Harnessing transcriptome signatures and CD103+CD8+ immune infiltration for prognosis and treatment outcomes in anal squamous cell carcinoma.

3524 Background: Anal squamous cell carcinoma (ASCC) is a rare malignancy linked to high-risk HPV, with rising incidence among younger adults. While immunotherapy advances have improved outcomes in metastatic ASCC, treatment for localized disease has remained unchanged for decades, with high recurrence rates. This study investigates molecular biomarkers and immune mechanisms predictive of chemoradiotherapy outcomes in non-metastatic ASCC. Methods: This retrospective study analyzed 94 stage I-III non-metastatic anal squamous cell carcinoma (ASCC) patients treated with curative chemoradiotherapy (CRT) at Hôpital Paris Saint Joseph (2010–2017) in France. Treatment response (CR) was assessed at 24 weeks by RECIST v1.1. Molecular analyses included whole-exome and RNA sequencing on FFPE samples to evaluate somatic mutations, tumor mutational burden (TMB), and gene expression profiles. Immunohistochemistry assessed immune markers (CD8, CD103). Statistical analyses identified predictors of CR, progression-free (DFS), and overall survival (OS). Results: Complete response (CR) was achieved in 71% of cases, with no significant differences between treatment regimens (p > 0.05). Mutational analysis identified 172 alterations in novel (SLAMF7 and GOLGA6L9) and previously described cancer driver genes (KMT2C, KMT2D, and PIK3CA), with higher mutational burdens showing a non-significant trend toward CR. Transcriptomic profiling revealed 350 differentially expressed genes among CR vs. NCR patients (p-value < 0.01; FC > 2). CR was associated with modulation of immune-related pathways, including TNFα/NFkB signaling (p < 0.01). Immune infiltrate analysis showed enrichment of CD8+ central memory T cells (p = 0.008) and CD4+ resting memory B cells (p = 0.01) in CR cases, correlating with improved OS (p = 0.0026) and DFS (p = 0.0098). CD103+CD8+ tumor-infiltrating lymphocytes emerged as the strongest predictor of survival (OS: p = 0.011; DFS: p = 0.003), underscoring their potential as prognostic biomarkers and therapeutic targets in ASCC. Conclusions: These findings underscore the potential of integrating molecular and immune markers into clinical practice to better predict treatment response and guide personalized therapies for CRT efficacy for ASCC patients. Further validation in independent cohorts is necessary to confirm the clinical relevance of these biomarkers and their application in therapeutic decision-making.