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Abstract Recent updates to the monocyte count thresholds for diagnosing chronic myelomonocytic leukemia (CMML) have recognized oligomonocytic CMML (OM-CMML) as an early stage within the CMML spectrum. However, the clinical validity of these revised criteria remains uncertain without considering biological factors and might misclassify myelodysplastic syndromes (MDS) with mild monocytosis as CMML. To characterize the genomic landscape of OM-CMML, we evaluated OM-CMML (n=249), MDS (n=304) and myelodysplastic CMML (MD-CMML, n=358) patients (pts) treated at three academic institutions. Targeted next-generation sequencing was performed on bone marrow (BM) at initial diagnosis or first referral. OM-CMML was characterized by lower frequencies of biallelic TET2 (biTET2) and RAS pathway (RASp) gene mutations (BRAF, CBL, KRAS, NF1, NRAS and PTPN11) compared to MD-CMML (p<0.001) but higher frequencies than MDS (p<0.001). BM monocyte frequencies, assessed morphologically, were higher in OM-CMML than in MDS (p<0.001) but lower than in MD-CMML (p<0.001), indicating that OM-CMML is a heterogenous group including true CMML and distinct MDS subsets. Unsupervised hierarchical clustering based on genomic alterations across all cases (n=911) identified three distinct clusters: 1) TP53 alterations (predominantly multihit TP53) and complex karyotype (TP53-complex); 2) biTET2 or TET2-SRSF2 co-mutation (biTET2/SRSF2); 3) a heterogenous group lacking these features (not otherwise specifiable [NOS]). Among OM-CMML pts, 11%, 34% and 55% belonged to these clusters, respectively. Model-based unsupervised clustering further identified 9 unique clusters that could be consolidated into 6 groups: 1) TP53-complex; 2) biTET2/SRSF2; 3) SF3B1 (without biTET2 or SRSF2); 4) SRSF2 without TET2 and frequent IDH2 mutations (SRSF2-IDH2); 5) ASXL1 and U2AF1 mutations and copy number alterations (CNA-ASXL1/U2AF1); 6) no somatic mutations (No mutation). biTET2/SRSF2 group exhibited monocytic bias with higher AMC, PB and BM monocyte % even among OM-CMML cases. This bias was not attributed to higher RAS pathway mutation frequencies, as biTET2/SRSF2 cases had similar RASp mutation frequencies but lower median variant allele frequencies for RASp mutations compared to NOS cases (9% vs. 21%, p=0.029). PB monocyte partitioning using flow cytometry (FC) in a subset of OM-CMML pts (n=50) demonstrated higher frequency of classical monocytosis (>94% CD14+/CD16- monocytes) in biTET2/SRSF2 vs other OM-CMML. To further determine if biTET2/SRSF2 genotype defines a distinct biological subgroup, we performed RNA-sequencing of BM CD34+ cells from healthy donors (n=15) and pts with OM-CMML (n=12), MD-CMML (n=14) and MDS (n=55). Unsupervised clustering based on gene expression profiles revealed that biTET2/SRSF2 clustered irrespective of their diagnostic group with greater dispersion being observed based on diagnosis. MDS and CMML with biTET2/SRSF2 had less differentially expressed genes compared to MDS and CMML with biTET2/SRSF2 relative to other MDS and CMML genotypes. Among 138 OM-CMML pts in whom progression to overt CMML was evaluable, 37 (27%) progressed to overt CMML of which 26 (70%) belonged to the biTET2/SRSF2 group. Competing risk analysis confirmed higher cumulative incidence and shorter time to CMML progression among biTET2/SRSF2 OM-CMML. Using ElasticNet regression we identified BM monocytosis and 14 genomic variables (including biTET2 and TET2-SRSF2) as predictors of overt CMML progression among OM-CMML. We developed a score-based model able to predict progression with high reliability (AUC 0.805, sensitivity 0.70, specificity 0.76). Model accuracy was confirmed by optimism-corrected bootstrap internal validation (AUC 0.668). Monocytic bias, BM classical monocytosis (>94% CD16- monocytes) and higher overt CMML progression in biTET2/SRSF2 was confirmed in a validation cohort (28, 66 and 22 pts with OM-CMML, MDS and MD-CMML, respectively). BM monocytosis of biTET2/SRSF2 was confirmed in this cohort with morphologic and FC monocyte frequency quantification having high concordance (r=0.737, p<0.001). Genomic subgroup analysis discriminated survival outcomes more reliably than diagnostic categories alone. Our findings suggest that the presence of biTET2 or TET2-SRSF2 co-mutations, combined with BM monocytosis, can more reliably identify true CMML cases within the OM-CMML spectrum. Integrating genomic determinants into the CMML diagnostic framework enhances diagnostic accuracy.

Abstract Introduction: The 5th edition of the World Health Organization (WHO-5) and the International Consensus Classification (ICC) recognize TP53-mutated myeloid neoplasms (MN) as a distinct disease category, reflecting their uniformly dismal outcomes and the urgent need for novel therapeutic strategies. However, both classifications do not include TP53-non-mutatedcases with 17p (TP53 locus) loss, despite emerging evidence that such lesions may confer biologic and clinical behaviour similar to TP53-mutated disease. Our preliminary work demonstrated that 17p loss without TP53 mutation in therapy-related MN (t-MN) is associated with poor survival, approximating that of TP53-mutated cases (Hiwase et al., Blood 2023). Yet, the true frequency and prognostic impact of 17p loss in TP53-non-mutatedMN remains largely undefined, representing a critical gap in current disease classification and risk stratification. To address this critical knowledge gap, we aim to define the frequency and prognostic impact of 17p loss across the TP53 locus in TP53-non-mutatedMN, including both myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Finally, we compared the survival outcomes of this cohort with those of TP53-mutated MN. Method This international collaborative study was designed to evaluate the frequency and prognostic significance of 17p loss in TP53-non-mutatedMN. We included MDS and AML patients who had conventional cytogenetic analysis and did not have TP53-mutation(TP53-non-mutated)by next-generation sequencing (NGS) with sensitivity of ≥2% variant allele frequency (VAF). These cases were systematically reviewed for 17p loss using karyotype data, and where available, fluorescence in situ hybridization (FISH) and SNP array were used to confirm the 17p/TP53 locus deletion. To assess the clinical impact, we compared the overall survival (OS) of TP53-non-mutatedcases with 17p loss against a reference cohort of TP53-mutated MNs with VAF ≥2% (n=603) (South Australian Myeloid Neoplasm Registry (SA-MN) and Mayo Clinic, Rochester, USA). Results We screened 8876 cases of TP53-non-mutatedMN cases across an international cohort comprising the SA-MN (Australia; n=1046), the Düsseldorf MDS Registry (Germany; n=545), Munich Leukaemia Laboratory (Germany; n=5785), and King's College Hospital, (United Kingdom; n=1500). We identified 85 (1%) cases with 17p loss across the TP53 locus, representing the largest international cohort to define the frequency of 17p loss in TP53-non-mutatedMN. Complex karyotype was present in 40% (n=34) of cases, frequently co-occurring with del(5q) and del(7q). Clinically, most cases were MDS (n=69; 81.2%), including MDS-IB1 (n=19), MDS-IB2 (n=15), MDS-LB (n=16), t-MDS (n=8), MDS/MPN overlap (n=3), CCUS (n=1), or unspecified MDS (n=7), while AML accounted for 16 cases (18.8%). Among evaluable cases, the majority were male (78.6%) and had a median age of 66.5 years at diagnosis. The most frequent cytogenetic abnormality causing 17p loss across the TP53 locus was monosomy 17 (39%), followed by i(17q) (29%), add (17p) (14%), derivative chromosome 17 from unbalanced translocations (11%), and del(17p) (7%). The majority represented de-novo MN (60%), with t-MN in 40% of cases. Median OS of evaluable 17p loss in TP53-non-mutatedcases (n=28; 10.2 months) was poor and comparable to TP53-mutated MN (n=603, 8.7 months; P=0.42). Similarly, survival censored for a stem cell transplant was also equally poor (10.4 vs. 8.7 months; P=0.34) in both groups. Complex vs. non-complex karyotype (7.7 vs. 10.7 months; P=0.68) did not significantly influence the OS of 17p loss. Median OS of MDS cases tended to be longer compared to AML (16.7 vs. 9.1 months; P=0.11), although not statistically significant. Conclusion: In this largest international study, 1% of TP53-non-mutatedMNs harbor 17p loss across the TP53 locus, displaying poor survival equivalent to TP53-mutated disease. Exclusive reliance on NGS to identify TP53 mutations misses these high-risk structural TP53 abnormalities, which are readily detectable by conventional karyotyping. These findings provide compelling evidence to include TP53-non-mutatedcases with 17p loss in the next revision of the classifications, enabling accurate prognostication and equitable access to curative and clinical trials of novel therapies.

Abstract Background: TP53-mutated (TP53mut) myeloid neoplasms (MN) are a biologically and clinically distinct subgroup of blood cancers associated with extremely poor outcomes and limited response to conventional therapies. In some patients, MN are preceded by clonal cytopenia of undetermined significance (CCUS). Prior cytotoxic exposure (treatment related, TR) is an independent predictor for survival in CCUS (Li et al, Blood Adv. 2024). As TP53mut CCUS and MN are enriched in TR cases, the interaction between cytotoxic exposure and TP53mut status on the risk of progression and survival remains unclear. Methods: Adult patients with CCUS who were evaluated at the Mayo Clinic or South Australian myelodysplastic syndrome (MDS) Registry (Adelaide, Australia) were included. CCUS and MDS were defined according to the 5th edition of the WHO classification for MN (Khoury et al, 2022), except for the exclusion of cases harboring MDS-defining cytogenetics. Next-generation sequencing was performed using a targeted panel covering genes commonly mutated in MN. In cases with >1 TP53mut, VAFs were summed. Survival outcomes were assessed by Kaplan-Meier with log-rank testing. Univariate and multivariate analyses were estimated using Cox proportional hazards regression. Results: Of 320 CCUS cases, 50 (15.6%) harbored TP53mut (TP53mut CCUS), whereas 270 (84.4%) were wild-type (TP53wt CCUS). Median age at diagnosis was 71 years (range 16-99). TP53mut CCUS had a greater proportion of females (60% vs 33.7%, p<0.001) and TR (66% vs 23%, p<0.001) compared to TP53wt. Hematologic parameters including hemoglobin, red cell distribution width, mean corpuscular volume, white blood count, absolute neutrophil count, platelets, and bone marrow blasts were comparable between the two groups. Almost all TP53mut (n=48, 96%) were located in the DNA-binding domain and the median variant allele frequency (VAF) was 9% (range 2-46%). Five (10%) cases harbored 2 TP53mut. The distribution of TP53mut VAF among patients was as follows: ≤2.5% (n=2), 2.5-5% (n=10), 5-10% (n=19), 10-20% (n=11), and ≥20% (n=7). The proportion of cases with cytogenetic abnormalities was smaller in TP53mut compared to TP53wt CCUS(12% vs 25.9%, p=0.035). Both cohorts had a comparable median number of mutations: 2 (range 0-7) in TP53mut and 2 (range 0-5) in TP53wt. The most common co-mutations in TP53mut CCUS were TET2 (n=8, 17.8%), PPMD1 (n=7, 15.6%), DNMT3A (n=6, 13.3%), ASXL1 (n=6, 13.3%), and SRSF2 (n=4, 8.9%). At last follow up, the rate of progression was comparable between TP53mut and TP53wt CCUS (12% vs 22%, p=0.13). Median time to MN progression was not reached in TP53mut and 6.25 months in TP53wt CCUS (p=0.5197). A significantly higher proportion of TP53mut cases acquired complex cytogenetics compared to TP53wt cases (33% vs 2%, p=0.024) at MN progression. Of 20 deaths observed in the TP53mut cohort, non-myeloid hematologic malignancies (n=6, 30%), infections (n=5, 25%), and TP53mut MN (n=4, 20%) were the 3 most common causes. With a median follow-up of 4 years (IQR 1.2-4.8), median OS was longer in de novo (dn) cases (8.7 years for dn TP53wt; not reached for dn TP53mut) and shorter in TR cases (3.3 years for TR TP53wt; 4.8 years for TR TP53mut). Notably, TP53mut showed no independent prognostic impact in the entire cohort, or when stratified by TR vs dn CCUS. On multivariate analysis that included TP53mut status, factors independently associated with inferior OS included prior therapy (HR 2.21, 95% CI 1.41-3.45, p<0.001), anemia (HR 4.1, 95% CI 1.88-8.99, p<0.001), and thrombocytopenia (HR 2.38, 95% CI 1.43-3.95, p<0.001). Similarly, TR CCUS (HR 1.6, 95% CI 1.06-2.4, p=0.03) and anemia (HR 2.15, 95% CI 1.25-3.7, p=0.006) were independently associated with inferior progression free survival. Conclusion: TP53 mut in CCUS did not independently predict inferior survival or increase leukemic transformation. While TP53mut CCUS were less likely to harbor cytogenetic abnormalities at diagnosis, significantly more cases harbored genomic instability at leukemic transformation. The poor outcomes attributed to TP53mut CCUS were driven by prior cytotoxic exposure, cytopenia – particularly anemia, and non-leukemic causes of death. These findings suggest risk stratification in CCUS should prioritize clinical context over TP53mut status alone.

Abstract Background: Isocitrate dehydrogenase (IDH) mutations are frequently implicated in the development of acute myeloid leukemia (AML). In a recent study (Salman MY, Blood Adv 2025), it was reported that IDH2 mutation subtypes, R140 and R172, may respond differently to intensive chemotherapy. In this study, we evaluated mid-cycle and end-of-cycle bone marrow blast responses in AML patients with IDH2 R140, IDH2 R172, and IDH1 R132 mutations, aiming to determine whether these subtypes exhibit different response dynamics. Methods: We reviewed the charts of AML patients diagnosed at Mayo Clinic, Rochester, between the years 2016 - 2023 that had available next generation sequencing (NGS) data. Institutional review board approval was obtained. All data were recorded at the time of AML diagnosis. Patients without molecular data or not treated with intensive chemotherapy (7+3 or CPX-351) were excluded from this study. Bone marrow blast percentages were assessed at mid-induction (days 14-21) and end of cycle (counts recovery). Blast clearance dynamics and complete remission rates were compared across IDH mutation subgroups and correlated with outcomes. GraphPad Prism 10.0.0 was used for statistical analysis. Results: Out of 381 AML patients, 31 had IDH2 mutations (23 with R140 and 8 with R172), and 8 had IDH1 mutations (all R132). Among patients with IDH2 mutations, those with R172 had numerically higher median mid-cycle bone marrow blast percentage compared to R140 (10% vs. 4%, p = 0.37). There was no significant difference in mid-cycle blast percentages between patients with IDH1 and IDH2 mutations (4.5% vs. 4%, p = 0.8). At mid-cycle, 68% of patients with IDH2 R140 had blast counts <5%, compared to 38% of those with IDH2 R172, suggesting a trend for faster mid-cycle clearance in the R140 group, though not statistically significant (OR 3.57, p = 0.2). In a broader comparison, 60% of IDH2-mutated patients and 50% of IDH1-mutated patients achieved blast clearance (blasts <5%) at mid-cycle, though not statistically significant (p = 0.7). At the end of the induction, patients with IDH2 R140 had significantly lower median bone marrow blast percentages compared to those with R172mutations (2% vs. 4%, p = 0.01). Similarly, patients with IDH1 mutations showed low blast counts compared to the IDH2 group (1.5% vs. 3%, p = 0.1). A higher proportion of IDH2 R140 patients achieved blast clearance compared to those with R172 mutations (100% vs. 75%, p = 0.06), while proportion of patients who achieved blast clearance were similar between IDH1 and IDH2 patients (87.5% vs 93.1%, P = 0.5). Analysis of overall complete remission rates (including both complete remission [CR] and CR with incomplete hematologic recovery [CRi]) within the IDH2-mutated group showed a higher rate in patients with the R140 mutation compared to those with R172 (87% vs. 75%), though the difference was not statistically significant (p = 0.5). When comparing IDH1- and IDH2-mutated patients, overall complete remission rates were 75% and 84%, respectively, with no significant difference observed (p = 0.6). Kaplan–Meier analysis of time to end-of-induction blast clearance revealed no statistically significant difference between IDH2 R140 and R172 patients (median time to clearance: 31 vs. 37 days, p = 0.3). Similarly, no difference was observed between IDH1 and IDH2-mutated patients (33 vs. 31 days, p = 0.9). Overall survival analyses showed no significant difference between IDH2 R140 and R172 groups (median 66.8 months vs not reached, P = 0.4). Likewise, no difference was observed between IDH1 and IDH2 patients (44.4 months vs not reached, P = 0.7), indicating similar overall survival across mutation subtypes. Conclusion: IDH2-R140 was associated with more effective blast cytoreduction following intensive induction compared to IDH2-R172. Although not statistically significant, IDH2 R172consistently showed trends toward higher blast percentages, delayed response, and lower overall complete remission rates. These findings align with the recent study reporting significantly slower blast reduction and delayed response in R172-mutated AML. Also, no significant differences were found between IDH1- and IDH2-mutated patients in terms of response kinetics or overall survival. Together, these findings highlight potential biological differences among IDH mutation subtypes that may impact treatment response and require further investigation in larger prospective cohorts.

Abstract Background Red blood cell distribution width (RDW) is an easily accessible diagnostic test that is underutilized as a prognostic marker, despite substantial supporting evidence in cancer, cardiovascular diseases, and helathy individuals (Pilling et al. PLoS One 2028;13:e0203504). The association between increased RDW and incident acute myeloid leukemia (AML) has long been suggested in healthy volunteers (Abelson et al. Nature 2019;559:400). We looked into a similar phenomenon in patients with polycythemia vera (PV) or essential thrombocythemia (ET) in large discovery and validation cohorts. Methods A machine learning (ML) approach was used to identify baseline clinical and laboratory parameters that were predictive of leukemic transformation (LT) and develop a predictive model in a large discovery cohort of PV and ET, obtained from electronic medical records of the Maccabi Healthcare Services (MHS; Tel-Aviv, Israel). Receiver operating characteristic (ROC) curve analysis was utilized to determine the optimal cutoff points for RDW and other continuous variables. Mayo Clinic databases (Rochester, MN, USA) for patients with PV and ET were utilized as validation cohorts. Identification of study patients in the latter was according to the International Consensus Classification (Arber et al. Blood 2022; 140:1200) while in the discovery cohort was based on presence of ICD-9 codes associated with documentation of disease-specific driver mutations or treatment records that are consistent. Statistical analyses were performed using the R statistical software 4.4.2 (November, 2024) (Foundation for Statistical Computing, Vienna, Austria) or JMP Pro 18.0.0 software (SAS Institute, Cary, NC, USA). Results The discovery cohorts from Israel included 4,592 patients with PV (median age 63 yesrs; 53% males) and 5,968 with ET (median age 61 yesrs; males 40%). The validation cohorts from the Mayo Clinic included 633 PV (median 65 years; 51% males) and 634 ET (median 60 years; females 64%) patients that were informative for RDW. In the discovery cohorts, median follow-up (incident AML) was 6.1 years (N=186; 4.1%) in PV and 6.1 years (N=85; 1.4%) in ET. The corresponding values for the validation cohorts were 6.6 years (N=24; 3.8%) in PV and 7.0 years (N=22; 3.5%) in ET. Discovery cohort analyses Covariate-adjusted Cox regression analysis was utilized to estimate associations between LT and several clinical variables followed by machine learning pipeline to predict LT in a given timeframe. Conversion from continuous to binary variables was accompliched by ROC analysis. Shapley Additive exPlanations (SHAP) application enabled selection of top-ranked features that included RDW >15.5 (HR 3.2, 1.1-9.5; p=0.03), age >60 years (HR 2.9, 1.4-5.9; p<0.01), and LDH >1.5 x UNL (HR 4.0 (1.6-10.0; p<0.01). A similar analysis in ET also identified RDW >15.5 (p<0.01), age >60 years (p=0.01) as well as male sex (p=0.03) as independent risk factors for LT. Subseqent risk models based on these risk factors were externally validated and enabled robust risk stratification with 10-20 year AUCs rangening from 0.75 to 0.81. Validation cohorts analyses The indpendent predictive value of increased RDW observed in the discovery cohort was confirmed in the validation cohorts for both PV and ET and further gender-specified: female/male RDW ≥16.4/15.8 in PV (HR 7.9, 1.9-33.8) and ≥16.1/14.4 in ET (HR 5.1, 2.2-11.9). Also confirmed were the additional prognostic contributions of age ≥60 years (HR 3.8, 1.3-10.9) and LDH >1.5 x UNL (HR 3.9, 1.6-9.4), in PV, while no other clinical risk factor showed RDW-independent prognostic relevance in ET. The prognostic impact of RDW in ET was most apparent in females (p<0.01), as opposed to males (p=0.32) while the effect was equally significant in PV. In multivariable analysis that included previously recognized high risk mutations in PV (SRSF2) and ET (SF3B1), both RDW and mutations sustained significance (p<0.05). Conclusion The current study identifies higher RDW as a key risk factor for LT in both PV and ET, independent of other clinical or genetic risk factors. Possible underlying mechanisms include increased RDW as a marker of long-term pre-leukemic clones with sub-clinical ineffective erythropoiesis or an associated pro-leukemic inflammatory state.

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.