Plasma Free Metanephrines Measured by LC-MS/MS: A New Diagnostic Tool for the Diagnosis of Neuroblastoma Giuliana Cangemi (Presenter) PhD, Istituto Giannina Gaslini >> POSTER (PDF) Presenter Bio: Giuliana Cangemi, graduated in Biological Sciences, specialization in laboratory immunology and PhD in biochemistry,Head of the chromatography-mass spectrometry section of the Central Laboratory of Analysis of G.Gaslini Institute, a tertiary care children hospital in Italy. She is the author of more than 60 scientific papers in international journals and speaker at several congresses.

Authors: Sebastiano Barco, Maria Valeria Corrias, Stefania Sorrentino, Alberto Garaventa, Gino Tripodi and Giuliana Cangemi
Istituto Giannina Gaslini, Genova, Italy

In this work we have investigated the diagnostic role of plasma free metanephrines (PFM):metanephrine (MN), normetanephrine (NMN) and 3-methoxytiramine (3-MT)quantified by LC-MS/MS, for neuroblastoma (NB), the most common extra-cranial solid tumor in children. 3-MT and NMN showed an excellent diagnostic performance. The determination of PFM should be taken in consideration as a new diagnostic and prognostic tool and further validated in clinical prospective studies in comparison to urinary catecholamines and metanephrines.

Introduction

Plasma free metanephrines (PFM) are well established diagnostic biomarkers of pheochromocytoma (1). Their quantification is challenging because of their polar nature, their low molecular weight and the very low physiological concentration in human plasma (in the order of ng /L). A complete kit by LC-MS / MS (Clinmass ®) was recently introduced by Recipe (Munich, Germany) based on SPE separation or on-line SPE with deuterated internal standards, 5-level calibrators and 3 controls. In this work we describe the analytical performance of this new test on our LC-MS / MS system (Thermofisher TSQ Quantiva coupled to UHPLC Ultimate 3000) and its diagnostic performance in patients with NB at the onset. NB is the most common extra-cranial solid tumor in pediatrics and the determination of urinary catecholamines (in particular the two metabolites homovanillic, HVA, and vamillylmandelic, VMA acids) represents the first level diagnostic standard, followed by imaging (CT, MRI and MIBG) and histo-pathological confirmation (2, 3). The diagnostic role of plasma metanephrines in NB has never been investigated on an adequate number of cases until now (4).

Methods

A partial validation of the method has been performed following EMA guidelines (5) for linearity, LLOQ, intra- and inter-run accuracy and reproducibility. Fifty-four plasma samples of patients with NB at the onset (4 stage 1, 16 stage 3, 24 stage 4 and 10 stage 4S) and 49 age-matched controls (0-5 years) were analyzed. The samples of patients with NB were collected between 2012 and 2016 from different italian centers and biobanked at GianninaGaslini Institute, a tertiary care pediatric hospital, italian national reference center for the biochemistry of NB. The study has been approved by the Local Ethical Committee and informed consent was obtained from all patients'guardians.

Results

The method is linear over a wide range of concentrations (2.83-5094 ng/L for 3-MT, 2.76-19860ng/L for NMN, 2.91-20970ng/L for MN) and has been shown to be highly accurate and reproducible (intra- and inter-assay CV% <5% and RE 95-105 for all the analytes) with a rapid runtime (4.5 min). Reference ranges were obtained in controls with a non-parametric test (CLSI 28-A3, non-normal distribution) and the results were as follows: 3MT: N.D-9.3; MN: N.D.-75.4; NMN: 11.4-169.6 ng / L. Results were comparable with literature data (3). Differently from MN, 3-MT and NMN showed very high sensitivity and specificity (3MT: 90.5 and 100% with AUC = 0.939 and NMN: 79.555 and 95.7% with AUC = 0.893 respectively) and were able to discriminate between NB and controls (Mann Whitney p <0.001). The concentration of 3-MT has been shown to increase from stage I to IV. Interestingly, 3/54 NB patients only resulted negative for all the 3 plasma markers and were also negative for urinary HVA and VMA. Accordingly, 3/54 patients that were negative for urinary HVA and VMA resulted also negative for PFM.

Conclusions & Discussion

The new quantitative LC-MS/MS assay for PFM was easily applicable to our LC-MS/MS system and its analytical performance allows us to routinely use it in the laboratory workflow. Its diagnostic performance on NB patients was tested in a cohort of well characterized samples of NB patients and matcjed controls. The analyte with the best performance was 3-MT followed by NMN. MN did not show any significant result and the differences displayed with 3-MT and NMN could be explained by the different biochemical pathways to which these three metabolites belong.In conclusion, PFM showed an excellent performance for the diagnosis of NB and can also be very useful in addition, or as an alternative, to urinary catecholamine profile. Their eventual prognostic role in identifying patients with an unfavorable outcome should be investigated in a larger cohort of patients. The results of this cohort study should be confirmed with a prospective study with extensive case studies.

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2) Swift CC1, Eklund MJ1, Kraveka JM1, Alazraki AL1.Updates in Diagnosis, Management, and Treatment of Neuroblastoma.Radiographics. 2018 Mar-Apr;38(2):566-580.

3) Brodeur GM, Pritchard J, Berthold F, et al. Revisions of the international criteria for neuroblastoma diagnosis, staging, and response to treatment. J ClinOncol. 1993;11(8):1466–1477.

4) Franscini LC, Vazquez-Montes M, Buclin T, Perera R, Dunand M, Grouzmann E, Beck-Popovic M. Pediatric reference intervals for plasma free and total metanephrines established with a parametric approach: relevance to the diagnosis of neuroblastoma.Pediatr Blood Cancer. 2015 Apr;62(4):587-93.

5) European Medicines Agency. Guideline on Bioanalytical Method Validation. http://www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2011/08/WC500109686.pdf, 2015