Genomic analysis of circulating tumor cells (CTC) from patients with castration-resistant prostate cancer (CRPC) as predictive biomarkers.

120 Background: Identification, enumeration, and genomic analysis of circulating tumor cells (CTCs) may allow for a better understanding of the mechanisms of resistance to therapies in metastatic castration-resistant prostate cancer (mCRPC). The Vitatex VitaAssay platform captures invasive CTCs (iCTCs) in a cell surface marker-independent fashion based on their ability to invade a fluorescently-labeled cell-adhesion matrix (CAM), allowing for the analysis of multiple CTC subpopulations. Here we sought to estimate epithelial, mesenchymal, and stem-like iCTC subpopulation diversity in men with CRPC starting abiraterone acetate therapy, to compare the genomic profiles of iCTCs to matched metastatic biopsies, and to explore the potential for 2D and 3D CTC culture. Methods: iCTCs were isolated from men with mCRPC using the CAM platform, and paired metastatic biopsies were performed. iCTCs were defined as CAM+/CD45-/CD14-/DAPI+, mesenchymal iCTCs as vimentin+/CAM+/CD45-/CD14-/DAPI+, and stem-like iCTCs as CD44+/CAM+/CD45-/CD14-/DAPI+. iCTCs were enumerated and purified using FACS. Agilent array comparative genomic hybridization (aCGH) of iCTCs and paired biopsies was performed, and to explore the potential for ex-vivo cell expansion and spheroid formation, iCTCs were cultured separately in CAM and in matrigel for up to 10 days. Results: iCTCs were isolated using the CAM platform from 29 men, of whom seven have undergone paired metastatic biopsy. The median pre-FACS purity was 1.06% (range 0.11%-10.16%). Post-FACS purity was increased to greater than 90%, and a median of 60 (range 2 to 1,314) iCTCs/7.5ml were detected by FACS. Both vimentin+ and CD44+ iCTCs are detectable, and compromise between 10 to 50% of total iCTCs. iCTC aCGH profiles resemble paired soft tissue biopsy, in vitro iCTCs culture is feasible, and iCTC spheroids were observed. Conclusions: Multiple CRPC iCTC subpopulations are identifiable from men starting abiraterone therapy, and cell sorting techniques increase iCTC purity. iCTCs resemble metastatic CRPC tissue and can be expanded in culture. Further enumeration, genomic profiling, and clinical correlation of paired iCTCs taken from men with abiraterone-resistant CRPC is underway, and may shed light on mechanisms of abiraterone resistance.

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Genomic analysis of circulating tumor cells (CTCs) from men with metastatic castration resistant prostate cancer (mCRPC) in the context of enzalutamide therapy.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.4_suppl.65 ◽

2014 ◽

Vol 32 (4_suppl) ◽

pp. 65-65

Author(s):

Andrew J. Armstrong ◽

Jing Li ◽

Joshua Beaver ◽

Rhonda Lynn Bitting ◽

Simon Gregory

Keyword(s):

Prostate Cancer ◽

Exome Sequencing ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Copy Number ◽

Predictive Biomarkers ◽

Castration Resistant Prostate Cancer ◽

Dna Copy Number ◽

Pten Loss ◽

Castration Resistant

65 Background: Given the evolving treatments available in metastatic castration resistant prostate cancer (mCRPC), predictive biomarkers are desirable that maximize benefit and minimize harms and costs.The goal of this study was to determine the feasibility of DNA copy number and whole exome sequencing (WES) analysis of circulating tumor cells (CTCs) from men with mCRPC receiving enzalutamide. Methods: We collected CTCs from men with mCRPC in the context of enzalutamide therapy. CTCs were isolated from EDTA blood through red cell lysis, CD45 depletion, and flow sorting on EpCAM/CD45 expression. Whole genomic amplification and array based comparative genomic hybridization (CGH) was performed using Qiagen Repli-Gene Single Cell kit, multiple displacement amplification, and Agilent microarray analysis. CTC copy number changes were compared with patient leukocyte DNA and reference metastatic PC datasets. CTC AR amplification and PTEN loss was confirmed with FISH. WES on REPLI-g amplified CTC and leukocyte DNA was performed using GeneWiz and TruSeq Exome Capture Kit, and sequenced with Illumina HiSeq 2000 (20x). Results: A novel method for CTC array CGH was developed that reproducibly identified genomic lesions previously reported in metastatic CRPC including: AR amplification or focal deletions, deletions of CHD1, Rb, PHLLP, FGFR2, FOXA1, and NCOA2, and amplifications of EZH2 and MYC. AR amplification was noted in a man with mCRPC who subsequently responded to enzalutamide, with loss of AR amplification and gain of MYCN and c-MET amplification noted at progression. CGH analysis was feasible down to 10 to 20 cells using spiked cell lines. Interpatient tumor specific genomic heterogeneity was observed. FISH confirmed AR changes and PTEN loss heterogeneity. WES demonstrated acquired PTEN, MAGI1, SMAD4, and RB1 mutations in a patient who progressed through enzalutamide therapy, in addition to AR region deletion detected by CGH. Conclusions: Whole genome DNA copy number and exome sequencing analysis from CTCs in men with mCRPC is feasible in men with high CTCs and identified previously validated and novel genomic lesions and suggest the potential to identify predictive biomarkers of enzalutamide efficacy and resistance in the clinic.

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