Intra- and Inter-Tumor Heterogeneity of BRAFV600EMutations 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 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.

Intratumoral Molecular Heterogeneity in a BRAF-Mutant, BRAF Inhibitor-Resistant Melanoma: A Case Illustrating the Challenges for Personalized Medicine

“…However, the current method of assessing heterogeneity in a subset of sampled lesions is unlikely to adequately predict tumor heterogeneity in vivo, nor reflect the ongoing genetic changes that occur during treatment (13,15). Thus, the strategy of biopsying metastatic disease to decide the next systemic therapy after progression on a targeted therapy will be complicated by heterogeneity.…”

Streamlining Molecular Pathobiology of Malignant Melanoma

“…Genes potentially involved in CMM metastases include BRAF and RAS of the MAPK pathway [89]. CMM therapies targeting the BRAF and cKIT pathways call for genotyping CMM in order to select the proper patients [90][91][92][93][94][95].…”

Next Generation Sequencing in Cancer Research and Clinical Application

“…Variations between tumors are referred to as intertumor heterogeneity, while variations within a single tumor are intratumor heterogeneity. Intertumor heterogeneity is recognized by different morphological phenotype, expression profiles and mutation and copy number variation patterns, categorizing tumors into different subtypes [27][28][29][30][31]. The mRNA-expression subtype was found to be associated with somatic mutation landscapes in the recent TCGA and Eillis et al's studies.…”

“…For example, whole genome sequencing identified a novel insertional fusion that created a classic bcr3 PML-RARA fusion gene for a patient with acute myeloid leukemia and the findings altered the treatment plan for the patient [33]. By sequencing the tumor genome of a patient, clinicians are able to design patient-specific probes that uses DNA in the patient's blood serum to monitor the progress of a patient's treatment and detect for any signs of relapse [27][28][29][30][31]. The discovery of more biomarkers and the development of target-therapies will be essential in helping a clinician choose the best personalized treatment for his or her patients.…”