Development and extensive analytical validation of deep amplicon sequencing for detecting KRAS and NRAS mutations in metastatic colorectal cancer samples
Language English Country Slovakia Media print-electronic
Document type Journal Article
PubMed
34881628
DOI
10.4149/neo_2021_210907n1276
PII: 210907N1276
Knihovny.cz E-resources
- MeSH
- Exons MeSH
- GTP Phosphohydrolases genetics MeSH
- Colorectal Neoplasms * genetics MeSH
- Humans MeSH
- Membrane Proteins genetics MeSH
- Mutation MeSH
- Proto-Oncogene Proteins p21(ras) * genetics MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- GTP Phosphohydrolases MeSH
- KRAS protein, human MeSH Browser
- Membrane Proteins MeSH
- NRAS protein, human MeSH Browser
- Proto-Oncogene Proteins p21(ras) * MeSH
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.
CGB Laboratory Inc Ostrava Czech Republic
Department of Neurology University Hospital Olomouc Czech Republic
Department of Oncology Faculty of Medicine and Dentistry Palacky University Olomouc Czech Republic
Department of Oncology University Hospital Olomouc Czech Republic
Laboratory of Experimental Medicine University Hospital Olomouc Czech Republic
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