Isolation of cell-free circulating DNA (cfDNA) from blood and detection of presence of circulating tumor DNA (ctDNA) in it are two main parts of liquid biopsy diagnosis technique. The analysis of circulating tumor DNA (ctDNA) among total isolated circulating nucleic acid is challenging and requires highly sensitive techniques due to the small fraction of tumor-specific DNA crowded with wild-type cell-free DNA and some amount of genomic DNA from white blood cells.
Poor detection coverage of cell-free DNA may lose tumor DNA fragments and can lead to false negative results.Variety of classical and advanced techniques are being used for the analysis of cell-free DNA and have their own advantages and disadvantage in using such techniques. Classical methods for analyzing cell-free DNA includes quantitative real-time polymerase chain reaction (PCR)-based, fluorescence-based, and spectrophotometric approaches.
Very recently new technologies are emerged in order to overcome poor sensitivity problem in ctDNA detection and to improve identification of genetic alterations in ctDNA. Digital PCR has now emerged as a sensitive tool to detect point mutations in ctDNA at low allele fractions, which comprises droplet-based systems, microfluidic platforms for parallel PCR, and an approach called BEAMing (beads, emulsions, amplification and magnetics).
Next-generation sequencing technologies are also showing noticeable application in this field. This is a low cost high-throughput technique and covers all ctDNA alterations across wide genomic regions. Targeted deep sequencing approaches have been used to analyze specified genomic regions in plasma DNA, including PCR-based targeted deep sequencing such as TamSeq, SafeSeq, and Ion-AmpliSeq™ and capture-based targeted deep sequencing such as CAPP-Seq.
Remarkably, whole genome sequencing provides novel opportunities for comprehensive characterization of the alteration profiles, not just limited to predefined or existing mutations in plasma DNA. Genome-wide detection of chromosomal rearrangements and CNVs can be characterized in ctDNA, serving as tumor biomarkers with excellent sensitivity and specificity. Two genome-wide methods, personalized analysis of rearranged ends (PARE) and digital karyotyping can be applied to ctDNA detection.
PARE is a method for identifying specific somatic rearrangements in tumor tissue and subsequently developing PCR-based assays to detect these tumor biomarkers in the circulation. Digital karyotyping is a genome-wide technique used to quantify the DNA copy number and novel sequences on a genomic scale. It has been applied to detect previously uncharacterized chromosomal changes and exogenous sequences in human tumors.
Moreover, recent implementation of whole-genome sequencing allows direct application to ctDNA analysis, and has provided an unprecedented view of somatic chromosomal alterations and CNVs on a genome-wide scale. Undoubtedly, with continuous improvements in the sensitivity of genomic approaches, next generation sequencing techniques will play a pivotal role in ctDNA analysis for future clinical applications.
Evaluation of cell-free DNA integrity index is a different approach to identify ctDNA alterations and constitutes an independent indicator different from any specific genomic changes. DNA integrity index is measured as the ratio of long to short DNA fragments. Circulating cell-free DNA released from apoptotic cells is uniformly truncated into 185- to 200-bp fragments, whereas cell-free DNA released from necrotic tumor cells varies in length, which may lead to elevation of DNA with long fragments in plasma or serum. A study by Leon et al. suggested that the cell-free DNA concentration was significantly increased in cancer patients compared with that in healthy individuals. Similar findings have also been demonstrated in several cancers such as periampullary cancer, breast cancer, colorectal cancer, esophageal cancer, head and neck cancer, renal cancer, melanoma, and prostate cancer.
Qin, Zhen, et al. “Cell-free circulating tumor DNA in cancer.” Chinese journal of cancer 35.1 (2016): 1.