Tumor-specific transcriptional targeting of suicide gene therapy.

Transcriptional targeting of gene expression has been plagued by the weakness of tissue-specific promoters. Thus, to increase promoter strength while maintaining tissue specificity, we constructed a recombinant adenovirus containing a binary promoter system with a tumor-specific promoter (CEA; carcinoembryonic antigen) driving a transcription transactivator, which then activates a minimal promoter to express a suicide gene (HSV-tk; herpes simplex virus thymidine kinase). This ADV/binary-tk induced equal or greater cell killing in a CEA-specific manner in vitro compared with the CEA-independent killing of a vector with a constitutive viral promoter driving HSV-tk (ADV/RSV-tk). To monitor adenovirus-mediated HSV-tk gene expression in vivo, we employed noninvasive nuclear imaging using a radioiodinated nucleoside analog ([((1)31)I]-FIAU) serving as a substrate for HSV-tk. [((1)31)I]-FIAU-derived radioactivity accumulated after intratumoral injection of ADV/binary-tk only in the area of CEA-positive tumors with significantly less spread to the adjacent liver tissue than after administration of the universally expressed ADV/RSV-tk. Both viruses exhibited similar antitumor efficacy upon injection of liver metastases. Importantly, in vivo dose escalation studies demonstrated significantly reduced toxicity after intravenous administration of ADV/binary-tk versus ADV/RSV-tk. In summary, the increased therapeutic index of this novel, amplified CEA-driven suicide gene therapy vector is a proof of principle for the powerful enhancement of a weak tissue-specific promoter for effective tumor restricted gene expression.

Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo.

To evaluate the efficiency of gene delivery in gene therapy strategies for malignant brain tumors, it is important to determine the distribution and magnitude of transgene expression in target tumor cells over time. Here, we assess the time- and vector dose-dependent kinetics of recombinant herpes simplex virus (HSV)-1 vector-mediated gene expression and vector replication in culture and in vivo by a recently developed radiotracer method for noninvasive imaging of gene expression (J. G. Tjuvajev et al., Cancer Res., 55: 6126-6132, 1995). The kinetics of viral infection of rat 9L gliosarcoma cells by the replication-conditional HSV-1 vector, hrR3, was studied by measuring the accumulation rate of 2-[14C]-fluoro-5-iodo-1-beta-D-arabinofuranosyl-uracil (FIAU), a selective substrate for viral thymidine kinase (TK). The level of viral TK activity in 9L cells was monitored by the radiotracer assay to assess various vector doses and infection times, allowing vector replication and spread. In parallel, viral yields and levels of Escherichia coli beta-galactosidase activity were assessed quantitatively. To study vector replication, spread and HSV-1-tk and lacZ gene coexpression in vivo, first- or second-generation recombinant HSV-1 vectors (hrR3 or MGH-1) were injected into s.c. growing rat 9L or human U87 deltaEGFR gliomas in nude rats at various times (8 h to 8 days) and at various vector doses [1 x 10(6) to 2 x 10(9) plaque-forming units (PFUs)] prior to imaging. For noninvasive assessment of HSV-1-tk gene expression (124I-labeled FIAU % dose/g), 0.15 mCi of 124I-labeled FIAU was injected i.v. 8 h after the last vector administration, and FIAU positron emission tomography (PET) was performed 48 h later. For the assessment of HSV-1-tk and lacZ gene coexpression, 0.2 mCi of 131I-labeled FIAU was injected i.v. 24 h after the last vector administration. Forty-eight h later, animals were killed, and tumors were dissected for quantitative autoradiographical and histochemical assessment of regional distribution of radioactivity (TK expression measured as 131I-labeled FIAU % dose/g) and coexpressed lacZ gene activity. The rates of FIAU accumulation (Ki) in hrR3-infected 9L cells in culture, which reflect the levels of HSV-1-tk gene expression, ranged between 0.12 and 3.4 ml/g/min. They increased in a vector dose- and infection time-dependent manner and correlated with the virus yield (PFUs/ml), where the PFUs:Ki ratios remained relatively constant over time. Moreover, a linear relationship was observed between lacZ gene expression and FIAU accumulation 5-40 h after infection of 9L cells with a multiplicity of infection of 1.5. At later times (> 52 h postinjection), high vector doses (multiplicity of infection, 1.5) led to a decrease of FIAU accumulation rates, viral yield, and cell pellet weights, indicating vector-mediated cell toxicity. Various levels of HSV-1-tk gene expression could be assessed by FIAU-PET after in vivo infection of s.c. tumors. The levels of FIAU accumulation were comparatively low (approximately ranging from 0.00013 to 0.003% injected dose/g) and were spatially localized; this may reflect viral-induced cytolysis of infected tumor cells and limited lateral spread of the virus. Image coregistration of tumor histology, HSV-1-tk related radioactivity (assessed by autoradiography), and lacZ gene expression (assessed by beta-galactosidase staining) demonstrated a characteristic pattern of gene expression around the injection sites. A rim of lacZ gene expression immediately adjacent to necrotic tumor areas was observed, and this zone was surrounded by a narrow band of HSV-1-tk-related radioactivity, primarily in viable-appearing tumor tissue. These results demonstrate that recombinant HSV-1 vector-mediated HSV-1-tk gene expression can be monitored noninvasively by PET, where the areas of FIAU-derived radioactivity identify the viable portion of infected tumor tissue that retains FIAU accumulation ability, and that the accumulation rate of FIAU in culture, Ki, reflects the number of HSV-1 viral particles in the infected tumor cell population [4.1 +/- 0.6 x 10(6) PFUs/Ki unit (PFUs divided by ml/min/g)]. Moreover, time-dependent and spatial relationships of HSV-1-tk and lacZ gene coexpression in culture and in vivo indicate the potential for indirect in vivo imaging of therapeutic gene expression in tumor tissue infected with any recombinant HSV-1 vector where a therapeutic gene is substituted for the lacZ gene.

Imaging TCR-dependent NFAT-mediated T-cell activation with positron emission tomography in vivo.

A noninvasive method for molecular imaging of T-cell activity in vivo would be of considerable value. It would aid in understanding the role of specific genes and signal transduction pathways in the course of normal and pathologic immune responses, and could elucidate temporal dynamics and immune regulation at different stages of disease and following therapy. We developed and assessed a novel method for monitoring the T-cell receptor (TCR)-dependent nuclear factor of activated T cells (NFAT)-mediated activation of T cells by optical fluorescence imaging (OFI) and positron emission tomography (PET). The herpes simplex virus type 1 thymidine kinase/green fluorescent protein [HSV1-tk/GFP (TKGFP)] dual reporter gene was used to monitor NFAT-mediated transcriptional activation in human Jurkat cells. A recombinant retrovirus bearing the NFAT-TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting NFAT-specific enhancer. Transduced Jurkat cells were used to establish subcutaneous infiltrates in nude rats. We demonstrated that noninvasive OFI and nuclear imaging of T-cell activation is feasible using the NFAT-TKGFP reporter system. PET imaging with [(124)I]FIAU using the NFAT-TKGFP reporter system is sufficiently sensitive to detect T-cell activation in vivo. PET images were confirmed by independent measurements of T-cell activation (e.g., CD69) and induction of GFP fluorescence. PET imaging of TCR-induced NFAT-dependent transcriptional activity may be useful in the assessment of T cell responses, T-cell-based adoptive therapies, vaccination strategies and immunosuppressive drugs.

Imaging adenoviral-mediated herpes virus thymidine kinase gene transfer and expression in vivo.

The feasibility of noninvasive imaging of adenoviral-mediated herpes virus type one thymidine kinase (HSV1-tk) gene transfer and expression was assessed in a well-studied animal model of metastatic colon carcinoma of the liver. Tumors were produced in syngeneic BALB/c mice by intrahepatic injection of colon carcinoma cells (MCA-26). Seven days later, three different doses (3 x 10(8), 1 x 10(8), and 3 x 10(7) plaque-forming units (pfu) of the recombinant adenoviral vector ADV. Rous sarcoma virus (RSV)-tk bearing the HSV1-tk gene were administered by intratumoral injection in separate groups of mice. Two control groups of tumor-bearing mice received intratumoral injections of the control adenoviral vector dl-312 or buffer alone, respectively. T2-weighted magnetic resonance (MR) images of mice were obtained before administering the virus and provided an anatomical reference of hepatic tumor localization. Eighteen h after the virus injection, one group of animals was given i.v. injections of 300 microCi of no-carrier-added 5-[131I]-2'-fluoro-1-beta-D-arabinofuranosyluracil (FIAU) and imaged 24 h later with a gamma camera. In some animals, the tumors were sampled and processed for histology and quantitative autoradiography (QAR). The gamma camera images demonstrated highly specific localization of [131I]FIAU-derived radioactivity to the area of ADV.RSV-tk-injected tumors in the liver, which was confirmed by coregistering the gamma camera and T2-weighted MR images. There was no accumulation of [131I]FIAU-derived radioactivity in tumors that were injected with the control vector or injection solution alone. A more precise distribution of radioactivity in the area of transfected tumor was obtained by histological and QAR comparisons. A heterogeneous pattern of radioactivity distribution in transfected tumors was observed. A punctate pattern of radioactivity distribution was observed in peritumoral liver tissue in animals given injections of 3 x 10(8) and 1 x 10(8) pfu of ADV.RSV-tk but not in animals given injections of 3 x 10(7) pfu nor in control animals. A QAR-microscopic comparison showed that the punctate areas of radioactivity colocalized with cholangial ducts. The level of [131I]FIAU-derived radioactivity accumulation (HSV1-tk expression) in the transfected tumors was viral dose-dependent. The viral dose-dependency of radioactivity accumulation was more pronounced in peritumoral liver, which was confirmed by reverse transcription-PCR analysis. A separate group of tumor-bearing animals received different doses of ADV.RSV-tk vector followed by treatment with ganciclovir (GCV), 10 mg/kg i.p. b.i.d. for 6 days. The ADV.RSV-tk transfected tumors significantly regressed with GCV treatment; the control tumors continued to grow. During the GCV treatment, the levels of liver transaminases (ALT and AST) were significantly increased in animals that received injections of 3 x 10(8) and 1 x 10(8) pfu of ADV.RSV-tk but not in animals that received injections of 3 x 10(7) pfu and in control animals. The observed liver toxicity confirms the results of gamma camera and QAR imaging, which demonstrated an unwanted spread of ADV.RSV-tk vector and HSV1-tk expression in peritumoral and remote liver tissue at higher doses. These and our previous results indicate that noninvasive imaging of adenoviral-mediated HSV1-tk gene expression is feasible for monitoring cancer gene therapy in patients.

Herpes simplex virus thymidine kinase as a marker/reporter gene for PET imaging of gene therapy.

Imaging transgene expression with radiopharmaceuticals is feasible and has been demonstrated with a gamma camera and by positron emission tomography (PET) in experimental animals. An important consideration in the development of the imaging paradigm was the selection of an appropriate transgene and radiopharmaceutical. The herpes simplex virus thymidine kinase gene (HSV1-tk) was selected as an example of a "marker gene", and radiolabeled 5-iodo-2'-fluoro-2'deoxy-1-beta-D-arabino-furanosyl-uracil (FIAU) was shown to be a substantially better "marker substrate" for the HSV1-TK enzyme than other nucleoside analogues, including radiolabeled ganciclovir and acyclovir. The magnitude of FIAU accumulation in different HSV1-tk transduced cell lines and in tumors derived from these cell lines, was highly correlated with independent measures of HSV1-tk expression; namely, to the level of HSV1-tk mRNA in the corresponding cell lines and to their level of sensitivity to the antiviral drug, ganciclovir. We have demonstrated for the first time that highly specific non-invasive images of HSV1-tk expression in experimental animal tumors can be obtained using radiolabeled FIAU and a clinical gamma camera or a PET system. Given the level of FIAU accumulation in the transduced tumors, it is likely that a clinically applicable method for imaging HSV1-tk gene expression can be implemented using existing clinical imaging techniques. Our results point towards the potential for a wider application of HSV1-tk as a "marker" gene for "indirect" imaging of other therapeutic transgenes. The use of multi-gene vector constructs, where imaging a "marker gene" can be used to assess the level of "therapeutic gene" expression, will be increasingly developed over the next decade. The ability to image the location (distribution) and the level of transgene expression over time will provide new and useful information for monitoring clinical gene therapy protocols in the future.

Noninvasive quantitation of cytosine deaminase transgene expression in human tumor xenografts with in vivo magnetic resonance spectroscopy.

Analysis of transgene expression in vivo currently requires destructive and invasive molecular assays of tissue specimens. Noninvasive methodology for assessing the location, magnitude, and duration of transgene expression in vivo will facilitate subject-by-subject correlation of therapeutic outcomes with transgene expression and will be useful in vector development. Cytosine deaminase (CD) is a microbial gene undergoing clinical trials in gene-directed enzyme prodrug gene therapy. We hypothesized that in vivo magnetic resonance spectroscopy could be used to measure CD transgene expression in genetically modified tumors by directly observing the CD-catalyzed conversion of the 5-fluorocytosine (5-FC) prodrug to the chemotherapeutic agent 5-fluorouracil (5-FU). The feasibility of this approach is demonstrated in subcutaneous human colorectal carcinoma xenografts in nude mice by using yeast CD (yCD). A three-compartment model was used to analyze the metabolic fluxes of 5-FC and its metabolites. The rate constants for yCD-catalyzed prodrug conversion (k(1)(app)), 5-FU efflux from the observable tumor volume (k(2)(app)), and formation of cytotoxic fluorinated nucleotides from 5-FU (k(3)(app)) were 0.49 +/- 0.27 min(-1), 0.766 +/- 0.006 min(-1), and 0.0023 +/- 0.0007 min(-1), respectively. The best fits of the 5-FU concentration data assumed first-order kinetics, suggesting that yCD was not saturated in vivo in the presence of measured intratumoral 5-FC concentrations well above the in vitro K(m). These results demonstrate the feasibility of using magnetic resonance spectroscopy to noninvasively monitor therapeutic transgene expression in tumors. This capability provides an approach for measuring gene expression that will be useful in clinical gene therapy trials.

Functional coexpression of HSV-1 thymidine kinase and green fluorescent protein: implications for noninvasive imaging of transgene expression.

Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1-tk genes was generated (tkgfp gene) and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS). TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2'-fluoro-2'-deoxy-1beta-D-arabinofuranosyl-5-iodo-uracil (FIAU), in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT]), and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.

A general approach to the non-invasive imaging of transgenes using cis-linked herpes simplex virus thymidine kinase.

Non-invasive imaging of gene expression opens new prospects for the study of transgenic animals and the implementation of genetically based therapies in patients. We have sought to establish a general paradigm to enable whole body non-invasive imaging of any transgene. We show that the expression and imaging of HSV1-tk (a marker gene) can be used to monitor the expression of the LacZ gene (a second gene) under the transcriptional control of a single promoter within a bicistronic unit that includes a type II internal ribosomal entry site. In cells bearing a single copy of the vector, the expression of the two genes is proportional and constant, both in vitro and in vivo. We demonstrate that non-invasive imaging of HSV1-tk gene accurately reflects the topology and activity of the other cis-linked transgene.

Imaging herpes virus thymidine kinase gene transfer and expression by positron emission tomography.

We report a series of studies that assess the feasibility and sensitivity of imaging of herpes virus type one thymidine kinase (HSV1-tk) gene transfer and expression with [124I]-5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil ([124I]-FIAU) and positron emission tomography (PET) and the ability of [124I]-FIAU-PET imaging to discriminate different levels of HSV1-tk gene expression. Studies were performed in rats bearing multiple s.c. tumors derived from W256 rat carcinoma and RG2 rat glioma cells. In the first set, we tested the sensitivity of [124I]-FIAU-PET imaging to detect low levels of HSV1-tk gene expression after retroviral-mediated gene transfer. HSV1-tk gene transduction of one of preestablished wild-type W256 tumor in each animal was accomplished by direct intratumoral injection of retroviral vector-producer cells (W256-->W256TK* tumors). Tumors produced from W256 and W256TK+ cells served as the negative and positive control in each animal. Highly specific images of [124I]-FIAU-derived radioactivity were obtained in W256TK* tumors (that were transduced in vivo) and in W256TK+ tumors but not in nontransduced wild-type W256 tumors. The level of "specific" incorporated radioactivity in transduced portions of both W256TK* and W256TK+ tumors was relatively constant between 4 and 50 h. In the second set, we tested whether [124I]-FIAU and PET imaging can measure and discriminate between different levels of HSV1-tk gene expression. Multiple s.c. tumors were produced from wild-type RG2 cells and stably transduced RG2TK cell lines that express different levels of HSV1-tk. A highly significant relationship between the level of [124I]-FIAU accumulation [% injected dose/g and incorporation constant (Ki)] and an independent measure of HSV1-tk expression (sensitivity of the transduced tumor cells to ganciclovir, IC50) was demonstrated, and the slope of this relationship was defined as a sensitivity index. We have demonstrated for the first time that highly specific noninvasive images of HSV1-tk expression in experimental animal tumors can be obtained using radiolabeled 2'-fluoro-nucleoside [124I]-FIAU and a clinical PET system. The ability to image the location (distribution) of gene expression and the level of expression over time provides new and useful information for monitoring clinical gene therapy protocols in the future.

Tumor growth modulation by sense and antisense vascular endothelial growth factor gene expression: effects on angiogenesis, vascular permeability, blood volume, blood flow, fluorodeoxyglucose uptake, and proliferation of human melanoma intracerebral xenografts.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, has been investigated as a potent mediator of brain tumor angiogenesis and tumor growth. We evaluated the effect of VEGF expression on the pathophysiology of tumor growth in the brain. Human SK-MEL-2 melanoma cells, with minimal VEGF expression, were stably transfected with either sense or antisense mouse VEGF cDNA and used to produce intracerebral xenografts. Vascular permeability, blood volume, blood flow, and tumor fluorodeoxyglucose metabolism were assessed using tissue sampling and quantitative autoradiography. Tumor proliferation was assessed by measuring bromodeoxyuridine labeling indices. Tumor vascular density and morphological status of the blood-brain barrier were evaluated by immunohistochemistry. SK-MEL-2 cells transfected with sense VEGF (V+) expressed large amounts of mouse and human VEGF protein; V+ cells formed well-vascularized, rapidly growing tumors with minimal tumor necrosis. V+ tumors had substantial and significant increases in blood volume, blood flow, vascular permeability, and fluorodeoxyglucose metabolism compared to wild-type and/or V- (antisense VEGF) tumors. VEGF antisense transfected V- expressed no detectable VEGF protein and formed minimally vascularized tumors. V- tumors had a very low initial growth rate with central necrosis; blood volume, blood flow, vascular permeability, and glucose metabolism levels were low compared to wild-type and V+ tumors. A substantial inhibition of intracerebral tumor growth, as well as a decrease in tumor vascularity, blood flow, and vascular permeability may be achieved by down-regulation of endogenous VEGF expression in tumor tissue. VEGF-targeted antiangiogenic gene therapy could be an effective component of a combined strategy to treat VEGF-producing brain tumors.

Noninvasive imaging of herpes virus thymidine kinase gene transfer and expression: a potential method for monitoring clinical gene therapy.

Noninvasive imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression is possible with a clinical gamma camera and by single-photon emission tomography (SPECT) using 131I-labeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodo-uracil (FIAU). Studies were performed in rats bearing s.c. tumors. Tumors were produced by injection of wild-type RG2 glioma or W256 mammary carcinoma cells into one flank and RG2TK+ glioma or W256TK+ mammary carcinoma cells (that had been transduced in vitro with the HSV1-tk gene) into the opposite flank. In some animals, HSV1-tk gene transduction of the pre-established wild-type tumors was accomplished in vivo by direct intratumoral injection of retroviral vector-producer cells. Imaging studies were performed 2 weeks after tumor transduction to allow time for production and spread of the retroviruses through the tumor and for sufficient growth and increase in size of the tumors to facilitate imaging. The gamma camera and SPECT images revealed highly specific localization of [131I]FIAU-derived radioactivity to areas of HSV1-tk gene expression at 24, 36, and 48 h after i.v. administration of 1.6-2.8 mCi of [131I]FIAU. Comparative analysis of quantitative autoradiographic images obtained from the same tumors confirmed that the high levels of [131I]FIAU-derived radioactivity (> 1% dose) were localized to areas of HSV1-tk gene expression demonstrated by immunohistochemical staining for HSV1-tk protein. In contrast, significantly lower levels of [131I]FIAU-derived radioactivity (< 0.01%) were observed in the surrounding nontransduced tumor tissue, contralateral wild-type tumors, and other tissues that showed no immunohistochemical staining for the HSV1-tk protein. The magnitude of FIAU accumulation in RG2TK+, W256TK+, and wild-type tumors corresponded to the in vitro ganciclovir sensitivity of the cell lines used to produce these tumors, which indicates that the magnitude of FIAU accumulation reflects the level of HSV1-tk gene expression. We suggest that "clinically relevant" levels of HSV1-tk gene expression in transfected tissue can be imaged with [131I]FIAU and a gamma camera or SPECT, and that a significant improvement in imaging sensitivity and resolution is expected with [124I]FIAU and PET.

Imaging the expression of transfected genes in vivo.

Imaging the expression of successful gene transduction has been demonstrated in vivo for the first time by using an appropriate combination of "marker gene" and "marker substrate" in an experimental animal model. The herpes simplex virus 1 thymidine kinase (HSV1-tk) gene was selected as an example of a marker gene, and the recombinant STK retrovirus containing HSV1-tk was used to transduce RG2 glioma cells in vitro and in vivo. RG2TK+ cell lines expressing the HSV1-tk gene and three potential marker substrates for the HSV1-TK enzyme were evaluated. Radiolabeled 5-iodo-2'-fluoro-2'deoxy-1-beta-D-arabinofuranosyluracil (FIAU) was shown to be a substantially better marker substrate for the HSV1-TK enzyme than 5-iodo-2'-deoxyuridine or ganciclovir. The magnitude of FIAU accumulation in different RG2TK+ clones corresponded to their sensitivity to ganciclovir and to the level of HSV1-tk mRNA expression. Imaging the expression of HSV1-tk in transduced RG2 tumor cells was demonstrated in animals using quantitative autoradiography; 2-[14C]FIAU accumulation was shown to be high in RG2TK+ brain tumors growing in one hemisphere and very low in nontransduced RG2 tumors in the contralateral hemisphere. Transduction of RG2 tumor cells with the HSV-tk gene in vivo resulted in tumors which accumulated FIAU to high levels and produced clearly defined images. Given the level of FIAU accumulation in the transduced tumors, it is likely that a clinically applicable method for imaging HSV1-tk gene expression can be implemented using existing clinical imaging techniques.

Imaging of brain tumor proliferative activity with iodine-131-iododeoxyuridine.

METHODS: Iodine-131-iododeoxyuridine (IUdR) uptake and retention was imaged with SPECT at 2 and 24 hr after administering a 10-mCi dose to six patients with primary brain tumors. The SPECT images were directly compared to gadolinium contrast-enhanced MR images as well as to [18F]fluorodeoxyglucose (FDG) PET scans and 201Tl SPECT scans. RESULTS: Localized uptake and retention of IUdR-derived radioactivity was observed in five of six patients. The plasma half-life of [131I]IUdR was short (1.6 min) in comparison to the half-life of total plasma radioactivity (6.4 hr). The pattern of [131I]IUdR-derived radioactivity was markedly different in the 2-hr compared to 24-hr images. Radioactivity was localized along the periphery of the tumor and extended beyond the margin of tumor identified by contrast enhancement on MRI. The estimated levels of tumor radioactivity at 24 hr, based on semiquantitative phantom studies, ranged between < 0.1 and 0.2 microCi/cc (< 0.001% and 0.002% dose/cc); brain levels were not measurable. CONCLUSIONS: Iodine-131-IUdR SPECT imaging of brain tumor proliferation has low (marginal) sensitivity due to low count rates and can detect only the most active regions of tumor growth. Imaging at 24 hr represents a washout strategy to reduce 131I-labeled metabolites contributing to background activity in the tumors, and is more likely to show the pattern of [131I]IUdR-DNA incorporation and thereby increase image specificity. Iodine-123-IUdR SPECT imaging at 12 hr and the use of [124I]IUdR and PET will improve count acquisition and image quality.

Mutant herpes simplex virus induced regression of tumors growing in immunocompetent rats.

Herpes simplex virus (HSV) mutants kill dividing tumor cells but spare non-proliferating, healthy brain tissue and may be useful in developing new treatment strategies for malignant brain tumors. Two HSV mutants, a thymidine kinase deficient virus (TK-) and a ribonucleotide reductase mutant (RR-), killed 7/7 human tumor cell lines in tissue culture. The TK-HSV killed Rat RG2 glioma and W256 carcinoma lines but not the rat C6 glioma in culture. TK-HSV replication (12 pfu/cell) was similar to wild-type HSV (10 pfu/cell) in rapidly dividing W256 cells in tissue culture, but was minimal (< 1 pfu/cell) in serum-starved cells, suggesting that the proliferative activity of tumor cells at the site and time of TK-HSV injection may influence efficacy in vivo. Subcutaneous W256 tumors in male Sprague-Dawley rats were injected with TK-HSV or free inoculum. A significant effect of TK-HSV therapy on W256 tumor growth was demonstrated compared to controls (p = 0.002). Complete regression was observed in 4/9 experimental tumors, with no recurrence over 6 months. Tumor growth in the remaining 5/9 animals was attenuated during the first 3 to 5 days after treatment, but not beyond 5 days compared to 9 matched control animals; no tumor regression was observed in any of the control animals. These results suggest that HSV mutants are potentially useful as novel therapeutic agents in the treatment of tumors in immunocompetent subjects.