Al‐Kali, Aref

Introduction Chronic myelomonocytic leukemia (CMML) is associated with a 15-30% risk for blast transformation (BT) over 3-5 years. Post-BT survival remains dismal with a median overall survival (mOS) of ~6 months (mo), even with intensive therapies. Although genetic drivers are identified in >40% of BT-CMML cases (Carr, 2021), mechanisms of BT remain to be elucidated. This multicenter study examines risk factors for BT in CMML and predictors of post-BT survival. Methods CMML patients at Mayo Clinic (n = 400, 47%) and MD Anderson Cancer Center (n = 449, 53%) were included with IRB approval. Analyses used clinical and molecular data at diagnosis or first referral in chronic phase (CP) and again at BT. Categorical variables were compared by Chi squared and continuous by Mann-Whitney U or Kruskal-Wallis tests. Multivariate analyses used Cox proportional hazards models with binarization via receiver operator curves. Kaplan-Meier analyses were censored at hematopoietic cell transplantation (HCT). P < 0.05 was considered significant. Calculations used BlueSky (v10.3.1) or Prism (v10.2.3). Results Of 849 CMML patients (median age 71 years, 32% female, 90% white) included in the study, 133 (16%) underwent BT. In comparison to CP-CMML without BT, those with BT were more likely (p < 0.05) to have had higher WBC counts (15.1 vs 11.4 x109/L), higher absolute monocyte counts (2.9 vs 2.2 x109/L), lower platelet counts (84 vs 102 x109/L), have circulating immature myeloid cells (52% vs 37%), and be classified as myeloproliferative (MP)-CMML (58% vs 44%) or CMML-2 (33% vs 17%) at diagnosis/first referral in CP. More mutations were detected in BT-CMML than in CP-CMML (mean 3.2 vs 2.8, p = 0.002). Mutations in RAS (NRAS, KRAS, BRAF, CBL, PTPN11; 51% vs 40%, p = 0.02) and signaling (CSF3R, FLT3, JAK2, SH2B3; 15% vs 7%, p = 0.004) pathway genes were more frequent in BT-CMML. Conversely, there were fewer BT-CMML patients with the favorable ASXL1WT/TET2MT genotype compared to CP-CMML (22% vs 33%, p = 0.004). Multivariate analysis identified MP-CMML (hazard ratio [HR] 1.5, p = 0.04), CMML-2 (HR 2.4, p < 0.001), and the presence of ≥4 somatic gene mutations (HR 1.8, p = 0.002) as risk factors for BT whereas the ASXL1WT/TET2MTgenotype was protective (HR 0.45, p = 0.002). The ASXL1WT/TET2MT genotype was also protective in the MP-CMML (HR 0.49, p = 0.04) and RAS-mutated (HR 0.35, p = 0.005) subgroups. Amongst BT-CMML cases, the median time to BT was 10 mo from diagnosis/referral. Compared to their CP assessment, patients in an overlapping Mayo Clinic BT-CMML cohort (n = 156, median age 69 years, 35% female) were more likely to have NPM1 (24% vs 3%, p < 0.001) and IDH2 (14% vs 1%, p = 0.01) mutations at BT. Amongst cases with paired molecular evaluations (n = 44), 7 (16%) acquired new mutations, including FLT3-ITD (n = 2) and NPM1 (n = 2). Conversely, 6 (14%) lost mutations, including TET2 (n = 3), SRSF2 (n = 2), CBL, NRAS, or PTPN11 (n = 1 each). There were no differences in variant allele fractions in driver genes present at CP and BT. Abnormal karyotypes were more prevalent at BT than in CP (60% vs 34%, p < 0.001), and 26 (25%) patients with a normal karyotype in CP acquired structural (65%) and/or numerical (69%) changes at BT, including 6 (23%) that acquired a complex karyotype. Acute myeloid leukemia defining translocations were seen in 4 (4%) cases: 2 with t(8;21) and 1 each with inv(3) and t(10;11)(KMT2A::TET1). After considering mutation and karyotype changes, only bone marrow blasts ≥30% (HR 1.7, p = 0.04) were independently prognostic for OS after BT (mOS 4 mo). Compared to supportive care (n = 50, mOS 1.3 mo), OS was improved by hypomethylating agents (HMA, n = 14, mOS 16 mo, HR 0.3, p < 0.001) or induction chemotherapy (n = 56, mOS 8 mo, HR 0.3, p < 0.001), but not by HMA + venetoclax (n = 11, mOS 6 mo, HR 0.7, p = 0.4). Allogeneic HCT (n = 28) improved mOS to 46 vs 3 mo (p < 0.001). Amongst 4 patients with paired whole genome sequencing before and after BT, 3 had increased copy number alterations (including 2 with loss of heterozygosity, 2 with gains, and 3 with deletions) while the 4th acquired novel likely pathogenic somatic mutations (MUC4, BAX, WNK2). Conclusions Survival in CMML after BT remains exceptionally poor. In this multicenter cohort, MP-CMML, CMML-2, and the presence of ≥4 somatic gene mutations were identified as risk factors for BT, with the ASXL1WT/TET2MT genotype being protective. Following BT, bone marrow blast burden ≥30% was an independent adverse predictor of survival.

Background: TP53-mutated (TP53mut) myeloid neoplasms (MN) have particularly poor survival. Discrepancies in defining TP53mut MN have impact on diagnostic work up, therapeutic decisions, and clinical trial enrolment. Methods: MN cases harboring TP53mut (variant allele frequency, VAF ≥2%) were classified based on bone marrow (BM) and peripheral blood (PB) blast % using the 4th revision of the WHO classification (WHO-4R). Biallelic loss was defined as (i) ≥2 mutations each with VAF ≥2%; (ii) single TP53mut VAF ≥2% plus 17p loss detected on karyotype, FISH, or SNP; or (iii) single TP53mut VAF ≥50%, whereas monoallelic loss was defined as single TP53mut VAF <50% without 17p loss. Primary endpoint was overall survival (OS) from diagnosis. Conditional Inference Trees (CIT) analysis was used to determine the true effect of a predictor. Results: We identified 580 cases including WHO-4R defined acute myeloid leukemia (AML, BM/PB blasts ≥20%, n=219, 37.8%), myelodysplastic syndrome (MDS) with excess blasts-2 (EB-2, BM 10-19% or PB 5%-19%, n=92, 15.9%), MDS with excess blasts-1 (EB-1, BM 5-9% or PB 2-4%, n=75, 12.9%), and the remaining were MDS with low blasts (-LB, BM <5% and PB ≤1%, n=194, 33.4%). Of the 578 cases evaluable for allelic status, 437 (75.6%) and 141 (24.4%) had biallelic and monoallelic TP53 loss, respectively. Higher frequency of biallelic loss was observed with increasing blasts. 63.2%, 73.3%, 78% and 86.3% of cases with MDS-LB, MDS-EB1, MDS-EB2, and AML had bi-allelic TP53 loss (P<0.0001). In a multivariable analysis, WHO-4R categories, VAF ≥10%, and biallelic loss were independent predictors of poor survival. CIT analysis further stratified the cohort into 4 groups with distinct survival: (1) MDS-LB (n=194, 33.4%); (2) MDS-EB1/EB2 and AML with VAF <10% (n=38, 6.6%); (3) MDS-EB1/EB2 with VAF ≥10% (n=149, 25.7%); and (4) AML with VAF ≥10% (n=199, 34.3%). MDS-LB cases could be further stratified based on allelic status and complex karyotype (CK): OS was poor for cases with biallelic TP53 loss and monoallelic TP53mut with CK compared to monoallelic TP53mut without CK, who had the most favorable survival (12.7 vs. 13 vs. 34.8 months, P<0.0001).MDS-EB1/EB2/AML with VAF<10% could also be stratified by CK: MDS-EB1/EB2/AML with VAF<10% cases without CK had the favorable survival compared to their counterparts with CK and MDS-EB1/EB2 with VAF ≥10% cases (26.2 vs. 5.6 vs. 6.3 months, P=0.003).MDS-EB1/EB2 with VAF ≥10% was a homogenous subgroup: Biological characteristics including the proportion of cases with biallelic TP53 loss, CK (P>0.9), monosomal karyotype (MK, P>0.9), del 5q (P=0.6), del 7q (P=0.4), and del 17p (P=0.2) were comparable between MDS-EB1 and -EB2 with VAF ≥10%. Importantly, median OS of MDS-EB1 and EB2 with VAF ≥10% was comparable (9.6 vs. 7.2 months, P=0.12). OS in this group was comparable regardless of the blast %, allelic status, or presence of CK.AML with VAF ≥10% had extremely poor survival: Median OS was 3.9 months. Survival of AML was equally poor irrespective of the mono- or biallelic (3.6 vs. 3.9, P=0.89) and non-CK vs. CK (1.8 vs. 3.9 months, P=0.09). Combined, our model would acknowledge poor survival of 514 (89.1%) cases. In contrast, retrospective application of the 5th edition of the WHO classification (WHO-5) and International Consensus Classification (ICC) would classify only 211 (36.4%) and 468 (80.7%) cases as biallelic/multi-hit TP53mut MN, respectively. Finally, the Harrell's c-index of the proposed model, WHO-5, and ICC was 0.66, 0.571, and 0.617, respectively. Conclusion: We provide evidence for subsequent refinements in classifying MN harboring TP53mut. Our salient findings include: (i) acknowledgement of CK as ‘biallelic equivalent’ in MDS-LB; as well as EB1/EB2/AML with VAF 2 to <10% cases; and (ii) demonstration of poor survival of MDS-EB1 and -EB2 with VAF ≥10% regardless of the allelic status. In conclusion, our proposed model acknowledged poor survival of a higher proportion of cases as TP53mut MN compared to the current models with a higher c- index.

Introduction: Somatic mutations in RAS pathway genes are present in ~30% of patients with chronic myelomonocytic leukemia (CMML) and are usually associated with myeloproliferative features. Whether mutations in distinct RAS pathway genes share similar genomic context and have similar implications remains unknown. Such an understanding might inform future studies evaluating genotype-specific therapies (RAS-codon specific and pan-RAS inhibitors) or cooperative leukemogenic mechanisms. This multicenter study examines the landscape of RAS pathway mutations (RASMT) in CMML. Methods: Newly diagnosed CMML patients evaluated at MD Anderson Cancer Center (n=449, 54%) and Mayo Clinic (n=383, n=46%) were included after IRB approval. Clonal relationships were tested using Pearson goodness-of-fit. Clones with the highest variant allele frequency (VAF), or with VAF close to 40% were defined as dominant, and those present at VAF <20% in the presence of another dominant clone were defined as subclonal. RASMT genes included NRAS, KRAS, BRAF, CBL, CBLC, PTPN11 and NF1. Standard statistical measures were applied. Results: Among 832 CMML patients (median age 71 years, 68% male) 387 (47%) had myeloproliferative (MP) and 445 (53%) had myelodysplastic (MD) CMML. A total of 350 (42%) patients had RASMT with higher prevalence among MP-CMML compared to MD-CMML (55% vs 31%, p<0.001). Among patients with RASMT, 262 (32%), 59 (7%), 20 (2%), 6 (<1%) and 3 (<1%) had 1, 2, 3, 4 or 5 distinct RASMT, respectively. Importantly, 17 patients harbored a RASmt as the only detectable somatic mutation, with KRASMT being the most frequent (n=10, 59%), including 3 (18%) patients with >1 RASMT. Among RASMT patients, a trend for higher hemoglobin (12.7 vs 10.8 g/dL, p=0.054) was observed in NF1MT CMML and lower WBC (14.4 vs 27.9 x109/L, p=0.067) values in CBLMT CMML. In total, 479 somatic mutations in RAS pathway genes were identified the most frequent involving NRAS (n=169, 35%) followed by CBL (n=120, 25%), KRAS (n=102, 21%), PTPN11 (n=38, 8%), NF1 (n=34, 7%), BRAF (n=11, 2%) and CBLC (n=5, 1%). Most frequently involved codons for each RAS pathway gene were as follows: G594 (n=3) for BRAF, R420 (n=22) and C404 (n=15) for CBL, G12 (n=41; G12D: 13, G12C: 3) and A146 (n=16) for KRAS, G12 (n=116; G12D: 74, G12C: 5) for NRAS and A72 (n=8) for PTPN11. CBLMT CMML had higher ASXL1 (58% vs 44%, p=0.011), BRAF (8% vs 4%, p=0.017), IDH2 (10% vs 4%, p=0.024), KIT (5% vs 1%, p=0.038), SRSF2 (54% vs 40%, p=0.004), TET2 (59% vs 47%, p=0.019) and ZRSR2 (10% vs 4%, p=0.014) co-mutation frequency. Of note, DNMT3A mutations were mutually exclusive of CBLMT CMML (0% vs 7%, p=0.007), and multihit TET2 was more common among TET2MT/CBLMT vs TET2MT/CBLWT CMML (82% vs 67%, p =0.039). CMML with NF1 mutations had higher BCORL1 (8% vs 0%, p=0.005), PTPN11 (15% vs 3%, p=0.014) and SETBP1 (23% vs 8%, p=0.022) mutational cooccurrences. NRASMT CMML was enriched for ASXL1 (53% vs 43%, p=0.042), ETNK1 (5% vs 1%, p=0.004), GATA2 (4% vs 1%, p=0.034) and STAG2 (4% vs 1 %, p=0.023) mutations but had lower JAK2 (1% vs 6%, p=0.036) and TP53 (1% vs 5%, p=0.022) mutational frequency. PTPN11 mutations were associated with higher NF1 (29% vs 7%, p=0.014), SETBP1 (27% vs 9%, p=0.01) and WT1 (6% vs <1%, p=0.014) mutational cooccurrences and lower SRSF2 (23% vs 42%, p=0.05) frequency. We evaluated RAS mutant clonal dominance in 253 (72%) RASMT patients. NRAS (63% vs 38%), KRAS (63% vs 38%) and CBL (65% vs 35%) mutations were more frequently observed as dominant clones while PTPN11 (44% vs 56%), BRAF (25% vs 75%) and NF1 (35% vs 65%) mutations were more frequently subclonal events, particularly of TET2, ASXL1 or SRSF2 mutant clones. Presence of a RAS pathway mutation was associated with shorter LFS (36 vs 53 months, HR 1.5, 95% CI 1.1-2.2, p=0.020), with presence of multiple RAS mutations shortening LFS further (53, 43, 27 months for 0, 1 and >1, p=0.017). PTPN11 mutations predicted for shorter OS (18 vs 39 months, HR 1.7, 95% CI 1.0-2.8, p=0.035) and LFS (14 vs 36 months, HR 1.9, 95% CI 1.2-2.9, p=0.008). Presence of >1 RAS pathway mutation was associated with shorter OS in ASXL1MT/TET2MT CMML compared to those with no RAS mutations (24 vs 53 months, HR 4.2, 95% CI 1.6-10.8, p=0.004). Conclusions: RAS mutations exhibit distinct genomic features and clonal hierarchy in CMML and cooperate with other somatic mutations to drive clinical outcomes. This study forms the foundation for assessment of novel RAS pathway inhibitors in CMML.

Background Venetoclax + hypomethylating agent (Ven-HMA) therapy results in favorable response rates (CR/CRi 66%) and overall survival (OS; median 14.7 months) in unfit patients with newly-diagnosed acute myeloid leukemia (ND-AML; N Engl J Med 2020;383). However, there is substantial heterogeneity in individual patient outcomes based on response to treatment. The objective of the current study was to develop a genetic risk model to predict survival outcome in treatment responders and non-responders, analyzed separately, and also account for allogeneic stem cell transplant (ASCT). Methods Treatment-naïve AML patients receiving Ven-HMA, outside of a clinical trial, at the Mayo Clinic, USA (MN, AZ, FL) were retrospectively recruited. Molecular studies (48-gene panel) were performed by next-generation sequencing. Response was assessed according to the 2022 European LeukemiaNet (ELN) criteria (Blood 2022;140). Standard statistical analyses were performed using JMP Pro (Version 17.0.0). Results 400 ND-AML patients (median age 73 years, 64% male, 60% de novo) received Ven-HMA. ELN cytogenetic risk included favorable 2%, intermediate 60%, or adverse 38%. Mutations involved TP53 in 26%, TET2 19%, RUNX1 19%, SRSF2 18%, ASXL1 18%, DNMT3A 15%, K/NRAS 13%, NPM1 12%, IDH2 12%, FLT3-ITD 10%, IDH1 7% and DDX41 in 4% of informative cases. 153 (38%) patients achieved CR, 94 (24%) CR with incomplete count recovery (CRi), resulting in CR/CRi in 247 (62%) patients. CR/CRi rates were higher with NPM1 (86% vs 59%), IDH2 (77% vs 60%), and DDX41 mutations (93% vs 61%) and lower with TP53 (45% vs 67%), FLT3-ITD (41% vs 64%), RUNX1 (49% vs 65%) mutations and adverse karyotype (48% vs 71%), with all retaining significance in multivariable analysis. CR/CRi rate was highest (87%) in 75 patients with one or more favorable mutations (NPM1, IDH2, DDX41) and no unfavorable mutation (TP53, FLT3-ITD, RUNX1). CR/CRi rate was lowest in 171 patients with at least one unfavorable mutation and no favorable mutation (44%; p<0.01). After a median follow up of 10.5 months (0.5-66), 105 relapses (43% of patients in CR/CRi), 248 (62%) deaths and 65 (16%) ASCT were documented. Univariate analysis for transplant-censored OS disclosed superior survival in patients achieving CR/CRi (median 21 vs 3.6 months; HR 5.4, 95% CI 4.1-7.1). Among patients achieving CR/CRi, IDH2 mutation was associated with superior (median not reached vs 20.5 months) and male gender (median 17.6 vs 29.2 months), TP53 mutations (median 11.9 vs 24.3 months) and adverse karyotype (median 12.6 vs 29.4 months) with inferior survival; multivariable analysis resulted in HRs (95% CI) of 2.1 (1.3-3.4) for male gender, 2.1 (1.2-3.8) for adverse karyotype, and 2.5 (1.1-5.7) for absence of IDH2 mutations. Male gender did not show an association with karyotype, or TP53 mutations (p>0.1). Accordingly, an HR-weighted genetic risk model was generated with assignment of one point each to male gender, adverse karyotype, and absence of IDH2 mutation: high risk = 3 risk factors (n=38; median 9.6 months; 3-year survival 0%); intermediate risk = 2 risk factors (n=129; median 22 months; 3-year survival 38%); and low-risk = ≤1 risk factor (n=78; median not reached; 3-year survival 69%; p<0.01). In patients bridged to ASCT (n=60), post-transplant survival was inferior in the presence of ≥ 2 risk factors (n=42; median 46 months) vs. ≤1 risk factor (n=18; median not reached; p=0.1). In patients not achieving CR/CRi, risk factors for OS included male gender (median 3.4 vs 6.1 months), adverse karyotype (median 3.3 vs 4.5 months) and TP53 mutation (median 3.1 vs 4.5 months) while those with IDH2 mutations lived longer (median 8.4 vs 3.4 months). In multivariable analysis, HRs (95% CI) were 1.9 (1.3-2.9) for male gender, 2.0 (1.4-2.9) for adverse karyotype, and 2.1 (1.1-4.3) for absence of IDH2 mutations. OS was shortest for non-responders harboring 3 risk factors (n=45; median 3 months; 1-year survival 0%) and longer for those harboring two (n=80; median 6 months, 1-year OS 15%) or ≤ 1 risk factor (n=25; median 14 months; 1-year OS 56%; p<0.01). Conclusion The current study describes a molecular profile for predicting response to Ven-HMA in ND-AML and also introduces a novel approach to survival prediction that considers treatment response as the starting point. In both responders and non-responders, male gender and adverse karyotype were identified as unfavorable and IDH2 mutation favorable risk factor for OS.

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid stem cell neoplasm with few effective therapies and poor prognosis. This disease is characterized by an expansion of malignant monocytes which express high and homogeneous levels of the homing chemokine receptor CCR2. We have developed a CCR2 targeting Cytotoxicity Targeting Chimera (CCR2-CyTAC, STX-0712), which potently binds CCR2 on cells, and when combined with the CyTAC platform antibody, eliminates target cells via enhanced effector mediated killing. We present here the characterization of CCR2 expression in CMML patient samples, ex vivo depletion of CMML cells via treatment with STX-0712, and the dose dependent depletion of MO1 monocytes (classical monocytes) in non-human primates, supporting CMML as a promising indication for STX-0712. Methods: IRB approval was obtained from Mayo Clinic for patient sample use. For CCR2 characterization and depletion experiments, we used bone marrow biopsy specimens and peripheral blood mononuclear cells (PBMCs) obtained from CMML patients and controls from healthy donors. CCR2 expression and monocyte depletion were evaluated via flow cytometry, as previously described. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) studies were conducted in cynomolgus monkeys. Results: First, we evaluated CCR2 expression in peripheral blood and bone marrow in CMML samples by flow cytometry. In 6/6 (100%) peripheral blood samples CCR2 was highly expressed on >98% of the monocytes. Also, in 3/3 (100%) bone marrow samples, CCR2 was expressed on a subset of CD34+ progenitors, while low/no expression was observed in healthy controls. This progenitor subpopulation had characteristics of the inflammatory granulocyte-macrophage progenitor (GMP)-like cells. Next, STX-0712 was added to freshly isolated PBMCs from CMML donors and CCR2+ monocyte depletion, mediated by endogenous Fc gamma receptor 3a positive (FcγRIIIa) effector cells, was assessed. In 5/5 (100%) CMML patient samples, the drug selectively induced depletion of CD14+, CCR2+ cells with an average potency of 3nM and 66-91% target cell killing vs no drug control. Finally, we evaluated the effects of STX-0712 in cynomolgus monkeys to evaluate PK and PD effects on CCR2 MO1 monocytes in vivo. STX-0712 demonstrated PK supportive of dosing in man. In 9/9 (100%) animals, STX-0712 effectively depleted >95% of CCR2+ monocytes which returned after drug levels dropped below ~10ng/mL. All doses were well tolerated and the findings of comprehensive immunophenotyping were to be as expected, predictable and mechanism directed. Conclusion: These results demonstrate high and homogenous CCR2 expression on malignant monocytes, ex vivo human target engagement and pharmacodynamics, and in vivo depletion of CCR2 MO1 monocytes in a potent and dose dependent manner. Based on this data we plan a First in Human, phase 1 clinical trial for patients with relapsed/refractory CMML.

Background Venetoclax + hypomethylating agent (Ven-HMA) therapy has demonstrated improved outcomes in elderly/unfit patients with newly-diagnosed acute myeloid leukemia (ND-AML) (N Engl J Med 2020;383). However, ~50% of patients who respond to Ven-HMA, eventually relapse (Am J Hematol 2024;99). In the current study we examined the prognostic contribution of gene mutations, karyotype, measurable residual disease (MRD) status and clinical risk factors for relapse in patients with ND-AML treated with Ven-HMA therapy. Methods Our study population of ND-AML was retrospectively recruited from Mayo Clinic (MN, AZ, FL), after institutional review board approval and based on documentation of complete remission with/without count recovery (CR/CRi) following Ven-HMA therapy. Relapse-free survival was determined from the time of remission to relapse or last follow-up. Survival analysis was censored for transplant. The 2022 European LeukemiaNet (ELN) criteria was used to assign cytogenetic risk and classify treatment response (Blood 2022;140). Standard statistical analyses were performed using JMP Pro (Version 17.0.0). Results 247 ND-AML patients treated with Ven-HMA (median age 73 years, 62% male, 64% de novo) who achieved CR (n=153), or CRi (n=94) were included. MRD by multiparameter flow cytometry (sensitivity 0.01%) performed at the time of CR/CRi, was negative in 117 (70%) of 166 informative cases. ELN cytogenetic risk categories at diagnosis included favorable 2%, intermediate 69%, or adverse 29%. Mutations involved TET2 21%, SRSF2 21%, TP53 19%, ASXL1 18%, DNMT3A 18%, NPM1 17%, IDH2 15%, RUNX1 15%, K/NRAS 12%, IDH1 8%, FLT3-ITD 7%, and DDX41 in 6% of informative cases. Relapse was documented in a total of 99 (40%) patients who achieved CR (39%) or CRi (41%) after a median remission duration of 6 months (1-31). Patients that relapsed within the first-year vs those that did not, were more likely to be males (75% vs 55%), MRD positive (44% vs 24%), and harbor TP53 mutations (23% vs 16%). Univariate analysis for RFS identified male gender (12 vs 29 months; p=0.02), secondary AML (10 vs 21 months; p<0.01), adverse karyotype (9 vs 22 months; p<0.01), TP53 mutations (6 vs 18 months, p<0.01) and MRD positive remission (13 months vs not reached; p<0.01) as risk factors for relapse; on the other hand, IDH2 mutations (not reached vs 15 months; p<0.01) were associated with a lower risk of relapse. Multivariable analysis confirmed male gender (HR 2.2), TP53 mutations (HR 3.0) and MRD positive remission (HR 1.7) as unfavorable predictors of relapse. Subsequently, in a three-tiered relapse prediction model, RFS was significantly inferior in the presence of three risk factors (male gender, TP53 mutations, MRD positive remission) (n=10, median 4 months), vs two (n=34, median 10 months) vs ≤ 1 risk factor (n=122, median 31 months) (p<0.01); 1-year cumulative incidence of relapse was 100%, 56%, and 26% in the presence of three, two and ≤ 1 risk factors, respectively. 50 of 99 (65%) patients received salvage therapy which included cladribine-cytarabine-Ven (n=19), intensive induction chemotherapy (n=9), FLT3 inhibitors (n=5), Ven-HMA-FLT3 inhibitor (n=1), IDH1/2 inhibitors (n=5), glasdegib-cytarabine (n=4), gemtuzumab (n=3), lenalidomide (n=1), or investigational therapies (n=3); in addition, Ven-HMA was resumed in 14 patients in whom treatment had been discontinued for a median of 5 months (2-12). Overall, 20 (31%) of patients achieved second CR/CRi and response rates were similar with intensive vs less intensive therapy (CR/CRi; 44% vs 29%; p=0.37). Median follow up after relapse was 3.4 months (1-66), during which 73 (74%) deaths, and 4 (5%) ASCT were recorded. Median transplant-censored survival was 4.2 months (1-year; 22%), and inferior in patients with adverse karyotype (median 4.6 vs 3.4 months; p=0.02), and TP53 mutations (3.6 vs 4.4; p=0.11); on the other hand, male gender (3.5 vs 6.8 months; p=0.51), MRD positive remission (4.6 vs 4.2; p=0.56), and IDH2 mutations (6.7 vs 4.2; p=0.28), did not influence post-relapse survival. Conclusions The current study identifies male gender, TP53 mutations, and MRD status as powerful predictors of relapse and proposes a relapse prediction model for ND-AML patients receiving Ven-HMA therapy. Furthermore, relapse following Ven-HMA was associated with uniformly poor outcomes, regardless of salvage treatment strategy.

Introduction: DDX41, DEAD-box RNA helicase 41 gene located on chromosome 5q25.3, is one of the most mutated genes in patients with germline predisposition to myeloid neoplasms. Germline and somatic mutations often have different locations and patterns of mutation, with some hot-spots displaying diversity based on ethnicity. We aimed to explore clinical outcomes in patients with various DDX41 hot-spots. Methods: A retrospective study of patients at Mayo Clinic with DDX41 mutation identified through Next Generation Sequencing (NGS) between 2016 and 2024. Patients classified as germline DDX41 variants or somatic variants based on confirmed germline testing results. Data regarding mutations recorded at time of patients' first in-house NGS. We completed unadjusted comparisons using continuous or categorical variables, and survival rates assessed using Kaplan-Meier method and cox regression analysis. Median overall survival calculated from time of NGS to last follow-up or death using BlueSky Statistics V.10.3.4 Results: Clinical Characteristics: Eighty-two patients analyzed, predominantly male (69.5%), with median age of diagnosis of 68 years (IQR 62-75). Most common diagnosis was MDS (50%), followed by AML (22%) and myeloproliferative neoplasm (MPN) (7.3%). Fourteen percent were DDX41 mutation carriers, 4.9% clonal cytopenia of undetermined significance (CCUS), and 1.2% chronic neutrophilic leukemia (CNL). MDS-IB2 was the prevalent MDS subtype (56.1%), and 83.3% of AML patients were intermediate risk. Cytogenetics normal in 73.8% of patients, with del20 being the most common abnormality (7.5%). Twenty-five (30.5%) of patients underwent allogeneic hematopoietic cell transplantation (alloHCT). Forty-nine patients (59.8%) had pathogenic DDX41 mutation only, 15 (18.3%) had only VUS, and 18 (22.0%) had both. Molecular Characteristic s: Mutation hot-spots included 12.2% with p.Arg525His only, 28% p.M1I only, 9.8% p.Asp140GlyFs*2 only, 9.8% p.Arg525 with co-mutation other than p.M1I/p.Asp140. Two patients (2.4%) had p.M1I co-mutation with non-p.Arg525 and p.Asp140, while 1 patient (1.2%) had p.M1I and p.Arg525, and 1 patient (1.2%) had p.Asp140 and p.Arg525. Lastly, 33 patients (40.2%) had mutations in hot-spots other than specified in this study. Median hemoglobin (Hg) was lowest in patients with p.Arg525 hotspot mutation (9.9 g/dl), compared to median Hg of 11.2 g/dl in group with p.M1I and 10.4 g/dl in p.Asp140 only group (p=0.87). The most common cytogenetics in patients with only p.Arg525 and only p.M1I hot-spot mutation was normal cytogenetics (70% and 90.9%, p=0.5). In the 16 patients with proven germline DDX41 mutation, 7 (43.8%) had mutations in only p.M1I. Lastly, median (m) VAF was significantly lower in the group with only p.Arg525 hot-spot mutation (9.5%, IQR 5.5-13.8), compared to mVAF of 48% in patients with only p.M1I (IQR 48.0-49.5, p<0.001). Co-mutations: The most frequent co-mutation was DNMT3A (11.0%), followed by JAK2 (9.8%).Mean number of co-mutations in p.Arg525 only was 0.6, 0.3 in p.M1I only, and 0.5 in p.Asp140 only (p=0.8). Most common co-mutation in p.M1I was ASXL1, with 13.0% of patients with p.M1I only having ASXL1 co-mutation (p=0.062). The most common co-mutation in p.Asp140 was DNMT3A (p=0.7). Survival: Median overall survival (mOS) was 21.5 months (IQR 12.4-38.6). mOS for p.Arg525 only group was 31.3, compared to 41.2 months in p.M1I only (p=0.197). At 12- and 24-months, survival rate was 88.9% and 59.3% in the p.Arg525 only group, compared to p.M1I group with 100% survival rate at 12 months and 89.5% at 24 months. p.Asp140 had 87.5% survival rate at both 12 months and 24 months. Males in p.M1I group had lower survival rate at 24 months (81.8%), compared to 100% survival in females. p.M1I only group with alloHCT also had lower 24 month survival at 60% compared to 100% in group who did not undergo alloHCT. Conclusions: The p.M1I hot-spot was commonly seen in patients with germline DDX41 mutation, likely resulting in significantly higher VAF percentage. Co-mutations frequency differed by hot spot mutation while p.Arg525 as second mutation was seen mainly in cases other than p.M1I/pArg140Gly. Overall survival appears to be higher in p.M1I/pArg140Gly compared to p.Arg525His hot-spot mutation, though males with p.M1I and those who underwent alloHCT had lower survival. Findings suggest implications for clinical outcomes based on DDX41 mutation hot-spots.

DDX41 MT Germline Predisposition Syndrome (GPS) is the most common adult GPS for myeloid neoplasms (MN), including MDS and AML. Although there have been reports of non-myeloid neoplasms, particularly lymphoid malignancies, in DDX41MT-GPS, these occurrences are infrequent and poorly characterized. We carried out this study in a large cohort of DDX41MT-GPS patients to address this deficiency. Following IRB approval, we retrospectively analyzed our database of 174 patients with DDX41MT-GPS to determine the rates of non-myeloid malignancies. Our previous data revealed that certain DDX41MT genotypes classified as variants of uncertain significance (VUS) exhibit a similar phenotypic spectrum to those classified as pathogenic (Badar et al Hematologica). Consequently, we included all these variants in our analysis. The median age of the cohort was 68.6 yrs (range, 13-4-93.4); 66% (n=114) male, 93% (n=162) Caucasian, with a median follow-up of 1.4 yrs (range, 0.03-5.6) from DDX41MT-GPS detection. The cohort included 34 (19.5%) asymptomatic carriers, 17 (9.8%) with clonal hematopoiesis (CH) or clonal cytopenias (CCUS), and 123 (70.7%) with MN. Eighteen (10%) patients had a lymphoid neoplasm (78% male, 100% Caucasian), with a median age at lymphoid neoplasm diagnosis of 67.7 yrs (range, 12.2-85.9). The most common lymphoid neoplasm was chronic lymphocytic leukemia (CLL) seen in 2.3% (n=), followed by follicular lymphoma (FL) and acute lymphoblastic leukemia (ALL) present in 1.7% each (n=3; 1 T-ALL and 2 B-ALL: 1 Ph+ and1 Ph-like). Additionally, there was 1 patient each with monoclonal B cell lymphocytosis (MBL), Waldenstroms macroglobulinemia , peripheral T cell lymphoma , large granular lymphocytic leukemia, and marginal zone lymphoma. Germline genotypes were classified as pathogenic in 50% (n=9, D140Gfs*2 x2, K331del, M1I x3, M316Dfs*31, P258L, Y340N) and VUS in 50% (n=9, E426K, E69D, G313D, K184R, P206A, P70S, Q246E, R164W, c.572-3T>C. Among these 18, 9 (50%) patients had co-existing myeloid disorders (CCUS x3, MDS x5, AML x1), and 5 (27.8%) patients were later diagnosed with therapy-related myeloid neoplasms (TMN) at a median of 5.9 yrs (range,1.6-38.7) after lymphoid neoplasm diagnosis and treatment. Seven (4%) patients had plasma cell dyscrasias (PCD; 3 with monoclonal gammopathy of undetermined significance(MGUS) and 4 with multiple myeloma (MM)); 57% (n=4) were male, with a median age of 61.3 yrs (range, 41.4-83.8) at PCD diagnosis. Subtypes of MGUS included IgG Kappa (n=2) and IgG Lambda (n=1), subtypes of MM included IgG Kappa (n=2), IgA Kappa (n=1), and Kappa light chain only (n=1). DDX41MT-GPS genotypes were pathogenic in 28.6% (n=2, G67Vfs*13, G313S) and VUS in 71.4% (n=5, A555T, R164W, Q246E x2, M307T). All patients with MM (n=4) received an autologous stem cell transplant, with 3 (75%) being subsequently diagnosed with a t-MN; median 3.6yrs after PCD diagnosis (range, 3.2-12.3). Seventeen percent (n=31) had visceral malignancies; 2.8% (n=5) having >1malignancy, 61% (n=19) male, 93.5% (n=29) Caucasian and 6.5% (n=2) Hispanic. Prostate cancer was most common (n=8, 4.6%), followed by breast (n=7, 4.0%), colon (n=5, 2.9%), squamous cell carcinoma (n=4, 2.3%, 3 of the head/neck and 1 of the leg), bladder (n=4, 2.3%), testicular (n=3, 1.7%), gynecologic (n=3, 1.7%), lung (n=2, 1.2%), thyroid (n=1, 0.6%) and pancreatic neuroendocrine tumor (PNET, n=1, 0.6%), respectively. The median age at first solid tumor diagnosis was 62.4 yrs (range, 10.9-81.9). DDX41MT-GPS was pathogenic in 64.5% (n=20, D140Gfs*2 x3, E113Kfs*14, G67Vfs*13, I396T, L283Cfs*21, M1I x4, M316Dfs*31 x2, p.M1?, P258L, Q368*, Q41* x2, R159*, R311*) and VUS in 35.5% (n=11, D30Y, H203Y, I215L, I337V, M155I, p.?, S289L, T13I, V318G, A555T). Of the 31 patients with visceral malignancies , 51.6% (n=16) were diagnosed with t-MN at a median of 4.9 yrs (range, 1.1-34.3) after malignancy diagnosis. While DDX41MT-GPS are clearly associated with an increased risk of myeloid neoplasms, we demonstrate potential associations with other neoplasms, especially lymphoproliferative disorders (10%). We for the first-time report associations with PCD (4%) and visceral malignancies (17%) and also highlight a worrisome trend for t-MN in DDX41MT-GPS treated with genotoxic agents. Given limitations of a retrospective analysis, this study underscores the need for a comprehensive natural history study in patients with DDX41MT-GPS .

Background The prognostic relevance of TP53 mutations (TP53MT) in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) has been extensively studied and the information accessed for defining the new International Consensus Classification (ICC) category of “myeloid neoplasms with mutated TP53” (Blood 2022;140:1200). The latter designation requires ≥10% TP53 variant allele frequency (VAF) for AML and MDS/AML and in case of MDS, multi-hit TP53 or ≥10% VAF associated with complex karyotype (CK). We examined the prognostic relevance of TP53MT in myeloproliferative neoplasms (MPN), specified by subtype and disease stage. Methods The current study was conducted under an institutional review board approved minimum risk protocol. Additional informative cases from the University of Florence, Italy and the South Australian Myeloid Neoplasm Registry were included. Diagnostic criteria and assignment of TP53 allelic state were according to the ICC. Next-generation sequencing (NGS) and cytogenetic studies were performed according to institutional standard protocols. Conventional statistical methods were employed. Survival analyses were censored for allogeneic stem cell transplantation (ASCT) and were calculated from the time of first detection of TP53MT. Results 115 patients with MPN and documented pathogenic TP53MT were considered (median age 68.5 years). ICC-defined subtype designations, at time of TP53MT detection, were polycythemia vera (PV) or essential thrombocythemia (ET) (N=9), chronic phase myelofibrosis (MF-CP; N=63), accelerated phase MPN (MPN-AP; N=16), and blast phase MPN (MPN-BP; N=27). Driver mutation distribution was JAK2 64%, CALR 18%, MPL 8%, and triple-negative 10%. Karyotype was monosomal (MK) in 42%, CK 9%, and normal (NK) 35%. Frequent co-mutations were ASXL1 (28%), TET2 (14%), and SRSF2 (14%). TP53MT were classified as ICC-defined multihit in 67 (58%) cases; karyotype in the latter was MK (58%), CK (16%), non-MK/CK (13%), or NK (13%). TP53 VAF was ≥50% in 27 (24%) patients and <10% in 25 (22%). TP53MT included one (N=96) or ≥2 (N=19). MPN subtype- and stage-specific distributions for multihit TP53MT were 91% for MPN-BP, 88% MPN-AP, 44% MF-CP, and 0% ET/PV. In order to examine disease subtype- and stage-specific prognostic contribution of TP53MT, the study population was divided into 10 operational groups: 1) PV/ET non-multihit/non-MK/CK (N=8; VAF range 3-32%); 2) MF-CP non-multihit/non-MK/CK with TP53MT VAF <10% (N=10); 3) MF-CP non-multihit VAF ≥10% and non-MK/CK (N=11); 4) PV/ET/MF-CP non-multihit with MK/CK (N=5); 5) MF-CP multihit/non-MK/CK (N=8); 6) MF-CP multihit and MK/CK (N=20); 7) MPN-BP/AP non-multihit/non-MK/CK (N=2); 8) MPN-BP/AP non-multihit with MK/CK (N=2); 9) MPN-BP/AP multihit/non-MK/CK (N=9); and 10) MPN-BP/AP multihit with MK/CK (N=30). The corresponding median survivals were “not reached”, 55, 42, 6, 18, 10, “not reached”, 4, 4, and 6 months (p<0.01). Survival was similar between groups 1, 2, and 3 (p>0.2 for all comparisons) now referred to as “PV/ET/MF-CP non-multihit/non-MK/CK (N=39)”. Survival was similarly dismal for groups 8, 9, and 10 (p values > 0.3 for all comparisons) now referred to as “MPN-BP/AP with or without multihit TP53MT or MK/CK (N=43)”. Survival was also similar between groups 4, 5, and 6 (p values >3 for all comparisons) now referred to as “PV/ET/MF-CP with multihit TP53MT or MK/CK (N=33)”. The three consolidated groups were subsequently compared for survival that showed a significantly worse outcome for “MPN-BP/AP with or without multihit TP53MT or MK/CK” with median survival of 4 months, compared to 12 months for “PV/ET/MF-CP with multihit TP53MT or MK/CK” (HR 2.3; 95% CI; 1.3-3.9) and 55 months for “PV/ET/MF-CP non-multihit/non-MK/CK” (10.9; 95% CI 5.3-22.4); survival in the latter two groups was also significantly different, in favor of PV/ET/MF-CP non-multihit/non-MK/CK (HR 4.8; 95% CI 2.3-10.1). Conclusions: The presence of multi-hit TP53MT or MK/CK, in MF-CP, and possibly in ET/PV, warrants their inclusion in the ICC category of “myeloid neoplasms with mutated TP53”. By contrast, non-multihit TP53MT in ET/PV/MF-CP, regardless of VAF and in the absence of MK/CK, does not appear to compromise short-term survival. The observations from the current study also suggest a prognostic hierarchy with blast percentage being the most unfavorable, followed by MK/CK and multihit TP53MT.

Introduction: Response assessment is used to evaluate effectiveness of hypomethylating agent (HMA)-based therapy in myelodysplastic syndromes/neoplasms (MDS) in clinical practice and trials. Recently, the clinical response criteria (IWG 2023) and the clinical-molecular risk assessment (IPSS-M) in MDS were published. However, it remains unknown whether the IWG 2023 response criteria have an independent impact on overall survival (OS) when adjusting for baseline IPSS-M risk. Additionally, the association of IWG 2023 response criteria with OS in patients (pts) undergoing allogeneic stem cell transplantation (allo-HCT) versus those who do not is unknown. Lastly, it is unclear if optimal time-to-transplant impacts OS in MDS pts who achieve a IWG 2023 defined remission with HMA therapy. We examined these questions in a large population of MDS pts within the multi-institutional international consortium for MDS (icMDS) VALIDATE database. Methods: High-risk MDS pts with IPSS ≥1.5 or IPSS-R >3.5 treated with HMA-based therapy were included in the study. Baseline IPSS-M risk was stratified into very low/low/moderate low (VL+L+ML), moderate-high/high (MH+H), and very high (VH). Response to treatment was evaluated using the IWG 2023 criteria and grouped into: composite complete response (cCR) and no cCR. cCR included: complete response (CR), CR equivalent (CRequ), CR bilineage (CRbi), CR unilineage (CRuni), and CR with partial hematologic recovery (CRh). We analyzed the differential impact of achieving cCR on HMA-based therapy prior to transplant on OS in pts who either did or did not undergo allo-HCT, while adjusting for their baseline IPSS-M risk in Cox regression analysis. Optimal time-to-transplant was determined based on age and IPSS-M category as described by Tentori et al. (JCO 2024). Pts proceeding to allo-HCT were stratified on whether they underwent transplant within the optimal time-to-transplant timeframe after MDS diagnosis or not. VALIDATE protocol was exempted, acknowledged, or approved by all centers' IRBs. VALIDATE is supported by an independent grant from AbbVie. Results: We analyzed 762 MDS pts. The median age at MDS diagnosis was 68 years (range: 19-95 years) and 64% of pts were male. Distribution of the baseline IPSS-M risk was: VL+L+ML in 110 (14%) pts, MH+H in 347 (46%) pts, VH in 305 (40%) pts. Pts received azacitidine monotherapy, decitabine monotherapy, and HMA-based combination therapy (e.g. HMA + venetoclax) in 52%, 21%, and 28% of cases, respectively. A total of 282/762 (37%) pts achieved cCR as their best response to HMA-based therapy. The median OS for all pts was 18.7 months (95% CI: 16.6-21.3 months). Pts who did not undergo allo-HCT (n=436, 57%) had an improved median OS if they achieved cCR when compared to pts in the no cCR group in all IPSS-M risk groups (VL+L+ML: 23.5 months vs. 10.7 months; MH+H: 20.8 months vs. 11.6 months; VH: 13.8 months vs. 7.5 months). Cox analysis demonstrated an independent effect of cCR on OS in these pts when adjusted for baseline IPSS-M risk (cCR vs. no cCR: HR 0.60, 95% CI 0.48-0.76, p<0.001). In contrast, in pts who subsequently received an allo-HCT (n=326, 43%), achieving cCR with HMA prior to allo-HCT did not provide an additional OS benefit, when adjusted for baseline IPSS-M risk (cCR vs. no cCR: HR 1.00, 95% CI 0.73-1.38, p=0.986). The median time-to-transplant was 6.7 months and 34% of transplanted MDS pts received their allo-HCT during the optimal timeframe. Among pts who achieved cCR with HMA-based therapy prior to allo-HCT, optimal time-to-transplant was associated with significantly improved OS (optimal vs. non-optimal timing: HR 0.55; p=0.033). Conclusion: Baseline IPSS-M risk and allo-HCT had an independent impact on OS, while IWG 2023 defined cCR had an independent effect on OS only in non-transplanted MDS pts. While this retrospective analysis occurred at large academic centers with specific expertise in the management of MDS, our data may have an impact on clinical practice: (i) failure to achieve cCR on HMA therapy prior to transplant should not impact the decision to proceed to allo-HCT, (ii) upfront allo-HCT without prior HMA therapy might also be an option if the clinical situation and availability of donors allow, and (iii) allo-HCT should ideally be performed during the timeframe that is considered optimal as defined by pt age and baseline IPSS-M.