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Background: Chimeric antigen receptor T-cell (CAR-T) therapy has shown substantial promise in treatment of hematological malignancies, including non-Hodgkin Lymphoma (NHL). A subset of patients develop prolonged cytopenia, comprising quality of life, increasing healthcare utilization, and imparting considerable morbidity and mortality. Therapy-related myeloid neoplasms (t-MN), typically seen in patients who receive DNA-damaging agents such as radiotherapy and alkylators, have also been observed in CAR-T recipients. CAR-Hematotox score predicts the development of hematotoxicity in CAR-T recipients using laboratory parameters obtained at lymphodepleting (LD) chemotherapy, a timepoint when the patient has already gone through leukapheresis thus committed to the CAR-T process. Therefore, it is critical to identify patients at the highest risk of prolonged cytopenia and/or t-MN prior to apheresis, making it possible for a better triage of those who will proceed with CAR-T and those who might benefit from other strategies. Methods: A retrospective analysis was conducted in NHL patients who received commercial CAR-T products between January 2018 and March 2024 at Mayo Clinic Rochester, Arizona and Florida. Clinical and laboratory data including age, hemoglobin (Hb), absolute neutrophil count (ANC), platelet (PLT), ferritin, and LDH, obtained at the initial CAR-T evaluation, typically within 1 week of leukapheresis, were used for analysis. Prior lines of therapies including autologous stem cell transplant (ASCT) were collected. Prolonged cytopenias were defined as Hb < 8 g/dL, ANC < 0.5 x 109/L, and/or PLT < 50 x 109/L at 3 months after CAR-T. t-MN was diagnosed per WHO 2016. For prolonged cytopenia, the optimal cutoff points for continuous variables were determined by maximizing the sum of sensitivity and specificity. Logistic regression was then used to determine the factors associated with cytopenia at 3 months post-CAR-T. For t-MN, maximally selected log-rank statistics was used to determine the optimal cutoffs. We used competing risk analysis to determine the factors associated with development of t-MN. Death without t-MN was considered a competing risk. For both analyses, the factors with P < 0.1 in univariate analysis (UVA) were included in multivariate analysis (MVA). Results: Among the 387 NHL patients, with a median follow-up of 11 months (95% CI 8.7 - 12.2 months), 56 (14.4%) developed (41, 10.5% with 1 cytopenia; 14, 3.6% with 2 and 1, 0.2% with cytopenia in all 3 lineages), 20 (5.2%) developed and 315 (81.2%) did not have either. Factors associated with prolonged cytopenia: were age >, PLT ≤ 150, ferritin ≥ 600, and LDH ≥ 250. Univariate analysis identified baseline Hb, ANC, PLT and ferritin to be significantly associated with prolonged cytopenia. MVA identified Hb ≤ 11 (HR = 1.98; 95% CI 1.03-3.85, P = 0.042) and PLT ≤ (HR = 2.56; 95% CI 1.39-4.78; P = 0.003) to be associated with the development of prolonged cytopenia. Factors associated with t-MN:were age > 65 years, Hb ≤ 9 g/dl, ANC < 3.8, PLT ≤ 80, Univariate analysis identified age, Hb, ANC, PLT, ferritin, and ≥ 3 prior lines of treatment to be significantly associated with t-MN. On MVA, age ≥ 65 (HR = 3.91; 95% CI 1.52-10.03; P = 0.004), Hb ≤ 9 (HR = 3.49; 95% CI 1.38-8.85; P = 0.008), ferritin ≥ 300 (HR = 3.06 ; 95% CI 1.08-8.67; P = 0.035) and ≥ 3 prior lines of therapy (HR = 3.25; 95% CI 1.33-7.94 ; P = 0.010) were associated with t-MN development. Association of prolonged cytopenia with t-MN development: Prolonged cytopenia was noted in 45% of patients who developed t-MN and 15% of those who did not (P = 0.002). Cumulative incidence of t-MN at 1 year was 3.3% and the presence of prolonged cytopenia was associated with a higher 1of t-MN (HR 8.93, 95% CI 2.15 - 37.1, P = 0.003). Conclusions: Readily accessible clinical and lab data such as blood count and inflammatory markers at the time of CAR-T evaluation were associated with the development of prolonged cytopenias. Similarly, advanced age, anemia, high ferritin levels and history of ≥3 lines of therapy were associated with the development of t-MN. These findings, when validated in larger dataset, can be used for patient counseling and therapy selection by identifying patients at high-risk of developing hematotoxicity and/or t-MN development.

Background Venetoclax + hypomethylating agent (Ven-HMA) therapy results in favorable response rates (CR/CRi 66%) and overall survival (OS; median 14.7 months) in unfit patients with newly-diagnosed acute myeloid leukemia (ND-AML; N Engl J Med 2020;383). However, there is substantial heterogeneity in individual patient outcomes based on response to treatment. The objective of the current study was to develop a genetic risk model to predict survival outcome in treatment responders and non-responders, analyzed separately, and also account for allogeneic stem cell transplant (ASCT). Methods Treatment-naïve AML patients receiving Ven-HMA, outside of a clinical trial, at the Mayo Clinic, USA (MN, AZ, FL) were retrospectively recruited. Molecular studies (48-gene panel) were performed by next-generation sequencing. Response was assessed according to the 2022 European LeukemiaNet (ELN) criteria (Blood 2022;140). Standard statistical analyses were performed using JMP Pro (Version 17.0.0). Results 400 ND-AML patients (median age 73 years, 64% male, 60% de novo) received Ven-HMA. ELN cytogenetic risk included favorable 2%, intermediate 60%, or adverse 38%. Mutations involved TP53 in 26%, TET2 19%, RUNX1 19%, SRSF2 18%, ASXL1 18%, DNMT3A 15%, K/NRAS 13%, NPM1 12%, IDH2 12%, FLT3-ITD 10%, IDH1 7% and DDX41 in 4% of informative cases. 153 (38%) patients achieved CR, 94 (24%) CR with incomplete count recovery (CRi), resulting in CR/CRi in 247 (62%) patients. CR/CRi rates were higher with NPM1 (86% vs 59%), IDH2 (77% vs 60%), and DDX41 mutations (93% vs 61%) and lower with TP53 (45% vs 67%), FLT3-ITD (41% vs 64%), RUNX1 (49% vs 65%) mutations and adverse karyotype (48% vs 71%), with all retaining significance in multivariable analysis. CR/CRi rate was highest (87%) in 75 patients with one or more favorable mutations (NPM1, IDH2, DDX41) and no unfavorable mutation (TP53, FLT3-ITD, RUNX1). CR/CRi rate was lowest in 171 patients with at least one unfavorable mutation and no favorable mutation (44%; p<0.01). After a median follow up of 10.5 months (0.5-66), 105 relapses (43% of patients in CR/CRi), 248 (62%) deaths and 65 (16%) ASCT were documented. Univariate analysis for transplant-censored OS disclosed superior survival in patients achieving CR/CRi (median 21 vs 3.6 months; HR 5.4, 95% CI 4.1-7.1). Among patients achieving CR/CRi, IDH2 mutation was associated with superior (median not reached vs 20.5 months) and male gender (median 17.6 vs 29.2 months), TP53 mutations (median 11.9 vs 24.3 months) and adverse karyotype (median 12.6 vs 29.4 months) with inferior survival; multivariable analysis resulted in HRs (95% CI) of 2.1 (1.3-3.4) for male gender, 2.1 (1.2-3.8) for adverse karyotype, and 2.5 (1.1-5.7) for absence of IDH2 mutations. Male gender did not show an association with karyotype, or TP53 mutations (p>0.1). Accordingly, an HR-weighted genetic risk model was generated with assignment of one point each to male gender, adverse karyotype, and absence of IDH2 mutation: high risk = 3 risk factors (n=38; median 9.6 months; 3-year survival 0%); intermediate risk = 2 risk factors (n=129; median 22 months; 3-year survival 38%); and low-risk = ≤1 risk factor (n=78; median not reached; 3-year survival 69%; p<0.01). In patients bridged to ASCT (n=60), post-transplant survival was inferior in the presence of ≥ 2 risk factors (n=42; median 46 months) vs. ≤1 risk factor (n=18; median not reached; p=0.1). In patients not achieving CR/CRi, risk factors for OS included male gender (median 3.4 vs 6.1 months), adverse karyotype (median 3.3 vs 4.5 months) and TP53 mutation (median 3.1 vs 4.5 months) while those with IDH2 mutations lived longer (median 8.4 vs 3.4 months). In multivariable analysis, HRs (95% CI) were 1.9 (1.3-2.9) for male gender, 2.0 (1.4-2.9) for adverse karyotype, and 2.1 (1.1-4.3) for absence of IDH2 mutations. OS was shortest for non-responders harboring 3 risk factors (n=45; median 3 months; 1-year survival 0%) and longer for those harboring two (n=80; median 6 months, 1-year OS 15%) or ≤ 1 risk factor (n=25; median 14 months; 1-year OS 56%; p<0.01). Conclusion The current study describes a molecular profile for predicting response to Ven-HMA in ND-AML and also introduces a novel approach to survival prediction that considers treatment response as the starting point. In both responders and non-responders, male gender and adverse karyotype were identified as unfavorable and IDH2 mutation favorable risk factor for OS.

INTRODUCTION While prognostic understanding (PU) is important for all patients (pts) considering hematopoietic stem cell transplantation (HCT), it may be particularly important for older (>65 years), medically infirm, and/or frail pts deciding whether to undergo allogeneic HCT (allo-HCT). This abstract reports on PU and concordance with transplant physician (hereafter MD) PU for pts participating in an ongoing seamless, phase II-III randomized clinical trial (ACE-BMT) comparing 4-arms: a pt-administered comorbidity management intervention (CM), specialist-administered palliative care (PC), a combined arm of CM and PC to standard of care. Here we report preliminary data on the concordance between pt and MD PU on the chance of cure 1-year (yr) post allo-HCT. METHODS Pt PU was compared to MD PU (gold standard) to determine concordance. MD and pt answers were compared on a single validated question asked at enrollment to the study (typically 2 weeks prior to HCT). Both pts and MD's estimated the chance of cure 1 yr after allo-HCT by selecting one of six possible response options: very good (more than 90%), good (75-90%), better than 50% (50-74%), worse than 50% (25-49%), bad (10-24%) or very bad (less than 10%). PU was “concordant” or “accurate” if MD and pt selected the same response option. Pts with discordant PU were labeled “pessimistic” if the pt selected a response option with a lower chance of cure than the MD (and “optimistic” if a higher chance). Response options were further simplified to greater than or less than 50% chance of cure and MD and pt PU were compared again. Descriptive statistics, chi-square for categorical and t-tests for continuous variables were used. Logistic regression was used to determine the association between pt prognostic concordance and pt socio-demographic and clinical characteristics. RESULTS All pts consented to date were included (n=358), except those who did not answer the prognosis question (n=61). 54.2% (n=194) had HCT-Comorbidity Index (HCT-CI) of ≥3, 67.9% (n=243) were older than 65 yrs and 4.5% (n=16) were frail, defined by walk speed <0.8 m/s. Median age was 68 yrs (range 20-80). 39.7% (n=141) were female. 89.9% (n=322) identified as White, 4.2% (n=15) Asian, and 1% each as Black/African American and Native Hawaiian/Other Pacific Islander. 5.3% (n=19) identified Hispanic. 11.5% (n=44) had high school education or less. 54.1% (n=183) made less than $100,000/yr. 40.1% (n=136) had acute myelogenous leukemia, 25.4% (n=86) had high-risk myelodysplastic syndrome and 10.6% (n=36) had a myeloproliferative cancer. 72.1% (n=245) had matched, 82.5% (n=283) unrelated and 95.0% (n=36) peripheral blood allo-HCT. 9% (n=31) had KPS of 100%, 35% (n=121) KPS 90%, and 47.5% (n=147) KPS 80%. Pts reported having a very good, good, better than 50%, worse than 50% and bad/very bad chance of cure 1 yr post allo-HCTL 34.4% (n=123), 30.7% (n=110), 26.0% (n=93), 7.3% (n=26) and 1.7% (n=6), respectively. 91.1% (n=326) of pts felt they had a >50% chance of being cured 1 yr post allo-HCT. MD answers were missing for 159/358 (44.4%) pts. MDs reported pt had a very good, good, better than 50%, worse than 50% and bad/very bad chance of being cured 1 yr post allo-HCT, 3.0% (n=6), 6.5% (n=13), 52.8% (n=105), 35.7% (n=71) and 2% (n=4) respectively. 62.3% (n=124) of MDs felt the pt had a >50% chance of being cured 1 yr post allo-HCT. For the subset of pts with MD prognostic data (n=199), pt's PU was concordant 20.6% (n=41) of the time when compared across all 6 response options. Overall, 71.9% (n=143) of pts erred toward optimism, while 7.5% (n=15) of pts were pessimistic. 60.8% of MD and pts agreed on prognosis when dichotomized to better or worse than 50% chance of being cured 1 yr post allo-HCT. Prognostic concordance was not significantly associated with any pt socio-demographic factors, HCT-CI or KPS via descriptive statistics or regression analysis. CONCLUSIONS In this prospective randomized trial focusing on a population of older, medically infirm, and/or frail pts receiving allo-HCT, pts' PU was not concordant with MD prognosis approximately 80% of the time. While some pts were pessimistic, the majority overestimated the likelihood they would be cured of disease compared to MD estimates. Future studies should investigate the true accuracy of both MD and pt prognosis, as well as how to improve communication about prognosis and pt PU (if inaccurate based on best available information) prior to HCT.

Background Venetoclax + hypomethylating agent (Ven-HMA) therapy has demonstrated improved outcomes in elderly/unfit patients with newly-diagnosed acute myeloid leukemia (ND-AML) (N Engl J Med 2020;383). However, ~50% of patients who respond to Ven-HMA, eventually relapse (Am J Hematol 2024;99). In the current study we examined the prognostic contribution of gene mutations, karyotype, measurable residual disease (MRD) status and clinical risk factors for relapse in patients with ND-AML treated with Ven-HMA therapy. Methods Our study population of ND-AML was retrospectively recruited from Mayo Clinic (MN, AZ, FL), after institutional review board approval and based on documentation of complete remission with/without count recovery (CR/CRi) following Ven-HMA therapy. Relapse-free survival was determined from the time of remission to relapse or last follow-up. Survival analysis was censored for transplant. The 2022 European LeukemiaNet (ELN) criteria was used to assign cytogenetic risk and classify treatment response (Blood 2022;140). Standard statistical analyses were performed using JMP Pro (Version 17.0.0). Results 247 ND-AML patients treated with Ven-HMA (median age 73 years, 62% male, 64% de novo) who achieved CR (n=153), or CRi (n=94) were included. MRD by multiparameter flow cytometry (sensitivity 0.01%) performed at the time of CR/CRi, was negative in 117 (70%) of 166 informative cases. ELN cytogenetic risk categories at diagnosis included favorable 2%, intermediate 69%, or adverse 29%. Mutations involved TET2 21%, SRSF2 21%, TP53 19%, ASXL1 18%, DNMT3A 18%, NPM1 17%, IDH2 15%, RUNX1 15%, K/NRAS 12%, IDH1 8%, FLT3-ITD 7%, and DDX41 in 6% of informative cases. Relapse was documented in a total of 99 (40%) patients who achieved CR (39%) or CRi (41%) after a median remission duration of 6 months (1-31). Patients that relapsed within the first-year vs those that did not, were more likely to be males (75% vs 55%), MRD positive (44% vs 24%), and harbor TP53 mutations (23% vs 16%). Univariate analysis for RFS identified male gender (12 vs 29 months; p=0.02), secondary AML (10 vs 21 months; p<0.01), adverse karyotype (9 vs 22 months; p<0.01), TP53 mutations (6 vs 18 months, p<0.01) and MRD positive remission (13 months vs not reached; p<0.01) as risk factors for relapse; on the other hand, IDH2 mutations (not reached vs 15 months; p<0.01) were associated with a lower risk of relapse. Multivariable analysis confirmed male gender (HR 2.2), TP53 mutations (HR 3.0) and MRD positive remission (HR 1.7) as unfavorable predictors of relapse. Subsequently, in a three-tiered relapse prediction model, RFS was significantly inferior in the presence of three risk factors (male gender, TP53 mutations, MRD positive remission) (n=10, median 4 months), vs two (n=34, median 10 months) vs ≤ 1 risk factor (n=122, median 31 months) (p<0.01); 1-year cumulative incidence of relapse was 100%, 56%, and 26% in the presence of three, two and ≤ 1 risk factors, respectively. 50 of 99 (65%) patients received salvage therapy which included cladribine-cytarabine-Ven (n=19), intensive induction chemotherapy (n=9), FLT3 inhibitors (n=5), Ven-HMA-FLT3 inhibitor (n=1), IDH1/2 inhibitors (n=5), glasdegib-cytarabine (n=4), gemtuzumab (n=3), lenalidomide (n=1), or investigational therapies (n=3); in addition, Ven-HMA was resumed in 14 patients in whom treatment had been discontinued for a median of 5 months (2-12). Overall, 20 (31%) of patients achieved second CR/CRi and response rates were similar with intensive vs less intensive therapy (CR/CRi; 44% vs 29%; p=0.37). Median follow up after relapse was 3.4 months (1-66), during which 73 (74%) deaths, and 4 (5%) ASCT were recorded. Median transplant-censored survival was 4.2 months (1-year; 22%), and inferior in patients with adverse karyotype (median 4.6 vs 3.4 months; p=0.02), and TP53 mutations (3.6 vs 4.4; p=0.11); on the other hand, male gender (3.5 vs 6.8 months; p=0.51), MRD positive remission (4.6 vs 4.2; p=0.56), and IDH2 mutations (6.7 vs 4.2; p=0.28), did not influence post-relapse survival. Conclusions The current study identifies male gender, TP53 mutations, and MRD status as powerful predictors of relapse and proposes a relapse prediction model for ND-AML patients receiving Ven-HMA therapy. Furthermore, relapse following Ven-HMA was associated with uniformly poor outcomes, regardless of salvage treatment strategy.

Introduction: DDX41, DEAD-box RNA helicase 41 gene located on chromosome 5q25.3, is one of the most mutated genes in patients with germline predisposition to myeloid neoplasms. Germline and somatic mutations often have different locations and patterns of mutation, with some hot-spots displaying diversity based on ethnicity. We aimed to explore clinical outcomes in patients with various DDX41 hot-spots. Methods: A retrospective study of patients at Mayo Clinic with DDX41 mutation identified through Next Generation Sequencing (NGS) between 2016 and 2024. Patients classified as germline DDX41 variants or somatic variants based on confirmed germline testing results. Data regarding mutations recorded at time of patients' first in-house NGS. We completed unadjusted comparisons using continuous or categorical variables, and survival rates assessed using Kaplan-Meier method and cox regression analysis. Median overall survival calculated from time of NGS to last follow-up or death using BlueSky Statistics V.10.3.4 Results: Clinical Characteristics: Eighty-two patients analyzed, predominantly male (69.5%), with median age of diagnosis of 68 years (IQR 62-75). Most common diagnosis was MDS (50%), followed by AML (22%) and myeloproliferative neoplasm (MPN) (7.3%). Fourteen percent were DDX41 mutation carriers, 4.9% clonal cytopenia of undetermined significance (CCUS), and 1.2% chronic neutrophilic leukemia (CNL). MDS-IB2 was the prevalent MDS subtype (56.1%), and 83.3% of AML patients were intermediate risk. Cytogenetics normal in 73.8% of patients, with del20 being the most common abnormality (7.5%). Twenty-five (30.5%) of patients underwent allogeneic hematopoietic cell transplantation (alloHCT). Forty-nine patients (59.8%) had pathogenic DDX41 mutation only, 15 (18.3%) had only VUS, and 18 (22.0%) had both. Molecular Characteristic s: Mutation hot-spots included 12.2% with p.Arg525His only, 28% p.M1I only, 9.8% p.Asp140GlyFs*2 only, 9.8% p.Arg525 with co-mutation other than p.M1I/p.Asp140. Two patients (2.4%) had p.M1I co-mutation with non-p.Arg525 and p.Asp140, while 1 patient (1.2%) had p.M1I and p.Arg525, and 1 patient (1.2%) had p.Asp140 and p.Arg525. Lastly, 33 patients (40.2%) had mutations in hot-spots other than specified in this study. Median hemoglobin (Hg) was lowest in patients with p.Arg525 hotspot mutation (9.9 g/dl), compared to median Hg of 11.2 g/dl in group with p.M1I and 10.4 g/dl in p.Asp140 only group (p=0.87). The most common cytogenetics in patients with only p.Arg525 and only p.M1I hot-spot mutation was normal cytogenetics (70% and 90.9%, p=0.5). In the 16 patients with proven germline DDX41 mutation, 7 (43.8%) had mutations in only p.M1I. Lastly, median (m) VAF was significantly lower in the group with only p.Arg525 hot-spot mutation (9.5%, IQR 5.5-13.8), compared to mVAF of 48% in patients with only p.M1I (IQR 48.0-49.5, p<0.001). Co-mutations: The most frequent co-mutation was DNMT3A (11.0%), followed by JAK2 (9.8%).Mean number of co-mutations in p.Arg525 only was 0.6, 0.3 in p.M1I only, and 0.5 in p.Asp140 only (p=0.8). Most common co-mutation in p.M1I was ASXL1, with 13.0% of patients with p.M1I only having ASXL1 co-mutation (p=0.062). The most common co-mutation in p.Asp140 was DNMT3A (p=0.7). Survival: Median overall survival (mOS) was 21.5 months (IQR 12.4-38.6). mOS for p.Arg525 only group was 31.3, compared to 41.2 months in p.M1I only (p=0.197). At 12- and 24-months, survival rate was 88.9% and 59.3% in the p.Arg525 only group, compared to p.M1I group with 100% survival rate at 12 months and 89.5% at 24 months. p.Asp140 had 87.5% survival rate at both 12 months and 24 months. Males in p.M1I group had lower survival rate at 24 months (81.8%), compared to 100% survival in females. p.M1I only group with alloHCT also had lower 24 month survival at 60% compared to 100% in group who did not undergo alloHCT. Conclusions: The p.M1I hot-spot was commonly seen in patients with germline DDX41 mutation, likely resulting in significantly higher VAF percentage. Co-mutations frequency differed by hot spot mutation while p.Arg525 as second mutation was seen mainly in cases other than p.M1I/pArg140Gly. Overall survival appears to be higher in p.M1I/pArg140Gly compared to p.Arg525His hot-spot mutation, though males with p.M1I and those who underwent alloHCT had lower survival. Findings suggest implications for clinical outcomes based on DDX41 mutation hot-spots.

DDX41 MT Germline Predisposition Syndrome (GPS) is the most common adult GPS for myeloid neoplasms (MN), including MDS and AML. Although there have been reports of non-myeloid neoplasms, particularly lymphoid malignancies, in DDX41MT-GPS, these occurrences are infrequent and poorly characterized. We carried out this study in a large cohort of DDX41MT-GPS patients to address this deficiency. Following IRB approval, we retrospectively analyzed our database of 174 patients with DDX41MT-GPS to determine the rates of non-myeloid malignancies. Our previous data revealed that certain DDX41MT genotypes classified as variants of uncertain significance (VUS) exhibit a similar phenotypic spectrum to those classified as pathogenic (Badar et al Hematologica). Consequently, we included all these variants in our analysis. The median age of the cohort was 68.6 yrs (range, 13-4-93.4); 66% (n=114) male, 93% (n=162) Caucasian, with a median follow-up of 1.4 yrs (range, 0.03-5.6) from DDX41MT-GPS detection. The cohort included 34 (19.5%) asymptomatic carriers, 17 (9.8%) with clonal hematopoiesis (CH) or clonal cytopenias (CCUS), and 123 (70.7%) with MN. Eighteen (10%) patients had a lymphoid neoplasm (78% male, 100% Caucasian), with a median age at lymphoid neoplasm diagnosis of 67.7 yrs (range, 12.2-85.9). The most common lymphoid neoplasm was chronic lymphocytic leukemia (CLL) seen in 2.3% (n=), followed by follicular lymphoma (FL) and acute lymphoblastic leukemia (ALL) present in 1.7% each (n=3; 1 T-ALL and 2 B-ALL: 1 Ph+ and1 Ph-like). Additionally, there was 1 patient each with monoclonal B cell lymphocytosis (MBL), Waldenstroms macroglobulinemia , peripheral T cell lymphoma , large granular lymphocytic leukemia, and marginal zone lymphoma. Germline genotypes were classified as pathogenic in 50% (n=9, D140Gfs*2 x2, K331del, M1I x3, M316Dfs*31, P258L, Y340N) and VUS in 50% (n=9, E426K, E69D, G313D, K184R, P206A, P70S, Q246E, R164W, c.572-3T>C. Among these 18, 9 (50%) patients had co-existing myeloid disorders (CCUS x3, MDS x5, AML x1), and 5 (27.8%) patients were later diagnosed with therapy-related myeloid neoplasms (TMN) at a median of 5.9 yrs (range,1.6-38.7) after lymphoid neoplasm diagnosis and treatment. Seven (4%) patients had plasma cell dyscrasias (PCD; 3 with monoclonal gammopathy of undetermined significance(MGUS) and 4 with multiple myeloma (MM)); 57% (n=4) were male, with a median age of 61.3 yrs (range, 41.4-83.8) at PCD diagnosis. Subtypes of MGUS included IgG Kappa (n=2) and IgG Lambda (n=1), subtypes of MM included IgG Kappa (n=2), IgA Kappa (n=1), and Kappa light chain only (n=1). DDX41MT-GPS genotypes were pathogenic in 28.6% (n=2, G67Vfs*13, G313S) and VUS in 71.4% (n=5, A555T, R164W, Q246E x2, M307T). All patients with MM (n=4) received an autologous stem cell transplant, with 3 (75%) being subsequently diagnosed with a t-MN; median 3.6yrs after PCD diagnosis (range, 3.2-12.3). Seventeen percent (n=31) had visceral malignancies; 2.8% (n=5) having >1malignancy, 61% (n=19) male, 93.5% (n=29) Caucasian and 6.5% (n=2) Hispanic. Prostate cancer was most common (n=8, 4.6%), followed by breast (n=7, 4.0%), colon (n=5, 2.9%), squamous cell carcinoma (n=4, 2.3%, 3 of the head/neck and 1 of the leg), bladder (n=4, 2.3%), testicular (n=3, 1.7%), gynecologic (n=3, 1.7%), lung (n=2, 1.2%), thyroid (n=1, 0.6%) and pancreatic neuroendocrine tumor (PNET, n=1, 0.6%), respectively. The median age at first solid tumor diagnosis was 62.4 yrs (range, 10.9-81.9). DDX41MT-GPS was pathogenic in 64.5% (n=20, D140Gfs*2 x3, E113Kfs*14, G67Vfs*13, I396T, L283Cfs*21, M1I x4, M316Dfs*31 x2, p.M1?, P258L, Q368*, Q41* x2, R159*, R311*) and VUS in 35.5% (n=11, D30Y, H203Y, I215L, I337V, M155I, p.?, S289L, T13I, V318G, A555T). Of the 31 patients with visceral malignancies , 51.6% (n=16) were diagnosed with t-MN at a median of 4.9 yrs (range, 1.1-34.3) after malignancy diagnosis. While DDX41MT-GPS are clearly associated with an increased risk of myeloid neoplasms, we demonstrate potential associations with other neoplasms, especially lymphoproliferative disorders (10%). We for the first-time report associations with PCD (4%) and visceral malignancies (17%) and also highlight a worrisome trend for t-MN in DDX41MT-GPS treated with genotoxic agents. Given limitations of a retrospective analysis, this study underscores the need for a comprehensive natural history study in patients with DDX41MT-GPS .

Background: Previously published risk variables for chronic myelomonocytic leukemia (CMML) included age, gender, CMML subtype, red blood cell (RBC) transfusion status, hemoglobin, leukocyte, monocyte, lymphocyte and platelet counts, immature myeloid cells, peripheral blood (PB) or bone marrow blast percentage, and mutations including ASXL1, RUNX1, NRAS, and SETBP1. Recent studies have reported on additional prognostically favorable (TET2) or unfavorable (DNMT3A, ASXL1, NRAS, SETBP1) mutations. In the current study, we took advantage of a clinically well-characterized and molecularly annotated cohort of CMML patients, in order to revisit the prognostic interaction between the aforementioned risk factors, with emphasis on mutations. Methods: The current study was conducted under an institutional review board approved protocol that allowed retrospective collection and analysis of data from Mayo Clinic patient records. Mutations were screened by multi-gene next-generation sequencing (NGS). Diagnostic criteria were according to the International Consensus Classification (Blood 2022; 140:1200). Conventional statistical methods were employed (JMP Pro 17.0.0 software SAS Institute, Cary, NC, USA). Survival analysis was censored for allogeneic stem cell transplantation (ASCT). Receiver Operating Characteristic (ROC) plots and Akaike Information Criterion (AIC) were used to compare predictive performance of the new model (MMMv2) vs. the Mayo Molecular Model (MMM) vs. the CMML-specific prognostic scoring system (CPSS). Results: The study cohort included 416 patients with CMML that were fully annotated for mutations. Cytogenetic information was available in 404 patients. At a median follow-up of 18 months, 302 (72%) deaths, 72 (18%) leukemic transformations, and 44 (11%) ASCTs were documented. Age-adjusted multivariable analysis (MVA) of mutations identified the following as risk factors: DNMT3A (HR 5.5; p<0.01), PHF6 (HR 0.3; p<0.01), TET2 (HR 0.6; p<0.01), PTPN11 (HR 2.5; p=0.01), NRAS (HR 1.6; p<0.01), ASXL1 (HR 1.3; p=0.02), and SETBP1 (HR 1.8; p=0.01). A separate MVA of clinical and cytogenetic variables identified RBC transfusion need, leukocyte count ≥13 x 109/L, peripheral blood blast ≥1%, abnormal karyotype, older age, and male sex to be independently associated with inferior survival (p<0.04 in all instances). All-inclusive MVA identified the following as independent risk factors for survival (p <0.01): DNMT3A mutation (HR 3.7; 95% CI 2.0-6.7), absence of PHF6 mutation (HR 2.8; 1.3-5.8), RBC transfusion need (HR 2.3; 1.7-3.1), leukocyte count ≥13 x 109/L (HR 2.0; 1.5-2.6), abnormal karyotype (HR 1.6; 1.2-2.2), and absence of TET2 mutation (HR 1.4,1.1-1.8). An HR based prognostic model was subsequently constructed by assigning 3 points to DNMT3A mutation, 2 points each for RBC transfusion need, leukocyte count ≥13 x 109/L, and absence of PHF6 mutation and one point each for abnormal karyotype and absence of TET2 mutation, resulting in a 4-tiered survival risk model (MMMv2): low risk (0-2 points; N=103) with median survival of 74 months; intermediate-1 (3 points; N=65) with median 37 months; intermediate-2 (4-5 points; N=126) with median 19 months; and high (6 points; N=88) with median 12 months (p<0.01). Independent genetic risk factors associated with inferior leukemia-free survival included DNMT3A (HR 5.5), PHF6 (HR 0.2), abnormal karyotype (HR 2.0), PTPN11 (HR 5.2), ASXL1 (HR 2.0), and BCOR (HR 8.7). Leukocyte count ≥13 x 109/L (p=0.01) but not RBC transfusion need (p=0.2) was additionally significant. However, presence of immature myeloid cells (p<0.01) and circulating blasts ≥1% (p<0.01) were prognostically more relevant than leukocyte count ≥13 x 109/L and independently predicted leukemic transformation, along with DNMT3A, PTPN11 and ASXL1 mutations. Predictive performance was superior for MMMv2 (AUC 87, AIC 137) vs. MMM (AUC 74, AIC 194) vs. CPSS (AUC 76, AIC 177). Conclusion: The revised Mayo Molecular Model (MMMv2) provides a contemporary risk model for CMML that recognizes DNMT3A as the most unfavorable and PHF6 as the most favorable mutation and confirms the mutation-independent prognostic contribution from red cell transfusion need and sustained leukocytosis (≥13 x 109/L). Mutations were also important in predicting leukemic transformation, notably DNMT3A, PTPN11 and ASXL1.

Introduction Somatic mutations in TET2 are present in ~60% of chronic myelomonocytic leukemia (CMML) patients and are associated with improved median overall (mOS) and acute leukemia free survival (mLFS). Multiple TET2 mutations are detected in ~50% of such cases and are thought to confer additional benefit. Whether these benefits apply to the molecularly distinct myelodysplastic (MD) and myeloproliferative (MP) CMML subtypes is unknown. This study assesses the impact of the number and type of TET2 mutations across the MD and MP-CMML subtypes. Methods CMML patients seen at Mayo Clinic (n = 400, 47%) and MD Anderson Cancer Center (n = 449, 53%) were included with IRB approval. Analyses considered the clinical and molecular data at diagnosis or first referral. Truncating (frameshift or nonsense) mutations in TET2 were further annotated by location in either the proximal genome targeting domain (aa 1 - 1128) or distal catalytic domain (aa 1129 - 1936) as described (Coltro, 2020). Categorical variables were compared by Pearson Chi squared and continuous by Mann-Whitney U or Kruskal-Wallis tests. Univariate and multivariate analyses utilized Cox proportional hazards models with continuous variables binarized by receiver operator curves. Kaplan-Meier analyses were censored at hematopoietic cell transplantation (HCT). P < 0.05 was considered significant. Calculations used BlueSky (v10.3.1) or Prism (v10.2.3). Results In a cohort of 849 CMML patients (median age 71 years, 32% female, 90% white), 454 (54%) were classified as MD-CMML and 394 (46%) as MP-CMML. Cumulatively, 664 somatic TET2 mutations were identified across the MD (n = 415, 63%) and MP-CMML (n = 249, 38%) subgroups, with ≥1 TET2 mutation in 57% vs 44% (p < 0.001) and ≥2 TET2mutations in 33% vs 20% (p = 0.001) , respectively. The median (range) number of TET2 mutations in MD vs MP-CMML was 1 (0 - 4) vs 0 (0 - 3; p < 0.001). Truncating frameshift or nonsense mutations comprised 86% vs 78% of mutations in each group with hypomorphic missense mutations comprising 13% vs 20% (p = 0.03). Proximal (48% vs 43%) and distal (38% vs 35%) truncations were evenly distributed between MD and MP-CMML (p = 0.91). In both the MD and MP subgroups as well as the overall cohort (p < 0.01 each), increasing number of TET2 mutations (0 vs 1 vs ≥2) was associated with higher median hemoglobin (10.3 vs 11.4 vs 12.0 g/dL), fewer mean peripheral blasts (1.3% vs 0.5% vs 0.3%), and a higher proportion of normal karyotypes (60% vs 76% vs 79%). Likewise, co-mutations were increasingly frequent in SRSF2 (36% vs 45% vs 53%) and less frequent in IDH2 (9% vs 1% vs 1%) and SETBP1 (14% vs 4% vs 0%) throughout the cohort. TET2 mutations were mutually exclusive with ETV6, GATA2, IDH1, and WT1 mutations. Uniquely, MD-CMML cases with TET2 mutations (0 vs 1 vs ≥2) were enriched for co-mutations in ZRSR2 (5% vs 13% vs 9%) and had fewer co-mutations in both ASXL1 (47% vs 34% vs 28%) and U2AF1 (11% vs 6% vs 3%). In MP-CMML, co-mutations in CBL (11% vs 20% vs 27%) were increased while co-mutations in BCOR (6% vs 1% vs 0%) were decreased. Accordingly, the favorable ASXL1WT/TET2MT genotype was more common in MD (39% vs 22%), while the adverse ASXL1MT/TET2WT genotype was more common in MP-CMML (20% vs 33%; p < 0.001). In multivariate analyses, the presence of ≥2 TET2 mutations was favorably associated with OS and LFS in both MD and MP-CMML (hazard ratios 0.53 - 0.67, p < 0.04) independent of several clinical and genetic features including ASXL1 mutations and abnormal karyotype. Beyond the number of TET2 mutations, however, neither the mutation class (truncating vs hypomorphic) nor location (proximal vs distal) further affected OS or LFS. Likewise, although the median variant allele fraction (VAF) was lower in MD vs MP-CMML (42% vs 46%, p < 0.001), VAF did not impact survival. Accordingly, the mOS for patients with 0, 1, or ≥2 TET2 mutations was 29, 58, and 75 months in MD-CMML and 19, 26, and 39 months in MP-CMML, respectively (p < 0.001 each). The mLFS was similarly improved to 26, 52, and 67 months in MD-CMML (p < 0.001) and 16, 22, and 31 months in MP-CMML (p = 0.003). CONCLUSIONS This multicenter study demonstrates that the favorable impact of multiple TET2 mutations in CMML extends to both the MD and MP subtypes. In MD-CMML, multiple TET2 mutations segregate against adverse ASXL1 mutations while, in MP-CMML, they appear to offset the impact of adverse RAS pathway mutations. Thus, multiple TET2 mutations may identify a lower-risk subgroup of MP-CMML patients.

Background TP53-mutant (mt) myelodysplastic syndromes (MDS) are high-risk (HR) neoplasms, with high rates of AML progression and suboptimal response to hypomethylating agents (HMA). Given the lack of effective therapies for HR-MDS, azacitidine (AZA) was combined with venetoclax (VEN) in a phase Ib MDS study demonstrating safety and early efficacy (Garcia et al. ASH abstract 2023). We retrospectively sought to explore clinical outcomes comparing VEN + HMA, to HMA alone in TP53mt HR-MDS. Methods Among 221 TP53mt MDS pts treated at the Mayo Clinic between January 2007 and April 2024, we reviewed outcomes of pts who received HMA alone (n=98) or HMA+VEN combination (n=34) as their first-line chemotherapy regimen. MDS diagnosis was based on the 5th edition of WHO classification of hematolymphoid tumors (Khoury et al. Leukemia 2022). Responses were assessed according to the International Working Group 2006, MDS response criteria (Cheson et al Blood 2006). Results Baseline characteristics and response The median age (years [yrs]) was 65 (R,37-80) and 70 (range [R], 19-87) yrs in the HMA + VEN and HMA alone groups (gps) (p= 0.42), respectively. The median bone marrow (BM) blast % (9.5 [R, 0-18] vs 5 [R, 0-16], p= 0.01) and TP53 variant allele frequency (VAF) (42 [R, 6-96] vs 37 [R, 2-94], p= 0.03) were significantly higher in HMA + VEN compared to HMA gps, respectively. However, proportion of pts with biallelic (BA)/multihitTP53mt (85% vs 79%, p= 0.61) and complex cytogenetics (CG) (85% vs 81%, p= 0.85) were not different between HMA + VEN and HMA alone gps. The distribution of pts with frequently occurring myeloid co-mutations (ASXL1, TET2, DNMT3A, RAS, BCOR and splicing factor) were not significantly (NS) different between the two gps. IPSS-M scores were assessed in 108 (83%) pts; HR/very HR-MDS as per IPSS-M were NS different between HMA + VEN and HMA gps (100% vs 91%, p= 0.12). While the overall response rate (ORR) (76% vs 49%, p= 0.01) and marrow complete remission (mCR) rates (21% vs 7%, p= 0.05) were higher with HMA+VEN compared to HMA, CR rates were similar (17% vs 12%, p= 0.47). Higher proportion of pts were bridged to allogeneic stem cell transplantation (allo-HCT) (41% vs 20%, p= 0.02), while AML progression rates (29% vs 31%, p= >0.99) were comparable between HMA + VEN and HMA gps, respectively. The non-relapse mortality (NRM) at day 60 (3% vs 3%, p=0.96) and day 90 (3% vs 4%, p=0.75) were comparable between HMA + VEN and HMA gps, respectively. RFS and OS The median follow-up from the time of initiation of HMA or HMA + VEN was 9.7 months (mo) (R, 0.3-90.6). The median RFS (8.47 vs 9.90 mo, p= 0.83) and OS (16.33 vs 14.20 mo, p= 0.66) from the time of initiation of therapy were comparable between the two gps. Similarly, among transplanted pts, there was no OS difference between HMA (21.7 mo) and HMA + VEN (18.43 mo) gps, respectively (p= 0.72). Comparable results for OS and RFS were obtained in both gps after censoring pts at the time of allo-HCT. Multivariable analysis for RFS and OS We conducted multivariable analysis (MVA) for RFS and OS using baseline variables that were significant/ trended towards significance (p<0.1), on univariate analysis (UVA) censored at the time of allo-HCT. Achieving ORR (HR; 0.30, 95% CI: 0.14-0.62, p= 0.001) retained significance for better RFS in MVA. Interestingly CR (HR; 1.74, 95% CI; 0.63-4.77, p= 0.28), BA TP53mt (HR; 1.27, 95% CI; 0.13-11.72, p= 0.83) and complex CG (HR; 1.52, 95% CI 0.16-14.03, p= 0.70) did not retain significance for RFS in MVA. In MVA for OS, achievement of ORR (HR; 0.56, 95% CI; 0.30-1.05, p=0.07) and CR (HR; 0.32, 95% CI; 0.09-1.11, p=0.071) trended towards favorable OS. BA TP53mt status (p= 0.42) and complex CG (p= 0.19) did not significantly impact OS in UVA. Conclusion We conclude that while HMA+VEN combination therapy in comparison to HMA monotherapy led to improved response rates, with higher percentage of pts being bridged to allo-HCT, this was not associated with reduced risk of AML transformation, or improved RFS/OS. We eagerly await results from the prospective randomized trial of VEN + AZA in treatment-naïve HR-MDS (VERONA).

Background: While myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF), are usually sporadic, adult-onset neoplasms, an increasing number of germline risk alleles have been described (e.g. EGLN1, ETV6, SH2B3, and ATM) in these disorders. DDX41MT Germline Predisposition Syndrome (GPS) is the most common myeloid GPS in adults and while there is extensive data on MDS/AML, little is known regarding MPN occurring in this context. We carried out this study to define the prevalence and characteristics of DDX41MT-GPS associated MPN. Methods: After IRB approval, we retrospectively assessed the Mayo Clinic cohort of 174 patients with DDX41MT-GPS, which included both pathogenic and clinically relevant germline variants of undetermined significance (VUS), based on our previous analysis (Badar et al Haematologica 2023). Standard statistical measures were applied. Results: We identified 12 (6.9%) patients with MPN (ET-5, PV-2, PMF-4, MPN-NOS-1). The median age at MPN diagnosis was 61.2 years (range, 8.-70.4); 58.3% (n=7) were male, and 83.3% (n=10) were Caucasian. A family history of hematologic malignancy was present in 16.7% (n=2, AML and T-LGL), while 50% (n=6) had a family history of visceral malignancies and 41.7% (n=5) had no family history of malignancies. Among ET patients (n=5) with DDX41MT-GPS, JAK2V617F was the MPN driver in 4 (80%) while type 1-CALR (L367fs*46) was a driver in 1 (20%). This included a 9-year-old child diagnosed with JAK2V617FMT ET. The DDX41 variant was classified as pathogenic in 40% (n=2; E113Kfs*14, D140Gfs*2) and VUS in 60% (n=3; V544L, V591I, P38L). Bone marrow (BM) aspirates in the ET patients were hypercellular in 40% (n=2), normocellular in 40% (n=2) and hypocellular in 20% (n=1), and none had increased blasts, or reticulin fibrosis (RF). In addition to JAK2V617F, somatic mutations seen included SF3B1 (K666T, VAF 16%) in 1 patient and IDH2 (R140Q, VAF 6%) and DNMT3A (R882H, VAF 7%) in 1 patient, respectively. With a median follow-up from diagnosis of 3.9yrs (range 2.9-8.9), there were no thrombotic events and no disease progressions was documented. There were 2 patients with JAK2V167FMT PV in whom the DDX41MT were clinically significant VUS (c.138+5G>T and M155I). Both patients had hypercellular BM, with no increase in blasts, no RF, normal karyotypes and no additional somatic mutations. With a median follow up of 7.5yrs (range, 2.1-12.9), 1 patient developed a deep vein thrombosis (DVT) and there were no fibrotic or leukemic transformations. There were 4 patients with PMF; 3 (75%) with JAK2V617F and 1(25%) with a type 1-CALR driver mutation (L367fs*46). DIPPS-plus scores ranged from 1 to 4 (median 2.5). The DDX41 variants were pathogenic in 50% (n=2, E256K and P78Qfs*3) and VUS in 50% (n=2, P510S and M155I). The median grade of RF at PMF diagnosis was grade 2 (range, 2-3). BM were hypercellular in 75% (n=3) and hypocellular in 25% (n=1), and 1 patient had 3% peripheral blasts (and no BM blasts). Somatic co-mutations included TET2 in 1 patient with a JAK2V617F, and CBL, EZH2 and SRSF2 in 1 patient with a CALR driver-mutation, respectively. PMF patients were treated with ruxolitinib (n=3), stem cell transplant (n=1), and pIFNα (n=1). With a median follow up of 3.9yrs (range,1.1-21.9), all patients were alive, and none had leukemia transformation. There was 1 patient with a JAK2V617F MPN-NOS with trisomy 9, where the DDX41MT-GPS was a VUS (I215L). There were no somatic co-mutations and at last follow up (3.1 years), there was no evidence for progression. Conclusions: While associations between late onset MDS/AML and DDX41MT-GPS has been well established, we systematically describe the MPN landscape seen in this context. MPN was diagnosed in 7% of our cohort, with JAK2V617F being the most common driver mutation. Most importantly, unlike in AML/MDS, where somatic DDX41 mutations are frequently associated with neoplastic transformation (30-50%; R525H being most common), none of our MPN patients had a somatic DDX41 mutation. Our work underscores the importance of germline mutation testing in patients with MPN.