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      Use of the QIAGEN GeneReader NGS system for detection of KRAS mutations, validated by the QIAGEN Therascreen PCR kit and alternative NGS platform

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          Abstract

          Background

          The detection of somatic mutations in primary tumors is critical for the understanding of cancer evolution and targeting therapy. Multiple technologies have been developed to enable the detection of such mutations. Next generation sequencing (NGS) is a new platform that is gradually becoming the technology of choice for genotyping cancer samples, owing to its ability to simultaneously interrogate many genomic loci at massively high efficiency and increasingly lower cost. However, multiple barriers still exist for its broader adoption in clinical research practice, such as fragmented workflow and complex bioinformatics analysis and interpretation.

          Methods

          We performed validation of the QIAGEN GeneReader NGS System using the QIAact Actionable Insights Tumor Panel, focusing on clinically meaningful mutations by using DNA extracted from formalin-fixed paraffin-embedded (FFPE) colorectal tissue with known KRAS mutations. The performance of the GeneReader was evaluated and compared to data generated from alternative technologies (PCR and pyrosequencing) as well as an alternative NGS platform. The results were further confirmed with Sanger sequencing.

          Results

          The data generated from the GeneReader achieved 100% concordance with reference technologies. Furthermore, the GeneReader workflow provides a truly integrated workflow, eliminating artifacts resulting from routine sample preparation; and providing up-to-date interpretation of test results.

          Conclusion

          The GeneReader NGS system offers an effective and efficient method to identify somatic (KRAS) cancer mutations.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12885-017-3328-z) contains supplementary material, which is available to authorized users.

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          Most cited references24

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          KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab.

          Cetuximab is efficient in advanced colorectal cancer (CRC). We previously showed that KRAS mutations were associated with resistance to cetuximab in 30 CRC patients. The aim of this study was to validate, in an independent larger series of 89 patients, the prognostic value of KRAS mutations on response to cetuximab and survival. Eighty-nine metastatic CRC patients treated with cetuximab after treatment failure with irinotecan-based chemotherapy were analyzed for KRAS mutation by allelic discrimination on tumor DNA. The association between KRAS mutations and tumor response, skin toxicity, progression-free survival (PFS) and overall survival (OS) was analyzed. A KRAS mutation was present in 27% of the patients and was associated with resistance to cetuximab (0% v 40% of responders among the 24 mutated and 65 nonmutated patients, respectively; P < .001) and a poorer survival (median PFS: 10.1 v 31.4 weeks in patients without mutation; P = .0001; median OS: 10.1 v 14.3 months in patients without mutation; P = .026). When we pooled these 89 patients with patients from our previous study, the multivariate analysis showed that KRAS status was an independent prognostic factor associated with OS and PFS, whereas skin toxicity was only associated with OS. In a combined analysis, median OS times of patients with two, one, or no favorable prognostic factors (severe skin toxicity and no KRAS mutation) was of 15.6, 10.7, and 5.6 months, respectively. These results confirm the high prognostic value of KRAS mutations on response to cetuximab and survival in metastatic CRC patients treated with cetuximab.
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            Integrating sequencing datasets to form highly confident SNP and indel genotype calls for a whole human genome

            Clinical adoption of human genome sequencing requires methods with known accuracy of genotype calls at millions or billions of positions across a genome. Previous work showing discordance amongst sequencing methods and algorithms has made clear the need for a highly accurate set of genotypes across a whole genome that could be used as a benchmark. We present methods to make highly confident SNP, indel, and homozygous reference genotype calls for NA12878, the pilot genome for the Genome in a Bottle Consortium. We minimize bias towards any method by integrating and arbitrating between 14 datasets from 5 sequencing technologies, 7 mappers, and 3 variant callers. Regions for which no confident genotype call could be made are identified as uncertain, and classified into different reasons for uncertainty. Our highly confident genotype calls are publicly available on the Genome Comparison and Analytic Testing (GCAT) website to enable real-time benchmarking of any method.
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              Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes.

              To optimize the molecular diagnosis of hereditary breast and ovarian cancer (HBOC), we developed a next-generation sequencing (NGS)-based screening based on the capture of a panel of genes involved, or suspected to be involved in HBOC, on pooling of indexed DNA and on paired-end sequencing in an Illumina GAIIx platform, followed by confirmation by Sanger sequencing or MLPA/QMPSF. The bioinformatic pipeline included CASAVA, NextGENe, CNVseq and Alamut-HT. We validated this procedure by the analysis of 59 patients' DNAs harbouring SNVs, indels or large genomic rearrangements of BRCA1 or BRCA2. We also conducted a blind study in 168 patients comparing NGS versus Sanger sequencing or MLPA analyses of BRCA1 and BRCA2. All mutations detected by conventional procedures were detected by NGS. We then screened, using three different versions of the capture set, a large series of 708 consecutive patients. We detected in these patients 69 germline deleterious alterations within BRCA1 and BRCA2, and 4 TP53 mutations in 468 patients also tested for this gene. We also found 36 variations inducing either a premature codon stop or a splicing defect among other genes: 5/708 in CHEK2, 3/708 in RAD51C, 1/708 in RAD50, 7/708 in PALB2, 3/708 in MRE11A, 5/708 in ATM, 3/708 in NBS1, 1/708 in CDH1, 3/468 in MSH2, 2/468 in PMS2, 1/708 in BARD1, 1/468 in PMS1 and 1/468 in MLH3. These results demonstrate the efficiency of NGS in performing molecular diagnosis of HBOC. Detection of mutations within other genes than BRCA1 and BRCA2 highlights the genetic heterogeneity of HBOC.
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                Author and article information

                Contributors
                agusd2004@gmail.com
                Anne.Hein@qiagen.com
                Sascha.Strauss@qiagen.com
                Yi.Kong@qiagen.com
                Andrew.Sheridan@qiagen.com
                Dan.Richards@qiagen.com
                Eric.Lader@qiagen.com
                mdominska@gmail.com
                Timothy.Pelletier@qiagen.com
                danielle.lee.adams@gmail.com
                Austin.Ricker@qiagen.com
                Nishit.Patel@qiagen.com
                Andreas.Kuehne@qiagen.com
                Simon.Hughes@qiagen.com
                Dan.Shiffman@qiagen.com
                Dirk.Zimmermann@qiagen.com
                Kai.teKaat@qiagen.com
                Thomas.rothmann@qiagen.com
                Journal
                BMC Cancer
                BMC Cancer
                BMC Cancer
                BioMed Central (London )
                1471-2407
                22 May 2017
                22 May 2017
                2017
                : 17
                : 358
                Affiliations
                [1 ]QIAGEN Waltham, 35 Gatehouse Dr, Waltham, MA 02451 USA
                [2 ]QIAGEN Arhus, Silkeborgvej 2, 8000 Aarhus, Denmark
                [3 ]ISNI 0000 0004 0552 1382, GRID grid.420167.6, , QIAGEN GmbH, ; QIAGEN Strasse 1, 40724 Hilden, Nordrhein-Westfalen Germany
                [4 ]QIAGEN Redwood City, 1700 Seaport Blvd, Redwood, CA 94063 USA
                [5 ]QIAGEN Frederick, 6951 Executive Way, Frederick, MD 21703 USA
                [6 ]ISNI 0000 0004 0451 3823, GRID grid.474454.2, , QIAGEN Manchester, ; Skelton House Lloyd Street North, Manchester, M15 6SH UK
                [7 ]ISNI 0000 0004 0439 2056, GRID grid.418424.f, , Novartis Institutes for BioMedical Research, ; Cambridge, MA 02139 USA
                [8 ]T2 Biosystems, Lexington, MA 02421 USA
                [9 ]Macherey-Nigel, Bethlehem, PA 18020 USA
                Author information
                http://orcid.org/0000-0002-9100-5266
                Article
                3328
                10.1186/s12885-017-3328-z
                5441096
                28532404
                8bfff20b-69e6-4e53-8702-8d93a83e8f20
                © The Author(s). 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 28 November 2016
                : 5 May 2017
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2017

                Oncology & Radiotherapy
                genereader,kras,mutation,cancer,ngs
                Oncology & Radiotherapy
                genereader, kras, mutation, cancer, ngs

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