Case Report
A Case of Metastatic Adrenocortical Carcinoma Diagnosed with Steroidogenic Factor-1 in a Sprague-Dawley Rat
Yuichi Takai1*, Tomoya Sano1, Takeshi Watanabe1, and Ryo Fukuda1
1Drug Safety Research Laboratories, Takeda Pharmaceutical Company Limited, 26-1 Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
Abstract: This report describes the morphological and immunohistochemical characteristics of an adrenocortical carcinoma with dis- tant metastasis in a Sprague-Dawley rat. Macroscopically, a single large mass was observed in the adrenal gland, and multiple nodules were noted in the lung, liver and thyroid. Histologically, the adrenal tumor consisted of a solid growth of eosinophilic round cells with nuclear atypia. Vascular invasion was present, and multiple metastatic lesions were also observed in the lungs, liver, and mediastinal lymph nodes. Immunohistochemically, the nuclei of these tumor cells were positive for Steroidogenic Factor-1 (SF-1). In the thyroid, tumor cells histologically resembling adrenal cells were immunohistochemically negative for SF-1 but positive for calcitonin; thus the lesion was diagnosed as thyroid C-cell carcinoma. From these results, the present case was diagnosed as adrenocortical carcinoma with distant metastases. SF-1 could be a valuable marker for the differential diagnosis of adrenocortical tumors versus other endocrine tumors such as C-cell carcinoma. (DOI: 10.1293/tox.26.319; J Toxicol Pathol 2013; 26: 319-323)
Key words: adrenocortical carcinoma, immunohistochemistry, Steroidogenic Factor-1
Spontaneous adrenocortical carcinomas occur at a relatively low frequency in most rat strains with the excep- tion of some specific strains including Osborne-Mendel rats and highly inbred lines of Wistar rats1,2. In Sprague-Dawley rats, the incidence of adrenocortical carcinomas is reported to be around 0-1.2%3-6. In tumor-prone rats, metastasis to distant organs such as regional lymph nodes and the lungs has been described1,2; however, to our knowledge, there have been no reports regarding adrenocortical carcinoma with distant metastasis in Sprague-Dawley rats3-5. We en- countered a case of adrenocortical carcinoma with metas- tasis to the lungs, liver and mediastinal lymph node in an aged female Sprague-Dawley rat. Because this animal coin- cidently had a thyroid C-cell carcinoma, which consisted of a solid proliferation of round tumor cells resembling adre- nocortical carcinoma cells, appropriate differential diagno- sis especially for the metastatic sites was needed. Here, we report the histological features of this rare case of metastatic adrenocortical carcinoma and also demonstrate the utility of Steroidogenic Factor-1 (SF-1), a nuclear receptor with criti- cal roles in steroidogenesis7 as an immunohistochemical marker for adrenocortical tumors.
A thirteen-week-old female Sprague-Dawley rat was
purchased from Charles River Laboratories Japan (Hino, Japan), housed in a metal cage in an animal room at Takeda Rabics Limited (Yamaguchi, Japan) with a temperature of 20-26°C, 40-80% relative humidity and a 12-hour light/ dark cycle and fed a commercial diet (CR-LPF: Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum. At 108 weeks of age, the animal was transported from Takeda Rabics Limited to the Shonan Research Center of Takeda Pharmaceutical Company Limited (Kanagawa, Japan) and was immediately sacrificed by exsanguination from the abdominal aorta under inhalation anesthesia with isoflu- rane. The experimental procedures were approved by the Institutional Animal Care and Use Committees of Takeda Pharmaceutical Company Limited. There were no clinical signs before necropsy. At necropsy, a dark red mass approx- imately 30 × 25 × 20 mm in diameter was observed in the left adrenal gland (Fig. 1A); however, the presence of the contralateral (right) adrenal gland was not confirmed mac- roscopically. In addition, multiple yellow or white nodules less than 10 mm in diameter were found in all lobes of the lungs (Fig. 1B), two white nodules approximately 10 mm and 1 mm in diameter were found in the left lateral lobe and the caudal part of the caudate lobe of the liver, and a white nodule approximately 5 mm in diameter were found in the right side of the thyroid. The mediastinal lymph nodes were enlarged with dark red discoloration (Fig. 1B). Furthermore, a dark red focus was observed in the pituitary gland. There were no remarkable findings in the other organs and tis- sues. All of the gross lesions mentioned above were fixed in 10 vol% neutral buffered formalin, embedded in paraffin,
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sectioned and stained with hematoxylin and eosin (H.E.). For the differential diagnosis, sequential sections for each lesion except the pituitary lesion were immunohistochemi- cally stained with anti-human Steroidogenic Factor-1 mouse monoclonal antibody (diluted 1:1000, clone: N1665, Perseus Proteomics, Tokyo, Japan) and anti-human calcitonin rab- bit polyclonal antibody (diluted 1:100, GeneTex, Inc., Los Angeles, CA, U.S.A.), and the pituitary was immunostained with anti-rat LH rabbit polyclonal antibody (diluted 1:5000, Accurate Chemical & Scientific Corp., Westbury, NY, U.S.A.) and anti-human ACTH mouse monoclonal antibody (diluted 1:1000, clone: 56, Novocastra Laboratories Ltd., Newcastle, U.K.).
Microscopically, the adrenal mass consisted of a solid proliferation of tumor cells, and the remaining cortical tis- sue was significantly distorted by these tumor cells. The tumor cells were round, possessed abundant eosinophilic cytoplasm with occasional vacuolation and were arranged in nests or trabeculae, which were separated by a fibrovas- cular stroma. The nuclei were round and had a low chro- matin density and distinct single nucleoli. Anisokaryosis and mitotic figures were frequently observed (Fig. 2A), and massive necrosis and multifocal hemorrhage were also seen within the tumor. As findings that indicated malignancy, lo- cal and vascular invasions by the tumor cells were observed (Fig. 2B); therefore, the adrenal tumor was diagnosed as ad- renocortical carcinoma and was subclassified into the well- differentiated type due to the degree of differentiation of the tumor cells.
The histological features of the nodules in the lungs and liver and enlarged mediastinal lymph node were very similar to those in the adrenal mass. That is to say, the le- sions consisted of solid proliferations of round tumor cells with abundant eosinophilic cytoplasm, occasionally vacu-
olated, and round nuclei with nuclear atypia (Fig. 2C-E). Massive necrosis and multifocal hemorrhage were also fre- quently observed within these lesions. The thyroid mass also consisted of a solid proliferation of round tumor cells with abundant eosinophilic cytoplasm and round nuclei with a lower chromatin density; however, the cytoplasm was slightly pale, vacuolation was rare, and the nucleoli ap- peared to be indistinct compared with those in the adrenal tumor. Normal follicles were often seen within the tumor tissue (Fig. 2F). The cellular morphology and presence of normal follicles indicated that the origin of the tumor cells was thyroid C-cells, and since local invasion of tumor cells to the adjacent tissues was partly evident, the thyroid tumor was considered to be a C-cell carcinoma.
Immunohistochemically, almost all nuclei of the nor- mal adrenocortical cells as well as the tumor cells in the ad- renal gland, lungs, liver and mediastinal lymph nodes were positive for SF-1 (Fig. 3A-D), indicating that these tumors were derived from steroidogenic cells. Since there were no proliferative lesions in the steroidogenic tissues other than the adrenal gland, the lesions in the lungs, liver and medias- tinal lymph nodes were determined to be metastases of the adrenocortical carcinoma. In addition, all the tumor cells in these tissues were negative for calcitonin. On the other hand, all the nuclei of the thyroid tumor cells were negative for SF-1 (Fig. 3E), and a positive reaction for calcitonin in the cytoplasm of the tumor cells was confirmed (Fig. 3F). Hence, the thyroid tumor was definitely diagnosed as C-cell carcinoma.
To clarify whether there was any morphological change in the corticotrophs or not, we also examined the pituitary histopathologically. Two instances of focal hyperplasia and one adenoma, both consisting of amphophilic cells, were observed in the pituitary; however, all these cells were positively immunostained with LH antibody, and thus, they were diagnosed as gonadotroph hyperplasia and adenoma. Meanwhile, immunohistochemical staining with ACTH an- tibody revealed no obvious changes in the distribution and density of the corticotrophs in this animal.
Based on the results described above, the present case was diagnosed as an adrenocortical carcinoma with metas- tases to the lungs, liver and mediastinal lymph nodes and a thyroid C-cell carcinoma. It was unclear whether the adre- nal carcinoma was functional or not, since hormone mea- surements for corticosterone and ACTH were not conducted for this animal. However, it was suggested that the plasma ACTH levels remained normal, since histopathological ex- amination of the pituitary gland showed no obvious changes in the corticotrophs. Both adrenocortical and thyroid C- cell carcinomas are relatively rare in rats, and there is no report describing metastasis of adrenocortical carcinoma in Sprague Dawley rats3-5. Therefore, the present case is considered to be notably rare as a spontaneous tumor in Sprague-Dawley rats. Because the histological features of adrenal and thyroid tumors often closely resembled each other, immunohistochemical staining with SF-1 was enor- mously useful, especially for diagnosis of the lesions at the
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metastatic sites. SF-1 is a nuclear receptor with critical roles in steroidogenic tissues as a transcriptional factor7 that is distributed in not only the steroidogenic organs such as the
gonads and adrenal cortex including the X-zone and subcap- sular polygonal (type B) cells in mice7-10 but also in the ven- tromedial hypothalamic nucleus11,12 and pituitary gonado-
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tropes13. SF-1 is a major regulator of cholesterol metabolism in steroidogenic cells, as it stimulates the expression of near- ly all the factors involved in cholesterol mobilization and steroid hormone biosynthesis7. Recently in humans, SF-1
was revealed to be a highly valuable immunohistochemi- cal marker for determination of the adrenocortical origin of an adrenal mass with high sensitivity and specificity14, and its expression is of stage-independent prognostic value in
patients with adrenocortical carcinoma14. Currently, there is no reliable immunohistochemical marker for adrenocorti- cal tumors in rats; however, the present results demonstrate that SF-1 is also useful in the case of rats. Adrenocortical tumors have been induced experimentally with variety of agents including estrogens15, irradiation16-18, certain chlori- nated hydrocarbons19-21 and benzenes22. Therefore, prolif- erative changes in the adrenal cortex might be induced in rat carcinogenicity studies, and in some cases, the differential diagnosis of various types of endocrine tumors including thyroid C-cell, parathyroid and islet cell tumors, pheochro- mocytoma and metastatic hepatocellular tumor may be re- quired due to the histological similarities of these tumors and the morphological heterogeneity of adrenocortical tu- mors2,14. Although further investigation using the other type of tumors including poorly differentiated or anaplastic types may be necessary, immunohistochemistry for SF-1 is con- sidered to be a valuable marker for the differential diagnosis of adrenocortical tumors versus other tumors such as thy- roid C-cell carcinoma in rat carcinogenicity studies.
Acknowledgment: The authors would like to thank Ms. Harumi Kitaura and Ms. Yumiko Miyamoto for their sup- port during this work.
References
1. Duprat P, Snell KC, and Hollander CF. Tumours of the adre- nal gland. In: Pathology of Tumors in Laboratory Animals. Vol. 1. Tumors of the Rat. 2nd ed. Turusov V, and Mohr U (eds). IARC Scientific Publications, Lyon. 573-596. 1990.
2. Patterson DR, Hamlin MH II, Hottendorf GH, Gough A, and Brown WR. Proliferative lesion of the adrenal glands in rats. In: Guides for Toxicologic Pathology. STP/ARP/AFIP, Washington, D.C. 1-12. 1995.
3. Suzuki H, Mohr U, and Kimmerle G. Spontaneous endo- crine tumors in Sprague-Dawley rats. J Cancer Res Clin Oncol. 95: 187-196. 1979. [Medline]
4. Chandra M, Riley MG, and Johnson DE. Spontaneous neo- plasms in aged Sprague-Dawley rats. Arch Toxicol. 66: 496-502. 1992. [Medline]
5. Nakazawa M, Tawaratani T, Uchimoto H, Kawaminami A, Ueda M, Ueda A, Shinoda Y, Iwakura K, Kura K, and Sumi N. Spontaneous neoplastic lesions in aged Sprague-Dawley rats. Exp Anim. 50: 99-103. 2001. [Medline]
6. Imai K, and Yoshimura S. Spontaneous tumors in Sprague- Dawley (CO: Crj) rats. J Toxicol Pathol. 1: 7-12. 1988.
7. Hoivik EA, Lewis AE, Aumo L, and Bakke M. Molecular aspects of steroidogenic factor 1 (SF-1). Mol Cell Endocri- nol. 315: 27-39. 2010. [Medline]
8. Bielinska M, Parviainen H, Porter-Tinge SB, Kiiveri S, Genova E, Rahman N, Huhtaniemi IT, Muglia LJ, Heikin- heimo M, and Wilson DB. Mouse strain susceptibility to gonadectomy-induced adrenocortical tumor formation correlates with the expression of GATA-4 and luteinizing hormone receptor. Endocrinology. 144: 4123-4133. 2003.
[Medline]
9. Bielinska M, Genova E, Boime I, Parviainen H, Kiiveri S, Leppaluoto J, Rahman N, Heikinheimo M, and Wilson DB. Gonadotropin-induced adrenocortical neoplasia in NU/J nude mice. Endocrinology. 146: 3975-3984. 2005. [Med- line]
10. Beuschlein F, Keegan CE, Bavers DL, Mutch C, Hutz JE, Shah S, Ulrich-Lai YM, Engeland WC, Jeffs B, Jameson JL, and Hammer GD. SF-1, DAX-1, and acd: molecular de- terminants of adrenocortical growth and steroidogenesis. Endocr Res. 28: 597-607. 2002. [Medline]
11. Ikeda Y, Shen WH, Ingraham HA, and Parker KL. Devel- opmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endo- crinol. 8: 654-662. 1994. [Medline]
12. Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, Shiba H, Sasaki H, Osawa Y, Ninomiya Y, Niwa O, Morohashi K, and Li E. Developmental defects of the ventromedial hypo- thalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice. Dev Dyn. 204: 22-29. 1995. [Medline]
13. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachti- gal MW, Abbud R, Nilson JH, and Parker KL. The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev. 8: 2302-2312. 1994. [Med- line]
14. Sbiera S, Schmull S, Assie G, Voelker HU, Kraus L, Beyer M, Ragazzon B, Beuschlein F, Willenberg HS, Hahner S, Saeger W, Bertherat J, Allolio B, and Fassnacht M. High diagnostic and prognostic value of steroidogenic factor-1 expression in adrenal tumors. J Clin Endocrinol Metab. 95: E161-E171. 2010. [Medline]
15. Dunning WF, Curtis MR, and Segaloff A. Strain differ- ences in response to estrone and the induction of mammary gland, adrenal, and bladder cancer in rats. Cancer Res. 13: 147-152. 1953. [Medline]
16. Rosen VJ Jr, Castanera TJ, Jones DC, and Kimeldorf DJ. Islet-cell tumors of the pancreas in the irradiated and nonir- radiated rat. Lab Invest. 10: 608-616. 1961. [Medline]
17. Castanera TJ, Jones DC, Kimeldorf DJ, and Rosen VJ. The influence of whole-body exposure to x-rays or neutrons on the life span distribution of tumors among male rats. Cancer Res. 28: 170-182. 1968. [Medline]
18. Vogel HH Jr, and Zaldivar R. Malignant tumours of the rat adrenal gland induced by fission neutron irradiation. Int J Radiat Biol Relat Stud Phys Chem Med. 18: 267-270. 1970. [Medline]
19. Bioassay of chlorobenzilate for possible carcinogenicity. Natl Cancer Inst Carcinog Tech Rep Ser. 75: 1-107. 1978. [Medline]
20. Reuber MD. Carcinomas, sarcomas and other lesions in Osborne-Mendel rats ingesting endrin. Exp Cell Biol. 46: 129-145. 1978. [Medline]
21. Kimbrough RD. The carcinogenic and other chronic effects of persistent halogenated organic compounds. Ann NY Acad Sci. 320: 415-418. 1979. [Medline]
22. Mulay AS, and Eyestone WH. Transplantable adrenocorti- cal adenocardinomas in Osborne-Mendel rats fed a carcino- genic diet. J Natl Cancer Inst. 16: 723-739. 1955. [Medline]