The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene. With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality. BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18). Nineteen patients had tissues available from multiple metastatic sites. Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR. The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes. To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors. Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients. Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful.

BACKGROUND. In patients with T1b‐T3b cutaneous melanoma the utility of radiologic imaging at the time of diagnosis is unclear. Whether initial imaging led to a change in stage or treatment plan was investigated. METHODS. The melanoma database was searched for patients with T1b‐T3b primary lesions, clinically N0, and asymptomatic for metastatic disease. Radiologic studies conducted before wide local excision ± sentinel lymph node biopsy as well as all further imaging and investigations were analyzed. Outcome measures included upstaging, change in initial surgical management, true‐positive, false‐positive, true‐negative, and false‐negative rates of each imaging modality. RESULTS. In all, 344 preoperative imaging studies (chest x‐ray [CXR], computed tomography [CT], positron emission tomography [PET]/CT) were performed on 158 patients, resulting in 49 findings suspicious for metastatic melanoma and 134 findings suggestive of nonmelanoma pathology. Only 1 of 344 (0.3%) studies, a PET/CT, correlated with confirmed metastatic melanoma. The false‐positive rates were CXR 5 of 7 (71.4%), chest CT 21 of 24 (87.5%), abdomen/pelvis CT 10 of 11 (90.9%), head CT 2 of 2 (100.0%), PET/CT 3 of 5 (60.0%). No patient was upstaged or had a change in initial surgical management based on preoperative imaging. The cost of all initial imaging and imaging to follow‐up abnormal findings was estimated as $555,308 for the 158 patients studied. CONCLUSIONS. Imaging at the time of initial diagnosis of T1b‐T3b, clinically N0, M0 melanoma was of low yield with a high false‐positive rate, and did not lead to upstaging or change in initial surgical management. These findings suggest that imaging of asymptomatic patients at the time of diagnosis may not be warranted. Cancer 2007. © 2007 American Cancer Society.

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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;