Abstract 1471: Characterization of the molecular heterogeneity of circulating tumour cells in metastatic prostate cancer.
Authors: Oulhen Marianne , Massard Christophe , Valent Alexander , Young Rachel , Le Moulec Sylvestre , Fizazi Karim , Vielh Philippe , Farace Françoise 
Published: August 2014

Abstract
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 characterize the status of key molecular markers such as ERG gene rearrangement, and AR and c-myc amplifications, according to epithelial and/or mesenchymal marker expression in CTCs of patients with metastatic prostate cancer (PCa).
Patients and Methods: CTCs were isolated from the blood of 40 patients at day 0 and day 30 of abiraterone treatment. CTCs were detected using two methods, CellSearch® (Veridex LLC, Raritan, NJ, USA) and ISET (Isolation by Size of Epithelial Tumor cells). Molecular characterization of CTCs was performed on ISET filters by combining four color immunofluorescent staining and Filter Adapted-Fluorescent In situ Hybridization (FA-FISH®). To optimize pan-keratin detection, four pancytokeratin antibodies, including AE1/AE3, KL1, C11, and A45-B/B3, were tested on the LnCAP prostate cell line spiked in normal blood cells and on PCa CTCs enriched by ISET. Epithelial (cytokeratins, E-cadherin, EpCAM (Epithelial Cell Adhesion Molecule)), mesenchymal (vimentin, N-cadherin), PTEN, androgen receptor (AR) and Ki67 marker expression was detected by immunofluorescent staining on ISET filters according to a method we previously established. FA-FISH® was optimized for the detection of ERG gene rearrangement and AR and c-myc amplification or gain of copy number. A two step method combining immunofluorescent staining and FA-FISH® was established on an ARIOL automated scanner (LEICA) to precisely relocate FISH signals in phenotypically different CTC subsets.
Results: Higher CTC levels were detected by ISET compared to CellSearch®. Quantitative analysis of the intensity values for each tested pan-keratins antibodies showed that KL1 and A45-B/B3 clones gave the strongest immunofluorescent signals both on the LnCAP cell line and PCa CTCs. CTCs expressing epithelial, mesenchymal and hybrid (both epithelial and mesenchymal) markers were detected in most patients suggesting the existence of CTCs at different stages of the epithelial to mesenchymal transition (EMT) process. The loss of PTEN expression was also observed using hematopoietic cells present on ISET filters as a positive control of PTEN expression. ERG gene rearrangement, AR and c-myc amplifications were detected. The presence of these markers within CTC subsets expressing epithelial and/or mesenchymal markers is currently studied.
Conclusion: Several molecular markers such as ERG gene rearrangement, AR and c-myc amplifications were detected in PCa CTCs. The presence of these markers in CTCs according to their EMT status will be presented.
Citation Format: Marianne Oulhen, Christophe Massard, Alexander Valent, Rachel Young, Sylvestre Le Moulec, Karim Fizazi, Philippe Vielh, Françoise Farace. Characterization of the molecular heterogeneity of circulating tumour cells in metastatic prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1471. doi:10.1158/1538-7445.AM2013-1471