title: Co-existence of BRAF and NRAS driver mutations in the same melanoma cells results in heterogeneity of targeted therapy resistance.
abstract: Acquired chemotherapeutic resistance of cancer cells can result from a Darwinistic evolution process in which heterogeneity plays an important role. In order to understand the impact of genetic heterogeneity on acquired resistance and second line therapy selection in metastatic melanoma, we sequenced the exomes of 27 lesions which were collected from 3 metastatic melanoma patients treated with targeted or non-targeted inhibitors. Furthermore, we tested the impact of a second NRAS mutation in 7 BRAF inhibitor resistant early passage cell cultures on the selection of second line therapies.We observed a rapid monophyletic evolution of melanoma subpopulations in response to targeted therapy that was not observed in non-targeted therapy. We observed the acquisition of NRAS mutations in the BRAF mutated patient treated with a BRAF inhibitor in 1 of 5 of his post-resistant samples. In an additional cohort of 5 BRAF-inhibitor treated patients we detected 7 NRAS mutations in 18 post-resistant samples. No NRAS mutations were detected in pre-resistant samples. By sequencing 65 single cell clones we prove that NRAS mutations co-occur with BRAF mutations in single cells. The double mutated cells revealed a heterogeneous response to MEK, ERK, PI3K, AKT and multi RTK - inhibitors.We conclude that BRAF and NRAS co-mutations are not mutually exclusive. However, the sole finding of double mutated cells in a resistant tumor is not sufficient to determine follow-up therapy. In order to target the large pool of heterogeneous cells in a patient, we think combinational therapy targeting different pathways will be necessary.
authors: Department of Dermatology, University of Zurich, University Hospital Zürich, Switzerland.
authors: Marieke I G
authors: MI
authors: Raaijmakers
authors: Department of Dermatology, University of Zurich, University Hospital Zürich, Switzerland.
authors: Daniel S
authors: DS
authors: Widmer
authors: American Museum of Natural History, New York, New York, USA.
authors: Apurva
authors: A
authors: Narechania
authors: Department of Dermatology, University of Zurich, University Hospital Zürich, Switzerland.
authors: Ossia
authors: O
authors: Eichhoff
authors: Department of Dermatology, University of Zurich, University Hospital Zürich, Switzerland.|Department of Dermatology, Skin and Endothelium Research Division, Medical University of Vienna, Austria.
authors: Sandra N
authors: SN
authors: Freiberger
authors: Department of Dermatology, University of Zurich, University Hospital Zürich, Switzerland.|Department of Dermatology, Skin and Endothelium Research Division, Medical University of Vienna, Austria.
authors: Judith
authors: J
authors: Wenzina
authors: Department of Dermatology, University of Zurich, University Hospital Zürich, Switzerland.
authors: Phil F
authors: PF
authors: Cheng
authors: Department of Pathology, University of Zurich, University Hospital Zürich, Switzerland.
authors: Daniela
authors: D
authors: Mihic-Probst
authors: American Museum of Natural History, New York, New York, USA.
authors: Rob
authors: R
authors: Desalle
authors: Department of Dermatology, University of Zurich, University Hospital Zürich, Switzerland.
authors: Reinhard
authors: R
authors: Dummer
authors: Department of Dermatology, University of Zurich, University Hospital Zürich, Switzerland.
authors: Mitchell P
authors: MP
authors: Levesque
mesh_terms: D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D019143:Evolution, Molecular; D005260:Female; D020558:GTP Phosphohydrolases / Q000235:genetics*; D006801:Humans; D008297:Male; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D009362:Neoplasm Metastasis; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D059010:Single-Cell Analysis; D000073359:Exome Sequencing
chemical_list: D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human
keywords: MAPK pathway; heterogeneity; melanoma; mutation; targeted therapy resistance