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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 14  |  Issue : 3  |  Page : 162-165

Correlation of Ki-67 labeling index in cervical intraepithelial neoplasia with histomorphologic grading system


Department of Pathology, Jawaharlal Nehru Medical College, DMIMS(DU), Sawangi(M), Maharashtra, India

Date of Submission05-Jun-2019
Date of Decision30-Jun-2019
Date of Acceptance30-Jul-2019
Date of Web Publication2-May-2020

Correspondence Address:
Dr. Pratibha Dawande
Meghe Heights, Building No. 1, Jawaharlal Nehru Medical College, Sawangi (Meghe), Wardha, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_44_19

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  Abstract 


Background: Worldwide cervical cancer ranks as a major health problem for women. The cervical carcinoma is preceded by premalignant dysplastic changes in normal squamous epithelial layer which is called as cervical intraepithelial neoplasia (CIN). Aim: The aim of the study is to assess the utility of Ki-67 labeling index (LI) in grading of CIN. Objectives: The objective of the study is to identify potential corelation between histomorphological grading and Ki-67 LI of CIN. Materials and Methods: The present study was carried out as an observational type of study. The study was carried out from August 1, 2016, to July 31, 2018, in the Department of Pathology in co-ordination with the Department of Obstetrics and Gynecology, Jawaharlal Nehru Medical College, Sawangi (M), Wardha, Maharashtra. A total of 82 specimens which include cervical biopsies and hysterectomy specimen formed a part of the present study, of which 59 cases were prospective and remaining 23 cases considered were retrospective. Results: Tumor cell proliferation as measured by Ki-67 proliferative marker shows a linear correlation with histopathological grading of cervical dysplasia as per the WHO classification. With increasing grades of CIN, Ki-67 LI also increases. Conclusion: It can be stated that to determine the grades of cervical dysplasia, Ki-67 antigen should be used as an adjunct with histopathological grading which remains as the “Gold Standard.“

Keywords: Cervical intraepithelial neoplasia, histomorphological grading of CIN, Ki-67 labeling index


How to cite this article:
Gupta N, Dawande P, Shukla S, Bhake A. Correlation of Ki-67 labeling index in cervical intraepithelial neoplasia with histomorphologic grading system. J Datta Meghe Inst Med Sci Univ 2019;14:162-5

How to cite this URL:
Gupta N, Dawande P, Shukla S, Bhake A. Correlation of Ki-67 labeling index in cervical intraepithelial neoplasia with histomorphologic grading system. J Datta Meghe Inst Med Sci Univ [serial online] 2019 [cited 2020 Aug 5];14:162-5. Available from: http://www.journaldmims.com/text.asp?2019/14/3/162/283589




  Introduction Top


Worldwide cervical cancer ranks as a major health problem for women. Although currently we have the best available therapies, a significant proportion of women experience metastasis and eventually death. Hence, there is a real need of more effective and specific methods for the diagnosis and treatment for women with cervical cancer to prolong the life of patient and increase their survival rate. The cervical carcinoma is preceded by premalignant dysplastic changes in normal squamous epithelial layer which is called as cervical intraepithelial neoplasia (CIN).[1]

Over the years, many classifications have been given for the premalignant dysplastic conditions of cervical carcinoma.[2] Out of all, the WHO classification remained as the most popular and followed classification by pathologist and clinicians. The WHO has classified CIN into three grades, i.e., CIN I, CIN II, and CIN III.[3] Cancer of the cervix is the third most common cancer among the women worldwide with an estimated 527,624 new cases and 265,672 deaths in 2012, according to GLOBOCAN statistics.[3] In India, cervical carcinoma ranked the second most frequent cancer among women.[4]

Microscopy of the cervix has a disadvantage that shows only epithelial features those are visible by standard hematoxylin and eosin (H and E) staining and thereby not giving any other possibly important information like proliferative rate. These are the most important causes of intraobserver and interobserver reproducibility, which for grading of CIN is far from perfect. This brings the need of adjuvant method to interpret the actual morphological impression of CIN in reference to dynamic terms rather than static morphological grades.[5],[6],[7] In our setup, Ki-67 is the most widely and easily available biomarker and it is cost effective too, which is important as our hospital is a rural-based hospital, and most of our patients belong to lower socioeconomic class [Figure 1], [Figure 2], [Figure 3], [Figure 4].
Figure 1: Linear correlation between cervical intraepithelial neoplasia grading and Ki-67 labeling index

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Figure 2: (a) Section stained with H and E shows mild dysplasia in lower one-third of cervical epithelium (×40), (b) immunohistochemically stained slide shows low Ki-67 proliferative index (10%–30%) (×40)

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Figure 3: (a) Section stained with H and E shows moderate dysplasia in middle and lower one-third of cervical epithelium (×40), (b) immunohistochemically stained slide shows moderate Ki-67 proliferative index (30%–50%) (×40)

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Figure 4: (a) Section stained with H and E shows severe dysplasia in upper, middle, and lower one-third of cervical epithelium (×40), (b) immunohistochemically stained slide shows high Ki-67 proliferative index (>50%) (×40)

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  Materials and Methods Top


The present study includes retrospective cases of 2 years from August 2014 to July 2016 and prospective cases of 2 years from August 2016 to July 2018. The study was observational and analytical type and was carried out with retrospective and prospective cases. A total of 82 specimens which include cervical biopsies and hysterectomy specimen formed a part of the present study, of which 59 cases were prospective and the remaining 23 cases considered were retrospective.

These retrospective cases were diagnosed cases of CIN in the Department of General Pathology, Jawaharlal Nehru Medical College and AVBRH, Sawangi (M). Prospective cases included were clinically suspected cases of CIN, attending a clinic in the Department of Obstetrics and Gynaecology, AVBRH, and were examined grossly, per-vaginum and per-speculum. These patients underwent colposcopic-guided punch biopsy. These biopsy specimens were sent to histopathology and stained with H and E.

A detailed microscopic examination was carried out wherein diagnosis and grading of CIN were made on histopathology according to the WHO classification. In all the cases diagnosed with CIN, immunohistochemistry was carried out using Ki-67 kit (Dako).

Inclusion criteria

  • Cervical biopsies of suspected cases of CIN
  • Posthysterectomy specimens with associated CIN.


Exclusion criteria

  • Already treated cases of CIN
  • Cervical pathogenesis other than dysplasias.


After receiving the cervical biopsies as a tissue specimen in the histopathology division, the tissue was then kept in 10% neutral buffered formalin solution overnight (12 h) for fixation. According to the standard protocol in histopathology division, sections were taken to automated tissue processor for 16 h cycle. The paraffin blocks were prepared with the help of Leukhart's mold. Using rotary microtome, 5 μm thickness sections were taken on the clean glass slides and then subjected to routine H and E staining for the confirmation of diagnosis of CIN.

Control used for Ki-67 labeling index

  • Positive control for Ki-67 labeling index (LI): reactive lymph node was used as positive control
  • Negative control for Ki-67 LI: adipose tissue was used as negative control.


Grading system of Ki-67 proliferating marker

Grade of Ki-67 LI was done by counting nucleus of 200 epithelial cells across the epithelial layer at 400×. Positive Ki-67 staining results in brown nuclear staining. This brown granular nucleus reactivity gets dispersed over nucleus with focal denser nucleoli. Ki-67 LI is defined as percentage of positively stained epithelial cells per 100 neoplastic cells after counting at least 200 cells. LI = (number of cells showing positive staining/total number of cells) ×100.[8] Based on the LI, the sections were scored from Grades 1+, 2+, and 3+ for Ki-67 expression [Table 1].[9]
Table 1: Grading system for Ki-67 labeling index

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  Results Top


The maximum number of patients in the present study is of more than 40 years, which includes approximately 81% (67) of the patients, and hence, the present study shows cervical dysplasia commonly in older age groups. Maximum patients, i.e., 31 (37.80%), presented with complaints of abnormal vaginal bleeding, whereas 2 (2.44%) patients diagnosed with CIN had abdominal complaints. Maximum patients in our study were multiparous and grand multiparous, i.e., 69 (84%). Most of the patients were of lower and upper lower, i.e., 52 (63%). Least patients in the present study were from upper socioeconomic class.

Of total 82 cases, 28 (34.15%) patients were of Grade CIN I, 36 (43.90%) patients were of Grade CIN II, and 18 (21.95%) patients were of Grade CIN III. Of total 82 cases, 28 cases were of CIN I which has a mean Ki-67 LI of 24.14%, 36 cases were of CIN II which has a mean Ki-67 LI of 39.66%, and 18 cases were of CIN III which has a mean Ki-67 LI of 60.83% [Table 2]. Of total 82 cases, 34.15% cases were of CIN I among which 75% were reported as Grade 1+, 14.28% of the cases were Grade 2+, and 10.72% cases were Grade 3+. About 43.90% cases were of CIN II among which 5.55% were reported as Grade 1+, 88.88% of the cases were Grade 2+, and 5.55% of the cases were Grade 3+. Nearly 21.95% cases were of CIN III among which none were reported as Grade 1+ and Grade 2+ and 100% cases were Grade 3+ [Table 3]. There is linear correlation of increasing expression of Ki-67 LI with increasing histopathological grades.
Table 2: Mean Ki-67 labeling index in histomorphological grades

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Table 3: Patients with Ki-67 grades in different histomorphological grades of cervical intraepithelial neoplasia

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  Discussion Top


In the present study, maximum patients (38 patients) were of the age group of 40–49 years with a mean age of 48.56 years. The most common symptomology of the patients diagnosed with cervical dysplasia was more than one with maximum number (37.80%) as abnormal vaginal bleeding. Most of the cases in our study were multiparous (52) and grand multiparous irrespective of grades of cervical dysplasia (17). In the present study, most of the patients (52) with cervical dysplasia seem to belong to lower and upper lower socioeconomic class according to Kuppuswamy classification. These findings are similar to the findings of study done by Archana et al.,[10] Sansanwal et al.,[11] Muñoz et al.,[12] Brinton et al.,[13] and Chauhan et al.[14]

In the present study, the mean Ki-67 LI of 28 cases of CIN I is 24.14, the mean Ki-67 LI of 36 cases of CIN II is 39.66, and the mean Ki-67 LI of 18 cases of CIN III is 60.83. In our study, we found higher LI with high grades and lower indices with CIN I and CIN II when compared to CIN III. In few cases, it has been seen that low grades of cervical dysplasia reported on histopathology have shown high grades of proliferative index by Ki-67 LI. These cases need to be followed up regularly because they have higher changes of progression to frank cervical carcinoma and were missed alone by histopathology. These findings are in concordance with studies done by Chauhan et al.,[14] Gupta et al.,[15] and Payne et al.[16]


  Conclusion Top


A correlation between Ki-67 LI and CIN grade was established. Patients with CIN were mostly age group of 40–49 years and presented with complaints of abnormal vaginal bleeding. There was an increasing grade of cervical dysplasia with increasing age of patient. CIN was more commonly reported in multiparous women with lower socioeconomic status. Tumor cell proliferation as measured by Ki-67 proliferative marker shows a linear correlation with histopathological grading of cervical dysplasia as per the WHO classification. Thus, it can be stated that to determine the cervical dysplasia, Ki-67 antigen should be used as an adjunct with histopathological grading which remains as the “Gold Standard.“

Limitations

Subjective limitations

The diagnosis of CIN on histopathology is considered to be gold standard, but sometimes, the pathologist faces diagnostic dilemmas not only because of overlap in morphological features among different grades of CIN but also due to interobserver and intraobserver variability especially during the grading of CIN which affects the prognosis of the patient. For prognostication and grading of tumors, an application of immunohistochemical proliferative marker like Ki-67 becomes imperative.

Technical limitations

The immunohistochemical staining is usually influenced by fixative used and antigen retrieval procedures.

Recommendations

The “Gold standard” method used for diagnosing and grading CIN is histopathology because traditional/conventional method of grading cervical dysplasia by an experienced pathologist remains the best. As Ki-67 LI has more accurate and has better predictive value, this technique can be used as an adjunct for correlation with the histopathological grading of CIN and can be routinely used as a prognostic marker in CIN. For prognostication and predicting the behavior of CIN, it is definitely recommended to use nuclear proliferative marker (like Ki-67) as an adjunct.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Stoler MH. Human papillomavirus biology and cervical neoplasia: Implications for diagnostic criteria and testing. Arch Pathol Lab Med 2003;127:935-9.  Back to cited text no. 1
    
2.
Martin CM, O'Leary JJ. Histology of cervical intraepithelial neoplasia and the role of biomarkers. Best Pract Res Clin Obstet Gynaecol 2011;25:605-15.  Back to cited text no. 2
    
3.
Syrjanen KJ. Natural history of genital papillomavirus infections. In: Lacey C, editor. Papillomavirus Reviews: Current Research on Papillomaviruses. Leeds: Leeds University Press; 1996. p. 189-206.  Back to cited text no. 3
    
4.
International Agency for Research on Cancer. GLOBOCAN 2012: Estimated cancer Incidence, Mortality and Prevalence Worldwide. International Agency for Research on Cancer; 2012.  Back to cited text no. 4
    
5.
Bruni L, Barrionuevo-Rosas L, Albero G, Aldea M, Serrano B, Valencia S, et al. Human Papillomavirus and Related Diseases in the World. ICO Information Centre on HPV and Cancer (HPV Information Centre); 23 December, 2015.  Back to cited text no. 5
    
6.
Panjković M, Ivković-Kapicl T. Ki-67 expression in squamous intraepithelial lesions of the uterine cervix. Arch Oncol 2006;14:23-5.  Back to cited text no. 6
    
7.
Ross W, Hall PA. Ki67: From antibody to molecule to understanding? Clin Mol Pathol 1995;48:M113-7.  Back to cited text no. 7
    
8.
Yim EK, Park JS. Biomarkers in cervical cancer. Biomark Insights 2007;1:215-25.  Back to cited text no. 8
    
9.
Ancuţa E, Ancuţa C, Cozma LG, Iordache C, Anghelache-Lupaşcu I, Anton E, et al. Tumor biomarkers in cervical cancer: Focus on Ki-67 proliferation factor and E-cadherin expression. Rom J Morphol Embryol 2009;50:413-8.  Back to cited text no. 9
    
10.
Archana S, Eswari V, Prakash G. Ki-67 expression in squamous intraepithelial lesions and carcinoma cervix by immunohistochemistry. Int J Sci Res 2016;5:204-7.  Back to cited text no. 10
    
11.
Sansanwal P, Sankala M, Singh G, Kathuriya M. Immunohistochemical detection of proliferative tumor cells in cervical cancer using monoclonal antibody Ki-67. Int J Health Sci Res 2015;5:166-71.  Back to cited text no. 11
    
12.
Muñoz N, Franceschi S, Bosetti C, Moreno V, Herrero R, Smith JS, et al. Role of parity and human papillomavirus in cervical cancer: The IARC multicentric case-control study. Lancet 2002;359:1093-101.  Back to cited text no. 12
    
13.
Brinton LA, Herrero R, Reeves WC, de Britton RC, Gaitan E, Tenorio F, et al. Risk factors for cervical cancer by histology. Gynecol Oncol 1993;51:301-6.  Back to cited text no. 13
    
14.
Sansanwal P, Sankala M, Singh G, Kathuriya M. Role of apoptotic index, mitotic index and MIB-1 antibody expression as biomarker in preneoplastic and neoplastic lesions of uterine cervix. Int J Res Med Sci 2016;4:2093-100.  Back to cited text no. 14
    
15.
Gupta K, Alam K, Maheshwari V, Khan R, Sharma R. Apoptotic index and Mib-1 antibody expression in premalignant and malignant lesions of uterine cervix. Gynecol Obstet 2013;3:173. [doi: 10.4172/2161-0932.1000173].  Back to cited text no. 15
    
16.
Payne S, Kernohan NM, Walker F. Proliferation in the normal cervix and in preinvasive cervical lesions. J Clin Pathol 1996;49:667-71.  Back to cited text no. 16
    


    Figures

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

  [Table 1], [Table 2], [Table 3]



 

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