Litzow, Mark R.

Abstract Background: VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a hemato-inflammatory syndrome due to somatic mutations in UBA1 gene. Recently, DNA methyltransferase inhibitors (specifically, 5-azacitidine) and allogeneic hematopoietic stem cell transplantation (alloHSCT) have shown success in eradication of UBA1 clones and clinical remission in some patients. This study aims to provide a comparative efficacy analysis between DNA methyltransferase inhibitors (hypomethylating agents/HMA) versus alloHSCT in VEXAS syndrome patients. Methods: This is a multi-center, retrospective cohort study. UBA1 mutations were detected either through next generation sequencing [NGS, limit of detection (LOD) 2% variant allele fraction (VAF)] or quantitative droplet digital polymerase chain reaction testing (ddPCR, LOD 0.5% VAF). Outcomes of interest included overall survival (OS), glucocorticoid (GC) discontinuation/reduction in dose (prednisone < 10 mg/day), and complete molecular remission (CMR, defined as an undetectable UBA1 mutation). OS was calculated from the time of diagnosis to death or last follow up. Cumulative incidence of reduction in GC dose was calculated using competing risk analysis after accounting for death. Results: Between years 2020-2024, a total of thirty-nine male patients (median age 67, range 45-82 years) with VEXAS syndrome, necessity for therapy (GC-refractory inflammation and/or progressive marrow failure/myeloid neoplasm) and confirmed UBA1 pathogenic mutation were included. Of these, 18 (46%) received HMA therapy (5-azacitidine 72% and Decitabine 28%) with a median of 4 (range 1-36) cycles. and 21 (54%) underwent alloHSCT (MSD=4, MUD=12, MMUD=2, and haploidentical=3), all received reduced intensity conditioning (Flu/Mel =11, Flu/Bu=5, Flu/Cy/TBI=2, and TBF=3) and 91% received post-transplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis (only 2 patients in alloHSCT group received prior HMA without benefit). Baseline characteristics were well-balanced between HMA and alloHSCT groups; age at treatment (median 70 vs 65 years, p=0.2), and UBA1 M41V subtype (25% vs 40%, p=0.3), cytogenetic and non-UBA1 mutation abnormalities (p=0.4), prior lines of therapy (median 4 in both, p=0.5), time from diagnosis to therapy (median 7 vs 13 months, p=0.15), indication for therapy (p=0.12), Charlson CI scores (median 4 in both, p=0.4) and HCI-CI scores (median 4 in both, p=0.7). At a median follow-up of 45 (range 7-57) months, 11 (28%) deaths (alloHSCT=3 and HMA=8) were documented. Causes of death included infections (n=6), cardiac disease (n=2), poor graft function (n=2) and pulmonary GVHD (n=1). Compared to HMA therapy, alloHSCT group demonstrated higher rates of GC discontinuation (52% vs 6%, p<0.01) and CMR (evaluable= 22, 100% vs 50%, p<0.01). In the HMA group, 50% (n=9) discontinued therapy after a median of 4 months due to infectious complications or lack of benefit. In the alloHSCT group, 3 (14%) patients developed grade 2 acute GVHD (1-skin, 2-gut, no grade 3-4 acute GVHD) and 4 (19%) experienced moderate to severe chronic GvHD (2-lung, 2-mucocutaneous). In multivariable analysis (MVA) adjusted for age and HCT-CI, alloHSCT was identified as the strongest favorable predictor for OS (HR 0.18 95% CI 0.04-0.8) with similar results when analysis was restricted to transplant-eligible patients < 75 years of age (HR 0.1, 95% CI 0.03-0.6). In a 6-month post-treatment landmark analysis (n=31 evaluable), patients who underwent alloHSCT (n=17) exhibited numerically superior GC discontiuation rate (29% vs 14%, p=0.049) and subsequent OS compared to HMA (2-years OS: 84% vs. 61%, median not reached vs 25 months, 95% CI: 0.06–1.7). In a post-treatment competing risk [GC dose reduction (death)] analysis alloHSCT was associated with superior 2-, and 3-year rates of 57% (10%), and 76% (10%), compared to HMA 22% (24%), and 31% (42%) [p<0.01 (0.04)]. AlloHSCT was also associated with higher rate of CMR (evaluable= 21, alloHSCT N=16 and HMA N=5, 100% vs 60%, p=0.01). At the last follow up, none of the patients who achieved CMR had re-emergence of the UBA1 clone (despite early mixed chimerism). Conclusions: This study represents the largest comparative analysis of alloHSCT versus HMA therapy in VEXAS syndrome, and confirms superiority of alloHSCT in achieving molecular remission, GC discontinuation/dose reduction, and overall survival. Prospective clinical trial confirmation is warranted.

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 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 Copy Number Alterations (CNAs), including chromosomal gains and losses, shape the genetics and clinical management of acute myeloid leukemia (AML), particularly in complex karyotypes (e.g. CK-AML). Traditionally, AML associated CNAs have been studied using cytogenetic approaches, i.e., karyotyping, that are limited in resolution and sensitivity. Here, low-coverage, sparse whole genome sequencing followed by machine learning applied to over 600 AML patient samples (ECOG-ARCIN trials: E1900, E3999) reveals novel genetic, biologic, and clinical correlates in AML disease. We find that multi-variate Cox-regression analysis applied to the data identifies select patients exhibiting focal and cryptic hemizygous deletions at 17q, 21q, and 3p, in otherwise diploid genomes, associate with inferior survival compared to other patients (median overall survival; OS = 65.5 compared to 421 days, Chi-square test pval=6x10-11), independent of factors such as age, treatment, and karyotype complexity status. In addition, we confirm that amplifications at 11q23, encoding the KM2TA gene, are associated with inferior outcome, and further identify unique breakpoint event features as well as transcriptional signatures suggestive of distinctive KM2TA-translocation independent biology. Applying machine learning approaches to the data, more specifically Non-negative Matrix Factorization (NMF), we develop a prognostic model that captures clinically adverse, cryptic variation such as focal 5q deletions, missed by conventional chromosome analysis and thereby, adds novel, complementary information to traditional, cytogenetic based risk-stratification (e.g. ELN). The model also associates gain of chromosome 8 in Intermediate risk patients with worse outcome compared to other Intermediate risk patients (median OS = 186 compared to 446 days, Chi-square test pval=0.005) and identifies a subgroup of patients within the adverse risk category that lack deletions on chromosomes 5, 7, and 17, and that fare better than the rest of the adverse risk patients (median OS = 188 compared to 97.5 days, Chi-square test pval=0.02), likely reflecting previously identified “atypical” CK-AML patients. Last, we develop a prognostic classifier based on a combination of Neural-Network and Random-Forest based learning, that predicts disease outcome with high accuracy and is based on a three genomic feature list on chromosome bands 5q31.2, 7q31.33, and 8q24, suggesting potential for clinical implementation. Altogether, our results show that low coverage, sparse whole genome sequencing combined with advanced learning methods can be applied clinically to support the diagnosis and clinical management of AML disease as well as serve as a powerful tool to further a basic understanding of the genetics and biology of CNAs in myeloid neoplasms and other cancers.

Abstract INTRODUCTION Allogeneic hematopoietic cell transplantation (alloHCT) is indicated in patients (pts) with FLT3-ITD mutated AML. The randomized BMT CTN 1506 trial (Levis et al. JCO) demonstrated that maintenance with FLT3 inhibitor gilteritinib (gilt) significantly improved relapse-free survival (RFS) among pts with detectable peri-alloHCT minimal residual disease (MRD) but was also associated with a greater degree of myelosuppression and infections. In older pts, aged >60 years, increasing post-HCT toxicity may adversely impact outcomes. On this basis, we performed a post-hoc analysis of pts aged > 60 years enrolled onto BMT CTN 1506 to examine the impact of gilteritinib maintenance in older pts. METHODS Pts ≥60 years randomized on BMT CTN 1506 (gilt=47, placebo=57; N=104) were included. The primary endpoint of this post-hoc analysis was RFS. We additionally assessed overall survival (OS), incidence of relapse, non-relapse mortality (NRM) in all pts and in pts with detectable MRD. RESULTS Baseline characteristics were balanced. Age was similar (65.2 vs 64.7 years; p = 0.12), as was prior TKI exposure, and NPM1 mutation status. Myeloablative conditioning (MAC) was used in 8 (17%) pts randomized to gilt and 18 (32%) pts randomized to placebo (p=0.08). Median f/u of survivors was 43.8 months (range:1-61 mo.). Estimated RFS and OS at 48 months were not different between groups (RFS: gilt: 57.0%, 95%CI 44.1-73.7%, placebo: 57.1%, 95%CI 45.4-71.8%; OS: gilt: 56.9%, 95%CI 42.9-46.7%, placebo: 58.4%, 95%CI 46.7-73.1%). While gilt assignment did not affect survival, several predictors of poorer survival were identified including use of reduced intensity conditioning (RIC) (HR = 3.46; p = 0.014), use of calcineurin inhibitor/MTX for graft-vs-host disease (GVHD) prophylaxis (HR = 2.29; p = 0.041), high-risk cytogenetics (HR = 2.64; p = 0.021), and MRD (+) pre-HCT (HR = 2.29; p = 0.018). Finally, relative to gilt assignment, opposing rates of relapse and NRM were observed. The incidence of relapse was lower with gilt (16%, 95%CI 6.8-28% vs 32%, 95%CI 20-45%, p=0.046) but NRM was higher (27%, 95%CI 15-41% vs 11%, 95% CI 4.3-21%, p=0.033). We next analyzed the 49 pts (gilt=22, placebo=27) with detectable peri-HCT MRD. RFS and OS again were not different (RFS: gilt: 44.4%, 95%CI 27.1-72.7%, placebo: 43.8%, 95%CI 28.4-67.5%, OS: gilt: 49.2%,95%CI, 31.3-77.4%, placebo: 55.6%, 95%CI 39.4-77.8%). The incidence of relapse trended lower in gilt pts (25%, 95%CI, 5.3-44.7% vs 45% (95%CI, 25-63%), p=0.2) while NRM trended higher (31%, 95%CI 12-52% vs 11%, 95%CI, 2.7-26%, p=0.12). Seventeen (36%) patients completed planned 2 year maintenance with gilt compared to 29 (51%) completing therapy with placebo. Median time on gilt was 13.2 months. The most common reason for discontinuation was adverse events (n=19, 40%), of which cytopenias (n=8) and infections (n=4) were most frequent. Non-relapse causes of death among gilt treated pts included infections (n=8), intracranial hemorrhage related to thrombocytopenia (n=1), second cancer (n=1), and Guillain-Barre syndrome (n=1). Most infections (n=5) were attributed to GVHD as an underlying cause. CONCLUSION Among pts aged ≥60 years, gilt maintenance did not improve RFS or OS. Gilt use was associated with reduced relapse regardless of peri-HCT MRD status demonstrating disease effect; however, higher NRM limited overall benefit. In older FLT3 mutated pts, employing strategies to mitigate HCT related toxicity, e.g. alternative GVHD prophylaxis strategies, may better accommodate use of maintenance therapy including gilt. Lower doses of gilt (particularly with azole co-use) may be worth exploring to mitigate drug AEs, improve compliance, thus further improving benefit.

Abstract Background: The addition of the CD19/CD3 bispecific T-cell engager, blinatumomab, to standard consolidation chemotherapy in adults with ALL who achieved a measurable residual disease negative (MRDneg) remission (CR), demonstrated an improvement in 3-year overall survival (OS, 85% vs 68%, HR 0.41, P=0.002) and 3-year relapse-free survival (RFS, 80% vs 64%, HR 0.53) compared to conventional chemotherapy in the ECOG-ACRIN E1910 study (Litzow et al, NEJM 2024). Given the complex therapy in this study, treatment delays and modifications are expected. We herein explored the impact of such delays on patient outcomes. Methods: In the phase 3 ECOG-ACRIN E1910 trial (NCT02003222), adults aged 30-70 years with newly diagnosedALL who had achieved MRDneg CR following induction and intensification were randomized to consolidation chemotherapy with blinatumomab (blina arm) or chemotherapy alone (chemo arm). We examined the association between the timing of the initiation of maintenance therapy and OS and RFS. The expected duration of consolidation was 252 days vs 126 days for patients on the blina and chemo arms, respectively. For this analysis, participants were classified into two groups (no/short delay or long delay) depending on whether they started on time/before or after the median time between the beginning of consolidation and start of maintenance therapy for their treatment group (medians: 335 days, range, 181-457 days vs 176 days, range, 139-265 days for blina and chemo arms, respectively). Multivariate analyses of OS and RFS were performed to examine the effect of no/short delay vs. long delay in starting maintenance. Patients were stratified according to their assigned consolidation treatment arm, and the models were further adjusted for age, sex, white blood cell count, platelets, hemoglobin, peripheral blood blasts, bone marrow blasts, performance status and genetic-genomic risk at diagnosis. Results: Of the 488 patients enrolled, 224 MRDneg patients were randomized to consolidation chemotherapy with or without blinatumomab and 135 of the 224 went on to maintenance therapy on study (N= 66 on blina arm and 69 on chemo arm, respectively). The median follow-up from the time of maintenance registration was 43.4 months. There were no significant differences in baseline characteristics between patients in either arm who experienced no/short delay or long delay. In the blina group, 60 of 66 patients (90.9%) received all 8 cycles of consolidation therapy of which 31 (45.6%) had long delays. Sixty-eight of 69 patients on the chemo arm (98.5%) received all cycles of consolidation therapy, of which 34 (50%) had a long delay. Strikingly, patients who had a long delay before initiation of maintenance had a significant improvement in OS in multivariate analysis compared to patients who had no/short delay (HR 0.31, 95% CI: 0.11-0.91; P =0.03). However, within each treatment group, OS benefit did not reach statistical significance (blina arm: HR 0.04, 95% CI: 0.001-1.64; P =0.09; chemo arm: HR 0.32, 95% CI: 0.08-1.38; P =0.13). Similar results were observed for RFS in multivariate analysis (HR 0.40, 95% CI: 0.16-1.01; P =0.05), though this impact on improved RFS was noted in the chemo arm but not in blinatumomab treated patients (blina arm: HR 0.56, 95% CI: 0.10-3.24; P =0.52; chemo arm: HR 0.19, 95% CI: 0.04-0.83; P=0.03). Additionally, patients who had no/short delay before starting maintenance experienced a longer median duration of maintenance treatment (blina arm, 548 days; chemo arm, 708 days) compared to patients who had a long delay (blina arm 488 days; chemo arm 598 days) before initiation of maintenance (P=0.0008). Conclusion: In the phase 3 randomized ECOG-ACRIN E1910 clinical trial, long delays between initiation of consolidation therapy and start of maintenance therapy were associated with improved OS compared to no/short delays. However, within each treatment group (blina or chemo), no significant difference of OS was observed. We postulate that these longer delays allowed more patients to receive all of the recommended protocol-assigned therapy thereby resulting in improved survival. Furthermore, patients who had no/short delays had a significantly longer duration of maintenance therapy. These data indicate that treatment delays during consolidation do not worsen survival. Further studies are being done to better understand why patients with long delays had better overall survival.

Abstract Background: Relapsed or refractory (R/R) acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL) remain challenging to treat, particularly in older patients and those with a T-cell phenotype. Venetoclax (VEN), a selective BCL-2 inhibitor, has shown preclinical synergy with vincristine (VCR) in multiple preclinical ALL models. Following the previously reported establishment of the recommended Phase II dose (RP2D), we now report the results of the Phase II (Ph2) portion of EA9152 (VEN 600 mg daily with weekly L-VCR 2.25 mg/m² IV or VCR 1.5 mg/m2 IV capped at 2 mg), evaluating efficacy and safety in this high-risk population. Methods: This open-label, multicenter Phase II study enrolled adults (≥18 years) with R/R B- or T-cell ALL or LL. The phase II portion of this trial was a single-arm, open-label study evaluating the combination of VEN (600 mg po D1–28) with either L-VCR (2.25 mg/m² IV weekly x4) or vincristine sulfate (VCR) (1.4 mg/m² IV weekly x4). Following the market withdrawal of L-VCR in the United States in April 2022, no new patients were initiated on this formulation and patients already receiving L-VCR were transitioned to VCR at the start of their next treatment cycle or sooner if necessitated by drug availability. Prior VCR but not VEN exposure was permitted in Ph2. The primary endpoint was achievement of a complete response or complete response with incomplete count recovery CR + CRi rate by end of cycle 3. Secondary endpoints included measurable residual disease (MRD) response, progression free survival (PFS), overall survival (OS), and safety. The association of phenotype with response and time-to-event were evaluated using Fisher's exact test and the Cox proportional hazards model, respectively. Results: Twenty-one eligible patients were treated (B-cell ALL/LL: n=12; T-cell ALL/LL: n=9). The trial was terminated early after 22/56 patients were accrued on account of withdrawal of sponsor support. Median age was 50 years and 76.2% were male. Performance status was 0–1 in 85.7%. All patients had received prior chemotherapy; 47.6% had undergone prior transplant. Median number of treatment cycles was 1 (2 for B-cell, 1 for T-cell). By end of cycle 3, no patients in the B-cell cohort (0/12; 90% CI, 0.00–0.22) achieved CR or CRi, 8 had stable disease (SD). In the T-cell cohort, 1 of 9 patients (11.1%; 90% CI, 0.01–0.43) achieved CR, 1 achieved PR and 1 had SD. There was no statistically significant difference in CR + CRi rate between phenotypes (p=0.43). None of the patients achieved an MRD-negative CR/CRi by flow cytometry or molecular methods. At the time of this analysis, 16 of 21 patients had died. Median overall survival (OS) was 4.3 months (95% CI, 1.5–22.0) for B-cell and 10.2 months (95% CI, 2.0–16.4) for T-cell patients (HR 1.32, p=0.60). Median PFS was 2.9 months (95% CI, 1.7–3.7) for B-cell and 1.2 months (95% CI, 0.7–4.6) for T-cell cohorts (HR 2.18, p=0.18). Grade 3–5 treatment-related adverse events occurred in most patients. One T-cell patient experienced grade 5 respiratory failure. Among 16 patients enrolled under the revised protocol using VCR, only 1 experienced grade 3 peripheral motor neuropathy, and no peripheral sensory neuropathy was reported (combined AE rate 0.06; 90% CI, 0.00–0.26). Tumor lysis syndrome was not observed in this cohort. Conclusions: The combination of VEN at the established RP2D and L-VCR/VCR was feasible and had an acceptable safety profile in heavily pretreated patients with R/R ALL/LL. However, response rates, including CR/CRi and MRD negativity, were limited, particularly in the B-cell cohort. Median OS and PFS remain poor in both subtypes, highlighting the need for improved strategies in this high-risk population. Venetoclax, in combination with other chemotherapy regimens, has shown encouraging response rates warranting continued exploration of its benefit in ALL.

Abstract Introduction: MyeloMATCH (mM), is an National Cancer Institute (NCI)-sponsored precision medicine initiative for newly diagnosed AML/MDS. It consists of an initial screening study (Master Screening and Reassessment Protocol, or MSRP) that involves centralized testing of bone marrow/whole blood, leading to assignment into several genetically driven treatment subprotocols within the mM portfolio. MSRP employs karyotype, 8-15 probe fluorescence in situ hybridization (FISH) and NCI-myeloid (NMAv2) assays to determine ploidy, translocations and mutational status needed for accurate disease subclassification, risk stratification as well as for theragnostic determination. NMAv2, a rapid next generation sequencing assay (TAT<48 hrs), interrogates 45 DNA and 35 RNA fusion driver genes to report 1661 hotspot DNA mutations and 779 targeted RNA fusions, including all relevant fusions in AML except Inv3/t(3;3)/MECOM rearrangement (Yeung et al, 2025). The initial mM MSRP workflow relied on karyotype and FISH assay results to identify fusion positive AML/MDS as per the current testing guidelines/ recommendations as well as to abide by the FDA's requirements under the investigational device exemption (IDE). Here we present concordance analysis between karyotype, FISH and NMAv2 assay to identify relevant fusions in AML/MDS. Methods: First 400 participants enrolled in the mM MSRP were included in this analysis. We examined the results from karyotype analysis analyzing 20 metaphase cells, 8-15 probe FISH analyzing 500 nuclei per probe, and NMAv2 curating fusion read counts greater than 100, performed at the mM labs to determine the prevalence of fusions in this cohort. Concordance analysis was performed to determine clinical utility of the NMAv2 assay to report relevant fusions at the time of initial diagnosis. The concordance analysis excluded biomarkers including Inv3/t(3;3)/MECOM rearrangement, KMT2A-PTD and other rare fusions that are beyond the scope of detection by NMAv2 and/or FISH assay. Results: Of the 400 patients analyzed in this cohort (median age 64, [range 20-95]; 43% female; 15.5% non-white; 9.5% Hispanic), translocations/fusions were reported in 67, 73 and 95 patients by karyotype, 8-15 probe FISH and NMAv2, respectively. The karyotypic analysis reported inv(16)/t(16;16) [15], t(11q23) [13], t(9;22) [10], inv3/t(3;3) [9], t(8;21) [6], t(15;17) [5], t(6;9) [4], t(21q22) [3], t(9q34) [1] and t(10;11) [1]. The FISH reported inv(16)/t(16;16) CBFB::MYH11 [15], t(11q23) KMT2A rearrangement [14], t(9;22) BCR::ABL1[10], inv3/t(3;3) MECOM rearrangement [10], t(8;21) RUNX1::RUNX1T1 [7], t(15;17) PML::RARA [5], t(6;9) DEK::NUP214 [5], t(21q22) RUNX1 rearrangement [3], t(11p15.4) NUP98 rearrangement [3], and t(9q34) ABL1 rearrangement [1]. The NMAv2 reported KMT2A-PTD [26], CBFB::MYH11 [15], KMT2A fusion [14], BCR::ABL1[12], RUNX1::RUNX1T1 [8], PML::RARA [5], DEK::NUP214 [5], RUNX1 fusion [4], NUP98 fusion[3], t(9q34) ABL1 fusion [1], PICALM::MLLT10 [1] and ETV6::MECOM [1]. The concordance analysis between NMAv2 and karyotype assay demonstrated positive percent agreement(PPA) of 100%, negative percent agreement(NPA) of 96.81% and overall agreement (OA) of 97.25% when using karyotype as reference. The concordance analysis between NMAv2 and FISH assay demonstrated PPA of 100%, NPA of 98.81% and OA of 99% when using FISH as reference. Of the 12 cases with discrepant results, 7 were due to presence of cryptic translocations missed by karyotype assay, 3 had suboptimal specimen submitted for karyotype/FISH analysis, 1 had false positive BCR::ABL1 fusion reported by NMAv2 due to low level contamination and 1 had minor subclonal population of leukemic blast with BCR::ABL1 fusion detected only by NMAv2. Conclusion: NMAv2 shows high concordance with karyotype and FISH assays which are the most commonly utilized assays to identify fusions/translocations in AML/MDS. NMAv2 is a faster, relatively cheaper and sample conserving method to identify fusions and is less prone to false negative results than karyotype/FISH assay from suboptimal specimen acquisition and testing. Based on the results above, mM MSRP workflow has been amended with FDA concurrence such that a combination of NMAv2, karyotype and MECOM breakapart FISH assay will be used to identify relevant fusion variants in all patients enrolled in mM, whereas additional specific FISH assay will be limited to cases where discrepant results between karyotype and NMAv2 are reported.

Abstract Aims BMT CTN 1506 (“MORPHO”) was a global phase 3 study of post-hematopoietic cell transplantation (HCT) maintenance with gilteritinib versus placebo for patients with FLT3-ITD-mutated AML in CR1. The study did not meet its primary endpoint of improved relapse-free survival (RFS) for post-HCT gilteritinib, but subgroup analysis indicated a benefit for participants with pre-HCT measurable residual disease (MRD). Subgroup analysis also indicated a statistically significant benefit of post-HCT gilteritinib for participants in North America, but no benefit for those in Europe or Asia. RFS for the control arms in Europe and Asia was higher than anticipated, suggesting that participants with different disease biology may have accrued to the study in these different geographic regions. In the primary analysis of the study, pre-HCT FLT3 inhibitor use and MRD across the entire study population were both found to have impacted outcomes. Time from diagnosis to HCT, a reflection of local practice patterns, is also known to impact outcomes for FLT3-ITD AML. We hypothesized that local practice patterns of pre-HCT FLT3 inhibitor use and time from AML diagnosis to HCT could have influenced the disease biology of study pts at the time of enrollment. This would be predicted to result in pre-HCT MRD levels and could provide an explanation for the regional differences observed in the study outcome. Methods We conducted a post-hoc analysis of the data focusing on days from AML diagnosis to HCT, pre-HCT FLT3 inhibitor use, and FLT3-ITD MRD assessed by a high-sensitivity assay. Eligibility requirements included FLT3-ITD-mutated AML in first remission after intensive chemotherapy and HCT occurring within 12 months of achieving remission. Patients refractory to induction therapy, or those with a history of relapse, were ineligible. Results Accrual took place in 16 different countries, with 5 countries accounting for 79.7% of the total accrual: USA 42.7%; Japan 15.7%; Korea 8.4%; UK 6.7%; Germany 6.2%; other countries 20.2%. In two countries, USA and Germany (48.9% of total enrollment), participants underwent HCT a median of 119 days after AML diagnosis, and 90.2% were treated with a FLT3 inhibitor pre-HCT. In contrast, in Japan, Korea, the UK and the other 11 countries all combined, participants underwent HCT a median of 152.5 days after AML diagnosis, and only 30.8% received any FLT3 inhibitor pre-HCT. Amongst all participants, those transplanted < 120 days from AML diagnosis and/or those treated with FLT3 inhibition pre-HCT were more likely to have improved RFS from post-HCT gilteritinib. RFS was improved with post-HCT gilteritinib in the US and Germany (HR 0.397; CI 0.220-0.718), whereas no benefit was evident in all other countries combined (HR 1.184; CI 0.685-2.048). Pre-HCT MRD levels were significantly higher (P = 0.011) in participants transplanted within 120 days from diagnosis as well as in those treated with a FLT3 inhibitor pre-HCT and also transplanted within 120 days (P = 0.019). Pre-HCT MRD was dependent on both FLT3 inhibitor use and time to HCT, as participants treated with successive courses of chemotherapy + FLT3 inhibition had successively lower MRD by the time of HCT. Conclusions The time from AML diagnosis to HCT and FLT3 inhibitor use pre-HCT both appeared to impact MRD levels immediately prior to HCT, and geographic differences in these two practice patterns likely accounted for the regional differences in benefit from post-HCT gilteritinib observed in BMT CTN 1506. Successive courses of chemotherapy without FLT3 inhibition appears to have selected a more favorable risk (e.g., MRD-lower) population for enrollment. Successive courses of chemotherapy combined with FLT3 inhibition progressively eradicated MRD, such that a majority of participants undergoing HCT after 3 or 4 courses were MRD negative and therefore unlikely to benefit from post-HCT gilteritinib. These findings have implications for clinical practice. Increasing the number of courses of chemotherapy combined with FLT3 inhibition before HCT may lower MRD sufficiently to eliminate the need for post-HCT inhibition for some patients but, alternatively, the additional delay could increase the risk of pre-HCT relapse, preventing patients from receiving HCT in first remission.

Abstract Background: Acute myeloid leukemia (AML) with TP53 alteration (TP53alt), including deletion (TP53del) detected by cytogenetics (CG) (karyotype, FISH, or genomic microarray) and/or TP53 mutation (TP53mut) identified by next-generation sequencing, is associated with poor prognosis. However, a subset of patients may achieve long-term survival after allogeneic hematopoietic cell transplantation (allo-HCT). Patient-, disease-, and transplant-related variables may inform risk stratification and improve treatment decision-making in this high-risk population. We used the CIBMTR registry to investigate outcomes of patients with TP53alt AML undergoing allo-HCT. Methods: We included adult patients with 2017 ELN adverse-risk AML who underwent first allo-HCT between 2013–2019 and had available CG and molecular data. TP53del was centrally reviewed by expert cytogeneticist. Patients were stratified into three TP53alt groups: Group 1 (TP53mut+/ TP53del+), Group 2 (TP53mut −/ TP53del+), and Group 3 (TP53mut+/ TP53del −). The control group comprised patients with 2017 ELN adverse-risk AML negative for both TP53mut and TP53del. Patients from embargoed centers, lacking consent or comprehensive research data, or missing CG and molecular information were excluded. Results: Of 6,474 patients with adverse-risk AML, 703 patients with TP53alt met inclusion criteria: Group 1 (n=203), Group 2 (n=271), Group 3 (n=229). The control group included 2,763 patients. Median age was 58 years (44% ≥60); 44% were female; 16% had Karnofsky score <90, and 52% had HCT-CI ≥3. Secondary AML was more frequent in TP53alt groups (32%, 27%, 27%) versus control (21%) (p<.01). Complex and monosomal karyotypes were most common in Group 1 (61%), followed by Group 2 (54%), Group 3 (31%), and control (6%), p<.01. More patients in Groups 1 and 2 (28% each) underwent HCT in primary induction failure/relapse (PIF/Rel) versus Group 3 (19%) and control (19%), p<.01. Median time from diagnosis to transplant was 5 months (1-331). Donor type was matched related in 23%, matched or mismatched unrelated in 50%, haploidentical in 18%, and umbilical cord blood in 7%. Conditioning intensity was myeloablative (MA) in 51%; reduced-intensity (RIC) in 36% , and 13% non-MA (NMA). Post-transplant cyclophosphamide (PTCy) was used in 27%; and in vivo T-cell depletion (ATG or alemtuzumab) in 26%. After median follow-up of 66 months (3-130), 5-yr OS was 18% (Group 1), 18% (Group 2), 35% (Group 3) vs 46% in controls (p<0.001). 5-yr LFS was 14%, 15%, and 28%, respectively, vs 38% (p<0.001). 5-yr CIR was 69%, 66%, and 52% vs 43% (p<0.001). Transplant-related mortality (TRM) was similar across groups (1-year: 12–16%). On multivariate analysis, compared with controls, TP53del (with or without TP53mut) was independently associated with higher relapse risk: Group 1 HR 1.97, Group 2 HR 1.91, Group 3 HR 1.45 (all p<0.001). These findings translated into inferior OS (HR 1.94, 1.87, and 1.45, respectively, p<0.001). PIF/Rel and CR2+ were also associated with worse survival (HR 2.58 (p<0.001) and 1.18 (p<0.001, respectively). No significant difference was observed between MA and RIC for relapse, while non-myeloablative regimens (HR 1.65; p<0.001) were associated with higher CIR. In vivo T-cell depletion increased relapse risk (HR 1.22; p=0.004). TRM was not influenced by TP53alt status; it was impacted by age, KPS, time from diagnosis to HCT, and GVHD prophylaxis regiment (non-PTCy). Conclusions: This large, registry-based study with long term median follow-up showed that among patients with adverse-risk AML, TP53alt is a strong independent predictor of poor post-HCT outcomes in AML, particularly when TP53del is present. Patients with TP53del , with or without concurrent TP53mut, experienced the worst outcomes, followed by those with isolated TP53mut. Despite the limitations of registry data including lack of information on timing of TP53mut, variant allele frequency (VAF), number of mutations, or accurate information on measurable residual disease (MRD), these findings reinforce the adverse prognostic impact of TP53alt and support its inclusion in risk stratification frameworks. While some patients with TP53alt —particularly those with isolated mutations—may benefit from allo-HCT, relapse remains the dominant cause of failure. Future studies incorporating TP53 functional status, VAF, clonal architecture, specific CG, MRD, and novel transplant approaches are warranted.