Mutational profile of oropharyngeal cancer in relation to HPV, tobacco smoking and prognosis with validation in the DAHANCA 19 randomized trial.

Introduction<br/>This study aimed to identify prognostic biomarkers in oropharyngeal squamous cell carcinoma (OPSCC). Additionally, we investigated potential molecular markers of tobacco exposure, as smoking has been hypothesized to induce specific genetic alterations and contribute to the poorer prognosis observed in smokers.<br/>First, we characterized the molecular profiles of OPSCC patients, focusing on HPV-induced tumors and their correlation with smoking history. Subsequently, we sought to independently validate our findings and assess the prognostic significance of molecular alterations in patients treated with curative (chemo-)radiotherapy in a randomized controlled trial (RCT).<br/>Materials and methods<br/>The exploration cohort included 56 patients with previously untreated OPSCC. We analyzed HPV status and the expression of genes related to hypoxia, tumor subtype, and radiosensitivity, alongside next-generation sequencing (NGS) of a broad panel of cancer-related genes. Based on these findings, we designed a custom NGS panel, to validate our findings and explore effect on prognosis in 162 OPSCC patients from the DAHANCA 19 RCT.<br/>Results<br/>In the exploration cohort of 56 patients (40 HPV+, 79%), the most frequent molecular alterations in HPV+ tumors were mutations or alterations in the PIK3CA and ATR genes, along with amplification of chromosome 3q. Mutations in ATR and CREBBP were more frequent in patients with more than 10 pack-years. However, these findings could not be reproduced in the DAHANCA 19 cohort. No distinct mutational signature of tobacco exposure was identified, and no association with TP53 mutations was observed.<br/>In the DAHANCA 19 cohort there were 17 locoregional failures (LRF) in 128 patients with p16+ tumors, corresponding to a cumulative incidence at five years of 11% (95% CI: 6 - 16%). Overall survival at five years was 88% (95% CI: 81-93).<br/>No common defining features were identified among these patients, although mutations in NFE2L2 and CASP8 as well as amplifications in genes located at 3q (BCL6, SOX2) were associated with LRF.<br/>Conclusions<br/>In HPV+ OPSCC, no mutations or molecular alterations that were prognostic in a high-quality cohort, and very few seem to be molecular drivers, or suitable as prognostic biomarkers. We identified no molecular features of tobacco exposure, contrary to previously proposed mechanisms.<br/>On this basis, we propose to refute the hypothesis of molecular alterations driving the worse prognosis in smokers with HPV+ OPSCC.