Ph-negative Myeloproliferative Neoplasms Research Articles

Development of an Artificial-Intelligence-Based Tool for Automated Assessment of Cellularity in Bone Marrow Biopsies in Ph-Negative Myeloproliferative Neoplasms.

The cellularity assessment in bone marrow biopsies (BMBs) for the diagnosis of Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs) is a key diagnostic feature and is usually performed by the human eyes through an optical microscope with consequent inter-observer and intra-observer variability. Thus, the use of an automated tool may reduce variability, improving the uniformity of the evaluation. The aim of this work is to develop an accurate AI-based tool for the automated quantification of cellularity in BMB histology. A total of 55 BMB histological slides, diagnosed as Ph- MPN between January 2018 and June 2023 from the archives of the Pathology Unit of University "Luigi Vanvitelli" in Naples (Italy), were scanned on Ventana DP200 or Epredia P1000 and exported as whole-slide images (WSIs). Fifteen BMBs were randomly selected to obtain a training set of AI-based tools. An expert pathologist and a trained resident performed annotations of hematopoietic tissue and adipose tissue, and annotations were exported as .tiff images and .png labels with two colors (black for hematopoietic tissue and yellow for adipose tissue). Subsequently, we developed a semantic segmentation model for hematopoietic tissue and adipose tissue. The remaining 40 BMBs were used for model verification. The performance of our model was compared with an evaluation of the cellularity of five expert hematopathologists and three trainees; we obtained an optimal concordance between our model and the expert pathologists' evaluation, with poorer concordance for trainees. There were no significant differences in cellularity assessments between two different scanners.

A lasso and random forest model using flow cytometry data identifies primary myelofibrosis.

Thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic/early (pre-PMF), and overt fibrotic PMF (overt PMF) are classical Philadelphia-Negative (Ph-negative) myeloproliferative neoplasms (MPNs). Differentiating between these types based on morphology and molecular markers is challenging. This study aims to clarify the application of flow cytometry in the diagnosis and differential diagnosis of classical MPNs. This study retrospectively analyzed the immunophenotypes, clinical characteristics, and laboratory findings of 211 Ph-negative MPN patients, including ET, PV, pre-PMF, overt PMF, and 47 controls. Compared to ET and PV, PMF differed in white blood cells, hemoglobin, blast cells in the peripheral blood, abnormal karyotype, and WT1 gene expression. PMF also differed from controls in CD34+ cells, granulocyte phenotype, monocyte phenotype, percentage of plasma cells, and dendritic cells. Notably, the PMF group had a significantly lower plasma cell percentage compared with other groups. A lasso and random forest model select five variables (CD34+CD19+cells and CD34+CD38- cells on CD34+cells, CD13dim+CD11b- cells in granulocytes, CD38str+CD19+/-plasma, and CD123+HLA-DR-basophils), which identify PMF with a sensitivity and specificity of 90%. Simultaneously, a classification and regression tree model was constructed using the percentage of CD34+CD38- on CD34+ cells and platelet counts to distinguish between ET and pre-PMF, with accuracies of 94.3% and 83.9%, respectively. Flow immunophenotyping aids in diagnosing PMF and differentiating between ET and PV. It also helps distinguish pre-PMF from ET and guides treatment decisions.

Chronic myeloproliferative neoplasms with concomitant CALR mutation and BCR::ABL1 translocation: diagnostic and therapeutic implications of a rare hybrid disease.

Myeloproliferative neoplasms (MPNs) are subdivided into Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) and Ph-negative MPNs. BCR::ABL1 translocation is essential for the development and diagnosis of CML; on the other hand, the majority of Ph-negative MPNs are characterized by generally mutually exclusive mutations of Janus kinase 2 (JAK2), calreticulin (CALR), or thrombopoietin receptor/myeloproliferative leukemia (MPL). CALR mutations have been described essentially in JAK2 and MPL wild-type essential thrombocythemia and primary myelofibrosis. Rarely coexisting CALR and MPL mutations have been found in Ph-negative MPNs. BCR::ABL1 translocation and JAK2 mutations were initially considered mutually exclusive genomic events, but a discrete number of cases with the combination of these genetic alterations have been reported. The presence of BCR::ABL1 translocation with a coexisting CALR mutation is even more uncommon. Herein, starting from a routinely diagnosed case of CALR-mutated primary myelofibrosis subsequently acquiring BCR::ABL1 translocation, we performed a comprehensive review of the literature, discussing the clinicopathologic and molecular features, as well as the outcome and treatment of cases with BCR::ABL1 and CALR co-occurrence.

Co-occurrence of JAK2-V617 F mutation and BCR::ABL1 translocation in chronic myeloproliferative neoplasms: a potentially confounding genetic combination.

Myeloproliferative neoplasms (MPNs) are classified into Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) and Ph-negative MPNs. BCR::ABL1 translocation is the key genetic event of CML, whereas JAK2/MPL/CALR mutations are molecular aberrations of Ph-negative MPNs. Despite initially considered mutually exclusive genetic aberrations, the co-occurrence of BCR::ABL1 and JAK2 has been reported in a limited number of cases. The two genetic alterations may be identified either at the same time or JAK2 aberration may be detected in patients with a previous CML treated with tyrosine kinase inhibitors or, finally, BCR::ABL1 translocation occurs in patients with a history of JAK2-positive MPN. This combination of genomic alterations is potentially confounding with clinical manifestations often misinterpreted either as disease progression or drug resistance, therefore leading to inappropriate patient's treatment. Our systematic review aims to improve hematologist and pathologist knowledge on this rare subset of patients. Starting from the presentation of two additional cases from our routine daily practice, we focus mainly on clinical, laboratory, and bone marrow histological findings, which may represent useful clues of BCR::ABL1 and JAK2 co-occurrence. The interaction between JAK2 and BCR::ABL1 clones during the disease course as well as therapy and outcome are presented.

Myeloid Neoplasia in Patients with Hereditary Hemoglobinopathies and Hemolytic Anemias (HHAs)

Background: Studies have shown an increased incidence of myeloid neoplasia (MN) in patients with sickle cell disease (SCD). It is hypothesized that increased erythropoietic stress may influence acquisition of molecular drivers of MN in SCD. However, present understanding of MN presentation and outcomes in SCD and other hereditary hemoglobinopathies and hemolytic anemias (HHAs) is limited to case reports. Systematic evaluation of MNs in patients with HHAs will better contextualize the risk of MN in this population relative to non-HHA controls. Methods: Patients with HHAs were identified from the Mass General Brigham Research Patient Data Repository using ICD-10 codes. Evaluated HHAs included sickle cell disease (SCD), sickle cell trait (SCT), thalassemia major, thalassemia trait, other hemoglobinopathy traits, and hereditary spherocytosis or elliptocytosis. A final HHA-MN cohort included patients with laboratory confirmed HHA diagnoses as well as incident myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), Ph+ chronic myelogenous leukemia (Ph+ CML), Ph negative myeloproliferative neoplasms (Ph- MPNs) or acute myeloid leukemia (AML). A control cohort of non-HHA MN patients was obtained from the Dana-Farber Hematologic Malignancies Data Repository. Clinical, molecular, and survival data were summarized and compared between HHA-MN and controls. Results: The HHA-MN cohort included 62 patients with HHAs and incident MN (Table 1). HHAs included SCD (n = 6, 9.7%), SCT (n = 9, 14.5%), thalassemia trait (n = 36, 58.1%), thalassemia major (n = 2, 3.2%), hereditary spherocytosis or elliptocytosis (n = 4, 6.5%), and other hemoglobinopathy traits (n = 5, 8.1%). MN was diagnosed at a median age of 61 years [IQR: 48, 70] in the HHA-MN cohort and incident MNs included AML (n = 12, 19.4%), MDS/CMML (n = 22, 35.4%), Ph+ CML (n = 6, 9.7%), and Ph- MPNs (n = 22, 35.4%). A control population of 3388 MN patients without HHA included cases of AML (n = 754, 22.2%), MDS/CMML (n= 1272, 37.5%), Ph+ CML (n = 354, 10.4%), and Ph- MPNs (n = 1008, 29.7%). Compared to HHA patients, control patients were older at MN diagnosis (median age 65 years [IQR: 56, 73], p = 0.0071), driven by significantly younger ages at AML diagnosis for HHA-MN patients (median age 50 years [IQR: 33, 63] vs 65 years [IQR: 57, 73], p =0.0044). AML was the most common MN in SCD (n = 4, 66.7%) whereas incident MDS/CMML was the predominant MN among individuals with thalassemia trait (n = 12, 33.3%) or hereditary spherocytosis and elliptocytosis (n = 2, 50%). Diagnostic cytogenetic data were available for 45 patients in the HHA-MN cohort (72.6% of cohort). Complex karyotype, defined as 3 or more chromosomal aberrations, was observed in 3 of 11 (27.3%) AML, 3 of 19 (15.8%) MDS/CMML and 1 of 17 (5.9%) Ph- MPN cases. Of the 33 HHA-MN patients with available diagnostic next generation sequencing, 60.6% had 2 or more pathogenic mutations at MN diagnosis. Including 2 cases of TP53-mutant AML in patients with SCD, the majority (90%) of AML and 38.9% of MDS/CMML diagnosed in HHA-MN patients were intermediate to adverse risk by ELN criteria or intermediate to very high risk by IPSS-R, respectively. High risk disease was also common in our control MN cohort, where 88.3% of AML cases were intermediate or adverse risk by ELN criteria and 55.9% of MDS/CMML cases were intermediate to very high risk by IPSS-R. Survival varied by subtype of HHA and MN (Figure 1). Survival was lowest for SCD (33.3% at 60-months) and highest for thalassemia traits (67.1% at 60 months). In HHA patients compared to controls, 60-month survival was 65.0% overall (vs 56.4%), 14.3% for AML (vs 23.8%), 51.0% for MDS/CMML (vs 43.9%), 90.5% in Ph- MPN (vs 80.6%), and 100% in Ph+ CML (vs 89.4%). Within the limitations of our sample size, survival differences were not statistically significant. Conclusion: In this retrospective analysis, we observe younger age of diagnosis of myeloid malignancy for patients with HHAs compared to non-HHA controls. Inferior survival in SCD and thalassemia major is largely explained by a higher proportion of AML diagnoses in patients with SCD. AML and MDS/CMML diagnosed in HHA patients tended to be intermediate or high-risk disease, with survival similar to the non-HHA control population that was also enriched for higher risk patients. Validation of our findings in an expanded cohort of HHA patients is currently underway.

The incidence of Ph-negative chronic myeloproliferative neoplasms in Belarus

Introduction. Ph-negative myeloproliferative neoplasms (MPN) are chronic clonal disorders characterized by the proliferation of mature cells from one or more myeloid cell lines. These disorders include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The incidence of classical Ph-negative MPN varies significantly throughout the world. These differences may be due to geographical or population features, as well as due to differences in methods of diagnosis and recording. Aim: to determine incidence of Ph-negative MPN in Belarus. Materials and methods. Data on all cases of newly diagnosed Ph-negative MPNs (PV, ET and PMF) for 2014-2018 were collected from central regional medical institutions with a hematology specialization. Population epidemiological analysis of incidence was carried out by determining crude incidence rates per 100 000 population per year with distribution by genderand age groups at diagnosis, age-standardized rates, as well as average annual percent change. Results. The overall annual standardized incidence rate of classical Ph-negative MPN in Belarus was 1.96 (1.86–2.07), PV — 0.93 (0.86–1.01), ET — 0.63 (0.57–0.70) and PMF — 0.40 (0.36–0.45) per 100 000 population per year, respectively. The overall crude incidence rate was 3.26 (3.10–3.43) per 100 000 population. The crude incidence rates increased steadily with the increase in age of patients with a peak value of 12.78 (10.97–14.81) in the age group of 70–74 years. No significant changes were revealed in the overall incidence of the MPN and for each of the studied diseases separately during the study period. Conclusion. For the first time the nationwide features of incidence of the Ph-negative MPN in Belarus were estimated, which made it possible to provide substantial information for public health management. The incidence of MPN in Belarus is generally comparable to the levels determined in the analysis of the populations of other countries.

Global tests in patients with Ph-negative myeloproliferative neoplasms

Introduction: A frequent clinical manifestation of Ph-negative myeloproliferative neoplasms (MPN) is the development of thrombosis. To identify the state of hypercoagulation it is relevant and promising to introduce global tests for evaluating the hemostasis — the thrombin generation test (TGT) and thromboelastography (TEG). Aim: to evaluate the parameters of thrombin generation and thromboelastography in patients with Ph-negative MPN. Material and methods. In total, 62 patients with MNP were included in the study: 27 with polycythemia vera (PV), 14 with essential thrombocythemia (ET) and 21 with primary myelofibrosis (PMF). The control group included 55 practically healthy individuals, comparable in gender and age (19 people in the study of TEG, 36 people in the study of TGT). The TEG was performed using a “TEG 5000” thromboelastograph. TGT was measured with Calibrated Automated Thrombinography. Results: Ly30 and Ly60 in TEG in patients were significantly lower (0.35 (0.20–0.48), 0.00 (0.00–0.40) and 0.00 (0.00 — 0.43) and 3.15 (2.45–3.60), 1.25 (0.10–3.58) and 0.60 (0.00–3.05) respectively), than in the control (1.60 (1.05–2.75) and 6.20 (4.15–8.30), respectively), which indicates the ineffectiveness of fibrinolysis. The values of MA and G in patients with ET and PV significantly exceeded the control (69.15 (67.98–70.78) mm and 65.20 (59.65–63.83) mm versus 62.00 (57.75–6.75) mm and 11.20 (10.60–12.15) din/cm2 and 9.40 (7.40–11.60) din/cm2 versus 8.20 (6.85–8.75) din/cm2, respectively. The most pronounced change in sensitivity to TM was observed in patients with ET (27.94 (17.35–43.58) % and 13.29 (-3.48–23.60) %, respectively; p < 0.05). A significant decrease in ETP was observed in patients with PV and PMF. Conclusion. The study of hemostasis in patients with MPN using TEG and TGT revealed the presence of multidirectional changes associated with the disease. The TEG showed that an increase in the time required for the onset of fibrin formation is combined with increased clot strength and inhibited fibrinolysis, which are risk factors for the development of thromboembolic complications. The study of TGT determined a decrease in the quantitative characteristics of thrombin generation and at the same time the failure of the anticoagulant system of protein C, leading to the development of hypercoagulation.

Management of classical Philadelphia chromosome-negative myeloproliferative neoplasms in Asia: consensus of the Asian Myeloid Working Group.

Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized clinically by the proliferation of one or more hematopoietic lineage(s). The classical Philadelphia-chromosome (Ph)-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The Asian Myeloid Working Group (AMWG) comprises representatives from fifteen Asian centers experienced in the management of MPN. This consensus from the AMWG aims to review the current evidence in the risk stratification and treatment of Ph-negative MPN, to identify management gaps for future improvement, and to offer pragmatic approaches for treatment commensurate with different levels of resources, drug availabilities and reimbursement policies in its constituent regions. The management of MPN should be patient-specific and based on accurate diagnostic and prognostic tools. In patients with PV, ET and early/prefibrotic PMF, symptoms and risk stratification will guide the need for early cytoreduction. In younger patients requiring cytoreduction and in those experiencing resistance or intolerance to hydroxyurea, recombinant interferon-α preparations (pegylated interferon-α 2A or ropeginterferon-α 2b) should be considered. In myelofibrosis, continuous risk assessment and symptom burden assessment are essential in guiding treatment selection. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in MF should always be based on accurate risk stratification for disease-risk and post-HSCT outcome. Management of classical Ph-negative MPN entails accurate diagnosis, cytogenetic and molecular evaluation, risk stratification, and treatment strategies that are outcome-oriented (curative, disease modification, improvement of quality-of-life).

Clinical Impact of JAK2V617F Allele Burden in Philadelphia-Negative Myeloproliferative Neoplasms

The impact of JAK2V617F allele burden on clinical course in Philadelphia-negative (Ph-negative) myeloproliferative neoplasms (MPNs) is not clear. We analyzed the clinical impact of JAK2V617F allele burden in a relatively large series of patients with Ph-negative MPNs and long-term follow-up. A total of 228 patients with Ph-negative MPNs, including 118 with essential thrombocythemia (ET), 84 with primary myelofibrosis (PMF), and 26 with polycythemia vera (PV), were analyzed. The JAK2 MutaScreen assay was used to quantify JAK2V617F allele burden in genomic DNA. In PV cases, high JAK2V617F allele burden was associated with a trend towards inferior overall survival. In ET, high JAK2V617F allele burden was associated with lower hemoglobin and hematocrit levels, higher lactate dehydrogenase (LDH) levels, larger spleen size, and increased bleeding and mortality rates. In PMF, high JAK2V617F allele burden was associated with higher leukocyte counts and larger spleen size. In the entire cohort, high allele burden was associated with higher leukocyte and lower platelet counts, higher LDH levels, larger spleen size, higher percentage of bleeding events, higher death rate, and inferior overall survival. Our results suggest that high JAK2V617F allele burdens are associated with more severe disease in PV and ET. In PMF, high JAK2V617F allele burdens were associated with more pronounced myeloproliferative phenotypes. In Ph-negative MPNs, high allele burdens were associated with more aggressive phenotypes. Our data with a long follow-up period support the possibility of JAK2V617F allele burden being used as a marker for predicting clinical phenotype in cases of Ph-negative MPNs.

POSTER: MPN-386 The Role of Variants of Uncertain Significance From NGS Data for Patients With pH-Negative Myeloproliferative Neoplasms

POSTER: MPN-386 The Role of Variants of Uncertain Significance From NGS Data for Patients With pH-Negative Myeloproliferative Neoplasms

MPN-386 The Role of Variants of Uncertain Significance From NGS Data for Patients With pH-Negative Myeloproliferative Neoplasms

MPN-386 The Role of Variants of Uncertain Significance From NGS Data for Patients With pH-Negative Myeloproliferative Neoplasms

Cutaneous involvement in Ph-negative myeloproliferative neoplasms: from extramedullary hematopoiesis to myeloid metastasis with histiocytic differentiation. A systematic review of the literature.

Myeloid neoplasms may metastasize to the skin, presenting a wide range of clinical-pathological features that often lead to a reduction in patients' survival. The presentation varies depending on the category of myeloid neoplasm and its prognostic significance. The literature has specifically focused on the features of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and chronic myelomonocytic leukemia (CMML). In this article, we aimed to uncover the peculiarities of clonal skin proliferations in the course of Ph-negative myeloproliferative neoplasms (MPNs). We conducted a systematic review and statistical analysis of the literature data. MPN patients mainly exhibited cutaneous extramedullary hematopoiesis, while a minority displayed cutaneous histiocytic lesions. Furthermore, these patients showed lower survival rates compared to the median survival of MPN patients, especially when calculating survival from the appearance of cutaneous lesions. Our work highlights, for the first time, the prognostic relevance and histological heterogeneity of cutaneous lesions in MPN. Moreover, it emphasizes the importance of dermatological and histological examinations when cutaneous lesions are present.

Transcriptomic comparison of bone marrow CD34 + cells and peripheral blood neutrophils from ET patients with JAK2 or CALR mutations

BackgroundEssential thrombocythemia (ET) is one of the most common types of Ph-negative myeloproliferative neoplasms, an infrequent group of blood cancers that arise from a CD34 + hematopoietic stem cell (HSC) in the bone marrow (BM) primarily due to driver mutations in JAK2, CALR or MPL. These aberrations result in an overproduction of mature myeloid cells in peripheral blood (PB). To date, no targeted therapies have been approved for ET patients, so the study of the molecular mechanisms behind the disease and the identification of new therapeutic targets may be of interest. For this reason, in this study, we have compared the transcriptomic profile of undifferentiated CD34 + cells and mature myeloid cells from ET patients (CALR and JAK2-mutated) and healthy donors deposited in publicly available databases. The study of the similarities and differences between these samples might help to better understand the molecular mechanisms behind the disease according to the degree of maturation of the malignant clone and the type of mutation and ultimately help identify new therapeutic targets for these patients.ResultsThe results show that most of the altered hallmarks in neutrophils were also found in CD34 + cells. However, only a few genes showed a similar aberrant expression pattern in both types of cells. We have identified a signature of six genes common to patients with CALR and JAK2 mutations (BPI, CRISP3, LTF, MMP8, and PTGS1 upregulated, and PBXIP1 downregulated), a different signature of seven genes for patients with CALR mutations (BMP6, CEACAM8, ITK, LCN2, and PRG2 upregulated, and MAN1A1 and MME downregulated) and a signature of 13 genes for patients with JAK2 mutations (ARG1, CAST, CD177, CLEC5A, DAPP1, EPS15, IL18RAP, OLFM4, OLR1, RIOK3, SELP, and THBS1 upregulated, and IGHM downregulated).ConclusionsOur results highlight transcriptomic similarities and differences in ET patients according to the degree of maturation of the malignant clone and the type of mutation. The genes and processes altered in both CD34 + cells and mature neutrophils may reveal altered sustained processes that could be studied as future therapeutic targets for ET patients.

Regional features of the incidence of chronic Ph-negative myeloproliferative neoplasms in Belarus

According to published data, there are differences in the registered incidence of chronic Ph-negative myeloproliferative neoplasms (MPN) in some regions of the world. In this study, for the first time, we compare the Ph- MPN incidence in the regions of Belarus in order to determine the regional features.It was shown that for the period 2014-2018 the highest rates of standardized incidence of polycythemia vera were noted in the Brest region – 1,17 and Minsk – 1,11, the lowest incidence rate was determined in the Vitebsk region – 0,68 per 100,000 population per year, p<0,05. The maximum incidence rates of essential thrombocythemia were recorded in Minsk city – 1,04 and Gomel region – 0,86, which is significantly higher than in Minsk region – 0,27, Vitebsk region – 0,35 and Grodno region – 0,44 per 100 000 population per year. The highest incidence rates of primary myelofibrosis were noted in Gomel – 0,63 and Vitebsk regions – 0,53, they were significantly higher than in Grodno – 0,14 and Minsk regions – 0,24 per 100 000 population per year.Differences in the incidence of Ph-MPN between the regions of Belarus were revealed, while the overall incidence rates in Minsk city, Gomel, Brest and Mogilev regions exceeded those in Vitebsk, Grodno and Minsk regions. In addition, variation in incidence rates for individual nosologies was noted. The information obtained can be used to make management decisions when organizing medical observation and treatment of patients with Ph- MPN in Belarus.

Issue highlights-May 2023.

In the era of personalized medicine, the list of targeted therapeutic drugs has been greatly expanded, many of which are employed to treat hematolymphoid neoplasms. These include drugs specifically targeting immune checkpoint signaling epitopes such as programmed cell death 1 (PD1) and its ligand PDL1 to inhibit T-cell activation. Alternatively, these drugs might target the surface antigens expressed by tumor cells such as CD19 and CD22 in B-cell neoplasms. Of the latter, targeted immunotherapies include monoclonal antibodies with or without drug conjugates, bispecific T-cell engagers, or chimeric antigen receptor (CAR) T-cell therapy. The binding sites of these therapeutic antibodies are often identical or in proximity with the binding sites of the diagnostic antibodies. Therefore, use of these therapies pose great challenges for clinical cytometry labs in the assessment of post-treatment samples, especially in the detection of measurable/minimal residual disease (MRD). In this issue, Chen, Gao, et al. (2023) provided an overview of MRD detection in B-lymphoblastic leukemia/lymphoma in the era of immunotherapy, and Gao et al. (2023) focused their review on the impact of targeted therapy on mature B- and plasma cell neoplasms utilizing flow cytometry assessments. In both reviews, the authors illustrated the challenges, identified the problems, and provided a list of available options and solutions. For each of the above-mentioned disease categories, optimal gating and analysis strategies were illustrated with literature review and inputs from the authors' experience and insights. The utility and interpretation of additional B-cell markers other than CD19 and CD20 for mature or immature B-cell neoplasms such as CD22, CD24, and cCD79a (Mikhailova et al., 2022) were studied as well as VS38, CD229, and CD319 (Pojero et al., 2016; Soh et al., 2021) for plasma cell neoplasms. The authors recommended that the flow cytometry assays in the era of targeted therapies must contain significant redundancy in the antibody panels allowing the detection of the cells of interest and additionally should be ready to utilize several gating strategies for accurate and consistent population identification. Neoplastic mature B-cells often show restricted kappa or lambda light chain expression; however, light chain expression may be absent in around 5%–10% of mature B-cell lymphoma (Li et al., 2002). It is suggested that mature B-cells lacking surface light chain expression can be used as a surrogate marker to diagnose mature B-cell lymphomas. Huang et al. (2023) reported a series of 89 cases of surface light chain negative B-cell lymphoma which consisted primarily of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Interestingly, the authors also reported no detectable light chain expression in normal/reactive mature B cells collected from body fluids and cystic fluids from 14 patients with no clinical and pathological evidence of lymphoma/leukemia. Body fluids are often proteaceous; thus additional washing prior to kappa/lambda staining is often required to obtain adequate light chain staining and caution is needed in the interpretation of light chain expression in B-cells collected from body fluids. It is known that out of the context of chronic myeloid leukemia (CML) (Soma et al., 2016), lymphoblastic crisis is extremely rare in other chronic myeloid neoplasms, such as myelodysplastic syndrome (MDS) (Xie et al., 2019) and Philadelphia negative myeloproliferative neoplasms (MPN). Chan et al. (2023) reported the detection of a low level of abnormal B-lymphoblasts ranging from 0.012% to 3.6% in 9 out of 1262 MDS or Ph-negative MPN. Cell sorting coupled with next-generational sequencing showed that the abnormal B- lymphoblasts in myeloid neoplasms were often, but not always, clonally related to the myeloid compartment, and the molecular genetic findings suggested a mutant multipotent progenitor cell with different lineage output that might be determined by the specific mutations and the particular cell stage where the mutations occurred. Importantly, unlike CML, the presence of abnormal B-lymphoblasts in MDS and Ph-neg MPNs did not necessarily indicate blast crisis, and therapy for the underlying myeloid neoplasms with close monitoring is suggested to be a reasonable approach. The last article in this issue (Chen, Zhang, et al., 2023) studied mucosal-associated invariant T cells (MAIT) (Godfrey et al., 2019) in aplastic anemia (AA), a type of bone marrow failure directly linked to abnormal immune responses. MAIT cells exhibit an effector-memory phenotype and express several NK receptors such as NKG2D and CD161 and play an important role in the development of autoimmune diseases. The authors showed an increased frequency of MAIT cells with an activation and effector function-related cell surface marker profile in patients with aplastic anemia. The specific property of NKG2D expression on MAIT cells suggests that these cells may have a distinct mechanism involved in AA immune pathogenesis.

Immunohistochemistry of bone marrow extracellular matrix in ph-negative myeloproliferative diseases

Introduction Evaluation of immunohistochemical expression of matrix metalloproteinases 2 and 9 (MMP2, MMP9), their inhibitors (TIMP1, TIMP2), fibroblast growth factor (FGF2), transforming growth factor beta (TGFB1) and collagen type III in the bone marrow of patients with Ph-negative myeloproliferative neoplasms (MPN) is of great importance.The aim of the study was the evaluation of expression of extracellular matrix components (MMP-2, MMP-9, TIMP1, TIMP-2, FGF2, TFGB1, Collagen III) involved in myelofibrosis progression in bone marrow trepan biopsies depending on mutational status of patients with CMPD.Materials and methods We analyzed 108 bone marrow biopsies of patients with MPN, which were divided into 3 groups: JAK2-positive (n=62), CARL-positive (n=25) and triple-negative (n=21). Whole-slide sections were immunostained using antibodies against MMP-2, MMP-9, TIMP-1, TIMP-2, FGF2, TGFB1, collagen type III and scored by ImageJ plugin software. We used Kruskal- Wallis test and Mann-Whitney U-test for comparisons of differences in medians. Spearman’s rank order correlation was calculated. Statistical significance was set at p<0,05.Results and Discussion MMP2 expression was observed in megakaryocytes. MMP9 expression was observed in neutrophils, macrophages and the bone marrow extracellular matrix (EM). TIMP1 expression was observed in the EM. TIMP-2, FGF2, TGFB1 and collagen type III expression was observed in megakaryocytes and the EM. Kruskal-Wallis test determined the differences between all 3 groups (MMP- 2 p< 0,001, MMP-9 p=0,023, TIMP-1 p< 0,001, TIMP-2 p< 0,001, FGF2 p< 0,001, TGFB1 p< 0,001, collagen type III p< 0,001). Mann-Whitney U-test determined the most differences between JAK2- and CALR-groups (MMP-2 p=0,001, MMP-9 p=0,001, TIMP-1 p=0,001, FGF2 p=0,001, TGFB1 p=0,001, collagen type III p=0,001), except TIMP-2. There was the weak and moderate positive correlation between JAK2-mutation and the immunohistochemistry expression of EM components, also the weak negative correlation between CALR-mutation and the immunohistochemistry expression of EM components.Conclusion The bone marrow immunohistochemistry expression of MMP-2, MMP-9, TIMP-1, TIMP-2, FGF2, TGFB1, collagen type III depends on driver mutations. It may be useful for understanding of fibrosis pathogenesis and prognosis estimate of Ph-negative MPN.

The Role of Leukocytes in the Formation of Neutrophil Extracellular Traps and Thrombosis in Ph-Negative Myeloproliferative Neoplasms: A Literature Review

Thrombotic complications often cause death in patients with chronic Ph-negative myeloproliferative neoplasms (MPNs). In spite of numerous studies, the pathogenesis of thrombus formation in MPN patients remains unclear. Its mechanism is complex and is determined by many factors. One of the essential phases in thrombogenesis is characterized by the activation of cell mechanisms and formation of neutrophil extracellular traps (NETs). NETs consist of DNA strands, histones, granular proteins and along with pathogen destruction provide an ideal matrix for platelet and clotting mechanism activation.

Transcriptomic Analysis Reveals JAK2/MPL-Independent Effects of Calreticulin Mutations in a C. elegans Model.

There is growing evidence that Ph-negative myeloproliferative neoplasms (MPNs) are disorders in which multiple molecular mechanisms are significantly disturbed. Since their discovery, CALR driver mutations have been demonstrated to trigger pathogenic mechanisms apart from the well-documented activation of JAK2/MPL-related pathways, but the lack of experimental models harboring CALR mutations in a JAK2/MPL knockout background has hindered the research on these non-canonical mechanisms. In this study, CRISPR/Cas9 was performed to introduce homozygous patient-like calreticulin mutations in a C. elegans model that naturally lacks JAK2 and MPL orthologs. Whole-genome transcriptomic analysis of these worms was conducted, and some of the genes identified to be associated with processes involved in the pathogenesis of MPNs were further validated by qPCR. Some of the transcriptomic alterations corresponded to typically altered genes and processes in cancer and Ph-negative MPN patients that are known to be triggered by mutant calreticulin without the intervention of JAK2/MPL. However, interestingly, we have also found altered other processes described in these diseases that had not been directly attributed to calreticulin mutations without the intervention of JAK2 or MPL. Thus, these results point to a new experimental model for the study of the JAK2/MPL-independent mechanisms of mutant calreticulin that induce these biological alterations, which could be useful to study unknown non-canonical effects of the mutant protein. The comparison with a calreticulin null strain revealed that the alteration of all of these processes seems to be a consequence of a loss of function of mutant calreticulin in the worm, except for the dysregulation of Hedgehog signaling and flh-3. Further analysis of this model could help to delineate these mechanisms, and the verification of these results in mammalian models may unravel new potential therapeutic targets in MPNs. As far as we know, this is the first time that a C. elegans strain with patient-like mutations is proposed as a potential model for leukemia research.

Current experience with ropeginterferon Alfa-2b in Ph negative myeloproliferative neoplasm at the Department of Internal Medicine – Haematology and Oncology in Brno

Current experience with ropeginterferon Alfa-2b in Ph negative myeloproliferative neoplasm at the Department of Internal Medicine – Haematology and Oncology in Brno

Molecular prognostication in Ph-negative MPNs in 2022

The application of genomic techniques, including cytogenetics and DNA sequencing, to decipher the molecular landscape of patients with myeloproliferative neoplasms (MPNs) has radically modified diagnostic approach and management through improved risk stratification. Three driver mutated genes (JAK2, MPL, CALR) are variably harbored by >80% of patients and associated with clinical characteristics, as well as major disease-related complications and different survival outcomes. Therefore, JAK2 V617F mutation is included in the revised International Prognosis Score of Thrombosis for Essential Thrombocythemia score for prediction of thrombosis in patients with essential thrombocythemia and prefibrotic primary myelofibrosis, while a CALR type 1 mutated genotype constitutes a favorable variable for survival in patients with myelofibrosis (MF). Novel, integrated clinical and cytogenetic/mutation scores (Mutation-Enhanced International Prognostic Score System for Transplantation-Age Patients with Primary Myelofibrosis [MIPSS70/v2], genetically inspired prognostic scoring system [GIPSS], Myelofibrosis Secondary to PV and ET- Prognostic Model [MYSEC-PM]) have been devised that guide selection of stem cell transplantation candidates with MF or help predict the risk associated with the transplant procedure (Myelofibrosis Transplant Scoring System), with greater performance compared with conventional scores based on hematologic and clinical variables only. On the other hand, several clinical needs remain unmet despite the great amount of molecular information available nowadays. These include the prediction of evolution to acute leukemia in a clinically actionable time frame, the identification of patients most likely to derive durable benefits from target agents, in primis JAK inhibitors, and, conversely, the significance of molecular responses that develop in patients receiving interferon or some novel agents. Here, we discuss briefly the significance and the role of genomic analysis for prognostication in patients with MPNs from a clinician's point of view, with the intent to provide how-to-use hints.