Quantitative detection of micrometastases in the lymph nodes of gastric cancer patients with real-time RT-PCR: a comparative study with immunohistochemistry.

Abstract

Histologic examination lacks the sensitivity to detect micrometastases in gastric cancer lymph nodes. In the present study, we applied a real-time RT-PCR approach to the quantitative detection of micrometastases in gastric cancer lymph nodes and compared diagnostic power with routine histology and immunohistochemistry. We studied 392 lymph nodes from 21 gastric cancer patients who underwent curative surgery. Real-time quantitative RT-PCR was performed on a LightCycler instrument using a hybridization probe for carcinoembryonic antigen (CEA) and cytokeratin-20 (CK20) as marker genes. Immunohistochemistry with antibodies to wide-keratin was also performed in the lymph nodes to compare the sensitivity and specificity. Median (average) values of CEA mRNA in lymph nodes in patients with histology(+), immunohistochemistry(+)/histology(-), immunohistochemistry(-)/histology(-) and negative control results were 4600 (16000), 200 (400), 0 (9.8) and 0 (0.6), respectively. There were some false-negative results with simple CEA and CK20 real-time RT-PCR due to the presence of low gene-expressing gastric cancers as revealed by CEA and CK20 immunohistochemistry. CEA in combination with CK20 (duplex) real-time RT-PCR partially covered this weakness. Consequently, all 71 histology(+) lymph nodes were positive for duplex real-time RT-PCR as well as wide-keratin immunohistochemistry. Positivity rates by histology, wide-keratin immunohistochemistry and duplex real-time RT-PCR were 18.0% (71/392), 20.9% (82/392) and 25.8% (101/392), respectively. In 2 of 8 patients with pT1N0, positive lymph nodes were observed by real-time RT-PCR but not by immunohistochemistry. These results indicate that duplex quantitative real-time RT-PCR is the most sensitive method for detecting micrometastases and useful for evaluating the prognostic significance of lymph node micrometastasis in gastric cancer patients.