Abstract Resolving the lineage history of cells is crucial for understanding tissue development, stem cell dynamics, and cancer, but is hampered by methods that are invasive, low-resolution, or prohibitively expensive for human studies. Clonal expansion of a single stem cell lineage is a hallmark of pre-cancer in both solid tissue tumors and hematological malignancies. Here, we describe a native, in vivo lineage tracing system that utilizes the stochastic (de)methylation of CpGs in the protocadherin (PCDH) gene cluster. By phasing these CpGs in bulk methylation sequencing we construct in situ evolvable methylation “barcodes” that uniquely mark stem cell clones. We longitudinally tracked these barcodes in annual blood samples from 50 individuals and age-matched controls up to a decade prior to acute myeloid leukemia (AML) diagnosis. We show that polyclonal tissues have high barcode diversity—reflecting a broad stem-cell pool—which then collapses during a clonal sweep. The fractional abundance of dominant barcodes provides a quantitative measure of clone size that precisely tracks genetic estimates from somatic mutations. However, 10% of pre-AML donors harbored expanded barcodes but lacked detectable somatic driver mutations, suggesting the presence of clonal expansions with cryptic drivers years before diagnosis. Barcode trajectories also reveal subclonal competition and interference. Because these barcodes evolve by stochastic (de)methylation at approximately 1% per CpG per year, heritable diversity is generated in clonal populations enabling the tracking and quantification of subclonal growth dynamics. We then demonstrate that barcodes containing dozens to hundreds of PCDH CpGs produced from long-read sequencing allow for deep phylogenetic reconstruction from a single time point. Finally, we observed expanded barcodes in solid tumor tissues, including prostate and kidney. PCDH methylation barcodes therefore represent a scalable, cost-effective, and pan-tissue tool for high-resolution lineage tracing in humans, opening new avenues for studying somatic evolution in health and disease. Citation Format: Christopher T. Boniface, Samuel Hackett, Adriana V. Fonseca, Akemi Ramos-Yamasaki, Caroline Watson, Joanna Baxter, Jyoti Nangalia, Sadik Esener, Hisham Mohammed, Jamie Blundell. In vivo clonal lineage tracing using methylation barcodes in the protocadherin gene cluster 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 696.
Boniface et al. (Fri,) studied this question.