Significant evidence has accumulated that DNA-methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene can serve as a prognostic and predictive biomarker in breast cancer. PITX2 DNA-methylation data have been obtained so far from microarray and polymerase chain reaction (PCR)-based research tests. The availability of an analytically validated in vitro methylation-specific real-time PCR assay format (therascreen PITX2 RGQ PCR assay) intended for the determination of the percent methylation ratio (PMR) in the (PITX2) promoter 2 prompted us to investigate whether the clinical performance of these different assay systems generate comparable clinical outcome data. Mathematically converted microarray data of a previous breast cancer study (n = 204) into PMR values leads to a PITX2 cut-off value at PMR 14.73. Recalculation of the data to experimentally equivalent PMRs with the PCR PITX2 assay leads to a cut-off value at PMR 12 with the highest statistical significance. This cut-off predicts outcome of high-risk breast cancer patients to adjuvant anthracycline-based chemotherapy (n = 204; Hazard Ratio 2.48; p < 0.001) comparable to microarray generated results (n = 204; Hazard ratio 2.32; p < 0.0001). The therascreen PITX2 RGQ PCR assay is an analytically validated test with high reliability and robustness and predicts outcome of high-risk breast cancer patients to anthracycline-based chemotherapy.

The therascreen PITX2 RGQ PCR assay is an optimized in vitro DNA methylation-specific quantitative real-time PCR test intended for the determination of the percent methylation ratio (PMR) in the pituitary homeobox 2 (PITX2) transcription factor gene promoter 2. The test uses bisulfite-converted genomic DNA derived from formalin-fixed paraffin-embedded tumor tissues of breast cancer patients. The PMR will aid clinicians in selecting adjuvant systemic chemotherapy, e.g. anthracycline-based chemotherapy in high-risk lymph node-positive, estrogen receptor-positive, HER2-negative breast cancer patients. The assay is intended to be used by qualified users, such as technicians, molecular biologists/clinical chemists, or physicians, trained in molecular biology techniques and in vitro diagnostic procedures. The complete workflow is streamlined for medium sample throughput with highly reliable and robust readout and can be performed in two working days.