Fractionated small cell-free DNA increases possibility to detect cancer-related gene mutations in advanced colorectal cancer.

BACKGROUND AND AIM

Liquid biopsy is a method that can efficiently detect tumor genetic abnormalities from body fluids such as blood and urine. Detection sensitivity and the available number of mutations in cell-free DNA (cfDNA) are limited. In this study, we develop a highly sensitive and comprehensive method to detect mutations from cfDNA by concentrating tumor fractions of small cfDNA in advanced colorectal cancers.

METHODS

Biopsied specimens and 37 serum samples were collected from 27 patients with advanced colorectal carcinoma. A serum-extracted cfDNA was divided into enriched fractionated small cfDNA and unfractionated cfDNA. Both cfDNAs were subjected to digital polymerase chain reaction (PCR) to evaluate their , and status. Consequently, their mutant allele frequencies (MAFs) were compared and analyzed by next-generation sequencing (NGS) in conjunction with tissue-derived DNA.

RESULTS

NGS analyses revealed mutations in (63%), (63%), (30%), and (22%). Digital PCR could detect mutations in 25 of 27 samples (93%) of unfractionated cfDNA, a rate that increased to 100% when samples were enriched with fractionated small cfDNA (6.8 10.7%,  < 0.001). NGS also showed increased MAFs in fractionated small cfDNA compared to unfractionated cfDNA (16.3 18.8%, = 0.012) and a tendency to detect a greater number of cancer-related genes in fractionated cfDNA.

CONCLUSIONS

Fractionated small cfDNA increased MAFs of gene mutations and increases the possibilities to detect cancer-related genes even in advanced cancer patients from whom it is difficult to obtain tissue samples.