Badar, Talha

Abstract Venetoclax + hypomethylating agent (Ven‐HMA) is currently the standard frontline therapy for older/unfit patients with newly diagnosed acute myeloid leukemia (ND‐AML). Our objective in the current retrospective study of 301 adult patients (median age 73 years; 62% de novo) with ND‐AML was to identify molecular predictors of treatment response to Ven‐HMA and survival; European LeukemiaNet (ELN) genetic risk assignment was favorable 15%, intermediate 16%, and adverse 69%. Complete remission, with (CR) or without (CRi), count recovery, was documented in 182 (60%) patients. In multivariable analysis, inclusive of mutations only, “favorable” predictors of CR/CRi were NPM1 (86% vs. 56%), IDH2 (80% vs. 58%), and DDX41 (100% vs. 58%) and “unfavorable” TP53 (40% vs. 67%), FLT3‐ ITD (36% vs. 63%), and RUNX1 (44% vs. 64%) mutations; significance was sustained for each mutation after adjustment for age, karyotype, and therapy‐related qualification. CR/CRi rates ranged from 36%, in the presence of unfavorable and absence of favorable mutation, to 91%, in the presence of favorable and absence of unfavorable mutation. At median follow‐up of 8.5 months, 174 deaths and 41 allogeneic stem cell transplants (ASCT) were recorded. In multivariable analysis, risk factors for inferior survival included failure to achieve CR/CRi (HR 3.4, 95% CI 2.5–4.8), adverse karyotype (1.6, 1.1–2.6), TP53 mutation (1.6, 1.0–2.4), and absence of IDH2 mutation (2.2, 1.0–4.7); these risk factors were subsequently applied to construct an HR‐weighted risk model that performed better than the ELN genetic risk model (AIC 1661 vs. 1750): low ( n = 130; median survival 28.9 months), intermediate ( n = 105; median 9.6 months), and high ( n = 66; median 3.1 months; p < .001); survival in each risk category was significantly upgraded by ASCT. The current study identifies genotype signatures for predicting response and proposes a 3‐tiered, CR/CRi‐based, and genetics‐enhanced survival model for AML patients receiving upfront therapy with Ven‐HMA.

Background: First-line therapies for chronic-phase (CP) CML include the tyrosine kinase inhibitors(TKIs) dasatinib, nilotinib, bosutinib or imatinib, while ponatinib and asciminib are indicated only in later lines. There are typically high response rates to first-line therapy in CP-CML, with responses to second- and third-line therapy lower and highly dependent on the specific TKI. Our goal in this study is to evaluate the role of ponatinib in the treatment of patients with CML using data from routine clinical practice. Study design and methods: Data will come from the H. Jean Khoury Cure CML Consortium (HJKC3) CML registry, a North American multi-site research database. Participating sites include the HJKC3 members (curecml.org). Patient inclusion criteria include a diagnosis CML in chronic phase, receiving second line therapy after initial therapy with a second generation TKI (dasatinib, nilotinib, or bosutinib) or receiving third or later line TKI therapy for CML. Participating patients provide consent to collect their data both retrospectively and prospectively for 1 year. Clinical data, including toxicities and complications of TKI therapy, are abstracted from the electronic health record. Participating patients are invited to complete a questionnaire to assess health-related quality of life. Data are managed using REDCap, a secure web application. Study Objectives: The primary objective is to compare the outcome of patients treated with ponatinib vs. other TKIs as second or later line therapy. Secondary objectives are to evaluate patient-reported outcomes (PRO) for CML treated with second or later line therapy and to determine if there are any patient or disease characteristics that can predict outcomes. Statistical plan for clinical outcomes: Descriptive statistics will be used to compare baseline demographic characteristics. Categorical variables will be compared using the Chi-square test. Continuous variables will be compared using ANOVA. Overall survival will be calculated from the time of diagnosis to death from any cause and patients will be censored if they were alive at the last follow-up. Survival probabilities will be computed using the Kaplan-Meier method and compared with the log-ranktest. Cox regression analyses will be used to determine significant factors that influence survival, and hazard ratios with 95% confidence intervals will be presented. Event free survival (EFS) and transformation free survival (TFS) will be calculated for patients. For EFS, loss of complete hematologic remission, loss of major cytogenetic response, progression to accelerated or blast phase, or death from any cause at any time will be considered an event. For TFS, transformation to accelerated or blast phase while on therapy or death on study will be considered an event. Statistical plan for PRO: Different PRO domains will be analyzed separately (for example, depression separate from fatigue, etc.). Descriptive statistics for each PRO measure will be reported and compared to previously published studies. Differences by treatment type will be evaluated.

Background: Hypomethylating agent (HMA) and venetoclax (Ven) frontline therapy for acute myeloid leukemia (AML) is astandard of care for patients (pts) deemed to be poor candidates for intensive induction due to age or fitness. However, given its attractive risk-benefit ratio, there is an increasing interest to extend the preference for HMA + Ven to other pts, including those who are fit for intensive therapy but with adverse risk disease. Unfortunately, most pts treated in this fashion will require future therapies for relapsed/refractory (R/R) disease and intensive therapy is given consideration mostly for pts with a path to allogeneic hematopoietic stem cell transplantation (alloHCT). Little is known about the outcomes of pts who receive intensive induction therapy after use of a frontline less-intensive regimen in the modern era. In this multi-center study, we seek to describe the experience with such a patient population and identify predictors of differential clinical outcomes. Methods: We conducted a multi-center, retrospective study of pts with AML treated with frontline less-intensive chemotherapy, followed by intensive induction chemotherapy for R/R disease. We first described the baseline characteristics of this cohort. Nominal variables were summarized with frequency and percentage while continuous variables were summarized with median and interquartile range (IQR). Overall response rate (ORR) was defined as complete remission (CR), complete remission with incomplete count recovery, or morphologic leukemia free state. Differences in ORR were analyzed with Fisher's exact test for nominal variables and Wilcoxon rank-sum test was used for continuous variables. Kaplan-Meier analysis was used to determine median overall survival (OS) after induction chemotherapy. Log-rank testing and Cox proportional hazard models were used to determine survival differences for nominal and continuous variables, respectively. Statistical significance was determined as p-value &lt;0.05. Results: A total of 23 pts were included in this study. Median age at diagnosis was 64 years (IQR: 62-71). The majority of pts were male (n=12, 52%), white (n=20, 87%), and had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1 (n=18, 82%). The majority of pts (61%) at time of frontline therapy had AML arising out of MDS. As expected, the majority of pts had adverse risk ELN cytogenetics (n=16, 70%) with an enrichment for disease harboring TP53 mutation (n=6, 27%) and complex karyotype (n=9, 39%). Fifteen pts (65%) had frontline therapy with azacitidine backbone, 6 pts (26%) with decitabine; some pts received both before intensive therapy. The median number of cycles of frontline therapy was 2 (IQR: 2-5). Median WBC at time of R/R therapy was 1.9 x 10 9/L (IQR: 0.8-6.45), while hemoglobin was 8.4 g/dL (IQR: 7.5-9.45), platelets were 41 x 10 9/L (IQR: 19.5-116.5), and marrow blasts were 25% (IQR: 16.5-30%). The plurality of pts received CPX-351 as intensive R/R therapy (n=9, 39%). Other regimens included 7+3 (n=2, 9%), FLAG-Ida + Ven (n=2, 9%), HiDAC + Mitoxantrone +/- Ven (n=4, 17%) ( Table 1). ORR after intensive R/R therapy was 27% (n=6), with 23% achieving CR. CPX-351 after HMA + Ven was specifically associated with an ORR of 33% (CR rate 22%). Median OS after initiation was 5.8 months. Thirty- and 60-day mortality was 17% and 26%, respectively. Five patients (23%) proceeded to alloHCT ( Table 1). Univariate differences in response rate and median survival were analyzed for each of the aforementioned variables as illustrated in Table 1. While none of the covariates reached statistical significance for impacting ORR or OS, ECOG PS trended towards significantly impacting OS ( p=0.074) and the median OS among patients proceeding to alloHCT was numerically doubled (8.0 vs. 4.0 months, p=0.11) ( Figure 1). Conclusions: Intensive therapy for R/R AML after prior frontline less-intensive therapy was associated with an ORR rate of 27% and median OS of 5.8 months in our cohort. This approach may be an appropriate strategy for a subset of pts, particularly those eligible for alloHCT. The relatively small and heterogeneous nature of this cohort limits strong conclusions regarding efficacy. As the use of frontline HMA + Ven continues to expand, further studies with larger sample size are needed to determine which pts would and would not benefit from intensive induction therapy in the R/R setting.

Background and significance: There are typically high response rates to first line TKI therapies in chronic phase chronic myeloid leukemia (CP-CML). However, responses in later lines (second line and beyond) are diminished and highly dependent on the specific tyrosine kinase inhibitors (TKIs) used. Asciminib received FDA approval in October 2021 for the treatment of adult patients with Philadelphia chromosome-positive (Ph+) CML-CP who were previously treated with two or more TKIs, and in patients with Ph+ CML-CP with the T315I mutation. Our goal in this study is to evaluate the role of asciminib therapy for patients with CML beyond clinical studies, in routine clinical practice. Study Design and methods: Data for this study will be abstracted from the HJKC3 (H. Jean Khoury Cure CML Consortium) CML registry project (HJKC3-0005); the HJKC3 CML Registry is a collaborative academic multi-site registry. Patients will be consented to allow collection of both retrospective and prospective data for the HJKC3 CML Registry. The data forms and surveys will be built and managed through the Research Electronic Data Capture (REDCap), a secure web application managed by the Clinical & Translational Science Institute. Participating sites include members of the HJKC3 (curecml.org). This study will be an opportunity to evaluate asciminib usage in ‘real-world’ academic practices across the United States. This observational study will include cohorts of patients diagnosed with CP-CML previously treated with two or more tyrosine kinase inhibitors and/or receiving asciminib and/or those with a T315I mutation, not currently enrolled in a randomized controlled trial. The HJKC3 CML Registry intends to collect clinical and patient reported outcome (PRO) data. After obtaining patient consent, patients will be invited to complete PRO assessments. The study will attempt to collect PRO data regardless of disease or treatment status. PRO assessments will be primarily administered online, though study staff will offer alternatives (e.g., phone) for participants without internet access. Study subjects will be emailed a link to access a secure platform hosted by one of the study sites, Medical College of Wisconsin (MCW), using the REDCap platform. Study Objectives: The primary objective of the study is to describe characteristics of patients who were previously treated with two or more TKIs and treated with asciminib or with any other TKIs. The secondary objectives are to describe the clinical outcomes and PRO of those patients. Exploratory objectives include description of characteristics, treatment outcome and PRO for patients with T315I mutation. Statistical plan: The sample size of approximately 225 participants during the 5-year recruitment period is based on the available clinical expertise and ongoing recruitment data in the HJKC3 CML registry in lieu of statistical power analysis. We estimate that 150 patients in the HJKC3 CML registry will be treated with other TKI and 75 treated with asciminib. Among a total of 225 patients, we estimate that 75 patients with the T315I mutation will be enrolled in the registry. No formal statistical inference will be drawn to compare the outcomes between these two groups. Bayesian predictive probability (PP) will be utilized for the exploratory analysis of Major Molecular Response (MMR). Survival probabilities will be computed using the Kaplan-Meier method, and the p-values from the log-rank test will be provided. Different PRO domains of the patient's symptoms and functioning will be analyzed separately (e.g., depression separate from fatigue, separate from GI symptoms, etc.) since combining them into a summary score may dilute the effects of the individual (and not necessarily related) components and mask actual change. The main objective for the analyses of PRO data from the registry is to describe any differences by treatment type in patients treated with asciminib and other TKIs.

Background: Minimal residual disease (MRD) is an established, prognostic indicator for patients with acute lymphoblastic leukemia (ALL), including Philadelphia chromosome-positive (Ph+) ALL. The objective of this study was to evaluate the association between MRD and long-term outcomes (event-free survival [EFS] and overall survival [OS]) in adult patients with Ph+ ALL receiving first-line therapy. Methods: A systematic literature review (SLR) was performed to identify eligible studies, including interventional and observational studies reporting survival outcomes by MRD status in adults ≥18 years of age with Ph+ ALL receiving first-line therapy. Key outcomes and data elements of interest were EFS, OS, and MRD or MRD-negative complete remission (CR) at end of induction (EOI). Aggregate study-level and individual patient data (IPD) meta-analyses were conducted. The IPD approach included a log-level MRD analysis to evaluate the concordance of deepening levels of molecular response with long-term EFS or OS in patients with CR and an analysis evaluating the utility of MRD-negative CR over CR alone. The analyses were conducted using frequentist and Bayesian approaches with fixed-effects and random-effects models. Kaplan-Meier method with log-rank tests was used for survival analysis. Where applicable, Cox regression method with and without adjustment for prognostic factors were applied. Results: The SLR identified 19 publications (18 unique studies), 10 (9 unique studies) of which had sufficient MRD and EFS/OS data. Nine studies (n=704) were included in the aggregate study-level analysis; 3 of the 9 studies (Phase II AP24534-11-001 [Jabbour et al. 2015], Phase II GIMEMAINCB 84344-201 [Martinelli et al. 2017], and Phase III GRAAPH-2005 RCT [Chalandon et al. 2015]) had IPD available. MRD was assessed by polymerase chain reaction in all 9 studies. Findings from the aggregate study-level meta-analysis demonstrated a significant long-term survival benefit (EFS and OS) in favor of patients with MRD-negative CR. This significant benefit was generally consistent across analysis types (base-case or sensitivity analyses), analytic models (fixed- or random-effects), and approaches (frequentist or Bayesian). Evidence from the IPD analysis, based on patients with CR, was mostly consistent with the aggregate study-level meta-analysis, supporting MRD-negative CR at the EOI as being predictive of EFS or OS. Log-level MRD analysis showed that patients who are in CR with deeper molecular response ( BCR::ABL1/ABL1 ≤0.01%) at EOI have significantly better long-term EFS and OS compared with patients with other levels of MRD (ie, BCR::ABL1/ABL1 &gt;0.01%). This significant long-term survival benefit was also demonstrated in patients with MRD-negative CR ( BCR::ABL1/ABL1 ≤0.01%) at EOI compared with patients who were MRD-positive ( BCR::ABL1/ABL1 &gt;0.01%) ( Table 1) or based on achievement of CR alone (regardless of BCR::ABL1/ABL1 values). Results from the IPD analysis standardized for outcome definition, with and without adjustment for prognostic factors (age, gender, white blood cell count at baseline, and stem cell transplant), were consistent with the findings from the log-level MRD analysis. The analysis showed a significant association between MRD-negative/MRD-negative CR status (ie, BCR::ABL1/ABL1 ≤0.01% threshold) at EOI with improved long-term EFS and OS. The results were significant for EFS in both the adjusted and unadjusted models. The OS results were significant only in the unadjusted model. Conclusions: These analyses indicate that a deeper molecular response at EOI results in better long-term EFS and OS; MRD-negative CR has a greater prognostic value than CR and is strongly associated with long-term EFS and OS in patients with Ph+ ALL. Acknowledgement: The authors acknowledge APHP, who provided the individualized patients data for the GRAAPH study.

Background Venetoclax (Ven) in combination with hypomethylating agent (HMA) is the standard treatment for newly diagnosed elderly/unfit patients with acute myeloid leukemia (AML). Treatment options for patients with relapsed/refractory (RR) disease following Ven-HMA are limited in the absence of targetable mutations. In a recent phase 2 study, the addition of Ven to cladribine (CDA) plus low-dose cytarabine (LDAC), in newly-diagnosed elderly/unfit AML, resulted in complete remission with (CR) or without (CRi) count recovery rate of 93% ( J Clin Oncol 20;40(33)). In the current study, we share our experience with this regimen in patients relapsed or refractory to Ven-HMA, treated outside of clinical trials. Methods Under an institutional review board approved minimum risk protocol, the Mayo Clinic (MN, AZ, FL) database was searched to identify patients with AML who failed treatment with Ven-HMA and subsequently received at least one cycle of salvage therapy with Ven-CDA-LDAC: Ven 100-400 mg, dose adjusted, d1-21, CDA 5 mg/m 2 d1-5, LDAC 20 mg twice daily d1-10, outside of clinical trials. Cytogenetic and molecular studies were performed at the time of Ven-CDA-LDAC initiation in most cases, by conventional karyotype and next-generation sequencing, respectively. The 2022 European LeukemiaNet (ELN) criteria were applied to define risk and response. Survival was calculated from the time of Ven-CDA-LDAC treatment start to last follow-up or death. Results Patient characteristics A total of 39 RR AML patients (median age 65 years, 67% male, 64% de novo), with prior exposure to Ven-HMA, received Ven-CDA-LDAC (median 1 cycle; range 1-5). Study patients had received one ( n=16), two ( n=14), three ( n=5), four ( n=2), five ( n=1) or nine ( n=1) prior therapies, including Ven-HMA. Nine (23%) patients had relapsed following allogeneic hematopoietic stem cell transplant (AHSCT). ELN cytogenetic risk included intermediate (46%, n=18), and adverse (54%, n=21). Mutations involved TP53 in 9 patients (23%), K/ NRAS in 8 (21%), RUNX1 in 7 (18%), TET2 in 6 (15%), ASXL1 in 5 (13%), and STAG2 in 5 (13%). Response and toxicity Eleven (28%) patients achieved CR ( n=2; 5%) or CRi ( n=9; 23%); median time to response was 1 month and median response duration 3.4 months. In addition, 2 (5%) patients each experienced partial remission (PR) and bone marrow aplasia without fulfilling criteria for morphologic leukemia free state. Measurable residual disease (MRD) was negative by multiparametric flow cytometry in 2 of 7 (29%) informative CR/CRi cases. CR/CRi rates were higher in females (54% vs 15%; p=0.01), de novo vssecondary AML (40% vs 7%; p=0.02), and absence of adverse karyotype (50% vs 9%; p&lt;0.01), K/NRAS (35% vs 0%, p=0.01), ASXL1 (32% vs 0%, p=0.05), or STAG2 mutations (32% vs 0%, p=0.05) (Table 1). Multivariable analysis confirmed superior response in de novo AML (p&lt;0.01), absence of adverse karyotype, (p&lt;0.01), and absence of K/NRAS mutations (p=0.02). CR/CRi rates were not significantly affected by relapsed vs refractory setting ( p=0.95), number of prior therapies ( p=0.51), prior AHSCT ( p=0.64), or TP53 mutations ( p=0.64) (Table 1). Treatment-emergent toxicities included infections (49%, n=19), comprising bacteremia ( n=11), pneumonia ( n=10), peri-rectal abscess ( n=6), and laboratory evidence of tumor lysis syndrome (3%, n=1). Survival At a median follow-up of 3.9 months, from initiation of Ven-CDA-LDAC, 31 deaths (79%), 6 relapses (15%), and 7 AHSCT (18%; including 5 in CR/CRi, 1 in aplasia) were documented. Median survival following Ven-CDA-LDAC was 4.6 months, and superior in the presence vs absence of CR/CRi (8.1 vs 2.9 months; p=0.01), and in patients receiving AHSCT (10.5 vs 3.4 months; p=0.02). Absence of adverse karyotype was also associated with superior survival (6 vs 3.4 months; p=0.05). Multivariable analysis confirmed the favorable survival impact of CR/CRi ( p=0.03) and AHSCT ( p=0.05); survival impact was not apparent for secondary AML ( p=0.29) or K/NRAS ( p=0.70) or TP53 ( p=0.10) mutations. Conclusions The current study suggests transplant bridging value for Ven-CDA-LDAC in AML patients relapsed or refractory to Ven-HMA, especially in the absence of adverse karyotype or K/NRAS mutation. AHSCT and achievement of CR/CRi appear to be indispensable for favorable post-Ven-CDA-LDAC survival.

Background: Core-binding factor acute myeloid leukemia (CBF-AML) is characterized by the presence of inv(16)/t(16;16) or t(8;21), and is classified as favorable risk by the 2022 European LeukemiaNet (ELN) guidelines. High remission rates are observed in the setting of cytarabine- or anthracycline-based induction regimens. The CD33-targeted antibody-drug conjugate gemtuzumab ozogamicin (GO) is FDA-approved for use in newly-diagnosed CD33+ AML; a meta-analysis of 5 prospective randomized studies investigating intensive chemotherapy (IC) +/- GO demonstrated an improved overall survival (OS) with the addition of GO to IC for favorable-risk AML (Hills et al, Lancet Oncol 2014). The trials included in the meta-analysis did not restrict inclusion criteria to CBF-AML alone and there remains variation amongst IC regimens utilized in CBF-AML treatment. In addition, IC combined with the KIT inhibitor dasatinib has shown remission rates and promising overall survival in prospective studies (Paschka et al, Leukemia 2018; Marcucci et al, Blood Adv 2020). We set out to investigate the outcomes of patients (pts) with CBF-AML treated with IC +/- targeted agents (GO or KIT inhibitor) across 5 institutions that are part of the Consortium on Myeloid Malignancies and Neoplastic Diseases (COMMAND). Methods: Retrospective chart review was done to identify pts who were treated with IC with AML harboring inv(16)/t(16;16) or t(8;21) identified on cytogenetics, from January 2010 through April 2023, and were treated across 5 geographically-diverse academic institutions. Pt clinical characteristics, cytogenetics, molecular mutations, treatment, relapse, and OS data were collected. Relapse-free survival (RFS) and OS were both analyzed using Kaplan-Meier analysis. Results: One-hundred forty-nine consecutive pts with CBF-AML were identified, 76 (51%) with inv(16) and 73 (49%) with t(8;21). Median age of pts was 47 years (yrs) (range 18-80yrs), with 64% male pts, and 106 (71%) pts had an ECOG performance status of 0-1. One-hundred seven (72%) pts identified as non-Hispanic White, 22 (15%) identified as non-Hispanic Black, and 12 (8%) identified as Hispanic. Molecular testing via next-generation sequencing was available for 124 pts (83%); 49 (33%) pts had adverse-risk ELN cytogenetics or mutations when excluding CBF status, and 27 (18%) pts harbored KIT mutations. Other baseline pt characteristics are listed in Table 1A. All pts underwent induction with IC; 44 (30%) pts received IC + GO, 21 (14%) received IC + KIT inhibitor (14 received dasatinib, 7 received midostaurin), and the remaining 84 (56%) received IC without a targeted agent. One-hundred fourteen (77%) pts achieved a complete response (CR) to induction therapy, and 1 pt experienced induction-related mortality. Fluorescence in-situ hybridization (FISH) data was available in 82 pts at end of induction with 83% achieving FISH negativity. Measurable residual disease (MRD) testing was done by polymerase chain reaction (PCR) in 62 pts with 76% achieving MRD-negativity. One-hundred forty pts underwent consolidation with a median of 3 cycles of chemotherapy; 21 (15%) pts received GO with consolidation and 17 (12%) received a KIT inhibitor with consolidation. Forty-four (30%) pts underwent allogeneic stem cell transplantation (alloSCT), with 17 in first CR, and 27 after second-line or beyond therapy. With a median follow-up of 22.5 months, the observed 3-yr RFS was 51% (95% CI: 43-61%) and the 3-yr OS was 69% (95% CI: 61-78%). Pts treated with IC + KIT inhibitor had significantly improved 3-yr RFS of 85% (95% CI: 70-100%) compared to other pts, including those who received IC + GO who had a 3-yr RFS of 51% (95% CI: 37-70%) and those who received IC without targeted therapy who had a 3-yr RFS of 45% (95% CI: 35-58%) ( p = 0.02) ( Figure 1). Full survival statistics are shown in Table 1B and Figure 1. Conclusions: In this multi-center retrospective study of pts with CBF-AML undergoing induction with IC regimens with diverse national treatment practices, we find that the addition of GO to IC is not associated with improved outcomes. Pts treated with IC + KIT inhibitor had significantly improved RFS; of note, pts receiving a KIT inhibitor oftentimes continued on maintenance even after consolidation. These data suggest that there may be a role for targeted therapies other than GO to be explored in randomized prospective studies of intensive induction chemotherapy for CBF-AML.

Germline variants in DDX41 are the most common cause of inherited predisposition to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). DDX41 codes for an RNA helicase with many described functions, and thus the precise role of these variants in disease is uncertain. Approximately 2/3 of disease-associated DDX41 variants cause truncation of the DDX41 protein due to frameshift, loss of a translation start, or splice alterations, while the other 1/3 of DDX41 variants are missense. The effect of these missense variants on DDX41 function is largely unknown and thus they are typically categorized as variants of unknown significance (VUSs). Importantly, 30-50% of MDS/AML patients with either truncating or missense DDX41 variants acquire a somatic mutation in the other allele of DDX41, and this mutation has strong preference for the amino acid substitution R525H. Our previous work demonstrated that the R525H mutation causes loss of DDX41 function. To better understand how germline variants affect DDX41 function, we designed a genetic complementation strategy in immortalized hematopoietic progenitor cells with inducible deletion of DDX41. We screened six of the most common missense VUSs by expressing them from a lentivirus in DDX41-proficient cells and then inducing deletion of endogenous DDX41 to determine the effect of each VUS on proliferation and survival of the cells. Empty vector-transduced DDX41-deleted cells undergo cell-cycle arrest and apoptosis within 3-4 days. In contrast, viral expression of wild-type DDX41 rescues the survival and proliferation of the cells, while expression of the R525H mutant fails to rescue the cells. We screened the Y33H, M155H, G173R, R219H, P258L, and I396T variants of DDX41 in this assay, and all six of the mutants successfully rescued the survival and proliferation of DDX41-deleted cells long-term. This indicates that the mutant proteins coded for by these variants are functional. To test the effect of commonly observed truncating variants in the same assay, we expressed M1I, Q52fs, and D140Gfs variants of DDX41. Surprisingly, we saw complete rescue of cell proliferation and survival by the M1I and Q52fs variants. In contrast, the D140Gfs variant failed to rescue the cells. These data indicate that frameshift at D140 or later causes loss of DDX41 function. It is likely that translation from alternative methionine residues located at M127 or M132 of DDX41 accounts for the rescue of the cells by truncating variants affecting sites upstream of M127. In support of this theory, we targeted DDX41 for CRISPR-mediated knockout in MOLM13 cells and found that guide RNAs targeting exons 1-4, which are upstream of M127, produced homozygous frameshift mutations in proliferating cells from single-cell clones. However, guides targeting exon 6, which is downstream of M127 and M132, did not produce any surviving cells with homozygous frameshift mutations, despite screening 96 clones. This finding indicates that short DDX41 isoforms expressed from alternative start sites are functional and can support cell proliferation when full-length protein is lost. Collectively, these data raise important questions about the precise role of DDX41 variants in hematologic malignancy. Data from recently published patient cohorts indicates that patients with missense DDX41 variants have similar patient characteristics to those with truncating variants and thus these aberrations are thought to cause disease by similar mechanisms. While these cell line models may not fully recapitulate the pathogenic mechanism by which DDX41 variants cause MDS/AML predisposition, the results of this study demonstrate that the germline variants are not complete loss-of-function alleles in all cases and more complex mechanisms may be involved.

Introduction The advent of venetoclax (VEN) based chemotherapy has revolutionized the treatment of elderly acute myeloid leukemia (AML), with improvement in remission rates from historical 10%-20% to 60%-70%. However, remission duration is short-lived in patients (pts) with high-risk AML with consequent dismal outcome. As our knowledge of genomic landscape of AML expands and more immunotherapies become available, exploration of the immune tumor microenvironment (TME) of AML may allow identification of subsets of pts that may benefit from immune based therapy. We conducted an investigator initiated prospective study to evaluate TME of pts receiving VEN plus hypomethylating agent (HMA; azacitidine or decitabine) combination. We hypothesize that these pts will cluster into immune-infiltrated vs. immune-depleted sub-types by high-definition immune cell profiling. Methods In 26 newly diagnosed AML pts, who were planned to receive VEN plus HMA therapy, we isolated bone marrow (BM) and peripheral blood (PB) derived mononuclear cells (MC) by density gradient centrifugation and cryopreserved. We performed CyTOF (cytometry time-of-flight) analysis to study their immune signatures. Thawed cells were labeled with metal-conjugated antibodies that bind CD33, CD196, CD19, CD127, CD11b, CD4, IgD, CD11c, CD14, CD56, CD123, TCRgd, CD185/CXCR5, CD45RA, CD27, CD28, CD66b, CD183 (CXCR3), CD161, CD45RO, CD197 (CCR7), CD8a, CD25, CD3, CD20, CD38, HLA-DR, CD194 (CCR4), CD57, CD16 and CD45. We performed a Rphenograph clustering on 25 paired BMMC and PBMC samples (one patient only had PBMC) to create 35 unique clusters for each ( Figure 1). Then we broke down the distribution of cluster by two different groupings: pts with TP53 mutation (m) and those who achieve complete remission. Statistical significance of clusters calculated using t-test. We performed Pearson correlation test on the paired BMMC and PBMC samples to assess correlation between BMMC and PBMC samples. Results Baseline characteristics The median age of the pts was 74 years (range, 53-88), and 17 (65%) pts were male. Ten (37%) pts had secondary AML, and 12 (46%) pts had ELN adverse risk disease (n=6; TP53m). There was no significant difference in median age (p= &gt;0.99), proportion of pts with secondary AML (p= 0.64), acute myelomonocytic leukemia (p= 0.14) white blood cell count (WBC) ≥ 100 (10 9/L) (p= &gt;0.99), and frequency of commonly occurring co-mutations ( ASXL1 [p=0.35]), FLT3 ITD [p=0.54], splicing [p=0.29], IDH1/2 [p=0.28], DNMT3A [p=0.29], NPM1 [p=0.29], TET2 [p=0.35]) in TP53m and TP53 wild-type group. Response 12/20 (60%) evaluable pts achieved complete remission with or without count recovery (CR/CRi). There was no significant difference in median age (p= 0.06), proportion of pts with secondary AML (p= 0.16), therapy related AML (p= 0.60) WBC ≥ 100 (10 9/L) (p= 0.40), and frequency of commonly occurring mutations ( TP53 [p=0.60], ASXL1 [p &gt; 0.99]), FLT3 ITD [p=0.54], splicing [p &gt; 0.99], IDH1/2 [p=0.60], DNMT3A [p=&gt;0.99], NPM1 [p=0.61], TET2 [p &gt;0.99]) in CR/CRi and no-CR/CRi group. CyTOF analysis and Rphenograph clustering on 25 paired BMMC and PBMC samples We found significant clustering with high expression of B-cell activated (cluster 2; p= 0.005), NK cell rich (cluster 1; p= 0.005, cluster 35; p= 0.0009), CD8 T-cells (cluster 31; p= 0.01) and CD4 effector cells (cluster 17; p= 0.005) phenotype among TP53m AML ( Table 1). Among pts who achieved CR/CRi, significant clustering with low expression of B-cell activated (cluster 29; p= 0.01), CD8 cytotoxic (cluster 14; p= 0.01) CD4 activated (cluster 21; p= 0.03) phenotypes observed. Correlation between BMMC and PBMC clustering Among clusters which showed significance for a particular phenotype, we performed Pearson correlation (R 2) and observed strong correlation between BMMC and PBMC samples clustering in cluster 1 (R 2 = 0.99), cluster 2 (R 2 = 0.95), cluster 17 (R 2 = 0.96), cluster 21 (R 2 = 0.99) and cluster 29 (R 2 = 0.93). Conclusion In this single institutional study using CyTOF analysis we have identified novel clustering with immune- infiltrated phenotype among pts with TP53m AML and conversely immune-depleted phenotype among AML pts who responded to VEN plus HMA. Moreover, we found strong correlation in CyTOF analysis conducted from BMMC and PBMC samples. Future studies with larger sample size needed to validate our findings.

Introduction: MDS are primarily diseases of the elderly, with median age at diagnosis of 72 years. Focused data on clinical and molecular characteristics, as well as outcomes, in younger pts under age 60 years are limited. We conducted this multicenter retrospective study to describe pt characteristics, cytogenetic and molecular profiles, treatment modalities, and outcomes in younger pts with MDS, including adolescent and young adults (AYA). Methods: The study included pts diagnosed with MDS between 2002-2023 at 7 academic centers. We compared baseline characteristics, treatment, and outcomes of AYA (18-39 years) and younger adult (YA) pts (40-59 years). Responses were assessed using the modified IWG 2006 response criteria which included complete remission (CR), marrow CR (mCR), and hematologic improvement (HI). Wilcoxon rank sum test and Fisher's exact (and Chi-square) test were used to compare pt and disease characteristics for continuous and categorical variables, respectively. Overall survival (OS) was estimated using the Kaplan-Meier method and compared using log-rank test. Results: A total of 173 pts were identified (AYA, N=50; YA, N=123). Median age at diagnosis was 50 years (range, 18-59), with 51% females. Baseline characteristics are shown in Table 1. Therapy-related MDS (t-MDS) was observed in 40 pts (23%) with the remaining classified as de novo MDS (N=133; 77%). Inherited bone marrow failure syndromes were documented in 15 pts (9%), with Shwachman-Diamond Syndrome (N=4) and Fanconi anemia (N=4) being the most frequent. Using the WHO 2016 classification, the most common subtypes of MDS in both the AYA and YA cohorts were MDS with multilineage dysplasia (38% vs 19%), MDS with excess blasts-2 (MDS-EB) (24% vs 29%), and MDS-EB-1 (16% vs 20%). Similarly, using the WHO 2022 classification, the most common subtypes were MDS with low blasts (50% vs 21%), MDS with increased blasts-2 (MDS-IB-2) (24% vs 24%), and MDS-IB-1 (16% vs 18%). Stratifying pts using the IPSS-R cytogenetic (CG) risk group, the most common CG risk category identified was good CG (34% vs 52%, p=0.012). Poor/very poor CG were present in 34% and 37% of the AYA and YA groups, respectively. STAG2 and NRAS mutations occurred more frequently in the AYA pts compared to the YA group, while the YA cohort was enriched for the following mutations: TP53, DNMT3A, SF3B1, SRSF2, and TET2 (Figure 1). In the AYA population, 18 pts (38%) had very low/low, 10 (21%) intermediate, and 20 pts (42%) had high/very high risk MDS using IPSS-R. In the YA population, 32 pts (26%) had very low/low, 32 (26%) intermediate, and 57 pts (47%) had high/very high risk MDS using IPSS-R. Twenty-seven percent of pts with very low to intermediate-risk MDS using IPSS-R were upstaged to moderate high to very high-risk MDS using IPSS-M. The majority of pts (AYA: 75%; YA: 63%, p=0.5) were transfusion independent at baseline. Single-agent hypomethylating agents were the most frequently used frontline therapy, used in 17 AYA pts (34%) and 58 YA pts (48%), with more AYA pts receiving upfront allogeneic stem cell transplantation (Allo-SCT) compared to YA pts (28% vs 5%). After frontline therapy (Table 1), an additional 14 AYA (28%) and 52 YA (42%) pts received Allo-SCT. In pts with IPSS-R intermediate to very high-risk, response rates (HI+CR+mCR+mCR with HI) to frontline therapies were 46% and 54.5% in the AYA and YA groups, respectively. After median follow-up of 24 months [range, 1-218], median OS was numerically longer in the AYA versus YA group (155 vs 53 months, p=0.2). AML transformation occurred in 24% and 20% of the pts in the AYA and YA groups, respectively. Median OS was significantly longer in de novo compared to t-MDS pts (56 vs 27 months, p=0.03). Median OS among Allo-SCT compared to non-SCT pts were not reached vs 155 months ( p=0.9) in the AYA group and 53 vs 54 months ( p=0.4) in the YA group. IPSS-R (very low+low vs intermediate vs high+very high) was able to distinguish differences in OS in the YA (133 vs 53 vs 17 months, p=0.0002) and AYA (155 vs not reached vs 18 months, p=0.006) pts, with the exception of the intermediate-risk group in the AYA cohort. Conclusions: In this study, d e novoMDS was seen in the majority of younger pts with MDS (both AYA and YA). Mutations in STAG2 and NRAS were more common in the AYA pts, while YA pts had MDS more enriched for TP53, DNMT3A, TET2 and splicing mutations. This study is still ongoing with a plan to compare the abovementioned groups to older pts (≥60 years) with MDS.