for effective
antigen-specific immune responses. Unlike the constitutively
expressed CD28, ICOS is induced on the T-cell surface and does not
upregulate the production of IL-2, but induces the synthesis of IL-4. Aim.
The extended study was undertaken to evaluate the association between
the T17int3C CD28 gene polymorphism, microsatelite (GT)n polymorphism
in intron 4 of the ICOS gene and susceptibility to B-CLL in a Polish
population. Methods. One hundred twenty four B-CLL patients and
202 healthy subjects were studied. Allele identification was achieved by
12 th Congress of the European Hematology Association
PCR amplification. The amplified product for SNP (single-nucleotide
polymorphism) loci was purified and minisequenced using the commercial
kit SnapShot of PE Applied Biosystems. The dinucleotide repeat
polymorphism was studied by PCR and fuorescence based technique.
The products were analyzed on the ABI PRISM 310 Genetic Analyzer
(ABI PRISM 310 capilary elctrophoresis system). Results. The distribution
of the CD28IVS3+17C/T alleles and genotypes presented significant differences
in B-CLL patients and healthy controls. The presence of
CD28IVS3 + 17C allele and CD28IVS3+17(C/C+C/T) genotype increased
the odds of B-CLL by 1.97 and 2.07 [p=0.0019, 1.2755~95%CI ~3.0313
and p=0.0033, 1.2686~95%CI ~3.3778, respectively]. Global distribution
of ICOS alleles was significantly different in B-CLL patients and
healthy controls (p= 0.000005). The results showed that (GT)9 allele
increased risk of B-CLL 2,3 times [p=0.0294, OR =2.345,
1.0700~95%CI~5.1419] while frequency of the (GT)12 was significantly
decreased in patients [p=0.0002, OR =0.4307, 0.4307]. Moreover, distribution
of genotypes differed between patients and healthy controls
(p=0.0076). The GT9/GT11 genotype was overexpressed in B-CLL
patients compared with control subjects (p=0.03, OR= 2.3457,
1.0700~95%CI ~5.1419), while genotypes GT12/GT12, was more frequent
in control subjects [p=0.0024, OR=0.0831, 0.0195~95%CI
~0.6306] Summary. Our results suggest that the CD28 T17int3C gene
polymorphism and microsatelite ICOS gene (GT)n polymorphism contribute
to genetic susceptibility to B-CLL
1112
TRYPTASE AS NOVEL TUMORMARKER IN CLINICAL HEMATOLOGY: EVALUATION OF 1041
PATIENTS
W.R. Sperr, 1 A. El-Samahi, 1 M. Girschikofsky, 2 S. Winkler, 1
H. Semper, 1 H. Agis, 1 C. Sillaber, 1 G. Endler, 1 H. Rumpold, 1 D. Lutz, 2
P. Valent1 1 2 Medical University of Vienna, VIENNA; KH der Elisabethinen, Dept of Int
Med I, LINZ, Austria
Background. Tryptase is a term used for a family of trypsin-like serine
proteases primarily produced and stored in mast cells. Recent data suggest,
that tryptase is expressed in neoplastic cells in various myeloid neoplasms.
The aim of the present study was to determine whether tryptase
may serve as a marker of disease in patients with hematologic neoplasms.
Aims. The aim of this study was to determine the diagnostic and prognostic
significance of tryptase in patients (pts) with hematologic neoplasms.
Methodes. Tryptase levels were determined in 906 pts with hematologic
malignancies, including myeloproliferative disorders (MPD,
n=153), myelodysplastic syndromes (MDS, n=233), acute myeloid
leukaemia (AML, n=317), and systemic mastocytosis (SM, n=71), and in
137 pts with reactive leukocytosis/thrombocytosis or idiopathic cytopenia,
by fluoroenzyme-immunoassay. Moreover, tryptase was analyzed in
165 healthy subjects, 80 pts with non hematologic disorders. Results. For
this purpose, serum tryptase levels were determined in 906 patients (pts)
with hematologic malignancies, including myeloproliferative disorders
(MPD, n=153), myelodysplastic syndromes (MDS, n=233), acute myeloid
leukaemia (AML, n=317), and systemic mastocytosis (SM, n=71), and in
137 pts with reactive leukocytosis/thrombocytosis or idiopathic cytopenia,
by fluoroenzyme-immunoassay. Moreover, tryptase was analyzed in
165 healthy subjects, 80 pts with non hematologic disorders. In 90% of
healthy controls serum tryptase levels ranged between 2.4 ng/mL and
9.5 ng/mL (median: 5.7 ng/mL; range in all pts: >0.1-18 ng/mL). Among
pts with non hematologic disorders, slightly elevated serum tryptase levels
(up to 24 ng/mL) were detectable in 6/18 pts with severe renal failure
and in 3/29 pts with helminth infections. In the majority of pts with reactive
leukocytosis/thrombocytosis or idiopathic cytopenia serum tryptase
was within the normal range (″15 ng/mL), as were most pts with lymphoid
neoplasms. Among myeloid neoplasms, elevated tryptase levels
were recorded in 83% of the pts with SM, 37% of pts with AML, 34%
of the pts with CML, and 24% of the pts with MDS. The highest tryptase
levels were found in pts with SM or AML-M4eo. In pts with CML treated
with imatinib, elevated tryptase levels were found to be associated
with an unfavourable prognosis concerning survival (tryptase ″15 ng/mL:
100%; tryptase >15 ng/mL: 71%, after 60 months, p