*3.3. Methylation Pattern of RUNX1 in Tumor Tissue Is Similar to that in Bronchial Biopsy Specimen*

To test if bronchial washing, sputum, and bronchial biopsy specimens could be used as surrogate samples for analyzing *RUNX1* methylation in the lung, we compared the methylation levels of the CpG (cg04228935) in 42 tumors and matched normal tissues, 136 bronchial washings, 12 sputum samples, and 6 bronchial biopsy specimens. The clinical and pathological characteristics of the NSCLC patients were previously reported [13]. The methylation levels of the CpG in lung tumor tissues were not significantly different from those in bronchial biopsy specimens from lung cancer patients (*p* > 0.05, Wilcoxon rank sum test), unlike bronchial washings and sputum samples (Figure 3A). The CpG methylation levels were further compared between paired bronchial washing and sputum samples from 12 NSCLC patients (Figure 3B) and between bronchial washing and paired bronchial biopsies from 6 NSCLC patients (Figure 3C). The methylation levels of the CpG were found to be similar between bronchial washings and sputum samples but significantly higher in bronchial biopsy than in bronchial washing (*p* < 0.05, Wilcoxon signed-rank test). These findings suggest that a detection model for NSCLC using abnormal methylation of *RUNX1* is applicable to bronchial biopsy specimens.

*J. Clin. Med.* **2020**, *9*, 1694

**Figure 3.** Comparison of *RUNX1* methylation levels among different types of specimens. (**A**) The methylation levels of a CpG (cg04228935) selected for NSCLC prediction were compared among bronchial biopsies from 6 lung cancer patients (biopsy), bronchial washing samples from 60 healthy individuals (bronchial washing normal, BWN) and 76 lung cancer patients (bronchial washing tumor, BWT), tumor (lung tumor tissue, LTT) and matched normal (lung normal tissue, LNT) tissues from 42 NSCLC patients, and sputum specimens from 12 lung cancer patients (sputum). (**B**,**C**) Methylation levels of a CpG (cg04228935) were compared using parallel coordinate plots between paired bronchial washing and sputum specimens from 12 NSCLC patients (**B**) and between paired bronchial washing and biopsy samples from six NSCLC patients (**C**). Methylation levels in bronchial washings were similar to those in sputum samples but were significantly low compared with those in bronchial biopsy (Wilcoxon signed-rank test). Y-axis indicates β-values from the 450 K array.
