Indian Journal of Cancer, Vol. 47, No. 3, July-September, 2010, pp. 304-307
Role of Ki-67 as a proliferative marker in lesions of thyroid
Pujani M, Arora B, Pujani M1,2, Singh SK1 , Tejwani N1
Department of Pathology, Pt. B D Sharma PGIMS, Rohtak, 1 Lady Hardinge Medical College, 2 Kalawati Saran Childrens Hospital, New Delhi, India
Code Number: cn10073
Background: Specific criteria are used to diagnose thyroid neoplasms; however, the distinction between certain neoplasms, such as follicular adenoma and carcinoma, could be difficult. Thus, additional diagnostic features that can assist in this distinction would have great clinical usefulness.
Aims: To evaluate the role of the proliferative marker Ki-67 in nonneoplastic and neoplastic lesions of the thyroid, with a special emphasis on the distinction between follicular adenoma and follicular carcinoma.
Settings and Design: A retrospective study from a tertiary care center.
Materials and Methods: One hundred cases of thyroid lesions, including 50 nonneoplastic and 50 neoplastic lesions, were retrieved from the archives of the Department of Pathology, Pt. BD Sharma PGIMS, Rohtak, Ki-67 immunostaining was performed by peroxidase-antiperoxidase method and compared with mitotic counts.
Results: Ki-67 labeling index (LI) showed a progressive rise from multinodular goiter to benign to malignant neoplasms. A statistically significant difference was observed in Ki-67 counts between multinodular goiter vs papillary carcinoma (P < 0.05) and follicular adenoma vs follicular carcinoma (P < 0.05). The correlation between mitotic counts and Ki-67 LI was found to be significant.
Conclusions: In the present study, Ki-67 was found to be useful in differentiating between follicular adenoma and follicular carcinoma, but since the sample size of our study was small, larger studies are needed to confirm this observation as well as to assign a cutoff value for differentiating benign from malignant tumors.
Keywords: Ki-67, proliferative marker, thyroid
Thyroid neoplasms represent a broad spectrum of lesions with different histopathologic features. Although specific criteria are used to diagnose these neoplasms, the distinction between certain neoplasms, such as follicular adenoma and follicular carcinoma, can be difficult. Thus, additional diagnostic features that can assist in this distinction would have great clinical usefulness. 
Ki-67 is a novel proliferative marker that can be readily detected by immunohistochemistry. Gerdes et al. have shown that Ki-67 is expressed in all stages of the cell cycle, except G0, whereas resting cells entering from G0 lack Ki-67 in the early part of G1. ,
This study was conducted to assess the utility of Ki-67 as a proliferation marker in nonneoplastic and neoplastic lesions of thyroid as well as to evaluate the usefulness of Ki-67 in distinguishing follicular adenoma from follicular carcinoma. Prior studies evaluating the role of Ki-67 in the thyroid have produced variable results. However, to the best of our knowledge, we did not come across any such literature from India. Hence, the present study was conducted to evaluate the role of Ki-67 in the Indian population.
Materials and Methods
In this retrospective study, 100 formalin-fixed, paraffin-embedded surgical specimens of lobectomies, thyroidectomies, and excision biopsies were retrieved from the archives of the Department of Pathology, Pt. BD Sharma PGIMS,Rohtak, over a period of 3 years. Ethical clearance was obtained from the Institutional Review Board. There were 50 cases of nonneoplastic lesions (multinodular goiter, thyroiditis, and others) and 50 cases of neoplastic lesions (follicular adenoma and different types of carcinomas). The consecutively received thyroid cases were included in the study to avoid selection bias. All the cases were reviewed separately by 2 pathologists.
Immunohistochemistry was performed on 3-΅m thick sections on poly-l-lysine-coated slides. Antigen retrieval was done using microwave in citrate buffer at pH 6. Monoclonal antibody MIB1 (Novocastra, code no. NCL-Ki-67-MM1) (Newcastle, UK) was used for Ki-67 antigen detection by standard streptavidin-biotin technique, using Novostain Universal Detection Kit (Novocastra, code no. NCL-RTU-D). Sections from a reactive lymph node were taken as positive control [Figure - 1]a, whereas sections treated with tris-buffer solution instead of primary antibody were used as negative control.
Brown granular nuclear reactivity was positive. An area with the maximum proliferation was chosen to evaluate the labeling index (LI, expressed as percentage of positively stained cells per 100 follicular epithelial cells) after counting at least 1000 cells in each case.
Data were analyzed using the statistical software package SPSS 12 (Chicago, USA). Statistical analysis of the data was performed using ANOVA (analysis of overall variance) test. P value of less than 0.05 was considered statistically significant.
Ki-67 immunostaining gave brown granular nuclear reactivity, finely dispersed over the nucleus with focal denser nucleoli-like positivity [Figure - 1]. The age of the patients ranged from 17 to 80 years with a female to male ratio of 10:1. Mitotic counts were found to be 0.20/10 high-power fields (hpf) (mean) in the nonneoplastic group, whereas in the neoplastic group, the mean mitotic count was 0.99/10 hpf.
Ki-67 LI was found to be the highest for undifferentiated carcinoma, followed by medullary carcinoma, follicular carcinoma, and papillary carcinoma. In the nonneoplastic group, Ki-67 LI was 0.21 (mean).
A statistically significant difference was observed in Ki-67 counts between multinodular goiter vs papillary carcinoma (P < 0.05) and follicular adenoma vs follicular carcinoma (P < 0.05). The correlation between mitotic counts and Ki-67 LI was found to be significant.
However, no statistically significant difference was observed between Ki-67 counts of multinodular goiter and follicular adenoma.
Ki-67 is an IgG1 class, murine monoclonal antibody raised against a crude nuclear fraction of Hodgkin′s disease-derived cell line L-428. The antibody was named after its place of production in Kiel, West Germany, and because the clone producing the antibody was grown in the 67 th well of tissue culture plate. Ki-67 serves as a prognostic indicator in lymphoproliferative disorders (non-Hodgkin′s lymphoma), breast carcinoma, and central nervous system tumors.  Saad et al. determined the proliferative rate of normal human thyroid cells in different age groups using Ki-67 (MIB-1 antibody) and found Ki-67 LI to be 7.4 ± 6.10% in 25 fetal thyroids, 0.23 ± 0.15% in 55 childhood thyroids, and 0.08 ± 0.04% in 37 adult thyroids at autopsy. 
In the present study, the mean values of Ki-67 LI increased progressively from multinodular goiter to follicular adenoma, papillary carcinoma, follicular carcinoma, and medullary carcinoma, and were the highest in undifferentiated carcinoma. Ki-67 LI of various lesions of thyroid are shown in [Table - 1]. A significant difference was observed in the mean values of multinodular goiter vs papillary carcinoma and follicular adenoma vs follicular carcinoma. These findings are in close agreement with those of Erickson et al.  They observed the highest values for Ki-67 LI in anaplastic carcinoma followed by follicular and papillary carcinoma. Kjellman et al. studied MIB-1 index in various thyroid tumors comprising 30 papillary thyroid carcinomas (PTC), 10 follicular thyroid carcinomas (FTCs), 8 anaplastic thyroid carcinomas (ATCs), and 96 follicular thyroid adenomas (FTAs), and found a median MIB-1 index of 0.5% in FTA, 1.9% in PTC, 2.7% in FTC, and 16.2% in ATC. 
Saiz et al. studied the immunohistochemical expression of cyclin D1, E2F-1, and Ki-67 in benign and malignant thyroid lesions and found the highest expression of all the 3 markers in malignant tumors, particularly papillary carcinoma. 
According to Tallini et al. downregulation of p27Kip1 and Ki-67/MIB-1 LI supports the classification of thyroid carcinoma into 3 prognostically relevant categories.  Siironen et al studied immunohistochemical expression of Bcl2, Ki-67, and p21 in patients with PTC. They found that Ki-67 expression increases significantly with age, which may explain the poor prognosis in these patients. 
Lloyd et al. studied TGF-β , TGF-β receptors, Ki-67, and p27Kip1 expression in variants of PTC in order to determine if differences in behavior of these tumors were related to the expression of growth factors/cell cycle proteins. There was a marked increase in the Ki-67 LI in the columnar cell tumors compared with that of other groups, but this difference was not significant because of the small number of tumors in this group. 
Tisell et al. studied 71 tumor specimens from 36 cases of medullary thyroid carcinomas (MTC), including 36 primary tumors, 21 primary lymph node metastases, and 14 recurrent lymph node metastases. The mean (SD) (median) of the Ki67 indices for all the 71 tumors was 1.71% (3.3) (median 0.61%), the Ki67 indices of the primary tumors, primary metastases, and recurrent metastases were 0.74% (0.96) (median 0.41%), 1.35% (1.49) (median 0.85%), and 4.73% (6.39) (median 1.65%), respectively. They showed that the Ki67 index is a very suitable prognostic marker for MTC. 
The results in the present study were not in accordance with those of Wallin et al. according to whom Ki-67 has no role in differentiating between benign and malignant lesions. They observed a Ki-67 LI of 0-1.1% in nonneoplastic lesions, 0-3.1% in benign lesions, and 0.2-3.9% in malignant lesions. 
Hori et al, using flow cytometry, assessed Ki-67-positive fractions in 17 benign and 33 malignant thyroid tumors. The percentage of Ki-67-positive fractions in malignant tumors (39.9 ± 3.9%) was significantly higher than in benign tumors (9.4 ± 2.1%).  Erickson et al. established that p27Kip1 expression distinguishes papillary hyperplasia in Graves disease from PTC. However, no significant difference in Ki-67 or topoisomerase IIa expression were identified between the 2 groups.  Resnick et al. investigated p27Kip1 expression in a series of 87 benign and malignant thyroid neoplasms and correlated its expression with Ki-67 LI and other prognostic factors. Poorly differentiated carcinoma had the lowest p27 staining frequency than that of all carcinomas examined. p27 Staining of papillary carcinoma was significantly lower than that of follicular carcinoma (P < 0.001). However, there was no significant difference in the Ki-67 LI between the 2 groups. 
Rigaud and Bogomoletz used the classical Ki-67 antibody for assessment of the proliferative activity of thyroid tumor cells. They did not find any correlation between the percentages of Ki-67-positive cells and histologic typing of thyroid tumors. 
Ziad et al. studied immunoexpression of thyroid transcription factor-1 (TTF-1) and Ki-67 in a coexistent ATC and FTC and found a significantly higher Ki-67 LI (30 ± 5) in anaplastic areas in comparison with the follicular areas (2 ± 1). They suggested that in thyroid cancers, TTF-1 and Ki-67 could provide useful information on the differentiation activities of thyroid tumor cells and may be helpful to distinguish well-differentiated and undifferentiated areas in a mixed thyroid cancer. 
Certain recent studies have indicated the role of novel markers, using tissue microarray technique. Bryson et al. evaluated the role of antibodies galectin-3, autotaxin, intestinal trefoil factor 3 (TFF3), extracellular matrix metalloproteinase inducer (EMMPRIN), and growth arrest and DNA damage-inducible protein 153 (GADD153) in distinguishing follicular adenoma from carcinoma, using tissue microarrays. A significantly higher percentage of FTC cells were stained with galectin-3, EMMPRIN, and GADD153.
The limitations of the present study are the small sample size (n = 100) comprising a limited number of cases of follicular adenoma and follicular carcinoma. Therefore, the cutoff value for Ki-67 for distinguishing between benign and malignant follicular neoplasms could not be ascertained. Due to cost constraints, the correlation of Ki-67 expression with other markers, such as p27 and bcl2 could not be assessed. However, to the best of our knowledge, this is the first study on the role of Ki-67 in thyroid lesions in India. Despite the above-mentioned limitations, Ki-67 was found to be helpful to differentiate follicular adenoma from carcinoma. Since the sample size of our study was small, larger studies are needed to confirm this observation as well as to assign a cutoff value for differentiating benign from malignant tumors.
Copyright 2010 - Indian Journal of Cancer
The following images related to this document are available:
Photo images[cn10073t1.jpg] [cn10073f1.jpg]