M. N. Lub-de Hooge

Molecular imaging of breast cancer.

Molecular imaging of breast cancer can potentially be used for breast cancer screening, staging, restaging, response evaluation and guiding therapies. Techniques for molecular breast cancer imaging include magnetic resonance imaging (MRI), optical imaging, and radionuclide imaging with positron emission tomography (PET) or single photon emission computed tomography (SPECT). This review focuses on PET and SPECT imaging which can provide sensitive serial non invasive information of tumor characteristics. Most clinical data are gathered on the visualization of general processes such as glucose metabolism with the PET-tracer [(18)F]fluorodeoxyglucose (FDG) and DNA synthesis with [18F]fluoro-L-thymidine (FLT). Increasingly more breast cancer specific targets are imaged such as the estrogen receptor (ER), growth factors and growth factor receptors. Imaging of the ER with the PET tracer 16-alpha-[(18)F]fluoro-17-beta-estradiol (FES) has shown a good correlation between FES tumor uptake and ER density. (111)In-trastuzumab SPECT to image the human epidermal growth factor receptor 2 (HER2) showed that in most patients with metastatic HER2 overexpressing disease more lesions were detected than with conventional staging procedures. The PET tracer (89)Zr-trastuzumab showed excellent, quantifiable, and specific tumor uptake. (111)In-bevacizumab for SPECT and (89)Zr-bevacizumab for PET-imaging have been developed for vascular endothelial growth factor (VEGF) imaging as an angiogenic marker. Lastly, tracers for the receptors EGFR, IGF-1R, PDGF-betaR and the ligand TGFbeta are under development. Although molecular imaging of breast cancer is still not commonly used in daily clinical practice, its application portfolio is expanding rapidly.

Bevacizumab-Induced Normalization of Blood Vessels in Tumors Hampers Antibody Uptake

In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of (89)Zr-trastuzumab (SKOV-3 and OE19), (89)Zr-bevacizumab (SKOV-3), or (89)Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment.

Molecular imaging: what can be used today

Biochemical cellular targets and more general metabolic processes in cancer cells can be visualised. Extensive data are available on molecular imaging in preclinical models. However, innovative tracers move slowly to the clinic. This review provides information on the currently available methods of metabolic imaging, especially using PET in humans. The uptake mechanisms of tracer methods and a brief discussion of the more ‘molecular’ targeted methods are presented. The main focus is on the different classes of tracers and their application in various types of cancer within each class of tracers, based on the current literature and our own experience. Studies with [18F]FDG (energy metabolism), radiolabelled amino acids (protein metabolism), [18F]FLT (DNA metabolism), [11C]choline (cell membrane metabolism) as general metabolic tracer methods and [18F]DOPA (biogenic amine metabolism) as a more specific tracer method are discussed. As an example, molecular imaging methods that target the HER2 receptor and somatostatin receptor are described.

Targeting breast cancer through its microenvironment: current status of preclinical and clinical research in finding relevant targets.

It is increasingly evident that not only breast cancer cells, but also the tissue embedding these cells: the tumor microenvironment, plays an important role in tumor progression, metastasis formation and treatment sensitivity. This review focuses on the current knowledge of processes by which the microenvironment affects breast cancer, including formation of the metastatic niche, metabolic stimulation, stimulation of tumor cell migration, immune modulation, angiogenesis and matrix remodeling. The number of drugs targeting key factors in these processes is expanding, and the available clinical data is increasing. Therefore current strategies for intervention and prediction of treatment response are outlined. At present, targeting the formation of the metastatic niche and metabolic stimulation by the breast cancer microenvironment, are already showing clinical efficacy. Intervening in the stimulation of tumor cell migration and immune modulation by the microenvironment upcoming fields of great research interest. In contrast, targeting microenvironmental angiogenesis or matrix remodeling appears to be of limited clinical relevance in breast cancer treatment so far. Further research is warranted to optimize intervention strategies and develop predictive tests for the relevance of targeting involved factors within the microenvironment in order to optimally personalize breast cancer treatment.

Development of a radioiodinated apoptosis-inducing ligand, rhTRAIL, and a radiolabelled agonist TRAIL receptor antibody for clinical imaging studies.

BACKGROUND AND PURPOSE The TNF‐related apoptosis inducing ligand (TRAIL) induces apoptosis through activation of the death receptors, TRAIL‐R1 and TRAIL‐R2. Recombinant human (rh) TRAIL and the TRAIL‐R1 directed monoclonal antibody mapatumumab are currently clinically evaluated as anticancer agents. The objective of this study was to develop radiopharmaceuticals targeting the TRAIL‐R1, suitable for clinical use to help understand and predict clinical efficacy in patients.

Associate editor: B. TeicherTargeting breast cancer through its microenvironment: Current status of preclinical and clinical research in finding relevant targets

It is increasingly evident that not only breast cancer cells, but also the tissue embedding these cells: the tumor microenvironment, plays an important role in tumor progression, metastasis formation and treatment sensitivity. This review focuses on the current knowledge of processes by which the microenvironment affects breast cancer, including formation of the metastatic niche, metabolic stimulation, stimulation of tumor cell migration, immune modulation, angiogenesis and matrix remodeling. The number of drugs targeting key factors in these processes is expanding, and the available clinical data is increasing. Therefore current strategies for intervention and prediction of treatment response are outlined. At present, targeting the formation of the metastatic niche and metabolic stimulation by the breast cancer microenvironment, are already showing clinical efficacy. Intervening in the stimulation of tumor cell migration and immune modulation by the microenvironment upcoming fields of great research interest. In contrast, targeting microenvironmental angiogenesis or matrix remodeling appears to be of limited clinical relevance in breast cancer treatment so far. Further research is warranted to optimize intervention strategies and develop predictive tests for the relevance of targeting involved factors within the microenvironment in order to optimally personalize breast cancer treatment.

Biodistribution and pharmacokinetics of radiolabeled trastuzumab in HER2-positive metastatic breast cancer

3063 Background: Trastuzumab combined with chemotherapy can induce remissions in HER2 positive metastatic breast cancer (MBC), but also cardiac dysfunction. The aim of this study is to evaluate whether scintigraphy using radiolabeled trastuzumab can predict cardiotoxicity, and identify tumor lesions and response. METHODS In an ongoing study, patients (pts) with HER2 positive MBC, suitable for Herceptin (d1, 8 and 15) plus paclitaxel (d1), underwent gammacamera imaging and blood sampling from 15 min to 7d after injection of 150 MBq 111In-DTPA-trastuzumab, prior to and after 4 therapy cycles. In known lesions (CT/ultrasound/Xray/bone scan), the degree of 111In-DTPA-trastuzumab tumor uptake was expressed as tumor/ background ratio (tumor vs. background uptake intensity) and as % injected dose index (tumor vs. total body (TB) counts). RESULTS Up to now, 11 pts have been enrolled. Plasma T½α was 14.7±6.97h, T½β 115.9±46.71h (n=8). Variable uptake in tumor lesions was present in all pts after 5 and 7d, starting from d1. At d1, tumor/background ratio was 2.0 (1.4-3.5), 0.7% TB (0.1-5.5%) uptake. At d7 these figures were 2.1 (1.7-9.3) and 0.9%TB (0.2-5.5%). One or multiple lesions were visualized in every pt, although not all known lesions. In 5 pts previously unknown lesions were found. Visualization of liver metastases was difficult (missed in 2/ 6 pts), due to high liver uptake (d5 24.3%, 17.4-33.6%). Uptake in organs such as, spleen and kidneys, and bloodpool activity decreased between d1 and 7, while tumor and liver uptake increased. Myocardial uptake occurred in 1 pt, who had cardiac arrhythmias prior to treatment. CONCLUSIONS Preliminary results show that radiolabeled trastuzumab scintigraphy can identify HER2 positive tumor lesions, which may assist in identifying metastases and in predicting tumor response. Defining predictive value for cardiotoxicity and tumor response requires a larger pt number. This study continues to recruit. [Table: see text].

Abstract A85: Preclinical evaluation of the radiolabeled bispecific T-cell engager 89Zr-AMG 211 targeting CEA-positive tumors

Background: AMG 211 is a novel, bispecific single-chain antibody of the bispecific T-cell engager (BiTE® antibody construct) class. AMG 211 targets carcinoembryonic antigen (CEA) on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex on T-cells. CEA is a glycosylated human oncofetal antigen and is abundantly expressed by a variety of tumors; especially those in the gastrointestinal tract. We radiolabeled AMG 211 with zirconium-89 (89Zr) to study the tumor targeting and tissue distribution of AMG 211 in preclinical mouse tumor xenograft models. Material and Methods: AMG 211 was conjugated with desferal for 89Zr labeling and quality control was performed. A tracer amount of 10 μg (5 MBq) 89Zr-AMG 211 was administered to mice bearing subcutaneously-implanted CEA-expressing LS174T human colorectal adenocarcinoma. MicroPET imaging was performed at 0.5, 3, 6, 24, 48 and 72 h after tracer injection. A dose-escalation biodistribution study was performed 6 h after injection of 2, 10, 50, 100 or 500 μg 89Zr-AMG 211. 89Zr-AMG 211 was also evaluated in a CEA-negative HL60 promyelocytic leukemia xenograft. Furthermore, the non-CEA-binding BiTE® antibody construct, MEC 14, served as a negative control in LS174T tumors. Ex vivo gel electrophoresis and autoradiography was used to determine 89Zr-AMG 211 integrity in plasma and tumor lysate. Results: In vitro analysis of desferal-conjugated AMG 211 showed similar CEA binding as unlabeled AMG 211. Radiochemical purity of 89Zr-AMG 211 exceeded 95%, with a maximum specific activity of 500 MBq/mg. Size-exclusion high-performance liquid chromatography showed no degradation products and less than 5% of aggregates. MicroPET imaging revealed time-dependent tumor uptake of 89Zr-AMG 211 in LS174T bearing mice with the highest uptake 3 h after injection and a prolonged imageable tumor retention up to 48 h. 89Zr-AMG 211 rapidly decreased in blood (T1/2 = 1 h). Dose-escalation showed a dose-dependent tumor uptake 6 hours after injection with the highest tumor uptake observed with 2 μg (7.5 ± 1.5%ID/g) and the lowest tumor uptake with 500 μg (3.9 ± 0.1%ID/g). Mice bearing CEA-positive LS174T versus CEA-negative HL60 tumors showed higher tumor uptake (6.0 ± 1.3 vs 0.5 ± 0.1%ID/g) and tumor-to-blood ratios (21.0 ± 4.0 vs 1.6 ± 0.2) after injection of 10 μg 89Zr-AMG 211. In addition, the non-specific 89Zr-MEC 14 showed low tumor (0.5 ± 0.2%ID/g) uptake and tumor-to-blood ratio (5.5 ± 2.1) that is similar to tissue background in the LS174T model. Ex vivo autoradiography showed intact 89Zr-AMG 211 in all plasma samples. Moreover, in line with biodistribution data, intact 89Zr-AMG 211 was present only in the LS174T xenograft lysates and not detected in HL60 lysate. 89Zr-MEC14 did not bind CEA and was not detected in LS174T lysates. Conclusions: This study showed dose-dependent CEA-specific targeting of 89Zr-AMG 211. In addition, intact labeled AMG 211 was present in plasma and LS174T tumor lysates indicating in vivo tracer integrity. Our data support the use of 89Zr-ImmunoPET in the clinical setting to assess the distribution and tumor-targeting properties of AMG 211 and BiTE® antibody constructs. Citation Format: S.J.H. Waaijer, F.J. Warnders, M.N. Lub-de Hooge, S. Stienen, M. Friedrich, A. Sternjak, P.C. Pieslor, K. Cheung, A.G.T. Terwisscha van Scheltinga, C.P. Schroder, E.G.E. de Vries. Preclinical evaluation of the radiolabeled bispecific T-cell engager 89Zr-AMG 211 targeting CEA-positive tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A85.

Clinical value of Zr-89-trastuzumab PET in HER2-positive breast cancer patients with a clinical dilemma

Background: Information on human epidermal growth factor receptor 2 (HER2) is essential for management of metastatic breast cancer (mBC). In patients suspected of HER2-positive mBC, standard work up may fail to clarify whole body HER2 status. We aimed to assess whether 89Zr-trastuzumab PET can support treatment decisions in patients posing this clinical dilemma. Methods: 89Zr-trastuzumab PET was performed as described earlier (Gaykema et al, Clin Cancer Res 2014) in patients in whom standard work up with bone scan, FDG PET, CT and if feasible a biopsy, failed to evaluate HER2 status of their disease. 89Zr-trastuzumab PET was defined positive, when at least a dominant part of the tumor load showed substantial tracer uptake (Gebhart et al, Ann Oncol 2015), when tumor tracer uptake in single lesions (except brain) was ≥ normal liver uptake or when brain metastases had a tracer uptake > background. Circulating tumor cell (CTC) analysis prior to tracer injectionwas performed using the CellSearch System (Janssen Diagnostics LLC) and CTC HER2 status was assessed immunofluorescently. Questionnaires about treatment decisions were completed before, directly after and ≥3 months after 89Zr-trastuzumab PET. Results: Twenty patients were enrolled: 8 with two primary cancers (HER2-positive and HER2-negative BC or BC and non-BC), 7 with metastases inaccessible for biopsy, 4 with prior HER2-positive and HER2-negative metastases, 1 with primary BC with equivocal HER2 status (average 4.23 HER2 gene copies/nucleus). 89Zr-trastuzumab PET was positive in 12 patients, negative in 7 and equivocal in one patient. In 15/20 patients 89Zr-trastuzumab PET supported treatment decision. The scan altered treatment of 8 patients, increased physicians9 confidence without affecting treatment in 10, and improved physicians9 understanding of disease in 18 patients. Ten patients had 1-99 CTCs, 6 with HER2 expression. There was no correlation between HER2 expression by CTCs and 89Zr-trastuzumab PET results or subsequent treatment decision. Conclusion: 89Zr-trastuzumab PET, but not CTC analysis, supports clinical decision making in BC patients in whom standard work up fails to evaluate HER2 status. (Funded by the Dutch A Sister9s Hope). Citation Format: Schroder CP, Bensch F, Brouwers AH, Lub-de Hooge MN, de Jong JR, van der Vegt B, Sleijfer S, de Vries EG. Clinical value of 89Zr-trastuzumab PET in HER2-positive breast cancer patients with a clinical dilemma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-03-06.

1627PPET-IMAGING WITH 89ZR-LABELED ANTI-MESOTHELIN (MSLN) ANTIBODY IN PATIENTS WITH PANCREATIC CANCER (PC) OR OVARIAN CANCER (OC)

Aim: The tumor antigen MSLN is frequently overexpressed in PC and OC. A 89Zr-PET study (NCT01832116) with MMOT0530A, an anti-MSLN antibody, was initiated in conjunction with a phase 1 study of the antibody-drug conjugate DMOT4039A (containing MMOT0530A linked to the anti-mitotic agent MMAE, NCT01469793). This imaging study aims to investigate antibody tumor uptake, whole body distribution and organ pharmacokinetics and to explore the relation between uptake and MSLN expression and response to DMOT40392A treatment in patients with unresectable PC or platinum-resistant OC. Methods: Before receiving DMOT4039A, patients were injected with 37 MBq 89Zr-MMOT0530A +/- additional unlabeled MMOT0530A, followed by PET/CT imaging 2, 4 and 7 days post injection (pi). Tracer uptake was quantified with standardized uptake value (SUV) and expressed as mean (±SD). MSLN expression was determined in archival tumor tissue with an exploratory immunohistochemical (IHC) assay. Results: 7 PC and 4 OC patients were included. MSLN expression varied from 0 to 3+. The optimal antibody protein dose resulting in sufficient circulating tracer was 10 mg MMOT0530A and the optimal imaging time was 4 or 7 days pi. Tumor tracer uptake was observed in 37 quantifiable tumor lesions (all patients) with mean SUV of 10.7 (±6.3) on PET 4 days pi. The mean SUV per patient (1-8 lesions/patient) was 10.9 (±5.7), with 9.2 (±4.5) in PC and 11.9 (±7.4) in OC lesions on PET 4 days pi. Within patients, a mean 2.4-fold (±1.10) difference in tumor uptake between lesions was found. Two measurable lesions on diagnostic CT (according to RECIST 1.1) were not visible on PET. Uptake in blood, liver, kidneys, spleen and intestine reflected normal antibody distribution with mean SUV at day 4 pi of 5.6, 7.8, 6.1, 4.1 and 3.2, respectively, while low uptake was observed in muscle, lung, brain and bone (0.6, 1.0, 0.2 and 0.7, respectively). Tracer tumor uptake was lower in the 2 patients with IHC scores 0 and 1. Best response on DMOT4039A was stable disease in ten patients. An association between iPET tumor uptake and clinical response could not be determined. Conclusions: 89Zr-MMOT0530A-PET shows antibody uptake in primary and metastatic PC and OC tumor lesions. This technique can potentially guide antibody-based therapy development.