Hypomethylating Agents Treatment Research Articles

Epigenetic priming by hypomethylation enhances the immunogenic potential of tolinapant in T-cell lymphoma.

Programmed cell death mechanisms are important for the regulation of tumor development and progression. Evasion of and resistance to apoptosis are significant factors in tumorigenesis and drug resistance. Bypassing apoptotic pathways and eliciting another form of regulated cell death, namely necroptosis, an immunogenic cell death (ICD), may override apoptotic resistance. Here, we present the mechanistic rationale for combining tolinapant, an antagonist of the inhibitor of apoptosis proteins (IAP), with decitabine, a hypomethylating agent (HMA), in T-cell lymphoma (TCL). Tolinapant treatment alone of TCL cells in vitro and in syngeneic in vivo models demonstrated that ICD markers can be upregulated, and we have shown that epigenetic priming with decitabine further enhances this effect. The clinical relevance of ICD markers was confirmed by the direct measurement of plasma proteins from peripheral TCL patients treated with tolinapant. We showed increased levels of necroptosis in TCL lines, along with the expression of cancer-specific antigens (such as cancer testis antigens) and increases in genes involved in interferon signaling induced by HMA treatment, together deliver a strong adaptive immune response to the tumor. These results highlight the potential of a decitabine and tolinapant combination for TCL and could lead to clinical evaluation.

Clinical activity, pharmacokinetics, and pharmacodynamics of oral hypomethylating agents for myelodysplastic syndromes/neoplasms and acute myeloid leukemia: A multidisciplinary review.

To review the pharmacokinetic (PK)-pharmacodynamic (PD) profiles, disease setting, dosing, and safety of oral and parenteral hypomethylating agents (HMAs) for the treatment of myelodysplastic syndromes/neoplasms (MDS) and acute myeloid leukemia (AML), and to provide a multidisciplinary perspective on treatment selection and educational needs relating to HMA use. Clinical and real-world data for parenteral decitabine and azacitidine and two oral HMAs: decitabine-cedazuridine (DEC-C) for MDS and azacitidine (CC-486) for AML maintenance therapy. Differences in the PK-PD profiles of oral and parenteral HMA formulations have implications for their potential toxicities and planned use. Oral DEC-C (decitabine 35 mg and cedazuridine 100 mg) has demonstrated equivalent systemic area under the concentration-time curve (AUC) exposure to a 5-day regimen of intravenous (IV) decitabine 20 mg/m2 and showed no significant difference in PD. The AUC equivalence of oral DEC-C and IV decitabine means that these regimens can be treated interchangeably (but must not be substituted within a cycle). Oral azacitidine has a distinct PK-PD profile versus IV or subcutaneous azacitidine, and the formulations are not bioequivalent or interchangeable owing to differences in plasma time-course kinetics and exposures. Clinical trials are ongoing to evaluate oral HMA combinations and novel oral HMAs, such as NTX-301 and ASTX030. Treatment with oral HMAs has the potential to improve quality of life, treatment adherence, and disease outcomes versus parenteral HMAs. Better education of multidisciplinary teams on the factors affecting HMA treatment selection may help to improve treatment outcomes in patients with MDS or AML.

Development of a Companion Diagnostic for a Targeted Protein Degradation Therapy: Ultrasensitive and Minimally-Invasive Detection of SALL4 Oncoproteins for Therapeutic Monitoring

Abstract Introduction/Objective Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 expression is normally repressed in most adult organs but is reactivated in a multitude of cancers and upon treatment with hypomethylating agents (HMA). Research in the past 20 years established SALL4 as a novel, druggable cancer target. SALL4 targeting therapies (inhibitors and degraders) are under pre-clinical development. There are currently no FDA/CLIA approved SALL4 diagnostic tests beyond tissue immunohistochemistry (IHC). Therefore, there is outstanding clinical need for the development and validation of a less invasive and sufficiently-sensitive SALL4 expression assay which can be used for selecting, and monitoring patients on SALL4-targeting therapy such as molecular glue degraders, as well as monitoring patients on HMA treatment. Methods/Case Report Using the Simoa (Single Molecule Array) platform, a bead-based digital enzyme-linked immunosorbent assay, we cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations, using the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. For quantitative assay development, we successfully expressed and purified full-length SALL4 which will be used to calibrate the standard curve and determine the assay’s limit of quantification. Results (if a Case Study enter NA) The best antibody pair can detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform. We successfully optimized detection of SALL4 in bone marrow and peripheral blood matrix, and tested the utility of the assay for quantifying the efficacy of SALL4 degrader drugs and with hypomethylating agent treatment, using cell culture system. We also optimized multiple pre-analytic steps, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. Conclusion We developed an ultrasensitive SALL4 expression assay will be useful for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders. There is ongoing effort to use MGB Biobank peripheral blood samples to validate SALL4 Simoa for patients with myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML), and liver cancer.

Ultrasensitive Detection of SALL4 Oncoproteins in Peripheral Blood and Bone Marrow Aspirate: Companion Diagnostics and Therapeutic Monitoring

Spalt-Like Transcription Factor 4 (SALL4), a member of the SALL family, is a regulator of embryonic stem cell development that plays a crucial role in cell renewal and proliferation. SALL4 is a tumor driver, as shown by gain-of-function and loss-of-function studies; high SALL4 expression invariably correlates with worse prognosis. These studies established SALL4 as a novel, druggable target, such as in liver, lung, and blood cancers. In myelodysplastic syndrome (MDS), SALL4 can be upregulated by hypomethylating agents (HMAs), correlating with worse patient outcomes. Despite its well-established function on oncogenesis, SALL4 targeting therapies (inhibitors and degraders) are under pre-clinical development. There are currently no FDA/CLIA approved SALL4 diagnostic tests beyond tissue immunohistochemistry. Therefore, there is outstanding clinical need for the development and validation of a less invasive and sensitive SALL4 expression assay via peripheral blood, which can be used for monitoring patients on HMA treatment and SALL4-targeting therapy such as molecular glue degraders. We developed a protein based SALL4 assay, compatible with peripheral blood and bone marrow aspirates samples, using the SIMOA (Single Molecule Array) technology, a bead-based digital enzyme-linked immunosorbent assay (aka digital ELISA). Previously, circulating SALL4 oncoproteins have been detected in the peripheral blood of patients with liver cancer using a conventional ELISA kit, but it is only semi-quantitative with limited sensitivity. We cross-tested 5 commercial SALL4 antibodies and 13 antibody pair combinations. After antibody screening, we used the best antibody pair to test the matrix effects of human plasma, buffy coats, and bone marrow aspirates. For quantitative assay development, we successfully expressed and purified full-length SALL4 for the first time, which was used to calibrate the standard curve and determine the assay's limit of quantification. The top bead and detector antibody pair could detect both SALL4A and SALL4B isoforms, with stronger signal detected with the SALL4A isoform in two different cell lysis buffers. Assay sensitivity was then tested. The Simoa assay captured endogenous SALL4 expression with greater sensitivity compared to the Western blot. The Western blot's limit of detection for SALL4A protein was 0.08 µg/µL whereas the Simoa assay continued until 3 ng/µL. Pre-analytic steps were then optimized, including our cell lysis protocol, isolation of buffy coats using density gradient separation, and sample collection method comparison. We assessed the effect of sample age for downstream processing and signal recovery as well as the possible matrix effect and interference from the buffy coat and plasma components of the peripheral blood and bone marrow samples. Our data showed no evidence of interference from the peripheral blood components, and interference from bone marrow aspirate can be further optimized. Antibody validation was then conducted. The preliminary data showed that the SALL4 Simoa assay can be used as a proof of principle to monitor SALL4 upregulation on HMA treatment for blood cancers and for SALL4 targeting therapy with two cell lines (SNU398 and H661) for solid cancers. To validate assay accuracy and precision, a retrospective cohort analysis is being conducted from patient peripheral blood samples. We obtained MGB Biobank samples for MDS, acute myeloid leukemia (AML), and liver cancer. We will use these samples to test for digital SALL4 protein detection. Further plans include expanding these analyses to breast and lung cancer. Additionally, we will conduct prospective studies to explore the role of the SALL4 protein biomarker in risk stratification and treatment monitoring, especially in MDS/AML patients undergoing HMA treatments. In conclusion, we have developed an ultrasensitive SALL4 protein expression diagnostic assay for use in monitoring patient treatment response and for guiding targeted therapy by analyzing peripheral blood for both solid and liquid tumors. Future steps include validating the diagnostic assay for specificity and sensitivity in a retrospective cohort analysis and conducting prospective studies to explore the role of the SALL4 protein biomarker for risk stratification, monitoring on HMA treatment, and guiding targeted therapy, ideally filing for approval as a laboratory developed test (LDT) in the near future

Azacitidine Treatment in MDS: A Systematic Literature Review and Meta-Analysis Comparing the Efficacy of Real World Data with Randomized Controlled Trials

Background: Myelodysplastic Syndromes (MDS) are a group of myeloid malignancies affecting the hematopoietic stem and progenitor cells and occurring mainly in elderly individuals. Two hypomethylating agents (HMA), Azacitidine (AZA) and Decitabine (DAC), have shown efficacy in treatment of MDS patients and received approval based on randomized controlled trials (RCTs) more than a decade ago. Since then, many publications have reported real world experience on HMA treatment in MDS patients, which showed a reduced efficacy of HMA than the RCT. The reasons for this difference have been extensively discussed but a systematic review never performed. Aim: We performed a systematic literature review and meta-analysis to compare the efficacy of real world data with randomized controlled trials and to identify potential cofactors for different outcomes. Material and Methods: We performed a systematic literature search from observational studies (OBSTs) describing real world experience of AZA treatment in MDS patients using Embase, Ovid Medline, and Web of Science databases. Patient characteristics, IPSS/IPSS-R, and outcome (response rates, overall survival) were collected from OBSTs that were performed between 2003 and 2023. Kaplan-Meier curves with log-rank tests were used to assess differences in survival outcomes. An inverse variance weighted meta-analysis was performed by using restricted mean survival times or proportions (response rate outcome). The restricted mean survival time and the standard error at 12, 24 and 36 months was calculated by using the R package survival. The pooled effect and prediction intervals from random effect models were reported. Results: In our systematic literature search, data from OBSTs describing real world experience of AZA treatment in MDS patients could be extracted and compared to the only RCT published with AZA (Fenaux P et al., Lancet Oncology 2009). Patient and disease characteristics were similar for AZA-RCT and OBST (respect of gender ratio, median age, IPPS/IPSS-R). Restricted mean survival time and standard error of the OBSTs and AZA-RCT showed no significant difference for the time points at 12 months (10.0 [95%-CI: 9.6; 10.3] months (OBSTs) vs 10.0 [95%-CI: 9.5; 10.5] months (AZA-RCT); figure part A) and 24 months (15.5 [95%-CI: 14.6; 16.5] months (OBSTs) vs. 16.9 [95%-CI: 15.7; 18.2] months (AZA-RCT); figure part B). At 36 months, the restricted mean survival time of OBSTs was 18.7 [95%-CI: 17.3; 20.1] months compared to 22.3 [95%-CI: 20.2; 24.5] months of the AZA-RCT (figure part C), which was different in favor of the RCT. Using random effect models, the AZA treatment response rate was 0.51 [95%-CI: 0.47; 0.56] for the OBSTs and 0.49 [95%-CI: 0.42; 0.57] for the AZA-RCT (figure part D). Conclusion: Compared to AZA-RCT data, restricted mean survival time (for time points 12 months and 24 months) and response rate of OBSTs describing real world data of AZA treatment in MDS patients are comparable. Regarding the observation time point at 36 months, the lower restricted mean survival time of the OBSTs compared to AZA-RCT may be most likely explained by study patient selection bias.

Treatment Outcomes of Stem Cell Transplant in Patients with Acute Myeloid Leukemia Treated with Venetoclax in Combination with Hypomethylating Agents: Experience from Routine Clinical Practices in the United States

Background: Venetoclax (Ven) in combination with hypomethylating agents (HMAs) is approved in the United States (US) for treatment in patients (pts) with newly diagnosed (ND) acute myeloid leukemia (AML) who are ≥75 years old or unfit for intensive chemotherapy. In a Phase Ib open-label trial (NCT02203773) and the Phase III VIALE-A trial (NCT02993523), a total of 33 pts with ND AML who were treated with Ven+HMAs underwent a stem cell transplant (SCT). The observed 12-month overall survival (OS) was 74.3% post-SCT, and the median OS was 29.9 months for pts who were in remission at the time of SCT (Pratz et al. EHA 2023). This study aims to evaluate the treatment patterns and outcomes of pts with ND AML treated with Ven+HMAs who underwent SCT post-remission from routine clinical practices in the US. Methods: This was a retrospective cohort study using the Flatiron Health electronic health record-derived, US nationwide, de-identified database. Pts aged ≥18 years with ND AML who initiated first-line Ven+HMAs treatment within 30 days from AML diagnosis (Dx) between July 1, 2018, and November 30, 2022, with ≥2 visits recorded within 3 months of AML Dx, and ≥3 months of potential data available before the study end date (February 28, 2023) were included. Pts with acute promyelocytic leukemia, t(9;22), treated with HMAs before AML Dx and pts who received Ven treatment more than 14 days prior to AML Dx were excluded. Descriptive analysis on patient characteristics was conducted. The proportion of pts who achieved real-world response (defined as <5% bone marrow [BM] blast), median time from Ven+HMAs treatment initiation to last administration (TTLA), and median time from Ven+HMAs initiation to SCT were reported. For pts who received SCT post-remission, 100-day real-world OS (rwOS), 12-month rwOS, and 15-month rwOS were evaluated using Kaplan-Meier analyses. The starting point of Kaplan-Meier analyses was SCT, and pts were censored at the last activity date. Results: Overall, 988 pts with ND AML treated with Ven+HMAs met the study criteria. A total of 51% (n=503/988) of pts achieved real-world response, of which 11% (n=57/503) subsequently underwent SCT post-remission. In pts with SCT post-remission (n=57) vs pts without SCT post-remission (n=446), the median age was 67 years vs 76 years, 30% (n=17/57) vs 30% (n=134/446) had secondary AML, and 60% (n=34/57) vs 24% (n=107/446) were treated at academic centers, respectively. Excluding pts with unknown cytogenetic and molecular data, the European LeukemiaNet (ELN) 2017 risk in pts with vs without SCT post-remission was favorable for 16% (n=7/43) vs 21% (n=76/355), intermediate for 33% (n=14/43) vs 26% (n=93/355), and adverse for 51% (n=22/43) vs 52% (n=186/355), respectively. Among pts with available molecular testing, FLT3 mutation in pts with SCT vs pts without SCT post-remission was 19% (n=5/26) vs 20% (n=48/245); IDH1/2 mutation was 33% (n=9/27) vs 35% (n=95/275); and TP53 mutation was 47% (n=9/19) vs 28% (n=54/192), respectively. Of the 57 pts who underwent SCT post-remission, the median (interquartile range [IQR]) TTLA on Ven+HMAs was 4.0 (3.0-5.3) months and the median (IQR) time from Ven+HMAs initiation to SCT was 5.4 (4.4-6.8) months. At a median follow-up of 8 months from SCT post-remission, the 100-day rwOS was 92%, 12-month rwOS was 52%, 15-month rwOS was 49%, and the median rwOS was 13.0 (95% confidence interval: 8.2-not reached) months ( Figure). Conclusions: In this study, similar to findings from clinical trials, we observed a subset of pts with ND AML treated with Ven+HMAs underwent SCT post-remission in routine clinical setting. Compared with pts who did not undergo SCT post-remission, pts who underwent SCT post-remission were younger, and more of these pts received care in academic centers. Of the Ven+HMAs-treated pts who underwent SCT post-remission, more pts treated in the routine clinical setting had the poor prognostic TP53 mutation than pts enrolled in the aforementioned clinical trials. In this study, most pts remained alive 100 days after SCT. The rwOS results observed in this study suggest that Ven+HMAs post-remission is a potential bridge to SCT in pts who are ineligible for intensive chemotherapy.

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.

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.

Novel Mechanisms of Venetoclax Resistance in Acute Myeloid Leukemia Based on Genomic Rearrangements

Introduction: Venetoclax (VEN), a BCL-2 inhibitor, effectively induces apoptosis in acute myeloid leukemia (AML) cells. Resistance to VEN in the treatment of AML can occur through various mechanisms. Some of the identified resistance mechanisms include: expansion or gain of clones with activation of the receptor tyrosine kinase (RTK) pathway; disruption of TP53 function, a tumor suppressor gene known to play a critical role in cell cycle regulation and apoptosis; selection of AML populations with monocytic differentiation, possibly altering cellular characteristics and response to treatment; overexpression of the MCL1 gene, which encodes for a protein that can counteract the pro-apoptotic effects of venetoclax; switch to lipid metabolism, an adaptive response by leukemia cells that may confer resistance to venetoclax; mutations in BCL-2 and BAX genes, which could affect the activity of these key regulators of apoptosis. While these mechanisms have been extensively studied, the role of rearrangements, often combined with genomic events, has not been fully explored. The INTHEMA protocol (NCT04298892) aimed to identify new VEN resistance mechanisms in patients with relapsed/refractory AML treated with VEN in combination with hypomethylating agents and to provide addressable molecular pathways useful for restoring drug sensitivity or offer novel therapeutic opportunities. Methods: Informed consent was obtained from 21 consecutive patients. Samples were collected from these patients with relapse or refractory disease after VEN + hypomethylating agent treatment. TruSight RNA Pan-Cancer analysis was performed to investigate fusion genes (using capture technology covering 1385 genes), RNA expression, and expressed mutations for each sample. Results: Among the 21 patients, 17 (81%) showed at least one putative mechanism of VEN resistance (as illustrated in the figure). These mechanisms included co-occurring mutations and genomic rearrangements. RTK mutations were found in 14 patients (67%), with specific mutations observed in NRAS, PTPN11, CBL, FLT3, JAK2, cKIT, EPHB6, FGFR and PDGFRb. One patient had a BAX mutation (p.G67R; BH3 domain), three patients exhibited a switch to a monocytic phenotype, and two patients had marker chromosomes. Gene fusions were identified in eight patients (38%): in particular, we discovered a cryptic rearrangement of MECOM (TBL1XR1-MECOM and ETV6_MECOM) in 2 patients, a novel BCL11B fusion ( BCL11B-GSDMC), a CCND2-ETV6 BIRC6-LTBP1, the reciprocal NUP98-NSD1, the canonical CBFB-MYH11 and additional transcripts with less defined roles ( PSPC1-ZMYM2, PER1-DDX5, ELL-SUGP2). Interestingly, most of the genes involved in traslocations, with a potential driver role in therapy resistance ( MECOM, ETV6, CCND2, BCL11B, NUP98) also showed high expression levels in patients harboring the traslocation. Unsupervised clustering of RNA expression data revealed two main patient groups. The first group (24%) demonstrated an “activating-like” signature, characterized by overexpression of CSF3R and IKZF, suggesting a potential target for tyrosine kinase inhibitors. The second group exhibited a “self-renewal like” signature, suggesting a possible benefit from histone deacetylase inhibitors, as previously published. Patients with gene fusions were predominantly clustered within the “self-renewal like” group, particularly in a subgroup (subgroup 4) showing overexpression of HOXA genes. Conclusions: Through deep transcriptomic characterization combined with conventional diagnostics, this analysis uncovered novel mechanisms of VEN resistance while confirming established ones. The distinct gene expression patterns may help tailor targeted therapies, with patients showing the “activating-like” signature potentially benefiting from tyrosine kinase inhibitors and those with the “self-renewal like” signature possibly responding well to histone deacetylase inhibitors. Furthermore, HOXA gene overexpression presents an exciting therapeutic opportunity for selected patients. Further research and clinical validation are needed to translate these findings into actionable clinical strategies.

Efficacy of Primary Prophylaxis in Patients with MDS or AML Treated with Non-Intensive Agents: Systematic Review with Meta-Analysis

Background: Infections are common among patients with myelodysplastic syndromes/neoplasms (MDS) and acute myeloid leukemia (AML) due to neutropenia, which leads to increase in hospitalization, morbidity and mortality. Antibiotic prophylaxis has been proven to reduce the incidence of infections in oncologic and hematooncologic patients receiving intensive chemotherapies. Accordingly, most hematology and oncology societies advice for its use especially in high-risk patients. These recommendations, however, cannot be directly extrapolated to elderly MDS or AML patients treated with less intensive regimens such as single agent hypomethylating agents (HMA) or low-dose cytarabine (LDC), as controlled prospective randomized trials are not available in this context. Aim: The aim of this systematic review was to summarize published data on the efficacy and safety of primary antibiotic prophylaxis in patients with MDS or AML treated with non-intensive agents and perform a meta-analysis. Methods: We performed a systematic review using PubMed and Embase databases on 14th September 2022 without time-restrictions concerning publication data. Inclusion criteria comprised demographics (adult), type of study (retrospective and prospective cohort studies, case control studies, randomized controlled trials), study population (patients with MDS, MDS/MPN or AML), and treatment (hypomethylating agent (HMA), low-dose cytarabine (LDC)). Study outcomes/endpoints included information on efficacy (reduction of infection or fever episodes requiring antibiotic treatment (bacterial, viral, fungal), hospitalizations, intensive care unit admissions or leading to dearth) and safety (antibiotic side-effects and resistance) of primary antibiotic prophylaxis. Meta-analysis was performed and odds ratios with 95% confidence intervals shown in a Forrest Plot. Results: From 233 screened studies, we identified no randomized or prospective studies and only two retrospective studies with 68 patients and 346 treatment cycles that matched our inclusion/exclusion criteria. ( A). One study included AML (n=40, Bainschab et al, 2016) and the other MDS patients (n=28 , Lee et al, 2011). Patients recived primary antibiotic prophylaxes in 79/215 (25%) and 95/131 (72.5%) treatment cycles, respectively (B). The antibiotics used included fluorquinolones (92.5% and 95%), penicillins (6.5% and 0%) and cephalosporins (1% and 5%). Infections episodes were reported in 53/215 (25%) and 15/131 (11%) treatment cycles. A meta-analysis of the selected studies suggests a significant benefit in reducing the incidence of infection or febrile episodes (OR 0.43 [0.25-0.75], p<0.003) with potential reduction in hospital admission, especially in patients with low ANC (< 0.5 G/l). As additional risk factors for infectious complications we identified severe cytopenia (grade ≥3) in at least one lineage, earlier therapy cycles, transfusion dependency, increased LDH and high Charlson Comorbidty Index. Data regarding the safety and tolerability of antibiotic prophylaxis was not available. Conclusion: Our investigation with only few eligible studies, patients and treatment cycles underlines an unmet need for prospective controlled trials in this field.Our systematic review with meta-analysis suggests a significant benefit of antibiotic prophylaxis in MDS and a tendency in AML patients undergoing single agent HMA or LDC treatment. Those individuals with high-risk features seem to have the greatest benefit. Prospective, controlled and randomized trials are, however, lacking and should be encouraged, even more as combinations with venetoclax are becoming the standard of care in AML and potentially also in MDS with higher risk of neutropenic infekctions.

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

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

Development of Oral Azacitidine with Cedazuridine for Myelodysplastic Syndrome (MDS) and Myeloproliferative Neoplasms (MPN) Including CMML (Chronic Myelomonocytic Leukemia) By Targeting Pharmacokinetic AUC Equivalence Vs Subcutaneous Azacitidine

Introduction/ Background: Hypomethylating agents (HMAs) such as azacitidine (AZA) and decitabine (DEC) are FDA-approved for patients with MDS/CMML and acute myeloid leukemia (AML). Treatment is generally administered parenterally as a daily injection or infusion over 5-7 days per four-week cycle. The oral fixed-dose combination of decitabine with cedazuridine, ASTX727, was approved by regulators in the USA and Canada for patients with MDS and CMML based upon the demonstration of biologically equivalent pharmacokinetics (PK) area under the curve (AUC) vs IV decitabine. ASTX030 is a combination of AZA and cedazuridine (CED), a cytidine deaminase inhibitor (CDAi), which enables oral AZA to achieve systemic AUC similar to that of parenteral (subcutaneous) AZA. Development and approval of an oral AZA providing equivalent PK exposure to the parenteral therapy has the potential to markedly reduce the treatment burden associated with parenteral treatment. This Phase I trial is designed to determine the dose combination for oral AZA plus CED to replicate SC AZA AUC exposures in subjects with MDS and MDS/MPN, including CMML. Methods: Adult patients with confirmed MDS, CMML, and other MDS/MPN or AML who are candidates to receive benefit from single agent AZA were enrolled in this open label Phase 1 trial. In addition to assessing safety and efficacy of each combination, the primary PK endpoint was to achieve oral AZA PK AUC 0-24 equivalence versus subcutaneous AZA (at the approved dose of 75 mg/m 2 over the span of 7 days). In the first cycle, patients received oral AZA alone on Day -3, followed by SC AZA at 75 mg/m 2 on Day 1. Subsequently, oral ASTX030 (combination of AZA with CED) was given at varying dose combinations of AZA and CED for each cohort. After cycle 1, patients received oral ASTX030 on Days 1-7 for each 28-day cycle. Dose levels for cohorts in escalation were determined by the data safety and review committee based on safety and PK results. Pharmacodynamic (PD) impact was assessed by changes in LINE-1 DNA methylation in peripheral blood cells. Clinical response assessments were based upon International Working Group (IWG) 2006 criteria for MDS patients and IWG 2015 criteria for MDS/MPN and CMML patients. Results: As of 05 JUL 2023, 65 patients were treated across 8 different dose combinations, with ≥6 patients per cohort. The median age was 72 years old (range 26-87), 36.9% (n=24) were female, 6.2% (n=4), had previously received HMA treatment (unlimited). Of the enrolled patients, 69% (n=45) had MDS, 25%(n=16) had CMML, and 6% (n=4) had MDS/MPN (other). Evaluated dose levels for AZA ranged from 60-136mg and CED dose ranged from 20-100 mg. In cohorts 1-2, tablet formulation was tested first (AZA ranging 80-100mg with CED ranging 80-100mg). Starting with cohort 3, capsule formulation was introduced for AZA to optimize the interaction effect of CDA inhibition by CED. In both tablet and capsule formulation, grade ≥3 AE's (regardless of causality) were observed in 75.9% of patients. The most common Grade ≥3 AE's regardless of relation to drug were leukopenia (25%), thrombocytopenia (20%), and anemia (20%). Gastrointestinal toxicity were similar (for example, nausea 70% ASTX030 vs 71% SC AZA) to those reported for SC AZA (VIDAZA USPI)). No dose limiting toxicity was observed in any of the cohorts. The CED dose of 20 mg resulted in ~100% bioavailability for AZA, suggesting inhibition in a linear range. LINE-1 demethylation results were similar to those reported historically with SC AZA. Although a clinical response comparable to SC AZA was observed, the immature nature of the data requires continued analysis to yield results on treatment efficacy. Conclusion: ASTX030 successfully achieved the primary endpoint of PK equivalence versus SC AZA based on total cycle AUC. The dose combinations evaluated were well tolerated, and the safety profile similar to that of SC AZA, with no unique AE's. LINE-1 demethylation and clinical response confirm the clinical activity of ASTX030. Phase 1B dose expansion for the ASTX030 study at the recommended dose combination of 144mg AZA with 20mg CED will begin soon to confirm PK equivalence for this dose level. An orally bioequivalent AZA based HMA offers significant opportunity for novel combinations and improved convenience for patients with high grade myeloid malignancy.

Anti-PD-1 Antibody (Sintilimab) Plus Decitabine As First Line Treatment for Patients with Higher-Risk Myelodysplastic Syndrome (MDS): Primary Results from a Single-Arm,Open-Label,Phase II Study

Background: Hypomethylating agents (HMAs) are the preferred treatment for untreated patients with higher-risk Myelodysplastic Syndrome (MDS). However, the survival rate of patients after HMA treatment remains poor. Studies have shown that HMAs upregulate the expression of PD-1/PD-L1 in MDS patients, making the combination of HMAs with PD-1 antibodies a compelling approach for MDS treatment. Consequently, we conducted this single-arm, open-label clinical trial (ChiCTR2100044393) to assess the safety and efficacy of sintilimab in combination with decitabine for patients with higher-risk MDS. Methods: Adult patients with higher-risk MDS, according to the IPSS-R, were eligible for enrollment in this study. The treatment regimen consisted of daily intravenous administration of decitabine at a dose of 20mg/m 2 for 5 consecutive days, along with intravenous administration of sintilimab at 200mg on the first and 22nd day of a 42-day treatment cycle. Treatment continued until disease progression, death, unacceptable toxicity, withdrawal of consent, or other protocol-defined reasons, with a maximum of 8 cycles allowed. The primary endpoint of this study was the overall response rate (ORR). To determine the sample size, we utilized Simon's optimal two-stage design. Secondary endpoints included the assessment of safety and various survival outcomes. Additionally, we investigated the relationship between the expression levels of immune checkpoints and the efficacy of the combination therapy and other potential biomarkers through genomic profiling. Results: As of the data cut-off on June 16, 2023, 53 patients were enrolled, with a median follow-up time of 14 months, and 52 patients were eligible for response evaluation. The median age of patients was 65 years (range 19-83), other characteristics were summarized in Table 1.The ORR was 76.9%, and 15 patients achieved complete response (CR), 13 marrow complete response(mCR),7 mCR+ hematologic improvement(HI), and 5 HI. Among the 40 responders, six patients (4 with CR and 2 with mCR) underwent allogeneic hematopoietic stem cell transplantation. The median overall survival (OS) has not reached, and the median event-free survival (EFS) was 16 months. The 24-month OS and EFS rates were 54.8% (95%CI: 39.5-76.2) and 46.2% (95%CI: 31.2-68.3), respectively. The most common grade 3 or higher treatment-emergent adverse events (TEAEs) reported in 10% or more of the patients were febrile neutropenia (67.9%) and pulmonary infection (20.8%). A total of 17 patients (32.1%) experienced immune-related adverse events (irAEs), with the most common irAEs being rash (17.0%), thyroid dysfunction (9.4%), and pneumonia (7.5%), all resolved after glucocorticoid treatment. Regarding genetic mutations, the most frequently mutated genes were ASXL1 (34.0%), RUNX1 (30.2%), TET2 (17.0%), and U2AF1 (17.0%). Conclusion: To the best of our knowledge, this is the first study evaluating the efficacy and safety of the combination therapy with sintilimab and decitabine in previously untreated patients with higher-risk MDS. This regimen showed an encouraging anti-tumor activity with a manageable safety profile.

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.

Venetoclax (VEN) Improves Response Rates but Not Survival in Patients with Chronic Myelomonocytic Leukemia (CMML) Treated with Hypomethylating Agents (HMA): A Multicenter, Propensity Score Analysis

Introduction HMAs are the only approved therapy for CMML. In order to improve response rates and survival, the addition of VEN has been utilized in both CMML and after blast transformation (CMML-BT) with conflicting results in small single center studies. To clarify the role of upfront HMA+VEN in CMML and CMML-BT, we performed a large multicenter analysis. Methods We included patients with HMA-naïve CMML and CMML-BT treated with combination HMA and VEN from 4 academic centers. The primary outcome was overall survival (OS) with secondary outcomes including overall response rate (ORR) by 2015 MDS/MPN IWG criteria for CMML and 2022 ELN criteria for CMML-BT. To better appreciate the benefit of adding VEN to HMA treatment, we also performed 1:1 propensity score matched (PSM) analysis of patients treated with HMA alone as upfront therapy for CMML or CMML-BT. Matching was based on age, sex, ECOG performance status, CPSS-Mol, blood counts, peripheral and bone marrow blasts. Results Eighty-nine patients were treated with frontline HMA+VEN, 51 CMML and 38 CMML-BT (5 had received HMA alone for prior CMML). The median age was 71 years, 70% were male, and 83% had ECOG 0-1. Among CMML patients, 55% had MP-CMML subtype, 65% had CMML-2 and 12%, 30%, 58% were CPSS-mol intermediate-1, intermediate-2 and high risk, respectively. In the entire cohort, ASXL1 (63%), TET2 (47%), SRSF2 (39%) and RUNX1 (27%) were the most commonly mutated genes and 55% of patients harbored a RAS mutation ( KRAS [20%], NRAS [18%], CBL [13%], NF1 [11%], PTPN11 [9%]). For HMA combination partner, 46 (52%) were treated with azacitidine, 35 (39%) with decitabine, and 8 (9%) with both during their VEN course. During a median follow-up time of 15.8 months (mos) 52 patients (58%) died: 23 (45%) with CMML and 29 (76%) with CMML-BT. Among CMML patients, the ORR was 90%, including complete remission (CR) in 22%, complete cytogenic remission (CCR) in 4%, optimal marrow response (CMR) in 50%, and partial marrow response (PMR) in 6%. The OS of CMML patients was 25.1 mo (95% CI 15.7 - NR). A PSM population of HMA patients matched to HMA+VEN treated patients showing similar baseline characteristics. ORR was significantly higher with HMA+VEN (95%) as compared those treated with HMA alone (46%), p < 0.001. However, there was no significant OS difference between HMA+VEN vs HMA alone (19.1 mos vs 19.1 mos, p = 0.85, Figure 1). For CMML-BT patients, the ORR of HMA+VEN was 81% including CR in 17%, CR with partial hematologic recover (CRh) in 3%, CR with incomplete hematologic recovery (CRi) in 28%, morphologic leukemia-free state (MLFS) in 22% and partial response (PR) in 11%. The OS of CMML-BT patients was 8 mos (95% CI 4.6 - 26.2). A PSM population of CMML-BT patients treated with HMA alone showed a significantly increased ORR with HMA+VEN (82% vs 40%, p = 0.039). However, there was no difference in OS (7.49 mos vs 9.26 mos, p = 0.83, Figure 2). Among HMA-VEN treated patients, achievement of a CR was associated with an improved OS in the CMML (HR 0.31, 95% CI 0.13 - 0.75) and CMML-BT (HR 0.37, 95% CI 0.19 - 0.74). No predictors of response were identified, including the presence of a RAS mutation. In terms of safety, 7 patients (7.9%) had a grade 3/4 (G3/4) neutropenic infection and 2 patients (2.2%) had a G3/4 hemorrhagic event while being treated with HMA+VEN. A total of 29 patients went on to receive allogeneic transplant (alloSCT): 22 (43%) with CMML and 7 (18%) with CMML-BT. CMML patients who underwent alloSCT after HMA+VEN were younger as compared to non-transplanted patients (64 vs 74 years, p < 0.0001), but had otherwise similar baseline characteristics including stage, CPSS-mol category and prevalence of RAS mutations. Similarly, CMML-BT patients bridged to alloSCT were younger (66 vs 74 years, p < 0.002) and had a better ECOG performance status (p = 0.047). Of patients who received alloSCT, 10 patients (34%) died, 5 with CMML and 5 with CMML-BT. The median OS after alloSCT was 26.7 mo. Conclusions Although the use of VEN with HMA in the upfront treatment of CMML and AML secondary to CMML results in higher response rates compared to HMA alone, this combination does not improve survival based on this multi-center PSM analysis. The current sample size of CMML-BT may limit detection of OS differences between treatment groups. The role of VEN to reduce blasts and serve as a bridge to alloSCT, particularly after HMA failure, requires further investigation in larger studies in both the CMML and CMML-BT populations.

Leukemic Cell-Intrinsic Innate Nucleic Acid Receptor Signaling Governs Efficacy of Hypomethylating Agents and Immunosurveillance in AML

Introduction: Hypomethylating agents (HMA), such as Azaciditine (AZA), are a long-established treatment option for acute myeloid leukemia (AML). Besides epigenetic reprogramming of malignant cell clones, HMAs are recognized to facilitate antineoplastic immunity. In vitro assessments in solid tumor models suggest that a major mechanism underlying AZA-triggered immune responses may be the activation of innate nucleic acid sensing pathways. As such, the RNA- and DNA-sensing receptor systems, RIG-I/MDA-5 and cGAS/STING - and subsequent type I interferon (IFN-I) signaling seem to be triggered by the transcriptional upregulation of otherwise DNA-hypermethylated and thus silenced endogenous retroviruses (ERVs). In AML, it is unknown whether similar processes are involved and/or by which molecular mechanisms, HMAs modulate anti-leukemic immunity against AML blasts, and how these processes are affected during acquired HMA resistance. Methods: Murine (c1498 and Wehi-3B) and human (MOLM-13) AML cell lines were exposed to AZA in vitro and analyzed for expression of ERVs. Nucleic acid receptor sensing pathways and IFN-I genes are not usually DNA methylated by AZA at promoter regions. However, the re-expression of endogenous nucleic acids or ERVs could trigger their activation. Protein and transcriptional expression of major components of nucleic acid receptor sensing pathways and IFN-I genes were, therefore, analysed. To understand the importance of AML cell-intrinsic nucleic acid receptor signaling in HMA-induced immunosurveillance, syngeneic c1498 and Wehi-3B AML models were employed. Mice received 2 cycles of 2.5mg/kg AZA (6 days on, 5 days off, 6 days on) intraperitoneally to mimic the principal treatment course used in patients. To address the contribution of the nucleic acid-sensing pathways to AZA treatment efficacy in vitroand in vivo, AML cells with acquired AZA resistance (AZA-R) or gene engineered AML cells deficient in RNA receptors (RIG-I/MDA-5), or the RNA- and DNA-sensing adapter proteins (MAVS and STING) were employed. Results: A targeted ERV screen revealed upregulation of various ERV transcripts in c1498 and Wehi-3B cells and was similarly observed in MOLM-13 AML cells following AZA exposure in vitro. Increased RNA transcript and protein expression of RIG-I/MAVS and (to a lesser degree) STING was observed, resulting in activation of downstream constituents including higher phosphorylation of the transcription factors IRF3/7 and NF-kB, IFN-I, and other ISGs. This was abrogated in RIG-I and MAVS, but not STING, deficient murine and human AML cells. Interestingly, expression of RIG-I/MDA-5, STING and all other downstream constituents, but not MAVS, were diminished in AZA-R AML cells. In mice harboring MAVS deficient AML (c1498), HMA treatment failed to show anti-leukemic activity resulting in rapid disease progress and death of recipient animals. Loss of STING in leukemic cells also impacted AZA treatment efficacy, although to a much less pronounced degree. These findings were similarly reproduced with Wehi-3B AML-engrafted mice. Following antibody-mediated depletion of T cells but not NK cells, mice rapidly succumbed to AML disease and demonstrated no anti-leukemic effect of AZA treatment. CyTOF and flow cytometric analysis also revealed increased bone marrow and spleen infiltration as well as activation of CD4 +/CD8 + T cells, NK cells, and MHC-II + APCs/Myeloid cells in AZA treated mice bearing wildtype AML, compared to untreated. This was not observed in AZA-treated mice bearing MAVS-deficient or AZA-R AML. Conclusion: Our findings indicate that nucleic acid receptor signaling (specifically RIG-I/MAVS) within leukemic cells is imperative for AZA efficacy and immunosurveillance in AML. Whether these RNA-sensing receptors are triggered by ERVs or alternate (endogenous) RNAs remains to be determined. Elucidating the molecular mechanisms of how nucleic acid sensors within leukemic blasts shape treatment efficacy of AZA may guide development of systematic immunotherapeutic strategies for AZA-R AML by targeted combinatorial approaches.

Real-World Outcomes with CPX-351 Induction in Adults with Newly Diagnosed Acute Myeloid Leukemia

Introduction: CPX-351 is a dual-drug liposomal encapsulation of daunorubicin and cytarabine with a fixed molar ratio of 1:5. CPX-351 is approved for the treatment of newly diagnosed, therapy-related acute myeloid leukemia (t-AML) or AML with myelodysplasia related changes (AML-MRC) in the U.S. and Europe for adult patients (pts) eligible for intensive chemotherapy. In a randomized phase 3 study of older pts ages 60-75, CPX-351 induction showed improved CR rates and OS compared to conventional 7+3 induction, with the greatest benefit seen in pts with t-AML and those without prior hypomethylating agent (HMA) use. There is limited data on the use of CPX-351 in younger pts, although the U.S. FDA approval was not age-restricted. This study examined real-world CPX-351 usage, including patient selection, clinical outcomes, and complications during induction hospitalizations. Methods: We identified previously untreated AML pts who received CPX-351 induction chemotherapy at the University of Kansas Medical Center via retrospective chart review from August 2017 - May 2023. Determination of t-AML and AML-MRC was made by patient history, bone marrow morphology, and available cytogenetic and molecular information at the time of therapy selection. We excluded pts who received CPX-351 as part of a clinical trial. Data was collected for baseline demographics, disease characteristics, outcomes, and safety profile. The composite complete remission rate (CRc) was defined as CR+CRi+CRh at recovery marrow after induction. Primary refractory disease included pts who failed to achieve CR, CRi, or CRh after 2 courses of intensive induction and excluded those who died in aplasia or due to an indeterminate cause. Results: 74 pts with previously untreated AML who received CPX-351 were identified. Median age was 64 years (21-78): 29% of pts were <60 years old, 50% were males, 84% were Non-Hispanic White, 32% had t-AML, 68% had AML-MRC, 20% had prior HMA treatment, and 22% had prior anthracycline exposure. ELN 2022 risk categories were 13% favorable, 20% intermediate, and 68% adverse. 21 pts had complex karyotype (28%), and 16% were TP53 mutant (Table 1). Indications for induction with CPX-351 versus 7+3 included history of prior chemotherapy (n=24), prior MDS (n=32), or MDS-related changes in bone marrow morphology (n=50). The CRc for the total study population was 61% (45/74). Of these, 29% (13/45) achieved MRD negativity by multiparameter flow cytometry <10 -4. CRc in the ELN adverse risk group was 54% (27/50), 73% (11/15) in the intermediate-risk group, and 78% (7/9) in the favorable risk group. Among TP53 mutant pts, CRc was 42% (5/12). CRc in the t-AML group was 71% (17/24), 56% (28/50) in the AML-MRC group, and 53% (8/15) in the prior HMA treatment group. The CRc in pts ≥60 was 63%, 15% were primary refractory, and 37% underwent alloSCT in CR1. In pts <60, the CRc was 54%, 32% were primary refractory, and 27% underwent an alloSCT in CR1 (Table 2). A second course of induction therapy was given for persistent disease at day 14 or similar time point in 36% (n=27) of pts. Among those, 55% (15/27) had primary refractory disease, 20% of the cohort. At a median follow-up of 11 mos, the mOS was 10.8 mos (0.4-54). The mOS for t-AML was 13 mos, whereas the mOS for the group with prior HMA treatment was 10.4 mos. For pts that achieved CR, mOS was 13.6 mos; for pts that achieved MRD negativity, mOS was 13.8 mos. The mOS for pts <60 was 16 mos, and for pts ≥60 was 10 mos (p=0.39). The mOS for pts that achieved CRc in the group <60 was 20.3mos, and for ≥60 was 11.2 mos (p=0.27). Pts that underwent alloSCT in CR1 <60 had mOS of 25 mos; for ≥60, it was 14.2 mos (p=0.37). During induction hospitalization, febrile neutropenia was the most common adverse event in 84% (n=62). ICU treatment was required in 14% (N=5). There were 2 deaths during induction: one due to respiratory failure and one attributed to sepsis. Conclusion: The use of CPX-351 demonstrated efficacy across subgroups, especially in t-AML and pts without prior HMA exposure, as shown in larger studies. In pts <60 years, even though CRc rates were lower, CPX-351 led to longer mOS in those who achieved CRc and in those who underwent alloSCT compared to pts ≥60 years, although it didn't reach statistical significance in this small cohort. Induction therapy outcomes and treatment-related complications in our study corroborate the data for prior CPX-351 studies.

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.

Ratio of stemness to interferon signalling as a biomarker and therapeutic target of myeloproliferative neoplasm progression to acute myeloid leukaemia.

Progression to aggressive secondary acute myeloid leukaemia (sAML) poses a significant challenge in the management of myeloproliferative neoplasms (MPNs). Since the physiopathology of MPN is closely linked to the activation of interferon (IFN) signalling and that AML initiation and aggressiveness is driven by leukaemia stem cells (LSCs), we investigated these pathways in MPN to sAML progression. We found that high IFN signalling correlated with low LSC signalling in MPN and AML samples, while MPN progression and AML transformation were characterized by decreased IFN signalling and increased LSC signature. A high LSC to IFN expression ratio in MPN patients was associated with adverse clinical prognosis and higher colony forming potential. Moreover, treatment with hypomethylating agents (HMAs) activates the IFN signalling pathway in MPN cells by inducing a viral mimicry response. This response is characterized by double-stranded RNA (dsRNA) formation and MDA5/RIG-I activation. The HMA-induced IFN response leads to a reduction in LSC signature, resulting in decreased stemness. These findings reveal the frequent evasion of viral mimicry during MPN-to-sAML progression, establish the LSC-to-IFN expression ratio as a progression biomarker, and suggests that HMAs treatment can lead to haematological response in murine models by re-activating dsRNA-associated IFN signalling.

Exploring preferences of different modes of administration of hypomethylating agent treatments among patients with acute myeloid leukemia.

About half of patients with Acute Myeloid Leukemia (AML) are not eligible for Standard Induction Chemotherapy (SIC). Hypomethylating Agents (HMAs) intravenously (IV) or subcutaneously (SC) in a clinical setting are typically offered as an alternative. However, injectable HMAs may be burdensome for patients given the frequent hospital visits and side effects. This study explored patient treatment preferences for different modes of administration (MOA) and the relative importance of treatment-related characteristics that influence treatment decisions. Semi-structured 1:1 interviews were conducted with 21 adult patients with AML in Germany, the United Kingdom, and Spain, who are not eligible for SIC, had experience with HMAs or were scheduled to be treated with HMAs. After discussing their experience of living with AML and its treatments, patients were presented with hypothetical treatment scenarios to explore their preferences, and a ranking exercise to assess the relative importance of treatment characteristics that influence their treatment-decisions for AML. Most patients reported an overall preference for oral administration over parenteral routes (71%), mostly due to convenience. Those preferring IV or SC routes (24%) reasoned with faster speed of action and onsite monitoring. When presented with a hypothetical situation of a patient having to choose between two AML treatments that were identical except for their MOA, the majority preferred the oral route (76%). Regarding treatment characteristics that influence treatment decisions, patients most frequently reported efficacy (86%) and side effects (62%) as important, followed by mode of administration (29%), daily life impacts (24%) and location of treatment (hospital versus home) (14%). However, only efficacy and side effects were rated as number one deciding factors (67% and 19%, respectively). Patients most frequently rated dosing regimen (33%) as least important. The insights gained from this study may help support patients with AML who are receiving HMA treatment instead of SIC. A potential oral HMA with similar efficacy and tolerability profiles to injectable HMAs could influence treatment decisions. Furthermore, an oral HMA treatment might decrease the burden of parenteral therapies and improve patients' overall quality of life. However, the extent of influence MOA has on treatment decisions requires further investigation.