Prevalence of Human Papillomavirus and Co-Infection with Epstein-Barr Virus in Oral and Oropharyngeal Squamous Cell Carcinomas

Background: Head and neck squamous cell carcinoma is one of the most important malignancies, worldwide. Oncogenic viruses, such as human papilloma virus (HPV) and Epstein-Barr virus (EBV), are linked to these cancers and studies suggest a possible interaction between HPV and EBV during co-infections to promote oncogenesis. Nonetheless, these reports are controversial and demand more investigations in this regard. The present work to assessed the prevalence of HPV and co-infection with EBV in oral and oropharyngeal squamous cell carcinomas. Methods: Formalin-fixed paraffin-embedded tissues were collected from 166 archived oral and oropharyngeal squamous cell carcinoma samples from Ahvaz Imam Khomeini hospital, Ahvaz, Iran, from March 2013 and December 2019. Nested-PCR was used to detect the viruses and type-specific PCR/nested-PCR and sequencing were performed for virus genotyping. Results: Out of the 166 specimens, 84.33% and 16.42% were from oral cavity and oropharynx, respectively; of which, 32 cases (19.3%) were HPV-positive (16.42% of oral cavity and 34.6% of oropharynx). HPV was detected in 36.36%, 25%, and 16.42% of base of tongue, tonsil, and oral tongue tumors, respectively. HPV was more associated with well differentiated tumors (24;18.04%) in compared to moderately and poorly differentiated ones. Regarding HPV-16 genotyping, 7 (21.8%) out of the 32 samples were found to be HPV-16 (4/26 (15.38%) for oropharynx and 3/140 (2.14%) for oral cavity). Moreover, 90 samples were evaluated for EBV infection and co-infection; of which, 4 (4.4%) subjects tested positive for EBV, including two cases with HPV co-infection. All the positive cases were EBV type B, from oral cavity, and histologically well differentiated. Conclusions: HPV was more associated with oropharyngeal cancer. This association has been linked to various factors such as repeated oral and oropharyngeal exposure to HPV due to change in patterns of sexual behaviors; a phenomenon that may demand routine HPV vaccination.


Introduction
Head and neck squamous cell carcinoma (HNSCC), known as the sixth most frequent malignancy worldwide, is a heterogeneous group of epithelial cancers, developed from the oral cavity, oropharynx, nasopharynx, hypopharynx, and larynx (Vigneswaran et al., 2014). Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) are the most prevalent malignancies of the oral cavity and oropharynx, respectively, accounting for more than 90% of oral and oropharyngeal cancers (Mirzaei et al., 2018). The etiology of HNSCCs is a multifactorial process, with various risk RESEARCH ARTICLE

Prevalence of Human Papillomavirus and Co-Infection with Epstein-Barr Virus in Oral and Oropharyngeal Squamous Cell Carcinomas
factors such as alcohol drinking and tobacco smoking (Kumar et al., 2016). However, despite a decrease in exposure to these factors during the past three decades, the number of new OSCC and OPSCC cases has continued to increase, suggesting other etiological factors, such as infection with human papilloma virus (HPV) and Epstein-Barr virus (EBV), for these cancers (Elrefaey et al., 2014). In fact, HPV and EBV, as two important human oncogenic viruses, have also been implicated in the development of OSCC and OPSCC (Mirzaei et al., 2020); so that, in 2009, the International Agency for Research on Cancer (IARC) classified the sexually transmitted oncogenic viral pathogen HPV-16 as an independent risk factor for OPSCC (Bouvard et al., 2009). Moreover, studies suggest a possible interaction between HR-HPVs (such as HPV-16) and EBV during their co-infections to promote oncogenesis by various mechanisms (Cyprian et al., 2018;Shi et al., 2016). HPVs are DNA viruses with up to 200 genotypes, categorized into two high-and low-risk viruses, according to their tumorigenic potential (Ghedira et al., 2016). While HPV is a well-known etiological factor for OPSCC, its causative role in OSCC has remained controversial (Mirzaei et al., 2018). On the other hand, EBV is also a well-established etiological factor for several cancers with two subtypes (EBV-A and -B), whose causative role in HNSCCs has remained highly controversial (Higa et al., 2002) and needed to be more investigated. In this way, the present work aimed to evaluate the prevalence of HPV and co-infection with EBV among 166 OSCC and OPSCC samples from Ahvaz, Iran, for the first time.

Specimens
In this cross-sectional study, 141 OSCC and 25 OPSCC formalin-fixed paraffin-embedded (FFPE) tissues diagnosed from March 2013 and December 2019 were retrieved from the pathology archive of Ahvaz Imam Khomeini hospital, maffiliated to the Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran, after obtaining ethical approval (IRAJUMS REC 1398233) from the Ethics Committee of AJUMS. This work used FFPE tissues due to the rarity of HNSCC and the lack of fresh tissues. Tissues were reviewed by an expert pathologist and selected based on their quality for molecular assays. Patient's information, including gender, age, anatomical site, and histological grades were collected from the patient records. Samples were categorized as OSCC (tumors of the oral tongue, hard palate, floor of mouth, and gingiva) and OPSCC (tumors from the back one-third of the tongue, the soft palate, uvula, and tonsils) (Mirzaei et al., 2018).

DNA extraction
After cutting the samples for 15-μm-thick sections, the microtome blade was cleaned repeatedly using 10% chlorox to reduce cross contamination. Moreover, empty paraffin blocks were sectioned after every 10-15 tissue blocks and investigated for HPV DNA intersample contamination. Deparaffinization was done by incubating at 37°C for 1 h with 1 mL xylene, and then, washing with a descending ethanol concentration (100%, 70%, and 50%) for 30 minutes. After drying at 65°C, samples were lysed by adding 300 μL lysis buffer containing 30 µl proteinase K (Bioneer, Korea) and incubation at 37 overnight. The proteins were removed from the suspension by using an equal volume of Phenol;Chloroform;Isoamyl Alcohol (Sigma-Alrdich, USA). The extracted DNA was precipitated via absolute ethanol and checked for concentration and purity by NanoDrop(Thermo Fisher, USA).

PCR assay Human β-globin gene
To check for DNA quality, a 110 bp human β-globin gene amplicon was amplified by using PCO3/PCO4 primers (Table 1), under the following condition; first denaturation at 95°C for 4 min, 35 cycles with denaturation at 95°C for 30s, annealing at 55°C for 30s, and extension at 72°C for 30s, and finally an extension at 72°C for 4 min. The positive samples were selected for further analysis. All the amplifications from this work used a 25μL PCR reaction containing 12.5μL mastermix (Ampliqon, Denmark), 1μL of forward/revers primers (10pmol), 9.5μL sterile water, and 1μL DNA sample (0.5μL for nested amplification).

HPV Detection and HPV-16 Genotyping
A nested PCR was done to detect HPV L1 gene by using two primer pairs (outer primers; MY09/11 (450 bp) and nested primers; GP5/6 (150 bp)) ( Table 1). The amplifications were done under the following conditions: for outer PCR; 35 cycles of 95°C for 30s, 50°C for 1min, and 72°C for 1min, with a pre-denaturation at 95°C for 5min and an additional extension at 72°C for 8min, and for nested-PCR; 5min at 95°C followed by 30 cycles of 95°C for 30s, 50°C for 30s, 72°C for 30s, and finally 8min at 72°C. For HPV-16 genotyping, the HPV-positive samples were evaluated by a HPV-16 E7 gene typespecific nested-PCR to amplify a 183 bp fragment of HPV-16 E7 via two primer pairs (Table 1). To perform this, after a pre-denaturation at 94°C for 3min, the first and second amplifications were done through 35 cycles at 94°C for 30s, 45°C for 30s (for nested-PCR, 30s at 52°C), and 72°C for 30s, and also 5min at 72°C as final extension. For HPV-16 sequencing, the L1 gene was first amplified by SB01/02 primers for a 495 bp product (under the following condition; 5min at 95°C as an initial denaturation, 10 cycles of denaturation at 95°C for 1min, annealing at 42°C for 1 min, extension at 72°C for 1min, followed by 28 cycles of 95°C for 20s, 42°C for 40s, 72°C for 1min, and a final extension at 72°C for 8min), and then by MY09/11 primers. Moreover, a PUC57 plasmid containing full-length HPV-16 E7 gene, supplied by Karbalaie Niya et al. (Niya et al., 2018) was used as positive controls. The presence of PCR products was checked using agarose gel electrophoresis and visualized by UV transilluminator(GENE FLASH , UK).

EBV Amplifications
For EBV detection, 90 samples (including 58 and 32 HPV-negative and -positive samples, respectively) were tested by two EBV-EBNA-1 gene primer pairs (Table 1). After 4min at 94°C, the first and second amplifications were performed through 35 cycles at 94°C for 45s, 54°C for 45s (for nested-PCR, 45s at 58°C), and 72°C for 45s, and also 5 min at 72°C as final extension. For EBV typing, a strain-specific region of EBNA-3C gene(246 and 153 bp for EBV type B and type A, respectively) was amplified by a primer pair (Table 1) and evaluated by agarose gel electrophoresis. The amplification condition was the same as the EBNA-1 gene protocol (the EBNA-3C annealing temperature was 53°C).

EBV results
Ninety samples (including 58 and 32 HPV-negative and -positive samples, respectively) were evaluated

Sequencing
The HPV-16-PCR products were sequenced in two directions using MY09/11 primers, by a 3130xl Applied Biosystems ABI sequencer(Applied Biosystems, Foster City, CA). SnapGene(GSL Biotech) software was employed to analyze the results. The sequences were submitted in GenBank database with the following accession numbers; MW078991-MW078993 and MW079001.

Phylogenetic Analysis
Four HPV-16 L1 sequences were blasted by online tools (www.ncbi.nlm.nih.gov/BLAST). The phylogenetic tree was constructed by Neighbor joining method by using Mega 5.0 software.

Statistical analysis
This was done by IBM SPSS Software(Version 22), using the Independent Samples T Test and Fisher's Exact Test. A P value<0.05 was considered statistically significant.

Discussion
HPV-associated HNSCC has emerged as a distinct entity in compared to HPV-unrelated ones. In fact, HPV is currently known as a well-established etiological factor for a subset of HNSCCs, in particular for those originated from the oropharynx (Vokes et al., 2015), so that it is suggested about 30% of OPSCCs are mediated by HPV (Tumban, 2019), where HR-HPV-16 is the most prevalent type, accounting for more than 90% of all HPVpositive OPSCCs (Kobayashi et al., 2018). While HPV is a well-known etiological factor for OPSCC, its causative role in OSCC is still poorly understood (Mirzaei et al., 2018). This work aimed to evaluate the HPV prevalence among 166 OSCCs and OPSCCs from Ahvaz, Iran. The   current work found that 19.3% of the cases were positive