Age-related Clonal Hematopoiesis Research Articles

Abstract LB227: Accelerated intra-clonal transcriptomic diversification of hematopoietic stem cells during aging fuels leukemia evolution

Abstract Cancers are diseases of aging. Many cellular processes characteristic of cancer, e.g., epigenetic dysregulation, are similarly observed during aging. Acute myeloid leukemia (AML) is emblematic of this interplay. As a blood cancer stemming from hematopoietic stem and progenitors (HSPC), its median age of diagnosis is 65. AML is linked to age-related clonal hematopoiesis (CH), a condition resulting from the expansion of HSPC, marked by mutations in leukemia-associated genes. We aim to clarify the intrinsic role of aging in HSPC as it causes CH and leukemic transformation. Hypothesis: aging amplifies the heritable phenotypic variations in hematopoietic stem cells (HSC), enhancing epigenetic heterogeneity. This acts as a substrate for natural selection, driving leukemic transformation. Results: We found a significant increase in epigenetic and transcriptomic heterogeneity in long-term HSC (LT-HSC) from old mice compared to their younger counterparts via single-cell RNA-seq (scRNA-seq)s. This process occurs in an aged bone marrow with higher inflammation and altered HSPC support. Of relevance, we previously show that the rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 loss and Flt3 ITD precedes leukemic transformation. We further employed CellTag Indexing and scRNA-seq to track the fate of specific HSC subclones. We tracked HSPC cell culture (in vitro) after two-weeks and two-months following transplantation into recipient mice of matching ages (in vivo). Cells of the same clone manifest higher transcriptomic similarities than those from different clones, validating CellTag in mapping clonal evolution. We computed the progeny output activity (Ai) by the ratio of clone i abundance in the committed progenitors to its frequency in the HSC compartment. We found that young mice exhibit significantly higher variability and a higher mean of progeny output activity Ai, indicating the efficiency in self-renewal and cell differentiation. While both young and old mice have low-output (higher stemness) clones, only young mice had significant differential expression of key genes in low-output clones compared to high-output clones, e.g., Dpp4, Eps8, and Col5a1. Depite lower progeny output Ai variation, LT-HSCs from older mice exhibited greater clonal transcriptome diversification both in vitro and in vivo , evidenced by intra-clonal transcriptomic correlation. This pattern also existed in ST-HSC and MPP from older mice. Genes with higher expression variability in older mice highlighted pathways pivotal for HSPC fitness, e.g., ribosome synthesis (RS), inflammation response mechanisms (JAK-STAT), hematopoietic cell lineage, AML and cancer pathways (GSEA). The RS pathway were remarkably enriched in the megakaryocytic-erythroid progenitors, directly before differentiation where substantial protein translation is required. Conclusion: our results suggest that the synergy of aging and somatic mutations in epigenetic regulators may enhance cell-to-cell variations, offering additional heritable phenotypic variations. These, in turn, may facilitate clonal hematopoiesis and leukemic transformation. Future work will delineate specific epigenetic and transcriptomic configurations for mutant HSPC clonal expansion. This study provides a cohesive framework, linking the principles governing aging to cancer. Citation Format: Lamis Naddaf, Marco De Dominici, Xiaowen Chen, Travis Roeder, Hideyuki Oguro, Shilpita Karmakar, Eric Pietras, James DeGregori, Sheng Li. Accelerated intra-clonal transcriptomic diversification of hematopoietic stem cells during aging fuels leukemia evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB227.

Abstract 4398: TET2 accelerates clonal hematopoiesis in an age-dependent manner

Abstract Aging is a process of systemic deterioration and the most significant risk factor for cancers. Clonal hematopoiesis (CH) commonly occurs with aging and links to higher mortality, leukemia risk, and cardiovascular diseases. Age-related CH involves the abnormal clonal expansion of hematopoietic stem cells (HSCs) bearing somatic mutations in genes frequently mutated in leukemia, including genes encoding epigenetic regulators, such as the DNA demethylase TET2. While such mutations are known to alter the HSC epigenome, the mechanisms through which these mutations drive HSC self-renewal, myeloid transformation, and inflammatory response remain elusive. In this study, we hypothesize that aging and CH mutations cooperatively reshape the HSC transcriptomic landscape and enhancing HSC competitive advantage that facilitates clonal expansion. Recently, TET2-mutant CH in humans has been strongly associated with epigenetic age acceleration. Using single-cell multi-omic analyses and flow-cytometric phenotyping, we demonstrated, for the first time, that HSC aging processes at transcriptomic, epigenomic, and cellular levels are accelerated by Tet2 deficiency in mice, in an age-dependent manner. Tet2-deficient HSCs increase the HSC aging scores at an older age of greater than or equal to 16 months, but not at a young or mid-age, based on gene expression and chromatin accessibility at a single-cell resolution. This age acceleration was further validated by epigenetic clock analysis of DNA methylation mapping. Moreover, we found that Tet2 deficiency drives a significant expansion of HSCs, a cellular hallmark of HSC aging, in an age-dependent manner. Specifically, we observed increased expression of S100a6, Cdk6, Egr1 in Tet2-deficient HSCs from older mice, suggesting that Tet2 deficiency hijacks the HSC expansion strategy during HSC aging by enhanced self-renewal. We also observed that the differentially expressed genes between Tet2-deficient older HSCs and control older HSCs are associated with open chromatin accessibility. Many of these genes are enriched in the targets of specific transcription factors (TFs), such as Egr1, Creb1, Stat family and Hoxb8. These target genes play critical roles in inflammatory pathways and HSC functions. In conclusion, our findings reveal that Tet2 deficiency significantly contributes to the acceleration of HSC aging, and hijacks the HSC expansion strategy (e.g., self-renewal) during HSC aging, which exacerbates age-related clonal hematopoiesis. By elucidating the transcriptomic and epigenomic alterations in Tet2-deficient HSCs, our study provides novel insights into how age and somatic mutations collaborate to promote the pathogenesis of age-related hematological diseases. These discoveries not only enhance our understanding of HSC aging mechanisms but also offer potential biomarkers for early detection and targets for therapeutic intervention in age-associated clonal disorders. Citation Format: Shilpita Karmakar, Yang Liu, Efthymios Motakis, Kaustav Sengupta, Travis Roeder, Kristina Mujica, Jennifer Trowbridge, Hideyuki Oguro, Sheng Li. TET2 accelerates clonal hematopoiesis in an age-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4398.

Abstract B031: Accelerated intra-clonal diversification of hematopoietic stem cells during aging fuels leukemia evolution

Abstract Cancer are diseases of aging. Many cellular processes characteristic of cancer, e.g., epigenetic dysregulation, are similarly observed during aging. Acute myeloid leukemia (AML) is emblematic of this interplay. As a blood cancer stemming from hematopoietic stem and progenitors (HSPC), the median age of AML diagnosis is 65. This is linked to age-related clonal hematopoiesis (CH), a condition resulting from the expansion of hematopoietic stem and progenitor cells, typically marked by mutations in leukemia-associated genes. Our investigation thus seeks to clarify the intrinsic role of aging in HSPCs as they relate to CH and leukemic transformation. Hypothesis: Aging amplifies the inheritable phenotypic variations in hematopoietic stem cells, enhancing epigenetic heterogeneity. This acts as a substrate for natural selection, driving leukemic transformation. HSPC Epigenomic and transcriptomic heterogeneity increase in old age: we profiled the single-cell transcriptome (scRNA-seq) and single-nuclear ATAC-seq (snATAC-seq) data from Lineage-c-Kit+ HSPCs isolated from mice at two age groups. Applying Shannon entropy, our analysis revealed a significant increase in epigenetic and transcriptomic heterogeneity in middle-aged LT-HSC compared to their younger counterparts. This process occurs in an aged bone marrow (BM) microenvironment characterized by inflammation and altered HSPC support. Interestingly, we previously show that the rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 loss (Tet2 MT) and Flt3 ITD precedes leukemic transformation. Diversification of LT-HSC Transcriptome: We employed CellTag Indexing to track the fate of specific HSC subclones. With a barcode detection rate of 80%, our findings indicate that a significant majority (97%) of clones emerged from an HSC. Furthermore, we observed that cells of the same clone manifest higher transcriptomic similarities than those from different clones, underscoring the technique's capability in mapping HSPC clonal evolution. Then we applied scRNA-seq to tagged HSPCs after 16 days of ex vivo expansion. We found that LT-HSCs from older mice exhibited greater clonal diversification, evidenced by intra-clonal transcriptomic correlation. ST-HSC and MPP from older mice also exhibt this pattern. Functional Relevance: Our GSEA analysis of genes with increased expression variability in older mice highlighted several pathways pivotal for HSPC fitness, encompassing ribosome synthesis, inflammation response mechanisms (e.g., JAK-STAT), hematopoietic cell lineage, and AML/cancer pathways. Conclusion: our results suggest that the combined effects of aging and somatic mutations in epigenetic regulators may enhance cell-to-cell variations, offering additional heritable phenotypic variations. These, in turn, may facilitate clonal hematopoiesis and leukemic transformation. Future work will delineate specific epigenetic and transcriptomic configurations that enable mutant HSPC clonal expansion. This study provides a cohesive framework, linking the principles governing aging to cancer. Citation Format: Lamis Naddaf, Marco De Dominici, Xiaowen Chen, Parveen Kumar, James Chavez, Travis Roeder, Shilpita Karmakar, Eric Pietras, Hideyuki Oguro, James DeGregori, Sheng Li. Accelerated intra-clonal diversification of hematopoietic stem cells during aging fuels leukemia evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B031.

Cells in Atherosclerosis: Focus on Cellular Senescence from Basic Science to Clinical Practice.

Aging is a major risk factor of atherosclerosis through different complex pathways including replicative cellular senescence and age-related clonal hematopoiesis. In addition to aging, extracellular stress factors, such as mechanical and oxidative stress, can induce cellular senescence, defined as premature cellular senescence. Senescent cells can accumulate within atherosclerotic plaques over time and contribute to plaque instability. This review summarizes the role of cellular senescence in the complex pathophysiology of atherosclerosis and highlights the most important senotherapeutics tested in cardiovascular studies targeting senescence. Continued bench-to-bedside research in cellular senescence might allow the future implementation of new effective anti-atherosclerotic preventive and treatment strategies in clinical practice.

Clinical and Molecular Spectrum of Somatic Mosaic States in DDX41 mutant Germline Predisposition Syndromes

Introduction: Germline DEAD-box RNA helicase-1 mutant (DDX41) predisposition syndrome ( DDX41-MT-GPS) is one of the most frequent hereditary predisposition syndromes in adults with myeloid neoplasms (MN-2-5%). Somatic mosaicism involving the unmutated DDX41 allele (e.g., R525H) as a potential mechanism for myeloid evolution has been reported in 39-50% of patients (pts). In addition, given the later age of onset, somatic mosaic states composed of age-related clonal hematopoiesis (ARCH) (e.g., DNMT3A, ASXL1, TET2, and TP53), with reported frequencies less than expected in MDS and AML, have been documented. We conducted this study to define the somatic mosaic landscape in DDX41-MT-GPS . Methods: After approval from the Mayo Clinic IRB the prospective GPS database was queried for pts with DDX41-MT GPS. Germline confirmation was conducted on skin fibroblast-, or hair follicle-derived DNA. Somatic CH testing was conducted using a targeted NGS panel as previously described. Given the high frequency of variants of undetermined significance (VUS), comparisons were made between pathogenic ( DDX41path) and DDX41VUS. Results: We identified 106 pts with DDX41-MT-GPS, of which 83 (78%) met criteria for MN, 16 (15%) for CH/CCUS (clonal cytopenias of undetermined significance), while 7 (6%) were unaffected carriers. Somatic mosaicism in DDX41-GPS patients without MN Sixteen DDX41-MT pts without MN met criteria for CH/CCUS ( DDX41path [n=4] and DDX41VUS [n=12]; median age 69 years (yrs) (range [R], 19-78) ( DDX41path [69 yrs] and DDX41VUS [67 yrs], p=0.85), with a male predilection (75%). Four (25%) pts had somatic DDX41 MT (R525H [n=3] and A376T [n=1]) with median variant allele frequency (VAF) 10.5% (R, 5-27); 2 (50%) pts with germline DDX41path had R525H (VAF; 5% and 13%) and 1 (8%) pt each with germline DDX41VUS had R525H (VAF 5%) and A376T (VAF 27%) somatic variants, respectively. Abnormal cytogenetics (CG) were observed in 2/13 evaluable (15%) pts ([+8 and del20q]) in the germline DDX41path group only (p= 0.05). ARCH co-mutations were observed in 11 (69%) pts, not significantly (NS) different between DDX41path (75%) and DDX41VUS (75%), p= 0.66. Individual co-mutations: DNMT3A (25% vs 16%, p= > 0.99), ASXL1 (25% vs 8%, p=0.71), TET2 (25% vs 8%, p= >0.99), and JAK2 (0% vs 8%, p= >0.99) were NS different between the two groups. Somatic mosaicism in DDX41-GPS patients with MN Eight-three pts met criteria for MN ( DDX41path [n=43] and DDX41VUS [n=40]; median age 67 yrs (range [R], 27-92), with a male predilection (58%). Fifty-four (65%) pts had MDS ( DDX41path [55%] and DDX41VUS [45%], p=0.55) and 29 (35%) had AML ( DDX41path [41%] and DDX41VUS [59%], p=0.65). Overall, 15 (18%) pts had somatic DDX41 MT; 12 (80%) pts with DDX41path and 3 (20%) pts with DDX41VUS (p= 0.003). The median VAF of somatic DDX41 MT was 12% (R, 5-37), NS different between DDX41path (10.5%) and DDX41VUS (12%) (p=0.08). The most common somatic DDX41 MT was R525H (66% [n=10]), significantly associated with DDX41path (9/10 [90%]), p=0.01. ARCH was observed in 17 (20%) pts, significantly higher in DDX41path (76%) compared to DDX41VUS (24%), p= 0.03. Abnormal CG were observed in 10/73 evaluable (14%) pts (-Y [n=2], del 20 [n=3], del 5q [n=2], del 7q [n=1], t (3;8) [n=1] and complex CG [n=1]), higher in DDX41path (70%) compared to DDX41VUS (30%) with p value >0.99. We compared the spectrum of CH between DDX41 MT CH/CCUS and MN pts ( Table 1) and found no difference in the proportion of pts with somatic DDX41 MT (p= 0.52) and ARCH/myeloid MT (p= 0.56). The individual occurrences of DNMT3A (p= 0.49), ASXL1 (p= 0.71), TET2 (p=0.59), JAK2 (p>0.99) and TP53 (p= 0.56) mutations were NS different between the two groups ( Figure 1). Conclusion: We define the somatic mosaic landscape in DDX41-MT GPS and demonstrate that somatic mutations involving the other allele, especially R525H, are seen in ~20% of pts (>80% of R525H present in MN). The frequency and composition of ARCH and myeloid driver mutations was lower than expected for the age of presentation, in comparison to de novo and secondary MDS/AML and occurred more frequently in DDX41path compared to DDX41VUS. Forty-two % (n=41), including 37% with MN, did not have somatic mosaicism, underscoring the fact that mechanisms of leukemia progression remain to be defined in subsets of affected pts.

Serum Amyloid A1 (SAA1) Secreted By the Stromal Microenvironment Drives Malignant Clonal Proliferation in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Drivers of malignant hematopoietic stem cell clonal proliferation from age-related clonal hematopoiesis (ARCH) to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) are not well understood. However, a pro-inflammatory and immune tolerant aging bone marrow (BM) microenvironment is thought to contribute to disease development. Serum Amyloid A1 (SAA1) has been identified as a novel pro-inflammatory oncoprotein the levels of which progressively increase with MDS and AML progression. SAA1 also selectively promotes growth of patient-derived MDS and AML cells independent of cytogenetic or mutational profile. We aimed to characterize the mechanism by which SAA1 drives oncogenesis within the microenvironment. Herein we further stratified the disease marking properties of SAA1 by clarifying the association of its BM levels with disease progression in MDS and AML. SAA1 levels were significantly elevated in BM aspirates from MDS and AML patients as compared to those from healthy age-matched subjects (p=0.0002 and p=0.0052, respectively). The effects of SAA1 on mutated hematopoietic cells were assessed in a model of age-related clonal hematopoiesis (ARCH). Utilizing a CRISPR-Cas9 system, ASXL1-mutant CD34+ hematopoietic stem cells (HSCs) were generated; the replating capacity of both mutated and healthy CD34+ cells were assessed in the presence or absence of SAA1. Notably, SAA1 increased the replating capability assessed by an increase in the number of colonies formed on serial replating of ASXL1-mutant CD34+ cells, but not those of SAA1-treated healthy CD34+ or vehicle treated ASXL1-mutated CD34+ cells, suggesting that the presence of SAA1 favors stemness of mutated HSCs, and implicating SAA1 as a driver of malignant clonality. To investigate the mechanism of action of SAA1 BM mononuclear cells from AML patients or healthy age-matched subjects were treated with SAA1 or vehicle overnight and single-cell RNA sequencing analysis was performed. Seurat and cell cluster annotation analysis showed that AML samples treated with SAA1 had increased numbers of hematopoietic stem and progenitor cells (HSPCs) and plasmacytoid dendritic cells as compared to matched untreated AML samples. Further, Enrichr analysis showed that pathways upregulated by SAA1 in AML samples included pro-inflammatory molecules, anti-apoptotic, and antiviral defense mechanisms (p<0.0001). Shared genes upregulated in these pathways in HSPCs include interferon-induced signals including JAK2, STAT1, and OAS3, and genes implicated in resistance to chemotherapy, including CD44 and CD36, as well as the anti-phagocytosis signal CD47. Lastly, we initiated the development of a blocking monoclonal antibody against SAA1 as a means of inhibiting its potent oncogenic effects on malignant HSCs and examining its potential therapeutic activity. Utilizing a RAW 264.7 NFκB-luciferase reporter cell line, we screened 51 clones for their ability to selectively block SAA1-mediated NFκB activation. 7 optimal candidates were identified which demonstrated dose-dependent reduction in NFκB activity upon SAA1 challenge. Importantly, these candidate clones were specific for SAA1 and did not affect NFκB activation mediated by lipopolysaccharide (LPS)-mediated activation. We observed up to an 85% reduction of SAA1-mediated activity using supernatants from further subcloning of previously selected candidates. Moreover, cell proliferation of a human AML cell line (OCI-AML3) stimulated by SAA1 was markedly reduced in the presence of candidate antibodies, suggesting anti-proliferative activity. Our findings show that SAA1 levels correlates with disease severity in MDS and AML progression, selectively promotes stemness and proliferative potential of ASXL1-mutant cells, and demonstrates unique immune-related mechanisms selecting for malignant clonality. Further development and purification of a novel inhibitory antibody against SAA1 for future in vivo and ex vivo testing is underway as a promising novel target to reduce SAA1-mediated, niche-driven effects with broad implications in the treatment of hematological malignancies.

Combined Myeloid and Lymphoid Malignancies in 13 Elderly Patients

Disclosures Myeloid malignancies such as myelodysplasia (MDS) often evolve from age related clonal hematopoiesis (CH) with progression of variant allele frequencies. The Philadelphia negative myeloproliferative neoplasms (MPN) polythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF) are associated with mutations in JAK2, CALR, or MPL. Multiple myeloma (MM) and monoclonal gammopathies (MGUS) are felt to be age related, and the malignant cell is of lymphoid origin. Burkitt's Lymphoma is associated with EBV exposure and dysregulation of c-myc. However the etiology of most lymphoproliferative disorders (LPD) is not defined, but immune dysregulation may be causative. The combination of myeloid and lymphoid malignancies are not recognized to be a common occurrence; however within a three year period 13 patients with combined myeloid and lymphoid malignancies were detected in a single community practice. DATA Five patients had combined myeloid and lymphoid malignancies diagnosed concurrently. Three had a lymphoid disorder and then developed a myeloid disorder. Five had a myeloid disorder followed by a lymphoid disorder. Three patients had multiple myeloma (MM) and two had MGUS. Initially all patients responded to standard therapies, One patient at 87 years died from end stage renal disease likely related to her myeloma. Another patient with CLL and PV died at 92 years from an epithelial malignancy. Concurrent diagnoses Age at diagnosis (years) CLL ET 76MGUS MDS 77MM MDS 78MM MDS (RSBA) 85 5) DLBCL MF. 60 LPD/MM followed by a Age at initial diagnosis (years) and time to second diagnosis myeloid disorder. 1) MZL MF 58 15 months 2) MM ET 83 7 months 3) CLL PV 86 4 years Myeloid disorder followed by a LPD/MGUS MDS DLBCL 76 3 monthsMDS MGUS 78 30 monthsTP CLL 15 60 yearsET MyF 77 65 monthsET CLL 60 15 years (CLL chronic lymphocytic leukemia, DLBCL diffuse large B cell lymphoma, MF myelofibrosis, MyF mycosis fungoides, MZL mantle zone lymphoma, RSBA ringed sideroblastic anemia, TP inherited thrombocytopenia) JAK2 mutations were found in three of the four ET patients and in the one PV patient. One MF patient and one ET patient were negative for the JAK2 and CALR mutations. The mutation status of the other MF patient was not known. At diagnosis the patient with inherited thrombocytopenia was suspected to have a myeloid disorder. Combined myeloid and lymphoid malignancies is not well defined and is a seemingly uncommon event. Yet an Italian study demonstrated that MPN patients with JAK2 mutations have a 2.8-3.4 fold increased risk of developing a LPD compared with an age matched population (1). The linkage is unclear. Immune senescence may be important for the development of LPD observed in the elderly. Also long term latency of many years between the JAK2V617F mutation acquisition and MPN presentation has been documented (2). The occurrence of both myeloid disorders and LPD is not well recognized. Future studies may provide insights into the mechanisms underlying these disorders. Increasing genetic complexity and immune senescence associated with aging likely plays a major role. The limitations of this study are its small size, single community investigator, retrospective clinical nature, and the lack of gene sequencing and other molecular studies in most if not all of the patients. This observation was noted over three years in a single community practice which strongly implies that combined occurrence may be more common than recognized and suggests that further investigation is warranted. Boxer none, Denes none

Sole DNMT3A/TET2/ASXL1 Mutations Define a Distinct Clinical Trajectory for Patients with Clonal Hematopoiesis

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is a clinically defined entity that is recognized by the 2022 International Consensus Classification and the 5 th Edition of the WHO. While the Clonal Hematopoiesis Risk Score (CHRS) incorporates 8 variables that underlie a prognostic framework for prediction of risk for progression to myeloid neoplasm (MN) (Weeks LD et al., NEJM Evidence 2023), there remains debate as to the role of mutations in epigenetic regulators, such as DNMT3A, TET2 and ASXL1 (“DTA”), with respect to clinical outcomes in the real-world setting. Although DTA mutations often occur early in myeloid pathogenesis, the persistence of these mutations as age-related clonal hematopoiesis (CH) might not significantly influence clinical course (Jongen-Lavrencic M et al., NEJM 2018). We have previously observed impaired clearance of DTA-mutant clones with hypomethylating agent or intensive chemotherapy for myelodysplastic neoplasms and acute myeloid leukemia (ASH abstract 4122; Blood (2022) 140 (Supplement 1): 9150-9151), and we now seek to understand the impact of sole DTA mutations in patients with CH. Methods: We studied a pre-selected group of patients who had an indication for undergoing bone marrow evaluation. Such indications included cytopenia(s), elevated blood cell count(s), staging/workup for lymphoma, and radiographic marrow abnormalities. Patients with CH were identified via concurrent morphologic, cytogenetic, and molecular diagnostics of bone marrow aspirates reviewed by UMass and Stanford Hematopathology from 2013 to 2023. CH was defined as presence of a somatic mutation with variant allele frequency (VAF) ≥ 2% on molecular diagnostics or non-myelodysplastic syndrome (MDS)-defining clonal cytogenetic aberration in a patient without morphologic evidence of MN. A total of 79 patients with CH were identified, then segregated based on presence or absence of sole DTA mutations. Results: We assessed the clinical trajectory of patients with CH who did not exclusively have DTA mutations (“non-sole DTA;” n = 58) (Panel A) compared to patients with CH who had DTA mutations alone without any other concurrent mutations (“sole DTA;” n = 21) (Panel B). The median age at detection of CH for sole DTA vs. non-sole DTA was 71.0 ± 2.11 years vs. 71.5 ± 1.45 years, respectively ( p = 0.454). Median number of mutations for sole DTA vs. non-sole DTA patients was 1.24 ± 1.2 vs. 2.24 ± 0.18, respectively ( p < 0.0001). Median follow-up for all patients was 450 days, and the median overall survival was not reached for either group. For non-sole DTA patients, 44 of 58 patients (76%) did not progress, while 14 of 58 patients (24%) progressed to frank MN at the time of analysis. Types of frank MN in the 14 progressors included MDS (50%), MDS/myeloproliferative neoplasm (MPN) overlap (28.6%), AML (14.3%), and MPN (7.1%). For sole DTA patients, 0 of 21 patients (0%) progressed. Regarding distribution of VAFs for sole DTA patients vs. non-sole DTA patients, mean VAF was 13.5% ± 1.91% vs. 31.6% ± 2.18%, respectively ( p < 0.001). For patients with both DTA mutations plus additional mutations, mean VAF for non-progressors vs. progressors was 30.4% ±1.47% vs. 38.6% ± 2.97%, respectively ( p = 0.206). For patients without any DTA mutations but with other somatic mutations, mean VAF for non-progressors vs. progressors was 29.7% ±1.67% vs. 35.2% ± 2.26%, respectively ( p = 0.160). Conclusion: In this study of pre-selected patients with CH, DTA mutations alone were insufficient for progression to frank MN. Progressors included patients with either (1) DTA mutations plus concurrent mutations, or (2) patients without DTA mutations but presence of mutations in other gene clusters. The clinical trajectory of patients with CH in our study differs from that of patients from large consortia possibly because our patients presented with an indication for testing, reflecting real-world data. A limitation of our analysis is the variability in time to recognition and diagnosis of CH, which reflects the high heterogeneity of these patients. Still, our data suggest the need for large-scale studies in the real-world setting to better prognosticate the relevance of DTA mutations for this patient population.

Involvement of the JAK-STAT Pathway in the Molecular Landscape of Fusion-Free Myeloid Neoplasms with Eosinophilia

Introduction. Gene fusions leading to the constitutive activation of tyrosine kinases (TK) such as PDGFRA, PDGFRB or FGFR1 have been the first recurrent genetic defects involved in myeloid hypereosinophilic syndromes (HES). Although activation of the Janus kinase (JAK)/Signal Transducer and Activator of Transcription(STAT) pathway is critical for eosinophil production and survival, genes involved in the JAK/STAT pathway are not included in most next-generation sequencing (NGS) panels used for the etiological workup of hypereosinophilia (HE). Methods. A custom 149-gene NGS panel including subunits of the IL3/IL5/GM-CSF receptors, TK ( PDGFRA/B, FGFR1, ABL1, FLT3, KIT), intracellular proteins of the JAK-STAT and RAS-MAPK pathways was performed in 64 consecutive adult patients (experimental group) referred between March 2012 to June 2023 to the French Reference Center for Hypereosinophilic Syndromes (CEREO) for HE/HES displaying at least one clinic-biological feature suggestive of myeloid neoplasm ( i.e. splenomegaly, other unexplained CBC abnormality besides HE, increased serum tryptase and/or vitamin B12 levels, corticosteroid-refractory HE and/or sensitivity to either TK inhibitors or JAK inhibitors). All of them were negative by PCR and/or FISH analyses for gene rearrangements involving PDGFRA, PDGFRB or FGFR1. Patients with lymphocytic HES (n=7), idiopathic HES (n=26) and HE of undetermined significance (n=11) were used as controls (total, n=44). Results. Concordant with the latest recommendations of the international cooperative working group on eosinophil-associated disorders, at least one mutation was reported in 50/64 (78%) patients of the experimental group versus 8/44 (18%) patients in the control group (p<0.001) when applying a threshold of at least 3% of variant allele frequency (VAF). Mutations in the control group implied genes involved in age-related clonal hematopoiesis ( e.g.DNMT3A, TET2), with low VAF in almost all cases. All 35 patients with at least one mutation involving the JAK/STAT pathway belonged to the experimental group, among whom all 22 patients treated with steroids were refractory to therapy. Eighteen patients had STAT5B mutations, including 13 (72%) with the somatic N642H mutational hotspot. Two patients harbored JAK2Ex13InDel mutations, including one with eosinophilia and erythrocytosis. Previously unreported molecular alterations were also evidenced, including seven patients with JAK1 mutations and three STAT5A-mutated patients who shared common features i.e. the co-occurrence of BCOR mutations, high hemoglobin levels and eosinophil hyperplasia. Of note, the latter mutations were not reported both in a public database (GnomAD) as well as in a second cohort of 613 samples referred for suspicion of myeloid malignancies (yet without HE) studied with the same NGS workflow, thereby strongly supporting their association with myeloid HES. Deciphering the data from bulk sequencing also suggests that JAK-STAT mutations were frequently preceded by (or associated with) myelodysplasia-related gene mutations, with SF3B1 (12/36) and ASXL1 (10/36) mutations being the most common. Overall, both KIT D816V (n=2) and RAS/MAPK pathway activation mutations (n=3) were rare. No mutation of either PDGFRA/B, FGFR1 or IL3/IL5/GM-CSF-receptor genes was evidenced.In the experimental group, 17/18 (94%) patients (including 12 with JAK-STAT mutations) treated with ruxolitinib and with > 3 months of follow-up responded to treatment (12 complete and 5 partial hematological responses). Discussion. These dataemphasize the usefulness of NGS in daily practice for the workup of fusion-free HES patients harboring features suggestive of myeloid neoplasms. In such patients, druggable mutations involving the JAK-STAT pathway (including yet unidentified STAT5A mutations) are frequent. Most JAK/STAT mutations occur in the setting of a preexisting myelodysplastic or myelodysplastic/myeloproliferative disease harboring mutations in RNA-splicing genes or chromatin modifiers. These findings provide a rationale for refining treatment algorithms in fusion-free myeloid HES patients, supporting the use of JAK inhibitors as frontline therapy. More data are warranted to assess whether JAK inhibition enables sustained molecular remission in all disease subtypes.

Mutational Changes in Solid Cancer Cells Induce DNA Damage Repair Deficiency in Hematopoietic Cells Leading to a Distinct Spectrum of Clonal Hematopoiesis Independent from Age-Related Clonal Hematopoiesis

Clonal hematopoiesis (CH) is characterized by the acquisition of defined somatic mutations in hematopoietic stem cells (HSC). It is well known that CH contributes to the development of hematological malignancies but also increases the risk of developing non-malignant conditions such as cardiovascular, pulmonary, and inflammatory liver disease. However, less is known about the association between clonal hematopoiesis and solid cancer. Reports suggest that patients with solid cancer have a higher incidence of clonal hematopoiesis at an earlier age and an inferior prognosis. To investigate whether the presence of solid cancer itself can drive the development of clonal hematopoiesis, we analyzed 392 paired tumor and blood samples from patients with advanced cancer. In our cohort, all patients had evidence of clonal hematopoiesis based on a variant allele frequency (VAF) of at least 2%. With respect to a defined set of the 20 most frequently CH-associated genes, we discovered an increased CH rate of 23% of all patients (90 cases, median age of 61 years, defined by a VAF from 2% to 30%). The frequency of mutated genes differed greatly from published data from the general population and cancer patients, with the most common mutations being PABPC1 (39%), KMT2C (28%), RUNX1 (18%), and DNMT3A (10%). In addition, we observed a strong correlation between certain mutations in tumor samples and the frequency of CH. The mutational signature of CH was not mainly associated with a typical chemotherapy-dependent mutational signature, but with an impaired DNA repair capacity. For further investigation, we performed additional experiments in mice, which showed that the presence of malignant cells impairs DNA repair capacity in HSC through inflammatory changes in the stroma. In summary, we were able to show that patients with advanced or recurrent solid tumors have a significantly increased frequency of CH, which is not only the result of prior chemotherapy exposure but the result of diminished DNA repair capacity. This suggests a possible role of solid cancer in promoting the development of CH independent from chemotherapy exposure, which is supported by our additional murine experiments.

DNA Damage Response Constrains Cell Growth and Drives Clonal Hematopoiesis in Telomere Biology Disorders

Telomere biology disorders (TBD) are inherited diseases caused by genetic defects in telomere maintenance. Patients with TBD have critically shortened telomeres, leading to bone marrow failure (BMF) and an increased risk of myeloid neoplasms (MN). We conducted a study to understand the genetic changes driving clonal evolution in TBD patients and their distinct mechanisms compared to normal aging and sporadic myelodysplastic syndrome (MDS). We analyzed acquired genetic alterations in hematopoietic cells from 105 TBD patients. The diagnosis of TBD was made using a combination of clinical history, germline mutation status, and telomere length. Germline mutations were identified in 84% of patients, a family history consistent with TBD was seen in 46%, and 76% of patients had lymphocyte telomere lengths (TL) <1st %ile for their age, while 22% fell between the 1st and 10th %ile. Complications observed in our cohort included BMF (42%), myeloid neoplasms (17%), solid tumors (17%), cirrhosis (14%), and interstitial lung disease (34%). Clonal hematopoiesis was detected in 44 of 105 patients (42%), including 8 of 27 (30%) patients 18 years or younger. Somatic genetic alterations primarily involved the DNA damage response (DDR) pathway, with ATM and TP53 being the most frequently mutated genes (11 ATM mutations in 5 of 80 patients; 5 TP53 mutations in 4 of 84 patients; Fig. 1). Mutations typical of age-related clonal hematopoiesis were seen in 4 patients (3 ASXL1, 1 TET2). Somatic reversion of TBD germline lesions was found in 7 patients. Additionally, 11 somatic mutations were detected in genes involved in DDR, telomere maintenance, and cell senescence, including KAT6B, PALB2, TELO2, TOP1, and RTEL1. MDS-associated gene mutations were found in 15 patients (18%), 8 in the setting of MN. The most common mutations affected spliceosome genes with U2AF1 p.S34F and SF3B1 being most notable, including 1 U2AF1 S34F identified in a 15-year-old. Mutations in NPM1, RUNX1, WT1, SF3B1, and FLT3 were identified only after malignant transformation. Acquired cytogenetic changes were observed in 16 of 87 evaluable patients (18%), 8 in the setting of MN, and included gain of 1q in 5 patients (5.7%). Two patients with TP53 mutations developed a complex karyotype (Fig 1). To understand selective pressures contributing to clonal hematopoiesis development in TBD, we analyzed transcriptional changes in bone marrow (n=2) and peripheral blood (n=1) mononuclear cells (MNCs) of 3 TBD patients with different germline lesions using single cell RNA sequencing (scRNASeq). MNCs from TBD patients exhibited significant differences compared to healthy controls, showing downregulation of MYC targets, reduced translation, cell cycle arrest, and upregulation of ATM-dependent DDR pathway and FAS pathway (Fig 2). We hypothesized that mutations in DDR pathway genes, particularly somatic ATM loss, enable TBD hematopoietic stem and progenitor cells to overcome telomere-induced DDR induction and premature senescence. Consistent with our hypothesis, low dose ATM inhibition in TBD patients' fibroblasts significantly enhanced growth rate of TERC-mutated and DKC1-mutated cells, whereas control fibroblasts were unaffected. Somatic mutations in ATM in our cohort were typically observed in patients with TERC-mutated TBD (3 of 5, 3 of 34 with TERC-mutated TBD), very low TL, and extra-hematopoietic manifestations. Of the 11 ATM mutations, 3 were predicted loss of function and 4 were missense kinase domain mutations, known or predicted to result in loss of kinase activity. Somatic ATM loss occurred both as heterozygous or compound heterozygous mutations. The development of ATM mutations alone was not causative of malignancy; however, 2 patients with somatic ATM mutations developed myeloid neoplasms after acquisition of additional mutations (subclonal mutation in NPM1 in one patient and clonal replacement with a TP53-mutant clone in another patient). In conclusion, our findings highlight the role of telomere dysfunction-induced DDR and cellular senescence in bone marrow failure and clonal selection in TBD. ATM loss selectively enhances growth of TBD cells by inhibiting DDR activation which may ameliorate marrow failure but has the potential to increase the risk of myeloid neoplasms after acquisition of additional genetic events. Future studies should explore the possibility of a therapeutic window involving very low doses of ATM inhibition in TBD.

TET2-Mediated Dysregulation of Heterochromatin in Age-Related Clonal Hematopoiesis

Aging is one of the major negative factors of normal hematopoiesis, characterized by a decline in the self-renewal capabilities of hematopoietic stem and progenitor cells (HSPCs), alongside a myeloid lineage bias, thereby sabotaging the immune system and increasing the risk of malignant transformation. Epigenetic deregulation is a hallmark feature representative of aging and significantly contributes to age-related dysfunction in multiple systems. DNA methylation drifting is commonly observed during aging, especially within the partially methylated domains (PMDs). PMDs are intriguing heterochromatin regions featured by their late-replicating, lamina-associated, and B-compartment localized properties in HiC analysis. While heterochromatin is generally considered devoid of genes, it nonetheless plays a pivotal role in maintaining genomic stability. However, the link between age-related DNA methylation loss in PMDs and heterochromatin dysfunction remains elusive. In this study, we juxtapose the influence of aging on both wild-type (WT) and Tet2 knockout (KO) HSPCs. Tet2 depletion was found to counteract the functional deterioration typically associated with aging by preserving robust self-renewal and repopulation capacities in aging HSPCs. This resistance to functional decay may account for the age-related clonal hematopoiesis observed in Tet2KO HSPCs. At a molecular level, we identified distinct epigenetic regulatory mechanisms mediated by Dnmt3a and Tet2 at heterochromatin. These mechanisms are associated with alterations in 3D genome architecture during HSPC aging. Age-related dysregulation of heterochromatin leads to the upregulation of endogenous retroviruses (ERVs), which subsequently activate intracellular innate immune response and contribute to the functional decline of aging HSPCs. The application of reverse transcriptase inhibitors was shown to suppress ERV production and interferon-stimulated genes (ISGs) expression, thereby ameliorating age-related defects in aged HSPCs. Our findings provide compelling evidence supporting the intricate interplay between DNMT and TET in regulating DNA (de)methylation equilibrium in heterochromatin and euchromatin during stem cell aging. Moreover, our study offers critical insights into the mechanisms underlying age-related heterochromatin dysfunction, which contributes to the functional deterioration of HSPCs during aging.

Age-Related Clonal Hematopoiesis Mutations Detected at the Time of Stopping Tyrosine Kinase Inhibitor Therapy Predict the Achievement of Treatment-Free Remission for Patients with CML

Background The gain of mutations with a competitive advantage in hematopoietic cells of healthy individuals is common with aging and termed clonal hematopoiesis of indeterminate potential (CHIP). Through sequencing studies we detected CHIP related mutants in chronic myeloid leukemia (CML) patients in deep molecular response. CHIP mutants may provide a selective growth advantage to non-leukemic cells, driving their clonal expansion in remission and potentially influencing treatment-free remission (TFR) if CHIP clones outcompete an emerging BCR:: ABL1 clone. Aim To determine if CHIP mutants detected at the time of stopping tyrosine kinase inhibitor (TKI) therapy could influence achievement of TFR. Methods An RNA-based sequencing method targeting 17 of the most frequently mutated CHIP genes was developed and applied to blood samples of 150 patients who attempted TFR. The median follow up of patients in TFR was 71 months. CHIP mutants met strict criteria for pathogenicity based on their potential to confer growth and survival advantages, i.e. the same criteria as cancer driver mutations. Variant allele frequency (VAF) of ≥0.4% was reproducibly detected. CHIP at the time of cessation, termed CHIP TOC, was defined as 1) ≥1 CHIP mutant with VAF ≥2.0% (n=30 patients) or 2) a novel criterion for patients with multiple CHIP mutants where the combined VAF was ≥2.0% (n=7). Overall, 25/37 patients (68%) with CHIP TOC had multiple mutations, range 2-7. Results CHIP TOC was detected in 37/150 patients (25%). The average age of patients with CHIP TOC was 66.3 years vs 58.0 for those without, P=.0004, which is consistent with the age related nature of CHIP. No patient aged <40 had CHIP TOC, whereas 44% of patients ≥70 had CHIP TOC. Twelve genes were mutated and the most frequent were TET2, DNMT3A, ASXL1 and PPM1D. The overall probability of TFR was 60.4% at 12 months and 53.0% at 84 months. CHIP TOC was associated with TFR. At 12 months, 83.6% of patients with CHIP TOC maintained TFR versus 52.9% of patients without CHIP TOC, P=.002. At 84 months, 62.9% of patients with CHIP TOC maintained TFR versus 49.6% of patients without CHIP TOC, P=.014. We previously demonstrated that e13a2 transcripts and a slow initial decline of BCR:: ABL1 after starting frontline TKI therapy, measured as the number of days over which BCR:: ABL1 halved, were our strongest predictors of relapse. Prognostic factors for the achievement of TFR at 12 and 84 months were examined in multivariable analysis. Candidate prognostic factors were CHIP TOC, BCR:: ABL1 halving time, BCR:: ABL1 transcript type, age at diagnosis, gender, duration of MR4.5 and time on TKI before TKI stop. The independent predictors of TFR at 12 and 84 months were the presence of CHIP TOC at TKI stop, a shorter BCR:: ABL1 halving time (indicating a more rapid initial BCR:: ABL1 decline) and the e14a2 (plus e14a2/e13a2) BCR:: ABL1 transcript type (Table 1). The rate of late relapse, defined as loss of MMR >12 months after cessation, was higher in patients with CHIP TOC. Overall, 85 patients had TFR at 12 months and late relapse occurred in 8. A landmark analysis demonstrated that the probability of maintaining TFR at 84 months for these 85 patients was 93.8% for those without CHIP TOC (n=57) and 75.2% for those with CHIP TOC (n=28), P=.007. The only variable associated with late relapse among the patients with CHIP TOC was fluctuation of BCR:: ABL1 within the first 12 months after cessation, defined as irregular rise and fall comprising 2 consecutive BCR:: ABL1 ratios with loss of MR4.5, or a single loss of MR4. Overall, 13 patients had fluctuation within the first 12 months and 5 of these had late relapse. All 5 had CHIP TOC. Patients with CHIP TOC and stable MR4.5 by 12 months had a 93.8% probability of TFR at 84 months (Table 2). Conclusion The presence of CHIP TOC at TKI cessation, a shorter BCR:: ABL1 halving time and e14a2 BCR:: ABL1 transcripts were independent predictors of TFR at 12 and 84 months. Delayed loss of MMR that occurred for a minority of patients with CHIP TOC may be associated with competition between CHIP clones and a residual leukemic clone. These patients had fluctuating BCR:: ABL1 over the first 12 months after cessation and prior to late relapse. Novel strategies may be warranted to reduce the risk of late relapse for these patients. Patients who sustained TFR by 12 months in stable MR4.5 had a reassuringly low risk of late relapse. CHIP status could be a key variable to guide TFR decision-making and monitoring.

Abstract 009: Age-related Clonal Hematopoiesis Predisposes To Hypertension In Mice Through An Inflammasome Mechanism

Hypertension incidence increases with age and represents one of the most prevalent risk factors for cardiovascular disease (CVD). Clonal events in the hematopoietic system resulting from somatic mutations in “driver” genes such as Tet2 are prevalent in elderly individuals in the absence of overt hematologic disorders. This condition is referred to as age-related clonal hematopoiesis (ARCH), and it is a newly recognized risk factor for CVD. It is unknown whether ARCH and hypertension in the elderly are causally related and, if so, what are the mechanistic features. Objective: Given that the aging process increases the prevalence of hypertension and ARCH, the current experimental study was designed to test the effect of ARCH in hypertension incidence. Methods and Results: A murine model of adoptive bone marrow transplantation to nonconditioned mice was employed to examine the interplay between ARCH and hypertension. Mice carrying Tet2-deficiency in hematopoietic cells resulted in elevated systolic (SBP) and diastolic blood pressure (DBP) as early as one day after challenge with a sub-pressure dose of angiotensin II (Ang II) 200ng/kg/min; SBP (day 1 Tet2 -/- : 148 mmHg± 4 vs. WT: 129±2 mmHg, P=0.002); day 9 Tet2 -/- 159±7 mmHg vs. WT: 139±5 mmHg, P<0.05), and DBP (day 1, Tet2 -/- : 108 ±3 mmHg vs. WT: 100±1 mmHg, P<0.005; day 9, Tet2 -/- : 111 ±4 mmHg vs. WT 107±3 mmHg). The ARCH Tet2 model of CH led to greater macrophage infiltration in the kidney, aorta and mesentery after Ang II compared with WT-treated mice. The Tet2-/- CH condition led to renal NLRP3 inflammasome activation and elevated renal levels of IL (interleukin)-1β (Tet2 -/- 1.4 vs. WT: 0.96, P<0.005) and IL-18 (Tet2 -/- 1.56 vs. WT: 1.18, P<0.005) after Ang II administration. Interestingly, administration of the inflammasome inhibitor MCC950 reversed hypertension seen in the mouse model of Tet2 -/- CH. Conclusion: Our data show that a sub-pressor dose of Ang II promotes hypertension due to elevated renal immune cell infiltration in a Tet2-CH model, promoting IL1-β and IL-18. These data suggest that Tet2 -/- CH carriers could be at elevated risk for the development of hypertension, and that NLRP3 inhibitors could be useful for treating hypertension in this population.

Prognostic relevance of clonal hematopoiesis in myeloid neoplastic transformation in patients with follicular lymphoma treated with radioimmunotherapy.

While novel radioisotope therapies continue to advance cancer care, reports of therapy-related myeloid neoplasms (t-MN) have generated concern. The prevalence and role of clonal hematopoiesis (CH) in this process remain to be defined. We hypothesized that: (i) CH is prevalent in relapsed follicular lymphoma and is associated with t-MN transformation, and (ii) radiation in the form of radioimmunotherapy (RIT) plays a role in clonal progression. In this retrospective cohort study, we evaluated the prevalence and prognostic impact of CH on clinical outcomes in 58 heavily pre-treated follicular lymphoma patients who received RIT. Patients had been given a median of four lines of therapy before RIT. The prevalence of CH prior to RIT was 46%, while it was 67% (P=0.15) during the course of RIT and subsequent therapies in the paired samples. Fourteen (24%) patients developed t-MN. Patients with t-MN had a higher variant allele fraction (38% vs. 15%; P=0.02) and clonal complexity (P=0.03) than those without. The spectrum of CH differed from that in age-related CH, with a high prevalence of DNA damage repair and response pathway mutations, absence of spliceosome mutations, and a paucity of signaling mutations. While there were no clear clinical associations between RIT and t-MN, or overall survival, patients with t-MN had a higher mutant clonal burden, along with extensive chromosomal abnormalities (median survival, afer t-MN diagnosis, 0.9 months). The baseline prevalence of CH was high, with an increase in prevalence on exposure to RIT and subsequent therapies. The high rates of t-MN with marked clonal complexities and extensive chromosomal damage underscore the importance of better identifying and studying genotoxic stressors accentuated by therapeutic modalities.

Abstract A028: Incidental detection of age-related somatic genomic alterations in blood samples from dogs with and without cancer

Abstract The concept of age-related somatic alterations in the blood or bone marrow of humans, commonly referred to as clonal hematopoiesis of indeterminate potential (CHIP) or age-related clonal hematopoiesis (ARCH), has been well characterized. In humans, these abnormalities tend to be associated with advanced age, often exhibit consistent signal over time, and typically involve recurrent locations in the genome. Though these somatic alterations may be associated with an increased risk of cancer, they do not originate from the tumor when cancer is concurrently present in the body. This study describes early findings that suggest similar age-related somatic alterations may also be present in dogs. Recently, next-generation sequencing-based liquid biopsy testing was developed for cancer detection in dogs. The clinical validation of this test involved 1,100 cancer-diagnosed and presumably cancer-free client-owned dogs. The test has also been performed commercially in thousands of additional dogs since 2021. This recent ability to test large numbers of dogs using liquid biopsy affords an unprecedented opportunity to study the genomic profiles of a broad population of canine subjects. Blood samples from over three thousand dogs ranging in age from 1 to >15 years were used in this analysis. Cell-free DNA (cfDNA) was extracted from the plasma, and genomic DNA (gDNA) was extracted from the white blood cells present in the buffy coat. Both cfDNA and gDNA were analyzed using next-generation sequencing to identify somatic genomic alterations. In a subset of patients, tumor tissue was also available for evaluation. Recurrent variants (e.g., involving CFA6 and CFA25, among others) were identified in the gDNA of a small fraction of patients. These findings occurred more commonly in older dogs and were typically persistent in gDNA across subsequent timepoints (when available) with no significant change in signal over time. When a clinical cancer evaluation was pursued, the majority of dogs with these findings had no evidence of cancer. For those in which cancer was identified, and tumor tissue was available for testing, the genomic profiles of the tumor tissue and gDNA were uncorrelated in almost all cases, suggesting the concomitant presence of age-related somatic alterations was incidental to the patient’s cancer. Additionally, regardless of whether cancer was present in the patient, the variants detected in gDNA were typically not encountered in cfDNA. These findings may be early evidence for the existence of age-related somatic alterations in dogs that potentially resemble the phenomenon of CHIP/ARCH previously observed in humans. Additional studies are ongoing to determine if these incidental findings may represent a risk factor for cancer development in dogs. Citation Format: Dana W.Y. Tsui, Allison L. O'Kell, Todd A. Cohen, Katherine M. Lytle, Kristina M. Kruglyak, Maggie A. Marshall, Carlos A. Ruiz-Perez, John A. Tynan, Susan C. Hicks, Jill M. Rafalko, Daniel S. Grosu, Jason Chibuk, Ilya Chorny, Angela L. McCleary-Wheeler, Andi Flory. Incidental detection of age-related somatic genomic alterations in blood samples from dogs with and without cancer [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A028.

Abstract PR001: Dissecting the role of aging and inflammation on clonal hematopoiesis

Abstract Aging is associated with the appearance in multiple tissues of somatic mutant clones often harboring mutations observed in cancers. In the hematopoietic system this phenomenon is named clonal hematopoiesis of indeterminant potential (CHIP) or “age-related clonal hematopoiesis”. The incidence of CHIP increases exponentially with aging, and has been associated with increased risk of myeloid malignancies, cardiovascular disease, and overall mortality. While mutations are thought to be acquired at a constant rate during life, whether aging plays a role in driving the expansion of these mutant clones is of crucial importance, as it can inform on the potential to prevent or halt the expansion of CHIP clones by intervening with the specific hallmark of aging fueling such expansion. To determine the effect of aging on the expansion of CHIP clones, we have established a method that relies on in vitro manipulation of mouse hematopoietic stem and progenitor cells (HSPC) in order to introduce by CRISPR-Cas9 selected mutations, followed by transplant into young and old recipient mice. Our method has several advantages: i) it allows rapid introduction of the desired mutations from mice of any background, including aged mice, bypassing the need to create transgenic lines; ii) by expanding the cultured cells in previously developed PVA media, it allows efficient engraftment in mildly (busulfan) conditioned recipient mice, avoiding the damaging effects on the bone marrow microenvironment that result from irradiation; iii) by manipulating HSPC with gRNAs targeting either the gene of interest (GFP tagged) or the safe harbor Rosa26 locus (dTomato tagged) prior to pooling, we can assess the competitive advantage of the mutation of interest relative to the Rosa26 control cells and determine their fitness advantage at various time points. We used this technique to generate Dnmt3a and Tet2 mutant clones and tested their expansion in young and aged recipient mice. Our data reveal that Dnmt3a exhibits a relatively mild fitness advantage and no difference in the rate of expansion between young and aged mice. By contrast Tet2-KO HSPC show a significantly faster expansion in old compared to young recipient mice, indicating a greater fitness advantage in the aged background. Tet2 mutant HSPC were previously shown to preferentially expand in inflammatory conditions such as stimulation with microbial products, IL-6 and TNFa. We have preliminary data that shows that co-housing old mice with young syngeneic mice alters their gut microbiome and results in a reduction of inflammation and restoration of more youthful gene expression profiles in stem and progenitor cells in the bone marrow. We are currently testing whether altering the gut microbiome or applying genetic models of reduced inflammation prevents the expansion of Tet2-KO cells, with associated “omics” analyses to explore underlying mechanisms. In all, these studies should contribute to our understanding of the aging-dependent occurrence of CHIP and suggest potential interventions to limit its prevalence. Citation Format: Marco De Dominici, Johannes Menzel, James V. DeGregori. Dissecting the role of aging and inflammation on clonal hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR001.

Abstract A026: History of blood transfusion and the risk of developing hematologic cancers in the United States: A cross-sectional study using the NHANES database. [R

Abstract Blood transfusion has long been regarded as a determinant of cancer, plausibly because it influences inflammatory and immunological changes in the recipient's body. Recently, there has been evidence of a role of donor-derived malignancy in donors of autologous stem cell transplants, supporting the possible cancer-driving role of age-related clonal hematopoiesis. Given that routine blood transfusion is more ubiquitous than stem cell transplantation, we hypothesize an association between blood transfusion and the occurrence of hematologic malignancies. We conducted a secondary analysis using data from the 2017-2018 survey of the National Health and Nutrition Examination Survey (NHANES). A total of 4,917 participants were included in our final analytic sample, of whom 587 (11.9%) had ever received a blood transfusion. Out of the participants receiving a blood transfusion, six (1.1%) were diagnosed with hematologic cancer versus 9 (0.21%) from the non-transfusion group. Specifically, three (0.51%) participants from the transfusion group were diagnosed with leukemia versus 2 (0.046%) from the non-transfusion group. Using multivariable logistic regression, we have found that, after adjustment of other predictors, people with a history of blood transfusion were 4.08 (95%CI= 1.18-14.05), 2.09 (95%CI= 0.47-9.27), and 6.76 (95%CI= 1.13-40.44) times more likely to report a history of hematologic cancers, Hodgkin lymphoma, and leukemia, respectively than those who were never transfused. In conclusion, we found a possible significant association between blood transfusion and the risk of developing hematological cancers (particularly leukemia) in the United States. Prospective studies are needed to confirm the temporal sequence of blood transfusion and hematologic cancers in that regard. Citation Format: Amr Ebied, Jason Salemi, Chighaf Bakour, Nancy Gillis. History of blood transfusion and the risk of developing hematologic cancers in the United States: A cross-sectional study using the NHANES database. [R] [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A026.

Immune pathophysiology of bone marrow failure

Bone marrow (BM) failure is a condition characterized by peripheral pancytopenia due to decreased BM function. It includes conditions such as acquired aplastic anemia (AA), myelodysplastic syndrome (MDS), and paroxysmal nocturnal hemoglobinuria (PNH). AA is characterized by pancytopenia and BM hypoplasia, and is primarily caused by an autoimmune mechanism involving cytotoxic T cells that damage hematopoietic stem cells (HSCs). Recent genomic research has revealed that patients with AA often exhibit clonal hematopoiesis by HSCs with genetic alterations, such as PIGA, DNMT3A, ASXL1, BCOR/BCORL1, copy-number neutral LOH of chromosome 6p (6pLOH), and HLA class I allele mutations. The genomic landscape of AA is distinct from MDS and age-related clonal hematopoiesis. Most notably, the presence of PNH-type cells and HLA class I allele-lacking cells indicates the presence of HSCs that have escaped from autoimmunity. We recently identified a common nonsense mutation at codon19 (c.19C>T, p.R7X) in exon1 (Exon1mut) of different HLA-A and HLA-B alleles, and HLA-DR loss of hematopoietic stem progenitor cells in AA patients carrying HLA-DR15. These results provide important clues for understanding the immune pathophysiology of BM failure.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.