Investigation on the presence of polyomavirus, herpesvirus, and papillomavirus sequences in colorectal neoplasms and their association with cancer

Studies in literature suggest that some human viruses, including JC polyomavirus (JCV),1-3 human cytomegalovirus (HCMV),4 human papillomaviruses (HPVs),5-8 and Epstein-Barr virus (EBV),9, 10 have a pathogenic role in starting or promoting colorectal cancer. However, a revision of the literature indicates that this association between colorectal tumors and viral infection is still matter of debate11-15 and further studies using robust methods to detect viral infection are required. In fact, the prevalence of viral infection in colorectal cancer samples varies widely among series, and this variability appears to be mainly related to technical aspects, such as in the case of JCV, for which the prevalence in colorectal tumors has been reported to range from 011 to 90%1-3, 16 in different studies. In our study, we used sensitive quantitative real-time PCR analysis and PCR and sequencing to investigate the prevalence of genome sequences not only of JCV, but also of other polyomaviruses which have been involved in human infection, i.e., BK virus (BKV), Simian virus 40 (SV40), and the newly discovered Washington University polyomavirus (WUV), Karolinska Institute polyomavirus (KIV) and Merkel cell carcinoma polyomavirus (MCV),17 in a large series of colorectal adenocarcinomas, adenomas and normal mucosa samples. Moreover, in the same samples, we investigated the presence DNA sequences of the herpesviruses HCMV and EBV and HPVs, which have been also suggested to be associated with colorectal tumorigenesis. A detailed description of methods is reported in the supporting information table. Patients gave informed consent to the study, which was approved by the local ethics committee and performed according to Declaration of Helsinki guidelines. Originally, the aim of our study was to investigate the prevalence of JCV infection in colorectal cancers from different age groups. To this aim, we first analyzed 144 archival samples of formalin-fixed paraffin-embedded tissues (group 1, Table I), collected at the Department of Medical Diagnostic Sciences and Special Therapies of the University of Padova in the period ranging from 1998 to 2007. Group 1 included colorectal adenocarcinomas and adjacent mucosa, from 72 patients, who were selected on the basis of age (36 patients were ≤45 years, age range 32–45 years; 36 patients were ≥75 years, age range 75–89 years) and matched for gender (14 males and 22 females in both subgroups), tumor site (28 in sigmoid, 30 in ascendent, 11 in descendent, and 3 in rectal colon in both subgroups), and stage (11 stage I-II, 17 stage III, 8 stage 4 in both subgroups). Quite unexpectedly, all samples of group 1 tested negative for JCV DNA at real-time PCR analysis. So, we tried other 3 validated primers and probe sets to detect JCV DNA and investigated whether genome sequences of other polyomaviruses, including the newly discovered viruses, were detectable in colorectal samples. But, no BKV, SV40, KIV, WUV and MCV DNA was detectable in any sample (Table I). Regarding the study of herpesviruses, 3 cancers and 1 cancer-adjacent mucosa from group 1 were positive for EBV DNA; likewise, 2 cancers and 2 cancer-adjacent mucosa samples were HCMV DNA-positive, without significant differences between age groups and among tumor sites and stages (Table I). Analysis of HPV infection demonstrated that 2 sigmoidal cancers from a male and a female patient <45 years of age were positive for HPV-16 DNA (Table I). The low frequency of viral DNA detection in this group of archival tumor samples prompted us to subsequently investigate fresh-frozen tissue samples to obtain better quality DNA. In particular, we investigated colorectal biopsies and tissue samples, which were consecutively collected during colonoscopy or surgery performed at the Department of Surgical and Gastroenterological Sciences of the University of Padova in the period ranging from January 2007 to July 2007 (group 2, Table II). Group 2 samples were obtained from 22 patients with colorectal adenomas (2 hyperplastic, 15 tubular, 5 tubulovillous) and 28 patients with colorectal adenocarcinoma (1 stage 0, 5 stage I, 8 stage II, 10 stage III and 4 stage IV). Mean age was 71.6 years (range, 41–92 years), 24 were males and 26 females. Overall, 5 tumors were located in caecum, 8 in ascendent, 1 in transvers, 4 in descendent, 17 in sigmoid colon and 15 in rectum. From each patient, pathologic tissues and tissues at about 3 and 10-cm distance from the lesions were collected. In addition, we analyzed biopsies of rectal, ascending and transverse colonic mucosa, obtained from 15 control subjects without medical history of colorectal pathology and without detection of lesions at colonoscopy (mean age, 58 years; age range, 36–82 years; 10 males, 5 females) (group 3). Like in group 1, no polyomavirus DNA sequences were detected in any sample of groups 2 and 3 (Table II). Regarding herpesviruses, EBV-DNA was detected in 11 colorectal cancers (including 2 with positive adjacent mucosa), in 5 adenomas (including 4 with positive adjacent mucosa), in 2 samples of 3-cm-adjacent mucosa, and in 1 sample of 10-cm-adjacent mucosa (with negative associated cancer or adenoma samples); HCMV-DNA was detected in 3 carcinomas (including 1 with positive adjacent mucosa) and in 1 sample of 10-cm-adjacent mucosa, but not in adenoma samples (Table II). EBV and HCMV-DNA load in tissue samples, calculated as the number of viral genome equivalents per cell, was very low in both tumor samples and adjacent mucosa (median EBV DNA load, 50 copies/106 cells; range, 10–10,000 copies/106 cells; median HCMV DNA load, 70 copies/106 cells; range, 10–30000 copies/106 cells) and consistent with lymphocyte/macrophage infiltration, rather than infection of tumor cells. The rate of EBV-DNA detection in carcinoma and adenoma samples was significantly higher than in adjacent mucosa (χ2-test, p < 0.05) and was explained by a greater inflammatory cell infiltration in neoplastic tissues than in normal mucosa. In the control group (group 3), EBV-DNA was detected in 5 out of 15 subjects (in the rectal mucosa in 2 cases, in the ascending colon in 2, and in all 3 colon samples in one subject); HCMV-DNA was detected in the rectal mucosa from a patient who was also EBV-positive. High-risk HPV-16 DNA was detected by both nested PCR/sequencing and real-time PCR in 2 rectal carcinomas and in a sigmoid adenoma of group 2, but not in control group 3 samples (Table III). In the 3 positive cases, the adjacent mucosa was also HPV-16 DNA-positive. In the literature, JCV genome sequences were detected by PCR amplification and Southern blot hybridization in 90% of colorectal cancers and in the adjacent normal colonic epithelium, with a 10-fold higher viral load in cancers than in adjacent mucosa,1, 2 as well as in 82% of sporadic adenomatous polyps.18 In colorectal mucosa, JCV was suggested to promote tumorigenesis through a direct interaction of its large T antigen (Tag) with β-catenin and p53.2, 3 Since a variant of the JCV Mad-1 strain was preferentially detected in colon cancers, it was hypothesized that this strain might be selectively activated in colonic epithelial cells.19 It cannot however be excluded that detection of a unique JCV strain represented a laboratory contamination, since the same Mad-1 strain was used as positive control in the laboratory.11 In our study, we used real-time PCR to detect viral sequences. This method is very sensitive and specific, but much less susceptible to amplicon contamination than PCR followed by Southern-blot, which was employed in the other studies. A study on large series of colorectal cancer/normal tissue pairs,11 which also employed real-time PCR, failed to demonstrate the presence JCV-DNA sequences in all samples, in agreement with our results. Immunostaining for SV40 TAg has been reported to give problems of false positive results,20 so it cannot be excluded that reported JCV TAg detection in colorectal tissues is also a technical artefact, since the same antibodies against SV40 TAg were employed to investigate JCV expression, exploiting their cross-reactivity with JCV Tag.2, 18 Reports regarding detection of BKV and SV40 in colorectal cancers are conflicting, with negative results in 1 study,21 in agreement with our data, but positive findings in a recent Italian report which demonstrated BKV and SV40 DNA in 6 and 10 out of 66 colorectal carcinomas, respectively.22 Problems of PCR contamination cannot be excluded even in this study, since the primers employed could amplify SV40 DNA sequences which are present in some laboratory plasmids.22 Our data seem also to exclude WUV, KIV and MCV polyomavirus have any role in colorectal tumorigenesis. HCMV has also been implicated in colorectal tumorigenesis because of its detection in about 80% of tumors, but not in adjacent non-neoplastic mucosa,4, 23 and because of the demonstration that HCMV infection of colorectal cancer cell lines in vitro resulted in induction Bcl2 and cycloxygenase-2 proteins, both of which are involved in important pathways of colorectal cancer progression.4 In contrast, other studies failed to detect HCMV DNA sequences in tumour samples.12-14 Regarding EBV, it has been associated with gastric adenocarcinoma and gastric lymphoepithelioma, whereas its involvement in colorectal tumorigenesis seems to be excluded by several studies which, generally, did not detect EBV-positive cancer cells, even though some EBV-positive tumor infiltrating B lymphocytes were frequently found.9, 24-26 However, we do not rule out the possibility that the occasional presence of EBV and HCMV in infiltrating lymphocytes/monocytes in colorectal mucosa may promote the production and release of cytokines and growth factors or deregulate tumor suppressor genes and thus cooperate with cancer development. Among candidate oncogenic viruses for colorectal tumorigenesis, HPV is the most reliable, as indicated by the epidemiological evidence of high-risk HPV infection in a relatively high percentage of colorectal tissues from patients with cancer, but not in control subjects without cancer.5-8 Accordingly, we also identified HPV-16 DNA in some cases of distally localized colorectal cancers. Persistent high-risk HPV infection has been definitely demonstrated to be a necessary cause for development of cervical cancer and other anogenital cancers27, 28 and might also be responsible for some cases of colorectal cancer. While waiting for further epidemiological data and for experimental studies to confirm this hypothesis, we can predict anti-HPV vaccination might have an impact on colorectal cancer prevention. In conclusion, our study, which for the first time systematically investigated the presence of genomic sequences of several putative oncogenic viruses in a large series of colorectal neoplasms, seems to exclude JCV, BKV, SV40, WUV, KIV, MCV, HCMV and EBV have a prominent role in the pathogenesis of colorectal cancer, whereas it suggests high-risk HPV could be involved in some cases of distally localized colorectal carcinoma. Yours sincerely, Valentina MILITELLO, Marta TREVISAN, Laura SQUARZON, Maria Angela BIASOLO, Massimo RUGGE, Carmelo MILITELLO, Giorgio PALÚ and Luisa BARZON Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.