Coxsackie-adenovirus Receptor Expression Research Articles

Abstract CT233: A phase 3, single-arm study of CG0070 in subjects with non-muscle invasive bladder cancer (NMIBC) unresponsive to Bacillus Calmette-Guerin (BCG)

Abstract IntroductionCG0070 is a serotype 5 adenovirus engineered to express GM-CSF and replicate in cells with mutated or deficient RB, with response rates (RR) of approximately 45% observed in patients with recurrent NMIBC after BCG (J Urol. 2012;188:2391-7; Urol Oncol. 2018;36(10):440-7). This single arm phase 3 study (NCT04452591) was launched to confirm the clinical activity of CG0070 in patients with BCG Unresponsive NMIBC.Methods110 patients with BCG-unresponsive CIS with or without concurrent Ta or T1 disease will be treated with intravesical (IVe) CG0070 at a dose of 1x1012 vp. CG0070 will be administered as follows: induction weekly x 6 followed by weekly x 3 maintenance instillations at months 3, 6, 9, 12, and 18. Patients with persistent CIS or HG Ta at 3 months (m) may receive re-induction with weekly x 6 CG0070. Assessment of response will include q 3 m cystoscopy with biopsy of areas suspicious for disease, urine cytology, CTU/MRU, and mandatory bladder mapping at 12 m. Detection of high grade disease within the bladder will be enumerated as recurrence or non-response.ResultsThe primary endpoint of the study is CR at any time on study as assessed by biopsy (directed to cystoscopic abnormalities and mandatory mapping at 12 m), urine cytology, and radiography, as above. Secondary endpoints include CR at 12 m, duration of response, progression free survival, cystectomy free survival and safety. Correlative assessments include changes in the tumor immune microenvironment, systemic immune induction as reflected in the peripheral blood and urine, as well as viral replication and transgene expression. Baseline expression of coxsackie adenovirus receptor, E2F transcription factor as well as anti-adenovirus antibody titer will be correlated with tumor response.ConclusionThe study an ongoing trial, currently enrolling in the United States, Japan, South Korea, Australia, and Taiwan.Refs. Burke JM, Lamm DL, Meng MV, Nemunaitis JJ, Stephenson JJ, Arseneau JC, Aimi J, Lerner S, Yeung AW, Kazarian T, Maslyar DJ, McKiernan JM. A first in human phase 1 study of CG0070, a GM-CSF expressing oncolytic adenovirus, for the treatment of nonmuscle invasive bladder cancer. Citation Format: Ed Uchio, Donald Lamm, Neil Shore, Trinity Bivalacqua, Ashish Kamat, Mark Tyson, Paul Anderson, Paola Grandi, James Burke. A phase 3, single-arm study of CG0070 in subjects with non-muscle invasive bladder cancer (NMIBC) unresponsive to Bacillus Calmette-Guerin (BCG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT233.

A phase 3, single-arm study of CG0070 in subjects with nonmuscle invasive bladder cancer (NMIBC) unresponsive to Bacillus Calmette-Guerin (BCG).

TPS598 Background: CG0070 is a serotype 5 adenovirus engineered to express GM-CSF and replicate in cells with mutated or deficient RB, with response rates (RR) of approximately 45% observed in patients with recurrent NMIBC after BCG (J Urol. 2012;188:2391–7; Urol Oncol. 2018;36(10):440–7). This single arm phase 3 study (NCT04452591) was launched to confirm the clinical activity of CG0070 in patients with BCG Unresponsive NMIBC. Methods: 110 patients with BCG-unresponsive CIS with or without concurrent Ta or T1 disease will be treated with intravesical (IVe) CG0070 at a dose of 1x1012 vp. CG0070 will be administered as follows: induction weekly x 6 followed by weekly x 3 maintenance instillations at months 3, 6, 9, 12, and 18. Patients with persistent CIS or HG Ta at 3 months (m) may receive re-induction with weekly x 6 CG0070. Assessment of response will include q 3 m cystoscopy with biopsy of areas suspicious for disease, urine cytology, CTU/MRU, and mandatory bladder mapping at 12 m. Detection of high grade disease within the bladder will be enumerated as recurrence or non-response. The primary endpoint of the study is CR at any time on study as assessed by biopsy (directed to cystoscopic abnormalities and mandatory mapping at 12 m), urine cytology, and radiography, as above. Secondary endpoints include CR at 12 m, duration of response, progression free survival, cystectomy free survival and safety. Correlative assessments include changes in the tumor immune microenvironment, systemic immune induction as reflected in the peripheral blood and urine, as well as viral replication and transgene expression. Baseline expression of coxsackie adenovirus receptor, E2F transcription factor as well as anti-adenovirus antibody titer will be correlated with tumor response. Study enrollment globally is ongoing including in North America, Taiwan, Japan, and South Korea. Refs. Burke JM, Lamm DL, Meng MV, Nemunaitis JJ, Stephenson JJ, Arseneau JC, Aimi J, Lerner S, Yeung AW, Kazarian T, Maslyar DJ, McKiernan JM. A first in human phase 1 study of CG0070, a GM-CSF expressing oncolytic adenovirus, for the treatment of nonmuscle invasive bladder cancer. Clinical trial information: NCT04452591.

Expression and clinicopathologic significance of coxsackie–adenovirus receptor in oral squamous cell carcinoma

This study aimed to explore the relationship between the expression of the coxsackie-adenovirus receptor (CAR) in oral squamous cell carcinoma (OSCC) and the clinicopathologic parameters associated with the disease. The diagnostic and prognostic potential of CAR in OSCC was also investigated. Immunohistochemistry was performed on human tissue microarrays, containing 42 oral mucosa, 69 dysplasia, and 176 OSCC tissue sections, to reveal the expression pattern of CAR. Statistical analysis was used to determine the correlation between CAR expression and the patient survival rate as a measure of the prognostic value of CAR. CAR was overexpressed in human OSCC tissues (P = .002), and higher expression of CAR was associated with a lower survival rate, which was not statistically significant (P = .123). In addition, patients with OSCC in the human papillomavirus (HPV)-positive group showed significantly higher CAR expression compared with the HPV- negative group (P = .0491). This study indicated that CAR expression was upregulated in human OSCC and that patients with OSCC with higher expression of CAR had a lower survival rate. Moreover, CAR expression may be associated with HPV infection.

Gold nanoparticle coatings as efficient adenovirus carriers to non-infectable stem cells.

Mesenchymal stem cells (MSCs) are adult pluripotent cells with the plasticity to be converted into different cell types. Their self-renewal capacity, relative ease of isolation, expansion and inherent migration to tumors, make them perfect candidates for cell therapy against cancer. However, MSCs are notoriously refractory to adenoviral infection, mainly because CAR (Coxsackie-Adenovirus Receptor) expression is absent or downregulated. Over the last years, nanoparticles have attracted a great deal of attention as potential vehicle candidates for gene delivery, but with limited effects on their own. Our data showed that the use of positively charged 14 nm gold nanoparticles either functionalized with arginine–glycine–aspartate (RGD) motif or not, increases the efficiency of adenovirus infection in comparison to commercial reagents without altering cell viability or cell phenotype. This system represents a simple, efficient and safe method for the transduction of MSCs, being attractive for cancer gene and cell therapies.

Effect of Combined Epigenetic Treatments and Ectopic NIS Expression on Undifferentiated Thyroid Cancer Cells.

Poorly differentiated (PDTC) and anaplastic thyroid (ATC) cancer cells are characterized by the acquisition of epigenetic abnormalities, leading to the silencing of both the sodium iodide co-transporter and the Coxsackie adenovirus receptor. As aberrant histone acetylation and DNA methylation represent epigenetic mechanisms involved in neoplastic transformation, our study investigated the anticancer properties of epigenetic modifiers in thyroid carcinoma. The cytotoxicity and gene expression modulation of histone deacetylase and DNA methyltransferase inhibitors were evaluated in both PDTC and ATC. Epigenetic treatments were cytotoxic to tumor thyrocytes and restored sodium iodide co-transporter and Coxsackie adenovirus receptor, expression as well as radioiodine uptake, in PDTC but not in ATC. However, ectopic expression sodium iodide co-transporter re-activated radioiodine incorporation in ATC. The ability of epigenetic treatments to interfere with tumor proliferation and induce Coxsackie adenovirus receptor expression, coupled with the ability of ectopic sodium iodide co-transporter to restore radioiodine uptake, raise the possibility that these therapeutic approaches may provide clinical benefit to patients with thyroid carcinoma refractory to radioiodine treatment.

Effects of the MAPK pathway and the expression of CAR in a murine model of viral myocarditis.

The pathogenesis of viral myocarditis (VMC) is not fully understood. This study aimed to examine the relationship between coxsackie-adenovirus receptor (CAR) and the p38 mitogen activated protein kinase (MAPK) pathway mechanisms in a mouse model. Three groups of mice were established: 5 mice in a control group injected with saline, 15 in the model group injected with coxsackie virus B3 (CVB) and 15 in the intervention group injected with CVB3 but treated with the p38 MAPK inhibitor SB203580. Mice were sacrificed at days 1, 5, 10, 15 and 30 and cardiac tissues were isolated to perform the tests. Quantitative PCR and western blot analysis showed CAR mRNA and protein expression levels were highest in the model group at all time-points (P<0.05). The expression levels of p38 MAPK protein by western blot analysis at days 1, 5 and 10 were obviously higher in the model group (P>0.05). H&E staining used to observe myocardial pathological changes showed the inflammatory infiltration was also higher in the model group at all the time-points (P<0.05). Our results show a direct relationship between CAR and p38 MAPK levels, and since the p38 MAPK inhibitor treatment resulted in reduced levels of CAR as well as lower inflammatory infiltration, it is possible that the signaling pathway may mediate CAR expression during the pathogenesis of VMC.

Coxsackie–adenovirus receptor expression is enhanced in pancreas from patients with type 1 diabetes

ObjectivesOne of the theories connecting enterovirus (EV) infection of human islets with type 1 diabetes (T1D) is the development of a fertile field in the islets. This implies induction of...

Abstract 3739: Efficacy of CD46-mediated Ad5/35 chimeric adenoviral gene therapy in colorectal cancers

Abstract CD46 is a complement inhibitor membrane cofactor protein and also acts as a receptor for certain pathogenic microbes, including group B denovirus (Ad). Whereas many Ads infect cells through coxsackie-adenovirus receptor (CAR), CAR expression is downregulated in many cancers preventing effective therapeutics of Ad-based therapies. There have been increasing numbers of studies in cancer gene therapy by modification of Ad fiber knobs to utilize more ubiquitously expressed CD46. However, very limited information is available on expression status of CD46 in many cancers. Our tissue microarray data demonstrated that CD46 was generally overexpressed to cancers from prostate and colon. To seek the evidence whether colorectal cancers (CRC) are good target for Ad-mediated gene therapy, chimeric Ad5 vectors that are capable of targeting CD46 were employed. CD46-ovrexpressed cells showed significantly higher response not only to Ad5/35-GFP but to Ad5/35-TK/GCV suicide therapy. While CRC cells express variable levels of CD46, CD46 expression was positively correlated with Ad5/35-mediated GFP fluorescence and cell killing, demonstrating that HCT-116, DLD-1, and Caco-2 cells are highly responsive. Furthermore, injection of Ad5/35-TK/GCV caused significantlyhigher anti-cancer effects in nude mice bearing CD46-overexpressed cancer cells compared to mice with mock cells. Using 80 selected CRC, immunohistochemical studies showed that there is inverted correlation between CD46 expression and clinico-pathological parameters in terms of differentiation, invasion, metastasis, T stage, and survival, suggesting that adenoviral gene therapy may not be as effective to the patients with highly advanced colorectal cancers. Taken altogether, our study demonstrated that CD46 is generally overexpressed in CRC in which group B-based adenoviral gene therapy seems to be suitable approach but careful consideration needs to be given to select cancer types and cancer status for effective cancer gene therapies. Citation Format: Young Seok Cho, Hung Manh Do, Sang-Jin Lee, Silvio Hemmi, Young-Eun Joo, Chaeyong Jung. Efficacy of CD46-mediated Ad5/35 chimeric adenoviral gene therapy in colorectal cancers. [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 3739.

Efficacy of CD46-targeting chimeric Ad5/35 adenoviral gene therapy for colorectal cancers.

CD46 is a complement inhibitor membrane cofactor which also acts as a receptor for various microbes, including species B adenoviruses (Ads). While most Ad gene therapy vectors are derived from species C and infect cells through coxsackie-adenovirus receptor (CAR), CAR expression is downregulated in many cancer cells, resulting inefficient Ad-based therapeutics. Despite a limited knowledge on the expression status of many cancer cells, an increasing number of cancer gene therapy studies include fiber-modified Ad vectors redirected to the more ubiquitously expressed CD46. Since our finding from tumor microarray indicate that CD46 was overexpressed in cancers of the prostate and colon, fiber chimeric Ad5/35 vectors that have infection tropism for CD46 were employed to demonstrate its efficacy in colorectal cancers (CRC). CD46-overexpressed cells showed a significantly higher response to Ad5/35-GFP and to Ad5/35-tk/GCV. While CRC cells express variable levels of CD46, CD46 expression was positively correlated with Ad5/35-mediated GFP fluorescence and accordingly its cell killing. Injection of Ad5/35-tk/GCV caused much greater tumor-suppression in mice bearing CD46-overexpressed cancer xenograft compared to mock group. Analysis of CRC samples revealed that patients with positive CD46 expression had a higher survival rate (p=0.031), carried tumors that were well-differentiated, but less invasive and metastatic, and with a low T stage (all p<0.05). Taken together, our study demonstrated that species B-based adenoviral gene therapy is a suitable approach for generally CD46-overexpressed CRC but would require careful consideration preceding CD46 analysis and categorizing CRC patients.

Surmounting limited gene delivery into primary immune cell populations: Efficient cell type-specific adenoviral transduction by CAR.

Ectopic gene expression studies in primary immune cells have been notoriously difficult to perform due to the limitations in conventional transfection and viral transduction methods. Although replication-defective adenoviruses provide an attractive alternative for gene delivery, their use has been hampered by the limited susceptibility of murine leukocytes to adenoviral infection, due to insufficient expression of the human coxsackie/adenovirus receptor (CAR). In this issue of the European Journal of Immunology, Heger etal. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] report the generation of transgenic mice that enable conditional Cre/loxP-mediated expression of human CAR. The authors demonstrate that this R26/CAG-CAR∆1(StopF) mouse strain facilitates the faithful monitoring of Cre activity in situ as well as the specific and efficient adenoviral transduction of primary immune cell populations in vitro. Further tweaking of the system towards more efficient gene transfer in vivo remains a future challenge.

Paclitaxel resistance increases oncolytic adenovirus efficacy via upregulated CAR expression and dysfunctional cell cycle control

Resistance to paclitaxel chemotherapy frequently develops in ovarian cancer. Oncolytic adenoviruses are a novel therapy for human malignancies that are being evaluated in early phase trials. However, there are no reliable predictive biomarkers for oncolytic adenovirus activity in ovarian cancer. We investigated the link between paclitaxel resistance and oncolytic adenovirus activity using established ovarian cancer cell line models, xenografts with de novo paclitaxel resistance and tumour samples from two separate trials. The activity of multiple Ad5 vectors, including dl922-947 (E1A CR2-deleted), dl1520 (E1B-55K deleted) and Ad5 WT, was significantly increased in paclitaxel resistant ovarian cancer in vitro and in vivo. This was associated with greater infectivity resulting from increased expression of the primary receptor for Ad5, CAR (coxsackie adenovirus receptor). This, in turn, resulted from increased CAR transcription secondary to histone modification in resistant cells. There was increased CAR expression in intraperitoneal tumours with de novo paclitaxel resistance and in tumours from patients with clinical resistance to paclitaxel. Increased CAR expression did not cause paclitaxel resistance, but did increase inflammatory cytokine expression. Finally, we identified dysregulated cell cycle control as a second mechanism of increased adenovirus efficacy in paclitaxel-resistant ovarian cancer. Ad11 and Ad35, both group B adenoviruses that utilise non-CAR receptors to infect cells, are also significantly more effective in paclitaxel-resistant ovarian cell models. Inhibition of CDK4/6 using PD-0332991 was able both to reverse paclitaxel resistance and reduce adenovirus efficacy. Thus, paclitaxel resistance increases oncolytic adenovirus efficacy via at least two separate mechanisms – if validated further, this information could have future clinical utility to aid patient selection for clinical trials.

Abstract 17775: Foxo3a Regulates Response to Coxsackievirus B3 Myocarditis via microRNA-155 Triggered NK-cell Activity

Background: FoxO3a is a transcription factor crucial in regulation of cell metabolism, stress resistance and immunity. Recently polymorphisms of FOXO3a have been associated with favorable outcome in inflammatory disease. However, mechanistic insight in FoxO3a effects is still limited. Here, we investigated the role of FoxO3a on NK-cell responses in viral myocarditis. Methods: A mouse model of Coxsackie Virus B3 (CVB3) myocarditis in wild-type and FoxO3a-/- mice was used to investigate the effects of FoxO3a on viral load and inflammation. Characterization of NK cells was performed by cytotoxic assay, surface marker- and interferon expression. Results: FoxO3a-/- mice showed significantly reduced myocardial inflammation, lower viral titers and attenuated expression of pro-inflammatory cytokines compared to wild-type mice 7 days post infection. Viral titers were significantly lower in FoxO3a-/- mice at day 3 while IFN γ and NKp46 expression were up-regulated within the heart suggesting early viral control by enhanced NK cell activity. There were no differences in expression of CAR (coxsackie-adenovirus receptor). Additionally NKp46+ splenic NK cells of FoxO3a-/- mice exhibited a superior functional status ex vivo determined by enhanced expression of CD69, higher frequencies of CD11b+CD27+ effector NK cells and increased cytotoxicity compared to NK cells of wild-type littermates. These effects were not due to different levels of apoptosis. MicroRNA-155 expression, recently reported to be essential in NK-cell activation, was significantly elevated in FoxO3a-/- NK cells while its inhibition led to diminished production of IFNγ in Foxo3a deficient animals. Moreover analysis of healthy human donors for the gain of function FoxO3a SNP rs9400239 revealed significantly reduced CD107a dependent degranulation activity in donors homozygous for the allelic variant. Conclusion: Our results implicate FoxO3a directly in regulation of NK-cell function and suggest an important role in innate immune responses to viral infection. While the anti-inflammatory effects of FoxO3a seem beneficial in chronic inflammation the suppression of innate immunity in acute viral myocarditis could have detrimental effects.

Correlations among persistent viral infection, heart function and Chinese medicine syndromes in dilated cardiomyopathy patients.

To investigate the correlations among persistent viral infection, heart function and Chinese medicine (CM) difined-syndromes in patients with dilated cardiomyopathy (DCM). Fifty patients with DCM in the First Affiliated Hospital of Zhejiang Chinese Medical University from October 2009 to December 2011 were selected as the research subjects, and 30 healthy people were simultaneously selected as the normal control group to detect persistent viral infections after admission. The CM syndrome type and grade of heart function were then evaluated. The expression level of Coxsackie adenovirus receptor (CAR) was detected using the flow cytometry (FCM) technique, coxsackie virus RNA (CVB-RNA) using reverse transcription polymerase chain reaction (RTPCR), and the plasma brain natriuretic peptide (BNP) level with a Triage meter plus diagnosis instrument. Finally, the parameters such as left ventricular end diastolic diameter (LVEDd) and left ventricular ejection fraction (LVEF) were measured by ultrasonic cardiogram. Person correlation analysis was used for measured data, Spearman correlation analysis for rating data, and the Chi-square test for numerical data. CVB-RNA was positive in 22 patients (44%) with DCM, while only 6 cases (20%) were CVB-RNA-positive in the normal control group, with a significant difference between the two groups (P<0.01). The expression level of CAR was significantly elevated in the DCM group compared with the normal control group (P<0.01). In CVB-RNA-positive patients (22 cases), the expression level of CAR was significantly higher than in CVB-RNA-negative patients (28 cases; P<0.01). In the DCM patients, there was a positive correlation between the CAR expression and the BNP level (r=0.34, P<0.05), while no significant difference was found between the CAR expression and the LVEF and LVEDd (r=-0.32, 0.30, P>0.05). There was no clear correlation between virus infection and the CM syndrome types in DCM patients (r=-0.22, P>0.05). According to the sequence of syndrome types: phlegm → qi deficiency → blood stasis → hydroretention with asthenic yang (from low to high), a positive correlation was existed between the BNP levels and CM syndrome types (r=0.139, P<0.05). The expression of CAR on the surface of white cells could be used to detect persistent viral infection. The expression level of CAR and heart function in DCM patients were highly correlated. The expression level of BNP may serve as an objective index for differentiating CM syndromes for patients with DCM.

Assessment of clinical-instrumental, morphological data and expression of coxsackie adenovirus receptor in patients with inflammatory cardiac pathology

In 22 patients with heart failure and/or ventricular arrhythmias presumably of inflammatory etiology the results of clinical and instrumental investigation were analyzed and compared to the endomyocardial biopsy data. In the subgroup of patients with left bundle branch block (LBBB) we revealed features indicative of lesser contribution of inflammatory destruction in pathogenesis of cardiomyopathy. The only virus, detected in biopsy samples, was parvovirus B19. Its persistence in myocardium was not related to activity of inflammation and severity of clinical course. Increased expression of Coxsackie adenovirus receptor (CAR) was found in 20 patients. It was not related to inflammatory cells infiltration and virus persistence in myocardium. Patients with most prominent CAR expression were characteried by right heart dilatation, more severe heart failure and absence of LBBB. Enhancement of CAR expression could reflect the attempt of organism to repair intercellular communications between cardiomyocites and to protect cells from the products of necrotic lysis during long standing inflammation.

Flow cytometry analysis of coxsackievirus B receptors expression in human CaCo-2 cells

AbstractA subset of coxsackieviruses B (CV-B) is able to initiate intestinal infection via the attachment to two cell surface proteins, decayaccelerating factor (DAF) and coxsackie adenovirus receptor (CAR). The aim of the present study was to investigate the expression pattern of these receptors in the polarized CaCo-2 cell line using flow cytometry. The expression of CAR-specific mRNA and proteins was analyzed by reverse transcriptase polymerase chain reaction and western blotting, respectively. Flow cytometry analysis was used to study the surface expression patterns of CAR and DAF. CAR and DAF were well detected at the surface of CaCo-2 cells by flow cytometry. Despite the fact that CAR was susceptible to the action of trypsin, a few amounts of the latter enzyme and a precise dilution did not impair its correct detection by flow cytometry. This technique was used to demonstrate that the density of cells did not influence the expression of CAR at the cell surface. CaCo-2 cells express high levels of CAR and DAF at their surface. Flow cytometry, if used adequately, represents a helpful tool for the study of the interactions between these cells and various viral targets.

Abnormal Expression Of The Major Coxsackie-Adenovirus Receptor CAR On Immature Dysplastic CD105+ Erythroid Progenitor Cells In Patients With MDS and Related Bone Marrow Neoplasms

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of myeloid neoplasms characterized by peripheral cytopenia, bone marrow (BM) dysplasia in one or more hematopoietic lineages, and clonal instability with an increased risk to transform to secondary acute myeloid leukemia (AML). In most patients, the erythroid lineage is affected. During the past few years our knowledge about the pathogenesis of MDS, has increased substantially. However, most of these studies focused on CD34+ stem and progenitor cells or granulomonocytic cells, whereas only little is known about genes aberrantly expressed in dysplastic erythroid cells in MDS.In an attempt to define abnormal gene expression patterns and molecular pathways contributing to erythroid dysplasia in MDS and to identify disease-specific phenotypic and molecular abnormalities in dysplastic erythoid cells, we compared gene expression profiles of erythroid progenitor cells (EryPC) in 10 patients with low-risk MDS (IPSS-R score≤7) and 15 control BM samples. EryPC were defined as CD45low/CD105+ cells and purified from BM samples by multicolor flow cytometry and cell sorting (purity>95%). Gene expression levels were analyzed by Affymetrix array technology (GeneChip U133 Plus 2.0 arrays) and confirmed for a panel of select genes by qPCR. A stringent cut-off (p<0.01; fold-change >3.0) was used to define differences in mRNA expression levels. A total number of 1,180 mRNA species were found to be differently expressed in EryPC in MDS (72 up-regulated, 1,108 down regulated) compared to normal BM EryPC. Among these, a number of genes regulating proliferation and differentiation in lymphohematopoietic cells, including TCEB1 transcription elongation factor B (SIII), the transcriptional repressor YY1, E2F3, and the ATP-dependent RNA helicase DDX5, were found to be downregulated in EryPC in MDS compared to normal BM.In the validation phase of our project, we identified the major Coxsackie-Adenovirus Receptor (CAR) as a rather specifically regulated protein-product in dysplastic EryPC. In fact, as assessed by flow cytometry, a significantly lower or even absent expression of CAR was found on EryPC in 20 of 30 patients with MDS (67%), including 3/4 patients with RCUD, 1/4 with RARS, 6/10 with RCMD, 8/9 with RAEB, and 2/3 with 5q- syndrome, compared to normal BM or non-neoplastic controls (p<0.001). In addition, reduced expression of CAR on EryPC was seen in 11/25 patients with myeloproliferative neoplasms (MPN) with dysplastic BM cells, in 4/8 patients with MDS/MPN overlap syndromes, and in 14/19 patients with AML. By contrast, expression levels of CAR on EryPC were normal in almost all non-neoplastic conditions, including renal anemia (n=6), hepatopathy (n=1), drug-related anemia (n=10), folic acid or vitamin B12 deficiency (n=3), intestinal bleeding (n=1), autoimmune hemolytic anemia (n=2), aplastic anemia (n=2), spherocytosis (n=1), chronic inflammation (n=4), and viral infections (n=2). Moreover, CAR expression on EryPC remained normal in most anemic patients suffering from lymphoid malignancies (n=36). Within the group of patients suffering from infection-related anemia, the lowest expression of CAR was observed in a case of adenovirus infection mimicking MDS. An overview of staining results obtained with anti-CAR antibody in various groups of patients and in healthy controls (normal BM, n=20) is shown in Figure 1. [Display omitted] In summary, our data show that the major Coxsackie-Adenovirus Receptor CAR is expressed at abnormally low levels on dysplastic EryPC in a substantial subset of patients with MDS and in most patients with related BM neoplasms. Since CAR expression remains normal in almost all reactive conditions, deficiency states and lymphoid malignancies, abnormal CAR expression may contribute as a potential indicator of (clonal) dysplastic erythropoiesis in myeloid malignancies, including MDS. The biochemical basis of abnormal expression and the possible role CAR may play in the pathogenesis of MDS and in related BM neoplasms, is currently under investigation. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

Correlation between virus persistent infection and cardic function in patients with dilated cardiomyopathy

In our study, 50 patients with dilated cardiomyopathy (DCM) were selected to investigate the correlation between virus persistent infection and cardic function. We found that 44% of patients with DCM were coxsackie virus B-RNA (CVB-RNA) positive, significantly different from that (20%) of the normal control group (P<0.05). The expression levels of coxsackie adenovirus receptor (CAR) in patients with DCM were significantly higher than those in the normal control group (P<0.01). In CVB-RNA-positive patients, expression levels of CAR were significantly higher than those in CVB-RNA-negative patients (P<0.01). There was a positive correlation between CAR expression and brain natriuretic peptide (BNP) level in patients with DCM, but no significant correlations between the CAR expression level and left ventricular ejection fraction (LVEF) or left ventricular end diastolic diameter (LVEDd). These results showed that expression levels of CAR on the surface of white cells can be used as an indicator for detecting persistent virus infection. We found that expression levels of CAR and heart function in patients with DCM were highly correlated.

The combination of an oxygen-dependent degradation domain-regulated adenovirus expressing the chemokine RANTES/CCL5 and NK-92 cells exerts enhanced antitumor activity in hepatocellular carcinoma

Oncolytic adenoviruses are modified based on adenovirus serotype 5 (Ad5), which belongs to subgroup C and depends on Coxsackie-adenovirus receptor (CAR) to recognize target cells. However, expression of CAR is generally low or lost in certain tumors including hepatocellular carcinoma (HCC). By contrast, CD46 is highly expressed in various types of malignant tumor cells. Therefore, we constructed an adenovirus vector expressing the human RANTES/CCL5 gene regulated by oxygen-dependent degradation domain (ODD) and analyzed its antitumor effects in vitro and in vivo. The human RANTES/CCL5 gene was fused with ODD by PCR and the recombinant oncolytic adenovirus containing RANTES-ODD, SG511-CCL5-ODD, was constructed by the Gateway system, which infected cells by binding CD46. Viral replication experiments were performed to evaluate the selective replication ability of SG511-CCL5-ODD. RANTES expression was determined by ELISA. The chemotactic test was used to analyze the ability of the expressed RANTES to recruit NK92 cells. The antitumor effects of SG511-CCL5-ODD were examined in HCC xenografts in nude mice. A chimeric oncolytic adenovirus, SG511-CCL5-ODD, was constructed successfully. Cells infected with the recombinant virus were able to express RANTES selectively in different environments controlled by ODD and the expressed RANTES was able to recruit NK92 cells by its chemotactic effect in vitro and improve the anticancer immune response in HCC xenografts in nude mice. The chimeric adenovirus SG511-CCL5-ODD highly expressed the RANTES-ODD fusion gene in the hypoxia of HCC under the control of the ODD and effectively attracted NK92 cells and a high number of immunocytes. These factors had complementary advantages and, in combination, exerted enhanced antitumor efficacy.

Expression of the coxsackie adenovirus receptor in neuroendocrine lung cancers and its implications for oncolytic adenoviral infection

Coxsackie adenovirus receptor (CAR) is the primary receptor to which oncolytic adenoviruses have to bind for internalization and viral replication. A total of 171 neuroendocrine lung tumors in form of multitissue arrays have been analyzed resulting in a positivity of 112 cases (65.5%). Immunostaining correlated statistically significant with histopathology and development of recurrence. The subtype small cell lung cancer (SCLC) showed the highest CAR expression (77.6%), moreover the CAR level was correlated to the disease-free survival. Further, high CAR expression level in SCLC cell lines was found in vitro and in vivo when cell lines had been transplanted into immunodeficient mice. A correlation between CAR expression in the primary tumors and metastases development in the tumor model underlined the clinical relevance. Cell lines with high CAR level showed a high infectivity when infected with a replication-deficient adenovirus. Low levels of CAR expression in SCLC could be upregulated with Trichostatin A, a histone deacetylase inhibitor. As a result of the unaltered poor prognosis of SCLC and its high CAR expression it seems to be the perfect candidate for oncolytic therapy. With our clinically relevant tumor model, we show that xenograft experiments are warrant to test the efficiency of oncolytic adenoviral therapy.

Coxsackie-adenovirus receptor as a novel marker of stem cells in treatment-resistant non-small cell lung cancer

BackgroundTreatment resistance resulting from the presence of cancer stem cells (CSCs) remains a challenge in cancer treatment. Little is known about possible markers of CSCs in treatment-resistant non-small cell lung cancer (NSCLC). We explored the coxsackie-adenovirus receptor (CAR) as one such marker of CSCs in models of treatment-resistant NSCLC. Materials and methodsResistant H460 and A549 cell lines were established by repeated exposure to paclitaxel or fractionated radiation. CSC markers were measured by Western blotting and flow cytometry. We also established stable CAR-overexpressing and stable shRNA-CAR-knockdown cell lines and assessed their survival, invasiveness, and tumorigenic capabilities with clonogenic, telomerase, Matrigel, and tumor formation assays. ResultsCAR expression was associated with CSC phenotype both in vitro and in vivo. CAR-overexpressing cells were more treatment-resistant, self-renewing, and tumorigenic than were parental cells, and shRNA-mediated knockdown of CAR expression was sufficient to inhibit these functions. CAR expression also correlated with the epithelial–mesenchymal transition. ConclusionsWe showed for the first time that CAR is a marker of CSCs and may affect the activities of CSCs in treatment-resistant NSCLC. CAR may prove to be a target for CSC treatment and a predictor of treatment response in patients with NSCLC.