Citations

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: Telomere biology disorders (TBD) are multisystemic accelerated aging syndromes due to abnormal telomere length (TL) shortening and increased cancer risk. Pediatric-onset TBD is characterized by severely shortened TL and specific associations with germline pathogenic variants mainly in DKC1, TINF2, PARN genes and characteristic clinical features [bone marrow failure (BMF), abnormal nail/skin/oral mucosa]. Adult-onset TBD is genetically distinct (predominantly, TERT, TERC, and RTEL1 mutations) with varied organ involvement [interstitial lung disease (ILD), BMF, cirrhosis, or bone disease] and heterogenous outcomes. In our previous study of predominantly adult patients with short telomeres, multiple organ involvement (Mangaonkar A et al. Blood Cancer J 2021) predicted poor overall survival (OS). We sought to validate these findings in a larger, well annotated adult cohort of TBD patients. Methods: Patients with suspected TBD underwent comprehensive clinical and molecular evaluation through our TBD-dedicated clinic after regulatory approval. TL was assessed through clinical flow cytometry fluorescence in-situ hybridization (FlowFISH).Clinical and laboratory features, including pulmonary imaging, lung volumes/function testing, hepatic imaging (MR elastography/fibroscan) and bone density assessments were retrospectively abstracted. Germline and somatic genetic testing was performed on peripheral blood through targeted panels for known TBD and myeloid genes respectively. Cox proportional hazard model was used to identify significant variables that also retained independent prognostic impact. Results: Overall, 122 patients met criteria for TBD as defined by lymphocyte TL (LTL) <=10th percentile and/or a presence of a germline-TBD variant; 34 patients LTL <1st percentile, 84 1st and the 10th percentile (27 with TBD-germline variants) and 4 with only TBD-germline variants. Median age at diagnosis was 56 (18-77) years, with 72 (59%) males. Clinical features included presence of ILD (n=81, 66%), BMF (n=39, 32%), cirrhosis/hepatic fibrosis (n=19, 38%), bone disease osteopenia/osteoporosis/fractures, n=53, 70%), MDS/AML (n=11, 9%), non-hematological cancer (n=38, 31%), and head and neck cancer (n=2). Germline pathogenic variants were detected in 22 (18%), most frequently in TERT (n=10), RTEL1 (n=7) and TERC (n=2), while variants of uncertain significance (VUS) were seen in 35 (29%) patients, especially in TERT (n=20), TERC/RTEL1(n=6), NAF1 (n=4). Somatic variants in hematopoietic cells were detected in 37 (30%) patients with frequent alterations seen in DNA damage response and repair (PPM1D/TP53, n=11), and splicing component (U2AF1/SF3B1/ZRSR2, n=9) genes. At last follow-up [median of 3 (0-18) years], there were 26 (21%) deaths (most commonly, progressive ILD-18, cancer of lung/breast/pancreas-3, transplant-related complications-2). After censoring patients at the time of any transplantation (lung transplant-26, liver transplant-5, bone marrow transplant-8), the Kaplan-Meier estimate of median OS was not reached. Univariate survival analysis found adverse OS predicted by older age at diagnosis (P=0.006), presence of ILD (P=0.0002), hepatic fibrosis/cirrhosis (P=0.02), and somatic DNA repair variants (P=0.03). Multivariate model analysis found that only presence of ILD retained independent prognostic impact (HR=2, P=0.02). Among radiographic ILD subtypes, UIP was the most common (51%), followed by NSIP (10%), and others (6%), with median FEV1 of 2L (range: 0.5-4.6), FVC 2.4L (0.7-6.2), and TLC 4L (1.9-7.4). TBD-ILD patients were older (P<0.0001) with a male preponderance (P=0.03), 52 (64%) with multi-organ involvement, and had a less severe hematological phenotype [lower incidence of BMF (P<0.0001) and fetal hemoglobin elevation (P=0.01)], more frequent bone disease (P=0.0009), relatively longer telomeres in lymphocytes (P=0.04), and no particular association with any germline or somatic genetic alterations. Conclusions: Presence of ILD is an independent and significant predictor of adverse survival in adult TBD. Within adult TBD, patients with ILD represent a unique clinical subtype without any obvious genetic or pathologic correlates. Focusing on definitive therapeutic measures in this subgroup of adult TBD has the best chance to alter natural history and improve outcomes.

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.

Introduction: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is an adult-onset autoinflammatory disorder defined by somatic UBA1 mutations in hematopoietic cells. VEXAS is associated with a high frequency of clonal hematopoiesis (CH- 60%); with the clonal landscape being dominated by DNMT3A mutations. In our prior study (Gutierrez-Rodrigues Blood 2023), we showed that DNMT3A mutations in VEXAS associated with increased mortality, but without a higher risk of MDS/AML. As DNMT3A is a critical epigenetic regulator, we conducted this study to define the epigenetic deregulation and impact on mortality of DNMT3Amt (mutant) CH in VEXAS. Methods: After IRB approval, 88 VEXAS patients from the Mayo Clinic and the NIH were retrospectively assessed, including 81 screened for CH by error-corrected DNA sequencing (ECS). DNA methylation changes were identified using the Illumina Infinium MethylationEPIC BeadChip on peripheral blood samples (PB). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed using the 10x Chromium 3' GEX platform; 16,600 pooled cells were subjected to UMAP dimensionality reduction, followed by Azimuth based cell identification. Genotyping of targeted loci with chromatin accessibility (GoTChA) was conducted on two PB DNMT3Amt samples from CCUS individuals (Izzo et al Nature 2024). Results: Patients' median age was 70 years (range: 56 - 89); 95% were white. Twelve out of 81 patients screened by ECS were found with a DNMT3Amt (15%), with a median variant allele fraction (VAF) of 42% (range: 6-46%). In this cohort, while the presence of any CH mutation did not impact survival, DNMT3Amt CH had an age-independent negative impact on mortality (5.5 years vs 8.7 years, p =0.005), without an increase in risk for MDS/AML. Changes in DNA methylation were profiled in 19 and 9 VEXAS patients without CH (Median Age: 68) or with DNMT3Amt CH (Median Age: 74), respectively. VEXAS samples with DNMT3Amt CH had >4,000 differentially methylated CpGs with 95% of the CpGs being hypomethylated compared to those without CH. Using the Roadmap Epigenomics project, we found that this hypomethylation was enriched at enhancer (Enh) sites and actively transcribed states (TxWk). Pathway analysis revealed that the hypomethylated sites were in or near genes involved in the development and differentiation of leukocytes, with IL-13 and IL-15 being identified as upstream regulators of the methylation changes. Bulk RNA-seq performed on PB of 2 and 3 VEXAS patients with and without DNMT3Amt, respectively, identified >1,500 differentially expressed genes in DNMT3Amt samples, 78% of these being overexpressed. Pathway analysis was enriched in gene pathways involved in the development of leukocytes, with IL-13 and IL-15 once again being identified as upstream regulators. Integration of methylation and transcriptomic data identified upregulation of several genes within the IL-13 and IL-15 pathways, with these regions correlating with a decrease in methylation. To assess if the epigenetic dysregulation is linked to DNMT3A mutations, we performed GoTChA-seq on two DNMT3Amt clonal cytopenia samples (CCUS). The comparison of the number of cutsites in DNMT3A wild type vs mt cells from the same individual demonstrated increased open chromatin within the IL-13 and IL-15 pathways in mt cells, both globally and in a genomic locus specific manner. CITE-seq performed on 3 VEXAS samples (2 DNMT3Amt CH vs 1 no CH) further showed that expression of genes involved in IL-13 and IL-15 pathway is significantly increased in CD4+ and CD8+ T cells from DNMT3Amt samples, suggesting a paracrine regulatory effect of T lymphocytes (mostly UBA1 and DNMT3A wild-type) in VEXAS. Pathway analysis of DEGs in these cells also revealed an enrichment of genes involved in inflammation and lymphocyte proliferation. Conclusions: Our study validates the age-independent, negative prognostic impact of DNMT3Amt CH on survival in VEXAS syndrome. DNMT3Amt mediates epigenetic and transcriptional priming associated with upregulation of IL-13/15 pathway in CD4/CD8 T cells, which are mostly DNMT3A and UBA1 wild-type, suggesting that the contribution of T lymphocytes to inflammation may be secondary to CH in the myeloid compartment.

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.

Background. Study of myeloid neoplasms (MN) has been rapidly transformed by genome characterization. Gene mutations have been reported to influence disease phenotype and risk of disease progression, and the evaluation of mutational status can provide valuable insights to improve the decision-making process. In this international study, we evaluated the clinical impact of mutational screening in patients with CMML, which is characterized by significant clinical and genomic heterogeneity, including a substantial proportion of patients with poor outcomes and unmet needs. Currently, only limited molecular information has been incorporated into CMML classification and prognostication. Methods. We retrospectively collected 3182 CMML patients. We used MOSAIC, an AI-based framework for multimodal analysis in rare cancers (PMID: 38875514) to develop innovative, molecular-based tools for classification and prognostication. An independent validation of the results on 516 prospectively collected patients was performed. Results. Bayesian Networs and Hierarchical Dirichlet Processes were used to identify genomic associations and define a CMML molecular classification. We identified 11 clusters with distinct clinical and genomic features, including splicing machinery, signal transduction and tyrosine kinase pathways aberrations, and high-risk molecular signatures (i.e., AML-like and TP53 mutations). Up to 15% of patients showed clear genomic overlap with other MN. Importantly, extensive multi-color flow cytometry on T lymphocytes, NK and myeloid cells (BD FACSymphony A5 Cell Analyzer) revealed specific immunologic and inflammation signatures associated with molecular subgroups. We then integrated molecular and clinical information to build an international CMML Prognostic Scoring System (iCPSS). We assessed the performance of different statistical and AI-based methods; L1-penalized Cox Model resulted as the best performing method. Selected features included hematological parameters (WBC, Hb, PLT and marrow blasts), cytogenetic abnormalities, and mutations in 10 genes (ASXL1, DNMT3A, EZH2, NRAS, RUNX1, SETBP1, STAG2, TET2, TP53, U2AF1). By Bayesian thresholds optimization, we identified 5 risk classes (i.e., very-low, low, intermediate, high and very-high) with median overall survival (OS) ranging from 99 to 9 months (P<.001), and median leukemia-free survival (LFS) ranging from 210 to 18 months (P<.001). iCPSS provided better patient discrimination across all clinical endpoints compared to currently available prognostic tools, with a Concordance Index (CI) for OS of 0.75 versus 0.62-0.64, respectively. Notably, compared to available scoring systems, up to 40% of patients were reassigned to higher or lower risk classes by the iCPSS. External validation on an independent prospective cohort confirmed that the performance of iCPSS is superior to existing prognostic models (CI for OS 0.71 vs 0.54-0.61, respectively) To demonstrate the clinical utility of iCPSS, we focused on 753 patients who underwent allogeneic stem cell transplantation (HSCT). In multivariable analysis, iCPSS stratified the probability of OS post-HSCT (P<.001) and identified groups of patients with different probabilities of disease relapse, ranging from 9% to 62% (P<.001). We employed a clinical- and genomic-based decision support system (PMID: 38723212) to determine the optimal timing of HSCT. Preliminary analyses indicated that higher-risk patients according to iCPSS (i.e., intermediate, high, and very high) benefit from an immediate HSCT strategy. Lower-risk patients (i.e., very low and low) had life expectancy maximized with delayed HSCT. Modeling decision analysis using iCPSS compared to conventional scores, resulted in a change in transplantation policy for a significant proportion of patients (up to 25%). Conclusion. Molecular information significantly improves the classification of CMML patients, providing a basis for refining diagnostic boundaries with other MN and for a more rational inclusion of patients in clinical trials. The iCPSS demonstrated superior performance over currently available scoring systems and improved HSCT decision making process.