Study of immunohistochemical expression of Ki-67 in squamous cell carcinoma of cervix.
Cervical cancer is the most common cancer cause of death among women in developing countries.  It can involve the uterine corpus by direct extension or through parametrium by lymphatic invasion to the uterine wall.  Mortality due to cervical cancer is also an indicator of health inequities, as 86% of all deaths due to cervical cancer are in developing, low and middle income countries.  Every year in India, 122,844 women are diagnosed with cervical cancer and 67,477 die from the disease. India has a population of 432.2 million women aged 15 years and older who are at risk of developing cancer. It is the second most common cancer in women aged 15-44 years.  India also has the highest age standardised incidence of cervical cancer in South Asia at 22, compared to 19.2 in Bangladesh, 13 in Sri Lanka, and 2.8 in Iran. 
The Ki-67 protein is a cellular marker for proliferation which can be detected within the cell nucleus. In all active phases of the cell cycle Ki-67 is present i.e. G1, S, G2, and M, but absent in resting cells (G0).  To determine the growth fraction of a given cell population Ki-67 is an excellent marker.  Ki-67 is of great importance in the tumour grading, proliferative degrees and prognosis.  Ki-67 expression is normally confined to the parabasal and basal layers in the normal cervical epithelium.  Some studies have showed that Ki-67 protein could be a biomarker in the evaluation of the proliferative activity and progressive potential of normal, dysplastic and neoplastic changes.  An Asian study observed Ki-67 may serve as a helpful adjunct to routinely-stained histological sections in differentiating SCC. 
The present study aims at assessing the Ki-67 expression in Squamous Cell Carcinoma (SCC) of the cervix and correlating its expression with the histological grades of SCC.
MATERIALS AND METHODS
A total of 63 histologically proven cases of cervical squamous cell carcinoma received in the Department of Pathology, Sri Ram Murti Smarak Institute of Medical Sciences, Bareilly, from January 2015 to December 2015 were included in the study. The study was approved by the institute's ethical committee.
Immunostaining for Ki-67 (BioGenex) was carried out. All slides were deparaffinised in xylene, rehydrated in graded alcohols, washed in PBS buffer, boiled in EDTA solution (pH 6.0, 100[degrees]C, 2.5 minutes) for antigen retrieval and cooled down. Following endogenous peroxide and protein blocking step, the slides were incubated with primary antibodies. After brief washes, incubation in a cocktail of biotinylated rabbit anti-mouse IgG/IgM for 30 min. was performed.  The sections were then washed and incubated with streptavidinbiotinylated horseradish peroxidase complex for 30 min., reacted with 3-amino-9-ethylcarbazole and hydrogen peroxide. To visualise the end product haematoxylin was used as a counter stain.
Ki-67 labelling index was calculated by the number of positive cells per 200 cervical epithelial cells under X400 magnification. Positive nuclei were expressed as the percentage of total nuclei counted. 
Modified Broder's grading system was used for grading squamous cell carcinomas. Carcinomas were divided into three grades: well-differentiated squamous cell carcinoma (WDSCC), moderately differentiated squamous cell carcinoma (MDSCC) and poorly differentiated squamous cell carcinoma (PDSCC). WDSCC showed abundant intercellular bridging, cytoplasmic keratinisation and keratin pearls, nuclear pleomorphism was minimal and mitotic figures were <2/hpf. In MDSCC individual cell keratinisation, moderate nuclear pleomorphism and mitotic figures up to 4/hpf were present. In PDSCC immature tumour cells with scant cytoplasm, marked nuclear pleomorphism, and mitotic figures, >4/hpf were present. 
All the data was entered in database file and analysis was done using SPSS 20 version. ANOVA test was applied to see correlation of Ki-67 expression with different grades of SCC. P value less than 0.05 was considered as significant.
The mean Ki-67 Index % observed was 54.0 [+ or -] 2.64 for those diagnosed as well-differentiated squamous cell carcinoma (SCC-WD), 63.0 [+ or -] 4.17 for patients diagnosed as moderately differentiated squamous cell carcinoma (SCC-MD), and 85.5 [+ or -] 9.3 for patients diagnosed for poorly differentiated squamous cell carcinoma (SCC-PD), and the difference in the Ki-67 Index among different forms of squamous cell carcinoma was highly statistically significant P<0.001 (Table 1).
Increase in Ki-67 expression was seen with the increasing grade of SCC. All 5 patients of SCC-PD were highly proliferative with an expression of Ki-67 in 70% or more of the cells. Among the SCC- MD lesions, 34/49 (69.4%) expressed Ki-67 in 30 to <70% of the cells and 9 out of 49 (18.4%) cases showed Ki-67 expression in 70% and more than of the cells. In SCC-WD lesions, mostly 5 patients out of 9 (55.5%) had expression of Ki-67 in 30 to <70% of the cells. (Table 2).
In the present study, total number of cases was 63. Among them, most (77.7%) were diagnosed as Moderately Differentiated Squamous Cell Carcinoma (SCC-MD) followed by 14.2% cases of Well-Differentiated Squamous Cell Carcinoma (SCC-WD) and 7.9% cases of Poorly Differentiated Squamous Cell Carcinoma (SCC-PD) respectively. Similar results were found by Hemlata Panwar et al  where out of the 78 cases of biopsy-proven invasive carcinoma, 54 cases had moderately differentiated squamous cell carcinoma, followed by 10 cases of well-differentiated SCC and 5 cases of poorly differentiated SCC.
Our cases showed an increase in the Ki-67 expression in relation to increase in the grade of the lesion. The mean Ki-67 Index % observed was 54.0 [+ or -] 2.64 for patients diagnosed as Well Differentiated Squamous Cell Carcinoma (SCC-WD), 63.0 [+ or -] 4.17 for patients diagnosed as Moderately Differentiated Squamous Cell Carcinoma (SCC-MD), and 85.5 [+ or -] 9.3 for patients diagnosed as Poorly Differentiated Squamous Cell Carcinoma (SCC-PD), and the difference was highly significant statistically (P<0.001). Similar result was found by Gupta K et al14 in their study which reported the mean LI in WD SCC was 55.333 [+ or -] 7.789; in MDSCC was 43.976 [+ or -] 3.152 and in PDSCC was 80 [+ or -] 5. The study conducted by Pahujaet al  showed an increase in LI from WDSCC to PDSCC with maximum mean value in PDSCC similar to the present study. The study conducted by Nam et al  also showed an increase in LI from WD SCC to PD SCC.
Mehrotra A et al  reported Ki-67 labelling index (LI) increased from dysplasia to carcinoma group. Statistical analysis showed that Ki-67 LI was significantly higher in diseased group as compared to normal group (P<0.001). Carreras R et al  showed increase in the Ki-67 expression in relation to increases in the severity of the lesions which is similar to our study.
In SCC-WD lesions, 5 patients out of 9 (55.5%) had expression of Ki-67 in 30 to <70% of the cells, 4/9 (44.4%) had expression of Ki-67 in 10 to <30%. None of the cells showed expression of 0 to <10% and in between 70 to 100%. Among the SCC- MD lesions, 34/49 (69.4%) showed expression of Ki-67 in 30 to <70% of the cells, 9 out of 49 (18.4%) cases showed Ki-67 expression in 70% and more than of the cells and 6 out of 49 (12.24%) cases showed Ki-67 expression in 10 to <30%. None of the cases showed Ki-67 expression in 0 to <10%. All 5 patients of SCC-PD were highly proliferative with an expression of Ki-67 in 70% or more of the cells. None of the cases showed Ki-67 expression in 0 to <10%, 10 to <30% and 30 to <70% category (Table 2). Reuschenbach M et al  found the proportion of lesions with Ki-67 expression in 70% or more of the cells was somewhat higher in the carcinomas (21/46, 45.7%) than in the CIN3/carcinoma in situ (CIS) group of cases.
We conclude that MIB-1 staining shows an increasing trend in the Ki-67 labelling index with increasing grades of SCC.
 Denny L. Cervical cancer: prevention and treatment. Discov Med 2012;14(75):125-31.
 Parkin DM, Whelan SL, Ferlay J, et al. Cancer incidence in five continents. vol. 8. Lyon: IARC Press, IARC Scientific Publications 2002:P 155.
 Aswathy S, Javed R, Dinesh A. Epidemiology of cervical cancer with special focus on India. Int J Women's Health 2015;7:405-14.
 ICO Information Centre on HPV and cancer. Human papillomavirus and related diseases in India 2014.
 Ross W, Hall PA. Ki67: from antibody to molecule to understanding? Clin Mol Pathol 1995;48(3):M113-7.
 Gerdes J, Schwab U, Lemke H, et al. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 1983;31(1):13-20.
 Li PL, Tan HZ. Expression of PPARy, p27 and Ki67 in cervical cancer and its clinical significance. J Int Transl Med 2015;3(1):513-9.
 Bulten J, Laak VDJA, Gemmink JH, et al. MIB-1, a promising marker for the classification of cervical intraepithelial neoplasia. J Pathol 1996;178(3):268-73.
 Kruse AJ, Baak JPA, Bruin PC, et al. Ki-67 immunoquantitation in cervical intraepithelial neoplasia (CIN): a sensitive marker for grading. J Pathol 2001;193(1):48-54.
 Tan GC, Sharifah NA, Shiran MS, et al. Utility of Ki-67 and p53 in distinguishing cervical intraepithelial neoplasia 3 from squamous cell carcinoma of the cervix. Asian Pac J Cancer Prev 2008;9(4):781-4.
 Mehrotra A, Goel MM. Assessment of monoclonal antibody MIB-1 labeling indices in cervical intraepithelial lesions of the uterine cervix in paraffin section. J Obstet Gynecol India 2008;58(4):327-32.
 Ng AB, Atkin NB. Histological cell type and DNA value in the prognosis of squamous cell cancer of uterine cervix. Br J Cancer 1973;28(4):322-31.
 Panwar H, Khurana U, Gupta K, et al. A clinicopathological study of neoplastic and preneoplastic lesions of cervix along with HPV genotyping in biopsy proven preneoplastic lesions. Indian Journal of Obstetrics and Gynecology Research 2016;3(4):400-6.
 Gupta K, Alam K, Maheshwari V, et al. Apoptotic index and MIB-1 antibody expression in premalignant and malignant lesions of uterine cervix. Gynecol Obstet 2013;3:173.
 Pahuja S, Choudhury M, Gupta U. Ki-67 labelling as a proliferation marker in pre-invasive squamous epithelial lesions of cervix. Indian J Pathol Microbiol 2003;46(3):402-4.
 Nam JH, Kim JH, Huh J, et al. Correlation of lesion grade in cervical neoplasia with cell proliferation and apoptosis. Int J Gynecol Cancer 1998;8(4):279-86.
 Carreras R, Alameda F, Mancebo G, et al. A study of Ki67, c-erbB2 and cyclin D-1 expression in CIN-I, CIN-III and squamous cell carcinoma of the cervix. Histol Histopathol 2007;22(6):587-92.
 Reuschenbach M, Seiz M, Doeberitz VKC, et al. Evaluation of cervical cone biopsies for coexpression of p16INK4a and Ki-67 in epithelial cells. Int J Cancer 2012;130(2):388-94.
Rashmi Priyadarshni (1), Tanu Agrawal (2), Ankit Ojha (3)
(1) Junior Resident, Department of Pathology, Shri Ram Murti Smarak Institute of Medical Sciences, Bareilly.
(2) Professor, Department of Pathology, Shri Ram Murti Smarak Institute of Medical Sciences, Bareilly.
(3) Junior Resident, Department of Pathology, Shri Ram Murti Smarak Institute of Medical Sciences, Bareilly.
Financial or Other, Competing Interest: None.
Submission 06-03-2017, Peer Review 08-04-2017, Acceptance 14-04-2017, Published 20-04-2017.
Dr. Rashmi Priyadarshni, Department of Pathology, Shri Ram Murti Smarak Institute of Medical Sciences, Nainital Road, Bhojipura, Bareilly-243202, Uttar Pradesh, India.
Caption: Figure 1. Photomicrograph of SCC-WD Showing Ki-67 Positivity (IHC Ki-67, 40X)
Caption: Figure 2. Photomicrograph of SCC-MD Showing Ki-67 Positivity (IHC Ki-67, 40X)
Caption: Figure 3. Photomicrograph of SCC-PD Showing Ki-67 Positivity (IHC Ki-67, 40X)
Table 1. Expression of mean Ki-67 Index (%) in Squamous Cell Carcinoma Diagnosis KI-67 Index % P value (SCC)(N=63) (Mean [+ or -] SD) SCC-WD (n=9) 54.0 [+ or -] 2.64 SCC-MD (n=49) 63.0 [+ or -] 4.17 <0.001 SCC-PD (n=5) 85.5 [+ or -] 9.3 Table 2. Ki-67 expression in Squamous Cell Carcinoma KI-67 Index Diagnosis (%) SCC-WD SCC-MD SCC-PD (n=9) (n=49) (n=5) >0 to <10% 0 (0.0%) 0 (0.0%) 0 (0.0%) 10 to <30% 4 (44.4%) 6 (12.24%) 0 (0.0%) 30 to <70% 5 (55.5%) 34 (69.38%) 0 (0.0%) 70 to 100% 0 (0.0%) 9 (18.36%) 5(100%)
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||Original Research Article|
|Author:||Priyadarshni, Rashmi; Agrawal, Tanu; Ojha, Ankit|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Apr 20, 2017|
|Previous Article:||Flow cytometric immunophenotyping of acute leukaemia with special reference to mixed phenotypic acute leukaemia.|
|Next Article:||Study of peri-operative complications in asymptomatic smokers posted for day care surgery.|