Reverse phase protein microarrays advance to use in clinical trials

Abstract

Individualizing cancer therapy for molecular targeted inhibitors requires a new class of molecular profiling technology that can map the functional state of the cancer cell signal pathways containing the drug targets. Reverse phase protein microarrays (RPMA) are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and non-phosphorylated) forms of cellular proteins from a limited amount of sample. This class of microarray can be used to interrogate tissue samples, cells, serum, or body fluids. RPMA were previously a research tool; now this technology has graduated to use in research clinical trials with clinical grade sensitivity and precision. In this review we describe the application of RPMA for multiplexed signal pathway analysis in therapeutic monitoring, biomarker discovery, and evaluation of pharmaceutical targets, and conclude with a summary of the technical aspects of RPMA construction and analysis.

Figure 1

Maturation of reverse phase protein microarray technology. Within the past 20 years, concepts regarding miniaturization of immunoassays, molecular profiling, gene expression microarrays, and protein microarrays have evolved into the “antigen‐down” class of reverse phase protein microarrays. Technological improvements in robotic arrayers and protein binding substratum, in combination with the availability of phospho‐specific antibodies and commercial laser capture microdissection instruments, enabled the production of high‐throughput reverse phase protein microarrays generated from a small microdissected tissue sample. Basic science applications provided proof‐of‐concept in multiple tissues and disease states. Independent laboratories validated the RPMA technology, and the array format was widely adopted for pre‐clinical studies using microdissected and non‐microdissected tissue, cells lines, serum, and body fluids. Enabling technologies such as phosphoprotein preservative solutions have helped mature the RPMA into a robust, reproducible research clinical trial tool for assessing the state of cell signaling proteins, predicting therapy response, and prospectively correlating outcome with proteomic profiles.

Figure 2

Protein microarray formats. Reverse phase protein microarrays have been referred to as ‘protein microarrays’, ‘lysate arrays’, and ‘tissue lysate arrays’ in the literature but not all protein microarrays are reverse phase microarrays. In general, 3 classes of protein microarrays exist: forward phase, sandwich, and reverse phase. In a forward phase, or antibody array, multiple antibodies are immobilized on a surface to capture proteins from a sample. Sandwich arrays require a pair of antibodies to capture the protein of interest and to detect the analyte. Each antibody of a sandwich assay must be able to detect unique epitopes of the same analyte on the sample. The reverse phase format consists of immobilizing the analyte protein on a surface and probing the array with a single antibody directed against the analyte of interest.

Figure 3

Clinical workflow for personalized medicine. Reverse phase protein microarrays were originally developed to profile cell signaling proteins from microdissected tissue samples. In current ongoing clinical trials a patient's biopsy is preserved in a novel preservative solution that retains both cellular antigenicity and histomorphology. Cells of interest are procured by laser capture microdissection, lysed, and printed on a reverse phase protein microarray. Multiple independent arrays are analyzed with a single antibody per array in order to map the state of the cellular signaling network. The final result can be an individualized therapy report or protein network analysis. (ArcturusXT laser capture microdissection photo courtesy of Applied Biosystems/Life Technologies).

Figure 4

Reverse phase protein microarray technology summary. Lysis and Printing: reverse phase protein microarrays are constructed by preparing samples in a microtiter plate prior to deposition on a series of identical nitrocellulose coated slides by a robotic arrayer. Each sample is printed in a dilution series, shown here as a series of four 2‐fold dilutions. Each spot represents the protein composition of the sample at the time of stabilization after sample procurement. Each individual array represents a self‐contained assay because it includes controls, calibrators and samples on the same array. Staining and Measuring: a single array slide is stained with one antibody that has been previously validated and shown to be specific for the protein of interest. Detection chemistries can be fluorescent, chemiluminescent, or chromogenic. Spot intensity is analyzed by a suitable platform based on the detection chemistry. Examples of fluorescent spot analysis include planar waveguide imaging and confocal laser scanning. Chromogenic spot analysis is performed with a high‐resolution flatbed scanner. Analysis: spot finding software programs convert pixel density for each spot into numerical values. Histograms of spot morphology can also be reviewed. The quantitative pixel intensity can be further normalized and applied to statistical or bioinformatics analysis.