J Toxicol Pathol 2008; 21: 105–108

Case Report Spontaneous Pituitary Carcinoma of the Pars Intermedia in a B6C3F1 Mouse

Yuzo Yasui1, Yoshihide Ueda1, Kayoko Nagai1, Ryo Yamashita1, Mayumi Ishizaki1,#, Tohru Kihara1, Kazushige Hasegawa1, Masayo Hosoi1, Rumiko Miyajima1, Atsushi Shiga1, Hijiri Iwata1,##, and Kiyoshi Imai1

1 Biosafety Research Center, Foods Drugs and Pesticides (An-Pyo Center), 582–2 Shioshinden, Iwata, Shizuoka 437–1213, Japan # Present: Pharmaceutical Research Laboratories, Ajinomoto Co., Inc., 1–1 Suzuki-cho, Kawasaki-ku, Kawasaki 210–0681, Japan ## Present: RCC Ltd., Zelgliweg 1, 4452, Itingen, Switzerland

Abstract: We encountered one pituitary carcinoma derived from the pars intermedia in an untreated female B6C3F1 mouse in a carcinogenicity study. A white nodule (6 × 5 × 3 mm) was observed in the base of the brain at necropsy. Tumor cells were histologically arranged in a solid sheet or nest-like structures, and had round or oval nuclei and pale eosinophilic granular cytoplasm. Sporadic mitotic figures were seen in the tumor while necrosis was a prominent feature. In addition, distinct infiltration into the brain parenchyma was observed. Immunohistochemically, tumor cells were positive for α-melanocyte stimulating hormone (α-MSH), γ-melanocyte stimulating hormone (γ-MSH), β- endorphin and adenocorticotropic hormone (ACTH), but were negative for prolactin. However, there were no histological abnormalities such as adrenocortical hyperplasia related to the pars intermedia tumor. These results suggest that the tumor cells might not have actively secreted (POMC)-derived peptides. The present report provides additional histopathological evidence of pituitary carcinoma of the pars intermedia in aging mice. (J Toxicol Pathol 2008; 21: 105–108) Key words: pituitary carcinoma, pars intermedia, mouse, infiltration, immunohistochemistry

Pituitary tumors of the pars distalis are common meninges7. Another report8 described the critical points for neoplasms in aging mice of some strains such as B6C3F1. differential diagnosis between carcinoma and adenoma of Pituitary adenomas of the pars distalis occur more often in the pars intermedia as being distinct infiltration into the female than in male mice, and a similar situation exists in brain. In addition, although these tumor cells demonstrated rats1–4. For example, the incidences of pars distalis neoplasm immunoreactivity for adenocorticotropic hormone (ACTH), in B6C3F1 mice are reported as being approximately 0.2% detailed morphological and immunohistochemical features and 15% for males and females, respectively, in the National have remained unclear. Herein, we describe our experience Toxicology Program historical control database of the with a single pituitary tumor suspected to be a carcinoma previous 2-year carcinogenicity studies3. In contrast, derived from the pars intermedia in an aged mouse at our pituitary adenomas of the pars intermedia are uncommon center. The present report describes the histological and neoplasms1,5,6, and pituitary carcinomas of the pars immunohistochemical characteristics of the tumor. intermedia are extremely rare tumors in aging mice3,7. The animal was a female Crj:B6C3F1 (C57BL/6N × Previous reports have described the criteria for pituitary C3H/HeN) mouse in the untreated group of a carcinogenicity carcinoma of the pars intermedia as follows: (i) projection study. The animal was housed individually in an aluminum into a large cystic space; (ii) abutting against brain or bone cage with a stainless steel wire mesh front and floor, under without infiltrating brain or bone; (iii) infiltration into the barrier conditions of 23 ± 3°C room temperature, 55 ± 20% relative humidity, and a 12 hour light-dark cycle. The animal had free access to a standard radiation-sterilized Received: 25 September 2007, Accepted: 21 February 2008 laboratory diet (CRF-1, Oriental Yeast Co. Ltd., Tokyo, Mailing address: Yuzo Yasui, Biosafety Research Center, Foods Drugs Japan) and tap water via an automatic water supply system. and Pesticides (An-Pyo Center), 582–2 Shioshinden, Iwata, Shizuoka All animal care and procedures were performed in 437–1213, Japan accordance with the Guidances for Care and Use of TEL: 81-538-58-2965 FAX: 81-538-58-1343 Laboratory Animals at the Biosafety Research Center, Foods E-mail: [email protected] 106 Pituitary Intermedia Carcinoma in a Mouse

Drugs and Pesticides (An-Pyo Center). The animal was under ether anesthesia at 68 weeks of age. observed for clinical signs twice a day (in the morning and At necropsy, a white nodule (6 × 5 × 3 mm) was afternoon). The animal showed the clinical sign of observed in the base of the brain and the was emaciation at 64 weeks of age, and piloerection, staggering, not recognizable. After gross postmortem examinations, the swelling of the head and decreased locomotor activity nodule was fixed in 10% neutral-buffered formalin, became apparent at 68 weeks of age. Therefore, the animal embedded in paraffin, sectioned at 3 µm and stained with was euthanatized by exsanguination via the abdominal aorta hematoxylin and eosin (H&E) for routine histopathological Yasui, Ueda, Nagai et al. 107 examination. Next, serial sections were obtained from the addition, distinct infiltration into the brain parenchyma was nodule, and were immunohistochemically stained with anti- observed in some areas (Fig. 4). However, distant α-melanocyte stimulating hormone (α-MSH, Phoenix metastases were not observed. Pharmaceuticals, Inc., CA, USA, dilution 1:100), anti-γ- Immunohistochemically, tumor cells were positive for melanocyte stimulating hormone (γ-MSH, Phoenix α-MSH (Fig. 5), γ-MSH (Fig. 6), β-endorphin (Fig. 7) and Pharmaceuticals, dilution 1:100), anti-β-endorphin ACTH (Fig. 8), but negative for prolactin. However, there (PROGEN Biotechnik GmbH, Heidelberg, Germany, were no histological abnormalities such as adrenocortical dilution 1:50), anti-ACTH (clone: 02A3, DAKO, CA, USA, hyperplasia related to this pars intermedia tumor. As in the dilution 1:50), anti-prolactin (R&D Systems, Inc., MN, case of this animal, it was reported that mice and rats with USA, dilution 1:10) and anti-proliferating cell nuclear pars intermedia adenomas had no endocrinological antigen (PCNA, clone: PC10, DAKO, Denmark, dilution abnormalities in the other organs including the adrenal 1:50), using a labelled streptavidin-biotin method (DAKO glands6. On the other hand, there are interesting reports of LSAB2 System-HRP, CA, USA). Tissue sections of typical pars intermedia adenoma affecting other organs and causing of the pars intermedia in aging mice were functional changes in horses and dogs. Pituitary adenomas concurrently stained for control of immunohistochemistry. of the pars intermedia in horses immunohistochemically We chose these antibodies because the relevant hormones, express POMC, α-MSH, β-endorphin and ACTH10. High except prolactin and PCNA, are proopiomelanocortin levels of plasma ACTH and/or adrenocortical hyperplasia (POMC)-derived peptides9. Furthermore, the intermediate have been reported in some horses with pituitary lobe of the pituitary gland is a major part of the POMC adenomas11,12. In the pars intermedia of dogs, most cells system in mice and rats6. demonstrated immunoreactivity to either ACTH or α- The white nodule grossly resembled a tumor arising MSH13. Adenomas of the pars intermedia in dogs were from the brain because the nodule adhered to and merged endocrinologically inactive and associated with varying into the brain tissue. Histologically, the tumor was degrees of hypopituitarism and diabetes insipidus, or were considered to have been derived from the pars intermedia of endocrinologically active and secreted excessive amounts of the pituitary gland because the pars distalis was separated ACTH, leading to bilateral adrenocortical hyperplasia and a from the tumor mass and compressed at the base of the brain. syndrome of cortisol excess14. Our histopathological and The tumor of the pars intermedia had grown expansively, immunohistochemical results suggest that the tumor cells and showed infiltrative growth into the brain parenchyma might not have actively secreted POMC-derived peptides, (Fig. 1). Clinical signs including swelling of the head were though we have no data on the serum concentrations of these considered to be attributable to compression of and damage hormones in this animal. In conclusion, the mouse described to the base of the brain, including the by the above was found to have a pituitary carcinoma of the pars pituitary tumor. The tumor consisted of polygonal or intermedia. The present report provides additional elongated cells, and tumor cells were arranged in a solid histopathological evidence of pituitary carcinoma of the pars sheet or nest-like structures intermingled with fibrovascular intermedia in aging mice. stroma. Tumor cells had round or oval nuclei and pale eosinophilic granular cytoplasm (Fig. 2), and showed slight Acknowledgments: The authors would like to thank Dr. cellular pleomorphism. Sporadic mitotic figures were seen Makoto Enomoto for reviewing this manuscript. We also in the tumor while necrosis was a prominent feature. A large would like to thank Dr. Mari Iida for her helpful comments. number of PCNA-positive nuclei was observed in the tumor compared with adenoma of the pars intermedia (Fig. 3). In

Fig. 1. Low-power magnification of the pituitary tumor of the pars intermedia. The tumor shows expansive growth and infiltrative growth into the brain parenchyma (arrows). Necrosis (asterisks) is a prominent feature of the tumor. PT: pituitary tumor, A: Ammon’s horn, C: cerebral cortex, H&E. Bar = 1 mm. Fig. 2. High-power magnification of the pituitary tumor of the pars intermedia. The tumor consists of polygonal or elongated cells, and tumor cells are arranged in a solid sheet or nest-like structures intermingled with fibrovascular stroma. Tumor cells have round or oval nuclei and pale eosinophilic granular cytoplasm. H&E. Bar = 50 µm. Fig. 3. A large number of PCNA-positive nuclei is observed in the tumor. Inset: the number of PCNA-positive nuclei in adenoma is far fewer than the present case. Bar = 100 µm. Fig. 4. High-power magnification of the pituitary tumor of the pars intermedia. Note the distinct infiltration into the brain parenchyma. H&E. Bar = 50 µm. Fig. 5. Tumor cells are positive for α-MSH by immunohistochemical staining. Bar = 50 µm. Fig. 6. Tumor cells are positive for γ-MSH by immunohistochemical staining. Bar = 50 µm. Fig. 7. Tumor cells are positive for β-endorphin by immunohistochemical staining. Bar = 50 µm. Fig. 8. Tumor cells are positive for ACTH by immunohistochemical staining. Bar = 50 µm. 108 Pituitary Intermedia Carcinoma in a Mouse

References Scientific Publifications, Lyon. France. 527–563. 1994. 8. Capen CC, Karbe E, Deschl U, George C, Germann P-G, 1. Frith CH and Ward JM. . In: Color Atlas of Gopinath C, Hardisty JF, Kanno J, Kaufmann W, Krinke G, Neoplastic and Non-neoplastic Lesions in Aging Mice. Küttler K, Kulwich B, Landes C, Lenz B, Longeart L, Elsevier, Amsterdam. 33–48. 1988. Paulson I, Sander E, and Tuch K. Endocrine system. In: 2. Mackenzie WF and Boorman GA. Pituitary gland. In: International Classification of Rodent Tumors, The Mouse. Pathology of the Fischer Rat, Reference and Atlas. GA U Mohr, P Greaves, N Ito, CC Capen, JF Hardisty, PH Long, Boorman, SL Eustis, MR Elwell, CA Montgomery Jr, and DL Dungworth, Y Hayashi, and G krinke (eds). Springer- WF. Mackenzie (eds). Academic Press, San Diego, CA. Verlag, Berlin, Germany. 269–322. 2001. 485–500. 1990. 9. Jones TC, Hunt RD, and King NW. Pituitary (hypophysis) 3. Mahler JF and Elwell MR. Pituitary gland. In: Pathology of and hypothalamus. In: Veterinary Pathology, 6th ed. the Mouse, Reference and Atlas, RR Maronpot, GA Williams & Wilkins, Baltimore, MD. 1223–1232. 1997. Boorman, and BW Gaul (eds). Cache River Press, Vienna, 10. Heinrichs M, Baumgärtner W, and Capen CC. Immuno- IL. 491–507. 1999. cytochemical demonstration of proopiomelanocortin-derived 4. Stefaneanu L and Kovacs K. Changes in structure and peptides in pituitary adenomas of the pars intermedia in function of the pituitary. In: Pathology of the Aging Rat, horses. Vet Pathol. 27: 419–425. 1990. Vol. 2. U Mohr, DL Dungworth, and CC Capen (eds). ILSI 11. Moore JN, Steiss J, Nicholson WE, and Orth DN. A case of Press, Washington DC. 173–191. 1994. pituitary adenocorticotropin-dependent Cushing’s syndrome 5. Hirouchi Y, Iwata H, Yamakawa S, Kato M, Kobayashi K, in the horse. . 104: 576–582. 1979. Yamamoto T, Inoue H, and Enomoto M. Historical data of 12. Wilson MG, Nicholson WE, Holscher MA, Sherrell BJ, neoplastic and non-neoplastic lesions in B6C3F1 (C57BL/ Mount CD, and Orth DN. Proopiolipomelanocortin peptides 6CrSlc × C3H/HeSlc) mice. J Toxicol Pathol. 7: 153–177. in normal pituitary, pituitary tumor, and plasma of normal 1994. and Cushing’s horses. Endocrinology. 110: 941–954. 1982. 6. Oishi Y, Matsumoto M, Yoshizawa K, and Fujihira S. 13. Capen CC. The endocrine glands. In: Pathology of Domestic Spontaneous pituitary adenoma of the pars intermedia in Animals, 3rd ed. Vol. 3, KVF Jubb, PC Kennedy, and N mice and rat: histopathological and immunocytochemical Palmer (eds). Academic Press, San Diego, CA. 237–303. studies. J Toxicol Pathol. 5: 223–231. 1992. 1985. 7. Liebelt AG. Tumours of the pituitary gland. In: Pathology of 14. Capen CC. Tumors of the endocrine glands. In: Tumors in Tumours in Laboratory Animals, Tumours of the Mouse, Domestic Animals, 4th ed. DJ Meuten (ed). Iowa State 2nd ed. Vol. 2, VS Turusov and U Mohr (eds). IARC Press, IA. 607–696. 2002.