Abstract
BackgroundFull RNA-Seq is a fundamental research tool for whole transcriptome analysis. However, it is too costly and time consuming to be used in routine clinical practice. We evaluated the transcript quantification agreement between RNA-Seq and a digital multiplexed gene expression platform, and the subtype call after running the PAM50 assay in a series of breast cancer patients classified as triple negative by IHC/FISH. The goal of this study is to analyze the concordance between both expression platforms overall, and for calling PAM50 triple negative breast cancer intrinsic subtypes in particular.ResultsThe analyses were performed in paraffin-embedded tissues from 96 patients recruited in a multicenter, prospective, non-randomized neoadjuvant triple negative breast cancer trial (NCT01560663). Pre-treatment core biopsies were obtained following clinical practice guidelines and conserved as FFPE for further RNA extraction. PAM50 was performed on both digital multiplexed gene expression and RNA-Seq platforms. Subtype assignment was based on the nearest centroid classification following this procedure for both platforms and it was concordant on 96% of the cases (N = 96). In four cases, digital multiplexed gene expression analysis and RNA-Seq were discordant. The Spearman correlation to each of the centroids and the risk of recurrence were above 0.89 in both platforms while the agreement on Proliferation Score reached up to 0.97. In addition, 82% of the individual PAM50 genes showed a correlation coefficient > 0.80.ConclusionsIn our analysis, the subtype calling in most of the samples was concordant in both platforms and the potential discordances had reduced clinical implications in terms of prognosis. If speed and cost are the main driving forces then the preferred technique is the digital multiplexed platform, while if whole genome patterns and subtype are the driving forces, then RNA-Seq is the preferred method.
Highlights
Full RNA-Seq is a fundamental research tool for whole transcriptome analysis
Developed on microarray data, Prediction analysis for microarrays (PAM50) is being successfully used in digital multiplexed gene expression platforms such as NanoString nCounter®, which is the basis for the Prosigna® test
The goal of the study was assessing the reproducibility of PAM50 intrinsic subtype when using RNA-Seq and NanoString nCounter® data from formalin-fixed paraffin-embedded (FFPE) tissue obtained from a triple negative breast cancer (TNBC) patient cohort
Summary
Full RNA-Seq is a fundamental research tool for whole transcriptome analysis. it is too costly and time consuming to be used in routine clinical practice. Developed on microarray data, PAM50 is being successfully used in digital multiplexed gene expression platforms such as NanoString nCounter®, which is the basis for the Prosigna® test. The latter includes the PAM50 assay in combination with a clinical factor (i.e. tumor size) and has been approved for the risk of distant relapse estimation in postmenopausal women with hormone receptorpositive, node negative or node positive early stage breast cancer patients; and is a daily-used tool assessing the indication of adjuvant chemotherapy [5, 6]
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