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

Summary

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.

Introduction

Few topics in cancer research have been as difficult and contentious as the role of simian virus 40 (SV40) in human tumours; the most extensively studied cancers are mesotheliomas, bone tumours, brain tumours, and lymphomas.1, 2, 3 In view of the potential importance of the findings, the continuing appearance of contradictory reports on SV40 detection in human tumour tissues, often followed by vigorous correspondence, remains troubling.4, 5 Various explanations have been proposed for the inconsistencies, but a broadly held consensus has yet to emerge.3, 6, 7

SV40 is a small, circular, 5243 bp, double-stranded DNA polyomavirus of monkey origin. The SV40 large T antigen is a 708-aminoacid multidomain protein that has proven oncogenic activity in some animals (mainly rodents). The primary mechanism of this oncogenic effect is thought to be through binding and inactivation of the nuclear proteins p53 and Rb.⁸ It is encoded by two exons corresponding to nucleotides 2691–4571 and 4918–5163 of the SV40 genome. SV40 causes various tumours when injected into hamsters, including mesotheliomas. The finding that SV40 was a widespread contaminant of poliovirus and adenovirus vaccines in the 1950s and 1960s was proposed as a plausible origin for widespread SV40 infection in human populations.1, 9 However, the age ranges and incidence trends of cancers in which SV40 has been reported have been inconsistent with a simple or robust role for contaminated vaccines as sources of oncogenic SV40 infections.¹⁰ After the recommendations of an international meeting organised in 1997 by the US National Institutes of Health and the Food and Drug Administration,11, 12 two multicentre studies of SV40 detection in mesotheliomas were set up to assess definitively the presence of SV40 in mesotheliomas by DNA PCR.13, 14 Although many samples were positive, both studies were beset by difficulties with reproducibility between laboratories, involving three of 12 mesothelioma samples tested by four laboratories in the first study and all of 25 mesothelioma samples tested by nine laboratories in the second study.13, 14 Such inconsistencies are important because at present the call for further studies of SV40 as a human carcinogen is not based on epidemiological evidence (which is weak or negative) but is primarily driven by the many reports of SV40 detection by PCR-based assays in various human tumours.¹ Furthermore, serological and epidemiological evidence within the past year has not been consistent with an oncogenic effect of exposure to SV40, adding to the controversy.15, 16, 17, 18, 19

In parallel with these studies of SV40 in mesothelioma, cytogenetic and molecular studies by several groups over the past decade have identified recurrent somatic genetic alterations in this tumour, the most common being homozygous deletion of the 9p21 locus within a cluster of genes that includes the tumour suppressor CDKN2A, seen in 70–80% of cases.20, 21, 22 CDKN2A encodes two cell-cycle regulatory proteins, p16 and p14ARF.²³ p16 acts through CDK4/CDK6 to block phosphorylation of the Rb protein and control cell-cycle progression. p14ARF binds MDM2, preventing it from mediating p53 degradation; hence loss of p14ARF results in functional impairment of the p53 pathway.²³ Having completed an analysis of 95 pleural mesotheliomas in which we confirmed the high prevalence (74%) of homozygous deletion of CDKN2A,²² we recently examined the relation between CDKN2A deletion and SV40 status in these tumours. We reasoned that since both the CDKN2A homozygous deletion and SV40 T antigen are thought to result in functional inactivation of the p53 and Rb pathways, they might have an inverse relation to each other because functionally redundant changes are generally not observed in human tumours. Such a model of SV40 infection and CDKN2A deletion as alternative mechanisms of coinactivation of p53 and Rb in mesotheliomas has been proposed by others²⁴ but never systematically studied in a single series of primary tumours. However, we could detect no statistically significant relation between SV40 positivity (with primers SV5-SV6 and SV5-SV6b described below) and CDKN2A deletion in 80 mesotheliomas with adequate frozen tissue for DNA extraction (unpublished; http://image.thelancet.com/extras/03art8144webtable1.pdf). This biologically puzzling finding prompted us to question our initial SV40 DNA PCR results and to investigate possible sources of false-positive results, as well as to obtain supporting data on SV40 status at the RNA and protein levels.