Alkhateeb, Hassan B.

Abstract Introduction: DDX41 (DEAD-box helicase 41) mutations (mt) represent the most prevalent germline predisposition syndrome (GPS) in adult patients (pts) with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This molecular subtype is typically associated with late-onset disease, runs an indolent clinical course, with a relative absence of high-risk co-mutations. While several retrospective studies have reported favorable responses to venetoclax (VEN)-based therapies, anecdotal observations suggest variable outcomes. We aimed to evaluate the clinical characteristics and treatment outcomes of patients with DDX41-mt MDS/AML treated at the Mayo Clinic. Methods: We conducted a retrospective study of pts diagnosed with DDX41-mt germline predisposition syndrome with concurrent myeloid neoplasms at our institution. Germline testing was conducted under an IRB approved protocol (Mayo Clinic IRB# 16-004173; clinical-trials gov. Identifier: NCT02958462). Of 198 pts with DDX41-mt myeloid neoplasms, 77 had high-risk disease—43 with MDS with increased blasts (MDS-IB1, n = 11; IB2, n = 32) and 34 with AML (WHO 2022). We assessed how clinical, and disease features influenced outcomes in this subgroup. Among these pts, 69 (90%) pts had likely pathogenic/pathogenic variant (DDX41path) variants and 8 (10%) pts had variant of undetermined significance (DDX41VUS) according to variant classification criteria from the American College of Medical Genetics/ the Association of Medical Genetics/ the Association for Molecular Pathology (ACMG/AMP) guidelines. Results: The median age was 68 years (yrs) (range [R],34-92), 70% were male and the median bone marrow blast % was 15 (R,5-60). The most common DDX41 germlinevariants (> 5% cases) included p.D140fs (26%), p.M1I (22%), and p.P258L (6.5%). 74% had DDX41 truncating variants. Thirty-one (40%) acquired a somatic DDX41-mt (R525H [81%]) and 43 (56%) had other somatic mutations. Among these, the most frequent were ASXL1 (n= 9, 21%), TP53 (n= 8, 10%), splicing factor (n= 7, 9%), and DNMT3A (n= 5, 6%). Twenty-six, (34%), 26 (34%), 15 (19%) and 9 (12%) pts were treated with hypomethylating agents (HMA), HMA+VEN, intensive chemotherapy (IC) and active surveillance/supportive care, respectively. Overall, the composite complete remission rate (cCR) in high-risk MDS and AML was 56%. The cCR rates (41%, 59% and 68.7%, p= 0.10) and median duration of response (DOR; 43.7, 44.9 and 28.9 months [mo], p= 0.46) were not significantly different with HMA, HMA+VEN, and IC induction. Thirty-one (40%) pts received allogeneic stem cell transplantation (allo-HCT); 4 (36%), 15 (47%) and 12 (35%) in MDS-IB1, MDS-IB2 and AML groups, respectively (p= 0.56). With a median follow up of 34.7 mo (R, 0.7-136.7), the median overall survival (OS) from the time of diagnosis was 56.9 mo (95% CI, 50.7-63.0) and was not significantly (NS) different between MDS-IB1 (not reached, 70% at 3 yrs), MDS-IB2 (56.9 mo, 86% at 3 yrs) and AML (55.7 mo, 60% at 3 yrs) groups, p= 0.16). The median OS was NS different between DDX41path and DDX41VUS (58.6 vs 55.7 mo, p= 0.50), respectively. We conducted a multivariable analysis for OS using variables that showed significance or trend towards significance in univariable analysis. Concurrent splicing factor mutations (HR; 5.29, 95% CI: 1.29-21.6, p= 0.02) remained negatively prognostic, while there was a trend towards better survival outcomes with allo-HCT (HR; 0.48, 95% CI: 0.21-1.07, p= 0.07). Bone marrow blast ≥ 20% (HR; 1.87, p= 0.12) and TP53-mt (HR; 2.14, p= 0.12) did not retain significance. Conclusion: In our cohort of patients with germline DDX41-mt high-risk MDS and AML, VEN-based regimens were not superior to HMA alone or IC, in terms of response rates and outcomes. On multivariable analysis, the presence of concurrent splicing factor mutations independently and negatively impacted survival with a trend towards better outcomes with allo-HCT. These findings highlight the heterogeneity within DDX41-mt myeloid neoplasms and underscore the need for future studies in larger cohorts to better define predictors of response and survival.

Abstract Introduction: The DEAD-box helicase 41 (DDX41) gene, located on chromosome 5q35, is a tumor suppressor gene involved in RNA processing. Germline mutations in this gene can predispose patients to myeloid malignancies. Studies have suggested a more favorable prognosis in those with germline mutations in DDX41, with progression of disease being a frequently encountered cause of death. In this retrospective study, we explored the causes of death in a cohort of patients with pathogenic DDX41 mutations. Methods: This was a retrospective study of patients diagnosed with myeloid neoplasms between 2009-2024 and found to have DDX41 mutation (DDX41m) via next generation sequencing (NGS) at our institution. Myeloid NGS was obtained at or after the diagnosis of myeloid disease. Germline testing (when available) was performed through buccal/skin samples. Carriers of DDX41m without a myeloid neoplasm diagnosis and patients with only DDX41m VUS were excluded. Baseline characteristics at the time of myeloid neoplasm diagnosis included age at diagnosis, DDX41m variant allele frequency (VAF), blood counts (hemoglobin, white blood cell count, platelets), cytogenetics, bone marrow blast percentage, number of co-mutations, and treatment received. Unadjusted comparisons were made using continuous or categorical variables. Median overall survival (OS) was calculated from the time of diagnosis with myeloid neoplasm to the time of death, using BlueSky Statistics V.10.3.4. Results: Of the 112 patients identified to have pathogenic DDX41m, 29 (26%) patients were deceased at the time of data analysis. In this cohort of 29 patients (median age at diagnosis 72 years, 17 (59%) males), disease groups at initial diagnosis included 17 (59%) patients with MDS, 9 (31%) AML, and 3 (10%) MPN. Fourteen of the 17 (82%) of the patients with MDS were classified as MDS-EB1 or MDS-EB2. Seven of the 17 (41%) patients with MDS progressed to AML at the time of death, with 57% of the patients having more than 30% bone marrow blasts at time of progression. Blood counts at diagnosis in the cohort included a median hemoglobin of 9.8 g/dL, platelets of 83 x 109/L, and white blood cell count of 2.1 x 109/L. Twenty-one (72%) of patients had normal cytogenetics. Nine (31%) patients had both a pathogenic mutation and a variant of uncertain significance (VUS) in DDX41, with the remaining 20 patients harboring only a pathogenic DDX41 mutation. Eight (27%) patients underwent germline testing, with 7 confirmed to harbor a germline DDX41m. In the deceased cohort, 9 (31%) patients underwent HSCT. Three of the patients with AML and 1 patient with MDS were observed and did not undergo treatment. Median overall survival of the deceased cohort was 29 months. The median time to AML progression in the group with MDS was 14 months. Causes of death included infection in 11 (38%) patients, 9 (31%) with disease progression/relapse, 3 (10%) with acute gastrointestinal graft versus host disease (GVHD), and 1 (3%) from cerebral bleed. The cause of death was unknown in the remaining 5 (17.2%) patients. Infections included pneumonia, bacteremia, neutropenic enterocolitis, fungal, and unknown etiology. Of the 4 patients who were observed, 2 died from unknown cause and 2 died from disease progression. Of the 16 patients with AML at time of death, 9 (56%) died from disease progression or relapse, 3 (19%) from infection, 2 (13%) from GVHD, and 2 (13%) from unknown causes. Of the 10 deaths in the MDS group, 7 (70%) were from infection, 1 (10%) from GVHD, 1 (10%) from cerebral bleed, and 1 (10%) from an unknown cause. Three cases of infection in the MDS group occurred during remission. Infection was a more common cause of death in the MDS group compared to the AML group, where disease progression prevailed as a cause of death (70% vs 19%, p=0.01). Eight (80%) of the patients with MDS who died from infection were neutropenic prior to death, and 4 (50%) of those patients were on neutropenic antimicrobial prophylaxis. Conclusions: In this cohort of 29 deceased patients with a DDX41-mutated myeloid neoplasm, we characterized causes of death. Disease progression/relapse was a significantly more common cause of death in those with AML, compared to infection in patients with MDS. Our work underscores the need for additional studies looking into non-leukemic causes of death in patients with DDX41m myeloid diseases and potential strategies to mitigate infection-related mortality in this population.

Abstract Background: Clonal cytopenia of undetermined significance (CCUS) is defined as clonal hematopoiesis in the absence of a hematologic neoplasm, along with unexplained cytopenia. While several studies have identified risk factors and developed scores to predict progression to overt myeloid neoplasms, the clinical and molecular features during the progression process remain poorly understood. Aim: Evaluate the clinical and clonal characteristics of patients with CCUS, with a focus on the longitudinal evolution of clonal and clinical parameters over time. Methods: After IRB approval, we retrospectively reviewed charts of patients with unexplained cytopenia (2015-2024), recording data at the time of CCUS diagnosis (WHO 2022 diagnostic criteria) and at time of follow-up bone marrow biopsy after diagnosis. Follow-up CBC and karyotype data were recorded for patients who had repeat bone marrow biopsies (n= 111), while follow-up clonal parameters and risk scores were calculated for those who underwent repeat NGS (n= 74). A reduction or expansion in clone size was defined as a change exceeding 5% (CE5%), or change exceeding 10% (CE10%) from the baseline value. Overall survival (OS) was calculated from diagnosis to last follow-up. For statistical analysis, we used BlueSky Statistics V10.3.1. Results: 214 CCUS patients (median age 73, 68% males) were included. At baseline, 30% of patients had abnormal cytogenetics, with del (20q) being the most common one (8%; n= 18), followed by trisomy 8 (6%; n= 13), and loss of chromosome Y (5%; n= 10). The median number of mutations was 2 (range, 1-5), with the most common mutations TET2 (38%), SRSF2 (21.5%), ASXL1 (21%), ZRSR2 (18.5%), DNMT3A (9.5%), and U2AF1 (9%). Most patients had high risk CHRS (60%), followed by intermediate (30%), and low risk (10%). On the other hand, 41% had high risk CCRS, 29% had intermediate risk, and 30% had low risk. After a median follow-up of 46 months, 62 patients died (median OS not reached). A decrease in the white blood cell (WBC) count from the time of diagnosis to follow-up was more frequently seen among CCUS patients who progressed to MN (70% vs 50%, p= 0.04), however, changes in hemoglobin and platelets did not differ between those who progressed and those who did not (p> 0.05). Although the frequency of abnormal karyotypes at diagnosis was similar between patients who eventually progressed (28%) and those who did not (31%) (p= 0.6), 4 (2%) patients with initially normal karyotypes acquired chromosomal abnormalities during follow-up, and all progressed to MDS. Among the 74 patients who had follow-up NGS, 31% acquired new mutations, clonal expansion of pre-existing mutations were seen in 54% using CE5% and 19% per CE10%, and 16% developed new variants of uncertain significance (VUS). Acquiring new pathogenic mutations was similar in patients who progressed to MN compared to those who did not progress (34% vs 28%, p= 0.6). On the other hand, clonal expansion was significantly more observed in patients who progressed compared to those who did not, using both CE5% (73% vs 31%, p = <0.001) and CE10% (27% vs 9%, p= 0.03). At follow-up, ASXL1 was the most commonly acquired mutation among patients who did not progress (4 cases), followed by TET2 (2 cases), and U2AF1, GATA2, DNMT3A, SF3B1, ZRSR2, TP53, and CBL (1 case each). On the other hand, SRSF2, SETBP1, CBL, JAK2, TET2, and RUNX1 were more frequently acquired among those who progressed to myeloid neoplasms (each appeared in 2 patients). When recalculating CCRS, CHRS, IPSS-M, and IPSS-R at follow-up for patients who had repeat NGS, there was no significant difference in score changes - either upward or downward - between those who progressed and those who did not (p> 0.05). Conclusion: In CCUS patients who progressed to myeloid neoplasms, WBC decline, clonal expansion (more frequent), and evolving chromosomal aberrations (less frequent) were observed, supporting the value of longitudinal genomic and clinical monitoring in CCUS.

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 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 Background: TP53-mutated (TP53mut) myeloid neoplasms (MN) are a biologically and clinically distinct subgroup of blood cancers associated with extremely poor outcomes and limited response to conventional therapies. In some patients, MN are preceded by clonal cytopenia of undetermined significance (CCUS). Prior cytotoxic exposure (treatment related, TR) is an independent predictor for survival in CCUS (Li et al, Blood Adv. 2024). As TP53mut CCUS and MN are enriched in TR cases, the interaction between cytotoxic exposure and TP53mut status on the risk of progression and survival remains unclear. Methods: Adult patients with CCUS who were evaluated at the Mayo Clinic or South Australian myelodysplastic syndrome (MDS) Registry (Adelaide, Australia) were included. CCUS and MDS were defined according to the 5th edition of the WHO classification for MN (Khoury et al, 2022), except for the exclusion of cases harboring MDS-defining cytogenetics. Next-generation sequencing was performed using a targeted panel covering genes commonly mutated in MN. In cases with >1 TP53mut, VAFs were summed. Survival outcomes were assessed by Kaplan-Meier with log-rank testing. Univariate and multivariate analyses were estimated using Cox proportional hazards regression. Results: Of 320 CCUS cases, 50 (15.6%) harbored TP53mut (TP53mut CCUS), whereas 270 (84.4%) were wild-type (TP53wt CCUS). Median age at diagnosis was 71 years (range 16-99). TP53mut CCUS had a greater proportion of females (60% vs 33.7%, p<0.001) and TR (66% vs 23%, p<0.001) compared to TP53wt. Hematologic parameters including hemoglobin, red cell distribution width, mean corpuscular volume, white blood count, absolute neutrophil count, platelets, and bone marrow blasts were comparable between the two groups. Almost all TP53mut (n=48, 96%) were located in the DNA-binding domain and the median variant allele frequency (VAF) was 9% (range 2-46%). Five (10%) cases harbored 2 TP53mut. The distribution of TP53mut VAF among patients was as follows: ≤2.5% (n=2), 2.5-5% (n=10), 5-10% (n=19), 10-20% (n=11), and ≥20% (n=7). The proportion of cases with cytogenetic abnormalities was smaller in TP53mut compared to TP53wt CCUS(12% vs 25.9%, p=0.035). Both cohorts had a comparable median number of mutations: 2 (range 0-7) in TP53mut and 2 (range 0-5) in TP53wt. The most common co-mutations in TP53mut CCUS were TET2 (n=8, 17.8%), PPMD1 (n=7, 15.6%), DNMT3A (n=6, 13.3%), ASXL1 (n=6, 13.3%), and SRSF2 (n=4, 8.9%). At last follow up, the rate of progression was comparable between TP53mut and TP53wt CCUS (12% vs 22%, p=0.13). Median time to MN progression was not reached in TP53mut and 6.25 months in TP53wt CCUS (p=0.5197). A significantly higher proportion of TP53mut cases acquired complex cytogenetics compared to TP53wt cases (33% vs 2%, p=0.024) at MN progression. Of 20 deaths observed in the TP53mut cohort, non-myeloid hematologic malignancies (n=6, 30%), infections (n=5, 25%), and TP53mut MN (n=4, 20%) were the 3 most common causes. With a median follow-up of 4 years (IQR 1.2-4.8), median OS was longer in de novo (dn) cases (8.7 years for dn TP53wt; not reached for dn TP53mut) and shorter in TR cases (3.3 years for TR TP53wt; 4.8 years for TR TP53mut). Notably, TP53mut showed no independent prognostic impact in the entire cohort, or when stratified by TR vs dn CCUS. On multivariate analysis that included TP53mut status, factors independently associated with inferior OS included prior therapy (HR 2.21, 95% CI 1.41-3.45, p<0.001), anemia (HR 4.1, 95% CI 1.88-8.99, p<0.001), and thrombocytopenia (HR 2.38, 95% CI 1.43-3.95, p<0.001). Similarly, TR CCUS (HR 1.6, 95% CI 1.06-2.4, p=0.03) and anemia (HR 2.15, 95% CI 1.25-3.7, p=0.006) were independently associated with inferior progression free survival. Conclusion: TP53 mut in CCUS did not independently predict inferior survival or increase leukemic transformation. While TP53mut CCUS were less likely to harbor cytogenetic abnormalities at diagnosis, significantly more cases harbored genomic instability at leukemic transformation. The poor outcomes attributed to TP53mut CCUS were driven by prior cytotoxic exposure, cytopenia – particularly anemia, and non-leukemic causes of death. These findings suggest risk stratification in CCUS should prioritize clinical context over TP53mut status alone.

Abstract Background: Isocitrate dehydrogenase (IDH) mutations are frequently implicated in the development of acute myeloid leukemia (AML). In a recent study (Salman MY, Blood Adv 2025), it was reported that IDH2 mutation subtypes, R140 and R172, may respond differently to intensive chemotherapy. In this study, we evaluated mid-cycle and end-of-cycle bone marrow blast responses in AML patients with IDH2 R140, IDH2 R172, and IDH1 R132 mutations, aiming to determine whether these subtypes exhibit different response dynamics. Methods: We reviewed the charts of AML patients diagnosed at Mayo Clinic, Rochester, between the years 2016 - 2023 that had available next generation sequencing (NGS) data. Institutional review board approval was obtained. All data were recorded at the time of AML diagnosis. Patients without molecular data or not treated with intensive chemotherapy (7+3 or CPX-351) were excluded from this study. Bone marrow blast percentages were assessed at mid-induction (days 14-21) and end of cycle (counts recovery). Blast clearance dynamics and complete remission rates were compared across IDH mutation subgroups and correlated with outcomes. GraphPad Prism 10.0.0 was used for statistical analysis. Results: Out of 381 AML patients, 31 had IDH2 mutations (23 with R140 and 8 with R172), and 8 had IDH1 mutations (all R132). Among patients with IDH2 mutations, those with R172 had numerically higher median mid-cycle bone marrow blast percentage compared to R140 (10% vs. 4%, p = 0.37). There was no significant difference in mid-cycle blast percentages between patients with IDH1 and IDH2 mutations (4.5% vs. 4%, p = 0.8). At mid-cycle, 68% of patients with IDH2 R140 had blast counts <5%, compared to 38% of those with IDH2 R172, suggesting a trend for faster mid-cycle clearance in the R140 group, though not statistically significant (OR 3.57, p = 0.2). In a broader comparison, 60% of IDH2-mutated patients and 50% of IDH1-mutated patients achieved blast clearance (blasts <5%) at mid-cycle, though not statistically significant (p = 0.7). At the end of the induction, patients with IDH2 R140 had significantly lower median bone marrow blast percentages compared to those with R172mutations (2% vs. 4%, p = 0.01). Similarly, patients with IDH1 mutations showed low blast counts compared to the IDH2 group (1.5% vs. 3%, p = 0.1). A higher proportion of IDH2 R140 patients achieved blast clearance compared to those with R172 mutations (100% vs. 75%, p = 0.06), while proportion of patients who achieved blast clearance were similar between IDH1 and IDH2 patients (87.5% vs 93.1%, P = 0.5). Analysis of overall complete remission rates (including both complete remission [CR] and CR with incomplete hematologic recovery [CRi]) within the IDH2-mutated group showed a higher rate in patients with the R140 mutation compared to those with R172 (87% vs. 75%), though the difference was not statistically significant (p = 0.5). When comparing IDH1- and IDH2-mutated patients, overall complete remission rates were 75% and 84%, respectively, with no significant difference observed (p = 0.6). Kaplan–Meier analysis of time to end-of-induction blast clearance revealed no statistically significant difference between IDH2 R140 and R172 patients (median time to clearance: 31 vs. 37 days, p = 0.3). Similarly, no difference was observed between IDH1 and IDH2-mutated patients (33 vs. 31 days, p = 0.9). Overall survival analyses showed no significant difference between IDH2 R140 and R172 groups (median 66.8 months vs not reached, P = 0.4). Likewise, no difference was observed between IDH1 and IDH2 patients (44.4 months vs not reached, P = 0.7), indicating similar overall survival across mutation subtypes. Conclusion: IDH2-R140 was associated with more effective blast cytoreduction following intensive induction compared to IDH2-R172. Although not statistically significant, IDH2 R172consistently showed trends toward higher blast percentages, delayed response, and lower overall complete remission rates. These findings align with the recent study reporting significantly slower blast reduction and delayed response in R172-mutated AML. Also, no significant differences were found between IDH1- and IDH2-mutated patients in terms of response kinetics or overall survival. Together, these findings highlight potential biological differences among IDH mutation subtypes that may impact treatment response and require further investigation in larger prospective cohorts.

Abstract Introduction: The ZRSR2 gene, located at Xp22.2, encodes a protein component of the RNA spliceosome. Alterations in ZRSR2 have been reported in various myeloid neoplasms (MN) and their precursors. They are frequently accompanied by other co-mutations, most commonly TET2. Isolated ZRSR2 mutations, however, are less commonly encountered and their clinicopathologic features have not been well documented. Aim: To describe the clinicopathologic features of MN and their precursors with isolated ZRSR2 mutations. Methods: Next-generation sequencing (NGS) was performed on peripheral blood (PB) or bone marrow (BM) samples from patients with confirmed/suspected MN using a 35-47 gene MN-targeting NGS panel. MN and clonal cytopenia of undetermined significance (CCUS) cases with isolated likely pathogenic/pathogenic (LP/P) ZRSR2 mutations were identified. Pathology diagnoses were rendered following the 2022 WHO/ICC classification and the slides were reviewed by three pathologists. Electronic medical records were reviewed for collection of relevant laboratory data. This study was approved by our institution's Institutional Review Board. Results: Of 164 ZRSR2-mutated cases, 10 cases with isolated LP/P mutations in ZRSR2 were identified. Patients were all male, with a median age 69.5 years. Three patients were diagnosed with CCUS, five with myelodysplastic syndrome (MDS), one with chronic myelomonocytic leukemia (CMML, MDS/MPN) and one with mixed phenotype acute leukemia (MPAL). Six patients had additional variants of undetermined significance (VUS) detected in ASXL1, CALR, EZH2, IDH2 and TET2. The other four patients had no additional variants identified by NGS but had cytogenetic abnormalities. One patient with MPAL had MECOM and IGH gene rearrangements detected on cytogenetic analysis. All patients had macrocytic anemia. One CMML patient with accompanying VUS in TET2 also had absolute monocytosis. BM cellularity and erythroid and granulocytic morphology were variable, ranging from unremarkable to left-shifted and dysplastic morphology. Nine (90%) cases showed atypical megakaryocyte morphology, characterized by hypolobated forms and osteoclast-like forms with separated and eccentrically located nuclear lobes. Interestingly, this megakaryocytic morphology was also observed in CCUS cases, albeit with lower frequency, below the 10% threshold necessary for the diagnosis of MDS. Megakaryocyte distribution was normal. Conclusions: Isolated LP/P ZSRS2 mutations are seen in a spectrum of myeloid neoplasms, including CCUS. They show a strong association with macrocytic anemia and megakaryocytic atypia/dysplasia. The presence of additional cytogenetic alterations and variants of undetermined significance is a confounding factor in determining the role of ZRSR2 mutations in driving the pathogenesis and morphologic features of these diseases.

Abstract Prognosis in acute myeloid leukemia (AML) patients is influenced by various factors, most importantly, patient clinical status and cytogenetic/molecular abnormalities. Within those factors, elevated lactate dehydrogenase (LDH) is considered a poor prognostic indicator in AML patients. Our study aims at exploring the different factors associated with elevated LDH and their impact on overall survival (OS). After obtaining IRB approval, we reviewed the charts of 381 AML patients diagnosed at Mayo Clinic, Rochester, between the years of 2016 and 2023. All data was recorded at the time of AML diagnosis. Patients with no cytogenetic and molecular data were excluded from this study. LDH levels were recorded if measured within 10 days of diagnosis and treatment for AML was not yet given. Both risk stratification and response evaluation were done following the 2022 European LeukemiaNet (ELN) guidelines. OS was calculated from the date of AML diagnosis to the date of last follow-up or death. We used BlueSky Statistics V 10.3.4 for statistical analysis. We identified 299 patients (78.4%) who had elevated LDH levels (normal ≤ 222 U/L) at the time of diagnosis with a median of 417 U/L (range, 223-7989). The median age for those patients was 68 years (range, 18-94), with 78 (26.1%) of them having a prior myeloid disease (MDS, MPN, or MDS/MPN) before AML diagnosis. By risk stratification, more than half of the patients had an adverse risk profile (n=171, 57.2%), while 71 (23.7%) and 57 (19.1%) had an intermediate and favorable risk, respectively. Most of those patients had an abnormal karyotype (n=204, 68.7%), with complex karyotype (n=103, 34.7%) being the most common abnormality, followed by monosomy 7 (n=50, 16.8%) and trisomy 8 (n=46, 15.5%). Next-generation sequencing (NGS) was performed on 268 (89.6%) patients with elevated LDH, and the most common mutations were TP53 (n=60, 22.3%), FLT3-ITD (n=59, 21.1%), and NPM1 (n=51, 18.5%). TP53 mutations were more common in patients with normal/low white blood cell count (WBC) in contrast with FLT3 and NPM1, which were the most common mutations in patients with high WBC. After grouping the patients according to their LDH levels (group 1: 223-444 U/L, group 2: 444-666 U/L, group 3: > 666 U/L), we found that FLT3-ITD mutations were more likely to occur at higher LDH levels i.e. groups 2 and 3 (p= 0.002). The majority of patients with elevated LDH (n=262, 87.6%) received therapy following diagnosis, with the most common treatment regimens being a combination of a hypomethylating agent and venetoclax (HMA+VEN) (n=97, 37%) and a (7+3)-based induction therapy (n=77, 29.4%). When comparing patients with elevated LDH (n=299, 78.4%) to patients who had non-elevated LDH (n=82, 21.5%), no difference in both median age at diagnosis and risk stratification was found. We noticed an increase in WBC (median, 7.7 vs. 2.1) (p<0.001) along with an increase in peripheral blasts (median, 28% vs. 7%) (p<0.001) in elevated LDH patients. Among patients with non-elevated LDH, 21% (n=17) had a complex karyotype compared to 34.7% (n=103) in the elevated LDH group (p=0.022). The molecular profile was different between the 2 groups, where the most common mutations in patients with non-elevated LDH were IDH2 (n=19, 25.3%), RUNX1 (n=17, 23%), and DNMT3A (n=16, 21.6%). All patients (n=82, 100%) in the non-elevated LDH group received therapy after diagnosis. There was no difference in terms of treatment regimens used between the 2 groups. Moreover, no statistical difference in remission rates after one line of therapy was found between those groups where non-elevated LDH patients had a 65.9% remission rate compared to the 55.3% remission rate in patients with elevated LDH (p=0.098). Patients with elevated LDH had a shorter OS than patients with non-elevated LDH (9.5 vs. 26.4 months, p=0.0008). However, median OS was similar across the different groups of elevated LDH. Using univariate analysis in our cohort (n=381), age, HSCT, risk stratification, FLT3 mutation, and LDH elevation all had an impact on OS. However, when comparing those variables using multivariate analysis, age and FLT3 mutation lost their significance (p-value of 0.25 and 0.84, respectively).Complex karyotype and FLT3 mutations were more common in patients with elevated LDH. These patients tend to have higher WBC and peripheral blasts. Elevated LDH had a negative impact on patients' OS regardless of patients' risk classification.