Figueroa, María E.

Abstract Introduction: Standardized reporting of clinical trials results for MDS is essential to improve clinical interpretation and cross-study comparison. However, reporting of patient characteristics, response criteria, and endpoints in publications is often inconsistent; there is no systemic analysis of MDS trial reporting available. We conducted a systematic review of published MDS trial manuscripts on behalf of the international consortium for MDS (icMDS) to assess variability in reporting practices. Methods We searched ClinicalTrials.gov to identify all clinical trials registered for adults (≥18 years) with MDS that reported results between 2015-2024. Clinical trials that included acute myeloid leukemia (AML) or MDS/myeloproliferative neoplasms (MPN) were included if they enrolled ≥5 MDS patients. We excluded trials without an MDS cohort; non-therapeutic trials; trials focused on transplant interventions; or those which enrolled solid malignancies or other hematologic malignancies (chronic myeloid leukemia or lymphoid diseases). Finally, trials were required to have a manuscript available on Larvol, PubMed, or Google Scholar. Trials were categorized by disease risk (lower-risk [LR] vs. higher-risk [HR]) and trial phase (early phase [EP] vs. late phase [LP]). Results We identified 502 MDS trials on ClinicalTrials.gov, of which 351 did not meet inclusion criteria and 79 lacked a manuscript. A total of 72 trials (32 EP, 40 LP) with 80 publications (34 EP, 46 LP) were analyzed. Frequently reported baseline characteristics included age (100% EP, 100% LP), disease risk (74% EP, 74% LP), Eastern Cooperative Oncology Group performance status (85% EP, 72% LP), prior treatments (71% EP, 67% LP), and RBC transfusion dependency (32% EP, 70% LP). Less frequently reported characteristics included blood counts (hemoglobin [Hb; 38% EP, 54% LP], platelet count [38% EP, 54% LP], neutrophil count [24% EP and LP]) and bone marrow blasts (29% EP, 43% LP). Although disease risk was widely reported, definitions varied: IPSS and IPSS-R were each reported in 54% of manuscripts, while IPSS-M appeared in only 3%. Cytogenetic risk was separately reported in 39% of manuscripts (38% EP, 39% LP), and mutational data in 45% (47% EP, 43% LP). Responses per IWG 2006 criteria were reported in 84% of manuscripts. The primary endpoint involved safety/tolerability in 85% of EP manuscripts, though definitions were variable; recommended phase 2 dose was the most common (26% of EP manuscripts). In LP trials, primary endpoints were risk-specific: transfusion independence (TI) in 77% of LR, overall response rate (ORR) in 28% of HR, and complete remission (CR) or overall survival (OS) in 22% of HR. In HR manuscripts, CR was reported in 100%, ORR in 73%, hematologic improvement (HI) in 48%, RBC-TI in 23%, and OS in 80%. However, seven different definitions were used for ORR: CR + marrow CR + partial remission + HI per IWG 2006 was most common (35%). Event-free survival was reported in 25%; progression-free survival and relapse-free survival in 13%; and leukemia-free survival in 5%. Early mortality rate was reported in 35% (18% EP, 56% LP) and transplant rate in 58% (50% EP, 67% LP). Only 47% of HR and 90% of LR manuscripts identified HI-eligible patients. In LR-MDS, RBC-TI was reported in 72% of manuscripts but used six definitions. The most common were RBC-TI ≥8 weeks at any time during treatment (24%), ≥8 weeks within 28 weeks of treatment (13%), ≥8 weeks with Hb increase ≥1.0 (4%), ≥12 weeks with Hb increase ≥1.5 (4%), and ≥16 weeks within 24 weeks of treatment (12%). The remainder did not report RBC-TI, did not define it, or used a custom definition not used in another trial. HI was reported in 80% of LR papers with most of those reporting HI using IWG 2006 criteria (90%). Conclusion Reporting of baseline characteristics, response criteria, and outcomes is inconsistent in MDS clinical trials, with wide variability in response definitions, including ORR and TI. This heterogeneity limits interpretability, hampers historical comparisons and may obscure efficacy signals. Our findings highlight the need for standardized reporting guidelines to ensure clarity and consistency in MDS trial publications. As a next phase of this effort, we will conduct a formal Delphi process among icMDS experts to establish consensus recommendations for minimal and optimal MDS clinical trial reporting in manuscripts by disease risk (LR/HR) and trial phase (EP/LP).

Abstract Excessively restrictive inclusion and exclusion criteria in clinical trials are one of many barriers to clinical trial enrollment for patients with myelodysplastic syndromes/neoplasms (MDSs). Many organizations are developing efforts to increase clinical trial eligibility; yet, several recent publications focused on patients with MDS suggest that many patients with this disease may be excluded from clinical trials unnecessarily. Clinical trial eligibility should reflect the phase of the study and risks of the agent being studied. Phase 3 trials should be less restrictive than early-phase trials to represent the real-world population as closely as possible. We hypothesize that many clinical trials, particularly phase 3 trials, have unnecessarily restrictive eligibility criteria. This study aims to evaluate the most common eligibility criteria according to phase of trial and to determine whether criteria correspond with drug safety signals. We identified MDS clinical trials registered on ClinicalTrials.gov from 1 January 2000 to 1 September 2023 and analyzed the eligibility criteria of 191 therapeutic MDS trials. We found that categorical inclusion and exclusion criteria are remarkably similar in representation across trial phases. Additionally, only 13% of trials are concordant with drug safety signals, suggesting that the eligibility criteria are often arbitrary. On behalf of the icMDS (International Consortium for Myelodysplastic Syndromes), an association of international MDS experts, we provide a position statement on restrictive eligibility criteria for MDS clinical trials that should be avoided with the aim of removing barriers to clinical trial enrollment.

Abstract Chronic idiopathic myelofibrosis (MF) is a clonal hematopoietic disorder that leads to progressive marrow fibrosis and peripheral cytopenias. Very little is known about the role of aberrant DNA methylation in the pathobiology of this disease. Whole genome methylation was analyzed by a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 9 patients with IMF were compared to 9 age-matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays using cDNA from the same samples. Methylation analysis showed that myelofibrosis samples clustered separately from normal and anemic controls when grouped by unsupervised clustering based on Pearson’s correlation coefficient. On the other hand, global gene expression demonstrated no clear cut differences between myelofibrosis and control samples suggesting that methylation profiling has greater discriminatory power when compared to conventional gene expression profiling. A high correlation (r=0.88–0.96) was observed between whole genome methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can act as a valid surrogates for epigenomic analysis. Further analysis showed that genes aberrantly methylated in all myelofibrosis samples included v-myc, histone 2A, TNF, TNF Receptor1, FGF14 and others. Functional pathway analysis by Ingenuity showed that pathways involved in Inflammation and Cell signaling were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes for chemokine CXCL13, APC, IL-3, STAT2 and others. The pathways most activated by hypomethylation were involved in hematopoiesis and cell growth and proliferation demonstrating the biological validity of our analysis. Thus, our data demonstrates that myelofibrosis is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and lead to translational studies with agents targeting DNA methylation.

Abstract Abstract 851 Clinical studies have demonstrated that acute myeloid leukemia (AML) is heterogeneous with respect to presentation and to clinical outcome, and many studies have shown that cytogenetics can be used to improve prognostication. More recently, it has been shown that FLT3, NPM1, and CEBPA mutations can be used to further risk stratify AML patients. Although recent studies have identified novel, recurrent somatic mutations in AML in genes including TET2, ASXL1, IDH1, IDH2, and PHF6, their prognostic value has not yet been evaluated among patients treated on a large phase III clinical trial. Here we report full-length DNA resequencing of FLT3, NPM1, CEBPA, H/K/NRAS, KIT, WT1, TET2, ASXL1, IDH1/2, TP53, RUNX1, PTEN, and PHF6 in pre-treatment genomic DNA from 398 patients with de novo AML younger than 60 years of age enrolled in the ECOG E1900 protocol. We could identify a clonal alteration, defined as a somatic mutation or cytogenetic abnormality, in 91.2% of all patients; 42% had 1 somatic alteration, 36.4% had 2 alterations, 11.3% had 3 alterations and 1.5% had 4 or more somatic alterations. Mutational Analysis: We identified somatic mutations in FLT3 (37% total; 30% ITD, 7% TKD), NPM1 (14%), CEBPA (10%), TET2 (10%), NRAS (10%), WT1 (10%), KIT (9%), IDH2 (8%), IDH1 (6%), RUNX1 (6%), ASXL1 (4%), PHF6 (3%), KRAS (2.5%), TP53 (2%), and PTEN (1.5%) (Figure 1A). We then performed correlation analysis to determine the frequency of different mutational combinations in AML patients. In addition to previously identified mutational correlations (FLT3 and NPM1, KIT and core binding factor leukemia), we found that FLT3 and ASXL1 mutations were mutually exclusive in this large cohort (p =0.0008). In addition, we found that IDH1/IDH2 mutations were mutually exclusive of both TET2 (p=0.02), and WT1 (p=0.01) mutations consistent with overlapping roles in AML pathogenesis (Figure 1B). Integration of mutational data with response data and outcome: Mutations in ASXL1 (CR rate 33.3% versus 66.5% for ASXL1-WT patients; p= 0.009) were enriched in patients who did not respond to induction chemotherapy. We found that mutations in FLT3 (p=0.0005), ASXL1 (p=0.005; Figure 1C), and PHF6 (p=0.02) were associated with reduced OS. Notably, the adverse effects of ASXL1 and PHF6 mutations on outcome were restricted to patients treated with standard dose daunorubicin (p<0.01 for each mutational cohort); ASXL1 and PHF6 mutational status did not affect outcome in patients treated with high-dose daunorubicin, demonstrating that increased intensity induction chemotherapy can improve outcomes in genetically defined poor risk AML subsets. We also found that mutations in CEBPA (p= 0.04) and in IDH2 (p=0.003) were associated with improved OS; the favorable impact of IDH2 mutations on outcome was exclusive to patients with IDH2 R140Q mutations (Figure 1D). Within the subset of patients with cytogenetically defined intermediate risk AML, we found, as expected, that FLT3-ITD mutations were associated with reduced OS and CEBPA mutations were associated with improved OS. Importantly, we found that IDH2 R140Q mutations were associated with improved OS (p=0.003) in patients with intermediate risk AML regardless of FLT3 mutational status. In contrast, TET2 mutations (P=0.0002) and PHF6 (p<0.0001) were associated with reduced OS in intermediate risk, FLT3-WT, AML. Summary: These data provide important clinical implications of genetic alterations in AML and provides insight into the multistep pathogenesis of adult AML. We identified novel mutations that impact response to induction chemotherapy and predict overall outcome, including ASXL1, PHF6, TET2 and IDH2, suggesting these mutations can be used to improve AML risk stratification and therapeutic decisions in AML patients 18–60 years of age. Most importantly, we identify TET2, IDH1/2, and WT1 mutations as mutually exclusive somatic alterations that form a novel mutational complementation group that contributes to AML pathogenesis. Figure 1. A) Circos diagram depicting relative frequency and pairwise co-occurrence of mutations in de novo AML patients enrolled in the ECOG protocol E1900. B) Circos diagram showing mutational events in IDH1/2, TET2, and WT1 mutant AML patient. C) Overall survival in AML patients with and without ASXL1 mutations (p=0.005). D) Overall survival in AML patients with and without IDH2 R172 and R140 mutations (p=0.003). Figure 1. A) Circos diagram depicting relative frequency and pairwise co-occurrence of mutations in de novo AML patients enrolled in the ECOG protocol E1900. B) Circos diagram showing mutational events in IDH1/2, TET2, and WT1 mutant AML patient. C) Overall survival in AML patients with and without ASXL1 mutations (p=0.005). D) Overall survival in AML patients with and without IDH2 R172 and R140 mutations (p=0.003). Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Despite advances, the majority of patients (pts) with acute myeloid leukemia (AML) will die of their disease. Current genetics-based risk classification schemes inadequately predict outcome. We sought to determine if a novel methylation-based biomarker could enhance risk stratification in AML. Methods: Using a novel methylation assay (xMELP) validated for the clinical laboratory, we performed DNA methylation assessment at 17 previously identified prognostic loci to calculate a summary methylation statistic (M-score, Wertheim et al. Clin Chem 2015) for 166 pts with de novo AML treated at the University of Pennsylvania (UPENN). Targeted next-generation sequencing (NGS) of 33 hematologic malignancy-associated genes was performed for a 136 pt subset. The association of M-score with other prognostic variables and outcome [complete remission (CR) and overall survival (OS)] was evaluated. Median follow-up was 68.1 mos (range 1.4-150.2) among 38 survivors and 10.5 mos (range 0.1-95.2) among those (n=128) deceased. The optimal M-score for identifying groups with differing OS in the total UPENN cohort defined a binary classifier. The classifier was validated in UPENN subgroups and in a separate ECOG ACRIN E1900 trial cohort. Results: The mean and median M-score for the UPENN cohort was 92.3 (95% confidence interval [CI], 87.4-97.2) and 91.4 (range, 30.8-97.3), respectively; the M-score was not significantly associated with age, gender, specimen type, blast percent, NPM1, or FLT3-ITD. Patients with favorable cytogenetics had a lower mean M-score than those with non-favorable cytogenetics; there was no difference between intermediate and unfavorable cytogenetic groups. Univariate analyses demonstrated that a 10-unit increase in M-score was associated with a 10% increase in the hazard of death (HR 1.1; P<.0001) and a 20% increase in the odds of failing to achieve CR (OR 1.2, P<.0001). In multivariable Cox analysis, higher M-score (HR 1.1, P=.011) and older age (P=.001) were significantly associated with increased hazard of death, while NPM1+/FLT3-ITD-status (P=.031) was associated with decreased hazard of death. In a multivariable logistic analysis, higher M-score (HR, 1.1, P=.034) and older age (P=.007) were associated with increased odds of failing to achieve CR, while favorable cytogenetics (P=.030) was associated with achievement of CR. Based on the maximization of the log-rank statistic, the optimal M-score cutpoint was 86, which defined a binary risk classifier (hazard of death for high vs low M-score: unadjusted HR 2.5, P<.0001; adjusted HR 1.9, P=.003). Median OS was 26.6 vs. 10.6 mos for low and high M-score groups (Figure). The CR rates for low and high M-score groups were 84% and 61%, respectively. The performance of the M-score classifier was confirmed in pts ≤ 60 yrs with intermediate cytogenetics (log-rank P=.001, OS in high vs low M-score groups: 36.4 vs 14.0 mos) and among pts who achieved CR with initial therapy (log-rank P<.00001, OS 43.9 vs 17.2 mos). The M-score classifier identified groups with different outcome regardless of allogeneic transplant. The M-score classifier was validated in the E1900 cohort (n=383). The mean and median M-scores were similar to the UPENN cohort and M-score was significantly associated with OS on multivariable analysis (P<0.0001). The M-score classifier identified E1900 subgroups with different OS (log-rank P<.00001; OS 29.5 vs 12.6 mos), a finding confirmed in the intermediate cytogenetics group, in those aged <50 and ≥50 yrs, and among pts assigned to both standard and high dose daunorubicin. Notably, high dose daunorubicin benefited patients with high M-scores (P=.001), but not those with low M-scores (P=.328). We also evaluated the prognostic value of the M-score in the context of an extended molecular profile. Random forest analysis of the UPENN cohort showed that M-score and age are the most robust predictors of OS, while a subset of recurrent mutations (FLT3-ITD, NPM1, IDH2, TET2, TP53, NRAS, CEBPA) also contribute to prognosis, but to a lesser degree. Conclusion: The M-score and associated classifier represent promising tools for clinical decision-making in AML and deserve further investigation. The prognostic value of the M-score may be superior to mutational analysis. Optimal methods for integration of M-score, clinical and genetic information are being defined. Figure 1. Figure 1. Disclosures Loren: Merck: Research Funding. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

BACKGROUND. Variable response to chemotherapy in acute myeloid leukemia (AML) represents a major treatment challenge. Clinical and genetic features incompletely predict outcome. The value of clinical epigenetic assays for risk classification has not been extensively explored. We assess the prognostic implications of a clinical assay for multilocus DNA methylation on adult patients with de novo AML. METHODS. We performed multilocus DNA methylation assessment using xMELP on samples and calculated a methylation statistic (M-score) for 166 patients from UPENN with de novo AML who received induction chemotherapy. The association of M-score with complete remission (CR) and overall survival (OS) was evaluated. The optimal M-score cut-point for identifying groups with differing survival was used to define a binary M-score classifier. This classifier was validated in an independent cohort of 383 patients from the Eastern Cooperative Oncology Group Trial 1900 (E1900; NCT00049517). RESULTS. A higher mean M-score was associated with death and failure to achieve CR. Multivariable analysis confirmed that a higher M-score was associated with death ( P = 0.011) and failure to achieve CR ( P = 0.034). Median survival was 26.6 months versus 10.6 months for low and high M-score groups. The ability of the M-score to perform as a classifier was confirmed in patients ≤ 60 years with intermediate cytogenetics and patients who achieved CR, as well as in the E1900 validation cohort. CONCLUSION. The M-score represents a valid binary prognostic classifier for patients with de novo AML. The xMELP assay and associated M-score can be used for prognosis and should be further investigated for clinical decision making in AML patients.

AbstractTherapy‐related myeloid neoplasms (tMN) are serious late effects of the treatment of cancer with poor response to conventional treatment. Azacitidine (AZA) has been used to treat patients with tMN but current data are retrospective. We present here 47 tMN patients prospectively enrolled as a specific cohort in the E1905 study. TheE1905 study was a randomized phase 2 study (NCT00313586) testing 10 d of AZA (50 mg/m2/d) +/− the histone deacetylase inhibitor entinostat (4 mg/m2/d PO day‐3 and day‐10). A total of 47 patients [29 therapy‐related myelosyspastic syndrome (t‐MDS) and 18 therapy‐related acute myeloid leukaemia (t‐AML)] were recruited to the study. 24 patients were treated with AZA monotherapy and 23 with AZA+entinostat. The median number of administered cycles was 4, significantly higher in patients treated with AZA (6 cycles vs. 3 cycles, P = 0·008). Haematological normalization rates were 46% in monotherapy and 17% in the combination arm. Median overall survivals were 13 and 6 months, respectively. The novel 50 * 10 schedule of azacitidine appears effective, with response rates, when given as single agent, comparable to those for patients with de novo MDS/AML treated on the same protocol. However, the combination of AZA and entinostat was associated with increased toxicity and could not be recommended for treatment of tMN.

Key PointsDNA methylation changes during the development of DS-AMKL occur in sequential waves of opposing losses and gains of methylation. Each wave of DNA methylation abnormalities targets specific gene networks that contribute to distinct biological features of the disease.

Abstract We determined the prognostic relevance of CD25 (IL-2 receptor-α) expression in 657 patients (≤ 60 years) with de novo acute myeloid leukemia (AML) treated in the Eastern Cooperative Oncology Group trial, E1900. We identified CD25POS myeloblasts in 87 patients (13%), of whom 92% had intermediate-risk cytogenetics. CD25 expression correlated with expression of stem cell antigen CD123. In multivariate analysis, controlled for prognostic baseline characteristics and daunorubicin dose, CD25POS patients had inferior complete remission rates (P = .0005) and overall survival (P < .0001) compared with CD25NEG cases. In a subset of 396 patients, we integrated CD25 expression with somatic mutation status to determine whether CD25 impacted outcome independent of prognostic mutations. CD25 was positively correlated with internal tandem duplications in FLT3 (FLT3-ITD), DNMT3A, and NPM1 mutations. The adverse prognostic impact of FLT3-ITDPOS AML was restricted to CD25POS patients. CD25 expression improved AML prognostication independent of integrated, cytogenetic and mutational data, such that it reallocated 11% of patients with intermediate-risk disease to the unfavorable-risk group. Gene expression analysis revealed that CD25POS status correlated with the expression of previously reported leukemia stem cell signatures. We conclude that CD25POS status provides prognostic relevance in AML independent of known biomarkers and is correlated with stem cell gene-expression signatures associated with adverse outcome in AML.