Abstract 3079: Characterization of the molecular heterogeneity of circulating tumor cells in metastatic prostate cancer
Authors: Massard Christophe , Oulhen Marianne , Valent Alexander , Lemoulec Sylvestre , Fizazi Karim , Vielh Philippe , Farace Françoise 
Published: May 2015

Purpose: CTCs represent an alternative source of tumor material potentially usable as a surrogate tissue marker in the context of targeted treatments. The objective of the present study was to analyze the status of key molecular markers such as ERG gene rearrangement and AR amplification in tumor tissues (primitive tumor specimens and metastasis biopsies) and CTCs of patients with metastatic prostate cancer (PCa).

Patients and Methods: CTCs were detected using two methods, CellSearch (Veridex LLC, Raritan, NJ, USA) and filtration (ISET, isolation by Size of Epithelial Tumor cells) combined to four-color immunofluorescent staining. Epithelial (cytokeratins, EpCAM (Epithelial Cell Adhesion Molecule)) and mesenchymal (vimentin, N-cadherin) markers were tested. Molecular characterization of CTCs was performed on filters by a two step method combining four color immunofluorescent staining and Filter Adapted-Fluorescent In situ Hybridization (FA-FISH). Image analysis was performed using an ARIOL automated scanner (LEICA) to precisely relocate FISH signals in phenotypically different CTC subsets. ERG gene rearrangement and AR copy number gains were examined both in CTCs enriched on filters and in the cell fractions recovered from the magnests after CellSearch and spread on slides for FISH. Primitive tumor specimens and metastasis biopsies were also collected and analyzed by FISH.

Results: Higher levels of CTCs were detected by filtration enrichment combined to four-color immunofluorescent staining compared to CellSearch. Filtration enriched CTCs expressed epithelial, mesenchymal and hybrid (both epithelial and mesenchymal) markers suggesting the existence of CTCs at different stages of the epithelial to mesenchymal transition (EMT) process in most PCa patients. A higher level of gene variability and tumor heterogeneity was observed in filtration enriched CTCs compared to those captured by the CellSearch. ERG gene rearrangement and AR amplification in prostate tissue, metastases, and filtration enriched CTCs were analysed by FISH. ERG gene rearrangement and AR copy number gain status present within the tumor tissue was in concordance with that found in filtration enriched CTCs. However, FISH experiments also revealed a much higher degree of cell-to-cell variability and tumor heterogeneity in filtration enriched CTCs compared to tumor tissues.

Conclusion: We show here that prostate CTCs are highly heterogeneous in term of EMT markers and ERG gene rearrangement and AR amplification marker status. The high degree of cell-to-cell variability in ERG and AR gene patterns present in CTCs reveals important tumor heterogeneity and could have important implications for developing personalized anticancer strategies based on the use of CTCs in PCa patients.

Citation Format: Christophe Massard, Marianne Oulhen, Alexander Valent, Sylvestre Lemoulec, Karim Fizazi, Philippe Vielh, Françoise Farace. Characterization of the molecular heterogeneity of circulating tumor cells in metastatic prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3079. doi:10.1158/1538-7445.AM2014-3079