Abstract 5140: Development and evaluation of a liquid bead array assay for the expression of VEGF121, VEGF165 and VEGF189 splice variants in non-small cell lung cancer (NSCLC)

Abstract

Abstract Introduction. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a key regulator of angiogenesis. The aim of our study was to develop and validate a novel liquid bead array assay for the expression of VEGF121, VEGF165 and VEGF189 splice variants, based on the Luminex technology and evaluate it in NSCLC. Materials and methods. We developed a multiplexed PCR-coupled liquid bead array to detect the expression of VEGF splice variants VEGF121, VEGF165 and VEGF189. We included PBGD as a reference gene, in order to evaluate sample RNA quality. The developed assay is based on liquid bead array technology (Luminex® xMAP® and xTAG®). The assay is based on the following procedure: total RNA isolation from samples, cDNA synthesis, multiplex PCR, treatment with ExoSAP-IT® reagent, target specific primer extension (TSPE), biotinylation of PCR products, hybridization of the biotinylated products to xMAP microspheres, addition of detection reagent (Streptavidin- phycoerythrin) and measurement in the Luminex analyzer. Before processing to patients samples an extensive optimization of every step of the method was performed in terms of analytical sensitivity, specificity, repeatability and reproducibility. The clinical performance of the assay was evaluated in 20 pairs of fresh frozen cancerous and corresponding noncancerous adjacent tissue samples originating from patients with NSCLC. The results were compared with RT-qPCR using our previously developed methodology for VEGF splice variants, (Zygalaki et al, Clin Chem, 2007). Results. When using the tumor cell line MCF-7 the developed method can detect down to 10 cells, is highly specific and is characterized by satisfactory repeatability and reproducibility for each VEGF splice variant. Comparison between the developed liquid bead array for VEGF splice variants and RT-qPCR has shown an accordance of 16/20 pairs (80%) for PBGD, 16/20 for VEGF121 (80%), 18/20 for VEGF165 (90%) and 17/20 for VEGF189 (85%). Conclusions. The developed liquid bead array can be used to detect VEGF splice variants simultaneously in clinical samples with high specificity and sensitivity. The method can be extended to analyze additional gene targets, while the use of an internal control could further enable quantification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5140. doi:10.1158/1538-7445.AM2011-5140