Validation of a multiplex immunoassay for serum angiogenic factors as biomarkers for aggressive prostate cancer
Highlights
► Performance of the Bio-Plex Human Cancer Biomarker Panel 1 was evaluated. ► All 16 assays had good reproducibility, sensitivity, and wide dynamic ranges. ► sTIE-2 was elevated in serum of prostate cancer patients with GS of 8, 9 or 10.
Introduction
Prostate cancer is currently the most common cancer in American men. There were an estimated 240,890 new cases of prostate cancer per year [1]. The fact that only 33,720 of them died of prostate cancers annually suggests that majority of the cancers does not progress [2]. Thus, development of accurate prognostic markers to identify patients with aggressive prostate cancers who need to be aggressively treated is urgently needed.
A prognostic biomarker should possess a clear biological significance. Prostate cancer progression and metastasis are pathological events closely linked to angiogenesis, the formation of new blood vessels from the pre-existing ones [3], [4], [5], [6]. This neovascularization process supplies oxygen and nutrients to tumor outgrowth, and contributes to tumor progression from in situ lesions to locally invasive, and eventually to metastatic tumors. In prostate cancer, intensity of angiogenesis, measured as microvessel density (MVD), was found to be higher when compared to benign glands [4], [5]. Furthermore, angiogenesis was found to be increased in the primary tumors of patients with metastasis compared to patients with localized disease [3].
The tumor angiogenic process is controlled by pro- and anti-angiogenic factors released from tumor cells, tumor-associated inflammatory cells, and/or from the extracellular matrix. Mechanistically driven by tumor progression, these factors may be present in serum, reflecting the overall angiogenic activity of the tumors. Tumor progression and patient survival in many cancers, including malignant melanoma [7], end-stage epithelial ovarian cancer [8], and pancreatic cancer [9], have been correlated with serum angiogenic factors. As a result, serum angiogenic factors are ideal candidates as prognostic biomarkers in prostate cancer.
Assays used for the discovery of biomarkers should be robust and high-throughput, capable of analyzing a sufficiently large number of samples over a sufficiently long period of time with good precision. Poor precision not only increases the required sample size for discovery, but also hinders the true revelation of differential expression of biomarkers in the case and control populations [10]. In this study, we discovered prognostic biomarkers for aggressive prostate cancer by composite profiling of serum angiogenic factors using the Bio-Plex Pro™ Human Cancer Biomarker Panel 1, a 16-plex multiplex immunoassay. This multiplex approach conserved specimen, limited sample handling, increased throughput, and reduced labor costs. We evaluated the analytical performance of the panel, including limit of quantification, linearity, and precisions. After the analytical evaluation, we applied the panel to assess the potentials of the 16 angiogenic factors for identifying aggressive prostate cancer in serum.
Section snippets
Samples and reagents
The 37 serum specimens analyzed in this study were from men with prostate cancer. All men had a prostate biopsy. Blood was collected before radical prostatectomy and sera stored frozen at − 70 °C before analysis. Informed consent was obtained under institutional review board approved and Health Insurance Portability and Accountability Act compliant protocols. Human pooled serum was from Sigma. Samples were assayed with the Bio-Plex Pro™ Human Cancer Biomarker Panel 1, 16-plex kit (171-AC500M)
Results and discussion
Multiplex immunoassays, developed either in-house or by commercial vendors, have been widely used for biomarker discovery and validation. For commercially available assays, information on analytical performance from the manufacturer is a useful reference point, but assay performance in the hands of customers may be different. Variations in the procedures, such as incubation times and washing conditions, affect reproducibility of the assays. The materials used by the manufacturer to establish
Conclusions
We evaluated the analytical performance of the Bio-Plex Pro™ Human Cancer Biomarker Panel 1, a 16-plex multiplex immunoassay, in serum for composite profiling of angiogenic factors. Our 5-day evaluation indicated good reproducibility (total precisions over 5 independent plates in 5 days of less than 20%), adequate sensitivity in serum for reliable detection of these 16 angiogenic factors in serum (LOQs of majority of the assays less than 100 pg/ml), and wide dynamic ranges (linearity of the
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