Role of SV40 in childhood Lymphomas: Detection by real time quantitative TaqMan assay
Centre for Women's and Children's Health, Clinic and Polyclinic for Paediatric Haematology and Oncology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, Hamburg, Germany
AIM: Simian virus 40 (SV40) is a DNA virus of monkey origin, known to posess strong oncogenic potential, based on the large T-antigen (TAG). Many independent studies have detected SV40 in a variety of human malignancies, so in lymphomas, but with significant discrepancies in its frequency. Differences in DNA quality, as well as differences in virus detection methods hamper a controlled and standardised analysis. Our aim was to establish a RQ-PCR based TaqMan assay for rapid and highly reproducible detection and quantification of SV40 and to use this method for analysing DNA samples from childhood lymphoproliferative disorders and from healthy people giving new facts for ongoing discussion of the role SV40 could play in human malignancies. METHODS: Genomic DNA was extracted from fresh or frozen cells using the Quiagen spin column method. RQ-PCR primer were selected for the highly conserved region of the retinoblastoma pocket binding domain of TAG. The internal TaqMan probe was labelled with FAM at the 5`end and TAMRA at the 3`end. Quantity and quality of the DNA was tested with ß-globin gene. Only samples with CT values of 20 +/- 1 cycle were selected. RQ-PCR was performed in 20µl reactions with 500ng template DNA, 1 U Taq-Polymerase, and annealing temperature of 60oC. DNA was serial diluted (10-1 to 10-6) and tested double or triple. Positive samples were analysed additionally on agarosegels, and sequenced by using the Abi Prism BigDye terminator cycle sequencing kit. RESULTS: We investigated 26 DNA samples from childhood disorders (14 B-ALL, 7 BCP-ALL, 5 T-ALL). Twenty of them showed SV40 positivity down to 10-4 fold dilution. Two cell lines, SV80 (episomal SV40) and COS-7 (with one integrated SV40 copy) served as positive controls. Additionally 149 DNA samples derived from Buffy coat of healthy people were tested for SV40: 2 out of 149 were SV40 positive. Here we present a RQ-PCR based assay for standardised detection and quantification of SV40 TAG sequences which can easily be expand to other regions of the SV40 genome. The use of 500ng template DNA in each reaction and the exact quantification as well as the routine amplification of a control gene allows the sensitive detection of SV40 sequences in a wide range (4 log). CONCLUSIONS: In our investigation we present with 76,9% a very high SV40 positivity, which is much higher than with 43% the highest SV40 incidence found by other investigators in lymphomas from adults. In Germany the SV40 incidence in healthy people with 1,3% is very low. Although we analysed up to now only a small number of samples, the high positivity in our investigation powerful supports SV40 to be connected with childhood lymphomagenesis. Therefore our data give tool for further discussion on the putative role of SV40 in human malignancies.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 791 (Viral Infections).