Clinical Analytical Assay for AFP-L3 Using Multiple Reaction Monitoring-Mass Spectrometry for Diagnosing Hepatocellular Carcinoma Youngsoo Kim (Presenter) Seoul National University College of Medicine Presenter Bio: Current position (2002-present): Professor, Department of Biomedical Sciences, Seoul National University College of Medicine, and Department of Biomedical Engineering, Seoul National University College of Medicine 9/1987 - 5/1992: The University of Texas at Austin,Department of Chemistry and Biochemistry, Austin, Texas. Ph.D. in Chemistry and Biochemistry, 1992 2/1992 - 9/1994: Postdoctoral associate, Yale University, Department of Molecular Biophysics and Biochemistry

Authors: Hyunsoo Kim (1), Areum Sohn (2), Injun Yeo (1), Su Jong Yu (3), Jung-Hwan Yoon (3), and Youngsoo Kim (1,2)
Departmets of (1) Biomedical Engineering, (2) Biomedical Sciences and (3) Internal Medicine, Seoul National University College of Medicine, Yongon-Dong, Seoul 110-799, Republic of Korea

We have developed and validated an MRM-MS assay for quantifying AFP-L3 in human serum to diagnose early-stage hepatocellular carcinoma. LiBA, the current standard method, cannot measure AFP-L3 concentrations accurately due to its low sensitivity. We addressed this issue with immunoprecipitation in conjunction with fractionation with LCA lectin. Consequently, the MRM-MS assay identified hepatocellular carcinoma patients that were missed by LiBA. In addition, we validated this approach in accordance with several multinational guidelines such as FDA, EMA, and CLSI. Our results demonstrated that this assay is robust and immediately applicable in clinical practice.

Introduction

Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein (AFP-L3) is a serum biomarker for hepatocellular carcinoma (HCC). AFP-L3 is typically measured by liquid-phase binding assay (LiBA)[1]. However, LiBA does not always reflect AFP-L3 concentrations, due to its low sensitivity. Thus, we aimed to develop an analytically sensitive multiple reaction monitoring-mass spectrometry (MRM-MS) assay to quantify AFP-L3 in serum[2].

Methods

Our MRM-MS assay entailed the addition of a protein analog as an internal standard, the enrichment of AFP using a monoclonal antibody, the fractionation of AFP-L3 using lens culinaris agglutinin (LCA) lectin, deglycosylation, trypsin digestion, online desalting, and MRM-MS analysis [3]. The performance of the MRM-MS assay was compared with that of LiBA in 400 human serum samples (100 chronic hepatitis, 100 liver cirrhosis, and 200 HCC). Integrated multinational guidelines were followed to validate the assay for clinical implementation.

Results

The lower limit of quantification (LLOQ) of the MRM-MS assay for AFP-L3 was, below that of LiBA. Thus, AFP-L3, which was not observed by LiBA in HCC samples (n = 39), was detected by the MRM-MS assay, improving the clinical value of AFP-L3 as a biomarker by switching to a more sensitive platform. The method was validated, meeting all of the criteria in integrated multinational guidelines.

Conclusions & Discussion

We have developed and validated an MRM-MS assay that has greater sensitivity for AFP-L3 than LiBA. The MRM-MS assay performs satisfactorily for clinical use. This method has potential application for quantifying other (glyco)protein biomarkers with higher sensitivity.

[1] Kurosawa T, Watanabe M. Development of on-chip fully automated immunoassay system "mutaswako i30" to measure the changes in glycosylation profiles of alpha-fetoprotein in patients with hepatocellular carcinoma. Proteomics 2016;16:3056-61.

[2] Hoofnagle AN, Becker JO, Oda MN, Cavigiolio G, Mayer P, Vaisar T. Multiple-reaction monitoring-mass spectrometric assays can accurately measure the relative protein abundance in complex mixtures. Clin Chem 2012;58:777-81.

[3] Kim H, Kim K, Jin J, Park J, Yu SJ, Yoon JH, Kim Y. Measurement of glycosylated alpha-fetoprotein improves diagnostic power over the native form in hepatocellular carcinoma. PLoS One 2014;9:e110366.