Malene Brandt-Larsen

Comparison of two new angiogenesis PET tracers 68Ga-NODAGA-E[c(RGDyK)]2 and 64Cu-NODAGA-E[c(RGDyK)]2; in vivo imaging studies in human xenograft tumors

INTRODUCTION The aim of this study was to synthesize and perform a side-by-side comparison of two new tumor-angiogenesis PET tracers (68)Ga-NODAGA-E[c(RGDyK)](2) and (64)Cu-NODAGA-E[c(RGDyK)](2) in vivo using human xenograft tumors in mice. Human radiation burden was estimated to evaluate potential for future use as clinical PET tracers for imaging of neo-angiogenesis. METHODS A (68)Ge/(68)Ga generator was used for the synthesis of (68)Ga-NODAGA-E[c(RGDyK)](2). (68)Ga and (64)Cu labeled NODAGA-E[c(RGDyK)](2) tracers were administrated in nude mice bearing either human glioblastoma (U87MG) or human neuroendocrine (H727) xenograft tumors. PET/CT scans at 3 time points were used for calculating the tracer uptake in tumors (%ID/g), integrin αVβ3 target specificity was shown by blocking with cold NODAGA-E[c(RGDyK)](2), and biodistribution in normal organs were also examined. From biodistribution data in mice human radiation-absorbed doses were estimated using OLINDA/EXM software. RESULTS (68)Ga-NODAGA-E[c(RGDyK)](2) was synthesized with a radiochemical purity of 89%-99% and a specific activity (SA) of 16-153 MBq/nmol. (64)Cu-NODAGA-E[c(RGDyK)](2) had a purity of 92%-99% and an SA of 64-78 MBq/nmol. Both tracers showed similar uptake in xenograft tumors 1h after injection (U87MG: 2.23 vs. 2.31%ID/g; H727: 1.53 vs. 1.48%ID/g). Both RGD dimers showed similar tracer uptake in non-tumoral tissues and a human radiation burden of less than 10 mSv with an administered dose of 200 MBq was estimated. CONCLUSION (68)Ga-NODAGA-E[c(RGDyK)](2) and (64)Cu-NODAGA-E[c(RGDyK)](2) can be easily synthesized and are both promising candidates for PET imaging of integrin αVβ3 positive tumor cells. (68)Ga-NODAGA-E[c(RGDyK)](2) showed slightly more stable tumor retention. With the advantage of in-house commercially (68)Ge/(68)Ga generators, (68)Ga-NODAGA-E[c(RGDyK)](2) may be the best choice for future clinical PET imaging in humans.

First-in-human uPAR PET: Imaging of Cancer Aggressiveness

A first-in-human clinical trial with Positron Emission Tomography (PET) imaging of the urokinase-type plasminogen activator receptor (uPAR) in patients with breast, prostate and bladder cancer, is described. uPAR is expressed in many types of human cancers and the expression is predictive of invasion, metastasis and indicates poor prognosis. uPAR PET imaging therefore holds promise to be a new and innovative method for improved cancer diagnosis, staging and individual risk stratification. The uPAR specific peptide AE105 was conjugated to the macrocyclic chelator DOTA and labeled with 64Cu for targeted molecular imaging with PET. The safety, pharmacokinetic, biodistribution profile and radiation dosimetry after a single intravenous dose of 64Cu-DOTA-AE105 were assessed by serial PET and computed tomography (CT) in 4 prostate, 3 breast and 3 bladder cancer patients. Safety assessment with laboratory blood screening tests was performed before and after PET ligand injection. In a subgroup of the patients, the in vivo stability of our targeted PET ligand was determined in collected blood and urine. No adverse or clinically detectable side effects in any of the 10 patients were found. The ligand exhibited good in vivo stability and fast clearance from plasma and tissue compartments by renal excretion. In addition, high uptake in both primary tumor lesions and lymph node metastases was seen and paralleled high uPAR expression in excised tumor tissue. Overall, this first-in-human study therefore provides promising evidence for safe use of 64Cu-DOTA-AE105 for uPAR PET imaging in cancer patients.

uPAR Targeted Radionuclide Therapy with 177Lu-DOTA-AE105 Inhibits Dissemination of Metastatic Prostate Cancer

The urokinase-type plasminogen activator receptor (uPAR) is implicated in cancer invasion and metastatic development in prostate cancer and provides therefore an attractive molecular target for both imaging and therapy. In this study, we provide the first in vivo data on an antimetastatic effect of uPAR radionuclide targeted therapy in such lesions and show the potential of uPAR positron emission tomography (PET) imaging for identifying small foci of metastatic cells in a mouse model of disseminating human prostate cancer. Two radiolabeled ligands were generated in high purity and specific activity: a uPAR-targeting probe ((177)Lu-DOTA-AE105) and a nonbinding control ((177)Lu-DOTA-AE105mut). Both uPAR flow cytometry and ELISA confirmed high expression levels of the target uPAR in PC-3M-LUC2.luc cells, and cell binding studies using (177)Lu-DOTA-AE105 resulted in a specific binding with an IC50 value of 100 nM in a competitive binding experiment. In vivo, uPAR targeted radionuclide therapy significantly reduced the number of metastatic lesions in the disseminated metastatic prostate cancer model, when compared to vehicle and nontargeted (177)Lu groups (p < 0.05) using bioluminescence imaging. Moreover, we found a significantly longer metastatic-free survival, with 65% of all mice without any disseminated metastatic lesions present at 65 days after first treatment dose (p = 0.047). In contrast, only 30% of all mice in the combined control groups treated with (177)Lu-DOTA-AE105mut or vehicle were without metastatic lesions. No treatment-induced toxicity was observed during the study as evaluated by observing animal weight and H&E staining of kidney tissue (dose-limiting organ). Finally, uPAR PET imaging using (64)Cu-DOTA-AE105 detected all small, disseminated metastatic foci when compared with bioluminescence imaging in a cohort of animals during the treatment study. In conclusion, uPAR targeted radiotherapy resulted in a significant reduction in the number of metastatic lesions in a human metastatic prostate cancer model. Furthermore, we have provided the first evidence of the potential for identification of small metastatic lesions using uPAR PET imaging in disseminated prostate cancer, illustrating the promising strategy of uPAR theranostics in prostate cancer.

Safety, dosimetry and tumor detection ability of 68Ga-NOTA-AE105 - a novel radioligand for uPAR PET imaging: first-in-humans study.

The overexpression of urokinase-type plasminogen activator receptors (uPARs) represents an established biomarker for aggressiveness in most common malignant diseases, including breast cancer (BC), prostate cancer (PC), and urinary bladder cancer (UBC), and is therefore an important target for new cancer therapeutic and diagnostic strategies. In this study, uPAR PET imaging using a 68Ga-labeled version of the uPAR-targeting peptide (AE105) was investigated in a group of patients with BC, PC, and UBC. The aim of this first-in-human, phase I clinical trial was to investigate the safety and biodistribution in normal tissues and uptake in tumor lesions. Methods: Ten patients (6 PC, 2 BC, and 2 UBC) received a single intravenous dose of 68Ga-NOTA-AE105 (154 ± 59 MBq; range, 48–208 MBq). The biodistribution and radiation dosimetry were assessed by serial whole-body PET/CT scans (10 min, 1 h, and 2 h after injection). Safety assessment included measurements of vital signs with regular intervals during the imaging sessions and laboratory blood screening tests performed before and after injection. In a subgroup of patients, the in vivo stability of 68Ga-NOTA-AE105 was determined in collected blood and urine. PET images were visually analyzed for visible tumor uptake of 68Ga-NOTA-AE105, and SUVs were obtained from tumor lesions by manually drawing volumes of interest in the malignant tissue. Results: No adverse events or clinically detectable pharmacologic effects were found. The radioligand exhibited good in vivo stability and fast clearance from tissue compartments primarily by renal excretion. The effective dose was 0.015 mSv/MBq, leading to a radiation burden of 3 mSv when the clinical target dose of 200 MBq was used. In addition, radioligand accumulation was seen in primary tumor lesions as well as in metastases. Conclusion: This first-in-human, phase I clinical trial demonstrates the safe use and clinical potential of 68Ga-NOTA-AE105 as a new radioligand for uPAR PET imaging in cancer patients.

Urokinase-Type Plasminogen Activator Receptor as a Potential PET Biomarker in Glioblastoma

Glioblastoma is one of the most malignant types of human cancer, and the prognosis is poor. The development and validation of novel molecular imaging biomarkers has the potential to improve tumor detection, grading, risk stratification, and treatment monitoring of gliomas. The aim of this study was to explore the potential of PET imaging of the urokinase-type plasminogen activator receptor (uPAR) in glioblastoma. Methods: The uPAR messenger RNA expression of tumors from 19 glioblastoma patients was analyzed, and a cell culture derived from one of these patients was used to establish an orthotopic xenograft model of glioblastoma. Tumor growth was monitored using bioluminescence imaging. Five to six weeks after inoculation, all mice were scanned with small-animal PET/CT using two new uPAR PET ligands (64Cu-NOTA-AE105 and 68Ga-NOTA-AE105) and, for comparison, O-(2-18F-fluoroethyl)-l-tyrosine (18F-FET). One MRI scan was obtained for each mouse to confirm tumor location. The uPAR specificity of 64Cu-NOTA-AE105 was confirmed by alignment of hematoxylin- and eosin-stained and uPAR immunohistochemistry–stained slides of the brain with the activity distribution as determined using autoradiography. Results: uPAR expression was found in all 19 glioblastoma patient tumors, and high expression of uPAR correlated with decreased overall survival (P = 0.04). Radiolabeling of NOTA-AE105 with 64Cu and 68Ga was straightforward, resulting in a specific activity of approximately 20 GBq/μmol and a radiochemical purity of more than 98% for 64Cu-NOTA-AE105 and more than 97% for 68Ga-NOTA-AE105. High image contrast resulting in clear tumor delineation was found for both 68Ga-NOTA-AE105 and 64Cu-NOTA-AE105. Absolute uptake in tumor was higher for 18F-FET (3.5 ± 0.8 percentage injected dose [%ID]/g) than for 64Cu-NOTA-AE105 (1.2 ± 0.4 %ID/g) or 68Ga-NOTA-AE105 (0.4 ± 0.1 %ID/g). A similar pattern was observed in background brain tissue, where uptake was 1.9 ± 0.1 %ID/g for 18F-fluorothymidine, compared with 0.05 ± 0.01 %ID/g for 68Ga-NOTA-AE105 and 0.11 ± 0.02 %ID/g for 64Cu-NOTA-AE105. The result was a significantly higher tumor-to-background ratio for both 68Ga-NOTA-AE105 (7.6 ± 2.1, P < 0.05) and 64Cu-NOTA-AE105 (10.6 ± 2.3, P < 0.01) than for 18F-FET PET (1.8 ± 0.3). Autoradiography of brain slides confirmed that the accumulation of 64Cu-NOTA-AE105 corresponded well with uPAR-positive cancer cells. Conclusion: On the basis of our translational study, uPAR PET may be a highly promising imaging biomarker for glioblastoma. Further clinical exploration of uPAR PET in glioblastoma is therefore justified.

Use of cis-[18F]fluoro-proline for assessment of exercise-related collagen synthesis in musculoskeletal connective tissue.

Protein turnover in collagen rich tissue is influenced by exercise, but can only with difficulty be studied in vivo due to use of invasive procedure. The present study was done to investigate the possibility of applying the PET-tracer, cis-[18F]fluoro-proline (cis-Fpro), for non-invasive assessment of collagen synthesis in rat musculoskeletal tissues at rest and following short-term (3 days) treadmill running. Musculoskeletal collagen synthesis was studied in rats at rest and 24 h post-exercise. At each session, rats were PET scanned at two time points following injection of cis-FPro: (60 and 240 min p.i). SUV were calculated for Achilles tendon, calf muscle and tibial bone. The PET-derived results were compared to mRNA expression of collagen type I and III. Tibial bone had the highest SUV that increased significantly (p<0.001) from the early (60 min) to the late (240 min) PET scan, while SUV in tendon and muscle decreased (p<0.001). Exercise had no influence on SUV, which was contradicted by an increased gene expression of collagen type I and III in muscle and tendon. The clearly, visible uptake of cis-Fpro in the collagen-rich musculoskeletal tissues is promising for multi-tissue studies in vivo. The tissue-specific differences with the highest basal uptake in bone are in accordance with earlier studies relying on tissue incorporation of isotopic-labelled proline. A possible explanation of the failure to demonstrate enhanced collagen synthesis following exercise, despite augmented collagen type I and III transcription, is that SUV calculations are not sensitive enough to detect minor changes in collagen synthesis. Further studies including kinetic compartment modeling must be performed to establish whether cis-Fpro can be used for non-invasive in-vivo assessment of exercise-induced changes in musculoskeletal collagen synthesis.

Angiogenesis PET Tracer Uptake (68Ga-NODAGA-E[(cRGDyK)]2) in Induced Myocardial Infarction in Minipigs

Angiogenesis is part of the healing process following an ischemic injury and is vital for the post-ischemic repair of the myocardium. Therefore, it is of particular interest to be able to noninvasively monitor angiogenesis. This might, not only permit risk stratification of patients following myocardial infarction, but could also facilitate development and improvement of new therapies directed towards stimulation of the angiogenic response. During angiogenesis endothelial cells must adhere to one another to form new microvessels. αvβ3 integrin has been found to be highly expressed in activated endothelial cells and has been identified as a critical modulator of angiogenesis. 68Ga-NODAGA-E[c(RGDyK)]2 (RGD) has recently been developed by us as an angiogenesis positron-emission-tomography (PET) ligand targeted towards αvβ3 integrin. In the present study, we induced myocardial infarction in Göttingen minipigs. Successful infarction was documented by 82Rubidium-dipyridamole stress PET and computed tomography. RGD uptake was demonstrated in the infarcted myocardium one week and one month after induction of infarction by RGD-PET. In conclusion, we demonstrated angiogenesis by noninvasive imaging using RGD-PET in minipigs hearts, which resemble human hearts. The perspectives are very intriguing and might permit the evaluation of new treatment strategies targeted towards increasing the angiogenetic response, e.g., stem-cell treatment.

Angiogenesis PET Tracer Uptake (68Ga-NODAGA-E[(cRGDyK)]2) in Induced Myocardial Infarction and Stromal Cell Treatment in Minipigs

Angiogenesis is considered integral to the reparative process after ischemic injury. The αvβ3 integrin is a critical modulator of angiogenesis and highly expressed in activated endothelial cells. 68Ga-NODAGA-E[(cRGDyK)]2 (RGD) is a positron-emission-tomography (PET) ligand targeted towards αvβ3 integrin. The aim was to present data for the uptake of RGD and correlate it with histology and to further illustrate the differences in angiogenesis due to porcine adipose-derived mesenchymal stromal cell (pASC) or saline treatment in minipigs after induction of myocardial infarction (MI). Three minipigs were treated with direct intra-myocardial injection of pASCs and two minipigs with saline. MI was confirmed by 82Rubidium (82Rb) dipyridamole stress PET. Mean Standardized Uptake Values (SUVmean) of RGD were higher in the infarct compared to non-infarct area one week and one month after MI in both pASC-treated (SUVmean: 1.23 vs. 0.88 and 1.02 vs. 0.86, p < 0.05 for both) and non-pASC-treated minipigs (SUVmean: 1.44 vs. 1.07 and 1.26 vs. 1.04, p < 0.05 for both). However, there was no difference in RGD uptake, ejection fractions, coronary flow reserves or capillary density in histology between the two groups. In summary, indications of angiogenesis were present in the infarcted myocardium. However, no differences between pASC-treated and non-pASC-treated minipigs could be demonstrated.

Abstract P5-01-04: uPAR PET imaging in breast cancer: First-in-humans studies using 64Cu-DOTA-AE105 and 68Ga-NOTA-AE105

Objective The urokinase-type plasminogen activator receptor (uPAR) is a well-established prognostic biomarker in many cancer types including breast cancer (BC). Numerous studies using immunohistochemically evaluation of uPAR expression in tissue samples from BC patients have shown that not only is uPAR consistently overexpressed, but also carries strong prognostic value and is associates with overall survival. Accordingly, uPAR is an obvious target for identifying BC and for phenotyping aggressiveness in BC. Using whole body Positron Emission Tomography (PET) imaging rather than tissue specimens circumvents possible sampling error and allows for staging. Our objective was therefore to perform first-in-humans studies of uPAR-specific PET imaging in BC using either 64Cu-DOTA-AE105 or 68Ga-NOTA-AE105 Methods Six patients with BC and scheduled for surgery were included. Prior to operation, patients were either PET/CT scanned 1, 3 and 24 h after injection of the uPAR PET ligand 64Cu-DOTA-AE105 (n=3; half life of 64Cu: 13 h) or PET/CT scanned 10 min, 1 h or 2 h after injection of 68Ga-NOTA-AE105 (n=3; half life of 68Ga: 1 h). PET Images were visually analyzed for visible tumor uptake of 64Cu-DOTA-AE105 or 68Ga-NOTA-AE105 and Standardized Uptake Values (SUV) were obtained by manually drawing volumes of interest (VOIs) around the primary tumor as well as identified metastases. Results are given as SUVmax. Tumor-to-background ratios relative to liver, kidney, blood and muscle were also calculated. Surgical tumor specimens were obtained from all patients during subsequent surgery. In addition to routine pathological examination, tissue was analyzed for ex vivo uPAR expression as target validation. Results Both primary tumors and metastases were visually detectable. For 64Cu-DOTA-AE105 SUVmax values were 2.9–4.0., and 2.9-4.0 after 1 and 3 h, respectively. Tumor-to-background ratios after 1 h were 0.91 (tumor-liver), 1.65 (tumor-kidney), 0.96 (tumor-blood) and 8.9 (tumor-muscle), respectively. Tumor-to-background ratios after 3 h were 0.50 (tumor-liver), 0.96 (tumor-kidney), 4.2 (tumor-blood) and 11.4 (tumor-muscle), respectively. Ex vivo analysis by immunohistochemistry confirmed uPAR expression in all primary cancer lesions. For 68Ga-NOTA-AE105, SUVmax was 5.0, 3.8 and 4.2 after 10 min, 1 h and 3 h, respectively (first patient analyzed). Tumor-to-background ratios after 10 min were 2.8 (tumor-liver), 0.4 (tumor-kidney), 1.6 (tumor-blood) and 8.4 (tumor-muscle), respectively. Tumor-to-background ratios after 1 h were 3.2 (tumor-liver), 0.6 (tumor-kidney), 1.7 (tumor-blood) and 7.1 (tumor-muscle), respectively. Conclusion This is the first study in humans using PET imaging of uPAR in BC. Both primary tumors and metastases were clearly visible with robust PET tracer uptake and a high and sufficient contrast between tumors and background. Our data supports continuation into phase II clinical studies using uPAR PET for staging and risk stratification, which potentially may be used for selection of treatment strategy in BC. Citation Format: Kjaer A, Persson M, Skovgaard D, Brandt-Larsen M, Christensen C, Madsen J, Nielsen CH, Loft A, Berthelsen AK, Kroman N, Hojgaard L. uPAR PET imaging in breast cancer: First-in-humans studies using 64Cu-DOTA-AE105 and 68Ga-NOTA-AE105. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-01-04.