SV40 and p53 as team players in childhood lymphoproliferative disorders.

Simian virus 40 (SV40) is known to be potently oncogenic and can induce several types of tumours, such as lymphoma. p53 was discovered as a cellular partner of the SV40 large T-antigen, the oncoprotein of this virus. There have not been many studies on SV40 and p53 in lymphomas and the ones that exist, are controversial. A comparison of these two components in lymphoma has not been reported previously. We examined 91 lymphomas [60 B-cell non-Hodgkin's lymphomas (B-NHLs), 19 B-cell acute lymphoblastic leukemias (B-ALLs), 7 B-cell precursor acute lymphoblastic leukemias and 5 T-cell acute lymphoblastic leukemias] for the presence of SV40. Overall, 40 samples from 12 B-NHL/19 B-ALL patients were additionally investigated for p53 mutation in the hot-spot exons 5 to 8. Overall, we found 62/91 lymphomas to be SV40-positive, among them 16/19 B-ALLs and 38/60 B-NHLs. SV40 was absent in 147 of the 149 blood control samples. We found 11 p53 mutations in 19 B-ALL patients: 5 in exon 5 (codons 132, 141, 143, 155 and 181), 4 in exon 7 (codons 236, 238 and 248), 2 in exon 8 (codon 273). In B-NHL patients we found p53-mutations in 9/12 samples: 6 of these in 3 lymph nodes (LNs). One LN harboured 3 different p53 mutations: Exon 5 (codon 132), exon 6 (codon 213) and exon 8 (codon 288). Another LN showed 2 different p53 mutations: Exon 6 (codon 213) and exon 8 (codon 285). Except for 1 nonsense mutation in an LN of a B-NHL patient, all 20 mutations were missense mutations, 2 were homozygous, both found in B-NHL-samples, and one of these (codon 175) is known to cause the global denaturation of p53. All occur in the DNA-binding domain of p53. All specimens showing a p53 mutation, were SV40-positive. p53 mutaions found in LNs of B-NHL patients harbour high SV40 copy numbers. Our data strongly support an important role for SV40, as well as a strong association of SV40 and p53 in childhood lympho-proliferative disorders.

Evaluation of SV40 in osteosarcoma and healthy population: a Hungarian-German study.

Simian virus 40 (SV40) has been linked to human cancer, as has osteosarcoma (OS). Although significant discrepancies exist in the frequency, evidence that the virus plays a causative role in some malignancies is mounting. The large-T-antigen and significant SV40 oncoprotein, bind and inactivate the tumour suppressor genes p53 and pRb, which play a key role in osteosarcoma development. We analysed 277 OS samples from two different European countries (154 OS samples from Hungary and 123 from Germany). To ascertain the prevalence of SV40 in the general population, additional blood samples from healthy volunteers from the two countries (166 Hungarian and 149 German) were analysed. To reach an appropriate detection level, we investigated a real-time quantitative PCR-based assay for the detection and quantification of SV40 <10 copies in 500 ng of genomic DNA. We detected SV40 in 52% (143/277) of the OS samples analysed. In detail, we saw differences in the distribution of SV40 between the two countries. Of the Hungarian OS samples, 74% (114/154) showed high amounts of SV40 (>100 copies), whereas only 22% (29/123) of the German OS samples harbour small amounts of SV40 ( approximately 10 copies). SV40 was detected in 8 of 60 German tumour samples (14%) and 21 of 63 German blood samples (33%) from OS patients. In the peripheral blood of healthy volunteers we found only weak SV40 positivity (<10 copies) in the two countries with a somewhat higher frequency in Hungarian 30/166 (18%) than in German blood samples 2/149 (1.3%). No sequence variations were found in SV40-positive samples from the two countries. Our data demonstrate significant differences in the prevalence and quantity of SV40 in OS of these different European geographical origins with a lower frequency in the blood samples of healthy volunteers from the two countries, underlining again the geographical differences previously described by other investigators.

Determination of the prognostic value of loss of heterozygosity at the retinoblastoma gene in osteosarcoma.

The retinoblastoma (RB) tumour suppressor gene is implicated in the development of several malignancies including osteosarcoma. Recent studies postulated its loss of heterozygosity (LOH) to be a poor prognostic factor at diagnosis of osteosarcoma (OS). It remains unclear whether LOH of the RB gene is suitable as a prognostic factor at diagnosis in patients with osteosarcoma. In this study we aimed to determine the early prognostic value of RB-LOH as well as the ability of denaturating high performance liquid chromatography (DHPLC) to detect LOH at this gene locus in comparison to classical PAGE. We therefore analysed 41 samples of OS on restriction fragment length polymorphisms in introns 1, 17 and 25, and variable numbers of tandem repeats (VNTRs) in intron 20. PCR fragments were separated on 1.5% agarose gel electrophoresis. VNTRs with length differentiation of only a few base pairs were analysed by 8% PAA/Spreadex gels and additionally by DHPLC. One-hundred percent concordance was observed between the results obtained by classical PAGE and DHPLC. The latter improved intron 20 analysis as a sensitive and high throughput method for detecting LOH. Overall we found 16 RB-LOH in 41 OS (39%). Three tumours exhibited additional microsatellite instability. There was no significant correlation of the event-free- and overall-survival rate or response to chemotherapy with RB-LOH found in our study. LOH positivity was associated with a significantly younger age at diagnosis. In conclusion RB-LOH could not be verified as a poor prognostic factor for osteosarcoma in the present study.

Standardized detection of Simian virus 40 by real-time quantitative polymerase chain reaction in pediatric malignancies.

BACKGROUND AND OBJECTIVES: Recent studies have detected Simian virus 40 (SV40) DNA in specific human tumors albeit with significant discrepancies in frequency. Possible inefficiency of DNA isolation and different detection methods do not allow comparable and precise quantification of SV40 in human samples. A standardized and common detection method is, therefore, essential for further routine SV40 analysis. DESIGN AND METHODS: We established a real time quantitative (RQ-) polymerase chain reaction (PCR) based TaqMan assay on the LightCycler system for reproducible detection and quantification of SV40 DNA. We used 500 ng of the COS-1 cell line, containing one single integrated copy of SV40 DNA, as the quantification standard. Amplification of b-globin served as the quality control for DNA integrity. For the extraction of the episomal form of SV40 we compared a column and a precipitation-based DNA extraction method. DNA samples from 149 healthy controls, from 26 fresh frozen childhood cases of acute lymphoblastic leukemia (ALL) (B-, BCP- and T-ALL) and from 12 paraffin-embedded osteosarcomas were investigated. RESULTS: The RQ-PCR assay had a linear amplification rate from 10 to 100,000 copies of SV40 in 500 ng genomic DNA. Very low copy numbers of SV40 DNA were detectable in 2/149 (1,3%) blood samples from healthy German controls. Various amounts of SV40 were detectable in 20/26 (77%) childhood ALL samples of German origin and, in part, high amounts were visible in 11/12 (92%) paraffin embedded Hungarian osteosarcomas. The column-based DNA isolation method allowed the detection of both, the integrated and the episomal forms of SV40. INTERPRETATION AND CONCLUSIONS: Our assay provides a standardized and reproducible quantification of SV40 DNA in a wide spectrum of specimens. Exact quantification strongly depends on the source, as well as on the quality, of the DNA used. Quantification of paraffin-embedded DNA generally leads to lower sensitivity of SV40 DNA detection. We strongly recommend this RQ-PCR assay for standardized detection of SV40.

Absence of simian virus 40 DNA sequences in primary central nervous system lymphoma in HIV-negative patients.

Simian virus 40 (SV40) is known to induce primary brain tumors and lymphomas in animal models. Recently, it was also associated with the pathogenesis of human non-Hodgkin's lymphomas. In the present study, we investigated primary central nervous system lymphomas (PCNSL), a defined subgroup of diffuse large B-cell lymphoma confined to the central nervous system, for the presence of SV40 DNA. Frozen tissue samples of 23 PCNSL derived from human immunodeficiency virus-negative patients were analyzed by two different, fully nested polymerase chain reaction protocols. SV40 DNA sequences could not be detected in any of these samples. Thus, SV40 can be added to the list of viruses that have already been excluded as pathogenetically relevant cofactors in PCNSL.