Adrienne C. Scheck

NFKBIA Deletion in Glioblastomas

BACKGROUND Amplification and activating mutations of the epidermal growth factor receptor (EGFR) oncogene are molecular hallmarks of glioblastomas. We hypothesized that deletion of NFKBIA (encoding nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor-α), an inhibitor of the EGFR-signaling pathway, promotes tumorigenesis in glioblastomas that do not have alterations of EGFR. METHODS We analyzed 790 human glioblastomas for deletions, mutations, or expression of NFKBIA and EGFR. We studied the tumor-suppressor activity of NFKBIA in tumor-cell culture. We compared the molecular results with the outcome of glioblastoma in 570 affected persons. RESULTS NFKBIA is often deleted but not mutated in glioblastomas; most deletions occur in nonclassical subtypes of the disease. Deletion of NFKBIA and amplification of EGFR show a pattern of mutual exclusivity. Restoration of the expression of NFKBIA attenuated the malignant phenotype and increased the vulnerability to chemotherapy of cells cultured from tumors with NFKBIA deletion; it also reduced the viability of cells with EGFR amplification but not of cells with normal gene dosages of both NFKBIA and EGFR. Deletion and low expression of NFKBIA were associated with unfavorable outcomes. Patients who had tumors with NFKBIA deletion had outcomes that were similar to those in patients with tumors harboring EGFR amplification. These outcomes were poor as compared with the outcomes in patients with tumors that had normal gene dosages of NFKBIA and EGFR. A two-gene model that was based on expression of NFKBIA and O(6)-methylguanine DNA methyltransferase was strongly associated with the clinical course of the disease. CONCLUSIONS Deletion of NFKBIA has an effect that is similar to the effect of EGFR amplification in the pathogenesis of glioblastoma and is associated with comparatively short survival.

Gene expression analysis of glioblastomas identifies the major molecular basis for the prognostic benefit of younger age

BackgroundGlioblastomas are the most common primary brain tumour in adults. While the prognosis for patients is poor, gene expression profiling has detected signatures that can sub-classify GBMs relative to histopathology and clinical variables. One category of GBM defined by a gene expression signature is termed ProNeural (PN), and has substantially longer patient survival relative to other gene expression-based subtypes of GBMs. Age of onset is a major predictor of the length of patient survival where younger patients survive longer than older patients. The reason for this survival advantage has not been clear.MethodsWe collected 267 GBM CEL files and normalized them relative to other microarrays of the same Affymetrix platform. 377 probesets on U133A and U133 Plus 2.0 arrays were used in a gene voting strategy with 177 probesets of matching genes on older U95Av2 arrays. Kaplan-Meier curves and Cox proportional hazard analyses were applied in distinguishing survival differences between expression subtypes and age.ResultsThis meta-analysis of published data in addition to new data confirms the existence of four distinct GBM expression-signatures. Further, patients with PN subtype GBMs had longer survival, as expected. However, the age of the patient at diagnosis is not predictive of survival time when controlled for the PN subtype.ConclusionThe survival benefit of younger age is nullified when patients are stratified by gene expression group. Thus, the main cause of the age effect in GBMs is the more frequent occurrence of PN GBMs in younger patients relative to older patients.

The ketogenic diet reverses gene expression patterns and reduces reactive oxygen species levels when used as an adjuvant therapy for glioma

BackgroundMalignant brain tumors affect people of all ages and are the second leading cause of cancer deaths in children. While current treatments are effective and improve survival, there remains a substantial need for more efficacious therapeutic modalities. The ketogenic diet (KD) - a high-fat, low-carbohydrate treatment for medically refractory epilepsy - has been suggested as an alternative strategy to inhibit tumor growth by altering intrinsic metabolism, especially by inducing glycopenia.MethodsHere, we examined the effects of an experimental KD on a mouse model of glioma, and compared patterns of gene expression in tumors vs. normal brain from animals fed either a KD or a standard diet.ResultsAnimals received intracranial injections of bioluminescent GL261-luc cells and tumor growth was followed in vivo. KD treatment significantly reduced the rate of tumor growth and prolonged survival. Further, the KD reduced reactive oxygen species (ROS) production in tumor cells. Gene expression profiling demonstrated that the KD induces an overall reversion to expression patterns seen in non-tumor specimens. Notably, genes involved in modulating ROS levels and oxidative stress were altered, including those encoding cyclooxygenase 2, glutathione peroxidases 3 and 7, and periredoxin 4.ConclusionsOur data demonstrate that the KD improves survivability in our mouse model of glioma, and suggests that the mechanisms accounting for this protective effect likely involve complex alterations in cellular metabolism beyond simply a reduction in glucose.

Tumor Necrosis Factor-α–Induced Protein 3 As a Putative Regulator of Nuclear Factor-κB–Mediated Resistance to O6-Alkylating Agents in Human Glioblastomas

Purpose Pre-existing and acquired drug resistance are major obstacles to the successful treatment of glioblastomas. Methods We used an integrated resistance model and genomics tools to globally explore molecular factors and cellular pathways mediating resistance to O6-alkylating agents in glioblastoma cells. Results We identified a transcriptomic signature that predicts a common in vitro and in vivo resistance phenotype to these agents, a proportion of which is imprinted recurrently by gene dosage changes in the resistant glioblastoma genome. This signature was highly enriched for genes with functions in cell death, compromise, and survival. Modularity was a predominant organizational principle of the signature, with functions being carried out by groups of interacting molecules in overlapping networks. A highly significant network was built around nuclear factor-κB (NF-κB), which included the persistent alterations of various NF-κB pathway elements. Tumor necrosis factor-α–induced protein 3 (TNFAIP3) was...

The ketogenic diet is an effective adjuvant to radiation therapy for the treatment of malignant glioma.

Introduction The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that alters metabolism by increasing the level of ketone bodies in the blood. KetoCal® (KC) is a nutritionally complete, commercially available 4∶1 (fat∶ carbohydrate+protein) ketogenic formula that is an effective non-pharmacologic treatment for the management of refractory pediatric epilepsy. Diet-induced ketosis causes changes to brain homeostasis that have potential for the treatment of other neurological diseases such as malignant gliomas. Methods We used an intracranial bioluminescent mouse model of malignant glioma. Following implantation animals were maintained on standard diet (SD) or KC. The mice received 2×4 Gy of whole brain radiation and tumor growth was followed by in vivo imaging. Results Animals fed KC had elevated levels of β-hydroxybutyrate (p = 0.0173) and an increased median survival of approximately 5 days relative to animals maintained on SD. KC plus radiation treatment were more than additive, and in 9 of 11 irradiated animals maintained on KC the bioluminescent signal from the tumor cells diminished below the level of detection (p<0.0001). Animals were switched to SD 101 days after implantation and no signs of tumor recurrence were seen for over 200 days. Conclusions KC significantly enhances the anti-tumor effect of radiation. This suggests that cellular metabolic alterations induced through KC may be useful as an adjuvant to the current standard of care for the treatment of human malignant gliomas.

Anticancer activity of extracts derived from the mature roots of Scutellaria baicalensis on human malignant brain tumor cells

BackgroundFlavonoid-rich extracts from the mature roots of Scutellaria baicalensis have been shown to exhibit antiproliferative effects on various cancer cell lines. We assessed the ability of an ethanolic extract of S. baicalensis root to inhibit the proliferation of malignant glioma cells.MethodsCell lines derived from primary and recurrent brain tumors from the same patient and cells selected for resistance to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) were used to identify antiproliferative effects of this extract when used alone and in conjunction with BCNU.Results and discussionResults indicated that Scutellaria baicalensis not only inhibits cellular growth in recurrent and drug resistant brain tumor cell lines, but also demonstrates an increased inhibitory effect when used in conjunction with BCNU.ConclusionThe results of this study support the efficacy of S. baicalensis as an anticancer agent for glioblastomas multiforme and a potential adjuvant treatment to current chemotherapeutic agents used in the treatment of both primary and recurrent GBMs. Further studies of the effects of individual flavonoids alone and in combination with each other and with currently used therapies are needed.

Use of in vivo near-infrared laser confocal endomicroscopy with indocyanine green to detect the boundary of infiltrative tumor

OBJECT Infiltrative tumor resection is based on regional (macroscopic) imaging identification of tumorous tissue and the attempt to delineate invasive tumor margins in macroscopically normal-appearing tissue, while preserving normal brain tissue. The authors tested miniaturized confocal fiberoptic endomicroscopy by using a near-infrared (NIR) imaging system with indocyanine green (ICG) as an in vivo tool to identify infiltrating glioblastoma cells and tumor margins. METHODS Thirty mice underwent craniectomy and imaging in vivo 14 days after implantation with GL261-luc cells. A 0.4 mg/kg injection of ICG was administered intravenously. The NIR images of normal brain, obvious tumor, and peritumoral zones were collected using the handheld confocal endomicroscope probe. Histological samples were acquired from matching imaged areas for correlation of tissue images. RESULTS In vivo NIR wavelength confocal endomicroscopy with ICG detects fluorescence of tumor cells. The NIR and ICG macroscopic imaging performed using a surgical microscope correlated generally to tumor and peritumor regions, but NIR confocal endomicroscopy performed using ICG revealed individual tumor cells and satellites within peritumoral tissue; a definitive tumor border; and striking fluorescent microvascular, cellular, and subcellular structures (for example, mitoses, nuclei) in various tumor regions correlating with standard clinical histological features and known tissue architecture. CONCLUSIONS Macroscopic fluorescence was effective for gross tumor detection, but NIR confocal endomicroscopy performed using ICG enhanced sensitivity of tumor detection, providing real-time true microscopic histological information precisely related to the site of imaging. This first-time use of such NIR technology to detect cancer suggests that combined macroscopic and microscopic in vivo ICG imaging could allow interactive identification of microscopic tumor cell infiltration into the brain, substantially improving intraoperative decisions.

Monosomy of Chromosome 10 Associated With Dysregulation of Epidermal Growth Factor Signaling in Glioblastomas

CONTEXT Glioblastomas--uniformly fatal brain tumors--often have both monosomy of chromosome 10 and gains of the epidermal growth factor receptor (EGFR) gene locus on chromosome 7, an association for which the mechanism is poorly understood. OBJECTIVES To assess whether coselection of EGFR gains on 7p12 and monosomy 10 in glioblastomas promotes tumorigenic epidermal growth factor (EGF) signaling through loss of the annexin A7 (ANXA7) gene on 10q21.1-q21.2 and whether ANXA7 acts as a tumor suppressor gene by regulating EGFR in glioblastomas. DESIGN, SETTING, AND PATIENTS Multidimensional analysis of gene, coding sequence, promoter methylation, messenger RNA (mRNA) transcript, protein data for ANXA7 (and EGFR), and clinical patient data profiles of 543 high-grade gliomas from US medical centers and The Cancer Genome Atlas pilot project (made public 2006-2008; and unpublished, tumors collected 2001-2008). Functional analyses using LN229 and U87 glioblastoma cells. MAIN OUTCOME MEASURES Associations among ANXA7 gene dosage, coding sequence, promoter methylation, mRNA transcript, and protein expression. Effect of ANXA7 haploinsufficiency on EGFR signaling and patient survival. Joint effects of loss of ANXA7 and gain of EGFR expression on tumorigenesis. RESULTS Heterozygous ANXA7 gene deletion is associated with significant loss of ANXA7 mRNA transcript expression (P = 1 x 10(-15); linear regression) and a reduction (mean [SEM]) of 91.5% (2.3%) of ANXA7 protein expression compared with ANXA7 wild-type glioblastomas (P = .004; unpaired t test). ANXA7 loss of function stabilizes the EGFR protein (72%-744% increase in EGFR protein abundance) and augments EGFR transforming signaling in glioblastoma cells. ANXA7 haploinsufficiency doubles tumorigenic potential of glioblastoma cells, and combined ANXA7 knockdown and EGFR overexpression promotes tumorigenicity synergistically. The heterozygous loss of ANXA7 in approximately 75% of glioblastomas in the The Cancer Genome Atlas plus infrequency of ANXA7 mutation (approximately 6% of tumors) indicates its role as a haploinsufficiency gene. ANXA7 mRNA transcript expression, dichotomized at the median, associates with patient survival in 191 glioblastomas (log-rank P = .008; hazard ratio [HR], 0.667; 95% confidence interval [CI], 0.493-0.902; 46.9 vs 74.8 deaths/100 person-years for high vs low ANXA7 mRNA expression) and with a separate group of 180 high-grade gliomas (log-rank P = .00003; HR, 0.476; 95% CI, 0.333-0.680; 21.8 vs 50.0 deaths/100 person-years for high vs low ANXA7 mRNA expression). Deletion of the ANXA7 gene associates with poor patient survival in 189 glioblastomas (log-rank P = .042; HR, 0.686; 95% CI, 0.476-0.989; 54.0 vs 80.1 deaths/100 person-years for wild-type ANXA7 vs ANXA7 deletion). CONCLUSION Haploinsufficiency of the tumor suppressor ANXA7 due to monosomy of chromosome 10 provides a clinically relevant mechanism to augment EGFR signaling in glioblastomas beyond that resulting from amplification of the EGFR gene.

The ketogenic diet for the treatment of malignant glioma

Advances in our understanding of glioma biology has led to an increase in targeted therapies in preclinical and clinical trials; however, cellular heterogeneity often precludes the targeted molecules from being found on all glioma cells, thus reducing the efficacy of these treatments. In contrast, one trait shared by virtually all tumor cells is altered (dysregulated) metabolism. Tumor cells have an increased reliance on glucose, suggesting that treatments affecting cellular metabolism may be an effective method to improve current therapies. Indeed, metabolism has been a focus of cancer research in the last few years, as many pathways long associated with tumor growth have been found to intersect metabolic pathways in the cell. The ketogenic diet (high fat, low carbohydrate and protein), caloric restriction, and fasting all cause a metabolic change, specifically, a reduction in blood glucose and an increase in blood ketones. We, and others, have demonstrated that these metabolic changes improve survival in animal models of malignant gliomas and can potentiate the anti-tumor effect of chemotherapies and radiation treatment. In this review we discuss the use of metabolic alteration for the treatment of malignant brain tumors.

Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model.

INTRODUCTIONRecent developments in optical science and image processing have miniaturized the components required for confocal microscopy. Clinical confocal imaging applications have emerged, including assessment of colonic mucosal dysplasia during colonoscopy. We present our initial experience with handheld, miniaturized confocal imaging in a murine brain tumor model. METHODSTwelve C57/BL6 mice were implanted intracranially with 105 GL261 glioblastoma cells. The brains of 6 anesthetized mice each at 14 and 21 days after implantation were exposed surgically, and the brain surface was imaged using a handheld confocal probe affixed to a stereotactic frame. The probe was moved systematically over regions of normal and tumor-containing tissue. Intravenous fluorescein and topical acriflavine contrast agents were used. Biopsies were obtained at each imaging site beneath the probe and assessed histologically. Mice were killed after imaging. RESULTSHandheld confocal imaging produced exquisite images, well-correlated with corresponding histologic sections, of cellular shape and tissue architecture in murine brain infiltrated by glial neoplasm. Reproducible patterns of cortical vasculature, as well as normal gray and white matter, were identified. Imaging effectively distinguished between tumor and nontumor tissue, including infiltrative tumor margins. Margins were easily identified by observers without prior neuropathology training after minimum experience with the technology. CONCLUSIONMiniaturized handheld confocal imaging may assist neurosurgeons in detecting infiltrative brain tumor margins during surgery. It may help to avoid sampling error during biopsy of heterogeneous glial neoplasms, with the potential to supplement conventional intraoperative frozen section pathology. Clinical trials are warranted on the basis of these promising initial results.