High Level of CD8+PD-1+ Cells in Patients with Chronic Myeloid Leukemia Who Experienced Loss of MMR after Imatinib Discontinuation.

Treatment-free remission (TFR) is achieved in approximately half of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The mechanisms responsible for TFR maintenance remain elusive. This study aimed to identify immune markers responsible for the control of residual CML cells early in the TFR (at 3 months), which may be the key to achieving long-term TFR and relapse-free survival (RFS) after discontinuation of imatinib. Our study included 63 CML patients after imatinib discontinuation, in whom comprehensive analysis of changes in the immune system was performed by flow cytometry, and changes in the BCR::ABL1 transcript levels were assessed by RQ-PCR and ddPCR. We demonstrated a significant increase in the percentage of CD8+PD-1+ cells in patients losing TFR. The level of CD8+PD-1+ cells is inversely related to the duration of treatment and incidence of deep molecular response (DMR) before discontinuation. Analysis of the ROC curve showed that the percentage of CD8+PD-1+ cells may be a significant factor in early molecular recurrence. Interestingly, at 3 months of TFR, patients with the e13a2 transcript had a significantly higher proportion of the PD-1-expressing immune cells compared to patients with the e14a2. Our results suggest the important involvement of CD8+PD-1+ cells in the success of TFR and may help in identifying a group of patients who could successfully discontinue imatinib.

Response to anti-SARS-CoV-2 mRNA vaccines in multiple myeloma and chronic lymphocytic leukemia patients.

Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) patients have increased morbidity and mortality rates of COVID-19 due to immunosuppression associated with the disease and ongoing therapy. The same immune impairment accompanying CLL and MM also affects suboptimal vaccine response. The study assessed the effectiveness of the humoral and T cell-mediated immunity following mRNA COVID-19 vaccination (using either BNT162b2 or mRNA-1273) in short-term (2-5 weeks after second dose) and long-term follow-up (12 weeks after vaccination). Between March and August 2021, blood samples were obtained from 62 CLL and 60 MM patients from eight different hematology departments in Poland. Total anti-RBD antibodies were detected in 37% MM patients before vaccination, increased to 91% and 94% in short- and long-term follow-up, respectively. In CLL, serological responses were detectable in 21% of patients before vaccination and increased to 45% in the short-term and 71% in long-term observation. We detected a tendency to higher frequencies of specific CD8+ T cells against SARS-CoV-2 after vaccination compared to samples before vaccination in MM patients and no changes in frequencies of specific T cells in CLL patients. Our study provides novel insights into mRNA vaccination efficacy in immunocompromised MM and CLL patients, and our findings highlight that specific CD8+ T cells against SARS-CoV-2 might be induced by vaccination but do not correlate positively with serological responses.

The role of NPM1 alternative splicing in patients with chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with heterogeneous clinical course. Recent studies revealed a link between NOTCH1 mutation and the overexpression of MYC and MYC-related genes involved in ribosome biogenesis and protein biosynthesis, such as nucleophosmin-1 (NPM1), in CLL cells. In the present study, we aim to evaluate the impact of the NOTCH1 mutation on the MYC and MYC induced NPM1 expression in CLL cells via quantification of their transcripts. METHODS: Using qRT-PCR, we analyzed the levels of MYC and three main NPM1 splice variants in 214 samples collected from CLL patients. We assessed the impact of each splice variant on CLL prognostic markers, including the IGHV, TP53, NOTCH1, SF3B1, and MYD88 mutational status, cytogenetic aberrations, and laboratory features. RESULTS: Significantly higher levels of NPM1.R1 transcripts in patients with unmutated compared to mutated IGHV status were found. The median time to first treatment (TTFT) in patients with a high level of NPM1.R1 was significantly shorter compared to the group with low NPM1.R1 levels (1.5 vs 33 months, p = 0.0002). Moreover, in Multivariate Cox Proportional Hazard Regression Model NPM1.R1 splice variant provided an independent prognostic value for TTFT. CONCLUSION: In conclusion, our study indicates the prognostic significance of the level of NPM1.R1 expression and suggests the importance of splicing alterations in the pathogenesis of CLL.

Harmonization of Flow Cytometric Minimal Residual Disease Assessment in Multiple Myeloma in Centers of Polish Myeloma Consortium.

Minimal residual disease (MRD) status is now considered as one of the most relevant prognostic factors in multiple myeloma (MM) while MRD negativity became an important endpoint in clinical trials. Here, we report the results of the first study evaluating the reproducibility of high-sensitivity flow cytometry MM MRD assessment in four laboratories in Poland. EuroFlow protocols for instrument setting standardization and sample preparation in MM MRD assessment were implemented in each laboratory. In the inter-laboratory reproducibility study, 12 bone marrow samples from MM patients were distributed and processed in participant laboratories. In the inter-operator concordance study, 13 raw data files from MM MRD measurements were analyzed by five independent operators. The inter-laboratory study showed high 95% overall concordance of results among laboratories. In the inter-operator study, 89% of MRD results reported were concordant, and the highest immunophenotype interpretation differences with regard to expression of CD27, CD45, CD81 were noticed. We confirmed the applicability and feasibility of the EuroFlow protocol as a highly sensitive method of MRD evaluation in MM. Results of our inter-center comparison study demonstrate that the standardization of MM MRD assessment protocols is highly desirable to improve quality and comparability of results within and between different clinical trials.

Differential Function of a Novel Population of the CD19+CD24hiCD38hi Bregs in Psoriasis and Multiple Myeloma.

Psoriasis (Ps), an autoimmune disease, and multiple myeloma (MM), a blood neoplasm, are characterized by immune dysregulation resulting from the imbalance between the effector and regulatory cells, including B regulatory (Breg) lymphocytes. Peripheral blood samples from 80 Ps patients, 17 relapsed/refractory MM patients before and after daratumumab (anti-CD38 monoclonal antibody) treatment, 23 healthy volunteers (HVs), and bone marrow samples from 59 MM patients were used in the study. Bregs were determined by flow cytometry using CD19, CD24, and CD38. Intracellular production of interleukin-10 (IL-10) was assessed by flow cytometry after CD40L, LPS, and CpG stimulation. IL-10 serum or plasma concentrations were tested using ELISA method. The percentage of CD19+CD24hiCD38hi Bregs was not different whereas the production of IL-10 in Bregs was significantly higher in Ps patients in comparison with HVs. The percentage of CD19+CD24hiCD38hi Bregs in MM patients was significantly higher than in HVs (p < 0.0001). The percentage of CD19+CD24hiCD38hi Bregs was significantly higher in MM patients with the ISS stage I (p = 0.0233) while IL-10 production in Bregs was significantly higher in ISS stage III (p = 0.0165). IL-10 serum or plasma concentration was significantly higher in Ps and MM patients when compared to HVs (p < 0.0001). Following the treatment with daratumumab the percentages of CD19+CD24hiCD38hi Bregs significantly decreased (p < 0.0003). Here, in the two opposite immune conditions, despite the differences in percentages of Bregs in Ps and MM we have identified some similarities in the IL-10 producing Bregs. Effective treatment of daratumumab besides the anti-myeloma effect was accompanied by the eradication of Bregs.

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.

Prognostic impact of NOTCH1, MYD88, and SF3B1 mutations in Polish patients with chronic lymphocytic leukemia.

INTRODUCTION    Currently available prognostic factors determining the course of chronic lymphocytic leukemia (CLL) are not fully efficient, especially for newly diagnosed patients. Investigation of molecular changes may help clarify the reasons for the heterogeneity of the disease. Apart from already confirmed TP53 mutations, the novel candidates: NOTCH1, SF3B1, and MYD88 might represent clinically relevant biomarkers. OBJECTIVES    The aim of this study was to evaluate the mutational status of NOTCH1, MYD88, and SF3B1 and to compare the results with confirmed prognostic factors: ZAP­70, CD38, and immunoglobulin heavy­chain variable region (IGHV) mutation in CLL. The study assessed also prognostic significance in terms of the time to first treatment (TTFT) and subset analysis. PATIENTS AND METHODS The study was conducted on samples of 370 newly diagnosed patients with CLL. The analysis was performed using high­resolution melting, Sanger sequencing, and polymerase chain reaction methods. RESULTS    Patients harboring the NOTCH1 mutation were significantly more often found among patients with an unmutated IGHV gene status and high expression of CD38 and ZAP­70. The MYD88 mutation was equally distributed in patients with mutated and unmutated IGHV status (5 vs 7 patients). For MYD88 and SF3B1, there were no significant differences in the levels of CD38 and ZAP­70 expression. The tendency for lower median TTFT was revealed in patients with mutated SF3B1 (P = 0.08). The analysis showed the presence of 14 different types of the subsets of IGHV in 50 of 345 patients (14.5%). The most frequent were subsets #1 and #2. CONCLUSIONS    The NOTCH1 and SF3B1 mutations accompany biological markers of unfavorable prognosis in patients with CLL. The mutations may contribute to the identification of patients with high­risk CLL.

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 we found CFL1 overexpression and low expression of MYH9 in comparison with healthy volunteers. We detected specific cytotoxic immune responses for peptides derived from MYHIIA in 66·7%, VIM in 87·5% and CFL1 in 62·5% CLL patients in an Enzyme-Linked ImmunoSpot assay. Low frequencies of autoreactive peptide-specific T cells were detected against MYHIIA, VIM and CFL1 in CLL patients ex vivo; most of the detected cells had an effector-memory phenotype. Our findings support the existence of cytotoxic immune responses against three autoantigens that have been identified as targets of CLL clonotypic B-cell receptors. The presence of autoreactive CD8(+) T cells against MYHIIA, VIM and CFL1 in CLL patients indicates the involvement of antigen-specific autoreactive T cells in the pathogenesis of CLL.

Accumulation of CD5+CD19+ B lymphocytes expressing PD-1 and PD-1L in hypertrophied pharyngeal tonsils.

Programmed death-1 (PD-1) is one of the most important inhibitory co-receptors expressed predominantly on activated T and B lymphocytes whose expression could be sustained by permanent antigenic stimulation accompanying chronic or recurrent tonsillitis. The expression of PD-1 and PD-1L was analyzed using flow cytometry on hypertrophied tonsils collected from 57 children. We observed high expression of PD-1 and PD-1L on certain lymphocytes subpopulations of hypertrophied tonsils; among T cells, the expression of PD-1 on protein level was higher on CD4+ cells (70.3 %) than on CD8+ cells (35 %). Interestingly, a limited expression of PD-1 was observed on CD19+ B lymphocytes (6.5 %), while CD5+CD19+ B cells overexpressed PD-1 (52.5 %). Moreover, the expression of PD-1L was also higher on CD5+CD19+ B cells (16.5 %) than on CD19+ B cells (3.5 %) and on CD4+ T cells (20 %) than on CD8+ T cells (10 %). PD-1 and PD-1L expressions correlated only on CD5+CD19+ cells. In conclusion, high expression of PD-1 and PD-1L on T and B cells could represent hallmark of immune system adaptation to chronic antigenic exposition in patients with tonsillitis.

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 PDL1 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.

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 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.

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.

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.