BM Blasts Research Articles

Application of serum tumor specific protein 70 for prognostic stratification in acute myeloid leukemia

Objective: To assess the value of serum tumor specific protein 70 (SP70) for prognostic stratification in acute myeloid leukemia (AML). Methods: A cohort study design was adopted. 129 newly diagnosed AML patients from September 2022 to January 2024 at the Hematology Department of the First Affiliated Hospital of Nanjing Medical University were included, as well as a control group consisted of 120 healthy individuals and 7 cases with benign hematologic diseases during the same period (total 127 cases). Clinical data were collected from Electronic Medical Records. According to the 2023 edition of the Chinese Leukemia Diagnosis and Treatment Guidelines, AML patients with good or moderate prognosis were categorized as low-to-intermediate risk, while those with poor prognosis were high-risk group. Univariate and multivariate logistic regression analyses were used to identify variables significantly associated with AML prognostic risk. ROC analysis was used to evaluate diagnostic performance. A nomogram for predicting patient prognostic risk was constructed by R 4.0.2 software, and the internal validation was performed using bootstrapping. Results: Among 129 AML patients, there were 71 males (55.0%) and 58 females (45.0%), with 42 (32.6%) classified as high-risk and 87 (67.4%) as low-intermediate risk. The high-risk group had a significantly higher median age [62 (48, 67) years] compared to the low-intermediate risk group [50 (35, 63) years, Z=-2.381, P=0.017], and a significantly higher proportion of males (30 patients, 71.4%) compared to the low-intermediate risk group (41 patients, 47.1%, χ2=6.760, P=0.009). Multivariate logistic regression analysis indicated that serum SP70 (OR=2.54, 95%CI: 1.68-3.84, P<0.001), hemoglobin (HB) (OR=0.96, 95%CI: 0.93-0.99, P<0.05), and bone marrow blast (BM blast) (OR=1.07, 95%CI: 1.02-1.13, P<0.05) were independent risk factors for high-risk prognosis in AML patients. ROC analysis showed that the area under the curve (AUC) for SP70 predicting high-risk patients was 0.908 (cut-off value of 5.74 ng/ml, 95%CI: 0.845-0.952, sensitivity 90.5%, specificity 82.8%). The combined model of serum SP70, HB, and BM blasts had an AUC of 0.931 (95%CI: 0.890-0.973); C-index=0.925 (95%CI: 0.876-0.963),with no statistically significant difference compared to serum SP70 alone (Z=1.693,P>0.05). Conclusion: Serum SP70 may be a promising non-invasive molecular biomarker for prognostic stratification in AML.

Low Levels of Natural Killer Cell in Newly Diagnosed Myelodysplastic Syndromes Patients May Confer Poor Prognosis: A Retrospective Cohort Study.

Immune imbalance appears to have a critical role in tumor growth according to emerging research. Peripherallymphocyte subsets are considered to reflect the systemic immune response and clinical prognosis. The prognostic value of lymphocyte subpopulations in myelodysplastic syndrome (MDS) patients remains unclear. A total of 94 MDS patients were enrolled for the study. X-tile software was performed to determine the prognostic significance of various lymphocyte subpopulations, CD3, CD4, CD8, CD4/CD8 ratio, natural killer cell (NK) and CD19. Among them, the appropriate threshold of NK percent could be found only. Patients were divided into the high NK percent group and the low NK percent group. The prognostic significance was determined by univariate and multivariate Cox hazard models. MDS patients with lower NK level had significantly shorter overall survival (OS). Based on univariate analysis, male gender (P = 0.030), lower HB (<10 g/dl, P = 0.029), higher BM blast (>5%, P < 0.0001), higher-risk IPSS-R cytogenetic (P = 0.032) and lower NK percent (P < 0.0001) were significantly associated with shorter OS. Multivariate Cox proportional hazards regression analysis indicated that low NK was also independent adverse prognostic factor for OS in MDS. Decreased NK level predicts poor prognosis independent of the IPSS-R and provide a novel evaluation factor for MDS patients.

Childhood myelodysplastic syndromes: Is cytoreductive therapy useful before allogeneic hematopoietic stem cell transplantation?

For most patients with childhood myelodysplastic syndrome (cMDS), allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative option. In the case of increased blasts (cMDS-IB), the benefit of pretransplant cytoreductive therapy remains controversial. In this multicenter retrospective study, the outcomes of all French children who underwent allo-HSCT for cMDS reported in the SFGM-TC registrybetween 2000 and 2020 were analyzed (n = 84). The median age at transplantation was 10.2 years. HSCT was performed from matched sibling donors (MSD) in 29% of the cases, matched unrelated donors (MUD) in 44%, haploidentical in 6%, and cord blood in 21%. Myeloablative conditioning was used in 91% of cases. Forty-eight percent of patients presented with cMDS-IB at diagnosis (median BM blasts: 8%). Among them, 50% received pretransplant cytoreductive therapy. Five-year overall survival (OS), cumulative incidence of nonrelapse mortality (NRM), and relapse were 67%, 26%, and 12%, respectively. Six-month cumulative incidence of grade II-IV acute graft-versus-host disease was 46%. Considering the whole cohort, age under 12, busulfan/cyclophosphamide/melphalan conditioning or MUD were associated with poorer 5-year OS. In the cMDS-IB subgroup, pretransplant cytoreductive therapy was associated with a better OS in univariate analysis. This seems to be mainly due to a decreased NRMsince no impact on the incidence of relapse was observed. Overall, those data may argue in favor of cytoreduction for cMDS-IB. They need to be confirmed on a larger scale and prospectively.

Validation of the Composite Complete Response (cCR) Definitions in the International Working Group (IWG) 2023 Criteria in Patients (Pts) with Higher-Risk Myelodysplastic Syndromes/Neoplasms (HR-MDS) Treated with Hypomethylating Agents (HMA) - a Large, Multicenter, Retrospective Analysis from the Validate Database

JPB and TK are co-first authors; RK and AMZ are co-senior authors Introduction: Efficacy of therapies used in HR-MDS pts has long been assessed using IWG 2006 MDS criteria, but important limitations in clinical utility and practical application of these criteria have become apparent. The revised IWG response criteria for HR-MDS were proposed in 2023 (Zeidan et al., Blood 2023) were proposed to address these gaps. IWG 2023 re-defined CR by lowering hemoglobin (Hb) threshold from 11 to 10 g/dL and bone marrow [BM] blasts from ≤5% to &amp;lt;5%. Further, CR with partial hematologic recovery (CRh; &amp;lt;5% BM blasts, no Hb threshold, platelets ≥50 × 10 9/L; neutrophils ≥0.5 × 10 9/L; peripheral blood [PB] blasts 0%), CR with limited count recovery (CR-L; &amp;lt;5% BM and 0% PB blasts + PB parameters for CR met in 1 [CRuni] or 2 lineages [CRbi]), and CR equivalent (CRequ; defined for pts with baseline BM of &amp;lt;5%.) were all proposed as provisional endpoints. Per IWG 2023 hierarchical response assessment, if a pt met criteria for both CR-L and CRh, they would be recorded as CR-L. We sought to validate the IWG 2023 definitions of cCR (CR+CR-L+CRh+CRequ) in a large, multi-center dataset of HR-MDS pts who received HMAs. Methods: The VALIDATE database includes HR-MDS pts treated with HMA-based therapies in frontline setting from 14 specialized centers. HR-MDS was defined as IPSS ≥1.5 or IPSS-R &amp;gt;3.5 (n=213 pts excluded). Pts were excluded (N=90) if age at diagnosis was &amp;lt;18 years, BM blasts ≥20% or unknown at HMA initiation, or if survival status, follow-up time, HMA type or initiation date were unknown. To preserve validity of response assessments, pts also were excluded (N=290) if no BM evaluation was performed or if first BM assessment was &amp;gt;180 days after HMA initiation, except in cases of overt disease progression. Best responses were assessed based on IWG 2006 and 2023 criteria. Kaplan-Meier analysis estimated OS from HMA initiation, and the log-rank test was used to compare subgroups. Cox multivariable regression models identified predictors of OS. This study was supported by an independent research grant from AbbVie. Results: Of 1,223 pts in VALIDATE database, 629 were met eligibility. Median age was 68 years, 27.7% and 27.4% were red blood cell and platelet transfusion-dependent, respectively, 38.1% had TP53 mutations, and 45.3% underwent allogeneic transplantation (allo-HCT). Most pts (71.6%) received HMA monotherapy (51.7% azacitidine, 19.9% decitabine), while 28.4% received HMA-based combinations. Median duration of therapy was 4 cycles (Range: 1-94). Median OS of different subgroups pts are shown in Figure 1. Median OS for pts with IWG 2023-defined cCR (CR+CR-L+CRh+CRequ; N = 230) was 26.5 months (mo; 95% CI: 20.7 - 32.2 mo) vs 14.3 mo (95% CI: 12.5 - 16.9 mo) for pts who did not achieve cCR (p=0.002). Median OS for pts who achieved IWG 2023-defined CR (N = 90) was 29.8 (95% CI: 23.1 - 41.8) vs. 26.4 mo (95% CI: 22.0 - 41.3) for IWG 2006-defined CR (p=0.71). The 124 pts (19.7%) who achieved CR-L had a median OS of 26.4 mo (95% CI: 17.6 - 36.2 mo); of those 67 pts achieved CRbi and had a median OS of 29.1 mo (95% CI: 20.7 - not reached), while 57 pts achieved CRuni and had a median OS of 18.7 mo (95% CI: 15.5 - 54.4 mo), with no significant difference in OS between CRbi and CRuni (Hazard ratio: 1.24; 95% CI: 0.78-1.98, p=0.71). Given the hierarchical prioritization of CR-L designation over CRh in IWG 2023 if a pt meets both response definitions, only 6 pts achieved CRh which precluded a reliable estimate of their OS. Similarly, CRequ was achieved only in 10 pts limiting a reliable assessment of OS in these pts. In a multivariable Cox model that adjusted for sex, HMA type, age, allo-HCT, complex karyotype, TP53 mutation, and IPSS-M risk group, achieving a cCR per IWG 2023 criteria was statistically significantly associated with improved OS was associated with improved OS compared to those who did not achieve cCR (HR: 1.64; 95% CI: 1.30 - 2.08; p&amp;lt;0.001, Figure 2). Discussion: In this real-world analysis of HR-MDS pts treated with HMA-based therapy, cCR according to IWG 2023 were associated improved OS. In particular, CR and CRL (including CRbi and CR uni) were associated with improved OS, supporting their inclusion in the overall response rate in clinical trials. The number of pts who achieved CRh and CRequ was too small to reliably assess their specific association with OS. Additional pts are being added to the database and detailed analyses will be presented in the meeting.

Treatment of Molecular Relapses in Pediatric AML Saves Toxicities Prior to Hematopoietic Stem Cell Transplantation (HSCT) - Results of AMoRe2017 Trial

Introduction The long-term survival for children with AML has increased to 70 % - but relapsed AML patients still have a poor prognosis (about 30 %). Recent data suggest that in AML residual leukemic cells below the 5 % limit required for a morphologic CR can adversely affect outcome and that increasing levels of Minimal Residual Disease (MRD) are almost always followed by clinical relapse. Early MRD detection provides an opportunity to implement pre-emptive therapies as a bridge-to-transplant approach, to potentially prevent hematological relapse and increase the probability of successful HSCT. A promising approach is the use of Azacitidine (Aza) for treatment of molecular relapse which we evaluated within the clinical trial “AMoRe2017”. Aza has been shown to reverse epigenetic changes in malignant cells, restoring the normal function of genes critical for differentiation and cell cycle control. The trials' primary objective was the evaluation of the effect of Aza treatment in AML subjects at molecular relapse after CR1 regarding molecular response prior to further treatment (reinduction/HSCT). Patients and Methods AMoRe2017 enrolled children with AML (N=20), &amp;gt;3 months &amp;lt; 21 yrs., of which n=1 dropped out due to laboratory measurement error. The median age was 10.9 yrs. (range 1.7-18.9 yrs.). N=10 (53 %) had a CBFB-AML, n=5 (27 %) KMT2A-rearrangement, n=1 (5 %) NUP98::NSD1, n=1 (5 %) FLT3-ITD, n=1(5 %) PICALM::MLLT10 and n=1 (5 %) NPM1. N=8 (42 %) were classified to subgroup M2, n=4 (21 %) to M5, n=3 (16 %) into M1 and n=4 (21 %) to M4/M4 Eo. At baseline, the white blood cell count averaged 3.8x10 9/l (range 0.1-6.6x10 9/l), without leukemic blasts in PB or BM. Each patient had at least one quantifiable genetic marker (rt-PCR), achieved CR with molecular remission, following initial therapy, and subsequently had a molecular relapse (defined as an increase of the MRD level by at least 1 log, less than 5 % BM myeloblasts and absence of proven extramedullary disease). Patients were treated for up to 6 cycles with 75 mg/m 2 i.v. Aza on days 1 to 7 in a 28-day cycle. Molecular response was categorized as molecular stabilization (&amp;lt; 1 log decrease or increase from baseline), molecular progression (≥ 1 log increase from baseline without hematological relapse), molecular improvement (≥ 1 log decrease from baseline), and hematological relapse (at least 5 % blasts in PB and/or BM and/or proven histological extramedullary relapse). Results During Aza treatment, 10 patients (53 %) had an open relapse, which occurred in n=9 after 1 st or 2 nd cycle. These patients were monitored for KMT2A::SEPTIN6 (n=1), KMT2A::MLLT3 (n=2), PICALM::MLLT10 (n=1), FLT3-ITD (n=1), NUP98::NSD1 (n=1), CBFB::MYH11 (n=1) and RUNX1::RUNX1T1 (n=3). 9 patients (47 %; RUNX1::RUNX1T1 n=5, CBFB::MYH11 n=1, NPM1 n=1, KMT2A::ELL n=2) showed a molecular response to Aza and proceeded to HSCT without receiving intensive re-induction treatment. 16 out of the 19 patients are alive (overall survival 84 %), 2 died from HSCT-associated side effects (VOD, infection), and 1 from disease progression. Considering genetics, 7 out of 11 patients with favorable genetics (core-binding leukemia n=10 or NPM1 n=1) responded to the Aza therapy ( RUNX1::RUNX1T1 n=5, CBFB::MYH11 n=1, NPM1 n=1). By contrast, in cases with KMT2A-rearrangements, NUP98::NSD1 or FLT3-ITD, 2 out of 8 patients showed improvement or stabilization. In total 152 adverse events (AEs) associated to Aza were reported, the majority classified as grade 1 or 2 (n= 114; 75 %). Mainly affected was the hematopoietic system (40 %), the gastrointestinal tract (15 %), kidney and liver (14 %). Cardiotoxicity was not reported. Discussion The use of Aza as pre-emptive “bridge-to-transplant” approach treating the molecular relapse showed efficacy in patients with RUNX1::RUNX1T1, CBFB::MYH11, NPM1, and KMT2A::ELL, who could be transplanted directly. This pilot provided proof of principle that molecular relapses could be treated, and MRD levels be reversed, at least in subgroups. In addition, avoiding intensive re-induction therapy prior to HSCT is feasible. Conversely, the relatively slow-acting mechanism of epigenetic therapy seems to be ineffective in highly proliferative subgroups of AML with KMT2A-rearrangements or FLT3-ITD. Further studies are mandatory to define treatment options for molecular relapses of non-favorable pediatric AML and to confirm potential improvement of outcome post-HSCT.

Safety and Efficacy Results from CLI120-001 a Phase 1 Study in RR-AML and HR-MDS: Update from Higher Dose Levels

Background: RVU120, a first-in-class CDK8/19 kinase inhibitor, showed antileukemic activity mediated by inhibition of STAT5 phosphorylation in AML cell lines and patient-derived cells.Clinical activity in a population of patients with relapsed/refractory AML and HR-MDS with poor prognostic factors was shown in a currently ongoing phase 1 dose escalation study (NCT04021368) consistent with the preclinical evidence of both cytotoxic and erythroid and myeloid differentiation effects in cell and mouse models. Aims: The primary objective of the study is to determine the safety profile and the recommended phase 2 dose (RP2D) of RVU120 as a single agent in R/R AML and HR-MDS. Secondary objectives include the characterization of PK, antitumor activity, and exploratory pharmacodynamic (PD) effects. Methods: CLI120-001 is an open-label, multi-center, dose escalation study. RVU120 is administered orally every other day, for a total of 7 doses, in a 3-week treatment cycle until disease progression or unacceptable toxicity. Adverse events are graded according to NCI-CTCAE v.5.0. DLTs are assessed at the completion of Cycle 1. Disease evaluation is performed according to Dohner 2017 and Cheson 2006 response criteria for AML and MDS respectively. Pharmacodynamic assessments (PD) include a flow cytometry assay to assess target engagement by evaluating changes in phosphorylated STAT5. Results: As of 26 Jul 2023, 31 patients received RVU120 at doses between 10 and 175 mg, median age 71 years, median of 3 prior lines of therapy. ECOG PS was 2 in 8 pts, 1 in 20 pts, and 0 in 3pts. No severe drug reactions, no DLT have been reported, with manageable nausea and vomiting being the most frequent treatment-emergent adverse events. Twelve out of the 24 evaluable patients showed clinically relevant benefit. Four patients achieved a reduction of blasts to &amp;lt;5% in the BM: One patient with NPM1 and FLT3 mutated AML, achieved a CR, 3 patients with HR-MDS had a marrow CR, one of those had G3 BM fibrosis and is currently in C3 of treatment. 3 additional patients treated across different dose levels experienced a sustained BM blast reduction: 2 patients with TP53+ AML with 37% and 74% BM blast reduction, 1 patient with AML-MRC and monosomy 7 with 28% blast reduction; 1 patient with AML-MRC failing 4 lines of therapy received allogeneic BM transplantation after 6 cycles of RVU120 treatment. 5 patients achieved hematology improvement including: 1 patient with HR-MDS failing 2 lines of therapy, 1 patient with AML secondary to MF, became RBC transfusion independent (TI) and is still ongoing after &amp;gt;18 months with nearly normal Hgb and Plts values. 2 patients with AML-MRC, with baseline RBC and Plt transfusion dependance (TD), became TI and reached normal Plts values. 1 patient with AML-MRC with RBC and Plt TD and severe neutropenia showed RBC and Plt TI and ANC recovery and is currently in C7 of treatment. A correlation between pSTAT5 inhibition and RVU120 exposure was observed. Conclusion: In the ongoing phase 1 trial, RVU120 shows clinical activity in both AML and HR-MDS, inducing RBC transfusion independence and blast reduction with a tolerable safety profile. Clearance of BM blasts including a formal CR were observed in patients treated at different dose levels. Relevant target inhibition is achieved from 110 mg onwards with expected higher pSTAT 5 inhibition with further dose escalation.

Adoptively Infused Memory-like Natural Killer Cells Impact Adaptive Immune Responses in Patients with Acute Myeloid Leukemia

Natural killer (NK) therapies have been challenging owing to antigen escape, restricted trafficking, and limited tumor infiltration, translating into modest clinical benefit. We have shown that memory-like (ML)-NK cells acquire an in vivo differentiated phenotype distinct from conventional NK cells and are well-tolerated without cytokine release syndrome, GVHD, or neurotoxicity. WU-NK-101 (W-NK) is an allogeneic cytokine-reprogrammed, expanded, cryopreserved ML-NK cell product derived from healthy human peripheral blood mononuclear cells, with scalable manufacturability, that is currently in clinical development in relapsed/refractory (R/R) AML (NCT# 05470140). We conducted a multi-modal analysis of the immune tumor microenvironment (TME) in 13 patients with R/R AML receiving ML-NK cells (NCT#01898793) with the aim to determine whether TME modifications induced by ML-NK cells correlate with clinical benefit. NK cells were purified from leukapheresate, and activated with IL-12, IL-15, and IL-18 for 12-16 hours. Patients were lymphodepleted with fludarabine and cyclophosphamide. NK cells were infused in escalating doses: 0.5 × 10 6/kg, 1.0 × 10 6/kg, and all NK cells from a single leukapheresis, capped at 10 × 10 6/kg. Response was defined as &amp;lt;5% BM blasts on d+28. BM trephines for immune gene expression profiling (nCounter IO360®) and spatial proteomics (GeoMx™ DSP; n=53 immuno-oncology [IO] targets; NanoString Technologies, Seattle) were collected at baseline (BL) and on treatment (OT). Immune infiltration, NK and T cell number from BM samples was quantified manually on immunofluorescent imaging. Oncomine™ TCR Pan-Clonality assay (Thermo Fisher) was used to profile TCRb and g repertoires by NGS. The Shannon index was used to describe TCR repertoire diversity, and clonal evenness was used as a measure of the similarity of clone sizes. BM infiltration with CD8 + effectors, enhanced glycolysis and cell cycling was higher in BM samples from responders (CR) to ML-NK cells. By contrast, OXPHOS RNA scores and gene signatures of immune senescence and terminal T-cell exhaustion were higher in non-responders (NR). We employed machine learning to construct an 8-gene predictor of response which encompassed T/NK markers, such as CD7, and IFN regulatory factors and TLRs. This score was significantly higher at baseline in CR and positively correlated with response duration. Principal component analysis (PCA) of spatially resolved IO proteins separated CR from NR, particularly in OT specimens, where T-cell markers CD3/CD8 and inhibitory receptors CTLA4/LAG3 were among the top contributing features. CD34, type I IFN signaling molecule STING1 and PARP1 were significantly higher at baseline compared with OT, with OT showing higher expression of cytotoxicity markers and inhibitory receptors (e.g., Tim-3 and CD276). This observation strongly suggests modulation of the immune TME after the infusion of ML-NK cells. Response to ML-NK cells was associated with coordinated up-regulation of T cell, NK cell and B cell protein markers. To further substantiate the co-localization of T cells and NK cells in the BM FFPE sections (Fig. A), and its correlation with clinical efficacy we annotated the 93 ROIs in our spatial proteomics dataset based on a median split of CD3 and CD56 protein barcodes. ROIs from CR were largely CD3 highCD56 high, both at BL and OT. Conversely, ROIs from BM sections of NR to ML-NK cells had a predominantly CD3 lowCD56 low phenotype. Quantitative analysis of T-cell infiltration post ML-MK dosing showed a significant association with response, 0.9 (PD) vs. 5.3 (CR)-fold-increase ( P=0.01). Interestingly, data demonstrate selected intra-BM clonal expansion as evidenced by decreased TCR diversity (mean ± SEM, 4.6±0.3 vs. 3.3±0.4, pre vs. post; P=0.013)and increased clonality (Fig. B). Thus, pre-existing T-cell responses appear to be co-opted in the AML TME of CR, suggesting the potential for durable effectiveness of ML-NK cells. Multi-modal analyses of WU-NK-101 highlight unique transcriptional and functional states, which could underpin in vivo potency and persistence. WU-NK-101 are currently being evaluated in a phase 1 study of R/R AML (#NCT05470140).

Clonal Dynamics of Gene Mutations during Oral Azacitidine Maintenance Therapy in Patients with Acute Myeloid Leukemia (AML): Outcomes from the QUAZAR AML-001 Trial

Background: Acute myeloid leukemia (AML) is a genetically heterogeneous disease and despite some patients (pts) achieving remission with frontline intensive chemotherapy (IC), most eventually relapse. In the QUAZAR trial (NCT01757535), oral azacitidine (Oral-AZA) prolonged overall survival and relapse-free survival (RFS) vs placebo (PBO) in older pts with AML in remission post IC (Wei et al, N Engl J Med 2020). Here, we studied the molecular landscape and clonal dynamics of pts treated with Oral-AZA/PBO in the QUAZAR study. The mutational landscape for older patients with AML in remission after IC may be confounded by persistence of pre-leukemic (PL) or age-related clonal hematopoietic (CH) variants. The implications of these residual mutations on disease relapse and treatment (Tx) outcome are largely unknown. Aims: 1) characterize the mutational landscape from remission bone marrow at baseline (BL) prior to Oral-AZA maintenance (i.e., in remission post-IC); 2) determine the fate of variants over time and at relapse in the Oral-AZA vs PBO arms; 3) examine associations between mutational landscape and relapse risk between Tx arms. Methods: In QUAZAR, 472 pts (≥55 years) with AML with intermediate- or poor-risk cytogenetics in remission after IC (BL) were randomized 1:1 to Oral-AZA or PBO. Among pts who consented to biomarker analyses (n=310), targeted NGS (37 myeloid genes) was performed on bone marrow DNA at BL (Oral-AZA/PBO: n=165/145), cycle 6 (n=107/79) and relapse (n=83/77). Mean NGS coverage was 13K reads and median minimal detectable variant allele frequency (VAF) was 0.12% (range: 0.02-2.79). Clonal variants were categorized by the longitudinal association between VAF and blast percentage (slope of leukemic variants &amp;gt;0.1; PL/CHIP &amp;lt;0.1). RFS was computed from time of randomization to relapse (≥5% BM blasts) or death, estimated by Kaplan-Meier methods. Hazard ratios (HR) and 95% confidence intervals (CI) were obtained from Cox regression models. Nominal P values were derived from log-rank tests. Results: In the NGS cohort (n=310), median RFS (mRFS) for Oral-AZA vs PBO was 10.2 vs 4.7 months (mo), respectively. At BL, prior to maintenance Tx, 221 (71.3%) had detectable mutations, the most frequently occurring mutations (&amp;gt;5% of pts) were in DNMT3A (28.4%), TP53 (15.5%), IDH2 (12.3%), TET2 (11.9%), SRSF2 (11.0%), IDH1 (6.1%) and ASXL1 (5.5%). At BL, 110/310 (35.5%) pts had VAF &amp;gt;5%, potentially representing persistence of PL/CH variants in remission. Comparative analysis of all gene variants at BL and relapse revealed that some VAFs increased with blast frequency, while other variants remained largely static. Applying a variant classification algorithm, 97/258 pts had potential leukemic variants detected at BL. These involved DNMT3A (16.1%), SRSF2 (7.1%), TP53 (7.1%) and IDH2 (5.7%). PL/CH variants included DNMT3A (17.5%), TP53 (8%), IDH2 (5.7%) or TET2 (5.7%). TP53 was detected in 19.4% PL/CH variants (mean VAF 1.7%; range 0.2-13.4). Notably, when analyses were limited to potential leukemic variants at BL (&amp;lt;5% VAF), their presence was correlated with worse RFS (PBO: mRFS for 0, 1 or 2+ mutations was 6.1, 4.7 or 1.9 mo, respectively). The presence of ≥2 leukemic mutations was associated with shorter RFS only in PBO arm (mRFS vs &amp;lt;2 mutations: 1.9 vs 5.7 mo, P=0.02; Oral-AZA: 10.2 vs 10.2 mo). RFS favored Oral-AZA in pts with low mutational burden at BL (&amp;lt;2 mutations: mRFS 10.2 vs 5.7 mo [ P=0.003]; n=145 vs 122 [PBO]), and in a small subset of pts with higher mutational burden (≥2 mutations: mRFS 10.2 vs 1.9 mo [ P=0.008]; n=14 vs 13 [PBO]). Oral-AZA prolonged RFS vs PBO in pts across most mutational subtypes, when assessed for mutations that were deemed potentially leukemic (Figure). At relapse, the frequency of mutations, hotspots variants and co-mutations were comparable between Tx arms. Mutation-based pathway analysis indicated Ras pathway genes were enriched at relapse in PBO (28.6%) vs Oral-AZA arm (14.5%, P=0.03). Summary: In pts with AML in remission post IC, post-hoc analyses showed that Oral-AZA improved RFS vs PBO regardless of the mutational landscape at baseline. The spectrum of mutations at relapse was similar between Tx arms, suggesting that Oral-AZA maintenance prolongs remission without altering mutational heterogeneity.

Somatic Mutations and DNA Hypermethylation at Enhancers and Promoters Identify Distinct Subtypes within Lower-Risk Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of heterogeneous disorders caused by the accumulation of somatic mutations in the hematopoietic stem and progenitor compartment. Besides del(5q), SF3B1 or TP53 mutations, referred to as defining genetic abnormalities, mutation patterning hardly structured the classification of MDS. Mutations in epigenetic factors occur early in the development of clonal hematopoiesis leading to precocious alterations of DNA methylation implicated in oncogenesis. Convergent DNA methylation patterns related to both mutations and microenvironment imprinting may induce specific gene expression profiles and contribute to phenotypic variations. To refine a pathophysiological classification of lower-risk MDS, we combined genetic profiling to DNA methylation and transcriptome data. Methods: We enrolled 543 lower-risk treatment-naïve MDS patients at diagnosis and performed DNA-sequencing of 24 genes. DNA methylation and RNA-sequencing data were generated in a representative subset of 175 cases with available material (75 MDS-SLD/MLD, 40 MDS-EB1, 53 MDS-RS-SLD/MLD, 5 MDS del(5q), 2 MDS-U) and 7 age-matched healthy controls. IPSS-R was very low, low or intermediate in 87.4%. During a median follow-up of 2 years, 16% of MDS cases progressed to AML (MDSp). Infinium EPIC 850K array allowed after filtering the examination of methylation of 723,612 CpG sites with enrichment at enhancers and promoters. Differentially methylated regions with differentially methylated CpG sites (DMR-CpG) between cases and controls were defined with a minimum of 2 probes, a Benjamini-Hochberg-adjusted P-value cut-off of 0.05, a false discovery rate of 0.001 and a mean Δβ-value &amp;gt;20% at CpG sites. Consensus motifs for transcription factors were identified using Homer. Results: Unsupervised clustering on genetic profiling of 543 patients identified distinct subsets of lower-risk MDS with different clinical features: one cluster (group A) was enriched in del(5q), three clusters were characterized by SF3B1 mutations, either isolated (group B) or associated with DNMT3A (group E) or TET2 (group F), while the two remaining clusters were enriched in either complex pattern of mutations (group C) or SRSF2 mutations (group D). Genetic clusters showed significantly different clinical outcomes, clusters C, D and E having lower overall survival and higher risk of progression to high risk MDS or AML compared to other clusters. In the subset of 175 cases, a total of 2,953 unique DMR-CpGs allowed a robust repartition of patient samples in 4 groups using the k-means method. The 4 methylation groups were clinically distinct in terms of age, hemoglobin, neutrophils, monocytes, platelets, BM blasts, WHO classification, IPSS-R, and mutation pattern. A significant correlation was found between genetic and methylation groups ( P&amp;lt;0.001). Hypomethylated CpGs defined group 4 which was enriched in MDS-RSwith low blast count and few co-mutations, while groups 1 and 3 demonstrated hypermethylated profiles driven by TET2/ IDH1-2 mutations and TET2/ SRSF2 mutations, respectively. TET2-mutated cases exhibited an increased proportion of hypermethylated DMR-CpGs at enhancers (DME) significantly enriched in C/EBP and ETS family transcription factor motifs. In MDS without evidence of progression, we identified 1,418 DME and their gene targets of which those significantly deregulated in RNA-seq were involved in the control of translation and response to TGF-β. By contrast, in MDS with rapid progression to AML, DMR-CpGs were significantly enriched at CpG islands and promoters ( P&amp;lt;0.0001) and a set of 10-20 genes downstream of these promoters were specifically deregulated. Conclusion: Somatic mutations and DNA methylation at enhancers and promoters identify distinct subsets of lower-risk MDS with different clinical behavior. Hypermethylation at enhancers with C/EBP or ETS family motifs is a hallmark of MDS and may account for changes in transcription factor recruitment and cell fate. Hypermethylation of promoters with downstream effects on gene expression may indicate a propensity to rapid evolution to AML.

Real-World Outcomes with Intensive Chemotherapy in Acute Myeloid Leukemia Patients Deemed “Unfit” for Remission Induction By Landmark Trials' Criteria

Background: Induction mortality rates in acute myeloid leukemia (AML) ranges from 7.1% to 40% across Indian tertiary care centers (Kayal S, ASH 2022) exceeding those in the western countries, possibly due to treatment delay, multidrug resistant infections, and inadequate social support. Furthermore, patients seen in our clinics are not representative of those in landmark clinical trials, as a majority of them would be considered unfit for intensive induction based on the trial criteria. Consequently, these patients are left with treatment options, such as less intensive hypomethylating agents, BCL2 inhibitors, and newer targeted therapies. However, these drugs often remain unfeasible due to high out-of-pocket costs and regulatory approval delays, so a majority of patients receive intensive induction with antibiotics. In this retrospective study, we evaluate the effectiveness and toxicities associated with the utilization of such a strategy in the Indian context. Method: This retrospective study was conducted at a single tertiary care cancer center in India. It included patients aged ≥15 years, with acute myeloid leukemia, who received intensive induction chemotherapy as decided by the multidisciplinary joint clinic between January 01, 2017, and December 31, 2019. Patient data were extracted from the Indian acute leukemia research database [INwARD], which is a prospective registry of multiple centers, maintained and audited by the Clinical Data Management Center [CDMC], Vellore for the Hematology cancer consortium and is compliant with ICH-GCP regulations. Ethical approval was obtained from the institutional ethics committee, and the study was registered in the Clinical Trial Registry - India (CTRI/2020/02/023455). Patients were categorized based on the established criteria for “unfit for intensive chemotherapy” derived from landmark trials (VIALE-A, NCT02203773, VIALE-C), and subsequent analyses were performed. Outcomes assessed included 30-day induction mortality, documented infection at diagnosis (defined as fever with radiological, microbiological, or clinical focus of infection) and fungal infections according to EORTC and MSG classifications. Post-induction bone marrow response was defined as complete response/complete response with incomplete marrow recovery (CR/CRi) with ≤5% BM blasts, platelet count &amp;lt;100 × 10 9/L, and absolute neutrophil count &amp;lt;1.0 × 10 9/L. Kaplan-Meier analyses were conducted to assess event-free survival and overall survival. Results: Among 1326 eligible patients, 385 received intensive chemotherapy during the study period (49 received less intensive therapy, 442 referred outside, 210 on palliative care, 212 lost to follow up and 28 died). Demographic and baseline characteristics are detailed in Table 1. Cytarabine and daunorubicin (98.9%) were the most common first-line treatments. Of the 385 patients receiving intensive chemotherapy, 188 were categorized as unfit according to the study criteria (Refer Table 1). The unfit cohort exhibited higher 30-day induction mortality compared to the fit cohort (20.5% vs. 6%, P&amp;lt;0.0001) with a trend for higher incidence of proven and probable fungal infections (18.4% vs. 11.2%, P=0.054). Complete remission rates (CR+CRi) were lower in the unfit cohort (52.4% vs. 68%, P&amp;lt;0.001), while no difference in documented bloodstream infections was observed (P=0.754). Univariate analysis revealed that induction mortality was most influenced by age&amp;gt;50 (P=0.019), ECOG ≥2 (P=0.002) and infection at diagnosis (P&amp;lt;0.0001). However, upon conducting multivariate analyses, infection at diagnosis emerged as the strongest predictor of induction mortality (OR:2.91, 95%CI, 1.51 to 5.59, P=0.001). The median follow up was 15.7 months (range, 0.2-73.8). The 2-year estimated event-free survival (EFS) and overall survival (OS) of the entire cohort was 42.9% and 69.2% respectively. For the unfit and fit cohort, the 2-year EFS was 42.2% vs 43.9% (P=0.065), and the 2-year OS was 62.5% vs 75.1% (P=0.001) respectively. Conclusion: Unfit patients who received intensive chemotherapy had higher induction mortality and lower overall survival. Furthermore, it underscores the importance of improving accessibility to novel therapies, to provide a safe and viable alternative for nearly half of our AML patient population.

Phase I Study of the T-Cell Receptor-like Antibody, Hu8F4 in Patients with Relapsed and Refractory Myeloid Malignancies

Background: Relapse after conventional chemotherapy for newly diagnosed AML is responsible for the majority of treatment failure. Relapsed (R/R) disease is frequently refractory to salvage attempts with further chemotherapy. The effectiveness of stem cell transplant and associated graft vs. leukemia effect implies an important role for immune-based therapy in producing long lasting remissions. Existing approaches using immunotherapy have failed to establish a suitable surface target or treatment paradigm that is effective in myeloid malignancies. Hu8F4 is a humanized T-cell receptor-like monoclonal antibody that binds to the conformational epitope of PR1 bound to HLA-A2, which is highly, differentially expressed on the surface of AML compared to normal progenitors. Methods: This is a first-in-human, Phase I dose escalation trial of Hu8F4 in patients (pts) with myeloid malignancies. Pts with R/R AML, MDS, CMML, and myeloid blast phase of CML with adequate organ function and PS ≤ 2 were eligible. Pts were treated on 7 escalating dose levels, ranging from 0.01 mg/kg to 10 mg/kg IV on D1 &amp; 15. Initial dose levels required 1 pt per dose (0.01, 0.03, 0.1, 0.3, 1), followed by 3 pts per dose (3, 10). Results: Fifteen pts with R/R AML have been enrolled, with a median age of 62 years (range, 23-77 years), including 7 females (47%). Pts had received a median of 4 prior therapies; 5 pts (33%) had a PS of 2. At enrollment, the median WBC was 2 (0.1 - 18.4), median BM blasts were 21% (0 - 76); 9 pts (60%) had complex karyotype and 4 (27%) had a TP53 mutation. All pts had &amp;gt; 98% surface expression of PR1 on the myeloid blasts. Hu8F4 C max ranged up to 160,000 ng/mL with t 1/2 of 48 hours and clearance of 2.61 hr*ng/mL at the highest dose. Weak anti-drug antibodies were observed after week 4 in 2 of 3 pts treated at 3 mg/kg. With a median follow up of 5 months (1.1 - 9.9), pts have received a median of 1 (1-4) cycle of therapy. No DLTs were observed at the 3 mg/kg cohort.; 4 additional pts who were on concomitant tacrolimus were enrolled at this level to assess safety with concurrent use, and no DLTs were observed. Three pts were enrolled at the 10 mg/kg level, and 1 experienced dose limiting thrombocytopenia, triggering expansion to 6 pts. Two pts had a decline in BM blasts and 4 had stable disease. There was rapid decline in peripheral blasts immediately after infusion of Hu8F4 on D1 and 15 with associated elevation in serum LDH in some pts and a rise in normal granulocytes, consistent with drug-mediated anti-leukemia effects. The pharmacokinetic parameters and transient blast reduction indicated a possible sink effect mediated by high levels of circulating blasts. SAEs documented on study were mostly disease-related and included infections, cytopenias, hemoptysis, pneumonia, and GI bleeding. Treatment related AEs were temporally related to the drug infusion, including hypotension (Grade 2: N=2), and rigors (Grade 2: N=10; Grade 1: N=1). Infusion reactions were observed at all dose levels, more common at 3 and 10 mg/kg, but were transient, and managed with steroids and antihistamines. All pts proceeded with their next dose without further issues. Neither cytokine release syndrome nor neurologic toxicities were observed. Correlative studies support antibody dependent cellular cytotoxicity and phagocytosis as important mechanisms of anti-AML activity, which was more effective at lower dose levels. Conclusion: Hu8F4, a first-in-class TCR mimic antibody, was well tolerated with expansion planned at more frequent dose intervals. On-target peripheral blast reduction temporally related to infusion suggests biological activity. Real-time pharmacokinetic data indicate a possible sink effect that may be overcome by a more frequent dosing strategy that is planned.

Blinatumomab in Combination with Immune Checkpoint Inhibitors (ICIs) of PD-1 and CTLA-4 in Adult Patients with Relapsed/Refractory (R/R) CD19 Positive B-Cell Acute Lymphoblastic Leukemia (ALL): Results of a Phase I Study

Background: Blinatumomab (blina) improves outcomes in R/R CD19+ ALL compared to chemotherapy. However, the overall response rate to blina was 44%, and median overall survival (OS) is just 7.7 mos (Kantarjian. NEJM. 2017). Preclinical studies show increased PD-L1 expression on leukemic blasts potentially contributes to relapse after blina. The addition of PD-1 +/- CTLA-4 blockade to blina leads to increased in vitro T cell proliferation and enhanced cytotoxicity (Feucht. Oncotarget 2016). Thus, blockade of co-inhibitory pathways may enhance blina efficacy in vivo. We describe results of a multi-center phase I study combining blina with immune checkpoint inhibitors (ICIs) targeting PD-1 (nivolumab) +/- CTLA-4 (ipilimumab). Methods: In a dose-escalation study, we evaluated the safety, tolerability, and preliminary efficacy of blina with nivolumab (nivo) +/- ipilimumab (ipi) using a 3+3 design. Pts ³16 years-old with R/R CD19+ B-ALL or mixed phenotype acute leukemia (MPAL) were eligible. Pts ³60 years could be untreated. Table 1 presents the dose escalation schema. Expansion cohorts of 6 pts were planned at the maximum tolerated dose (MTD) for blina + nivo AND blina + nivo + ipi. Pts received up to 5 cycles of blina and 1 year of ICIs. Pts removed from the study during the blina lead-in (days 1-10) were replaced. Dose-limiting toxicities (DLTs) were defined as grade 3+ non-hematologic toxicities requiring the permanent discontinuation of treatment in the first 42 days. Primary endpoints were toxicities and MTD. Secondary endpoints included complete remission (CR), MRD-negativity at a sensitivity of 0.01%, duration of response and OS from treatment initiation. Results: Twenty-seven pts were enrolled to 3 dose levels (14 DLA1, 3 DLB1, and 10 DLB-1) from September 2017-December 2022. Six pts were replaced (4 prior to ICI dosing and 2 requiring subsequent therapy in the DLT window). The median age of enrolled pts was 55 (range 24-84), 14 were male (52%), and median baseline BM blasts were 60% (range 0.2-98%). Baseline characteristics are presented in Table 2. A single DLT (G4 infusion reaction and G3 hypotension) occurred among 6 pts at DLA1. Based on this safety and efficacy data, blina + nivo was expanded to include 6 additional pts without additional DLTs. There were 2 DLTs at DLB1 (G5 pneumonitis and G2 GVHD), which led to de-escalation to DLB-1, where there was 1 DLT (G3 delirium). Grade 2+ immune-related adverse events attributable to ICIs included pneumonitis (G2+G5), rash (G2+G3), transaminitis (G2+G3), colitis (G3), and hypothyroidism (G2). Grade 3+ adverse events attributable to blina included neutropenia (5-G4+3-G3), dysphasia (2-G3), weakness (G3), seizure (G4), and transaminitis (1-G4+4-G3). Among 22 pts evaluable for response, the CR rate was 68% (100% MRD-negative). Among 13 pts with &amp;gt;50% BM blasts at baseline, 62% achieved CR. Pts with B-myeloid MPAL (0/2) and a history of extramedullary disease (1/4) were less likely to respond. Nine responders subsequently relapsed including 3 with isolated extramedullary disease and 2 with CD19-negative disease. As shown in Figure 1, relapse-free survival (RFS) at 1 year was 27% (95% CI 10-46), while OS at 1 year was 63% (95% CI 38-79). Twelve pts underwent allogeneic blood or marrow transplant (alloBMT) following study treatment. At 1 year for alloBMT pts, RFS was 51% (95% CI 19-76) and OS was 61% (95% CI 26-83). Ongoing analyses of changes in T cell subpopulations, co-signaling molecule expression, and single cell RNA seq results will be presented. Conclusions: Combination therapy with blina and ICIs in R/R ALL is safe, feasible, and associated with a high MRD-negative response rate. Long-term survival was promising in comparison to prior results with blina monotherapy, especially following consolidation with alloBMT. A randomized trial of blina +/- nivolumab is needed to confirm the benefit of this combination.

Measurable Residual Disease Results & Survival with Augmented Early Intensification Therapy in Intermediate-Risk & High-Risk Group Pediatric Acute Lymphoblastic Leukemia Patients Treated with ALL IC-BFM 2009 Protocol: A Single-Center Study from India

Introduction Measurable residual disease (MRD) has proven to be one of the most effective disease monitoring responses in ALL. Multiple treatment groups have demonstrated End-of-Induction (day 33) flow MRD of &amp;lt;0.01% has excellent disease-free survival (DFS). Patients with end-of-consolidation MRD of &amp;lt;0.01% has an overall DFS of 75 % as per COG AALL1131 including the very high-risk B-ALL genotypes. We conducted a study to assess the efficacy &amp; toxicity of augmented early intensification therapy (phase IB) in ALL IC-BFM 2009 protocol for intermediate-risk &amp; high-risk group pediatric ALL patients. Objectives To evaluate efficacy of augmented early intensification therapy (phase IB) in ALL IC-BFM 2009 in terms of achievement of day 78 MRD negative status. To determine the event-free survival in intermediate-risk &amp; high-risk group patients. To appraise the adverse events (AEs) of augmented phase IB Induction. Methodology The study enrolled treatment-naïve ALL patients of 1-25 years' age between April 2021 to February 2023 at AIIMS Rishikesh, India. Patients were risk stratified into Standard (SR), Intermediate (IR) &amp; High risk (HR) groups as per ALL IC BFM 2009 protocol. All patients received Augmented phase IB protocol. The regimen comprised of cyclophosphamide (2 doses), four blocks of cytarabine &amp; 6-mercaptopurine, augmented with vincristine (4 doses), and L-Asparaginase (12 doses). Day 78 bone marrow flowcytometric MRD was performed in all patients irrespective of day 33 marrow morphology &amp; MRD. The study defined complete remission as BM blasts &amp;lt;5% with no extramedullary disease and MRD negativity as &amp;lt;0.01%. The AEs was monitored as per NCI-CTCAE v5. As per the study protocol, patients of B-ALL with day 33 MRD &amp;gt;0.01%, ph-positive ALL, hypodiploidy and T-ALL with day 78 MRD &amp;gt; 0.01% was considered as HR &amp; treated with High-risk blocks consolidation followed by transplant or delayed intensification &amp; maintenance. Results A total of 75 patients enrolled in the study. The M: F ratio was 1.6:1. The median age was 8 years (range 1-25 yrs). B &amp; T-ALL constituted 80% &amp; 20% respectively. ph-positivity accounted 22% of B-ALL. The median Lansky performance was 40 &amp; ECOG was 3 among the participants. The median hb, total white counts, and platelets were 5.9 g/dl, 28 x10 9/L and 30 x10 9/L respectively. At diagnosis, 28% of patients had hyperleukocytosis, 15% with bacterial sepsis, 8% with invasive fungal infections, and 8% with CNS leukemia. At baseline, the distribution of patients in SR, IR &amp; HR was 15%, 68% &amp; 17% respectively. The final risk strata at end-of-induction were 8%, 49% &amp; 43% in SR, IR &amp; HR groups. CNS leukemia accounted for 8% of all patients. Of the total evaluable patients, 86% achieved day 8 good steroid response (absolute blasts count &amp;lt;1000/cu.mm), 73% &amp; 38% achieved day 15 marrow M1 response (&amp;lt;5%blasts) and MRD &amp;lt; 0.1%, 97% &amp; 77% achieved day 33 M1 marrow response and day 33 MRD &amp;lt;0.01% respectively. Among the study participants who were MRD positive on day 15, three-fourths (75%) of them accomplished MRD negativity at day 33. Of the 23% patients with day 33 MRD positivity, 80% achieved day 78 MRD negativity of &amp;lt;0.01% with Augmented phase IB with 3 participants being persistent MRD positive. The median follow-up was 24 months and the overall survival was 74%. The mean survival time was 16.7 months (12.7-20.6) for HR &amp; 21.2 months (18.3-24.1) for IR patients. The subgroup analysis demonstrated an overall survival of 80% for SR, 84% for IR &amp; 64% for HR groups. Nevertheless, the proportion of patients in the SR group (8%) was lower compared to the others. The commonest hematological adverse events were hypofibrinogenemia (22%), followed by febrile neutropenia (19%), anemia (19%), and thrombocytopenia (18%) respectively. The commonest non-hematological AEs were peripheral neuropathy (5%) and typhlitis (5%). Conclusion In our single-center experience, the use of augmented early intensification therapy (phase IB) in ALL IC-BFM 2009 protocol in intermediate-risk &amp; high-risk pediatric ALL patients was associated with achievement of day 78 MRD negative status in 80% patients who had day 33 MRD-positivity without significant treatment-related toxicity, and encouraging event-free survival in this patient population.

Updated Results from Phase 1 Study of Targeted Radiotherapy with Lintuzumab-Ac225 in Combination with Venetoclax in Relapsed/Refractory AML

Background: Resistance to venetoclax, a BCL2 inhibitor, is mediated by co-expression of other BCL2 proteins including BCLXL and MCL1. In vitro data show that DNA damage reduces the level of MCL1, a known mediator of venetoclax resistance. The monoclonal antibody radioconjugate, lintuzumab-Ac225, is a highly cytotoxic alpha-radiation emitter that selectively targets CD33, a cell surface antigen expressed on the majority of AML cells. Clinical studies have shown that the high-energy alpha-particle emissions from lintuzumab-Ac225 elicit single and double-stranded DNA breaks in targeted tumor cells. In venetoclax-resistant cell lines in vitro, lintuzumab-Ac225 promotes MCL1 degradation through DNA damage resulting in increased cell sensitivity to venetoclax. Similarly, mice xenografted with venetoclax-resistant AML tumor lines demonstrated tumor regression and increased survival when treated with venetoclax and lintuzumab-Ac225. The aims of this phase I/II study are to assess the safety, tolerability and efficacy of lintuzumab-Ac225 in combination with venetoclax in R/R AML. Study Design: The Phase I portion of the study used a 3+3 dose-escalation design to determine the maximum tolerated dose (MTD) of lintuzumab-Ac225 combined with venetoclax. The dose levels for lintuzumab-Ac225 were 0.5, 0.75, 1.0 µCi/kg, 1.5 µCi/kg, and 2.0 µCi/kg (expansion cohort). The Phase II portion of the study was designed to enroll up to an additional 20 patients at the recommended phase II dose (RP2D) with venetoclax to determine the best overall response (CR+CRh+CRi) up to 6 months after starting treatment. Eligible patients included R/R AML patients aged 18 years and older with adequate organ function, ECOG performance status 0-2, and more than 25% CD33 positive leukemic blasts by flow cytometry. Patients with antecedent myelodysplastic syndromes, myeloproliferative neoplasms, or therapy-related AML were eligible. Venetoclax was given at a dose of 400 mg daily on days 1 to 21 (cohorts 1-3) or days 2-22 (cohort 4). Lintuzumab-Ac225 was administered as a single dose of each cycle on day 5 (cohorts 1-3) or day 1 (cohort 4). Results: In total, eighteen R/R AML patients received lintuzumab-Ac225 at 0.5 µCi/kg (n=3), 0.75 µCi/kg (n=6), 1.0 µCi/kg (n=3), 1.5 µCi/kg (n=3) and 2.0 µCi/kg (n=3) with venetoclax in the phase I dose escalation portion. The median age was 73 years (range 46-87); 7/18 (39%) had refractory AML and 9/18 (50%) had ELN unfavorable risk. In the dose escalation phase, one DLT with grade 3 prolonged thrombocytopenia occurred in 1 of 6 patients received lintuzumab-Ac225 at 0.75 µCi/kg plus venetoclax. No DLTs were observed at 1.0 µCi/kg (Cohort 2), 1.5 µCi/kg (Cohort 3) and 2.0 µCi/kg (Cohort 4) of lintuzumab-Ac225 plus venetoclax. Grade 3 or higher adverse events related to lintuzumab-Ac225 treatment included hematologic AEs and one count of non-hematologic event (hyponatremia) (Table 1). In cohort 4 at 2.0 µCi/kg of lintuzumab-Ac225 with the dosing schedule modification, reduction of BM blasts was observed in 2 of 3 patients. One patient achieved MLFS with 93% reduction of BM blasts. Conclusion: The combination of lintuzumab-Ac225 and venetoclax had a manageable safety profile and no early mortality at day 30. The MTD was not reached and no significant toxicities have been reported during the follow-up period. Modified dosing schedule in Cohort 4 of the combination demonstrated improved anti-leukemic effects. The data observed supports the development of further trials to evaluate the safety and anti-leukemic efficacy of lintuzumab-Ac225 in combination with venetoclax-based treatment in the R/R AML population.

Efficacy and Tolerance of Brexucabtagene Autoleucel in Adults with Relapsed/Refractory B-Cell Precursor Acute Lymphoblastic Leukemia : A Graall Study from the Descar-T Registry

Background : Brexucabtagene autoleucel (brexu-cel) was recently approved for adult patients with relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) based on the results of the ZUMA-3 phase 2 study. After a median follow-up of 39 months, brexu-cel showed a complete remission (CR)/CR with incomplete hematologic recovery (CRi) rate of 71% and a median overall survival of 26 months (Shah B, et al. ASCO 2023). In the present study, we aimed at investigating the efficacy and safety of brexu-cel in a real-life cohort of adult BCP-ALL patients included in the French early access program. Methods : In this Group for Research on Adult Acute Lymphoblastic leukemia (GRAALL) study, we performed an analysis of the DESCAR-T registry which collects real-life data of patients treated with CAR T-cells since July 2018 in France. All patients who underwent a leukapheresis with an intent to manufacture brexu-cel were included (N=80). Efficacy and safety analyses were performed in the 64/66 infused patients with available response assessment after infusion. The data cut-off was June 2023. The present study reports on best overall response, overall survival (OS), relapse-free survival (RFS), as well as immune effector cell-associated neurotoxicity syndrome (ICANS), and cytokine release syndrome (CRS) incidence and grading (according to ASTCT Consensus). Results : Between May 2019 and February 2023, 80 adults with R/R BCP-ALL from 22 French centers had a leukapheresis for brexu-cel. Among them, 14 patients (18%) were not infused because of death (8 pts), progression (1 pt), manufacturing failure (2 pts), or other causes (3 pts). Among the 64 infused patients with available response assessment, median age was 43.5 yrs (range, 22-69). A Philadelphia(Ph)-chromosome was found in 20/64 (31%). Patients had received a median of 3 (range, 1-6) prior lines of treatment including blinatumomab, inotuzumab ozogamicin and allogeneic hematopoietic stem cell transplantation (allo-HSCT) in 67%, 19% and 70% of patients respectively. Twelve patients (19%) had an active CNS disease (CNS2/3) prior to apheresis and 18/40 (45%) had bone marrow blasts ≥ 25% before lymphodepletion (LD). ECOG before infusion was ≥2 in 6/64 (9%) patients. All patients received fludarabine and cyclophosphamide as LD. Median time from apheresis to infusion was 40 days (range, 27-99). CRS and ICANS occurred in 48/64 (77%, grade 3+ in 6%) and 28/64 (45%, grade 3+ in 8%) of patients respectively and were reversible. A CR was achieved in 49/64 patients (77%) of whom 23/25 (92%) were MRD-negative. An older age, a prior allo-HSCT, and the occurrence of CRS were associated with significantly higher CR rates. The median follow-up was 13 months. Three patients were bridged to an allo-HSCT in continuous complete remission (second allo-HSCT for 2 of them). The median RFS and OS were 12.9 months and 15.6 months respectively (Figure). In patients who achieved a CR, median OS was 25.8 months (Figure). Patients with CNS involvement before leukapheresis had a significantly shorter RFS (HR 5.4, 95%CI 1.9-15.8, p=0.002) and OS (HR 3.3, 95%CI 1.4-8.2, p=0.008). Number of prior treatment lines, prior inotuzumab, prior blinatumomab, or pre-LD BM blast% were not associated with outcome. A better OS was observed in patients with prior allo-HSCT (HR .4, 95%CI 0.2-0.9, p=0.04). Conclusion : This multicenter real-life study confirms the efficacy and acceptable safety profile of brexu-cel in adult patients with R/R BCP-ALL. It also suggests that patients with CNS involvement, that were not eligible to ZUMA-3 study, have a limited benefit of brexu-cel when compared to other patients.

A Phase 1 Dose-Escalation Study of the Cladribine Added to CPX-351 in Patients with Relapsed/Refractory Acute Myeloid Leukemia (AML) - the Polish Adult Leukemia Group AML-1/2018 Study

AW and AP equally contributed to the study Introduction: There is no standard salvage therapy in relapsed/refractory AML (R/R AML). CPX-351, a dual-drug liposomal encapsulation of daunorubicin (DNR) and cytarabine (AraC) in a synergistic 1:5 molar ratio, shows effective antileukemia activity ( Lancet JE, JCO 2018). Favorable results of cladribine-based salvage regimens (CLAG-M, CLAG) ( Wierzbowska A; Eur J Hematol 2008, Scheckel CJ, Leuk Res. 2020) as well as first-line therapies ( Hołowiecki J, JCO 2012; Kadia TM Lancet Haematol 2021, Kadia TM JCO 2022) confirmed a value of cladribine in the treatment of AML. Aim: This phase I study was designed to explore safety (MTD), toxicity and efficacy of increasing doses of cladribine in combination with standard dose of CPX-351 in R/R AML patients (pts). (EudraCT number: 2020-002535-29). Results: Twelve pts were enrolled with a median age 65 (range 61-69) years. Importantly, 8 pts (67%) were refractory to the former first- or second- line treatment. Nine pts (75%) were in the adverse risk group according to ELN 2022 including 4 pts with TP53 mut. Four pts (33%) were refractory to venetoclax (VEN)-based therapy and 3 pts (25%) had previous alloHCT (Table 1). Three patients (2 in DL1 and 1 in DL2) required two inductions. One patient in DL3 experienced a CNS and GI hemorrhage due to thrombocytopenia refractory to platelet transfusion, which was considered a DLT requiring enrollment of additional 3 pts. No patients discontinued therapy due to intolerance. Toxicities of grade ≥3 during the induction cycle were mainly infections. All patients developed infections during 1-st cycle of treatment. Blood stream infections (n=5; 42%), febrile neutropenia (n=4; 33%), and pneumonia (n=3; 25%) were the most common infectious events. One episode of grade 5 hemorrhage occurred in a patient with thrombocytopenia refractory to platelet transfusions. Cytopenias in responding pts treated with CPX-351 and cladribine were similar to that observed with CPX-351 alone. The composite CR (cCR=CR+CRi+CRp) rate was 50% (6/12 pts), 5 pts (42%) not responded and 1 patient died due to CNS hemorrhage before response evaluation but with significant BM blasts clearance at D14. cCR was achieved in 2 out of 3 pts (66%) with intermediate-risk and 4/9 pts (44%) with adverse-risk according ELN 2022. Within the high-risk population, pts with TP53 mut had lower probability to achieve remission (1/4; 25%) than pts with myelodysplasia related mutations (3/5; 60%). No remissions were observed in pts refractory or relapsed after VEN-based therapy. Measurable residual disease (MRD)-negative cCR assessed via multiparameter flow cytometry was achieved in 60% ( n = 3/5) of MRD-evaluable pts, including all pts (n = 3/3; 100%) in DL3. All pts achieving cCR had post remission therapy with one (n=1; 8%) or two (n=5; 92%) courses of CPX-351+ cladribine consolidation and 3 pts proceeded to alloHCT. After a median (Me) follow-up of 12.8 months (mos), Me overall survival (OS) was 6.6 mos (95% CI, 2.3-not reached [NR]) for all pts and 9.8 mos (95% CI, 8.07-NR) for those who achieved cCR. Median event-free survival (EFS) was 3.28 mos (95% CI, 0.8-NR) for the study population. Conclusions: Combination of cladribine with standard dose of CPX-351 is well tolerated. Cladribine dose 5 mg/m 2 was selected as RP2D. Safety profile does not indicate any significant additional myelosuppression. Cladribine with CPX-351 shows satisfying clinical activity with cCR rate of 50%, cCR-MRD neg of 60% and durable remission in some R/R AML patients. Further evaluation of the efficacy and safety of this regimen is needed.

Unmet Medical Needs Amongst Older Patients Aged ≥60 Years with Newly Diagnosed Acute Myeloid Leukemia Receiving Intensive Chemotherapy: Experience from a Predominately United States Community Setting

Background: Venetoclax (Ven) in combination with hypomethylating agents is approved for treatment in patients (pts) with newly diagnosed (ND) acute myeloid leukemia (AML) who are ≥75 years old or unfit for intensive chemotherapy (IC), based on the longer median overall survival (OS) with Ven+azacitidine (Aza) compared with Aza in combination with placebo in the Phase III VIALE-A study (NCT02993523). Several studies are now evaluating Ven-based regimens in pts eligible for IC, for example Ven+Aza compared with IC (NCT04801797). Additionally, Ven in combination with cladribine (CLAD) and low-dose cytarabine (LDAC) is currently under investigation in a Phase II single-arm study (NCT03586609) amongst older pts (aged ≥60 years) with ND AML. An interim analysis of this trial evaluated 60 pts where complete remission (CR) and CR with incomplete count recovery (CRi) was observed in 93% of pts, and the median OS, overall and by transplant status, was not reached; 1-year OS was 72.9% and 2-year OS was 63.5% (Kadia et al. J Clin Oncol 2022). The aim of this study is to better understand treatment outcomes amongst older pts (aged ≥60 years) with ND AML receiving IC in a routine clinical care setting in the United States (US). Methods: This was a retrospective cohort study using the Flatiron Health electronic health record-derived, US nationwide, de-identified database. Pts aged ≥60 years with ND AML who initiated IC within 60 days of AML diagnosis (Dx) between January 1, 2014, and November 30, 2022, with ≥2 visits recorded within 3 months of AML Dx, and ≥3 months of follow-up before study end date (February 28, 2023) were included. Clinical trial participants and pts with acute promyelocytic leukemia or core binding factor were excluded. Descriptive analysis on patient characteristics was conducted. The proportion of pts who achieved real-world response (defined as &amp;lt;5% bone marrow [BM] blasts), real-world CR/CRi (defined as &amp;lt;5% BM blast with either platelet count &amp;lt;100 x 10 9/L or absolute neutrophil count &amp;lt;1000 x 10 9/L) and received a stem cell transplant (SCT) post-remission were reported. Thirty-day mortality, 60-day mortality, 1-year real-world OS (rwOS), and 2-year rwOS were evaluated using Kaplan-Meier (KM) analyses. The starting points for KM analyses were IC treatment initiation (all pts), SCT (pts who underwent SCT post-remission), and remission (pts who did not undergo SCT post-remission). Pts were censored at the last activity date or SCT date (whichever occurred earliest) for the overall patient cohort, and the last activity date for pts with or without SCT post-remission. Results: A total of 1105 pts met the study criteria. The median (IQR) age was 67 (64-71) years, 57% were male, 27% had secondary AML, 48% had BM blast &amp;gt;50% at Dx, and 66% were treated in the community setting. Excluding 340 pts with unknown cytogenetic and molecular data, 19%, 32%, and 48% of pts had European LeukemiaNet 2017 favorable, intermediate, and adverse risk, respectively. At a median (IQR) follow-up of 13 (6-28) months from AML Dx, 74% (n=821) of pts achieved real-world response, including 66% (n=724) who achieved real-world CR/CRi. For all pts, the 30-day mortality was 3%, 60-day mortality was 6%, 1-year rwOS was 58%, 2-year OS was 34%, and the median rwOS was 14.3 months ( Figure A). A total of 37% (n=306/821) of pts underwent SCT post-remission; 98% (n=300/306) underwent allogeneic-SCT and 2% (n=6/306) underwent autologous-SCT. The median rwOS was 40.4 months for pts undergoing SCT post-remission, and 13.8 months for pts without SCT post-remission ( Figure B). Conclusions: In this large real-world data study, we observed older pts (aged ≥60 years) with ND AML receiving IC treatment had a median OS of &amp;lt;14 months and &amp;lt;35% of pts remained alive 2 years after IC initiation. Findings from this study suggest the need for other novel treatment options, including Ven based regimens, to improve OS in older pts (aged ≥60 years) with ND AML who are eligible for IC. Future studies are warranted to conduct randomized controlled trials to comparatively evaluate IC vs other novel treatment options in older pts (aged ≥60 years) with ND AML, or conduct the appropriate statistical adjustment to minimize confounding in indirect treatment comparisons from different studies.

Genomic Landscape of Ccus Compared to MDS Indicates a Potential Applicability of the IPSS-M

Background: The 5 th edition of the WHO classification newly included myeloid precursor lesions introducing CCUS as an entity. CCUS is defined as clonal hematopoiesis (CH) in the presence of unexplained, persistent cytopenia requiring detection of either somatic mutation in certain CH associated genes or clonal chromosomal abnormalities. Aim: Characterize a large CCUS cohort with respect to cytogenetics and molecular genetics and compare the findings to an MDS cohort to evaluate differences in the genomic landscape. Methods: The CCUS cohort comprised 222 cases (median age: 76 y; female: 33%), the MDS cohort 698 cases (median age: 73 y; female: 43%). The diagnoses were established following WHO 2022. All samples were analyzed by cytomorphology, cytogenetics, targeted NGS panel (median coverage 1500x) and whole genome (median coverage 100x) sequencing. Mutation (MUT) status of 59 genes associated with myeloid malignancies were analyzed in detail (54 CH associated; 5 relevant for IPSS-M). Results: Significant differences in age, gender, BM blasts, blood parameters and number and types of cytopenias were detected. CCUS patients (pts) were older, had more WBC, less PLT, harbored a higher HB, less BM blasts and were even more predominantly male compared to MDS pts (all p&amp;lt;0.05). Within the CCUS cohort 29 pts (13%) showed cytogenetic abnormalities (CA) only (thereof 69% loss of chromosome Y (Y-loss)), while the remaining 193 pts (87%) showed at least one MUT (VAF ≥2%) in any of the 54 CH associated genes (thereof 84% MUT only; 16% MUT+CA). Regarding cytogenetics 162 CCUS pts (73%) harbored normal karyotypes while CA were detected in 27% (MDS: 43%; p&amp;lt;0.001) with Y-loss being most frequent (39/60; 65%). Y-loss was more frequently found in CCUS than in MDS (17% vs. 5%; p&amp;lt;0.001), while complex karyotypes and del(5q) were more frequent in MDS (11% and 16% vs. 1% and 0%; both p&amp;lt;0.001). Based on WGS data additional pts with Y-loss (n=7; confirmed by FISH) and recurrent CN-LOH (4q: n=4; 7q: n=5) were observed. With respect to somatic MUTs, 356 MUTs were found within the entire CCUS cohort (in 28 CH genes, 3 non-CH genes). Overall, CCUS pts showed less MUTs (median: 1 [0-6]) compared to MDS (median: 2 [0-12]; p&amp;lt;0.001). CCUS pts most frequently harbored MUTs in DNMT3A (32%), TET2 (28%) and ASXL1 (14%) followed by MUTs in splicing genes and TP53 (Fig. 1A). MUTs in DNMT3A and PPM1D were more frequent in CCUS than MDS while MUTs in ASXL1, TP53, SF3B1, STAG2, RUNX1, NRAS, CUX1 were less frequent in CCUS (each p&amp;lt;0.05). Notably, biallelic TP53 MUTs were less frequent in CCUS compared to MDS ( TP53bi of mutated: 14% vs. 55% p=0.008). Within the 15 most frequently mutated genes in CCUS, MUTs in 6 genes ( DNMT3A, SF3B1, TP53, CBL, STAG2, PPM1D) showed a median VAF &amp;lt;10%. Nine of the top 15 genes showed significantly lower median VAFs than in MDS ( DNMT3A, TET2, ASXL1, SRSF2, U2AF1, SF3B1, TP53, PPM1D, IDH2; each p&amp;lt;0.05). Thus, the frequency and VAF of ASXL1, TP53, SF3B1 MUTs were significantly lower in CCUS than in MDS, while DNMT3A and PPM1D MUTs were significantly more often detected in CCUS but at a lower VAF than in MDS. Combining cytogenetics and mutational analysis we calculated the IPSS-M, a patient-specific risk score for MDS resulting in six risk categories, and detected a clear skewing towards low risk categories in CCUS (Fig. 1B). The frequency of categories very low (VL) and low (L) were significantly higher in CCUS than in MDS (30% vs. 15%; 50% vs. 40%; both p&amp;lt;0.013), while high and very high categories were more rare (1% vs. 13%; 1% vs. 14%; both p&amp;lt;0.001). Although CCUS follow-up data were short (median: 1.5 y), no differences in overall survival were observed between CCUS VL+L and MDS VL+L, while within CCUS survival differed between VL+L and the remaining cases ( p=0.05). Conclusions: Our data confirm the comparable mutational spectrum between CCUS and MDS but clearly show major differences in the frequency and VAF of distinct gene mutations. These biological differences hint towards different subgroups within CCUS with cases closer to MDS than others. A combined risk score for MDS and CCUS would reflect this continuous spectrum and has the potential to derive an objective risk assessment irrespective of observer-dependent grading of dysplasia. Our study indicates plausible short term results for CCUS patients stratified according to the IPSS-M. However, the definite applicability of the IPSS-M needs to be confirmed in larger CCUS studies with longer follow-up.

Uproleselan Added to Cladribine Plus Low Dose Cytarabine (LDAC) in Patients with Treated Secondary Acute Myeloid Leukemia (TS-AML)

AML arising from HMA-treated myeloid disorders, also known as “treated-secondary AML” (ts-AML) is associated with an adverse prognosis (Complete Remission [CR] rates 15-30% and median Overall Survival [OS] 6-10 months). E-selectin, a marker of cell survival and resistance to chemotherapy, is highly expressed in the leukemic microenvironment. Exposure of leukemic blasts to HMAs has been shown to increase its expression. Uproleselan is an e-selectin antagonist that has demonstrated antileukemic activity in AML. We sought to study the combination of low-intensity chemotherapy with Cladribine + LDAC (CLAD/LDAC) with uproleselan to overcome local and microenvironmental resistance. This Phase Ib/II clinical trial (NCT04848974) evaluate the safety, tolerability, and efficacy of Uproleselan added to Cladribine and LDAC. A 3+3 dose-escalation approach was implemented to evaluate 2 different dose levels for Cladribine (CLAD)+ LDAC; each 4-week cycle consists of Uproleselan (at fixed dose of 800mg intravenously [IV]). Induction and reinduction if required, included IV uproleselan twice a day from day 1-12; consolidation was given same days but once a day if patients(pts) achieved response (complete response[CR] with incomplete hematological recovery [CRi] or morphologic leukemia-free state [MLFS]). CLAD was IV for 5 days (3.75mg/m2 and 5mg/m2; level -1 and 1 respectively) and subcutaneous LDAC twice daily 10 days (15mg, and 20mg; level -1 and 1 respectively) during induction; consolidation was similar except it was with 3-days of CLAD, for up to 6 cycles, pts aged ≥18 years with a diagnosis of ts-AML with adequate organ function, who have not received therapy for their AML were enrolled. ts-AML is defined as AML arising from previously treated myeloid neoplasm. Presence of the e-selectin was assessed as exploratory analysis. 20 pts have been treated, 18 pts evaluable: 14 (70%) male, median age was 72 yrs (range, 58-86); at the start of therapy, the median bone marrow blasts were 26.5% (20-82%), median WBC was 2.1x10 9/L (0.6-26.4), and median platelets were 26x10 9/L (4-667). Pts had received a median of 1 (1-6) treatments prior to AML transformation. Prior diagnoses were: Chronic Myelomonocytic Leukemia (CMML), MDS, and MDS/MPN in 3 (25%), 14 (70%), and 1 (5%) respectively; all had received HMA, 11 (55%) additionally had received Ven, and 5 (25%) had stem cell transplantation (SCT) prior to enrolling. All pts had unfavorable cytogenetics by ELN 2022. The most frequent mutations were: TET2, TP53, RUNX1 and SRSF2 in 9 (45%), 7 (35%), 6 (30%) and 6 (30%) pts respectively. 18 pts were evaluable for e-selectin; median plasmatic concentration was 26.2ng/mL (15.3-131 ng/mL). Most common AEs were grade(gr) ≥3 neutropenic fever (13 pts, 65%; including 2 gr 5 events), gr 3 bleeding (10%) and gr 2 thrombosis (5%). There were no dose-limiting toxicities observed on dose levels -1 or 1. The chosen dose level was 1 as described above. 2/3 pts who received dose level -1 died during the study follow-up due to sepsis in the first 4-week after induction. Median time to 0.5x10 9/L neutrophil and 50x10 9/L platelets recovery was 30 (17-52) and 38 (20-69) days, respectively. The response rates were 2 (11%) CRi, 1 (6%) CRp, and 4 (22%) MLFS. There was a reduction in BM blasts in 13 pts (72%) (Figure 1). The ORR was 50%(5/10) ( p=0.60) and 20% (1/5) ( p=0.06) among pts who had prior Ven exposure or prior SCT, respectively. The median cycles received was 1.5 (1-4), median cycles at which the best response was achieved was 1 (1-2). 12 pts were taken off protocol due to progression, 3 for death, 3 for allogeneic SCT and 1 continued in remission. The pt who achieved negative MRD-negative by flow cytometry underwent SCT and is still alive. The median follow-up is 8.1 months. Median OS and EFS were 5.3 and 1.4 months respectively; 4-month RFS (CRi, CRp and MLFS) was 30% (Fig 2). In the univariate analysis by Cox regression for plasmatic E-selectin concentration the HR for OS was 1.016 (95% CI 1.002-1.031). Ts-AML, progressing from previously treated antecedent myeloid disorders, is associated with low rates of remission and poor OS. Uproleselan combined with Cladribine + LDAC was well-tolerated with minimal therapy-related AEs - allowing a safe approach to marrow blast reduction and disease control in preparation for a potential allogeneic SCT. We are currently determining the relationship of plasmatic e-selectin concentration and response to treatment.

Non-Canonical Double Mutant DNMT3A mutations: Clinical and Biological Significance in Myeloid Neoplasms

Introduction: DNMT3A is well accepted as an ancestral lesion in clonal hematopoiesis. Canonical R882 mutations ( MT) enriched in acute myeloid leukemia are considered dominant negative. In contrast, the functional impact of non-R882 lesions is quite heterogeneous and largely present in clonal hematopoiesis of indeterminate potential (CHIP) and myeloid neoplasms (MN). While it has been shown that a single non-R882 DNMT3AMT may not be as functionally impactful as R882, little is known about whether double mutant non-R882 DNMT3A (monoallelic or hemi/heterozygous biallelic) has a similar functional/biological impact as R882 canonical lesion. Studying this aspect is only feasible in a larger cohort of sequencing studies. We performed an extensive analysis to compare the prognostic outcome between single canonical and non-canonical double mutant DNMT3AMT in MN. Methods: Multiple datasets were used to construct a larger cohort of 16,565 MN patients with sequencing data. This included MN patients from Karmanos Cancer Institute and a publicly available metanalytic cohort including Awada et al., Blood 2021, Kewan et al., Nature Communications 2023, cBioPortal (Cerami et al., 2012) and AACR GENIE (v 13.1). Patients with known DNMT3A mutational status were included in the final analysis. Baseline clinical and molecular characteristics were noted. The chi-square test was used to study various parameters described, and the Kaplan-Meier curves were used for survival analyses. Results: DNMT3A was mutated in 18% (2903/16,565) of all MN which included primary acute myeloid leukemia (pAML, n=1910), secondary (sAML, n=531), myeloproliferative neoplasms (MPN, n=100), myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN, n=57), myelodysplastic syndrome (MDS, n=305) in the whole cohort. We analyzed 2903 DNMT3AMT MN and further classified them; 41%(n=1191/2903) had single R882, 48%(n=1407/2903) with single non-R882, 8%(n=226/2903) had double mutations at non-R882 locus, 1%(n=39/2903) was homozygous for R882 and 1% (n=40/2903) with mutations at R882 and non-R882 positions. Among non-R882 mutations, missense was more common [38%(85/226)] than nonsense [4%(9/226)], frameshift [1%(2/226)], and all others [57%(130/226)] (including splice-site, insertions, deletions, and other combinations in double non-R882 mutations). Double non-R882 were common in older MN patients [median age 68(37-89) vs. 63(18-96) yrs. in single R882 DNMT3AMT, p=&amp;lt;0.0001]. Double non-R882 compared to single R882 DNMT3AMT were less enriched in pAML and more in sAML [67 vs. 78%, p=0.0012 and 20 vs. 14%, p=0.010 resp]. Double non-R882 was more associated with abnormal karyotype, had lower WBC and BM blast percentage vs. single R882 DNMT3AMT [45 vs. 25%, p&amp;lt;0.0001, median WBC 11(0.5-280) vs 28 (0.1-427)x10 3/mm 3, p&amp;lt;0.010 and median BM blasts 55(0-100) vs 70(0-100),%, p&amp;lt;0.015 resp.]. Median overall survival (OS) was better in single R882 [17 vs. 13 mo. in double non-R882 DNMT3AMT patients, p=0.0013]. Single R882 with normal cytogenetics had better median OS than double non-R882 with normal and abnormal cytogenetics [20 vs. 15 mo., p=0.047 and 20 vs. 11 mo., p=&amp;lt;0.0001 resp.]. Median OS in single R882 was better than in double non-R882 DNMT3AMT p&amp;sAML patients [16 vs. 13 mo., p=0.0267 and 15 vs. 11 mo., p=0.098 resp]. IDH2 (33% vs. 17%, p=&amp;lt;0.0001), BCOR (14% vs. 8%, p=0.003) and ASXL1 (14% vs. 7%, p=0.003) were the most common associated mutations in double non-R882 whereas FLT3 (25% vs. 37%, p=0.0009) and NPM1 (19% vs. 42%, p=&amp;lt;0.0001) were common in single R882 DNMT3AMT MN. Also, a minority [20/226(9%)] of double non-R882were homozygous and had poor median OS [11.5 vs 13.5 mo., p=0.69)] than double non-homozygous non-R882 DNMT3AMT. Our analysis reveals a poor prognosis for double non-R882 vs. single R882 DNMT3AMT patients. Double non-R882 DNMT3AMT patients were older, more enriched with abnormal cytogenetics, and co-mutated with other poor prognostic mutations such as BCOR and ASXL1. BCOR and ASXL1 co-mutated patients are observed to have worse outcomes in myeloid neoplasms (data not shown here). Our ongoing analysis, to be presented at the ASH meeting, will determine if treatment and transplant may alter poor outcomes in this unique subgroup of DNMT3AMT patients. Biallelic homozygous non-R882 DNMT3AMT is rare and carries a worse prognosis, and further studies are needed to elucidate this unique subgroup of AML.