Abstract 4966: High-definition single cell analysis (HD-SCA) reveals enrichment in androgen receptor (AR) expression in tumor cell clusters in bone marrow and blood of metastatic castration-resistant prostate cancer (mCRPC) patients
Authors: Zurita Amado J. , Carlsson Anders , Troncoso Patricia , Hicks James , Logothetis Christopher J. , Kuhn Peter 
Published: July 2016

Abstract
Introduction. The analysis of tumor cells in the circulation (CTC) provides an opportunity for non-invasive characterization of mCRPC and monitoring of therapy response, but it is the bone where prostate cancer cells almost consistently migrate to, expand and adapt to therapy. We adapted the HD-CTC assay to bone marrow aspirates (BMA) to discern features most significant to the tumorigenicity and the dynamics of metastatic progression in this disease, and to allow for serial comparison of tumor cells in the circulatory and bone compartments. Since available experimental data suggests that tumor cells organized in clusters are more important contributors to metastasis than single CTC, we evaluated their presence and characteristics in the 2 compartments.
Methods. Peripheral blood and BMA obtained through the posterior iliac crest were synchronously collected from individual mCRPC patients (n = 89) while progressing on therapy, and tumor cells in them were detected and characterized using the HD-SCA high-content platform. This flexible non-enrichment-based approach allows for quantification of cell morphometric and protein expression parameters and provides easy access to individual cells and cell clusters for single cell genomics and proteomics analysis. Candidate tumor cells based on cell size and morphology were presented and manually classified as DAPI and cytokeratin-positive and CD45-negative. Clusters were defined as two or more tumor cells in direct contact. AR protein expression and sub-cellular localization were also examined for each tumor cell and tumor cell cluster.
Results. Metastatic tumor cells were detected in 24/61 (39%) available BMAs. A greater number of tumor cells were organized in clusters, and clusters were generally larger, in BMAs than in blood (50% vs. 33%, and 20% vs. 5% with 6 or more cells, respectively). In 14 patient-matched and synchronously collected blood and BMA specimens with at least 1 tumor cell, we found 10 (71%) with clusters in the BMA (13-357 clusters/case, with the exception of 1 case that had 1 cluster), while only 3 (21%) had CTC clusters (2-4 clusters/case) (p = 0.02). The 4 cases that had no clusters in the marrow did not have any clusters in the blood. Patients with larger cluster size did significantly worse both in terms of progression-free (p = 0.002) and overall survival (p = 0.02). AR expression in individual cells was significantly higher in those forming part of cell clusters than in single cells, and was positively correlated with cluster size, both in blood and BMAs.
Conclusions. Tumor cell clusters are frequent in the circulation and especially in the bone marrow of mCRPC patients whose disease has progressed to therapy. In this setting, the increasing AR expression observed in tumor cell clusters is of probable biological relevance.
Citation Format: Amado J. Zurita, Anders Carlsson, Patricia Troncoso, James Hicks, Christopher J. Logothetis, Peter Kuhn. High-definition single cell analysis (HD-SCA) reveals enrichment in androgen receptor (AR) expression in tumor cell clusters in bone marrow and blood of metastatic castration-resistant prostate cancer (mCRPC) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4966.