Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel

Real-time RT-PCR is a relatively new technology that uses an online fluores cence detection system to determine gene expression levels. It has the pote ntial to significantly improve detection of breast cancer metastasis by vir tue of its exquisite sensitivity, high throughput capacity and quantitative readout system. To assess the utility of this technology in breast cancer staging, we determined the relative expression levels of 12 cancer-associat ed genes (mam, PIP, mamB, CEA, CK19, VEGF, erbB2, mucl, c-myc, p97, vim and Ki67) in 51 negative-control normal lymph nodes and in 17 histopathology-p ositive ALNs. We then performed a receiver operating characteristic (ROC) c urve analysis to determine the sensitivity and specificity levels of each g ene. Areas under the ROC curve indicated that the most accurate diagnostic markers were mom (99.6%), PIP (93.3%), CK19 (91.0%), mamB (87.9%), mucl (81 .5%) and CEA (79.4.0%). mam was overexpressed in 16 of 17 lymph nodes known to contain metastatic breast cancer at levels ranging from 22- to 2.8 x 10 (5)-fold above normal mean expression, whereas PIP was overexpressed from 3 0- to 2.2 x 10(6)-fold above normal in 13 lymph nodes. Real-time RT-PCR ana lysis of pathology-negative LN from breast cancer patients revealed evidenc e of overexpression of PIP (6 nodes), mom (3 nodes) and CEA (1 node) in 8 o f 21 nodes (38%). Our results provide evidence that mom, PIP, CK19, mamB, m ud and CEA can be applied as a panel for detection of metastatic and occult micrometastatic disease. (C) 2001 Wiley-Liss, Inc.