Detection of mutated BRAFV600E variant in circulating DNA of stage III–IV melanoma patients

Abstract

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. © 2007 Wiley‐Liss, Inc.

Detection of BRAF mutations in the tumour and serum of patients enrolled in the AZD6244 (ARRY-142886) advanced melanoma phase II study

“…The concordance rate of cfDNA BRAF mutations with tumour BRAF mutations was 56%, which is consistent with that of other reports (Daniotti et al, 2007;Yancovitz et al, 2007). Although other groups have demonstrated the feasibility of detecting BRAF mutations in serum and plasma of patients with melanoma (Daniotti et al, 2007;Shinozaki et al, 2007;Yancovitz et al, 2007), this is the first study that compares tumour and cfDNA results from a large cohort of patients and demonstrates the potential clinical application of cfDNA mutation detection for patient selection within clinical trials.…”

“…Of 17 available tissue samples, the concordance of results was 10 of 17 (59%). Daniotti et al compared cfDNA and tumour BRAF mutations in 20 patients and found that cfDNA was positive for a BRAF mutation in 5 of 13 cases in which the tumour harboured a BRAF mutation (Daniotti et al, 2007). Shinozaki et al demonstrated BRAF mutations in 38 of 103 (37%) patients with melanoma (Shinozaki et al, 2007).…”

“…Yancovitz et al and Daniotti et al both identified two patients in whom BRAF mutations were detected in cfDNA but not in tumour DNA (Daniotti et al, 2007;Yancovitz et al, 2007). Often, tumour BRAF status is derived from a primary lesion that occurred months or years earlier.…”

“…Abbreviations: BRAF þ , BRAF mutation positive; cfDNA, cell-free DNA; HR, hazard ratio; CI, confidence interval. Detection of BRAF mutations in tumour and serum samples RE Board et al stage IV melanoma patients but in none of 4 stage I/II patients (Daniotti et al, 2007). This 'stage-dependency' may restrict cfDNA as a detection method in trials of agents for advanced disease, although more sensitive technologies may alter this in the future.…”

“…Previous studies have demonstrated the feasibility of detecting tumour-specific mutations in cfDNA from patients with cancer, including detection of epidermal growth factor receptor mutations in patients with non-small-cell lung cancer (Kimura et al, 2006(Kimura et al, , 2007 and KRAS mutations in patients with pancreatic and colorectal cancers (Sorenson, 2000). More recently, BRAF mutations have been detected in cfDNA of patients with melanoma (Daniotti et al, 2007;Shinozaki et al, 2007;Yancovitz et al, 2007). However, these initial studies included a small number of patients with only limited numbers of matched tumour samples.…”