Retinoblastoma gene mutations detected by whole exome sequencing of Merkel cell carcinoma

Abstract

Merkel cell carcinoma is a highly aggressive cutaneous neuroendocrine tumor that has been associated with Merkel cell polyomavirus in up to 80% of cases. Merkel cell polyomavirus is believed to influence pathogenesis, at least in part, through expression of the large T antigen, which includes a retinoblastoma protein-binding domain. However, there appears to be significant clinical and morphological overlap between polyomaviruspositive and polyomavirus-negative Merkel cell carcinoma cases. Although much of the recent focus of Merkel cell carcinoma pathogenesis has been on polyomavirus, the pathogenesis of polyomavirus-negative cases is still poorly understood. We hypothesized that there are underlying human somatic mutations that unify Merkel cell carcinoma pathogenesis across polyomavirus status, and to investigate we performed whole exome sequencing on five polyomavirus-positive cases and three polyomavirus-negative cases. We found that there were no significant differences in the overall number of single-nucleotide variations, copy number variations, insertion/deletions, and chromosomal rearrangements when comparing polyomavirus-positive to polyomavirus-negative cases. However, we did find that the retinoblastoma pathway genes harbored a high number of mutations in Merkel cell carcinoma. Furthermore, the retinoblastoma gene (RB1) was found to have nonsense truncating protein mutations in all three polyomavirus-negative cases; no such mutations were found in the polyomavirus-positive cases. In all eight cases, the retinoblastoma pathway dysregulation was confirmed by immunohistochemistry. Although polyomavirus-positive Merkel cell carcinoma is believed to undergo retinoblastoma dysregulation through viral large T antigen expression, our findings demonstrate that somatic mutations in polyomavirus-negative Merkel cell carcinoma lead to retinoblastoma dysregulation through an alternative pathway. This novel finding suggests that the retinoblastoma pathway dysregulation leads to an overlapping Merkel cell carcinoma phenotype and that oncogenesis occurs through either a polyomavirusdependent (viral large T antigen expression) or polyomavirus-independent (host somatic mutation) mechanism. Modern Pathology (2014) 27, 1073-1087; doi:10.1038/modpathol.2013.235; published online 10 January 2014Keywords: Merkel cell carcinoma; retinoblastoma; whole exome sequencing; polyomavirus Merkel cell carcinoma is a rare neuroendocrine tumor of the skin with an aggressive clinical course and an increased prevalence in the elderly and immunosuppressed. 1 The incidence of Merkel cell carcinoma has increased in the last several decades, and the United States has an estimated incidence rate of 0.32 per 100 000 persons per year. 2 Merkel cell carcinoma has a predilection for sun exposed areas, most often occurring in the head and neck region. 3 There is an overall 5-year survival rate of 40%, with stage being a significant prognosticator. 3,4 Merkel cell polyomavirus was discovered in Merkel cell carcinoma and found to be clonally integ...

Age and Gender Associations of Virus Positivity in Merkel Cell Carcinoma Characterized Using a Novel RNA In Situ Hybridization Assay

“…MCPyV is a DNA virus that likely mediates tumorigenesis via large T antigen (LTAg) binding to the tumor suppressor RB1 and small T antigen (sTAg) upregulation of oncoprotein stability and mTOR activation (1,2). Unlike MCPyV-positive tumors, MCPyV-negative MCC tumors display high mutation burdens, TP53 and RB1 mutations, and UV-signature mutational profiles, suggesting a molecular dichotomy between MCPyV-positive and MCPyV-negative tumors that may have translational relevance (4)(5)(6)(7).…”

Cytokeratin 20-negative Merkel cell carcinoma is infrequently associated with the Merkel cell polyomavirus

“…31 Merkel cell carcinoma with squamous elements or coexisting squamous cell carcinoma is often Merkel cell polyomavirus negative. 12,23,25,[33][34][35][36] Recent findings indicate that Merkel cell polyomavirusnegative tumors may be associated with RB1 inactivating mutations 37 and (in a subset) PIK3CA activating mutations. 38 Hence, although additional study is needed to clarify differences between Merkel cell polyomavirus-positive and Merkel cell polyomavirus-negative tumors, current data suggest that molecular differences exist that may have implications for immune-based or targeted therapy.…”

Next-generation sequencing of salivary high-grade neuroendocrine carcinomas identifies alterations in RB1 and the mTOR pathway

“…Within virus-positive tumors, several groups have demonstrated non-recurrent clonal integration of the virus into the host genome, where a mutated viral large T antigen (LTA) is expressed that binds to and induces a conformational change in RB1 interfering with its cell cycle regulation (Angermeyer et al, 2013;Chang and Moore, 2012;Cheng et al, 2013;Demetriou et al, 2012;Houben et al, 2012Houben et al, , 2010Sastre-Garau et al, 2009;Shuda et al, 2008;Sihto et al, 2011). Recently, Cimino et al (2014) demonstrated by whole exome sequencing in a small cohort of 3 cases in our laboratory that MCPyV-negative Merkel cell carcinomas have protein truncating mutations in RB1 (Cimino et al, 2014).…”

“…One distinguishing feature, however, is that while up to 80% of Merkel cell carcinomas harbor the Merkel cell polyomavirus (MCPyV), the virus appears to be absent or rare in salivary high-grade NECs, including those of the 'Merkel cell type' (Chernock et al, 2011;de Biase et al, 2012;Duncavage et al, 2009;Ellis and Auclair, 2008;Feng et al, 2008). Retinoblastoma protein 1 (RB1) inactivation appears to be important in the pathogenesis of both MCPyV-positive and MCPyV-negative Merkel cell carcinomas (Cimino et al, 2014). Within virus-positive tumors, several groups have demonstrated non-recurrent clonal integration of the virus into the host genome, where a mutated viral large T antigen (LTA) is expressed that binds to and induces a conformational change in RB1 interfering with its cell cycle regulation (Angermeyer et al, 2013;Chang and Moore, 2012;Cheng et al, 2013;Demetriou et al, 2012;Houben et al, 2012Houben et al, , 2010Sastre-Garau et al, 2009;Shuda et al, 2008;Sihto et al, 2011).…”