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      Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA

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          Abstract

          Introduction

          Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes.

          Materials and methods

          We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material.

          Results

          The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA.

          Conclusions

          These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.

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          Most cited references12

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          Safety and efficacy of vemurafenib in BRAF(V600E) and BRAF(V600K) mutation-positive melanoma (BRIM-3): extended follow-up of a phase 3, randomised, open-label study.

          In the BRIM-3 trial, vemurafenib was associated with risk reduction versus dacarbazine of both death and progression in patients with advanced BRAF(V600) mutation-positive melanoma. We present an extended follow-up analysis of the total population and in the BRAF(V600E) and BRAF(V600K) mutation subgroups. Patients older than 18 years, with treatment-naive metastatic melanoma and whose tumour tissue was positive for BRAF(V600) mutations were eligible. Patients also had to have a life expectancy of at least 3 months, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and adequate haematological, hepatic, and renal function. Patients were randomly assigned by interactive voice recognition system to receive either vemurafenib (960 mg orally twice daily) or dacarbazine (1000 mg/m(2) of body surface area intravenously every 3 weeks). Coprimary endpoints were overall survival and progression-free survival, analysed in the intention-to-treat population (n=675), with data censored at crossover. A sensitivity analysis was done. This trial is registered with ClinicalTrials.gov, NCT01006980. 675 eligible patients were enrolled from 104 centres in 12 countries between Jan 4, 2010, and Dec 16, 2010. 337 patients were randomly assigned to receive vemurafenib and 338 to receive dacarbazine. Median follow-up was 12·5 months (IQR 7·7-16·0) on vemurafenib and 9·5 months (3·1-14·7) on dacarbazine. 83 (25%) of the 338 patients initially randomly assigned to dacarbazine crossed over from dacarbazine to vemurafenib. Median overall survival was significantly longer in the vemurafenib group than in the dacarbazine group (13·6 months [95% CI 12·0-15·2] vs 9·7 months [7·9-12·8]; hazard ratio [HR] 0·70 [95% CI 0·57-0·87]; p=0·0008), as was median progression-free survival (6·9 months [95% CI 6·1-7·0] vs 1·6 months [1·6-2·1]; HR 0·38 [95% CI 0·32-0·46]; p<0·0001). For the 598 (91%) patients with BRAF(V600E) disease, median overall survival in the vemurafenib group was 13·3 months (95% CI 11·9-14·9) compared with 10·0 months (8·0-14·0) in the dacarbazine group (HR 0·75 [95% CI 0·60-0·93]; p=0·0085); median progression-free survival was 6·9 months (95% CI 6·2-7·0) and 1·6 months (1·6-2·1), respectively (HR 0·39 [95% CI 0·33-0·47]; p<0·0001). For the 57 (9%) patients with BRAF(V600K) disease, median overall survival in the vemurafenib group was 14·5 months (95% CI 11·2-not estimable) compared with 7·6 months (6·1-16·6) in the dacarbazine group (HR 0·43 [95% CI 0·21-0·90]; p=0·024); median progression-free survival was 5·9 months (95% CI 4·4-9·0) and 1·7 months (1·4-2·9), respectively (HR 0·30 [95% CI 0·16-0·56]; p<0·0001). The most frequent grade 3-4 events were cutaneous squamous-cell carcinoma (65 [19%] of 337 patients) and keratoacanthomas (34 [10%]), rash (30 [9%]), and abnormal liver function tests (38 [11%]) in the vemurafenib group and neutropenia (26 [9%] of 287 patients) in the dacarbazine group. Eight (2%) patients in the vemurafenib group and seven (2%) in the dacarbazine group had grade 5 events. Inhibition of BRAF with vemurafenib improves survival in patients with the most common BRAF(V600E) mutation and in patients with the less common BRAF(V600K) mutation. F Hoffmann-La Roche-Genentech. Copyright © 2014 Elsevier Ltd. All rights reserved.
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            Erlotinib as maintenance treatment in advanced non-small-cell lung cancer: a multicentre, randomised, placebo-controlled phase 3 study.

            First-line chemotherapy for advanced non-small-cell lung cancer (NSCLC) is usually limited to four to six cycles. Maintenance therapy can delay progression and prolong survival. The oral epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitor erlotinib has proven efficacy and tolerability in second-line NSCLC. We designed the phase 3, placebo-controlled Sequential Tarceva in Unresectable NSCLC (SATURN; BO18192) study to assess use of erlotinib as maintenance therapy in patients with non-progressive disease following first-line platinum-doublet chemotherapy. Between December, 2005, and May, 2008, 1949 patients were included in the run-in phase (four cycles of platinum-based chemotherapy). At the end of the run-in phase, 889 patients who did not have progressive disease were entered into the main study, and were randomly allocated using a 1:1 adaptive randomisation method through a third-party interactive voice response system to receive erlotinib (150 mg/day; n=438) or placebo (n=451) until progression or unacceptable toxicity. Patients were stratified by EGFR immunohistochemistry status, stage, Eastern Cooperative Oncology Group performance status, chemotherapy regimen, smoking history, and region. Co-primary endpoints were progression-free survival (PFS) in all analysable patients irrespective of EGFR status, and PFS in patients whose tumours had EGFR protein overexpression, as determined by immunohistochemistry. This study is registered with www.ClinicalTrials.gov, number NCT00556712. 884 patients were analysable for PFS; 437 in the erlotinib group and 447 in the placebo group. After a median follow-up of 11.4 months for the erlotinib group and 11.5 months for the placebo group, median PFS was significantly longer with erlotinib than with placebo: 12.3 weeks for patients in the erlotinib group versus 11.1 weeks for those in the placebo group (HR 0.71, 95% CI 0.62-0.82; p<0.0001). PFS was also significantly longer in patients with EGFR-positive immunohistochemistry who were treated with erlotinib (n=307) compared with EGFR-positive patients given placebo (n=311; median PFS 12.3 weeks in the erlotinib group vs 11.1 weeks in the placebo group; HR 0.69, 0.58-0.82; p<0.0001). The most common grade 3 or higher adverse events were rash (37 [9%] of 443 patients in the erlotinib group vs none of 445 in the placebo group) and diarrhoea (seven [2%] of 443 patients vs none of 445). Serious adverse events were reported in 47 patients (11%) on erlotinib compared with 34 patients (8%) on placebo. The most common serious adverse event was pneumonia (seven cases [2%] with erlotinib and four [<1%] with placebo). Maintenance therapy with erlotinib for patients with NSCLC is well tolerated and significantly prolongs PFS compared with placebo. First-line maintenance with erlotinib could be considered in patients who do not progress after four cycles of chemotherapy. F Hoffmann-La Roche Ltd. Copyright 2010 Elsevier Ltd. All rights reserved.
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              About the possible origin and mechanism of circulating DNA apoptosis and active DNA release.

              In addition to cell lysis, apoptosis has been advanced as the origin of circulating DNA on the basis of several observations. Plasma or serum DNA often presents a ladder pattern reminiscent of that displayed by apoptotic cells when subjected to electrophoresis. However, the phenomenon of active release of DNA from cells might also be expected to result in a ladder pattern on electrophoresis. Non-dividing cells, such as lymphocytes, frog auricles and cultured cell lines including HL-60, spontaneously release a nucleoprotein complex within a homeostatic system in which newly synthesized DNA is preferentially released. In relation to DNA synthesis, the phenomenon of extracellular DNA in different culture conditions favors apoptosis or spontaneous active DNA release.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – review & editing
                Role: InvestigationRole: ValidationRole: Writing – review & editing
                Role: Investigation
                Role: Investigation
                Role: InvestigationRole: MethodologyRole: Validation
                Role: InvestigationRole: Validation
                Role: InvestigationRole: Validation
                Role: Supervision
                Role: Supervision
                Role: Formal analysis
                Role: ValidationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: Data curationRole: Formal analysis
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Methodology
                Role: Supervision
                Role: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Role: Investigation
                Role: Resources
                Role: Funding acquisitionRole: Project administrationRole: Resources
                Role: InvestigationRole: MethodologyRole: Project administrationRole: VisualizationRole: Writing – review & editing
                Role: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole: SoftwareRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                16 March 2018
                2018
                : 13
                : 3
                : e0194630
                Affiliations
                [1 ] Inivata Ltd, Granta Park, Cambridge, United Kingdom
                [2 ] Inivata Inc, Research Triangle Park, NC, United States of America
                [3 ] LGC, Teddington, Middlesex, United Kingdom
                [4 ] School of Biosciences & Medicine, Faculty of Health & Medical Science, University of Surrey, Guildford, United Kingdom
                [5 ] Horizon Discovery, Waterbeach, Cambridge, United Kingdom
                CNR, ITALY
                Author notes

                Competing Interests: DG, AL, KH, MM, BD, SS, JCa, SB, GJ, JCl, PD, MP, SW, ME, EG, TF, VP and NR are current or former employees, officers, consultants and/or share-holders of Inivata Ltd or Inivata Inc. Inivata, InVision, TAm-Seq and eTAm-Seq are UK registered trademarks of Inivata Ltd. DG, TF and NR are co-founders of Inivata Ltd. NR is a Board member of Inivata. DG, SW, NR and TF are co-inventors or contributors on patent applications that describe methods for the analysis of DNA fragments and applications of circulating tumor DNA. AF-G, AW, JH, CF, GMJ and AD are current or former employees of LGC. HR-A and KS are former employees of Horizon Discovery. This does not alter our adherence to PLOS ONE policies on sharing data and materials, with restrictions described in the Data Availability Statement.

                Author information
                http://orcid.org/0000-0002-4521-8199
                Article
                PONE-D-17-31337
                10.1371/journal.pone.0194630
                5856404
                29547634
                e154d343-8d9e-4ea5-91c0-ab66970b1ab9
                © 2018 Gale et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 25 August 2017
                : 19 February 2018
                Page count
                Figures: 4, Tables: 2, Pages: 18
                Funding
                The analytical validation study was funded by Inivata. Inivata provided support in the form of salaries for authors DG, AL, KH, MM, BD, SS, JCa, SB, GJ, JCl, PD, MP, SW, ME, EG, TF, VP and NR, and participated in study design, data collection and analysis, decision to publish, and preparation of the manuscript. The work on the novel full-process control material performed by LGC was funded by the UK government Department for Business, Energy & Industrial Strategy (BEIS) and Innovate UK under the project ‘Enabling stratified medicine through cell and tissue reference standards for minimally invasive cancer testing’ (Project Number: 101862) in partnership with Horizon Discovery. LGC provided support in the form of salaries for authors AF-G, AW, JH, CF, GMJ and AD, and Horizon Discovery provided support in the form of salaries for authors HR-A and KS. LGC and Horizon Discovery did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of all authors are articulated in the ‘author contributions’ section.
                Categories
                Research Article
                Biology and Life Sciences
                Genetics
                Gene Identification and Analysis
                Mutation Detection
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
                Biology and Life Sciences
                Physiology
                Body Fluids
                Blood
                Blood Plasma
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Blood
                Blood Plasma
                Medicine and Health Sciences
                Surgical and Invasive Medical Procedures
                Biopsy
                Biology and life sciences
                Biochemistry
                Biomarkers
                Circulating tumor DNA
                Medicine and health sciences
                Diagnostic medicine
                Cancer detection and diagnosis
                Circulating tumor DNA
                Medicine and health sciences
                Oncology
                Cancer detection and diagnosis
                Circulating tumor DNA
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Mutagenesis and Gene Deletion Techniques
                Mutational Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Mutagenesis and Gene Deletion Techniques
                Mutational Analysis
                Biology and Life Sciences
                Genetics
                Mutation
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                DNA sequencing
                Next-Generation Sequencing
                Research and analysis methods
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                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Transcriptome Analysis
                Next-Generation Sequencing
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Transcriptome Analysis
                Next-Generation Sequencing
                Custom metadata
                All relevant data are within the paper and its Supporting Information files. S4 Table describes all allele fractions (shown as percentages) for the common low frequency variants validated/quantified in Horizon Tru-Q 7 and in the InVision liquid biopsy panel for both laboratories (one tab per laboratory). S5 Table details the list of variants analyzed in the Horizon Tru-Q 6 and Horizon Tru-Q 7 dilution study. S6 Table shows the sensitivity analysis for all samples. All reportable calls (excluding SNPs and non-coding changes) made using eTAm-Seq technology to analyse the novel full-process control are available in S7 Table. In addition, a completed dPCR dMIQE checklist and assay information files have been provided for the full-process control study.

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