Recent findings shed light into the relationship between tumor development and infections by several viruses. This culminates in the development
of preventive vaccinations against cervical carcinoma associated with HPV high-risk types [2]. A causal relationship between the development of CRC and viral infections has repeatedly been suggested and this could, if proved, open new avenues for similar therapeutic and preventive approaches.
Our study aimed at deciphering a potential involvement of several oncogenic DNA viruses in CRC development. The starting hypothesis was that if viruses would have an active role in the development of CRC, their footsteps in form of virus-encoded nucleotide sequences should be present in patients’ CRC cells. In order to avoid any bias by non-tumor cells present in the tumor microenvironment, we focused on patient-individual, low passage CRC cell lines; but corresponding patient tissue samples were analyzed in addition. Using highly sensitive PCR approaches, we investigated the natural occurrence of the polyomaviruses SV40, JCV and BKV as well as the herpesvirus EBV – the most frequently discussed viruses with a potential role in CRC development [9, 15, 21, 13, 10, 23, 24, 25, 26]. First, both patient-individual CRC cell lines (n = 49) and corresponding tissue samples from patients (normal and tumor tissue, as well as liver, lung and peritoneal metastases) were analyzed for the presence of viral sequences by end-point PCR. But in none of the tested samples, viral sequence amplification was successful.
Regarding a potential SV40 infection, none of the 49 low-passage patient-derived cell lines was positively tested for SV40. This finding excludes SV40 infection at least as a frequent event in CRC cells. It is well known that SV40 might act as a cofactor in the carcinogenesis of several carcinomas; but its involvement in CRC oncogenesis has been examined poorly up to now [27]. Only one case control study by Campello et al. analyzed the involvement of SV40 in CRC pathogenesis [9]. However, only six out of 94 analyzed patients’ tumors were tested positive for SV40 and the authors could not delineate a relationship between viral infection and CRC. An explanation for the differences to the results of the present study could be the extracellular presence of viral particles – either between the cells, more likely in infiltrating lymphocytes or macrophages, and possibly even in the lumen of the gut.
An infection of the analyzed cell lines with the polyomaviruses JCV and BKV could be ruled out likewise. Hitherto existing data on prevalence of JCV infections in CRC are very heterogeneous. Several small case control studies were reported. A study from Portugal could show a prevalence of 90% JCV infections in samples of CRC lesions [15] and in another study from Greek about 60% of the samples tested positive for JCV [28]. A Chinese study of 137 samples described a rate of infection in CRC of 40.9% [13]. But in contrast, a large American study failed to prove JCV infections in 233 patient samples [29] and a study from Iran described a positive detection rate of only 1.46% (2 of 70 patients) [30]. Our own results are pretty much in line with the latter findings and also indicate that JCV is unlikely involved in CRC development.
Worldwide ninety percent of adults have been exposed to BKV. BKV DNA has been detected in several human cancers, including carcinoma of the lung, urogenital tract, liver, head and neck, in rhabdomyosarcoma and in brain tumors [17, 31]. However, reports on detection of BKV DNA in CRC tissues are very scarce. A study by Giuliani and colleagues could detect BKV DNA in 9% out of 66 samples [23] and Casini et al. found BKV DNA in several CRC tissues [10]. Again, in our study population in none of the examined cell lines or tissue samples BKV DNA could be detected.
Finally, all cell lines and primary materials were examined for the presence of EBV. Besides non-specific PCR products, presence of EBV DNA could not be verified. All analyzed samples must therefore be considered as EBV negative. Militello et al. described a very low positive detection frequency for EBV DNA in cancerous and cancer-adjacent mucous samples [32] and a study by Cho et al. could not detect EBV in 274 CRC specimen [33]. In contrast, several studies reported a high EBV DNA positivity of CRC tissues [24, 25, 26. Interestingly, a study by Fiorina et al. demonstrated EBV in 23 of 44 CRC samples but they could prove that this was restricted to latency in the lymphoid infiltrate of the tumors [34]. This latter observation delivers the best explanation for the inconsistencies between the before mentioned results. EBV most effectively infects B cells and rarely other lymphoid cell populations. In cultured epithelial-derived cells with no contaminating lymphocytes left over from the initial culturing process, a cross-contamination of the PCR results can safely be excluded. Of note, such a large panel of patient-individual cell lines with corresponding tissue samples has never been tested before. The primary CRC tissues included into our analysis were obtained from the invasive margins which are especially rich in (vital) tumor cells – but infiltrating T cells and also B cells are also readily detectable [35]. Thus, we cannot exclude sporadic positive results in EBV testing due to EBV-positive lymphocytes present in the tumor microenvironment, if a larger sample set of our primary CRC tissue collection would be analyzed.
Based on the data obtained in this work for SV40, JCV, BKV and EBV, it can be concluded that none of the tested viruses are likely to have an obvious general role in CRC development. Possible reasons for the variation in the detection rate of these viruses in our and several of the aforementioned studies, are beside the lymphocytic contamination of the specimens [36], differences in the detection limit of the applied detection systems but possibly also the genetic background of the different patient populations.
In order to verify the general negative findings on a more functional level, we tested exemplary the susceptibility of the CRC cell line HROC257 T0 M1 to JCV and BKV infection. However, even after an extended cultivation period subsequent to viral inoculation, no evidence for a successful viral infection could be provided. This led us to conclude that CRC cells are not susceptible to JC or BK virus infection and thus further supports the negative screening results.