Single-Cell Analysis of BRAFV600E and NRASQ61R Mutation Status in Melanoma Cell Lines as Method Generation for Circulating Melanoma Cells

Joseph W Po; Yafeng Ma; Alison W S Luk; David Lynch; Bavanthi Balakrishnar; Daniel Brungs; Farhad Azimi; Adam Cooper; Erin Saricilar; Vinay Murthy; Paul de Souza; Therese M Becker

doi:10.1007/978-1-0716-1205-7_21

Single-Cell Analysis of BRAF^V600E and NRAS^Q61R Mutation Status in Melanoma Cell Lines as Method Generation for Circulating Melanoma Cells

Methods Mol Biol. 2021:2265:277-286. doi: 10.1007/978-1-0716-1205-7_21.

Authors

Joseph W Po^{1

2

3}, Yafeng Ma^{4

5

6}, Alison W S Luk^{4

7}, David Lynch^{4

5

8}, Bavanthi Balakrishnar⁹, Daniel Brungs^{4

5

10}, Farhad Azimi⁹, Adam Cooper^{5

8

9}, Erin Saricilar^{8

6

9

11}, Vinay Murthy^{4

5

8

9}, Paul de Souza^{4

8

6

9

12}, Therese M Becker^{4

5

8

6}

Affiliations

¹ Centre for Circulating Tumour Cell Diagnostics & Research at the Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia. joseph.po@health.nsw.gov.au.
² Centre for Oncology Education and Research Translation (CONCERT), Liverpool, NSW, Australia. joseph.po@health.nsw.gov.au.
³ School of Medicine, Western Sydney University, Campbelltown, NSW, Australia. joseph.po@health.nsw.gov.au.
⁴ Centre for Circulating Tumour Cell Diagnostics & Research at the Ingham Institute for Applied Medical Research, Liverpool, NSW, Australia.
⁵ Centre for Oncology Education and Research Translation (CONCERT), Liverpool, NSW, Australia.
⁶ School of Medicine, University of New South Wales, Kensington, NSW, Australia.
⁷ Charles Perkins Centre, University of Sydney, Camperdown, NSW, Australia.
⁸ School of Medicine, Western Sydney University, Campbelltown, NSW, Australia.
⁹ Liverpool Hospital, Liverpool, NSW, Australia.
¹⁰ Illawarra Cancer Centre, Wollongong Hospital, Wollongong, NSW, Australia.
¹¹ University of Sydney, Camperdown, NSW, Australia.
¹² School of Medicine, University of Wollongong, Wollongong, NSW, Australia.

PMID: 33704722
DOI: 10.1007/978-1-0716-1205-7_21

Abstract

Molecular testing of tumor biopsies allows for the identification of the key mutations driving a patient's cancer. However, this is limited to singular nodes and may not accurately reflect cancer heterogeneity. Circulating tumor cell (CTC) analyses offer a noninvasive method of interrogating the genomic profile of patient-derived tumor material. To date, molecular analysis of CTCs has relied on the characterization of bulk or pooled CTC lysates, limiting the detection of minor tumorigenic CTC subclones. Here, we show a workflow enabling BRAF^V600E/NRAS^Q61R mutation detection from single cultured melanoma cells by combining micromanipulation and genomic material amplification methods. This workflow can be directly integrated into circulating tumor cell analysis applications.

Keywords: Cell lines; Circulating tumor cells (CTC); Liquid biopsy; Melanoma; Whole-genome amplification (WGA).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Substitution
Cell Line, Tumor
GTP Phosphohydrolases / genetics*
Humans
Melanoma / genetics*
Melanoma / pathology
Membrane Proteins / genetics*
Mutation, Missense*
Neoplastic Cells, Circulating*
Proto-Oncogene Proteins B-raf / genetics*
Single-Cell Analysis*

Substances

Membrane Proteins
BRAF protein, human
Proto-Oncogene Proteins B-raf
GTP Phosphohydrolases
NRAS protein, human