Utility of a corticotropin‐releasing hormone test to differentiate pituitary‐dependent hyperadrenocorticism from cortisol‐producing adrenal tumors in dogs

Abstract Background Hyperadrenocorticism (HAC) is a common endocrine disorder in dogs; however, there are no reports on the use of the corticotropin‐releasing hormone test (CRHT) to differentiate between pituitary‐dependent hyperadrenocorticism (PDH) and cortisol‐producing adrenal tumors (CPATs), both causative of HAC. Objectives To evaluate the usefulness of CRHT as a tool to differentiate between PDH and CPAT in dogs and to determine the reference intervals for CRHT in healthy, PDH, and CPAT dogs. Animals Dogs diagnosed with PDH (n = 21), CPAT (n = 6), and healthy beagle dogs (n = 33). Methods This prospective study included dogs with a definitive diagnosis of PDH and CPAT and healthy beagle dogs, in which CRHT was performed, were prospectively evaluated. We investigated the correlations of CRHT (endogenous adrenocorticotropic hormone [ACTH] concentration, endogenous ACTH concentration [EAC], and poststimulation ACTH concentration [PAC]) with pituitary‐to‐brain ratio (PBR) (in PDH) and with indices of adrenal ultrasonography (smaller and larger adrenal gland dorsoventral thickness in PDH and CPAT). Results For EAC, the area under the curve (AUC) was 0.95, with a cutoff value of 26.3 pg/mL (sensitivity: 90.62%, specificity: 87.50%). The AUC for PAC was 0.96 with a cutoff value of 54.5 pg/mL (sensitivity: 100.00%, specificity: 66.67%). The 95% reference interval for CRHT in healthy (control) dogs ranged 5.00 to 79.8 pg/mL (1.10‐17.57 pmol/L) for EAC, and 1.92 to 153.42 pg/mL (0.42‐33.78 pmol/L) for PAC. There was no significant correlation between PBR and CRHT, nor adrenal size and CRHT. Conclusions and Clinical Importance CRHT appears to be a rapid and reliable test for differentiating PDH from CPAT in dogs.

Conclusions and Clinical Importance: CRHT appears to be a rapid and reliable test for differentiating PDH from CPAT in dogs.

K E Y W O R D S
ACTH, canine, cortisol, Cushing syndrome, diagnosis

| INTRODUCTION
In small animal medicine, hyperadrenocorticism (HAC) is a common endocrine disorder, with incidence in dogs of approximately 1 to 2 cases/1000 dogs/year. 1 Approximately 80% to 85% of HAC in dogs is caused by pituitary-dependent hyperadrenocorticism (PDH) resulting from pituitary adrenocorticotropic hormone (ACTH)producing adenomas, and the remaining 15% to 20% is caused by unilateral or bilateral cortisol-producing adrenal tumors (CPATs). 2 Ectopic ACTH-producing adenomas account for 5% to 15% of HACs occurring in tissues other than the pituitary gland in human medicine, 3-5 but to our knowledge, in small animals, there is only one report of a definitive diagnosis in a dog. 3 Considering such a background of HAC in small animal medicine, it is essential to accurately differentiate between PDH and CPAT to ensure that the appropriate treatment is implemented. The goals of treating hypercortisolism in dogs would optimally be to eliminate the source of either ACTH or autonomous cortisol excess, achieve normocortisolism, eliminate the clinical signs, reduce long-term complications and death, and improve the quality of life. 4 Achieving these treatment goals require definitive treatments, such as transsphenoidal hypophysectomy or radiotherapy for PDH, and adrenalectomy in the case of CPAT. 4 PDH and CPAT are generally differentiated using a combination of imaging modalities, such as abdominal ultrasonography and MRI, and hormonal testing. 5,6 In veterinary medicine, the first choice of hormone test to differentiate PDH from CPAT is the highdose dexamethasone suppression test (HDDST). 5,6-8 However, HDDST requires blood sample, either 4 or 8 hours after loading, which is time-consuming. Furthermore, in HDDST, response resistance has been reported in 25% of PDH dogs, limiting the reliability of the results. 5 In contrast, in human medicine, several basic and clinical studies have been conducted using the human corticotropin-releasing hormone test (CRHT) and the data obtained indicate that CRHT is useful as a diagnostic tool, especially in differentiating between PDH and CPAT, which are disorders of the hypothalamic-pituitary-adrenal axis. [9][10][11][12][13][14][15] Corticotropin-releasing hormone (CRH) is a hypothalamic hormone that stimulates the synthesis and secretion of ACTH in the cranial pituitary gland, and was first isolated and identified from ovines. 16 The structure of human CRH was subsequently clarified. 17 In small animal medicine, there have been several reports of CRHT using ovine CRH in clinical cases and experimental dogs, but to the best of our knowledge, no reports focused on the differentiation between PDH and CPAT or establishing reference intervals for CRHT in dogs. 3,[18][19][20] Therefore, we conducted this study to evaluate the potential usefulness of CRHT as a tool to differentiate between PDH and CPAT in dogs, and examine preliminary reference intervals of CRHT in healthy, PDH, and CPAT dog cases.

| Dogs
Dogs diagnosed with PDH (n = 21) or CPAT (n = 6) and not treated medically or surgically for HAC were included in this study at the

| Endocrine tests and sample collection
All PDH and CPAT dogs were food with held overnight before their visit to Veterinary Medical Teaching Hospital, Nippon Veterinary and Life Science University. After arriving at the hospital, they were placed on cage rest for 1 hour, and CRHT was performed at 10:00 am. For the control group, CRHT was performed at 10:00 am the day after overnight, once the animals had fully adapted to the environment in which they were managed at our university. All PDH and CPAT dogs underwent CRHT and ACTHST on different days under similar conditions. Thus, ACTHST was performed first, followed by CRHT. The median interval between ACTHST and CRHT was 20 days (range, 3-43 days) in the PDH group and 19.5 days (range, 6-42 days) in the CPAT group. None of the dogs in the PDH or CPAT groups were treated before ACTHST and CRHT were performed.
Blood samples for hormone measurements were collected, at rest, from the jugular vein. The ACTHST was performed by collecting a blood sample to measure the endogenous cortisol concentration (ECC) immediately before IV administration of synthetic ACTH The CRHT was performed by collecting a blood sample to measure the endogenous ACTH concentration (EAC) immediately before IV administration of 1.5 μg ovine corticotropin-releasing factor (ovine CRF) (Peptide Institute, Inc, Osaka, Japan), as well as the poststimulation ACTH concentration (PAC) at 30 minutes after administration. 18,22,23 Blood for the plasma ACTH assay was dispensed into ice-cold tubes containing edetate disodium (EDTA-2Na), and blood for the serum cortisol assay was dispensed into plain ice-cold tubes.
Plasma and serum were centrifuged at 1000g for 15 minutes at 4 C and stored at À80 C until measurements were performed. The sample storage time at À80 C was limited to 1 week in this study, with just one freeze-thaw cycle before measuring the analytes. Serum cortisol (intra-assay coefficients, 1.9%; interassay coefficients, 4.0%) and plasma ACTH (intra-assay coefficients, 1.9%; interassay coefficients, 4.3%) concentrations were analyzed in each sample individually at Veterinary Medical Teaching Hospital, Nippon Veterinary and Life Science University using a chemiluminescent enzyme immunoassay (IMMULITE 1000, Siemens Healthineers, Erlangen, Germany). 24,25 The reference intervals for these assays were ECC: 1.0 to 7.8 μg/dL cage rest for 2 hours after administration of ovine CRF. 19 In the PDH and CPAT groups, the time from the onset of clinical signs to the date of CRHT and ACTHST was investigated and compared between groups.

| Abdominal ultrasonography
All PDH and CPAT dogs underwent abdominal ultrasonography at first visit using an ultrasound system (LOGIQ S7 Expert, GE Healthcare, Tokyo, Japan; or LOGIQ 7, GE Healthcare) to measure the size of the left and right adrenal glands. Specifically, the dogs were gently restrained in the supine position without sedation. Ultrasonography was performed using a linear probe (11L-D probe, GE Healthcare). Ultrasound images were obtained using the same settings for all dogs, except for the overall gain, which was adjusted for each dog. Color Doppler ultrasonography was performed to identify the vessels adjacent to the adrenal glands as anatomical references. In longitudinal images of each dog's left and right adrenal glands, the maximum adrenal thickness perpendicular to the long axis of the T A B L E 1 Characteristics of dogs in this study including the control (33 dogs), PDH (21 dogs), and CPAT (6 dogs adrenal gland was measured using electronic calipers. 26

| Magnetic resonance imaging
Magnetic resonance imaging was performed only in the PDH group.
The cranial MRI scans were performed using a 1.5-T superconducting

| Statistical analysis
All data were subjected to the Shapiro-Wilk test to assess normality and are expressed as median (range 3 | RESULTS

| Adrenocorticotropic hormone stimulation test
The results of the ACTHST in the PDH and CPAT groups are shown in Tables 2 to 4. The ROC curves for EAC and PAC in the control, PDH, and CPAT groups are shown in Figure 1. Note: There was no significant difference in either the EAC (P = .14) or PAC (P = 1.000) between the control and CPAT groups. The reference intervals for these assays were cortisol concentration, 1.0 to 7.  The 95% reference interval for EAC and PAC in the three groups is shown in Table 5.

| Corticotropin-releasing hormone test
The median time from onset of clinical signs to CRHT was 8 months No dogs had adverse events before CRHT or at 2 hours afterovine CRF administration, and no owners reported any adverse effects after their dogs returned home.

| Abdominal ultrasonography
In the PDH group, the median LAGDT was 9.3 mm (range,

| DISCUSSION
In this study, we investigated the effect of CRH administration on plasma ACTH concentration by measuring EAC and PAC in the control, PDH, and CPAT groups. The results of this study suggest that CRHT, which is useful in human medicine, is also a valid method for distinguishing PDH from CPAT in dogs and might help guide appropriate treatment strategies for both.
Regarding the CRHT (EAC and PAC) results in this study, there was a significant difference between the control and PDH groups, and between the PDH and CPAT groups; however, no significant difference was observed between the control and CPAT groups. Therefore, CRHT might be a useful hormone test to differentiate between PDH and CPAT in cases of confirmed HAC in dogs. Indeed, our study indicated that the CRHT has a high discriminatory ability to differentiate PDH from CPAT. Besides, CRHT might be useful in differentiating pituitary ACTH-producing microadenomas from ectopic ACTHproducing tumors in humans 35,36 and dogs. 3 Specifically, in human medicine, PAC/EAC increases by more than 1.5 times in many patients with PDH, 37 while there is little change in patients with ectopic ACTH-producing tumors, 35,36 which is a similar to in dogs with PDH 20 and ectopic ACTH-producing tumor. 3 Therefore, in cases of suspected PDH without obvious enlargement of the pituitary gland, it might be useful to perform CRHT to rule out the possibility of an ectopic ACTH-producing tumor. However, the CRHT is not intended to replace conventional screening tests for HAC in dogs. Therefore, in patients with suspected HAC, it is recommended that an endocrinological screening test such as ACTHST or low-dose dexamethasone suppression test be performed first, followed by CRHT as a means of performing a differential diagnosis between PDH and CPAT.
The results of the 95% reference interval for CRHT in this study might be used as an example of a reference interval for CRHT in dogs.
In this study, endocrine tests were performed at 10:00 am. Therefore, due to a few reports that support diurnal ACTH and cortisol concentrations, 38,39 it is recommended that CRHT be performed at a similar time of day if using these as reference values. The CRHT is advantageous as it takes a significantly shorter time to perform compared to the HDDST, 3,18,19 and it is more convenient and practical in the clinical setting.
Compared to our study in which the EAC threshold was 5.0 pg/ mL (1.10 pmol/L) which did not discriminate well between PDH and CPAT, previous studies indicated good discriminatory ability at this threshold. 40 One of the possible reasons for the potential differences between our study and others might be that the PDH and CPAT dogs were under various stresses due to coming to the veterinary hospital which might have resulted in higher EAC levels. 41,42 In fact, in the report showing the above thresholds, the median EAC was 30 pg/mL (range, 6-1250 pg/mL) in the PDH group and 5 pg/mL (range, below the limit of measurement to 5 pg/mL) in the CPAT group, which is T A B L E 5 Preliminary reference intervals for endogenous and poststimulation adrenocorticotropic hormone concentration with 95% reference interval and 90% confidence interval generated by receiver operating characteristic curve in control (33 dogs There are several limitations to this study. First, there was a significant difference in age between the control and case groups, and some previous veterinary reports have shown increased cortisol and ACTH concentrations in older dogs compared to younger dogs, 41,48 whereas another study reported that older dogs had significantly lower cortisol and ACTH concentrations compared to younger dogs. 49 Therefore, a possible age-related effect due to the significant differences in age between our groups in this study cannot be excluded.
Second, we cannot rule out the possibility that the smaller sample size in the CPAT group than that in the PDH group might have lowered the statistical power of the study. Third, pituitary imaging was not performed in CPAT cases and thus, the possibility of pituitary tumors could not be ruled out in dogs classified as CPAT.
In conclusion, CRHT might be a rapid and reliable test for differentiating PDH and CPAT, which account for the majority of HAC in dogs.
EAC alone was able to differentiate the patients with high sensitivity and specificity, but considering that the patients were under various stresses when they came to the hospital, evaluating PAC might be beneficial for understanding the condition of the patients. Additionally, for future research, the 95% CI for CRHT results from this study might be used as an example of a preliminary reference interval for CRHT in dogs.

ACKNOWLEDGMENT
This research was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (project number: 17K08114). The institution was not involved in any stage of this study. We thank Akiko Yasuda, DVM and Kae Shigihara, DVM from Veterinary Medical Teaching Hospital, Nippon Veterinary and Life Science University for their help in collecting the cortisol-producing adrenal tumors cases for this study. We also thank EDITAGE (www. editage.jp) for editing a draft of this manuscript.

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.