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

Michael E. Clark, Helen Rizos, Michelle R. Pereira, Ashleigh C. McEvoy, Gabriela Marsavela, Leslie Calapre, Katie Meehan, Olivia Ruhen, Muhammad A. Khattak, Tarek M. Meniawy, Georgina V. Long, Matteo S. Carlino, Alexander M. Menzies, Michael Millward, Melanie Ziman, Elin S. Gray*

*Corresponding author for this work

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
Pages (from-to)4016-4027
Number of pages12
JournalOncotarget
Volume11
Issue number44
DOIs
Publication statusPublished - 3 Nov 2020

Bibliographical note

Copyright the Author(s) 2020. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Keywords

  • BRAF splicing
  • Drug resistance
  • Extracellular vesicles
  • Melanoma
  • Targeted therapy

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