Thijs H. Oude Munnink

89Zr-Bevacizumab PET of Early Antiangiogenic Tumor Response to Treatment with HSP90 Inhibitor NVP-AUY922

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

Zr-89-trastuzumab and Zr-89-bevacizumab PET to Evaluate the Effect of the HSP90 Inhibitor NVP-AUY922 in Metastatic Breast Cancer Patients

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 89Zr-trastuzumab PET (for HER2-positive breast cancer) or 89Zr-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/m2 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), 89Zr-trastuzumab, or 89Zr-bevacizumab. Response evaluation was performed according to RECIST1.0. FDG, 89Zr-trastuzumab, and 89Zr-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 89Zr-trastuzumab PET was heterogeneous and related to size change on CT after 8 weeks treatment (r2 = 0.69; P = 0.006). Tumor response on 89Zr-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 89Zr-trastuzumab PET was positively associated with change in size of individual lesions assessed by CT. Clin Cancer Res; 20(15); 3945–54. ©2014 AACR.

89Zr-Bevacizumab PET Visualizes Heterogeneous Tracer Accumulation in Tumor Lesions of Renal Cell Carcinoma Patients and Differential Effects of Antiangiogenic Treatment

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 89Zr-bevacizumab, a VEGF-A–binding PET tracer, in mRCC patients before and during antiangiogenic treatment in a pilot study. Methods: Patients underwent 89Zr-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: 89Zr-bevacizumab PET scans visualized 125 evaluable tumor lesions in 22 patients, with a median SUVmax (maximum standardized uptake value) of 6.9 (range, 2.3–46.9). Bevacizumab/interferon-α induced a mean change in tumor SUVmax 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 SUVmax was −14.3% at 2 wk (range, −80.4 to +269.9; P = 0.006), but at 6 wk the mean change in tumor SUVmax was +72.6% (range, −46.4 to +236%; P < 0.0001) above baseline. SUVmax was not related to plasma VEGF-A at all scan moments. A baseline mean tumor SUVmax 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 89Zr-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 SUVmax was associated with longer time to progression.

PET with the 89Zr-Labeled Transforming Growth Factor-β Antibody Fresolimumab in Tumor Models

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

Lapatinib and 17AAG Reduce Zr-89-Trastuzumab-F(ab')(2) Uptake in SKBR3 Tumor Xenografts

Human epidermal growth factor receptor-2 (HER2) directed therapy potentially can be improved by insight in drug effects on HER2 expression. This study evaluates the effects of the EGFR/HER2 tyrosine kinase inhibitor lapatinib, the heat shock protein-90 inhibitor 17AAG, and their combination, on HER2 expression with in vivo HER2-PET imaging. Lapatinib and 17AAG effects on EGFR and HER2 membrane expression were determined in vitro using flow cytometry of human SKBR3 tumor cells. Effect of lapatinib on HER2 internalization was studied in vitro by (89)Zr-trastuzumab-F(ab)(2) internalization. For in vivo evaluation, (89)Zr-trastuzumab-F(ab)(2) μPET imaging was performed two times with a 7 day interval. Lapatinib was administered for 6 days, starting 1 day after the baseline scan. 17AAG was given 1 day before the second (89)Zr-trastuzumab-F(ab)(2) injection. Imaging data were compared with ex vivo biodistribution analysis and HER2 immunohistochemical staining. 17AAG treatment lowered EGFR expression by 41% (P = 0.016) and HER2 by 76% (P = 0.022). EGFR/HER2 downregulation by 17AAG was inhibited by lapatinib pretreatment. Lapatinib reduced internalization of (89)Zr-trastuzumab-F(ab)(2) with 25% (P = 0.0022). (89)Zr-trastuzumab-F(ab)(2) tumor to blood ratio was lowered 32% by lapatinib (P = 0.00004), 34% by 17AAG (P = 0.0022) and even 53% by the combination (P = 0.011). Lapatinib inhibits HER2 internalization and 17AAG lowers HER2 membrane expression. Both drugs reduce (89)Zr-trastuzumab-F(ab)(2) tumor uptake. Based on our findings, supported by previous preclinical data indicating the antitumor potency of lapatinib in combination with HSP90 inhibition, combination of these drugs deserves further investigation.

TGF-β antibody uptake in recurrent high grade glioma imaged with 89Zr-fresolimumab PET

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 89Zr-fresolimumab PET in mice. The aim of this study was to investigate the fresolimumab uptake in recurrent high-grade gliomas using 89Zr-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 89Zr-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. 89Zr-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 89Zr-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: 89Zr-fresolimumab penetrated recurrent high-grade gliomas very well but did not result in clinical benefit.

Everolimus Reduces Zr-89-Bevacizumab Tumor Uptake in Patients with Neuroendocrine Tumors

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 89Zr-bevacizumab PET scans before and at 2 and 12 wk during everolimus treatment. 89Zr-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 89Zr-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 (r2 = 0.51, P < 0.05, and r2 = 0.61, P < 0.01, respectively). Conclusion: This study demonstrates variable 89Zr-bevacizumab PET tumor uptake in NET patients. 89Zr-bevacizumab tumor uptake diminished during everolimus treatment. Serial 89Zr-bevacizumab PET might be useful as an early predictive biomarker of anti–VEGF-directed treatment in NET patients.

Transforming growth factor (TGF)-β expression and activation mechanisms as potential targets for anti-tumor therapy and tumor imaging.

Cancer remains one of the leading causes of death in the developed countries and cancer mortality is expected to rise globally. Despite encouraging developments regarding targeted drugs, the most prevalent cancer mortality remains metastatic disease. Therefore, drugs that target cancer progression, invasion and metastasis are clearly needed. One of the most interesting targets in this setting is transforming growth factor β (TGF-β). TGF-β can promote tumor growth, invasion and metastasis. However, TGF-β also has a physiological, opposing role: maintaining tissue homeostasis and suppression of tumor progression. The window of effective TGF-β targeting is therefore evidently small, which poses a clear challenge in selecting patients at the right time. Despite this complexity, several TGF-β inhibitors are currently in clinical development, modulating TGF-β production, activation or signaling. Still, specificity and long term toxicity remain unclear, emphasizing the importance of careful monitoring of clinical trials. Development and application of these drugs in the clinic require adequate insight and evaluation methods for the role of TGF-β during tumor invasion and metastasis. In this review, presently available methods for clinical evaluation will be discussed, such as an ex vivo stimulation assay, TGF-β response signature and molecular imaging techniques. Future clinical trials incorporating the validation of these evaluation methods will show which method will be most predictive and suitable for clinical application.

Toward Molecular imaging-Driven Drug Development in Oncology

With current testing strategies, the number of novel targeted anticancer agents will exceed our drug selection capacity. Molecular imaging is a powerful additional tool that can assist us in selecting effective drugs and help patients benefit from targeted agents. Moreover, measurement of the functional effects of such targeted agents could permit dynamic tuning of treatment selection at the earliest time point at which loss of functional effects is observed.

Multidrug resistance in oncology and beyond: from imaging of drug efflux pumps to cellular drug targets

Resistance of tumor cells to several structurally unrelated classes of natural products, including anthracyclines, taxanes, and epipodophyllotoxines, is often referred as multidrug resistance (MDR). This is associated with ATP-binding cassette transporters, which function as drug efflux pumps such as P-glycoprotein (Pgp) and multidrug resistance-associated protein 1 (MRP1). Because of the hypothesis in the early eighties that blockade of these efflux pumps by modulators would improve the effect of chemotherapy, extensive effort has been put to visualize these pumps using nuclear imaging with several specific tracers, using both SPECT and PET techniques. The methods and possibilities to visualize these pumps in both the tumor and the blood-brain barrier will be discussed. Because of the fact that the addition of Pgp or MRP modulators has not shown any clinical benefit in patient outcome, these specific MDR tracers are not routinely used in clinical practice. Evidence emerges that combination of chemotherapeutic drugs involved in MDR with the so-called targeted agents can improve patient outcome. The concept of molecular imaging can also be used to visualize the targets for these agents, such as HER2/neu and angiogenic factors such as vascular endothelial growth factor (VEGF). Potentially visualizing molecular drug targets in the tumor can function as biomarkers to support treatment decision for the individual patient.