Joshua Carson

Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus.

IntroductionOncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153.MethodsGLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free 124I radiotracer.ResultsGLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via 124I-PET.ConclusionInsertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.

Irreversible electroporation is a surgical ablation technique that enhances gene transfer

BACKGROUND Reversible electroporation has long been used to transfer macromolecules into target cells in the laboratory by using an electric field to induce transient membrane permeability. Recently, the electric field has been modulated to produce permanent membrane permeability and cell death. This novel technique, irreversible electroporation (IRE), is being developed for nonthermal cancer ablation. We hypothesize that outside the central zone of IRE exists a peripheral zone of reversible electroporation where gene transfer may occur. METHODS IRE of the liver was performed in a Yorkshire pig model with administration of a plasmid expressing the marker gene green fluorescent protein (GFP) by bolus or primed infusion through the hepatic artery or portal vein. After 6 hours, livers were harvested for fluorescent microscopy and histologic examination. RESULTS Of 36 liver specimens treated with IRE and the GFP plasmid, 31 demonstrated strong green fluorescence. Liver ablation by IRE was demarcated clearly on histology. CONCLUSION IRE is a promising technique not only for operative tissue ablation but also for gene therapy. Because IRE ablation may leave behind intact tumor antigens, these findings encourage clinical studies of tumor ablation with delivery of immunostimulatory plasmids for combined local eradication and systemic immunotherapy.

Imaging Characteristics, Tissue Distribution, and Spread of a Novel Oncolytic Vaccinia Virus Carrying the Human Sodium Iodide Symporter

Introduction Oncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS. Methods GLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide 131I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP) and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via 124I-positron emission tomography (PET). Detection of systemic administration of virus was investigated with both 124I-PET and 99m-technecium gamma-scintigraphy. Results GLV-1h153 successfully facilitated time-dependent intracellular uptake of 131I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P<0.05). In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 109 plaque-forming unit (PFU)/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean %ID/gm of 3.82±0.46 (P<0.05) 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV-1h153-infected tumors were detected via 124I-PET and 99m-technecium-scintigraphy. Conclusion GLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic cancer cells, facilitating detection by PET with both intratumoral and systemic administration. Therefore, GLV-1h153 is a promising candidate for the noninvasive imaging of virotherapy and warrants further study into longterm monitoring of virotherapy and potential radiocombination therapies with this treatment and imaging modality.

Tunneling nanotubes: an alternate route for propagation of the bystander effect following oncolytic viral infection

Tunneling nanotubes (TNTs) are ultrafine, filamentous actin-based cytoplasmic extensions which form spontaneously to connect cells at short and long-range distances. We have previously described long-range intercellular communication via TNTs connecting mesothelioma cells in vitro and demonstrated TNTs in intact tumors from patients with mesothelioma. Here, we investigate the ability of TNTs to mediate a viral thymidine kinase based bystander effect after oncolytic viral infection and administration of the nucleoside analog ganciclovir. Using confocal microscopy we assessed the ability of TNTs to propagate enhanced green fluorescent protein (eGFP), which is encoded by the herpes simplex virus NV1066, from infected to uninfected recipient cells. Using time-lapse imaging, we observed eGFP expressed in infected cells being transferred via TNTs to noninfected cells; additionally, increasing fluorescent activity in recipient cells indicated cell-to-cell transmission of the eGFP-expressing NV1066 virus had also occurred. TNTs mediated cell death as a form of direct cell-to-cell transfer following viral thymidine kinase mediated activation of ganciclovir, inducing a unique long-range form of the bystander effect through transmission of activated ganciclovir to nonvirus-infected cells. Thus, we provide proof-of-principle demonstration of a previously unknown and alternative mechanism for inducing apoptosis in noninfected recipient cells. The conceptual advance of this work is that TNTs can be harnessed for delivery of oncolytic viruses and of viral thymidine kinase activated drugs to amplify the bystander effect between cancer cells over long distances in stroma-rich tumor microenvironments.

Role of MAPK in oncolytic herpes viral therapy in triple-negative breast cancer

Triple-negative breast cancers (TNBCs) have poor clinical outcomes owing to a lack of targeted therapies. Activation of the MEK/MAPK pathway in TNBC has been associated with resistance to conventional chemotherapy and biologic agents and has a significant role in poor clinical outcomes. NV1066, a replication-competent herpes virus, infected, replicated in and killed all TNBC cell lines (MDA-MB-231, HCC1806, HCC38, HCC1937, HCC1143) tested. Greater than 90% cell kill was achieved in more-sensitive lines (MDA-MB-231, HCC1806, HCC38) by day 6 at a multiplicity of infection (MOI) of 0.1. In less-sensitive lines (HCC1937, HCC1143), NV1066 still achieved >70% cell kill by day 7 (MOI 1.0). In vivo, mean volume of flank tumors 14 days after treatment with NV1066 was 57 versus 438 mm3 in controls (P=0.002). NV1066 significantly downregulated p-MAPK activation by 48 h in all cell lines in vitro and in MDA-MB-231 xenografts in vivo. NV1066 demonstrated synergistic effects with a MEK inhibitor, PD98059 in vitro. We demonstrate that oncolytic viral therapy (NV1066) effectively treats TNBC with correlation to decreased MEK/MAPK signaling. These findings merit future studies investigating the potential role of NV1066 as a sensitizing agent for conventional chemotherapeutic and biologic agents by downregulating the MAPK signaling pathway.

Irreversible Electroporation Facilitates Gene Transfer of a GM-CSF Plasmid With a Local and Systemic Response

BACKGROUND Electroporation uses an electric field to induce pores in the cell membrane that can transfer macromolecules into target cells. Modulation of electrical parameters leads to irreversible electroporation (IRE), which is being developed for tissue ablation. We sought to evaluate whether the application of IRE may induce a lesser electric field in the periphery where reversible electroporation may occur, facilitating gene transfer of a granulocyte macrophage colony-stimulating factor (GM-CSF) plasmid to produce its biologic response. METHODS Yorkshire pigs underwent laparotomy, and IRE of the liver was performed during hepatic arterial infusion of 1 or 7 mg of a naked human GM-CSF plasmid. The serum, liver, lymph nodes, and bone marrow were harvested for analysis. RESULTS Human GM-CSF level rose from undetectable to 131 pg/mL in the serum at 24 hours after IRE and plasmid infusion. The liver demonstrated an ablation zone surrounded by an immune infiltrate that had greater macrophage intensity than when treated with IRE or plasmid infusion alone. This dominance of macrophages was dose dependent. Distant effects of GM-CSF were found in the bone marrow, where proliferating myeloid cells increased from 14% to 25%. CONCLUSION IRE facilitated gene transfer of the GM-CSF plasmid and brought about a local and systemic biologic response. This technique holds potential for tumor eradication and immunotherapy of residual cancer.

Regional Liver Therapy Using Oncolytic Virus to Target Hepatic Colorectal Metastases

The mortality of colorectal carcinoma often results from the progression of metastatic disease, which is predominantly hepatic. Although recent advances in surgical, locoregional, and systemic therapies have yielded modest improvements in survival, treatment of these aggressive lesions is limited to palliation for the vast majority of patients. Oncolytic viral therapy represents a promising novel therapeutic modality that has achieved tumor regression in several preclinical and clinical models. Evidence further suggests that locoregional viral administration may improve viral efficacy while minimizing toxicity. This study will review the theories behind hepatic arterial infusion of oncolytic virus, as well as herpes viral design, preclinical data, and clinical progress in regional liver therapy using oncolytic virus to treat hepatic colorectal carcinoma metastases.

Lung Cancer Staging: An Overview of the New Staging System and Implications for Radiographic Clinical Staging

The Cancer Commission of the League of Nations Health Organization established the Radiological Sub-Commission in 1929 with the primary mandate of creating a common cancer staging system in order to facilitate effective communication across institutions around the world. Shortly thereafter by Dr. Pierre Denoix developed the TNM system used in almost all solid cancer staging today, and he continued to broaden and refine his TNM based staging approach through a series of articles published in the 1940’s and 50’s.1 Denoix’s approach gained its first institutional endorsement when the 7th International Congress of Radiology adopted TNM staging for laryngeal and breast cancer in 1953. Subsequently, as chairman of the Union Contre Le Cancer (UICC) staging committee, Denoix oversaw the publication of a series of “fascicles” (brochures) released during the 1960’s establishing standardized TNM staging for 23 solid organ cancers. A tradition of international consensus in cancer staging was established in 1969, when the American Joint Committee for Cancer Staging and End Results Reporting (renamed American Joint Committee on Cancer in 1980) and the UICC agreed that all subsequent staging recommendations by either body would be published in consultation with the other.2 Lung cancer staging was incorporated into the UICC TNM staging system in 1966. The first substantial revision of the lung cancer TNM staging was proposed by Dr. Mountain et al. in 1973 with the publication of “A Clinical Staging System for Lung Cancer”. This work was based on the outcomes of 2,155 cases of lung cancer, 1,712 of which were non-small cell lung cancer. It further specified the T, N and M descriptors and introduced stage groupings for the first time.3 Over the next 25 years, the lung cancer TNM staging system underwent three major revisions, all based on Dr. Mountain’s growing database, which had increased to 5,319 cases by 1996.4 It was at this point that the International Association for the Study of Lung Cancer (IASLC) initiated the IASLC Lung Cancer Staging Project. The IASLC Lung Staging Project sought to revise lung cancer staging to by using a data set more powerful, current, and universal than any single institutional database. From 1997 until 2009, this group developed a database incorporating patient data gathered from participating institutions around the world, collecting over 81,000 cases of lung cancer. The majority of cases were from Europe and the United States (58% and 21%, respectively), but a significant number of patients from Asia and Australia were included. Patients treated with all modalities were accepted into the database (including surgery, chemotherapy and radiation therapy) in order to better sample the true lung cancer population.2 The 7th edition of the lung cancer staging system thus represents the culmination of a remarkably comprehensive review process. Improvements in surgical, medical and radiation treatments, surveillance, radiographic staging, post-operative care and minimally invasive surgery have all changed lung cancer treatment and survival in the last decade. These new factors have not been accounted for in the previous staging schema and impact treatment decisions. Based on outcomes data from patients treated with these improved modalities, the TNM staging is more representative of current treatment patterns and helps better guide the clinician. The IASLC Lung Cancer Staging Project set out to accomplish a series of goals: Aid the clinician in planning treatment Give some indication of prognosis for each stage Assist in the evaluation of the results of treatment Facilitate the exchange of information between centers across the globe To contribute to the continuing investigation of human cancer.2 This article will review the AJCC 7th edition Cancer Staging for lung cancer and will compare it to the 6th edition, with emphasis on operable disease. Clinical staging, its strengths and weakness, along with minimally invasive staging supplements will be discussed to aid the clinician in the preoperative staging of lung cancer.

Abstract 929: Tunneling nanotube conduits facilitate the bystander effect after oncolytic viral infection

Background: Oncolytic viruses have come to the forefront of cancer therapeutics following FDA approval for treatment of metastatic melanoma in 2015. Efficacy of viral therapy is enhanced by the bystander effect, a cellular phenomenon that amplifies the effects of the virus following activation of the prodrug ganciclovir (GCV) by virally-expressed thymidine kinase (TK) and intercellular spread of GCV via gap junctions. In the complex and stroma-rich tumor microenvironment, gap junctions may not completely account for cell-to-cell communication. Tunneling nanotubes (TNTs) are a novel and recently characterized alternative form of direct cell-to-cell communication in the tumor matrix. TNTs are fine, long, F actin-based cell extensions that serve as short and long-range conduits for efficient transfer of cellular cargo. Here, we investigate the ability of TNTs to mediate a TK-based bystander effect after oncolytic viral infection and administration of GCV. Methods: We infected 3 mesothelioma cell lines with NV1066, a mutant replication-competent strain of herpes simplex virus-1 (HSV-1) that encodes viral eGFP and viral TK. Confocal microscopy and time-lapse imaging were performed 12-36 hours later. A modified Transwell assay was used to separate infected cells from uninfected cells to assess TNT propagation of eGFP-tagged NV1066. GCV was added to infected cells in the top chamber to assess TNT propagation of TK-activated GCV. Apoptosis was measured using TUNEL assay in the bottom chamber to quantify the extent of the bystander effect. Results: Confocal microscopy demonstrated effective intercellular transfer of GFP-tagged virus between cells via TNTs prior to oncolysis. Quantification of TUNEL-positive cells at 48 hours indicated that addition of NV1066 to the top chamber resulted in 33% of the initially uninfected cells in the bottom chamber dying by 48 hours. The addition of GCV to virally infected cells in the top chamber significantly increased apoptosis in recipient cells in the bottom chamber, from 33% to 71%, producing a 2.3-fold increase in cell killing attributed to TNT transfer of viral TK-activated GCV (p = 0.007). Thus, TNTs were shown to transfer viral TK-activated GCV to non-infected cells, leading to cell death via a long-range form of the bystander effect. Conclusions: Here we demonstrate that TNTs provide a previously unknown and alternative mechanism for the bystander effect in which viral TK-activated GCV is transferred via TNTs and induces apoptosis in non-infected recipient cells. Citation Format: Emil Lou, Justin Ady, Venugopal Thayanithy, Kelly Mojica, Joshua Carson, Prassanna Rao, Yuman Fong. Tunneling nanotube conduits facilitate the bystander effect after oncolytic viral infection. [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 929.

Cancer of the Pancreas: Distal Resections and Staging of Pancreatic Cancer

Laparoscopy for pancreatic cancer, either for staging initially or for resection as part of a treatment plan, has gained more and more acceptance in the surgical community over the past few years. Using laparoscopy and diagnosing extensive disease, we are able to spare far more patients an unnecessary laparotomy than we were in the past. This chapter describes workup, sheds some light on still controversial topics like port-site metastasis and palliation, and gives a step-by-step explanation of how to perform a partial pancreatic resection.