Recent observations raise possibility for constitutively active, mutated JAK2 to modulate expression of RAS genes in CMPD. We analyzed the expression of AGT, renin, AT2R1 and ACE genes in normal and bone marrows of PV and ET patients with the respect to the presence of V617F JAK2 mutation. PV and ET had different expression patterns of major RAS components compared to normal BM which was primarily associated with the JAK2V617F mutation and less with PV or ET disease phenotype. However, AT2R1 was exclusively markedly upregulated only in PV, while ET showed moderate expression irrespective of the JAK2 mutational status.

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3607 Background: In B-cell chronic lymphocytic leukemia (B-CLL) there is a well documented intraclonal and interclonal variability of B-CLL cells in different lymphoid compartments with respect to the expression of a number of surface and intracellular molecules (for example CD38 and ZAP-70). This variability in part may reflect a number of interactions of malignant B-CLL clone with supporting microenviroment including cells (T-cells, nurse-like cells, etc.), cytokines, chemokines and stroma. One of the key interactions of B-CLL clone is with T-cells, through CD154/CD40 system. It is important pathway modulating survival, drug resistance and immunity. It is known that CD154 is transiently expressed on CD4+ T cells, as well as that CD154 can be coexpressed on B-CLL cells with CD40 in a subpopulation of B-CLL patients. Its expression on B-CLL cells can be induced by gene therapy and lenalidomide, being in part responsible for their therapeutic effects. Aim of this study was to determine the level of expression of CD154 and CD40 in vivo on B-CLL cells and T lymphocytes and to evaluate intra and interclonal differences due to different microenvironment, i.e. peripheral blood, bone marrow and lymph nodes. Methods: peripheral blood, (PB), bone marrow (BM) and lymph node (LN) samples were taken by conventional techniques (venepuncture and fine needle aspiration) on the same day. The expression level of CD154 and CD40 molecules on CD19+CD5+ B-CLL cells and CD19-CD5+ T cells was analyzed by flow cytometry. Results were expressed as mean fluorescence intensity (MFI) and analyzed by paired tests. Results: samples taken from 21 typical B-CLL patients with median age of 72 years were analyzed. There were 9 males and 12 females. Mean beta-2 microglobuin was 4.3mg/l, mean Total Tumor Mass size was 8.9 and mean Tumor Distribution pattern was 0.75. There were 2, 14 and 5 patients in Rai stage 0, I+II and III+IV, respectively. There were 6 previously treated patients (but off therapy 3 months before sampling). The expression level of CD154 was absent/low on T-cells and in 14/21 patients on B-CLL cells. However in 7/21 patients B-CLL cells had higher CD154 expression (“CD154 positive” patients). There was no detectible difference in CD154 expression on T cells between compartments, while on B-CLL cells there was highest expression in lymph nodes and lowest in peripheral blood (p<0.01). CD40 expression on B-CLL cells was significantly higher than CD154, i.e. all cases were positive, and there was no significant difference between lymphoid compartments. There was no significant difference between CD154 positive and negative patients in measured disease parameters. Conclusions: our results show that CD154 expression on T-cells is absent/low and not significantly different between lymphoid compartments regardless of different microenvironment milieu. CD40 expression on B-CLL cells is high and comparable through compartments. In subset of patients there is CD154 positivity on B-CLL cells and shows strong association with lymphoid compartments possibly indicating microenviroment influence on CD154/CD40 system in B-CLL in vivo. These results warrant further studies to indentify the role of CD154 expression on B-CLL cells in pathologic process and its regulation and may eventually uncover novel or modulate existing innovative therapeutic approaches (like gene therapy or immunomodulatory agents like lenalidomide). Disclosures: No relevant conflicts of interest to declare.

3877 B-cell chronic lymphocytic leukemia (B-CLL) is characterized by variable clinical presentation with different involvement of various lymphoid compartments i.e. peripheral blood, bone marrow and lymphoid organs such as lymph nodes and spleen. Also there is a well documented intraclonal and interclonal variability of B-CLL cells in different microenvironments regarding a number of surface and intracellular molecules (for example CD38 and ZAP-70). This variable distribution of tumor mass has strong association with prognosis and a well documented influence on response to antibodies like rituximab and alemtuzumab. There is a well documented efficacy of rituximab in cases with with 11q deletion (associated with significant lymphadenopathy), and known resistance to alemtuzumab in pateints with bulky lymphadenopaty (>5cm). Aim of this study was to evaluate level of expression of CD20 and CD52 along with key chemokine receptor CXCR-4 and proliferation marker Ki-67 on B-CLL lymphocytes and intra and interclonal differences dependent on different microenvironment, ie peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). PB, BM and LN samples were taken by conventional techniques (venepuncture and fine needle aspiration) on the same day. The expression levels of CD20, CD52, CXCR-4 and Ki-67 molecules on CD19+CD5+ B-CLL cells were analyzed by flow cytometry. Results were expressed as mean fluorescence intensity (MFI) and analyzed by paired tests. We have analyzed samples taken from 25 typical B-CLL patients with median age of 72 years. There were 14 males and 11 females. Mean beta-2 microglobulin was 4.3mg/l, mean TTM size was 9.8 and mean TD was 0.74. There were 13, 5 and 7 patients in Binet stage A, B and C, respectively. There were 4 previously treated patients (patients were not treated 3 months before sampling) of whom one patient was previously treated with both rituximab and alemtuzumab. Among included patients there were patients with 11q deletion and with 17p deletion. Median expression level (MFI) of CD52 on B-CLL cells was 171, 193, and 352 for PB, BM and LN respectively (p<0.05) and on T cells was 224, 171 and 137 (p<0.05). Median expression level (MFI) of CD20 on B-CLL cells was 7.5, 5.7 and 4.7 for PB, BM and LN respectively (p<0.05). These results were very consistent in this clinically and cytogenetically heterogenous group of B-CLL patients showing the same pattern in almost all patients. Median expression level (MFI) of CXCR-4 was 9.6, 5.9 and 2.6 (p<0.05) and Ki-67 was 1.08, 1.27 and 1.64 (p<0.05) for PB, BM and LN respectively. There was no correlation of CXCR-4 with CD20 and C52 expression in any compartment and there is positive correlation of Ki-67 with both CD20 and CD52 in PB but not in other compartments. Relatively unexpected results demonstrating the lowest level of expression of CD20 on B-CLL cells in lymph nodes compared to PB and BM and the highest expression of CD52 on B-CLL cells in LN compared to PB and BM (but with the opposite pattern on T cells) is inversely related to known efficacy of agents (ie rituximab and alemtuzumab) targeting these molecules in these lymphoid compartments. These results indicate that other factors in selected microenvironment (beside number of molecules on cell surface) regulate sensitivity of B-CLL cells on rituximab and alemtuzumab in vivo. These may include other cells (like T cells) and soluble factors. Also in this study levels of expression were not clearly related to molecules involved in recirculation (CXCR-4) and proliferation (Ki-67) indicating that other factors in microenvironment may be important for the observed expression levels. These results warrant further studies to indentify these factors which may eventually uncover novel therapeutic targets. Disclosures: No relevant conflicts of interest to declare.