Figure S1. Landscape of somatic mutations and cytogenetics in Moffitt MF cohort. Figure S2. Effects of enforced MYC expression in HSCs in vivo. Figure S3. MYC-induced changes in hematopoietic sub-populations and colony forming potential. Figure S4. The JAK/STAT, PI3K/AKT, MEK/ERK, and alarmin pathways in MYC-driven MF.

Supplementary Figure 8. Impact of TP53 Mutation and Complex Karyotype on Multivariable Coefficients and Collinearity.

Background Venetoclax, a potent B‐cell leukemia/lymphoma‐2 inhibitor, is an antineoplastic agent used in various hematologic malignancies, including acute myeloid leukemia (AML). The aim of the study is to evaluate the incidence, risk factors, and outcomes of major adverse cardiac events (MACEs) among patients with newly diagnosed AML receiving venetoclax‐based therapy. Methods We conducted a retrospective, single‐center cohort study of 214 patients with newly diagnosed AML treated with venetoclax. MACE was defined as a composite of new‐onset heart failure, heart failure exacerbation requiring hospitalization, myocardial infarction/coronary revascularization, or stroke/transient ischemic attack during active venetoclax‐based therapy. The Fine–Gray subdistribution hazard model determined the association between baseline clinical characteristics and MACEs, with noncardiovascular death as a competing risk. A Cox proportional hazard model determined the association between time‐dependent MACEs and overall survival. Results Among 214 patients (mean age, 71.5 years; 41% women), MACEs occurred in 14 (6.5%) patients. Patients with non–de novo (secondary /treatment‐related) AML had a higher incidence of MACEs compared with de novo AML (85.7% versus 14.3%; subdistribution hazard ratio [HR], 7.1 [95% CI, 1.5–33]; P<0.01). Time‐dependent MACE was independently associated with inferior overall survival (adjusted HR, 2.75 [95% CI, 1.41–5.35]). Conclusions In conclusion, MACE is a clinically significant event that occurs in 6.5% of patients with AML treated with venetoclax and is a higher‐risk event for patients with secondary AML or treatment‐related AML.

Supplementary Figure 17. Comparison of Risk Classification Logic Across Models.

Supplementary Figure 16. Effect of Allo-SCT and IDH1/2 inhibitors on OS.

Supplementary Table 4. Statistical Significance of Risk Reclassification Between Moffitt and Comparator Models.

Comparison of gene expression profiles of Mx1-Cre+/-;Rosa26LSL-MYC/LSL-MYC vs. Mx1-Cre+/-;Rosa26+/+ mouse.

6504 Background: Hypomethylating agents (HMAs; azacitidine/decitabine) plus venetoclax (VEN) are standard for newly diagnosed acute myeloid leukemia (AML) unfit for intensive chemotherapy; however FMS-like tyrosine kinase-3 internal tandem duplication ( FLT3-ITD ) confers resistance and poor overall survival (OS). Addition of quizartinib (QUIZ), a potent FLT3 inhibitor, may improve outcomes. Methods: Phase I/II study of QUIZ+decitabine+VEN in two cohorts: newly diagnosed FLT3-ITD AML pts unfit for intensive chemotherapy and relapsed/refractory (R/R) FLT3-ITD AML pts. Primary endpoints were maximum tolerated dose (MTD) and overall response rate (ORR). Results: Eighty-eight patients (pts) were enrolled (frontline N=42; R/R N=46). QUIZ 26.5mg/day was selected as the recommended phase II dose (RP2D). In the frontline cohort, median age was 70y (62–85); 15 pts (36%) were ≥75y; 25 (60%), 12 (N=28%), and 5 (12%) had de novo, secondary, and therapy-related AML, respectively. DNMT3A (43%), NPM1 (33%), and RUNX1 (31%) were the most common co-mutations. Complete remission (CR), CR with incomplete count recovery (CRi), morphologic leukemia-free state (MLFS), end-of-cycle-1 (EOC1) measurable residual disease (MRD) negativity by multicolor flow cytometry (MFC; 0.01%), and next-generation sequencing (NGS) FLT3-ITD negativity (5×10⁻⁵) were 72% (N=29), 17% (N=7), 2% (N=1), 58% (19/32), and 27% (5/18), respectively; 2 pts in cycle 1 were not evaluable. Best MRD negativity by MFC and FLT3-NGS were 68% (23/34) and 83% (15/18). Median time to absolute neutrophil count (ANC) >500, ANC >1000, and platelets >50K were 40 (19–72), 41 (14–72), and 35 (16–71) days, respectively. The median relapse-free survival (RFS) and OS were 24.7 and 36.5mo. Fifteen pts (36%) proceeded to allogeneic stem cell transplant (ASCT) in CR1, with OS not reached vs 36.6mo without ASCT (p=0.65, landmark analysis). Median 3 cycles (1–39) were delivered; 11 pts remain on study. Thirty-one pts discontinued protocol due to ASCT (N=15), relapse (N=9), physician/pt choice (N=3), induction death (N=1), death in CR (N=1), death with disease (N=1), or hospice (N=1). Grade ≥3 non-hematologic adverse events (>5%) included febrile neutropenia (37%), pneumonia (33%), infections (17%), pain (10%), ALT increase (10%), fracture (10%), sepsis (9%), hypertension (7%), hypotension (7%), hyponatremia (7%), gait disturbance (7%), oral mucositis (7%), and pleural effusion (7%). In the R/R cohort, CR/CRi was 28% (N=13) with 33% (N=15) MLFS; 37% (N=17) proceeded to ASCT; median OS was 6.3mo (further data will be provided at the time of presentation). Conclusions: QUIZ+decitabine+VEN combination resulted in high CR/CRi, deep MRD responses, and encouraging survival in older pts with newly diagnosed FLT3-ITD AML. Clinical trial information: NCT03661307 .

Myelodysplastic neoplasms (MDS) with TP53 multihit alterations are associated with dismal outcomes. MDS with isolated del(5q) present favorable prognosis but is defined by the absence of TP53 multihit alterations. However, whether TP53 multihit alterations exert the same adverse impact in this genetic context remains uncertain. We retrospectively analyzed the characteristics and outcome of 43 patients with MDS with isolated del(5q) harboring TP53 multihit alterations (MDS-del(5q) TP53 multihit) and compared with 68 patients with low-blast MDS with TP53 multihit and without isolated del(5q) (MDS-LB TP53 multihit). Patients with MDS-del(5q) TP53 multihit showed significantly higher platelet counts, more frequent SF3B1 mutations, were less often classified as high-risk by IPSS-R or IPSS-M, and had significantly better outcomes than patients with MDS-LB TP53 multihit: overall survival of 70.2 vs 13.9 months, and time to acute myeloid leukemia progression (AML) of 31.9 vs 7.2 months, respectively. Moreover, the superior outcomes of MDS-del(5q) TP53 multihit patients persisted significant even when compared with MDS-LB TP53 multihit cases without complex karyotype (survival of 70.2 vs 39.9 months; time to AML progression of 31.9 vs 11.4 months). These findings indicate that, in MDS-del(5q) the adverse impact of TP53 multihit alterations may be less important than in other MDS subtypes.

Supplementary Table 8. Baseline clinical and genetic data for the study cohort.

Supplementary Figure 13. Risk Group Reclassification Patterns Between Moffitt Model and Comparator Models.

Outcomes are poor for TP53 mutant myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients who undergo allo-HSCT. Notably, minimal data exist on the impact of TP53 MRD monitoring post allo-HSCT. Thus, we completed duplex TP53 MRD sequencing for all patients on our prospective eprenetapopt (APR-246) + azacitidine maintenance study (n=14). Bone marrow aspirates were obtained prior to allo-HSCT, prior to the start of investigational therapy (day +30 to day +100) and after cycle 3 and cycle 12 of therapy. To assess low allele frequency mutations in TP53, a custom-targeted sequencing panel was used with duplex sequencing, targeting 30,000-70,000X duplex coverage to detect variants at a frequency as low as .005%. For all analyses, TP53 MRD negativity cutoff was 0.01%. All study patients had significant TP53 positivity prior to allo-HSCT and 57% post-HSCT. MRD evaluation after end of maintenance (12 cycles) was the strongest predictor of outcomes. Specifically, MRD negativity after cycle 12 strongly predicted OS (33.9 vs 20.4 months; P=.005) and EFS (33.9 vs 10.1 months; P=.004) with a trend for RFS (32.6 vs 13.5 months; P=.06). TP53 MRD was strongly predictive of outcomes, supporting incorporation of this assay in future novel strategies.

Frameshift mutations in exon 12 of nucleophosmin 1 (NPM1 mut) are among the most common mutations in acute myeloid leukemia (AML) and have historically been considered favorable-risk in the absence of FLT3-ITD. In the European LeukemiaNet (ELN) 2024 risk-classification for patients treated with hypomethylating agents plus venetoclax (HMA + VEN), NPM1 mut is not considered favorable when co-occurring with signaling gene (SG) mutations (i.e., FLT3-ITD, NRAS, KRAS). However, due to limited numbers in the original analysis, the prognostic impact of SG mutations in NPM1-mutant AML remains unclear. We evaluated the prognostic significance of NPM1 mut with and without SG mutations in two independent cohorts of patients ≥ 60 years with ELN 2024 favorable- or intermediate-risk AML treated with HMA + VEN. Cohort 1 included 322 patients treated in the academic setting. NPM1 mut (n = 61) was associated with a nonsignificantly longer overall survival (OS) compared to NPM1 wild-type (NPM1 wt) (median, 53.05 vs. 17.03 months, p = 0.10). In multivariable analysis (MVA), SG mutations were not independently prognostic within the NPM1 mut subgroup. Cohort 2 included 816 patients from a real-world community-treated cohort. NPM1 mut (n = 124) had a longer OS compared with NPM1 wt (median, 15.3 vs. 14.4 months, p = 0.03). In MVA, NRAS, KRAS, and FLT3-ITD were independent unfavorable prognostic factors; NPM1 mut with, compared to without, SG co-mutation had a shorter OS (median, 9.4 vs. 31.6 months, p = 0.001). These findings suggest SG mutations negate the favorable impact of NPM1 mut in older patients treated with HMA + VEN. Prospective clinical trials are needed to investigate the use of combination therapies to improve outcomes in this high-risk subgroup.

Mosaic chromosomal alterations (mCAs) are a prevalent but poorly understood form of clonal hematopoiesis (CH). Whether mCAs contribute to disease independently of CHIP, and whether their large-scale genomic effects can be resolved to actionable targets, remain unknown. In 452,594 UK Biobank participants, we show that mCAs confer multimorbidity and mortality risk independent of CHIP. Notably, mCA-CHIP co-occurrence defines a very high-risk clonal state with synergistically elevated mortality, identifying a population not captured by CHIP screening alone. To resolve large mCAs to specific disease mechanisms, a cytoband-level mapping framework was developed that links mCAs to discrete genomic loci and candidate effector genes. Functional validation using single-cell transcriptomics and mouse models prioritized MYC (chr8 gain) and S100A9 (chr1 gain) as key drivers of systemic inflammation and multiorgan pathology. These findings establish mCAs as independent, synergistic, and genetically-resolvable drivers of age-related disease, with immediate implications for screening, risk stratification, and therapeutic development.

Myelodysplastic syndromes with isolated deletion of chromosome 5q [MDS-del(5q)] constitute a distinct biological entity traditionally associated with favorable outcomes, although up to one quarter of patients progress to acute myeloid leukemia (AML). Existing prognostic models, developed in heterogeneous MDS populations, may not adequately capture risk within this subgroup. We assembled an international cohort of 682 patients with MDS-del(5q) to evaluate the performance of the IPSS-R and IPSS-M, identify prognostic variables, and develop a disease-specific prognostic tool, the IPSS-del(5q). Most patients were classified as lower-risk by IPSS-R (94.4%) and IPSS-M (85.5%), yet both systems showed limited discriminatory ability (C-indices ≈0.5). Independent adverse prognostic factors included age ≥70 years, male sex, anemia (hemoglobin ≤10 g/dL), thrombocytopenia (platelets ≤100×10⁹/L), the presence of one additional chromosomal abnormality, ≥2 gene mutations, SF3B1 mutations, and high-risk TP53 status. Six variables were included in the IPSS-del(5q), stratifying patients into standard-risk (74.3%) and high-risk (25.7%) groups with significantly different LFS (69.2 vs. 32.0 months; p<0.01). Moreover, this model reclassified 19.1% of lower-risk IPSS-R and 14.6% of lower-risk IPSS-M patients into the high-risk IPSS-del(5q) group. However, its discriminative power remained modest, with a C-index of 0.60. Overall, this study provides the most comprehensive prognostic evaluation of MDS-del(5q) to date, demonstrates the limited discriminatory capacity of existing MDS scores in this entity, and underscores the need to develop refined disease-specific prognostic approaches for this MDS subtype.

Venetoclax (VEN) combined with hypomethylating agents (HMA) improves outcomes for patients with newly diagnosed acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy, yet overall survival (OS) remains variable. We analyzed 506 patients with AML treated with first-line HMA/VEN at Moffitt Cancer Center to develop a genetics-based prognostic model. In multivariate analysis, mutations in TP53, KRAS, JAK2, U2AF1, CBL, and cytogenetic lesions del(7q)/-7, del(17p)/-17/i(17q), del(20q), and MECOM rearrangements predicted inferior OS, whereas IDH1/2 mutations were favorable. A point-based system stratified patients into low-, intermediate-, and high-risk groups with median OS of 54.2, 22.3, and 7.5 months, respectively [P < 0.0001; concordance index (C-index) 0.648]. External validation (n = 126) retained prognostic separation (median OS 24.7, 17.4, and 4.3 months, P = 0.0005; C-index 0.626). Compared with existing HMA/VEN-specific models, our model demonstrated superior low- versus intermediate-risk discrimination (31.9-month separation, P = 0.002; hazard ratio = 0.45, P = 0.003), with a comparable C-index. Our model supports personalized risk stratification for HMA/VEN-treated AML, pending broader validation. SIGNIFICANCE:This study identifies key mutational and cytogenetic markers associated with treatment response and OS in patients with AML receiving HMA/VEN therapy. By integrating these genetic markers, our new prognostic model offers improved risk stratification, guiding personalized treatment strategies for patients ineligible for intensive chemotherapy. See related commentary by Lachowiez and Loghavi, p. 339.

Introduction Allogeneic hematopoietic stem cell transplant (allo-HSCT) can improve survival in select patients (pts) ≥60 years (yrs) old with acute myeloid leukemia. Venetoclax (VEN) with a hypomethylating agent (HMA) is standard for older (≥60 yrs) or medically unfit pts unable to tolerate 7+3. The impact of VEN/HMA vs 7+3 on outcomes post-HSCT is unclear in this age group. Methods Eleven institutions in the Myeloid Malignancy Association on Rapid Research Outcomes Working Group (MARROW) consortium collected data on newly diagnosed pts aged 60-80 yrs treated with VEN/HMA or 7+3 followed by allo-HSCT. Composite complete remission (CRc) was defined per ELN 2022. Categorical and continuous variables were compared by chi-square/Fisher's exact test and Wilcoxon rank-sum test, respectively. Kaplan-Meier and log-rank methods estimated disease-free (DFS) and overall survival (OS). Cox proportional hazards models estimated hazard ratios (HR). Results Of 251 transplanted pts, 201 (80%) received 7+3 and 50 (20%) VEN/HMA. Median follow-up was 2.8 yrs. Full pt and disease characteristics are in Table 1. VEN/HMA pts were older (median 70 vs 66 yrs, p<0.001). Forty-four percent (n=111) had abnormal karyotype and 9% (n=22) had complex karyotype. Per ELN 2022, majority (54%, n=100) were adverse risk. Per ELN 2024, VEN/HMA had more favorable risk (76% vs 56%, p=0.006). Most common mutations were in NPM1, DNMT3A, and IDH2 (frequencies in Table 1). VEN/HMA pts had more mutations in FLT3-TKD (p=0.004), SRSF2 (p=0.007), and IDH1 and IDH2 (p=0.002 each).Most (90%, n=225) had CRc (91% 7+3 and 86% VEN/HMA, p=0.43). Median DFS in 7+3 was 3.2 yrs vs not reached in VEN/HMA (p=0.04; Figure 1A). The 7+3 group had more relapse post-SCT (33% vs 14%, p=0.01). On multivariate analysis (MVA), VEN/HMA predicted superior DFS (HR=0.28, p<0.005), while complex karyotype (HR=3.68, p<0.001) and FLT3-TKD (HR=3.51, p=0.003) predicted inferior DFS.VEN/HMA predicted superior OS on MVA (HR=0.33, p=0.02) while complex karyotype (HR=2.8, p=0.003) predicted inferior OS. Median OS in 7+3 was 5.6 yrs vs not reached in VEN/HMA (p=0.25; Figure 1B); this difference became significant (p=0.04; Figure 1C) after excluding pts with complex karyotype. PTPN11 (HR=3.1, p=0.03) and TP53(HR=2.3, p=0.04) mutations also predicted inferior OS on MVA. Discussion In this real-world, multicenter study, VEN/HMA did not compromise post-HSCT survival. This is especially important to consider in older adults, in whom the risks of intensive chemotherapy may outweigh the benefits. In our cohort, VEN/HMA pts had less relapse post-HSCT, superior DFS, and, after exclusion of complex karyotype, superior OS. This is consistent with VEN/HMA and complex karyotype being independent predictors of superior and inferior OS, respectively, on MVA. Prospective trials are warranted to better understand the impact of VEN/HMA on post-HSCT outcomes in this age group.

Supplementary Figure 4. Total cycles of HMA/VEN treatment.

BACKGROUND:Myelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by multistep molecular evolution and a variable risk of leukemic transformation. Given this prognostic heterogeneity, accurate risk stratification is essential for clinical decision-making. We developed ProgEvo, a proprietary framework that infers molecular evolutionary trajectories and integrates them with clinical data to improve prognostic accuracy. METHODS:ProgEvo was trained on 2519 patients in cBioPortal (https://www.cbioportal.org) and validated using two external cohorts: Genomed4All (2043 patients) and a Moffitt Cancer Center (MCC) cohort (2157 patients). Directional evolutionary routes were inferred and selected for prognostic modeling if they were consistently associated with leukemia-free survival. A multivariable feature selection strategy was applied to integrate evolution-consistent variables into the existing IPSS-M model. RESULTS:ProgEvo identified 1765 gene co-occurrences aggregated into 45 directional evolutionary routes. Of these, 18 were validated in the Genomed4All cohort. Five evolution-informed variables, two directional routes (Additional Sex Combs-Like 1 [ASXL1]→KRAS Proto-Oncogene [KRAS] and Serine and Arginine-Rich Splicing Factor 2 [SRSF2]→NRAS Proto-Oncogene [NRAS]), one co-occurrence (NRAS/RUNX Family Transcription Factor 1 [RUNX1]), and two early mutations (ATRX [ATRX Chromatin Remodeler] and Janus Kinase 2 [JAK2]) were integrated into IPSS-M to generate IPSS-M-Evo. The model with "-Evo" improved discrimination for both leukemia-free survival and overall survival, with over 40% of patients restratified in the Genomed4All data. The performance of the model was further confirmed in the MCC cohort. CONCLUSIONS:ProgEvo enabled inference of a molecular evolution model and integration of evolution-informed covariates into clinical prognostic frameworks, supporting the development of the IPSS-M-Evo model. A free web-based tool allows clinicians to calculate the IPSS-M-Evo score and match individual mutational profiles to cohort-derived evolutionary trajectories (https://evoclin.unimib.it/tools/evolution-graphs.html and https://evoclin.unimib.it/tools/ipssmevo.html). (Funded by the European Union and others.).