Clinical Utility of Plasma POMC and AgRP Measurements in the Differential Diagnosis of ACTH-Dependent Cushing's Syndrome

Abstract

Context:

Distinguishing between pituitary [Cushing's disease (CD)] and ectopic causes [ectopic ACTH syndrome (EAS)] of ACTH-dependent Cushing's syndrome can be challenging. Inferior petrosal sinus sampling (IPSS) best discriminates between CD and occult EAS but is a specialized procedure that is not widely available. Identifying adjunctive diagnostic tests may prove useful. In EAS, abnormal processing of the ACTH precursor proopiomelanocortin (POMC) and the accumulation of POMC-derived peptides might be expected and abnormal levels of other neuropeptides may be detected.

Objective:

The objective of the study was to evaluate the diagnostic utility of POMC measurements for distinguishing between CD and occult EAS in patients referred for IPSS. Another objective of the study was to evaluate in parallel the diagnostic utility of another neuropeptide, agouti-related protein (AgRP), because we have observed a 10-fold elevation of AgRP in plasma in a patient with EAS from small-cell lung cancer.

Design and Participants:

Plasma POMC and AgRP were measured in 38 Cushing's syndrome patients presenting for IPSS, with either no pituitary lesion or a microadenoma on magnetic resonance imaging, and in 38 healthy controls.

Results:

Twenty-seven of 38 patients had CD; 11 of 38 had EAS. The mean POMC was higher in EAS vs CD [54.5 ± 13.0 (SEM) vs 17.2 ± 1.5 fmol/mL; P < .05]. Mean AgRP was higher in EAS vs CD (280 ± 76 vs 120 ± 16 pg/mL; P = .01). Although there was an overlap in POMC and AgRP levels between the groups, the POMC levels greater than 36 fmol/mL (n = 7) and AgRP levels greater than 280 pg/mL (n = 3) were specific for EAS. When used together, POMC greater than 36 fmol/mL and/or AgRP greater than 280 pg/mL detected 9 of 11 cases of EAS, indicating that elevations in these peptides have a high positive predictive value for occult EAS.

Conclusions:

Expanding upon previous observations of high POMC in EAS, this study specifically demonstrates elevated POMC levels can identify occult ectopic tumors. Elevations in AgRP also favor the diagnosis of EAS, suggesting AgRP should be further evaluated as a potential neuroendocrine tumor marker.

ACTH-dependent Cushing's syndrome (CS) is characterized by ACTH excess, arising from tumors of the pituitary, ectopic tumors like small-cell lung carcinomas, medullary thyroid carcinomas, and pheochromocytomas as well as thymic, pancreatic, and bronchial carcinoids, which are often indolent (1). Although cases of ectopic ACTH syndrome (EAS) associated with overt malignancies and cases of Cushing's disease (CD) secondary to large ACTH-secreting pituitary tumors rarely pose diagnostic dilemmas, distinguishing between pituitary and occult ectopic ACTH-secreting tumors can sometimes be quite challenging. Occult EAS refers to ACTH-dependent CS of nonpituitary origin, present for longer than 6 months' duration without the emergence of an obvious source (2). Often the culprit tumors are small and difficult to locate, and similarities in the clinical and biochemical presentations of occult EAS and CD further complicate the differential diagnosis. Traditional diagnostic biochemical tests, including the high-dose dexamethasone suppression and the CRH stimulation test, lack sensitivity and specificity (3–5), and only 50% of the pituitary tumors that cause CD are detected by magnetic resonance imaging (MRI) (6). Although inferior petrosal sinus sampling (IPSS) is the gold standard test for distinguishing between pituitary and ectopic ACTH-secreting tumors, this invasive and technically rigorous procedure is not widely available and false-positive and –negative results have been described (7–9). Given these limitations, additional diagnostic tools that capitalize on tumor-specific differences in peptide processing and synthesis may prove clinically useful.

ACTH derives from the 31-kDa precursor, proopiomelanocortin (POMC). In the anterior pituitary, POMC is enzymatically cleaved at the C terminal of ACTH by prohormone convertase 1 (PC1) to give rise to β-lipotrophin and pro-ACTH. Pro-ACTH is further cleaved by PC1 into N-terminal POMC, joining peptide, and ACTH (10). In overt EAS, early chromatographic studies demonstrated high concentrations of ACTH precursors in plasma (11–14), and subsequent studies using immunoradiometric assays to directly measure plasma ACTH precursors have confirmed that elevations in POMC levels are commonly associated with clinically obvious EAS, in contrast to pituitary Cushing's (13, 15, 16). The accumulation of these precursors in EAS has been shown to result from aberrant POMC processing suggested by the presence of markedly elevated ACTH precursor to ACTH ratios in many ectopic tumors (13, 16, 17). Although elevated plasma POMC levels have also been observed in some cases of CD caused by pituitary macroadenomas, they are rarely associated with ACTH-secreting pituitary microadenomas because these traditionally well-differentiated tumors have been shown to process the peptide precursor more effectively, like the corticotrophs of the anterior pituitary (12, 14–16, 18–20). These observations suggest that the clinical utility of plasma POMC measurements may lay in their potential ability to identify occult ectopic tumors in diagnostically challenging cases of ACTH-dependent CS, such as those referred for IPSS. Although Oliver et al (21) prospectively compared the accuracy of POMC measurements to IPSS in a cohort of CS patients, reporting a high sensitivity and specificity, given there were only four ectopic tumors in the cohort, a more rigorous evaluation of the utility of this marker in this important population is clearly indicated.

In addition to POMC, the identification of other tumor markers that are differentially expressed by ectopic and pituitary tumors could further facilitate the differential diagnosis of ACTH-dependent CS. Agouti-related protein (AgRP) may be one such tumor marker. We have observed, for the first time, very high levels of AgRP in a case of EAS from small-cell lung cancer. Although AgRP is most well known as a hypothalamic neuropeptide that regulates energy balance, AgRP expression has also been detected in the lungs, adrenals, kidneys, and gonads and so may be synthesized by neuroendocrine tumors originating from these tissues (22). We have therefore analyzed AgRP levels, in parallel with POMC, in peripheral plasma samples from CS patients referred for IPSS to evaluate the utility of these measurements in the differential diagnosis of ACTH-dependent CS.

Subjects and Methods

Subjects

We studied 38 patients over the age of 18 years with ACTH-dependent CS, recruited from the Neuroendocrine Unit at Columbia University (Table 1). All patients had active hypercortisolism and plasma ACTH levels greater than 9 pg/mL and were being referred for IPSS (23). Fifteen of the 38 patients had a microadenoma of 7 mm or less on MRI, whereas 23 had no pituitary tumor on MRI. Diagnostic classifications for patients with CS were based on surgical pathology, surgical cure, and/or IPSS results in which a central to peripheral plasma ACTH gradient of 2:1 or greater before CRH administration or 3:1 or greater after CRH was considered diagnostic of a pituitary ACTH source. Twenty-seven patients aged 42.9 ± 3.5 years had CD based on the IPSS results. Eighteen of 27 were women; 9 of 27 were men. All underwent transsphenoidal surgery and achieved initial surgical cure. Eleven of the 38 patients aged 46.7 ± 3.5 years had EAS based on IPSS results. Six of 11 underwent curative resection of bronchial carcinoids. One of 11 had an ACTH-secreting adrenal neuroendocrine tumor removed. In 4 of 11 cases of EAS, the tumor remained occult. Thirty-eight healthy subjects over the age of 18 years recruited from Columbia University and the surrounding community were evaluated for comparison. Healthy subjects had no history of depression, anxiety, or significant alcohol intake and were on no medications. Individuals using oral or transdermal contraceptives or progesterone-containing intrauterine devices were excluded. Premenopausal subjects had regular menstrual cycles. Half of the healthy volunteers were men and half were women. This study was approved by the Columbia University Institutional Review Board and appropriate written informed consent was obtained.

Table 1.

Subject Characteristics

Variable	CD (n = 27)	EAS (n = 11)	Normal Subjects (n = 38)
Age, (y)	42.9 ± 3.5	46.7 ± 3.5	33.7 ± 1.6
Sex
Male, %	33%	64%	50%
Female, %	67%	36%	50%
MRI
Visible microadenoma	52% (14/27)	9% (1/11)	N/A
No microadenoma	48% (13/27)	91% (10/11)	N/A

Abbreviation: N/A, not available. Age is reported as mean ± SE.

Protocol

Peripheral EDTA plasma samples were collected from all participants. All patient blood samples were collected prior to or at the time of IPSS. Blood samples from healthy volunteers were collected the morning after an overnight fast, in the early follicular phase of the menstrual cycle where applicable. POMC and ACTH levels were measured in peripheral plasma samples from all 38 patients. AgRP levels were measured in plasma samples from 24 of 38 patients, specifically in 10 of 11 cases of EAS. Plasma samples from each healthy volunteer were assayed for POMC, ACTH, and AgRP for comparison.

Assays

POMC was assayed using an in-house, two-site ELISA using antibodies that were produced and characterized as previously described (24, 25). The capture monoclonal antibody is directed against ACTH (10–18) and the detection antibody is directed against γ-MSH. There is 100% cross-reactivity with 22K pro-ACTH. There is no cross-reactivity with ACTH, α-MSH, γ-MSH, or β-EP (15). Affinity purified human 31K POMC was used for standards (26). Assay sensitivity is 7 fmol/mL. AgRP was assayed by ELISA (R&D Systems) using a recombinant full-length human AgRP standard. There is 17% cross-reactivity with AgRP (83–132). Assay sensitivity is 4 pg/mL. ACTH was assayed using a solid-phase, two-site sequential chemiluminescent immunometric assay using the Immulite 1000 immunoassay analyzer (Siemens). Assay sensitivity is 9 pg/mL.

Characterization of POMC and AgRP immunoactivity in plasma

POMC immunoactivity was characterized by gel filtration in plasma from a patient with an aggressive ACTH-secreting pituitary macroadenoma. Plasma was chromatographed on a Sephadex G-75 (superfine) column in 0.04 N HCl and 0.1% BSA and fractions were assayed for POMC by ELISA. The column was calibrated with affinity purified 31K human POMC standard and ACTH standard. All of the detected POMC immunoactivity eluted in two peaks in the position of POMC and pro-ACTH (Figure 1A).

Figure 1.

A, G-75 Sephadex chromatography of a plasma sample from a patient with a large ACTH-secreting macroadenoma. Fractions were assayed for POMC by an ELISA. Most immunoactivity was in the position of affinity-purified 31K POMC and pro-ACTH. B, Characterization of AgRP in the plasma of a patient with EAS and small-cell lung carcinoma by HPLC. Fractions were assayed for AgRP by an ELISA. Most of the immunoactivity eluted in the position of full-length AgRP.

AgRP immunoactivity was characterized by HPLC as reported previously (27, 28) in plasma from a patient with EAS (Figure 1B). Plasma was extracted using a Sep-Pak C18 cartridge and was then subjected to reverse-phase HPLC with a gradient of 80% acetonitrile containing 0.1% trifluoroacetic acid, and fractions were assayed for AgRP by ELISA. The columns were calibrated with 5 ng of human AgRP (83–132) (Phoenix Pharmaceuticals Inc) and 5 ng of human full-length AgRP (R&D Systems). Most immunoactivity eluted in the position of full-length AgRP (Figure 1B).

Data capture and statistical analysis

Age, gender, hormone, and peptide concentrations, size of MRI lesions, and histology were recorded for all patients. Age, gender, and peptide concentrations were recorded for all healthy volunteers. Values of POMC and AgRP below the limit of detection were analyzed as the numerical value of the limit. Means are reported ± SEM throughout. Differences in POMC and AgRP between groups were compared using the Wilcoxon rank-sum test and the diagnostic cutoff levels for POMC and AgRP that optimally separate CD from EAS were identified. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of these markers for the diagnosis of EAS were calculated.

Results

POMC

POMC levels among normal healthy individuals ranged from less than 7.0 to 32.0 fmol/mL. POMC levels in the 38 CS patients presenting for IPSS are shown in Figure 2A. Mean POMC levels were significantly higher in EAS compared with CD [54.5 ± 13.0 (SEM) vs 17.2 ± 1.5 fmol/mL] and healthy controls (13.6 ± 0.87 fmol/mL; P < .0001). All patients with a POMC level greater than 36 fmol/mL had EAS (n = 7) (Figure 2C): five of seven identified had bronchial carcinoids and two of seven had occult tumors (Table 2). ACTH levels ranged from 10 to 111 pg/mL in patients with CD (41.1 ± 4.93 pg/mL) compared with 39–240 pg/mL in patients with EAS (125 ± 19.9 pg/mL), and there was significant overlap in ACTH levels between the two groups.

Figure 2.

Mean POMC (A) and AgRP (B) levels in patients with CD, EAS, and normal controls (*, P < .05 and **, P = .01) and scatterplots of POMC (C) and AGRP (D) levels in patients with CD (▾), EAS (●), and normal controls (■). The dashed horizontal lines represent the diagnostic cutoff values, above which patients with EAS are identified.

Table 2.

Tumor Type and Biomarker Level in Studied EAS Cases

EAS Case Number	Ectopic Tumor Type	POMC, fmol/mL	AgRP, pg/mL
1	Bronchial carcinoid	7.0	118
2	Bronchial carcinoid	132	168
3	Bronchial carcinoid	107	QNS
4	Bronchial carcinoid	57	154
5	Bronchial carcinoid	78	171
6	Bronchial carcinoid	84	150
7	Occult tumor	39	119
8	Occult tumor	64	479
9	Occult tumor	9.5	445
10	Occult tumor	7.0	140
11	Neuroendocrine adrenal tumora	14	855

Abbreviation: QNS, quantity not sufficient for assaying. Bold print indicates biomarker value above diagnostic cutoff: POMC greater than 36 fmol/mL and AgRP greater than 280 pg/mL.

^aTumor specimen not available for pathological confirmation of tumor type.

AgRP: novel tumor marker

We observed, for the first time, marked plasma elevations of the neuropeptide AgRP (3005 pg/mL; 42–118 pg/mL, normal range) in a case of EAS secondary to metastatic small-cell lung carcinoma. In the cohort of CS patients subsequently evaluated, AgRP levels ranged from 118 to 855 pg/mL among patients with EAS and from 51 to 280 pg/mL among patients with CD. Although there was significant overlap, AgRP levels greater than 280 pg/mL identified 3 of 10 cases: one adrenal neuroendocrine tumor and two occult tumors (Figure 2D and Table 3). Additionally, the mean AgRP levels were significantly higher in EAS compared with CD (280 ± 76 vs 120 ± 16 pg/mL; P = .01) (Figure 2B). The mean AgRP levels were also higher in CD compared with normal subjects (73.6 ± 3.1 pg/mL; P = .02) (Figure 2D). We went on to measure AgRP levels in CD IPSS samples to evaluate for a potential pituitary AgRP source, but we did not observe a central to peripheral AgRP gradient (data not shown).

Table 3.

Plasma POMC Levels and Clinical Characteristics of Two Representative Cases of CS From This Cohort With Similar Presentations

	CD	EAS (Table 2, Case 2)
Patient information	31-year-old male	29-year-old male
Symptoms/signs	Weight gain, bruisability, striae	Puffiness, leg weakness, striae
Morning cortisol	30 μg/dL	26.9 μg/dL
UFC	1460 μg per 24 h	632 μg per 24 h
MRI	No pituitary tumor	No pituitary tumor
ACTH	42 pg/mL	55 pg/mL
POMC	22 fmol/mL	132 fmol/mL
IPSS results	Central-to-peripheral gradient > 3:1	No central-to-peripheral gradient
Diagnosis	CD	Bronchial carcinoid

Abbreviation: UFC, urinary free cortisol.

The tumor-specific origin of AgRP in EAS was supported by the measurement of AgRP levels prior to and after bilateral adrenalectomy in a patient with EAS, which was ultimately thought to be secondary to an adrenal neuroendocrine tumor (Table 2, case 11). Bilateral adrenalectomy was pursued to control cortisol levels. Prior to surgery the plasma AgRP was markedly elevated at 855 pg/mL and the ACTH level was 195 pg/mL. Postoperatively, AgRP concentrations fell to 45 pg/mL, and the ACTH level became undetectable. Given adrenalectomy in patients with ACTH-secreting tumors typically has either no impact or a stimulatory impact on ACTH levels (29, 30), this case suggests that the source of ectopic ACTH and AgRP secretion was likely an adrenal neuroendocrine tumor that was serendipitously removed at time of adrenalectomy. Surgery was performed at an outside hospital, and the tumor specimen was not available for pathological confirmation of ACTH or AgRP immunoactivity. When bilateral adrenalectomy was performed to control cortisol levels in a second patient in this cohort with occult EAS (Table 2, case 8), ACTH and AgRP levels remained elevated postoperatively and the tumor remained occult.

Tumor marker sensitivity, specificity, PPV, and NPV for the diagnosis of EAS

The sensitivity, specificity, PPV, and NPV were greatest when AgRP and POMC were used in conjunction as diagnostic markers because 9 of 11 patients with EAS were identified if either POMC and/or AgRP were elevated above the deemed diagnostic cutoff values (>36 fmol/mL and > 280 pg/mL, respectively), whereas the criteria excluded all 27 cases of CD, yielding a sensitivity and specificity of 82% [48–97, 95% confidence interval (CI)] and 100% (83–100, 95% CI) respectively, a PPV of 100%, and a NPV of 93%. Among the two EAS cases that were not identified by elevated biomarkers, one was a well-differentiated bronchial carcinoid and one was an occult tumor. The sensitivity for the diagnosis of EAS decreased markedly when two elevated biomarkers were required. When evaluated alone, POMC elevations above a diagnostic cutoff level of 36 fmol/mL had the best diagnostic accuracy, identifying 7 of 11 of the cases of EAS, corresponding to a sensitivity and specificity of 64% (32–88, 95% CI) and 100% (83–100, 95% CI), respectively. AgRP elevations identified 3 of 10 cases of EAS, but this marker may have tissue specificity.

Discussion

The differential diagnosis of ACTH-dependent CS remains one of the more challenging issues in clinical endocrinology. Although EAS can occur secondary to overt malignancies like small-cell lung cancer, it is commonly associated with occult neoplasms and indolent carcinoids, whose clinical features often overlap with those of ACTH-secreting pituitary tumors. Using highly sensitive assays, we have demonstrated the diagnostic value of plasma POMC and AgRP measurements for distinguishing between CD and occult EAS specifically in clinically challenging cases in which there is either no tumor or a small microadenoma on pituitary MRI and IPSS is indicated. In this study, POMC greater than 36 fmol/ml and/or AgRP greater than 280 pg/mL identified 9 of 11 cases of EAS, indicating that although POMC and AgRP levels overlap among those with CS, elevations in these peptides above a diagnostic cutoff have a high PPV for EAS.

POMC elevations alone identified 7 of 11 of the occult ectopic tumors in this unique cohort of EAS patients. This finding is noteworthy, given the marked elevations in ACTH precursors and plasma POMC in EAS that have previously been described in the literature have overwhelmingly been in cases of clinically obvious ectopic tumors (11–16). It has even been suggested that POMC measurements have little diagnostic utility in the case of typical bronchial carcinoids, the most frequent cause of EAS (16). In contrast, we demonstrate plasma POMC elevations in five typical bronchial carcinoids and two occult tumors (cases 2–8). Our findings are consistent with those of Oliver et al (21), who described elevations in ACTH precursors in four case of EAS from nonmalignant carcinoids among a CS cohort presenting for IPSS.

Yet although we have shown that plasma POMC elevations have a high specificity for EAS (96%–100%), our findings suggest that POMC measurements have a limited sensitivity. The secretion of intact ACTH precursors was not a consistent finding in our ectopic cohort, and four of 11 of the ectopic tumors (Table 2, cases 1 and 9–11) had frankly low POMC levels (ranging from 7.0 to 14.0 fmol/mL) that were comparable with the concentrations seen among CD cases and normal controls. The identification of low POMC levels in this subset of EAS cases parallels the findings of Raffin-Sanson et al (16), who observed undetectable POMC levels in all four patients with typical bronchial carcinoids in their EAS cohort. However, it should be noted that in contrast to our two-site ELISA, their group used a POMC IRMA that detected POMC alone (not pro-ACTH), which may not have been sensitive enough to measure subtle, but potentially diagnostically useful, elevations in ACTH precursors. Nonetheless, it is clear that not all well-differentiated ectopic ACTH secreting tumors underprocess POMC. In fact, some bronchial carcinoid tumors undergo a process of differentiation that mirrors that of normal corticotrophic cells, expressing the POMC gene and processing POMC effectively (31). Other carcinoid tumors have been found to exhibit subtle processing aberrations characterized by the secretion of small peptides, like corticotrophin-like intermediary lobe peptide and human β-MSH_5–22 (32), possibly resulting from the expression of enzymes not typically found in the anterior pituitary (prohormone convertase 2) (33).

The observed peptide heterogeneity in EAS reflects the breadth of the histologic and clinical behaviors that characterize the array of tumors that give rise to the condition. Clearly, even among carcinoids and other occult ectopics, there can be marked variability in the degree of neuroendocrine differentiation and in their subsequent clinical behavior (16, 31). Regardless of the peptide heterogeneity that characterizes subtle cases of EAS, the ability of our highly sensitive two-site ACTH precursor ELISA to identify most bronchial carcinoids and other occult tumors in this EAS cohort is important, given these tumors commonly mimic CD and often elude detection. In the evaluated CS cohort, the clinical utility of POMC measurements were well illustrated by two cases of young men with CS and markedly similar clinical and biochemical presentations who were clearly distinguished by POMC measurements alone (Table 3). Although we believe that our POMC findings alone support further consideration of the clinical application of this assay to the differential diagnosis of ACTH-dependent CS, the identification of other peripheral neuroendocrine markers that can be measured concomitantly may enhance the detection of occult ectopic ACTH-secreting tumors.

Toward this end, we measured AgRP levels in our cohort as well, demonstrating, for the first time higher mean AgRP levels in EAS compared with CD. Despite the overlap between the two groups, we observed elevated plasma AgRP in several cases of EAS. AgRP levels greater than a diagnostic cutoff level of 280 pg/mL independently identified 3 of 10 cases of EAS. Of the three cases identified, one tumor was an ACTH-secreting adrenal neuroendocrine tumor, and the other two were ectopic tumors that remain occult. When used in combination, AgRP greater than 280 pg/mL and/or POMC levels greater than 36 fmol/mL identified 9 of 11 cases of EAS. It is important to note that an AgRP greater than 280 pg/mL identified two of three ectopic tumors that would have been diagnostically missed had we measured POMC levels alone (Table 2). Interestingly, only one ectopic ACTH-secreting tumor had both a high AgRP level and a high POMC level (Table 2, case 8).

To date, we have detected marked AgRP elevations (>280 pg/mL) in tumors of adrenal, lung (small cell lung cancer), and occult origin. Although the concentration of AgRP mRNA is known to be highest in the hypothalamus, in humans the second highest concentration of AgRP mRNA is detected in the adrenal glands followed by the lungs (22). Although AgRP levels do not appear to be as sensitive as POMC levels for the identification of EAS, this marker could be specific for certain tumor types, although further investigation is needed. The reason for the observed differences in AgRP concentrations among those with CD and those with EAS has not been definitively established, but it appears that some ectopic tumors make AgRP. Although future histological studies confirming AgRP immunoactivity in ACTH-secreting ectopic tumors associated with high plasma AgRP levels are needed, the pronounced decline in plasma AgRP levels from 855 pg/mL to 45 pg/mL in parallel with a fall in ACTH levels after the resection of the ACTH-secreting adrenal neuroendocrine tumor in case 11 (Table 2) supports a tumor-derived AgRP source. It remains to be determined whether there are differences in AgRP processing in ectopic tumors that secrete AgRP. It should be noted that although C-terminal AgRP 83–132 is the predominant form in the brain (28), it is primarily the full-length peptide that we found to be elevated when we performed HPLC on a patient with EAS from a small-cell lung carcinoma (Figure 1B). It is of interest that AgRP, like POMC, is posttranslationally cleaved by PC1 to generate AgRP 83–132 (34). It is currently unknown whether there are differences in AgRP processing that are affected not only by tumor type and the degree of tumor cell differentiation but also by the absence of certain processing enzymes.

Although the primary purpose of the study was to evaluate the use of these biomarkers for distinguishing between EAS and CD, we found that AgRP levels were significantly higher in CD than in normal volunteers. Overstimulation of the adrenal glands in ACTH-dependent CS may mediate this particular finding. It has been previously suggested that the adrenal glands are a source of elevated plasma AgRP in states of cortisol excess, and glucocorticoid stimulation has been shown to stimulate adrenal AgRP mRNA expression in rats (35–38). This observation is worthy of future exploration because it could also have diagnostic implications.

Overall, our data support the clinical use of plasma POMC and AgRP measurements in the differential diagnosis of ACTH-dependent CS in clinically challenging cases presenting for IPSS. Although we acknowledge that abnormal POMC processing and AgRP expression are not constant features of occult EAS, significant elevations in either POMC and/or AgRP have a high specificity for EAS and can provide useful diagnostic information. The potential utility of these biomarkers becomes evident when considered in the context of the limitations of other available dynamic noninvasive tests for the differential diagnosis of ACTH-dependent CS, namely the CRH test and the high-dose dexamethasone suppression test (HDDST). Even when the most rigid discriminating criteria are used, up to 14% of CD patients fail to respond as expected to CRH stimulation (39), and although long considered the biochemical test of choice, the shortcomings of the HDDST, particularly for distinguishing between CD and occult ectopics, are now better understood. In a series of 41 CS patients evaluated at the National Institutes of Health, Dichek et al (40) demonstrated that both the standard HDDST and the overnight 8-mg HDDST have limited diagnostic accuracy, with the more commonly used 8-mg test exhibiting a sensitivity and specificity of 88% and 57%, respectively. Accordingly, in a larger cohort of CS patients, in which most of those with EAS had bronchial carcinoids, there was extensive overlap in the response to HDDST between the two groups. The reported overall sensitivity and specificity among the 73 patients tested were 81% and 66%, respectively (lower than the pretest probability of CD), leading the authors to question the grounds for the continued clinical use of this test (3).

Although we recognize the limited sensitivity of POMC and AgRP measurements, the specificity of these markers for occult EAS may provide clinically useful information. Although larger studies are needed to determine the most appropriate place for these markers in the diagnostic algorithm, there are several contexts in which they could provide benefit. Given the technical difficulty and limited availability of the diagnostic gold standard, IPSS, and the high pretest probability of CD, neurosurgery is sometimes pursued in cases of ACTH-dependent CS, even when a definitive diagnosis has not been made. Our clinical experience suggests that in such cases, the preoperative measurement of POMC and AgRP may prevent unnecessary neurosurgery because elevations in these biomarkers may motivate endocrinologists to pursue IPSS, even when it is available only remotely. Similarly, in diagnostically challenging cases in which IPSS is desired but contraindicated, elevated POMC and/or AgRP levels may lead clinicians to consider advanced imaging techniques to identify potential ectopic tumors before pursuing neurosurgery. Lastly, in cases in which traditional biochemical tests are inconclusive, these biomarkers could provide additional information to facilitate clinical decision making about who should be referred for IPSS.