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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 3  |  Page : 518-524
P16INK4A and MIB-1: An immunohistochemical expression in preneoplasia and neoplasia of the cervix


Department of Pathology, King George Medical University, K. G. M. U, Lucknow, India

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Date of Web Publication22-Oct-2010
 

   Abstract 

Aim: To evaluate the potential of p16INK4A and MIB-1 and to compare the expression and interrelationship of these markers in cervical preneoplasias and neoplasias. Materials and Methods: Immunohistochemical analysis of p16 and MIB-1 was performed in n = 63 tissue sections and by matching the corresponding Papanicolaou smears. Staining intensity for p16 was determined using the 0-3 grading system. For MIB-1, labelling indices (LI) were calculated and grading was performed using the I-III scoring system. Results: No positive staining of p16 was observed in the normal cervical epithelium. With increasing severity of cervical intraepithelial neoplasias (CIN), the p16 expression increased progressively. Significant up-regulation of p16 was observed in carcinoma cervix. MIB-1 LI was observed to increase with increasing grades of CIN, and significant overexpression of MIB-1 was observed in carcinoma cervix. Correlation between grades of p16 and that of MIB-1 among cervical neoplasias showed an increasing p16 expression with consistently increasing MIB-1 LI in the groups of increasing severity. Conclusion: This pattern of overexpression of p16 and MIB-1 demonstrate their use as a diagnostic marker for cervical neoplastic lesion. Therefore, p16 and MIB-1 markers in tissue sections can be used as an adjunct to definitively diagnose preneoplastic and neoplastic lesions in the cervix.

Keywords: P16INK4A, MIB-1, immunohistochemistry, cervix carcinoma, Papanicolaou smear

How to cite this article:
Srivastava S. P16INK4A and MIB-1: An immunohistochemical expression in preneoplasia and neoplasia of the cervix. Indian J Pathol Microbiol 2010;53:518-24

How to cite this URL:
Srivastava S. P16INK4A and MIB-1: An immunohistochemical expression in preneoplasia and neoplasia of the cervix. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Jul 18];53:518-24. Available from: http://www.ijpmonline.org/text.asp?2010/53/3/518/68301



   Introduction Top


Cervical cancers are the second most frequent type of female cancer, responsible for about 5% of cancer deaths in females' worldwide. [1] In India, cervical cancer ranks the first most frequent cancer among females between 15 and 44 years of age. Current estimates indicate that every year 132,082 women are diagnosed with cervical cancer and that 74,118 die from this disease in India alone. [1] Having said this, however, no form of cancer better documents the remarkable effects of prevention, early diagnosis and curative therapy on the mortality rate than does cancer cervix. The potential threat of cancer however is central to Papanicolaou (Pap) smear screening programmes and histologic interpretation of biopsy specimen by the pathologist, which has significantly reduced the mortality of cervical cancers. However, the Pap test is not very accurate due to subjective test criteria. This limits the present screening programmes and emphasizes the need for the identification of specific biomarkers for dysplastic epithelial cells to aid in primary screening and lesion diagnosis.

The etiologic role of Human Papilloma Virus (HPV) in cervical carcinoma is well known. HPV is known to be a major causative agent in cervical neoplasia and invasive cervical carcinoma. [2] Many different HPV types associated with cervical neoplasia have been discovered and they have been divided into high- and low-risk categories based on their association with invasive cervical carcinoma. [3] Central to the carcinogenesis of cervix is the role of HPV, esp. types 16 and 18. Experimental data indicate that viral E6 and E7 genes of high-risk HPV E7 protein specifically bind to and inactivate pRB (retinoblastoma gene product).

P16INK4A

P16INK4A (inhibitor of kinase 4A, from now on referred to as p16), is a tumor suppressor protein and inhibitor of cyclin-dependant kinase 4 and 6. The phosphorylation of pRB (retinoblastoma protein) is a molecular "ON-OFF" switch for the cell cycle. In the hypophosphorylated form, pRB binds to transcription factors responsible for cell cycle progression. p16 inhibits the cyclin-dependant kinases and thereby prevents the phosphorylation of RB, keeping it in the hypophosphorylated form, i.e. its active form. However, in HPV infection, the viral gene E7 binds to RB protein and functionally inactivates it. This results in accumulation of p16 protein because, normally, RB inhibits the transcription of p16. [4],[5],[6] Because this protein is not expressed in the normal cervical epithelium, p16 overexpression will allow to specifically identify dysplastic lesions and will reduce interobserver disagreement of conventional histological or cytological tests.

MIB-1

MIB-1 (Molecular Immunology Borstel) is a proliferative marker. It 1990, it was demonstrated that the MIB-1 antibody detects Ki-67 antigen in the G 1 , S, G 2 and M phase, but it is absent in the G 0 phase. [7] Therefore, this antibody may be a useful marker of proliferation in dysplastic lesions, particularly in cervical smears, and, in addition, can be of prognostic value. [8] VonHoven [9] in 1996 suggested it as a sensitive biological indicator of progression in cervical intraepithelial neoplasias (CIN) lesions. However,cervical cancer screening remains a labor-intensive complex method, the outcome of which depends on human judgment. [10] The failure of Pap test to eradicate this potentially preventable disease emphasizes the need for the identification of specific biomarkers for dysplastic epithelial cells of the cervix. The use of specific biomarkers of dysplasia in conjunction with the current cytological or histological procedures could greatly improve the accuracy, precision and sensitivity of cervical screening programmes. The present study is, therefore, aimed at p-16 and MIB-1 expression in tissue sections and cervical smears and of CINs and carcinoma to assess whether these markers could be used as a diagnostic adjunct to cervical  Pap smear More Detailss.


   Materials and Methods Top


Case Selection

Histopathological tissues (n = 63) were taken from females, age ranging from 20 to 70 years, presenting with cervicovaginal discharge and unhealthy cervix during the period of 2005-2006. Tissue sections with inadequate study material and with extensive necrosis and hemorrhage were excluded from the study. Additional Papanicolaou smears were obtained from these patients wherever possible. Pap smears with infectious etiology were not included in the study. Informed formal consent was taken from each patient and the study was approved by the hospital medical ethics committee. Histopathological examination of biopsies for grading and typing of lesions was performed in the Postgraduate Department of Pathology, and these were divided into Cervical intraepithelial neoplasia (CIN) I, II, III and carcinoma cervix according to the FIGO classification. [11] Cytological examination of Papanicolaou smears for grading and typing of lesions was carried out and these were divided into low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL) and carcinoma cervix according to the revised Bethesda classification. [12]

Immunohistochemistry

In brief, immunohistochemistry was performed on 63 tissue biopsies and 38 Pap smears. Matched Pap smears with tissue biopsies were obtained wherever possible. Four-micrometer-thin sections were cut and placed on slides. Sections were then deparaffinized and rehydrated through graded alcohols followed by distilled water. Antigen retrieval was carried out at 120΀C in citrate buffer (pH = 6.0) for 15 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Sections were then treated with blocking antibody provided in the kit. P16 immunostaining was performed using the mouse monoclonal antibody RTU-p16-432 (clone6H12) (Novacastra Lab., Newcastle, UK). MIB-1 immunostaining was performed using the mouse antihuman clone Ki-67, MIB-1 antibody (Dako Corporation, Glostrup, Germany), USA). The above-mentioned primary antibodies were applied and the sections were incubated overnight at 4΀C. Link antibody was applied for 90 min followed by enzyme conjugate (streptavidin horse radish peroxidise) for 45 min. Color development was accomplished with concentrated Diaminobenzidine solution diluted to 50-times and the sections were counterstained with 10% hematoxylin. A histological section and smear of squamous cell carcinoma cervix was used as positive control in each batch of staining. For negative control, 1% nonimmune serum was used instead of primary antibody. For immunocytochemistry, Pap smears were destained and treated similarly, except for the step of deparaffinization.

Interpretation

For p16, immunopositivity was considered when there was diffuse, strong, nuclear and/or cytoplasmic staining. Heterogeneous or focal moderate nuclear staining was also considered positive. Weak cytoplasmic staining was considered negative. Grading was then performed for each case by the number of positive cells in different epithelial clusters as Grade 0, 1, 2 and 3, based on the number of positive cells, 1-10%, 10-50% >50%, respectively. [13]

For MIB-1, immunopositivity was considered when there was strong nuclear staining. Because basal staining is a normal finding, the slides were first assessed for basal staining (lower one-third versus suprabasal). Staining in the upper two-third of the epithelium was considered positive. Labelling indices (LI) were calculated for each case by evaluating the percentage positive nuclei and the cases were divided into three groups, I, II and III, according to 0-10%, 10-20% and >20% positive nuclei. [8]

Statistical Analysis

Data analysis was carried out by STATA 9.2 Statistical Software Package. The means among more than two groups were compared using the Kruskall Wallis test. This was followed by the Mann-Whitney test to test the level of significance between the groups.


   Results Top


p16 immunohistochemistry revealed that all normal cervical tissue containing epithelial, metaplastic, endocervical, reactive and inflammatory regions were not stained with p16 antibody. All cervical intraepithelial neoplasias and carcinoma cervix showed both nuclear and cytoplasmic staining and focal staining of nuclei or cytoplasm [Table 1] and [Table 2] [Figure 1]. As we moved from the normal to the invasive carcinome cervix group, the percentage of tumor cells showed progressive increase in positive staining [Table 3]. MIB-1 immunohistochemistry revealed that MIB-1 expression was confined to the basal layer in the normal cervical epithelium. All CIN and carcinoma showed nuclear staining [Figure 2]. As we moved from the normal to the carcinoma group, the LI increased with increasing severity of intraepithelial neoplasias to the carcinoma group [Table 4]. [Table 5] and [Table 6] show the statistical analysis by two-sample Wilcoxon rank-sum (Mann-Whitney test) in tissue biopsy and cervical smears. A difference between two values was considered significant if P<0.05.
Table 1: Overview of the results of immunohistochemistry and immunocytochemistry of p16 and MIB-1

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Table 2: Immunohistochemistry and immunocytochemistry of individual cases of P16INK4A and MIB-1

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Figure 1: (a– c) p16 immunostaining in cervix ti ssue biopsy in cervical intraepithelial neoplasias (CIN) I, CIN II and carcinoma cervix. (d) p16 immunostaining in the HSIL cervical smear (IHC, ×400)

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Table 3: Results of immunocytochemical analysis of p16

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Figure 2: The top panel shows MIB-1 nuclear immunostaining in cervix tissue biopsy in cervical intraepithelial neoplasias (CIN) I, CIN II and carcinoma cervix. The lower panel shows MIB-1 immunostaining in cervical smears in LSIL, HSIL and carcinoma cervix (IHC, ×400)

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Table 4: Results of immunocytochemical analysis of MIB-1

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Table 5: Statistical analysis by two sample Wilcoxon rank-sum (Mann-Whitney Test) in tissue biopsy

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Table 6: Statistical analysis by two sample Wilcoxon rank-sum (Mann-Whitney Test) in Pap smears

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   Discussion Top


Wide arrays of potential biomarkers have been evaluated for the diagnostic usefulness of cervical cancer and its precursors. Two markers that have shown a potential in this direction are p16 and MIB-1. p16 is a tumor-suppressor protein that is expressed in dysplastic cervical epithelial cells only, while MIB-1 is a marker of active dividing cells, normally not shed in cervical smears. Therefore, presence of p16 and MIB-1 positivity in cervical Pap smear is a marker of cervical dyskaryosis. In a tropical country like India, any perimenopausal women presenting in the gynecological outpatient department with any complaint is subjected to a single Pap smear test. However, single Pap test is subject to suboptimal sensitivity, limited reproducibility and, many a times, is with a high rate of false-positive and false-negative results along with equivocal results. To compensate for the aforementioned deficiencies, a screening programme with repeated testing and follow-up of positive cases is warranted. Moreover, colposcopic biopsy is directed in any suspicious-appearing acetowhite area. This subjects the patient to unnecessary surgical intervention. Therefore, additional diagnostic and prognostic markers for the detection of cervical cancers and precursors are required, which could save the patients from surgical intervention and the high screening cost associated with repeated testing. This study was therefore conducted to ascertain these two biomarkers as a diagnostic adjunct.

p16 INK4A immunohistochemistry revealed that there was a significant overexpression and up-regulation in different groups as we moved from normal cervical epithelia to dysplasia of varying severity to carcinoma. This was found to be a statistically significant finding on making a comparison between control versus different groups (P < 0.05). However, on making an intergroup comparison, this was found to be statistically insignificant (P > 0.05). We found no detectable p16 positivity in normal cases of cervical epithelia. Cytoplasmic staining was observed in two cases of LSIL .

Our findings are similar to those of Klaes et al. (2001), who observed overexpression of p16 INK4A in all CIN I lesions (n = 47), all CIN II lesions (n = 32), all CIN III lesions (n = 60) and 56 of 58 invasive Squamous cell carcinoma (SCC). On the contrary, no detectable expression of p16 was observed in the normal cervical epithelium, inflammatory lesions and CIN I lesions associated with a low risk of HPV types. [5] Similarly, in the present study, p16 INK4A overexpression was seen in all CIN I lesions (15/15), all CIN II lesions (15/15), all CIN III lesions (3/3) and all cases of carcinoma cervix (15/15) of tissue biopsies. In Pap smears, p16 positivity was seen in CIN I/LSIL (8/10), CIN II/HSIL (5/5), CIN III/HSIL (3/3) and carcinoma cervix (15/15). No detectable p16 expression was observed in normal cervical epithelium in both Pap smears and tissue biopsies. p16 is a negative regulator of normal proliferation, working through a negative feedback loop to down-regulate CDK4. This function is bypassed by HPV-7, causing p16 up-regulation in proliferating cells. The detection of elevated levels of p16 is a clear indicator of abnormal proliferation. It can thus optimize the interobserver agreement in the diagnosis of cervical intraepithelial neoplasm. In our study, we observed diffuse staining, both nuclear and cytoplasmic, in all cases, with the exception of two cases where only cytoplasmic staining was observed. p16 is basically a nuclear protein and hence immunohistochemistry should show nuclear staining. However, in dysplasia, both nuclear and cytoplasmic staining with p16 is observed possibly because of posttranscriptional modification or overproduction of p16 protein, forcing its transfer into the cytoplasm. [13] In two cases of CIN I/LSIL, only cytoplasmic staining was seen. Similar findings have been reported by Volgerava in his study of cervical cancer cell lines. [14] No valid explanation for this observation could be found in the literature. Two Pap smears with LSIL showed negative p16 staining, whereas it was positive in the corresponding CIN lesion of the tissue section. p16 may rarely be negative in cervical dyskaryosis, which may have important implications for the use of p16 staining as a stand-alone test and support the use of a combination of markers of cervical dyskaryosis. [15] However, in our study, we did not find any dysplasia negative for p16 in tissues biopsies. p16 staining in LSIL was found to be negative in 20% of the Pap smears, which could possibly be due to the technical error as their corresponding sections showed consistent positivity.

In our study, it was seen that p16 overexpression was restricted to CIN I, II, III and carcinoma cervix. No positive staining was observed in the adjacent normal cervical epithelia. The p16 INK 4A-positive samples increased in the following order: CIN I, CIN II, CIN III and carcinoma cervix. Therefore, p16 immunostaining allowed precise identification of even small CIN or cervical cancer lesions in biopsy sections and Pap smears and helped to reduce interobserver variation and also reduce false-positive and false-negative interpretation and, thereby, significantly improve the cervical cancer and precancer detection.

MIB-I immunohistochemistry revealed that there was a significant overexpression of MIB-1 in different groups and as we move from normal cervical epithelia to varying severity of CINs to carcinoma, with increase in the MIB-1 positivity. This was found to be a statistically significant finding on making a comparison between control versus different groups (P < 0.05). However, on making an intergroup comparison, this was found to be statistically insignificant (P > 0.05). MIB-1 antibody detects Ki-67 antigen in G 1 , S, G 2 and M phases, but it is absent in the G0 phase. Therefore, this antibody may be a useful marker of the proliferative activity of premalignant and malignant lesions of the cervix.

MIB-I positivity was seen in 14 of 15 cases of CIN I, 15 of 15 cases of CIN II, 3 of 3 cases of CIN III and 15 of 15 cases of carcinoma cervix. As we moved from the normal to the carcinoma group via the varying degrees of CIN, the LI of positively stained nuclei increased with the severity of CIN to carcinoma group. Mravunac et al., in their study on MIB-1 immunostaining in microbiopsy specimens from inconclusive or unsatisfactory cervical smears, observed that high MIB-1 index LI was indicative of high-grade lesion, but low MIB-1 index did not rule out high-grade lesion. The microbiopsy specimens were thick tissue fragments removed from cytological smears and processed for histological sections. [16] We did not observe any HSIL lesion with a low proliferative index in our study. The aforementioned studies, although performed in a different settings than ours, have drawn more or less similar inference regarding the use of MIB-1 in conjunction with routine Pap staining, and lend support to our observation that MIB-1 immunostaining can be a diagnostic adjunct to cervical Pap smears.

Correlation between grades of p16 and MIB-1 among cervical neoplasia showed an increasing p16 expression with consistently increasing MIB-1 LI in the groups of increasing severity of cervical neoplasia.

To conclude, we recommend that for LSIL, because the sensitivity of the p16 marker is 80%, the marker should be evaluated together with MIB-1 or HPV test. For HSIL, the sensitivity and specificity of the p16 marker is 100% and thus it can be used as a stand-alone test.

We also recommend that with a careful interpretation of immunostaining with morphological characteristic in the conventional Pap smears, the immunostaining with p16 and MIB-1 markers may be a diagnostic adjunct, reducing the need of tissue biopsy. This is simple, reliable and easily applicable in routine cytosmears.

The following conclusions were drawn from the above study:

  • p16 and MIB-1 expression were observed in CIN I/LSIL, CIN II/HSIL and CIN III/HSIL
  • Significant up-regulation of p16 and MIB-1 was observed in carcinoma cervix and, with increasing severity of CINs/SILs, the p16 expression increased progressively
  • No positive staining of p16 was observed in normal cervical epithelium
  • Correlation between grades of p16 and that of MIB-1 among cervical neoplasia showed an increasing p16 expression with consistently increasing MIB-1 LI in the groups of increasing severity

   Acknowledgment Top


The authors would like to acknowledge the help of the technical staff of the Department of Pathology, KGMU, Lucknow, India.

 
   References Top

1.WHO/ICO Information Centre on HPV and Cervical Cancer. Available from: http://www.who.int/hpvcentre . [last cited on 2009 May 5].   Back to cited text no. 1      
2.Agoff SN, Lin P, Morihara J, Mao C, Kiviat NB, Koutsky LA, et al. p16 expression correlates with degree of cervical neoplasia: A comparison of Ki67 expression and detection of high risk HPV types. Mod Path 2003;16:665-73.   Back to cited text no. 2      
3.Lorincz AT, Reid R, Jenson AB, Greenberg MD, Lancaster W, Kurman RJ et al. Human Papillomavirus infection of the cervix:relative risk associations of 15 common anogenital types. Obstet Gynecol 1992;79:328-37.  Back to cited text no. 3  [PUBMED]    
4.Sano T, Oyama T, Kashiwabara K, Fukuda T, Nakajima T. Expression status of p16 protein is associated with HPV oncogenic potential in cervical and genital lesions. Am J Pathol 1998;153:1741-8.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]  
5.Klaes R, Friedrich T, Spitkovsky D, Ridder R, Rudy W, Petry U, et al. Over expression of p16 as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri. Int J Cancer 2001;92:276-84.   Back to cited text no. 5  [PUBMED]  [FULLTEXT]  
6.Keating JT, Cviko A, Riethdorf S, Riethdorf L, Quade BJ, Sun D, et al. Ki-67, Cyclin E, and p16 INK4a Arc complimentary surrogate biomarkers for human papilloma virus - related cervical neoplasia. American Journal of Surgical Pathology 2001;25:884-91.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]  
7.Gerdes J. Ki-67 and other proliferation markers useful for immunohistological diagnostic and prognostic evaluation in human malignancies. Semin Cancer Biol 1990;1:199-206.  Back to cited text no. 7  [PUBMED]    
8.Goel MM, Mehrotra A, Singh U, Gupta HP, Misra JC. MIB-1 and PCNA immunostaining as a diagnostic adjunct to cervical pap smear. Diagn Cytopathol 2005;33:15-9.  Back to cited text no. 8      
9.Von Hoven KH, Kovatich AJ, Oliver RE, Nobel M, Dunton CJ. Immunocytochemical detection of squamous intraepithelial lesion in cervical smears. Mod Pathol 1996;9:407-11.  Back to cited text no. 9      
10.Stoler MH, Schiffman M. Interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ASCUS-LSIL. Triage Study. JAMA 2001;285:1500-5.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]  
11.Shepherd JH. FIGO staging for gynecological cancer. BR J Obstet Gynaecol 1989;96:889-92.  Back to cited text no. 11  [PUBMED]    
12.National Cancer Institute. Available from: http://www.Bethesda2001.cancer.gov. [last accessed on 2002 Jun 29].  Back to cited text no. 12      
13.Murphy N, Ring M, Killalea AG, Uhlmann V, O′Donovan M, Mulcahy F, et al. p16 INK4a as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and thin prep smears. J Clin Pathol 2003;56:56-63.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]  
14.Volgareva 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.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]  
15.Murphy N, Ring M, Heffron CC, King B, Killalea AG, Hughes C, et al. p16 INK4a, CDC6 and MCM5: predictive biomarkers in cervical preinvasive neoplasia and cervical cancer. J Clin Pathol 2005;58:525-34.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]  
16.Mravunac M, Smedts F, Philippi A, Remerij D, Krul A, Schrik M et al. Interpreting micro biopsies in cervical smears. A cytohistologic approach. Acta Cytol 2000;44:752-9.  Back to cited text no. 16      

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Correspondence Address:
Supriya Srivastava
Cancer Science Institute, National University Singapore, 28 Medical Drive, Level 2, Singapore - 117 456, Singapore

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.68301

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