Clonal origin analysis of secondary lesions reveals metastatic predominance and a role for FHL2 in head and neck squamous cell carcinomas

Patients with head and neck squamous cell carcinoma (HNSCC) often develop secondary squamous lesions in the esophagus or lungs, yet discerning whether such lesions represent metastases or independent second primary tumors (SPTs) remains a key diagnostic challenge. We aimed to map their clonal and immune landscapes and uncover mediators of dissemination. We analyzed 71 tumor samples from 20 patients with multifocal squamous cell carcinomas using whole-exome sequencing (WES) and multiplex immunofluorescence (mIF) to reconstruct clonal relationships and characterize immune heterogeneity. Complementary in vitro and in vivo experiments, alongside analyses of clinical specimens, were performed to investigate the role of tetramer half-LIM domain protein 2 (FHL2) in metastatic HNSCC and its impact on tumor progression. Clonal reconstruction revealed that most thoracic secondary squamous lesions share a common origin with primary head and neck tumors, indicating metastatic dissemination rather than independent second primaries. Focusing on metastatic progression, FHL2 was identified as enriched in metastatic clones and a potential driver gene. Mechanistically, FHL2 directly interacts with EGFR to enhance its phosphorylation, sustain PI3K-AKT signaling, and promote cytoplasmic retention of FOXO3, thereby suppressing its tumor-suppressive transcriptional program and driving tumor growth and dissemination. In a lung metastasis model, EGFR-targeted therapy (cetuximab) markedly reduced metastatic burden in tumors overexpressing FHL2. Clinical sample analysis further demonstrated that higher FHL2 expression showed a potential association with response to cetuximab in our cohort. Metastatic lesions exhibited reduced CD4 + and CD8 + T-cell infiltration together with altered spatial distributions of PD-L1 and CD138 + cells. In addition, high FHL2 expression was associated with lower CD4 + and CD8 + T-cell infiltration. This study provides initial genomic evidence supporting the molecular characterization of metastatic lesions and second primary tumors in head and neck-thoracic squamous carcinomas. The findings suggest that FHL2 may be implicated in metastatic progression and may represent an exploratory candidate biomarker associated with response to EGFR-targeted therapy.