Stephanie K. Carlson

The sodium iodide symporter (NIS) as an imaging reporter for gene, viral, and cell-based therapies.

Preclinical and clinical tomographic imaging systems increasingly are being utilized for non-invasive imaging of reporter gene products to reveal the distribution of molecular therapeutics within living subjects. Reporter gene and probe combinations can be employed to monitor vectors for gene, viral, and cell-based therapies. There are several reporter systems available; however, those employing radionuclides for positron emission tomography (PET) or singlephoton emission computed tomography (SPECT) offer the highest sensitivity and the greatest promise for deep tissue imaging in humans. Within the category of radionuclide reporters, the thyroidal sodium iodide symporter (NIS) has emerged as one of the most promising for preclinical and translational research. NIS has been incorporated into a remarkable variety of viral and non-viral vectors in which its functionality is conveniently determined by in vitro iodide uptake assays prior to live animal imaging. This review on the NIS reporter will focus on 1) differences between endogenous NIS and heterologously-expressed NIS, 2) qualitative or comparative use of NIS as an imaging reporter in preclinical and translational gene therapy, oncolytic viral therapy, and cell trafficking research, and 3) use of NIS as an absolute quantitative reporter.

Sodium Iodide Symporter (NIS)-Mediated Radiovirotherapy for Pancreatic Cancer

OBJECTIVE We have previously shown the therapeutic efficacy of an engineered oncolytic measles virus expressing the sodium iodide symporter reporter gene (MV-NIS) in mice with human pancreatic cancer xenografts. The goal of this study was to determine the synergy between MV-NIS-induced oncolysis and NIS-mediated (131)I radiotherapy in this tumor model. MATERIALS AND METHODS Subcutaneous human BxPC-3 pancreatic tumors were injected twice with MV-NIS. Viral infection, NIS expression, and intratumoral iodide uptake were quantitated with (123)I micro-SPECT/CT. Mice with MV-NIS-infected tumors were treated with 0, 37, or 74 MBq (131)I and monitored for tumor progression and survival. Additional studies were performed with stable NIS-expressing tumors (BxPC-3-NIS) treated with 0, 3.7, 18.5, 37, or 74 MBq of (131)I. RESULTS Mice treated with intratumoral MV-NIS exhibited significant tumor growth delay (p < 0.01) and prolonged survival (p = 0.02) compared with untreated mice. Synergy between MV-NIS-induced oncolysis and NIS-mediated (131)I ablation was not seen; however, a significant correlation was observed between NIS-mediated intratumoral iodide localization (% ID/g) and peak tumor volume reduction (p = 0.04) with combination MV-NIS and (131)I therapy. Stably transduced NIS-expressing BxPC-3 tumors exhibited rapid regression with > or = 18.5 MBq (131)I. CONCLUSION Delivery of (131)I radiotherapy to NIS-expressing tumors can be optimized using micro-SPECT/CT imaging guidance. Significant hurdles exist for NIS as a therapeutic gene for combined radiovirotherapy in this human pancreatic cancer model. The lack of synergy observed with MV-NIS and (131)I in this model was not due to a lack of radiosensitivity but rather to a nonuniform intratumoral distribution of MV-NIS infection.

Quantitative Molecular Imaging of Viral Therapy for Pancreatic Cancer Using an Engineered Measles Virus Expressing the Sodium-Iodide Symporter Reporter Gene

OBJECTIVE Our objectives were to, first, determine the oncolytic potential of an engineered measles virus expressing the sodium-iodide symporter gene (MV-NIS) for intratumoral (i.t.) therapy of pancreatic cancer and, second, evaluate NIS as a reporter gene for in vivo monitoring and quantitation of MV-NIS delivery, viral spread, and gene expression in this tumor model. MATERIALS AND METHODS Cultured human pancreatic cancer cells were infected with MV-NIS. Light microscopy, cell viability, and iodide uptake assays were used to confirm viral infection and NIS gene expression and function in vitro. Human pancreatic tumor xenografts were established in mice and infected via i.t. MV-NIS injections. NIS-mediated i.t. iodide uptake was quantitated by (123)I micro-SPECT/CT. i.t. MV-NIS infection was confirmed by immunohistochemistry of excised pancreatic xenografts. The oncolytic efficacy of MV-NIS was determined by measurement of tumor growth and mouse survival. RESULTS Infection of human pancreatic cancer cell lines with MV-NIS in vitro resulted in syncytia formation, marked iodide uptake, and ultimately cell death. Tumor xenografts infected with MV-NIS concentrated radioiodine, allowing serial quantitative imaging with (123)I micro-SPECT/CT. i.t. MV-NIS therapy of human pancreatic cancer xenografts resulted in a significant reduction in tumor volume and increased survival time of the treated mice compared with the control mice. CONCLUSION MV-NIS efficiently infects human pancreatic tumor cells and results in sufficient radioiodine uptake to enable noninvasive serial imaging and quantitation of the intensity, distribution, and time course of NIS gene expression. MV-NIS also shows oncolytic activity in human pancreatic cancer xenografts: Tumor growth is reduced and survival is increased in mice treated with the virus.

In Vivo Quantitation of Intratumoral Radioisotope Uptake Using Micro-Single Photon Emission Computed Tomography/Computed Tomography

PurposeThis study was undertaken to determine the ability of micro-single photon emission computed tomography (micro-SPECT)/computed tomography (CT) to accurately quantitate intratumoral radioisotope uptake in vivo and to compare these measurements with planar imaging and micro-SPECT imaging alone.ProceduresHuman pancreatic cancer xenografts were established in 10 mice. Intratumoral radioisotope uptake was achieved via intratumoral injection of an attenuated measles virus vector expressing the NIS gene (MV-NIS). On various days after MV-NIS injection, 123I planar and micro-SPECT/CT imaging was performed. Tumor activity was determined by dose calibrator measurements and region-of-interest (ROI) image analysis. Agreement and reproducibility of tumor activity measurements were assessed by Bland–Altman plots and Lin’s concordance correlation coefficient (CCC).ResultsIntratumoral radioisotope uptake was detected in all mice. Scatterplots demonstrate strong agreement (CCC = 0.93) between micro-SPECT/CT ROI image analysis and dose calibrator tumor activity measurements. The differences between dose calibrator activity measurements and those obtained with ROI image analysis of micro-SPECT alone and planar imaging are less accurate and more variable (CCC = 0.84 and 0.78, respectively).ConclusionsMicro-SPECT/CT can be used to accurately quantify intratumoral radioisotope uptake in vivo and is more reliable than planar or micro-SPECT imaging alone.

Safety Studies on Intrahepatic or Intratumoral Injection of Oncolytic Vesicular Stomatitis Virus Expressing Interferon-β in Rodents and Nonhuman Primates

Toxicology studies were performed in rats and rhesus macaques to establish a safe starting dose for intratumoral injection of an oncolytic vesicular stomatitis virus expressing human interferon-beta (VSV-hIFNbeta) in patients with hepatocellular carcinoma (HCC). No adverse events were observed after administration of 7.59 x 10(9) TCID(50) (50% tissue culture infective dose) of VSV-hIFNbeta into the left lateral hepatic lobe of Harlan Sprague Dawley rats. Plasma alanine aminotransferase and alkaline phosphatase levels increased and platelet counts decreased in the virus-treated animals on days 1 and 2 but returned to pretreatment levels by day 4. VSV-hIFNbeta was also injected into normal livers or an intrahepatic McA-RH7777 HCC xenograft established in Buffalo rats. Buffalo rats were more sensitive to neurotoxic effects of VSV; the no observable adverse event level (NOAEL) of VSV-hIFNbeta in Buffalo rats was 10(7) TCID(50). Higher doses were associated with fatal neurotoxicity and infectious virus was recovered from tumor and brain. Compared with VSV-hIFNbeta, toxicity of VSV-rIFNbeta (recombinant VSV expressing rat IFN-beta) was greatly diminished in Buffalo rats (NOAEL, >10(10) TCID(50)). Two groups of two adult male rhesus macaques received 10(9) or 10(10) TCID(50) of VSV-hIFNbeta injected directly into the left hepatic lobe under computed tomographic guidance. No neurological signs were observed at any time point. No abnormalities (hematology, clinical chemistry, body weights, behavior) were seen and all macaques developed neutralizing anti-VSV antibodies. Plasma interleukin-6, tumor necrosis factor-alpha, and hIFN-beta remained below detection levels by ELISA. On the basis of these studies, we will be proposing a cautious approach to dose escalation in a phase I clinical trial among patients with HCC.

Radiation Dose Levels for Interventional CT Procedures

OBJECTIVE The purpose of this study was to determine typical radiation dose levels to patients undergoing CT-guided interventional procedures. MATERIALS AND METHODS A total of 571 patients undergoing CT interventional procedures were included in this retrospective data analysis study. Enrolled patients underwent one of five procedures: cryoablation, aspiration, biopsy, drain, or injection. With each procedure, two scan modes were used, either intermittent (no table increment) or helical mode. Skin dose was estimated from the volumetric CT dose index (CTDI(vol)) and phantom measurements. Effective dose was calculated by multiplying dose-length product (DLP) and conversion factor (k factor) for helical mode, and using Monte Carlo organ dose coefficients for intermittent mode. RESULTS The mean (± SD) skin doses were 728 ± 382, 130 ± 104, 128 ± 81, 152 ± 105, and 195 ± 147 mGy, and the mean effective doses were 119.7 ± 50.3, 20.1 ± 11.0, 13.8 ± 9.2, 25.3 ± 15.4, and 9.1 ± 5.5 mSv for each of the five procedures, respectively. The maximum skin dose was 1.95 Gy. The mean effective dose across all procedure types was 24.1 mSv, with 2.3 mSv from intermittent scans and 21.8 mSv from helical scans. CONCLUSION Substantial dose differences were observed among the five procedures. The risk of deterministic effects appears to be very low, because the maximum observed skin dose did not exceed the threshold for transient skin erythema (2 Gy). The average risk of stochastic effects was comparable to that of 1-10 abdomen and pelvis CT examinations. Although the intermittent mode can contribute substantially to skin dose, it contributes minimally to the effective dose because of the much shorter scan range used.

Pinhole micro-SPECT/CT for noninvasive monitoring and quantitation of oncolytic virus dispersion and percent infection in solid tumors

The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to: 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). Sodium iodide symporter (NIS) RNA level and dispersion pattern were determined in control and MV-NIS-infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with 123I or 99TcO4 micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r2=0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS-infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from non-infected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection, and can replace more time-consuming and expensive analyses (for example, autoradiography and IHC) that require animal killing.

Diseases of the cecum: a CT pictorial review

Abstract.Cecal pathology is commonly encountered and may represent a diagnostic challenge in patients with either acute or chronic clinical presentations. Although appendicitis accounts for the majority of surgical conditions presenting with right lower quadrant pain, readers should be aware of the broad spectrum of cecal pathologies and characteristic CT findings, which can be useful in establishing the correct diagnosis.

Concordant activity of transgene expression cassettes inserted into E1, E3 and E4 cloning sites in the adenovirus genome

Expression cassettes can be inserted at several positions into recombinant adenoviral genomes but the implications of this choice for transgene expression level have not been determined. Knowledge of the relative expression levels of transgenes inserted at different sites in the adenoviral genome is of particular significance for transgene expression monitoring approaches that rely on the concordant expression of a marker transgene inserted elsewhere in the viral genome.

Development of monoclonal antibody-based immunoassays for detection of Helicobacter pylori neutrophil-activating protein

Neutrophil-activating protein (NAP) is a major virulence factor expressed by Helicobacter pylori isolates associated with severe chronic gastroduodenal inflammation and peptic ulcers. NAP is one of the main protective antigens and a target for vaccine development against Helicobacter infection. In addition, NAP is a potent immune stimulator with potential application as a general vaccine adjuvant and in treatment of allergic diseases and cancer immunotherapy. NAP-specific immunoassays are needed for both H. pylori diagnostics and characterization of NAP-based vaccines and immunomodulatory preparations. We generated a panel of NAP-specific monoclonal antibodies (MAbs) by immunization of BALB/c mice with synthetic NAP peptides. The antibody reactivity against recombinant or native NAP antigen was characterized by enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence. A sensitive capture ELISA was developed using MAbs 23C8 and 16F4 (directed against different NAP epitopes) for detection of native or measles virus (MV) vector-expressed recombinant NAP in a concentration range of 4 ng/ml to 2000 ng/ml. MAb 23C8 antigen-binding depends on Tyr101 in a variable amino acid sequence of the NAP molecule, indicating the existence of antigenic variants among H. pylori strains. MAb 16F4 reacted with NAP from different H. pylori strains and was a sensitive tool for detection of small amounts of isolated NAP antigen or whole bacteria by immunoblotting or immunofluorescence. In conclusion, MAb-based immunoassays are highly specific and sensitive for detection of native NAP antigen and recombinant NAP immunostimulatory transgenes expressed by replication competent virus vectors.