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Abstract Background Although myeloproliferative neoplasm-unclassifiable (MPN-U) stands as a distinct entity in the International Consensus Classification (ICC) for myeloproliferative neoplasms, its diagnosis and management remain challenging due to the overlapping clinico-pathological characteristics with other MPNs. In the current study, we describe a well-defined cohort of patients with MPN-U and compare their presentation and outcome with those of a separate cohort with essential thrombocythemia (ET). Methods Diagnostic criteria were according to the ICC (Blood 2022; 140:1200). Non-driver mutations were screened by next-generation sequencing (NGS) and were available for subsets of patients with MPN-U or ET. Conventional statistical methods were employed, using JMP Pro 18.0.0 software SAS Institute, Cary, NC, USA. Results Thirty Mayo Clinic patients who met the ICC criteria for MPN-U (median age 64 years; range 33-94; 60% males) and 658 with ET (median age 60 years; range 18-90; 36% males) were included in the current study. Table 1 outlines a comparative list of presenting characteristics (including mutations), clinical outcomes, and treatment details between patients with MPN-U vs. ET. JAK2 mutations were significantly more prevalent in MPN-U cohort (87% vs. 63% in ET; p<0.01) and CALR mutations less common (3% vs. 25%; p<0.01). Other somatic mutations, including ASXL1, SRSF2, and EZH2 were more frequently observed in the MPN-U group (p<0.05 in all). Abnormal karyotype was reported in 14% of patients with MPN-U vs. 8% with ET (p=0.3). Compared to patients with ET, those with MPN-U were more likely to be males (60% vs. 36%; p<0.01) and, as expected from disease presentation and indications for diagnostic work up of suspected cases, present with venous thrombosis (43% vs. 10%; p<0.01) and palpable splenomegaly (40% vs. 8.5%; p<0.01). Similarly, they were less likely to display thrombocytosis or leukocytosis (p<0.01 for both). Portal, superior mesenteric, and hepatic vein thromboses constituted 77% (vs. 27% in ET; p<0.01) of the venous events in MPN-U; in contrast, the incidences of pre-diagnosis arterial events were similar between the two groups (10% vs. 12%; p=0.7). At a median follow-up of 2.8 years in the MPN-U cohort, 6 (20%) deaths and 1 (3%) leukemic transformation were documented while 3 (10%) patients progressed to meet the diagnostic criteria for polycythemia vera. Four (13%) patients with MPN-U experienced venous thrombo-embolic events (VTEs) including 2 with recurrent VTEs, despite active therapy with systemic anticoagulation and aspirin. The ET cohort was followed for a median of 7.3 years with 161 (24%) deaths, 23 (3%) leukemic transformations, and 66 (10%) VTEs. Among the latter, 27 (41%) had recurrent episodes despite treatment with systemic anticoagulation, aspirin, and/or cytoreductive drugs. Notably, patients with MPN-U were less likely to receive aspirin and cytoreductive therapy during the clinical course (77% vs. 91% and 37% vs. 79%; p<0.01 for both, respectively). Long-term survival rates were similar between MPN-U and ET (15-year survival 70% vs. 65%; p=0.1). Consistent with previous associations seen in ET, patients with JAK2-mutated MPN-U were more likely to be males (58% vs. 30%; p<0.01, Table 2), and display a lower platelet and leukocyte count at the time of diagnosis (p<0.01 for both). These patients were also more likely to present with venous thrombosis (50% vs. 12% in JAK2 wild-type cases; p<0.01) and palpable splenomegaly (46% vs. 9%; p<0.01). Of note, the incidences of pre- and post-diagnosis arterial and post-diagnosis venous events were similar between JAK2-mutated MPN-U vs. JAK2-mutated ET patients (12% vs. 14%; p=0.7, 4% vs. 12.5%; p=0.1, and 15% vs. 12%, p=0.6, respectively). Similarly, long-term outcomes including deaths (15% vs. 26%; p=0.2), leukemic transformations (4% vs. 4%; p=0.9), and 15-year survival rates (78% vs. 60%; p=0.9) were also comparable between the two groups. Conclusion The findings from the current study were not unexpected and underline the ongoing challenges that face formal classification systems in formulating accurate diagnostic criteria for patients with MPN-U whose clinical presentation is biased by disease definitions. Regardless, it is comforting to document a favorable long-term outcome that appeared to be similar to that of ET while venous thrombosis remains a treatment challenge in both groups.

Abstract Background: JAK2-unmutated erythrocytosis constitutes a spectrum of hereditary and acquired entities. We assessed the diagnostic yield and utility of next generation sequencing panel for hereditary erythrocytosis (NGS-HEP) in adults with JAK2-unmutated and otherwise unexplained erythrocytosis (OUE). Additionally, we describe the clinical phenotype, genetic findings, treatment approaches, and thrombotic outcomes in this cohort. Methods: Adults (≥18 years) withOUE, defined as hemoglobin (Hgb) >16.5 g/dL or hematocrit (Hct) >49% in males and Hgb >16 g/dL or Hct > 48% in females, negative for JAK2 exon 12-15mutations, absence of acquired cause, and high-oxygen affinity variants, underwent NGS-HEP; the gene panel included ACO1, ANKRD26, BHLHE41, BPGM, CYB5A, CYB5R3, EGLN1, EGLN2, EGLN3, EPAS1, EPO, EPOR, GFI1B, HIF1A, HIF1AN, HIF3A, JAK2, KDM6A, PFKM, PIEZO1, PKLR, SH2B3, SOCS3 and VHL, including deletion/duplication analysis.Variant curation was performed using ACMG-AMP guidelines and classified as pathogenic or variants of uncertain significance (VUS). Family and thrombosis history, and symptoms were recorded. Results: A total of 44 adult patients with JAK2-unmutated OUE (median age 48 years, range 21–79; 34 males [77%]) underwent NGS-HEP at the Mayo Clinic between March 2023 and June 2025. Variants were identified in 27 patients (61%), with a single variant detected in 19 (70%) and two distinct variants in 8 (30%). 26 variants (96%) were classified as heterozygous VUS and involved PIEZO1 (n=9, 20%; median variant allele frequency [VAF] 47.1%), HIF1A (n=8, 18%;46.2%), ANKRD26 (n=4, 9%;43.7%), SH2B3 (n=3, 7%; 49.9%), HIF3A (n=3, 7%;51.4%), EGLN2 (n=2, 5%;47.9%), BPGM (n=1, 2%; 50.4%), VHL (n=1, 2%; 41.8%), EPO (n=1, 2%; 45.4%) and BHLHE41 (n=1, 2%; 54.2%), while a pathogenic mutation in PKLR was detected in one patient. Presenting median Hgb/Hct values for males and females, respectively were 18.3 g/dL/53.5% and 16.9 g/dl/51.2% in variant- positive cases, and 17.8 g/dl/53.1% and 16.1 g/dl/49.3% in variant-negative cases (p>0.05). Median serum erythropoietin (sEpo) levels were 8.35 mIU/mL (range: <1-52.9) vs 8.8 mIU/mL (range: 1.6-54.7), respectively (p=0.67). Notably, sEpo was < 1 mIU/mL in a patient with ANKRD26/HIF3A VUS. Family history of erythrocytosis was documented in 5 patients (11%). Hyperviscosity-related symptoms were reported in 24 patients (55%), with no significant difference in prevalence between variant-positive and variant-negative cases (59% vs 47% p=0.42). Also, there was no significant association between the presence of symptoms and Hgb or Hct levels (p>0.05). Eight thrombotic events occurred in 7 patients (16%; median age 44 years; 71% males; 4 arterial and 4 venous; 3 provoked deep venous thromboses). Five (71%) of the 7 patients harbored VUS in PIEZO1, PIEZO1/HIF1A, ANKRD26, SH2B3, or BHLHE41. Notably, two younger male patients experienced arterial events in the absence of identifiable risk factors: a 32-year-old with PIEZO1/HIF1A VUS (VAF 46.2%/47.6%; sEpo 10.3 mIU/mL) suffered a myocardial infarction, and a 35-year-old with SH2B3 VUS (VAF 50.2%; sEpo 3.7 mIU/mL) had recurrent cerebrovascular accident despite phlebotomy, aspirin and apixaban. Overall, there was no significant association between thrombosis and the presence of a variant (19% in variant-positive vs 12% in variant-negative, p=0.54). Active therapies included phlebotomy in 22 patients (50%), antiplatelet agents in 23 (52%), systemic anticoagulation in 5 (11%), and hydroxyurea in one patient (2%). Among those who received phlebotomy, 10 patients (45%) reported improvement in symptoms, however, symptom relief did not correlate with Hgb or Hct levels (p>0.05). A separate analysis of 9 patients with heterozygous PIEZO1 VUS, none of whom had undergone splenectomy, showed a numerically higher incidence of symptoms (67% vs 51%) and thrombosis (22% vs 14%) compared to those without PEIZO1 variants, although the differences were not statistically significant (p>0.05). Conclusion: NGS-HEP yielded a molecular diagnosis in 61% of adults with JAK2-unmutated and OUE with heterozygous PIEZO1 variants being the most frequent. No consistent association was observed between variant status and presence of hyperviscosity-related symptoms or thrombotic events. These findings underscore the importance of NGS-HEP testing in the diagnostic workup of JAK2-unmutated and OUE and continued investigation into novel candidate genes.

Abstract Background: The International Prognostic Scoring System (IPSS; Cervantes et al. Blood 2009;113) and dynamic IPSS (DIPSS; Passamonti et al. Blood 2010;115:1703) were developed about 15 years ago, based on 5 clinical risk factors: age >65 years, hemoglobin <10 g/dl, white blood cell count (WBC) >25 x 109/L, peripheral blood (PB) blasts ³1%, and constitutional symptoms. DIPSS-independent genetic risk factors have since been incorporated into molecular risk models with higher predictive accuracy (Guglielmelli et al. JCO 2018;36:310; Tefferi et al JCO 2018;36:1769). Because of its accessibility, DIPSS is often preferred for use in patient selection for clinical trials. The objective of the current study was to revise DIPSS by incorporating new and refined clinical risk variables and excluding those that are subjective (e.g., symptoms) or could exaggerate the need for therapy for older adults (e.g., age). Methods: Diagnostic criteria were according to the International Consensus Classification (Arber et al. Blood 2022; 140:1200). Severity of anemia was adjudicated by the Revised International Working Group-European LeukemiaNet criteria (Blood 2024; 144:1813). Receiver operating characteristic curve analysis was utilized to determine the optimal cutoff points for age, WBC, PB blast percentage, absolute monocyte count (AMC), and platelet count. Data were collected retrospectively corresponding to the time of first referral, which, in the majority of cases, coincided within one year of initial diagnosis and before initiation of cytoreductive therapy. Survival analysis was censored for allogenic stem cell transplantation (ASCT). Time-specific AUC was computed at pre-defined landmark time points and used to compare model performance between DIPSS and the revised model (DIPSS-R). Results: The discovery cohort from the Mayo Clinic included 1,073 patients (median age 65 years; 61% males) and the validation cohort from Italy 635 patients (median age 63 years; 60% males). In the discovery cohort, median follow-up (% deaths, AML, ASCTs) was 45 months (49%, 10%, 17%) and in the validation cohort 60 months (42%, 11%, 5%). In univariate analysis, the following correlated with inferior survival, in both the discovery and validation cohorts, with significance confirmed by multivariable analysis (MVA) in the discovery cohort: severe anemia (transfusion-dependent or hemoglobin level <8 g/dL in women or <9 g/dL in men), moderate anemia (hemoglobin level 8 to <10 in women or 9 to <11 in men), marked leukocytosis (WBC ≥25 x 109/L), moderate leukocytosis (WBC 11 to <25 x 109/L), age >65 years, PB blast ≥2%, AMC ≥1 x 109/L, platelet count <150 x 109/L, constitutional symptoms, and male sex. After excluding age, sex, and constitutional symptoms, MVA-derived HRs (95% CI) were 3.5 (2.8-4.4) for severe anemia, 2.0 (1.5-2.5) moderate anemia, 2.4 (1.8-3.3) marked leukocytosis, 1.5 (1.2-1.9) moderate leukocytosis, 1.7 (1.4-2.1) PB blast ≥2%, 1.5 (1.2-2.0) AMC ≥1 x 109/L, and 1.5 (1.2-1.8) platelet count <150 x 109/L. An HR-weighted risk model was subsequently developed (Figure 1) and validated (Figure 2) using the discovery and validation cohorts, respectively: low risk (0 points; N=205; median survival 226 months), intermediate-1 risk (1 point; N=253; 131 months), intermediate-2 risk (2-3 points; N=457; 62 months), and high risk (4-7 points; N=153; 31 months); the corresponding median survival estimates in the validation cohort were “not reached”, 205 months, 96 months, and 44 months (inter-risk category p values <0.01 in all instances). Ten-year predictive accuracy was comparable between DIPSS-R (AUC 0.78) and DIPSS (AUC 0.78), despite the exclusion of age and constitutional symptoms, as risk variables, from the revised model. Furthermore, predictive performance was superior for DIPSS-R vs. DIPSS when analysis was stratified by age: >65 years (AUC 0.77 vs. 0.74) and ≤65 years (AUC 0.71 vs. 0.66). MVA that included high (ASXl1, SRSF2, U2AF1) and low (CALR type 1/like) risk mutations, karyotype, and DIPSS-R variables showed independent prognostic contribution from all except thrombocytopenia (p=0.2; Figure 3). Conclusion: DIPSS-R utilizes disease-related, as opposed to host-related, predictors of survival whose prognostic contribution was independent and complementary to genetic risk factors. The model preserves global applicability and allows for a more accurate assessment of disease severity for practice and research purposes.

Summary Chronic myelomonocytic leukaemia (CMML) is a morphological hybrid between myelodysplastic and myeloproliferative neoplasms and is characterized by clonal monocytosis. Myeloid neoplasm‐associated somatic mutations are present in >90% of patients with CMML but are not diagnostically specific but a subset are prognostically relevant. Current drug therapy in CMML is mostly palliative and not disease‐modifying. Allogeneic haematopoietic stem cell transplantation is a potentially curative treatment modality in CMML and its timing is determined by formal prognostic models including the recently developed BLAST and BLAST‐molecular risk models.

ABSTRACT Disease Overview JAK2 unmutated erythrocytosis encompasses a heterogeneous spectrum of hereditary and acquired entities. Diagnosis The foremost step is excluding polycythemia vera (PV) with JAK2 mutation screening (exons 12–15). Apparent polycythemia such as physiological outliers or relative polycythemia secondary to volume contraction should be considered. A historical overview of hematocrit (Hct) and hemoglobin (Hgb) levels helps distinguish longstanding from acquired erythrocytosis. Serum erythropoietin (Epo) levels are variably informative. Hereditary Erythrocytosis Hereditary erythrocytosis should be considered in longstanding erythrocytosis with a positive family history; causes include EPOR mutations (subnormal Epo), high oxygen affinity hemoglobin variants, PIEZO1 mutations, 2,3‐bisphosphoglycerate deficiency, methemoglobinemia, and germline oxygen sensing pathway mutations ( HIF2A ‐ PHD2 ‐ VHL ). Acquired Erythrocytosis Acquired erythrocytosis results from central (cardiopulmonary disease) or peripheral (renal artery stenosis) hypoxia, Epo‐producing tumors (renal cell carcinoma) or drugs (testosterone, sodium glucose co‐transporter‐2 inhibitors (SGLT2‐i), erythropoiesis stimulating agents). Idiopathic Erythrocytosis Idiopathic erythrocytosis is an ill‐defined terminology that presumes the existence of an increased Hgb/Hct level without an identifiable etiology. Management Cytoreductive therapy should be avoided. Phlebotomy should be considered for symptom control. Cardiovascular risk optimization and low‐dose aspirin are advised, while the role of HIF2A inhibitors remains unclear. Recent Advances EPO mutations which produce hyperactive, hepatic‐like Epo were identified. In cases with negative workup but high clinical suspicion, an expanded next generation sequencing panel for hereditary erythrocytosis is recommended. Among drugs, SGLT2‐i‐associated erythrocytosis is increasingly recognized. Future Directions Advances in molecular hematology are expected to improve the characterization of “idiopathic erythrocytosis”. Results from prospective studies are needed to elucidate the underlying pathology and guide management.