Clonal integration of a polyomavirus in human Merkel cell carcinoma

Abstract

Merkel cell carcinoma (MCC) is a rare but aggressive human skin cancer that typically affects elderly and immunosuppressed individuals, a feature suggestive of an infectious origin. We studied MCC samples by digital transcriptome subtraction and detected a fusion transcript between a previously undescribed virus T antigen and a human receptor tyrosine phosphatase. Further investigation led to identification and sequence analysis of the 5387-base-pair genome of a previously unknown polyomavirus that we call Merkel cell polyomavirus (MCV or MCPyV). MCV sequences were detected in 8 of 10 (80%) MCC tumors but only 5 of 59 (8%) control tissues from various body sites and 4 of 25 (16%) control skin tissues. In six of eight MCV-positive MCCs, viral DNA was integrated within the tumor genome in a clonal pattern, suggesting that MCV infection and integration preceded clonal expansion of the tumor cells. Thus, MCV may be a contributing factor in the pathogenesis of MCC.

Fig. 1

(A) MCC is an aggressive skin cancer derived from Merkel mechanoreceptor cells that expresses neuroendocrine and perinuclear cytokeratin 20 markers, distinguishing it from other small round cell tumors (MCC349, left, hematoxylin and eosin; right, cytokeratin 20 staining, 40×. Scale bar represents 10 μm). (B) Discovery of Merkel cell polyomavirus transcripts in (MCC). 3′-RACE mapping of an MCC fusion transcript between the MCV T antigen and human PTPRG. A cDNA corresponding to a polyomavirus-like T antigen transcript was found by DTS analysis of MCC. This T antigen cDNA was extended by 3′-RACE to map three mRNA sequences (arrows), one of which terminates at a viral polyadenylation site and two of which extend into flanking human sequence and terminate in intron 1 of the human PTPRG gene on chromosome 3p14, indicative of viral DNA integration into the tumor cell genome. The two viral-human chimeric transcripts were generated by read-through of a weak polyadenylation signal in the viral T antigen gene. Identical RACE products were also sequenced from a lymph node metastasis of this primary tumor.

Fig. 2

(A) Schematic of MCV genome. Genome walking was used to clone the full MCV genome from tumor MCC350. The genome encodes typical features of a polyomavirus, including large T (purple) and small T (blue) open reading frames. Also shown are predicted VP1 (green) and overlapping VP2 (orange) and VP3 (yellow) genes. DTS1 and DTS2 (red) represent cDNA fragments originally identified by DTS screening. The former was used to identify MCV, and the latter is a spliced transcript with no homology to known polyomavirus sequences. (B) Neighbor-joining trees for putative MCV large T, small T, VP1, and VP2 proteins. The four known human polyomaviruses (BKV, JCV, KIV, and WUV) cluster together in the SV40 subgroup (blue), whereas MCV is most closely related to MuPyV subgroup viruses (red). Both subgroups are distinct from the avian polyomavirus subgroup (orange). Scale bars indicate an evolutionary distance of 0.1 amino acid substitutions per position in the sequence.

Fig. 3

Clonal MCV integration in MCC tumors detected by direct Southern hybridization. (A) DNA digested with BamHI (left) or EcoRI (right) and Southern-blotted with MCV DNA probes reveals different banding patterns in each tumor, including >5.4-kb bands. Open arrowhead shows the expected position for MCV episomal or concatenated-integrated genome (5.4 kb) with corresponding bands present in tumors MCC344 and 350. Tumors MCC339, 345, 347, 348, and 349 have different band sizes and doublet bands (solid arrowheads), consistent with genomic monoclonal integration. MCC352 has a prominent 5.4-kb band as well as higher and lower molecular weight monoclonal integration bands (BamHI), consistent with an integrated concatemer. Tumors MCC337, 343, and 346 have no MCV DNA detected by Southern blotting [bands at 1.5 kb (kidney) and 1.2 kb (MCC346) are artifacts]. (B) Viral and cellular monoclonality in MCC347 and 348. Tumor MCC347 and its metastasis MCC348 were digested with SacI and NheI and Southern-blotted with unique human flanking sequence probe [Chr3 (red), left] or viral probes [LT1 and LT2 (yellow), right]. The wild-type human allele is present in all samples at 3.1 kb (left). The MCC tumors, however, have an additional 3.9-kb allelic band formed by MCV DNA insertion into chromosome 3p14. Hybridization with probes for MCV T antigen sequence (yellow, right) generates an identical band.