Infection with a high-risk human papillomavirus (HR-HPV) type is necessary for the development of cervical carcinoma ([@B15]).  Among these, types 16 and 18 are the most common found in cervical cancer (CC) tumours ([@B14]); they are associated with approximately 76% of CC and 80% of anal cancers ([@B22], de [@B12]).  In total, 5.2% of the worldwide cancer burden is attributable to HPV ([@B27]).  HPV DNA has been detected in tumour tissues of patients with head and neck cancers ([@B18]), oral cancer ([@B19]), oesophageal cancer ([@B8]), some skin cancers ([@B7]), and lung cancer ([@B20], [@B28]).  Over the last few years, a possible correlation between HPV infection and colorectal cancer (CRC) has been suggested, based on several researchers detecting HPV DNA in these cancers, using different techniques ([@B4], [@B9], [@B16], [@B5]). 

CRC is the fourth most common cancer worldwide.  It is estimated to be responsible for approximately 1,360,602 new cases and 693,881 deaths annually ([@B11]).  Approximately 95% of CRC are adenocarcinomas, with adenomas as the main precursor lesions ([@B25].  The progression of adenoma to adenocarcinoma is inﬂuenced by environmental and lifestyle factors, sequential genetic changes, and possibly infections ([@B3]. 

Findings of the present study indicate the presence of HPV DNA in colorectal tumours and suggest a role for oncogenic HPV types in the establishment of colorectal adenocarcinomas.  These results may contribute to an understanding of the aetiology of sporadic CRC. 

To explore the possible relationship between HPV infection and colorectal carcinogenesis, we investigated the presence of HPV DNA in CRC tissues from Cuban patients and examined the relationship between HR-HPV genotypes and viral loads and adenocarcinomas. 

MATERIALS AND METHODS
=====================

*Study population* - For this purpose, a cross-sectional study to detect HPV infection in colorectal tissues was performed.  Study participants were outpatients at the Cuban National Institute of Gastroenterology between April and August 2014.  Patients contributed only one sample for viral detection.  The inclusion criteria were no prior surgery, radiation, or cytotoxic therapy for colorectal adenocarcinoma.  Diagnoses of samples from colorectal adenomas, adenocarcinomas, and tissues with unremarkable pathological changes (a non-malignant control group) were confirmed by pathologists through standard criteria.  In the study, 63 colorectal paraffin-embedded tissues were included: 42 from patients with CRC and 21 from individuals without CRC.  Of the 42 tumours sampled, 24 were adenocarcinomas and 18 adenomas.  Twenty-one specimens diagnosed as chronic inflammatory infiltrates of colorectal mucosa due to benign colitis were used as the non-malignant control group. 

Colorectal tissues were obtained from the rectum, rectosigmoid junction, and sigmoid colon.  Complete clinical and pathological data were recorded, including patients' clinical histories and demographics (age at the time of sampling, gender, skin colour), family antecedents, grade of tumour differentiation, and tumour location. 

*DNA extraction* - *HPV detection and genotyping* - Formalin-fixed paraffin-embedded (FFPE) colorectal tissues were obtained.  Between seven and 10 slides of approximately 5-µM wide sections of tissue were deparaffinised in xylene and absolute ethanol, and then DNA was obtained using a QIAamp DNA FFPE Tissue Kit (Qiagen, Germany), following the manufacturer's protocol.  The potential for sample-to-sample cross-contamination was limited by performing DNA extractions from only six tissue samples per day.  DNA quality was evaluated by polymerase chain reaction (PCR) amplification of a fragment from the human β-globin gene using primers PC04/GH20 ([@B13]). 

Quantitative real-time PCR (qRT-PCR) was performed as previously described ([@B21], [@B23]) to identify the most frequent genotype of HR-HPV and quantify viral loads.  PCR primers and corresponding TaqMan probes were used to amplify the LCR/E6/E7 region of HPV-16, -18, -31, -33, -45, -52, and -58 genomes ([@B21], [@B23]). 

HPV 16 and 18 standard curves were constructed from purified genomic DNA from HPV cultures in SiHa and HeLa cell lines, respectively.  The curves showed good linear correlation (r = 0,99) and low error values throughout the range of the six target DNA concentrations.  The assay had a detection limit of 10 copies for HPV DNA for all genotypes tested.  The detection limit for HPV18 and 45 was also of 10 copies of viral DNA.  No cross reactions between HPV and other DNA viruses were observed ([@B21], [@B23]). 

To identify HR-HPV types and quantify viral loads, single PCR reactions were performed for each HPV type.  To control for DNA quality, β-globin was amplified in each sample.  PCR was performed using a LightCycler 1.5 platform (Roche Diagnostics, Indianapolis, USA).  The PCR reaction mixture comprised 5 µL of DNA (up to 50 ng), 4 µL of Quantitative PCR TaqMan Master Mix (Roche Diagnostics), 10 pmol of each primer, and 1-5 pmol of each probe in a final volume of 25 µL. The initial denaturation step at 94ºC for 10 min was followed by 45 PCR cycles at 94ºC for 15 s, 50ºC for 20 s, and 60ºC for 40 s each. 

*Statistical analysis* - Epidemiological and clinical data were collected in one-on-one interviews in a private room, using a standardised questionnaire.  Data were processed using IBM SPSS Statistics 20.  Proportions were compared by Chi-squared testing.  Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated in a univariate logistic regression to estimate the relative risk of HPV detection associated with different variables, including age at the time of sampling, gender, skin colour, family antecedents, grade of tumour differentiation, and tumour location.  Comparisons between viral loads from groups of patients with different histological classifications or HPV genotypes were made using Kruskall-Wallis and Mann-Whitney tests.  All statistical tests were considered to be significant at a p-value \< 0.05. 

*Ethics* - This study was approved by the Ethics Committee of the Institute of Tropical Medicine Pedro Kourí and complies with the principles established in the Helsinki Declaration of 1975 and revised in 1983 ([@B26]).  Written informed consent was obtained from each study participant before the interview, sample collection, and testing. 

RESULTS
=======

Overall, HPV DNA was detected in 23.8% (15/63) of the samples tested.  HPV infection was detected in 41.7% of adenocarcinoma (10/24) and 27.7% of adenoma cases (5/18), but was not detected in any of the negative cases (p = 0.002).  An association between histological diagnosis of adenocarcinoma and HPV infection was observed (OR = 4.85, 95% CI = 1.40-16.80, p = 0.009).  HPV infections were exclusively detected in the rectum (60%, 9/15) and in rectosigmoid junction (40%, 6/15) of colorectal samples ([Table I](#t1){ref-type="table"}). 

TABLE IAnalysis of epidemiological data and histology, and their association with human papillomavirus (HPV) infectionVariables Total (N = 63) n (%)HPV positive (N = 15) % (n/N)HPV negative (N = 48) % (n/N)p-valueOR (CI 95%)Age (years) n = 63≤ 40 41-50 51-60 \> 607 (11.1) 12 (19.1) 13 (20.6) 31 (49.2)6.7 (1/15) 26.7 (4/15) 20 (3/15) 46.6 (7/15)12.5 (6/48) 16.7 (8/48) 20.8 (10/48) 50 (24/48)1.000 0.457 1.000 0.4720.50 (0.05-4.52) 1.81 (0.46-7.17) 0.95 (022-4.02) 0.80 (0,25-2.57)Sex n = 63F M31 (49.2) 32 (50.8)66.7 (10/15) 33.3 (5/15)43.8 (21/48) 56.2 (27/48)0.1202.57 (0.76-8.67)Color of the skin n = 61Black Mulatto White41(67.2) 7 (11.5) 13 (21.3)57.1 (8/14) 7.2 (1/14) 35.7 (5/14)70.2 (33/47) 12.8 (6/47) 17.0 (8/47)1.000 0.580 0.7101.13 (0.20-6.39) 0.75 (0.64-0.87) 1.83 (0.35-9.47)Histology results n = 63Negative Adenoma Adenocarcinoma21 (33.3) 18 (28.6) 24 (38.1)0 (0/15) 33.3 (5/15) 66.7 (10/15)43.8 (21/48) 27.1 (13/48) 29.1 (14/48)0.002 0.746 0.009- 1.34 (0.38-4.69) 4.85 (1.40-16.09)Colorectal tissue location n = 63Rectum Rectum-sigmoid Sigmoid34 (54) 18 (28.6) 11 (17.4)60 (9/15) 40 (6/15) 052.1 (25/48) 25 (12/48) 22.9 (11/48)0.591 0.330 0.0531.38 (0.42-4.48) 2.00 (0.58-6,79) -Tumor differentiation n=23Well Moderate8 (34.8) 15 (65.2)44.4 (4/9) 55.6 (5/9)28.6 (4/14) 71.4 (10/14)0.4030.40 (0.07-2.18)Family antecedents n = 63Yes11 (17.5)20 (3/15)16.7 (8/48)0.7141.25(0.28-5.46)[^2]

Among HPV-positive cases, 66.7% had adenocarcinomas (10/15) and 33.3% had adenomas (5/15) ([Table II](#t2){ref-type="table"}).  The only genotypes identified were HPV 16 and 33.  HPV 16 was detected in 70% (7/10) of HPV-positive adenocarcinomas and in 100% of HPV-positive adenomas (5/5).  HPV 33 infection was only identified in adenocarcinoma cases (60%, 6/10).  Dual infections with HPV 16 and 33 were identified in 30% (3/10) of HPV-positive adenocarcinomas.  Adenocarcinomas were significantly associated with HPV 33 infection (p = 0.002).  Other variables such as tumour differentiation or anatomical location of the colorectal lesion were not found to be associated with a specific HPV type ([Table III](#t3){ref-type="table"}). 

TABLE IIComparison of age groups with histological results and human papillomavirus (HPV) infectionAge group N = 63Negative for tumor N = 21Adenomas N = 18Adenocarcinomas N = 24 HPV positive n = 0HPV negative n = 21Total n (%)HPV positive n = 5HPV negative n = 13Total n (%)HPV positive n = 10HPV negative n = 14Total n = 24\< 40 (n = 7)033 (14.3)022 (11.1)112 (8.3)41-50 (n = 12)077 (33.3)112 (11.1)303 (12.5)51-60 (n = 13)055 (23.8)224 (22.2)134 (16.7)\> 60 (n = 31)066 (28.6)2810 (55.6)51015 (62.5)

TABLE IIIHuman papillomavirus (HPV) 16 and 33 infections and colorectal lesion characteristicsVariablesHPV 16 positive N = 12p-valueHPV 33 positive N = 6p-valueHistology results n = 63Adenoma Adenocarcinoma5 (41.7%) 7 (58.3%)0.299 0.1850 (0%) 6 (100%)0.170 0.002Colorectal tissue n = 63Rectum Rectum-sigmoid Sigmoid8 (66.7) 4 (33.3) 0 (0%)0.358 0.729 0.1043 (50%) 3 (50%) 0 (0%)1.000 0.341 0.579Adenocarcinoma differentiation n = 23Well Moderate3 (25%) 4 (33.3%)0.6471 (16.7%) 5 (83.36%)0.621Family antecedents n = 63Yes3 (25%)0.4250 (0%)0.579

Viral loads were analysed and compare between the different histological classifications, adenomas and adenocarcinomas ([Figure](#f01){ref-type="fig"}).  The median viral loads were similar in adenocarcinoma and adenoma cases.  The maximum viral load in adenomas was lower than that in adenocarcinomas (2.5 × 10^4^ copies/µL versus 3 × 10^6^ copies/µL, respectively).  HPV 16 viral loads were higher than those of HPV 33 (3 × 10^6^ copies/µL versus 2.5 × 10^4^ copies/µL, respectively) ([Figure](#f01){ref-type="fig"}). 

Comparison of the median (A) human papillomavirus (HPV) viral loads according to histological classification and (B) HPV 16 and 33 viral loads in colorectal tissues. ( C) HPV 16 viral load according to histological classification. 

[Table I](#t1){ref-type="table"} shows the relationship between HPV infection and variables analysed in the study population.  HPV infection was more frequent in black individuals (57.1%, 8/14), in females (66.7%, 10/15), and in those over 60 years of age (46.6%, 7/15). 

In the present study, the prevalence of HPV infection was higher in the group of patients over 60 years of age than in other age groups.  However, no statistical association was found (p \< 0.05) between HPV infection and age when proportions were compared by Chi-squared test ([Table I](#t1){ref-type="table"}). 

The majority of patients with both colorectal lesions, adenomas and adenocarcinomas, were over 60 years of age (55.6% \[10/18\] and 62.5% \[15/24\], respectively).  Furthermore, among patients with colorectal adenocarcinomas, HPV infection was more frequent in those over 60 years (33.3%, 5/15) ([Table II](#t2){ref-type="table"}). 

Family antecedents of familial adenomatous polyposis were associated with the presence of adenomas (OR = 6.52, 95% CI = 1.61-26.38, p = 0.009). 

DISCUSSION AND CONCLUSIONS
==========================

Our study showed that oncogenic HPV types can be detected in colorectal adenocarcinoma tissues from Cuban patients.  The absence of HPV DNA in non-malignant tissues suggests that the presence of HPV is not merely coincidental in colorectal carcinomas, but that it is a possible cofactor in development of the disease.  In our study, an association between a histological diagnosis of adenocarcinoma and HPV infection was observed. [ @B10] published a meta-analysis of case-control studies that indicated a high prevalence of colorectal HPV infection in patients with CRC, as well as a significant increase in CRC risk associated with the presence of the virus. 

Recently, [@B1] conducted a meta-analysis of 37 studies.  Of 2630 adenocarcinoma cases included, the pooled HPV prevalence was 11.2% (95% CI = 4.9-19.6%).  The meta-analysis conducted by [@B1] showed that studies in which five or more HPV were tested and five or more adenocarcinoma cases were HPV positive, the prevalence of HPV 16 in HPV positive adenocarcinoma cases ranged widely, from 18.2-85.7%.  Applying the same criteria, the prevalence of HPV 33 ranged widely also; from 0-85.7%.  According to this meta-analysis, both genotypes were the most frequently detected in adenocarcinoma. 

A few studies have also investigated the presence of HPV in premalignant adenomatous polyps (adenomas).  Data on the relationship between HPV infection and other factors, including genetic and molecular changes, and the development of CRC from adenomas are not conclusive. [ @B6] found HPV DNA in 28% of 109 paraffin-embedded adenomas.  In contrast, [@B3] did not find HPV DNA in an assessment of 167 colorectal adenomas, 87 hyperplasic polyps, and 250 polyp-free controls. 

According to our results, HPV infection was more frequent in females. [ @B1] also reported on the sex-specific HPV prevalences in colorectal adenocarcinomas.  In two studies analysed, a non-significant tendency toward a higher HPV prevalence in women than men was found.  In the remaining studies, the HPV prevalence in colorectal adenocarcinoma cases was similar between women and men. 

In the present study, the prevalence of HPV infection was higher in patients over 60 years of age than in other age groups.  However, no statistical association was found (p \< 0.05).  Furthermore, in the group of patients over 60 years of age, a higher proportion of HPV-positive cases had a histologic diagnosis of colorectal adenocarcinoma.  According to demographic data analysis by [@B17], more than 90% of individuals with both CRC and HPV infections are older than 50 years of age.  Our study shows an epidemiological pattern of CRC and HPV infection in a Cuban population that is similar to what was found in other studies ([@B10], [@B17]. 

In the present study, family antecedents of familial adenomatous polyposis were associated with the presence of adenomas.  Approximately 95% of CRCs are believed to have evolved from adenomatous polyps (adenomas).  Various genetic and molecular changes occur as these polyps transform from benign to malignant.  Familial adenomatous polyposis is a Mendelian dominant syndrome with an incidence of 1:11,000 that is caused by an alteration in the APC gene.  The syndrome is characterised by the presence of adenomatous polyps in the gastroenteric tract, mostly in the colorectal-junction and duodenum, with a demonstrated adenoma-carcinoma sequence ([@B2]).  There is no evidence to associate HPV infection with familial adenomatous polyposis ([@B24]). 

In our study, HPV infections were exclusively detected in the rectum and rectosigmoid junction, suggesting that HPV infection may be a result of retrograde viral transmission from the anogenital area.  However, this result differs from those of most previously published research, which have shown no relationship between HPV infection and the anatomic location of tumours.  In these studies, rates of HPV detection in tissues collected from the rectum or sigmoid colon were similar to those in tissues obtained from the cecum or ascending colon.  Because HPV infection is mainly transmitted by cell surface contact, the route of viral transmission to the colon remains to be determined ([@B5], [@B17]). 

According to [@B10], oncogenic HPV types may be associated with development of CRC.  However, it should be noted that the detection of HPV DNA alone is not sufficient to establish a role for HPV in colorectal carcinogenesis. 

Although further studies are needed to reach a definitive conclusion on the involvement of oncogenic HPV types in the establishment of colon adenocarcinomas, our study provides data that suggests a potential role for HR-HPV infection in the pathogenesis of CRC.  Establishing a link between HPV infection and colorectal carcinogenesis, in addition to contributing to our understanding of the pathogenesis of this disease, could have important implications for patient management and prevention of CRC.  If infection with HPV is found to have a causal role in colorectal carcinogenesis, then prophylactic vaccines against HPV could be used to prevent a large proportion of sporadic CRC. 

[^1]: YS, CML and LG contributed equally to this work. 

[^2]: CI: confidence interval; OR: odd ratio.