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Year : 2021  |  Volume : 13  |  Issue : 1  |  Page : 8-12

HPV-16 Detection and Quantitation in Whole Mouth Fluid of Oral and Cervical Cancer Patients

Department of Oral Pathology, Ragas Dental College, Chennai, Tamil Nadu, India

Date of Submission04-Feb-2021
Date of Acceptance31-May-2021
Date of Web Publication06-Aug-2021

Correspondence Address:
Bose Divya
Department of Oral Pathology, Ragas Dental College, Chennai, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jofs.jofs_38_21

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Introduction: The association of human papillomavirus (HPV) in cervical and oropharyngeal cancer is well established. There is a growing evidence that HPV could possibly contribute to oral cancer along with alcohol and tobacco use. The aim of this study was to evaluate and quantify HPV-16 in the whole mouth fluid of patients with oral squamous cell carcinoma (OSCC), cervical cancer, and normal controls. Materials and Methods: The whole mouth fluid of 20 patients with OSCC (Group I), 10 with cervical cancer (Group II), and 10 normal individuals (Group III) was collected by the spit technique. The DNA was extracted and quantified by quantitative polymerase chain reaction. Data were analyzed by SPSS 21 (IBM Corp. Released 2012, Armonk, NY: IBM Corp) software and analysis of variance test was used. Results: The mean age of Group I patients was 50.9 ± 13.5 years, Group II was 51.6 ± 8.07 years, and Group III was 46 ± 8.5 years. In Group I, 15 were males and 5 were females. In Group III, 5 were males and 5 were females. Eight patients belonging to Group I, four of Group II, and seven of Group III had HPV-16 in their whole mouth fluid. Conclusion: HPV-16 was identified in the whole mouth fluid of OSCC, patients with cervical cancer, and even in normal controls. However, the mean viral loads were highest in patients with cervical cancer followed by patients with OSCC and normal controls.

Keywords: Cervical cancer, HPV-16, normal individuals, oral cancer, polymerase chain reaction, whole mouth fluid

How to cite this article:
Divya B, Rao UK, Thavarajah R, Joshua E, Ranganathan K. HPV-16 Detection and Quantitation in Whole Mouth Fluid of Oral and Cervical Cancer Patients. J Orofac Sci 2021;13:8-12

How to cite this URL:
Divya B, Rao UK, Thavarajah R, Joshua E, Ranganathan K. HPV-16 Detection and Quantitation in Whole Mouth Fluid of Oral and Cervical Cancer Patients. J Orofac Sci [serial online] 2021 [cited 2023 Jun 9];13:8-12. Available from:

  Introduction Top

Oncogenic viruses such as high-risk human papillomavirus (HPV) and Epstein–Barr virus have a tumorigenic effect on oral epithelium. HPV integrates into the host genome and induces carcinogenesis. HPV infection has been established as the cause for cervical cancer.[1]

The HPV-associated head and neck squamous cell carcinoma (HNSCC) is an entity with unique clinical and molecular characteristics. More than 90% HPV-associated HNSCCs are caused by HPV-16.[2] Among HNSCC, HPV is closely associated with oropharyngeal carcinoma.[3] Recently, Food and Drug Administration has approved the use of Gardasil®-9 vaccine in the prevention of oropharyngeal and other HNSCCs caused by HPV types 16, 18, 31, 33, 45, 52, and 58.[4] Prophylactic vaccination can also be effective in HPV-associated oral squamous cell carcinoma (OSCC).

The HPV can be transmitted by sexual contact, autoinoculation, or vertical transmission during the perinatal period. HPV is also associated with benign oral lesions such as warts, squamous cell papilloma, condyloma, and focal epithelial hyperplasia. Oral cavity can serve as a reservoir for HPV and the oral exfoliated cells harbor HPV DNA. HPV has been detected in the saliva of patients with oral cancer which may allow early cancer detection and monitoring of disease progression. The association between the presence of HPV in cervical region and oral cavity remains unclear.[5]

The present study was conducted to quantify and compare HPV-16 in the whole mouth fluid of patients with OSCC, cervical cancer, and normal matched controls.

  Materials and Methods Top

The study was carried out after obtaining ethical clearance (Ethical clearance number − 20140546) from the Institutional Ethical Committee of Ragas Dental College, Chennai on 5th May 2014. All the procedures were performed in accordance with the 1964 Helsinki Declaration and its later amendments. The sample size was calculated as 36 using G power software version 3.1, for analysis of variance (ANOVA) test, with effect size 0.7, alpha error 0.05, and power 0.95. The study involved three groups consisting of 20 patients with OSCC (Group I), 10 patients with cervical cancer (Group II), and 10 individuals who were apparently healthy (Group III). Informed consent was obtained from all participants involved in the study. The whole mouth fluid was obtained from patients in each group by spitting method as described by Navazesh and Christensen.[6] The collected samples were then analyzed by quantitative real-time polymerase chain reaction (qPCR) for HPV-16 DNA.

The patients were recruited for the study by convenience sampling. Patients with a histopathologic diagnosis of primary oral or cervical cancer were only included in the study. Clinical staging of oral cancer was carried out by tumor, node, and metastasis (TNM) system,[7] and International Federation of Gynecology and Obstetrics system[8] was followed for staging of cervical cancer. Patients with a history of prior malignancy, autoimmune disease, hepatitis, immunodeficient states, and patients who were under any treatment for OSCC or cervical cancer were excluded.

The DNA extraction was carried out using DNA extraction kit (Swift saliva kit™, Shrimpex Biotech Services Pvt. Ltd, Chennai, India) by spin column method. The extracted DNA was then quantified by spectrophotometer and determined to be more than 2 ng which was considered sufficient for evaluation of HPV by previous studies.[9],[10] GenoRime realtime HPV-16 kit™ (shrimpex Biotech Services Pvt. Ltd, Chennai, India) was used to detect the HPV-16 genome, E7 region. The details of the primers and probes used are summarized in [Table 1]. The negative control consisted of PCR grade water and the positive control consisted of synthetic HPV-16 DNA containing 83 bp nucleotides. Absolute quantification was performed using standards of known concentration and of composition similar to the target amplicon. PCR amplification plot [Figure 1] was obtained and standard curve [Figure 2] was used to determine the starting amount of each unknown template based on its threshold value.
Table 1 Properties of probes and primers used

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Figure 1 Amplification plot.

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Figure 2 Standard curve.

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Data entry, database management, and analysis were performed using SPSS 21 (IBM Corp. Released 2012, Armonk, NY: IBM Corp). ANOVA test was used to analyze continuous variables. A P-value of ≤0.05 was considered as statistically significant.

  Results Top

The age and gender of the study population are summarized in [Table 2]. Among patients with OSCC, 90% had the habit of either smoking or tobacco chewing or alcohol consumption and two patients did not have any habits. About 40% of the patients had OSCC in buccal mucosa, 35% in tongue, 10% in alveolar mucosa, 10% in hard palate, and 5% in retromolar region. About 50% had stage IV oral cancer, 30% had stage III, 15% had stage II, whereas 1% had stage I cancer. Among Group II patients, 60% (n = 6) had stage II cervical cancer, 20% (n = 2) had stage IV cancer, 10% (n = 1) had stage I cancer, and 10% (n = 1) had stage III cancer.
Table 2 Demographics and HPV-16 status of the study population (N = 40)

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The HPV-16 was detected in 40% of Group I patients, 40% of Group II, and 60% of Group III patients [Figure 3]. In Group I patients with HPV-16 positivity, 63% had stage IV oral cancer, 25% had stage III cancer, and 13% had stage II cancer.
Figure 3 Distribution of HPV-16 positivity in whole mouth fluid of study groups (N = 40).

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Group I patients had a mean HPV-16 viral load of 2673.75 ± 5449.11 copies/mL of saliva, Group II had 11,884.25 ± 21,916.92 copies/mL of saliva, and Group III patients had 685.14 ± 1250.35 copies/mL of saliva. The difference in the mean viral load between the groups was not statistically significant [Table 2]. The mean HPV-16 viral load was compared among Group I patients based on TNM staging of oral cancer [Table 3].
Table 3 Comparison of mean HPV-16 viral load based on TNM staging of oral cancer in Group I patients (N = 8)

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  Discussion Top

The HPVs are double-stranded, circular DNA viruses which infect the squamous epithelial cells. The prevalence of oral HPV infection widely varies from 0 to 70% in normal mucosa depending on the type of sample collected, sample size of the study, HPV detection method, and primers used.[10] Real-time qPCR enables a more precise quantification of HPV DNA[11] and primers targeting E6/E7 region provides the optimum choice for detection of HPV.[12] Hence, in our study, HPV-16 was quantitated by qPCR using E7 primer.

Among patients with OSCC, 40% of them had HPV-16 in their whole mouth fluid and among normal controls, 70% of them were positive for HPV-16. In a study conducted in Karnataka, Grewal et al. used nested multiplex PCR, which is highly specific to detect HPV. HPV was detected in the saliva of 19 out of 47 (40%) patients with OSCC and HPV-16 in 15 out of 47 (32%) patients with OSCC.[13] In another study by Kulkarni et al. conducted in North Karnataka, PCR-based high-risk HPV genotyping was performed to detect HPV in 60 patients with cervical cancer, 34 salivary rinse samples of patients with OSCC, and 396 salivary rinse samples of normal individuals. HPV-16 was found in 42% of patients with cervical cancer, 42% of the patients with OSCC, and 3% of the normal individuals.[14] Detection of HPV DNA in saliva does not translate to oral HPV infection and persistence of HPV is essential for cancer development. Since our study did not elicit the sexual behavior of the study population, the precise cause for the presence of HPV-16 in the whole mouth fluid of normal individuals could not be determined.

Peixoto et al. from Brazil analyzed HPV DNA in buccal swabs obtained from 100 women with histopathologic diagnosed cervical HPV infection using PCR and observed that women with genital HPV infection are at a greater risk of having subclinical HPV infection in the oral mucosa.[15] In the present study also none of the patients with cervical cancer had oral lesions suggestive of HPV infection. Several studies have shown that the prevalence of concurrent oral and cervical HPV infections is low, suggesting that self-inoculation is not common.[16],[17] In the study carried out in Germany by Meyer et al., oral HPV prevalence was evaluated in women with genital HPV infection using PCR and only 6% of the genital HPV positive women had HPV in the oral cavity.16 In another similar study in Germany by Eggersmann et al., oral HPV infection was identified in only one woman among 144 female participants (0.7%).[17]

In the current study, HPV-16 was positive in whole mouth fluid 40% of the patients with cervical cancer which is similar to the observation by Sánchez-Vargas et al. In their study, HPV-16 was detected in oral swabs of 35% of the Mexican patients with cervical intraepithelial neoplasia, which suggested that women with persistent HPV-16 infections had higher risk of their detection in the oral cavity.[18]

In our study, the mean HPV-16 viral load was highest among the patients with cervical cancer supporting the role of HPV-16 in pathogenesis of cervical cancer. Several studies have shown association between high HPV viral load in the cervical region and the risk of cervical cancer.[19],[20] The high viral load can result from few cells with large number of virions or large number of cells with few virions. Hence, the viral load interpretation could be difficult.[21]

The mean HPV-16 viral load was higher among patients with OSCC when compared to normal controls. In contrast, a study from Japan by Shigeishi et al. showed the high HPV-16 DNA copy number in saliva of patients with OSCC was comparable to the normal individuals without cancer.[22] This discrepancy could be explained by the difference in study population and the primer used. It has also been reported that HPV DNA copy number by itself is not an accurate measure of HPV positivity in patients with OSCC.[23] HPV may be present in normal mucosa in low copy numbers since persistent or transient HPV infection of the oral mucosa occur frequently.[24]

In our study, among the patients with OSCC positive for HPV-16, 63% had stage IV cancer with high mean viral load supporting the fact that HPV-positive tumors tend to present mostly at advanced stage.[2] A similar finding was observed by Chuang et al. who conducted a study which included 59 patients diagnosed with HNSCC from Baltimore. HPV was detected in 95% patients who presented at advanced stage (stages III and IV) in contrast to 5% of patients at stages I and II.[25] More than half of the HPV-associated oropharyngeal cancers are diagnosed at an advanced stage since they present at an early T stage and more advanced nodal stage.[26] Herrero et al. conducted a multicenter case–control study of cancer of the oral cavity and oropharynx in which they observed that HPV DNA was prevalent in oropharyngeal cancer in stages III and IV (22%) compared to oropharyngeal cancer in stages 0 and I (7%).[27]

Though there was a difference in the mean viral load among our study groups, it was not statistically significant. This can be explained by the presence of outliers in our group due to the extreme values of the HPV-16 viral load and also small sample size. Further large-scale studies are required to evaluate the association between HPV viral loads and development of oral or oropharyngeal cancer.

From the present study, it can be concluded that HPV-16 can be detected in the whole mouth fluid of patients with oral cancer, patients with cervical cancer, and even in the normal individuals. The mean HPV-16 viral load was highest in patients with cervical cancer followed by patients with oral cancer and normal controls. An elevated viral load in patients with oral cancer when compared with the controls, highlights the fact that HPV along with other risk factors such as smoking, tobacco chewing, and alcohol use can contribute to oral carcinogenesis. However, it is essential to establish the cause and effect relationship to confirm the etiologic role of HPV-16 in the pathogenesis of oral cancer.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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Grewal RK, Sircar K, Bhat KG, Grewal DS, Tyagi KK, David S. Detection of human papilloma virus-E6/E7 proteins of high-risk human papilloma virus in saliva and lesional tissue of oral squamous cell carcinoma patients using nested multiplex polymerase chain reaction: a comparative study. J Oral Maxillofac Pathol 2018;22:318.  Back to cited text no. 13
[PUBMED]  [Full text]  
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Terai M, Hashimoto K, Yoda K, Sata T. High prevalence of human papillomaviruses in the normal oral cavity of adults. Oral Microbiol Immunol 1999;14:201-5.  Back to cited text no. 24
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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]


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