161 Detection of non-metastatic non-small cell lung cancer in urine -------> recurrence, treatment, and patient survival are also represented for each case. Methylation levels are depicted as square root transformed Ct ratios. The upper quartile value of methylation levels in control samples is visualized on the y-axes to illustrate an arbitrarily defined threshold (1.2 for CDO1 and 1.3 for SOX17) for preoperatively elevated methylation levels. Of note, in patient 12, the primary tumor was not completely resected, indicated by an R1 classification of the surgical specimen. For this reason, this patient underwent radiotherapy after which no residual disease was determined during follow-up. DISCUSSION This prospective biomarker study demonstrated that urine of non-metastatic NSCLC patients contains elevated levels of the DNA methylation markers CDO1 and SOX17, as compared with urines of sex- and age-matched controls. When combined, the two methylation markers yielded a cross-validated AUC of 0.71 for the detection of non-metastatic NSCLC. The results from the present study are amongst the first to demonstrate that detection of NSCLC-specific ctDNA in urine is feasible through DNA methylation analysis. In a 2020 pioneer study, Liu and Hulbert et al. demonstrated that urinary DNA methylation analysis in cancer-specific loci, including CDO1, SOX17, TAC1, and HOXA9, was significantly associated with the diagnosis of NSCLC (19). By combining with plasma DNA methylation analysis, high accuracy could be achieved. In the present study, SOX17 was the most discriminating marker, as was the case in the pioneering study (AUC 0.72 and AUC 0.78, respectively). Methylation marker TAC1 was not increased in NSCLC patients in this study, while it had an equal performance as CDO1 in the study by Liu et al. This finding could be explained by technical differences and differences in the source populations of cases and controls. In this respect, also stage of disease may be of importance as in our previous study on metastatic NSCLC patients, opposed to non-metastatic NSCLC in present study, TAC1 was found to be increased in urine as compared to healthy controls. Yet, TAC1 showed a lower reproducibility as compared to the markers CDO1 and SOX17 (25). Surprisingly, methylation levels of SOX17 were highest in urine from stage I NSCLC patients. This could be partly due to the differences in number of cases per stage of which the majority (64%) were stage I tumors. Yet, in the abovementioned pioneer study of Liu et al. (19), also no differences were found when comparing both plasma and urine of low (stage I and II) versus high stage (stage III and IV) NSCLC tumors. Although counterintuitive, the absence of ctDNA in advanced cancer patients with a high tumor burden and presence of metastasis has been remarked previously (32). A study on colorectal cancer detection in urine showed that the presence of the primary tumor may influence the detection of methylated DNA, with higher methylation levels in 6
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