Detection of potential biomarkers
Plasma samples collected on W1D1, week 5 day 1 (W5D1) and week 9 day 1 (W9D1) were tested for potential biomarkers, including HGF, MET, GAS6, and AXL (the protein level at W1D1 was defined as baseline). Detection was achieved using commercially available enzyme-linked immunosorbent assay (ELISA) kits and the following procedures: GAS6 (Human Gas 6 DuoSet ELISA, R&D Systems, Inc., Minneapolis, USA), AXL (Human Axl DuoSet ELISA, R&D Systems, Inc., Minneapolis, USA), HGF (Human HGF Immunoassay, R&D Systems, Inc., Minneapolis, USA), and c-MET (Human c-Met Assay Kit MCM, Immuno-Biological Laboratories Co., Ltd., Gunma, Japan). ELISA kits were validated by using test samples over the range of the standard curve, testing for precision and accuracy, examining diurnally, assessing between-day reproducibility and dilution reproducibility, undertaking freeze–thaw tests and stability tests at room temperature, as well as assessing storage stability for up to 6 months. Validation and measurements were conducted by LSI Medicine Corporation (Tokyo, Japan). Plasma protein concentrations (ng/mL) were obtained by fitting to the standard curve.