Detection Of Micrometastasic Tumor Cells In Lymphadenectomy Specimens Of Patients With NSCLC By Real-time RT-PCR

Introduction: Lung cancer is the most common cancer over the world, also is a one of the leading causes of cancer death. Disseminated individual tumor cells in regional lymph nodes can be found in 25-40% of patients with operable non-small cell lung cancer (NSCLC) and are significantly associated with a poor prognosis. Detection of disseminated tumor cells in mediastinoscopic biopsies could improve staging prior to resection and might be helpful concerning indications for neoadjuvant therapy regimens. However, the most common method for the detection of tumor cells in lymph nodes – immunohistochemistry - is laborious, time consuming and observer-dependent. This prospective study was performed to evaluate a simple and observer-independent polymerase chain reaction (PCR) based method for the detection of tumor cells in clinical specimens of regional lymph nodes. Material and methods: Regional lymph nodes of 30 consecutive patients without neoadjuvant therapy were removed by systematic lymphadenectomy during resection of primary NSCLC. 95 lymph nodes which were tumor free by routine histopathology method, were examined subsequently using real-time reverse transcriptase PCR (real-time RT-PCR). Real-time RT-PCR amplification of Melanoma antigen gene A (MAGE-A) and cytokeratin 19 (CK19) transcripts in a light-cycler instrument were applied for the detection of tumor cell-specific RNA in lymph nodes. 8 lymph nodes of patients with benign lung diseases served as negative controls. 15 primary tumor tissues of patients with NSCLC served as positive controls. Results: In the positive control group, 80.0%(12/15) tumor tissues express the MAGE-A mRNA, in which there were 7 squamous carcinoma samples, 3 adenocarcinoma samples and 2 large cell carcinoma samples. For the expression of CK19mRNA, the positivity is 93.3 %(14/15), only one large cell carcinoma sample is negative. In 95 lymph nodes which were tumor-free by histopathology, the expression of MAGE-A is 17.9 %,in which squamous carcinoma is 25.0%,adenocarcinoma is 6.1%,large cell carcinoma is 22.2% respectlly. The expression of CK19 mRNA is 28.4%, in which squamous carcinoma is 34.1%,adenocarcinoma is 21.2%,large cell carcinoma is 27.8%。Histopathology showed that 22 (73.3%) patients had a pN0 status,MAGE-A and CK19 real-time RT-PCR detected disseminated tumor cells in 8 (36.4%) and 13(59.1%) of these pN0 patients. The expression of MAGE-A and CK19 mRNA were detected in 10 patients。There were obviously differences in the expression of two tumor markers by Chi-square test. (p-value is 0.019,<0.05). Conclusion: MAGE-A and CK19 real-time RT-PCR are sensitive and specific tools for the detection of disseminated tumor cells in regional lymph nodes of patients with operable NSCLC. The false positive results can be avoided and the sensitivity can be improved by using over two marker genes, there is a little chance of encountering significant illegitimate expression of more than one gene at a time. Further studies are required to assess if this molecular method might improve mediastinoscopic staging.