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Low Frequency of Human Papillomavirus and Epstein-Barr Virus DNA in Ameloblastoma of Thai Patients

Ab s t r Ac t Aim: This preliminary study aimed to evaluate the frequency of human papillomavirus (HPV) and Epstein-Barr virus (EBV) DNA in solid/multicystic ameloblastoma (AM). Materials and methods: A total of 20 fresh-frozen AM tissue was investigated for HPV and EBV DNA using polymerase chain reaction (PCR) at L1 of HPV and LMP1 of EBV. Results: It was found that 2 out of 20 cases (10%) were positive for HPV PCR, whereas 3 out of 20 cases (15%) were positive for EBV PCR. Importantly, a mix of HPV and EBV DNA was observed in one case. Conclusion: Our study presented a low frequency of HPV and EBV DNA in AM. Further studies with different cohorts and larger sample sizes are necessary for a better understanding of the frequency of both viruses in AM. Clinical significance: HPV and EBV may not be involved in ameloblastogenesis.


IntroductIon
Ameloblastoma (AM) is a benign neoplasm derived from residual epithelial components of tooth development and generally present in the jawbone.The majority of AM occurs in patients between 20 and 40 years of age, although they can occur at any age. Ameloblastoma was observed in 1% of maxillofacial tumors and approximately 35% of odontogenic tumors in Thailand AM-solid/ multicystic type. [1][2][3] This tumor is often asymptomatic, slow-growing but shows locally aggressive behavior and has high recurrence potential among odontogenic tumors.The mechanisms involved in the pathogenesis of AM have not been completely elucidated. Factors such as trauma, tooth eruption, and viral infection have been reported with AM etiopathogenesis. [3][4][5] To date, several studies reported an association between the presences of viral DNA in many cancers. Both human papillomavirus (HPV) and Epstein-Barr virus (EBV) are known as oncogenic viruses. 6,7 Human papillomavirus infection is the most common sexually transmitted infection and about 9-13% of the world's population is already infected. 8 High-risk HPV (hrHPV) is an established cause of various human neoplasms including cervical, vulvar, sinonasal, oral, and penile cancers. [8][9][10][11] In addition, EBV is the first human tumor virus that is observed in more than 90% of the world's adult population. 6 Epstein-Barr virus can contact and cause infectious mononucleosis during childhood. In addition, EBV infection could be linked to several other types of human malignancies, particularly nasopharyngeal cancer (NPC), gastric cancer (GC), Burkitt's lymphoma, some Hodgkin's lymphoma, and B-cell lymphoma. 6,12 Epstein-Barr virus and HPV implicate to cellular immortalization and oncogenesis. Their genetic materials can produce viral oncoproteins that inhibit tumor suppressor gene and/or integrate viral DNA to the human genome. 13,14 Considering the ameloblastoma pathogenesis, the relationship with HPV and EBV is needed to be substantiated. In this study, we aimed to evaluate the frequency of HPV and EBV DNA in solid/multicystic AM which could contribute to the knowledge concerning viral-associated AM patients in Thailand.

Ethical Statement and Sample Recruitment
Ethical consideration of this cross-sectional study was reviewed and approved by the Institutional Review Board of Faculty of the Dentistry/Faculty of Pharmacy, Mahidol University, Bangkok, Thailand (Approval number: 2018/005.1101). An informed consent was obtained from all patients before the collection of specimens. Data were analyzed anonymously. A total of 20 fresh-frozen tissue samples were obtained from the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Mahidol University, during January 1, 2018, to December 31, 2019. These AM patients were Thai-12 males and 8 females with a median age of 48.5 years (range 8-73 years). Most cases were located in the mandible while only four cases were located in the maxilla. All cases were histologically confirmed as solid multicystic AM 8 follicular and 12 plexiform histological subtypes by two oral pathologists based on current World Health Organization (WHO) guidelines. 15 Clinical and pathological data such as age, sex, anatomic location of the lesions, and histological subtypes are summarized in Table 1.

DNA Isolation
Fresh-frozen tissues were lysed at 50°C overnight with a lysis buffer (0.75 mol/L NaCl, 0.024 mol/L EDTA, pH 8.0) that was mixed with 10% sodium dodecyl sulfate (SDS) and 20 mg/mL proteinase K (Sigma-Aldrich, St. Louis, MO, USA). Next, the DNA purification was done using a standard phenol-chloroform extraction protocol. 16 Thereafter, the purified DNA was air-dried, eluted with distilled water, and stored below −20°C for subsequent use. The DNA concentration was measured using a NanoDrop 2000 spectrophotometer (ND-2000 Spectrophotometer, NanoDrop Technologies, Wilmington, DE, USA). The optical density 260/280 ratio was greater than 1.8, which is acceptable for DNA purity and PCR.

Detection of EBV and HPV DNA by Polymerase Chain Reaction
Polymerase chain reaction (PCR) was performed using the primers, displayed sequences, and conditions as shown in Table 2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control to test the quality of DNA. The L1 HPV primer (GP5+/GP6) 17 that encodes major capsid protein of HPV was tested to detect HPV DNA. The LMP-1 EBV primer 18 that encodes EBV latent membrane protein was tested to detect EBV DNA.
The PCR mixtures of all reactions contained 1× PCR buffer, 200 mM dNTPs, 0.2 mM primers, 0.5 U Taq DNA polymerase (Qiagen, San Diego, CA, USA), and 50 ng of template DNA. DNA from the B958 and HeLa cell lines was used as the positive controls of EBV PCR and HPV PCR, respectively. Distilled water was used as the negative control in every PCR reaction. Following amplification, the PCR products were separated by gel electrophoresis using a 2%agarose gel in

results
As shown in Table 1, the frequency of EBV and HPV was 3/20 (15%) and 2/20 (10%), respectively. From the histopathological feature, koilocytic changes due to viral infection were not observed in all samples. Among EBV-positive cases (AM1, AM4, and AM6), there were two males and one female. All were located in the mandible with histological plexiform subtype. Two HPV positive cases (AM1 and AM7) consisted of one male and one female. Both of them were located in the mandible, with individual histological plexiform and follicular subtypes. After sequencing and blasting, both DNA matched to the HPV 18 (KU298886.1) completely. Our results found a noteworthy case (AM1) showing mixed EBV and HPV DNA (Fig. 1).

dIscussIon
The EBV and HPV are common cancer-associated viruses. Both viruses have permissive cells and induce tumorigenicity in specific cell types. 14 Race is one of the dependent factors that affect frequencies of EBV and HPV DNA in each individual population. 12 We have summarized the varied frequencies of EBV and HPV DNA in AM from acquired previous studies in Tables 3 and 4. Since, there is no data about the frequency of HPV and EBV DNA of AM in Thai population (PubMed Database, Scopus, and Web of Science), this preliminary study was carried out to evaluate both viruses' frequencies in Thai patients. Here, we found low frequencies of both viruses, which might suggest their minor involvement in a group of AM patients. We observed 15% of EBV DNA in AM that was similar to the previous study by Fujita et al. 19 and in range with other studies (0, 26.6, and 48% by Khalele et al., 21 Badrawy et al., 11 and Jang et al., 20 respectively) ( Table 3). The differences of frequency may be affected by the EBV detection technique and racial involvement. In general, the gold standard technique for EBV detection is by in situ hybridization (ISH) of EBER; however, the cost of this technique is very expensive. The PCR is more convenient and highly sensitive, but it might cause false-positive results that require confirmation by immunohistochemistry (IHC) using EBV antibodies or ISH EBV-EBER. 20 To ensure the results of this study, future investigations by the other techniques may be needed.
The EBV-associated Burkitt's lymphoma has a high prevalence in sub-Sahara area of Africa, whereas EBV-associated nasopharyngeal cancer has a high prevalence in South China area, 20 suggesting that the incidence of EBV-associated cancer may be attributed to racial and/or geographic distribution. The current study in Thai AM patients showed the same frequency as that in Japanese AM patients 19 but, however, was lower than that in Korean AM patients 20 (Table 3).Due to the small sample size in our study, investigation of EBV and HPV DNA of AM in a larger population may increase the strength of this epidemiology. It is worth noting that two studies in Egypt showed unequal results that may be caused by different detection techniques. 11,21  Another key oncogenic virus is HPV. Since 1989, more than 10 studies have reported the frequency of HPV DNA in AM that varied from 0 to 40% as summarized in Table 4. Our exploration is the first study in Southeast Asia and the HPV DNA frequency is 10% that was similar to the report of Migaldi et al. 26 Considering for HPV typing in Table 4, several studies encountered high-risk HPV and HPV 18 were mostly observed. [22][23][24][25] Nevertheless, some studies displayed low-risk and intermediate-risk HPV types whichever may not involve in AM oncogenesis. In the present study, it is to be noted that both positive cases were HPV 18, the hrHPV that may be directly involved in AM oncogenesis.
Both HPV and EBV DNA have been remarkably observed in one case of AM. Regarding to the recent study reviewed the evidence of EBV and HPV co-infection in several cancers including cancer of nasopharynx, cervix, breast and prostate gland. 31 Apolipoprotein B mRNA editing enzyme catalytic polypeptide3 (APOBEC3) deaminase was proven to involve virus innate immunity response and may promote viral coinfection. 32 APOBEC3B mRNA levels were higher in oral squamous cell carcinoma compared to normal oral epithelium, 33 but there is no study on APOBEC3B in AM. The study of APOBEC3 in this case may elucidate whether the coinfection of EBV and HPV plays any role in the development of AM.
The EBV and HPV association in AM oncogenesis has been curiously underlined. In this study, we did not observe koilocytic changes in both HPV positive samples, meaning that no morphological appearance of HPV infection like what observed in the study of Migaldi et al. 26 We also did not observe syncytial formation in EBV-positive cases. Both viruses may transform normal odontogenic epithelium to ameloblastoma epithelial cells without any feature of viral cytopathologic changes. It was also suggested that HPV in AM may represent a background infection without AM oncogenesis development. 28 Sensitivity of PCR technique should also be a concern. Both HPV and EBV DNA might contaminate in normal epithelium or from the surgically acquired contamination during the procedure. In the other aspects, the common permissive cells of EBV are lymphocytes and epithelial cells, while of HPV are epithelial cells. 8,31,34 Several reports exhibited HPV and EBV positive cases derived from the in situ hybridization technique and demonstrated EBV and HPV infected in AM cells. 5,19,22,23,26,28 (Tables 3 and 4). We hypothesize that AM may possess EBV and HPV receptors, which need to be explored in further studies. To evaluate if EBV and HPV were related to AM oncogenesis, the PCR positive cases must be confirmed by ISH or IHC.

conclusIon
The low frequencies of EBV and HPV DNA in a group of Thai AM patients may suggest their minor participation in the biological events related to AM pathogenesis. Nevertheless, it could not be concluded that the virus was involved in the etiology and pathogenesis of the lesions. These studies are preliminary in nature involving limited number of AM patients and hence further work with a larger sample size comprising various techniques is required. Finally, the studies of in-depth mechanisms in the role of viruses in AM oncogenesis were suggested for further prevention and treatment of virus-associated ameloblastoma.

clInIcAl sIgnIfIcAnce
HPV and EBV may not be involved in ameloblastogenesis.

AvAIlAbIlIty o f dAtA A n d MAterIAls
The data analyzed during this present study are available from corresponding author on reasonable request.