Gene Expression Detection Assay for Cancer Clinical Use

Narrandes, Shavira; Xu, Wayne

The Digital Analyzer uses a CCD camera through a microscope objective lens to magnify and image the immobilized Reporters, capturing hundreds of images per sample and outputting hundreds of thousands of counts; it processes the images and exports the data as comma separated (.csv) output files which can then be used as input for the nSolver TM Analysis software. The nSolver TM software performs quality control (QC) and data normalization and analysis. 36, 38 Normalization is needed to account for variations in hybridization, purification, binding efficiency, and other experimental factors. NanoString recommends applying the internal positive controls in each CodeSet to remove variability. In addition to the positive control and reference (housekeeping) gene normalization, global and Plex 2 normalization may also be performed. 38 Cancer clinical PROSIGNA is a well-known 50-gene NanoString nCounter assay for measuring the risk of relapse (ROR), a prognostic factor for relapse-free survival in breast cancer patients with node-negative tumors who have not received adjuvant systemic therapy. 31, 39 One study used a custom designed nCounter CodeSet for 414 gene elements for studying colon cancer and discovered a subset of genes serving as potential biomarkers for cancer prognosis in FFPE tissue samples. There was a moderate correlation between the nCounter and microarray platforms. The assay included multiple published gene signatures for colon cancer prognosis and several candidate genes elements from ongoing studies in intestinal stem cell biology and epithelial-to-mesenchymal transition (EMT). 40 In another study, the NanoString nCounter technology was chosen to assay RNA extracted from FFPE samples from stage II gastric cancer patients based on its ability to evaluate the expression levels of up to 800 genes. After screening 186 patients for the identification of prognostic genes and evaluating the strength of the prognostic algorithms, 8 genes (LAMP5, CDC25B, CDK1, CLIP4, LTB4R2, MATN3, NOX4, and TFDP1) were identified that, together, efficiently predicted a patient's outcome for the recurrence of cancer post-surgery, regardless of prior adjuvant chemotherapy. There is hope this prognostic signature will be validated in a phase III trial. 41 Through the extraction and analysis of 100 ng of RNA from fresh-frozen tissue and FFPE samples, a 25-gene signature for medulloblastoma patients was established using a specific NanoString nCounter CodeSet through demonstrating a high concordance between the nCounter assay and an Affymetrix expression array data using 101 medulloblastoma samples and a second group of 130 non-overlapping medulloblastomas of known subgroup. Three laboratories in Canada, Switzerland, and the United States demonstrated reproducibility of the assay. 42