Clinically relevant glioblastoma patient-derived xenograft models to guide drug development and identify molecular signatures.

Glioblastoma (GBM) heterogeneity, aggressiveness and infiltrative growth drastically limit success of current standard of care drugs and efficacy of various new therapeutic approaches. There is a need for new therapies and models reflecting the complex biology of these tumors to analyze the molecular mechanisms of tumor formation and resistance, as well as to identify new therapeutic targets. We established and screened a panel of 26 patient-derived subcutaneous (s.c.) xenograft (PDX) GBM models on immunodeficient mice, of which 15 were also established as orthotopic models. Sensitivity toward a drug panel, selected for their different modes of action, was determined. Best treatment responses were observed for standard of care temozolomide, irinotecan and bevacizumab. Matching orthotopic models frequently show reduced sensitivity, as the blood-brain barrier limits crossing of the drugs to the GBM. Molecular characterization of 23 PDX identified all of them as IDH-wt (R132) with frequent mutations in EGFR, TP53, FAT1, and within the PI3K/Akt/mTOR pathway. Their expression profiles resemble proposed molecular GBM subtypes mesenchymal, proneural and classical, with pronounced clustering for gene sets related to angiogenesis and MAPK signaling. Subsequent gene set enrichment analysis identified hallmark gene sets of hypoxia and mTORC1 signaling as enriched in temozolomide resistant PDX. In models sensitive for mTOR inhibitor everolimus, hypoxia-related gene sets reactive oxygen species pathway and angiogenesis were enriched. Our results highlight how our platform of s.c. GBM PDX can reflect the complex, heterogeneous biology of GBM. Combined with transcriptome analyses, it is a valuable tool in identification of molecular signatures correlating with monitored responses. Available matching orthotopic PDX models can be used to assess the impact of the tumor microenvironment and blood-brain barrier on efficacy. Our GBM PDX panel therefore represents a valuable platform for screening regarding molecular markers and pharmacologically active drugs, as well as optimizing delivery of active drugs to the tumor.

Role of mTOR and VEGFR Inhibition in Prevention of Metastatic Tumor Growth in the Spine.

Objective: Spinal metastatic disease remains a major problem of oncological diseases. Patients affected may suffer pain, spinal instability, and severe neurological deficits. Today, palliative surgery and radiotherapy are the mainstays of therapy. In contrast, preventive treatment strategies or treatment concepts for an early stage are lacking. Here, we have used a syngeneic, experimental spine metastases model in the mouse to test the efficacy of mTOR inhibition and anti-angiogenesis on the formation and progression of spinal melanoma metastases. Methods: We used our previously established syngeneic spinal metastases mouse model by injecting luciferin-transfected B16 melanoma cells into the common carotid artery. Following injection, mice were treated with everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) complex, axitinib, a tyrosine kinase inhibitor, that blocks vascular endothelial growth factor receptors (VEGFR) 1-3, as well as placebo. Animals were followed-up daily by neurological assessment and by repeat in vivo bioluminescence imaging. With occurrence of neurological deficits, a spinal MRI was performed, and mice were sacrificed. The whole spine was dissected free and analyzed by immunohistochemical techniques. Results: Overall survival was 23 days in the control group, significantly prolonged to 30 days (p = 0.04) in the everolimus group, and to 28 days (p = 0.04) in the axitinib group. While 78% of mice in the placebo group developed symptomatic metastatic epidural spinal cord compression, only 50% did so in the treatment groups. The mean time to manifestation of paralysis was 22 days in the control group, 26 days (p = 0.10) in the everolimus group, and 27 days (p = 0.06) in the axitinib group. Screening for spinal metastases by bioluminescence imaging on two different time points showed a decrease in metastatic tumor formation in the treatment groups compared to the controls. Immunohistochemical analysis confirmed the bioactivity of the two compounds: The Ki67 proliferation labeling index was reduced in the everolimus group and numbers of CD31 positive endothelial cells were reduced in the axitinib group. Conclusion: Both, the mTOR inhibitor everolimus as well as antiangiogenetic effects by the VEGFR inhibitor axitinib showed potential to prevent and retard formation of symptomatic spinal metastases. However, the therapeutic efficacy was only mild in this experimental model.

Treatment with 5-azacitidine delay growth of glioblastoma xenografts: a potential new treatment approach for glioblastomas.

PURPOSE: Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults. The epigenetically active ribonucleoside analog 5-azacitidine is a new therapy option that changes tumor cell chromatin, which is frequently modified by methylation and deacetylation in malignant gliomas. METHODS: In vitro, we analyzed cell viability, cell apoptosis, and migration of human GBM cells. In vivo, we established subcutaneous and intracerebral GBM mouse models originating from U87MG, U373MG, and primary GBM cells as well as one patient-derived xenograft. Xenografts were treated with 5-azacitidine as well as valproic acid, bevacizumab, temozolomide, and phosphate buffered saline. The tumor sizes and Ki67 proliferation indices were determined. Glioma angiogenesis was examined immunohistochemically by expression analysis of endothelial cells (CD31) and pericytes (PDGFRß). RESULTS: In vitro, 5-azacitidine treatment significantly reduced human glioblastoma cell viability, increased cellular apoptosis, and reduced cellular migration. In vivo, 5-azacitidine significantly reduced growth in two intracerebral GBM models. Notably, this was also shown for a xenograft established from a patient surgery sample; whereas, epigenetically acting valproic acid did not show any growth reduction. Highly vascularized tumors responded to treatment, whereas low-vascularized xenografts showed no response. Furthermore, intracerebral glioblastomas treated with 5-azacitidine showed a clearly visible reduction of tumor angiogenesis and lower numbers of endothelial cells and tumor vessel pericytes. CONCLUSIONS: Our data show significant growth inhibition as well as antiangiogenic effects in intracerebral as well as patient-derived GBM xenografts. This encourages to investigate in detail the multifactorial effects of 5-azacitidine on glioblastomas.

Catheter-tip Granulomas Associated with Intrathecal Drug Delivery--A Two-Center Experience Identifying 13 Cases.

BACKGROUND: Intrathecal (IT) drug therapy with implanted pumps is an effective treatment modality for chronic pain and/or spasticity, especially after non-invasive treatment has failed. Long-term use of intrathecal opioids may cause formation of inflammatory masses at the tip of intrathecal catheters, possibly leading to neurological deficits and/or catheter revision. OBJECTIVE: We aimed to identify risk factors for catheter-tip granuloma (CG) formation. STUDY DESIGN: Retrospective study. SETTING: Tertiary Spine Centers in Germany and Switzerland. METHODS: We retrospectively reviewed data at 2 Swiss centers (Kantonsspital St. Gallen, Swiss Paraplegic Centre Nottwil) between 01/1994 and 10/2013. Collected data were age at operation, gender, smoking status, previous spinal operations, spinal level of catheter-tip, clinical symptoms, catheter testing with contrast agent, applied drugs, drug concentration, as well as cumulative daily drug dosages. RESULTS: Thirteen patients with a mean age of 52.6 years and CG formation after a mean of 6.9 years of follow-up were identified and compared to 54 patients of similar age and length of follow-up (48.6 years, P = 0.535; follow-up 5.3 years, P = 0.236) without CG. In the analysis of risk factors, catheter ending in the middle thoracic spine (Th4-8; 38.5 vs. 6.5%; P = 0.010), previous spinal surgery (75 vs. 41%; P = 0.051), and chronic pain as an underlying primary symptom for IT drug therapy (100 vs. 56%, P = 0.003) were associated with CG formation. IT drug therapy for spasticity appeared to be much less associated with CG formation (0 vs. 44%, P = .0003). As the symptomatology is closely related to the medical treatment applied, patients with CG were more likely to be treated with IT morphine (77 vs. 20%; P < 0.001), and as tendency with IT clonidine (54 vs. 26%; P = 0.092) and IT bupivacaine (46 vs. 20%; P = 0.077). Average in-pump morphine concentration (30.3 vs. 19.5 mg/mL; P = 0.05) as well as average daily dose of morphine (12.5 vs. 6.2 mg/d; P = 0.037) were significantly higher in the CG group. Smoking could not be identified as risk factor for CG formation. LIMITATIONS: Limitations include the retrospective approach, the limited group size of granuloma patients, as well as missing data in the investigated patient groups. CONCLUSION: Our patient cohort with CG differed in some features, of which some like catheter localization, choice, dosage, and the concentration of drugs are potentially modifiable. These results could contribute to the prevention of CG in the future.

[Low-grade gliomas in adults]. / Niedrig-gradige Gliome im Erwachsenenalter.

Low-grade gliomas (LGG) are brain tumors with a low or intermediate biological aggressiveness. According to histopathological features, they are further specified as grade I or II by WHO criteria. Diffuse astrocytomas, oligodendrogliomas, and mixed gliomas are the most common LGG. They mainly affect young patients in their 3rd to 5th decade and often manifest with epileptic seizures. A macroscopically complete or near-complete tumor resection that does not induce additional neurological deficits, is recommended as first line therapy in surgically accessible tumors, as a significant benefit for overall survival has been demonstrated. The indication for adjuvant chemo- or radiotherapy must be discussed interdisciplinary in each case. MGMT promotor methylation, LOH 1p/19q, as well as the status of somatic mutations within IDH1/2 gene constitute biomarkers that may predict response to adjuvant therapy and may correlate to overall survival. These and other biomarkers could be of benefit in future managing plans to offer patients with LGG an individually tailored, optimal treatment.

Les gliomes de bas grade (GBG) sont des lésions tumorales cérébrales primaires considérées à potentiel malin faible à modéré; l'Organisation Mondiale de la Santé les a classés de grades I et II. Les astrocytomes, les oligodendrogliomes et les gliomes mixtes constituent des sous-groupes. Les GBG sont principalement retrouvés chez les jeunes patients et se manifestent souvent par des crises épileptiques. La résection tumorale complète, dans la mesure où elle ne cause pas de dysfonction neurologique, est considérée comme le traitement de première ligne, car elle améliore significativement la survie globale. L'indication à une radiothérapie et/ou une chimiothérapie adjuvantes doit être discutée au cas par cas à la consultation multidisciplinaire. La connaissance de biomarqueurs tels que le MGMT (et de son éventuelle méthylation), et le status, muté ou non, de 1p/19q et IDH1/2- peuvent être utiles en raison de la répercussion sur le traitement et la survie globale. À l'avenir, ces marqueurs (et d'autres encore) pourraient être utilisés pour fournir un traitement personnalisé, par conséquent avec un résultat thérapeutique optimal pour les patients atteints de GBG.

Overexpression of protease-activated receptor type 1 (PAR-1) in glioblastoma multiforme WHO IV cells and blood vessels revealed by NCAM-assisted glioblastoma border labeling.

Glioblastomas are neuroepithelial tumors with lost cellular differentiation and tenfold increased growth rates compared to low-grade gliomas. Despite of very aggressive treatment options based on surgery, irradiation, and chemotherapy, the prognosis of affected patients has remained poor and showed only slight improvements during the last 30 years. Research on glioblastoma border zone was hindered by the tumor's intense invasion into the brain parenchyma and the lack of suitable tumor cell markers. Nevertheless, the compact tumor mass and tumor invasion zone are composed of distinct cell types that need to be distinguished from each other to be addressed selectively. As the isoform 140 of the neural cell adhesion molecule (NCAM-140) was recently demonstrated to be lost in human gliomas with rising WHO grade, human multiform glioblastomas were characterized as a NCAM-140 negative entity displaying three main distinct invasion patterns. Evaluation of putative therapy targets within the tumor tissue and tumor invasion zone has been made possible through NCAM-140 negativity. In the present study, brain tissue controls and human glioblastoma samples with compact tumor mass and invasion areas were analyzed for their vascularization at the tumor border and the expression of thrombin receptor protease-activated receptor type 1 (PAR-1) within tumor tissue and vascular vessel walls. Use of NCAM-140 enabled the identification of the tumor invasion zone and its experimental investigation. Tissue vascularization was found to be significantly increased in the compact tumor mass of glioblastomas compared to their invasion zone and tumor-free controls with a significantly high and specific overexpression of PAR-1 within tumor cells and within tumor blood vessels depending upon the tumor area. This suggests thereby a functional role of the thrombin receptor PAR-1 in glioma cell malignancy and glioblastoma neoangiogenesis.