ROLE OF SV40 IN HUMAN CANCER
Experimental Approaches [Ο ΡΟΛΟΣ ΤΟΥ SV40 ΣΤΟΝ ΚΑΡΚΙΝΟ ΤΟΥ ΑΝΘΡΩΠΟΥ Πειραματικές Προσεγγίσεις]
During the last decade, many studies have shown the presence of SV40 large T-ag DNA or other viral markers in primary human brain and bone cancers, malignant mesotheliomas, and NHL (Fig. (Fig.6).6). Sequence analyses (Fig. (Fig.33 and and5)5) and detection of T-ag protein (Fig. (Fig.7)7) ruled out laboratory contamination of tumor samples. Importantly, infectious SV40 was isolated from a primary brain cancer of a 4-year-old child (62). An important consideration when evaluating the molecular biology data is the sensitivity of methods used to detect SV40 in human tumor samples. Early studies (before 1992) identified SV40-positive neoplasms by using indirect immunofluorescence for viral proteins or DNA hybridization techniques (55, 74, 130), whereas studies after 1992 generally used PCR-based assays.
Detection of SV40 T-ag DNA in NHLs. (A) PCR-amplified polyomavirus sequences after agarose gel electrophoresis and staining with ethidium bromide (upper panel) and after Southern blotting with oligonucleotide probes specific for individual polyomaviruses (lower panels). Lane M, molecular weight marker. Positive control reactions included plasmids specific for SV40 (SV40+), JCV (JCV+), and BKV (BKV+). The negative control (NC) was a reaction without added DNA template. CA, cancer control samples (colon and breast cancers). Hybridization of the PCR products to an oligonucleotide probe specific for SV40 identified the lymphoma-associated viral sequences as SV40 specific; JCV and BKV were not detected. (B) Sequence analysis of the PCR product from the N terminus of the T-ag gene. The sequence from the lymphoma sample (NHL) is identical to that of SV40 and distinguishable from those of JCV and BKV. From reference 125; used with permission.
(A) Immunohistochemical detection of SV40 T-ag in a diffuse large B-cell NHL. (B) Negative control specimen (a reactive lymph node). Specimens were stained by using a monoclonal antibody, PAb416. Note the strong nuclear staining of the majority of lymphoma cells. Magnification, ×400.
During the last three decades more than 60 original studies have reported the detection of SV40 in primary brain and bone cancers, malignant mesothelioma, and NHL, whereas a few studies have described an absence of SV40 in those malignancies (16, 33, 34, 44, 48, 70, 113). However, the small numbers of samples tested, the histologic types of malignancies examined, and the laboratory methodologies employed in some cases limit the significance of the results in those studies reported to be negative. Indeed, several steps need to be considered when performing molecular studies of human specimens (1, 50, 61, 107). First, the extraction step of nucleic acids determines whether tissues yield adequate and suitable DNA or RNA for analysis. Unfortunately, with formalin-fixed and paraffin-embedded specimens, degradation of nucleic acids and proteins is a common problem, and the quality of recovered DNA may be poor. If only small amounts of paraffin-embedded tissues are available, the yield of nucleic acids may be inadequate for analysis. Primers directed to a human cellular gene should be used to establish the suitability of a sample for PCR analysis. Because of the sensitivity of PCR-based assays, it is important to rigorously guard against laboratory contamination of samples and controls during processing or testing. Tissue processing and PCR assay setup should be performed in different facilities, from which positive controls (i.e., plasmids) are excluded. Negative tissue controls, extracted and analyzed in parallel, should be included in each experiment to monitor for reagent contamination. The selection of primers and PCR conditions greatly influences the sensitivity and reliability of the assay. Another factor is that tumor specimens usually contain mixtures of normal and malignant cells, in varying proportions. Variations in one or more of these important parameters may explain, at least in part, the ranges in positivity observed among some positive studies and the results obtained in some negative studies.
Summary and Meta-Analysis of Controlled Studies
Table Table22 provides a timeline for landmark discoveries associating the polyomavirus SV40 and human malignancies. Although numerous studies have detected SV40 in human primary brain and bone cancers, malignant mesothelioma, and NHL, the small sample sizes and the lack of a control group in some studies made it difficult to make conclusions about the extent to which SV40 may be associated with those human cancers. For this reason, we conducted a meta-analysis of controlled studies (122), an approach which can provide a more balanced and less biased estimate of the evidence than individual studies (57). For inclusion in the meta-analysis, reports had to meet the following criteria: studies were conducted among patients with primary malignancies, the investigation of SV40 was performed on primary cancer specimens and not on cultured cells, the analysis included a control group, and the same laboratory technique was used for both case and control samples. These criteria were established because the use of appropriate controls is crucial in the proper analysis of tissue for viral DNA, especially considering the sensitivity of PCR techniques (38). Thirty-five independent studies met these inclusion criteria. In total, data from 1,793 patients with primary malignancies were evaluated to determine whether SV40 is significantly associated with primary brain cancer, malignant mesothelioma, bone cancer, and NHL.
TABLE 2.
Timeline for discoveries associating SV40 and human malignancies
| Yr | Discovery |
|---|---|
| 1970s-1980s | Pre-PCR: SV40 and brain cancers |
| 1992 | SV40 DNA (PCR) and expression of large T-ag in brain cancers |
| 1994 | SV40 DNA and expression of large T-ag in malignant mesotheliomas |
| 1995 | Infectious SV40 isolated from a brain cancer of a 4-year-old child |
| 1996 | SV40 DNA in bone cancers |
| 2002 | SV40 DNA in lymphomas |
| 2002 | Institute of Medicine concluded that “SV40 exposure could lead to cancer in humans under natural conditions” |
Thirteen studies fulfilled the criteria for the investigation of primary brain cancers (Table (Table3).3). The combined odds ratio (OR) of the studies used in the analysis was 3.9 (95% confidence interval [CI], 2.6 to 5.8). This effect was based on specimens from a total of 1,143 patients, of which 661 were primary brain cancer samples and 482 were control specimens. A modifier detected was the type of sample analyzed (paraffin embedded versus frozen). The adjusted OR was 3.8 (95% CI, 2.6 to 5.7). For malignant mesothelioma, 15 studies fulfilled the criteria; the combined OR of analysis was 16.8 (95% CI, 10.3 to 27.5) and was based on 528 patients with malignant mesothelioma and 468 controls (Table (Table4).4). Modifiers detected were the type of control tissue and the method of detection of SV40. The adjusted OR was 15.1 (95% CI, 9.2 to 25.0). The combined OR of the analysis of bone cancers and SV40 was 24.5 (95% CI, 6.8 to 87.9) and was based on 303 patients with bone tumors and 121 controls from four reports (122). The OR for NHL was 5.4 (95% CI, 3.1 to 9.3) and represented 301 cases and 578 controls included in three studies (Table (Table5).5). Because there were only three studies that fulfilled the inclusion criteria, further examination of modifying variables was not possible for NHL.
TABLE 3.
SV40 in primary brain tumorsa
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TABLE 4.
SV40 in malignant mesotheliomasa
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TABLE 5.
SV40 in NHLsa
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This analysis of published reports found a significant excess risk of SV40 associated with human primary brain cancers, malignant mesotheliomas, bone cancers, and NHL compared to control samples. Therefore, the major types of human malignancies associated with SV40 are the same as those induced by SV40 in animal models. Although the proportion of human cancers containing SV40 varied from study to study, viral prevalence was always greater among primary tumors than among control tissues. Importantly, analysis of data indicated that SV40 may be etiologically meaningful in the development of a specific subset of human cancers. Multiple studies have shown the expression of SV40 mRNA and/or T-ag in cancer cells, the integration of SV40 sequences in some cancers, and SV40 T-ag protein complexed with p53 and pRb in some tumor specimens (1, 10, 13, 39, 50, 76, 122). These findings are compatible with current understanding of how SV40 T-ag mediates oncogenesis. Moreover, microdissection of human malignant mesothelioma samples followed by PCR detected SV40 T-ag DNA only in cancer cells and not in adjacent nonmalignant cells (1, 39, 104). These results from different experimental studies support the conclusion of the Institute of Medicine (111) that “the biological evidence is of moderate strength that SV40 exposure could lead to cancer in humans under natural conditions.”
