Merkel-cell polyomavirus and B-cell chronic lymphocytic leukemia

Introduction: Some authors described a possible association between CLL and Merkel cell polyomavirus (MCPyV) the pathogenetic agent of the Merkel-cell carcinoma, a rare and aggressive carcinoma of the skin. This association suggests the possibility that MCPyV may play a role in the pathogenesis of CLL as well as in the progression of the disease. Given several contradictory reports on the role of MCPyV in CLL, the presence of this virus as well of the other polyomaviruses JCV, BKV and of other lymphotropic viruses i.e. EBV and SV40 was investigated in highly purified malignant cells of a large number of CLL patients in different stages of the disease. Methods: Blood samples were obtained from 66 patients fulfilling diagnostic criteria for CLL. In each case the IGHV gene mutational status was previously assessed using standard methods and CD38 and ZAP-70 expressions were determined by flow cytometry. Interphase FISH was performed on nuclei preparations of PBMC and each case was investigated for 13q deletion, 11q deletion, 17p deletion or presence of trisomy 12. The samples were processed immediately after blood withdrawl and processed by Ficoll-Hypaque density gradient resulting in purity of > 98% of CD19+/CD5+ CLL cells as assessed by expression analysis on flow cytometry. DNA was extracted by using a commercial and stored at -80 C° until the time of analysis. A multiple Q-PCR assay was run to amplify both Polyomavirus JCV, BKV and MCPyV. Real time Q-PCR for MCPyV Tag sequences, viral loads and the cellular beta-globin gene (as reference gene) were performed in each sample. Infection of EBV was evaluated by specific quantitative real time PCR (Q-PCR) assay using a commercially available molecular kit (Nanogen, Italy). The lowest limit of EBV detection assays was 100 copies/reaction and for Polyomavirus 10 copies/reaction. Q-PCR assays were run on the AB PRISM 7900 Sequence detection System (Applied Biosystems, Milan, Italy). Multiple negative controls (containing water instead of DNA templates), and positive controls (containing plasmid with the entire viral genome) were included with each assay batch. Results: Among the 66 patients one case only (1.5%) was positive for MCPyV, while no one was positive for SV40, JCV and BKV. Five cases (7.6%) were positive for EBV replication in PBMC. The EBV positivity was independent from age, IGVH mutational status or from ZAP70 and CD38 expression or from the presence of 17p deletion, while correlates with previous treatments. The patient positive for MCPyV was affected by Merkel Cell skin carcinoma. This patient at diagnosis was negative for both MCPyV and EBV and underwent 4 therapy lines before positivity. Conclusions: The low frequency of detection of MCPyV DNA in B-CLL patient samples, points against a direct involvement of MCPyV in CLL pathogenesis. Likely, the immune-depression of CLL, exacerbated by chemio- and immune-therapies, results in reactivation of both EBV and MCPyV.