Abstract 2126: Single-cell sequencing of early-stage lung adenocarcinomas reveals prominent intratumoral heterogeneity and epithelial plasticity programs

Decoding the complex molecular and cellular processes during lung adenocarcinoma (LUAD) development is needed to devise early intervention strategies. To comprehensively capture LUAD neoplastic heterogeneity and cellular plasticity, we performed single-cell RNA-sequencing (scRNA-seq) of 257,481 enriched epithelial cells (EPCAM+ sorting) from 16 early-stage LUADs, each with 3 matched normal lung (NL) samples at defined spatial proximities to the tumor (n=47). 29,076 LUAD-derived cells clustered by patient and harbored distinct gene expression features (e.g., oxidative stress response), signifying interpatient LUAD heterogeneity. We also identified, using whole exome sequencing (WES) of matching lung and germline control samples, recurrent oncogenic driver alterations (e.g., EGFR, TP53, KRAS). Transcriptomic features of malignant cells were shared between LUADs (e.g., loss of lineage-specific gene expression) or private such as those associated with driver mutation status (e.g., KRAS). Indeed, clusters of malignant cells were overall segregated based on driver mutations (e.g., KRAS, EGFR). Malignant cells from KRAS-mutant LUADs (KM-LUADs) had increased activation of NF-kB, estrogen and hypoxia signaling, comprising a unique gene module (GM) that correlated with a less differentiated state. We also found hallmark pathways (cholesterol metabolism, DNA replication, cell fate decision) specific to EGFR-mutant LUADs (EM-LUADs). Notably, cells from one EM-LUAD and its 3 multiregion NL tissues clustered closely and had activated pro-tumor lymphoid signatures (CD4 naïve, Treg). Mutation burden increased with tumor proximity and intriguingly, EGFR exon20 mutation was evident in the tumor (VAF = 0.29) and its most proximal NL (VAF = 0.05), signifying a mutational field effect. Copy number variations (CNVs) derived from WES of all samples were overall consistent with those inferred from scRNA-seq data. Relative to EM-LUADs, malignant cells from KM-LUADs displayed lower CNV burdens. Interpatient CNV heterogeneity was prominent even among LUADs harboring the same oncogenic drivers. Notably, intratumor heterogeneity (ITH) was high among epithelial cells within single regions from the same LUAD. Among LUADs, malignant cell clades with KRAS mutations and lower CNV scores displayed less differentiated states. To investigate biological pathways driving ITH, we derived 6 GMs with tumor-relevant functional features, including a transcription/translation regulation GM that consi