High concordance of a closed-system, RT-qPCR breast cancer assay for HER2 mRNA, compared to clinically determined immunohistochemistry, fluorescence in situ hybridization, and quantitative immunofluorescence

Abstract

Background Historically, mRNA measurements have been tested on several commercially available platforms, but none have gained broad acceptance for assessment of HER2. An mRNA measurement, as a continuous value, has the potential for use in adjudication of the equivocal category. Here we use a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay in a closed, single-use cartridge, automated system. 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 with a FFPE lysis kit to create lysates that were tested with the automated RT-qPCR assay. Results for IHC and FISH were extracted from the pathology reports and quantitative immunofluorescence (QIF) for each case was measured as previously described. Results Quality control testing showed that the GX platform RT-qPCR 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. Concordance (OPA) between RT-qPCR and QIF was 94% (sensitivity = 0.90; specificity = 0.96; PPV = 0.93; NPV = 0.94) using dCt ≥ −1 and a previously defined cut-point for positivity by QIF. Conclusions The closed system RT-qPCR assay shows greater than 90% concordance with the ASCO/CAP HER2 IHC/FISH scoring. Additionally, the RT-qPCR assay is highly concordant (94%) 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 fast, closed system molecular assays may have future value for the adjudication of the ASCO/CAP HER2 equivocal category or possibly routine usage in time constrained or low resource settings.

Comparison of central laboratory assessments of ER, PR, HER2, and Ki67 by IHC/FISH and the corresponding mRNAs (ESR1, PGR, ERBB2, and MKi67) by RT-qPCR on an automated, broadly deployed diagnostic platform

“…STRAT4 has already been shown to be highly concordant with automated quantitative analysis of IHC (AQUA) [ 20 ]. From a LMIC perspective, ESR1 and ERBB2 measurements have the greatest relevance currently.…”

Quantitative assessments and clinical outcomes in HER2 equivocal 2018 ASCO/CAP ISH group 4 breast cancer

“…The range and average QIF AQUA score in a series of unselected breast carcinomas was 135.5 to 15087.5 and 3282.8, with 7281.3 the average score for carcinomas classified as positive by QIF. 12 By chance due to random errors associated with any test results close to the cut points could be considered false positive or false negative results compared to conventional testing. Adjudication of these results into negative or positive by repeat testing with multiple methods or different arbitrary cut points is unlikely to be meaningful.…”

“…The Breast Cancer STRAT4 Research Use Only (RUO) assay was performed as previously described. 11,12 STRAT4 is a CE-IVD (Conformité Européene In-vitro Medical Device) product that is available in some, but not all, European countries and is not available in the United States. Where the STRAT4 assay is not available under CEIVD, evaluations of its performance using specimens prepared under local pre-analytical sample handling procedures can be supported under collaborative research agreements using a RUO version.…”

Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer

“…The BC STRAT4 assay was performed as previously described. 12 Briefly, 5μM thick FFPE tissue sections were collected either as a whole tissue section, non m-d, or macrodissected, m-d, to collect tumor only. Samples were lysed using 5μl Proteinase K and 260μl FFPE lysis reagent and incubated for 30 minutes at 80°C.…”