A Multiplex Technology Platform For The Rapid Detection Of Relevant Mutations In The Egfr Signaling Pathway
JOURNAL OF CLINICAL ONCOLOGY(2011)
Abstract
386 Background: Determination of the mutational status of key gene effectors along the EGFR signaling pathway plays a critical role in the management of metastatic colorectal cancer (mCRC) and other cancer types. We previously reported the rapid, multiplex, and sensitive detection of 7 common KRAS mutations in mCRC FFPE specimens using the Signature KRAS Mutations (RUO) kit. We show here that the same technology platform can be applied to the detection of additional clinically relevant mutations in the EGFR, BRAF, KRAS, NRAS and HRAS genes. Methods: Mutation panels were designed for multiplex PCR amplification of an internal control gene and discrete mutation regions in independent target genes. PCR products were hybridized on a liquid bead array carrying target-specific probes and sorted by flow cytometry using a Luminex 200 system. Genomic DNA was extracted from cell lines and representative flash frozen, FFPE or FNA clinical specimens using laboratory-validated extraction methods. Results: Using model plasmids and cell lines we developed specific assays for distinct mutations in KRAS codon 12/13, KRAS 3' UTR (rs61764370), BRAF codon 600/601, HRAS codon 12/61, NRAS codon 61, and EGFR exon 19/20/21. The various assays reached 0.1 to 5% analytical sensitivity and could be further combined in multiplex panels relevant to specific applications. The Signature KRAS/BRAF Mutations (RUO) kit, an assay for 12 KRAS mutations in codon 12/13 and BRAF V600E, was validated in over 250 mCRC FFPE specimens with >99% agreement with clinically validated methods. Specific detection of these 13 mutations and 6 HRAS/NRAS mutations was also demonstrated in thyroid nodule FNA specimens. Single-well detection of EGFR deletions in exon 19 together with the point mutations L858R and T790M was also successfully evaluated in lung specimens. Conclusions: The Signature technology platform is a specific, sensitive and flexible tool for the assessment of various mutations in different tissue types. The development and validation of broader mutation panels would likely facilitate the rapid molecular characterization of individual tumor specimens and the optimization of personalized treatments for mCRC and other cancer types. [Table: see text]
MoreTranslated text
Key words
egfr,multiplex technology platform,relevant mutations,signaling
AI Read Science
Must-Reading Tree
Example
![](https://originalfileserver.aminer.cn/sys/aminer/pubs/mrt_preview.jpeg)
Generate MRT to find the research sequence of this paper
Chat Paper
Summary is being generated by the instructions you defined