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

Introduction: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. Objective: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). Methods:Five patients with ESCC who underwent preoperative treatment followed by esophagectomy were examined. The frequency of TP53 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. Results:TP53 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%. Conclusions: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.