BACKGROUND: The BRAF gene has been identified as an oncogene in human cancer and the V600E mutation has been shown to be associated with clinico pathological features of primary invasive melanomas. As BRAF may be an attractive therapeutic target, it is crucial to have a sensitive method for detecting mutated DNA in biological samples. Our aim was to investigate COLD-PCR (co-amplification at lower denaturation temperature-PCR) as a new approach for the pre-analytical enrichment of the BRAFV600E variant in formalin fixed paraffin embedded (FFPE) melanoma tissues. METHODS: COLD-PCR was used to selectively amplify BRAFV600E minority alleles from mixtures of wild-type and mutated sequences, and from biological samples. The method shows higher specificity than other conventional PCR-based methods in detecting somatic mutations. RESULTS: We used COLD-PCR to increase the theoretical sensitivity of three different post-PCR methods: sequencing, pyrosequencing and HRMA. The gain in sensitivity seems to be more evident for HRMA, which allows the detection of 3.1% mutated alleles. More than 20% of patients initially classified negative for BRAFV600E were found positive after COLD-PCR. CONCLUSIONS: COLD-PCR was confirmed as a suitable method for the enrichment of mutated alleles, particularly for samples in which the percentage of tumor cells is very low.
BACKGROUND: The BRAF gene has been identified as an oncogene in humancancer and the V600E mutation has been shown to be associated with clinico pathological features of primary invasive melanomas. As BRAF may be an attractive therapeutic target, it is crucial to have a sensitive method for detecting mutated DNA in biological samples. Our aim was to investigate COLD-PCR (co-amplification at lower denaturation temperature-PCR) as a new approach for the pre-analytical enrichment of the BRAFV600E variant in formalin fixed paraffin embedded (FFPE) melanoma tissues. METHODS: COLD-PCR was used to selectively amplify BRAFV600E minority alleles from mixtures of wild-type and mutated sequences, and from biological samples. The method shows higher specificity than other conventional PCR-based methods in detecting somatic mutations. RESULTS: We used COLD-PCR to increase the theoretical sensitivity of three different post-PCR methods: sequencing, pyrosequencing and HRMA. The gain in sensitivity seems to be more evident for HRMA, which allows the detection of 3.1% mutated alleles. More than 20% of patients initially classified negative for BRAFV600E were found positive after COLD-PCR. CONCLUSIONS: COLD-PCR was confirmed as a suitable method for the enrichment of mutated alleles, particularly for samples in which the percentage of tumor cells is very low.
Authors: Carrie C Lubitz; Sareh Parangi; Tammy M Holm; M Jordana Bernasconi; Aislyn P Schalck; Hyunsuk Suh; Konstantinos P Economopoulos; Viswanath Gunda; Samuel E Donovan; Peter M Sadow; Lori J Wirth; Ryan J Sullivan; David J Panka Journal: J Mol Diagn Date: 2015-11-26 Impact factor: 5.568
Authors: L Heinzerling; S Kühnapfel; D Meckbach; M Baiter; E Kaempgen; P Keikavoussi; G Schuler; A Agaimy; J Bauer; A Hartmann; F Kiesewetter; R Schneider-Stock Journal: Br J Cancer Date: 2013-04-11 Impact factor: 7.640