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Understanding which patients with human epidermal growth factor receptor 2 (HER2)-negative or -low metastatic breast cancer (MBC) benefit from HER2-targeted strategies is urgently needed. We assessed the whole-body heterogeneity of HER2 expression on 89 Zr-trastuzumab PET (HER2 PET) and the diagnostic performance of HER2 PET in a large series of patients, including HER2-negative and -low MBC. Methods: In the IMPACT-MBC study, patients with newly diagnosed and nonrapidly progressive MBC of all subtypes were included. Metastasis HER2 status was determined by immunohistochemistry and in situ hybridization. 89 Zr-trastuzumab uptake was quantified as SUV max and SUV mean HER2 immunohistochemistry was related to the quantitative 89 Zr-trastuzumab uptake of all metastases and corresponding biopsied metastasis, uptake heterogeneity, and qualitative scan evaluation. A prediction algorithm for HER2 immunohistochemistry positivity based on uptake was developed. Results: In 200 patients, 89 Zr-trastuzumab uptake was quantified in 5,163 metastases, including 186 biopsied metastases. With increasing HER2 immunohistochemistry status, uptake was higher (geometric mean SUV max of 7.0, 7.6, 7.3, and 17.4 for a HER2 immunohistochemistry score of 0, 1, 2, or 3+, respectively; P < 0.001). High uptake exceeding 14.6 (90th percentile) was observed in one third of patients with a HER2-negative or -low metastasis biopsy. The algorithm performed best when lesion site and size were incorporated (area under the curve, 0.86; 95% CI, 0.79-0.93). Conclusion: HER2 PET had good diagnostic performance in MBC, showing considerable whole-body HER2 heterogeneity and uptake above background in HER2-negative and -low MBC. This provides novel insights into HER2-negative and -low MBC compared with standard HER2 immunohistochemistry on a single biopsy., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

The purpose of this study was to quantify any differences between the SUVs of 89 Zr immuno-PET scans obtained using a PET/CT system with a long axial field of view (LAFOV; Biograph Vision Quadra) compared to a PET/CT system with a short axial field of view (SAFOV; Biograph Vision) and to evaluate how LAFOV PET scan duration affects image noise and SUV metrics. Methods: Five metastatic breast cancer patients were scanned consecutively on SAFOV and LAFOV PET/CT scanners. Four additional patients were scanned using only LAFOV PET/CT. Scans on both systems lasted approximately 30 min and were acquired 4 d after injection of 37 MBq of 89 Zr-trastuzumab. LAFOV list-mode data were reprocessed to obtain images acquired using shorter scan durations (15, 10, 7.5, 5, and 3 min). Volumes of interest were placed in healthy tissues, and tumors were segmented semiautomatically to compare coefficients of variation and to perform Bland-Altman analysis on SUV metrics (SUV max , SUV peak , and SUV mean ). Results: Using 30-min images, 2 commonly used lesion SUV metrics were higher for SAFOV than for LAFOV PET (SUV max , 16.2% ± 13.4%, and SUV peak , 10.1% ± 7.2%), whereas the SUV mean of healthy tissues showed minimal differences (0.7% ± 5.8%). Coefficients of variation in the liver derived from 30-min SAFOV PET were between those of 3- and 5-min LAFOV PET. The smallest SUV max and SUV peak differences between SAFOV and LAFOV were found for 3-min LAFOV PET. Conclusion: LAFOV 89 Zr immuno-PET showed a lower SUV max and SUV peak than SAFOV because of lower image noise. LAFOV PET scan duration may be reduced at the expense of increasing image noise and bias in SUV metrics. Nevertheless, SUV peak showed only minimal bias when reducing scan duration from 30 to 10 min., (© 2023 by the Society of Nuclear Medicine and Molecular Imaging.)

The mesothelin (MSLN)-targeted 227 Th conjugate is a novel α-therapy developed to treat MSLN-overexpressing cancers. We radiolabeled the same antibody-chelator conjugate with 89 Zr to evaluate whether PET imaging with 89 Zr-MSLN matches 227 Th-MSLN tumor uptake, biodistribution, and antitumor activity. Methods: Serial PET imaging with protein doses of 4, 20, or 40 μg of 89 Zr-MSLN and 89 Zr-control was performed up to 168 h after tracer injection in human tumor-bearing nude mice with high (HT29-MSLN) and low (BxPc3) MSLN expression. 89 Zr-MSLN and 227 Th-MSLN ex vivo tumor uptake and biodistribution were compared at 6 time points in HT29-MSLN and in medium-MSLN-expressing (OVCAR-3) tumor-bearing mice. 89 Zr-MSLN PET imaging was performed before 227 Th-MSLN treatment in HT29-MSLN and BxPc3 tumor-bearing mice. Results: 89 Zr-MSLN PET imaging showed an SUV mean of 2.2 ± 0.5 in HT29-MSLN tumors. Ex vivo tumor uptake was 10.6% ± 2.4% injected dose per gram at 168 h. 89 Zr-MSLN tumor uptake was higher than uptake of 89 Zr-control ( P = 0.0043). 89 Zr-MSLN and 227 Th-MSLN showed comparable tumor uptake and biodistribution in OVCAR-3 and HT29-MSLN tumor-bearing mice. Pretreatment SUV mean was 2.2 ± 0.2 in HT29-MSLN tumors, which decreased in volume on 227 Th-MSLN treatment. BxPc3 tumors showed an SUV mean of 1.2 ± 0.3 and remained similar in size after 227 Th-MSLN treatment. Conclusion: 89 Zr-MSLN PET imaging reflected MSLN expression and matched 227 Th-MSLN tumor uptake and biodistribution. Our data support the clinical exploration of 89 Zr-MSLN PET imaging together with 227 Th-MSLN therapy, both using the same antibody-chelator conjugate., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

BiTE ® (bispecific T-cell engager) molecules exert antitumor activity by binding one arm to CD3 on cytotoxic T-cells and the other arm to a tumor-associated antigen. We generated a fully mouse cross-reactive mesothelin (MSLN)-targeted BiTE molecule that is genetically fused to a Fc-domain for half-life extension, and evaluated biodistribution and tumor targeting of a zirconium-89 ( 89 Zr)-labeled MSLN HLE BiTE molecule in 4T1 breast cancer bearing syngeneic mice with positron emission tomography (PET). Biodistribution of 50 µg 89 Zr-MLSN HLE BiTE was studied over time by PET imaging in BALB/c mice and revealed uptake in tumor and lymphoid tissues with an elimination half-life of 63.4 hours. Compared to a non-targeting 89 Zr-control HLE BiTE, the 89 Zr-MLSN HLE BiTE showed a 2-fold higher tumor uptake and higher uptake in lymphoid tissues. Uptake in the tumor colocalized with mesothelin expression, while uptake in the spleen colocalized with CD3 expression. Evaluation of the effect of protein doses on the biodistribution and tumor targeting of 89 Zr-MSLN HLE BiTE revealed for all dose groups that uptake in the spleen was faster than in the tumor (day 1 vs day 5). The lowest dose of 10 µg 89 Zr-MSLN HLE BiTE had higher spleen uptake and faster blood clearance compared to higher doses of 50 µg and 200 µg. 89 Zr-MSLN HLE BiTE tumor uptake was similar at all doses. Conclusion: The MSLN HLE BiTE showed specific tumor uptake and both arms contributed to the biodistribution profile. These findings support the potential for clinical translation of HLE BiTE molecules., (Copyright © 2021 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

18 F-BMS-986192, an adnectin-based human programmed cell death ligand 1 (PD-L1) tracer, was developed to noninvasively determine whole-body PD-L1 expression by PET. We evaluated the usability of 18 F-BMS-986192 PET to detect different PD-L1 expression levels and therapy-induced changes in PD-L1 expression in tumors. Methods: In vitro binding assays with 18 F-BMS-986192 were performed on human tumor cell lines with different total cellular and membrane PD-L1 protein expression levels. Subsequently, PET imaging was performed on immunodeficient mice xenografted with these cell lines. The mice were treated with interferon γ (IFNγ) intraperitoneally for 3 d or with the mitogen-activated protein kinase kinase inhibitor selumetinib by oral gavage for 24 h. Afterward, 18 F-BMS-986192 was administered intravenously, followed by a 60-min dynamic PET scan. Tracer uptake was expressed as percentage injected dose per gram of tissue. Tissues were collected to evaluate ex vivo tracer biodistribution and to perform flow cytometric, Western blot, and immunohistochemical tumor analyses. Results: 18 F-BMS-986192 uptake reflected PD-L1 membrane levels in tumor cell lines, and tumor tracer uptake in mice was associated with PD-L1 expression measured immunohistochemically. In vitro IFNγ treatment increased PD-L1 expression in the tumor cell lines and caused up to a 12-fold increase in tracer binding. In vivo, IFNγ affected neither PD-L1 tumor expression measured immunohistochemically nor 18 F-BMS-986192 tumor uptake. In vitro, selumetinib downregulated cellular and membrane levels of PD-L1 in tumor cells by 50% as measured by Western blotting and flow cytometry. In mice, selumetinib lowered cellular, but not membrane, PD-L1 levels of tumors, and consequently, no treatment-induced change in 18 F-BMS-986192 tumor uptake was observed. Conclusion: 18 F-BMS-986192 PET imaging allows detection of membrane-expressed PD-L1 as soon as 60 min after tracer injection. The tracer can discriminate a range of tumor cell PD-L1 membrane expression levels., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Bispecific T-cell engager (BiTE) molecules are designed to engage and activate cytotoxic T cells to kill tumor cells. Little is known about their biodistribution in immunocompetent settings. Methods: To explore their pharmacokinetics and the role of the immune cells, BiTE molecules were radiolabeled with the PET isotope 89 Zr and studied in immunocompetent and immunodeficient mouse models. Results: PET images and ex vivo biodistribution in immunocompetent mice with [ 89 Zr]Zr-DFO- N -suc-muS110, targeting mouse CD3 (dissociation constant [K D ], 2.9 nM) and mouse epithelial cell adhesion molecule (EpCAM; K D , 21 nM), and with [ 89 Zr]Zr-DFO- N -suc-hyS110, targeting only mouse CD3 (K D , 2.9 nM), showed uptake in the tumor, spleen, and other lymphoid organs, whereas the human-specific control BiTE [ 89 Zr]Zr-DFO- N -suc-AMG 110 showed similar tumor uptake but lacked spleen uptake. [ 89 Zr]Zr-DFO- N -suc-muS110 spleen uptake was lower in immunodeficient than in immunocompetent mice. After repeated administration of nonradiolabeled muS110 to immunocompetent mice, 89 Zr-muS110 uptake in the spleen and other lymphoid tissues decreased and was comparable to uptake in immunodeficient mice, indicating saturation of CD3 binding sites. Autoradiography and immunohistochemistry demonstrated colocalization of [ 89 Zr]Zr-DFO- N -suc-muS110 and [ 89 Zr]Zr-DFO- N -suc-hyS110 with CD3-positive T cells in the tumor and spleen but not with EpCAM expression. Also, uptake in the duodenum correlated with a high incidence of T cells. Conclusion: [ 89 Zr]Zr-DFO- N -suc-muS110 biodistribution is dependent mainly on the T-cell-targeting arm, with a limited contribution from its second arm, targeting EpCAM. These findings highlight the need for extensive biodistribution studies of novel bispecific constructs, as the results might have implications for their respective drug development and clinical translation., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Recently, N -(4- 18 F-fluorobenzoyl)-interleukin-2 ( 18 F-FB-IL2) was introduced as a PET tracer for T cell imaging. However, production is complex and time-consuming. Therefore, we developed 2 radiolabeled IL2 variants, namely aluminum 18 F-fluoride-(restrained complexing agent)-IL2 ( 18 F-AlF-RESCA-IL2) and 68 Ga-gallium-(1,4,7-triazacyclononane-4,7-diacetic acid-1-glutaric acid)-IL2 ( 68 Ga-Ga-NODAGA-IL2), and compared their in vitro and in vivo characteristics with 18 F-FB-IL2. Methods: Radiolabeling of 18 F-AlF-RESCA-IL2 and 68 Ga-Ga-NODAGA-IL2 was optimized, and stability was evaluated in human serum. Receptor binding was studied with activated human peripheral blood mononuclear cells (hPBMCs). Ex vivo tracer biodistribution in immunocompetent BALB/cOlaHsd (BALB/c) mice was performed at 15, 60, and 90 min after tracer injection. In vivo binding characteristics were studied in severe combined immunodeficient (SCID) mice inoculated with activated hPBMCs in Matrigel. Tracer was injected 15 min after hPBMC inoculation, and a 60-min dynamic PET scan was acquired, followed by ex vivo biodistribution studies. Specific uptake was determined by coinjection of tracer with unlabeled IL2 and by evaluating uptake in a control group inoculated with Matrigel only. Results: 68 Ga-Ga-NODAGA-IL2 and 18 F-AlF-RESCA-IL2 were produced with radiochemical purity of more than 95% and radiochemical yield of 13.1% ± 4.7% and 2.4% ± 1.6% within 60 and 90 min, respectively. Both tracers were stable in serum, with more than 90% being intact tracer after 1 h. In vitro, both tracers displayed preferential binding to activated hPBMCs. Ex vivo biodistribution studies on BALB/c mice showed higher uptake of 18 F-AlF-RESCA-IL2 than of 18 F-FB-IL2 in liver, kidney, spleen, bone, and bone marrow. 68 Ga-Ga-NODAGA-IL2 uptake in liver and kidney was higher than 18 F-FB-IL2 uptake. In vivo, all tracers revealed uptake in activated hPBMCs in SCID mice. Low uptake was seen after a blocking dose of IL2 and in the Matrigel control group. In addition, 18 F-AlF-RESCA-IL2 yielded the highest-contrast PET images of target lymph nodes. Conclusion: Production of 18 F-AlF-RESCA-IL2 and 68 Ga-Ga-NODAGA-IL2 is simpler and faster than that of 18 F-FB-IL2. Both tracers showed good in vitro and in vivo characteristics, with high uptake in lymphoid tissue and hPBMC xenografts., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Optical molecular imaging using fluorescently labeled monoclonal antibodies is of significant added value in guiding surgical or endoscopic procedures. However, development of tracers for clinical trials is complex, and implementation in the clinic is therefore slow. We present a roadmap for development and translation of monoclonal antibody tracers into a drug product compliant with current good manufacturing processes (cGMPs). Methods: The production process for cetuximab-800CW and trastuzumab-800CW was optimized with regard to dye-to-protein ratio and formulation buffer. Promising formulations were produced under cGMP conditions and advanced to a full-scale stability study. Tracers were analyzed for stability by size-exclusion high-pressure liquid chromatography, pH measurement, osmolality, visual inspection, and sterility, as required by the European Pharmacopeia and cGMP guidelines. Results: Seven formulations were investigated for cetuximab-800CW and 10 for trastuzumab-800CW. On the basis of the formulation study results, we chose 2 formulations per antibody for investigation during the full-scale stability study. These formulations all performed well, showing good compliance with the acceptance criteria set for each product. Conclusion: We designed a roadmap to standardize the development, formulation, and cGMP translation of molecular fluorescent tracers. Using our standardized approach, we developed 2 stable antibody-based tracers for clinical use. The proposed roadmap can be used to efficiently develop a cGMP-compliant formulation and improve the translation of newly developed optical tracers to first-in-human use., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

Development of new oncology drugs has increased since the improved understanding of cancer's complex biology. The oncology field has become the top therapeutic research area for new drugs. However, only a limited number of drugs entering clinical trials will be approved for use as the standard of care for cancer patients. Molecular imaging is increasingly perceived as a tool to support go/no-go decisions early during drug development. It encompasses a wide range of techniques that include radiolabeling a compound of interest followed by visualization with SPECT or PET. Radiolabeling can be performed using a variety of radionuclides, which are preferably matched to the compound on the basis of size and half-life. Imaging can provide information on drug behavior in vivo, whole-body drug target visualization, and heterogeneity in drug target expression. This review focuses on current applications of molecular imaging in the development of small molecules, antibodies, and antihormonal anticancer drugs., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

In theranostics, radiolabeled compounds are used to determine a treatment strategy by combining therapeutics and diagnostics in the same agent. Monoclonal antibodies (mAbs) and antibody-related therapeutics represent a rapidly expanding group of cancer medicines. Theranostic approaches using these drugs in oncology are particularly interesting because antibodies are designed against specific targets on the tumor cell membrane and immune cells as well as targets in the tumor microenvironment. In addition, these drugs are relatively easy to radiolabel. Noninvasive molecular imaging techniques, such as SPECT and PET, provide information on the whole-body distribution of radiolabeled mAbs and antibody-related therapeutics. Molecular antibody imaging can potentially elucidate drug target expression, tracer uptake in the tumor, tumor saturation, and heterogeneity for these parameters within the tumor. These data can support drug development and may aid in patient stratification and monitoring of the treatment response. Selecting a radionuclide for theranostic purposes generally starts by matching the serum half-life of the mAb or antibody-related therapeutic and the physical half-life of the radionuclide. Furthermore, PET imaging allows better quantification than the SPECT technique. This information has increased interest in theranostics using PET radionuclides with a relatively long physical half-life, such as 89 Zr. In this review, we provide an overview of ongoing research on mAbs and antibody-related theranostics in preclinical and clinical oncologic settings. We identified 24 antibodies or antibody-related therapeutics labeled with PET radionuclides for theranostic purposes in patients. For this approach to become integrated in standard care, further standardization with respect to the procedures involved is required., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

The human epidermal growth factor receptor 3 (HER3) is an interesting target for antitumor therapy. For optimal HER3 signaling inhibition, a biparatopic Nanobody construct (MSB0010853) was developed that binds 2 different HER3 epitopes. In addition, MSB0010853 contains a third HER3 epitope that binds albumin to extend its circulation time. MSB0010853 is cross-reactive with HER3 and albumin of mouse origin. We aimed to gain insight into MSB0010853 biodistribution and tumor uptake by radiolabeling the Nanobody construct with 89 Zr. Methods: MSB0010853 was radiolabeled with 89 Zr. Dose- and time-dependent tumor uptake was studied in nude BALB/c mice bearing a subcutaneous HER3 overexpressing H441 non-small cell lung cancer xenograft. Dose-dependent biodistribution of 89 Zr-MSB0010853 was assessed ex vivo at 24 h after intravenous injection. Protein doses of 5, 10, 25, 100, and 1,000 μg were used. Time-dependent biodistribution of MSB0010853 was analyzed ex vivo at 3, 6, 24, and 96 h after intravenous administration of 25 μg of 89 Zr-MSB0010853. PET imaging and biodistribution were performed 24 h after administration of 25 μg of 89 Zr-MSB0010853 to mice bearing human H441, FaDu (high HER3 expression), or Calu-1 (no HER3 expression) tumor xenografts. Results: Radiolabeling of MSB0010853 with 89 Zr was performed with a radiochemical purity of greater than 95%. Ex vivo biodistribution showed protein dose- and time-dependent distribution of 89 Zr-MSB0010853 in all organs. Uptake of 89 Zr-MSB0010853 in H441 tumors was only time-dependent. Tumor could be visualized up to at least 96 h after injection. The highest mean SUV of 0.6 ± 0.2 was observed at 24 h after injection of 25 μg of 89 Zr-MSB0010853. 89 Zr-MSB0010853 tumor uptake correlated with HER3 expression and was highest in H441 (6.2 ± 1.1 percentage injected dose per gram [%ID/g]) and lowest in Calu-1 (2.3 ± 0.3 %ID/g) xenografts. Conclusion: 89 Zr-MSB0010853 organ distribution and tumor uptake in mice are time-dependent, and tumor uptake correlates with HER3 expression. In contrast to tumor uptake except for kidney uptake, organ distribution of 89 Zr-MSB0010853 is protein dose-dependent for the tested doses. 89 Zr-MSB0010853 PET imaging gives insight into the in vivo behavior of MSB0010853., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Currently, biomarkers that predict the efficacy of everolimus in metastatic renal cell carcinoma (mRCC) patients are lacking. Everolimus inhibits vascular endothelial growth factor A (VEGF-A) expression. We performed PET scans on mRCC patients with 89 Zr-bevacizumab, a VEGF-A-binding antibody tracer. The aims were to determine a change in tumor tracer uptake after the start of everolimus and to explore whether 89 Zr-bevacizumab PET can identify patients with early disease progression. Methods: 89 Zr-bevacizumab PET was done before and 2 and 6 wk after the start of everolimus, 10 mg/d, in mRCC patients. Routine CT scans were performed at baseline and every 3 mo thereafter. Tumor tracer uptake was quantified using SUV max The endpoints were a change in tumor tracer uptake and treatment response on CT after 3 mo. Results: Thirteen patients participated. The median SUV max of 94 tumor lesions was 7.3 (range, 1.6-59.5). Between patients, median tumor SUV max varied up to 8-fold. After 2 wk, median SUV max was 6.3 (1.7-62.3), corresponding to a mean decrease of 9.1% ( P < 0.0001). Three patients discontinued everolimus early. At 6 wk, a mean decrease in SUV max of 23.4% compared with baseline was found in 70 evaluable lesions of 10 patients, with a median SUV max of 5.4 (1.1-49.4, P < 0.0001). All 10 patients who continued treatment had stable disease at 3 mo. Conclusion: Everolimus decreases 89 Zr-bevacizumab tumor uptake. Further studies are warranted to evaluate the predictive value of 89 Zr-bevacizumab PET for everolimus antitumor efficacy., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Unlabelled: Patients with von Hippel-Lindau disease (VHL) are at risk to develop multiple tumors. The growth of lesions is unpredictable, and regular surveillance is critical for early treatment to control local damage. Vascular endothelial growth factor A (VEGF-A) produced locally is supposed to play an important role in development of disease manifestations and is a target for antiangiogenic therapy with the monoclonal antibody bevacizumab. We aimed to assess whether VHL manifestations can be visualized with (89)Zr-bevacizumab PET and to explore whether (89)Zr-bevacizumab PET can differentiate progressive from nonprogressive lesions., Methods: VHL patients with at least 1 measurable hemangioblastoma were eligible. (89)Zr-bevacizumab (37 MBq) was administered intravenously 4 d before the scan. Maximum standardized uptake values were calculated. PET scans were fused with routine MRI of the central nervous system and abdominal MRI or CT. Progressive lesions were defined as new lesions, lesions that became symptomatic, and lesions ≥ 10 mm that increased ≥ 10% and ≥ 4 mm on repeated anatomic imaging within 12 mo., Results: Twenty-two patients were enrolled. At baseline, anatomic imaging showed 311 lesions. (89)Zr-bevacizumab PET visualized 59 VHL manifestations, 0-17 per patient. The median of maximum standardized uptake values was 8.5 (range, 1.3-35.8). The detection rate for lesions ≥ 10 mm was 30.8%. Seven additional hotspots without substrate on baseline anatomic imaging were found; 2 were also detected with anatomic imaging during follow-up. Nine of 25 progressive lesions were visible on PET and 27 of 175 nonprogressive lesions, corresponding to a positive predictive value of 25% and a negative predictive value of 90%. SUVmax was similar in progressive and nonprogressive lesions (median, 4.8; range, 0.9-8.9 vs. median, 6.7; range, 1.3-35.8, P = 0.14)., Conclusion: VHL manifestations can be visualized with (89)Zr-bevacizumab PET with a striking heterogeneity in tracer accumulation. (89)Zr-bevacizumab uptake does not predict progression within 12 mo. In one third of the lesions, the drug target VEGF is available and accessible. (89)Zr-bevacizumab PET might offer a tool to select VHL patients for anti-VEGF therapy., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Unlabelled: AMG 110, a bispecific T cell engager (BiTE) antibody construct, induces T cell-mediated cancer cell death by cross-linking epithelial cell adhesion molecule (EpCAM) on tumor cells with a cluster of differentiation 3 ε (CD3ε) on T cells. We labeled AMG 110 with (89)Zr or near-infrared fluorescent dye (IRDye) 800CW to study its tumor targeting and tissue distribution., Methods: Biodistribution and tumor uptake of (89)Zr-AMG 110 was studied up to 6 d after intravenous administration to nude BALB/c mice bearing high EpCAM-expressing HT-29 colorectal cancer xenografts. Tumor uptake of (89)Zr-AMG 110 was compared with uptake in head and neck squamous cell cancer FaDu (intermediate EpCAM) and promyelocytic leukemia HL60 (EpCAM-negative) xenografts. Intratumoral distribution in HT-29 tumors was studied using 800CW-AMG 110., Results: Tumor uptake of (89)Zr-AMG 110 can be clearly visualized using small-animal PET imaging up to 72 h after injection. The highest tumor uptake of (89)Zr-AMG 110 at the 40-μg dose level was observed at 6 and 24 h (respectively, 5.35 ± 0.22 and 5.30 ± 0.20 percentage injected dose per gram; n = 3 and 4). Tumor uptake of (89)Zr-AMG 110 was EpCAM-specific and correlated with EpCAM expression. 800CW-AMG 110 accumulated at the tumor cell surface in viable EpCAM-expressing tumor tissue., Conclusion: PET and fluorescent imaging provided real-time information about AMG 110 distribution and tumor uptake in vivo. Our data support using (89)Zr and IRDye 800CW to evaluate tumor and tissue uptake kinetics of bispecific T cell engager antibody constructs in preclinical and clinical settings., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Molecular imaging with PET is a rapidly emerging technique. In breast cancer patients, more than 45 different PET tracers have been or are presently being tested. With a good rationale, after development of the tracer and proven feasibility, it is of interest to evaluate whether there is a potential meaningful role for the tracer in the clinical setting-such as in staging, in the (early) prediction of a treatment response, or in supporting drug choices. So far, only (18)F-FDG PET has been incorporated into breast cancer guidelines. For proof of the clinical relevance of tracers, especially for analysis in a multicenter setting, standardization of the technology and access to the novel PET tracer are required. However, resources for PET implementation research are limited. Therefore, next to randomized studies, novel approaches are required for proving the clinical value of PET tracers with the smallest possible number of patients. The aim of this review is to describe the process of the development of PET tracers and the level of evidence needed for the use of these tracers in breast cancer. Several breast cancer trials have been performed with the PET tracers (18)F-FDG, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT), and (18)F-fluoroestradiol ((18)F-FES). We studied them to learn lessons for the implementation of novel tracers. After defining the gap between a good rationale for a tracer and implementation in the clinical setting, we propose solutions to fill the gap to try to bring more PET tracers to daily clinical practice., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Unlabelled: Transforming growth factor-β (TGF-β) signaling is involved in glioma development. The monoclonal antibody fresolimumab (GC1008) can neutralize all mammalian isoforms of TGF-β, and tumor uptake can be visualized and quantified with (89)Zr-fresolimumab PET in mice. The aim of this study was to investigate the fresolimumab uptake in recurrent high-grade gliomas using (89)Zr-fresolimumab PET and to assess treatment outcome in patients with recurrent high-grade glioma treated with fresolimumab., Methods: Patients with recurrent glioma were eligible. After intravenous administration of 37 MBq (5 mg) of (89)Zr-fresolimumab, PET scans were acquired on day 2 or day 4 after tracer injection. Thereafter, patients were treated with 5 mg of fresolimumab per kilogram intravenously every 3 wk. (89)Zr-fresolimumab tumor uptake was quantified as maximum standardized uptake value (SUVmax). MR imaging for response evaluation was performed after 3 infusions or as clinically indicated., Results: Twelve patients with recurrent high-grade glioma were included: 10 glioblastomas, 1 anaplastic oligodendroglioma, and 1 anaplastic astrocytoma. All patients underwent (89)Zr-fresolimumab PET 4 d after injection. In 4 patients, an additional PET scan was obtained on day 2 after injection. SUVmax on day 4 in tumor lesions was 4.6 (range, 1.5-13.9) versus a median SUVmean of 0.3 (range, 0.2-0.5) in normal brain tissue. All patients showed clinical or radiologic progression after 1-3 infusions of fresolimumab. Median progression-free survival was 61 d (range, 25-80 d), and median overall survival was 106 d (range, 37-417 d)., Conclusion: (89)Zr-fresolimumab penetrated recurrent high-grade gliomas very well but did not result in clinical benefit., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Unlabelled: No validated predictive biomarkers for antiangiogenic treatment of metastatic renal cell carcinoma (mRCC) exist. Tumor vascular endothelial growth factor A (VEGF-A) level may be useful. We determined tumor uptake of (89)Zr-bevacizumab, a VEGF-A-binding PET tracer, in mRCC patients before and during antiangiogenic treatment in a pilot study., Methods: Patients underwent (89)Zr-bevacizumab PET scans at baseline and 2 and 6 wk after initiating either bevacizumab (10 mg/kg every 2 wk) with interferon-α (3-9 million IU 3 times/wk) (n = 11) or sunitinib (50 mg daily, 4 of every 6 wk) (n = 11). Standardized uptake values were compared with plasma VEGF-A and time to disease progression., Results: (89)Zr-bevacizumab PET scans visualized 125 evaluable tumor lesions in 22 patients, with a median SUV(max) (maximum standardized uptake value) of 6.9 (range, 2.3-46.9). Bevacizumab/interferon-α induced a mean change in tumor SUV(max) of -47.0% (range, -84.7 to +20.0%; P < 0.0001) at 2 wk and an additional -9.7% (range, -44.8 to +38.9%; P = 0.015) at 6 wk. In the sunitinib group, the mean change in tumor SUV(max) was -14.3% at 2 wk (range, -80.4 to +269.9; P = 0.006), but at 6 wk the mean change in tumor SUV(max) was +72.6% (range, -46.4 to +236%; P < 0.0001) above baseline. SUV(max) was not related to plasma VEGF-A at all scan moments. A baseline mean tumor SUV(max) greater than 10.0 in the 3 most intense lesions corresponded with longer time to disease progression (89.7 vs. 23.0 wk; hazard ratio, 0.22; 95% confidence interval, 0.05-1.00)., Conclusion: Tumor uptake of (89)Zr-bevacizumab is high in mRCC, with remarkable interpatient and intrapatient heterogeneity. Bevacizumab/interferon-α strongly decreases tumor uptake whereas sunitinib results in a modest reduction with an overshoot after 2 drug-free weeks. High baseline tumor SUV(max) was associated with longer time to progression., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Unlabelled: Everolimus increases progression-free survival in patients with advanced neuroendocrine tumors (NETs). Currently, no biomarkers are available for early selection of patients who will benefit from everolimus. Everolimus can reduce vascular endothelial growth factor A (VEGF-A) production by tumor cells. Therefore, we aimed to investigate the effect of everolimus on tumor uptake of the radioactive-labeled VEGF-A antibody bevacizumab with PET in NET patients., Methods: Patients with advanced progressive well-differentiated NETs underwent (89)Zr-bevacizumab PET scans before and at 2 and 12 wk during everolimus treatment. (89)Zr-bevacizumab uptake was quantified by the maximum standardized uptake value (SUVmax). Tumor response and the percentage change in the sum of target lesion diameters were determined according to Response Evaluation Criteria in Solid Tumors 1.1 on CT (3 monthly)., Results: In 4 of the 14 patients entered, no tumor lesions were visualized with (89)Zr-bevacizumab PET. In the remaining patients, 19% of tumor lesions 1 cm or greater known by CT were visualized. Tumor SUVmax decreased during everolimus treatment, with a median of -7% at 2 wk (P = 0.09) and a median of -35% at 12 wk (P < 0.001). The difference in SUVmax at 2 and 12 wk with respect to SUVmax at baseline correlated with percentage change on CT at 6 mo (r(2) = 0.51, P < 0.05, and r(2) = 0.61, P < 0.01, respectively)., Conclusion: This study demonstrates variable (89)Zr-bevacizumab PET tumor uptake in NET patients. (89)Zr-bevacizumab tumor uptake diminished during everolimus treatment. Serial (89)Zr-bevacizumab PET might be useful as an early predictive biomarker of anti-VEGF-directed treatment in NET patients., (© 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Unlabelled: Anticalins are a novel class of biopharmaceuticals, displaying highly desirable attributes as imaging agents. The anticalin PRS-110 was rationally engineered to target the oncogene MET with high affinity and specificity. The aim of this study was to visualize MET expression and analyze biodistribution of (89)Zr-labeled PRS-110 in human tumor-bearing mice., Methods: (89)Zr-PRS-110 was generated. For biodistribution studies (96 h after injection of tracer) 10 μg of (89)Zr-PRS-110 (with 0-490 μg of unlabeled PRS-110) were injected into BALB/c mice bearing high MET-expressing H441 non-small cell lung cancer xenografts. Further characterization with PET imaging was performed at 6, 24, 48, and 96 h after injection of 50 μg of (89)Zr-PRS-110 into mice bearing H441, primary glioblastoma U87-MG (intermediate MET), or ovarian cancer A2780 (low MET) xenografts. Drug distribution was also analyzed ex vivo using fluorescently labeled PRS-110., Results: Biodistribution analyses showed a dose-dependent tumor uptake of (89)Zr-PRS-110, with the highest fractional tumor uptake at 10 μg of (89)Zr-PRS-110, with no unlabeled PRS-110. Small-animal PET imaging supported by biodistribution data revealed specific tumor uptake of (89)Zr-PRS-110 in the MET-expressing H441 and U87-MG tumors whereas the MET-negative A2780 tumor model showed a lower uptake similar to a non-MET binder anticalin control. Tumor uptake increased up to 24 h after tracer injection and remained high, whereas uptake in other organs decreased over time. Ex vivo fluorescence revealed intracellular presence of PRS-110., Conclusion: (89)Zr-PRS-110 specifically accumulates in MET-expressing tumors in a receptor density-dependent manner. PET imaging provides real-time noninvasive information about PRS-110 distribution and tumor accumulation in preclinical models.

Unlabelled: Vascular endothelial growth factor (VEGF)-A is overexpressed in most malignant and premalignant breast lesions. VEGF-A can be visualized noninvasively with PET imaging and using the tracer (89)Zr-labeled bevacizumab. In this clinical feasibility study, we assessed whether VEGF-A in primary breast cancer can be visualized by (89)Zr-bevacizumab PET., Methods: Before surgery, breast cancer patients underwent a PET/CT scan of the breasts and axillary regions 4 d after intravenous administration of 37 MBq of (89)Zr-bevacizumab per 5 mg. PET images were compared with standard imaging modalities. (89)Zr-bevacizumab uptake was quantified as the maximum standardized uptake value (SUV max). VEGF-A levels in tumor and normal breast tissues were assessed with enzyme-linked immunosorbent assay. Data are presented as mean ± SD., Results: Twenty-five of 26 breast tumors (mean size ± SD, 25.1 ± 19.8 mm; range, 4-80 mm) in 23 patients were visualized. SUV max was higher in tumors (1.85 ± 1.22; range, 0.52-5.64) than in normal breasts (0.59 ± 0.37; range, 0.27-1.69; P < 0.001). The only tumor not detected on PET was 10 mm in diameter. Lymph node metastases were present in 10 axillary regions; 4 could be detected with PET (SUV max, 2.66 ± 2.03; range, 1.32-5.68). VEGF-A levels in the 17 assessable tumors were higher than in normal breast tissue in all cases (VEGF-A/mg protein, 184 ± 169 pg vs. 10 ± 21 pg; P = 0.001), whereas (89)Zr-bevacizumab tumor uptake correlated with VEGF-A tumor levels (r = 0.49)., Conclusion: VEGF-A in primary breast cancer can be visualized by means of (89)Zr-bevacizumab PET.

Unlabelled: Placental growth factor (PlGF) is a member of the proangiogenic vascular endothelial growth factor family, which is upregulated in many tumors. RO5323441, a humanized monoclonal antibody against PlGF, showed antitumor activity in human tumor xenografts. We therefore aimed to radiolabel RO5323441 and preclinically validate this tracer to study drug tumor uptake and organ distribution by PET imaging. (89)Zr-RO5323441 was tested for stability and immunoreactivity in vitro., Methods: The tumor uptake and organ distribution for 10, 50, and 500 μg of (89)Zr-RO5323441 was assessed in mice bearing human PlGF-expressing hepatocellular cancer (Huh7) xenografts or human renal cell carcinoma (ACHN) xenografts without detectable human PlGF expression. The effect of pretreatment with RO5323441 (20 mg/kg) on (89)Zr-RO5323441 tumor uptake was analyzed in Huh7 xenografts. (111)In-IgG served as a control for nonspecific tumor uptake and organ distribution. Cy5-RO5323441 was injected to study the intratumor distribution of RO5323441 with fluorescence microscopy., Results: (89)Zr-RO5323441 showed a time- and dose-dependent tumor accumulation. Uptake in Huh7 xenografts at 10 μg of (89)Zr-RO5323441 was 8.2% ± 1.7% injected dose (ID)/cm(3) at 144 h after injection, and in ACHN xenografts it was 5.5 ± 0.3 %ID/cm(3) (P = 0.03). RO5323441 pretreatment of Huh7 xenograft-bearing mice reduced (89)Zr-RO5323441 tumor uptake to the level of nonspecific (111)In-IgG uptake. Cy5-RO5323441 was present in the tumors mainly in the microenvironment., Conclusion: The findings show that RO5323441 tumor uptake is PlGF-specific and time- and dose-dependent.

Unlabelled: Transforming growth factor-β (TGF-β) promotes cancer invasion and metastasis and is therefore a potential drug target for cancer treatment. Fresolimumab, which neutralizes all mammalian active isoforms of TGF-β, was radiolabeled with (89)Zr for PET to analyze TGF-β expression, antibody tumor uptake, and organ distribution., Methods: (89)Zr was conjugated to fresolimumab using the chelator N-succinyldesferrioxamine-B-tetrafluorphenol. (89)Zr-fresolimumab was analyzed for conjugation ratio, aggregation, radiochemical purity, stability, and immunoreactivity. (89)Zr-fresolimumab tumor uptake and organ distribution were assessed using 3 protein doses (10, 50, and 100 μg) and compared with (111)In-IgG in a human TGF-β-transfected Chinese hamster ovary xenograft model, human breast cancer MDA-MB-231 xenograft, and metastatic model. Latent and active TGF-β1 expression was analyzed in tissue homogenates with enzyme-linked immunosorbent assay., Results: (89)Zr was labeled to fresolimumab with high specific activity (>1 GBq/mg), high yield, and high purity. In vitro validation of (89)Zr-fresolimumab showed a fully preserved immunoreactivity and long (>1 wk) stability in solution and in human serum. In vivo validation showed an (89)Zr-fresolimumab distribution similar to IgG in most organs, except for a higher uptake in the liver in all mice and higher kidney uptake in the 10-μg group. (89)Zr-fresolimumab induced no toxicity in mice; it accumulated in primary tumors and metastases in a manner similar to IgG. Both latent and active TGF-β was detected in tumor homogenates, whereas only latent TGF-β could be detected in liver homogenates. Remarkably high (89)Zr-fresolimumab uptake was seen in sites of tumor ulceration and in scar tissue, processes in which TGF-β is known to be highly active., Conclusion: Fresolimumab tumor uptake and organ distribution can be visualized and quantified with (89)Zr-fresolimumab PET. This technique will be used to guide further clinical development of fresolimumab and could possibly identify patients most likely to benefit.

Unlabelled: Angiogenesis is a critical step in tumor development, in which vascular endothelial growth factor (VEGF) is a key growth aspect. Heat shock protein 90 (HSP90), a molecular chaperone, is essential for the activity of key proteins involved in VEGF transcription. Currently, no biomarkers to predict the effect of, or monitor, HSP90 inhibition therapy in individual patients exist. (89)Zr-bevacizumab PET provides a noninvasive tool to monitor tumor VEGF levels. The aim of this study was to investigate (89)Zr-bevacizumab PET for early antiangiogenic tumor response evaluation of treatment with the new HSP90 inhibitor NVP-AUY922. In xenografts of A2780 and its cisplatin-resistant CP70 human ovarian cancer subline, (89)Zr-bevacizumab small-animal PET was performed before and after NVP-AUY922 treatment and verified with histologic response and ex vivo tumor VEGF levels. Compared with pretreatment values, 2 wk of NVP-AUY922 treatment decreased (89)Zr-bevacizumab uptake by 44.4% (P = 0.0003) in A2780 xenografts, whereas tumor uptake was not affected in CP70 xenografts. The same pattern was observed in A2780 and CP70 tumor VEGF levels, measured with enzyme-linked immunosorbent assay, and mean vessel density after NVP-AUY922 treatment. These findings coincided with reduction in the proliferation rate, assessed by Ki67 staining, in A2780 tumor tissue only., Conclusion: (89)Zr-bevacizumab PET was in line with the antiangiogenic response and direct antitumor effects after NVP-AUY922 treatment, supporting the specificity of (89)Zr-bevacizumab PET as a sensitive technique to monitor the antiangiogenic response of HSP90 inhibition in vivo.

Unlabelled: The anti-human epidermal growth factor receptor 2 (HER2/neu) antibody trastuzumab is administered to patients with HER2/neu-overexpressing breast cancer. Whole-body noninvasive HER2/neu scintigraphy could help to assess and quantify the HER2/neu expression of all lesions, including nonaccessible metastases. The aims of this study were to develop clinical-grade radiolabeled trastuzumab for clinical HER2/neu immunoPET scintigraphy, to improve diagnostic imaging, to guide antibody-based therapy, and to support early antibody development. The PET radiopharmaceutical (89)Zr-trastuzumab was compared with the SPECT tracer (111)In-trastuzumab, which we have tested in the clinic already., Methods: Trastuzumab was labeled with (89)Zr and (for comparison) with (111)In. The minimal dose of trastuzumab required for optimal small-animal PET imaging and biodistribution was determined with human HER2/neu-positive or -negative tumor xenograft-bearing mice., Results: Trastuzumab was efficiently radiolabeled with (89)Zr at a high radiochemical purity and specific activity. The antigen-binding capacity was preserved, and the radiopharmaceutical proved to be stable for up to 7 d in solvent and human serum. Of the tested protein doses, the minimal dose of trastuzumab (100 microg) proved to be optimal for imaging. The comparative biodistribution study showed a higher level of (89)Zr-trastuzumab in HER2/neu-positive tumors than in HER2/neu-negative tumors, especially at day 6 (33.4 +/- 7.6 [mean +/- SEM] vs. 7.1 +/- 0.7 percentage injected dose per gram of tissue). There were good correlations between the small-animal PET images and the biodistribution data and between (89)Zr-trastuzumab and (111)In-trastuzumab uptake in tumors (R(2) = 0.972)., Conclusion: Clinical-grade (89)Zr-trastuzumab showed high and HER2/neu-specific tumor uptake at a good resolution.

Unlabelled: Vascular endothelial growth factor (VEGF), released by tumor cells, is an important growth factor in tumor angiogenesis. The humanized monoclonal antibody bevacizumab blocks VEGF-induced tumor angiogenesis by binding, thereby neutralizing VEGF. Our aim was to develop radiolabeled bevacizumab for noninvasive in vivo VEGF visualization and quantification with the single gamma-emitting isotope 111In and the PET isotope 89Zr., Methods: Labeling, stability, and binding studies were performed. Nude mice with a human SKOV-3 ovarian tumor xenograft were injected with 89Zr-bevacizumab, 111In-bevacizumab, or human 89Zr-IgG. Human 89Zr-IgG served as an aspecific control antibody. Small-animal PET and microCT studies were obtained at 24, 72, and 168 h after injection of 89Zr-bevacizumab and 89Zr-IgG (3.5 +/- 0.5 MBq, 100 +/- 6 microg, 0.2 mL [mean +/- SD]). Small-animal PET and microCT images were fused to calculate tumor uptake and compared with ex vivo biodistribution at 168 h after injection. 89Zr- and 111In-bevacizumab ex vivo biodistribution was compared at 24, 72, and 168 h after injection (2.0 +/- 0.5 MBq each, 100 +/- 4 microg in total, 0.2 mL)., Results: Labeling efficiencies, radiochemical purity, stability, and binding properties were optimal for the radioimmunoconjugates. Small-animal PET showed uptake in well-perfused organs at 24 h and clear tumor localization from 72 h onward. Tumor uptake determined by quantification of small-animal PET images was higher for 89Zr-bevacizumab-namely, 7.38 +/- 2.06 %ID/g compared with 3.39 +/- 1.16 %ID/g (percentage injected dose per gram) for human 89Zr-IgG (P = 0.011) at 168 h and equivalent to ex vivo biodistribution studies. Tracer uptake in other organs was seen primarily in liver and spleen. 89Zr- and 111In-bevacizumab biodistribution was comparable., Conclusion: Radiolabeled bevacizumab showed higher uptake compared with radiolabeled human IgG in a human SKOV-3 ovarian tumor xenograft. Noninvasive quantitative small-animal PET was similar to invasive ex vivo biodistribution. Radiolabeled bevacizumab is a new tracer for noninvasive in vivo imaging of VEGF in the tumor microenvironment.

Unlabelled: Serum amyloid P component (SAP) binds to amyloid. (123)I-SAP scintigraphy is used to evaluate the extent and distribution of amyloid in systemic amyloidosis and has great clinical value in the detection of systemic amyloidosis. The aim of the study was to assess during scintigraphy the diagnostic performance and prognostic value of a simple parameter describing extravascular (123)I-SAP retention in systemic amyloidosis., Methods: Two hundred megabecquerels of (123)I-labeled human SAP was injected intravenously for scintigraphy in 20 controls and in 189 consecutive patients with systemic and localized amyloidosis. Extravascular retention of (123)I-SAP was quantified from serum and urine measurements after 24 h (EVR(24)) and 48 h. Sensitivity and specificity were assessed, and retention was correlated with kidney, heart, liver, and nerve involvement and with survival., Results: The cutoff value representing a desired specificity of 90% of EVR(24) was 50%. The associated sensitivity of EVR(24) for detecting reactive systemic, immunocyte-derived (AL), and hereditary amyloidosis was 65%, 61%, and 22%, respectively, using a cutoff point of 50%. In AL amyloidosis, the EVR(24) increased with the number of organs involved (from a mean of 43% for 1 organ to a mean of 81% for 4 organs). The EVR(24) correlated with serum alkaline phosphatase (r = 0.63) and with creatinine clearance (r = -0.36). In AL amyloidosis, both cardiac involvement (hazard ratio, 3.9; 95% CI, 2.0-7.8) and EVR(24) (hazard ratio, 2.0; 95% CI, 1.1-3.9) were independent predictors of survival., Conclusion: In AL amyloidosis, the EVR(24) is strongly associated with organ involvement and with prognosis and might serve as an indicator of the body amyloid load. Quantification of SAP retention using the EVR(24) has no additional value over (123)I-SAP scintigraphy in the detection of systemic amyloidosis.