A 13-Steroid Serum Panel Based on LC-MS/MS: Use in Detection of Adrenocortical Carcinoma
David R. Taylor,1* Lea Ghataore,1 Lewis Couchman,1,2 Royce P. Vincent,1 Ben Whitelaw,3 Dylan Lewis,4 Salvador Diaz-Cano,5 Gabriele Galata,6 Klaus-Martin Schulte,6 Simon Aylwin,3 and Norman F. Taylor1
BACKGROUND: Adrenocortical carcinoma (ACC) is a rare malignancy, with an annual incidence of 1 or 2 cases per million. Biochemical diagnosis is challenging because up to two-thirds of the carcinomas are biochemically silent, resulting from de facto enzyme deficiencies in steroid hormone biosynthesis. Urine steroid profiling by GC-MS is an effective diagnostic test for ACC because of its capac- ity to detect and quantify the increased metabolites of steroid pathway synthetic intermediates. Corresponding serum assays for most steroid pathway intermediates are usually unavailable because of low demand or lack of immunoassay specificity. Serum steroid analysis by LC- MS/MS is increasingly replacing immunoassay, in partic- ular for steroids most subject to cross-reaction.
METHODS: We developed an LC-MS/MS method for the measurement of serum androstenedione, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate, preg- nenolone, 17-hydroxypregnenolone, progesterone, 17- hydroxyprogesterone, and testosterone. Assay value in discriminating ACC from other adrenal lesions (phae- ochromocytoma/paraganglioma, cortisol-producing ad- enoma, and lesions demonstrating no hormonal excess) was then investigated.
RESULTS: In ACC cases, between 4 and 7 steroids were increased (median = 6), and in the non-ACC groups, up to 2 steroids were increased. 11-Deoxycortisol was mark- edly increased in all cases of ACC. All steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC from non- ACC adrenal lesions.
CONCLUSIONS: Serum steroid paneling by LC-MS/MS is useful for diagnosing ACC by combining the measure-
ment of steroid hormones and their precursors in a single analysis.
@ 2017 American Association for Clinical Chemistry
LC-MS/MS is recognized as the method of choice for low molecular weight analytes such as steroids in the clinical laboratory (1-3). Among its advantages over steroid im- munoassay is its superior analytical specificity and ability to measure multiple steroids in a single analysis (4-8). To date, steroid paneling by LC-MS/MS has been ap- plied to congenital adrenal hyperplasia screening (9), in- vestigation of polycystic ovary syndrome (10), primary hyperaldosteronism subtyping (11, 12), and subclinical cortisol-producing adenoma (13).
Adrenocortical carcinoma (ACC)7 is a rare malig- nancy of the steroid-producing adrenal cortex with an annual incidence of 1 or 2 cases per million (14). Al- though associated with a number of familial syndromes, including Li-Fraumeni, Beckwith-Wiedemann, and Lynch syndromes and multiple endocrine neoplasia type 1, the majority of cases of ACC present sporadically, most frequently in the fifth or sixth decade of life and more commonly in women (15, 16).
The diagnosis of ACC is challenging. Patients al- most always present with tumors of ≥4 cm (17, 18), but this only offers a clinical specificity of 61% (19). Clinical evidence of steroid overproduction (e.g., Cushing’s syn- drome, androgen excess in females) is evident in <50% of cases (20). Nonetheless, up to two-thirds of cases of ACC show biochemical evidence of hormone excess, with hypercortisolism most common (15). The Euro- pean Network for the Study of Adrenal Tumors recom- mends a biochemical workup for suspected ACC that includes serum cortisol (basal and postdexamethasone),
1 Department of Clinical Biochemistry (Viapath), King’s College Hospital NHS Foundation Trust, London, UK; 2 Department of Toxicology (Viapath), King’s College Hospital NHS Foundation Trust, London, UK; 3 Department of Endocrinology, King’s College Hospital NHS Foundation Trust, London, UK; 4 Department of Radiology, King’s College Hospital NHS Foundation Trust, London, UK; 5 Department of Histopathology, King’s College Hospital NHS Foundation Trust, London, UK; 6 Department of Surgery, King’s College Hospital NHS Foundation Trust, London, UK.
* Address correspondence to this author at: Department of Clinical Biochemistry, Viapath, First Floor, Bessemer Wing, King’s College Hospital, Denmark Hill, London SE5 9RS, UK. Fax +0203-299-5888; e-mail: davidtaylor8@nhs.net.
@ 2017 American Association for Clinical Chemistry
7 Nonstandard abbreviations: ACC, adrenocortical carcinoma; DHEAS, dehydroepiandros- terone sulfate; IS, internal standard; IQC, internal quality control; PPC/PGL, phaeochro- mocytoma/paraganglioma; LLoQ, lower limit of quantification; ULoQ, upper limit of quantification; IQR, interquartile range; NFAA, nonfunctioning adrenal adenoma.
aldosterone (if hypokalemic or demonstrating arterial hy- pertension), 17-hydroxyprogesterone, dehydroepiandro- sterone sulfate (DHEAS), androstenedione, testosterone, and 17ß-estradiol (men and postmenopausal women). An alternative approach is the measurement of steroid metabolites in urine by GC-MS (20-22). Two studies report clinical sensitivities of 90% and 100% and clinical specificities of 90% and 99% for GC-MS results in diag- nosing ACC, respectively (20, 22), and that the 11- deoxycortisol metabolite, tetrahydro-11-deoxycortisol, provides the greatest diagnostic yield. However, 11- deoxycortisol is rarely measured in serum and assays for this, and other steroid synthetic pathway intermediates such as 17-hydroxypregnenolone and pregnenolone, me- tabolites of which provide useful markers of malignancy in urine (20), are not widely available.
Here we present an LC-MS/MS method for the pan- eling of 13 steroids in serum, which we assessed for its ability to differentiate samples from patients with ACC from those of other adrenal lesions in the setting of a tertiary referral center for adrenal pathology.
Materials and Methods
MATERIALS AND METHODS AND INSTRUMENTATION
The liquid chromatography (LC) instrumentation was an Aria Transcend TLX-II system, and the tandem mass spectrometer (MS/MS) used was a TSQ Vantage (both ThermoFisher Scientific). An Accucore™M reversed-phase C18 column (RP-MS 100 × 2.1 mm i.d., 2.6-um total particle size) fitted with a 0.5-um precolumn filter was used (Fisher Scientific). Column temperature (40 ℃) was maintained using a Hot- Pocket (ThermoFisher). HPLC-grade methanol, ace- tonitrile, acetone, and 2-propanol were from Rath- burn. Water was deionized in-house (18 ml2, Elga). Androstenedione, cortisone, 11-deoxycortisol, 21- deoxycortisol, 11-deoxycorticosterone, pregnenolone, 17- hydroxyprogesterone, and 17-hydroxypregenenolone were from Steraloids. DHEAS was from ResearchPlus. Cortisol, corticosterone, progesterone, testosterone, testosterone-16,16,17-D3, and formic acid were from Sigma-Aldrich. Androstenedione-2,2,4,6,6,16,16-D7, cortisol-9,11,12,12-D4, cortisone-1,2-D2, corticosterone- 2,2,4,6,6,17,21,21-D8, 11-deoxycortisol-21,21-D2, 21- deoxycortisol-2,2,4,6,6,21,21,21-Dg, DHEAS-16,16-D2, DOC-2,2,4,6,6,17,21,21-Dg, pregenenolone-17,21,21,21- D4, progesterone-2,2,4,6,6,17a,21,21,21-D9, 17- hydroxyprogesterone-2,2,4,6,6,21,21,21-Dg, and 17- hydroxypregnenolone-21,21,21-D3 were from CDN isotopes. Double charcoal-stripped human serum was from BBI solutions. Vacuette 4-mL Z Serum Separa- tor Clot Activator and 3-mL K2 EDTA tubes were from Greiner Bio-One. External quality assurance
(EQA) samples were from the United Kingdom Exter- nal Quality Assurance Scheme (UKNEQAS).
CALIBRATOR, INTERNAL QUALITY CONTROL, AND INTERNAL STANDARD SOLUTIONS
Individual 1000-mg/L stock solutions for all analytes and internal standards (IS) were prepared in methanol and combined to prepare working solutions containing all analytes or IS for calibration, internal quality control (IQC), and IS purposes. To make the working solutions, appropri- ate volumes of stock solution were added to a 100 × 15 mm, 10-mL glass tube and then evaporated under nitrogen at 60 ℃, followed by reconstitution in 1 mL of ethanol. Calibrator and IQC working solutions contained the fol- lowing steroid concentrations: DHEAS, 2000 µg/mL; cor- tisol, 200 µg/mL; 17-hydroxypregnenolone, 160 µg/ml; 17-hydroxyprogesterone, 120 µg/mL; androstenedione, 80 ug/ml; pregnenolone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, and cortisone, each 40 µg/mL; testoster- one, 8 µg/ml; and 11-deoxycorticosterone, 4 µg/mL. These solutions were further diluted in ethanol as follows: 3 + 20 (v/v), 1 + 39 (v/v), and 1:199 (v/v). All 4 working solutions were used to make calibrator/IQC solutions by dilution in double charcoal-stripped serum (see Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol63/ issue12). After thorough mixing and equilibration (24 h, 2-8 ℃), calibrators and IQC were portioned (250 [L) into 1.5-mL polypropylene tubes (Eppendorf) and stored at -20 ℃. The deuterated IS stock solution contained the following steroid concentrations: DHEAS-D2, 25 000 µg/mL; cortisol-D4, 2500 µg/ml; 17-hydroxypregnenolone-D3, 2000 µg/ml; 17-hydroxyprogesterone-D8, 1500 µg/mL; androstenedione-D7, 1000 µg/ml; pregenenolone-D4> corticosterone-D8, 11-deoxycortisol-D2, 21-deoxycortisol-D8, and cortisone-D2, each at 500 µg/ml; testosterone-D3, 100 µg/mL; and DOC-D8, 50 µg/mL. An IS working solution was prepared before each batch by dilution of 100 µL of the IS stock solution in acetonitrile to 20 mL.
CLINICAL SAMPLES
Samples (minimum volume 2 mL into an EDTA tube and 3 mL into a serum separator tube plus either random or 24-h urine samples) were collected by endocrine nurses from patients attending scheduled appointments in the Programed Investigation Unit at King’s College Hospital NHS Foundation Trust. At this appointment, informed consent was obtained from each patient to al- low biochemical testing (including steroid measure- ments) for clinical evaluation of their adrenal lesions in accordance with a Trust standard operating procedure for suspected adrenal cancer. All samples were collected between 9:00 and 11:30 AM. Samples were subsequently taken immediately to the laboratory for processing. EDTA and serum separator tubes were centrifuged at
Simultaneous Quantification of 13 Serum Steroids by LC-MS/MS
2163g for 10 min, and serum and plasma were aliquoted and either analyzed immediately or stored frozen at -20 ℃ before analysis. Urine aliquots were acidified to a pH <2 for urine metanephrine analysis. Patients were categorized in the adrenal multidisciplinary meeting us- ing combinations of biochemistry, radiology, and histol- ogy (if available). There were 10 ACC cases (all histolog- ically proven) and 15 with phaeochromocytoma/ paraganglioma (PCC/PGL, all histologically proven); 7 had adenoma with glucocorticoid excess; and 16 adrenal lesions demonstrated no biochemical evidence of adrenal cortical or medullary excess (NFAA group). Surgical cases were defined according to standard pathological criteria (23-26). In nonsurgical cases, conventional im- aging criteria were applied for stratification of benign or malignant neoplasms (27).
ROUTINE BIOCHEMICAL ASSESSMENTS
Routine steroid immunoassays were serum cortisol, testos- terone, progesterone, and 17ß-estradiol (Siemens Centaur XPi); serum androstenedione and DHEAS (Siemens Im- mulite); and EDTA plasma aldosterone and renin mass (Diasorin Liaison). Serum 17-hydroxyprogesterone was measured at St. Thomas’ Hospital, London by LC-MS/MS. We measured plasma metanephrines in EDTA plasma by LC-MS/MS (28) and urine metanephrines by HPLC with electrochemical detection (Chromsystems). Urine steroid profiling used GC-MS (29).
SPECIMEN PROCESSING FOR LC/MS/MS
Portions of frozen calibrators, IQC, and unknown pa- tient/EQA samples were thawed and mixed; then 250 µLL was transferred into a 1.5-mL polypropylene tube. Sub- sequently, 250 µL of IS working solution and 500 µL of ice-cold acetonitrile were added, and tubes were then vortex-mixed for 30 s. Precipitated protein was pelleted by centrifugation (12000g, 10 min), and the supernatant was transferred to a 10-mL glass tube containing 300 µL of deionized water (dH2O). Ethyl acetate (1 mL) was added, and the tube was vortex-mixed for 5 min. Following centrif- ugation (161g, 1 min), the top organic layer was removed to a clean 75- × 10-mm, 4.5-mL glass tube. Extracts were evaporated to dryness under nitrogen at 60 ℃ and recon- stituted in 200 µL of a 65 + 35 (v/v) mixture of dH2O/ methanol and transferred to an autosampler vial.
LC-MS/MS PROCEDURE
Extracts were injected (100 (L) onto the LC column at a flow rate of 0.40 mL/min. Mobile phases were (A) dH2O and (B) methanol, each containing 0.1% (v/v) formic acid. The LC system was controlled using Aria MX (version 1.1, ThermoFisher). The gradient elution is summarized in Table 2 of the online Data Supplement. The total analysis time was 19.7 min, including column reequilibration. Elu- ent flow was diverted to waste for the first 3 min.
MS/MS was carried out using Xcalibur (version 2.2, ThermoFisher) in the positive mode using atmospheric pressure chemical ionization. Data were collected in high resolution (0.40 m/z full width at half maximum) in the multiple reaction monitoring mode, with 2 m/z transi- tions per analyte and 1 mlz per IS (see Table 3 in the online Data Supplement). Postanalysis processing used LC QuanTM (version 2.6, ThermoFisher). For assay cal- ibration, peak area ratios (analyte quantifier to IS) were used to construct calibration graphs, with lines fitted by linear regression. The intercepts were not forced through zero, and line weighting was applied (1/concentration).
METHOD VALIDATION
To validate the developed liquid-liquid extraction (LLE) LC-MS/MS assay, the recovery, linearity, and lower and upper limits of quantification (LLoQ and ULoQ) were determined in accordance with US Food and Drug Ad- ministration Center for Drug Evaluation and Research guidance for bioanalytical method validation.
To assess LLE recovery using ethyl acetate, 2 exper- iments were performed using IQC material. First, abso- lute recovery was evaluated by directly comparing analyte peak area from protein-precipitated samples that had un- dergone LLE with samples undergoing protein precipita- tion only. Second, relative recovery was assessed using IS-corrected peak area ratios in the same samples. To test linearity, charcoal-stripped serum was spiked with ste- roids at concentrations covering physiological and path- ological ranges and tested in triplicate. LLoQ and ULOQ were defined for each analyte as the lowest concentration at which the imprecision (%CV) was <20% (LLOQ) or <15% (ULoQ), with the measured concentration within ±20% of the nominal value. Method precision was assessed using IQC material at 3 target values, either analyzed 6 times in 1 batch (intraassay precision) or in singlicate within 6 batches on different days (interassay precision). Matrix effects were assessed by the postcol- umn infusion method (30) using an IS working solution infusion via a tee-piece during the analysis of extracted patient samples (n = 5), as well as monitoring IS intraas- say precision during sample analysis. Two steroid stabil- ity experiments were performed using IQC material. First, freeze-thaw stability was assessed on samples un- dergoing freeze-thaw cycles on 3 consecutive days. Sec- ond, postextraction stability was evaluated in samples left either refrigerated (4 ℃) or at room temperature for 7 days before analysis. In each case, analysis was performed against fresh calibrators.
Method comparison with cortisol, testosterone, progesterone, DHEAS, androstenedione, and 17- hydroxyprogesterone UKNEQAS samples was per- formed (n = 30 for each). Results were compared with the LC-MS/MS users group mean value for all steroids except progesterone. No UKNEQAS-registered labora-
tories perform progesterone analysis by LC-MS/MS, so results were compared with the all-laboratory immuno- assay mean. Anonymized excess serum samples obtained in primary care were used to determine steroid reference ranges (n = 200).
STATISTICAL ANALYSIS
Statistical analysis was performed using Analyze-It® (ver- sion 4.65.3). Good method agreement was defined by (a) Deming regression analysis demonstrating a slope of ap- proximately 1 with 95% CIs bracketing 1 with an inter- cept of approximately 0 (95% CI bracketing 0) and (b) the Altman-Bland plot giving a bias with a confidence limit spanning 0. Clinical data were found not to be normally distributed using the Shapiro-Wilk test. Pair- wise comparisons were performed using Mann-Whitney U-tests, with post hoc Bonferroni correction. Data are reported as median and interquartile ranges (IQR). Val- ues of P < 0.05 were defined as statistically significant.
Results
Chromatographic resolution of 13 steroids was achieved within 14.5 min (Fig. 1). This extended time was necessary to achieve baseline separation of the tar- geted isobaric steroids 21-deoxycortisol, corticosterone and 11-deoxycortisol, and 11-deoxycorticosterone and 17-hydroxyprogesterone.
Recovery of steroids after protein precipitation and subsequent LLE was assessed in both absolute and rela- tive terms. For all steroids, absolute extraction recovery was >50%, with relative extraction recoveries, evaluated after IS correction, between 90% and 110% (see Table 4 in the online Data Supplement).
The developed method was linear over several orders of magnitude (r ≥ 0.99) for all steroids (Table 1). We established ULoQs for each steroid that permitted mea- surement at the high pathological concentrations ex- pected in ACC (Table 1), whereas the LLOQs were suf- ficiently low to allow quantification of most steroids in healthy individuals. Intraassay and interassay precisions were ≤10% for all steroids (see Table 5 in the online Data Supplement). No analytically significant ion sup- pression/enhancement was observed, as evidenced by in- fusion studies (suppression <15% for all steroids stud- ied) and IS peak area precision <20% during analysis of extracted patient samples (Table 1). All steroids were sta- ble through 3 freeze-thaw cycles, whereas extracted sam- ples were stable at room temperature and at 4 ℃ for 7 days, with concentrations for all steroids within ±10% of the original value measured in each stability experiment. There was a good agreement between the developed method and EQA consensus values (see Fig. 1 in the online Data Supplement).
100
sk
E - RT 5.08
50
m/z 361.1 > 163.1
362 395 479
5.66
0
5.84
7.42 779 900 1031 11.37 1274 1343 1483
100
9H3
F - RT 5.65
50
m/z 363.2 > 121.0
5.09
0
305 359 478
621 7:32 835
9.63 1031 11.72 1219 1414 14,25
100
CK
21-DF - RT 6.94
50
153 801
m/z 347.1 > 105.1
0
320 356 472 509 565 630
N
9.01
10.07 11:54 11.77 1243
14.01
100
6.95
B - RT 7.53
50
₦ 8 01
m/z 347.1 > 105.1
0
320 356 472 509 565 630
1
9.01 10 07 11:54 11.77 1243
14.01
100
Mª
DHEAS - RT 7.53 m/z271.1 > 105.0
50
0
269 302 341 400 508 565 618 7.16
816 979 10:30 1099 1151 1276 1373 1492
Relative abundance
100
**
S - RT 8.02
50
m/z 347.1 > 109.0
0
3.02 2 4.16 509 563 610
6.95
A
8.90 952 10:16 11.36 1250 14 00 1469
100
Fİ
44A - RT 8.90
50
m/z 287.1 > 97.0
0
260 302 344 415 524 564 695 774 800
10 09 10.48 11.24 1200 1262 13.70 1493
100
1
DOC - RT 9.71
50
m/z 331.2 > 97.0
0
320 4.11 508 565 6:90 7:32 8 00 8.62
971
1072 2 1216 1337 1424 1469
100
T - RT 10.08
*
10.08
50
m/z 289.2 > 97.0
0
252 302 341 447 492 563 625 7:03 7.62 798
90
992
11.77 1271 1344 1412
100
1083
17OHP - RT 10.30
50
m/z 331.2 > 97.0
0
320 411 508 565 6:90 7:32 800 8 62
971
10,72
1216 13:37 1424 1469
100
1550
17AP - RT 10.51
14.91
50
m/z 297.0 > 105.0 5166
1461
0
258 302 345 486 A 670 754 857 923 1015
11.07
12.50
13:52
100;
1280
P- RT 12.50
50
m/z 315.1 > 970
0
258 303 364 437 507 360 583 764 840 967 10:50 11,13
1341 14.01
100
1313
Preg - RT 13.90
50
m/z 299.2 > 105.11028
10.49
11.82
1322
279 302 397 5.07
6.94
7.59 801
9 06
9.41
1481
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time (min)
Simultaneous Quantification of 13 Serum Steroids by LC-MS/MS
| Table 1. Linearity, LLoQ, ULoQ, and IS precision. | ||||||
|---|---|---|---|---|---|---|
| Calibration range, ng/ml | Recovery, % | Linear regression y = ax + c (R)b | LLoQ, ng/ml | ULoQ, ng/ml | IS typical intrabatch CV, % | |
| E | 0-200 | 99 ± 9.8 | y = 1.0063x - 0.2482 (0.99) | 0.61 | 200 | 13.2 |
| F | 0-1000 | 98 ± 9.7 | y = 1.0252x - 7.3743 (0.99) | 1.25 | 1000 | 9.0 |
| 21-DF | 0-200 | 102 ± 9.9 | y = 1.0144x - 0.3055 (0.99) | 0.12 | 200 | 16.2 |
| B | 0-200 | 101 ± 9.7 | y = 1.0361x - 0.9363 (0.98) | 0.25 | 200 | 13.1 |
| S | 0-200 | 99 ± 9.1 | y = 0.9837x+0.825(0.99) | 0.12 | 200 | 9.6 |
| 44A | 0-400 | 98 ± 8.6 | y = 1.0053x - 1.3789 (0.99) | 0.25 | 400 | 17.2 |
| DHEAS | 0-5000 | 101 ± 9.8 | y = 0.9877x + 17.918 (0.99) | 12.9 | 5000 | 7.4 |
| DOC | 0-10 | 99 ± 9.6 | y = 1.0048x - 0.0279 (0.99) | 0.1 | 150 | 13.9 |
| T | 0-20 | 100 ± 9.8 | y = 0.9878x+0.2064 (0.99) | 0.05 | 60 | 6.7 |
| 17OHP | 0-300 | 99 ± 8.6 | y = 1.018x - 1.4606 (0.99) | 0.38 | 600 | 13.3 |
| 17AP | 0-400 | 101 ± 9.9 | y = 1.0191x - 1.766 (0.99) | 0.50 | 400 | 9.8 |
| P | 0-50 | 98 ± 8.8 | y = 1.0077x - 0.2138 (0.99) | 0.13 | 100 | 12.1 |
| Preg | 0-100 | 101 ± 9.5 | y = 0.9961x + 0.0224 (0.99) | 1.27 | 200 | 10.1 |
a Describes the percentage of measured vs expected concentration expressed as mean ± SD, n = 30, n = 3 for each concentration.
b y = ax + c; y, measured concentration; x, analyte peak area/IS peak area ratio.
B, corticosterone; DOC, 11-deoxycorticosterone; P, progesterone; Preg, pregnenolone; E, cortisone; F, cortisol; S, 11-deoxycortisol; 170HP, 17-hydroxyprogesterone; 44A, andro- stenedione; 21DF, 21-deoxycortisol; T, testosterone; 17AP, 17-hydroxypregnenolone.
Demographic and clinical characteristics of the ACC, cortisol-producing adenoma, PPC/PGL, and NFAA groups are summarized in Table 2. Groups were well matched for age and sex, although the PCC/PGL group tended to be younger (Table 2). ACC cases pre- sented with larger tumors. All patients with ACC and PPC/PGL underwent surgery, whereas only 57% of those with cortisol-producing adenoma and 12.5% of the NFAA group underwent more surgery.
For the ACC cases, 5 of 10 presented with clinical features of steroid hormone excess. Three females had signs of Cushing’s syndrome, 1 female had androgen ex- cess, and 1 male presented with uncontrolled hyperten- sion. Of the remaining cases, 2 females presented with abdominal symptoms, 1 male with weight loss, and 1 male with hematuria; the initial clinical presentation was not documented for 1 male. Diagnostic workup of the
ACC group using existing routine biochemical methods was standard and in line with European Network for the Study of Adrenal Tumors guidance. Urine steroid profil- ing was performed in 9 of 10 cases, and all profiles were consistent with ACC. Random cortisol concentration was increased in 6 of 10 cases, whereas cortisol failed to suppress to 1.8 µg/dL in 3 patients undergoing overnight dexamethasone suppression testing. Serum androgens were increased in 7 patients (increased testosterone in 2 females, androstenedione in 4 of 9 cases, and DHEAS in 5 of 9 cases). 17-Hydroxyprogesterone was increased in 6 of 9 cases tested. Progesterone was detected in 2 males (usually <1.6 ng/ml). The aldosterone/renin ratio was normal in all cases tested.
The non-ACC adrenal group was divided according to biochemical and radiological criteria: Overt biochem- ical glucocorticoid excess was defined by the failure of
| Table 2. Demographics and clinical characteristics of the adrenal tumor patients. | ||||
|---|---|---|---|---|
| ACC group | Cortisol-producing adenoma | PPC/PGL | NFAA | |
| Number | 10 | 7 | 15 | 16 |
| Sex, male/female | 4/6 | 4/3 | 8/7 | 6/10 |
| Age (range), years | 59 (47-69) | 68 (66-70) | 50 (44-66) | 62 (48-72) |
| Maximum diameter of tumor at time of serum collection, median (range), mm | 100 (75-160) | 40 (38-44) | 56 (40-76) | 17 (13-46) |
| Surgical removal of adrenal tumor (%) | 10 (100) | 4 (57) | 15 (100) | 2 (12.5) |
cortisol to suppress to <5 µg/dL in the overnight dexa- methasone suppression testing or a urine free cortisol concentration above the reference range (>71 µg/dL per 24 h). In these cases, radiology demonstrated lipid-rich pathology in 6 of 7 cases, with lipid-poor adenoma in the other case. In 15 patients with radiological features of PPC/PGL, catecholamine excess was confirmed by in- creased plasma and/or urine metanephrines. Patients were included in the NFAA group if they (a) were proven normal on histology or the mass was shown to be stable on imaging after >12 months follow-up and (b) had no clinical evidence of hormone excess, a normal aldoste- rone/renin ratio (or normal blood pressure), a normal overnight dexamethasone suppression test (cortisol <1.8 ug/dL), and normal plasma metanephrine and normeta- nephrine. In this group, radiology fell into 2 categories: Hounsfield units <10 or defined by a radiologist as a lipid-poor adenoma with no features of malignancy.
Comparison of LC-MS/MS steroid data between the ACC and non-ACC groups revealed striking differ- ences (Table 3). Across the non-ACC adrenal lesion groups, only up to 2 steroid concentrations were in- creased above the reference ranges given in Table 3 in individual cases, whereas in ACC between 4 and 7 ste- roids were increased (median = 6 steroids). 11- Deoxycortisol was increased in all ACC cases (median, 6.2 ng/ml; IQR, 2.5-9.0; normal range, <0.9 ng/ml). Other steroids increased in ACC were androstenedione and DHEAS (6 cases), cortisol, pregnenolone, and 17- hydroxypregnenolone (5 cases), corticosterone (4 cases), and 17-hydroxyprogesterone, 11-deoxycorticosterone, and cortisone (3 cases). Testosterone was increased in 2 females and progesterone was detectable in 3 males with ACC (normally <0.13 ng/ml by LC-MS/MS). In 1 in- stance, 17-hydroxypregnenolone could not be reliably quantified because of the presence of an interfering peak. Steroid heterogeneity in ACC was demonstrated when data for each steroid in each ACC was plotted as the multiple of the median value calculated from the non- ACC adrenal lesion group (see Fig. 2 in the online Data Supplement).
Discrimination of the non-ACC adrenal lesion and ACC groups was possible using several steroids (Table 3). Whereas pregnenolone and 21-deoxycortisol could not be detected in the non-ACC adrenal lesion group, preg- nenolone was measurable in 5 ACC cases and 21- deoxycortisol was detectable in 2 ACC cases. All other ste- roids except cortisone, corticosterone, and male testosterone showed significant increases in ACC when compared with non-ACC adrenal lesion groups. 11-Deoxycortisol and 17- hydroxypregnenolone provided the best discrimination be- tween ACC and the non-ACC adrenal lesions (Table 3, Fig. 2). 17-Hydroxyprogesterone and androstenedione were in- creased in the ACC group, but the IQRs overlapped the normal reference ranges of these steroids (Table 3, Fig. 2).
For DHEAS, 4 cases of ACC showed concentrations in the lower half of the reference range, whereas in the other cases its concentration was dramatically increased. The DHEAS concentration was lower in the cortisol-producing adenoma group than in the PPC/PGL and NFAA groups.
Because the values in ACC were so variable, the mul- tivariate technique of principal component analysis was applied, both to the European Network for the Study of Adrenal Tumors-recommended sex-independent steroids (cortisol, androstenedione, DHEAS, and 17- hydroxyprogesterone) and to all sex-independent serum steroid panel steroids (all minus testosterone and proges- terone). Both positive and negative correlations among variables after principal component analysis were ob- served in biplot graphs (Fig. 3). Using the European Net- work for the Study of Adrenal Tumors-recommended steroid measurements, all but 2 of the ACC patients were separated from the other adrenal lesions (Fig. 3A). When full panel data were included, complete separation was achieved (Fig. 3B). However, the ACC cases did not cluster, reflecting the heterogeneity of tumor steroid pro- duction, which is the hallmark of this disorder (20).
Discussion
In this study we showed that serum steroid paneling by LC-MS/MS is a useful tool to discriminate ACC from other non-ACC adrenal tumor lesions. Previous practice for selection of biochemical investigations has been dic- tated by the clinical presentation, e.g., signs of cortisol or androgen excess. This only characterizes subpopulations of ACC; < 50% of cases of ACC present with clinical symptoms of hormone excess (20). In contrast, serum steroid paneling allows the investigation of adrenal masses more comprehensively by offering measurement of all major steroid biosynthetic intermediates. It is both the number of steroids increased and the marked in- creases of several synthetic intermediates without biolog- ical activity that appear particularly useful in discriminat- ing ACC from other adrenal lesions, validating the paneling approach to adrenal mass investigation. The DHEAS concentration was lower in cortisol-producing adenoma than in other adrenal lesions, in keeping with previous observations (13, 31, 32).
The cortisol precursor 11-deoxycortisol was most discriminating for differentiating ACC from non-ACC adrenal lesions, an observation consistent with previous studies demonstrating the usefulness of measuring its uri- nary metabolite tetrahydro-11-deoxycortisol by GC-MS (20, 22). In blood, 11-deoxycortisol is known to be in- creased in benign and malignant adrenal tumors in chil- dren, although it was not reported whether 11-deoxycortisol discriminated benign from malignant disease (33). That 11-deoxycortisol is such a useful marker suggests a critical change in 11ß-hydroxylase activity in ACC. 11ß-
| Table 3. Comparison of serum steroid concentrations between ACC and non-ACC adrenal lesions.ª | ||||||||
|---|---|---|---|---|---|---|---|---|
| Steroid | Normal reference range | Steroid concentration, median (IQR), ng/ml | P value | |||||
| ACC (n = 10) | Cortisol-producing adenoma (n = 7) | NFAA | PPC/PGL | ACC vs cortisol-producing adenoma | ACC vs PPC/PGL | ACC vs NFAA | ||
| T | M: 2.9-8.6 | 2.8 (2.1-3.5) | 1.9 (0.9-3.6) | 3.2 (2.3-4.3) | 2.9 (2.3-3.6) | NS | NS | NS |
| F: < 1.70 | 0.35 (0.28-0.86) | 0.07 (0.06-1.00) | 0.15 (0.12-0.32) | 0.17 (0.12-0.22) | NS | NS | NS | |
| A4 | <2.0 | 2.2 (1.5-3.5) | 0.8 (0.7-0.9) | 0.6 (0.5-0.8) | 0.6 (0.4-0.9) | 0.04 | 0.005 | 0.004 |
| DHEAS | 148-3852 | 6333 (861-10 159) | 303 (215-513) | 1176 (469-2116) | 1222 (701-1547) | 0.04 | NS | NS |
| P | M: < 0.13 | 3 cases: 0.25, 0.47, 0.31 | <0.13 all cases | <0.13 all cases | <0.13 all cases | N/A | N/A | N/A |
| F: < 9.4 | 3 cases: 0.22, 0.25, 1.2 | <0.13 all cases | <0.13 all cases | <0.13 all cases | N/A | N/A | N/A | |
| 17OHP | <1.7 | 1.17 (0.9-1.8) | 0.7 (0.6-0.9) | 0.6 (0.5-0.9) | 0.7 (0.6-1.0) | 0.01 | 0.003 | 0.004 |
| Preg | <1.3 | 3.1 (2.1-6.8) | <1.3 all cases | <1.3 all cases | <1.3 all cases | N/A | N/A | N/A |
| 17AP | <7.3 | 7.5 (3.4-19.3) | 0.6 (0.5-0.7) | 1.3 (0.8-1.9) | 0.8 (0.6-1.6) | 0.001 | <0.0001 | 0.004 |
| S | <0.9 | 6.2 (2.5-9.0) | 0.7 (0.2-0.9) | 0.3 (0.14-0.3) | 0.2 (0.2-0.3) | 0.004 | <0.001 | <0.001 |
| 21-DF | <0.12 | 3 cases: 0.12, 0.16, 0.16 | <0.12 in all cases | <0.12 in all cases | <0.12 in all cases | N/A | N/A | N/A |
| DOC | <0.5 | 0.4 (0.3-0.6) | 0.2 (0.2-0.3) | 0.2 (0.1-0.2) | 0.2(0.1-0.2) | NS | 0.01 | 0.002 |
| B | 1.2-20.5 | 7.0 (1.7-25.5) | 2.6 (2.3-5.8) | 2.4 (0.8-4.8) | 2.4 (1.3-4.3) | NS | NS | NS |
| F | 46-207 | 185 (118-270) | 88 (84-148) | 43 (35-81) | 73 (65-137) | NS | 0.05 | 0.004 |
| E | 7.1-27.9 | 21.0 (18.7-29.0) | 15.1 (14.1-18.8) | 14.9 (9.3-17.9) | 15.0 (13.0-17.5) | NS | NS | NS |
a Steroid concentrations expressed as median (IQR). P < 0.05 was defined as significant. NS, nonsignificant. Progesterone, pregnenolone, and 21-deoxycortisol were excluded from statistical analyses because of insufficient data points. T, testosterone; A4, androstenedione; P, progesterone; 170HP, 17-hydroxyprogesterone; Preg, pregnenolone; 17AP, 17-hydroxypregnenolone; S, 11-deoxycortisol; 21DF, 21-deoxycortisol; DOC, 11-deoxycorticosterone; B, corticosterone; F, cortisol; E, cortisone.
17-Hydroxyprogesterone (ng/ml) >>
B
3.0
600
2.5
500
0.004
Cortisol (ng/ml)
2.0
400
0.003
1.5
300
0.01
0.05
1.0
200
NS
0.004
0.5
100
0.0
0
ACC
Cortisol-producing adenoma
PPC/ PGL
NFAA
ACC
Cortisol-producing adenoma
PPC/ PGL
NFAA
C
D
18 000
4.5
16 000
DHEAS (ng/ml)
Androstenedione (ng/ml)
4.0
14 000
3.5
12 000
3.0
10 000
2.5
8000
2.0
0.005
6000
1.5
0.04
0.004
4000
NS
NS
1.0
2000
0.04
0.5
0
0.0
ACC
Cortisol-producing adenoma
PPC/ PGL
NFAA
ACC
Cortisol-producing adenoma
PPC/ PGL
NFAA
17-Hydroxypregnenolone (ng/ml) m
F
100
18.0
90
11-Deoxycortisol (ng/mL)
16.0
80
14.0
70
12.0
60
10.0
50
8.0
40
30
6.0
20
4.0
0.004
10
0.001
<0.001
<0.004
2.0
<0.001
<0.001
0
0.0
ACC
Cortisol-producing adenoma
PPC/ PGL
NFAA
ACC
Cortisol-producing adenoma
PPC/ PGL
NFAA
Fig. 2. Comparison of steroid concentrations between ACC and non-ACC adrenal lesions.
17-Hydroxyprogesterone (A); cortisol (B); DHEAS (C); androstenedione (D); 17-hydroxypregnenolone (E); and11-deoxycortisol (F). On each box-and-whisker plot, post hoc Bonferroni corrected P values are indicated. NS, nonsignificant.
Hydroxylase catalyzes 11-deoxycortisol conversion to cortisol within the inner mitochondrial membrane, un- der the control of corticotropin. Most of the other steroid pathway enzymes are located in the smooth endoplasmic reticulum. Disruption of mitochondrial oxidative phos-
phorylation is common in cancer, termed the “Warburg effect” (34), so 11ß-hydroxylase activity may be espe- cially impaired in ACC. Alternatively, increased concen- trations of steroid precursors could interfere with corti- cotropin release (22). CYP11B1 expression has been
Simultaneous Quantification of 13 Serum Steroids by LC-MS/MS
A
4
.
3
.
2
PC 2 (21.2%)
1
XD
Cortisol
0
17-Hydroxyprogesterone - Androstenedione
DHEAS
-1
-2
.
-3
.
-4
-2
-1
PC 1 (55.3%)
0
1
2
3
B
4
3
.
.
2
.
PC 2 (20.0%)
.
1
21-Deoxycortisol / DHEAS / 17-Hydroxypregnenolone
Pregnenolone
0
Androstenedione
~11-Deoxycortisol / 17-Hydroxyprogesterone
Cortisone
* Cortisol
00
11-Deoxycorticosterone / Corticosterone
-1
.
.
-2
.
-3
-2
-1
PC 1 (40.4%)
0
1
2
3
shown to be downregulated in ACC, along with several other steroidogenic enzymes (35). It may be that the heterogeneity of steroidogenesis observed in the current study is a reflection of variable loss of steroid synthetic pathway enzyme expression in each tumor. Whether this heterogeneity predicts pathological features or disease prognosis warrants further investigation.
Further studies are needed to evaluate the similari- ties and differences in qualitative and quantitative data produced by urine steroid profiling and serum steroid paneling. Quantification of serum pregnenolone and 17- hydroxypregnenolone were useful in ACC in the current study; however, the relative concentrations did not reflect the large amounts of their metabolites pregnanediol and pregnanetriol often seen in urine. This discrepancy may be because these 3ß-hydroxy-5-ene steroids are largely present in serum as sulfates, analogous to DHEAS. Other than DHEAS, the sulfated 3B-hydroxy-5-ene steroids are not measured by the LC-MS/MS method, but their sul- fated metabolites are measured by GC-MS, as free com- pounds after enzymatic hydrolysis (29). Nonetheless, the current study suggests that unconjugated pregnenolone and 17-hydroxypregnenolone are still useful ACC markers.
Urine steroid metabolite measurement may offer greater clinical sensitivity over single blood measure- ments because 24-h collections reflect steroid production throughout the day (20). Nonetheless, accurate 24-h col- lections are often not easily obtained and may be incon- venient to patients. Serum steroid paneling by LC- MS/MS offers a viable alternative and may also be more easily interpretable for clinicians because it targets the smaller number of major circulating steroids rather than the large number of urinary steroid metabolites. In many institutions, plasma metanephrine measurement is fa- vored for PCC/PGL exclusion in patients with large ad- renal masses in which ACC is in the differential diagno- sis. Combined plasma metanephrine and serum steroid panel measurements may be sufficient for the biochemi- cal exclusion of ACC or PCC/PGL.
Further work is needed to clarify the effects of diur- nal variation (36) and age and sex (37) on serum steroid paneling for ACC diagnosis. Our study used age- matched adrenal tumor groups with all samples collected in the morning to minimize these effects. Nonetheless, in
most cases, concentrations of the most useful ACC mark- ers exceed variations attributable to age, gender, or time of day; such increases are only otherwise encountered in forms of congenital adrenal hyperplasia (9).
The inherent limitations of steroid immunoassays for adrenal tumor evaluation are demonstrated again in our study. Although progesterone was detected by LC- MS/MS in 2 patients who had tested positive by immuno- assay, concentrations were much smaller. Pregnenolone and 17-hydroxypregenenolone sulfates are known 17- hydroxyprogesterone immunoassay interferences (38) and are potential progesterone immunoassay interferents. There was also evidence for interference in the androstenedione and 17-hydroxyprogesterone immunoassay results per- formed in the ACC cohort. Prediction of potential cross- reacting steroids is difficult because of steroid secretion het- erogeneity in ACC.
In summary, LC-MS/MS serum steroid paneling of- fers a potentially important advancement in the clinical workup of patients with adrenal lesions by combining the measurement of both common and rarely measured ste- roids in a single analysis. It supports the published con- clusions from urine steroid profiling that it is the in- creased concentrations of steroid synthetic pathway intermediates that best allow discrimination of ACC from non-ACC adrenal lesions.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 require- ments: (a) significant contributions to the conception and design, acquisi- tion of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon man- uscript submission, all authors completed the author disclosure form. Dis- closures and/or potential conflicts of interest:
Employment or Leadership: K .- M. Schulte, King’s College Hospital. Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: None declared.
Expert Testimony: None declared.
Patents: None declared.
Role of Sponsor: No sponsor was declared.
References
1. Grebe SK, Singh RJ. LC-MS/MS in the clinical laboratory- where to from here? Clin Biochem Rev 2011;32:5- 31.
2. Leung KS, Fong BM. LC-MS/MS in the routine clinical laboratory: has its time come? Anal Bioanal Chem 2014;406:2289-301.
3. Soldin SJ, Soldin OP. Steroid hormone analysis by tan- dem mass spectrometry. Clin Chem 2009;55:1061-6.
4. Couchman L, Vincent RP, Ghataore L, Moniz CF, Taylor
NF. Challenges and benefits of endogenous steroid analysis by LC-MS/MS. Bioanalysis 2011;3:2549-72.
5. Wooding KM, Auchus RJ. Mass spectrometry theory and application to adrenal diseases. Mol Cell Endocrinol 2013;371:201-7.
6. Guo TD, Taylor RL, Singh RJ, Soldin SJ. Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photo spray ionization tandem mass spectrometry. Clin Chim Acta 2006;372:76-82.
7. Fanelli F, Belluomo I, Di Lallo VD, Cuomo G, De Iasio R, Baccini M, et al. Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults. Steroids 2011;76:244-53.
8. Stolze BR, Gounden V, Gu J, Elliott EA, Masika LS, Abel BS, et al. An improved micro-method for the measure- ment of steroid profiles by APPI-LC-MS/MS and its use in assessing diurnal effects on steroid concentrations and
Simultaneous Quantification of 13 Serum Steroids by LC-MS/MS
optimizing the diagnosis and treatment of adrenal in- sufficiency and CAH. J Steroid Biochem Mol Biol 2016; 162:110-6.
9. Janzen N, Peter M, Sander S, Steuerwald U, Terhardt M, Holtkamp U, Sander J. Newborn screening for congen- ital adrenal hyperplasia: additional steroid profile using liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab 2007;92:2581-9.
10. O’Reilly MW, Taylor AE, Crabtree NJ, Hughes BA, Cap- per F, Crowley RK, et al. Hyperandrogenemia predicts metabolic phenotype in polycystic ovary syndrome: the utility of serum androstenedione. J Clin Endocrinol Metab 2014;99:1027-36.
11. Peitzsch M, Dekkers T, Haase M, Sweep FC, Quack I, Antoch G, et al. An LC-MS/MS method for steroid profil- ing during adrenal venous sampling for investigation of primary aldosteronism. J Steroid Biochem Mol Biol 2015;145:75-84.
12. Eisenhofer G, Dekkers T, Peitzsch M, Dietz AS, Bidlingmaier M, Treitl M, et al. Mass spectrometry- based adrenal and peripheral venous steroid profil- ing for subtyping primary aldosteronism. Clin Chem 2016;62:514-24.
13. Di Dalmazi G, Fanelli F, Mezzullo M, Casadio E, Rinaldi E, Garelli S, et al. Steroid profiling by LC-MS/MS in non- secreting and subclinical cortisol-secreting adrenocor- tical adenomas. J Clin Endocrinol Metab 2015;100: 3529-38.
14. Fassnacht M, Kroiss M, Allolio B. Update in adrenocor- tical carcinoma. J Clin Endocrinol Metab 2013;98: 4551-64.
15. Creemers SG, Hofland LJ, Korpershoek E, Franssen GJ, van Kemenade FJ, de Herder WW, Feelders RA. Future directions in the diagnosis and medical treatment of adrenocortical carcinoma. Endocr Relat Cancer 2016; 23:R43-69.
16. Challis BG, Kandasamy N, Powlson AS, Koulouri O, An- namalai AK, Happerfield L, et al. Familial adrenocortical carcinoma in association with Lynch syndrome. J Clin Endocrinol Metab 2016;101:2269-72.
17. Boland GW, Blake MA, Hahn PF, Mayo-Smith WW. Inci- dental adrenal lesions: principles, techniques, and al- gorithms for imaging characterization. Radiology 2008;249:756-75.
18. Icard P, Goudet P, Charpenay C, Andreassian B, Car- naille B, Chapuis Y, et al. Adrenocortical carcinomas: surgical trends and results of a 253-patient series from the French Association of Endocrine Surgeons study group. World J Surg 2001;25:891-7.
19. Hamrahian AH, Ioachimescu AG, Remer EM, Motta- Ramirez G, Bogabathina H, Levin HS, et al. Clinical util- ity of noncontrast computed tomography attenuation value (Hounsfield units) to differentiate adrenal adeno- mas/hyperplasias from nonadenomas: Cleveland Clinic experience. J Clin Endocrinol Metab 2005;90: 871-7.
20. Arlt W, Biehl M, Taylor AE, Hahner S, Libé R, Hughes BA, et al. Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumors. J Clin En- docrinol Metab 2011;96:3775-84.
21. Tiu SC, Chan AO, Taylor NF, Lee CY, Loung PY, Choi CH, Shek CC. Use of urinary steroid profiling for diagnosing and monitoring adrenocortical tumours. Hong Kong Med J 2009; 15:463-70.
22. Kerkhofs TM, Kerstens MN, Kema IP, Willems TP, Haak HR. Diagnostic value of urinary steroid profiling in the evaluation of adrenal tumors. Horm Cancer 2015;6: 168-75.
23. Blanes A, Diaz-Cano SJ. Histologic criteria for adreno- cortical proliferative lesions: value of mitotic figure vari- ability. Am J Clin Pathol 2007;127:398-408.
24. Blanes A, Sanchez-Carrillo JJ, Diaz-Cano SJ. Topo- graphic molecular profile of pheochromocytomas: role of somatic down-regulation of mismatch repair. J Clin Endocrinol Metab 2006;91:1150-8.
25. Diaz-Cano SJ. Clonality studies in the analysis of adre- nal medullary proliferations: application principles and limitations. Endocrine Pathol 1998;9:301-16.
26. Diaz-Cano SJ, de Miguel M, Blanes A, Tashjian R, Galera H, Wolfe HJ. Clonal patterns in pheochromocytomas and MEN-2A adrenal medullary hyperplasias: histo- logic and kinetic correlates. J Pathol 2000; 192:221-8.
27. Nieman LK. Approach to the patient with an adrenal incidentaloma. J Clin Endocrinol Metab 2010;95: 4106-13.
28. He X, Gabler J, Yuan C, Wang S, Shi Y, Kozak M. Quan- titative measurement of plasma free metanephrines by ion-pairing solid phase extraction and liquid
chromatography-tandem mass spectrometry with po- rous graphitic carbon column. J Chromatogr B Analyt Technol Biomed Life Sci 2011;879:2355-9.
29. Taylor NF. Urine steroid profiling. Methods Mol Biol 2013;1065:259-76.
30. Bonfiglio R, King RC, Olah TV, Merkle K. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug com- pounds. Rapid Commun Mass Spectrom 1999;13: 1175-85.
31. Dennedy MC, Annamalai AK, Prankerd Smith O, Free- man N, Vengopal K, Graggaber J, et al. Low DHEAS: a sensitive and specific test for detection of subclinical hypercortisolism in adrenal incidentalomas. J Clin En- docrinol Metab 2017;102:786-92.
32. Yamaji T, Ishibashi M, Sekihara H, Itabashi A, Yanaihara T. Serum dehydroepiandrosterone sulfate in Cushing’s syndrome. J Clin Endocrinol Metab 1984;59:1164-8. 33. Doerr HG, Sippell WG, Drop SL, Bidlingmaier F, Knorr D. Evidence of 11 beta-hydroxylase deficiency in child- hood adrenocortical tumors. The plasma corticosterone/ 11-deoxycorticosterone ratio as a possible marker for ma- lignancy. Cancer 1987;60:1625-9.
34. Wallace DC. Mitochondria and cancer. Nat Rev Cancer 2012;12:685-98.
35. Ragazzon B, Assié G, Bertherat J. Transcriptome analy- sis of adrenocortical cancers: from molecular classifica- tion to the identification of new treatments. Endocr Relat Cancer 2011;18:R15-27.
36. Stolze BR, Gounden V, Gu J, Abel BS, Merke DP, Skaru- lis MC, Soldin SJ. Use of micro-HPLC-MS/MS method to assess diurnal effects on steroid hormones. Clin Chem 2015;61:556-8.
37. Eisenhofer G, Peitzsch M, Kaden D, Langton K, Pampo- raki C, Masjkur J, et al. Reference intervals for plasma concentrations of adrenal steroids measured by LC-MS/ MS: impact of gender, age, oral contraceptives, body mass index and blood pressure status. Clin Chim Acta 2017;470:115-24.
38. Wong T, Shackleton CH, Covey TR, Ellis G. Identification of the steroids in neonatal plasma that interfere with 17 alpha-hydroxyprogesterone radioimmunoassays. Clin Chem 1992;38:1830-7.