Asbestos is a potent carcinogen and the most important single cause of mesothelioma, a mostly fatal cancer of the pleura 123. Only about 20% of mesothelioma cases occur in non-exposed individuals 45. Previous studies have shown that mesothelioma mortality rates correlate with past asbestos consumption rates (defined by production minus export plus import) in industrialized countries 16. During the past 40 to 50 years, asbestos consumption varied considerably in European countries, with low per capita use in less industrialized countries, such as Bulgaria, and vast use in ship building and other insulating industries,e.g. in the U.K. Since the time lag between asbestos exposure and tumor development is 30 to 45 years in most cases, the recent increase in mesothelioma incidence should therefore reflect the intensified use of asbestos during this particular period 3789. Indeed, the production of asbestos peaked worldwide in the late 1970s and early 1980s 10. Likewise, the mesothelioma incidence is expected to reach maximum levels between 2010 and 2020 in industrialized countries 6. About half of the cases will occur in construction and shipbuilding workers, as in these professions asbestos exposure was particularly common 6. Incidence is much lower in women, as they were generally, not involved in asbestos-related activities 11.

The fact that traces of simian virus 40 (SV40) were repeatedly demonstrated in a significant proportion of mesothelioma samples led to the notion that this virus may act as either a co-carcinogen or tumor promoter 121314. SV40 was introduced unintentionally into millions of people via contaminated poliomyelitis virus vaccines between 1955 and 1963. However, some vaccines produced later may have not been entirely SV40-free, as evidenced by the fact that SV40 DNA has been recently detected in archival polio vaccines produced in 1966 and 1969 by a major Eastern European manufacturer 15. SV40 was present in both the attenuated (oral) polio vaccine (OPV) and the inactivated polio vaccine (IPV), since formaldehyde treatment, which was used to inactivate the poliomyelitis virus, failed to inactivate SV40 12.

SV40 DNA was subsequently detected in human brain and bone tumors as well as lymphoma samples (reviewed in 16). Intriguingly, SV40 causes the same tumor types in hamsters 4. The most important step in the process of carcinogenesis is the inactivation of tumor suppressor p53 and members of the retinoblastoma family of proteins through the SV40 large T antigen 17. In addition, other tumor suppressor genes become methylated and are shut down. These gene modifications have not only been observed in vitro, but also in an analogous fashion in SV40-positive lymphoma samples 18.

However, it is important to note that SV40 DNA was not detected in several studies of tumor samples from particular populations, whereas it was readily detectable in appropriate control samples from the USA 1920. In particular, SV40 DNA was not detected in mesothelioma, brain tumor and bone tumor samples from Austria, Finland and Turkey, countries which apparently had never used contaminated polio vaccines 192021222324. These findings were therefore taken as evidence that the population-specific and linked geographic differences were genuine and that they reflected the heterogeneous use of SV40-contaminated polio vaccines in the respective countries 4. However, definitive epidemiological proof for the presence or absence of an association between (past) SV40 exposure and cancer is lacking so far, mostly because the infected cohorts can no longer be identified unambiguously 4.

The presence of virus-specific antibodies in serum is a well-established biomarker of viral infection. Antibodies to SV40 have been measured in humans by plaque inhibition neutralization assays and enzyme immunoassays. However, the studies have been mostly negative or detected only low levels of SV40 serum antibodies 25262728. Cross-reactivity between SV40 and the BK polyomavirus has been proposed as an explanation for the detection of low levels of SV40-reactive antibodies in human serum samples and may complicate the interpretation of positive assay results 27. In a recent population-based case-control study published by Engels et al. 26 using competitive assays to analyze SV40-specific reactivity, it has been shown that an estimate of 1% to 1.6% individuals from the U.S. born before 1963 had SV40 specific antibodies, whereas those born later showed no SV40 specific reactivity. The authors stated that these results point to the possibility that exposure to SV40 could have led to antibody responses that declined over the decades, probably due to lack of virus replication, and that SV40 seems not to be a common cause of infection in humans 26.

However, the past exposure of humans to SV40 is undoubted. We therefore set out to investigate whether mesothelioma (pleural cancer) incidence and mortality rates might be higher in those European countries (including the former Communist countries), in which at least some circumstantial evidence indicates that a population-wide exposure had taken place. For this purpose, we first investigated to what extent pleural cancer mortality rates correlate with those of past asbestos use. We then compared country-specific asbestos consumption and mesothelioma rates with the appropriate likelihood of SV40 contamination. To ascertain whether the respective SV40 exposure was frequent, low or absent in specific countries, we used data from publications that dealt with the analysis of SV40 DNA in human tissue samples. In addition, we also collected all the available information about which type of poliomyelitis virus vaccines had been used in these countries and whether they were SV40-contaminated or not.

The main purpose of this ecological analysis was to explore the potential effect of a country-wide presence of SV40 on the respective pleural cancer mortality rate, presuming that SV40 has the long adjudicated tumor-inducing or -promoting role in humans. A country-wide presence of SV40 was inferred from previous vaccination programs with contaminated vaccines or the detection of virus DNA in tumor samples. Although the results of our analyses confirmed the previous well-established association between the male pleural cancer mortality rate and the extent of the asbestos consumption 25 to 30 years earlier, we failed to detect any discernible effect on the respective male or female pleural cancer mortality rates that could have been allocated to SV40.

The oncogenic potential of SV40 and the mode of its introduction into the human population via contaminated vaccines are well documented (for review see 12). However, whether viral infection indeed also represents a risk factor for tumor development in humans is still a rather controversial issue, which at present is primarily supported by spurious molecular genetic studies, but not by firm epidemiological evidence 106. However, even the question of whether SV40 is actually present in human tumors and if so, what role it might play in tumor development, is currently unsolved and a matter of ongoing debate, especially since two multi-center studies reached different conclusions 107108. Epidemiological studies about tumors developing in recipients of SV40-contaminated vaccines are mainly hampered by the fact that the infected cohorts are difficult to identify, because SV40 has been found in tumors of patients that were too young to have been exposed to contaminated vaccines 4.

To differentiate between SV40-contaminated and SV40-free European countries, we collected all eligible studies dealing with this topic and extracted the relevant information. However, we excluded all studies which had merely used immunological and serological detection procedures, because SV40 and the human BK and JC polyomaviruses are known to cross-react to a high degree 109110111112113. For the same reason, we also excluded reports that dealt with serum antibodies against SV40. We were nevertheless able to identify three countries (Austria, Finland and Turkey), in which exclusively SV40-free polio vaccines had been used. Incidentally, these were the same countries in which molecular genetic studies failed to detect SV40 DNA in a representative large number of tumor samples 192021222324.

Although we were unable to find any epidemiological evidence for a potential association between SV40 and pleural cancer, the following limitations of our analysis have to be taken into consideration. First of all, we were unable to account for different types and particular usage of asbestos. Instead, we used the asbestos data from a single source, the U.S. Geological Survey 10. Naturally occurring asbestos is found in soils or rocks in certain villages in southeast Turkey, where it causes endemic occurrence of mesothelioma 114, and also in other parts of the world, e.g. in California 115. However, it was not possible to correct for the impact of natural asbestos deposits in the cohorts used in this study. Even if Turkey is excluded from the analysis, no significant association between SV40 and pleural cancer is found, neither in males (asbestos-corrected rates), nor in females (data not shown).

Second, differences in cancer registration exist and increasing diagnostic awareness in the past 20 years may have affected the reported mortality rates. Miscoding might have occurred in countries where only death certificates were used for mesothelioma registration 34. To optimize comparability, we therefore used the WHO database as our only source of information. In the majority of cases, we used mortality data from 1985 to 1989. More recent mortality rates from the WHO mortality database would not allow comparability of mortality rates with former country-wide asbestos consumption data because of the unification or division of certain European countries after the end of the Cold War (about 1989). Data from the International Agency for Research on Cancer (IARC) database 116 were not suitable, because for many countries the incidence rates are indicated on a district-basis only, and not in a country-based fashion. However, the important limitation of possibly incorrect pleural cancer rates could not be overcome in the current study. Therefore, improvements in cancer registration are important for future studies addressing these questions.

Third, methodological differences in SV40 screening procedures (e.g. different PCR primers, frozen versus paraffin embedded tissues) lead to unequal sensitivities 117. An unknown proportion of positive SV40 DNA results may have been related to contamination or to other problems with the PCR methods 118. Optimized laboratory procedures for SV40 detection have been only recently defined 119. However, as is suggested, mostly by the laboratory investigators themselves, there might be a relationship between country-specific PCR results and geographic variations in SV40 occurrence 4 and hence mesothelioma incidence. Therefore, to study geographic variations, we chose to assume the accuracy of the prior positive PCR results. The different sample sizes of the SV40 detection studies were taken into account by assessing the number of samples with positive SV40 nucleic acid detection versus the whole number analyzed samples for each eligible study. Further issues that also have to be considered in such analyses are, of course, that potential but yet undefined specific genetic factors might influence the likelihood of mesothelioma development in different populations 120.

In the United States, a potentially contaminated poliomyelitis virus vaccine was used between 1955 and 1963 nationwide on about 90% of children and 60% of adults 16121. In Europe, approximately 60% of the population received a potentially contaminated vaccine 121. However, the SV40 prevalence in many European countries is more mosaic-like, because it still mirrors the pattern of when and with which vaccine mass vaccination programs were initiated by local governments. At the beginning of our investigation, we were confident that we could obtain the necessary SV40 data from the vaccine producing companies, which would have been the most efficient and accurate way of determining the pattern of use of contaminated vaccines. However, many of our requests remained unanswered and in some instances the relevant information had apparently already been destroyed, unclear statements were frequent. Therefore, national health bulletins (like those issued in Austria and Spain) became a particularly important resource 9192101. Reports on historical SV40 contamination of vaccines may be of varying accuracy, depending on the detection methods used and on the number and source of samples (batches) tested. However, all data were assumed to be of equal weight because weighting of such heterogenous data was not possible. Moreover, other routes of transmission may exist. Epidemiological evidence suggests that SV40 may be contagiously transmitted in humans by horizontal infection, independent of the earlier administration of SV40-contaminated polio vaccines 113. Recently, even infection through laboratory strains of SV40 has been proposed 122.

Although little is known about the severity of SV40 infection in humans (systemic or non-systemic), some data indicate that there might be biological differences depending on the type of exposure (IPV vs. OPV). After oral administration of SV40 in humans, there was no antibody response, indicating that it most likely did not result in systemic infection, which would most likely be necessary for cancer induction (for review see 88). On the other hand, after subcutaneous inoculation of SV40-contaminated inactivated vaccines, antibody titers were high and remained high or declined slightly over a 3-year follow-up 88. After 1961, IPV was replaced in many countries by OPV, although a few continued to use IPV and others subsequently reintroduced it. We identified four countries that used only IPV between 1957 and 1963 and recalculated differences in mean pleural cancer rates taking into account the type of vaccine used in the country. However, whether SV40-contaminated OPV or IPV was used in a specific country had no impact on asbestos-corrected (male) or raw (female) pleural cancer rates. Many countries used IPV to a variable extent before mass vaccinations with OPV. Unfortunately, hardly any information about these vaccination programs remains available. Therefore, the level of exposure to IPV could not be exactly determined for every country.

We used two different approaches to analyze the association between SV40 prevalence and pleural cancer mortality in European countries. First, we used data on historical SV40 exposure. However, the infected cohorts are diluted by individuals not vaccinated during the possible time of infection or vaccinated with SV40-free vaccines. Second, we screened the literature for molecular genetic evidence of SV40 in tumor samples from the different countries. The methods applied would detect only a strong carcinogenic or co-carcinogenic effect of SV40 on the pleura (in males aggregated by country) because of the crude adjustments for asbestos consumption, without distinguishing between chrysotile and amphibole asbestos. However, the results of this study, the negative SV40 DNA reports, and the serologic data argue against a major role of SV40 in mesothelioma carcinogenesis.

Finally, despite all the shortcomings and problems, our comprehensive data collection provides the first account of the diverse usage of different types of poliomyelitis virus vaccines from all available data from European countries. In conjunction with the records obtained from the molecular genetic screening for SV40 DNA in tumor samples derived from the respective populations, it provides the best achievable distribution map available to date on SV40 prevalence in Europe and the basis for future reassessments of epidemiological SV40 data, whenever new information becomes available. This ecological analysis makes an association of male pleural cancer with SV40 unlikely, but this needs to be confirmed by case control studies and cohort studies.

The author(s) declare that they have no competing interests.

K.L., A.L., and O.A. designed the study and edited the manuscript, K.L. and P.R. performed the statistical analysis. H.C. has made substantial contributions to acquisition of data. A.W., R.R., P.K., R.W., and H.O. have been involved in drafting the manuscript or revising the manuscript. All authors read and approved the final manuscript.