Background: Lung adenocarcinoma frequently presents with EGFR mutations, often progressing on EGFR tyrosine kinase inhibitors (TKls) despite an initial response. Progression is frequently driven by additional genetic changes, including mutations in tumor suppressor genes (TSGs). Understanding the role of these concurrent TSG mutations can help elucidate resistance mechanisms and guide the development of more effective treatment approaches.

Materials and methods: We examined survival outcomes in 483 EGFR-mutant (mEGFR) patients from the GENIE BPC non-small-cell lung cancer (SCLC) dataset. To understand the mutational landscape and clonal dynamics, whole exome sequencing (WES) was carried out on 48 tumor samples from 16 mEGFR patients at both baseline and post-relapse.

A comprehensive gene panel was applied to 200 liquid biopsy samples obtained longitudinally from 25 patients to track clonal evolution.

Results: mEGFR patients with co-occurring TSG mutations exhibited significantly worse outcomes. In the GENIE dataset, overall survival (OS) was shorter [51.11 versus 99.3 months; hazard ratio (HR) 1.8, confidence interval (CI) 1.22-2.75, P = 0.003] and progression-free survival (PFS) was reduced (9.83 versus 11.48 months; HR 1.4, CI 1.03-1.91, P=0.026). WES analysis revealed 17 TSG mutations that were retained and showed clonal enrichment, particularly in early relapse (progression within 10 months of TKI initiation) or intermediate-stage relapse (relapse occurred between 10 and 20 months), indicated by increased variant allele frequency and their presence was strongly linked to early relapse. Longitudinal clonal studies further confirmed that TSG mutations co-occurring with mEGFR were often truncal, predominantly in early relapsers. Survival analysis using this subset of 17 TSGs showed significantly

shorter OS (55.26 versus 99.3 months; HR 1.7, CI 1.12-2.65, P = 0.011) and PFS (9.67 versus 13.12 months; HR 1.5,

CI 1.08-2.10, P = 0.013).

Conclusions: A set of 17 co-occurring TSG mutations has been identified as key biomarkers for early relapse in mEGFR lung adenocarcinoma. Longitudinal genomic monitoring, with a focus on clonal evolution, offers valuable insights that can inform personalized treatment strategies and potentially improve patient outcomes.

Key words: lung adenocarcinoma, tyrosine kinase inhibitor, whole exome sequencing, comprehensive gene panel, tumor suppressor genes