Molecular Prevalence of Human Papillomavirus Types 16 and 18 in Oral Squamous Cell Carcinoma using Real-time PCR

1. Introduction

Oral squamous cell carcinoma (OSCC) is a malign disease worldwide with a dramatic effect on health and life quality, with more than 377,700 cases worldwide in 2020 (1). The OSCC is known as the most common type of oral cancer, with high morbidity and mortality in patients with head and neck cancers (2). Various risk factors have been connected to oral cancer; among them, alcohol consumption and tobacco smoking are the most typical connected risk factors of OSCC in 74% of cases with OSCC (3). The Human papillomaviruses (HPVs) are a group of various and prevalent DNA viruses that infect epithelial cells of the mucosa and skin. The HPV lesions are usually clinically imperceptible, but enduring infection with high-risk HPV types resulted in malignancies at diverse anatomic locations in the neck, head, and anogenital region (4). The HPV16 and HPV18, as major high-risk types, are closely connected to malignant tumors (5). Although the association between OSCC and HPV was first proposed in 1983, the existence of HPV DNA was approved two years after, using in situ hybridization. The interesting point is that HPV types 16 and 18 contribute to more than 70% of cases of all HPV-related cancers (6). The HPV types 16 and 18 raise tumorigenesis via expression of oncoproteins, including the E6 and E7, which inactivate the tumor suppressors P53 and RB1, respectively (7). The HPV role in the growth of head and neck cancers is not obvious, mainly because its prevalence is not closely 100% as in cervical carcinoma. The HPV incidence is variant in head and neck cancers (8). In Iran, the prevalence of HPV in oral cancers ranges from 9.0% in Shiraz to 50% in Rasht. These differences in HPV prevalence are related to the intraoral location, geographical area, type of lesion, and grading (9). Initial studies indicate that the prevalence of HPV-positive oral cancers is increasing in Iran (10). Despite the presence of considerable preventative policies, HPV-related cancer remains one of the causes of mortality in different regions of the world, especially in developing countries. There is no denying that increasing knowledge about the prevalence of HPV is necessary to put a better plan into practice for vaccination and treatment. Therefore, in this study, we estimate the prevalence of HPV types 16 and 18 in OSCC in the Qazvin province, Iran.

2. Materials and Methods

2.1. Study area and study population

This study was carried out in Qazvin, located in north-central Iran, in a wide fertile plain at the southern foot of the Elburz Mountains with a population of 1,284,000.

2.2. Sample collection

In this cross-sectional study, 38 archived formalin-fixed and paraffin-embedded OSCC biopsy specimens were tested at the Oral Pathology Laboratory located in the Faculty of Dentistry of Qazvin University of Medical Sciences as a province referral laboratory. These specimens were received from 20 (52.6%) women and 18 (47.4%) men with ages ranging from 31-95 years (mean age of 64±17 years).

2.3. Extraction of DNA

Genomic DNA was isolated using a purification kit (Roche High Pure FFPE DNA isolation kit, Germany). Before DNA extraction, deparaffinization was perfirmed by the xylene-ethanol deparaffinization method (11). Following, all extracted DNA was stored at -80^◦C until the time of molecular tests.

2.4. Quality control of DNA Extraction

The DNA quality was assessed by PCR using primers PC04 (5′-CAACTTCATCCACGTTCACC-3′) and GH20 (5′-GAAGAGCCAAGGACAGGTAC-3′) that amplify a 268 bp product from the human β-Globin gene (12).

2.5. Molecular identification of HPV

Positive samples of β-Globin were subjected to HPV detection using SYBR green-based real-time PCR by primers GP5+: 5'-TTTGTTACTGTGGTAGATACTAC-3' and GP6+: 5' AAAAATAAACTGTAAATCATATTC-3' that amplifies a 150 bp product by targeting the L1 region of HPV (13). Following real-time PCR amplification, each reaction was performed in a final volume of 20 μL, containing 10 μL SYBR green PCR Master mix (TAKARA BIO INC Japan), 0.5 μL 10 pM of each forward and reverse primer, 2 ml of DNA template, 6.6 μL PCR grade water, and 0.4 ml ROX Reference Dye. Initial denaturation was at 95°C for 2 min, followed by 40 cycles, each cycle including denaturation at 94°C for 30 sec, annealing at 40°C for 2 min, and extension at 72°C for 90 sec.

2.6. HPV genotype identification

The SYBR green-based real-time PCR assay was performed using two specific primers (HPV16F: 5'-TGCTAGTGCTTATGCAGCA-3', HPV16R: 5'-TTACTGCAACATTGGTACATGG-3' and HPV18F: 5'-CGCCACGTCTAATGTTTCTG-3', HPV18R: 5'-CCTGTGATAAAGGACGCGA-3') on all positive tested samples in the previous section for the detection of HPV types 16 and 18. A final reaction volume consisted of 20 μL containing 10 μL SYBR green PCR Master mix (TAKARA BIO INC Japan), 0.5 μL 10 pM of each forward and reverse HPV16F and HPV18 primers and 2 ml of DNA template, 6.6 μL PCR grade water and 0.4 ml ROX Reference Dye. DNA was amplified by real-time PCR (Applied Biosystems, CA, USA) with SYBR green dye for detection, and each cycle consists of 2 min at 95^◦C, 40 cycles of 10 sec at 95^◦C, 30 sec at 60^◦C and 72°C for 30 sec.

2.7. Statistical Analysis

The association between HPV infection in OSCC and age, sex, and anatomic location was analyzed using Fisher’s exact tests and Chi-square with the SPSS software (version 16). A P-value less than 0.05 was considered statistically significant.

3. Results

3.1. General

The study group included 38 archived formalin-fixed and paraffin-embedded OSCC biopsy specimens tested at the School of Dentistry Qazvin University of Medical Sciences. Out of 38 patients with OSCC, 18 )47.4%( were men and 20) 52.6%( were women, with a mean age of 64 ± 17, and a distribution range of 31-95, classified into seven groups (Fig 1). The OSCC lesions’ locations were in the jaw bone 6/38 (15.8%), buccal mucosa 7/38 (18.4%), oral floor 3/38 (7.9%), tongue 16/38 (42.1%), lip 3/38 (7.9%), and other areas 3/38 (7.9%).

3.2. Prevalence of HPV types 16 and 18 using real-time PCR

From a total of 38 formalin-fixed and paraffin-embedded

OSCC after molecular test, 13/38 (34.2%) cases were HPV positive. The prevalence of HPV among women and men was estimated 40.0% (8/20) and 27.8 % (5/18), respectively. The age group of 61-70 (10.5%) years had the highest HPV infection rate. Although HPV infection was prevalent in female compared to male patients, the difference was not statistically significant (40.0% vs. 27.8%, P=0.428). Our findings also showed non-significant associations between age and HPV infection (P=0.714). The HPV16 and HPV18 were detected in 8/38 (21.0%) and 4/38 (10.5%) cases, respectively. Of them, 1/38 (2.6%) was related to other viral subtypes. The overall prevalence of HPV in total in different locations, including tongue 6/38 (15.8%), jaw bone 3/38 (7.9%), buccal mucosa 3/38 (7.9%), and oral floor 1/38 (2.6%) is presented in Figure 2. It should be noted that the prevalence of HPV within the location of lesions is mentioned in Table 1. Although statistically no significant difference was shown between HPV and lesion location (P=0.536), HPV was more prevalent in tongue samples.

4. Discussion

Although numerous studies have confirmed the causative role of HPV infection in oral squamous cell carcinoma worldwide, its role as a risk factor in OSCC has been contentious (14). In the current study, the prevalence of HPV and its genotypes were estimated using real-time PCR in OSCC. These days, a considerable interest exists in using molecular methods in epidemiologic studies to estimate the prevalence of pathogens, including high-risk HPV types (15), which are considered the gold standard method for HPV detection in clinical specimens (16). Many studies have also shown that the prevalence of HPV

 in OSCC differed from 0% in Japan (17) and 64.5% in India (18). This broad spectrum variability might be described by various factors, including sample collection techniques, disease stage, sensitivity of the used methods, and demographic factors (19). There is no denying the fact that HPV is regarded as one of the risk factors of OSCC; accordingly, estimating the prevalence rate of HPV and its genotypes in OSCC seems to be necessary. The findings of this study revealed that 34.2% (13/38) of formalin-fixed and paraffin-embedded OSCCs tested were positive for HPV. Of these 13 positive samples, 61.5% (8/13), 30.8% (4/13), and 7.6% (1/13) were found to be positive for HPV 16, HPV 18, and other HPV types, respectively. Findings of a study by Hadi Razavi Nikoo (16) showed the prevalence of HPV-linked OSCC was 36%, which is in agreement with our study. In another study conducted by Tabatabai, they reported that HPV prevalence was 43%, and they also estimated that HPV16 is the most prevalent type, which is approximately in line with our results (20). In another study conducted in 2018 in Iran on oral samples, the HPV prevalence rate was reported at 13.0%, which was less than the current study findings (21); these differences in the prevalence of HPV can be related to geographical differences (9). As well as in a parallel study to our findings conducted in Korea, the prevalence of HPV was evaluated at 36%, and the more common HPV type was HPV-16 (22). In the current study, HPV was more prevalent in women (40.0%) than men (27.8%) groups, which was similar to the study by Seifi (23). In a similar study reported by Tsimplaki, HPV infection was more prevalent in women compared to men, and the presence of HPV was not associated with age (24). The findings of this study were against the research by Ashraf and Kreimer that the prevalence of HPV in men is higher than in women (25). The prevalence of high-risk HPV highly varies in different studies between women and men in different geographic regions (26). Heterogeneity among studies about this can be also associated with the sexual behavior of patients (20). In our study, this non-significant correlation may be related to our patient's ages that majority of them were above 50 years old. Tongue is the most prevalent location in OSCC (27). In this study, HPV prevalence in OSCC was more prevalent in tongue samples similar to Ashraf et al. and Makvandi studies conducted in Iran (26, 28); in those studies, HPV was more prevalent in tongue compared to other locations of the lesions. In general, it seems that due to strong cultural changes in lifestyle and sexual behaviors, studies related to the prevalence of HPV in OSCC need to be repeated and re-evaluated. Therefore, the results of the present study would be important from this point of view. The high prevalence of HPV16 and 18 in OSCC suggests that there is a pressing need for future implementations of public health interventions, including vaccination, increasing people's awareness, sanitation, sexual health, appropriate healthcare, and ongoing screening of HPV. Furthermore, HPV DNA testing with high sensitivity and positive predictive value should become a main tool in OSCC screening.