Detection of BRAF splicing variants in plasma-derived cell-free nucleic acids and extracellular vesicles of melanoma patients failing targeted therapy therapies

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. splicing variants are known to confer melanoma resistance to BRAF inhibitors. We developed a test to screen cell-free RNA (cf...

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Published in:Oncotarget 2020-11, Vol.11 (44), p.4016-4027
Main Authors: Clark, Michael E, Rizos, Helen, Pereira, Michelle R, McEvoy, Ashleigh C, Marsavela, Gabriela, Calapre, Leslie, Meehan, Katie, Ruhen, Olivia, Khattak, Muhammad A, Meniawy, Tarek M, Long, Georgina V, Carlino, Matteo S, Menzies, Alexander M, Millward, Michael, Ziman, Melanie, Gray, Elin S
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Language:English
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Research Paper
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Summary: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. 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 splicing variants. Custom droplet digital PCR assays were designed for the detection of 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 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. p61 was also detectable in plasma of one of four patients with confirmed splicing variants in their progressing tumours. Isolation and analysis of RNA from extracellular vesicles (EV) from resistant cell lines and patient plasma demonstrated that 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.
ISSN:1949-2553
1949-2553
DOI:10.18632/oncotarget.27790