Dana Haddad

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.

The immunologic aspects of poxvirus oncolytic therapy

The concept of using replicating oncolytic viruses in cancer therapy dates to the beginning of the twentieth century. However, in the last few years, an increasing number of pre-clinical and clinical trials have been carried out with promising preliminarily results. Novel, indeed, is the suggestion that viral oncolytic therapy might not operate exclusively through an oncolysis-mediated process but additionally requires the “assistance” of the host’s immune system. Originally, the host’s immune response was believed to play a predominant obstructive role against viral replication, hence limiting the anti-tumor efficacy of viral vectors. Recent data, however, suggest that the immune response may also play a key role in promoting tumor destruction in association with the oncolytic process. In fact, immune effector pathways activated during oncolytic virus-induced tumor rejection seem to follow a similar pattern to those observed when the broader phenomenon of immune-mediated tissue-specific rejection occurs in other immune-related pathologies. We recently formulated the “Immunologic Constant of Rejection” hypothesis, emphasizing commonalties in transcriptional patterns observed when tissue-destruction occurs: whether with a favorable outcome, such as in tumor rejection and pathogen clearance; or a destructive one, such as in allograft rejection or autoimmunity. Here, we propose that a similar mechanism induces clearance of virally infected tumors and that such a mechanism is primarily dependent on innate immune functions.

Novel therapy for anaplastic thyroid carcinoma cells using an oncolytic vaccinia virus carrying the human sodium iodide symporter

BACKGROUND Anaplastic thyroid carcinoma (ATC) is fatal with resistance to radiotherapy because of the loss of intrinsic human sodium iodine symporter (hNIS). We determined whether vaccinia virus carrying hNIS kills and induces hNIS reexpression in ATC cells, facilitating deep-tissue imaging. METHODS Vaccinia virus (GLV-1h153) carrying hNIS was tested against ATC lines for killing and replication via cytotoxicity and viral plaque assays. Cellular radiouptake was determined using radiouptake assays. GLV-1h153-infected ATC xenografts were imaged via (99m)Tc-pertechnetate. RESULTS GLV-1h153 infected, replicated in, and killed all ATC cell lines. GFP expression confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1.0, GLV-1h153 reached near 100% cytotoxicity in 8305c and FRO by day 5 and 70% in the least sensitive cell line, 8505c. GLV-1h153-infected ATC cells had a 14-fold increase of hNIS-specific radiouptake compared with uninfected control 24 hours after infection at an MOI of 1.0. In vivo, GLV-1h153 facilitated imaging of hNIS expression in 8505c tumors using (99m)Tc-pertechnetate. CONCLUSION GLV-1h153 is an effective oncolytic agent against ATC. The results show hNIS-specific radiouptake in infected ATC cells, facilitating deep-tissue imaging. GLV-1h153 is a promising candidate for treatment and imaging, and potentially enhancing susceptibility to radioiodine therapy by converting non-hNIS-expressing cells into hNIS-expressing ATC cells.

A Vaccinia Virus Encoding the Human Sodium Iodide Symporter Facilitates Long-Term Image Monitoring of Virotherapy and Targeted Radiotherapy of Pancreatic Cancer

To assess therapeutic response and potential toxicity of oncolytic virotherapy, a noninvasive, deep-tissue imaging modality is needed. This study aimed to assess the feasibility, parameters, and determining factors of serial imaging and long-term monitoring of virotherapy and the therapeutic response of pancreatic cancer xenografts treated with a vaccinia virus carrying the human sodium iodide symporter GLV-1h153. Methods: Pancreatic cancer xenografts (PANC-1) in nude mice were treated systemically or intratumorally with GLV-1h153 and serially imaged using 124I PET at 1, 2, 3, and 5 wk after viral injection. Signal intensity was compared with tumor therapeutic response and optical imaging, and tumors were histologically analyzed for morphology and the presence of virus. Autoradiography was performed using technetium-pertechnetate and γ-scintigraphy to assess determining factors for radiouptake in tumors. Finally, the enhanced therapeutic effect of combination therapy with GLV-1h153 and systemic radioiodine was assessed. Results: GLV-1h153 successfully facilitated serial long-term imaging of virotherapy, with PET signal intensity correlating to tumor response. GLV-1h153 colonization of tumors mediated radioiodine uptake at potentially therapeutic doses. Successful radiouptake required the presence of virus, adequate blood flow, and viable tissue, whereas loss of signal intensity was linked to tumor death and necrosis. Finally, combining systemically administered GLV-1h153 and 131I led to enhanced tumor kill when compared with virus or 131I alone (P < 0.01). Conclusion: GLV-1h153 is a promising oncolytic agent for the treatment, long-term imaging, and monitoring of therapeutic response in a xenograft model of pancreatic cancer. GLV-1h153 provided insight into tumor biologic activity and facilitated enhanced tumor kill when combined with systemic targeted radiotherapy. These results warrant further investigation into parameters and potential synergistic effects of combination 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.

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.

Oncolytic herpes simplex virus kills stem-like tumor-initiating colon cancer cells

Stem-like tumor-initiating cells (TICs) are implicated in cancer progression and recurrence, and can be identified by sphere-formation and tumorigenicity assays. Oncolytic viruses infect, replicate in, and kill a variety of cancer cells. In this study, we seek proof of principle that TICs are susceptible to viral infection. HCT8 human colon cancer cells were subjected to serum-free culture to generate TIC tumorspheres. Parent cells and TICs were infected with HSV-1 subtype NV1066. Cytotoxicity, viral replication, and Akt1 expression were assessed. TIC tumorigenicity was confirmed and NV1066 efficacy was assessed in vivo. NV1066 infection was highly cytotoxic to both parent HCT8 cells and TICs. In both populations, cell-kill of >80% was achieved within 3 days of infection at a multiplicity of infection (MOI) of 1.0. However, the parent cells required 2-log greater viral replication to achieve the same cytotoxicity. TICs overexpressed Akt1 in vitro and formed flank tumors from as little as 100 cells, growing earlier, faster, larger, and with greater histologic atypia than tumors from parent cells. Treatment of TIC-induced tumors with NV1066 yielded tumor regression and slowed tumor growth. We conclude that colon TICs are selected for by serum-free culture, overexpress Akt1, and are susceptible to oncolytic viral infection.

Abdominal imaging post bariatric surgery: predictors, usage and utility

PURPOSE A lack of well-defined postoperative imaging guidelines for bariatric patients may lead to false-positive findings, radiation exposure, additional cost, and patient anxiety. We investigated our institutional usage and utility of nonroutine postoperative abdominal imaging. METHODS AND MATERIALS Laparoscopic gastric bypass and sleeve gastrectomy patients over a 5-year period were retrospectively identified. All bariatric-related nonroutine initial and all subsequent prompted abdominal and pelvic imaging was included. RESULTS A total of 578 patients were included (399 gastric bypass, 179 sleeve gastrectomy); 907 nonroutine studies in 69% of patients were performed, and 36% patients underwent computed tomography (CT). Only 20.3% of findings were symptom-related, 26% had benign incidental findings, and 50% were negative. Incidental findings prompted 71 additional studies. Bypass procedure (sleeve versus bypass, odds ratio [OR] .3), older age (median 43 versus 48 years), and lower initial body mass index (BMI) (median 43 versus 45) increased the likelihood of imaging. History of prior abdominal surgery and dyspepsia increased the probability of undergoing CT by an odds ratio of 1.8 and 2.0, respectively (P<.05). History of ulcer (OR .6) or reflux on routine upper gastrointestinal imaging (OR .3) decreased probability (P<.05). Patients who underwent CT were more likely to undergo other abdominal imaging (3 versus 1 study per patient, P<.01). CONCLUSIONS Postoperative abdominal imaging in the bariatric population is common, with almost 70% of patients undergoing imaging and 70% of findings not related to patient symptoms. Bypass procedure, older age, and lower initial BMI were associated with a higher likelihood of patients undergoing imaging. Heightened understanding of this important subject is necessary to help streamline cost-effective imaging protocols for these patients.