Simplified urinary steroid profiling by LC-MS as diagnostic tool for malignancy in adrenocortical tumors.

OBJECTIVES: Preoperative identification of malignant adrenal tumors is challenging. 24-h urinary steroid profiling by LC-MS/MS and machine learning has demonstrated high diagnostic power, but the unavailability of bioinformatic models for public use has limited its routine application. We here aimed to increase usability with a novel classification model for the differentiation of adrenocortical adenoma (ACA) and adrenocortical carcinoma (ACC). METHODS: Eleven steroids (5-pregnenetriol, dehydroepiandrosterone, cortisone, cortisol, α-cortolone, tetrahydro-11-deoxycortisol, etiocholanolone, pregnenolone, pregnanetriol, pregnanediol, and 5-pregnenediol) were quantified by LC-MS/MS in 24-h urine samples from 352 patients with adrenal tumor (281 ACA, 71 ACC). Random forest modelling and decision tree algorithms were applied in training (n = 188) and test sets (n = 80) and independently validated in 84 patients with paired 24-h and spot urine. RESULTS: After examining different models, a decision tree using excretions of only 5-pregnenetriol and tetrahydro-11-deoxycortisol classified three groups with low, intermediate, and high risk for malignancy. 148/217 ACA were classified as being at low, 67 intermediate, and 2 high risk of malignancy. Conversely, none of the ACC demonstrated a low-risk profile leading to a negative predictive value of 100% for malignancy. In the independent validation cohort, the negative predictive value was again 100% in both 24-h urine and spot urine with a positive predictive value of 87.5% and 86.7%, respectively. CONCLUSIONS: This simplified LC-MS/MS-based classification model using 24-h-urine provided excellent results for exclusion of ACC and can help to avoid unnecessary surgeries. Analysis of spot urine led to similarly satisfactory results suggesting that cumbersome 24-h urine collection might be dispensable after future validation.

Gas Chromatography-Mass Spectrometry Analysis of Urinary Steroid Metabolomics for Detection of Early Signs of Adrenal Neoplasm Malignancy in Patients with Cushing's Syndrome.

The metabolomics of urinary steroids was studied by gas chromatography-mass spectrometry in 25 patients with Cushing's syndrome without malignant potential and in 12 patients with malignant potential of adrenal neoplasms (Weiss score 1-3). Patients with adrenocortical adenoma (N=24) constituted the control group. In patients with Cushing's syndrome and malignant potential, increased urinary excretion of 16-oxo-androstendiol, tetrahydro-11-deoxycortisol, and 16-hydroxypregnendiol, which had 100% specificity and sensitivity >90% for the diagnosis of malignant potential. Additionally, non-classical 5-ene-pregnenes (16-OHpregnenolone, 21-OH-pregnenolone, 3ß,16,20-pregnentriol, and 3ß,17,20-pregnentriol) were identified. The revealed changes in the metabolomics of steroids can be early signs of malignant potential in patients with Cushing's syndrome. In patients with malignant potential, three signs of reduced activity of 11ß-hydroxysteroid dehydrogenase type 2 were detected and in patients without malignant potential, one sign was found. In patients with and without malignant potential, three signs increased activity of 5ß-reductase were found.

High-Resolution, Accurate-Mass (HRAM) Mass Spectrometry Urine Steroid Profiling in the Diagnosis of Adrenal Disorders.

BACKGROUND: Steroid profiling is a promising diagnostic tool with adrenal tumors, Cushing syndrome (CS), and disorders of steroidogenesis. Our objective was to develop a multiple-steroid assay using liquid-chromatography, high-resolution, accurate-mass mass spectrometry (HRAM LC-MS) and to validate the assay in patients with various adrenal disorders. METHODS: We collected 24-h urine samples from 114 controls and 71 patients with adrenal diseases. An HRAM LC-MS method was validated for quantitative analysis of 26 steroid metabolites in hydrolyzed urine samples. Differences in steroid excretion between patients were analyzed based on Z-score deviation from control reference intervals. RESULTS: Limits of quantification were 20 ng/mL. Dilution linearity ranged from 80% to 120% with means of 93% to 110% for all but 2 analytes. Intraassay and interassay imprecision ranged from 3% to 18% for all but 1 analyte. Control women had lower excretion of androgen and glucocorticoid precursors/metabolites than men (P < 0.001), but no difference in mineralocorticoids was seen (P = 0.06). Androgens decreased with age in both sexes (P < 0.001). Compared with patients with adrenocortical adenoma (ACA), patients with adrenocortical carcinoma (ACC) had 11 steroids with increased Z scores, especially tetrahydro-11-deoxycortisol (14 vs 0.5, P < 0.001), pregnanetriol (7.5 vs -0.4, P = 0.001), and 5-pregnenetriol (5.4 vs -0.4, P = 0.01). Steroid profiling also demonstrated metabolite abnormalities consistent with enzymatic defects in congenital adrenal hyperplasia and differences in pituitary vs adrenal CS. CONCLUSIONS: Our HRAM LC-MS assay successfully quantifies 26 steroids in urine. The statistically significant differences in steroid production of ACC vs ACA, adrenal vs pituitary CS, and in congenital adrenal hyperplasia should allow for improved diagnosis of patients with these diseases.

Diagnosis of a malignant adrenal mass: the role of urinary steroid metabolite profiling.

PURPOSE OF REVIEW: Adrenal masses are highly prevalent, found in 5% of the population. Differentiation of benign adrenocortical adenoma from adrenocortical carcinoma is currently hampered by the poor specificity and limited evidence base of imaging tests. This review summarizes the results of studies published to date on urine steroid metabolite profiling for distinguishing benign from malignant adrenal masses. RECENT FINDINGS: Three studies have described cohorts of at least 100 patients with adrenal tumors showing significant differences between urinary steroid metabolite excretions according to the nature of the underlying lesion, suggesting significant value of steroid metabolite profiling as a highly accurate diagnostic test. SUMMARY: Steroid profiling is emerging as a powerful novel diagnostic tool with a significant potential for improving the management for patients with adrenal tumors. Although the current studies use gas chromatography-mass spectrometry for proof of concept, widespread use of the method in routine clinical care will depend on transferring the approach to high-throughput tandem mass spectrometry platforms. The use of computational data analysis in conjunction with urine steroid metabolite profiling, that is, steroid metabolomics, adds accuracy and precision.

Different Types of Urinary Steroid Profiling Obtained by High-Performance Liquid Chromatography and Gas Chromatography-Mass Spectrometry in Patients with Adrenocortical Carcinoma.

Urinary steroid profiling (USP) was studied using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) methods in 108 patients with adrenocortical adenoma (ACA) and in 31 patients with adrenocortical carcinoma (ACC). Thirteen ACC and Cushing's syndrome (ACC-CS) patients had two types of USP as well as 18 ACC patients without hypercortisolism. These four types differed by androgen and glucocorticoid secretion of the adrenal cortex. Fifteen main ACC features were observed by GC-MS. Urinary excretion of dehydroepiandrosterone (DHEA) was increased in 67.7 % of ACC patients and tetrahydro-11-deoxycortisol (THS) in 74.2 %. By combination of the following parameters: THS >900 µg/24 h and/or DHEA >1500 µg/24 h with ratios of 3α,16,20-pregnentriol/3ß,16,20-pregnentriol (3α,16,20dP3/3ß,16,20dP3) less than 6.0 and 3α,17,20dP3/3ß,17,20dP3 less than 9.0 and the detection of "non-classical" 5-en-pregnens, not found in ACA and healthy persons, 100 % sensitivity and specificity of ACC and ACA differential diagnosis were achieved. Features of 21-hydroxylase and 11ß-hydroxylase deficiency were observed by GC-MS in 32.2 and 61.3 % of the ACC patients, respectively. Additional features for ACC-CS diagnostic were increased urinary excretion of 6ß-hydroxycortisol, 18-hydroxycorticosterone, the sum (UFF + UFE) obtained by HPLC, tetrahydrocorticosterone, and the sum (THF + THE + allo-THF) obtained by GC-MS.

Enhanced Soluble Serum CD40L and Serum P-Selectin Levels in Primary Aldosteronism.

Primary aldosteronism (PA) is one of the most frequent forms of secondary hypertension, associated with atherosclerosis and higher risk of cardiovascular events. Platelets play a key role in the atherosclerotic process. The aim of the study was to evaluate the platelet activation by measuring serum levels of soluble CD40L (sCD40L) and P-selectin (sP-selectin) in consecutive PA patients [subgroup: aldosterone-secreting adrenal adenoma (APA) and bilateral adrenal hyperplasia (IHA)], matched with essential hypertensive (EH) patients. The subgroup of APA patients was revaluated 6-months after unilateral adrenalectomy. In all PA group, we measured higher serum levels of both sP-selectin (14.29±9.33 pg/ml) and sCD40L (9.53±4.2 ng/ml) compared to EH patients (9.39±5.3 pg/ml and 3.54±0.94 ng/ml, respectively; p<0.001). After removal of APA, PA patients showed significant reduction of blood pressure (BP) values, plasma aldosterone (PAC) levels and ARR-ratio, associated with a significant reduction of sP-selectin (16.74±8.9 pg/ml vs. 8.1±3.8 pg/ml; p<0.01) and sCD40L (8.6±1 ng/ml vs. 5.24±0.94 ng/ml; p<0.001). In PA patients, we found a significant correlation between sP-selectin and sCD40L with PAC (r=0.52, p<0.01; r=0.50, p<0.01, respectively); this correlation was stronger in APA patients (r=0.54; p<0.01 r=0.63; p<0.01, respectively). Our results showed that PA is related to platelet activation, expressed as higher plasma values of sCD40L and sP-selectin values. Surgical treatment and consequent normalization of aldosterone secretion was associated with significant reduction of sCD40L and sP-selectin values in APA patients.

Adrenocortical adenomas with regression and myelolipomatous changes: urinary steroid profiling supports a distinctive benign neoplasm.

BACKGROUND: Adrenocortical neoplasms are classically divided into adenomas (ACA) and carcinomas (ACC). Heterogeneous appearance and greater size are criteria to suggest malignancy, along with the urinary steroid profile (USP). The presence of regression and myelolipomatous changes in adenomas (ACA-RML) can contribute to confusion with ACC and its USP remains unknown. OBJECTIVE: To evaluate the features of ACA-RML in comparison with other adrenocortical neoplasms. METHODS: We selected consecutive ACA (11), ACA-RML (7) and ACC (13) cases for which USP analysis was performed before surgery and tissue was available for histological evaluation (King's College Hospital, 2005-2012). Cases were classified according to WHO and Armed Forces Institute of Pathology criteria. USPs were obtained by gas chromatography/mass spectrometry. Total excretion of individual steroids and indices (sums and ratios chosen to reflect steroid metabolic activity) were compared between ACA-RML, ACA and ACC. RESULTS: In comparison with ACA, tumours in ACA-RML were significantly larger (8·5 ± 2·4 vs 3·5 ± 1·0, P = 0·002), presented in older patients and showed relatively higher incidence in males. Mitotic figure counts were significantly lower (0·39 ± 0·04 vs 0·93 ± 0·11 in ACA, P = 0·001) and revealed higher frequency of apoptotic cells (100% vs 9% in ACA, P = 0·001). The USP of ACA-RML showed no diagnostic features of ACC. No differences from ACA were significant, but there was a tendency towards lower dehydroepiandrosterone DHA and DHA metabolites. CONCLUSIONS: ACA-RML reveals distinctive histological features and lack of USP markers of malignancy. More cases of this rare tumour may confirm differences from ACA in steroid excretion. It is important to recognize ACA-RML because its size and heterogeneous appearance raise the possibility of ACC.

Plasma and urinary metanephrines determined by an enzyme immunoassay, but not serum chromogranin A for the diagnosis of pheochromocytoma in patients with adrenal mass.

Adrenal pheochromocytomas are neoplasms characterized by catecholamine excess. Determination of metanephrines by high-pressure liquid chromatography has been well established for the diagnosis of pheochromocytomas, demonstrating high sensitivity and specificity. This study evaluates the diagnostic value of newly available enzyme immunoassays for metanephrines in plasma and urine. Chromogranin A was studied as a potential additional diagnostic tool. Spontaneous blood samples and 24-h urine samples were collected in 149 subjects, including 24 histologically proven pheochromocytomas, 17 aldosterone-secreting and 21 cortisol-secreting adrenal adenomas, 30 nonfunctioning adrenal masses, 15 patients with essential hypertension and 42 healthy normotensive volunteers. Plasma and urinary metanephrine and normetanephrine as well as chromogranin A were determined and putative thresholds were calculated by ROC analysis. Plasma free normetanephrine was found to be the best single parameter with the highest sensitivity (89.5%) and specificity (98.3%) using a threshold of 167 pg/ml. Analysis of the combination of plasma free metanephrines revealed a similar sensitivity with lower specificity of 90.0%. Considering both urinary parameters demonstrated a slightly higher sensitivity (92.9%) with lower specificity (77.6%). ROC analysis revealed a threshold of 215 µg/l for chromogranin A with rather low sensitivity (73.9%) and specificity (74.2%). A weak positive correlation was found between the tumor size of pheochromocytomas and plasma metanephrine (r = 0.53, p ≤ 0.05) as well as chromogranin A (r = 0.60, p ≤ 0.01). In conclusion, plasma free and urinary metanephrines measured by enzyme immunoassays are convenient and reliable parameters for the diagnosis of pheochromocytoma. In contrast, CgA demonstrated poor sensitivity and specificity.

Pattern of adrenal hormonal secretion in patients with adrenal adenomas: the relevance of aldosterone in arterial hypertension.

CONTEXT: Approximately 10% of hypertensives are considered to exhibit autonomous aldosterone secretion (AAS). Although adrenal incidentalomas (AI) can be found in up to 19% of hypertensive individuals, data on the incidence of AAS in hypertensive patients with AI remain scarce. OBJECTIVE: The aim was to study adrenal aldosterone (ALD) secretory pattern in patients with adrenal adenomas with and without arterial hypertension. DESIGN AND SETTING: We conducted a case-control study in a tertiary general hospital. PATIENTS AND MAIN OUTCOME MEASURES: We investigated 72 normotensive subjects with normal adrenal morphology and 191 subjects divided in three groups: 46 normotensive individuals with an AI (NAI), 89 hypertensive patients with an AI (HAI), and 56 hypertensive patients with an adrenal adenoma identified after investigation for arterial hypertension (HAA). Evaluation of autonomous cortisol secretion was based on a low-dose dexamethasone suppression test. Autonomous ALD secretion was based on a modified saline infusion test (MSI). Normal cutoff levels were obtained from the control matched population. RESULTS: Post-MSI ALD levels and the ALD/renin (REN) ratios were significantly elevated in HAI and HAA patients compared to NAI subjects. To evaluate the prevalence of AAS, we applied the combination of post-MSI ALD level and the ALD/REN ratio simultaneously (post-MSI cutoffs, ALD levels, 2.41 ng/dl; ALD/REN ratio, 0.35 ng/dl/µU/ml). Based on these cutoffs, 12% of NAI, 36.4% of HAI, and 54.2% of HAA patients had AAS. The prevalence of autonomous cortisol secretion did not differ among the three groups. CONCLUSIONS: Using a MSI test, we found a remarkably increased prevalence of AAS in hypertensive patients with adrenal adenomas, even when the latter represented an incidental finding.

Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumors.

CONTEXT: Adrenal tumors have a prevalence of around 2% in the general population. Adrenocortical carcinoma (ACC) is rare but accounts for 2-11% of incidentally discovered adrenal masses. Differentiating ACC from adrenocortical adenoma (ACA) represents a diagnostic challenge in patients with adrenal incidentalomas, with tumor size, imaging, and even histology all providing unsatisfactory predictive values. OBJECTIVE: Here we developed a novel steroid metabolomic approach, mass spectrometry-based steroid profiling followed by machine learning analysis, and examined its diagnostic value for the detection of adrenal malignancy. DESIGN: Quantification of 32 distinct adrenal derived steroids was carried out by gas chromatography/mass spectrometry in 24-h urine samples from 102 ACA patients (age range 19-84 yr) and 45 ACC patients (20-80 yr). Underlying diagnosis was ascertained by histology and metastasis in ACC and by clinical follow-up [median duration 52 (range 26-201) months] without evidence of metastasis in ACA. Steroid excretion data were subjected to generalized matrix learning vector quantization (GMLVQ) to identify the most discriminative steroids. RESULTS: Steroid profiling revealed a pattern of predominantly immature, early-stage steroidogenesis in ACC. GMLVQ analysis identified a subset of nine steroids that performed best in differentiating ACA from ACC. Receiver-operating characteristics analysis of GMLVQ results demonstrated sensitivity = specificity = 90% (area under the curve = 0.97) employing all 32 steroids and sensitivity = specificity = 88% (area under the curve = 0.96) when using only the nine most differentiating markers. CONCLUSIONS: Urine steroid metabolomics is a novel, highly sensitive, and specific biomarker tool for discriminating benign from malignant adrenal tumors, with obvious promise for the diagnostic work-up of patients with adrenal incidentalomas.

Use of urinary steroid profiling for diagnosing and monitoring adrenocortical tumours.

It has been suggested that urinary steroid profiling may be used to provide information aiding the diagnosis and monitoring of adrenocortical carcinoma. Nonetheless, the abnormal patterns suggestive of adrenal malignancy are not well defined. We retrospectively studied the urinary steroid profiles of five patients with adrenocortical carcinoma at presentation and at follow-up, and compared these results with those from 76 patients with benign adrenocortical adenoma and 172 healthy controls. Three abnormal patterns of urinary steroid excretion were identified in patients with adrenocortical carcinoma at presentation and/or follow-up of residual disease: (1) hypersecretion in multiple steroid axes; (2) excretion of unusual metabolites, notably 5-pregnene-3alpha,16alpha,20alpha-triol, 5-pregnene-3beta,16alpha,20alpha-triol, and neonatal steroid metabolites in the post-neonatal period; (3) increase of tetrahydro-11-deoxycortisol relative to total cortisol metabolites. These preliminary findings offer ways in which urinary steroid profiling performed using gas chromatography-mass spectrometry can be helpful in the diagnosis and monitoring of adrenocortical carcinoma.

Adipokine levels and cardiovascular risk in patients with adrenal incidentaloma.

Adrenal incidentalomas (AIs) have been associated with an increased incidence of several cardiovascular risk factors, similar to overt Cushing syndrome. Data about the involvement of the adipokines in the development of insulin resistance and atherosclerosis in AI are completely lacking. The aim of the present study was to evaluate plasma interleukin 6 (IL-6), adiponectin, resistin, tumor necrosis factor alpha (TNF-alpha), and monocyte chemoattractant protein 1 (MCP-1) levels in patients with AI. Plasma IL-6, adiponectin, resistin, TNF-alpha, and MCP-1 levels were measured in 20 healthy subjects (6 males; 14 females; age, 58.5 +/- 2.2 years; body mass index, 28.1 +/- 0.9 kg/m(2)) and in 20 patients (5 males; 15 females; age, 57.9 +/- 2.0 years; body mass index, 28.0 +/- 0.8 kg/m(2)) with AI and typical computed tomographic features of cortical adenoma, who were not affected by diabetes mellitus, hypertension, or other relevant diseases. All patients underwent anthropometric measurements and determination of basal corticotropin, cortisol, and urinary free cortisol excretion. Overnight dexamethasone test and 250-microg corticotropin test were performed in all cases. A subclinical Cushing syndrome was found in 3 patients, whereas the others had apparently nonfunctioning masses. Plasma IL-6, adiponectin, resistin, TNF-alpha, and MCP-1 levels were higher in patients than in controls (64.4 +/- 2.8 vs 5.5 +/- 0.6 pg/mL, 13.7 +/- 1.3 vs 3.6 +/- 0.5 microg/mL, 12.5 +/- 1.9 vs 5.1 +/- 0.2 ng/mL, 27.0 +/- 1.5 vs 22.2 +/- 1.5 pg/mL, 172.5 +/- 20.0 vs 104.4 +/- 19.5 pg/mL, respectively; P < .05) and apparently not affected by the presence of visceral obesity. Plasma IL-6 levels were negatively correlated with urinary free cortisol (r = -0.461, P < .05), and TNF-alpha levels were positively correlated with cortisol after the administration of 1 mg dexamethasone (r = 0.636, P < .01). In conclusion, patients with AI may show increased levels of adipokines (apparently not related to the presence of diabetes, hypertension, or obesity), which may be affected by the presence of the adrenal adenoma. For some adipokines, a direct production from the adrenal gland may be hypothesized even if other studies are needed to better investigate the role of adipokines in states of altered cortisol secretion.

A functioning black adenoma of the adrenal gland.

A 53-year-old female had clinical and laboratory findings suggestive of Cushing's syndrome. In contrast to the Cushing's syndrome caused by cortical adenoma, a high level of urinary 17-ketosteroids (17-KS) was also noted. Imaging studies revealed a right adrenal tumor. Right adrenectomy was performed; the surgical specimen revealed a black adenoma consisting of compact cells with numerous pigments which seemed to be lipofuscin in nature. The present case indicates that black adenoma as well as adrenocortical carcinoma should be suspected, when patients with Cushing's syndrome show an increased level of urinary 17-KS excretion.

[Pre-toxic adrenocortical adenoma ("pre-Cushing syndrome"): role of 17-hydroxyprogesterone dosage under ACTH analog stimulation. Apropos of a case]. / Adénome surrénalien pré-toxique ("syndrome de pré-Cushing"): intérêt du dosage de la 17-hydroxyprogestérone sous stimulation par analogue de l'ACTH a propos d'un cas.

We report on a case of a 63-year-old male patient who presented with an adrenal incidentaloma corresponding to a pre-toxic adrenocortical adenoma ("pre-Cushing's syndrome"). Nycthemeral cortisol cycle and free urinary cortisol were within the normal range. Basal ACTH and dehydroepiandrosterone sulfate levels were decreased and the 17-hydroxyprogesterone (17OHP) response under ACTH stimulation (tetracosactide) was increased. The ACTh test appears mandatory in the incidentaloma work-up to order to identify the pre-toxic adenomas. Its elevation reflects likely intra-tumoral enzymatic defects. Pre-Cushing's syndrome therapeutic management is still debated. No surgery was performed in our patient. A 3-year follow-up did not show overt Cushing's syndrome features.

An unusual profile of endocrine study in a patient with pre-Cushing's syndrome.

A unilateral adrenal tumour was incidentally detected in a 39-year-old woman with no characteristic features of Cushing's syndrome. Basal levels of glucocorticoids were within normal limits. However, abnormal pattern of plasma cortisol and ACTH was observed. The dexamethasone suppression test and the metyrapone test showed also abnormal response. Adrenocortical scintigram demonstrated high accumulation of the radiopharmaceutical in the tumour region alone. Final diagnosis was "pre-Cushing's syndrome" and a solitary adenoma was removed from the left adrenal gland.