Detection of genes from five gene expression panels (GEPs) in breast tumor samples using NanoString nCounter analysis

The aim of this study was to examine the effect of intra-tumor heterogeneity (ITH) on detection of genes within gene expression panels (GEPs), and the subsequent ability to predict prognostic risk. Multiplexed barcoded RNA analysis was used to measure the expression of 141 genes from five GEPs (Oncotype Dx, MammaPrint, PAM50, EndoPredict, and BCI) in breast cancer tissue sections and tumor-rich cores from 71 ER positive node-negative tumors. Tumor regions with high Ki67 and/or low PR were also used to punch cores, potentially representing aggressive areas of a tumor. In addition to examining the effect of ITH on measurement of GEPs, the effect of ITH on prediction of prognosis in the Oncotype Dx assay was also assessed. For each patient, the FFPE block that was used for the clinical Oncotype Dx test was selected, and a 5µM section, and a 0.6mm core from a tumor-rich representative part of the block was cut. Additional cores were cut from tumors that had foci of high Ki67 (n=26), low PR (n=13), or both (n=5). In total, 181 samples were processed for NanoString nCounter analysis.