Abstract
There are differences in the responsiveness to differential diagnostic tests for Cushing’s disease (CD), corticotroph tumor size, and the somatostatin receptor (SSTR) 5 expression in corticotroph tumors between CD patients. The differences in SSTR5 expression are particularly significant for identifying therapeutic targets for CD. However, prospective predictors of SSTR5 expression remain unclear. Thus, our objective was to elucidate the relationships among these clinical characteristics of CD, including SSTR5 expression. In 27 hospitalized patients with CD at Osaka University Hospital, Osaka, Japan, associations between corticotroph tumor diameter, the response of ACTH and cortisol to differential diagnostic tests for CD (CRH, desmopressin [DDAVP], and high-dose dexamethasone suppression test [HDDST]), the ACTH/cortisol index, and the SSTR5 immunoreactive score were retrospectively investigated. The response to differential diagnostic tests, ACTH/cortisol index, tumor diameter, and SSTR5 expression were significantly related (vs. tumor diameter [CRH: r = –0.54; DDAVP: r = –0.54; HDDST r = –0.67; ACTH/cortisol index: r = 0.76; SSTR5: r = –0.61], vs. CRH [DDAVP: r = 0.63, HDDST: r = 0.72, ACTH/cortisol index: r = –0.45; SSTR5: r = 0.56], vs. DDAVP [HDDST: r = 0.66; ACTH/cortisol index: r = –0.46; SSTR5: r = 0.76], vs. HDDST [ACTH/cortisol index: r = –0.62; SSTR5: r = 0.77], ACTH/cortisol index vs. SSTR5: r = –0.67). The areas under the receiver operating characteristic curve for the prediction of high SSTR5 expression via the CRH test, DDAVP test, HDDST, ACTH/cortisol index, and tumor diameter were 0.79, 0.87, 0.80, 0.71, and 0.71, respectively. Tests for differential diagnosis of CD, the ACTH/cortisol index, and the corticotroph tumor diameter have the potential for identifying SSTR5 expression in corticotroph tumors. These parameters may reflect the biological characteristics of corticotroph tumors.
Introduction
Cushing’s disease (CD) is a rare disorder classified as chronic hypercortisolism due to corticotroph tumors [1]. CRH, desmopressin (DDAVP) tests, and high-dose dexamethasone suppression tests (HDDSTs) are available for the differential diagnosis of CD and ectopic ACTH syndrome (EAS). Patients with CD exhibit a greater response of ACTH to CRH and DDAVP, as well as greater inhibition of cortisol by the HDDST, than patients with EAS. However, certain patients diagnosed with CD exhibit a lower response to these stimuli. Therefore, the differential diagnosis of CD is sometimes difficult to evaluate.
The pathophysiology of the difference in responsiveness to differential diagnostic tests between patients with CD remains to be fully elucidated, although some reports on responsiveness have been published previously. For example, it has been reported that corticotroph microtumors have a greater cortisol response to CRH, greater cortisol suppression during the HDDST, and a higher cortisol-to-ACTH ratio [2]. However, no work to date has presented a linear relationship between the response to stimuli and tumor size. In addition, the tumor size, increased response to DDAVP, increased somatostatin receptor (SSTR) 5 expression, and ACTH inhibition induced by pasireotide may be associated with each other, as mutations in ubiquitin-specific peptidase 8 (USP8) have been shown to be related to these factors [3-5]. However, the relationships between responsiveness to differential diagnostic tests and SSTR5 expression have not been elucidated.
Previously, it was reported that a greater GH response to oral glucose and thyrotropin-releasing hormone (TRH) was associated with a smaller pituitary tumor size and SSTR2 ligand effectiveness in patients with acromegaly [6-8]. Since both somatotroph and corticotroph tumors originate from Rathke’s pouch stem cells, they may share common characteristics, such as a smaller tumor size, greater responsiveness to differential diagnostic tests, and elevated expression of different receptors.
Thus, our objective was to comprehensively elucidate the relationships between clinical characteristics, including diagnostic tests for CD and SSTR5 expression, in corticotroph tumors. Additionally, we evaluated whether the clinical characteristics of CD patients predict SSTR5 expression.
Subjects and Methods
Patients
We reviewed the medical records of all patients clinically diagnosed with CD who had undergone transsphenoidal surgery between September 2003 and November 2020 at Osaka University Hospital, Osaka, Japan. CD has been determined to be in compliance with diagnostic criteria in Japan [9]. The diagnosis was subsequently validated postoperatively via pathological examination by confirming pituitary tumor immunostaining for ACTH. Two patients were excluded because of the unavailability of tumor specimens in hospital repositories. In the other two patients, the size of the tumor sample appeared to be insufficient, and SSTR5 immunohistochemical assessment was not feasible. Overall, we investigated 27 subjects.
The study was approved as a retrospective observational study by the Human Ethics Committee of Osaka University (16136) and was conducted in accordance with the Helsinki Declaration.
Endocrinology evaluation
The CRH and DDAVP tests employed the use of 100 μg CRH and 4 μg DDAVP intravenously, respectively. In these tests, plasma ACTH levels were measured before each test and 15, 30, 60, 90, and 120 minutes after stimulant injection. The degree of ACTH responsiveness was calculated by setting an increase rate (expressed in percentages [%]) between the pretest value and the maximum response value. For the HDDST, the response degree of cortisol under 8 mg dexamethasone was defined as the decrease rate (expressed in percentages [%]) between the pretest value and the maximum suppression value.
Furthermore, we evaluated the preoperative ACTH/cortisol index in our cohort as ACTH negative feedback control by cortisol [10]. The ACTH/cortisol index was determined as the ratio of the basal plasma ACTH value to the basal serum cortisol value measured at admission. Due to diurnal variation in ACTH and cortisol levels, two separate measurements of the ACTH/cortisol index were evaluated.
Plasma ACTH levels were measured via an immunoradiometric assay kit until November 2009 and then via an electrochemiluminescence immunoassay (ECLusys ACTH kit, Roche Diagnostics, Tokyo, Japan) until the end of the study. The ACTH levels measured by the first kit were converted to values consistent with those of the second kit via the following formula: y = 0.76x + 1.54. Cortisol levels were measured by an immunoradiometric assay kit until April 2007, by a chemiluminescent enzyme immunoassay (Access Cortisol Kit, Beckman Coulter, Tokyo, Japan) until April 2017, by a chemiluminescent enzyme immunoassay (Accuraseed Cortisol Kit, FUJIFILM Wako Pure Chemical, Osaka, Japan) until August 2019, and by a chemiluminescent enzyme immunoassay (Accuraseed Cortisol Kit (B), FUJIFILM Wako Pure Chemical, Osaka, Japan) until the end of the study. The cortisol levels measured by the first kit were converted to values consistent with those of the second kit via the following formula: y = 0.89x – 0.31. Additionally, the values of the second kit were converted to values consistent with those of the third kit via the following formula: y = 1.08x + 1.34. The values of the third kit were converted to values consistent with those of the fourth kit via the following formula: y = 0.66x + 0.05.
Assessment of pituitary tumors via magnetic resonance imaging (MRI)
Two authors, K.B. and K.M., individually measured the maximum pituitary tumor diameter on MRI for all patients prior to pituitary surgery. The measurements conducted by both methods yielded the same result. A macrotumor was defined as a tumor with a maximum diameter ≥10 mm. Furthermore, we estimated the tumor volume in each case, expressed in milliliters (FUJIFILM 3D workstation SYNAPSE VINCENT, Tokyo, Japan).
Pathology and Immunohistochemical Evaluation
Slides were stained via the DAKO Autostainer Link 48 system (EnVision FLEX Mini Kit, High pH; Dako A/S, Glostrup, Denmark). The sections were incubated with the clone UMB-4 antibody, which was diluted 1:800. The immunohistochemical reactions were carried out using diaminobenzidine (Dako A/S). The sections were incubated with an anti-SSTR5A (Abcam ab109495 UMB4) antibody. SSTR5 protein expression levels were scored via the immunoreactive score (IRS). IRS (scored 0–12), as in previously published reports [4, 11], was the final result of the combination of the staining intensity (0, no staining; 1, weak; 2, moderate; and 3, strong) multiplied by the proportion of immunoreactive cells (0, 0%; 1, <10%; 2, 10–50%; 3, 51–80%; or 4, >80%).
Statistical Methods
Data presented as patients’ clinical characteristics are shown as the median and interquartile range (IQR; first and third quartiles). Similarly, the minimum to maximum range was presented to outline each parameter’s distribution. Since the dataset was initially nonnormally distributed, it was transformed logarithmically to normally distributed models to test associations. Parametric and nonparametric regression analyses have been utilized to evaluate the correlation between clinical parameters such as differential diagnosis tests, pituitary tumor diameter, ACTH/cortisol index, and SSTR5 expression (represented by the IRS). Furthermore, the proportion of graph marker sizes was adjusted according to the maximum pituitary tumor diameter on MRI presented in the cohort. Finally, receiver operating characteristic (ROC) curves were generated to evaluate the discriminatory value of clinical markers for high SSTR5 expression (≥8 [the median IRS in our cohort]), as SSTR5 has the potential for use in receptor-targeted treatment. The optimal cutoffs were obtained via the Youden index. However, it is clinically important for personalized medicine practitioners to avoid missing patients with high SSTR5-expressing pituitary tumors. Higher sensitivity of SSTR5 expression is required to ensure that the expression is not missing. Therefore, we analyzed the cutoffs with a sensitivity of 100%. P < 0.05 was considered the minimum limit of statistical significance. JMP Pro Software, version 16.2.0, for Windows (SAS Institute, Inc. Cary, NC, USA), was used to conduct all the statistical analyses.
Results
Patient characteristics
The main features of the recruited cohort are summarized in Table 1. A total of 77.8% of the patients were female. Patients presented with a median age of 47 years (IQR, 37 to 56) and a body mass index of 23.2 kg/m2 (IQR, 21 to 27.8). The baseline biochemical parameters at the time of admission were ACTH 16 pmol/L (12.6–21.5) and cortisol 437 nmol/L (352–717). Regarding pituitary radiological findings, microtumor cases constituted the majority of patients, as opposed to macrotumors (81.5% [22 cases] vs. 18.5% [5 cases]). The median maximum tumor diameter was 4.9 mm (3.3–8.7). The first and third quartiles of the SSTR5 IRS were 4 and 10, with a median score of 8 in the studied group.
Table 1 Clinical and histopathological characteristics of the recruited patients
| No. of patients n |
27 |
| Sex: |
| Female n (%) |
21 (77.8%) |
| Male n (%) |
6 (22.2%) |
| Age at diagnosis (years) |
| Median |
47 |
| Range |
16–84 |
| IQR |
37–56 |
| BMI (kg/m2) |
| Median |
23.2 |
| Range |
17.9–40 |
| IQR |
21–27.8 |
| Surgical remission n (%) |
20 (74.1%) |
| Laboratory findings |
| Baseline ACTH (pmol/L) |
| Median |
16 |
| Range |
4.4–40.7 |
| IQR |
12.6–21.5 |
| Baseline cortisol (nmol/L) |
| Median |
437 |
| Range |
199–1,082 |
| IQR |
352–717 |
| Na serum level (mmol/L) |
| Median |
142 |
| Range |
136–147 |
| IQR |
139–143 |
| K serum level (mmol/L) |
| Median |
3.9 |
| Range |
2.5–4.7 |
| IQR |
3.4–4.1 |
| Glycated hemoglobin (%) |
| Median |
6.4 |
| Range |
5–13.1 |
| IQR |
5.6–7.5 |
| Radiological findings maximum tumor diameter (mm) |
| Median |
4.9 |
| Range |
2.2–28.6 |
| IQR |
3.3–8.7 |
| microtumor n (%) |
22 (81.5%) |
| macrotumor n (%) |
5 (18.5%) |
| IRS classification |
| Median |
8 |
| Range |
0–12 |
| IQR |
4–10 |
Data are expressed as n (%) or median (IQR).
Abbreviations: IQR, interquartile range; BMI, body mass index; IRS, immunoreactive score
Macrotumor diameter was defined as the maximum tumor diameter ≥10 mm.
Regarding tumor size (Fig. 1), the corticotroph tumor diameter was negatively correlated with the increase in ACTH induced by both CRH and DDAVP: [(tumor diameter vs. CRH: p = 0.0039, r = –0.54) (Fig. 1a); (tumor diameter vs. DDAVP: p = 0.0071, r = –0.54) (Fig. 1b)]. Similarly, there was a significant correlation between tumor diameter and the decrease in cortisol levels during the HDDST (p = 0.0004, r = –0.67) (Fig. 1c). The ACTH/cortisol index and tumor diameter were positively related (p = 0.0001, r = 0.76) (Fig. 1d). Tumor diameter was correlated with basal ACTH levels but not with basal cortisol levels (Supplementary Table 1). Similarly, the relationship between the ACTH/cortisol index and tumor diameter was validated upon analysis via separate measurements of ACTH and cortisol (data not shown).
Additionally, the correlation patterns of tumor volume were similar to those of tumor diameter (Supplementary Fig. 1).
Correlations between hormonal parameters in CD patients
Fig. 2 shows the relationships between the response rates of differential diagnostic tests. The CRH-induced increase in ACTH was positively correlated with the DDAVP-induced increase in ACTH (p = 0.001, r = 0.63) (Fig. 2a). Similarly, the decrease in cortisol levels in the HDDST was significantly correlated with the increase in CRH and DDAVP-induced ACTH levels [(HDDST vs. CRH: p = 0.0001, r = 0.72) (Fig. 2b); (HDDST vs. DDAVP: p = 0.0010, r = 0.66) (Fig. 2c)]. Basal ACTH levels were related to the responsiveness of differential diagnostic tests but not that of basal cortisol (Supplementary Table 1).
Fig. 3 shows correlations between the ACTH/cortisol index and differential diagnostic tests. A negative correlation between the ACTH/cortisol index and ACTH increase was observed in both the DDAVP and CRH tests [(ACTH/cortisol index vs. DDAVP: p = 0.0227, r = –0.46) (Fig. 3a); (ACTH/cortisol index vs. CRH: p = 0.0189, r = –0.45) (Fig. 3b)]. The decrease in cortisol during the HDDST was in accordance with previous results (ACTH/cortisol index vs. HDDST: p = 0.0016, r = –0.62) (Fig. 3c). The ACTH/cortisol index was correlated with the cortisol levels in the serum but not in the urine (ACTH/cortisol index vs. basal serum cortisol: p = 0.0396, r = –0.40) (Fig. 3d); (ACTH/cortisol index vs. urine free cortisol: p = 0.1281, r = –0.31) (Fig. 3e). Additionally, these correlations were validated upon analysis via separate measurements of ACTH and cortisol (data not shown).
Relationships between pituitary tumor SSTR5 expression and clinical characteristics
We analyzed the correlations between SSTR5 expression and clinical variables, as presented in Fig. 4. Both the CRH- and DDAVP-induced increases in ACTH correlated positively with the SSTR5 IRS [(CRH vs. SSTR5: p = 0.0024, r = 0.56) (Fig. 4a); (DDAVP vs. SSTR5: p = 0.0001, r = 0.76) (Fig. 4b)]. In addition, the decrease in cortisol levels caused by the HDDST was significant (p = 0.0001, r = 0.77) (Fig. 4c). However, the ACTH/cortisol index was negatively correlated with the SSTR5 IRS (p = 0.0002, r = –0.67) (Fig. 4d). The correlation between the ACTH/cortisol index and receptor expression was validated upon analysis via separate measurements of ACTH and cortisol (data not shown).
Corticotroph tumor diameter was also negatively correlated with the SSTR5 IRS (p = 0.0008, r = –0.61) (Fig. 4e), similar to the correlation between corticotroph tumor volume and the SSTR5 IRS (Supplementary Fig. 1). The summary shown in Table 2 presents the diagnostic potential of different clinical parameters for the prediction of high SSTR5 expression. The optimal cutoffs according to the Youden index were as follows: CRH test: 142.71% increase in ACTH (AUC = 0.79; sensitivity [Se] 64.29%, specificity [Sp] 84.62%); DDAVP test: 24.51% increase in ACTH (AUC = 0.87; Se 100%, Sp 66.67%); HDDST: 75.22% decrease in cortisol (AUC = 0.80; Se 75%, Sp 90.91%); ACTH/cortisol index: 6.38 (AUC = 0.71; Se 100%, Sp 46.15%); and corticotroph tumor diameter: 6.7 mm (AUC = 0.71; Se 92.86%, Sp 46.15%). On the other hand, the cutoffs with a sensitivity of 100% were as follows: CRH test: 39.69% increase in ACTH (Sp 30.77%); DDAVP test: 50.44% increase in ACTH (Sp 66.67%); HDDST: 50.44% decrease in cortisol (Sp 27.27%); ACTH/cortisol index: 6.38 (Sp 46.15%); and corticotroph tumor diameter: 9.49 mm (Sp 38.46%).
Table 2 The diagnostic utility of the CRH test, DDAVP test, HDDST, ACTH/cortisol index, and tumor size for the prediction of high SSTR5 expression
| Diagnostic method |
AUC |
Cut-off point |
Sensitivity
(%) |
Specificity
(%) |
CRH test
(% ACTH increase) (%) |
0.79 |
142.71 |
64.29 |
84.62 |
| 39.69 |
100 |
30.77 |
DDAVP testa
(% ACTH increase) (%) |
0.87 |
24.51 |
100 |
66.67 |
HDDSTb
(% cortisol decrease) (%) |
0.80 |
75.22 |
75 |
90.91 |
| 50.44 |
100 |
27.27 |
| ACTH/cortisol index |
0.71 |
6.38 |
100 |
46.15 |
| Tumor diameter (mm) |
0.71 |
6.7 |
92.86 |
46.15 |
| 9.49 |
100 |
38.46 |
Abbreviations: AUC, area under the curve; CRH, corticotropin-releasing hormone; DDAVP, desmopressin; HDDST, high-dose dexamethasone suppression test.
The upper and lower rows represent the data based on the Youden index and a sensitivity of 100%. The data from the DDAVP test and ACTH/cortisol index are represented in one row because the Youden index provides a sensitivity of 100%.
a patients, n = 24
b patients, n = 23
Discussion
The present study demonstrated mutual associations between responsiveness to CRH, DDAVP, and HDDST, the ACTH/cortisol index, tumor size, and somatostatin receptor 5 expression in corticotroph tumors (Graphical Abstract). These associations could predict SSTR5 expression via differential diagnostic tests, indices, and tumor size. This information may be valuable for planning CD treatments that consider SSTR5 expression. Additionally, our findings suggest that these clinical markers and SSTR5 expression can reflect the biological properties of corticotroph tumors.
The present study investigated the responsiveness of patients to differential diagnostic tests for CD. Although it has not been shown whether the responsiveness to CRH and HDDST corresponds to the expression of the CRH receptor (CRHR) and glucocorticoid receptor (GR), the correlation between corticotroph tumor AVPR2 expression and the response of ACTH to DDAVP has been reported previously [12]. Thus, our findings suggest that certain corticotroph tumors present high coexpression of AVPR and SSTR5.
Receptor-targeted treatments for various types of tumors are making consistent progress. For example, an antagonist (bicalutamide), an antibody-drug conjugate (trastuzumab deruxtecan), and peptide receptor radionuclide therapy (lutetium oxodotreotide) are used to treat prostate cancer, breast cancer, and neuroendocrine tumors, respectively. Therefore, treatments targeting CRHR, AVPR, GR, and SSTR5 could be beneficial in patients with CD, with increased responsiveness to CRH, DDAVP, and HDDST and a decreased ACTH/cortisol index. With respect to SSTR5, the association between SSTR5 expression and pasireotide efficacy in CD remains unclear [13]. To date, no in vivo reports have been described. However, USP8-mutant corticotroph tumors presented increased SSTR5 expression and pasireotide-induced ACTH inhibition in vitro [3]. Therefore, the predictors of SSTR5 expression in the present study and USP8 mutation may be useful in pasireotide treatment for CD. However, some CD patients have no corticotroph tumor specimens because of the difficulty in their surgical removal due to the small tumor size. In such cases, the predictors in the present study were available without the specimen but not the USP8 mutation. Moreover, predictors can be routinely obtained at the differential diagnosis of Cushing’s syndrome. Finally, the noninvasiveness and cost performance of these methods would be more favorable than the investigation of USP8 mutations.
The ACTH/cortisol index is likely influenced by cortisol-negative feedback, although a previous study suggested that ACTH activity is lower [2]. For example, when negative feedback is strong, ACTH secretion is greatly inhibited. As a consequence, the ACTH/cortisol index becomes low in value. Overall, both the HDDST and the ACTH/cortisol index are associated with responsiveness to glucocorticoids.
We also demonstrated that corticotroph tumor size linearly corresponds to responsiveness to differential diagnostic tests for CD, the ACTH/cortisol index, ACTH levels, and SSTR5 expression. Similarly, several studies reported that corticotroph tumor size was associated with the response to CRH [2, 14], ACTH levels [2], and SSTR5 expression [15]. However, they did not demonstrate whether size is linearly related to responsiveness to differential diagnostic tests for CD and SSTR5 expression. The linear relationships of our findings may be useful for CD diagnosis in cases where MRI reveals undetectable corticotroph microtumors with a high response rate to diagnostic tests or large corticotroph tumors with a low response rate to diagnostic tests. However, further research is needed to differentiate between CD syndrome and ectopic ACTH syndrome, as comparing the two is beyond the scope of this study. Further, since SSTR5 is known to suppress tumor cell proliferation [16, 17], corticotroph tumor size may be attributed to SSTR5 expression.
The present study has certain limitations. Since CD is a rare disease [18], the number of patients is limited. In addition, the study was restricted to subjects with pituitary tumor specimens because the aim of study was to evaluate SSTR5 expression. Moreover, the efficacy of pasireotide could not be investigated because of the limited number of patients receiving pasireotide. The study is retrospective, and potential selection bias could not be completely eliminated. Furthermore, the diurnal variation may influence the analysis results of the ACTH/cortisol index. However, two individual measurements of the ACTH/cortisol index produced comparable results, suggesting that the ACTH/cortisol index may accurately represent the biological characteristics of a corticotroph tumor. Additionally, multiple assays were utilized because our cohort covered a relatively long time period. Multiple assays may affect the value of the ACTH/cortisol index, as each ACTH/cortisol index can be calculated via different assays. However, since the responses to differential diagnostic tests were evaluated as a response rate of ACTH and cortisol by the same assay, employing multiple assays may not affect these results. Finally, our study was a retrospective observational study. Therefore, we could not assess the USP8 mutation due to a lack of ethical committee permission for a prospective genetic analysis. Therefore, further study is needed.
In conclusion, responsiveness to CRH, DDAVP, and HDDST; the ACTH/cortisol index; corticotroph tumor size; and the SSTR5 expression in corticotroph tumor are related to each other. The introduced predictors have potential in future CD treatment planning that considers SSTR5 expression. These findings may reflect the biological characteristics of corticotroph tumors and be useful in the diagnosis and treatment of CD.
Acknowledgments
Conceptualization: Karolina Budzen and Kosuke Mukai; Data curation: Karolina Budzen; Formal analysis: Karolina Budzen; Funding acquisition: Kosuke Mukai; Investigation: Karolina Budzen, Kosuke Mukai, and Masaharu Kohara; Methodology: Masaharu Kohara; Project administration: Kosuke Mukai; Resources: Satoru Oshino, Youichi Saitoh, Michio Otsuki, Kosuke Mukai, and Eiichi Morii; Supervision: Iichiro Shimomura and Atsunori Fukuhara; Writing - original draft: Karolina Budzen, Kosuke Mukai, Atsunori Fukuhara and Iichiro Shimomura; Writing - review and editing: all authors.
This study received support from the Center for Medical Research and Education, Graduate School of Medicine, Osaka University.
Disclosure
The authors report no conflicts of interest related to this work. Michio Otsuki is a member of Endocrine Journal’s Editorial Board.
Funding statement
This study was supported by the Ministry of Health, Labour and Welfare FC Program Grant Number JPMH20FC1021.
Data availability statement
The datasets analyzed in this study are not publicly available but may be available from the corresponding author upon reasonable request.
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