title: Functional profiling of live melanoma samples using a novel automated platform.
abstract: OBJECTIVE
  This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform.
  
  
  METHODS
  A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma.
  
  
  RESULTS
  Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy.
  
  
  CONCLUSIONS
  Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients.
authors.affiliation: BioMarker Strategies, 855 N. Wolfe St., Baltimore, MD 21205, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Schayowitz
authors.forename: Greg
authors.initials: G
authors.lastname: Bertenshaw
authors.forename: Emiko
authors.initials: E
authors.lastname: Jeffries
authors.forename: Timothy
authors.initials: T
authors.lastname: Schatz
authors.forename: James
authors.initials: J
authors.lastname: Cotton
authors.forename: Jessie
authors.initials: J
authors.lastname: Villanueva
authors.forename: Meenhard
authors.initials: M
authors.lastname: Herlyn
authors.forename: Clemens
authors.initials: C
authors.lastname: Krepler
authors.forename: Adina
authors.initials: A
authors.lastname: Vultur
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.forename: Gordon H
authors.initials: GH
authors.lastname: Yu
authors.forename: Lynn
authors.initials: L
authors.lastname: Schuchter
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Clark
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D057205:Automation, Laboratory; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D005260:Female; D006801:Humans; D007211:Indoles / Q000493:pharmacokinetics / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008545:Melanoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D058990:Molecular Targeted Therapy; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors*; D013449:Sulfonamides / Q000493:pharmacokinetics / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D007211:Indoles; C528407:PLX 4720; D013449:Sulfonamides; D048493:Proto-Oncogene Proteins B-raf; D020928:Mitogen-Activated Protein Kinases