Abstract
We report the performance evaluation of a non-quantitative reverse-hybridization assay (KRAS-BRAFStripAssay) designed for the simultaneous detection of 10 mutations in codons 12 and 13 of theKRASgene andBRAFmutation V600E. Dilution experiments using DNA from tumor cell lines or from formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) tissue were performed to assess assay sensitivity. Using 50 ng of total DNA (mutant and wild-type), theKRAS-BRAFStripAssay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. With respect toBRAFV600E, theKRAS-BRAFStripAssay was evaluated using 60 FFPE CRC samples previously analyzed by high resolution melting (HRM). Test strip hybridization identified 2/60 (3%) samples to carry theBRAFV600E mutation, and results were in agreement with those obtained by HRM analysis. This work demonstrates theKRAS-BRAFStripAssay to be a robust and sensitive method for the detection of commonKRAS/BRAFmutations in genomic DNA isolated from FFPE tissue samples.
Highlights
Testing for KRAS mutation is an accepted prerequisite to select patients with colorectal cancer (CRC) for epidermal growth factor receptor (EGFR)-targeted therapies; typically, only individuals with wild-type KRAS respond to anti-EGFR therapy [1]
Because KRAS and BRAF mutations are mutually exclusive in CRC [11], BRAF V600E testing may further empower the selection of patients eligible for anti-EGFR antibody treatment [2]
The KRAS-BRAF StripAssay under investigation here has been modified to contain 10 mutations in codons 12 and 13 of the KRAS gene and BRAF mutation V600E. It combines mutant-enriched PCR based on peptide nucleic acid (PNA) clamping and reverse-hybridization of biotinylated PCR products to nitrocellulose test strips containing a parallel array of oligonucleotide probes
Summary
Testing for KRAS mutation is an accepted prerequisite to select patients with colorectal cancer (CRC) for epidermal growth factor receptor (EGFR)-targeted therapies; typically, only individuals with wild-type KRAS respond to anti-EGFR therapy [1]. Patients with BRAF V600E colorectal tumors show impaired responsiveness to the anti-EGFR therapeutic antibodies cetuximab and panitumumab [10]. Because KRAS and BRAF mutations are mutually exclusive in CRC [11], BRAF V600E testing may further empower the selection of patients eligible for anti-EGFR antibody treatment [2]. In conjunction with the very recently described PI3KCA activating mutations and the inactivation of PTEN phosphatase (a PI3K inhibitor), Bardelli et al established the term “quadruple negative” tumors as having the highest probability of response to anti-EGFR therapies [2]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
