Tumor evolutionary trajectories during the acquisition of invasiveness in early stage lung adenocarcinoma

Lung adenocarcinoma (LUAD) is a leading cause of cancer death globally. Early-stage LUAD prognosis is heterogeneous, with invasive status significantly impacting outcome. While surgical resection offers high survival rates for pre-invasive stages (atypical adenomatous hyperplasia (AAH) and adenocarcinoma in situ (AIS)), early invasive LUAD has a worse prognosis and recurrence rate. Minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma (IAC) are important prognostic indicators. However, the evolutionary patterns from pre-invasive to invasive LUAD remain unclear. Previous studies have shown genetic differences between AAH, AIS, and LUAD, but not within single MPNs. EGFR and KRAS are frequent driver mutations in LUAD, with EGFR mutations often associated with better prognosis in early stages. This study aimed to delineate driver molecular events and early invasive progression in MPNs, combining micro-dissection and genomic sequencing of 53 stage I LUAD cases with data from 496 T1 stage patients.

The literature extensively covers the genetic landscape of LUAD, highlighting the roles of EGFR and KRAS mutations. Studies have demonstrated the prognostic significance of invasive status in early-stage LUAD. However, a comprehensive understanding of the evolutionary trajectories from pre-invasive lesions to invasive disease within individual nodules has been lacking. This study bridges this gap by investigating the genomic heterogeneity within individual malignant pulmonary nodules.

Fifty-three stage I LUAD cases with MPNs were selected for micro-dissection and genomic sequencing. Paired pre-invasive and invasive components were separated, and panel-genome sequencing was performed using two different panels (phase 1 and 2). Phylogenetic analysis was used to reconstruct the evolutionary relationships between the pre-invasive and invasive components within each MPN. The study also included data from 496 T1 stage patients from the Boston Lung Cancer Study (BLCS) for survival analysis and validation. Specific methodologies included whole exome sequencing, targeted sequencing, variant calling, copy number variation analysis, and phylogenetic tree construction. Immunohistochemistry (IHC) was used to assess immune cell infiltration. Statistical analysis included Kaplan-Meier curves, log-rank test, and Fisher's exact test.

Three evolutionary trajectories were identified: EM1 (no shared driver mutations between components), EM2 (shared driver mutations with private variants in invasive components), and EM3 (shared driver mutations with private variants in pre-invasive components). EGFR, TP53, KRAS, and STK11 were recurrently mutated driver genes. EGFR mutations were frequently found in truncal mutations, but their abundance significantly decreased in the invasive component compared to the pre-invasive component, suggesting a strong selective pressure. This decrease was associated with increased B cell infiltration in the invasive component. Survival analysis showed a better prognosis for EGFR-mutated patients compared to KRAS/STK11-mutated patients. The study also analyzed circulating cell-free DNA (cfDNA) and found no EGFR mutations in these samples.

This study provides novel insights into the evolutionary dynamics of early-stage LUAD. The identification of three distinct evolutionary trajectories reveals the complexity of the progression from pre-invasive to invasive disease. The finding that EGFR mutations are associated with a decrease in abundance during invasiveness and increased B cell infiltration is intriguing and suggests that interactions between tumor cells and the immune microenvironment play a crucial role in the selection and evolution of clones. The improved prognosis associated with EGFR mutations highlights the importance of targeted therapies.

This study demonstrates the complexity of early-stage LUAD evolution with three distinct trajectories. The observed strong selective pressure on EGFR-mutated clones during invasiveness, potentially driven by B cell infiltration, provides new understanding of tumor-immune interactions. Further research should investigate the role of B cells in this process and explore potential therapeutic implications.

The study is limited by its sample size and the use of different sequencing panels in the two phases, which might influence the detection of certain mutations. The focus on specific driver mutations might not capture the full complexity of LUAD evolution. The study's findings need to be validated in larger, independent cohorts.