An Observational Study Depicting Role of p16 in Detecting Human Papilloma Virus in Oral and Oropharyngeal Cancer and Associated Factors

Abstract

The longstanding phrase, traditional risk factors like—smoking, tobacco and alcohol are the only causative agents of oral and oropharyngeal squamous cell carcinoma (SCC) is changing now. Etiological divergence is taking place which we have to know to reach upon an exact cause of the disease. Human papilloma virus (HPV) infection is a well-established cause of oral cancer. Escalating incidence of HPV associated cancer has a strong impact on its management. So, a concern clinician needs to get aware for it. Among various types of HPV, HPV-16 is more frequently associated with oral cancers. HPV detection will become a game changer in management of oral cancers. HPV infection and p16 protein expression has a strong and consistent correlation. Therefore, immunohistochemical analysis of p16 protein can be a surrogate biomarker in high risk groups. This hospital based prospective observational study recruited 180 subjects of oral and oropharyngeal SCC. We underwent immunohistochemical analysis of p16 in biopsy specimen of subjects and studied HPV status and associated environmental, clinical and behavioral factors. We observed 9.4% (17/180) incidence of p16 positivity with female predominance. Our observations showed that its prevalence was higher in urban patients who were non tobacco users, had sexually transmitted disease, early stage (T1–T2), poorly differentiated SCC with nodal metastasis. We conclude that keeping in mind HPV as a cause of oral and oropharyngeal SCC, p16 should be use as a biomarker for its detection in high risk cases.

Introduction

Squamous cell carcinoma of oral cavity represents eleventh most common malignancy worldwide with oropharyngeal cancer (OPSCC) at first rank [1]. Recent studies quote that approximately 15–20% of patients of the western population with oropharyngeal squamous cell carcinoma (OPSCC) occur without conventional risk factors, like—tobacco, smoking. This fact suggests that somewhere there is participation of non-traditional behavioral and environmental risk factors in its occurrence [2]. Epidemiological data from the Surveillance, Epidemiology, and End Results Program (SEER) show that while overall incidence of HNSCC has decreased in conjunction with a decrease in smoking and drinking, oropharyngeal squamous cell cancers (OPSCC) have increased both in the United States and other developed nations internationally. Human papilloma virus (HPV) is now a well-established causative agent of OPSCC. HPV is a sexually transmitted infection, and currently recognized as the most prevalent sexually transmitted infection in the United States, especially among younger populations. There are nine HPV subtypes considered carcinogenic, but HPV16 accounts for 90–95% of HPV positive OPSCC cases [3].

Detection of high-risk HPV has become a powerful and widely used biomarker of improved clinical outcome for patients with oropharyngeal squamous cell carcinoma. A strong correlation between HPV infection and p16 protein expression has consistently been reported in OPSCC. Therefore, p16 can be presumed to be a surrogate biomarker for HPV infection in these high-risk tumors [4–8]. Immunohistochemical (IHC) assessment of p16 protein has recently been considered as a practical alternative for HPV testing based on a high correlation between the HPV detection and p16 overexpression. The simplicity, cost effectiveness, and high sensitivity of p16 IHC analysis have prompted consideration of this test as a diagnostic tool in suspected cases [9].

HPV status may guide treatment modality, once diagnosed and has a significant and favorable effect on prognosis. Thus, in cases of OPSCC detection of HPV will form one of the standard choices of diagnostic tool. This thought sheds light on association of HPV and oropharyngeal carcinoma, and draws attention of clinicians towards use of p16 as a routine test in concern high risk groups. To the best of our knowledge there are various literatures which determine presence of HPV in oral cancer but very few are there which determine its correlation with oropharyngeal cancer. Here we studied HPV status in both oral and oropharyngeal squamous cell carcinoma patients and various environmental, clinical and behavioral factors associated with it.

Methods

This is a hospital based, prospective, observational study conducted in department of otorhinolaryngology with co-operation of department of pathology of our institute from March 2017 to November 2018. All patients who were newly diagnosed as oral squamous cell carcinoma (oral cavity and oropharyngeal) histopathologically were included in the study after taking informed and written consent. Those who had previously received treatment in form of surgery or chemoradiation, recurrent case of oral cancer, other than squamous cell carcinoma and who were not willing to participate in the study were excluded. Total 180 subjects of both sexes were enrolled in the study. Age range of the study population was 20–89 years. The study was approved from the institutional review and ethical board.

Demographic, exposure, behavioral and clinical data were obtained using a prospective self-administered questionnaire at enrollment. Immunohistochemical (IHC) marker study was done using p16 as a biomarker in department of pathology of our institute. As our patients are already diagnosed as oral squamous cell carcinoma, we used same biopsy specimen for marker study. IHC will be performed on four-micron thick sections with P16ink4 ready to use antibody with standard protocol. Antigen retrieval was performed by heat induced epitope retrieval method with the help of de-cloaking chamber. Slides were kept at room temperature for cooling (30 min) and arrange on rack of moisture chamber. Slides were examined twice with tris buffer (ph = 9) and mark the sections by pap pen. Background snipper was applied for 5 min and primary antibody for 1 h 30 min then slides were washed twice with tris buffer. Then polymer was applied for 30 min and again slides were washed with tris buffer (twice). DAB was applied for 1 min 30 s and washed with distilled water then running tap water. Slides were kept in hematoxylin for 5–7 dips and kept in running tap water for 2 min. Then acetone applied two times with 1 min each time and xylene two times for 10 min and now slides were mounted and examined. The nuclear positivity with or without cytoplasmic positivity was considered as positive result for p16 and only cytoplasmic positivity was considered as negative.

The procedures followed were in accordance with the ethical standards of the institutional ethics and research committee and with the Helsinki Declaration of 1975, as revised in 2000.

Data collected was entered into MS Excel spread sheet. Nominal/categorical variables were summarized as frequency and percentage and were analyzed using Chi square test/Fischer Exact test as applicable. Continuous variables were summarized as mean and standard deviation and were analyzed using independent sample t test. A p value < 0.05 was taken as statistically significant. All statistical analyses were done using Epi info version 7.2.1.0.

Results

Total 180 subjects were included in the study with age range of 20–89 years. Mean age range was 51.09 ± 12.93 years. Among these, there were 151 males and 29 females. Thus, this study shows male preponderance (M:F = 5.2:1). Out of 180 subjects 102 subjects were belong to rural area and 78 to urban area.

In our study we included patients who had squamous cell carcinoma of oral cavity and oropharynx. Among 180 subjects 129 had carcinoma of oral cavity and rest 51 had carcinoma of oropharynx (Fig. 1). When we subdivided these subjects according to subsites than our data indicated that maximum 69 subjects had involvement of buccal mucosa (Fig. 2). Other are as follows:

Fig. 1.

Distribution of study subjects according to their site of tumor

Fig. 2.

p16 (HPV) status in relation to site of tumor

Oral hygiene was divided into good (25), satisfactory (87) and poor (68) on the basis of clinical appearance.

We also measured the size of malignant lesion in all subjects and we found that in 113 subjects it was > 4 cm and 67 subjects it was < 4 cm in size.

We clinically examined the nodal metastasis and 119 subjects had nodal metastasis and rest 61 had no nodal metastasis.

According to histopathological differentiation 94 subjects had moderately differentiated carcinoma, 80 had well differentiated carcinoma and 6 had poorly differentiated carcinoma.

In this study population 80 patients were tobacco addict, 42 were smokers and 25 were both tobacco addict and smokers. 24 subjects had addiction of other than these substances. Only 9 subjects had no history of any substance abuse.

Out of 180 subjects 166 had no sexually transmitted disease (STD) and 14 subjects were diagnosed as a case of STD.

Biopsy specimen of all subjects underwent immunohistochemical marker study specifically for p16 which indicates Human papilloma virus (HPV) is one of the etiological factors. We observed that among 180 subjects, 17 (9.4%) subjects were p16 positive i.e. HPV was associated with malignancy in those subjects.

Further correlation of p16 positivity with our study factors and results are mentioned in Table 1.

Table 1.

Various study factors and their correlation with p16 (HPV)

S. no	Factors	Total (N) = 180	p16 positive	p16 negative
1.	Sex
	Male	151	13	138
	Female	29	4	25
2.	Locality
	Rural	102	5	97
	Urban	78	12	66
3.	Sites involved
	Buccal mucosa	69	4	65
	Palate	9	0	9
	Tongue	51	3	48
	Tonsils	20	6	14
	Base of tongue	31	4	27
4.	Oral hygiene
	Good	25	1	24
	Satisfactory	87	10	77
	Poor	68	6	62
5.	Size of malignant lesion
	< 4 cm (T1–T2)	67	8	59
	> 4 cm (T3–T4)	113	9	104
6.	Nodal metastasis
	Present	119	14	105
	Absent	61	3	58
7.	Differentiation
	WDSCC	80	6	74
	MDSCC	94	10	84
	PDSCC	6	1	5
8.	Substance abuse
	Tobacco	80	8	72
	Smoking	42	3	39
	Tobacco + smoking	25	2	23
	Others	24	4	20
	No abuse	9	0	9
9.	STD
	Present	14	3	11
	Absent	166	14	152

Discussion

Previous studies show downward trend in HNSCC which was roughly parallel with trends of smoking. This great optimism has recently been shaken by specifically increase in incidence of oropharyngeal squamous cell carcinoma (OPSCC). Literatures shed light on this fact that incident trends for HNSCC have generally paralleled smoking trends. The escalating incidence of oropharyngeal carcinoma in the absence of a parallel rise in smoking and alcohol consumption suggests that nontraditional behavioral and environmental factors are driving this aberration. HPV, particularly type 16, has been established as a causative agent in up to 70% of oropharyngeal cancers [10].

A case–control study done by D’souza et al. concluded that there is association between HPV and a subgroup of oropharyngeal cancers. They found that HPV-16 DNA was specifically localized to tumor-cell nuclei in 72% of 100 paraffin-embedded specimens of oropharyngeal cancers, a finding corroborated by the high prevalence of antibodies for HPV-16 oncoprotein E6, E7, or both (64%) in the patients with oropharyngeal cancer. This study establishes association between HPV and oropharyngeal cancer regardless of tobacco and alcohol use [11].

Kane et al. did a retrospective analysis of 124 oral cancer patients. They did immunohistochemical analysis of p16 in pretreatment local site biopsy specimen and they found 16 (12.9%) patients were HPV positive [12]. Similarly, Murthy et al. conducted a study on 170 head and neck squamous cell carcinoma cases and found 20% (34/170) cases were p16 positive [13].

There are many more literatures which indicate p16 positivity in OPSCC. So, we have to change our vision and should focus on this aspect of disease to get better prognosis.

Our findings differ from Patel et al., who studied prevalence of HPV in 97 oral malignancy cases and found none of them was HPV positive [14]. It doesn’t reveal that HPV has no relation with oral malignancy, in fact we need a large study population to disclose this fact.

In our study prevalence of p16 positivity (HPV) was higher in females 4/29 (13.8%) as compare to males 13/151 (8.6%). These results are similar to various studies in which female patients show higher prevalence of p16 positivity (HPV) [12, 15, 16].

In our study prevalence of p16 positivity (HPV) was higher in urban area 12/78 (15.4%) as compared to rural area 5.9% (5/102). This difference was found to be statistically significant (p = 0.034). Bahl et al. in their study also had 16/46 (34.7%) urban and 8/59 (13.6%) rural with urban preponderance [17]. Our study concurs with the above study.

We observed that prevalence of p16 positivity (HPV) was more in malignancy of oropharynx (19.6%, 10/51) as compared to oral cavity (5.4%, 7/129). Most common site was tonsil (30%, 6/20) followed by base of tongue (12.9%, 4/13), tongue (5.9%, 3/51), and buccal mucosa (5.8%, 4/59). This difference was found to be statistically significant (p = 0.011). Adilbay et al. [18] observed that prevalence of p16 positivity (HPV) was 25.7% (9/35) in oropharyngeal cancer patients and 12.2% (5/41) in oral cavity cancer patients. Kane et al. [12] concluded that the commonest site was site other than tongue (14.3%, 13/91) followed by tongue (9.09%, 3/33). Our results concur with these studies. But some studies show results which are not in favor of our study. Koppikar et al. [16] observed that HPV positive tumor was most common of oral cavity origin (33.7%), as compare to oropharynx (33.3%). D’costa et al. [15] found that there was significantly higher prevalence of HPV-16 in the tongue cancer group (43%, 6/14), compared to tumors of the buccal mucosa (7%, 4/57).

In this study prevalence of p16 positivity (HPV) was more in patients who didn’t used tobacco (12.9%) as compared to those with history of tobacco use (8.7%). This difference was however not found to be statistically significant (p = 0.699). Similarly, in study of Kane et al. [12] and Murthy et al. [13] HPV-positive oral cancer was more prevalent in non-tobacco users as compare to tobacco users. In a comparative study by Koppikar et al. [16] prevalence of HPV positive oral cancer was more in habitual tobacco users (30%) as compare to non-users (6%). Our results don’t favor this study.

When we classified cases as per ‘T’ stage we observed that prevalence of p16 positivity (HPV) was more in early stage oral cancer i.e. T1–T2 (11.9%, 8/67) as compared to patients with later stage i.e. T3–T4 (8%, 9/113). This difference was statistically insignificant (p = 0.511). Similarly, Murthy et al. [13] observed p16 (HPV) positivity was more common in stage T1–T2 patients (10.5%) in comparison of stage T3–T4 patients (8.33%).

When we focused on nodal metastasis, we found that prevalence of p16 positivity (HPV) was more in oral cancer with nodal metastasis (11.8%, 14/119) as compared to those without nodal metastasis (4.9%, 3/61). This difference was however not found to be statistically significant (p = 0.956). Kane et al. [12] observed 14.7% prevalence of HPV positivity in cases with nodal metastasis and 4.5% in node negative cases. Murthy et al. [13] observed HPV positivity in 23.5% cases with nodal metastasis and 14.06% cases without nodal metastasis. Our results are nearly identical with these studies which denote higher prevalence of p16 positivity (HPV) with nodal involvement.

In our study prevalence of p16 positivity (HPV) was highest in PDSCC grade of malignancy (16.7%, 1/16) followed by MDSCC (10.6%, 10/94) and WDSCC (7.5%, 6/80). D’costa et al. [15] done an almost similar study and observed that prevalence of HPV associated oral cancer was highest in poorly differentiated grade (23.5%) followed by well differentiated (14.3%) and moderately differentiated (12.7%) squamous cell carcinoma. Thus, both studies quote similar results in aspect of tumor grade as highest prevalence of HPV positivity was found in poorly differentiated squamous cell carcinoma.

We observed that prevalence of p16 positivity (HPV) was more in oral cancer with STD 21.4% (3/14) as compared to those without STD 8.4% (14/166). This difference was however not found to be statistically significant (p = 0.110), probably because of smaller number of STD patients. In study carried out by Schwartz et al. [19] genital warts (STD) was more often reported by male case subjects than male control subjects, leading to an approximately two-fold odd ratio (OR) following adjustment for cigarette smoking and alcohol use. There was no association with genital warts among females. 4 case subjects (1.4%) and 3 control subjects (0.6%) reported a history of oral warts (OR = 1.6; 95% CI = 0.3–8.6). The ORs for HPV type 16 capsid seropositivity were 2.3 [95% confidence interval (CI) = 1.6–3.3] for all oral SCCs and 6.8 (95% CI = 3.0–15.2) for oral SCCs containing HPV type 16 DNA.

Conclusion

This study reveals that Human papilloma virus is a well-established causative agent of both oral and oropharyngeal squamous cell carcinoma. P16 is a sensitive biomarker which can be used for diagnosis of HPV. We recommend the use of p16 as a diagnostic tool among other investigations in suspected high-risk cases of oral and oropharyngeal malignancy. We need more studies with large study population for further clearance of our conclusion.

Compliance with Ethical Standards

Conflict of interest

Authors declare that they have no conflict of interests.

Ethical Approval

All procedures performed in presented study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed Consent

Informed and written consent was obtained from all individual participants included in the study.