Abstract
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.
Highlights
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
DNA quantification was performed with the QIAxpert spectrophotometer (Qiagen, Hilden, Germany) using the QIAamp DNA plugin with internal blank calibration for elution buffer ATE with the OD260 readout of total nucleic acids
Fractional polynomial approach was used to analyze the correlation between percent methylation ratio (PMR) data calculated from generalized log transformation (gLOG) values of methylation-specific microarray data (PMRcalc) and PMR data measured with the therascreen PITX2 RGQ assay (PMRexp) values to determine the best-fitting function19. gLog(2) fluorescence intensity values) of PITX2 on a methylation specific oligonucleotide microarray[20] were transformed into PMR values (PMRcalc) by the formula: PMRcalc = 2exp(gLOG) to allow for comparability with PMR data (PMRexp) from the therascreen PITX2 RGQ polymerase chain reaction (PCR) assay according to the respective probe Genomic DNA (gDNA) yield
Summary
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. 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 anthracyclinebased 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 St. Gallen classification includes steroid hormone receptor status and human epithelial growth factor receptor 2 (HER2) status, nodal status, tumor size and other clinicopathological factors to classify low- (10%), medium(65%) and high-risk (25%) patient groups and help to guide therapy decision[2,3]. Until now biomarkers to predict outcome to anthracycline-based chemotherapy are of high unmet medical need
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