Causality of mesothelioma: SV40 question

Thorac Surg Clin 14 (2004) 497 – 504

Causality of mesothelioma: SV40 question Keerti V. Shah, MD, DrPH Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, USA

Occupational exposure to asbestos is the major established risk factor for mesothelioma [1,2]. Workers in the insulation industries and in industries employed in the construction, maintenance, and demolition of buildings are a high-risk group. In the United States and elsewhere, large numbers of workers in the expanded shipbuilding industry were exposed to asbestos around World War II [3]. In Great Britain, the peak of asbestos imports occurred during the 1960s and 1970s [4]. The duration between initial exposure to asbestos and peak mortality as a result of mesothelioma is 35 to 40 years. Almost no cases are seen within 20 years of initial exposure. Tumors rarely arise before the age of 40 years, and the age-specific incidence reaches its peak at the age of 65 to 75. In the United States, the increasing incidence of mesothelioma since the 1950s has been linked to increased exposure to asbestos in prior decades. It is anticipated that the incidence will decrease in the coming years, likely as a result of the control measures taken in the 1960s and 1970s to reduce exposure to asbestos and because the cases linked to asbestos exposure around World War II will have occurred by then. The studies to investigate human mesothelioma for simian virus 40 (SV40) etiology were triggered by an experimental observation from Dr. Carbone’s laboratory that 100% of 21-day-old Syrian hamsters inoculated with SV40 directly into the pleural cavity and more than 50% of those inoculated by the intracardiac route developed mesothelioma [5]. Spe-

This article was supported in part by Cancer Research Grant F402 from the Maryland Cigarette Restitution Fund. E-mail address: [email protected]

cifically, tumors in hamsters developed 3 to 6 months after inoculation with 108.5 plaque-forming units of SV40. Examination by Southern hybridization of the tumor DNA treated with different restriction enzymes indicated that the SV40 genome was integrated into the cellular DNA. In studies of cell lines derived from hamster tumors, SV40 T antigen was immunoprecipitated from lysates of tumor cells and detected in virtually every tumor cell by immunofluorescence tests. These data were consistent with the known oncogenicity of SV40 for laboratory animals and they provided unequivocal evidence that SV40 caused mesothelioma in hamsters after experimental direct inoculation of a large quantity of SV40. In 1994, Carbone et al [6] reported that SV40 sequences were recovered from 60% of human mesothelioma tissues and suggested that the SV40contaminated polio vaccines administered in the late 1950s and early 1960s may have been the source of SV40 infection of humans. Subsequently, studies to examine the possible association of SV40 with human mesothelioma and other human cancers have been conducted on several continents, and some investigators have concluded that SV40 contributes to the development of mesothelioma, pediatric and adult brain tumors, osteosarcoma, and non-Hodgkin’s lymphoma (NHL) [7 – 9]. Other investigators have failed to detect SV40 sequences in mesothelioma or find epidemiologic evidence consistent with an etiologic relationship between SV40 and human mesothelioma or other cancers. The Institute of Medicine Report [10] listed 29 studies in which mesothelioma tissues were screened for SV40 genomic sequences, with the reported prevalence of SV40 DNA ranging from 0% to 100%. Some of these studies sought to characterize SV40-positive tumors for SV40 transcripts, SV40 T protein, and molecular and clinical

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K.V. Shah / Thorac Surg Clin 14 (2004) 497 – 504

correlates of SV40 positivity. Epidemiologic investigations were performed to assess whether exposure to SV40 through contaminated polio vaccines was associated with an increased risk of subsequent mesothelioma. More recently, new serologic assays have been developed to use SV40-reactive antibodies as another marker of SV40 infection. The results of these human studies do not provide a coherent framework of how SV40 infection might be occurring in human communities and how it might contribute to the development of mesothelioma. The cumulative data argue against this possibility.

Recovery of simian virus 40 DNA from mesothelioma tissues is inconsistent and not reproducible The most persuasive evidence that disputes the SV40 – mesothelioma link came from the National Institutes of Health/US Food and Drug Administration – sponsored multicenter evaluation of assays to detect SV40 DNA in mesothelioma specimens [11]. This study, conducted by the International SV40 Working Group, was methodologically rigorous, as illustrated by the following features. All specimens were masked so that the laboratories were unaware of whether they were examining mesothelioma or control specimens. The 25 mesothelioma tissues that were examined were fresh-frozen and were shown to contain adequate amounts of tumor tissue. Each mesothelioma specimen was tested in duplicate as a control on the reproducibility of results in each laboratory. An equal number of lung tissues was tested as controls. There were no polymerase chain reaction (PCR) inhibitors in the DNA extracts as judged by tests on some specimens. The masked specimens included reference panels for estimation of specificity and sensitivity. Among the nine laboratories that participated, some had previously reported positive data, others had previously reported negative data, and still others had not worked on this research question before. All aspects of the investigation were monitored by a nongovernmental, independent study supervisor. The study was designed to be bimmuneQ to manipulations. Eight laboratories used PCR methodology, all employing one common assay, and five of the laboratories used additional assays. The analyses shown here are from results of the common assay from seven PCR laboratories. One of the eight PCR laboratories (Laboratory 1) reported that its primers were contaminated. The results from this laboratory were excluded.

Sensitivity The whole SV40 genome was diluted in tenfold steps to represent a range of copy numbers from 50,000 to 5. Two replicates of each dilution were tested by each laboratory (Table 1). Four of the laboratories consistently detected five or more genome copies, one laboratory detected 50 or more copies, and two laboratories detected 5000 and more copies. Each PCR reaction contained approximately 0.5 mg of tumor DNA, approximately a 67,000 diploid cell equivalent. The sensitivities of the assays in the different laboratories were satisfactory and ranged from one SV40 genome copy per approximately 10 cells to one copy per hundreds of cells. Specificity Each laboratory tested two batches of specificity controls (negative controls), and each batch comprised five samples. One batch was inadvertently contaminated with SV40 at its point of origin. Of the 35 specimens in the contaminated batch, 24 were found to be positive, with six of the seven laboratories contributing 2 to 5 positive specimens. Of the 35 specimens in the uncontaminated batch, 2 were positive, both in a single laboratory. The inadvertent contamination of one of the two batches of negative controls illustrates the wellrecognized pitfall of the PCR technology. It does not invalidate the negative results of the study, however. Reproducibility Four laboratories (Sites 2, 5, 7, and 8) scored both duplicates as negative for all 25 mesothelioma specimens (Table 2). Two laboratories (Sites 3 and 4) scored both duplicates as negative for 24 specimens but had discordant results for one specimen each. One laboratory (Site 6) scored both duplicates

Table 1 Sensitivity estimate in the National Institutes of Health – US Food and Drug Administration study Numbers of copies detected consistently* 5 50 500 5000

Laboratory sites 3, 4, 7, 8 2 5,6

* All specimens at that genome copy number, and all specimens with greater copy number, were positive.

K.V. Shah / Thorac Surg Clin 14 (2004) 497 – 504 Table 2 Intralaboratory reproducibility in the National Institutes of Health – US Food and Drug Administration study: 25 mesothelioma specimens were tested in duplicate Number DNA-positive Laboratory site

0

1

2

2 3 4a 5 6 7 8a

25 24 24 25 19 25 25

0 1 1 0 5 0 0

0 0 0 0 1 0 0

a

Two results scored as bnot interpretableQ are considered negative.

as negative for 19 specimens and both duplicates as positive for one specimen, and it had discordant results for five specimens. A single specimen (designated Q) was found to be positive in both duplicate samples in a single laboratory. That specimen was not positive in any other laboratory. Not one of the 25 mesothelioma specimens was reproducibly positive for SV40 sequences. The frequency of SV40 DNA recovery in mesotheliomas, normal lung tissue, and the uncontaminated negative controls in the study is shown in Table 3. The results from all seven laboratories are combined. There were twice as many tests for mesothelioma as for normal lung, because the mesothelioma tissues were tested in duplicate. Overall, a small proportion of the specimens (2.3% – 5.7%) was positive. SV40 DNA was not recovered any more frequently from mesothelioma tissues than from normal lung or negative controls. The authors of the study discussed several possibilities for the negative results, including geographic variation in the prevalence of SV40 in mesothelioma and chance sampling of SV40-negative mesotheliomas. A third possibility proposed a selective loss of viral DNA during the extraction procedure. This question was not directly examined in the study, because the human DNA used for laboratory-prepared positive controls was spiked with SV40 DNA after—rather than before—DNA extraction. We conducted an experiment to evaluate this question (R.W. Daniel, K.V. Shah, unpublished data). High molecular weight human DNA was mixed with an SV40 virus pool grown in BSC-1 cells. One aliquot of this mixture was digested with proteinase K followed by inactivation of the enzyme at 95C. Another aliquot was extracted in the same way as the study specimens by the commercially available Qiagen DNeasy tissue

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kit (Qiagen, Valencia, California). Each preparation was titrated, and the dilutions were amplified separately by PCR for b-globin and SV40 sequences. Gels of the amplification products of the two preparations showed identical results, which indicated that the extraction by the tissue kit had not resulted in the selective loss of either the high molecular weight human DNA or the low molecular weight viral DNA. The extraction method was unlikely to have been responsible for the negative data. Although most of the early studies reported a high prevalence of SV40 sequences in mesothelioma tissues, the more recent studies have not confirmed the earlier results. Hubner and Van Marck [12] retested 12 frozen mesothelioma specimens, many of which were previously reported to be positive for SV40 sequences. They used five extraction methods and tested the DNA with four primer pairs targeted at different regions of the SV40 genome. SV40 sequences were not recovered from any of the tissues. They remarked that the hybridization conditions may account for false-positive results in some previous studies. In another study, Gordon et al [13] examined 35 fresh-frozen mesothelioma tissues by quantitative SV40 PCR in the first reported use of quantitative SV40 PCR in mesothelioma studies. Thirty-three tissues tested negative; in the two positive tissues, the authors estimated that there was less than one copy of SV40 per 100 tumor cells. Mayall et al [14] failed to detect SV40 sequences in 56 mesothelioma tissues from New Zealand and England. In the most recent investigation, the results of which are available currently only in an abstract, Lopez-Rios et al [15] did not find significant evidence for the presence of SV40 sequences or SV40 early region transcripts in 71 mesothelioma tissues. On an examination of the inconsistencies in their results with different sets of primers targeting regions of the SV40 T antigen region, they concluded that contamination with common laboratory plasmids that contain SV40 sequences (especially in the nucleotide 4100 – 4713 region) may have contributed to some of the positive results reported in earlier studies.

Table 3 Frequency of detection of simian virus 40 sequences in the National Institutes of Health – US Food and Drug Administration study Mesothelioma Normal lung Negative controla a

No. of tests

No. (%) positive

350 175 35

9 (2.6) 4 (2.3) 2 (5.7)

In the uncontaminated batch.

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A closer look at the studies that reported SV40 sequences in human tissues reveals numerous inconsistencies. Carbone et al [6] reported SV40 T-reactive antibodies in sera of all 13 patients who had SV40-positive mesothelioma but also in sera of all 13 patients who had SV40-negative mesothelioma. Galateau-Salle et al [16] reported SV40 sequences not only in mesotheliomas (48%) but also in bronchopulmonary carcinomas (29%) and normal lungs (21%), which raises questions about the specificity of the SV40 – mesothelioma association. A review of all the data relating to the detection of SV40 sequences in mesothelioma leads to two alternative possibilities: (1) the assays in the negative studies were insensitive or (2) the positive results may represent laboratory artifacts. The artifacts may arise from nonspecific bands, contamination from other specimens or from PCR products, or from SV40 sequences in cloning vectors, use of high number of amplification cycles, or use of nested primers. In the positive studies, a small amount of SV40 sequences (far fewer than one copy per tumor cell) was recovered, and the viral genome could not be localized consistently to tumor cells. In summary, the SV40 – mesothelioma association described for human tumors in the positive studies does not in any way resemble that in experimentally induced hamster tumors described earlier in this article.

Epidemiologic studies do not show an increased risk of mesothelioma in populations exposed to simian virus 40 In contrast to asbestos exposure that has occurred over several decades and currently is continuing at low levels, the major documented human exposure to SV40 occurred in a defined time period (1955 – 1963) during which asbestos exposure was also prevalent. Between 1955 and 1963, SV40 was a contaminant of some lots of the formalin-inactivated polio vaccines [17]. Although approximately 100 million US residents were immunized during this time, not all were exposed to SV40. The vaccinees may have received lots that were free of SV40 or contained only formalin-inactivated noninfectious SV40 or formalin-inactivated and live residual SV40. The only group at risk was the one that received live SV40. Although the size and other characteristics of this group are not known, the numbers were probably large. The vaccine was administered in three doses (which would not all necessarily be from the same lot); therefore, the vaccinee had more than one opportunity to receive contaminated vaccine. The

only data available on the distribution of polio vaccines in the United States that contained live SV40 indicate that most of the children immunized in the period of May through July 1955 received at least some amount of vaccine that contained live SV40 [18]. Strickler et al [19] compared the incidence of mesothelioma (and other tumors reported to contain SV40 sequences) in three birth cohorts: (1) persons born between 1956 and 1962 (exposed as infants or children), (2) persons born between 1947 and 1952 (exposed as children), and (3) persons born between 1964 and 1969 (not exposed). There was no significant increase in the age-specific incidence rates for mesothelioma or for other cancers (eg, ependymoma, brain cancers, and osteosarcomas) related to exposure to contaminated vaccine. The authors recognized that the estimates for mesothelioma were based on small numbers (a total of 71 cases) and were imprecise. They also noted that at the time of the follow-up, the oldest of the individuals in the cohorts were only 46 years old and had not reached the age when mesothelioma incidence is high. In a study of similar design, Engels et al [20] examined whether childhood exposure to SV40 was associated with an increase in the incidence of certain cancers in Denmark. All of the vaccines used until 1962 in Denmark were contaminated with SV40 and contained live SV40. They found that SV40 exposure was not linked to an increased incidence of mesotheliomas. As in the US study, the number of mesothelioma cases was small (a total of 57 cases), and the exposed children had not reached the age of peak incidence of mesothelioma. In a subsequent study, Strickler et al [21] took advantage of the fact that the probability of having received contaminated vaccine in the United States differed by age. For example, 80% to 90% or more of US residents aged 1 to 20 years in 1961 but only 20% or less of US residents aged 50 years or older in 1961 were exposed to potentially SV40-contaminated vaccine. They calculated age- and sex-specific incidence rates of mesothelioma between 1975 and 1997 for the different birth cohorts of the vaccinees. A large number of mesothelioma cases were recorded in this time period. Overall, the incidence increased in men and women, but the incidence in men was sixfold higher than in women. They found that the greatest increase in mesothelioma incidence occurred in the oldest age groups, the groups that had a low probability of having received SV40-contaminated vaccine. In contrast, the mesothelioma incidence declined or remained stable in the younger age groups, which had a high probability of having

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received SV40-contaminated vaccine. They further examined this relationship in an age period cohort model and found that the incidence rates of mesothelioma were unrelated to level of exposure to SV40-contaminated vaccine. Two additional observations are in order. First, if SV40 exposure is an independent risk factor for mesothelioma with a specific latency period, similar to that seen with asbestos exposure, then mesothelioma cases should occur at younger ages; many individuals were exposed to SV40 as young children, in contrast to occupational asbestos exposure in adulthood. Such a change in the age distribution of the disease has not been noted. Second, mesothelioma incidence is much lower in women than in men, which may reflect differential occupational exposure

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to asbestos by gender. SV40 exposure was similar in men and women. If SV40 were an independent risk factor for mesothelioma, one would anticipate an acceleration of mesothelioma incidence in women. Such a trend has not been observed [21].

Serologic data call into question the association of simian virus 40 with any human cancer In the studies to date of the SV40 – human cancer association, the detection of SV40 genomic sequences has been used almost exclusively as the marker of SV40 exposure. Immune response is a time-honored and robust marker of viral infection, and polyomavirus infections generate excellent antibody responses

Fig. 1. Reactivity of sera of rhesus macaques (A, B) and humans (C, D) to SV40 and BKV VLPs in ELISA. (Courtesy of R. Viscidi.)

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in their natural hosts. Early studies of individuals who do not have a known exposure to SV40 have shown that a small proportion of human sera neutralize SV40 and that the antibody titers of the positive sera are low [22]. It was not clearly established if this lowlevel reactivity to SV40 represents human infection with SV40 or cross-reactivity to the closely related human polyomaviruses BK virus (BKV), and JC virus (JCV). It is possible to address this question with the newly developed ELISA assays based on the use of SV40, BKV, and JCV virus-like particles (VLPs) as antigen [23]. A detailed review of the serologic data would not be appropriate in this article, but the main findings are summarized later. Serologic studies do not provide evidence that SV40 is circulating in human communities [23 – 25]. The low level of SV40 reactivity of human sera is likely to be a result of cross-reactivity with antibodies to human polyomaviruses. Sera from rhesus macaques react strongly with SV40 VLPs (Fig. 1A), but they also bind to BKV VLPs, to a lesser degree (Fig. 1B), thus providing clear evidence of immunologic cross-reactivity between SV40 and BKV [23]. Conversely, human sera react strongly to BKV VLPs (Fig. 1D), but they also react to a much lesser degree with SV40 VLPs (Fig. 1C) (R. Viscidi et al, personal communication, 2004). The SV40 reactivity of human sera correlates positively with the strength of BKV reactivity of the same sera and is reduced by adsorption with BKV VLPs [23]. It is likely that all or most of the SV40 reactivity of human sera can be explained by cross-reactivity with antibodies to human polyomaviruses [23,25]. Serologic data also fail to show that mesothelioma patients have greater exposure to SV40 than controls. If SV40 were the cause of mesothelioma in an individual, the following events likely would occur: multiplication of the virus at the site of entry (subcutaneous tissue if introduced by the vaccine), transport of the virus to the mesothelium via blood, virus multiplication in the mesothelium, and malignant transformation of the mesothelium. These events (ie, virus multiplication and viremia) would be expected to generate a significant antibody response to SV40 capsid antigens (and perhaps to SV40 T protein), which would result in higher prevalence and higher titers of antibodies in cases as compared with controls. In a serologic study of masked specimens from mesothelioma cases and controls, neutralizing antibodies to SV40 were rare in both groups, and the positive sera had low titers [26]. The SV40 capsid antibody profile in mesothelioma cases was no different from that which has been seen in noncancerous populations.

Summary The fragility of the evidence for SV40 association with human cancer is seen in studies of NHL. A publication in 1999 stated that SV40 is rarely present in NHL [27]. In 2002, two laboratories reported SV40 sequences in 42% to 43% of cases of NHL [28,29]. One of these laboratories also detected SV40 sequences in small proportions of pediatric tumors (eg, Wilm’s tumor, hepatoblastoma, rhabdomyosarcoma, medulloblastoma, osteosarcoma, and retinoblastoma) and adult carcinomas (eg, lung, colon, breast, and prostate) [29]. These positive results were not confirmed in subsequent studies published in 2003. Capello et al [30] and Mackenzie et al [31] failed to detect SV40 sequences in NHL tissues. Sanjose et al [32] examined sera from patients with NHL and from controls for antibodies reactive to SV40 VLPs, and they detected no significant differences between the two groups. The association of SV40 with NHL is in doubt. An etiologic link between a virus and a cancer becomes plausible when evidence from different lines of enquiry (eg, epidemiology, pathogenesis, and molecular mechanisms) is mutually reinforcing and together provides a coherent picture that can connect the biology of the virus to the characteristics of the disease. The associations of human papillomaviruses

Box 1. Weaknesses in the evidence linking mesothelioma with simian virus 40: a summary 1. Mesothelioma incidence did not increase in cohorts exposed to contaminated vaccines [19,20] 2. The low mesothelioma incidence in women exposed to contaminated vaccines did not accelerate, an indication that SV40 is not an independent risk factor for mesothelioma [19] 3. Tumor cell-SV40 relationship in human mesothelioma [7,13] does not resemble that in experimentally induced hamster mesothelioma [5] 4. SV40 cannot be reproducibly detected in mesothelioma tissues [11] 5. Patients with mesothelioma do not have serologic evidence of exposure to SV40 [26]

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with cervical cancer and hepatitis B and C viruses with hepatocellular carcinoma are examples in which the etiologic link is clear. With SV40 and mesothelioma, the data on viral sequences in tumors is inconsistent and disputed, and serologic evidence does not support any association. The epidemiologic data do not show that documented exposures to SV40 increase the risk of mesothelioma. It seems improbable that a single virus (which cannot be conclusively demonstrated to be present in the community) contributes to the development of such a wide variety of tumors, spanning all age groups and histologic types. The weaknesses in the evidence linking SV40 with mesothelioma are summarized in Box 1. It seems unlikely that infection with SV40 contributes to the development of human mesothelioma or any other human cancer.

[12]

[13]

[14]

[15]

[16]

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