Detection of mutated BRAFV600E variant in circulating DNA of stage III-IV melanoma patients.
BRAFV600E is the most represented somatic point mutation in cutaneous melanoma, thus providing a unique molecular marker for this disease. The development of efficient methods for its detection in free circulating DNA of patients may lead to the improvement of diagnostic and prognostic tools. With this aim, we evaluated whether BRAFV600E represents a detectable marker in the plasma/serum from melanoma patients in a pilot study. Circulating cell-free DNA was extracted from the serum or plasma of 15 healthy donors and 41 melanoma patients at different clinical stages and obtained either presurgery or after surgery during follow-up. Quantitative analysis showed higher levels of circulating free DNA in patients compared to controls, with the highest levels detected in samples obtained presurgery and at stage IV. Four different PCR methods were compared for their capacity to amplify a few copies of BRAFV600E in wild-type DNA. BRAFV600E was detectable in circulating DNA of 12 patients and in none of the controls; only 1 PCR method reproducibly amplified BRAFV600E. Positive samples were obtained from 8/13 patients at stage IV and from 4/24 patients at stage III, but not in 4 patients at stage I-II; half of the positives were obtained presurgery and half at follow-up. Correspondence between circulating DNA and related tumors were examined for 20 patients, and a correlation was found for stage IV patients. In conclusion, this method can be utilized for monitoring the disease in stage IV melanoma patients but it appears unsatisfactory for the early detection of melanoma.
Daniotti
Maria
M
Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Vallacchi
Viviana
V
Rivoltini
Licia
L
Patuzzo
Roberto
R
Santinami
Mario
M
Arienti
Flavio
F
Cutolo
Gianluca
G
Pierotti
Marco A
MA
Parmiani
Giorgio
G
Rodolfo
Monica
M
D001483:Base Sequence; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000097:blood*; D006801:Humans; D008545:Melanoma / Q000097:blood* / Q000235:genetics; D010865:Pilot Projects; D017354:Point Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*
D017931:DNA Primers; D004273:DNA, Neoplasm; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf