Abstract
Abstract 4578
In recent years, several authors suggested an association between Merkel-cell carcinoma (MCC), a rare neuroendocrine carcinoma of the skin, and B chronic lymphocytic leukemia (B-CLL). This association is particularly interesting in light of the discovery that the DNA of a novel virus, named as Merkel-cell polyomavirus (MCPyV), was detected in the majority of MCC. Since some studies have reported conflicting results on the frequency and potential pathogenetic role of MCPyV in B-CLL, we planned this study to further investigate the occurence of MCPyV infection in B-CLL patients.
Peripheral blood mononuclear cells (PBMC) were obtained from a group of 50 B-CLL patients, including naïve patients (31 cases) and previously treated, and relapsed cases (19 cases). In each case the IGHV gene mutational status was 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.
Genomic DNA was extracted using the EZ1 DNA Tissue kit (Qiagen, Hilden, Germany), eluted in 100 μl TE buffer and stored at -20°C until analyzed. Quantitative PCR (Q-PCR) assay was performed to amplify the cellular b-globin gene to verify the suitability of the DNA sample, and as a reference gene to determine the human cell equivalents present in each DNA sample (Euro-RT Polyoma Panel kit; Eurospital, Trieste, Italy). Q-PCR for Tag sequences of MCPyV was performed according standard methods. The assay can reproducibly detect 1 copy/reaction for each target gene. EBV viral load was examined by commercial kit following the manufacture's instructions (Nanogen Advances Diagnostic, Torino, Italy).
While six out of 50 B-CLL patient samples investigated were positive for EBV, only one patient was positive for MCPyV. The clinical history of this patient is of interest: he had been treated with several courses of chemotherapy including FC-R, R-CNOP, FluCam and high dose methyl-prednisolone for recurrent generalized lymphadenopathy. Five years after the discovery of B-CLL, he had surgery for a moderately differentiated squamous cell carcinoma with infiltrated adjacent structures of the scalp, but before any treatment he died for pneumonia. At the time of death, the squamous cell carcinoma of the scalp and the tonsil as well as the circulating PBMC were positive for MCPyV, while previous samples were negative.
Although the high sensitivity of our Q-PCR assay might detect incidentally infected cells in tissues, we found an extremely low frequency of positivity for MCPyV DNA (4.0 %) in the group of B-CLL patients investigated in the present study. Our current data, showing a very low frequency of detection of MCPyV DNA in B-CLL patients, coupled with the delayed appearance of this virus in only one case of B-CLL, point against a direct involvement MCPyV in B-CLL pathogenesis. In fact, MCPyV DNA was detected in tonsils and in squamous cell carcinoma as well as in PBMC of one patient only after the development of severe immuno-suppression due to several courses of chemo-immunotherapy.
Disclosures:
No relevant conflicts of interest to declare.
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