—NOTE— Immunohistochemical Characterization of Parafollicular Cell Adenoma, Medullary Carcinoma and Follicular Adenocarcinoma in of Horses

Toshifumi OYAMADA1*, Hideaki UEKI1, Hiroyuki KOWATARI1, Park CHUN-HO1, Yoshinari KATAYAMA2, Masa-aki OIKAWA2 and Hiroyasu YOSHIKAWA1 1Department of Veterinary Pathology, School of Veterinary Medicine and Animal Sciences, Kitasato University, 35–1 Higashi 23 bancho, Towada city, Aomori 034-8628, 2 Equine Research Institute, Japan Racing Association, 321-4 Tokami-cho, Utsunomiya, Tochigi 320-0856, Japan

An Immunohistochemical examination was conducted using commercially available primary J. Equine Sci. antibodies to evaluate their utility for differential diagnosis of equine thyroid tumors i.e. non- Vol. 16, No. 1 functional C-cell adenoma, medullary carcinoma and follicular adenocarcinoma. The pp. 11–17, 2005 primary antibodies used in the study were antibodies against , calcitonin-gene- related peptide (CGRP), thyroglobulin, chromogranin A, neuron specific enolase (NSE), synaptophysin and . Calcitonin, NSE and synaptophysin were found to be useful to identify the origin of tumors as C-cells and thyroglobulin as follicular epithelial cells. In addition, it was found that detection of CGRP and chromogranin A is related with the functional status of C-cells. The carcino-related-gene (CRG) product, bcl-2, was positive in medullary carcinoma and follicular adenocarcinoma, and weakly positive to negative in non- functional C-cell adenoma. Another CRG product, p53, was positive in non-functional C-cell adenoma and follicular adenocarcinoma, and negative in medullary carcinoma. These results suggest that the expression pattern of 9 antigens used may be of help for the differential diagnosis of these tumors. Key words: horse, Immunohistopathology, thyroid, tumor

Tumors occurring in the thyroid gland are primarily either pathomorphological examination by HE derived from 2 types of cells: the follicular epithelial staining or ultrastructural examination are at present, cell and the parafollicular cell (C-cell). There are the only methods for differential diagnosis. Previously, extensive studies on immunohistochemical we reported that thyroid tumors frequently found in characterization of thyroid tumors in humans, dogs aged horses are derived from C-cells, but not from and rats [5, 6, 8, 12, 18, 19, 21], although there are only follicular epithelial cells as previously believed [15]. a few studies in horses because malignant thyroid During the previous study, we also came across other tumors are especially rare in this species. Generally, types of malignant thyroid tumors: medullary follicular epithelial cells are positive for thyroglobulin, carcinoma derived from C-cells, and follicular and C-cells are positive for calcitonin. In addition to adenocarcinoma derived from follicular epithelial these markers, somatostatin [5, 8] and calcitonin-gene- cells. In the present study, we conducted related peptide (CGRP) [3, 6] are useful for the immunohistochemical examinations using 9 identification of C-cells in some animal species. commercially available primary antibodies in order to Nevertheless, immunohistochemical examination of determine the most useful method for the differential thyroid tumors in horses has rarely been done. Hence, diagnosis of these thyroid tumors. Expression of carcinomatous character was investigated by two This article was accepted November 18, 2004 carcino-related gene products of bcl-2 and p53. Finally, *Corresponding author. useful primary antibodies for identification of follicular 12 T. OYAMADA, H. UEKI, H. KOWATARI ET AL. epithelial cells and C-cells in horses were evaluated the right and left lobes of the thyroid were fixed in 10% based on the findings of the characterization of the neutral buffered formalin and embedded in paraffin tumor cells. wax by the routine procedures. Thin sections (6 µm) were stained with hematoxylin and eosin (HE). Immunohistochemistry: Primary antibodies consisted Materials and Methods of antisera against calcitonin (Zymed Laboratories, San Francisco, USA), calcitonin-gene-related peptide Horses: As has already has been reported, twelve non- (CGRP) (Affiniti Research products, Mamhead, UK), functional C-cell adenomas, one medullary carcinoma thyroglobulin (DakoCytomation, Kyoto, Japan), and one follicular adenocarcinoma were examined chromogranin A (Nichirei, Tokyo, Japan) , neuron [15]. Age, sex and breed of the horses and diagnosis of specific enolase (NSE) (Zymed Laboratories), thyroid tumors are listed in Table 1. Slices (5 mm) of synaptophysin (DakoCytomation) and somatostatin

Table 1. Age, sex and breed of horses, and diagnosis of the thyroid tumors Age Sex Breed Diagnosis neoplastic thyroid 14 gelding unknown non-functional C cell adenoma 15 male Anglo-arab non-functional C cell adenoma 15 female Thoroughbred non-functional C cell adenoma 20 male Thoroughbred non-functional C cell adenoma 20 male Thoroughbred non-functional C cell adenoma 21 female Thoroughbred non-functional C cell adenoma 22 male Thoroughbred non-functional C cell adenoma 22 female Thoroughbred non-functional C cell adenoma 24 male Thoroughbred non-functional C cell adenoma 24 female Thoroughbred non-functional C cell adenoma 25 gelding Thoroughbred non-functional C cell adenoma 26 male Anglo-arab non-functional C cell adenoma 27 gelding Anglo-arab medullary carcinoma 29 male Anglo-arab follicular adenocarcinoma non-neoplastic thyroid 10 male Drought horse 11 male Thoroughbred 13 female Thoroughbred 13 female Thoroughbred 13 male Thoroughbred 13 male Thoroughbred 13 female Thoroughbred 14 male Drought horse 14 female Thoroughbred 15 female Thoroughbred 15 female Thoroughbred 15 female Thoroughbred 15 male Thoroughbred 16 female unknown 17 female Thoroughbred 17 female Thoroughbred 17 female Thoroughbred 17 female Thoroughbred 17 female unknown 18 female Thoroughbred 18 gelding Thoroughbred 18 male Thoroughbred 18 female unknown 20 male Anglo-arab IMMUNOHISTOCHEMICAL CHARACTERIZATION OF THYROID TUMORS 13

Table 2. Antisera used for immunohistochemistry Antiserum to Source Supplier Dilution Pretreat for antigen retrieval Human calcitonin Rabbit Zymed 1:50 in PBS non Human CGRP Rabbit Affiniti 1:4000 in PBS non Human thyroglobulin Rabbit DakoCytomation prediluted waterbath (95–99°C, 40 min. in citrate buffer pH6.0) Human chromogranin A Rabbit Nichirei prediluted microwave (500W, 15 min. in citrate buffer pH6.0) Human NSE Mouse Zymed prediluted microwave (500W, 15 min. in citrate buffer pH6.0) Human Synaptophysin Rabbit DakoCytomation prediluted proteinase K (room temp. 10 min.) Human Somatostatin Rabbit DakoCytomation prediluted microwave (500W, 15 min. in citrate buffer pH6.0) Human bcl-2 Mouse Nichirei prediluted autoclave (121°C, 5 min. in citrate buffer pH7.0) Human p53 Mouse DakoCytomation prediluted autoclave (121°C, 5 min. in citrate buffer pH7.0)

(DakoCytomation) (Table 2). Antigen retrievals were for calcitonin, CGRP, chromogranin A, and NSE. conducted by heating in a waterbath (95–99°C, 40 min) Follicular epithelial cells were negative for all markers. for thyroglobulin methodology, in a microwave oven Intrafollicular colloid was strongly positive for (500 W, 15 min) for chromogranin A, NSE, thyrogloburin and weakly positive for somatostatin. somatostatin assays or in a protenase K treatment (10 Non-functional C-cell adenoma (Fig. 1): Tumor cells min) for synaptophysin assay. Immunolabelling was were weakly positive for calcitonin, strongly positive for performed with the polymer reagents Simplestain MAX NSE, weakly positive for synaptophysin, marginally PO (Nichirei) and EnVision+ (DakoCytomation). To positive or negative for CGRP, and negative for identify the immunoreaction, red substrates (new thyroglobulin and chromogranin A. In the C-cells in fuchsin [Nichirei]) or 3-amino-9-ethyl carbazole [AEC] the extranodular normal tissues, the staining pattern of [Nichirei]) were used, because all samples contained calcitonin of the tumor cells was generally weak, large amounts of yellow-brown lipofuscin deposits. although a very few cells were strongly positive. Some Sections were counterstained with hematoxylin. tumor cells were occasionally weakly positive for Detection of carcino-related-gene products: Bcl-2 and p53- somatostatin. Follicular epithelial cells were positive in gene-protein (bcl-2 [Nichirei], p53 [DakoCytomation]) rare cases for thyroglobulin and somatostatin, but were investigated by the immunohistochemical method negative with other markers. Colloid was positive for according to the manufacturer’s. Prior to incubation thyroglobulin and weakly positive for somatostatin. with the primary anti-sera, deparaffinized slides were Medullary carcinoma (Fig. 2): Tumor cells were placed in 0.3% H2O 2 to consume endogenous strongly positive for calcitonin, NSE, and peroxidase activity, which were subjected to autoclave synaptophysin, weakly positive for CGRP, and weakly to antigen retrieval by immersion in citrate buffer. strongly positive for chromogranin A. They were Immunolabelling was performed with the EnVision+ negative for thyroglobulin and somatostatin. Follicular (DakoCytomation) and AEC (Nichirei) were used to epithelial cells were negative for all markers, although identify the immunoreaction. Sections were intrafollicular colloid was positive for thyroglobulin, counterstained with hematoxylin (Table 2). and weakly positive for somatostatin. Twenty four normal from adult horses of Follicular adenocarcinoma (Fig. 3): Follicular epithelial various age were used for immunohistochemical cells undergoing neoplastic proliferation were positive controls and reaction utilities of these antibodies had for thyroglobulin and somatostatin. Intrafollicular already been confirmed [15]. colloid and fibrinoid substances were also positive with both antibodies. Cells positive for calcitonin, chromogranin A and NSE were identical, with positive Results reactions observed scattered in the follicular cells and always located along the basement of the follicular epithelial cells, but not adjacent to the follicular lumen. Immunohistochemical characterization There were no cells positive for CGRP or Non-neoplastic thyroid: C-cells were strongly positive synaptophysin. 14 T. OYAMADA, H. UEKI, H. KOWATARI ET AL.

Fig. 1. Immunohistochemistry of non-functional C-cell adenoma. The tumorous C cells are positive for calcitonin, NSE and synaptophysin and negative for other four markers. AEC chromogen. Haematoxylin counterstain. Bars=50 µm.

Fig. 2. Immunohistochemistry of medullary carcinoma. The tumorous C-cells are positive for calcitonin, CGRP, NSE and synaptophysin, weakly positive for chromogranin A, and negative for thyroglobulin and somatostatin. AEC chromogen. Haematoxylin counterstain. Bars=50 µm.

Fig. 3. Immunohistochemistry of follicular adenocarcinoma. The tumorous epithelial cells are positive for thyroglobulin and somatostatin, but were negative for the other five markers. AEC chromogen. Haematoxylin counterstain. Bars= 50 µm.

Fig. 4. Immunohistochemical expression of CRG products. Non-functional C-cell adenoma is weakly positive for bcl-2 (a) and p53 (b). Medullary carcinoma is positive for bcl-2 (c) but negative for p53 (d). Follicular adenocarcinoma is positive both for bcl-2 (e) and p53 (f). AEC chromogen. Haematoxylin counterstain. Bars=50 µm. IMMUNOHISTOCHEMICAL CHARACTERIZATION OF THYROID TUMORS 15

Expression of Carcino-related-gene products cells in horses, calcitonin gave a better detection Non-functional C-cell adenoma (Figs. 4a and 4b): sensitivity than CGRP or chromogranin A [11, 22]. Expression of bcl-2 in tumor cells was negative to weakly Both non-functional C-cell adenoma and medullary positive, depending on each case. There were no carcinoma are positive for NSE and synaptophysin. strongly positive cells, which are frequently observed in NSE is an isomer of glycol enolase residing in the medullary carcinoma and follicular adenocarcinoma. cytoplasm (cytosol) of nerve cells and neuroendocrine Expression of p53 was weakly positive to strongly cells [14, 17] and synaptophysin is one of the main positive. Other than tumor cells, many follicular essential proteins localized on the membranes of epithelial cells in the tumor mass were positive for p53. presynaptic vesicles in nerve cells and neuroendocrine Medullary carcinoma (Figs. 4c and 4d): The tumor cells cells [20]. Both NSE and synaptophysin are widely used were positive for bcl-2 and negative for p53. as markers for neuroendocrine tumors, for which C- Follicular adenocarcinoma (Figs. 4e and 4f): Tumor cells cells are obviously positive. In the present study, tumor were positive for both bcl-2 and p53. cells in non-functional C-cell adenoma and medullary carcinoma were positive for both NSE and synaptophysin, suggesting that these cells are derived Discussion from C-cells. Thus, the immunoreactivity for calcitonin, NSE and synaptophysin should be considered in combination in order to identify C-cell Utility of commercially available antibodies derived tumors, because the detection sensitivity of the In this study, both neoplastic and non-neoplastic C- antibodies against NSE and synaptophysin, may vary cells were found to be positive for calcitonin. Moreover, depending on the fixation conditions [2, 13]. both follicular epithelial cells and the corresponding By immunohistochemical staining for somatostatin, neoplastic cells were positive for thyroglobulin, D-cells in the islets of Langerhans were specifically demonstrating that two typical proteins in thyroid stained in normal pancreas in horses. Somatostatin is a gland can be specifically detected in horses. In some peptide hormone secreted from D-cells in the islets of species, it is possible to use antibodies against CGRP or Langerhans in the pancreas, but is also known to reside somatostatin, in addition to calcitonin, as specific in C-cells in the thyroid gland in some species [4]. primary antibodies for identifying C-cells [3, 4, 6, 8]. In Therefore, somatostatin is used as well as calcitonin as a horses, the utility of commercially available primary specific marker for the diagnosis of medullary antibodies was determined as follows: as reported carcinoma in the thyroid glands in humans and previously, the negative staining reaction to CGRP and experimental animals [8, 9]. chromogranin in non-functional C-cell adenoma is In this study, C-cells in normal thyroid glands were associated with an inactive secretion of calcitonin [15]. also stained, although the number of cells was smaller In medullary carcinoma, CGRP was positive and most than the number of calcitonin positive cells. cells were weakly but occasionally strongly positive for Intrafollicular colloid in normal thyroid glands in chromogranin A. Hence, it is likely that, in medullary horses, as well as C-cells, was also found to be weakly carcinoma, the calcitonin gene is activated to synthesize positive. In non-functional C-cell adenoma and calcitonin. In other words, cells in the process medullary carcinoma, somatostatin was found to be synthesizing calcitonin show a weakly positive reaction, occasionally weakly positive in tumor cells and in and those with mature secretory granules show a colloid, and, rarely weakly positive in follicular strongly positive reaction for chromogranin A. epithelial cells. Therefore, in horses, the staining Therefore, secretion of calcitonin seems to be activated pattern of somatostatin in C-cell derived cells was in medullary carcinoma (functional tumor). Leblanc et similar to that of calcitonin. Nevertheless, about al. (1991) [6] reported that CGRP is more useful than follicular epithelial cells and colloid, the staining calcitonin for the detection of C-cells in dogs. In pattern was similar to that of thyroglobulin in follicular horses, Ueki et al. (2004) [15] reported that non- epithelial cells and tumor cells of follicular epithelial functional C-cell adenoma occurs frequently with adenocarcinoma. Follicular epithelial cells in horses negative reactions for CGRP and chromogranin A. In may have the ability to secret somatostatin. These this study, when these commercially available findings are similar to that of thyroid tumors in dogs by antibodies of human origin were used to identify C- Leblanc et al. (1991) [6]. They considered that poor 16 T. OYAMADA, H. UEKI, H. KOWATARI ET AL. immunoreactivity of somatostatin to C-cell adenoma require pretreatment). In addition, CGRP and was due to a species difference. Considering the very chromogranin should be considered in combination as specific positive reaction for somatostatin in normal markers to evaluate whether the C-cells are functional pancreas, it is unlikely that the result in the present or not. study was due to a non-specific reaction. At present, it appears that commercially available antibodies against somatostatin are not useful for identifying C-cells in Acknowledgments horses. This study was supported by a grant-in-aid from the Expression of Carcino-related-geness Equine Research Institute, Japan Racing Association. In hereditary medullary carcinoma of humans, the bcl-2 gene is highly expressed, but the expression of the p53 gene is not detected so frequently [18, 19, 23]. It References has been suggested that expression of the bcl-2 gene plays a very important role in the pathogenesis of 1. Donghi, R., Longoni, A., Pilotti, S., Michieli, P., medullary carcinoma in humans [7]. There has been Della Porta, G., and Pierotti, M.A. 1993. Gene p53 only one report on C-cell derived malignant tumor muteation are restricted to poorly differentiated (medullary carcinoma) in horses [16], and its and undifferentiated carcinomas of the thyroid pathogenic mechanism is not known. In our case of gland. J. Clin. 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