Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR

Document Type

Journal Article

Publisher

Elsevier

Faculty

Faculty of Health, Engineering and Science

School

School of Medical Sciences

RAS ID

18946

Funders

National Health and Medical Research Council

Grant Number

NHMRC Number : 1013349

Comments

Reid, A. L., Freeman, J. B., Millward, M., Ziman, M., & Gray, E. S. (2015). Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR. Clinical biochemistry, 48(15), 999-1002. Available here

Abstract

Objectives: Defining the BRAF mutation status in metastatic melanoma patients is critical to selecting patients for therapeutic treatment with targeted therapies. Circulating tumour cells (CTCs) can provide an alternative source of contemporaneous tumour genetic material. However methodologies to analyse the presence of rare mutations in a background of wild-type DNA requires a detailed assessment. Here we evaluate the sensitivity of two technologies for cancer mutation detection and the suitability of whole genome amplified DNA as a template for the detection of BRAF-V600 mutations. Design and methods: Serial dilutions of mutant BRAF-V600E DNA in wild-type DNA were tested using both competitive allele-specific PCR (castPCR) and droplet digital PCR (ddPCR), with and without previous whole genome amplification (WGA). Using immunomagnetic beads, we partially enriched CTCs from blood obtained from metastatic melanoma patients with confirmed BRAF mutation positive tumours and extracted RNA and DNA from the CTCs. We used RT-PCR of RNA to confirm the presence of melanoma cells in the CTC fraction then the DNAs of CTC positive fractions were WGA and tested for BRAF V600E or V600K mutations by ddPCRs. Results: WGA DNA produced lower than expected fractional abundances by castPCR analysis but not by ddPCR. Moreover, ddPCR was found to be 200 times more sensitive than castPCR and in combination with WGA produced the most concordant results, with a limit of detection of 0.0005%. BRAF-V600E or V600K mutated DNA was detected in 77% and 44%, respectively, of enriched CTC fractions from metastatic melanoma patients carrying the corresponding mutations. Conclusions: Our results demonstrate that using ddPCR in combination with WGA DNA allows the detection with high sensitivity of cancer mutations in partially enriched CTC fractions.

DOI

10.1016/j.clinbiochem.2014.12.007

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