Improvement of the sensitivity of circulating tumor DNA-based liquid biopsy: current approaches and future perspectives

Abstract

Liquid biopsy (LB) is a complex of procedures aimed at the detection of tumor-derived fragments (nucleic acids, proteins, cells, etc.) persisting in the blood or other body fluids. It can be utilized for early cancer diagnosis, analysis of biomarkers of tumor drug sensitivity and prognosis, monitoring of minimal residual disease (MRD), etc. Circulating tumor DNA (ctDNA) is an accessible and reliable LB analyte as it may contain tumor-specific mutations and is amenable to efficient detection by next-generation sequencing (NGS) or droplet digital PCR (ddPCR). High level of ctDNA is typically associated with increased tumor burden and poor prognosis, whereas treatment-related ctDNA clearance increases the probability of a favorable disease outcome. Major efforts have been invested in enhancing the analytical performance of ctDNA detection. Stimulation of apoptosis of tumor cells by irradiation of cancer lumps has been shown to result in a transient but modest increase in ctDNA concentration. There are several sophisticated modifications of ultra-deep NGS protocols, which discriminate between "true" low-copy mutation-specific signals and sequencing artifacts. Slowing physiological ctDNA decay by interfering with liver macrophages and circulating nucleases has shown promise in animal experiments. Reproducibility of ctDNA-based LB assays remains insufficient for samples with ultra-low content of ctDNA; hence, interlaboratory harmonization of ctDNA testing procedures is of paramount importance.

Keywords: Liquid biopsy; analytical performance; cancer therapy; circulating tumor DNA; circulating tumor DNA assays; next-generation sequencing; reproducibility.

Figure 1

Improvement of the analytical performance of ctDNA-based liquid biopsy. (A) Ultra-sensitive methods of detection of mutated DNA sequences; (B) pre-analytical factors affecting the quality and yield of ctDNA; (C) slowing the ctDNA clearance in the blood (black arrows indicate the inhibition of two routes of ctDNA clearance); (D) transient shedding of DNA from tumors. BCT: blood collection tubes; BEAM: beads, emulsions, amplification, and magnetics; ccfDNA: circulating cell-free DNA; ctDNA: circulating tumor DNA; ddPCR: droplet digital PCR; EDTA: ethylenediaminetetraacetic acid; NGS: next-generation sequencing; TKI: tyrosine kinase inhibitors; VAF: variant allele frequency; WGS: whole-genome sequencing. Some images were adapted using free resources from Flaticon.com

Figure 2

UMI-barcoding strategy for background noise reduction. e: sequencing error; M: true mutation. NGS: next-generation sequencing; UMIs: unique molecular identifiers. Some images were adapted using free resources from Flaticon.com

Figure 3

Beyond plasma ctDNA: the reliability of LB can be increased by expanding the range of analytes and involving biological fluids which are proximal to the tumor site. circRNA: circular RNA; CTC: circulating tumor cell; ctDNA: circulating tumor DNA; EMT-CTC: circulating tumor cell undergoing epithelial-to-mesenchymal transition; LB: liquid biopsy; miRNA: microRNA; mRNAs: messenger RNAs