Adverse Risk Disease Research Articles

Diagnostic genomic analysis is prognostic in AYA patients with ALL treated on an MRD-stratified pediatric protocol

AbstractOver the last decade, next-generation sequencing has enabled classification of multiple new recurrent genomic drivers of acute lymphoblastic leukemia (ALL). The aim of this study was to describe the genomic drivers of ALL in an adolescent and young adult (AYA) cohort (ALL06; target age, 15-39 years; recruited age, 16.6-39 years), treated uniformly on a pediatric-inspired protocol (the Australasian Leukaemia and Lymphoma Group ALL06 study). ALL06 assessed the safety and efficacy of adapting a pediatric chemotherapy protocol in older patients. Genomic risk classification of patients with B-cell ALL (B-ALL) and T-cell ALL (T-ALL) enrolled to the study was based on the use of multiple assays: messenger RNA sequencing, multiplex ligation-dependent probe amplification, immunophenotyping, and cytogenetics. Using this approach, 36 of 40 (90%) patients with B-ALL and 13 of 17 (76.5%) patients with T-ALL were classified according to genomic risk. A strong correlation existed between adverse genomic risk and minimal residual disease (MRD) at the end of consolidation, translating to inferior overall and relapse free survival. Patients with adverse risk genomics who achieved negative MRD status had improved responses compared with those with persistent MRD. Patients with standard-risk genomics had excellent responses regardless of MRD status. This is, to our knowledge, the first report of the impact of genomics in an individual cohort of AYA patients treated on a single protocol. These data argue strongly for incorporation of a genomic risk classification into future ALL treatment paradigms at the time of diagnosis, and also for the rigorous assessment of risk assignments in a group of patients who are not children and not older adults. This trial was registered at https://anzctr.org.au/ as #ACTRN12611000814976.

Clinical outcomes of patients receiving venetoclax and hypomethylating agents for AML in academic and academic-community partnerships.

e23235 Background: Prior studies have reported improved outcomes for acute myeloid leukemia (AML) in academic over nonacademic settings potentially due to the availability of multidisciplinary teams, clinical trials and increased resources (1,2). Our institution has partnered with community practices to further evaluate this disparity. In this study, we analyzed treatment outcomes when nonacademic centers partnered with academic centers to treat AML using a regimen of venetoclax and a hypomethylating agent. Methods: We reviewed 92 patients with newly diagnosed AML treated with venetoclax and either decitabine or azacitidine at an academic center (n = 57) or community practice with academic center support (n = 35). When partnering with the community, patients received the first cycle of therapy at an academic center. Before discharge, our team completed financial screening, scheduled follow up appointments and provided prescriptions. The primary objective was to evaluate the overall response rate (ORR) with a secondary evaluation of differences in chemotherapy management and complications. Results: Patients in the academic setting tended to have more adverse risk disease (80.7% vs. 74.3%, p = 0.36) according to the European LeukemiaNet (ELN) (table). The overall survival (OS) of all patients was 10.7 months (95% CI 9.4-15.7) with no significant difference between the groups. The ORR was similar between the community and academic settings (40.0% vs. 36.8%, p = 0.46). The complete remission (CR) rate was 11.4% in the community versus 19.3% in the academic setting (p = 0.67), and the CR rates were improved in the academic group versus the community after cycle 2 (12.9% vs. 0%, p = 0.72) and cycle 3 (25.0% vs. 0%, p = 0.46). Adverse events were comparable, except for increased rates of fatigue (80.7 vs. 54.3%, p = 0.01) and diarrhea (33.3 vs. 11.4%, p = 0.04) in the academic setting. Conclusions: Contrary to prior studies, our evaluation and ongoing partnerships support the transition of AML treatment to a community setting with academic consultation. Although there were some differences in adverse events, the OS and ORR were similar. Furthermore, this study did not identify any significant safety concerns that could prevent patients from receiving this regimen in either setting. Our goal is to continue evaluating this partnership, as we design prospective studies to improve the care of this patient population. 1. Giri S et al. OS of AML Treated at Academic Center Vs Nonacademic Center. Blood 2015. 2. Halpern AB et al. Practice patterns & outcomes for adults with AML. Hematol Am Soc 2020.[Table: see text]

Evaluation of the toxicity and outcomes of the combination of midostaurin and CLAG-M in patients with FLT3-mutated acute myeloid leukemia (AML): A multi-center retrospective analysis.

6523 Background: FLT3-mutated AML is associated with poor outcomes. Addition of midostaurin (multi-targeted kinase inhibitor) to standard “7+3” with cytarabine and daunorubicin significantly prolongs overall and event-free survival for these patients (Stone 2017). At the University of Washington/Fred Hutchinson Cancer Center (UW/FHCC), the standard regimen for newly diagnosed (ND) and relapsed/refractory (r/r) AML is cladribine, high-dose cytarabine, GCSF, and mitoxantrone (CLAG-M); midostaurin is added if FLT3-mutated (Halpern 2018). There is currently no peer-reviewed literature to support this combination for these patients. The purpose of this study is to evaluate the safety and efficacy of the combination of midostaurin with CLAG-M in FLT3-mutated AML patients and compare the toxicity profile to midostaurin plus 7+3. Methods: This is a retrospective multi-center review conducted at three major cancer centers. Through pharmacy records and/or an institutional AML database, we identified adults with FLT3-mutated AML undergoing (re)induction chemotherapy who received either CLAG-M (UW/FHCC, Swedish) or 7+3 (OHSU, Swedish). The primary outcome was toxicity profile of the combination as measured by rate of adverse events ([AE], CTCAE Version 5.0). Secondary outcomes included disease response per ELN2017 AML working group criteria and 28-day mortality (TRM) (Döhner 2017). Patients treated on a clinical trial and in whom midostaurin was started > 30 days after day 1 of chemotherapy were excluded. Rates of AEs were compared using Fisher’s exact test. Results: Eighty patients treated between 10/2022-12/2023 were included; 35 patients received CLAG-M, and 44 patients 7+3. Baseline characteristics were similar across all institutions, with most patients having adverse or intermediate risk disease. AE rates were similar between the two cohorts, (Table 1) except diarrhea and bleeding events were more common in the 7+3 vs. CLAG-M cohort (p = 0.004 and p = 0.037, respectively). The rate of complete remission (CR) plus CR with incomplete blood count recovery (CRi) did not significantly differ between the two cohorts: CLAG-M, 86% versus 7+3, 70% for 7+3 (p =0.11). No TRM occurred. Conclusions: The toxicity profile of CLAG-M combined with midostaurin is comparable to the combination of 7+3 with midostaurin, and induces high remissions rates in adults with FLT3-mutated AML. [Table: see text]

Abstract 3352: A CRISPR-enabled function genomics profiling platform in acute leukemia: Updates from a pilot study of Function (Fx) Heme utilizing fresh patient samples

Abstract Introduction: In this study we utilized a novel primary cell CRISPR/Cas9 profiling approach to identify gene dependencies in fresh acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) patient samples. The primary aim of this prospective study was to demonstrate the feasibility of the CRISPR-based personalized functional genomic testing with fresh patient samples in the clinical setting. Methods: AML and ALL patient peripheral blood samples (10-16 mL) were obtained at time of initial diagnosis and/or at relapse, and if feasible post-treatment. Primary cells were transduced with lentivirus harboring Cas9 enzyme and a ‘drug targets’ sgRNA panel. Transduced cells were harvested at early (input) and Day 8 timepoints, and sgRNA distribution was assessed by amplicon sequencing of DNA barcodes. Changes in barcode abundance were quantitated and aggregated to calculate gene-level phenotype scores, enabling identification of gene dependencies. Results: 37 patients were included in the study, 23 with AML, 9 with Philadelphia (Ph) (+) ALL, 5 with Ph (-) ALL. The majority of patients had adverse or poor risk disease. The average age of patients was 52.6, range 18-84. 23 were male, 14 female; 20 identified as white, 17 as other. 20 patients were sampled at initial diagnosis, 12 were sampled at time of relapse and 5 at time of refractory disease. Serial samples were collected from 9 patients. 34 patients received treatment following sample collection. The majority of patients received intensive chemotherapy or a hypomethylating agent with venetoclax, however 15 patients received some form of targeted therapy. At the time of the abstract submission, Fx Heme results were available for 23 patients and among these, we identified potential treatment targets in 10 samples (43%). Importantly, 60% of the positive samples have a dependency on a gene clinically targetable by an approved leukemia drug. In addition, our profiling platform enabled the discovery of novel druggable dependencies in 100% of the positive samples. Conclusions: With this pilot study we demonstrated that utilizing small volume fresh patient samples to assess CRISPR-defined gene dependencies on targeted therapies in AML and ALL is feasible, timely, and results in clear Fx Heme dependency gene output. Our data indicates that CRISPR-based comprehensive functional profiling has the potential to add valuable information beyond traditional NGS methods to determine the optimal therapy for patients with AML and ALL. Future directions include translating further into the clinical setting and correlating Fx Heme dependence with response to targeted treatment in acute leukemia patients. Citation Format: Kaitlyn C. Dykes, Tuyet R. Tan, Karl Hodel, Kathleen Tran, Maci Meyers, Leonardo Hagmann, Michele Baltay, Peter Kilfeather, Yali Li, Christian Schmedt, Srinath Sampath, Srihari Sampath, Tiffany N. Tanaka, Stephane Corbel. A CRISPR-enabled function genomics profiling platform in acute leukemia: Updates from a pilot study of Function (Fx) Heme utilizing fresh patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3352.

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

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

Association between BMI and Survival in Adolescents and Young Adults (AYA) with Acute Myeloid Leukemia Treated with Intensive Chemotherapy

Introduction: Obesity is linked to a higher incidence of acute myeloid leukemia (AML) in older adults as well as B-cell acute lymphoblastic leukemia in children and adolescents and young adults (AYA). To date, however, the relationship between weight and clinical outcomes in AYA patients (pts) diagnosed with AML has not been clearly established. We hypothesized that obesity reflected by higher body mass index (BMI) would confer worse prognosis in AYA pts treated with intensive chemotherapy and/or allogeneic stem cell transplant (SCT) for AML. Methods: We conducted a single institute retrospective chart review of AYA patients aged 18 to 39 years diagnosed with AML between Jan 2006 and Oct 2022. All patients received intensive chemotherapy with cytarabine- and anthracycline-based induction and consolidation chemotherapy followed in some cases by SCT. BMI was calculated based on height and weight on the date of AML diagnosis. Additional information including European Leukemia Net (ELN) 2022 risk classification, therapy response (complete remission (CR), CR with incomplete count recovery [CRi]), receipt of SCT, relapsed disease, event-free (EFS), overall survival (OS) and percentage of pts alive at 2- and 5-years after diagnosis were collected. All data was de-identified and procured on an IRB-approved protocol. Results: Seventy-six AYA patients were identified. Median age was 25.2 (range 18-39) years. 39 (51%) were female. The majority (65, 86%) were Caucasian. Per ELN 2022 risk classification, 24 (32%) had favorable, 36 (47%) had intermediate, and 16 (21%) had adverse risk disease. The CR rates based on ELN 2022 risk classification were 86%, 72% and 50%, respectively. Median OS for all pts was 791 days and per risk group was 1379, 569, and 595 days, respectively. Pts were then categorized per initial BMI into 4 categories. The majority of pts (44, 58%) had above average BMI (≥ 25), with 21 (28%) considered overweight with BMI 25-29.9 and 23 (30%) obese defined as BMI >30. Only 3 pts (4%) were underweight with BMI <18.5 (Table 1). Of note, 43% of obese pts had ELN favorable risk vs 33% of overweight pts and 24% of normal weight pts. Overall response rates in each BMI category were similar, ranging from 96-100% except in overweight pts where only 6 of 21 pts (29%) obtained CR. Relapse occurred in approximately one third (29-35%) of pts in the three highest BMI categories. Two thirds of all pts with BMI ≥18.5 underwent SCT. Excluding underweight pts due to small numbers (n=3), there was a trend to decreased survival in pts with BMI ≥18.5. Median overall survival among normal, overweight, and obese pts was 1403, 1295, and 777 days, respectively, with a significant difference between healthy and obese pts (p-value 0.0274 by log rank test). The percentage of pts alive after 5 years was 41%, 29%, and 17%, respectively (Table 2). Conclusion: Our real-world single-institute retrospective analysis suggests that AYA pts aged 18-39 yrs old diagnosed with AML and with higher BMI have decreased overall survival following intensive chemotherapy and/or allogeneic transplant. Obese patients with the highest BMI (>30) had a markedly shorter overall survival (median 777 days) and lower percentage of pts (17%) alive after five years following therapy than AYA AML pts with normal BMI (median 1403 days, 41% 5-year survival). This was despite over 40% of obese pts having favorable ELN risk. Potential reasons for this include biological heterogeneity in BMI cohorts, chemotherapy dosing, weight-related medical co-morbidities (i.e. cardiovascular disease) and/or transplant-related complications leading to shorter survival. Additional analyses to rule out the contribution of these and other factors and to confirm these results in a larger cohort are ongoing.

Presence of Recurrent Somatic Mutations in Mesenchymal Stromal Cell Fractions Isolated from Acute Myeloid Leukemia As an Evidence of Clonality

Introduction: Acute myeloid leukemia (AML) involves somatic mutations in hematopoietic stem cells (HSCs). Recent evidence suggests the bone marrow microenvironment, particularly mesenchymal stromal cells (MSCs), also influences AML development. Abnormalities in MSCs from AML patients have been observed (von der Heide et al., 2016), potentially impacting leukemia progression. This study aims to identify MSC somatic mutations and their consequences, further exploring their role in AML. Understanding MSCs' impact on the leukemic microenvironment may shed light on AML pathophysiology and guide targeted therapies, particularly benefiting patients with adverse risk disease. Methods: A total of 28 bone marrow cases were collected from AML patients with adverse risk, including those with complex karyotype, monosomy karyotype, chromosome 5/7/17 abnormalities, TP53 mutation, or relapsed/refractory AML. From these cases, 14 paired MSC and leukemic cell (LC) fractions were analyzed and are presented here. Magnetic bead sorting was used to isolate CD33+ LCs, while the plastic adherence method was employed for MSC culture. Following magnetic isolation, CD33- cells were plated in DMEM-LG with FBS and pen/strep, and MSC colonies formed within 48 hours. Contaminating cells were removed through media changes, and passaging was performed at 70-80% confluency. Flow cytometry at passage 3 characterized MSCs based on cell surface expression of CD105, CD73, and CD90, in accordance with the ISCT guideline. DNA and RNA were extracted from MSCs at passage 3 and subjected to whole exome sequencing (WES) using the Illumina platform, along with the LC fractions. WES aimed for 200X depth for the paired DNA samples. A standard bioinformatics pipeline was used for sequence alignment and variant calling, with variants requiring sufficient read depth (≥30x) and variant allele frequency in the case (>5%) and in the control (<5%). Synonymous variants were filtered out, and gene ontology and pathway enrichment analyses explored the biological significance and potential involvement in leukemogenesis of the identified mutations. Whole transcriptomics for gene expression analyses was also conducted for both MSC and LC fractions. Results: In 14 analyzed cases, a total of 548 somatic variants were identified in the MSC fraction, affecting 480 genes, including 518 nonsynonymous single nucleotide variants (SNVs), 7 stop-gain SNVs, 1 stoploss, 1 non-frameshift insertion, 2 frameshift deletions, 1 frameshift insertion, and 2 non-frameshift deletions, with 16 unknown variants. The average read depth was 75 (range: 15 - 887), and the average variant allele frequency was 50% (range: 11% - 90%). On average, 38 genes were mutated per MSC case, with 8 genes detected in at least 3 cases, 25 in exactly 2 cases, and 447 genes found in only one case. In the leukemic fraction, 686 somatic variants in 595 genes were identified, with an average read depth of approximately 75 (range: 10 - 2596) and an average variant allele frequency of 47% (range: 8% - 85%). On average, 48 mutations were detected per leukemic case, with 12 mutations found in at least 3 cases, 40 in 2 cases, and 543 in only one case. Among the 14 cases, 10 cases (71%) had mutations in known driver genes for AML ( FLT3, NPM1, IDH1, IDH2, DNMT3A, RUNX1, TP53) exclusively in the leukemic fraction. As seen in Figure 1., of the 480 genes mutated in the MSC fraction, 69 overlapped with the 595 genes mutated in the leukemic fraction, while 411 genes were uniquely mutated in the MSC fraction. Further analysis is ongoing, and pathway analysis will be conducted once the remaining 14 cases are processed. Conclusions: This study identifies distinct somatic mutations in AML patients' MSC and leukemic cell fractions, revealing genomic complexity and crosstalk impacting leukemia progression. Understanding the functional implications of these mutations is crucial for unraveling their roles in leukemogenesis and developing personalized therapeutic interventions targeting MSC somatic mutations.

Cladribine and Low-Dose Cytarabine-Based Salvage Therapy for Relapsed/Refractory AML in a Predominantly Venetoclax-Exposed Cohort

Introduction: Relapsed/refractory acute myeloid leukemia (R/R AML) remains challenging to treat. Unfit patients receive either targeted therapy for actionable mutations or low-intensity chemotherapy, often a hypomethylating agent with venetoclax (HMA/ven). Aggressive regimens, such as a purine analog with high-dose cytarabine, are reserved for fit patients. A regimen using similar drug classes, comprised of cladribine (clad), low-dose cytarabine (LDAC), and venetoclax (clad/LDAC/ven), has demonstrated favorable efficacy and safety for frontline AML treatment in older patients, according to recent phase 2 studies [Kadia 2018 Lancet Hematology; Kadia 2022 Journal of Clinical Oncology]. Interestingly, monocytic differentiation is a proposed mechanism of venetoclax resistance, and monocytic leukemic stem cells rely on purine metabolism, thus suggesting unique cladribine sensitivity [Pei 2023 Cancer Discovery]. The utility of clad/LDAC with or without venetoclax in R/R AML is not well characterized, especially in today's evolving treatment landscape. Methods: In this single-center retrospective cohort study, we report efficacy and safety outcomes of a salvage regimen using a clad/LDAC backbone in adult patients with R/R AML between 2019 and 2023. For induction, patients received cladribine 5 mg/m2 IV daily days 1 to 5 and cytarabine 20 mg/m2 IV daily days 1 to 10. A third agent, such as venetoclax at a target dose of 400 mg oral daily days 1 to 21, was added at the provider's discretion. Dose reductions were allowed based on performance status and organ function. Re-induction was considered if the bone marrow biopsy demonstrated inadequate response. Patients who responded received clad/LDAC-based consolidation or proceeded to allogeneic hematopoietic cell transplant (alloHCT). Results: A total of 35 patients received clad/LDAC-based salvage induction for R/R AML. Median age was 68 years old, and patients had a median of 1 prior line of therapy. Half of patients had secondary AML, and most had adverse risk disease. The most common regimen received was clad/LDAC (49%), followed by clad/LDAC/ven (43%). Among 32 patients evaluable for response, the overall response rate (ORR) was 38%, with complete response (CR) of 16% and complete response with incomplete count recovery (CRi) of 22%. The median progression-free survival (mPFS) was 1.4 months (range 0.4-22.8), and median overall survival (mOS) was 3.4 months (range 0.4-22.8). At 6 months, PFS was 11% and OS was 26%. At 12 months, PFS was 11% and OS was 15%. For responders, 33% achieved negative measurable residual disease (MRD), 33% proceeded to alloHCT, mPFS was 3.3 months (range 0.9-22.8), and mOS was 6.4 months (0.9-22.8). Mutational profiling of responders revealed ASXL1, CSF3R, NF1, SETBP1, and U2AF1 to be most the common. In a subgroup of 21 patients who had progressive or refractory disease with frontline HMA/ven, clad/LDAC-based salvage resulted in ORR 37%, CR 16%, CRi 21%, MRD negativity 57% among responders, mPFS 1.4 months (range 0.4-20.9), and mOS 2.3 months (range 0.4-21.8). Clad/LDAC-based induction was well tolerated, with no cases of treatment-related mortality. Common adverse effects included febrile neutropenia (74%), grade 1 or 2 elevated bilirubin (49%), grade 1 transaminitis (48%), rash (43%), nausea/vomiting (37%), and grade 1 acute kidney injury (31%). One third of patients received clad/LDAC-based regimen in the outpatient setting. Conclusions: Clad/LDAC and clad/LDAC/ven represent feasible salvage induction strategies for R/R AML that may be administered outpatient and associated with minimal toxicity. Favorable response rates occurred in a subgroup of patients who failed frontline HMA/ven, in which there is an unmet need.

Comparable Survival of Treatment Naïve TP53 Mutated Acute Myeloid Leukemia Treated with Hypomethylating Agent Compared to Hypomethylating Agent Plus Venetoclax Based Therapy

Background TP53-mutated (m) acute myeloid leukemia (AML) is an adverse risk disease, which continues to have dismal outcome in the contemporary era of novel therapies. Venetoclax (VEN) in combination with hypomethylating agents (HMA; azacitidine or decitabine) have shown to improve complete remission (CR) rates compared to HMA alone, however no significant differences in overall survival (OS) have been observed. Methods We conducted a retrospective study through the COMMAND consortium (a collaboration of acute leukemia experts from 10 US academic institutions) to analyze the impact on survival outcomes of HMA plus (+) VEN compared to HMA-based therapies in adult (≥18 years) pts with TP53m AML. The AML Database of 381 pts with TP53m AML diagnosed between 2012-2022 was queried and 154 (40%) (HMA [n=50] and HMA +VEN [n=104]) pts were identified eligible for this analysis. Continuous variables were summarized as median (range) while categorical variables were reported as frequency (percentage). The Kaplan-Meier method was used to estimate OS, defined as time from diagnosis to death or last follow-up. Logistic regression models were used to determine the univariate and multivariate of OS. Multivariable models included all significant univariate predictors. All tests were two-sided with a p value <0.05 considered statistically significant. Results Baseline characteristics and responses The median age (years [yrs]) was 74 (range [R], 38-87) and 71 (R,29-88) yrs in the HMAand HMA + VEN groups (gps) (p= 0.23), respectively ( Table 1). Thirty-four (68%) and 57 (55%) patients (pts) were ≥ 70 yrs in the HMA and HMA + VEN gps, respectively (p= 0.16). Twenty-six percent vs 31% ( p=0.57), 86% vs 90% ( p=0.58) and 68% vs 73% ( p=0.56) of pts had secondary AML, had complex cytogenetics (CG), and had multi-hit TP53m in the HMA and HMA + VEN gps, respectively. The proportion of pts with most frequently occurring (> 5%) myeloid co-mutations ( RUNX1, ASXL1, TET2, DNMT3A, RAS, PTPN11 and JAK2) were not significantly (NS) different between the HMA and HMA + VEN gps, as shown in Table 1. The early treatment related mortality in first 30 days of induction was higher in the HMA+VEN gp (25%) when compared with the HMA gp (6%), p=0.004. The complete remission rates (CR/CRi) were higher in the HMA + VEN gp compared to the HMA gp (35% vs 18%, p=0.05). Similarly, the proportion of pts receiving allogeneic stem cell transplant (allo-HCT) after induction was higher in the HMA + VEN gp when compared with the HMA gp (13% vs 4%; p=0.14). Duration of response and overall survival The median follow up was 9.8 months (mo) (R, 1-56) and 5.80 mo (R, 1-30.4) in the HMA and HMA + VEN gps, respectively ( p=0.12). The median duration of response (DOR) was higher in the HMA +VEN gp (15.6 mo) when compared with the HMA gp (7.93 mo), but NS ( p=0.26). The median OS was NS different when comparing the HMA and HMA + VEN gps (9.23 vs 7.3 mo, p=0.80 [ Figure 1]). Multivariable analysis for duration of response and overall survival We did multivariable analysis (MVA) for DOR and OS using baseline variables ( Table 1) that showed significance/trend towards significance in univariate analysis ( p<0.1). Allo-HCT (HR; 0.11, 95% CI: 0.02-0.52, p=0.005) retained significance for better DOR, whereas age ≥ 70 yrs (HR; 1.7, 95% CI: 0.63-4.65, p=0.28) and complex CG (HR; 3.05, 95% CI: 0.79-11.73, p=0.10) did not retain significance in MVA. In MVA for OS, achievement of CR/CRi (HR; 0.35, 95% CI; 0.20-0.59, p=<0.001) and allo-HCT after induction (HR; 0.06, 95% CI; 0.09-0.46, p=0.007) retained favorable significance for OS, whereas complex CG (HR; 2.36, 95% CI: 1.11-4.99, p=0.02) retained significance for shorter OS. PTPN11m (HR; 1.57, 95%CI: 0.75-3.28, p=0.23) and TET2m (HR; 1.02, 95% CI: 0.56-1.87, p= 0.92) did not retain significance in MVA. Conclusion In this multi-center real-world study, while HMA+VEN led to improvement in CR rates and a higher proportion of pts were bridged to allo-HCT, it did not associate with an improvement in OS when compared with HMA monotherapy. More effective treatment strategies are warranted to improve outcome of pts with TP53m AML.

Tumor Lysis Syndrome in Patients with AML Initiated on Venetoclax-Based Treatment without a Dose Ramp-up

Introduction Treatment with the combination of a hypomethylating agent (HMA) and venetoclax (VEN) is the standard of care for older patients (pts) with acute myeloid leukemia (AML). Venetoclax, an oral BCL-2 inhibitor, was initially approved for the treatment of chronic lymphocytic leukemia (CLL). In the early, pivotal studies assessing VEN safety, tumor lysis syndrome (TLS) was found to be a dose-limiting, potentially fatal toxicity. Dosing modifications were made to allow for safer initiation of the drug and, ultimately, a dose ramp-up was included in the dosing recommendation for CLL. Given the risk for TLS with VEN initiation for CLL, it was hypothesized that the risk would be the same, or potentially greater, when treating pts with AML. Therefore, studies of VEN in AML have included a dose ramp-up. Although reported rates of tumor lysis syndrome in these studies have been much lower than expected, a dose ramp-up for pts with AML is included in the package insert for VEN and continues to be utilized in clinical trials. Methods We conducted a retrospective analysis of pts ≥18 years of age with newly diagnosed or relapsed/refractory AML treated with a standard dose hypomethylating agent (HMA), either azacitidine or decitabine, and VEN without a dose ramp-up (initiated at 400mg daily or 100mg daily if on concomitant voriconazole or posaconazole) at Thomas Jefferson University Hospital from January 1, 2018- August 1, 2021. The primary objective of this study was to determine the rate of laboratory and clinical TLS within 72 hours after chemotherapy initiation, as defined by the Cairo-Bishop criteria (Table 1). The secondary objectives were (1) to identify clinical and laboratory risk factors for the development of TLS in pts with AML who were treated with HMA-VEN without a VEN dose ramp-up and (2) to compare rates of TLS in pts with AML who were treated with HMA-VEN without a VEN dose ramp-up to historical rates of TLS in pts who underwent a VEN dose ramp-up. In the Phase 3 study of azacitidine and venetoclax the rate of TLS during the VEN dose ramp-up period (days 1-3 of treatment) was 1% (DiNardo CD, Jonas BA, Pullarkat V, et al. Azacitidine and venetoclax in previously untreated acute myeloid leukemia. N Engl J Med. 2020;383(7):617-629.). Results Ninety pts were included in the analysis. Seventy-one pts had newly diagnosed AML and 19 had relapsed/refractory disease. The majority of pts (58/90, 64%) had adverse risk disease by 2017 European LeukemiaNet Criteria. Median baseline white blood cell count at the time of therapy initiation was 3.25 (range 0.5-161.4). Sixty-one pts (68%) were receiving allopurinol prophylaxis at the time of HMA-VEN administration. Eighteen pts (20%) had a history of underlying renal disease. Fifteen pts (17%) were on medications, such as thiazide diuretics, aspirin, or levodopa, that can increase the risk of TLS at the time of treatment. Zero pts met the defined criteria for laboratory or clinical TLS. One pt developed acute kidney injury and one pt developed a cardiac arrhythmia (atrial fibrillation) but neither had concomitant evidence of laboratory TLS. Discussion In this retrospective study of 90 pts with newly diagnosed or relapsed/refractory AML who were treated with HMA-VEN without a VEN dose ramp-up no pts developed clinical or laboratory tumor lysis syndrome. This is comparable to the rate of TLS (1%) seen in the registration trial of HMA-VEN where pts were treated with VEN 100 mg on day 1, 200 mg on day 2, and 400 mg on day 3 and thereafter. Starting pts with AML on full dose VEN on day 1, regardless of white blood cell count, renal function, or concomitant medications, appears safe and could be implemented in clinical practice and clinical trials moving forward. This would simplify dosing for pts, pharmacists, and providers and allow for easier initiation of therapy.

Treatment Outcomes with Hypomethylating Agents and Venetoclax for AML in the Community Compared to an Academic Setting

Background: The treatment of acute myeloid leukemia (AML) in older and/or unfit patients consists of a combination of a hypomethylating agent (HMA), such as decitabine and azacitidine, with venetoclax, which can be safely administered in the ambulatory setting. In this study, we aimed to compare the outcomes of the treatment of patients with AML who received chemotherapy with venetoclax and an HMA in an ambulatory setting at an academic center or at a community practice with academic center support. Methods: This was a retrospective review of patients with de novo or relapsed/refractory AML treated with a combination of venetoclax and either decitabine or azacitidine between February 2018 and June 2023. Patients were included if they received treatment at an academic center or if they received their treatment at a community practice with ongoing consultation at an academic center. Drugs were administered per the standard of care and local practice. The primary objective was to evaluate the overall response rate (ORR). Secondary objectives were to evaluate differences in chemotherapy management between locations, potential socioeconomic disparities, and to identify adverse events that affect the dosing or administration of therapy. Results: A total of 129 patients received venetoclax plus HMA for AML for a minimum of one cycle as seen in table 1. Of these patients, 77 (60%) received treatment at an academic center, and 52 (40%) received treatment in the community setting. The average age at treatment initiation was 67 years. Disease risk was stratified according to the 2022 European LeukemiaNet (ELN) guidelines. While not statistically significant, there was a tendency for more patients at the academic center to have adverse risk disease (77.9% vs. 69.2%, p=0.364). They were also more likely to have had exposure to a previous HMA (n=10 vs. n=7, p=1) and prior AML treatment (n=20 vs. n=17, p=0.529). Additionally, patients were more likely to have been diagnosed with secondary AML in an academic setting (n=29 vs. n=18, p=0.868). In a multivariate Cox regression model, patients in the middle- and high-income groups, defined as a median household income greater than $52,000, had a 17% lower relative risk regarding survival probability than those in the lower-income group (HR=0.83, 95% CI 0.39-1.73, p=0.622). Overall, patients in the academic setting had a 33% higher relative risk regarding survival probabilities than the community-treated with covariate consideration of performance status, ELN risk, poverty status and exposure to prior-treatments (HR=1.33, 95% CI 0.78-2.26, p=0.285). The OS of all patients was 9.67 months (95% CI 8.33-12.7), with no significant difference between the groups as seen in figure 1. Additionally, the overall response rates (ORR) of the two groups were similar at 38.5% (n=20) and 35.1% (n=27) in community and academic settings, respectively. The complete remission (CR) rate in the community was 11%, while there was an increase of 18% in the academic setting. There were also increased CR rates in the academic compared to the community setting after cycle 1 (4.7 vs. 2.9%, p=0.33), cycle 2 (11.1 vs. 4.8%, p=0.858), and cycle 3 (18.2 vs. 0%, p=0.27). Adverse events in the study were similar except for increased rates of fatigue in the academic setting (76.6% vs. 55.8%, p=0.021). Only 67% of patients underwent a bone marrow biopsy after cycle 1 in the community versus 83% in the academic setting (p=0.101). Lastly, 67% of patients underwent dose reduction with the decitabine with venetoclax regimen in the community setting versus 31% in the academic setting (p=0.012). Conclusion: This study shows that the use of venetoclax plus HMA allows patients to safely receive AML treatment in the community with support from an academic center. Although there are some significant differences in management, such as increased rates of dose reduction in the community setting, the OS and ORR are similar. However, CR rates remain lower in the community setting. It is important to note that the patients in our study, who were treated at an academic center, had disease characteristics that were more resistant to treatment. Ultimately, this study identifies our efforts in creating community partnerships. Our goal is to further evaluate this partnership with quality improvement projects to bridge the gap in AML care between the community and academic practices.

Phase I Trial Combining Venetoclax with G-CSF, Cladribine, Cytarabine, and Mitoxantrone (CLAG-M) for Patients with High-Grade Myeloid Neoplasms

Background: Venetoclax in combination with hypomethylating agents or low dose cytarabine is a safe and effective treatment for newly diagnosed older, unfit patients with acute myeloid anemia (AML). Combining venetoclax with high intensity combination chemotherapy may increase response rates and eventual chance for cure. Previous studies have combined venetoclax with fludarabine, cytarabine, idarubicin, and G-CSF (FLAG-ida) with 89% of patients achieving a composite complete remission (CR), of which 93% were MRD negative (DiNardo et. al, AJH, 2022). We report the preliminary results of a phase 1 study examining the addition of venetoclax to a high intensity induction regimen, CLAG-M (cladribine, cytarabine, mitoxantrone, G-CSF), for fit patients with newly diagnosed adverse risk or relapsed/refractory high-grade myeloid neoplasms. Methods: Subjects with AML or other high-grade myeloid neoplasm (≥10% myeloid blasts in peripheral blood or marrow) were eligible if they had (1) new diagnosis with adverse risk disease by European LeukemiaNet 2017 criteria or (2) relapsed/refractory (R/R) disease. Venetoclax was administered on day 1-14 with a 4-day ramp-up. All patients received standard neutropenic prophylaxis and venetoclax was dose-adjusted for concomitant use of CYP3A4 inhibitors. Dose level (DL) is defined in Table 1. Dose limiting toxicity (DLT) was defined as grade >4 non-heme toxicity, prolonged aplasia > 49 days, or grade >3 non-heme toxicity lasting >48h and resulting in a treatment delay beyond day 56. Two subjects were started at DL 2 and a Bayesian optimal interval (BOIN) design was used for subsequent doses, with a true targeted DLT rate of 30%. Re-induction was allowed for persistent disease after the first cycle; subjects with CR were eligible to receive up to three cycles of venetoclax plus cladribine and cytarabine (CLAG) at the same dose level as induction. Target accrual is 20 subjects. Events were defined as death, relapse, or failure to achieve CR/CRi on post induction evaluation. The study was approved by the local IRB, and subjects were consented in accordance with the Declaration of Helsinki. Results: At time of data cutoff (7/6/23), 15 subjects (5 with newly diagnosed and 10 with R/R AML) were enrolled. Median age was 65 (range: 30-74) years. Five were newly diagnosed and 10 were R/R. Two subjects were enrolled at DL 1, 6 at DL 2, 5 at DL 3, and 2 at DL 4 as per the BOIN design. Two DLTs occurred: grade 4 respiratory failure at DL 2 and grade 5 sepsis at DL 3. Treatment related grade 3 adverse events (AE) included febrile neutropenia (5 subjects), dyspnea (2), mucositis (1), hypoxia (1), delirium (1), syncope (1), tumor lysis syndrome (1), and sepsis (1). Non-treatment related grade 3 AEs included hypokalemia (1 patient) and pericarditis (1). 28-day mortality was 7%. Twelve subjects received 1 cycle and 3 subjects received 2. Overall response rate (CR + CRi) was 73%. Two subjects achieved CR without MRD, 1 CR with MRD, 3 CRi without MRD, 5 CRi with MRD, 1 MLFS without MRD, 2 had persistent disease, and 1 died before evaluation. Median overall survival from treatment start was 7.4 months (95% CI 4.2 months - not reached [NR]). Median follow up of survivors was 6 months. Median event free survival was 3.3 months (95% CI 1.2 months - NR). Four subjects (1 newly diagnosed, 3 R/R) went on to receive allogeneic hematopoietic cell transplant (HCT). The administration of venetoclax presented challenges for inpatient and outpatient dosing, as medication diaries showed subjects underdosing venetoclax when taking concomitant posaconazole due to the complexity of the dosing regimen. Discussion: Preliminary results show that venetoclax can be safely added to a high intensity regimen for fit, high-risk patients without adversely affecting standard therapies including subsequent allogeneic HCT. Grade 3 and higher toxicity was common. In this preliminary analysis, the overall response rate was 73%, which is comparable to that observed with other chemotherapy regimens with the addition of venetoclax. A protocol amendment is in process to simplify venetoclax dosing with concomitant CYP3A4 inhibitors. This study will ultimately determine the MTD for subsequent phase II study to evaluate the therapeutic efficacy of venetoclax and CLAG-M.

Impact of Recent Advancements in Relapsed/Refractory Acute Myeloid Leukemia on Overall Survival in Clinical Practice

Introduction: The treatment landscape for relapsed or refractory acute myeloid leukemia (RR AML) is quickly evolving, guided by routine genetic profiling at diagnosis and relapse. In recent years, there has been a surge in FDA approvals for novel therapies, including venetoclax in combination with a hypomethylating agent (VEN + HMA), gilteritinib, ivosidenib, and enasidenib. Additionally, newer multi-agent combination chemotherapy regimens, such as fludarabine, cytarabine, idarubicin, and venetoclax (FLAG-Ida + VEN), have been increasingly used for RR disease. The year-by-year impact of the availability of these therapies on overall survival remains unclear. Methods: A cohort of 257 subsequent patients aged > 18 years with R/R AML treated at VCU Massey Comprehensive Cancer Center was retrospectively studied from 2015 to 2019. Only 2015-2019 were studied to avoid confounding from effects of the COVID-19 pandemic. Data was obtained from the medical record including cytogenetic and molecular profile, treatment regimens, allogeneic stem cell transplantation status (HCT), and survival. Disease was categorized by ELN 2022 genetic risk stratification. Median overall survival (mOS) was compared across individual calendar years using the Kaplan-Meier method. Categorical variables were analyzed using Fisher's exact test. Results: The median age of all patients with R/R AML was 61.4 years (range, 19.9-84.8) and median ECOG performance status was 1 (range, 0-4). The median Charlson Comorbidity Index was 4 (range, 0-14). Of the 248 patients for whom data on ELN 2022 genetic risk was available, 30 (12.1%) patients had ELN favorable risk disease, 65 (26.2%) had intermediate risk, and 151 (60.9%) had adverse risk disease. The median OS in 2015 was 5.8 months for all patients compared to 6.9 months in 2019 (p=0.38). When stratified by ELN risk category, patients with R/R favorable risk disease in 2019 had improved mOS compared to those in 2015 (mOS not reached vs. 7.1 months, respectively, HR 0.30 [95% CI 0.08-1.13], p=0.0400) (Fig. 1). There was no significant difference in mOS for the intermediate or adverse risk R/R AML cohorts over time. The proportions of patients with relapsed disease in 2019 who received alloSCT (39.2%) were numerically higher than in 2015 (26.3%), but were not statistically significant (p=0.259). Next, we studied relapsed/refractory therapeutic regimen usage over time. Coinciding with the approval of venetoclax, there was a marked increase in the percentage of patients who received a VEN + HMA beginning in 2018-2019, which corresponded to a decline in HMA monotherapy, as well as increased use of targeted therapeutics (Fig. 2). Conclusions: Prognosis for relapsed/refractory AML remains poor despite advances in novel therapeutics. While modest improvements in survival have been achieved, this is largely for ELN 2022 favorable-risk disease. Our work highlights the need for novel therapeutics aimed at those with relapsed or refractory disease. Our findings demonstrate reduced use of conventional chemotherapy in RR AML, with increased reliance on small molecule inhibitors and targeted therapies.

Recent Advancements in Acute Myeloid Leukemia Have Led to Incremental Improvement in Overall Survival

Background: Treatment approaches in acute myeloid leukemia are rapidly evolving due to a dynamic treatment landscape, routine molecular profiling, and the detection of measurable residual disease. Recently, there has been a surge in FDA approvals for novel therapies (e.g., midostaurin, gilteritinib, CPX-351, enasidenib, ivosidenib, and venetoclax), as well as a departure from a “one-size-fits-all” treatment approach. It is unclear, however, what effect these changes have had on overall survival over time. Since 2017, we have witnessed a significant introduction of novel therapeutics. We hypothesized that patients diagnosed after 2017 may have improved survival due to recent therapeutic advances. Methods: We retrospectively analyzed 627 consecutive patients in the Project ERIS database aged > 18 years with AML treated at VCU Massey Comprehensive Cancer Center from 2015 to 2022. We limited the analysis to January 1, 2015 to December 31, 2019 to avoid confounding from the COVID-19 pandemic. We obtained cytogenetic and molecular characteristics, induction regimens, allogeneic stem cell transplant status, and survival data. The median overall survival (mOS) was compared across calendar years using the Kaplan-Meier method. Categorical variables were analyzed using Fisher's exact test. Results: The median age at AML diagnosis was 62.9 years (range, 19.6-93.0), and the median ECOG performance status was 1 (range, 0-4). The median Charlson Comorbidity Index score was 5 (range, 0-14). There were 139 (22.2%) patients with ELN 2022 favorable-risk disease, 199 (31.7%) with intermediate risk, and 260 (41.5%) with adverse risk. The median OS of all patients diagnosed with AML in 2015 was 8.8 months. In 2016, the mOS was 14.5 months. There was a statistically significant improvement in mOS in 2016 compared to 2015 (HR 0.62 [95% CI 0.40-0.95], p = 0.027). The mOS in 2017, 2018, and 2019 was 12.9 months, 11.1 months, and 19.7 months, respectively. A significant improvement in OS was noted between 2015 and 2019 (HR 0.55 [95% CI 0.36-0.85], p = 0.003) in the entire cohort (Figure 1). When stratified by ELN 2022 genetic risk, clinically relevant improvement in OS was noted across all risk categories from 2015 to 2019, which reached statistical significance in the adverse risk group - favorable risk mOS: 16.4 months vs not reached (p = 0.141); intermediate risk: 13 months vs 18.7 months (p = 0.064); adverse risk: 7.2 months vs 13 months (p = 0.02). We observed expected changes in front-line treatment in this timeframe with decreased single-agent hypomethylating agent (HMA) usage in favor of HMA + venetoclax and increased intensive induction therapies outside of 7+3 (Figure 2). Strikingly, increased procession to allogeneic stem cell transplantation (HCT) was noted in the same timeframe (18.9% in 2015 vs 35.1% in 2019, p = 0.049). Conclusions : Our data demonstrate an emerging trend of clinically significant improvement in overall survival in patients with AML. The introduction of a multitude of therapeutic options may be associated with significantly improved survival over time. In addition to the advent of novel therapeutics leading to improved outcomes, the refinement of SCT conditioning regimens has likely led to the improved accessibility of HCT. In summary, we present an encouraging and significant trend of improved survival and access to HCT, particularly for patients with adverse-risk disease.

The Molecular Landscape of Acute Myeloid Leukemia with Trisomy 8: A Comprehensive Cancer Center Analysis of 627 Patients

1. Introduction Trisomy 8 is one of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). Trisomy 8 frequently occurs with additional aberrant or complex karyotypes; it can also occur as the sole cytogenetic abnormality. While prior studies have shown that trisomy 8 cooperates with other somatic genetic mutations (e.g., RUNX1), there is persistent controversy about the prognostic impact of AML with trisomy 8. We analyzed the relationship between trisomy 8 and additional molecular lesions, the ELN 2022 revision, and the prognostic impact of trisomy 8 across modern treatment strategies. 2. Methods We analyzed 627 patients from the Project ERIS database with newly diagnosed AML treated from January 1, 2013 to April 18, 2023 at VCU Massey Comprehensive Cancer Center. We identified 86 patients with trisomy 8 independent of the presence of complex cytogenetics and a cohort of 541 historical controls without trisomy 8. We recorded baseline patient-related and disease characteristics, including age, ECOG, Charlson comorbidity index (CCI) scores, molecular profiling and ELN 2022 cytogenetic risk, dates of regimen initiation, and survival. We used the D'Agostino & Pearson method for normality testing and the t-test or Mann-Whitney (as applicable) tests for between-group comparisons. Categorical comparisons used Fischer's exact test. We applied the Bonferroni correction if multiple comparisons were made. The Wilson-Brown method was used for 95% confidence intervals. We analyzed survival by the Kaplan-Meier method with significance determined by the log-rank test. The event for calculating the overall survival (OS) was the date of death. Patients were otherwise censored at the date of last contact. 3. Results The most common cytogenetic abnormalities associated with trisomy 8 were the presence of a complex karyotype (45.2%, 95% CI: 35.0-55.9), loss of 5 (15.5%, 95% CI, 9.3-24.7%), loss of 7 (8.3%, 95% CI, 4.1-16.2), and -17p (13.1%, 95% CI, 7.5-21.9). ASXL1 mutations tended to occur significantly more frequently in trisomy 8 compared to those without (27.5% vs 13.0%, p = 0.029, Table A). Similarly, TP53 mutations occurred significantly more frequently in trisomy 8 than those without (27.5% vs 13.0%, p = 0.029). Conversely, DNMT3A mutations were less likely to be associated with trisomy 8, which approached statistical significance (10.0% vs 23.47%, p = 0.063). NPM1 mutations were significantly less likely to be associated with trisomy 8 compared to those without (7.5% vs 30.3%, p = 0.002). Consequently, trisomy 8 was significantly more likely to be associated with ELN 2022 adverse risk disease (odds ratio: 2.754, p < 0.0001). Next, we compared response and survival with intensive chemotherapy using conventional 7+3 in those with and without trisomy 8. In 51 patients with trisomy 8, the composite complete response rate (CRc; CR + CRi + CRh) was 34.1% (95% CI, 21.9-48.9), significantly worse compared to 330 patients without trisomy 8 (55.1%, 95% CI, 49.4-60.6, p = 0.010). The median overall survival was significantly worse in patients with trisomy 8 treated with 7+3 compared to those without trisomy 8 (7.1 m. vs 19.2 m., p = 0.009, Figure B). In 13 patients treated with venetoclax and a hypomethylating agent (HMA), the CRc was 25.0% (95% CI, 8.9-53.2), which was nonsignificantly worse compared with 87 patients without trisomy 8 that demonstrated a CRc of 48.4% (95% CI, 36.4-60.6, p = 0.206). The overall survival of patients treated with venetoclax + HMA nonsignificantly favored patients without trisomy 8 (8.2 m. vs 5.0 m., p = 0.268). 4. Discussion AML with trisomy 8 is significantly enriched in ASXL1 and TP53 mutations; conversely, trisomy 8 is significantly less likely to harbor NPM1 mutations. Patients with trisomy 8 are significantly more likely to have ELN 2022 adverse-risk disease. The CRc rate and median overall survival are significantly worse in patients with trisomy 8 treated with intensive chemotherapy. We observed no significant difference in response or survival in patients with or without trisomy 8 treated with venetoclax + HMA.

T-Cells Immune Cluster Analysis Using Cytof Identifies Unique Patient Subgroups with TP53 Mutation and Predicts Complete Remission (CR) in Patients with Acute Myeloid Leukemia (AML) Receiving Venetoclax Plus Hypomethylating Agent Therapy

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

Improved MRD Negativity Rates in Adverse Genomic Risk B-ALL Patients with Chemotherapy / Blinatumomab Induction: Experience from the Australasian Leukaemia Lymphoma Group (ALLG) ALL06/09 Studies

Introduction: Despite the benefits conferred by pediatric treatment protocols, outcomes in adolescent and young adult (AYA) acute lymphoblastic leukemia (ALL) remain inferior to children. Poor outcomes in AYA ALL have been associated with reduced tolerability of intensive cytotoxics and higher incidence of adverse risk disease. Our earlier ALLG ALL06 study demonstrated intensive pediatric induction/consolidation could be delivered in a similar time frame as in children suggesting tolerability was not a major contributor to poor outcome in AYA ALL. ALL06 established post consolidation (TP2=d79) minimal residual disease (MRD) positivity (MRD+) and high BMI was predictive of inferior disease free- and overall survival (OS). In order to improve TP2 MRD negativity (MRD-) in B-cell disease, the ALLG ALL09 study substituted standard consolidation for blinatumomab (blin) in phase I and II of a BFM-style protocol . We present the impact of blin on outcomes in genomic cohorts from ALL09 and compare these to similar cohorts from ALL06 treated with standard chemotherapy. Methods: Between Sep 2020 and Apr 2022, 55 patientswere enrolled to ALL09. Of these, 45 had appropriate samples for genomic analyses and 39/45 had an MRD marker identified. These 39 patients formed the genomic cohort presented below. Genomic subtyping was performed using whole transcriptome sequencing, MLPA, karyotype and FISH analyses, and disease was classified into genomic adverse (AR) vs standard (SR) risk based on contemporary literature classifiers. Bone marrow MRD was assessed using ASO-PCR for IGH and TCR rearrangements, at d33 post induction (TP1), and TP2 post blin consolidation. Differences in TP2 MRD were assessed, with estimated 3 yr relapse free survival (RFS) and OS calculated using the Kaplan-Meier method. Results: In the ALL09 cohort, median follow up was 629 days with 3 yr RFS 84.5% and OS 87.1%. In the genomic cohort, median follow up was 602 days with 3 yr RFS 88.8% and OS 93.2%, respectively. These compared favorably to 3 yr RFS and OS (both 67.5%) in the B-cell genomic cohort from ALL06. Between ALL09 and ALL06 there were similar proportions of AR patients (73% and 62.5%, respectively). In ALL09, the most frequent subtypes were PAX5alt (18%) and PAX5 p.P80R (11%) versus KMT2Ar and DUX4r (both 17.5%) in ALL06. In ALL09, Ph-like disease represented 13% vs 12.5% in ALL06. In the ALL09 genomic cohort, the MRD+ rate reduced between TP1 and TP2 (p< 0.001), with the reduction in the rate of positivity being more pronounced in the AR cohort (AR MRD+ TP1=85% vs AR TP2=39%; p< 0.001). In contrast, in ALL06 MRD+ in the AR cohort reduced by only 28% between TP1 and TP2 (p >0.05). This suggests a positive impact of blin consolidation in AR patients in ALL09. Despite improved AR MRD+ rates overall, not all genomic subtypes in ALL09 followed this trend (Table 1). To date, there are 5 relapses in the ALL09 cohort, 4 from the genomic cohort. Of these, 1 was hypodiploid and strikingly the remaining 3 patients harbored a TCF3 rearrangement ( TCF3r). All 4 relapses were significantly associated with MRD+ at TP2 (RFS: MRD-, 100% (n=14) vs MRD+, 71% (n=25); p= 0.013), and all were observed in the AR genomic group. Of interest, the fifth case, outside of the genomic cohort, was classified as PAX5alt. In ALL09, high-risk (HR) patients proceeded to intensive HR block chemotherapy with the aim of eliminating MRD prior to allogeneic transplant (SCT). In the genomics cohort 15 patients proceeded to HR therapy, n=12 were considered AR. Sustained MRD+ through HR blocks 1 and 2 was noted in patients harboring PAX5 p.P80R (3/3), KMT2Ar (2/2) and TCF3r (2/3). Of the 9 patients who proceeded to SCT, 3 had PAX5 p.P80R 1 had PAX5alt, 2 were KMT2Ar and there was 1 each of DUX4r, hypodiploid and TCF3r. The 3 patients who remained MRD+ in the SR cohort had PAX5 p.P80R and all 3 proceeded to SCT. Conclusion: This is the first report of the impact of blin consolidation on genomic subsets of de novo AYA ALL. Our study suggests blin sensitivity as assessed by TP2 MRD response may be genomic subtype dependent, with TCF3r associated with sustained MRD+, leading to eventual relapse (3/3 patients). In addition, the PAX5 p.P80R subset was also associated with sustained high levels of TP2 MRD+ (3/4 with an MRD marker) and SCT (3/5 patients). These results may assist with identification of AYA ALL subsets that could benefit from alternative approaches as blin becomes increasingly incorporated into front-line protocols.

Validation of the Charlson Comorbidity Index Model in Acute Myeloid Leukemia Treated with a Hypomethylating Agent and Venetoclax

1. Introduction The Charlson comorbidity index (CCI) model is a scoring model that combines comorbidities and age; it has been shown to predict mortality in patients with acute myeloid leukemia (AML). However, treatment-specific comorbidity score thresholds associated with increased patient mortality remain unclear, particularly for newer treatment approaches. The aim of this study is to identify and update CCI score thresholds for patients undergoing treatment with venetoclax and a hypomethylating agent (decitabine or azacitidine; VEN + HMA) to refine the selection of treatment candidates in the first-line and salvage settings. 2. Methods We analyzed 183 patients with newly diagnosed or relapsed or refractory AML treated with HMA + VEN from January 1, 2018 to April 18, 2023 at VCU Massey Comprehensive Cancer Center. We recorded baseline patient-related and disease characteristics, including Charlson comorbidity index (CCI) scores at the time of initial diagnosis, dates of regimen initiation, and survival. We used the D'Agostino & Pearson method for normality testing and the Mann-Whitney test for between-group comparisons. Categorical comparisons used Fischer's exact test. We applied the Bonferroni correction if multiple comparisons were made. We analyzed survival by the Kaplan-Meier method with significance determined by the log-rank test. The event for calculating the overall survival (OS) was the date of death. Patients were otherwise censored at the date of last contact. 3. Results We analyzed 100 (54.6%) patients in the first-line setting treated with HMA + VEN. Starting from a CCI score threshold of 4, we incrementally analyzed CCI cohorts (e.g., ≤4 vs >4; ≤5 vs >5, and so on) until we reached a significant survival difference between cohorts at a CCI score threshold of 6. CCI score thresholds of 4 and 5 were not significant (p = 0.946 and p = 0.840, respectively). In patients treated with first-line HMA + VEN, those with a CCI score threshold of ≤6 had significantly improved overall survival compared with patients with a CCI score of >6: (8.8 m. vs 3.4 m., p = 0.013, Figure A). Next, we analyzed 89 (48.6%) patients in the relapsed or refractory setting; six (3.3%) patients received HMA + VEN as both first-line and as salvage therapy. We observed a significant survival difference at the first model increment analyzed: patients with a CCI score of 4 or lower had a median overall survival of 9.4 m compared to 5.2 months for CCI scores greater than 4 (p = 0.048, Figure B). In light of these findings, we conducted an exploratory analysis to account for any significant between-group differences independent of the assigned CCI score. In the first-line setting, patients with CCI scores ≤6 were significantly younger compared to those with scores >6 (71 vs 75 y., p = 0.002). The median ECOG score was 2 in both groups at the specified CCI score threshold of 6 (p = 0.939). There was no difference in the proportion of ELN 2022 adverse-risk disease between the ≤6 and >6 cohorts (55.4% vs 61.8%, p = 0.669), and there was no significant difference in the receipt of allogeneic stem cell transplant (3.0% vs 0%, p = 0.547). In the salvage setting, we observed that age was also significantly different between the ≤4 and >4 cohorts (54 y. vs 70 y., p < 0.0001), and there was no significant difference in the ECOG scores at the time of relapse in the ≤4 vs >4 cohorts (1 vs 1, p = 0.769) or in the proportion of ELN 2022 adverse-risk disease (54.4% vs 61.9%, p = 0.522). 4. Discussion This study validates new Charlson comorbidity index score thresholds for patients treated with HMA + VEN. We did not identify any significant confounding factors outside of the variables analyzed in the CCI model; age was the only significant finding, which is accounted for in the CCI scoring model. In the first-line setting, we identified a novel lower CCI score threshold of 6 that predicts superior survival in patients treated with HMA + VEN. In the salvage setting, the CCI score threshold appears lower at 4, which likely reflects a selection for patients able to tolerate multiple lines of therapy due to a decreased comorbidity burden. These CCI score thresholds can be used as a predictive guide and a clinical tool to assess fitness for lower-intensity venetoclax-based therapies in AML.

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

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

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

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