Optimisation of robust singleplex and multiplex droplet digital PCR assays for high confidence mutation detection in circulating tumour DNA

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.

Capturing the primordial Kras mutation initiating urethane carcinogenesis

“…ddPCR was performed using the QX200 AutoDG Droplet Digital PCR System (Bio-Rad) following the manufacturer's protocol in a 22 µl ddPCR reaction containing 11 µl of 2x ddPCR SuperMix for probes (no dUTP) (Bio-Rad), 66 ng template DNA, 450 nM forward and reverse primers, and 250 nM FAM-and HEX-labeled probes. The primer and probe oligonucleotides were synthesized (IDT) based on sequences previously described 76 with minor modifications. The sequences for the primers are Kras_Q61_For: 5ʹ-ATGGAGAAACCTGTCTCTTGG-3ʹ and Kras_Q61_Rev: 5ʹ-CTCATGTACTGGTCCCTCATT-3ʹ.…”

Feasibility of monitoring peripheral blood to detect emerging clones in children with acute lymphoblastic leukemia†

“…Another limitation of our study was that individual assays were performed for single targets. However, multiplexing is possible, as demonstrated by the detection of four rare mutations at 0.09% in myelomonocytic leukemia patient samples 12,13 …”

Molecular Pathology in the New Age of Personalized Medicine

“…The analysis of broader genomic regions using ddPCR is not feasible. However, discriminatory multiplex ddPCR assays can be developed, which enable very rapid and cost-effective monitoring for a limited number of mutations in serial plasma samples [28].…”