ABSTRACT
Liquid biopsy testing utilising Next Generation Sequencing (NGS) is rapidly moving towards clinical adoption for personalised oncology. However, before NGS can fulfil its potential any novel testing approach must identify ways of reducing errors, allowing separation of true low-frequency mutations from procedural artefacts, and be designed to improve upon current technologies. Popular NGS technologies typically utilise two DNA capture approaches; PCR and ligation, which have known limitations and seem to have reached a development plateau with only small, stepwise improvements being made. To maximise the ultimate utility of liquid biopsy testing we have developed a highly versatile approach to NGS: Adaptor Template Oligo Mediated Sequencing (ATOM-Seq). ATOM-Seq's strengths and versatility avoid the major limitations of both PCR- and ligation-based approaches. This technology is ligation free, simple, efficient, flexible, and streamlined, and it offers novel advantages that make it perfectly suited for use on highly challenging clinical material. Using reference and clinical materials, we demonstrate detection of known SNVs down to allele frequencies of 0.1% using as little as 20-25 ng of cfDNA, as well as the ability to detect fusions from RNA. We illustrate ATOM-Seq's suitability for clinical testing by showing high concordance rates between paired cfDNA and FFPE clinical samples.
Subject(s)
Circulating Tumor DNA/genetics , Colonic Neoplasms/diagnosis , High-Throughput Nucleotide Sequencing/methods , Lung Neoplasms/diagnosis , RNA, Neoplasm/genetics , Alleles , Base Sequence , Circulating Tumor DNA/blood , Colonic Neoplasms/blood , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , DNA Primers/chemical synthesis , DNA Primers/metabolism , Gene Frequency , Gene Library , Humans , Liquid Biopsy , Lung Neoplasms/blood , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Polymorphism, Single Nucleotide , RNA, Neoplasm/blood , Sensitivity and SpecificityABSTRACT
Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive and accurate method for quantification of nucleic acid sequences. We used absolute quantification of mutated v-Ki-ras2 Kirsten rat sarcoma viral oncogene homology gene (KRAS) by ddPCR to investigate the prognostic role of mutated KRAS in patients with KRAS-mutated lung adenocarcinomas. Pre-treatment plasma samples from 60 patients with stages I-IV KRAS-mutated lung adenocarcinomas were analysed for KRAS mutations. The associations between survival, detectable KRAS mutations in plasma, and the plasma concentration of mutated KRAS were assessed. Overall, 23 of 60 (38%) patients had detectable KRAS mutation in plasma. The percentage of patients with detectable mutation was 8% in stage I, 30% in stage II, 71% in stage III, and 73% in stage IV. Estimated overall median progression-free survival (PFS) and overall survival (OS) were 26.2 months [95% confidence interval (CI) 12.5-39.9] and 50.8 months (95% CI 0-107.3), respectively. Patients with detectable mutations in plasma had significantly worse median PFS compared to patients with undetectable mutation (13.1 versus 70.1 months) and shorter median OS (20.7 versus not reached). High circulating tumour DNA (ctDNA) concentrations of mutated KRAS were significantly associated with shorter PFS [hazard ratio (HR) 1.008, 95% CI 1.004-1.012] and OS (HR 1.007, 95% CI 1.003-1.011). All associations remained statistically significant in multivariable analyses. In conclusion, ddPCR is an accurate and easily feasible technique for quantification of KRAS mutations in ctDNA. The presence of detectable KRAS mutation in plasma at baseline was associated with worse PFS and OS. High concentration of mutated KRAS in ctDNA was an independent negative prognostic factor for both PFS and OS.
