Mutations in the RAS/MAPK signaling pathway are recurrent in acute myeloid leukemia (AML), primarily involving NRAS and KRAS. In contrast, mutations in the gene encoding an effector protein, BRAF, occur at relatively lower frequencies in AML and are associated with poor outcomes. To date, no comprehensive analysis has assessed the clinical and molecular characteristics of BRAF-mutated AML. In this study, we report the identification of canonical and non-canonical BRAF mutations in ~1% of 5,779 consecutive clinically and molecularly fully-annotated AML patients treated at two major United States Cancer Centers (50/5779 AML patients: 21 newly diagnosed AML; 9 relapsed/refractory; 20 newly diagnosed secondary AML). We performed single-cell multiomic analysis on a subset of AML samples. BRAF mutations were enriched in myelodysplasia-related AML (AML-MR), and most mutations were located outside the V600 hotspot. Single-cell multiomic profiling delineated BRAF mutation class-specific patterns of co-mutations, clonality, and immunophenotypes. Notably, BRAF mutations and other signaling co-mutation(s) could be found in the same cell, a finding that significantly diverges from prior studies of RAS-mutant AML. In this cohort, BRAF-mutant AML patients had poor overall survival with currently available treatments, including venetoclax-based regimens. Drug sensitivity data suggest possible avenues for targeted treatment of BRAF-mutated AML.

Gene mutations and chromosome abnormalities are important components of prognostication in acute myeloid leukemia (AML). Here we assessed whether DNA methylation patterns in AML patients can augment risk assessments provided by genetic and other markers to better predict outcomes. Unsupervised DNA methylation patterns separating patients into 13 DNA methylation subtypes (epitypes) were used to classify 1,262 patients with de novo AML. Epitypes were predominantly comprised of a predominant genetic alteration; however, some patients within epitypes lacked cardinal alterations and were termed genetic alteration-like. Interestingly, patients displaying alteration-like DNA methylation patterns of CEBPAbZIP, FLT3-ITD, core-binding factor, KMT2A-rearrangements and other abnormalities displayed outcomes similar to patients with actual cardinal alterations. We further derived a DNA methylation signature enriched in patients with FLT3-ITD mutations that involved hypomethylation of STAT binding sites, termed the STAT hypomethylation signature (SHS). SHS positivity identified patients with inferior outcomes further adding to the prognostic significance of FLT3-ITD. Machine learning modeling revealed these DNA methylation signatures together significantly added to genetic, demographic and clinical markers to predict remission, relapse and overall survival. In summary, DNA methylation signatures capture patients who mimic cardinal genetic mutations providing additional prognostic information that may be used to in concert with standard genetic markers.

Immunoglobulins (IGs) made by chronic lymphocytic leukemia (CLL) B cells are unique in that they bind themselves (homo-dimerize). This interaction leads to signal transduction with functional consequences that depend on the affinity of homo-dimerization. We have studied the antigen-binding properties of the IGs from a subset of patients with CLL (Subset #4) that homo-dimerize at high affinity. Previously, we had found that subset #4 IGs bound viable lymphocytes. Our new studies, probing an array of >8,000 antigens, indicate that these IGs also bind influenza virus. Because of the IGs high-affinity homo-dimerization, we asked if the defined foreign- and self-antigenic interactions were mediated by conventional B-cell receptor (BCR) domains or a non-conventional receptor created by homo-dimerization. The studies indicated the latter since abrogation of homo-dimerization eliminated binding to influenza virus and its hemagglutinin and to viable lymphocytes. Using these findings, we modeled a developmental path whereby a naive IgM+ B cell with subset #4 heavy and light chain variable domains used the conventional BCR to interact with auto- and foreign antigens and acquire homo-dimerization capacity to create the non-conventional antigen-receptor when transitioning to a leukemic cell. Future studies will determine if this process is an idiosyncratic occurrence or a physiologic principle.

BACKGROUND:Acute lymphoblastic leukemia (ALL) in adults is aggressive, with long-term outcomes impacted by treatment resistance and toxicity. CD52 is expressed in most cases of B- and T-lineage ALL. Alemtuzumab, a humanized immunoglobulin G1 monoclonal antibody that targets CD52, was identified as a potential agent to improve treatment efficacy without increasing toxicity. METHODS:In this phase 1/2 study (Cancer and Leukemia Group B [CALGB] 10102, NCT00061945), a course of single-agent alemtuzumab was intercalated into CALGB 19802 backbone chemotherapy after the third course of intensive chemotherapy in those who were CD52+ at diagnosis. Phase 1 tested three dose levels of subcutaneous alemtuzumab (10, 20, and 30 mg 3 times weekly for 4 weeks/12 doses) and demonstrated that 30 mg was tolerable. Phase 2 established feasibility. RESULTS:The study enrolled 295 evaluable patients (115 in phase 1, 180 in phase 2); 206 (69.8%) were CD52+. Among evaluable CD52+ patients, 43.7% (90/206) completed the first three treatment modules; 97.8% (88 of 90) were treated with alemtuzumab. Alemtuzumab was associated with cytomegalovirus viremia, which occurred in 23.3% (14 of 60) of patients during and 29.2% (19 of 65) after alemtuzumab treatment. With a median follow-up of 101.2 months, median overall survival (OS) was 26.3 months (3-year rate, 44%; 5-year rate, 36%; 10-year rate, 31%). Landmark analysis at the start of the fourth course of treatment demonstrated no difference in OS or disease-free survival between patients who did and who did not receive alemtuzumab. CONCLUSION:Alemtuzumab was feasible to administer in adults with ALL receiving intensive chemotherapy, but was without evidence of benefit.

Acute myeloid leukemia (AML) is a hematopoietic neoplasm characterized by uncontrolled proliferation of myeloid blast cells. AML is the most common acute leukemia, with over 20, 000 new U.S. cases diagnosed annually. The 5-year survival rate varies greatly by age, from 63% in younger patients to 11% in older patients. AML is a genetically heterogeneous disease, with over 25 genes implicated in classification. Clinical next-generation sequencing (NGS) is crucial for AML management, aiding in relapse risk assessment, therapy guidance, and understanding of the genetic landscape. In AML, this typically involves sequencing a panel of 50-500 genes. In practice, only diagnostic samples are tested, without germline controls. In research settings, whole genome sequencing (WGS) is performed on paired diagnosis and remission samples, with remission serving as a germline control. This study assessed the accuracy and utility of AML clinical NGS reports by comparing with matched WGS data. We explored the benefits of germline controls and how discrepancies impact risk classification. 65 AML patients treated in the Northwell Health System and enrolled in an IRB-approved biospecimen study were analyzed. Clinical NGS testing was performed using FoundationOne Heme (n=51) or GenPath OnkoSight (n=14). We analyzed mutation data from clinical NGS reports (median 12 variants per report; range 1-26), identifying DNMT3A, FLT3, TP53, and NPM1 as the most frequently mutated genes, consistent with prior studies. Matched WGS data from 15 patients classified clinically reported variants as somatic (27%; n=57), germline (65%; n=135), or not observed (8%; n=17), using variant allele frequencies (VAFs). There was a strong but imperfect correlation between somatic/germline labels and known/unknown status, provided in the clinical NGS reports: 89% of germline variants were of unknown significance (n=120), while 83% of somatic variants were of known significance (n=43). Clinical NGS reports lacked data such as population variant frequency, pathogenicity scores, and VAFs. Germline and unknown significance variants had higher population frequencies and lower pathogenicity scores than somatic and known significance variants (p<0.0001). European LeukemiaNet (ELN) risk groups assigned using clinical NGS data (Adverse: 51%, n=33; Favorable: 29%, n=19) were affected by the inclusion of germline or unobserved variants; excluding those reclassified two patients from Adverse to Intermediate risk. This study finds general agreement between AML clinical NGS and WGS in the overlapping genes. The clinical NGS reports studied here lack annotation of germline and subclonal variant status. We highlight the limitations of clinical NGS without germline controls and its implications for AML classification, clinical decision making, and broader scientific understanding. Annette A. Prah, Derek Van Delden, Joan Alexander, Asya Stepansky, Erin Boyle, Nicholas Chiorazzi, Jonathan Kolitz, Stephanie Boisclair, Steven L. Allen, David Chitty, Andrea B. Moffitt. Comparison of clinical next generation sequencing and whole genome sequencing for acute myeloid leukemia characterization and classification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7171.

The FLT3 gene frequently undergoes mutations in acute myeloid leukemia (AML), with internal tandem duplications (ITD) and tyrosine kinase domain (TKD) point mutations (PMs) being most common. Recently, PMs and deletions in the FLT3 juxtamembrane domain (JMD) have been identified, but their biological and clinical significance remains poorly understood. We analyzed 1660 patients with de novo AML and found FLT3-JMD mutations, mostly PMs, in 2% of the patients. Patients with FLT3-JMD mutations had a higher relapse rate and shorter disease-free survival than those with FLT3-TKD, whereas their relapse rate, disease-free and overall survival were not significantly different from those of FLT3-ITD-positive patients. In vitro experiments showed that FLT3-JMD PMs transformed hematopoietic cells and responded well to type I and II FLT3 inhibitors. Molecular dynamics simulations were used to explore the conformational changes of JMD PMs relative to wild-type FLT3. These mutations exhibited constrained domain motions with wider gate openings, potentially enhancing drug binding. Altered residue interactions and structural changes shed light on their unique functional mechanisms, with increased allosteric pathways suggesting reduced interactions with other residues. We conclude that patients with FLT3-JMD PMs represent uncommon but important subset with distinct molecular and biological features, and may benefit from FLT3 inhibitors.

Importance:Therapeutic responses in acute myeloid leukemia (AML) demonstrate considerable variability both across and within established risk stratifications and age groups. Moreover, significant racial disparities persist, with Black patients experiencing inferior survival outcomes compared with their White counterparts. Objective:To validate the association of the previously reported 10 single nucleotide variant (SNV)-based ara-C pharmacogenomics score (ACS10) with survival outcomes in a large cohort of pediatric AML patients; to evaluate whether ACS10 remains relevant in an adolescent and young adult (AYA) population of patients with AML treated with similar intensive induction chemotherapy protocols; and to assess the association of ACS10 with race and treatment outcomes in both cohorts. Design, Setting, and Participants:This cohort study included patients from the Children's Oncology Group's AAML1031 trial, a multicenter, open-label randomized clinical trial that enrolled pediatric patients with newly diagnosed, treatment-naive primary AML from June 2011 to July 2017 (aged 0 to 29.5 years) and from the Alliance for Clinical Trials in Oncology frontline protocols, which included AYA patients from 9 different trials that enrolled patients with newly diagnosed AML from 1992 to 2010. Data were analyzed from September 2022 to March 2025. Exposures:Patients in the AAML1031 trial were randomized to 2 arms, standard chemotherapy alone or standard chemotherapy with the addition of bortezomib. Patients in the Alliance for Clinical Trials in Oncology cohorts were treated with similar intensive induction chemotherapy protocols. Main Outcomes and Measures:ACS10 scores were evaluated for association with outcomes according to race, treatment arm, and hematopoietic stem cell transplant (HSCT) status. Results:The study included 1086 patients with AML. There were 717 patients from the pediatric AML cohort (median [range] age, 9.6 [0.04-29.2 years]; 379 [53%] male; 33 [5%] Asian, 84 [12%] Black, and 522 [73%] White) and 369 AYA patients with AML from the Alliance for Clinical Trials in Oncology group (median [range] age, 30 [17-39] years; 196 [53%] male; 7 [2%] Asian, 32 [9%] Black, and 288 [78%] White). Within the standard treatment arm of AAML1031, patients in the low ACS10 group had significantly worse event-free survival (EFS) compared with those in the high ACS10 group (all patients: hazard ratio [HR], 1.42; 95% CI, 1.05-1.95; P = .02; non-HSCT cohort: HR, 1.48; 95% CI, 1.06-2.07; P = .02). The ACS10 score remained significantly associated with EFS in multivariable analysis after adjusting for age, race, risk group and white blood cell count, within the standard treatment arm (HR, 1.44; 95% CI, 1.03-2.02; P = .03). In the Alliance for Clinical Trials in Oncology AYA non-HSCT cohort, the low ACS10 score group had significantly inferior overall survival (OS) and a higher point estimate for EFS compared with patients with a high ACS10 score (OS: HR, 1.50; 95% CI, 1.05-2.14; P = .03; EFS: HR, 1.32; 95% CI, 0.95-1.83; P = .10). A higher number of early deaths was observed in the low ACS10 group compared with the high ACS10 group, but the difference was not statistically significant (death within 30 days of treatment initiation: 6 of 112 [5%] vs 2 of 257 [1%]; P = .07). Across both cohorts, a low ACS10 score was significantly more abundant in Black patients compared with White patients (eg, in Alliance for Clinical Trials in Oncology cohort, 27 of 32 Black patients [84%] had low ACS10 scores compared with 64 of 288 White patients [22%]; P < .001) and inferior survival was observed in Black patients (eg, OS of Black compared with White patients in AAML1031 cohort: HR, 1.47; 95% CI, 1.02-2.13; P = .04). In the AAML1031 cohort, there were no significant differences in EFS or OS between Black and White patients receiving augmented treatment, suggesting that the addition of bortezomib was associated with benefit for Black patients. Conclusions and Relevance:In this study of 717 pediatric and 369 AYA patients with AML, the ACS10 score was associated with EFS in pediatric and AYA patients when treated with a standard induction regimen. There was a higher abundance of low ACS10 scores in Black patients, and Black patients treated with augmented therapy (ie, the addition of bortezomib) seemed to have improved outcomes. Integrating the ACS10 score into a prospective clinical trial to personalize induction therapy based on an individual's genetic profile has the potential to improve treatment outcomes.

The outcome of patients with acute myeloid leukemia (AML) worsens with increasing age. Dichotomization into “younger” and “older” patients is clinically routine and often dictates treatment options. We aimed to delineate whether molecular genetic features and/or outcome measures support assorting patient populations by age, including division into “younger” and “older” groups. We analyzed 2823 adult AML patients enrolled onto frontline chemotherapy-based clinical protocols of two cooperative study groups from USA and Germany who were profiled molecularly via targeted sequencing platforms. Frequencies of gene mutations and cytogenetic findings were depicted in 5-year age increments. Clinical outcomes of 2756 AML patients were analyzed with respect to molecular features, genetic-risk groups and age. Age-associated distributions of gene mutations and cytogenetic abnormalities were similar in both cohorts. There was almost linear shortening of overall survival with increasing age among all patients (P < 0.001) and within 2022 European LeukemiaNet-defined genetic-risk groups, with survival decreasing as age increased (favorable-risk, P < 0.001; intermediate-risk, P < 0.001; adverse-risk, P < 0.001). Although mutational profiles and outcomes of the youngest patients differed from those of older patients, there was no age cut-off identifying “younger” and “older” patients. These findings support more age-associated flexibility for drug approval and trial eligibility.

Although chronic lymphocytic leukemia (CLL) is diagnosed by identifying a circulating B-cell clone that exceeds 5x103/μL, additional distinct clones (ADC) have been identified in various studies. Notably, the numbers of ADC documented in these studies has increased as the various technologies evolved. To better define the frequency and the characteristics of ADC in CLL, we used a next-generation sequencing platform that affords high sequencing depth along with steps that limit overcounting to analyze IGHV-IGHD-IGHJ gene rearrangements in circulating CD5+ B cells from 57 patients. Notably, all patients had at least one ADC, in addition to the clinically relevant clone. In 46 patients for whom lymphocyte count data were available, 44 had at least one ADC above the threshold of 1 B cell/μL and, remarkably, the average number of ADC was 12 per patient. Notably, in two patients, the predominant ADC qualified clinically as a separate CLL clone and in the remaining cases as low/high-count monoclonal B-cell lymphocytosis clones. Moreover, in 11 patients studied longitudinally, predominant ADC were persistent and often increased in number. ADC in patients with CLL exhibited 4-fold more stereotyped IGHV-IGHD-IGH rearrangements than found in CD5+ B cells from healthy individuals, and IGHV use, somatic mutations, and Ig isotype distribution were similar between predominant ADC and clinically relevant clones. Thus, finding multiple expanded clones within the CD5+ B cells is the rule in patients with CLL, indicating that leukemogenesis is a multiclonal process that likely involves competition among B cells with special B-cell receptor features.

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with demographic and genetic features. Although the associations of acquired genetic alterations with patients’ sex have been recently analyzed, their impact on outcome of female and male patients has not yet been comprehensively assessed. We performed mutational profiling, cytogenetic and outcome analyses in 1726 adults with AML (749 female and 977 male) treated on frontline Alliance for Clinical Trials in Oncology protocols. A validation cohort comprised 465 women and 489 men treated on frontline protocols of the German AML Cooperative Group. Compared with men, women more often had normal karyotype, FLT3 -ITD, DNMT3A , NPM1 and WT1 mutations and less often complex karyotype, ASXL1 , SRSF2 , U2AF1 , RUNX1 , or KIT mutations. More women were in the 2022 European LeukemiaNet intermediate-risk group and more men in adverse-risk group. We found sex differences in co-occurring mutation patterns and prognostic impact of select genetic alterations. The mutation-associated splicing events and gene-expression profiles also differed between sexes. In patients aged <60 years, SF3B1 mutations were male-specific adverse outcome prognosticators. We conclude that sex differences in AML-associated genetic alterations and mutation-specific differential splicing events highlight the importance of patients’ sex in analyses of AML biology and prognostication.

Genomic profiles and prognostic biomarkers in patients with acute myeloid leukemia (AML) from ancestry-diverse populations are underexplored. We analyzed the exomes and transcriptomes of 100 patients with AML with genomically confirmed African ancestry (Black; Alliance) and compared their somatic mutation frequencies with those of 323 self-reported white patients with AML, 55% of whom had genomically confirmed European ancestry (white; BeatAML). Here we find that 73% of 162 gene mutations recurrent in Black patients, including a hitherto unreported PHIP alteration detected in 7% of patients, were found in one white patient or not detected. Black patients with myelodysplasia-related AML were younger than white patients suggesting intrinsic and/or extrinsic dysplasia-causing stressors. On multivariable analyses of Black patients, NPM1 and NRAS mutations were associated with inferior disease-free and IDH1 and IDH2 mutations with reduced overall survival. Inflammatory profiles, cell type distributions and transcriptional profiles differed between Black and white patients with NPM1 mutations. Incorporation of ancestry-specific risk markers into the 2022 European LeukemiaNet genetic risk stratification changed risk group assignment for one-third of Black patients and improved their outcome prediction. Analysis of exomes and transcriptomes from 100 African American patients with acute myeloid leukemia identifies ancestry-related variation in mutation profiles and survival. Refined risk classification suggests clinical relevance of these ancestry-associated differences.

TCR mediated T cell activation is less robust in patients with CLL than healthy controls (HC), contributing to an immune deficiency that impairs quality and longevity of life. In HCs, T cell activation begins with myeloid or B cells acquiring and processing antigen and then presenting antigenic peptide-MHCII complexes to T cells. Activated B cells are more effective antigen-presenters than resting cells. In CLL, the process is defective, particularly for immune synapse formation with T cells. Most CLL cells and unswitched normal B cells express surface membrane (sm) IgM and IgD. Since both molecules bear the same antigen binding site, each initiates the antigen processing and presentation activity. However, documentation that both do this in the same way is lacking. We investigated if delivery of foreign antigen to CLL and normal B cells selectively by sm IgM or by smIgD influences antigen delivery, processing, and presentation, and subsequent T-cell activation. We recombinantly modified two murine mAbs of comparable affinities for IgM or for IgD so they bear human IgG4 constant regions to obviate Fc receptor interaction (r-αIgM, r-αIgD), and so they carry (or not) the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (r-αIgM-RBD, r-αIgD-RBD). Using the CXCR4DimCD5Bright, proliferative fraction (PF) of CLL cells, which is enriched in antigen presentation molecules and capacities, we assessed the efficiency of internalization of smIg and of associated RBD, and the ability of preferentially targeted B cells to present RBD peptides to T cells; we used flow cytometry to measure induction of sm CD69, CD134, CD137, and intracellular (ic) IFNγ and IL-4. For confocal microscopy, we used pHrodo labelled r-αIgM (Deep Red) and r-αIgD (Green) to determine if IgM and IgD deposit antigen in distinct ic compartments. Exposing CLL cells to the mAbs at 4oC, showed although the PF has significantly more smIgM than smIgD (P=0.0141), preferential engagement of sm IgD or IgM at 37oC led to significantly higher levels of ic IgD than IgM (P=0.0402). Similar data were found for normal B cells (P=0.0302). Next, we tested if delivery of RBD selectively to smIgM or smIgD led to differences in T cell activation based on increases in activation antigen and Th1 and Th2 cytokine levels. Consistent with previous observations, incubation of resting T cells with PF cells led to increases in T cell numbers (Total: P=0.0370; CD4: P=0.0322; CD8: P=0.0420). So this value was used as the baseline for all comparisons. Exposure of T+PF to r-αIgM or r-αIgD did not significantly change the 5 readouts (not shown). Notably, engagement of smIgM on PF cells by r-αIgM-RBD never led to increases in T cell numbers, T cell activation antigen expression, or cytokine production over baseline. In contrast, engagement of smIgD with r-αIgD-RBD increased T cell numbers and in 3 T cell readouts over the same baseline (CD8+P=0.0445; CD4+CD134+P= 0.368; CD8+CD134+P=0.0385). Additionally, significant differences were found after comparing r-αIgD vs r-αIgD-RBD for multiple readouts (CD3+P=0.0087; CD4+P=0.0161; CD8+P=0.0029; CD4+CD69+P=0.0013; CD4+CD134+P=0.0010; CD4+CD137+P=0.0172; CD4+IL-4+P=0.0334; CD8+CD69+P=0.0020; CD8+CD134+P=0.0008; CD8+CD137+P=0.0006; CD8+IFNg+P=0.0008; CD8+IL-4+P=0.0012). This was never the case for r-αIgM vs r-αIgM-RBD. Finally, T cell activation was significantly greater when comparing r-αIgD-RBD to r-αIgM-RBD (CD3+P=0.0070; CD4+P=0.0045; CD8+P=0.0062; CD4+CD69+P=0.0012; CD4+CD134+P=0.0014; CD4+CD137+P=0.0278; CD8+CD69+P=0.0042; CD8+CD134+P=0.0004; CD8+CD137+P=0.0004; CD8+IFNg+P=0.0157; CD8+IL-4+P=0.0206). Thus, antigen delivered by smIgD is more stimulatory to T cells than if delivered by smIgM, and these activating events are occurring on RBD-specific T cells. Similar results were observed in normal B cells. To determine if the ic “handling” of antigens differed based on the route of entry, we incubated CLL cells with r-αIgM and r-αIgD, each labeled with a distinct fluorophore that emits at acid pH. This showed that r-αIgM and r-αIgD entered distinct compartments. CLL B cells activated through IgD more effectively induce T-cell activation than when activated through IgM, suggesting the presence of a distinct B cell activation pathway in CLL and normal B cells. Using this immunostimulatory pathway might allow enhanced immune responses to foreign and potentially tumor antigens.

Abstract Although chronic lymphocytic leukemia (CLL) is diagnosed by identifying in a patient’s blood a B-cell clone that exceeds 5x106/µL, additional clones have on occasion been identified. To characterize such clones in patients with CLL, we analyzed by NGS the IGHV-IGHD-IGHJ gene rearrangements in circulating CD5+ B cells from 57 untreated patients. In every patient, we identified additional distinct clonotypes (ADCs) from the clinically relevant clone (CRC). Even when defining clonal expansion using a rigorous threshold, 68% of patients had at least one expanded ADC. The predominant ADC (pADC) reached numbers qualifying as a CLL clone in two patients and qualifying as low/high-count monoclonal B-cell lymphocytosis in each other case. Over time, pADCs enlarged or shrank, suggesting an ongoing selection process. Stereotyped IGHV-D-J rearrangements were enriched in ADCs and IGHV usage and mutations revealed similarities specifically between pADCs and CRCs, while minor ADCs more closely resembled normal CD5+ B-cells. Thus, finding multiple expanded clones within the CD5+ B-cell population of patients with CLL is more the rule rather than the exception. Thus, leukemogenesis involves a competitive selection for dominance of more than one B-cell clone, with the process being influenced to some degree by the structure of the B-cell receptor.

Venetoclax (Ven) combined with a hypomethylating agent (HMA) enhances survival in elderly/unfit acute myeloid leukemia (AML) patients, yet often necessitates regimen modifications due to intolerance. However, it is unclear how these modifications affect patient outcome. This retrospective cohort study evaluates the impact of post-induction HMA/Ven regimen modifications on disease progression and survival. This study reviewed 142 AML patients treated with HMA/Ven within the Northwell Health System from January 2019 to December 2022. To assess the impact of post-induction regimen modifications, patients were grouped according to median days between cycles (≤34 or ≥35 days cycle intervals) and median Ven days per cycle (≤14 or ≥15 days/cycle) based on only cycle 3 and beyond. Kaplan-Meier and Cox proportional hazard regression analyses were employed for univariate and multivariate assessments, respectively. There was no significant difference in median progression-free survival (mPFS)(11.6 vs 11.8 months, p = 0.73) or median overall survival (mOS)(15.1 vs 21.8 months, p = 0.16) between cycle interval groups. However, there was a clinically and statistically significant advantage in mPFS (15.8 vs 8.7 months, p = 0.01) and mOS (24.7 vs 11.3 months, p = 0.006) for patients with a median of ≤14 Ven days/cycle compared to ≥15 Ven days/cycle. Multivariate analysis demonstrated that ≤14 days of Ven for cycle 3 and beyond was an independent predictor of decreased mortality (HR 0.18, CI 0.07-0.48, p = 0.0007). Extended cycle intervals did not adversely affect mortality while reduced Ven duration per cycle post-induction was associated with improved survival in elderly AML patients.

CLL B cells express elevated pro-survival BCL2, and its selective inhibitor, venetoclax, significantly reduces leukemic cell load, leading to clinical remission. Nonetheless, relapses occur. This study evaluates the hypothesis that progressively diminished BCL2 protein in cycling CLL cells within patient lymph node niches contributes to relapse. Using CFSE-labeled, purified CLL populations known to respond with vigorous cycling in d6 cultures stimulated with TLR9-activating ODN (oligodeoxynucleotide) + IL15, we show that BCL2 protein progressively declines during consecutive cell divisions. In contrast, MCL1 and survivin are maintained/slightly elevated during cycling. Delayed pulsing of quiescent and activated CLL cultures with selective inhibitors of BCL2 or survivin revealed selective targeting of noncycling and cycling populations, respectively, raising implications for therapy. To address the hypothesis that BCL2-repressive miRs (miR15a/miR16-1), encoded in Chr13, are mechanistically involved, we compared BCL2 protein levels within ODN + IL15-stimulated CLL cells, with/without del(13q), yielding results suggesting these miRs contribute to BCL2 reduction. In support, within ODN-primed CLL cells, an IL15-driven STAT5/PI-3K pathway (required for vigorous cycling) triggers elevated p53 TF protein known to directly activate the miR15a/miR16-1 locus. Furthermore, IL15 signaling elicits the repression of BCL2 mRNA within 24 h. Additional comparisons of del(13q)+ and del(13q)−/− cohorts for elevated p53 TF expression during cycling suggest that a documented miR15a/miR16-1-mediated negative feedback loop for p53 synthesis is active during cycling. Findings that robust CLL cycling associates with progressively decreasing BCL2 protein that directly correlates with decreasing venetoclax susceptibility, combined with past findings that these cycling cells have the greatest potential for activation-induced cytosine deaminase (AICDA)-driven mutations, suggest that venetoclax treatment should be accompanied by modalities that selectively target the cycling compartment without eliciting further mutations. The employment of survivin inhibitors might be such an approach.

Background: The addition of midostaurin (M), an oral multi-kinase inhibitor, to intensive chemotherapy (IC) prolongs survival in newly diagnosed (ND) FLT3 mutated (m) AML. It is not known if there is a benefit to more potent and selective FLT3 inhibitors. Among these is Gilteritinib (G), now approved as a single agent in relapsed/refractory FLT3m AML. Combined with IC, G also leads to high measurable residual disease (MRD)negative (-) composite complete remission (CRc) in FLT3 Internal Tandem Duplication (ITD)m AML. The aim of the PrECOG 0905 study was to compare FLT3m clearance and CRc rates of G vs M with IC in adults with ND FLT3m AML. Methods: We conducted a randomized, open-label, phase 2 trial comparing G to M in combination with IC during induction and consolidation in ND AML pts with centrally PCR detected FLT3 ITD or TKD mutations. Pts age 18-70 with non M3 AML and no known core binding factor rearrangement were eligible. Pts were stratified by mutation type (TKD only vs. ITD (+/- TKD)), NPM1 mutation status, and FLT3-ITD allelic ratio. Induction consisted of cytarabine 100 mg/m2 by continuous infusion daily, on day (d) 1-7 and daunorubicin 90 mg/m2 IV on d 1-3. G 120 mg daily or M 50 mg twice daily was given orally on d8-21. Consolidation (up to 4 cycles) began within 60 d of induction with Cytarabine (1.5-3 g/m2 IV) X 6 doses and G or M (as per randomization) on d8-21. Pts could proceed to transplant (HCT) at any time. Maintenance was not included. The primary outcome was FLT3m MRD (-) CRc after induction. MRD for TKD mutations was tested by PCR (sensitivity 10-2); ITD MRD was tested by amplicon-based NGS (sensitivity 10-4). Safety and efficacy were analyzed in all eligible pts who received treatment. Secondary endpoints included comparison of CRc, MRD by flow cytometry (sensitivity 10-3), and survival. Results: From 11/2019 to 11/2022, 722 pts were screened at 37 centers, 180 pts randomized, and 177 pts (58% female, 77% white, 8.5% black, 8.5% hispanic) treated on the G (n=90) or M (n=87) arm. Median age was 54, with 27.7% > age 60. FLT3-ITD was present in 80% in the G arm and 78.2% in the M arm. Of those with FLT3-ITD, NPM1 was detected in 56.9% pts in G arm and 58.8% in M arm. 24.4% G and 20.7% M pts had co-occuring FLT3-ITD, NPM1 and DNMT3A mutations. Adverse karyotype was seen in 3.4% G and 9.3% M while 10% G and 13.8% M were ELN 2022 adverse risk. For pts on G arm, 85.6% achieved CRc compared to 72.4% for M (p=0.042). 5 (5.6%) pts on G arm and 6 (6.9%) on M arm received 2 cycles in induction. Post induction, the FLT3m- CRc rate was 40% for G vs 47.1% for M (p=0.366). FLT3-TKD only mutated pts achieved CRc in 88.9 % with G and 63.2% with M and FLT3m- CRc in 72.2% and 63.2% respectively. FLT3-ITDm (+/- TKDm) pts achieved CRc/FLT3m- CRc in 84.7%/31.9% with G and 75%/42.6% with M, respectively. In pts with FLT3-ITD, NPM1 and DNMT3A mutations, 95.5% G and 72.2% M achieved CRc and 50% G vs 33.3% on M achieved FLT3m- CRc. Flow cytometry MRD negative CRc was documented in 64.4% on G arm and 59.8% on M arm after induction (p=0.539). On G arm 67 (74%) patients received at least one cycle of consolidation compared to 53 (61%) pts on M arm. On exploratory analysis, 54 (66%) G and 40 (46%) M were reported to proceed to HCT in first remission. FLT3m testing was done post consolidation cycle 1 in 27 pts in FLT3m+ CRc after induction and 15/18 (83%) on G and 4/9 (44.4%) on M converted to FLT3m-. On multivariate logistic regression analysis for FLT3m-CRc post-induction, FLT3-ITD allelic ratio, NPM1m, WBC and hemoglobin at baseline were significant at the 0.10 two-sided level, while treatment arm, and 2017 ELN risk were not. There were no deaths from treatment-related (TR) adverse events (TRAEs) while grade >=3 TRAE was reported in 73%G vs 70% M during induction and 79%G vs 73%M pts during consolidation. Conclusion: Induction therapy with daunorubicin 90 mg/m2 X 3 days, cytarabine 100 mg/m2 and gilteritinib results in an excellent CRc rate with no TR deaths in pts with ND FLT3m AML up to age 70. Compared to M, G increased the CRc but not the FLT3m- CRc rate after induction. More post induction FLT3m+CRc pts who received G became FLT3m- after consolidation cycle 1 and more G pts succeeded in proceeding to HCT. Future survival data will help evaluate the clinical impact of G and MRD assessments in ND FLT3mAML. Larger studies will be needed to establish most predictive timing of MRD and for definitive comparisons of these drugs in patients with specific mutation profiles ( ie TKD, ITD/NPM1/DNMT3).

Introduction The revised 5th edition of the World Health Organization Classification of Haematolymphoid Tumours and the 2022 European LeukemiaNet (ELN) genetic-risk classification have led to profound changes in the classification, risk assignment, and associated treatment options for AML pts. One of the major changes affects AML-MR. Pts with MR-mutations are now considered to have adverse-risk, except when MR-mutations co-occur with favorable-risk AML subtypes. However, AML-MR is a clinically and molecularly heterogeneous group with diverse clinical outcomes when treated with intensive chemotherapy.Although gene mutations and recurrent cytogenetic abnormalities are equally included as AML-MR-defining genetic events, the associations of specific genetic lesions with pretreatment features and clinical outcome may differ. Thus, data-driven support for the definition of AML-MR is needed to improve risk assignment and subsequent treatment guidance for pts. We hypothesized that better portrayal of tumor biology characterized by gene expression-based genetic phenotype may help refine AML-MR classification. Methods We performed transcriptome-based gene-expression profiling and t-distributed stochastic neighbor embedding (t-SNE) data visualization on a clinically and molecularly well-characterized cohort of 971 newly diagnosed AML pts who were similarly treated with intensive chemotherapy on CALGB/Alliance frontline chemotherapy-based protocols. Using transcriptome-based gene-expression profiles of pts diagnosed with myelodysplastic neoplasms, we defined an MDS similarity score, which was almost exclusively found in a relatively large, central t-SNE cluster enriched for pts with AML-MR-defining genetic lesions. We recognized 3 pt groups (n=377 pts): pts with both AML-MR-defining genetic lesions and MR-expression profile (n=149), pts who had the MR-expression profile but not AML-MR-defining genetic lesions (n=99) and pts who harbored AML-MR-defining genetic lesions but did not have the MR-expression profile (n=129). Clinical and molecular characteristics and treatment outcomes of pts in these 3 groups were then compared. Results Treatment outcomes of pts with MR-expression profile with and of those without AML-MR-defining genetic lesions were equally poor, whereas pts with AML-MR-defining genetic lesions but without the MR-expression profile had superior complete remission (CR) rates (43% vs 45% vs 69%, P<.001), and overall survival (OS; 3-y rates: 18% vs 14% vs 39%, P<.001). Because the outcome of pts with MR-expression profile who harbored AML-defining genetic lesions was very similar to outcome of pts with MR-expression profile who did not, we next combined these 2 pt groups into one MR-expression group (termed “MR-e”, n=248) and compared this group with the “mutation only” (“MR-mut”, n=129) group. Pts in the MR-e group were older than pts in the MR-mut group (median, 57 vs 48 y, P<.001). We next performed cell type quantifications using Dampened Weighted Least Squares (DWLS) based on our recently established single cell labels (Lasry et al, 2022) for deconvolution. Here, MR-e patients had a more HSC-like phenotype and a larger fraction of CD4+ T cells. In comparative survival analyses, MR-e patients had lower CR rates (44% vs 69%, P<.001), and worse OS (3-y rates: 15% vs 39% P<.001) than MR-mut pts. Within the 2022 ELN Favorable genetic-risk group, although pts with MR-expression profile had similar CR rates (60% vs 81%, P=.21), their OS was worse (3-y rates: 11% vs 60%, P<.001) than OS of MR-mut pts. In multivariable analyses, the presence of MR-e independently associated with lower CR rates (P=.004), adjusting for SF3B1 mutations and shorter OS (P<.001), adjusting for IDH1/2 mutations). Co-existing IDH1/2 mutations were the only identified genetic feature outweighing the negative prognostic effect of MR-e. Conclusions We have identified an expression-based MDS-similarity score (MR-e) as a possible phenotypic correlate for AML-MR pts that also includes patients without AML-MR defining genetic lesions. Its presence portends very poor treatment outcome, especially in pts classified in the 2022 ELN Favorable genetic-risk group. In contrast, the absence of MR-e associates with better CR and OS in pts carrying AML-MR-defining lesions. Validation of these findings in an independent data set of intensive chemotherapy treated AML-MR patients is ongoing.