Study of Association of AgNOR vs Ki-67 in Thyroid Lesions

The thyroid is an important endocrine gland, which plays a major role in the functions of almost all normal metabolic processes. In present study, by focusing the sections at very high magnification (100x, oil immersion level) AgNOR dots are visible as black dots inside the nucleus. Our studies which have reported that thyroid lesions with less AgNOR counts are more likely to regress as compared to those with high AgNOR counts and that showed its prognostic significance. Thus, the AgNOR technique can be useful as a supportive tool in both the prognosis and therapeutic value in thyroid lesions most commonly in malignant lesions that are stained by routinely performed haematoxylin and eosin. The Present Study is aim to find out the prevalence of thyroid lesions in the patients and also to Study and find out the different types of neoplastic and nonneoplastic thyroid lesions and their relation with AgNOR and Ki-67 proliferation makers. The Present Study focuses on to evaluate the association of AgNOR vs Ki-67 in the prognostic value of thyroid lesions.


INTRODUCTION
Thyroid nodules which are more commonly encountered during routine medical care and their prevalence increases with age, with the average of 4−7% in adult population [1]. Thyroid carcinoma, which accounts for approximately 1% of all the cancers, is the most common malignancy among all the endocrine organs [2]. Approximately 5% occur as thyroid cancers among all the thyroid nodules, irrespective of their size [3]. Based on histopathology, thyroid carcinomas are classified into follicular thyroid cancer (FTC), papillary thyroid cancer (PTC), medullary thyroid cancer (MTC), anaplastic thyroid cancer and poorly differentiated thyroid cancer [4]. It has been documented that 90 people were diagnosed with thyroid cancer out of 1,00,000 people and among these patients,6.8 people were died in china. It has also been reported that 1,980 people died of the disease in 2016 and it has been estimated that there will be 64,300 new patients of thyroid cancer in USA [4]. The diagnosis of thyroid cancer is often based on magnetic resonance imaging, ultrasound, computed tomography, fine needle aspiration and radionuclide imaging. Although various methods are well applied in clinics, patients often have poor outcome. Reports have shown that the patients' age when being diagnosed, sex, tumour size, lymph node, distant metastases and pathologic differentiation of the cancer are the risk factors of prognosis in thyroid cancer.
Of all thyroid cancers, PTC is found as the most common form of thyroid cancer. Though it is found difficult to differentiate PTC from benign papillary hyperplasia of the thyroid gland based on their morphology, it is important to differentiate benign ones from malignant ones for early treatment and better patient management. Due to overlapping of morphological features, distinction of certain neoplasms was found difficult. Though characteristic nuclear changes were found important for the diagnosis of papillary thyroid carcinoma (PTC), but when these features are present focally along with papillary structures then they cause diagnostic dilemma in distinguishing it from other thyroid lesions [5].
Histomorphologic examination of slides which are routinely stained by Haematoxylin and Eosin (H&E) is used as the standard diagnosis currently. This has been documented in literature that interobserver or intraobserver disagreements in the diagnosis of follicular and papillary thyroid lesions [6]. A large number of molecular alterations in thyroid cancer have been documented in distinguishing malignant from benign thyroid lesions, in the recent years.Alterations of some proteins have been observed in thyroid cancer, such as, Ki67, CK19, Calcitonin, TG, TTF-1, RET, HBME-1, BRAF, FHL1, galectin-3, TfR1/CD71 and SERPINA1 [7] and have been translated into clinical practice which were used for the significant improvement in the preoperative diagnosis of thyroid cancer [8]. Among these markers, this has been found that, Ki67 and cytokeratin-19 (CK-19), galectin-3, and TG have been used most frequently in thyroid pathology.
Several newer Immunohistochemistry (IHC) markers have been described and validated for differentiating benign from malignant ones and Follicular Variant of Papillary Carcinoma (FVPTC) from Follicular Carcinoma (FCa) or Follicular Adenoma (FA) [8]. However, none of the studies showed the markers had prognostic value in thyroid cancer. Cell proliferative activity is an important factor in cancer biologic behaviour. Different proliferation markers such as Ki67 [9], proliferating cell nuclear antigen (PCNA) [10] and argyrophilic nucleolar organizer regions (AgNORs) [11] have been examined and observed their importance in thyroid tumours. Even though the diagnostic and prognostic values of Ki-67/MIB-1 in thyroid cancer remain unclear, but it has been observed that Ki-67/MIB-1 has an effect on the clinical progression and prognosis in cancers with the growing evidence [4]. Ki-67, which is considered as a nuclear antigen, is expressed in all the cell nuclei, except those in the G0 phase. MIB-1 acts as a monoclonal antibody and that increases against Ki-67 [4]. In recent years, Ki-67 has been studied in many cancers, including cervical cancer, [12] lung cancer, [13] breast cancer [14] and thyroid cancer [15]. This has been reported that Ki-67 is also found as an important and independent prognostic factor in thyroid cancer patients, in many studies [16][17][18][19][20] The nucleolar organizer regions (NORs) which are DNA segments, that encode ribosomal RNA [21]. NORs are associated with some nucleolar proteins which are usually called as AgNOR proteins or AgNORs, that are stained black with silver methods [22,23]. It has also been reported that other than Ki-67, AgNORs are also related to the rate of cell proliferative activity [24]. As they also found useful in distinction, so they can be used in distinguishing different lesions [25]. Many studies show a linear correlation between AgNOR and Ki-67 in different malignancies [26,27].

Sources of Data
This study was carried out in the Department of Pathology, Sree Balaji Medical College & Hospital, Bharath Institute of Higher Education and Research during my study period of 17 months that is between February 2017 to July 2018. A total of 78 specimens of hemi thyroidectomy and total thyroidectomy were received for histopathological examination from Department of Surgery. Out of 78 thyroid specimens, 60 specimens were selected for study by excluding the remaining non-neoplastic lesions of thyroid like Hashimoto's thyroiditis, autoimmune thyroiditis and other thyroiditis to avoid errors of statistical analysis.

Inclusion Criteria
A total of 60 cases were included in this study and according to WHO guidelines, all these cases were diagnosed. The cases included were: 25 PTC (in which 12 were of classic variant, 7 were of follicular variant, 2 were of solid variant, 4 were of oncocytic variant), 3 FTC (in which 2 were of oncocytic variant), 10 benign neoplasms (in which 8 were follicular micro and macroadenoma, 2 were hurthle cell adenoma), 22 nodular goitres (NG).

Exclusion Criteria
The cases with medullary, anaplastic carcinomas and poorly differentiated carcinoma cases were excluded from the current study as any of these cases were diagnosed in our department in the study period of 17 months and also excluded few non-neoplastic lesions like Hashimoto's thyroiditis, autoimmune thyroiditis and other thyroiditis which were diagnosed in our department to avoid errors of statistical analysis.

Method of Data Collection
Out of the 78 thyroid specimens which were received for histopathological examination following initial cytological evaluation by fine needle aspiration cytology, 60 gross specimens were taken for study. These biopsy specimens were routinely fixed in 10% formalin and processed in paraffin wax.5 microns thick Sections were cut and taken for the study and hematoxylin and eosin staining of sections was done. Histopathological examination of these sections were done. Special stain (AgNOR Stain) and Immunohistochemical study using Ki-67 monoclonal antibody as a proliferation marker was done in 60 cases.

Nucleus: stained blue colour
Connective tissue and cytoplasm stained: pink colour

AgNOR Staining
AgNOR staining was performed by adopting both previous and also modified procedures. In previous methods counter stain (Neutral red) was used. But in this study, neutral red counter stain was not used. Table:1) Solution A: This solution was prepared by dissolving 500 mg gelatin powder in 25 ml deionized water at 37 degrees C and then 250 micro L formic acid was added. Continuous shaking for about 10 min at 37 degrees C was sufficient to dissolve the gelatin and a clear solution was obtained.

Solution B:
It was prepared with silver nitrate and deionized water.

Working solution
This was prepared by mixing one part by volume of solution A with two parts by volume of

AgNOR Staining Procedure
The AgNOR technique was adapted from the study as mentioned earlier and also from the study which was described by Crocker and Nar. [28] 

AgNORs Quantitative Analysis21
From the selected areas, an average of 5-10 microscopic fields, were analysed from each case, at magnification 100x (Under oil immersion). 100 nuclei were analysed in each case, whole nuclei were taken as actual lesion.
Each overlapped and fragmented nuclei were counted and taken as 1.

Ki-67 Immunohistochemistry and Determination of the Labeling Index (LI)
IHC of Ki-67 technique was adopted from Path in Situ IHC Protocol:

KI-67 Labelling Index
Ki-67 labelling index was analyzed in 5 different high-power microscopic fields (40x) and positive cells that showed brown nuclear reactivity were counted22. Generally, Ki-67 LI was observed higher in the peripheral portion than in the central portion of the lesion23. Values were expressed in percentages.

Statistical Analysis
 A statistical analysis was performed with the use of SPSS version  For multiple comparisons, post hoc Bonferroni test was used to detect the exact significance between the groups. The P value of ≤0.05 was taken as significant.

RESULTS
The present study consists of 60 cases of thyroid masses which were removed surgically and were sent for the Histopathological Examination to our department (Department of Pathology, SBMCH). All these specimens were received from the Department of Surgery, SBMCH.
 Nodules may be present and also reactive fibrosis surrounding nodule present but without thick capsule.
 Papillary projections (Sanderson polsters) may be seen in cystically dilated follicles; and they may mimic papillary carcinoma but they lack nuclear features of PTC.
 Degenerative changes like haemorrhage, cystic change, foamy histiocytes and giant cell formation in the goitre.   There will be no necrosis, no squamous metaplasia and no/rare lymphatic invasion25. During 17 months study period, total of 2893 biopsy specimens were received in our department for histopathological examination. Among these cases, a total of 1172 cases were received from surgery department, out of which 78 cases were thyroid specimens. The prevalence of thyroid specimens coming to our department from all the departments is 2.70%, out of the specimens from the surgery department, the prevalence of thyroid specimens is 6.68%.

HISTOPATHOLOGICAL EVALUATION OF THYROID LESIONS
The histopathological diagnosis was done for all the 78 thyroid cases, out of them 60 cases were taken for study by excluding few Non-neoplastic lesions like inflammatory lesions27 as mentioned earlier and their distribution in the current study was shown in Table:3 and chart:1.

Fig. 1. Graphical representation showing diagnosis of different cases
Among the distribution of thyroid lesions, the prevalence of papillary carcinoma of thyroid cases were reported as more common which were of 41.67% in this study followed by nodular colloid goitre of 36.67%. Follicular carcinoma was of least prevalence of 5%. No medullary carcinoma and anaplastic carcinoma cases were received during this study period.

DISCUSSION
During the last decade, there have been increasing attempts to find out additional criteria to make accurate diagnosis of thyroid nodules. The simple and low-cost antibody can be incorporated in most of the laboratories if they are proved successful, for routin of thyroid nodules [1]. It is urgent to find out the reliable and repeatable IHC markers to distinguish between benign thyroid nodules and malignant lesions. Many studies have been tried to classify various biomarkers for thyroid carcinoma till date, on the basis of their gene expression profiles. Alterations of some proteins such as CK19, TG, galectin-3, Ki67, Calc TTF-1, BRAF, RET, TfR1/CD71, FHL1, HBME 1, SERPINA1 were found in thyroid cancer. Some of these proteins can be applied and used in clinical practice to differentiate thyroid cancer from benign thyroid lesions [8].
Widespread application of AgNORs in tumour histopathology has been reported in assessing the growth potential and malignant potential of tumours [28], to distinguish between benign and malignant neoplasms, to assess the prognosis and also to evaluate the risk of recurrence Present study highlighted the use of Ki AgNOR counts in differentiating benign and malignant thyroid lesions [30].

Fig. 1. Graphical representation showing diagnosis of different cases
Among the distribution of thyroid lesions, the prevalence of papillary carcinoma of thyroid cases were reported as more common which were of 41.67% in this study followed by nodular colloid goitre of 36.67%. Follicular carcinoma 5%. No medullary carcinoma and anaplastic carcinoma cases were During the last decade, there have been increasing attempts to find out additional criteria to make accurate diagnosis of thyroid nodules.
cost antibody-based tests can be incorporated in most of the laboratories if they are proved successful, for routine evaluation It is urgent to find out the reliable and repeatable IHC markers to benign thyroid nodules and malignant lesions. Many studies have been tried to classify various biomarkers for thyroid carcinoma till date, on the basis of their gene expression profiles. Alterations of some proteins 3, Ki67, Calcitonin, 1, BRAF, RET, TfR1/CD71, FHL1, HBME-1, SERPINA1 were found in thyroid cancer. Some of these proteins can be applied and used in clinical practice to differentiate thyroid cancer AgNORs in tumour histopathology has been reported in assessing the growth potential and malignant potential of , to distinguish between benign and malignant neoplasms, to assess the prognosis and also to evaluate the risk of recurrence [29]. esent study highlighted the use of Ki-67 and AgNOR counts in differentiating benign and

Incidence of Thyroid Lesions
60 gross specimens were taken for study which were received for histopathological after initial cytological evaluation by FNAC (fine needle aspiration cytology) in our department of pathology, Sree Balaji Medical College, Chennai. In present study, Non-neoplastic lesions accounts for 22 cases and neoplastic lesions accounts for 38cases.
The ratio between non-neoplastic and neoplastic thyroid lesions in this study is 1:1.72. During 17 months study period, total of 2893 biopsy specimens were received in our department for histopathological examination. Among these cases, a total of 1172 cases were received from surgery department, out of which 78 cases were thyroid specimens. The histopathological diagnosis was done for all the 78 thyroid cases, out of them 60 cases were taken for study by excluding few Non-neoplastic lesions inflammatory lesions which were received for histopathological examination following initial cytological evaluation by fine needle aspiration cytology in our department of pathology in Sree Balaji Medical College, Chennai.
In present study, Non-neoplastic lesions accounts for 22 cases and neoplastic lesions accounts for 38 cases. The ratio between non neoplastic and neoplastic thyroid lesions in this study is 1:1.72. The prevalence of thyroid specimens coming to our department from all the departments is 2.70%, out of the specimens from the surgery department, the prevalence of thyroid specimens is 6.68%.
Among the distribution of thyroid lesions, the prevalence of papillary carcinoma cases were ; Article no.JPRI.66762

Fig. 1. Graphical representation showing diagnosis of different cases
Thyroid Lesions 60 gross specimens were taken for study which were received for histopathological examination after initial cytological evaluation by FNAC (fine needle aspiration cytology) in our department of pathology, Sree Balaji Medical College, Chennai. neoplastic lesions accounts for 22 cases and neoplastic lesions neoplastic and neoplastic thyroid lesions in this study is 1:1.72. During 17 months study period, total of 2893 biopsy specimens were received in our department for histopathological examination. Among these tal of 1172 cases were received from surgery department, out of which 78 cases were thyroid specimens. The histopathological diagnosis was done for all the 78 thyroid cases, out of them 60 cases were taken for study by neoplastic lesions like inflammatory lesions which were received for histopathological examination following initial cytological evaluation by fine needle aspiration cytology in our department of pathology in Sree lastic lesions accounts for 22 cases and neoplastic lesions accounts for 38 cases. The ratio between nonneoplastic and neoplastic thyroid lesions in this study is 1:1.72. The prevalence of thyroid specimens coming to our department from all the s is 2.70%, out of the specimens from the surgery department, the prevalence of thyroid Among the distribution of thyroid lesions, the prevalence of papillary carcinoma cases were reported as more common which were of 41.67% in this study followed by nodular colloid goitre of 36.67%. Follicular Adenoma cases were reported as 16.67%. Follicular carcinoma cases were reported as least prevalence of 5 %. A total of 60 patients belonging to the age group of 19-70 years with the mean age of 43.08 years, were subjected to histopathological study of thyroid lesions. Female patients were of 81.67% and male patients were of 18.33% and with female to male ratio was of 4.5:1.
The present study showed that mean mAgNOR in case of nodular goitre was 1.59 (SD ± 0.13) with a range of 1.4-1.75, in benign lesions i.e.; Follicular Adenoma / Hurthle cell Adenoma was 3.63 (SD ±0.41) with a range of 2.9 -4.05, in malignant lesions i.e.; in papillary carcinoma was 2.77 (SD ± 0.56) with a range of 1.5 -4.05 and in follicular carcinoma was 4.92 (SD± 0.27) with a range of 4.68 -5.21, highest in FTC followed by FA.
Although overlapping in range values were seen in both follicular adenoma and Papillary carcinoma, with the mean mAgNOR values and pAgNOR values, still it can be suggested that mAgNOR count and pAgNOR values can differentiate follicular adenoma and papillary carcinoma and also can differentiate nonneoplastic, benign and malignant lesions of thyroid.

CONCLUSION
The data which is currently available established that the evaluation of the quantity of interphase AgNORs is an independent prognostic factor in human tumour types. They are useful in discriminating between benign and malignant conditions being significantly higher in neoplastic cells than in normal cells and also useful as a significant prognostic indicator in malignant lesions. As the mean number of AgNORs is proportional to the rapidity of cell duplication, it was also found that it was related to the proliferation rate of tumour cell population. It has also been suggested that the number of AgNORs might have few diagnostic and prognostic values in different neoplasms. Although, many studies show a linear correlation between AgNOR and Ki-67 in different malignancies [26,27], the counting of mean AgNOR is more sensitive, simple and cost effective as compared to Ki-67 LI for differentiating between benign and malignant thyroid follicular neoplasms. Therefore, use of both proliferation markers can be suggested to differentiate thyroid lesions in difficult situations.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
As per international standard or university standard ethical approval has been collected and preserved by the authors.