Reliable detection of Merkel cell polyomavirus in Merkel cell carcinoma by whole exome next-generation sequencing.

Abstract

e15071 Background: Studies indicate that Merkel cell polyomavirus (MCPyV) infection is a causative factor for the development of virus-positive Merkel cell carcinoma (VP MCC). This neuroendocrine neoplasm (NEN) is sometimes undiagnosed or misdiagnosed when MCPyV infection is not properly identified; accurate diagnosis is essential, as research suggests the utility of immunotherapy in the treatment of MCC compared to other NENs. Consequently, we seek to establish whole exome next-generation sequencing (WES) as a reliable method for finding MCPyV in tumors. Specifically, we propose a novel assay incorporating MCPyV detection into standard-of-care molecular cancer profiling, therefore ensuring proper diagnosis of VP MCC. Methods: WES was validated on DNA extracted from previously diagnosed VP MCC tumor samples submitted to Caris Life Sciences utilizing NovaSeq 6000 technology (Illumina, Inc.). A hybrid pull-down panel of sequencing baits was used to provide enhanced 1500x depth of coverage for 720 cancer-related genes and 500x coverage for the remaining exome. Given bait availability, the panel was also designed to cover the maximal possible amount of the MCPyV genome—82.4%. The presence of virus was suggested if ≥1,000 sequencing reads specific to MCPyV were detected. Orthogonal IHC testing using CM2B4 antibody (Santa Cruz Biotechnology, Inc.) was used to confirm the MCPyV infection status detected by WES. Clinical utility of the sequencing assay was demonstrated by testing primary and metastatic tumor specimens previously classified as NENs not otherwise specified, so as to identify potentially undiagnosed or misdiagnosed MCC. Results: We performed WES on 835 NEN specimens that were not previously diagnosed as MCC. MCPyV was detected in 10/835 (1.2%), suggesting that these cases might have been undiagnosed or misdiagnosed. To verify the validity of the sequencing results, we performed WES on 70 samples with a known diagnosis of MCC, and MCPyV was detected in 49 tumors. Conversely, orthogonal IHC testing identified virus in 45 of the 70 specimens. The presence of viral sequences was confirmed by the Basic Local Alignment Search Tool (BLAST, National Institutes of Health) in 49 of the cases, including the four tumors discordant by IHC, indicating that WES is more sensitive in detecting the virus (100% sensitivity [95% CI: 91.4-100%] and 84.0% specificity [95% CI: 63.9-95.5%]) compared to IHC. Conclusions: This study presents a robust method for identifying MCPyV in VP MCC utilizing standard-of-care cancer genomic profiling for precision oncology. By confirming the presence of viral sequences within neoplastic tissue specimens, we have demonstrated the superior sensitivity of WES in detecting MCPyV compared to IHC. Moreover, our sequencing assay can assist in properly classifying tumors, therefore ensuring that patients receive appropriate treatment based on the diagnosis of MCC.