Abstract Clonal hematopoiesis (CH) is a common age-associated phenomenon whereby blood cells derived from a somatically mutated hematopoietic stem cell expand, in the absence of hematological disorders. CH is linked to an increased risk of hematological and non-hematological malignancies, including lung cancer. We recently identified tumor-infiltrating CH (TI-CH) as a promoter of non-small cell lung cancer (NSCLC) progression, but its temporal dynamics and effects on the tumor microenvironment remain undefined. To address this gap, we integrated whole exome sequencing and longitudinal cell-free DNA sequencing with single-cell multimodal analyses to characterize CH dynamics in NSCLC patients. Using pre-surgical blood samples from over 800 patients in the TRACERx cohort, we detected CH-associated mutations in 32% of patients through whole exome sequencing, most frequently in DNMT3A and TET2. Of these patients, 46% had TI-CH. Patients with TI-CH exhibited reduced disease-free survival compared to patients without CH (HR = 1.53, 95% CI = 1.20-1.95, p = 0.0006) and to patients with blood-only CH (HR = 1.41, 95% CI = 1.04-1.90, p = 0.03). In 424 patients with longitudinal cell-free DNA from blood plasma samples, we tracked single-nucleotide variants (SNV) across a median of six timepoints per patient using patient-specific liquid biopsy panels. 38 SNV-CH mutations in 35 patients were covered through variant calling. Mixed-effects modeling revealed a modest but significant global expansion of CH clones (Estimate = 0.00022, p = 0.027), with ASXL1 and TET2 mutations showing the strongest positive trends. Adjuvant therapy modestly attenuated clone growth. To extend our analysis to molecular mechanisms, we used two single-cell multimodal frameworks to gain a detailed view of lung tumor microenvironments in the presence and absence of TI-CH. We examined lung tumor immune infiltrates from two early-stage NSCLC patients with mutations in TET2 using TARGET-seq, integrating gene expression and high sensitivity genotyping at single-cell resolution. Additionally, in six patients (3 with TI-CH and 3 without CH) we employed DOGMA-seq to profile gene regulation in single cells across three distinct modalities, including chromatin accessibility, mRNA levels and cell surface proteins. While differences between mutated and non-mutated cells were nuanced, genes involved in inflammasome activation and regulation were upregulated in TET2Mut mononucleated phagocytes compared to those that were TET2WT. Collectively, these data show that CH clones expand over time in NSCLC and reshape the tumor immune microenvironment toward an inflammatory state that may potentiate disease progression. Citation Format: Aino-Maija Leppä, Oriol Pich, James R. Black, Maria Zagorulya, Elsa Bernard, Sophia Ward, Andrew Rowan, Hubert Slawinski, Lydia Y. Liu, Charlotte Grieco, Ramy Slama, Carlos Martinez Ruiz, Ariana Huebner, Takahiro Karasaki, Cristina Naceur-Lombardelli, Selvaraju Veeriah, TRACERx Consortium, Mariam Jamal-Hanjani, Alexandar M. Frankell, Adam Mead, Charles Swanton. The temporal dynamics and molecular impact of clonal hematopoiesis in non-small cell lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6821.
Leppä et al. (Fri,) studied this question.