Background: Avelumab, an FDA-approved human anti–PD-L1 IgG1 monoclonal antibody for patients (pts) with metastatic Merkel cell carcinoma, showed an objective response rate (ORR; by RECIST v1.1) of 31.8% in a second-line phase 2 trial (NCT02155647). We assessed the association of tumor mutational burden (TMB; nonsynonymous somatic variants/megabase), PD-L1 expression, Merkel cell polyomavirus (MCPyV) status, and CD8+ tumor-infiltrating T-cell density with ORR and survival. Molecular profiles (RNAseq and WEX) also were analyzed. Methods: Baseline tumors (n = 36) were profiled using RNAseq and WEX sequencing. PD-L1 expression (≥1% cutoff), MCPyV status, and CD8+ T-cell density at the tumor invasive margin were evaluated by IHC. MHC locus expression was measured with OptiType and loss of heterozygosity (LOH) with LOHHLA. Results: Of 36 pts profiled, 12 had a response, 27 were PD-L1+, and 23 were MCPyV+. The TMB upper tertile and quartile values were 1.34 and 3.16, respectively. Consistent with literature, MCPyV− pts had a higher median TMB (2.72) than MCPyV+ pts (0.49). PD-L1+ tumors trended toward a higher TMB. An empirical cohort-specific TMB cutoff of ≥2 was chosen to include sufficient pts per subgroup. Pts with TMB ≥2 vs TMB < 2 had higher ORR (5 of 11 [45.5%] vs 7 of 25 [28.0%]) and 6-mo PFS rates (60% vs 38%). Among pts with TMB ≥2, the highest ORRs were reported in MCPyV− (4 of 7 pts), PD-L1+ (5 of 9 pts), and CD8+ T-cell density higher than median (5 of 6 pts) subgroups. MHC expression trended with ORR and survival. Higher mean MHC expression was found in pts with CD8+ T-cell density higher than median (p < 0.05). Mutations in antigen presentation genes were detected: LOH at the HLA locus in 9 of 30 pts (28%), including 4 with a response; an NK cell activation signature was also associated with response. These data may suggest that ADCC contributes to response. Factorial analysis of gene signature scores identified signatures (eg, IFNγ, TP53 pathway) associated with MCPyV status and response. Conclusions: Responses in this data set were not attributed to any specific biomarker alone. Future analysis is focused on validating these results and identifying rational drug combinations with avelumab. Clinical trial information: NCT02155647