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.

Programmed Cell Death-1 and Its Ligands as Targets for Therapy of Multiple Myeloma Patients.

Purpose: Among hematological malignancies, the expression profile of programmed cell death-1 (PD-1) and its ligands in multiple myeloma (MM) is still debated by numerous research groups. In current study, we characterized the expression of PD-1 and its ligands both on RNA and protein levels in MM patients. We have also attempted to analyze whether daratumumab therapy might overcome CD38-mediated immunosuppression that inhibits in particular CD8+ T-cell function. Patients and Methods: This study included 149 newly diagnosed MM patients and 15 relapsed/refractory MM patients before and after daratumumab treatment. The mRNA levels of PDCD1, PDCD1LG1, PDCD1LG2 and their splicing variants was assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Flow cytometry was used to characterize the surface expression of PD-1 and its ligands on plasma cells, B and T cells. The surface expression of PD-1 on T cells was assessed by flow cytometry before and after daratumumab treatment. Results: The mRNA expression of PDCD1LG1, PDCD1LG2 and their splicing variants were higher in plasma cells as compared to bone marrow mononuclear cells (BMMCs). Our results show that the percentage of plasma cells expressing PD-L1 was significantly higher than plasma cells expressing PD-L2 (p<0.0001) in bone marrow (BM) of MM patients. There was no significant difference between the percentage of plasma cells expressing PD-1 and B cells expressing PD-1 in BM of MM patients (11.19% vs 8.91%). We also found that the percentage of CD8+PD-1+ T cells was significantly higher than CD4+PD-1+T cells in BM (p<0.0001) of MM patients. Here, we observed no change in PD-1 expression on CD4+ and CD8+ T cells after the daratumumab treatment. Conclusion: The PD-1 and its ligands might represent an interesting target for MM immunotherapy, as one would target both malignant plasma cells as well as the immune cells that play a key role in tumor escape mechanisms.

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.

Cofilin-1 Maintains Prosurvival Signaling in Chronic Lymphocytic Leukemia Cells.

BACKGROUND/AIM: Despite numerous studies, the etiology of chronic lymphocytic leukemia (CLL) remains unknown. A hypothesis of autoantigen stimulation in leukemic clone selection might explain 'stereotypy' of B-cell receptors. In healthy cells, cofilin-1 (CFL1) has multiple functions. Its role was described in several malignancies. The aim of this study was characterization of the role of CFL1 in CLL. Materialas and Methods: Cells from peripheral blood of 180 patients and 42 healthy volunteers (HVs) were isolated. Gene expression was assessed with reverse transcription polymerase chain reaction (RT-qPCR); western blot was performed for determination of protein level and activity. After silencing of CFL1 gene, cell ability for migration and chemotaxis was investigated with Transwell method. Post-silencing, apoptosis and cell cycle was determined by flow cytometry. RESULTS: In RT-qPCR, we observed significantly higher expression of CFL1. Higher activity of protein in CLL cells when compared to HVs was detected. Knock-down of CFL1 led to decreased chemotaxis and migration of CLL cells versus cells from HVs. Apoptosis was increased amongst cells with silenced CFL1 and correlated with higher proportion of cells in the G2/M phase. CONCLUSION: Significantly higher expression of CFL1 mRNA in CLL and higher protein activity might indicate high utilization of CFL1 in malignant cells, maintaining their viability, as its inhibition affected viability, cell-cycle progression and motility of leukemia cells.

Expression and Clinical Significance of Neuropilin-1 in Patients With Multiple Myeloma.

BACKGROUND: Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor A (VEGFA), and has been reported to be overexpressed in several malignancies. Since angiogenesis plays an important role in pathogenesis of multiple myeloma (MM) and the role of NRP1 in MM has not been studied yet, we characterized the expression of NRP1 in this disease. MATERIALS AND METHODS: The expression level of NRP1 was measured in 140 patients newly diagnosed with MM and 28 healthy controls by flow cytometry and quantitative reverse transcriptase polymerase chain reaction. RESULTS: Expression of NRP1 was significantly reduced on plasma cells (median=2.05%) compared to that on B-cells (median=10.05%, p<0.0001) in bone marrow of patients with MM. In MM, the expression of NRP1 was high on plasmacytoid dendritic cells (median=85.85%) and low on regulatory T-cells (median=0.6%). CONCLUSION: In MM, NRP1 is regulated differentially as compared to other B-cell malignancies at both the RNA and protein level.

Decreased blood CD4+PD-1+ and CD8+PD-1+ T cells in psoriatic patients with and without arthritis.

INTRODUCTION: Psoriasis with and without arthritis have common immunological mechanisms which among others involve the interactions between cytokines produced by T cells, including Th1, Th17 and Th22. Although quite a lot is known about psoriasis pathogenesis, the cause of chronic immune activation and response in the disease remains unclear. One of the negative regulators of the immune system is programmed death 1 (PD-1). AIM: To assess the expression level of PD-1 in the peripheral T cells of psoriatic patients with and without arthritis. MATERIAL AND METHODS: The study included 23 psoriatic patients with arthritis, 52 psoriatic patients without arthritis and 52 healthy controls. The percentages of CD3+, CD4+, CD8+, CD4+PD-1+ and CD8+PD-1+ T cells were analyzed using flow cytometry. RESULTS: The percentages of CD4+PD-1+ as well as CD8+PD-1+ T cells in the psoriatic patients both with and without arthritis were significantly lower than in the control group. The percentages of CD4+PD-1+ as well as CD8+PD-1+T cells were not significantly different between the psoriatic patients with and without arthritis. A significant positive correlation between PD-1 expression on the CD4+ and CD8+ T cells was found in the psoriatic patients without arthritis. CONCLUSIONS: Impairment of the negative co-stimulation from PD-1 may be another common characteristic of psoriasis both with and without arthritis.

The expression of selected molecular markers of immune tolerance in psoriatic patients.

BACKGROUND: Psoriasis is a chronic autoinflammatory disease whose underlying molecular mechanisms remain unclear. The disease is mediated by the cells and molecules of both the innate and adaptive immune systems. Some T cell surface molecules, including neuropilin-1 (NRP1), programmed death 1 (PD-1) and the human leukocyte antigen G (HLA-G), are known to play a role in the maintenance of immune tolerance. OBJECTIVES: The aim of this study was to investigate HLA-G, NRP1 and programmed cell death gene (PDCD1) mRNA expression in psoriatic patients. MATERIAL AND METHODS: The study included 72 psoriatic patients and 35 healthy individuals. Twentyone patients (29.17%) suffered from concomitant psoriatic arthritis. The mRNA expression of HLA-G, NRP1, and PDCD1 were determined using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The severity of skin lesions was assessed by means of the Psoriasis Area and Severity Index (PASI), Body Surface Area (BSA), the Patient Global Assessment (PGA), and the Dermatology Life Quality Index (DLQI). RESULTS: The median value of the PASI was 11.5, and of BSA was 15.8%. The expressions of NRP1 and PDCD1, but not HLA-G, were significantly lower in psoriatic patients in comparison with the control group. The expression of HLA-G, NRP1 and PDCD1 were not significantly different in the psoriatic arthritis and psoriasis vulgaris patients. CONCLUSIONS: The results of this study suggest that the molecular markers of immune tolerance, i.e., HLA-G, NRP1, and PD-1, may be involved in the immune response in psoriatic patients.

Suppressed Programmed Death 1 Expression on CD4+ and CD8+ T Cells in Psoriatic Patients.

Psoriasis is a chronic inflammatory disease mediated by T cell immunity. Programmed death 1 (PD-1), a coinhibitory receptor, plays an important role in immune regulation and maintaining peripheral tolerance. The aim of the study was to compare the expression of PD-1 on the peripheral T cells between psoriatic patients and healthy controls. The study included 75 psoriatic patients and 52 healthy volunteers. The percentages and absolute numbers of CD3+, CD4+, CD8+, CD4+PD-1+, and CD8+PD-1+ T cells were analyzed using flow cytometry. The absolute numbers and percentages of CD4+PD-1+ and CD8+PD-1+ T cells were significantly decreased in the psoriatic patients in comparison with the control group. No significant correlations were found between the absolute numbers and percentages of CD4+PD-1+ or CD8+PD-1+ T cells and clinical characteristics of psoriasis. Decreased PD-1 expression on the T cells may be responsible for impaired negative regulation of immune response in psoriasis pathogenesis.

Differential expression of programmed death 1 (PD-1) on CD4+ and CD8+ T cells in rheumatoid arthritis and psoriatic arthritis.

INTRODUCTION    Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are characterized by chronic inflammatory processes mediated by proinflammatory cytokines that affect the synovial lining. Programmed death 1 (PD­1) is a critical regulator of T­cell activation by downregulating immune responses.  OBJECTIVES    The aim of the study was to investigate whether the expression of PD­1 on CD4+ and CD8+ T cells differs between patients with RA and those with PsA. PATIENTS AND METHODS    The study included 100 patients with RA, 31 patients with PsA, and 52 healthy controls. The percentages, absolute numbers, and mean fluorescence intensity (MFI) of CD4+PD­1+ and CD8+PD­1+T cells from peripheral blood were analyzed using flow cytometry. RESULTS    The percentages and absolute numbers of CD4+ and CD8+ T cells with PD­1 expression were significantly higher in patients with RA than in controls. In patients with PsA, the percentages of CD4+PD­1+ and CD8+PD­1+ T cells were significantly lower than in controls. Because of the high frequency of PD-1­positive T cells in RA and their low frequency in PsA, we analyzed the expression level by analyzing the MFI. The median MFI of PD­1 on CD4+ and CD8+ T cells was significantly higher in patients with RA (median, 421 and 437, respectively) in comparison with patients with PsA (median, 222 and 198, respectively) and controls (median, 205 and 187, respectively). CONCLUSIONS    The differential expression of PD­1 in RA and PsA suggests that PD­1 might be involved in autoimmune mechanisms in RA and autoinflammatory mechanisms in PsA in a different manner.

Novel prognostic molecular factors: a quantum leap in the field of chronic lymphocytic leukemia.

Cytogenetic lesions do not completely explain clinical heterogeneity of chronic lymphocytic leukemia (CLL). The 2016 revision of the World Health Organization classification 2008 indicated that molecular lesions of TP53, NOTCH1, SF3B1 and BIRC3 have potential clinical relevance and could be integrated into an updated risk profile. The negative clinical implications of TP53 disruptions are well constituted and patients with these mutations should be considered for novel, small molecule signal transduction inhibitors therapies. Mutations of NOTCH1, SF3B1 and BIRC3 are associated with poor prognosis. Patients with mutated SF3B1 or NOTCH1 genes present shorter time to first treatment compared to unmutated group. NOTCH1 mutations are related to a high risk of Richter's syndrome transformation, especially in case of TP53 disruptions' coexistence. Large studies on MYD88 mutations in CLL have not explained clearly their clinical importance.The aim of this paper is to provide a comprehensive review on novel molecular aberrations identified in CLL.

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.