Evaluation of ER/PR and HER2 status by RNA sequencing in tissue core biopsies from preoperative clinical trial specimens.

Abstract

46Background: Next-generation sequencing for measuring RNA (RNASeq) offer increased sensitivity, dynamic range and provide unbiased detection of all transcripts. To evaluate the clinical utility of such methods, we sequenced entire transcriptomes from fresh-frozen biopsies in a cohort of 120 patients enrolled on a preoperative therapy trial receiving carboplatin, nab paclitaxel and either bevacizumab (HER2-) or trastuzumab (HER2+).Methods: Total RNA was extracted and amplified from frozen breast core biopsies and libraries constructed using TruSeq (Illumina). Sequencing was performed on the Illumina GAII platform. 75bp reads were mapped using Tophat and transcript abundance in FPKM units (Fragments per kilo-base of mRNA per million reads) calculated using Cufflinks. CLIA approved assays were performed for ER, PR, HER2 (IHC+/- FISH) on patient tumors. Four tumors from each subtype (ER +ve/HER2 -ve; HER2 +ve; ER/HER2 -ve) were analyzed for correlation with clinical status. PAM50 classification will be provided for verification of molecular subtypes.Results: RNA-Seq library construction/sequencing were successful in 12/12 samples with 50-90% reads mapped. ER +ve tumors ranged in FPKM values from 1.76-22.67 and ER -ve tumors ranged from 0.00-0.79. i.e. ER RNASeq measurements can separate clinical ER status. HER2 +ve tumors ranged in FPKM values from 2.62-21.71 and HER2 -ve tumors from 0.21-1.79. Of note, 7/8 HER2 -ve tumors ranged from 0.21-0.87 with one ‘outlier’ at 1.79±0.3. This outlier was HER2 IHC 2+, FISH ratio 1.1 with 45% of tumor cells with polysomy chromosome 17. Correspondence of ER/PR and HER2 status with molecular subtyping by PAM50 analysis will be presented.Conclusions: RNASeq has potential to provide in depth analysis of the breast cancer transcriptome and a single analysis test for standard markers. In addition, RNASeq may uncover unexpected expression patterns in conventionally-defined HER2 -ve tumors. If reproducible in larger datasets, this technology may provide both standard and novel information previously unavailable to oncologists and their patients.