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INTRODUCTION: The Stromal Antigen 2 ( STAG2), on Xq25, is the most common cohesin complex gene mutated in myeloid neoplasms (MN) (5-10%). STAG2 mutations ( STAG2m) were incorporated in the ELN2022 as AML-MDS defining genetic mutations inferring poor prognosis and is a genetic marker in IPSS-M risk stratification. The PHD-finger protein 6 ( PHF6) gene, on Xq26.3, is a tumor suppressor gene, mutated in 3% of MN, and is more common in T-ALL. PHF6m is believed to be a late event in MN with little data on its prognostic implications in MN. Both STAG2m and PHF6m are more prevalent in males, but their implications on the clinical presentation of MN have not been compared prior. METHOD: Our study is a multicenter study at Mayo Clinic (Rochester, Florida, Arizona). After IRB approval, we retrospectively analyzed charts of 7935 patients who had NGS between 2016-2022. STAG2m was found in 96 patients, and PHF6m in 116 (including 3 with concurrent STAG2m and PHF6m). We excluded 4 PHF6m patients who had T-ALL. Data was captured at time of NGS. MDS risk stratification was by IPSS-M and AML by ELN2022. Overall survival (OS) was calculated from date of NGS to date of last follow-up. BlueSky Software was used for statistical analysis. RESULTS: Characteristics: We compared 96 STAG2m vs 112 PHF6m MN patients. Of STAG2m cases, 78% were male vs 76% in PHF6m MN. Median age was 72 vs 73 years, respectively. Diagnoses included AML (29% of STAG2m vs 34% of PHF6m MN), MDS (54% vs 28%), MDS/MPN (10% vs 21%) and MPN (3% vs 9%). MDS was more common among STAG2m MN (p<.001) while MDS/MPN was more common among PHF6m MN (=.05). MDS-IB was the most common subtype in both (69% vs 52% in STAG2m vs PHF6m MDS). MDS-LB and 5q subtypes were only found in PHF6m MDS, and there was more PHF6m MDS-RS (13% vs 2%). Median IPSS-M score was 0.9 vs 0.8 in STAG2m vs PHF6m MDS (both high-risk). Majority of STAG2m AML were adverse risk (96%) while PHF6m AML were intermediate (79%) (p<.001). AML cases included 43% vs 30% secondary AML (sAML) in STAG2m vs PHF6m AML. Among STAG2m and PHF6m MN, 17% of each were tMN. Cytogenetic abnormalities were more common in PHF6m MN (50% vs 31%, p=.005). The median bone marrow (BM) blasts count among STAG2m MN was 7% vs 4% in PHF6m MN (p=.03). Genetic characteristics: STAG2m were nonsense, frameshift and splice site (54%, 36% and 11%). PHF6m were nonsense, frameshift, splice site, missense and start loss variant mutations (44%, 33%, 12%, 10% and 1%). The median variant allele frequency (VAF) of STAG2m MN was 50% compared to 32% in PHF6m MN (p=.008). Co-mutations: The median number of co-mutations was 3 in STAG2m and 4 in PHF6m MN (p=.04). The most common were TET2m (37% in STAG2m MN vs 45% in PHF6m MN), RUNX1m (31% in both), SRSF2m (39% vs 20%), ASXL1m (66% vs 38%), BCORm (19% vs 6%), U2AF1m (13% vs 14%) and IDH2m (18% vs 7%). ASXL1m, SRSF2m, BCORm, IDH2m and SF3B1m were more common among STAG2m MN (p=<.001, .003, .006, .02, .09). JAK2m, IDH1m are more common among PHF6m MN (p=.02, .06). Survival: Of STAG2m and PHF6m MN, 70% and 72% of patients received treatment. HSCT was performed in 23 (24%) and 23 (21%) of STAG2m and PHF6m patients, including AML (n=9, n=10), MDS (n=12, n=8), MDS/MPN (n=2, n=2). OS among STAG2m and PHF6m MN was 20.5 and 22.1 months, respectively. OS among AML cases was 13.5 vs 21 months (p=.6) in STAG2m vs PHF6m, was NR vs 22 months (p=.4) in MDS, 11.7 vs 17.6 months in MDS/MPN (p=.4), and 10.4 vs 27.4 months (p=.5) in MPN. There was no difference between males and females in either STAG2m or PHF6m MN. HSCT led to longer OS among both STAG2m (NR vs 19.9 months, p=.01) and PHF6m MN patients (NR vs 17.6 months, p=.07). Among AML patients, HSCT did not significantly impact OS (18 vs 9.6 months, p=.3 and 25.1 vs 21 months, p=.2, in STAG2m and PHF6m). HSCT improved OS in STAG2m MDS (NR vs 20.5 months, p=.03). In PHF6m MDS, OS was 22.1 vs 13.1 months in HSCT vs non-HSCT (p=.3). CONCLUSION: STAG2m and PHF6m are 2 X-linked mutations that occur in MN. Both commonly occur in elderly males. MDS was the most common diagnosis in STAG2m MN. STAG2m co-occurred particularly with SRSF2m ASXL1m and BCORm, which are associated with poor prognosis. In our cohort, STAG2m AML carried a poorer prognosis than PHF6m AML. Our study confirms the ELN2022 AML risk stratification. MDS risk classification and OS did not differ between STAG2m and PHF6m in our cohort. A larger cohort is required to confirm our findings and may provide stronger validation.

Introduction: Germline DEAD-box RNA helicase-1 mutant (DDX41) predisposition syndrome ( DDX41-MT-GPS) is one of the most frequent hereditary predisposition syndromes in adults with myeloid neoplasms (MN-2-5%). Somatic mosaicism involving the unmutated DDX41 allele (e.g., R525H) as a potential mechanism for myeloid evolution has been reported in 39-50% of patients (pts). In addition, given the later age of onset, somatic mosaic states composed of age-related clonal hematopoiesis (ARCH) (e.g., DNMT3A, ASXL1, TET2, and TP53), with reported frequencies less than expected in MDS and AML, have been documented. We conducted this study to define the somatic mosaic landscape in DDX41-MT-GPS . Methods: After approval from the Mayo Clinic IRB the prospective GPS database was queried for pts with DDX41-MT GPS. Germline confirmation was conducted on skin fibroblast-, or hair follicle-derived DNA. Somatic CH testing was conducted using a targeted NGS panel as previously described. Given the high frequency of variants of undetermined significance (VUS), comparisons were made between pathogenic ( DDX41path) and DDX41VUS. Results: We identified 106 pts with DDX41-MT-GPS, of which 83 (78%) met criteria for MN, 16 (15%) for CH/CCUS (clonal cytopenias of undetermined significance), while 7 (6%) were unaffected carriers. Somatic mosaicism in DDX41-GPS patients without MN Sixteen DDX41-MT pts without MN met criteria for CH/CCUS ( DDX41path [n=4] and DDX41VUS [n=12]; median age 69 years (yrs) (range [R], 19-78) ( DDX41path [69 yrs] and DDX41VUS [67 yrs], p=0.85), with a male predilection (75%). Four (25%) pts had somatic DDX41 MT (R525H [n=3] and A376T [n=1]) with median variant allele frequency (VAF) 10.5% (R, 5-27); 2 (50%) pts with germline DDX41path had R525H (VAF; 5% and 13%) and 1 (8%) pt each with germline DDX41VUS had R525H (VAF 5%) and A376T (VAF 27%) somatic variants, respectively. Abnormal cytogenetics (CG) were observed in 2/13 evaluable (15%) pts ([+8 and del20q]) in the germline DDX41path group only (p= 0.05). ARCH co-mutations were observed in 11 (69%) pts, not significantly (NS) different between DDX41path (75%) and DDX41VUS (75%), p= 0.66. Individual co-mutations: DNMT3A (25% vs 16%, p= > 0.99), ASXL1 (25% vs 8%, p=0.71), TET2 (25% vs 8%, p= >0.99), and JAK2 (0% vs 8%, p= >0.99) were NS different between the two groups. Somatic mosaicism in DDX41-GPS patients with MN Eight-three pts met criteria for MN ( DDX41path [n=43] and DDX41VUS [n=40]; median age 67 yrs (range [R], 27-92), with a male predilection (58%). Fifty-four (65%) pts had MDS ( DDX41path [55%] and DDX41VUS [45%], p=0.55) and 29 (35%) had AML ( DDX41path [41%] and DDX41VUS [59%], p=0.65). Overall, 15 (18%) pts had somatic DDX41 MT; 12 (80%) pts with DDX41path and 3 (20%) pts with DDX41VUS (p= 0.003). The median VAF of somatic DDX41 MT was 12% (R, 5-37), NS different between DDX41path (10.5%) and DDX41VUS (12%) (p=0.08). The most common somatic DDX41 MT was R525H (66% [n=10]), significantly associated with DDX41path (9/10 [90%]), p=0.01. ARCH was observed in 17 (20%) pts, significantly higher in DDX41path (76%) compared to DDX41VUS (24%), p= 0.03. Abnormal CG were observed in 10/73 evaluable (14%) pts (-Y [n=2], del 20 [n=3], del 5q [n=2], del 7q [n=1], t (3;8) [n=1] and complex CG [n=1]), higher in DDX41path (70%) compared to DDX41VUS (30%) with p value >0.99. We compared the spectrum of CH between DDX41 MT CH/CCUS and MN pts ( Table 1) and found no difference in the proportion of pts with somatic DDX41 MT (p= 0.52) and ARCH/myeloid MT (p= 0.56). The individual occurrences of DNMT3A (p= 0.49), ASXL1 (p= 0.71), TET2 (p=0.59), JAK2 (p>0.99) and TP53 (p= 0.56) mutations were NS different between the two groups ( Figure 1). Conclusion: We define the somatic mosaic landscape in DDX41-MT GPS and demonstrate that somatic mutations involving the other allele, especially R525H, are seen in ~20% of pts (>80% of R525H present in MN). The frequency and composition of ARCH and myeloid driver mutations was lower than expected for the age of presentation, in comparison to de novo and secondary MDS/AML and occurred more frequently in DDX41path compared to DDX41VUS. Forty-two % (n=41), including 37% with MN, did not have somatic mosaicism, underscoring the fact that mechanisms of leukemia progression remain to be defined in subsets of affected pts.

Abstract We surveyed the performance of ponatinib, as salvage therapy, in a real-world setting of chronic phase chronic myeloid leukemia (CML-CP). Among 55 consecutive patients (median age 49 years) with relapsed/refractory CML-CP, 35 (64%) had failed ≥ 3 tyrosine kinase inhibitors (TKIs), 35 (64%) were pre-treated with nilotinib, and 14 (28%) harbored ABL1T315I. At start of ponatinib (median dose 30 mg/day), 40 patients were already in complete hematologic (CHR), 4 in complete cytogenetic (CCyR), 3 in major molecular (MMR) remission, while 8 had not achieved CHR (NR). Ponatinib improved the depth of response in 13 (33%), 3 (75%), 2 (66%), and 4 (50%) patients with CHR, CCyR, MMR, and NR, respectively (p = 0.02). At a median follow-up of 42 months, 13 (23%) deaths, 5 (9%) blast transformations, and 25 (45%) allogeneic transplants were recorded. Five/10-year post-ponatinib survival was 77%/58% with no significant difference when patients were stratified by allogeneic transplant (p = 0.94), ponatinib-induced deeper response (p = 0.28), or a post-ponatinib ≥ CCyR vs CHR remission state (p = 0.25). ABL1T315I was detrimental to survival (p = 0.04) but did not appear to affect response. Prior exposure to nilotinib was associated with higher risk of arterial occlusive events (AOEs; 11% vs 0%; age-adjusted p = 0.04). Ponatinib starting/maintenance dose (45 vs 15 mg/day) did not influence either treatment response or AOEs. Our observations support the use of a lower starting/maintenance dose for ponatinib in relapsed/refractory CML-CP but a survival advantage for deeper responses was not apparent and treatment might not overcome the detrimental impact of ABL1T315I on survival. The association between prior exposure to nilotinib and a higher risk of post-ponatinib AOEs requires further validation.

AbstractETV6 mutations are rare but recurrent somatic events in myeloid neoplasms and are negatively prognostic in myelodysplastic syndrome. We set out to examine the clinical and molecular characteristics of patients undergoing investigation for myeloid neoplasms, found to have deleterious ETV6 mutations. ETV6 mutations occurred in 33 of 5793 (0.6%) cases investigated and predominantly in high‐risk disease entities including MDS with increased blasts, primary myelofibrosis and AML, myelodysplasia‐related. In three cases, isolated iso (17q) karyotype was concurrently detected, an otherwise rare karyotype in myeloid neoplasms. ETV6 mutations were frequently subclonal and never occurred as an isolated abnormality with ASXL1 (n = 22, 75%), SRSF2 (n = 14, 42%) and SETBP1 (n = 11, 33%) the predominant co‐mutations. Restricting to patients with MDS, higher rates of ASXL1, SETBP1, RUNX1 and U2AF1 mutations occurred in ETV6 mutated cases, relative to a consecutive control cohort with wild‐type ETV6. The median OS of the cohort was 17.5 months. This report highlights the clinical and molecular associations of somatic ETV6 mutations in myeloid neoplasms, suggests their occurrence as a later event, and proposes further translational research questions for their role in myeloid neoplasia.

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