Extensive molecular profiling of squamous cell anal carcinoma in a phase 2 trial population: Translational analyses of the “CARACAS” study

Background: Molecular characteristics of squamous cell anal carcinoma (SCAC) are poorly explored. Immune checkpoint inhibitors showed limited activity in phase I/II trials, but predictive and prognostic biomarkers are lacking. Patients and methods: In the phase II randomised trial CARACAS (NCT03944252), avelumab alone (Arm A) or with cetuximab (Arm B) was tested in pre-treated advanced SCAC , with overall response rate being the primary end-point. On pre-treatment tumour tissue samples, we assessed Human papillomavirus status, programmed-death ligand 1 (PD-L1) expression, mismatch repair proteins expression, tumour mutational burden (TMB) and comprehensive genomic profiling by FoundationOne CDx. Tumour-infiltrating lymphocytes were characterised on haematoxylin-eosine-stained samples. Primary objective was to describe response to immunotherapy in the CARACAS trial population according to molecular and histological characteristics. Secondary objectives were to assess progression-free survival (PFS) and overall survival (OS) according to molecular biomarkers. Results: High PD-L1 (>40 with combined positive score) was significantly more frequent in patients with disease control (p = 0.0109). High TMB (>10 mutations per megabase) was related to better OS (hazard ratio (HR) = 0.09; 95%confidence interval (CI) 0.01-0.68; p = 0.019) and PFS (HR = 0.44; 95%CI = 0.15-1.27; p = 0.129). High expression of PD-L1 conferred longer OS (HR = 0.46; 95%CI = 0.19-1.08; p = 0.075) and PFS (HR = 0.42; 95%CI = 0.20-0.92; p = 0.03). Neither OS (HR = 1.30; 95%CI = 0.72-2.36; p = 0.39) or PFS (HR = 1.31; 95%CI = 0.74-2.31; p = 0.357) was affected by high (>1.2) Tumour-infiltrating lymphocytes count. High TMB and PD-L1identified patients were with significantly better OS (HR = 0.33; 95%CI = 0.13-0.81; p = 0.015) and PFS (HR = 0.48; 95%CI = 0.23-1.00; p = 0.015). Conclusions: To our knowledge, TranslaCARACAS is the first study to document prognostic role of TMB and PD-L1 in advanced SCAC patients treated with immune checkpoint inhibitors.