title: Intra- and inter-tumor heterogeneity of BRAF(V600E))mutations in primary and metastatic melanoma.
abstract: 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 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.
authors.affiliation: Department of Dermatology, New York University Langone Medical Center, New York, New York, United States of America.
authors.forename: Molly
authors.initials: M
authors.lastname: Yancovitz
authors.forename: Adam
authors.initials: A
authors.lastname: Litterman
authors.forename: Joanne
authors.initials: J
authors.lastname: Yoon
authors.forename: Elise
authors.initials: E
authors.lastname: Ng
authors.forename: Richard L
authors.initials: RL
authors.lastname: Shapiro
authors.forename: Russell S
authors.initials: RS
authors.lastname: Berman
authors.forename: Anna C
authors.initials: AC
authors.lastname: Pavlick
authors.forename: Farbod
authors.initials: F
authors.lastname: Darvishian
authors.forename: Paul
authors.initials: P
authors.lastname: Christos
authors.forename: Madhu
authors.initials: M
authors.lastname: Mazumdar
authors.forename: Iman
authors.initials: I
authors.lastname: Osman
authors.forename: David
authors.initials: D
authors.lastname: Polsky
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000473:pathology / Q000556:secondary*; D042282:Microdissection; D009154:Mutation; D009362:Neoplasm Metastasis; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf