Detection of somatic mutations in circulating cell-free DNA from patients with esophageal squamous cell carcinoma

Background: The aim of this study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in circulating cell-free DNA (cfDNA) from patients with esophageal squamous cell carcinoma (ESCC). Methods: Paired multi-regional tumor tissues, cfDNA and white blood cells (WBCs) collected from five ESCC patients before treatment from a prospective study (NCT02395705). Of them, samples from Cohort 1 (E102 and E110) were sequenced by whole-exome sequencing (WES) and those from Cohort 2 (E104, E111 and E121) were sequenced by targeted captured sequencing with a panel of 560 cancer-related genes respectively. To call somatic single nucleotide variations (SNVs) by comparing the solid tumor or cfDNA with matched WBCs, the minimal variant allele frequency (VAF min ) as 0.1% and P value <0.05 were allowed. Results: Genomic DNA (gDNA) and plasma-derived cfDNA from 26 samples were successfully sequenced. In Cohort 1, 596 (596/712, 83%) and 562 (562/796, 71%) were heterogeneous SNVs in E102 and E110 respectively. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA ( R 2 = 0.78, P <0.0001). In Cohort 2, 296 (296/323, 92%), 384 (384/423, 91%) and 331 (331/357, 93%) were heterogeneous SNVs in E104, E111 and E121respectively. cfDNA could recover an average of 60.7% (31/51; range, 35.7%-76.2%) of somatic mutations present in matched solid tumors. The correlation of VAFs between cfDNA and matched solid tumor was significantly positive (r 2 =0.92, P <0.0001). Conclusions: Both sequencing approaches revealed the highly intratumoral heterogeneity in ESCC and enabled the detection of both ubiquitous and heterogeneous mutations in cfDNA. Further validation in cfDNA is required to define its potential utility for ESCC in clinical practice. Trial registration All patients selected in this study were from the registered clinical trial from ClinicalTrials.gov (NCT02395705). Date of registration: March 24, 2015.