Somatotroph Pituitary Tumors in Budgerigars ( Melopsittacus undulatus )

Abstract

A series of 11 pituitary tumors in budgerigars were classified on the basis of their clinical, gross, microscopic, and immunohistochemical characteristics. Affected birds were young to middle-aged. Clinically, neurologic signs—including difficulties flying, ataxia, and blindness—were most commonly reported. Additional clinical signs included weight loss, abnormal feathers or molting, increased respiratory efforts, and exophthalmos. Nine birds were diagnosed with chromophobic pituitary adenomas, and 2 birds had chromophobic pituitary carcinomas. Only 1 tumor was delimited to the pituitary gland; the other 10 variably invaded the brain, skull, and retrobulbar space. Distant metastases were identified in 2 birds. All tumors were immunohistochemically strongly positive for growth hormone, consistent with the diagnosis of somatotroph tumors. The common occurrence and early onset may suggest a genetic predisposition of budgerigars to develop somatotroph pituitary tumors with a high incidence of local invasion and with metastatic potential.

Pituitary tumors are common in budgerigars (Melopsittacus undulatus). ^12,13,16 Historically, pituitary tumors have been classified as acidophil, basophil, or chromophobe on the basis of their tinctorial characteristics. ⁸ In budgerigars, most pituitary tumors have been reported as chromophobic adenomas based on their microscopic appearance, ¹² but data about their hormonal characterization are lacking. The purpose of this study was to characterize a series of pituitary tumors in budgerigars based on their clinical, gross, microscopic, and immunohistochemical appearance.

Material and Methods

Between 1995 and 2008, Northwest ZooPath identified 17 proliferative pituitary neoplastic lesions in the 665 budgerigar submissions received (relative prevalence = 2.5%). Eleven of these pituitary tumors were included in this study. For all cases, prosection was done by the referring veterinarian, and either the tumor was sampled at that time or the entire fixed head submitted. Specimens were fixed in 10% neutral-buffered formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin. For those cases in which the head was submitted, transverse serial sections of the head were made following decalcification with a hydrochloride acid–based solution (Rapid-Cal, BBC Advanced Biomedical Reagents and Technologies, Stanwood, WA). All tumors were examined microscopically and classified for the purpose of this article as benign or malignant according to the current World Health Organization classification of human and mammalian pituitary tumors. ^1,8 Immunohistochemistry was performed on sections of all pituitary tumors for adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), melanocyte-stimulating hormone (MSH), growth hormone (GH), follicle-stimulating hormone (FSH), and β-endorphin using a Bond-maX autostainer (Leica, Bannockburn, IL) and for oxytocin using a Dako Autostainer (Dako, Carpenteria, CA). Primary antibodies were rabbit polyclonal anti-human ACTH (A0571, Dako; 1:500), mouse monoclonal anti-human TSH (M3503, Dako; 1:100), rabbit polyclonal anti-α-MSH (16110, Progen, Heidelberg, Germany; 1:100), rabbit polyclonal anti-human GH (A0570, Dako; 1:1000), guinea-pig polyclonal anti-ovine FSH β (National Hormone and Peptide Program, Harbor UCLA Research and Education Institute, Torrance, CA; 1:50), rabbit polyclonal anti-β-endorphin (16017, Progen; 1:50), and rabbit polyclonal anti-oxytocin (16083, Progen; 1:800). The Bond Polymer Refine Detection system (Leica) was used for ACTH, TSH, and MSH, and the Bond Intense R Detection system (Leica) was used for GH, FSH β, and β-endorphin. For oxytocin, the streptavidin-biotin 2 detection system (LSAB2, Dako) was used. Diaminobenzidine was used as the chromogen, with hematoxylin as the counterstain. Sections of normal budgerigar pituitary gland were used as positive controls. Negative controls consisted of sections of normal budgerigar pituitary gland treated identically except that the primary antibodies were replaced by buffer.

Results

Pituitary tumors of 11 budgerigars were included in this study. Birds included 8 males, 1 female, and 2 of undetermined sex, varying in age from 2 to 5 years old. Eight birds had reportedly neural signs, including difficulty flying, ataxia, seizurelike activity, abnormal posture, blindness, head tilt, and circling. Additional reported clinical signs consisted of weight loss (n = 4), abnormal feathers or molting (n = 2), increased respiratory effort (n = 2), and unilateral exophthalmos (n = 1). Six birds were found dead, and 5 were humanely euthanized. Clinical disease was generally short, for up to 1 week, but 1 bird was reportedly a poor flier, with abnormal molting and gradual weight loss, for approximately 15 months.

Birds with history of weight loss were variably thin at necropsy, with reduced pectoral muscle mass and scant body fat. The pituitary gland in normal budgerigars lays within the sella turcica in the dorsal surface of the basisphenoid bone, as it does in mammals (Fig. 1). In the birds with pituitary neoplasms, the gland was replaced by a soft pale pink mass that measured up to 1 cm and variably extended into the brain, calvarium, and circumjacent soft tissues (Fig. 2). Histologically in all budgerigars, the pituitary gland was completely replaced by neoplastic cells. One tumor was histologically well delimited and encapsulated, expanding the sellar cavity and compressing the overlying brain. The other 10 tumors were poorly delimited, were partially encapsulated or not at all, and variably invaded the circumjacent hard and soft tissues, including the calvarial bone and the regional skeletal muscles (Fig. 3). In the bird with reported exophthalmos, the tumor extended along both optic nerves into the retrobulbar space, displacing the most affected eye cranially. The neoplasms comprised compact packets and nests of polygonal cells interspersed by delicate fibrovascular septae. Individual cells had indistinct cell margins, scant to moderate eosinophilic fine granular cytoplasm, and vesicular oval nuclei with variably prominent nucleoli. Anisokaryosis varied from mild to marked, with most nuclei ranging in size from 8 to 15 μm. Cells with bizarre and giant nuclei measuring up to 100 μm were common in 2 cases (Fig. 4). Many karyomegalic cells had intranuclear cytoplasmic invaginations (pseudoinclusions). Mitotic figures were generally rare (1 per 5 high-power fields), but tumors with high nuclear pleomorphism had 1 to 2 mitoses per single high-power field (400×). In the 10 cases with invasive neoplasms, the cells extended into the surrounding brain and meninges as well as into the marrow and air spaces of the bones forming the calvarium and nasal septum, variably effacing the preexisting woven bone in these areas. Two birds had distant metastases in the liver (Fig. 5); 1 of them also had metastasis in the midbrain and air sacs. Based on the occurrence of the metastases, the pituitary tumors in these 2 birds were diagnosed as pituitary carcinomas. Following the current human and mammalian pituitary tumor classification, ^1,8 which had been previously applied to budgerigar pituitary tumors, ¹² 2 coauthors (I.M.L. and M.K.) classified the other 9 tumors as adenomas despite the extensive invasiveness of 8 tumors into the surrounding bone and muscles. On the basis of their tinctorial characteristics, all tumors were classified as chromophobe.

Figure 1.

Head, midsagittal section, budgerigar. Normal pituitary (*) in the sella turcica at the base of the brain. Figure 2. Head, coronal section, budgerigar. The pituitary gland is replaced by a large mass that extends far beyond the sella turcica into the hard and soft tissues at the base of the brain (*). Figure 3. Pituitary adenoma, budgerigar. Neoplastic cells are packeted by delicate fibrovascular stroma, as typically seen with endocrine neoplasms. Note bony invasion of the tumor in the adjacent calvarium. HE. Figure 4. Pituitary adenoma, budgerigar. Cells with giant and bizarre nuclei are common in this neoplasm. HE. Figure 5. Liver, budgerigar. A metastatic nodule of a pituitary carcinoma replaces part of the parenchyma in the liver (L). Remaining hepatocytes are swollen and vacuolated. HE. Figure 6. Orbit, budgerigar. Sheets of neoplastic cells of an invasive pituitary adenoma extend into the retrobulbar space along the optic nerve, abutting the caudal aspect of the eye (E). Neoplastic cells have strong cytoplasmic immunolabeling for growth hormone. Inset: High-power view of the growth hormone-positive neoplastic cells.

Additional alterations noted histologically in the budgerigars included acute pulmonary congestion and hemorrhage (n = 4), chronic proventriculitis (n = 4) with intralesional Macrorhabdus ornithogaster yeasts (n = 2), hepatocellular (nonlipid) vacuolar degeneration (n = 3), hepatic lipidosis (n = 1), hepatic cholestasis and anisokaryosis (n = 1), mesangioproliferative glomerulonephritis (n = 3), renal tubular necrosis (n = 3), interstitial nephritis (n = 2) with urate stasis (n = 1), renal tubular epithelial adenoviral inclusions (n = 1), renal tubular adenocarcinoma (n = 1), metastatic fibrosarcoma in the medullary cavity of the long bones and vertebrae (n = 2), visceral gout (n = 1), splenic lymphoid depletion (n = 1), crop epithelial hyperplasia and hyperkeratosis (n = 1), and mild feather dysplasia (n = 1).

In birds, the normal pituitary gland consists of 2 distinct parts: the adenohypophysis and the neurohypophysis. The adenohypophysis is further divided into 2 parts: the pars distalis and the pars tuberalis. ¹⁷ In the cross sections of normal pituitary and overlying brain used as a control for this study, the distribution of the various secretory cell types was similar to that described in human for most hormones: ⁵ oxytocin, the hypothalamus; GH and β-endorphin, throughout the lateral wings and singly or in small random clusters in the central region of the pars distalis; ACTH and MSH, throughout the central portion and lateral wings of the pars distalis; and FSH, singly or in small random clusters in the pars distalis. Only the distribution of the TSH-producing cells differed in that these cells formed peripheral continuous aggregates in the budgerigar control section rather than singly or in small clusters in the anteromedial portion of the human gland.

All pituitary tumors had 75% to 95% cells with strong positive cytoplasmic immunolabeling for GH (Fig. 6). Seven tumors also had scattered neoplastic cells with positive cytoplasmic labeling for ACTH and FSH, and 5 had scattered positive cells for β-endorphin. The cells that labeled positive for these other hormones were usually located at the periphery of the tumors. Neoplastic cells were negative for TSH, MSH, and oxytocin. Results of the immunohistochemical labeling indicate that the pituitary masses in budgerigars are somatotroph tumors.

Discussion

Consistent expression of GH (somatotropin) indicates that the pituitary masses in budgerigars are somatotroph tumors, which are uncommon in domestic animals. ⁸ The results of this study are consistent with previous indication by bioassays of a growth-promoting effect of a transplantable pituitary tumor in budgerigars. ¹⁴ Nestling budgerigars with pituitary tumor transplants had increased body and organ weights after neoplastic cell inoculation, ¹⁴ and hypophysectomized rats inoculated with budgerigar pituitary tumor extracts had increased tibial epiphyseal width. ¹³ Additionally, significant plasma albumin increase, fatty degeneration of the liver, and hyperglycemia were reported in pituitary tumor-bearing budgerigars. ^13,15 In this study, hepatic lipidosis was reported in only 1 bird, and 4 birds reportedly had weight loss rather than increased weight. Although clinical signs were said to be usually short, several budgerigars included in this study came from large flocks, and clinical signs may therefore have been evident only in advanced stages of the disease, when birds were emaciated.

The most commonly reported history was that of neurologic signs, which were attributed to variable compression and extension of the neoplasm into the surrounding brain. Exophthalmos and blindness, noted in 2 of the birds in this study, have been reported as the most frequent signs in affected budgerigars in association with tumor extension into the retrobulbar space. ¹² Despite such extensive tumor growth, affected birds can still live for several weeks ¹² or even months, as indicated by the 15-month clinical history in 1 budgerigar. Abnormal molting and feathers were observed in 2 birds in this study. Retarded feather growth and feather abnormalities are seen with reduced thyroxin levels. ¹⁰ It is possible that the replacement of the normal pituitary gland, including that of TSH-producing cells, by tumor cells is the reason for the feathering abnormalities reported in this study.

Pituitary tumors in budgerigars reportedly occur with approximate equal frequency in males and females. ¹² In this study, 8 of the 11 budgerigars were male. A reason for the overrepresentation of males in this study is undetermined.

A gradual transition from pituitary hyperplasia to neoplasia in budgerigars has been suggested. ¹² Exposure to prolonged or more intense light has been thought to be the underlying cause for this alteration. ¹² Additionally, pituitary cell proliferation following apoptosis has been shown to occur in laying hens after temporary feed withdrawal to induce egg-laying cessation and molting. ⁶ Thus, feed management could play a role in the induction of proliferative lesions in the pituitary gland of birds. Interestingly, whereas pituitary tumors have been determined to be one of the most common tumors in budgerigars in North America, ¹² they are reportedly far less common in the United Kingdom. ³ In a survey of the causes of mortality and morbidity in budgerigars in the United Kingdom, the pituitary gland had often not been examined, ² which could account at least in part for the reduced reported prevalence of this lesion in British birds.

According to the current classification of human and mammalian pituitary tumors and a previous study of pituitary tumors in budgerigars, the sole morphologic criterion to distinguish benign from malignant pituitary tumors is the presence of metastases. ^1,8,12 In this study, only 2 birds (18%) had distant metastases to the liver and air sac. This is still a higher incidence, as in a previously reported study of 50 affected budgerigars, where metastases were noted in the kidney and liver of 2 birds (4%). ¹² Furthermore, the majority of cases (10 of 11) variably invaded the soft and bony tissues surrounding the pituitary. It is interesting that a budgerigar with a pituitary tumor transplant in the sixth passage developed metastases in the liver. ⁷ Given the large number of invasive pituitary tumors with extensive destruction of bone and extension into the overlying brain in these birds, it is possible that these tumors present in general with a more aggressive behavior and a higher likelihood for malignant transformation than in mammals. It is also possible that more of the birds in our report had metastatic lesions that were not detected in the limited tissue sets submitted for histologic examination. One author (M.M.G.) felt strongly that the current classification of pituitary tumor behavior for mammals is not pertinent for budgerigars and that these tumors should be classified as malignant based on their highly invasive behavior and potential for metastasis.

Most tumors in this study were composed of a monomorphic to mildly pleomorphic cell population with a typical neuroendocrine pattern characterized by cell packeting; however, one of the neoplasms included in this study had initially been misdiagnosed as glioblastoma multiforme due to the prominent pleomorphism of the neoplastic cells. Interestingly, a case of glioblastoma multiforme with similar clinical and morphologic characteristics was reported in a budgerigar. ¹¹ Immunohistochemistry for growth hormone in these cases may be helpful in differentiating such tumors.

In 3 cases, the budgerigars had neoplasms in other organs. One bird had a renal adenocarcinoma, and 2 had intraosseous metastatic fibrosarcoma. The primary site of the fibrosarcomas was undetermined but may have been the wing because this is where most of them start (M. M. Garner, personal communication). The concurrent occurrence of renal adenocarcinoma and pituitary tumor in budgerigars was regarded as noteworthy. ¹² Mutations in oncogenes or tumor suppressor genes would be a plausible explanation for this; however, at least in human pituitary adenomas, somatic gene mutations or deletions identified in other malignancies are absent, such as those of the retinoblastoma (Rb), p53, and c-erbB2/neu genes. ¹ It remains to be determined whether the concurrent development of various tumors in budgerigars is the result of mutations in oncogenes or tumor suppressor genes or is only a coincidental concurrence of tumors that are common in budgerigars. ⁹

In conclusion, whereas in mammals, pituitary tumors usually have an expansile growth, ⁸ the majority of the pituitary tumors in the budgerigars were invasive and some metastasized. And while pituitary tumors affect most commonly older mammals, ⁴ budgerigars with spontaneous pituitary tumor were young to middle-aged. The relatively common occurrence and early onset of somatotroph pituitary tumors in budgerigars suggest that these birds are genetically predisposed to developing such tumors.