Detection Of Lymph-Micrometastisis Of Pationts With NSCLC By Using Real-Time RT-PCR

[Objective]The prospective study was to detect micrometastases of the tissue near .carcinoma and regional lymph node in non-small cell lung cancer (NSCLC) patients by using Lunx mRNA and vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor-3 (VEGFR-3) by using fluorescence-based real-time reverse transcriptase-polymerase Chain Reaction (real-time RT-PCR) , to evaluate their sensitivity and specificity ,and the practicality of the technology. [Methods]1. Study SamplesAll the 30 patients were selected randomly from the Department of Thoracic Surgery, The Second Hospital of Shan Dong University, who underwent complete resection, from September 2004 to October 2005. Of the 30 patients, 20 were male and 10 were female. The patients who were cardiac insufficiency, hepatic inadequacy or renal inadequacy, were excluded. All the patients have pathologic staging from stage I to stage III( according to TNM classification of UICC for lung cancer in 1997[3] , 10 patients were classified in stage 1,9 in stage II and 11 in stage III .The control group included 20 regional lymph nodes obtained from20 patients with benign lung disease, who were selected from the paleontology department and chest surgery department of The Second Hospital of Shan Dong University at the same period. All patients gave their written consent.2. The Detection of Lunx mRNA. VEGF-C and VEGFR-3 mRNA1) VEGF-C mRNA and VEGFR-3 mRNA of the tissue near carcinoma obtained from 30 patients with NSCLC was detected by real-time RT-PCR. 20 control specimens were obtained from patients with benign pulmonary diseases.2) SYBR Green I real-time RT-PCR was used to check-up lunx mRNA in 60 regional lymph nodes obtained from 30 patients with NSCLC ,compared to 20 regional lymph nodes obtained from patients with benign lung disease. The results were compared to that of regular histopathology.3) Comparing the positive expression of Lunx mRNA between the COVEGF-C-3 (+) group and COVEGF-C-3 (-) group, the relationship between the expression of VEGF-C mRNA, VEGFR-3 mRNA in the tissue near carcinoma and the expression of lunx in lymph nodes ,was assessed.[Results]1) VEGF-C mRNA and VEGFR-3 mRNA real-time RT-PCR detected disseminated tumor cells in 18 (60%),23(77%),respectively, while histopathology showed that 11 (37%) pulmonary tissue near carcinoma were tumor-positive. The expression rate of VEGF-C and VEGFR-3 of the tissue near carcinoma in the patients with NSCLC was significantly higher than the histopathological tumor-positive in them. (X~2=4, P<0.05; X~2=6.72, P<0.01)2) Lunx real time RT-PCR detected disseminated tumor cells in 47 (78.3%) of 60 lymph nodes obtained from patients with NSCLC, while histopathology showed that 33 lymph nodes were tumor-positive. The sensitivity of Lunx real time RT-PCR showed higher than that of histopathology (X~2=7.68, P<0.01).3) When the tissue near carcinoma express VEGF-C(+)andVEGFR-3 (+) , positive rate of Lunx expression is 73.3%,in the lymph nodes from the patient (44/60), significantly high than VEGF-C(-)and VEGFR-3 (-) from the same site. Lunx expression rate of the tissue near carcinoma is just 5% (3/60), they have a significant deviation (X~2 =6.50, P<0.05) , the expression of COMVEGF-C-3 has a signification correlation with lymph nodes with cancer migration. [Conclusion] :Quantitative real-time RT-PCR for Lunx, VEGF-C and VEGFR-3 can be applied for detection of Lymph-micrometastasis for patients with NSCLC. This technique may be an appropriate tool in diagnosing earlier period Lung cancer metastasis. Lunx mRNA, VEGF-C mRNA and VEGFR-3 mRNA are appropriate target genes for detection of micrometastasis from NSCLC.