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SPOP mutation identified in this cohort.

Purpose: CHD1 deletions and SPOP mutations frequently cooccur in prostate cancer with lower frequencies reported in castration-resistant prostate cancer (CRPC). We monitored CHD1 expression during disease progression and assessed the molecular and clinical characteristics of CHD1-deleted/SPOP-mutated metastatic CRPC (mCRPC).Experimental Design: We identified 89 patients with mCRPC who had hormone-naive and castration-resistant tumor samples available: These were analyzed for CHD1, PTEN, and ERG expression by IHC. SPOP status was determined by targeted next-generation sequencing (NGS). We studied the correlations between these biomarkers and (i) overall survival from diagnosis; (ii) overall survival from CRPC; (iii) duration of abiraterone treatment; and (iv) response to abiraterone. Relationship with outcome was analyzed using Cox regression and log-rank analyses.Results: CHD1 protein loss was detected in 11 (15%) and 13 (17%) of hormone-sensitive prostate cancer (HSPC) and CRPC biopsies, respectively. Comparison of CHD1 expression was feasible in 56 matched, same patient HSPC and CRPC biopsies. CHD1 protein status in HSPC and CRPC correlated in 55 of 56 cases (98%). We identified 22 patients with somatic SPOP mutations, with six of these mutations not reported previously in prostate cancer. SPOP mutations and/or CHD1 loss was associated with a higher response rate to abiraterone (SPOP: OR, 14.50 P = 0.001; CHD1: OR, 7.30, P = 0.08) and a longer time on abiraterone (SPOP: HR, 0.37, P = 0.002, CHD1: HR, 0.50, P = 0.06).Conclusions: SPOP-mutated mCRPCs are strongly enriched for CHD1 loss. These tumors appear highly sensitive to abiraterone treatment. Clin Cancer Res; 24(22); 5585–93. ©2018 AACR.

C4-2 and LAPC4 cells were subjected to treatment protocol (Fig.1A); the canonical AR target genes including KLK3 and NKX3.1 were assessed by real-time PCR analysis. Data from three independent

Supplementary figure and table legends

Epithelial-to-mesenchymal plasticity (EMP) has been linked to metastasis, stemness, and drug resistance. In prostate cancer, EMP has been associated with both suppression and activation of the androgen receptor (AR) signaling. Here we investigated the effect of the potent AR antagonist enzalutamide on EMP in multiple preclinical models of prostate cancer and patient tissues. Enzalutamide treatment significantly enhanced the expression of EMP drivers (ZEB1, ZEB2, Snail, Twist, and FOXC2) and mesenchymal markers (N-cadherin, fibronectin, and vimentin) in prostate cancer cells, enhanced prostate cancer cell migration, and induced prostate cancer transformation to a spindle, fibroblast-like morphology. Enzalutamide-induced EMP required concomitant suppression of AR signaling and activation of the EMP-promoting transcription factor Snail, as evidenced by both knockdown and overexpression studies. Supporting these findings, AR signaling and Snail expression were inversely correlated in C4-2 xenografts, patient-derived castration-resistant metastases, and clinical samples. For the first time, we elucidate a mechanism explaining the inverse relationship between AR and Snail. Specifically, we found that AR directly repressed SNAI1 gene expression by binding to specific AR-responsive elements within the SNAI1 promoter. Collectively, our findings demonstrate that de-repression of Snail and induction of EMP is an adaptive response to enzalutamide with implications for therapy resistance. Cancer Res; 77(11); 3101–12. ©2017 AACR.

PC3 and PC-3(AR)2 cells were subjected to the treatment protocol (Fig.1A). After 6 days of treatment, the monolayer surface was scratched carefully and washed. Wound healing at 24 hours under normal growth media conditions was quantitated. Data are expressed as percentage of wound healing (WH %) calculated by dividing migrated distance by scratched distance.

Additional Information on molecular analyses determining CHD1 and SPOP status

10 million C4-2 cells were injected subcutaneously into two locations into 8 nude mice. 4 mice (8 tumors) were subject to surgical castration, and the other 4 mice were left intact. Six days after surgery, the mice were sacrificed and the tumors were subjected to H&E (B) and IHC evaluation for E-Cadherin and Fibronectin (C).

Sample overview

Primers sequence information for qPCR and ChIP-qPCR

A) Validation of consititutive ARv567es binding to FASN and TSC2 AR binding sites in R1-D567 cells by ChIP-PCR; Examination of ARv567es binding events at putative AR binding sites within SNAI1 promoter region in R1-D567 by ChIP-PCR. Data from three independent experiments mean{plus minus}SD; *, p

C4-2 cells were subjected to the treatment protocol (Fig.1A). The mesenchymal marker Zeb1 expression was evaluated by Western Blotting

Correlation between EMP markers and AE signaling in MSKCC and TCGA datasets

Combined SPOP/ CHD1 status and time/ response to abiraterone.

Liquid biopsy components from blood, such as cell free DNA (cfDNA) and circulating tumor cells (CTCs), are prognostic for overall survival in advanced prostate cancer patients and allow the study of clonal evolution. cfDNA is easily obtained and has been widely used for molecular characterization and reflects pooled genomic profiles in a patient, but has limitations regarding gene copy number calls. CTC single-cell genomic studies generate precise gene copy number calls and elucidate intrapatient intercellular genomic heterogeneity. The main limitation of CTC analyses has been the low CTC count found in many cancer patients. We elected to study whether liquid biopsy by apheresis in advanced prostate cancer patients increases the yield of CTC to study tumor genomics, intrapatient heterogeneity, and ex vivo organotypic 3D models. Advanced metastatic prostate cancer patients being considered for clinical trials were invited to consent to apheresis. Apheresis CTC counts using CellSearchTM (Menarini) were acquired from 16 patients. The contents of the CellSearch cartridges were sorted into pure single cells by fluorescence-activated cell sorting and subsequently assessed by array comparative genomic hybridization (aCGH, Agilent Technology) for copy number aberrations (CNA). Exome and aCGH from tissue biopsies were compared to the single cell aCGH results. We generated patient-derived organoid (PDOs) cultures from apheresis products by preenrichment using density gradient (Lymphoprep) and subsequent CTC enrichment by EpCAM positive selection (EasySep StemCell Technologies). PDOs were characterized by immunofluorescence (IF) as DAPI+/CK+/EpCAM+ and CD45- cells and subsequently by aCGH for CNA. All sixteen patients (median age of 70 years; range 60-77 years) tolerated apheresis without any adverse effects. CTC counts from peripheral blood (PB) prior to apheresis ranged from 13 to 711 (median = 96), and did not significantly change post apheresis. The estimated CTC yield per apheresis ranged from 660-35473 per apheresis product (median = 3351). This constitutes an increase of 102-fold when compared to median CTC capture from 7.5mL of PB. A total of 170 single CTCs from 15 apheresis patients were genomically profiled and the copy number aberration profiles confirmed prostate cancer with multiple genomic hallmarks including CNAs such as AR amplification, chromosome 8q gain (MYC locus), and PTEN, RB1, BRCA2, TP53, CHD1 loss. CNA profiles of PDOs showed similar genomic aberrations to same patient CTCs and also reflected intrapatient heterogeneity detected by single CTC analysis. In conclusion, apheresis from advanced prostate cancer patients is a well-tolerated procedure and in our study increased the CTC yield by 102-fold when compared to PB. CTC and PDOs from apheresis products shared similar CNA profile compared with tissues biopsies and furthermore gave us an insight of the tumor heterogeneity and clonal evolution. Citation Format: Maryou B.K. Lambros, Veronica Gil, Mateus Crespo, Mariane S. Fontes, Alan Mackay, Gemma Folwer, Gemma Folwer, Berni Ebbs, Rui Neves, Penny Flohr, Susana Miranda, Semini Sumanasuriya, Daniel N. Rodrigues, Rita Pereira, Geroge Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Niven Mehra, Jane Goodall, Claudia Bertan, Suzanne Carreira, Nikolas H. Stoecklein, Leon W.M.M. Terstappen, Gunther Boysen, Joahnn S. De Bono. Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A051.

To improve early diagnosis of prostate cancer to aid clinical decision-making. Diffusion-weighted magnetic resonance imaging (DW-MRI) is sensitive to water diffusion throughout tissues, which correlates with Gleason score, a histological measure of prostate cancer aggressiveness. In this study the ability of DW-MRI to detect prostate cancer onset and development was evaluated in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice.T2 -weighted and DW-MRI were acquired using a 7T MR scanner, 200 mm bore diameter; 10 TRAMP and 6 C57BL/6 control mice were scanned every 4 weeks from 8 weeks of age until sacrifice at 28-30 weeks. After sacrifice, the genitourinary tract was excised and sectioned for histological analysis. Histology slides registered with DW-MR images allowed for validation of DW-MR images and the apparent diffusion coefficient (ADC) as tools for cancer detection and disease stratification. An automated early assessment tool based on ADC threshold values was developed to aid cancer detection and progression monitoring.The ADC differentiated between control prostate ((1.86 ± 0.20) × 10(-3) mm(2) /s) and normal TRAMP prostate ((1.38 ± 0.10) × 10(-3) mm(2) /s) (P = 0.0001), between TRAMP prostate and well-differentiated cancer ((0.93 ± 0.18) × 10(-3) mm(2) /s) (P = 0.0006), and between well-differentiated cancer and poorly differentiated cancer ((0.63 ± 0.06) × 10(-3) mm(2) /s) (P = 0.02).DW-MRI is a tool for early detection of cancer, and discrimination between cancer stages in the TRAMP model. The incorporation of DW-MRI-based prostate cancer stratification and monitoring could increase the accuracy of preclinical trials using TRAMP mice.