Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma
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Ashleigh C. McEvoy1, Leslie Calapre1, Michelle R. Pereira1, Tindaro Giardina2, Cleo Robinson2,3,4, Muhammad A. Khattak3,5, Tarek M. Meniawy3,6, Antonia L. Pritchard7, Nicholas K. Hayward7, Benhur Amanuel2,3, Michael Millward3,6, Melanie Ziman1,4 and Elin S. Gray1
1School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia, Australia
2Anatomical Pathology, PathWest, QEII Medical Centre, Nedlands, Western Australia, Australia
3School of Medicine and Pharmacology, The University of Western Australia, Crawley, Western Australia, Australia
4School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia, Australia
5Department of Medical Oncology, Fiona Stanley Hospital, Murdoch, Western Australia, Australia
6Department of Medical Oncology, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
7QIMR Berghofer Medical Research Institute, Herston, Brisbane, QLD, Australia
Elin S. Gray, email: [email protected]
Keywords: droplet digital PCR (ddPCR), TERT, melanoma, cancer
Received: June 06, 2017 Accepted: July 25, 2017 Published: August 18, 2017
Background: Currently mainly BRAF mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. TERT promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of BRAF wild-type cases. Therefore, a sensitive method for detection of TERT promoter mutations would increase the number of patients that could be monitored through ctDNA analysis.
Methods: A droplet digital PCR (ddPCR) assay was designed for the concurrent detection of chr5:1,295,228 C>T and chr5:1,295,250 C>T TERT promoter mutations. The assay was validated using 39 melanoma cell lines and 22 matched plasma and tumor samples. In addition, plasma samples from 56 metastatic melanoma patients and 56 healthy controls were tested for TERT promoter mutations.
Results: The established ddPCR assay detected TERT promoter mutations with a lower limit of detection (LOD) of 0.17%. Total concordance was demonstrated between ddPCR and Sanger sequencing in all cell lines except one, which carried a second mutation within the probe binding-site. Concordance between matched plasma and tumor tissue was 68% (15/22), with a sensitivity of 53% (95% CI, 27%-79%) and a specificity of 100% (95% CI, 59%-100%). A significantly longer PFS (p=0.028) was evident in ctDNA negative patients. Importantly, our TERT promoter mutations ddPCR assay allowed detection of ctDNA in 11 BRAF wild-type cases.
Conclusions: The TERT promoter mutation ddPCR assay offers a sensitive test for molecular analysis of melanoma tumors and ctDNA, with the potential to be applied to other cancers.
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