Joanna Zaleska

Programmed Death-1 and Its Ligand Are Novel Immunotolerant Molecules Expressed on Leukemic B Cells in Chronic Lymphocytic Leukemia

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.

Expression of Programmed Death 1 Ligand in Different Compartments of Chronic Lymphocytic Leukemia.

Background: The programmed death 1 (PD-1) receptor pathway is responsible for the negative regulation of both T and B lymphocytes upon activation of these cells. There is growing evidence that chronic lymphocytic leukemia (CLL) cells exploit the PD-1 ligand (PD-L1) to resist antitumor immune reactions and maintain their survival by shaping their own microenvironment. Methods: We used a quantitative RT-PCR method to analyze PD-L1 gene expression in bone marrow and peripheral blood mononuclear cells, representing the proliferation and accumulation compartments of CLL. Results: PD-L1 expression was found to be significantly higher in 112 CLL patients than in controls. Levels of PD-L1 expression in bone marrow and peripheral blood were comparable and showed a positive correlation. Furthermore, expression of PD-L1 strongly correlated with expression of PD-1 receptor in mononuclear cells from the same compartment, and was not affected by incubation with immunomodulatory drug thalidomide. Conclusion: PD-L1 expression is shared between CLL cells localized in distinct disease compartments, demonstrating that PD-1/PD-L1 a universal target for therapy.

The function of a novel immunophenotype candidate molecule PD-1 in chronic lymphocytic leukemia

Programmed death-1 (PD-1) is a negative receptor expressed on lymphocytes including malignant B cells in chronic lymphocytic leukemia (CLL). In this work, we found that patients with CLL had a higher expression of PD-1 transcript (PDCD1) than healthy volunteers (p < 0.0001). PDCD1 expression was comparable between CLL cells from accumulation (peripheral blood) and proliferation (bone marrow) disease compartments. In blood samples of patients with mutated IGHV genes PDCD1 expression was higher than with unmutated IGHV (p = 0.0299). We demonstrated that phosphorylation of SYK and LYN, key B-cell receptor signaling kinases, was independent of PD-1 expression in patients with CLL, while ZAP-70 phosphorylation in negative tyrosine residue 292 showed strong inverse correlation (r = − 0.8, p = 0.0019). No associations between five single nucleotide polymorphisms of PDCD1, their expressions and susceptibility to CLL were found. In conclusion, PD-1 might be an independent, universal marker of CLL cells and a part of their activated phenotype, and subsequently might modulate the function of ZAP-70.

Analysis of NPM1 splice variants reveals differential expression patterns of prognostic value in acute myeloid leukemia

Mutations of the nucleophosmin-1 (NPM1) gene in cytogenetically normal (CN) acute myeloid leukemia (AML) identify a group of patients with more favorable prognosis. NPM1 encodes three main alternatively spliced isoforms R1(B23.1), R2(B23.2), and R3(B23.3). The expression of splice variants R1, R2 and R3 were higher in AML patients compared to normal cells of healthy volunteers (HVs), although RNA-seq analysis revealed enhanced R2 expression also in less differentiated cells of HVs as well as in AML cells. The variant R2, which lacks exons 11 and 12 coding for the nucleolar localization domain, might behave similar to the mutant form of NPM1 (NPM1mut). In accordance, in CN-AML high R2 expression was associated with favorable impact on outcome. Moreover, functional studies showed nucleolar localization of the eGFP-NPM1 wildtype and cytoplasmic localization of the eGFP-NPM1 mut protein. While the eGFP-NPM1 R2 splice variant localized predominantly in the nucleoplasm, we also could detect cytoplasmic expression for the R2 variant. These results support a unique biological consequence of R2 overexpression and in part explain our clinical observation, where that high R2 variant expression was associated with a better prognosis in CN-AML patients.

Specific cytotoxic T-cell immune responses against autoantigens recognized by chronic lymphocytic leukaemia cells

Mounting evidence suggests that autoreactivity and inflammatory processes are involved in the pathogenesis of chronic lymphocytic leukaemia (CLL). Cytoskeletal proteins, including non‐muscle myosin heavy chain IIA (MYHIIA), vimentin (VIM) and cofilin‐1 (CFL1), exposed on the surface of apoptotic cells have been identified as autoantigens that are recognized by the specific B‐cell receptors of the CLL cells. In 212 CLL patients analysed with quantitative reverse transcriptase‐polymerase chain reaction

Indirect induction of regulatory T cells accompanies immune responses during peptide vaccination of chronic lymphocytic leukaemia patients.

Keywords: chronic lymphocytic leukaemia; thalidomide; T regulatory cells; interleukin 2; peptide vaccination

Cytotoxic Activity of Valproic Acid on Primary Chronic Lymphocytic Leukemia Cells

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 VPAs 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.

Expression of CD274 (PD-L1) is associated with unfavourable recurrent mutations in AML

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Immunotherapeutical approaches for multiple myeloma

ABSTRACT Multiple myeloma (MM) is characterized by deep immunodeficiency caused by many factors including uncontrolled production of monoclonal immunoglobulin and inhibitory effect of microenvironment. Despite the use of different therapeutic strategies as drug treatment, chemotherapy and stem cell transplantation still remains an incurable disease. Novel treatment modalities introduced for MM significantly increased overall survival, but it still reaches not more than 4 years. Therefore there is a necessity to generate novel therapeutical strategies that successfully improve quality of MM patients life and cause complete recovery. Tumor-associated antigens (TAA) are potential targets for cancer immunotherapy due to their limited expression on normal tissues or restriction to tumor cells. Epitopes derived from TAA induce specific, cytotoxic T lymphocytes which are able to recognize and eradicate myeloma cells with good efficacy. These features allow to construct various types of peptide-based vaccines, which could prolong MM patients life and lead to complete remission. In this work we have characterized (C/T), human telomerase reverse transcriptase (hTERT), X-box binding protein 1 (XBP-1), PAS domain-containing protein 1 (PASD-1), receptor for hyaluronic acid-mediated motility (RHAMM), mucin 1 (MUC-1), Wilms Tumor-1 (WT1), that might represent a target for peptide-based immunotherapy. Results from first clinical trials on immunotherapy in MM were also characterized.