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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 30  |  Issue : 4  |  Page : 367-371

Evaluation of human papillomavirus as an independent risk factor in known patients of oral squamous cell carcinoma using immunohistochemistry


1 Department of Oral Medicine and Radiology, Sri Aurobindo College of Dentistry, Indore, Madhya Pradesh, India
2 Department of Oral Medicine and Radiology, Saraswati Dental College, Lucknow, Uttar Pradesh, India
3 Department of Dentistry, Saraswati Medical College, Unnao, Uttar Pradesh, India

Date of Submission24-Sep-2018
Date of Acceptance25-Sep-2018
Date of Web Publication17-Jan-2019

Correspondence Address:
Dr. Siddharth K Singh
Department of Oral Medicine and Radiology, Saraswati Dental College, 233 Tiwari Ganj, Faizabad Road, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_122_18

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   Abstract 


Introduction: Oral cancer (OC), ever since its discovery has not failed to claim lives of many. However, in between 1960s and 1980s, some astonishing trends started to emerge pertaining to OC, which almost entirely changed the face of this ailment. Almost a fourfold ascent in the number of patients diagnosed with OC was seen in the age group of 30–39 years! Authors, after several studies and meticulous observations got a clear picture of the phenomenal changes. These changes, which were almost entirely attributed to a rise of human papillomavirus (HPV) after few years became a global issue and did not leave Asian countries, including India unscathed. Rigorous studies were conducted in India, although each one yielded different and somewhat contrasting results. While some authors could not demonstrate a single OC case with HPV, some concluded otherwise. Aims: 1. To identify the role of HPV as an independent risk factor for oral squamous cell carcinoma (OSCC). 2. To calculate the prevalence of HPV in local population. Settings and Design: Biopsy specimens of 100 patients were analyzed in the study. They were tested for the presence of surrogate marker p16 to predict the involvement of HPV. Materials and Methods: OC samples with already proven histopathological diagnosis of (of all grades) without habits were taken as cases and were subjected to immunohistochemistry. Statistical Analysis Used: Descriptive statistics were taken in use along with inferential statistics. Results: Out of 100 patients, 6 patients showed positive results for the presence of p16 or HPV. The P value was P = 0.012, which was statistically significant. Conclusions: It can be concluded that despite the absence of any other risk factors, HPV can result in OSCC even in a developing and conservative nation like India.

Keywords: Human papillomavirus, immunohistochemistry, oral cancer, oral squamous cell carcinoma, surrogate marker, tumour marker


How to cite this article:
Verma H, Singh SK, Phulambrikar T, Gupta A. Evaluation of human papillomavirus as an independent risk factor in known patients of oral squamous cell carcinoma using immunohistochemistry. J Indian Acad Oral Med Radiol 2018;30:367-71

How to cite this URL:
Verma H, Singh SK, Phulambrikar T, Gupta A. Evaluation of human papillomavirus as an independent risk factor in known patients of oral squamous cell carcinoma using immunohistochemistry. J Indian Acad Oral Med Radiol [serial online] 2018 [cited 2019 Apr 21];30:367-71. Available from: http://www.jiaomr.in/text.asp?2018/30/4/367/250244




   Introduction Top


Ever since the time, in as early as 3000 BC[1] when Edwin Smith Papyrus diagnosed the very first case of cancer, the growth rate of cancer has not stopped. Oral cancer (OC) too, right from its discovery, has not failed to claim lives of many, the evidence of which lies in the fact that it is now the seventh most common malignancy worldwide.[2] Conventional studies in OC show a higher prevalence among men, tongue being the most frequently affected site, the mean age being 64.5 years and so forth.[3] However, in between 1960s and 1980s, some astonishing trends started to emerge pertaining to OC (Chen et al.), which almost entirely changed the face of this ailment that was known previously. Almost a fourfold ascent in the number of patients diagnosed with OC was seen in the age group of 30–39 years[4]!

Authors, after several studies and meticulous observations got a clear picture of the phenomenal changes. These changes, which were almost entirely attributed to a rise of human papillomavirus (HPV), after few years became a global issue and did not leave Asian countries, like India unscathed. Rigorous studies were conducted in India, although each one yielded different and somewhat contrasting results. While some authors (Laprise et al.)[5] could not demonstrate a single OC case with HPV, some (Balaram et al. and D'Costa et al.)[6],[7] concluded otherwise.

The aim of this study is to determine the role that HPV plays in the development of OC, specifically in the absence of other risk factors like tobacco and alcohol in a developing country like India, where habits contribute as a key role in development of OC and at the same time to evaluate the current trends of the role of HPV in the disease.


   Subjects and Methods Top


This study received approval from the Institutional Review Board of the Sri Aurobindo Institute of Medical Sciences. The design of the study was a comparative, case control analysis, with a sample size of 100, of which, 50 were cases and 50 controls, all >18 years of age. The cases included patients who were already diagnosed with oral squamous cell carcinoma (OSCC) [Figure 1] while controls included patients who were devoid of any pathology and who visited the institute for procedures like flap surgery, extraction and so on. It was ensured that only the individuals who did not have any habit of tobacco (in either of its form of consumption) and alcohol within a span of 15 years were counted as cases in the study. There was no specificity for selection of cases and controls, however, it was ensured that age and gender were matched. Pregnant and lactating women were excluded from the study in entirety.
Figure 1: Haematoxylin and eosin-stained section of oral squamous cell carcinoma

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Samples, once collected, were taken to the Surgipath Diagnostic Laboratory, Indore for further processing and evaluation of the study.

Statistical analysis

Descriptive and inferential statistics were utilized. The descriptive statistics were taken in use to project the main features and characteristics of the patients who were diagnosed clinically with oral squamous cell carcinoma irrespective of age, sex and grade of the disease. Inferential statistics were utilized to determine the test of significance based on pre-determined objectives of the study.

The Pearson's Chi-square test was used to observe the association of presence of HPV with groups. The probability value from P < 0.05 to P < 0.02 was considered statistically significant while from P < 0.01 to P < 0.001 was considered as statistically highly/strongly significant.


   Results Top


Among 100 subjects, mean age lied between 47.69 ± 6.73 years. While tongue was the most common site of lesion, affecting one-third (32.0%) of the patients, alveolus remained second most commonly inflicted site (in more than one-fourth – 28.0%), followed by buccal mucosa (affecting 12 – 24.0% of the patients). Gingivobuccal sulcus, nasopharynx, palate and lower lip were revealed to be least-affected sites. Elaborate details about patient distribution as per site can be seen in [Table 1].
Table 1: Distribution of patients according to site of lesion in experimental group

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More than half (28, 56.0%) of the patients had Grade II of OSCC, making it the most common grade in our study, followed by Grade I, which was revealed in 13 (26.0%) patients. The less common grades were Grade III (8, 16.0%) and IV (1, 2.0%). What is more interesting is the fact that 100% patients who showed positivity for p16 had lesions of Grade II. The grades of OC in our patients are explained in [Figure 2].
Figure 2: Diagram presenting the distribution of grading of oral squamous cell carcinoma for selected patients

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Of the total patients who were positive for p16 (6), 4 (66.7%) were female patients while 2 (11.1%) were male patients, indicating that the presence of HPV did not depend much on sex.

Of the 16 patients in our study who had lesion on tongue, 4 (16.0%) were positive for p16; both the lesions on nasopharynx (100.0%) also confirmed the presence of HPV. Thus, it can be asserted that there is a co-relation between HPV and site of the lesion, indicated by above-mentioned results.HPV was most commonly prevalent among 19.2% patients who were clinically diagnosed with OSCC within age from 45 to 55 years.

Overall prevalence of HPV in cases of OSCC was found to be 12.0%. The details about prevalence of p16 according to the site of lesion are mentioned in [Table 2] and [Figure 3].
Table 2: Prevalence of HPV with site of lesion

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Figure 3: Bar diagram presenting the prevalence of human papillomavirus according to site of lesion in experimental group

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The strongly positive-stained p16 section is shown in [Figure 4].
Figure 4: Strongly positive-stained section for p16 surrogate marker

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   Discussion Top


While tobacco, its products and alcohol have been known to be recognized as the classical causes of cancer of the oral cavity since time immemorial, since a few decades HPV has emerged as an aetiological agent in a subset of oropharyngeal cancers. This association was first depicted by Syrjänen et al. in 1983,[8] following which, many trends of HPV-associated OC have been observed. In the western culture, HPV contributes to a significant proportion of OC. In a developing country like India, the involvement of HPV in OC might be an issue of controversy for two reasons – one, as the habit of consuming tobacco is much more common in India, it accounts for majority of cases of OC; the second cause lies in the beliefs and culture of the nation, where practices like oral sex which are the main cause of HPV-associated OC, are believed to be not much common. Even though the literature shows copious data concerning HPV and OC, it is difficult to infer that in the absence of any other aetiological factors, HPV can result in OC, more so in a developing nation like India. Out of >120 HPV strains known, 12 strains are identified in oral cavity, while 16 (maximum risk), 18, 6 and 11 are the most commonly isolated ones.[9],[10],[11]

The findings in our study regarding the age showed maximum patients belong to 35–45 years age group. These findings were rather similar to those by Smith et al. (2004),[12] Sharma and Singh (2016)[13] and Chaturvedi et al. (2008),[14] as their results too asserted that HPV-associated OC to be found more commonly in a comparatively younger population.

Tongue was found to be the most common site for OC in our study, which was in accordance with previous studies done by Sharma and Singh (2016),[13] Sasaki et al. (2005)[15] and Elango et al.[16] in 2006.

The sex of patients was not found to affect positivity for p16 in our study. However, the same cannot be said about site of the lesion. There was a definite relationship between site of the lesion and positivity for p16. This can be verified with high HPV positivity (66.7%) on tongue and nasopharynx (100%). Thus the inference that site affects presence of HPV can be drawn. Similar results have been obtained in studies by D'Costa et al. (1998),[7] Smith et al. (2004)[12] and Schwartz et al. (2001).[17]

Many studies in the past have shown lower grades of OC to be associated with HPV-induced carcinomas [Schwartz et al. (2001),[17] Campisi et al. (2006)[18] and Hung et al. (2012)].[19] In our study as well it was the lower and less severe grades of OC which were more common (Grade II followed by III), however because of limited sample size, it is difficult to directly infer a particular relationship between the grades of OC and severity of HPV-associated OC. This conclusion can only be evaluated on much large-scale studies with a bigger sample size.

Out of 50 OC cases in our study, HPV was seen only in 6 (12%) patients, which lied somewhere in between the results of studies carried out previously (Miguel et al. in 1998 – 8%,[20] D'Costa et al. in 1998 – 15%,[7] Kojima et al. in 2002 – 66%[21] and Kulkarni et al. in 2011 – 70.59%[22]). This variance of prevalence can be attributed almost entirely to the sexual practices in various regions of the world.

The polymerase chain reaction (PCR), immunohistochemistry (IHC) and in situ hybridization (ISH) are common methods used to detect HPV DNA. Considering the cost, high sensitivity[11] and convenience to perform the laboratory analysis, IHC was chosen to detect HPV in this study. Since p16 is a surrogate marker for tumours induced due to HPV infection, IHC for p16 was employed in this study.

Buajeeb W et al. 2008,[23] Lewis JS et al. 2010[24] and Patil et al. 2014[25] successfully used p16 IHC to demonstrate the presence of HPV in OSCC.

Pannone G et al. 2012,[26] Thomas J and Primeaux 2012[27] in their studies analyzed various methods to find HPV in tissue samples to conclude IHC to be a better method over PCR and ISH.

Thus keeping the cost in mind, IHC was an overall feasible method for our study. As per the results obtained in our study, it can be concluded that HPV is an independent factor for OC even in a conservative and developing nation like India.


   Conclusion Top


A larger study group is needed to refine these findings, as due to the cost factor the number of subjects considered in the present study were restricted. For more elaborate results, other diagnostic methods like PCR can also be employed. Nevertheless, what needs to be considered is an emphasis on the fact that a clinician should not overlook suggesting an HPV analysis, especially to those patients who belong to the younger age group so that better treatment strategies can be planned, ensuring better prognosis of OC, as OC resulting from HPV in India is no more an impossibility.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

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