Joyce T. Au

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.

Gold Nanoparticles Provide Bright Long-Lasting Vascular Contrast for CT Imaging

OBJECTIVE Iodinated contrast agent for CT has a short half-life in the vasculature. As the field of interventional procedures expands, a more durable contrast agent would be highly useful. Our study investigated whether gold nanoparticles are feasible as a long-lasting vascular contrast agent for CT. MATERIALS AND METHODS Gold nanoparticles were synthesized by a modified Turkevich method, coated with methoxy-polyethylene glycol-thiol chains, and compared with an iodine-based contrast agent used in mice. Contrast agents were imaged in tubes by CT at 40, 60, and 140 kVp and then were tested in vivo by tail vein injection. Nine mice received gold nanoparticles, two received iodine-based contrast agent, and one received saline. CT of mice was performed at 60 kVp immediately, 6 hours, and 24 hours after injection. RESULTS In an isolated form in tubes, gold nanoparticles had greater radiographic density than did iodine-based contrast agent at 40 kVp and were comparable at the other CT voltages. In mice, gold nanoparticles provided bright contrast enhancement that enabled clear visualization of the abdominal aorta and renal arteries, which could not be distinguished without contrast agent. This persisted up to 24 hours, which was the last time point assessed. Contrast enhancement of the vasculature by iodine-based contrast agent was present immediately after injection but had disappeared by 6 hours. CONCLUSION Gold nanoparticles can provide clear and durable contrast enhancement of the vasculature even at 24 hours. These findings merit further study of gold nanoparticles for their potential as a contrast agent in CT and CT-guided interventional procedures.

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.

Irreversible electroporation ablation of the liver can be detected with ultrasound B-mode and elastography.

BACKGROUND Irreversible electroporation (IRE) is a novel ablation technique that induces permanent membrane permeability and cell death. We are interested in ultrasound B-mode and elastography to monitor IRE ablation in the liver. METHODS Yorkshire pigs underwent IRE ablation of the liver and were imaged with ultrasound B-mode and elastography. Histologic evaluation of cell death by triphenyltetrazolium chloride and hematoxylin and eosin staining was performed. RESULTS Elastography showed that liver ablated by IRE exhibited increased tissue stiffness with a peak strain ratio of 2.22. The IRE lesion had a discrete border without bubble artifact, and the lesion size significantly correlated with area of cell death on histology. IRE ablation was unaffected by presence of large blood vessels or bile ducts. CONCLUSION IRE ablation led to increased tissue stiffness that was detectable by elastography and indicative of cell death. Elastography may complement B-mode ultrasonography to monitor IRE ablation of the liver.

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.

Endoscopic spray cryotherapy for genitourinary malignancies: safety and efficacy in a porcine model.

Objective: To examine the effects and safety of using endoscopic spray cryotherapy (ESC) on bladder, ureteral, and renal pelvis urothelium in a live porcine model. Subjects and methods: ESC treatments were systematically applied to urothelial sites in the bladder, ureter, and renal pelvis of eight female Yorkshire swine in a prospective trial. Freeze–thaw cycles ranged from 5 to 60 s/cycle for one to six cycles using a 7 French cryotherapy catheter. Tissue was evaluated histologically for treatment-related effects. Acute physiologic effects were evaluated with pulse oximetry, Doppler sonography, and postmortem findings. Results: In bladder, treatment depth was inconsistent regardless of dose, demonstrating urothelial necrosis in one, muscularis propria depth necrosis in two, and full thickness necrosis in all remaining samples. In ureter, full thickness necrosis was seen in all samples, even with the shortest spray duration (5 s/cycle for six cycles or 30 s/cycle for one cycle). Treatment to the renal pelvis was complicated by adiabatic gas expansion of liquid nitrogen to its gaseous state, resulting in high intraluminal pressures requiring venting to avoid organ perforation, even at the lowest treatment settings. At a planned dose of 5 s/cycle for six cycles of the first renal pelvis animal, treatment was interrupted by sudden and unrecoverable cardiopulmonary failure after three cycles. Repeated studies replicated this event. Ultrasound and immediate necropsy confirmed the creation of a large gaseous embolism and reproducible cardiopulmonary effects. Conclusion: ESC in a porcine urothelial treatment model results in full-thickness tissue necrosis in bladder, ureter, and renal pelvis at a minimal treatment settings of 5 s/cycle for six cycles. Adiabatic gas expansion may result in fatal pyelovenous gas embolism and collateral organ injury, as seen in both animals receiving treatment to the renal pelvis in this study. These results raise safety concerns for use of ESC as a treatment modality in urothelial tissues with current device settings.

Trichobezoar: A rare cause of bowel obstruction.

A bezoar is an intraluminal mass formed by the accumulation of undigested material in the gastrointestinal tract. A trichobezoar is a bezoar made up of hair and is a rare cause of bowel obstruction of the proximal gastrointestinal tract. They are seen mostly in young women with trichotillomania and trichotillophagia and symptoms include epigastric pain, nausea, loss of appetite and bowel or gastric outlet obstruction. We herein describe a case of a trichobezoar that presented as a gastric outlet obstruction and was subsequently successfully removed via a laparotomy.