Abstract P1-03-07: High concordance of a closed system, near point of care, RT-qPCR breast cancer assay for HER2 (ERBB2) mRNA compared to both IHC/FISH and quantitative immunofluorescence

Background Reliable assessment of HER2 receptor status in breast cancer by either IHC or FISH does not unequivocally define receptor expression, due to their semi-quantitative nature, and as many as 10-15% of cases fall into the ASCO/CAP “equivocal” category. Historically, RNA measurements by PCR, including using several commercially available platforms, have been tested, but have not gained broad acceptance for assessment of HER2. However, RNA measurement, as a continuous value, has potential for use for adjudication of the equivocal category. In the current study, we used a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay (GeneXpert ® Breast Cancer Stratifier RUO Assay, Cepheid, Sunnyvale, CA, USA) for ERBB2 (HER2) mRNA on the GeneXpert ® (GX) platform (Cepheid), which utilizes a closed-system, single-use cartridge, automated system. The RT-qPCR results from GX were then compared with results from clinical HER2 IHC/FISH assays following ASCO/CAP 2013 HER2 testing guidelines (Wolff et al JCO 2013) and quantitative immunofluorescence (QIF). Methods Multiple cores (1mm in diameter) were retrospectively collected from 80 formalin-fixed paraffin-embedded (FFPE) tissue blocks with invasive breast cancer seen by Yale Pathology Labs between 1998 and 2011. Tissue cores were processed as lysates for testing at Yale in the automated GX assay. Briefly, gene-specific reverse transcription was performed, followed by RT-qPCR (TaqMan) and ERBB2 mRNA results were expressed as the difference in cycle threshold values (delta Ct) between the endogenous control transcript (CYFIP1) and the ERBB2 mRNA transcript. Results from IHC and FISH were extracted from the pathology reports for the Yale CLIA lab and QIF for each case was measured as previously described (Carvajal et al, JNCI 2015). Results Quality control testing showed that the GX platform shows no case to case cross contamination on material from routine histology practices. Concordance between RT-qPCR and IHC/FISH was 91.25% (sensitivity = 0.87; specificity = 0.94; PPV = 0.89; NPV = 0.92) using a pre-defined delta Ct cut-off (dCt ≥ -1) for HER2 (+) based on prior concordance studies with HER2 IHC/FISH. Concordance between RT-qPCR and QIF was 99% (sensitivity = 0.97; specificity = 1.0; PPV = 1.0; NPV = 0.98) using dCt ≥ -1 and the pre-defined cut-point for positivity by QIF. Conclusions The GX closed system RT-qPCR assay shows greater than 90% concordance with the ASCO/CAP 2013 HER2 IHC/FISH scoring. Additionally, the GX RT-qPCR assay is highly concordant (99%) with the continuous variable HER2 QIF assay, and may better reflect the true continuum of HER2 receptor status in invasive breast cancer. These initial results suggest that rapid, closed system molecular assays may have future value for the adjudication of the ASCO/CAP HER2 equivocal category. This pilot study did not include ASCO/CAP 2013 “equivocal” cases, but that effort is underway. Citation Format: Wasserman B, Carvajal-Hausdorf D, Ho K, Wong W, Wu N, Chu VC, Lai EW, Weidler JM, Bates M, Neumenister V, Rimm DL. High concordance of a closed system, near point of care, RT-qPCR breast cancer assay for HER2 (ERBB2) mRNA compared to both IHC/FISH and quantitative immunofluorescence [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 P1-03-07.