Quantification of the lung cancer tumor marker CYFRA 21-1 using protein precipitation, immunoaffinity bottom-up LC-MS/MS

Sylvia A.A.M. Genet (Corresponding author), Sebastian A.H. van den Wildenberg, Maarten A.C. Broeren, Joost L.J. van Dongen, Luc Brunsveld, Volkher Scharnhorst, Daan van de Kerkhof (Corresponding author)

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Abstract

Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0-100 » ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.

Original languageEnglish
Pages (from-to)720-728
Number of pages9
JournalClinical Chemistry and Laboratory Medicine
Volume62
Issue number4
Early online date30 Oct 2023
DOIs
Publication statusPublished - 25 Mar 2024

Bibliographical note

© 2023 Walter de Gruyter GmbH, Berlin/Boston.

Funding

Research funding : The study was supported by The Netherlands Organization for Scientific Research (NWO) via LIFT grant 731.017.405. Roche Diagnostics partially sponsored the CYFRA 21-1 reagents used in the study. The funding organizations did not play a role in the design of the study, choice of enrolled patients, review and interpretation of data or preparation of approval of manuscripts.

FundersFunder number
Nederlandse Organisatie voor Wetenschappelijk Onderzoek731.017.405

    Keywords

    • cytokeratin 19 fragment 21-1
    • immunoaffinity (IA)
    • liquid chromatography-tandem mass spectrometry (LC-MS/MS)
    • lung cancer diagnostics
    • protein precipitation
    • tryptic digestion
    • Chromatography, Liquid/methods
    • Antigens, Neoplasm
    • Humans
    • Tandem Mass Spectrometry/methods
    • Lung Neoplasms/diagnosis
    • Biomarkers, Tumor
    • Carcinoma, Non-Small-Cell Lung/diagnosis
    • Keratin-19
    • Liquid Chromatography-Mass Spectrometry

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