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Year : 2011  |  Volume : 54  |  Issue : 3  |  Page : 472-475
Diagnostic utility of ki67 and p53 immunostaining on solitary thyroid nodule - A cytohistological and radionuclide scintigraphic study

Department of Pathology, Lady Hardinge Medical College and Smt S.K. Hospital, New Delhi, India

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Date of Web Publication20-Sep-2011


Background: For management of thyroid nodules, distinction between benign and malignant tumors is essential. Present study was undertaken to differentiate between benign and malignant lesions by using Ki-67 and p53 immunostaining and radionuclide perfusion scan. Materials and Methods: Study comprised of 25 prospective and 25 retrospective cases of solitary thyroid nodules. Fine needle aspiration was done on 25 prospective cases, which was correlated with histopathological diagnosis in 24 surgically excised cases. Immunostaining for p53 and Ki-67 was put on histopathological sections of 25 retrospective and 24 prospective cases. Radionuclide perfusion scan was performed and vascularity patterns were compared with their pathological nature to differentiate between benign and malignant nodule. Results: Cytohistological correlation was present in 80% of cases. On immunostaining, significant difference in mean value of Ki67 positivity was found between benign and malignant nodules (P < 0.05). On p53 immunostaining significant difference was observed in counts of benign and malignant lesions (P = 0.037). On radionuclide perfusion scan mean of difference between maximum and minimum perfusion activity between benign and malignant nodules was found to be statistically significant (P = 0.04), however there was no correlation between perfusion patterns and antigenic characteristics. Conclusions: P53 and Ki-67 immunostaining along with radionuclide perfusion scan appears to be useful tools to differentiate between benign and malignant lesions in solitary thyroid nodule; however, more studies are needed to confirm this observation.

Keywords: Immunohistochemistry, radionuclide, thyroid, tumor

How to cite this article:
Choudhury M, Singh S, Agarwal S. Diagnostic utility of ki67 and p53 immunostaining on solitary thyroid nodule - A cytohistological and radionuclide scintigraphic study. Indian J Pathol Microbiol 2011;54:472-5

How to cite this URL:
Choudhury M, Singh S, Agarwal S. Diagnostic utility of ki67 and p53 immunostaining on solitary thyroid nodule - A cytohistological and radionuclide scintigraphic study. Indian J Pathol Microbiol [serial online] 2011 [cited 2023 Oct 2];54:472-5. Available from:

   Introduction Top

Thyroid nodules are commonly encountered during routine medical care. The prevalence of thyroid nodule increases with age, average 4-7% in adult population but it is much higher (19-67%) when subclinical nodules are also considered. [1],[2] Thyroid cancer represents 5-24% of all these nodules. [3] Fine needle aspiration (FNA) is the best initial diagnostic tool to evaluate thyroid nodules. However, distinction between certain neoplasms can be difficult due to overlapping morphological features. In this study two immunomarkers Ki-67 and p53 were used on histological sections of benign and malignant thyroid lesions along with radionuclide perfusion scan to evaluate their diagnostic accuracy in differential diagnosis of thyroid nodules.

   Materials and Methods Top

The present, prospectivem and retrospective study comprises of 50 cases. Prospective group comprised of total 25 cases presenting clinically as solitary nodule of thyroid. Clinical details were recorded in all the cases.

Radionuclide perfusion scan using Technetium 99 were performed on 13 prospective cases. Tc99 pertechnitate was injected into antecubital vein and then arterial perfusion of thyroid was studied using gamma camera and on-line computer. Sixteen to twenty rapid sequence scintigrams were recorded at the rate of one frame every 3 sec. Region of interest (ROI) was drawn over the nodule, another ROI, containing the same number of pixels as the cold nodule, was drawn over normal adjoining thyroid tissue and in the background. Images showing maximum perfusion and minimum perfusion as well as static image at 20 min were analyzed. Ratio of difference between maximum and minimum activity were analyzed in each case.

Fine needle aspiration was performed using 23 G needle. Two slides were stained using Giemsa and Papanicolaou stain. Twenty four of the 25 prospective cases underwent surgery and histopathological examination along with immunohistochemistry was performed on these 24 prospective cases.

Retrospective group comprised of 25 cases presenting clinically as solitary nodule of thyroid over a period of 5 years. Paraffin-embedded tissue of these patients were taken for study. All cases were reviewed separately by two pathologists.

Immunohistochemistry was performed on these 25 retrospective cases as well as 24 prospective cases, which underwent surgical excision, using 3-mm thick sections on poly-l-lysine-coated slides. Antigen retrieval was done using microwave in citrate buffer at pH. 6. Monoclonal antibodies MIB1 (DAKO M7240) and p53 (DAKO M 7001) in dilution of 1:70 each were used for antigen detection by standard streptavidin avidin biotin kit (K 0679). Sections from a reactive lymph node were taken as positive control for Ki67 and melanoma for p53, whereas sections treated with tris-buffer solution instead of primary antibody were used as negative control. Brown nuclear reactivity was considered positive.Ki-67 was assessed by counting the number of positive and negative cells and the strongest stained tumor areas were chosen to evaluate labeling index (LI) (expressed as percentage of positively stained cells per 100 follicular epithelial cell) after counting at least 100 cells in each case. P53 scoring was done as 0 = 0 to 5%, 1+ = 6-10%, 2+ = 11-30%, and 3+ = >30% of positive tumor cell nuclei.

Statistical Analysis

Data were analyzed using statistical software package SPSS12. Statistical analysis of the data was performed using ANOVA (analysis of overall variance) test. P value of less than 0.005 as considered statistically significant.

   Results Top

Age of patients varied from 15 to 40 years with female to male ratio of 5:1. Size of nodule varied from 2-5 cm with 68% of them occurring in right lobe. In prospective group distribution of patients according to cytological diagnosis is summarized in [Table 1].
Table 1: Distribution of 25 prospective cases according to cytological diagnosis

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Cytohistological correlation was seen in 19 of 24 cases (80%). Out of eight cases with cytological diagnosis of nodular colloid goiter, one case turned out to be follicular thyroid adenoma (FTA) and one as Hashimoto's thyroiditis. Of three cases with cytologic diagnosis of hyperplastic nodule of colloid goiter, one case was found to have a focus of micropapillary carcinoma on histopathology and one was hyalinizing trabecular adenoma. One case of follicular neoplasm turned out to be multinodular goiter on histopathology.

Results of Ki-67 on immunohistochemistry are summarized in [Table 2]. A significant difference in mean value of Ki-67 positivity was found between benign and malignant nodules (P < 0.05). A statistically significant difference was observed in Ki-67 count between goiter versus follicular thyroid carcinoma (FTC) [Figure 1] and papillary thyroid carcinoma (PTC) (P < 0.05). Statistically significant difference was also observed between Ki- 67 count of follicular adenoma and carcinoma (P < 0.05). One case of hyperplastic nodule showed high Ki67 LI as an unexpected finding, although it is a benign lesion.
Table 2: Histopathological diagnosis and ki67 counts in histopathological sections

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Figure 1: Ki67 nuclear positivity in case of follicular carcinoma (IHC, x400)

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P53 immunostaining was positive in 2 cases of PTC and 1 case of FTC only. However, on statistical analysis, p valve was found to be significant (P = 0.037) in differentiating between benign and malignant lesions.

On radionuclide perfusion scan mean value of the difference between maximum and minimum nodular activity in benign nodules was 1.27 (S.D = 1.34), whereas in malignant nodules it was 3.47 (S.D = 2.11) and this difference was found to be statistically significant (P value = 0.044) [Table 3] and [Table 4] [Figure 2]. This suggests that decline in nodular activity in malignant cases after achieving a maximum level, is more as compared to benign cases. Values of nodular activity were also correlated with Ki-67 and p53 level. However, no correlation could be established.
Table 3: Nodular activity in benign cases compared to background

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Table 4: Nodular activity in malignant cases compared to background

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Figure 2: Radionuclide scan in a cold nodule

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   Discussion Top

Currently, the standard diagnostic tool for thyroid nodules is FNA cytology. FNA has a sensitivity of 25% to 42% for detection of FTC and hurthle cell carcinoma. [4] Therefore, benign and malignant follicular thyroid lesions cannot be distinguished by FNA cytology alone. In the present study, cytohistological correlation was found in 80% cases. This value is slightly lower than figure reported by Das et al. [5] in which case it was 88.9%.

During the last decade, there have been increasing attempts to find additional criteria to make accurate diagnosis of thyroid nodules. If successfully developed, a simple and low cost antibody based tests could be incorporated in most laboratories for routine evaluation of thyroid nodules. Present study highlights the use of Ki-67 and p53 in differentiating benign and malignant thyroid lesions. P53 is an important tumor suppressor gene as it integrates multiple stress signals and regulates cell response to DNA damage and is capable of inhibiting cell proliferation and transformation. [6] In thyroid malignancies, it serves as an independent prognostic factor for overall survival of patients. [7] We found p53 positivity in three cases on immunohistochemistry with diagnosis of FTC with widespread metastasis in one case and PTC in other two cases. This low positivity was probably due to lack of undifferentiated thyroid tumors in present study. Also, it has been proposed that p53 detection by immunohistochemistry, may be present in few cells in early primary tumors and only appears as a late event as compared to other techniques like polymerase chain reaction. Our findings are in agreement with those of Pollina et al. [8] and Okayasu et al. [9]

Ki-67 is recognized with monoclonal antibody MIB1. It is expressed in active phase of cell cycle and it rapidly degrade as the cell enters non proliferative state. [5] Various authors have successfully used MIB1 antibodies for separating benign and malignant thyroid tumor. [10],[11],[12] In the present study, significant differences in Ki-67 mean values of colloid goiter versus FTC and PTC were seen. Significant differences were also seen between FTA and FTC. Highest value of Ki-67 positivity was seen in cases of FTC followed by PTC. These findings are in close agreement with those of Erichson et al. [7] In present study, though there was significant difference in mean values between FTA and FTC, cut-off value cannot be given due to small sample size. Kjellman et al. [13] suggested a cut off of MIB1 index as 1.9 or more for discriminating malignant and benign follicular thyroid lesions. An index of 1.85 or more was found to be independently significant risk factor for a less favorable clinical course in papillary thyroid carcinoma.

However, these results are in contrast to those by Wallin et al. [14] who observed a Ki-67 LI of 0-1.1% in non neoplastic lesions, 0-3.1% in benign lesions, and 0.2-3.9% in malignant lesions. Rigaud et al. [15] mentioned that low rate of cell proliferation as seen by the MIB-1 staining was not helpful in establishing a malignant profile, but are consistent with an indolent rate of growth. Thus, according to them Ki-67 has no role in differentiating between benign and malignant lesion.

We also assessed radionuclide perfusion scan on 13 prospective cases. This scan, which assesses the vascularity of nodules using Tc99 pertechnitate, is a safe, simple and non expensive technique. It classifies nodules as hypervascular, equally vascular or avascular according to vascularity patterns of nodules. Various authors concluded that nodules showing hyperperfusion should be operated on where as patients with equally vascular or avascular nodules can be given a trial period of conservative clinical management as they are usually benign. [16] However, no definite diagnosis can be assigned. In our study, it was observed that radioactivity in malignant vascular nodule decay to lower levels after achieving peak. This decay is comparatively less in benign nodules as compared to malignant nodules. This difference in decay pattern was found to be statistically significant. However, larger studies are needed to confirm this fact as well as to establish the cut off level between benign and malignant nodules. Results of radionuclide perfusion studies were also correlated with levels of Ki-67 and p53 immunostaining. However, no correlation could be demonstrated. This suggests that increase in antigen levels due to proliferation and p53 mutation in malignant nodules have no effect on vascular pattern of nodules.

In conclusion, Ki-67 and p53 are candidate markers for differential diagnosis between benign and malignant thyroid lesions. Along with radionuclide perfusion scan, they help the clinicians in separation of FTC from FTA. However, since the sample size was small, larger studies are needed to confirm this observation as well as to assign the cut-off value for differentiating benign from malignant nodules.

   References Top

1.Rosai J, Carcangiu ML, Delellis RA. Tumors of the thyroid gland. Atlas of tumor pathology. Washington, DC: Armed Forces Institute of Pathology; 1992. p. 1-34.  Back to cited text no. 1
2.Tan GH, Gharib H. Thyroid incidentalomas: Management approaches to nonpalpable nodules discovered incidentally on thyroid imaging. Ann Intern Med 1997;126:226-31.  Back to cited text no. 2
3.Hundahl SA, Fleming ID, Fremgen AM, Menck HR. A national cancer data base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985-1995. Cancer 1998;83:2638-48.  Back to cited text no. 3
4.Yeh MW, Demircan O, Ituarte P, Clark OH. False-negative fine-needle aspiration cytology results delay treatment and adversely affect outcome in patients with thyroid carcinoma. Thyroid 2004;14:207-15.  Back to cited text no. 4
5.Das DK, Khanna CM, Tripathi RP, Pant CS, Mandal AK, Chandra S, et al. Solitary nodular goiter. Review of cytomorphologic features in 441 cases. Acta Cytol 1999;43:563-72.  Back to cited text no. 5
6.Ozolins A, Narbuts Z, Strumfa I, Volanska G, Gardovskis J. Diagnostic utility of immunohistochemical panel in various thyroid pathologies. Langenbecks Arch Surg 2010;395:885-91.  Back to cited text no. 6
7.Dobashi Y, Sakamoto A, Sugimura H, Mernyei M, Mori M, Oyama T, et al. Overexpression of p53 as a possible prognostic factor in human thyroid carcinoma. Am J Surg Pathol 1993;17:375-81.  Back to cited text no. 7
8.Pollina L, Pacini F, Fontanini G, Vignati S, Bevilacqua G, Basolo F. Bcl- 2, p53 and proliferating cell nuclear antigen expression is related to the degree of differentiation in thyroid carcinomas. Br J Cancer 1996;73:139-43.  Back to cited text no. 8
9.Okayasu I, Osakabe T, Onozawa M, Mikami T, Fujiwara M. p53 and p21(waf1) expression in lymphocytic thyroiditis and thyroid tumors. Clin immunol and Immunopathol 1998;88:183-91.  Back to cited text no. 9
10.Erickson LA, Jin L, Wollan PC, Thompson GB, van Heerden J, Lloyd RV. Expression of p27 kip1 and Ki-67 in benign and malignant thyroid tumors. Mod Pathol 1998;11:169-74.  Back to cited text no. 10
11.Saiz AD, Olvera M, Rezk S, Florentine BA, McCourty A, Brynes RK. Immunohistochemical expression of cyclin D1, E2F-1, and Ki-67 in benign and malignant thyroid lesions. J Pathol 2002;198:157-62.  Back to cited text no. 11
12.Horii A, Yoshida J, Sakai M, Okamoto S, Honjo Y, Mitani K, et al. Ki-67 Positive Fractions in Benign and Malignant Thyroid Tumours: Application of Flow Cytometry. Acta Otolaryngol 1999;119:617-20.  Back to cited text no. 12
13.Kjellman P, Wallin G, Hoog A, Auer G, Larsson C, Zedenius J. MIB-1 Index in Thyroid Tumors: A Predictor of the Clinical Course in Papillary Thyroid Carcinoma. Thyroid 2003;13:371-80.  Back to cited text no. 13
14.Wallin G, Backdahl M, Christensson B, Grimelius L, Auer G. Nuclear protein content and Ki-67 immunoreactivity in nonneoplastic and neoplastic thyroid cells. Anal Quant Cytol Histol 1992;14:296-303.  Back to cited text no. 14
15.Rigaud C, Bogomoletz WV. Apparent lack of usefulness of monoclonal antibody Ki-67 in thyroid tumor pathology: Relation to histological typing and classification. Pathol Res Pract 1991;187:198-200.  Back to cited text no. 15
16.Prakash R, Lakshmipathi N, Jena A, Narayanan RV, Behari V. Computer-assisted radionuclide perfusion study in solitary cold thyroid nodules for diagnosis of malignancy. Eur J Nucl Med 1985;11:143-6.  Back to cited text no. 16

Correspondence Address:
Smita Singh
Department of Pathology, Lady Hardinge Medical College and Smt S.K. Hospital, Shaheed Bhagat Singh Marg, New Delhi - 110 001
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.85077

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  [Table 1], [Table 2], [Table 3], [Table 4]

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