Updated threshold, renewed problems: should the diagnostic criteria of polycythemia vera be reconsidered? A retrospective cross-sectional cohort study.

OBJECTIVE: This aim of this study was to evaluate hemoglobin and hematocrit values of polycythemia vera and secondary polycythemia patients with updated World Health Organization thresholds. In addition, by determining our own threshold values, we aimed to demonstrate the necessity of bone marrow biopsy and genetic analysis to be used for further diagnosis in patients with high-normal hematocrit and hemoglobin values. METHODS: A cross-sectional and retrospective study was performed with the medical records of patients from Eskisehir City Hospital hematology clinics and outpatient clinics between July 1, 2019 and July 1, 2020. The study included patients with polycythemia, divided into two groups according to polycythemia vera and secondary polycythemia. A bone marrow biopsy was performed on patients with either Janus kinase mutation positivity and/or subnormal erythropoietin levels. Receiver operating characteristics analysis was used to find threshold values, and the diagnostic efficiency of these values in differentiating World Health Organization thresholds in 2008 and 2016 was evaluated. RESULTS: A total of 73 patients were included. The median age was 43.5 years (min: 18; max: 84). The hematocrit value of 54.1 was predicted to diagnose polycythemia vera with a sensitivity of 45% and a specificity of 80%. Subsequent analysis revealed that an hemoglobin value of 17.7 was indicative of diagnosing polycythemia vera with a sensitivity of 60% and a specificity of 63%. The mean follow-up length was 6.4 months (2-12). CONCLUSION: Our study demonstrated that modified World Health Organization criteria might lead to unnecessary additional tests for polycythemia vera patients with high-normal hemoglobin and hematocrit values.

Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Proinflammatory phenotype of iPS cell-derived JAK2 V617F megakaryocytes induces fibrosis in 3D in vitro bone marrow niche.

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery.

Morphology of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are a group of clonal haematological malignancies first described by Dameshek in 1957. The Philadelphia-negative MPN that will be described are polycythaemia vera (PV), essential thrombocythaemia (ET), pre-fibrotic myelofibrosis and primary myelofibrosis (PMF). The blood and bone marrow morphology are essential in diagnosis, for WHO classification, establishing a baseline, monitoring response to treatment and identifying changes that may indicate disease progression. The blood film changes may be in any of the cellular elements. The key bone marrow features are architecture and cellularity, relative complement of individual cell types, reticulin content and bony structure. Megakaryocytes are the most abnormal cell and key to classification, as their number, location, size and cytology are all disease-defining. Reticulin content and grade are integral to assignment of the diagnosis of myelofibrosis. Even with careful assessment of all these features, not all cases fit neatly into the diagnostic entities; there is frequent overlap reflecting the biological disease continuum rather than distinct entities. Notwithstanding this, an accurate morphologic diagnosis in MPN is crucial due to the significant differences in prognosis between different subtypes and the availability of different therapies in the era of novel agents. The distinction between "reactive" and MPN is also not always straightforward and caution needs to be exercised given the prevalence of "triple negative" MPN. Here we describe the morphology of MPN including comments on changes with disease evolution and with treatment.

Increase in Frequency of Myeloid-Derived Suppressor Cells in the Bone Marrow of Myeloproliferative Neoplasm: Potential Implications in Myelofibrosis.

The Philadelphia-negative myeloproliferative neoplasms (MPNs), defined as clonal disorders of the hematopoietic stem cells, are characterized by the proliferation of mature myeloid cells in the bone marrow and a chronic inflammatory status impacting the initiation, progression, and symptomatology of the malignancies. There are three main entities defined as essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), and genetically classified by JAK2V617F, CALR, or MPL mutations. In MPNs, due to the overproduction of inflammatory cytokines by the neoplastic cells and non-transformed immune cells, chronic inflammation may provoke the generation and expansion of myeloid-derived suppressors cells (MDSCs) that highly influence the adaptive immune response. Although peripheral blood MDSC levels are elevated, their frequency in the bone marrow of MPNs patients is not well elucidated yet. Our results indicated increased levels of total (T)-MDSCs (CD33+HLA-DR-/low) and polymorphonuclear (PMN)-MDSCs (CD33+/HLA-DRlow/CD15+/CD14-) in the bone marrow and peripheral blood of all three types of MPNs malignancies. However, these bone marrow MDSCs-increased frequencies did not correlate with the clinical parameters, such as hepatomegaly, leukocytes, hemoglobin, or platelet levels, or with JAK2 and CALR mutations. Besides, bone marrow MDSCs, from ET, PV, and PMF patients, exhibited immunosuppressive function, determined as T-cell proliferation inhibition. Notably, the highest T-MDSCs and PMN-MDSC levels were found in PMF samples, and the increased MDSCs frequency strongly correlated with the degree of myelofibrosis. Thus, these data together indicate that the immunosuppressive MDSCs population is increased in the bone marrow of MPNs patients and may be implicated in generating a fibrotic microenvironment.

Myelofibrosis-type megakaryocyte dysplasia (MTMD) as a distinct category of BCR::ABL-negative myeloproliferative neoplasms. Challenges and perspectives.

In this Perspective, we discuss criteria for defining a new disease entity or variant of a recognized disease or disorder. We do so in the context of the current topography of the BCR::ABL-negative myeloproliferative neoplasms (MPNs) where two new variants are reported: clonal megakaryocyte dysplasia with normal blood values (CMD-NBV) and clonal megakaryocyte dysplasia with isolated thrombocytosis (CMD-IT). The cardinal feature of these variants is bone marrow megakaryocyte hyperplasia and atypia corresponding the WHO histological criteria for primary myelofibrosis (myelofibrosis-type megakaryocyte dysplasia-MTMD). Persons with these new variants have a different disease course and features from others in the MPN domain. In a broader context we suggest myelofibrosis-type megakaryocyte dysplasia defines a spectrum of related MPN variants including CMD-NBV, CMD-IT, pre-fibrotic myelofibrosis and overt myelofibrosis, which differ from polycythemia vera and essential thrombocythemia. Our proposal needs external validation and we stress the need for a consensus definition of the megakaryocyte dysplasia which is the hallmark of these disorders.

JAK2 V617F allele burden in polycythemia vera: burden of proof.

Polycythemia vera (PV) is a hematopoietic stem cell neoplasm defined by activating somatic mutations in the JAK2 gene and characterized clinically by overproduction of red blood cells, platelets, and neutrophils; a significant burden of disease-specific symptoms; high rates of vascular events; and evolution to a myelofibrosis phase or acute leukemia. The JAK2V617F variant allele frequency (VAF) is a key determinant of outcomes in PV, including thrombosis and myelofibrotic progression. Here, we critically review the dynamic role of JAK2V617F mutation burden in the pathogenesis and natural history of PV, the suitability of JAK2V617F VAF as a diagnostic and prognostic biomarker, and the utility of JAK2V617F VAF reduction in PV treatment.

Clinicopathologic characteristics of myeloproliferative neoplasms with JAK2 exon 12 mutation.

The presence of JAK2 exon 12 mutation was included by the 2016 World Health Organization (WHO) Classification as one of the major criteria for diagnosing polycythemia vera (PV). Few studies have evaluated the clinical presentation and bone marrow morphology of these patients and it is unclear if these patients fulfill the newly published criteria of 5th edition WHO or The International Consensus Classification (ICC) criteria for PV. Forty-three patients with JAK2 exon 12 mutations were identified from the files of 7 large academic institutions. Twenty patients had complete CBC and BM data at disease onset. Fourteen patients met the diagnostic criteria for PV and the remaining six patients were diagnosed as MPN-U. At diagnosis, 9/14 patients had normal WBC and platelet counts (isolated erythrocytosis/IE subset); while 5/14 had elevated WBC and/or platelets (polycythemic /P subset). We found that hemoglobin and hematocrit tended to be lower in the polycythemia group. Regardless of presentation (P vs IE), JAK2 deletion commonly occurred in amino acids 541-544 (62 %). MPN-U patients carried JAK2 exon 12 mutation, but did not fulfill the criteria for PV. Half of the patients had hemoglobin/hematocrit below the diagnostic threshold for PV, but showed increased red blood cell count with low mean corpuscular volume (56-60 fL). Three cases lacked evidence of bone marrow hypercellularity. In summary, the future diagnostic criteria for PV may require a modification to account for the variant CBC and BM findings in some patients with JAK2 exon 12 mutation.

Continuous Indexing of Fibrosis (CIF): improving the assessment and classification of MPN patients.

The grading of fibrosis in myeloproliferative neoplasms (MPN) is an important component of disease classification, prognostication and monitoring. However, current fibrosis grading systems are only semi-quantitative and fail to fully capture sample heterogeneity. To improve the quantitation of reticulin fibrosis, we developed a machine learning approach using bone marrow trephine (BMT) samples (n = 107) from patients diagnosed with MPN or a reactive marrow. The resulting Continuous Indexing of Fibrosis (CIF) enhances the detection and monitoring of fibrosis within BMTs, and aids MPN subtyping. When combined with megakaryocyte feature analysis, CIF discriminates between the frequently challenging differential diagnosis of essential thrombocythemia (ET) and pre-fibrotic myelofibrosis with high predictive accuracy [area under the curve = 0.94]. CIF also shows promise in the identification of MPN patients at risk of disease progression; analysis of samples from 35 patients diagnosed with ET and enrolled in the Primary Thrombocythemia-1 trial identified features predictive of post-ET myelofibrosis (area under the curve = 0.77). In addition to these clinical applications, automated analysis of fibrosis has clear potential to further refine disease classification boundaries and inform future studies of the micro-environmental factors driving disease initiation and progression in MPN and other stem cell disorders.

Neutrophil-lymphocyte ratio and carotid plaque burden in patients with essential thrombocythemia and polycythemia vera.

BACKGROUND AND AIMS: Chronic inflammation plays a critical role in the pathogenesis of myeloproliferative neoplasm (MPN), and inflammatory conditions are closely related to the development and exacerbation of atherosclerosis. This study aimed to compare carotid plaque burden and neutrophil-lymphocyte ratio (NLR) in the essential thrombocythemia (ET)/polycythemia vera (PV) and control groups. METHODS AND RESULTS: We retrospectively assessed carotid plaque burden and NLR in patients with ET/PV between January 2010 and September 2021 and propensity-score matched these patients to control subjects from the general population. All patients underwent carotid imaging using carotid ultrasonography for atherosclerosis screening. After 3:1 propensity-score matching, 140 patients in the control group were matched to 51 patients in ET/PV group. The mean NLR was significantly higher in the MPN group than in the control group (4.77 ± 3.96 vs. 1.93 ± 1.03, p < 0.001). The carotid plaque score was also higher in MPN group than in the control group (2.37 ± 1.47 vs. 1.94 ± 1.17, p = 0.038). CONCLUSION: Patients with PV/ET show a higher NLR and carotid plaque burden than the normal population. This reflected that PV/ET was a highly inflammatory and atherosclerotic condition expressing potentially increased cardiovascular risk.

Red Blood Cell Morphodynamics in Patients with Polycythemia Vera and Stroke.

Polycythemia vera (PV) is a Ph-negative myeloproliferative neoplasm (MPN) which is characterized by erythrocytosis and a high incidence of thrombotic complications, including stroke. The study aimed to evaluate red blood cell (RBC) morphodynamic properties in PV patients and their possible association with stroke. We enrolled 48 patients with PV in this cross-sectional study, 13 of which have a history of ischemic stroke. The control group consisted of 90 healthy subjects. RBC deformability and aggregation analysis were performed using a laser-assisted optical rotational red cell analyzer. The following parameters were calculated: aggregation amplitude (Amp), RBC rouleaux formation time constant (Tf), time of formation of three-dimensional aggregates (Ts), aggregation index (AI), rate of complete disaggregation (y-dis), and the maximal elongation of RBC (EImax). Statistical analysis was performed with the R programming language. There were significant differences in RBCs morphodynamics features between patients with PV and the control group. Lower EImax (0.47 (0.44; 0.51) vs. 0.51 (0.47; 0.54), p < 0.001) and γ-dis (100 (100; 140) vs. 140 (106; 188) s-1, p < 0.001) along with higher amplitude (10.1 (8.6; 12.2) vs. 7.7 (6.6; 9.2), p < 0.001) was seen in patients with PV compared with control. A statistically significant difference between PV patients with and without stroke in aggregation amplitude was found (p = 0.03). A logistic regression model for stroke was built based on RBC morphodynamics which performed reasonably well (p = 0.01). RBC alterations may be associated with overt cerebrovascular disease in PV, suggesting a possible link between erythrocyte morphodynamics and increased risk of stroke.

Clinical and Pathological Features of Renal Presentations in Polycythemia Vera.

BACKGROUND: Polycythemia vera (PV) is a common type of Philadelphia chromosome-negative chronic myeloproliferative disorder. PV-associated kidney disease is rarely reported and remains poorly understood. It has been observed that chronic kidney disease could be a risk factor for poor prognosis in PV. METHODS: We retrospectively analyzed the clinicopathological features of renal presentations in eight patients with confirmed PV-associated kidney disease. RESULTS: The eight patients were 6 males and 2 females, with a mean age of 46.4 ± 16.8 years. Six patients had a history of PV, with a duration range 0.5-16 years. The other two patients were newly diagnosed with PV simultaneously with glomerular disease. Seven patients conducted a JAK2 V617F mutation test, with a positive result in five. Proteinuria and renal dysfunction were the patients' main complaints, with only one having nephrotic syndrome and three having microscopic hematuria. The level of proteinuria ranged from 0.52-10.96 g/day. Three patients had advanced chronic kidney disease (CKD), two in stage 3b and one in stage 4, but only one patient had anemia. Three patients had monoclonal immunoglobulinemia, one patient with immunoglobulin (Ig) G kappa plus light chain lambda, one patient with IgG kappa, and one patient with IgG lambda. Five patients underwent a renal biopsy. The pathological diagnosis was IgA nephropathy in three, non-IgA mesangial proliferative glomerulopathy in one, and glomerular hypertrophy with ischemic renal injury in one patient. Glomerular ischemia, ischemic shrinkage, focal segmental sclerosis, and glomerulomegaly were common pathological features. Glomerular crescents and endocapillary proliferation were also observed. All patients were administered hydroxyurea, and seven were administered renin-angiotensin system inhibitors. During follow-up, one patient with uncontrolled PV developed secondary myelofibrosis and died, three patients were lost to follow-up, and four patients remained alive with CKD. CONCLUSIONS: Patients with untreated or uncontrolled PV could have massive proteinuria and advanced CKD, pathologically showing ischemic, sclerosing glomerular lesions with hypercellurity, glomerular crescents and endocapillary proliferation. IgA nephropathy was most commonly diagnosed. These findings deserve attention because early screening and effective control of PV may benefit the long-term kidney prognosis.

Platelet transcriptome identifies progressive markers and potential therapeutic targets in chronic myeloproliferative neoplasms.

Predicting disease progression remains a particularly challenging endeavor in chronic degenerative disorders and cancer, thus limiting early detection, risk stratification, and preventive interventions. Here, profiling the three chronic subtypes of myeloproliferative neoplasms (MPNs), we identify the blood platelet transcriptome as a proxy strategy for highly sensitive progression biomarkers that also enables prediction of advanced disease via machine-learning algorithms. The MPN platelet transcriptome reveals an incremental molecular reprogramming that is independent of patient driver mutation status or therapy. Subtype-specific markers offer mechanistic and therapeutic insights, and highlight impaired proteostasis and a persistent integrated stress response. Using a LASSO model with validation in two independent cohorts, we identify the advanced subtype MF at high accuracy and offer a robust progression signature toward clinical translation. Our platelet transcriptome snapshot of chronic MPNs demonstrates a proof-of-principle for disease risk stratification and progression beyond genetic data alone, with potential utility in other progressive disorders.

The Power of Extracellular Vesicles in Myeloproliferative Neoplasms: "Crafting" a Microenvironment That Matters.

Myeloproliferative Neoplasms (MPN) are acquired clonal disorders of the hematopoietic stem cells and include Essential Thrombocythemia, Polycythemia Vera and Myelofibrosis. MPN are characterized by mutations in three driver genes (JAK2, CALR and MPL) and by a state of chronic inflammation. Notably, MPN patients experience increased risk of thrombosis, disease progression, second neoplasia and evolution to acute leukemia. Extracellular vesicles (EVs) are a heterogeneous population of microparticles with a role in cell-cell communication. The EV-mediated cross-talk occurs via the trafficking of bioactive molecules such as nucleic acids, proteins, metabolites and lipids. Growing interest is focused on EVs and their potential impact on the regulation of blood cancers. Overall, EVs have been suggested to orchestrate the complex interplay between tumor cells and the microenvironment with a pivotal role in "education" and "crafting" of the microenvironment by regulating angiogenesis, coagulation, immune escape and drug resistance of tumors. This review is focused on the role of EVs in MPN. Specifically, we will provide an overview of recent findings on the involvement of EVs in MPN pathogenesis and discuss opportunities for their potential application as diagnostic and prognostic biomarkers.

Lower response to BNT162b2 vaccine in patients with myelofibrosis compared to polycythemia vera and essential thrombocythemia.

In a population of 42 Philadelphia negative myeloproliferative neoplasm patients, all on systemic active treatment, the likelihood of responding to anti-SARS-CoV-2 BNT162b2 vaccine at 2 weeks after the second dose was significantly lower in the ten patients with myelofibrosis compared to the 32 with essential thrombocythemia (n = 17) and polycythemia vera (n = 15) grouped together, both in terms of neutralizing anti-SARS-CoV-2 IgG titers and seroprotection rates (32.47 AU/mL vs 217.97 AU/mL, p = 0.003 and 60% vs 93.8%, p = 0.021, respectively). Ruxolitinib, which was the ongoing treatment in five patients with myelofibrosis and three with polycythemia vera, may be implicated in reducing vaccine immunogenicity (p = 0.076), though large prospective study is needed to address this issue.

Improving the investigative approach to polycythaemia vera: a critical assessment of current evidence and vision for the future.

Polycythaemia vera is a challenging disease to study given its low prevalence and prolonged time-to-event for important clinical endpoints such as thrombosis, progression, and mortality. Although researchers in this space often rise to meet these challenges, there is considerable room for improvement in the analysis of retrospective data, the development of risk-stratification tools, and the design of randomised controlled trials. In this Viewpoint, we review the evidence behind the contemporary approach to risk stratification and treatment of polycythaemia vera. Frameworks for using data more efficiently, constructing more nuanced prognostic models, and overcoming challenges in clinical trial design are discussed.

Repeat exit site infection in peritoneal dialysis patient with polycythemia vera - a case report.

BACKGROUND: Infectious complications of peritoneal dialysis (PD) remain a common cause of catheter loss and discontinuation of PD. Exit site infection (ESI) constitutes a significant risk factor for PD-related peritonitis and determination of predisposing states is relevant. We here present a case of repeat ESI due to Pseudomonas aeruginosa in a PD patient with skin changes in the course of polycythemia vera (PV). CASE PRESENTATION: A 73-year-old PD patient with chronic kidney disease secondary to renal amyloidosis and ankylosing spondylitis, presented to the nephrology unit with signs of ESI. In 2006 he was diagnosed with PV and since then has was successfully treated with hydroxyurea; however, he reported recurrent episodes of developing skin nodules in the course of the disease. Exit site swab yielded Pseudomonas aeruginosa and the infection developed in the ulcerated PV nodule that appeared in exit site 2 weeks earlier. Patient was treated with intraperitoneal amikacin and oral ciprofloxacin, however, due to neurological complications, the treatment had to be interrupted and finally catheter was removed. Similar episode of ESI with Pseudomonas aeruginosa developed in the patient two years earlier and also required catheter removal. CONCLUSION: This is the first case report demonstrating the development of ESI on the polycythemia vera skin lesion in this area. Skin manifestations of PV might be a predisposing factor to ESI in PD patients.

Ruxolitinib discontinuation in polycythemia vera: Patient characteristics, outcomes, and salvage strategies from a large multi-institutional database.

Ruxolitinib is approved for the treatment of patients with polycythemia vera (PV) who are intolerant or resistant to hydroxyurea. While ruxolitinib discontinuation in myelofibrosis is associated with dismal outcomes, the analogous experience in PV has not been reported. Using a large, multi-institutional database of PV patients, we identified 93 patients with PV who were treated with ruxolitinib, of whom 22 discontinued therapy. Adverse events were the primary reason for discontinuation. After a median follow-up of 18.2 months following ruxolitinib discontinuation, no patients experienced a thrombotic event. One patient died 20.8 months after discontinuation. As compared with the 71 patients who were still receiving treatment with ruxolitinib at last follow up, patients who discontinued ruxolitinib were older at time of treatment initiation (67.5 versus 64.8 years, p = 0.0058), but had similar patient and disease characteristics. After discontinuation, only 4 patients (18 %) received subsequent cytoreductive therapy, including hydroxyurea in one patient and pegylated interferon α-2a in three patients. In stark contrast to the experience in myelofibrosis, discontinuation of ruxolitinib in PV was associated with generally favorable outcomes. However, there is a lack of available salvage therapies, highlighting the need for further therapeutic development in PV.