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 BRAF(V600E) as a therapeutic target, we investigated intra- and inter-
tumor heterogeneity in
melanoma using detection of the BRAF(V600E) 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 (nā=ā18). 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 BRAF(V600E) 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 BRAF(V600E)-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAF(V600E)and BRAF(wild-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 BRAF(V600E) mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAF(V600E) 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.