Comparison of three 18F-labeled 2-nitroimidazoles for imaging hypoxia in breast cancer xenografts: [18F]FBNA, [18F]FAZA and [18F]FMISO.

BACKGROUND: Tumour hypoxia is associated with increased metastasis, invasion, poor therapy response and prognosis. Most PET radiotracers developed and used for clinical hypoxia imaging belong to the 2-nitroimidazole family. Recently we have developed novel 2-nitroimidazole-derived PET radiotracer [18F]FBNA (N-(4-[18F]fluoro-benzyl)-2-(2-nitro-1H-imidazol-1-yl)-acet-amide), an 18F-labeled analogue of antiparasitic drug benznidazole. The present study aimed to analyze its radio-pharmacological properties and systematically compare its PET imaging profiles with [18F]FMISO and [18F]FAZA in preclinical triple-negative (MDA-MB231) and estrogen receptor-positive (MCF-7) breast cancer models. METHODS: In vitro cellular uptake experiments were carried out in MDA-MB321 and MCF-7 cells under normoxic and hypoxic conditions. Metabolic stability in vivo was determined in BALB/c mice using radio-TLC analysis. Dynamic PET experiments over 3 h post-injection were performed in MDA-MB231 and MCF-7 tumour-bearing mice. Those PET data were used for kinetic modelling analysis utilizing the reversible two-tissue-compartment model. Autoradiography was carried out in tumour tissue slices and compared to HIF-1α immunohistochemistry. Detailed ex vivo biodistribution was accomplished in BALB/c mice, and this biodistribution data were used for dosimetry calculation. RESULTS: Under hypoxic conditions in vitro cellular uptake was elevated in both cell lines, MCF-7 and MDA-MB231, for all three radiotracers. After intravenous injection, [18F]FBNA formed two radiometabolites, resulting in a final fraction of 65 ± 9 % intact [18F]FBNA after 60 min p.i. After 3 h p.i., [18F]FBNA tumour uptake reached SUV values of 0.78 ± 0.01 in MCF-7 and 0.61 ± 0.04 in MDA-MB231 tumours (both n = 3), representing tumour-to-muscle ratios of 2.19 ± 0.04 and 1.98 ± 0.15, respectively. [18F]FMISO resulted in higher tumour uptakes (SUV 1.36 ± 0.04 in MCF-7 and 1.23 ± 0.08 in MDA-MB231 (both n = 4; p < 0.05) than [18F]FAZA (0.66 ± 0.11 in MCF-7 and 0.63 ± 0.14 in MDA-MB231 (both n = 4; n.s.)), representing tumour-to-muscle ratios of 3.24 ± 0.30 and 3.32 ± 0.50 for [18F]FMISO, and 2.92 ± 0.74 and 3.00 ± 0.42 for [18F]FAZA, respectively. While the fraction per time of radiotracer entering the second compartment (k3) was similar within uncertainties for all three radiotracers in MDA-MB231 tumours, it was different in MCF-7 tumours. The ratios k3/(k3 + k2) and K1*k3/(k3 + k2) in MCF-7 tumours were also significantly different, indicating dissimilar fractions of radiotracer bound and trapped intracellularly: K1*k3/(k2 + k3) [18F]FMISO (0.0088 ± 0.001)/min, n = 4; p < 0.001) > [18F]FAZA (0.0052 ± 0.002)/min, n = 4; p < 0.01) > [18F]FBNA (0.003 ± 0.001)/min, n = 3). In contrast, in MDA-MB231 tumours, only K1 was significantly elevated for [18F]FMISO. However, this did not result in significant differences for K1*k3/(k2 + k3) for all three 2-nitroimidazoles in MDA-MB231 tumours. CONCLUSION: Novel 2-nitroimidazole PET radiotracer [18F]FBNA showed uptake into hypoxic breast cancer cells and tumour tissue presumably associated with elevated HIF1-α expression. Systematic comparison of PET imaging performance with [18F]FMISO and [18F]FAZA in different types of preclinical breast cancer models revealed a similar tumour uptake profile for [18F]FBNA with [18F]FAZA and, despite its higher lipophilicity, still a slightly higher muscle tissue clearance compared to [18F]FMISO.

A comparative PET imaging study of 44gSc- and 68Ga-labeled bombesin antagonist BBN2 derivatives in breast and prostate cancer models.

INTRODUCTION: Radiolabeled peptides play a central role in nuclear medicine as radiotheranostics for targeted imaging and therapy of cancer. We have recently proposed the use of metabolically stabilized GRPR antagonist BBN2 for radiolabeling with 18F and 68Ga and subsequent PET imaging of GRPRs in prostate cancer. The present work studied the impact of 44gSc- and 68Ga-labeled DOTA complexes attached to GRPR antagonist BBN2 on the in vitro GRPR binding affinity, and their biodistribution and tumor uptake profiles in MCF7 breast and PC3 prostate cancer models. METHODS: DOTA-Ava-BBN2 was radiolabeled with radiometals 68Ga and 44gSc. Gastrin-releasing peptide receptor (GRPR) affinities of peptides were assessed in PC3 prostate cancer cells. GRPR expression profiles were studied in human breast cancer tissue samples and MCF7 breast cancer cells. PET imaging of 68Ga- and 44gSc-labeled peptides was performed in MCF7 and PC3 xenografts as breast and prostate cancer models. RESULTS: Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava BBN2 were prepared in radiochemical yields of 70-80% (decay-corrected), respectively. High binding affinities were found for both peptides (IC50 = 15 nM (natGa) and 5 nM (natSc)). Gene expression microarray analysis revealed high GRPR mRNA expression levels in estrogen receptor (ER)-positive breast cancer, which was further confirmed with Western blot and immunohistochemistry. However, PET imaging showed only low tumor uptake of both radiotracers in MCF7 xenografts ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.27 ± 0.06); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.20 ± 0.03)). In contrast, high tumor uptake and retention were found for both radiopeptides in PC3 tumors ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.46 ± 0.07); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.51 ± 0.11)). CONCLUSIONS: Comparison of 68Ga- and 44gSc-labeled DOTA-Ava-BBN2 peptides revealed slight but noticeable differences of the radiometal with an impact on the in vitro GRPR receptor binding properties in PC3 cells. No differences were found in their in vivo biodistribution profiles in MCF7 and PC3 xenografts. Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava-BBN2 displayed comparable tumor uptake and retention profiles with rapid blood and renal clearance profiles in both tumor models. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: The favorable PET imaging performance of [44gSc]Sc-DOTA-Ava-BBN2 in prostate cancer should warrant the development of an [43Sc]Sc-DOTA-Ava-BBN2 analog for clinical translation which comes with a main γ-line of much lower energy and intensity compared to 44gSc.

Comparison of scandium-44 g with other PET radionuclides in pre-clinical PET phantom imaging.

PURPOSE: The decay characteristics of radionuclides in PET studies can impact image reconstruction. 44gSc has been the topic of recent research due to potential theranostic applications and is a promising radiometal for PET imaging. In this study, the reconstructed images from phantom measurements with scandium in a small-animal PET scanner are compared with 18F and two prominent radiometals: 64Cu and 68Ga METHODS: Three phantoms filled with 18F, 64C, 68Ga, and 44gSc were imaged in the Siemens Inveon PET scanner. The NEMA image quality phantom was used to determine the recovery coefficients (RCs), spill-over ratios (SORs), and noise (%SD) under typical pre-clinical imaging conditions. Image contrast was determined using a Derenzo phantom, while the coincidence characteristics were investigated using an NEC phantom. Three reconstruction algorithms were used, namely filtered back projection (FBP), ordered subset expectation maximization (OSEM), and maximum a-posteriori (MAP). RESULTS: Image quality parameters were measured for 18F, 64Cu, 68Ga, and 44gSc respectively; using FBP, the %SD are 5.65, 5.88, 7.28, and 7.70; the RCs for the 5-mm rod are 0.849, 1.01, 0.615, and 0.825; the SORs in water are 0.0473, 0.0595, 0.141, 0.0923; and the SORs in air are 0.0589, 0.0484, 0.0525, and 0.0509. The contrast measured in the 2.5-mm rods are 0.674, 0.637, 0.196, and 0.347. The NEC rate with 44gSc increased at a slower rate than 18F and 68Ga as a function of activity in the field of view. CONCLUSION: 44gSc demonstrates intermediate behavior relative to 18F and 68Ga with regard to RC and contrast measurements. It is a promising radionuclide for preclinical imaging.

Activity cross-calibration of unsealed radionuclides utilizing a portable ion chamber.

PURPOSE: To present and evaluate an ion chamber-based method for the cross-calibration between sites of activity measurements of unsealed radionuclides. Notably, the method allows direct comparison of short lived (i.e., clinically used) radioisotopes and the cross-calibration of radionuclide activity meters (also known as "dose calibrators"). METHODS: A portable ion chamber has been designed which is easily shipped between sites, e.g., between a standards laboratory and a nuclear medicine department. The cylindrical chamber accommodates a syringe filled with unsealed radionuclide. Low background and staff shielding are achieved by designing the ion chamber small enough to fit into the well of a dose calibrator. The chamber's sensitivity for the clinically important unsealed radioisotopes 99mTc, 131I, and 18F was measured and compared to Monte Carlo calculations. The influence of syringe fill volume, positioning, and construction (wall diameter, length) was also investigated using Monte Carlo simulations. The chamber's linearity was measured over 5.5 orders of magnitude and its constancy tested over a period of >14.5 months. An overall uncertainty budget is presented. RESULTS: Measured chamber sensitivity was 12.1 pA/100 MBq, 12.5 pA/100 MBq and 29.4 pA/100 MBq for 131I, 99mTc, and 18F, respectively. The uncertainty budget for the ion chamber alone yields an overall uncertainty of less than 1%, with the greatest contribution arising from constancy and linearity (0.5% each). Strategies to further reduce uncertainties are discussed. CONCLUSIONS: The investigation presented in this paper confirms the feasibility of the concept introduced here. To optimize its practical implementation, the concept would benefit from computerization for the purpose of data acquisition, evaluation, processing, and storage of measured values.

Delivered dose uncertainty analysis at the tumor apex for ocular brachytherapy.

PURPOSE: To estimate the total dosimetric uncertainty at the tumor apex for ocular brachytherapy treatments delivered using 16 mm Collaborative Ocular Melanoma Study (COMS) and Super9 plaques loaded with (125)I seeds in order to determine the size of the apex margin that would be required to ensure adequate dosimetric coverage of the tumor. METHODS: The total dosimetric uncertainty was assessed for three reference tumor heights: 3, 5, and 10 mm, using the Guide to the expression of Uncertainty in Measurement/National Institute of Standards and Technology approach. Uncertainties pertaining to seed construction, source strength, plaque assembly, treatment planning calculations, tumor height measurement, plaque placement, and plaque tilt for a simple dome-shaped tumor were investigated and quantified to estimate the total dosimetric uncertainty at the tumor apex. Uncertainties in seed construction were determined using EBT3 Gafchromic film measurements around single seeds, plaque assembly uncertainties were determined using high resolution microCT scanning of loaded plaques to measure seed positions in the plaques, and all other uncertainties were determined from the previously published studies and recommended values. All dose calculations were performed using plaque simulator v5.7.6 ophthalmic treatment planning system with the inclusion of plaque heterogeneity corrections. RESULTS: The total dosimetric uncertainties at 3, 5, and 10 mm tumor heights for the 16 mm COMS plaque were 17.3%, 16.1%, and 14.2%, respectively, and for the Super9 plaque were 18.2%, 14.4%, and 13.1%, respectively (all values with coverage factor k = 2). The apex margins at 3, 5, and 10 mm tumor heights required to adequately account for these uncertainties were 1.3, 1.3, and 1.4 mm, respectively, for the 16 mm COMS plaque, and 1.8, 1.4, and 1.2 mm, respectively, for the Super9 plaque. These uncertainties and associated margins are dependent on the dose gradient at the given prescription depth, thus resulting in the changing uncertainties and margins with depth. CONCLUSIONS: The margins determined in this work can be used as a guide for determining an appropriate apex margin for a given treatment, which can be chosen based on the tumor height. The required margin may need to be increased for more complex scenarios (mushroom shaped tumors, tumors close to the optic nerve, oblique muscle related tilt, etc.) than the simple dome-shaped tumor examined and should be chosen on a case-by-case basis. The sources of uncertainty contributing most significantly to the total dosimetric uncertainty are seed placement within the plaques, treatment planning calculations, tumor height measurement, and plaque tilt. This work presents an uncertainty-based, rational approach to estimating an appropriate apex margin.

Detecting functional changes with [(18)F]FAZA in a renal cell carcinoma mouse model following sunitinib therapy.

BACKGROUND: The multitargeting tyrosine kinase inhibitor (TKI) sunitinib is currently the first-line drug therapy for metastasizing renal cell carcinoma (RCC). TKIs have profound effects on tumor angiogenesis, leading to modifications of the tumor microenvironment. The goal of this study was to determine whether these treatment-induced changes can be detected with [(18)F]FAZA. METHODS: The present study utilized positron emission tomography (PET) to analyze tumor oxygenation status during and after sunitinib therapy in the murine Caki-1 RCC tumor model. Dynamic and static scans were performed, as well as ex vivo biodistributions at 3 h post injection (p.i.). Immunohistochemical analysis of tumor tissue was carried out for the quantification of pimonidazole binding and the hypoxia-associated factors CD-31, Ki-67, and Von Willebrand factor (VWF). In addition, in vitro cellular uptake studies were done to analyze the direct effects of sunitinib on the Caki-1 cells. RESULTS: During therapy with sunitinib (40 mg/kg/day), uptake of [(18)F]FAZA into Caki-1 mice decreased by 46 ± 5% (n = 4; 5 days) at 3 h post injection (p.i.) during the first study and 22 ± 5% (n = 8; 9 days) during the long-term study, indicating a decrease in the tumor's hypoxia level. However, when drug therapy was stopped, this effect was reversed completely, and the tumor [(18)F]FAZA uptake increased to 126 ± 6% (n = 6) of the control tumor uptake, indicative of an even higher level of tumor hypoxia compared to the therapy starting point. Sunitinib had no direct effect on [(18)F]FAZA uptake into Caki-1 cells in vitro. CONCLUSION: [(18)F]FAZA PET could be used to monitor drug response during sunitinib therapy in RCC and may guide combination therapies based on the tumor's hypoxia status.

NEMA NU 4-2008 comparison of preclinical PET imaging systems.

UNLABELLED: The National Electrical Manufacturers Association (NEMA) standard NU 4-2008 for performance measurements of small-animal tomographs was recently published. Before this standard, there were no standard testing procedures for preclinical PET systems, and manufacturers could not provide clear specifications similar to those available for clinical systems under NEMA NU 2-1994 and 2-2001. Consequently, performance evaluation papers used methods that were modified ad hoc from the clinical PET NEMA standard, thus making comparisons between systems difficult. METHODS: We acquired NEMA NU 4-2008 performance data for a collection of commercial animal PET systems manufactured since 2000: microPET P4, microPET R4, microPET Focus 120, microPET Focus 220, Inveon, ClearPET, Mosaic HP, Argus (formerly eXplore Vista), VrPET, LabPET 8, and LabPET 12. The data included spatial resolution, counting-rate performance, scatter fraction, sensitivity, and image quality and were acquired using settings for routine PET. RESULTS: The data showed a steady improvement in system performance for newer systems as compared with first-generation systems, with notable improvements in spatial resolution and sensitivity. CONCLUSION: Variation in system design makes direct comparisons between systems from different vendors difficult. When considering the results from NEMA testing, one must also consider the suitability of the PET system for the specific imaging task at hand.

Biodistribution and uptake of 3'-deoxy-3'-fluorothymidine in ENT1-knockout mice and in an ENT1-knockdown tumor model.

UNLABELLED: (18)F-3'-Deoxy-3'-fluorothymidine ((18)F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for (18)F-FLT uptake in normal tissues and tumors. METHODS: ENT1-knockout (ENT1(-/-)) mice were generated and compared with wild-type (ENT1(+/+)) mice using small-animal (18)F-FLT PET. In addition, ENT1(+/+) mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by (18)F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography-mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and (18)F-FLT uptake using real-time polymerase chain reaction and (3)H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by (18)F-FLT small-animal PET. RESULTS: Compared with noninjected ENT1(+/+) mice, ENT1(+/+) mice injected with NBMPR-P and ENT1(-/-) mice displayed a reduced percentage injected dose per gram (%ID/g) for (18)F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for (18)F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1(-/-) mice displayed 1.65-fold greater plasma thymidine levels than did ENT1(+/+) mice. Spleen tissue from ENT1(+/+) and ENT1(-/-) mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold (3)H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo gamma-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for (18)F-FLT uptake at 1 h after tracer injection. CONCLUSION: Loss of ENT1 activity significantly affected (18)F-FLT biodistribution in mice and (18)F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of (18)F-FLT uptake in normal and transformed cells.

Investigation of a 3D system distortion correction method for MR images.

Interest has been growing in recent years in the development of radiation treatment planning (RTP) techniques based solely on Magnetic Resonance (MR) images. However, it is recognized that MR images suffer from scanner-related and object-induced distortions that may lead to an incorrect placement of anatomical structures. This subsequently may result in a reduced accuracy in delivering treatment dose fractions in RTP. To accomplish the precise representation of anatomical targets required by RTP, distortions must be mapped and the images rectified before being used in the clinical process. In this work, we investigate a novel, phantom-based method that determines and corrects for 3D system-related distortions. The algorithm consists of two key components: an adaptive control point identification and registration tool and an iterative method that finds the best estimate of 3D distortion. It was found that the 3D distortions were successfully mapped to within the voxel resolution of the raw data for a 260 x 260 x 240 mm3 volume.