1145 - Sensitive detection of BRAF mutations using mutant-enriched PCR and reverse-hybridization teststrips

Date 28 September 2012
Event ESMO Congress 2012
Session Publication Only
Topics Biomarkers
Diagnostics
Melanoma and other Skin Tumours
Presenter Michael Novy
Authors M. Novy, G. Kriegshäuser, B. Rauscher, C. Oberkanins
  • Research - Development, ViennaLab Diagnostics GmbH, 1120 - Vienna/AT

Abstract

Background

BRAF plays a key role in growth factor receptor induced signalling pathways. Somatic BRAF mutations are involved in oncogenesis and are found in various types of tumors, including colorectal, thyroid and skin cancer. Activating BRAF mutations confer resistance to anti-EGFR monoclonal antibody therapy. Moreover mutated BRAF is a prominent target of the drug vemurafenib.

Materials and methods

We have developed a reverse-hybridization StripAssay detecting nine BRAF mutations: c.1799T > C (V600A), c.1799_1800TG > AT (V600D), c.1799T > A (V600E), c.1799_1800TG > AA (V600E), c.1799T > G (V600G), c.1798_1799GT > AA (V600K), c.1798G > A (V600M), c.1798_1799GT > AG (V600R) and c.1801A > G (K601E). The test is based on mutant-enrichted PCR in the presence of a BRAF wild-type suppressor, followed by hybridization of PCR products to teststrips presenting a parallel array of allele-specific oligonucleotide probes. Bound sequences are visualized using streptavidin-alkaline phosphatase conjungate and colour substrates. The hybridization and detection steps can be carried out fully automated using commercially available instrumentation.

Results

Plasmid clones served as reference DNA templates to control for specificity. StripAssay performance was evaluated on genomic DNA obtained from cultured cell lines and formalin-fixed paraffine-embedded (FFPE) tissue. Using normal DNA spiked with serial dilutions of DNA from the BRAF-mutant tumor cell lines HT29 or IGR-1, the mutations c.1799T > A (V600E) and c.1798_1799GT > AA (V600K) were shown to be detectable at a level of 1%.

Conclusions

The simultaneous detection of nine different mutations with high sensitivity will make the StripAssay a very useful tool for the assessment of the BRAF mutation status of cancer patients.

Disclosure

All authors have declared no conflicts of interest.