Mariska Sie

Anti-angiogenic therapy in pediatric brain tumors: An effective strategy?

Brain tumors are still the leading cause of cancer morbidity and mortality among children, despite different therapeutic options including neurosurgery, chemotherapy and radiation. As angiogenesis is highly crucial in brain tumor growth and progression, numerous clinical trials evaluating diverse anti-angiogenic agents have been described. In the present review, we aimed to answer the question if anti-angiogenic therapy is an effective strategy in the treatment of children with brain tumors. Although some encouraging results have been published of anti-angiogenic therapy targeting vascular endothelial growth factor (VEGF)/VEGF receptor signaling or epidermal growth factor receptor (EGFR), still more insight is warranted to be highly conclusive about the efficacy of anti-angiogenic therapy with currently potential upcoming anti-angiogenic agents in pediatric brain tumors. However, given the need for new therapeutic strategies, multi targeted therapy with anti-angiogenic agents anticipating on possible tumor escape mechanisms could be effective in the future treatment of pediatric brain tumors.

Tumor vessel biology in pediatric intracranial ependymoma Clinical article

OBJECT This study aimed to characterize the pediatric intracranial ependymoma vasculature in terms of angiogenic activity and maturation status so as to provide indications for the applicability of vessel-targeted therapy in cases of pediatric intracranial ependymoma. METHODS Tumor samples obtained in patients with ependymomas were immunohistochemically (double) stained for Ki 67/CD34, caspase 3a/CD34, vascular endothelial growth factor (VEGF)-A, -B, -C, -D, collagen Type IV, and smooth muscle actin to determine microvessel density, tumor and endothelial cell proliferation and apoptotic fraction, the relative expression of VEGF family members, and the coverage of the tumor endothelial cells by basal membrane and pericytes. Messenger RNA expression of angiopoietin-1 and -2 was analyzed by real-time reverse transcriptase polymerase chain reaction. These data were compared with those obtained in a glioblastoma series. RESULTS Despite a low endothelial cell turnover, the microvessel density of ependymomas was similar to that of glioblastomas. In ependymomas the expression of VEGF-A was within the range of the variable expression in glioblastomas. The staining intensities of VEGF-B, -C, and -D in ependymomas were significantly lower (p < 0.001). The expression of angiopoietin-1 was higher in ependymomas than in glioblastomas (p = 0.03), whereas angiopoietin-2 expression was similar. The coverage of tumor endothelial cells with basal membrane and pericytes was more complete in ependymomas (p = 0.009 and p = 0.022, respectively). CONCLUSIONS The ependymoma vasculature is relatively mature and has little angiogenic activity compared with malignant gliomas. Therefore, the window for vessel normalization as a therapeutic aim might be considered small. However, the status of the tumor vasculature may not be a reliable predictor of treatment effect. Therefore, possible benefits of antiangiogenic treatment cannot be excluded beforehand in patients with ependymomas.

Growth-Factor-Driven Rescue to Receptor Tyrosine Kinase (RTK) Inhibitors through Akt and Erk Phosphorylation in Pediatric Low Grade Astrocytoma and Ependymoma

Up to now, several clinical studies have been started investigating the relevance of receptor tyrosine kinase (RTK) inhibitors upon progression free survival in various pediatric brain tumors. However, single targeted kinase inhibition failed, possibly due to tumor resistance mechanisms. The present study will extend our previous observations that vascular endothelial growth factor receptor (VEGFR)-2, platelet derived growth factor receptor (PDGFR)β, Src, the epidermal growth factor receptor (ErbB) family, and hepatocyte growth factor receptor (HGFR/cMet) are potentially drugable targets in pediatric low grade astrocytoma and ependymoma with investigations concerning growth-factor-driven rescue. This was investigated in pediatric low grade astrocytoma and ependymoma cell lines treated with receptor tyrosine kinase (RTK) inhibitors e.g. sorafenib, dasatinib, canertinib and crizotinib. Flow cytometry analyses showed high percentage of cells expressing VEGFR-1, fibroblast growth factor receptor (FGFR)-1, ErbB1/EGFR, HGFR and recepteur d’origine nantais (RON) (respectively 52-77%, 34-51%, 63-90%, 83-98%, 65-95%). Their respective inhibitors induced decrease of cell viability, measured with WST-1 cell viability assays. At least this was partially due to increased apoptotic levels measured by Annexin V/Propidium Iodide apoptosis assays. EGF, HGF and FGF, which are normally expressed in brain (tumor) tissue, showed to be effective rescue inducing growth factors resulting in increased cell survival especially during treatment with dasatinib (complete rescue) or sorafenib (partial rescue). Growth-factor-driven rescue was less prominent when canertinib or crizotinib were used. Rescue was underscored by significantly activating downstream Akt and/or Erk phosphorylation and increased tumor cell migration. Combination treatment showed to be able to overcome the growth-factor-driven rescue. In conclusion, our study highlights the extensive importance of environmentally present growth factors in developing tumor escape towards RTK inhibitors in pediatric low grade astrocytoma and ependymoma. It is of great interest to anticipate upon these results for the design of new therapeutic trials with RTK inhibitors in these pediatric brain tumors.

Abstract B4: Growth-factor-driven rescue to receptor tyrosine kinase inhibitors in pediatric low-grade astrocytoma and ependymoma

Introduction: Brain tumors are still the leading cause of cancer morbidity and mortality among children, despite different therapeutic options including neurosurgery, chemotherapy and radiation. Up to now, several clinical studies have been started investigating the importance of receptor tyrosine kinase (RTK) inhibitors upon progression free survival in various pediatric brain tumors. However, it has been shown that single targeted kinase inhibition failed due to tumor resistance mechanisms. The present study will extend our previous observations that vascular endothelial growth receptor (VEGFR) 2, platelet derived growth factor receptor (PDGFR) β, Src, the epidermal growth factor receptor (ErbB) family, and hepatocyte growth factor receptor (HGFR/cMet) are potential drugable targets in pediatric low grade astrocytoma and ependymoma with investigations concerning growth-factor-driven rescue. Material and methods: Expression of various RTKs in pediatric low grade astrocytoma (Res-186, Res-259, UW-467) and ependymoma (Res-196) cell lines were determined using flow cytometry analyses. Growth-factor-driven rescue during RTK inhibition with agents including sorafenib, dasatinib, canertinib and crizotinib were analyzed on cell viability level measured with WST-1 cell viability assays and on phosphorylation level of the crucial downstream PI3K/Akt and MAPK/Erk survival signaling pathways using western blot analyses. Results: VEGFR-1, fibroblast growth factor receptor (FGFR) 1, ErbB-1, HGFR and recepteur d9origine nantais (RON) were highly expressed (respectively 52-77%, 34-51%, 63-90%, 83-98%, 65-95%) and their respective inhibitors showed induced decrease of tumor cell viability. EGF, HGF and FGF, which are normally environmentally expressed in brain (tumor) tissue, showed to be effective rescue inducing growth factors resulting in increased cell survival especially during treatment with dasatinib (complete rescue) or sorafenib (partial rescue) in both low grade astrocytoma and ependymoma. Growth-factor-driven rescue was less prominent when canertinib or crizotinib were used. Rescue was underscored by activating downstream Akt and/or Erk phosphorylation. Combination treatment showed to be able to overcome the growth-factor-driven resistance. Conclusions: In conclusion, our study highlights the extensive importance of environmentally present growth factors in developing resistance towards RTK inhibitors. It is of great interest to anticipate upon these results for the design of new therapeutic trials with RTK inhibitors in pediatric low grade astrocytoma and ependymoma. Citation Format: Mariska Sie, Wilfred F.A. den Dunnen, Harm Jan Lourens, Tiny G.J. Meeuwsen-de Boer, Frank J.G. Scherpen, Kim R. Kampen, Eelco W. Hoving, Eveline S.J.M. de Bont. Growth-factor-driven rescue to receptor tyrosine kinase inhibitors in pediatric low-grade astrocytoma and ependymoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B4.

Less tumor engraftment after anti-VEGF therapy in pediatric low grade astrocytoma

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction. Low grade astrocytomas are the most frequent brain tumors in children. Although children can benefit from treatments including neurosurgery, chemotherapy and radiotherapy, still children die due to tumor progression. Furthermore, morbidity can be serious. So new therapeutic strategies are warranted for these patients. Previously, we showed that angiogenesis is a characteristic of pediatric low grade astrocytoma (Sie et al, NAN 2010). Therefore, vascular endothelial growth factor (VEGF), the most critical angiogenic factor, may be a potential therapeutic target. The present study aimed to analyze the effect of VEGF inhibition in vitro and in vivo in pediatric low grade astrocytoma. Materials and methods. Three different pediatric low grade astrocytoma cell lines were used: Res-186 (WHO grade I astrocytoma), Res-259 and UW-467 (WHO grade II astrocytoma). Effects in vitro of the anti-VEGF monoclonal antibodies, bevacizumab (0 - 50 ug/ml, Avastin®, anti human VEGF) and B20-4.1.1 (0 - 200 ug/ml, anti human and mouse VEGF) were studied using a WST-1 cell viability assay and a cell proliferation assay with BRDU. Res-259 cells were orthotopically implanted in NOD-scid IL2Rgnull mice. After 6 weeks of treatment intraperitoneal twice weekly with bevacizumab (15 mg/kg), B20-4.1.1 (5 mg/kg) or phosphate buffered saline as control group, mice were euthanized and tumor engraftment was studied in brain slides using Aperios imagescope viewer. Results. In vitro cell growth was not changed as measured by WST-1 and BRDU incorporation. In vivo results showed less tumor engraftment in bevacizumab (n = 10) and B20-4.1.1 (n = 9) treated mice compared with the control group (n = 10) (resp. 70%, 44%, 100%). In the bevacizumab and B20-4.1.1 treated mice tumor mass was lower as compared with controls (resp. P = 0.062, P = 0.001). Conclusion(s). This study showed less tumor engraftment after anti-VEGF therapy in a newly developed pediatric low grade astrocytoma mouse model. In these tumors anti-VEGF seems to work especially on tumor microenvironment. Suggesting that anti-VEGF could have a potential role in the therapeutic strategy for children with low grade astrocytoma, thoughtfulness on possible tumor escape mechanisms that may arise will be crucial. [W.F.A. den Dunnen and E.S.J.M. de Bont shared senior authorship.] Citation Format: Mariska Sie, Arend H. Sikkema, Frank J.G. Scherpen, Arja ter Elst, Kim R. Kampen, Eelco W. Hoving, Wilfred F.a. den Dunnen, Eveline S.j.m. de Bont. Less tumor engraftment after anti-VEGF therapy in pediatric low grade astrocytoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5085. doi:10.1158/1538-7445.AM2013-5085

Overlapping high pro- and anti-angiogenic VEGF-A isoform ratio between pediatric pilocytic astrocytoma and adult glioblastoma suggests possibilities for anti-angiogenic therapy in pilocytic astrocytoma

Introduction. Pilocytic astrocytoma (WHO grade I), the most frequent brain tumor in children, showed a critical overlap with the angiogenic profile and vessel immaturity/instability of glioblastoma (WHO grade IV). However, the highly vascular pilocytic astrocytoma had fewer but wider vessels compared with glioblastoma,(Sie et al. NAN 2010) possibly due to different VEGF-A isoform expression. So this study aimed to analyze VEGF-A isoforms in correlation with tumor vasculature in pilocytic astrocytoma and glioblastoma. As it is hypothesized that tumors with relatively higher pro- (VEGF-A XXX a) than anti-angiogenic (VEGF-A XXX b) isoform expression will be more sensitive to anti-angiogenic therapy, because VEGF-A XXX b inhibits the effect of anti-VEGF-A antibodies like bevacizumab, we secondly aimed to determine the VEGF-A XXX a: VEGF-A XXX b ratio. Materials and methods. VEGF-A isoforms were analyzed in tumor tissue of 15 pediatric pilocytic astrocytoma and 12 adult glioblastoma patients using Real Time RT-PCR. Microvessel density, vessel maturity in terms of basement membrane and pericyte coverage, and endothelial and tumor cell turnover were evaluated in immunohistochemically stained slides with CD34, collagen IV, smooth muscle actin, Ki67/CD34 and caspase-3/CD34 respectively. The angiopoietin-1/-2 balance as indicator for vessel stability was assessed using Real Time RT-PCR. Results. All pro-angiogenic VEGF-A isoforms (VEGF-A 121 a, 145 , 148 , 165 , 183 , 189 ) were lower expressed in pilocytic astrocytoma compared with glioblastoma. However, analyzing these isoforms relative to VEGF-A 121 a as it is expressed by normal cells, no significant differences were found between both tumors. Pilocytic astrocytoma showed an association between VEGF-A 165 a and their dilatated vessels, while VEGF-A 189 a was associated with the characteristic small blood vessels in glioblastoma. In both tumors, VEGF-A 189 a was correlated with less vessel immaturity (r: 0.621, P = 0.024; r: 0.697, P = 0.025). Surprisingly, pilocytic astrocytoma and glioblastoma showed an overlapping VEGF-A XXX a: VEGF-A XXX b ratio with relatively higher VEGF-A 165 a and VEGF-A 189 a expression (P = 0.486; P = 0.456). Conclusion(s). This study showed that VEGF-A isoforms were associated with the different vessel architecture in pilocytic astrocytoma and glioblastoma. Interestingly, a clear overlap was found between both tumors in the VEGF-A isoform ratios with relatively high VEGF-A 165 a and VEGF-A 189 a expression. These findings suggest encouraging possibilities for anti-angiogenic therapy, for instance with bevacizumab, in pilocytic astrocytoma, especially for those children with unresectable tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1381. doi:1538-7445.AM2012-1381

Abstract 1306: Tumor vasculature and angiogenic profile of pediatric pilocytic astrocytoma

Introduction: Pilocytic astrocytomas (WHO, grade I astrocytomas) are the most frequent brain tumors in children. Despite a 5-year survival of 90%, outcome is not always favourable, mainly because of surgical morbidity and tumor progression. To reduce this morbidity as well as mortality, new therapeutic modalities are needed. As target in cancer treatment, angiogenesis is extensively investigated, and formed an effective and well tolerated treatment in recurrent adult glioblastomas (WHO, grade IV astrocytomas). However, little is known about angiogenesis in low grade pediatric astrocytomas. So this study aimed to characterize tumor vasculature and the angiogenic profile of 59 pediatric pilocytic astrocytomas to obtain insights into potential angiogenic related therapeutic targets in these tumors. Materials and methods: Microvessel density (MVD), vessel maturity in terms of basement membrane and pericytes coverage, and turnover of both endothelial and tumor cells, and vascular endothelial growth factor (VEGF) expression were evaluated in tumor tissue, immunohistochemically stained with respectively CD34, collagen IV, Smooth Muscle Actin (SMA), Ki67/CD34, caspase-3/CD34, and VEGF(-A-D). Collagen IV and SMA slides were quantified using morphometry software. As indicator for vessel stabilization, the angiopoietin (ANGPT)-1/ANGPT-2 balance was calculated using Real Time RT-PCR. These data were compared with previously described 62 adult glioblastomas,(Sie et al, J Neurosurg 2009) often seen as a model for studying angiogenesis. Results: Pilocytic astrocytoma had fewer but wider vessels than glioblastoma. Slightly thicker pericyte coverage and a higher ANGPT-1/ANGPT-2 balance were seen in pilocytic astrocytoma (respectively p = 0.016, and p = 0.001). Turnover of endothelial and tumor cells were lower in pilocytic astrocytoma (p Conclusion: Despite the fact that pilocytic astrocytomas showed slightly less immature and instable vasculature compared with glioblastoma, a large overlap in the angiogenic profile was seen between both tumors. These findings suggest promising possibilities for targeting angiogenesis with anti-VEGF as therapeutic strategy in pilocytic astrocytoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1306.