MAGE A1-A6 RT-PCR and MAGE A3 and p16 methylation analysis in induced sputum from patients with lung cancer and non-malignant lung diseases

Abstract

The melanoma antigen gene (MAGE) A1-A6 RT-PCR system was developed for the detection of lung cancer cells in the sputum. However, we identified MAGE expression in some patients with non-malignant lung diseases. To understand these patterns of MAGE expression, we performed MAGE A3 methylation-specific PCR (MSP) and p16 MSP. We collected 24 biopsy specimens of lung cancer tissue and performed MAGE A1-A6 RT-PCR, MAGE A3 MSP and p16 MSP. RNA and DNA were simultaneously extracted from induced sputum specimens of 133 patients with lung diseases and 30 random sputum specimens of healthy individuals and the 3 molecular analyses were performed. The patients were diagnosed as 65 cases of lung cancer and 68 of benign lung diseases. Positive rates of MAGE A1-A6 RT-PCR, MAGE A3 MSP and p16 MSP were as follows: in lung cancer tissue, 87.5, 58.3 and 70.8%; in the sputum of lung cancer patients, 50.8, 46.2 and 63.1%; benign lung diseases, 10.3, 30.9 and 39.7%; and healthy individuals, 3.3, 6.7 and 3.3%. Of the 40 MAGE-positive cases, 33 were diagnosed with lung cancer and 7 as having benign lung diseases. From the 7 cases of MAGE-positive benign lung diseases, 6 cases showed methylation abnormalities. The MAGE-positive group revealed significantly higher rates of methylation abnormalities. Of the 40 MAGE-positive cases, 39 cases were found to be lung cancer or benign lung diseases with abnormal methylation. Thus, MAGE expression in the sputum suggests the presence of lung cancer cells or pre-cancerous cells.

Figure 1

MAGE A3 methylation specific PCR (MSP) results in sputum of patients with lung diseases. (A), MAGE A3 MSP; (B), MAGE A3 unmethylation specific PCR; SM, Size marker; lanes 1-10, number of patient's matched specimen.

Figure 2

p16 methylation specific PCR (MSP) results in the sputum of patients with lung diseases. (A), p16 MSP; (B), p16 unmethylation specific PCR; SM, size marker; lanes 1-16, number of patient's matched specimen.

Figure 3

Positive rates of MAGE A3 and p16 methylation-specific PCR according to MAGE results in the sputum. The Chi-square test was performed to compare the positive rates.