Tumor Cells In Prostate Cancer Research Articles

Re: The Initial Detection and Partial Characterization of Circulating Tumor Cells in Neuroendocrine Prostate Cancer

Re: The Initial Detection and Partial Characterization of Circulating Tumor Cells in Neuroendocrine Prostate Cancer

Abstract 4955: Isolation and characterization of circulating tumor cells (CTCs) in breast and prostate cancer: Comparison of Harpoon CTC assay performance with the CellSearch CTC Test

Abstract Circulating tumor cells (CTCs) can provide important information about a patient's prognosis. Currently, the CELLSEARCH® CTC Test (hereafter referred to as CS) is the only US Food and Drug Administration-cleared assay for isolating and enumerating CTCs. This technology uses a CTC antigen-dependent method to isolate CTCs that may limit its ability to detect CTCs with low or no epithelial cell adhesion molecule (EpCAM) expression. We evaluated the Harpoon CTC Isolator and Chip, which uses an antigen-independent, negative depletion method to enrich CTCs from patient blood samples. As a result of the negative depletion method, the final Harpoon Product (ie, CTC-enriched cell suspension) contains CTCs that express cytokeratin (CK) and EpCAM (canonical CTCs) as well as those with low or no expression of these antigens (non-canonical CTCs). After isolation, CTCs were spun onto microscope slides and imaged using a Vectra Multispectral Imaging System. We obtained performance data on an initial prototype Harpoon assay (Harpoon + Vectra [Hrp+Vec]) and compared CTC enumeration between Hrp+Vec and CS using blood samples from 36 breast and prostate cancer patients. From 7.5 mLs of blood, the Hrp+Vec assay recovered 79.0% to 93.5% of canonical EpCAM+/CK+ CTCs recovered by CS. The Hrp+Vec assay also successfully detected non-canonical CTCs (EpCAM-/CK+ or EpCAM+/CK-) in these samples. At the CS clinical cutoff for prognostic value, where ≥5 CTCs is associated with poor prognosis, the Hrp+Vec assay was 97% concordant with CS. Hrp+Vec also detected CTCs in 12.5% (2/16) more patient samples than CS. Finally, a consensus analysis of all Vectra scan results (including both canonical and non-canonical CTCs) demonstrated that the Hrp+Vec assay detected between 90.5% and 108.9% of the number of CTCs as identified by CS. These results indicate that, when compared with CS, the prototype Hrp+Vec assay detected CTCs with similar efficiency and identified more samples as CTC-positive in these breast and prostate cancer patients. Importantly, the Hrp+Vec assay did enrich non-canonical CTCs thought to be missed by CS, and ongoing efforts are aimed to further characterize these cell populations. Citation Format: Mark Connelly, David Chianese, Carrie Morano, Thai Bui, Shemeeakah Powell, Noel Ngoubilly, Renouard Sanders, Tom Barber, Ravi Kapur, Shyamala Maheswaran, Mehmet Toner, Daniel Haber. Isolation and characterization of circulating tumor cells (CTCs) in breast and prostate cancer: Comparison of Harpoon CTC assay performance with the CellSearch CTC Test. [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 4955.

MP07-19 NEW MOUSE XENOGRAFT MODELS FOR HUMAN PROSTATE CANCER BONE AND BRAIN METASTASES

You have accessJournal of UrologyProstate Cancer: Markers II1 Apr 2016MP07-19 NEW MOUSE XENOGRAFT MODELS FOR HUMAN PROSTATE CANCER BONE AND BRAIN METASTASES Qinlong Li, Lijuan Yin, Michael Lewis, Peng Duan, Lawrence W. Jones, Quanlin Li, Leland W.K. Chung, Isla Garraway, and Haiyen E. Zhau Qinlong LiQinlong Li More articles by this author , Lijuan YinLijuan Yin More articles by this author , Michael LewisMichael Lewis More articles by this author , Peng DuanPeng Duan More articles by this author , Lawrence W. JonesLawrence W. Jones More articles by this author , Quanlin LiQuanlin Li More articles by this author , Leland W.K. ChungLeland W.K. Chung More articles by this author , Isla GarrawayIsla Garraway More articles by this author , and Haiyen E. ZhauHaiyen E. Zhau More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2222AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PC) bone and brain metastases are terminal. In contrast to PC bone metastasis, PC brain metastasis is largely detected by autopsies with a prevalence of 0.2-11.1%. We correlated the expression of cytokeratin 13 (KRT13), an intermediate filament protein, in clinical primary and metastatic PC specimens with its function in cancer metastasis using human PC cell lines. This epithelial stem cell differentiation marker has biologically directive roles in human PC bone and brain metastases. METHODS We evaluated KRT13 protein expression in primary cancer tissues from 44 PC cases with well-documented overall survival by quantitative multiplexing quantum dot labelling (mQDL) (Hu et al. Plos One 6: e28670, 2011). We confirmed the expression of KRT13 in both clinical specimens and experimental PC bone and brain metastases. We genetically overexpressed KRT13 in three AR-positive human PC cell lines, LNCaP, ARCaPE and CWR22Rv1, and studied the metastatic behaviors and associated cell growth, migration, invasion and cell signaling pathways of these cells in mice after intracardial inoculation, and in cell culture. RESULTS KRT13 expression in primary PC correlates with patient overall survival and bone metastasis (p<0.05). It is increased in clinical cancer bone and brain metastasis specimens and PC tumor cell models with increased propensity for bone and brain metastases; drives PC bone and brain metastases in LNCaP, ARCaPE and CWR22Rv1 cell models; promotes differential expression of genes associated with EMT, stemness, neuromimicry and osteomimicry in PC cells; and increases PC cell migration, invasion and soft agar colony formation. CONCLUSIONS Expression of the intermediate filament protein KRT13 was associated with PC bone and brain metastases in human PC primary and metastatic specimens. KRT13 expression in primary PC specimens quantified by mQDL correlated with patient overall survival and bone metastasis. KRT13 drives cancer metastases via osteomimicry and neuromimicry related genes resulting in downstream cell signaling network convergence among EMT, stemness, cell growth, and survival pathways. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e84 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Qinlong Li More articles by this author Lijuan Yin More articles by this author Michael Lewis More articles by this author Peng Duan More articles by this author Lawrence W. Jones More articles by this author Quanlin Li More articles by this author Leland W.K. Chung More articles by this author Isla Garraway More articles by this author Haiyen E. Zhau More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Dormant Circulating Tumor Cells in Prostate Cancer: Therapeutic, Clinical and Biological Implications.

Circulating Tumor Cells (CTCs) are a valuable prognostic factor in several solid tumors. By understanding the biological characteristics of CTCs we could better understand the biology of metastasis. CTCs usually adopt a dormant state that is believed to be a strategy to survive in extreme conditions. To enter a dormant state, CTCs undergo numerous phenotypic, genetic and functional mutations that significantly affect the efficacy of the therapies used to kill dormant CTCs. Hence, understanding the biological events involved in the dormancy process of CTCs would allow the identification of new therapeutic targets. Some experimental studies or preclinical models have explored these biological events, as well as the molecular factors that contribute to the maintenance of and release from dormancy. However, few studies have assessed the effects of anticancer therapies on dormant cells. This study reviews current the data currently available on cell dormancy mechanisms in prostate cancer, with a special focus on the functional, genetic and phenotypic plasticity of CTCs and their potential implications in the clinical and therapeutic management of prostate cancer.

The Initial Detection and Partial Characterization of Circulating Tumor Cells in Neuroendocrine Prostate Cancer.

The transition of prostate adenocarcinoma to a predominantly androgen receptor (AR) signaling independent phenotype can occur in the later stages of the disease and is associated with low AR expression +/- the development of small-cell or neuroendocrine tumor characteristics. As metastatic tumor biopsies are not always feasible and are difficult to repeat, we sought to evaluate noninvasive methods to identify patients transitioning toward a neuroendocrine phenotype (NEPC). We prospectively studied a metastatic tumor biopsy, serum biomarkers, and circulating tumor cells (CTC, Epic Sciences) from patients with castration-resistant prostate cancer (CRPC) including those with pure or mixed NEPC histology present on biopsy. CTCs labeled with the patient's clinical status were used to learn features that discriminate NEPC patients, which was then applied to an independent cohort. Twenty-seven patients with CRPC including 12 NEPC and 5 with atypical clinical features suggestive of NEPC transition were studied. CTCs from NEPC patients demonstrated frequent clusters, low or absent AR expression, lower cytokeratin expression, and smaller morphology relative to typical CRPC. A multivariate analysis of protein and morphologic variables enabled distinguishing CTCs of NEPC from CRPC. This CTC classifier was applied to an independent prospective cohort of 159 metastatic CRPC patients and identified in 17/159 (10.7%) of cases, enriched in patients with high CTC burden (P < 0.01) and visceral metastases (P = 0.04). CTCs from patients with NEPC have unique morphologic characteristics, which were also identified in a subset of CRPC patients with aggressive clinical features potentially undergoing NEPC transition.

Differential IKK/NF-κB Activity Is Mediated by TSC2 through mTORC1 in PTEN-Null Prostate Cancer and Tuberous Sclerosis Complex Tumor Cells.

The serine/threonine protein kinase Akt plays a critical role in regulating proliferation, growth, and survival through phosphorylation of different downstream substrates. The mTOR is a key target for Akt to promote tumorigenesis. It has been reported that Akt activates mTOR through phosphorylation and inhibition of the tuberous sclerosis complex (TSC) protein TSC2. Previously, it was demonstrated that mTOR activates IKK/NF-κB signaling by promoting IκB kinase (IKK) activity downstream of Akt in conditions deficient of PTEN. In this study, the mechanistic role of the tumor-suppressor TSC2 was investigated in the regulation of IKK/NF-κB activity in PTEN-null prostate cancer and in TSC2-mutated tumor cells. The results demonstrate that TSC2 inhibits IKK/NF-κB activity downstream of Akt and upstream of mTORC1 in a PTEN-deficient environment. However, TSC2 promotes IKK/NF-κB activity upstream of Akt and mTORC1 in TSC2 mutated tumor cells. These data indicate that TSC2 negatively or positively regulates IKK/NF-κB activity in a context-dependent manner depending on the genetic background. This study provides fundamental insight for understanding the molecular details by which TSC2/mTOR regulates NF-κB signaling in different tumors.

High-Resolution Genomic Profiling of Disseminated Tumor Cells in Prostate Cancer

Circulating tumor cells and disseminated tumor cells (DTCs) are of great interest because they provide a minimally invasive window for assessing aspects of cancer biology, including tumor heterogeneity, a means to discover biomarkers of disease behavior, and a way to identify and prioritize therapeutic targets in the emerging era of precision oncology. However, the rarity of circulating tumor cells and DTCs poses a substantial challenge to the consistent success in analyzing their molecular features, including genomic aberrations. Herein, we describe optimized and robust methods to reproducibly detect genomic copy number alterations in samples of 2 to 40 cells after whole-genome amplification with the use of a high-resolution single-nuclear polymorphism-array platform and refined computational algorithms. We have determined the limit of detection for heterogeneity within a sample as 50% and also demonstrated success in analyzing single cells. We validated the genes in genomic regions that are frequently amplified or deleted by real-time quantitative PCR and nCounter copy number quantification. We further applied these methods to DTCs isolated from individuals with advanced prostate cancer to confirm their highly aberrant nature. We compared copy number alterations of DTCs with matched metastatic tumors isolated from the same individual to gain biological insight. These developments provide high-resolution genomic profiling of single and rare cell populations and should be applicable to a wide-range of sample sources.

CCR 20th Anniversary Commentary: Circulating Tumor Cells in Prostate Cancer.

Circulating tumor cells (CTC) have substantial promise for multipurpose biomarker studies in prostate cancer. The IMMC-38 trial conducted by de Bono and colleagues, which was published in the October 1, 2008, issue of Clinical Cancer Research, demonstrated for the first time that CTCs are the most accurate and independent predictor of overall survival in metastatic prostate cancer. Since the publication of prospective trials demonstrating prognostic utility, CTCs have been utilized for nucleic acid analyses, for protein analyses, and in intermediate endpoint studies. CTC studies are also now facilitating the analysis of intrapatient heterogeneity. See related article by de Bono et al., Clin Cancer Res 2008;14(19) October 1, 2008;6302-9.

Correlation Between MRI-Derived Quantitative Biomarkers and Circulating Tumor Cells in Prostate Cancer

Correlation Between MRI-Derived Quantitative Biomarkers and Circulating Tumor Cells in Prostate Cancer

Single Cell Analysis and Circulating Tumor Cells in Prostate Cancer: Experience of an Observership at Cold Spring Harbor Laboratory, CSH and New York Presbyterian Hospital (Cornell Campus), New York

Single Cell Analysis and Circulating Tumor Cells in Prostate Cancer: Experience of an Observership at Cold Spring Harbor Laboratory, CSH and New York Presbyterian Hospital (Cornell Campus), New York

Abstract 631: Utilizing Vortex Chip for enumeration and determination of single-cell heterogeneity of circulating tumor cells in prostate cancer

Abstract Background: Circulating tumor cells (CTCs) are cells that break away from either the primary tumor or metastatic sites and circulate in the peripheral blood. CTCs are increasingly used as an accessible source of tumor cells (i.e. “liquid biopsy”). We are using a CTC capture technology known as the Vortex Chip, which allows for rapid isolation of highly purified CTCs in less than 1 hour. Moreover, because the Vortex Chip isolates CTCs based on their size, this platform does not require antibody based capture that can potentially bias the isolation of CTC subpopulations to cells that express a specific surface marker, like EpCAM. A new version of the Vortex Chip, Vortex HT, has been used for an increased capture of prostate CTCs. Methods: The Vortex HT Chip was fabricated using standard lithographic techniques with polydimethylsiloxane (PDMS) polymer. Samples were run using a syringe pump setup to deliver blood to the chip at a constant flow rate, with large cells (including CTCs) trapped in the vortices present within each chamber. Trapped cells were released using PBS rinse buffer and available for immunofluorescence staining (CK, PSA, CD45, DAPI) or other downstream analysis. Individual cells could also be isolated for total RNA amplification and RNA sequencing. Results: With the new HT chip design, we achieved greater than 10,000-fold enrichment of the cells and an overall purity of greater than 50%. Individual CTCs from patients with advanced metastatic castration resistant prostate cancer (mCRPC) were isolated, immunostained and enumerated: among 13 patients, 100% had more than 1.5 CTCs/mL, while among 9 healthy donors, all had less than 1.5 CTCs/mL; we thus set 1.5 CTC/mL as the cutoff. Proof-of-principle single-cell RNA seq transcriptome analysis was performed on cells isolated from the chip. We isolated three CTCs from two distinct patients and performed RNA sequencing and compared the results to single cell RNA sequencing of three VCaP cell-line cells. Conclusions: The Vortex Chip HT design has been successfully applied to prostate cancer with an increased throughput. CTCs have been collected from patient samples within less than 1 hour. The individual cells have sufficient RNA for individual single cell RNA seq analysis. Further optimization of these techniques will be necessary to validate these methods and to gain additional insight into heterogeneity of prostate cancer as reflected in CTCs. Citation Format: Edward Pao, James Che, Elodie Sollier, Andrew King, Guoping Fan, Jiaoti Huang, Dino Di Carlo, Matthew B. Rettig, Rajan P. Kulkarni. Utilizing Vortex Chip for enumeration and determination of single-cell heterogeneity of circulating tumor cells in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 631. doi:10.1158/1538-7445.AM2015-631

A glycolytic phenotype is associated with prostate cancer progression and aggressiveness: a role for monocarboxylate transporters as metabolic targets for therapy.

Metabolic adaptation is considered an emerging hallmark of cancer, whereby cancer cells exhibit high rates of glucose consumption with consequent lactate production. To ensure rapid efflux of lactate, most cancer cells express high levels of monocarboxylate transporters (MCTs), which therefore may constitute suitable therapeutic targets. The impact of MCT inhibition, along with the clinical impact of altered cellular metabolism during prostate cancer (PCa) initiation and progression, has not been described. Using a large cohort of human prostate tissues of different grades, in silico data, in vitro and ex vivo studies, we demonstrate the metabolic heterogeneity of PCa and its clinical relevance. We show an increased glycolytic phenotype in advanced stages of PCa and its correlation with poor prognosis. Finally, we present evidence supporting MCTs as suitable targets in PCa, affecting not only cancer cell proliferation and survival but also the expression of a number of hypoxia‐inducible factor target genes associated with poor prognosis. Herein, we suggest that patients with highly glycolytic tumours have poorer outcome, supporting the notion of targeting glycolytic tumour cells in prostate cancer through the use of MCT inhibitors. © 2015 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Circulating tumor cells (CTCs) in systemic prostate cancer.

e22019 Background: The purpose of the study was to evaluate quantitatively CTCs in peripheral blood of patients with stage III-IV prostate cancer. Methods: 30 patients with systemic prostate cancer...

The Role of Osteoclasts in Early Dissemination of Prostate Cancer Tumor Cells.

Prostate cancer (PCa) is one of the most common neoplasms that metastasize to bone. The aim of this study was to determine if osteoclasts play a role in the seeding of disseminated tumor cells to the bone marrow by mobilizing hematopoietic stem cells (HSCs) out of their marrow niche. Human PC-3Luc cells were introduced into male SCID mice by intracardiac (i.c.) injection after mice were treated with the antiresorptive agent Zoledronic Acid (bisphosphonate (BP)) and/or AMD3100, which mobilizes HSCs out of the marrow. Short term homing of PC-3 was assessed at 24 hours by QPCR for human Alu and luciferase and HSC number was determined by FACS. Mice also received pre and/or post treatments of BP by intraperiteneal (i.p.) injections, in addition to PC-3 luc by intratibial (i.t.) injections. TRAP assays were used to determine the osteoclast (OC) number in both studies. AMD3100 enhanced the release of HSCs from the bone marrow, while BP increased the retention of HSCs. PCa entry into bone was facilitated in AMD3100, BP, and AMD3100+BP treatments. Before PCa injection, the number of TRAP+ OC was increased in mice treated with AMD3100, while treatment with BP resulted in relatively lower TRAP+ OCs. TRAP+ OCs were not detected in the AMD3100 + BP treatment. After PCa injection, however, the number of TRAP+ OCs was dramatically increased, but did not differ significantly amongst the treatment groups. The pre and post BP treatments in the Nude mice decreased the size of PCa lesions in the tibia compared to the control. The results indicate that OC activation is not necessary for PCa metastasis to bone at the earliest stages. These findings are critical in proving that OCs' contribution to metastasis occur during the growth phase of the tumor rather than at the initiation phase.

Forced LIGHT expression in prostate tumors overcomes Treg mediated immunosuppression and synergizes with a prostate tumor therapeutic vaccine by recruiting effector T lymphocytes.

LIGHT, a ligand for lymphotoxin-β receptor (LTβR) and herpes virus entry mediator, is predominantly expressed on activated immune cells and LTβR signaling leads to the recruitment of lymphocytes. The interaction between LIGHT and LTβR has been previously shown to activate immune cells and result in tumor regression in a virally-induced tumor model, but the role of LIGHT in tumor immunosuppression or in a prostate cancer setting, where self antigens exist, has not been explored. We hypothesized that forced expression of LIGHT in prostate tumors would shift the pattern of immune cell infiltration toward an anti-tumoral milieu, would inhibit T regulatory cells (Tregs) and would induce prostate cancer tumor associated antigen (TAA) specific T cells that would eradicate tumors. Real Time PCR was used to evaluate expression of forced LIGHT and other immunoregulatory genes in prostate tumors samples. For in vivo studies, adenovirus encoding murine LIGHT was injected intratumorally into TRAMP-C2 prostate cancer cell tumor bearing mice. Chemokine and cytokine concentrations were determined by multiplex ELISA. Flow cytometry was used to phenotype tumor infiltrating lymphocytes and expression of LIGHT on the tumor cell surface. Tumor-specific lymphocytes were quantified via ELISpot assay. Treg induction and Treg suppression assays determined Treg functionality after LIGHT treatment. LIGHT in combination with a therapeutic vaccine, PSCA TriVax, reduced tumor burden. LIGHT expression peaked within 48 hr of infection, recruited effector T cells that recognized mouse prostate stem cell antigen (PSCA) into the tumor microenvironment, and inhibited infiltration of Tregs. Tregs isolated from tumor draining lymph nodes had impaired suppressive capability after LIGHT treatment. Forced LIGHT treatment combined with PSCA TriVax therapeutic vaccination delays prostate cancer progression in mice by recruiting effector T lymphocytes to the tumor and inhibiting Treg mediated immunosuppression. Prostate 75:280-291, 2015. © 2014 Wiley Periodicals, Inc.

Detection of circulating tumor cells in prostate cancer based on carboxylated graphene oxide modified light addressable potentiometric sensor

Circulating tumor cells (CTCs) are a group of rare cancer cells that have detached from a primary tumor and circulate in the bloodstream. Herein, light addressable potentiometric sensor (LAPS) was exploited in the label-free detection of CTCs in the prostate cancer. To this end, the mouse anti-human epithelial cell adhesion molecule (anti-EpCAM) monoclonal antibody was selected as the probe to capture CTCs according to our western blot experiments, and therefore the anti-EpCAM was immobilized on the surface of carboxylated graphene oxide (GO-COOH) modified LAPS. Spiking experiments confirmed that LAPS' voltage decreased with the increasing of CTCs' concentration both in phosphate buffer (PBS) and blood, and as few as 10 CTCs in 1ml of blood could be detected, illustrating the high sensitivity of the proposed strategy. The analysis of healthy blood samples revealed no change in electrical signal, confirming the specificity of the system. Ultraviolet–visible (UV–vis) spectroscopy, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and immunofluorescent assay (IFA) were conducted to characterize GO-COOH, testify its existence on LAPS and validate CTCs' capturing by anti-EpCAM grafted on GO-COOH modified substrates. It is indicated that LAPS could be a potential platform for CTCs detection and may provide a powerful tool for downstream analysis.

Abstract 3079: Characterization of the molecular heterogeneity of circulating tumor cells in metastatic prostate cancer

Abstract Purpose: CTCs represent an alternative source of tumor material potentially usable as a surrogate tissue marker in the context of targeted treatments. The objective of the present study was to analyze the status of key molecular markers such as ERG gene rearrangement and AR amplification in tumor tissues (primitive tumor specimens and metastasis biopsies) and CTCs of patients with metastatic prostate cancer (PCa). Patients and Methods: CTCs were detected using two methods, CellSearch (Veridex LLC, Raritan, NJ, USA) and filtration (ISET, isolation by Size of Epithelial Tumor cells) combined to four-color immunofluorescent staining. Epithelial (cytokeratins, EpCAM (Epithelial Cell Adhesion Molecule)) and mesenchymal (vimentin, N-cadherin) markers were tested. Molecular characterization of CTCs was performed on filters by a two step method combining four color immunofluorescent staining and Filter Adapted-Fluorescent In situ Hybridization (FA-FISH). Image analysis was performed using an ARIOL automated scanner (LEICA) to precisely relocate FISH signals in phenotypically different CTC subsets. ERG gene rearrangement and AR copy number gains were examined both in CTCs enriched on filters and in the cell fractions recovered from the magnests after CellSearch and spread on slides for FISH. Primitive tumor specimens and metastasis biopsies were also collected and analyzed by FISH. Results: Higher levels of CTCs were detected by filtration enrichment combined to four-color immunofluorescent staining compared to CellSearch. Filtration enriched CTCs expressed epithelial, mesenchymal and hybrid (both epithelial and mesenchymal) markers suggesting the existence of CTCs at different stages of the epithelial to mesenchymal transition (EMT) process in most PCa patients. A higher level of gene variability and tumor heterogeneity was observed in filtration enriched CTCs compared to those captured by the CellSearch. ERG gene rearrangement and AR amplification in prostate tissue, metastases, and filtration enriched CTCs were analysed by FISH. ERG gene rearrangement and AR copy number gain status present within the tumor tissue was in concordance with that found in filtration enriched CTCs. However, FISH experiments also revealed a much higher degree of cell-to-cell variability and tumor heterogeneity in filtration enriched CTCs compared to tumor tissues. Conclusion: We show here that prostate CTCs are highly heterogeneous in term of EMT markers and ERG gene rearrangement and AR amplification marker status. The high degree of cell-to-cell variability in ERG and AR gene patterns present in CTCs reveals important tumor heterogeneity and could have important implications for developing personalized anticancer strategies based on the use of CTCs in PCa patients. Citation Format: Christophe Massard, Marianne Oulhen, Alexander Valent, Sylvestre Lemoulec, Karim Fizazi, Philippe Vielh, Françoise Farace. Characterization of the molecular heterogeneity of circulating tumor cells in metastatic prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3079. doi:10.1158/1538-7445.AM2014-3079

Abstract 2092: Elevated PKCα expression and its association with Notch signaling in localized and metastatic prostate cancers

Abstract Background: Conventional protein Kinase C alpha (PKCα) is linked to the regulation of cell proliferation, differentiation, motility, survival, and apoptosis. Elevated PKCα expression has been reported in human early prostate cancer (PCa) and rat prostatic tumor cell lines. In this study, the expression level of PKCα was evaluated by immunohistochemistry (IHC) and correlated with the expression levels of Notch1 and Jagged1 in localized and metastatic PCa. Methods: : IHC for PKCα was performed on tissue microarray (TMA) slides containing 156 formalin-fixed and paraffin-embedded (FFPE) prostatic tissues specimens, including 39 benign prostatic changes, 97 localized prostate cancers and 20 metastatic prostate cancers. The expression level of PKCα was reported as a final IHC score calculated as staining extent score (0-3) multiplied by intensity score (0-3) with a maximal score of 9. ANOVA was employed to reveal the differences in IHC scores among pathologic categories, followed by Tukey-adjusted pairwise comparisons. Spearman correlation coefficient was evaluated to measure correlation between expression levels of PKCα, Notch1, and Jagged1. Results: IHC staining for PKCα was predominantly observed in cytoplasm (in 94% specimens). The score of PKCα cytoplasmic staining in metastatic PCa (5.48 ± 1.84, Mean ±SD) was significantly higher than those in localized PCa (3.30 ± 2.10, P = 2.95E-05) and in benign prostatic tissue (1.92 ± 1.53, P = 6.45E-09). Similarly, IHC score in localized PCa was significantly higher than that observed in benign prostatic changes (P = 0.001). Correlation analysis revealed that the expression level of cytoplasmic PKCα was significantly correlated with the expression levels of cytoplasmic Notch1 (r = 0.396, P = 5.85E-05), cytoplasmic Jagged1 (r = 0.610, P = 3.42E-11), and membranous Jagged1 (r = 0.354, P = 3.76E-04, respectively) in localized and metastatic prostate cancers. Conclusions: Our results indicate that up-regulation of PKCα plays a role not only in PCa formation and progression, but also in PCa metastasis. The evidence of significant correlation between PKCα and Jagged1-Notch1 signaling pathway supports the notion that synergy of PKCα with Jagged1-Notch1 signaling may play an important role in modulating the tumorgenesis and metastasis in human PCa. Citation Format: Xinchun Zhou, He Zhu, Xu Zhang, Jack Lewin, Lucio Miele. Elevated PKCα expression and its association with Notch signaling in localized and metastatic prostate cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2092. doi:10.1158/1538-7445.AM2014-2092

Circulating tumor cells in prostate cancer: a better predictor of survival than prostate specific antigen?

Prostate specific antigen has been extensively studied and represents the most common biomarker across the spectrum of prostate cancer. Circulating tumor cells are a more recent method for monitoring patients with metastatic prostate cancer and have shown great promise in predicting outcomes. We review the prognostic capabilities of these markers to determine if circulating tumor cells should unseat prostate specific antigen as the test of choice in prostate cancer.

In vitro modeling of HER2-targeting therapy in disseminated prostate cancer.

Prostate cancer (PCa) is the most common cancer type among men. Treatments against advanced PCa are limited and in many cases only palliative. In a later, androgent independent, stage of PCa androgen receptors can be activated without interaction with ligand, i.e., by receptors of tyrosine kinase (RTK) family in the outlaw pathway. Human epidermal growth factor receptors HER2 and EGFR belong to RTK-family. HER2 is one of the main actors in the outlaw pathway with EGFR as the preferable heterodimerizing partner. We hypothesized that information on HER2 expression in advanced PCa could be useful for selection of patients for anti-RTK therapy and monitoring of therapy response. A panel of PCa cell lines (LNCap, PC3, DU-145) was subjected to a 8-week treatment using drugs influencing the RTK: trastuzumab(anti‑HER2), 17-DMAG (Hsp90 inhibitor), alone or in combination, and their HER2 and EGFR expressions were compared with non-treated cells. Treatment with trastuzumab decreased proliferation of LNCap and DU-145 cell lines, while 17-DMAG and trastuzumab/17‑DMAG combination affected all three cell lines. HER2 expression was significantly increased in PC3 cells, the most resistant cell line. On the contrary, in responding cells (LNCap and DU-145) HER2 expression decreased, accompanied by increased EGFR expression. However, additional treatment of cells with cetuximab (anti‑EGFR) did not give any additive effect to trastuzumab. In this study the response to anti-RTK therapy proved to vary between different PCa cell lines. We have demonstrated that RTK targeting treatments may affect the phenotypic profile of PCa tumor cells that correlates with therapy outcome. Observation of such changes during treatment could be used for monitoring and an improved therapy outcome.