Detection of Merkel cell polyomavirus with a tumour-specific signature in non-small cell lung cancer

Abstract

Background: We searched for a viral aetiology for non-small cell lung cancer (NSCLC), focusing on Merkel cell polyomavirus (MCPyV).

Methods: We analysed 112 Japanese cases of NSCLC for the presence of the MCPyV genome and the expressions of RNA transcripts and MCPyV-encoded antigen. We also conducted the first analysis of the molecular features of MCPyV in lung cancers.

Results: PCR revealed that 9 out of 32 squamous cell carcinomas (SCCs), 9 out of 45 adenocarcinomas (ACs), 1 out of 32 large-cell carcinomas, and 1 out of 3 pleomorphic carcinomas were positive for MCPyV DNA. Some MCPyV DNA-positive cancers expressed large T antigen (LT) RNA transcripts. Immunohistochemistry showed that MCPyV LT antigen was expressed in the tumour cells. The viral integration sites were identified in one SCC and one AC. One had both episomal and integrated/truncated forms. The other carried an integrated MCPyV genome with frameshift mutations in the LT gene.

Conclusion: We have demonstrated the expression of a viral oncoprotein, the presence of integrated MCPyV, and a truncated LT gene with a preserved retinoblastoma tumour-suppressor protein-binding domain in NSCLCs. Although the viral prevalence was low, the tumour-specific molecular signatures support the possibility that MCPyV is partly associated with the pathogenesis of NSCLC in a subset of patients.

Figure 1

Representative results for the detection of MCPyV genomes by PCR. The LT1, LT3, and VP1 primers produced amplicons with sizes of 440, 309, and 352 bp, respectively. The DNA samples were subjected to amplification in parallel with the housekeeping gene β-globin (110 bp), which was detected in all samples. Lane 1: DNA extracted from tumours; lanes 2 and 3: DNAs extracted from two different sites in non-neoplastic tissues; lane N: water as the negative control; and lane P: positive control DNA from MCPyV-positive MCC tumour. Case numbers and molecular weight markers are shown on the left.

Figure 2

Expression of the LT and VP1 gene transcripts. DNase-treated RNAs were reverse transcribed and the cDNAs were PCR amplified with primers complementary to the regions corresponding to the LT or VP1 genes. All cDNAs were also subjected to amplification in parallel with the housekeeping gene β-globin, which was expressed at similar levels in all samples. Case numbers are indicated on the top. Molecular weight markers are shown on the left.

Figure 3

Immunohistochemical detection of the MCPyV LT antigen. (A) Haematoxylin and eosin staining of histological specimens containing tumour cells (original magnification × 200). (B) Immunohistochemical analysis with the CM2B4 monoclonal antibody, showing immunoreactivity in the tumour cells. Insets show higher magnification views of the tumour cells. (C) Immunohistochemical analysis with an isotype-matched negative control antibody (mouse IgG2b) for CM2B4, showing no immunoreactivity.

Figure 4

Amino-acid sequence alignment of the MCPyV LT antigen. The LT genes in the MCPyV-positive tumours were predicted to encode 817 amino-acid proteins. The amino-acid sequences were compared with reference sequences from the non-tumour-derived MCPyV isolate Appendix206 (GenBank accession number JN038578) and the MCC-tumour-derived isolate MCC350 (GenBank accession number EU375803). The position of the LxCxE motif, which is essential for Rb binding, is shown by the upper line. The psycho motif, which influences Rb activities, is represented with grey boxes. This motif, interrupted by amino-acids at positions 103–209, is shown in a white box with black lines. The arrows indicate the position of the helicase domain. The bold lines indicate the truncated regions of the LT antigen found in MCC350 and the MCPyV strain in tumour AC43. The numbers indicate the amino-acid positions.