Christopher J. LaRocca

Oncolytic adenovirus expressing interferon alpha in a syngeneic Syrian hamster model for the treatment of pancreatic cancer

BACKGROUND The addition of interferon (IFN) alpha to adjuvant chemoradiotherapy regimens resulted in remarkable improvements in survival for pancreatic cancer patients. However, systemic toxicities and insufficient levels of IFN at the tumor sites have limited its widespread adoption in treatment schemes. We have previously developed an IFN-expressing conditionally replicative oncolytic adenovirus and demonstrated its therapeutic effects both in vitro and in vivo. Here, the same vectors were tested in a syngeneic and immunocompetent Syrian hamster model to better understand the roles of adenoviral replication and of the pleiotropic effects of IFN on pancreatic tumor growth suppression. METHODS Oncolytic adenoviruses expressing human or hamster IFN were designed and generated. Viral vectors were tested in vitro to determine qualitative and quantitative cell viability, cyclooxygenase 2 (Cox2) promoter activity, and IFN production. For the in vivo studies, subcutaneous hamster pancreatic cancer tumors were treated with 1 intratumoral dose of virus. Similarly, 1 intraperitoneal dose of virus was used to prolong survival in a carcinomatosis model. RESULTS All cell lines tested demonstrated Cox2 promoter activity. The oncolytic potential of a replication competent adenovirus expressing the IFN cytokine was clearly demonstrated. These viruses resulted in significant tumor growth suppression and survival increases compared with controls in a hamster model. CONCLUSION The profound therapeutic potential of an IFN-expressing oncolytic adenovirus for the treatment of pancreatic cancer was demonstrated in a syngeneic Syrian hamster model. These results strongly suggest the potential application of our viruses as part of combination regimens with other therapeutics.

Showing the Way: Oncolytic Adenoviruses as Chaperones of Immunostimulatory Adjuncts

Oncolytic adenoviruses (OAds) are increasingly recognized as vectors for immunotherapy in the treatment of various solid tumors. The myriads of advantages of using adenovirus include targeted specificity upon infection and selective replication, which lead to localized viral burst, exponential spread of OAds, and antitumor effect. OAds can also induce a strong immune reaction due to the massive release of tumor antigens upon cytolysis and the presence of viral antigens. This review will highlight recent advances in adenoviral vectors expressing immunostimulatory effectors, such as GM-CSF (granulocyte macrophage colony-stimulating factor), interferon-α, interleukin-12, and CD40L. We will also discuss the combination of OAds with other immunotherapeutic strategies and describe the current understanding of how adenoviral vectors interact with the immune system to eliminate cancer cells.

Oncolytic adenoviruses targeted to Human Papilloma Virus-positive head and neck squamous cell carcinomas

OBJECTIVES In recent years, the incidence of Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) has markedly increased. Our aim was to design a novel therapeutic agent through the use of conditionally replicative adenoviruses (CRAds) that are targeted to the HPV E6 and E7 oncoproteins. METHODS Each adenovirus included small deletion(s) in the E1a region of the genome (Δ24 or CB016) intended to allow for selective replication in HPV-positive cells. In vitro assays were performed to analyze the transduction efficiency of the vectors and the cell viability following viral infection. Then, the UPCI SCC090 cell line (HPV-positive) was used to establish subcutaneous tumors in the flanks of nude mice. The tumors were then treated with either one dose of the virus or four doses (injected every fourth day). RESULTS The transduction analysis with luciferase-expressing viruses demonstrated that the 5/3 fiber modification maximized virus infectivity. In vitro, both viruses (5/3Δ24 and 5/3CB016) demonstrated profound oncolytic effects. The 5/3CB016 virus was more selective for HPV-positive HNSCC cells, whereas the 5/3Δ24 virus killed HNSCC cells regardless of HPV status. In vivo, single injections of both viruses demonstrated anti-tumor effects for only a few days following viral inoculation. However, after four viral injections, there was statistically significant reductions in tumor growth when compared to the control group (p<0.05). CONCLUSION CRAds targeted to HPV-positive HNSCCs demonstrated excellent in vitro and in vivo therapeutic effects, and they have the potential to be clinically translated as a novel treatment modality for this emerging disease.

Combination of interferon-expressing oncolytic adenovirus with chemotherapy and radiation is highly synergistic in hamster model of pancreatic cancer

Recent clinical trials utilizing Interferon-alpha (IFN) in combination with chemoradiation have demonstrated significant improvements in the survival of patients with pancreatic cancer. However, efficacy was limited by the systemic toxicity of IFN and low intratumoral levels of the cytokine. We sought to address these drawbacks by using an Oncolytic Adenovirus expressing IFN (OAd-hamIFN) in combination with chemotherapy and/or radiation in regimens mimicking the IFN-based therapies used in clinical trials. IFN expressed from OAd-hamIFN potentiated the cytotoxicity of radiation and chemotherapy (5-FU, Gemcitabine, and Cisplatin), and enhanced pancreatic cancer cell death in both in vitro and in vivo experimental settings. Notably, synergism was demonstrated in therapeutic groups that combined the interferon-expressing oncolytic virus with chemotherapy and radiation. In an in vivo immunocompetent hamster model, treatment regimens combining oncolytic virus therapy with 5-FU and radiation demonstrated significant tumor growth inhibition and enhanced survival. This is the first study to report synergism between an IFN-expressing oncolytic adenovirus and chemoradiation-based therapies. When combined with an IFN-expressing OAd, there is a significant enhancement of radiation and especially chemoradiation, which may broaden the application of this new therapeutic approach to the pancreatic cancer patients who cannot tolerate existing chemotherapy regimens.

The Use of Imaging in Gallbladder Disease

Diseases of the gallbladder and biliary tree are conditions that physicians routinely encounter, and they can present multiple diagnostic and treatment dilemmas. Cholelithiasis, choledocholithiasis, and acute cholecystitis are only some of the many potential diagnoses. Recent advances in imaging modalities have provided numerous options to aid clinicians in confirming or clarifying a particular diagnosis. For most cases of acute cholecystitis, ultrasound remains the initial diagnostic imaging of choice. It is inexpensive, readily available, and easily repeatable. HIDA scans also can be helpful, especially if an initial ultrasound is equivocal. These scans also have a role in the post-operative setting to evaluate for biliary leaks. MRI and MRCP provide high-resolution images of the hepatobiliary system with excellent tissue contrast. Currently, this modality is especially useful in the evaluation choledocholithiasis, and it will likely play an increasing role in the diagnosis of acute cholecystitis as MRI technology improves and becomes more widely available. CT scans are available to most patients, but have the downside of delivering ionizing radiation. They are most useful when the clinical picture is unclear or when there are additional symptoms outside of the right upper quadrant. By tailoring the selection of radiographic images to the individual patient scenario, clinicians can maximize diagnostic utility and mitigate unnecessary costs and radiation exposure.

Cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy for colorectal cancer: choosing the right candidates

Author for correspondence: Department of Surgery, University of Minnesota, 420 Delaware Street SE, MMC 195, Minneapolis, MN 55455, USA Tel.: +1 612 625 2991 Fax: +1 612 625 4406 tuttl006@umn.edu Despite advances in systemic chemotherapy, the survival of patients with peritoneal metastases from colon cancer remains poor. In contrast, the median overall survival rate after complete cytoreduction, hyperthermic intaperitoneal chemotherapy (HIPEC) and systemic chemotherapy is about 30–50 months, and the 5-year survival rate is about 30–50%. Treatment with cytoreductive surgery (CRS) plus HIPEC is associated with significant morbidity and potential mortality, so careful patient selection is critical to maximize the potential success of this treatment. The best candidates have the following characteristics: few co-morbidities, excellent performance status, no extra-abdominal disease, low peritoneal cancer index (PCI) score (<20), limited small bowel involvement, prolonged disease-free interval between primary cancer treatment and peritoneal metastasis and completely resectable disease (CC-0/1). For patients with highgrade colorectal cancers (including signet ring) or extensive peritoneal disease on imaging studies, preoperative systemic chemotherapy is recommended and may increase the proportion of patients who can receive complete cytoreduction. Diagnostic laparoscopy is also useful in selecting appropriate patients and avoiding non-therapeutic laparotomy in patients with borderline characteristics. Finally, depending upon the outcomes of ongoing randomized trials, prophylactic HIPEC may be useful for patients with high-risk cancers who do not have established metastases.

519. Intravenous Application of CXCR4 Targeted Conditionally Replicative Adenovirus with Fiber and Hexon Modifications to Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in the United States. Most newly diagnosed patients have unresectable disease due to local spread or early aggressive metastasis and have a medial survival of 6 months. Cancer stem cells (CSC) have been found to be involved in the development of metastatic disease. CSC markers such as C-X-C chemokine receptor 4 (CXCR4) and CD133 are overexpressed on the invasive front of PDAC. In this study, we tested a CXCR4-targeted conditionally replicative adenovirus (CRAd) with fiber and hexon modifications toward systemic treatment of PDAC. Due to poor infectivity of CRAd to PDAC cells, our vector contains a modified chimeric fiber which incorporates adenovirus 5 shaft and adenovirus 3 knob (Ad5/3) in order to target PDAC cells via an alternative receptor. The CXCR-4 targeted CRAd with Ad5/3 fiber modification showed strong replication in CD133(+) primary cells isolated from human PDAC tumors. The virus copy number in CD133(+) cells was more than 5 times higher than in CD133(-) cells (p<0.0005). Virus replication capability as measured by luciferase expression from Ad major late promoter was more than double in CD133(+) cells (p<0.05). This data indicates Ad5/3 modified oncolytic adenovirus shows enhanced replication in CSC-rich CD133(+) population. To overcome the issue of adenoviral sequestration upon systemic injection by non-target organs (ie liver, lung), our vector was additionally modified by replacing the Ad5 hexon hypervariable regions 5 and 7 with those from Ad3. We assessed the hexon modified adenovirus in a human Ad replication-permissive model that is most frequently used in IND-directed distribution/toxicology studies, the Syrian hamster. When the virus copy number was assayed after systemic injection via the saphenous vein, the hexon modified virus showed decreased liver sequestration, while showing no difference in the lungs. On the other hand, the major late promoter driven luciferase expression showed much more significant reductions in both liver and lung. The difference between the assays may indicate more contribution of macrophages to the copy number. Next, antitumor effect of the fiber and hexon modified CRAd was assessed in patient-derived xenografts. After intratumoral administration, the fiber-modified CRAd showed antitumor effect compared to the untreated group regardless of the presence (AdCXCR4 E1 F5/3 H5/H3) or absence of (AdCXCR4 E1 F5/3) hexon modification (p<0.01 and 0.05, respectively). On the contrary, with intravenous infection, only the fiber and hexon modified CRAd (AdCXCR4 E1 F5/3 H5/H3) showed significant antitumor effect. We believe that our CXCR4-targeted chimeric fiber and hexon modified CRAd may be employed in advanced PDAC, and that hexon modification of oncolytic adenovirus can be advantageous with systemic administration in the clinical setting.

639. Oncolytic Adenovirus Expressing IFN Alpha Works Synergistically with Chemoradiation

Aside from curative resection, there is no curative treatment against pancreatic adenocarcinoma (PDAC) at present. Late diagnosis and high recurrence results in five-year survival of 6%. Notably, Phase II trials based on adjuvant therapy combining systemic IFN Alpha (IFN) and chemoradiation reported 30-50% increase in two-year survival and impressive 35% increase in five-year survival of PDAC patients. Despite promising results, trial drawbacks included high patient dropout due to IFN systemic toxicity and low IFN levels in tumors. Low intratumoral IFN hampered the full potential of the therapy while IFN is known to induce tumor apoptosis, chemoradio sensitization, and decreased tumor neo-vasculatization.Aiming to improve efficacy and tolerability of IFN therapy, we have developed an oncolytic adenovirus expressing human IFN (OAd-IFN). Vector has Ad5/3 fiber modification and overexpresses Adenoviral Death Protein respectively contributing to increased infectivity and oncolysis. Taking advantage of Cox-2 up-regulation in PDAC, the Cox-2 promoter was included in the upstream of Adenovirus E1 region, restricting vector replication to cancer cells. Human IFN-alpha gene was placed in the Adenovirus E3 region in the way that its expression is controlled by the adenovirus major late promoter. Therefore, IFN expression in this vector is replication dependent. To test the vector in an immunocompetent syngeneic hamster model of pancreatic cancer, OAd-IFN expressing hamster IFN was generated. Vector contains RGD fiber modification enhancing its infectivity in hamster cells.MTS and crystal violet assays demonstrated sensitization of PDAC cells to chemotherapy (5-FU, Cisplatin, and Gemcitabine), and radiation (4 and 8Gy) by OAd-IFN. Comparison between OAd-IFN and control vector not expressing IFN (OAd-LUC) indicated that IFN expressed by OAd-IFN is functional in combination therapy sensitizing PDAC cells to chemoradiation. Colony formation assay showed that combinations of OAd-IFN with chemotherapy, radiation, or chemoradiation are synergistic and exhibit superior killing effect compared to groups without OAd-IFN.In vivo studies using immunocompetent syngeneic hamster model of pancreatic cancer showed that combinations including OAd-IFN resulted in augmented tumor shrinkage and survival compared to groups treated with chemotherapy, radiation, chemoradiation, or OAd-LUC + radiation. Hexon staining and viral DNA quantification by qPCR show OAd-IFN effectively replicates and spreads in tumors.Our data suggests OAd-IFN synergistically improves chemoradioation in PDAC cells, and shows superior therapeutic effect when treating immunocompetent model of pancreatic cancer. Vector capacity to express high levels of IFN intratumorally and the strong synergism between OAd-IFN and radiation, chemotherapy, and chemoradiation indicates OAd-IFN will contribute to more olerable and effective IFN therapy. Strong synergism between OAd-IFN and chemoradiation combined with focal expression of IFN can help to reduce not only IFN toxicity, but also dose-dependent toxicity of chemoradiation. Considering systemic IFN injection combined with chemoradiation is one of the few therapies to effectively treat pancreatic cancer, usage of OAd-IFN in combination with chemoradiation has great potential to result in more effective and tolerable therapy agaisnt pancreatic cancer.

A Case of Pseudocholinesterase Deficiency Resulting From Malnutrition.

Pseudocholinesterase deficiencies occur because of both genetic and acquired factors. We present the case of a patient with a history of bariatric surgery and severe malnutrition who subsequently developed prolonged neuromuscular blockade after succinylcholine administration. She had markedly decreased pseudocholinesterase levels at the time of the incident, but her motor function returned to normal with supportive care. After aggressive nutritional support over multiple weeks, her pseudocholinesterase levels drastically improved. For those patients in a poor nutritional state who experience an unexpected episode of apnea or prolonged neuromuscular blockade, practitioners must always consider malnutrition-induced pseudocholinesterase deficiency as a possible etiology.

321. Oncolytic Adenovirus Expressing IFN as a Tool To Eliminate Pancreatic Cancer Stem Cells

Pancreatic cancer is the 4th leading cause of cancer related death in the US, and curative resection of tumors is the most effective treatment against the disease. Unfortunately, the majority of patients are diagnosed in the advanced stage of the disease, and palliative treatment with Gemcitabine is the therapy of choice. In both cases, curative resection of tumors or chemotherapy, effectiveness can be limited by the presence of pancreatic cancer stem cells. Cancer stem cells (CSC) are correlated with drug resistance, tumor recurrence, and metastasis. CSC are usually located in the hypoxic center of the tumors in a quiescent stage and are not affected by conventional chemotherapeutics that target highly replicating cells, such as Gemcitabine. CSC are also called tumor initiating cells (TIC), and as low as 100 cells are reported to form tumors in pre-clinical models.Inclusion of IFN alpha (IFN) in combination therapy protocols against pancreatic cancer can be highly beneficial to tackle this problem. IFN is reported to be cytotoxic to cancer cells, have antiangiogenic properties, stimulate anti-tumor immunity, and sensitize cancer cells to chemoradiation. In addition, IFN is reported to induce activation of quiescent CSC making them susceptible to chemotherapy drugs that target highly replicating cells. Phase II and III clinical trials combining IFN with 5-FU and radiation in an adjuvant therapy setting reported a 35% increase in the five year overall survival of pancreatic cancer patients. Stimulated by the promising results reported in IFN clinical trials we developed an oncolytic adenovirus expressing IFN (OAd-IFN). Our aim is to use the virus to improve IFN therapeutic effects in combination therapy by restricting high levels of IFN expression to the tumor. In vitro data testing triple combination of OAd-IFN + radiation+ 5-FU using MIA PACA-2 and S2103 pancreatic cancer cells in colony formation assay (CFA) showed that OAd-IFN combination was more efficacious in inhibiting colony formation than triple combination with OAd-LUC, a counterpart of OAd-IFN expressing luciferase. Triple combination with OAd-IFN was also more efficient than double therapies with 5-FU+radiation, OAd-LUC + radiation, and OAd-LUC+5-FU. As CFA is an assay frequently used to access the proliferating capacity of single cells in vitro, our data suggests that IFN expressed virus decreased the number of TIC/CSC. This is supported by our in vivo studies showing that inclusion of OAd-IFN in combination with radiation or with 5-FU+radiation results in superior tumor shrinkage than all therapies not involving IFN or including OAd-LUC. In addition, tumors treated with OAd-IFN combination have a longer recurrence interval compared with all other treatments suggesting reduced levels of TIC.Although further studies are necessary to understand the impact of our OAd-IFN virus in pancreatic cancer stem cells, we believe IFN expressed by our virus made quiescent CSC more susceptible to treatments. Because our virus can express high levels of IFN restricted to the tumor site, we believe that a higher percentage of these cells will be affected by IFN delaying clinical recurrence of tumors, and improving therapy effectiveness.