Microarray-based determination of ER, PR and HER2 receptor status: validation and comparison with IHC assessments.

Abstract

#3007 Background
 In breast cancer patients the level of expression of estrogen receptor (ER), progesterone receptor (PR) and HER2 is predictive for prognosis and/or treatment response. However, differences in assessment methods and interpretation can substantially affect the accuracy and reproducibility of the results. Previously, we have determined the association between immunohistochemistry (IHC) and mRNA levels for ER, PR and HER2, and have confirmed the accuracy of microarray readout on >400 samples. In the current study we describe the use of this microarray based readout on prospectively collected samples. We compared these readouts with multiple IHC and fluorescent in situ hybridization (FISH) assessments generated in various hospitals and a CLIA-certified reference laboratory and developed a microarray based test called TargetPrint™.
 Methods
 Gene expression data for ER, PR and HER2 were obtained by analysis of 100 breast carcinomas that have been collected prospectively within the RASTER study. Samples were stratified as receptor positive or negative using thresholds for ER, PR and HER2 mRNA levels. IHC assessment was performed (1) according to local standards of the hospital from where the sample originated, (2) by the central laboratory of the Netherlands Cancer Institute, and (3) at an independent reference laboratory using FDA-approved procedures and ASCO/CAP guidelines. A tumor was classified positive for ER and PR when ≥10% of tumor cells showed positive staining. HER2 IHC status was scored as 0, 1+, 2+ or 3+; a score of 3+ was considered positive. In case of 2+ samples, a FISH was performed to assess final HER2 amplification status. The cohort used in this study was pre-selected to include about two-third ER and PR positive samples and one-third HER2 positive samples.
 Results
 Multiple microarray readouts were highly reproducible (Pearson correlation 0.991) and resulted in 67, 61 and 39 percent positive samples for ER, PR and HER2, respectively. Comparison of microarray results with IHC (including FISH for HER2) performed at the three centers indicated highly similar results for receptor readout with a concordance of 92, 93 and 92% for ER; 84, 81 and 86% for PR; and 93, 95 and 94% for HER2. Overall misclassification rates between microarray and IHC readout were low for ER (0.08) and HER2 (0.06) and quite low for PR (0.14), and were comparable to the misclassification rates between the three IHC methods.
 Conclusion
 A microarray-based assessment of ER, PR and HER2 in relation to mRNA levels gives results comparable to multiple IHC methods and FISH and provides an objective and more quantitative assessment of tumor receptor status than IHC alone. Using TargetPrint™ for microarray readouts for hormone and HER2 receptor in addition to standard IHC will improve molecular characterization of breast cancer tissue. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3007.

Comparison of MammaPrint and TargetPrint results with clinical parameters in German patients with early stage breast cancer

“…The microarraybased read-out TargetPrint relies on controlled and objective quantification of receptor mRNA levels showing a high concordance with IHC, as well as fluorescence in situ hybridization (FISH) or chromogenic in situ hybridisation (CISH) assessments. Concordant results between TargetPrint and IHC/CISH were detected in 93% for ER and 96% for HER2 (15) indicating that this method provides accurate measurement of important prognostic factors and, therefore, is a reliable addition to current techniques.…”

A diagnostic gene profile for molecular subtyping of breast cancer associated with treatment response

“…The ER, PR, and HER2 status of cohorts 1, 2, and 3 were determined by IHC and by TargetPrint (Agendia; see overview in Table 1) [14,23]. Detailed procedures for centrally performed IHC and TargetPrint of the samples from cohorts 1-3 have been previously reported [14].…”

“…Detailed procedures for centrally performed IHC and TargetPrint of the samples from cohorts 1-3 have been previously reported [14].…”

“…We used the TargetPrint assay [14] to quantify ER, PR, and HER2 mRNA levels in a training cohort of 295 breast cancer samples. We then used IHC/CISH to measure ER, PR, and HER2 protein levels in the same 295 samples for the same three receptors.…”

“…TargetPrint), rely on the presence of protein and mRNA, respectively, but neither assay determines whether the protein, or mRNA is functional in making full length and functional receptor proteins. As such, both methods have an inherent uncertainty in predicting whether a tumor is truly positive for functional ER, PR, or HER2 protein [14]. One method to circumvent this problem is to develop gene signatures that measure the expression of groups of genes that correlate with the presence of the gene(s) of interest [15,16].…”