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

0294
MYCOSIS FUNGOIDES. IMMUNOHYSTOCHEMISTRY ANALYSIS OF LYMPHOID AND
MICROENVIREMENT CELLS BY MACROTISSUE ARRAY
C. Del Agua, 1 A. Rubio-Martinez, 2 F Felipo, 3 M.A. Piris, 4 P. Giraldo 2
1 HU Miguel Servet, ZARAGOZA; 2 Haematology Department. HU Miguel
Servet, ZARAGOZA; 3 Pathology Department. HU Miguel Servet,
ZARAGOZA; 4 CNIO, MADRID, Spain
Background. Cutaneous lymphomas (CL) represent a unique group of
lymphomas and are the second most frequent extranodal lymphomas.
CL probably is the result of a multifactor and multistep process. Firstly
an inflammatory reactive process is developing secondary to various
chronic stimuli that may be genetic, environmental, infectious and
immunologic. The consequences are the negative effects in cell regulation
and deregulation of oncogenes and/or suppressor genes later promotes
transition from pre-neoplastic conditions to neoplasia. Detailed
molecular expression analysis of cutaneous T-cell lymphoma is not available.
Some oncogenic alterations have been demonstrated, such as functional
inactivation of the Fas receptor, constitutive activity of STAT 3, or
the inactivation of the p16 gene via deletion or promoter hypermethylation.
Objective: To study the expression of p16, c-myc, Ki67, bcl-2,
CD1a, CD123, TCL1, CD68, STAT 3, STAT 4 and MAL1 in an homogeneous
group of. CL diagnosed in one tertiary Hospital in order to
know more about the characteristics of this neoplastic process. Methods.
We have study 30 CL diagnosed consecutively as Mycosis Fungoides (14
early and 16 advanced stages) between January 2005-December 2006. By
macrotissue array techniques and immunohistochemistry protocol with
p16, c-myc, Ki67, bcl-2, CD1a, CD123, TCL1, CD68, STAT 3, STAT 4 y
MAL1 was applied in all paraffin histological samples. Results. In 28 samples
(92%) we have observed p16 positive over expression, the two negative
samples corresponding to early affectation. In 14 samples from early
stages (48%) c-myc was negative. In 29 samples (96%) CD1a was positive
in dermal and epidermal layer. CD123 (interleukine 3 receptor) was
negative in 16 samples (52%) and TCL1 was positive in 12 cases (39%)
in small cells with oval and cleaved nucleus and scarce cytoplasm. Over
expression of MAL1 was observed in advanced patients with aggressive
CL. Conclusions. In our study an over expression of p16 is observed in the
majority of cases and high c-myc expression in advanced stages. Probably
dendritic plasmacytic cells are involved in the pathogenesis of skin
lymphoproliferative disorders with cutaneous T cell infiltration. MAL1
could be a predictor of agressive CL but it is necessary more studies and
more cases in order to confirm it.
0295
EXPRESSION OF NPM-ALK LEADS TO ONCOGENIC EFFECTS OF JUNB
P. Vesely, 1 P. Staber, 1 C. Fuchs, 1 I. Bambach, 1 S. Schauer, 1 D. Sternberg, 2
L. Kenner, 3 G. Hoefler1 1 Medizinische Universitaet Graz, GRAZ, Austria; 2 Mount Sinai School of Medicine,
NEW YORK, USA; 3 Medizinische Universität Wien, VIENNA, Austria
Background. The oncogene NPM-ALK arises due to a chromosomal
rearrangement in human T-cells, resulting in activation of MAPK, PI3K
and PLCy signaling pathways. Enhanced activity of multiple signalling
cascades and subsequently ALC(L)- lymphoma formation are the result.
High expression of CD30 and JunB are a hallmark of NPM-ALK positive
ALCL. In contrast to the prototypic AP-1 transcription factor c-Jun, JunB
exerts an antioncogenic function in most cell types. Its functional role in
the context of NPM-ALK remains uncertain. Results. Here we show that
aberrant NPM-ALK expression leads to IL-3 independent outgrowth of
Ba/F3 cells. Furthermore NPM-ALK expression induces JunB and CD30
expression, which is undetectable in the corresponding wild type cells
and can be reversed by MEK-inhibition. Interruption of the NPM-ALK
kinase domain impedes JunB and CD30 expression. Specific down-modulation
of JUNB mRNA using small hairpin (sh) RNA avoids CD30
expression and arrests the cell cycle in G1 phase of NPM-ALK expressing
cells. Conversely, ectopic JunB expression in NPM-ALK transgenic
Ba/F3 cells leads to enhanced proliferation in the absence of IL3, whereas
ectopic JunB expression in WT Ba/F3 is not sufficient to provoke IL3
independence and even leads to reduced proliferation in the presence of
IL3. Conclusions. Thus, both, NPM-ALK and JunB are essential to induce
CD30 expression and malignant transformation. The presence of NPM-
ALK determines the oncogenic role of JunB.
12 th Congress of the European Hematology Association
0296
RITUXIMAB ENHANCES CELL-MEDIATED CYTOTOXICITY IN PATIENTS WITH B-CELL
LYMPHOMA: IN VIVO STUDY OF NK/T-LYMPHOCYTES ACTIVITY
V. Prochazka, M. Novak, Z. Pikalova, T. Papajik, K. Indrak
Teaching Hospital, OLOMOUC, Czech Republic
Background. Rituximab dramatically improves the outcome of patients
with B-cell lymphomas. The major mechanism of its action is antibodydependent
cellular cytotoxicity (ADCC) mediated by degranulation of
cytotoxic lymphocytes (CTL) after binding to the Fc receptor. Although
rituximab has been routinely used for 10 years, the published studies of
natural killer (NK) and T-lymphocyte cytotoxic activity are limited to in
vitro and ex vivo studies. The novel flow cytometry method is capable of
visualizing the degranulation of T/NK cells via surface expression of
lysosomal-associated membrane protein 1 (CD107a). Aims. To investigate
changes in NK/T lymphocytes subsets and in vivo anti-lymphoma
activity of normal NK/T lymphocytes in patients with B-cell lymphoma
during the administration of chemoimmunotherapy. Methods. We investigated
peripheral blood samples from 44 patients with newly diagnosed
lymphomas (diffuse large B-cell lymphoma, DLBCL=21, follicular lymphoma,
FL=16, mantle cell lymphoma=3, marginal zone lymphoma=2,
B-cell lymphoma, unspecified=2). The patients were treated with
chemoimmunotherapy (CHOP-like regimen=42, fludara-based regimen=2)
repeated every 21 days. Rituximab was given in standard dose
of 375 mg/m2 . Blood samples were obtained during the 4th therapy cycle
- before and within 1 hour after the administration of rituximab. Patients
with active infection or inflammation were excluded from the study.
Flow cytometric analysis was performed on a FACSCalibur cytometer
(BD Biosciences). Anti-human fluorescein-conjugated monoclonal antibodies:
CD 3 FITC/ CD16+56+ PE, CD 4 FITC/CD 8 PE, CD 16 FITC/
CD 56 PE/ CD 3 EDC, (all Beckman Coulter) and CD 107a PE-Cy5 (BD
Biosciences) were used for multi-color sample analysis. Antibody concentrations
were adjusted according to the manufacturer’s protocol. The
results were expressed as the percentage of cells in a gated lymphocyte
region. The collected data were analyzed using the Cell Quest Pro software
by BD Biosciences. Results. We did not find any difference in the
numbers of T/NK cell subsets when comparing patients with FL and
DLBCL. The administration of rituximab is connected with a significant
increase of all NK subsets (CD16 + 12.8±9.1 vs 20.8±14.4, p=0.007; CD
56 + CD16 + 14.9±7.4 vs 23.1±13, p=0.03), the proportion of CD8+ lymphocytes
did not change, CD4 + cells decreased (14.5±2.2 vs 12.1±1.8,
p=0.01). The proportion of degranulated CTL increased more than twice
(CD107a+ 3.07±1.9 vs 6.37±3.8, p