Human Epithelial Cell Adhesion Molecule Research Articles

ImmunoPET imaging of EpCAM in solid tumours with nanobody tracers: a preclinical study.

Epithelial cell adhesion molecule (EpCAM) is a potential therapeutic target and anchoring molecule for circulating and disseminated tumour cells (CTC/DTC) in liquid biopsy. In this study, we aimed to construct EpCAM-specific immuno-positron emission tomography (immunoPET) imaging probes and assess the diagnostic abilities in preclinical cancer models. By engineering six single-domain antibodies (e.g., EPCD1 - 6) targeting EpCAM of different binding properties and labelling with 68Ga (T1/2 = 1.1 h) and 18F (T1/2 = 110 min), we developed a series of EpCAM-targeted immunoPET imaging probes. The probes' pharmacokinetics and diagnostic accuracies were investigated in cell-derived human colorectal (LS174T) and esophageal cancer (OE19) tumour models. Based on in vitro binding affinities and in vivo pharmacokinetics of the first three tracers ([68Ga]Ga-NOTA-EPCD1, [68Ga]Ga-NOTA-EPCD2, and [68Ga]Ga-NOTA-EPCD3), we selected [68Ga]Ga-NOTA-EPCD3 for tumour imaging which showed an average tumour uptake of 2.06 ± 0.124%ID/g (n = 3) in LS174T cell-derived tumour model. Development and characterisation of [18F]AIF-RESCA-EPCD3 showed comparable tumour uptake of 1.73 ± 0.0471%ID/g (n = 3) in the same tumour model. Further validation of [68Ga]Ga-NOTA-EPCD3 in OE19 cell-derived tumour model showed an average tumour uptake of 4.27 ± 1.16%ID/g and liver uptake of 13.5 ± 1.30%ID/g (n = 3). Near-infrared fluorescence imaging with Cy7-EPCD3 confirmed the in vivo pharmacokinetics and relatively high liver accumulation. We further synthesized another three 18F-labeled nanobody tracers ([18F]AIF-RESCA-EPCD4, [18F]AIF-RESCA-EPCD5, and [18F]AIF-RESCA-EPCD6) and found that [18F]AIF-RESCA-EPCD6 had the best pharmacokinetics with low background. [18F]AIF-RESCA-EPCD6 showed explicit uptake in the subcutaneously inoculated OE19 tumour model with an average uptake of 4.70 ± 0.26%ID/g (n = 3). In comparison, the corresponding tumour uptake (0.17 ± 0.25%ID/g, n = 3) in the EPCD6 blocking group was substantially lower (P < 0.001), indicating the targeting specificity of the tracer. We developed a series of 68Ga/18F-labeled nanobody tracers targeting human EpCAM. ImmunoPET imaging with [18F]AIF-RESCA-EPCD6 may facilitate better use of EpCAM-targeted therapeutics by noninvasively displaying the target's expression dynamics.

Chimeric antigen receptor-T cells targeting epithelial cell adhesion molecule antigens are effective in the treatment of colorectal cancer

ObjectiveTo construct chimeric antigen receptor (CAR)-T cells targeting epithelial cell adhesion molecule (EpCAM) antigen (anti-EpCAM-CAR-T).MethodsA third-generation CAR-T cell construct used a single-chain variable fragment derived from monoclonal antibody against human EpCAM. Peripheral blood mononuclear cells were extracted from volunteers. The proportion of cluster of differentiation 8 positive (CD8+) and CD4 + T cells was measured using flow cytometry. Western blot was used to detect the expression of EpCAM-CAR. The killing efficiency was detected using the MTT assay and transwell assay, and the secretion of killer cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected using the ELISA. The inhibitory effect of EpCAM-CAR-T on colorectal cancer in vivo was detected using xenografts.ResultsIt was found that T cells expanded greatly, and the proportion of CD3+, CD8 + and CD4 + T cells was more than 60%. Furthermore, EpCAM-CAR-T cells had a higher tumour inhibition rate in the EpCAM expression positive group than in the negative group (P < 0.05). The secretion of killer cytokines TNF-α and IFN-γ in the EpCAM expression positive cell group was higher than that in the negative group (P < 0.05). In the experimental group treated with EpCAM-CAR-T cells, the survival rate of nude mice was higher (P < 0.05), and the tumour was smaller than that in the blank and control groups (P < 0.05). The secretion of serum killer cytokines TNF-α and IFN-γ in tumour-bearing nude mice in the experimental group treated with EpCAM-CAR-T cells was higher than that in the blank and control groups (P < 0.05).ConclusionThis study successfully constructed EpCAM-CAR cells and found that they can target and recognise EpCAM-positive tumour cells, secrete killer cytokines TNF-α and IFN-γ and better inhibit the growth and metastasis of colorectal cancer in vitro and in vivo than unmodified T cells.

Co-infusion of CAR T cells with aAPCs expressing chemokines and costimulatory ligands enhances the anti-tumor efficacy in mice

Chimeric antigen receptor-modified T (CAR-T) cell therapy has shown curable efficacy for treating hematological malignancies, while in solid tumors, the immunosuppressive microenvironment causes poor activation, expansion and survival of CAR-T cells, accounting mainly for the unsatisfactory efficacy. The artificial antigen-presenting cells (aAPCs) have been used for ex vivo expansion and manufacturing of CAR-T cells. Here, we constructed a K562 cell-based aAPCs expressing human epithelial cell adhesion molecule (EpCAM), chemokines (CCL19 and CCL21) and co-stimulatory molecular ligands (CD80 and 4-1BBL). Our data demonstrated that the novel aAPCs enhanced the expansion, and increased the immune memory phenotype and cytotoxicity of CAR-T cells recognizing EpCAM, in vitro. Of note, co-infusion CAR-T and aAPC enhances the infiltration of CAR-T cells in solid tumors, which has certain potential for the treatment of solid tumors Moreover, IL-2-9-21, a cytokine cocktail, prevents CAR-T cells from entering the state of exhaustion prematurely after continuous antigen engagement and boosts the anti-tumor activity of CAR-T cells co-infused with aAPCs. These data provide a new strategy to enhance the therapeutic potential of CAR-T cell therapy for the treatment of solid tumors.

Dual electroactive AgM (M=Ru, Pt) NPs for double electroanalysis of HER2 and EpCAM

Parallel multiple detections of tumor markers is of significant for the efficient and accurate early monitoring of related cancers. Electrochemical aptasensors possess excellent selectivity and sensitive quantitative detection while maintaining multiple detection ability. The study of high and different electrochemical beacons is urgent to achieve multiple-level parallel detection. In this work, electroactive AgRu NPs and AgPt NPs were explored to exhibit electrooxidation peaks at 0.8 V and − 0.7 V, respectively. Plasmonic AgRu NPs and AgPt NPs harvested light energy and exhibited amplified electrooxidation activity under local surface plasmon resonance (LSPR) excitation. Plasmon-enhanced electrooxidation principle was proposed for AgM (M=Ru, Pt) NPs owing to the distinct imaginary part of dielectric constants between Ag and platinum group metals (PGM). The strong and non-interference electrooxidation peaks at 0.8 V and − 0.7 V enabled AgRu NPs and AgPt NPs to be different electroactive beacons for the sensitive and reliable quantitative dual detection of human epidermal growth factor receptor-2 (HER2) and epithelial cell adhesion molecule (EpCAM). The LODs were 0.3 pg mL−1 (7.5 amol mL−1) for EpCAM and 0.6 pg mL−1 (3.2 amol mL−1) for HER2. The proposed plasmon-promoted electrooxidation principle provides a novel route for the design of multiple electroactive plasmonic metal-PGM composite electrochemical beacons for simultaneous electroanalysis applications.

Tufting Enteropathy: A Review of Clinical and Histological Presentation, Etiology, Management, and Outcome.

Congenital tufting enteropathy (CTE), also named intestinal epithelial dysplasia, is a rare, autosomal recessive enteropathy with persistent and life-threatening intractable diarrhea early in life. Intractable diarrhea is present independent of breast or formula feeding. Most CTE patients require total parenteral nutrition (TPN), and in severe cases, small bowel transplantation is needed. In the last decade, we have seen remarkable progress in certain aspects, such as the pathogenesis and diagnostic methods of the disease. Rapidly developing molecular analysis techniques have improved the diagnostic methods for CTE and reduced invasive and expensive procedures. Mutations in the gene encoding human epithelial cell adhesion molecule (EpCAM) were identified in the typical form of CTE, which usually exhibits isolated refractory diarrhea. Moreover, the syndromic form of CTE features anal and choanal atresias as well as ophthalmologic signs, which are associated with mutations in the gene encoding Serine Peptidase Inhibitor Kunitz Type 2 (SPINT2). This article reviews CTE disease based on its clinical and histological presentation, etiology and pathogenesis, and management and outcome.

Functions of EpCAM in physiological processes and diseases (Review)

EpCAM (epithelial cell adhesion molecule) is a type I transmembrane glycoprotein, which was originally identified as a tumor-associated antigen due to its high expression level in rapidly growing epithelial tumors. Germ line mutations of the human EpCAM gene have been indicated as the cause of congenital tufting enteropathy. Previous studies based on cell models have revealed that EpCAM contributes to various biological processes including cell adhesion, signaling, migration and proliferation. Due to the previous lack of genetic animal models, the in vivo functions of EpCAM remain largely unknown. However, EpCAM genetic animal models have recently been generated, and are useful for understanding the functions of EpCAM. The authors here briefly review the functions and mechanisms of EpCAM in physiological processes and different diseases.

Eradication of Established Tumors by Chemically Self-Assembled Nanoring Labeled T Cells.

Our laboratory has developed chemically self-assembled nanorings (CSANs) as prosthetic antigen receptors (PARs) for the nongenetic modification of T cell surfaces. PARs have been successfully employed in vitro to activate T cells for the selective killing of leukemia cells. However, PAR efficacy has yet to be evaluated in vivo or against solid tumors. Therefore, we developed bispecific PARs that selectively target the human CD3 receptor and human epithelial cell adhesion molecule (EpCAM), which is overexpressed on multiple carcinomas and cancer stem cells. The αEpCAM/αCD3 PARs were found to stably bind T cells for >4 days, and treating EpCAM+ MCF-7 breast cancer cells with αEpCAM/αCD3 PAR-functionalized T cells resulted in the induction of IL-2, IFN-γ, and MCF-7 cytotoxicity. Furthermore, an orthotopic breast cancer model validated the ability of αEpCAM/αCD3 PAR therapy to direct T cell lytic activity toward EpCAM+ breast cancer cells in vivo, leading to tumor eradication. In vivo biodistribution studies demonstrated that PAR-T cells were formed in vivo and persist for over 48 h with rapid accumulation in tumor tissue. Following PAR treatment, the production of IL-2, IFN-γ, IL-6, and TNF-α could be significantly reduced by an infusion of clinically relevant concentrations of the FDA-approved antibiotic, trimethoprim, signaling pharmacologic PAR deactivation. Importantly, CSANs did not induce naïve T cell activation and thus exhibit a limited potential to induce naïve T cell anergy. In addition, murine immunogenicity studies demonstrated that CSANs do not induce a significant antibody response nor do they activate splenic cells. Collectively, our results demonstrate that bispecific CSANs are able to nongenetically generate reversibly modified T cells that are capable of eradicating targeted solid tumors.

A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM.

Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids).

MOC31PE immunotoxin - targeting peritoneal metastasis from epithelial ovarian cancer.

Peritoneal metastasis (PM) is an important feature of epithelial ovarian cancer (EOC) and is a frequent site of drug resistant disease recurrence, identifying PM-EOC an important clinical challenge. The MOC31PE immunotoxin targets and kills tumor cells expressing the epithelial cell adhesion molecule (EpCAM), which is highly expressed in EOC, and MOC31PE is being investigated for use in treatment of PM-EOC.The efficacy of MOC31PE treatment alone and in combination with cytotoxic drugs was investigated in two human EpCAM expressing EOC cell lines, B76 and MDHA-2774, in vitro and in corresponding mouse models mimicking PM-EOC. MOC31PE efficaciously killed tumor cells alone and showed equal or superior activity in vitro (paclitaxel, cisplatin, carboplatin) and in vivo (paclitaxel, mitomycin C) compared to the investigated cytotoxic drugs. Additive, or importantly, no antagonistic effects were observed in combination experiments.In ex vivo cell culture, the cytotoxic effect of MOC31PE was studied on freshly isolated surgical EOC samples. All investigated fresh EOC samples expressed EpCAM and MOC31PE effectively reduced cell viability in ex vivo cultures.In conclusion, these results, together with our previous preclinical and clinical experience, support development of MOC31PE for treatment of PM-EOC in combination with currently used cytotoxic drugs.

Cleavage and cell adhesion properties of human epithelial cell adhesion molecule (HEPCAM).

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.

Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.

Quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging

Surface plasmon resonance imaging (SPRi) is most frequently used for the label-free measurement of biomolecular interactions. Here we explore the potential of SPRi to measure antibody production of individual hybridoma cells. As a model system, cells from a hybridoma, producing monoclonal antibodies recognizing epithelial cell adhesion molecule (EpCAM), were used. Recombinant human EpCAM protein was immobilized on an SPR sensor and hybridoma cells were introduced into an IBIS MX96 SPR imager and the SPRi response was followed for 10h. SPRi responses were detected on the spots of the sensor only where ligands of the produced antibody were present. By measuring the SPRi signals on individual cells the antibody production of the individual cells was measured and production rates were calculated. For 53 single EpCAM hybridoma cells the production ranged from 0.16 to 11.95pg (mean 2.96pg per cell, SD 2.51) over a period of 10h. Antibody excretion per cell per hour ranged from 0.02 to 1.19pg (mean 0.30, SD 0.25). Here we demonstrate for the first time that antibody production of individual cells can be measured and quantified by SPRi, opening a new avenue for measuring excretion products of individual cells.

Hepatitis B virus X protein induces EpCAM expression via active DNA demethylation directed by RelA in complex with EZH2 and TET2.

Chronic hepatitis B virus (HBV) infection is a major risk factor for developing hepatocellular carcinoma (HCC), and HBV X protein (HBx) acts as cofactor in hepatocarcinogenesis. In liver tumors from animals modeling HBx- and HBV-mediated hepatocarcinogenesis, downregulation of chromatin regulating proteins SUZ12 and ZNF198 induces expression of several genes, including epithelial cell adhesion molecule (EpCAM). EpCAM upregulation occurs in HBV-mediated HCCs and hepatic cancer stem cells, by a mechanism not understood. Herein we demonstrate HBx induces EpCAM expression via active DNA demethylation. In hepatocytes, EpCAM is silenced by PRC2 and ZNF198/LSD1/Co-REST/HDAC1 chromatin modifying complexes. Cells with stable knockdown of SUZ12, an essential PRC2 subunit, upon HBx expression demethylate a CpG dinucleotide located adjacent to NF-κB/RelA half-site. This NF-κB/RelA site is in a CpG island downstream from EpCAM transcriptional start site (TSS). Chromatin immunoprecipitation (ChIP) assays demonstrate HBx-dependent RelA occupancy of NF-κB half-site, while RelA knockdown suppresses CpG demethylation and EpCAM expression. TNF-α activates RelA, propagating demethylation to nearby CpG sites, shown by sodium bisulfite sequencing. RelA-dependent demethylation occurring upon HBx expression requires methyltrasferase EZH2, TET2 a key player in cytosine demethylation, and inactive DNMT3L, shown by knockdown assays and sodium bisulfite sequencing. Co-immunoprecipitations and sequential ChIP assays demonstrate that RelA in the presence of HBx forms a complex with EZH2, TET2 and DNMT3L, although the role of DNMT3L remains to be understood. Interestingly, the human EpCAM gene also has a CpG island downstream from its TSS, and a NF-κB binding site flanked by CpGs. HepG2 cells derived from human HCC exhibit demethylation of these NF-κB-flanking CpG sites, and HBV replication propagates demethylation to nearby CpG sites. DLK1, another PRC2 target gene, also upregulated in HBV-mediated HCCs, is demethylated in liver tumors at CpG dinucleotides flanking the NF-kB binding sequence, supporting that this active DNA demethylation mechanism functions during oncogenic transformation.

Crystal structure and its bearing towards an understanding of key biological functions of EpCAM.

EpCAM (epithelial cell adhesion molecule), a stem and carcinoma cell marker, is a cell surface protein involved in homotypic cell-cell adhesion via intercellular oligomerization and proliferative signalling via proteolytic cleavage. Despite its use as a diagnostic marker and being a drug target, structural details of this conserved vertebrate-exclusive protein remain unknown. Here we present the crystal structure of a heart-shaped dimer of the extracellular part of human EpCAM. The structure represents a cis-dimer that would form at cell surfaces and may provide the necessary structural foundation for the proposed EpCAM intercellular trans-tetramerization mediated by a membrane-distal region. By combining biochemical, biological and structural data on EpCAM, we show how proteolytic processing at various sites could influence structural integrity, oligomeric state and associated functionality of the molecule. We also describe the epitopes of this therapeutically important protein and explain the antigenicity of its regions.

Hepatic progenitor cells express SerpinB3

BackgroundIn the setting of liver injury hepatic progenitor cells are activated, counterbalancing the inhibited regenerative capacity of mature hepatocytes. Chronic activation of this compartment may give rise to a subset of liver tumours with poor prognosis. SerpinB3, a serpin over-expressed in injured liver and in primary liver cancer, has been shown to induce apoptosis resistance, epithelial to mesenchymal transition and to increase TGF-beta and Myc expression. Aim of the present study was to explore the presence of SerpinB3 in hepatic progenitor cells in human livers and in a mouse model of liver stem/progenitor cell activation.Hepatic progenitor cells were analysed in foetal and adult livers at protein and transcriptional levels. To induce experimental activation of the liver stem/progenitor compartment, C57BL/6J mice were injected with lipopolysaccharide plus D-galactosamine and were sacrificed at different time points. Liver cDNA was amplified using specific primers for mouse-homologous SerpinB3 isoforms and automatically sequenced.ResultsThe presence of SerpinB3 in the progenitor cell compartment was detected in sorted human foetal and adult epithelial cell adhesion molecule (EpCAM) positive liver cells. By immunohistochemistry SerpinB3 was found in human cirrhotic livers in portal areas with progenitor cell activation showing ductular proliferation. CK-7, CK-19, EpCAM and CD-90 positive cell were also positive for SerpinB3. In the animal model, time course analysis in liver specimens revealed a progressive increase of SerpinB3 and a parallel decrease of activated caspase 3, which was barely detectable at 20 hours. Transcription analysis confirmed the presence of SerpinB3-homologous only in the liver of injured mice and sequence analysis proved its belonging to mouse Serpinb3b.ConclusionSerpinB3 is highly expressed in hepatic stem/progenitor cell compartment of both foetal and adult livers.

Selection of DNA Aptamers against Epithelial Cell Adhesion Molecule for Cancer Cell Imaging and Circulating Tumor Cell Capture

Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and is an ideal antigen for clinical applications in cancer diagnosis, prognosis, imaging, and therapy. Currently, most of the EpCAM-based diagnostic, prognostic, and therapeutic strategies rely on the anti-EpCAM antibody. However, the use of EpCAM antibody is restricted due to its large size and instability. In this study, we have successfully identified DNA aptamers that selectively bind human recombinant EpCAM protein. The aptamers can specifically recognize a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM but not bind to EpCAM-negative cells. Among the aptamer sequences identified, a hairpin-structured sequence SYL3 was optimized in length, resulting in aptamer sequence SYL3C. The Kd values of the SYL3C aptamer against breast cancer cell line MDA-MB-231 and gastric cancer cell line Kato III were found to be 38 ± 9 and 67 ± 8 nM, respectively, which are better than that of the full-length SYL3 aptamer. Flow cytometry analysis results indicated that the SYL3C aptamer was able to recognize target cancer cells from mixed cells in cell media. When used to capture cancer cells, up to 63% cancer cell capture efficiency was achieved with about 80% purity. With the advantages of small size, easy synthesis, good stability, high binding affinity, and selectivity, the DNA aptamers reported here against cancer biomarker EpCAM will facilitate the development of novel targeted cancer therapy, cancer cell imaging, and circulating tumor cell detection.

922 SURVIVIN EXPRESSING CIRCULATING TUMOR CELLS IN NON-MUSCLE INVASIVE BLADDER CANCER: THEIR IDENTIFICATION AND CHARACTERIZATION WITH CELLECTION DYNABEADS ANALYSIS

You have accessJournal of UrologyBladder Cancer: Basic Research (II)1 Apr 2013922 SURVIVIN EXPRESSING CIRCULATING TUMOR CELLS IN NON-MUSCLE INVASIVE BLADDER CANCER: THEIR IDENTIFICATION AND CHARACTERIZATION WITH CELLECTION DYNABEADS ANALYSIS Gian Maria Busetto, Paola Gazzaniga, Chiara Nicolazzo, Arianna Petracca, Riccardo Giovannone, Vincenzo Gentile, and Ettore De Berardinis Gian Maria BusettoGian Maria Busetto Rome, Italy More articles by this author , Paola GazzanigaPaola Gazzaniga Rome, Italy More articles by this author , Chiara NicolazzoChiara Nicolazzo Rome, Italy More articles by this author , Arianna PetraccaArianna Petracca Rome, Italy More articles by this author , Riccardo GiovannoneRiccardo Giovannone Rome, Italy More articles by this author , Vincenzo GentileVincenzo Gentile Rome, Italy More articles by this author , and Ettore De BerardinisEttore De Berardinis Rome, Italy More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.499AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prognosis of high risk non-muscle invasive bladder cancer is highly variable and unpredictable on the basis of clinical and pathological variables. Survivin, a small protein belonging to æinhibitor of apoptosisÆ family, is frequently overexpressed in bladder tumours but not detected in normal differentiated transitional epithelium. Thus, the presence of survivin transcripts in bladder tumours is considered a marker of bladder cancer progression. The evasion of apoptosis through the overexpression of survivin also represents one of the mechanisms by which tumour cells acquire the ability to enter and survive in the blood flow; consistent with this hypothesis, it was shown that survivin is often expressed by circulating tumour cells (CTCs), a subset of cancer cells that escape from the primary tumour into the bloodstream and travel through the circulation to distant sites, where are thought to possibly develop into secondary tumours. Main objective was to evaluate the presence of circulating tumor cells (CTCs) in T1G3 tumors and to characterize them by the expression of survivin. METHODS 141 patients with T1G3 non muscle invasive bladder cancer (NMIBC) were enrolled. Additional inclusion criteria were: tumor size<3cm; absence of CIS and multifocality. Planned follow up was 36 mo. Survivin was evaluated by RT-PCR in tumoral tissues. CTCs were isolated from blood by CELLection™ Dynabeads coated with the monoclonal antibody towards the human Epithelial Cell Adhesion Molecule. Cells were lysed and Dynabeads Oligo(dT) was used to capture poly A+ mRNA. cDNA was synthesised and analysed for the expression of CD45, CK8 and Survivin. The primary end point was disease free survival (DFS); the favourable group at 36 mo was defined as that without any clinical evidence of disease (NED); the unfavourable group was that with evidence of recurrent disease (RD) or progressive disease (PD). Tumoral Survivin expression and presence of CTC were correlated to DFS. RESULTS Survivin was found in 50% of tumors. Survivin - patients showed a longer DFS than Survivin + (÷2: 4.572; p =0.029). CTCs were found in 48/141 patients (34%); 92% of CTC were Survivin expressing. The difference in DFS between CTC - and CTC + patients was statistically significant (÷2: 28.098; p <0.001). CTC presence was found an independent prognostic factor of DFS (p<0.001). CONCLUSIONS CTC presence is an independent prognostic factor in high risk NMIBC patients. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e379 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gian Maria Busetto Rome, Italy More articles by this author Paola Gazzaniga Rome, Italy More articles by this author Chiara Nicolazzo Rome, Italy More articles by this author Arianna Petracca Rome, Italy More articles by this author Riccardo Giovannone Rome, Italy More articles by this author Vincenzo Gentile Rome, Italy More articles by this author Ettore De Berardinis Rome, Italy More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Abstract B49: Autologous T cell responses against patient-derived colorectal cancer xenografts.

Abstract Prognosis in colorectal cancer (CRC) correlates closely with the density of tumor infiltrating T lymphocytes (TIL) in the primary tumor, suggesting that the adaptive cellular immune system can influence the natural history of this disease. However, it is unclear if TIL in CRC comprise a non-specific inflammatory response, or if T cells in TIL are specifically reactive with antigens expressed on CRC tumor cells. To date the study of TIL interaction with autologous CRC cells has been hampered by the small numbers of tumor cells and TIL that can be obtained from CRC surgical samples. We have developed reliable methods for expanding primary human CRC tumor cells as xenografts in immune-deficient mice. Moreover, we have adapted next generation DNA sequencing of T cell receptor β chain CDR3 regions to define the clonal composition of CRC TIL with exquisite precision and depth. In combination with standard procedures for expanding T cells in vitro, we are using these techniques to dissect TIL-CRC tumor cell interactions at the cellular and molecular level. The CD4+/CD8+ surface phenotype and TCRβ CDR3 sequence repertoire of TIL from 7 CRC tumor samples (a primary colon tumor from one patient, an ovarian metastasis from a second patient, and 4 non-contiguous hepatic metastases from a third patient), and from T cells in blood or normal adjacent tissue was evaluated by flow cytometry and next-generation sequencing, respectively. Flow cytometry revealed that CD4+ T cells comprised the majority of TIL expanded from the tumors from all 3 patients. Comparison of the TCRβ CDR3 sequence repertoires in the tumor and normal tissues demonstrated that the TIL were clonally distinct from the T cells present in blood or normal adjacent tissue. Moreover, extensive overlap in the repertoire of TIL isolated from the 4 hepatic metastases in patient 3 was observed, suggesting the presence in all 4 metastases of a population of T cells with similar antigenic specificity. We designed TCRβ CDR3 sequence-specific PCR primers to determine whether specific TCRβ sequences identified by next-generation sequencing were primarily found in the CD4+ or CD8+ subsets of TIL. The most common CDR3 sequences observed in TIL from the primary colon tumor of patient 1 were primarily found in the CD4+ subset, while the most common CDR3 sequences in the ovarian metastasis from patient 2 and the hepatic metastases from patient 3 were primarily found in the CD8+ subset. The reactivity of CD8+ and CD4+ T cells expanded from CRC tumor, tumor-associated ascites fluid, normal adjacent tissue, and blood against autologous CRC tumor cells (freshly harvested from xenografts) and autologous dermal fibroblasts was assessed for in a series of 5 patients, including patients 1-3. T cells expanded from the 4 tissue compartments were evaluated by interferon-γ (IFN-γ) ELISPOT for reactivity with xenograft-derived autologous tumor cells or autologous dermal fibroblasts. No significant IFN-γ reactivity was seen when T cells from any compartment were cultured with autologous dermal fibroblasts. In all 5 patients, IFN-γ reactivity with autologous human epithelial cell adhesion molecule (EpCAM) sorted tumor cells was observed. This reactivity was more prominent in purified CD4+ T cells than in CD8+ T cells. No significant difference in the reactivity of T cells from various compartments against autologous tumor cells was observed, but T cells expanded from tumor associated ascites fluid showed overall lower levels of reactivity against all target cells. Collectively, these results demonstrate that CRC TIL comprise a clonally distinct population of T cells containing both CD4+ and CD8+ T cells that are specifically reactive with autologous CRC tumor cells. However, T cells reactive with autologous tumor cells can also be isolated from other compartments such as normal adjacent tissue and peripheral blood. Citation Format: Jeffrey Chou, Christie-Lynn L. Mortales, Sheila Ojeaburu, Raymond SW Yeung, Edus H. Warren. Autologous T cell responses against patient-derived colorectal cancer xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B49.

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes.

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via “wick-in-needle” technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.

1060 SURVIVIN EXPRESSING CIRCULATING TUMOR CELLS IN NON-MUSCHE INVASIVE BLADDER CANCER A CELLECTION™ DYNABEADS ANALYSIS

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20121060 SURVIVIN EXPRESSING CIRCULATING TUMOR CELLS IN NON-MUSCHE INVASIVE BLADDER CANCER A CELLECTION™ DYNABEADS ANALYSIS Gian Maria Busetto, Arianna Petracca, Chiara Nicolazzo, Paola Gazzaniga, Vincenzo Gentile, and Ettore De Berardinis Gian Maria BusettoGian Maria Busetto Roma, Italy More articles by this author , Arianna PetraccaArianna Petracca Roma, Italy More articles by this author , Chiara NicolazzoChiara Nicolazzo Roma, Italy More articles by this author , Paola GazzanigaPaola Gazzaniga Roma, Italy More articles by this author , Vincenzo GentileVincenzo Gentile Roma, Italy More articles by this author , and Ettore De BerardinisEttore De Berardinis Roma, Italy More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1166AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The prognosis of T1G3 bladder cancer is highly ariable and not predictable basing upon clinical and pathological prognostic factors. There is need for improvement in risk stratification in this population; understanding the molecular profile of individual patients could provide a more personalized and tailored treatment. Main objective was to evaluate the prognostic significance of Survivin in tumor tissues and that of Survivin expressing circulating tumor cells (CTCs) in T1G3 tumors. METHODS 161 patients with T1G3 non muscle invasive bladder cancer (NMIBC), including CIS associated, were enrolled. Planned follow up was 24 mo. Survivin was evaluated by RT-PCR in tumoral tissues. CTCs were isolated from blood by CELLection™ dynabeads coated with the monoclonal antibody towards the human Epithelial Cell Adhesion Molecule. Cells were lysed and Dynabeads Oligo(dT) was used to capture poly A+ mRNA. cDNA was synthesised and analysed for the expression of CD45, CK8 and Survivin. The primary end point was disease free survival (DFS); the favourable group at 24 mo was defined as that without any clinical evidence of disease (NED); the unfavourable group was that with evidence of recurrent disease (RD) or progressive disease (PD). Tumour survivin expression and presence of CTC were correlated with DFS. Multivariate analysis was used to investigate whether the presence of CTC was an independent indicator of DFS. RESULTS Survivin was found in about 50% of tumors. Survivin-patients showed a longer DFS than Survivin + tumors (X2=6.222, p=0.019). CTCs were found in 84/161 patients (52%); 96% of CTC were Survivin expressing. The difference in DFS between CTC - and CTC + patients was statistically significant (X2=29.565; p=0.001). CTC presence was found an independent prognostic factor of DFS (p=0.001). CONCLUSIONS To date, for bladder cancer, there is the lack of a realible marker able to predict the prognosis and with the capability to remark the biological bahaviour of hign-risk NMIBC. The presence of CTC is an independent prognostic factor in patients with T1G3 bladder cancer and in the future could be an important parameter to evaluate. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e430 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gian Maria Busetto Roma, Italy More articles by this author Arianna Petracca Roma, Italy More articles by this author Chiara Nicolazzo Roma, Italy More articles by this author Paola Gazzaniga Roma, Italy More articles by this author Vincenzo Gentile Roma, Italy More articles by this author Ettore De Berardinis Roma, Italy More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...