Background: Epithelial Plasticity (EP) is a proposed mechanism of immune escape, resistance to programmed cell death, resistance to specific drugs, and promotion of metastasis. EP has been extensively explored in mathematical, cell biological, and animal models, which predict the presence of these tumor cells in circulation. Most studies on CTCs in human subjects are based on enrichment of cells expressing EpCAM prior to enumeration or molecular interrogation, which precludes analysis of cells that might be EP. The non-enrichment Epic Sciences platform was utilized to identify CTCs in metastatic castration-resistant prostate cancer patient samples phenotypically consistent with EP: CTCs that do not express cytokeratins (CK), but do express malignant or EP biomarkers: Androgen Receptor (AR), AR splice variant 7 isoform (AR-V7) or N-cadherin. Phenotypic and genotypic analyses of individual CTCs were performed to assess the malignant origin of CK- CTCs and their prevalence associated with overall patient survival.
Methods: 221 pre-therapy mCRPC blood samples were collected at Memorial Sloan-Kettering Cancer Center and shipped to Epic Sciences. Patients represent a diverse number of previous therapies and tumor burdens. All nucleated cells were plated to glass slides and subjected to DAPI and immunofluorescent (IF) staining of cytokeratin (CK), CD45 and another tumor marker (AR, AR-V7, or N-cadherin), followed by CTC identification by fluorescent scanners and malignant morphology. All samples were stained for AR, with a subset of samples stained for AR-V7 or N-Cadherin as well. Additionally, 83 AR(+)/CK(-) and 9 AR-V7(+)/CK(-) CTCs were analyzed for copy number variation (CNV) by low pass whole genome single CTC sequencing. Abnormal genomes were identified by amplifications and deletions of known oncogenes and tumor suppressors.
Results: 3931 AR(+) CTCs were detected, of which 2996 (76%) expressed CK and 935 (24%) did not. The AR(+)/CK(-) CTCs had a lower median AR signal, less nucleoli, and more nuclear speckles by digital pathology vs. CK(+) CTCs. AR(+)/CK(-) and AR-V7(+)/CK(-) CTCs had gross genomic alterations consistent with malignant prostate origin (AR gain, PTEN loss, RB1 loss, 8q gain, 8p loss, etc), often sharing the same genomic alterations with CK(+) CTCs in the same patients, with some having unique CNV profiles Presence of AR(+)/CK(-) CTCs was associated with worse overall survival (median 13.7 mo vs. 33.9 mo, p < 0.0001) and remained additive and independent to treatment Line, pre-therapy PSA, and therapy type in multivariate models.
Conclusions: Similar to CTCs expressing epithelial markers, CK(-) CTCs have gross genomic aberrations consistent with metastatic prostate cancer and are a negative prognostic factor associated with worse overall survival in both iterative threshold and multivariable analyses.
Citation Format: Ryon P. Graf, Yipeng Wang, Nicole Schreiber, Brigit McLaughlin, Stephanie Greene, Angel Rodriguez, Adam Jendrisak, Jerry Lee, Mark Landers, Ryan Dittamore, Howard I. Scher. Phenotypic, genomic, and clinical associations of Circulating Tumor Cells (CTCs) lacking epithelial biomarkers in metastatic Castration Resistant Prostate Cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1740. doi:10.1158/1538-7445.AM2017-1740