Helle Broholm

Cetuximab, bevacizumab, and irinotecan for patients with primary glioblastoma and progression after radiation therapy and temozolomide: a phase II trial

The aim of this clinical trial was to investigate safety and efficacy when combining cetuximab with bevacizumab and irinotecan in patients with recurrent primary glioblastoma multiforme (GBM). Patients were included with recurrent primary GBM and progression within 6 months of ending standard treatment (radiotherapy and temozolomide). Bevacizumab and irinotecan were administered IV every 2 weeks. The first 10 patients received bevacizumab 5 mg/kg, but this was increased to 10 mg/kg after interim safety analysis. Irinotecan dose was based on whether patients were taking enzyme-inducing antiepileptic drugs or not: 340 and 125 mg/m(2), respectively. Cetuximab 400 mg/m(2) as loading dose followed by 250 mg/m(2) weekly was administered IV. Forty-three patients were enrolled in the trial, of which 32 were available for response. Radiographic responses were noted in 34%, of which 2 patients had complete responses and 9 patients had partial responses. The 6-month progression-free survival probability was 30% and median overall survival was 29 weeks (95% CI: 23-37 weeks). One patient had lacunar infarction, 1 patient had multiple pulmonary embolisms, and 3 patients had grade 3 skin toxicity, for which 1 patient needed plastic surgery. One patient was excluded due to suspicion of interstitial lung disease. Three patients had deep-vein thrombosis; all continued on study after adequate treatment. Cetuximab in combination with bevacizumab and irinotecan in recurrent GBM is well tolerated except for skin toxicity, with an encouraging response rate. However, the efficacy data do not seem to be superior compared with results with bevacizumab and irinotecan alone.

Endocannabinoid metabolism in human glioblastomas and meningiomas compared to human non-tumour brain tissue

The endogenous levels of the two cannabinoid receptor ligands 2‐arachidonoyl glycerol and anandamide, and their respective congeners, monoacyl glycerols and N‐acylethanolamines, as well as the phospholipid precursors of N‐acylethanolamines, were measured by gas chromatography‐mass spectrometry in glioblastoma (WHO grade IV) tissue and meningioma (WHO grade I) tissue and compared with human non‐tumour brain tissue. Furthermore, the metabolic turnover of N‐acylethanolamines was compared by measurements of the enzymatic activity of N‐acyltransferase, N‐acylphosphatidylethanolamine‐hydrolysing phospholipase D and fatty acid amide hydrolase in the same three types of tissue. Glioblastomas were characterized by enhanced levels of N‐acylethanolamines (eightfold, 128 ± 59 pmol/μmol lipid phosphorus) including anandamide (17‐fold, 4.6 ± 3.1pmol/μmol lipid phosphorus) and several species of N‐acylphosphatidylethanolamines (three to eightfold). This was accompanied by a more than 60% reduction in the enzyme activities of N‐acylphosphatidylethanolamine‐hydrolysing phospholipase D and fatty acid amide hydrolase. By contrast, meningiomas were characterized by a massively enhanced level of 2‐monoacyl glycerols (20‐fold, 2293 ± 361 pmol/μmol lipid phosphorus) including 2‐arachidonoyl glycerol (20‐fold, 1524 ± 361 pmol/μmol lipid phosphorus). This was accompanied by an enhanced in vitro conversion of phosphatidylcholine to monoacyl glycerol (fivefold). The enhanced level of the 2‐arachidonoyl glycerol, anandamide and other N‐acylethanolamines detected in the two types of tumour tissue may possibly act as endogenous anti‐tumour mediators by stimulation of both cannabinoid and non‐cannabinoid receptor‐mediated mechanisms.

Vascular endothelial growth factor (VEGF) receptor neuropilin‐1's distribution in astrocytic tumors

Neuropilin‐1 is a VEGF165‐ and semaphorin receptor expressed by endothelial cells and tumor cells. The specific function of neuropilin‐1 is not fully known, but in the developing nervous system neuropilin, as a semaphorin receptor, has been shown to influence neuronal guidance. The expression of neuropilin‐1 was studied in low‐grade and high‐grade astrocytic tumors, the latter characterized by extensive angiogenesis. We examined 20 low‐grade astrocytomas (WHO grade II) and 46 glioblastomas (WHO grade IV) immunohistochemically for neuropilin‐1, p53 and EGFR. The glioblastomas were according to the p53 and EGFR expression classified as 35 primary –de novo– glioblastomas, 9 secondary glioblastomas, and 2 uncertain cases. Furthermore, the presence of mast cells was evaluated to search for any potential function in angiogenesis. The glioblastomas expressed neuropilin‐1 in the endothelial cells of the proliferating vessels and the majority of the glioblastomas had immunoreactive neoplastic astrocytes, with no difference between the glioblastoma subgroups. Six out of twenty of the low‐grade astrocytomas were negative in the endothelial cells and 8 out of 20 in the tumor cells for neuropilin‐1. Mast cells were observed in the collagen matrix around larger vessels in the leptomeninges, but not adjacent to malignant tumor vessels or as part of the tumor process itself. Increased expression of neuropilin‐1 is shown in endothelial cells and in neoplastic astrocytes of glioblastomas. Less neuropilin‐1 expression is found in about half of the low‐grade astrocytomas in both neoplastic astrocytes and endothelial cells. The results suggest a correlation between neuropilin‐1 and vascularity in human astrocytic tumors and a possible role for neuropilin‐1 as a receptor for VEGF‐induced angiogenesis.

Prospective evaluation of angiogenic, hypoxic and EGFR-related biomarkers in recurrent glioblastoma multiforme treated with cetuximab, bevacizumab and irinotecan

Hasselbalch B, Eriksen JG, Broholm H, Christensen IJ, Grunnet K, Horsman MR, Poulsen HS, Stockhausen M‐T, Lassen U. Prospective evaluation of angiogenic, hypoxic and EGFR‐related biomarkers in recurrent glioblastoma multiforme treated with cetuximab, bevacizumab and irinotecan. APMIS 2010; 118: 585–94.

Maintenance of EGFR and EGFRvIII expressions in an in vivo and in vitro model of human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common, and most aggressive primary brain tumor among adults. A vast majority of the tumors express high levels of the epidermal growth factor receptor (EGFR) as a consequence of gene amplification. Furthermore, gene amplification is often associated with mutation of EGFR, and the constitutive activated deletion variant EGFRvIII is the most common EGFR mutation found in GBM. Activated EGFR signaling, through overexpression and/or mutation, is involved in increased tumorigenic potential. As such, EGFR is an attractive target for GBM therapy. However, clinical studies with EGFR inhibitors have shown inconsistent results, and as such, further knowledge regarding the role of EGFR and EGFRvIII in GBM is needed. For this, an appropriate in vivo/in vitro tumor model is required. Here, we report the establishment of an experimental GBM model in which the expressions of EGFR and EGFRvIII are maintained both in xenograft tumors growing subcutaneously on mice and in cell cultures established in stem cell conditions. With this model it will be possible to further study the role of EGFR and EGFRvIII, and response to targeted therapy, in GBM.

Aberrant expression of miR-218 and miR-204 in human mesial temporal lobe epilepsy and hippocampal sclerosis—Convergence on axonal guidance

Mesial temporal lobe epilepsy (MTLE) is one of the most common types of the intractable epilepsies and is most often associated with hippocampal sclerosis (HS), which is characterized by pronounced loss of hippocampal pyramidal neurons. microRNAs (miRNAs) have been shown to be dysregulated in epilepsy and neurodegenerative diseases, and we hypothesized that miRNAs could be involved in the pathogenesis of MTLE and HS.

Genome-wide high-density SNP linkage search for glioma susceptibility loci: results from the Gliogene Consortium

Gliomas, which generally have a poor prognosis, are the most common primary malignant brain tumors in adults. Recent genome-wide association studies have shown that inherited susceptibility plays a role in the development of glioma. Although first-degree relatives of patients exhibit a two-fold increased risk of glioma, the search for susceptibility loci in familial forms of the disease has been challenging because the disease is relatively rare, fatal, and heterogeneous, making it difficult to collect sufficient biosamples from families for statistical power. To address this challenge, the Genetic Epidemiology of Glioma International Consortium (Gliogene) was formed to collect DNA samples from families with two or more cases of histologically confirmed glioma. In this study, we present results obtained from 46 U.S. families in which multipoint linkage analyses were undertaken using nonparametric (model-free) methods. After removal of high linkage disequilibrium single-nucleotide polymorphism, we obtained a maximum nonparametric linkage score (NPL) of 3.39 (P = 0.0005) at 17q12-21.32 and the Z-score of 4.20 (P = 0.000007). To replicate our findings, we genotyped 29 independent U.S. families and obtained a maximum NPL score of 1.26 (P = 0.008) and the Z-score of 1.47 (P = 0.035). Accounting for the genetic heterogeneity using the ordered subset analysis approach, the combined analyses of 75 families resulted in a maximum NPL score of 3.81 (P = 0.00001). The genomic regions we have implicated in this study may offer novel insights into glioma susceptibility, focusing future work to identify genes that cause familial glioma.

Excitatory amino acid transporters EAAT‐1 and EAAT‐2 in temporal lobe and hippocampus in intractable temporal lobe epilepsy

Intractable temporal lobe epilepsy (TLE) is an invalidating disease and many patients are resistant to medical treatment. Increased glutamate concentration has been found in epileptogenic foci and may induce local over‐excitation and cytotoxicity; one of the proposed mechanisms involves reduced extra‐cellular clearance of glutamate by excitatory amino acid transporters (EAAT‐1 to EAAT‐5). EAAT‐1 and EAAT‐2 are mainly expressed on astroglial cells for the reuptake of glutamate from the extra‐cellular space. We have studied the expression of EAAT‐1 and EAAT‐2 in the hippocampus and temporal lobe in 12 patients with TLE by immunhistochemistry and densitometry. The expression of EAAT‐1 and EAAT‐2 was reduced to approximately 40% and 25%, respectively, in CA1 of the hippocampus. In the same area, an increased expression of glial fibrillary acid protein (GFAP) at 90% reflected molecular rearrangements and upregulation of GFAP in the existing astrocytes as Ki‐67 staining failed to demonstrate any signs of astrocytic proliferation. The aetiology of the reduced expression of EAAT‐1 and EAAT‐2 remains unclear. The downregulation of EAAT‐1 and EAAT‐2 may be an adaptive response to neuronal death or it may be a causative event contributing to neuronal death. Further studies of the EAATs and their function are needed to clarify the mechanisms and significance of EAAT‐1 and EAAT‐2 disappearance in TLE.

Neural tube defects and associated anomalies in a fetal and perinatal autopsy series

Neural tube defects (NTDs) are congenital malformations of the central nervous system (CNS) secondary to abnormal closure of the neural tube during embryonic development. This study provides information on NTD rate, distribution, associated morphologic anomalies and organ weights in a Danish fetal and perinatal autopsy series during a 16 year period. The data were extracted from the autopsy reports of a consecutive series of 1984 fetal and perinatal autopsies from the Copenhagen area performed in the period 1989–2004. Registered parameters included: The location and morphology of the NTD, associated morphological anomalies, and organ weights. Organ weights were evaluated according to new fetal autopsy standards and grouped as low, normal or high. Ninety‐seven NTD cases were found (4.9%): Spina bifida (38 cases), cephalocele (17 cases) and anencephaly (42 cases). 63% of NTD cases had associated morphologic anomalies. Among the most common were hydrocephalus, NTD in another region, and anomalies in the urogenital system. 58% of the NTD cases had abnormal weight of one or more organs. Most notable was low adrenal weight not only in anencephalic fetuses but also in cases with cephalocele, suggesting a possible association.

MNS16A minisatellite genotypes in relation to risk of glioma and meningioma and to glioblastoma outcome

The human telomerase reverse transcriptase (hTERT) gene is upregulated in a majority of malignant tumours. A variable tandem repeat, MNS16A, has been reported to be of functional significance for hTERT expression. Published data on the clinical relevance of MNS16A variants in brain tumours have been contradictory. The present population‐based study in the Nordic countries and the United Kingdom evaluated brain‐tumour risk and survival in relation to MNS16A minisatellite variants in 648 glioma cases, 473 meningioma cases and 1,359 age, sex and geographically matched controls. By PCR‐based genotyping all study subjects with fragments of 240 or 271 bp were judged as having short (S) alleles and subjects with 299 or 331 bp fragments as having long (L) alleles. Relative risk of glioma or meningioma was estimated with logistic regression adjusting for age, sex and country. Overall survival was analysed using Kaplan–Meier estimates and equality of survival distributions using the log‐rank test and Cox proportional hazard ratios. The MNS16A genotype was not associated with risk of occurrence of glioma, glioblastoma (GBM) or meningioma. For GBM there were median survivals of 15.3, 11.0 and 10.7 months for the LL, LS and SS genotypes, respectively; the hazard ratio for having the LS genotype compared with the LL was significantly increased HR 2.44 (1.56–3.82) and having the SS genotype versus the LL was nonsignificantly increased HR 1.46 (0.81–2.61). When comparing the LL versus having one of the potentially functional variants LS and SS, the HR was 2.10 (1.41–3.1). However, functionality was not supported as there was no trend towards increasing HR with number of S alleles. Collected data from our and previous studies regarding both risk and survival for the MNS16A genotypes are contradictory and warrant further investigations.