Trials In Acute Myeloid Leukemia Research Articles

Abstract PO4-14-01: Synergistic Activity of CDK8/19 Inhibitor RVU120 and MEK Inhibitors in Hormone-Negative Breast Cancer: Implications for Targeted Therapy

Abstract Breast cancer (BC) is a complex disease with distinct subtypes, including triple-negative breast cancer (TNBC), which lacks targeted therapies. CDK8 and CDK19, components of the mediator complex involved in transcriptional regulation, have emerged as potential therapeutic targets in BC. Transcriptomic analysis revealed that elevated CDK8 expression was associated with poor prognosis across all BC subtypes. Furthermore, previous studies demonstrated that sensitivity to CDK8/CDK19 inhibition correlated with high STAT3 phosphorylation and enrichment of TNF/NF-kB and STAT target genes in responder cells. In this study, we evaluated the responsiveness of TNBC cell lines to RVU120, a highly selective and potent CDK8/CDK19 inhibitor currently undergoing clinical trials for acute myeloid leukemia (AML), high-risk myelodysplastic syndrome (HR-MDS), and solid tumors. Notably, the sensitivity of TNBC cell lines to RVU120 varied depending on the culture conditions, with the presence of epidermal growth factor (EGF) identified as a major determinant of response variability. Considering the role of EGFR in activating downstream signaling pathways, including MAPK and STAT, and previous observations of synergy between small molecule CDK8 inhibitors and MEK inhibitors in neuroblastoma, we investigated MEK inhibitors as potential combination partners in hormone-negative BC. Among the 15 tested BC cell lines, a synergy between CDK8/CDK19 inhibitors and MEK inhibitors was observed in 5 cell lines, antagonism in another 5 cell lines, and no synergy in the remaining 5 cell lines, based on the analysis with Combenefit software (Loewe additivity model). Importantly, cell lines where synergy of the combination therapy has been observed, exhibited EGFR amplification and markers of activated RAS pathway. This pattern of response was consistent across different MEK and MEK/RAF inhibitors, including selumetinib, trametinib, and avutometinib. These findings highlight the synergistic potential of combining RVU120 with MEK inhibitors in hormone-negative BC, particularly in TNBC with EGFR amplification and an active RAS pathway. Ongoing studies involving RNAseq analysis and organoid screening aim to further elucidate the underlying mechanisms and confirm the efficacy of this combination therapy. Furthermore, in vivo efficacy experiments are necessary to validate in vitro activity of both compounds. Successful development of this therapeutic concept relies on the validation and implementation of selected partner drugs targeting RAS pathway, as well as the identification of suitable biomarkers such as EGFRamp or RAS-score. Promising findings of this study provide a rationale for exploring the combination of CDK8/CDK19 inhibitor RVU120 and MEK inhibitors as a potential targeted therapy strategy for hormone-negative BC. Citation Format: Urszula Pakulska, Adrianna Moszynska, Justyna Martyka, Jerzy Woznicki, Elzbieta Adamczyk, Marta Obacz, Kinga Keska Izworska, Katarzyna Wiklik, Agata Stachowicz, Hendrik Nogai, Milena Mazan, Tomasz Rzymski. Synergistic Activity of CDK8/19 Inhibitor RVU120 and MEK Inhibitors in Hormone-Negative Breast Cancer: Implications for Targeted Therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-01.

Abstract SY16-01: What IDH mouse models have taught us about hematological diseases, from leukemia to T cell lymphoma

Abstract SY16-01: What IDH mouse models have taught us about hematological diseases, from leukemia to T cell lymphoma

Abstract 5178: Guided by a predictive ex vivo test: Bringing the PLK1 inhibitor volasertib back into the clinic for venetoclax-HMA relapsed/refractory acute myeloid leukemia patients

Abstract INTRODUCTION: Volasertib, a polo-like kinase 1 (PLK1) inhibitor, has a roughly 30% CR/CRi response rate in de novo acute myeloid leukemia (AML) patients when combined with low-dose cytarabine. However, its development was halted due in part to toxicity in a Phase 3 study. We have built a flow cytometry-based, high-throughput predictive precision medicine platform to forecast clinical responses to various treatments. Here, we present our design plan for a Phase 2a clinical trial with volasertib in relapsed/refractory (R/R) AML using a modified protocol to reduce toxicity and the development of a companion diagnostic to predict responders in a follow-up Phase 2b study. METHODS: We will initiate an open-label clinical trial in AML with volasertib in venetoclax-hypomethylating agent (HMA) R/R patients while co-developing a companion diagnostic to selectively enroll predicted responders in a follow-up, Phase 2b, study. Primary and secondary objectives will focus on efficacy/safety of intravenous volasertib and ex vivo responses, respectively. To reduce the toxicity seen in the Phase 3 study, specific strategies like tailored dosing will be implemented. To develop the clinical trial assay, peripheral blood or bone marrow samples were collected from AML patients and treated ex vivo with volasertib for 3 days. Surviving blast cells were enumerated by flow cytometry. Using a dynamic separation model restricted to the 35% most sensitive samples, we estimated optimal blast cutoffs for volasertib sensitivity. RESULTS: In single-agent experiments with 41 primary AML samples (31 de novo, 9 R/R, 1 with disease status unclear), we observed dose-response profiles consistent with volasertib’s role as a cell cycle inhibitor. Irrespective of the disease status, median and mean EC50 values were about 10 nM (1st-3rd quartile from 7-12 nM). To identify an optimal concentration of volasertib for patient stratification, we compared the ex vivo resistant blast fractions at each concentration with the areas under the dose-response curves (AUCs), assuming that in our case, AUCs most accurately reflect clinical responses. At 31.6 and 100 nM, the Pearson correlation coefficients were highest (r > 0.8), suggesting that these are optimal stratifying doses. With these concentrations and corresponding blast cutoff values, 32-33% of the de novo and 25% of the R/R patients would have been predicted to be responders were they given volasertib, assuming a positive predictive value of 100%. CONCLUSION: Volasertib’s EC50 values clustered within a narrow range, suggesting that EC50 may not be a suitable metric to stratify patients into responders and nonresponders, in contrast to AUCs and residual blast fractions at 31.6 and 100 nM of volasertib, which displayed more heterogeneous distributions. Limitations include small sample sizes, especially for the R/R group. Citation Format: Markus Daniel Lacher, Glenn Michelson, Allen Ling, Wade Anderson, Jeaneille Vinas, Christine J. Gu, Chris Leonardi, Joseph Wagner, Gabriel N. Mannis. Guided by a predictive ex vivo test: Bringing the PLK1 inhibitor volasertib back into the clinic for venetoclax-HMA relapsed/refractory acute myeloid leukemia patients [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 5178.

Abstract 5069: Retrospective analysis of acute myeloid leukemia using next generation cytogenomics identifies variants missed at diagnosis

Abstract Cytogenetics forms the cornerstone of diagnostic genetic testing in leukemia, providing both prognostic and diagnostic information to help guide treatment. Currently, standard-of-care cytogenetics in acute myeloid leukemia (AML) is composed of a combination of karyotyping, FISH testing, and in some cases chromosomal microarray analysis, each test offering a unique combination of advantages and weaknesses. A particular challenge for this combination of tests is the delivery of results in a timely and comprehensive manner. To overcome these limitations, we have developed OncoTerra, a proximity-ligation-sequencing based method which can rapidly detect all classes of variants detected by cytogenetics tests in a single assay. We applied OncoTerra to a cohort of more than 100 samples from a SWOG AML trial (S0106), benchmarking sensitivity and specificity of the variants. We observed an overall >0.95 sensitivity and a specificity of 1 for the variants identified by cytogenetics and identified numerous cryptic variants. Overall, we identified variants that changed the diagnostic risk stratification of 15% of the patient involved in the trial based on the ELN 2022 criteria. This translated into better overall prediction of outcomes of patients in the trial and has identified both known and novel variants missed by standard-of-care tools. Citation Format: Stephen Eacker, Maika Malig, Mary Wood, Alexander Muratov, Olga Sala-Torra, Ivan Liachko, Cecilia Yeung, Jerald Radich. Retrospective analysis of acute myeloid leukemia using next generation cytogenomics identifies variants missed at diagnosis [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 5069.

A critical evaluation of the EFS endpoint in AML: Does induction treatment failure timing have a profound impact on study design and results?

Despite emerging novel therapies, treating acute myeloid leukemia (AML) remains challenging. Complexities persist in designing pivotal clinical trials and establishing acceptable endpoints for AML. Recent FDA guidance for drug and biological products development for AML outlines considerations for trial design. The guidance defines overall survival (OS) and event-free survival (EFS) as endpoints representing clinical benefit for AML therapies without curative intent. We highlight the EFS definition, particularly the assignment of day 1 as the event date for patients with induction treatment failures (ITFs), as recommended in the guidance. Through a comprehensive simulation study, our results show that the guidance EFS definition performs adequately with high complete remission (CR) rates but may pose challenges for low CR rates. When the experimental arm CR rate is 5% or less over the control, the use of the ITF events at day 1 for EFS definition leads to a critical power decrease, hampering the ability to predict survival benefit for a moderate OS duration. We further expand upon the EFS definition with the event date at ITF period end. Our goal is to inform investigators and regulatory agencies about the implications and limitations of various EFS definitions for future pivotal trials in AML.

Mezigdomide is effective alone and in combination with menin inhibition in preclinical models of KMT2A-r and NPM1c AML

AbstractSmall molecules that target the menin-KMT2A protein-protein interaction (menin inhibitors) have recently entered clinical trials in lysine methyltransferase 2A (KMT2A or MLL1)–rearranged (KMT2A-r) and nucleophosmin-mutant (NPM1c) acute myeloid leukemia (AML) and are demonstrating encouraging results. However, rationally chosen combination therapy is needed to improve responses and prevent resistance. We have previously identified IKZF1/IKAROS as a target in KMT2A-r AML and shown in preclinical models that IKAROS protein degradation with lenalidomide or iberdomide has modest single-agent activity yet can synergize with menin inhibitors. Recently, the novel IKAROS degrader mezigdomide was developed with greatly enhanced IKAROS protein degradation. In this study, we show that mezigdomide has increased preclinical activity in vitro as a single-agent in KMT2A-r and NPM1c AML cell lines, including sensitivity in cell lines resistant to lenalidomide and iberdomide. Further, we demonstrate that mezigdomide has the greatest capacity to synergize with and induce apoptosis in combination with menin inhibitors, including in MEN1 mutant models. We show that the superior activity of mezigdomide compared with lenalidomide or iberdomide is due to its increased depth, rate, and duration of IKAROS protein degradation. Single-agent mezigdomide was efficacious in 5 patient-derived xenograft models of KMT2A-r and 1 NPM1c AML. The combination of mezigdomide with the menin inhibitor VTP-50469 increased survival and prevented and overcame MEN1 mutations that mediate resistance in patients receiving menin inhibitor monotherapy. These results support prioritization of mezigdomide for early phase clinical trials in KMT2A-r and NPM1c AML, either as a single agent or in combination with menin inhibitors.

Disparities in the Enrollment of Racialized, Ethnic Minority, and Older Adults in Randomized Trials of Acute Myeloid Leukemia: A Systematic Review

Introduction: There are significant racial and ethnic disparities in incidence and survival for patients diagnosed with hematologic malignancies. Understanding discrepancies in enrollment in randomized controlled trials based on race, ethnicity, and age is important to better understand access to care and clinical trial conduct. Objective: To systematically review the literature on enrollment of racialized, ethnic minority, and older adults in randomized controlled trials (RCTs) of acute myeloid leukemia (AML) and to provide enrollment estimates and compare these to population characteristics. Methods: MEDLINE was searched from inception through to June 2023. No restrictions based on language or publication date were used. Pairs of reviewers independently screened titles, abstracts, and full texts of records. Inclusion criteria were phase II and III RCTs of disease modifying therapy for AML in adults (≥18 years) reporting efficacy and safety outcomes. We excluded single arm trials, trials with unpublished results, conference abstracts, follow-up reports, subgroup/post-hoc/exploratory analyses, and supportive care trials, except those evaluating disease-modifying therapies and reporting clinical efficacy. A standardized form was pilot-tested and used to extract data related to trial characteristics. The proportion of trials reporting racial and ethnic subgroups (African American/Black, Asian, American Indian/Alaskan native, Native Hawaiian or other Pacific Islander, White, Hispanic), and age ≥65 were determined. For US trials, we calculated the enrollment incidence ratios (EIRs), the ratio of trial proportions of members of a racial and ethnic subgroup divided by US population-based incidence in the corresponding racial and ethnic subgroup using the Surveillance, Epidemiology, and End Results (SEER 20) database. We conducted a random-effects meta-analysis to pool EIRs. Results: After screening 7,759 titles and abstracts and 157 full texts, we included 90 studies, of which 14 (15%) were US trials. Overall, there were 21 (23.3%) trials that reported race or ethnicity and 26 (28.9%) that reported the enrollment proportion of ≥65 years. Of the trials with data on race or ethnicity, 15 (71.4%) had data on African American/Blacks, 21 (100%) Whites, 14 (66.7%) Asian or Pacific Islanders, 2 (9.52%) American Indian and Alaskan Native, 4 (19.0%) Hispanics. Of trials reporting on race, 176 (3.6%) of participants were African American/Black, 360 (7.2%) Asian or Pacific Islander, 5 (0.1%) American Indian and Alaskan Native, 3,914 (79.6%) White, and 39 (0.8%) Hispanic. Of the 14 US trials, 4 (28.6%) reported race and 4 (28.6%) reported enrolled proportion of older adults. Hispanic patients (EIR 0.20; 95%CI 0.07 to 0.59, I 2=86%), and Asian patients (EIR 0.24; 95%CI 0.07 to 0.86, I 2=77%) were significantly underrepresented while White patients (EIR 1.33, 95%CI 1.07 to 1.66, I 2=99%) were significantly overrepresented. Confidence intervals were wide for EIR of Black patients (EIR 0.96, 95%CI 0.42 to 2.2, I2=91%). Conclusion: Most trials did not report data on race and ethnicity or on enrollment proportion of participants ≥65 years. Only a small proportion of trial participants were from racial and ethnic minority groups. Hispanic and Asian patients were significantly underrepresented while White patients were overrepresented.

Nucleophosmin (NPM1) Type D Genotype Is Associated with Distinct Outcome in Acute Myeloid Leukemia - an AIEOP-BFM and COG-SWOG Intergroup Collaboration

Background: Nucleophosmin (NPM1) is a nucleo-cytoplasmic shuttling protein, predominantly located in the nucleolus that regulates a multiplicity of different biological processes. NPM1 localization in the cell is finely tuned by the nuclear export signal domain, where two tryptophan residues (Trp) are essential for the nucleolar localization. In acute myeloid leukemia (AML), several genomic subsets of NPM1 frameshift mutations of 4 base pair insertions have been identified, with the type A variant (insertion of TCTG) being the most common, followed by type B (CATG) and D (CCTG). More recently, a series of different and rarer frameshifts have been found in adult and pediatric AML. We collected and studied the NPM1 insertion genotypes in a large cohort of children with de novo AML enrolled in the AIEOP, BFM, ELAM02, NOPHO, DCOG, COG trials. Methods: NPM1 mutations were sequenced in a group of 345 pediatric patients with de novo AML. 166 patients were enrolled in recent AIEOP, BFM, ELAM02, NOPHO, DCOG trials, and 179 in the CCG/COG AML trials (CCG-2961, AAML03P1, AAML0531 and AAML1031). A cohort of 75 patients of SWOG young adult (age 18-60) AML trial (S0106) were also included as validation cohort. The Kaplan-Meier method was used to estimate the probabilities of overall survival (OS) and event-free survival (EFS). Results: In this cohort of 345 pediatric patients sequencing of exon 12 demonstrated multiple 4 bp insertion subtypes, with type A variant detected in 188 patients (55%), type B in 64 cases (19%) and type D mutation in 26 patients (7.5%). Other variants with the loss of the 2 Trp residues (namely A-like) were found in 42 patients (12%), whereas in 25 patients (7.5%) we identified mutations with the loss of 1 Trp residue (namely non-A-like). Comparison of disease characteristics demonstrated that NPM1 variants were prevalent in older patients (median age=13.3 years). WBC count was not significantly different among subgroups (Type A=30.3; Alike=25.2; B=16.8; D=41; non-A-like= 14.8x10 3/ul, p=0.14). FLT3-ITD prevalence was higher in Type A, B and D compared to the other Type A-like variants (42%, 44%, 46% vs. 26%); interestingly, FLT3-ITD was absent in non-A-like variants. Evaluation of clinical outcome based on NPM1 genotypes showed that although patients with type A, A-Like, non-A-like and type B had favorable outcomes, those with Type D had a significantly worse outcome with OS of 63% versus 86% for other genotypes (p=0.0053). Type D variant adverse outcome was confirmed in most of the trials and will be further examined considering different treatments. Each NPM1 variant mediated a different EFS, with most of early events occurring within the D group (EFS 51.4%), and type A (EFS 68.8%) compared to the non-A-like NPM1, A like and B mutations (79.8%, 73%, and 75.5%, respectively) (p=ns). Validation data from adult SWOG trial (SWOG0106) demonstrated similar adverse outcome with EFS of 40% for type D vs. 61.4% for other NPM1 variants, indicating D as a peculiar NPM1 genotype /Figure A). Evaluation of overlapping mutations demonstrated that except for lack of FLT3-ITD in non-A-like genotype, there was similar distribution of high-risk mutations (FLT3-ITD, WT1, etc.) in those with different genotypes, thus not explaining the underlying biology responsible for the Type D adverse outcome. Even if the aminoacidic sequence of D and A variants is the same, the different prognosis suggests a further layer of regulation. We inquired whether Type D genotype might have a different NPM1 localization pattern compared to that in Type A variant. Immunofluorescence staining of primary AML cells from patients with Type D vs. Type A variant demonstrated that, in contrast to the canonical Type A variant with aberrant cytoplasmic localization, Type D variant maintains nuclear localization (Figure B). Further studies will provide the underlying biology of Type D being a silent mutation mediating a clinical outcome like NPM1 wild type AML. Conclusion: This Eu/COG/SWOG collaborative study of over 400 pediatric and adult patients suggest a different functional biology behind NPM1 genotype where those with Type D variants have a significantly inferior outcome resembling feature of non-NPM1 mutated AML. Our data suggest that the specific genotype of NPM1 mutations can contribute to a more accurate risk-stratification, with D cases eventually shifting from standard to higher risk groups.

Racial Disparities in Acute Myeloid Leukemia and Myelodysplastic Syndrome US FDA Drug Approval Trials

Introduction Young black patients with acute myeloid leukemia (AML) have remarkably worse outcomes than white patients of the same age. Compared to white patients, the death rate of young black patients is fivefold more within 30 days of starting treatment and twofold more within five years. Here we studied the demographic representation of different races in the AML and myelodysplastic syndrome (MDS) clinical trials that led to drug approval by the US Food and Drug Administration (FDA). Methods Data were collected from all the trials submitted to the FDA to support the approval of AML and MDS drugs from 2006 to 2022. A total of 17 trials were screened, and 10 trials that did not mention the race or ethnicity of the participants were excluded. Our analysis included six trials of AML drugs and one trial of MDS drugs. The methodological quality of the included studies was evaluated using the NIH quality assessment tool. The inter-study variance was calculated using the Der Simonian-Laird Estimator. Analysis for this study was performed using R version 4.1.2 and R Studio version 1.1.423 (R Project for Statistical Computing). Results: Seven FDA trials reported the race or ethnicity of the participants, and a total of 2484 patients from these trials were analyzed. (Table 1) Olutasidenib, glasdegib plus low dose cytarabine, gemtuzumab-ozogamicin, liposome-encapsulated combination of daunorubicin and cytarabine, ivosidenib, and midostaurin were approved for AML. An oral combination of decitabine and cedazuridine was approved for MDS. The median age of the participants was 69.5 years (0.003-94). The median follow-up time was 20.9 months (0-85.2), and 51% (n=1267) of the participants were male and female participants were 49 % (n=1217). The racial analysis of included patients showed 63.4% (n=1574) white, 5.7% (n=141) black, 3.9% (n=97) Asian, 0.2% (n=4) American Indian/Alaska Native, and 0.1% (n=2) Native Hawaiian or other Pacific Islander while 5.9% (n=147) of the patients belonged to other races. Data about race was not reported for 20.8% (n=519) of the patients. Cortes et al. (2018) reported the highest racial disparity with 97.7% (n=129) white patients and only 0.76% black patients (n=1), while Gamis et al. (2014) reported the highest number of black patients (n=116). Conclusion: Despite being affected adversely to a significant degree by AML, black patients are underrepresented in drug trials. More efforts should be made to include them in trials so that better treatment plans can be formulated for this underrepresented group of patients.

Cardiotoxicity Related Mortality Among Children with Acute Myeloid Leukemia in Low Middle Income Countries: Is It Treatment or Sepsis Related?

Background: Heart disease and cancer are two major causes of morbidity and mortality worldwide, accounting for at least 70% of the medical reasons for mortality across the globe. Modern treatment strategies led to improvements in cancer survival, however, these gains might be offset by the potential negative effect of cancer therapy on cardiovascular health. Over the last few decades, cooperative oncology group trials for pediatric acute myeloid leukemia (AML) have improved overall survival (OS) to nearly 65% by combining intensive chemotherapy with improved supportive care. Despite their efficacy, anthracyclines are linked to an increased risk of cardiotoxicity. Objectives:The study aimed to describe the incidence of cardiotoxicity among pediatric patients with acute myeloid leukemia, assess the effect of cardiotoxicity on treatment outcome in terms of overall survival (OS) and event free survival (EFS) and to determine the different risk factors (Age, gender, body mass index, cumulative doses of anthracyclines and sepsis) that may contribute to cardiotoxicity. Patient and Methods: A retrospective study, conducted at the National Cancer Institute (NCI), Cairo University, Egypt.,that included all pediatric patients ≤ 18 years old diagnosed and treated as Acute Myeloid Leukemia (AML) during the period from the first of January 2015 to the end of December 2019. Retrospective analysis of the different features of cardiotoxicity associated with the treatment of AML. Results : The study included 142 patients with a median age of 8.5 years old and M/F ratio 1.2:1.Out of the total study population, Eighty four patients (59.2%) developed cardiotoixicity in which 59.5% were acute onset cardiotoxicity and 40.5 % were chronic early cardiotoxicity and there was no detected cases of chronic late cardiotoxicity.Cardiac dysfunction was detected in 65/142 (45.8%) patients at time of death in association with sepsis. The cumulative incidence of cardiotoxicity was higher in the presence of blood stream infection with p value (p<0.001).55/84 (65.5%) patients with cardiotoxicity showed progressive cardiac dysfunction and they all died due to sepsis that was associated with cardiac dysfunction. At the end of the study the OS and EFS were 16.2 % and the median survival time was 1.9 month.The cumulative OS,EFS and the median survival time were reduced in the presence of acute cardiotoxicity with statistical significant p-value (p<0.001). Conclusion: The incidence of cardiotoxicity is high among pediatric AML patients and the most important risk factor for cardiotoxcity is blood stream infection. The Cumulative OS, EFS and median survival time are reduced in patients with acute cardiotoxicity which represents a major challenge that warrants more attention to the field of cardio-oncology.

Alignment of Later Phase Clinical Trial Eligibility Criteria with Drug Safety in Acute Leukemia: Minimizing Unnecessary Patient Risk and Exclusion

Introduction Clinical trial eligibility criteria can unfairly exclude patients or expose patients to risk if they are not justified by drug safety. Our prior analysis of acute myeloid leukemia (AML) trials identified unjustified variability in the use of criteria and in the limits placed when criteria were used (ASCO 2023 Abstract #6525). As additional safety data are available in later phase studies, we sought to assess if criteria were more justified in phase III versus phase II trials, and we extended our analysis to include acute lymphoblastic leukemia (ALL). Methods Trials and criteria were identified using ClinicalTrials.gov. Trials were included if they treated patients in the United States, were interventional, therapeutic, phase II or III, treated previously untreated adults with AML and/or ALL, were not cellular therapy trials, and had study start dates from 1/1/2010-12/31/2019. Drug safety data from labels, trial protocols, and previous study results available as of study start dates, as identified in the ClinicalTrials.gov record, were compared to systematically coded eligibility criteria following FDA publications.Primary endpoints were differences in discordant criteria use (presence of criteria without a known, relevant drug safety signal or absence of criteria with a signal) and limits (numerical values of drug safety- or criteria-based limits) between phase II and III studies assessed via Fisher's exact, rank sum, and median absolute deviation (MAD) testing. Regression modeling assessed differences in infectious disease (ID) exclusions by phase adjusted for disease type and trial sponsor, year, size, and drug safety profiles (drug-drug interaction and association with viral reactivation). Results There were 250 eligible trials, of which 203 were phase II and 47 phase III; 190 trials were in AML and 60 ALL. Similar proportions of phase II and III trials had exclusion criteria (with specific limits when applicable) for low left ventricular ejection fraction (LVEF; 36 vs 25%), high corrected QT interval (QTc; 21 vs 27%), presence of human immunodeficiency virus (HIV; 57 vs 47%), and hepatitis B (43 vs 38%) and C (42 vs 38%). More phase II than phase III trials had renal function (70 vs 53%; p=0.04), bilirubin (73 vs 49%; p<0.01), and alanine transaminase/aspartate transaminase (AST/ALT; 26 vs 64%; p<0.01) based exclusions. Table 1 shows proportions and odds ratios (OR) of discordant criteria by trial phase. OR of discordance for renal function (1.90), AST/ALT (2.30), and bilirubin (2.45) were significantly higher for phase III studies (all p<0.05); no criteria were less discordant for phase III studies. Drug safety- and trial-based limits for renal and liver function are shown in Figure 1. Median differences between limits numerically improved from phase II to phase III (renal: 0.62 to 0.20; AST/ALT: 0.50 to 0; bilirubin: 0.20 to 0); only the change in renal function was significant (rank sum p=0.03). The dispersion of these differences was numerically smaller for renal function (phase III to III MAD ratio: 0.65) and bilirubin (0.91) and higher for AST/ALT (1.17); no changes in dispersion were significant. In multivariable regressions adjusted for other trial and drug safety factors, phase III trials were equally likely to exclude persons diagnosed with HIV and hepatitis B and C as phase II (adjusted OR 1.48, 1.37, 1.41; all p>0.05). Conclusions Despite increasing understanding of drug safety profiles in later phase trials, the use of eligibility criteria for phase III acute leukemia clinical trials were at least as discordant with known drug safety as earlier phase studies. When drug safety- and trial-based limits were present, phase III criteria were not consistently more aligned with drug safety and were equally likely to exclude persons diagnosed with HIV and hepatitis. Future phase III trials in acute leukemia should standardize and rigorously justify eligibility criteria to enhance patient representativeness and welfare.

Roles of Ribosomal S6 Kinases in Acute Leukemia and Normal Hematopoiesis

Ribosomal S6 kinases (RSKs) are serine-threonine kinases that are activated by the Ras/mitogen activated protein kinase signaling pathways and regulate cell proliferation and survival. There are 4 isoforms of RSK (RSK1, 2, 3, 4), and there is evidence that the isoforms mediate tissue-specific cellular functions. We hypothesized that RSK isoforms have distinct roles in hematopoiesisand evaluated RSK as a target for the treatment of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). To investigate isoform expression in hematopoiesis, we sorted cord blood mononuclear cells into hematopoietic progenitor stages and quantified RSK isoform mRNA by RT-qPCR. RSK1 expression remained consistent throughout hematopoiesis, whereas RSK2 expression decreased in the more differentiated populations. RSK3 was elevated in the lymphoid-primed multipotential progenitor and common myeloid progenitor stages and was greatly reduced in the more mature megakaryocyte-erythroid progenitor population. These results suggest that RSK proteins have isoform-specific roles in hematopoiesis. To analyze the importance of each isoform on hematopoiesis, we transduced cord blood CD34+ cells with lentiviral constructs expressing isoform-specific or control shRNAs. After liquid culture for 5 days, the cells were plated in methylcellulose media containing SCF, IL-3, GM-CSF, and EPO to determine the colony-forming potential. RSK1 and RSK2 knockdown decreased the colony-forming unit - erythroid colonies and increased the granulocyte/macrophage colony numbers. RSK3 knockdown increased the burst-forming unit - erythroid colonies and total colony numbers. After liquid culture days 8, 11, and 14, the erythroid and myeloid populations were analyzed by flow cytometry. RSK1 knockdown increased the CD11b+ and CD14+ myeloid populations and decreased the CD71+ erythroid population. RSK3 knockdown increased the erythroid population and decreased the myeloid populations. These data demonstrate that RSK1 and RSK3 have opposing effects on hematopoietic lineage determination. Overexpression of RSK proteins leads to aberrant cell survival and proliferation in many cancers including leukemia. We have previously reported that RSK and phosphorylated RSK are elevated in pediatric AML and are associated with a worse prognosis. There are only two orally bioavailable RSK inhibitors that have moved to phase I clinical trials: TAS0612 and PMD-026. TAS0612 is a multi-kinase inhibitor of RSK, AKT, and S6K. PMD-026 has high specificity to all four RSK isoforms. We evaluated the effects of both compounds in AML and ALL. To determine the effects of the RSK inhibitors on AML, we treated AML cell lines and patient samples with a range of doses of PMD-026, TAS0612, or 0.1% DMSO. After 3 days, we performed CellTiter Glo assays to determine the relative viable cell number and calculate the concentration of drug that inhibits cell growth by 50% (IC50). TAS0612 had an IC50 of 0.096-0.64 µM in AML cell lines Molm13, U937, AML193, and MV4;11. To test the cytotoxicity of TAS0612 on normal hematopoiesis, we plated healthy bone marrow mononuclear cells with TAS0612 in methylcellulose media. Colony formation was not adversely affected by TAS0612 up to 1 µM. PMD-026 was effective in Molm13, Kasumi-1, and MV4;11 with an IC50 of 0.34-0.55 µM and in AML patient samples 3123 and 4192 with an IC50 of 1.5-2.3 µM. PMD-026 did not affect healthy bone marrow colony formation up to 5 µM for a 2.5- to 10-fold therapeutic window. Furthermore, we tested the effects of RSK inhibitors in ALL. While PMD-026 was not effective, we found that B-ALL cell lines (REH, Nalm-6) and T-ALL cell lines (Loucy, Jurkat) were sensitive to RSK inhibition by TAS0612 with an IC50 of 0.056-0.19 µM. Western blot analysis of RSK protein revealed that RSK1, RSK2, and phospho-RSK proteins are elevated in ALL cell lines compared to healthy peripheral blood mononuclear cells. We are investigating the role of RSK and RSK isoforms in ALL. We found that RSK isoform expression is modified during hematopoiesis. Isoform expression influences early-fate decisions to differentiate into erythroid and myeloid lineages. RSK inhibitors PMD-026 and TAS0612 are effective at targeting AML and ALL cells with limited cytotoxicity to healthy cells, making them promising candidates for translation into clinical trials for AML and ALL.

Influence of Pre-Treatment Features and Therapy Choice By Physicians on Overall Survival in Older Adults with Acute Myeloid Leukemia: A Report from the Beat AML Master Trial

Background: Acute myeloid leukemia (AML) therapy in older patients (age ≥ 60) has undergone a transformation with the introduction of multiple targeted therapies, which has allowed more patients to receive therapy than before. The most successful has been the combination of venetoclax + azacitidine (VA) which generated higher overall complete response rates in phase 2 studies than would be expected with azacitidine alone, leading to accelerated FDA approval on 21Nov2018. The VIALE-A phase 3 study confirmed the improved overall survival (OS, median 14.7 versus 9.6 months) of VA, leading to full FDA approval for marketing. This retrospective analysis provides real world data on the impact of baseline clinical and genomic markers of individuals receiving intensive chemotherapy versus VA. Methods: The precision medicine Beat AML Master Trial (NCT03013998) assigns patients to biomarker specific sub-study treatments based on targeted DNA sequencing and cytogenetics. However, a large subset of patients did not enroll on a sub-study, but were followed for off-study treatment and OS. From this subset, patients enrolled onward from 21Nov2018, when VA received accelerated approval, were examined for differences in clinical/genetic characteristics and OS in those receiving venetoclax + hypomethylating agent (V/HMA), any form of intensive chemotherapy (IC), alternative non-intensive therapy (NIT), or no therapy. The genetic mutations identified in this study are characterized by a variant allele frequency of 20%+, as defined in the trial for determining the dominant clone. The method of Kaplan-Meier was used to estimate OS, and the Cox model was fit to associate patient characteristics with OS. Results: From 21Nov2018, a total of 468 AML patients consented to the Beat AML trial and did not enroll to a sub-study. Treatment was chosen by the investigator based upon available clinical and genomic data. Of these 468 patients, 62 did not receive treatment, 2 had treatment data missing, 226 were treated with V/HMA, 112 with IC, and 66 with NIT. Demographics for all patients include median age 71 (60-90), 40% female, performance status (PS) (0-20%; 1-55%; 2-22%; 3-3%), median white blood cell (WBC) 4.1 (range 0.3-298.6), complex karyotype (CK) 20.6%, core binding factor (CBF) 7.8%, KMT2A-rearranged 3.6%, NPM1-mutated (m) 15.6%, IDH2m 14.6%, TP53m 13.2%, FLT3-ITD/TKD 12.9%, and NRASm/ PTPN11m/ KRASm/ NF1m/ CBLm 23.6%. Demographics that were significantly different (p<0.01) among the three groups (V/HMA, IC, and NIT) include age (younger in IC), PS (worse in NIT), AST/ALT (worse in NIT), CBF (more in IC), CK and TP53m (less in IC). Of the 404 patients who underwent treatment with V/HMA, IC, or NIT, 208 have died with those surviving having a median follow-up of 22.3 months. Figure 1 summarizes the OS of each treatment group. The median OS (95% CI) from time of initiating therapy is 13.6 (10.9-16.8) for V/HMA, 33.8 (20.7-not reached) for IC, and 11.6 (5.2-21.3) months for NIT. Univariable analysis for OS was significant at p<0.05 for increased age, WBC, PS, hemoglobin, CBF, CK, NPM1m, TP53m, TET2m and IC versus V/HMA. Multivariable analysis was significant at p<0.05 (hazard ratio) for increased age (1.14), PS (1.83), WBC (1.08), hemoglobin (0.91), and select genomic aberrations including CBF (0.37), NPM1m (0.35), and TP53m (2.1). Conclusions: These results from a large cohort of older AML patients treated with V/HMA, IC, or NIT show that their outcome is best defined by pre-treatment clinical features previously identified including age, performance status, WBC and hemoglobin along with limited genomic characteristics including CBF, NPM1m, and TP53m. While univariate analysis of OS favored IC over V/HMA and NIT, multivariable analysis supports that this advantage was most likely due to the favorable clinical and genomic features. The OS of V/HMA patients in this cohort is similar to that in the VIALE-A registration study of VA, providing further justification for this treatment for older AML patients deemed ineligible for intensive chemotherapy. Additionally, this data supports use of the patient cohort in this study for ongoing work in understanding and/or validating biomarkers associated with survival with V/HMA.

Methods of Estimating Renal Function for Use in Clinical Trial Eligibility Criteria Widely Vary in Phase II and III Clinical Trials in Acute Myeloid Leukemia

Background Renal function is an important parameter to inform drug-dosing in oncology. In acute myeloid leukemia (AML), commonly used therapies or their metabolites are renally cleared. Several equations are commonly used to estimate renal function, such as Cockcroft-Gault (CG) to estimate creatinine clearance (CrCl) and the Modification of Diet in Renal Disease (MDRD) or Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equations to calculate estimated glomerular filtration rates (eGFR). These equations frequently use endogenous serum markers, such as creatinine (Cr) and cystatin-C due to their ready availability but can have limitations. For example, Cr can be affected by muscle mass, diet, and age. Current ADDIKD international consensus guidelines recommend using the CKD-EPI creatinine formula to calculate eGFR to guide anti-cancer drug dosing unless direct GFR measurement is indicated. Renal function criteria are commonly used in clinical trial eligibility. In this study, we analyzed the types of reported renal eligibility criteria in phase II and III trials in AML and the methodology specified for renal function estimation, which can significantly impact treatment options for patients and potentially confound toxicity evaluation. Methods We examined the clinicaltrials.gov database to review publicly reported renal eligibility criteria for interventional phase II and III clinical trials recruiting adults with AML between 2021 to 2023. Trials including pediatric patients and exclusive phase I studies were excluded. Chi-square tests were used to evaluate for potential associations between eGFR cutoff criteria and disease variables. Results A total of 134 trials met criteria for review, including 84 (63%) trials specifically for AML and 50 (37%) allowing additional diagnoses. Of the total study cohort, 107 trials (80%) were evaluating chemotherapy, small molecule inhibitors, immunotherapy and/or combinations of these whereas 27 (20%) were evaluating primarily cell therapy and/or transplant-based regimens. Among the 134 trials, 21 (16%) did not mention renal or general organ function in eligibility criteria; 16 (12%) mentioned renal or organ function in inclusion/exclusion criteria but did not specify an eGFR cutoff value; and 15 (11%) specified a serum Cr cutoff value without a specific eGFR cutoff value. Among 82 (61%) studies specifying eGFR cutoffs: 18 required eGFR ≥60 ml/min and 64 specified cutoffs between 30 ml/min to 50 ml/min or greater (table 1). We found an association between studies requiring an eGFR of 60 ml/min or greater and studies investigating cell therapy or transplant-based regimens (p<0.01). We did not find associations between eGFR cutoff requirements and trials based on disease status (relapsed/refractory AML versus other AML states, p=0.24). We next evaluated methodologies reported in eligibility criteria to estimate renal function. Among the 113 trials commenting on renal function criteria: 15 (13%) did not report either an eGFR cutoff or methodology of determination; 67 (59%) specified eGFR cutoffs without specifying the methodology of determination and/or provided serum Cr-based cutoffs; and 31 (27%) specified either a method of direct measurement or equations to estimate eGFR (including CG-based in 25, CKD-EPI in 1, MDRD-based in 3 and radioisotope methodology in 1) (table 1). No trials mentioned cystatin-C as part of renal function determination. Conclusion In contemporary phase II/III clinical trials for adult patients with AML, we identified that while most trials include renal function in inclusion/exclusion criteria, there was tremendous heterogeneity in methodologies specified for determination of renal function which may make efficacy and toxicity comparisons challenging and impact patient eligibility across institutions. Several trials specified serum Cr cutoffs which can be impacted by factors besides renal function such as age or muscle mass. This study was limited by what trial criteria were publicly available and additional information specifying methodologies to calculate renal function may be available in full trial protocols. Further research is needed to determine the most suitable method for assessing eGFR in patients with AML to optimize treatment exposures for patients and to help reduce confounding of trial results or trial eligibility by institutional practice variation.

A Phase 1a/b Trial of Luxeptinib (CG-806) in Patients with Relapsed/Refractory B-Cell Malignancies or Acute Myeloid Leukemia and Evaluation of New G3 Formulation

INTRODUCTION: Luxeptinib (CG-806; LUX) is an orally active noncovalent kinase inhibitor of Bruton's tyrosine kinase (BTK), wild type and mutant forms of Fms-like tyrosine kinase 3 (FLT3) (including the internal tandem duplicates (ITD), tyrosine kinase domain (TKD), and F691L mutant forms), and growth receptors like KIT, CSF1R, PDGFRα, and TRKs. In prior studies, LUX has been shown to suppress aberrant proliferative signaling in B cell malignancies and acute myeloid leukemia (AML) via regulation of BTK, LYN, SYK, AKT, ERK, and MAPK. LUX is cytotoxic to primary AML cells insensitive to other FLT3 inhibitors and to malignant B-cells insensitive to ibrutinib, at pM and nM concentrations, respectively. AIMS: The primary objectives of these studies are to assess the safety, tolerability, and pharmacokinetics (PK) of LUX and determine recommended phase 2 doses for relapsed or refractory (R/R) AML and B cell malignancy patients. METHODS: In two studies (NCT04477291; NCT03893682), LUX (original formulation, G1) was administered continuously as oral capsules BID in 28-day cycles of ascending cohorts (relapsed or refractory de novo, secondary, or therapy-related AML or higher risk myelodysplastic syndrome (MDS), and relapsed and refractory for B cell lymphoma and chronic lymphocytic/small lymphocytic leukemia). A novel generation 3 (G3) formulation of LUX designed to increase bioavailability was tested at a single-dose (sub-study) for relative bioavailability (RBA), followed by continuous dosing in subsequent R/R AML patients. RESULTS: In the B-cell study (as of 15 th May 2023), LUX has been administered to 36 patients at dose levels from 150 mg to 900 mg BID in patients with a median of 3 lines of prior treatment, with 47.2% having received a BTK-inhibitor. Only one (2.8%) patient had a DLT of hypertension and 14 (38.9%) experienced at least one drug related ≥Grade3 treatment emergent adverse event (TEAE). Two patients (900 mg) are currently on study with no DLT and no ≥Grade 3 related TEAEs. A Follicular lymphoma patient is at cycle 28, and achieved a best response as partial response (PR) with a decrease in lesion size of 76.2%; an SLL patient is at cycle 13, and achieved stable disease (SD) showing a decrease in lesion size of 43.1% (Figure A). The overall best response achieved among the 17 patients who have been on treatment for more than 12 weeks are stable disease (SD) (n=11), partial response (PR) (n=3), minor response (n=2) and complete response (CR) (n=1). As of 5 th June 2023, in the AML trial, a total of 40 patients (16 (40%) FLT3-ITD, 21 (52.5%) FLT3-WT, 2 (5.0%) FLT3-TKD, and 1 (2.5%) unknown mutations) with a median of 3 prior treatments (range, 1 - 10) have been treated with LUX G1 formulation at dose levels from 450 mg - 900 mg BID (n=34) or with 50 mg BID of G3 formulation (n=6). Four (10%) experienced drug related SAE and 7 (17.5%) had a drug related grade ≥3 TEAEs. The most common related TEAEs were lymphocyte count decrease, platelet count decrease, and anemia (n=2; 5% patients each). One (2.5%) patient at 450 mg dose, reported complete remission without minimum residual disease (CRmrd) and remained on study for 56 weeks. In the RBA sub-study, G3 (50 mg) produced comparable exposures to G1 (450-900 mg range) (Figure B) and was, therefore, selected as the starting dose for use in the R/R AML study. Six patients were treated with a continuous dosing of 50 mg BID G3; with plasma levels of 274 ng/mL and 195 ng/mL LUX observed by the end of C1D15 and C1D22 respectively. No drug related Grade ≥3 TEAEs or DLTs were observed in 50 mg BID G3 LUX treated patients; with no new safety signals observed for the G3 formulation. Based on these findings and expected exposures for higher dose levels, the cohort safety review committee has approved escalation of G3 dosing to 200 mg BID at which dosing is ongoing. CONCLUSIONS: LUX has a favorable safety profile in patients at all tested dose levels, for multiple cycles, for both studies. Antitumor activity was observed in a heavily pretreated relapsed AML patient and in multiple B-NHL subtypes and CLL/SLL patients including several with prior ibrutinib exposure. Continuous dosing of patients with R/R AML and Higher-Risk MDS with the G3 formulation is ongoing; with updated clinical data (200 mg dose level) to be presented at the meeting.

Impact-AML: A European Master Framework for Cohort Studies and Pragmatic Clinical Trials in Relapse or Refractory Acute Myeloid Leukemia

Background and Significance: The treatment outcomes for acute myeloid leukemia (AML) have improved in recent decades, particularly for patients who can tolerate intensified treatment approaches. However, current therapies only cure a small portion of patients, with therapy resistance and relapse being the main causes of death. Relapsed or refractory (R/R) AML has a poor prognosis, and allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option. However, there is no common therapeutic strategy for R/R AML, and most of the clinical trials have primarily focused on first-line treatments. The lack of standardized approaches leads to a wide range of variations in treatment choices across centers. Study Design and Methods: Founded by the European Union (EU) through Mission Cancer in the Horizon Europe research and innovation program, we will create STREAM, an inclusive, prospective, master framework for the design and conduction of pragmatic trials in R/R AML. In STREAM, we will enroll patients across Europe, proficiently acquiring an unselected population for clinical trials and monitoring outcomes of neglected cohorts. STREAM will involve more than 200 institutions and will represent a first of a kind large accrual network; online registration of patients will made available at participating sites through a user-friendly form designed in cooperation with the European Rare Blood Disorders Platform (ENROL), an EuroBloodNet initiative of ERN-EuroBloodNet. Within STREAM, we will conduct a proof-of-concept prospective randomized pragmatic clinical trial (RPCT) that will compare the classical “high intensity” rescue chemotherapy with biology-driven, “low intensity” rescue to obtain real world data on the benefit of one of the two different strategies in term of survival. In the RPCT we will also consider and gather patients and caregivers' preferences also through patient-reported outcomes, assess accessibility barriers and social cost of the disease. The RPCT will aim to evaluate the effectiveness of real-world clinical alternatives in routine care; in addition to retaining the high internal validity of traditional randomized trials, it will maximize external validity, i.e. generalizability of results to many settings. In this context, the inclusion of an RPCT in the master framework will allow dynamic inclusion and the collection of the excluded population as an instrument to predict the real-life applicability of clinical trial results. Risk-oriented monitoring and technical developments will pave the way for academy driven clinical trials in the setting. Standard operative procedures for decentralized mesurable residual disease assessment and samples biobanking will be provided. With the aim to generate evidences also in rare subsets, we will run single arm cohort studies that will allow the inclusion of rare patients and will be available for molecular-oriented cohort studies, studies in elderly or fragile patients, and studies in minorities. Single cohort studies external validity will be maximized compared with the STREAM general registered population. The project will receive the support of patients themselves in the creation of both STREAM and clinical trials through patient-directed living labs and patient advocates advice. Conclusion: Maximizing outcomes in R/R AML involves aiming for deep complete remission while minimizing adverse events. Unfortunately, it is not feasible in most patients, and - in treatment-eligible patients -the best path to reach this objective is unclear. IMPACT-AML represents an innovative platform to foster prospective generation of evidence in R/R AML, with a special focus on personalized medicine and innovative treatments for neglected cohorts. IMPACT-AML is funded by the European Union with the grant agreement n. 101104421 in the Mission Cancer cluster of projects on “Diagnosis and treatment”.

Prognostic Value of Measurable Residual Disease in High-Risk Myelodysplastic Syndromes with Intensive Chemotherapy Treatment: Analysis of HOVON-SAKK Studies

Diagnosis of myelodysplastic syndromes (MDS) and accurate risk assessment to tailor treatment remains challenging. High-risk MDS (hrMDS) patients who are fit enough may benefit from intensive chemotherapy followed by post-remission consolidation. The HOVON-SAKK (HO) acute myeloid leukemia (AML) trials, designed to test novel drug combinations with an intensive treatment regimen, included hrMDS patients (RAEB and RAEB-T). Since these HO-trials have been executed with large patient cohorts for many years, they offer the unique possibility to study the outcome, risk classification, and relevance of measurable residual disease (MRD) in hrMDS. A pooled database is available from HOVON-SAKK of patients who were treated with intensive chemotherapy (studies containing MDS patients with IPSS ≥ 1.5 HO42A, HO81, HO92, HO102, and IPSS-R > 4.5 HO103, HO132). The database contained 3268 patients; 11% (364/3268) were hrMDS scored according to IPSS and IPPS-R. Three-year overall survival (OS) for hrMDS was 32% (standard error; SE 2%) compared to AML 46% (SE 1%). In total, 1074 AML and 75 hrMDS had reached complete remission (CR/CRi) and received a second induction cycle (C2). MRD after C2 based on multiparameter flow cytometry (Cloos et al., 2018, J. Vis.) with a cut-off of ≥0.1% for MRD positivity was assessed to evaluate the MRD status. We observed a trend of a higher MRD positivity rate for MDS compared to AML patients (MDS vs. AML: 32% (23/73) vs. 22% (232/1064), P = 0.055). Kaplan-Meier OS analysis showed a significant prognostic value of MRD in MDS, but not in the cumulative incidence of relapse (CIR) (log-rank test 0.011, Gray's test P = 0.124). Since the MDS classification has been changed, we re-classified MDS based on the ICC 2022 classification into AML, AML/MDS, and MDS. We reclassified 3035 patients at diagnosis, including 2605 AML, 258 MDS/AML, and 172 MDS. The three-year OS was 47% (SE 1%) for AML, 29% (SE 3%) for MDS/AML, and 33% (SE 4%) for MDS. After C2, 2215 patients had reached CR/CRi. A total of 1074 patients containing 984 AML, 57 AML/MDS, and 33 high-risk MDS patients with CR/CRi after C2, with a suitable bone marrow sample, were included in the MRD analysis. Most patients were treated according to HO132 (40-48%). As expected, MDS patients were older (median age, range; 60, 32-74), had lower bone marrow blast counts, and were predominantly of adverse risk. A trend of a higher proportion of MRD positivity after C2 was observed in the MDS group (33%; 11/33) as compared to AML (22%; 215/984) or MDS/AML (18%; 10/57). Three-year OS was 61% for AML (SE 1.6%), 42% (SE 6.6%), and 49% (8.7%) for AML/MDS and MDS, respectively (Figure 1A). Kaplan-Meier OS analysis and CIR showed a significant prognostic value of MRD for MDS (log-rank test P<0.001, Gray's test P = 0.015) and AML ( P<0.001; P<0.001) but not for MDS/AML patients ( P = 0.5, P = 0.342)(Figure 1B). Remarkably, the three-year OS of MRDpos in MDS was 18% (SE 12%) compared to MRDpos in AML 40% (SE 16%). The limitation of this study is the relatively small number of MDS patients according to the new ICC 2022 guidelines. In summary, MRD detection in the HOVON-SAKK trials shows the potential of MRD as a prognostic value in MDS patients treated on intensive induction regimens with a clinically validated and standardized assay.

Engineering Off-the-Shelf Gamma Delta CAR T Cells for the Treatment of Acute Myeloid Leukemia

Chimeric antigen receptor T cell therapy (CART) therapy has shown remarkable success in the treatment of B cell acute lymphoblastic leukemias (B-ALL) and lymphomas. However, CART therapies for acute myeloid leukemia (AML), where 5-year survival rates are significantly lower than for B-ALL, are only in their infancy. CD33-CART have potent activity against AML in preclinical models and a first-in-child/first-in-human phase 1/2 CD33-CART clinical trial for AML is ongoing in the Pediatric Oncology Branch of the National Cancer Institute (NCT03971799). Nonetheless, published outcomes suggest a modest efficacy of approximately 50% (Shahzad et al., Front Immunol 2023), highlighting the critical need to develop new strategies to improve CART accessibility and a more robust anti-AML response. We hypothesized that off-the-shelf gamma delta (γδ) CD33 CART cells could potentially overcome current challenges for the treatment of AML. γδ lineage T cells are unconventional lymphocytes whose functions are not restricted to MHC-mediated antigen presentation; they are primed for immediate responses, including tumor killing. Furthermore, allogeneicγδ T cells have the potential to induce robust anti-tumor cytotoxicity without causing graft versus host disease (GVHD). Here, we generated γδ CAR T cells from healthy donor elutriated lymphocytes by activation with zoledronic acid and IL-2 for 7-14 days. Within 9 days post stimulation, the vast majority of lymphocytes were Vδ2+ and 30-40% were successfully transduced with a lentiviral CD33 CAR construct harboring the 4-1BB costimulatory domain. Importantly, and unlike conventional alpha beta (ab) T lymphocytes, >98% of these γδ CD33CAR T cells expressed IFNγ under basal conditions. This characteristic likely accounted for the efficient in vitro killing of AML cell lines by untransduced γδ T lymphocytes under conditions of high effector/target (E/T) ratios. While untransduced γδ T cells did not exhibit cytotoxicity following repeat AML stimulations, γδ CD33CAR T lymphocytes exhibited proficient in vitro cytotoxicity, with killing rates that were more rapid than those initiated by ab CD33 CART (Figure 1). These characteristics were associated with a prolonged metabolic activity of γδT cells; γδ CD33 CART expressed high levels of the GLUT1 glucose transporter for >14 days post activation whereas GLUT1 levels on ab CD33 CART returned to resting within 10 days. High GLUT1 levels were linked to efficient killing under conditions of basal glucose levels. Most notably, γδ CD33CAR T lymphocytes achieved high in vivo cytotoxicity, assessed using bioluminescent AML cell line xenografts in humanized NSG mice. Together, these data highlight the feasibility of generating allogeneic γδ CD33CART with a strong anti-AML cytotoxic response.

Exclusion of Acute Myeloid Leukemia Patients with Central Nervous System Involvement from Clinical Trials: An Analysis of the National Institutes of Health Clinical Trials Registry from 2012 to 2022

Introduction: Central nervous system (CNS) involvement in acute myeloid leukemia (AML) can be successfully treated with intrathecal chemotherapy and carries debatable prognostic impact. However, patients with CNS involvement are commonly excluded from clinical trials at an unknown rate. We systematically evaluated exclusion criteria of AML clinical trials based on CNS involvement and determined associations with clinical trial characteristics. Methods: The National Institutes of Health Clinical Trials Registry was searched for interventional adult AML trials between 2012 and 2022 that were phase 1, 2, or 3 and relevant trial characteristics were extracted. Results: 1,270 trials were included in the analysis with 790 trials (62.1%) explicitly excluding CNS involvement. There was no significant change in rates of CNS exclusion over the past decade. CNS exclusion was higher in trials that included the non-transplant population compared to trials exclusive to the transplant population (66.9% vs. 43.8%, p < 0.01). Non-transplant trials were also more likely to exclude patients with a history of or ambiguous timing of CNS involvement (p < 0.01). Phase 3 trials were associated with more liberal definitions of CNS exclusion (history or ambiguous timing) as compared to phase 1 and 2 trials that had higher rates of excluding patients with only active CNS involvement (p < 0.01). Conclusion: A majority of AML clinical trials, particularly in the non-transplant setting, exclude patients with CNS involvement. Many of these trials, most notably phase 3 trials, exclude patients not only with active but also with any history of CNS involvement. Further research is needed to determine optimal management of these patients in order to increase representation in large clinical trials.

TAK-981, a SUMOylation inhibitor, suppresses AML growth immune-independently.

Acute myeloid leukemia (AML) generally has an unsatisfactory prognosis despite the recent introduction of new regimens, including targeted agents and antibodies. To find a new druggable pathway, we performed integrated bioinformatic pathway screening on large OHSU and MILE AML databases, discovered the SUMOylation pathway, and validated it independently with an external data set (totaling 2959 AML and 642 normal sample data). The clinical relevance of SUMOylation in AML was supported by its core gene expression which is correlated with patient survival, European LeukemiaNet 2017 risk classification, and AML-relevant mutations. TAK-981, a first-in-class SUMOylation inhibitor currently under clinical trials for solid tumors, showed antileukemic effects with apoptosis induction, cell-cycle arrest, and induction of differentiation marker expression in leukemic cells. It exhibited potent nanomolar activity, often stronger than that of cytarabine, which is part of the standard of care. TAK-981's utility was further demonstrated in invivo mouse and human leukemia models as well as patient-derived primary AML cells. Our results also indicate direct and cancer cell-inherent anti-AML effects by TAK-981, different from the type 1 interferon and immune-dependent mechanism in a previous solid tumor study. Overall, we provide a proof-of-concept for SUMOylation as a new targetable pathway in AML and propose TAK-981 as a promising direct anti-AML agent. Our data should prompt studies on optimal combination strategies and transitions to clinical trials in AML.