Mette K. Nedergaard

Targeting the Epidermal Growth Factor Receptor in Solid Tumor Malignancies

The epidermal growth factor receptor (EGFR) is over-expressed, as well as mutated, in many types of cancers. In particular, the EGFR variant type III mutant (EGFRvIII) has attracted much attention as it is frequently and exclusively found on many tumor cells, and hence both EGFR and EGFRvIII have been proposed as valid targets in many cancer therapy settings. Different strategies have been developed in order to either inhibit EGFR/EGFRvIII activity or to ablate EGFR/EGFRvIII-positive tumor cells. Drugs that inhibit these receptors include monoclonal antibodies (mAbs) that bind to the extracellular part of EGFR, blocking the binding sites for the EGFR ligands, and intracellular tyrosine kinase inhibitors (TKIs) that block the ATP binding site of the tyrosine kinase domain. Besides an EGFRvIII-targeted vaccine, conjugated anti-EGFR mAbs have been used in different settings to deliver lethal agents to the EGFR/EGFRvIII-positive cells; among these are radio-labelled mAbs and immunotoxins. This article reviews the current status and efficacy of EGFR/EGFRvIII-targeted therapies.

The Use of Longitudinal 18F-FET MicroPET Imaging to Evaluate Response to Irinotecan in Orthotopic Human Glioblastoma Multiforme Xenografts

Objectives Brain tumor imaging is challenging. Although 18F-FET PET is widely used in the clinic, the value of 18F-FET MicroPET to evaluate brain tumors in xenograft has not been assessed to date. The aim of this study therefore was to evaluate the performance of in vivo 18F-FET MicroPET in detecting a treatment response in xenografts. In addition, the correlations between the 18F-FET tumor accumulation and the gene expression of Ki67 and the amino acid transporters LAT1 and LAT2 were investigated. Furthermore, Ki67, LAT1 and LAT2 gene expression in xenograft and archival patient tumors was compared. Methods Human GBM cells were injected orthotopically in nude mice and 18F-FET uptake was followed by weekly MicroPET/CT. When tumor take was observed, mice were treated with CPT-11 or saline weekly. After two weeks of treatment the brain tumors were isolated and quantitative polymerase chain reaction were performed on the xenograft tumors and in parallel on archival patient tumor specimens. Results The relative tumor-to-brain (T/B) ratio of SUVmax was significantly lower after one week (123±6%, n = 7 vs. 147±6%, n = 7; p = 0.018) and after two weeks (142±8%, n = 5 vs. 204±27%, n = 4; p = 0.047) in the CPT-11 group compared with the control group. Strong negative correlations between SUVmax T/B ratio and LAT1 (r = −0.62, p = 0.04) and LAT2 (r = −0.67, p = 0.02) were observed. In addition, a strong positive correlation between LAT1 and Ki67 was detected in xenografts. Furthermore, a 1.6 fold higher expression of LAT1 and a 23 fold higher expression of LAT2 were observed in patient specimens compared to xenografts. Conclusions 18F-FET MicroPET can be used to detect a treatment response to CPT-11 in GBM xenografts. The strong negative correlation between SUVmax T/B ratio and LAT1/LAT2 indicates an export transport function. We suggest that 18F-FET PET may be used for detection of early treatment response in patients.

Urokinase-Type Plasminogen Activator Receptor as a Potential PET Biomarker in Glioblastoma

Glioblastoma is one of the most malignant types of human cancer, and the prognosis is poor. The development and validation of novel molecular imaging biomarkers has the potential to improve tumor detection, grading, risk stratification, and treatment monitoring of gliomas. The aim of this study was to explore the potential of PET imaging of the urokinase-type plasminogen activator receptor (uPAR) in glioblastoma. Methods: The uPAR messenger RNA expression of tumors from 19 glioblastoma patients was analyzed, and a cell culture derived from one of these patients was used to establish an orthotopic xenograft model of glioblastoma. Tumor growth was monitored using bioluminescence imaging. Five to six weeks after inoculation, all mice were scanned with small-animal PET/CT using two new uPAR PET ligands (64Cu-NOTA-AE105 and 68Ga-NOTA-AE105) and, for comparison, O-(2-18F-fluoroethyl)-l-tyrosine (18F-FET). One MRI scan was obtained for each mouse to confirm tumor location. The uPAR specificity of 64Cu-NOTA-AE105 was confirmed by alignment of hematoxylin- and eosin-stained and uPAR immunohistochemistry–stained slides of the brain with the activity distribution as determined using autoradiography. Results: uPAR expression was found in all 19 glioblastoma patient tumors, and high expression of uPAR correlated with decreased overall survival (P = 0.04). Radiolabeling of NOTA-AE105 with 64Cu and 68Ga was straightforward, resulting in a specific activity of approximately 20 GBq/μmol and a radiochemical purity of more than 98% for 64Cu-NOTA-AE105 and more than 97% for 68Ga-NOTA-AE105. High image contrast resulting in clear tumor delineation was found for both 68Ga-NOTA-AE105 and 64Cu-NOTA-AE105. Absolute uptake in tumor was higher for 18F-FET (3.5 ± 0.8 percentage injected dose [%ID]/g) than for 64Cu-NOTA-AE105 (1.2 ± 0.4 %ID/g) or 68Ga-NOTA-AE105 (0.4 ± 0.1 %ID/g). A similar pattern was observed in background brain tissue, where uptake was 1.9 ± 0.1 %ID/g for 18F-fluorothymidine, compared with 0.05 ± 0.01 %ID/g for 68Ga-NOTA-AE105 and 0.11 ± 0.02 %ID/g for 64Cu-NOTA-AE105. The result was a significantly higher tumor-to-background ratio for both 68Ga-NOTA-AE105 (7.6 ± 2.1, P < 0.05) and 64Cu-NOTA-AE105 (10.6 ± 2.3, P < 0.01) than for 18F-FET PET (1.8 ± 0.3). Autoradiography of brain slides confirmed that the accumulation of 64Cu-NOTA-AE105 corresponded well with uPAR-positive cancer cells. Conclusion: On the basis of our translational study, uPAR PET may be a highly promising imaging biomarker for glioblastoma. Further clinical exploration of uPAR PET in glioblastoma is therefore justified.

Combined EGFR- and notch inhibition display additive inhibitory effect on glioblastoma cell viability and glioblastoma-induced endothelial cell sprouting in vitro

BackgroundFor Glioblastoma (GBM) patients, a number of anti-neoplastic strategies using specifically targeting drugs have been tested; however, the effects on survival have been limited. One explanation could be treatment resistance due to redundant signaling pathways, which substantiates the need for combination therapies. In GBM, both the epidermal growth factor receptor (EGFR) and the notch signaling pathways are often deregulated and linked to cellular growth, invasion and angiogenesis. Several studies have confirmed cross-talk and co-dependence of these pathways. Therefore, this study aimed at testing a combination treatment strategy using inhibitors targeting the notch and EGFR pathways.MethodsFor evaluation of cell viability a standard MTT assay was used. Western blotting (WB) and Q-RT-PCR were employed in order to assess the protein- and mRNA expression levels, respectively. In order to determine angiogenic processes, we used an endothelial spheroid sprouting assay. For assessment of secreted VEGF from GBM cells we performed a VEGF-quantikine ELISA.ResultsGBM cells were confirmed to express EGFR and Notch and to have the capacity to induce endothelial cell sprouting. Inhibition of EGFR and Notch signaling was achieved using either Iressa (gefitinib) or the gamma-secretase inhibitor DAPT. Our data showed that DAPT combined with Iressa treatment displayed increased inhibitory effect on cell viability and abrogated expression and activation of major pro-survival pathways. Similarly, the combinational treatment significantly increased abrogation of GBM-induced endothelial cell sprouting suggesting reduced GBM angiogenesis.ConclusionThis study finds that simultaneous targeting of notch and EGFR signaling leads to enhanced inhibitory effects on GBM-induced angiogenesis and cell viability, thereby stressing the importance of further evaluation of this targeting approach in a clinical setting.

VEGF-C sustains VEGFR2 activation under bevacizumab therapy and promotes glioblastoma maintenance

Abstract Background Glioblastoma ranks among the most lethal cancers, with current therapies offering only palliation. Paracrine vascular endothelial growth factor (VEGF) signaling has been targeted using anti-angiogenic agents, whereas autocrine VEGF/VEGF receptor 2 (VEGFR2) signaling is poorly understood. Bevacizumab resistance of VEGFR2-expressing glioblastoma cells prompted interrogation of autocrine VEGF-C/VEGFR2 signaling in glioblastoma. Methods Autocrine VEGF-C/VEGFR2 signaling was functionally investigated using RNA interference and exogenous ligands in patient-derived xenograft lines and primary glioblastoma cell cultures in vitro and in vivo. VEGF-C expression and interaction with VEGFR2 in a matched pre- and post-bevacizumab treatment cohort were analyzed by immunohistochemistry and proximity ligation assay. Results VEGF-C was expressed by patient-derived xenograft glioblastoma lines, primary cells, and matched surgical specimens before and after bevacizumab treatment. VEGF-C activated autocrine VEGFR2 signaling to promote cell survival, whereas targeting VEGF-C expression reprogrammed cellular transcription to attenuate survival and cell cycle progression. Supporting potential translational significance, targeting VEGF-C impaired tumor growth in vivo, with superiority to bevacizumab treatment. Conclusions Our results demonstrate VEGF-C serves as both a paracrine and an autocrine pro-survival cytokine in glioblastoma, promoting tumor cell survival and tumorigenesis. VEGF-C permits sustained VEGFR2 activation and tumor growth, where its inhibition appears superior to bevacizumab therapy in improving tumor control.

Inhibition of Notch signaling alters the phenotype of orthotopic tumors formed from glioblastoma multiforme neurosphere cells but does not hamper intracranial tumor growth regardless of endogene Notch pathway signature

Background Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in the devastating brain tumor glioblastoma multiforme (GBM). bCSC are proposed a central role in tumor initiation, progression, treatment resistance and relapse and as such present a promising target in GBM research. The Notch signaling pathway is often deregulated in GBM and we have previously characterized GBM-derived bCSC cultures based on their expression of the Notch-1 receptor and found that it could be used as predictive marker for the effect of Notch inhibition. The aim of the present project was therefore to further elucidate the significance of Notch pathway activity for the tumorigenic properties of GBM-derived bCSC. Methods Human-derived GBM xenograft cells previously established as NSC-like neurosphere cultures were used. Notch inhibition was accomplished by exposing the cells to the gamma-secretase inhibitor DAPT prior to gene expression analysis and intracranial injection into immunocompromised mice. Results By analyzing the expression of several Notch pathway components, we found that the cultures indeed displayed different Notch pathway signatures. However, when DAPT-treated neurosphere cells were injected into the brain of immunocompromised mice, no increase in survival was obtained regardless of Notch pathway signature and Notch inhibition. We did however observe a decrease in the expression of the stem cell marker Nestin, an increase in the proliferative marker Ki-67 and an increased number of abnormal vessels in tumors formed from DAPT-treated, high Notch-1 expressing cultures, when compared with the control. Conclusion Based on the presented results we propose that Notch inhibition partly induces differentiation of bCSC, and selects for a cell type that more strongly induces angiogenesis if the treatment is not sustained. However, this more differentiated cell type might prove to be more sensitive to conventional therapies.

Abstract 2805: A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs

Background: Patients with glioblastoma multiforme (GBM) have a poor prognosis and few treatment options; hence new treatments are needed. Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, orthotopic implantation confers a translational advantage as the cancer develops in a microenvironment more closely mimicking that of the original patient tumor. Also the major impact of the blood brain barrier that must be taking into account when targeting brain tumors as GBM in terms of drug bioavailability is better represented in the orthotopic models. The aim of this study was therefore to develop a panel of orthotopic GBM PDX models for pre-clinical efficacy studies of new drugs. The models were then used to study the efficacy of standard of care such as temozolomide (TMZ) and external radiation therapy (XRT). Methods: Low passage subcutaneous tumors from six different PDX GBM models designated ST108, ST112, ST146, ST545, ST610 and ST2473 were digested and used for intracranial stereotactic injection in nude mice. Tumor take and growth was determined by T2-weighted magnetic resonance imaging (MRI). At confirmed tumor take mice were either treated with TMZ (100mg/kg/day for 5 days) or whole brain XRT (2 Gy/day for 5 days). Control groups receiving vehicle or sham XRT were included depending on treatment regiment. Final endpoint was survival by humane endpoints and tumors were fixed in formalin for histological evaluation. Results: MRI confirmed tumor take in all models within 5 weeks of implantation. The take rate was > 80% across all models. TMZ showed efficacy in the orthotopic ST610 GBM PDX model evaluated by MRI on day 14 (16.2±2.9 mm3 vs. 76.8±13.1 mm3, p=0.016), whereas the ST146 model displayed resistance to TMZ on day 14 (12.7±5.6 mm3 vs. 26.5±11.9 mm3, p=0.26). The median survival was 60 days vs. 14 days in the ST610 model (TMZ vs. vehicle, p=0.0005) and 27 days vs. 13 days in the ST146 model (TMZ vs. vehicle, p=0.007). XRT showed efficacy in the orthotopic ST2473 model. Tumor volume was significantly smaller in treated vs. sham animals 11 days after inclusion (6.9±1.4 mm3 vs. 28.9±3.3 mm3, p=0.001). Also, a survival benefit was observed in XRT treated animals compared to sham. Histology confirmed the presence of orthotopic tumors and typical GBM pathology characteristics such as pseudopalisading tumor cells surrounding necrosis and micro vascular proliferation were identified. Conclusion: Six different orthotopic GBM PDX models were established from low passage subcutaneous PDX models. Models sensitive and resistant to TMZ were identified and histological GBM characteristics were identified. Together, the established panel of orthotopic PDX models can be used as a relevant translational platform for testing of new drugs in a setting that more closely mimics the GBM tumor microenvironment and the impact of the blood brain barrier in patients. Citation Format: Mette M. Jensen, Camilla S. Knudsen, Lotte K. Kristensen, Mette K. Nedergaard, Michael J. Wick, Kyriakos P. Papadopoulos, Anthony W. Tolcher, Andreas Kjaer, Carsten H. Nielsen. A panel of orthotopic glioblastoma multiforme (GBM) patient derived xenograft (PDX) mouse models for efficacy evaluation of drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2805. doi:10.1158/1538-7445.AM2017-2805

Abstract P3-03-01: Intracranial PDX models of breast cancer metastasis

Background: Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast cancer is an independent factor for development of brain metastases. Up to 37% of patients with HER2 positive disease relapse intracranially despite control of extra-cranial metastatic disease. Inability of anti-cancer agents to cross an intact blood-brain barrier (BBB) is a possible explanation for the increased incidence of brain metastases. Subcutaneous (SQ) patient-derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, orthotopic PDX models may confer a translational advantage as the patient tumor microenvironment is more closely mimicked. Especially when targeting brain tumors, the major impact of the BBB on drug bioavailability must be taken into consideration. The aim of this study was therefore to develop a panel of intracranial PDX models of breast cancer brain metastases for pre-clinical efficacy studies of new anticancer drugs. Methods: SQ tumors from three different HER2 positive PDX breast cancer models designated ST340, ST1339 and ST1616B were enzymatically digested and used for intracranial stereotactic injection in nude mice. Contrast-enhanced T1- and T2-weighted Magnetic Resonance Imaging (MRI) were used to determine tumor take. Intracranial tumor growth was monitored using MRI and positron emission tomography (PET) in conjunction with the amino acid radio tracer 18F-FET. Results: MRI confirmed tumor take in one model as early as 2 weeks after intracranial implantation. Increased 18F-FET uptake was detected in all models. MRI could be effectively used to monitor tumor growth and the corresponding 18F-FET PET images demonstrated increased 18F-FET uptake over time. Conclusion: Three different HER2 positive intracranial PDX breast metastases models were established from low passage SQ PDX models. We suggest, that using these intracranial PDX models of brain metastases, new drugs for advanced breast cancer can be evaluated in preclinical models that more closely mimic the microenvironment and the BBB in patients. In addition, translational imaging techniques can be evaluated during preclinical testing and the potential of tracers like 18F-FET as imaging biomarkers of therapeutic response can be assessed. Together, the established SQ and orthotopic PDX models of breast cancer and brain metastases can be used as a relevant translational platform for testing of new drugs. Citation Format: Nielsen CH, Nedergaard MK, Wick MJ, Papadopoulos K, Tolcher AW, Kjaer A. Intracranial PDX models of breast cancer metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-03-01.

Abstract 5187: PET imaging of trastuzumab emtansine (T-DM1) drug delivery to intracranial patient derived xenograft (PDX) models of breast cancer metastasis

Background: It is estimated that 10-30% of all breast cancer patients at some point develop brain metastases. Overexpression of the human epidermal growth factor receptor 2 (HER2) is a independent risk factor for development of brain metastases. Up to 37% of patients with HER2-positive metastatic breast cancer develop brain metastases and half of these patients die as a result of failure to control the intracranial disease. A reason for this is the challenge to obtain efficient drug delivery across the blood brain barrier (BBB). Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, when targeting brain tumors or metastases, the major impact of the BBB on drug bioavailability must be taken into consideration. Clinical PET imaging with 64Cu or 89Zr labeled trastuzumab has previously been able to visualize breast cancer brain metastases. The aim of our study was to investigate if PET imaging with 64Cu-labeled trastuzumab was predictive of the efficacy of trastuzumab emtansine (T-DM1) in a HER2 positive breast cancer PDX model established as an intracranial brain metastases model. Methods: The intracranial PDX model was established by stereotactic intracranial injection of enzymatically digested ST1339 tumor tissue. At confirmed tumor take, mice were randomized into two arms: control and T-DM1 (10 mg/kg/week x4). Treatment response was monitored by contrast-enhanced T1- and T2-weighted Magnetic Resonance Imaging (MRI) and positron emission tomography (PET) with the amino acid radiotracer O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET). PET/CT imaging with 64Cu-trastuzumab was performed in animals with confirmed intracranial ST1339 tumors prior to treatment with T-DM1 (10 mg/kg/week x4). Results: T-DM1 treatment with 10 mg/kg/week x4 of mice with intracranial tumors inhibited tumor growth and prolonged survival compared to non-treated animals. A variable response within the treatment group was observed. Forty percent had tumor shrinkage while 60% exhibited tumor growth within the duration of the therapy. The intracranial tumors were clearly visible on the 64Cu-trastuzumab PET images co-registered with T2-weighted MR images for anatomical localization. Interestingly, the intracranial tumor uptake between the animals was rather heterogeneous. Conclusion: A treatment response to T-DM1 was observed in an intracranial ST1339 HER2 positive breast cancer PDX model. PET imaging with 64Cu-trastuzumab confirmed delivery of trastuzumab to the tumors. Further quantitative image analysis of the intracranial 64Cu-trastuzumab tumor uptake will reveal if the drug delivery measured by 64Cu-trastuzumab PET imaging is predictive of the efficacy of T-DM1 in a HER2 positive PDX breast cancer brain metastases model. Citation Format: Carsten Haagen Nielsen, Mette K. Nedergaard, Lotte K. Kristensen, Camilla S. Knudsen, Michael J. Wick, Kyri Papadopoulos, Anthony Tolcher, Andreas Kjaer. PET imaging of trastuzumab emtansine (T-DM1) drug delivery to intracranial patient derived xenograft (PDX) models of breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5187.

Abstract A15: Efficacy of trastuzumab emtansine (T-DM1) in subcutaneous and intracranial patient derived xenograft models of breast cancer metastasis

Background: It is estimated that 10-30% of all breast cancer patients at some point develop brain metastases. Overexpression of the human epidermal growth factor receptor 2 (HER2) is a independent risk factor for development of brain metastases. Up to 37% of patients with HER2-positive metastatic breast cancer develop brain metastases and half of these patients die as a result of failure to control the intracranial disease. A reason for this is the challenge of efficient drug delivery across the blood brain barrier (BBB). Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, when targeting brain tumors or metastases, the major impact of the BBB on drug bioavailability must be taken into consideration. The aim of this study was therefore to compare the efficacy of trastuzumab emtansine (T-DM1) in a HER2 positive breast cancer PDX model established subcutaneously and as an intracranial brain metastases model. Methods: Mice were implanted subcutaneously with the HER2 positive breast cancer PDX model designated ST1339 and randomized into 3 treatment arms: Control, T-DM1 (5 mg/kg/week x4) and T-DM1 (10 mg/kg/week x4). Treatment response of subcutaneous tumors was monitored by caliper measurements. The intracranial PDX model was established by stereotactic intracranial injection of enzymatically digested ST1339 tumor tissue. At confirmed tumor take, mice were randomized into two arms: Control and T-DM1 (10 mg/kg/week x4). Treatment response was monitored by contrast-enhanced T1- and T2-weighted Magnetic Resonance Imaging (MRI) and positron emission tomography (PET) with the amino acid radiotracer O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET). Results: T-DM1 at 10 mg/kg/week x4 effectively inhibited tumor growth in the subcutaneous model whereas treatment with 5 mg/kg/week x4 did not have any effect on tumor growth. T-DM1 treatment at 10 mg/kg/week x4 of mice with intracranial tumors inhibited tumor growth and prolonged survival compared to non-treated animals. Conclusion: A treatment response to T-DM1 was observed in both the subcutaneous and intracranial ST1339 HER2 positive breast cancer PDX model. With the combination of subcutaneous and intracranial PDX models of breast cancer and breast cancer brain metastases new drugs can thus be tested in preclinical models that more closely mimic the microenvironment and the challenges of drug delivery across the BBB in patients. Citation Format: Carsten H. Nielsen, Mette K. Nedergaard, Lotte K. Kristensen, Camilla S. Knudsen, Michael J. Wick, Kyri Papadopoulos, Anthony W. Tolcher, Andreas Kjaer. Efficacy of trastuzumab emtansine (T-DM1) in subcutaneous and intracranial patient derived xenograft models of breast cancer metastasis. [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 A15.