Background: Advanced squamous cell anal cancer (aSCAC) is a rare and aggressive disease, accounting for poor prognosis and high morbidity. No targeted therapies are currently available and, after the first line, no standard treatments are approved. Immune checkpoint inhibitors (ICI) showed signs of activity in previous phase I/II trials, but predictive and prognostic biomarkers are lacking. Anti-EGFR have been tested given the rarity of KRAS mutations in aSCAC, with encouraging results. Earlier preclinical evidence suggests possible synergism between cetuximab (cet) and ICI. Methods: In the phase II randomized trial CARACAS (NCT03944252), we tested avelumab (ave) alone (Arm A) or with cet (Arm B) in pretreated aSCAC; overall response rate (ORR) was the primary endpoint. With one-sided a error set at 0.05 and power of 80%, at least 4 responses out of 27 patients (pts) per arm had to be observed to declare the study positive. On pre-treatment tumor tissue samples, we assessed HPV status, PD-L1 expression, microsatellite status, tumor mutational burden (TMB) and performed next generation sequencing (NGS) via FoundationOne CDx. Primary objective was to describe the clinical outcomes of ICI in SCAC in the CARACAS trial population according to molecular analyses. Secondary objectives were to assess progression-free survival (PFS) and overall survival (OS) according to molecular characteristics to individuate new prognostic biomarkers in SCAC. Cox regression was used to investigate the effect of the main variables analysed on survival. Translational analyses were performed on the 100% of the study population since all the pts received ICI. Results: In the clinical trial, the Arm B reached the primary endpoint (ORR 17%, 95% CI 5·6-34·7). High TMB (≥10 mutations per megabase) was related with longer OS (HR=0.09; 95% CI 0.01-0.68; p=0.019), showing the same trend in PFS (HR=0.44; 95%CI=0.15-1.27; p=0.129). As well, tumors with high expression of PD-L1 (>40 measured with combined positive score, CPS) showed significantly longer OS (HR=2.19; 95% CI=0.92-5.19; p=0.075) and PFS (HR=2.35; 95%CI=1.09-5.1; p=0.03). High TILs (>1.2) did not affect significantly OS (HR=0.77; 95% CI=0.42-1.4; p=0.39) nor PFS (HR=1.19; 95%CI=0.57-2.48; p=0.645). When combined together and with high TILs, high TMB and PD-L1identified pts with significantly better prognosis in OS (HR=0.43; 95% CI=0.21-0.87; p=0.019) and PFS (HR=0.48; 95%CI=0.23-1.00; p=0.051). Remarkable responses were also observed in pts with high PD-L1 expression and TMB. Conclusions: TranslaCARACAS study documented prognostic role of high TMB and PD-L1 in mSCAC treated with ICI with or without anti-EGFR. Stratifying per high TMB, PD-L1 and TILs, a subgroup of pts with particularly favorable prognosis and deep responses were detected. Further investigation in larger cohorts is warranted to confirm our findings.