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Abstract Background: The primary goal of induction therapy in acute myeloid leukemia (AML) is to induce complete remission with (CR) or without (CRi) hematologic recovery. In order to accomplish this, re-induction might be necessary based on D14-residual AML. The current study examines the impact of re-induction on relapse, transplant-censored survival, and post-transplant survival in a consecutive series of AML patients who have achieved CR/CRi following intensive chemotherapy. The study also examined the impact of variations in consolidation chemotherapy including the use of anthracycline-based chemotherapy (AC) in the first consolidation cycle and the use of high (HiDAC) vs. intermediate (IDAC) dose cytarabine. Methods: The current study was conducted under IRB-approved minimum risk protocols that allowed retrospective collection and analysis of patient data. Formal criteria were used for AML diagnosis, genetic risk stratification, and adjudication of response (Blood 2022;140:1345). Patients who received less intensive induction regimens or failed to achieve CR/CRi were excluded. Relapse-free (RFS) and overall survival (OS) analysis was calculated from the date of documented CR/CRi. Patients who underwent allogeneic stem cell transplant (ASCT) were censored at the time of transplant. Conventional statistical methods were utilized (JMP Pro 17.1.0, SAS Institute, Cary, NC, USA). Results: A total of 279 AML patients diagnosed between 2004 and 2020 were included: median age 58 years, males 60%, de novo 75%, post-chronic myeloid neoplasm (post-CMN) 16%, and therapy-related 9%. Induction regimens included idarubicin (Ida) + cytarabine (Cyt) in 183 (66%) patients, daunorubicin (Da) 60 mg/m² + Cyt in 76 (27%), and Da 90 mg/m2 + Cyt in 19 (7%). Re-induction was utilized to achieve CR/CRi in 72 (27%) at a median of 18 days from start of first induction regimen. The indication for re-induction was based on residual bone marrow blast (median 30%; range 4-90) on day14 (N=53) or day 21-28 (N=15), persistent extramedullary disease (N=2) or the presence of circulating blast (N=2). At a median transplant censored follow-up of 26 months (mos), 93 (33%) deaths, 117 (42%) relapses, and 134 (48%) ASCTs were documented; ASCTs were performed in CR1 (50%) or CR2 (50%). Median RFS was 18 mos, with respective 1-, 2-, 3- year RFS of 63%, 42% and 35%. In multivariable analysis (MVA), age ≥65 years (HR 1.5, p=0.04), European LeukemiaNet (ELN) adverse karyotype (vs. intermediate HR 1.7, p=0.02; vs. favorable HR 2.8, p<0.01), and the need for re-induction to achieve CR/CRi (HR 1.7, p=0.01) were independently predictive of relapse. RFS was not impacted by NPM1 mutation ( HR 0.5, p=0.09) or FLT3-ITD (HR 1.3, p=0.3). The median OS was 80 mos, with 5-year OS of 53%. In MVA, age ≥65 years (HR 2, p=0.01), ELN adverse karyotype (vs. intermediate HR 3.3, p<0.01; vs. favorable HR 4.7, p<0.01), FLT3-ITD (HR 1.9, p=0.04), and the need for re-induction (HR 1.9, p=0.03) independently predicted inferior OS. RFS was similar among patients who received AC (N=84; Idarubicin 9 mg/m2 or Da 60 mg/m2 days 1 to 2/3 + 100 mg/m2 Cyt days 1 to 5/7; median 18 mos) as their first consolidation cycle vs. those who received HiDAC (N=99; 3 g/m2 q12 hours on days 1,3 and 5; median 16 mos) or IDAC (N=58; IDAC; 1-2 g/m2 q12 hours on days 1, 3 and 5; median 24 mos; p=0.9); the corresponding median OS were 125, 85, and 40 mos (p=0.4). In patients aged ≥65 years, but not younger patients, both RFS (p<0.01; median 23 (N=36) vs. 8 (N=24) mos) and OS (median 40 vs. 12 mos, p<0.01) were significantly longer with IDAC vs. AC as their first consolidation cycle. In the AC consolidation cohort, patients who received 2-3 additional HiDAC/IDAC cycles displayed longer OS (p<0.01) and RFS (p=0.047), compared to those who received less. In both the HiDAC and IDAC cohorts, RFS and OS were not significantly different between those who received 3-4 versus 1-2 consolidation cycles. The need for re-induction did not affect post-transplant survival (p=0.5), which was superior in patients receiving Ida + Cyt induction (vs. Da 60mg/m2 + Cyt, HR 0.6, p=0.04) and in those transplanted in CR1 (HR 0.5, p<0.01). Conclusions: The current study signals the need for re-induction to achieve CR1 as a negative predictor of outcome in AML patients achieving CR/CRi following intensive induction chemotherapy. The particular information is potentially actionable in terms of the choice of consolidation strategy.

Abstract Background Momelotinib, a small-molecule Janus kinase 1 and 2 (JAK1/2) inhibitor (JAKi), is FDA-approved (September 15, 2023) for use in anemic patients with myelofibrosis (MF). The drug appears to have an edge over other FDA-approved JAKis in regard to its unique erythropoietic effect that has been attributed to its inhibitory activity on activin A receptor type-1 (ACVR1). In clinical trials, anemia response to momelotinib in JAKi-naïve MF patients has been reported in more than a third of patients with or without transfusion-dependent anemia (TDA). The current study examines the drug's efficacy and toxicity in JAKi-naïve patients treated outside of clinical trials and after the drug was commercially approved. Methods The current study was conducted under an institutional review board approved minimum risk protocol. Diagnostic criteria were according to the International Consensus Classification (Arber et al. Blood 2022; 140:1200). Study inclusion criteria included a hemoglobin level of <10 g/dL and absence of previous treatment with JAKi. Responses were adjudicated by the Revised International Working Group-European LeukemiaNet criteria (IWG-ELN; Blood 2024; 144:1813). Conventional statistical methods were used (JMP Pro 18.0.0 software SAS Institute, Cary, NC, USA). Results A Mayo Clinic enterprise-wide search identified 60 patients (median age 73 years; 55% males) with primary or secondary MF who received momelotinib outside of clinical trials as their first JAKi for treatment of anemia and associated symptoms. Driver mutation distribution included JAK2 42%, CALR 30%, MPL 18%, and triple-negative 10%. Table 1 outlines clinical and laboratory findings at the time of momelotinib treatment initiation; Dynamic International Prognostic Scoring System (DIPSS)-plus risk distribution included high (72%) and intermediate 1/2 (28%) risk. Twenty-seven (45%) patients were transfusion-requiring, 24 (40%) displayed peripheral blood (PB) blast ≥2%, 10 (17%) platelet count <100 x 109/L, and 14 (25%) marked palpable splenomegaly (≥20cm). Unfavorable karyotype was seen in 9 (16%) of 58 patients. NGS-derived mutations were assessed in 57 cases and included ASXL1 (35%), TET2 (19%), SF3B1 (16%), SRSF2 (14%) and 7% (N=4) each for EZH2, IDH2, SH2B3, and ZRSR2. Baseline plasma cytokine levels were assessed in 17 patients with elevated TNF-a in 88%, IL-2 soluble 65%, IL-6 29% and IL-18 53%. Anemia response and predictors Fourteen (23%) patients met revised IWG-ELN criteria for anemia response (TDA 15% and non-TDA 30%; p=0.15). The anemia response was within 3 months in 10 (71%) patients. Median (range) durations of momelotinib exposure and anemia response were 8 (1-23) and 5 (1-13) months, respectively. The drug was discontinued in 29 (48%) patients (39% due to side effects; Table 2). In age-adjusted univariate analysis, predictors of anemia response included circulating blasts <2% (33% [12/36] vs. 8% [2/24]; p=0.02), presence of SH2B3 mutation (100% [4/4] vs. 19% [10/53]; p<0.01), and elevated IL-2 soluble (36% [4/11] vs. 0% [0/6]; p=0.02). The correlation with SH2B3 mutation remained significant (p<0.01) after accounting for circulating blasts. Treatment emergent neuropathy and nephropathy Sensory peripheral neuropathy (PN) was reported by 12 (20%) patients with a median onset of 2 months (range 1-7); CTCAE v5.0 grades 1 (N=8), 2 (N=3), and 3 (N=1); reversibility was documented only for grade 1 severity and did not appear to be consistently affected by drug discontinuation or dose reduction (Table 3). Seventeen (29%) patients experienced a >20% treatment-emergent decrease in estimated glomerular filtration rate from baseline, including 14 (23%) whose serum creatinine level crossed from the normal to abnormal range with pre- and post-treatment median (range) values of 1.05 (0.60-1.26) and 1.53 (1.1-3.31), respectively, with the majority reverting back to the normal range upon drug discontinuation. Conclusion In routine practice, momelotinib therapy might alleviate anemia in about a quarter of JAKi-naive patients with MF but is associated with non-trivial rates of treatment-emergent peripheral neuropathy and nephropathy. The latter appeared to be reversible on treatment discontinuation. Our novel observation regarding association of anemia response with SH2B3 mutation requires validation in a larger group of informative cases.

Abstract Background A previous multicenter study of patients with TP53-mutated myeloproliferative neoplasms showed a significantly shorter survival in chronic phase myelofibrosis (MF-CP) that included primary (PMF) and secondary MF and was mostly based on research-based mutation screening and was not systematically controlled for patients without TP53 mutations (AJH 2025;100:552). The objective of the current study was to examine the prognostic impact of TP53 mutations (VAF ≥2%) that were discovered during clinical NGS testing among consecutive patients with PMF-CP. Impact on overall (OS) and leukemia-free (LFS) survival was analyzed in the context of other genetic risk factors and calculated from the time of mutation detection. Methods The current retrospective study includes consecutive patients with PMF-CP who underwent clinical NGS testing at the Mayo Clinic between 2015 and 2025. Diagnoses of PMF-CP and leukemic transformation (LT) were retrospectively fitted to be in accordance with the International Consensus Classification criteria (Blood 2022;140:1200). Mutation and cytogenetic analyses were performed according to institutional protocols for clinical use and reported in conformity with international guidelines. OS and LFS analyses were censored for treatment with allogeneic stem cell transplantation (ASCT) and calculated from the date of NGS. Conventional statistical methods were utilized (JMP Pro 17.1.0, SAS Institute, Cary, NC, USA). Results A total of 633 patients with PMF-CP underwent clinical NGS testing prior to LT and prior to ASCT (median age 65 years; males 59%). Median interval from initial diagnosis/referral to NGS testing was 2 months; 357 (56%), 392 (62%), and 429 (68%) patients had NGS performed within 3, 6 and 12 months of diagnosis, respectively. Risk distribution according to the dynamic International Prognostic Scoring System (DIPSS; Blood 2010;115:1703) were low (19%), intermediate-1 (44%), intermediate-2 (31%), or high (6%). TP53 mutations (TP53MUT) with variant allele frequency (VAF) of ≥2% were detected in 28 (4.4%) patients with a median VAF of 25.3% (range 2.4-85; ICC-defined multihit 43%). Patients with TP53MUT were more likely to harbor IDH2MUT (14% vs. 3%; p<0.01), EZH2MUT (14% vs. 4%; p=0.03), and unfavorable karyotype (36% vs. 19%; p=0.047) and less likely to harbor CALR type 1/like (4% vs. 18%; p=0.02) and SF3B1MUT (0% vs. 6%; p=0.07). The association with unfavorable karyotype was mainly driven by multihit configuration. No significant associations with clinical features were apparent. At a median follow-up of 40 months for surviving patients, 157 (25%) deaths, 48 (8%) LTs and 135 (21%) ASCTs were documented. A near-two-fold lower, albeit not statistically significant OS was observed in the presence of TP53MUT (median 123 vs. 228 months; HR 1.9, 95% CI 0.94-3.7; p=0.074); the difference reached significance for multihit TP53MUT (median 81 months; HR 3.6, 1.7-7.7; p<0.01) but not for non-multihit TP53MUT (median not reached; HR 0.7, 0.2-2.8; p=0.6), both compared to TP53WT (median 228 months). The significant impact of multihit TP53MUT on OS was confirmed in multivariable analysis (MVA) that included other high molecular risk (HMR) mutations and unfavorable karyotype: multihit TP53MUT (HR 2.5, 1.1-5.5; p=0.02), SRSF2MUT (2.4, 1.5-3.8; p<0.01), ASXL1MUT (1.8, 1.2-2.6; p<0.01), U2AF1MUT-Q157 (4.8, 2.6-8.6; p<0.01), and absence of CALR type 1/like (4.0, 2.0-7.8; p<0.01); additional prognostic contribution was not evident for unfavorable karyotype (p=0.17), EZH2MUT (p=0.3), IDH1MUT (p=0.6), IDH2MUT (p=0.7), or any other HMR mutation. Multihit TP53MUT was also associated with inferior LFS (10-year risk 62% vs. 32% for non-multihit TP53MUT vs. 15% for TP53WT; p=0.03) but significance was lost during MVA that included SRSF2MUT, which was the only HMR mutation showing a significant effect on LFS (HR 5.0, 2.5-9.8; p<0.01). Post-ASCT survival among 135 transplanted patients was significantly shorter for patients with multihit TP53MUT (N=4; median 5 months) vs. those with non-multihit TP53MUT (N=7; median not reached) vs. patients with TP53WT (N=124; median not reached) [p=0.02]. Conclusion The current study implicates multihit TP53MUT as an HMR mutation-independent prognostic factor for OS in PMF-CP. By contrast, prognosis in PMF-CP patients with non-multihit TP53MUT was more likely to be influenced by other HMR mutations, as was the case for those with TP53WT.

Abstract Background In our first exploration of mutations in essential thrombocythemia (ET; N=183), we described somatic variants other than JAK2/CALR/MPL in 53% (N=96) of patients with the most frequent being TET2 (16%), ASXL1 (11%), DNMT3A (6%), and SF3B1 (5%) [Blood Adv 2016;1:21]. In a subsequent report (N=270; BJH 2020;189:291), we highlighted the prognostic relevance of SF3B1, SRSF2, and EZH2 mutations for overall survival (OS), SRSF2, EZH2, and TP53 for leukemia-free survival (LFS), and SF3B1 and U2AF1 for myelofibrosis-free survival (MFFS). We have now expanded our NGS-informative patient cohort to 500 and examined the distribution and frequencies of specific mutations at different stages of the disease and corresponding prognostic correlates. Methods Molecularly-annotated ET patients diagnosed at the Mayo Clinic between January 1980 and May 2025 were considered. Diagnoses of chronic phase ET (ET-CP), post-ET MF, and post-ET acute myeloid leukemia (AML) were retrospectively fitted to be in accordance with the International Consensus Classification criteria (Blood 2022;140:1200). Mutation and cytogenetic analyses were performed according to institutional protocols for clinical use that included multi-gene next-generation sequencing (NGS); in a subset of patients, historically archived samples were accessed for analysis in a research setting. Patients were monitored until death or time of last follow-up, determined through electronic medical records or direct communication with patients or healthcare providers. Conventional statistical methods were utilized (JMP Pro 17.1.0, SAS Institute, Cary, NC, USA). Results A total of 500 ET patients (median 57 years; Females 61%) were informative for NGS-derived mutations that were obtained in the chronic (N=401; 80.2%; group A), post-ET MF (N=88; 17.6%; group B), or post-ET AML (N=11; 2.2%; group C) phase of the disease; group C included 9 (1.8%) patients who transformed directly to AML without transitioning through the MF phase. The most frequent mutations with at least 1% (N=5) mutational frequency were (approximately) TET2 (10%), ASXL1 (10%), DNMT3A (7%), SF3B1 (4%), TP53 (5%), SRSF2 (3%), EZH2 (2%), U2AF1 (2%), CBL (1%), IDH1 (1%), IDH2 (1%), RUNX1 (1%), ZRSR2 (1%), and PPM1D (1%). Comparison of distribution of mutations and their frequencies based on timing of NGS (AML phase vs. MF phase vs. ET-CP) revealed significant differences for TP53MUT (72.7% vs. 11.4% vs. 1%; p<0.01), ASXL1MUT (18.2% vs. 25% vs. 7.2%; p<0.01), and EZH2MUT (18.2% vs. 6.8% vs. 0.3%; p<0.01), respectively; borderline significance was also apparent for RUNX1MUT (9.1% vs. 0% vs. 1.3%; p=0.10) and ZRSR2MUT (9.1% vs. 2.3% vs. 0.5%; p=0.08). Notably, among 9 patients in whom NGS was performed at the time of direct transition from ET to AML, without an intermediary MF phase, 8 (88.9%) displayed TP53MUT and only one (11.1%) EZH2MUT and none (0%) ASXL1MUT. At a median follow-up of 66 months, 93 (18.6%) deaths, 26 (5.2%) leukemic transformations (LT), and 108 (21.6%) MF transformations were reported. In group A patients, age-adjusted multivariable analysis (MVA) of mutations identified ASXL1MUT (N=29; HR 3.8, 1.9-7.7; p<0.01) and SF3B1MUT (N=12; HR 3.9, 1.4-11.0; p=0.01) to be associated with inferior OS, ASXL1MUT (HR 4.9, 1.01-24.2; p=0.048) and SRSF2MUT (N=11; HR 11.9, 1.3-104.8; p=0.03) with inferior LFS, and ASXL1MUT (HR 9.0, 2.8-29.5; p<0.01) and SF3B1MUT (HR 15.5, 3.1-77.5; p<0.01) with inferior MFFS, all calculated from time of NGS. A similar analysis in group B patients identified ASXL1MUT (N=22; HR 3.7, 1.2-11.7; p=0.03) to be associated with inferior OS and TP53MUT (N=10; HR 13.4, 1.7-108.6; p=0.02) with inferior LFS. Age-adjusted MVA combining groups A and B (N=489) confirmed the association between inferior OS and ASXL1MUT (N=51; p<0.01) and SF3B1MUT (N=18; p<0.01) while inferior LFS was associated with TP53MUT (N=14; p<0.01) and ASXL1MUT (p=0.03). Conclusions The majority (⁓73%) of patients with post-ET AML harbor TP53MUT, at the time of LT. By contrast, TP53MUT are infrequent in ET-CP (⁓1%) and did not appear to predict LT, as was the case with post-ET MF. ASXL1MUT was shown to be an independent risk factor for OS, MFFS, and LFS in ET-CP and for OS in post-ET MF. SF3B1MUT was associated with inferior OS and MFFS in ET-CP and SRSF2 with inferior LFS in ET-CP. Identifying ET patients with adverse mutations allows for closer surveillance and potentially more tailored therapeutic interventions.

Abstract Background: The recently published BLAST clinical and BLAST molecular (BLAST-Mol) risk models for chronic myelomonocytic leukemia (CMML) incorporate clinical and molecular variables, respectively, for prediction of overall survival (OS), while BLAST-BT (blast transformation) risk model was predictive of BT free survival (BTFS) [Blood 2025;doi:]. In the current study, the Mayo Clinic (USA) CMML cohort used in the aformentioned study was expanded from 457 to 600 patients and merged with an external cohort of 501 patients from the Humanitas Research Hospital (HRH; Italy) in order to validate the BLAST, BLAST-Mol and BLAST-BT risk models. Methods: CMML diagnosis and sub-categorization were according to the International Consensus Classification, which included CMML-1 (BM/PB blasts <10%/<5%) vs. CMML-2 (BM/PB blasts ≥10%/≥5%) and CMML-MP (leukocytes ≥13 x 10⁹/L) vs. CMML-MD (leukocytes <13 x 10⁹/L) [Blood 2022;140:1200]. The BLAST score is based on clinical risk factors including PB blasts ≥2%, leukocytes ≥13 x 109/L, and severe or moderate anemia resulting in BLAST low, intermediate, and high risk groups. BLAST-Mol also accounts for mutations and karyotype and identifies PHF6 and TET2 as “favorable” and DNMT3A, U2AF1, BCOR, SETBP1, ASXL1, NRAS, PTPN11, RUNX1, TP53, as “unfavorable” mutations with adverse karyotype also considered unfavorable. BLAST-BT variables include absence of PHF6MUT, BM blasts ≥10%, presence of DNMT3AMUT, PB blasts ≥2%, leukocyte count ≥13 x 109 /L, and ASXL1MUT. OS analyses were censored for allogeneic stem cell transplant (ASCT). Conventional statistical methods were employed, using JMP Pro 18.0.0 software SAS Institute, Cary, NC, USA. Results: A total of 1,101 patients (median age 73 years; 67% males) with newly-diagnosed CMML were considered: Mayo cohort 600 and HRH cohort 501. Compared to the HRH cohort, the Mayo cohort was more likely to include CMML-MP (44% vs. 28%; p<0.01), CMML-1 (87% vs. 81%; p<0.01), ASXL1MUT (48% vs. 35%; p<0.01), SRSF2MUT (43% vs. 33%; p<0.01), NRASMUT (15% vs 9%; p<0.01), PHF6MUT (7% vs. 4%; p<0.01), and less likely to harbor TET2MUT (50% vs. 58%; p<0.01), and SF3B1MUT (4% vs. 10%; p<0.01) [Table 1]. At a median follow-up of 55 months (range 0-202), 686 (62%) deaths, 218 (20%) BTs, and 142 (13%) ASCTs were documented. CPSS-mol risk categories at diagnosis included low (N=190; median OS 67 months), intermediate-1(N=271; median 38 months), intermediate-2 (N=407; median 26 months), and high (N=231; median 16 months; p<0.01) [AUC at 5 years 0.75]. BLAST clinical risk distribution was low (N=356; median OS 65 months), intermediate (N=393; median 30 months), and high (N= 352; median 16 months; p<0.01) [Figure 1; AUC at 5 years 0.79]. The corresponding AUCs calculated for the Mayo and HRH cohorts, separately, were 0.81 and 0.76, respectively. BLAST-Mol risk assignments included low (N=357; median OS 66 months), intermediate (N=512; median 27 months) and high (N=232; median 15 months; p<0.01) [Figure 2; AUC at 5 years 0.79]. The corresponding AUCs for the Mayo and HRH cohorts were 0.81 and 0.77, respectively. The 3-year cumulative incidence of BT (accounting for death from another cause) stratified by BLAST-BT risk model included 8% for very low risk (N=54), 11% for low risk (N=332), 20% for intermediate-risk (N=281), 27% for high risk (N=250), and 34% for very high risk (N=176) [p<0.01; Figure 3]. Multivariable analysis of the combined Mayo-Humanitas cohort (N=1,101) identified age >65 years (HR 1.7, 95% CI 1.3-2.1), CMML-2 subtype (HR 2.0; 95% CI 1.6-2.5), and a number of other mutations or co-mutations with BLAST-Mol-independent and CPSS-mol-independent prognostic contribution (to be elaborated further at the time of meeting presentation). Conclusions: The current study validates the predictive performance of the BLAST, BLAST-Mol and BLAST-BT risk models in a large two-center international cohort of patients with CMML. The possibility of further enhancement of the BLAST-Mol model is suggested by the demonstration of independent prognostic relevance from additional mutations and co-mutations.

Abstract Background: The coexistence of systemic mastocytosis (SM) and lymphoid neoplasms is rare and remains poorly defined. These cases are classified under SM with associated hematologic neoplasm (SM-AHN) in the WHO framework, though the ICC distinguishes SM with associated myeloid neoplasm (SM-AMN) as a separate entity. Emerging evidence points to SM with associated lymphoid neoplasms (SM-ALN) as a biologically distinct and clinically indolent subgroup (Aperna F et al, 2025). We performed a multisite retrospective study to characterize the clinico-molecular features and outcomes of SM-ALN. Methods: We conducted a retrospective analysis across Mayo Clinic sites, reviewing records from January 1980 to December 2024 to identify patients with SM-ALN. IRB approval was obtained. Of 22 cases identified, 4 were excluded due to co-occurring myeloid neoplasms. The final cohort included 18 patients. Variables analysed included age, sex, SM subtype, KIT mutation, symptoms, treatment, and survival outcomes. Results: Eighteen patients were included (median age: 61.5 years; range: 38–80; 11 female). Most had indolent SM (ISM, n=16); two had aggressive SM (ASM). SM preceded the lymphoid neoplasm in 4 cases (22%), followed it in 6 (33%), and was concurrent in 8 (44%). Low-grade lymphomas (n=12, 67%) included follicular lymphoma (n=2), CLL (, n=2), marginal zone (n=1), lymphoplasmacytic (n=1), and mantle cell (n=1). High-grade lymphomas (n=6, 33%) included diffuse large B-cell lymphoma (DLBCL, n=3), Hodgkin lymphoma (n=2), and T-cell acute lymphoblastic leukemia (n=1). SM-related symptoms were present in 16 of 18 patients (89%). The most common manifestations included dermatologic symptoms (rash/pruritus, 56%), systemic complaints (fatigue/weight loss, 39%), GI symptoms (17%), and anaphylaxis (6%). KIT D816V testing mutation was positive in 10 of 16 patients tested (63%). Tryptase was elevated (>20 ng/mL) in 15 of 18 (83%). NGS data was available for 9 patients: among the two ASM cases, both harbored SRSF2 mutation; one also had JAK2, DNMT3A, RUNX1, TET2, and TP53, while the other carried DNMT3A and IDH1. Mutations observed in ISM cases included CCND3, CXCR4, NOTCH2, SF3B1, MYD88 L265P, ASXL1, and TET2. SM management was divided into two categories: (1) Conservative approach, including observation alone in 4 patients (22%) and symptomatic therapies such as antihistamines or leukotriene inhibitors in 7 (39%); and (2) Cytoreductive treatments, comprising avapritinib in 5 patients (28%), cladribine in 1 (6%), and imatinib in 1 (6%). Management of lymphoid neoplasms included intensive regimens such as R-CHOP and hyper-CVAD for high-grade subtypes (e.g., DLBCL and T-ALL), while indolent forms (e.g., CLL and follicular lymphoma) were treated with bendamustine-rituximab or observation depending on disease burden. Overall, 13 patients (72%) received systemic therapy. At a median follow-up of 81 months (6.7 years), 17 of 18 patients (94%) were alive and progression-free. Median OS and PFS were not reached. The sole death occurred in a patient with ASM, while all ISM cases remained alive. ASM was associated with a higher (though nonsignificant) risk of death (OR 33.0; 95% CI, 0.89–1220.9; p = 0.111). Among patients with high-grade lymphomas (n=5), 80% were alive at follow-up, compared to 100% with indolent lymphomas (n=13). Discussion: Our findings support SM-ALN as a clinically indolent and biologically distinct subset within SM-AHN. Most patients had ISM and low-grade lymphoid neoplasms, with favorable outcomes regardless of lymphoma subtype or diagnostic sequence. High-risk mutations were confined to ASM cases, reinforcing underlying biologic differences. These findings align with prior Mayo Clinic data showing longer survival in SM-ALN compared to SM-AMN (median OS 8.1 vs 2.0 years; p<0.01). Treatment was primarily directed by the lymphoid malignancy, with limited need for cytoreduction in ISM. Larger, prospective studies are needed to confirm these observations and optimize management strategies. Limitations include the study's retrospective, single-center design and limited racial diversity. Multi-institutional studies are warranted to validate these findings and guide optimal management. Conclusion: SM-ALN is a rare but distinct SM subtype characterized by indolent behavior, favorable treatment responses, and prolonged survival. Recognition of this subset is critical to avoid over-treatment and to guide risk-adapted management strategies.

Abstract Background Myeloproliferative neoplasms (MPNs) can progress from chronic phase to accelerated or blast phase (MPN-AP/BP), a transition with poor prognosis and limited treatment options. While some abnormalities have been described, the genomic and clonal drivers of transformation remain poorly defined. We characterized mutational and structural alterations in MPN-AP/BP, leveraging paired samples to trace clonal evolution. Methods We profiled 186 samples from 160 individuals, including 159 MPN-AP/BP and 27 chronic-phase MPN samples (26 MF, 1 PV), using targeted sequencing of a 588 gene panel (mean depth: 709x). Paired pre- and post-transformation samples were available for 26 patients (n= 52). A consensus pipeline was used to detect somatic mutations, copy number variations (CNVs), and copy-neutral loss of heterozygosity (cnLOH). Clinical cytogenetic data were integrated when available. Single-cell validation is ongoing. Results Genomic complexity was widespread: 99% of patients harbored ≥1 oncogenic mutation (median 4; range 1–10). In total, 859 mutations were identified, most commonly in JAK2 (68%), ASXL1 (28%), TET2 (26%), SRSF2 (24%), TP53 (22%), and RUNX1 (22%). 684 structural alterations were identified in 86% of patients (median 3; range 1-20), including arm-level (22.5%), segmental (>5 Mb; 60%), and focal (<2 Mb; 9%) alterations. Recurrent events included 9p cnLOH (16%), del(5q) (13%), del(17p) (10%), and focal deletion of 21q22.12 involving RUNX1 (10%). Complex karyotypes (≥3 abnormalities) were observed in 55% of patients. Biallelic mutations accounted for 21% (183/859) of mutations, affecting 58% of patients across 21 genes, most frequently JAK2 V617F (28%), TP53 (17%) and TET2 (14%). Patients with biallelic mutations in these genes had significantly higher blast percentages compared to those with monoallelic mutations (p<0.05), highlighting an association with aggressive disease. In 26 patients with longitudinal samples (chronic phase MPN and MPN-AP/BP), 87% (125/144) of mutations were already detectable during the chronic phase, on average more than 7 years prior to transformation. Only 11% (16/144) of mutations were newly acquired, including additional RUNX1 and TP53 hits, or structural changes that converted pre-existing JAK2 V617F and TP53 R248Q mutations from monoallelic to biallelic via 9p cnLOH or 17p loss, respectively. These findings suggest transformation is often driven by outgrowth of pre-existing high-risk clones, indicating that these clones likely have a competitive advantage and that the mutations within these clones have a cooperative biological interaction which underlies this competitive advantage. To investigate this hypothesis, we examined whether patterns of recurrent co-mutations were identifiable in this cohort. We identified recurrent co-occurring mutations in SRSF2-IDH2, ASXL1-SRSF2, and ASXL1-EZH2, which were enriched at transformation (p < 0.05; Fisher exact test). Importantly, presence of these co-mutations was consistently associated with a higher transformation risk in two independent cohorts of MF patients from Mayo Clinic (n= 405) and University of Florence (n= 518). Clonal architecture analysis revealed that these co-mutations frequently arose within the same malignant clone: 100% for SRSF2-IDH2, 80% for ASXL1-SRSF2, and 85% for ASXL1-EZH2, and are often within the dominant clone. Prior studies have demonstrated mechanistic interplay between SRSF2 and IDH2 (Yoshimi et al.), as well as ASXL1 and SRSF2 (Sui et al.) mutations in myeloid malignancies. We assessed functional synergy of ASXL1-EZH2 using a murine model and identified that dual deletion caused a rapidly fatal myeloid neoplasm with shorter survival than single-gene loss. These results reinforce that functional synergy emerges within a shared cellular context, supporting clonal co-dependence as a driver of progression. Single-cell RNA sequencing of ASXL1-EZH2 mutant cases is ongoing and will be presented. Conclusions Transformation to MPN-AP/BP is driven by gradual clonal remodeling and expansion of pre-existing high-risk abnormalities. Biallelic and multi-hit alterations in TP53, JAK2, TET2, and RUNX1 promote clonal dominance and blast progression. Recurrent co-mutations such as IDH2-SRSF2 and ASXL1-EZH2 arise within the same clone, exhibit functional synergy, and are linked to poor outcomes. These findings support the clinical utility of early clonal and co-mutational profiling to guide risk-adapted intervention.

Abstract Introduction Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with overlapping features of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). Blast transformation (BT) is a frequent complication of CMML with a 3-year risk of approximately 31%. MPN driver mutations (i.e., JAK2, CALR, MPL) are known to occur in CMML but their impact on overall survival (OS) or BT remains uncertain. Methods The current study was conducted under institutional review board approved minimum risk protocols that allowed retrospective collection and analysis of patient data. Diagnostic criteria were according to the International Consensus Classification (Blood 2022; 140:1200). For survival analysis, patients were censored at the time of allogeneic stem cell transplant (ASCT). All statistical analyses were conducted using JMP Pro 17.0.0 software (SAS Institute, Cary, NC, USA) and Bluesky Statistics Software (Worldwide Headquarters, Chicago, IL, USA). Results A total of 612 CMML patients were considered (median age 71 years; 68% males). MPN driver mutations (MPNMUT) were detected in 56 (9%) patients, including 43 (77%) with JAK2, 10 (18%) MPL, and 3 (5%) CALR. The median variant allele frequencies were 44% for JAK2 (range, 0.3–100%), 49% for MPL (range, 0.3–97%), and 50% for CALR (range, 20–80%). Age and sex distributions and CMML-1 vs. CMML-2 representation were similar between CMML patients with and without MPNMUT. On the other hand, CMML patients with MPNMUT were more likely to display higher hemoglobin level (p <0.01), platelet count (p <0.01), absolute neutrophil count (p = 0.02) and leukocytosis (≥13 ×109/L; p <0.01); they were also less likely to be red blood transfusion dependent (p <0.01), display platelet counts of <100 ×109/L (p <0.01) or abnormal karyotype (p= 0.03), present with severe anemia (p= 0.03) or harbor NRAS mutation (p = 0.02). Higher Mutational frequencies of SRSF2 (median 53% vs. 41%; p = 0.01), TP53 (median 8% vs. 2%; p = 0.02) and IDH2 (median 13% vs. 4%; p = 0.01) were noted in CMML patients with MPNMUT. During a median follow-up of 23 months (inter-quantile range 11-51 months) for all 612 CMML patients, 402 (67%) deaths, 118 (19%) BT and 108 (18%) ASCT were documented. Of these, 36 (9%) deaths, 18 (15%) BT and 13 (12%) ASCT occurred in patients with MPNMUT. Median OS for the entire group (N=612) was 32 months with 1-, 2- and 3-year OS rates of 78%, 58% and 47%, respectively. There was no significant difference in OS between CMML patients with or without MPNMUT (median 41 vs 31 months; p = 0.27). Thrombotic events were reported in 53 (9%) patients after diagnosis of CMML, including 48 venous and 5 arterial events. There was no significant difference in arterial (p = 0.10) or venous (p = 0.76) thrombosis-free survival between CMML patients with or without MPNMUT. Among all 612 patients with CMML, followed for a median of 18 months for living patients, 118 (19%) experienced BT, with 1-, 3-, and 5-year BT rates of 10.8%, 23.1%, and 31%, respectively. Cumulative incidence analysis function (CIF) was calculated in order to assess the BT-free survival while accounting for death as a competing risk; 3-year CIF was 30% for CMML patients with vs. 18% for those without MPNMUT (Gray's test; p = 0.01). This observation was confirmed in univariate analysis of time to BT (HR 1.7; p=0.03) as well as the corresponding multivariable analysis involving other mutations and clinical risk factors: MPNMUT (HR 1.8; 0.036), ASXL1MUT (HR 2.3; p<0.01), DNMT3AMUT (HR 2.6; p<0.01), CMML-2 vs. CMML-1 (HR 3.4; p<0.01), and leukocyte count ≥13 ×109/L (HR 2.0; p<0.01). SRSF2, IDH2, and TP53 mutations did not impact BT. Notably, the aforementioned risk factors for BT, including ASXL1MUT and DNMT3AMUT, were no longer significant when analysis was restricted to patients with MPNMUT. Conclusions The current study suggests a higher risk of BT in CMML patients with MPNMUT, independent of other molecular or clinical risk factors for BT. On the other hand, overall and thrombosis-free survival did not appear to be similarly influenced. The differential impact of previously recognized risk factors for BT in CMML (e.g., ASXL1MUT, DNMT3AMUT, CMML-2, CMML-MP), in the presence (lost significance) vs. absence (remained significant) of MPNMUT was intriguingly noted.

Abstract Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder caused by PIGA mutations, resulting in complement-mediated hemolysis. Myeloproliferative neoplasms (MPNs) are also clonal hematopoietic stem cell disorders driven by mutations in JAK2, CALR, or MPL, and include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Although the coexistence of PNH and and MPN is rare, both entities are associated with anemia and an increased risk of thrombosis. The main objectives of the current study were to characterize the clinical phenotype, and evaluate treatment outcomes, including response to complement inhibition in patients with treatment-requiring PNH and MPN. Methods A Mayo Clinic database search was conducted following Institutional Review Board approval to identify patients with PNH and MPN. Between January 2000 and June 2025, 11 patients had a pathologic diagnosis of MPN per the International Consensus Classification, and PNH requiring therapy. Clinical features, including hemolysis and/or thrombosis, and therapeutic interventions were recorded. Results The study cohort included 11 patients (median age 63 years; 73% male) with MPN (6 with PMF, 3 ET, 1 post-ET MF, 1 post-PV MF) in whom PNH was diagnosed concurrently (n=5), or at a median of 9 years (range: 2-15) after MPN diagnosis (n=6). In 9 of 11 patients (82%), PNH workup was initiated due to hemolytic anemia, while in 2 patients (18%), it was prompted by splanchnic vein thrombosis. Transfusion-dependent anemia was present in 6 patients (54%). PNH clone size [median (range)] was 0.58% (0–10.96%) in red blood cells, 18.82% (0–91.2%) in granulocytes, and 23.27% (0–98.8%) in monocytes. MPN driver mutations were evaluated in 8 cases and identified mutations in JAK2 (n=3), CALR (n=4), and MPL (n=1); two additional cases were negative for JAK2 mutations. Next-generation sequencing (NGS) was performed in 6 patients and identified ASXL1, ZRSR2, and SF3B1 mutations in one case each. Cytogenetic analysis (n =10) demonstrated an abnormal karyotype in 3 patients (30%), all of which were categorized as favorable risk. First-line treatment for PNH included complement inhibitors in 9 patients (82%), with eculizumab (n=8), or ravulizumab (n=1). Hemolysis was controlled in only 2 of 9 (22%) patients; one patient treated with eculizumab achieved durable anemia response for five years, while another patient achieved transfusion-independence within three months of eculizumab initiation. 3 patients underwent allogeneic stem cell transplant (ASCT); 2 achieved remission with follow up exceeding 5 years, while the third patient experienced PNH recurrence five months after transplant. The latter received donor lymphocyte infusions and eculizumab was re-initiated resulting in eradication of the PNH clone. Among patients treated with complement inhibitor and not transplanted, 4 of 6 (67%) were transfusion-dependent at last follow-up. Notably, one patient with PNH clone size of 91.19 % in granulocytes, had ongoing hemolysis despite dual complement blockade with ravulizumab and danicopan. Treatment for MPN included hydroxyurea (n=5), ruxolitinib (n=4), pacritinib (n=1), and erythropoiesis stimulating agents (n=4). Additionally, two patients each received antiplatelet therapy alone, anticoagulation alone, or a combination of antiplatelet and anticoagulation. During a median follow-up of 8.4 years (range, 0.6-19), 2 patients (18%) experienced venous thrombosis, and 1 patient (9%) had progression to myelodysplastic syndrome. 5 patients (45.5%) died, with sepsis as the cause of death in 3 cases. The incidence rate of thrombosis was 22 events per 1,000 person-years. Recurrent thrombosis was observed in a 68-year-old male with CALR-mutated MF and PNH clone size of 14.65% in granulocytes, with history of portal vein thrombosis. Despite treatment with eculizumab and low-molecular weight heparin, he developed thrombosis involving the inferior vena cava, common iliac, and hepatic veins. The second event occurred in a patient who developed deep venous thrombosis, while in remission and not on anticoagulation. Conclusion The current study represents the largest series of patients with treatment-requiring PNH and MPN. It reveals a predominance of MF patients, particularly among those harboring CALRmutations, and underlines suboptimal efficacy of complement inhibitor therapy in this patient population.

Abstract Background Blast transformation (BT) occurs in approximately 15-30% of patients with chronic myelomonocytic leukemia (CMML) and remains a leading cause of death. Allogeneic stem cell transplantation (ASCT) is currently the only treatment modality with the potential to cure the disease or prolong survival. Optimal timing of ASCT is critical for maximizing benefit while minimizing risks. To that end, contemporary risk models have focused on the prognostic relevance of individual, as opposed to concurrent mutations. In the current study, we looked into the possibility of prognostic prominence from concurrent mutations in predicting BT in CMML. Methods CMML diagnostic criteria were according to the International Consensus Classification (Arber et al. Blood 2022; 140:1200). A machine-learning hierarchical clustering algorithm was developed and tailored for patient stratification using survival outcomes and co-expression of genomic alterations. To reduce complexity and improve interpretability, we generalized each cluster using existing rule induction algorithms, including the in Trees framework and JRip. The final output was a set of mutation-based cluster definitions, each representing a distinct patient subgroup with similar survival trajectories. Competing risk analysis and cumulative incidence functions were used in downstream evaluation to validate the clinical distinctiveness of the clusters. For all survival analysis, patients were censored at the time of ASCT. Time-to-BT was calculated from the date of diagnosis to the date of BT or ASCT or last contact. BT-free-survival was calculated from the date of diagnosis to the date of BT, ASCT, death, or last contact. Results The core study cohort included 605 patients from the Mayo Clinic, USA, and the external validation cohort 501 patients from Humanitas Cancer Center, Milan, Italy. Using the patient cohort from the Mayo Clinic, machine-learning algorithms identified five molecular clusters with 3-year blast transformation (BT) rates ranging from 0% to 100% (AUC at 3 years 0.78): the order of molecular signature assignment (probability of BT/death from another cause) was i) PHF6MUT/ASXL1WT (0%/17% at 3 years; N=32), ii) NPM1MUT OR BCORMUT/ASXL1MUT OR SETBP1MUT/NRASMUT (48%/30% at 1 year; N=24), iii) RUNX1MUT/ASXL1MUT OR SRSF2MUT/NRASMUT OR EZH2MUT/ASXL1MUT OR SETBP1MUT OR BCORMUT (31%/55% at 3 years; N=132), iv) ASXL1MUT/TET2MUT OR DNMT3AMUT OR JAK2MUT (24%/28% at 3 years; N=123), and v) all other permutations (10%/40% at 3 years; N=294) [Figure 1]. Additional analysis confirmed significant differences in survival between RUNX1MUT/ASXL1MUT vs. RUNX1MUT/ASXL1WT (p<0.01) OR RUNX1WT/ASXL1WT (p<0.01) OR RUNX1WT/ASXL1MUT (p=0.038) [Figure 2]. Similar patterns of differences in survival were also documented for NRAS/SETBP1, ASXL1/EZH2, and NRAS/SRSF2 mutation combinations (Figure 2). A subsequent Cox regression analysis confirmed independent prognostic contributions from “PHF6MUT/ASXL1WT” (HR 5.43e-10; p<0.01), NPM1MUT (HR 26.7; p<0.01), “SETBP1MUT/NRASMUT” (HR 12.7; p<0.01), BCORMUT(HR 5.8; p<0.01), “RUNX1MUT/ASXL1MUT” (HR 2.3, p<0.01), JAK2MUT (HR 2.1; p<0.01), and “ASXL1MUT/TET2MUT” (HR 1.7; p=0.02). The prognostic relevance of “SETBP1MUT/NRASMUT”, “RUNX1MUT/ASXL1MUT”, NPM1MUT, and BCORMUT was validated in the external cohort from Italy (N=501). In the Mayo Clinic cohort, presence of any of the latter mutations was associated with 1-, 3-, and 5-year BT (death from other cause) rates of 27% (21%), 44% (49%), and 44% (51%), respectively (Figure 3). The corresponding values in the absence of high risk mutations were 7% (15%), 15% (37%), and 18% (52%) [Gray's p value <0.01 for both BT and death from another cause; Figure 3]. Similarly, the 1-, 3-, and 5-year BT (death from another cause) rates in the Italian cohort were 21% (19%), 37% (47%), and 42% (54%) in the presence and 8% (14%), 20% (35%), and 24% (45%) in the absence of high-risk mutations (Gray's p value <0.01 for BT and 0.18 for death from another cause; Figure 4). Conclusions In the current study, machine-learning algorithms enabled the discovery of concurrent mutations in CMML that were shown to be prognostically more significant than their individual constituents. Such prognostic interaction might have contributed to some of the discrepancies noted in current literature regarding the prognostic relevance of certain mutations in CMML and should be accounted for in the development of future risk models.