Thomas Urup

A comprehensive profile of recurrent glioblastoma

In spite of relentless efforts to devise new treatment strategies, primary glioblastomas invariably recur as aggressive, therapy-resistant relapses and patients rapidly succumb to these tumors. Many therapeutic agents are first tested in clinical trials involving recurrent glioblastomas. Remarkably, however, fundamental knowledge on the biology of recurrent glioblastoma is just slowly emerging. Here, we review current knowledge on recurrent glioblastoma and ask whether and how therapies change intra-tumor heterogeneity, molecular traits and growth pattern of glioblastoma, and to which extent this information can be exploited for therapeutic decision-making. We conclude that the ability to characterize and predict therapy-induced changes in recurrent glioblastoma will determine, whether, one day, glioblastoma can be contained in a state of chronic disease.

The impact of bevacizumab treatment on survival and quality of life in newly diagnosed glioblastoma patients.

Glioblastoma multiforme (GBM) remains one of the most devastating tumors, and patients have a median survival of 15 months despite aggressive local and systemic therapy, including maximal surgical resection, radiation therapy, and concomitant and adjuvant temozolomide. The purpose of antineoplastic treatment is therefore to prolong life, with a maintenance or improvement of quality of life. GBM is a highly vascular tumor and overexpresses the vascular endothelial growth factor A, which promotes angiogenesis. Preclinical data have suggested that anti-angiogenic treatment efficiently inhibits tumor growth. Bevacizumab is a humanized monoclonal antibody against vascular endothelial growth factor A, and treatment has shown impressive response rates in recurrent GBM. In addition, it has been shown that response is correlated to prolonged survival and improved quality of life. Several investigations in newly diagnosed GBM patients have been performed during recent years to test the hypothesis that newly diagnosed GBM patients should be treated with standard multimodality treatment, in combination with bevacizumab, in order to prolong life and maintain or improve quality of life. The results of these studies along with relevant preclinical data will be described, and pitfalls in clinical and paraclinical endpoints will be discussed.

Angiotensinogen and HLA class II predict bevacizumab response in recurrent glioblastoma patients

Bevacizumab combination therapy is among the most frequently used treatments in recurrent glioblastoma and patients who achieve response to bevacizumab have improved survival as well as quality of life. Accordingly, the aim of this study was to identify predictive biomarkers for bevacizumab response in recurrent glioblastoma patients.

18F-FET MicroPET and MicroMRI for Anti-VEGF and Anti-PlGF Response Assessment in an Orthotopic Murine Model of Human Glioblastoma

Background Conflicting data exist for anti-cancer effects of anti-placental growth factor (anti-PlGF) in combination with anti-VEGF. Still, this treatment combination has not been evaluated in intracranial glioblastoma (GBM) xenografts. In clinical studies, position emission tomography (PET) using the radiolabeled amino acid O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) and magnetic resonance imaging (MRI) add complementary but distinct information about glioma growth; however, the value of 18F-FET MicroPET combined with MicroMRI has not been investigated preclinically. Here we examined the use of 18F-FET MicroPET and MicroMRI for evaluation of anti-VEGF and anti-PlGF treatment response in GBM xenografts. Methods Mice with intracranial GBM were treated with anti-VEGF, anti-PlGF + anti-VEGF or saline. Bioluminescence imaging (BLI), 18F-FET MicroPET and T2-weighted (T2w)-MRI were used to follow tumour development. Primary end-point was survival, and tumours were subsequently analysed for Ki67 proliferation index and micro-vessel density (MVD). Further, PlGF and VEGFR-1 expression were examined in a subset of the xenograft tumours and in 13 GBM patient tumours. Results Anti-VEGF monotherapy increased survival and decreased 18F-FET uptake, BLI and MVD, while no additive effect of anti-PlGF was observed. 18F-FET SUVmax tumour-to-brain (T/B) ratio was significantly lower after one week (114±6%, n = 11 vs. 143±8%, n = 13; p = 0.02) and two weeks of treatment (116±12%, n = 8 vs. 190±24%, n = 5; p = 0.02) in the anti-VEGF group as compared with the control group. In contrast, T2w-MRI volume was unaffected by anti-VEGF. Gene expression of PlGF and VEGFR-1 in xenografts was significantly lower than in patient tumours. Conclusion 18F-FET PET was feasible for anti-angiogenic response evaluation and superior to T2w-MRI; however, no additive anti-cancer effect of anti-PlGF and anti-VEGF was observed. Thus, this study supports use of 18F-FET PET for response evaluation in future studies.

Development and validation of a prognostic model for recurrent glioblastoma patients treated with bevacizumab and irinotecan

Abstract Background Predictive markers and prognostic models are required in order to individualize treatment of recurrent glioblastoma (GBM) patients. Here, we sought to identify clinical factors able to predict response and survival in recurrent GBM patients treated with bevacizumab (BEV) and irinotecan. Material and methods A total of 219 recurrent GBM patients treated with BEV plus irinotecan according to a previously published treatment protocol were included in the initial population. Prognostic models were generated by means of multivariate logistic and Cox regression analysis. Results In multivariate analysis, corticosteroid use had a negative predictive impact on response at first evaluation (OR 0.45; 95% CI 0.22–0.93; p = 0.03) and at best response (OR 0.51; 95% CI 0.26–1.02; p = 0.056). Three significant (p < 0.05) prognostic factors associated with reduced progression-free survival and overall survival (OS) were identified. These factors were included in the final model for OS, namely corticosteroid use (HR 1.70; 95% CI 1.18–2.45; p = 0.004), neurocognitive deficit (HR 1.40; 95% CI 1.04–1.89; p = 0.03) and multifocal disease (HR 1.56; 95% CI 1.15–2.11; p < 0.0001). Based on these results a prognostic index able to calculate the probability for OS at 6 and 12 months for the individual patient was established. The predictive value of the model for OS was validated in a separate patient cohort of 85 patients. Discussion and conclusion A prognostic model for OS was established and validated. This model can be used by physicians to risk stratify the individual patient and together with the patient decide whether to initiate BEV relapse treatment.

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.

Biomarkers in Recurrent Grade III Glioma Patients Treated with Bevacizumab and Irinotecan

ABSTRACT Predictive biomarkers and prognostic models are required to identify recurrent grade III glioma patients who benefit from existing treatment. In this study of 62 recurrent grade III glioma patients, a range of clinical and paraclinical factors are tested for association with progression-free survival, overall survival, and response to bevacizumab and irinotecan therapy. Significant factors from univariate screening are included in multivariate analysis. Biomarkers previously advanced as predictive or prognostic in the first-line setting did not affect outcome in this patient cohort. Based on the optimized model for overall survival, comprising performance status and p53 expression, a prognostic index is established.

DNA Methylation Levels of the ELMO Gene Promoter CpG Islands in Human Glioblastomas

Complete surgical resection of glioblastoma is difficult due to the invasive nature of this primary brain tumor, for which the molecular mechanisms behind remain poorly understood. The three human ELMO genes play key roles in cellular motility, and have been linked to metastasis and poor prognosis in other cancer types. The aim of this study was to investigate methylation levels of the ELMO genes and their correlation to clinical characteristics and outcome in patients diagnosed with glioblastoma. To measure DNA methylation levels we designed pyrosequencing assays targeting the promoter CpG island of each the ELMO genes. These were applied to diagnostic tumor specimens from a well-characterized cohort of 121 patients who received standard treatment consisting of surgery, radiation therapy, plus concomitant and adjuvant chemotherapy. The promoter methylation levels of ELMO1 and ELMO2 were generally low, whereas ELMO3 methylation levels were high, in the tumor biopsies. Thirteen, six, and 18 biopsies were defined as aberrantly methylated for ELMO1, ELMO2, and ELMO3, respectively. There were no significant associations between the methylation status of any of the ELMO gene promoter CpG islands and overall survival, progression-free survival, and clinical characteristics of the patients including intracranial tumor location. Therefore, the methylation status of the ELMO gene promoter CpG islands is unlikely to have prognostic value in glioblastoma.

Abstract A25: Predictive biomarkers for bevacizumab response in recurrent glioblastoma patients

BACKGROUND: Bevacizumab (BEV) plus chemotherapy has shown high response rates in recurrent glioblastoma (GBM) and patients who achieve response have an improved overall survival as well as quality of life. Recent retrospective analysis of the randomized phase III trial, AVAglio, indicate that patients with the proneural GBM subtype have a survival benefit when treated with BEV in combination with standard treatment. However, no validated biomarkers able to predict BEV response have been identified and the biology reflecting a clinical BEV response is poorly understood. The primary objective of this study was to evaluate the predictive and prognostic value of GBM subtypes in recurrent GBM patients treated with BEV therapy. The secondary objective was to identify biomarkers able to predict response to BEV therapy in recurrent GBM patients. METHODS: A total of 90 recurrent GBM patients treated with BEV combination treatment according to a previously published treatment protocol were included. Inclusion criteria: BEV plus irinotecan treatment in the period between May 2005-2011; available GBM tissue (according to WHO); response evaluable (RANO). RNA from tumor tissue was analyzed by the NanoString platform covering 800 genes. Raw data was assigned to molecular subtypes for each of the samples using the PAMR classifier model, previously trained on the AVAglio dataset. In order to identify novel candidate biomarkers able to predict response, differentially expressed genes (fold-change difference > 1.5) between patients responding versus progressing on BEV were identified using a t-test. Biomarkers significantly (P Citation Format: Thomas Urup, Signe Regner Michaelsen, Lars Ronn Olsen, Anders Toft, Ib Jarle Christensen, Kirsten Grunnet, Helle Broholm, Michael Kosteljanetz, Shohreh Issazadeh-Navikas, Hans Skovgaard Poulsen, Ulriik Lassen. Predictive biomarkers for bevacizumab response in recurrent glioblastoma patients. [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 A25.

Abstract B3: Prognostic and predictive biomarkers in recurrent WHO grade 3 malignant glioma patients treated with bevacizumab and irinotecan

BACKGROUND Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor A (VEGF-A), has demonstrated activity in the treatment of recurrent malignant glioma. High response rates have been observed, but particularly in WHO grade 3 gliomas, efforts to identify predictors of clinical response have been limited. Predictive markers and prognostic models are required in order to individualize treatment for this patient population. The primary endpoint of this study was to identify predictive biomarkers associated with response to bevacizumab therapy. The secondary endpoint was to identify prognostic factors associated with progression-free survival (PFS) and overall survival (OS). METHODS A total of 62 consecutive, recurrent grade 3 glioma patients were retrospectively evaluated. Eligible patients from our center had a WHO performance status of 0-2 and were administered bevacizumab and irinotecan between December 2005 and November 2014 according to a previously published clinical protocol. Baseline factors screened for potential prognostic and predictive value included: Age, gender, PS, WHO grade 3 diagnosis, tumor size and location, multifocal disease, extent of resection, number of prior chemotherapy regiments, response to prior chemotherapy, first-line treatment, number of previous recurrences, neurological deficit, corticosteroid use, necrosis, vascular proliferation, neutrophil-to-lymphocyte ratio, and expression of p53, EGFR, MIB-1, MGMT, IDH1 and ATRX. Candidate factors with p-values below 5% were considered for multivariate analysis. Prognostic models were generated by logistic regression and Cox regression, modelling response and survival endpoints. RESULTS Twenty-two patients (35.5%) demonstrated a response according to the RANO criteria. Responders had significantly prolonged OS (p = 0.007) and trended toward longer PFS (p = 0.067) as compared to non-responders (OS: 12.4 vs 4.3 months, PFS: 5.6 vs 3.2 months). Presence of necrosis (OR: 0.17, CI: 0.04-0.68, p = 0.012) and a WHO performance status (PS) of more than 1 (OR: 0.04, CI: 0.002-0.89, p = 0.042) were more common in non-responders than responders. Female gender (HR: 0.48, CI: 0.28-0.82, p = 0.008) and a PS of 0-1 (HR: 0.20, CI: 0.10-0.41, p CONCLUSIONS A favorable baseline PS and absence of necrosis were positively associated with response to bevacizumab treatment in recurrent grade 3 glioma patients. Low PS, female gender and p53 negativity are prognostic of improved outcome in this patient group. Citation Format: Anders Toft, Thomas Urup, Ib J. Christensen, Signe R. Michaelsen, Babloo S. Lukram, Kirsten Grunnet, Michael Kosteljanetz, Vibeke A. Larsen, Ulrik Lassen, Helle Broholm, Hans S. Poulsen. Prognostic and predictive biomarkers in recurrent WHO grade 3 malignant glioma patients treated with bevacizumab and irinotecan. [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 B3.