Clinical Chemistry 63:12 000-000 (2017)
Steroid Profiling for Adrenocortical Disorders: A Pathway for Omics-Based Diagnostics
Graeme Eisenhofer1* and Martin Fassnacht2
During the past 15 years, a considerable investment of effort and funds has been directed to the use of omics- based technologies for biomarker identification and de- velopment. The outcome in terms of laboratory tests ap- plicable to the routine environment has been slow to materialize, in some cases corrupted by poorly conceived and conducted work (1, 2). This disappointing return is in part attributable to the time and expertise required to meet the many steps and processes required from bio- marker discovery to validation and acceptance for clinical use. Compounding the problem, application of an omics- based panel of multiple measurements requires considerably more complex approaches for test validation and interpreta- tion than those required for more commonly used single biomarkers. In this issue of Clinical Chemistry, 2 groups, 1 at King’s College (3) and a second at the Mayo Clinic (4), highlight the potential applications of mass spectrometry-based steroid profiling (“ste- roidomics”) for disorders of the adrenal cortex. With respective panels of 12 and 26 steroids, the outlined methods illustrate pathways for validation of omics- based signatures for diagnostics and stratification of disease for therapeutic intervention.
Application of steroid profiling for the diagnosis of adrenocortical disorders has a long history going back >50 years, first with use of laborious chromatographic separation and colorimetric or immunoassay procedures (5-7) that have subsequently been replaced by methods using gas chromatography or liquid chromatography combined with mass spectrometry (8, 9). GC-MS meth- ods, in particular, have proven extremely useful for ste- roid profiling, but because of the need for complex sam- ple preparation, they are not well suited for the routine laboratory environment. Consequently, methods using liquid chromatography at the front end of mass spec- trometry instruments have come to the forefront for rou-
tine laboratories. Whereas the King’s College panel of 13 serum steroids uses an LC-MS/MS instrument common to many routine laboratories, the Mayo Clinic panel of 26 urinary steroids uses liquid chromatography in con- junction with Orbitrap mass spectrometry technology, more common to the research environment.
Today’s new generation of hybrid Orbitrap or time- of-flight instruments combines the high mass resolving power of those technologies with accurate quantification and selectivity of quadrupole instruments. These hybrid instruments thereby allow both untargeted discovery and targeted quantitative omics applications. The result for targeted applications, such as that from the Mayo Clinic, is substantially improved resolution of ions with similar masses. Nevertheless, the instruments do not allow dis- crimination of isobaric isomers, which, as shown in Table 1 of the report from the Mayo Clinic, are common to numerous steroids in the panel. Presumably there are many other steroids not in the panel that may further contribute to potential problems related to separation of isobaric isomers. Therefore, chromatographic separation remains crucial so that both applications involve total run times of 20 to 35 min. As outlined in the report from the Mayo Clinic, some reduction in run times may be achieved by multiplexing. A potential alternative ap- proach could be to include ion mobility, either alone or as a second dimension of rapid separation following a short- ened liquid chromatography step. Either way, in terms of run times, the high mass resolution method does not appear to offer any advantage over standard LC-MS/MS methods. Perhaps of more importance for the high mass resolution method are the more easily interpretable ion spectra in a highly complex sample matrix present even after specimen cleanup.
Another difference between the 2 methods concerns choice of sample matrix. Because of highly dynamic di- urnal changes, measurements of steroids in serum or plasma should be determined at a specific time during the day, with reference intervals matching to that period (10). The ideal time for sampling is at the nadir in the diurnal rhythm, typically close to midnight, which is im- practical for outpatient blood sampling. The more prac- tical time for patients is in the morning, but this is when plasma steroids are at their peak and when it can be more difficult to distinguish patients with adrenocortical hy- perfunction from those with normal function. Twenty- four-hour urine collections have the advantage of pro-
1 Institute of Clinical Chemistry & Laboratory Medicine and Department of Internal Medi- cine III, Technische Universität Dresden, Dresden, Germany; 2 Department of Internal Medicine and Central Laboratory, University Hospital of Würzburg, Würzburg, Germany.
* Address correspondence to this author at: Institute of Clinical Chemistry and Laboratory Medicine and the Department of Medicine III, University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany. Fax +49- 351-458-6398; e-mail graeme.eisenhofer@uniklinikum-dresden.de.
Received September 12, 2017; accepted September 12, 2017.
Previously published online at DOI: 10.1373/clinchem.2017.281048 @ 2017 American Association for Clinical Chemistry
viding an integrated measure of steroid output but also involve practical difficulties for patients. Completeness of collections is always a concern. Spot first or early morning urine collections may offer an alternative ap- proach with the potential added advantage of collections over the nadir in the diurnal rhythm.
As outlined in the report from the Mayo Clinic, another disadvantage of urine collections is the need to consider sulfate and glucuronide conjugates, which, as major urinary end products of steroid metabolism, re- quire a deconjugation step before sample cleanup. With- out conjugated steroids for both calibration and quality control purposes, there is potential for error and uncer- tainty in final measurements. As pointed out by the King’s College group and apparent in the report from the Mayo Clinic, the need to consider a wide array of largely obscure urinary metabolites rather than better known steroids that comprise the backbone of the biosynthetic pathway is a further factor that might be limiting to clin- ical acceptance.
In terms of clinical applications, mass spectrometry- based diagnostic methods have the potential to markedly improve the treatment of patients with adrenocortical disorders. Adrenal tumors, in particular, with a preva- lence of at least 3% in individuals >50 years old, are the most frequently encountered neoplasms in humans and a substantial problem for diagnosis and management. Al- though the majority of these tumors are benign hormon- ally inactive adenomas, a small proportion of up to 8%, dependent on size, are extremely aggressive malignant adrenocortical carcinomas (11). The clinical challenge is that there is no single diagnostic tool that can discrimi- nate benign from malignant tumors. Adrenocortical car- cinoma has a 5-year survival rate of <50%, and only early detection at a localized stage can lead to surgical cure (12, 13). Otherwise, the median survival period is <15 months (14). Thus, improved diagnostic tools are ur- gently needed.
Both reports in this issue of Clinical Chemistry indi- cate distinct patterns of steroids potentially useful for distinguishing patients with adrenocortical carcinoma from those with other adrenocortical lesions. Among steroids of the serum panel, 11-deoxycortisol, 17- hydroxypregnenolone, and other intermediates in steroid metabolism were the most prominently increased in pa- tients with adrenocortical carcinoma. These findings were mirrored by increases in respective urinary metabo- lites reported by the Mayo Clinic group. Although the consistent results from both studies are encouraging and in line with a previous European multicenter study that used GC-MS (8), the small patient numbers for both studies limit enthusiasm. Nevertheless, the heteroge- neous nature of serum steroid patterns among patients with adrenocortical carcinoma also raises the possibility
that differences in signatures may have prognostic signif- icance that might impact treatment decisions.
Clearly further larger series are required, ideally in- volving carefully planned prospective trials with detailed clinical annotations and follow-up that can establish not only utility for diagnosis but also stratification of patients for therapy. With rare diseases such as adrenocortical carcinoma, any prospective trial would best take advan- tage of established multicenter networks, such as the Eu- ropean Network for the Study of Adrenal Tumors, to enable recruitment of sufficient patients and collection of data using already established procedures and registries.
Suitably large-sized reference populations are also required to fully characterize both sex differences and the highly dynamic age-related changes in steroids (11). The Mayo Clinic group, to their credit, adjusted results for individual patients according to sex and age. Neverthe- less, the reference populations outlined in both reports are insufficiently large to provide truly useful age- and sex-specific reference data. Adding to the complexity of test interpretation and validation of biomarker panels are not only requirements to consider age- and sex-specific reference intervals but also the need in omics-based anal- yses to consider patterns of test results.
For interpretation of patterns of results in test pan- els, it is necessary to develop computational models and tools to deal with large sets of generated data. Appropri- ate and easily understandable reporting of results for cli- nicians represents another challenge to the development and clinical implementation of omics-based laboratory tests. For all this it is important to have the involvement of multidisciplinary teams well versed in the develop- ment and validation of tests for subsequent translation to the clinic. Both the teams at King’s College and the Mayo Clinic are suitably experienced and well situated for bringing their methods into the routine clinical environ- ment. The Mayo Clinic laboratory group headed by Ravin- der Singh, a pioneer in the field of clinical mass spectrome- try, is supported by the Bancos team with complementary expertise in clinical endocrinology and information technol- ogy. The Taylor laboratory team is also well supported by strong clinical partnerships at King’s College. Such teams promise a pathway model for others in the omics-based bio- marker field to follow. More specifically for adrenocortical disorders, the outlined methods and data from the 2 labo- ratories represent the first steps of important progress in bringing mass spectrometry-based steroidomics closer to hand for the routine clinical laboratory.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intel- lectual 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: No authors declared any potential conflicts of interest.
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