Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies

Abstract

Cell-free DNA (cfDNA) refers to short fragments of acellular nucleic acids detectable in almost all body fluids, including blood, and is involved in various physiological and pathological phenomena such as immunity, coagulation, aging, and cancer. In cancer patients, a fraction of hematogenous cfDNA originates from tumors, termed circulating tumor DNA (ctDNA), and may carry the same mutations and genetic alterations as those of a primary tumor. Thus, ctDNA potentially provides an opportunity for noninvasive assessment of cancer. Recent advances in ctDNA analysis methods will potentially lead to the development of a liquid biopsy tool for the diagnosis, prognosis, therapy response monitoring, and tracking the rise of new mutant sub-clones in cancer patients. Over the past few decades, cancer-specific mutations in ctDNA have been detected using a variety of untargeted methods such as digital karyotyping, personalized analysis of rearranged ends (PARE), whole-genome sequencing of ctDNA, and targeted approaches such as conventional and digital PCR-based methods and deep sequencing-based technologies. More recently, several chip-based electrochemical sensors have been developed for the analysis of ctDNA in patient samples. This paper aims to comprehensively review the diagnostic, prognostic, and predictive potential of ctDNA as a minimally invasive liquid biopsy for cancer patients. We also present an overview of current advances in the analytical sensitivity and accuracy of ctDNA analysis methods as well as biological and technical challenges, which need to be resolved for the integration of ctDNA analysis into routine clinical practice.

Exosomes for Non‐Invasive Cancer Monitoring

“…As a result, this approach under‐values the dynamic nature of protein modification and tumor evolution during therapy. On the other hand, more informative serial tumor biopsies are prohibitively invasive and costly . A promising new approach to cancer diagnosis exploits circulating biomarkers that can be repeatedly and conveniently assessed with minimal complications.…”

Liquid biopsy in pancreatic ductal adenocarcinoma: current status of circulating tumor cells and circulating tumor DNA

“…BEAMing quantifies independently the fluorescently labeled particles, which is able to detect the rare variants with allele frequency < 0.01%. This method enables the counting of error rate of DNA polymerases (Gorgannezhad et al ., ). The ddPCR platform performs PCR amplification within water‐in‐oil emulsion droplets where individual DNA molecules are dispersed in.…”

“…Using fluorescently labeled probes, droplets can be identified as a binary (mutant‐positive or mutant‐negative) system. The Bio‐Rad QX‐200 platform produces 20 000 droplets and is one of the most commonly used dPCR systems for ctDNA detection (Gorgannezhad et al ., ).…”

“…Based on PCR technology, new technologies including real‐time quantitative PCR (qPCR) (Brown, ), amplification‐refractory mutation system (ARMS)‐based qPCR (Zhang et al ., ), competitive allele‐specific TaqMan PCR (cast‐PCR) (Ashida et al ., ; Reid et al ., ), coamplification at lower denaturation temperature PCR (COLD‐PCR) (Milbury et al ., ) have been introduced. More recently, digital PCR (dPCR), which uses droplets to compartmentalize individual DNA strands, reached the high sensitivity ranging from 0.1% to 0.001% and is therefore beneficial to detect low allele frequency variants (Gorgannezhad et al ., ; Vogelstein and Kinzler, ). dPCR includes droplet PCR, Bio‐Rad droplet dPCR (ddPCR) platform (Hindson et al ., ), and BEAMing (beads, emulsion, amplification and magnetics) (Chen et al ., ): This method is currently among the most promising of targeted approaches, which focuses on the detection of rare mutations in DNA samples with prior knowledge of genetic changes at specific loci of the tumor (e.g., KRAS, BRCA2, ERBB2, and EGFR) (Alix‐Panabieres and Pantel, ; Cheng et al ., ) and exhibits high analytical sensitivity.…”

A High-Throughput Protocol for Isolating Cell-Free Circulating Tumor DNA from Peripheral Blood

“…A recent comprehensive review published by Gorgannezhad etal . outlined opportunities, challenges and advances made in the field of ctDNA research [5]. In particular, Gorgannezhad etal .…”