Utility of Simple and Non-Invasive Strategies Alternative to Inferior Petrosal Sinus Sampling and Peripheral CRH Stimulation in Differential Diagnosis of ACTH-Dependent Cushing Syndrome

Abstract

We aimed to evaluate the utility of simple, cost-effective, and non-invasive strategies alternative to BIPSS and peripheral CRH stimulation in differential diagnosis of ACTH-dependent CS. First, we performed ROC analysis to evaluate the performance of various tests for differential diagnosis of ACTH-dependent CS in our cohort (CD, n=76 and EAS, n=23) and derived their optimal cut-offs. Subsequently, combining various demographic (gender), clinical (hypokalemia), biochemical (plasma ACTH, HDDST, peripheral CRH stimulation) and imaging (MRI pituitary) parameters, we derived non-invasive models with 100% PPV for CD. Patients with pituitary macroadenoma (n=14) were excluded from the analysis involving non-invasive models. Relative percent ACTH (AUC: 0.933) and cortisol (AUC: 0.975) increase on peripheral CRH stimulation demonstrated excellent accuracy in discriminating CD from EAS. Best cut-offs for CD were plasma ACTH<97.3 pg/ml, HDDST≥57% cortisol suppression, CRH stimulation≥77% ACTH increase and≥11% cortisol increase. We derived six models that provided 100% PPV for CD and precluded the need for BIPPS in 35/85 (41.2%) patients with ACTH-dependent CS and no macroadenoma (in whom BIPSS would have otherwise been recommended). The first three models included basic parameters and avoided both peripheral CRH stimulation and BIPSS in 19 (22.4%) patients, while the next three models included peripheral CRH stimulation and avoided BIPSS in another 16 (18.8%) patients. Using simple and non-invasive alternative strategies, BIPSS can be avoided in 41% and peripheral CRH stimulation in 22% of patients with ACTH-dependent CS and no macroadenoma; such patients can be directly referred for a pituitary surgery.

Keywords

Cushing disease - ACTH-dependent Cushing syndrome - BIPSS - CRH stimulation - non-invasive

Introduction

Endogenous Cushing Syndrome (CS) is a state of glucocorticoid hypersecretion characterized by the loss of normal diurnal rhythm of cortisol secretion and the normal negative feedback suppression of hypothalamic-pituitary-adrenal axis. Adrenocorticotrophic hormone (ACTH)-dependent CS accounts for 80–85% of endogenous CS. Differentiation between the two subtypes of ACTH-dependent CS, that is, Cushing disease (CD) and ectopic ACTH syndrome (EAS), is a clinical challenge [1]. This is especially true when magnetic resonance imaging (MRI) of pituitary is negative/ambiguous (e. g., absent lesion or a lesion<6 mm, a common finding in>60% patients with CD) [2], or when the results of endocrine dynamic tests [e. g., high-dose dexamethasone suppression test (HDDST) and peripheral corticotropin-releasing hormone (CRH) stimulation test] are discordant [3] [4].

Bilateral inferior petrosal sinus sampling (BIPSS) is considered as the gold standard in the differential diagnosis between CD and EAS. This procedure involves the catheterization of bilateral inferior petrosal sinuses and the demonstration of presence or absence of an ACTH gradient between the central venous effluent and the peripheral vein [5]. According to a recent international consensus, all patients with ACTH-dependent CS and no or equivocal adenoma or microadenoma (size<6–9 mm) on pituitary MRI are candidates for BIPSS. On the other hand, patients with a macroadenoma (size ≥10 mm) can be presumed to have CD and skip BIPSS [6]. Given that only a small proportion (around 10–15%) of patients with CD present pituitary macroadenoma, BIPSS may be needed in a majority [7] [8]. However, BIPSS is invasive, cost-intensive and is available only at selected centers of expertise. Furthermore, BIPSS can have false negative and false positive results and the accuracy in clinical practice is far from 100% [5] [9]. For instance, in a recent study, using a post-CRH IPS: P ACTH cut-off≥2.1, Detomas et al., reported 9 false negative results out of 103 CD cases (sensitivity 91%) and 1 false positive result out of 14 EAS cases (specificity 93%) [10]. Overall, the sensitivity and specificity of BIPSS varies between 88–100% and 67–100%, respectively [5]. Notably, the availability of human CRH, employed as a stimulating agent in BIPSS as well as peripheral CRH test, is presently dwindling globally [11] [12]. Thus, there is a need for simple, easily available, cost-effective, and non-invasive alternatives that can be of immense value to resource-rich and resource-poor settings alike. With this background, the current study aimed to evaluate the utility of strategies alternative to BIPSS and peripheral CRH stimulation in the differential diagnosis of ACTH-dependent CS. For this purpose, we evaluated data from our cohort of 99 patients with treatment naïve ACTH-dependent CS (EAS, n=23 and CD, n=76), the clinical and biochemical profile of which have been published previously [13].

Materials and Methods

Settings and study design

The data were collected as a part of an ambispective observational study performed in the department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi, India between 2021 and 2022. Case files for patients admitted with features of endogenous CS between January 2013 and March 2021 were reviewed retrospectively, while a prospective review was made for patients admitted between March 2021 and June 2022. Based on this review, a proforma was filled documenting the relevant demographic, clinical, biochemical, and radiological details. The protocol was approved by the institutional ethics committee (IEC No. IECPG 265/24.03.2021) and informed consent was obtained from all patients enrolled prospectively in the study.

Study objectives

The objectives of our study were: a) to evaluate the performance of various parameters in the differential diagnosis of ACTH-dependent CS, b) to derive cohort-specific cut-offs for biochemical parameters and dynamic tests used in the differential diagnosis, and c) to derive simple and non-invasive strategies/models that provide 100% specificity and positive predictive value (PPV) for CD, thus precluding the need for BIPSS alone or both BIPSS and peripheral CRH stimulation.

Data collection and study protocol and definitions

We collected data for 146 patients with ACTH-dependent CS, of whom 29 patients with an occult ACTH source and 18 patients with a history of prior surgery or radiotherapy were excluded. Thus, the current analysis involved 99 patients with treatment naïve ACTH-dependent CS, including 23 with EAS and 76 with CD. For evaluating the differential performance of various tests and deriving cohort-specific cut-offs, we included all 99 patients with ACTH-dependent CS. On the other hand, for the analysis involving non-invasive models, we excluded patients with pituitary macroadenoma (n=14), and the sample size was 85 (see section “development of non-invasive models”).

The protocol for diagnosis of ACTH-dependent hypercortisolism and its subsequent evaluation as well as the study definitions for CD and EAS have been provided in our previous publication [13]. Briefly, a plasma 8 AM ACTH level>10 pg/ml was used to subtype endogenous CS as ACTH-dependent [14]. Such patients underwent a number of inpatient tests for differential diagnosis, including contrast-enhanced MRI pituitary (n=92; CD, n=76 and EAS, n=16), HDDST (n=76; CD, n=59 and EAS, n=17), peripheral CRH stimulation (n=36; CD, n=30 and EAS, n=6), contrast-enhanced computed tomography (CT) scan of neck to pelvis (n=56; CD, n=35 and EAS, n=21), 68-Ga-DOTANOC-PET/CT scan (n=60; CD, n=38 and EAS, n=22) and CRH-stimulated BIPSS (bilateral and simultaneous approach with central and peripheral samples collected at baseline and at 3, 5, 10, and 15 minutes after 100 μg human CRH injection; n=17; CD, n=14 and EAS, n=3). For peripheral CRH stimulation, venous samples for cortisol and ACTH were collected at baseline and at 15, 30, 45, 60, 90, and 120 minutes after intravenous human CRH injection. The percentage ACTH/cortisol response was calculated as the difference between peak and baseline ACTH/cortisol level divided by the baseline ACTH/cortisol level, × 100. For HDDST, a single tablet of 2 mg dexamethasone was administered every 6 hours for 48 hours (9–3–9–3 or 12–6–12–6 regimen) and the venous sample was collected at 8 AM (2 or 5 hours after the last dose). The percentage cortisol suppression was calculated as the difference between post-HDDST and basal 8 am serum cortisol divided by basal 8 AM serum cortisol, × 100. Hypokalemia was defined as lowest serum potassium level during admission<3.5 mmol/l.

The diagnosis of CD was based on histopathological evidence of ACTH immunostaining pituitary adenoma (n=61) and/or biochemical remission following adenoma resection (early remission, n=51 and delayed remission, n=10) or evolution of corticotroph tumor progression after bilateral adrenalectomy/Nelson’s syndrome (CTP-BADX/NS; n=2). The diagnosis of EAS was based on unambiguous findings on ⁶⁸Ga-DOTANOC PET/CT (n=21) and/or other imaging modalities (n=19) and/or resolution of hypercortisolism following surgery of ectopic tumor source (n=5). Thus, the differential diagnosis into CD and EAS in this study was not based on the results of BIPSS or peripheral CRH stimulation tests, which lack 100% accuracy [4] [5] [9].

Hormone analysis

Cortisol was measured in blood and saliva samples and ACTH in blood samples using an electrochemiluminescence immunoassay on Cobas e411 autoanalyzer (Roche Diagnostics, Germany). The reference ranges provided by the manufacturer are 8 AM serum cortisol (5th–95th percentile): 6.0–18.4 μg/dl, late-night salivary cortisol:<0.274 μg/dl (95th percentile) and<0.410 μg/dl (97.5th percentile), and 8 AM plasma ACTH (5th–95th percentile): 7.2–63.3 pg/ml.

Development of non-invasive models

We developed a total of six non-invasive models combining both qualitative and quantitative variables; each model was individually associated with 100% specificity and PPV for CD and thus precluded the need for BIPSS in patients fulfilling all its components. Because, as per current recommendations, patients with ACTH-dependent CS and pituitary macroadenoma are considered to have CD and spared of BIPSS [6], such patients (n=14) were not included in the analysis pertaining to the non-invasive models and the final sample on which these models were applied was 85.

For quantitative variables employed in the models, we selected both cohort-specific thresholds (e. g., plasma ACTH level<97 pg/ml, HDDST cortisol suppression≥57%, peripheral CRH stimulation ACTH rise≥77%, peripheral CRH stimulation cortisol rise≥11%) as well as higher thresholds described in the literature that are associated with a greater specificity for CD (e. g., HDDST cortisol suppression>80% and peripheral CRH stimulation ACTH rise>100%).

The first three models included components other than peripheral CRH stimulation test (model 1: female gender, absence of hypokalemia, plasma ACTH<97 pg/ml and HDDST cortisol suppression≥57% ; model 2: absence of hypokalemia, plasma ACTH<97 pg/ml and HDDST cortisol suppression>80%; model 3: absence of hypokalemia, plasma ACTH<97 pg/ml, HDDST cortisol suppression≥57% and definitive lesion on CEMRI pituitary), while the next three models (model 4: HDDST cortisol suppression>80% and peripheral CRH stimulation ACTH rise≥77%; model 5: peripheral CRH stimulation ACTH rise>100% alone; model 6: peripheral CRH stimulation cortisol rise≥11% alone) additionally included this dynamic test. We derived the total number of patients with ACTH-dependent CS (and no macroadenoma) fulfilling all the components in a given model as well as the number of individuals exclusive to a given model [for example, 13 patients fulfilled all the components in model no. 2 (i. e., they had no hypokalemia, plasma ACTH was<97 pg/ml and HDDST cortisol suppression was>80%), however, 10 of them also satisfied the preceding model (no. 1), and therefore, the number exclusive to this model was 3].

Non-invasive models: rationale for selected variables

The variables selected for deriving the non-invasive models were the ones that are relatively simple and more easily available compared to BIPSS and individually associated with high PPV (>80%) for CD in our analysis. We chose MRI pituitary and not CT/Ga-DOTANOC PET/CT in imaging parameters, because MRI pituitary is the first line of investigation for patients with ACTH-dependent CS and was more uniformly available in study participants.

Statistical analysis

Statistical analysis was performed using Stata 15.0 (StataCorp LP, Texas, USA). Qualitative data were represented as number (percentage) and quantitative data as mean±SD or median (P₂₅–P₇₅). To evaluate the performance of various tests in differential diagnosis of ACTH-dependent CS, receiver operating characteristic (ROC) curves were drawn and area under curve (AUC; 95% CI) values were derived. Using the ROC analysis, the optimal cut-offs were derived and the corresponding sensitivity (95% CI), specificity (95% CI), positive predictive value (PPV; 95% CI), negative predictive value (NPV; 95% CI) and likelihood ratio positive (LR+; 95% CI) were reported. Subsequently, using a combination of various demographic (female gender), clinical (presence or absence of hypokalemia), biochemical (plasma ACTH, HDDST cortisol suppression, peripheral CRH stimulation ACTH and cortisol response) and imaging (MRI pituitary) parameters, we derived non-invasive models with 100% specificity and PPV for CD. A p-value<0.05 was considered statistically significant.

Results

Baseline characteristics of study participants

The detailed clinical, demographic, and biochemical profile of this cohort has been published previously [13]. The salient parameters are provided in [Table 1]. Patients with CD (n=76) were more likely to be females and less likely to manifest hypokalemia compared to those with EAS (n=23). The median 8 am serum cortisol, late-night salivary cortisol and 8 AM plasma ACTH levels were significantly higher in EAS group. Among patients with CD, 14 had pituitary macroadenoma.

Table 1 Baseline characteristics of study participants.

Variable	EAS (n=23)	CD (n=76)	p-Value
Age (years)	24.5 (20–40)	29.0 (23.1–37.5)	0.27
Female (n, %)	8 (34.8%)	55 (72.4%)	<0.001
BMI (kg/m2)	25.3 (21.9–31.5)	27.3 (24.7–32.0)	0.051
Hypertension (n, %)	19 (82.6%)	64 (84.2%)	0.855
Diabetes (n, %)	13 (56.5%)	39 (51.3%)	0.661
Hypokalemia (n, %)	19 (82.6%)	16 (21.0%)	0.001
8 AM serum cortisol (μg/dl)	49.7 (29.6–63.4)	26.7 (22.6–37.9)	<0.001
11 pm serum cortisol (μg/dl)	40.7±18.2	26.9±12.1	<0.001
Late-night salivary cortisol (μg/dl)	3.7 (2.4–10.3)	1.08 (0.68–1.86)	<0.001
ONDST cortisol (μg/dl)	25.3 (22.6–82.0)	20.1 (14.9–23.7)	0.02
8 AM plasma ACTH (pg/ml)	151.4 (94.7–279.0)	76.4 (46.2–102.8)	<0.001

Data are presented as number (%), mean±SD or median (IQR). ACTH: Adrenocorticotropic hormone; BMI: Body mass index; CD: Cushing disease; EAS: Ectopic ACTH syndrome; ONDST: Overnight dexamethasone suppression test.

Differential diagnosis of ACTH-dependent CS

The sensitivity, specificity, PPV, NPV and LR+of different demographic, clinical and biochemical variables (at standard cut-offs described in the literature) for differential diagnosis of ACTH-dependent CS have been provided in [Table 2].

Table 2 Sensitivity, specificity, positive and negative predictive values of different demographic, clinical and biochemical parameters for Cushing disease.

Parameter	Sensitivity (95% CI)	Specificity (95% CI)	PPV (95% CI)	NPV (95% CI)	LR+(95% CI)
Female gender	72.4 (61.4–81.2)	65.2 (44.9–81.2)	87.3 (76.9–93.4)	41.7 (27.1–57.8)	2.1 (1.6–2.7)
No hypokalemia	79.0 (68.5–86.6)	82.6 (62.8–93.0)	93.8 (85.0–97.5)	54.3 (38.2–69.5)	4.5 (2.8–7.5)
Plasma ACTH<90 pg/ml	65.8 (54.6–74.5)	82.6 (62.9–93.0)	92.6 (82.5–97.1)	42.2 (28.9–56.7)	3.8 (2.3–6.3)
HDDST:>50% cortisol suppressiona	69.5 (56.8–80)	58.8 (36–78)	85.4 (72.8–92.7)	35.7 (20.7–54.1)	1.7 (1.2–2.3)
HDDST:>80% cortisol suppressiona	40.7 (29–53.4)	82.4 (59.0–93.8)	88.9 (72.0–96.1)	28.6 (17.8–42.4)	2.3 (1.1–5.0)
Peripheral CRH:>50% ACTH riseb	93.3 (78.6–98.1)	83.3 (43.6–97.0)	96.6 (82.8–99.3)	71.4 (35.8–91.7)	5.6 (0.78–40)
Peripheral CRH:>100% ACTH riseb	73.3 (55.5–85.8)	100 (60.9–100)	100 (85.1–100)	42.9 (21.3–67.4)	–
Peripheral CRH:>20% cortisol riseb	86.7 (70.3–94.7)	100 (61–100)	100 (87.1–100)	60.0 (31.2–83.1)	–
Presence of lesion on CEMRI pituitaryc	81.6 (71.4–88.7)	93.8 (71.6–98.9)	98.4 (91.5–99.7)	51.7 (34.4–68.6)	13.1 (1.8–93.3)
Absence of lesion on CECT scand	97.1 (85.5–99.5)	90.5 (71.1–97.3)	94.4 (81.9–98.5)	95.0 (76.4–99.1)	10.2 (3.8–27.2)
Absence of lesion on 68-Ga-DOTANOC-PET/CT scane	94.7 (82.7–98.5)	95.5 (78.2–99.2)	97.3 (86.2–99.5)	91.3 (73.2–97.6)	20.8 (2.9–148.4)

ACTH: Adrenocorticotropic hormone; CECT: Contrast enhanced computed tomography; CEMRI: Contrast enhanced magnetic resonance imaging; CRH: Corticotropin releasing hormone; HDDST: High dose dexamethasone suppression test; LR+: Likelihood ratio positive; NPV: Negative predictive value; PPV: Positive predictive value. ^a HDDST performed in 59 patients with Cushing disease and 17 with ectopic ACTH syndrome. ^b Peripheral CRH stimulation performed in 30 patients with Cushing disease and 6 with ectopic ACTH syndrome. ^c CEMRI pituitary performed in all 76 patients with Cushing disease and 16 with ectopic ACTH syndrome. ^d CECT scan performed in 35 patients with Cushing disease and 21 with ectopic ACTH syndrome. ^e 68-Ga-DOTANOC-PET/CT scan performed in 38 patients with Cushing disease and 22 with ectopic ACTH syndrome.

Notably, variables with high PPV (> 80%) for CD included: female gender, absence of hypokalemia, plasma ACTH levels<90 pg/ml, HDDST cortisol suppression>50%/80%, peripheral CRH>50% ACTH increase, presence of a lesion on MRI pituitary, absence of a peripheral lesion on CT imaging and absence of a peripheral lesion on 68-Ga-DOTANOC PET/CT imaging. Furthermore, an ACTH rise>100% and a cortisol rise>20% following CRH stimulation provided 100% specificity and PPV for CD.

Performance of various biochemical and dynamic tests and optimal cohort-specific cut-offs

The results of ROC analysis, the area under the curve (AUC) for different biochemical variables and dynamic tests in differential diagnosis and the optimal cohort-specific cut-offs (best criteria) with corresponding sensitivity, specificity, PPV, NPV and LR+are presented in [Table 3].

Table 3 Performance of different biochemical parameters and dynamic tests and optimal cohort-specific cut-offs for diagnosis of Cushing disease.

Parameter	AUC (95% CI)	Optimal cut-off	Sensitivity (95% CI)	Specificity (95% CI)	PPV 95% CI)	NPV (95% CI)	LR+(95% CI)
Serum 8 AM cortisol (μg/dl)	0.730 (0.60–0.85)	<34.1	67.1 (55.9–76.6)	65.2 (44.9–81.2)	86.4 (75.5–93.0)	37.5 (24.2–53.0)	1.9 (1.5–2.5)
Plasma 8 AM ACTH (pg/ml)	0.800 (0.68–0.91)	<97.3	71.1 (60.0–80.0)	69.6 (49.1–84.4)	88.5 (78.2–94.3)	42.1 (27.9–57.8)	2.3 (1.7–3.1)
HDDST: % cortisol suppression	0.702 (0.56–0.84)	≥57%	61.0 (48.3–72.4)	58.8 (36.0–78.4)	83.7 (70.0–91.9)	30.3 (17.4–47.3)	1.5 (1.1–2.0)
Peripheral CRH: % ACTH rise	0.933 (0.84–1.0)	≥77%	86.7 (70.3–94.7)	83.3 (43.7–97.0)	96.3 (81.7–99.3)	55.6 (26.7–81.1)	5.2 (0.72–37.4)
Peripheral CRH: % cortisol rise	0.975 (0.92–1.0)	≥11%	96.7 (83.3–99.4)	100 (61–100)	100 (88.3–100)	85.7 (48.7–97.4)	–

ACTH: Adrenocorticotropic hormone; AUC: Area under curve; CRH: Corticotropin releasing hormone; HDDST: High dose dexamethasone suppression test; LR+: Likelihood ratio positive; NPV: Negative predictive value; PPV: Positive predictive value.

Serum 8 AM cortisol and relative percent cortisol suppression on HDDST showed poor accuracy, plasma 8 AM ACTH showed good accuracy and relative percent ACTH and cortisol rise on peripheral CRH stimulation showed excellent accuracy in discriminating between CD and EAS. The optimal cut-offs for diagnosis of CD in our cohort were plasma ACTH<97.3 pg/ml, HDDST relative cortisol suppression≥57%, peripheral CRH relative ACTH increase≥77% and relative cortisol increase≥11%.

Simple and non-invasive alternative models/strategies in differential diagnosis

We derived a total of six non-invasive models that provided 100% specificity and PPV for CD ([Table 4]). These models precluded the need for BIPPS in 35/85 (41.2%) patients with ACTH-dependent CS in whom the procedure would have otherwise been recommended (on account of absence of pituitary macroadenoma). Of these, the first three models included basic parameters other than peripheral CRH stimulation and precluded the need for both peripheral CRH stimulation and BIPSS in 19 (22.4%) participants. On the other hand, the last three models included peripheral CRH stimulation and precluded the need for BIPSS in another 16 (18.8%) participants.

Table 4 Non-invasive strategies/models for diagnosis of Cushing disease in study cohort (n=85, after excluding patients with pituitary macroadenoma).

Entry	Parameter used	Model/Strategy	PPV	N*	n*
1	Gender Serum Potassium Plasma ACTH HDDST	Female gender Absence of hypokalemia Plasma ACTH<97 pg/ml, and HDDST≥57% cortisol suppression	100%	15	15
2	Serum Potassium Plasma ACTH HDDST	Absence of hypokalemia Plasma ACTH<97 pg/ml, and HDDST>80% cortisol suppression	100%	13	3
3	Serum Potassium Plasma ACTH HDDST CEMRI pituitary	Absence of hypokalemia Plasma ACTH<97 pg/ml HDDST≥57% cortisol suppression, and Definitive lesion on CEMRI pituitary	100%	16	1
4	HDDST Peripheral CRH stimulation	HDDST:>80% cortisol suppression and Peripheral CRH stimulation:≥77% ACTH rise	100%	8	2
5	Peripheral CRH stimulation	Peripheral CRH stimulation:>100% ACTH rise	100%	19	11
6	Peripheral CRH stimulation	Peripheral CRH stimulation:≥11% cortisol rise	100%	25	3
Total	–	–	–	–	35/85 (41.2%)

ACTH: Adrenocorticotropic hormone; CEMRI: Contrast enhanced magnetic resonance imaging; CRH: Corticotropin releasing hormone; HDDST: High dose dexamethasone suppression test; PPV: Positive predictive value. ^*“N” is the total number of individuals with ACTH-dependent CS fulfilling all the variables in a given model and “n” is the number of individuals exclusive to that model. For example, 13 individuals (N) fulfilled all the variables in model no. 2, however, 10 of them also fulfilled all the variables in the preceding model (no. 1), and therefore, the number exclusive to this model was 3 (n). Similarly, 19 individuals (N) satisfied model no. 5, however, 8 of them also satisfied the preceding models (no. 1 to 4), and therefore, the number exclusive to this model was 11 (n).

Discussion

The differential diagnosis of ACTH-dependent CS is challenging, especially given that patients with benign EAS often have an indolent course, at times clinically and biochemically indistinguishable from CD. Pituitary imaging and endocrine dynamic tests are useful but have their own limitations and cannot be completely relied upon to differentiate between an ectopic and pituitary source [3] [4] [5]. Thus, BIPSS remains the most accurate procedure in the differential diagnosis and recommended in most patients except for those harboring a pituitary macroadenoma [6]. In this study, we evaluated the diagnostic performance of various tests in the differential diagnosis of ACTH-dependent CS and proposed simple and non-invasive strategies that preclude the need for more invasive and resource-intensive procedures such as BIPSS in a large proportion of patients. We found that peripheral CRH stimulation test (both relative percent ACTH and cortisol rise) provided excellent discrimination between EAS and CD and that the cortisol rise≥11% and the ACTH rise>100% individually excluded the possibility of EAS. We derived six non-invasive models that combined various demographic, clinical, biochemical, and imaging parameters and provided 100% specificity and PPV for CD, thus precluding the need for BIPSS in 41% participants in whom this procedure would have otherwise been recommended (on account of absence of a pituitary macroadenoma) [6]. Importantly, the first three models included basic clinical and biochemical parameters other than peripheral CRH stimulation test and precluded the need for BIPSS as well as CRH test in 22% participants.

Recently, Frete et al. in their cohort of 194 patients with ACTH-dependent CS (167 with CD and 27 with EAS) evaluated a strategy of performing intravenous human CRH and desmopressin stimulation tests (consecutively on different days and in a random order) alongside pituitary MRI in all patients, followed by CT scan of neck to pelvis where diagnosis of CD is not clear [15]. The authors reported 100% PPV for CD in patients with positive response to CRH (37% ACTH and 17% cortisol increase) and desmopressin (33% ACTH and 18% cortisol increase) tests with a negative pituitary MRI and a negative CT scan and 100% NPV for CD in patients with a negative response to CRH and desmopressin tests with a negative pituitary MRI and a positive CT scan. They concluded that using this approach, BIPSS could be potentially avoided in 53/112 (47%) patients where it would have been recommended. Similarly, in our cohort, using simple and non-invasive strategies that achieved 100% specificity and PPV for CD, BIPSS could be spared in 35/85 (41.2%) patients where it would have been recommended according to the current international consensus [6]. Furthermore, including 14 patients with pituitary macroadenoma who are presumed to be CD and do not require BIPSS, 49/99 patients (49.5%, i. e., nearly 1 in every 2 patients with ACTH-dependent CS) can be directly referred for a pituitary surgery. BIPSS is invasive, expensive, and only available at selected centers of excellence; the non-invasive strategy outlined here simplifies the diagnostic algorithm and has the potential to limit BIPSS to only a selected proportion of patients (nearly 1 in every 2 patients with ACTH-dependent CS). In a retrospective analysis of 264 patients with CD and 47 patients with EAS, Lyu et al., similarly reported a non-invasive scoring model (score ranging from –14 to 14) comprising of simple variables such as gender, plasma ACTH, MRI pituitary, HDDST and hypokalemia to minimize the need for BIPSS. The non-invasive model yielded a higher diagnostic accuracy than HDDST (AUC: 0.915 vs. 0.756) and scores of≥−10 and≥13 provided 100% sensitivity and specificity, respectively, for diagnosis of CD. The authors concluded that BIPSS may only be performed in patients with scores between –10 and 12, as scores below and above these were associated with a high probability of EAS and CD, respectively [16].

Recently, the availability of human CRH has also been a cause of major concern globally [11] [12]. Desmopressin has been suggested as an effective and less expensive alternative to CRH during BIPSS [17] [18]; however, peripheral desmopressin test has lower accuracy compared to CRH test in differentiating between CD and EAS [19] [20] [21]. To add, intravenous desmopressin is not available in India and other countries. We found that using a combination of basic parameters (other than peripheral CRH test; models 1–3), CD could be diagnosed with 100% specificity and PPV in 19/85 (22.4%) patients; such patients could not only be spared of BIPSS, but also of a peripheral CRH stimulation test. Furthermore, including 14 patients with pituitary macroadenoma who are presumed to be CD, 33/99 patients (33.3%, i. e., nearly 1 in every 3 patients with ACTH-dependent CS) can be directly referred for a pituitary surgery without a BIPSS and peripheral CRH test.

The value of HDDST in clinical practice for the differential diagnosis of ACTH-dependent CS has been debated [22] [23]. Nearly 20–30% patients with EAS and a similar proportion of patients with CD can present false positive and false negative cortisol suppression (>50%) on HDDST. Thus, the overall diagnostic accuracy of this test is 70–80%, lower than the pretest probability of 85–90% for CD in a patient with ACTH-dependent CS [4] [22]. We also noted that HDDST yielded poor accuracy (AUC: 0.702) in discriminating between EAS and CD. As expected, the higher cut-off>80% cortisol suppression (vs.>50%) yielded higher specificity (82% vs. 59%) for CD, but at the cost of lower sensitivity (41% vs. 70%) and the most optimal cut-off was derived at≥57% cortisol suppression (sensitivity 61%, specificity 59%). While the performance of HDDST as a standalone test was suboptimal, when used in certain models (models 1–4) in combination with various clinical, biochemical, and radiological variables, the test yielded 100% PPV for CD. In a recent study, Gupta et al. also highlighted the importance of using HDDST in their limited invasive protocols alongside MRI pituitary and CT imaging to avoid the need for BIPSS in 36–62% of patients with ACTH-dependent CS [24]. In our study, hypokalemia also served as an important discriminator between CD and EAS, especially in combination with other parameters (models 1–3). Owing to the higher severity of hypercortisolism, hypokalemia is more commonly reported in EAS (up to 85% of patients) than CD (up to 20% of patients) and is related to overwhelming of the 11-β-hydroxysteroid dehydrogenase type 2 enzyme by excess cortisol [13] [25]. This results in inadequate conversion of active cortisol into inactive cortisone and an exaggerated action of the former at mineralocorticoid receptor.

Peripheral CRH stimulation is useful in discriminating between the ectopic and pituitary forms, however, the results may be unreliable in 7–15% patients, most often due to false negative results in CD [4]. The sensitivity and specificity depend on the stimulating agent (ovine vs. human CRH) used and the cut-off criteria chosen; a range of 35–105% for the increase of ACTH and 14–50% for the increase of cortisol above basal levels has been used, resulting in a sensitivity of 70–93% and specificity of 95–100% for ACTH response, and 50–91% and 88–100% for cortisol response [4] [26]. Thus, several thresholds have been published for CRH test, but none is universal. In our study, peripheral CRH stimulation (using human CRH) was performed in 36 patients (30 CD and 6 EAS) and both ACTH (AUC: 0.933) and cortisol (AUC: 0.975) response yielded excellent discrimination between EAS and CD. The most optimal cut-off was derived at≥77% for ACTH (sensitivity 87%, specificity 83%) and≥11% for cortisol (sensitivity 97%, specificity 100%). The loss of specificity for ACTH response at this cut-off was accounted by a single patient in the EAS group, who demonstrated 97% ACTH rise following CRH stimulation. It is well known that some neuroendocrine tumors causing EAS express functional CRH receptors, resulting in false positive response to CRH stimulation [15] [27]. Our findings are consistent with a previous study by Newell-Price et al. that reported sensitivity and specificity estimates of 70% and 100%, respectively for>105% ACTH response, and 85% and 100%, respectively for>14% cortisol response following human CRH stimulation [28]. Recent studies, including the ones by Detomas et al. (≥31% ACTH rise, sensitivity, and specificity: 83% and 85%, respectively;≥12% cortisol rise: 82% and 89%, respectively) and Ceccato et al. (≥31% ACTH rise: 91% and 80%, respectively;≥20% cortisol rise: 86% and 80%, respectively) have demonstrated similar performance, albeit at different cut-offs [29] [30].

The strengths of our study include its relevance to both resource-rich and resource-limited settings in limiting the use of more invasive and resource-intensive procedures such as BIPSS and peripheral CRH stimulation. We acknowledge certain limitations. First, a major component of data collection was retrospective, with its inherent limitations. Second, we excluded patients with occult ACTH source from the present analysis; it will be of interest to re-evaluate the current strategies once the final diagnosis is available for these patients. Third, the results of peripheral CRH stimulation were available in only 36 patients. Considering that this test demonstrated excellent performance in differentiating between CD and EAS and precluded the need for BIPSS in a large proportion of patients (16/36) who took the test, a more uniform prescription could have potentially added to the numbers fulfilling the non-invasive strategy. Finally, the sample size was relatively small, and the study patients were derived from a single center, with implications for generalizability to other cohorts. We recognize that there is no one-size-fits-all approach to differential diagnosis of ACTH-dependent hypercortisolism and the diagnostic protocol may vary from one center to another depending upon several factors including the expertise and experience of the treating team, the availability of resources and the nature of patient presentation. Thus, similar non-invasive protocols should be evaluated and validated in other settings using these and other emerging modalities such as desmopressin stimulation test, and CRH-receptor targeted molecular imaging (68Ga-CRH-PET-CT) [31] [32]. Recent studies have also shown subtype-specific differences in hematological parameters between CD and EAS [33] [34]; further validation of these findings and incorporation in future non-invasive models would add value.

To conclude, using simple and non-invasive alternative strategies, BIPSS could be avoided in 41% and peripheral CRH stimulation in 22% of patients with ACTH-dependent CS and no macroadenoma, in whom these procedures would have otherwise been recommended. These strategies should be validated in other settings and the outcomes of treatment with the non-invasive vis-à-vis BIPSS-based invasive strategy should be evaluated in future prospective studies.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgement

The authors acknowledge residents and faculty from the Department of Endocrinology and Metabolism and other related departments for contributing to the clinical care of study patients.

• References

• 1 Nieman LK. Molecular derangements and the diagnosis of ACTH-dependent Cushing's syndrome. Endocr Rev 2022; 43: 852-877
• 2 Ferrante E, Barbot M, Serban AL. et al. Indication to dynamic and invasive testing in Cushing's disease according to different neuroradiological findings. J Endocrinol Invest 2022; 45: 629-637
• 3 Nieman LK, Biller BMK, Findling JW. et al. Treatment of Cushing’s syndrome: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2015; 100: 2807-2831
• 4 Hayes AR, Grossman AB. The ectopic adrenocorticotropic hormone syndrome: rarely easy, always challenging. Endocrinol Metab Clin North Am 2018; 47: 409-425
• 5 Zampetti B, Grossrubatscher E, Dalino Ciaramella P. et al. Bilateral inferior petrosal sinus sampling. Endocr Connect 2016; 5: R12-R25
• 6 Fleseriu M, Auchus R, Bancos I. et al. Consensus on diagnosis and management of Cushing's disease: a guideline update. Lancet Diabetes Endocrinol 2021; 9: 847-875
• 7 Ammini AC, Tandon N, Gupta N. et al. Etiology and clinical profile of patients with Cushing's syndrome: A single center experience. Indian J Endocrinol Metab 2014; 18: 99-105
• 8 Nishioka H, Yamada S. Cushing's disease. J Clin Med 2019; 8: 1951
• 9 Swearingen B, Katznelson L, Miller K. et al. Diagnostic errors after inferior petrosal sinus sampling. J Clin Endocrinol Metab 2004; 89: 3752-3763
• 10 Detomas M, Ritzel K, Nasi-Kordhishti I. et al. Bilateral inferior petrosal sinus sampling with human CRH stimulation in ACTH-dependent Cushing’s syndrome: results from a retrospective multicenter study. Eur J Endocrinol 2023; 188: 448-456
• 11 Ferring-CRH GHRH. Published 2022. https:// www. ese-hormones.org/ media/ 4911/ ferring-pharmaceuticals-response- crh-ghrh-december- 2022
• 12 Ceccato F, Barbot M, Ceccato F. et al. Shortage of hCRH for the diagnosis of endogenous CS: the end of an era or the beginning of a new journey?. J Endocrinol Invest 2023; 46: 2189-2191
• 13 Attri B, Goyal A, Kalaivani M. et al. Clinical profile and treatment outcomes of patients with ectopic ACTH syndrome compared to Cushing disease: a single-center experience. Endocrine 2023; 80: 408-418
• 14 Meier CA, Biller BM. Clinical and biochemical evaluation of Cushing's syndrome. Endocrinol Metab Clin North Am 1997; 26: 741-762
• 15 Frete C, Corcuff JB, Kuhn E. et al. Non-invasive diagnostic strategy in ACTH-dependent Cushing's syndrome. J Clin Endocrinol Metab 2020; 105: dgaa409
• 16 Lyu X, Zhang D, Pan H. et al. A noninvasive scoring model for the differential diagnosis of ACTH-dependent Cushing's syndrome: a retrospective analysis of 311 patients based on easy-to-use parameters. Endocrine 2022; 78: 114-122
• 17 Valizadeh M, Ahmadi AR, Ebadinejad A. et al. Diagnostic accuracy of bilateral inferior petrosal sinus sampling using desmopressin or corticotropic- releasing hormone in ACTH-dependent Cushing's syndrome: a systematic review and meta-analysis. Rev Endocr Metab Disord 2022; 23: 881-892
• 18 Chen S, Chen K, Wang S. et al. The optimal cut-off of BIPSS in differential diagnosis of ACTH-dependent Cushing's syndrome: is stimulation necessary?. J Clin Endocrinol Metab 2020; 105: dgz194
• 19 Ceccato F, Barbot M, Mondin A. et al. Dynamic testing for differential diagnosis of ACTH-dependent Cushing syndrome: a systematic review and meta-analysis. J Clin Endocrinol Metab 2023; 108: e178-e188
• 20 Vassiliadi DA, Tsagarakis S. Diagnosis of endocrine disease: the role of the desmopressin test in the diagnosis and follow-up of Cushing's syndrome. Eur J Endocrinol 2018; 178: R201-R214
• 21 Terzolo M, Reimondo G, Alì A. et al. The limited value of the desmopressin test in the diagnostic approach to Cushing's syndrome. Clin Endocrinol (Oxf) 2001; 54: 609-616
• 22 Aron DC, Raff H, Findling JW. Effectiveness versus efficacy: the limited value in clinical practice of high dose dexamethasone suppression testing in the differential diagnosis of adrenocorticotropin-dependent Cushing's syndrome. J Clin Endocrinol Metab 1997; 82: 1780-1785
• 23 Lila AR, Sarathi V, Jagtap VS. et al. Cushing's syndrome: Stepwise approach to diagnosis. Indian J Endocrinol Metab 2011; 15: S317-S321
• 24 Gupta R, Walia R, Ahuja C. et al. Limited invasive protocol: optimizing diagnostic modalities in corticotropin mediated Cushing syndrome. Endocr Pract 2022; 28: 767-773
• 25 Young J, Haissaguerre M, Viera-Pinto O. et al. Management of endocrine disease: Cushing's syndrome due to ectopic ACTH secretion: an expert operational opinion. Eur J Endocrinol 2020; 182: R29-R58
• 26 Newell- Price J, Trainer P, Besser M. et al. The diagnosis and differential diagnosis of Cushing’s syndrome and pseudo-Cushing’s states. Endocr Rev 1998; 19: 647-672
• 27 de Keyzer Y, Lenne F, Auzan C. et al. The pituitary V3 vasopressin receptor and the corticotroph phenotype in ectopic ACTH syndrome. J Clin Invest 1996; 97: 1311-1318
• 28 Newell-Price J, Morris DG, Drake WM. et al. Optimal response criteria for the human CRH test in the differential diagnosis of ACTH-dependent Cushing’s syndrome. J Clin Endocrinol Metab 2002; 87: 1640-1645
• 29 Detomas M, Ritzel K, Nasi-Kordhishti I. et al. Outcome of CRH stimulation test and overnight 8 mg dexamethasone suppression test in 469 patients with ACTH-dependent Cushing’s syndrome. Front Endocrinol 2022; 13: 955945
• 30 Ceccato F, Tizianel I, Vedolin CK. et al. Human corticotropin-releasing hormone tests: 10 years of real-life experience in pituitary and adrenal disease. J Clin Endocrinol Metab 2020; 105: dgaa564
• 31 Wright K, van Rossum EFC, Zan E. et al. Emerging diagnostic methods and imaging modalities in cushing's syndrome. Front Endocrinol (Lausanne) 2023; 14: 1230447
• 32 Walia R, Gupta R, Bhansali A. et al. Molecular imaging targeting corticotropin-releasing hormone receptor for corticotropinoma: a changing paradigm. J Clin Endocrinol Metab 2021; 106: e1816-e1826
• 33 Detomas M, Deutschbein T, Tamburello M. et al. Erythropoiesis in Cushing syndrome: sex-related and subtype-specific differences. Results from a monocentric study. J Endocrinol Invest 2023;
• 34 Detomas M, Altieri B, Chifu I. et al. Subtype-specific pattern of white blood cell differential in endogenous hypercortisolism. Eur J Endocrinol 2022; 187: 439-449

Publikationsverlauf

Eingereicht: 13. November 2023

Angenommen nach Revision: 28. Dezember 2023

Accepted Manuscript online:
28. Dezember 2023

Artikel online veröffentlicht:
16. Februar 2024