Finke, Christy M.

Abstract RAS pathway (RASp) mutations induce proliferative features, and promote transformation in chronic myelomonocytic leukemia (CMML). However, the unique clonal landscape and hierarchy of distinct RASp mutations remain unexplored. To characterize the landscape, architecture, and implications of unique RASp mutations in CMML, we evaluated a cohort of 814 patients with CMML. We identified 461 RASp mutations among 342 patients (42%). N/KRAS and CBL mutations were the most common, frequently involved the P-loop or RING domains, respectively, and frequently appeared as dominant events (63% and 65%, respectively). BRAF, NF1, and PTPN11 mutations spanned throughout the gene structure, and frequently appeared as subclonal events (75%, 64%, and 59%, respectively). CBL mutations frequently occurred in codominance with SRSF2 and multihit TET2, and were enriched for KIT mutations. PTPN11 mutations more frequently co-occurred with SETBP1 and DNMT3A mutations, and were infrequently codominant with TET2 or ASXL1. RASp mutations predicted for shorter overall survival (hazard ratio [HR], 1.55; 95% confidence interval [CI], 1.15-2.07; P = .0075) and leukemia-free survival (LFS; HR, 1.67; 95% CI, 1.26-2.20; P = .0011), influenced outcomes of myelodysplastic and TET2 mutant CMML, and cooperated with IDH2 and RUNX1 mutations to induce shorter LFS. These data set the bases for refined genomic classifications of CMML, and underscore the need to develop RAS-directed therapies for patients with CMML.

We sought to develop a survival model in chronic myelomonocytic leukemia (CMML) that is primarily based on clinical variables and examine additional impact from mutations and karyotype. 457 molecularly-annotated patients were considered. Multivariable analysis identified circulating Blasts ≥2% (1 point), Leukocytes ≥13 x 109/L (1 point), and severe (2 points) or moderate (1 point) Anemia as preferred risk variables in developing a clinical risk Stratification Tool for overall survival (OS), acronymized to "BLAST": low-risk (0 points; median 63 months); intermediate-risk (1 point; median 28 months; HR 2.2, 95% CI 1.6-3.0), and high-risk (2-4 points; median 13 months; 5.4, 4.1-7.3); the corresponding 3/5 year OS rates were 68%/53%, 43%/18%, and 12%/1%. BLAST model performance (AUC 0.77/0.85 at 3/5-years) was shown to be comparable to that of the molecular CMML-specific prognostic scoring system (CMML-mol; AUC 0.73/0.75) and the international prognostic scoring system-molecular (IPSS-M; AUC 0.73/0.74). Multivariable analysis of mutations and karyotype identified PHF6MUT and TET2MUT as being "favorable" and DNMT3AMUT, U2AF1MUT, BCORMUT, SETBP1MUT, ASXL1MUT, NRASMUT, PTPN11MUT, RUNX1MUT, TP53MUT, and adverse karyotype, "unfavorable". Molecular information was subsequently encoded in a combined clinical-molecular risk model (BLAST-mol; AUC 0.80/0.86 at 3/5-years) that included the aforementioned BLAST clinical risk variables and a 3-tiered molecular risk score. BLAST and BLAST-mol were subsequently validated by two separate external cohorts. Independent risk factors for blast transformation included DNMT3AMUT, ASXL1MUT, PHF6WT, leukocytes ≥13 x 109/L, and ≥2% circulating or ≥10% bone marrow blasts. The current study proposes an easy to implement, globally applicable, and molecularly adaptive risk model for CMML.

SummaryGermline variants in DDX41 (DDX41MT‐germline predisposition syndrome [GPS]) are associated with predisposition to haematological malignancies (HM), including lymphoid and myeloid neoplasms (MN). We retrospectively analysed the clinical and molecular features of 195 patients diagnosed and treated at Mayo Clinic with DDX41MT‐GPS. Patients with germline DDX41 pathogenic variants (42.3%) and variants of unknown significance (VUS, 57.6%) were included. The median age was 68.6 years (16.2–93.4). Ninety‐two per cent were Caucasian, 64.1% were male and 30.8% had a family history of HM. There were 92 distinct germline variants among our cohort, and the most common was p.Met1? (15.9%), followed by p.Asp140Glyfs*2 (9.2%). Clinical diagnoses included asymptomatic carriers (10.2%), clonal cytopenia of undetermined significance (CCUS, 6.1%), myeloproliferative neoplasms (6.7%), myelodysplastic syndrome (40.5%), acute myeloid leukaemia (20.5%), lymphoid neoplasms (9.2%), plasma cell dyscrasias (6.1%) and solid tumours (22.5%). Patients with MN were older (median age 70 vs. 63.5 years) and more likely to be male (M:F ratio 2.3 vs. 1.0) and most patients (78.8%) with MN had a normal karyotype. The most common somatic variants involved DDX41 (34.4%), followed by TET2 (11.2%), DNMT3A (9.6%) and ASXL1 (9.2%). In summary, we have comprehensively described the spectrum of clinical phenotypes within the Mayo Clinic DDX41MT‐GPS cohort.

Abstract Background: DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for hematological neoplasms. In this study, we make use of a natural inflammatory response occurring during COVID-19 to define the impact of these mutations on inflammation and outcomes. Methods: A cohort of 243 community-based patients (Olmsted County, MN) with COVID-19 were identified. CH status was determined on PBMC using error corrected sequencing (lower detection limit 0.5% VAF). In a subset of patients, we use multi-omics interrogation of the genome, transcriptome, and epigenome at single cell (sc) resolution along with DNA methylation, in PBMC, from patients with COVID-19 and CH. Results: We detected 97 CH mutations in 72 (29.6%) of 243 patients with COVID-19. The most frequent being DNMT3A (n=30, 30%) and TET2 (n=26, 28%). The presence of CH negatively impacted overall survival when restricted to TET2 and DNMT3A mutations. However, only DNMT3Amt CH retained age and comorbidity independent prognostication (HR 2.84, p = 0.022). We then assessed for changes in DNA methylation using Illumina MethylationEPIC array on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=4) CH. Site specific DNA methylation changes revealed significant loss of methylation in DNMT3Amt CH in comparison to TET2mt patients, with 10,944 hypomethylated and 1,160 hypermethylated sites. We found that actively transcribed states (Tx, TxWk) were more commonly hypomethylated in DNMT3Amt CH while enhancers (Enh) and promoters (TssA, TssAFlnk) were more commonly hypomethylated in TET2mt CH. We then conducted scDNA-seq in DNMT3Amt (n=3) and TET2mt (n=1) CH in COVID-19 and observed that while in TET2mt CH, mutations were largely restricted to classical and intermediate monocytes, in DNMT3Amt CH, mutations were seen in myeloid and lymphoid lineage cells. We performed scRNA-seq on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=6) CH. Differential gene expression analysis identified 1,569 upregulated genes in DNMT3Amt patients and 205 downregulated. Pathway analysis demonstrated an upregulation of genes involved in T cell survival, function, and inflammation in DNMT3Amt patients compared to TET2mt patients with a significant overexpression of GIMAP1, GIMAP4, IRF2, IL32 in multiple cell types. We correlated transcriptomic changes with chromatin accessibility using the 10X Genomics Multiome platform to profile both gene expression and open chromatin from the same samples. Analysis of global distribution of cut sites and differentially accessible peaks showed increased chromatin accessibility in DNMT3Amt CH, especially CD4+ T lymphocytes and NK cells. Conclusion: We demonstrate an age, sex, and comorbidity-independent adverse impact on mortality associated with DNMT3Amt CH in the context of COVID-19 and highlight transcriptomic and epigenetic differences between DNMT3A and TET2mt CH, with single cell resolution. Citation Format: Jenna A Fernandez, Wazim Mohammed Ismail, Moritz Binder, Terra Lasho, Susan M. Geyer, Amelia Mazzone, Christy M. Finke, Abhishek Mangaonkar, Jeong-Heon Lee, Liguo Wang, Vernadette A. Simon, Fariborz Rakhshan Rohakthar, Amik Munankarmy, Jonathan J. Harrington, Melissa R. Snyder, Keith D. Robertson, Alexandre Gaspar-Maia, Mrinal Patnaik. Divergent impacts of DNMT3A and TET2 mutant clonal hematopoiesis on COVID-19 related outcomes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A012.

Abstract Background: Age-related clonal hematopoiesis (ARCH) is a precursor state to blood cancers and is associated with epigenetic age acceleration (EpiAgeAccel). While ARCH is associated with increased all-cause mortality and cardiovascular disease, not much is known about these risks with therapy-related clonal hematopoiesis (t-CH) – CH occurring in the context of prior cytotoxic chemotherapy/radiation exposure. Methods: Patients were prospectively recruited for CH and EpiAge assessment from Mayo Clinic between 2022 and 2024. CH status was determined using error corrected sequencing on peripheral blood (PB) DNA (sensitivity 0.5% VAF). Patients with CH were classified as t-CH if they had received chemotherapy or radiation prior to CH detection. All other CH cases were classifed as ARCH. For EpiAge analysis, PB DNA was analyzed using the Illumina MethylationEPIC array. The resulting beta values were used to obtain epigentic age estimates using Horvath’s clock, Hannum’s Clock, PhenoAge, DunedinPACE, and estimated telomere lengths (DNAmTL). Results: 106 (62%) of 170 patients assessed were found to have CH. CH patients were classified into ARCH-63 (59%) and t-CH-43 (41%). While the median chronological age (69 vs 67 years) was similar between ARCH and t-CH, t-CH patients had lower Hgb [10.9g/dL vs. 13g/dL (p=0.0001)], lower platelet [146x109/L vs. 192x109/L (p=0.0132)] and lower WBC [4.4x109/L vs. 5.2x109/L (p=0.0345)]. The 3 most common ARCH mutations were TET2 (20%), DNMT3A (19%), and ASXL1 (14%), while PPM1D (27%), TET2 (18%), and DNMT3A (14%) were most frequent in t-CH. There was no significant difference in median EpiAge via Horvath (68 vs 67.5years p=0.2826), Hannum (65 vs 66 years p=0.9333), or PhenoAge (55 vs 56 years p=0.618) between ARCH and t-CH. However, DunedinPACE of aging was higher in t-CH (1.27 vs 1.18 bioyear/chronoyear p=0.0148) and median DNAmTL was shorter in t-CH (6.35 vs 6.49kb p=0.0247), compared to ARCH (Note patients with telomere biology disorders were excluded). Multivariable regression analysis adjusting for chronologic age, smoking status, and male sex showed that both ARCH and t-CH had EpiAgeAccel via Horvath(5.8years p=0.0001, 3.4 years p=0.051), Hannum(5years p=0.001, 4.5 years p=0.007), and PhenoAge (6.7years p=0.002, 7.6 years p=0.002) clocks. t-CH more than doubled DunedinPACE (0.2 bioyear/chronoyear, p<0.001) of aging compared to ARCH (0.08 bioyear/chronoyear, p=0.026). t-CH also demonstrated a significant decrease in DNAmTL (0.34kb, p<0.001) in comparison to ARCH (0.17kb, p=0.015). With a median follow-up of 20 months, t-CH was associated with worse overall survival in comparison to ARCH, and non-CH patients (p=0.013, 93% vs. 90% alive at 10 months, 87 vs. 72% alive at 20 months). Conclusion: Our prospective CH cohort study validates the association between CH and EpiAgeAccel using several different methylation clocks and for the first time highlights an increased pace of aging and shorter DNAmTL in the context of inferior survival outcomes in t-CH. Citation Format: Marissa Li, Jenna Fernandez, Kristina Kirschner, Tamir Chandra, Kristen McCullough, Sarah Aug, Terra Lasho, Christy Finke, James Foran, Cecilia Arana Yi, Yael Kusne, Talha Badar, Abhishek Mangaonkar, Naseema Gangat, Rachel Simon, Alejandro Ferrer, Mrinal Patnaik. A prospective cohort study of biological aging and epigenetic age acceleration in 106 patients with age and therapy-related clonal hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A015.

Background: Therapy-related myeloid neoplasms (t-MN) occur after exposure to DNA-damaging agentsincluding cytotoxic chemo and radiation therapy and are associated with dismal outcomes. TP53 and PPM1D (protein phosphatase Mn2+/Mg2+-dependent 1D) mutations (MT) disrupt the DNA damage response and repair (DDR) pathway, are often co-mutated (25-35%), and although enriched in t-MN (20%), can also be seen in age-related clonal hematopoiesis (CH) (5-10%). While progressive genomic instability with biallelic inactivation has been described as a mechanism of MN progression with TP53 MT, similar mechanisms remain to be elucidated for PPM1D MT. We conducted this study to assess the clinical phenotype and outcomes of TP53 and PPM1D co-mutated (PPM1DTP53m) versus PPM1D MT and wild type TP53 (PPM1DTP53wt) CH and MN. Methods: After approval from the Mayo Clinic IRB, the CH and MN databases were queried and 119 pts with PPM1D MT (CH= 26, clonal cytopenia of unknown significance [CCUS]= 64, myelodysplastic syndrome [MDS]=16, myeloproliferative neoplasm [MPN]=8, acute myeloid leukemia [AML]= 5) were identified. We analyzed baseline characteristics, karyotypic changes, somatic mutations, and survival outcomes between the PPM1DTP53m (n= 22) and PPM1DTP53wt (n= 97) groups (gp). Results: Baseline characteristics The median age in years (yrs) was 68.5 (range [R], 43-89) and 71 (R,30-92) in the PPM1DTP53m and PPM1DTP53wt gp (p= 0.65). 22% vs 18% (p=0.78), 58% vs 32% (p=0.03), 9% vs 32% (p=0.01), 7% vs 4.5% (p=>0.99) and 3% vs 14% (p=0.07) pts met criteria for CH, CCUS, MDS, MPN and AML in the PPM1DTP53wt and PPM1DTP53m gp, respectively. Similarly, 19% vs 18% (p=>0.99), 47% vs 32% (p=0.23), 7% vs 36% (p=0.001), 2% vs 0% (p=>0.99) and 2% vs 14% (p=0.04) of pts met criteria for t-CH, t-CCUS, t-MDS, t-MPN and t-AML in the PPM1DTP53wt and PPM1DTP53m gp, respectively. All PPM1D variants were truncating, located in exon 6 of the gene, with median variant allele frequency (VAF) (8% [R,1-64] vs 5.5% [R,2-24], p= 0.10) and number of PPM1D MT (1 [R,1-5] vs 1 [R, 1-3], p=>0.99) being similar between the two gp. While proportion of pts with t-MN were similar between the two gps (77% vs 86%, p= 0.40), cytogenetic (CG) abnormalities (67% vs 31%, p= 0.006), including monosomies (28% vs 2%, p= <0.001) and complex CG (53% vs 4%, p= <0.001) were more frequent in PPM1DTP53m gp compared to PPM1DTP53wt gp, respectively. The median TP53 MT VAF was 14% (R, 1-14); 50% had multi-hit TP53 MT. Somatic mutations in PPM1D MT myeloid neoplasm with and without TP53 MT Somatic co-mutations excluding TP53 were seen in 71% of pts; 73.5% in PPM1DTP53wt and 59% in PPM1DTP53m gp (p= 0.20). Apart from CBL (0% vs 9%,p=0.03), MT involving DNMT3A (20% vs 14%, p= 0.56), TET2 (15% vs. 14%, p= >0.99), IDH1 (6% vs 19%, p= 0.07), splicing factors (8% vs 14%, p=0.08), ASXL1 (11% vs 4.5%, p=0.46), and JAK2 (9% vs 0%,p=0.20) were comparable between PPM1DTP53wt and PPM1DTP53m gps, respectively. The proportion of pts with ≥ 2 somatic co-mutations excluding TP53 MT were numerically higher in PPM1DTP53wt gp (43%) compared to PPM1DTP53m gp (23%) (p= 0.09). In the PPM1DTP53wt gp, other somatic driver mutations included RUNX1 (n= 7 [7%]; CH [n=1], CCUS [n=2], MDS [n=3], MPN [n=1]), NRAS (n=1 [1%]; CCUS), BCOR (n=1 [1%]; CCUS), and PTPN11 (n=1 [1%]; CCUS). Leukemic transformation and survival With a median follow up of 6.43 months (mo) (R, 0.23-36.4), median overall survival (mOS) in mo among high-risk MDS/AML (16.4 vs 15.0, p= 0.66) and CHIP/CCUS pts (35.79 vs 26.28, p= 0.93) was similar between PPM1DTP53wt and PPM1DTP53m gps, respectively. CHIP/CCUS pts harboring multiple vs single PPM1D MT had an inferior mOS (23.9 vs 36.7 mo, p= 0.05). Three (2.5%) MDS pts underwent AML transformation; 1 (1%) and 2 (10%) in PPM1DTP53wt and PPM1DTP53m gp, respectively (p= 0.07). Conclusions In our study, 18% of pts had concurrent TP53 and PPM1D MT (82% t-MN), with the PPM1DTP53wt genotype being more frequent in CCUS, while the PPM1DTP53m genotype was more frequent in MN. The PPM1DTP53m genotype had a higher frequency of CG abnormalities, including complex CG. Survival outcome was inferior and comparable in PPM1D MT MN with or without TP53 MT. While 73.5 % of the PPM1DTP53wt gp had somatic driver MT and 31% had karyotypic abnormalities, 22% had no additional MT or CG changes (all CH/CCUS), underscoring the need to understand how PPM1D MT contribute to hematopoietic dysfunction and MN.

Background: The majority of patients with chronic myelomonocytic leukemia (CMML) harbor somatic mutations with the most frequent being TET2 (56%), SRSF2 (42%), and ASXL1 (41%) (Leukemia 2020;34:1407). The prognostic relevance of mutations in CMML has been studied extensively with consistent observations suggesting favorable survival impact from TET2 and unfavorable from DNMT3A, ASXL1, NRAS, and SETBP1 mutations. Among the latter, DNMT3A mutation stands out as being the strongest predictor of inferior overall (OS) and leukemia-free (LFS) survival (AJH 2017;92:56). The current study was inspired by observations from exploratory analyses of a large Mayo Clinic CMML cohort. Methods: The current retrospective study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from patient records (Mayo Clinic, Rochester, MN; Scottsdale, AZ; and Jacksonville, FL). 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 for comparisons of groups and survival analysis (JMP Pro 17.0.0 software SAS Institute, Cary, NC, USA). Results: Exploratory analysis of a well-characterized cohort of 398 Mayo Clinic patients with CMML suggested an unusually long median survival of 96 months for 7 patients harboring PHF6 mutation vs. 27 months for those without the mutation. A subsequent institutional database search focusing on PHF6-mutated CMML identified 20 more cases, expanding the total number of study patients to 418, including 27 PHF6-mutated cases. PHF6 mutations clustered with (co-mutation frequency) TET2 (93%) and less likely to be co-mutated with TP53 (0%), PTPTN11 (0%) and SF3B1 (0%) mutations. PHF6-mutated patients often presented with thrombocytopenia (platelet count <100 x 10(9)/L; 85% vs. 51% in PHF6 wild-type cases; p<0.01). Median time from initial presentation with thrombocytopenia to CMML diagnosis was 4 years (0-14 years). In order to isolate the prognostic impact of PHF6 mutation without associated DNMT3A mutation and independent of TET2 mutation, we considered 4 mutational categories: i) DNMT3A-mutated (N=19; 2 co-expressed PHF6 and 7 TET2 mutations), ii) PHF6-mutated with wild-type DNMT3A (N=25; 23 co-expressed TET2 mutation), iii) TET2-mutated without DNMT3A or PHF6 co-expression (N=169), and iv) wild-type for DNMT3A, TET2, and PHF6 (N=205). Compared to the other groups, PHF6-mutated patients with wild-type DNMT3A were less likely to display leukocytosis (p<0.01) or red cell transfusion need at presentation (p<0.01). At a median follow-up of 18 months, 302 deaths, 78 leukemic transformations, and 44 allogenic stem cell transplants were documented. Univariate analysis for OS disclosed superiority in favor of PHF6-mutated patients with wild-type DNMT3A (median OS 90 months) vs. the other three groups with median OS ranging from 10 months for DNMT3A-mutated patients (HR 20.7, 95% CI 8.1-52.7; p<0.01) to 40 months for TET2-mutated cases with wild-type DNMT3A (HR 3.5, 95% CI 1.7-7.6; p<0.01). Results were similar for multivariable analysis of LFS, where, remarkably, none (0%) of the 25 PHF6-mutated patients with wild-type DNMT3A experienced leukemic transformation vs. 15-30% risk of leukemic transformation in the other three groups (p<0.01). Multivariable analysis for both OS and LFS, which included other risk factors such as red cell transfusion need, leukocytosis (leukocyte count ≥13 x 109/L) and karyotype, confirmed the independent and favorable impact of PHF6 mutation/wild-type DNMT3A (p<0.01 in both instances). Finally, all-inclusive multivariable analysis of karyotype and mutations, in DNMT3A wild-type patients, confirmed the highly favorable and independent prognostic impact of PHF6 mutation (HR 0.18, 95% CI 0.09-0.37). Conclusion: The current study proposes a 2-gene genomic signature (PHF6 mutated and DNMT3A wild-type) to identify a unique subset of CMML patients who often present with thrombocytopenia and whose clinical course is characterized by a very low risk of leukemic transformation, resulting in superior overall survival. Additional studies are warranted to validate these findings and include the information in contemporary risk models.

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.

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.

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.