Abstract
Liquid biopsies offer the potential to monitor cancer response and resistance to therapeutics in near real-time. However, the plasma cell free DNA (cfDNA) level can be low and the fraction of circulating tumour DNA (ctDNA) bearing a mutation – lower still. Detection of tumour-derived mutations in ctDNA is thus challenging and requires highly sensitive and specific assays. Droplet digital PCR (ddPCR) is a technique that enables exquisitely sensitive detection and quantification of DNA/RNA markers from very limiting clinical samples, including plasma. The Bio-Rad QX200 ddPCR system provides absolute quantitation of target DNA molecules using fluorescent dual-labelled probes. Critical to accurate sample analysis are validated assays that are highly specific, reproducible, and with known performance characteristics, especially with respect to false positives. We present a systematic approach to the development and optimisation of singleplex and multiplex ddPCR assays for the detection of point mutations with a focus on ensuring extremely low false positives whilst retaining high sensitivity. We also present a refined method to determine cfDNA extraction efficiency allowing for more accurate extrapolation of mutational levels in source samples. We have applied these approaches to successfully analyse many ctDNA samples from multiple clinical studies and generated exploratory data of high quality.
Highlights
Monitoring of response and resistance to therapies in cancer patients at the molecular level presents many challenges
Various methods are in use to purify cell free DNA (cfDNA) from plasma
Purification of cfDNA is a critical step prior to any genomic assay, increasing number of publications report that extraction efficiencies can vary greatly, depending on several factors, from the kind of sample collection tubes to extraction kits[21,22,23,24,25]
Summary
Monitoring of response and resistance to therapies in cancer patients at the molecular level presents many challenges. A few sets of primers and probes targeting several genomic regions can be mixed together in one PCR reaction to produce multiplex PCR This multiplexing, comes with multiple challenges such as varying efficiency of individual assays, different primer annealing temperatures, possible oligonucleotide cross-dimerisation and accurate separation of fluorescent signals from a limited number of reporter dyes with overlapping emission spectra[15]. It is possible to develop discriminatory multiplex ddPCR assays that enable very rapid and cost-effective monitoring for a limited number of mutations in serial plasma samples Such assays can be very informative for serial analysis after target mutations are identified via broader sequence profiling of tumour tissue or a baseline ctDNA sample. We present a refinement of a previously reported method for calculating an accurate quantification of mutant copies in the original plasma sample utilising an externally spiked control DNA to correct for variation in cfDNA extraction efficiencies[16,17]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
