Author of

• 14612

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  P2.06 - Poster Session/ Screening and Early Detection (ID 219)

  • Event: WCLC 2015
  • Type: Poster
  • Track: Screening and Early Detection
  • Presentations: 1
  • Moderators:
  • Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)

  • 14625

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    P2.06-013 - Verification of the Biomarker Candidates for Non-Small-Cell Lung Cancer Using a Targeted Proteomics Approach (ID 706)

    09:30 - 09:30  |  Author(s): B. Domon
    • Abstract

    Background:
    Lung cancer, with its high metastatic potential and high mortality rate, is the worldwide leading cause of cancer-related deaths. High-throughput “omics”-based platforms have accelerated the discovery of biomarkers for lung cancer, and the resulting candidates are to be evaluated for their diagnostic potential as non-invasive biomarkers. The evaluation of the biomarker candidates involves the quantitative measurement of large numbers of proteins in bodily fluids using advanced mass spectrometric techniques. In this study, a robust method based on targeted proteomics was developed for biomarker verification in plasma samples and applied to verifying lung cancer biomarker candidates.
    
    Methods:
    Sample acquisition: Blood samples were obtained from 72 patients diagnosed with non-small-cell lung cancer (NSCLC) (stages I-IV), and 30 healthy volunteers with the approval of the National Research Ethics Committee. Sample processing and Liquid Chromatography-Selected Reaction Monitoring (LC-SRM): Two most-abundant plasma proteins were depleted from each sample. Proteins were digested by trypsin to generate peptide mixtures. Peptides representing the potential biomarker proteins were selected. Stable isotope-labeled (SIL) peptides of the selected target peptides were synthesized to be used as internal standards, and spiked-in the processed plasma samples. A peptide banking system equipped with a local spectral library of synthetic peptides was used to facilitate automatic generation of LC-SRM methods. Multiplexed LC-SRM assays for >100 potential markers for NSCLC were generated to screen the plasma samples.
    
    Results:
    In the first set of screening for 190 peptides, a total of 60 peptides corresponding to 44 proteins were detectable by LC-SRM in the plasma. Among them, 17 proteins exhibited higher expression levels in the NSCLC patients compared to the control. For those proteins, additional peptides were prepared in order to increase the coverage of the protein sequence, and the number of samples were expanded (72 NSCLC and 30 controls). After differential analysis of the SRM results, 17 proteins were finally verified as potential diagnostic markers. The verified targets include ACTN1, ALDOA, ENO1, FLNA, G6PD, GPI, HSP90B1, ICAM1, ILK, LDHB, MSN, PGK1, PKM2, SPP1, TALDO1, THBS1, and ZYX. The expression levels were cross-validated by ELISAs if available. A novel plasma-based biomarker, ZYX, showed a potential of early diagnostics as its plasma level increases from the early stages (stages I and II). The overall pattern of the plasma levels of four ZYX peptides and the ELISA results were correlated. The role of ZYX in cancer has been recently discussed as a key player in epithelial-mesenchymal transition mechanism, and the association to lung cancer was reported in several studies. To the best of our knowledge, the potential use of ZYX protein as a tumor biomarker in plasma for lung cancer has been verified for the first time in this study.
    
    Conclusion:
    A targeted proteomics-based, analytical pipeline was designed for a large-scale biomarker verification and successfully applied to verifying a set of potential biomarkers for NSCLC. The robust workflow is critical to the early-stage screening where the attrition rate tends to be high (68% in this study). Several novel targets were verified as plasma-based NSCLC biomarkers, and ZYX showed a potential of early diagnostics.