Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens

Abstract

BackgroundNanoString’s Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories.MethodsAnalytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test.ResultsThe measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area).ConclusionsThe analytical performance of NanoString’s Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories.

Molecular signatures in breast cancer

“…These gene expression panel assays are risk predictors and examples include: MapQuantDX™ (Genomic Grade Index, GGI) [36], ProSigna® [24] [37], Mammaprint® [38], OncotypeDX® [39] and EndoPredict® [40] (Table 1). Each predictor was derived in a different manner and using a unique algorithm, but collectively they classify the risk of recurrence of breast cancer, and hence provide insight into whether a patient might require adjuvant chemotherapy or not.…”

“…ProSigna® is the commercial iteration of the PAM50 subtyping tool, and generates a risk of recurrence score primarily to predict chemotherapy benefit [21,24,37,48].…”

An mRNA Gene Expression–Based Signature to Identify FGFR1-Amplified Estrogen Receptor–Positive Breast Tumors

“…49 The RNA samples were hybridized with the code set and processed using the NanoString nCounter Analysis system, as described in our previously published studies. 47,50 Six positive control genes at six concentrations, eight negative control genes, and three housekeeping genes (MRPL19, SF3CA1, and PUM1) were profiled together with the nine candidate genes. Quality control and preprocessing of the NanoString nCounter raw counts on the UBC-TAM cohort samples were performed according to the manufacturer's instruction guide, through the following sequential processing steps: i) remove samples with a binding density >2.25 or <0.1; ii) remove samples with three or more positive control genes having zero counts; iii) calculate a positive control normalization factor per sample as the mean summed counts of the six positive control genes across all samples divided by the summed counts of the six positive control genes per sample; iv) remove samples with the positive control normalization factor >3 or <0.3; v) calculate the mean and SD across negative control genes per sample and subtract each gene expression value by the background, defined as mean þ 2 Â SD; vi) calculate the housekeeping genes geometric mean per sample and the averaged geometric mean across samples, and the housekeeper gene normalization factor is calculated as the averaged geometric mean divided by per-sample geometric mean; vii) gene expression values per sample, multiply the sample-specific housekeeper gene normalization factor; viii) take log2 transformation of the gene expression.…”

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

“…36 A handful of miRNA-based tests aimed at guiding therapeutic management have been already approved by FDA for applications such as classification of cancer of unknown primary origin (CUP), lung cancer type classification, thyroid cancer stratification, and breast cancer metastasis and recurrence analysis, based on the differential expression of miRNA biomarker sets. [37][38][39][40][41][42][43][44][45] Unlike the tests for viral RNA, which are offered as kits with a companion platform, cancer testing is offered as a service to healthcare providers. Most studies so far have dealt measuring the concordance of test results with immunohistochemistry and pathology in cancer typing and stratification.…”