JAK2V617F mutation and circulating extracellular vesicles in essential thrombocythemia.

The clinical course of essential thrombocythemia (ET) is complicated with thrombosis which significantly impacts patients' mortality. Studies have identified JAK2V617F mutation as an independent risk factor for thrombosis. Circulating extracellular vesicles (EVs) were evaluated in several studies regarding myeloproliferative neoplasms and thrombosis as potential biomarkers. The present study investigates the relationship between JAK2V617F mutation and EVs levels in 119 ET patients. Our analyses revealed that JAK2V617F-positive patients are at a significantly increased risk of thrombosis within five years before the ET diagnosis (hazard ratio [95% CI]: 11.9 [1.7-83.7], P = 0.013), and that JAK2V617F mutation is an independent risk factor for thrombosis at ET diagnosis or during the follow-up (hazard ratio [95% CI]: 3.56 [1.47-8.62], P = 0.005). ET patients have higher levels of platelet-EVs, erythrocyte-EVs and procoagulant activity of EVs than the healthy population. Absolute and relative counts of platelet-EVs are increased in the presence of JAK2V617F mutation (P = 0.018, P = 0.024, respectively). In conclusion, our results support the role of JAK2V617F mutation in the pathogenesis of thrombosis in essential thrombocythemia through enhancing platelet activation.

More Than 2% of Circulating Tumor Plasma Cells Defines Plasma Cell Leukemia-Like Multiple Myeloma.

PURPOSE: Primary plasma cell leukemia (PCL) is the most aggressive monoclonal gammopathy. It was formerly characterized by ≥ 20% circulating plasma cells (CTCs) until 2021, when this threshold was decreased to ≥ 5%. We hypothesized that primary PCL is not a separate clinical entity, but rather that it represents ultra-high-risk multiple myeloma (MM) characterized by elevated CTC levels. METHODS: We assessed the levels of CTCs by multiparameter flow cytometry in 395 patients with newly diagnosed transplant-ineligible MM to establish a cutoff for CTCs that identifies the patients with ultra-high-risk PCL-like MM. We tested the cutoff on 185 transplant-eligible patients with MM and further validated on an independent cohort of 280 transplant-ineligible patients treated in the GEM-CLARIDEX trial. The largest published real-world cohort of patients with primary PCL was used for comparison of survival. Finally, we challenged the current 5% threshold for primary PCL diagnosis. RESULTS: Newly diagnosed transplant-ineligible patients with MM with 2%-20% CTCs had significantly shorter progression-free survival (3.1 v 15.6 months; P < .001) and overall survival (14.6 v 33.6 months; P = .023) than patients with < 2%. The 2% cutoff proved to be applicable also in transplant-eligible patients with MM and was successfully validated on an independent cohort of patients from the GEM-CLARIDEX trial. Most importantly, patients with 2%-20% CTCs had comparable dismal outcomes with primary PCL. Moreover, after revealing a low mean difference between flow cytometric and morphologic evaluation of CTCs, we showed that patients with 2%-5% CTCs have similar outcomes as those with 5%-20% CTCs. CONCLUSION: Our study uncovers that ≥ 2% CTCs is a biomarker of hidden primary PCL and supports the assessment of CTCs by flow cytometry during the diagnostic workup of MM.

Involvement of Small Non-Coding RNA and Cell Antigens in Pathogenesis of Extramedullary Multiple Myeloma.

Extramedullary multiple myeloma (EMD) is an aggressive disease; malignant plasma cells lose their dependence in the bone marrow microenvironment and migrate into tissues. EMD is a negative prognostic factor of survival. Using flow cytometry and next-generation sequencing, we aimed to identify antigens and microRNAs (miRNAs) involved in EMD pathogenesis. Flow cytometry analysis revealed significant differences in the level of clonal plasma cells between MM and EMD patients, while the expression of CD markers was comparable between these two groups. Further, miR-26a-5p and miR-30e-5p were found to be significantly down-regulated in EMD compared to MM. Based on the expression of miR-26a-5p, we were able to distinguish these two groups of patients with high sensitivity and specificity. In addition, the involvement of deregulated miRNAs in cell cycle regulation, ubiquitin-mediated proteolysis and signaling pathways associated with infections or neurological disorders was observed using GO and KEGG pathways enrichment analysis. Subsequently, a correlation between the expression of analyzed miRNAs and the levels of CD molecules was observed. Finally, clinicopathological characteristics as well as CD antigens associated with the prognosis of MM and EMD patients were identified. Altogether, we identified several molecules possibly involved in the transformation of MM into EMD.

The Clinical Significance of Circulating Microparticles Concerning Thrombosis in BCR/ABL1-negative Myeloproliferative Neoplasms.

BACKGROUND/AIM: This work aimed to prospectively evaluate the clinical significance of circulating microparticles (MPs) in relation to thrombotic risk factors and thrombotic complications in patients with BCR/ABL1-negative myeloproliferative neoplasms (MPN). PATIENTS AND METHODS: In a cohort of 206 patients with MPN, MPs' procoagulant activity was measured by the Zymuphen functional assay in 429 samples, while platelet- and erythrocyte-MPs were enumerated by flow cytometry in 558 samples. RESULTS: MPN patients had higher MP levels than the control group. The levels of MPs were higher in male patients, smokers, and those who were older than 60 years, and in the presence of JAK2V617F mutation, history of thrombosis, platelets >400×109/l, hematocrit >45%, or leukocytes >10×109/l. Cytoreductive treatment reduced MP levels, with anagrelide being associated with lower MP levels than hydroxyurea. CONCLUSION: The relationship with thrombotic risk factors indicates a possible role of MPs in the complex thrombotic mechanism, though cytoreductive treatment seems to affect this role through reducing MP levels.

Necessity of flow cytometry assessment of circulating plasma cells and its connection with clinical characteristics of primary and secondary plasma cell leukaemia.

Plasma cell leukaemia (PCL) is a rare and very aggressive plasma cell disorder. Preventing a dismal outcome of PCL requires early diagnosis with appropriate analytical tools. Therefore, the investigation of 33 patients with primary and secondary PCL was done when the quantity of circulating plasma cells (PCs) using flow cytometry (FC) and morphology assessment was evaluated. The phenotypic profile of the PCs was also analysed to determine if there is an association with clinical outcomes and to evaluate the prognostic value of analysed markers. Our results revealed that FC is an excellent method for identifying circulating PCs as a significantly higher number was identified by FC than by morphology (26·7% vs. 13·5%, P = 0·02). None of secondary PCL cases expressed CD19 or CD20. A low level of expression with similar positivity of CD27, CD28, CD81 and CD117 was found in both PCL groups. A decrease of CD44 expression was detected only in secondary PCL. Expression of CD56 was present in more than half of PCL cases as well as cytoplasmic nestin. A decreased level of platelets, Eastern Cooperative Oncology Group score of 2-3 and lack of CD20+ PC were associated with a higher risk of death. FC could be incorporated in PCL diagnostics not only to determine the number of circulating PCs, but also to assess their phenotype profile and this information should be useful in patients' diagnosis and possible prognosis.

Plasmacytoid Dendritic Cells in Patients with MGUS and Multiple Myeloma.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) play prominent roles in mediating innate and adaptive immune responses. However, it is unclear how pDCs contribute to the immunosuppressive tumor microenvironment described in multiple myeloma (MM). METHODS: Newly diagnosed myeloma patients (MM, n = 37) were analyzed to determine the pDC counts in comparison to peripheral blood (PB, n = 53) and bone marrow (BM, n = 10) samples of age-matched healthy donors (HD) using flow cytometry. Second, proliferation of myeloma tumor cells in the presence of freshly isolated pDCs was examined. Third, production of IFNα by pDCs co-cultured with MM cells was determined by intracellular staining. RESULTS: We found a highly significant reduction of circulating pDCs (p < 0.0001) and in bone marrow (p < 0.0001) of MM patients compared to HD. We also observed a significant decrease of pDCs (p = 0.004) in BM in patients with monoclonal gammopathy of undetermined significance (MGUS, n = 12). Importantly, we determined that pDCs promote proliferation specifically of MM cells and not the stromal cells and that pDCs secrete IFNα upon co-culture with MM tumor cells. CONCLUSIONS: Our results show altered pDC frequencies in the BM microenvironment in MGUS and MM patients at diagnosis. We showed the tumor-promoting function of pDCs that may mediate immune deficiencies affecting long-term disease control and treatment outcome.

Minimal residual disease assessment by multiparameter flow cytometry in transplant-eligible myeloma in the EMN02/HOVON 95 MM trial.

Minimal residual disease (MRD) by multiparameter flow cytometry (MFC) is the most effective tool to define a deep response in multiple myeloma (MM). We conducted an MRD correlative study of the EMN02/HO95 MM phase III trial in newly diagnosed MM patients achieving a suspected complete response before maintenance and every 6 months during maintenance. Patients received high-dose melphalan (HDM) versus bortezomib-melphalan-prednisone (VMP) intensification, followed by bortezomib-lenalidomide-dexamethasone (VRd) versus no consolidation, and lenalidomide maintenance. Bone marrow (BM) samples were processed in three European laboratories, applying EuroFlow-based MFC protocols (eight colors, two tubes) with 10-4-10-5 sensitivity. At enrollment in the MRD correlative study, 76% (244/321) of patients were MRD-negative. In the intention-to-treat analysis, after a median follow-up of 75 months, 5-year progression-free survival was 66% in MRD-negative versus 31% in MRD-positive patients (HR 0.39; p < 0.001), 5-year overall survival was 86% versus 69%, respectively (HR 0.41; p < 0.001). MRD negativity was associated with reduced risk of progression or death in all subgroups, including ISS-III (HR 0.37) and high-risk fluorescence in situ hybridization (FISH) patients (HR 0.38;). In the 1-year maintenance MRD population, 42% of MRD-positive patients at pre-maintenance became MRD-negative after lenalidomide exposure. In conclusion, MRD by MFC is a strong prognostic factor. Lenalidomide maintenance further improved MRD-negativity rate.

Prognostic significance of lymphocyte patterns in multiple myeloma patients after autologous transplant.

Despite the high efficacy of current induction regimens, most multiple myeloma (MM) patients relapse over time. The link between changes in the immune system and the prognosis of the disease is still not entirely clear. Therefore, we analyzed whether the pattern of bone marrow (BM) lymphocytes during routine BM examination after autologous stem cell transplant (ASCT) is related to disease prognosis or MRD negative complete remission. From 2009 to 2018, 98 MM patients underwent routine BM testing after the first ASCT. Using multi-parametric flow cytometry, twelve BM lymphocyte subtypes were analyzed. In 60% of patients who achieved a complete response (CR), MRD by flow cytometric analysis (sensitivity threshold 10-6) was evaluated. We found an association of relative proportion of BM lymphocyte subtypes with treatment response, progression-free survival (PFS), overall survival (OS), and minimal residual disease (MRD) negativity. Higher relative proportion of memory B cells was associated with inferior median PFS [HR 1.089 (95% CI: 1.023-1.160), p=0.008] and median OS [HR 1.170 (95% CI: 1.074-1.274), p<0.001]. In non-responding patients (minimal response and worse), higher proportion of memory B cells was found when compared to patients achieving CR [3.8% (range 0.5-35.0) vs. 1.0% (range 0.1-12.5); p=0.001]. No significant association of BM lymphocyte subtypes proportion with MRD negative CR was found. Our results show that changes in BM lymphocyte subsets including memory B cells may have prognostic value in MM patients after ASCT.

The impact of lymphocytosis and CD4/CD8 ratio on the anti-JCV antibody index and clinical data in patients treated with natalizumab.

BACKGROUND: Natalizumab is an effective therapy in the treatment of relapsing-remitting multiple sclerosis; it induces lymphocytosis (NIL, natalizumab-induced lymphocytosis) and changes the peripheral lymphocyte pattern. METHODS: This study aims to evaluate NIL, peripheral blood lymphocyte subsets, CD4/CD8 ratio, and their impacts on JCV index and clinical data-No Evidence of Disease Activity (NEDA-3) and annualized relapse rate (ARR) in patients treated with natalizumab. RESULTS: Forty-one patients (33 women) were included in the study. The mean duration of follow-up on natalizumab treatment was 6.7 ± 3.2 years. Significant increases in relative lymphocytosis after 1 month, with a median of 40.4% (- 34.1 to + 145.5%) (p < 0.001), and after 1 year (49.0% (- 9.3 to + 127.6%)) (p < 0.001) were found. Significant differences were found after 1 month when comparing NIL between patients JCV-seroconverting (20.6% (- 17.7 to 72.7%)) and stable JCV-seronegative ones (43.5% (- 6.3 to +96.3%)) (p = 0.04). No significant difference NIL level was found between the patients exhibiting NEDA-3 status and those without it. ARR on natalizumab treatment correlated with CD4/CD8 ratio (r = 0.356; p = 0.021); patients who maintained NEDA-3 status over the whole treatment period exhibited a lower CD4/CD8 ratio (1.89 ± 1.08 vs. 2.5 ± 0.73; p < 0.04). CONCLUSION: This contribution reports the CD4/CD8 ratio as a possible biomarker for better clinical efficacy of natalizumab in patients exhibiting a lower CD4/CD8 ratio. NIL did not correlate with long-term therapeutic efficacy in patients treated with natalizumab, but was demonstrated as lower in patients JCV-seroconverting in the course of follow-up.

Heparin-induced thrombocytopenia: a case report and literature overview.

Heparin-induced thrombocytopenia (HIT) is an immunologically-mediated complication, which usually follows heparin exposition, less frequently exposition to other drugs or even occurs spontaneously. The type of heparin, its dose and mode of application as well as the exposition time, major trauma or operation, and obesity represent the main risk factors for HIT. The probability of HIT correlates with so-called 4T-score. A confirmatory laboratory diagnostic should be exclusively reserved for patients with a medium to a high probability of HIT development (more than 3 points in 4T-score). The screening method is based on serological detection of antibodies against heparin-platelet factor-4 complexes; confirmation tests aim to identify the activation of platelets. The treatment of HIT requires an immediate interruption of heparin application and rigorous antithrombotic treatment with an alternative agent. Herein authors describe a clinical case of HIT manifested as an extreme urticarial reaction in the location of nadroparin application as well as thrombosis of deep subcutaneous veins in a polymorbid obese patient with an extensive and infected burn. Due to timely diagnosis and fondaparinux treatment, no more severe thrombotic events occurred in this patient.

Dynamics of tumor-specific cfDNA in response to therapy in multiple myeloma patients.

OBJECTIVES: Progress in multiple myeloma treatment allows patients to achieve deeper responses, for which the assessment of minimal residual disease (MRD) is critical. Typically, bone marrow samples are used for this purpose; however, this approach is site-limited. Liquid biopsy represents a minimally invasive and more comprehensive technique that is not site-limited, but equally challenging. METHODS: While majority of current data comes from short-term studies, we present a long-term study on blood-based MRD monitoring using tumor-specific cell-free DNA detection by ASO-qPCR. One hundred and twelve patients were enrolled into the study, but long-term sampling and analysis were feasible only in 45 patients. RESULTS: We found a significant correlation of quantity of tumor-specific cell-free DNA levels with clinically meaningful events [induction therapy (P = .004); ASCT (P = .012)]. Moreover, length of cfDNA fragments is associated with better treatment response of patients. CONCLUSIONS: These results support the concept of tumor-specific cell-free DNA as a prognostic marker.

Systemic inflammatory response with high CRP values as the dominant symptom of multiple myeloma.

A man aged 60 years was examined for intense inflammatory response, night sweats, subfebrile and later febrile temperatures and a weight loss of 18 kg in 7 months. CRP was 270 mg / l, i.e. more than 20 times the upper limit of the physiological range. Reactive leukocytosis (10 × 109/l), thrombocytosis (530 × 109/l), increased fibrinogen (greater than 7 g/l), and anemia with hemoglobin of 80 g/l were present. No infection or systemic autoimmune disease has been proven. The patient had normal renal function and had no osteolytic deposits detectable by FDG-PET/CT. The procalcitonin level was not elevated. The bone marrow examination revealed a 30-40% infiltration of proplasmacyte type with admixture of plasmablasts, expressing light chains λ. Monoclonal immunoglobulin IgA λ was at a low concentration of about 8 g/l and the ratio of free light chains κ/λ was 0.13. The extent of bone marrow infiltration and anemia met the criteria for the diagnosis of symptomatic multiple myeloma. Following initiation of the combination therapy using thalidomide, bortezomib and dexamethasone, the maximum decrease in the concentrations of monoclonal immunoglobulin, free light chains and CRP was observed already after the first 2 cycles of treatment. Later, during the following two 2 cycles, the disease began to progress again. The patient underwent successful stem cell collection after the application of cyclophosphamide 2.5 g/m 2 and leukocyte growth factor (G-CSF), and high-dose chemotherapy (melphalan 200 mg/m 2) with the support of stem cell transplantation. At 2 months following high-dose chemotherapy, CRP levels of the physiological range decreased, the blood count was normalized, and monoclonal immunoglobulin was not detectable. Conclusion: The chronic inflammatory response may be due to plasmocytary bone marrow infiltration even if there are no other symptoms of multiple myel-oma present, except for anemia which, however, also involves the inflammatory reaction. In this case, the systemic inflammatory reaction with high CRP levels signalled aggressive behaviour of the disease. Key words: CRP - multiple myeloma - procalcitonin - systemic inflammatory response.

Waldenström's macroglobulinemia: Two malignant clones in a monoclonal disease? Molecular background and clinical reflection.

Waldenström's macroglobulinemia (WM) is a complex disease characterized by apparent morphological heterogeneity within the malignant clonal cells representing a continuum of small lymphocytes, plasmacytoid lymphocytes, and plasma cells. At the molecular level, the neoplastic B cell-derived clone has undergone somatic hypermutation, but not isotype switching, and retains the capability of plasmacytic differentiation. Although by classical definition, WM is formed by monoclonal expansion, long-lived clonal B lymphocytes are of heterogeneous origin. Even more, according to current opinion, plasma cells also conform certain population with pathogenic and clinical significance. In this article, we review the recent advances in the WM clonal architecture, briefly describe B-cell development during which the molecular changes lead to the malignant transformation and mainly focus on differences between two principal B-lineage clones, including analysis of their genome and transcriptome profiles, as well as immunophenotype features. We assume that the correct identification of a number of specific immunophenotypic molecular and expression alterations leading to proper aberrant clone detection can help to guide patient monitoring throughout treatment and successfully implement therapy strategies directed against both B- and plasma cell tumor WM clones.

A first Czech analysis of 1887 cases with monoclonal gammopathy of undetermined significance.

INTRODUCTION: Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition with a risk of malignant conversion. PATIENTS AND METHODS: With the aim to estimate the cumulative risk MGUS progression to hematologic malignancies, we analyzed a nationwide population-based cohort of 1887 MGUS patients from the Czech Registry of Monoclonal Gammopathies (RMG) between 2007 and 2013. RESULTS: During the follow-up period (median 4 years; range 0.6-34.8), progression to hematologic malignancies was observed in 8.6% (162 of 1887) of patients. Factors associated with progression were as follows: M-protein concentration ≥1.5 g/dL, pathological sFLC (<0.26 or >1.65) ratio, bone marrow plasma cells (BMPCs) in cytology >5%, immunoparesis, age ≥69 years, and the level of serum hemoglobin at baseline <12.0 g/dL. Combining these factors, we propose a new risk model (CMG model). The risk of progression at 10 years was 1.6%, 16.9%, 22.9%, 39.4%, and 52.3%, respectively, if 0 (reference group), one, two, three, or four to five risk factors are present (P<.001) with HR 63 times higher compared to the reference MGUS group. CONCLUSION: The new CMG model was established with an advantage for better identification of MGUS patients at low risk.

Activity of aldehyde dehydrogenase in B-cell and plasma cell subsets of monoclonal gammopathy patients and healthy donors.

BACKGROUND: Aldehyde dehydrogenase (ALDH) is highly active in physiological stem cells as well as in tumor-initiating cells of some malignancies including multiple myeloma (MM). Finding higher activity of ALDH in some cell subsets in monoclonal gammopathies (MG) could identify potential source of myeloma-initiating cells (MICs). METHODS: Bone marrow of 12 MM, 9 monoclonal gammopathy of undetermined significance (MGUS), and 10 healthy donors (HD) were analyzed by flow cytometry. ALDH activity of B-cells and plasma cells (PC) was analyzed using Aldefluor. RESULTS: Similar changes of ALDH activity were found during B-cell development in HD and MG. Decreasing of ALDH activity from immature to naïve B-cells was found. In postgerminal stages, the activity started to increase, and in PCs, the ALDH activity was the same as in immature B-cells. Increased ALDH activity of all PC subsets compared to naïve B-cells was found in MM as well as in HD, while in MGUS, only CD19- PCs have higher ALDH activity. In HD, ALDH activity was higher in CD19+ PCs compared with MG. CONCLUSIONS: Our results indicate that changes of ALDH activity are the natural phenomenon in B-cell development; thus, high ALDH activity as a single marker is not appropriate for MICs identification.

Do human B-lymphocytes avoid aging until 60 years?

Broad changes in human innate and adaptive immunity are associated with advanced age. The age-related alteration of gene expression was reported for both T and B lymphocytes. We analysed the genome-wide expression profiles (n=20) of naive and whole B cell populations from young and early aged healthy donors under 60 years. We revealed large homogeneity of all analysed genome-wide expression profiles but did not identified any significant gene deregulation between young (30-45 years) and early aged healthy donors (50-60 years). We argue that B cells avoid the aging program on molecular level until 60 years of age. Our results demonstrate the potential of hematopoietic stem cells to generate uncompromised lymphocytes in early elderly. These are very encouraging findings for the general health and the immunity maintenance would not need any intervention to naive B cells. Rather, a suitable immune stimulation in healthy body environment warrants further research into aging of older elderly.

Flow cytometry in immunoglobulin light chain amyloidosis: Short review.

Flow cytometry (FCM) has found its application in clinical diagnosis and evaluation of monoclonal gammopathies (MG). Although, research has been mainly focused on multiple myeloma (MM), nowadays FCM becomes to be potential tool in the field of AL amyloidosis. Clonal plasma cells identification and specific phenotype profile detection is important for diagnosis, monitoring and prognosis of AL amyloidosis. Therefore, FCM could be a perspective method for study not only MM but also AL amyloidosis. This review provides an overview and possibilities of FCM application in AL amyloidosis.