|Year : 2016 | Volume
| Issue : 1 | Page : 232-237
Assessment and clinicopathological correlation of p16 expression in head and neck squamous cell carcinoma
Megha Ralli1, Sunita Singh1, S.P.S. Yadav2, Nisha Sharma1, Renuka Verma1, Rajeev Sen1
1 Department of Pathology, Pandit Bhagwat Dayal Sharma Postgraduate Institute of Medical Sciences, Rohtak, Haryana, India
2 Department of Ear, Nose, Throat, Pandit Bhagwat Dayal Sharma Postgraduate Institute of Medical Sciences, Rohtak, Haryana, India
|Date of Web Publication||13-Apr-2016|
Pathology, 30/32 West Patel Nagar, New Delhi - 110 008
Source of Support: None, Conflict of Interest: None
Introduction: Oral squamous cell carcinoma is a major cause of death throughout the developed world. It is associated with smoking and alcohol consumption. Human papillomavirus (HPV) type 16 has also been suggested to play a role in etiology of head and neck squamous cell carcinoma (HNSCC). p16 expression is now being used as a surrogate marker of HPV infection in squamous cell carcinoma and provides important prognostic information and future therapy planning.
Materials and Methods: In this prospective study, total of 75 cases of HNSCC were taken. Tumor grade was determined according to World Health Organization (WHO) criteria. p16 expression was determined by immunohistochemical staining. The obtained results were analyzed and evaluated using Chi-square test (Statistical Package for Social Sciences (SPSS) version 20), value of P <0.05 was taken significant.
Results: Out of 75 cases, 78.7% cases were positive for p16 (inclusive of all grades), while 21.3% cases were negative. Expression of p16 was higher in nonsmokers and nonalcohol consumers and significantly associated with paan chewing habit. No significant correlation was seen with history of abnormal sexual habits, but p16 expression was significantly correlated in cases with multiple sexual partners (P = 0.003), with increasing histological grade (P = 0.045) and in cases with lymph node metastasis (P = 0.03).
Conclusion: As HPV integration with transcription of viral oncoprotein induces overexpression of p16, immunohistochemical expression of p16 can be used as a surrogate marker of HPV. This approach can be implemented in diagnostic laboratories and can provide support for vaccination program in high risk group.
Keywords: Head and neck squamous cell carcinoma, human papillomavirus, Immunohistochemistry, p16
|How to cite this article:|
Ralli M, Singh S, Yadav S, Sharma N, Verma R, Sen R. Assessment and clinicopathological correlation of p16 expression in head and neck squamous cell carcinoma. J Can Res Ther 2016;12:232-7
|How to cite this URL:|
Ralli M, Singh S, Yadav S, Sharma N, Verma R, Sen R. Assessment and clinicopathological correlation of p16 expression in head and neck squamous cell carcinoma. J Can Res Ther [serial online] 2016 [cited 2021 Jan 24];12:232-7. Available from: https://www.cancerjournal.net/text.asp?2016/12/1/232/151447
| > Introduction|| |
Oral squamous cell carcinoma (OSCC) consistently ranks as one of the top seven cancers worldwide., In India, it ranks among the top three types of cancer. Squamous cell carcinoma of the upper aerodigestive tract typically occurs in older patients in their fifth to 7th decade of life and older. Commonly, there is a prolonged history of tobacco exposure and alcohol abuse. The risk of cancer development in this population is both dose- and time-dependent. Less frequently, patients with little or no exposure to known risk factors develop squamous cell carcinoma of the oral cavity, pharynx, and larynx. The identification of a high-risk oncogenic human papillomavirus (HPV) type 16 in a case of squamous cell carcinoma of the oral cavity in 1985 suggested a possible role of the virus in the etiology of head and neck squamous cell carcinoma (HNSCC). Several studies using a variety of techniques, including immunohistochemistry, in situ hybridization, dot blot and southern blot hybridization, and polymerase chain reaction (PCR), have since been able to demonstrate the presence of HPV genome in the cells of some cases of HNSCC. According to thecurrent literature, the risk factors of HNSCC are surprisinglysimilar to those of cervical cancer and cervical intraepithelial neoplasia (CIN), including thenumber of sexual partners, younger age at first sexualintercourse, practice of oral sex, history of genital warts, andyounger age., HPV status is associated withp16 expression, andHPV positive tumors are less likely to harbor p53 mutations.
The abrogation of p16 occurs frequently in human oral cancers. The loss of expression of p16 has been observed in oral premalignant lesions and primary tumors of the oral cavity. Mechanisms of inactivation include homozygous gene deletion, gene mutation, and hypermethylation of upstream CpG island regions. HPV-associated cancers are caused by expression of HPV's E6 and E7 proteins that bind to and inactivate tumor suppressor proteins p53 and retinoblastoma protein (pRb), respectively, leading to malignant transformation of HPV infected cells  pRb is functionally inactivated by binding of viral protein and no longer acts as a cell cycle inhibitor. HPV E7 binds to hypophosphorylated form of Rb. The binding occurs in Rb pocket that sequesters E2F transcription factors. Thus pRb, unable to bind the E2F transcription factors, is functionally inactivated and transcription factors are free to cause cell cycle progression.
p16 expression is now being used as a surrogate marker of HPV infection in squamous cell carcinoma, and thus is expected to be showing differences depending on endemicity of HPV infection. This may help in providing important prognostic information and future therapies aimed at targeting this pathway of HPV tumorigenesis. The present study has been undertaken to study expression of p16 in HNSCC.
| > Materials and Methods|| |
The present study was conducted in our hospital during the period 2012–2014. In this prospective study, 75 cases of HNSCC pertaining to oral cavity, oropharynx, larynx, and hypopharynx were studied. Patients with other than HNSCC such as adenocarcinoma, melanoma, sarcoma, metastasis, etc., were excluded. Data obtained were analyzed with other clinicopathological parameters including age of patient, history of tobacco use, paan chewing, alcohol abuse, other relevant irritants, involvement of abnormal sexual habits, site of lesion, grade of tumor, and lymph node metastasis.
The tissue was fixed in buffered formalin (pH = 7.0), and embedded in paraffin. The tissue block was sectioned at 4–5 µm and the sections were stained for hematoxylin and eosin and examined. H istopathologic grading was done according to World Health Organization criteria based on three parameters (1) flattened polyhedral, round, or ovoid epithelial cells; (2) intracellular or extracellular keratinization; and (3) intercellular bridges.
Grade I: Well-differentiated
Grade II: Moderately differentiated
Grade III: Poorly-differentiated or anaplastic.
Immunohistochemical (IHC) profile of the tumor was assessed by subjecting one representative section from tumor block to p16 (BioGenex). Immunohistochemistry was performed on 4 µm thick sections from 10% formalin-fixed paraffin-embedded specimens, according to the streptoavidin-biotin immunoperoxidase technique. Positive and negative controls were run simultaneously. Strong brown nuclear immunoreactivity was considered as positive staining.
Interpretation of results
The IHC expression of p16 was classified according to nuclear and cytoplasmic positivity. The biopsies were scored as positive when more than 5% cells (cut-off) stain positive and was graded as negative (0–5% of nuclei and cytoplasm positive), sporadic (5–10% of nuclei and cytoplasm with weak and scattered positivity), focal (>–30% of labeled nuclei and cytoplasm strongly positive, spreading in one tissue area), and diffuse (>–85% of labeled cells with strong positivity, spreading in several tissue areas). Biopsies with diffuse pattern were considered to have high IHC expression of p16 (Grade III). Focal distribution was considered as moderate expression (Grade II) and sporadic positivity as low expression (Grade I).
A descriptive study was carried out for all the variables included in the study. The whole data was entered in Microsoft Excel master sheet and analyzed using Statistical Package for Social Sciences (SPSS) version 20 software. As the data was qualitative, Chi-square test was used to assess the association between these parameters. A value of P < 0.05 was taken as significant and <0.01 as highly significant; whereas, P > 0.05 was taken as nonsignificant.
The age of the patients ranged from 27 to 75 years with a mean of 54.3 ± 11.37 years. It was observed that maximum cases (26.67%) were in the age group 61–70 years. The youngest patient was 27-years-old and oldest was 75 years of age.
Out of total 75 cases, 65 (86.67%) of the patients were smokers and 46 (61.33%) patients had history of alcohol consumption. Twenty-eight percent cases had history of paan chewing and 10.67% of the HNSCC patients had history of abnormal sexual habits like practice of oral sex, while 67 (89.33%) cases did not provide any such history. It was observed that 22.67% of the HNSCC patients had multiple sexual partners, while 58 (77.33%) cases had no such history. It was analyzed that 14.67% of the HNSCC patients had lymph node metastasis.
Oropharynx was the most common site (80%) for occurrence of HNSCC, while in larynx and oral cavity incidence was 10.7 and 9.3%, respectively. World Health Organization (WHO) histological grading was done for all cases of HNSCC. Majority cases (82.67%) were Grade II [Figure 1] followed by seven (9.33%) cases of Grade III. Grade I tumors were least common constituting six (8%) cases. Rare type included one case of papillary SCC which was categorized under Grade I.
|Figure 1: Moderately-differentiated squamous cell carcinoma (Grade II) showing keratin pearls and invasion of tumor cells in surrounding tissue (a) (hematoxylin and eosin (H and E), ×100) and mild-to-marked nuclear pleomorphism in tumor cells with 5–10 mitotic figures/hpf (b) (H and E, ×200)|
Click here to view
It was observed that 59 (78.67%) cases were positive for p16 (inclusive of all grades), while 11 (21.33%) cases were negative. Out of 78.67% positive cases, 11 (14.67%) showed Grade I (low expression) [Figure 2], 22 (29.33%) showed Grade II (moderate expression) [Figure 2], and 26 (34.67%) showed Grade III (strong expression).
|Figure 2: Moderately-differentiated squamous cell carcinoma showing p16 nuclear and cytoplasmic positivity in 5–10% tumor cells (Grade I) (immunohistochemical (IHC), ×200) (a) and p16 nuclear and cytoplasmic positivity in 10–30% tumor cells (Grade II) (IHC, ×40) (b)|
Click here to view
[Table 1] illustrates correlation of p16 expression with various clinical parameters. Out of 26 cases with Grade III expression of p16, 16 (61.54%) cases belonged to age more than 50 years, 22 (84.62%) were males, and 21 (80.7%) occurred in oropharynx. No significant association was seen between p16 expression and age (P = 0.462), sex (P = 0.331), and site of tumor (P = 0.334).
[Table 2] illustrates correlation of p16 expression with various risk factors. Though majority of our patients were smokers and had history of alcohol consumption, p16 expression was not significantly associated with these risk factors (P = 0.346 and 0.308, respectively). However, amongst the nonsmokers and nonalcohol consumers, expression of p16 was significantly higher.
History of paan chewing was present in 21 (28%) cases in our study. However, p16 expression was significantly seen in these cases (P = 0.03). A significant correlation of p16 expression was also seen in cases with multiple sexual partners (P = 0.003).
[Table 3] illustrates correlation of p16 expression with histological grades of HNSCC. Out of 62 cases of Grade II (MDSCC), 25 (40.32%) cases showed strong p16 staining (Grade III), while only 11 (17.8%) cases were p16 negative. Out of 26 cases showing strong p16 staining (Grade III), maximum cases belonged to MDSCC category. A significant correlation was observed in p16 staining with histological grading (P = 0.045).
[Table 4] illustrates correlation of p16 expression with lymph node metastasis. Of the 64 cases who were node negative at the time of presentation showed different grade p16 expression in 45 (85.3%) cases. A significant correlation was observed in p16 staining with presence of lymph node metastasis (P = 0.03).
| > Discussion|| |
In the present study, Grade III p16 expression was observed in 26 cases (34.7%), Grade II in 22 cases (29.3%), and Grade I in 11 cases (14.7%); whereas, 16 cases (21%) were negative for p16 and scored Grade 0. No significant association was seen between p16 expression and age (P = 0.462) as well as sex distribution (P = 0.331). Majority of the cases included in the present study were smokers (86.67%), alcohol consumers (61.33%), and non-paan chewers (72%). Paan chewing which is considered a high risk factor for oral cancer, is less prevalent in Haryana state as compared to the neighboring states of Uttar Pradesh, Delhi, etc., The 75 cases of HNSCC analyzed for expression of p16 showed no statistically significant association between p16 expression and smoking (P = 0.346) and alcohol consumption (P = 0.308). However, statistically significant association between p16 expression and patients with history of paan chewing was observed (P = 0.03). Similar to our study, Lazarus et al., found no statistically significant association between p16 expression and tobacco use. In contrast, Smith et al., found a statistically significant association of p16 expression with alcohol consumption and tobacco use (P < 0.05).
Hypermethylation of p16 with loss of p16 expression has been associated with smoking; whereas, upregulation of p16 with overexpression is associated with active HPV. As most of our patients were smokers, loss of p16 expression is expected. Though statistically nonsignificant, still presence of p16 expression in 59 of 75 cases (78.6%) points towards an additional risk factor that is HPV. The variability and difference in expression in our study as compared to other studies may be due to geographic distribution and difference in prevalence of various risk factors.
Many studies on head and neck cancer have utilized p16 as a surrogate marker for HPV DNA.,,,, According to a study conducted by Herrero et al., in 2003, HPV DNA was detected more frequently in oral cancer biopsies of patients who reported more than one sexual partner or who practiced oral sex. Keeping in view the above studies, history of multiple sexual partners and practice of abnormal sexual activity was taken in our study. In the present study, history of abnormal sexual habits was obtained in eight (10.67%) cases and history of multiple sexual partners was obtained in 17 (22.67%) cases. Association of HPV with p16 expression was correlated with patient's history of abnormal sexual habits like practice of oral sex and with history of multiple sexual partners. A significant association was seen between p16 expression and history of multiple sexual partners (P = 0.003). According to studies conducted by Fregonesi et al., Smith et al., Klussmann et al., König et al., Singhi et al., and Pannone et al., p16 expression was strongly associated with HPV infected HNSCC.,,,,,
No statistically significant association of abnormal sexual practices like oral sex was observed. This can be attributed to social and cultural difference amongst the two societies or small number of patients providing the history of such practices as extracting such history from the patients in our society was not very easy as it is considered a social taboo.
Most HNSCC arise in the hypopharynx, larynx, oral cavity, and oropharynx. In the western world, HNSCC of the oral cavity and oropharynx are becoming more prevalent which may be related to an increase in oral cavity and oropharynx HPV infections. Seventy-five cases of HNSCC were categorized according to American Joint Committee on Cancer (AJCC) site grading. The commonest site of involvement in our study was oropharynx in 63 (84%) cases, with maximum cases from tonsils and base of tongue. Though oral cavity was the most common site of involvement in most of the studies, the difference in percentage could be because of different geographical location and prevalence of risk factors., The highest incidence of p16 positive tumors was seen in tonsils (53.9%). No significant association was seen between p16 and tumor site (P = 0.334), which was in concordance with the study of Smith et al., which also showedno statistical difference in p16 expression and tumor site (P = 0.4).
Histological grade is a means of quantitating the degree of differentiation by applying a set of histological criteria. Usually well-differentiated tumors are low grade lesions, whereas poorly-differentiated tumors are high grade neoplasms. Grade is a strong and independent factor associated with distant metastasis in head and neck carcinomas. Thus, it adds important information to clinical and pathologic staging. It helps to identify patients at high risk for distant metastasis for whom an efficient systemic treatment is mandatory. In our study, maximum number of cases belonged to Grade II (82.67%). Grade I and III tumors constituted 8% and 9.33% of cases, respectively. In the present study, p16 expression had a significant correlation with histological grade of the tumor (P = 0.045). While none of six well-differentiated tumors showed higher p16 expression (Grade III), the corresponding values for moderately- and poorly-differentiated tumors were 40 and 14%, respectively [Figure 3] and [Figure 4]. Our findings are in agreement with the study of Smith et al., (P = 0.02) and Muirhead et al., (P = 0.001), who also observed that p16 over expression was more likely to be detected with later stage and higher grade., They hypothesized that tumors that exhibited p16 expression had an effect on cell differentiation. The cells are probably arrested at a stage within the process of differentiation leading to tumor comprised predominantly of poorly-differentiated nonkeratinizing areas. These differences could also be attributed to difference in sample size, distribution of tumor site, different scoring criteria, and different type of antibodies used by different authors. However, no significant association between p16 expression and tumor grade was observed by Yuen et al., and Dragomir et al.,
|Figure 3: Well-differentiated squamous cell carcinoma showing keratinization in <75% tumor cells (a) (H and E, × 100) and weak p16 staining (Grade 0) (b) (IHC, × 100)|
Click here to view
|Figure 4: Poorly-differentiated squamous cell carcinoma showing keratinization in <25% tumor cells (a) (H and E, ×100) and diffuse p16 staining (Grade III) (b) (IHC, × 100)|
Click here to view
Lymph node involvement being an important prognostic variable was assessed in all cases. In the present study, lymph node metastasis was seen in 11 (14.67%) cases and 64 (85.33%) cases were node negative. Fifty-three of the 64 (82.8%) lymph node negative cases showed positive staining of p16 (P = 0.03). This is in accordance with study conducted by Smith et al., and study by Lingbao et al., (P < 0.0001) who opined that as p16 protein is an important cell cycle regulatory protein, the under expression/weak expression of this protein allows cancer cells to proliferate without control and over expression of p16 protein blocks cell proliferation at G1-S phase, and hence has impact on the stage of tumor including tumor size and nodal metastasis.,
However, Muirhead et al., and Yuen et al., did not observe any statistically significant association between p16 expression and lymph node metastasis status and concluded that expression of p16 significantly contribute to cell proliferation and tumor size, but it has no prognostic significance for nodal metastasis and survival.,
| > Conclusion|| |
p16 immunohistochemistry is advocated as a surrogate marker of HPV, based on the findings that HPV integration with transcription of viral oncoprotein induces over expression of p16. This approach is very practical and can be readily implemented in most diagnostic pathology laboratories. Over expression of p16 has been significantly seen in patients of HNSCC who did not have the history of paan chewing (a major risk factor in patients with HNSCC) and had the history of multiple sexual partners. Finding of high incidence of high risk HPV provide support for a vaccination program for risk group. Informed education of patients and their close contacts (like sexual partners and family members) will also require knowledge of the HPV status.
Significant expression of p16 in higher histological grade of tumor and in node negative patients may guide the type and intensity of the therapy in patients with HNSCC.
HPV assessment may play some role in early cancer detection, tumor localization, posttreatment surveillance, and informed consultation of patients and their partners in future. It is impressed upon that HPV status must be included as a stratification factor in future trials. Separate trials in HPV positive and HPV negative oropharyngeal carcinoma will be required to determine the optimal treatment for each of the distinct entities.
| > References|| |
Rodrigues VC, Moss SM, Tuomainen H. Oral cancer in the UK: To screen or not screen. Oral Oncol 1998;34:454-65.
Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108.
Elango JK, Gangadharan P, Sumithra S, Kuriakose MA. Trends of head and neck cancers in urban and rural India. Asian Pac J Cancer Prev 2006;7:108-12.
El-Mofty SK, Lu DW. Prevalence of human papillomavirus type 16 DNA in squamous cell carcinoma of the palatine tonsil, and not the oral cavity, in young patients: A distinct clinicopathologic and molecular disease entity. Am J Surg Pathol 2003;27:1463-70.
Hennessey PT, Westra WH, Califano JA. Human papillomavirus and head and neck squamous cell carcinoma: Recent evidence and clinical implications. J Dent Res 2009;88:300-6.
Herrero R, Castellangue X, Pawlita M, Lissowska J, Kee F, Balaram P, et al
. IARC Multicenter Oral Cancer Study Group. Human papillomavirus and oral cancer: The International agency for research on cancer multicenter study. J Natl Cancer Inst 2003;95:1772-83.
Syrajanen S. The role of human papillomavirus infection in head and neck cancers. Ann Oncol 2010;21:243-5.
Vairaktaris E, Yapijakis C, Psyrri A, Spyridonidou S, Yannopoulos A, Lazaris A, et al
. Loss of tumour suppressor p16 expression in initial stages of oral oncogenesis. Anticancer Res 2007;27:979-84.
Smeets SJ, van der Plas M, Schaaij-Visser TB, van Veen EA, van Meerloo J, Braakhuis BJ, et al
. Immortalisation of oral keratinocytes by functional inactivation of p53 and pRb pathways. Int J Cancer 2011;128:1596-605.
Strickler TP, Kumar V. Neoplasia. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cotran Pathologic Basis of Disease. 8th
ed. Saunders; 2011. p. 259-330.
Barnes L, Chiosea SI, Seethala RR. Squamous cell carcinoma-variants. Head and neck pathology. 3 ed. New York: Demos medical publishing LLC; 2011. p. 1-18.
Fregonesi PA, Teresa DB, Duarte RA, Neto CB, de Oliveira MR, Soares CP. p16INK4A
immunohistochemical overexpression in premalignant and malignant oral lesions infected with human papillomavirus. J Histochem Cytochem 2010;51:1291-7.
Lazarus P, Sheikh SN, Ren Q, Schantz SP, Sten JC, Richie JP, et al
. p53, but not p16 mutations in oral squamous cell carcinomas are associated with specific CYP1A1 and GSTM1 polymorphic genotypes and patient tobacco use. Carcinogenesis 1998;19:509-14.
Smith EM, Rubenstein LM, Hoffman H, Haugen TH, Turek LP. Human papillomavirus, p16 and p53 expression associated with survival of head and neck cancer. Infect Agent Cancer 2010;5:4.
Klussmann JP, Gultekin E, Weissenborn SJ, Wieland U, Dries V, Dienes H, et al
. Expression of p16 protein identifies a distinct entity of tonsillar carcinomas associated with human papillomavirus. Am J Pathol 2003;162:747-53.
König F, Krekeler G, Honig JF, Cordon-Cardo C, Fischer G, Korabiowska M. Relation between human papillomavirus positivity and p16 expression in head and neck carcinomas – A tissue microarray study. Anticancer Res 2007;27:283-8.
Singhi Ad, Westra WH. Comparison of human papillomavirus in situ
hybridization and p16 immunohistochemistry in the detection of human papillomavirus- associated head and neck cancer based on a prospective clinical experience. Cancer 2010;116:2166-73.
Pannone G, Rodolico V, Santoro A, Lo Muzio L, Franco R, Botti G, et al
. Evaluation of combined triple method to detect causative HPV in oral and oropharyngeal carcinomas: p16 immunohistochemistry, consensus PCR HPV-DNA, and in situ
hybridization. Infect Agent Cancer 2012;7:4-17.
Johnson N, Franceschi S, Ferley J, Ramadas K, Schmid S, MacDonald DG, et al
. Tumors of the oral cavity and oropharynx. In: Barnes L, Eveson JW, Reichart P, Sidransky D, editors. World Health Organization classification of tumors. Pathology and genetics of head and neck tumors. France: IARC Press; 2005. p. 168-75.
American Joint Committee On Cancer. Head and neck. In: Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A, editors. AJCC cancer staging manual. 7th
ed. New York: Springer; 2010. p. 33-126.
Ai Lingbao, Stephenson KK, Ling W, Zuo C, Mukunyadzi P, Suen JY, et al
. The p16 (CDKN2a/INK4a) tumor-suppressor gene in head and neck squamous cell carcinoma: A promoter methylation and protein expression study in 100 cases. Mod Pathol 2003;16:944-50.
Fortin A, Couture C, Doucet R, Albert M, Allard J, Tetu B. Does histologic grade have a role in the management of head and neck cancers? J Clin Oncol 2001;19:4107-10.
Muirhead DM, Hoffman HT, Robinson RA. Correlation of clinicopathological features with immunohistochemical expression of cell cycle regulatory proteins p16 and retinoblastoma: Distinct association with keratinisation and differentiation in oral cavity squamous cell carcinoma. J Clin Pathol 2006;59:711-5.
Yuen P, Man M, Lam KY, Kwong YL. Clinicopathological significance of p16 gene expression in the surgical treatment of head and neck squamous cell carcinomas. J Clin Pathol 2002;55:58-60.
Dragomir LP, Simionescu C, Margaritescu C, Stepan A, Dragomir IM, Popescu MR. p53, p16 and Ki-67 immunoexpression in oral squamous carcinomas. Rom J Morphol Embryol 2012;53:89-93.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]