Role of HPV-16 in Pathogenesis of Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma and Correlation of p16INK4A Expression in HPV-16 Positive Cases: An Immunohistochemical Study

The objective of current study is to evaluate the role of HPV-16 in the pathogenesis of oral epithelial dysplasias (OED) and oral squamous cell carcinoma (OSCC) by immunohistochemistry (IHC) and to know whether HPV-16 participates in disruption of the regulation of p16 INK4A suppressor protein in OED and OSCC by IHC. Histopathologically diagnosed 20 cases of OED and 20 cases of OSCC were selected from amongst the patients attending the OPD of Vasantdada Patil Dental College and Hospital, Sangli. Biopsy tissue section were then tested for HPV-16 by IHC. HPV-16 positive tissue sections were then again tested by p16 by IHC. Overall 22.5% of cases in our study were found to be positive for HPV 16 which includes 10% of cases of OED and 35% cases of OSCC. Amongst the HPV 16 positive cases, more than 60% of cells were positive for p16INK4A IHC in OED (50%) and OSCC (85.71%). Thus, HPV 16 participates in disruption of the regulation of p16INK4A suppressor protein and can be used as surrogate biomarker for detection of HPV infection in OED and OSCC.


Introduction
Cancer is a disease of cells that escape the control mechanisms of orderly cell growth and acquire the ability to proliferate, invade normal tissues and metastasize [1]. Head and neck cancer is the sixth most common cancer worldwide, with an estimated annual burden of 563,826 incident cases (including 274,850 oral cavity cancers, 159,363 laryngeal, and 52,100 oropharyngeal cancers) [2,3]. In India oral cancer constitute around 9.8% of total cancer cases & ranks first among the all cancer cases in males & third most common among females. Age standardized incidence rate varies between 7 and 17/1000,000 person-years. The incidence rate is higher than the rate of 3 to 4/1000,000/year found in western countries [4][5][6]. India has one of the world's highest incidences of head and neck cancers which were primarily caused by tobacco habits and alcohol consumption [5].
The association between high risk human papillomavirus [HR HPV] and risk of oral cancer development dates to back 1983, when HPV16 was detected in oral squamous cell tumors. Since then HPV DNA has repeatedly been found in a variable proportion of head and neck cancers [6][7][8][9].
HPV is a ∼7.9 kb, nonenveloped, double-stranded, circular DNA virus that has been implicated in a variety of anogenital & aerodigestive diseases, ranging from common warts to laryngeal papilloma to cervical cancer. The first isolation of this virus particle was performed in 1933 in rabbit papillomatosis [10].
The oral cavity is covered by an epithelium that has characteristics similar to those of the genital region, and both are exposed to strange bodies and microorganisms, some of which could be potentially carcinogenic. Considering that in the genital region this virus is implicated in the development of cancer of uterine cervix, it is possible that this link would also be present in relation to oral cancer. Therefore it has become important to know the frequency of this virus in the oral cavity and its probable relationship with the presence of the premalignant and malignant neoplasm in the region. Papillomavirus are considered to be persistent viruses capable of residing latently within host cells for extended periods of time [6].
The oncogenic potential of HPV is thought to be due to two oncoproteins, E6 and E7 [11][12][13]. The E6 protein of oncogenic HPV strains has been shown to interact with the p53 protein and promote its degradation by means of a ubiquitin-dependent pathway. The E7 oncoprotein can similarly complexed with retinoblastoma (Rb) and inactivate it.
Any single method of detection has some limitations. Thus, use of algorithms that combine the strength of complementary assays is needed. The recent reports have suggested that overexpression of the p16INK4A protein can act as a surrogate biomarker of HPV-induced carcinomas [16]. HPVinduced carcinogenesis is associated with low pRb protein levels which leads to subsequent p16 upregulation. Positive p16INK4A immunostaining of HPV-associated tumors is 100% sensitive but only 79% specific [3,17].
Considering all the previous facts this study focused on combining specificity of HPV immunohistochemistry with sensitivity of p16 immunohistochemistry for detection of role of HPV 16 in pathogenesis of OED & OSCC. (II) To evaluate whether HPV-16 participates in disruption of the regulation of p16INK4A suppressor protein in OED and OSCC by IHC.

Materials and Methods
Histopathologically diagnosed 20 cases of OED and 20 cases of OSCC constituted the study group. HPV 16 positive tissue sections were then tested for expression of p16INK4A by IHC. Primary antibody used was p16INK4A with immunoglobulin fraction diluted in PBS. Clone Used: G175-405, Mouse Monoclonal, Biogenix, CA.
The German semiquantitative scoring system [16,19,20] was adopted with some modification for scoring the staining results of p16. All the slides were interpreted for nuclear as well as cytoplasmic positivity.

Results and Statistical Analysis
Our study showed that in 20 cases of OED, 2 (10%) cases were positive for HPV 16 whereas out of 20 cases of OSCC, 7 (35%) cases were positive for HPV 16.
Total HPV 16 positivity including OED and OSCC was found to be 22.5%.
All the observations and results were categorized as

HPV 16 Associations in Oral Epithelial Dysplasias.
The study constituted 20 cases of OED. The age range was 20 yrs to 75 yrs with mean age of 52.58 yrs. Out of total cases, 40% cases were in age group of 51 to 60 yrs. HPV 16 positivity was found to be mainly in 51 to 70 yrs of age group and this finding was statistically significant. Genderwise distribution of cases of OED showed female predominance in HPV 16 positivity with 2 out of 3 females being positive for HPV 16. HPV 16 positivity was seen in buccal mucosa (50%) & alveolus (50%) which was statistically significant. HPV16 IHC showed a significant difference between proportions of mild & severe grading of OED ( < 0.05), & no significant difference was found between proportions of moderate grading for HPV 16 positivity.
All the patients were exposed to tobacco and mixed tobacco & smoking habits. None of the patients were exposed to specific occupational or environmental carcinogens except tobacco (Table 1 and Figure 2).    Histopathological grading of OSCC cases showed degree of differentiation to be 13 (65%) well, 5 (25%) moderate, and 2 (10%) poor. HPV 16 positivity on the basis of degree of differentiation was found to be 71.44% of well, 14.28% of moderate, and 14.28% of poor.

HPV 16 Associations in Oral
By applying test of difference between two proportions statistically significant difference was found between proportions of histopathological grading ( < 0.05) ( Table 2 and Figure 3).
Thus, HPV 16 positivity was found to be related to the degree of differentiation.
TNM staging of OSCC showed 9 cases (45%) in Stage I, 7 cases (35%) in Stage II, and 4 cases (20%) in Stage III. HPV 16 association on TNM staging showed positivity in 51.15% of Stage I, 28.5% of Stage II, and 14.28% of Stage III. This finding was statistically significant after applying test of difference (Table 3).

HPV 16
Positivity & p16INK4A Expression. The study constituted total of 40 cases which included 20 cases of OED & OSCC, respectively. All the 40 cases underwent IHC evaluation of HPV 16, out of which 9 cases were positive for HPV 16. 7 cases out of 9 HPV 16 positive cases, that is, 77.77%, showed strong positivity for p16INK4A. German semiquantitative scoring system suggests that overall positivity for p16 was 60.62% which scores as 3. It also suggests focal positivity for p16 by IHC. On the basis of the previous findings, immunohistochemical results are Overall mean and SD of all 9 slides are 60.62% and 12.56%, respectively.
suggestive of strong correlation between positivity of HPV 16 and p16INK4A (Table 4, Figures 2 and 3).

Discussion
The mechanism which leads to development of malignancy is always difficult to evaluate. Transformation of normal tissue into malignancy occurs through intermediate stages of premalignancy. The etiology or risk factors for initiation development of malignancy and always been a challenge for investigators. Tobacco and alcohol consumption is the most common risk factor for developing oropharyngeal cancers. In addition to this, certain viral infections play an important role in malignancy [4,8,21]. The participation of HPV in oral and oropharyngeal carcinoma was first proposed by K. Syrjänen et al. [22] in 1983 and then supported by several other authors [8,9,23,24]. According to the studies carried out to explain etiological role of HPV in cancer of the head and neck region, HPV has been found to be both in an episomal form and in an integral form. It has also been suggested that HPV may be latent for a long time in the episomal format in the oral mucosa, hence being responsible for initiation and development of a tumoral growth. This tumoral growth may occur as a result of a multicarcinogenic interaction together with some other carcinogens and cocarcinogens [3,5,12]. K. Syrjänen et al. in 1983 first demonstrated HPV 16 DNA in 6 of 21 cases of OED [22]. Later, one more study by them in 1986 found HPV 16 antigen expression in 66.7% of cases of leukoplakia with majority of cases with mild dysplasia [11]. Another study by Fregonesi et al. found HPV 16/18 positivity of 40% in oral premalignant lesions with various grades of dysplasia [24]. Shroyer investigated cases of OED for presence of HPV 16/18 as one of the risk factors for carcinogenesis. Their results showed positivity of 17% [25]. Some investigators like Rafik Abdelsayed [26] (0%) and Carmen Maria Lazzari et al. (8.8%) found no significant association between epithelial dysplasia and HPV status. Recently, systematic review from 1966 to 2010 by S. Syrjänen et al., expressed statisfactory significant association between HPV 16 positivity and OED ranging from 18.8% to 48.4% [27]. There was high variability of HPV positivity in OED ranging between 0% to 80% by various investigators [11,22,27,28].
Our study showed 2/20, that is, 10% HPV 16 positivity in OED, which is very low but in accordance with the literature.
There are more than 40 HPV types, which were detected in anogenital invasive tumors and their precursor lesions. The occurrence of low risk type HPV in precursor lesions rarely progresses to malignancy. Therefore, HR HPV like types 16 and 18 has major contribution in papillomavirus-associated carcinogenesis [12,19,20].
In contradiction to the previous studies, Young and Min et al. [32] and Tsuchiya et al. [36] were unable to find such association between HPV 16 and OSCC. Recently, systematic review by Syrjänen et al. [27] in 2011 in their review from 1966 to 2010 showed strong correlation between the presence of HPV DNA, specifically with HPV16 and OSCC. Estimated risk for HPV was found to vary from 0.32 to 363.0 in 33 studies included by them. In our study 35% (7/20) of OSCC cases were positive for HPV 16 by IHC, which is in accordance with the literature review so far. In current study, agewise distribution of HPV 16 positive and negative cases was not showing any statistically significant correlation which is in accordance with Kurose et al. [37] and in contradiction to Cruz et al. [38] In genderwise distribution of HPV positive OSCC cases, Werness et al. [13] found statistical correlation with male predominance. Other investigators like Cruz et al. [38] and Priya Koppikar et al. [5] concluded that men were more likely to be positive for HPV than females. Our study had no such correlation related to gender. Sitewise distribution of OSCC cases amongst HPV16 positive cases in our study showed no significant correlation, which is in accordance with Jalal et al. [39] & Cruz et al. [38] In the current study we found that there was significant association between HPV 16 and histopathologic grades of differentiation, with HPV 16 positivity being highest in cases of WDSCC (71.44%). Our results are in accordance with Abdelsayed [26] and in contradiction to Schlecht et al. [31] and Badaracco et al. [34] HPV 16 association in OSCC with clinical staging was studied by several investigators like Mellin et al. [20], Tachezy et al. [7], Badaracco et al. [34], and so forth. Out of them Mellin et al. [20] found significant correlation between TNM staging and HPV positivity. Our results are in accordance with the literature review, showing majority of positive cases from Stage I (51.44%).
Establishment of exact HPV association in carcinogenesis has always been a challenge due to absence of universal standardization detection method. The variety of methods in current use include PCR, ISH, detection of serum antibodies directed against HPV epitope, IHC for detection of HPV ORF like L 1 , E 6 , E 7 , and p16 immunohistochemistry. Any single method of detection has some limitations. Thus use of algorithms that combine the strength of complementary assay is needed. According to Guimaraes et al. [40], Fregonesi et al. [24], Cunningham et al. [17], Negri et al. [28], and Samama et al. [40] overexpression of p16INK4A protein by immunohistochemistry can act as surrogate biomarker of HPV-induced carcinomas.
In accordance with the review so far, our study showed a strong correlation between HPV positivity and disruption of regulation of p16INK4A suppressor protein which resulted in overexpression of p16INK4A in OED (50%) and OSCC (85.71%). We also found that overall 60.62% cells showed positive expression of p16INK4A in those cases. This suggests focal positivity for p16INK4A which has an association with carcinogenesis by HR HPV [24,40].
This clearly reveals that p16INK4A immunohistochemistry can be used as surrogate biomarker for HPV-associated carcinogenesis.

Conclusion
Immunohistochemical detection of HPV 16 and p16INK4A was performed using Super Sensitive Polymer HRP IHC detection system (Biogenix, CA). Overall 22.5% of cases in our study were found to be positive for HPV 16 which includes 10% of cases of OED and 35% of cases of OSCC. A significant correlation was found in degree of differentiation of tumor and TNM staging amongst HPV 16 positive and negative cases. Amongst the HPV 16 positive cases, more than 60% of cells were positive for p16INK4A IHC in OED (50%) and OSCC (85.71%). There are a wide range of molecular assays available for the detection of HPV which includes IHC, ISH, PCR, Southern blotting, and p16INK4A IHC. All these methods have some advantages as well as disadvantages.
Immunohistochemical detection of HPV 16 is based on viral integration in tissue. It mainly detects viral capsid proteins of papillomavirus.
HPV IHC lacks sensitivity. So an attempt was made in our study to combine specificity of HPV 16 IHC with sensitivity of p16INK4A IHC for detection of HPV infection in OED & OSCC.
On the basis of the previous relation we can conclude that, (i) HPV 16 does have a role in pathogenesis of OED and OSCC and can be studied effectively by immunohistochemistry; (ii) HPV 16 participates in disruption of the regulation of p16INK4A suppressor protein and thus can used as surrogate biomarker for detection of HPV infection in OED & OSCC.