Pre-PCR Mutation-Enrichment Methods for Liquid Biopsy Applications

Abstract

Liquid biopsy is having a remarkable impact on healthcare- and disease-management in the context of personalized medicine. Circulating free DNA (cfDNA) is one of the most instructive liquid-biopsy-based biomarkers and harbors valuable information for diagnostic, predictive, and prognostic purposes. When it comes to cancer, circulating DNA from the tumor (ctDNA) has a wide range of applications, from early cancer detection to the early detection of relapse or drug resistance, and the tracking of the dynamic genomic make-up of tumor cells. However, the detection of ctDNA remains technically challenging, due, in part, to the low frequency of ctDNA among excessive circulating cfDNA originating from normal tissues. During the past three decades, mutation-enrichment methods have emerged to boost sensitivity and enable facile detection of low-level mutations. Although most developed techniques apply mutation enrichment during or following initial PCR, there are a few techniques that allow mutation selection prior to PCR, which provides advantages. Pre-PCR enrichment techniques can be directly applied to genomic DNA and diminish the influence of PCR errors that can take place during amplification. Moreover, they have the capability for high multiplexity and can be followed by established mutation detection and enrichment technologies without changes to their established procedures. The first approaches for pre-PCR enrichment were developed by employing restriction endonucleases directly on genomic DNA in the early 1990s. However, newly developed pre-PCR enrichment methods provide higher sensitivity and versatility. This review describes the available pre-PCR enrichment methods and focuses on the most recently developed techniques (NaME-PrO, UVME, and DEASH/MAESTRO), emphasizing their applications in liquid biopsies.

CRISPR Technology for Diagnosis: A Brief Review

“…CUT-PCR, as a CRISPR-mediated method, are accurately able to detect a few amount of mutation among DNA sequences in tumor cells. Recently, it was reported that there is 80% applicability of CUT-PCR method for detecting cancer-inducing mutations which are registered in Catalogue of Somatic Mutations in Cancer (COSMIC) database 10 .…”

Clinical Utility and Application of Liquid Biopsy Genotyping in Lung Cancer: A Comprehensive Review

“… 29 ctDNA can be detected in plasma even if present at low levels: the detection limits of modern kits are as low as an allelic fraction of 0.1% or lower, with a median allelic fraction of 0.5%. 30 , 31 …”

“…29 ctDNA can be detected in plasma even if present at low levels: the detection limits of modern kits are as low as an allelic fraction of 0.1% or lower, with a median allelic fraction of 0.5%. 30,31 Overall, high specificity and high positive-predicted value are core features of ctDNA, meaning that the detection of targetable genomic alterations, even in low allelic fractions, can be clinically relevant. 32 This has been proven with single-gene PCR (specificity of PCR-based EGFR tests was close to 98% 27,33 ) and NGS assay (specificity of 97% 34 ) when dealing with targetable variants.…”

Enzymatic Methods for Mutation Detection in Cancer Samples and Liquid Biopsies

“…For example, profiling somatic mutations in circulating DNA has been difficult because of their extremely low frequency in the presence of high excess WT alleles. To this end, the availability of sensitive mutation enrichment and detection technologies is important for realizing the clinical utility of liquid biopsies [ 77 ]. NGS-based approaches for accurate enumeration of mutations as low as 0.01–0.001% have been reported [ 51 , 74 , 75 , 76 ], but NGS still requires advanced instrumentation, resources, and time.…”