Programmed death-1 (PD-1) is an immunoreceptor predominantly expressed on exhausted T cells, which through an interaction with its ligand (PD-L1), controls peripheral tolerance by limiting effector functions of T lymphocytes. qRT-PCR for PD-1, PD-L1 and their splicing forms as well as flow cytometric assessment of surface expression was performed in a cohort of 58 chronic lymphocytic leukemia (CLL) patients. In functional studies, we assessed the influence of the proliferative response of leukemic B-cells induced by IL-4 and CD40L on PD-1 transcripts and expression on the protein level. The median level of PD-1, but not PD-L1, transcripts in CLL patients was higher in comparison to healthy volunteers (HVs, n = 43, p = 0.0057). We confirmed the presence of PD-1 and PD-L1 on the CLL cell surface, and found the expression of PD-1, but not PD-L1, to be higher among CLL patients in comparison to HVs (47.2% vs. 14.8%, p<0.0001). The Kaplan-Meier curves for the time to progression and overall survival in groups with high and low surface expression of PD-1 and PD-L1 revealed no prognostic value in CLL patients. After stimulation with IL-4 and CD40L, protein expression of PD-1 was significantly increased in samples that responded and up-regulated CD38. PD-1, which is aberrantly expressed both at mRNA and cell surface levels in CLL cells might represent a novel immunotolerant molecule involved in the pathomechanism of the disease, and could provide a novel target for future therapies.

Recently novel treatment modalities has focused on targeted therapies. Tyrosine kinases represent a good target for cancer treatment since they are involved in transferring phosphate groups from ATP to tyrosine residues in specific substrate proteins transducing intracellular signals engaged in the many mechanisms, playing an important role in the modulation of growth factors signaling that are strongly related to carcinogenesis. Deregulation of tyrosine kinases activity was also found in hematological malignancies, particularly overexpression of tyrosine kinases was observed in chronic myeloid leukemia or acute lymphoblastic leukemia. Herein we show that tyrosine kinase inhibitors have revolutionized hematology malignancies therapy in a very short period of time and they still remain one of the most interesting anticancer compounds that could give a hope for cure and not only long-lasting complete remission. This manuscript summarizes current view on the first generation tyrosine kinase inhibititor--imatinib, second generation--dasatinib, nilotinib and bosutnib as well as new generation tyrosine kinase inhibititors--ponatinib and danusertib in hematooncology.

Background. Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in western civilization. The accumulation of CD5 + CD19 + B lymphocytes in peripheral blood is due to a defect in the apoptotic pathway rather than excessive proliferation in the bone marrow and lymph nodes. Despite a number of treatments, CLL remains an incurable disease. Valproic acid (VPA) activity, as a histone deacetylase inhibitor, could restore the epigenetic changes underlying the pathogenesis of CLL and thus induce cell death. Objectives. In the present study we hypothesized that VPA could induce CLL primary cells death through activation of apoptosis. Material and Methods. Peripheral blood samples were obtained from 53 CLL patients. Peripheral blood mononuclear cells were isolated through density gradient centrifugation and were the subject of a 24-hour cell culture with 10 mM of VPA. The cytotoxic effect of VPA was evaluated with an XTT test and thereafter confirmed using Annexin V-FITC/PI staining and flow cytometry techniques. Results. In this study, a median VPA cytotoxicity of 13.88% with a range of 0-54.65% was observed. Annexin V/PI staining confirmed that the demonstrated cytotoxicity was caused by increased apoptosis in the VPA treated cells as compared to control cells. Statistical analysis showed that VPA's effect on CLL cells depends on lactate dehydrogenase serum levels, but is independent of all other prognostic markers. Conclusions. The results of the present experiments found that VPA at a clinically applicable concentration significantly induces apoptosis independently of the disease stage and might be a valuable therapeutic agent for all CLL patients (Adv Clin Exp Med 2015, 24, 1, 55-62).

Dasatinib inhibits the breakpoint cluster region-Abelson murine leukemia 1 ( BCR-ABL1 ) gene along with other kinases known to be overexpressed and abnormally active in patients with chronic lymphocytic leukemia (CLL). The current study used primary leukemic cells obtained from 53 patients with CLL that were treated with dasatinib. A 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay and Annexin V staining was performed to assess the cytotoxic effects of dasatinib treatment. The XTT assay revealed that the median cytotoxicity of dasatinib was 8.30% (range, 0.00–77.89%). Due to high dispersion of dasatinib activity, patients were divided into sensitive (n=27; 50.94%; median cytotoxicity, 22.81%) and resistant groups (n=26; 49.06%; median cytotoxicity, 0.00%). A median cytotoxicity of 8.30% was selected as a cut off value. Using Annexin V staining and flow cytometry on exemplary sensitive and resistant CLL samples, it was revealed that 17.71 and 1.84% of cells were apoptotic, respectively. The current study presented a case of a patient with concomitant occurrence of CLL and chronic myeloid leukemia (CML) with a major molecular response after dasatinib treatment. A simultaneous reduction of circulating CLL cells indicated in vivo anti-CLL activity induced by dasatinib. After an in vitro culture of the patient's mononuclear cells with subsequent dasatinib treatment, a higher percentage of CLL cells undergoing apoptosis was obsevered when compared with untreated samples (38.19 vs. 21.99%, respectively). Similarly, the percentage of CLL apoptotic cells (ΔΨm low ) measured by chloromethyl-X-rosamine was higher after incubation with dasatinib (7.28%) than in the negative control (2.86%). In conclusion, dasatinib induced antileukemic effects against CML and CLL cells. The results of the current study indicated that dasatinib may induce apoptosis ex vivo, in vitro and in vivo in CLL.

4608 In chronic lymphocytic leukemia (CLL) thalidomide was found to significantly decrease the percentage of regulatory T cells (Tregs) as well the number of CLL cells in vivo. In combination with fludarabine, thalidomide was effective both in refractory/relapse and naïve CLL patients. In our recent clinical trial, we also observed a reduction of vascular endothelial growth factor (VEGF) levels during therapy that were correlated with the reduction of Tregs (r2=0.47, p<0.05). Furthermore, gene expression profiles associated with thalidomide response in CLL revealed several genes involved in angiogenesis (Giannopoulos et al. Leukemia 2009). To further characterize the thalidomide mechanism of action in CLL we assessed expression of neuropilin 1 (NRP1), which is a membrane-bound coreceptor to the tyrosine kinase receptor for both vascular endothelial growth factor (VEGF) and semaphorin (SEMA3A) family members. NRP1 plays versatile roles in angiogenesis, cell survival, migration, and invasion. Recently, NRP1 was also found expressed on plasmacytoid dendritic cells (PDC) as well as on Tregs, two immune cell subpopulations involved in tolerance mechanisms commonly deregulated in tumorigenesis. Our analysis showed NRP1 expression on CLL cells, Tregs and PDC of 38 CLL patients. Using five parameter flow cytometry we found increased expression of NRP1 in CLL when compared to cells derived from healthy volunteers. NRP1 expression was 22.7% on CD5+CD19+ CLL cells vs. 6.2% on CD19+ B cells from controls, p=0.03. Furthermore, we found expression of NRP1 on Tregs as well as PDC with a median expression of NRP1 on Tregs of 42.6 % (range: 10 – 100%). NRP1 was expressed on almost all PDC with a median expression of 100% (range: 98.2 – 100%). In functional studies, we found that NRP1 expression might be regulated by VEGF expression levels. Magnetically separated CLL cells increased expression of NRP1 after cell culture with VEGF. Here, VEGF levels of 0.1 – 0.5ng/ml, which are also observed in primary CLL patient samples, effectively induced expression of NRP1. In accordance, we also observed that VEGF upregulates NRP1 expression on magnetically separated Tregs. However, higher VEGF concentrations inhibited NRP1 expression in CLL cells probably due to a negative feedback loop. In conclusion, we found expression of NRP1 on CLL cells, Tregs as well as PDC in patients with CLL, and we could demonstrate that the expression of NRP1 is regulated by VEGF expression levels. Thus, our previously observed thalidomide-induced reduction of VEGF levels along with a reduced percentage of Tregs in CLL might in part be explained by down-regulation of the NRP1 expression. Disclosures: Stilgenbauer: Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Travel Grants.