ENDOCRINE SOCIETY
OXFORD
Clinical Research Article
Plasma Steroid Profiling in Patients With Adrenal Incidentaloma
Kristina Berke, 1 Georgiana Constantinescu,1 Jimmy Masjkur,1 Otilia Kimpel,2 Ulrich Dischinger,2 Mirko Peitzsch,3 Aleksandra Kwapiszewska,4 Piotr Dobrowolski,4 Svenja Nolting,5,6 Martin Reincke,6 Felix Beuschlein,5,6 Stefan R. Bornstein,1 Aleksander Prejbisz,4 Jacques W. M. Lenders,1,7 Martin Fassnacht2 and Graeme Eisenhofer1,3
1Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany; 2Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Würzburg, 97082 Würzburg, Germany; 3Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, 01307 Dresden, Germany; 4Department of Hypertension, National Institute of Cardiology, 04-828 Warsaw, Poland; 5Department of Endocrinology, Diabetology and Clinical Nutrition, University Hospital Zurich (USZ) and University of Zurich (UZH), 8057 Zurich, Switzerland; 6Department of Medicine IV, University Hospital, Ludwig Maximilian University of Munich, 80539 Munich, Germany; and 1Department of Internal Medicine, Radboud University Medical Center, 6500 HB Nijmegen, the Netherlands
ORCID numbers: 0000-0002-7304-2557 (G. Constantinescu); 0000-0002-8283-7861 (0. Kimpel); 0000-0002-2472-675X (M. Peitzsch); 0000-0002-9817-9875 (M. Reincke); 0000-0001-7826-3984 (F. Beuschlein); 0000-0001-7085-0244 (A. Prejbisz); 0000- 0001-6170-6398 (M. Fassnacht); 0000-0002-8601-9903 (G. Eisenhofer).
Abbreviations: ACC, adrenocortical carcinoma; ACS, autonomous cortisol secretion; CT, computed tomography; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone-sulfate; DST, dexamethasone suppression test; ENS@T, European Network for the Study of Adrenal Tumors; LC-MS/MS, liquid chromatography with tandem mass spectrometry; NFAI, nonfunctional adrenal incidentaloma; NPV, negative predictive value; PA, primary aldosteronism; PHEO, pheochromocytoma, PMT, Prospective Monoamine-producing Tumor study; PPV, positive predictive value; PROSALDO, PROspective study on the diagnostic value of Steroid profiling in primary ALDOsteronism; ROC, receiver operating characteristic.
Received: 8 July 2021; Editorial Decision: 11 October 2021; First Published Online: 19 October 2021; Corrected and Typeset: 6 November 2021.
Abstract
Context: Most patients with adrenal incidentaloma have nonfunctional lesions that do not require treatment, while others have functional or malignant tumors that require intervention. The plasma steroid metabolome may be useful to assess therapeutic need. Objective: This work aimed to establish the utility of plasma steroid profiling combined with metanephrines and adrenal tumor size for the differential diagnosis of patients with adrenal incidentaloma.
ISSN Print 0021-972X ISSN Online 1945-7197
@ The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved.
Methods: This retrospective cross-sectional study, which took place at 7 European tertiary- care centers, comprised 577 patients with adrenal incidentaloma, including 19, 77, 65, 104 and 312 respective patients with adrenocortical carcinoma (ACC), pheochromocytoma (PHEO), primary aldosteronism (PA), autonomous cortisol secretion (ACS), and nonfunctional adrenal incidentaloma (NFAI). Mesaures of diagnostic performance were assessed (with [95% CIs]) for discriminating different subgroups of patients with adrenal incidentaloma.
Results: Patients with ACC were characterized by elevated plasma concentrations of 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone, androstenedione, and dehydroepiandrosterone-sulfate, whereas patients with PA had elevations of aldosterone, 18-oxocortisol, and 18-hydroxycortisol. A selection of those 8 steroids, combined with 3 others (cortisol, corticosterone, and dehydroepiandrosterone) and plasma metanephrines, proved optimal for identifying patients with ACC, PA, and PHEO at respective sensitivities of 83.3% (66.1%-100%), 90.8% (83.7%-97.8%), and 94.8% (89.8%-99.8%); and specificities of 98.0% (96.9%-99.2%), 92.0% (89.6%-94.3%), and 98.6% (97.6%-99.6%). With the addition of tumor size, discrimination improved further, particularly for ACC (100% [100%-100%] sensitivity, 99.5% [98.9%-100%] specificity). In contrast, discrimination of ACS and NFAI remained suboptimal (70%-71% sensitivity, 89%-90% specificity).
Conclusion: Among patients with adrenal incidentaloma, the combination of plasma steroid metabolomics with routinely available plasma free metanephrines and data from imaging studies may facilitate the identification of almost all clinically relevant adrenal tumors.
Key Words: adrenal incidentaloma, adrenocortical carcinoma, pheochromocytoma, primary aldosteronism, autono- mous cortisol secretion, steroids
Adrenal incidentalomas are commonly defined as adrenal masses found incidentally during abdominal imaging per- formed for indications other than the suspected adrenal dis- ease (1-3). Incidentalomas are now regularly found thanks to improvements in imaging technology and increased use of imaging studies in routine clinical practice, particularly computed tomography (CT) and magnetic resonance im- aging. The overall prevalence of adrenal incidentaloma in imaging series lies between 2% and 5%, but increases with age (2, 4). Incidentally discovered adrenal masses are rela- tively rare in patients younger than age 30 years and show a peak incidence in the fifth to seventh decades, reaching a prevalence of up to 10% in older individuals (5).
The majority of adrenal incidentalomas are nonfunctional adenomas that usually do not require thera- peutic intervention. More rare forms of nonfunctional adrenal incidentalomas (NFAIs) include myelolipoma, hem- angioma, and metastasis from other malignancies. Those that are either functional or malignant include adrenal cortical carcinoma (ACC), pheochromocytoma (PHEO), aldosterone-producing adenomas in primary aldosteronism (PA), and more commonly adenomas that feature autono- mous cortisol secretion (ACS). With the exception of the
latter, these tumors invariably require surgical resection or pharmacotherapy when resection is not possible or where there is metastatic involvement. In cases of ACS, some form of follow-up is required and under current guidelines sur- gical intervention is usually recommended only when there is evidence of Cushing syndrome or excess comorbidity due to cortisol hypersecretion (5).
Although imaging characteristics-including mass size and lipid content assessed by CT-based attenuation or contrast-enhanced washout-can assist in the diagnostic workup of adrenal tumors (6, 7), these features alone cannot be used to establish hormonal functionality, ma- lignancy, or specific requirements for therapeutic interven- tion and patient management. Thus, in addition to clinical evaluation of signs and symptoms of adrenal hormone ex- cess, clinical practice guidelines stipulate the importance of hormonal assessments in virtually all patients with ad- renal incidentaloma (5). The dexamethasone suppression test (DST) is important to rule out ACS. Measurements of plasma or urinary fractionated metanephrines are re- commended to exclude PHEO, while in patients with hypertension or unexplained hypokalemia, the plasma aldosterone-to-renin ratio provides the first step for
identifying PA. For ACC there are a number of steroid bio- markers that can be useful, though none alone provide suf- ficient accuracy for reliable diagnosis.
For a significant proportion of patients with adrenal incidentaloma the process of reaching a final diagnosis involves an arduous multistep process with extensive la- boratory testing and follow-up studies that can remain in- conclusive. Laboratory evaluation of ACC, PA, and ACS is in particular fraught with difficulties indicating a need for new and improved diagnostic strategies. One such strategy involves mass spectrometric-based measurements of panels of steroids-steroid metabolomics-in plasma or urine (8, 9). This multidimensional approach to diagnostics has been trialed with promising results for evaluation of ACC (10-13), PA (14-16), and ACS or Cushing syndrome (17-20). In these studies, including those involving patients with adrenal incidentaloma (11, 20), the focus was directed to identifying or subtyping specific adrenal disorders. In the present study we hypothesized that a single panel of selected steroids could be applied in patients with adrenal incidentaloma to facilitate the simultaneous identification of ACC, PA, ACS, and NFAI. We further hypothesized that identification could be improved and extended to include PHEO by the respective inclusion of imaging data and add- itional measurements of plasma free metanephrines.
Material and Methods
Patient Recruitment
This retrospective, cross-sectional diagnostic study included patients with adrenal incidentaloma registered in ENS@T (European Network for the Study of Adrenal Tumors) from 7 European centers as detailed in Supplementary Table 1 (21). Patients were recruited under 3 clinical protocols: the Prospective Monoamine-producing Tumor (PMT) study, the PROspective study on the diagnostic value of Steroid profiling in primary ALDOsteronism (PROSALDO), and the ENS@T registry and biobanking protocol. Entry cri- teria for the 3 protocols included incidental findings of an adrenal mass. All patients provided written informed con- sent under these protocols, which were approved by the ethics committees at each center.
Inclusion and Exclusion Criteria
Apart from an incidental finding of a mass discovered for reasons other than suspicion of an adrenal disorder, in- clusion in the study also required availability of plasma samples for steroid profiling. On this basis 614 patients, recruited between January 2011 and May 2021, were in- cluded in the study (Fig. 1). The subsequent exclusion of 37 patients was based on several criteria, including insufficient
PMT
ENSAT
PROSALDO
Inclusion based on incidental finding of a mass and available plasma for steroid profiling
INCLUSIONS
n=444
n=95
n=75
n=614
EXCLUSIONS n=19 Dexamethasone
EXCLUSIONS
n=32
n=3
n=2
n=11 Classification unclear
n=6 Non-adrenal mass
n=1 ACTH secreting PHEO
FINAL POPULATION
n=577
CLASSIFICATIONS
ACC n=19
ACS n=104
NFAI n=312
PA
PHEO n=77
n=65
data to allow a diagnosis, an extra-adrenal location of the incidentally found mass, and measurable plasma concen- trations of dexamethasone indicating that blood samples were taken after a DST.
Data Collection
Patient data were retrieved from electronic case report forms or registries for the 3 protocols, supplemented by searches of hospital information systems directed to la- boratory, radiology, surgical and pathology reports. Data included age, sex, body weight, height, and blood pressure. Adrenal mass location and size, usually only 1 dimension for masses smaller than 1.5 cm and 2 to 3 dimensions for larger tumors, were derived from imaging data. Owing to variable imaging procedures, other imaging characteristics (eg, tissue attenuation) could not be consistently collected.
Classification of patients into 5 groups-ACC, ACS, NFAI, PA, and PHEO-was based on established proced- ures and guidelines (5, 22, 23) supplemented by follow-up of patients according to medical records as detailed in Supplementary Methods (21). A plasma cortisol greater than 1.8 ug/dL (> 50 nmol/L) after a 1-mg DST defined
ACS; no attempt was made to subclassify ACS according to a DST cortisol between 51 and 138 nmol/L and greater than 138 nmol/L. Diagnosis of PA required an elevated aldosterone-to-renin ratio and a plasma aldosterone greater than 61 ng/ml (> 170 nmol/L) for the saline infusion test. Diagnosis of PHEO and ACC was based on histopathology. After exclusions, patients with NFAI mainly included those with nonfunctional adenomas (n = 255); however, to be inclusive other cases (n = 57) with nonfunctional adrenal lesions included patients with metastases to the adrenals (n = 22), myelolipoma (n = 15), hemangiomas (n = 6), ad- renal cysts (n = 5), ganglioneuroma (n = 3), 1 case each of an angiomyolipoma, leiomyoma, arteriovenous malforma- tion, and 3 that remained unidentified.
Plasma Steroids and Metanephrines
Collection of blood samples took place between 8 AM and 11 AM with sampling in the supine position for meas- urements of metanephrines. Plasma was separated and stored at -80 ℃ until assay. Samples were shipped on dry ice to the central laboratory where steroids were measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS) according to an established method (24). The panel included 19 steroids: aldosterone, 18-oxocortisol, 18-hydroxycortisol, 11-deoxycortico- sterone, corticosterone, cortisol, 11-deoxycortisol, cortisone, 21-deoxycortisol, progesterone, 17-hydroxyprogesterone, 18-hydroxycorticosterone, 11-dehydrocorticosterone, androstenedione, dehydroepiandrosterone (DHEA), DHEA- sulfate (DHEAS), testosterone, dihydrotestosterone, and dexamethasone. Measurements of plasma normetanephrine and metanephrine were by routine LC-MS/MS methods established at 3 of the participating centers and validated for harmonized measurements as described elsewhere (25). Age- and sex-specific reference intervals for steroids and plasma metanephrines are described elsewhere (26, 27).
Statistical Analysis
Statistical analysis was performed using the JMP stat- istics software package version 15 (SAS Institute Inc). Steel-Dwass all pairs test was used for nonparametric comparisons of continuous data of the five groups. Data with nonnormal distributions were logarithmically trans- formed before parametric statistical analyses. Significance was defined as P less than .05. Data in figures containing plasma concentrations of steroids and metanephrines are provided as geometric least square means corrected for age and sex. The Tukey honestly significant difference post hoc test was used in multivariable analyses with age and sex as covariates to establish significance of differences
between groups. Logistic regression and discriminant ana- lyses were used to generate receiver operating character- istic (ROC) curves according to features that were selected by stepwise regression (eg, steroids) or that were hypothe- sized to improve discrimination (eg, plasma metanephrines and tumor size). In patients with more than one adrenal lesion, volumes of each lesion (4/3xr3) were summed and the mean diameter was derived from the summed volume. For discriminant analyses, values for selected analytes were normalized for age and sex according to data from a refer- ence population as outlined in the Supplementary Methods and Supplementary Table 2 (21). Differences between areas under ROC curves and data from confusion matrices were used to assess discriminatory value according to diagnostic sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Data for these variables are shown with 95% CIs.
Results Study Population and Demographics
The final study population comprised 577 patients, including 19 with ACC, 77 with PHEO, 65 with PA, 104 with ACS, and 312 with NFAI (see Fig. 1). Sex was almost equally distributed, with a tendency to more women than men except in patients with ACC (Table 1). Median ages of patient groups ranged between 54 and 64 years, with the highest ages in patients with ACS. Body mass index was lowest in patients with PHEO, whereas blood pressure was highest in PA patients, who also showed the highest preva- lence of hypertension. Mean diameters of adrenal masses were significantly different between all 5 patient groups, with the largest diameters recorded in patients with ACC, ranging from 3.5 to 12.5 cm (Supplementary Table 3) (21).
Steroid Profiles and Metanephrines
Differences in the plasma steroid metabolome and metanephrines among different groups of patients with adrenal incidentaloma were observed according to both nonparametric comparisons (Supplementary Table 3 (21) and parametric analyses of least square geometric means corrected for age and sex (Figs. 2 and 3).
Patients with PA were characterized by more than 3.5- and 4.5-fold higher (P < . 05) plasma concentrations of al- dosterone and 18-oxocortisol than other groups (see Fig. 2). These patients also had significantly higher plasma con- centrations of 18-hydroxycortisol, though the differences were less marked than for aldosterone and 18-oxocortisol. Similarly, plasma 18-hydroxycorticosterone concentrations were significantly higher in patients with PA than NFAI, ACS, and ACC (Supplementary Table 3) (21). Plasma
| ACC | ACS | NFAI | PA | PHEO | |
|---|---|---|---|---|---|
| Demographics | |||||
| No. | 19 | 104 | 312 | 65 | 77 |
| Women | 7 (36.8%) | 54 (54.8%) | 169 (54.2%) | 34 (52.3%) | 43 (55.8%) |
| Age, y | 56 (47-70) | 64 (56-71)ª | 59 (52-68)6 | 54 (43-59) | 56 (47-63) |
| BMI | 27.1 (24.9-28.4) | 28.8 (24.7-34.0) | 27.7 (24.7-32.0) | 26.8 (23.9-30.5) | 24.4 (21.9-27.6)€ |
| Hypertension& | 8/19 (57.9%) | 84/104 (80.7%) | 226/310 (72.9%) | 63/65 (96.9%)e | 60/77 (77.9%) |
| BP | |||||
| Systolic BP, mm Hg | 135 (130-153) | 145 (132-159) | 140 (126-133) | 150 (137-162)d | 138 (123-150) |
| Diastolic BP, mm Hg | 80 (80-87) | 86 (80-103) | 84 (79-92) | 93 (84-101)e | 85 (78-92) |
| Tumor or lesion data | |||||
| Right adrenal | 9 (47%) | 27 (26%) | 101 (33%) | 23 (36%) | 44 (64%) |
| Left adrenal | 10 (53%) | 57 (55%) | 166 (54%) | 36 (55%) | 19 (27%) |
| Bilateral | 0 (0%) | 20 (19%) | 42 (13%) | 6 (9%) | 6 (9%) |
| Diameter, cmb | 7.6 (5.8-9.9)f | 2.8 (1.8-3.6)f | 1.8 (1.2-2.7)f | 1.4 (1.1-1.7)f | 3.6 (2.6-5.1)f |
| Volume, cc | 232 (105-524)f | 10.9 (3.2-24.4)f | 2.8 (1.5-9.2)f | 1.5 (0.7-2.8)f | 23.9 (9.1-70.2)f |
With the exception of tumor diameter, categorical data are shown as numbers and percentages, whereas continuous data are displayed as medians and interquartiles. Abbreviations: ACC, adrenocortical carcinoma; ACS, autonomous cortisol secretion; BMI, body mass index; BP, blood pressure; NFAI, nonfunctional adrenal incidentaloma; PA, primary aldosteronism; PHEO, pheochromocytoma.
ªP less than .0001 higher than PA and PHEO.
bP less than .002 higher than PA.
‘P less than .01 lower than NFAI, ACS, and PA.
dP less than .005 higher than NFAI and PHEO.
“P less than .05, higher than ACC, ACS, NFAI, and PHEO.
(P less than .005 different from all other patient groups. Missing data: for BMI 5 NFAI, 2 ACS, and 4 PHEO; for BP 6 NFAI, 2 ACS, 1 ACC, and 3 PHEO; for adrenal locations 3 NFAI and 8 PHEO; for tumor diameter 4 NFAI.
“Presence of hypertension was established from a combination of an established history of hypertension, a record of antihypertensive therapy, and measurements of office BP above 140 mm Hg systolic and/or 90 mm Hg diastolic.
“Tumor diameter was calculated from tumor volume using available dimensions; for more than one adrenal lesion, volumes were summed before calculation of mean tumor diameter.
concentrations of 11-deoxycorticosterone were also signifi- cantly higher in patients with PA than those with NFAI and PHEO (see Fig. 2).
With least squares correction for age and sex, patients with ACC were distinguished from all other groups by sig- nificantly higher plasma concentrations of 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone and androstenedione, DHEAS, and progesterone (see Fig. 2). The largest differences were observed for 11-deoxycortisol, which in patients with ACC were 7.7- to 11.2-fold higher than in other groups.
Although patients with ACS had considerably lower plasma concentrations of 11-deoxycortisol than those with ACC, this steroid was significantly higher in this group than in those with NFAI and PHEO (see Fig. 2). Patients with ACS also had 40% higher plasma concentrations of 11-deoxycorticosterone than those with NFAI. However, the main characteristic features of patients with ACS were the significantly lower plasma concentrations of DHEAS and DHEA than in the other groups.
As expected, patients with PHEO were characterized by considerably higher (P <. 001) plasma concentrations
of normetanephrine, metanephrine and methoxytyramine than other groups (see Fig. 3).
Subgroup Discrimination
Stepwise variable selection in a logistic regression model for distinguishing patients with ACC, ACS, NFAI, PA, and PHEO indicated 9 plasma steroids most optimal for identification of the 5 patient groups in the following rank order: 11-deoxycortisol, 18-oxocortisol, andro- stenedione, 18-hydroxycortisol, corticosterone, DHEAS, cortisol, 17-hydroxyprogesterone, and aldosterone. The addition of plasma normetanephrine and metanephrine significantly improved diagnostic performance for distinguishing patients with PHEO (P <. 001), NFAI (P <. 0001), and ACS (P =. 01) from all other groups combined, but had little impact on distinguishing patients with ACC and PA (Supplementary logistic regression and Supplementary Fig. 1) (21). With the addition of adrenal lesion diameter, there was a trend for improved identifi- cation of patients with ACC, but this failed to reach sig- nificance (P = . 06).
*
*
1.8
*
0.20
0.10
1.5
Aldosterone ng/ml
0.15
18-Oxocortisol ng/ml
18-Hydroxycoritsol ng/mL
0.08
1.3
1.0
0.10
**
0.05
0.8
**
0.05
0.5
0.03
I
T
0.3
T
0.00
0.00
0.0
*
2.5
*
*
1.25
0.15
11-Deoxycortisol ng/ml
11-Deoxycorticosterone ng/ml
17-Hydroxyprogesterone ng/ml
2.0
1.00
1.5
0.10
+
0.75
1.0
1
**
0.50
+
0.05
0.5
**
0.25
T
I 1
I
0.0
0.00
0.00
*
2.0
*
1.75
3.0
Androstenedione ng/ml
1.50
1.5
2.5
1.25
DHEAS µg/mL
DHEA ng/ml
2.0
1.00
1.0
0
1.5
0.75
0.50
1.0
T
0.5
0.25
0.5
0.00
0.0
0.0
150
0.30
*
2.5
125
0.25
Cortisol ng/ml
Corticosterone ng/ml
100
2.0
Progesterone ng/ml
0.20
75
1.5
0.15
50
1.0
0.10
25
0.5
0.05
0
ACC
ACS
NFAI
PA
PHEO
0.0
ACC
ACS
NFAI
PA
PHEO
0.00
ACC
ACS
NFAI
PA
PHEO
With discriminant analysis, using models that included the aforementioned and 2 further steroids (11-deoxycor- ticosterone and DHEA) and correction of measured plasma
variables for age and sex, areas under ROC curves for the steroid panel improved with addition of metanephrines and adrenal lesion size (Fig. 4). With steroids alone these areas
900
*
200
*
20
*
800
175
18
Normetanephrine pg/mL
700
Metanephrine pg/mL
150
Methoxytyramine pg/mL
16
600
14
125
500
12
100
10
400
8
300
75
6
200
50
4
100
25
I
2
0
ACC
ACS
NFAI
PA
PHEO
0
ACC
ACS
NFAI
PA
PHEO
0
ACC
ACS
NFAI
PA
PHEO
indicated the highest performance for identification of pa- tients with ACC (see Fig. 4A). Corresponding data from confusion matrices indicated that based on steroids alone, 14 of 19 patients with ACC were identified (73.7[53.9- 93.5]% sensitivity) at a specificity of 98.0% (96.9%-99.2%) (Table 2). With the addition of plasma metanephrines and data for lesion size, all patients with ACC were identified (100% [100%-100%] sensitivity) with only 3 false-positive test results restricted to patients with NFAI (99.5% [98.9%- 100%] specificity). Corresponding values for PPV and NPV were 85.7% (70.7%-100%) and 100% (100%-100%), respectively.
As expected for identification of patients with PHEO, diagnostic performance was markedly improved by the addition of plasma metanephrines reaching a sensitivity of 94.8% (89.8%-99.8%) at a specificity of 98.6% (97.6%- 99.6%) (see Table 2). With the additional inclusion of lesion size in the model, all except 2 of 77 patients with PHEO were correctly identified (97.4% [93.8%-100%] sensitivity) with only 4 false-positives (99.2% [98.4%- 100%] specificity); respective values for PPV and NPV were 94.9% (90.1%-99.8%) and 99.6% (99.0%-100%) and for PPV fell to 85.2% at a prevalence of 5% with little change in NPV (Supplementary Figs. 2 and 3) (21).
For PA, the use of the steroid panel alone indicated cor- rect identification of 54 of 65 patients (83.1% [74.0%- 92.2%] sensitivity) at a specificity of 91.8% [89.4%-94.2%] (see Table 2). With the addition of metanephrines and ad- renal lesion size, sensitivity increased to 92.3% (85.8%- 98.8%) at a specificity of 91.3% (88.9%-93.8%). The corresponding NPV was 98.9% (98.9%-99.9%), but be- cause of a relatively large number of false-positive (n = 44) compared to true-positive (n = 60) results, the PPV reached only 57.7% (48.2%-67.2%) and was lower still (35.9%) at a prevalence of 5% (Supplementary Fig. 2) (21).
The diagnostic performance of steroid profiles, with and without inclusion of metanephrines and adrenal lesion size, was poorest for patients with ACS and NFAI; among these 2 groups many with a classification of NFAI were identi- fied as having ACS and vice versa (see Table 2). The diag- nostic performance for patients with ACS showed some improvement after inclusion of metanephrines and lesion size (70.2% [61.4%-79.0%] sensitivity, 89.8% [87.1%- 92.5%] specificity) compared to the use of the steroid panel alone (66.4% [57.3%-75.4%] sensitivity, 88.0% [85.1%- 90.9%] specificity). In contrast, for patients with NFAI, diagnostic sensitivity increased from 44.2% (38.7%- 49.7%) to 70.5% (65.4%-75.5%) after the inclusion of plasma metanephrines and lesion size; however, specificity (89.4% [85.7%-93.1%]) remained unchanged.
Discussion
This study presents novel data about distinguishing fea- tures among different groups of patients with inciden- tally discovered adrenal masses. Using LC-MS/MS-based plasma steroid profiling and measurements of plasma metanephrines, we show that these features combined with information about adrenal lesion size may be used to fa- cilitate discrimination of different types of adrenal tumors. This multidimensional approach for the simultaneous diag- nosis of multiple adrenal pathologies offers several oppor- tunities for improving on traditional approaches that target only single types of adrenal tumors. First and foremost is the opportunity provided by mathematical pattern recog- nition to more efficiently screen and subtype disease in a single step compared to the multiple sequential steps usu- ally required with unidimensional diagnostic approaches.
The advantages of a multidimensional approach to screening and subtype classification are perhaps most
A
100%
90%
80%
70%
Sensitivity
60%
50%
40%
AUC
30%
ACC
0.9683
20%
ACS
0.8623
NFAI
0.7723
10%
PA
0.9599
PHEO
0.7963
0%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90% 100
1-Specifity
B
100%
90%
80%
70%
Sensitivity
60%
50%
40%
AUC
30%
ACC
0.9899
ACS
0.9028
20%
NFAI
0.8928
10%
PA
0.9717
PHEO
0.9961
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1-Specifity
C
100%
90%
80%
70%
Sensitivity
60%
50%
40%
AUC
30%
ACC
0.9992
ACS
0.9127
20%
NFAI
0.9136
10%
PA
0.9787
PHEO
0.9969
0%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1-Specifity
apparent in the present analysis for patients with ACC. Among this group, all patients were correctly identified by the combination of variables; although the PPV reached only 85.7%, the associated NPV remained high even without considerations of tumor size (99%), indicating the promise of steroid profiling alone for exclusion of disease. In agreement with 2 previous studies employing LC-MS/MS steroid profiling (12, 13), the plasma steroids most clearly elevated in our patients with ACC were 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone, andro- stenedione, and DHEAS. These findings are also in line with multiple studies employing MS-based measurements of urinary steroids (10, 28-33), which together have clari- fied a diversity of urinary steroid metabolite biomarkers in patients with ACC, including tetrahydro-11-deoxycortisol and metabolites of androgens and precursor steroids.
One advance of the present analysis over previous studies that considered ACC involves correction for the confounding influences of sex and age using data from a large reference population of more than 500 patients (26). This obviates the need for separating groups according to age or sex, which for the former can involve up to 3-fold higher concentrations of steroids in young compared to older patients. Nevertheless, for some steroids such as pro- gesterone and estradiol, which can show marked elevations in patients with ACC (12), influences of the menstrual cycle are so large that these steroids can be considered only on an individual basis rather than as part of a profile applied to all patients.
Most recently urinary steroid profiling has been com- bined with information on tumor size and CT imaging characteristics in a machine-learning approach to achieve a PPV of 76.4% at an NPV of 99.7% for discriminating pa- tients with ACC from others with adrenal cortical tumors (11). In the present study we collected data on adrenal le- sion size from almost all patients, but were unable to con- sistently collect data on imaging characteristics. While there were highly significant differences in sizes of adrenal lesions among the patient groups, introduction of this vari- able into the discriminant model overall only modestly im- proved diagnostic performance; as expected, improvement was most pronounced for patients with ACC who had the largest tumors. It would of course be preferable to identify ACC at an earlier stage when tumors are smaller and when prognosis may be better. Imaging characteristics, such as unenhanced CT tissue attenuation, would be a more appro- priate variable than tumor size for earlier identification of such patients. Nevertheless, steroid profiles alone provided reasonable performance for identifying most patients with ACC and excluding disease in almost all others.
Although the present study was not prospective and con- sequently did not allow for consistent collection of imaging characteristics, and although it involved a smaller patient
| A. Steroid panel | Confusion matrices | Diagnostic performance | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Predicted group | Percentages and 95% CI | |||||||||
| ACC | ACS | NFAI | PA | PHEO | Sensitivity | Specificity | PPV | NPV | ||
| Actual group | ACC | 14 | 0 | 2 | 2 | 1 | 74 (54-94) | 98 (97-99) | 56 (37-76) | 99 (98-100) |
| ACS | 0 | 69 | 12 | 7 | 16 | 66 (57-75) | 88 (85-91) | 54 (46-63) | 92 (90-95) | |
| NFAI | 9 | 46 | 138 | 28 | 91 | 44 (39-50) | 89 (86-93) | 83 (77-89) | 58 (53-62) | |
| PA | 1 | 3 | 3 | 54 | 4 | 83 (74-92) | 92 (89-94) | 56 (46-66) | 98 (96-99) | |
| PHEO | 1 | 9 | 11 | 5 | 51 | 66 (56-77) | 78 (75-82) | 31 (24-38) | 94 (92-96) | |
| Predicted group | Percentages and 95% CI | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ACC | ACS | NFAI | PA | PHEO | Sensitivity | Specificity | PPV | NPV | ||
| Actual group | ACC | 15 | 0 | 2 | 1 | 0 | 83 (66-100) | 98 (97-99) | 58 (39-77) | 99 (99-100) |
| ACS | 1 | 74 | 23 | 4 | 2 | 71 (62-80) | 89 (87-92) | 59 (50-67) | 94 (91-96) | |
| NFAI | 9 | 49 | 214 | 35 | 5 | 69 (63-74) | 89 (85-92) | 88 (84-92) | 71 (66-75) | |
| PA | 1 | 2 | 3 | 59 | 0 | 91 (84-98) | 92 (90-94) | 59 (49-67) | 99 (98-100) | |
| PHEO | 0 | 1 | 2 | 1 | 73 | 95 (90-100) | 99 (98-100) | 91 (85-97) | 99 (98-100) | |
| Predicted group | Percentages and 95% CI | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ACC | ACS | NFAI | PA | PHEO | Sensitivity | Specificity | PPV | NPV | ||
| Actual group | ACC | 18 | 0 | 0 | 0 | 0 | 100 (100-100) | 99 (99-100) | 86 (71-100) | 100 (100-100) |
| ACS | 0 | 73 | 24 | 6 | 1 | 70 (61-79) | 90 (87-92) | 59 (51-68) | 93 (91-96) | |
| NFAI | 3 | 48 | 217 | 37 | 3 | 71 (65-76) | 89 (86-93) | 89 (85-93) | 72 (67-77) | |
| PA | 0 | 2 | 3 | 60 | 0 | 92 (86-99) | 91 (89-94) | 58 (48-67) | 99 (98-100) | |
| PHEO | 0 | 0 | 1 | 1 | 75 | 97 (94-100) | 99 (98-100) | 95 (90-100) | 100 (99-100) | |
The 11 steroids in the panel include aldosterone, 18-oxo-cortisol, 18-hydroxycortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione, dehydroepi- androsterone, dehydroepiandrosterone-sulfate, 11-deoxycorticosterone, corticosterone, and cortisol.
Abbreviations: ACC, adrenocortical carcinoma, ACS, autonomous cortisol secretion, NFAI, nonfunctional adrenal incidentaloma; NPV, negative predictive value; PA, primary aldosteronism; PHEO, pheochromocytoma; PPV, positive predictive value.
population than the study by Bancos et al (11), overall numbers of patients were considerably larger than other relevant studies on steroid profiling (12, 13, 28-33). As de- tailed in the Supplementary Material (21), the enrollment of patients was not population based so patients with PA and PHEO were overrepresented among the incidentaloma population. Nevertheless, this and the large overall popula- tion size enabled the application of the method to multiple subgroups of patients with functional and nonfunctional adrenal masses. For the functional tumors, and in agree- ment with previous observations (19, 20), patients with ACS were distinguished from other groups by lower plasma concentrations of DHEA and DHEAS; also concentrations of 11-deoxycortisol and 11-deoxycorticosterone, while lower than those for patients with ACC, were still higher than for patients with NFAI.
In agreement with previous observations (8, 34, 35), pa- tients with PA were characterized by high plasma concen- trations of aldosterone, 18-oxocortisol, 18-hydroxycortisol, and 18-hydroxycorticosterone. Diagnostic performance of steroid profiles to distinguish patients with PA from others was higher in the present than a past study (8). This likely reflects the present focus on patients with adrenal incidentaloma in whom unilateral adenomas predominated compared to the earlier study, which included many pa- tients with bilateral PA in whom steroid profiling is less effective for diagnosis.
As expected, discrimination of patients with PHEO from other groups was nearly exclusively dependent on measurements of plasma metanephrines. Although the metanephrines had little if any impact on the discrim- ination of PA and ACC from other groups, inclusion of
metanephrines significantly improved discrimination of pa- tients with ACS and NFAI, particularly the latter.
The NFAI group was composed mainly of nonfunctional adenomas, which for pragmatic reasons were combined with other nonfunctional adrenal lesions. Discrimination was poorest between NFAI and ACS, which may in part reflect suboptimal performance of the DST for defining patients with ACS compared to those with nonfunctional adenomas (36). More specifically, a DST plasma cortisol between 51 and 138 nmol/L has limited PPV and indicates only possible ACS (5), which without careful follow-up is a limitation of our retrospective study. Relevant to this, the smaller adrenal lesion size in patients with NFAI than ACS may underlie differences in functionality that could be ex- plored prospectively by follow-up (37). In this way those patients with NFAI who were classified with ACS, or even ACC, using steroid profiles may in fact show functionality with enlarging nodule size. Compared with improved im- plementation of the DST as outlined previously (36), those patients classified with ACS based on the DST might be reclassified as NFAI on follow-up.
Follow-up was an integral component of the PMT study (38), but this was initially directed at excluding or con- firming PHEO rather than other adrenal tumors. Even with this, and as detailed in the Supplementary Material (21), there was one patient with a 1.5-cm incidentaloma and a solitary slightly elevated plasma metanephrine in whom PHEO was wrongly excluded based on imaging char- acteristics. Ten years after the initial presentation of that incidentaloma, a 2.2-cm tumor was resected and patho- logically confirmed to be a PHEO. Although imaging char- acteristics can be helpful in excluding PHEO (6, 7), there are clearly exceptions. Similarly, in the PROSALDO study there have been problems with incorrect diagnosis of PA based on routine confirmatory diagnostics (39); thus, it is likely in the present retrospective series that, similarly to ACS and NFAI, some classifications of PA are incorrect, at least for patients included under the PMT protocol. These cases clarify the importance of careful follow-up (see Supplementary Material) (21), ideally with alterna- tives to routine laboratory diagnostics, for final disease classification.
Apart from missing data about imaging characteris- tics, the retrospective design involving collections of data from 3 studies also meant that recruitment of patients was not population based; as detailed in the Supplementary Material (21), this resulted in an overrepresentation of pa- tients with PA and PHEO, a shortcoming addressed by as- sessments of PPV and NPV according to disease prevalence. Other limitations of this study included incomplete collec- tions of some data and the overall low number of patients with ACC. The latter limitation reflects the low incidence of this disease and did not permit application of machine
learning. The other limitations either precluded use of those variables in models or resulted in a fall out of patient num- bers among different models. A limitation of measurements of the plasma compared to the urinary steroid metabolome is that the former provides for only a single time point ra- ther than time-integrated measurements. Sampling of blood in the morning hours for steroid profiling is also likely to be less effective for the identification of adrenal cortical disorders than sampling in the evening hours. However, 24-hour urine collections are cumbersome for patients. These and other pros and cons of plasma vs urinary steroid profiling have been covered elsewhere (40).
In summary, the present study stands out from others that have examined steroid profiles in patients with ad- renal incidentaloma by inclusion of sufficient numbers of patients to allow comparisons of steroid profiles and other data in 5 groups of patients with malignant, func- tional, and nonfunctional adrenal tumors. From this it was possible to build models to explore the potential of a multidimensional approach for the simultaneous diag- nosis of multiple adrenal pathologies in patients with ad- renal incidentaloma. Realizing this potential will require further studies with larger numbers of patients under a prospective design, and with machine-learning algo- rithms in place.
Acknowledgments
Thanks are extended to Katharina Langton, Carola Kunath, Catleen Conrad, Denise Kaden, Yvonne Möhres, Katharina Wang, Susanne Schmidt, and Thomas Baumgartner for technical support or support with patients.
Financial Support: This work was supported by the Deutsche Forschungsgemeinschaft (DFG, Projektnummer: 314061271-TRR/ CRC 205-1/2 to M.P., S.N., M.R., F .. B, S.B., S.R.B., J.W.M.L., M.F., and G.E.) and by the Clinical Research Priority Program of the University of Zurich (for the CRPP HYRENE to F.B.).
Additional Information
Correspondence: Graeme Eisenhofer, PhD, Department of Medi- cine III and Institute for Clinical Chemistry and Laboratory Medicine, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany. Email: Graeme.Eisenhofer@uniklinikum-dresden.de.
Disclosures: The authors have nothing to disclose.
Data Availability: Some or all data sets generated during and/or analyzed during the present study are not publicly available but are available from the corresponding author on reasonable request.
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