SUMMARY Toward development of a precision medicine framework for metastatic, castration resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53 and PTEN were frequent (40–60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified novel genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin and ZBTB16/PLZF. Aberrations of BRCA2, BRCA1 and ATM were observed at substantially higher frequencies (19.3% overall) than seen in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides evidence that clinical sequencing in mCRPC is feasible and could impact treatment decisions in significant numbers of affected individuals.

An increasingly recognized resistance mechanism to androgen receptor (AR)-directed therapy in prostate cancer involves epithelial plasticity, wherein tumor cells demonstrate low to absent AR expression and often neuroendocrine features. The etiology and molecular basis for these “alternative” treatment-resistant cell states remain incompletely understood. Here, by analyzing whole exome sequencing data of metastatic biopsies from patients, we observed significant genomic overlap between castration resistant adenocarcinoma (CRPC-Adeno) and neuroendocrine histologies (CRPC-NE); analysis of serial progression samples points to a model most consistent with divergent clonal evolution. Genome-wide DNA methylation revealed marked epigenetic differences between CRPC-NE and CRPC-Adeno that also designated cases of CRPC-Adeno with clinical features of AR-independence as CRPC-NE, suggesting that epigenetic modifiers may play a role in the induction and/or maintenance of this treatment-resistant state. This study supports the emergence of an alternative, “AR-indifferent” cell state through divergent clonal evolution as a mechanism of treatment resistance in advanced prostate cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer that most commonly evolves from preexisting prostate adenocarcinoma (PCA). Using Next Generation RNA-sequencing and oligonucleotide arrays, we profiled 7 NEPC, 30 PCA, and 5 benign prostate tissue (BEN), and validated findings on tumors from a large cohort of patients (37 NEPC, 169 PCA, 22 BEN) using IHC and FISH. We discovered significant overexpression and gene amplification of AURKA and MYCN in 40% of NEPC and 5% of PCA, respectively, and evidence that that they cooperate to induce a neuroendocrine phenotype in prostate cells. There was dramatic and enhanced sensitivity of NEPC (and MYCN overexpressing PCA) to Aurora kinase inhibitor therapy both in vitro and in vivo, with complete suppression of neuroendocrine marker expression following treatment. We propose that alterations in Aurora kinase A and N-myc are involved in the development of NEPC, and future clinical trials will help determine from the efficacy of Aurora kinase inhibitor therapy.

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Geometrically-enhanced differential immunocapture (GEDI) and an antibody for prostate-specific membrane antigen (PSMA) are used for high-efficiency and high-purity capture of prostate circulating tumor cells from peripheral whole blood samples of castrate-resistant prostate cancer patients.Prostate circulating tumor cells (PCTCs) are often found in the blood of patients suffering from metastatic prostate cancer 1, 2. While these PCTCs are rare, as few as one cell per 10 9 hematologic cells in blood 3,4 , they are theorized to contribute to metastatic progression 3,5 . Currently, the enumeration of PCTCs is used clinically as a prognostic indicator of patient survival 2,6,7 . Capture of peripheral blood PCTCs may enable early clinical assessment of metastatic process and chemotherapeutic response, as well as genetic and pharmacological evaluation of cancer cells.Current approaches to isolate circulating tumor cells are complex and produce low yields and purity 5 . Existing commercial and research devices for the immunocapture of rare cancer cells use EpCAM antibodies 2,8,9 , which capture many circulating endothelial cells and large numbers of leukocytes. As a result, purity of captured cells is widely variable and often below 50%. In addition, while previous devices use 3D antibody-coated surfaces for immunocapture 8,9 , these devices are not designed to induce a size-dependent collision frequency. Devices focused on size-dependent particle transport are typically focused on sorting 10 , separation 11,12 , or filtration 13 .In this communication, we demonstrate high-efficient and high-purity capture of PCTCs from peripheral blood samples of castrate-resistant prostate cancer patients using an antibody for prostate-specific membrane antigen (PSMA), a highly prostate-specific cellsurface antigen 14 . In addition, we describe a theoretical framework for the use of staggered § To whom correspondence should be addressed: The GEDI µdevice geometry was designed to maximize streamline distortion and thus bring desired cells in contact with the immunocoated obstacle walls for capture. Blood is a dense heterogeneous cell suspension consisting of cells of various sizes ranging from approximately 4 to 18 µm in size16. PCTCs, in contrast, are larger and range from 15 to 25 µm in diameter 16 . Relative obstacle alignment was chosen so that the displacement caused by cell impact with obstacles (which ranges from zero to one cell radius) increases the likelihood of future cell impacts for large cells more than for small cells. Thus when cellobstacle impact does not lead to capture, larger cells are displaced onto streamlines that impinge onto the next obstacle, while smaller cells are displaced onto streamlines that do not impinge ( Figure 1A). Cell advection was modeled in silico (computational details in supplementary information) to determine obstacle array geometries that optimize PCTCwall interactions and minimize wall shear forces to maximize PCTC capture. For a given obstacle geometry, the frequency of cell-wall...

Prostate cancer progression requires active androgen receptor (AR) signaling which occurs following translocation of AR from the cytoplasm to the nucleus. Chemotherapy with taxanes improves survival in patients with castrate resistant prostate cancer (CRPC). Taxanes induce microtubule stabilization, mitotic arrest, and apoptotic cell death, but recent data suggest that taxanes can also affect AR signaling. Here, we report that taxanes inhibit ligand-induced AR nuclear translocation and downstream transcriptional activation of AR target genes such as prostate-specific antigen. AR nuclear translocation was not inhibited in cells with acquired b-tubulin mutations that prevent taxane-induced microtubule stabilization, confirming a role for microtubules in AR trafficking. Upon ligand activation, AR associated with the minus-end-microtubule motor dynein, thereby trafficking on microtubules to translocate to the nucleus. Analysis of circulating tumor cells (CTC) isolated from the peripheral blood of CRPC patients receiving taxane chemotherapy revealed a significant correlation between AR cytoplasmic sequestration and clinical response to therapy. These results indicate that taxanes act in CRPC patients at least in part by inhibiting AR nuclear transport and signaling. Further, they suggest that monitoring AR subcellular localization in the CTCs of CRPC patients might predict clinical responses to taxane chemotherapy. Cancer Res; 71(18); 6019-29. Ó2011 AACR.