In:
Molecular Cancer Therapeutics, American Association for Cancer Research (AACR), Vol. 10, No. 11_Supplement ( 2011-11-12), p. C139-C139
Abstract:
Background: BRAF is one of the most frequently activated oncogenes in melanoma comprising 40–60% of all mutations. About ninety percent of activating BRAF mutations occur at the V600E position enabling specific therapeutic targeting with inhibitors such as vemurafenib. Detection of the BRAF V600E mutation is therefore important for guiding treatment; however, access to tissue for testing is limited. We have recently shown that microvesicles (exosomes) isolated from serum/plasma contain RNA (exoRNA) and that tumor-derived exoRNA can be extracted from microvesicles circulating in peripheral blood. Thus the detection of the BRAF V600E mutation in blood represents a potentially useful biomarker for non-invasive diagnosis and monitoring therapeutic response. Methods: ExoRNA was extracted from the serum and plasma of 18 patients with biopsy confirmed grade IV melanoma and 12 healthy controls. Patients were consented and samples were collected in accordance with protocols approved by the IRB at MGH and LMU. Some patients included in the study were undergoing chemotherapy or drug treatment during blood draw (13/18), while others did not receive any treatment (5/18). BRAF V600E mutation in the isolated exoRNA from serum/plasma was measured by two methods. qPCR based assay: This assay used Taqman MGB™ probes and ARMS primers. The assay was able to detect mutations as low as & lt;0.5% and could detect to as little as 10 mutant copies in a wild-type background. Ultra-deep Sequencing assay using Illumina HiSeq technology: Eight melanoma patient samples were used for this assay (3 from group I and 5 from group II). Using PCR, a 100 bp region of BRAF encompassing the V600E mutation locus was amplified. Illumina library preparation protocol and bioinformatics methodology was then optimized to identify BRAF V600E mutations from ultra-high coverage amplicon sequencing data. To establish the sensitivity of the sequencing platform, we also made admixtures of synthetic DNA oligos with known sequences diluting the V600E variant in up to 10,000 copies of the wild type. We found that the lower detection limit for the rare mutant variant is about 1 in 5000 wild type molecules. Results: qPCR results indicate that 66% patients had concordance with biopsy. The healthy controls tested were all negative for V600E (0/12), giving the assay a specificity of 100% Ultra deep sequencing of exoRNA indicated that 62% patients had concordance with biopsy. By this method 2 million sequencing reads were generated per sample. The fraction of V600E to wild type variant was estimated to range from 0.05% to 10% in the samples. Conclusions: Both qPCR and ultra-deep sequencing have identified the BRAF V600E mutation from microvesicles circulating in peripheral blood in patients with malignant melanoma. Ultra-deep sequencing further allows accurate estimation of the fraction of tumor-derived microvesicles which may be used as a surrogate marker for the actual tumor burden. ExoRNA from peripheral blood has the potential to act as a novel class of diagnostic tests for both mutation detection and therapy response monitoring without the need for invasive biopsy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C139.
Type of Medium:
Online Resource
ISSN:
1535-7163
,
1538-8514
DOI:
10.1158/1535-7163.TARG-11-C139
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2011
detail.hit.zdb_id:
2062135-8
SSG:
12
