|Ahead of print publication
Salivary detection of high-risk human papillomavirus 16 in oral squamous cell carcinoma using polymerase chain reaction in the South Indian population
Soujanya L Krishnappa1, Chaya M David1, BK Ramnarayan1, Alekhya Kanaparthi2, Suprith L Krishnappa3, Divya Dukkireddy4
1 Department of Oral Medicine and Radiology, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka, India
2 Department of Oral Medicine and Radiology, MNR Dental College and Hospital, Sangareddy, Telangana, India
3 Department of ENT, Mandya Institute of Medical Sciences, Mandya, Karnataka, India
4 Department of Public Health Dentistry, Dayananda Sagar College of Dental Sciences, Bengaluru, Karnataka, India
|Date of Submission||07-Nov-2019|
|Date of Acceptance||27-Jan-2020|
|Date of Web Publication||28-Oct-2020|
Department of Oral Medicine and Radiology, MNR Dental College and Hospital, Sangareddy - 502 001, Telangana
Source of Support: None, Conflict of Interest: None
Introduction: Human papilloma virus (HPV) has been associated with oral squamous cell carcinoma (OSCC) as a potential carcinogen. There are several types of HPV, of which type 16 has been strongly implicated in carcinogenesis. HPV16 in saliva can potentially facilitate early detection of subclinical cases that may warrant further diagnosis, monitoring and intervention.
Aim: The aim of this study was to evaluate the presence of HPV 16 in saliva and lesional tissue of OSCC and to determine the use of saliva as an alternative non invasive diagnostic tool in HPV16 identification.
Materials and methods: 30 cases of Histopathologically confirmed OSCC with HPV positive on ELISA were taken up for the study. The tumour tissue and saliva sample of each patient were obtained to detect the presence of specific HPV16 genotype by polymerase chain reaction (PCR). The data was subjected to statistical analysis using Student t-test.
Results: In our study we found 28/30, 26/30 positive for HPV 16 in tissue and saliva samples respectively on PCR analysis. The P value was statistically significant (0.00).
Conclusion: The study revealed significant prevalence of HPV 16 among both tissue and salivary specimens of OSCC patients in south Indian population. Though, the yielded content was relatively less in saliva, it can be concluded that, saliva being a non invasive tool proved to be as useful as tissue specimen and can be used as an alternative indicator of HPV16 positivity in OSCC.
Keywords: Biomarkers, cancer, DNA probes, human papilloma virus, polymerase chain reaction, saliva
|How to cite this URL:|
Krishnappa SL, David CM, Ramnarayan B K, Kanaparthi A, Krishnappa SL, Dukkireddy D. Salivary detection of high-risk human papillomavirus 16 in oral squamous cell carcinoma using polymerase chain reaction in the South Indian population. J Can Res Ther [Epub ahead of print] [cited 2020 Dec 2]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=299464
| > Introduction|| |
Oral cancer represents a major health issue worldwide due to its morbidity and mortality. It is ranked eleventh among all sites of cancer. Viral infections have been considered as one of the major risk factors for cancer. Viruses, such as human papillomavirus (HPV), hepatitis C virus, human herpesvirus (mainly herpes simplex virus, Epstein–Barr virus, human immunodeficiency virus), have been strongly implicated in the development of malignant tumors of the squamous epithelium., HPV has been implicated as a cause of cervical cancer worldwide by the World Health Organization. Literature studies have shown a clear association between the HPV and oral carcinogenesis.,,,,,,
Certain HPV types, such as HPV 16, 18, 31, 33, 35, and 39 can be referred as “high-risk” types found to be associated with oropharyngeal and oral squamous cell carcinoma (OSCC)., Some authors have reported the presence of HPV, particularly of type 16, as a factor contributing to the onset of squamous cell carcinoma of the oropharynx, but its role in oral cancer is still controversial.,,,,,,
Conventionally, the samples for testing were collected from the tumor tissue. However, advancements in the biochemical science have added zeal to the probability of using the DNA profiling by enabling the use of noninvasive diagnostics. The oral fluid meets the demand for being noninvasive, as it is easily accessible bio-fluid of human body that has a wide spectrum of biological analytics, informative for clinical diagnostic application.
HPV diagnosis includes various methods such as polymerase chain reaction (PCR), HPV in situ hybridization, and immunohistochemistry. PCR is an enzymatic assay, which allows the amplification of a specific DNA fragment from a complex pool of DNA. Hence, PCR is the method of choice to characterize the sequence context of genome-integrated viroid DNA and can hardly be replaced by any alternative procedure. The prevalence reports in India, however, are not consistent with the global statistics. In India, the incidence of HPV in oral cancers ranges from 0% to 74%.
With this background, this study attempted to evaluate HPV association in OSCC in the south Indian population and to assess the role of saliva as a noninvasive diagnostic fluid in the detection of HPV 16 in OSCC using PCR.
| > Materials and Methods|| |
The study was conducted in the Department of Oral Medicine and Radiology, Private Dental College and other apex cancer centers. It was approved by the Institutional Ethical Committee (DSCDS/2015-16/294A). The selection of participants was based on the patient's history and a thorough clinical examination. Patients who agreed to sign the information and consent form were considered for the study and patients with recurrent OSCC, salivary gland disorders, and those undergoing radiotherapy or chemotherapy were excluded.
After obtaining their consent, salivary, tissue samples were collected from subjects who satisfied the inclusion criteria under aseptic condition. The tissue samples confirming OSSC were subjected to ELISA test for the initial screening of HPV. A total of 30 patients who were identified positive for HPV and histopathologically confirmed OSCC were included in the study. Group A included tissue specimens, and Group B included their respective salivary samples.
During the study, 103 patients with OSCC were examined for the study, 32 patients had a previous history of surgical treatment, radiotherapy or chemotherapy for OSCC, A total of 13 patients refused to participate in the study, and 28 salivary samples yielded DNA content <1 μg hence could not be taken up for the study. Consequently, only 30 histopathologically confirmed cases of OSCC that are HPV positive on ELISA were selected for the study [Figure 1].
|Figure 1: Selection of oral squamous cell carcinoma cases for the evaluation of human papilloma virus type 16|
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Saliva samples were collected from the patients prior to biopsy. Spit method was used to collect 5 ml of unstimulated whole saliva sample from OSCC patients, between 9 a.m. and 11 a.m. The individuals were advised to avoid intake of any food or beverage including smoking, chewing gum, and intake of coffee (water exempted) 1 h before the procedure and were asked to stop the medications that might affect the salivary secretion, at least 1 day prior to collection.
The individuals were asked to rinse the mouth with deionized (distilled) water and then to relax for 5 min. Saliva was allowed to accumulate in the floor of the mouth, and the subjects were then asked to spit it out into the graduated test tube every 60 s or when the subject experienced an urge to swallow the fluid accumulated on the floor of the mouth. The individuals were instructed not to spit forcibly so as to avoid blood contamination, if any, from the inflamed gingival tissue or any ulcerated lesion [Figure 2]. The saliva collected was put in tubes and stored in a freezer at −20°C and later placed in a thermocol box with dry ice to maintain −4°C and transferred to the laboratory for biochemical analysis.
Collection of tissue specimen
Incisional biopsy was done under aseptic conditions [Figure 3]. Tissue specimens were collected in 10% neutral formalin and phosphate buffer saline. The tissue specimen was processed and later formalin-fixed paraffin-embedded blocks were prepared, from which 4 to 5 paraffin sections (5 μm each) were placed in a mouth sterile plastic container that was later dewaxed. The patients who were histopathologically confirmed with OSCC were graded and the remaining tissue samples of the confirmed cases were transferred to the laboratory for DNA extraction [Figure 4].
|Figure 3: Representative image of incisional biopsy (a) presurgical view, (b) specimen of lesion, (c) postsurgical view with suture placement|
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|Figure 4: Tissue specimen in formalin container and saliva sample in collecting tubes|
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Salivary samples were centrifuged at 1000 rpm for 10 min, and the top layer was collected. DNA from tissues and saliva were extracted with phenol–chloroform, precipitated in 100% ethanol, centrifuged at 5100 rpm for 45 min, washed in 70% ethanol, dissolved in lysis buffer (10 mM TRIS hydrochloride, 1 mM ethylenediaminetetraacetic acid buffer, pH 8), and stored at −20°C.
DNA extraction from the tissue specimens was done using a modified proteinase K method and was stored at −80°C till further analyzed. The eluted DNA of the tissues was subjected to PCR for the detection of HPV Type 16. Following which eluted DNA of saliva samples of the corresponding 28 patients who were positive for the HPV TYPE 16 and 2 patients who were negative for HPV TYPE 16 (negative control) were considered for PCR. The samples were analyzed by PCR (Taqman HT 7900; Applied Biosystems) [Figure 5]. Specific primers and probes previously designed to amplify HPV-16 using the consensus primers MY09/MY11were used [Figure 6] by a PCR assay, which amplify a fragment of about 450 base pairs within the L1 gene of the viral genome. The amplified products were analyzed through gel electrophoresis, stained with ethidium bromide and visualized with ultraviolet (UV) transilluminator for band formation. The presence of positive band at 457 bp indicated HPV16 positivity [Figure 7]. The data collected were tabulated and subjected to statistical analysis.
|Figure 5: DNA purification kit (a) 3B MTB/NTM DNA extraction kit, (b) reagents, (c) proteinase K|
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|Figure 7: Gel Dox image tissue and saliva samples – wells which shows presence of bands are positive for HPV type 16 DNA and wells which are blank are negative|
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The IBM SPSS Statistics for Window, version 22.0 (Armonk, NY, USA) was used for data analysis. Paired t-test was used to evaluate the Type 16 specific HPV prevalence between saliva and lesional tissue.
| > Results|| |
The present study comprised of 4 females and 26 males out of 30 OSCC individuals. The minimum age was 30 years, and the maximum age was 51 years, with a mean age of 36.66 years. In our study, of 30, we found 26 (86.6%) tobacco users and 4 (13.3%) nontobacco users. Lesions were found most commonly on the tongue 12 (30.76%), followed by Buccal mucosa 9 (23.07%), oropharyngeal 7 (17.94%), lip 4 (10.25%), gingivobuccal sulcus 4 (10.25%), and retromolar pad region 3 (7.69%). Of 30 cases, 21 cases (70%) were well-differentiated, 6 cases (20%) were moderately differentiated, and 3 cases (10%) were poorly differentiated on histopathological examination.
The extraction of DNA from both tissue and saliva was considered-quantity (μ g/250 ml) and quality of DNA (A°260/280). In the present study, DNA yield (quantity) in Group A was found to be a minimum of 10 μg/250 ml and maximum of 22 μg/250 ml; the mean DNA yield was found to be 14.62 with a standard deviation of 2.884. DNA yield in Group B was found to be a minimum of 2 μg/250 ml and maximum of 9 μg/250 ml; the mean DNA yield was found to be 5.04 with a standard deviation of 1.944 [Table 1] and [Graph 1].
The quality of DNA was measured at A°260/280. Group A was found to be between 1.80 and maximum of 1.89. The mean was 1.8487 with a standard deviation of. 03224. In Group B, the minimum was 1.80 and maximum was 1.89. The mean was found to be 1.8407with a standard deviation of. 03331 [Table 2] and [Graph 2].
Paired t-test was done between the DNA yielding and quality of DNA to check the correlation of the samples and data taken from the same individuals. On applying the paired t-test Group A showed a mean of 9.585 with standard deviation of 2.928 and Group B showed a mean of 0.00800 with standard deviation of 0.04937. On comparison DNA Yielding between two groups P = 0.00 which is highly significant and Quality of DNA between two groups P = 0.382 [Table 3].
Of 30 tissue samples amplification of HPV type 16 DNA was present in 28 (93.3%) and absent in 2 (6.7%) and in salivary samples, it was present in 26 (86.7%) and absent in 4 (13.3%). On comparison between Group A and Group B using the Chi-square test, value is 13.929 and the level of significance is 0.00, which is highly significant [Table 4].
|Table 4: Comparison of amplification of human papillomavirus Type 16 in tissue and Saliva|
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| > Discussion|| |
In India, oral and oropharyngeal carcinoma is the most common cancer in men and the third most common cancer in women. Several studies have supported viral infection as a potent carcinogen, and this proposal was under tremendous research. Syrjänen et al. in 1983, was the first to propose the etiological role of HPV in head and neck carcinogenesis.
The most prevalent high-risk types are HPV 16 and 18, which have been detected in the majority of malignant oral lesions worldwide.,,,,,, Studies by Tabatabai et al., Miller and Johnstone  and Koh et al. have shown high risk HPV type 16 prevalence of 82%, 69.2%, and 68%, respectively. It has been reported that HPV oncoproteins E6 and E7 inactivates the host tumor suppressor gene products p53 and pRB, molecular mechanisms in the epithelial cells of oral carcinogenesis. However, the rate of HPV detection appears to be highly variable, based on the study population, specimen site, and molecular methodology used.
To diagnosis, high-risk HPV tumor tissue samples are the gold standard in OSCC. However, the challenges faced, for individual assessment, large scale epidemiological screenings and inadequate health-care facilities suggested the use of biofluids as an alternative source of diagnosis. Among them, Saliva is a noninvasive biofluid proved to be an alternative source for quantifying a variety of biomarkers that can be collected, stored, and transported. Salivary examination for the detection of HPV in OSCC may be beneficial due to its direct contact with oral cancer lesions and the presence of exfoliated cells from the lesion.,,,,,,,,,
Literature studies to assess the prevalence of HPV in saliva by Grewal et al., SahebJamee et al., Smith et al., and Turner et al. showed HPV prevalence of 15%, 2.6%, 40.9%, and 40.42%, respectively.
Molecular analysis technique using PCR has been considered as one of the sensitive methods for the detection of HPV genome by sequencing specific DNA strands. A systematic review by Gupta and Gupta in their review included 50 studies of HPV in OSCC, premalignant, benign and malignant lesions. They inferred that most of the authors validated PCR for examination of HPV-DNA. In this study, we used salivary amplification of DNA and PCR allowed extraction of DNA from small amount of saliva. It also allowed amplification of specific HPV16 genotype allowing direct detection of the viral oncogenes along with the extension of assay. We observed a high specificity using PCR technique and results of our study are in accordance with previously reported studies.
SahebJamee et al. reported similar rates of HPV prevalence in the saliva of patients of OSCC (40.9%) of the patients and out of which the (27.3%) were positive for HPV16 in saliva. and a study by Chuang et al. to evaluate HPV16, E6, and E7 DNA copy number in salivary rinses of OSCC found a 33.9% prevalence of HPV 16. Similarly, Adamopoulou et al. also analyzed saliva samples OSCC for high risk-HPV and reported the prevalence of 10.3% out of which 50% was HPV 16. Zhao et al. used PCR to detect HPV16, E6 and E7 DNA level in primary tumors and salivary rinses from patients with head and neck squamous cell cancer (HNSCC) and reported total of 45.6% of primary HNSCC and 32.6% of saliva rinse samples from HNSCC patients had detectable HPV. Grewal et al. used nested multiplex PCR to evaluate the prevalence of HPV in saliva. They reported HPV positivity was 40.42% (19/47), of which HPV16 (31.91%).
The results of our study are in accordance with the previous studies. In our study, we got a higher number of positives in saliva compared to previously published studies. This could be attributed to the strain-specific primers used in the current study. Previously reported studies used a broad spectrum of HPV, primers that may have lowered the sensitivity.
Chuang et al., in 2008, assessed HPV-DNA shed postoperatively into the saliva in HNSCC concluded that HPV16 presence in follow-up salivary rinses preceded clinical detection of disease recurrence by an average of 3.5 months and patients with the presence of HPV16 DNA in surveillance salivary rinses for recurrence. Ahn et al. in 2014, investigated retrospectively the feasibility of HPV-16 DNA detection in pretreatment and posttreatment plasma and saliva as a prognostic marker in 93 patients. They inferred that HPV-16 DNA status in saliva and plasma posttreatment was 90.7% specific and 69.5% sensitive in predicting recurrence within 3 years. Suggesting analysis of HPV-16 DNA in saliva and plasma after primary treatment may allow early detection of recurrence in patients with HPV-16–positive oropharyngeal carcinoma. Thus, they concluded that the quantitative assessment of salivary HPV16 DNA has a promising outcome for early detection and surveillance of OSCC. Chai et al. reported that 39 out of 40 (92.9%) had oral rinse samples with detectable HPV-16 DNA, using end-point and quantitative PCR.
In the current study, the prevalence of HPV16 in lesional tissue was found to be 93.3%, and in saliva, it was found to be 86.72% (26/30). The positivity of lesional tissue and/or saliva could be explained by epitheliotropism hypothesis explained by Syrjänen et al. who demonstrated that the presence of HPV in epithelium progressively increases from the basal cell layer to the superficial layers. Furthermore, vegetative replication may lead to shedding of HPV into superficial cells and consequent shedding into saliva.,,,
Although tumor tissue samples are the gold standard to the diagnosis of HPV in OSCC, Examination of saliva can give the greatest benefit due to its direct contact with oral cancer lesions and presence of exfoliated cells from the tumor lesion.
In our study, of 30 subjects, we found 26 were tobacco users. An Indian study by Kumar et al. demonstrated that tobacco may act as risk factors for high-risk HPV infection of about 31.13%. Nagpal et al. evaluated the association of tobacco chewing for HPV 16 and 18 infections of oral cancer. They reported HPV in 37/110 patients (33.6%), among which the presence of HPV-16, 18 and 16/18 co-infection is 22.7%, 14.5%, and 10%, respectively. On the contrary, Goud et al. in 2017 evaluated if tobacco smoking is associated with the prevalence of HPV 16 and HPV 18 in saliva of chronic smokers and nonsmokers in the Malaysian population. They opined that smoking alone is not associated with the HPV 16 and HPV 18 prevalence in saliva. Further studies in this realm need to be explored.
The analysis of the DNA at Š260 and Š280 is a direct measure for the assessment of its purity because DNA absorbs UV light at 260 nm and proteins absorb light at 280 nm. In the present study, quality of DNA was measured at A°260/280. In our study, in Group A, it was between 1.80 and 1.89. The mean was 1.8487. In Group B, it was 1.80–1.89. The mean was found to be 1.8407. In a study done by Turner et al., the purity of the DNA isolates, averaged between 1.87 and 2.05 which is in concurrence with values in our study.
The limitations of the current study were small sample size and lack of patient follow-up to determine the prognosis of the HPV-positive OSCC. Further studies are recommended to multicenterr evaluation of HPV type 16 prevalence and also the presence of HPV 16 in Potentially malignant disorder's for early diagnosis, monitoring and intervention of progression of such lesions to frank carcinoma.
| > Conclusion|| |
Although tremendous research in the recent decades to understand the carcinogenic potential of HPV has been done, it is important to emphasize that there is a lack of awareness about the nature of the disease, risk factors, mode of transmission, clinical presentation, vaccination, and therapeutic protocols among the general public. Conducting, oral and cervical screening campaigns may be of great value in the early detection and progression of carcinoma.
In our study, we found saliva to be equally potent to tissue specimen in assessing HPV type 16 DNA and can be used as a noninvasive screening tool of “high-risk patients” to detect early lesions and educate them about the risk factors that may warrant further monitoring and intervention. We also found that DNA extraction using PCR method is a useful, reliable and a sensitive method of identification of HPV 16 in cells from OSCC.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4]