High-Risk Human Papillomaviruses and Epstein–Barr Virus in Colorectal Cancer and Their Association with Clinicopathological Status

Colorectal cancer (CRC) is a common malignancy with a high mortality rate worldwide. It is a complex, multifactorial disease that is strongly impacted by both hereditary and environmental factors. The role of microbes (e.g., viruses) in the pathogenesis of CRC is poorly understood. In the current study, we explored the status of high-risk human papillomaviruses (HPV) and Epstein–Barr virus (EBV) in a well-defined CRC cohort using immunohistochemistry and polymerase chain reaction assays. Our data showed that high-risk HPVs were common (~80%) and EBV had a low presence (14–25%) in the CRC samples. The most common high-risk HPVs are HPV16, 31, 18, 51, 52 and 45 genotypes. The co-presence of high-risk HPV and EBV was observed in ~16% of the sample population without any significant association with the clinicopathological variables. We conclude that high-risk HPVs are very prevalent in CRC samples while EBV positivity is relatively low. The co-expression of the two viruses was observed in a minority of cases and without any correlation with the studied parameters. Further studies are necessary to confirm the clinical relevance and potential therapeutic (preventive) effects of the observations reported herein.


Introduction
Colorectal cancer (CRC) is the third most common cancer reported worldwide, and corresponds to about 10% of all cancer cases [1]. It is considered to be second most common cause of mortality in both developed and developing countries, affecting men and women equally [2].
CRC is a complex disease involving multiple factors including hereditary (family history and genetic diseases such as familial adenomatous polyposis (FAP) and hereditary non-polyposis CRC (Lynch) syndromes), (epi)genetic, lifestyle, environmental and viral factors [3].
Among viral factors, microbial-epithelial interactions are assumed to instigate oncogenic activity, which could lead to the onset and development of CRC [3]. Viral infections by high-risk human papillomaviruses (HPVs) and Epstein-Barr virus (EBV) have been linked with the initiation and progression of several human carcinomas [4][5][6].

Clinicopathological Characteristics of the Cohort
The clinicopathological characteristics of the cohort are summarized in Table 1. The study included samples from 64 (60%) male and 42 (40%) female patients. The mean age of the patients was 65 years (range, 41-86 years) and the vast majority of specimens were taken from the rectum (102 cases, 96%). The remaining four samples were from the sigmoid colon and other parts of the large bowel (Table 1).  (21) Stage II 23 (22) Stage III 54 (52) Stage IV 5 (5)  Sigmoid colon 2 (2) Other parts of colon 2 (2) † Numbers may not add up to the total number of subjects because of some missing values. * The staging was according to the American Joint Committee on Cancer (AJCC) TNM system (8th edition). SD = Standard deviation.
Pathogens 2020, 9, x FOR PEER REVIEW 4 of 15 Only one case was well-differentiated (grade 1) rectal adenocarcinoma (Table 1). Notably, twenty-one cases (20%) had been previously treated with neoadjuvant chemotherapy or combined chemo-and radiotherapy. None of the cases had received targeted therapies (e.g., anti-EGFR, antiangiogenic or immune checkpoint inhibitors) prior to the surgery.

The Status of High-Risk HPV Subtypes and EBV by PCR
The PCR data for HPVs and EBV and their association with the clinicopathological parameters are summarized in Table 2. Only one case was well-differentiated (grade 1) rectal adenocarcinoma (Table 1). Notably, twenty-one cases (20%) had been previously treated with neoadjuvant chemotherapy or combined chemo-and radiotherapy. None of the cases had received targeted therapies (e.g., anti-EGFR, anti-angiogenic or immune checkpoint inhibitors) prior to the surgery.
On the other hand, 15/106 patients (14.1%) were positive for EBNA1 alone and 26/106 (24.5%) were positive for the LMP1 gene of EBV; 15/106 (14.1%) of the cases were positive for both (EBNA1 and LMP1) sequences. Although all the EBNA1-positive tumors were LMP1-positive tumors, the same did not hold for LMP1-positive tumors. Only 16% (17/106) of the colorectal cases were positive for both high-risk HPVs and EBV.

Immunohistochemical Expression of E6 (HPVs) and LMP1 (EBV)
The immunohistochemical (IHC) data for HPV and EBV and their relationship to the clinicopathological parameters are summarized in Table 4.
Interpretable results by IHC were obtained for 58 cases of HPVs. Forty-five cases (77.5%) were positive for E6 above the threshold of 1% of positive cancer cells; in contrast, LMP1 of EBV expression was found in 11% of the cases (7/63) (Table 4). Four out of seven LMP1-positive cases also co-expressed E6 of HPV ( Figure 1C-F). When positive, both E6 of HPV and LMP1 of EBV expression was diffused (>50% cancer cells were positive) in the majority of the cases. Notably, adjacent normal mucosa was devoid of E6 while LMP1 expression was occasionally seen in tumor-infiltrating lymphocytes (TIL) and lymphatic aggregates within the tumor and adjacent structures.
Positive samples for E6 of HPV showed a statistically significant association with cancers of rectal origin (p = 0.024). In addition, LMP1 of EBV positive samples by IHC seemed to be associated with grade 2 adenocarcinomas (p = 0.035) ( Table 4).
PCR and IHC data were in good concordance for HPV analysis while discrepant data were observed in EBV assessment; however, the inter-reliability rating between PCR and IHC was fair (Kappa = 0.31; p = 0.005).

Discussion
There are various types of HPV-associated cancers including cervical, vulvar, vaginal, penile, rectal, anal and oropharyngeal cancer [29]. From a clinical and therapeutic point of view, exploring HPV status in cancers could be highly relevant due to the prophylactic vaccines that have been shown to be effective in preventing common HPV-associated cancers such as cervical cancer [29].
In this investigation we explored for the first time, the co-presence of high-risk HPVs and EBV in human CRC in the Bosnian population; our study found a high prevalence of high-risk HPVs and a low positivity of EBV in CRC samples. The most frequent HPV types in our cohort are 16, 31, 18, 51, 52, and 45. This is also, to the best of our knowledge, the first report regarding the distribution of high-risk HPVs in CRC samples from Bosnia. Our data are similar to those reported for a large cohort of cervical cancer patients by de Sanjose et al. [30] and from Bosnian cervical cancer patients [31][32][33].
Among our HPV-positive rectal cases, HPV16 and HPV18 were the most prevalent genotypes (~50%), which is in line with the HPV distribution in cervical cancer samples that were previously reported in the Bosnian population [31][32][33]. In Polish CRC samples, HPV16 and 18 were also the most common HPV serotypes (~60%) [34]. Another study done on Italian CRC patients revealed HPV in 33% of the cases [35]. A meta-analysis study based on the European population showed that HPV18 was present in 47% of CRC cases [36]. The overall HPV16+/18+ prevalence in our study was~23%, which is similar to the findings in cervical cancer samples from the Croatian population (21%) [37]. HPVs 16, 31 and 18 were also the most common high-risk HPVs in cervical cancer samples in Serbian women [38]. In our study, the other expressed HPV-types included HPV−45, −51 and −52, which have been previously reported in cervical cancer in Bosnia [33]. Moreover, HPV45 has been previously found in colorectal cancer [25,39]. Studies done in the USA and Iran identified HPV-51 as the most frequent HPV subtype in colorectal cancer [39,40].
The highest reported EBV-positivity in CRC was up to 46%, with the majority of the studies reporting EBV positivity of 20-40% [19,28,41,42]. Our study indicated the individual presence of EBNA1 and LMP1 genes of EBV in 14% and 25% of the samples, respectively. EBV consists of two strains, B95-8 (EBV type 1) and AG876 (EBV type 2), which also known as type A and type B, respectively [43]. These EBV variants are differentiated based upon genetic differences in EBNA sequences [43], and discrepancies in EBV variants have previously been described in breast cancer samples [44]. This can be explained by the different roles of EBNA and LMP genes. In general, EBNA1 acts as a transcriptional activator that binds to the FR element, thereby enhancing the expression of the viral Cp promoter and LMP genes. This in turn leads to transcriptional activation of viral LMP genes (LMP-1 and -2) promoting cellular proliferation [45][46][47]. During latency 0, no EBNA protein is expressed, while only one EBNA (EBNA1) is expressed during latency I and II, and during latency III, all six EBNAs are expressed [48][49][50]. Further, the co-presence of both EBNA1 and LMP1 in our cohort can be explained by the fact that in EBV-infected cells, in the latent phase, EBNA1 and LMP1 are expressed in a cancer-specific manner [51]. Thus, the most restricted pattern of viral latent gene expression (latency 1: EBNA1 and EBERs) is expressed in Burkitt lymphomas [51,52] while the unrestricted pattern of viral gene expression (latency II and III: EBNA1, LMPs and EBERs) is expressed in Hodgkin lymphomas, diffused large B cell lymphomas and EBV-positive gastric and nasopharyngeal carcinomas [51,52]. Regardless of the latency pattern, virtually all EBV-associated malignancies express EBNA-1 and EBERs [52]. In addition, the functional variance can be explained by the genetic diversity of the LMP-1 gene, which shows a higher degree of polymorphisms than most EBV genes [53,54]. Ten main LMP1 variants (China 1, China 2, China 3, NC (North Carolina), Mediterranean+ (Med+), Med-, GD1, SEA 1, and SEA 2) have been defined in different regions and diseases [55][56][57].
Karpinski et al. reported that 19% of the CRC cases from Poland were EBV positive [58]. In an Italian CRC cohort, the EBV-positivity rate ranged from 3-29% according to RT-PCR and sequencing assays [59]. Another study in Italy did not find any significant involvement of any oncogenic virus (HPV, HTLV, HHV-8, JCV, BKV and Merkel cell polyomavirus), except for EBV (52%) [60]. However, a Czechoslovakian study failed to detect any oncogenic virus (EBV, HPV and CMV) in adenocarcinoma of the colon [61]. A Chinese cohort of colorectal carcinomas was reported to be EBV positive (LMP1) in 27% of the samples [62].
The combined oncogenic effects of viral infections have been recognized as potential oncogenic drivers in various cancers [6]. Oncoviruses can lead to the onset and progression of cancer via commonly shared pathways including WNT/β-catenin, JAK/STAT/SRC, PI3k/Akt/mTOR, and/or RAS/MEK/ERK signaling pathways [6,8,14,41]. A study conducted by Guidry and Scott [63] showed that co-infection by HPV and EBV enhances EBV persistence either via latency or increased viral replication or by aggregating HPV oncogene expression. Our group has previously demonstrated that oncoproteins of EBV and high-risk HPVs can interact during the onset of human carcinomas, and subsequently result in metastasis progression via epithelial-mesenchymal transition [14]. Epithelial differentiation stimulates both the productive and lytic phases of HPV and EBV, respectively; high-risk HPV stabilizes the EBV genome and induces EBV lytic reactivation in differentiating epithelial cells, which suggests that co-infection with HPV may increase EBV-mediated pathogenesis of CRC [64]. Additionally, HPV and EBV may work together to promote the proliferation of cultured cervical [65] and prostate cells [66], suggesting the same for colorectal epithelial cells. In our study, co-expression of EBV and HPV was seen in only~16% of CRCs. This co-expression was not associated with any noted clinicopathological parameters. The lack of any association may be due to the relatively small sample size as well as low EBV positivity in the cohort.

Sample Collection and DNA Extraction
The study included 106 formalin-fixed paraffin-embedded (FFPE) tissue samples that were diagnosed in 2010-2017 at the Department of Pathology, University Clinical Center Sarajevo, Bosnia and Herzegovina. For the study, all cases were de-identified and patients' information anonymized. The study was conducted based on the IBC approval of the local research committee (#IBC-2019/005).
Prior to molecular assays, all cases were re-reviewed by board-certified pathologists (F.S. and S.V.) to confirm the diagnosis and select appropriate FFPEs for tissue microarray, immunohistochemistry and PCR assays.
DNA was extracted from FFPE tissue samples using the Thermo Scientific GeneJET FFPE DNA Purification Kit according to the manufacturer's instructions (ThermoFisher Scientific, USA). Briefly, FFPE sections were subjected to enzymatic digestion using 200 µL of digestion buffer. After digestion, 20 µL Proteinase K solution was added to lyse and release genomic DNA, which was de-crosslinked by heat incubation (90 • C) for 40 min. The obtained solution was further centrifuged and the supernatant containing DNA was mixed with 200 µL binding buffer. After addition of ethanol (96%), the lysate was loaded onto the purification column and the adsorbed DNA was subjected to washing (wash Buffers 1 and 2) to eliminate contaminants. DNA was eluted with 60µL elution buffer.

EBV and HPV Detection by PCR
Twenty-five nanograms of purified genomic DNA from each sample was analyzed for HPV and EBV by polymerase chain reaction (PCR) as previously described [67] using specific primers for HPV types: 16,18,31,33,35,39,45,51,52,56  were used as internal control. All the primers and analyses were performed as previously described [68].
PCR was performed using the Invitrogen Platinum II Hot-Start Green PCR Master Mix (2X) (ThermoFisher Scientific, Waltham, MA, USA). Briefly, HPV gene was amplified for an initial denaturation at 94 • C for 2 min followed by 40 cycles of 94 • C for 30 s, annealing at temperatures ranging from 50 to 62 • C for 30 s depending on each primer's melting temperature as previously described [68], and 72 • C for 30 s. In parallel, EBNA1 and LMP1 genes were amplified for an initial denaturation at 94 • C for 2 min followed by 40 cycles of 94 • C for 30 s, 61 • C for 30 s, and 72 • C for 30 s. The samples were finally incubated for 10 min at 72 • C for a final extension. The PCR product from each exon was resolved using 1.5% agarose gel electrophoresis.

Tissue Microarray (TMA)
Tissue microarray was constructed as described previously by our group [17]. Briefly, cancer samples and controls were embedded into a virgin paraffin TMA block using a manual tissue arrayer (Beecher Instruments, Silver Spring, MD, USA).
Following the sampling of two TMA cores of 1.0 mm in diameter, sections of 4 µm were cut and stained with hematoxylin and eosin (H&E) on the initial slides to verify the histopathological diagnosis. Next, slides of completed blocks were used for immunohistochemical assays.

Immunohistochemistry (IHC)
Immunohistochemical assays for E6 (HPV) and LMP1 (EBV) expression were performed using procedures as previously described [17]. For TMA construction, each slide was deparaffinized in graded alcohol, rehydrated and boiled (microwave) in 10 mM citrate sodium solution (pH 6.0) for 10 min. Endogenous peroxidase activity within the rehydrated tissue was blocked with a solution of 3% hydrogen peroxide in methanol for 10 min at room temperature. TMA slides were further incubated for 35 min at 37 • C with primary monoclonal antibodies for E6 (clones 1-4 and C1P5, Dako Agilent, Carpinteria, CA, USA and Calbiochem, Canada) and LMP1 (clone CS1-4, Abcam) using a fully automated immunostainer (Ventana Medical System, Tucson, AZ). The fully automated Ventana Medical System uses an indirect biotin-avidin system with a universal biotinylated immunoglobulin secondary antibody. The slides were counterstained with hematoxylin prior to mounting. The staining procedures were completed according to the manufacturer's recommendations. Negative controls were obtained by omitting specific primary antibodies for E6 and LMP1 as well as specific blocking peptides from Santa Cruz Biotechnology.
The tumors were considered positive for E6 and LMP1 if cancer cells exhibited positivity ≥1% of the cells [69]. In the case of LMP1 protein expression, we also evaluated the presence of viral infection in tumor-infiltrating lymphocytes and stromal cells [69,70].

Statistical Analysis
The Spearman Correlation Rank test was used to assess the significance of HPV and EBV associations. The Kolmogorov-Smirnov test and histograms were used to assess normal distribution. Categorical variables were compared using Chi square and Kruskal-Wallis tests while Cramer's V test was used to assess a possible association between nominal and ordinal variables in asymmetric tables. HPV and EBV IHC status were included as the dependent variables in a multiple regression analysis to test the relationship with other independent variables such as age, gender, tumor location, stage, and grade. The values for missing samples were excluded from the regression analysis. Cohen's kappa coefficient was used to assess inter-rater reliability for categorical variables. The results were considered statistically significant if p-values were ≤0.05 in two-tailed tests. All the statistical analyses were performed using IBM Statistical Package for the Social Sciences (version 25).

Conclusions
We conclude that high-risk HPVs are prevalent in CRC samples while EBV positivity is relatively low. The co-presence of the two viruses was observed in a minority of the cases and without any correlation with the studied parameters. Further studies are necessary to confirm the clinical and preventive relevance of the observed findings.