Based on our results in this study, the assessment of SHOX2 and RASSF1A DNA methylation identified 11 of 17 malignant puncture biopsy specimens with no signs of malignancy by conventional pathology were referred to surgical biopsies, which were confirmed with lung cancer, making this assay an applicable and relative fast method to detect malignant in puncture biopsy specimens and improved sensitivity of lung cancer detection with small biopsy specimens. This evidence concerns the gene RASSF1 and lung cancer.