ddPCR Assay

cfDNA, DNA from primary tumor, and DNA from metastatic tissue were interrogated for the presence of TP53 indel (c.560-7_560-2delCTCTTAinsT) by a ddPCR custom assay developed from BioRad (Bio-Rad Laboratories, Hercules, CA, USA). ddPCR was performed using primers and specific TaqMan probes targeting the wild-type TP53 sequence and the aberrant TP53 sequence bearing the indel c.560-7_560-2delCTCTTAinsT. As reference mutated control, a synthetic oligonucleotide bearing the TP53 indel c.560-7_560-2delCTCTTAinsT was used (Sigma Aldrich, St. Louis, MO, USA). As a reference wild-type control, a germline DNA in which the presence of the TP53 indel (c.560-7_560-2delCTCTTAinsT) was excluded through next-generation sequencing (NGS) was used. Droplet generation was performed using QX200™ Droplet Generator™ (Bio-Rad Laboratories, Hercules, CA, USA), and fluorescence emitted from droplets was measured using QX200™ Droplet Reader (Bio-Rad Laboratories, Hercules, CA, USA). Sample analysis was performed using QuantaSoft v1.7.4.0917 software (Bio-Rad Laboratories, Hercules, CA, USA). Details concerning ddPCR conditions and data analysis are reported in Supplementary Methods.