Abstract 5409: Assessment of the MammaPrint 70-gene profile using RNA sequencing technology
Authors: Mittempergher Lorenza , Spangler Jacob B. , Snel Mireille H. , Delahaye Leonie J. , Rink Iris de , Tian Sun , Glas Annuska M. , Bernards Rene 
Published: July 2017

Abstract
Introduction: Improvements in RNA processing have enabled microarray diagnostics for formalin-fixed, paraffin-embedded (FFPE) tissue. Recently, MammaPrint, a prognostic 70-gene profile for early-stage breast cancer, was successfully translated to FFPE tissue showing to be substantially equivalent to fresh tissue. In recent years, RNA-sequencing (RNA-Seq) became the standard method for transcriptome analysis, because of its low background signal and its ability of quantifying a large dynamic range of expression levels. Here we report a preliminary analysis of the FFPE MammaPrint 70-gene profile using RNA-Seq technology and the comparison with the MammaPrint® microarray diagnostic test in a series of FFPE samples.
Methods: RNA-Seq was carried out using a strand-specific RNA library preparation followed by target enrichment of the coding region of the human transcriptome without relying on the presence of poly-A tail. RNA sequencing libraries were prepared starting from a minimal amount of 20 ng of total RNA based on the DV200 metric assessment. The library pools were single-end sequenced on the Illumina HiSeq 2500 instrument at the length of 65bp. The resulting sequences were mapped to the human reference genome (build 38) using TopHat v2.1. Tophat was guided by using a transcriptome index from Ensembl (version 77). The HTSeq-count tool was used to generate the total number of uniquely mapped reads for each gene. Gene expressions were normalized with Count Per Million (CPM) normalization and log2 transformed afterwards. Microarray data of the sample were available for analysis comparison.
Results: On average, we obtained 22 million reads assigned to gene per sample (min=15M, max=28M). The number of reads assigned to genes vary from 61% to 70% of the total number of reads. Between 80% and 90% of the reads assigned to genes mapped to protein coding genes which is comparable to fresh frozen material. The 70-gene signature was successfully mapped to the RNA-Seq genes. A median raw read-count of 384 was observed for the 70-gene profile among the samples. Importantly, we observed a high concordance (R2 Pearson correlation=0.97) between the MammaPrint index calculated using the RNA-Seq data and the correspondent Microarray MammaPrint index. Additionally, the BluePrint profile, a microarray diagnostic test for breast cancer molecular subtyping, was successfully translated to the RNA-Seq platform. As with the MammaPrint profile, BluePrint showed high concordance between the two technologies with high correlation values for each of the subtypes (Luminal R2 Pearson correlation=0.98, Basal R2 Pearson correlation=0.97, HER2 R2 Pearson correlation=0.77). Conclusions: Next Generation RNA-sequencing is a feasible technology to assess diagnostic signatures, such as the 70 gene MammaPrint and BluePrint profiles.
Citation Format: Lorenza Mittempergher, Jacob B. Spangler, Mireille H. Snel, Leonie J. Delahaye, Iris de Rink, Sun Tian, Annuska M. Glas, Rene Bernards. Assessment of the MammaPrint 70-gene profile using RNA sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5409. doi:10.1158/1538-7445.AM2017-5409