Hypomethylating Agent Therapy Research Articles

Survival and Prognosis of Patients with Acute Myeloid Leukemia with Myelodysplasia-Related Changes Transformed from Myelodysplastic Syndrome

To explore the risk factors affecting the survival and efficacy of patients with acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) transformed from myelodysplastic syndrome (MDS). The clinical data of 60 patients with AML-MRC transformed from MDS who hospitalized in The Third Affiliated Hospital of Soochow University from January 2010 to December 2021 were retrospectively analyzed. The demographic data and laboratory parameters, cytogenetic karyotypes, target genes of AML detected by next generation sequence, risk stratification, treatment regimen, therapeutic efficacy and survival outcome were documented. Rank sum test and Chi-square test or Fisher exact test were used to compare the survival and efficacy. The effects of clinical parameters, risk stratification and treatment regimens on the survival and efficacy of the AML-MRC patients were analyzed by univariate and multivariate analysis. The median overall survival (OS) of the AML-MRC patients was 4.5 months, the 1-year OS rate was 28.3%, and the complete remission (CR) rate after treatment was 33.3%. The univariate analysis showed that age≥60 years, leukocytosis, severe thrombocytopenia, poor-risk group and only accepted hypomethylating agents(HMAs) or supportive therapy were the risk factors affecting OS. COX multivariate analysis showed that thrombocytopenia ( HR=4.46), HMAs therapy (compared to transplantation, HR=10.47), supportive therapy (compared to transplantation, HR=25.80) and poor-risk group (compared to medium-risk group, HR=13.86) were independent hazard factors for median OS of patients with AML-MRC. The univariate analysis showed that the risk factors affecting 1-year OS in patients with AML-MRC were age≥60 years, thrombocytopenia, time of transformation from MDS to AML (TTA)≥3 months, fibrinogen-albumin ratio index (FARI)≥0.07, CONUT score≥5, poor-risk group and supportive therapy. Binary logistic regression analysis showed that the independent risk factors for 1-year OS in AML-MRC patients were age≥60 years ( HR=11.23), thrombocytopenia ( HR=8.71), FARI≥0.07 ( HR=5.19) and poor-risk group ( HR=14.00). The risk factors affecting CR of AML-MRC patients in univariate analysis were age≥60 years, thrombocytopenia, FARI≥0.1, CONUT score≥5, poor-risk group and supportive therapy, while binary logistic regression analysis showed that age≥60 years( HR=7.35), CONUT score≥5 ( HR=9.60), thrombocytopenia ( HR=12.05) and poor-risk group ( HR=32.5) were independent risk factors affecting CR of the patients. The OS of AML-MRC patients is poor, old age(≥60 years old), supportive therapy, HMA therapy, poor-risk, thrombocytopenia, FARI≥0.07 and CONUT score≥5 may be associated with poor prognosis.

Venetoclax in the treatment of acute myeloid leukemia: Beyond VIALE-A.

Venetoclax and hypomethylating agent therapy in acute myeloid leukemia.

Clinical Efficacy of Hypomethylating Agent Therapy in Patients with Chronic Myelomonocytic Leukemia

To observe the clinical efficacy and safety of hypomethylating agent therapy in chronic myelomonocytic leukemia (CMML). From February 2014 to June 2021, the clinical data, efficacy, survival time and safety of CMML patients diagnosed in the Second Hospital of Hebei Medical University and treated with hypomethylating agent therapy were retrospectively analyzed. A total of 25 CMML patients received hypomethylating agent therapy, including 18 cases treated with decitabine (DEC) and 7 cases treated with azacytidine (AZA) as the basic treatment. Among them, 20 patients responded, and 7 patients got complete remission (CR). All patients with CR were treated with DEC as the basic treatment. Five cases of CR occurred in the first 4 courses of treatment. After a median follow-up of 16.4 (9.4-20.5) months, 4 patients with CR progressed to acute myeloid leukemia (AML). The median overall survival (OS) time of 25 CMML patients was 17.4 months (95%CI: 12.437-22.363). According to MD Anderson prognostic scoring system (MDAPS), CMML-specific prognostic scoring system (CPSS), CPSS molecular (CPSS-mol), Mayo molecular model (MMM), risk stratification of patients was performed, and the difference only between different risk stratification of MDAPS and survival time was statistically significant. Common adverse reactions of hypomethylating agent therapy in CMML patients included infection, gastrointestinal reaction, hematological toxicity, skin allergy and liver function damage. All patients' symptoms were improved after corresponding treatment. Hypomethylating agent therapy is effective and safe for CMML patients. CR mostly occurs in the first 4 courses of treatment, and hypomethylating agent therapy combined with low-dose chemotherapy can be used for patients who do not respond. Hypomethylating agent therapy can delay the disease, but can't prevent progression.

The approach of HMA plus VEN with or without BMT for all patients with AML.

Treatment options for acute myeloid leukemia (AML) have expanded over the last 5 years. New regimens are increasing the options for patients who previously may not have been offered any antineoplastic therapy. The use of the hypomethylating agent (HMA) decitabine or azacitidine combined with the BCL2 inhibitor venetoclax (HMA-VEN) has improved overall survival in an older and unfit population compared to HMA therapy alone. Delivering these regimens outside academic centers allows more patients with AML to be treated, though support and collaboration with allogeneic stem cell transplant (SCT) centers should still be considered to determine eligibility and promptly initiate a donor search for potential transplant candidates. Expanding the use of HMA-VEN to younger and fit patients who are also candidates for intensive chemotherapy (IC) is being studied prospectively and is not recommended at this time outside of a clinical trial. Retrospective studies suggest populations that may benefit from HMA-VEN over IC, but this is not yet confirmed prospectively. Utilizing HMA-VEN prior to allogeneic SCT is also under investigation, and some retrospective data show feasibility and the ability to achieve measurable residual disease negativity pretransplant. Upcoming prospective randomized clinical trials aim to answer the comparability or superiority of HMA-VEN vs IC in fit populations and its potential use as a standard pretransplant induction regimen.

Clinicopathologic Predictors of Hypomethylating Agent Failure in Patients with Myelodysplastic Syndromes

Background: Hypomethylating agents (HMA), such as decitabine, azacitidine, and oral decitabine/cedazuridine, are standard-of-care therapies for patients with myelodysplastic syndromes (MDS). Though they improve peripheral blood cytopenia and delay disease progression, most patients eventually lose response to these agents, a phenomenon known as HMA failure (HMA-F). Survival outcomes are poor with no Federal Drug Administration-approved treatment options in the HMA-F setting. Here, we assess MDS patients treated with HMA for potential clinical and pathological predictors of HMA-F. Methods: We retrospectively evaluated all untreated patients with MDS seen at a single tertiary cancer center between July 2017 and July 2021 and identified those who received HMA therapy after diagnosis. Baseline patient characteristics and bone marrow (BM) data, including morphology, cytogenetics, and mutations, were collected. Genomic DNA was extracted from whole BM aspirate samples and subject to 81-gene target PCR-based sequencing using a next-generation sequencing platform. Survival data was updated in July 2023. Results: A total of 799 untreated MDS patients were identified, and 455 patients (57%) eventually underwent treatment with HMA. With a median follow-up time of 46.8 months (95% confidence interval (CI): 44.4, 52.4), 174 patients (38%) were refractory to or later progressed on HMA therapy. Figure 1 depicts the differences in baseline clinical characteristics in those who remained on HMA therapy and those who developed HMA-F. HMA-treated patients who eventually developed HMA-F were more likely to be older (p=0.003), have higher BM blasts (p=0.044), and higher-risk by both IPSS-R (p=0.003) and IPSS-M (p<0.001) scoring systems. No differences were observed by HMA treatment type, number of cycles, and response. More patients underwent allogeneic stem cell transplantation while on frontline HMA therapy than after development of HMA-F (p<0.001). Figure 2 shows the distribution of mutations detected in at least 10 patients between individuals who remained on HMA therapy and those who developed HMA-F. TP53 mutations tended to be more prevalent in those who developed HMA-F (p=0.067). No mutations were detected in 31 patients (11%) on HMA therapy and 13 patients (8%) with HMA-F (p=0.222). Interestingly, patients who remained on HMA therapy more frequently had multiple mutations in the same gene than those who developed HMA-F (58 patients (21%) vs 12 patients (7%), p<0.001). The median overall survival from MDS diagnosis was 35.9 months (95% CI: 31.9, 55.3) in those who remained on HMA therapy vs 22.5 months (95% CI: 18.9, 25.7) in those who developed HMA-F (p<0.001). When only accounting for patients who underwent SCT, there was no difference in survival between the two groups (current HMA therapy 53.9 months vs HMA-F 58.1 months, p=0.541). In the HMA-F cohort, the median overall survival from the time of HMA-F was 6.8 months (95% CI: 5.5, 9.6). Conclusions: In this group of MDS patients treated with HMA, patients who eventually developed HMA-F were older with higher BM blasts and higher-risk disease by IPSS-R and IPSS-M. No statistically significant differences were observed by HMA therapy details, cytogenetics, and specific mutations, but those with multiple mutations in the same gene tended to remain on HMA therapy. Further investigation into predictors of HMA-F and potential other treatment modalities is warranted.

Impact of Hypomethylating Agents on Cardiovascular Disease Risk Among Patients with Myelodysplastic Syndromes

Introduction: The association between clonal hematopoiesis (CH) and cardiovascular disease (CVD) risk has drawn attention to cardiovascular outcomes in myelodysplastic syndromes (MDS). CVD accounts for 25% of deaths in MDS, potentially via CH-mediated accelerated atherosclerosis from inflammatory vascular responses. We previously demonstrated that MDS was independently associated with increased risk of CVD in older adults from SEER-Medicare. We subsequently hypothesized that treatment with hypomethylating agents (HMA) could reduce CVD risk by ameliorating CH clones and reducing the inflammation. In this study, we aimed to define the impact of HMA on incident cardiovascular events among older adults with MDS. Methods: We identified subjects ≥66 years diagnosed with MDS from 2007 to 2017 in SEER-Medicare. Exclusion criteria including lacking continuous Medicare parts A, B, and D, enrolled in HMO, or incomplete follow-up. Demographics included age, sex, race, rurality, socioeconomic quintile, marital status, and geographic region. Baseline CVD risk factors, defined from ICD-9/10 codes, included hypertension, hyperlipidemia, diabetes, atrial fibrillation, pre-existing cardiac disease, obesity, and smoking. MDS factors, defined using HCPCS codes, ICD-O-3 codes and prescription records, included MDS subtype and histologic risk stratification, transfusion dependence and treatment with hypomethylating agents (HMA), lenalidomide, or erythropoietin-stimulating agents (ESA) as time-varying variables. Incident CVD was defined as new myocardial infarction (MI) and/or ischemic stroke occurring after MDS diagnosis, using published ICD-9/10 algorithms. We controlled for confounding using 2 complementary analytic approaches. First, we assessed the association between HMA use and CVD using propensity score adjusted cause-specific competing risk Cox hazard models with death as competing risk, Second, we used a case-crossover design with conditional logistic regression, in which subjects with CVD served as their own controls, using a 3-month HMA treatment window and a control period 6 months prior to the incident event. Results: We identified a cohort of 15,227 eligible patients with MDS. Of these, 1,125 patients had an MI and/or ischemic stroke within 1 year before MDS diagnoses and were excluded. We analyzed 14,102 MDS patients, with median age 82 years (IQR 77 - 86), who were predominantly male (53.8%), non-Hispanic white (88.6%), and from urban/suburban areas (86.2%). MDS was of low, intermediate, and high histologic risk in 14.7%, 72.8%, and 12.5%, respectively, and 28.6% were transfusion dependent. Pre-existing CVD risk factors at time of MDS diagnosis were present in 12,538 patients, with baseline cardiovascular risk defined as low, intermediate, and high in 11.1%, 32.3%, and 56.6% of the cohort. The cumulative incidences of MI, ischemic stroke and composite CVD events were 11.3%, 10.6% and 20.4%, respectively. The propensity score (PS) conditional probability for being treated with HMA included demographic variables, CVD risk factors, MDS histology and transfusion dependence. Our PS model adequately predicted HMA treatment propensity (AUC 0.76). Cause-specific regression with death as competing risk, adjusted for PS and other MDS-specific treatments (ESA and lenalidomide), showed no significant effect of HMA on the risk of composite CVD events, MI, or ischemic stroke (Table 1). Conditional logistic regression in the case-crossover analysis of 1,907 patients with composite CVD events showed no significant effect of HMA. Results were similar for analysis of patients with MI and ischemic stroke (Table 2). Conclusions: No study has previously investigated the effect of HMA therapy on cardiovascular outcomes in patients with MDS. We demonstrate that HMA therapy is not associated with a significant risk reduction in cardiovascular events among patients with MDS patients from the SEER-Medicare database. These results show that HMA therapy is unlikely to reduce cardiovascular mortality and underscore the need for alternative therapeutic strategies targeting cardiovascular health patients with MDS.

Clinical Implications of TP53 Mutations/Allelic State in Patients (Pts) with Myelodysplastic Syndromes/Neoplasms (MDS) Treated with Hypomethylating Agents (HMA)- a Multicenter, Retrospective Analysis from the Validate Database

TK and JPB Co-first; RK, DS, and AMZ are Co-senior authors. Introduction MDS with TP53 mutations ( TP53 MT) are associated with high-risk disease features and rapid progression to AML. Effective treatment for this aggressive subtype is still not available. As compared to monoallelic TP53 MT, biallelic MT have been associated with adverse outcomes and worse overall survival (OS), but the impact of allelic status on treatment response and OS after HMA therapy are not well established. In this study, we analyzed TP53 allelic state for a large well annotated cohort of pts with MDS who were treated with HMAs. Methods The VALIDATE database includes pts with MDS treated with frontline HMA from 14 specialized centers. Pts were considered to have biallelic TP53 MT according to the ICC definition (Arber et al. 2022, Blood) if they meet any of the following: a. two TP53 MT (variant allelic frequency [VAF]>10% for each) b. single TP53 MT with copy neutral loss of heterozygosity (CN-LOH, available for 308 pts) c. TP53 MT with del17p and d. Single TP53 MT with VAF >50%. Time to event analyses were estimated using Kaplan-Meier estimator and groups were compared by the log-rank test from the time of HMA initiation. Only higher risk (HR)-MDS pts (IPSS>1 or IPSS-R>3.5) with BM biopsy within 180 days after HMA initiation were assessed for complete response (CR) and overall response rate (ORR) according to the IWG 2023 criteria (Zeidan et al, Blood 2023) (n=480). OS was calculated from time of HMA therapy initiation. This study was supported by an independent research grant from AbbVie. Results A total of 816 pts met eligibility and were included in this analysis. Median age was 68 years (IQR: 61-74) and 64% were men. MDS excess blast (EB) 1/2 was the most common MDS subtype (62%). Overall, 78% of the pts were treated with HMA monotherapy (26% decitabine [DEC] and 74% azacitidine [AZA]) and 22% received HMA as part of combination therapy. In total, 40% of the pts underwent allogenic HSCT. Median HMA treatment time was 5 months (mo), IQR:3-11. Median follow up time was 17 mo (IQR:9-31). TP53 MT were detected in 253 pts (31%) at time of MDS diagnosis, of whom 153 (60%) pts had biallelic TP53 MT and 100 (40%) pts had monoallelic TP53 MT. Missense MTs were identified in 80% of pts with monoallelic TP53 MT. TP53 MT/del17p (33%), TP53 MT/CN-LOH (28%), and multiple TP53 MT (25%) were the most common combinations in pts with biallelic TP53 MT. Pts with TP53 MT had higher rates of MDS-EB (74% vs. 61%, p<0.001). Complex karyotype was observed in 112 (73%) pts with biallelic TP53 MT and 81 (81%) pts with monoallelic TP53 MT (p=0.154). Median number of HMA cycles among pts with TP53 MT was 8 (IQR:5-12) and higher compared to pts without TP53 MT (5 cycles, IQR:3-9), p=0.032 ( Panel-A). In a logistic regression model for CR, TP53 MT was not associated with probability of achieving CR (1.4, 0.89-2.29). CR rates for pts with biallelic TP53 MT (18%) and monoallelic TP53 MT (14%) were not significantly different, p=0.37. There were also no significant differences in ORR observed between biallelic TP53 MT (59%) and monoallelic TP53 MT (54%) pts, p=0.39. Among TP53 MTpts treated with HMA monotherapy, pts treated with AZA compared to DEC had similar odds for ORR (OR:1.1, 95%CI: 0.7-1.6)/CR (OR: 0.8, 95%CI: 0.4-1.3) and hazard for OS (HR: 1.1, 0.9-1.4). As expected, the median OS (mo, 95%CI) for pts with TP53 MT (12, 10-13) was significantly lower than pts without TP53 MT (28, 25-31), p<0.001. Importantly, pts with biallelic TP53 MT (11, 9-14) had no significant difference in median OS compared to monoallelic TP53 MT (13, 11-17), p=0.268 (Panel-B). In a multivariable cox proportional hazard regression model for OS (hazards ratio, 95%CI) adjusted for age (1.0, 0.9-1.0), HSCT (0.4, 0.3-0.4), IPSS-M score (1.2, 1.1-1.3), and complex karyotype (1.4, 1.1-1.8), TP53 MT status (1.8, 1.4-2.3) was still associated with OS. However, biallelic TP53 MT had comparable risk to monoallelic TP53 MT (1.2, 0.9-1.7). Conclusions To our knowledge, VALIDATE is the largest database of MDS pts with TP53 MT who were treated with HMA. We confirm that TP53 MT have significant negative impact on survival, but not on response to HMA therapy. Importantly, we did not observe OS differences between monoallelic and biallelic TP53 MT. We also show that the type of HMA monotherapy used does not impact outcome of these pts. Overall, pts with TP53 MT have very poor outcomes and are in desperate need of new, effective therapies.

Single-Cell Multiomics Analysis Reveals Potential Drivers of Response to the Anti-TIM3 Inhibitor Sabatolimab Combined with Azacitidine in MDS and CMML

Background The median survival for patients with newly diagnosed (ND) higher risk myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) remains poor. The potential curability of MDS and CMML with allogeneic hematopoietic stem cell transplantation supports the concept of anti-tumor immunity and has led to interest in evaluating immune-based therapeutic approaches in myeloid neoplasms. The immune checkpoint molecule TIM-3 (encoded by the gene HAVCR2) is a target of interest in myeloid neoplasms given its expression on leukemic stem cells as well as several subsets of immune cells including T cells, monocytes, dendritic cells and NK cells. Sabatolimab (MBG453) is an investigational IgG4 anti-TIM-3 antibody currently under evaluation for myeloid neoplasms. In this exploratory study, we sought to characterize the effects of sabatolimab combined with the hypomethylating agent (HMA), azacitidine, on the immune landscape using single cell sequencing of samples from subjects with MDS and CMML treated with the combination from a previously reported phase 1b study (NCT03066648; Brunner et al 2022). Methods Following IRB approval, single-cell RNA sequencing (scRNA-seq) and associated proteomic cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) was performed on both blood (BLD) and bone-marrow (BM) derived samples from subjects with: 1) MDS (n=3) or CMML (n=2) treated with HMA therapy alone as part of standard care; 2) ND MDS (n=5) or CMML (n=3) treated with azacitidine combined with sabatolimab; and 3) relapsed/refractory MDS (n=3) treated with sabatolimab alone. Paired BLD and BM samples as well as samples from serial treatment time points were selected whenever feasible (Fig 1). A total of 206,183 cells from BM and 172,421 cells from BLD post-quality control filtering were analyzed. Further, scRNAseq data from normal BLD(200,000 cells) and BM(240,650 cells) generated as part of the human cell atlas (https://data.humancellatlas.org/explore/projects/cc95ff89-2e68-4a08-a234-480eca21ce79) were integrated with this dataset to assist with immune cell subset characterization, enable downstream comparisons to healthy hematopoietic cells, and to help define cell subsets associated with disease states. Results In baseline samples from subjects with MDS treated with azacitidine-sabatolimab, we found that an increased baseline abundance of interferon-responsive CD8 T cells in both the BM and BLD (FDR<0.1) was associated with response to therapy. An increased baseline abundance of plasmacytoid dendritic cells and a granulocyte population also suggested an association with response to therapy. Differential gene expression analysis of baseline samples from responding versus non-responding subjects with MDS treated with azacitidine-sabatolimab showed a higher baseline expression of TNF and IFNG in BM CD8 T cells and up-regulated expression of MHC-II machinery ( HLA-DRB1, HLA-DPA1, CD74) in myeloid cell subsets in responding subjects (FDR <0.1), suggesting that pre-treatment ability of these cells to be involved in antigen-presentation may play a role in response. In subjects with CMML treated with azacitidine-sabatolimab, we observed dynamic changes in cellular abundances and gene expression when comparing baseline to post-therapy timepoints. Specifically, BM CD8 T cell subsets showed an up-regulation of cytotoxicity genes ( GZMA), interferon response genes ( IFIT2, IFITM1, IFITM2) and IL32 cytokine post-therapy (FDR <0.1). When evaluating myelomonocytic cell populations, we observed an increase in CD16 monocytes post-treatment. Additionally, dendritic cells and monocytes showed an up-regulation of interferon response genes ( IFIT1, OAS1, IFI27). Conversely, we observed a down-regulation of metallothionein genes ( MT1E, MT1G, MT2A) as well as a collection of transcription factors ( NR4A1, FOSL2, JUN, CEBPB, CEBPD), NF-κB inhibitors ( NFKBIZ, NFKBIA) and CXCL8 in the post-treatment samples (FDR <0.1). Conclusions Our study provides one of the most comprehensive evaluations of the cellular dynamics of anti-TIM3 immunotherapy in patients to date, allowing for the nomination of novel putative predictive biomarkers of response and identification of potential immunomodulatory mechanisms induced by the combination of sabatolimab with azacitidine in MDS and CMML for further future analysis.

Real-World Treatment Patterns Among Patients with Myelodysplastic Syndromes Initiating Oral Decitabine and Cedazuridine or Intravenous/Subcutaneous Hypomethylating Agents

Introduction: Hypomethylating agents (HMAs) are recommended standard of care for patients with higher-risk myelodysplastic syndromes (HR-MDS). HMA therapies include intravenous (IV) or subcutaneous (SC) decitabine and azacitidine; however, parenteral administration of HMAs has been associated with increased patient burden. The oral HMA decitabine and cedazuridine (DEC-C) was FDA-approved in July 2020 for the treatment of MDS, and offers an alternative to IV/SC HMAs, with similar pharmacokinetic exposure and response rates compared to IV decitabine. This study examined treatment patterns among patients with MDS receiving oral DEC-C compared with IV/SC HMAs in real-world clinical practice. Methods: This was a retrospective analysis using the US Cerner Enviza claims database; medical and prescription claims data for patients were linked to mortality data from Datavant. Adults aged ≥18 years, who were diagnosed with MDS (ICD-10-CM code D46.x; all risk levels), and who had initiated a HMA (≥1 claim; August 1, 2020─August 31, 2022) were included; index date was defined as the first pharmacy or medical claim for DEC-C or IV/SC HMAs, respectively. Patients had variable follow-up after index and were followed until end of enrollment, death, or end of study. Propensity score matching (PSM; 1:1) was performed to balance for confounding factors (age, sex, acute myeloid leukemia [AML] diagnosis, Charlson Comorbidity Index [CCI] score, and red blood cell [RBC] transfusion); adjusted bivariate analysis was used to compare outcomes between treatment groups. Longitudinal persistence was assessed according to the number of cycles of therapy received during follow-up, where a cycle was defined as either 3-10 days of administration of index IV/SC HMA or 1 claim for oral DEC-C, within a 28-day cycle. Results: Of 1569 patients included, 160 (10.2%) received DEC-C and 1409 (89.8%) received IV/SC HMAs. In the DEC-C cohort, median age was 72.5 years and 66.9% were male; in the IV/SC cohort, median age was 69.0 years and 58.2% were male. Two-thirds of patients newly initiated HMAs during the study period (69.4% for DEC-C and 72.2% for IV/SC HMAs); 30.6% of the DEC-C cohort had switched from prior IV/SC HMA treatment. Both groups had high levels of comorbidity; 55.6% of patients in the DEC-C cohort and 58.4% in the IV/SC HMA cohort had CCI scores of ≥3, and 14.4% and 23.8%, respectively, had an AML diagnosis. During the 6-month pre-index period, half (55.3%) of patients in both groups received RBC transfusions, and one quarter (25.9%) received platelet transfusions. After matching, there were 158 patients in each treatment cohort and all covariates included in the PSM were balanced (standardized mean difference <0.2). Median age was 72.0 years and 74.0 years for the DEC-C and IV/SC HMAs matched cohorts, respectively, and 66.5% and 69.6%, respectively, were male. Mean CCI scores were 3.45 for DEC-C and 3.15 for IV/SC HMA groups and 14.6% and 15.8%, respectively, had an AML diagnosis pre index. Longitudinal persistence was comparable between the matched DEC-C and IV/SC cohorts during the first 6 months post-index, with similar proportions receiving the maximum number of treatment cycles (based on a 28-day cycle) at each month following index (73.8% vs 71.1%, 46.5% vs 48.7%, 28.6% vs 24.8%, for 2, 4, 6 months, respectively; Figure). However, a trend towards improved persistence with DEC-C versus IV/SC HMAs was observed in patients receiving treatment beyond 6 months (25.0% vs 17.0%, 18.5% vs 9.0%, 11.4% vs 7.6%, for 8, 10, 12 months, respectively). Mean time to discontinuation of treatment was also numerically higher for DEC-C compared with the IV/SC HMA group (87.7 vs 82.0 days); however, differences were not statistically significant. Conclusions: This study, which is to our knowledge the largest real-world report of oral DEC-C use to date, reveals similar but high levels of disease burden and comorbidities among patients with MDS receiving oral DEC-C and parenteral HMA therapy. Trends in treatment patterns suggest comparable persistence with oral DEC-C and IV/SC HMAs at early stages of therapy and improved persistence with oral DEC-C beyond 6 months. These data support the consideration of oral DEC-C as an alternative to chronic parenteral HMA therapy, with the potential for oral DEC-C to reduce the treatment burden associated with administration of IV/SC HMAs.

Survival and Prognosis Among 301 Patients with Newly-Diagnosed Acute Myeloid Leukemia Following Venetoclax Plus Hypomethylating Agent Therapy

Background: Venetoclax (Ven) in combination with hypomethylating agent (HMA) is the preferred treatment for elderly/unfit patients with newly-diagnosed acute myeloid leukemia (AML). Current prognostication for AML is based on European LeukemiaNet (ELN) genetic risk stratification which has limited applicability in patients treated with Ven-HMA ( Blood, 2022). Accordingly, our primary objective was to determine predictors of treatment response and survival outcomes in treatment-naïve AML patients receiving Ven-HMA. Methods: Our study population was recruited from Mayo Clinic (MN, FL, AZ), after institutional review board approval and based on documentation of newly-diagnosed AML treated with Ven-HMA outside of clinical trials between November 2018 and April 2023. Cytogenetic and molecular studies were performed by conventional karyotype, and next-generation sequencing, respectively. Response was assessed according to the 2022 ELN criteria Results: Patient characteristics A total of 301 newly-diagnosed AML patients (median age 73 years, 66% male, 62% de novo) received a median of 3 cycles (range 1-48) of azacitidine 75 mg/m2 days 1-7 ( n=100) or decitabine 20 mg/m2 days 1-5 ( n=201) with Ven. ELN 2022 cytogenetic risk included favorable (1%, n=4), intermediate (61%, n=184) or adverse (38%, n=113). Mutations involved TP53 in 77/301 (25%), ASXL1 in 54/279 (19%), RUNX1 in 54/296 (18%), NPM1 in 44/299 (15%), DNMT3A in 40/296 (13%), K/NRAS in 41/296 (14%), IDH1 in 20/301 (6%), IDH2 in 30/301 (10%), FLT3-ITD in 25/301 (8%) and DDX41 in 9/247 (4%) of informative cases. Predictors of response Complete remission, with (CR) or without (CRi), count recovery, was documented in 182 (60%) patients, including CR in 36% and measurable residual disease (MRD) negative by flow cytometry in 77% of 105 informative cases that achieved CR/CRi. Median time to CR/CRi was 1.4 months. In univariate analysis, CR/CRi was more likely to occur in the presence of NPM1 mutation, (86% vs. 56%; p < .01), IDH2 mutation, (80% vs. 58%; p = .02), DNMT3A mutation, (78% vs. 57%; p = .01), or DDX41 mutation, (100% vs. 58%; p <.01), and in the absence of adverse karyotype, (71% vs. 42%; p < .01), TP53 mutation, (67% vs. 40%; p <.01), FLT3-ITD mutation, (63% vs. 36%; p = .01), or RUNX1 mutation, (64% vs. 44%; p = .01); in multivariable analysis, adverse karyotype, ( p < .01), FLT3-ITD ( p < .01), RUNX1 ( p < .01), NPM1 ( p = .03), IDH2 ( p = .04), and DDX41, ( p = .01) mutations remained significant. Predictors of survival After a median follow-up of 8.5 months (range 0.5-56), 13.2 months for living patients, 174 (58%) deaths, 73 relapses (40% of those achieving CR/CRi), and 41 (14%; including 35 in CR/CRi) allogeneic hematopoietic stem cell tranplants (AHSCT) were documented. In univariate analysis of pre-treatment variables, thrombocytopenia < 100 x 10 9/l, (HR 1.5, 95% CI 1.03-2.2; p = .03), adverse karyotype, (HR 2.8, 95% CI 2.1-3.8; p < .01) TP53 mutation, (HR 2.5, 95% CI 1.8-3.5; p < .01), or absence of IDH2 mutation, (HR 3.5, 95% CI 1.7-7.6; p < .01) predicted inferior survival. Failure to achieve CR/CRi (HR 4.5, 95% CI 3.3-6.1; p < .01) was also associated with inferior survival and in multivariable analysis, failure to achieve CR/CRi, (HR 3.4, 95% CI 2.5-4.8; p< .01), presence of adverse karyotype, (HR 1.6, 95% CI 1.1-2.6; p = .02), TP53 mutation, (HR 1.6, 95% CI 1.002-2.4; p = .04), and absence of IDH2 mutation (HR 2.2, 95% CI 1.004-4.7; p = .04) were identified as risk factors for survival. Subsequent HR-weighted scoring resulted in three-tiered risk stratification: low (0-1 point; n= 130), intermediate (2-3 points; n= 105), and high (4-5 points; n= 66), with respective median survival (3-year rate) of 28.9 (48%), 9.6 (6%), and 3.1 (0%) months ( p < .01) (Figure). AHSCT had an independent favorable impact on survival (HR 0.3, 95% CI 0.1-0.6; p < .01), most apparent in low ( p =0.04), and intermediate ( p <.01), as opposed to high ( p = .06) risk. Conclusions: In the current single institutional series of Ven-HMA treated newly-diagnosed AML, response to Ven-HMA was the foremost predictor of survival. Additional risk factors for survival included adverse karyotype, presence of TP53 and absence of IDH2 mutations. These observations allowed for a three-tiered genetics-enhanced survival model with CR/CRi as a backbone, and also confirmed survival advantage from AHSCT, regardless of risk category.

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 Overall Survival Among Luspatercept Responders and Predictors of Response

Introduction Luspatercept (luspa) is a first-in-class erythroid-maturation agent approved for red blood cells transfusion dependent (RBC-TD) lower risk myelodysplastic syndrome (LR-MDS) with ring sideroblast (RS) based on MEDALIST clinical trial. We aimed to explore impact of luspa on overall survival (OS) and predictors of response in a large cohort of pts treated with luspa, collected from Moffitt Cancer Center (MCC) and from Fondazione Italiana Sindromi Mielodisplastiche (FISiM). Methods We evaluated all pts with LR-MDS-RS treated with luspa from MCC cohort since FDA approval, and from a multicenter observational trial of FISiM for pts treated on a compassionate use program . Baseline RBC transfusion burden (TB) was defined as: non-transfusion dependent (NTD) (0 units in 8 weeks prior luspa), low TB (LTB) (1-5 units/8 weeks ) and high TB (HTB) (≥ 6 units/8 weeks). A hematological response (HI) was defined as an (i) objective Hgb increase of > 1.5 g/dl in NTD, and (ii) RBC-TI with Hgb increase of 1.5 g/dl, or RBC-TI without Hgb 1.5 g/dl increase, or >50% reduction in RBC TB among RBC-TD. Somatic mutation (MT) data were available for 137 pts. Results Between January 2020 and March 2023, 331 pts were treated with Luspa (Table-1). The median age was 69 years; 308 pts (93%) had MDS-RS, 86,1% were intermediate, low or very low risk MDS by R-IPSS and 82% (105/128) were moderate low (ML), low (L) or very low (VL) by IPPS-M. SF3B1 MT was detected in 86.3% (120/139) with a median VAF of 37.7. The mean Hgb level was 7.97 g/dl and 93.7% were RBC-TD. Majority had prior erythroid stimulating agents (ESA) (93.6%). In the MCC cohort 42.9% (52/121) had prior hypomethylating agents (HMA) therapy, and 31.4% (38/121) had prior lenalidomide (len). With a median follow up of 14.6 mos from starting luspa treatment, the median OS was not reached (NR) among responders to luspa compared to 24.5 mos for non-responders (p <.001) (Figure-1). The median OS benefit was more pronounced among pts with RBC-TI and Hgb> 1.5 g/dl increase compared to other categories of response. In the NTD group at baseline, the median OS among luspa responders was NR compared to 22.7 mos among non-responders (P< .001). In the LTB group at baseline median OS was NR as well among luspa responders compared to 29.9 mos in non-responders (p=.002) and finally among HTB group at baseline, the median OS among luspa responders was 35.4 mos compared to 21.2 mos in non-responders (p=.007). In multivariable analysis adjusting for IPSS-M and baseline RBC-TD, response to luspa was independently associated with improved OS, HR .39 (95% CI .18-.83), p= .015 The overall HI rate to luspa was 40.6% (134/330) (Table-1), 85% of pts needed dose escalation, HI correlated with baseline RBC-TB where 80% (16/20) NTD, 60.2% (65/108) LTB and 41.8% HTB (77/184) achieved HI respectively, p< .001. By IPSS-M, there was a trend for lower HI with higher risk disease (100% in VL, 60.3% in L, 36.7% in ML, 56.3% in MH, 16.7% in H risk pts, p=.051). There was a mild trend for higher response among SF3B1 MT pts compared to wild type, 54% (64/119) versus 42% (8/19), p=.34.The segregation of SF3B1 MT cases into three independent groups based on IPSS-M, showed that HI rate was better in SF3B1 β ( SF3B1 and any gene from BCOR, BCORL1, NRAS, RUNX1, SRSF2 or STAG2) and SF3B1 α (as any other mutant) than SF3B1 5q(concomitantpresence with isolated del5q): 50% (7/14), 58.8% (57/97), and 0% (0/5) respectively, p=.03. Among SF3B1 5qpts, 4 had been treated with len before luspa and only 1 had achieved an HI with prior len. Among pts with SF3B1 MT, we observed a trend for higher response in pts with the hotspot K700E, 62.7% (37/59) vs 47.4% (27/57) in non-K700E, p=.097. In pts with SF3B1 MT, divided by median VAF, we observed a trend for better HI with VAF was >38, 63.5% (33/52) vs 45.3% (24/53), p=.062. Among responders, the median duration of response was 14.1 mos. As of last follow up, 85% pts (191/224) discontinued treatment. Reason of discontinuation was lack of response in 126 pts, loss of response in 26 patients and adverse events in 6 pts. The most common reported adverse events were fatigue (n=10), GI symptoms (n=4), shortness of breath and arthralgia (1 each). Conclusions Our RWD in the largest luspa treated cohort demonstrates OS benefit to luspa response. Low baseline RBC-TB dependency, lower risk IPSS-M, SF3B1 MT, SF3B1-K700E mutation and SF3B1 α co-mutations correlated with higher response rates. Our data emphasizes the impact of RBC-TI in LR-MDS.

Clonal Dynamics and Deterministic Clinical Fate Mapping of Patients with Myelodysplastic Neoplasms and Acute Myeloid Leukemia with TP53 Disruption

Background: Myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML) with multi-hit TP53 comprise the highest risk subgroup of all myeloid malignancies. TP53 aberration is recognized as a diagnostic category in the 2022 International Consensus Classification (ICC) and the 5 th edition of the WHO. However, these systems do not discern among the subtypes of TP53 aberrations regarding response to therapy. In this study, we explore the clonal dynamics that underlie such prognostic heterogeneity within distinct subgroups of TP53-aberrant MDS and AML, with a specific focus on the effect of hypomethylating agent (HMA) vs. araC-based chemotherapy on TP53 variant allele frequency (VAF). Methods: The UMass Leukemia Registry from the UMass Data Lake identified 76 patients harboring TP53 aberration(s) plus ICD-10 code of either D46.9 (MDS and its subentities) or C92.00 (AML and its subentities) between 2011-2023. Data was integrated with hematopathology records. TP53 exome sequencing was performed with coverage depth 500X to 6500X, and VAFs were adjusted for copy number variation (CNV), as previously described (Patel SA et al., Leuk Lymphoma 2021; 62: 3348-60). Patients with serial bone marrow biopsy samples were included. We defined multi-hit TP53 status as per the ICC: two or more distinct TP53 mutations with VAF ≥ 10%, or a single TP53 mutation plus one of the following abnormalities: (1) del(17p13.1), (2) TP53 VAF >50%, (3) copy-neutral loss-of-heterozygosity, or (4) any complex karyotype. Clonal dynamics were assessed for MDS vs. AML and for varying TP53 allelic states as a function of therapeutic intervention. Clinical fate mapping was performed based on management decisions at various points of clinical care. Results: For patients with TP53-aberrant MDS, 17 patients underwent serial bone marrow biopsies during treatment. Of these, 11 (64.7%) had multi-hit status. Eight patients (47.1%) underwent hematopoietic cell transplant (HCT), and 7 of these 8 patients had received HMA-based front-line therapy. For patients with MDS, we assessed the effect of HMA-based therapy on TP53 VAF. The mean TP53 VAF (sum of VAFs) was significantly lower after HMA-based treatment (35% vs. 12.6%) ( p = 0.0075) (Panel A). For patients with TP53-aberrant AML, 19 patients underwent serial bone marrow biopsies. Of these, 14 (73.7%) had multi-hit status. Only 2 patients (10.5%) underwent HCT. Five patients (26.3%) received araC-based induction, while 11 patients (57.9%) initially received HMA-based induction. For patients with AML with multi-hit TP53, we assessed the effect of HMA-based vs. araC-based front-line therapy on TP53 VAF. HMA therapy led to mean fold reduction in TP53 VAF (pre-treatment to post-treatment) of 11.7 ± 5.8 ( p = 0.019), while araC-based therapy actually led to mean fold increase (but not statistically significant) in TP53 VAF (pre-treatment to post-treatment) of 2.6 ± 1.05 ( p = 0.319) (Panel B). HMA-based treatment resulted in significantly greater reduction in the VAF in aberrant clones and subclones compared to araC-based treatment. Compared to AML, most patients with MDS (62.9%) proceeded with palliative intent therapy at the time of diagnosis. Although patients with AML (53.7%) attempted to proceed with curative intent at the time of diagnosis, only 14.6% eventually proceeded with HCT, largely due to clinical decompensation or disease progression during front-line therapy. Among HCT recipients for MDS ( n =10), 7 (70%) remained in remission. Among HCT recipients for AML (n = 6), 4 (66.7%) remained in morphologic leukemia-free state. Conclusion: There is limited literature on clonal and subclonal diversity in patients with MDS or AML with TP53 aberrations. Such considerations constitute an important therapeutic issue, as certain interventions may only eliminate a fraction of a patient's mutant cells. In our study, HMA- and araC-based regimens had differential effects on various subclones: HMA was more effective in reducing the TP53 mutational burden and increasing patient ability to proceed with HCT. AraC-based therapy appeared less effective, perhaps due to selection pressure in favor of TP53-mutant cells. Prospective trials are required to identify optimal regimens that lead to the best long-term outcomes, and single-cell resolution might assist with more definitively assessing dynamics of clonal response to treatment.

Performance of the Molecular International Prognostic Scoring System (IPSS-M) in Hypomethylating Agent-Treated Patients with Myelodysplastic Syndromes

Background: Patients with myelodysplastic syndromes (MDS) undergo risk stratification with various prognostication tools, such as the International Prognostic Scoring System (IPSS) and Revised IPSS (IPSS-R). Recently, the Molecular IPSS (IPSS-M) was developed incorporating genomic data in addition to clinical and cytogenetic characteristics. Though IPSS-M has been shown to improve prognostication in all MDS patients at baseline, its utility in specifically patients undergoing hypomethylating agent (HMA) therapy remains unknown. Here, we assess the efficacy of IPSS-M in HMA-treated patients with MDS. Methods: We retrospectively evaluated all untreated patients with MDS seen at a single tertiary cancer center from July 2017 to July 2021 and identified those who later received HMA therapy. Patient characteristics and bone marrow (BM) data, including morphology, cytogenetics, and mutations, were assessed at diagnosis. Genomic DNA was extracted from whole BM aspirate samples and subject to 81-gene target PCR-based sequencing using a next-generation sequencing platform. Survival data was updated in July 2023. Results: Out of 799 untreated MDS patients, 455 patients (57%) eventually underwent treatment with HMA. At diagnosis, the median age was 69 (range: 22-90) with 289 (64%) male patients. Patients were generally high risk, with therapy-related MDS in 160 (35%), complex cytogenetics in 167 (37%), and TP53 mutations in 168 (37%). By IPSS-R, 259 patients (58%) had high- or very high-risk disease, and 340 patients (75%) had higher-risk disease (moderate high-, high-, and very high-risk) by IPSS-M. A total of 291 patients (64%) were treated with HMA monotherapy, and lower-risk MDS patients received more cycles of HMA (p < 0.001). By IPSS-R, the median overall survival was 60.0 months (95% CI: 46.0, not estimable (NE)) in very low-, 87.4 months (95% CI: 59.9, NE) in low-, 36.2 months (95% CI: 30.0, 58.1) in intermediate-, 23.7 months (95% CI: 19.6, 34.8), in high-, and 12.8 months (95% CI: 11.0, 16.7) in very high-risk patients (p < 0.0001) with a concordance index of 0.669. Figure 1 depicts the reclassification of patients between IPSS-R and IPSS-M. Risk stratification by IPSS-M (Figure 2) provided similar results: not reached (95% CI: 56.8, NE) in very low-, 60.0 months (95% CI: 49.8, NE) in low-, 40.8 months (95% CI: 22.8, NE) in moderate low-, 44.2 months (95% CI: 27.8, NE) in moderate high-, 33.4 months (95% CI: 22.8, 44.5) in high-, and 16.6 months (95% CI: 13.8, 19.4) in very high-risk patients (p < 0.0001). The concordance index of IPSS-M in all HMA-treated MDS patients was 0.648. When analyzing patients with MDS who were more likely to undergo HMA as per standard-of-care treatment (those with IPSS-M moderate low-, moderate high-, high-, and very high-risk disease), the IPSS-M remained statistically significant (p < 0.0001) though less discerning in the patients with moderate-risk MDS, as seen by the concordance index of 0.620. Conclusions: In this group of MDS patients treated with HMA, the IPSS-M did not provide additional prognostic power over the IPSS-R though median overall survival remained inversely proportional to IPSS-M risk category. Concordance indices for survival remained below 0.7, likely due to the overlapping curves in moderate-risk patients by IPSS-M. Further development and validation of prognostic scoring systems in patients treated with HMA are warranted.

Downregulation of Mitochondrial Complex II (MC II) in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of diseases of hematopoietic stem cells (HSCs) that occur in the aging population and following therapy for unrelated cancers. The standard of care for MDS involves hypomethylating agents (HMAs) that invariably result in resistance and disease progression. We previously determined the role of mitochondrial metabolism in MDS pathophysiology, whereby suppression of mitochondrial activity leads to the accumulation of oncometabolites that competitively inhibit α-ketoglutarate-dependent dioxygenases (2OGDDs). Consequently, hypermethylation of epigenomes and hypoxia-inducible factor 1A (HIF1A) activation ensues a pseudohypoxia state that drives MDS pathogenesis. Our recent informatics analyses of primary MDS samples identified a patient subset with high TGF-β signaling in which mitochondrial complex II (MC II) genes were downregulated. MC II, or succinate dehydrogenase (SDH) is the smallest complex of the oxidative phosphorylation system that oxidizes succinate to fumarate and thereby donates electrons to reduce FAD to FADH 2 and the ubiquinone pool. Its downregulation results in the accumulation of Krebs cycle intermediate metabolites that can act as antagonists for 2OGDDs. To understand whether SDH dysregulation alone can contribute to MDS pathobiology, we generated a doxycycline-inducible and reversible mouse model (SDH-KD) that expresses shRNAs against the SDH subunits (SDHA, B, and D) in HSCs. These mice showed a gradual buildup of intracellular succinate in c-Kit + bone marrow (BM) cells and the peripheral blood, and a corresponding decrease of fumarate levels after induction for 2 weeks, 4 weeks, and 2 months. We found that knocking down Sdh was sufficient to induce pancytopenia and trilineage dysplasia in SDH-KD mice, while intracellular iron granules that surround the nucleus of erythroblasts, similar to ring sideroblasts commonly observed in MDS patients were also detected in BM and spleen smears. Of note, removing doxycycline and restoring Sdh expression rescued macrocytic anemia among other MDS phenotypes. Furthermore, treating SDH-KD mice with HMAs restored mitochondrial respiration and reversed HIF1A activation through the upregulation of SDH subunits. Using another MDS model, MllPTD/WT/ Vav1-Cre/Runx1 Flox/Flox, we found that HMA-treated HSCs differentiate towards CD11b +Gr1 + mature myeloid cells and that this requires Sdh expression. In conclusion, our results indicate that pseudohypoxia- and TGF-β signaling-associated changes in metabolic regulation caused by SDH downregulation underlie MDS pathogenesis. Elucidating the role of MC II in MDS pathobiology may provide alternative targets that can be combined with HMA or luspatercept therapy for MDS.

Myelodysplastic Syndrome Hematopoietic Stem Cells Can Undergo Differentiation Trajectory Rewiring to Escape Venetoclax Vulnerability

Myelodysplastic syndromes (MDS) are incurable neoplasms that arise from early hematopoietic stem cells (HSCs). The standard of care for MDS is hypomethylating agent (HMA) therapy. However, the disease invariably fails these agents, resulting in fatal cytopenias or progression to acute myeloid leukemia (AML) with a dismal prognosis. We previously showed that the cellular hierarchy of MDS HSCs predicts the mechanisms of progression after HMA failure and can guide the design or choice of specific therapeutic approaches targeting these cells (Ganan-Gomez et al., Nature Medicine 2022). MDS HSCs in one of two differentiation states, long-term HSCs or lymphoid-primed multipotent progenitors, have a “common myeloid progenitor (CMP) pattern” or “granulocytic-monocytic progenitor (GMP) pattern” of differentiation. HSCs in these 2 differentiation states persist throughout the disease course and, driven by the recurrent activation of BCL2- or NF-κB-mediated survival pathways, respectively, expand at disease progression. In our preclinical studies, pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden, which suggests that only patients with “CMP pattern” MDS may benefit from venetoclax-based therapies. Accordingly, patients with “CMP pattern” MDS, whose disease progressed after frontline HMA failure, enrolled in clinical trials of venetoclax-based therapy (NCT04160052, NCT04550442, or NCT04655755; n=28: 12 “CMP pattern” MDS and 16 “GMP pattern” MDS) had a shorter cumulative time to complete remission (P=0.012) and a longer relapse-free survival duration (14.0 months vs 5.2 months) than those with “GMP pattern” MDS did. Consistent with the hypothesis that venetoclax-based therapy selectively targets different HSC populations after HMA therapy failure, bone marrow (BM) HSC frequencies were significantly decreased at the time of remission after venetoclax-based therapy in “CMP pattern” MDS patients, but not in those with “GMP pattern” MDS. These results explain why “GMP pattern” MDS are resistant to venetoclax therapy. However, 8 of the 12 patients with “CMP pattern” MDS relapsed during venetoclax treatment and/or progressed to AML after an initial remission. In 2 patients with “CMP pattern” MDS, sequential flow cytometry and next generation sequencing analyses showed that the HSC hierarchy switched to “GMP pattern” MDS before venetoclax failure. This immunophenotypic hierarchical change was associated with the detection or expansion of STAG2- or RUNX1-mutant clones with isolated trisomy 8. To evaluate whether the immunophenotypic switch resulted from the distinct differentiation potential of early HSCs or from the aberrant expression of cell surface markers, we performed sequential single-cell RNA sequencing analyses of BM mononuclear cells isolated from the 2 “CMP pattern” MDS patients whose disease changed hierarchy before venetoclax failure. Our analyses revealed that after undergoing significant depletion during disease remission, HSCs expanded at therapy failure. Differential expression analysis showed that the expansion of STAG2- or RUNX1-mutant clones in the HSC compartment not only rewired MDS HSCs' differentiation state towards a granulo-monocytic-biased trajectory but also changed HSCs' survival dependency from BCL2-mediated anti-apoptotic pathways to TNFa-induced pro-survival NF-κB signaling, thus enabling HSCs to evade the cytotoxic effects of venetoclax (Fig. 1). The anti-apoptotic gene MCL1, a direct transcriptional target of NF-κB, was also significantly upregulated in HSCs at the time of progression, which suggests that the combination of venetoclax with an MCL1 inhibitor can overcome secondary failure to venetoclax. Together, these results reveal a novel mechanism of venetoclax resistance. HSCs under venetoclax therapy undergo survival pressure, which results in the expansion or acquisition of clones carrying specific mutations that change HSCs' survival dependencies (Fig. 2). Our study suggests that MDS patients receiving venetoclax-based therapy should be monitored at the molecular level and enrolled in clinical trials of other targeted agents before their disease undergoes clonal evolution and HSC hierarchical rewiring.

Post Allogeneic Stem Cell Transplant Outcomes Following Response to Hypomethylating Agent Therapy in Myelodysplastic Syndromes Are Predicted By Persistent International Prognostic Scoring System-Molecular Risk

Introduction: The International Prognostic Scoring System-Molecular (IPSS-M) ( Bernard NEJM Evidence 2022) and the 2023 International Working Group (IWG) response criteria for myelodysplastic syndrome (MDS) ( Zeidan Blood 2023) are used to more accurately assess prognosis and therapeutic response in MDS. However, it is unknown if these tools can be used to predict outcomes post-allogeneic hematopoietic stem cell transplant (HCT). We sought to understand the impact of pre-HCT IPSS-M on post-HCT outcomes in patients (pts) with MDS who responded to hypomethylating agent (HMA) therapy. Methods: Pts with MDS treated with HMA (azacitidine or decitabine) who received an HCT post-HMA at Dana-Farber Cancer Institute from January 2014 to December 2020 were included. Response to HMA was assessed by 2023 IWG response criteria and was defined as complete remission (CR) + CR with bi-lineage blood count recovery (CRbi) + CR with uni-lineage blood count recovery (Cruni) + CR with partial hematological recovery (CRh). Combined clinical and molecular risk was assessed by IPSS-M at times of diagnosis and of HCT, the latter using the bone marrow biopsy and sequencing data collected closest to HCT. High risk was defined as IPSS-M moderately high, high, and very high, whereas low risk was defined as IPSS-M very low, low, and moderately low. Results: A total of 148 pts with MDS who received HMA and underwent subsequent HCT were included. Median age was 64 years (range 26-79) and 61.5% were men. Pts were diagnosed with MDS-EB1 (26.4%), MDS-EB2 (45.3%), and other MDS subtypes (28.3%). IPSS-M at time of diagnosis was very low (2%), low (10.1%), moderate low (6.1%), moderate high (8.8%), high (31.8%), and very high (22.3%). IPSS-M pre-HCT was very low (12.8%), low (8.8%), moderate low (10.8%), moderate high (12.8%), high (20.3%), and very high (10.8%). IPSS-M at diagnosis and pre-HCT could not be calculated because of missing molecular data in 18.9% and 23.6% of pts, respectively. Pts received a median of 4 HMA cycles (range 1-20) and were treated with azacitidine for 7 days (54.1%), decitabine for 5 days (39.2%), and other schedules (6.7%). Prior to HCT, IWG 2023 responses were: CR (15.5%), CRbi (14.9%), CRuni (19.6%), CRh (0.7%), partial remission (PR: 1.4%), hematological improvement (HI: 9.5%), and no response (38.5%). Most pts received HCT from either a matched unrelated (60.8%) or matched related donor (18.2%) with reduced intensity conditioning (74.3%). Pts received graft versus host disease prophylaxis with tacrolimus (tac)/methotrexate (MTX) (54.7%), tac/MTX/sirolimus (19.6%), post-transplant cyclophosphamide/mycophenolate mofetil/tac (15.5%), tac/sirolimus (8.1%), other (2%). Median HCT-comorbidity index (CI) was 2 (range 0-13) and 48.6% pts had HCT-CI ≥ 3. For the entire cohort, the median follow-up time among ongoing survivors was 48.3 months (range 5.5-101.3). Among pts who responded to HMA per IWG 2023 criteria (CR/CRbi/CRuni/CRh), those who had high risk by IPSS-M prior to HCT had significantly shorter median overall survival (OS) (27 months; 95% CI 7.5-51) compared to pts with a low risk by IPSS-M (not reached; p=0.016) ( Figure A). Cumulative incidence of relapse (CIR) at 4 years was 66% for pts with high risk and 31% for low risk (p=0.034) ( Figure B). Pts with response to HMA with high risk by IPSS-M had lower OS (4-year OS: 27% versus 53%; p=0.016) and progression-free survival (4-year PFS: 19% versus 50%; p=0.018) but similar non-relapse mortality (NRM) (4-year NRM: 16% vs. 19%; p=0.66) post-HCT compared to pts with low risk at time of HCT. Conclusion: For pts with MDS who achieve a response to HMA prior to HCT, combined clinical/molecular risk, as assessed by IPSS-M, has an important prognostic impact on post-HCT outcomes. Pre-HCT risk should be evaluated for prognostication and to guide patient care, including future prospective studies evaluating novel agents for post-HCT therapy.

Comprehensive Characterization of Evolution of Genomic Complexity By Structural Variant and Mutational Profiling in Myelodysplastic Syndrome Patients with Hypomethylating Agent Failure

Introduction MDS patients (pts) who develop resistance to hypomethylating agent therapy (HMA-F) have dismal outcomes. Genomic aberrations underlying progression to HMA-F remains poorly understood. By sequencing, studies have demonstrated acquisition of new mutations affecting transcription factors such as FLT3 and RAS-MAPK pathway genes in a subset of cases. Until now, evolution of genomic complexity in HMA-F by acquisition of new structural variants (SVs) has not been systematically investigated. This is due to lack of genome-wide technologies to quantitatively identify all types of SVs at a gene/exon-level resolution (deletions/insertions/inversions/fusions etc..). In this study, we optical genome mapping (OGM), a novel, non-sequencing-based approach for accurate high-resolution SV detection, combined with ultra-deep coverage targeted NGS to comprehensively analyze genome-wide changes affecting both SVs and somatic mutations using paired baseline and progression samples from 21 MDS pts who developed HMA-F. Methods We identified all MDS pts with sufficient (1.5 million) cryopreserved, viable bone marrow (BM) mononuclear cells at both diagnosis (baseline) and HMA-F. All pts underwent BM morphologic exam, traditional banding analysis (TBA), and 81-gene targeted NGS panel (2500X coverage; LOD: 2% VAF; myeloid genes). We excluded pts with AML-defining abnormalities by standard-of-care work-up (per WHO 2022 criteria) and CMML pts. OGM was performed using standard procedures: extraction of ultra-long DNA molecules, sequence-specific fluorophore labeling and stain, followed by loading on to nanochannels and imaging (Saphyr; Bionano Genomics). Rare Variant Pipeline was used for detection of clinically significant SVs [minimum 300X coverage; 5000 bp size cut-off). We used bidirectional paired genome-wide analysis for identifying unique SVs seen exclusively at either baseline or HMA-F. Results There were 21 pts [11 men; 10 women; median age: 73 (43-89)] with available cryopreserved cells for comprehensive mutational and SV profiling. Median BM blast% was 5 (1-18). The median time to HMA failure was 11 (6-21) months. All pts had at least 1 genomic aberration: 14 (66%) by TBA and 20 (95%) by NGS (median: 3 mutations per pt.). Seven (35%) pts had normal karyotype (NK), 6 (30%) pts had complex karyotype (CK). Most frequent gene mutations included TET2, ASXL1 TP53 [each seen in 7 pts, 33%], SRSF2 [6 (30%)], DNMT3A, RUNX1 and STAG2 (5 pts each, 25%). Baseline OGM identified multiple clinically significant SVs, 6 of which, seen across 4 (19%) pts, were cryptic by TBA. These included CBFB::MYH1 and KMT2A partial tandem duplication. OGM at HMA-F showed increased genomic complexity compared to baseline in 9 (43%) pts. Paired analysis comparing genome-wide SV burden at HMA-F with matched baseline sample revealed 306 new unique SVs at HMA-F not seen at baseline ( Fig 1). These included 30 deletions (8 pts), duplications (6 pts), inversions, intrachromosomal and intrachromosomal translocations (5 pts each). Recurrent aberrations included segmental deletions of chr 17, 1, 7, 9, 12 and 21, affecting ETV6, CDKN1B, IKZF1, RUNX1, FHIT and HOXA11 genes, exon-spanning duplication in RUNX1, and FANCA::NF1 and YTHDF3-SETBP1 fusions. Three (14%) pts (2 with CK, 1 with NK), showed a cataclysmic increase in the numbers of SVs (translocations/ inversions) indicative of chromoanagenesis ( Fig 2). In contrast, there were 29 SVs seen exclusively at baseline and not detected at HMA-F [intrachromosomal translocations affecting chr 3 ( FOXP1) and chr 7 ( CUX1), low-level IGH::BCL2, deletions and insertions]. Sequential NGS and TBA was available in 20 pts. At HMA-F, 13 (62%) pts developed additional mutations, most frequent in NRAS, followed by TP53, TET2, CSF3R, KIT, second mutation in CEBPA, DDX41 and WT1 genes. The median number of mutations per pt. was 4. The highest increase in SV burden by OGM was noted in pts who did not acquire new mutations by NGS at HMA-F (5/7; 71%). Seven (33%) pts showed cytogenetic clonal evolution at HMA-F by TBA. OGM identified new SVs at HMA-F in 2 (50%) pts who did not show clonal evolution by NGS or TBA. Conclusions HMA-F MDS is characterized by increased genomic complexity by acquisition of new somatic mutations (NGS) and SVs (OGM), highlighting their complementary role in MDS progression. OGM further highlighted cataclysmic genomic changes by chromoanagenesis in a subset of MDS pts at HMA-F.

Decitabine/Cedazuridine in the Management of Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia in Canada.

The management of myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) is limited and remains an unmet need. Decitabine/cedazuridine (DEC-C, ASTX727) is Canada's first and only approved oral hypomethylating agent for MDS and CMML. We characterized the real-world use of DEC-C through a Canadian compassionate use program. Demographic and clinical data from 769 patients enrolled in Taiho Pharma Canada's Patient Support Program were collected and analyzed. These patients represent a collection period from 10 November 2020 to 31 August 2022 with a median age of 76 years. Among 651 patients who started DEC-C, the median treatment duration was 4.2 cycles. The median overall and progression-free survival were 21.6 and 10.7 months, respectively. Among 427 patients who discontinued treatment, the majority (69.5%) stopped due to death (n = 164) or disease progression (n = 133). Multivariable cox regression showed that age, province of residence, blast counts, antibiotic prophylaxis, and number of dose reductions and delays were not significantly associated with overall and progression-free survival. DEC-C is a promising alternative to parenteral hypomethylating agent therapy, and it likely addresses an important unmet need for effective and convenient therapies in this setting.

Efficacy and safety of venetoclax combined with hypomethylating agents for relapse of acute myeloid leukemia and myelodysplastic syndrome post allogeneic hematopoietic stem cell transplantation: a systematic review and meta-analysis

BackgroundCurrently, there is no standard treatment for managing relapse in patients with acute myeloid leukemia and myelodysplastic syndrome (AML/MDS) after allogeneic hematopoietic cell transplantation. Venetoclax-based therapies have been increasingly used for treating post-transplantation relapse of AML. The aim of this systematic review and meta-analysis was to evaluate the efficacy and adverse events of Venetoclax combined with hypomethylating agents (HMAs) for AML/MDS relapse post-transplantation.MethodsWe searched PubMed, Web of Science, Excerpta Medica Database, Cochrane Library, and Clinical. gov for eligible studies from the inception to February 2022. The Methodological Index for Non-Randomized Studies was used to evaluate the quality of the included literatures. The inverse variance method calculated the pooled proportion and 95% confidence interval (CI).ResultsThis meta-analysis included 10 studies involving a total of 243 patients. The pooled complete response and complete response with incomplete blood count recovery rate of Venetoclax combined with HMAs for post-transplantation relapse in AML/MDS was 32% (95% CI, 26-39%, I2 = 0%), with an overall response rate of 48% (95% CI, 39-56%, I2 = 37%). The 6-month survival rate was 42% (95% CI, 29-55%, I2 = 62%) and the 1-year survival rate was 23% (95% CI, 11-38%, I2 = 78%).ConclusionThis study demonstrated a moderate benefit of Venetoclax in combination with HMAs for patients with relapsed AML/MDS post-transplantation (including those who have received prior HMAs therapy), and may become one of treatment options in the future. Large-scale prospective studies are needed to confirm the potential benefit from venetoclax combined with HMAs.