Chronic hypoxia remodels the tumor microenvironment to support glioma stem cell growth.

Cerebral organoids co-cultured with patient derived glioma stem cells (GLICOs) are an experimentally tractable research tool useful for investigating the role of the human brain tumor microenvironment in glioblastoma. Here we describe long-term GLICOs, a novel model in which COs are grown from embryonic stem cell cultures containing low levels of GSCs and tumor development is monitored over extended durations (ltGLICOs). Single-cell profiling of ltGLICOs revealed an unexpectedly long latency period prior to GSC expansion, and that normal organoid development was unimpaired by the presence of low numbers of GSCs. However, as organoids age they experience chronic hypoxia and oxidative stress which remodels the tumor microenvironment to promote GSC expansion. Receptor-ligand modelling identified astrocytes, which secreted various pro-tumorigenic ligands including FGF1, as the primary cell type for GSC crosstalk and single-cell multi-omic analysis revealed these astrocytes were under the control of ischemic regulatory networks. Functional validation confirmed hypoxia as a driver of pro-tumorigenic astrocytic ligand secretion and that GSC expansion was accelerated by pharmacological induction of oxidative stress. When controlled for genotype, the close association between glioma aggressiveness and patient age has very few proposed biological explanations. Our findings indicate that age-associated increases in cerebral vascular insufficiency and associated regional chronic cerebral hypoxia may contribute to this phenomenon.

Concurrent functional and metabolic assessment of brain tumors using hybrid PET/MR imaging.

To evaluate diagnostic accuracy of perfusion weighted imaging (PWI) and positron emission tomography (PET) using an integrated PET/MR system in tumor grading as well as in differentiating recurrent tumor from treatment-induced effects (TIE) in brain tumor patients. Twenty patients (Group A: treatment naïve, 9 patients with 16 lesions; Group B: post-therapy, 11 patients with 18 lesions) underwent fluorine 18 ((18)F) fluorodeoxyglucose (FDG) brain PET/MR with PWI. Two blinded readers predicted low versus high-grade tumor (for Group A) and tumor recurrence versus TIE (for Group B) based solely on tumor rCBV (regional cerebral blood volume) and SUV (standardized uptake values). Tumor histopathology at resection was the reference standard. Using rCBV(mean) ≤ 1.74 as a cut-off, 100% sensitivity and 74% specificity were observed, whereas 75% sensitivity and 89.7% specificity were observed with SUV(mean) ≤ 4.0 as a cut-off to classify patients as test positive for low-grade tumors (Group A) and TIE (Group B). Diagnostic accuracy for detection of low-grade tumors was 90% using PWI and 40% using PET in Group A (p = 0.056); for detection of TIE in Group B, diagnostic accuracy was 94.1% using PWI and 55.6% using PET (p = 0.033). No significant correlation was demonstrated between rCBV parameters and SUV in Group A (mean values: p > 0.403), Group B (p > 0.06) and in the entire population (p > 0.07). Best overall sensitivity and specificity were obtained using rCBV(mean) ≤ 1.74 and SUV(mean) ≤ 4.0 cut-off values. PWI demonstrated better diagnostic accuracy in both groups. Poor correlation was observed between FDG and rCBV parameters.

Glioma Angiogenesis and Perfusion Imaging: Understanding the Relationship between Tumor Blood Volume and Leakiness with Increasing Glioma Grade.

BACKGROUND AND PURPOSE: The purpose of this study was to investigate imaging correlates to the changes occurring during angiogenesis in gliomas. This was accomplished through in vivo assessment of vascular parameters (relative CBV and permeability surface-area product) and their changing relationship with increasing glioma grade. MATERIALS AND METHODS: Seventy-six patients with gliomas underwent preoperative perfusion CT and assessment of relative CBV and permeability surface-area product. Regression analyses were performed to assess the rate of change between relative CBV and permeability surface-area product and to test whether these differed for distinct glioma grades. The ratio of relative CBV to permeability surface-area product was also computed and compared among glioma grades by using analysis of variance methods. RESULTS: The rate of change in relative CBV with respect to permeability surface-area product was highest for grade II gliomas followed by grade III and then grade IV (1.64 versus 0.91 versus 0.27, respectively). The difference in the rate of change was significant between grade III and IV (P = .003) and showed a trend for grades II and IV (P = .098). Relative CBV/permeability surface-area product ratios were the highest for grade II and lowest for grade IV. The pair-wise difference among all 3 groups was significant (P < .001). CONCLUSIONS: There is an increase in relative CBV more than permeability surface-area product in lower grade gliomas, whereas in grade III and especially grade IV gliomas, permeability surface-area product increases much more than relative CBV. The rate of change of relative CBV with respect to permeability surface-area product and relative CBV/permeability surface-area product ratio can serve as an imaging correlate to changes occurring at the tumor microvasculature level.

An in silico screen links gene expression signatures to drug response in glioblastoma stem cells.

Cancer stem cells (CSCs) are thought to promote resistance to chemotherapeutic drugs in glioblastoma, the most common and aggressive primary brain tumor. However, the use of high-throughput drug screens to discover novel small-molecule inhibitors for CSC has been hampered by their instability in long-term cell culture. We asked whether predictive models of drug response could be developed from gene expression signatures of established cell lines and applied to predict drug response in glioblastoma stem cells. Predictions for active compounds were confirmed both for 185 compounds in seven established glioma cell lines and 21 compounds in three glioblastoma stem cells. The use of established cell lines as a surrogate for drug response in CSC lines may enable the large-scale virtual screening of drug candidates that would otherwise be difficult or impossible to test directly in CSCs.

Neuromuscular junction toxicity with tandutinib induces a myasthenic-like syndrome.

BACKGROUND: Tandutinib (MLN 518, Millennium Pharmaceuticals, Cambridge, MA) is an orally active multitargeted tyrosine kinase inhibitor that is currently under evaluation for the treatment of glioblastoma and has been used in the treatment of leukemia. In prior clinical and animal studies, a dose-dependent muscular weakness has been observed with this drug, though the etiology of the weakness has not been defined. METHODS: Standard neurophysiologic techniques, including repetitive nerve stimulation, needle EMG, and single-fiber EMG, were used to evaluate patients who developed weakness while being treated with tandutinib and bevacizumab (Avastin, Genentech, South San Francisco, CA) for glioblastoma (NCT00667394). RESULTS: Six patients were observed to develop a reversible weakness that correlated with the administration of the tandutinib. The onset of weakness after starting tandutinib occurred within 3 to 112 days and in less than 15 days in 3 patients. Electrophysiologic studies showed that all patients developed abnormal repetitive nerve stimulation studies. Four patients had short duration motor unit potentials. Two of these patients also had abnormal single-fiber EMG, as did a third patient who did not have standard needle EMG. The clinical and electrophysiologic abnormalities improved with the termination or reduction in the dose of tandutinib. CONCLUSION: These observations suggest that tandutinib is toxic to the neuromuscular junction, possibly by reversibly binding to a molecule on the postsynaptic acetylcholine receptor complex. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that tandutinib 500 mg twice daily induces reversible muscle weakness and electrophysiologic changes consistent with neuromuscular junction dysfunction.

Cellular origin of fundus autofluorescence in patients and mice with a defective NR2E3 gene.

AIM: To characterise new clinical features in a family with enhanced S-cone syndrome (ESCS) and investigate the pathogenesis of these clinical features in the homozygous Nr2e3(rd7) (rd7) mutant mice. METHODS: Four patients from an affected family were included for genotypic and phenotypic study. Eye tissues from rd7 mice were used to detect a possible relationship between macrophages and autofluorescent material by immunohistochemistry (IHC) staining. RESULTS: Homozygous mutation in R311Q in NR2E3 was detected in this family. Colour photographs revealed that white dots do not correlate to hyperautofluorescent spots seen in autofluorescence imaging of the macula. OCT showed rosette-like lesions similar to those found in rd7 mice histology sections. From IHC analysis, we observed that F4/80 (a pan macrophage marker) and autofluorescence were colocalised to the same cells within the retina rosettes. CONCLUSIONS: The retinal structure of a young ESCS patient with homozygous R311Q mutation in the NR2E3 gene is similar to that seen in the rd7 mice. The macrophages were found to contain autofluorescent materials in the retinal rosettes of rd7 mice. These data are consistent with macrophage infiltration contributing to the hyperautofluorescent spots found in our patients.

Single-nucleotide polymorphisms in selected cytokine genes and risk of adult glioma.

A role of immunological factors in glioma etiology is suggested by reports of an inverse relationship with history of allergy or autoimmune disease. To test whether single-nucleotide polymorphisms (SNPs) in cytokine genes were related to risk of adult glioma, we genotyped 11 SNPs in seven cytokine genes within a hospital-based study conducted by the National Cancer Institute and an independent, population-based study by the National Institute for Occupational Safety and Health (overall 756 cases and 1190 controls with blood samples). The IL4 (rs2243248, -1098T>G) and IL6 (rs1800795, -174G>C) polymorphisms were significantly associated with risk of glioma in the pooled analysis (P trend = 0.006 and 0.04, respectively), although these became attenuated after controlling for the false discovery rate (P trend = 0.07 and 0.22, respectively). Our results underscore the importance of pooled analyses in genetic association studies and suggest that SNPs in cytokine genes may influence susceptibility to glioma.

Season of birth and risk of brain tumors in adults.

BACKGROUND: Recent studies demonstrated an excess of winter births in children with brain tumors and in adults with various neurologic or psychiatric diseases relative to the general population. OBJECTIVE: To investigate a possible association between month of birth and risk of brain tumors in adults using data from a large, hospital-based case-control study. METHODS: Cases were patients with incident glioma (n = 489) or meningioma (n = 197) diagnosed at hospitals in Boston, MA, Phoenix, AZ, and Pittsburgh, PA. Controls (n = 799) were patients hospitalized for a variety of nonmalignant conditions and frequency matched to cases by hospital, age, sex, race/ethnicity, and distance of residence from hospital. Odds ratios (ORs) were calculated using multivariate unconditional logistic regression allowing for cyclic variation in risk with month of birth. RESULTS: A relationship between month of birth and risk of adult glioma and meningioma was found, best described by a 12-month periodic function with peaks in February and January and troughs in August and July. The association between month of birth and risk of glioma differed significantly by handedness, with left-handed and ambidextrous subjects born during late fall through early spring being at particularly high risk of adult glioma as compared with those born at other times of the year. CONCLUSION: These findings suggest the importance of seasonally varying exposures during the pre- or postnatal period in the development of brain tumors in adults.

Occupation and the risk of adult glioma in the United States.

OBJECTIVE: Previous studies have observed increased glioma incidence associated with employment in the petroleum and electrical industries, and in farming. Several other occupations have also been associated with increased risk, but with inconsistent results. We evaluated associations between occupational title and glioma incidence in adults. METHODS: Cases were 489 patients with glioma diagnosed from 1994 to 1998 at three United States hospitals. Controls were 799 patients admitted to the same hospitals for non-malignant conditions. An experienced industrial hygienist grouped occupations that were expected to have similar tasks and exposures. The risk of adult glioma was evaluated for those subjects who ever worked in an occupational group for at least six months, those who worked longer than five years in the occupation, and those with more than ten years latency since starting work in the occupation. RESULTS: Several occupational groups were associated with increased glioma incidence for having ever worked in the occupation, including butchers and meat cutters (odds ratio [OR] = 2.4; 95% confidence limits [CL]: 1.0, 6.0), computer programmers and analysts (OR = 2.0; 95% CL: 1.0, 3.8), electricians (OR = 1.8; 95% CL: 0.8, 4.1), general farmers and farmworkers (OR = 2.5; 95% CL: 1.4, 4.7), inspectors, checkers, examiners, graders, and testers (OR = 1.5; 95% CL: 0.8, 2.7), investigators, examiners, adjustors, and appraisers (OR = 1.7; 95% CL: 0.8, 3.7), physicians and physician assistants (OR = 2.4; 95% CL: 0.8, 7.2), and store managers (OR = 1.6; 95% CL: 0.8, 3.1), whereas occupation as a childcare worker was associated with decreased glioma incidence (OR = 0.4; 95% CL: 0.2, 0.9). These associations generally persisted when the subjects worked longer than five years in the occupation, and for those with more than ten years latency since starting to work in the occupation. CONCLUSIONS: This is our first analysis of occupation and will guide future exposure-specific assessments.

Bone marrow-derived, endothelial progenitor-like cells as angiogenesis-selective gene-targeting vectors.

Human and mouse proliferation-competent, bone marrow or peripheral circulation derived endothelial progenitor-like cells (EPCs) were isolated, expanded and genetically engineered ex vivo to express the beta galactosidase (beta-gal), green fluorescence protein or thymidine kinase (TK) genes using retrovirus-mediated gene transfer. Genetically labeled EPCs were transplanted into sublethally irradiated tumor-bearing mice and were found to migrate to and incorporate into the angiogenic vasculature of growing tumors while maintaining transgene expression. Ganciclovir treatment resulted in significant tumor necrosis in animals previously administered TK-expressing EPCs with no systemic toxicity. These results demonstrate the feasibility of using genetically modified EPCs as angiogenesis-selective gene-targeting vectors and the potential of this approach to mediate non-toxic and systemic antitumor responses.

Aqueous humor and plasma vascular endothelial growth factor in uveitis-associated cystoid macular edema.

PURPOSE: To determine the association between cystoid macular edema and vascular endothelial growth factor concentration in the aqueous humor and plasma of uveitis patients. METHODS: Cross-sectional study. Vascular endothelial growth factor concentrations were measured by enzyme-linked immunosorbent assay in the aqueous humor of 20 uveitis patients (9 with and 11 without cystoid macular edema), and in the plasma of 40 uveitis patients (20 with and 20 without cystoid macular edema) and 20 healthy volunteers. RESULTS: Mean aqueous humor vascular endothelial growth factor concentrations for uveitis patients with and without cystoid macular edema were 152.3 and 109.5 pg/ml, respectively, P =.044. Mean plasma vascular endothelial growth factor concentrations in uveitis patients with and without cystoid macular edema and in healthy volunteers were 32.2, 29.6, and 55.0 pg/ml, respectively. Uveitis patients had lower plasma vascular endothelial growth factor levels than did healthy volunteers, P =.0002. CONCLUSION: In uveitis patients, vascular endothelial growth factor concentration is increased in the aqueous humor of eyes with cystoid macular edema. It may be useful to investigate vascular endothelial growth factor antagonists as a treatment for uveitis-associated cystoid macular edema.

Therapeutic effects of viral vector-mediated antiangiogenic gene transfer in malignant ascites.

Malignant ascites is a common complication of advanced intraabdominal neoplasms for which standard treatments are suboptimal. Evidence suggests that tumor-mediated angiogenesis and enhanced vascular permeability in the peritoneal wall due to high levels of vascular endothelial growth factor play a fundamental role in the pathogenesis of malignant ascites. To explore the advantage of viral vector-mediated "targeted antiangiogenic therapy" in ascites formation, we constructed and administered adenoviral vectors encoding several different antiangiogenic proteins (angiostatin, endostatin, platelet factor 4, and a fusion protein between angiostatin and endostatin) alone or in combination intraperitoneally in mice with peritoneal carcinomatosis from breast cancer (TA3 cells) and ovarian cancer (SKOV-3 i.p. and ES-2 cell lines) to explore the potential of additive or synergistic activity. Our data demonstrated statistically significant downregulation of ascites formation, tumor growth, vascularity, and prolongation of animal survival after intraperitoneal treatment with antiangiogenic adenoviral vectors in three different ascites tumor models. Combined treatment proved to be more effective than treatment with one vector alone. Reduced ascites formation was accompanied by decreased microvascular density in the peritoneal wall and increased apoptosis of tumor cells after administration of antiangiogenic vectors in vivo. Of interest was the observation that AdPF4 caused a significant decrease in the level of VEGF secreted by tumor cells both in vitro and in TA3 ascites tumor-bearing animals in vivo. These data suggest that adenoviral vector-mediated delivery of genes encoding antiangiogenic proteins may represent a potentially new treatment modality for malignant ascites.

Frosted branch angiitis in a child with HIV infection.

PURPOSE: In adults with human immunodeficiency virus (HIV) infection, frosted branch angiitis is commonly associated with cytomegalovirus retinitis and responds to anti-cytomegalovirus therapy. We describe the first pediatric case of HIV-associated frosted branch angiitis. METHODS: Case report. RESULTS: A 7-year-old HIV-infected male with frosted branch angiitis was refractory to induction doses of intravenous ganciclovir and foscarnet over a 2-month period. Although cytomegalovirus antigenemia resolved, the angiitis only improved after subsequent treatment with systemic corticosteroids. CONCLUSION: Frosted branch angiitis in this patient was not attributed to cytomegalovirus. The pathogenesis of HIV-associated frosted branch angiitis may differ between children and adults.

Cellular-telephone use and brain tumors.

BACKGROUND: Concern has arisen that the use of hand-held cellular telephones might cause brain tumors. If such a risk does exist, the matter would be of considerable public health importance, given the rapid increase worldwide in the use of these devices. METHODS: We examined the use of cellular telephones in a case-control study of intracranial tumors of the nervous system conducted between 1994 and 1998. We enrolled 782 patients through hospitals in Phoenix, Arizona; Boston; and Pittsburgh; 489 had histologically confirmed glioma, 197 had meningioma, and 96 had acoustic neuroma. The 799 controls were patients admitted to the same hospitals as the patients with brain tumors for a variety of nonmalignant conditions. RESULTS: As compared with never, or very rarely, having used a cellular telephone, the relative risks associated with a cumulative use of a cellular telephone for more than 100 hours were 0.9 for glioma (95 percent confidence interval, 0.5 to 1.6), 0.7 for meningioma (95 percent confidence interval, 0.3 to 1.7), 1.4 for acoustic neuroma (95 percent confidence interval, 0.6 to 3.5), and 1.0 for all types of tumors combined (95 percent confidence interval, 0.6 to 1.5). There was no evidence that the risks were higher among persons who used cellular telephones for 60 or more minutes per day or regularly for five or more years. Tumors did not occur disproportionately often on the side of head on which the telephone was typically used. CONCLUSIONS: These data do not support the hypothesis that the recent use of hand-held cellular telephones causes brain tumors, but they are not sufficient to evaluate the risks among long-term, heavy users and for potentially long induction periods.

Phase II trial of the antiangiogenic agent thalidomide in patients with recurrent high-grade gliomas.

PURPOSE: Little progress has been made in the treatment of adult high-grade gliomas over the last two decades, thus necessitating a search for novel therapeutic strategies. Malignant gliomas are vascular or angiogenic tumors, which leads to the supposition that angiogenesis inhibition may represent a potentially promising strategy in the treatment of these tumors. We present the results of a phase II trial of thalidomide, a putative inhibitor of angiogenesis, in the treatment of adults with previously irradiated, recurrent high-grade gliomas. PATIENTS AND METHODS: Patients with a histologic diagnosis of anaplastic mixed glioma, anaplastic astrocytoma, or glioblastoma multiforme who had radiographic demonstration of tumor progression after standard external-beam radiotherapy with or without chemotherapy were eligible. Patients were initially treated with thalidomide 800 mg/d with increases in dose by 200 mg/d every 2 weeks until a final daily dose of 1,200 mg was achieved. Patients were evaluated every 8 weeks for response by both clinical and radiographic criteria. RESULTS: A total of 39 patients were accrued, with 36 patients being assessable for both toxicity and response. Thalidomide was well tolerated, with constipation and sedation being the major toxicities. One patient developed a grade 2 peripheral neuropathy after treatment with thalidomide for nearly a year. There were two objective radiographic partial responses (6%), two minor responses (6%), and 12 patients with stable disease (33%). Eight patients were alive more than 1 year after starting thalidomide, although almost all with tumor progression. Changes in serum levels of basic fibroblastic growth factor (bFGF) were correlated with time to tumor progression and overall survival. CONCLUSION: Thalidomide is a generally well-tolerated drug that may have antitumor activity in a minority of patients with recurrent high-grade gliomas. Future studies will better define the usefulness of thalidomide in newly diagnosed patients with malignant gliomas and in combination with radiotherapy and chemotherapy. Additionally, studies will be needed to confirm the potential utility of changes in serum bFGF as a marker of antiangiogenic activity and/or glioma growth.

Systemic metastasis in glioblastoma may represent the emergence of neoplastic subclones.

Glioblastomas only rarely metastasize to sites outside the central nervous system, for reasons that are poorly understood. We report the clinicopathological and molecular genetic findings in 6 patients with metastatic glioblastoma. Four patients were under the age of 32 and all but 1 patient died within 2 yr of diagnosis. The number of metastases ranged from 1 to 3. At the time of death, 3 patients had apparent tumor control at their primary site. We evaluated DNA from both primary and metastatic glioblastomas for genetic alterations commonly found in glioblastomas: TP53 mutations, CDKN2A/p16 deletions, EGFR amplification, and allelic loss of chromosomes 1p, 10q and 19q. Four of 6 cases had TP53 mutations and only single cases had EGFR amplification, CDKN2A/p16 deletions, or allelic loss of 1p, 10q and 19q; 2 cases had no detectable genetic alterations. In 2 cases, the primary and metastatic tumors had identical genotypes. Remarkably, however, 2 cases had different TP53 alterations in the primary and metastatic lesions, or among the metastatic tumors, which suggests that some metastatic deposits may represent emergence of subclones that were not necessarily dominant in the primary tumor. The present observations and a review of the recent literature demonstrate that metastatic glioblastomas tend to occur in younger adults who do not follow long clinical courses, and may be characterized by TP53 mutations and differential clonal selection.

Treatment of patients with primary glioblastoma multiforme with standard postoperative radiotherapy and radiosurgical boost: prognostic factors and long-term outcome.

OBJECT: To assess the value of stereotactic radiosurgery (SRS) as adjunct therapy in patients suffering from glioblastoma multiforme (GBM), the authors analyzed their experience with 78 patients. METHODS: Between June 1988 and January 1995, 78 patients underwent SRS as part of their initial treatment for GBM. All patients had undergone initial surgery or biopsy confirming the diagnosis of GBM and received conventional external beam radiotherapy. Stereotactic radiosurgery was performed using a dedicated 6-MV stereotactic linear accelerator. Thirteen patients were alive at the time of analysis with a median follow-up period of 40.8 months. The median length of actuarial survival for all patients was 19.9 months. Twelve- and 24-month survival rates were 88.5% and 35.9%, respectively. Patient age and Radiation Therapy Oncology Group (RTOG) class were significant prognostic indicators according to univariate analysis (p < 0.05). Twenty-three patients aged younger than 40 years had a median survival time of 48.6 months compared with 55 older patients who had 18.2 months (p < 0.001). Patients in this series fell into RTOG Classes III (27 patients), IV (29 patients), or V (22 patients). Class III patients had a median survival time of 29.5 months following diagnosis; this was significantly longer than median survival times for Classes IV and V, which were 19.2 and 18.2 months, respectively (p = 0.001). Only patient age (< 40 years) was a significant prognostic factor according to multivariate analysis. Acute complications were unusual and limited to exacerbation of existing symptoms. There were no new neuropathies secondary to SRS. Thirty-nine patients (50%) underwent reoperation for symptomatic necrosis or recurrent tumor. The rate of reoperation at 24 months following SRS was 54.8%. CONCLUSIONS: The addition of a radiosurgery boost appears to confer a survival advantage to selected patients.