Plasma sodium and potassium concentrations after hypophysectomy in dogs with corticotroph adenomas.

BACKGROUND: Electrolyte abnormalities, especially hypernatremia, are frequent complications after transsphenoidal hypophysectomy in dogs with pituitary-dependent hypercortisolism. OBJECTIVES: To describe electrolyte abnormalities after transsphenoidal hypophysectomy and to investigate possible associations between postoperative hypernatremia and clinical and surgical variables as well as with postoperative outcome. ANIMALS: One hundred and twenty-seven client-owned dogs. METHODS: Dogs with pituitary corticotroph adenomas that underwent transsphenoidal hypophysectomy were retrospectively included. Plasma sodium and potassium concentrations were measured -2, +2, +8, +24, and +48 hours from hypophysectomy. Clinical (breed, age, body weight, skull type, urinary cortisol/creatinine ratio, percentage of suppression to dexamethasone) and surgical variables (duration of anesthesia and surgery, pituitary dimensions) were compared to the development of hypernatremia. RESULTS: Postoperative hypernatremia developed in 46.5% (57/127) of dogs and hyponatremia in 6.3% (8/127). Plasma sodium concentration increased after surgery and peaked at 8 hours after surgery, normalizing after 24 to 48 hours. Plasma potassium concentration increased without exceeding the reference limit. No significant associations were found between clinical and surgical variables and hypernatremia, or between hypernatremia and postoperative death, long-term survival or recurrence. Surgery time was significantly longer in dogs that developed persistent diabetes insipidus (P = .02) and persistent diabetes insipidus occurred more frequently in dogs with enlarged pituitary glands (P = .01). CONCLUSION AND CLINICAL IMPORTANCE: Hypernatremia remains a frequent postoperative complication after transsphenoidal hypophysectomy but did not appear to have an impact on postoperative outcome. No predisposing factor to postoperative hypernatremia was identified. Variations in plasma potassium concentrations do not seem to influence postoperative outcome.

Pituitary tumour types in dogs and cats.

Pituitary tumours are common in dogs and are being increasingly recognized in cats. Pituitary tumours are usually classified as adenomas and should only be classified as carcinomas when there is evidence of metastatic spread of the tumour, which is rare. Despite the benign nature of most pituitary tumours, they can still compress or invade neighbouring tissues. Pituitary tumours can be functional (hormonally active) or non-functional (hormonally silent). The aim of this review was to provide an overview of the different pituitary tumour types in dogs and cats that have been reported in the literature. In dogs, the most common pituitary tumour type is the corticotroph adenoma, which can cause pituitary-dependent hypercortisolism. In cats, the most common pituitary tumour is the somatotroph adenoma, which can cause hypersomatotropism, and the second-most common is the corticotroph adenoma. A lactotroph adenoma has been described in one dog, while gonadotroph, thyrotroph and null cell adenomas have not been described in dogs or cats. Hormonally silent adenomas are likely underdiagnosed because they do not result in an endocrine syndrome. Tools used to classify pituitary tumours in humans, particularly immunohistochemistry for lineage-specific transcription factors, are likely to be useful to classify canine and feline pituitary tumours of unknown origin. Future studies are required to better understand the full range of pituitary adenoma pathology in dogs and cats and to determine whether certain adenoma subtypes behave more aggressively than others. Currently, the mechanisms that underlie pituitary tumorigenesis in dogs and cats are still largely unknown. A better understanding of the molecular background of these tumours could help to identify improved pituitary-targeted therapeutics.

The mRNA expression of PTTG1 is a strong prognostic indicator for recurrence after hypophysectomy in dogs with corticotroph pituitary adenomas.

Pituitary-dependent hypercortisolism (PDH) is a common endocrinopathy in dogs, but the promotors and initiators of the tumourigenesis of corticotroph pituitary adenomas remain unknown. Based on human data, we investigated mRNA expression of pituitary tumour transforming gene 1 (PTTG1) with quantitative RT-PCR in canine corticotroph pituitary adenomas. PTTG1 was overexpressed in adenomas approximately 3-fold. A strong association was observed between PTTG1 expression and disease-free interval; dogs with high PTTG1 expression had a significantly (4 times; P=0.02) shorter disease-free interval than dogs with low PTTG1 expression. This paper shows that PTTG1 expression is a negative prognosticator in relation to disease-free interval and recurrence in dogs undergoing transsphenoidal hypophysectomy as treatment for PDH.

Protein expression of somatostatin receptor 2, somatostatin receptor 5 and dopamine D2 receptor in normal pituitary gland and ACTH-secreting pituitary adenoma in dogs.

PURPOSE: To evaluate the protein expression of somatostatin receptor (SSTR) 2, SSTR5 and dopamine D2 receptor (DA2R)-targets of somatostatin analogs and dopamine agonists-in normal canine pituitary and canine adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. METHODS: Six normal canine pituitary glands and 14 canine ACTH-secreting pituitary adenoma cases were included in this study. The protein expression of SSTR2, SSTR5 and DA2R was determined by double-label immunofluorescence staining of these receptors and ACTH. RESULTS: SSTR2, SSTR5, and DA2R proteins were expressed in the anterior and intermediate lobes of normal canine pituitary glands. In the anterior pituitary lobes, the percentages of SSTR2-, SSTR5-, and DA2R-positive cells among the ACTH-positive population were 27.0 ±â€¯8.6%, 27.9 ±â€¯5.9%, and 34.0 ±â€¯9.4%, respectively. In contrast, the corresponding percentages in the intermediate pituitary lobes were 97.8 ±â€¯1.5%, 94.1 ±â€¯4.4%, and 96.1 ±â€¯6.6%, respectively. Of the 14 ACTH-secreting pituitary adenoma cases, 11, 12, and 6 cases expressed SSTR2, SSTR5, and DA2R, respectively. Additionally, four cases showed strong positive staining for both SSTR2 and SSTR5. Two of these were ACTH-secreting pituitary adenomas likely derived from the intermediate pituitary lobe, because these are α-Melanocyte-stimulation hormone (α-MSH)-positive stains. CONCLUSION: Immunohistological detection and characterization of SSTR2, SSTR5 and DA2R may provide useful additional information for determining treatment options when an ACTH-secreting pituitary adenoma cannot be completely resected, or in the case of recurrence.

Bone morphogenetic protein 4 and bone morphogenetic protein receptor expression in the pituitary gland of adult dogs in healthy condition and with ACTH-secreting pituitary adenoma.

The purpose of this study was to investigate the expression of bone morphogenetic protein 4 (BMP4) and its receptors, bone morphogenetic protein receptor I (BMPRI) and BMPRII, in the pituitary gland of healthy adult dogs and in those with ACTH-secreting pituitary adenoma. Quantitative polymerase chain reaction analysis showed that the BMP4 messenger RNA expression level in the ACTH-secreting pituitary adenoma samples was significantly lower than that in the normal pituitary gland samples (P = 0.03). However, there were no statistically significant differences between samples with respect to the messenger RNA expression levels of the receptors BMPRIA, BMPRIB, and BMPRII. Double-immunofluorescence analysis of the normal canine pituitary showed that BMP4 was localized in the thyrotroph (51.3 ± 7.3%) and not the corticotroph cells. By contrast, BMPRII was widely expressed in the thyrotroph (19.9 ± 5.2%) and somatotroph cells (94.7 ± 3.6%) but not in the corticotroph cells (P < 0.001, thyrotroph cells vs somatotroph cells). Similarly, in ACTH-secreting pituitary adenoma, BMP4 and BMPRII were not expressed in the corticotroph cells. Moreover, the percentage of BMP4-positive cells was also significantly reduced in the thyrotroph cells of the surrounding normal pituitary tissue obtained from the resected ACTH-secreting pituitary adenoma (8.3 ± 7.9%) compared with that in normal canine pituitary (P < 0.001). BMP4 has been reported to be expressed in corticotroph cells in the human pituitary gland. Therefore, the results of this study reveal a difference in the cellular pattern of BMP4-positive staining in the pituitary gland between humans and dogs and further revealed the pattern of BMPRII-positive staining in the dog pituitary gland. These species-specific differences regarding BMP4 should be considered when using dogs as an animal model for Cushing's disease.

Lack of Ubiquitin Specific Protease 8 (USP8) Mutations in Canine Corticotroph Pituitary Adenomas.

PURPOSE: Cushing's disease (CD), also known as pituitary-dependent hyperadrenocorticism, is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumours. Affected humans and dogs have similar clinical manifestations, however, the incidence of the canine disease is thousand-fold higher. This makes the dog an obvious model for studying the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite certain similarities identified at the molecular level, the question still remains whether the two species have a shared oncogenetic background. Recently, hotspot recurrent mutations in the gene encoding for ubiquitin specific protease 8 (USP8) have been identified as the main driver behind the formation of ACTH-secreting pituitary adenomas in humans. In this study, we aimed to verify whether USP8 mutations also play a role in the development of such tumours in dogs. METHODS: Presence of USP8 mutations was analysed by Sanger and PCR-cloning sequencing in 38 canine ACTH-secreting adenomas. Furthermore, the role of USP8 and EGFR protein expression was assessed by immunohistochemistry in a subset of 25 adenomas. RESULTS: None of the analysed canine ACTH-secreting adenomas presented mutations in the USP8 gene. In a subset of these adenomas, however, we observed an increased nuclear expression of USP8, a phenotype characteristic for the USP8 mutated human tumours, that correlated with smaller tumour size but elevated ACTH production in those tumours. CONCLUSIONS: Canine ACTH-secreting pituitary adenomas lack mutations in the USP8 gene suggesting a different genetic background of pituitary tumourigenesis in dogs. However, elevated nuclear USP8 protein expression in a subset of tumours was associated with a similar phenotype as in their human counterparts, indicating a possible end-point convergence of the different genetic backgrounds in the two species. In order to establish the dog as a useful animal model for the study of CD, further comprehensive studies are needed.

The prognostic value of perioperative profiles of ACTH and cortisol for recurrence after transsphenoidal hypophysectomy in dogs with corticotroph adenomas.

BACKGROUND: Transsphenoidal hypophysectomy is an effective treatment for dogs with pituitary-dependent hypercortisolism (PDH). However, long-term recurrence of hypercortisolism is a well-recognized problem, indicating the need for reliable prognostic indicators. OBJECTIVES: The aim of this study was to evaluate the prognostic value of perioperative plasma ACTH and cortisol concentrations for identifying recurrence of hypercortisolism after transsphenoidal hypophysectomy. ANIMALS: A total of 112 dogs with PDH that underwent transsphenoidal hypophysectomy met the inclusion criteria of the study. METHODS: Hormone concentrations were measured preoperatively and 1-5 hours after surgery. Both absolute hormone concentrations and postoperative concentrations normalized to preoperative concentrations were included in analyses. The prognostic value of hormone concentrations was studied with Cox's proportional hazard analysis. RESULTS: Median follow-up and disease-free period were 1096 days and 896 days, respectively. Twenty-eight percent of patients had recurrence, with a median disease-free period of 588 days. Both absolute and normalized postoperative cortisol concentrations were significantly higher in dogs with recurrence than in dogs without recurrence. High ACTH 5 hours after surgery, high cortisol 1 and 4 hours after surgery, high normalized ACTH 3 hours after surgery, high normalized cortisol 4 hours after surgery and the random slope of cortisol were associated with a shorter disease-free period. CONCLUSIONS AND CLINICAL IMPORTANCE: Individual perioperative hormone curves provide valuable information about the risk of recurrence after hypophysectomy. However, because no single cutoff point could be identified, combination with other variables, such as the pituitary height/brain area (P/B) ratio, is still needed to obtain a good estimate of the risk for recurrence of hypercortisolism after hypophysectomy.

EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas.

Cushing disease is a condition in which the pituitary gland releases excessive adrenocorticotropic hormone (ACTH) as a result of an adenoma arising from the ACTH-secreting cells in the anterior pituitary. ACTH-secreting pituitary adenomas lead to hypercortisolemia and cause significant morbidity and mortality. Pituitary-directed medications are mostly ineffective, and new treatment options are needed. As these tumors express EGFR, we tested whether EGFR might provide a therapeutic target for Cushing disease. Here, we show that in surgically resected human and canine corticotroph cultured tumors, blocking EGFR suppressed expression of proopiomelanocortin (POMC), the ACTH precursor. In mouse corticotroph EGFR transfectants, ACTH secretion was enhanced, and EGF increased Pomc promoter activity, an effect that was dependent on MAPK. Blocking EGFR activity with gefitinib, an EGFR tyrosine kinase inhibitor, attenuated Pomc expression, inhibited corticotroph tumor cell proliferation, and induced apoptosis. As predominantly nuclear EGFR expression was observed in canine and human corticotroph tumors, we preferentially targeted EGFR to mouse corticotroph cell nuclei, which resulted in higher Pomc expression and ACTH secretion, both of which were inhibited by gefitinib. In athymic nude mice, EGFR overexpression enhanced the growth of explanted ACTH-secreting tumors and further elevated serum corticosterone levels. Gefitinib treatment decreased both tumor size and corticosterone levels; it also reversed signs of hypercortisolemia, including elevated glucose levels and excess omental fat. These results indicate that inhibiting EGFR signaling may be a novel strategy for treating Cushing disease.

Ki-67 and minichromosome maintenance-7 (MCM7) expression in canine pituitary corticotroph adenomas.

Pituitary-dependent hyperadrenocorticism (PDH) caused by pituitary corticotroph adenoma is a common endocrine disorder in dogs. The ratio between pituitary height and the area of the brain (P/B) has been used to evaluate the pituitary size. A P/B ratio > 0.31 indicates an enlarged pituitary, whereas a P/B ratio ≤ 0.31 indicates a nonenlarged pituitary. The aim of this study was to investigate the expression of proliferation markers Ki-67 and minichromosome maintenance-7 (MCM7) in canine corticotroph adenomas in enlarged and in nonenlarged pituitaries and to evaluate their relation with the size of canine pituitary corticotroph adenomas. Ki-67 and MCM7 expression in ACTH-positive tumor cells was determined by dual-labeling immunohistochemistry in resected corticotroph adenomas from 15 dogs with PDH. The mean ± SD Ki-67 labeling index (LI) was 0.55% ± 0.59% in corticotroph adenomas with nonenlarged pituitaries and 1.6% ± 0.6% in adenomas with enlarged pituitaries. The MCM7 LI in corticotroph adenomas with nonenlarged pituitaries and in adenomas with enlarged pituitaries was 2.9% ± 2.2% and 10.9% ± 3.7%, respectively. The Ki-67 LI and MCM7 LI were significantly greater in the adenomas with enlarged pituitaries than in the adenomas with nonenlarged pituitaries (P < 0.01 and P < 0.01, respectively). The MCM7 LI was significantly greater than the Ki-67 LI in adenomas (P < 0.01). The Ki-67 LI was positively correlated with the MCM7 LI (r = 0.820, P < 0.01), and the P/B ratio was positively correlated with the Ki-67 LI (r = 0.560, P = 0.03) and the MCM7 LI (r = 0.854, P < 0.01). In conclusion, canine corticotroph adenomas in enlarged pituitaries show greater proliferation potential than do adenomas in nonenlarged pituitaries. MCM7 expression was significantly greater than Ki-67 expression in canine pituitary corticotroph adenomas. Thus, MCM7 may be superior to Ki-67 as a proliferation marker in pituitary tumors.

Type II muscle fibers atrophy associated with silent corticotroph adenoma in a dog.

The Silent Corticotroph Adenoma (SCA) is a pituitary adenoma variant characterized by the immunoreactivity for adrenocorticotropic hormone (ACTH) and related peptides, without the clinical signs of Cushing's disease. SCA has been postulated to either secrete structurally abnormal ACTH that is inactive but detectable by immunohistochemistry or radioimmunoassay, or to secrete ACTH intermittently or at low levels continuously. Excess of ACTH has been associated to type II muscle atrophy. We describe a case of type II muscle fibers atrophy associated with silent corticotroph adenoma in a dog. The dog showed moderate to severe proximal muscle wasting and weakness with normal levels of muscle-associated enzymes. In the limb muscle biopsies, type II fibers were uniformly smaller than type I fibers. In temporalis muscles, there were few atrophic fibers, and several irregular areas of loss of enzymatic activity observed in NADH, SDH and COX stains. The tumour showed a trabecular growth pattern and immunohistochemical analysis demonstrated the presence of cytoplasmic immunoreactivity for ACTH. The muscle atrophy was considered to be related to an excess of inactive ACTH. Studying spontaneous occurring rare diseases in animals could help to understand the mechanism of similar diseases in human has well.

Corticotroph adenoma in the dog: pathogenesis and new therapeutic possibilities.

The corticotrophinoma, causing pituitary dependent hypercortisolism, represents the highest percentage of pituitary tumours in the dog. The mechanism by which it develops is currently unknown and two theories are postulated: the hypothalamic and the monoclonal. It is not clear either what factors are involved in the tumour genesis; nevertheless, firm candidates are the Rb1 gene, proteins p27, p21 and p16, as are also defects in the glucocorticoid receptor and Nur77/Nurr1. The role of BMPs remains to be evaluated in greater depth. Although at present the chosen treatment in human is surgical, there are various pharmacological treatments already in use that have favourable results and others, still under research, also showing promising results.

Expression of leukemia inhibitory factor and leukemia inhibitory factor receptor in the canine pituitary gland and corticotrope adenomas.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 family that activates the hypothalamic-pituitary-adrenal axis and promotes corticotrope cell differentiation during development. The aim of this study was to investigate the expression of LIF and its receptor (LIFR) in the canine pituitary gland and in corticotrope adenomas, and to perform a mutation analysis of LIFR. Using immunohistochemistry, immunofluorescence, and quantitative expression analysis, LIF and LIFR expression were studied in pituitary glands of control dogs and in specimens of corticotrope adenoma tissue collected through hypophysectomy in dogs with pituitary-dependent hypercortisolism (PDH, Cushing's disease). Using sequence analysis, cDNA was screened for mutations in the LIFR. In the control pituitary tissues and corticotrope adenomas, there was a low magnitude of LIF expression. The LIFR, however, was highly expressed and co-localized with ACTH(1-24) expression. Cytoplasmatic immunoreactivity of LIFR was preserved in corticotrope adenomas and adjacent nontumorous cells of pars intermedia. No mutation was found on mutation analysis of the complete LIFR cDNA. Surprisingly, nuclear to perinuclear immunoreactivity for LIFR was present in nontumorous pituitary cells of the pars distalis in 10 of 12 tissue specimens from PDH dogs. These data show that LIFR is highly co-expressed with adrenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) in the canine pituitary gland and in corticotrope adenomas. Nuclear immunoreactivity for LIFR in nontumorous cells of the pars distalis may indicate the presence of a corticotrope adenoma.

Expression of Ki-67, PCNA, and p27kip1 in canine pituitary corticotroph adenomas.

Pituitary-dependent hypercortisolism (PDH), which is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas, is a common endocrinopathy in dogs. Dogs with non-enlarged pituitaries harboring a microadenoma have a better prognosis than those with enlarged pituitaries. The aim of this study was to investigate the expression of the proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) and the cell-cycle inhibitor p27kip1 in corticotroph adenomas in enlarged and non-enlarged pituitaries. The expression of Ki-67, PCNA, and p27kip1 was analyzed by immunohistochemical staining of 17 pituitary adenoma samples harvested during pituitary surgery in dogs with PDH. The labeling index was calculated by counting the number of immunopositive cells per 1,000 cells. The mean (+/- standard deviation) labeling index for Ki-67 was 8.4%+/-14.2% for the group with enlarged pituitaries, and 8.8%+/-5.5% for the group with non-enlarged pituitaries; that for PCNA was 35.5%+/-12.2% and 37.0%+/-15.5%; and that for p27kip1 was 29.3%+/-22.6% and 42.5%+/-27.9%, respectively. No significant differences in Ki-67, PCNA, and p27kip1 labeling indices were found between enlarged and non-enlarged pituitaries. However, a trend toward significance was observed when comparing the expression of p27kip1 in enlarged pituitaries versus normal pituitary tissue. It is concluded that Ki-67 and PCNA are not useful as proliferative markers for studying the pathobiology of pituitary corticotroph adenomas in dogs.

A comparative study by age and gender of the pituitary adenoma and ACTH and alpha-MSH secretion in dogs with pituitary-dependent hyperadrenocorticism.

Pituitary-dependent hyperadrenocorticism (PDH) is frequent in dogs. Little is known about its presentation in different age groups and its characteristics. Dividing the population under study (n=107) into three age groups we observed that 11.2% were young, 51.4% adults and 37.4% aged. Using magnetic resonance, pituitary tumours were intra-sellar (IS) in 30.8% and extra-sellar (ES) in 62.6% and the pars intermedia (PI) was affected in 6.5%. ES are predominant in females and IS in males (p<0.0001). In the adult-aged population, the ES and PI are predominant, while in the young, the IS predominate (p<0.0001). ACTH concentration was greater in the ES vs. IS (p<0.05). alpha-MSH did not present significant differences according to tumour size, showing a negative correlation (r=-0.47; p<0.01) vs. ACTH. Differences in adenoma size according to gender and their age-related frequency of apparition could be because of different origins of the corticotrophinoma.

Coexistence of corticotroph adenoma and thyrotroph hyperplasia in a dog.

Pituitary thyrotroph hyperplasia results from prolonged primary hypothyroidism in humans, mice and rats. In dogs with Cushing's disease, many cases have low serum thyroid hormones concentrations due to euthyroid sick syndrome. A 6-year-old castrated male Beagle diagnosed with Cushing's disease had a high serum thyroid stimulating hormone (TSH) concentration that was treated by hypophysectomy. On histological examination, the resected pituitary gland contained both a corticotroph adenoma and thyrotroph hyperplasia. The TSH-positive cell ratio in this case was greater than that of healthy Beagles. In the present case, the pituitary thyrotroph hyperplasia was probably caused by primary hypothyroidism. In conclusion, this Beagle is the first histological confirmation of the coexistence of a corticotroph adenoma and thyrotroph hyperplasia.

Expression of genes related to corticotropin production and glucocorticoid feedback in corticotroph adenomas of dogs with Cushing's disease.

Cushing's disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to negative feedback by glucocorticoids. In this study, we examined gene expression related to adrenocorticotropic hormone (ACTH) production and secretion, and the negative feedback by glucocorticoids in canine corticotroph adenoma. We used resected corticotroph adenomas from 10 dogs with Cushing's disease. In order to investigate the alteration of gene expression between corticotroph adenoma and normal corticotrophic cells, ACTH-positive cells in the anterior lobe were microdissected using a laser-capture microdissection system, and mRNA levels of proopiomelanocortin (POMC), corticotropin releasing hormone receptor 1 (CRHR1), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11 beta hydroxysteroid dehydrogenase (11HSD) type 1 and type 2 were determined using real-time RT-PCR. POMC, CRHR1, and 11HSD2 mRNA levels in corticotroph adenoma were greater than those in normal corticotrophic cells (POMC, 5.5-fold; CRHR1, 4.9-fold; 11HSD2, 4.2-fold, P<0.01, respectively). MR and 11HSD1 mRNA levels in corticotroph adenoma were lower than those in normal corticotrophic cells (MR, 2.2-fold; 11HSD1, 2.9-fold, P<0.01, respectively). GR mRNA levels did not differ between corticotroph adenoma and normal corticotrophic cells. Our results may help to understand the increased ACTH production and the resistance to negative feedback suppression by glucocorticoids in canine corticotroph adenomas. These changes in gene expression may have a role in the growth of canine corticotroph adenoma, and help elucidate the pathophysiology of dogs with Cushing's disease.

Expression and mutation analysis of Tpit in the canine pituitary gland and corticotroph adenomas.

Pituitary-dependent hyperadrenocorticism (PDH) in dogs is caused by a pituitary corticotroph adenoma. Although PDH is a common disorder in dogs, little is known about the underlying pathogenesis. In the pituitary glands of humans and mice, the pro-opiomelanocortin (POMC)-expressing cell lineages, the corticotrophs and melanotrophs, have a specific marker in common, the T-box transcription factor Tpit (Tbx19), which is obligate for POMC expression. Tpit also regulates the late differentiation of the corticotrophs and melanotrophs, and therefore may contribute to the pathogenesis of the corticotroph adenomas. The aim of this study was to perform an expression and mutation analysis of Tpit in the normal canine pituitary and in corticotroph adenomas. The distribution of the Tpit protein in the pituitary gland was studied with immunohistochemistry and the expression of the gene with RT-PCR. The coding region of Tpit cDNA from 14 dogs with PDH was screened for mutations. Tpit was expressed in corticotroph and melanotroph cells of the normal and adenomatous canine pituitary, and remained present in non-adenomatous corticotrophs of pituitaries from PDH dogs. No tumor-specific mutation in the Tpit cDNA from the corticotroph adenomas was found. However, a missense polymorphism in the highly conserved DNA-binding domain, the T-box, was discovered in one dog. It is concluded that Tpit can be used as a reliable marker for the corticotroph and melanotroph cells in the canine pituitary tissue and that mutations in the Tpit gene are unlikely to play a major role in the pathogenesis of canine corticotroph adenomas.