Simian Virus 40 Is Present in Most United States Human Mesotheliomas, but It Is Rarely Present in Non-Hodgkin’s Lymphoma: Materials and Methods

1 Oct

In this study, we investigated non-Hodgkin’s human lymphomas obtained from nonimmunocompromised patients to determine if they contained SV40 DNA sequences. Because viruses may spread more efficiently and be more pathogenic in immunocompromised individuals, we also tested for SV40 lymphoproliferative disorders that had developed in immunocompromised patients.
Materials and Methods
DNA Detection
Frozen tumor samples were obtained from 29 non-Hodgkin’s lymphoma patients who were classified according to the Revised European-American Lymphoma classification: 26 large cell, B lineage; 2 large cell high-grade T-cell lymphoma, and 1 large cell B, Burkitt’s-like lymphoma. In the immunocompromised group, we studied 25 patients with posttransplant lymphoproliferative disorders and 5 AIDS patients with large cell lymphoma, B-cell origin. DNA was extracted and purified from frozen lymphoma or mesothelioma tumor tissue as previously described in detail. in detail
All of the DNA samples were tested for suitability of amplification with primers specific for a 268-base pair (bp) fragment of the P-globin gene; all DNA samples could be amplified. PCR reactions were performed using the hot-start technique as described. The GeneAmp PCR reagent kit containing Amplitaq polymerase (Perkin-Elmer Biosystems; Norwalk, CT) and the Ampliwax PCR gems (Perkin-Elmer Biosystems) were utilized for the analysis. DNA samples from tumors were amplified using the SV5 and SV6 set of primers which amplifies the retinoblastoma (RB)-pocket binding domain of SV40 tumor antigen (Tag), as previously described. The samples were further analyzed with primers R1/R2. This set of primers amplifies the regulatory region and the origin of replication of the virus. The total volume for each PCR reaction was 100 μL; the concentrations of MgCl2 and that of the primers were 2.5 mM and 0.5 μM, respectively (the concentration of each primer was calculated from the equation 1 OD = 20 μg/mL). One μg of DNA was used per each PCR. All other reagents were used according to the recommendations of the manufacturer. Negative controls were included in each PCR experiment to test for PCR contamination.