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This nine-laboratory multicenter investigation was designed to assess the sensitivity, specificity, and reproducibility of previously described assays for detection of SV40 DNA with three goals, i.e., (a) to compare methods for testing human tissues, (b) to examine the ability of these methods to detect SV40 in human mesotheliomas, and (c) to uncover assay differences that could explain conflicting findings in some past investigations. Each laboratory received, in a masked fashion, paired replicate DNA samples extracted from 25 fresh frozen mesotheliomas (50 samples) and one from each of 25 normal human lungs. Interspersed were masked positive (titrations of the SV40 genome) and negative control samples. Preliminary studies confirmed the adequacy of the samples for testing high molecular weight double-stranded linear DNA targets. All 15 PCR-based assays detected 5,000 copies or less of the SV40 genome spiked into 2 microg of WI-38 DNA. A high level of specificity and reproducibility was found among the PCR assays performed in most laboratories. However, none of the selected normal human lung tissue or the 25 mesothelioma tumor specimens obtained from archival samples at a single center was reproducibly positive for the presence of SV40 DNA. Further studies are needed to reconcile these results with previous reports of detection of SV40 DNA in tumor specimens.

Serological markers of squamous intraepithelial lesions (SILs), the precursors of cervical cancer, have not been studied extensively. To screen for antibody responses that might be associated with SILs, we measured IgG and/or IgA to nine antigens based on papillomaviruses, the infectious cause of SIL and cervical cancer, using an ELISA format. Cases were 59 women with low grade SIL (LSIL) and 38 with high grade SIL (HSIL). Controls were 50 women chosen to minimize the possibility that they ever had SILs [individuals who had no history of SIL and repeatedly tested negative for cervical human papillomavirus (HPV) DNA], frequency age-matched to cases. The data showed that five antibodies had strong positive associations with SILs and that one was inversely related to SILs. By studying these antibodies in pairs, furthermore, we found that case-control differences were enhanced. In particular, the combination of IgG to an epitope in the E6 protein of HPV 16 (E6:10) and IgA to HPV 16 virus-like particles (VLPs) was detected in 53% of LSILs and 65% of HSILs but only 9% of controls. These same responses were both negative in just 6% of LSILs and zero HSILs, compared to 59% of controls. Notably, E6:10 IgG and HPV 16 VLP IgA were not correlated with each other, and the other antibody responses positively associated with SILs could be broken into two groups: those correlated with E610 IgG and those correlated with HPV 16 VLP IgA. Overall, the data suggest that several papillomavirus antibodies may be strongly related to SILs, and that they can be divided into at least two independent groups of humoral immune reactions.

It has been reported that DNA of SV40, a virus of Asian macaques that is tumorigenic for rodents and can transform human cells in vitro, is present in pleural mesotheliomas and in several other cancers. To verify these observations, we tested paraffin sections from mesothelioma tissues of 50 patients for SV40 DNA using PCR with two separate sets of primers. The analytic sensitivity for detection of SV40 DNA was 1-10 genome copies. We also tested the specimens for beta-globin by PCR to assess the suitability of the specimen DNAs for amplification. beta-Globin amplification was detected in 48 of the 50 specimens, but SV40 DNA was not detected in any tumors, with either of two SV40 primer sets. Furthermore, sera from 34 additional patients with mesothelioma, 33 patients with osteosarcoma (another cancer reported to be SV40-related) and 35 controls were tested for SV40 antibodies by a plaque neutralization assay. The serological data, like the DNA results, did not support an association of SV40 with mesothelioma or with osteosarcoma; antibodies to SV40 were detected in three mesothelioma patients, in one osteosarcoma patient, and in one control. These findings call into question the association of SV40 with mesothelioma.

Neopterin, a marker of cellular immune activation, was elevated in patients who had cervical cancer in previous studies. To examine neopterin in the presence of precursors to cervical cancer (i.e., cervical intraepithelial neoplasia) we measured serum levels in 185 colposcopy patients in Jamaica, a country with high cervical cancer incidence, and in 72 age-matched Jamaican women selected from a large population-based sample. We also measured serum levels of beta-2-microglobulin, another commonly used marker of immune activation. Neopterin and beta-2-microglobulin levels were not elevated in colposcopy patients; neither were they related to severity of cervical neoplasia. In multivariable analysis, neither adjustments for detection of cervical human papillomavirus DNA by PCR nor detection of antibodies to human T-cell lymphotropic virus type I (a retrovirus endemic to Jamaica) altered our findings. The absence of a serologically detectable increase in cellular immune activation linked to cervical intraepithelial neoplasia suggests that the immunological response to cervical intraepithelial neoplasia does not involve substantial systemic cellular immune activation.