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Simple Summary  Lung cancer causes the largest number of cancer-related deaths worldwide. Circulating tumor DNA (ctDNA) has been suggested as a diagnostic and prognostic biomarker in non-small cell lung cancer, but the optimal target for measuring ctDNA has not been established. We aimed to compare a gene methylation biomarker with a gene mutation biomarker in order to determine the mutual agreement. Mutation analysis requires a broad and expensive test like next-generation sequencing, while methylation analysis can be performed by the less expensive droplet digital PCR. We found a good correlation between methylated HOXA9 and mutated KRAS in plasma from patients with lung adenocarcinoma.Abstract  Circulating tumor DNA (ctDNA) has been suggested as a biomarker in non-small cell lung cancer. The optimal target for measuring ctDNA has not yet been established. This study aimed to investigate methylated Homeobox A9 (meth-HOXA9) as an approach to detect ctDNA in advanced lung adenocarcinoma and compare it with mutated Kirsten rat sarcoma viral oncogene homolog (mut-KRAS) in order to determine the mutual agreement. DNA was purified from formalin-fixed, paraffin-embedded non-malignant lung tissue and lung adenocarcinoma tissue, and plasma from healthy donors and lung adenocarcinoma patients, respectively. KRAS mutations in tumor tissue were identified by next-generation sequencing and quantified in tumor and plasma by droplet digital polymerase chain reaction (ddPCR). The meth-HOXA9 analysis was based on bisulfite-converted DNA from tumor and plasma and quantified by ddPCR. Samples consisted of 20 archival non-malignant lung tissues, 48 advanced lung adenocarcinomas with matched plasma samples, and 100 plasma samples from healthy donors. A KRAS mutation was found in the tumor in 34/48 (70.8%) adenocarcinoma patients. All tumors were positive for meth-HOXA9, while none of the non-malignant lung tissues were. Meth-HOXA9 was detected in 36/48 (75%) of plasma samples, and the median level was 0.7% (range of 0–46.6%,  n  = 48). Mut-KRAS was detected in 29/34 (85.3%) of the plasma samples, and the median level was 1.2% (range of 0–46.1%,  n  = 34). There was a good correlation between meth-HOXA9 and mut-KRAS in plasma (Spearman’s rho 0.83,  p  < 0.001). Meth-HOXA9 is present in tissue from incurable lung adenocarcinoma but not in non-malignant lung tissue. It may be used as an approach for detecting ctDNA. The results demonstrated a high agreement between meth-HOXA9 and mut-KRAS in patients with advanced lung adenocarcinoma.

Comparison of Mutated KRAS and Methylated HOXA9 Tumor-Specific DNA in Advanced Lung Adenocarcinoma