Xie, Zhuoer

Background: Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative overlap neoplasm (MDS/MPN), further categorized as MD (WBC < 13 x 109/L) and MP (≥ 13 x 109/L) subtypes with an inherent risk for AML transformation (Am J Hematol 2024; 99: 1142). Hypomethylating agents (HMA), decitabine (DAC) and azacitidine, are the only FDA approved agents for the management of CMML, with complete remission rates of <20%, and a recent prospective study showing no event free survival benefit of DAC over hydroxyurea in MP-CMML (J Clin Oncol 2023; 41: 1888). TET2 mutations (TET2MT) occur in approximately 60% of CMML cases. TET2 converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), resulting in DNA demethylation (Nat Rev Gen 2017; 18: 517). IV ascorbic acid (AA) at pharmacologic concentrations demonstrates anti-cancer activity via H2O2-induced oxidative stress and DNA demethylation mediated by TET-activation. AA, a cofactor of TET2, binds to its catalytic domain, restores, and enhances TET1/2/3 activity. Given the synergistic impact of IV AA and HMA on DNA methylation, we designed a phase II clinical trial testing the safety and efficacy of standard dose decitabine and high dose IV AA in HMA-naïve CMML patients. Methods: LS1781 (NCT03418038) is an investigator-initiated, prospective, single-arm, single-institution, phase II trial assessing the safety and preliminary efficacy of high-dose IV AA in combination with DAC for patients with TET2MT CMML. DAC 20 mg/m2 Days 1-5 of each 28-day cycle is given as standard of care. With each cycle, IV AA (1g/kg, maximum 100g) is given 3 times on Days 1, 3, 5 via central line within 2 hours after DAC. Patients are encouraged to take oral AA 1000 mg once daily on non-treatment days. The primary endpoint is overall response rate determined by IWG MDS/MPN 2015 criteria after 4 cycles of therapy. Secondary endpoints include safety and side effects. Exploratory endpoints include the impact of AA on TET2MT variant allele fraction, plasma cytokine levels, DNA methylation and hydroxymethylation (as measured by Infinium MethylationEPIC array), and hematopoietic progenitor colony forming assay assessment (colony counts and impact on differentiation). Inclusion criteria include patients ≥ 18 years with TET2MT CMML, ≤1 cycle of HMA or ESA (hydroxyurea allowed), platelets ≥ 20,000/m3, ANC ≥ 500/mm3, and Cockcroft-Gault creatinine clearance ≥ 60 mL/min/m2. Twenty-three patients will be enrolled at Mayo Clinic Rochester and the Mayo Clinic Health System, based on power calculations to demonstrate enhanced complete response and overall response rates, in comparison to those seen historically with decitabine alone. Dose modifications have been established and will be considered based on adverse events. Treatment will be discontinued for disease progression or in the event of ≥ Grade 3 adverse events that do not resolve promptly. After completion of up to 12 cycles, patients with stable disease or better may continue single agent DAC with or without oral AA. Enrollment to the trial began in February 2024, with 2 patients enrolled at the time of abstract submission.

Abstract This phase 2 trial assessed high-dose IV ascorbic acid in TET2 mutant clonal cytopenia. Eight of 10 patients were eligible for response assessment, with no responses at week 20 by International Working Group Myelodysplasia Syndromes/Neoplasms criteria. This trial was registered at www.clinicaltrials.gov as #NCT03418038.

Abstract Clonal cytopenia of undetermined significance (CCUS) represents a distinct disease entity characterized by myeloid-related somatic mutations with a variant allele fraction of ≥2% in individuals with unexplained cytopenia(s) but without a myeloid neoplasm (MN). Notably, CCUS carries a risk of progressing to MN, particularly in cases featuring high-risk mutations. Understanding CCUS requires dedicated studies to elucidate its risk factors and natural history. Our analysis of 357 patients with CCUS investigated the interplay between clonality, cytopenia, and prognosis. Multivariate analysis identified 3 key adverse prognostic factors: the presence of splicing mutation(s) (score = 2 points), platelet count of <100 × 109/L (score = 2.5), and ≥2 mutations (score = 3). Variable scores were based on the coefficients from the Cox proportional hazards model. This led to the development of the clonal cytopenia risk score (CCRS), which stratified patients into low- (score of <2.5 points), intermediate- (score of 2.5 to <5), and high-risk (score of ≥5) groups. The CCRS effectively predicted 2-year cumulative incidence of MN for low- (6.4%), intermediate- (14.1%), and high-risk (37.2%) groups, respectively, by the Gray test (P < .0001). We further validated the CCRS by applying it to an independent CCUS cohort of 104 patients, demonstrating a c-index of 0.64 (P = .005) in stratifying the cumulative incidence of MN. Our study underscores the importance of integrating clinical and molecular data to assess the risk of CCUS progression, making the CCRS a valuable tool that is practical and easily calculable. These findings are clinically relevant, shaping the management strategies for CCUS and informing future clinical trial designs.

Introduction: MDS are primarily diseases of the elderly, with median age at diagnosis of 72 years. Focused data on clinical and molecular characteristics, as well as outcomes, in younger pts under age 60 years are limited. We conducted this multicenter retrospective study to describe pt characteristics, cytogenetic and molecular profiles, treatment modalities, and outcomes in younger pts with MDS, including adolescent and young adults (AYA). Methods: The study included pts diagnosed with MDS between 2002-2023 at 7 academic centers. We compared baseline characteristics, treatment, and outcomes of AYA (18-39 years) and younger adult (YA) pts (40-59 years). Responses were assessed using the modified IWG 2006 response criteria which included complete remission (CR), marrow CR (mCR), and hematologic improvement (HI). Wilcoxon rank sum test and Fisher's exact (and Chi-square) test were used to compare pt and disease characteristics for continuous and categorical variables, respectively. Overall survival (OS) was estimated using the Kaplan-Meier method and compared using log-rank test. Results: A total of 173 pts were identified (AYA, N=50; YA, N=123). Median age at diagnosis was 50 years (range, 18-59), with 51% females. Baseline characteristics are shown in Table 1. Therapy-related MDS (t-MDS) was observed in 40 pts (23%) with the remaining classified as de novo MDS (N=133; 77%). Inherited bone marrow failure syndromes were documented in 15 pts (9%), with Shwachman-Diamond Syndrome (N=4) and Fanconi anemia (N=4) being the most frequent. Using the WHO 2016 classification, the most common subtypes of MDS in both the AYA and YA cohorts were MDS with multilineage dysplasia (38% vs 19%), MDS with excess blasts-2 (MDS-EB) (24% vs 29%), and MDS-EB-1 (16% vs 20%). Similarly, using the WHO 2022 classification, the most common subtypes were MDS with low blasts (50% vs 21%), MDS with increased blasts-2 (MDS-IB-2) (24% vs 24%), and MDS-IB-1 (16% vs 18%). Stratifying pts using the IPSS-R cytogenetic (CG) risk group, the most common CG risk category identified was good CG (34% vs 52%, p=0.012). Poor/very poor CG were present in 34% and 37% of the AYA and YA groups, respectively. STAG2 and NRAS mutations occurred more frequently in the AYA pts compared to the YA group, while the YA cohort was enriched for the following mutations: TP53, DNMT3A, SF3B1, SRSF2, and TET2 (Figure 1). In the AYA population, 18 pts (38%) had very low/low, 10 (21%) intermediate, and 20 pts (42%) had high/very high risk MDS using IPSS-R. In the YA population, 32 pts (26%) had very low/low, 32 (26%) intermediate, and 57 pts (47%) had high/very high risk MDS using IPSS-R. Twenty-seven percent of pts with very low to intermediate-risk MDS using IPSS-R were upstaged to moderate high to very high-risk MDS using IPSS-M. The majority of pts (AYA: 75%; YA: 63%, p=0.5) were transfusion independent at baseline. Single-agent hypomethylating agents were the most frequently used frontline therapy, used in 17 AYA pts (34%) and 58 YA pts (48%), with more AYA pts receiving upfront allogeneic stem cell transplantation (Allo-SCT) compared to YA pts (28% vs 5%). After frontline therapy (Table 1), an additional 14 AYA (28%) and 52 YA (42%) pts received Allo-SCT. In pts with IPSS-R intermediate to very high-risk, response rates (HI+CR+mCR+mCR with HI) to frontline therapies were 46% and 54.5% in the AYA and YA groups, respectively. After median follow-up of 24 months [range, 1-218], median OS was numerically longer in the AYA versus YA group (155 vs 53 months, p=0.2). AML transformation occurred in 24% and 20% of the pts in the AYA and YA groups, respectively. Median OS was significantly longer in de novo compared to t-MDS pts (56 vs 27 months, p=0.03). Median OS among Allo-SCT compared to non-SCT pts were not reached vs 155 months ( p=0.9) in the AYA group and 53 vs 54 months ( p=0.4) in the YA group. IPSS-R (very low+low vs intermediate vs high+very high) was able to distinguish differences in OS in the YA (133 vs 53 vs 17 months, p=0.0002) and AYA (155 vs not reached vs 18 months, p=0.006) pts, with the exception of the intermediate-risk group in the AYA cohort. Conclusions: In this study, d e novoMDS was seen in the majority of younger pts with MDS (both AYA and YA). Mutations in STAG2 and NRAS were more common in the AYA pts, while YA pts had MDS more enriched for TP53, DNMT3A, TET2 and splicing mutations. This study is still ongoing with a plan to compare the abovementioned groups to older pts (≥60 years) with MDS.

Background: Clonal cytopenia(s) of undetermined significance ( CCUS) is defined as persistent cytopenias arising in the context of myeloid neoplasm ( MN)-associated somatic mutations ( MT) in hematopoietic stem cells. Patients ( PTs) with TET2MT CCUS have a high probability of progression to MN. To date, no FDA-approved therapies exist for CCUS, and PTs often have similar cytopenias/transfusion needs as those with myelodysplastic syndromes ( MDS). TET2 is an ascorbic acid (AA)-dependent dioxygenase that catalyzes the conversion of 5-methylcytosine ( 5mC) to 5-hydroxymethylcytosine ( 5hmC), providing rationale to study high-dose IV (HI)-AA in TET2MT CCUS. Here we report the final results of this pilot trial. Methods: This is an investigator-initiated, single-institutional, phase II trial assessing the safety and efficacy of HI-AA in PTs with TET2MT CCUS (NCT03418038). PTs≥ 18 years with ≥1 TET2MT with or without additional somatic MTs, without prior treatment, and with any of the following laboratory criteria: (1) hemoglobin ( Hb) ≤10g/dL, (2) absolute neutrophil count ( ANC) ≤1(10^9/L), (3) platelet count ( PLT)≤100 (10^9/L) were eligible. HI-AA (1g/kg, maximum 100g) was given 3 times weekly for 12 weeks. The primary endpoint was hematologic response rates determined by MDS IWG 2018 criteria at week 20. (Platzbecker Blood 2018) and reported as hematologic improvement ( HI)- erythropoietic ( HI-E), platelets ( HI-P), neutrophils ( HI-N). Secondary endpoints include safety and adverse events ( AEs), graded by NCI-CTCAE v4.03. Correlative studies include changes in TET2MT variant allele fraction ( VAF), in vitro colony formation and differentiation, global and sequence-specific DNA methylation/hydroxymethylation, and quantitative 5hmC estimated by IHC in bone marrow biopsy specimens. Results: Ten patients were enrolled, with a median age of 71.4 (range: 65, 79) years, 8 (80%) males. The median number of mutations was 3, with 9 (90%) having co-mutations. All PTs but one [-Y] had normal karyotype. Baseline Hb levels were normal in all but one who was red blood transfusion dependent. Five (50%) PTs were thrombocytopenic, and 5 (50%) PTs had ANC<1 (10^9/L). ( Table). HI-AA was well tolerated with the most common AEs being infusion-related polyuria (40%) and polydipsia (40%, 3 Grade 1, 1 Grade 2). One (10%) PT had constipation and headaches (both Grade 1), and 1 (10%) experienced dyspepsia. No treatment-related Grade 3 or 4 AEs' or deaths were reported. The median follow-up duration was 16.8 months (range: 9.4, 24.1). Overall, no PTs met the criteria for HI. There were no significant differences with regards to median Hb, PLT, and ANC values at baseline, week 20, and 1-year (median Hb: 13.3, 12.5, and 12.7 g/dL, p=0.93; PLT: 97 vs. 120 vs. 92.5X10^9/L, p>0.99, and ANC: 0.9 vs. 1.2 vs. 1.3X10^9/L, p=0.99). By CTCAE grading criteria for cytopenias, 3 (30%) PTs had an improvement in severity of cytopenias, while 2 had worsening. One (10%) PT had an improvement in PLT (baseline vs. week 20: 62 vs 192X10^9/L) but was morphologically deemed to meet criteria for CMML-1 at week 20. Four (40%) PTs met criteria for disease progression (PT_4, 7,8,10); CMML-1- 2 and MDS-2. The median time to progression was 5 months (95% CI: 4.9, unreached). Two PTs had mutational clonal evolution, with 1 (PT_8) acquiring a RUNX1 (VAF: 20%) and 1 (PT_9) acquiring 2 CBL mutations (both VAF:4%) at one-year follow-up. There were no significant differences regarding the clinical characteristics or the TET2 variants between PTs who had stable disease vs. progression. There were no significant differences in TET2 VAF at baseline, week 20, and 1-year (median: 39% vs. 43% vs. 42%, p=0.54). We profiled changes in 5-mC and 5-hmC (Ilumina EpicArray) in PTs pre- and post-IV AA treatment and did not observe global changes. However, we were able to highlight site-specific changes in differentially methylated regions, largely resulting in hypomethylation at enhancer sites and affecting actively transcribed states in PTs with an improvement in cytopenia(s) severity by CTACE criteria ( Figure). Conclusion: We report the final safety and efficacy analysis of HI-AA in PTs with TET2 MT CCUS. The trial data suggest that HI-AA is safe and well tolerated. Although there were no significant responses by IWG MDS criteria, alleviation in severity of cytopenias in a subset of PTs (n=3) along with correlative epigenetic changes in enhancer regions, needs further exploration.

Background. The inclusion of gene mutations and chromosomal abnormalities in the 2022 WHO and ICC Classifications of MDS has enhanced diagnostic precision and is expected to improve clinical decision-making process. Although these two systems share similarities, clinically relevant discrepancies still exist and potentially cause inconsistency in their adoption in a clinical setting. In this study on behalf of the International Consortium for MDS (icMDS), we adopted a data-driven approach to provide a harmonization roadmap between the 2022 WHO and ICC classification for MDS. A modified Delphi Process consensus approach is currently ongoing among icMDS experts to finalize a harmonized MDS classification scheme. Methods. We analyzed retrospective international cohorts of patients with a diagnosis of MDS (n=7017) and AML (n=1002) according to WHO 2016 criteria. Hierarchical Dirichlet Processes were applied to define clusters capturing broad dependencies among all gene mutations and cytogenetic abnormalities. To investigate the features of importance and their impact on the clustering process, we employed the SHapley Additive exPlanations approach (SHAP). This allowed to define harmonized labels for each clinical entity. The clinical relevance of the unsupervised clustering was assessed through the analysis of phenotypic features and outcomes among each group. ( Blood 2022;140: 9828-9830) Results. Patients' characteristics are summarized in Table 1. We identified 9 clusters, defined by specific genomic features. The cluster of highest hierarchical importance was characterized by biallelic inactivation of TP53 (biTP53). According to SHAP analysis, bi TP53 was defined as 2 or more TP53 mutations, or 1 mutation with copy number loss or cnLOH. Most patients assigned to bi TP53 cluster had TP53 VAF>10% (77.9%) and complex karyotype (70.1%). Assignment to bi TP53 cluster was irrespective of blast count. Patients with monoallelic TP53 mutation segregated into other clusters. Hierarchically, the second cluster included patients with del(5q). SHAP analysis highlighted 5q deletion alone, or with one other chromosomal abnormality other than -7, and absence of bi TP53, as the most relevant features. Most of these patients had blast counts <5% (88.1%). The third distinct cluster included patients with SF3B1 mutations (in the absence of concurrent del(7q), abn3q26.2, complex karyotype or RUNX1 mutation). Most patients with MDS and SF3B1 mutation had <5% blasts (94.2%). Common co-mutated variants in the SF3B1 cluster included mutant DNMT3A (25.2%) and TET2 (38.3%). Morphologically defined MDS cases (i.e., not meeting criteria for bi TP53, del(5q) or SF3B1) were preferentially assigned to the following additional clusters: SF3B1 and concurrent higher-risk mutations (e.g., RUNX1 and ASXL1); SRSF2 and concomitant TET2 mutations; U2AF1 mutations with del(20q), del(7q) or -7; SRSF2 with TET2 mutations and co-mutational patterns including RUNX1 and ASXL1; and AML-like genomic signatures. Our analyses suggest that morphologically defined MDS is characterized by a large heterogeneity in terms of mutation profiles, not entirely captured by the presence of unilineage versus multilineage dysplasia, percentage of bone marrow blasts, and presence of hypocellularity and fibrosis. To better investigate the continuum between high risk MDS (i.e., patients with ≥10% blasts) and AML, an exploratory comparison was made using a cohort of AML (defined according to WHO 2016) patients analyzed using the same statistical methods. Only a partial overlap in genetic signatures was observed between MDS with ≥10% blasts and AML. However, similarities were observed between the AML-like MDS clusters (characterized by mutant NPM1, bZIP CEBPA, and Core Binding Factor abnormalities) and AML clusters defined by the same genetic signature, thus supporting the classification of these entities as AML, irrespective of blast count. Conclusion. Our study demonstrated the utility of a data-driven approach based on advanced statistical methods to generate a harmonized classification for MDS. Table 2 shows a provisional, hierarchical classification algorithm. Further refinement of entity labels and classification criteria is the subject of the ongoing modified Delphi Process consensus approach among icMDS experts.

VEXAS is caused by somatic mutations in UBA1 (UBA1mut) and characterized by heterogenous systemic auto-inflammation and progressive hematologic manifestations, meeting criteria for myelodysplastic syndrome (MDS) and plasma cell dyscrasias. The landscape of myeloid-related gene mutations leading to typical clonal hematopoiesis (CH) in these patients is unknown. Retrospectively, we screened 80 VEXAS patients for CH in their peripheral blood (PB) and correlated findings with clinical outcomes in 77. UBA1mutwere most common at hotspot p.M41 (median variant allele frequency/VAF = 75%). Typical CH mutations co-occurred with UBA1mut in 60% of patients, mostly in DNMT3A and TET2, and were not associated with inflammatory or hematologic manifestations. In prospective single-cell proteogenomic sequencing (scDNA), UBA1mutwas the dominant clone, present mostly in branched clonal trajectories. Based on integrated bulk and scDNA analyses, clonality in VEXAS followed two major patterns: with either typical CH preceding UBA1mutselection in a clone (Pattern 1), or occurring as an UBA1mutsubclone or in independent clones (Pattern 2). VAF in PB differed markedly between DNMT3A and TET2 clones (median VAF of 25% vs 1%). DNMT3A and TET2 clones associated with hierarchies representing patterns 1 and 2, respectively. Overall survival for all patients was 60% at 10 years. Transfusion-dependent anemia, moderate thrombocytopenia, and typical CH mutations, each correlated with poor outcome. In VEXAS, UBA1mut cells are the primary cause of systemic inflammation and marrow failure, being a new molecularly defined somatic entity associated with MDS. VEXAS-associated MDS is distinct from classical MDS in its presentation and clinical course.

Background: Clonal hematopoiesis (CH) of indeterminate potential (CHIP) is a risk factor for cardiovascular disease. The relationship between CHIP and coronary microvascular dysfunction (CMD) is unknown. The current study examines the association between CHIP and CH with CMD and the potential relationships in risk for adverse cardiovascular outcomes. Methods: In this retrospective observational study, targeted next-generation sequencing was performed for 177 participants with no coronary artery disease who presented with chest pain and underwent routine coronary functional angiogram. Patients with somatic mutations in leukemia-associated driver genes in hematopoietic stem and progenitor cells were examined; CHIP was considered at a variant allele fraction ≥2%; CH was considered at a variant allele fraction ≥1%. CMD was defined as coronary flow reserve to intracoronary adenosine of ≤2. Major adverse cardiovascular events considered were myocardial infarction, coronary revascularization, or stroke. Results: A total of 177 participants were examined. Mean follow-up was 12±7 years. A total of 17 patients had CHIP and 28 had CH. Cases with CMD (n=19) were compared with controls with no CMD (n=158). Cases were 56±9 years, were 68% women, and had more CHIP (27%; P =0.028) and CH (42%; P =0.001) than controls. CMD was associated with independent risk for major adverse cardiovascular events (hazard ratio, 3.89 [95% CI, 1.21–12.56]; P =0.023), and 32% of this risk was mediated by CH. The risk mediated by CH was ≈0.5× as large as the direct effect of CMD on major adverse cardiovascular events. Conclusions: In humans, we observe patients with CMD are more likely to have CHIP, and nearly one-third of major adverse cardiovascular events in CMD are mediated by CH.