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With the increasing number of sequencing projects involving families, quality control tools optimized for family genome sequencing are needed. However, accurately quantifying contamination in a DNA mixture is particularly difficult when genetically related family members are the sources. We developed TrioMix, a maximum likelihood estimation (MLE) framework based on Mendel's law of inheritance, to quantify DNA mixture between family members in genome sequencing data of parent–offspring trios. TrioMix can accurately deconvolute any intrafamilial DNA contamination, including parent–offspring, sibling–sibling, parent–parent, and even multiple familial sources. In addition, TrioMix can be applied to detect genomic abnormalities that deviate from Mendelian inheritance patterns, such as uniparental disomy (UPD) and chimerism. A genome-wide depth and variant allele frequency plot generated by TrioMix facilitates tracing the origin of Mendelian inheritance deviations. We showed that TrioMix could accurately deconvolute genomes in both simulated and real data sets.

Hematopoietic stem cells (HSCs) with age-associated somatic mutations that disproportionally contribute to hematopoiesis generate the condition known as clonal hematopoiesis (CH). While CH conveys increased risk of hematologic cancer, there is also strong association between CH and cardiovascular disease (CVD). Accumulating evidence suggests that inflammation mechanistically links CH to CVD, and we hypothesized that CH may be a predictive biomarker of CVD in conditions of chronic inflammation. One such patient population comprises people living with HIV (PLWH) who also have substantially increased incidences of CVD and CH . We studied the association between CH and CVD in PLWH using samples from ACTG Study A5001 (or ALLRT), a prospective clinical trial of HIV-infected persons with long-term follow-up. We observed a positive association between CH and CVD in PLWH independent of traditional CVD risk factors. Moreover, in CVD cases, the CH clone was identifiable in the blood years before CVD diagnosis, unlike in PLWH with CH who did not have CVD. With the life span of PLWH increasing because of advances in treatment, our results indicate that the presence of CH and its clonal dynamics could be used as a prognostic biomarker of the risk for CVD in PLWH.

Purpose:To identify molecular subclasses of clear cell ovarian carcinoma (CCOC) and assess their impact on clinical presentation and outcomes.Experimental Design:We profiled 421 primary CCOCs that passed quality control using a targeted deep sequencing panel of 163 putative CCOC driver genes and whole transcriptome sequencing of 211 of these tumors. Molecularly defined subgroups were identified and tested for association with clinical characteristics and overall survival.Results:We detected a putative somatic driver mutation in at least one candidate gene in 95% (401/421) of CCOC tumors including ARID1A (in 49% of tumors), PIK3CA (49%), TERT (20%), and TP53 (16%). Clustering of cancer driver mutations and RNA expression converged upon two distinct subclasses of CCOC. The first was dominated by ARID1A-mutated tumors with enriched expression of canonical CCOC genes and markers of platinum resistance; the second was largely comprised of tumors with TP53 mutations and enriched for the expression of genes involved in extracellular matrix organization and mesenchymal differentiation. Compared with the ARID1A-mutated group, women with TP53-mutated tumors were more likely to have advanced-stage disease, no antecedent history of endometriosis, and poorer survival, driven by their advanced stage at presentation. In women with ARID1A-mutated tumors, there was a trend toward a lower rate of response to first-line platinum-based therapy.Conclusions:Our study suggests that CCOC consists of two distinct molecular subclasses with distinct clinical presentation and outcomes, with potential relevance to both traditional and experimental therapy responsiveness.See related commentary by Lheureux, p. 4838

Supplementary Data from Molecular Subclasses of Clear Cell Ovarian Carcinoma and Their Impact on Disease Behavior and Outcomes

Supplementary Data from Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia

Purpose:In this era of precision-based medicine, for optimal patient care, results reported from commercial next-generation sequencing (NGS) assays should adequately reflect the burden of somatic mutations in the tumor being sequenced. Here, we sought to determine the prevalence of clonal hematopoiesis leading to possible misattribution of tumor mutation calls on unpaired Foundation Medicine NGS assays.Experimental Design:This was a retrospective cohort study of individuals undergoing NGS of solid tumors from two large cancer centers. We identified and quantified mutations in genes known to be frequently altered in clonal hematopoiesis (DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2, SF3B1, CBL, JAK2) that were returned to physicians on clinical Foundation Medicine reports. For a subset of patients, we explored the frequency of true clonal hematopoiesis by comparing mutations on Foundation Medicine reports with matched blood sequencing.Results:Mutations in genes that are frequently altered in clonal hematopoiesis were identified in 65% (1,139/1,757) of patients undergoing NGS. When excluding TP53, which is often mutated in solid tumors, these events were still seen in 35% (619/1,757) of patients. Utilizing paired blood specimens, we were able to confirm that 8% (18/226) of mutations reported in these genes were true clonal hematopoiesis events. The majority of DNMT3A mutations (64%, 7/11) and minority of TP53 mutations (4%, 2/50) were clonal hematopoiesis.Conclusions:Clonal hematopoiesis mutations are commonly reported on unpaired NGS testing. It is important to recognize clonal hematopoiesis as a possible cause of misattribution of mutation origin when applying NGS findings to a patient's care.See related commentary by Pollyea, p. 5790

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Supplementary Figure 2, Part 1 from Assessment of Automated Image Analysis of Breast Cancer Tissue Microarrays for Epidemiologic Studies

Supplementary Tables S1-S6, Supplementary References. Supplementary Table S1. Characteristics of included studies; S2. Complete-case analysis of ten years estimated hazard ratios (HR) for all cause death in patients with ovarian cancer. S3. Complete-case analysis of ten years estimated subhazard ratios (SHR) for ovarian cancer death in patients with ovarian cancer. S4. Ten years estimated subhazard ratios (SHR) for ovarian cancer death in patients with ovarian cancer (with multiple imputation for missing grade and stage). S5. Complete-case analysis of ten years estimated hazard ratios (HR) for all cause death in patients with high grade serous ovarian cancer. S6. Ten years estimated hazard ratios (HR) for all cause death in patients with high grade serous ovarian cancer (with multiple imputation for missing stage).

Supplementary Figure from Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia

Supplementary Figure 2, Part 2 from Assessment of Automated Image Analysis of Breast Cancer Tissue Microarrays for Epidemiologic Studies

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Supplementary Figure 1 from Assessment of Automated Image Analysis of Breast Cancer Tissue Microarrays for Epidemiologic Studies

Background: Genome-wide association studies (GWAS) for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy, have identified novel susceptibility loci. GWAS for survival after EOC have had more limited success. The association of each single-nucleotide polymorphism (SNP) individually may not be well suited to detect small effects of multiple SNPs, such as those operating within the same biologic pathway. Gene set analysis (GSA) overcomes this limitation by assessing overall evidence for association of a phenotype with all measured variation in a set of genes.Methods: To determine gene sets associated with EOC overall survival, we conducted GSA using data from two large GWAS (N cases = 2,813, N deaths = 1,116), with a novel Principal Component-Gamma GSA method. Analysis was completed for all cases and then separately for high-grade serous histologic subtype.Results: Analysis of the high-grade serous subjects resulted in 43 gene sets with P < 0.005 (1.7%); of these, 21 gene sets had P < 0.10 in both GWAS, including intracellular signaling pathway (P = 7.3 × 10−5) and macrolide binding (P = 6.2 × 10−4) gene sets. The top gene sets in analysis of all cases were meiotic mismatch repair (P = 6.3 × 10−4) and macrolide binding (P = 1.0 × 10−3). Of 18 gene sets with P < 0.005 (0.7%), eight had P < 0.10 in both GWAS.Conclusion: This research detected novel gene sets associated with EOC survival.Impact: Novel gene sets associated with EOC survival might lead to new insights and avenues for development of novel therapies for EOC and pharmacogenomic studies. Cancer Epidemiol Biomarkers Prev; 21(3); 529–36. ©2012 AACR.

Supplementary Figure Legend for Figure 2 from Assessment of Automated Image Analysis of Breast Cancer Tissue Microarrays for Epidemiologic Studies

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Supplementary data file

Background: A major challenge in studies of etiologic heterogeneity in breast cancer has been the limited throughput, accuracy, and reproducibility of measuring tissue markers. Computerized image analysis systems may help address these concerns, but published reports of their use are limited. We assessed agreement between automated and pathologist scores of a diverse set of immunohistochemical assays done on breast cancer tissue microarrays (TMA).Methods: TMAs of 440 breast cancers previously stained for estrogen receptor (ER)-α, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), ER-β, and aromatase were independently scored by two pathologists and three automated systems (TMALab II, TMAx, and Ariol). Agreement between automated and pathologist scores of negative/positive was measured using the area under the receiver operating characteristics curve (AUC) and weighted κ statistics for categorical scores. We also investigated the correlation between immunohistochemical scores and mRNA expression levels.Results: Agreement between pathologist and automated negative/positive and categorical scores was excellent for ER-α and PR (AUC range = 0.98-0.99; κ range = 0.86-0.91). Lower levels of agreement were seen for ER-β categorical scores (AUC = 0.99-1.0; κ = 0.80-0.86) and both negative/positive and categorical scores for aromatase (AUC = 0.85-0.96; κ = 0.41-0.67) and HER2 (AUC = 0.94-0.97; κ = 0.53-0.72). For ER-α and PR, there was a strong correlation between mRNA levels and automated (ρ = 0.67-0.74) and pathologist immunohistochemical scores (ρ = 0.67-0.77). HER2 mRNA levels were more strongly correlated with pathologist (ρ = 0.63) than automated immunohistochemical scores (ρ = 0.41-0.49).Conclusions: Automated analysis of immunohistochemical markers is a promising approach for scoring large numbers of breast cancer tissues in epidemiologic investigations. This would facilitate studies of etiologic heterogeneity, which ultimately may allow improved risk prediction and better prevention approaches. Cancer Epidemiol Biomarkers Prev; 19(4); 992–9. ©2010 AACR.

ARID1A (BAF250a) is a component of the SWI/SNF chromatin modifying complex, plays an important tumour suppressor role, and is considered prognostic in several malignancies. However, in ovarian carcinomas there are contradictory reports on its relationship to outcome, immune response, and correlation with clinicopathological features. We assembled a series of 1,623 endometriosis-associated ovarian carcinomas, including 1,078 endometrioid (ENOC) and 545 clear cell (CCOC) ovarian carcinomas through combining resources of the Ovarian Tumor Tissue Analysis (OTTA) Consortium, the Canadian Ovarian Unified Experimental Resource (COEUR), local, and collaborative networks. Validated immunohistochemical surrogate assays for ARID1A mutations were applied to all samples. We investigated associations between ARID1A loss/mutation, clinical features, outcome, CD8+ tumour-infiltrating lymphocytes (CD8+ TIL), and DNA mismatch repair deficiency (MMRd). ARID1A loss was observed in 42% of CCOC and 25% of ENOC. We found no associations between ARID1A loss and outcomes, stage, age, or CD8+ TIL status in CCOC. Similarly, we found no association with outcome or stage in endometrioid cases. In ENOC, ARID1A loss was more prevalent in younger patients (p=0.012), and associated with MMRd (p

Patients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Pre-leukemic clones (i.e., clonal hematopoiesis; CH) are detectable years before the development of these aggressive malignancies, though the genomic events leading to transformation and expansion are not well-defined. Here, leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of pre-chemotherapy samples, we reconstruct the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy is revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan are hypermutated and enriched for complex structural variants (i.e., chromothripsis) while neoplasms with non-mutagenic chemotherapy exposures are genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to a discrete clinical exposure in each patient's life, we estimate that several complex events and genomic drivers are acquired after chemotherapy is administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes melphalan exposure and is selected following reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA-damage. Overall, we reveal a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select pre-existing CH, but also promote the acquisition of recurrent genomic drivers.

Purpose: To identify molecular subclasses of clear cell ovarian carcinoma (CCOC) and assess their impact on clinical presentation and outcomes. Experimental Design: We profiled 421 primary CCOCs that passed quality control using a targeted deep sequencing panel of 163 putative CCOC driver genes and whole transcriptome sequencing of 211 of these tumors. Molecularly defined subgroups were identified and tested for association with clinical characteristics and overall survival. Results: We detected a putative somatic driver mutation in at least one candidate gene in 95% (401/421) of CCOC tumors including ARID1A (in 49% of tumors), PIK3CA (49%), TERT (20%), and TP53 (16%). Clustering of cancer driver mutations and RNA expression converged upon two distinct subclasses of CCOC. The first was dominated by ARID1A-mutated tumors with enriched expression of canonical CCOC genes and markers of platinum resistance; the second was largely comprised of tumors with TP53 mutations and enriched for the expression of genes involved in extracellular matrix organization and mesenchymal differentiation. Compared with the ARID1A-mutated group, women with TP53-mutated tumors were more likely to have advanced-stage disease, no antecedent history of endometriosis, and poorer survival, driven by their advanced stage at presentation. In women with ARID1A-mutated tumors, there was a trend toward a lower rate of response to first-line platinum-based therapy. Conclusions: Our study suggests that CCOC consists of two distinct molecular subclasses with distinct clinical presentation and outcomes, with potential relevance to both traditional and experimental therapy responsiveness. See related commentary by Lheureux, p. 4838

Stably acquired mutations in hematopoietic cells represent substrates of selection that may lead to clonal hematopoiesis (CH), a common state in cancer patients that is associated with a heightened risk of leukemia development. Owing to technical and sample size limitations, most CH studies have characterized gene mutations or mosaic chromosomal alterations (mCAs) individually. Here we leverage peripheral blood sequencing data from 32,442 cancer patients to jointly characterize gene mutations (n = 14,789) and mCAs (n = 383) in CH. Recurrent composite genotypes resembling known genetic interactions in leukemia genomes underlie 23% of all detected autosomal alterations, indicating that these selection mechanisms are operative early in clonal evolution. CH with composite genotypes defines a patient group at high risk of leukemia progression (3-year cumulative incidence 14.6%, CI: 7–22%). Multivariable analysis identifies mCA as an independent risk factor for leukemia development (HR = 14, 95% CI: 6–33, P, Patients with solid cancers have high rates of clonal haematopoiesis associated with increased risk of secondary leukemias. Here, by using peripheral blood sequencing data from patients with solid non-hematologic cancer, the authors profile the landscape of mosaic chromosomal alterations and gene mutations, defining patients at high risk of leukemia progression.

PURPOSECoronavirus-2019 (COVID-19) mortality is higher in patients with cancer than in the general population, yet the cancer-associated risk factors for COVID-19 adverse outcomes are not fully characterized.PATIENTS AND METHODSWe reviewed clinical characteristics and outcomes from patients with cancer and concurrent COVID-19 at Memorial Sloan Kettering Cancer Center until March 31, 2020 (n = 309), and observed clinical end points until April 13, 2020. We hypothesized that cytotoxic chemotherapy administered within 35 days of a COVID-19 diagnosis is associated with an increased hazard ratio (HR) of severe or critical COVID-19. In secondary analyses, we estimated associations between specific clinical and laboratory variables and the incidence of a severe or critical COVID-19 event.RESULTSCytotoxic chemotherapy administration was not significantly associated with a severe or critical COVID-19 event (HR, 1.10; 95% CI, 0.73 to 1.60). Hematologic malignancy was associated with increased COVID-19 severity (HR, 1.90; 95% CI, 1.30 to 2.80). Patients with lung cancer also demonstrated higher rates of severe or critical COVID-19 events (HR, 2.0; 95% CI, 1.20 to 3.30). Lymphopenia at COVID-19 diagnosis was associated with higher rates of severe or critical illness (HR, 2.10; 95% CI, 1.50 to 3.10). Patients with baseline neutropenia 14-90 days before COVID-19 diagnosis had worse outcomes (HR, 4.20; 95% CI, 1.70 to 11.00). Findings from these analyses remained consistent in a multivariable model and in multiple sensitivity analyses. The rate of adverse events was lower in a time-matched population of patients with cancer without COVID-19.CONCLUSIONRecent cytotoxic chemotherapy treatment was not associated with adverse COVID-19 outcomes. Patients with active hematologic or lung malignancies, peri–COVID-19 lymphopenia, or baseline neutropenia had worse COVID-19 outcomes. Interactions among antineoplastic therapy, cancer type, and COVID-19 are complex and warrant further investigation.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1-3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

The acquisition of mutations in hematologic stem cells (clonal hematopoiesis) is common with normal aging and can be identified as an incidental finding through clinical genetic testing. Clonal hematopoiesis is associated with a heightened risk of developing hematologic neoplasms (especially myeloid) and accelerated atherosclerotic cardiovascular disease. This article discusses a multidisciplinary clinical approach to the management of patients with clonal hematopoiesis. Key areas of research needed to establish evidence-based clinical care guidelines and intervention strategies for individuals with clonal hematopoiesis are discussed.

Clonal hematopoiesis (CH) mutations are common among individuals without known hematologic disease. CH mutations have been associated with numerous adverse clinical outcomes across many different studies. We systematically reviewed the available literature for clinical outcomes associated with CH mutations in patients without hematologic disease. We searched PubMed, EMBASE, and Scopus for eligible studies. Three investigators independently extracted the data, and each study was verified by a second author. Risk of bias was assessed using the Newcastle-Ottawa Scale. We identified 32 studies with 56 cohorts that examine the association between CH mutations and clinical outcomes. We conducted meta-analyses comparing outcomes among individuals with and without detectable CH mutations. We conducted meta-analyses for cardiovascular diseases (nine studies; HR=1.61, 95% CI=1.26-2.07, p= .0002), hematologic malignancies (seven studies; HR=5.59, 95% CI=3.31-9.45, p&nbsp

Background: Clonal hematopoiesis (CH), the presence of acquired mutations in leukemia driver genes, promotes systemic inflammation and is common among aging men. Prostate cancer, a subset of which is lethal, also develops in aging men. We hypothesized that CH contributes to development of lethal prostate cancer. Methods: We conducted nested case-cohort studies for metastatic prostate cancer and prostate cancer-specific death (lethal prostate cancer) within the prospective Health Professionals Follow-up Study. First, we followed 1155 men free of prostate cancer and cardiovascular disease at blood draw (1993-1995) for development of lethal prostate cancer over up to 26 years. Second, we followed 532 men with incident non-metastatic prostate cancer for development of lethal prostate cancer. We sequenced blood DNA from 1488 participants for putative CH driver mutations in the 9 most common CH-defining genes with a custom targeted panel (VariantPlex, Invitae, Inc.) at ultra-high depth (mean, 18,000x), employing unique molecular identifiers for error correction. CH variant calling used a novel ensemble calling approach, ArCCH, validated with in-silico tumor dilutions and blinded technical replicates, which had high accuracy for variant allele frequencies (VAFs) as low as 0.1%. We estimated hazard ratios (HRs) with 95% confidence intervals (CIs) in proportional hazards regression with Prentice case-cohort weights. Results: In a random sample of 968 men initially free of prostate cancer and cardiovascular disease (median age at blood draw 60 years, interquartile range 52 to 67), 80% of men had CH at a variant allele frequency (VAF) of >0.1%, 15% had VAFs >2%, and 3% had VAFs >10%; 75% of men had variants in epigenetic modifier genes (DNMT3A, TET2, ASXL1), and 21% had variants in DNA repair genes (PPM1D, TP53, CHEK2). CH burden was strongly age-associated, with approximately one additional CH variant per decade of age at blood draw (mean difference 1.07 variants, 95% CI 0.93 to 1.21). Among men initially free from prostate cancer, after adjusting for age at blood draw, CH clones between 0.1% and 10% VAF were not related to lethal prostate cancer (206 events total; HR 0.93, 95% CI 0.49-1.76 for VAFs 2-10% vs. no variants detected at >0.1% VAF). Results for epigenetic modifiers and DNA repair genes were similar. While inconclusive, data were compatible with positive associations among younger men (< 65 years) or for VAFs >10%. Among men initially diagnosed with non-metastatic prostate cancer, results were similarly null for progression to lethal prostate cancer (164 events). Conclusions: This large prospective study with long-term follow-up suggests that low-level CH is unlikely a major contributor to and not well suited for early detection of lethal prostate cancer. Citation Format: Konrad H. Stopsack, Irenaeus C. Chan, Evelyn Schmidt, Alex Panchot, Samantha McNulty, Nicole A. Schreiber, Yiwen Zhang, Kathryn L. Penney, Michael F. Berger, Luis A. Diaz, Ross L. Levine, Kelly L. Bolton, Lorelei A. Mucci, Philip W. Kantoff. Clonal hematopoiesis and risk of lethal prostate cancer: a prospective cohort study with long-term follow-up. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P011.

A novel, common, and potent cardiovascular risk factor has recently emerged: clonal hematopoiesis of indeterminate potential (CHIP). CHIP arises from somatic mutations in hematopoietic stem cells that yield clonal progeny of mutant leukocytes in blood. Individuals with CHIP have a doubled risk of coronary heart disease and ischemic stroke, and worsened heart failure outcomes independent of traditional cardiovascular risk factors. The recognition of CHIP as a nontraditional risk factor challenges specialists in hematology/oncology and cardiovascular medicine alike. Should we screen for CHIP? If so, in whom? How should we assess cardiovascular risk in people with CHIP? How should we manage the excess cardiovascular risk in the absence of an evidence base? This review explains CHIP, explores the clinical quandaries, strives to provide reasonable recommendations for the multidisciplinary management of cardiovascular risk in individuals with CHIP, and highlights current knowledge gaps.

Purpose: Therapy-related myelodysplastic syndrome and acute leukemias (t-MDS/AL) are a major cause of nonrelapse mortality among pediatric cancer survivors. Although the presence of clonal hematopoiesis (CH) in adult patients at cancer diagnosis has been implicated in t-MDS/AL, there is limited published literature describing t-MDS/AL development in children. Experimental Design: We performed molecular characterization of 199 serial bone marrow samples from 52 patients treated for high-risk neuroblastoma, including 17 with t-MDS/AL (transformation), 14 with transient cytogenetic abnormalities (transient), and 21 without t-MDS/AL or cytogenetic alterations (neuroblastoma-treated control). We also evaluated for CH in a cohort of 657 pediatric patients with solid tumor. Results: We detected at least one disease-defining alteration in all cases at t-MDS/AL diagnosis, most commonly TP53 mutations and KMT2A rearrangements, including involving two novel partner genes (PRDM10 and DDX6). Backtracking studies identified at least one t-MDS/AL-associated mutation in 13 of 17 patients at a median of 15 months before t-MDS/AL diagnosis (range, 1.3–32.4). In comparison, acquired mutations were infrequent in the transient and control groups (4/14 and 1/21, respectively). The relative risk for development of t-MDS/AL in the presence of an oncogenic mutation was 8.8 for transformation patients compared with transient. Unlike CH in adult oncology patients, TP53 mutations were only detectable after initiation of cancer therapy. Last, only 1% of pediatric patients with solid tumor evaluated had CH involving myeloid genes. Conclusions: These findings demonstrate the clinical relevance of identifying molecular abnormalities in predicting development of t-MDS/AL and should guide the formation of intervention protocols to prevent this complication in high-risk pediatric patients.

Background: Ovarian clear cell carcinoma (OCCC) is a rare ovarian cancer histotype that tends to be resistant to standard platinum-based chemotherapeutics. We sought to better understand the role of DNA methylation in clinical and biological subclassification of OCCC. Methods: We interrogated genome-wide methylation using DNA from fresh frozen tumors from 271 cases, applied nonsmooth nonnegative matrix factorization (nsNMF) clustering, and evaluated clinical associations and biological pathways. Results: Two approximately equally sized clusters that associated with several clinical features were identified. Compared with Cluster 2 (N = 137), Cluster 1 cases (N = 134) presented at a more advanced stage, were less likely to be of Asian ancestry, and tended to have poorer outcomes including macroscopic residual disease following primary debulking surgery (P < 0.10). Subset analyses of targeted tumor sequencing and IHC data revealed that Cluster 1 tumors showed TP53 mutation and abnormal p53 expression, and Cluster 2 tumors showed aneuploidy and ARID1A/PIK3CA mutation (P < 0.05). Cluster-defining CpGs included 1,388 CpGs residing within 200 bp of the transcription start sites of 977 genes; 38% of these genes (N = 369 genes) were differentially expressed across cluster in transcriptomic subset analysis (P < 10−4). Differentially expressed genes were enriched for six immune-related pathways, including IFNα and IFNγ responses (P < 10−6). Conclusions: DNA methylation clusters in OCCC correlate with disease features and gene expression patterns among immune pathways. Impact: This work serves as a foundation for integrative analyses that better understand the complex biology of OCCC in an effort to improve potential for development of targeted therapeutics.

Clonal hematopoiesis (CH), defined as the clonal expansion of mutated hematopoietic stem cells (HSCs), is a common aging process. CH is known to be strongly associated with the development of hematologic malignancies, most commonly myeloid neoplasms (MN) including acute myeloid neoplasm and myelodysplastic syndrome (MDS).1–6 Recent work has elucidated how the development and cellular fitness of CH is shaped by human aging, environmental exposures, and the germline (inherited) composition of an individual. This in turn provides valuable insights into the pathogenesis of MNs including MDS.

Tumor protein p53 (TP53) is the most frequently mutated gene in cancer1,2. In patients with myelodysplastic syndromes (MDS), TP53 mutations are associated with high-risk disease3,4, rapid transformation to acute myeloid leukemia (AML)5, resistance to conventional therapies6–8 and dismal outcomes9. Consistent with the tumor-suppressive role of TP53, patients harbor both mono- and biallelic mutations10. However, the biological and clinical implications of TP53 allelic state have not been fully investigated in MDS or any other cancer type. We analyzed 3,324 patients with MDS for TP53 mutations and allelic imbalances and delineated two subsets of patients with distinct phenotypes and outcomes. One-third of TP53-mutated patients had monoallelic mutations whereas two-thirds had multiple hits (multi-hit) consistent with biallelic targeting. Established associations with complex karyotype, few co-occurring mutations, high-risk presentation and poor outcomes were specific to multi-hit patients only. TP53 multi-hit state predicted risk of death and leukemic transformation independently of the Revised International Prognostic Scoring System (IPSS-R)11. Surprisingly, monoallelic patients did not differ from TP53 wild-type patients in outcomes and response to therapy. This study shows that consideration of TP53 allelic state is critical for diagnostic and prognostic precision in MDS as well as in future correlative studies of treatment response. Clinical sequencing across a large prospective cohort of patients with myelodysplasic syndrome uncovers distinct associations between the mono- and biallelic states of TP53 and clinical presentation

Acquired genetic mutations in hematopoietic stem or progenitor cells can lead to clonal expansion and imbalanced blood cell production. Clonal hematopoiesis is exceptionally common with human aging, confers a risk of evolution to overt hematologic malignancy, and increases all-cause mortality and the risk of cardiovascular disease. The degree of risk depends on the specific mutant allele driving clonal expansion, number of mutations, mutant allele burden, and concomitant nongenetic risk factors (eg, hypertension or cigarette smoking). People with clonal hematopoiesis may come to clinical attention in a variety of ways, including during the evaluation of a possible hematologic malignancy, as an incidental discovery during molecular analysis of a nonhematologic neoplasm, after hematopoietic cell transplantation, or as a result of germline testing for inherited variants. Even though the risk of clonal progression or a cardiovascular event in an individual patient with clonal hematopoiesis may be low, the possibility of future clinical consequences may contribute to uncertainty and worry, because it is not yet known how to modify these risks. This review summarizes clinical considerations for patients with clonal hematopoiesis, including important points for hematologists to consider discussing with affected persons who may understandably be anxious about having a mutation in their blood that predisposes them to develop a malignancy, but which is significantly more likely to result in a myocardial infarction or stroke. The increasing frequency with which people with clonal hematopoiesis are discovered and the need for counseling these patients is driving many institutions to create specialized clinics. We describe our own experience with forming such clinics.

Cancer-associated venous thromboembolism (CAT) is a well-described complication of cancer and a leading cause of death in cancer patients. The purpose of this study was to assess potential associations of molecular signatures with CAT, including tumor-specific mutations and the presence of clonal hematopoiesis. We analyzed deep-coverage targeted DNA-sequencing data of >14,000 solid tumor samples using the MSK-IMPACT™ platform to identify somatic alterations associated with VTE. Endpoint was defined as the first instance of cancer-associated pulmonary embolism and/or proximal/distal lower extremity deep vein thrombosis. Cause-specific Cox proportional hazards regression was used, adjusting for pertinent clinical covariates. Of 11,695 evaluable individuals, 72% had metastatic disease at time of IMPACT. Tumor-specific mutations in KRAS (HR=1.34 [1.09-1.64]; adjusted p=0.08), STK11 (HR=2.12 [1.55-2.89]; adjusted pKEAP1 (HR=1.84 [1.21-2.79]; adjusted p=0.07), CTNNB1 (HR=1.73 [1.15-2.60]; adjusted p=0.09), CDKN2B (HR= 1.45 [1.13-1.85], adjusted p=0.07) and MET (HR=1.83 [1.15-2.92]; adjusted p=0.09) were associated with a significantly increased risk of CAT independent of tumor type. Mutations in SETD2 were associated with a decreased risk of CAT (HR=0.35 [0.16-0.79], adjusted p=0.09). The presence of clonal hematopoiesis was not associated with an increased VTE rate. This is the first large-scale analysis to elucidate tumor-specific genomic events associated with CAT. Somatic tumor mutations of STK11, KRAS, CTNNB1, KEAP1, CDKN2B and MET were associated with an increased risk of VTE in solid tumor patients. Further analysis is needed to validate these findings and identify additional molecular signatures unique to individual tumor types.Key PointsTumor mutations in STK11, KRAS, CTNNB1, KEAP1, CDKN2B, MET and SETD2 modulate the risk of cancer-associated thrombosis.The presence of clonal hematopoiesis does not affect the risk of cancer-associated thrombosis.

Acquired mutations are pervasive across normal tissues. However, understanding of the processes that drive transformation of certain clones to cancer is limited. Here we study this phenomenon in the context of clonal hematopoiesis (CH) and the development of therapy-related myeloid neoplasms (tMNs). We find that mutations are selected differentially based on exposures. Mutations in ASXL1 are enriched in current or former smokers, whereas cancer therapy with radiation, platinum and topoisomerase II inhibitors preferentially selects for mutations in DNA damage response genes (TP53, PPM1D, CHEK2). Sequential sampling provides definitive evidence that DNA damage response clones outcompete other clones when exposed to certain therapies. Among cases in which CH was previously detected, the CH mutation was present at tMN diagnosis. We identify the molecular characteristics of CH that increase risk of tMN. The increasing implementation of clinical sequencing at diagnosis provides an opportunity to identify patients at risk of tMN for prevention strategies. Environmental exposures shape patterns of selection for mutations in clonal hematopoiesis. Cancer therapies promote the growth of clones with mutations that are strongly enriched in treatment-related myeloid neoplasms.

Purpose: In this era of precision-based medicine, for optimal patient care, results reported from commercial next-generation sequencing (NGS) assays should adequately reflect the burden of somatic mutations in the tumor being sequenced. Here, we sought to determine the prevalence of clonal hematopoiesis leading to possible misattribution of tumor mutation calls on unpaired Foundation Medicine NGS assays. Experimental Design: This was a retrospective cohort study of individuals undergoing NGS of solid tumors from two large cancer centers. We identified and quantified mutations in genes known to be frequently altered in clonal hematopoiesis (DNMT3A, TET2, ASXL1, TP53, ATM, CHEK2, SF3B1, CBL, JAK2) that were returned to physicians on clinical Foundation Medicine reports. For a subset of patients, we explored the frequency of true clonal hematopoiesis by comparing mutations on Foundation Medicine reports with matched blood sequencing. Results: Mutations in genes that are frequently altered in clonal hematopoiesis were identified in 65% (1,139/1,757) of patients undergoing NGS. When excluding TP53, which is often mutated in solid tumors, these events were still seen in 35% (619/1,757) of patients. Utilizing paired blood specimens, we were able to confirm that 8% (18/226) of mutations reported in these genes were true clonal hematopoiesis events. The majority of DNMT3A mutations (64%, 7/11) and minority of TP53 mutations (4%, 2/50) were clonal hematopoiesis. Conclusions: Clonal hematopoiesis mutations are commonly reported on unpaired NGS testing. It is important to recognize clonal hematopoiesis as a possible cause of misattribution of mutation origin when applying NGS findings to a patient's care. See related commentary by Pollyea, p. 5790

Poly (ADP-ribose) polymerase (PARP) inhibitors are an important new class of anti-cancer therapies. Therapy-related myeloid neoplasia (tMN) has been reported following PARPi therapy and is associated with adverse outcomes. We have previously shown, in retrospective data, that prior chemotherapy increases the incidence of clonal hematopoiesis (CH), especially in DNA damage response (DDR) pathway genes including TP53, PPM1D, and CHEK2 and is associated with progression to tMN. In particular, patients who receive PARPi therapy are more likely to have CH compared to other therapies or untreated patients. In the IMPACT study of CH in 10,156 cancer patients, exposure to PARPi were more likely to have CH (33%) compared to untreated patients (16%). This was particularly pronounced for DDR gene mutations, with 25% of PARPi treated patients with DDR CH compared to 2% of untreated patients. In multivariate analysis accounting for demographics and exposure to other chemotherapy or radiation therapy, exposure to PARPi conferred an increased risk of DDR CH (OR = 3.6, 95% CI 1.5-8.5, p = 0.004). From these data, we hypothesize that mutations in DDR pathway genes provide a fitness advantage to hematopoietic stem/progenitor cells (HSPCs) following PARPi treatment, leading to clonal hematopoiesis. A major limitation, however of our previous work in retrospective clinical samples, is the inability to completely adjust for the confounding effect of prior exposure to cytotoxic therapy (in particular platinum therapies) and germline BRCA1/2 mutations; both which have been shown or hypothesized to increase the risk of tMN. To test whether PARPi exposure might provide a fitness advantage to HSPCs independent of prior exposure to other therapies, we first examined the response of CRISPR-gene edited TP53-/- MOLM13 cells to the PARPi Olaparib and, as a control, Cisplatin. As expected, TP53-/- cells had increased resistance to both agents, though the response was much more pronounced in Cisplatin-treated cells (Figure 1A,B). Next, we implemented a mouse model of TP53-mutant clonal hematopoiesis, by generating mixed bone marrow chimeras transplanted with a 1:9 ratio of wildtype (CD45.1) to TP53 R172H+/- (CD45.2) cells. The "baseline" contribution of TP53 R172H+/- (CD45.2) cells to peripheral blood leukocytes 8 weeks after transplantation was determined by flow cytometry. Mice were then randomized into the following three cohorts: 1) Cisplatin (6mg/kg on days 1, 8, and 15); 2) Olaparib (50mg/kg daily for 3 weeks); and 3) vehicle alone. Peripheral blood chimerism was assessed 3, 9, and 12 weeks after initiating treatment. In addition, the contribution of TP53 R172H+/- to lineage -Sca1 +Kit + (LSK) cells in the bone marrow was determined. Cisplatin treatment resulted in a significant increase in the contribution of TP53 R172H+ to peripheral blood total leukocytes, granulocytes, and bone marrow LSK cells (Figure 1C-E). In contrast, Olaparib treated mice showed no change in CD45 chimerism. From these results we conclude that p53-deficiency does not confer a strong fitness advantage to mouse HSPCs in response to PARPi treatment. This suggests that the strong association observed between prior PARPi therapy, CH and tMN in clinical cohorts may in part be due to the confounding effects of prior (often heavy) exposure to platinum-based therapy. However, the majority of patients receiving PARPi have germline heterozygous BRCA1/2 mutations that could be contributing to their hematopoietic response to PARPi therapy. Experiments are underway to test this possibility by analyzing mixed bone marrow chimeras carrying heterozygous mutations of both Brca1 and Trp53. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Patients treated with cytotoxic chemotherapies and/or autologous stem-cell transplantation (ASCT) are at risk for therapy-related myeloid neoplasms (tMN). As these agents yield increased mutation burden in relapsed malignancies and leave evidence of exposure via mutational signatures, we studied the genomic and temporal relationship between chemo exposure and progression of clonal hematopoiesis (CH) to tMN. Methods: We analyzed 32 tMN whole genomes (WG) from 31 patients [27 acute myeloid leukemias (AML), 4 myelodysplastic syndromes]. For 7 patients with tMN post-high-dose melphalan/ASCT, we investigated the presence of antecedent CH using targeted sequencing (MSK-IMPACT; Bolton et al. Nat Gen 2020) on pre-melphalan blood mononuclear cells, granulocytes, or CD34+ apheresis samples. Results: TMN was diagnosed a median of 4.2 years (IQR, 2.6-6.6) following primary treatment. When compared to data from 200 de novo AML from TCGA (NEJM, 2013), tMNs had fewer mutations in FLT3 (9.7% v 28.0%; p = 0.028) and NPM1 (3.2% v 27.0%; p = 0.003). TP53 loss was enriched in tMNs (25.8% v 10.5%; p = 0.035 ). Mutational signature analysis revealed 5 known single base substitution (SBS) signatures in tMN: the hematopoietic stem-cell (SBS-HSC), aging (SBS1), melphalan (SBS-MM1), and platinum signatures (E-SBS1, E-SBS20) (Rustad et al. Nat Comm 2020, Pich et al. Nat Gen 2019). Complex structural variants (SV), defined as ≥3 breakpoint pairs involved in simultaneous copy number changes (Rustad et al. Blood Can Disc 2020), were observed in 7 tMNs; including chromothripsis in 6 tumors (19.4%), chromoplexy in 2 (6.5%), templated insertion in 1 (3.2%), and unspecified complex SV in 2 (6.5%). Chromothripsis has not been previously reported in de novo AML and, in 4 cases, involved chromosome 19 with hyper-amplification of the SMARCA4 locus (≥5 copies). CH variants that became clonal in tumor were seen in 5/7 pre-melphalan/ASCT samples and included mutations in TP53, RUNX1, NCOR1, NF1, CREBBP, DNMT3A, and PPM1D. Chemotherapy introduces hundreds of mutations, leaving each exposed cell with a unique catalogue (i.e., barcode). In fact, TMNs with evidence of chemo signatures had a higher mutation burden (median 1574 single nucleotide variants) than those without (median 938; p = 0.004). Detection of chemo signatures in bulk genome sequencing relies on one cell, with its catalogue of mutations, to expand to clonal dominance (Fig 1a, Landau et al. Nat Comm 2020). Given the long latency between exposure and tMN diagnosis, this single-cell expansion model was expected for all samples exposed to melphalan or platinum-based regimens (i.e., agents with a measurable signature). Strikingly, all patients with pre-tMN platinum exposure (n=7) had evidence of platinum SBS signatures whereas only 2 of 7 patients with prior melphalan/ASCT had a melphalan signature (SBS-MM1). As all platinum-exposed tMN had mutational evidence of exposure, a CH clone must have existed prior to exposure, supporting a single-cell expansion model. Absence of a chemo signature for 5/7 post-melphalan/ASCT tumors despite exposure implies tumor progression driven either by multiple clones in parallel (Fig 1b) or by an unexposed clone. As latency largely excludes the former, this suggests pre-tMN CH clones were re-infused during SCT, thus avoiding chemo exposure (Fig 1c). This is supported by two lines of evidence: 1) tMNs from 2 patients exposed to sequential platinum and melphalan/ASCT had platinum but not melphalan signatures confirming single-cell expansion of the pre-tMN CH clone post-platinum but with escape from exposure to melphalan in the ASCT (Fig 1d); 2) targeted sequencing of pre-tMN samples from melphalan/ASCT patients identified tMN genomic mutations at the CH level in 5/7 cases, including in all 3 tested apheresis samples - one of which (TP53) expanded to dominance without a melphalan signature. Conclusion: WG sequencing identified novel features of tMN revealing the key driver role of complex SV. Mutational signature analyses and targeted sequencing of pre-tMN samples can increase our understanding of tMN pathogenesis and demonstrate that tMNs arising post-ASCT are often driven by CH clones that re-engraft after escaping melphalan exposure. This mode of expansion suggests that a permissive, immunosuppressed, post-transplant environment might play a more important role than chemotherapy-induced mutagenesis in tMN pathogenesis. Figure 1 Figure 1. Disclosures Diamond: Sanofi: Honoraria; Medscape: Honoraria. Watts: Rafael Pharmaceuticals: Consultancy; Genentech: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Aptevo Therapeutices: Research Funding. Kazandjian: Arcellx: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bradley: AbbVie: Consultancy, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Bolli: Amgen: Honoraria; Takeda: Honoraria; Janssen: Consultancy, Honoraria; Celgene/BMS: Consultancy, Honoraria. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy. Scordo: Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; i3 Health: Other: Speaker; Omeros Corporation: Consultancy; Angiocrine Bioscience: Consultancy, Research Funding; McKinsey & Company: Consultancy. Lahoud: MorphoSys: Membership on an entity's Board of Directors or advisory committees. Stein: Jazz Pharmaceuticals: Consultancy; Foghorn Therapeutics: Consultancy; Blueprint Medicines: Consultancy; Gilead Sciences, Inc.: Consultancy; Abbvie: Consultancy; Janssen Pharmaceuticals: Consultancy; Genentech: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Agios Pharmaceuticals, Inc: Consultancy; Novartis: Consultancy; Astellas: Consultancy; Syndax Pharmaceuticals: Consultancy; PinotBio: Consultancy; Daiichi Sankyo: Consultancy; Syros Pharmaceuticals, Inc.: Consultancy. Sauter: Precision Biosciences: Consultancy; Kite/Gilead: Consultancy; Bristol-Myers Squibb: Research Funding; GSK: Consultancy; Gamida Cell: Consultancy; Celgene: Consultancy, Research Funding; Genmab: Consultancy; Novartis: Consultancy; Spectrum Pharmaceuticals: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding. Hassoun: Celgene, Takeda, Janssen: Research Funding. Mailankody: Bristol Myers Squibb/Juno: Research Funding; Physician Education Resource: Honoraria; Plexus Communications: Honoraria; Takeda Oncology: Research Funding; Jansen Oncology: Research Funding; Fate Therapeutics: Research Funding; Allogene Therapeutics: Research Funding; Legend Biotech: Consultancy; Evicore: Consultancy. Korde: Medimmune: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Hultcrantz: Daiichi Sankyo: Research Funding; Intellisphere LLC: Consultancy; Curio Science LLC: Consultancy; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding. Shah: Bristol Myers Squibb: Research Funding; Janssen: Research Funding. Shah: Janssen Pharmaceutica: Research Funding; Amgen: Research Funding. Park: Servier: Consultancy; Affyimmune: Consultancy; Autolus: Consultancy; Minerva: Consultancy; PrecisionBio: Consultancy; BMS: Consultancy; Novartis: Consultancy; Kura Oncology: Consultancy; Curocel: Consultancy; Artiva: Consultancy; Innate Pharma: Consultancy; Intellia: Consultancy; Amgen: Consultancy; Kite Pharma: Consultancy. Landau: Genzyme: Honoraria; Takeda, Janssen, Caelum Biosciences, Celgene, Pfizer, Genzyme: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding. Sekeres: BMS: Membership on an entity's Board of Directors or advisory committees; Takeda/Millenium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Ho: Blueprint Medicine: Membership on an entity's Board of Directors or advisory committees. Roshal: Celgene: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Physicians' Education Resource: Other: Provision of services. Lesokhin: pfizer: Consultancy, Research Funding; Janssen: Honoraria, Research Funding; Iteos: Consultancy; Serametrix, Inc: Patents & Royalties; Genetech: Research Funding; Trillium Therapeutics: Consultancy; bristol myers squibb: Research Funding; Behringer Ingelheim: Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees. Landgren: Janssen: Other: IDMC; Janssen: Research Funding; Amgen: Honoraria; Celgene: Research Funding; Janssen: Honoraria; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Maura: OncLive: Honoraria; Medscape: Consultancy, Honoraria.

Introduction An estimated 1.2 million individuals in the United States are living with HIV (www.hiv.gov). Thanks to modern antiretroviral therapies (ART), the life expectancies of individuals living with HIV now approach those of people without HIV (Marcus 2020). But with this, an increase in the proportion of deaths due to non-infectious causes has occurred including, predominately, cardiovascular disease (CVD). Multiple studies have shown that HIV infection is an independent risk factor for cardiovascular disease. This increased risk is multifactorial due to an increased prevalence of traditional CVD risk factors and HIV-specific risk factors including ART, chronic inflammation and immune activation (Hsue 2010). Clonal Hematopoiesis, characterized by the expansion of blood cells stemming from a mutant hematopoietic stem/progenitor cell (HSPCs) is an emerging risk factor for cardiovascular disease. Mechanistically, this is thought to be driven, at least in part, by CH-induced proinflammatory circulating leukocytes (Jaiswal 2017, Bick 2020). Recent data suggest that inflammatory states may also promote the expansion of DNMT3A and TET2 CH mutant HSPCs suggesting a potential feedback loop (Zheng 2019, King 2019). CH has been recently reported to be increased among individuals living with HIV (Dharan 2021). Whether CH is a risk factor for HIV-associated CVD is not known. Given evidence of an increased prevalence of CH among those with HIV, we hypothesized that CH would predict risk of CVD. Methods We performed a nested case-control study drawn from the AIDS Clinical Trials Group Longitudinal Linked Randomized Trials (ALLRT) observational study which was a long-term study of individuals with newly diagnosed HIV receiving ART (N=4,371). Cases who developed a cardiovascular event were matched to controls with no event based on age at blood draw and sex. Peripheral blood mononuclear cells (PMBCs) were isolated from the blood draw closest to the time of CVD event or time of censoring, for cases and controls respectively. Extracted DNA was subjected to whole exome sequencing (WES) at a median depth of 500x. We also included WES data from 267 children sequenced using the same platform as a technical panel of normal (PON). Mutation calling was performed using Mutect2, VarDict and Varscan2. We retained variants that met the following criteria: 1) passed by at least two callers 2) showed statistically significant higher variant allele fraction (VAF) compared to our PON 3) had VAF between 2-35% 4) at least 6 reads supporting the variant 5) passed additional post-calling filters for germline variants and sequencing artifacts 6) were annotated as a putative driver of CH based on previously defined criteria. Results Over 13 years of follow-up we observed 83 cardiovascular events: 4 cases of ASC, 14 of stroke, 25 of cerebrovascular accident, 37 of myocardial infarction, 2 of peripheral artery disease, and 1 heart failure. The mean age of our participants was 51.6 years, 81% were male, 35% percent were African-American, 17% were Hispanic and 2% were Asian. Among 161 participants (83 cases and 78 controls) we observed at least one CH mutation in 14% of participants (18% of cases and 10% of controls). The median VAF was 3.5% with 23 individuals harboring a median of 1 mutation (range 1-2). In a conditional logistic regression model adjusted for race, Atherosclerotic Cardiovascular Disease (ASCVD) Risk Score and stratified by case-control matched pairs, CH was significantly associated with CVD (OR=3.70, 95% CI 1.03-13.23 p=0.045). Because of the possible confounding effects of classes of ART therapy on the CH-CVD association, we explored whether the prevalence of CH differed based on prior exposure to ART sub-class(es). We did not observe differences in the frequency of CH among individuals with prior exposure to different ART regimens (Non-Nucleoside Reverse Transcriptase Inhibitors 14%, Nucleoside reverse transcriptase inhibitors 17%, Protease inhibitors 20%, Integrase inhibitors 18%) Conclusions Among individuals with HIV, CH is associated with a nearly four-fold increased risk of CVD. These findings highlight the relevance of CH to HIV-associated CVD and provide support for interventions targeting potential CH-induced pro-inflammatory states among patients with HIV. Disclosures Erlandson: Gilead Sciences: Consultancy, Research Funding; Viiv Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Bolton: bristol myers squibb: Research Funding.

Background: Advanced age, medical co-morbidities and a pro-inflammatory immunologic profile are associated with severe COVID-19. Acquired somatic mutations in hematopoietic stem cells (clonal hematopoiesis or CH) are common in elderly individuals. Previous studies have shown CH is associated with alterations in immunologic repertoire and function. Here, we investigate whether CH predisposes to severe COVID-19 or other infections. Methods: We performed a retrospective cohort study of 39,291 adult cancer patients with non-hematologic malignancies who had their tumor and blood sequenced using a panel of 468 known cancer driver genes. We identified individuals who tested positive for SARS-CoV-2 between March 8, 2020 and June 9, 2020. We determined the association between CH and severe COVID-19 (hypoxic event, hospitalization or death) using multivariable logistic regression models. We used an established aggregation schema for ICD-CM codes to identify distinct infection subtypes. The relationship between CH and risk of incident infections was determined using multivariable Cox regression. Results: In the study cohort, 436 patients were positive for SARS-CoV-2 and 165 developed severe COVID-19. COVID-19 patients with CH were more likely to develop severe disease compared to non-severe (OR=1.8; 95% CI: 1.1-2.9; p=0.01). CH was also more common among patients with severe COVID-19 compared to COVID-19 negative individuals (OR=1.6, 95% CI: 1.2-2.4, p=0.01). In 62,891 person-years of follow-up, 4,059 individuals developed an infection. CH was associated with risk of sepsis (HR=1.13; 95% CI 1.01-1.25, p=0.04) and bacterial enteritis (HR=1.8, 95% CI 1.1-2.8, p=0.01). Conclusions: CH is associated with severe COVID-19 and an increased risk of other infections in cancer patients. Disclosures Bolton: GRAIL: Research Funding. Jee:MDSeq Inc.: Patents & Royalties. Papaemmanuil:Celgene: Consultancy, Honoraria, Research Funding; Prime Oncology: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; MSKCC: Patents & Royalties; Kyowa Hakko Kirin: Consultancy, Honoraria; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Berger:Illumina: Research Funding; Grail: Research Funding; Roche: Consultancy. Levine:Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Novartis: Consultancy; Amgen: Honoraria; Gilead: Honoraria. Zehir:Illumina: Honoraria; Memorial Sloan Kettering Cancer Center: Current Employment.

Clonal hematopoiesis (CH), defined by expansion of clones in the hematopoietic system, has been linked to internal (aging) and external (smoking and oncologic therapy) factors in patients with advanced solid tumors. The effects of inherited variants, however, have yielded inconclusive results in few studies so far. While comparison of CH prevalence between monozygotic and dizygotic pairs failed to detect genetic predisposition to CH, others have shown associations with germline variants near TERT and TET2, and a potential link between pathogenic CHEK2 germline variants and CH has been proposed. Here, utilizing prospective tumor-blood paired sequencing data from a large institutional clinical cohort, we link inherited pathogenic variants in cancer predisposition genes to CH and show interactions between germline and somatic mutations in DNA damage repair (DDR) genes. We analyzed sequencing data from 32,748 cancer patients who have undergone MSK-IMPACT testing using paired tumor and blood samples to study the associations between CH and rare pathogenic and likely-pathogenic (P/LP) germline variants. P/LP germline mutations were identified in the blood using an in-house developed random forest algorithm, trained using known P/LP mutations. Following joint anonymization of P/LP germline mutations and CH variants, linear regression models by gene were used to test for associations, accounting for known CH confounders. In our cohort, 9,697 patients (29.6%) harbored at least one CH mutation, while P/LP germline variants were detected in 1,793 of these CH patients. P/LP germline variants in CHEK2 (OR=1.29, p=4.49x10-2) were found to be significantly associated with CH in a pan-cancer analysis. Interestingly, germline P/LP and CH co-mutations in CHEK2 were very rare; we identified only one patient with CH and germline mutations in CHEK2 out of 161 patients with germline CHEK2 mutations, compared to 304 patients with CH CHEK2 mutation when CHEK2 is wild-type in the germline. In contrast, ATM and TP53 showed increased number of patients with co-mutations in P/LP germline and CH mutations in the same gene, with 14 out of 84 patients with germline mutations in ATM compared to 254 when not mutated (OR=7.05, p=2.08x10-7) , and 3 out of 16 compared to 387 for TP53 (OR=10.94, p=5.44x10-3). Further, we found mutual exclusivity between CHEK2 P/LP germline variants and CH mutations in PPM1D (OR=0.18, p=1.79x10-2). This relationship was replicated by comparing CHEK2 germline mutations and somatic PPM1D mutations in the solid tumor samples within the same cohort and same pattern of mutual exclusivity also persisted in comparisons of other DDR pathway genes (i.e. ATM vs PPM1D and TP53 vs PPM1D). When studying specific cancer types, a significant association between CHEK2 P/LP germline variants and CH in breast cancer patients was also identified (OR=2.33, p=2.64x10-3). Furthermore, trends between germline mutations in ATM and CH rates in breast cancer (OR=1.84, p=9.82x10-2) and lung adenocarcinoma (OR=2.22, p=8.91x10-2) patients were observed. Our results support the already reported link between rare pathogenic germline mutations in CHEK2 with CH in cancer patients, with stronger association among breast cancer patients (Comen E. et al, 2019). Moreover, this study suggests a close relationship between inherited variants and CH mutations within the DDR genes in solid tumor patients and points out to interesting mutual exclusivity patterns between these same genes. Intriguingly, hematopoietic stem cells have been largely related to proficient DDR systems in order to regulate HSC maintenance and tissue homeostasis in the hematopoietic system. All in all, associations identified in this study might translate into enhanced clinical surveillance for CH and associated comorbidities cancer patients harboring these germline mutations. Disclosures Bolton: GRAIL: Research Funding. Papaemmanuil:Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Illumina: Consultancy, Honoraria; Prime Oncology: Consultancy, Honoraria; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Kyowa Hakko Kirin: Consultancy, Honoraria; MSKCC: Patents & Royalties. Levine:Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Novartis: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Berger:Illumina: Research Funding; Grail: Research Funding; Roche: Consultancy. Zehir:Illumina: Honoraria; Memorial Sloan Kettering Cancer Center: Current Employment.

Background Stably acquired mutations in hematopoietic cells represent substrates of selection that may lead to clonal hematopoiesis (CH), a common state in cancer patients that is associated with a heightened risk of leukemia development. Owing to technical and sample size limitations most CH studies have characterized gene mutations or mosaic chromosomal alterations (mCAs) individually. The relationship between acquired gene mutations and mCAs in CH and their joint roles in leukemia development have not been systematically investigated. Methods We developed a method to reliably map mCAs at low cell fractions from deep targeted sequencing data. We applied this method in a cohort of 32,442 solid tumor patients who have undergone prospective clinical sequencing (MSK-IMPACT). We characterized gene mutations in our patient cohort using an established variant calling procedure from our previous studies. Results We jointly characterized 383 mCA events (median aberrant cell fraction 32%, range 10%-90%) and 14,789 mutations across 457 genes. mCA was significantly associated with age (OR=1.8, P20% VAF, compared to 1% of the general cohort. We identify co-mutational patterns characteristic of diverse mechanisms of clonal selection. We observe that mutations in DNMT3A, TET2, JAK2, MPL, EZH2, TP53 and ATM form recurrent double-hits with deletions or CNLOHs, resulting in either oncogene mutant dosage adjustment or inactivation of tumor suppressors. Notably, certain mCA events were highly directed events acting on previously acquired gene mutations in the corresponding loci. Of six events of 7qCNLOH, all six co-localized with an EZH2 (7q36.1) mutation (q During patient follow-up, the 3-year cumulative incidence of leukemias was significantly higher in patients with composite CH genotypes (14.6%, CI: 7-22%) as compared to patients with either mCA, gene mutation alone or no CH, of which all had a 3-year cumulative incidence of Conclusions Our joint characterization of gene mutations and mCAs in a large prospective sequencing cohort reveals a previously unrecognized layer of complexity in the evolutionary dynamics of clonal hematopoiesis that converges towards characteristic genotypes associated with distinct leukemia subtypes. This puts mCAs in the context of the continuous evolutionary process of oncogenesis that can often span years and sheds new lights on its patterns of acquisition and progression. We demonstrate that the integration of chromosomal aberrations provides additional resolution to risk stratification as well as interpretation of clinical phenotypes and that mCAs should be screened in conjunction with gene mutations to improve existing CH surveillance programs in cancer patients. Disclosures Bolton: GRAIL: Research Funding. Medina:Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Mantha:MJH Associates: Honoraria; Physicians Education Resource: Honoraria. Solit:Pfizer: Honoraria; Loxo Oncology: Honoraria; Lilly Oncology: Honoraria; Illumina: Honoraria; Vivideon Therapeutics: Honoraria. Diaz:Neophore: Consultancy, Current equity holder in private company; Merck: Consultancy; Johns Hopkins University: Patents & Royalties; Jounce Therapeutics: Current equity holder in private company; Thrive Earlier Detection: Current equity holder in private company; Personal Genome Diagnostics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Berger:Illumina: Research Funding; Roche: Consultancy; Grail: Research Funding. Levine:Lilly: Consultancy, Honoraria; Janssen: Consultancy; Roche: Consultancy, Honoraria, Research Funding; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Prelude Therapeutics: Research Funding; Gilead: Honoraria; Amgen: Honoraria; Morphosys: Consultancy; Novartis: Consultancy; Astellas: Consultancy; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Zehir:Memorial Sloan Kettering Cancer Center: Current Employment; Illumina: Honoraria. Papaemmanuil:Celgene: Consultancy, Honoraria, Research Funding; Prime Oncology: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Kyowa Hakko Kirin: Consultancy, Honoraria; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; MSKCC: Patents & Royalties.

The acquisition of mutations in hematologic stem cells (clonal hematopoiesis) is common with normal aging and can be identified as an incidental finding through clinical genetic testing. Clonal hematopoiesis is associated with a heightened risk of developing hematologic neoplasms (especially myeloid) and accelerated atherosclerotic cardiovascular disease. This article discusses a multidisciplinary clinical approach to the management of patients with clonal hematopoiesis. Key areas of research needed to establish evidence-based clinical care guidelines and intervention strategies for individuals with clonal hematopoiesis are discussed.

SummaryMyeloid malignancies, including acute myeloid leukemia (AML), arise from the proliferation and expansion of hematopoietic stem and progenitor cells which acquire somatic mutations. Bulk molecular profiling studies on patient samples have suggested that somatic mutations are obtained in a step-wise fashion, where mutant genes with high variant allele frequencies (VAFs) are proposed to occur early in disease development and mutations with lower VAFs are thought to be acquired later in disease progression1–3. Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity and clone size, or offer definitive evidence of mutational order. To elucidate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is most commonly comprised of a small number of dominant clones, which in many cases harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We also used these data to map the clonal trajectory of each patient and found that specific mutation combinations (FLT3-ITD + NPM1c) synergize to promote clonal expansion and dominance. We combined cell surface protein expression with single cell mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of clonal architecture at a single cell level provide novel insights into the pathogenesis of myeloid transformation and how clonal complexity contributes to disease progression.