BRAFV600E detection in melanoma is highly improved by COLD-PCR.
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.
Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. p.pinzani@dfc.unifi.it
Pamela
P
Pinzani
Claudio
C
Santucci
Irene
I
Mancini
Lisa
L
Simi
Francesca
F
Salvianti
Nicola
N
Pratesi
Daniela
D
Massi
Vincenzo
V
De Giorgi
Mario
M
Pazzagli
Claudio
C
Orlando
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005557:Formaldehyde / Q000378:metabolism; D006801:Humans; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000473:pathology; D008875:Middle Aged; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D044366:Transition Temperature
D005557:Formaldehyde; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf