title: Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: School of Medical Sciences, Edith Cowan University, Perth, WA, Australia. Electronic address: a.reid@ecu.edu.au.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Reid
authors.affiliation: School of Medical Sciences, Edith Cowan University, Perth, WA, Australia. Electronic address: james.freeman@ecu.edu.au.
authors.forename: James B
authors.initials: JB
authors.lastname: Freeman
authors.affiliation: School of Medicine and Pharmacology, University of Western Australia, Crawley, WA, Australia. Electronic address: michael.millward@uwa.edu.au.
authors.forename: Michael
authors.initials: M
authors.lastname: Millward
authors.affiliation: School of Medical Sciences, Edith Cowan University, Perth, WA, Australia; School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, WA, Australia. Electronic address: m.ziman@ecu.edu.au.
authors.forename: Melanie
authors.initials: M
authors.lastname: Ziman
authors.affiliation: School of Medical Sciences, Edith Cowan University, Perth, WA, Australia. Electronic address: e.gray@ecu.edu.au.
authors.forename: Elin S
authors.initials: ES
authors.lastname: Gray
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D001667:Binding, Competitive; D014408:Biomarkers, Tumor / Q000097:blood* / Q000378:metabolism; D004273:DNA, Neoplasm / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D055106:Genome-Wide Association Study; D006784:Hospitals, Teaching; D006801:Humans; D057230:Limit of Detection; D008545:Melanoma / Q000097:blood* / Q000378:metabolism / Q000473:pathology / Q000556:secondary; D008832:Microchemistry / Q000379:methods; D050505:Mutant Proteins / Q000097:blood* / Q000378:metabolism; D059645:Mutation Rate; D009360:Neoplastic Cells, Circulating / Q000378:metabolism* / Q000473:pathology; D009693:Nucleic Acid Hybridization; D048493:Proto-Oncogene Proteins B-raf / Q000097:blood* / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012815:Signal Processing, Computer-Assisted; D014904:Western Australia
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D050505:Mutant Proteins; D012334:RNA, Neoplasm; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; Circulating tumour cells; Droplet digital PCR; Melanoma; WGA; castPCR