Shahriar S. Yaghoubi

Noninvasive detection of therapeutic cytolytic T cells with 18F-FHBG PET in a patient with glioma.

Background A 57-year-old man had been diagnosed with grade IV glioblastoma multiforme and was enrolled in a trial of adoptive cellular immunotherapy. The trial involved infusion of ex vivo expanded autologous cytolytic CD8+ T cells (CTLs), genetically engineered to express the interleukin 13 zetakine gene (which encodes a receptor protein that targets these T cells to tumor cells) and the herpes simplex virus 1 thymidine kinase (HSV1 tk) suicide gene, and PET imaging reporter gene.Investigations MRI, whole-body and brain PET scan with 18F-radiolabelled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (18F–FHBG) to detect CTLs that express HSV1 tk, and safety monitoring after injection of 18F–FHBG.Diagnosis MRI detected grade III–IV glioblastoma multiforme plus two tumors recurrences that developed after resection of the initial tumor.Management Surgical resection of primary glioblastoma tumor, enrollment in CTL therapy trial, reresection of glioma recurrences, infusion of approximately 1 × 109 CTLs into the site of tumor reresection, and 18F–FHBG PET scan to detect infused CTLs.

Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors.

Biodistribution, magnitude and duration of a therapeutic transgenes expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRGs expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic genes expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D2R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D2R express both reporter genes with good correlation (r2 = 0.93). Similarly, a high correlation (r2 = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D2R expression with 9-(4-[18F]fluoro-3-hydroxymethylbutyl) guanine ([18F]FHBG) and 3-(2-[18F]fluoroethyl)spiperone ([18F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.

Quantitative imaging of gene induction in living animals

Methods to repeatedly, non-invasively, and quantitatively image gene expression in living animals are rapidly emerging and should fundamentally change studies of gene expression in vivo. We previously developed assays utilizing positron emission tomography (PET) to image reporter gene expression. In this paper we: (1) describe a new bi-directional, tetracycline-inducible system that can be used to pharmacologically induce target gene expression and to quantitatively image induced expression by using a PET reporter gene; (2) demonstrate the potential of this system in transient and stable cell transfection assays; and (3) demonstrate the ability to repetitively and quantitatively image tetracycline and tetracycline analog induction of gene expression in living animals. We utilize the dopamine type-2 receptor (D2R) and the mutant herpes-simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter genes to validate this system. We utilize microPET technology to show that quantitative tomographic imaging of gene induction is possible. We find a high correlation (r2 = 0.98) between ‘target’ and reporter gene expression. This work establishes a new technique for imaging time-dependent variation of gene expression both from vectors with inducible promoters and in transgenic animals in which pharmacologic induction of gene expression must be monitored. These techniques may be applied both in gene therapy and for the study of gene expression in transgenic animals.

Imaging progress of herpes simplex virus type 1 thymidine kinase suicide gene therapy in living subjects with positron emission tomography

Molecular imaging of a suicide transgenes expression will aid the development of efficient and precise targeting strategies, and imaging for cancer cell viability may assess therapeutic efficacy. We used the PET reporter probe, 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)guanine ([18F]FHBG) to monitor the expression of a mutant Herpes Simplex Virus 1 thymidine kinase (HSV1-sr39tk) in C6 glioma tumors implanted subcutaneously in nude mice that were repetitively being treated with the pro-drug Ganciclovir (GCV). [18F]-Fluorodeoxyglucose ([18F]FDG), a metabolic tracer, was used to assess tumor cell viability and therapeutic efficacy. C6 glioma tumors stably expressing the HSV1-sr39tk gene (C6sr39) accumulated [18F]FHBG prior to GCV treatment. Significant declines in C6sr39 tumor volumes and [18F]FHBG and [18F]FDG accumulation were observed following 2 weeks of GCV treatment. However, 3 weeks after halting GCV treatment, the tumors re-grew and [18F]FDG accumulation increased significantly; in contrast, tumor [18F]FHBG concentrations remained at background levels. Therefore, [18F]FHBG can be used to detect tumors expressing HSV1-sr39tk, susceptible to regression in response to GCV exposure, and the effectiveness of GCV therapy in eradicating HSV1-sr39tk-expressing cells can be monitored by [18F]FHBG scanning. [18F]FHBG and [18F]FDG imaging data indicate that exposure of C6sr39 tumors to GCV causes the elimination of [18F]FHBG-accumulating C6sr39 cells and selects for re-growth of tumors unable to accumulate [18F]FHBG.

PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and to image intracellular molecular events and cell trafficking in living subjects. The expression of these PRGs can be imaged using 18F- or 124I-radiolabeled acycloguanosine or pyrimidine analog PET reporter probes (PRPs). This protocol describes the procedures for imaging HSV1-tk or HSV1-sr39tk PRG expression in living subjects with the acycloguanosine analog 9-4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG). [18F]FHBG is a high-affinity substrate for the HSV1-sr39TK enzyme with relatively low affinity for mammalian TK enzymes, resulting in improved detection sensitivity. Furthermore, [18F]FHBG is approved by the US Food and Drug Administration as an investigational new imaging agent and has been shown to detect HSV1-tk transgene expression in the liver tumors of patients. MicroPET imaging of each small animal can be completed in approximately 1.5 h, and each patient imaging session takes approximately 3 h.

Novel Strategy for a Cocktail 18F-Fluoride and 18F-FDG PET/CT Scan for Evaluation of Malignancy: Results of the Pilot-Phase Study

18F-FDG PET/CT is used for detecting cancer and monitoring cancer response to therapy. However, because of the variable rates of glucose metabolism, not all cancers are identified reliably. Sodium 18F was previously used for bone imaging and can be used as a PET/CT skeletal tracer. The combined administration of 18F and 18F-FDG in a single PET/CT study for cancer detection has not been reported to date. Methods: This is a prospective pilot study (November 2007–November 2008) of 14 patients with proven malignancy (6 sarcoma, 3 prostate cancer, 2 breast cancer, 1 colon cancer, 1 lung cancer, and 1 malignant paraganglioma) who underwent separate 18F PET/CT and 18F-FDG PET/CT and combined 18F/18F-FDG PET/CT scans for the evaluation of malignancy (a total of 3 scans each). There were 11 men and 3 women (age range, 19–75 y; average, 50.4 y). Results: Interpretation of the combined 18F/18F-FDG PET/CT scans compared favorably with that of the 18F-FDG PET/CT (no lesions missed) and the 18F PET/CT scans (only 1 skull lesion seen on an 18F PET/CT scan was missed on the corresponding combined scan). Through image processing, the combined 18F/18F-FDG scan yielded results for bone radiotracer uptake comparable to those of the 18F PET/CT scan performed separately. Conclusion: Our pilot-phase prospective trial demonstrates that the combined 18F/18F-FDG administration followed by a single PET/CT scan is feasible for cancer detection. This combined method opens the possibility for improved patient care and reduction in health care costs.

Preclinical Efficacy of the c-Met Inhibitor CE-355621 in a U87 MG Mouse Xenograft Model Evaluated by 18F-FDG Small-Animal PET

The purpose of this study was to evaluate the efficacy of CE-355621, a novel antibody against c-Met, in a subcutaneous U87 MG xenograft mouse model using 18F-FDG small-animal PET. Methods: CE-355621 or control vehicle was administered intraperitoneally into nude mice (drug-treated group, n = 12; control group, n = 14) with U87 MG subcutaneous tumor xenografts. Drug efficacy was evaluated over 2 wk using 18F-FDG small-animal PET and compared with tumor volume growth curves. Results: The maximum %ID/g (percentage injected dose per gram of tissue) of 18F-FDG accumulation in mice treated with CE-355621 remained essentially unchanged over 2 wk, whereas the %ID/g of the control tumors increased 66% compared with the baseline. Significant inhibition of 18F-FDG accumulation was seen 3 d after drug treatment, which was earlier than the inhibition of tumor volume growth seen at 7 d after drug treatment. Conclusion: CE-355621 is an efficacious novel antineoplastic chemotherapeutic agent that inhibits 18F-FDG accumulation earlier than tumor volume changes in a mouse xenograft model. These results support the use of 18F-FDG PET to assess early tumor response for CE-355621.

A Novel Method for Direct Site-Specific Radiolabeling of Peptides Using [18F]FDG

We have used the well-accepted and easily available 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG) positron emission tomography (PET) tracer as a prosthetic group for synthesis of (18)F-labeled peptides. We herein report the synthesis of [(18)F]FDG-RGD ((18)F labeled linear RGD) and [(18)F]FDG-cyclo(RGD(D)YK) ((18)F labeled cyclic RGD) as examples of the use of [(18)F]FDG. We have successfully prepared [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) in 27.5% and 41% radiochemical yields (decay corrected) respectively. The receptor binding affinity study of FDG-cyclo(RGD(D)YK) for integrin alpha(v)beta(3), using alpha(v)beta(3) positive U87MG cells confirmed a competitive displacement with (125)I-echistatin as a radioligand. The IC(50) value for FDG-cyclo(RGD(D)YK) was determined to be 0.67 +/- 0.19 muM. High-contrast small animal PET images with relatively moderate tumor uptake were observed for [(18)F]FDG-RGD and [(18)F]FDG-cyclo(RGD(D)YK) as PET probes in xenograft models expressing alpha(v)beta(3) integrin. In conclusion, we have successfully used [(18)F]FDG as a prosthetic group to prepare (18)F]FDG-RGD and [(18)F]FDG-cyclic[RGD(D)YK] based on a simple one-step radiosynthesis. The one-step radiosynthesis methodology consists of chemoselective oxime formation between an aminooxy-functionalized peptide and [(18)F]FDG. The results have implications for radiolabeling of other macromolecules and would lead to a very simple strategy for routine preclinical and clinical use.

Gene therapy imaging in patients for oncological applications

Thus far, traditional methods for evaluating gene transfer and expression have been shown to be of limited value in the clinical arena. Consequently there is a real need to develop new methods that could be repeatedly and safely performed in patients for such purposes. Molecular imaging techniques for gene expression monitoring have been developed and successfully used in animal models, but their sensitivity and reproducibility need to be tested and validated in human studies. In this review, we present the current status of gene therapy-based anticancer strategies and show how molecular imaging, and more specifically radionuclide-based approaches, can be used in gene therapy procedures for oncological applications in humans. The basis of gene expression imaging is described and specific uses of these non-invasive procedures for gene therapy monitoring illustrated. Molecular imaging of transgene expression in humans and evaluation of response to gene-based therapeutic procedures are considered. The advantages of molecular imaging for whole-body monitoring of transgene expression as a way to permit measurement of important parameters in both target and non-target organs are also analyzed. The relevance of this technology for evaluation of the necessary vector dose and how it can be used to improve vector design are also examined. Finally, the advantages of designing a gene therapy-based clinical trial with imaging fully integrated from the very beginning are discussed and future perspectives for the development of these applications outlined.

Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications

Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.