6 Serum steroid profiling by mass spectrometry in URRENT PINION adrenocortical tumors: diagnostic implications
Flaminia Fanelli and Guido Di Dalmazi
Purpose of review
Liquid chromatography-tandem mass spectrometry (LC-MS/MS), allowing the reliable measurement of large panels of steroids, opened a new era in the characterization of adrenal diseases. This review summarizes the most recent findings on serum steroid profile in benign adrenocortical tumors and provides a focus on the most promising analytical developments.
Recent findings
Recently developed LC-MS/MS assays included challenging compounds, providing new knowledge on adrenal steroid secretion. Pioneering studies highlighted the potential of incoming technologies in increasing measurement selectivity and implementing the steroidomic approach. In primary aldosteronism, several studies highlighted the signature of aldosterone-producing adenomas, mainly characterized by secretion of hybrid steroids. The combination of steroid panel and radiological data reached an agreement with adrenal vein sampling-based classification in more than 80% of the cases. The serum steroid profiling in patients with Cushing’s syndrome, mainly characterized by reduced androgens and increased 11- dexoycorticosterone in adrenal hypercortisolism, showed a good discriminant power for patients’ subtyping (90% correct classification rate). Finally, a selected panel of steroids, including 11-deoxycortisol as the main discriminant compound, was able to achieve a good separation of patients with and without adrenocortical carcinomas.
Summary
The constantly evolving serum steroid profiling by MS may improve the diagnosis of different types of adrenocortical tumors.
Keywords
adrenocortical tumors, mass spectrometry, serum steroid profiling
INTRODUCTION
In the last decade, the advent of serum steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) boosted the investiga- tion of adrenal cortex diseases. This technique sen- sibly improved the quality of the measurement of routine-assayed steroids while enlarging the spec- trum of measurable steroids on a multianalyte approach, thereby triggering a renewed interest on misknown or neglected steroid players. LC- MS/MS is now consolidating its value in the charac- terization and in the refinement of diagnostic para- digms, with important clinical implications for several adrenocortical diseases. This review summa- rizes the most recent advancement in the LC and MS analytical field and the clinical implications of serum steroid profiling in patients with benign adrenocortical tumors.
SERUM STEROID PROFILING BY MASS SPECTROMETRY
Advancements in liquid chromatography- tandem mass spectrometry
Analyte measurement by LC-MS/MS generally includes off-line and/or online serum extraction by polarity affinity, separation by reversed-phase
Department of Medical and Surgical Sciences, Endocrinology Unit and Center for Applied Biomedical Research (CRBA), S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy
Correspondence to Guido Di Dalmazi, MD, Department of Medical and Surgical Sciences, Endocrinology Unit, University of Bologna, via Mas- sarenti 9, 40138 Bologna, Italy. Tel: +39 051 2143009; e-mail: guido.didalmazi@unibo.it
Curr Opin Endocrinol Diabetes Obes 2019, 26:160-165 DOI:10.1097/MED.0000000000000475
KEY POINTS
. Novel advancement in the field of mass spectrometry is improving the measurement selectivity and implementing the steroidomic profiling.
· Steroid profiling in primary aldosteronism has shown implication for patients’ subtyping (unilateral versus bilateral forms) with potential future application in the diagnosis of the disease.
· The measurement of a single serum steroid panel in Cushing’s syndrome may provide good power in disease subtyping and potentially simplify the diagnostic workup of hypercortisolism.
· Patients with adrenocortical cancer have a specific steroid fingerprint, even though a large interindividual heterogeneity is frequently observed. Future studies are needed to address the potential implication of serum steroid profiling for diagnosis and tailored follow-up of the patients.
LC, soft ionization, targeted MS/MS detection of precursor-to-product ion transitions on triple quad- rupole instruments, and quantitation by isotopic dilution.
Together with aldosterone and cortisol, major androgens and progestogens, LC-MS/MS panels for the study of the adrenal secretion usually include 11-deoxycortisol, 21-deoxycortisol, corticosterone, and 11-deoxycorticosterone. The utility of these intermediates, rarely available in clinical laborato- ries using immunoassays, is consolidating in the characterization of different forms of adrenal tumors. Nonetheless, the thorough picture of adre- nal steroidogenesis still misses important actors. Recently developed large LC-MS/MS panels (up to 16 steroids) include the upper chain precursors pregnenolone and 17OH-pregnenolone [1-3]. How- ever, these hormones are still analytically challeng- ing and few and somehow discordant data are available on their circulating reference values [4]. Notably, two studies recently addressed the inter- mediates of glucocorticoid and mineralocorticoid pathways 18OH-11-deoxycorticosterone, 18OH- corticosterone, 11-dehydrocorticosterone [5], 18OHF, and 18oxoF [6], also providing values in the healthy children and adults [5,7].
Technological evolutions in chromatography and mass spectrometry
Owing to the countless number of metabolites, their extreme physicochemical similarities, and the impressive variability in the circulating levels, the quantification of steroid hormones is still a
challenge [8]. Triple quadrupole instruments display ideal sensitivity and linearity for quantitative scopes. However, they have low (unit) spectral reso- lution, meaning that they cannot distinguish between compounds with the same molecular mass. The complexity of the steroidome, that is, the total- ity of steroid compounds pertaining to all possible pathways, is such that isomeric and isobaric com- pounds occur with tremendous frequency. More- over, due to structural similarity, steroids most often generate overlapping fragmentation spectra. Therefore, in triple quadrupole MS/MS detection, steroid selectivity largely relies on chromatography. Keeping together utmost specificity, sensitivity, dynamic range, and practicability is a compromise for available technologies; nonetheless, irrepressible evolutions are on their way to solve these constrains.
Novel chromatographic approaches
Supercritical fluid chromatography (SFC) promises to improve LC specificity to a level close to gas chromatography (GC), while preserving its high throughput. In principle, the diffusivity of liquid pressurized CO2, serving as a mobile phase, induces effective separation of complex steroid mixtures without derivatization processes required by GC. Moreover, CO2 volatility results in enhancing the ionization at the MS interface. Applications of SFC- MS/MS to serum steroid profiling were recently reported. du Toit et al. [9] reported the measurement of a 31-steroid panel in 6 min, for profiling classical, alternative, backdoor, and 11OH-androstenedione adrenal pathways in a surrogate serum matrix. de Kock et al. [10] reported the sensitive quantitation of 19 among progestogens, corticosteroids, androgens, and estrogens in 50 ul of plasma in 5 min. However, profiling results in study cohorts are not yet available.
Novel mass spectrometry approaches
High-resolution accurate MS (HRMS) operated by hybrid Q-ion trap and Q-time-of-flight (QTOF) instruments are opening a novel analytical era com- bining quantitative and qualitative information into multidimensional data set. HRMS platforms can differentiate steroid isobars on the basis of mass differences in the fourth/fifth decimal place, allow- ing targeted quantitative experiments by single ion monitoring or by MS/MS. In addition, various acqui- sition modes allow for untargeted, that is, not a priori defined, profiling in the same run. Effective sample extraction and LC are still mandatory to reduce the background noise and to ensure selectivity among steroid isomers, that is, sharing the same elemental composition that cannot be distinguished by HRMS.
Matysik et al. [11”] developed the Q-ion trap MS/ HRMS quantitation of cortisone, cortisol, 11-deox- ycortisol, corticosterone, 17OH-progesterone, pro- gesterone, androstenedione, and testosterone using 100 ul serum in less than 6 min. Imprecision and sensitivity (0.02 ng/ml for cortisone to 2 ng/ml of cortisol) were comparable to most of the modern LC-MS/MS assays, allowing the analysis of authen- tic clinical samples. Notably, HRMS high selectivity was demonstrated in the detection of steroid typical fragment ions. Drotleff et al. [12”] illustrated the impressive potential of data-independent acquisi- tion with sequential acquisition of all theoretical fragment-ion mass spectra operated by QTOF-MS/ MS in providing specific and sensitive targeted abso- lute quantitation of estradiol and testosterone (down to 10 and 20 pg/ml, respectively), combined with the untargeted scan mode allowing postacqui- sition relative quantitation of a multitude of ste- roids, in 500 ul serum and 5 min run.
CLINICAL IMPLICATIONS OF SERUM STEROID PROFILING IN ADRENOCORTICAL TUMORS
Primary aldosteronism
In the last years, much research has been focused on understanding the genetic and molecular back- ground of adrenocortical tumors and hormone hypersecretion. In primary aldosteronism, the piv- otal discovery of somatic mutations of KCNJ5, ATP2B3, ATP1A1, CACNA1D, and CACNA1H have revealed the pathophysiological basis of most aldo- sterone-producing tumors (APA), which were largely unknown until recently [13]. Alongside the recent ‘genetic breakthrough,’ the analysis of large panels of steroids in patients with primary aldosteronisms has given an important contribution in understand- ing the mechanisms of aldosterone hyperproduction.
The potential clinical implications of measuring steroid precursors and 18-oxygenated metabolites as a diagnostic aid in primary aldosteronism have been postulated decades ago. However, the unavailability of practicable measurement procedures hindered the advancement in this field [14,15]. By applying LC-MS/MS for measurement of 18-oxocortisol and 18-hydroxycortisol in plasma derived from periph- eral and adrenal veins, during adrenal vein sampling (AVS), it was clear that measuring those hybrid steroids may improve the discrimination of APA from bilateral hyperplasia (BAH), being selectively elevated in unilateral tumors [16,17]. Those results were confirmed and extended in a recent study analyzing a large panel of steroids, including hybrid compounds, in blood taken from AVS and
peripheral vein specimens. Even though peripheral 18-oxocortisol was clearly elevated in patients with APA versus BAH, the diagnostic accuracy in subtype differentiation was severely improved by consider- ing the entire panel of 15 steroids, rather than single values of hybrid steroids [18]. This study confirmed that the potential usefulness of MS-based analysis of several steroids is higher when those compounds are analyzed as a whole, rather than individually. The association with peripheral serum steroid profile and adrenal morphology has been recently analyzed more in depth, in a study including 197 patients with AVS-based diagnosis of BAH and APA. After dividing the patients with APA in micro and macro- APA (maximum diameter less than 10 and at least 10 mm, respectively, based on histopathology find- ings), Yang et al. [19] identified a specific steroid fingerprint for those subtypes of disease, with aldo- sterone and 18-hydroxycortisol as major discrimi- nant markers. Additionally, 11-deoxycorticosterone and androgen precursors Dehydroepiandrosterone (DHEA) and Dehydroepiandrosterone Sulfate (DHEAs) were also significantly different between macro-APA and BAH. Interestingly, the whole 15- steroid profile showed a good agreement with AVS in identifying monolateral or bilateral diseases (89% of the cases). The analysis by random forest algo- rithm to predict the outcome of patients, defined according to a previously published international consensus [20], showed that the combination of computerized tomography (CT)-scan data with the 15-steroid profile had the same accuracy as AVS in 83% of the cases [19]. Therefore, if those criteria were applied to the total cohort as a part of the diagnostic workup, only a minority of the patients with discordant or inaccurate results would have needed AVS to define the subtype of primary aldo- steronism. Thus, if those results are confirmed by independent studies, it is tempting to speculate that a single measurement of a large panel of steroids and CT-scan data may produce sufficient amount of information to avoid performing AVS in most of the patents with primary aldosteronism.
Overall, the serum steroid profiling in patients with primary aldosteronism has reinforced the con- cept of the complexity of steroid secretion in this pathological entity, apart from aldosterone. The analysis of large panels of steroids highlighted piv- otal differences between APA and BAH and a revis- ited role of hybrid steroids, as markers of disease rather than compounds with a pathological role.
Overt and subclinical hypercortisolism
The serum steroid profiling has been recently extended also to cortisol-secreting tumors. Studies
investigating the steroid output in patients with Cushing’s syndrome highlighted the specific finger- print of hypercortisolism, with implication in diag- nosis and subtyping differentiation. The analysis of 84 patients with Cushing’s syndrome (21 because of adrenal tumors, 51 pituitary, and 12 ectopic) and 138 control patients showed increased baseline cor- tisol and glucocorticoid precursors in all patients with Cushing’s syndrome [21”]. When divided by different subtypes, patients with adrenal Cushing’s syndrome showed lower androgen precursors’ levels and higher 11-dexoycorticosterone, when com- pared with ACTH-dependent forms of hypercortis- olism. A 7-steroid panel including 11-deoxycortisol, 11-deoxcorticosterone, cortisol, aldosterone, 21- deoxycortisol, DHEA, and DHEAs showed a good diagnostic accuracy in identifying patients with Cushing’s syndrome, with 87% sensitivity and 89% specificity [21”]. Additionally, the discriminant analysis showed that an extended 10-steroid panel showed a good accuracy in discriminating the different subtypes of Cushing’s syndrome, with an overall 9.5% misclassification rate. Notably, the serum steroid profile alone showed a power close to that of the combination of all traditional tests used for subtyping Cuhsing’s syndrome [21”].
The analysis of serum steroid profiles of patients with adrenal incidentalomas associated with auton- omous cortisol hypersecretion has been largely underinvestigated so far. Indeed, one study pub- lished by our group addressed this topic by analyz- ing 94 patients with unilateral adrenal adenomas (28 with autonomous cortisol secretion with cortisol levels after dexamethasone suppression test above 50 nmol/l and 66 nonfunctioning tumors) and 188 control patients, showing that reduced androgen precursors were specifically associated with hyper- cortisolism [22]. The stimulation test with 1-24 ACTH 250 µg induced an increase in all steroids, with 21-deoxycortisol and 11-deoxycorticosterone as the compounds with the highest percentage increase from baseline in patients with autonomous cortisol secretion [22]. Interestingly, a recent study investigating the steroid profile after Synacthen test in a mixed population of 36 patients, including 24 women attending a fertility clinic and 12 healthy volunteers, confirmed that several steroids had a larger percentage increase from baseline than corti- sol, with corticosterone showing the highest relative increase (15.2) after stimulation [23]. Overall, those data highlighted the complexity of steroid secretion after a maximal stimulation and the greater hyper- responsiveness of functioning adrenocortical tumors than adrenal of (presumed) normal patients, raising the hypothesis that even mild stressful con- dition in patients with functioning adrenocortical
tumors may lead to severe alterations of the steroid profile, whose clinical consequences should be investigated in targeted studies. Indeed, considering that some steroid precursors like corticosterone may have implications on the cardiovascular system, through activation of the mineralocorticoid recep- tor [24], it is intriguing to understand whether stress-induced rise in those mineralo/glucocorticoid active steroids may contribute to the increased cardiovascular risk of patients with adrenal inciden- talomas and autonomous cortisol secretion.
Adrenocortical carcinoma
The potential role of analysis of adrenal steroids in detecting adrenocortical carcinoma (ACC) has been highlighted several decades ago [25]. Pioneering studies applying GC-MS to depict the 24-h urinary steroidome of a large cohort of adrenocortical tumors revealed a specific fingerprint for ACC. The steroid profile in 24-h urine showed a high sensitivity (90-100%) and specificity (90-99%) in discriminating ACC versus non-ACC lesions [26,27]. Even though a high heterogeneity of steroid secre- tion was commonly detected in those tumors, a few compounds proved to be more specific for ACC, with tetrahydro-11-deoxycortisol, a metabolite of 11-deoxycortisol, among the most discriminant ones. Nonetheless, the analysis of urinary steroids by GC-MS carries several limitations that do not allow the direct transfer of this technique into clini- cal practice, mainly because of GC-MS poor practi- cability and the inaccuracy of 24-h urine collection. Therefore, two recent studies attempted to analyze the steroid profiling in a single serum sample by LC- MS/MS in patients with ACC. The study by Taylor et al. [2] analyzed a 13-steroid panel of 10 ACC (50% associated with hormone hypersecretion) and 38 non-ACC lesions, including cortisol-producing adenoma, pheochromocytoma, and nonfunction- ing adrenal tumor. LC-MS/MS profile revealed that a median of six steroids was altered in ACC group, with a large heterogeneity among patients. Despite that, 11-deoxycortisol was confirmed invariably higher in plasma of ACC versus non-ACC patients. Principal component analysis achieved a complete separation between ACC and non-ACC group when using the whole 13-steroid panel, with 11-deoxy- cortisol and 17-hydroxypregnenolone as best dis- criminant markers [2]. More recently, Schweitzer et al. [28] extended those results by analyzing a 15-steroid panel with a commercial LC-MS/MS kit in a much larger cohort of patient with adreno- cortical tumors (42 ACC and 66 adrenocortical adenomas). By combining multiple statistics into a machine-learning model, it was possible to extract
a 6-steroid sex-specific signature of ACC, including, among other compounds, estradiol and progester- one in males and androgens in females. Intrigu- ingly, the presence of estradiol in male signature and dihydrotestosterone in female signature underlines the need for a comprehensive steroid profiling for discrimination of ACC versus non- ACC lesions.
CONCLUSION AND FUTURE DIRECTIONS
Multianalyte assays are prompting novel data han- dling approaches that overcome the traditional interpretation on the basis of single-hormone refer- ence or decisional levels. The uncoordinated perme- ation of LC-MS/MS in the clinical routine calls for the attention of the scientific community to the perennial issue of harmonization of steroid meas- urements. LC-MS/MS is a chance for achieving such a goal on single steroids and on steroid panels [29]. Establishing LC-MS/MS steroid assays harmoniza- tion is relevant for the advancement of disease comprehension and cure and a cornerstone for the validation of novel highly informative multidi- mensional analytical technologies.
In the next future, the measurement of a large panel of steroids on a routine basis may provide outstanding improvement in the selection of patients with primary aldosteronism, who are can- didate for invasive tests (i.e., AVS) or those who eventually deserve surgical treatment or medical therapy. On the same line, it will be of great interest to explore the utility of incorporating the steroid profiling into recently developed tools for predic- tion of outcomes in patients with primary aldoste- ronism [30]. Additionally, given the preliminary results in patients with hypercortisolism and in those with ACC, future studies should address the main criticisms in the diagnosis of Cushing’s syndrome, by proving the pivotal role of multi- steroid panels in simplifying the hormonal workup for disease subtyping, and should be able to iden- tify an individualized steroid fingerprint of ACC, to address a rapid diagnosis and a tailored follow-up.
Acknowledgements
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Conflicts of interest
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