Gangat, Naseema

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: 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.

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 Hepcidin, a central regulator of iron homeostasis, is pathologically elevated in patients with myelofibrosis (MF) and anemia. Chronic hepcidin elevation limits iron availability for red blood cell production and contributes to onset and severity of anemia, for which there is a large unmet need for safe and effective treatments. DISC-0974 is an investigational, first-in-class, monoclonal antibody that blocks hemojuvelin, a co-receptor in the bone morphogenetic protein-signaling pathway driving hepcidin expression. A completed healthy volunteer study demonstrated serum hepcidin reductions, serum iron increases, and increasing trends in hematologic parameters, with no safety signal identified. Aims This Phase 1b/2, open-label, multiple-ascending dose study (NCT05320198) assessed safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and initial efficacy of DISC0974 in patients with MF and anemia. Methods Eligible participants were ≥18 years of age with intermediate-2 or high-risk MF and anemia. Anemia was defined as baseline hemoglobin (Hgb) <10 g/dL with or without intermittent transfusion requirement (non-transfusion dependent [nTD]) or transfusion dependent (TD), as defined by the IWG-MRT. A stable dose of concomitant hydroxyurea and/or Janus kinase (JAK) inhibitor was allowed. Major exclusion criteria included liver iron concentration ≥7 mg/g dry weight and nutritional, genetic, infectious, or autoimmune anemia. Dose escalation was based on a Bayesian optimal interval design with accelerated titration. DISC-0974 was administered subcutaneously monthly for 6 doses. Primary endpoints included evaluating DISC-0974 safety and tolerability. Secondary endpoints included PK/PD markers of iron regulation and hematologic parameters. Hematologic response was defined for TD as transfusion independence (TI) during any 12-week consecutive period and for nTD as ≥1.5 g/dL Hgb increase from baseline at any time point. Durability was evaluated as mean Hgb ≥1.5 g/dL above baseline during any 12-week period. Data were summarized using descriptive statistics. Results At the time of data cut, 34 participants were enrolled at 5 dose levels: 14 mg (n=1), 28 mg (n=7), 50 mg (n=12), 75 mg (n=8), and 100 mg (n=6). Treatment with DISC-0974 resulted in meaningful and sustained hepcidin reductions with mean change from baseline (mean 88.2 ng/mL; range [8.7, 374.7]) reaching over 80% across treated participants. This corresponded to iron mobilization at all dose levels. Efficacy Hematologic responses were evaluated in participants dosed at 28 to 100 mg. Responses were achieved independent of baseline transferrin saturation (TSAT) values. Initial hematologic responses in nTD participants were observed around the time of administration of the second dose, with a rise in absolute reticulocyte count preceding responses. Mean erythropoietin values decreased from baseline (mean 68.2 mIU/mL; range [13.2, 1815]) in participants treated at 50 to 100 mg. Hemoglobin responses were achieved in 69% (20/29) of nTD participants; 60% of nTD participants (9/15) achieved a durable response. TSAT values above normal range (50%) were observed in all participants with a durable response. One of two evaluable TD participants achieved TI at the end of study. All evaluable participants with a baseline transfusion requirement (n=8) had ≥50% reduction in transfusions over a rolling 8-week window compared to baseline. Hematologic response was observed in 6 of 10 participants with concomitant JAK inhibitor therapy. Safety One adverse event (AE), diarrhea, was at least possibly related to DISC-0974 and reported in ≥2 participants. Grade 3 AEs included headache reported in 1 participant treated at 28 mg (not related to DISC-0974) and Grade 3 anemia reported in 2 participants treated at 28 mg and 4 participants treated at 50 mg (1 at 50 mg was deemed related to DISC-0974; all others were deemed not related). One serious AE of Grade 2 arthralgia was reported and deemed not related to DISC-0974. Summary/Conclusions DISC-0974 demonstrated acceptable safety and tolerability at all evaluated dose levels. Hematologic responses were achieved in nTD and TD participants, regardless of concomitant JAK inhibitor use. Sustained hepcidin reduction with DISC-0974 led to increased serum iron that preceded hematological response. Additional Phase 1b data, including longer follow-up, will be presented.

Background: Previously published risk variables for chronic myelomonocytic leukemia (CMML) included age, gender, CMML subtype, red blood cell (RBC) transfusion status, hemoglobin, leukocyte, monocyte, lymphocyte and platelet counts, immature myeloid cells, peripheral blood (PB) or bone marrow blast percentage, and mutations including ASXL1, RUNX1, NRAS, and SETBP1. Recent studies have reported on additional prognostically favorable (TET2) or unfavorable (DNMT3A, ASXL1, NRAS, SETBP1) mutations. In the current study, we took advantage of a clinically well-characterized and molecularly annotated cohort of CMML patients, in order to revisit the prognostic interaction between the aforementioned risk factors, with emphasis on mutations. Methods: The current study was conducted under an institutional review board approved protocol that allowed retrospective collection and analysis of data from Mayo Clinic patient records. Mutations were screened by multi-gene next-generation sequencing (NGS). Diagnostic criteria were according to the International Consensus Classification (Blood 2022; 140:1200). Conventional statistical methods were employed (JMP Pro 17.0.0 software SAS Institute, Cary, NC, USA). Survival analysis was censored for allogeneic stem cell transplantation (ASCT). Receiver Operating Characteristic (ROC) plots and Akaike Information Criterion (AIC) were used to compare predictive performance of the new model (MMMv2) vs. the Mayo Molecular Model (MMM) vs. the CMML-specific prognostic scoring system (CPSS). Results: The study cohort included 416 patients with CMML that were fully annotated for mutations. Cytogenetic information was available in 404 patients. At a median follow-up of 18 months, 302 (72%) deaths, 72 (18%) leukemic transformations, and 44 (11%) ASCTs were documented. Age-adjusted multivariable analysis (MVA) of mutations identified the following as risk factors: DNMT3A (HR 5.5; p<0.01), PHF6 (HR 0.3; p<0.01), TET2 (HR 0.6; p<0.01), PTPN11 (HR 2.5; p=0.01), NRAS (HR 1.6; p<0.01), ASXL1 (HR 1.3; p=0.02), and SETBP1 (HR 1.8; p=0.01). A separate MVA of clinical and cytogenetic variables identified RBC transfusion need, leukocyte count ≥13 x 109/L, peripheral blood blast ≥1%, abnormal karyotype, older age, and male sex to be independently associated with inferior survival (p<0.04 in all instances). All-inclusive MVA identified the following as independent risk factors for survival (p <0.01): DNMT3A mutation (HR 3.7; 95% CI 2.0-6.7), absence of PHF6 mutation (HR 2.8; 1.3-5.8), RBC transfusion need (HR 2.3; 1.7-3.1), leukocyte count ≥13 x 109/L (HR 2.0; 1.5-2.6), abnormal karyotype (HR 1.6; 1.2-2.2), and absence of TET2 mutation (HR 1.4,1.1-1.8). An HR based prognostic model was subsequently constructed by assigning 3 points to DNMT3A mutation, 2 points each for RBC transfusion need, leukocyte count ≥13 x 109/L, and absence of PHF6 mutation and one point each for abnormal karyotype and absence of TET2 mutation, resulting in a 4-tiered survival risk model (MMMv2): low risk (0-2 points; N=103) with median survival of 74 months; intermediate-1 (3 points; N=65) with median 37 months; intermediate-2 (4-5 points; N=126) with median 19 months; and high (6 points; N=88) with median 12 months (p<0.01). Independent genetic risk factors associated with inferior leukemia-free survival included DNMT3A (HR 5.5), PHF6 (HR 0.2), abnormal karyotype (HR 2.0), PTPN11 (HR 5.2), ASXL1 (HR 2.0), and BCOR (HR 8.7). Leukocyte count ≥13 x 109/L (p=0.01) but not RBC transfusion need (p=0.2) was additionally significant. However, presence of immature myeloid cells (p<0.01) and circulating blasts ≥1% (p<0.01) were prognostically more relevant than leukocyte count ≥13 x 109/L and independently predicted leukemic transformation, along with DNMT3A, PTPN11 and ASXL1 mutations. Predictive performance was superior for MMMv2 (AUC 87, AIC 137) vs. MMM (AUC 74, AIC 194) vs. CPSS (AUC 76, AIC 177). Conclusion: The revised Mayo Molecular Model (MMMv2) provides a contemporary risk model for CMML that recognizes DNMT3A as the most unfavorable and PHF6 as the most favorable mutation and confirms the mutation-independent prognostic contribution from red cell transfusion need and sustained leukocytosis (≥13 x 109/L). Mutations were also important in predicting leukemic transformation, notably DNMT3A, PTPN11 and ASXL1.

Introduction Somatic mutations in TET2 are present in ~60% of chronic myelomonocytic leukemia (CMML) patients and are associated with improved median overall (mOS) and acute leukemia free survival (mLFS). Multiple TET2 mutations are detected in ~50% of such cases and are thought to confer additional benefit. Whether these benefits apply to the molecularly distinct myelodysplastic (MD) and myeloproliferative (MP) CMML subtypes is unknown. This study assesses the impact of the number and type of TET2 mutations across the MD and MP-CMML subtypes. Methods CMML patients seen at Mayo Clinic (n = 400, 47%) and MD Anderson Cancer Center (n = 449, 53%) were included with IRB approval. Analyses considered the clinical and molecular data at diagnosis or first referral. Truncating (frameshift or nonsense) mutations in TET2 were further annotated by location in either the proximal genome targeting domain (aa 1 - 1128) or distal catalytic domain (aa 1129 - 1936) as described (Coltro, 2020). Categorical variables were compared by Pearson Chi squared and continuous by Mann-Whitney U or Kruskal-Wallis tests. Univariate and multivariate analyses utilized Cox proportional hazards models with continuous variables binarized by 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 In a cohort of 849 CMML patients (median age 71 years, 32% female, 90% white), 454 (54%) were classified as MD-CMML and 394 (46%) as MP-CMML. Cumulatively, 664 somatic TET2 mutations were identified across the MD (n = 415, 63%) and MP-CMML (n = 249, 38%) subgroups, with ≥1 TET2 mutation in 57% vs 44% (p < 0.001) and ≥2 TET2mutations in 33% vs 20% (p = 0.001) , respectively. The median (range) number of TET2 mutations in MD vs MP-CMML was 1 (0 - 4) vs 0 (0 - 3; p < 0.001). Truncating frameshift or nonsense mutations comprised 86% vs 78% of mutations in each group with hypomorphic missense mutations comprising 13% vs 20% (p = 0.03). Proximal (48% vs 43%) and distal (38% vs 35%) truncations were evenly distributed between MD and MP-CMML (p = 0.91). In both the MD and MP subgroups as well as the overall cohort (p < 0.01 each), increasing number of TET2 mutations (0 vs 1 vs ≥2) was associated with higher median hemoglobin (10.3 vs 11.4 vs 12.0 g/dL), fewer mean peripheral blasts (1.3% vs 0.5% vs 0.3%), and a higher proportion of normal karyotypes (60% vs 76% vs 79%). Likewise, co-mutations were increasingly frequent in SRSF2 (36% vs 45% vs 53%) and less frequent in IDH2 (9% vs 1% vs 1%) and SETBP1 (14% vs 4% vs 0%) throughout the cohort. TET2 mutations were mutually exclusive with ETV6, GATA2, IDH1, and WT1 mutations. Uniquely, MD-CMML cases with TET2 mutations (0 vs 1 vs ≥2) were enriched for co-mutations in ZRSR2 (5% vs 13% vs 9%) and had fewer co-mutations in both ASXL1 (47% vs 34% vs 28%) and U2AF1 (11% vs 6% vs 3%). In MP-CMML, co-mutations in CBL (11% vs 20% vs 27%) were increased while co-mutations in BCOR (6% vs 1% vs 0%) were decreased. Accordingly, the favorable ASXL1WT/TET2MT genotype was more common in MD (39% vs 22%), while the adverse ASXL1MT/TET2WT genotype was more common in MP-CMML (20% vs 33%; p < 0.001). In multivariate analyses, the presence of ≥2 TET2 mutations was favorably associated with OS and LFS in both MD and MP-CMML (hazard ratios 0.53 - 0.67, p < 0.04) independent of several clinical and genetic features including ASXL1 mutations and abnormal karyotype. Beyond the number of TET2 mutations, however, neither the mutation class (truncating vs hypomorphic) nor location (proximal vs distal) further affected OS or LFS. Likewise, although the median variant allele fraction (VAF) was lower in MD vs MP-CMML (42% vs 46%, p < 0.001), VAF did not impact survival. Accordingly, the mOS for patients with 0, 1, or ≥2 TET2 mutations was 29, 58, and 75 months in MD-CMML and 19, 26, and 39 months in MP-CMML, respectively (p < 0.001 each). The mLFS was similarly improved to 26, 52, and 67 months in MD-CMML (p < 0.001) and 16, 22, and 31 months in MP-CMML (p = 0.003). CONCLUSIONS This multicenter study demonstrates that the favorable impact of multiple TET2 mutations in CMML extends to both the MD and MP subtypes. In MD-CMML, multiple TET2 mutations segregate against adverse ASXL1 mutations while, in MP-CMML, they appear to offset the impact of adverse RAS pathway mutations. Thus, multiple TET2 mutations may identify a lower-risk subgroup of MP-CMML patients.

Background Previous studies have suggested a higher prevalence of autoimmune or inflammatory diseases (AID) in patients with myeloproliferative neoplasms (MPN) (Haematologica 2010; 95:1216); more recent studies have suggested the absence of impact on tumor genetics or disease outcome (Leukemia 2023; 37:1741). In the current study, we conducted a before and after MPN diagnosis survey, in order to examine the prevalence of AID and its impact on overall and transformation-free survival as well as the risk of thrombosis or other co-morbidities, such as cancer. Methods The current study was conducted under an institutional review board approved minimum risk protocol. Diagnostic criteria were according to the International Consensus Classification (Blood 2022; 140:1200). AID was operationally classified as occuring before or after diagnosis, with the latter requiring first diagnosis occuring ≥3 months after MPN diagnosis. Non-driver mutations were screened by next-generation sequencing (NGS) and was available for 620 patients. Cox proportional hazard regression model was used for univariate and multivariable analysis. Kaplan-Meier plots were used for time-to-event assessment, using JMP Pro 17.0.0 software SAS Institute, Cary, NC, USA. Results The study population included 1,968 consecutive patients with polycythemia vera (PV; N=1,001) or essential thrombocythemia (ET; N=967), seen at the Mayo Clinic between 1970 and 2024. Mutation information was available in 620 patients. AID was diagnosed before or after MPN diagnosis in 157 (8%) patients, including 121 (6%) before and 43 (4%) after. 134 (85%) patients had history of only one AID while 23 (15%) had ≥2 AIDs. The most prevalent diagnoses included organ-specific AID (48 cases before and 21 after MPN diagnosis), inflammatory arthritis (37 before and 6 after), connective tissue diseases (17 before and 7 after) and inflammatory dermatosis (20 before and 4 after). For each one of these four categories, thyroid disorders (21 before and 6 after), rheumatoid arthritis (22 before and 2 after), antiphospholipid syndrome (8 before and 1 after), and psoriasis (11 before and 4 after) were the most frequent, respectively. Patients with pre-MPN AID history were older (p=0.02) and were more likely to have ET vs. PV and be females (p<0.01); they were also more likely to present with venous (18% vs. 12%, p=0.04) but not arterial (p=0.2) thrombosis. Pre-MPN AID history was significantly associated with pre-MPN cancer history, with the latter including (25% vs 14%, p<0.01) or excluding (18% vs 10%, p<0.01) non melanoma skin cancers (NMSC). Pre-MPN AID history did not correlate with driver or other mutations (including TET2, ASXL1, DNMT3A), leukocyte, neutrophil, monocyte, or lymphocyte counts (p≥0.1 in all instances). Pre-MPN history of AID did not affect overall (p=0.08), myelofibrosis-free (p=0.3), leukemia-free (p=0.3), arterial thrombosis-free (p=0.9), or venous thrombosis-free (p=0.6) survival. Second cancer-free survival (SC-FS), including (p<0.01) or excluding (p<0.01) NMSC, was negatively affected by pre-MPN history of AID. Multivariate analysis confirmed the independent detrimental effect of a pre-MPN history of AID (HR 1.7, p<0.01) on SC-FS; other independent risk factors for SC-FS were older age (p<0.01), male sex (p<0.01) and pre-MPN history of cancer (p<0.01). The association between pre-MPN history of AID and second cancer was also apparent when the latter was limited to lymphoproliferative neoplasms, although with borderline significance (p=0.08), which was further confirmed by adjusting for age and sex (p=0.09). Median time from MPN diagnosis to post-MPN AID was 9 years (range 1-23), which was similar between PV and ET (p=0.5). Post-MPN development of AID was more likely in females (HR 2.3; p=0.01) and in patients with pre-MPN history of AID (HR 4.8; p<0.01) or hypertension (HR 2.1; p<0.03). These risk factors remained significant during multivariable analysis of post-MPN AID-free survival. Conclusions The current study suggests an increased risk of post-MPN AID and second cancers, in patients with pre-MPN history of AID. The study also showed a significant association between pre-MPN history of AID and venous thrombosis at time of MPN diagnosis. These observations reinforce the widely suggested role of tumor-extrinsic inflammation in exacerbating MPN-intrinsic thrombophilia and the development of cancer.