Distinct merkel cell polyomavirus molecular features in tumour and non tumour specimens from patients with merkel cell carcinoma

Abstract

Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell (34 vs 10 months, P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission (60% vs 11%, P = 0.00083). Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease.

Figure 1. Kaplan Meier analysis of survival in complete remission relative to primary tumour MCPyV load.

Patients with ≥1 genome copy per cell (n = 15, black curve) had longer survival in complete remission than patients with <1 genome copy per cell (n = 9, grey curve). VL : viral load. Open circles indicate patients censored at last follow-up visit.

Figure 2. Kaplan Meier analysis of overall survival relative to MCPyV detection in PBMC.

Patients who had all MCPyV-negative PBMC (n = 14, black curve) had a longer survival than patients who had at least one MCPyV-positive PBMC (n = 14, grey curve). Open circles indicate patients censored at last follow-up visit.

Figure 3. Characterization of MCC patients' strain and tumour signature in MCPyV LT.

A. Direct PCR sequencing of the second exon of MCPyV LT in MCC tumours. Twelve distinct representative cases (case number indicated on the left) are represented. Sequences from MCC (black), nasal swab (blue), urine (green) and PBMC (red) tissues reveal single nucleotide polymorphisms (SNPs), and tumour-specific mutations: open circle, synonymous; filled circle, non synonymous; black cross, premature stop codon; filled box, deletions. In cases 1 and 20, weak viral load in PBMC and nasal swab respectively prevented full length amplification of LT. Base pair designation as in Table 2. B. Diagram representation of main functional domains encoded by the second exon of LT (Rb  =  Rb binding domain, NLS  =  nuclear localization signal, OBD  =  origin binding domain and helicase domain). Arrows indicate premature truncating mutations determined by direct sequencing and arrowheads indicate the site of integrated viral DNA determined by 3′ DIPS-PCR. GenBank accession number: HM587427-HM587448.