Abstract LB314: Whole genome methylation sequencingstrongly suggests a novel, non-stem cell origin for some lung adenocarcinomas

Abstract Introduction: Lung adenocarcinoma (LuAd) is thought to arise from the type 2 alveolar epithelial (AT2) cell, the stem cell of the alveolus of the lung. However, we recently found in mice that driving oncogenic KRAS in the non-stem cell of the lung alveolus, the type 1 alveolar epithelial (AT1) cell, drove it to take on an AT2 phenotype and go on to form indolent tumors that modeled lepidic LuAd in humans. Given that it is perhaps the most stable epigenetic change, CpG methylation may be the best method to determine the lineage of human tumors. Using whole genome methylation sequencing, we validated this approach in a genetic mouse model with defined AT1 and AT2 origin tumors and then applied it to human non-small cell lung cancer (NSCLC) resection specimens. Methods: Fluorescence assisted cell sorting was used to purify AT1, AT2, and tumor cells from genetically modified mice and from human NSCLC resection specimens. DNA was converted for methylation sequencing using an enzymatic approach. Libraries were prepared and then sequenced to a target depth of approximately 5X for mouse specimens and 30X for human specimens. Results: AT1-derived tumor cell populations in mice had a significantly higher degree of AT1-specific methylation than AT2-derived tumors. Twelve LuAd, three squamous cell lung cancer (LuSC), four normal AT1 populations, and four normal AT2 populations were collected from specimens from the Stanford University Hospital operating rooms. In the LuAd specimens, EGFR was the most commonly mutated driver with KRAS, KDR, P53, BRAF, BRCA2, MET, RB1, ARID1A, KEAP1, NF1, and STK11 also represented. Driver mutations and abnormal copy number were verified in all LuAd populations, and purity was determined to be up to ∼99%. Comparison of the methylation profile of the tumors to an existing methylation atlas that included profiles of alveolar, bronchial, and upper airway epithelial cells suggested a basal cell origin for LuSc and an alveolar origin for LuAd. One LuSC and three LuAd had such extensive methylation derangement that they lost a methylation signature of any particular tissue origin. We then defined 1808 differentially methylated regions in AT1 versus AT2 cells. Of the remaining 9 LuAd that retained their lung methylation identity, 8 appeared to be of AT1 cell origin and 1 of AT2 cell origin. Higher AT1 methylation score correlated with lower clinical grade, more lepidic and acinar histology, and more ground glass appearance on CT scan. Conclusion: Whole genome methylation sequencing strongly suggests that human LuAd is derived from alveolar epithelial cells, with some tumors being derived from AT1 cells, the non-stem cell of the alveolus. The presence of non-stem cell methylation correlated with favorable clinical indices. Of note, our analysis includes only resectable LuAd, potentially biasing towards indolent tumors more likely to be derived from AT1 cells. Citation Format: Nicholas Juul, Diego Almanza, Maximilian Diehn, Tushar Desai. Whole genome methylation sequencingstrongly suggests a novel, non-stem cell origin for some lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB314.