Detection of BRAF splicing variants in plasma-derived cell-free nucleic acids and extracellular vesicles of melanoma patients failing targeted therapy therapies
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Michael E. Clark1,2, Helen Rizos3,4,5, Michelle R. Pereira1, Ashleigh C. McEvoy1, Gabriela Marsavela1, Leslie Calapre1, Katie Meehan6, Olivia Ruhen2, Muhammad A. Khattak1,7,8, Tarek M. Meniawy1,7,9, Georgina V. Long5,10,11, Matteo S. Carlino4,5,10, Alexander M. Menzies5,10,11, Michael Millward1,7,9, Melanie Ziman1,2 and Elin S. Gray1
1 School of Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia, Australia
2 School of Biomedical Science, University of Western Australia, Crawley, Western Australia, Australia
3 Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, New South Wales, Australia
4 Westmead Institute for Cancer Research, The University of Sydney, Sydney, New South Wales, Australia
5 Melanoma Institute Australia, Sydney, New South Wales, Australia
6 Department of Otorhinolaryngology, Head and Neck Surgery, Chinese University of Hong Kong, Hong Kong
7 School of Medicine, The University of Western Australia, Crawley, Western Australia, Australia
8 Department of Medical Oncology, Fiona Stanley Hospital, Murdoch, Western Australia, Australia
9 Department of Medical Oncology, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
10 Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia
11 Mater Hospital, North Sydney, New South Wales, Australia
|Elin S. Gray,||email:||[email protected]|
Keywords: melanoma; targeted therapy; drug resistance;
Received: August 07, 2020 Accepted: October 10, 2020 Published: November 03, 2020
The analysis of plasma circulating tumour nucleic acids provides a non-invasive approach to assess disease burden and the genetic evolution of tumours in response to therapy. BRAF splicing variants are known to confer melanoma resistance to BRAF inhibitors. We developed a test to screen cell-free RNA (cfRNA) for the presence of BRAF splicing variants. Custom droplet digital PCR assays were designed for the detection of BRAF splicing variants p61, p55, p48 and p41 and then validated using RNA from cell lines carrying these variants. Evaluation of plasma from patients with reported objective response to BRAF/MEK inhibition followed by disease progression was revealed by increased circulating tumour DNA (ctDNA) in 24 of 38 cases at the time of relapse. Circulating BRAF splicing variants were detected in cfRNA from 3 of these 38 patients; two patients carried the BRAF p61 variant and one the p55 variant. In all three cases the presence of the splicing variant was apparent only at the time of progressive disease. BRAF p61 was also detectable in plasma of one of four patients with confirmed BRAF splicing variants in their progressing tumours. Isolation and analysis of RNA from extracellular vesicles (EV) from resistant cell lines and patient plasma demonstrated that BRAF splicing variants are associated with EVs. These findings indicate that in addition to plasma ctDNA, RNA carried by EVs can provide important tumour specific information.
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