Higher prevalence and gene amplification of HPV16 in oropharynx as compared to oral cavity

ABSTRACT Objective The objective of this study was to clarify differences regarding HPV16 infection and gene amplification between the oral cavity and oropharynx in healthy individuals. Material and Methods The subjects were 94 healthy asymptomatic individuals (41 males, 53 females; mean age 58.6 years, range 16-97 years) who visited the Department of Oral and Maxillofacial Reconstructive Surgery of the Hiroshima University Hospital from 2014 to 2015. Oral epithelial cells were collected from oral rinse and pharynx gargle samples and placed in saline. The human endogenous retrovirus gene ERV3-1 was used as a reference to estimate the number of human cells in each sample. DNA samples were extracted from approximately 10,000 human cells and tested for HPV16 DNA by PCR using a type-specific primer. Similarly, we analyzed the HPV16 viral copy number in HPV16-positive cases using real-time PCR to examine genomic amplification. Results The percentage of HPV16-positive cases was higher in the gargle (28.7%) as compared to the rinse (16.0%) samples. In the oral rinse samples, males (26.8%) showed a significantly higher rate of HPV16 than females (7.5%) (P=0.021). Importantly, in older subjects (aged 60-89 years), gargle samples showed a significantly higher rate of HPV16 (33.3%) than oral rinse samples (13.7%) (P=0.034). The average number of viral copies was approximately 8 times higher in the gargle than in the oral rinse samples (0.16±0.27 vs. 1.35±1.26 copy numbers per cell), a significant difference (P<0.001). Conclusion Our findings suggest that the oropharynx is more susceptible to HPV16 infection as compared to the oral cavity, while HPV16 gene amplification is also more commonly found in the oropharynx.


INTRODUCTION
The human papilloma virus (HPV) consists of a circle of double-stranded DNA and belongs to the family of papillomaviridae, with infection generally occurring in the skin and mucosa 7,8 . HPV infection remains in basal cells, though is discharged outside the body upon differentiation of the infected cells into keratinocytes 8 . The virus, therefore, does not enter the bloodstream and is able to evade immune surveillance. The HPV genome consists of six early open reading frames (ORFs) (E1, E2, E4, E5, E6, E7), two late ORFs (L1, L2), and a non-coding long control region (LCR) 7,8 . According to the International Agency for Research on Cancer (IARC), 12 HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) 3 , of which in the early 1980s and considered to be the most important risk factor for development of cervical cancer 30 . In addition, persistent infection with oral HPV16 is also thought to drive the development of oropharyngeal cancer 12 . We previously reported that most precancerous lesions caused by HPV16 infection of the oral associated with malignant transformation of precursor lesions 24 . However, few epidemiologic studies have focused on oral HPV16 prevalence in healthy Japanese individuals and that has not been fully elucidated. In this study, we investigated oral rinse and gargle samples obtained from healthy individuals to clarify differences of HPV16 infection oropharynx.

Subjects
The subjects of this study were 94 healthy asymptomatic individuals (41 males, 53 females; mean age 58.6 years, range 16-97 years), who visited the Department of Oral and Maxillofacial Reconstructive Surgery of the Hiroshima University Hospital from 2014 to 2015. None had evidence of oral cancer or pre-malignant lesions. The study design was approved by the Ethics Committee of the Hiroshima University and all participants signed an informed consent form.

Oral rinse and gargle sample processing and DNA extraction
Oral rinse samples were used to evaluate HPV16 infection in the oral cavity while gargle samples were used to the evaluation of the oropharynx. Exfoliated epithelial cells were collected in samples obtained by a 30-second oral rinse and a separate 30-second gargle 9 their mouth with 10 mL of saline for 30 seconds and then spit into a sterile 50-mL falcon tube. Next, they gargled 10 mL of saline for another 30 seconds and spit that sample into a different 50-mL falcon tube. Immediately after collection, all samples were centrifuged at 3000x g for 10 minutes at 4°C, then the supernatant was decanted, the pellet resuspended in 10 ml of phosphate-buffered saline (PBS), and centrifugation repeated. Thereafter, the pellets were stored at -80°C until further processing.

Quantitation of human cell numbers
DNA amounts extracted from the oral rinse samples were not equivalent to the amount of human DNA, due to the quantity of non-human derived DNA. Therefore, we employed the human endogenous retrovirus group 3 member 1 (ERV3-1) gene to quantitate human cells using a real-time PCR assay, according to a previously reported method 9 . A 1.7-kb Hind III:Pst I fragment within the ERV3-1 genome was prepared and then cloned into a 2.7-kb pUC57 vector. Serial 10-fold dilutions of the pUC57 vector were made with copy numbers ranging from 10 0 to 10 9 . Calculation of number of copies was performed based on the DNA molecular weight of the plasmid, according to a previously reported method 29 . Next, we performed real-time PCR assays primer. Quantitation of DNA levels was done using a CFX connect real-time PCR detection system (BioRad, Hercules, CA, USA) and SYBR Green PCR Master Mix (TOYOBO, Tokyo, Japan), with a reaction mixture containing 1.0 μl of DNA, 12.5 μl of SYBR Green Mix, and 10 μmol of each pair of oligonucleotide primers. The primer sequences were as follows: ERV3-1; 5'-CATGGGAAGCAAGGGAACTAATG-3' (sense) and 5'-CCCAGCGAGCAATACAGAATTT -3' (antisense) 9 initial melting at 95°C for 5 minutes, then 40 cycles of denaturing at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. A standard curve indicating CT value versus ERV3-1 copy number was obtained ( Figure 1A). The number of cells, determined by use of the ERV3-1 diploid genome, was used to estimate the number of human cells in each sample.

HPV16 DNA detection by PCR
The HPV16 sequence was examined by PCR open reading frame using a PCR HPV Detection Set fragment with a length of 140 base pairs. Samples an oral cellular sample and those without G3PDH were excluded from analysis. A positive control containing HPV16 DNA extracted from Caski cells and a negative control containing DNA were used for the PCR reactions. We prepared DNA samples extracted from approximately 10,000 human cells in each PCR mixture. Each 10x PCR buffer (TOYOBO), dNTPs, Taq DNA polymerase, and HPV16 primers. The primer sequences for HPV16 were 5'-AAGGGCGTAACCGAAATCGGT-3' (sense) and 5'-GTTTGCAGCTCTGTGCATA-3' (antisense), and those for G3PDH were 5'-ACCACAGTCCATGCCATCAC-3' (sense) and 5'-TCCACCACCCTGTTGCTGTA-3' (antisense). The PCR program included initial melting at 95°C for 5 minutes, followed by 28 cycles at 95°C for 30 product was electrophoresed on 3% agarose gels HPV16 DNA sequence, we performed sequencing for HPV16. PCR product was analyzed by electrophoresis using 2% agarose gels. For the sequencing reaction, a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) was with an ABI PRISM 310 genetic analyzer (Applied Biosystems).

HPV16 viral copy quantitation
Real-time PCR assays were performed to determine the viral load of HPV16 DNA in the samples. We prepared a 200-bp fragment within the HPV16 E6 genome, which was then cloned into a 2.7-kb pUC57 vector. Serial 10-fold dilutions of the vector were made with copy numbers ranging from 10 0 to 10 9. Serial 10-fold dilutions were used to generate a standard curve, which indicated the CT value versus the copy number of HPV16 E6. We then prepared DNA samples extracted from approximately 10,000 human cells from each PCR mixture. Real-time PCR was performed using a (Takara). The PCR program was as follows: initial melting at 95°C for 5 minutes, followed by 40 cycles at 95°C for 30 seconds, 57°C for 2 minutes, and 72°C for 30 seconds. A standard curve was used to enumerate the copy number of HPV16 in each sample.

Statistical analysis
Fisher's exact test was used to examine the correlation between oral HPV16 infection and clinical parameters. Student's t-test was used to evaluate significant differences regarding HPV16 copy numbers. P values less than 0.05 were regarded as RESULTS HPV16 prevalence in oral rinse and pharynx gargle samples HPV16 prevalence was examined in a total of 94 oral rinse and gargle samples. The product of HPV16 DNA was detected in Caski cells (HPV16 positive), but not in HTB-31 cells (HPV negative), by PCR analysis ( Figure 1B). Next, the PCR products were

Quantitation of HPV16 viral copy number using real-time PCR
In HPV16-positive cases, we analyzed the HPV16 viral copy number using real-time PCR to examine Serial 10-fold dilutions of a plasmid vector containing HPV16 E6 DNA were used to generate a standard curve (Figure 2A), then we evaluated the number of HPV16 DNA copies as copy number per human cell. The average number of viral copies was approximately 8 times higher in the gargle samples than in the oral rinse samples (0.16±0.27 vs. 1.35±1.26 copy numbers per cell) ( Figure 2B). We found a significant difference regarding HPV16 E6 DNA copy number between the gargle and oral rinse samples (P<0.001). These is more commonly found in the oropharynx as compared to the oral cavity.

DISCUSSION
Oral HPV infection is considered to be transmitted through sexual behavior, thus marital status, number of sex partners, oral sex, and anal sex are thought to be related variables. Oral HPV prevalence is significantly higher in individuals who have participated in oral sex as compared to those who have not, indicating that as a risk factor for oral HPV infection 11 . As for other risk factors, smoking and poor oral hygiene related to such variables as periodontitis, gingivitis, and denture use have been found to be associated with oral HPV infection 12,15,18 .
As for smoking, the chemical substances contained in tobacco may be involved and suppress immune mechanisms in the oral cavity 11 , while the relationship of denture use to HPV infection suggests that oral mucosal injury caused by mechanical stimulation from denture wearing and poor oral hygiene due to unclean dentures may have an association 23 . Thus, HPV infection may be associated with oral environmental changes due to local effects, such as smoking and denture use. Importantly, our results showed that being male Although the reasons remain unclear, smoking, denture use, sexual activity, and other factors may be related to the high rate of oral infection seen in men.
In the subjects of this study, the prevalence rate of HPV16 was higher in the gargle samples as compared to the oral rinse samples. Gargle samples from the older groups also showed oral rinse samples from those subjects, indicating an important association between age and HPV16 infection in the oropharyngeal region, and that age distribution is likely to be a cause of differences between oral rinse and gargle samples. In addition, in the gargle samples. Despite cross-mixing of the samples between the oral cavity and oropharyngeal region, we considered that the collected oral rinse and gargle samples were associated with HPV DNA present primarily in the oral cavity and oropharyngeal region, respectively. Thus, our observations suggest that the oropharynx is more susceptible to HPV16 infection and HPV16 gene In a previous study of head and neck cancer cases, the positive rate of HPV was 10% in patients with oral cavity cancer, 83.2% in those with oropharyngeal cancer, and 44.4% in those with nasopharynx cancer, demonstrating the highest prevalence in oropharyngeal cancer cases, while approximately 90% of the HPV-positive oropharyngeal cancer patients also showed HPV16 infection 27 . Furthermore, Wang, et al. 28 (2012) reported that male oropharyngeal cancer patients showed a higher rate of HPV16 infection than female patients. Our results also showed that the gargle specimens from males had a higher rate of HPV16 infection as compared to those from females. Another study also reported that the HPV16 genome copy number was much higher in oropharyngeal cancer than oral cancer specimens 22 . Together, these results support the notion that the oropharynx is a more common site for HPV16 infection as compared to other head and neck regions. In addition, the variability of HPV16 infection rate among different sites (i.e., oral, oropharyngeal, laryngeal) suggests the biological characteristics of HPV-related cancer.
Findings for the prevalence of oral HPV vary substantially among reported studies, which has been attributed to differences in study populations, as well as approaches for specimen collection and detection methods 1,2,[4][5][6]13,14,16,17,[19][20][21]25,26 . In recent studies of oral HPV infection, the methods most frequently used for oral sample collection have included oral swabbing, oral rinsing, and gargling with mouthwash or saline, with rinsing with mouthwash or saline thought to be highly effective. PCR assays targeting the HPV L1 region have also been commonly performed in many studies, while direct HPV genotyping without HPV detection was utilized in some 1,2,[4][5][6]13,14,16,17,[19][20][21]25,26 . In those, HPV genotypes were determined by PCR using typehybridization frequently using the LINEAR ARRAY HPV Genotyping Test , as it is useful for detection of combined HPV infection, though less sensitive and positive rates may vary with different cut-off values 10 . Thus, we used a combination of PCR with detection to accurately determine HPV16 infection status in the present subjects.
Taken together, our observations indicate that HPV16 infection has a higher rate of prevalence in gargle samples. Thus, HPV16 is considered to be an important risk factor for development of head and neck cancer, especially oropharyngeal cancer. Future studies are needed to examine the risks of smoking, oral hygiene, and sexual behavior in relation to HPV infection, and will likely lead to a decrease in oral HPV infection rates, while elucidation of the molecular biology of HPV may reduce the occurrence of HPV-related head and neck cancer.

CONCLUSION
Our results indicate that the oropharynx is more susceptible to HPV16 infection as compared to the more commonly found in the oropharynx.