Molecular genetic characteristics of tumor-derived gene alterations in cfDNA

Introduction: Circulating cell-free DNA (cfDNA) exists in very small amounts and is fragmented in the bloodstream. In patients with malignant disease, cfDNA includes tumor-derived DNA (tDNA). Therefore, it has been postulated that cfDNA might be a tool for gene testing as a liquid biopsy. The aims of this study were to clarify the factors that define detection sensitivity and to determine whether it can detect intra-tumor or inter-tumor heterogeneity, which is necessary for clinical application of cfDNA gene testing.

Methods: Genetic alteration profiles using next generation sequencing (NGS) were analyzed in each tumor lesion, including metastases and cfDNA, from samples obtained from 6 post-mortem patients diagnosed with histologically-confirmed metastatic primary lung cancer. cfDNAs extracted from plasma samples were collected < 1 month before the patients’ death and were stored. The genetic alteration profiles from each tumor lesion were compared with those from cfDNA.

Results: Gene mutation profiles were analyzed from 6 patients. The mean number of detectable gene alterations per patient was 253 (range 99-1918) from tDNA samples and 218 (range 56-362) from cfDNA samples. Genetic alterations detected from cfDNA were shown to be present at significantly higher variant allele frequency (VAF) in tumor tissues compared with the alterations detected in cfDNA. Additionally, the truncal gene alterations that existed in all tumor lesions were more easily detected from cfDNA than the shared mutation which was in more than one tumor lesion, not in all lesions, and the individual mutations which in only one tumor lesion.

Conclusions: In genetic test using cfDNA, it was concluded that high VAF and truncal mutations were factors that defined the detection sensitivity.