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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.)

Monoclonal antibodies are important cancer medicines. The European Medicines Agency (EMA) approved 48 and the Food and Drug Administration (FDA) 56 anticancer monoclonal antibody-based therapies. Their high prices burden healthcare systems and hamper global drug access. Biosimilars could retain costs and expand the availability of monoclonal antibodies. In Europe, five rituximab biosimilars, six trastuzumab biosimilars, and eight bevacizumab biosimilars are available as anti-cancer drugs. To gain insight into the biosimilar landscape for cancer treatment, we performed a literature search and analysis. In this review, we summarize cancer monoclonal antibodies' properties crucial for the desired pharmacology and point out sources of variability. The analytical assessment of all EMA-approved bevacizumab biosimilars is highlighted to illustrate this variability. The global landscape of investigational and approved biosimilars is mapped, and the challenges for access to cancer biosimilars are identified., Competing Interests: E.G.E.d.V. reports institutional financial support for advisory boards/consultancy from NSABP, Daiichi Sankyo, and Crescendo Biologics and institutional financial support for clinical trials or contracted research grants from Amgen, Genentech, Roche, Bayer, Servier, Regeneron, and Crescendo Biologics, all outside this work., (© 2024 The Authors.)

Immuno-positron emission tomography (immunoPET) enables imaging of specific targets that play a role in targeted therapy and immunotherapy, such as antigens on cell membranes, targets in the disease microenvironment, or immune cells. The most common immunoPET applications use a monoclonal antibody labeled with a relatively long-lived positron emitter such as 89 Zr ( T 1/2  = 78.4 h), but smaller antibody-based constructs labeled with various other positron emitting radionuclides are also being investigated. This molecular imaging technique can thus guide the development of new drugs and may have a pivotal role in selecting patients for a particular therapy. In early phase immunoPET trials, multiple imaging time points are used to examine the time-dependent biodistribution and to determine the optimal imaging time point, which may be several days after tracer injection due to the slow kinetics of larger molecules. Once this has been established, usually only one static scan is performed and semi-quantitative values are reported. However, total PET uptake of a tracer is the sum of specific and nonspecific uptake. In addition, uptake may be affected by other factors such as perfusion, pre-/co-administration of the unlabeled molecule, and the treatment schedule. This article reviews imaging methodologies used in immunoPET studies and is divided into two parts. The first part summarizes the vast majority of clinical immunoPET studies applying semi-quantitative methodologies. The second part focuses on a handful of studies applying pharmacokinetic models and includes preclinical and simulation studies. Finally, the potential and challenges of immunoPET quantification methodologies are discussed within the context of the recent technological advancements provided by long axial field of view PET/CT scanners., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Mohr, van Sluis, Lub-de Hooge, Lammertsma, Brouwers and Tsoumpas.)

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.)

Immune checkpoint inhibitors (ICIs), by reinvigorating CD8 + T cell mediated immunity, have revolutionized cancer therapy. Yet, the systemic CD8 + T cell distribution, a potential biomarker of ICI response, remains poorly characterized. We assessed safety, imaging dose and timing, pharmacokinetics and immunogenicity of zirconium-89-labeled, CD8-specific, one-armed antibody positron emission tomography tracer 89 ZED88082A in patients with solid tumors before and ~30 days after starting ICI therapy (NCT04029181). No tracer-related side effects occurred. Positron emission tomography imaging with 10 mg antibody revealed 89 ZED88082A uptake in normal lymphoid tissues, and tumor lesions across the body varying within and between patients two days after tracer injection (n = 38, median patient maximum standard uptake value (SUV max ) 5.2, IQI 4.0-7.4). Higher SUV max was associated with mismatch repair deficiency and longer overall survival. Uptake was higher in lesions with stromal/inflamed than desert immunophenotype. Tissue radioactivity was localized to areas with immunohistochemically confirmed CD8 expression. Re-imaging patients on treatment showed no change in average (geometric mean) tumor tracer uptake compared to baseline, but individual lesions showed diverse changes independent of tumor response. The imaging data suggest enormous heterogeneity in CD8 + T cell distribution and pharmacodynamics within and between patients. In conclusion, 89 ZED88082A can characterize the complex dynamics of CD8 + T cells in the context of ICIs, and may inform immunotherapeutic treatments., (© 2022. The Author(s).)

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.)

The advent of immune checkpoint inhibitors has reinvigorated the field of immuno-oncology. These monoclonal antibody-based therapies allow the immune system to recognize and eliminate malignant cells. This has resulted in improved survival of patients across several tumor types. However, not all patients respond to immunotherapy therefore predictive biomarkers are important. There are only a few Food and Drug Administration-approved biomarkers to select patients for immunotherapy. These biomarkers do not consider the heterogeneity of tumor characteristics across lesions within a patient. New molecular imaging tracers allow for whole-body visualization with positron emission tomography (PET) of tumor and immune cell characteristics, and drug distribution, which might guide treatment decision making. Here, we summarize recent developments in molecular imaging of immune checkpoint molecules, such as PD-L1, PD-1, CTLA-4, and LAG-3. We discuss several molecular imaging approaches of immune cell subsets and briefly summarize the role of FDG-PET for evaluating cancer immunotherapy. The main focus is on developments in clinical molecular imaging studies, next to preclinical studies of interest given their potential translation to the clinic., Competing Interests: Competing interests: EDV reports an advisory role at Daiichi Sankyo, NSABP, and Sanofi and research funding from Amgen, AstraZeneca, Bayer, Chugai Pharma, Crescendo, CytomX Therapeutics, G1 Therapeutics, Genentech, Nordic Nanovector, Radius Health, Regeneron, Roche, Servier, and Synthon (paid to UMCG). AvdW reports an advisory role at Janssen, Takeda, and Boehringer-Ingelheim (paid to UMCG) and research funding from AstraZeneca, Boehringer-Ingelheim, Pfizer, Roche, and Takeda. ML-dH reports research funding from Merck, Bayer, and Amgen (paid to UMCG). SO reports research funding from Novartis, Pfizer and Celldex Therapeutics (paid to UMCG) and an advisory role at Bristol Myers Squibb (paid to the UMCG)., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Due to ethical and practical reasons, a knowledge gap exists on the pharmacokinetics (PK) of inflammatory bowel disease (IBD)-related drugs in pregnant women with IBD. Before evidence-based dosing can be proposed, insight into the PK has to be gained to optimize drug therapy for both mother and fetus. This systematic review aimed to describe the effect of pregnancy and IBD on the PK of drugs used for IBD. One aminosalicylate study, two thiopurine studies and twelve studies with biologicals were included. Most drugs within these groups presented data over multiple moments before, during and after pregnancy, except for mesalazine, ustekinumab and golimumab. The studies for mesalazine, ustekinumab and golimumab did not provide enough data to demonstrate an effect of pregnancy on concentration and PK parameters. Therefore, no evidence-based dosing advice was given. The 6-thioguanine nucleotide levels decreased during pregnancy to 61% compared to pre-pregnancy levels. The potentially toxic metabolite 6-methylmercaptopurine (6-MMP) increased to maximal 209% of the pre-pregnancy levels. Although the PK of the thiopurines changed throughout pregnancy, no evidence-based dosing advice was provided. One study suggested that caution should be exercised when the thiopurine dose is adjusted, due to shunting 6-MMP levels. For the biologicals, infliximab levels increased, adalimumab stayed relatively stable and vedolizumab levels tended to decrease during pregnancy. Although the PK of the biologicals changed throughout pregnancy, no evidence-based dosing advice for biologicals was provided. Other drugs retrieved from the literature search were mesalazine, ustekinumab and golimumab. We conclude that limited studies have been performed on PK parameters during pregnancy for drugs used in IBD. Therefore, more extensive research to determine the values of PK parameters is warranted. After gathering the PK data, evidence-based dosing regimens can be developed.

Background: Programmed cell death protein 1 (PD-1) antibody treatment is standard of care for melanoma and non-small-cell lung cancer (NSCLC). Accurately predicting which patients will benefit is currently not possible. Tumor uptake and biodistribution of the PD-1 antibody might play a role. Therefore, we carried out a positron emission tomography (PET) imaging study with zirconium-89 ( 89 Zr)-labeled pembrolizumab before PD-1 antibody treatment., Patients and Methods: Patients with advanced or metastatic melanoma or NSCLC received 37 MBq (1 mCi) 89 Zr-pembrolizumab (∼2.5 mg antibody) intravenously plus 2.5 or 7.5 mg unlabeled pembrolizumab. After that, up to three PET scans were carried out on days 2, 4, and 7. Next, PD-1 antibody treatment was initiated. 89 Zr-pembrolizumab tumor uptake was calculated as maximum standardized uptake value (SUV max ) and expressed as geometric mean. Normal organ uptake was calculated as SUV mean and expressed as a mean. Tumor response was assessed according to (i)RECIST v1.1., Results: Eighteen patients, 11 with melanoma and 7 with NSCLC, were included. The optimal dose was 5 mg pembrolizumab, and the optimal time point for PET scanning was day 7. The tumor SUV max did not differ between melanoma and NSCLC (4.9 and 6.5, P = 0.49). Tumor 89 Zr-pembrolizumab uptake correlated with tumor response (P trend = 0.014) and progression-free (P = 0.0025) and overall survival (P = 0.026). 89 Zr-pembrolizumab uptake at 5 mg was highest in the spleen with a mean SUV mean of 5.8 (standard deviation ±1.8). There was also 89 Zr-pembrolizumab uptake in Waldeyer's ring, in normal lymph nodes, and at sites of inflammation., Conclusion: 89 Zr-pembrolizumab uptake in tumor lesions correlated with treatment response and patient survival. 89 Zr-pembrolizumab also showed uptake in lymphoid tissues and at sites of inflammation., Competing Interests: Disclosure MJ reports consultancy fees from AstraZeneca (paid to UMCG). JBH reports consultancy roles for Achilles Therapeutics, BioNTech, BMS, GSK, Immunocore, Instil Bio, Molecular Partners, MSD, Merck Serono, Neogene Therapeutics, Novartis, Pfizer, PokeAcel, Roche/Genentech, Sanofi, T-Knife, and Third Rock Ventures and research grants from Amgen, Asher-Bio, BMS, BioNTech, MSD, Novartis, and Neogene Therapeutics (paid to the Netherlands Cancer Institute). WT reports fees from Merck, Sharp, Dohme, and Bristol-Myers-Squibb (paid to UMCG). EGEdV reports an advisory role at Daiichi Sankyo, NSABP, and Sanofi and research funding from Amgen, AstraZeneca, Bayer, Chugai Pharma, Crescendo, CytomX Therapeutics, G1 Therapeutics, Genentech, Nordic Nanovector, Radius Health, Regeneron, Roche, Servier, and Synthon (paid to UMCG). GAPH reports consulting and advisory role at Amgen, Roche, MSD, BMS, Pfizer, Novartis, and Pierre Fabry and research funding from BMS and Seerave (paid to UMCG). TJNH reports consultancy fees (paid to UMCG) from BMS, MSD, Merck, Boehringer, AstraZeneca, and Roche. AJvdW reports an advisory role at Janssen, Takeda, and Boehringer-Ingelheim (paid to UMCG) and research funding from AstraZeneca, Boehringer-Ingelheim, Pfizer, Roche, and Takeda. MNL-dH reports research funding from Merck, Bayer, and Amgen (paid to UMCG). HJMG reports an advisory role at Eli Lilly, MSD, BMS, and Novartis. All other authors have declared no conflicts of interest., (Copyright © 2021. Published by Elsevier Ltd.)

Macrophages can promote tumor development. Preclinically, targeting macrophages by colony-stimulating factor 1 (CSF1)/CSF1 receptor (CSF1R) monoclonal antibodies (mAbs) enhances conventional therapeutics in combination treatments. The physiological distribution and tumor uptake of CSF1R mAbs are unknown. Therefore, we radiolabeled a murine CSF1R mAb and preclinically visualized its biodistribution by PET. CSF1R mAb was conjugated to N -succinyl-desferrioxamine ( N -suc-DFO) and subsequently radiolabeled with zirconium-89 ( 89 Zr). Optimal protein antibody dose was first determined in non-tumor-bearing mice to assess physiological distribution. Next, biodistribution of optimal protein dose and 89 Zr-labeled isotype control was compared with PET and ex vivo biodistribution after 24 and 72 h in mammary tumor-bearing mice. Tissue autoradiography and immunohistochemistry determined radioactivity distribution and tissue macrophage presence, respectively. [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb optimal protein dose was 10 mg/kg, with blood pool levels of 10 ± 2% injected dose per gram tissue (ID/g) and spleen and liver uptake of 17 ± 4 and 11 ± 4%ID/g at 72 h. In contrast, 0.4 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R mAb was eliminated from circulation within 24 h; spleen and liver uptake was 126 ± 44% and 34 ± 7%ID/g, respectively. Tumor-bearing mice showed higher uptake of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb in the liver, lymphoid tissues, duodenum, and ileum, but not in the tumor than did 89 Zr-labeled control at 72 h. Immunohistochemistry and autoradiography showed that 89 Zr was localized to macrophages within lymphoid tissues. Following [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb administration, tumor macrophages were almost absent, whereas isotype-group tumors contained over 500 cells/mm 2 . We hypothesize that intratumoral macrophage depletion by [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb precluded tumor uptake higher than 89 Zr-labeled control. Translation of molecular imaging of macrophage-targeting therapeutics to humans may support macrophage-directed therapeutic development., Competing Interests: EV reports institutional financial support for her advisory role from Daiichi Sankyo, Merck, NSABP, Pfizer, Sanofi, and Synthon and institutional financial support for clinical trials or contracted research from Amgen, AstraZeneca, Bayer, Chugai Pharma, CytomX Therapeutics, G1 Therapeutics, Genentech, Nordic Nanovector, Radius Health, Roche, Synthon, and Servier. CS reports receiving unrestricted research grants from Novartis, Roche, Genentech, Pfizer, SNS Oncology, and G1 Therapeutics that were made available to UMCG. KdV reports receiving research grants from Roche and is a consultant for Third Rock Ventures. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Waaijer, Suurs, Hau, Vrijland, de Visser, de Groot, de Vries, Lub-de Hooge and Schröder.)

Purpose: CX-072, a PD-L1-targeting Probody therapeutic, is engineered to be activated by tumor proteases that remove a masking peptide. To study effects on biodistribution and pharmacokinetics, we performed 89Zr-CX-072 positron emission tomography (PET) imaging., Experimental Design: Patients received ∼1 mg, 37 MBq 89Zr-CX-072 plus 0, 4, or 9 mg unlabeled CX-072 and PET scans at days 2, 4, and 7. After that, treatment comprised 10 mg/kg CX-072 q2 weeks (n = 7) + 3 mg/kg ipilimumab q3w 4× (n = 1). Normal organ tracer uptake was expressed as standardized uptake value (SUV)mean and tumor uptake as SUVmax. PD-L1 expression was measured immunohistochemically in archival tumor tissue., Results: Three of the eight patients included received 10-mg protein dose resulting in a blood pool mean SUVmean ± SD of 4.27 ± 0.45 on day 4, indicating sufficient available tracer. Tumor uptake was highest at day 7, with a geometric mean SUVmax 5.89 (n = 113) and present in all patients. The median follow-up was 12 weeks (4-76+). One patient experienced stable disease and two patients a partial response. PD-L1 tumor expression was 90% in one patient and ≤1% in the other patients. Mean SUVmean ± SD day 4 at 10 mg in the spleen was 8.56 ± 1.04, bone marrow 2.21 ± 0.46, and liver 4.97 ± 0.97. Four patients out of seven showed uptake in normal lymph nodes and Waldeyer's ring. The tracer was intact in the serum or plasma., Conclusions: 89Zr-CX-072 showed tumor uptake, even in lesions with ≤1% PD-L1 expression, and modest uptake in normal lymphoid organs, with no unexpected uptake in other healthy tissues., (©2021 American Association for Cancer Research.)

This review describes acetaminophen pharmacokinetics (PK) throughout pregnancy, as analyzed by three methods (non-compartmental analyses (NCA), population PK, and physiologically based PK (PBPK) modelling). Eighteen studies using NCA were reported in the scientific literature. These studies reported an increase in the volume of distribution (3.5-60.7%) and an increase in the clearance (36.8-84.4%) of acetaminophen in pregnant women compared to non-pregnant women. Only two studies using population PK modelling as a technique were available in the literature. The largest difference in acetaminophen clearance (203%) was observed in women at delivery compared to non-pregnant women. One study using the PBPK technique was found in the literature. This study focused on the formation of metabolites, and the toxic metabolite N-acetyl-p-benzoquinone imine was the highest in the first trimester, followed by the second and third trimester, compared with non-pregnant women. In conclusion, this review gave an overview on acetaminophen PK changes in pregnancy. Also, knowledge gaps, such as fetal and placenta PK parameters, have been identified, which should be explored further before dosing adjustments can be suggested on an evidence-based basis.

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.)

Background: To better predict response to immune checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal antibodies is essential. Therefore, we aimed to study in vivo pharmacokinetics and whole-body distribution of zirconium-89 ( 89 Zr) labeled programmed cell death protein-1 (PD-1) targeting pembrolizumab with positron-emission tomography (PET) in humanized mice., Methods: Humanized (huNOG) and non-humanized NOG mice were xenografted with human A375M melanoma cells. PET imaging was performed on day 7 post 89 Zr-pembrolizumab (10 µg, 2.5 MBq) administration, followed by ex vivo biodistribution studies. Other huNOG mice bearing A375M tumors received a co-injection of excess (90 µg) unlabeled pembrolizumab or 89 Zr-IgG 4 control (10 µg, 2.5 MBq). Tumor and spleen tissue were studied with autoradiography and immunohistochemically including PD-1., Results: PET imaging and biodistribution studies showed high 89 Zr-pembrolizumab uptake in tissues containing human immune cells, including spleen, lymph nodes and bone marrow. Tumor uptake of 89 Zr-pembrolizumab was lower than uptake in lymphoid tissues, but higher than uptake in other organs. High uptake in lymphoid tissues could be reduced by excess unlabeled pembrolizumab. Tracer activity in blood pool was increased by addition of unlabeled pembrolizumab, but tumor uptake was not affected. Autoradiography supported PET findings and immunohistochemical staining on spleen and lymph node tissue showed PD-1 positive cells, whereas tumor tissue was PD-1 negative., Conclusion: 89 Zr-pembrolizumab whole-body biodistribution showed high PD-1-mediated uptake in lymphoid tissues, such as spleen, lymph nodes and bone marrow, and modest tumor uptake. Our data may enable evaluation of 89 Zr-pembrolizumab whole-body distribution in patients., Competing Interests: Competing interests: EGEdV reports grants from IMI TRISTAN (GA no.116106), during the conduct of the study; consulting and advisory role for NSABP, Daiichi Sankyo, Pfizer, Sanofi, Merck, Synthon Biopharmaceuticals; grants from Amgen, Genentech, Roche, Chugai Pharma, CytomX Therapeutics, Nordic Nanovector, G1 Therapeutics, AstraZeneca, Radius Health, Bayer, all made available to the institution, outside the submitted work., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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.)

Purpose: Probody therapeutic CX-072 is a protease-activatable antibody that is cross-reactive with murine and human programmed death-ligand 1 (PD-L1). CX-072 can be activated in vivo by proteases present in the tumor microenvironment, thereby potentially reducing peripheral, anti-PD-L1-mediated toxicities. To study its targeting of PD-L1-expressing tissues, we radiolabeled CX-072 with the PET isotope zirconium-89 ( 89 Zr)., Experimental Design: 89 Zr-labeled CX-072, nonspecific Probody control molecule (PbCtrl) and CX-072 parental antibody (CX-075) were injected in BALB/c nude mice bearing human MDA-MB-231 tumors or C57BL/6J mice bearing syngeneic MC38 tumors. Mice underwent serial PET imaging 1, 3, and 6 days after intravenous injection (pi), followed by ex vivo biodistribution. Intratumoral 89 Zr-CX-072 distribution was studied by autoradiography on tumor tissue sections, which were subsequently stained for PD-L1 by IHC. Activated CX-072 species in tissue lysates were detected by Western capillary electrophoresis., Results: PET imaging revealed 89 Zr-CX-072 accumulation in MDA-MB-231 tumors with 2.1-fold higher tumor-to-blood ratios at 6 days pi compared with 89 Zr-PbCtrl. Tumor tissue autoradiography showed high 89 Zr-CX-072 uptake in high PD-L1-expressing regions. Activated CX-072 species were detected in these tumors, with 5.3-fold lower levels found in the spleen. Furthermore, 89 Zr-CX-072 uptake by lymphoid tissues of immune-competent mice bearing MC38 tumors was low compared with 89 Zr-CX-075, which lacks the Probody design., Conclusions: 89 Zr-CX-072 accumulates specifically in PD-L1-expressing tumors with limited uptake in murine peripheral lymphoid tissues. Our data may enable clinical evaluation of 89 Zr-CX-072 whole-body distribution as a tool to support CX-072 drug development (NCT03013491)., (©2020 American Association for Cancer Research.)

Background: Bispecific antibodies redirecting T cells to the tumor obtain increasing interest as potential cancer immunotherapy. ERY974, a full-length bispecific antibody targeting CD3ε on T cells and glypican 3 (GPC3) on tumors, has been in clinical development However, information on the influence of T cells on biodistribution of bispecific antibodies, like ERY974, is scarce. Here, we report the biodistribution and tumor targeting of zirconium-89 ( 89 Zr) labeled ERY974 in mouse models using immuno-positron emission tomography (PET) imaging., Methods: To study both the role of GPC3 and CD3 on the biodistribution of [ 89 Zr]Zr-N-suc-Df-ERY974, 89 Zr-labeled control antibodies targeting CD3 and non-mammalian protein keyhole limpet hemocyanin (KLH) or KLH only were used. GPC3 dependent tumor targeting of [ 89 Zr]Zr-N-suc-Df-ERY974 was tested in xenograft models with different levels of GPC3 expression. In addition, CD3 influence on biodistribution of [ 89 Zr]Zr-N-suc-Df-ERY974 was evaluated by comparing biodistribution between tumor-bearing immunodeficient mice and mice reconstituted with human immune cells using microPET imaging and ex vivo biodistribution. Ex vivo autoradiography was used to study deep tissue distribution., Results: In tumor-bearing immunodeficient mice, [ 89 Zr]Zr-N-suc-Df-ERY974 tumor uptake was GPC3 dependent and specific over [ 89 Zr]Zr-N-suc-Df-KLH/CD3 and [ 89 Zr]Zr-N-suc-Df-KLH/KLH. In mice engrafted with human immune cells, [ 89 Zr]Zr-N-suc-Df-ERY974 specific tumor uptake was higher than in immunodeficient mice. Ex vivo autoradiography demonstrated a preferential distribution of [ 89 Zr]Zr-N-suc-Df-ERY974 to T cell rich tumor tissue. Next to tumor, highest specific [ 89 Zr]Zr-N-suc-Df-ERY974 uptake was observed in spleen and lymph nodes., Conclusion: [89Zr] Zr-N-suc-Df-ERY974 can potentially be used to study ERY974 biodistribution in patients to support drug development., Competing Interests: Competing interests: TI, YS and NS are employees of Chugai Pharmaceutical and have ownership interest (including stocks and patents) in Chugai Pharmaceutical. A research grant to EDV was obtained from Chugai and made available to the institution., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Immune checkpoint inhibitors (ICIs) have substantially changed the field of oncology over the past few years. ICIs offer an alternative treatment strategy by exploiting the patients' immune system, resulting in a T cell mediated anti-tumor response. These therapies are effective in multiple different tumor types. Unfortunately, a substantial group of patients do not respond to ICIs. Molecular imaging, using single-photon emission computed tomography (SPECT) and positron emission tomography (PET), can provide non-invasive whole-body visualization of tumor and immune cell characteristics and might support patient selection or response evaluations for ICI therapies. In this review, recent studies with 18 F-fluorodeoxyglucose-PET imaging, imaging of immune checkpoints and imaging of immune cells will be discussed. These studies are until now mainly exploratory, but the first results suggest that molecular imaging biomarkers could have a role in the evaluation of ICI therapy., Competing Interests: Competing Interests: E.G.E. de Vries reports institutional financial support for her advisory role from Daiichi Sankyo, Merck, NSABP, Pfizer, Sanofi, Synthon and for clinical trials or contracted research from Amgen, AstraZeneca, Bayer, Chugai Pharma, CytomX Therapeutics, G1 Therapeutics, Genentech, Nordic Nanovector, Radius Health, Roche, Synthon. S.F. Oosting reports institutional financial support for clinical trials or contracted research from Celldex, Pfizer, Novartis, Bristol Myers Squibb, Kura Oncology, MedImmune, Roche, Merck Sharp & Dohme. A.J. van der Wekken reports institutional financial support for his advisory role from Pfizer, Boehringer-Ingelheim, Roche (diagnostics), Astra Zeneca and institutional financial support for clinical trials from AstraZeneca. All remaining authors have declared no conflicts of interest., (© The author(s).)

Background: Molecular imaging of immune cells might be a potential tool for response prediction, treatment evaluation and patient selection in inflammatory diseases as well as oncology. Targeting interleukin-2 (IL2) receptors on activated T-cells using positron emission tomography (PET) with N-(4-[ 18 F]fluorobenzoyl)-interleukin-2 ([ 18 F]FB-IL2) could be such a strategy. This paper describes the challenging translation of the partly manual labeling of [ 18 F]FB-IL2 for preclinical studies into an automated procedure following Good Manufacturing Practices (GMP), resulting in a radiopharmaceutical suitable for clinical use., Methods: The preclinical synthesis of [ 18 F]FB-IL2 was the starting point for translation to a clinical production method. To overcome several challenges, major adaptations in the production process were executed. The final analytical methods and production method were validated and documented. All data with regards to the quality and safety of the final drug product were documented in an investigational medicinal product dossier., Results: Restrictions in the [ 18 F]FB-IL2 production were imposed by hardware configuration of the automated synthesis equipment and by use of disposable cassettes. Critical steps in the [ 18 F]FB-IL2 production comprised the purification method, stability of recombinant human IL2 and the final formulation. With the GMP compliant production method, [ 18 F]FB-IL2 could reliably be produced with consistent quality complying to all specifications., Conclusions: To enable the use of [ 18 F]FB-IL2 in clinical studies, a fully automated GMP compliant production process was developed. [ 18 F]FB-IL2 is now produced consistently for use in clinical studies.

Purpose: Biodistribution of bispecific antibodies in patients is largely unknown. We therefore performed a feasibility study in 9 patients with advanced gastrointestinal adenocarcinomas to explore AMG 211 biodistribution (also known as MEDI-565), an approximately 55 kDa bispecific T-cell engager (BiTE®) directed against carcinoembryonic antigen (CEA) on tumor cells and cluster of differentiation 3 (CD3) on T-cells., Experimental Design: 89 Zr-labeled AMG 211 as tracer was administered alone or with cold AMG 211, for PET imaging before and/or during AMG 211 treatment., Results: Before AMG 211 treatment, the optimal imaging dose was 200-μg 89 Zr-AMG 211 + 1,800-μg cold AMG 211. At 3 hours, the highest blood pool standardized uptake value (SUV) mean was 4.0, and tracer serum half-life was 3.3 hours. CD3-mediated uptake was clearly observed in CD3-rich lymphoid tissues including spleen and bone marrow (SUV mean 3.2 and 1.8, respectively), and the SUV mean decreased more slowly than in other healthy tissues. 89 Zr-AMG 211 remained intact in plasma and was excreted predominantly via the kidneys in degraded forms. Of 43 visible tumor lesions, 37 were PET quantifiable, with a SUV max of 4.0 [interquartile range (IQR) 2.7-4.4] at 3 hours using the optimal imaging dose. The tracer uptake differed between tumor lesions 5-fold within and 9-fold between patients. During AMG 211 treatment, tracer was present in the blood pool, whereas tumor lesions were not visualized, possibly reflecting target saturation., Conclusions: This first-in-human study shows high, specific 89 Zr-AMG 211 accumulation in CD3-rich lymphoid tissues, as well as a clear, inter- and intraindividual heterogeneous tumor uptake., (©2019 American Association for Cancer Research.)

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.)

Programmed cell death protein-1/ligand-1 (PD-1/PD-L1) blockade is effective in a subset of patients with several tumor types, but predicting patient benefit using approved diagnostics is inexact, as some patients with PD-L1-negative tumors also show clinical benefit 1,2 . Moreover, all biopsy-based tests are subject to the errors and limitations of invasive tissue collection 3-11 . Preclinical studies of positron-emission tomography (PET) imaging with antibodies to PD-L1 suggested that this imaging method might be an approach to selecting patients 12,13 . Such a technique, however, requires substantial clinical development and validation. Here we present the initial results from a first-in-human study to assess the feasibility of imaging with zirconium-89-labeled atezolizumab (anti-PD-L1), including biodistribution, and secondly test its potential to predict response to PD-L1 blockade (ClinicalTrials.gov identifiers NCT02453984 and NCT02478099). We imaged 22 patients across three tumor types before the start of atezolizumab therapy. The PET signal, a function of tracer exposure and target expression, was high in lymphoid tissues and at sites of inflammation. In tumors, uptake was generally high but heterogeneous, varying within and among lesions, patients, and tumor types. Intriguingly, clinical responses in our patients were better correlated with pretreatment PET signal than with immunohistochemistry- or RNA-sequencing-based predictive biomarkers, encouraging further development of molecular PET imaging for assessment of PD-L1 status and clinical response prediction.

Purpose: AMG 211, a bispecific T-cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T-cell receptor. AMG 211 was labeled with zirconium-89 ( 89 Zr) or fluorescent dye to evaluate the tumor-targeting properties. Experimental Design: 89 Zr-AMG211 was administered to mice bearing CEA-positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA-negative HL-60 promyelocytic leukemia xenografts. Biodistribution studies with 2- to 10-μg 89 Zr-AMG211 supplemented with unlabeled AMG 211 up to 500-μg protein dose were performed. A BiTE that does not bind CEA, 89 Zr-Mec14, served as a negative control. 89 Zr-AMG211 integrity was determined in tumor lysates ex vivo Intratumoral distribution was studied with IRDye800CW-AMG211. Moreover, 89 Zr-AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199 Results: 89 Zr-AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with a 2-μg dose, and the lowest tumor uptake was observed with a 500-μg dose. After 24 hours, higher uptake of 10-μg 89 Zr-AMG211 occurred in CEA-positive xenografts, compared with CEA-negative xenografts. Although the blood half-life of 89 Zr-AMG211 was approximately 1 hour, tumor retention persisted for at least 24 hours. 89 Zr-Mec14 showed no tumor accumulation beyond background level. Ex vivo autoradiography revealed time-dependent disintegration of 89 Zr-AMG211. 800CW-AMG211 was specifically localized in CEA-expressing viable tumor tissue. GMP-manufactured 89 Zr-AMG211 fulfilled release specifications. Conclusions: 89 Zr-AMG211 showed dose-dependent CEA-specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 in vivo behavior. Clin Cancer Res; 24(20); 4988-96. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Rationale: Knowledge on monoclonal antibody biodistribution in healthy tissues in humans can support clinical drug development. Molecular imaging with positron emission tomography (PET) can yield information in this setting. However, recent imaging studies have analyzed the behavior of single antibodies only, neglecting comparison across different antibodies. Methods: We compared the distribution of four 89 Zr-labeled antibodies in healthy tissue in a retrospective analysis based on the recently published harmonization protocol for 89 Zr-tracers and our delineation protocol. Results: The biodistribution patterns of 89 Zr-lumretuzumab, 89 Zr-MMOT0530A, 89 Zr-bevacizumab and 89 Zr-trastuzumab on day 4 after tracer injection were largely similar. The highest tracer concentration was seen in healthy liver, spleen, kidney and intestines. About one-third of the injected tracer dose was found in the circulation, up to 15% in the liver and only 4% in the spleen and kidney. Lower tracer concentration was seen in bone marrow, lung, compact bone, muscle, fat and the brain. Despite low tracer accumulation per gram of tissue, large-volume tissues, especially fat, can influence overall distribution: On average, 5-7% of the injected tracer dose accumulated in fat, with a peak of 19% in a patient with morbid obesity. Conclusion: The similar biodistribution of the four antibodies is probably based on their similar molecular structure, binding characteristics and similar metabolic pathways. These data provide a basis for a prospectively growing, online accessible warehouse of molecular imaging data, which enables researchers to increase and exchange knowledge on whole body drug distribution and potentially supports drug development decisions., Competing Interests: Competing Interests: The UMCG received grants form Roche and Genentech for conduct of the imaging trials. All other authors declare no competing interests.

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.)

Treatment of human epidermal growth factor receptor 2 (HER2)-driven breast cancer with tyrosine kinase inhibitor lapatinib can induce a compensatory HER3 increase, which may attenuate antitumor efficacy. Therefore, we explored in vivo HER3 tumor status assessment after lapatinib treatment with zirconium-89 ( 89 Zr)-labeled anti-HER3 antibody mAb3481 positron emission tomography (PET). Lapatinib effects on HER3 cell surface expression and mAb3481 internalization were evaluated in human breast (BT474, SKBR3) and gastric (N87) cancer cell lines using flow cytometry. Next, in vivo effects of daily lapatinib treatment on 89 Zr-mAb3481 BT474 and N87 xenograft tumor uptake were studied. PET-scans (BT474 only) were made after daily lapatinib treatment for 9 days, starting 3 days prior to 89 Zr-mAb3481 administration. Subsequently, ex vivo 89 Zr-mAb3481 organ distribution analysis was performed and HER3 tumor levels were measured with Western blot and immunohistochemistry. In vitro, lapatinib increased membranous HER3 in BT474, SKBR3 and N87 cells, and consequently mAb3481 internalization 1.7-fold (BT474), 1.4-fold (SKBR3) and 1.4-fold (N87). 89 Zr-mAb3481 BT474 tumor uptake was remarkably high at SUV mean 5.6±0.6 (51.8±7.7%ID/g) using a 10 μg 89 Zr-mAb3481 protein dose in vehicle-treated mice. However, compared to vehicle, lapatinib did not affect 89 Zr-mAb3481 ex vivo uptake in BT474 and N87 tumors, while HER3 tumor expression remained unchanged. In conclusion, lapatinib increased in vitro HER3 tumor cell expression, but not when these cells were xenografted. 89 Zr-mAb3481 PET accurately reflected HER3 tumor status. 89 Zr-mAb3481 PET showed high, HER3-specific tumor uptake, and such an approach might sensitively assess HER3 tumor heterogeneity and treatment response in patients.

Purpose: We evaluated biodistribution and tumor targeting of 89 Zr-lumretuzumab before and during treatment with lumretuzumab, a human epidermal growth factor receptor 3 (HER3)-targeting monoclonal antibody. Experimental Design: Twenty patients with histologically confirmed HER3-expressing tumors received 89 Zr-lumretuzumab and underwent positron emission tomography (PET). In part A, 89 Zr-lumretuzumab was given with additional, escalating doses of unlabeled lumretuzumab, and scans were performed 2, 4, and 7 days after injection to determine optimal imaging conditions. In part B, patients were scanned following tracer injection before (baseline) and after a pharmacodynamic (PD)-active lumretuzumab dose for saturation analysis. HER3 expression was determined immunohistochemically in skin biopsies. Tracer uptake was calculated as standardized uptake value (SUV). Results: Optimal PET conditions were found to be 4 and 7 days after administration of 89 Zr-lumretuzumab with 100-mg unlabeled lumretuzumab. At baseline using 100-mg unlabeled lumretuzumab, the tumor SUVmax was 3.4 (±1.9) at 4 days after injection. SUVmean values for normal blood, liver, lung, and brain tissues were 4.9, 6.4, 0.9 and 0.2, respectively. Saturation analysis ( n = 7) showed that 4 days after lumretuzumab administration, tumor uptake decreased by 11.9% (±8.2), 10.0% (±16.5), and 24.6% (±20.9) at PD-active doses of 400, 800, and 1,600 mg, respectively, when compared with baseline. Membranous HER3 was completely downregulated in paired skin biopsies already at and above 400-mg lumretuzumab. Conclusions: PET imaging showed biodistribution and tumor-specific 89 Zr-lumretuzumab uptake. Although, PD-active lumretuzumab doses decreased 89 Zr-lumretuzumab uptake, there was no clear evidence of tumor saturation by PET imaging as the tumor SUV did not plateau with increasing doses. Clin Cancer Res; 23(20); 6128-37. ©2017 AACR ., (©2017 American Association for Cancer Research.)

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.)

Purpose: To provide proof of principle of safety, breast tumor-specific uptake, and positive tumor margin assessment of the systemically administered near-infrared fluorescent tracer bevacizumab-IRDye800CW targeting VEGF-A in patients with breast cancer. Experimental Design: Twenty patients with primary invasive breast cancer eligible for primary surgery received 4.5 mg bevacizumab-IRDye800CW as intravenous bolus injection. Safety aspects were assessed as well as tracer uptake and tumor delineation during surgery and ex vivo in surgical specimens using an optical imaging system. Ex vivo multiplexed histopathology analyses were performed for evaluation of biodistribution of tracer uptake and coregistration of tumor tissue and healthy tissue. Results: None of the patients experienced adverse events. Tracer levels in primary tumor tissue were higher compared with those in the tumor margin ( P < 0.05) and healthy tissue ( P < 0.0001). VEGF-A tumor levels also correlated with tracer levels ( r = 0.63, P < 0.0002). All but one tumor showed specific tracer uptake. Two of 20 surgically excised lumps contained microscopic positive margins detected ex vivo by fluorescent macro- and microscopy and confirmed at the cellular level. Conclusions: Our study shows that systemic administration of the bevacizumab-IRDye800CW tracer is safe for breast cancer guidance and confirms tumor and tumor margin uptake as evaluated by a systematic validation methodology. The findings are a step toward a phase II dose-finding study aimed at in vivo margin assessment and point to a novel drug assessment tool that provides a detailed picture of drug distribution in the tumor tissue. Clin Cancer Res; 23(11); 2730-41. ©2016 AACR ., (©2016 American Association for Cancer Research.)

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.)

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and nonmalignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here, we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multiscale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels. Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labeled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging. Cancer Res; 77(3); 623-31. ©2016 AACR., (©2016 American Association for Cancer Research.)

Preclinical positron emission tomography (PET) imaging revealed a mismatch between in vivo epidermal growth factor receptor (EGFR) expression and EGFR antibody tracer tumor uptake. Shed EGFR ectodomain (sEGFR), which is present in cancer patient sera, can potentially bind tracer and therefore influence tracer kinetics. To optimize EGFR-PET, we examined the influence of sEGFR levels on tracer kinetics and tumor uptake of EGFR monoclonal antibody 89Zr-imgatuzumab in varying xenograft models. Human cancer cell lines A431 (EGFR overexpressing, epidermoid), A549 and H441 (both EGFR medium expressing, non-small cell lung cancer) were xenografted in mice. Xenografted mice received 10, 25 or 160 μg 89Zr-imgatuzumab, co-injected with equal doses 111In-IgG control. MicroPET scans were made 24, 72 and 144 h post injection, followed by biodistribution analysis. sEGFR levels in liver and plasma samples were determined by ELISA. 89Zr-imgatuzumab uptake in A431 tumors was highest (29.8 ± 5.4 %ID/g) in the 160 μg dose group. Contrary, highest uptake in A549 and H441 tumors was found at the lowest (10 μg) 89Zr-imgatuzumab dose. High 89Zr-imgatuzumab liver accumulation was found in A431 xenografted mice, which decreased with antibody dose increments. 89Zr-imgatuzumab liver uptake in A549 and H441 xenografted mice was low at all doses. sEGFR levels in liver and plasma of A431 bearing mice were up to 1000-fold higher than levels found in A549, H441 and non-tumor xenografted mice. 89Zr-imgatuzumab effectively visualizes EGFR-expressing tumors. High sEGFR levels can redirect 89Zr-imgatuzumab to the liver, in which case tumor visualization can be improved by increasing tracer antibody dose.

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.)

Purpose: Mesothelin (MSLN) is frequently overexpressed in pancreatic and ovarian cancers, making it a potential drug target. We performed an (89)Zr-PET imaging study with MMOT0530A, a MSLN antibody, in conjunction with a phase I study with the antibody-drug conjugate DMOT4039A, containing MMOT0530A bound to MMAE. The aim was to study antibody tumor uptake, whole-body distribution, and relation between uptake, response to treatment, and MSLN expression., Experimental Design: Before DMOT4039A treatment, patients received 37 MBq (89)Zr-MMOT0530A followed by PET/CT imaging 2, 4, and 7 days postinjection. Tracer uptake was expressed as standardized uptake value (SUV). MSLN expression was determined with immunohistochemistry (IHC) on archival tumor tissue., Results: Eleven patients were included, 7 with pancreatic and 4 with ovarian cancer. IHC MSLN expression varied from absent to strong. Suitable tracer antibody dose was 10 mg MMOT0530A and optimal imaging time was 4 and 7 days postinjection. Tumor tracer uptake occurred in 37 lesions with mean SUVmax of 13.1 (±7.5) on PET 4 days postinjection, with 11.5 (±7.5) in (N= 17) pancreatic and 14.5 (±8.7) in (N= 20) ovarian cancer lesions. Within patients, a mean 2.4-fold (±1.10) difference in uptake between tumor lesions existed. Uptake in blood, liver, kidneys, spleen, and intestine reflected normal antibody distribution. Tracer tumor uptake was correlated to IHC. Best response to DMOT4039A was partial response in one patient., Conclusions: With (89)Zr-MMOT0530A-PET, pancreatic and ovarian cancer lesions as well as antibody biodistribution could be visualized. This technique can potentially guide individualized antibody-based treatment., (©2015 American Association for Cancer Research.)

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.)

Mesothelin is a tumor differentiation antigen expressed by epithelial tumors, including pancreatic cancer. Currently, mesothelin is being targeted with an antibody-drug conjugate (ADC) consisting of a mesothelin-specific antibody coupled to a highly potent chemotherapeutic drug. Considering the toxicity of the ADC and reduced accessibility of pancreatic tumors, non-invasive imaging could provide necessary information. We therefore developed a zirconium-89 (89Zr) labeled anti-mesothelin antibody (89Zr-AMA) to study its biodistribution in human pancreatic tumor bearing mice. Biodistribution and dose-finding of 89Zr-AMA were studied 144 h after tracer injection in mice with subcutaneously xenografted HPAC. MicroPET imaging was performed 24, 72 and 144 h after tracer injection in mice bearing HPAC or Capan-2. Tumor uptake and organ distribution of 89Zr-AMA were compared with nonspecific 111In-IgG. Biodistribution analyses revealed a dose-dependent 89Zr-AMA tumor uptake. Tumor uptake of 89Zr-AMA was higher than 111In-IgG using the lowest tracer dose. MicroPET showed increased tumor uptake over 6 days, whereas activity in blood pool and other tissues decreased. Immunohistochemistry showed that mesothelin was expressed by the HPAC and CAPAN-2 tumors and fluorescence microscopy revealed that AMA-800CW was present in tumor cell cytoplasm. 89Zr-AMA tumor uptake is antigen-specific in mesothelin-expressing tumors. 89Zr-AMA PET provides non-invasive, real-time information about AMA distribution and tumor targeting.

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.)

Fluconazole is a first-line antifungal agent for the treatment and prophylaxis of invasive candidiasis in pediatric patients. Pediatric patients are at risk of suboptimal drug exposure, due to developmental changes in gastrointestinal and renal function, metabolic capacity, and volume of distribution. Therapeutic drug monitoring (TDM) can therefore be useful to prevent underexposure of fluconazole in children and infants. Children, however, often fear needles and can have difficult vascular access. The purpose of this study was to develop and clinically validate a method of analysis to determine fluconazole in oral fluid in pediatric patients. Twenty-one paired serum and oral fluid samples were obtained from 19 patients and were analyzed using a validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) method after cross-validation between serum and oral fluid. The results were within accepted ranges for accuracy and precision, and samples were stable at room temperature for at least 17 days. A Pearson correlation test for the fluconazole concentrations in serum and oral fluid showed a correlation coefficient of 0.960 (P < 0.01). The mean oral fluid-to-serum concentration ratio was 0.99 (95% confidence interval [CI], 0.88 to 1.10) with Bland-Altman analysis. In conclusion, an oral fluid method of analysis was successfully developed and clinically validated for fluconazole in pediatric patients and can be a noninvasive, painless alternative to perform TDM of fluconazole when blood sampling is not possible or desirable. When patients receive prolonged courses of antifungal treatment and use fluconazole at home, this method of analysis can extend the possibilities of TDM for patients at home., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Purpose: HSP90 chaperones have key client proteins that are involved in all hallmarks of breast cancer growth and progression. The primary aim of this clinical trial was to evaluate the feasibility of using (89)Zr-trastuzumab PET (for HER2-positive breast cancer) or (89)Zr-bevacizumab PET [for estrogen receptor (ER)-positive breast cancer] to determine in vivo degradation of client proteins caused by the novel HSP90 inhibitor NVP-AUY922., Experimental Design: Of note, 70 mg/m(2) NVP-AUY922 was administered intravenously in a weekly schedule to patients with advanced HER2 or ER-positive breast cancer. Biomarker analysis consisted of serial PET imaging with 2[18F]fluoro-2-deoxy-D-glucose (FDG), (89)Zr-trastuzumab, or (89)Zr-bevacizumab. Response evaluation was performed according to RECIST1.0. FDG, (89)Zr-trastuzumab, and (89)Zr-bevacizumab distributions were scored visually and quantitatively by calculating the maximum standardized uptake values (SUVmax). In blood samples, serial HSP70 levels, extracellular form of HER2 (HER2-ECD), and pharmacokinetic and pharmacodynamic parameters were measured., Results: Sixteen patients (ten HER2-positive and six ER-positive tumors) were included. One partial response was observed; seven patients showed stable disease. SUVmax change in individual tumor lesions on baseline versus 3 weeks (89)Zr-trastuzumab PET was heterogeneous and related to size change on CT after 8 weeks treatment (r(2) = 0.69; P = 0.006). Tumor response on (89)Zr-bevacizumab PET and FDG-PET was not correlated with CT response., Conclusions: NVP-AUY922 showed proof-of-concept clinical response in HER2-amplified metastatic breast cancer. Early change on (89)Zr-trastuzumab PET was positively associated with change in size of individual lesions assessed by CT., (©2014 American Association for Cancer Research.)

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.)

The humanized monoclonal antibody with high affinity for the human epidermal growth factor receptor (HER) 3, RG7116, is a glycoengineered, IgG1 class antibody. By labeling RG7116 with zirconium-89 ((89)Zr) we aimed to visualize in vivo HER3 expression and study the biodistribution of this antibody in human tumor-bearing mice. Biodistribution of (89)Zr-RG7116 was studied in subcutaneously xenografted FaDu tumor cells (HER3-positive). Dose-dependency of (89)Zr-RG7116 organ distribution and specific tumor uptake was assessed by administering doses ranging from 0.05 to 10 mg/kg RG7116 to SCID/Beige mice. Biodistribution was analyzed at 24 and 144 h after injection. MicroPET imaging was performed at 1, 3, and 6 days after injection of 1.0 mg/kg (89)Zr-RG7116 in the FaDu, H441, QG-56 and Calu-1 xenografts with varying HER3 expression. The excised tumors were analyzed for HER3 expression. Biodistribution analyses showed a dose- and time-dependent (89)Zr-RG7116 tumor uptake in FaDu tumors. The highest tumor uptake of (89)Zr-RG7116 was observed in the 0.05 mg/kg dose group with 27.5%ID/g at 144 h after tracer injection. MicroPET imaging revealed specific tumor uptake of (89)Zr-RG7116 in FaDu and H441 models with an increase in tumor uptake over time. Biodistribution data was consistent with the microPET findings in FaDu, H441, QG56 and Calu-1 xenografts, which correlated with HER3 expression levels. In conclusion, (89)Zr-RG7116 specifically accumulates in HER3 expressing tumors. PET imaging with this tracer provides real-time non-invasive information about RG7116 distribution, tumor targeting and tumor HER3 expression levels.

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.