Abstract In cancer-free tissues, somatic mutations accumulate in all of us over time. In most instances, these mutations are inconsequential to cellular fitness or to overall health. However, some mutations can result in a cell having a fitness advantage over its surroundings, enabling proliferation. In the blood, this proliferation - known as clonal hematopoiesis (CH) - is associated with an increased risk of hematologic malignancies, most notably leukemia, and is commonly observed in the elderly. People with Down syndrome (DS) also exhibit CH and have a markedly higher risk of leukemia (up to 400x); therefore, we investigated CH in people with DS to understand how mutations in the blood can lead to disrupted hematopoiesis and lead to varying susceptibility, onset, and severity of hematologic malignancies. To detect CH, I used a rare-mutation detection technique called DuplexSeq to analyze leukocytic genomic DNA from blood draws. This targeted-sequencing tool spans 37 genes implicated in leukemia - including leukemias seen in those with DS - and implements a double-stranded tag to incorporate mutational data from both DNA strands, enabling higher sensitivity than most sequencing methods. We characterized detected mutations using bioinformatic pipelines, various databases, and statistical methods. By comparing CH between people with and without DS, we observe an overrepresentation of protein-altering mutations in people with DS in genes important for hematopoiesis. Notably, people with DS exclusively harbor CH with JAK2 mutations, many of which have been associated with myeloid proliferative neoplasms. Mutations in NPM1 that have been previously seen in cancer are also exclusively observed in people with DS with potential to lead to differentiation block, a hallmark of leukemia. Finally, mutations with the highest variant allele frequency in TET2 are largely observed in people with DS, indicating preferential expansion in the DS context. These fledgling expansions in TET2 may be indicative of modified hematopoietic differentiation. Ongoing work is centered around understanding the consequences of these mutations and how they distinguish phenotypes among people with DS. Using a database for DS research, we are performing multi-omics analyses to understand which biological pathways may be disrupted in the same individuals with CH of interest. Additionally, we will use mouse models of DS to investigate the selective advantage of the observed mutations in the DS context, how they impact differentiation fate, and how cell intrinsic and extrinsic factors can influence clonal expansions. Overall, these studies offer a means by which risk of hematologic malignancies can be monitored by CH with elucidation of potential mechanisms. By characterizing CH, early markers of disrupted hematopoiesis can be established to determine susceptibility of leukemia and be used to monitor those who are most at risk. Citation Format: Edward J. Evans, Neetha Paul-Eduthan, Junxiao Hu, Dexiang Gao, Matthew Galbraith, Joaquin Espinosa, James DeGregori. Clonal hematopoiesis in Down syndrome and its potential impact on hematopoietic lineages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1510.