Abstract P1-03-09: Highly reproducible decentralized gene expression analysis of ESR1, PGR, ERBB2 and MKi67 on an automated, standardized molecular diagnostics platform, GeneXpert
Authors: Wong W , Ho KE , Wu N , Chu VC , Lalli P , Longshore JW , Klein J , Stonecypher M , Lykke C , Sherwood T , Davenport S , Weidler J , Bates M , Press MF 
Published: March 2017

Abstract
Background: Accurate assessment of ER, PgR, HER2, and Ki67 status using FFPE samples from patients with breast cancer is critical for appropriate patient management, yet immunohistochemistry (IHC), the most common method of assessing these markers, suffers from inherent variability due to pre-analytical/analytical factors and subjective interpretation by pathologists. Here we describe the GeneXpert (GX) Breast Cancer Stratifier RUO Assay (BC Strat), a real time quantitative PCR assay (RT-qPCR) kit which exhibits robust, highly reproducible mRNA measures of ESR1, PGR, ERBB2 (HER2) and MKi67.
The aims of this study were: 1) To assess the impact of variability contributed by pathologist-to-pathologist differences in the selection of the tumor area to be assayed, inter-laboratory assay performance, and macrodissection (MAC) vs. no macrodissection (nonMAC) on decentralized BC Strat results; and 2) to assess preliminary concordance of BC Strat with central IHC and FISH results.
Materials & Methods: The GX BC Strat is a cartridge-based RT-qPCR assay performed on the GeneXpert® Instrument (Cepheid) that automates RNA purification, RT-qPCR amplification and detection of mRNA of target genes (ESR1, PGR, ERBB2, and MKi67) and a control gene (CYFIP1) after sample preparation. Results are reported as delta cycle threshold (dCt) measurements (CYFIP1 Ct - target gene Ct) in less than 2 hrs.
Thirty-two invasive ductal carcinoma FFPE blocks were sourced based on varying levels of ER, PgR, HER2, and Ki67 expression and % tumor cell content/tumor area. Adjacent sections from each block were prepared as slides and sent to 3 external GX testing sites and a reference lab. Each site used its own pathologists/technicians to determine the % tumor cell content/tumor area, perform MAC or nonMAC, prepare lysates, and perform GX testing. Reference IHC/FISH was performed by Geneuity/MPLN (Maryville, TN, USA). Site-to-site concordance in GX results for MAC or nonMAC samples using pre-defined assay cutoffs per marker were analyzed, as were % tumor cell content/tumor area assessments between pathologists.
Results: BC Strat testing of 32 FFPE breast cancer samples with MAC demonstrated excellent GX site-to-site concordance in positive/negative status calls for ESR1 (100%), PGR (100%), ERBB2 (97%), and MKi67 (97%). In most cases, MAC vs. nonMAC had minimal impact on final positive/negative calls for GX, resulting in high overall concordance for MAC vs. nonMAC for ESR1 (91%), PGR (99%), ERBB2 (99%), and MKi67 (95%). The assay also demonstrated a strong overall concordance with IHC for ESR1 (97%), PGR (81%), ERBB2 (98%, IHC/FISH), and MKi67 (89%).
Conclusion: Decentralized performance of the GX BC Strat Assay is feasible and minimally affected by differences in tumor area selection and MAC techniques across tumors with a range of sizes, invasive tumor cell contents, and expression levels of ER, PgR, HER2, and Ki67. GX BC Strat dCt results across sites are highly reproducible and show good concordance of results with central lab IHC and HER2 FISH results. These results suggest standardized, decentralized testing of ESR1, PGR, ERBB2 and MKi67 by the GX BC Strat in local pathology labs is feasible.
Citation Format: Wong W, Ho KE, Wu N, Chu VC, Lalli P, Longshore JW, Klein J, Stonecypher M, Lykke C, Sherwood T, Davenport S, Weidler J, Bates M, Press MF. Highly reproducible decentralized gene expression analysis of ESR1, PGR, ERBB2 and MKi67 on an automated, standardized molecular diagnostics platform, GeneXpert [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-09.