Association of Human Papilloma Virus in Oral Squamous Cell Carcinoma: An Alarming Need for Human Papillomavirus 16 Screening in Cancer Patients.

BACKGROUND: The percentage of cancers of the tongue and palatine tonsils has continued to increase by 2%-4% among younger men. This increased prevalence of a subsection of oropharyngeal carcinoma can be associated with human papillomavirus (HPV). Among the head-and-neck cancers, a strong association with HPV infection is evident with oropharyngeal cancers, particularly tonsillar and basal tongue cancers.
OBJECTIVES: Oral carcinoma, with an overall incidence of 16.1 adults per 100,000, is one of the leading malignancies worldwide, presenting a noticeable geographic variation in its distribution. Squamous cell carcinoma (SCC) being the most common of all oral malignancies, the objective of the study is to detect the HPV antigen p16 over-expression in patients with oral SCC using immunohistochemistry (IHC).
MATERIALS AND METHODS: Oral SCC (OSCC) diagnosed formalin-fixed-paraffin embedded blocks were processed for IHC.
RESULTS: Out of 50 cases, 3 were deferred due to insufficient tumor sample and 2/47 cases were p16 positive and the site was the lateral border of the tongue.
CONCLUSION: The HPV antigen overexpression in patients with OSCC was investigated to detect the incidence of HPV in SCC of oral cavity. P16 was used as a marker for the detection of OSCC using IHC in HPV-induced OSCC. Positives were detected thus concluding the significance of studying HPV expression during diagnosis. Copyright:

INTRODUCTION

Oral carcinoma, with an overall incidence of 16.1 adults per 100,000, is one of the leading malignancies worldwide, presenting a noticeable geographic variation in its distribution. In India, it ranks number one among all cancers in males and third in females. Squamous cell carcinoma (SCC) is the most common of all oral malignancies.[1] One of the world's highest incidences of tobacco and alcohol-related head-and-neck cancers is recorded in India.[2] A continued rise of rates of tongue and palatine tonsil cancers by 2%–4% among young men may partly be substantiated by the increased prevalence of a subgroup of oropharyngeal carcinoma associated with human papillomavirus (HPV), frequently HPV 16.[3]

HPV is a ≅7.9 kb, nonenveloped, double-stranded, circular DNA virus associated with various anogenital and aerodigestive diseases, stretching from warts, laryngeal papilloma, to cervical cancer.[4] HPV prevalence rates in oral SCC (OSCC) are varying due to different geographical regions. The prevalence rates were higher, varying from 64% to 72% in Maryland.[5] HPV prevalence has been stated to be high in carcinomas of the tonsil and the base of the tongue, compared to the other oropharyngeal subsites.[6] Furthermore, a strong positive correlation has been presented between p16 and HPV.[7] About 25%–75% of oropharyngeal cancers were reported to be positive for HPV, with tonsillar cancer being the highest, followed by tongue and buccal mucosa cancers.[8]

MATERIALS AND METHODS

Sample selection

Fifty archival formalin-fixed-paraffin embedded (FFPE) specimens were retrieved from the Department of Oral and Maxillofacial Surgery and Oral Pathology of Sri Ramachandra Institute of Higher Education and Research, Chennai. The specimens included untreated, surgically resected tumors with complete clinicopathological data. Paraffin blocks were selected for tissue sectioning following a case review. The case history and clinicopathological data including age, gender, lesion site, lesion size, lesion status, lesion grading, habits, past medical and dental history, and drug intake were recorded for all cases [Table 1]. Out of 50 patients, 13 patients were female and 37 were male. Grossly identified oral squamous tissues were classified as “negative” or “positive” based on the histopathological diagnosis.

Table 1

Habit and demographic details

	Frequency (%)
Habits
Valid	21 (48.8)
Betel nut	7 (14.0)
Gutka	1 (0.2)
Pan chewing	28 (59.5)
Quid	2 (4.2)
Smoking	6 (12.7)
Nil	2 (4.2)
Tobacco	1 (2.1)
Total	47 (100.0)
Age range (years)
<40	14 (29.7)
40-50	24 (51.2)
>50	9 (19.1)
Total	47 (100.0)
Gender
Female	12 (25.6)
Male	35 (74.4)
Total	47 (100.0)

Immunohistochemical procedures

The biopsied tissues were immediately transferred to 10% buffered formalin and were embedded in paraffin after adequate fixation. Paraffin-embedded sections (4 μm thickness) were cut and used for routine hematoxylin and eosin and immunohistochemical staining. The slides were dewaxed in xylene and hydrated through three grades of alcohol and then water. The slides were washed in running tap water for 5 min. The slides were differentiated in 1% acid alcohol for 5 min followed by running tap water wash for 5 min and the tissue sections were stained in eosin for 30 s and washed again with running tap water for 1 min. The slides were dehydrated through alcohol, cleared, mounted, and viewed under a light microscope.

The ribbons of tissue sections were transferred onto the precoated slides from the tissue float bath, with one tissue bit on each slide. One of the tissue sections was labeled positive to which the primary antibody (p16 Mouse Monoclonal-Santacruz), secondary antibody (Bio SB Mouse/Rabbit Monoclonal), and chromogen were added and another tissue section was marked negative to which primary antibody was not added. Antibody-reactive sites were visualized with the chromogen substrate diaminobenzidine tetrachloride. Cervical cancer tissue sections were taken as positive control and a section showing no lesion was taken as negative.

Malignancy grading of oral squamous cell carcinoma

Sections were graded as well-differentiated (WD), moderately differentiated (MD), and poorly differentiated OSCC on the basis of Anneroth classification. This classification grades the malignancy of tumor-host relation based on the degree of keratinization, number of mitosis, nuclear polymorphism, pattern and stage of invasion, and lymphoplasmacytic infiltration.

Grading of oral squamous cell carcinoma

The study samples (n = 50) included 41 cases of WDSCCs (82%), 6 MDSCCs (12%), 2 poorly differentiated SCCs (4%) and 1 carcinoma in situ (2%).

RESULTS

The study was conducted with 50 cases. Out of the 50 cases, 3 were deferred due to insufficient tumor sample in the block. [Figures 1–3] represent the different nucleus–cytoplasm percentages observed. More than 50% nucleus and cytoplasm, which was considered positive was seen in only 2/47 cases with the lateral border of the tongue as the site. Furthermore, the age range of the patients who were p16 positive was around 35 years. Other observations included >50% of only cytoplasm in 5/47 cases; >10% of only cytoplasm in 14/47 cases; >10% of only nucleus in 3/47 cases; >10% nucleus and cytoplasm in 3/47 cases; < 10% nucleus and cytoplasm in 1/47 cases; < 25% only cytoplasm in 7/47 cases; < 50% only cytoplasm in 1/47 cases. The outcomes and the associated factors were studied. It was found that two of the HPV 16 positive cases were OSCC positive

Figure 1

Immunoexpression of Anti-p16 was found positive in >50% of cytoplasm (10x)

Figure 3

Immunoexpression of Anti-p16 was found negative (10x)

Immunoexpression of Anti-p16 was found positive in <25% of nucleus (10x)

DISCUSSION

The overexpression of p16 represents positivity in 5% of OSCC cases, i.e., 2/47 cases, expressed more than 75% tumor cells (nucleus and cytoplasm). The results of this study were highly in accordance with previous studies[2910] which concluded low prevalence of HPV in oral carcinoma.[11] Although the prevalence is low, according to a multicenter case–control study, the incidence of HPV has been reported to have a role in the etiology in various cancers of oropharynx and in a small subcategory of oral cavity cancers.[12] In our study, the presence of more than 50% nucleus and cytoplasm was considered positive. However, according to another study,[13] the slides were considered as p16 (INK4a) positive if the specimen showed continuous staining of at least 10% of tumor cells which corresponds to >10% only cytoplasm staining (30% cases), >10% only nucleus staining (6.3% cases), and >10% nucleus and cytoplasm staining (6.3% cases) in our study. P16 overexpression of more than 75% nucleus and cytoplasm, seen in two patients, was from the lateral border of the tongue. Habits have a serious effect on the incidence of OSCC. The detailed history of habits has been referred to in the present study and its significance is proven.

The OSCC patients showing HPV positivity differ in their response from the patients who do not show positivity to HPV 16. Butz et al. reported that survival rates were steadily higher in patients with HPV-positive oropharyngeal cancers. Furthermore, their response to radiation therapy and chemotherapy was better and was less likely to express progression and recurrence of tumors.[14] Similarly, a meta-analysis has revealed a lower risk of mortality and recurrence with HPV-positive head-and-neck SCC (HNSCC) patients compared to HPV-negative patients.[15] A positive correlation was observed between HPV 16 and oral tongue cancers.[16]

Overexpression of the p16INK4A protein by immunohistochemistry (IHC) can act as a surrogate biomarker of HPV-induced carcinomas.[17] The presence of HPV DNA (analyzed by polymerase chain reaction) on FFPE material and the overexpression of p16INK4a analyzed by IHC has been reported to be as sensitive and specific as defining HPV status by the golden standard.[18] In the present study, IHC was done to detect the p16 positivity and the results revealed that HPV was a significant criterion to be considered in the diagnosis of cancer patients. However, the use of HPV association as a prognostic indicator in Indian HNSCCs necessitates further studies. The identification of HPV 16 and 18 as high-risk types has resulted in the development of prophylactic vaccines based on their viral capsids. Cervarix™ and Gardasil® are already available in the market to prevent HPV-related diseases. The efficacy of these vaccines in preventing HPV-related-HNSCC is still under research. This study possibly indicates an alarming need to implement the HPV testing and vaccination mandatory in oral cancer patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.