Abstract P4-08-20: Pre-analytical effects of FFPE extraction methods on targeted and whole transcriptome sequencing assays for endocrine sensitivity in metastatic breast cancer
Authors: Marczyk M , Fu C , Lau R , Du L , Trevarton AJ , Sinn BV , Gould RE , Symmans WF , Hatzis C 
Published: May 2019

Abstract
Background: The clinical management of patients with metastatic HR-positive breast cancer is often uncertain due to decreased sensitivity to anti-estrogen therapy over time. Recently, we developed a targeted RNAseq based 18-transcript SET ER/PR assay of endocrine sensitivity from biopsies of metastatic cancer. In this work we assess the effect of pre-analytical factors, specifically RNA extraction methods for FFPE tissue samples, on the reliability of the targeted RNAseq assay.
Methods: FFPE blocks and matched fresh frozen (FF) sections from 12 tumors were collected at MD Anderson Cancer Center. RNA from FFPE slides was extracted in duplicate using three kits (Norgen, Qiagen, Roche), and RNAseq libraries from all samples were prepared using Kapa Total RNAseq kit. Targeted RNA libraries were prepared using droplet-based PCR (RainDance), and also by transcriptome-wide RNAseq for comparison. Reads were mapped to genomic sequence using STAR and expression was quantified using RSEM. Expression data were normalized based on expression of 10 reference genes. The effect of FFPE RNA extraction kit on the reliability of the SET index was assessed using linear mixed effects model (LME) analysis, and agreement with FF was assessed using the concordance correlation coefficient (CCC).
Results: Analysis of the whole transcriptome RNAseq data confirmed minimal 3'-end transcript bias from FFPE samples, irrespective of transcript size or FFPE kit. All 18 genes included in the SET index had high overall concordance between FFPE and FF (median CCC percentile=98.8, range 57.2-99.9 for Norgen; similar for the other two kits) and relatively consistent bias across genes, as estimated by the random effects of the LME model. Furthermore, compared to random 18-gene indices, concordance in the SET index values between FF and FFPE was higher than 99.8% of the random samples, verifying the analytical reliability of the selected genes. For the targeted RNAseq assay, RNA from FFPE extracted with the Norgen kit showed the highest concordance compared to FF (CCC=0.956, 95%CI 0.871-0.985). In general, the analytical variation of SET from FFPE samples was greater than that from FF (1.71-2.71 fold greater), with the lowest variation associated with the Norgen kit. The SET index values from targeted RNAseq for both FF and FFPE samples were consistently lower compared to transcriptome-wide RNAseq but were highly correlated, with the Norgen kit having the highest correlation between targeted and transcriptome-wide RNAseq (rho=0.915).
Conclusions: All three FFPE RNA extraction kits have excellent analytical performance compared to FF samples. The Norgen kit may be marginally better yielding higher concordance with FF and lower analytical variation between replicates. All genes in the SET ER/PR showed very good analytical performance in comparison to random indices and individual genes. Targeted gene RNA sequencing appears very promising as a platform for clinical deployment of quantitative assays, showing only a small (fixable) bias compared to RNAseq.
Citation Format: Marczyk M, Fu C, Lau R, Du L, Trevarton AJ, Sinn BV, Gould RE, Symmans WF, Hatzis C. Pre-analytical effects of FFPE extraction methods on targeted and whole transcriptome sequencing assays for endocrine sensitivity in metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-20.