Detection of Mutant BRAF Alleles in the Plasma of Patients with Metastatic Melanoma

Abstract

Mutations in the BRAF oncogene at amino acid 600 have been reported in 40 to 70% of human metastatic melanoma tissues, and the critical role of BRAF in the biology of melanoma has been established. Sampling the blood compartment to detect the mutational status of a solid tumor represents a highly innovative advance in cancer medicine, and such an approach could have advantages over tissue-based techniques. We report the development of a fluorescence-based polymerase chain reaction (PCR) assay to detect mutant BRAF alleles in plasma. A mutant-specific PCR assay was optimized to specifically amplify the mutant BRAF allele without amplifying the wild-type allele. Experiments mixing DNA from a BRAF mutant melanoma cell line with wild-type human placental DNA in varying proportions were performed to determine the threshold of this assay and to compare it with routine DNA sequencing. The assay was then applied to tissue and plasma specimens from patients with metastatic melanoma. The assay detected 0.1 ng of mutant DNA mixed in 100 ng of wild-type DNA and was 500-fold more sensitive than DNA sequencing. The assay detected mutant BRAF alleles in plasma samples from 14 of 26 (54%) metastatic melanoma patients. These data demonstrate the feasibility of blood-based testing for BRAF mutations in metastatic melanoma patients. (J Mol Diagn 2007, 9:178 -183;

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.…”

“…Yancovitz et al demonstrated BRAF mutations in cfDNA extracted from plasma of 14 of 26 (54%) stage IV melanoma patients (Yancovitz et al, 2007). Of 17 available tissue samples, the concordance of results was 10 of 17 (59%).…”

“…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.…”

“…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.…”

How detection of epigenetic alterations of blood-borne DNA could improve melanoma diagnosis

“…Furthermore, the genetic and epigenetic changes present in the tumor tissue are represented and can be detected in the blood-borne DNA. For example, BRAF mutations can be detected in the peripheral blood of melanoma patients [7,8]. The frequent (but not invariable) association of CGI methylation with cancer, together with the relative stability of methylated genomic DNA in peripheral blood, makes it an attractive potential source of biomarkers and detection in the serum of methylated DNA from several genes has been reported.…”