New Way Forward for the Diagnosis and Management of Gastroenteropancreatic Neuroendocrine Tumors with an LC-MS/MS Panel of Indole Biomarkers

Diagnosis and surveillance of gastroenteropancreatic neuroendocrine tumors (GEP-NETs)² involve a repertoire of several different biochemical tests with some differences in utility according to classifications and guidelines from the North American Neuroendocrine Tumor Society (1) and the European Neuroendocrine Tumor Society (2). Measured biomarkers include plasma chromogranin A, urinary or plasma 5-hydroxyindole acetic acid, platelet or whole blood serotonin, serum neuron-specific enolase, and, depending on presentation, a variety of peptides such as gastrin, vasoactive intestinal peptide, and several pancreatic-specific peptides (3). Measured alone for screening, these biomarkers have limited clinical sensitivity, but when positive they can be further useful for patient management, including therapeutic monitoring. Some of the more commonly measured biomarkers, such as chromogranin A, suffer from poor clinical specificity or may be rendered pointless to measure by medications (e.g., proton pump inhibitors) or associated clinical conditions (e.g., renal failure). At the routine laboratory level, maintaining multiplicities of tests for such relatively rare tumors can be cost-prohibitive. Thus, measurements available for clinicians to make the diagnosis or monitor patients can be limited.

To address the above issues, van Faassen and colleagues present in this issue of Clinical Chemistry a novel LC-MS/MS method for the measurement of four indole biomarkers in platelet-rich plasma (4). The method is made possible by a derivatization step utilizing propionic anhydride. The question is why use derivatization when one of the advantages of liquid over gas chromatographic front ends in mass spectrometry is to escape such extra steps. First, however, as clarified by the authors, the derivatization step is performed directly in the sample and simply involves mixing for 15 min before centrifugation and solid-phase extraction, which they perform online. Such in situ derivatization steps utilizing a wide array of agents are well established (5), but few have been applied to LC-MS/MS measurements of indoles or biogenic amines. Currently described procedures have invariably used derivatizations after sample clean-up and dry-down (6–10), including one method involving propionic anhydride (8). The method described by van Faassen and colleagues is novel in that it is directed at indoles and involves in situ use of propionic anhydride, a relatively simple step, which results in derivatization of both hydroxyl and amine groups.

As described in the article (4), as well as in others (5–10), there are several advantages to derivatization before LC-MS/MS that generally relate to the resulting reduced polarity of derivatized analytes. Consequent enhancement of ionization efficiencies and improved signal-to-noise ratios enable small sample volume applications such as in neonatal pediatrics (8) or microdialysis (9). Thus, the currently described method for indoles requires only 50 μL of platelet-rich plasma. The relatively nonpolar nature of derivatized analytes also allows for improved chromatographic separation, which is particularly important for mass spectrometric applications in which methods to enhance separation of polar analytes are limited. Finally, and of most importance to the present application directed to patients with GEP-NETs, the nonpolar nature of derivatized analytes allows for a simpler sample clean-up procedure. This in turn allows for targeting a wide array of analytes at similarly high extraction efficiencies, in the present method enabling simultaneous purification and quantification of several indoles with differing functional groups.

The capacity of LC-MS/MS for multiplex measurements of panels of biomarkers is one of several major advantages of mass spectrometry over other laboratory methods such as immunoassays that usually allow measurements of only a single analyte. For the panel of four indoles described by van Faassen and colleagues, the advantage for the laboratory is a single method for multiple analytes. At the clinic and for patients, the benefits are multiple. First, measurements of the serotonin precursor, 5-hydroxytryptophan, may be useful for diagnosis or even disease monitoring of patients with tumors that produce no or minimal serotonin owing to absent or reduced expression of aromatic amino acid decarboxylase (AADC), which converts 5-hydroxytryptophan to serotonin. As the authors show, this seems to be particularly useful for foregut tumors, which tend to be deficient in AADC. Second, for tumors that express AADC, measurements of platelet-rich plasma serotonin, normalized to platelet count, and of its deaminated acid metabolite, 5-hydroxyindole acetic acid, provide complementary information, with the former measurements more clinically sensitive for diagnosis and the latter more useful for disease monitoring. Third, the panel of indoles also incorporates the amino acid tryptophan, which is derived from the diet; this source of tryptophan can be rate limiting as a building block in downstream metabolism, including for the synthesis of nicotinic acid (niacin). Because patients with extensive GEP-NET disease burden can suffer from depletion of tryptophan and associated niacin deficiency (11–13), the additional measurement of tryptophan can be useful for treatment of patients in whom dietary supplementation can be important for clinical care.

Beyond the aforementioned advantages for diagnosis and patient care, it is possible that different patterns of the various indole biomarkers might be useful in other ways for stratification of disease. Unlike pheochromocytoma and paraganglioma, for which tumor location, size, mutations, and tumorigenic pathways are clearly linked to different patterns of catecholamine production (14), there has been minimal progress in linking differing patterns of indoles and amine biomarkers to the considerable intertumor heterogeneity displayed by GEP-NETs. The presently described LC-MS/MS panel of indoles provides potential for facilitating such progress, especially if other kynurenine pathway metabolites and amines can be incorporated into the profile. With such profiles, it should become possible to break free of present-day constraints imposed by conventional unidimensional thinking in diagnostics and start to take advantage of 21st century computational mathematics and artificial intelligence for multidimensional approaches in laboratory medicine (15).

Although the LC-MS/MS panel of indoles described by van Faassen and colleagues provides a valuable advance over existing methods, it is unlikely to offer a single-test alternative for all currently used biomarkers of GEP-NETs, particularly chromogranin A. Nevertheless, additional mass spectrometric methods that simultaneously target granin family members and various gastrointestinal and pancreatic polypeptides may offer a further solution. In this way, it should be possible using just two laboratory methods to comprehensively cover all useful biomarkers for more effective diagnosis and treatment of patients with GEP-NETs than currently possible.

The senior and corresponding author of the article, Ido Kema, has a strong, long, and rich history in research into GEP-NET tumors and innovative use of mass spectrometry in diagnostic laboratory medicine. In keeping with this history, the newly described method provides an important advance to substantially improve current diagnosis and surveillance of a neoplasia that has become increasingly recognized to be not as rare as previously appreciated, but still difficult to effectively identify, stratify, and monitor for best therapeutic management.

2 Nonstandard abbreviations

 
• GEP-NET

  gastroenteropancreatic neuroendocrine tumor

 
• AADC

  aromatic amino acid decarboxylase.

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