Lotte K. Kristensen

PET Imaging of Tissue Factor in Pancreatic Cancer Using 64Cu-labeled Active Site Inhibited Factor VII

Tissue factor (TF) is the main initiator of the extrinsic coagulation cascade. However, TF also plays an important role in cancer. TF expression has been reported in 53%–89% of all pancreatic adenocarcinomas, and the expression level of TF has in clinical studies correlated with advanced stage, increased microvessel density, metastasis, and poor overall survival. Imaging of TF expression is of clinical relevance as a prognostic biomarker and as a companion diagnostic for TF-directed therapies currently under clinical development. Factor VII (FVII) is the natural ligand to TF. The purpose of this study was to investigate the possibility of using active site–inhibited FVII (FVIIai) labeled with 64Cu for PET imaging of TF expression. Methods: FVIIai was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with 64Cu (64Cu-NOTA-FVIIai). Longitudinal in vivo PET imaging was performed at 1, 4, 15, and 36 h after injection of 64Cu-NOTA-FVIIai in mice with pancreatic adenocarcinomas (BxPC-3). The specificity of TF imaging with 64Cu-NOTA-FVIIai was investigated in subcutaneous pancreatic tumor models with different levels of TF expression and in a competition experiment. In addition, imaging of orthotopic pancreatic tumors was performed using 64Cu-NOTA-FVIIai and PET/MRI. In vivo imaging data were supported by ex vivo biodistribution, flow cytometry, and immunohistochemistry. Results: Longitudinal PET imaging with 64Cu-NOTA-FVIIai showed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3, and 2.4 ± 0.3 percentage injected dose per gram at 1, 4, 15, and 36 h after injection, respectively. An increase in tumor–to–normal-tissue contrast was observed over the imaging time course. Competition with unlabeled FVIIai significantly (P < 0.001) reduced the tumor uptake. The tumor uptake observed in models with different TF expression levels was significantly different from each other (P < 0.001) and was in agreement with the TF level evaluated by TF immunohistochemistry staining. Orthotopic tumors were clearly visible on the PET/MR images, and the uptake of 64Cu-NOTA-FVIIai was colocalized with viable tumor tissue. Conclusion: 64Cu-NOTA-FVIIai is well suited for PET imaging of tumor TF expression, and imaging is capable of distinguishing the TF expression level of various pancreatic tumor models.

In vivo imaging of therapy response to a novel Pan-HER antibody mixture using FDG and FLT positron emission tomography

Purpose Overexpression of the human epidermal growth factor receptor (HER) family and their ligands plays an important role in many cancers. Targeting multiple members of the HER family simultaneously may increase the therapeutic efficacy. Here, we report the ability to image the therapeutic response obtained by targeting HER family members individually or simultaneously using the novel monoclonal antibody (mAb) mixture Pan-HER. Experimental design and results Mice with subcutaneous BxPC-3 pancreatic adenocarcinomas were divided into five groups receiving vehicle or mAb mixtures directed against either EGFR (HER1), HER2, HER3 or all three receptors combined by Pan-HER. Small animal positron emission tomography/computed tomography (PET/CT) with 2′-deoxy-2′-[18F]fluoro-D-glucose (FDG) and 3′-deoxy-3′-[18F]fluorothymidine (FLT) was performed at baseline and at day 1 or 2 after initiation of therapy. Changes in tumor uptake of tracers were quantified and compared to reduction in tumor size. Imaging results were further validated by immunohistochemistry and qPCR. Mean FDG and FLT uptake in the Pan-HER treated group decreased by 19±4.3% and 24±3.1%, respectively. The early change in FDG and FLT uptake correlated with tumor growth at day 23 relative to day 0. Ex vivo molecular analyses of markers associated with the mechanisms of FDG and FLT uptake confirmed the in vivo imaging results. Conclusions Taken together, the study supports the use of FDG and FLT as imaging biomarkers of early response to Pan-HER therapy. FDG and FLT PET/CT imaging should be considered as imaging biomarkers in clinical evaluation of the Pan-HER mAb mixture.

Quantitative PET Imaging of Tissue Factor Expression Using 18F-Labeled Active Site–Inhibited Factor VII

Tissue factor (TF) is upregulated in many solid tumors, and its expression is linked to tumor angiogenesis, invasion, metastasis, and prognosis. A noninvasive assessment of tumor TF expression status is therefore of obvious clinical relevance. Factor VII is the natural ligand to TF. Here we report the development of a new PET tracer for specific imaging of TF using an 18F-labeled derivative of factor VII. Methods: Active site–inhibited factor VIIa (FVIIai) was obtained by inactivation with phenylalanine–phenylalanine–arginine–chloromethyl ketone. FVIIai was radiolabeled with N-succinimidyl 4-18F-fluorobenzoate and purified. The corresponding product, 18F-FVIIai, was injected into nude mice with subcutaneous human pancreatic xenograft tumors (BxPC-3) and investigated using small-animal PET/CT imaging 1, 2, and 4 h after injection. Ex vivo biodistribution was performed after the last imaging session, and tumor tissue was preserved for molecular analysis. A blocking experiment was performed in a second set of mice. The expression pattern of TF in the tumors was visualized by immunohistochemistry and the amount of TF in tumor homogenates was measured by enzyme-linked immunosorbent assay and correlated with the uptake of 18F-FVIIai in the tumors measured in vivo by PET imaging. Results: The PET images showed high uptake of 18F-FVIIai in the tumor regions, with a mean uptake of 2.5 ± 0.3 percentage injected dose per gram (%ID/g) (mean ± SEM) 4 h after injection of 7.3–9.3 MBq of 18F-FVIIai and with an average maximum uptake in the tumors of 7.1 ± 0.7 %ID/g at 4 h. In comparison, the muscle uptake was 0.2 ± 0.01 %ID/g at 4 h. At 4 h, the tumors had the highest uptake of any organ. Blocking with FVIIai significantly reduced the uptake of 18F-FVIIai from 2.9 ± 0.1 to 1.4 ± 0.1 %ID/g (P < 0.001). The uptake of 18F-FVIIai measured in vivo by PET imaging correlated (r = 0.72, P < 0.02) with TF protein level measured ex vivo. Conclusion: 18F-FVIIai is a promising PET tracer for specific and noninvasive imaging of tumor TF expression. The tracer merits further development and clinical translation, with potential to become a companion diagnostics for emerging TF-targeted therapies.

18F-FDG PET/CT-based early treatment response evaluation of nanoparticle-assisted photothermal cancer therapy

Within the field of nanoparticle-assisted photothermal cancer therapy, focus has mostly been on developing novel heat-generating nanoparticles with the right optical and dimensional properties. Comparison and evaluation of their performance in tumor-bearing animals are commonly assessed by changes in tumor volume; however, this is usually a late-occurring event. This study implements 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging to perform early evaluation of the treatment outcome of photothermal therapy. Silica-gold nanoshells (NS) are administered intravenously to nude mice bearing human neuroendocrine tumor xenografts and the tumors are irradiated by a near-infrared laser. The animals are positron emission tomography scanned with 2-deoxy-2-[F-18]fluoro-D-glucose one day before and one day after treatment. Using this setup, a significant decrease in tumor uptake of 2-deoxy-2-[F-18]fluoro-D-glucose is found already one day after therapy in the group receiving NS and laser treatment compared to control animals. At this time point no change in tumor volume can be detected. Moreover, the change in tumor uptake, is used to stratify the animals into responders and non-responders, where the responding group matched improved survival. Overall, these findings support the use of 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging for preclinical and clinical evaluation and optimization of photothermal therapy.

T‐cells and macrophages peak weeks after experimental stroke: Spatial and temporal characteristics

The activities of the central and peripheral immune systems impact neurological outcome after ischemic stroke. However, studies investigating the temporal profile of leukocyte infiltration, especially T‐cell recruitment, are sparse. Our aim was to investigate leukocyte infiltration at different time points after experimental stroke in mice. Permanent middle cerebral artery occlusion was performed on 11 weeks old C57BL/6J mice, allowed to survive for 1, 3, 8, 14 or 28 days. In addition to infarct size measurements, detailed immunohistochemical analyses of T‐cell and macrophage influx were performed. A recently introduced F‐19 MR probe (V‐sense), designed to track macrophages, was furthermore tested. Fourteen and 28 days after permanent middle cerebral artery occlusion a significant increase in CD3+ T‐cells was found within the ipsilateral hemisphere compared to controls, especially within the infarct core and the corpus callosum. The number of CD68+ cells within the infarct core was significantly increased at days 8, 14 and 28. This temporal pattern was also seen in MRI. After experimental stroke within the infarcted cortex we found a delayed (day 14) infiltration of T‐cells and macrophages. Furthermore, our data show that T‐cells are present in higher numbers in the corpus callosum compared to the rest of the brain (except from the infarct core where they were highest).

Site-Specific 64Cu Labeling of the Serine Protease, Active Site Inhibited Factor Seven Azide (FVIIai-N3), Using Copper Free Click Chemistry

A method for site-specific radiolabeling of the serine protease active site inhibited factor seven (FVIIai) with 64Cu has been applied using a biorthogonal click reaction. FVIIai binds to tissue factor (TF), a trans-membrane protein involved in hemostasis, angiogenesis, proliferation, cell migration, and survival of cancer cells. First a single azide moiety was introduced in the active site of this 50 kDa protease. Then a NOTA moiety was introduced via a strain promoted azide-alkyne reaction and the corresponding conjugate was labeled with 64Cu. Binding to TF and the stability was evaluated in vitro. TF targeting capability of the radiolabeled conjugate was tested in vivo by positron emission tomography (PET) imaging in pancreatic human xenograft cancer mouse models with various TF expressions. The conjugate showed good stability (>91% at 16 h), an immunoreactivity of 93.5%, and a mean tumor uptake of 2.1 ± 0.2%ID/g at 15 h post injection. In conclusion, FVIIai was radiolabeled with 64Cu in single well-defined position of the protein. This method can be utilized to prepare conjugates from serine proteases with the label at a specific position.

Neodymium-140 DOTA-LM3: Evaluation of an In Vivo Generator for PET with a Non-Internalizing Vector

140Nd (t1/2 = 3.4 days), owing to its short-lived positron emitting daughter 140Pr (t1/2 = 3.4 min), has promise as an in vivo generator for positron emission tomography (PET). However, the electron capture decay of 140Nd is chemically disruptive to macrocycle-based radiolabeling, meaning that an in vivo redistribution of the daughter 140Pr is expected before positron emission. The purpose of this study was to determine how the delayed positron from the de-labeled 140Pr affects preclinical imaging with 140Nd. To explore the effect, 140Nd was produced at CERN-ISOLDE, reacted with the somatostatin analogue, DOTA-LM3 (1,4,7,10- tetraazacyclododecane, 1,4,7- tri acetic acid, 10- acetamide N - p-Cl-Phecyclo(d-Cys-Tyr-d-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)d-Tyr-NH2) and injected into H727 xenograft bearing mice. Comparative pre- and post-mortem PET imaging at 16 h postinjection was used to quantify the in vivo redistribution of 140Pr following 140Nd decay. The somatostatin receptor-positive pancreas exhibited the highest tissue accumulation of 140Nd-DOTA-LM3 (13% ID/g at 16 h) coupled with the largest observed redistribution rate, where 56 ± 7% (n = 4, mean ± SD) of the in situ produced 140Pr washed out of the pancreas before decay. Contrastingly, the liver, spleen, and lungs acted as strong sink organs for free 140Pr3+. Based upon these results, we conclude that 140Nd imaging with a non-internalizing vector convolutes the biodistribution of the tracer with the accumulation pattern of free 140Pr. This redistribution phenomenon may show promise as a probe of the cellular interaction with the vector, such as in determining tissue dependent internalization behavior.

Abstract 2805: A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs

Background: Patients with glioblastoma multiforme (GBM) have a poor prognosis and few treatment options; hence new treatments are needed. Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, orthotopic implantation confers a translational advantage as the cancer develops in a microenvironment more closely mimicking that of the original patient tumor. Also the major impact of the blood brain barrier that must be taking into account when targeting brain tumors as GBM in terms of drug bioavailability is better represented in the orthotopic models. The aim of this study was therefore to develop a panel of orthotopic GBM PDX models for pre-clinical efficacy studies of new drugs. The models were then used to study the efficacy of standard of care such as temozolomide (TMZ) and external radiation therapy (XRT). Methods: Low passage subcutaneous tumors from six different PDX GBM models designated ST108, ST112, ST146, ST545, ST610 and ST2473 were digested and used for intracranial stereotactic injection in nude mice. Tumor take and growth was determined by T2-weighted magnetic resonance imaging (MRI). At confirmed tumor take mice were either treated with TMZ (100mg/kg/day for 5 days) or whole brain XRT (2 Gy/day for 5 days). Control groups receiving vehicle or sham XRT were included depending on treatment regiment. Final endpoint was survival by humane endpoints and tumors were fixed in formalin for histological evaluation. Results: MRI confirmed tumor take in all models within 5 weeks of implantation. The take rate was > 80% across all models. TMZ showed efficacy in the orthotopic ST610 GBM PDX model evaluated by MRI on day 14 (16.2±2.9 mm3 vs. 76.8±13.1 mm3, p=0.016), whereas the ST146 model displayed resistance to TMZ on day 14 (12.7±5.6 mm3 vs. 26.5±11.9 mm3, p=0.26). The median survival was 60 days vs. 14 days in the ST610 model (TMZ vs. vehicle, p=0.0005) and 27 days vs. 13 days in the ST146 model (TMZ vs. vehicle, p=0.007). XRT showed efficacy in the orthotopic ST2473 model. Tumor volume was significantly smaller in treated vs. sham animals 11 days after inclusion (6.9±1.4 mm3 vs. 28.9±3.3 mm3, p=0.001). Also, a survival benefit was observed in XRT treated animals compared to sham. Histology confirmed the presence of orthotopic tumors and typical GBM pathology characteristics such as pseudopalisading tumor cells surrounding necrosis and micro vascular proliferation were identified. Conclusion: Six different orthotopic GBM PDX models were established from low passage subcutaneous PDX models. Models sensitive and resistant to TMZ were identified and histological GBM characteristics were identified. Together, the established panel of orthotopic PDX models can be used as a relevant translational platform for testing of new drugs in a setting that more closely mimics the GBM tumor microenvironment and the impact of the blood brain barrier in patients. Citation Format: Mette M. Jensen, Camilla S. Knudsen, Lotte K. Kristensen, Mette K. Nedergaard, Michael J. Wick, Kyriakos P. Papadopoulos, Anthony W. Tolcher, Andreas Kjaer, Carsten H. Nielsen. A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2805. doi:10.1158/1538-7445.AM2017-2805

Abstract 4771: PET imaging of tissue factor using 64Cu-labeled active site-inhibited factor VII: A potential companion diagnostic for tissue factor targeted cancer therapies

Introduction: The transmembrane glycoprotein tissue factor (TF) is the primary initiator of coagulation. In addition to its physiological role, TF is associated with a variety of pathophysiological processes including tumor growth, tumor angiogenesis and metastasis. Increased TF expression has been reported in 53-89% of all pancreatic adenocarcinomas and clinically correlates with advanced stage and poor survival. Non-invasive imaging of tumor TF status holds great clinical potential as a companion diagnostic for anti-cancer therapy targeting TF. The goal of our study was to develop and evaluate a PET tracer for imaging of TF expression in pancreatic cancer by utilizing the natural ligand for TF, coagulation factor VII. Experimental procedures: Active site-inhibited factor VII (FVIIai) was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) followed by radiolabeling with 64Cu (64Cu-NOTA-FVIIai). Mice bearing subcutaneous pancreatic adenocarcinoma tumors (BxPC-3) were subjected to PET imaging 1, 4, 15 and 36 hours post-injection of 64Cu-NOTA-FVIIai for longitudinal assessment. The in vivo specificity of 64Cu-NOTA-FVIIai towards TF was evaluated in a competition blocking experiment and in a panel of subcutaneous pancreatic tumor models exhibiting low, medium and high TF expression. Additionally, the ability of 64Cu-NOTA-FVIIai to visualize TF expression in orthotopically implanted BxPC-3 tumors was evaluated by PET/MRI. Ex vivo biodistribution, immunohistochemistry and flow cytometry was performed to verify the in vivo imaging data. Results: PET imaging at 1, 4, 15 and 36 hours after injection of 64Cu-NOTA-FVIIai revealed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3 and 2.4 ± 0.3% injected dose per gram (%ID/g), respectively. A graduate increase in image contrast (tumor to background ratio) was observed over the imaging time course with no further increase beyond 15 hours. Competition with excess unlabeled FVIIai significantly reduced tumor uptake of 64Cu-NOTA-FVIIai (p Conclusions: 64Cu-NOTA-FVIIai is well suited for specific PET imaging of TF in pancreatic cancer and its uptake is related to the tumor TF expression level. The data supports further development of 64Cu-NOTA-FVIIai as a companion diagnostic and theranostic agent. Citation Format: Lotte K. Kristensen, Carsten H. Nielsen, Troels E. Jeppesen, Mette M. Jensen, Jacob Madsen, Bo Wiinberg, Lars C. Petersen, Andreas Kjaer. PET imaging of tissue factor using 64Cu-labeled active site-inhibited factor VII: A potential companion diagnostic for tissue factor targeted cancer therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4771.

Abstract 5187: PET imaging of trastuzumab emtansine (T-DM1) drug delivery to intracranial patient derived xenograft (PDX) models of breast cancer metastasis

Background: It is estimated that 10-30% of all breast cancer patients at some point develop brain metastases. Overexpression of the human epidermal growth factor receptor 2 (HER2) is a independent risk factor for development of brain metastases. Up to 37% of patients with HER2-positive metastatic breast cancer develop brain metastases and half of these patients die as a result of failure to control the intracranial disease. A reason for this is the challenge to obtain efficient drug delivery across the blood brain barrier (BBB). Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, when targeting brain tumors or metastases, the major impact of the BBB on drug bioavailability must be taken into consideration. Clinical PET imaging with 64Cu or 89Zr labeled trastuzumab has previously been able to visualize breast cancer brain metastases. The aim of our study was to investigate if PET imaging with 64Cu-labeled trastuzumab was predictive of the efficacy of trastuzumab emtansine (T-DM1) in a HER2 positive breast cancer PDX model established as an intracranial brain metastases model. Methods: The intracranial PDX model was established by stereotactic intracranial injection of enzymatically digested ST1339 tumor tissue. At confirmed tumor take, mice were randomized into two arms: control and T-DM1 (10 mg/kg/week x4). Treatment response was monitored by contrast-enhanced T1- and T2-weighted Magnetic Resonance Imaging (MRI) and positron emission tomography (PET) with the amino acid radiotracer O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET). PET/CT imaging with 64Cu-trastuzumab was performed in animals with confirmed intracranial ST1339 tumors prior to treatment with T-DM1 (10 mg/kg/week x4). Results: T-DM1 treatment with 10 mg/kg/week x4 of mice with intracranial tumors inhibited tumor growth and prolonged survival compared to non-treated animals. A variable response within the treatment group was observed. Forty percent had tumor shrinkage while 60% exhibited tumor growth within the duration of the therapy. The intracranial tumors were clearly visible on the 64Cu-trastuzumab PET images co-registered with T2-weighted MR images for anatomical localization. Interestingly, the intracranial tumor uptake between the animals was rather heterogeneous. Conclusion: A treatment response to T-DM1 was observed in an intracranial ST1339 HER2 positive breast cancer PDX model. PET imaging with 64Cu-trastuzumab confirmed delivery of trastuzumab to the tumors. Further quantitative image analysis of the intracranial 64Cu-trastuzumab tumor uptake will reveal if the drug delivery measured by 64Cu-trastuzumab PET imaging is predictive of the efficacy of T-DM1 in a HER2 positive PDX breast cancer brain metastases model. Citation Format: Carsten Haagen Nielsen, Mette K. Nedergaard, Lotte K. Kristensen, Camilla S. Knudsen, Michael J. Wick, Kyri Papadopoulos, Anthony Tolcher, Andreas Kjaer. PET imaging of trastuzumab emtansine (T-DM1) drug delivery to intracranial patient derived xenograft (PDX) models of breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5187.