|Year : 2018 | Volume
| Issue : 1 | Page : 22-26
|Role of calcium-sensing receptor, Galectin-3, Cyclin D1, and Ki-67 immunohistochemistry to favor in the diagnosis of parathyroid carcinoma
Nuran Sungu1, Hayriye Tatli Dogan1, Aydan Kiliçarslan1, Mehmet Kiliç2, Sefika Polat3, Mehmet Tokaç4, Soner Akbaba2, Ömer Parlak2, Serdar Balci1, Betül Ögüt5, Bekir Çakir3
1 Department of Pathology, Ankara Yıldırım Beyazit University, Faculty of Medicine, Ataturk Education and Research Hospital, Ankara, Turkey
2 Department of General Surgery, Ankara Yıldırım Beyazit University, Faculty of Medicine, Ataturk Education and Research Hospital, Ankara, Turkey
3 Department of Endocrinology and Metabolism, Ankara Yıldırım Beyazit University, Faculty of Medicine, Ataturk Education and Research Hospital, Ankara, Turkey
4 Department of General Surgery, Yeni Yüzyıl University, Gaziosmanpaşa Hospital, İstanbul, Turkey
5 Department of Pathology, Gazi University, Faculty of Medicine, Ankara, Turkey
Click here for correspondence address and email
|Date of Web Publication||22-Mar-2018|
| Abstract|| |
Background: As histopathological findings of parathyroid carcinoma are not certain, the diagnosis of tumors with degenerative changes may be difficult. In these cases, immunohistochemical markers are beneficial. We aimed to research the acceptability of calcium-sensing receptor (CaSR), Galactin-3, Cyclin D1, and Ki-67 as helpful markers in parathyroid tumors in cases which are difficult to diagnose. Materials and Methods: Those cases who had been diagnosed with atypical parathyroid adenoma and parathyroid carcinoma between 2010 and 2015 were reevaluated. İmmunohistochemical markers were applied to this cases. Results: About 21 cases were parathyroid adenoma, 14 were atypical adenoma, and 10 cases were parathyroid carcinoma. According to the immunohistochemical results, global loss of CaSR staining was seen in 50% (5/10) of the patients with carcinoma while there was no loss of staining in those with parathyroid adenoma (P = 0,001). Global loss of CaSR staining was found in only one out of 14 cases with atypical adenoma. The expression of Galactin-3 was found to be positive in 40% (4/10) of carcinoma cases, 71.4% (10/14) of those with atypical adenoma, and 14.3% (3/21) of those with adenoma (P = 0,002). Cyclin D1 expression was determined to be positive in 70% (7/10) of patients with carcinoma, 71.4% (10/14) of atypical adenoma cases, and 23.8% (5/21) of those with adenoma. The Ki-67 proliferation index was seen to be above 5% in 50% (5/10) of carcinoma cases and 35,7% (5/14) of those with atypical adenoma. Conclusion: In these studies, it has been emphasized that the global loss of CaSR staining was used as a negative marker in the diagnosis of carcinoma. In this study, we have also confirmed that the global loss of CaSR staining is a useful marker to determine potential increased malignancy.
Keywords: Atypical parathyroid adenoma, calcium-sensing receptor, parathyroid adenoma, parathyroid carcinoma
|How to cite this article:|
Sungu N, Dogan HT, Kiliçarslan A, Kiliç M, Polat S, Tokaç M, Akbaba S, Parlak &, Balci S, Ögüt B, Çakir B. Role of calcium-sensing receptor, Galectin-3, Cyclin D1, and Ki-67 immunohistochemistry to favor in the diagnosis of parathyroid carcinoma. Indian J Pathol Microbiol 2018;61:22-6
|How to cite this URL:|
Sungu N, Dogan HT, Kiliçarslan A, Kiliç M, Polat S, Tokaç M, Akbaba S, Parlak &, Balci S, Ögüt B, Çakir B. Role of calcium-sensing receptor, Galectin-3, Cyclin D1, and Ki-67 immunohistochemistry to favor in the diagnosis of parathyroid carcinoma. Indian J Pathol Microbiol [serial online] 2018 [cited 2019 Mar 21];61:22-6. Available from: http://www.ijpmonline.org/text.asp?2018/61/1/22/228201
| Introduction|| |
Parathyroid carcinomas are malignant tumors which are rarely observed and are extremely aggressive. They constitute <1% of primary hyperparathyroidism (HRPT) cases. According to the World Health Organization (WHO), the histopathological criteria of parathyroid carcinoma are vascular invasion, perineural invasion, and capsular penetration with extension into the adjacent tissue and/or metastasis. In addition, according to the WHO, broad fibrous bundles with or without hemosiderin-laden macrophages, mitosis, neoplastic cell groups in the thick fibrous capsula, which are the other criteria of parathyroid carcinoma that do not contain capsular or vascular invasion, are all evaluated as atypical adenoma., These heterogeneous morphological configurations cause difficulties in the diagnosis.
Different immunohistochemical markers have been used in these groups where the diagnosis is difficult. Most researchers have emphasized that multiple markers should be used rather than just one.
Calcium-sensing receptor (CaSR), which has recently increased in predictive and prognostic importance, does not only regulate the synthesis and secretion of the parathyroid hormone but also regulates the proliferation of parathyroid glands. According to recent studies, a decrease in or loss of CaSR expression has been observed in several malignancies such as parathyroid, colorectal, and neuroblastoma. In fact, an increase in CaSR expression has been shown in prostate, testicular, ovarian, and breast carcinomas. It has been demonstrated that CaSR acted as a tumor suppressor gene in the first situation and as an oncogene in the second. In reality, these mechanisms have so far not been clearly defined.
Galactin-3, Cyclin D1, and Ki-67 are the other important markers. Galactin-3 is a multifunctional protein and contains several biological functions such as tumor cell adhesion, proliferation, differentiation, angiogenesis, cancer progression, and metastasis. The overexpression of Galactin-3 has been correlated with thyroid and breast cancers.,,
K-67 is a proliferation marker which helps to differentiate between adenomas and carcinomas. When Ki-67 is above 5%, there is an increased risk of malignancy.,
Cyclin D1 which is one of the cell growth factors displays different expressions in adenomas and carcinomas. Studies have shown that there is heterogeneous staining with Cyclin D1 in benign and malignant parathyroid neoplasias., Cyclin D1 may signify overexpression in parathyroid carcinomas.
We aimed to determine the supporting roles of immunohistochemical markers such as CaSR, Cyclin D1, Galactin-3, and Ki-67 with any histopathological findings in the diagnosis of parathyroid carcinoma.
| Materials and Methods|| |
Those cases who had been diagnosed with atypical parathyroid adenoma and parathyroid carcinoma between 2010 and 2015 were extracted from the authors' archives and reevaluated. The diagnosis of parathyroid carcinoma is suspected by intraoperative features of local invasion and confirmed by the WHO (2004) histopathological criteria for parathyroid carcinoma. A total of 22 cases diagnosed with adenoma were included in the study as the control group. Ki-67, Galactin-3, Cyclin D1, and CaSR antibodies were immunohistochemically performed on whole surface sections which were prepared from formalin-fixed-paraffin-embedded tissues that included 14 atypical adenoma, 10 carcinoma, and control cases.
Archival sections were deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min. To unmask the antigen, heat-induced antigen retrieval was performed using 10 mm sodium citrate (pH 6) buffer microwaved for 10 min. Subsequently, antigen retrieval tissues were blocked in 3% bovine serum albumin-phosphate-buffered saline for 30 min. After blocking, the sections were incubated with antihuman CaSR monoclonal antibody (diluted 1:100, Abcam, clone 5C10, ab 19347), antihuman Galectin-3 (diluted 1:100, Novocastra, clone 9C4, NCL-GAL3), antihuman Cyclin D1 (diluted 1:100, Cell Marque, clone SP4) A, and with Ki-67 (diluted 1:100, Biocare, clone SP6). After washing, the slides were incubated with biotinylated secondary antibodies and streptavidin-horseradish peroxidase complex for 30 min each. Finally, staining was visualized using diaminobenzidine.
Normal parathyroid tissue was accepted as providing an internal positive control in CaSR staining. Strong complete membrane stainings that were seen in normal parathyroid tissue were accepted as positive. Samples scored as negative showed irregular and faint staining [Figure 1]b. Complete membrane staining of <10% was evaluated as global loss. Galectin-3 cytoplasmic or membranous staining above 30% were accepted as positive [Figure 2]. Cyclin D1 was scored as positive if specific nuclear staining above 5% was detected [Figure 3].
|Figure 1: Immunohistochemical analyses of calcium-sensing receptor expression. (a) Parathyroid adenoma. The majority of cells showed a membranous immunoreactivity for calcium-sensing receptor (×400 magnification). (b) Global loss of expression in parathyroid carcinoma (×200 magnification) (Yellow arrow: Normal parathyroid cells showed a diffuse membranous staining)|
Click here to view
|Figure 2: The majority of cells showed a cytoplasmic or membranous staining for Galectin-3 in parathyroid carcinoma (×400 magnification)|
Click here to view
|Figure 3: The most of cells seen a nuclear staining for Cyclin D1 in parathyroid carcinoma (×200 magnification)|
Click here to view
Chi-square and Fisher's exact test analysis was undertaken using the SPSS software (version 16, IBM corp. USA) to determine any association between categorical variables.
| Results|| |
There were 21 cases with parathyroid adenoma, 15 cases with atypical adenoma, and 9 cases with carcinoma. The mean length of follow-up of the 9 cases who were diagnosed with parathyroid carcinoma was 31 months. Relapse was seen during the follow-up in two cases who had previously been diagnosed with carcinoma. When these two cases were diagnosed 2 years later, hypercalcemia was confirmed, and bone metastases were observed in whole-body scintigraphy. The mean length of follow-up of the 15 cases who were diagnosed with atypical adenoma was 30 months. One case was reclassified as being carcinoma 2 years later because hypercalcemia was confirmed and cervical lymph node metastases were seen in whole-body scintigraphy. Thus, the number of carcinoma cases increased to 10. The mean age was 50.2 ± 12.6 years (min: 19, max: 80). No difference was found concerning the mean age between the carcinoma, adenoma, or atypical adenoma cases (P = 0, 5). Thirty-one (68.9%) of our cases were female. Gender distribution was no difference between cases with or without carcinoma (P = 0, 7).
About 40% (4/10) of the carcinoma cases had vascular invasion, 70% (7/10) had capsular invasion, and 50% (5/10) had common invasion to the surrounding tissue. Nuclear atypia was found in 50% of carcinoma cases and fibrous septation was found in all carcinoma patients. Mitosis was found in 50% of carcinoma and 28,6% of atypical adenoma cases per 50 high power microscopic fields [Table 1].
|Table 1: Distribution of histological features in different diagnostic histological criteria|
Click here to view
Global loss of staining of CaSR was seen in 5 (50%) carcinoma patients, but in only one case (3%) of the 35 with adenoma. This particular case, who demonstrated loss of staining, had atypical adenoma. Global loss of staining was not found in parathyroid adenoma cases which were not atypical [Figure 1]a. A significance concerning the global loss of staining of CaSR was found between those with adenoma, atypical adenoma, and carcinoma (P = 0,001) [Table 2] and [Table 3].
|Table 2: Global loss of calcium-sensing receptor staining, Cyclin D1 and Galectin-3 overexpression of cases|
Click here to view
|Table 3: Global loss of calcium-sensing receptor staining, Cyclin D1 and Galectin-3 overexpression of parathyroid adenoma and other parathyroid tumor (atypical adenoma and parathyroid carcinoma) cases|
Click here to view
The overexpression of Galactin-3 was found to be positive in 40% (4/10) of carcinoma cases, 71.4% (10/14) of atypical adenoma cases, and 14.3% (3/21) of those with adenoma. If carcinoma and atypical adenomas were evaluated together, then the rate (58.3%) was higher than among those with adenoma (14.3%) (P = 0,002) [Table 2] and [Table 3].
Cyclin D1 was found to be positive in 70% (7/10) of carcinoma patients, 71.4% (10/14) of atypical adenoma cases, and 23.8% (5/21) of those with adenoma. If carcinoma and atypical adenomas were evaluated together then the rate (70.8%) was higher than among those with adenoma (23.8%) (P = 0,002) [Table 2] and [Table 3].
Vascular invasion was observed in 2 cases and peripheral tissue invasion was seen in one out of 3 cases with recurrence/metastasis. In addition, global loss of CaSR and positive staining with Galactin-3 and Cyclin D1 were seen in both of these two cases who had vascular invasion.
The Ki-67 proliferation index was seen to be above 5% in 50% (5/10) of carcinoma cases and 35,7% of atypical adenoma cases [Figure 4]a and [Figure 4]b.
|Figure 4: Immunoreactivity for Ki67. (a) Low Ki 67 proliferation index in parathyroid adenoma (×400 magnification). (b) High Ki 67 proliferation index in parathyroid carcinoma (×200 magnification)|
Click here to view
There was no difference between the carcinoma and adenoma cases concerning preoperative PTH levels. Preoperative calcium levels were found to be above 11.5 in 78% of carcinoma cases. During the clinical follow-up, postoperative calcium and PTH levels were seen to be normal.
The tumor size was found to be higher in carcinoma cases (median 3.25) than among those with adenoma and atypical adenoma (median 2.5) (P = 0,017).
| Discussion|| |
In this study, we found that half of the carcinoma cases had global loss of CaSR staining. No global loss of staining was found in those with adenoma.
There are very few studies about CaSR in parathyroid carcinomas. We have not previously seen any study that researches CaSR in atypical adenomas. When we compared atypical adenomas with other adenomas in our study, we found that global loss of CaSR was not seen in any adenoma case and was seen in only one case of atypical adenoma. When we compared atypical adenomas with carcinomas, global loss of CaSR was seen in 50% of carcinomas and 7% of atypical adenomas. These results showed that CaSR is an important biomarker to help differentiate carcinomas from adenomas (atypical or not) rather than differentiate adenomas from atypical adenomas. Haven et al. found global loss of CaSR in 9 out of 29 carcinoma cases and one out of 104 adenoma cases in their study with tissue microarray sections in parathyroid carcinoma and adenoma cases. We also found loss of CaSR in only one adenoma case compatible with this study. Witteveen et al. found loss of CaSR in 7 out of 23 cases in their study with only parathyroid carcinoma case. We found a higher loss of CaSR in carcinoma cases. We thought the reason for this was that we had researched whole surface sections.
Downregulation of CaSR was found to be correlated with poor prognosis in other tumors, especially colorectal and gastric tumors. CaSR overexpression was determined as being a poor prognostic marker in prostate, breast, renal, and lung carcinomas. Downregulation of CaSR was found to be correlated with poor prognosis in parathyroid carcinoma. Witteveen et al. found both the 5-year disease-free survival and the 5-year overall survival were lower in patients with downregulation of CaSR expression compared with patients with normal CaSR expression (81 vs. 0% and 94 vs. 29%, respectively). In this study, global loss was found in 2 out of 3 relapse cases.
Galactin-3 is important in the diagnosis of follicular thyroid malignancies and in tumor development., Recent studies have determined Galactin-3 overexpression in parathyroid carcinomas., Fernandez-Ranvier et al. have emphasized that the loss of parafibromin, retinoblastoma expression, and Galactin-3 overexpression could be used to differentiate parathyroid carcinomas from other parathyroid tumors in their study using multiple immune markers. We observed Galactin-3 overexpression in both carcinomas and atypical adenomas.
Truran et al. and Cetani et al. have found heterogeneous staining with Cyclin D1 in both benign and malignant parathyroid neoplasia in their studies which included parafibromin and Cyclin D1., In fact, in our study, we found Cyclin D1 expression to be higher among atypical adenomas and carcinomas than in adenomas.
Ki 67 is usually lower in adenomas than in carcinomas. In fact, its diagnostic performance is limited. If the Ki-67 index is above 5%, there is an increased risk of malignancy. We found that half of the carcinoma cases had a Ki-67 proliferation index above 5%.
Parafibromin is one of the biomarkers that have been used in research as biomarkers of malignancy in parathyroid tumours. Parafibromin is the protein product of the HRPT2 gene, originally found to be mutated in the majority of sporadic parathyroid cancers. Recent studies have emphasized that loss of parafibromin is an extremely sensitive biomarker for parathyroid tumors. The loss of parafibromin in parathyroid tumors was found to be 46% by Truran et al., 72% by Erovic et al. and 100% by Wittewen et al.,,
Our results show that immunohistochemical markers are beneficial to determine whether parathyroid tumors are benign or malignant. Cyclin D1, Ki-67 and Galactin-3 expression can be used as helper markers to differentiate adenomas from atypical adenomas and carcinomas. According to our study, global loss of CaSR staining was observed to be a strong marker when differentiating adenomas from carcinomas because it was not seen in adenomas.
| Conclusion|| |
Histopathology is the gold standard method for diagnosing parathyroid carcinomas. CaSR can be used in cases where there is doubt concerning malignancy as in fact clinically these do not contain parathyroid carcinoma criteria histopathologically or who have adenomas with atypical characteristics. If these cases have global loss of CaSR staining, they should then be followed up closely because of the risk of relapse or metastasis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Erovic BM, Harris L, Jamali M, Goldstein DP, Irish JC, Asa SL, et al.
Biomarkers of parathyroid carcinoma. Endocr Pathol 2012;23:221-31.
Bondeson L, Grimelius L, DeLellis RA, Lioyd R, Akerstrom G, Larsson C, et al
. Parathyroid carcinoma. In: DeLellis RA, Lloyd RV, Heitz PU, Eng C, editors. World Health Organization Classification of Tumors, Pathology & Genetics Tumors of Endocrine Organs. Lyon: IARC Press; 2004. p. 124-7.
Grimelius L, DeLellis RA, Bondeson L, Akerstrom G, Arnold A, Franssila KO, et al
. Parathyroid adenoma. In: DeLellis RA, Lloyd RV, Heitz PU, Eng C, editors. World Health Organization Classification of Tumors, Pathology & Genetics Tumors of Endocrine Organs. Lyon: IARC Press; 2004. p. 128-32.
Haven CJ, van Puijenbroek M, Karperien M, Fleuren GJ, Morreau H. Differential expression of the calcium sensing receptor and combined loss of chromosomes 1q and 11q in parathyroid carcinoma. J Pathol 2004;202:86-94.
Witteveen JE, Hamdy NA, Dekkers OM, Kievit J, van Wezel T, Teh BT, et al.
Downregulation of CASR expression and global loss of parafibromin staining are strong negative determinants of prognosis in parathyroid carcinoma. Mod Pathol 2011;24:688-97.
Tennakoon S, Aggarwal A, Kállay E. The calcium-sensing receptor and the hallmarks of cancer. Biochim Biophys Acta 2016;1863:1398-407.
Fernandez-Ranvier GG, Khanafshar E, Tacha D, Wong M, Kebebew E, Duh QY, et al.
Defining a molecular phenotype for benign and malignant parathyroid tumors. Cancer 2009;115:334-44.
Bergero N, De Pompa R, Sacerdote C, Gasparri G, Volante M, Bussolati G, et al.
Galectin-3 expression in parathyroid carcinoma: Immunohistochemical study of 26 cases. Hum Pathol 2005;36:908-14.
Al-Sharaky DR, Younes SF. Sensitivity and specificity of galectin-3 and glypican-3 in follicular-patterned and other thyroid neoplasms. J Clin Diagn Res 2016;10:EC06-10.
Zhang H, Luo M, Liang X, Wang D, Gu X, Duan C, et al.
Galectin-3 as a marker and potential therapeutic target in breast cancer. PLoS One 2014;9:e103482.
Lloyd RV, Carney JA, Ferreiro JA, Jin L, Thompson GB, Van Heerden JA, et al.
Immunohistochemical analysis of the cell cycle-associated antigens ki-67 and retinoblastoma protein in parathyroid carcinomas and adenomas. Endocr Pathol 1995;6:279-87.
Kumari N, Chaudhary N, Pradhan R, Agarwal A, Krishnani N. Role of histological criteria and immunohistochemical markers in predicting risk of malignancy in parathyroid neoplasms. Endocr Pathol 2016;27:87-96.
Truran PP, Johnson SJ, Bliss RD, Lennard TW, Aspinall SR. Parafibromin, galectin-3, PGP9.5, ki67, and cyclin D1: Using an immunohistochemical panel to aid in the diagnosis of parathyroid cancer. World J Surg 2014;38:2845-54.
Cetani F, Ambrogini E, Viacava P, Pardi E, Fanelli G, Naccarato AG, et al.
Should parafibromin staining replace HRTP2 gene analysis as an additional tool for histologic diagnosis of parathyroid carcinoma? Eur J Endocrinol 2007;156:547-54.
Vasef MA, Brynes RK, Sturm M, Bromley C, Robinson RA. Expression of cyclin D1 in parathyroid carcinomas, adenomas, and hyperplasias: A paraffin immunohistochemical study. Mod Pathol 1999;12:412-6.
Brennan SC, Thiem U, Roth S, Aggarwal A, Fetahu IS, Tennakoon S, et al.
Calcium sensing receptor signalling in physiology and cancer. Biochim Biophys Acta 2013;1833:1732-44.
Farnebo F, Auer G, Farnebo LO, Teh BT, Twigg S, Aspenblad U, et al.
Evaluation of retinoblastoma and ki-67 immunostaining as diagnostic markers of benign and malignant parathyroid disease. World J Surg 1999;23:68-74.
Department of Pathology, Ankara Yildirim Beyazit University, Bilkent - 006800 Ankara
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]