Abstract 1598: Isolation and characterization of circulating melanoma cells by size filtration and fluorescent in situ hybridization

Abstract

Abstract Somatic mutations in BRAF are predictive of the clinical efficacy of BRAF and/or MEK inhibitors for advanced melanoma. Resistance to treatment is observed for most patients despite a high response rate to kinase inhibitors. While repeated tumor biopsy is the gold standard for evaluating genetic changes following treatment, this is not generally feasible. The analysis of circulating melanoma cells (CMCs) has the potential to be a non-invasive means of interrogating the biology underlying response and resistance. Here we report on the isolation, detection, and characterization of CMCs from patients using size filtration followed by immunoflourescence and/or fluorescent in situ hybridization (FISH) analyses. Blood from a cohort of 20 patients with advanced melanoma was collected in an initial proof of concept experiment. CMCs were identified in 14 out of 20 melanoma blood samples (70%) with a range of 2.5-99 CMCs per 3mL of blood by immunofluorescence (IF) using a cocktail of 4 melanoma specific antibodies (HMB45, MART-1, Tyrosinase and MiTF). Cells that appeared tumor like by microscopy, but that did not stain for CD45 or the cocktail of melanoma specific antibodies were also detected. To further characterize these cells, FISH was performed with a set of 4 melanoma-specific probes (RREB1, MYB, Centromere 6, CCND1). In a cohort of 13 patients with melanoma specimens that had paired blood specimens, CMCs were detected in all 13 specimens ranging from 7.2-76 CMCs per 3mL of blood with more identification by FISH in 10 out 13 samples. Additionally, cells that were double negative by IF were observed to be positive by FISH. CMCs are detectable in the majority of patients with advanced melanoma. Use of IF and FISH can help detect and characterize CMCs. These tools will be useful in characterizing treatment related changes in CMCs. Citation Format: Masahiko Yanagita, Jason J. Luke, Cloud P. Paweletz, Elena Ivanova, Paul T. Kirschmeier, Stephen F. Hodi, Pasi A. Jänne. Isolation and characterization of circulating melanoma cells by size filtration and fluorescent in situ hybridization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1598. doi:10.1158/1538-7445.AM2015-1598