Hypomethylating Agents Research Articles

Treatment Outcomes in Unfit Patients with Newly Acute Myeloid Leukemia According to IDH1 Mutational Status: Real World Evidence from the Pethema Epidemiologic Registry

Background: Current treatment of unfit acute myeloid leukemia (AML) patients include hypomethylating agents (HMA) with or without venetoclax, low-dose cytarabine (LDAC), and supportive care only. Recently, the combination of azacytidine with ivosidenib, an IDH1 inhibitor, has showed a significant improvement in survival in unfit patients with IDH1 mutated (IDH1mut) AML compared to azacytidine plus placebo. Real world studies analyzing outcomes of IDH1mut AML patients are scarce. Aims: This study aims to assess retrospectively the characteristics, therapeutic approaches, and outcomes of unfit patients with AML in an unselected population reported to the multicentric PETHEMA registry according to IDH mutational status. We present here a first interim analysis, as the study is aimed to enroll 3500 patients. Methods: AML unfit patients reported to PETHEMA registry between January 2015 and June 2022 were included in the study, regardless of their therapeutic approach. IDH1 mutational status was analyzed with next generation sequencing (NGS) and polymerase chain reaction (PCR) technics performed in central labs from PETHEMA group, as well as locally as per standard practice. All clinical records were reviewed from diagnosis to death/last follow-up and data were analyzed with R statistical software. Results: From 9398 patients reported to the PETHEMA registry between 2015 and 2022, 4533 patients had information on IDH1 mutational status. Of them, 2096 patients were unfit for intensive chemotherapy, and therapeutic approach was available in 1255 patients who were evaluable for this interim report. Median age was 75 years, 716 (57%) were male, 312 (35%) had adverse cytogenetic-risk. Overall, 141 (11%) had IDH1mut [106 (12.4%) by NGS and 77 (9.9%) by PCR only] and 1114 (89%) had no IDH1 mutation (IDH1wt). There were differences between IDH1mut vs IDH1wt cohorts regarding white blood cells (WBC) count (P=0.003), platelets count (P=0.002), bone marrow blasts percentage (P<0.001), MRC cytogenetic risk (P=0.02) and NPM1 mutational status (P<0.001). More detailed information is shown in Table 1. Regarding treatments, 176 (15%) patients received only best supportive care (BSC), 293 (23%) were included in non-intensive clinical trials, 433 (34%) received HMAs, 281 (22%) LDAC schedules and 62 (5%) venetoclax plus HMA or LDAC (VEN). From 818 patients with available response, 261 (32%) achieved complete remission (CR) or CR with incomplete recovery (CRi), 105 (13%) partial remission, 305 (37%) were resistant and 147 (18%) died before being assessed. No statistical differences were observed between IDH1mut and IDH1wt groups (P=0.18). Median overall survival (OS) was 5.9 months (CI95, 5.3-6.7), with no differences in IDH1mut and IDH1wt cohorts [median 7.7 months (CI95, 5.9-11.2) vs 5.6 (CI95, 5-6.4)], with a 2y-OS of 15% (9.7-24.5) vs 15% (12.9-18.2), respectively (P=0.24). Median OS was 7.9 (CI95, 6.8-9.3) in patients receiving active treatment. Patients receiving VEN schedules had a median OS of 11.1 months (CI95, 9.4-14.1) vs 10.5 (CI95, 8.2-13.0) in patients included in clinical trials vs 8.3 (CI95, 6.7-10.0) in those receiving HMA vs 5.5 (CI95, 4.5-6.8) treated with LDAC-based schemes vs 0.4 (CI95, 0.3-0.7) in BSC patients (P<0.001). Figure 1 shows OS according to therapeutic approach in all patients. According to the therapeutic approach, IDH1mut patients included in clinical trials had a median OS of 16.8 (CI95, 6.0-NA) vs 10.6 months (CI95, 9.9-NA) in patients receiving VEN schedules vs 9.0 (CI95, 6.5-14.5) in those receiving HMA vs 7.0 (CI95, 5.5-12.7) with LDAC-based vs 0.6 (CI95, 0.2-3.7) in BSC patients (P<0.001). Conclusion: We show a prevalence of 11% of IDH1 mutation among patients considered unfit to receive intensive chemotherapy. Although there were differences between IHD1mut and IDH1wt subgroups in some variables with demonstrated prognostic impact, no significant differences were observed in response to treatment or OS. This study was partially supported by Servier.

Development of the Treatment Preference in Myelodysplasia Questionnaire (TPMQ) for Clinicians (mTPMQ), Carers (cTPMQ), and Patients (pTPMQ)

Objective: Myelodysplastic syndrome (MDS) is a disease of the hematopoietic stem cells and leads to an increased risk of developing acute myeloid leukemia (AML).[1] The injectable hypomethylating agents (HMA) azacitdine and decibtabine have been shown to be superior to best supportive care in improving cytopenias in intermediate to high-risk patients.[2, 3] These injectable HMAs regimens are commonly administered in a healthcare facility, with patients traveling to the facility on consecutive days. The recent availability of an oral form of decitabine, ASTX 727 (a fixed-dose combination of decitabine and the cytidine deaminase inhibitor cedazuridine), may significantly impact patient, carer, and clinician choices. To assess preference for an injectable vs. orally administered HMA, three questionnaires were developed based on qualitative input from clinicians, patients, and carers: the Treatment Preference in Myelodysplasia Questionnaire (TPMQ) for clinicians (mTPMQ), carers (cTPMQ), and patients (pTPMQ). Methods: This was a non-interventional, cross-sectional qualitative interview study consisting of interviews with patients with MDS who were eligible for or were being/had been treated with injectable azacitidine, their carers and their treating clinicians. Data were collected from qualitative mixed method interviews composed of concept elicitation and cognitive debriefing. Two sites were identified to enrol participants. During concept elicitation, participants were asked about MDS and treatment options to best understand the constructs of interest relative to their specific circumstance and preferences. Cognitive debriefing occurred after participants reviewed a draft of the questionnaire. Based on the interviews, the questionnaires were revised to ensure the constructs of importance were captured and the questionnaires, response options, and instructions were all well understood. Results: 15 participants completed interviews (5 clinicians for the mTPMQ, 5 patients for the pTPMQ, and 5 carers for the cTPMQ). Concepts identified as important included across groups included: easier to be compliant, less travel time, less time in the clinic, less painful, costs and convenience (Table 1). The mTPMQ underwent 3 revisions. Overall, clinicians indicated the directions, items and response options were understandable and clear. Comments that significantly modified the questionnaire concerned splitting the last question from 1 to 2 questions, depending on which treatment the patient would be receiving. Patient participants thought the pTPMQ captured concepts of interest and was easy to understand. Modifications were made to the questionnaire either as a direct result of patient participant comments (e.g., changed skip directions to “turn to page X, question X”), study team discussions of the interviewer's comments and notes and making items consistent between questionnaires. Carer participants thought the cTPMQ captured the constructs of interest and the questions were clear and the questionnaire was easy to answer. Modifications were made to the questionnaire as a direct result of carer participant comments (e.g., changed “We spend less time in the clinic” to “It means less time in the clinic for the patient”), study team discussions of the interviewer's comments and notes (e.g., added “It makes it easier for the patient to continue treatment”) and making items consistent between questionnaires. Conclusions: The findings of the participant interviews suggest the concepts assessed in the assessments are complete. Respondents could respond to items and talk about all aspects assessed in the questionnaires in meaningful ways and were able to comprehend the instructions and response options on a categorical scale. The TPMQs are based on direct input from the population of interest and assess the constructs of importance to each group. 1. Bell, J.A., et al., Clin Lymphoma Myeloma Leuk, 2018. 18(4): p. e157-e166. 2. Kantarjian, H., et al., Cancer, 2006. 106(8): p. 1794-803. 3. Fenaux, P., et al., Lancet Oncol, 2009. 10(3): p. 223-32.

Combinatorial Optimization of Chemotherapeutic Agents on T-Cell Acute Lymphoblastic Leukemia

Introduction: Up to now, treatment options for T-cell acute lymphoblastic leukemia (T-ALL) remain quite limited. This study aims to assess the efficacy of monotherapies and combination of chemotherapeutic agents for T-ALL. Jurkat, CCRF-CEM are late T-cell progenitor (LTP) cell lines, while Loucy is early T-cell progenitor (ETP) cell line characterized by an immature phenotype. The drugs under evaluation include venetoclax, a selective BCL-2 inhibitor; cytarabine, a cytosine analog with established antineoplastic activity; bendamustine, an alkylating agent; and azacitidine, a DNA methyltransferase inhibitor. Given the notable tumor heterogeneity observed in T-ALL, the comprehensive assessment of these therapeutic agents on Jurkat, CCRF-CEM, and Loucy may render valuable insights for treatment strategies in T-ALL. Methods: Cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2. Venetoclax, bendamustine, and azacitidine were prepared as stock solutions in DMSO, while cytarabine was prepared in distilled water. Monotherapies (venetoclax, cytarabine, bendamustine and azacitidine) and combination therapies (venetoclax plus cytarabine, venetoclax plus bendamustine and venetoclax plus azacitidine) were administered. A control group received DMSO or distilled water only. Cell viability was assessed after 48 hours by using Trypan blue and colorimetric method (MTS assay) on cells treated with various drug concentrations. Statistical analysis was conducted using the SynergyFinder Plus and drc R package, with significance set at p < 0.05. Results: Our study demonstrated that T-ALL cell lines displayed a different pattern of drug sensitivities according to combination of chemotherapeutic agents. Loucy showed greater sensitivity to venetoclax while Jurkat and CCRF-CEM exhibited higher sensitivity to cytarabine, bendamustine, and azacitidine. When conducting drug combinations on Jurkat and CCRF-CEM, the addition of venetoclax to cytarabine, bendamustine, or azacitidine resulted in an additive effect, with Loewe synergic scores ranging from -10 to 10 for each combination. On the other hand, Loucy demonstrated a distinct pattern of response; the combination of venetoclax with cytarabine showed additivity (Loewe synergic score: 8.45 and 5.82 with MTS and trypan blue assays, respectively), while venetoclax combined with bendamustine or azacitidine showed a synergistic effect (Loewe synergic score > 10 with MTS assay). Notably, among these combination treatments on Loucy, the pairing of venetoclax with bendamustine showed the most significant synergistic effects, with a Loewe score of 16.6. Conclusion: Venetoclax-based chemotherapeutic combinations exhibited various responses in T-ALL cell lines. Of note, the combination of venetoclax plus bendamustine showed potent synergistic effects in all T-ALL cell lines. These findings warrant further study to develop fundamentally curative therapeutic strategies in T-ALL.

Impact of Therapy Sequence on Survival Outcomes Among Patients with Relapsed or Refractory Mature T and NK Cell Neoplasms: A Global Retrospective Cohort Study

Despite evolution of therapeutic strategies, there is no universal standard of care for relapsed or refractory (RR) mature T and NK-cell neoplasms (TNKL). Most patients receive multiple lines of therapy and cycle through many available options. 1-3 There is no data to inform optimal therapy sequence, however emerging data suggest that exposure to epigenetic modifiers (EM) can sensitize tumors to other therapies. 4-6 Here we report results of comparative analyses assessing survival outcomes based on therapy sequence using a global RR TNKL patient cohort with data from 15 centers across 6 continents. We conducted a retrospective target-trial 7,8 cohort study using an updated global patient cohort. 9 Patients were excluded from analyses if they had anaplastic large cell lymphoma (as EM is infrequently used 10,11), did not receive first line cytotoxic chemotherapy (CC), or no documented second line (2L) start date. Cohort assignment was based on 2L therapy received: EM (e.g. histone deacetylase or DNA methyltransferase inhibitors), small molecule inhibitors (SI; broad or selective), or CC. Antibody-drug conjugates were included in CC. Outcomes were overall survival (OS; time from 2L start to death) and real-world progression-free-survival-2 (rwPFS2; time from 2L start to fourth line start or death 12 to assess if 2L modifies the effect of third line [3L]). Planned subgroups included histologic subtype and those who received allogeneic hematopoietic transplant (HSCT) after 2L. For sequence analyses, included patients must have received 3L. Outcomes were assessed between all prespecified 2L to 3L sequences using a single comparator, and pairwise. Kaplan Meier curves were used, and adjusted hazard ratios (aHR) were estimated using Cox regression with previously described a priori covariates. 9 Maximal, minimal, and no covariate Cox models were compared using likelihood ratio tests and Akaike's information criterion. Of the 925 RR patients in the global cohort, 472 were included who received EM (n=89), SI (n=46), or CC (n=337) at 2L. The most common SI included immunomodulatory imide drug (26.1%), PI3K inhibitor (21.7%), investigational pathway inhibitor (19.6%) and JAK inhibitor (17.4%). Within EM, SI, and CC, 42.7%, 37%, and 47.2% of patients were primary refractory to first line, respectively. The most common histologies were PTCL-NOS (51.7%, 41.3%, and 57.3%) and AITL (43.8%, 54.3%, and 26.4%) in EM, SI, and CC, respectively. Most patients had a Prognostic Index for T-cell Lymphoma (PIT) score at diagnosis of 1 (27%, 34.8%, and 27%) or 2 (28.1%, 26.1%, and 25.8%) in EM, SI, and CC, respectively. In EM, 34.8% received first-line autologous HSCT, versus 28.2% in SI, and 15.4% in CC. Following 2L, 12.4%, 17.4%, and 9.8% received allogeneic HSCT in EM, SI, and CC, respectively. The Cox model containing histology, PIT, and primary refractory status was used as the final model (vs maximal model: p=0.88). Compared to CC at 2L, EM did not affect OS (aHR 0.88, 95% CI 0.63-1.24; p=0.46) or rwPFS2 (aHR 0.84, 95% CI 0.61-1.16; p=0.28), however SI improved OS (aHR 0.61, 95% CI 0.37-0.97; p=0.038) and rwPFS2 (aHR 0.61, 95% CI 0.38-0.90; p=0.038). In the allogeneic HSCT subgroup, there was no difference in OS (EM vs CC: aHR 0.32, 95% CI 0.04-2.40; p=0.27; SI vs CC: aHR 0.51, 95% CI 0.06-4.69; p=0.55) or rwPFS2 (EM vs CC: aHR 0.30, 95% CI 0.05-1.96; p=0.21; SI vs CC: aHR 0.64, 95% CI 0.05-3.90; p=0.46). There were no differences in PTCL-NOS patients in OS (EM vs CC aHR 0.80, 95% CI 0.51-1.25; p=0.33; SI vs CC: aHR 1.00, 95% CI 0.54-1.84; p=0.99) or rwPFS2 (EM vs CC: aHR 0.91, 95% CI 0.61-1.38; p=0.66; SI vs CC: aHR 0.90, 95% CI 0.49-0.97; p=0.16). In AITL, SI improved OS (aHR 0.34, 95% CI 0.16-0.76; p=0.009) and rwPFS2 (aHR 0.36, 95% CI 0.17-0.77; p=0.008) versus CC, but EM showed no difference in OS (aHR 0.80, 95% CI 0.43-1.46; p=0.46) or rwPFS2 (aHR 0.56, 95% CI 0.31-1.02; p=0.059). There was no difference in rwPFS2 or OS across sequences overall (Figure 1) or between pairwise sequence comparisons (Figure 2). Compared to CC, SI at 2L improved OS and rwPFS2 in AITL patients. Therapy sequence did not affect OS or rwPFS2. Advanced approaches such as machine learning and dynamic treatment regimes have been initiated to fully elucidate the effect of therapy sequence. Our study highlights several equally effective therapy sequences to treat RR TNKL allowing therapy to be individualized based on patient and disease characteristics, and drug access at a given time.

Homoharringtonine Overcomes the Negative Impact of Genetic Patterns on Venetoclax Plus Azacitidine Regimen in Relapsed/Refractory Acute Myeloid Leukemia: A Multi-Center, Cohort Study

Objective: Whether other agents added to venetoclax (VEN) plus hypomethylating agents (HMA) regimen could overcome the negative impact of genetic patterns on response in patients with relapsed/refractory acute myeloid leukemia (RR-AML) remains unclear. Aim of this study was to evaluate whether homoharringtonine (HHT) added to VEN plus azacitidine (AZA) regimen could improve the response and overcome the negative impact of genetic patterns in patients with RR-AML . Methods: A multi-center, cohort study of the response and the genetic patterns of response of VEN plus AZA and HHT (VAH) versus VEN plus AZA (VA) regimens as salvage treatment in the patients with RR-AML was performed. Patients were enrolled from four studies from October 2018 to December 2022 at nine medical centers in china. Results: A total of 321 patients were analyzed, including 150 females and 171 males, with a median age at 46 (IQR, 35-61) years. There were 172 patients in the VAH and 149 in the VA group. VAH significantly improved CRc rate (66.3% vs. 44.3%, P<0.001) and prolonged OS (median OS, not reach vs. 14.3 months, P=0.004), to compared with VA. Multivariate analysis showed that VAH was the independently protective factor for CRc and survival. In addition, VAH significantly overcame the negative impact of FLT3-ITD/TKD, N/KRAS, TET2, DNMT3A mutations, and t(8;21)/AML1-ETO, as well as non-adverse ELN risk, also apparently in adverse ELN risk or complex karyotype, on the response of VA regimen. Conclusion: The impact of genetic patterns on the response presented diversely in different VEN-based regimens. HHT added to VA regimen might improve the response and overcome the negative impact of part genetic patterns in RR-AML. Key words: homoharringtonine, venetoclax, relapsed/refractory, acute myeloid leukemia, genetic pattern

A Phase 1 Study of Avapritinib in Combination with Decitabine in Patients with Systemic Mastocytosis with an Associated Hematologic Neoplasm

Background and Rationale: Systemic mastocytosis (SM) is a clonal hematologic neoplasm characterized by abnormal accumulation of mast cells which can lead to symptoms related to mast cell degranulation and/or, in the case of advanced systemic mastocytosis (AdvSM), organ dysfunction due to mast cell infiltration. AdvSM is a category of SM comprised of aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), and SM with an associated hematologic neoplasm (SM-AHN). Among these, SM-AHN is the most common subtype, accounting for approximately 70% of AdvSM. A KIT D816V mutation found in up to 95% of SM cases is the primary oncogenic driver, making it a key therapeutic target. Avapritinib, a highly potent and selective inhibitor of D816V mutant KIT, has demonstrated high rates of rapid, deep, and durable responses in all AdvSM subtypes, including SM-AHN. However, a recent analysis of the EXPLORER study showed that avapritinib-treated patients were still at risk for progression of the AHN component, despite continued control of mast cell disease burden, indicating that combination therapy is likely to be required for optimal control of SM-AHN due to its inherent molecular complexity. Hypomethylating agents (HMA) form the backbone of therapy for advanced myelodysplastic syndrome (MDS) and myelodysplastic/myeloproliferative neoplasm (MDS/MPN), hematologic neoplasms most likely to co-occur with SM in patients (pts) with SM-AHN. To date, the combination of HMA and KIT inhibitors has not been clinically evaluated. In this study, we aim to assess the safety and tolerability of the combination of avapritinib and decitabine in patients with SM-AHN. Study design: The primary objective of the study is to establish the recommended dose (RP2D) of avapritinib in combination with decitabine based on dose-limiting toxicity, overall safety, and clinical activity data. A dose evaluation steering committee (DESC) comprised of participating investigators will review the totality of safety and efficacy data to determine RP2D. Secondary endpoints include SM overall response rate by the end of 6 cycles by the m-IWG consensus criteria and proposed ECNM-AIM criteria ( Gotlib et al., J Allergy Clin Immunol Pract, 2022), as well as incidence and frequency of adverse events. Exploratory endpoints will consist of patient reported outcomes, progression free survival, overall survival, and correlative biomarkers linked to mast cell disease or AHN. Adult pts with a diagnosis of SM-AHN by the WHO 2022 classification criteria will be eligible for inclusion. Eligible pts must have any of the following myeloid AHN per WHO 2022 criteria: MDS, chronic myelomonocytic leukemia, MDS/MPN with neutrophilia, MDS/MPN with SF3B1 mutation and thrombocytosis, or MDS/MPN- not otherwise specified. Patients with MDS must have intermediate, high- or very-high risk by IPSS-R. Patients with very-low or low-risk MDS by IPSS-R must be intolerant/refractory to ESA or have serum EPO > 200 U/L. A baseline platelet count of ≥ 75 x 10 9/L and ≥ 25 x 10 9/L will be required for inclusion in the dose-finding and the dose-expansion phases, respectively (Figure). Key exclusion criteria include a diagnosis of acute myeloid leukemia, history of intracranial hemorrhage or risk of major hemorrhage, prior treatment with avapritinib or decitabine with documented progression in SM or AHN component, respectively, or history of treatment with alternative KIT inhibitor or azacitidine within 4 weeks of study treatment initiation. In the dose finding part of the study, a Bayesian adaptive design will be employed to assess different doses of avapritinib (50 to 200 mg PO daily) in combination with fixed doses of decitabine (oral decitabine/cedazuridine: 35/100 mg/day or intravenous decitabine: 20 mg/m 2/day on days 1-5) in a 28-day treatment cycle. Close attention will be paid to treatment-emergent thrombocytopenia with protocol-driven treatment holds and dose modification strategies employed when platelet count drops below 50 x 10 9/L. After RP2D is determined, an additional 10 patients will be enrolled in the dose expansion phase. Patients with platelet count ≥ 25 x 10 9/L and < 75 x 10 9/L, will receive lead-in dosing with decitabine or decitabine/cedazuridine with the ability to add avapritinib if sufficient platelet thresholds are achieved. This study will open at 7 sites in the United States and is anticipated to open in January 2024.

Methylation Patterns of Fetal Hemoglobin Regulating Genes in High-Risk (HR) -MDS Patients Following Treatment with Azacytidine

Background: Current standard of care for HR-MDS patients, i.e. hypomethylating agent (HMAs) treatment with azacytidine (AZA) or decitabine (DAC), is associated with a 30-50% response rate, and many efforts have been made to identify predictive factors for response. Fetal hemoglobin (HbF) levels have been proposed as a prognostic index for DAC treatment (Stomper J. et al, 2019), but no study has examined its prognostic value in AZA treated patients, and the mechanism of γ-globin chain re-expression has not been investigated. Methods: Bone marrow (BM) aspirates of 28 HR-MDS or CMML patients were collected at baseline and at the first evaluation of response, following 6 cycles of AZA treatment. Methylation of Cytosine followed by Guanosine (CpG) sites of the γ-globin gene promoter and CpG 326 of ZBTB7A/LRF, a previously characterized epigenetic repressor of HbF (Chondrou V. et al, 2022), were detected with pyrosequencing analysis in total genomic DNA, extracted from BM samples, pre- and post-treatment. Response to AZA was characterized as marrow response (mCR), when blasts were <5% and were reduced >50% from baseline, and as hematological response (HI-R) when there was at least one lineage improvement. HbF was measured with HPLC from the peripheral blood of 20 patients, at 5 time-points (before 0, 1, 2, 4 and 6-7 cycles of treatment), to test its expression pattern and potential prognostic significance. HbF values >1% were considered induced, as in previous studies (Stomper J. et al, 2019). For statistical analysis IBM SPSS 28.0.0.0 program was used. One patient with extremely high HbF before treatment (HbF 0=13.4%) was excluded from the analysis, as well as the CpG site 3 of γ-globin gene promoter, which was 100% methylated pre- and post- AZA treatment. Results: Median baseline HbF levels were 0.6% (range 0 - 2.6), and they were increased to 0.8% (range 0 - 1.8) and to 1.1% (range 0 - 3.5), at 2 and 6 months, post-AZA treatment, respectively. Difference in the repeated measurements of HbF was of borderline statistical significance (p=0.068), specified between cycles 2 and 6 (p=0.038). Correlation analysis showed a significant association of mCR with induced HbF after 6 cycles of treatment (p=0.011, r s=0.618) and a strong positive correlation between elevation of HbF at cycle 2 and time to leukemia progression (p=0.019, r s=0.886). Overall, median methylation of the γ-globin gene promoter was 73.4% and 72.8% pre- and post-AZA treatment, respectively (p=0.159). However, patients exhibiting any kind of response showed reduced methylation at site 2 post- AZA, at the limit of statistical significance (p=0.095 for mCR patients and p=0.066 for HI-R), whereas site 4 was significantly hypermethylated among HI-R patients (p=0.042). A negative correlation between methylation of the γ-globin gene promoter and gene expression was also identified (p=0.008, r s=-0.427), particularly at the sites 2 and 5 (p 2=0.011, p 5=0.004). Methylation analysis of CpG 326 of ZBTB7A/LRF showed significant hypermethylation at site 1 (p=0.044) and reduction of methylation at site 22 (p=0.043), following AZA treatment. When analysis was restricted among responders only, there was significant hypermethylation at site 1 (p 1=0.020) and borderline significant hypermethylation at sites 5 and 11, among mCR patients (p 5=0.069, p 11=0.079). Conversely, sites 9 and 13 were significantly hypermethylated among non-responders (p 9=0.034, p 13=0.047). With Spearman's analysis, a significant negative correlation between methylation fold change in site 13 and mCR was observed (p=0.002, r s=-0.555). Although no correlation between HbF and CpG 326 methylation status pre- treatment was observed, after 6 cycles of AZA a borderline correlation was identified between HbF levels and fold change of mean methylation of CpG 326 (p=0.05, r s=0.482). Moreover, induced HbF levels were related at the limit of statistical significance to increase in methylation at sites 1,3,5,11 and 22 (p 1=0.077, p 3=0.052, p 5=0.087, p 11=0.061, p 22=0.060). Conclusions: In this study, a relationship of γ-globin gene promoter methylation status and HbF levels was found among HR-MDS patients, treated with AZA. Moreover, hypermethylation in CpG326 of ZBTB7A/LRF after AZA treatment was associated with HbF levels, and different methylation patterns were observed between responders and non-responders. Further research is needed to clarify the role of LRF in MDS response to AZA.

Venetoclax Exposure Reduction in Venetoclax-Hypomethylating Agents Combination Is Feasible and Has the Same Efficacy in Patients with Acute Myeloblastic Leukemia. Experience of a Single Center

BACKGROUND The combination of venetoclax (VEN) and azacitidine (AZA) is the actual standard of care for patients with acute myeloid leukemia (AML) not eligible for intensive treatment ( Di Nardo et al. N Engl J Med 2020; 383:617-629). Despite being considered a low-intensity treatment, dose adjustments are frequently needed in this group of patients due to prolonged myelosuppression and increased incidence of infection. Reducing the duration of VEN administration might improve the safety profile, although its efficacy is still unknown in this setting. AIM We present the experience of the Institut Català d'Oncologia-Hospital Duran i Reynals in patients with AML treated with VEN in combination with hypomethylating agents (HMA) to evaluate the efficacy of VEN exposure reduction in patients who require VEN adjustment due to myelotoxicity. METHODS We retrospectively analyzed 68 patients with AML treated with VEN+HMA as a first-line or as salvage therapy between May 2019 and April 2023. We specifically evaluated patients in complete remission (CR) after 1 to 3 cycles of VEN combination who require VEN scheme shortening to 7 (low dose) or 14-21 (intermediate dose) days every 28 days cycle due to myelotoxicity. Response to treatment was defined according to ELN2022 criteria. RESULTS Median age at diagnosis was 75 years (range 33-85 years). Forty-seven (69%) were male. Twenty-eight (41.2%) were classified according to the 2022 ELN classification as an adverse risk, 33 (48.5%) as intermediate risk and 7 (10.3%) as a favorable risk group. The median number of cycles was 7 (range 1-23). ORR of the whole series was 76.5%. Forty-six (67.6%) patients achieved a composite complete response (CRc) (CR, CR with partial hematologic recovery, and CR with incomplete hematologic recovery). Among the 46 responding patients, seven of them (15.2%) discontinued treatment because of patient's decision after achieving CR and they were not considered for further analysis. The remaining 39 patients reached the best response in the first 2 cycles. Twenty-eight (71.8%) patients received VEN+HMA as first-line treatment and 11 (28.2%), as salvage therapy. No significant differences were found between these two groups regarding sex, age or ELN2022 risk. Nine out of these 39 patients (23.1%) received VEN for 28 days every 28 days cycle (VEN28 group), 21 (53.8%) patients received VEN for 14 or 21 per cycle (VEN14-21 group), and 9 (23.1%) patients received VEN for 7 days per cycle (VEN7 group). With a median follow-up of 18.8 months (range 14.6-23.1), the median OS of the VEN14-21 group was 15.9 months but it was not reached for the VEN7 group. Regarding EFS, the median EFS of the VEN14-21 group was 11.4 months though it was not reached for the VEN7 group. No statistically significant differences were found comparing OS and EFS between the VEN14-21 group and the VEN7 group nor were both of them with the VEN28 group (Figure 1). CONCLUSION Shortening VEN exposure to 14-21 or 7 days per cycle with concomitant HMA administration seems to induce a similar response rate and is likely to have comparable efficacy to the 28-day cycle. Among responders, VEN dose reduction seems to be associated with favorable outcomes. Prospective clinical trials that further evaluate the efficacy and safety of shortened VEN administration are required.

Survival and Prognosis Among 301 Patients with Newly-Diagnosed Acute Myeloid Leukemia Following Venetoclax Plus Hypomethylating Agent Therapy

Background: Venetoclax (Ven) in combination with hypomethylating agent (HMA) is the preferred treatment for elderly/unfit patients with newly-diagnosed acute myeloid leukemia (AML). Current prognostication for AML is based on European LeukemiaNet (ELN) genetic risk stratification which has limited applicability in patients treated with Ven-HMA ( Blood, 2022). Accordingly, our primary objective was to determine predictors of treatment response and survival outcomes in treatment-naïve AML patients receiving Ven-HMA. Methods: Our study population was recruited from Mayo Clinic (MN, FL, AZ), after institutional review board approval and based on documentation of newly-diagnosed AML treated with Ven-HMA outside of clinical trials between November 2018 and April 2023. Cytogenetic and molecular studies were performed by conventional karyotype, and next-generation sequencing, respectively. Response was assessed according to the 2022 ELN criteria Results: Patient characteristics A total of 301 newly-diagnosed AML patients (median age 73 years, 66% male, 62% de novo) received a median of 3 cycles (range 1-48) of azacitidine 75 mg/m2 days 1-7 ( n=100) or decitabine 20 mg/m2 days 1-5 ( n=201) with Ven. ELN 2022 cytogenetic risk included favorable (1%, n=4), intermediate (61%, n=184) or adverse (38%, n=113). Mutations involved TP53 in 77/301 (25%), ASXL1 in 54/279 (19%), RUNX1 in 54/296 (18%), NPM1 in 44/299 (15%), DNMT3A in 40/296 (13%), K/NRAS in 41/296 (14%), IDH1 in 20/301 (6%), IDH2 in 30/301 (10%), FLT3-ITD in 25/301 (8%) and DDX41 in 9/247 (4%) of informative cases. Predictors of response Complete remission, with (CR) or without (CRi), count recovery, was documented in 182 (60%) patients, including CR in 36% and measurable residual disease (MRD) negative by flow cytometry in 77% of 105 informative cases that achieved CR/CRi. Median time to CR/CRi was 1.4 months. In univariate analysis, CR/CRi was more likely to occur in the presence of NPM1 mutation, (86% vs. 56%; p < .01), IDH2 mutation, (80% vs. 58%; p = .02), DNMT3A mutation, (78% vs. 57%; p = .01), or DDX41 mutation, (100% vs. 58%; p <.01), and in the absence of adverse karyotype, (71% vs. 42%; p < .01), TP53 mutation, (67% vs. 40%; p <.01), FLT3-ITD mutation, (63% vs. 36%; p = .01), or RUNX1 mutation, (64% vs. 44%; p = .01); in multivariable analysis, adverse karyotype, ( p < .01), FLT3-ITD ( p < .01), RUNX1 ( p < .01), NPM1 ( p = .03), IDH2 ( p = .04), and DDX41, ( p = .01) mutations remained significant. Predictors of survival After a median follow-up of 8.5 months (range 0.5-56), 13.2 months for living patients, 174 (58%) deaths, 73 relapses (40% of those achieving CR/CRi), and 41 (14%; including 35 in CR/CRi) allogeneic hematopoietic stem cell tranplants (AHSCT) were documented. In univariate analysis of pre-treatment variables, thrombocytopenia < 100 x 10 9/l, (HR 1.5, 95% CI 1.03-2.2; p = .03), adverse karyotype, (HR 2.8, 95% CI 2.1-3.8; p < .01) TP53 mutation, (HR 2.5, 95% CI 1.8-3.5; p < .01), or absence of IDH2 mutation, (HR 3.5, 95% CI 1.7-7.6; p < .01) predicted inferior survival. Failure to achieve CR/CRi (HR 4.5, 95% CI 3.3-6.1; p < .01) was also associated with inferior survival and in multivariable analysis, failure to achieve CR/CRi, (HR 3.4, 95% CI 2.5-4.8; p< .01), presence of adverse karyotype, (HR 1.6, 95% CI 1.1-2.6; p = .02), TP53 mutation, (HR 1.6, 95% CI 1.002-2.4; p = .04), and absence of IDH2 mutation (HR 2.2, 95% CI 1.004-4.7; p = .04) were identified as risk factors for survival. Subsequent HR-weighted scoring resulted in three-tiered risk stratification: low (0-1 point; n= 130), intermediate (2-3 points; n= 105), and high (4-5 points; n= 66), with respective median survival (3-year rate) of 28.9 (48%), 9.6 (6%), and 3.1 (0%) months ( p < .01) (Figure). AHSCT had an independent favorable impact on survival (HR 0.3, 95% CI 0.1-0.6; p < .01), most apparent in low ( p =0.04), and intermediate ( p <.01), as opposed to high ( p = .06) risk. Conclusions: In the current single institutional series of Ven-HMA treated newly-diagnosed AML, response to Ven-HMA was the foremost predictor of survival. Additional risk factors for survival included adverse karyotype, presence of TP53 and absence of IDH2 mutations. These observations allowed for a three-tiered genetics-enhanced survival model with CR/CRi as a backbone, and also confirmed survival advantage from AHSCT, regardless of risk category.

Selinexor and Chidamide in Patients with Relapsed/Refractory Acute Myeloid Leukemia Exposed to Venetoclax: A Single-Arm, Phase 2 Study

Background: Novel therapies for patients with relapsed/refractory acute myeloid leukemia (R/R AML) are imperative, particularly those who have experienced relapse or resistance to multiline chemotherapies or venetoclax-based regimens, thereby underscoring the need for more innovative therapies that are both effective and well-tolerated. Chidamide and Selinexor have shown potential synergistic anti-leukemia effects by targeting NF-Κb /c-FLIP signaling via suppressing HDAC1/XPO1 activity. Therefore, the aim of this study was to investigate the activity and tolerability of chidamide combined with selinexor regimen for R/R AML. Methods: We report the findings of a phase 2 study in patients with relapsed/refractory AML that combined chidamide with selinexor. Selinexor was administered orally twice weekly for 4 weeks at a dose of 40/60 mg, depending on the patent's weight (over or under 70 kg). Chidamide was taken orally once a day at a dose of 10mg for 28 days. The primary endpoint was composite complete remission rate [CRc, complete response (CR) plus complete response with incomplete blood count recovery (CRi)] after 1 cycles of treatment. The secondary endpoints include safety and survival. Results: Between December 2022 and April 2023, a total of 11 patients, median age 40 years (range, 22-54) were enrolled in the trail (ClinicalTrials.gov number, NCT05951855.). 8 out of 11 (72.7%) patients with R/R AML had received more than three prior lines of treatment. All of patients were previously exposed to venetoclax or hypomethylating agents (HMAs). Key baseline and clinical characteristics are summarized in Table 1. 10(90.9%) patients received the initial dose of 40 mg of selinexor twice a week, while one (9.1%) received 60 mg. Chidamide was administered to all patients at a daily dose of 10 mg daily. Five (45.5%) patients achieved a CRc (with 3 CR and 2 CRi), two (18.1%) achieved Partial remission (PR), and 5 (45.5%) had progressive disease for an overall response rate (ORR) of 63.6%. With a median follow-up of 147 days（range 59-234）, the median event-free survival (EFS) was 75 days and median overall survival (OS) was not yet reached (Figure 1). Among the seven patients who achieved CRc or PR, all proceeded to transplant after responding. One relapsed on day 56 following response and died 20 days later. Another patient died from an infection during the allogeneic hematopoietic stem cell transplantation (allo-HSCT). The remaining 5 patients are currently alive as of July 2023. No dose-limiting toxicities were observed. Common adverse events included neutropenia (72.7%), thrombocytopenia (63.6%), nausea/vomiting (63.6%), fatigue (45.5%), diarrhea (18.1%), bacteremia (9%). Conclusion: In this study, the combination of Selinexor and chidamide was administered as salvage treatment for R/R AML patients, resulting a high response rate and favorable safety profile. This offers a potential way for patients to bridge to transplantation. However, future studies with larger sample sizes and longer follow-up periods are needed to further validate its efficacy.

Single-Cell Multiomics Analysis Reveals Potential Drivers of Response to the Anti-TIM3 Inhibitor Sabatolimab Combined with Azacitidine in MDS and CMML

Background The median survival for patients with newly diagnosed (ND) higher risk myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) remains poor. The potential curability of MDS and CMML with allogeneic hematopoietic stem cell transplantation supports the concept of anti-tumor immunity and has led to interest in evaluating immune-based therapeutic approaches in myeloid neoplasms. The immune checkpoint molecule TIM-3 (encoded by the gene HAVCR2) is a target of interest in myeloid neoplasms given its expression on leukemic stem cells as well as several subsets of immune cells including T cells, monocytes, dendritic cells and NK cells. Sabatolimab (MBG453) is an investigational IgG4 anti-TIM-3 antibody currently under evaluation for myeloid neoplasms. In this exploratory study, we sought to characterize the effects of sabatolimab combined with the hypomethylating agent (HMA), azacitidine, on the immune landscape using single cell sequencing of samples from subjects with MDS and CMML treated with the combination from a previously reported phase 1b study (NCT03066648; Brunner et al 2022). Methods Following IRB approval, single-cell RNA sequencing (scRNA-seq) and associated proteomic cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) was performed on both blood (BLD) and bone-marrow (BM) derived samples from subjects with: 1) MDS (n=3) or CMML (n=2) treated with HMA therapy alone as part of standard care; 2) ND MDS (n=5) or CMML (n=3) treated with azacitidine combined with sabatolimab; and 3) relapsed/refractory MDS (n=3) treated with sabatolimab alone. Paired BLD and BM samples as well as samples from serial treatment time points were selected whenever feasible (Fig 1). A total of 206,183 cells from BM and 172,421 cells from BLD post-quality control filtering were analyzed. Further, scRNAseq data from normal BLD(200,000 cells) and BM(240,650 cells) generated as part of the human cell atlas (https://data.humancellatlas.org/explore/projects/cc95ff89-2e68-4a08-a234-480eca21ce79) were integrated with this dataset to assist with immune cell subset characterization, enable downstream comparisons to healthy hematopoietic cells, and to help define cell subsets associated with disease states. Results In baseline samples from subjects with MDS treated with azacitidine-sabatolimab, we found that an increased baseline abundance of interferon-responsive CD8 T cells in both the BM and BLD (FDR<0.1) was associated with response to therapy. An increased baseline abundance of plasmacytoid dendritic cells and a granulocyte population also suggested an association with response to therapy. Differential gene expression analysis of baseline samples from responding versus non-responding subjects with MDS treated with azacitidine-sabatolimab showed a higher baseline expression of TNF and IFNG in BM CD8 T cells and up-regulated expression of MHC-II machinery ( HLA-DRB1, HLA-DPA1, CD74) in myeloid cell subsets in responding subjects (FDR <0.1), suggesting that pre-treatment ability of these cells to be involved in antigen-presentation may play a role in response. In subjects with CMML treated with azacitidine-sabatolimab, we observed dynamic changes in cellular abundances and gene expression when comparing baseline to post-therapy timepoints. Specifically, BM CD8 T cell subsets showed an up-regulation of cytotoxicity genes ( GZMA), interferon response genes ( IFIT2, IFITM1, IFITM2) and IL32 cytokine post-therapy (FDR <0.1). When evaluating myelomonocytic cell populations, we observed an increase in CD16 monocytes post-treatment. Additionally, dendritic cells and monocytes showed an up-regulation of interferon response genes ( IFIT1, OAS1, IFI27). Conversely, we observed a down-regulation of metallothionein genes ( MT1E, MT1G, MT2A) as well as a collection of transcription factors ( NR4A1, FOSL2, JUN, CEBPB, CEBPD), NF-κB inhibitors ( NFKBIZ, NFKBIA) and CXCL8 in the post-treatment samples (FDR <0.1). Conclusions Our study provides one of the most comprehensive evaluations of the cellular dynamics of anti-TIM3 immunotherapy in patients to date, allowing for the nomination of novel putative predictive biomarkers of response and identification of potential immunomodulatory mechanisms induced by the combination of sabatolimab with azacitidine in MDS and CMML for further future analysis.

Utilizing Chemotherapy to Enhance the Anti-Tumor Properties of Ex Vivo Expanded Gamma Delta (γδ) T Cells Against Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the second most common type of blood cancer in children with a high rate of treatment failure and aggressive relapse. While allogeneic HSCT offers a cure, limitations arise in achieving remission prior to transplant. Immunotherapy-based approaches can induce remission, gaining interest in AML. Here, we explore the use of gamma delta (γδ) T cells as a promising tool to fulfill this unmet need. Unlike predominant alpha beta (αβ) T cells, γδ T cells do not require antigen presentation and identify their targets in a major histocompatibility complex (MHC) independent manner. Allogeneic γδ T cells thus have minimal risk of causing graft-versus-host disease (GvHD), creating ‘off-the-shelf’ cellular therapeutics. This would be ideal to target AML, especially given the aggressive nature of relapsed disease. Cytotoxic mechanisms used by γδ T cells include recognition of cellular stress molecules such as NKG2D and DNAM-1 receptor ligands on target cells, activation of death receptor pathway through Fas-FasL and TRAIL-R interactions, as well as γδ ΤCR mediated killing through recognition of butyrophilins (BTNs) on target cells. These stress molecules are expressed on AML cells and can be further upregulated through chemotherapeutic agents such as azacitidine (a hypomethylating agent) and venetoclax (BCL-2 antagonist), drugs currently in use to treat relapsed AML. Here, we tested these drugs in combination with γδ T cells to target AML. Methods Our studies explored four different AML cell lines (Kasumi-1, Nomo-1, MV4-11 and MOLM13). Each one was respectively treated at their corresponding inhibitory concentration 25 (IC 25) and 50 (IC 50) of azacitidine and/or venetoclax for 24 hours. After treatment, AML cells were assessed by flow cytometry for expression of NKG2D and DNAM-1 ligands, death receptor pathway molecules (CD95, TRAIL-R1/R2) as well as the butyrophillins (BTN2A and BTN3A1) to elucidate which pathways are over-expressed, aiding in γδ T cell targeting and cytotoxicity. Next, we co-cultured AML cells pre-treated with azacitidine and/or venetoclax with γδ T cells and measured cytotoxicity using a flow cytometry-based assay and a bioluminescence assay. Results: Each cell line expressed a unique pattern of markers at baseline. Cell lines treated with azacitidine had a robust expression of the NKG2D ligands ULBP1, ULBP2/5/6 and ULBP3. Azacitidine also increased CD155 and TRAIL-R2 in Nomo-1 while CD112 increased in MOLM13. Most venetoclax-treated AML cells over-expressed BTN2A and BTN3A1 along with CD95 in MOLM13. Cell lines were then treated with a combination of both drugs. Dual treatment of cells resulted in a significant increase in expression of most NKG2D markers as well the death receptor Fas (CD95). Additionally, increase in CD155 as well as BTNs was seen in several cell lines. Importantly, azacitidine and venetoclax had minimal effects on the viability of γδ Τ cells when treated for 24 hours. In vitro cytotoxicity studies indicate increased cell death across multiple AML cell lines when γδ T cells were co-cultured with cells pretreated with azacitidine and venetoclax. Conclusions: Azacitidine and venetoclax treatment resulted in upregulation of several markers involved in γδ Τ cell-mediated cytotoxicity. Ex vivo expanded γδ Τ cells combined with chemotherapy-treated AML cells resulted in increased overall cytotoxicity, which is likely both additive and synergistic. Our next steps include preclinical validation of this combinatorial approach in an in-vivo mouse study.

Decitabine/Cedazuridine (ASTX727) Combined with a Molecularly-Targeted Agent (Venetoclax, Gilteritinib, Ivosidenib, or Enasidenib) As Personalized Maintenance Therapy in Acute Myeloid Leukemia: First Results from a Phase 1b Study

Background: Disease relapse is the major cause of treatment failure and death in acute myeloid leukemia (AML). Currently, oral azacitidine (CC-486) is the only drug approved in the maintenance setting in patients with AML who are unable to complete standard therapy. The ECOG ACRIN E2906 trial suggested an overall survival (OS) benefit with 3-day IV decitabine maintenance. Decitabine/cedazuridine (ASTX727) is an oral formulation of decitabine that achieves systemic exposures as seen with IV decitabine. Multiple small-molecule agents are now available targeting key leukemic drivers (BCL-2, FLT3, and IDH1/IDH2 inhibitors). We developed a multi-arm phase 1b study (NCT05010772) to evaluate an oral maintenance regimen composed of an ASTX727 backbone combined with a molecularly-targeted agent (venetoclax, gilteritinib, ivosidenib, or enasidenib). Methods: Patients ≥ 18 years with AML in first complete remission (CR) or CR with incomplete blood count recovery (CRi) not currently eligible for stem cell transplantation (SCT) and unable/unwilling to complete standard therapy were eligible. Patients having received either intensive induction (at least 2 cycles of therapy based on intermediate to high dose cytarabine) or low-intensity induction (at least 3 cycles based on low-dose cytarabine or a hypomethylating agent) were permitted. Other inclusion criteria were ECOG ≤ 3, adequate hepatic/renal function, absolute neutrophil count > 0.5 x 10 9/L, and platelets > 50 x 10 9/L. Patients were enrolled in one of five arms per physician choice based on molecular profile. Arm A consisted of ASTX727 35/100 mg PO on D1-3. All other arms combined this ASTX727 backbone with either venetoclax 400 mg (adjusted for azoles) PO on D1-5 (arm B), gilteritinib 120 mg PO on D1-28 (arm C), enasidenib 100 mg PO on D1-28 (arm D), or ivosidenib 500 mg PO on D1-28 (arm E). Treatment was administered in 4-week cycles, up to 24 cycles. Each arm initially consisted of a safety lead-in cohort with predetermined dose reductions for dose-limiting toxicity (DLT) per a 3+3 dose de-escalation design. Prophylactic antibiotics, antifungals, and antivirals were encouraged. The primary objective was safety and tolerability. Secondary objectives included relapse-free survival (RFS) and OS. Patients becoming eligible for SCT were taken off study and censored at the time of SCT for time-to-event endpoints. Results: 23 patients, with a median age of 68 years (range 33-83), have been enrolled (3 on arm A, 19 on arm B, and 1 on arm C). 12 (52%) were enrolled following intensive induction and 11 (48%) after low-intensity induction. 16 (70%) had prior venetoclax. Best response at enrollment was CR in 22 (96%) patients and CRi in 1 (4%). 6/22 (27%) patients with available testing were measurable residual disease (MRD)-positive. 2 (9%) patients had antecedent myelodysplastic/myeloproliferative neoplasms and 1 (4%) had therapy-related AML. ELN 2022 risk was favorable in 10 (43%), intermediate in 3 (13%), and adverse in 10 (43%) patients. The most common grade 3/4 adverse events were neutropenia (96%), thrombocytopenia (74%), anemia (39%), lung infection (22%), hypertension (17%), and neutropenic fever (17%). Cycle 2 dose reductions due to myelosuppression occurred in 5/23 (22%) patients. No DLTs were observed. No deaths occurred on protocol. Two patients died in remission from SCT-related complications after going off maintenance. The current median follow-up time is 5.3 months (m). The median number of cycles given is 3 (1-15). Three relapses have occurred to date (1 in arm A, 2 in arm B). The median RFS for the full cohort is not reached (NR, 1-year 71%). The median OS for the full cohort (figure 1A) is NR (1-year 100%). The median RFS for arms A, B, and C are NR (1-year 67%), NR (1-year 84%), and NR, respectively (figure 1B). The median RFS is 14.5 m (1-year 50%) for patients who received intensive induction and NR (1-year 75%) for those who received low-intensity induction. The median RFS for patients who were MRD-negative and MRD-positive at enrollment were NR (6-month 93%) and NR (6-month 75%), respectively (p=0.50). Of the 6 MRD-positive patients, 2 (33%) cleared their MRD while on maintenance (both patients in arm B). Conclusions: A fully oral, targeted maintenance regimen is feasible in AML. Early results show encouraging RFS and OS. Further enrollment and follow-up are required to better assess the safety and efficacy of these regimens.

Impact of COVID-19 Pandemic on Overall Survival in Acute Myeloid Leukemia: A Comprehensive Cancer Center Experience of 627 Patients

Background: The COVID-19 pandemic impacted cancer care nationwide. Challenges with frequent clinic visits for lab draws, administration of multi-agent chemotherapy regimens, and scarcity of supportive blood products disrupted effective health care delivery. The magnitude of these factors on the survival of patients diagnosed with acute myeloid leukemia (AML) during the pandemic is incompletely described. Methods: We included 627 patients aged ≥18 years with AML treated at VCU Massey Comprehensive Cancer Center from January 1, 2013 to December 31, 2022; we recorded baseline demographics, molecular profiles, induction regimens, and survival data. We defined the pandemic era as March 7, 2020 (coinciding with the first confirmed case in our region) to July 1, 2021 (routine and widespread vaccination available). Survival analyses were performed using the Kaplan-Meier method and compared using the log-rank test. ECOG and Charlson comorbidity index (CCI) scores were compared using the Mann-Whitney test; we used the D'Agostino & Pearson method for normality testing. Results: The median age of patients who received intensive chemotherapy (7+3, CPX-351, FLAG-IDA, MEC, or HAM) in the COVID-19 pandemic cohort was 60 years (range, 28.5-69.6) vs 58 years (range, 19.6-81.4) for the non-COVID-19 cohort (p = 0.932). The median ECOG performance status in both cohorts was 1 (range, 0-4, p = 0.658). The median Charlson comorbidity index (CCI) score for patients for both groups was 4 (range, 0-9 vs 2-9, p = 0.909). Patients who were initiated on lower-intensity therapies (e.g., a hypomethylating agent with or without venetoclax) during the COVID-19 pandemic had a median age of 75 (range, 61.9-85.1) years vs 73 (range, 26.1-92.0) years in the non-COVID-19 cohort (p=0.438). The median ECOG performance status for the COVID-19 cohort was 2 (range, 0-3) vs 1 (range, 0-4) for the non-COVID-19 cohort (p=0.005). CCI score for both groups was 6 (range, 4-12 vs 2-14, p=0.789). We then compared the mOS between the COVID-19 and non-COVID-19 eras for both intensive and non-intensive treatment cohorts. For patients treated with intensive chemotherapy during the pandemic, the mOS was 25.1 months in the COVID-19 cohort vs 18.0 months (p = 0.173) in the non-COVID-19 cohort. For patients treated with non-intensive regimens, mOS was 6.6 months in the COVID-19 cohort vs 6.0 months in the non-COVID-19 cohort (p = 0.963). Patients with relapsed disease who were retreated with intensive chemotherapy during the COVID-19 pandemic had a mOS of 6.5 months compared to 8.2 months (p = 0.094) in the non-COVID-19 cohort. Relapsed patients in the lower-intensity COVID-19 cohort had a significantly shorter mOS of 1.8 months compared to 5.2 months in the non-COVID-19 cohort (p = 0.005). Regardless of therapy, the mOS was shorter in the COVID-19 cohort at 11.0 months compared to 13.4 months in the non-COVID-19 cohort (p = 0.191). Relapsed or refractory patients in the COVID-19 cohort had significantly shorter mOS at 5.8 months compared to 8.0 months in the non-COVID-19 cohort (p = 0.019). Discussion: The median overall survival appears shorter for patients diagnosed with AML during the COVID-19 pandemic. Patients with relapsed or refractory AML appeared to have significantly worse overall survival during the COVID-19 pandemic, particularly with lower-intensity therapies. Patients treated with less-intensive regimens are generally older with increased frailty, raising concern for lack of physiologic reserve. These results convey a lesson in preparation for future healthcare crises.

CUX1mut Acute Myeloid Leukemia As a Distinct Biological Entity: An Analysis of Clinical Outcomes and Implications

1. Introduction CUX1 is a tumor suppressor gene located on the long arm of chromosome 7. CUX1 mutations induce apoptosis evasion and are more commonly seen in therapy-related myeloid neoplasms. It is frequently mutated in myelodysplastic syndrome and less frequently in acute myeloid leukemia (AML). Preclinical investigations are ongoing to explore therapeutic approaches in CUX1-deficient leukemia cell lines. However, data on the clinical outcomes of patients with AML and mutated CUX1 are sparse. We aimed to characterize the biological and clinical outcomes of CUX1 mut AML. 2. Methods We analyzed next-generation sequencing from 318 patients in the Project ERIS database with newly diagnosed AML treated from January 1, 2013 to April 18, 2023 at VCU Massey Comprehensive Cancer Center. We identified 12 patients with CUX1 mut AML and compared them to a cohort of 306 CUX1 wild-type historical controls. We recorded baseline patient-related and disease characteristics, including age, ECOG, Charlson comorbidity index (CCI) scores, molecular profiling and ELN 2022 cytogenetic risk, dates of regimen initiation, and survival. We used the D'Agostino & Pearson method for normality testing and the t-test or Mann-Whitney (as applicable) tests for between-group comparisons. Categorical comparisons used Fischer's exact test. We applied the Bonferroni correction if multiple comparisons were made. The Wilson-Brown method was used for 95% confidence intervals. We analyzed survival by the Kaplan-Meier method with significance determined by the log-rank test. The event for calculating the overall survival (OS) was the date of death. Patients were otherwise censored at the date of last contact. 3. Results CUX1 mutations are found at a frequency of 3.9% (95% CI, 2.2-6.5) in AML. The median variant allele frequency of CUX1 mutations was 40.3% (range: 3.8-48.7), consistent with a heterozygous presentation. The median age at diagnosis of patients with CUX1 mut who received any anti-leukemic therapy was 57 years (range: 30-78 years), which was not significantly different from CUX1 wt at 65 years (range: 19-88, p = 0.312). Thirty-three (95% CI, 13.8-60.9) percent of patients had AML with myelodysplasia-related changes (AML-MRC) on the initial diagnostic bone marrow biopsy, compared with 29.2% (95% CI, 24.2-34.7, p=0.752) for CUX1 wt. CUX1 mut AML was found in cytogenetically normal specimens at a frequency of 30.0% compared with 49.3% for CUX1 wt (p = 0.337). The most frequent cytogenetic abnormality in CUX1 mut AML was -7 (20.0%), followed by +8 (10.0%) and complex cytogenetics (10.0%). The most frequent cooperating molecular lesions were RUNX1, SRSF2, STAG2, FLT3-ITD, and NRAS (25.0% each). Patients with CUX1 mutations were nonsignificantly more likely to have ELN 2022 adverse-risk disease (odds ratio: 2.013, p = 0.379). In patients treated with intensive cytarabine-based inductions, the median overall survival was 26.3 months in the CUX1 mut group and 17.6 months in the CUX1 wt cohort, which was not significantly different (p = 0.501). Two patients with CUX1 mut AML were treated with first-line venetoclax and a hypomethylating agent. The overall survival was 12.7 months in one patient and not reached at a follow-up time of 20.2 months in the second. 4. Discussion While definitive conclusions are limited by the rarity of the mutation, mutations in CUX1 are heterozygous and appear to be non-significantly more frequently associated with myelodysplastic-related changes and secondary-type mutations. These findings may explain a higher proportion of patients harboring CUX1 mutations that have ELN 2022 adverse-risk disease. While we did not detect any significant survival differences between patients with and without CUX1 mutations, larger cohorts are needed to confirm the prognostic impact of these rare mutations in AML.

Real-World Treatment Patterns and Overall Survival Among Newly Diagnosed and Relapsed/Refractory Acute Myeloid Leukemia (AML) Patients: A Retrospective Cohort Study Using Claims Data

Introduction: Acute myeloid leukemia (AML) is a malignant disorder of the bone marrow, characterized by abnormal clonal proliferation and differentiation arrest of myeloid progenitor cells. Prognosis is generally poor and worsens with age. While curative therapies are available, they are often unavailable to older patients and those with significant comorbidities due to high morbidity and mortality associated with intensive therapy. Since 2017, seven new systemic therapies have been approved for various AML patient populations, including those unfit for intensive therapy. This study aims to characterize first-line (1L) and second-line (2L) treatment patterns and outcomes in newly diagnosed (ND) and relapsed/refractory (R/R) AML patients, in the context of these newly available therapies. Methods: A large US administrative claims database (Optum) was used to identify ND AML patients between January 1, 2016, and August 31, 2022. The “ND index date” was defined as the first date a non-R/R AML diagnosis code was observed. To confirm that the initial AML diagnosis represented a true case of AML, ≥2 subsequent AML diagnoses within 60 days post-index were required. A washout period of 12 months was used to confirm no previous AML diagnosis. ND patients were required to have continuous enrollment from 1 year prior to the index until ≥30 days after the index date or death, whichever was earlier. A subgroup of R/R patients was then identified: all ND cohort members who 1) had an R/R AML diagnosis code, 2) had continuous enrollment between ND index date and ≥30 days after the RR index date, and 3) did not receive 1L hematopoietic stem cell transplant (HSCT) after the ND index date were eligible for inclusion. In this subgroup, the “RR index date” was defined as the first date an R/R AML diagnosis code was observed. Demographic characteristics, clinical characteristics, and treatment patterns over time were reported descriptively. Treatment regimens over time were described using the following classification: HSCT, intensive chemotherapy without targeted agents, intensive chemotherapy with targeted agents, hypomethylating agent (HMA) therapy only, other targeted therapy, Venetoclax-based therapy, unspecified/other treatment, no treatment/supportive care only. Overall survival (OS) was described using the Kaplan-Meier estimator. All analyses were stratified by line of therapy and transplant status. Results: We identified 5,135 ND AML patients and 934 R/R AML patients (ND cohort: mean age at initial AML diagnosis 73.5 years, mean follow-up time 6.1 months). ND patients receiving transplant in 1L tended to be younger and had fewer comorbidities than those not receiving transplant ( Table 1). Over the entire study period, 61% of ND patients and 69% of R/R patients received some treatment for AML; older patients were more likely to receive supportive care only. In 1L, Venetoclax-based and intensive chemotherapy without targeted agents were the most common treatment regimens (21% and 17% of all ND patients). In 2L, Venetoclax-based and HMA-only therapies were most common (17% and 16% of all R/R patients). Increases in the use of novel therapies (Venetoclax, intensive chemotherapy with targeted agents, or other targeted therapies) were observed in both 1L and 2L over the study period ( Figure 1). 1-year OS among ND patients was 32% in non-transplant recipients, and 84% in transplant recipients; among R/R patients, 1-year OS from RR index date was 21% in non-transplant recipients and 81% in transplant recipients. Conclusions: The findings from the present study indicate substantial disease burden and ongoing unmet need among ND and R/R AML patients in the USA. While treatment patterns for AML have changed significantly following the advent of novel therapies (with 41% of 1L patients and 31% of 2L patients being treated with novel therapies in 2022), OS remained low. Notably, around 1/3 of patients with RR AML did not receive active treatment; these patients tended to be older and were more frail. Increasing off-label use of Venetoclax in 2L further underscores high unmet need for patients who require more effective treatment options.

Optimizing FLT3 Inhibitor Use in Adult Acute Myeloid Leukemia with FLT3 Mutations Using Proteomics

CONCLUSION FLT3 mutations are the most frequent genetic alteration in adult Acute Myeloid Leukemia (AML). The use of FLT3 inhibitors (FLT3I) has improved outcome among FLT3 mutant patients, but not all benefit. We developed a proteomic-based approach to stratify patients according to their response to FLT3I therapy. The expression of 429 proteins (339 total, 90 post-translational modified) was measured by Reverse Phase Protein Arrays (RPPA) in fresh samples from 805 newly diagnosed AML patients, with levels normalized to normal CD34+ cells. Of 137 with FLT3 ITD or D835 mutations, 54 who were treated with FLT3I (Sorafenib=22, Quizartinib=11, Midostaurine=6, Gilteritinib=4 and Crenolanib=1) combined with other agents (Hypomethylating agents (HMA)=23, HMA+Venetoclax (VTX)=5, Idarrubicin+Cytarabine (AraC)+Purine Analogs (IA+Pur)=19, IA+Pur+VTX=2, AraC=3, Fludarabine+AraC (FA)=1, none=1) were selected for analyses. Proteins prognostic for Overall Survival (OS) and Complete Remission Duration (CRD) were identified using pairwise LogRank tests (p < 0.05) adjusted for False Discovery Rate, followed by unbiased hierarchical clustering. Continuous and categorial variables were compared with Wilcoxon, Kruskal-Wallis or Fisher's Exact test with simulated p-values (10000 replicates). Cox proportional hazards models (CoxPH) were developed for Uni-(UV) and Multi-variate (MV) analysis. Deep neural network analysis using random forest models identified proteins most predictive of cluster assignment. 28 proteins were individually prognostic for both OS and CRD and divided patients into 3 clusters (Fig. 1: C1=red, C2=blue, C3=green). The distribution of nearly all other clinical and molecular features did not differ between clusters, including: age, gender, race, cytogenetic risk and AML group. Co-occurrence with other mutations was not biased, except for FLT3+TET2 and FLT3+TET2+NPM1 mutants, more frequent in C1. OS was significantly longer (Fig. 2), in C1 (Median: C1>84mo, C2=14.8mo, C3=11.4mo; p<0.001) as was CRD (Median: C1>84mo, C2=8.6mo, C3=5.7mo; p<0.05). A similar OS pattern was observed for mutation combos, including with NPM1 or DNMT3a but not if IDH, CEBPa, TET2, or ASXL1 mutation were present. It was also seen when patients were divided by gender, age groups (except >70yo), primary, but not secondary AML, diploid karyotype and intermediary cytogenetic risk, and with Sorafenib or IA+PR therapies. In the UV CoxPH model for OS, C1, C2 and C3 were predictive (HR=1, 4.1, 7.5; p=ref., 0.037, 0.009), along with age, secondary AML, complex karyotype, Trisomy 6 and IDH1 mutation. In the MV model for OS, only C1, C2 and C3 (HR=1, 3.3, 4.8; p=ref., 0.011, <0.001), and age were predictive. In the CRD UV model, C1 and C3 (HR=1, 3.3, 4.8; p=ref., 0.072, 0.022), age, secondary AML, and mutations in ASLX1, PTPN11, RAS, TP53 and WT1 were predictive. Only C1 and C3 were predictive in the MV model for CRD (HR=1, 2.9, 6.3; p=ref., 0.16, 0.04). The predictive proteins, are biased towards specific cellular processes, mainly: proliferation and metabolism (RHEB, GSKA_B, SDHA, AKT1, AKT2 and TYRO3), cell cycle and DNA damage response (CDKN2A, DDB1, SIRT6, SLFN11 and WEE1), cellular adhesion and cytoskeleton organization (MYH9.pS1943, PAK4 and PXN), and apoptosis (AIFM1, BBC3 and BCL2L11). Key differences between clusters included: higher FOS in C1, and higher CDKN2A, MYH9.pS1943, PXN, PAK4, SDHA, BBC3 in both C2 and C3, with higher levels of these in C3. Since PAK4, PXN and MYH9 are connected to cellular motility/migration and inhibitors for those proteins are available, there is a therapeutic opportunity to overcome FLT3I resistance in C2/C3 patients. Moreover, we identified a set of 6 proteins (FOS, AKT1, PXN, SMARCA2, CDKN2A and DPF2) that predicted cluster assignment with 97.5% accuracy, making rapid patient stratification possible in the clinical setting. Protein profiling identified cohorts of FLT3 mutant patients that achieved the highest benefit from FLT3I addition, regardless of all other clinical or molecular characteristics. We identified targetable proteins that are elevated in adverse signatures, and hypothesized that these may be targets for modulation to improve FLT3I response. Moreover, a small set of 6 proteins accurately classifies patients and could be used clinically to triage patients for treatment recommendation.

Comparison of Response and Survival with Chemotherapy and Targeted Therapies between IDH mut Isoforms in Acute Myeloid Leukemia

1. Introduction Isocitrate dehydrogenase (IDH) mutations are among the most frequent genetic alterations in acute myeloid leukemia (AML) - they are detected in approximately 20% of patients. While ivosidenib and enasidenib have been approved as targeted therapy for IDH1 and IDH2-mutated AML, respectively, relative outcomes between the IDHmut isoforms remain unclear across various therapeutic options. The aim of this study was to compare response and survival between IDH-mutated cohorts of AML when treated with intensive chemotherapy, venetoclax-based strategies, or targeted therapies. 2. Methods We analyzed 93 patients with newly diagnosed or relapsed or refractory IDHmut AML from January 1, 2013 to April 18, 2023 at VCU Massey Comprehensive Cancer Center. We recorded baseline patient-related and disease characteristics, including dates of regimen initiation and survival. Categorical comparisons used Fischer's exact test. We excluded those with cooperating IDH1 and IDH2 mutations. We analyzed survival by the Kaplan-Meier method with significance determined by the log-rank test. The event for calculating the overall survival (OS) was the date of death. Patients were otherwise censored at the date of last contact. 3. Results We analyzed 43 patients with IDH1mut or IDH2mut AML treated with first-line intensive chemotherapy with conventional 7+3, CPX-351, or FLAG-IDA with or without venetoclax; we divided the patients into two cohorts: IDH1 and IDH2. We noted no significant differences in the presence of cooperating NPM1 mutations (25.0% vs 37.0%, p = 0.529) or in the proportion of ELN 2022 adverse-risk disease (45.0% vs 40.7%, p > 0.999). The composite complete remission (CRc; CR + CRi + CRh) rate for IDH1mut AML was 66.7% (95% CI, 43.7-83.7) compared to 60.0% (95% CI, 40.7-76.6, p = 0.755) for IDH2mut. The rate of MRD negativity was 50.0% (95% CI, 18.8-81.2) for IDH1mut and 30.0% for IDH2mut (95% CI, 10.8-60.3, p = 0.607). Strikingly, the median overall survival significantly favored the IDH2mut cohort at 25.5 months vs 11.3 months for IDH1mut (p = 0.047, Figure A). Next, we analyzed the outcomes of 19 patients with IDHmut AML treated with first-line venetoclax and a hypomethylating agent (VEN+HMA). We noted a lower proportion of cooperating NPM1 mutations in the IDH2mut cohort compared with the IDH1mut cohort (36.4% vs 75.0%, p = 0.170). Despite this, the CRc rate favored the IDH2mut cohort compared with the IDH1mut cohort (62.5%; 95% CI, 40.7-76.6, vs 20.0%; 95% CI, 30.6-86.3, p = 0.266). We noted no significant difference in overall survival between the IDH1mut or IDH2mut cohorts treated with HMA+VEN (12.2 months vs 14.8 months, p = 0.834). In the relapsed or refractory setting, we analyzed 17 patients that received either ivosidenib or enasidenib with or without azacitidine. The CRc for the ivosidenib cohort was 33.3% (95% CI, 5.9-70.0), compared with 28.6% (95% CI, 5.1-64.1, p > 0.999) for the enasidenib cohort. The median overall survival favored the ivosidenib cohort at 18.8 months compared to 2.7 months for the enasidenib cohort, which approached statistical significance (p = 0.069). 4. Discussion The median overall survival of IDH1mut AML is significantly worse compared to IDH2mut AML when treated with intensive chemotherapy. The significance of the survival disparity disappears with patients treated with HMA+VEN, suggesting both isoforms of IDHmut AML benefit from venetoclax. Investigators should be cautious when combining IDHmut AML cohorts while reporting the results of clinical trials in molecularly selected patients.

Trial in Progress: An Open Label Phase I/II, Multicenter Study Evaluating Safety, Pharmacokinetics and Efficacy of S65487, a BCL-2 Inhibitor Combined with Azacitidine in Adults with Previously Untreated Acute Myeloid Leukemia Ineligible for Intensive Treatment

Preclinical and clinical data support combining Bcl-2 inhibitors (Bcl-2i) with hypomethylating agents (HMAs) in the treatment of acute myeloid leukemia (AML). S65487, a potent intravenous Bcl-2i, has shown promising synergistic activity when combined with azacitidine against in vitro AML models. Based on those data, we initiated a Phase I/II multicenter study of S65487 combined with azacitidine for newly diagnosed patients aged 18 years or older with de novo or secondary AML ineligible for intensive chemotherapy in 6 countries in Europe and Asia (NCT04742101). Secondary AML was defined as those related to previous leukemogenic therapies or transformed from a myelodysplastic syndrome. Patients with AML originating from myeloproliferative disorders were excluded. Ineligibility to intensive chemotherapy was defined as age of at least 75 years or the presence of significant co-morbidities. Previous use of HMA was allowed in the first six dose levels tested in the Phase I portion of the study, but was excluded afterwards, including during the Phase II part. Other eligibility criteria were standard for this patient population. Each cycle of therapy lasts for 28 days. Subcutaneous or intravenous azacitidine is administered at a dose of 75mg/m 2 in 7 consecutive administrations per cycle starting on day (D) 1 with a 2-day interval during weekends. Three schedules of S65487 are planned to be tested during the Phase I part to increase its concurrent administration with azacitidine and their synergism. Schedule 1 consists of weekly administrations (doses on D1, 8, 15, and 22). Two more intensified schedules consist of S65487 administration on D1, D3, D5, D8 (schedule 2) or D1 through D5, D8, and D9 (schedule 3) on the same days of azacitidine dosing. S65487 is administered over 30 minutes. When administered on the same day, S65487 is given first followed by azacitidine within 30 to 60 minutes. Treatment should be discontinued due to disease progression, or if other standard withdrawal criteria are met. The Phase I portion of the study follows a standard design guided by an adaptive Bayesian Logistic Regression Model with overdose control to estimate the maximum tolerated dose (MTD) based on the occurrence of dose-limiting toxicities during cycle 1. Patient accrual for the Phase I portion of this study is ongoing. The planned testing of schedule 1 is limited due to the expected low synergism with azacitidine. Once the recommended Phase II dose (RP2D) or MTD are established for schedules 2 and 3, the Phase II part will start and will be comprised of a maximum of two cohorts, each containing up to 27 patients. A Bayesian optimal design based on complete remission rate will guide the Phase II. Interim futility analyses are planned after enrollment of 10 and 20 evaluable patients. Randomization will not be used to allocate patients to one of the two cohorts planned during the Phase II portion of the study. The primary objective during the Phase I is to determine the safety, tolerability, and the RP2D or MTD of S65487 in combination with azacitidine. The primary objective of the Phase II is to assess the efficacy of this therapy. Pharmacokinetic analyses are included as secondary objectives in both parts of the study. Measurable residual disease will be assessed as a secondary objective in the Phase II part using a well validated, standard flow-cytometry panel. Pharmacodynamic analyses are conducted as exploratory objectives in both parts of the study. In particular, the disruption of the BCL2/BIM complex is followed in peripheral-blood mononuclear cells to guide the estimation of the RP2D.

Outcomes in Patients with Acute Myeloid Leukemia Treated with CLAG (cladribine, cytarabine, filgrastim) and/or Mitoxantrone with Venetoclax

Introduction: CLAG (cladribine, cytarabine, filgrastim) with or without M (mitoxantrone) has anti-leukemic activity in newly diagnosed (ND) and relapsed/refractory (R/R) AML (Wierzbowska et al. 2008, Jaglal et al. 2014, Seiter et al. 2016). Venetoclax (Ven) with hypomethylating agents (HMA), approved for patients (pts) who are not candidates for intensive chemotherapy (IC), is increasingly being employed even for pts who are fit for IC. R/R disease remains a major cause of mortality in fit pts with AML, and thus improved IC regimen is an area of active research. Various combination of IC with Ven are ongoing with promising early findings (DiNardo et al. 2021, Reville et al. 2022). In this study, we examined the outcomes of AML patients treated with CLAG and/or mitoxantrone with Ven, CLAG(M)/Ven. Methods: This was a retrospective study of pts with AML who received CLAG(M)/Ven (Days 2-8) in 2022-2023. Clinical responses were reported using ELN 2022 criteria. Kaplan-Meier analyses were used to estimate overall survival (OS) from the date of CLAG(M)/Ven initiation. Results: This cohort of 19 pts (8M/11F with median age of 60 [23-74] at diagnosis) is comprised of 68.4% (13/19) R/R and 31.6% (6/19) ND AML who had prior MDS directed therapy. There were 57.9% (11/19) with secondary/therapy-related AML and when including de novo AML pts who had mutations highly specific for secondary AML ontogeny, it increased to 78.9% (15/19). Most of the pts (68.4%, 13/19) had adverse risk disease by ELN 2022. Common mutations being TP53 (26.3%, 5/19) followed by IDH1/2 (21.1%), ASXL1 (15.8%), and RUNX1 (15.8%). There were 36.8% (7/19) who had received allogeneic hematopoietic cell transplant (alloHCT) (Table 1). Similar number of pts received CLAG/Ven (47.5%) and CLAG-M/Ven (52.6%). Pts received on average 2.6 lines of treatment prior to CLAG(M)/Ven with 1 pt received 5 prior lines of treatment after AML diagnosis. Most of the pts, 78.9% (15/19), had prior HMA/Ven. The 30-day and 60-day morality was 5.3% (1/19) (disease status unknown at the time of death) and 26.3% (5/19) (disease status: 3 yes, 1 no, 1 unknown), respectively. Among these 5 early deaths within 60 days, 3 were ND AML that were heavily pretreated for prior MDS (average 3 lines of prior therapy) and 2 were R/R AML. All of them had prior HMA/Ven. Eighty percent (4/5) had adverse risk disease and received CLAG-M/Ven. Forty percent (2/5) had TP53 mutation. Objective response rate (ORR) of the entire cohort was 26.3% (5/19) (2 CR, 1 CRi, and 2 MLFS). Among those with evaluable response (n=10), ORR was 50%. Two CR/CRi pts achieved undetectable MRD by flow cytometry. MRD was not tested in the other 3 pts. Notably, ORR was higher in pts who received CLAG/Ven (44.4%, 4/9) compared to CLAG-M/Ven (10%, 1/10). No response in ND AML and 38.5% (5/13) ORR in R/R AML. Of the 47.4% (9/19) nonevaluable pts, 6 had aplastic marrow (2 died while getting induction, 2 transitioned to alloHCT, 1 moved onto next line of treatment, and 1 lost to follow up) and 3 had recovery marrow pending. For pts who achieved a response, 60% (3/5) underwent alloHCT (all of them alive without disease at data cutoff). The median time to count recovery was 42.5 days (27 days to never recovered). Prior HMA/Ven failure, high dose cytarabine failure, and mutations did not significantly impact response in this small cohort. The median OS from the time of CLAG(M)/Ven initiation was 5.5 months with a median follow up of 8.6 months (Figure 1). OS from the date of CLAG(M)/Ven was significantly shorter in pts with TP53 mutation vs. wild-type (2 vs. 11.2 months, p=0.031). Conclusions: Our findings showed the combination regimen CLAG(M)/Ven is feasible in pts with AML. It also provided better characterization in terms of early mortality and survivals in these pts, which can help inform future clinical trial design. ORR was similar to what was previously reported with CLAG(M); however, it is encouraging that those who achieved CR/CRi and had MRD testing also achieved MRD negativity. Outcomes of TP53-mutated subset is dismal suggesting IC-based approach in this setting is limited. Larger cohort and longer follow up is needed to confirm the impact of factors such as specific regimen, mutations, and prior therapies on outcomes.