Presence of simian virus 40 sequences in malignant pleural, peritoneal and noninvasive mesotheliomas (2000)

Malignant mesothelioma (MM) is a tumor associated with asbestos exposure and that originates from the serosal lining of the pleural, peritoneal or pericardial cavities (Antman et al., 1997). Simian virus 40 (SV40) is a well-known oncogenic DNA virus of papovaviridae class, whose oncogenic potential is associated with an early gene product, large T antigen (Tag) (Fanning and Knippers, 1992). SV40 causes MM (Cicala et al., 1993) and other cancers in hamsters. There have been several reports of SV40 DNA sequences in MM of pleural origin (Carbone et al.,1994, Carbone et al., 1999; Cristaudo et al., 1995; De Luca et al., 1997; Pass et al., 1998). However, some studies have not confirmed these findings (Linnainmaa, 1997; Mulatero et al., 1999; Strickler et al., 1996), and the viral association with MM is highly controversial. Two groups have published contradictory results from a single country, the UK (Mulatero et al., 1999; Pepper et al., 1996). In addition to this controversy, other aspects of the relationship between SV40 and MM remain to be explored. It is not known whether SV40 sequences are present in MMs of nonpleural origin. In addition, it is not known whether SV40 sequences can be detected at a noninvasive stage during the pathogenesis of MM. In this report, we address these questions and compare the frequencies of SV40 sequences in MMs arising in the USA and Germany. Formalin-fixed and paraffin-embedded samples of pleural, peritoneal and noninvasive mesotheliomas were obtained from Germany and the USA. Areas of malignant cells were either precisely microdissected under microscopic observation or scraped with a razor blade and used for DNA isolation. Corresponding normal lung or stromal tissue provided a source of constitutional DNA. SV40 Tag sequences were specifically amplified using PCR as described by and analyzed by gel electrophoresis and DNA sequencing (Carbone et al., 1999; Cristaudo et al., 1995; Sanger et al., 1977). Fisher's exact two-tailed test was used for statistical evaluation of the differences between SV40 Tag frequencies from 2 different groups. Probability values of p < 0.05 were regarded as statistically significant. As illustrated in Figure 1, 44/78 (56%) of pleural MMs were positive for SV40 Tag sequences. Our findings, using formalin-fixed, paraffin-embedded microslides, are similar to those reported by Carbone et al. (1994), who found a frequency of SV40 sequences in 60% in frozen samples of human MMs. Similar findings were reported by Pepper et al. (1996). There were no significant differences between the frequencies of SV40 TAG sequences in pleural MMs from the USA (11/21, 52%) or from Germany (33/57, 58%). A multi-institutional international study has confirmed that SV40 sequences are frequently present and expressed in mesotheliomas in the USA (Testa et al., 1998). However, no direct comparison of viral sequences in the tumors arising in different countries or continents has been previously reported. (a) Agarose gel electrophoresis of PCR-amplified SV40 Tag region from representative SV40-positive pleural MMs and corresponding normal stroma (cases 4, 9 and 25) and from representative SV40-positive peritoneal MMs and corresponding stromal tissue (cases 31 and 52). (b) Agarose gel electrophoresis of PCR-amplified SV40 Tag region from 2 SV40-positive noninvasive mesotheliomas (cases 2 and 10). N: normal stroma; R: reactive mesothelium; P: pre-invasive component; T: invasive component. (c) Agarose gel electrophoresis of PCR-amplified SV40 Tag region from representative lung carcinomas (cases 1–5 were small-cell lung carcinomas, while cases 6–10 were non-small-cell lung carcinomas). Peritoneal MMs are relatively rare tumors, occurring less frequently than pleural MMs (Antman et al., 1997). In men, both pleural and peritoneal MMs are attributable to asbestos exposure (Spirtas et al., 1994). Seven of 11 (64%) peritoneal MMs (all from Germany) were positive for SV40 Tag sequences (Fig. 1). These findings ascertain the association of DNA virus-like sequences with MMs of nonpleural origin. Because there is no mesothelial specific marker that discriminates consistently between normal, hyperplastic and neoplastic mesothelial tissue (Henderson et al., 1998), distinction between reactive mesothelial hyperplasia and epithelial mesothelioma in biopsy tissues can pose a major problem for the surgical pathologist. The diagnosis of noninvasive or in situ mesothelioma can be made with certainty only when invasive epithelial mesothelioma is demonstrable in the same specimen (Henderson et al., 1998). We examined 12 cases in which invasive and noninvasive components were present in pleural MM cases (all from Germany). In 4 cases, SV40 TAG sequences were present both in the invasive and noninvasive components (Figs. 1, 2). There were no examples of discordant expression between the 2 components. In one example, nonmalignant reactive epithelium was present in addition to invasive and noninvasive components of MM (Fig. 2). The invasive and noninvasive components of MM were positive for SV40 TAG sequences, while the adjacent reactive epithelium was negative (Fig. 1). We thus demonstrate the presence of SV40 sequences during the early stages of mesothelioma development. This observation suggests that SV40 sequences are present before the development of tumor invasion and that virus infection may play a role in MM pathogenesis. The presence of SV40 sequences may help differentiate reactive mesothelial hyperplasia and MM. Photomicrographs of an SV40-positive pleural mesothelioma showing reactive mesothelium (a) and pre-invasive (b) and invasive (c) components (Fig. 1b, case 2). In c, arrow indicates cancer cells invading stroma. Corresponding normal lung tissue or stroma from 54 MM cases (17 cases of pleural MMs from Germany, 21 cases of pleural MMs from USA, 4 cases of peritoneal MMs from Germany and 12 cases of noninvasive mesotheliomas from Germany) were negative for SV40 Tag sequences. In addition, 20 lung carcinomas from the USA (8 small-cell and 12 non-small-cell carcinomas) were also negative for SV40 Tag sequences. Representative examples of the PCR amplification of the 105 bp SV40 Tag and agarose gel electrophoresis are illustrated in Figures 2a–c. These findings contradict those of Galateau-Salle et al. (1998), who found SV40 sequences in bronchopulmonary carcinomas and nonmalignant lung tissues. However, our findings support those of Pepper et al. (1996), who demonstrated the lack of SV40 sequences in lung carcinomas and in nonmalignant lung, provided that the appropriate primer sequences were utilized. Our findings confirm and extend previous reports that: 1. SV40 sequences are frequently present in MMs and are absent in adjacent lung tissues and in lung carcinomas; 2. archival paraffin-embedded materials are suitable for study; 3. the frequencies of viral sequences in the USA and Germany are similar; 4. SV40 sequences are present in MMs of extra-pleural origin; and 5. viral sequences are present relatively early during the pathogenesis of MMs. Yours sincerely, Narayan Shivapurkar, Thorsten Wiethege, Ignacio I. Wistuba, Sara Milchgrub, Klaus-Michael Muller, Adi F. Gazdar [email protected]