Tissue samples were obtained from 100 patients with OSCC, including basaloid squamous cell carcinoma (BSCC), who underwent surgical resection at the Department of Oral and Maxillofacial Surgery at Ehime University Hospital (Ehime, Japan) between April 2004 and March 2013. Tumor staging was assessed according to the Union for International Cancer Control TNM Classification of Malignant Tumors 7th Edition and histological grading was performed according to the World Health Organization criteria for OSCC (24). The Institutional Review Board of Ehime University Hospital (Ehime, Japan) approved the present study.

Surgically resected OSCC specimens were fixed in 10% phosphate-buffered formalin for 24 h at room temperature and embedded in paraffin. A series of 4-µm thick sections were prepared from each sample. Immunohistochemical (IHC) staining was performed using the avidin-biotin-peroxidase complex method. Briefly, the sections were deparaffinized and heated at 121°C in an autoclave for 20 min in 10 mM citrate buffer (pH 6.0) to regenerate epitopes. The sections were incubated with 0.3% hydrogen peroxide in distilled water for 5 min at room temperature to block endogenous peroxidase activity. The sections were then incubated overnight at 4°C with a specific mouse monoclonal antibody to anti-human p16 (cat. no. 550834; diluted 1:50; BD Pharmingen; BD Biosciences). After washing with TBS+Tween-20 (TBS-T; Sigma-Aldrich; Merck KGaA), the sections were overlaid with biotinylated anti-mouse antibody (Maravai LifeSciences) at room temperature for 60 min, washed with TBS-T, then labeled with streptavidin-peroxidase complex (Maravai LifeSciences). The sections were subsequently counterstained with hematoxylin for 10 sec at room temperature, dehydrated with a series of graded ethanols (75, 95, 100 and 100%) for 5 min each at room temperature, treated with xylene and enclosed in synthetic resin. IHC staining was observed at ×40 and ×100 magnifications under a light microscope (Nikon Corporation). Positive expression of p16 protein was determined as when >70% of tumor cells showed strong and diffuse nuclear and cytoplasmic staining.

Total RNA was extracted using ISOGEN (Nippon Gene) from lysing the tissues, after homogenization, with a TissueLyser (Qiagen), according to the manufacturer's protocol.

The presence of HPV-16E6 mRNA in the tissues was determined using RT-qPCR and the TaqMan^® RNA-to-C_T™ 1-Step kit (Thermo Fisher Scientific, Inc.). PCR amplification was performed in a 10-µl final reaction mixture containing 0.25 µl TaqMan^® RT Enzyme Mix (40X), 5 µl TaqMan^® RT-PCR Mix (2X), 0.4 µl each forward and reverse primers (10 µM each), 0.2 µl TaqMan^® probe (10 µM) and 1 µl total RNA (100 ng/µl). The following thermocycling conditions were used: RT at 48°C for 15 min, then initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 min and 60°C for 1 min. Oropharyngeal cancer tissues from biopsy materials obtained from patients with tonsil cancer treated in the Department of Oral and Maxillofacial Surgery at Ehime University Hospital were used as the positive control. Written informed patient consent was obtained.

The expression of HPV-18E6 mRNA was detected using RT-qPCR and the SYBR^® system. PCR amplification was performed in a 10-µl final reaction mixture containing 5 µl 2X One Step SYBR^® RT-PCR Buffer 4, 0.4 µl PrimeScript^® One Step Enzyme Mix 2, 0.2 µl ROX reference Dye II (50X), 2.6 µl RNase-free distilled water (all from Takara Bio, Inc.), 0.4 µl forward and reverse primers (10 µM each) and 1 µl total RNA (100 ng/µl). The following thermocycling conditions were used: RT at 45°C for 5 min and 95°C for 10 sec, followed by 40 cycles at 95°C for 5 sec and 55°C for 30 sec.

Amplification and detection were performed using the ViiA™ 7 real-time PCR system (Thermo Fisher Scientific, Inc.). Hydroxymethylbilane synthase (HMBS) was used as an internal control. To confirm the amplicon size, the PCR products were electrophoresed on 3% agarose gels (Bio-Rad Laboratories), stained with ethidium bromide for 5 min at room temperature, and visualized under an ultraviolet transilluminator (FAS-III; Toyobo Life Science).

The following primers and TaqMan^® probe were used: HPV-16E6 forward, 5′-GAATGTGTGTACAAGCAACAG-3′, reverse, 5′-TGGATTCCCATCTCTATATACTATGCAT-3′ and TaqMan^® probe, 5′-CGACGTGAGGTATATGACTTTGCTTTTCGG-3′ (25); HPV-18E6 forward, 5′-CAGAAACCGTTGAATCCAGCA-3′ and reverse, 5′-TTTCTCTGCGTCGTTGGAGTC-3′ (25) and HMBS forward, 5′-CATGCAGGCTACCATCCATGTC-3′ and reverse, 5′-GTTACGAGCAGTGATGCCTACCAA-3′.

Genomic DNA was extracted from formalin fixed paraffin embedded (FFPE) OSCC tissues using a GeneRead DNA FFPE kit (Qiagen GmbH). Exonuclease V (ExoV; New England BioLabs, Inc.) digestion was performed, as previously described (26). For detecting HPV-16E6 DNA, qPCR amplification was conducted in a 10-µl final reaction mixture containing 5 µl 2X PowerTrack™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Inc.), 0.5 µl each forward and reverse primers (8 µM each), 3 µl nuclease-free distilled water and 1 µl genomic DNA (10 ng/µl) with or without ExoV digestion. The following thermocycling conditions were used: Enzyme activation at 95°C for 2 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Amplification and detection was performed using a ViiA™ 7 real-time PCR system (Thermo Fisher Scientific, Inc.). Human ribosomal 18S DNA (rDNA) was used as an internal control. To confirm the amplicon size, the PCR products were electrophoresed and visualized using an Agilent 2100 Bioanalyzer with the DNA 1000 kit (Agilent Technologies). The following primers were used: HPV-16E6 forward, 5′-GAGAACTGCAATGTTTCAGGACC-3′ and reverse, 5′-TGTATAGTTTGCAGCTCTGTGC-3′; rDNA forward, 5′-GCAATTCCCCATGAACG-3′ and reverse, 5′-GGGACTTAATCAACGCAAGC-3′ (26).

χ² or Fisher's exact tests was used to determine significant differences between 2 groups. The Kaplan-Meier method was applied for survival analysis after follow-up for 36 months. Differences in patient survival were determined using the log-rank test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using GraphPad Prism software (v5.04; GraphPad Software, Inc.).

The 100 OSCC cases included 54 men and 46 women, ranging in age from 39 to 93 years (median, 70.3 years). The primary tumor was located in the tongue (n=36), mandibular gingiva (n=31), maxillary gingiva (n=13), floor of the mouth (n=9), buccal mucosa (n=9), or lower lip (n=2). All the patients were histopathologically diagnosed with squamous cell carcinoma, including BSCC (n=1).

Positive expression of p16 protein was observed in 10 out of 100 OSCC cases (10%). Most p16-positive cases showed strong and diffuse staining in the nucleus and the cytoplasm of the tumor cells (Fig. 1A and B). The association between p16 immunohistochemistry expression in the tumors from 100 patients with OSCC and their clinicopathological parameters was investigated; however, the differences were not significant (Table I). Furthermore, following the analysis between p16 expression and survival using the Kaplan-Meier method, no significant association between overall and disease-free survival times was found (Fig. S1).

To clarify HPV-16 infection, the expression level of HPV-16E6 mRNA in 100 OSCC cases was determined using RT-qPCR. HPV-16E6 mRNA expression was only detected in one case (1%), which was also positive for p16 expression. PCR products of HPV-16E6 and HMBS were visualized using agarose gel electrophoresis (Fig. 2). HPV-18E6 mRNA expression was investigated in p16 positive OSCC tissues using RT-qPCR; however, it was not detected (data not shown). Furthermore, the possibility of persistent or silent infection with HPV-16 was investigated using genomic DNA derived from p16 positive OSCC cases using qPCR. HPV-16E6 DNA was detected in 3 out of 10 cases (Cases 1, 3 and 10; Fig. S2A). One of these cases expressed HPV-16E6 mRNA (Case 1), but the other cases did not (Case 3 and 10). Subsequently, genomic DNA was digested by ExoV, which preserved nicked and supercoiled DNAs but degraded linear DNAs, followed by qPCR for the detection of HPV-16E6 DNA. HPV-16E6 DNA was ExoV-resistant, suggesting that the circular episome was observed only in the HPV-16E6 DNA and mRNA positive case (Case 1; Fig. S2B). Since neither HPV-16E6 DNA nor RNA was detected in 7 of the 10 cases of p16 overexpression (Case 2, 4, 5, 6, 7, 8 and 9), the false-positive rate of HPV-16 infection was 70%. Finally, only the HPV-16E6 DNA- and mRNA-positive case (Case 1) was histopathologically determined as BSCC, which is a rare variant of OSCC. The other cases (Case 3 and 10) were determined to be keratinized squamous cell carcinomas.