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Abstract Background Blast transformation (BT) occurs in approximately 15-30% of patients with chronic myelomonocytic leukemia (CMML) and remains a leading cause of death. Allogeneic stem cell transplantation (ASCT) is currently the only treatment modality with the potential to cure the disease or prolong survival. Optimal timing of ASCT is critical for maximizing benefit while minimizing risks. To that end, contemporary risk models have focused on the prognostic relevance of individual, as opposed to concurrent mutations. In the current study, we looked into the possibility of prognostic prominence from concurrent mutations in predicting BT in CMML. Methods CMML diagnostic criteria were according to the International Consensus Classification (Arber et al. Blood 2022; 140:1200). A machine-learning hierarchical clustering algorithm was developed and tailored for patient stratification using survival outcomes and co-expression of genomic alterations. To reduce complexity and improve interpretability, we generalized each cluster using existing rule induction algorithms, including the in Trees framework and JRip. The final output was a set of mutation-based cluster definitions, each representing a distinct patient subgroup with similar survival trajectories. Competing risk analysis and cumulative incidence functions were used in downstream evaluation to validate the clinical distinctiveness of the clusters. For all survival analysis, patients were censored at the time of ASCT. Time-to-BT was calculated from the date of diagnosis to the date of BT or ASCT or last contact. BT-free-survival was calculated from the date of diagnosis to the date of BT, ASCT, death, or last contact. Results The core study cohort included 605 patients from the Mayo Clinic, USA, and the external validation cohort 501 patients from Humanitas Cancer Center, Milan, Italy. Using the patient cohort from the Mayo Clinic, machine-learning algorithms identified five molecular clusters with 3-year blast transformation (BT) rates ranging from 0% to 100% (AUC at 3 years 0.78): the order of molecular signature assignment (probability of BT/death from another cause) was i) PHF6MUT/ASXL1WT (0%/17% at 3 years; N=32), ii) NPM1MUT OR BCORMUT/ASXL1MUT OR SETBP1MUT/NRASMUT (48%/30% at 1 year; N=24), iii) RUNX1MUT/ASXL1MUT OR SRSF2MUT/NRASMUT OR EZH2MUT/ASXL1MUT OR SETBP1MUT OR BCORMUT (31%/55% at 3 years; N=132), iv) ASXL1MUT/TET2MUT OR DNMT3AMUT OR JAK2MUT (24%/28% at 3 years; N=123), and v) all other permutations (10%/40% at 3 years; N=294) [Figure 1]. Additional analysis confirmed significant differences in survival between RUNX1MUT/ASXL1MUT vs. RUNX1MUT/ASXL1WT (p<0.01) OR RUNX1WT/ASXL1WT (p<0.01) OR RUNX1WT/ASXL1MUT (p=0.038) [Figure 2]. Similar patterns of differences in survival were also documented for NRAS/SETBP1, ASXL1/EZH2, and NRAS/SRSF2 mutation combinations (Figure 2). A subsequent Cox regression analysis confirmed independent prognostic contributions from “PHF6MUT/ASXL1WT” (HR 5.43e-10; p<0.01), NPM1MUT (HR 26.7; p<0.01), “SETBP1MUT/NRASMUT” (HR 12.7; p<0.01), BCORMUT(HR 5.8; p<0.01), “RUNX1MUT/ASXL1MUT” (HR 2.3, p<0.01), JAK2MUT (HR 2.1; p<0.01), and “ASXL1MUT/TET2MUT” (HR 1.7; p=0.02). The prognostic relevance of “SETBP1MUT/NRASMUT”, “RUNX1MUT/ASXL1MUT”, NPM1MUT, and BCORMUT was validated in the external cohort from Italy (N=501). In the Mayo Clinic cohort, presence of any of the latter mutations was associated with 1-, 3-, and 5-year BT (death from other cause) rates of 27% (21%), 44% (49%), and 44% (51%), respectively (Figure 3). The corresponding values in the absence of high risk mutations were 7% (15%), 15% (37%), and 18% (52%) [Gray's p value <0.01 for both BT and death from another cause; Figure 3]. Similarly, the 1-, 3-, and 5-year BT (death from another cause) rates in the Italian cohort were 21% (19%), 37% (47%), and 42% (54%) in the presence and 8% (14%), 20% (35%), and 24% (45%) in the absence of high-risk mutations (Gray's p value <0.01 for BT and 0.18 for death from another cause; Figure 4). Conclusions In the current study, machine-learning algorithms enabled the discovery of concurrent mutations in CMML that were shown to be prognostically more significant than their individual constituents. Such prognostic interaction might have contributed to some of the discrepancies noted in current literature regarding the prognostic relevance of certain mutations in CMML and should be accounted for in the development of future risk models.

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 Recent updates to the monocyte count thresholds for diagnosing chronic myelomonocytic leukemia (CMML) have recognized oligomonocytic CMML (OM-CMML) as an early stage within the CMML spectrum. However, the clinical validity of these revised criteria remains uncertain without considering biological factors and might misclassify myelodysplastic syndromes (MDS) with mild monocytosis as CMML. To characterize the genomic landscape of OM-CMML, we evaluated OM-CMML (n=249), MDS (n=304) and myelodysplastic CMML (MD-CMML, n=358) patients (pts) treated at three academic institutions. Targeted next-generation sequencing was performed on bone marrow (BM) at initial diagnosis or first referral. OM-CMML was characterized by lower frequencies of biallelic TET2 (biTET2) and RAS pathway (RASp) gene mutations (BRAF, CBL, KRAS, NF1, NRAS and PTPN11) compared to MD-CMML (p<0.001) but higher frequencies than MDS (p<0.001). BM monocyte frequencies, assessed morphologically, were higher in OM-CMML than in MDS (p<0.001) but lower than in MD-CMML (p<0.001), indicating that OM-CMML is a heterogenous group including true CMML and distinct MDS subsets. Unsupervised hierarchical clustering based on genomic alterations across all cases (n=911) identified three distinct clusters: 1) TP53 alterations (predominantly multihit TP53) and complex karyotype (TP53-complex); 2) biTET2 or TET2-SRSF2 co-mutation (biTET2/SRSF2); 3) a heterogenous group lacking these features (not otherwise specifiable [NOS]). Among OM-CMML pts, 11%, 34% and 55% belonged to these clusters, respectively. Model-based unsupervised clustering further identified 9 unique clusters that could be consolidated into 6 groups: 1) TP53-complex; 2) biTET2/SRSF2; 3) SF3B1 (without biTET2 or SRSF2); 4) SRSF2 without TET2 and frequent IDH2 mutations (SRSF2-IDH2); 5) ASXL1 and U2AF1 mutations and copy number alterations (CNA-ASXL1/U2AF1); 6) no somatic mutations (No mutation). biTET2/SRSF2 group exhibited monocytic bias with higher AMC, PB and BM monocyte % even among OM-CMML cases. This bias was not attributed to higher RAS pathway mutation frequencies, as biTET2/SRSF2 cases had similar RASp mutation frequencies but lower median variant allele frequencies for RASp mutations compared to NOS cases (9% vs. 21%, p=0.029). PB monocyte partitioning using flow cytometry (FC) in a subset of OM-CMML pts (n=50) demonstrated higher frequency of classical monocytosis (>94% CD14+/CD16- monocytes) in biTET2/SRSF2 vs other OM-CMML. To further determine if biTET2/SRSF2 genotype defines a distinct biological subgroup, we performed RNA-sequencing of BM CD34+ cells from healthy donors (n=15) and pts with OM-CMML (n=12), MD-CMML (n=14) and MDS (n=55). Unsupervised clustering based on gene expression profiles revealed that biTET2/SRSF2 clustered irrespective of their diagnostic group with greater dispersion being observed based on diagnosis. MDS and CMML with biTET2/SRSF2 had less differentially expressed genes compared to MDS and CMML with biTET2/SRSF2 relative to other MDS and CMML genotypes. Among 138 OM-CMML pts in whom progression to overt CMML was evaluable, 37 (27%) progressed to overt CMML of which 26 (70%) belonged to the biTET2/SRSF2 group. Competing risk analysis confirmed higher cumulative incidence and shorter time to CMML progression among biTET2/SRSF2 OM-CMML. Using ElasticNet regression we identified BM monocytosis and 14 genomic variables (including biTET2 and TET2-SRSF2) as predictors of overt CMML progression among OM-CMML. We developed a score-based model able to predict progression with high reliability (AUC 0.805, sensitivity 0.70, specificity 0.76). Model accuracy was confirmed by optimism-corrected bootstrap internal validation (AUC 0.668). Monocytic bias, BM classical monocytosis (>94% CD16- monocytes) and higher overt CMML progression in biTET2/SRSF2 was confirmed in a validation cohort (28, 66 and 22 pts with OM-CMML, MDS and MD-CMML, respectively). BM monocytosis of biTET2/SRSF2 was confirmed in this cohort with morphologic and FC monocyte frequency quantification having high concordance (r=0.737, p<0.001). Genomic subgroup analysis discriminated survival outcomes more reliably than diagnostic categories alone. Our findings suggest that the presence of biTET2 or TET2-SRSF2 co-mutations, combined with BM monocytosis, can more reliably identify true CMML cases within the OM-CMML spectrum. Integrating genomic determinants into the CMML diagnostic framework enhances diagnostic accuracy.

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: The 5th edition of the World Health Organization (WHO-5) and the International Consensus Classification (ICC) recognize TP53-mutated myeloid neoplasms (MN) as a distinct disease category, reflecting their uniformly dismal outcomes and the urgent need for novel therapeutic strategies. However, both classifications do not include TP53-non-mutatedcases with 17p (TP53 locus) loss, despite emerging evidence that such lesions may confer biologic and clinical behaviour similar to TP53-mutated disease. Our preliminary work demonstrated that 17p loss without TP53 mutation in therapy-related MN (t-MN) is associated with poor survival, approximating that of TP53-mutated cases (Hiwase et al., Blood 2023). Yet, the true frequency and prognostic impact of 17p loss in TP53-non-mutatedMN remains largely undefined, representing a critical gap in current disease classification and risk stratification. To address this critical knowledge gap, we aim to define the frequency and prognostic impact of 17p loss across the TP53 locus in TP53-non-mutatedMN, including both myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Finally, we compared the survival outcomes of this cohort with those of TP53-mutated MN. Method This international collaborative study was designed to evaluate the frequency and prognostic significance of 17p loss in TP53-non-mutatedMN. We included MDS and AML patients who had conventional cytogenetic analysis and did not have TP53-mutation(TP53-non-mutated)by next-generation sequencing (NGS) with sensitivity of ≥2% variant allele frequency (VAF). These cases were systematically reviewed for 17p loss using karyotype data, and where available, fluorescence in situ hybridization (FISH) and SNP array were used to confirm the 17p/TP53 locus deletion. To assess the clinical impact, we compared the overall survival (OS) of TP53-non-mutatedcases with 17p loss against a reference cohort of TP53-mutated MNs with VAF ≥2% (n=603) (South Australian Myeloid Neoplasm Registry (SA-MN) and Mayo Clinic, Rochester, USA). Results We screened 8876 cases of TP53-non-mutatedMN cases across an international cohort comprising the SA-MN (Australia; n=1046), the Düsseldorf MDS Registry (Germany; n=545), Munich Leukaemia Laboratory (Germany; n=5785), and King's College Hospital, (United Kingdom; n=1500). We identified 85 (1%) cases with 17p loss across the TP53 locus, representing the largest international cohort to define the frequency of 17p loss in TP53-non-mutatedMN. Complex karyotype was present in 40% (n=34) of cases, frequently co-occurring with del(5q) and del(7q). Clinically, most cases were MDS (n=69; 81.2%), including MDS-IB1 (n=19), MDS-IB2 (n=15), MDS-LB (n=16), t-MDS (n=8), MDS/MPN overlap (n=3), CCUS (n=1), or unspecified MDS (n=7), while AML accounted for 16 cases (18.8%). Among evaluable cases, the majority were male (78.6%) and had a median age of 66.5 years at diagnosis. The most frequent cytogenetic abnormality causing 17p loss across the TP53 locus was monosomy 17 (39%), followed by i(17q) (29%), add (17p) (14%), derivative chromosome 17 from unbalanced translocations (11%), and del(17p) (7%). The majority represented de-novo MN (60%), with t-MN in 40% of cases. Median OS of evaluable 17p loss in TP53-non-mutatedcases (n=28; 10.2 months) was poor and comparable to TP53-mutated MN (n=603, 8.7 months; P=0.42). Similarly, survival censored for a stem cell transplant was also equally poor (10.4 vs. 8.7 months; P=0.34) in both groups. Complex vs. non-complex karyotype (7.7 vs. 10.7 months; P=0.68) did not significantly influence the OS of 17p loss. Median OS of MDS cases tended to be longer compared to AML (16.7 vs. 9.1 months; P=0.11), although not statistically significant. Conclusion: In this largest international study, 1% of TP53-non-mutatedMNs harbor 17p loss across the TP53 locus, displaying poor survival equivalent to TP53-mutated disease. Exclusive reliance on NGS to identify TP53 mutations misses these high-risk structural TP53 abnormalities, which are readily detectable by conventional karyotyping. These findings provide compelling evidence to include TP53-non-mutatedcases with 17p loss in the next revision of the classifications, enabling accurate prognostication and equitable access to curative and clinical trials of novel therapies.

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 and Significance: CMML is a myeloid neoplasm characterized by hematopoietic stem and progenitor cell (HSPCs) monocytic bias, leading to downstream monocyte expansion that plays a key role in disease maintenance and progression. Monocytes in CMML express high surface expression of the homing C-C chemokine receptor 2 (CCR2), which promotes malignant monocyte survival. Expansion of CCR2+ monocytic progenitors drives acute transformation in CMML and contributes to venetoclax resistance in AML. For these reasons, CCR2 is a potential novel therapeutic target in CMML and monocytic AML. The CyTAC platform was designed to enable targeting of cell surface antigens that demonstrate limited or ineffective binding to conventional therapeutic antibodies. A CyTAC is a heterobivalent small molecule that binds to both a target of choice and to a fully humanized monoclonal antibody. STX-0712 comprises a CCR2 CyTAC which potently binds to CCR2 on malignant cells, and a CyTAC humanized ADCC-enabling monoclonal antibody, which when combined eliminates target cells via enhanced effector-mediated killing in the presence of NK cells. We have demonstrated that treatment with STX-0712 ex vivo results in human target engagement and pharmacodynamic activity, and in vivo leads to depletion of CCR2 MO1 monocytes in a potent and dose-dependent manner in CMML and AML (ASH 2024). Therapeutic depletion of CCR2+ malignant monocytes presents a novel opportunity to develop a potential disease-modifying targeted therapeutic agent. We have designed a first-in-human, phase 1 clinical trial to evaluate the effect of CCR2 CyTAC in patients with refractory/resistant CMML, and relapsed/refractory monocytic or monocytic predominant AML. Study Design and Methods This is a phase 1, first-in-human, open-label, multicenter clinical trial based in the USA to evaluate STX-0712 as a monotherapy in patients with CMML and AML who have no remaining treatment options known to confer clinical benefit. STX-0712 will be administered as a single intravenous (IV) infusion every 3 weeks (additional regimen schedules may be explored) until evidence of disease progression, death, intolerance to study medication, or withdrawal of consent. The study consists of a dose-escalation and dose-expansion part and includes two cohorts: Cohort 1 (refractory/resistant CMML) and Cohort 2 (relapsed/refractory monocytic or monocytic predominant AML). Dose escalation will begin in participants with CMML using the Bayesian Optimal Interval (BOIN) design, with a minimum of three patients enrolled at each dose level. Backfilling of up to two cleared dose levels will be initiated once efficacious exposure levels are reached and at least one CMML participant has achieved a complete remission (CR) or a partial remission (PR), marrow response, or clinical benefit as defined by the MDS/MPN IWG 2015 criteria. In the AML cohort, backfilling may begin once at least one participant has achieved a response defined by ELN 2022 criteria. Dose expansion will occur upon completion of dose escalation in each cohort, using a Simon 2-stage adaptive design. Approximately 47 CMML and 58 AML patients may be enrolled in this phase 1 study. The primary objectives of the study are to evaluate the safety and tolerability of STX-0712 and to determine the minimal safe and biologically effective dose, recommended phase 2 dose (RP2D), and/or maximum tolerated dose (MTD), and to characterize dose-limiting toxicities (DLTs). Secondary objectives include evaluation of preliminary antitumor activity, pharmacokinetic (PK) parameters, and pharmacodynamic effects of STX-0712 on CCR2+ malignant cells in blood and bone marrow. Key exploratory endpoints include: (1) assessment of changes in CCR2+ tumor cells following STX-0712 dosing, (2) evaluation of candidate biomarkers, (3) characterization of T cells, B cells and NK cells at baseline and over the course of treatment, and (4) to explore the effects of STX-0712 on patient-reported outcomes using the CMML Symptoms Assessment Tool (MPN-SAF-TSS scoring system) and the EuroQoL 5 Dimension 5 level (EQ-5D-5L) for AML. Enrollment for Cohort 1 (CMML) began in March 2025, and the study is ongoing. Clinical Trial Information: NCT06950034