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.
Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA. We analyzed the origins of discrepancies in KRAS (Kirsten rat sarcoma viral oncogene homolog gene) mutation detection in six of ten samples amplified using the GenomePlex® Tissue Whole Genome Amplification kit 5 (WGA5; Sigma-Aldrich, St. Louis, MO, USA) and KRAS StripAssay® (KRAS SA; ViennaLab Diagnostics, Vienna, Austria). We undertook reextraction, reamplification, retyping, authentication, reanalysis, and reinterpretation to determine whether the discrepancies originated during the preanalytical, analytical, and/or interpretative phase of genotyping. We conclude that a combination of glass slide/sample heterogeneity and biased amplification due to stochastic effects in the early phases of whole genome amplification (WGA) may have adversely affected the results obtained. Our findings are relevant for both forensic genetics testing and massively parallel sequencing using preamplification.
- MeSH
- genom lidský genetika MeSH
- genomika metody MeSH
- genotypizační techniky metody MeSH
- geny ras genetika MeSH
- kolorektální nádory genetika MeSH
- lidé MeSH
- sekvenční analýza DNA MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
BACKGROUND: It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters. METHODS: We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits. RESULTS AND CONCLUSIONS: Our results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples.
- MeSH
- analýza nákladů a výnosů MeSH
- DNA nádorová analýza genetika MeSH
- kolorektální nádory * genetika sekundární MeSH
- lidé MeSH
- mutace MeSH
- nemalobuněčný karcinom plic genetika MeSH
- protoonkogenní proteiny genetika MeSH
- ras proteiny genetika MeSH
- sekvenční analýza DNA * ekonomika metody MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
The KRAS gene (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is an oncogene that encodes a small GTPase transductor protein called KRAS. KRAS is involved in the regulation of cell division as a result of its ability to relay external signals to the cell nucleus. Activating mutations in the KRAS gene impair the ability of the KRAS protein to switch between active and inactive states, leading to cell transformation and increased resistance to chemotherapy and biological therapies targeting epidermal growth factor receptors. This review highlights some of the features of the KRAS gene and the KRAS protein and summarizes current knowledge of the mechanism of KRAS gene regulation. It also underlines the importance of activating mutations in the KRAS gene in relation to carcinogenesis and their importance as diagnostic biomarkers, providing clues regarding human cancer patients' prognosis and indicating potential therapeutic approaches.
- MeSH
- lidé MeSH
- nádory genetika metabolismus terapie MeSH
- protoonkogenní proteiny MeSH
- ras proteiny MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
