Supplementary Material for: Molecular Barcode Sequencing for Highly Sensitive Detection of Circulating Tumor DNA in Patients with Esophageal Squamous Cell Carcinoma

<b><i>Introduction:</i></b>Next-generation sequencing (NGS) with molecular barcodes (MB) is a novel method that enables the highly sensitive detection of circulating tumor DNA (ctDNA) in a relatively wide range of genes. <b><i>Objective:</i></b>The aim of this study was to examine the utility of NGS with MB for detecting ctDNA in patients with esophageal squamous cell carcinoma (ESCC). <b><i>Methods:</i></b>Five patients with ESCC who underwent preoperative treatment followed by esophagectomy were examined. The frequency of <i>TP53</i> mutations in DNA extracted from tumor tissue and plasma at each time point during the treatment course was analyzed using NGS without MB. In 1 patient, additional analysis using NGS with MB was conducted to compare the sensitivities and to evaluate the clinical utility of this novel method. <b><i>Results:</i></b><i>TP53</i> mutations in tumor tissue were identified in 3 of 5 patients with ESCC. In 1 patient, the mutational allele frequency in plasma was 1.97% before preoperative treatment, and decreased to 0.09% after preoperative treatment. As the maximum frequency of background errors were 3.22% using NGS without MB and 0.08% with MB, which indicated that the sensitivity of ctDNA detection using NGS with MB was much higher than without MB. In 1 patient who had recurrence half a year after surgery, only NGS with MB could detect ctDNA even at 4 weeks after surgery, at a frequency of 0.20%. <b><i>Conclusions:</i></b>NGS with MB enabled comprehensive and highly sensitive detection of ctDNA in a patient with ESCC. This novel method may be useful for the clinical diagnosis of ESCC.