Abstract
Cushing’s syndrome, a clinical condition characterized by hypercortisolemia, exhibits distinct clinical signs and is associated with cyclic cortisol secretion in some patients. The clinical presentation of cyclic Cushing’s syndrome can be ambiguous and its diagnosis is often challenging. We experienced a 72-year-old woman with cyclic ACTH-dependent Cushing’s syndrome caused by a pulmonary carcinoid tumor. Diagnosis was challenging because of the extended trough periods, and the responsible lesion was initially unidentified. A subsequent follow-up computed tomography revealed a pulmonary lesion, and ectopic ACTH secretion from this lesion was confirmed by pulmonary artery sampling. Despite the short peak secretion period of ACTH (approximately one week), immunostaining of the surgically removed tumor confirmed ACTH positivity. Interestingly, stored plasma chromogranin A levels were elevated during both peak and trough periods. The experience in evaluating this patient prompted us to investigate the potential use of plasma chromogranin A as a diagnostic marker of ACTH-dependent Cushing’s syndrome. A retrospective study was conducted to determine the efficacy of plasma chromogranin A in three patients with ectopic ACTH syndrome (EAS), including the present case, and six patients with Cushing’s disease (CD) who visited our hospital between 2018 and 2021. Notably, plasma chromogranin A levels were higher in patients with EAS than in those with CD. Additionally, a chromogranin A level in the present case during the trough phase was lower than that in the peak phase, and was similar to those in CD patients. The measurement of plasma chromogranin A levels could aid in differentiating EAS from CD.
Introduction
Cushing’s syndrome is characterized by a chronic excess of glucocorticoids (hypercortisolemia) with distinctive clinical signs, such as a moon-like facial appearance. The etiology of Cushing’s syndrome can be classified into two categories: ACTH-dependent and ACTH-independent. In some cases, cyclic serum cortisol secretion has been observed. A diagnosis of cyclicity is established when serum cortisol levels display at least three peaks and two troughs [1]. The duration of these cycles varies, with trough periods often lasting from days to months [2]. Consequently, the clinical presentation of cyclic Cushing’s syndrome can vary, making the diagnosis challenging.
Chromogranin A is a protein present in secretory dense core granules of neuroendocrine tissues [3] and is co-secreted with amines and peptides present in neurosecretory granules [4], making it a useful serum marker for neuroendocrine tumors. Although Zemskova et al. investigated plasma chromogranin A levels for diagnostic insight into ACTH-dependent Cushing’s syndrome [5], its significance in cyclic ACTH-dependent Cushing’s syndrome remains unclear. In this study, we investigated the possibility of plasma chromogranin A as a diagnostic tool for ACTH dependent Cushing’s syndrome, especially for cyclic Cushing’s syndrome.
Case Presentation
A 72-year-old Japanese woman who was an inactive hepatitis B carrier presented at the hospital for routine follow-up. With no family history of endocrine diseases and only on calcium channel blockers for hypertension, the patient complained of general malaise. Blood tests revealed elevated plasma ACTH and serum cortisol (F) levels (ACTH 158 pg/mL, F 120.4 μg/dL). Computed tomography (CT) showed a small nodule in the S2b segment of the right lung and a micronodule in S5, which was suspected to be atelectasis (Fig. 1A, B), along with bilateral adrenal gland enlargement (Fig. 1C). Magnetic resonance imaging (MRI) of the pituitary gland suggested a microadenoma. The patient was referred to Hamamatsu University Hospital for further examination. Upon admission, ACTH and cortisol levels were within the reference ranges (Table 1), and she did not exhibit typical signs of Cushing’s syndrome. However, both ACTH and cortisol levels increased significantly during hospitalization (ACTH 213 pg/mL, F 87.5 μg/dL). The ACTH levels were unresponsive to both CRH and DDAVP loads (Table 2); therefore, ectopic ACTH syndrome (EAS) was suspected. However, neither 18F-fluoro-2-deoxy-D-glucose positron emission tomography (PET) nor 111In-pentetreotide scintigraphy revealed any abnormalities. Additionally, neither inferior petrosal sinus sampling (IPSS) nor pulmonary artery sampling performed at the peak phase confirmed ACTH hypersecretion from the pituitary gland or right pulmonary S2 node (Table 3); therefore, the responsible lesion could not be identified. The patient was discharged with spontaneously normalized levels.
Fig. 1
Radiological findings at initial admission. CT imaging reveals a small nodule in the S2b segment of the right lung (A), a micro nodule in S5 of the right lung (B), and bilateral adrenal gland enlargement (C).
Table 1 Laboratory data at the initial admission to our hospital
| Blood count |
Blood chemistry |
Endocrinological data |
| WBC |
4,760/μL |
Na |
145 mEq/L |
ACTH |
38.5 pg/mL |
| RBC |
451 × 104/μL |
K |
4.6 mEq/L |
Cortisol |
4.3 μg/dL |
| Hb |
14.0 g/dL |
Cl |
109 mEq/L |
DHEA-S |
91 μg/dL |
| Ht |
40.7% |
BUN |
12.2 mg/dL |
UFC |
174 μg/day |
| Plt |
20.5 × 104/μL |
Cr |
0.59 mg/dL |
|
|
|
|
AST |
19 IU/L |
|
|
|
|
ALT |
21 IU/L |
|
|
|
|
GGT |
17 IU/L |
|
|
|
|
LDL-C |
114 mg/dL |
|
|
|
|
HDL-C |
59 mg/dL |
|
|
|
|
TG |
77 mg/dL |
|
|
|
|
FPG |
109 mg/dL |
|
|
|
|
HbA1c |
5.9% |
|
|
WBC: white blood cell, RBC: red blood cell, Hb: hemoglobin, Ht: hematocrit, Plt: platelet, BUN: blood urea nitrogen, Cr: creatinine, AST: aspartate aminotransferase, ALT: alanine aminotransferase, GGT: gamma-glutamyl transpeptidase, LDL-C: low density lipoprotein cholesterol, HDL-C: high density lipoprotein cholesterol, TG: triglyceride, FPG: fasting plasma glucose, ACTH: adrenocorticotropic hormone, DHEA-S: dehydroepiandrosterone sulfate, UFC: urinary free cortisol.
Table 2 Results of CRH and desmopressin tests
| Time (min) |
0 |
15 |
30 |
60 |
90 |
120 |
| CRH test |
| ACTH (pg/mL) |
217 |
206 |
205 |
220 |
210 |
218 |
| Cortisol (μg/dL) |
72.2 |
74.0 |
74.0 |
75.6 |
76.8 |
77.8 |
| Desmopressin test |
| ACTH (pg/mL) |
166 |
229 |
220 |
180 |
170 |
171 |
| Cortisol (μg/dL) |
68.5 |
70.4 |
75.2 |
76.3 |
70.8 |
72.1 |
Table 3 Results of ACTH (pg/mL) in inferior petrosal sinus (IPS) sampling before and after intravenous administration of 100 μg CRH, and pulmonary artery (PA) sampling
| IPS sampling |
Right IPS |
Left IPS |
Cavernous sinus: right |
Cavernous sinus: left |
Peripheral vein |
| Baseline |
150 |
180 |
144 |
204 |
123 |
| +5 min CRH |
276 |
329 |
157 |
|
157 |
| +10 min CRH |
266 |
254 |
196 |
|
196 |
| +15 min CRH |
278 |
261 |
179 |
|
179 |
| PA sampling |
Inferior vena cava |
Superior vena cava |
Main PA |
Right A2 segment of PA |
Peripheral vein |
|
101 |
112 |
106 |
125 |
123 |
Eight months later, she was readmitted because of a disease flare-up, with no examinations possible because of the early intermittence of the disease. Another 15 months later, a similar episode led to hospitalization and a bromocriptine tolerance test, which indicated ACTH suppression (decreased to 33.5% of the previous value), leading to the initiation of dopamine agonist. However, the disease flared 13 months later, leading to the fourth hospital admission. CT showed a slight increase in the size of the S5 nodule in the right lung (Fig. 2A). Repeated pulmonary artery (PA) sampling confirmed an increase in ACTH levels, identifying the S5 lung lesion as the lesion responsible (PA/peripheral ratio, 5.97, Table 4). The lesion was surgically removed, and immunohistochemical staining confirmed that it was an ACTH-producing carcinoid tumor (Fig. 2B–G). Postoperatively, the patient was free of ACTH elevation, with no recurrence on imaging. Interestingly, stored plasma chromogranin A levels were elevated during both peak and trough periods, respectively. Written informed consent for publication was obtained from this patient.
Fig. 2
Radiological and pathological findings at fourth admission. CT imaging reveals a slight increase in the S5 nodule of the right lung (A). Pathological examination of the lung lesion shows tumor cells densely packed with relatively homogeneous, round nuclei, predominantly alveolar in shape, as seen in hematoxylin and eosin (HE) staining (B). Immunohistochemical staining demonstrates positivity for CD56 (C), TTF-1 (D), chromogranin A (E), synaptophysin (F), and ACTH (G). Scale bars represent 100 μm.
Table 4 Results of ACTH (pg/mL) in pulmonary artery (PA) sampling during the fourth hospitalization
| PA sampling |
Inferior
vena cava |
Superior
vena cava |
Main PA |
Right A5
segment of PA |
Peripheral vein |
|
109 |
117 |
114 |
687 |
115 |
The experience in evaluating this challenging case of cyclic EAS prompted us to investigate the potential use of plasma chromogranin A as a diagnostic marker for ACTH-dependent Cushing’s syndrome.
Methods
We retrospectively reviewed plasma chromogranin A levels in the present patient, in addition to two patients with non-cyclic EAS and six patients with non-cyclic Cushing’s disease (CD) who visited the Hamamatsu University Hospital between November 2018 and February 2021. The demographic and biochemical data of the patients are presented in Supplemental Table 1. The present case was the only patient who was diagnosed as cyclic Cushing’s syndrome among the nine cases. Tumor specimens which were surgically removed in each case were histologically confirmed as the responsible lesion by ACTH immunostaining. The pathological findings of the ACTH-producing tumor in two additional cases of non-cyclic EAS were both pancreatic neuroendocrine tumors, and also positive for immunohistochemical staining of chromogranin A. Additionally, three blood samples of pheochromocytoma/paraganglioma (PPGL) patients were subjected to analysis as a positive control. The study protocol was approved by the ethics committee of Hamamatsu University School of Medicine (15–326) and was conducted in accordance with the Declaration of Helsinki.
Blood samples subjected to plasma chromogranin A analysis were collected after at least 30 minutes of rest and stored in –80°C, respectively. The chromogranin A concentration was determined using Human Chromogranin A ELISA Kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and quantified spectrophotometrically (BioTek, Synergy H1). As a reference, the manufacturer of this ELISA kit stated that the measurement of chromogranin A had a mean of 251.4 ± 67.4 ng/mL for 15 samples of human plasma. Statistical analyses were conducted using the IBM SPSS Statistics software (version 27). For comparisons of two datasets, statistical significance was determined using the Student’s t-test. All results are expressed as means ± SD. Differences were considered statistically significant at p < 0.05.
Results
Plasma chromogranin A levels were significantly higher in EAS than in CD (EAS: 734.8 ± 59.0 ng/mL vs. CD: 371.7 ± 158.6 ng/mL: p = 0.007, Fig. 3A). Both EAS and CD exhibited levels higher than the mean value in human plasma measured by the manufacturer. The ratio of chromogranin A to ACTH (chromogranin A/ACTH) showed that EAS tended to secrete less chromogranin A than ACTH in ratio, but the difference was not statistically significant (EAS: 2.1 ± 1.0 vs. CD: 6.5 ± 2.1: p = 0.211, Fig. 3B). The product of ACTH and chromogranin A showed no significant difference between EAS and CD (EAS: 439,918 ± 235,968 vs. CD: 35,832 ± 14,481: p = 0.229, Fig. 3C); however, the product of EAS and CD did not overlap. Additionally, chromogranin A level was measured in three PPGL samples as controls, revealing markedly higher levels than in the EAS and CD groups (1,423.1 ± 217.2 ng/mL). Comparing plasma chromogranin A levels between the peak phase (694.6 ng/mL) and trough phase (279.2 ng/mL) of the present case showed a lower level in the trough phase (ACTH; 166.0 pg/mL vs. 51.4 pg/mL, respectively). The trough phase level of chromogranin A was comparable to levels observed in patients with CD.
Fig. 3
Plasma chromogranin A levels in patients with ectopic ACTH syndrome (EAS) and Cushing’s disease (CD). Examination results indicate that plasma chromogranin A levels are significantly higher in patients with EAS compared to those with CD (EAS: 734.8 ± 59.0 ng/mL vs. CD: 371.7 ± 158.6 ng/mL; p = 0.007) (A). The ratio of chromogranin A to ACTH (chromogranin A/ACTH) suggests that EAS tended to secrete less chromogranin A than ACTH in ratio (EAS: 2.1 ± 1.0 vs. CD: 6.5 ± 2.1: p = 0.211) (B). The product of ACTH and chromogranin A did not differ significantly (EAS: 439,918 ± 235,968 vs. CD: 35,832 ± 14,481: p = 0.229). However, the product of EAS and CD did not overlap (C).
Discussion
In this study, we investigated the efficacy of plasma chromogranin A as a diagnostic marker for cyclic ACTH-dependent Cushing’s syndrome.
Cyclic Cushing’s syndrome, exhibiting periodic changes in cortisol secretion, is more commonly seen in Cushing’s disease (CD) (54%), but is also reported in ectopic ACTH secretion (EAS) (26%) [1]. Currently in Japan, ACTH-dependent Cushing’s syndrome is screened by 0.5 mg overnight dexamethasone test (DST) and midnight serum cortisol over 5 μg/dL. The definite diagnosis will be made by CRH test, 8 mg overnight DST, pituitary imaging and IPSS [6]. In the Endocrine Society guideline, measurement of urine free cortisol or salivary cortisol are suggested in test choice for diagnosis when cyclic are suspected [7], and there is no suggestion in Japanese guideline. However, all of these tests will result in negative during the trough period of cyclic Cushing’s syndrome. Since the duration of the cycle is unpredictable, the diagnosis becomes challenging and sometimes test has to be repeated. The cyclic pattern in this case had a very short peak of about one week and a prolonged trough of almost a year, complicating diagnosis and treatment. Furthermore, even if the ACTH-dependent Cushing’s syndrome is biochemically confirmed, the differentiation of CD and EAS still remains.
EAS from non-pituitary tumors constitutes approximately 10–20% of Cushing’s syndrome cases [8]. In Japan, it has been reported that of 16 patients diagnosed with EAS, 3 (19%) had bronchial carcinoid as the etiology [9]. Biochemical testing such as CRH, dDAVP has been suggested to differentiate CD and EAS, but to date bilateral IPSS is still the gold standard [10]. IPSS is invasive and requires preparation and admission. If the biochemical test results suggest EAS, whole-body imaging is the next strategy for detection. Thin-slice whole-body CT was reported to detect 67% of EAS at initial presentation [11] but tumor less than 1cm required other imaging or delay in time for locating the tumor. A combination of functional and anatomic imaging modalities such as low octreotide scintigraphy and [(18)F] l-3,4-dihydroxyphenylalanine (F-DOPA)-PET scans has been shown to be useful in the localization of EAS [12], but the latter is not covered by insurance in Japan at the time of writing this paper. In the present case, pulmonary artery sampling during the peak period was crucial in localizing the source of ectopic ACTH secretion. The pulmonary artery sampling is a technique that can accurately reflect ectopically secreted hormones from the tumor by selectively sampling of the “wedged” blood collected via backflow to the artery or via the peritumoral capillaries [13]. Several case reports demonstrate the usefulness of pulmonary artery sampling in localizing EAS [13, 14]. Pulmonary arterial sampling is recommended for suspected pulmonary lesions with negative functional imaging as in the present case.
To investigate a non-invasive diagnostic biochemical marker for differential diagnosis for ACTH-dependent Cushing’s syndrome, we have focused on serum chromogranin A. Chromogranin A is released together with the co-stored hormones upon exocytosis from many normal and neoplastic neuroendocrine tissues [15]. Chromogranin A was first reported as a biomarker for a wide range of neuroendocrine tumors, and over the years it has been reported as diagnostic and prognostic marker for a cardiovascular, gastrointestinal, and inflammatory diseases. In pulmonary neuroendocrine (carcinoid) tumors measurement of plasma chromogranin A during diagnosis and follow up is recommended by European Neuroendocrine Tumor Society [16]. There have been only limited studies evaluating the efficacy of measuring chromogranin A in pituitary disease. Gussi et al. reported that measuring chromogranin A in non-functioning pituitary adenomas by novel assay was not useful [17]. In patients with ACTH-dependent Cushing’s syndrome, a cutoff serum chromogranin A of 225 ng/mL, measured by immunochemiluminometric assays using anti-human chromogranin A mouse monoclonal antibodies, was reported to be diagnostic for EAS with a positive predictive value of 83% and a negative predictive value of 70% [5]. Our data demonstrated significantly higher plasma chromogranin A levels in EAS compared to CD, indicating its potential utility in differentiating between the two. Previous reports have shown that plasma ACTH levels tend to be higher in EAS than in CD [18, 19]. In this study, the ACTH levels were not significantly different because of the small sample size, but this trend was also observed (Supplemental Fig. 1). Although EAS tended to secrete less chromogranin A than ACTH in ratio (Fig. 3B), the actual measurement of chromogranin A was higher, suggesting the potential of simultaneous hypersecretion of ACTH and chromogranin A. In our study, there was no significant difference in basal ACTH levels between the EAS and CD groups (p = 0.229, Supplemental Fig. 1) because of the small sample size of the EAS group. For the same reason, the product of ACTH and chromogranin A did not differ significantly (Fig. 3C). However, the product of EAS and CD did not overlap, suggesting it might be possible to clearly delineate EAS and CD if the sample size is increased. Although plasma chromogranin A is known to be affected by renal dysfunction [20], there was no difference in renal function between the two groups. Plasma chromogranin A during the trough phase was also elevated in the present case, which was comparable to CD group levels. This suggests that in order to differentiate EAS and CD, blood sample taken at the peak period must be subjected to analysis. The limitation of this study is that the sample size is small and further case accumulation is necessary for comprehensive investigation.
Conclusion
Investigating a non-invasive and simple procedure test for diagnosing cyclic Cushing’s syndrome is crucial, and plasma chromogranin A measurement shows promise for aiding in the etiological diagnosis.
Financial Support
This work was partially supported by the Japan Society for the Promotion of Science KAKENHI Grant (22K16410 for KK).
Disclosure
The authors have no conflicts of interest to declare.
Supplemental Table 1 Demographic and biochemical data of patients with ectopic ACTH syndrome (EAS) and Cushing’s disease (CD)
|
EAS group
(n = 3) |
CD group
(n = 6) |
| Female (%) |
66.6 |
66.6 |
| Age (mean ± SD) |
54.3 ± 18.6 |
52.5 ± 13.8 |
| ACTH (pg/mL) |
572.7 ± 492.3 |
86.5 ± 63.8 |
| F (μg/dL) |
45.2 ± 30.7 |
18.4 ± 3.4 |
| Urine free cortisol (μg/day) |
4,010.0 ± 4,341.3 |
368.3 ± 191.8 |
| Serum Creatinine (mg/dL) |
0.63 ± 0.21 |
0.77 ± 0.26 |
Supplemental Fig. 1
The basal ACTH levels between the EAS and CD groups. Although there was no significant difference between the two groups, plasma ACTH levels exhibited a tendency to be higher in the EAS than in the CD (EAS: 572.7 ± 492.3 vs. CD: 86.5 ± 63.8: p = 0.229).
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