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Development and extensive analytical validation of deep amplicon sequencing for detecting KRAS and NRAS mutations in metastatic colorectal cancer samples

The result's identifiers

  • Result code in IS VaVaI

    <a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00098892%3A_____%2F22%3A10157743" target="_blank" >RIV/00098892:_____/22:10157743 - isvavai.cz</a>

  • Alternative codes found

    RIV/61989592:15110/22:73614465

  • Result on the web

    <a href="http://www.elis.sk/index.php?page=shop.product_details&flypage=flypage.tpl&product_id=7476&category_id=180&option=com_virtuemart&vmcchk=1&Itemid=1" target="_blank" >http://www.elis.sk/index.php?page=shop.product_details&flypage=flypage.tpl&product_id=7476&category_id=180&option=com_virtuemart&vmcchk=1&Itemid=1</a>

  • DOI - Digital Object Identifier

    <a href="http://dx.doi.org/10.4149/neo_2021_210907N1276" target="_blank" >10.4149/neo_2021_210907N1276</a>

Alternative languages

  • Result language

    angličtina

  • Original language name

    Development and extensive analytical validation of deep amplicon sequencing for detecting KRAS and NRAS mutations in metastatic colorectal cancer samples

  • Original language description

    The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥ 0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA.

  • Czech name

  • Czech description

Classification

  • Type

    J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database

  • CEP classification

  • OECD FORD branch

    30204 - Oncology

Result continuities

  • Project

    <a href="/en/project/NV16-32198A" target="_blank" >NV16-32198A: Genetic and epigenetic predictive biomarkers of treatment success for angiogenesis inhibitors in colorectal carcinoma</a><br>

  • Continuities

    P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Others

  • Publication year

    2022

  • Confidentiality

    S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Data specific for result type

  • Name of the periodical

    Neoplasma

  • ISSN

    0028-2685

  • e-ISSN

    1338-4317

  • Volume of the periodical

    69

  • Issue of the periodical within the volume

    1

  • Country of publishing house

    SK - SLOVAKIA

  • Number of pages

    13

  • Pages from-to

    203-215

  • UT code for WoS article

    000823580800003

  • EID of the result in the Scopus database

    2-s2.0-85124497169