|Year : 2014 | Volume
| Issue : 3 | Page : 531-534
Lack of human papillomavirus DNA in colon adenocarcinama and adenoma
Arezoo Aghakhani1, Rasool Hamkar2, Amitis Ramezani1, Farahnaz Bidari-Zerehpoosh3, Shahram Sabeti3, Nastaran Ghavami2, Mohammad Banifazl4, Niloofar Rashidi1, Ali Eslamifar1
1 Department of Clinical Research, Pasteur Institute of Iran, Tehran, Iran
2 Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
3 Pathology Ward, Loghman Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Iranian Society for Support of Patients with Infectious Diseases, Tehran, Iran
|Date of Web Publication||14-Oct-2014|
Department of Clinical Research, Pasteur Institute of Iran,No 69, Pasteur Ave., Tehran, 13164
Source of Support: This project was financially supported by Pasteur Institute of Iran, Conflict of Interest: None
Background: Human papilloma viruses (HPV) have been detected in several types of cancers. Over the last few years, a possible correlation between HPV infection and colon cancers has been suggested. The aim of this study was to assess the presence of HPV-DNA in colon adenocarcinomas and adenomas to describe the relationship of HPV infection with these pathologic features.
Materials and Methods: The study included formalin-fixed and paraffin-embedded tissue samples of 70 patients with colon adenocarcinoma and 30 patients with adenoma (as study group) and 30 tumor adjacent tissues (as control). HPV-DNA was purified and first amplified through MY09/MY11 and GP5+/GP6+ primers. Subsequently, for more precision, another PCR was performed using PGMY09/11 L1 consensus primer system.
Results: All tested adenocarcinomas and adenomas as well as normal tumor adjacent tissues were negative for all types of HPV in two PCR assays.
Conclusion: Our results do not support the relationship between HPV infection and colon carcinoma or adenoma. Attributing a role to the HPV in the etiology of colon carcinogenesis will require further studies.
Keywords: Adenoma, adenocarcinoma, colon, human papilloma virus
|How to cite this article:|
Aghakhani A, Hamkar R, Ramezani A, Bidari-Zerehpoosh F, Sabeti S, Ghavami N, Banifazl M, Rashidi N, Eslamifar A. Lack of human papillomavirus DNA in colon adenocarcinama and adenoma. J Can Res Ther 2014;10:531-4
|How to cite this URL:|
Aghakhani A, Hamkar R, Ramezani A, Bidari-Zerehpoosh F, Sabeti S, Ghavami N, Banifazl M, Rashidi N, Eslamifar A. Lack of human papillomavirus DNA in colon adenocarcinama and adenoma. J Can Res Ther [serial online] 2014 [cited 2020 Feb 22];10:531-4. Available from: http://www.cancerjournal.net/text.asp?2014/10/3/531/137674
| > Introduction|| |
Colorectal cancer is a major cause of morbidity and mortality throughout the world with more than 1 million new cases diagnosed per year. , It accounts for over 9.4% of all cancer incidences.  It is the third most common cancer worldwide and the fourth most common cause of death.  Colorectal adenomatous polyps, or adenomas, are the most common type of polyps and make up about 70% of the polyps found in the colon. They are well-established precursor lesions; however, only about 5% of these adenomas ever progress to cancer. , Approximately 95% of colorectal cancers are adenocarcinomas, and they usually originate from pre-existing adenomas via the adenoma-carcinoma sequence.  The risk factors for colorectal carcinoma and adenoma are similar. 
Human papilloma viruses (HPV) have been detected in several types of epithelial cancers such as cervix, vulva, penis, anus, oral cavity, esophagus, and larynx. ,,,,, Over the last few years, a possible correlation between HPV infection and colorectal cancers and adenomas has been suggested, ,,,,,,, but results are inconsistent. Some studies reporting HPV-DNA in various prevalence in colon cancers and adenomas, ,,,, but some investigations failed to establish a link between HPV and colon malignancy. ,,, Therefore, the possible role of HPV infection in colon carcinogenesis is still a subject of great controversy.
Identification of HPV as a predisposing factor for colon cancer could be an important discovery and have significant implications in public health, identifying high risk groups, patient care, cancer prevention, and developing new therapies.  So, the purpose of current study was to assess the presence of HPV-DNA in colon adenocarcinomas and adenomas to describe the relationship of HPV infection with these pathologic features.
| > Materials and methods|| |
Formalin-fixed and paraffin-embedded tissue samples of 70 patients with colon adenocarcinoma and 30 patients with adenoma (as study group) and 30 tumor adjacent tissues (as control) were provided for analysis. The study was approved by Pasteur Institute of Iran Ethics Committee. Clinicopathological data including sex, age, histological differentiation, and tumor location were recorded.
Paraffin blocks were re-cut, and prepared slides were stained with hematoxylin and eosin for histopathological review, confirming the original diagnosis and circling areas for micro dissection.
Sections of 5-10 μm wide were prepared from each specimen, avoiding any cross-contamination between samples. Sections were subsequently deparaffinized by xylene and digested using digestion buffer containing proteinase K, followed by extensive extraction with phenol/chloroform. DNA quality was evaluated by PCR using primers PCO3/PCO4 that amplify a 110 bp product from the human β-globin gene. β-globin positive samples were subjected to nested PCR.
First, the samples were screened for the presence of HPV using the standard nested PCR approach consisting of the MY09/11 primer set (outer primers) and the GP5+/6+ primer set (inner primers).
The MY09/MY11 primer set [MY9 (5' - CGT CCA/C AA/GA/G GGA A/TAC TGA TC - 3')] and [MY11 (5' - GCA/C CAG GGA/T CTA TAA C/TAA TGG - 3)], which amplify the L1 gene of HPV was capable of amplifying a wide spectrum of HPV types to produce a PCR product of 450 bp. Second rounds of PCR were done using GP5+/GP6 + primers. The GP5+/GP6 + primer set [GP5+ (5΄- TTT GTT ACT GTG GTA GAT ACT AC-3΄)] and [GP6+ (5΄-AAA AAT AAA CTG TAA ATC ATA TTC-3΄)] is a non-degenerate primer set that detects a wide range of HPV types using a lower annealing temperature during PCR and produces a PCR product of approximately 150 bp. The PCR was performed as previously described by Aghakhani et al. 
As MY09/11 and GP5+/6 + may be demonstrated lower detection rates, specimens were also tested by amplifying 10 μl of the DNA extracts with the PGMY09/11 L1 consensus primer system, which are the preferred primer set in PCR screening of HPV in different clinical samples.  The PGMY primer PCR system amplifies mucosal HPV types, producing a 450 bp amplicon. This system also includes primers for a cellular target (HLA), producing an amplicon of 230 bp, which confirms that DNA has been extracted in sufficient amount and inhibitors are not preventing amplification. PGMY09/11 primers are listed in [Table 1].
In brief, each amplification contained 15 μl Hot start master mix, 3 mM MgCl2, 80 nm of each of PGMY09 primers, 80 nm of each of PGMY11 primers, 20-40 nm of each of HLAdQ Primers, in a 30 μl total volume. For every 10 samples, a negative control (H2O) and a positive control (HPV 16 genome extracted from human cervical cancer sample) were run to control for contamination and accuracy. The samples were amplified by the following amplification profile: 95°C hot start for 9 min, 95°C denaturation for 30 sec, 55°C annealing for 90 sec, and 72°C extension for 2 min for 45 cycles, followed by a 5-min terminal extension at 72°C and a hold step at 4°C. PCR products subjected to 1.5% gel electrophoresis to determine presence of HPV in samples.
The Chi-square was used with the SPSS 16 package program for statistical analysis (Chicago, IL, USA). Data was presented as mean ± SD or, when indicated, as an absolute number and percentage.
| > Results|| |
Formalin-fixed and paraffin-embedded tissue samples of 70 colon adenocarcinoma patients with mean age 61.6 ± 13.7 years (range: 40-86 year) and 30 adenoma cases with mean age 61.6 ± 12.8 years (range: 33-84 year) were analyzed for presence of HPV-DNA. Thirty tumor adjacent tissues (5 cm distance from the tumor) were also tested as control specimens. Histopathological characteristics and localization of the colon adenocarcinomas and adenomas are summarized in [Table 2] and [Table 3].
|Table 2: Demographic, tumor localization, and histopathological grade of the colon adenocarcinoma cases|
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|Table 3: Demographic, localization and histopathologic type of the colon adenomas cases|
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All tested adenocarcinomas and adenomas as well as normal tumor adjacent tissues were negative for all types of HPV in two PCR assays [Figure 1].
|Figure 1: PGMY09/11 PCR amplicons of colorectal adenocarcinoma samples with positive and negative controls. Lanes from left to right: 100 bp DNA marker, negative control, positive control, and samples. 450 bp amplicon represents of HPV positivity, and 230 bp amplicon represents of HLA and confirms that DNA has been extracted in sufficient amount|
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| > Discussion|| |
This study investigated the presence of HPV-DNA in colon adenocarcinomas and adenomas to determine the relationship between HPV infection and colon carcinogenesis. We didn't find HPV-DNA in colon adenocarcinoma or adenoma specimens regardless of tumor grade, location, or adenoma type.
Colorectal cancer is accounting for 9.4% of all cancers worldwide. This cancer is the third most common cancer among women and fourth most common among men.  Adenomatous polyps or adenomas are colonic benign lesions that potentially can develop into cancer. 
Oncogenic papilloma viruses, in particular HPV types 16 and 18, have been shown to be involved in malignant lesions of several sites, such as cervix, prostate, bladder, esophagus, larynx, and oral cavity. ,,, The evidence regarding a link between HPV infection and colon carcinoma or adenoma is gradually accumulating, ,,, but reports have been rather inconsistent. ,,,,
In 1990, Kirgan et al. demonstrated HPV genome in nearly 43% of colon carcinomas.  After that, a growing number of surveys have detected HPV in colon cancers with variable ranges from 14% to 84%. ,,,,, The most frequent HPV types detected were HPV-16 and HPV-18.  In the other hand, some scholars did not find any correlation between HPV infection and colon cancers. ,,, In current study, we didn't find any association between HPV infection and colon adenocarcinoma, which is in agreement with Shah, Boguszaková, Yavuzer, and Gornick et al. investigations, which reported no HPV-DNA in colon cancers. ,,,
Fewer studies have been performed on association between HPV infection and colon adenomas. McGregor et al. reported that 38% of adenomas were positive for HPV-DNA.  This rate was reported as 29.7% and 28% in two separate studies by Cheng et al. , In contrast, Yavuzer and Burnett-Hartman didn't find any HPV-DNA in colon adenomas. , We also did not detect HPV-DNA in adenomatous polyps, which is consistent with Yavuzer and Burnett-Hartman results. ,
One potential reason for these discrepancies can be due to HPV contamination of specimens that do not actually harbor pathogenic infections. In this study, we avoided any cross-contamination between samples by using separate disposable items such as blades, gloves, and tubes. Especially the first section of each specimen plus blade and gloves were discarded and new blade and gloves were used for main sectioning. We also used two separate PCR assays to analyze our specimens, and this improved the overall sensitivity for HPV detection. Specially using the PGMY primer system, which is more sensitive than other primer sets in HPV detection,  allows detection of low copies of HPV and a wider range of HPV types.
Other explanations for the divergent frequencies of HPV positivity in colon cancer specimens may be duo to environmental, geographical, and genetic heterogeneities in the study populations and differences in detection assays. Moreover, HPV has known tropism to squamous epithelium, and colon cancer is not squamous in origin; besides, colon is not an anatomic site of direct exposure to HPV. 
Another reason for our results may also be hidden in the overall low rate of HPV prevalence in our region. The studies on the prevalence of HPV-related cancers in the Middle East revealed that the HPV incidence is low in this area in comparison to the rest of the world. 
In conclusion, our results do not support the relationship between HPV infection and colon carcinoma or adenoma. Further studies in larger series are required to investigate the role of HPV in colon carcinogenesis. A conclusive result will provide new possibilities in prevention, diagnosis, and therapy of colon cancers.
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[Table 1], [Table 2], [Table 3]