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Year : 2021  |  Volume : 14  |  Issue : 1  |  Page : 26-30  

To correlate the immunohistochemical analysis of p16 and Ki-67 in oral squamous cell carcinoma with the background of oral submucous fibrosis

Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Vidyapeeth's, Dr. D.Y. Patil Dental College and Hospital, Pune, Maharashtra, India

Date of Submission17-Jan-2020
Date of Decision14-Aug-2020
Date of Acceptance14-Aug-2020
Date of Web Publication22-Jan-2021

Correspondence Address:
Roopa Yadahalli
Department of Oral Pathology and Microbiology, Dr. D.Y. Patil Vidyapeeth's, Dr. D.Y. Patil Dental College and Hospital, Pimpri, Pune - 411 018, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_18_20

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Aim: Oral submucous fibrosis (OSF) is a potentially malignant disorder with 7.6% of malignant transformation rate into oral squamous cell carcinoma (OSCC). The aim of the study is to assess the genetic markers p16 and ki-67 in OSF, OSCC, and OSCC with the background of OSF and control group. Materials and Methods: Ten cases of each group were stained with p16 and Ki-67 by immunohistochemistry technique. Results: Statistically significant results were found among the four groups with moderate-to-severe expression (P < 0.05) for Ki-67 and results were negative for p16 in all groups. Conclusion: According to our result, p16 is found not significant marker and Ki-67 can be useful as a proliferative index marker for malignancy development in oral precancerous conditions.

Keywords: Ki-67, oral squamous cell carcinoma in the background of oral submucous fibrosis, oral squamous cell carcinoma, oral submucous fibrosis, p16

How to cite this article:
Yadahalli R, Kheur S, Reddy M, Gupta A. To correlate the immunohistochemical analysis of p16 and Ki-67 in oral squamous cell carcinoma with the background of oral submucous fibrosis. Med J DY Patil Vidyapeeth 2021;14:26-30

How to cite this URL:
Yadahalli R, Kheur S, Reddy M, Gupta A. To correlate the immunohistochemical analysis of p16 and Ki-67 in oral squamous cell carcinoma with the background of oral submucous fibrosis. Med J DY Patil Vidyapeeth [serial online] 2021 [cited 2021 Feb 27];14:26-30. Available from: https://www.mjdrdypv.org/text.asp?2021/14/1/26/307664

  Introduction Top

Oral malignancy is one of the most common malignancies influencing Indian community. It is a multifactorial and complex entity associated with the sequential genetic alterations.[1] Habits of smoking and nonsmoking tobacco, chewing betel quid, alcohol intake, and oncogenic human papillomavirus (HPV) viruses are thought to be the main reasons of carcinogenesis in oral squamous cell carcinoma (OSCC).[2] Oral submucous fibrosis (OSF) is a precancerous condition with marked alteration of connective tissue fibers of the lamina propria and deeper parts. Around 7.6% of malignant transformation (MT) rate of OSF is noted over a 10-year period.[3]

Before the clinical and histopathological findings, changes at molecular level would occur.[4] The assembled genetic and epigenetic factors lead to modifications from normal oral epithelium to dysplastic and malignant condition.[5] In the OSF cases transforming to malignancy, an altered epithelial cells might keep hold of the genetic memory of earlier differentiation and maturation developing in better grade of tumor differentiation.[6] Biological molecular indicators have advocated to be of worth in the diagnostic and prognostic analysis of potentially malignant disorders.

The protein p16 is a cellular protein involved in cell cycle modulation. It is an inhibitor of cyclin-dependent kinase and initiates the cyclin D1 CKD4 and 6 complex, preventing phosphorylation of the retinoblastoma protein (pRb) and leading to cell cycle inhibition.[7],[8] The p16 protein in normal cells is expressed in very less extent and is almost undetectable by immunohistochemical staining (IHC). Due to the metamorphic action of E7 oncogene, p16 is strongly revealed by tumor cells affected by HPV and may be easily detected by IHC.[9] Immunohistochemically, p16 is a noted potential marker to determine dysplasia of oral cavity. Hence, p16 positivity correlates strongly with HPV positivity.[10] Ki-67 is a human nuclear protein connected with cell cycle seen in perichromosomal area commonly used in pathology as a proliferative marker to quantify the growth fraction of cells in human tumors.[11]

This study was conducted to investigate the presence of p16 protein and Ki-67 antigen in OSF, OSCC, and OSCC associated with OSF and to analyze the interrelation of the expression of these cell cycle regulative proteins and clinicopathological aspects. Analyzing these aforesaid biological markers and correlating it with clinical parameters would assist in the carcinogenesis pathway in OSCC involved with OSF. Recognition of risky oral premalignant disorders and intercession at early phase will establish keynote in bringing down the risk related to OSCC.

  Materials and Methods Top

Study cases included formalin-fixed, paraffin-embedded biopsy of OSF, OSCC, and OSF in the background of OSCC and normal tissue. Tissues were retrieved from the archives of the Department of Oral Pathology and Microbiology, Dr. D. Y. Patil Dental College, Pimpri, Pune. Ten paraffin blocks of each group were selected for the study. IHC technique was performed using the avidin–biotin–peroxidase protocol, according to Abrahao et al.[12] Permission for the study was obtained from the Institutional Ethical Committee.


The commercially available mouse monoclonal antibody for p16INK4a protein (BioGenex, USA) was used for the detection of p16 immunohistochemistry. IHC detection of Ki-67 was performed using AM297-5M Ki-67 antigen, proliferating cell, monoclonal (BGX-297), mouse, and IgG1 (BioGenex, USA) detection system. Positive control slides for p16 of breast carcinoma were taken to ascertain the validity of IHC kit and the accuracy of the technique. For negative control, the primary antibodies were omitted. The existence of brown color in cytoplasm and nucleus was suggestive of p16 positive and brown coloration of nucleus was indicative of Ki-67 positive immunoreactivity regardless of staining intensity.

For IHC staining, 3 μm thick sections was cut using rotatory manual microtome. Two consecutive sections were cut, one for immunohistochemistry and another stained with hematoxylin and eosin from each block. For IHC staining, precoated slides were used which were kept 30 min in hot air oven at 65°C, and deparaffinization for 10 minutes in xylene was done. Citrate buffer was used for antigen retrieval at 95°C for 5 min (4–5 times). The next exercises were conducted in a humid chamber after cooling down the slides. Further slides were washed with wash buffer (PBS, phosphate buffer solution), and then peroxide block (0.3% hydrogen peroxide in methanol) was added for 10 min. Then, it was followed by power block (supplied by the manufacturer) application for 10 min. Primary antibody (monoclonal mouse anti Ki-67 antibody [MIB-1] for Ki-67 and antihuman p16 protein [INK 4] for p16) was applied after power blocking and incubated for 30 min. Washing the slides and application of super enhancer (secondary antibody) for 30 min was done. Then, application of poly horse radish peroxidase enzyme polymer reagent for 30 min and washing in wash buffer of the slides was performed. Application of di-acetyl bromo acetic acid to the slide for 3–4 min and washing with buffer was performed. The slides were immersed in Mayor's hematoxylin for less than a minute and washed. Finally, the slides were dehydrated in ascending grades of alcohol and then to xylene and mounted with diphenylene phthalate xylene.

The slides were examined by three pathologists using a light microscope and the entire procedure was blinded to minimize the interobserver bias.

Evaluation of stained slides

The intenseness of IHC staining was graded upon subjective assessment of intensity of brown color presented by antigen, antibody, and chromogen complex as follows: negative (−no color), mild (+ light brown color), moderate (++ dark brown color), or intense (+++ very dark brown color). The allocation of staining was graded for Ki 67 as nuclear staining only with clear brown color as positive for basal layer of epithelial cells. The nuclear staining of all layers of epithelial cells regardless of staining intensity, were regarded for p16. The number of positive cells per 100 basal or parabasal cells was noted in percentage. The percentage of positive cells was scored as follows: 0 = 0%–5%; 2 = 6%–25%; 4 = 26%–60%; and 6 = 61%–99%. The area with maximum number of positive cells was considered in each section.[13] Positive and negative known IHC slides were used as controls.

The slides stained histopathologically and immunohistochemically were grouped into three groups such as OSCC associated with OSF group, OSCC not associated with OSF group, and OSF group. For the analysis of both p16 protein and Ki-67 expression, protocol followed was (1) distribution of expression pattern in the epithelial layers, (2) intensity of staining, and (3) the percentage of positive cells.

  Results Top

The groups investigated were of 10 OSCC, 10 OSCC in the background of OSF, 10 OSF, and 10 normal patients [Table 1].
Table 1: Expression of p16 and Ki-67 in the oral submucous fibrosis, oral submucous fibrosis with oral squamous cell carcinoma, and oral squamous cell carcinoma without oral submucous fibrosis

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P16 expression

In accordance with p16 expression, samples were categorized as negative, mild, moderate, and intense. All groups and normal group slides were negatively immunoreactive for p16. Of the OSF cases which were negatively expressed for p16, 5 cases were expressed less than 10% and other slides in between. In OSCC and OSCC in the background of OSF group, samples were all negatively expressed for p16, as immunoreaction seen was less than 10% of cells [Figure 1] and [Figure 2].
Figure 1: Negative expression of p16 in oral submucous fibrosis

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Figure 2: Negative expression of p16 in oral squamous cell carcinoma cases with the background of oral submucous fibrosis

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Ki-67 expression

In control groups, staining was mild to moderate and was limited to the basal cell layer, out of which 6 cases showed mild and 4 cases showed moderate staining. OSF cases showed only basal layer expression, with 3 cases mild staining and negative expression in 7 cases. All 10 cases of OSCC showed intense staining. The staining intensity was moderate in 3 cases and intense in 7 cases of OSCC in the background of OSF [Figure 3] and [Figure 4]. Statistically significant results were found between the normal with OSF, OSCC, and OSCC with OSF in expression with P < 0.05. Statistically highly significant (P = 0.000) results were found between OSF with OSCC and OSCC with OSF with P < 0.05. Similarly, highly significant (P = 0.000) results were found between OSCC and OSCC with OSF, P < 0.05.
Figure 3: Intense expression of Ki-67 in oral squamous cell carcinoma cases with the background of oral submucous fibrosis

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Figure 4: Mild expression of Ki-67 in oral squamous cell carcinoma cases with the background of oral submucous fibrosis

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  Discussion Top

Oral carcinogenesis being a multifactorial entity has peculiar genetic act, leading to malignancy of oral epithelium.[14] OSCC can be anticipated by potentially malignant conditions as OSF. For diagnostic and prognostic assessment of precancerous lesions, molecular biological markers have been implied to be of worth. Identifying the phenomenon significant to MT is extremely important. It can be proven advantageous to clinicians for evaluating the progressive risk of premalignant lesions against malignancy, and necessary precautionary measures can be applied.The studies have already proved that an oral carcinoma arising in the background to be of a better histological grade with lesser incidence of nodal metastasis.[6] Taking this in consideration, the current study was taken to assess the expression of p16 and Ki-67 in OSF, OSCC, and OSCC in the background of OSF.

The proliferation-related nuclear antigen is commonly analyzed by the proliferation condition of a cell or tissue with the expression of Ki-67 marker. It identifies proliferative-relevant nuclear antigen seen at all aspects of cell cycle other than G0 the resting phase.[15] In our study, each sample of control group exhibited positive staining with Ki-67 in basal cell layer of epithelium; this can be justified as the physiological proliferative feature of the basal cell layer. Three cases of OSF had mild parabasal staining as did OSCC. This pattern of parabasal staining has been reported to have a strong correlation with progression to cancer. The intensity of staining in OSF was more compared to normal but less than OSCC. Studies using various proliferation markers have shown the link with the proliferative index and tumor progression in oral mucosa.[16],[17],[18] In a recent study of expression of markers Ki 67, α SMA and CD105 alone or together, corresponded with the disease progression model of OSF and had a clinical outcome of OSCC in the background of OSF.[19]

In OSCC cases and OSCC with OSF, the staining was intense and statistical significant result was obtained. The intense staining might be because cells were less differentiated and were in more proliferating phase. This proliferative feature of tumor cells indicates the biological aggressiveness and is stated as a valuable prognostic marker. Ki 67 expresses not even the fact regarding the total fraction of proliferating cells, it also expresses regarding DNA synthesis and apoptotic activity.[20] With this study, it is suggested that the Ki-67 expression in OSCC is important and can be used to anticipate histologic grade of differentiation and prognostic condition of the lesion.

All the cell cycle stages are controlled by continuation of cyclin-dependent kinases (CDKs), initiated by their partner cyclins. The progression of cell cycle to the S phase by CDK4/CDK6 is advocated by cyclin D1. The cell progression from G1 to S phase is blocked by protein p16.[21] This p16 protein, a CDK inhibitor, inactivates cyclin D-CDK4/6 complexes following hamper of retinoblastoma protein (pRb).[22] Further transcriptional factors such as E2F are released by phosphorylation of pRb, which initiates an array of phenomenon that went to progress the cell into the S phase.[23] The release of E2F also activates p16 transcription. Therefore, inactivation of pRb by phosphorylation leads to p16 expression.[24] We found the negative expression of p16 protein in our study. Many of the previous studies also showed negative or minimum p16 immunoreactivity in head and neck cancer, OSCC cases, which is persistent with our study conclusion.[25],[26],[27]

Lots of variability in results is also seen in immunohistochemical studies of p16 in OSCCs and oral potentially malignant disorders. However, to understand the role of HPV in OSF studies is limited. Few studies based on polymerase chain reaction/DNA sequencing by Jalouli et al. have reported HPV positivity in 11/12 (91%) cases.[28] Chen et al. have reported the prevalence of HPV in 52.6% of OSF cases.[29] The study conducted by Sudhakaran et al. suggested that the percentage of p16 positive cells raised from normal to OSF to OSCC with and without OSF. In addition, a shift from nuclear to cytoplasmic expression from normal to OSCC was noted with a statistical significance (P < 0.001). They concluded that the presence of HPV using p16 was not detected in OSF but was positive in OSCC. Altered pattern of expression from normal to OSF to OSCC indicates promising use of p16 as a diagnostic marker.[30] Bazarsad et al. suggested that the p16 positivity could provide evidence for assessing high-risk OSF. Taken together, the proliferating activity of Ki-67 more than 8% and p16 expression of more than 5% can serve as promising biomarkers to assess the high risk of OSF.[31] Depending on the results, we obtained it prompted that p16 immunochemistry would not be an useful marker for disclosure of malignancy development.

  Conclusion Top

The presence of HPV was not detected in OSF and OSCC cases. OSF cases showed only basal layer expression and statistically significant results were seen between the normal with OSF, OSCC, and OSCC with OSF in Ki-67 expression. According to the results of the present study, it indicates that the Ki-67 expression in OSCC is of valuable and useful in evaluating histologic grade of differentiation and prognosis of the lesion.


I would like to acknowledge my departmental technician Mrs. Hemlatha Chougule for assisting in immunohistochemical staining of the slides.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Williams HK. Molecular pathogenesis of oral squamous carcinoma. Mol Pathol 2000;53:165-72.  Back to cited text no. 1
Azizi SA, Nik Mohd Abdul Nasser NF, Sailan AT, Ajura AJ, Ibrahim N. Expression of P53 and P16 at tumour invasive front in oral squamous cell carcinoma (OSCC). Cosmetol Oro Facial Surg 2016;2:205.  Back to cited text no. 2
Ray JG, Ranganathan K, Chattopadhyay A. Malignant transformation of oral submucous fibrosis: Overview of histopathological aspects. Oral Surg Oral Med Oral Pathol Oral Radiol 2016;122:200-9.  Back to cited text no. 3
Mehrotra R, Gupta A, Singh M, Ibrahim R. Application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions. Mol Cancer 2006;5:11.  Back to cited text no. 4
Mascolo M, Siano M, Ilardi G, Russo D, Merolla F, De Rosa G, et al. Epigenetic disregulation in oral cancer. Int J Mol Sci 2012;13:2331-53.  Back to cited text no. 5
Sarode SC, Sarode GS. Better grade of tumor differentiation of oral squamous cell carcinoma arising in background of oral submucous fibrosis. Med Hypotheses 2013;81:540-3.  Back to cited text no. 6
Gröbe A, Hanken H, Kluwe L, Schöllchen M, Tribius S, Pohlenz P, et al. Immunohistochemical analysis of p16 expression, HPV infection and its prognostic utility in oral squamous cell carcinoma. J Oral Pathol Med 2013;42:676-81.  Back to cited text no. 7
Rayess H, Wang MB, Srivatsan ES. Cellular senescence and tumor suppressor gene p16. Int J Cancer 2012;130:1715-25.  Back to cited text no. 8
Patil S, Rao RS, Amrutha N, Sanketh DS. Analysis of human papilloma virus in oral squamous cell carcinoma using p16: An immunohistochemical study. J Int Soc Prev Community Dent 2014;4:61-6.  Back to cited text no. 9
Kakei Y, Akashi M, Komatsubara H, Minamikawa T, Komori T. p16 overexpression in malignant and premalignant lesions of the oral and esophageal mucosa following allogeneic hematopoietic stem cell transplantation. Head Neck Oncol 2012;4:38.  Back to cited text no. 10
Raju K, Punnayanapalya S, Mariyappa N. Significance of p53, pRb and Ki-67 markers in Cervical intraepithelial lesion and Malignancy. Biomed Res Ther 2015;2:374-84.  Back to cited text no. 11
Abrahao KP, Quadros IM, Souza-Formigoni ML. Nucleus accumbens dopamine D1 receptors regulate the expression of ethanol-induced behavioural sensitization. Int J Neuropsychopharmacol 2011;14:175-85.  Back to cited text no. 12
Schlüter C, Duchrow M, Wohlenberg C, Becker MH, Key G, Flad HD, et al. The cell proliferation-associated antigen of antibody Ki-67: A very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J Cell Biol 1993;123:513-22.  Back to cited text no. 13
Rivera C, Venegas B. Histological and molecular aspects of oral squamous cell carcinoma (Review). Oncol Lett 2014;8:7-11.  Back to cited text no. 14
Kannan S, Chandran GJ, Pillai KR, Mathew B, Sujathan K, Nalinakumary KR, et al. Expression of p53 in leukoplakia and squamous cell carcinoma of the oral mucosa: Correlation with expression of Ki67. Clin Mol Pathol 1996;49:M170-5.  Back to cited text no. 15
Zoller J, Flentje M, Sinn P, Born IA. Evaluation of AgNOR and Ki67 antigen as cell kinetic parameters in oral dysplasias and carcinomas. Anal Cell Pathol 1994;7:77-88.  Back to cited text no. 16
Shin DM, Voravud N, Ro JY, Lee JS, Hong WK, Hittelman WN. Sequential increases in proliferating cell nuclear antigen expression in head and neck tumorigenesis: A potential biomarker. J Natl Cancer Inst 1993;85:971-8.  Back to cited text no. 17
Gonzalez-Moles MA, Ruiz-Avila I, Gil-Montoya JA, Esteban F, Bravo M. Analysis of Ki-67 expression in oral squamous cell carcinoma: Why Ki-67 is not a prognostic indicator. Oral Oncol 2010;46:525-30.  Back to cited text no. 18
Gadbail AR, Chaudhary MS, Sarode SC, Gondivkar SM, Belekar L, Mankar-Gadbail M. PKi67, CD105 and α-smooth muscle actin expression in disease progression model of oral submucous fibrosis. J Investig Clin Dent 2019;10:e12443.  Back to cited text no. 19
van Oijen MG, Medema RH, Slootweg PJ, Rijksen G. Positivity of the proliferation marker Ki-67 in noncycling cells. Am J Clin Pathol 1998;110:24-31.  Back to cited text no. 20
Uzawa N, Sonoda I, Myo K, Takahashi K, Miyamoto R, Amagasa T. Fluorecence in situ hybridization for detecting genomic alterations of cyclin D1 and p16 in oral squamous cell carcinomas. Cancer 2007;110:2230-9.  Back to cited text no. 21
Fregonesi PA, Teresa DB, Duarte RA, Neto CB, de Oliveira MR, Soares CP. p16 immunohistochamical overexpression in premalignant and malignant oral lesions infected with human papilloma virus. J Histochem Cytochem 2003;51:1291-7.  Back to cited text no. 22
Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 1993;366:704-7.  Back to cited text no. 23
Morgan DO. Principles of CDK regulation. Nature 1995;374:131-4.  Back to cited text no. 24
Ambrosch P, Schlott T, Hilmes D, Ruschenburg I. p16 alterations and retinoblastoma protein expression in squamous cell carcinoma and neighboring dysplasia from the upper aerodigestive tract. Virchows Arch 2001;438:343-9.  Back to cited text no. 25
Natarajan E, Saeb M, Crum CP, Woo SB, McKee PH, Rheinwald JG. Co-expression of p16(INK4A) and laminin 5 gamma2 by microinvasive and superficial squamous cell carcinomas in vivo and by migrating wound and senescent keratinocytes in culture. Am J Pathol 2003;163:477-91.  Back to cited text no. 26
Rocco JW, Sidransky D. p16(MTS-1/CDKN2/INK4a) in cancer progression. Exp Cell Res 2001;264:42-55.  Back to cited text no. 27
Jalouli J, Ibrahim SO, Mehrotra R, Jalouli MM, Sapkota D, Larsson PA, et al. Prevalence of viral (HPV, EBV, HSV) infections in oral submucous fibrosis and oral cancer from India. Acta Otolaryngol 2010;130:1306-11.  Back to cited text no. 28
Chen PC, Pan CC, Kuo C, Lin CP. Risk of oral nonmalignant lesions associated with human papillomavirus infection, betel quid chewing, and cigarette smoking in Taiwan: An integrated molecular and epidemiologic study. Arch Pathol Lab Med 2006;130:57-61.  Back to cited text no. 29
Sudhakaran A, Hallikeri K, Babu B. p16 as an independent marker for detection of high-risk HPV in oral submucous fibrosis and oral squamous cell carcinoma. Indian J Pathol Microbiol 2019;62:523-8.  Back to cited text no. 30
[PUBMED]  [Full text]  
Bazarsad S, Zhang X, Kim KY, Illeperuma R, Jayasinghe RD, Tilakaratne WM, et al. Identification of a combined biomarker for malignant transformation in oral submucous fibrosis. J Oral Pathol Med 2017;46:431-8.  Back to cited text no. 31


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

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