Characteristics and Outcomes of 241 Births to Women With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection at Five New York City Medical Centers.

OBJECTIVE: To describe the characteristics and birth outcomes of women with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection as community spread in New York City was detected in March 2020. METHODS: We performed a prospective cohort study of pregnant women with laboratory-confirmed SARS-CoV-2 infection who gave birth from March 13 to April 12, 2020, identified at five New York City medical centers. Demographic and clinical data from delivery hospitalization records were collected, and follow-up was completed on April 20, 2020. RESULTS: Among this cohort (241 women), using evolving criteria for testing, 61.4% of women were asymptomatic for coronavirus disease 2019 (COVID-19) at the time of admission. Throughout the delivery hospitalization, 26.5% of women met World Health Organization criteria for mild COVID-19, 26.1% for severe, and 5% for critical. Cesarean birth was the mode of delivery for 52.4% of women with severe and 91.7% with critical COVID-19. The singleton preterm birth rate was 14.6%. Admission to the intensive care unit was reported for 17 women (7.1%), and nine (3.7%) were intubated during their delivery hospitalization. There were no maternal deaths. Body mass index (BMI) 30 or higher was associated with COVID-19 severity (P=.001). Nearly all newborns tested negative for SARS-CoV-2 infection immediately after birth (97.5%). CONCLUSION: During the first month of the SARS-CoV-2 outbreak in New York City and with evolving testing criteria, most women with laboratory-confirmed infection admitted for delivery did not have symptoms of COVID-19. Almost one third of women who were asymptomatic on admission became symptomatic during their delivery hospitalization. Obesity was associated with COVID-19 severity. Disease severity was associated with higher rates of cesarean and preterm birth.

Risk factors associated with delayed discharge following robotic assisted surgery for gynecologic malignancy.

BACKGROUND: The risk factors for extended length of stay (LOS) have not been examined in a cohort of patients with complex social and medical barriers who undergo robotic assisted (RA) surgery for gynecologic malignancies. We sought to identify those patients with a LOS > 24 h after robotic surgery and the risk factors associated with delayed discharge. Then we aimed to develop a predictive model for clinical care and identify modifiable pre-operative risk factors. METHODS: After IRB approval, data was abstracted from medical records of all patients with a gynecologic malignancy who underwent a RA laparoscopic surgery from 2010 to 2015. Univariable and multivariable logistic regression was performed to identify independent risk factors associated with delayed discharge defined as LOS > 24 h. A multi-variable logistic regression model was performed using a stepwise backward selection for the final prediction model. All testing was two-sided and a p-value < 0.05 was considered statistically significant. RESULTS: Of the 406 eligible and evaluable patients, 194 (48%) had a LOS > 24 h. Age ≥ 60 years, a higher usage of narcotic medication, a longer surgical time, and a larger estimated blood loss were all associated with LOS > 24 h (p < 0.05). Many of these women had a social work consultation and went home with home care services despite no surgical or post-operative complications. Our prediction model has the potential to correctly classified 75% of the patients discharged within 24 h. CONCLUSIONS: The development of a pre-hospitalization risk stratification and anticipating the possible need for home care services pre-operatively shows promise as a strategy to decrease LOS in patients classified as high-risk. These findings warrant prospective validation through the use of this prediction model in our institution.

Racial Differences in Survival From Epithelial Ovarian Cancer Are Associated With Stage at Diagnosis and Use of Neoadjuvant Therapy: A 10-Year Single-Institution Experience With a Racially Diverse Urban Population.

OBJECTIVE: The aim of this study was to evaluate the racial/ethnic disparities in ovarian cancer survival in a diverse population. METHODS: We performed a retrospective cohort study evaluating all patients with epithelial ovarian cancer who received primary treatment at Montefiore Medical Center from 2005 to 2015. Clinicopathologic and survival data were abstracted from medical records. Two-sided statistical analyses were performed using SAS 9.3. RESULTS: Three hundred forty-four evaluable patients were identified: 85 (25%) black, 107 (31%) white, 74 (21%) Hispanic, and 78 (23%) other. Black patients were more likely to present with stage IV disease (P = 0.01) and receive neoadjuvant chemotherapy (P < 0.01). By Kaplan-Meier survival analysis, black race was associated with worse recurrence-free survival (P = 0.01) when compared with white race. In multivariate Cox regression model including treatment and stage, race was no longer associated with survival. In a separate multivariate analysis, utilization of neoadjuvant chemotherapy was associated with black race (odds ratio 4.03; 95% confidence interval, 1.56-10.38; P < 0.01) and stage IV disease (odds ratio 3.44; 95% confidence interval, 1.66-7.12; P < 0.01). CONCLUSIONS: In a racially/ethnically diverse population with ovarian cancer, black women had poorer disease-free survival than whites, although this was statistically accounted for by stage at diagnosis and use of neoadjuvant therapy. Research is needed to determine how differences in access/utilization of care and genetic differences in tumor biology may impact late stage diagnosis and use of neoadjuvant chemotherapy among black ovarian cancer patients.

Clinical activity of gemcitabine plus pertuzumab in platinum-resistant ovarian cancer, fallopian tube cancer, or primary peritoneal cancer.

PURPOSE: Pertuzumab is a humanized monoclonal antibody that inhibits human epidermal growth factor receptor 2 (HER2) heterodimerization and has single-agent activity in recurrent epithelial ovarian cancer. The primary objective of this phase II study was to characterize the safety and estimate progression-free survival (PFS) of pertuzumab with gemcitabine in patients with platinum-resistant ovarian cancer. PATIENTS AND METHODS: Patients with advanced, platinum-resistant epithelial ovarian, fallopian tube, or primary peritoneal cancer who had received a maximum of one prior treatment for recurrent cancer were randomly assigned to gemcitabine plus either pertuzumab or placebo. Collection of archival tissue was mandatory to permit exploration of biomarkers that would predict benefit from pertuzumab in this setting. RESULTS: One hundred thirty patients (65 per arm) were treated. Baseline characteristics were similar between arms. The adjusted hazard ratio (HR) for PFS was 0.66 (95% CI, 0.43 to 1.03; P = .07) in favor of gemcitabine + pertuzumab. The objective response rate was 13.8% in patients who received gemcitabine + pertuzumab compared with 4.6% in patients who received gemcitabine + placebo. In patients whose tumors had low HER3 mRNA expression (< median, n = 61), an increased treatment benefit was observed in the gemcitabine + pertuzumab arm compared with the gemcitabine alone arm (PFS HR = 0.32; 95% CI, 0.17 to 0.59; P = .0002). Grade 3 to 4 neutropenia, diarrhea, and back pain were increased in patients treated with gemcitabine + pertuzumab. Symptomatic congestive heart failure was reported in one patient in the gemcitabine + pertuzumab arm. CONCLUSION: Pertuzumab may add activity to gemcitabine for the treatment of platinum-resistant ovarian cancer. Low HER3 mRNA expression may predict pertuzumab clinical benefit and be a valuable prognostic marker.

Development of an optimized conditionally replicative adenoviral agent for ovarian cancer.

Human ovarian cancer is a highly lethal malignant neoplasm in woman with no effective treatment if conventional chemotherapy fails. In this regard, conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution to the development of CRAds was the introduction of tumor-selective viral replication to restrict amplification to the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells, killing the cells by cytolysis, leaving normal cells unaffected. However, to date, there have been limitations to the clinical application of these CRAd agents i.e. poor viral infectivity, poor tumor specificity and high toxicity. Here, we report the in vitro and in vivo comparison of four CRAd agents developed for ovarian cancer application, specifically, Ad-Delta24.F5/3, CRAd-C.F5/3, CRAd-M.F5/3 and CRAd-S.F5/3. All CRAd agents contained fiber knob chimeras of adenovirus serotype 3, which enhanced the viral infectivity at the transductional level via a non-Coxsackie-Adenovirus Receptor alternative pathway. In addition, these CRAds embodied distinct mechanisms for the achievement of replication specificity. Tumor cell killing was assessed by using an oncolytic assay and a cell viability assay (MTS) in vitro, while tumor growth was examined in a xenograft model in vivo by using a bioluminescent imaging assay. In addition, the replication rates of the CRAd agents were determined in human liver slices. Both the Ad-Delta24.F5/3 and CRAd-S.F5/3 were demonstrated to have higher tumor killing effects in tumor cells and a lower viral replication rate in human liver. These agents are thus excellent candidates for clinical trials of CRAd agents against human ovarian cancer.

HER dimerization inhibitors: developing pertuzumab as an anticancer agent in women's oncology.

BACKGROUND: Pertuzumab is a novel monoclonal antibody that blocks the dimerization domain of the human epidermal growth factor receptor (HER)-2/neu receptor, disabling its ability to form heterodimers with the other members of its family. OBJECTIVE: We review the background and scientific rationale, but more specifically cover current clinical trial outcomes of pertuzumab in solid tumors, with an emphasis on the work completed in women's cancers. METHODS: Clinical trial results published or presented at national meetings are included in this review. RESULTS: Pertuzumab shows promising activity with trastuzumab in the treatment of metastatic breast cancer. The results in ovarian cancer have been limited thus far but a post hoc analysis of the results of a completed randomized Phase II trial of gemcitabine with or without pertuzumab suggests that low HER3 levels may mark a group of women who may benefit from the addition of pertuzumab to chemotherapy. CONCLUSIONS: The efficacy of pertuzumab independent of HER-2/neu overexpression remains under investigation. Still, the benefits seen in HER-2/neu positive breast cancer is encouraging. Work in ovarian cancer remains preliminary but the possibility of using pertuzumab in a targeted population with low HER3 levels warrants further evaluation.

Improved anti-tumor therapy based upon infectivity-enhanced adenoviral delivery of RNA interference in ovarian carcinoma cell lines.

BACKGROUND: Hec1 (Highly Expressed in Cancer gene 1) has recently been shown to play an important role in the proper segregation of chromosomes during mitosis. Recently, an adenovirus delivery system carrying RNA interference (RNAi) of Hec1 has been reported in a cervical adenocarcinoma model. Adenoviral delivery systems, however, have the main limitation of poor viral infectivity due to lack of the native receptor, Coxsackie-Adenovirus Receptor (CAR), on the surface of tumor cells. We hypothesize that the viral infectivity of the adenovirus vector would be enhanced via a CAR-independent pathway by altering the targeting tropism, thus increasing the knockdown effect of Hec1 expression in ovarian carcinoma cells. METHODS: Two adenoviruses (Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3), along with a negative control (Ad-siRNA-GAPDH.F5/3), were created using homologous recombination. HEY and SKOV3.ip1 cell lines were used to perform experiments. The following assays were then used to determine RNAi knockdown efficiency: (1) quantitative PCR (QPCR), (2) Western blot, (3) MTS assay, (4) Annexin V-FITC FACS, (5) crystal violet staining. In all experiments, a negative control served as a baseline measure. RESULTS: QPCR demonstrated a 2-log viral infectivity enhancement with Ad-siRNA-Hec1.F5/3 over Ad-siRNA-Hec1. QPCR at 72 h revealed mRNA knockdown induced by Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cells, respectively (71%/60%, and 32%/78% mRNA knockdown compared to negative control). Western blot revealed translational inhibition induced by both Hec1 Ads with the least knockdown seen with Ad-siRNA-GAPDH.F5/3. FACS analysis revealed increased annexin V positivity in RNAi-infected cells, suggesting a higher rate of apoptosis. MTS assay indicated increased cell death 8 days post-infection with Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cell lines, respectively (75% vs. 35% and 43% vs. 12% viable cells). Crystal violet staining revealed increased cell death with Ad-siRNA-Hec1.F5/3 in all tested cell lines. CONCLUSIONS: RNAi against Hec1 results in gene expression knockdown and apoptosis in vitro. The infectivity-enhanced adenovirus as delivery mechanism shows potential application in future gene therapy models of RNAi in ovarian cancer.

Targeting of a conditionally replicative adenovirus agent to human squamous cell carcinomas of the head and neck.

Conventional cancer treatments are not adequate for the majority of most patients stricken with squamous cell carcinomas of the head and neck (SCCHN). Conditionally replicating adenoviruses (CRAds) represent a promising new modality for treating of neoplastic diseases, including SCCHN. Specifically, CRAd agents infect tumor cells and selectively replicate within them, thus causing their death while sparing surrounding normal cells in the host. Oncolysis results from the replicative life cycle of the virus, which lyses infected tumor cells and releases viral progeny for propagation of infection and resultant lysis of neighboring cancer cells, sparing normal host cells. However, to date there have been two main limitations to successful clinical application of these CRAd agents: poor infectivity and poor tumor specificity. Here we report the construction of a CRAd agent, CRAd-CXCR4.F5/3, in which the adenovirus E1 gene is driven by a tumor-specific CXCR4 promoter, and the viral infectivity is enhanced by a fiber modification, F5/3, containing an Ad3 knob chimeric fiber protein. As expected, this agent improved both of the viral infectivity and tumor specificity as evaluated in established SCCHN tumor cell lines and in primary tumor tissues from multiple patients. As an added benefit, the activity of the CXCR4 promoter was low in human liver as described previously. Based on these data, the CRAd-CXCR4.F5/3 is a promising novel CRAd agent for SCCHN targeting with low host toxicity.

Identifying the safety profile of a novel infectivity-enhanced conditionally replicative adenovirus, Ad5-delta24-RGD, in anticipation of a phase I trial for recurrent ovarian cancer.

OBJECTIVE: The purpose of this study was to evaluate the biodistribution and toxicity of the tropism-modified infectivity-enhanced conditionally replicative adenovirus, Ad5-delta24-arginine-glycine-aspartate (RGD). STUDY DESIGN: Cohorts of cotton rats were treated intravenously or intraperitoneally for 3 consecutive days with 5 x 10(8) to 5 x 10(11) particles/kg of Ad5-delta24-RGD or controls and killed on day 8, 17, or 56. For biodistribution studies, tissue samples from 14 organ sites and serum samples were evaluated for the presence of virus with the use of quantitative polymerase chain reaction analysis. For toxicity experiments, tissue samples from more than 30 organ sites and serum samples were obtained for the assessment of vector-related tissue or laboratory effects. RESULTS: Ad5-delta24-RGD was noted in tested samples at days 8 and 17 in animals that were treated intravenously and intraperitoneally with clearance by day 56. There were lower copies of vector noted in the blood and liver specimens of intraperitoneally treated animals. Mild peritonitis histopathologic findings were noted in rats that were treated intraperitoneally with Ad5-delta24-RGD; pathologic findings did not vary significantly with dose, over time, or in comparison to that noted in animals that were treated with Ad5-delta24. CONCLUSION: These studies provide critical insights regarding Ad5-delta24-RGD dosing and anticipated toxicity for a planned clinical trial for ovarian cancer.

Treatment of ovarian cancer with a novel dual targeted conditionally replicative adenovirus (CRAd).

OBJECTIVES: Current virotherapy strategies for ovarian cancer have been hampered by limitations in target cell infectivity and nonspecific tissue replication. In an effort to circumvent these limitations, we evaluated various CRAds modified to incorporate novel capsid targeting motifs (RGD and chimeric Ad5/3) with a novel tissue-specific promoter (CXCR4). METHODS: Two novel CRAds (Ad5-CXCR4-F5/3 and Ad5-CXCR4-RGD) were constructed via homologous recombination and verified by PCR and DNA sequencing. The infectivity and viral replication rates of these two CRAds were analyzed via quantitative real-time PCR (QRT-PCR) in cell line experiments using three ovarian cancer cell lines (SKOV3.ip1, Hey, and OV4) and compared to that achieved with a clinical grade CRAd (delta24-RGD) to be evaluated in a Phase I trial. Cytocidal effects were determined by crystal violet staining in these same cell lines infected with different concentrations of viral particles per cell (0, 0.1, 1, 10, 100, and 500). Additionally, viral replication was evaluated by QRT-PCR in primary ovarian cancer tissue slices from multiple patients with ovarian cancer as well as in primary human normal liver tissue slices in order to establish CRAd selectivity. All experiments incorporated appropriate controls and repeated in triplicate. RESULTS: Compared to RGD-capsid CRAds (delta24-RGD and CXCR4-RGD), the F5/3-capsid CRAd (CXCR4-F5/3) demonstrated significant improvements in infection rates (p=0.025, 0.006, and 0.006) in all ovarian cancer cell lines tested (SKOV3.ip1, Hey, and OV4, respectively). In addition to improved transduction of virus into the cells, the TSP CXCR4-based CRAds demonstrated improved viral replication. Specifically, CXCR4-F5/3 further enhanced viral replication 89-fold (p=0.009, 0.010, 0.003) in the same cancer cell lines. Furthermore, CXCR4-F5/3 showed a 4-log improvement in oncolytic potential over delta24-RGD. In the ex vivo primary ovarian tissue slices, CXCR4-F5/3 showed a 58-fold improvement in viral replication (p=0.005) compared to the clinical grade delta24-RGD. Both CXCR4-F5/3 and CXCR4-RGD demonstrated significant reduction of viral replication in normal liver slices (p=0.001). CONCLUSIONS: These data suggest that a dual targeted approach is feasible for the combined enhancement of infectivity and replication in ovarian cancer with a specificity that was attenuated in normal liver tissues. In fact, CXCR4-F5/3 outperformed our best CRAd agent to date nearly 60-fold in our most stringent ex vivo model of primary ovarian cancer tissue slices and suggests that this novel agent could be useful for the treatment of ovarian cancer.

Targeting lung cancer using an infectivity enhanced CXCR4-CRAd.

Conventional treatments are not adequate for the majority of lung cancer patients. Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of neoplastic diseases, including non-small cell lung cancer. Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis. Next, the progeny virions infect a new population of surrounding target cells, replicate again and eradicate the infected tumor cells while leaving normal cells unaffected. However, to date, there have been two main limitations to successful clinical application of these CRAd agents; i.e. poor infectivity and poor tumor specificity. Here we report the construction of a CRAd agent, CRAd-CXCR4.RGD, in which the adenovirus E1 gene is driven by a tumor-specific CXCR4 promoter and the viral infectivity is enhanced by a capsid modification, RGD4C. This agent CRAd-CXCR4.RGD, as expected, improved both of the viral infectivity and tumor specificity as evaluated in an established lung tumor cell line and in primary tumor tissue from multiple patients. As an added benefit, the activity of the CXCR4 promoter was low in human liver as compared to three other promoters regularly used for targeting tumors. In addition, this agent has the potential of targeting multiple other tumor cell types. From these data, the CRAd-CXCR4.RGD appears to be a promising novel CRAd agent for lung cancer targeting with low host toxicity.

Survivin promoter-based conditionally replicative adenoviruses target cholangiocarcinoma.

Cholangiocarcinoma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard was the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells by cytolysis, leaving normal cells unaffected. However, to date there have been two limitations to the clinical application of these CRAd agents, i.e. poor viral infectivity and tumor specificity. Here we report the construction of three new CRAd agents, CRAd-S.RGD, CRAd-S.F5/3 and CRAd-S.pk7, in which the tumor specificity is regulated by a tumor-specific promoter, the survivin promoter, and the viral infectivity is enhanced by incorporating a capsid modification (RGD, F5/3 or pk7) in the adenovirus fiber region. These CRAd agents effectively target cholangiocarcinoma cells, induce strong cytoxicity in these cells in vitro and inhibit tumor growth in a murine xenograft model in vivo. In addition, the survivin promoter has extremely low activity both in the non-transformed cell line, HMEC, and in human liver tissue. Our results suggest that the survivin-based CRAds are promising agents for targeting cholangiocarcinoma with low host toxicity. Such results should provide important insights into the identification of novel therapeutic strategies for cholangiocarcinoma.

Inducible resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand receptor 2-mediated apoptosis by generation of a blockade at the death domain function.

Induction of tumor cell resistance to therapeutics has been a major obstacle in cancer therapy. Targeting of the death receptors by a natural ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), or agonistic monoclonal antibodies against TRAIL receptor 1 (TRAIL-R1) or TRAIL receptor 2 (TRAIL-R2) has been thought to be a promising cancer therapy. To determine whether tumor cells are able to generate a resistance to apoptosis induced by an anti-TRAIL-R2 antibody, TRA-8, we examined the apoptotic response of human breast and ovarian cancer cell lines after treatment with TRA-8. Our results show that tumor cell resistance to TRA-8 can be induced by repeated treatment of tumor cells with low, non-apoptosis-inducing doses of TRA-8. Interestingly, the induced resistance to apoptosis was not due to a global apoptotic defect in tumor cells but rather a selective defect in the TRAIL-R2 signaling pathway. Whereas TRA-8-treated tumor cells developed a selective resistance to TRAIL-R2-mediated apoptosis, the apoptotic responses induced by TRAIL, an anti-TRAIL-R1 antibody (2E12), and other apoptotic stimuli were not impaired. The expression levels of cell surface TRAIL-R2 were not altered and mutations of TRAIL-R2 were not found in the resistant cells. The induced TRA-8 resistance was due to a selective blockade at the level of the death domain and could be reversed by a wide array of chemotherapeutic agents. Proteomic analysis of death-inducing signaling complex formation during TRA-8 treatment shows that the translocation of TRAIL-R2-associated apoptotic proteins was significantly altered. Our results suggest that the prevention of tumor cell resistance to therapeutic agents that target the death receptors must be taken into consideration.

Icodextrin enhances survival in an intraperitoneal ovarian cancer murine model utilizing gene therapy.

OBJECTIVE: Icodextrin, a novel glucose polymer solution utilized for peritoneal dialysis, has been demonstrated to have prolonged intraperitoneal (IP) instillation volumes in comparison to standard PBS solutions. In an animal model of ovarian cancer, we explored whether a survival advantage exists utilizing icodextrin rather than PBS as a delivery solution for an infectivity enhanced virotherapy approach. METHODS: Initial experiments evaluated whether icodextrin would adversely affect replication of a clinical grade infectivity enhanced conditionally replicative adenovirus (Delta24-RGD). Virus was added to prepared blinded solutions of PBS or icodextrin (20%) and then evaluated in vitro in various human ovarian cancer cell lines (SKOV3.ip1, PA-1, and Hey) and in vivo in a SKOV3.ip1 human ovarian cancer IP murine model. Viral replication was measured by detecting adenovirus E4 gene levels utilizing QRT-PCR. Survival was subsequently evaluated in a separate SKOV3.ip1 ovarian cancer IP murine model. Cohorts of mice were treated in blinded fashion with PBS alone, icodextrin alone, PBS+Delta24-RGD, or icodextrin+Delta24-RGD. Survival data were plotted on Kaplan-Meier curve and statistical calculations performed using the log-rank test. RESULTS: There was no adverse affect of icodextrin on vector replication in the ovarian cancer cell lines nor murine model tumor samples evaluated. Median survival in the IP treated animal cohorts was 23 days for the PBS group, 40 days for the icodextrin group, 65 days for the PBS+Delta24-RGD group, and 105 days for icodextrin+Delta24-RGD (p=0.023). Of note, 5 of the 10 mice in the icodextrin+Delta24-RGD group were alive at the end of the study period, all without evidence of tumor (120 days). CONCLUSIONS: These experiments suggest that the use of dialysates such as icodextrin may further enhance the therapeutic effects of novel IP virotherapy and other gene therapy strategies for ovarian cancer. Phase I studies utilizing icodextrin-based virotherapy for ovarian cancer are currently in development.

Targeting mesothelioma using an infectivity enhanced survivin-conditionally replicative adenoviruses.

Mesothelioma is a highly malignant neoplasm with no effective treatment. Conditionally replicative adenoviruses (CRAds) represent a promising new modality for the treatment of cancer in general. A key contribution in this regard is the introduction of tumor-selective viral replication for amplification of the initial inoculum in the neoplastic cell population. Under ideal conditions following cellular infection, the viruses replicate selectively in the infected tumor cells and kill the cells by cytolysis, leaving normal cells unaffected. However, to date there have been two limitations to clinical application of these CRAd agents; viral infectivity and tumor specificity have been poor. Herein we report on two CRAd agents, CRAd-S.RGD and CRAd-S.F5/3, in which the tumor specificity is regulated by a tumor-specific promoter, the survivin promoter, and the viral infectivity is enhanced by incorporating a capsid modification (RGD or F5/3) in the adenovirus fiber region. These CRAd agents effectively target human mesothelioma cell lines, induce strong cytoxicity in these cells in vitro, and viral replication in a H226 murine xenograft model in vivo. In addition, the survivin promoter has extremely low activity both in the non-transformed cell line, HMEC, and in human liver tissue. Our results suggest that the survivin-based CRAds are promising agents for targeting mesothelioma with low host toxicity. These agents should provide important insights into the identification of novel therapeutic strategies for mesothelioma.

Incorporating the survivin promoter in an infectivity enhanced CRAd-analysis of oncolysis and anti-tumor effects in vitro and in vivo.

Conditionally replicating adenoviruses (CRAds) represent a promising new modality for the treatment of cancer. A key contribution in this regard was the introduction of tumor-selective viral replication for amplification of the initial inoculum. Specifically, following cellular infection, the virus replicates selectively in the infected tumor cells and kills the cells by cytolysis. Next, the progeny virions infect surrounding target cells, replicate and eradicate the infected tumor cells, leaving normal cells unaffected. However, to date there have been two limitations to clinical application of these CRAd agents; i.e., both infectivity and tumor specificity are poor. Survivin protein is a novel member of the inhibitor of apoptosis (IAP) protein family, which plays an important role in the survival of cancer cells and progression of malignancies. Previous data have shown the survivin promoter has high activities in multiple cancer cells with a low activity in mouse liver. In this study, we propose an improved CRAd agent to circumvent the obstacles. We constructed a novel CRAd agent, CRAd-Survivin-RGD, which contains both the survivin promoter (either the short version, S-S, or the long version, S-L) to selectively drive E1 gene expression in tumor cells and a capsid modification and RGD4C to specifically enhance the tumor infectivity of CRAd agents. Both CRAd agents (S-S and S-L) showed high replication rates in the breast cancer cell line, MDA-MB-361, and low promoter activity in both normal mouse and human liver, thus signifying the CRAd agents have the phenotype of 'tumor on/liver off'. In cytocidal experiments, the CRAd agents demonstrated a high cytocidal effect on multiple cancer cell lines, including the breast cancer cell line, MDA-MB-231; the glioma cell line, D65, the melanoma cell line, MEL-28; and mesothelioma, Meso2374. The results also showed the tumor growth was dramatically inhibited by intertumoral administration of the CRAd agents in a breast cancer (MDA-MB-361) xenograft animal model. These data clearly demonstrate that CRAd-Survivin-RGD is a potential novel therapeutic agent for treatment in many, but not all, human cancers.

Targeted gene therapy for ovarian cancer.

Despite advances in therapy, advanced ovarian cancer maintains a dismal overall survival of 15-30%. Thus, the need for novel therapeutic modalities exists. Gene therapy represents one such approach and the purpose of this review is to present a logical rationale for the investigation of gene therapy for the treatment of ovarian cancer. The different strategies of gene therapy (molecular chemotherapy (prodrugs), mutation compensation, immunotherapy approaches, altered drug sensitivity, and virotherapy) for cancer treatment are discussed separately with attention to investigations with clinical applicability. Furthermore, the different viral vectors utilized for improvements in targeted therapy are presented. The advancements, discovery, and shortcomings are reviewed which lend itself to future directions. These future directions involve coxsackie-adenovirus receptor (CAR) independent pathways to improve infectivity and specificity to ovarian tumor cells, the potential of utilizing gene therapy as an imaging modality in detecting cancer, and incorporating the recently described technique of RNA interference. Due to the advancements in detection and targeting of ovarian cancer, coupled with the containment to the intraperitoneal cavity, gene therapy remains a promising treatment modality for ovarian cancer.

Transport across a polarized monolayer of Caco-2 cells by transferrin receptor-mediated adenovirus transcytosis.

Adenoviral vectors have a poor record of transgene delivery efficiency through physical barriers such as the epithelium or endothelium. We report here the construction of an adenoviral vector that has the capability to be transported across polarized epithelial monolayers of Caco-2 cells (a colon carcinoma cell line) by transcytosis. This transcytosis is transferrin receptor (TfR)-mediated with use of a bifunctional adaptor, soluble coxsackie adenovirus receptor (sCAR)-Tf, and is both temperature and iron dependent. Under experimental conditions, the adenoviral transcytosis was inhibited by pretreatment of Caco-2 cells with colchicine, an inhibitor of transcytosis, and was not enhanced by pretreatment with Brefeldin A (BFA), an enhancer of transcytosis. In these Caco-2 cells, the transcytosis rate was 0.3 +/- 1.3% (SD). The transcytosed adenoviruses remain biologically functional. These data suggest the potential clinical benefit under conditions where drug delivery is a challenge, such as within the airway epithelium, at the bladder lumen urothelial cell interface, and across the blood-brain barrier for clinical treatment of lung, urogenital, and brain disorders, respectively, by adenoviral transcytosis of transgene delivery.

Transcriptional targeting of tumors with a novel tumor-specific survivin promoter.

It has been demonstrated that survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, is expressed in human cancers but is undetectable in normal differentiated tissues. We employed a recombinant adenoviral vector (reAdGL3BSurvivin) in which a tumor-specific survivin promoter and a luciferase reporter gene were inserted into the E1-deleted region of adenovirus vector. Luciferase activity was measured in both multiple tumor cell lines and two primary melanoma cells infected with reAdGL3BSurvivin. Human fibroblast and mammary epithelial cell lines were used as negative controls. A reAdGL3CMV, containing the CMV promoter and luciferase gene, was used as a positive control to normalize the luciferase activity generated by the survivin promoter. Our data revealed that the survivin promoter showed high activity in both established tumor cell lines and the primary melanoma cells. In contrast, the in vivo studies indicated that the activities of survivin promoter were extremely low in the major mouse organs. The survivin promoter appears to be a promising tumor-specific promoter exhibiting a "tumor on" and "liver off" profile, and therefore, it may prove to be a good candidate for transcriptional targeting of cancer gene therapy in a wide variety of tumors.

Synergistic induction of tumor cell apoptosis by death receptor antibody and chemotherapy agent through JNK/p38 and mitochondrial death pathway.

Using two agonistic monoclonal antibodies specific for each death receptor of TRAIL, 2E12 (anti-human DR4) and TRA-8 (anti-human DR5), we examined the signal transduction of the death receptors in combination with or without chemotherapy agents such as Adriamycin (doxorubicin hydrochloride) and Cisplatin. Our results demonstrated that chemotherapy agents were able to enhance apoptosis-inducing activity of these antibodies against several different types of tumor cell lines through enhanced caspase activation. The combination of the antibodies and chemotherapy agents led to a synergistical activation of the JNK/p38 MAP kinase, which was mediated by MKK4. The combination also caused an increased release of cytochrome c and Smac/DIABLO from mitochondria in parallel with the profound loss of mitochondrial membrane potential. These results suggest that the enhanced activation of the JNK/p38 kinase and the mitochondrial apoptosis pathways play a crucial role in synergistic induction of the death receptor-mediated apoptosis by chemotherapy agents. Thus, the simultaneous targeting of cell surface death receptors with agonistic antibodies and the intracellular JNK/p38 and the mitochondrial death pathways with chemotherapy agents would enhance the efficacy and selectivity of both agents in cancer therapy.