Pituitary-dependent hypercortisolism (PDH) is one of the most frequent endocrinopathies in dogs, but prognostic factors are largely unknown. The aim of this retrospective case series study was to determine the prognostic value of different clinical and clinicopathological variables evaluated in dogs newly diagnosed with PDH that were subsequently treated with trilostane. Medical records from one referral centre were evaluated. Eighty-five dogs with PDH were included. The median survival time was 852 days (range 2–3210 days); 60/85 (70 per cent) and 25/85 (29 per cent) dogs survived more than one and three years, respectively. In multivariable model analysis the length of survival of older dogs (HR 1.24, 95% CI 1.09 to 1.40) and dogs with higher serum phosphate concentrations (HR 1.35, 95% CI 1.01 to 1.81) was shorter. Serum phosphate concentrations were above the reference range in 37/85 (44 per cent) of animals. Clinical signs, liver enzymes, serum cortisol concentrations of the endocrine tests, proteinuria, systolic hypertension, the presence of concomitant disorders, and the frequency of trilostane administration were not associated with survival time. Hyperphosphataemia is a common finding in dogs with newly diagnosed PDH and represents a negative prognostic factor.
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Spontaneously occurring hypercortisolism (HC), or Cushing's syndrome, is defined as the combination of physical and biochemical changes that result from chronic and pathologically high concentrations of circulating glucocorticoids. In most cases HC is due to inappropriate secretion of adrenocorticotropic hormone (ACTH) from the hypophysis (pituitary dependent-hypercortisolism (PDH)) or is caused by a primary adrenal disorder (adrenal-dependent hypercortisolism (ADH)). PDH accounts for 85 per cent of HC cases (Feldman 1983).
An excess of glucocorticoids causes various clinical signs, which are reviewed elsewhere (Feldman and Nelson 2004). The majority of clinical signs have a considerable impact on the animal's quality of life; this condition is therefore usually treated and there are no studies on the causes of death in a large series of untreated cases.
PDH is most commonly treated medically, although surgical options have been published (Hanson and others 2005). The destruction of the adrenal cortex with mitotane (o,p'DDD) has long been the medical treatment of choice for PDH in dogs. Trilostane (4,5-epoxy-17-hydroxy-3-oxoandrostan-2-carbonitrile), a competitive inhibitor of 3-β-hydroxysteroid dehydrogenase, has gained increasing acceptance in the treatment of dogs with PDH, and its efficacy has been reported in several studies (Neiger and others 2002, Ruckstuhl and others 2002).
Although the pathophysiological features, clinical aspects, diagnostic procedures and treatment options for dogs with PDH have been extensively reported, only a few studies (Neiger and others 2002, Barker and others 2005, Perez-Alenza and others 2006, Clemente and others 2007, Hanson and others 2007) have mentioned the life expectancy and/or prognostic factors of the disease in dogs treated with trilostane.
Precise data about the outcome and prognostic factors of PDH in dogs would help to characterise the disease better. Therefore, the aim of the present study was to determine the survival time and the prognostic value for survival of different variables retrieved from the history, signalment, physical examination and laboratory evaluations in a population of dogs newly diagnosed with PDH that were subsequently treated with trilostane.
Materials and methods
The medical records of all dogs with spontaneous HC admitted to the Department of Veterinary Medical Sciences, University of Bologna, Italy, between March 2003 and October 2013 were evaluated. Dogs were included in the study if they had newly diagnosed PDH, had not been treated for HC, were subsequently treated with trilostane, and had follow-up examinations until death or until the last re-evaluation for which records were evaluable. Dogs were excluded if, at diagnosis, the owner declined a complete diagnostic work-up or if the dogs had previously been treated by referring veterinarians. Dogs with ADH were also excluded.
Dogs were included in the study when clinical and laboratory findings were consistent with HC, the low dose dexamethasone suppression test (LDDST) and/or the ACTH stimulation test were positive for HC, a treatment with trilostane was subsequently carried out, and no other treatments for HC had previously been administered. A diagnosis of PDH or ADH was based on the ultrasonographic appearance of the adrenal glands, the results of the LDDST, and concentrations of endogenous ACTH (Behrend and others 2013).
Medical records review
Data obtained at the time of diagnosis from the medical records comprised breed, gender, bodyweight, age, history, physical examination findings, systolic blood pressure, routine haematology, biochemistry profile, urinalysis including urinary protein:creatinine ratio (UPC), ACTH stimulation test, LDDST, endogenous ACTH concentration, and abdominal ultrasonography. Any concurrent disease diagnosed at initial evaluation and the trilostane treatment dose and regimen (every 24 hours or every 12 hours) were recorded. Date of death or survival of all cases were recorded and entered into the database which was closed on October 15, 2013 before analysis. When necessary, owners and/or referral veterinarians were contacted.
An ACTH stimulation test and LDDST were performed by injecting intravenous tetracosactide esacetate (Synacthen, Novartis, Origgio, Italy) and dexamethasone (Dexadreson, Intervet, Peschiera Borromeo, Italy), respectively, as previously described (Feldman and Nelson 2004). Blood samples for the determination of endogenous ACTH concentrations were collected from the jugular vein or cephalic vein into EDTA-coated plastic tubes placed on ice. The samples were immediately centrifuged at 4°C, 500g for eight minutes, and plasma was immediately transferred to plastic tubes and stored at –80°C until analysis.
Serum cortisol and plasma ACTH concentrations were determined using kits (Immulite cortisol and Immulite ACTH, Diagnostic Product Corporation, Los Angeles, California, USA) that have previously been validated for use in dogs (Singh and others 1997, Scott-Moncrieff and others 2003). The reference intervals reported in the present study were obtained from a population of healthy adult dogs (>1 year) of different breeds as described in the Clinical and Laboratory Standards Institute (CLSI) guidelines (Horowitz 2010).
Systemic blood pressure measurement was determined by means of either an oscillometric device (BP-88 Next, Colin Corporation, Japan or petMAP graphic, Ramsey Medical, Inc, Tampa, Florida, USA) or by Doppler ultrasonic transducer (Minidop ES-100VX, Hadeco, Japan). The choice of method depended on the dog's bodyweight: Doppler was employed in dogs <20 kg bodyweight, and the oscillometric method was used in heavier dogs. Dogs were considered hypertensive whenever systolic blood pressure ≥160 mm Hg was found (Ortega and others 1996).
Treatment protocol and monitoring
Treatment with trilostane was started at the initial dose of about 1–6 mg/kg bodyweight once or twice daily. The decision on dose regimen was the responsibility of the clinician managing the case. As standard procedure at our clinic, dogs with HC were reassessed at 10 days, 4, 8, 13, 24 weeks, and then every 3–6 months. Each re-evaluation included an assessment of clinical signs (eg, decreased polyuria, polydipsia, polyphagia), biochemical profile and an ACTH stimulation test performed between two and three hours after trilostane administration (Burkhardt and others 2013). The dose of trilostane was changed based on the results of the ACTH stimulation test and the clinical signs. When the post-ACTH plasma cortisol concentration was <150 nmol/L, the dose was considered appropriate if the owner also reported resolution of the clinical signs of hypercortisolism, such as polyuria, polydipsia and polyphagia. When the post-ACTH plasma cortisol concentration was >150 nmol/L, and/or the clinical signs of hypercortisolism persisted, the dose was increased and a new re-evaluation was scheduled for three weeks later. If a dog's response to trilostane administered once daily was classified as poor and its post-ACTH stimulation serum cortisol concentration was <150 nmol/l, it was usually recommended to the owner that the frequency of trilostane administration be increased (twice daily). Excessive clinical control was defined as clinical signs of hypoadrenocorticism and a post-ACTH cortisol concentration <40 nmol/l. Treatment was temporarily discontinued if the patient showed clinical signs or serum electrolyte concentrations consistent with hypoadrenocorticism. When these signs had resolved, treatment was re-initiated at a lower dosage and/or the frequency of trilostane administration was decreased.
The median survival time was calculated using the Kaplan–Meier product limit method. The survival time was defined as the interval between the first presentation and the date on which the dog was last known to be alive, or the date of its death due to any cause. The following variables were investigated to determine their association with overall survival time: age, sex (male or female), reproductive status (intact or neutered), bodyweight, main clinical signs (polyuria, polydipsia, polyphagia, enlarged abdomen, weakness, dermatological abnormalities), systolic blood pressure, haematocrit, white blood cell count, neutrophil count, lymphocyte count, monocyte count, eosinophil count, platelet count, aspartate transaminase, alanine transaminase (ALT), alkaline phosphatase (ALP), γ glutamyl transferase (GGT), total bilirubin, total serum protein, albumin, cholesterol, urea, creatinine, glucose, total calcium, inorganic phosphorus, sodium, potassium, chloride, basal and post-ACTH cortisol, basal and four- and eight-hour cortisol of LDDST concentrations, urinary specific gravity, UPC, presence of concurrent diseases, frequency of administration of trilostane (every 24 hours v every 12 hours), and trilostane starting dose (<5 v ≥5 mg/kg/day). Weight and age were considered as continuous and binary variables based on median values (weight ≤10 v >10 kg; age ≤10 v >10 years). To assess the association between clinical signs at diagnosis and the survival time, our study population was also divided into two groups: dogs with <4 clinical signs of HC and dogs with ≥4 clinical signs of HC. For this classification the following clinical signs were used: polyuria and/or polydipsia (considered as a single clinical sign), polyphagia, enlarged abdomen, weakness, and dermatological signs (considered as a single clinical sign).
Univariate Cox proportional hazards regression analyses were used to screen potential predictors for subsequent inclusion in a multivariable model. Variables with a value <0.20 via univariate analysis were included in the final model-building process. Variables were then removed one at a time until the model with the best fit was identified. In the model-building process, the selection of variables that were strongly collinear (ie, creatinine and urea concentrations) were also considered. The HR and 95% CIs were calculated. A receiver-operating characteristic (ROC) curve was used to select the optimum cut-off value of variables associated with survival in the multivariate analysis to discriminate dogs with short-term survival from dogs with long-term survival. Differences between the Kaplan-Meier curves were tested with the log-rank test. The correlations between the various variables were performed using the Spearman's correlation. Data were analysed using a commercially available software program (MedCalc). The significance level was set at P<0.05.
Of the 153 cases of canine HC that were retrieved from the records, 85 dogs fulfilled all of the inclusion criteria and were used in the analysis. Twenty-six dogs were excluded because they were affected by ADH, 21 were excluded because the diagnosis had been made previously and they had been treated by private practitioners, 12 were excluded because the owners denied permission for a comprehensive diagnostic evaluation and/or refused treatment, and nine dogs were excluded because after a first period of treatment with trilostane they were subsequently treated with mitotane (five dogs) or hypophysectomy (four dogs).
Twenty-five different breeds were included. The most commonly represented breeds were mixed breed (31), dachshund (10), boxer (nine), and Yorkshire terrier (six). There were 27 entire females, 21 spayed females, 32 intact males, and five castrated males. The median age at diagnosis was 10 years (range 4–17 years). The median bodyweight was 10.0 kg (range 3.7–62.5 kg).
The most common clinical signs reported at diagnosis by the owners or observed at the physical examination were polyuria (73/85), polydipsia (72/85), polyphagia (72/85), enlarged abdomen (56/85), weakness (53/85), and dermatological abnormalities (50/85). The median systolic blood pressure was 180 mm Hg (range 152–205 mm Hg). In comparison with the laboratory reference range, the most frequent abnormalities on biochemistry profile and urinalysis were increased concentrations of serum ALT, ALP, GGT, decreased urinary specific gravity, and increased UPC (Table 1).
An ACTH stimulation test and LDDST were performed at diagnosis in 83 and 67 dogs, respectively. The post-ACTH cortisol concentration exceeded 600 nmol/L in 71/83 (86 per cent) and the eight-hour post-dexamethasone cortisol concentration exceeded 40 nmol/L in 59/67 (88 per cent). Eighty-one dogs were evaluated by abdominal ultrasonography in our referral centre. In four dogs the abdominal ultrasonography had already been performed by the referring veterinarian no more than one month before our first visit and was not repeated. All dogs showed either bilateral mild adrenomegaly (Barthez and others 1995) or normal adrenal glands.
Systemic blood pressure was ≥160 mmHg in 56/73 (77 per cent) of dogs. UPC was above the reference range in 47/77 (61 per cent) of animals. One or more concurrent disorders were documented in 20/85 (24 per cent) dogs, including nine with diabetes mellitus, six with hypothyroidism, seven with other neoplasia not related to hypercortisolism, two with mitral valve endocardiosis, and one with chronic bronchopathy. Trilostane was administered once or twice daily in 60 (71 per cent) and 25 (29 per cent) dogs, respectively. The median starting dose of trilostane was 3.75 mg/kg (range 1.11–5.94 mg/kg) and 1.21 mg/kg (range 0.94–3.80 mg/kg) for dogs treated once or twice daily, respectively.
At the time of censorship, 14 were alive, 55 dead and 16 had been lost to follow-up. Of the 55 dogs that had died, 28 were euthanased and 27 had died spontaneously. Of the 55 dogs that were dead at the time of censorship, the cause of death or reason for euthanasia was recorded when possible (Table 2). The cause of death was supported by necropsy only in 11 dogs.
The median survival time of the 85 dogs was 852 days (range 2–3210 days). Seventy-one of the 85 dogs (84 per cent) lived more than 6 months, 60/85 (71 per cent) more than one year, 46/85 (54 per cent) more than two years, and 25/85 (29 per cent) more than three years (Fig 1). Several variables showed a value of P<0.20 on univariate Cox regression analysis, including age, bodyweight, platelet count, serum albumin, creatinine, urea, ALP, phosphate and potassium concentrations (Table 3). On multivariate analysis only two variables retained a value of P<0.05; age (P=0.0009, HR 1.24, 95% CI 1.09 to 1.40) and increased serum phosphate concentrations (P=0.0424, HR 1.35, 95% CI 1.01 to 1.81) at diagnosis were significantly associated with a shorter survival time. At diagnosis serum phosphate concentrations were above the reference range in 37/85 (44 per cent) of cases. The ROC curve analysis showed that 1.45 mmol/l was the optimal cut-off of serum phosphate concentration to discriminate dogs with short-term survival from dogs with long-term survival. The median survival time calculated via Kaplan-Meier analysis was 983 days (range 2–3210 days) in dogs with serum phosphate concentrations ≤1.45 mmol/l and 656 days (range 34–2670 days) in dogs with serum phosphate concentrations >1.45 mmol/l (Fig 2); however, the difference was not significant (P=0.2530, log rank test). There was a significant positive correlation between the phosphate and ALT (r=0.34, P=0.002), post-ACTH cortisol (r=0.30, P=0.009), GGT (r=0.33, P=0.003), four-hour cortisol of LDDST (r=0.40, P=0.001), and eight-hour cortisol of LDDST (r=0.35, P=0.005) concentrations. There was also a significant negative correlation between phosphate concentrations and lymphocyte (r=–0.52, P<0.0001) and eosinophil (r=–0.29, P=0.01) counts and between phosphate and total calcium (r=–0.35, P=0.002) concentrations. Age was positively correlated with creatinine (r=0.39, P=0.0002) and urea (r=0.41, P=0.0001) concentrations.
The dogs in the present study had a median survival time of 852 days. This result is consistent with other studies in which dogs with PDH treated with trilostane survived 662 days (Barker and others 2005), 930 days (Perez-Alenza and others 2006) and 900 days (Clemente and others 2007). The survival time in dogs with ADH treated with trilostane is shorter and one study reported a median survival time of 353 days (Helm and others 2011).
The age and serum phosphate concentrations were significantly associated with survival; therefore, at the time of diagnosis, older dogs and dogs with a higher serum phosphate concentration had a shorter life expectancy. With regard to age, several previous studies have found the same statistical association (Barker and others 2005, Clemente and others 2007), probably because older dogs tend to have a naturally lower life expectancy than younger dogs and are more susceptible to different diseases. In the present study many dogs died of causes unrelated to the HC.
The results of this study support other studies showing that serum phosphate concentrations are increased in dogs with hypercortisolism (Ramsey and others 2005, Tebb and others 2005, Corbee and others 2012). Furthermore, it was previously observed that parathyroid hormone (PTH) is also increased in dogs with hypercortisolism (Ramsey and others 2005, Tebb and others 2005) but that there is no direct correlation between phosphate concentrations and PTH concentrations (Ramsey and others 2005). Treatment with trilostane results in a significant decline in serum phosphate and PTH concentrations (Tebb and others 2005). However, a recent study showed that PTH concentrations did not differ significantly between 12 dogs with PDH (before and after hypophysectomy) and control dogs (Corbee and others 2012). One possible explanation is that, in common with humans, the great variation in PTH concentrations in dogs with hyperadrenocorticism (HAC) may be due to the clinical stage of the disease (Kugai and others 1986). The pathogenesis of hyperphosphataemia in dogs with HC, a condition that is not observed in humans with Cushing's syndrome (Findling and others 1982, Faggiano and others 2003), remains unknown. In the present study we observed that, at diagnosis, higher serum phosphate concentrations in dogs with PDH are associated with shorter survival times. Therefore, understanding the reason for such phosphate abnormalities might have positive consequences for the management of the disease. Possible explanations for hyperphosphataemia are reduced renal excretion of phosphate, increased intestinal absorption of phosphate or mobilisation of phosphate from tissues (ie, bones). Increased concentrations of vitamin D in dogs with HC would explain the hyperphosphataemia; however, in a recent study 25-hydroxy-vitamin D, 24, 25-dihydroxy-vitamin D and 1, 25-dihydroxy-vitamin D concentrations were normal in dogs with PDH (Corbee and others 2012).
There was no association between frequency of trilostane administration and survival time. There are no papers directly comparing the survival time based on the frequency of trilostane administration; however, recent studies showed that there are only small practical differences between once or twice daily trilostane administration in the treatment of canine HC (Augusto and others 2012, Arenas and others 2013, Cho and others 2013). The data in our study support the proposal that the frequency of trilostane administration (once v twice daily) seems to have a minimal impact on the management of canine HC.
The present study has a number of limitations largely related to the fact that it was a retrospective study and incomplete records were occasionally present. One limitation is that the laboratory reference intervals were not obtained from an age-matched control population but were the ones provided by the laboratory for routine use. This could have influenced the percentage of dogs with abnormal laboratory findings observed in this study. The main limitation of the study is that the size of the pituitary gland was only assessed by CT or MRI in a few dogs and for this reason this variable could not be included in the survival analysis. Pituitary adenomas in dogs can be classified, based on their size, as either non-enlarged pituitaries containing micro-adenomas or enlarged pituitaries, which are also called macro-adenomas (Kiupel and others 2008). Although trilostane is effective at relieving clinical signs associated with HC, it does not reduce the volume of the pituitary tumour. A study performed in healthy dogs has shown that trilostane may even produce an increase in the volume of the pituitary gland (Teshima and others 2009). Dogs with a pituitary macro-adenoma therefore have a significantly shorter survival time compared to dogs with micro-adenoma if not treated with radiotherapy (Kent and others 2007) or other treatment options aimed at decreasing the pituitary mass effect, such as pituitary surgery (Hanson and others 2007). It is therefore possible that in our study dogs with macro-adenoma survived for a shorter time than dogs with micro-adenoma. A link between pituitary size and the serum phosphate concentration has never been investigated and cannot be excluded. One recent study performed in dogs with PDH showed that the plasma phosphate concentration was 1.6±0.3 mmol/l before hypophysectomy and 1.4±0.3 mmol/l after hypophysectomy (Corbee and others 2012); the difference was not significant but the study examined only 12 dogs and therefore the difference might have been significant if more dogs had been included. The possible connection between the dimensions of pituitary masses and the serum phosphate concentration is pure speculation; however, in light of the prognostic importance of phosphataemia observed in this study a further study aimed at investigating this potential correlation is recommended.
In our study population UPC was increased in 61 per cent of dogs, in accordance with other recent reports (71 per cent: Mazzi and others 2008; 68 per cent: Smets and others 2012). Proteinuria may promote progressive renal injury by several mechanisms that are described elsewhere (Bartges 2012). Based on our results proteinuria did not seem to influence the survival time in the study population. In proteinuric PDH dogs in one recent report, normalisation or a significant reduction in UPC was observed in most cases subsequently treated with trilostane or hypophysectomy (Smets and others 2012); this phenomenon might prevent the progression of renal injury induced by proteinuria. Furthermore, deaths related to renal diseases were uncommon in this study as in previous similar studies (Barker and others 2005, Perez-Alenza and others 2006, Clemente and others 2007).
In conclusion, most of the dogs with newly diagnosed PDH subsequently treated with trilostane survived for more than two years. The survival time was shorter in older dogs and in dogs with elevated serum phosphate concentrations. At diagnosis, proteinuria, hypertension and the presence of concomitant diseases were not associated with decreased survival time.
Provenance Not commissioned; externally peer reviewed
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