Indian Journal of Pathology and Microbiology

: 2010  |  Volume : 53  |  Issue : 1  |  Page : 7--11

Protein p 16INK4A expression in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of uterine cervix

Ruchi Gupta1, Radhika Srinivasan2, Raje Nijhawan2, Vanita Suri3, Radha Uppal2,  
1 Department of Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Obstetrics and Gynecology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Correspondence Address:
Radhika Srinivasan
Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012


The association of human papilloma virus (HPV) infection and cervical intraepithelial neoplasia (CIN) is well recognized. Interaction of HPV oncogenic proteins with cellular regulatory proteins leads to up regulation of p16 INK4A , a CDK inhibitor, which is a biomarker for HPV infection. We investigated p16 expression in CIN and invasive squamous cell carcinoma (SCC) which has not been reported in the Indian population previously. Materials and Methods: Retrospective analysis of 100 cases with 20 cases each of histologically normal cervical epithelium, CIN1, 2, 3 and invasive SCC for p16 expression was performed by immunohistochemistry using commercially available mouse monoclonal antibody to p16 (clone 6H12). Statistical Analysis: For differences in expression among groups, statistical analysis was carried out using ANOVA and post hoc test of Scheffe. Results: p16 immunoreactivity was found to be both nuclear and/or cytoplasmic. The normal cervical epithelium was predominantly negative for p16 (18/20). There was a progressive increase of p16 expression with the grade of CIN. In CIN 1, two cases (20%) showed nuclear and nucleocytoplasmic positivity respectively. In contrast, diffuse strong nuclear or nucleocytoplasmic expression was observed in 45 and 55% cases of CIN 2 and CIN 3 respectively. All except one squamous cell carcinoma stained strongly positive for p16. The difference in expression between CIN 2/3 and SCC versus normal cervix was found highly significant (p is equal to 0.008 and p less than 0.001). Conclusions: p16 expression correlates excellently with the grade of CIN and is a sensitive marker of cervical intraepithelial neoplasia.

How to cite this article:
Gupta R, Srinivasan R, Nijhawan R, Suri V, Uppal R. Protein p 16INK4A expression in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of uterine cervix.Indian J Pathol Microbiol 2010;53:7-11

How to cite this URL:
Gupta R, Srinivasan R, Nijhawan R, Suri V, Uppal R. Protein p 16INK4A expression in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of uterine cervix. Indian J Pathol Microbiol [serial online] 2010 [cited 2023 May 29 ];53:7-11
Available from:

Full Text


Carcinoma of the uterine cervix remains a major killer in women in the developing world including India. [1] According to recent data provided by the atlas of cancer in India project by the ICMR, cervical cancer is the second most common cancer in women after breast cancer in most urban population based registries in India. [2] The age-adjusted incidence rates for cervical cancer, in India, varied from 10.9 to 65.4 amongst various registries with an average incidence of around 25 per 100,000 women. [2]

The association of cervical cancer with high risk human papilloma virus (HPV) infection has long been established. However, in a substantial number of females, HPV produces transient infection which gets cleared off by host immune responses. [3] It has recently been shown that viral persistence is necessary to bring about a morphological phenotypic change in the squamous cell [4],[5] and subsequently, development of high grade cervical intraepithelial neoplasia. Genomic integration of these viral genes can disrupt several cellular proteins resulting in their up regulation. One of the consequences of this viral cellular protein interplay is the up regulation of a tumor suppressor gene p16 INK4A (also known as p16) which is a cyclin dependent kinase (CDK4) inhibitor. The protein p16 is integral to pRb (Retinoblastoma)- mediated control of the G1-S phase transition of the cell cycle; it puts a brake on the cell cycle by inactivating the CDKs that phosphorylate Rb protein. [6] A reciprocal relation between p16 INK4a and pRb expression has been observed. [7],[8] The E7 protein of HPV binds and inactivates Rb thereby causing release of E2F, a transcription factor, which in turn can activate genes required for entry into S-phase of the cell cycle. [7] The disruption of Rb by HPV E7 results in accumulation of the E2F and increased levels of p16 through negative feedback regulation. Accumulation of E2F has also been found to increase p16 transcription. [8] Thus, overexpression of p16 INK4a indicates already advanced interference of the viral oncoproteins with cellular proteins involved in cell cycle regulation.

The protein p16 INK4A serves as a surrogate marker for the oncogenic activities of HPV in replication-competent cells of cervical epithelia and its overexpression is well established in cervical intraepithelial neoplasia and invasive cancer by many studies. [9],[10],[11],[12] Till date, there are no reports from Indian literature, despite the fact that Indian females represent a major proportion of the affected population. The aim of this study was, therefore, to study p16 expression in cervical intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma (SCC) of the uterine cervix in our population.

 Material and Methods

This was a retrospective study of a total of 100 cases including 20 each of normal cervical histology, CIN1, CIN2, CIN3 and invasive squamous cell carcinoma diagnosed over a period of 2004-2006. Two sections were obtained on poly-L-lysine coated slides from the paraffin embedded tissue blocks. Antigen retrieval was carried out by the pressure cooker method, 0.01 M citrate solution at pH 6.0. Blocking reagent comprised of one part of hydrogen peroxide (diluted in 1:10 with distilled water) mixed with three parts of methanol. Procured from Novacastra, U.K., p16 mouse monoclonal antibody (clone 6H12), was applied at 1:20 dilution (diluted in Bovine serum albumin) on to the sections and incubated overnight at 4 o C. The secondary biotinylated rabbit antimouse antibody was applied at 1:100 dilution in bovine serum albumin for 30 minutes followed by Streptavidin conjugated with horse radish peroxidase (HRP) for another 30 minutes. Finally, the color was developed by treatment with diaminobenzidine (DAB) solution (Sigma, U.S.A.) and hydrogen peroxide. Sections were counter stained with hematoxylin and then dehydrated, cleared with xylene and mounted in DPX mountant. The final brown reaction product was evaluated by light microscopy. Cases of squamous cell carcinoma served as positive control slides. Positivity in the stromal fibroblasts served as an internal positive control. The negative control slides in each batch consisted of omission of the primary antibody and substitution by the buffer.

Evaluation for p16 Immunostaining

p16 immunoreactivity was evaluated taking into account the percentage positivity of tumor cells and staining intensity. Positivity was seen as a brown reaction product staining the nucleus or cytoplasm or both. Scoring of percentage positive tumor cells was carried out as following; 0% staining as negative, 0-5% as 1+, 5-25% as 2+ and over 25% as 3+. Intensity of immunostaining was taken as 1+, 2+ and 3+ depending upon the positivity. Immunohistochemistry score was obtained as a product of percentage positive tumor cells (0-3) and staining intensity score (0-3) thus achieving a maximum of 9.

Statistical Analysis

Data was analyzed using the statistical package SPSS version 10.0 for MS-Windows (SPSS Inc., Chicago, IL). The significance of p16 expression between and within the individual groups was analyzed using the Friedman ANOVA test and post hoc test of Scheffe respectively. Significance was assumed at a p value less than 0.05.


Both nuclear and/or cytoplasmic immunoreactivity was taken as positive for p16 immunostaining. Variable degree of p16 staining was seen in the cervical stromal fibroblasts which served as an internal control. There was a progressive increase in the percentage positivity as well as the staining intensity through increasing grades of cervical dysplasia and invasive cancer which is depicted in the panel in [Figure 1]. The expression of p16 in the various groups is summarized in [Table 1]. The normal cervical epithelium was essentially p16 negative [Figure 1]A except for two cases, one of which showed weak cytoplasmic immunoreactivity and the other showed nucleocytoplasmic reactivity. In CIN1, p16 expression was seen in 10 cases (50%) while in the rest it was negative [Figure 1]B. The positivity was focal and weak cytoplasmic in eight cases, nuclear in one [Figure 1]C and nucleocytoplasmic in another. The pattern of staining was seen to be basal one third in 50% cases and diffuse/full thickness in the other five cases. Interestingly, the two cases with nuclear positivity also showed a full thickness pattern of staining.

In contrast, majority of CIN 2 (n is equal to 9), CIN 3 (n is equal to 11) and SCC (n is equal to 18) showed a strong nuclear or nucleocytoplasmic positivity [Figure 1]D-F. All cases of CIN 2/3 showed full thickness immunoreactivity, except for three cases of CIN 2, in which positivity was seen in basal half of the squamous epithelium. In squamous cell carcinoma, all except one case showed strong diffuse nucleocytoplasmic p16 expression.

Statistical analysis: ANOVA was applied for comparison of the mean p16 immunoreactivity scores in each category and the results are depicted in the form of box plots in [Figure 2]. This was followed by the post hoc test of Scheffe, which was applied to ascertain the groups which showed the significant difference. It may be seen distinctly that p16 is markedly up regulated in the higher grades of intraepithelial neoplasia. As one progresses from CIN2/ CIN3 to squamous cell carcinoma the difference in p16 expression was statistically significant in comparison to normal cervix with p values being 0.021, 0.008 and 0.000 respectively. However, the increased expression in cases of CIN1 in comparison to normal epithelium was not found to be statistically significant (p is equal to 0.627).


Cervical cancer has a well recognized precancerous state, morphologically well recognized as cervical intraepithelial neoplasia (CIN), which is graded from CIN 1 to 3. Thus, the alterations of cellular proteins at different levels can be analyzed at every step of progression to cancer. It is now recognized that the higher grade CIN (2 and 3) is related to the persistence of HPV infection with its integration into the cellular genomic DNA. [4],[5] This results in disturbances in the expression of several cellular proteins, one of which is p16, an important cell cycle regulatory molecule. The up regulation of p16INK4A gene product has been shown to occur following HPV infection which has been very well documented in various studies in literature. Although there are several previous reports on the role of p16 in cervical carcinogenesis there is a paucity of them in Indian literature in spite of the fact that cervical cancer is one the most common cancers among females in India.

Our study is broadly in accordance with results of the previous publications; the major and relatively more recent studies [8],[11],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25],[26] have been compiled in [Table 2]. A minor difference was the p16 expression in 60-75% of CIN2/3 in our study as compared to 75-100% expression reported in most of the earlier studies. We have excluded technical reasons for negativity as many of these cases showed positive internal controls and further, these cases were immunostained at least twice with consistent results. The explanation for the same could be attributed to the use of a different clones of antibodies used in different studies which include 6H12 [27] (also used in the present study) and other JC8, [2],[8],[13] and E6H4. [17] The other possible explanation could be the difference in HPV types causing CIN. [29] p16 is considered a surrogate marker for high risk HPV infection according to several reports which have mainly examined HPV16 and 18 sub types. It is conjectural whether infection by other HPV types may result in similar p16 up regulation as with HPV 16 and 18 infections. In view of other HPV types prevalent in India, this aspect needs further studies. Moreover a substantial number of CIN 2 cases may be caused by low risk HPV types [29] making it a heterogeneous group. A third reason for p16 negativity in CIN 2/3 could be the epigenetic modification of the p16 gene promoter.

In a recent study by Kang et al. [10] 35.7% and 28% of CIN II and CIN III cases respectively were negative for p16 expression. This lack of immunoreactivity was correlated to promoter hypermethylation. They concluded that p16 gene silencing during CIN was not a rare event and also does not correspond with either HPV status or CIN grading. [10] Thus a proportion of high grades CIN are p16 negative as most recent reports indicate. [19],[23],[24],[26]

The immunoreactivity for p16 in squamous cell carcinoma is unequivocally shown by all studies till date and has been confirmed by this study as well. However, we had only one case which was negative for p16 protein, which is in concert with a study by Volgareva et al. [30] who showed that p16 INK4A -negative carcinomas did exist. The probable explanation could be p16 silencing through epigenetic mechanisms such as promoter methylation or through genetic mechanisms such as deletion or loss of heterozygosity. Tripathy et al. [31] from India, in a study on invasive cervical cancer, had shown p16 promoter hypermethylation and homozygous deletion in 6.5% and 8.7% samples respectively.

We observed nucleocytoplasmic strong p16 expression in a small proportion of CIN1. Some reports have suggested that p16 expression in CIN1 might have a correlation with viral persistence, and this approach may prove reliable to identify the low grade lesions with a tendency to progress to higher ones and thus allow appropriate and aggressive follow up and management in such patients. [16],[32],[33],[34],[35] However, with a limited follow-up period, it was not possible to derive any conclusions from p16 positivity in CIN1 lesions in the present study.

Thus p16 expression is a valuable marker for cervical intraepithelial neoplasia and should be incorporated into routine surgical pathology practice.


1Stoler MH. Human papillomavirus biology and cervical neoplasia: implications for diagnostic criteria and testing. Arch Pathol Lab Med 2003;127:935-9.
2Ferlay J, Bray F, Pisani P, DM. P. GLOBOCAN 2002 cancer incidence. Mortality and prevalence worldwide. In: IARC Cancer Base: Lyon IARC Press Cancer Base No. 5 version 2.0;2004.
3Ho GY, Burk RD, Klein S, Kadish AS, Chang CJ, Palan P, et al. Persistent genital human papillomavirus infection as a risk factor for persistent cervical dysplasia. J Natl Cancer Inst 1995;87:1365-71.
4Winer RL, Kiviat NB, Hughes JP, Adam DE, Lee SK, Kuypers JM, et al. Development and duration of human papillomavirus lesions, after initial infection. J Infect Dis 2005;191:731-8.
5Castle PE, Ashfaq R, Ansari F, Muller CY. Immunohistochemical evaluation of heat shock proteins in normal and preinvasive lesions of the cervix. Cancer Lett 2005;229:245-52.
6Tam SW, Shay JW, Pagano M. Differential expression and cell cycle regulation of the cyclin-dependent kinase 4 inhibitor p16Ink4. Cancer Res 1994;54:5816-20.
7Münger K, Howley PM. Human papillomavirus immortalization and transformation functions. Virus Res 2002;89:213-28.
8Tringler B, Gup CJ, Singh M, Groshong S, Shroyer AL, Heinz DE, et al. Evaluation of p16INK4a and pRb expression in cervical squamous and glandular neoplasia. Hum Pathol 2004;35:689-96.
9Masumoto N, Fujii T, Ishikawa M, Saito M, Iwata T, Fukuchi T, et al. P16 overexpression and human papillomavirus infection in small cell carcinoma of the uterine cervix. Hum Pathol 2003;34:778-83.
10Klaes R, Friedrich T, Spitkovsky D, Ridder R, Rudy W, Petry U, et al. Overexpression of p16 (INK4A) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri. Int J Cancer 2001;92:276-84.
11Kang S, Kim J, Kim HB, Shim JW, Nam E, Kim SH, et al. Methylation of p16 INK4a is a non-rare event in cervical intraepithelial neoplasia. Diagn Mol Pathol 2006;15:74-82.
12Kalof AN, Cooper K. p16INK4a immunoexpression: surrogate marker of high-risk HPV and high-grade cervical intraepithelial neoplasia. Adv Anat Pathol 2006;13:190-4.
13Ishikawa M, Fujii T, Saito M, Nindl I, Ono A, Kubushiro K, et al. Overexpression of p16 INK4a as an indicator for human papillomavirus oncogenic activity in cervical squamous neoplasia. Int J Gynecol Cancer 2006;16:347-53.
14Kong CS, Balzer BL, Troxell ML, Patterson BK, Longacre TA. p16INK4A immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia. Am J Surg Pathol 2007;31:33-43.
15Yildiz IZ, Usubütün A, Firat P, Ayhan A, Küçükali T. Efficiency of immunohistochemical p16 expression and HPV typing in cervical squamous intraepithelial lesion grading and review of the p16 literature. Pathol Res Pract 2007;203: 445-9.
16Focchi GR, Silva ID, Nogueira-de-Souza NC, Dobo C, Oshima CT, Stavale JN. Immunohistochemical expression of p16 (INK4A) in normal uterine cervix, nonneoplastic epithelial lesions, and low-grade squamous intraepithelial lesions. J Low Genit Tract Dis 2007;11:98-104.
17Benevolo M, Mottolese M, Marandino F, Vocaturo G, Sindico R, Piperno G, et al. Immunohistochemical expression of p16 (INK4a) is predictive of HR-HPV infection in cervical low-grade lesions. Mod Pathol 2006;19:384-91.
18Kalof AN, Evans MF, Simmons-Arnold L, Beatty BG, Cooper K. p16INK4A immunoexpression and HPV in situ hybridization signal patterns: potential markers of high-grade cervical intraepithelial neoplasia. Am J Surg Pathol 2005;29:674-9.
19Lorenzato M, Caudroy S, Bronner C, Evrard G, Simon M, Durlach A, et al. Cell cycle and/or proliferation markers: what is the best method to discriminate cervical high-grade lesions. Hum Pathol 2005;36:1101-7.
20Hu L, Guo M, He Z, Thornton J, McDaniel LS, Hughson MD. Human papillomavirus genotyping and p16 INK4a expression in cervical intraepithelial neoplasia of adolescents. Mod Pathol 2005;18:267-73.
21Wang JL, Zheng BY, Li XD, Angström T, Lindström MS, Wallin KL. Predictive significance of the alterations of p16 INK4A , p14ARF, p53, and proliferating cell nuclear antigen expression in the progression of cervical cancer. Clin Cancer Res 2004;10:2407-14.
22Murphy N, Heffron CC, King B, Ganuguapati UG, Ring M, McGuinness E, et al. p16 INK4A positivity in benign, premalignant and malignant cervical glandular lesions: a potential diagnostic problem. Virchows Arch 2004;445:610-5.
23Branca M, Ciotti M, Santini D, Di Bonito L, Giorgi C, Benedetto A, et al. p16 (INK4A) expression is related to grade of cin and high-risk human papillomavirus but does not predict virus clearance after conization or disease outcome. Int J Gynecol Pathol 2004;23:354-65.
24Agoff SN, Lin P, Morihara J, Mao C, Kiviat NB, Koutsky LA. p16(INK4a) expression correlates with degree of cervical neoplasia: a comparison with Ki-67 expression and detection of high-risk HPV types. Mod Pathol 2003;16:665-73.
25Klaes R, Benner A, Friedrich T, Ridder R, Herrington S, Jenkins D, et al. p16INK4a immunohistochemistry improves interobserver agreement in the diagnosis of cervical intraepithelial neoplasia. Am J Surg Pathol 2002;26:1389-99.
26Keating JT, Ince T, Crum CP. Surrogate biomarkers of HPV infection in cervical neoplasia screening and diagnosis. Adv Anat Pathol 2001;8:83-92.
27Guo M, Hu L, Baliga M, He Z, Hughson MD. The predictive value of p16 (INK4a) and hybrid capture 2 human papillomavirus testing for high-grade cervical intraepithelial neoplasia. Am J Clin Pathol 2004;122:894-901.
28Lambert AP, Anschau F, Schmitt VM. p16INK4A expression in cervical premalignant and malignant lesions. Exp Mol Pathol 2006;80:192-6.
29Snijders PJ, Steenbergen RD, Heideman DA, Meijer CJ. HPV-mediated cervical carcinogenesis: concepts and clinical implications. J Pathol 2006;208:152-64.
30Volgareva G, Zavalishina L, Andreeva Y, Frank G, Krutikova E, Golovina D, et al. Protein p16 as a marker of dysplastic and neoplastic alterations in cervical epithelial cells. BMC Cancer 2004;4:58.
31Tripathi A, Banerjee S, Roy A, Roychowdhury S, Panda CK. Alterations of the P16 gene in uterine cervical carcinoma from Indian patients. Int J Gynecol Cancer 2003;13:472-9.
32Negri G, Vittadello F, Romano F, Kasal A, Rivasi F, Girlando S, et al. p16 INK4a expression and progression risk of low-grade intraepithelial neoplasia of the cervix uteri. Virchows Arch 2004;445:616-20.
33Guimarães MC, Gonçalves MA, Soares CP, Bettini JS, Duarte RA, Soares EG. Immunohistochemical expression of p16INK4a and bcl-2 according to HPV type and to the progression of cervical squamous intraepithelial lesions. J Histochem Cytochem 2005;53:509-16.
34von Knebel Doeberitz M. Aspects of molecular pathogenesis of cervical cancer in establishing new tumor markers for early detection and diagnosis. Zentralbl Gynakol 2001;123:186-91.
35Sano T, Oyama T, Kashiwabara K, Fukuda T, Nakajima T. Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions. Am J Pathol 1998;153:1741-8.