Preliminary efficacy of [90Y]DOTA-biotin-avidin radiotherapy against non-muscle invasive bladder cancer.

PURPOSE: Bladder cancer represents 3% of all new cancer diagnoses per year. We propose intravesical radionuclide therapy using the ß-emitter 90Y linked to DOTA-biotin-avidin ([90Y]DBA) to deliver short-range radiation against non-muscle invasive bladder cancer (NMIBC). MATERIAL AND METHODS: Image-guided biodistribution of intravesical DBA was investigated in an animal model by radiolabeling DBA with the 68Ga and dynamic microPET imaging following intravesical infusion of [68Ga]DBA for up to 4 h and post-necropsy γ-counting of organs. The antitumor activity of [90Y]DBA was investigated using an orthotopic MB49 murine bladder cancer model. Mice were injected with luciferase-expressing MB49 cells and treated via intravesical administration with 9.2 MBq of [90Y]DBA or unlabeled DBA 3 days after the tumor implantation. Bioluminescence imaging was conducted after tumor implantation to monitor the bladder tumor growth. In addition, we investigated the effects of [90Y]DBA radiation on urothelial histology with immunohistochemistry analysis of bladder morphology. RESULTS: Our results demonstrated that DBA is contained in the bladder for up to 4 h after intravesical infusion. A single dose of [90Y]DBA radiation treatment significantly reduced growth of MB49 bladder carcinoma. Attaching 90Y-DOTA-biotin to avidin prevents its re-absorption into the blood and distribution throughout the rest of the body. Furthermore, immunohistochemistry demonstrated that [90Y]DBA radiation treatment did not cause short-term damage to urothelium at day 10, which appeared similar to the normal urothelium of healthy mice. CONCLUSION: Our data demonstrates the potential of intravesical [90Y]DBA as a treatment for non-muscle invasive bladder cancer.

Evaluating the Combined Anticancer Response of Checkpoint Inhibitor Immunotherapy and FAP-Targeted Molecular Radiotherapy in Murine Models of Melanoma and Lung Cancer.

Immunotherapy has dramatically improved outcomes for some cancer patients; however, novel treatments are needed for more patients to achieve a long-lasting response. FAP-targeted molecular radiotherapy has shown efficacy in both preclinical and clinical models and has immunomodulatory effects. Here, we studied if combined immunotherapy and radiotherapy could increase antitumor efficacy in murine models of lung cancer and melanoma and interrogated the mechanisms by which these treatments attenuate tumor growth. Using LLC1 and B16F10 murine models of lung cancer and melanoma, respectively, we tested the efficacy of 177Lu-FAPI-04 alone and in combination with immunotherapy. Alone, 177Lu-FAPI-04 significantly reduced tumor growth in both models. In animals with melanoma, combined therapy resulted in tumor regression while lung tumor growth was attenuated, but tumors did not regress. Combined therapy significantly increased caspase-3 and decreased Ki67 compared with immunotherapy alone. Flow cytometry demonstrated that tumor-associated macrophages responded in a tumor-dependent manner which was distinct in animals treated with both therapies compared with either therapy alone. These data demonstrate that 177Lu-FAPI-04 is an effective anticancer therapy for melanoma and lung cancer which mediates effects at least partially through induction of apoptosis and modulation of the immune response. Translational studies with immunotherapy and 177Lu-FAPI-04 are needed to demonstrate the clinical efficacy of this combined regimen.

Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054.

Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.

Real-Time Positron Emission Tomography Evaluation of Topotecan Brain Kinetics after Ultrasound-Mediated Blood-Brain Barrier Permeability.

Glioblastoma (GBM) is the most common primary adult brain malignancy with an extremely poor prognosis and a median survival of fewer than two years. A key reason for this high mortality is that the blood-brain barrier (BBB) significantly restricts systemically delivered therapeutics to brain tumors. High-intensity focused ultrasound (HIFU) with microbubbles is a methodology being used in clinical trials to noninvasively permeabilize the BBB for systemic therapeutic delivery to GBM. Topotecan is a topoisomerase inhibitor used as a chemotherapeutic agent to treat ovarian and small cell lung cancer. Studies have suggested that topotecan can cross the BBB and can be used to treat brain metastases. However, pharmacokinetic data demonstrated that topotecan peak concentration in the brain extracellular fluid after systemic injection was ten times lower than in the blood, suggesting less than optimal BBB penetration by topotecan. We hypothesize that HIFU with microbubbles treatment can open the BBB and significantly increase topotecan concentration in the brain. We radiolabeled topotecan with 11C and acquired static and dynamic positron emission tomography (PET) scans to quantify [11C] topotecan uptake in the brains of normal mice and mice after HIFU treatment. We found that HIFU treatments significantly increased [11C] topotecan brain uptake. Moreover, kinetic analysis of the [11C] topotecan dynamic PET data demonstrated a substantial increase in [11C] topotecan volume of distribution in the brain. Furthermore, we found a decrease in [11C] topotecan brain clearance, confirming the potential of HIFU to aid in the delivery of topotecan through the BBB. This opens the potential clinical application of [11C] topotecan as a tool to predict topotecan loco-regional brain concentration in patients with GBMs undergoing experimental HIFU treatments.

Effect of Propranolol on 18F-Fluorodeoxyglucose Uptake in Brown Adipose Tissue in Children and Young Adults with Neoplastic Diseases.

PURPOSE: To evaluate the effectiveness of propranolol at mitigating FDG uptake in brown adipose tissue (BAT) of pediatric patients with known or suspected malignancies. METHODS: PET/CT scans of 3 cohorts of patients treated from 2005 to 2017 were scored for the presence of FDG uptake by BAT at 7 sites: right or left neck/supraclavicular area, right or left axilla, mediastinum, posterior thorax, and abdomen/pelvis. Uptake was scored as follows: 0, none; 1, mild uptake < liver; 2, moderate uptake = liver; and 3, intense uptake > liver. Group 1 consisted of 323 patients (630 scans) who had no specific preparation to mitigate FDG uptake by BAT. Group 2 consisted of 345 patients (705 scans) who underwent only warming in an uptake room with a fixed temperature at 24 °C. Group 3 consisted of 622 patients (1457 scans) who underwent warming. In group 3, patients 8 years and older, 471 patients (1114 scans), were also pre-medicated with oral propranolol 60 min before injection of FDG. Generalized estimation equation, using the logit link method, was used to model the relationship between the incidence of BAT score > 0, in any site, as a function of age, sex, seasonal effect, and body surface area (BSA). RESULTS: In patients aged 8 years or older, the incidence of BAT uptake was 35-44 % and declined to 15 % with propranolol. BAT was most frequent in the neck (26 %), axilla (18 %), posterior thorax (18 %), mediastinum (14 %), and abdomen/pelvis (8 %); BAT was less common in warm months (p = 0.001). No substantial benefit was shown with pre-injection warming alone. No significant effect was found for age, sex, or BSA separately. When BAT uptake was present, it was usually intense. CONCLUSION: Propranolol preparation minimizes FDG uptake by BAT and should be considered routine for pediatric FDG PET/CT cancer-related protocols in children, adolescents, and young adults.

3D optical/CT as a preclinical companion imaging platform for glioblastoma drug development.

Multimodality 3D Optical Imaging (OI)/CT has the potential to play a major role in drug development for glioblastomas (GBM), as it is an accessible preclinical method. To demonstrate the potential of 3D OI/CT to visualize orthotopic GBM implantation, we labeled GBM cells with Cy7 and imaged their location using 3D OI/CT. To confirm the accuracy of the spatial localization and demonstrate the ability to image locoregionally delivered therapies, we labeled mouse albumin with Cy7 (Cy7ALB) and delivered it via locoregional infusion 1 mm or 3 mm into the brain and demonstrated correlation of signal between the 3D OI/CT and post necropsy brain slices. In addition, we demonstrated the potential of systemically delivered Cy7ALB contrast to detect blood-brain barrier (BBB) permeability caused by orthotopic GBMs using 3D OI/CT. We also tested the potential of 3D OI/CT to assess focal BBB permeability induced by high intensity focused ultrasound (HIFU), a methodology being used in clinical trials to noninvasively permeabilize the BBB for systemic therapeutic delivery to GBM. We demonstrated the ability of systemic Cy7ALB contrast together with 3D OI/CT to accurately assess real-time HIFU-induced BBB permeability, which correlated to post necropsy imaging of brains. Furthermore, we demonstrated that 3D OI/CT can also image the therapeutic distribution of a Cy7-labeled anti-PD-1 antibody, a prototype translational antibody therapy. We successfully imaged real-time antibody distribution after HIFU-induced BBB permeability, which correlated with post necropsy Cy7 signal and translational PET imaging after injection of [89Zr] anti-PD-1 antibody. Thus, we demonstrated the broad potential of using 3D OI/CT as an accessible preclinical tool to develop anti-GBM therapies.

Radiosynthesis and evaluation of [18F]FMTP, a COX-2 PET ligand.

BACKGROUND: The upregulation of cyclooxygenase-2 (COX-2) is involved in neuroinflammation associated with many neurological diseases as well as cancers of the brain. Outside the brain, inflammation and COX-2 induction contribute to the pathogenesis of pain, arthritis, acute allograft rejection, and in response to infections, tumors, autoimmune disorders, and injuries. Herein, we report the radiochemical synthesis and evaluation of [18F]6-fluoro-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine ([18F]FMTP), a high-affinity COX-2 inhibitor, by cell uptake and PET imaging studies. METHODS: The radiochemical synthesis of [18F]FMTP was optimized using chlorine to fluorine displacement method, by reacting [18F]fluoride/K222/K2CO3 with the precursor molecule. Cellular uptake studies of [18F]FMTP was performed in COX-2 positive BxPC3 and COX-2 negative PANC-1 cell lines with unlabeled FMTP as well as celecoxib to define specific binding agents. Dynamic microPET image acquisitionwas performed in anesthetized nude mice (n = 3), lipopolysaccharide (LPS) induced neuroinflammation mice (n = 4), and phosphate-buffered saline (PBS) administered control mice (n = 4) using a Trifoil microPET/CT for a scan period of 60 min. RESULTS: A twofold higher binding of [18F]FMTP was found in COX-2 positive BxPC3 cells compared with COX-2 negative PANC-1 cells. The radioligand did not show specific binding to COX-2 negative PANC-1 cells. MicroPET imaging in wild-type mice indicated blood-brain barrier (BBB) penetration and fast washout of [18F]FMTP in the brain, likely due to the low constitutive COX-2 expression in the normal brain. In contrast, a ~ twofold higher uptake of the radioligand was found in LPS-induced mice brain than PBS treated control mice. CONCLUSIONS: Specific binding to COX-2 in BxPC3 cell lines, BBB permeability, and increased brain uptake in neuroinflammation mice qualifies [18F]FMTP as a potential PET tracer for studying inflammation.

Granzyme B PET Imaging of the Innate Immune Response.

The human immune system is a complex system which protects against invaders and maintains tissue homeostasis. It is broadly divided into the innate and adaptive branches. Granzyme B is serine protease that plays an important role in both and can serve as a biomarker for cellular activation. Because of this, a granzyme B PET agent (GZP) has recently been developed and has been shown to be able to monitor response to immunotherapy. Here, we evaluated the utility of granzyme B PET imaging to assess the innate immune response. We subcutaneously administered LPS to mice to induce inflammation and performed granzyme B PET imaging after 24 and 120 h. We dissected out tissue in the region of injection and performed granzyme B immunofluorescence (IF) to confirm specificity of the GZP radiotracer. Granzyme B PET imaging demonstrated increased uptake in the region of LPS injection after 24 h, which normalized at 120 h. Granzyme B immunofluorescence showed specific staining in tissue from the 24 h time point compared to the PBS-injected control. These findings support the use of granzyme B PET for imaging innate immunity. In certain clinical contexts, the use of GZP PET imaging may be superior to currently available agents, and we therefore suggest further preclinical studies with the ultimate goal of translation to clinical use.

Multimodality molecular imaging of the alveolar-capillary barrier in lung disease using albumin based optical and PET tracers.

Inflammatory changes caused by viruses, bacteria, exposure to toxins, commonly used drugs and even surgical intervention have the potential of causing abnormal epithelial permeability, which is manifest as infiltrative processes on computed tomography (CT), including the widespread infiltrates seen in COVID-19 pneumonia and acute respiratory distress syndrome (ARDS). We utilized a previously published mouse model of ARDS, intranasal delivery of LPS, to induce the alveolar-capillary barrier permeability seen in lung disease. We intravenously injected mice with Cy7 or 68-Gallium (68Ga) labeled mouse albumin and imaged using optical imaging (OI)/CT and PET. We observed significantly increased lung levels of Cy7-albumin on 3D OI/CT, which matched the abnormal appearance on microCT. This uptake correlated with fluorescence seen on sectioned lungs. To examine the translational potential of these findings, we radiolabeled albumin with 68Ga. We found that in mice with LPS-induced lung injury, 68Ga-albumin PET correlated with our optical imaging findings and demonstrated abnormal activity in the lung fields, indicative of abnormal epithelial permeability. These findings indicate 68Ga-albumin can be utilized as a sensitive translational radiotracer for quantifying the abnormal epithelial permeability that is seen in various lung pathologies, including COVID-19 induced pneumonia and ARDS. The ability to use Cy7-albumin 3D OI/CT imaging as a preclinical translational surrogate for 68Ga-albumin offers an accessible high throughput means to rapidly screen potential therapeutics against lung diseases that clinically manifest with endothelial permeability.

Impact of MYCN status on response of high-risk neuroblastoma to neoadjuvant chemotherapy.

BACKGROUND/PURPOSE: MYCN-amplification in neuroblastoma is associated with an aggressive clinical phenotype. We evaluated the association of MYCN amplification with tumor response to neoadjuvant chemotherapy. METHODS: Primary tumor response, assessed by percentage volume change on CT scan and degree of tumor resection, assessed by the operating surgeon, were retrospectively compared in 84 high-risk neuroblastoma patients. There were thirty-four (40%) with MYCN-amplified tumors and fifty (60%) with non-amplified tumors treated at our institution from 1999 to 2016. Metastatic disease response was assessed on MIBG scan by change in Curie score. RESULTS: MYCN-amplification was associated with a greater mean percentage reduction in primary tumor volume after neoadjuvant chemotherapy (72.27% versus 46.83% [non-amplified tumors], p = 0.001). The percentage of patients with a Curie score > 2 at diagnosis who then had a score ≤ 2 after neoadjuvant chemotherapy was not significantly different (8 [61.5%] and 8 [34.8%], respectively, p = 0.37). Twenty-eight (85.7%) patients with MYCN-amplification had ≥90% surgical resection compared to 45 (91.84%) patients with non-amplified tumors (p = 0.303). CONCLUSIONS: MYCN-amplification in high-risk neuroblastoma was associated with a better response of the primary tumor to neoadjuvant chemotherapy, but not metastatic sites, than in patients with non-amplified tumors. This did not significantly impact the ability to resect ≥90% of the primary tumor/locoregional disease. TYPE OF STUDY: Treatment Study LEVEL OF EVIDENCE: Level III.

eIF4A supports an oncogenic translation program in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with limited treatment options. Although metabolic reprogramming is a hallmark of many cancers, including PDA, previous attempts to target metabolic changes therapeutically have been stymied by drug toxicity and tumour cell plasticity. Here, we show that PDA cells engage an eIF4F-dependent translation program that supports redox and central carbon metabolism. Inhibition of the eIF4F subunit, eIF4A, using the synthetic rocaglate CR-1-31-B (CR-31) reduced the viability of PDA organoids relative to their normal counterparts. In vivo, CR-31 suppresses tumour growth and extends survival of genetically-engineered murine models of PDA. Surprisingly, inhibition of eIF4A also induces glutamine reductive carboxylation. As a consequence, combined targeting of eIF4A and glutaminase activity more effectively inhibits PDA cell growth both in vitro and in vivo. Overall, our work demonstrates the importance of eIF4A in translational control of pancreatic tumour metabolism and as a therapeutic target against PDA.

Implications of Image-Defined Risk Factors and Primary-Site Response on Local Control and Radiation Treatment Delivery in the Management of High-Risk Neuroblastoma: Is There a Role for De-escalation of Adjuvant Primary-Site Radiation Therapy?

PURPOSE: The predictive value of Image-Defined Risk Factors (IDRFs) developed by the International Neuroblastoma Risk Group Task Force as it relates to primary-site management is undefined and may aid patient selection for de-escalation of adjuvant radiation therapy to the primary site in high-risk neuroblastoma. METHODS AND MATERIALS: Patients (N = 76) with high-risk neuroblastoma treated on prospective trials at our institution from 1997 to 2014 were eligible for inclusion. IDRFs were defined based on pretherapy imaging. Overall survival, progression-free survival, and locoregional failure-free survival (LRFFS) were described using the Kaplan-Meier estimator and tested across strata by using the log-rank test. RESULTS: Twenty of 76 patients (26%) experienced local (n = 6), regional (n = 6), or combined locoregional failure (n = 8) with or without distant failure. Ten (50%) of the locoregional failures had concurrent distant relapse. Of patients who completed all therapy, both those with no IDRFs and those with >90% resection had a 3-year LRFFS of 100%, with or without radiation therapy. Patients with either ≥1 IDRF or 90% tumor resection and no primary site IDRFs at diagnosis may be candidates for de-escalation of adjuvant primary-site radiation therapy, although validation of these findings in future studies is required.

Evaluation of 11C-Methionine PET and Anatomic MRI Associations in Diffuse Intrinsic Pontine Glioma.

The role of metabolic imaging in the diagnosis, treatment, and response assessment of diffuse intrinsic pontine glioma (DIPG) is poorly defined. We investigated the uptake of 11C-methionine in pediatric patients with newly diagnosed DIPG and evaluated the associations of 11C-methionine PET metrics with conventional MRI indices and survival outcomes. Methods: Twenty-two patients with newly diagnosed DIPG were prospectively enrolled on an institutional review board-approved investigational study of 11C-methionine PET. All patients underwent baseline 11C-methionine PET/CT, and initial treatment-response scans after chemotherapy or radiation therapy were obtained for 17 patients. Typical and atypical DIPGs were assessed clinically and radiographically and defined by multidisciplinary consensus. Three-dimensional regions of interest, reviewed by consensus between a nuclear medicine physician and a radiation oncologist, were delineated after coregistration of PET and MR images. Associations of 11C-methionine uptake intensity and uniformity with survival, along with associations between 11C-methionine uptake and conventional MRI tumor indices over time, were evaluated. 11C-methionine PET voxel values within regions of interest were assessed as threshold values across proportions of the study population, and 11C-methionine uptake at baseline was assessed relative to MRI-defined tumor progression. Results:11C-methionine uptake above that of uninvolved brain tissue was observed in 18 of 22 baseline scans (82%) and 15 of 17 initial response scans (88%). 11C-methionine avidity within MRI-defined tumor was limited in extent, with 11 of 18 positive baseline 11C-methionine PET scans (61%) showing less than 25% 11C-methionine-avid tumor. The increase in total tumor volume with 11C-methionine PET was relatively limited (17.2%; interquartile range, 6.53%-38.90%), as was the extent of 11C-methionine uptake beyond the MRI-defined tumor (2.2%; interquartile range, 0.55%-10.88%). Although baseline 11C-methionine PET intensity and uniformity metrics did not correlate with survival outcomes, initial 11C-methionine avidity overlapped with recurrent tumor in 100% of cases. A clinical diagnosis of atypical DIPG was associated with borderline significantly prolonged progression-free survival (P = 0.07), yet 11C-methionine PET indices at diagnosis did not differ significantly between atypical and typical DIPGs. Conclusion: Most newly diagnosed DIPGs are successfully visualized by 11C-methionine PET. Baseline 11C-methionine uptake delineates regions at increased risk for recurrence, yet intensity and uniformity metrics did not correlate with treatment outcomes in children with DIPG in this study.

Developmental Venous Anomalies Mimicking Neoplasm on 11C-Methionine PET and DSC Perfusion MRI.

Elevated relative cerebral blood volume on perfusion MRI and increased uptake on C-methionine PET can be used to diagnose and guide biopsy of brain tumors but are not specific. We report increased uptake on C-methionine PET associated with 4 developmental venous anomalies (DVAs) in 3 children with brain tumors, which could potentially mimic tumor and misdirect biopsy. Because DVAs are not readily visible on CT, prevention of misdirected biopsy in patients with focally elevated C-methionine uptake and relative cerebral blood volume relies on close correlation with contrast-enhanced anatomic MRI to exclude DVA or other nonneoplastic etiology.

Characterization of a c-Rel Inhibitor That Mediates Anticancer Properties in Hematologic Malignancies by Blocking NF-κB-Controlled Oxidative Stress Responses.

NF-κB plays a variety of roles in oncogenesis and immunity that may be beneficial for therapeutic targeting, but strategies to selectively inhibit NF-κB to exert antitumor activity have been elusive. Here, we describe IT-901, a bioactive naphthalenethiobarbiturate derivative that potently inhibits the NF-κB subunit c-Rel. IT-901 suppressed graft-versus-host disease while preserving graft-versus-lymphoma activity during allogeneic transplantation. Further preclinical assessment of IT-901 for the treatment of human B-cell lymphoma revealed antitumor properties in vitro and in vivo without restriction to NF-κB-dependent lymphoma. This nondiscriminatory, antilymphoma effect was attributed to modulation of the redox homeostasis in lymphoma cells resulting in oxidative stress. Moreover, NF-κB inhibition by IT-901 resulted in reduced stimulation of the oxidative stress response gene heme oxygenase-1, and we demonstrated that NF-κB inhibition exacerbated oxidative stress induction to inhibit growth of lymphoma cells. Notably, IT-901 did not elicit increased levels of reactive oxygen species in normal leukocytes, illustrating its cancer selective properties. Taken together, our results provide mechanistic insight and preclinical proof of concept for IT-901 as a novel therapeutic agent to treat human lymphoid tumors and ameliorate graft-versus-host disease.

Monitoring the induction of heat shock factor 1/heat shock protein 70 expression following 17-allylamino-demethoxygeldanamycin treatment by positron emission tomography and optical reporter gene imaging.

The cell response to proteotoxic cell stresses is mediated primarily through activation of heat shock factor 1 (HSF1). This transcription factor plays a major role in the regulation of the heat shock proteins (HSPs), including HSP70. We demonstrate that an [124I]iodide-pQHNIG70 positron emission tomography (PET) reporter system that includes an inducible HSP70 promoter can be used to image and monitor the activation of the HSF1/HSP70 transcription factor in response to drug treatment (17-allylamino-demethoxygeldanamycin [17-AAG]). We developed a dual imaging reporter (pQHNIG70) for noninvasive imaging of the heat shock response in cell culture and living animals previously and now study HSF1/HSP70 reporter activation in both cell culture and tumor-bearing animals following exposure to 17-AAG. 17-AAG (10-1,000 nM) induced reporter expression; a 23-fold increase was observed by 60 hours. Good correspondence between reporter expression and HSP70 protein levels were observed. MicroPET imaging based on [124I]iodide accumulation in pQHNIG70-transduced RG2 xenografts showed a significant 6.2-fold reporter response to 17-AAG, with a corresponding increase in tumor HSP70 and in tumor human sodium iodide symporter and green fluorescent protein reporter proteins. The HSF1 reporter system can be used to screen anticancer drugs for induction of cytotoxic stress and HSF1 activation both in vitro and in vivo.

Successful treatment of EBV-associated posttransplantation lymphoma after cord blood transplantation using third-party EBV-specific cytotoxic T lymphocytes.

Cellular therapy of Epstein-Barr virus (EBV)(+) posttransplantation lymphoproliferative diseases (PTLD) in cord blood transplant (CBT) recipients is limited by lack of donor access and the donor's naive neonatal immune system. We therefore used partially human leukocyte antigen-matched third-party in vitro expanded EBV-specific cytotoxic T lymphocytes (CTLs) to treat 2 CBT recipients with life-threatening, donor-derived monoclonal EBV(+) diffuse large B-cell lymphomas with extranodal involvement developing in the context of graft-versus-host disease. Both patients had failed immunosuppression taper and Rituximab. After 5 and 9 infusions of 10(6) EBV-CTL/kg, respectively, each patient achieved a sustained complete remission without toxicity or graft-versus-host disease. Each is alive without recurrence at 20 and 15 months, respectively, post-EBV-PTLD diagnosis. This approach demonstrates the efficacy of using "off-the-shelf," virus-specific third-party CTLs restricted by human leukocyte antigens expressed by the tumor to treat otherwise lethal EBV-PTLD. Such therapy may also be applicable to the treatment of other infections and residual or recurrent malignancy after CBT.

(124)I-iodopyridopyrimidinone for PET of Abl kinase-expressing tumors in vivo.

UNLABELLED: Because of the recent development of an iodopyridopyrimidinone Abl protein kinase inhibitor (PKI), (124)I-SKI-212230 ((124)I-SKI230), we investigated the feasibility of a PET-based molecular imaging method for the direct visualization of Abl kinase expression and PKI treatment. METHODS: In vitro pharmacokinetic properties, including specific and nonspecific binding of (124)I-SKI230 to its Abl kinase target and interaction with other PKIs, were assessed in cell-free medium and chronic myelogenous leukemia (CML) cells overexpressing BCR-Abl (K562), in comparison with BT-474 cells that are low in Abl expression. In a xenograft tumor model, we assessed the in vivo pharmacokinetics of (124)I-SKI230 using PET and postmortem tissue sampling. We also tested a paradigm of (124)I-SKI230 PET after treatment of the animal with a dose of Abl-specific PKI for the monitoring of the tumor response. RESULTS: In vitro studies confirmed that SKI230 binds to Abl kinase with nanomolar affinity, that selective uptake occurs in cell lines known to express Abl kinase, that RNAi knock-down supports specificity of cellular uptake due to Abl kinase, and that imatinib, an archetype Abl PKI, completely displaces SKI230. With SKI230, we obtained successful in vivo PET of Abl-expressing human tumors in a nude rat. We were also able to demonstrate evidence of substrate inhibition of in vivo radiotracer uptake in the xenograft tumor after treatment of the animal as a model of PKI treatment monitoring. CONCLUSION: These results support the hypothesis that molecular imaging using PET will be useful for the study of in vivo pharmacodynamics of Abl PKI molecular therapy in humans.

In vivo imaging and quantitation of adoptively transferred human antigen-specific T cells transduced to express a human norepinephrine transporter gene.

Sequential imaging of genetically marked effector cells after adoptive transfer in vivo has greatly enhanced analyses of their biodistribution, growth, and activity both in animal models and in clinical trials of cellular immunotherapy. However, the immunogenicity of cells expressing xenogeneic reporter constructs limits their survival and clinical utility. To address this limitation, we have evaluated a human norepinephrine transporter (hNET) permitting imaging of transduced cells in vivo with a previously approved clinical grade radiolabeled probe, metaiodobenzylguanidine (MIBG). The hNET gene cDNA was cloned from the SK-N-SH cell line and inserted into a bicistronic retroviral vector also encoding green fluorescent protein. Following transfection, human EBV-specific T lymphocytes seemed fully functional in vitro and also selectively accumulated [(123)I]MIBG. In nonobese diabetic/severe combined immunodeficient mice bearing human EBV lymphoma xenografts, as few as 10(4) transduced T cells injected into the tumors could be imaged by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) after i.v. infusion of [(123)I]MIBG or [(124)I]MIBG, respectively. When hNET(+) EBV-specific T cells were infused i.v., their migration and specific accumulation in EBV(+) tumors expressing their restricting HLA allele could be imaged by SPECT or PET over 28 days. Image intensity was closely correlated with the number of T cells accumulated in targeted tumors. The use of two reporter probes (MIBG and 2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl-5-iodouracil) permitted independent contemporaneous tracking of two distinct EBV-specific T-cell subpopulations expressing different reporter genes (hNET-CD4(+) T cells and HSV-TK-CD8(+) T cells) in the same animal using three-dimensional nuclear modalities (SPECT and PET). The hNET-based system described may thus have significant potential as a nonimmunogenic reporter for extended repeated quantitative in vivo imaging of transduced cells in man.

HSP70-inducible hNIS-IRES-eGFP reporter imaging: response to heat shock.

A retroviral vector pQHSP70/hNIS-IRES-eGFP (pQHNIG70) was constructed containing the hNIS-IRES-eGFP dual-reporter genes under the control of an inducible human heat shock protein (HSP)70 promoter and RG2-pQHSP70/hNIS-IRES-eGFP (RG2-pQHNIG70) transduced cells were generated. Heat-induced expression of both reporter genes in RG2-pQHNIG70 cells was validated by enhanced green fluorescent protein (eGFP) fluorescence-activated cell sorter, in vitro radiotracer assays, and immunoblot and immunocytochemistry. A 2.2- to 6.1-fold ((131)I(-)), a 6.1- to 14.4-fold ((99m)TcO(4)(-)), and a 5.1- to 39-fold (fluorescence) increase above baseline was observed in response to graded hyperthermia (39-43 degrees C). Increases in eGFP fluorescence and radiotracer uptake were first noted at 6 hours, reached a maximum at 24 hours, and fell toward baseline at 72 hours. A stable ratio of radiotracer uptake to eGFP fluorescence and to heat shock protein (HSP)70 protein was demonstrated over a wide range of expression levels, induced by different levels of heating. We also demonstrate that the local application of heat on RG2-pQHNIG70 xenografts can effectively induce hNIS and eGFP gene expression in vivo and that this expression can be efficiently visualized by fluorescence, scintigraphic, and micro-positron emission tomography imaging. Endogenous HSP70 protein and reporter expression was confirmed by postmortem tissue evaluations (immunoblot and immunohistochemistry). The pQHNIG70 reporter system can be used to study stress and drug responses in transduced cells and tissues.