| Every year, about 1 million people die due to lung cancer. In many countries, lung cancer is the leading reason of death due to carcinoma. Fiberoptic brochoscope is a useful instrument to diagnose lung cancer. Bronchial lavages using fiberoptic bronchoscope could not only improve the diagnosis of lung cancer, but also cost little. In addition, bronchial lavages, called"fluid biopsy", is helpful to diagnose primary of secondary carcinoma locating in respiratory tract, including peripheral lung cancer, bronchioalveolar carcinoma, pulmonary metastatic carcinoma and lymphoma. However, most of peripheral lung cancer can't be diagnosed by examination related to fiberoptic bronchoscope. It is necessary to find more sensitive molecular biology methods to diagnose lung cancer. We extracted DNA from bronchial lavages cells and supernatants, and made a quantitative analysis of free DNA from supernatants, then analyzed hypermethylation of three genes in bronchial lavages supernatants and cells. Our objective is to investigate the possibility of bronchial lavages supernatants used in molecular biology diagnosis of lung cancer, and the value of hypermethylation of gene in the diagnosis of primary lung cancer. The target genes include p16 and MGMT,which have a high frequency in non small cell lung cancer, and RASSF1A, which have a high frequency in small cell lung cancer.All patients were admitted into Beijing Thoracic Tumor & Tuberculosis Hospital. There were 51 patients with lung cancer, including 23 cases with squamous cancer, 20 cases with adenocarcinoma, 5 cases with small cell lung cancer and 3 cases with adenosquamous carcinoma. 27 patients were grouped as central lung cancer, and 24 patients were grouped as peripheral lung cancer. There are 32 men and 19 women with mean age (57.1±13.5) years. In addition, 27 patients were diagnosed as benign pulmonary lesions, including 17 cases with pulmonary tuberculosis, 5 cases with pulmonary infection, and 5 cases with pulmonary benign tumour. There are 16 men and 11 women with mean age (39.5±10.7) years. After extracting DNA from cells and supernatants of bronchial lavages, we investigated promoter hypermethlation of three genes above -mentioned by NMSP. In order to verify the NMSP results, representative bands from each target were cloned into T-vector , followed by automatic DNA sequencing. Then comparing with final diagnosis, we calculated the sensitivity and specificity of promoter hypermethylation in the diagnosis of lung cancer. Difference of cell-free DNA concentrations was analyzed by one-way analysis of variance using SPSS 10.0 software. There is a significant difference if p value was less than 0.05.The results showed that:1 DNA concentrations in cell-free lavages supernatants in central lung cancer, peripheral lung cancer and pulmonary benign lesions are (0.83±0.34)μg/ml, (0.66±0.31)μg/ml and (0.59±0.37)μg/ml, respectively. There is not a significant difference in these groups by one-way analysis of variance.2 Among 51 cases with lung cancer, 7 specimens showed malignant cells in bronchial lavages, including 4 cases in central lung cancer and 3 cases in peripheral lung cancer. Concordant results were observed in 20 cases. 6 specimens showed malignant cells on cytological analysis, and NMSP was positive in at least one gene tested. 14 samples did not show malignant cells on cytological analysis, and the NMSP results were correspondingly negative. The results of cytology and NMSP were discordant in 31 samples. 30 samples were cytologically negative, but NMSP positive in one or more genes. In addition, there was one case that the cytology was positive for malignant cells, but the NMSP was negative in all of the three gene tested.3 The investigation of cells of bronchial lavages with lung cancer showed hypermethylation of at least one gene in squamous cell carcinoma, adenocarcinoma, small cell lung cancer and adenosquamous carcinoma is 17/23(73.9%),13/20(65.0%),4/5 and 2/3, respectively. Combining with supernatants, hypermethylation of at least one gene is 18/23(78.3%),15/20(75.0%),4/5 and 3/3, respectively.4 By investigating hypermethylation of bronchial lavages cells, the sensitivity and specificity to diagnose lung cancer are 70.6% and 81.5%. For central lung cancer, sensitivity is 85.2%, but 54.2% for peripheral lung cancer. Combining with supernatants, the sensitivity and specificity to diagnose lung cancer are 78.4% and 70.4%. For central lung cancer, sensitivity is 88.9%, but 75.0% for peripheral lung cancer. Conclusion: Bronchial lavages supernatants are helpful for molecular diagnosis of lung cancer. Hypermethylation of anti-oncogene is a useful marker for diagnosis of lung cancer.
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