Abstract P2-03-03: A multicenter clinical study of Xpert® breast cancer STRAT4 demonstrates high concordance with central lab ER, PgR, HER2, and Ki67 IHC and HER2 FISH tests in FFPE breast tumor tissues
Authors: Wu NC , Wong E , Acca B , Birkmeier J , Tran L , Zhao S , Wong W , Chu VC , Ho K , Malek M , Lu C , Ge G , David K , Quigley NB , Beqaj SS , Davenport S , Weidler J , Bates M , Press M 
Published: February 2018

Abstract
The Xpert® Breast Cancer STRAT4 (STRAT4) is a CE-IVD marked, semi-quantitative, cartridge-based RT-qPCR assay for the detection of ESR1, PGR, ERBB2 (HER2), and MKi67 mRNAs from formalin fixed, paraffin embedded (FFPE) breast tumors. The assay is fast (&lt; 2 hrs), reproducible, robust, and easy to perform.
The aim of this multicenter clinical study was to assess the performance characteristics of the STRAT4 assay relative to central lab immunohistochemistry (IHC) for ER, PgR, HER2, and Ki67 and to fluorescence in situ hybridization (FISH) for HER2 gene amplification.
Methods: A total of 200 archived primary invasive breast cancer FFPE blocks were sourced from Indivumed for this study. From each block, twelve (12) adjacent tissue sections (4-µm thickness) on slides were prepared for pathological H&E confirmation to define tumor area, and for testing by STRAT4, IHC (ER, PgR, HER2,Ki67), and HER2 FISH. Standard STRAT4 lysate preparation using a single unstained slide per specimen and testing on N=84, N=68, and N=48 samples was performed at 3 independent sites, respectively (2 US and 1 EU). A single slide from each specimen was also processed using the recommended concentrated lysate procedure for STRAT4 testing at Cepheid. All IHC and FISH testing was performed by a central academic reference laboratory in the US. For a given sample, STRAT4 data generated using the standard lysate procedure was included for concordance analysis when all target gene test results were valid. In cases where the standard lysate preparation yielded indeterminate test results for any target, data from the concentrated lysate preparation was used for the data analysis. Receiver Operating Characteristic (ROC) analysis, overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA) between STRAT4 and IHC (IHC/FISH for HER2) were determined for ESR1,PGR, ERBB2, and MKi67.
Results: Of the 200 samples tested by STRAT4, all samples generated valid results for ESR1 and ERBB2, 199 of 200 samples were valid for PGR, and 198 of 200 samples were valid for MKi67 using the standard or concentrated lysate preparation protocol. One sample failed to generate results for both ER and PgR IHC. Twelve samples failed to yield HER2 FISH results.
The STRAT4 success rate and results concordance with IHC were comparable across study sites. OPA between STRAT4 and IHC was 97% for ESR1, 88.9% for PGR, 93.3% for HER2 (92.4% for IHC and FISH), and 90.7% for MKi67 (excluding IHC 10-20% staining). Areas under the ROC curves were 0.9922 for ESR1, 0.9509 for PGR, 0.9958 for ERBB2, and 0.9395 for MKi67.
Conclusion: STRAT4 measurements for ESR1, PGR, ERBB2 and MKi67 mRNA expression are robust and highly concordant with IHC (IHC/FISH for HER2). The technical portion of the assay is easily performed in &lt; 2 hrs including hands-on time using standard FFPE tissue sections. Xpert STRAT4 offers local pathology labs an alternative to centralized, subjective IHC/FISH tests that require a higher level of expertise. Further investigations correlating STRAT4 markers directly with clinical outcomes in independent cohorts are in progress.
Citation Format: Wu NC, Wong E, Acca B, Birkmeier J, Tran L, Zhao S, Wong W, Chu VC, Ho K, Malek M, Lu C, Ge G, David K, Quigley NB, Beqaj SS, Davenport S, Weidler J, Bates M, Press M. A multicenter clinical study of Xpert® breast cancer STRAT4 demonstrates high concordance with central lab ER, PgR, HER2, and Ki67 IHC and HER2 FISH tests in FFPE breast tumor tissues [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-03.