Gangat, Naseema

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 Introduction Blast transformation (BT) in CMML occurs in 15-30% of patients (pts) with a median overall survival (OS) <6 months (mo). While clonal evolution can be observed, biological mechanisms of BT remain unknown for many pts. Using paired samples from chronic phase (CP) and at BT, this study defines the cytogenetic and molecular landscape of BT-CMML. Methods BT-CMML pts from Mayo Clinic (n=153, 31%), MD Anderson (n=195, 40%), and 7 centers of the Groupe Francophone des Myélodysplasies (n=145, 29%) were included after IRB approval. ICC 2022 criteria defined CMML and AML. Pt data were collected at initial diagnosis or referral at CP and again at BT. Clonal dominance was inferred from a natural log-fold difference of mutation variant allele fractions (VAF). OS was censored at allogeneic stem cell transplant (alloSCT). Single-cell multiomic analyses used the Tapestri platform (Mission Bio). Statistics used R. Results Among 1,883 cases of CMML with a median follow up of 96 mo, 493 (26%) underwent BT. The median age at CP and BT was 69 and 71 years, respectively. BT-CMML pts were predominantly male (69%) and had proliferative CMML (53%) or CMML-1 (69%) in CP; 39% and 33% were considered high-risk by CPSS-Mol and BLAST-Mol criteria, respectively. The median time to BT was 16 mo (interquartile range 7-35 mo) and was not different in males vs females (p=0.60). Compared to their CP parameters (p<0.001 each), pts at BT had lower median hemoglobin (10.8 vs 9.2 g/dL) and platelets (100 vs 44 x109/L) but higher leukocytes (13.9 vs 16.3 x109/L), peripheral blasts (0% vs 14%), and bone marrow blasts (6% vs 32%). Blasts exhibited mostly monocytic (57%) or immature myeloid (40%) differentiation. Abnormal karyotypes (AK) were more prevalent at BT (25% vs 62%, p<0.001). Among 318 pts with paired karyotype data, 221 (69%) had a normal karyotype (NK) in CP, of which 123 (56%) remained normal at BT and 98 (44%) acquired new abnormalities. Conversely, 97 (31%) pts had an AK in CP, of which 67 (69%) acquired abnormalities or increased clonal metaphases (6%), 23 (24%) remained stable, and 7 (7%) reverted to a NK at BT. Overall, 46% acquired and 8% lost a clonal cytogenetic abnormality, while 46% remained unchanged. Numeric (37%) and structural (30%) changes were most common. Complex (12% vs 3%) and monosomal (5% vs 2%) karyotypes were more frequent at BT than in CP. Only 6 (2%) pts acquired an AML-defining rearrangement at BT. Mutational data were available for 248 paired cases. The number of somatic mutations was higher at BT (median 3 vs 4, p<0.001). Mutations in RUNX1 (27% vs 39%), NRAS (23% vs 33%), KRAS (11% vs 17%), FLT3 (6% vs 15%), NPM1 (0% vs 11%), IDH2 (5% vs 11%), and CEBPA (3% vs 10%) were more frequent at BT (all p<0.05). At BT, the average VAF of the dominant clone increased in 32%, decreased in 35%, and remained unchanged (delta ≤5%) in 33%. The most frequent dominant clones both in CP and at BT had mutations in TET2 (8% vs 4%) or co-dominant mutations involving SRSF2-TET2 (7% vs 3%). Mutational clonal evolution was observed in 72%, with mutations gained in 45% and lost from the dominant clone in 19%. The most gained mutations at BT were in NRAS (15%) and RUNX1 (13%). Single cell proteogenomic analyses in 4 selected CP and BT pts shed light on the impact of NRAS and RUNX1 mutations on clonal hierarchies and differentiation. Among 196 fully evaluable cases, a new cytogenetic and/or molecular aberration was uniquely gained in 106 (54%) cases and lost in 9 (5%), with 40 (20%) both gaining and losing a genetic alteration. Forty-one (21%) patients had no identifiable genetic change between CP and BT, suggesting driver alterations outside of that detected by routine analyses. Median OS was 31 mo from CMML diagnosis and 5.6 mo from BT. Compared to supportive care, treatment in CP did not prolong median time to BT (14 vs 17 mo, p=0.91) or OS (35 vs 29 mo, p=0.05), regardless of intensity. After BT, median OS was 1.2 mo with supportive care (n=83), 8.7 mo with a hypomethylating agent (HMA, n=39), 10 mo with HMA plus venetoclax (n=37), 7.8 mo with chemotherapy (n=91), and 31 mo in pts who underwent alloSCT (n=57). Conclusions In this large BT-CMML cohort, cytogenetic (46% gained, 8% lost) or mutational (45% gained, 19% lost) driver events were identified in 79% of pts, with 21% having no identifiable changes by conventional testing. Whole genome/transcriptome analyses are underway in this subgroup to identify novel drivers of BT.

Abstract Background: TP53-mutated myeloid neoplasms (MNs) represent one of the most lethal subgroups of blood cancers. The World Health Organization (WHO-5) recognize TP53-mutated (TP53mut) myelodysplastic syndrome (MDS) and International Consensus Classification (ICC) recognize TP53mut MDS and acute myeloid leukaemia (AML) as distinct disease entities, acknowledging their dismal prognosis and the urgent need for novel therapeutic strategies. However, key discrepancies exist between the classifications, creating uncertainty in diagnosis, influencing therapeutic decisions, and complicating clinical trial enrolment for patients with uniformly poor survival. Importantly, neither classifications included TP53-mutated myelofibrosis (MF), other myeloproliferative neoplasms (MPN), and MDS/MPN overlap syndromes, despite growing evidence that these cases carry similar adverse outcomes. To address these critical gaps, we conducted a comprehensive analysis across the full spectrum of TP53-mutated MN. Methods: We included TP53mut cases with variant allele frequency (VAF ≥2%) of AML, MDS, MDS/MPN overlap, myelofibrosis and other MPN managed at the Mayo clinic (Rochester, USA) and Local Health Network in South Australia (Australia). Cases were classified according to the revised 4th edition WHO classification (WHO-4R). Biallelic TP53 loss was defined as: (i) ≥2 mutations with VAF ≥2%; (ii) single TP53mut with VAF ≥2% plus 17p loss on karyotype, FISH, or SNP array; or (iii) single TP53mut with VAF ≥50% with or without 17p loss. Monoallelic loss was defined as a single TP53mut with VAF <50% without 17p loss or copy neutral loss of heterozygosity. The primary endpoint was overall survival (OS) from diagnosis, censored at allogeneic transplantation. Conditional inference tree analysis was performed to define the effect of individual predictors on OS. Results: We identified 853 TP53mutMN cases including WHO-4R-defined AML (BM/PB blasts ≥20%, n=314; 36.8%), MDS with excess blasts-2 (EB-2; BM 10–19% or PB 5–19%, n=129; 15.1%), MDS-EB-1 (BM 5–9% or PB 2–4%, n=100; 11.7%), and MDS with low blasts (LB; BM <5% and PB <1%, n=228; 26.7%). MF included chronic phase (MF-CP, n=48; 5.6%), accelerated phase (MF-AP, n=7; 0.8%), and blast phase (MF-BP, n=17; 2.0%). Additional cases comprised other MPN-chronic phase (n=3, 0.4%) and MDS/MPN overlap syndromes (n=7, 0.8%). Of 848 evaluable TP53mut MN, 616 (72.6%) harbored biallelic TP53 loss and 232 (27.4%) had monoallelic loss, with 42.4% (n=98) of monoallelic cases harboring a complex karyotype (CK). Biallelic loss was highly prevalent in AML (83.1%), MDS (70.4%), and MDS/MPN overlap (71.4%), but was less frequent in cases with MPN (33.3%) and MF-chronic phase (25.0%). Across the spectrum, the frequency of biallelic loss increased stepwise with disease progression, rising from MDS-LB (62.8%) to MDS-EB1 (76.0%), MDS-EB2 (79.5%), AML (83.1%), and MF-AP/BP (79.2%) (P<0.0001). In multivariable analysis, WHO-4R category, TP53mut VAF, biallelic loss and CK were independent predictors of poor survival in TP53mut MN. Conditional inference tree analysis stratified the cohort into seven prognostic groups with distinct median OS: (1) AML, MF-BP/AP, and MDS/MPN overlap regardless of mono/biallelic and CK status (n=335, 39.7%; median OS 4.0 months); (2) MDS-EB1/EB2 with biallelic loss or monoallelic plus CK (n=212, 25.1%; 7.2 months); (3) MDS-LB, MF-CP and MPN with CK (n=156, 18.5%, OS 10.2 months); (4) biallelic MDS-LB (n=28, 3.3%; OS 15.6 months), (5) monoallelic MDS-EB1/EB2 without CK (n=20, 2.4%; OS 26.2 months), (6) monoallelic MDS-LB without CK (n=57, 6.7%; OS 33.4 months), and (7) MF-CP and other MPN without CK (n=36, 4.3%; OS 37.0 months). This analysis defines a clear stepwise risk gradient, identifying subsets with extremely poor prognosis versus a relatively indolent disease. Conclusions: This is the first study to evaluate the prognostic impact of TP53mut MN across the entire MN spectrum, providing a framework for future refinement of TP53-driven disease classification. Key findings include: (i) MF-AP/BP and MDS/MPN overlap exhibit survival outcomes as dismal as AML, irrespective of the TP53 allele status; and (ii) complex karyotype is a surrogate for “biallelic equivalent”, refining risk interpretation in monoallelic cases. Overall, our study defines seven prognostic subgroups spanning all TP53mut MN, offering a foundation for risk-adapted clinical trial design and future classification updates.

Abstract Introduction The European Leukemia Net (ELN) 2022 stratified acute myeloid leukemia (AML) patients (pts) treated with intensive chemotherapy (IC) into three groups based on genetic features. ELN 2024 (Döhner et al, Blood) and Beat AML 2024 (Hoff et al, Blood Advances) proposed revised systems to better capture the impact of genetic features in pts receiving less-intensive therapy (LIT). Real-world validation across treatment settings, including allogeneic hematopoietic stem cell transplantation (allo-HCT), is warranted. Methods We evaluated the performance of ELN 2022, ELN 2024, and Beat AML 2024 among AML pts treated at Mayo Clinic from 2016 to 2025. Only treated pts with available molecular data included. Response was assessed according to the ELN 2022 criteria. Overall survival (OS) was calculated from diagnosis. Multivariable Cox proportional hazard (CPH) models were used to adjust for covariates. Model performances were assessed using Harrell's c-index. Results 426 AML pts (median age 68 years; 60% male) were included; 195 (46%) received IC and 231 (54%) received LIT, while Allo-HCT was performed in 133 (31%) pts. Complex karyotype was present in 126 (30%) and normal cytogenetics in 143 (34%) pts. Molecular mutations included TP53 (20%), TET2 (18%), DNMT3A (16%), SRSF2 (16%), RUNX1 (16%), ASXL1 (16%), and NPM1 (15%). Among evaluable pts (83%), complete remission (CR) after first-line therapy was achieved in 47%. Risk group assignments varied: ELN 2022 vs. ELN 2024 classified pts as favorable (20% vs. 55%), intermediate (20% vs. 24%), and adverse (60% vs. 20%); Beat AML 2024 categorized pts as favorable (41%), intermediate (21%), and adverse (38%). ELN 2024 reclassified 229 (54%) pts of whom 5% upstaged and 49% downstaged, primarily affecting ELN 2022 adverse (67%) and intermediate (46%) groups. Among LIT-treated, CR rates did not differ across groups in any classification. Per ELN 2024, CR was achieved in 43% (favorable), 67% (intermediate), and 29% (adverse) of the LIT-treated pts. However, among IC-treated pts, CR was higher in favorable/intermediate vs. adverse groups across all models (p<0.0001). Allo-HCT was most common in ELN 2024 intermediate (41%) and favorable (32%) vs. adverse (16%) groups. Median OS (months; 95% CI) for the entire cohort was 15.7 (12.5–20.9) with median follow-up time of 49.8 (40.9-55). By ELN 2022, OS of the entire cohort was 49.4 (30.7–NA) for favorable, 50.0 (29.2–NA) for intermediate, and 8.8 (7.7–11.2) for adverse (p<0.0001). Per ELN 2024, OS of the entire cohort was 25.4 (18.5–37.3), 20.3 (13.4–39.8), and 5.7 (3.4–8.2), respectively (p<0.0001). Beat AML 2024 stratified OS of the entire cohort as 42.5 (30.7–NA), 11.4 (9.3–16.7), and 5.6 (3.5–8.0) for favorable, intermediate, and adverse groups (p<0.0001). ELN 2024 stratified the OS of the pts treated with LIT as 15.7 (10.1-24.9), 8.0 (4.9-13.9), and 6.8 (3.4-10.2) for favorable, intermediate and adverse risk groups, (p <0.0001). Among ELN 2022 adverse-risk pts, those reclassified as favorable per ELN 2024 had superior OS (15.0, 9.8–22.7) compared to intermediate (7.8, 5.0–9.3) or adverse (5.4, 3.3–8.0) risk groups, p<0.0001. C-index (95% CI) for OS in the entire cohort was highest for Beat AML 2024 (0.658, 0.629–0.688), followed by ELN 2022 (0.627, 0.599–0.655), and ELN 2024 (0.600, 0.568–0.631). In LIT-treated pts, ELN 2024 (0.597, 0.558–0.637) and Beat AML 2024 (0.586, 0.545–0.627) outperformed ELN 2022 (0.559, 0.521–0.596). Among allo-HCT pts, regardless of treatment type, ELN 2022 (0.610, 0.539–0.681) performed better than ELN 2024 (0.568, 0.487–0.648). In multivariable CPH models adjusted for age, bone marrow blasts, treatment intensity, and complex karyotype, ELN 2022 and ELN 2024 adverse-risk groups had higher hazard of death (HR 2.4, 1.6–3.5 and HR 1.7, 1.1–2.5, respectively, vs. favorable). No significant difference was observed between intermediate and favorable risk for ELN 2022 (HR 1.4, 0.9–2.2) or ELN 2024 (HR 1.4, 0.9–1.9). Allo-HCT was associated with a lower hazard of death (HR 0.4, 0.3–0.5). Conclusions In this real-world AML cohort, ELN 2024 and Beat AML 2024 improved OS prediction in LIT-treated pts compared to ELN 2022. This advantage was not observed in allo-HCT pts. Multivariable analysis confirmed the adverse-risk group as having the worst prognosis across all models. These findings highlight the importance of aligning molecular risk stratification with treatment modalities to enhance prognostication in AML.

Abstract Background Missense mutations in IDH1 are well-established therapeutic targets in acute myeloid leukemia (AML). In contrast, the synonymous IDH1 variant p.Gly105Gly (rs11554137), present in 5–10% of AML cases, has been considered a benign polymorphism due to its high population frequency (5.7% overall, 0.7–10.5%) and lack of protein-altering effect. However, prior studies have associated this variant with inferior outcomes (Wagner, J Clin Oncol, 2010), though findings have been inconsistent. Few studies have examined its prognostic significance in the context of treatment intensity or allogeneic stem cell transplantation (SCT). Methods With IRB approval, clinical and genomic data were extracted from the Mayo Clinic AML database for patients diagnosed between Jan 1, 2018, and Jan 31, 2025. Patients were stratified by IDH1 Gly105Gly (rs11554137) status via NGS on bone marrow. Outcomes included co-mutation profiles, treatment response, measurable residual disease (MRD) by multiparametric flow cytometry, and survival. Multivariable Cox and logistic regression models for relapse-free survival (RFS) and overall survival (OS) were adjusted for age at diagnosis, ELN 2022 risk category, and SCT conditioning regimen. Results Among 379 AML patients, 36 (9.5%) harbored the IDH1 Gly105Gly (rs11554137) variant (cases) (median VAF 49%, IQR 48–49.2). In the full cohort, median age was 68 years (IQR 59–74), M:F ratio was 1.5; 75% had de novo AML, and median follow-up was 52 months. Among cases, common cytogenetic abnormalities included complex karyotype (28%), del(7q) (14%), and trisomy 8 (6%), while frequently mutated genes included DNMT3A (19%), IDH2 (17%), and BCOR (14%). Of 345 treated patients, 43% received intensive, and 56% received low-intensity regimens, including hypomethylating agent (HMA) alone (n=13), HMA+venetoclax (n=159), and targeted therapy (n=7). Among cases, complete remission (CR) rates were higher with intensive vs. low-intensity therapy (70% vs. 17%, p=0.03). MRD positivity post induction was more common in cases (66% vs. 26%, p=0.01), suggesting suboptimal leukemic clearance. Among 103 patients who underwent allogeneic SCT in CR1 (47% received myeloablative conditioning), morphologic relapse occurred in 64.3% of cases compared to 23.6% of controls (p = 0.004). Cases experienced significantly inferior median RFS (8 months [95% CI 5–NA] vs. not reached for controls, p <0.001), indicating more durable post-SCT remission in the control group. In a multivariable Cox proportional hazards model adjusting for age, ELN risk, conditioning intensity, MRD status, and complex karyotype, the IDH1 Gly105Gly variant remained independently associated with shorter RFS (HR 3.97, 95% CI: 1.74–9.05, p = 0.001). Complex karyotype was also an independent predictor of relapse (HR 3.35, 95% CI: 1.28–8.73, p = 0.014). Consistent with this, logistic regression revealed a significantly increased odds of post-SCT relapse among cases (OR 7.95, 95% CI: 2.19–35.27, p=0.001), independent of clinical covariates. Relapsed cases were significantly more likely to harbor FLT3-ITD mutations than relapsed controls (p=0.041). Pathway-level analysis revealed increased frequency of mutations in signal transduction genes (FLT3-ITD, CSF3R, NF1) (p=0.002). All CNS relapses among cases occurred in patients harboring at least one of these mutations. Despite the higher relapse rate, there were no significant differences in OS (14.4 vs. 11.3 months, p=0.36) between cases and controls. The IDH1 Gly105Gly variant was not independently associated with OS (HR 1.26, p=0.30). In contrast, age (p<0.0001) and adverse ELN risk (p<0.0001) were significant predictors of inferior OS. These findings suggest that the prognostic impact of IDH1 Gly105Gly is most pronounced in the post-SCT setting. Conclusion The synonymous IDH1 Gly105Gly variant (rs11554137) is an independent predictor of post-SCT relapse in AML, potentially mediated by co-occurring mutations in signal transduction pathways. Despite no observed impact on OS, likely due to effective salvage therapies, the variant's link to lower CR rates with low-intensity therapy and higher MRD burden suggests treatment intensity influences its clinical relevance. These findings support reevaluating the prognostic role of synonymous variants in AML and call for larger studies and mechanistic investigations into splicing, transcript stability, and translation.

Abstract Introduction: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is an adult-onset autoinflammatory disorder defined by somatic UBA1 mutations in hematopoietic stem cells (HSCs). Given the higher median age at onset and prevalent inflammation, approximately 60% of patients have concurrent clonal hematopoiesis (CH) involving epigenetic regulators, DNMT3A and TET2. We have previously demonstrated (Gutierrez-Rodrigues, Blood 2023) that DNMT3A and TET2 mutations in VEXAS are independently associated with increased mortality, without an increased risk of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). To define the role of CH in VEXAS, we investigated the impact of CH on mortality and related epigenetic dysregulation in primary VEXAS patient samples and validated these findings using an adenine base-editing (ABE) strategy to model cooccurring UBA1 and DNMT3A mutations in CD34+ HSCs. Methods: After IRB approval, 114 VEXAS patients from the Mayo Clinic and the NIH were retrospectively assessed, including 104 screened for CH by error-corrected DNA sequencing (ECS). DNA methylation changes were identified using the Illumina Infinium MethylationEPIC BeadChip and genome-wide using Biomodal 6-base sequencing on peripheral blood samples (PB). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed using the 10x Chromium 3' GEX platform; 21,114 pooled cells were subjected to UMAP dimensionality reduction, followed by Azimuth based cell identification. HSCs from different donors were base edited for UBA1 M41T mutation with and without cooccurring DNMT3A splice-site mutations. Levels of cytokines in cell culture supernatants were measured using Olink technology, transcriptomic changes were assessed using bulk RNA-seq. Statistical analyses were carried out using R. Results: Patients' median age was 68 (62-74) years; 95% were white and 99% were males. 39% had a pathogenic CH variant, with DNMT3A (18%; median VAF: 42%; range 6-46%) and TET2 (11.5%; median VAF: 11%; range: 2-37%) being most common (2% had both). While the presence of any CH mutation did not impact survival (7.7 vs 11.5yrs, p =0.69), DNMT3Amt CH had an age-independent negative impact on mortality (9.2 vs 5.6yrs, p=0.02), without an increased risk of MDS/AML (p=0.31). Changes in DNA methylation were profiled using the methylation array in 19 VEXAS patients without CH (Median Age: 68 years) and in 9 with DNMT3Amt CH (Median Age: 74 years), respectively. VEXAS samples with DNMT3Amt CH displayed a hypomethylation phenotype compared to VEXAS samples with no CH, with 4,096 unique hypomethylated CpGs, occurring mainly at gene enhancers (Enh) and transcriptional states (Tx, TxWk). This loss of DNA methylation was enriched in inflammatory pathways with extensive hypomethylation observed in the promoters and gene bodies of IL-1 family genes. CITE-seq performed on 7 VEXAS samples (4 DNMT3Amt CH vs 3 no CH) demonstrated an upregulation of gene expression predominantly in T-cell subsets. This increased expression was enriched in inflammatory genes, largely centered around IL-1 and IL-18 family signaling. Pathway analysis revealed an enrichment in respiratory electron transport and oxidative phosphorylation (oxphos), with the VEXAS samples with DNMT3Amt CH demonstrating significantly increased oxphos gene signatures. Base editing of CD34+ cells was done using an adenine base-editing (ABE) strategy to model dual UBA1 and DNMT3A splice-site mutations. We observed that the HSCs base edited for the UBA1 M41T recapitulated the cellular hallmarks of VEXAS demonstrating reduced ubiquitination, unfolded protein response (UPR) activation, and increased inflammation. The addition of cooccurring DNMT3A mutations resulted in decreased DNMT3A expression, further increasing inflammatory markers, confirming altered inflammatory and metabolic pathways. Conclusion Our study validates the age-independent, negative prognostic impact of DNMT3Amt CH on survival in VEXAS syndrome. DNMT3A mutations mediate epigenetic and transcriptional priming associated with upregulation of inflammatory and mitochondrial function pathways. These findings have been validated in a base edited cell model with UBA1 and DNMT3A co-mutations.

Abstract Introduction: DDX41 (DEAD-box helicase 41) mutations (mt) represent the most prevalent germline predisposition syndrome (GPS) in adult patients (pts) with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This molecular subtype is typically associated with late-onset disease, runs an indolent clinical course, with a relative absence of high-risk co-mutations. While several retrospective studies have reported favorable responses to venetoclax (VEN)-based therapies, anecdotal observations suggest variable outcomes. We aimed to evaluate the clinical characteristics and treatment outcomes of patients with DDX41-mt MDS/AML treated at the Mayo Clinic. Methods: We conducted a retrospective study of pts diagnosed with DDX41-mt germline predisposition syndrome with concurrent myeloid neoplasms at our institution. Germline testing was conducted under an IRB approved protocol (Mayo Clinic IRB# 16-004173; clinical-trials gov. Identifier: NCT02958462). Of 198 pts with DDX41-mt myeloid neoplasms, 77 had high-risk disease—43 with MDS with increased blasts (MDS-IB1, n = 11; IB2, n = 32) and 34 with AML (WHO 2022). We assessed how clinical, and disease features influenced outcomes in this subgroup. Among these pts, 69 (90%) pts had likely pathogenic/pathogenic variant (DDX41path) variants and 8 (10%) pts had variant of undetermined significance (DDX41VUS) according to variant classification criteria from the American College of Medical Genetics/ the Association of Medical Genetics/ the Association for Molecular Pathology (ACMG/AMP) guidelines. Results: The median age was 68 years (yrs) (range [R],34-92), 70% were male and the median bone marrow blast % was 15 (R,5-60). The most common DDX41 germlinevariants (> 5% cases) included p.D140fs (26%), p.M1I (22%), and p.P258L (6.5%). 74% had DDX41 truncating variants. Thirty-one (40%) acquired a somatic DDX41-mt (R525H [81%]) and 43 (56%) had other somatic mutations. Among these, the most frequent were ASXL1 (n= 9, 21%), TP53 (n= 8, 10%), splicing factor (n= 7, 9%), and DNMT3A (n= 5, 6%). Twenty-six, (34%), 26 (34%), 15 (19%) and 9 (12%) pts were treated with hypomethylating agents (HMA), HMA+VEN, intensive chemotherapy (IC) and active surveillance/supportive care, respectively. Overall, the composite complete remission rate (cCR) in high-risk MDS and AML was 56%. The cCR rates (41%, 59% and 68.7%, p= 0.10) and median duration of response (DOR; 43.7, 44.9 and 28.9 months [mo], p= 0.46) were not significantly different with HMA, HMA+VEN, and IC induction. Thirty-one (40%) pts received allogeneic stem cell transplantation (allo-HCT); 4 (36%), 15 (47%) and 12 (35%) in MDS-IB1, MDS-IB2 and AML groups, respectively (p= 0.56). With a median follow up of 34.7 mo (R, 0.7-136.7), the median overall survival (OS) from the time of diagnosis was 56.9 mo (95% CI, 50.7-63.0) and was not significantly (NS) different between MDS-IB1 (not reached, 70% at 3 yrs), MDS-IB2 (56.9 mo, 86% at 3 yrs) and AML (55.7 mo, 60% at 3 yrs) groups, p= 0.16). The median OS was NS different between DDX41path and DDX41VUS (58.6 vs 55.7 mo, p= 0.50), respectively. We conducted a multivariable analysis for OS using variables that showed significance or trend towards significance in univariable analysis. Concurrent splicing factor mutations (HR; 5.29, 95% CI: 1.29-21.6, p= 0.02) remained negatively prognostic, while there was a trend towards better survival outcomes with allo-HCT (HR; 0.48, 95% CI: 0.21-1.07, p= 0.07). Bone marrow blast ≥ 20% (HR; 1.87, p= 0.12) and TP53-mt (HR; 2.14, p= 0.12) did not retain significance. Conclusion: In our cohort of patients with germline DDX41-mt high-risk MDS and AML, VEN-based regimens were not superior to HMA alone or IC, in terms of response rates and outcomes. On multivariable analysis, the presence of concurrent splicing factor mutations independently and negatively impacted survival with a trend towards better outcomes with allo-HCT. These findings highlight the heterogeneity within DDX41-mt myeloid neoplasms and underscore the need for future studies in larger cohorts to better define predictors of response and survival.

Abstract Background: KMT2A-rearranged acute myeloid leukemia (KMT2Ar AML) is characterized by chromosomal translocation involving KMT2A gene at 11q23, resulting in fusion with variety of partner genes. It is usually associated with poor outcomes, high risk of relapses and suboptimal responses to conventional therapies. While NPM1-mutated (mt) AML is considered favorable risk, they are equally dismal in relapse refractory (RR) setting. Menin inhibitors have emerged as a promising therapeutic strategy, particularly in RR AML patients (pts) with KMT2Ar and NPM1-mt genotypes. While revumenib, a menin inhibitor approved for managing RR KMT2Ar acute leukemia, no menin inhibitor is currently approved for NPM1-mt AML. In this study, we evaluated clinical outcomes of adults with KMT2Ar AML and performed a comparative analysis of pts with RR KMT2Ar and NPM1-mt AML using data from the Mayo Clinic. Methods: After screening 102 AML pts with reported KMT2A abnormalities, we identified 33 adults with confirmed KMT2Ar diagnosed between 2012 and 2024 for retrospective analysis. Clinical and molecular/cytogenetics data, induction type (intensive chemotherapy [IC] vs non-intensive chemotherapy [NIC]), response, transplant use, and survival outcomes were reviewed. A separate analysis was performed comparing 20 RR KMT2Ar AML pts with 20 propensity score matched RR NPM1-mt AML pts from a pool of 125 NPM1-mt RR pts. Results: The median age was 50 years (yrs) (range [R], 19.0-75.0), and 60.6% were < 60 yrs. Most cases were de novo AML (64%), and the most frequent translocation was t(6;11)(q27;q23)/MLLT4 (27%), followed by t(10;11)(p12;q23)/MLLT10 (21%), t(9;11)(p21.3;q23.3)/MLLT3 (15%), and t(11;19)(q23;p13.1)/ELL (12%). Other KMT2A fusion partners were rare and collectively accounted for the remaining cases. The median follow-up was 57 months (mo) (R, 7.0-109.0). The overall response rate (ORR) was 91%, and composite complete remission (CRc, , including CR, CRi, and CRh) was 73%. The CRc rates were comparable between IC and NIC (80% vs 62%, p=0.24). CRc were not significantly different by disease type (de novo [71.4%] vs secondary [sAML] [75%], p= 0.82) or KMT2A fusion partner gene (60% for t[9;11]/MLLT3 vs. 57.1% for t[10;11]/MLLT10 vs 55.6% for t[6;11]/MLLT4, p=0.069). Seventeen (52%) pts received an allogeneic stem cell transplantation (allo-HCT). The median overall survival (mOS ) for the entire cohort was 16 mo (R, 11.0-34.0). In subset analysis, IC compared to NIC was associated with significantly longer mOS (30 vs 11 mo, p=0.014). The mOS was also superior in pts <60 vs ≥60 yrs (32 vs 8 mo, p=0.005). Similarly, de novo cases had better mOS than sAML (22.0 vs 9.5 mo, p=0.052). Among KMT2Ar subtypes, t(6;11)/MLLT4 showed the longest mOS (54 mo [R, 30.0-not reached (NR)]), while t(9;11)/MLLT3 had the shortest (11.0 mo, [R 9-NR]), but differences were not statistically significant (p=0.32). The mOS was significantly longer among allo-HCT pts (30 vs 11 mo, p=0.005). Among 29 pts who experienced RR disease, the median relapse free survival (RFS) was 3 mo (R, 2.0-10.0). First salvage therapy yielded a CRc of 43.8% in 16 evaluable pts. When compared to a propensity matched cohort of 20 RR NPM1-mt AML pts, the CRc rates was 57.9% vs 50% in RR NPM1-mt and KMT2Ar AML (p= 0.68). The mRFS was not significantly different between RR KMT2Ar and NPM1-mt AML, (3 vs 4 mo, p=0.9). However, KMT2Ar pts had inferior mOS compared to NPM-mt AML (12 vs 65 mo, p=0.004). We further compared survival between NPM1-mt and KMT2Ar pts stratified by allo-HCT status. Among those who underwent allo-HCT, NPM1-mt pts had longer mOS (NR vs 22 mo, p=0.037). Among non-allo-HCT pts, mOS was numerically higher in NPM1-mt vs KMT2Ar (19 vs 8 mo, p=0.11). Conclusions: This single institusion cohort of adult AML with KMT2Ar underscores the biological and clinical heterogeneity of this high-risk group. IC was associated with a survival benefit, particularly among younger and fit patients. However, the outcomes following relapse remained poor, highlighting the urgent need for improved post-remission and salvage therapies. Comparative analysis with matched RR NPM1-mt AML pts revealed similarly dismal survival outcomes. Emerging data from single-arm studies of menin inhibitors in both frontline and RR settings among pts with NPM1-mt and KMT2Ar AML are encouraging and may represent a paradigm shift in treatment.

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.