Komrokji, Rami S.

Abstract Background The expansion of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow is universally associated with adverse outcomes and disease progression in all chronic myeloid neoplasms, to include chronic myelomonocytic leukemia (CMML). However, HSPCs are a heterogeneous cellular compartment and the impact of its composition on clinical outcomes remains poorly defined. Single cell RNA-sequencing (scRNA-seq) represents an optimal strategy to annotate cellular composition but no large clinically annotated scRNA-seq datasets are currently available. To address this, we have embarked to generate a clinically annotated 500 patient CMML scRNA-seq atlas from CD34+ enriched BMNCs to establish among the largest clinically and genetically annotated single cell references for any chronic myeloid malignancy. Here we report the interim analysis of 113 clinically annotated cases that demonstrates massive reshaping of HSPC composition that is independently a predictor of overall survival. Methods Single-cell RNA expression profiling was performed on 113 CD34-enriched CMML BMNC samples using 10x Genomics GEM-X Universal 3' Gene Expression v4 (4-plex) chemistry. Libraries were sequenced on NovaSeq S4 flow cells in two runs, and data were processed using Cell Ranger to generate gene expression matrices. A total of 502,659 single cells from 113 samples were processed using Seurat. The upper threshold for gene count was defined as the mean number of detected genes plus two standard deviations data normalization was performed using the log-normalization method. Batch correction and integration were conducted with Harmony to mitigate technical variation across pools. Descriptive statistics were used to tabulate clinical and genetic parameters. KM curves, log-rank test, and cox regression were used to calculate survival, test for statistical differences, and perform multivariate analysis, respectively. Results To derive high resolution cell annotations, we employed the SingleR algorithm using a large hematopoietic reference of over 300,000 single cells (Zhang, X. Nature Immunology, 2024). Using this approach a diverse set of cell types were identified that included HSCs, 23 myeloid progenitors, 18 erythroid progenitors, and 18 lymphoid progenitors. This diverse cellular repertoire displayed massive reshaping relative to the normal CD34 enriched reference including a >53x expansion of multilineage GMPs and a >100x depletion of B-cell progenitors consistent with the known myeloid skewing seen in this disease. To understand whether expansion of specific progenitors was associated with outcomes we iteratively evaluated key progenitor populations and observed that expansions of HSC (HR 0.468 p<0.005) and MEP (HR 0.485, p<0.005) were associated with improved survival while GMP (HR 1.729, p=0.029) expansions were associated with inferior survival when using a cut point derived with a maximally selected ranks statistics approach. To achieve a more comprehensive metric of HSPC composition we explored GMP ratios to include GMP/HSC ratios and GMP/MEP ratios and observed that both were associated with inferior survival in univariate and multivariate when considering clinical and genetic features such as the CPSS-molecular prognostic score and FAB classification. When considering both GMP/HSC and GMP/MEP ratios in a multivariate model only GMP/MEP ratios and CPSS molecular remained independently prognostic. As an orthogonal approach, pseudotime projections were carried out using a reference map from Setty et al., 2019 to calculate differentiation potential and branch probabilities. This approach enabled both cell identity assignment and placement of cells along the hematopoietic differentiation trajectory. Samples were classified as monocyte-biased, MEP-biased, or normal-like, as previously described (Ferrall-Fairbanks et al. 2022) and demonstrated that increased GMP ratios are associated with a monocytic biased trajectory and that this trajectory is also associated with overall survival. Conclusion This study represents an interim analysis of among the largest scRNA-seq cohorts generated in chronic myeloid malignancies. This enabled us, for the first time, to demonstrate that HSPC composition is independently prognostic even when considering modern CMML prognostic models. As this cohort continues to increase, validation of these findings and examining the impact of therapy on HPSC composition in serial samples will be prioritized.

Abstract Introduction Defining the epidemiology of myelodysplastic syndromes/neoplasms (MDS) and precursor conditions, such as clonal cytopenia of undetermined significance (CCUS), is challenging due to inconsistent reporting, complex diagnostics, and evolving criteria. The National MDS Natural History Study (NHS) overcomes these challenges through central pathology review, prospective follow-up, and a nationally representative cohort enrolled at the time of suspected MDS, enabling robust analysis across the myeloid disease spectrum. We analyzed clinical, genetic, and pathologic features in NHS participants by race, ethnicity, and sex to characterize disease variation across populations. Methods The NHS (NCT02775383) enrolled individuals undergoing evaluation for suspected MDS from June 2016–July 2024 at 102 US sites. Participants underwent bone marrow assessment with centralized, adjudicated histopathology review. Those meeting WHO 2016 criteria for MDS, MDS/myeloproliferative neoplasm (MPN), CCUS, idiopathic cytopenia/dysplasia of undetermined significance (ICUS/IDUS), or oligoblastic AML were followed longitudinally. Demographics, comorbidities, exposures, and clinical data were collected at baseline and every 6 months. Deep DNA sequencing was performed on baseline marrow, with pathogenic variants manually curated (53 genes). Statistical analyses assessed associations using chi-squared, Fisher's exact, Kruskal-Wallis, logistic regression, and Cox models. A p-value <0.05 was considered significant. Results Among 2,021 participants, 1,174 (58%) were diagnosed with MDS or precursor conditions. Median age was 74 years (yrs) and 66% were male. Most identified as Non-Hispanic White (NHW, 92%), with 4% Black and 4% Hispanic. The most common diagnoses were MDS (42%) and CCUS (37%). Pathogenic somatic mutations were identified in 84% (774/918) of MDS/CCUS, with TET2 (26% of 918), DNMT3A (21%), and ASXL1 (16%) most frequent. Median follow-up was 1.8yrs and consistent across race, ethnicity, and sex (p≥0.6). Black, compared to NHW, participants were younger (69 vs 74 yrs, p=0.006) and trended toward more females (47% vs 34%, p=0.09) and more MDS diagnoses (51% vs 41%, p=0.07). Black participants showed more advanced disease (e.g., lower hemoglobin, higher peripheral blood blasts, higher variant allele frequencies (VAFs) of pathogenic mutations), yet received less MDS-directed treatment (14% vs 41%, p=0.01). Black, compared to NHW, participants had more mutations in RUNX1, TP53, BCOR, and DNMT3A, but fewer in TET2 and ASXL1. Hispanic, compared to Non-Hispanic, participants had fewer known pathogenic mutations, leading to more ICUS (19% vs 5%, p=0.02). Females, compared to males, were younger at diagnosis (72 vs 75yrs, p<0.001), had more ICUS/IDUS (20% vs 10%, p<0.001), fewer pathogenic mutations, especially in splicing/chromatin modifier genes (U2AF1, SRSF2, ASXL1, ZRSR2), more DNMT3A mutations, and lower VAFs (0.27 vs 0.33, p<0.001). Median overall survival (OS) was 6.4yrs, and shorter in MDS (4.2yrs) than CCUS (not reached). Worse OS was associated with age (adjusted hazard ratio (aHR) 1.06), MDS (vs CCUS; aHR 1.89), higher-risk disease by IPSS-R/CCRS (aHR 2.15), and TP53 mutations (aHR 2.17) (all p<0.001). A trend toward improved OS in Black participants was attenuated after adjustment (aHR 0.79, p=0.6). Progression (e.g., worsening of disease, increase in transfusion dependency, death) occurred in 53% of participants. Worse progression-free survival (PFS) was associated with MDS diagnosis (aHR 2.59), higher-risk disease by IPSS-R/CCRS (aHR 2.17), and TP53 mutations (aHR 1.86) (all p<0.001). Despite more advanced disease and less MDS therapy, Black participants trended toward improved PFS (aHR 0.62, p=0.14), while age was borderline significant (aHR 1.01, p=0.05). Conclusions For the first time, we show that among individuals evaluated for suspected MDS at sites across the US, 58% had MDS or precursor conditions. Differences across demographics underscore the heterogeneity of MDS. Black participants presented with more advanced MDS, received less treatment, but trended toward improved outcomes, particularly PFS, even after adjustment, suggesting biological rather than access-to-care differences driving disparities. These findings highlight the complex interplay of sex, race, and genetics in shaping MDS outcomes and reinforce the need for diverse, large-scale studies to improve risk stratification and personalized care.

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

Abstract Introduction TP53MT are associated with poor outcomes in MDS, especially in the context of biallelic MT. We have previously reported on outcomes of pts with HR-MDS with TP53MT who received HMA in the VALIDATE dataset (Kewan T et al, ASH 2023). Here we provide expanded analyses focusing on outcomes post allo-HCT, and we explore the prognostic significance of different classification methods for biallelic TP53MT. Methods The VALIDATE database includes 4,732 pts with MDS who received HMA-based frontline therapy from 32 international centers in 9 countries. For this study, only pts with HR-MDS with molecular data at time of diagnosis were included. Biallelic TP53MT status was assessed using three classification schemes: WHO 2022, ICC 2022, and a VAF-based Cleveland Clinic Foundation (CCF) method.1TP53 copy neutral loss of heterozygosity (CN-LOH) was evaluable in 386 pts (118 had TP53MT). Treatment responses were evaluated using IWG 2023 criteria (Zeidan A et al, Blood 2023). Composite complete remission (cCR) was defined as CR+CRbi+CRuni+CRh+CRequivalent. We calculated OS from time of HMA initiation to date of death or last follow up. Unsupervised clustering (MOSAIC framework) was applied to identify molecular subgroups within the TP53MT cohort. The study was funded by an independent grant from Abbvie. Results A total of 1,485 pts were included. Median age was 69 years (IQR: 62–76), with 66% male pts. Median bone marrow blast was 7%, and 28% had a complex karyotype. Treatment regimens included azacitidine (68%), decitabine (15%), HMA/Venetoclax (8%), and other HMA combinations (9%); 34% underwent allo-HCT. Overall, 392 (26%) had TP53MT with 430 TP53 mutations (missense: 316; truncating: 89). Biallelic TP53MT % varied by classification: 72% (WHO), 84% (ICC), and 85% (CCF). Compared to TP53 wild-type (TP53WT) pts, pts with TP53MT had worse OS (median: 11.5 vs. 23.9 months [mo], p< 0.001), were younger (68 vs. 70 years, p=0.039), and had lower counts at diagnosis. Complex karyotype (81% vs. 14%) and chromosomal abnormalities were more frequent in TP53MT pts. Pts with TP53MT also had fewer co-mutations in other genes. Response to HMA was evaluable in 1,094 pts: cCR and CR were 32% and 13%, respectively. TP53MT pts had higher cCR (39% vs. 29%, p=0.001) than TP53WT, but similar CR rates (16% vs. 12%, p=0.093). According to WHO 2022, biallelic TP53MT had higher CR rates than monoallelic TP53MT (18% vs. 8%, p=0.03), but similar cCR. ICC and CCF classifications did not differentiate response rates. Median OS did not differ by TP53MT allelic status using WHO or ICC definitions. However, using the CCF method, pts with biallelic TP53MT had shorter OS than monoallelic TP53MT (10.4 vs. 17.2 mo, p=0.038). In multivariable models adjusted for age, gender, and allo-HCT, only the CCF-defined biallelic TP53MT group had significantly worse OS (HR: 1.6; 95% CI: 1.1–2.4). TP53MT pts undergoing allo-HCT had improved OS regardless of allelic status. Median OS for TP53MT pts with allo-HCT was 18.9 vs. 9.2 mo without all-HCT. This benefit was observed across WHO, ICC, and CCF classifications. However, a TP53MT VAF ≥19% predicted worse OS even post allo-HCT (14.6 vs. 47.1 mo, p<0.001). At 1 year, the survival rate was 78% (95%CI: 69-89%) in TP53MT pts who underwent allo-HCT and had cCR vs. 68% (95%CI: 55-84%) in pts who did not have cCR. However, 1 year survival rate was significantly higher for pts with TP53MT who did not have allo-HCT and achieved cCR (60%, 95%CI: 48-76%) vs. no cCR (26%, 95%CI: 20-35%). Unsupervised clustering of 335 TP53MT pts identified 9 molecular clusters (MCs). MC2, characterized by low TP53MT VAF (median 17%), fewer co-mutations, and frequent del17p, was associated with significantly improved OS (17.9 mo) and predicted favorable outcome (HR for death: 0.6; 95% CI: 0.4–0.8). Other clusters were enriched for adverse features (e.g., high VAF, multi-hit TP53MT).Conclusions The CCF method appears to best classify biallelic TP53MT pts with worse survival in the absence of accurate assessment of LOH status. Treatment response per IWG 2023 for TP53MT pts was associated with OS only in non-transplanted pts. Allo-HCT improved survival across TP53MT subsets, but TP53 VAF ≥19% at diagnosis remained a strong negative prognostic factor. Integrative clustering revealed a less aggresive molecular subset among TP53MT pts, suggesting refined risk stratification may guide therapy in this high-risk population. Ref: 1.Bahaj et al. J Hematol Oncol 2023.

Abstract Background: Mutations in ethanolamine kinase-1 (ETNK1) have been identified in a wide range of myeloid neoplasms (MN), but their clinical significance remains poorly defined. Key gaps remain, including the true prevalence, variant- and disease-specific co-mutation patterns, cytogenetic patterns, and prognostic impact, highlighting the need for broader multi-institutional studies to refine our understanding and risk stratification. We present findings from an ongoing multi-institutional study of ETNK1-mutated (ETNK1mut) MN. Methods We analyzed 106 patients with ETNK1mut MN across the Mayo Clinic Comprehensive Cancer Center (Minnesota, Arizona, and Florida), H. Lee Moffitt Cancer Center (Tampa, FL), Massachusetts General Hospital (Boston, MA), and University of Milano-Bicocca (Monza, Italy), integrating clinical, hematologic, cytogenetic, and next-generation sequencing (NGS) data. Appropriate IRB approval was obtained. Diagnoses were assigned by the 5th edition of the World Health Organization and 2022 European LeukemiaNet criteria. A clone was considered dominant if it had the highest variant allele frequency (VAF) or VAF within 5% of the highest co-mutation. Overall survival (OS) was assessed by Kaplan-Meier. Comparison cohorts for OS included 83 ETNK1 wild-type (ETNK1wt) myelodysplastic syndrome (MDS) and 415 ETNK1wt acute myeloid leukemia (AML) patients without TP53 mutations. GraphPad Prism v10 was used for statistical analysis. Results Median age at diagnosis was 70 years (range 30.4-90), with a male predominance of 72.6% (n=77). ETNK1mut were identified in MDS (n=47, 44.3%), myelodysplastic/myeloproliferative neoplasms (MDS/MPN; n=23, 21.7%), myeloproliferative neoplasms (MPN; n=13, 12.3%), AML (n=12, 11.3%), clonal cytopenia of undetermined significance (CCUS; n=5, 4.7%), and other entities (n=6, 5.7%) including atypical chronic myeloid leukemia (n=1) and systemic mastocytosis (n=1). Over a median follow-up of 33.6 months (IQR 15-91.8), 49.1% (n=52) patients died and 19.4% (n=18 of 93 at risk) progressed to AML. Cytogenetics were available in 103 patients; 57.3% (n=59) had a normal karyotype, while 30.1% (n=31) had abnormal and 12.6% (n=13) had complex karyotypes. None harbored classical MDS-associated deletions such as del(5q), del(7q), or del(20q). The most frequent cytogenetic abnormalities included translocations (n=17), monosomy 7 (n=9), and trisomy 8 (n=8). Of 17 translocations, 8 (47%) involved chromosome 1, two of which (11.8%) involved der(1;7)(q10;p10). The most common ETNK1 mutation was N244S (n=70, 66.0%), followed by K378N (n=12, 11.3%) and G245 variants (n=9, 8.5%), including G245V and G245D. VAF data were available for 103 patients; the median ETNK1 VAF was 31.4% (range 2.0-54.8%). High VAF (≥40%) was observed in 27.2% (n=28) of patients. ETNK1 was the dominant mutation in 33% (n=34) of cases, shared equal dominance with a co-mutation in 13% (n=14), and was a subclone in 56% (n=58). Co-mutations were detected in 77% (n=82) of patients, with a median of 4 (range 0-13) co-mutated genes. A third of patients (n=37, 35%) had ≥5 co-mutations. The most frequently co-mutated genes were epigenetic regulators including ASXL1 (n=29, 27%), TET2 (n=17, 16%), EZH2 (n=12, 11%), and DNMT3A (n=12, 11%), followed by splicing factor genes including U2AF1 (n=13, 12%), SRSF2 (n=10, 9%), and ZRSR2 (n=10, 9%). Mutations in signaling genes (JAK2, NRAS, SETBP1) and transcription factors (RUNX1, BCOR) were also observed. High VAFs were seen in co-mutations of EZH2 (median 49%), TET2 (46%), and NRAS (46%). ASXL1 was most enriched in MDS/MPN (n=8, 35%) and MPN (n=3, 23%). Survival analysis showed better survival in ETNK1mut compared to ETNK1wt within AML (65 vs 19 months; p=0.2002) and MDS (107 vs 61 months; p=0.1993), although statistical significance was not reached. Conclusion ETNK1 mut frequently co-occur with mutations in epigenetic and splicing regulators, and demonstrate distinct molecular and cytogenetic features, including the absence of classic MDS-associated deletions and a high prevalence of translocations involving chromosome 1. ETNK1 was the dominant clone in a third of cases with a high co-mutation burden. Overall survival was better in ETNK1mut AML and MDS, but the difference was not statistically significant. These findings highlight the need for larger collaborative studies.

Abstract Introduction: Marrow complete remission (mCR) in 2006 IWG criteria for MDS (Cheson B et al, Blood 2006) reflects achieving bone marrow (BM) blast percentage below 5% but is frequently not associated with meaningful count recovery. While mCR + hematologic improvement (HI) does reflect wide ranges of count improvement or transfusion reductions, HI is difficult to measure accurately in routine clinical practice. The 2023 International Working Group (IWG) response criteria (Zeidan A et al, Blood 2023) eliminated the mCR category without meaningful count recovery and reclassified mCR associated with meaningful count recovery into specific “less-than-CR” categories. Still, many clinical trials continue to use mCR as a secondary efficacy endpoint or as a part of overall response rate (ORR). Within the icMDS VALIDATE database, we assessed the association of mCR with OS in hypomethylating agent (HMA)-treated higher risk (HR)-MDS patients among both (1) transplanted and (2) non-transplanted patients. Methods We included patients with HR-MDS (defined as IPSS ≥1.5 and/or IPSS-R >3.5) who received HMA-based therapies from the international icMDS VALIDATE dataset. We evaluated the best response by IWG 2006 criteria into CR, mCR, or no CR/mCR. We then reclassified patients with mCR per IWG 2023 criteria into either (A) having achieved a composite CR (cCR) defined as CR + CR equivalent (CRequ) + CR with bilineage count recovery (CRbi) + CR with unilineage count recovery (CRuni) + CR with partial hematologic recovery (CRh) or (B) not having achieved cCR (no cCR). OS was calculated from time of HMA initiation to date of death or last follow-up by using 6-month landmark analyses. We used Kaplan-Meier methods to estimate OS, and the Log-rank test to compare OS. VALIDATE is supported by an independent grant from AbbVie. Results A total of1049 patients met eligibility.Median age of patients was 69 (range 21-95), 64% were men. While 61% received azacitidine, 17% received decitabine, and 22% received HMA-based combination therapy. In total, 37% underwent subsequent allogenic stem cell transplantation (allo-HCT). Based on IWG 2006 criteria, 17%, 33%, and 50% achieved CR, mCR, and no CR/mCR as their best response, respectively. Patients achieving CR had superior median OS (25 months, 95% CI: 21-34 months) compared to those with mCR (16 months, 95% CI: 13-18 months; p=0.038) and no CR/mCR (15 months, 95% CI: 12-17 months; p=0.001). In patients who received a subsequent allo-HCT, the median OS for patients with mCR (30 months, 95% CI: 21-54 months; 1 year OS: 71%, 2 year OS: 55%) did not significantly differ compared to patients with CR (59 months, 95% CI: 25 months-not reached, p=0.80; 1 year OS: 73%, 2 year OS 62%) and patients with no CR/mCR (32 months, 95% CI: 21-45 months, p=0.46; 1 year OS: 69%, 2 year OS 54%). In contrast, in patients treated with HMA therapy alone without subsequent allo-HCT, median OS for patients with mCR (11 months, 95% CI: 9-15 months; 1 year OS: 46%, 2 year OS: 23%) did not significantly differ compared to patients with no CR/mCR (11 months, 95% CI: 9-14 months, p=0.85; 1 year OS: 47%, 2 year OS: 22%) but was significantly shorter compared to patients with CR (20 months, 95% CI: 17-28 months, p<0.001; 1 year OS: 69%, 2 year OS: 46%). Patients with mCR by IWG 2006 (N=347) were reclassified per IWG 2023 into having either achieved a cCR (N=219, 63%) or no cCR (N=128, 37%). Specifically, patients with mCR were reclassified per IWG 2023 into CR (4.9%), CRequ (0.6%), CRbi (26.8%), CRuni (29.4%), CRh (1.4%), PR (0%), HI (10%), no response (21.9%) and PD (4.9%). Patients with mCR who were reclassified into cCR had an improved median OS (16 months, 95% CI: 15-26 months; 1 year OS: 62%; 2 year OS: 43%) compared to patients reclassified into no cCR per IWG 2023 (13 months, 95% CI: 11-17 months, p=0.023; 1 year OS: 52%; 2 year OS: 31%). Conclusions Using the large icMDS VALIDATE dataset, we found that achievement of mCR by 2006 IWG criteria was not associated with improved OS compared to patients who did not achieve CR/mCR. Reclassification of mCR using IWG 2023 highlights the heterogeneity within the mCR group and suggests that IWG 2023 criteria stratifies outcomes in patients previously labeled as mCR by IWG 2006. IWG 2023 should be the standard used for ORR assessment in all future clinical trials involving patients with HR-MDS.

Abstract Background and Significance: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy with overlapping dysplastic and proliferative features, a high risk of leukemic transformation, and no disease-modifying therapies. Patients with proliferative CMML often present with symptomatic monocytosis, leukocytosis, splenomegaly, and systemic inflammation, which contribute to shortened survival and impaired quality of life. Hydroxyurea (HU), the standard cytoreductive agent, provides transient control of leukocytosis but has no known disease modifying properties. In the phase 3 DACOTA trial (Itzykson et al., JCO 2022), decitabine modestly delayed leukemic transformation compared to HU but did not improve event-free or overall survival and was associated with increased early mortality. HU achieved a clinical benefit rate (CBR) of approximately 15% (excluding stable disease), highlighting the urgent need for safer and more effective therapies. Pacritinib is a potent and selective multikinase inhibitor with a kinome profile that targets key mediators of clonal proliferation and inflammatory signaling relevant to CMML. It inhibits JAK2 (IC₅₀ = 6 nM) and IRAK1 (IC₅₀ = 13.6 nM)—critical drivers of cytokine-mediated growth and monocytic skewing—while sparing JAK1, potentially reducing myelosuppressive toxicity. Pacritinib also inhibits CSF1R (IC₅₀= 39.5 nM) which is involved in monocytic differentiation. Preclinical models demonstrate that dual inhibition of JAK2 and IRAK1 suppresses CMML colony formation and prolongs survival in xenografts. Furthermore, CSF1R inhibition may reduce monocyte-lineage bias in leukemic clones. Clinically, pacritinib is FDA-approved for intermediate- or high-risk myelofibrosis with severe thrombocytopenia (platelets <50 × 10⁹/L) based on evidence of significant spleen volume and symptom reduction with limited hematologic toxicity. These mechanistic and clinical data provide a strong rationale for evaluating pacritinib in advanced proliferative CMML, a setting characterized by both cytokine-driven proliferation and treatment-limiting cytopenias. Study Design and Methods PROSPERA (NCT07033598) is a collaborative multicenter, open-label, randomized phase 2 trial. Sixty-six patients with WHO-defined CMML-1 or CMML-2 will be enrolled. Eligible patients must have proliferative features (WBC ≥13 × 10⁹/L) and advanced disease as defined by ≥5 cm palpable splenomegaly, symptom burden (Myeloproliferative Neoplasm-Symptom Assessment Form Total Symptom Score MPNSAF-TSS ≥20), or thrombocytopenia (platelets <100 × 10⁹/L). Prior JAK inhibitor therapy is not allowed. The trial will be conducted at approximately six sites across the United States and United Kingdom, with enrollment anticipated to begin by the end of 2025. Participants will be randomized 2:1 to receive either pacritinib (200 mg orally twice daily) or HU (dosed as per investigator discretion) for 48 weeks. Patients in the HU arm may cross over to pacritinib at Week 24 if, in the opinion of the treating physician, they are not deriving clinical benefit (after assessment of the primary endpoint). Randomization will be stratified by prior cytoreductive therapy (none vs. prior HU or hypomethylating agents). The primary endpoint is CBR at Week 24, defined by modified IWG MDS/MPN criteria incorporating hematologic improvement, spleen volume reduction, and symptom response. Secondary endpoints include CBR at any time, duration of response, event-free survival, leukemia-free survival, and overall survival. Correlative studies will assess treatment-related changes in clonal dynamics, cytokine signaling, and hematopoietic composition using longitudinal peripheral blood and bone marrow sampling. Single-cell transcriptomic and immunophenotypic profiling will be employed to characterize pacritinib's impact on both the malignant clone and the surrounding immune microenvironment.

Abstract Background Myelodysplastic syndromes (MDS) develop from the acquisition of somatic mutations over years or decades. While some mutations are random stochastic events, others derive from extrinsic agents that promote genomic instability, including environmental exposures. Agent Orange (AO) is a phenoxy herbicide used by the U.S. military as a defoliant during the Vietnam War (1962-71), often diluted with gasoline to promote aerosolization, contaminated with the carcinogen dioxin during the manufacturing process, and linked to various cancers. We studied AO as a risk factor for MDS. Methods The prospective National Heart, Lung, and Blood Institute National MDS Natural History Study includes patients (pts) with suspected or recently diagnosed MDS enrolled from 6/2016-6/2024. Bone marrow samples underwent central pathology review, resulting in diagnoses including MDS, MDS/myeloproliferative neoplasm overlap (MDS/MPN), or clonal cytopenia of undetermined significance (CCUS). Pts completed detailed assessment for AO exposure and other risk factors for MDS development. Associations between AO exposure, mutation prevalence, and key covariates (demographics, diagnosis, military service, clinical history, cytogenetics and smoking) were assessed using univariate tests of association, multivariable regression and Cox regression models for survival and progression. Results Among 2115 enrolled pts, 1191 (56%) had a myeloid malignancy or precursor condition and 130 (6.1%) reported AO exposure, the majority (96%) male. Compared to unexposed pts, AO exposed pts were more likely to derive from the 1945-54 birth cohort (p<.001), of age to serve in Vietnam, and to have an ultimate diagnosis of MDS or CCUS vs. a non-clonal diagnosis (57% vs. 44%, p=.046). AO exposure was more common among Blacks (12% vs 6%, p=.02), specifically Black males (21% vs 9%, p=.006). AO exposure was marginally associated with International Prognostic Scoring System (IPSS)-R poor-risk cytogenetics (p=.066) and lower baseline platelet count (p=.072). In multiple regression analyses, risk of AO exposure was significantly higher for the 1945-1954 birth cohort (OR [95% CI]=10.3 [4.4-30.6], p<.001), among those with military service (OR=24.1 [11.9-55.8], p<.0001), and for Blacks vs Whites (OR=2.6 [1.2-5.5], p=.017). The crude risk of AO exposure trended higher for patients with MDS compared to idiopathic cytopenia of undetermined significance (ICUS; OR=2.1 [0.9-6.0],p=.13), but not in multivariable analysis (OR=1.3 [0.5-4.4, p=.60]). AO exposure trended toward an association with younger age at MDS diagnosis (p=.06) and toward having a prior primary malignancy (49% vs. 41%, p=.09): Compared to non-AO exposed pts, AO exposed pts had higher rates of prior prostate cancer (16% vs. 8%), notable given its documented association with AO exposure. AO exposure was not associated with a history of or intensity of cigarette smoking (p=.67). After accounting for age, diagnosis, sex, and military service, AO exposure was significantly associated with increased numbers of pathogenic variants in 53 genes (OR=1.2 [1.0-1.4], p=.02) and with having Very Poor/Poor risk IPSS-R cytogenetics (OR=3.7 [1.2-11.1], p=.02). AO exposed and non-exposed pts had similar overall survival (HR=1.1 [0.8-1.7], p=.50), but those exposed to AO had a higher risk of disease progression in the first two years following diagnosis (HR =1.9 [1.2-2.8], p=.004). AO exposed pts appeared more likely to have del (5q) (15% vs. 8%) and del (20q) (13% vs. 5%) karyotypic abnormalities, and TP53 (7.7% vs. 4.5%), STAG2 (5.1% vs. 2.8%), and NRAS mutations (3.4% vs. 1.7%), although sample sizes were small. Focusing on gene pathways, AO exposed pts appeared more likely to have abnormalities in RAS signaling (10.3% vs. 5.7%), cell cycle (7.7% vs. 4.5%), and cohesion complex pathways (6.0% vs. 3.1%), but less likely to have abnormalities in cytokine receptor pathways (0% vs. 4.5%). ConclusionsIn our representative national cohort of pts with at least one cytopenia, and for the first time, we demonstrated that AO exposure is associated with younger age at MDS diagnosis, ultimate MDS diagnosis, genetic complexity of MDS, increased risk of disease progression, and with Black race, despite Black individuals comprising just 16% of men who served in the Vietnam War and being less likely to have MDS than white individuals. AO should formally be recognized as a risk factor for the development of MDS.

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

Abstract Myelodysplastic syndromes/neoplasms (MDSs) are heterogeneous stem cell malignancies characterized by poor prognosis and no curative therapies outside of allogeneic hematopoietic stem cell transplantation. Despite some recent approvals by the US Food and Drug Administration, (eg, luspatercept, ivosidenib, decitabine/cedazuridine, and imetelstat), there has been little progress in the development of truly transformative therapies for the treatment of patients with MDS. Challenges to advancing drug development in MDS are multifold but may be grouped into specific categories, including criteria for risk stratification and eligibility, response definitions, time-to-event end points, transfusion end points, functional assessments, and biomarker development. Strategies to address these challenges and optimize future clinical trial design for patients with MDS are presented here.