Presence Of Clonal Hematopoiesis Research Articles

Single-cell transcriptome analyses reveal distinct gene expression signatures of severe COVID-19 in the presence of clonal hematopoiesis

Clonal hematopoiesis of indeterminate potential (CHIP), a common aging-related process that predisposes individuals to various inflammatory responses, has been reported to be associated with COVID-19 severity. However, the immunological signature and the exact gene expression program by which the presence of CHIP exerts its clinical impact on COVID-19 remain to be elucidated. In this study, we generated a single-cell transcriptome landscape of severe COVID-19 according to the presence of CHIP using peripheral blood mononuclear cells. Patients with CHIP exhibited a potent IFN-γ response in exacerbating inflammation, particularly in classical monocytes, compared to patients without CHIP. To dissect the regulatory mechanism of CHIP (+)-specific IFN-γ response gene expression in severe COVID-19, we identified DNMT3A CHIP mutation-dependent differentially methylated regions (DMRs) and annotated their putative target genes based on long-range chromatin interactions. We revealed that CHIP mutant-driven hypo-DMRs at poised cis-regulatory elements appear to facilitate the CHIP (+)-specific IFN-γ-mediated inflammatory immune response. Our results highlight that the presence of CHIP may increase the susceptibility to hyperinflammation through the reorganization of chromatin architecture, establishing a novel subgroup of severe COVID-19 patients.

Clinical significance of clonal hematopoiesis in the setting of autologous stem cell transplantation for lymphoma.

Autologous stem cell transplantation (ASCT) remains a key therapeutic strategy for treating patients with relapsed or refractory non-Hodgkin and Hodgkin lymphoma. Clonal hematopoiesis (CH) has been proposed as a major contributor not only to the development of therapy-related myeloid neoplasms but also to inferior overall survival (OS) in patients who had undergone ASCT. Herein, we aimed to investigate the prognostic implications of CH after ASCT in a cohort of 420 lymphoma patients using ultra-deep, highly sensitive error-correction sequencing. CH was identified in the stem cell product samples of 181 patients (43.1%) and was most common in those with T-cell lymphoma (72.2%). The presence of CH was associated with a longer time to neutrophil and platelet recovery. Moreover, patients with evidence of CH had inferior 5-year OS from the time of first relapse (39.4% vs. 45.8%, p= .043) and from the time of ASCT (51.8% vs. 59.3%, p= .018). The adverse prognostic impact of CH was not due to therapy-related myeloid neoplasms, the incidence of which was low in our cohort (10-year cumulative incidence of 3.3% vs. 3.0% in those with and without CH, p= .445). In terms of specific-gene mutations, adverse OS was mostly associated with PPM1D mutations (hazard ratio (HR) 1.74, 95% confidence interval (CI) 1.13-2.67, p= .011). In summary, we found that CH is associated with an increased risk of non-lymphoma-related death after ASCT, which suggests that lymphoma survivors with CH may need intensified surveillance strategies to prevent and treat late complications.

Effect of Clonal Hematopoiesis on Cardiovascular Disease in People Living with HIV

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.

Clonal hematopoiesis in men living with HIV and association with subclinical atherosclerosis.

People with HIV (PWH) are at increased risk for premature cardiovascular disease (CVD). Clonal hematopoiesis is a common age-related condition that may be associated with increased CVD risk. The goal of this study was to determine the prevalence of clonal hematopoiesis and its association with chronic inflammation and CVD in PWH. Cross-sectional study utilizing archived specimens and data from 118 men (86 PWH and 32 HIV-uninfected) from the Baltimore-Washington DC center of the Multicenter AIDS Cohort Study (MACS) who had had coronary computed tomography angiography (CTA) and measurement of 34 serologic inflammatory biomarkers. Clonal hematopoiesis was assessed on peripheral blood mononuclear cells utilizing targeted error-corrected next generation sequencing (NGS) focused on 92 genes frequently mutated in hematologic malignancies. Clinical and laboratory data were obtained from the MACS database. Clonal hematopoiesis with a variant allele frequency (VAF) greater than 1% was significantly more common in PWH [20/86 (23.3%)] than in HIV-uninfected men [2/32 (6.3%)] ( P = 0.035). PWH with clonal hematopoiesis (VAF > 1%) were more likely to have coronary artery stenosis of at least 50% than those without clonal hematopoiesis [6/20 (30%) vs. 6/64 (9%); P = 0.021]. Presence of clonal hematopoiesis was not significantly associated with serological inflammatory markers, except for significantly lower serum leptin levels; this was not significant after adjustment for abdominal or thigh subcutaneous fat area. Clonal hematopoiesis was more common in PWH and among PWH was associated with the extent of coronary artery disease. Larger studies are needed to further examine the biological and clinical consequences of clonal hematopoiesis in PWH.

Abstract 5901: Germline tissue sequencing in patients with solid tumors uncovers clonal hematopoiesis variants

Abstract The peripheral blood (PB) is broadly used as a source of germline tissue for next generation sequencing in both clinical and research settings. Incidental or secondary findings are often uncovered at the time of germline sequencing. Current guidelines focus on secondary findings as they relate to inherited cancer predisposition. However, sequencing done on the PB has the potential of uncovering cancer-associated somatic variants. Somatic variants in genes associated with hematologic malignancies could indicate the presence of clonal hematopoiesis, clonal cytopenias or in rare instances an undiagnosed hematologic malignancy. In this abstract, we describe leukemia-associated somatic variants identified through whole exome sequencing (WES) in the PB of genomic research participants. A total of 24 patients with clonal hematopoiesis were identified from our institutional cohort of the ORIEN avatar study of 807 patients with solid tumors. Pathogenic and likely pathogenic variants were selected from protein coding sequences and splice sites of 64 genes involved in hematologic malignancies. Matched tumor and PB samples allowed for inclusion of variants found exclusively in the PB sample. Chart reviews were performed, and blood indices were collected for patients with clonal hematopoiesis variants. The median age of patients in this cohort was 73 years old (range 42-82) and 54% (n=13) were still living at the time of chart review. There were 27 variants distributed amongst 24 patients with 8% (n=2) of patients having 2 or more variants identified. The median reported variant allele frequencies (VAF) was 0.447. Most variants were missense (n=14) followed by stop gain (n=5), frameshift (n=3), splice site (n=3), stop lost (n=1), and inframe insertion (n=1). TP53 and DNMT3A (n=4) variants were identified most frequently followed by JAK2, KRAS, MYD88 (n=3), and PTEN (n=2). The rest of the genes accounted for one variant each, including TET2 and IDH2. Complete blood count (CBC) values were recorded for WBC, Hgb, MCV, RDW, platelets, and ANC, 79% (n=19) of patients had abnormal CBCs. No patients in this cohort were seen by a hematologist. Our results indicate that approximately 3% of patients identified in a research based solid tumor cohort had clonal hematopoiesis. Most of these patients had abnormalities in their hematologic parameters and would likely benefit from referral to hematology. Our data demonstrate the importance of considering somatic variation when the PB is used as germline tissue in research settings. Clonal hematopoiesis has significant clinical consequences ranging from risk of blood cancers to cardiovascular disease. We argue for the need to establish accepted frameworks for identification and evaluation of clonal hematopoiesis in participants of genomics research. Further analysis of associations between incidence of CHIP, cancer type, and prior treatment are underway Citation Format: Jennie Vagher, Aaron Atkinson, Mikaela Larson, David Nix, Andrew Post, Courtney Scaife, Jonathan Tward, Cindy Matsen, Jason P. Hunt, Marcus Monroe, Mia Hashibe, Deborah M. Stephens, Nicola J. Camp, Wallace L. Akerley, Trudy Oliver, Douglas Grossman, Sheri L. Holmen, Howard Colman, Randy Jensen, Kevin B. Jones, Afaf E. Osman. Germline tissue sequencing in patients with solid tumors uncovers clonal hematopoiesis variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5901.

Impact of clonal hematopoiesis on tumor control following radiation therapy.

3145 Background: Clonal hematopoiesis (CH) has well established associations with adverse clinical outcomes including all-cause mortality, cardiovascular disease, and progression to hematologic malignancy. The presence of CH has also been demonstrated to adversely impact survival from non-hematologic cancers, however whether CH may modulate response to radiation therapy (RT) in solid tumors is not known. Here we investigate the potential impact of CH mutations on radiation outcomes. Methods: We analyzed data from two previously well annotated cohorts of patients with tumors harboring somatic ATM mutations (n = 358) and FAT1 mutations (n = 365) who received RT and underwent prospective tumor and matched WBC sequencing utilizing the MSK-IMPACT assay. CH variants were detected in the blood samples utilizing a well-validated variant detection and filtration pipeline. Given that pathogenic mutations in ATM have been shown to be strongly associated with improved response to RT, these patients were excluded to avoid confounding. Additionally, patients with blood sampling for CH assessment that occurred more than 6 months after RT were excluded to address the possibility of therapy-related CH. We compared outcomes including irradiated tumor progression in patients with and without CH. Results: The final analysis consisted of 412 patients who underwent 811 total courses of radiation. A wide spectrum of solid tumor types were represented, most commonly non-small cell lung cancer (32.5%) and breast cancer (11.9%). A total of 161 patients (39.0%) had CH, with the most commonly mutated genes being DNMT3A (25.6%), PPM1D (6.2%), TET2 (5.8%), and TP53 (5.0%), consistent with prior studies of CH. Patients with CH were older at blood sample collection (67.6 vs 60.2 years, p < 0.001), reflecting an expected increase in CH burden with age. Fine Gray competing risks analysis, with death treated as a competing event and with clustering around patient identifier, showed no difference in irradiated tumor progression between patients with and without CH (HR 1.03, 95% CI 0.69 – 1.53, p = 0.896). Similarly, subanalyses by CH variant allele frequency and putative CH-driver mutations did not reveal an association between CH and response to RT. A hypothesis generating subgroup analysis by common cancer types, however, suggested that CH was associated with increased risk of progression post-radiation in prostate (HR 4.68, 1.14 – 19.1) and thyroid (HR 3.13, 1.55 – 6.34) cancer cohorts, warranting further investigation. Conclusions: We found no difference in irradiated tumor progression among patients who did and did not have CH. There may be an association between CH and poor radiation outcomes in certain cancer types, and further studies are needed to clarify the specific clinical and genomic factors that may influence radiation response.

The Presence of Clonal Hematopoiesis Is Negatively Associated with Diabetic Peripheral Neuropathy in Type 2 Diabetes.

Background Clonal hematopoiesis of indeterminate potential (CHIP) has been reported to be associated with increased cardiovascular disease, aging and insulin resistance. Despite the debate of causal contribution of CHIP on metabolic diseases, we want to explore whether CHIP is related to diabetic peripheral neuropathy (DPN).Methods This study analyzed the prevalence of CHIP in patients with type 2 diabetes classified according to DPN status. Logistic regression analysis was used to evaluate the association between CHIP and DPN.Results CHIP was more prevalent in subjects without DPN than those with DPN (19.9% vs. 8.8%, respectively; P=0.013). Individuals having any CHIP, or DNA methyltransferase 3A (DNMT3A) CHIP were less likely to have any abnormality shown in DPN test; the adjusted odds ratio were 0.85 (95% confidence interval [CI], 0.73 to 1.00) and 0.70 (95% CI, 0.56 to 0.89), respectively. Interestingly, DNMT3A CHIP showed the negative association, but Tet methylcytosine dioxygenase 2 (TET2) CHIP showed the positive association with abnormal feet electrochemical skin conductance level.Conclusion On the contrary to expectations, CHIP was negatively associated with DPN. Functional linking between the mutation in hematopoietic cells and DPN, and the opposite role of DNMT3A and TET2 should be investigated.

Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia.

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. 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. 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. 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.

Clonal Hematopoiesis Is Frequent and Associated with Inferior Survival Irrespective of Transplantation Strategy in Patients with Newly Diagnosed Multiple Myeloma

Background: The acquisition of somatic clonal mutations in the blood of healthy individuals is referred to as clonal hematopoiesis (CH) and is associated with increased age, risk of hematologic malignancy, and non-malignant inflammatory outcomes. The presence of CH in patients with non-Hodgkin lymphoma (NHL) has been associated with adverse outcomes and decreased overall survival (OS) especially in the setting of autologous transplantation. In multiple myeloma (MM), the presence of CH at the time of stem cell collection was associated with decreased OS only in those patients who did not receive thalidomide analog maintenance. However, the clinical significance of CH in patients with newly diagnosed MM undergoing autologous transplant has not been assessed. We sought to determine the frequency and clinical outcome of CH in patients with newly diagnosed MM.The IFM 2009 study randomized 700 newly diagnosed patients with MM to lenalidomide, bortezomib and dexamethasone (RVd) versus RVd plus high-dose therapy (HDT). This patient cohort provides a unique opportunity to examine the prevalence of CH in newly diagnosed MM patients and assess its influence on clinical outcomes, including early transplantation, in a homogeneously treated population of MM patients.Methods: We performed targeted error-corrected next generation sequencing of 100 genes recurrently mutated in hematologic malignancies in 377 patients with newly diagnosed MM who were part of the IFM 2009 study (additional patient samples are pending analysis). CH was defined as the presence of any pathogenic mutation with a variant allele fraction (VAF) ≥ 0.005. Sample DNA was collected from peripheral blood of subjects at the time of screening/diagnosis. The median age at sample collection was 59 years. Median follow-up was 7.8 years.Results: We identified CH in 188 (48%) patients with VAF ≥ 0.005, in 119 (32%) patients with VAF ≥ 0.01 and in 59 (16%) patients with VAF ≥ 0.02. Because prior studies have primarily assessed clinical significance of VAF ≥ 0.02, further analysis was restricted to this threshold. Consistent with prior reports DNMT3A (30 patients), TET2 (11 patients) and ASXL1 (3 patients) were the most commonly mutated genes. The presence of CH was associated with increased median age (61 vs 59 years, p = 0.003), ISS stage III (p = 0.007) and increased LDH (p = 0.006), but not with sex, monoclonal protein isotype, b2-microglobulin level, or assignment to RVd or HDT arms.As previously reported, randomization to the early transplant arm was associated with increased relapse free survival but not with improved OS. Across the entire cohort, the presence of CH was associated with inferior OS (p = 0.003), but had no association with relapse-free survival or time to relapse. The decrement in OS was seen equally in both the RVd and HDT arms of the study. The presence of mutant clones smaller than a VAF < 0.02 was not associated with adverse clinical outcomes.Fourteen patients developed a second primary malignancy (SPM), only two of which were hematologic neoplasms. There was no difference in the frequency of SPM development in those patients with or without CH.Conclusions: In patients with newly diagnosed multiple myeloma, the presence of CH at a VAF ≥ 0.02 is frequently observed and is associated with decreased overall survival regardless of transplantation strategy. The presence of CH did not affect the risk of developing an SPM. Taken together, these data suggest that while CH is associated with decreased survival the presence of CH should not alter eligibility of patients for autologous transplantation. [Display omitted] DisclosuresSperling: Adaptive: Consultancy. Miller: Foundation Medicine: Consultancy. Moreau: Amgen: Honoraria; Celgene BMS: Honoraria; Abbvie: Honoraria; Janssen: Honoraria; Sanofi: Honoraria; Oncopeptides: Honoraria. Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Other: Stock ownership. Ebert: Celgene: Research Funding; Skyhawk Therapeutics: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy; Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Deerfield: Research Funding. Munshi: Amgen: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Legend: Consultancy; Bristol-Myers Squibb: Consultancy.

Clonal Hematopoiesis Is More Common in People Living with HIV and May be Associated with Increased Prevalence of Cardiovascular Disease

Background: Cardiovascular disease (CVD) is more common in people living with HIV (PLWH) and may be related to abnormal immune activation. With aging, the expansion of mutated hematopoietic clones or clonal hematopoiesis (CH) has been associated with CVD events in the general population and is hypothesized to be driven by systemic inflammation. We investigated whether CH was more common in PLWH compared to those without HIV, whether the distribution of CH mutations differed by HIV status, and whether CH was associated with subclinical coronary atherosclerosis in PLWH.Methods: Study participants were selected from men in the Baltimore -Washington DC center of the Multicenter AIDS Cohort Study (MACS) who had had coronary CT angiography (CTA) as part of the MACS Cardiovascular Study 2. The MACS is a prospective study of the natural history of HIV infection in men who have sex with men, and includes both PLWH and HIV-uninfected men. Since the prevalence of CH increases with age, the oldest participants were preferentially included in this study. To detect CH, DNA extracted from viably frozen peripheral blood mononuclear cells or cell pellets was subjected to targeted, error-corrected, ultra-deep next generation sequencing (NGS) which included 70 genes frequently mutated in hematologic malignancies. CTA results and inflammatory biomarker levels were already available from MACS data.Results: The current analysis was a cross-sectional study involving 118 participants: 86 (72.9%) PLWH and 32 (27.1%) HIV-uninfected men (HIV-). The groups were well-balanced in terms of known major risk factors for CH such as age and smoking. The median age was 53 and 54 years for PLWH and HIV- men, respectively (p=0.147). Caucasians represented 37 (43.0%) of PLWH and 21 (65.6%) of HIV- men. Out of 86 PLWH, 72 (83.7%) were on antiretroviral therapy (ART) at the time of the assessment; 41 (47.7%) on a PI-containing regimen and 31 (36.0%) on an NNRTI-containing regimen. The HIV viral load was under 400 copies/mL in 77 (89.5%) of PLWH, and the median (IQR) CD4+ count was 585 (397, 745)/mL. The prevalence of coronary artery stenosis of 50% or more was 12 (14.3%) in PLWH and 5 (16.1%) in HIV- men (p = 0.774).Since the minimum size of the biologically relevant CH is unknown, we applied variant allele frequency (VAF) cut-offs of > 0.5% and > 1%. For both cutoffs, CH was significantly more frequent in PLWH than in HIV- men (p=0.012 and p=0.036, respectively) (Figure 1A). Mutations in epigenetic modifiers (DNMT3A, TET2) were the most common mutations among both PLWH and HIV- men. Interestingly, DNMT3A mutations were more frequent in PLWH (Figure 1B). For both VAF cut-offs, PLWH had significantly more somatic mutations than HIV-uninfected men (p = 0.043 and 0.033, respectively) (Figure 1C).Since inflammation-mediated complications of CH become more apparent in people with larger clones, we focused on CH with VAF > 1% in order to determine the clinical consequences of CH in PLWH. As CH is known to be a risk factor for accelerated CVD in the general population, we asked whether CH was also associated with subclinical atherosclerosis in PLWH. Coronary artery stenosis > 50% (moderate/severe) was significantly more frequent in PLWH with CH compared to PLWH without CH (p = 0.032) (Figure 1 D). This difference remained significant in multivariable logistic regression models that adjusted for the Framingham coronary heart disease 10-year risk (p=0.017) and for the Framingham hard coronary heart disease 10-year risk (p<0.01).Additionally, we examined the association of serum concentrations of several inflammatory markers such as CRP, IL-1b, IL-6, as well as white blood cell count, with the presence of CH in PLWH, but we found no significant associations.Conclusions: CH was more common in PLWH compared to HIV-uninfected men, and PLWH had more somatic mutations. Moreover, the presence of CH was significantly associated with the presence of coronary artery stenosis > 50% (moderate/severe) in PLWH, even after adjusting for known CVD risk factors. The results from our exploratory analysis may provide a potential explanation for increased CVD in PLWH. Larger studies are warranted to further delineate the etiology of increased CH in PLWH and its impact on accelerated CVD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dnmt3a Mutations in the Hematopoietic System Promote Colitis-Associated Colon Cancer: A Model of Clonal Hematopoiesis in Solid Tumors

Clonal hematopoiesis (CH) is a condition wherein hematopoietic stem cells (HSCs) acquire mutations, often in presumed leukemia driver genes such as in DNMT3A, leading to clonal dominance and disproportionate contribution to peripheral blood without overt hematologic abnormalities (Jaiswal et al. 2014). In large cohorts of patients with solid tumors including colorectal cancer, presence of CH is associated with inferior overall survival due to progression of primary malignancy (Coombs et al. 2017). While potential involvement of CH in the pathogenesis of solid tumors driven by unrelated genetic alterations has far-reaching translational implications, the mechanistic understanding of this relationship is lacking.Colorectal cancer is a leading cause of cancer deaths worldwide and can arise from preceding inflammatory bowel disease. More than 20% of patients with colon cancer have detectable CH, which is notably more prevalent than in age-matched general population. We hypothesized that bone marrow-derived cells harboring alterations in DNMT3A, the most commonly disrupted gene in CH (up to 40% of cases), contribute to the pathogenesis of colorectal cancer. To address this, we combined a bone marrow transplantation-based model of CH driven by heterozygous loss of Dnmt3a with a well-established induction of colitis-associated colon cancer (CAC) by azoxymethane (AOM, a carcinogen) and dextran sodium sulfate (DSS, a colitogen). High resolution colonoscopy demonstrated increased colon wall opacity, visible bleeding, numerous fibrin patches and earlier onset of tumor formation with higher penetrance in animals with partial ablation of Dnmt3a in the hematopoietic compartment. Heightened colon pathology was reflected by modified murine endoscopic index of CAC severity (MEICS) (Fung and Putoczki 2018) (MEICS scores 7.5±2.0 in WT vs 10.0±3.2 in Dnmt3a +/-, p=0.05, n=14,13). We observed elevated incidence of tumors >2mm in diameter (40.2±22.2% in WT vs 74.2±15.7% in Dnmt3a +/-, p=0.03, in 5 independent trials) and a higher tumor burden in Dnmt3a +/- chimerae (7.4±7.8 mm 3 in WT vs 14.5±13.9 mm 3 in Dnmt3a +/-, p=0.0017, n=53,55) at end point. Histopathological analysis revealed marked immune infiltration, extensive ulceration and dysplasia of colonic epithelium, and more frequent adenocarcinoma formation with occasional submucosal invasion (histology score 20.2±4.4 in WT vs 24.6±6.9 in Dnmt3a +/-, p=0.03, n=10,9). To uncover signaling pathways deregulated by experimental Dnmt3a-driven CH, we surveyed the auto- and paracrine stimulatory loops by multiplex cytokine/chemokine assays and profiled transcriptomes of colon tumors by bulk RNA-seq. These studies identified gene signatures commonly associated with colon carcinogenesis including accentuated epithelial-to-mesenchymal transition (gene set enrichment analysis (GSEA) NES=2.42, padj=0.0096), Wnt/b-catenin (NES=2.07, padj=0.0096), and Vegf/angiogenesis (NES=1.83, padj=0.0096). Elevated proliferation (Ki67) and increased vascularization (CD31) were validated by immunohistochemistry. These aspects of tumor pathophysiology along with the composition and functional characteristics of the tumor-infiltrating leukocytes derived from animals with and without hematopoietic-specific Dnmt3a partial loss are being further investigated. Preliminary data showed bone marrow-derived macrophages (BMDMs) from Dnmt3a +/- chimera exhibit impaired ability to phagocytose syngeneic MC38 colon tumor cells, compared to WT.Our study provides new insights into the non-tumor-cell-autonomous mechanisms of carcinogenesis effected by alterations in the hematopoietic system and into the interplay between the immune system and the aggressive phenotype of colorectal cancer. Our findings may aid the development of novel therapeutic options to improve outcomes in solid tumor patients with CH. [Display omitted] DisclosuresBowman: Mission Bio: Honoraria, Speakers Bureau. Levine: Ajax: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Zentalis: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Lilly: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Astellas: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Roche: Honoraria, Research Funding.

Clonal Hematopoiesis Prevalence Increases throughout Treatment of Newly Diagnosed Multiple Myeloma Patients

Clonal Hematopoiesis Prevalence Increases throughout Treatment of Newly Diagnosed Multiple Myeloma Patients

Evaluation of the Relationship between Clonal Hematopoiesis and Severe COVID-19 Disease in Rhesus Macaques

Clinical manifestations of infection with the novel SARS-CoV-2 in humans are widely varied, ranging from asymptomatic to COVID-19 respiratory failure and multiorgan damage. Profound inflammation is the hallmark of severe COVID-19 disease, and commonly does not occur until the second week of infection. Although risk factors for this late hyperinflammatory disease have been identified, most notably age and pre-existing co-morbidities, even within high-risk groups the specific factors leading to severe COVID-19 illness remain elusive. Acquired somatic mutations in hematopoietic stem and progenitor cells (HSPCs), termed clonal hematopoiesis (CH), are associated with advanced age, and loss of function (LOF) mutations in certain genes, most commonly DNMT3A and TET2, have been linked to a marked hyperinflammatory phenotype as well as clonal expansion of mutant HSPCs. Given the similar age range of frequent CH and severe COVID-19 disease, the presence of CH could impact the risk of severe COVID-19. Several human cohort studies have suggested this relationship may exist, but results to date are conflicting. Rhesus macaques (RM) have been established as a model for SARS-CoV infection and are being utilized to test therapies and vaccine development, but up to now, macaques have not been reported to develop late hyperinflammatory COVID-19 disease. We have created a robust RM model of CH by introducing LOF TET2 mutations into young adult HSPC via CRISPR/Cas9 followed by autologous transplantation, recapitulating the clonal expansion and hyperinflammatory phenotype. Thus, we hypothesized that macaques with CH could develop severe late COVID-19 disease and be utilized as a model to study disease pathophysiology or test therapeutic approaches. Macaques with either engineered (n=2) or natural CH (n=1) along with age-matched transplanted controls (n=3) were inoculated with SARS-CoV-2 and monitored clinically and via laboratory studies until 12 days post-inoculation (dpi). Macaques normally clear infection and symptoms within 3-5 days of infection. No significant differences in clinical symptoms and blood counts were noted, however, an aged animal with natural DNMT3A CH died on 10 dpi. IL-6 levels were somewhat higher in sera of the CH animals until 12 dpi, and in BAL, mean concentrations of MCP-1, IL-6, IL-8 and MIP-1b were consistently higher in CH macaques compared to controls. Interestingly, we found the median copy number of subgenomic SARS-CoV-2 RNA was higher at every timepoint in the CH group as compared with the control group, in both upper and lower respiratory samples. Lung sections from euthanasia at 10 or 12 dpi showed evidence of mild inflammation in all animals. However, in the immunohistochemical analysis, the viral antigen was detected in the lung tissues of all three animals in the CH group even at the time of autopsy, whereas only one animal of three controls had detectable viral antigen. Although the striking inflammation and serious disease have not been observed, data so far provide evidence of potential pathophysiological differences with or without CH upon SARS-CoV-2 infection. We continue to expand sample size and conduct further analyses to draw a solid conclusion, but we believe this model may be of benefit to understand the relationship between COVID-19 disease and CH. DisclosuresNo relevant conflicts of interest to declare.

Clinical Significance of Clonal Hematopoiesis in the Setting of Autologous Stem Cell Transplantation for Lymphoma

Clinical Significance of Clonal Hematopoiesis in the Setting of Autologous Stem Cell Transplantation for Lymphoma

Level of Unique T-Cell Clonotypes Are Associated with Clonal Hematopoiesis and Survival in Patients with Lymphoma Intended for Autologous Stem Cell Transplant

The advent of novel immunotherapy (CAR-T cell therapy, bispecific CD20×CD3 antibodies) have highlighted the importance of T-cells in the treatment of lymphoma. However, overall T-cell characteristics have not been properly examined in patients receiving conventional chemotherapy. Next-generation sequencing (NGS) of the T-cell receptor (TCR) has enabled the possibility of identifying hundred thousands of unique T-cell clones in a single patient sample. Here we analyzed the impact of systemic TCR diversity and T-cell clonotypes in patients with Non-Hodgkin lymphoma (NHL) and Hodgkin-lymphoma (HL) receiving high-dose chemotherapy with stem cell support (HDT/ASCT).Autologous peripheral blood stem cell harvest samples from patients with lymphoma (predominantly B-cell NHL) were collected as part of a national population-based study (Husby et al. - Leukemia 2020). We performed high-throughput RNA-based sequencing of the V, D and J segment of the TCR β-chain to identify unique clonal rearrangements.To ensure supreme quality for TCR repertoire calculations, samples with less than 100.000 aligned reads to the TCR β chain were omitted from further analysis. By using the MiXCR bioinformatic pipeline we analyzed the number of unique clonotypes and TCR repertoire diversity, as calculated by the Simpson index. T-cell clonotype and diversity were for categorical analyses split in two groups by the median, respectively.A total of 96 patients with lymphoma who were intended for HDT/ASCT were included and analyzed for TCR characteristics. In brief, median age was 56 years, 64% were male and major subtypes were diffuse large B-cell lymphoma (37%), follicular lymphoma (24%), Hodgkin lymphoma (16%), and mantle cell lymphoma (14%). Median follow-up time was 6.7 years. Number of unique T-cell clonotypes was not associated with age (Fig. 1A), but low levels were highly associated with inferior survival (Fig. 1B, p=0.008), especially in the first year of follow-up. In contrast, elderly patients had a trend toward lower TCR diversity (Fig. 1C, p=0.08), but this did not impact overall survival (Fig. 1D).Low T-cell clonotype levels was also significantly associated with presence of clonal hematopoiesis (Fig. 1E, p=0.033). No association with clonal hematopoiesis was found with regard to TCR diversity (Fig. 1F). Furthermore, we investigated TCR repertoire in relation to subsequent severe infections (defined as sepsis, pneumonia, or invasive fungal infection). Number of unique T-cell clonotypes did not have an impact (Fig. 1F), but remarkably patients with a high T-cell diversity had significant increased incidence of severe infections in the first 500 days after sampling (Fig. 1G, p=0.029). This implies that patients who have a high T-cell diversity before high-dose chemotherapy, are more capable of mounting an immune response against infectious pathogens.These findings should be validated in larger homogenous cohorts. However, they imply the importance of inherent immune characteristics in patients with lymphoma. Although the immune response is exceedingly complex, we have identified systemic T-cell characteristics that associate with several important clinical variables. Assessment of systemic immunological parameters in patients with aggressive lymphoma may in the future inform on choice of optimal personalized therapy. [Display omitted] DisclosuresEl-Galaly: ROCHE Ltd: Ended employment in the past 24 months; Abbvie: Other: Speakers fee. Larsen: Odense University Hospital, Denmark: Current Employment; Celgene: Consultancy; BMS: Consultancy; Novartis: Consultancy; Gilead: Consultancy.

OAB-040: Clonal hematopoiesis is associated with increased risk of progression of asymptomatic Waldenström Macroglobulinemia

<h3>Background</h3> Clonal hematopoiesis (CH) is associated with adverse outcomes in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma undergoing autologous stem cell transplantation. Still, its implications for patients with indolent NHL have not been well studied. Here, we report the prevalence of CH in patients with Waldenström Macroglobulinemia (WM) and its association with clinical outcomes. <h3>Methods</h3> We retrospectively reviewed clinical data of 602 patients with IgM MGUS, smoldering WM and WM who had clinical next-generation sequencing (NGS) performed on bone marrow aspirates or peripheral blood obtained between October 2014 and February 2020 at the Dana-Farber Cancer Institute (DFCI). An Illumina Truseq amplicon-based NGS assay of 95 genes recurrently mutated in myeloid and lymphoid neoplasms was utilized. Each specimen yielded ~2 million reads and ~1500X average coverage with 90% of amplicons having >200X coverage. Pathogenic driver variants were identified based on mutation type, position, and frequency in published reports and public databases. In order to unambiguously differentiate CH mutations from those in the WM clone, CH was defined by the presence of somatic mutations in DNMT3A, TET2 or ASXL1 (CH-DTA). <h3>Results</h3> The cohort included 147 patients with MGUS or asymptomatic WM and 453 patients with symptomatic WM with a median age of 66 years (range: 40-89) and 68 years (range: 33-93), respectively, at time of first NGS assay. The prevalence of CH-DTA was 14% in symptomatic WM patients and did not differ significantly in MGUS and SWM (13% and 14%, respectively). Among precursor patients, there was an increased risk of progression to symptomatic WM in those with CH-DTA (7/20 patients with vs. 11/116 without CH-DTA progressed over a median of 54 months [p= 0.002]). In symptomatic WM patients CH-DTA was positively associated with older age (p<0.001) at time of NGS, with a median age of 72 vs. 67 years for patients with versus those without CH-DTA. CH-DTA was not associated with inferior OS with a relatively short median follow-up from diagnosis and NGS assay of 6.7 (95% CI: 6.1-7.6) and 2.5 (95% CI: 2.2-2.8) years, respectively. The most common cause of death was disease progression with no significant difference between those with or without CH-DTA. Patients with CH-DTA had an increased risk of cardiovascular disease (30% vs. 18%, p=0.036). <h3>Conclusion</h3> We demonstrate that CH is common in WM patients and is associated with increased risk of progression from precursor states but not with inferior survival. Further work is needed to determine how the presence of CH might promote progression to WM and whether it can be incorporated into future risk stratification models. Importantly, our data do not support changes in clinical management or alterations in therapy for patients with WM and coexistent CH and reinforce the need to interpret NGS results within their specific clinical context.

Prognostic Impact of Clonal Hematopoiesis in Acute Myeloid Leukemia Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation in Complete Remission

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is defined as presence of hematologic malignancy-associated mutations, e.g. in the genes DNMT3A, TET2, ASXL1, but absence of hematologic neoplasms & is a frequent phenomenon in healthy individuals with increasing age. Some individuals with CHIP eventually develop acute myeloid leukemia (AML) & persistent clonal hematopoiesis-associated mutations (CH-mutations) in complete remission (CR) may give rise to relapse. To date in AML little is known about the biological & clinical implications of the presence of CH-mutations in CR, especially in the context of allogeneic hematopoietic stem cell transplantation (HSCT).Patients & Methods: We analyzed peripheral blood samples of 113 AML patients (median age 63.6 years, range 31.9-75.8 years) in CR (CR1 61.9%, CR2 14.2%) or CR with incomplete peripheral recovery (CRi; 23.9%) for the presence of CH-mutations by targeted amplicon sequencing (mean amplicon coverage per sample 7205x) prior to HSCT. Genes evaluated for somatic CH-mutations based on database entries (dbSNP & COSMIC) & with a variant allele frequency (VAF) of ≥3% were DNMT3A, TET2, ASXL1, SRSF2, SF3B1, IDH1, IDH2, JAK2, PPM1D, &IKZF1 . Codon 646 ASXL1 mutations were validated using proof-reading polymerase Sanger sequencing. For 76 patients (67.3%) diagnostic bone marrow samples were available for applying a targeted amplicon panel comprising 54 recurrently mutated genes in myeloid malignancies (mean amplicon coverage per sample 8624x). At diagnosis, mutations in the genes NPM1, CEBPA, presence of FLT3 -ITD, & cytogenetics were determined using standard techniques. All patients received HSCT after non-myeloablative conditioning at our institution between 2001 & 2015. Samples were collected within 30 days before HSCT. Median follow-up for patients alive was 4.4 years after HSCT.Results: We identified 70 CH-mutations present in CR/CRi in 48 AML patients (42.5%) with a mean VAF of 19.1% (58.6% of mutations with VAF>10%). The genes DNMT3A (31.4%), TET2 (28.6%) &ASXL1 (14.3%) were found mutated most frequently. We observed no associations between present CH-mutations in CR/CRi & clinical characteristics, except that patients with ≥1 CH-mutation were more often transplanted in CR2 compared to patients without CH-mutation by trend (20.8% vs 9.2%, P=0.10). The presence of ≥1 CH-mutation in CR/CRi did not impact leukemia-free survival (LFS; P=0.95, Figure 1A) or OS (P=0.37, Figure 1B), whereas patients with ≥2 CH-mutations had longer LFS (P=0.02, Figure 1C) & OS (P=0.007, Figure 1D) compared to patients with no or 1 CH-mutation. In CR/CRi DNMT3A mutations did not impact LFS (P=0.73) or OS (P=0.71), the presence of TET2 mutations did not influence LFS (P=0.25) but associated with longer OS (P=0.06) &ASXL1 mutations associated with longer LFS (P=0.11) & OS (P=0.13) by trend. At diagnosis, we identified 83 CH-mutations in 56/76 AML patients (73.7%) of which 29 (34.9%) persisted in 19/76 patients (25.0%) in CR/CRi. Mutations in the genes JAK2 (80.0%), SRSF2 (57.1%) &DNMT3A (50.0%) were most likely to persist. We identified 9 CH-mutations (3 DNMT3A, 3 TET2, 2 ASXL1, 1 SF3B1 mutation) in CR/CRi in 8 patients (10.5%), which were not detectable at diagnosis, possibly following the expansion of subclones under chemotherapy selection pressure. Whether patients had persistent, newly detected or lost CH-mutation in CR/CRi did not impact LFS (P=0.22) or OS (P=0.83).Conclusion: In AML patients in CR/CRi CH-mutations are frequently present (42.5%) & show high VAFs (mean 19.1%) indicating presence of clonal hematopoiesis. Compared to diagnosis 34.9% of CH-mutations persisted in CR/CRi. 10.5% of patients had CH-mutations only detectable in CR/CRi prior to HSCT. While we did not observe a negative prognostic impact of presence of ≥1 CH-mutation in CR/CRi, the presence of distinct mutations (TET2, ASXL1) may positively influence outcome. Patients with ≥2 CH-mutations had longer LFS & OS. This finding may be explained by an increased immunogenic potential with higher number of CH-mutations leading to potent graft vs leukemia effects in the HSCT context. [Display omitted] DisclosuresLange:Novartis: Honoraria, Research Funding. Franke:Takeda: Consultancy; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Schwind:Novartis: Consultancy.

Clinical relevance of clonal hematopoiesis in metastatic gastrointestinal malignancies.

e16082 Background: Clonal hematopoiesis (CH) represents non-random clonal selection of bone marrow-derived cells marked by somatic mutations in certain genes. The presence of CH is associated with development of atherosclerosis and leukemia, and accelerated by toxic exposures (chemotherapy, radiation, smoking) and aging (Jaiswal et al. NEJM 2017; Abelson et al. Nature 2018). The impact of these genetic alterations on cellular function is unknown, especially in the broader context of immunity and in response to cancer therapy. To determine the contribution of CH to therapeutic response and hematologic toxicity in cancer patients, we examined the outcomes of patients treated with cytotoxic and immunotherapy in relationship to CH status. Methods: We evaluated patients with metastatic colorectal cancer (CRC) or esophagogastric cancer (EGC). DNA extracted from whole blood and tumor tissue were sequenced in tandem as part of the MSK-IMPACT hybridization capture-based sequencing assay. CH was defined as any mutation with a VAF of at least 2%, present in at least 10 reads, with at least 2:1 blood:tumor reads, or 1.5:1 blood:lymph node that was not found in gnomAD with a frequency &gt; 0.005. Additional filtering and putative driver definitions (CH-PD) were described by Bolton et al. Nature Genetics 2020. Multivariate survival analyses were performed using a Cox Proportional Hazard model correcting for CH, CH-PD, prior smoking, prior chemotherapy, prior radiation, MSI status, and age at cancer diagnosis. Results: 654 patients with EGC (n = 348) and CRC (n = 306) who began treatment between 2006 and 2020 were included in the analysis. CH was present in 34.5% and 24.4% of each group, and 17.2% and 12.9% harbored CH-PD, respectively. CH and CH-PD were both associated with older age and smoking history, and CH was also associated with prior radiation and MSI-high status (p &lt; 0.05). Patients with CH or CH-PD receiving first-line (1L) therapy for CRC or EGC demonstrated no difference in mPFS after multivariate analysis, though 1L EGC patients with CH-PD had inferior mOS (p = 9e-5). There was no difference in pre-1L WBC, ANC, or ALC, nor in G-CSF or PEG-G-CSF doses administered during 1L therapy between patients with CH or CH-PD versus those without. Similarly, presence of CH or CH-PD had no impact on mPFS or mOS in patients receiving immune checkpoint blockade (ICB) without concurrent chemotherapy after multivariate survival analysis. Conclusions: We confirmed that the mere presence of CH is not prognostic for overall survival, but that EGC patients with CH-PD mutations have inferior overall survival, which is consistent with previous findings. Presence of CH or CH-PD was not associated with differences in baseline leukocyte counts nor need for G-CSF support, nor did it impact PFS in either tumor type, suggesting limited utility of CH in solid tumor clinical decision-making.

Genomic profiling identifies somatic mutations predicting thromboembolic risk in patients with solid tumors

Venous thromboembolism (VTE) associated with cancer (CAT) is a well-described complication of cancer and a leading cause of death in patients with cancer. 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 Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets platform to identify somatic alterations associated with VTE. End point 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 analysis. Tumor-specific mutations in KRAS (hazard ratio [HR], 1.34; 95% confidence interval (CI), 1.09-1.64; adjusted P = .08), STK11 (HR, 2.12; 95% CI, 1.55-2.89; adjusted P < .001), KEAP1 (HR, 1.84; 95% CI, 1.21-2.79; adjusted P = .07), CTNNB1 (HR, 1.73; 95% CI, 1.15-2.60; adjusted P = .09), CDKN2B (HR, 1.45; 95% CI, 1.13-1.85; adjusted P = .07), and MET (HR, 1.83; 95% CI, 1.15-2.92; adjusted P = .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; 95% CI, 0.16-0.79; adjusted P = .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 patients with solid tumors. Further analysis is needed to validate these findings and identify additional molecular signatures unique to individual tumor types.

Erythrocytosis in the general population: clinical characteristics and association with clonal hematopoiesis.

Erythrocytosis is a common reason for referral to hematology services and is usually secondary in origin. The aim of this study was to assess clinical characteristics and clonal hematopoiesis (CH) in individuals with erythrocytosis in the population-based Lifelines cohort (n = 147 167). Erythrocytosis was defined using strict (World Health Organization [WHO] 2008/British Committee for Standards in Hematology) and wide (WHO 2016) criteria. Individuals with erythrocytosis (strict criteria) and concurrent leukocytosis and/or thrombocytosis were 1:2 matched with individuals with isolated erythrocytosis and analyzed for somatic mutations indicative of CH (≥5% variant allele frequency). One hundred eighty five males (0.3%) and 223 females (0.3%) met the strict criteria, whereas 4868 males (7.6%) and 309 females (0.4%) met the wide criteria. Erythrocytosis, only when defined using strict criteria, was associated with cardiovascular morbidity (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.2-2.6), cardiovascular mortality (hazard ratio [HR], 2.2; 95% CI, 1.0-4.6), and all-cause mortality (HR, 1.7; 95% CI, 1.2-2.6), independent of conventional risk factors. Mutations were detected in 51 of 133 (38%) evaluable individuals, with comparable frequencies between individuals with and without concurrent cytosis. The JAK2 V617F mutation was observed in 7 of 133 (5.3%) individuals, all having concurrent cytosis. The prevalence of mutations in BCOR/BCORL1 (16%) was high, suggesting aberrant epigenetic regulation. Erythrocytosis with CH was associated with cardiovascular morbidity (OR, 9.1; 95% CI, 1.2-68.4) in a multivariable model. Our data indicate that only when defined using strict criteria erythrocytosis is associated with cardiovascular morbidity (especially in the presence of CH), cardiovascular mortality, and all-cause mortality.