Extending Circulating Tumor DNA Analysis to Ultralow Abundance Mutations: Techniques and Challenges

Abstract

Liquid biopsies that analyze circulating tumor DNA (ctDNA) hold great promise in the guidance of clinical treatment for various cancers. However, the innate characteristics of ctDNA make it a difficult target: ctDNA is highly fragmented, and found at very low concentrations, both in absolute terms and relative to wildtype species. Clinically relevant target sequences often differ from the wildtype species by a single DNA base pair. These characteristics make analyzing mutant ctDNA a uniquely difficult process. Despite this, techniques have recently emerged for analyzing ctDNA, and have been used in pilot studies that showed promising results. These techniques each have various drawbacks, either in their analytical capabilities or in practical considerations, which restrict their application to many clinical situations. Many of the most promising potential applications of ctDNA require assay characteristics that are not currently available, and new techniques with these properties could have benefits in companion diagnostics, monitoring response to treatment and early detection. Here we review the current state of the art in ctDNA detection, with critical comparison of the analytical techniques themselves. We also examine the improvements required to expand ctDNA diagnostics to more advanced applications and discuss the most likely pathways for these improvements.

Liquid Biopsy in Oral Cancer

“…Although evidence exists regarding the value of ctDNA in different clinical scenarios, there are still some issues to solve before reaching general use at hospitals. Firstly, ctDNA is detected in a very low ratio in early stages, being undetectable without highly sensitive techniques [ 42 , 73 ]. Secondly, due to tumour heterogeneity and evolution, multiplexed assays are need to analyze several mutations simultaneously and to solve the limitation of the low levels of cfDNA present in some patients [ 73 ].…”

“…Firstly, ctDNA is detected in a very low ratio in early stages, being undetectable without highly sensitive techniques [ 42 , 73 ]. Secondly, due to tumour heterogeneity and evolution, multiplexed assays are need to analyze several mutations simultaneously and to solve the limitation of the low levels of cfDNA present in some patients [ 73 ]. Finally, several methods have been developed to detect ctDNA, but there is still a lack of a standardized method, which is essential for its clinical application together with the need of reducing the analysis cost.…”

High‐Performance Nucleic Acid Sensors for Liquid Biopsy Applications

“…Although cancer patients often have elevated levels of cfNAs relative to healthy controls, the levels may vary significantly in plasma or serum samples in each group . In addition, the precise quantitation of cfNAs is challenging owing to cfDNA fragmentation, RNA instability, and the low concentrations present in body fluids . Current research is therefore focused on the specific detection of tumour‐associated sequences for clinically meaningful analysis of cfNAs.…”

Sequence-specific preconcentration of a mutation prone KRAS fragment from plasma using ion-tagged oligonucleotides coupled to qPCR compatible magnetic ionic liquid solvents

“…Although SCODA is successful at preconcentrating low concentrations of ctDNA mutations with limits of detection of 0.001% mutation abundance without PCR amplification, the technique is expensive and has limited sample throughput. [8] Commercially-available streptavidin magnetic beads have been widely used in the extraction of ctDNA. [3,9] In this approach, target DNA anneals to a biotinylated DNA probe followed by the probe-target complex binding to streptavidin coated magnetic beads, which are collected using an external magnet.…”

“…[49] Therefore, to examine the selectivity of the ITOs towards target DNA, the T m of the ITO to a complementary oligonucleotide (20 nt), 1 nt mismatch (20 nt), and 2 nt mismatch (20 nt) was examined. Figure S9 shows that the T m significantly M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 20 decreased when examining the hybridization of the ITO to the 1 nt mismatch, and a T m could not be determined when hybridizing the [AOIM + ]-KRAS [PF -], [ABzIM + ]-KRAS [Br -], and [ABzIM + ]-KRAS [PF -] ITOs to the nt mismatch.Background cfDNA in cancer patients is approximately the same length as ctDNA (i.e, about 166 bp) and is present in high concentrations, typically 0-1000 ng mL -1 for cancer patients with mutation abundances less than 0.01% [4,8,50]. To examine the effect of background DNA on the ITO-MIL-DLLME method, stDNA was sheared to around 150 bp and spiked into the aqueous sample.…”