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

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61989592%3A15110%2F22%3A73614465" target="_blank" >RIV/61989592:15110/22:73614465 - isvavai.cz</a>

Alternative codes found

RIV/00098892:_____/22:10157743

Result on the web

<a href="https://pubmed.ncbi.nlm.nih.gov/34881628/" target="_blank" >https://pubmed.ncbi.nlm.nih.gov/34881628/</a>

DOI - Digital Object Identifier

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

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 (longRAS 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

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10608 - Biochemistry and molecular biology

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)

Publication year

2022

Confidentiality

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

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