Abstract P1-03-11: Analytical validation for the RT-qPCR based multiplex mRNA measurements of ER, PgR, HER2, and Ki67 from FFPE tumor tissue using the GeneXpert breast cancer stratifier assay
Authors: Chu VC , Wu N , Ho KE , Rizo A , Malek M , Weidler JM , Bates M , Wong W 
Published: March 2017

Abstract
BACKGROUND: Accurate assessment of ER, PgR, HER2 and Ki67 status is crucial for breast cancer therapy and patient management. Immunohistochemistry (IHC) assays have been standard diagnostic tools but they are complex and time-consuming to perform and may not be readily available in decentralized laboratories, particularly in low-to-middle income countries. Molecular diagnostics can be a sensitive and accurate alternative to the traditional IHC, and the GeneXpert Breast Cancer Stratifier assay RUO (BC Strat), a single use cartridge-based assay performed on the broadly distributed GeneXpert® Instrument (GX) platform, streamlines a technically demanding RT-qPCR process to provide easy, robust, and reproducible ESR1, PGR, ERBB2, and MKi67 mRNA measurements from a 4 µm thick, formalin-fixed paraffin embedded (FFPE) breast tumor section in less than 2 hours.
METHODS: Analytical validation of the BC Strat assay included studies of Linearity/Dynamic Range, Analytical Sensitivity (Minimum Assay Input), Specificity (Potential Interfering Substances), Carryover Contaminations, and Kit and Specimen Slide Stabilities. Both in-vitro RNA transcript (IVT) and/or clinical breast cancer tissues were used as sample input materials. Assay results for each analyte were generated from cycle threshold (Ct) values, and final positive/negative test results for each target were also reported as delta Ct values, where dCt = Ct [CYFIP1 Reference] – Ct [Target], using dCt cutoffs previously derived from a clinical sample cohort.
RESULTS: The BC Strat assay demonstrated ≥3 log Linear Dynamic Range covering 5-7 logs sample input for all 4 Target dCts with R2≥0.95 independently. The assay currently requires minimal sample input equivalent to CYFIP1 Ct≤35 (Ct=33.5 ±1.5Ct SD) from 20 replicates of 5-level serial sample dilutions using two independent assay lot materials. It is acceptably robust against non-tumor tissues, DCIS, necrotic and/or hemorrhagic cells, lymphocytes, and genomic DNA contaminants. No carryover contamination from the same GeneXpert module was observed over 20 repeat tests during 9 consecutive days. Current real-time data supports assay stability at 5, 30, 37, 45 and 50°C for at least 3 months with minimal performance impact. Sectioned FFPE breast tumor tissues generated consistent dCt results when stored at 4°C and 30°C for up to 1 month before BC Strat assay testing.
CONCLUSIONS: The analytical validations of the BC Strat assay demonstrate an easy and robust mRNA detection with high sensitivity, specificity, reproducibility, and stability in order to aid medical pathologists and clinicians to more rapidly and objectively determine ESR1, PGR, ERBB2, and MKi67 mRNA status in breast cancer. Although stability studies out to 37 months are ongoing, current data suggest the assay is stable for at least 3 months over a wide range of temperatures. The GeneXpert Breast Cancer Stratifier assay potentially offers a rapid, standardized, and cost-effective solution to streamlining complex molecular diagnostics available for use in local pathology laboratories worldwide.
Citation Format: Chu VC, Wu N, Ho KE, Rizo A, Malek M, Weidler JM, Bates M, Wong W. Analytical validation for the RT-qPCR based multiplex mRNA measurements of ER, PgR, HER2, and Ki67 from FFPE tumor tissue using the GeneXpert breast cancer stratifier assay [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 P1-03-11.