Allele specific Taqman-based real-time PCR assay to quantify circulating BRAFV600E mutated DNA in plasma of melanoma patients.
BACKGROUND
BRAF is the most frequently mutated oncogene in melanoma with BRAF(V600E) mutation accounting for 92% of all BRAF variants. As this event occurs early in melanoma progression, the quantification of BRAF-mutated alleles in plasma may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy.


METHODS
We propose an assay based on the use of a locked nucleic acid probe and an allele specific primer to measure plasma-circulating BRAF(V600E) concentration in patients affected by cutaneous melanoma (n=55) and non-melanoma skin cancers (n=13) as well as 18 healthy subjects. The assay is highly sensitive and accurate in detecting down to 0.3% of mutated allele in plasma.


RESULTS
A significant difference between the control group and invasive melanomas (p<0.01) was evidenced in BRAF(V600E) concentration, either as relative percentage or absolute values. ROC curve indicated that BRAF(V600E) absolute concentration has the maximal diagnostic relevance with 97% sensitivity and 83% specificity. Comparison of the results obtained in plasma with those found in the corresponding tissues indicated an 80% concordance.


CONCLUSIONS
The allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAF(V600E) mutated DNA in plasma.
Pinzani
Pamela
P
Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. p.pinzani@dfc.unifi.it
Salvianti
Francesca
F
Cascella
Roberta
R
Massi
Daniela
D
De Giorgi
Vincenzo
V
Pazzagli
Mario
M
Orlando
Claudio
C
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D001483:Base Sequence; D002474:Cell-Free System; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008545:Melanoma / Q000097:blood* / Q000235:genetics; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012878:Skin Neoplasms / Q000097:blood* / Q000235:genetics
D017931:DNA Primers; D004273:DNA, Neoplasm; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf