Anti-epithelial Cell Adhesion Molecule Research Articles

Selective cell capture and analysis using shallow antibody-coated microchannels

Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath et al., Nature 450, 1235-1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples.

Regression of liver metastases after treatment with intraperitoneal catumaxomab for malignant ascites due to breast cancer

Malignant ascites is quite rare in breast cancer and is mainly associated with a lobular histology. To date, no studies have evaluated locoregional therapy for malignant ascites in breast cancer. The anti-epithelial cell adhesion molecule (EpCAM), trifunctional antibody catumaxomab was approved in the European Union for the intraperitoneal (i.p.) treatment of malignant ascites in patients with EpCAM-positive carcinomas where standard therapy is not available or no longer feasible. We report the case of a 69-year-old female with pretreated breast cancer who received i.p. catumaxomab for the treatment of malignant ascites and showed a regression of liver metastases. The patient originally underwent a left mastectomy and ipsilateral axillary lymph node dissection for an invasive ductal carcinoma in 1995. Following several lines of treatment, she was enrolled in February 2010 in a phase IIIb study (CASIMAS) investigating the safety of a 3-h i.p. catumaxomab infusion. In addition to a local benefit, as shown by an improvement in malignant ascites and a prolongation of the paracentesis-free interval with i.p. catumaxomab, a computed tomography scan, performed some weeks after catumaxomab administration, showed a regression of liver metastases. In addition to a locoregional effect on EpCAM-positive disease, i.p. catumaxomab may also show systemic effects. The use of i.p. catumaxomab for the treatment of malignant ascites due to breast cancer should be explored further in appropriate clinical studies and its possible systemic effects should also be further investigated.

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.

Channel Surface Patterning of Alternating Biomimetic Protein Combinations for Enhanced Microfluidic Tumor Cell Isolation

Here, we report a new method for multicomponent protein patterning in a microchannel and also a technique for improving immunoaffinity-based circulating tumor cell (CTC) capture by patterning regions of alternating adhesive proteins using the new method. The first of two proteins, antiepithelial cell adhesion molecule (anti-EpCAM), provides the specificity for CTC capture. The second, E-selectin, increases CTC capture under shear. Patterning regions with and without E-selectin allows captured leukocytes, which also bind E-selectin and are unwanted impurities in CTC isolation, to roll a short distance and detach from the capture surface. This reduces leukocyte capture by up to 82%. The patterning is combined with a leukocyte elution step in which a calcium chelating buffer effectively deactivates E-selectin so that leukocytes may be rinsed away 60% more efficiently than with a buffer containing calcium. The alternating patterning of this biomimetic protein combination, used in conjunction with the elution step, reduces capture of leukocytes while maintaining a high tumor cell capture efficiency that is up to 1.9 times higher than the tumor cell capture efficiency of a surface with only anti-EpCAM. The new patterning technique described here does not require mask alignment and can be used to spatially control the immobilization of any two proteins or protein mixtures inside a sealed microfluidic channel.

Abstract 2388: Dendrimer-mediated multivalent binding strongly captures tumor cells

Abstract Multivalent interactions, simultaneous binding of multiple ligands toward their multiple receptors, commonly occur in physiological and pathological events, which have been utilized to enhance targeting efficacy. The multivalent binding can be leveraged to develop an extremely sensitive device for detection of rare cells such as circulating tumor cell (CTC). Accurate enumeration of CTCs is of clinical importance in diagnosis and prognosis of cancer metastasis; however the rareness of CTCs, comprising as few as one tumor cell in the background of one million-one billion of normal blood cells, has made the effective detection of the cells extremely difficult. Here we report a novel surface chemistry that utilizes the multivalent effect for a highly stable cancer cell capture. Poly(amidoamine) (PAMAM) dendrimers have been reported as an excellent multivalent binding mediator due to their deformability and multiple functional groups. PAMAM dendrimers were conjugated with anti-epithelial cell adhesion molecules (aEpCAM), a commonly used antibody for CTC detection. A direct, quantitative analysis using surface plasmon resonance (SPR) revealed that the aEpCAM-dendrimer conjugates exhibited a dramatically enhanced binding avidity by a million-fold, compared to free aEpCAM. Dendrimers and aEpCAM were successfully immobilized on the surfaces, as confirmed by x-ray photoelectron spectroscopy and fluorescence microscopy. The surfaces were tested using in vitro cell lines (MDA-MB-361, MCF-7, and MDA-MB-231 cells as a CTC model). Surface immobilization of the dendrimer-aEpCAM conjugates resulted in a significantly enhanced tumor cell detection than the surfaces with the linear PEG-aEpCAM conjugates by ∼1.9 fold with higher binding stability over agitation (up to 69.1-fold). The enhancement through dendrimer-mediated multivalent binding effect was further increased up to 7-fold with addition of E-selectin that recruits the cancer cells onto the surface and induces their rolling under flow. This study demonstrates a novel surface engineering approach to exploiting the strong multivalent binding for sensitive detection of tumor cells, which has great potential for clinically significant detection of CTCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2388. doi:1538-7445.AM2012-2388

Validation of a multiparameter flow cytometry method for the determination of phosphorylated extracellular‐signal‐regulated kinase and DNA in circulating tumor cells

A simple, selective, and sensitive multiparameter fluorescence activated cell sorting method utilizing density gradient centrifugation and magnetic antibody cell sorting was developed and validated for the determination of phosphorylated extracellular-signal-regulated kinase (pERK) and DNA in circulating tumor cells (CTCs). Cell preparation tubes (CPT) were used for peripheral blood collection and density gradient centrifugation, followed by phosphorylation of ERK with epidermal growth factor (EGF). After fixation with formaldehyde and methanol, magnetic anti epithelial cell adhesion molecule (EpCAM) micro-beads were used for the selective isolation of CTCs from the background, consisting of peripheral blood mononuclear cells and platelets. Subsequently, samples were stained with Hoechst 33342, and fluorescent antibodies against EpCAM, CD45, and pERK. Flow cytometry was used for identification and enumeration of CTCs and determination of their pERK and DNA content. The validation parameters included specificity, recovery, linearity, precision, sensitivity, and stability. The lower limit of quantification was two CTCs per 8 ml peripheral blood. Samples were stable for 4 months in storage at -80°C. The applicability of the method was demonstrated by successful enumeration of CTCs, and the determination of DNA, and pERK before and after stimulation with EGF in 8 ml peripheral blood samples from patients with metastatic cancer.

Toll-like receptor-4 expression by hepatic progenitor cells and biliary epithelial cells in HCV-related chronic liver disease

Notwithstanding numerous evidences implicating toll-like receptor-4 (TLR4) in the pathogenesis of chronic hepatitis C virus (HCV) infection, the localization and level of TLR4 expression in the liver of patients with hepatitis C have never been investigated. We aimed to evaluate, by means of immunohistochemistry and real-time PCR (rt-PCR), hepatic TLR4 expression in patients with chronic HCV infection. Fifty patients who had undergone liver biopsy and 11 patients transplanted because of chronic HCV infection, and 12 controls free of liver disease, were included in the study. Each case was analyzed by immunohistochemistry for TLR4, α–smooth muscle actin and cytokeratin-7 (CK-7), and a subgroup of patients and all controls by rt-PCR for TLR4. Immunohistochemistry for α-smooth muscle actin was used to derive a score of activation of hepatic stellate cells and portal/septal myofibroblasts, while immunohistochemistry for CK-7 was used to evaluate and count hepatic progenitor cells, interlobular bile ducts and intermediate hepatocytes. In patients, the parenchymal elements responsible for the highest TLR4 level of expression were hepatic progenitor cells and biliary epithelial cells of interlobular bile ducts. Double-labeling experiments between anti-TLR4 and anti-CK7, anti-CD133, anti-CD44, anti-neural cell adhesion molecule, anti-epithelial cell adhesion molecule and anti-sex determining region Y-box 9, confirmed these findings. TLR4-positive hepatic progenitor cells and interlobular bile ducts were significantly correlated with the stage of liver disease (P<0.001), the grade of inflammation (P<0.001), and the activity of portal/septal myofibroblasts (P<0.001). rt-PCR study confirmed an increased TLR4 expression in the 26 patients analyzed with respect to controls (P<0.001). TLR4 expression positively correlated with fibrosis (P<0.05) and inflammation (P<0.05). The present results suggest that TLR4 expression by hepatic progenitor cells and biliary epithelial cells contributes to the progression of liver damage in the course of chronic HCV-related infection.

Anti-epithelial cell adhesion molecule monoclonal antibody conjugated fluorescent nanoparticle biosensor for sensitive detection of colon cancer cells

In this paper, a sensitive and selective sensor for detecting colon cancer cells based on nanoparticle covalent modified anti-human epithelial cell adhesion molecule (EpCAM) antibody is developed. The transmission electron microscope (TEM) images showed that the nanoparticle and functionalized nanoparticle had good decentrality for application. The NaIO4 oxidation method, which was used as oxidizing antibody for immobilization of conjugating antibody on the silica-coated fluorescent nanoparticles, maintained the activities of antibodies very well. The fluorescence microscopy imaging and flow cytometer (FCM) experiments demonstrated that the nanosensor could increase the signal intensity obviously and distinguish three kinds of target cells (colo205, sw480 and NCM460) well. The membrane and nuclear staining showed the distribution and abundance of EpCAM in cells’ membrane. It also provides a possibility to quantify special membrane proteins on different regions of cells’ surface. At the end, the result of detecting a simple sample proved that colo205 cells were selected by anti-EpCAM antibody nanosensors in this environment, and made a good foundation for subsequent research.

SSA-MOA: a novel CTC isolation platform using selective size amplification (SSA) and a multi-obstacle architecture (MOA) filter

Circulating tumor cells (CTCs) have gained increasing attention as physicians and scientists learn more about the role these extraordinarily rare cells play in metastatic cancer. In developing CTC technology, the critical criteria are high recovery rates and high purity. Current isolation methods suffer from an inherent trade-off between these two goals. Moreover, ensuring minimal cell stress and robust reproducibility is also important for the clinical application of CTCs. In this paper, we introduce a novel CTC isolation technology using selective size amplification (SSA) for target cells and a multi-obstacle architecture (MOA) filter to overcome this trade-off, improving both recovery rate and purity. We also demonstrate SSA-MOA's advantages in minimizing cell deformation during filter transit, resulting in more stable and robust CTC isolation. In this technique, polymer microbeads conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) were used to selectively size-amplify MCF-7 breast cancer cells, definitively differentiating from the white blood cells (WBCs) by avoiding the size overlap that compromises other size selection methods. 3 μm was determined to be the optimal microbead diameter, not only for size discrimination but also in maximizing CTC surface coverage. A multi-obstacle architecture filter was fabricated using silicon-on-glass (SOG) technology-a first such application of this fabrication technique-to create a precise microfilter structure with a high aspect ratio. The filter was designed to minimize cell deformation as simulation results predicted that cells captured via this MOA filter would experience 22% less moving force than with a single-obstacle architecture. This was verified by experiments, as we observed reliable cell capture and reduced cell deformation, with a 92% average recovery rate and 351 peripheral blood leukocytes (PBL) per millilitre (average). We expect the SSA-MOA platform to optimize CTC recovery rates, purity, and stability, increasing the sensitivity and reliability of such tests, thereby potentially expanding the utilization of CTC technologies in the clinic.

Development of a GMP Phase III purification process for VB4-845, an immunotoxin expressed in E. coli using high cell density fermentation

VB4-845 is a recombinant immunotoxin comprised of an anti-epithelial cell adhesion molecule (EpCAM) scFv fused to a truncated form of the bacterial toxin, Pseudomonas exotoxin A. VB4-845, purified from TB fed-batch fermentation, showed clinical efficacy when administered locally to treat non-muscle invasive bladder cancer (NMIBC) and squamous cell carcinomas of the head and neck (SCCHN). Here, we describe the implementation of an Escherichia coli high cell density (HCD) cultivation and purification process for VB4-845. HCD cultivation was a prerequisite for achieving higher yields necessary for Phase III clinical trials and commercialization. Using this process, the VB4-845 titer in the supernatant was increased by 30-fold over the original TB fed-batch cultivation. To obtain clinical grade material, a process involving a five-step column purification procedure was implemented and led to an overall recovery of ∼40%. VB4-845 purity of >97% was achieved after the first three columns following the removal of low-molecular weight product-related impurities and aggregates. Endotoxins were effectively separated from VB4-845 on the Q-columns and by washing the Ni-column with a detergent buffer while host cell proteins were removed using ceramic hydroxyapatite. Comparability studies demonstrated that the purified product from the Phase III process was identical to the Phase II reference standard produced using TB fed-batch fermentation.

Expected clinical applications of circulating tumor cells in breast cancer

Tumor cell invasion and intravascular filtration lead to the presence of circulating tumor cells (CTCs) in peripheral blood. CTCs have, thus, been counted in patients with cancer to analyze metastatic mechanisms or in the hope of developing clinical applications for diagnosis and therapy; various CTC-related studies have been performed. However, the clinical significance of CTCs remains to be established because of the extremely small number of CTCs in peripheral blood as compared with the number of blood cells. Technical problems (e.g. reproducibility and reliability) in the detection of CTCs also remain to be solved. The use of flow cytometric analysis, which can be performed with tumor-cell markers such as anti-epithelial cell adhesion molecule antibodies and anti-cytokeratin antibodies and non-tumor-cell markers such as anti-CD45 antibodies has enhanced specificity for the detection of tumor cells. The CellSearch System(®) can detect 1 CTC in 7.5 mL of peripheral blood, with high reproducibility. Its detection rate and accuracy for CTCs have been confirmed. In the United States, clinical trials have used this system to detect CTCs in patients with metastatic breast cancer, metastatic colorectal cancer, and metastatic prostate cancer, and CTCs have been confirmed to be a useful prognostic factor. This system was also suggested to be useful for monitoring treatment response in patients with metastatic breast cancer and was approved by the United States Food and Drug Administration in 2004. Measuring CTC counts can facilitate the early prediction of treatment response and thereby avoid unnecessary therapy. CTCs may also be a useful biomarker for molecular targeted agents, enabling the identification of patients most likely to respond to a given treatment and facilitating treatment selection. However, the widespread use of CTC monitoring as a routine examination requires a further improvement in measurement sensitivity, the establishment of criteria for quantitative and qualitative evaluations, and additional clear-cut evidence supporting the clinical significance of CTCs. We expect that CTCs will be established to be a new diagnostic and therapeutic index for breast cancer.

Microchip-based immunomagnetic detection of circulating tumor cells

Screening for circulating tumor cells (CTCs) in blood has been an object of interest for evidence of progressive disease, status of disease activity, recognition of clonal evolution of molecular changes and for possible early diagnosis of cancer. We describe a new method of microchip-based immunomagnetic CTC detection, in which the benefits of both immunomagnetic assay and the microfluidic device are combined. As the blood sample flows through the microchannel closely above arrayed magnets, cancer cells labeled with magnetic nanoparticles are separated from blood flow and deposited at the bottom wall of the glass coverslip, which allows direct observation of captured cells with a fluorescence microscope. A polydimethylsiloxane (PDMS)-based microchannel fixed on a glass coverslip was used to screen blood samples. The thin, flat dimensions of the microchannel, combined with the sharp magnetic field gradient in the vicinity of arrayed magnets with alternate polarities, lead to an effective capture of labeled cells. Compared to the commercially available CellSearch™ system, fewer (25%) magnetic particles are required to achieve a comparable capture rate, while the screening speed (at an optimal blood flow rate of 10 mL h(-1)) is more than five times faster than those reported previously with a microchannel-based assay. For the screening experiment, blood drawn from healthy subjects into CellSave™ tubes was spiked with cultured cancer cell lines of COLO205 and SKBR3. The blood was then kept at room temperature for 48 hours before the screening, emulating the actual clinical cases of blood screening. Customized Fe(3)O(4) magnetic nanoparticles (Veridex Ferrofluid™) conjugated to anti-epithelial cell adhesion molecule (EpCAM) antibodies were introduced into the blood samples to label cancer cells, and the blood was then run through the microchip device to capture the labelled cells. After capture, the cells were stained with fluorescent labelled anti-cytokeratin, DAPI and anti-CD45. Subsequent immunofluorescence images were taken for the captured cells, followed by comprehensive computer aided analysis based on fluorescence intensities and cell morphology. Rare cancer cells (from ∼1000 cells down to ∼5 cells per mL) with very low tumor cell to blood cell ratios (about 1 : 10(7) to 10(9), including red blood cells) were successfully detected. Cancer cell capture rates of 90% and 86% were demonstrated for COLO205 and SKBR3 cells, respectively.

The Trifunctional Antibody Catumaxomab in Treatment of Malignant Ascites and Peritoneal Carcinomatosis

Peritoneal carcinomatosis remains an unsolved medical problem in modern oncologic treatment. Excruciating symptoms such as malignant ascites, ileus, nausea, vomiting, dyspnoea and pain deteriorate the quality of life for affected patients. There is still no effective standard treatment for peritoneal carcinomatosis. The trifunctional antibody catumaxomab (antiepithelial cell adhesion molecule x anti-CD3) is able to direct T lymphocytes and Fcg-receptor-positive accessory cells to epithelial cell adhesion molecule-positive tumor cells. Intraperitoneal catumaxomab therapy was shown to be the first effective therapy against accumulation of malignant ascites in patients with peritoneal carcinomatosis of epithelial cancer, reducing the need of paracentesis and prolonging puncture-free survival. This paper reviews the mode of action of catumaxomab and analyzes different fields of local immunotherapy in patients with peritoneal carcinomatosis. A summary of completed and ongoing studies is included. Catumaxomab is discussed to be an outstanding option for local control and therapy of peritoneal carcinomatosis, which could be an optimal modular therapy in addition to systemic chemotherapy and surgical tumor resection.

The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelial cancer: Results of a prospective randomized phase II/III trial

Malignant ascites is a common manifestation of advanced cancers, and treatment options are limited. The trifunctional antibody catumaxomab (anti-epithelial cell-adhesion molecule x anti-CD3) represents a targeted immunotherapy for the intraperitoneal (i.p.) treatment of malignant ascites secondary to epithelial cancers. In this phase II/III trial (EudraCT 2004-000723-15; NCT00836654), cancer patients (n = 258) with recurrent symptomatic malignant ascites resistant to conventional chemotherapy were randomized to paracentesis plus catumaxomab (catumaxomab) or paracentesis alone (control) and stratified by cancer type (129 ovarian and 129 nonovarian). Catumaxomab was administered as an i.p. infusion on Days 0, 3, 7 and 10 at doses of 10, 20, 50 and 150 μg, respectively. The primary efficacy endpoint was puncture-free survival. Secondary efficacy parameters included time to next paracentesis, ascites signs and symptoms and overall survival (OS). Puncture-free survival was significantly longer in the catumaxomab group (median 46 days) than the control group (median 11 days) (hazard ratio = 0.254: p < 0.0001) as was median time to next paracentesis (77 versus 13 days; p < 0.0001). In addition, catumaxomab patients had fewer signs and symptoms of ascites than control patients. OS showed a positive trend for the catumaxomab group and, in a prospectively planned analysis, was significantly prolonged in patients with gastric cancer (n = 66; 71 versus 44 days; p = 0.0313). Although adverse events associated with catumaxomab were frequent, they were manageable, generally reversible and mainly related to its immunologic mode of action. Catumaxomab showed a clear clinical benefit in patients with malignant ascites secondary to epithelial cancers, especially gastric cancer, with an acceptable safety profile.

Phase II Study of the Human Anti-Epithelial Cell Adhesion Molecule Antibody Adecatumumab in Prostate Cancer Patients with Increasing Serum Levels of Prostate-Specific Antigen after Radical Prostatectomy

Background: Rising serum levels of prostate-specific antigen (PSA) after radical prostatectomy are indicative of recurrent prostate cancer. This double-blind, placebo-controlled phase II study evaluated the anti-tumour activity of the anti-epithelial cell adhesion molecule (EpCAM) antibody adecatumumab in delaying biochemical disease progression. Patients and Methods: Prostate cancer patients with increasing serum PSA levels following radical prostatectomy were randomized to low- (2 mg/kg) or high-dose adecatumumab (6 mg/kg) or placebo. The primary efficacy endpoint was the mean change from baseline in total serum PSA at week 24. Secondary endpoints included PSA response rate, prolongation of serum PSA doubling time and time to biochemical disease progression. Results: The primary and secondary endpoints of the study were not met in the predefined analyses. In a retrospective analysis of patients with baseline PSA ≤1 ng/ml and a high EpCAM expression, both the mean increase in PSA from baseline to week 24 and the PSA doubling time at week 15 were significantly improved in the high-dose adecatumumab group compared with the placebo group. Most frequent treatment-related clinical adverse events were gastrointestinal (diarrhoea and nausea) or general events (chills), showing a dose dependency but no grade 3/4 intensity in any patient. Conclusion: In men with rising PSA levels after radical prostatectomy and no evidence of clinical relapse, adecatumumab delayed disease progression in a subgroup of patients with baseline PSA levels ≤1 ng/ml and high EpCAM-expressing tumours.

Engineering and Biological Characterization of VB6-845, an Anti-EpCAM Immunotoxin Containing a T-cell Epitope-depleted Variant of the Plant Toxin Bouganin

The clinical development of immunotoxins in the treatment of solid tumors has been impeded in part, by the induction of an immune response directed primarily against the toxin moiety. Bouganin, a type I ribosome inactivating protein isolated from the leaf of Bougainvillea spectabilis Willd, was mutated to remove the T-cell epitopes while preserving the biological activity of the wild-type molecule. The T-cell epitope-depleted variant of bouganin (de-bouganin) was genetically linked to an anti-epithelial cell adhesion molecule (EpCAM) Fab moiety via a peptidic linker containing a furin proteolytic site to create the fusion construct VB6-845. To determine the optimal construct design for VB6-845, several dicistronic units where de-bouganin was genetically linked to either the N-terminal or C-terminal of either the heavy or light chain were engineered. Only the C-terminal variants expressed the full-length molecule. An in vitro assessment of the biological activity of VB6-845 showed that it bound and selectively killed EpCAM-positive cell lines with a greater potency than many commonly used chemotherapeutic agents. In vivo efficacy was demonstrated using an EpCAM-positive human tumor xenograft model in SCID mice with the majority of the mice treated being tumor free at the end of the study.

Characterization of ERG, AR and PTEN Gene Status in Circulating Tumor Cells from Patients with Castration-Resistant Prostate Cancer

Hormone-driven expression of the ERG oncogene after fusion with TMPRSS2 occurs in 30% to 70% of therapy-naive prostate cancers. Its relevance in castration-resistant prostate cancer (CRPC) remains controversial as ERG is not expressed in some TMPRSS2-ERG androgen-independent xenograft models. However, unlike these models, CRPC patients have an increasing prostate-specific antigen, indicating active androgen receptor signaling. Here, we collected blood every month from 89 patients (54 chemotherapy-naive patients and 35 docetaxel-treated patients) treated in phase I/phase II clinical trials of an orally available, highly specific CYP17 inhibitor, abiraterone acetate, that ablates the synthesis of androgens and estrogens that drive TMPRSS2-ERG fusions. We isolated circulating tumor cells (CTC) by anti-epithelial cell adhesion molecule immunomagnetic selection followed by cytokeratin and CD45 immunofluorescence and 4',6-diamidino-2-phenylindole staining. We used multicolor fluorescence in situ hybridization to show that CRPC CTCs, metastases, and prostate tissue invariably had the same ERG gene status as therapy-naive tumors (n=31). We then used quantitative reverse transcription-PCR to show that ERG expression was maintained in CRPC. We also observed homogeneity in ERG gene rearrangement status in CTCs (n=48) in contrast to significant heterogeneity of AR copy number gain and PTEN loss, suggesting that rearrangement of ERG may be an earlier event in prostate carcinogenesis. We finally report a significant association between ERG rearrangements in therapy-naive tumors, CRPCs, and CTCs and magnitude of prostate-specific antigen decline (P=0.007) in CRPC patients treated with abiraterone acetate. These data confirm that CTCs are malignant in origin and indicate that hormone-regulated expression of ERG persists in CRPC.

A phase I clinical study of intratumorally administered VB4-845, an anti-epithelial cell adhesion molecule recombinant fusion protein, in patients with squamous cell carcinoma of the head and neck

VB4-845 is a novel recombinant fusion protein that targets the epithelial cellular adhesion molecule (EpCAM). This initial clinical trial was conducted to determine the maximum tolerated dose of intratumoral injections in patients with advanced squamous cell carcinoma of the head and neck and to assess pharmacokinetics and immunogenicity. Twenty-four patients with advanced, recurrent squamous cell carcinoma of the head and neck received two cycles of five daily intratumoral VB4-845 injections of 20, 40, 80, 130, 200, or 280 microg. The maximum tolerated dose was established to be 280 microg administered daily for 5 days. Common adverse events were pain due to intratumoral injection and reversibly elevated liver enzymes. Of the 24 patients, 15 had detectable blood levels with a mean drug half-life of 4.0 +/- 0.3 h. VB4-845 reduced or stabilized tumors in 71.4% of epithelial cell adhesion molecule-positive patients. VB4-845 intratumoral injection therapy was well tolerated and feasible.

Exosomal microRNA: A Diagnostic Marker for Lung Cancer

BackgroundTo date, there is no screening test for lung cancer shown to affect overall mortality. MicroRNAs (miRNAs) are a class of small noncoding RNA genes found to be abnormally expressed in several types of cancer, suggesting a role in the pathogenesis of human cancer. Their accumulation within the peripheral circulation appears to be unique to cancer. The genome-wide expression profiling of miRNAs has been shown to be significantly different among primary lung cancers and corresponding noncancerous lung tissues. In studies demonstrating diagnostic miRNA signatures of NSCLC, specific miRNAs were overexpressed compared with normal lung tissue (miR-17-3p, miR-21, miR-106a, miR-146, miR-155, miR-191, miR-192, miR-203, miR-205, miR-210, miR-212, and miR-214). In this study, we evaluate the levels of circulating tumor exosomes, the circulating levels of exosomal small RNA, and specific exosomal miRNAs in patients with and without lung adenocarcinoma, correlating the levels with the American Joint Committee on Cancer (AJCC) stage of disease to validate it as an acceptable marker for diagnosis and prognosis in patients with adenocarcinoma of the lung. Patients and MethodsPlasma from patients with lung adenocarcinoma and a control group without known lung cancer or other active cancer were collected. Exosomes were isolated from plasma samples by a 2-step procedure using size-exclusion chromatography and magnetic activated cell sorting (MACS). Plasma samples (1 mL) were separated on Sepharose 2B, monitoring elution at 280 nm, and using a modified MACS procedure, exosomes of tumor origin were isolated using anti—epithelial cell adhesion molecule. Small RNA was isolated from circulating tumor exosomes using mirVana isolation kit (Ambion, Austin, TX) and this low molecular RNA enriched fraction was used for miRNA profiling as defined by microarray analysis. Low molecular weight RNA (5 μg) was used for hybridization on miRNA microarray chips. These miRNA were identified as hsa-miR-17-3p, hsa-miR-21, hsa-miR-106a, hsa-miR-146, hsa-miR-155, miR-191, miR-192, miR-203, miR-205, miR-210, miR212, and hsa-miR-214. ResultsTo date, 28 patients and 9 controls, AJCC stages I-IV, ages 21–80 years, were enrolled in the study. Exosome concentration ranged from 1.02–9.24 mg/mL for the lung adenocarcinoma group versus 0.62–1.7 mg/mL in the control group. The total miRNA concentration ranged from 131.1–275 ug/mL for the lung adenocarcinoma group versus 44.9–131.1 ug/mL in the control group. The mean exosome value in the lung cancer group was 2.85 mg/mL (CI, 1.94–3.76) and 0.77 mg/mL (CI, 0.68–0.86; P < .001). The mean RNA value in the lung cancer group was 158.6 ug/mL (CI, 145.7–171.5) and 68.1 ug/mL (CI, 57.2–78.9; P < .001). The only patient in the control group who had an exosome concentration > 1.0 mg/mL and RNA concentration > 100 ug/mL had a history of vulvar cancer without evidence of active disease. No correlation between the levels and the stage of disease was found. To compare the presence of specific miRNAs between tumors and their corresponding circulating exosomes, miRNA fractions were isolated and profiled from circulating tumor exosomes and the original tumor. MicroRNA profiling was performed in duplicate, using microarrays containing probes for 467 human mature miRNA. Comparisons between peripheral circulation-derived exosomes and tumors indicated that the miRNA signatures were not significantly different. This approach confirmed that the 12 specific miRNA were elevated in NSCLC and that the associations of these 12 were mirrored in the circulating exosomes. The levels of tumor-derived miRNA profiles exhibited a strong correlation with the levels of peripheral blood-derived exosomal miRNAs (for miR-17-3p, r = 0.76; miR-21, r = 0.77; miR-146, r = 0.88; miR-155, r = 0.85; miR-191, r = 0.83; miR-203, r = 0.85; miR-205, r = 0.91; and miR-214, r = 0.71). ConclusionThe significant difference in total exosome and miRNA levels between patients with lung cancer and controls suggests that exosomal miRNA is a screening test for lung adenocarcinoma. There is no obvious correlation between the total exosomal miRNA levels and stage of disease; however, it has been suggested in miRNA profiling studies of tumor tissue, that miRNA expression might be critical for the development of cancer but not for its progression. If specific miRNA levels predict response to treatment and can add prognostic information in addition to conventional staging needs further study. While validation studies will be necessary before bypassing the use with tumor mass biopsies, the use of exosomal miRNA profiling could extend this approach to screening of asymptomatic individuals as well as for monitoring disease recurrence.

Acquisition of HER2/neu over-expression on circulating tumor cells (CTCs) in patients (pts) with advanced breast cancer (ABC) during chemotherapy

11017 Background: CTCs can be detected and isolated from the peripheral blood of women with ABC. In addition to enumeration, an exciting area of research involves the phenotyping and expression profiling of CTCs. In this regard, assessing the presence of target antigens on CTCs could be considered as a real-time biopsy allowing the possibility to evaluate the change in tumour phenotype during the clinical course of the disease. HER2/neu status of tumor cells detected in metastatic sites may differ from the original primary tumor in about 5–20% of cases. Furthermore, there is evidence that nearly one third of pts whose primary tumors were HER2/neu negative might have amplified HER2/neu on CTCs. Methods: We collected 7.5 ml blood samples from each of 40 pts with ABC. CTCs were isolated with the CellSearch System® (Immunicon Co.) by means of immunomagnetic separation, using ferrofluid nanoparticles binding anti-epithelial cell adhesion molecules (EpCAM), and fluorescently stained with Epithelial Cell Kit®. C...