Development of a molecular profiling assay for circulating tumor cells (CTCs) utilizing automated multiplexed in situ hybridization for metastatic castrate-resistant prostate cancer (mCRPC).

Abstract

39 Background: ERG gene rearrangements lead to overexpression of the ERG oncogene in 50% of localized and metastatic prostate cancers. Recent studies have indicated that this gene fusion correlates with androgen sensitivity in prostate cancer patients and an association with increased DNA Repair activity (PARP1). The PTEN tumor suppressor has also been shown to regulate the AKT pathway, and related to increased PARP1 activity. We hypothesized that future PARP1 inhibitors and other targeted therapies for mCRPC will likely require the development of a companion diagnostic assay for measuring ERG rearrangement and PTEN deletion status in CTCs. Methods: We isolated CTCs utilizing the CellSearch system from mCRPC patients for enumeration. Following enumeration, the cells were transferred to glass slides and interrogated utilizing the automated Benchmark staining platform with multiplexed Quantum Dot FISH inclusive of probes for 5’ERG, 3’ERG, PTEN, and Cen 10. FISH signals were visualized utilizing spectral imaging. Results: Successful transfer of the CTCs from the CellSearch to a glass slide were achieved In model cell line systems, in cultured cells (LnCaP and VCaP) either directly or after spiking to PBL and five out of 6 times we were able to successfully demonstrate simultaneous single copy 5’ and 3’ ERG and PTEN/Cen 10 labeled probes. Studies are in progress to extend the clinical utility of this assay to isolated CTC from cases with metastatic prostate cancer. Conclusions: CTCs are likely to be utilized clinically for monitoring disease progression mCRPC patients as measures for therapeutic efficacy. As more therapeutics are approved for use in mCRPC patients, the ability for oncologists to molecularly characterize the type of CTCs driving tumor burden is likely to play a significant role in optimizing patient management by determining the type and the sequence of future targeted therapies. We have for the first time demonstrated a platform to molecularly characterize patient CTCs on an automated platform that is amenable to standard clinical use.