Intra- and Inter-Tumor Heterogeneity of BRAFV600EMutations in Primary and Metastatic Melanoma

作者：Molly Yancovitz, Adam J. Litterman, Joanne Yoon, Elise Ng, Richard L. Shapiro, Russell S. Berman, Anna C. Pavlick, Farbod Darvishian, Paul J. Christos, Madhu Mazumdar, Iman Osman, David Polsky · 发表于：PLoS ONE · 年份：2012 · DOI：10.1371/journal.pone.0029336 · 被引用次数：286 · 研究领域：Melanoma and MAPK Pathways、Cutaneous Melanoma Detection and Management、CAR-T cell therapy research

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 (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 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 t...