Functional Profiling of Live Melanoma Samples Using a Novel Automated Platform

Abstract

AimsThis 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.MethodsA 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.ResultsMelanoma 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.ConclusionPharmacodynamic 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.

Three-dimensional organotypic culture: experimental models of mammalian biology and disease

“…The success of this therapeutic approach will require a high degree of standardization and reproducibility across experiments. In addition, patient tissue in 3D culture could be analysed for its dynamic molecular responses to standardized cellular and molecular cues, such as targeted therapies 150 .…”

“…Similarly, primary glioblastoma specimens show variable cell death rates and DNA damage responses following treatment with the chemotherapeutic drug temozolomide after irradiation 157 . Primary samples can also be challenged with molecular therapeutic agents and assayed for downstream signalling responses 150 . Validation of the predictive value of such assays will require clinical trials that test whether assay-guided clinical management results in better patient outcomes than the standard of care 154,155 .…”

“…Prognostic assays could be developed to measure functionally based outputs, such as morphological criteria (for example, the extent of invasion into a defined microenvironment 30 ) (see the figure, part b ) or molecular criteria (for example, expression of components of the MAPK pathway 150 ). These assays can potentially provide information that is independent of that derived from static phenotypic analyses that have been conducted on formalin-fixed paraffin-embedded sections.…”

Precision medicine for cancer with next-generation functional diagnostics

“…Although such a test can thus be used to determine the response to a specific agent, this assay could also be used to recommend novel therapies among a panel of potential targeted therapies by identifying targets with high activity in patient samples. A different technique that used an ex vivo platform to measure phosphorylated (activated) ERK responses of live melanoma samples to BRAF inhibitors identified and predicted a patient with a BRAF mutation who showed no response to a BRAF inhibitor 72 .…”

Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

“…This is supported by additional findings demonstrating that RPMI-7981 cells treated with the closely related BRAF inhibitor PLX4032/vemurafenib (a successor of PLX4720) also remain refractory to inhibitor treatment as assessed by annexin V staining ( Paraiso et al, 2012 ) and MTS assay ( Park et al, 2013 ). However, others have reported RPMI-7981 cells as exhibiting modest sensitivity to PLX4720 ( Schayowitz et al, 2012 ). In the latter case, ERK activity was reduced by 50% after incubation with inhibitor, although these differences in sensitivity may reflect the significantly shorter time course and experimental design used by Park and colleagues.…”