Analytical Validation of a Novel 6-Gene Signature for Prediction of Distant Recurrence in Estrogen Receptor-Positive, HER2-Negative, Early-Stage Breast Cancer

Tony Loughman, Stephen Barron, Chan-Ju Angel Wang, Peter Dynoodt, Bozena Fender, Cesar Lopez-Ruiz, Sharon Stapleton, Aurelie Fabre, Cecily Quinn, Bjorn Nodin, Karin Jirström, Fatemeh Razmara, Anthony O'Grady, Anne-Marie Baird, Steven G Gray, Ana Freixo, Cathy B Moelans, Paul J van Diest, Michael J Duffy, Desmond O'LearyJohn Crown, Adrian P Bracken, William M Gallagher

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Abstract

BACKGROUND: OncoMasTR is a recently developed multigene prognostic test for early-stage breast cancer. The test has been developed in a kit-based format for decentralized deployment in molecular pathology laboratories. The analytical performance characteristics of the OncoMasTR test are described in this study.

METHODS: Expression levels of 6 genes were measured by 1-step reverse transcription-quantitative PCR on RNA samples prepared from formalin-fixed, paraffin-embedded (FFPE) breast tumor specimens. Assay precision, reproducibility, input range, and interference were determined using FFPE-derived RNA samples representative of low and high prognostic risk scores. A pooled RNA sample derived from 6 FFPE breast tumor specimens was used to establish the linear range, limit of detection, and amplification efficiency of the individual gene expression assays.

RESULTS: The overall precision of the OncoMasTR test was high with an SD of 0.16, which represents less than 2% of the 10-unit risk score range. Test results were reproducible across 4 testing sites, with correlation coefficients of 0.94 to 0.96 for the continuous risk score and concordance of 86% to 96% in low-/high-risk sample classification. Consistent risk scores were obtained across a > 100-fold RNA input range. Individual gene expression assays were linear up to quantification cycle values of 36.0 to 36.9, with amplification efficiencies of 80% to 102%. Test results were not influenced by agents used during RNA isolation, by low levels of copurified genomic DNA, or by moderate levels of copurified adjacent nontumor tissue.

CONCLUSION: The OncoMasTR prognostic test displays robust analytical performance that is suitable for deployment by local pathology laboratories for decentralized use.

Original languageEnglish
Pages (from-to)837-847
Number of pages11
JournalClinical chemistry
Volume68
Issue number6
Early online date21 Mar 2022
DOIs
Publication statusPublished - 1 Jun 2022

Keywords

  • RT-qPCR
  • breast cancer
  • gene expression
  • prognostic biomarker

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