Marjolijn N. Lub-de Hooge

In Vivo VEGF Imaging with Radiolabeled Bevacizumab in a Human Ovarian Tumor Xenograft

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

Development and Characterization of Clinical-Grade 89Zr-Trastuzumab for HER2/neu ImmunoPET Imaging

The anti–human epidermal growth factor receptor 2 (HER2/neu) antibody trastuzumab is administered to patients with HER2/neu-overexpressing breast cancer. Whole-body noninvasive HER2/neu scintigraphy could help to assess and quantify the HER2/neu expression of all lesions, including nonaccessible metastases. The aims of this study were to develop clinical-grade radiolabeled trastuzumab for clinical HER2/neu immunoPET scintigraphy, to improve diagnostic imaging, to guide antibody-based therapy, and to support early antibody development. The PET radiopharmaceutical 89Zr-trastuzumab was compared with the SPECT tracer 111In-trastuzumab, which we have tested in the clinic already. Methods: Trastuzumab was labeled with 89Zr and (for comparison) with 111In. The minimal dose of trastuzumab required for optimal small-animal PET imaging and biodistribution was determined with human HER2/neu-positive or -negative tumor xenograft–bearing mice. Results: Trastuzumab was efficiently radiolabeled with 89Zr at a high radiochemical purity and specific activity. The antigen-binding capacity was preserved, and the radiopharmaceutical proved to be stable for up to 7 d in solvent and human serum. Of the tested protein doses, the minimal dose of trastuzumab (100 μg) proved to be optimal for imaging. The comparative biodistribution study showed a higher level of 89Zr-trastuzumab in HER2/neu-positive tumors than in HER2/neu-negative tumors, especially at day 6 (33.4 ± 7.6 [mean ± SEM] vs. 7.1 ± 0.7 percentage injected dose per gram of tissue). There were good correlations between the small-animal PET images and the biodistribution data and between 89Zr-trastuzumab and 111In-trastuzumab uptake in tumors (R2 = 0.972). Conclusion: Clinical-grade 89Zr-trastuzumab showed high and HER2/neu-specific tumor uptake at a good resolution.

Indium-111-Labeled Trastuzumab Scintigraphy in Patients With Human Epidermal Growth Factor Receptor 2-Positive Metastatic Breast Cancer

PURPOSE The cardiac and antineoplastic effects of trastuzumab may be related to specific uptake of trastuzumab in myocardium and tumor tissue, respectively. We evaluated whether indium-111 (111In)-labeled trastuzumab scintigraphy can predict cardiotoxicity and identify tumor lesions. In addition, we evaluated whether plasma markers for cardiac dysfunction can be used to predict cardiotoxicity. PATIENTS AND METHODS Patients with human epidermal growth factor receptor 2 (HER2) -positive metastatic breast cancer underwent gamma camera imaging from 15 minutes to 7 days after injection of 150 MBq 111In-diethylenetriamine penta-acetic acid anhydride (DTPA) -trastuzumab, after loading-dose trastuzumab, and after once-a-week trastuzumab doses for 11 weeks, and concomitant paclitaxel once every 3 weeks. Cardiac assessments were performed before treatment, and after four and six cycles. Plasma N-terminal probrain natriuretic peptide (NT-proBNP) and serum troponin I were measured with immunoassay. RESULTS Fifteen of the 17 patients were available for cardiac and tumor uptake analysis. On the first scan, myocardial 111In-DTPA-trastuzumab uptake was observed in one patient with pre-existing cardiac arrhythmias, who did not develop heart failure during treatment. Severe cardiotoxicity occurred in three patients, without initial myocardial uptake, whereas one showed weak myocardial uptake after four cycles. The detection rate of single tumor lesions was 45%. New tumor lesions were discovered in 13 of 15 patients. Pretreatment plasma NT-proBNP levels were higher in patients with than without heart failure (mean, 534 [standard deviation, 236] v 105 [standard deviation, 79] ng/L; P = .009). CONCLUSION Radiolabeled trastuzumab scintigraphy was not valuable in predicting trastuzumab-related cardiotoxicity in metastatic breast cancer patients, but can identify HER2-positive tumors. Measurement of plasma NT-proBNP is promising regarding prediction of trastuzumab-related cardiotoxicity.

Immuno-PET: A Navigator in Monoclonal Antibody Development and Applications

Monoclonal antibodies (mAbs) have been approved for use as diagnostics and therapeutics in a broad range of medical indications, but especially in oncology. In addition, hundreds of new mAbs, engineered mAb fragments, and nontraditional antibody-like scaffolds directed against either validated or novel tumor targets are under development. Immuno-positron emission tomography (PET), the tracking and quantification of mAbs with PET in vivo, is an exciting novel option to improve diagnostic imaging and to guide mAb-based therapy. In this review, recent technical advances leading to a jump ahead in mAb imaging are discussed. The availability of proper positron emitters, sophisticated radiochemistry, and advanced PET and PET-computed tomography scanners is crucial in these developments. Immuno-PET might play an important future role in cancer staging, in the improvement and tailoring of therapy with existing mAbs, and in the efficient development of novel mAbs. An overview of the preclinical and first clinical immuno-PET studies is provided.

Preclinical characterisation of 111In-DTPA-trastuzumab

Trastuzumab (Herceptin®) is a recombinant humanised IgG1 monoclonal antibody against the human epidermal growth factor receptor 2 (HER2), used for metastatic breast cancer treatment. Radiolabelled trastuzumab may have several future applications for diagnostic use. The aim of the present study was to develop clinical grade 111Indium (111In) radiolabelled trastuzumab, to evaluate the stability and immunoreactivity of the tracer and to perform a biodistribution study in human tumour‐bearing mice. Trastuzumab was radiolabelled with 111In using DTPA as a chelator. 111In‐DTPA‐trastuzumab (labelling yield 92.3±2.3%, radiochemical purity 97.0±1.5%) is stable in PBS when stored at 4°C for more than 14 days. The immunoreactive fraction determined by cell‐binding assays, using the HER2‐overexpressing human ovarian SK‐OV‐3 tumour cell line, was 0.87±0.06. Biodistribution and tumour targeting were studied in HER2 receptor‐positive and ‐negative tumour‐bearing athymic mice. The HER2‐positive tumour showed (9.77±1.14% injected dose per gram (ID g−1)) substantial uptake of the labelled antibody already after 5 h. The difference in uptake between HER2‐positive versus ‐negative tumours was even more pronounced 3 days after injection (16.30±0.64% ID g−1), and was visualised by radioimmunoscintigraphy. Liver, spleen and kidney showed marked tracer uptake. In summary, trastuzumab can be efficiently radiolabelled with 111In with high labelling yields and high stability. 111In‐DTPA‐trastuzumab selectively binds to the human HER2 receptor both in vitro and in vivo in animals. Therefore, 111In‐DTPA‐trastuzumab appears suitable for clinical use.

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.

VEGF-PET Imaging Is a Noninvasive Biomarker Showing Differential Changes in the Tumor during Sunitinib Treatment

Non-invasive imaging of angiogenesis could ease the optimization of antiangiogenesis treatments for cancer. In this study, we evaluated the role of VEGF-PET as a biomarker of dynamic angiogenic changes in tumors following treatment with the kinase inhibitor sunitinib. The effects of sunitinib treatment and withdrawal on the tumor was investigated using the new VEGF-PET tracer (89)Zr-ranibizumab as well as (18)F-FDG PET, and (15)O-water PET in mouse xenograft models of human cancer. The obtained imaging results were compared with tumor growth, VEGF plasma levels and immunohistologic analyzes. In contrast to (18)F-FDG and (15)O-water PET, VEGF-PET demonstrated dynamic changes during sunitinib treatment within the tumor with a strong decline in signal in the tumor center and only minimal reduction in tumor rim, with a pronounced rebound after sunitinib discontinuation. VEGF-PET results corresponded with tumor growth and immunohistochemical vascular- and tumor- markers. Our findings highlight the strengths of VEGF-PET imaging to allow serial analysis of angiogenic changes in different areas within a tumor.

89Zr-Bevacizumab PET Imaging in Primary Breast Cancer

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

89Zr-trastuzumab PET visualises HER2 downregulation by the HSP90 inhibitor NVP-AUY922 in a human tumour xenograft

NVP-AUY922, a potent heat shock protein (HSP) 90 inhibitor, downregulates the expression of many oncogenic proteins, including the human epidermal growth factor receptor-2 (HER2). Because HER2 downregulation is a potential biomarker for early response to HSP90-targeted therapies, we used the (89)Zr-labelled HER2 antibody trastuzumab to quantify the alterations in HER2 expression after NVP-AUY922 treatment with HER2 positron emission tomography (PET) imaging. The HER2 overexpressing human SKOV-3 ovarian tumour cell line was used for in vitro experiments and as xenograft model in nude athymic mice. In vitro HER2 membrane expression was assessed by flow cytometry and a radio-immuno assay with (89)Zr-trastuzumab. For in vivo evaluation, mice received 50mg/kg NVP-AUY922 intraperitoneally every other day. (89)Zr-trastuzumab was injected intravenously 6d before NVP-AUY922 treatment and after 3 NVP-AUY922 doses. MicroPET imaging was performed at 24, 72 and 144h post tracer injection followed by ex-vivo biodistribution and immunohistochemical staining. After 24h NVP-AUY922 treatment HER2 membrane expression showed profound reduction with flow cytometry (80%) and radio-immuno assay (75%). PET tumour quantification, showed a mean reduction of 41% (p=0.0001) in (89)Zr-trastuzumab uptake at 144h post tracer injection after NVP-AUY922 treatment. PET results were confirmed by ex-vivo (89)Zr-trastuzumab biodistribution and HER2 immunohistochemical staining. NVP-AUY922 effectively downregulates HER2, which can be monitored and quantified in vivo non-invasively with (89)Zr-trastuzumab PET. This technique is currently under clinical evaluation and might serve as an early biomarker for HSP90 inhibition in HER2 positive metastatic breast cancer patients.

Relative Blood Volume Changes Underestimate Total Blood Volume Changes during Hemodialysis

BACKGROUND Measurements of relative blood volume changes (DeltaRBV) during hemodialysis (HD) are based on hemoconcentration and assume uniform mixing of erythrocytes and plasma throughout the circulation. However, whole-body hematocrit (Ht) is lower than systemic Ht. During HD, a change in the ratio between whole-body to systemic Ht (F cell ratio) is likely to occur as a result of a net shift of low Ht blood from the microcirculation to the macrocirculation. Hence, DeltaRBV may differ significantly from total blood volume changes (DeltaTBV). Therefore, this study compared DeltaRBV and DeltaTBV during HD. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS Plasma and erythrocyte volumes were measured using (125)I- and (123)I-radioiodinated albumin and (51)Cr-labeled erythrocytes, respectively. After validation of the standardized method in two patients on a nondialysis day, seven patients completed the protocol during HD. (125)I-albumin and (51)Cr-labeled erythrocytes were administered 20 min before the start of HD. (123)I-albumin was administered at 160 min into the HD session to quantify and correct for (125)I-albumin leakage. DeltaRBV was measured continuously throughout HD. The F cell ratio was derived from whole-body and systemic Ht. RESULTS Total ultrafiltration volume was 2450 +/- 770 ml. TBV declined from 5905 +/- 824 to 4877 +/- 722 ml during HD. Thus, TBV declined 17.3 +/- 4.4%, whereas the RBV decline was only 8.2 +/- 3.7% (P = 0.001). The F cell ratio increased from 0.896 +/- 0.036 to 0.993 +/- 0.049 during HD (P = 0.002). CONCLUSIONS DeltaRBV significantly underestimates DeltaTBV during HD. The rise in F cell ratio strongly suggests that during HD, blood translocates from the microcirculation to the macrocirculation, probably as a cardiovascular compensatory mechanism in response to hypovolemia.