Signatures of early plasticity in the histological transformation of lung cancer.

8042 Background: Tumor plasticity, especially histological transformation, is an emergent mechanism of therapeutic resistance. Lung adenocarcinoma (LUAD) can transform to small cell lung cancer (SCLC), a more aggressive tumor type of neuroendocrine (NE) histology, leading to EGFR inhibitor resistance and a more aggressive disease phenotype. TP53 and RB1 loss are enriched but insufficient for NE transformation, and it remains unknown what additional molecular changes underlie plasticity. We use single-cell RNA sequencing (scRNA-seq) to profile the intratumoral transcriptional heterogeneity of NE transformation across multiple time points. Methods: We performed 10X scRNA-seq in patient samples to create an atlas of NE plasticity (47 transformed and admixed LUAD-SCLC, 10 EGFR/RB1/TP53 mut LUAD, 17 de novo SCLC, 4 normal lung). Our pipeline performed ambient RNA removal, outlier filtering, normalization, and batch correction. Tumor cells from transformed and admixed LUAD-SCLC tumors were divided into the LUAD compartment prior to transformation (pre-transformed LUAD) and the transformed SCLC compartment based on acquisition of NE marker expression. We performed copy-number inference, pseudotime analysis, and estimation of differentiation potential to establish dynamic changes in phenotypic and subclonal architecture across longitudinal timepoints. Results: We captured 12,105 (9%) pre-transformed LUAD cells from transformed and admixed LUAD-SCLC tumors. Canonical squamous cell carcinoma genes (KRT5, KRT6A, TP63, SERPINB4) were found to be enriched in this pre-transformed LUAD compartment. By projecting a force-directed layout of cells from these samples, we modeled the transition between LUAD, LUSC, and SCLC cell types. Genetic pathways promoting angiogenesis, inflammatory signaling, epithelial-to-mesenchymal transition, and RAS/RAF signaling were highly overexpressed during LUAD-LUSC transformation. Furthermore, we show that macrophages from transforming LUAD-LUSC tumors exhibit a pro-fibrotic phenotype. CNV inference revealed distinct subclonal populations in pre-transformed LUAD tumors. Subclones with high CNV burden and high differentiation potential represented early plastic cells whose lineage could be traced to matched longitudinal post-transformation samples. Conclusions: There is remarkable intratumoral heterogeneity within transforming LUAD. Squamous transformation, mediated by early RAS/RAF signaling, may represent a precursor to SCLC transformation. Identifying early hallmarks of plasticity can help stratify patients at high risk for transformation and reveal therapeutic vulnerabilities.