Evidence against a role for SV40 infection in human mesotheliomas and high risk of false-positive PCR results owing to presence of SV40 sequences in common laboratory plasmids

Our plain-English summary of Evidence against a role for SV40 infection in human mesotheliomas and high risk of false-positive PCR results owing to presence of SV40 sequences in common laboratory plasmids, by F. López-Ríos, P.B. Illei, V. Rusch, et al (Lancet, 2004).

The study

• Authors: F. López-Ríos, P.B. Illei, V. Rusch, M. Ladanyi
• Journal: Lancet
• Published: 2004;364(9440):1157-66
• Cited by: 146 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Background: PCR-based evidence of infection by simian virus 40 (SV40) has been reported in varying proportions of pleural mesotheliomas and other tumours, but data are conflicting and reproducibility limited. During a study of SV40 in relation to homozygous deletion of CDKN2A in mesotheliomas, we became concerned by inconsistent results and therefore used several independent techniques to investigate SV40 in these tumours.

Methods: High-quality DNA and RNA were extracted from 71 frozen mesothelioma samples. DNA PCR was done with four sets of primers for the SV40 T-antigen gene. RNA transcripts were examined by RT-PCR.

Findings: The first two primer sets for DNA PCR gave positive results in proportions similar to those reported in positive studies (56-62%) but there were unusual reproducibility difficulties. These primers were in a region of the T-antigen gene (nucleotides 4100-4713) that is present in many common laboratory plasmids. In assays with PCR primers not included within that region, only four cases (6%) showed products but these were too faint to suggest clonal infection. Further PCR assays confirmed that the SV40 sequences in the tumour samples had a deletion found only in plasmids, not in native functional SV40. Review of previous studies showed a similar pattern of discrepancies between SV40 T-antigen DNA PCR results obtained with primers within and beyond the region 4100-4713. All 71 mesotheliomas were negative for T-antigen transcripts by RT-PCR, and lacked T-antigen-positive tumour cells by immunohistochemistry.

Interpretation: Our data based on three independent experimental approaches do not support a significant role for SV40 in human mesotheliomas. The risk of false-positive results due to contamination by common laboratory plasmids containing SV40 sequences has been underestimated. Studies of SV40 based on PCR methods require careful primer design to reduce this risk.

Relevance To Practice: This paper presents several lines of evidence against the proposed link between SV40 infection and human mesotheliomas. Studies reporting a high prevalence of SV40 DNA in human tumours have been based on molecular assays prone to false-positive results. Because SV40 appears unlikely to have a major role, if any, in human mesotheliomas, clinicians should continue to consider asbestos exposure as the most likely and most thoroughly established aetiological factor in individuals with this cancer.

In plain English

This laboratory study tested 71 frozen mesothelioma samples, removed at surgery at Memorial Sloan-Kettering Cancer Center, New York, between 1990 and 2000, for simian virus 40 (SV40), a monkey virus some had proposed as a cause. Two DNA tests aimed at overlapping parts of the SV40 T-antigen gene were positive in 44 of 71 (62%) and 40 of 71 (56%) samples but disagreed in 14 cases (20%). Both targeted a stretch of SV40 found in many common laboratory plasmids. A test outside that stretch gave very weak, inconsistent results in four cases (6%). No sample showed SV40 T-antigen RNA transcripts or tumour cells with nuclear T-antigen staining; the authors judged cytoplasmic staining in many samples to be nonspecific. A test for a sequence found only in plasmids, not natural SV40, was positive in 16 of 69 samples (23%), which the authors read as laboratory contamination. They concluded that SV40 is at most only rarely present in human mesotheliomas, and wrote that asbestos exposure remains the most likely, most thoroughly established cause. The study did not measure asbestos exposure.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study of stored tumour samples using DNA PCR, RT-PCR (RNA) and immunohistochemistry (protein staining) to look for SV40
• Who was studied: Frozen mesothelioma samples taken at surgery at Memorial Sloan-Kettering Cancer Center, New York, between 1990 and 2000. Of 80 samples with DNA, 71 had good-quality RNA, and these 71 are the ones reported. Most patients were American; median age 62 years (range 30 to 78).
• Exposure: Presence of SV40 virus (T-antigen gene DNA, T-antigen RNA transcripts and T-antigen protein) in the tumours. Asbestos exposure was not studied.
• Main results:
  • With the widely used primer pair SV5-SV6, 44 of 71 mesothelioma samples (62%) tested positive for SV40 DNA
  • With the overlapping primer pair SV5-SV6b, which used a newly designed reverse primer, 40 of 71 samples (56%) tested positive
  • The two high-contamination-risk primer pairs disagreed in 14 cases (20%), and some no-DNA negative control reactions were occasionally positive
  • With a primer pair outside the plasmid-shared region (SVINTfor-SVINTrev), only 4 cases (6%) gave very weak bands, and these were not consistently positive on repeat testing
  • The SV40 T-antigen RT-PCR assay found no T-antigen transcripts in any RNA sample from the 71 mesotheliomas, although the positive control cell line amplified strongly
  • None of the 71 samples had tumour cells positive for SV40 T antigen by immunohistochemistry, and none showed nuclear staining even at a more concentrated antibody dilution
  • At the more concentrated antibody dilution, many samples showed weak to moderate cytoplasmic staining, which the authors interpreted as nonspecific because it also appeared in non-tumour control tissues
  • A plasmid-specific SV40 sequence (210 bp product), not the product expected from genuine SV40 (1539 bp), was found in 16 of 69 cases (23%). Most of these (14 of 16) had earlier been positive with one or both high-contamination-risk primer pairs.
• The authors' conclusion: The authors concluded that SV40 is at most only rarely present in human mesotheliomas, and that PCR data on SV40 in human tumours need to be carefully re-examined. In their summary they stated that asbestos exposure is the most likely and most thoroughly established cause of this cancer.
• Limitations the authors state:
  • The authors could not estimate the sensitivity of the RNA (RT-PCR) assay in the way they did for the DNA assay
  • The authors stated that the technical pitfalls they identified may not call into question all published data on SV40 in human tumour samples

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: SV40, Mesothelioma, Pleural Mesothelioma, Homozygous Deletion, CDKN2A, Immunohistochemistry, Asbestos Exposure.

Related studies

• Simian virus 40 sequences in blood specimens from healthy individuals of Casale Monferrato, an industrial town with a history of asbestos pollution — C. Pancaldi, V. Balatti, et al. (2009)
• SV40 multiple tissue infection and asbestos exposure in a hyperendemic area for malignant mesothelioma — M. Comar, C. Rizzardi, et al. (2007)
• Aberrant methylation profile of human malignant mesotheliomas and its relationship to SV40 infection — M. Suzuki, S. Toyooka, et al. (2005)
• Human mesotheliomas contain the simian virus-40 regulatory region and large tumor antigen DNA sequences — H.I. Pass, J.S. Donington, et al. (1998)
• SV40 enhances the risk of malignant mesothelioma among people exposed to asbestos: a molecular epidemiologic case-control study — A. Cristaudo, R. Foddis, et al. (2005)
• Simian virus 40 transformation, malignant mesothelioma and brain tumors — F. Qi, M. Carbone, et al. (2011)

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