Daniotti Maria, Vallacchi Viviana, Rivoltini Licia, Patuzzo Roberto, Santinami Mario, Arienti Flavio, Cutolo Gianluca, Pierotti Marco A, Parmiani Giorgio, Rodolfo Monica
Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Int J Cancer. 2007 Jun 1;120(11):2439-44. doi: 10.1002/ijc.22598.
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.
BRAFV600E是皮肤黑色素瘤中最常见的体细胞点突变，因此为这种疾病提供了一个独特的分子标记。开发在患者游离循环DNA中检测该突变的有效方法可能会改善诊断和预后工具。出于这个目的，我们在一项初步研究中评估了BRAFV600E是否是黑色素瘤患者血浆/血清中可检测到的标记物。从15名健康供体和41名处于不同临床阶段的黑色素瘤患者的血清或血浆中提取循环游离DNA，这些样本在术前或随访期间术后获得。定量分析显示，与对照组相比，患者的循环游离DNA水平更高，术前和IV期样本中检测到的水平最高。比较了四种不同的PCR方法在野生型DNA中扩增少量BRAFV600E拷贝的能力。12名患者的循环DNA中可检测到BRAFV600E，而对照组中均未检测到；只有1种PCR方法可重复性地扩增BRAFV600E。阳性样本来自IV期的8/13名患者和III期的4/24名患者，但I-II期的4名患者中未检测到；一半的阳性样本在术前获得，一半在随访时获得。对20名患者的循环DNA与相关肿瘤之间的对应关系进行了检查，发现IV期患者存在相关性。总之，这种方法可用于监测IV期黑色素瘤患者的疾病，但对于黑色素瘤的早期检测似乎并不令人满意。
