Limiting dietary carbohydrates inhibits glioma growth in preclinical models. Therefore, the ERGO trial (NCT00575146) examined feasibility of a ketogenic diet in 20 patients with recurrent glioblastoma. Patients were put on a low-carbohydrate, ketogenic diet containing plant oils. Feasibility was the primary endpoint, secondary endpoints included the percentage of patients reaching urinary ketosis, progression-free survival (PFS) and overall survival. The effects of a ketogenic diet alone or in combination with bevacizumab was also explored in an orthotopic U87MG glioblastoma model in nude mice. Three patients (15%) discontinued the diet for poor tolerability. No serious adverse events attributed to the diet were observed. Urine ketosis was achieved at least once in 12 of 13 (92%) evaluable patients. One patient achieved a minor response and two patients had stable disease after 6 weeks. Median PFS of all patients was 5 (range, 3–13) weeks, median survival from enrollment was 32 weeks. The trial allowed to continue the diet beyond progression. Six of 7 (86%) patients treated with bevacizumab and diet experienced an objective response, and median PFS on bevacizumab was 20.1 (range, 12–124) weeks, for a PFS at 6 months of 43%. In the mouse glioma model, ketogenic diet alone had no effect on median survival, but increased that of bevacizumab-treated mice from 52 to 58 days (p<0.05). In conclusion, a ketogenic diet is feasible and safe but probably has no significant clinical activity when used as single agent in recurrent glioma. Further clinical trials are necessary to clarify whether calorie restriction or the combination with other therapeutic modalities, such as radiotherapy or anti-angiogenic treatments, could enhance the efficacy of the ketogenic diet.

Raf are conserved, ubiquitous serine/protein kinases discovered as the cellular elements hijacked by transforming retroviruses. The three mammalian RAF proteins (A, B and CRAF) can be activated by the human oncogene RAS, downstream from which they exert both kinase-dependent and kinase-independent, tumor-promoting functions. The kinase-dependent functions are mediated chiefly by the MEK/ERK pathway, whose activation is associated with proliferation in a broad range of human tumors. Almost 10 years ago, activating BRAF mutations were discovered in a subset of human tumors, and in the past year treatment with small-molecule RAF inhibitors has yielded unprecedented response rates in melanoma patients. Thus, Raf qualifies as an excellent molecular target for anticancer therapy. This review focuses on the role of BRAF and CRAF in different aspects of carcinogenesis, on the success of molecular therapies targeting Raf and the challenges they present.

BackgroundEven in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells.MethodsTo investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model.ResultsThe ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival.ConclusionIn summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.

Cilengitide is a cyclic peptide antagonist of integrins alphavbeta3 and alphavbeta5 that is currently being evaluated as a novel therapeutic agent for recurrent and newly diagnosed glioblastoma. Its mode of action is thought to be mainly antiangiogenic but may include direct effects on tumor cells, notably on attachment, migration, invasion, and viability. In this study we found that, at clinically relevant concentrations, cilengitide (1-100 microM) induces detachment in some but not all glioma cell lines, while the effect on cell viability is modest. Detachment induced by cilengitide could not be predicted by the level of expression of the cilengitide target molecules, alphavbeta3 and alphavbeta5, at the cell surface. Glioma cell death induced by cilengitide was associated with the generation of caspase activity, but caspase activity was not required for cell death since ectopic expression of cytokine response modifier (crm)-A or coexposure to the broad-spectrum caspase inhibitor zVAD-fmk was not protective. Moreover, forced expression of the antiapoptotic protein marker Bcl-X(L) or altering the p53 status did not modulate cilengitide-induced cell death. No consistent effects of cilengitide on glioma cell migration or invasiveness were observed in vitro. Preliminary clinical results indicate a preferential benefit from cilengitide added to temozolomide-based radiochemotherapy in patients with O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation. Accordingly, we also examined whether the MGMT status determines glioma cell responses to cilengitide alone or in combination with temozolomide. Neither ectopic expression of MGMT in MGMT-negative cells nor silencing the MGMT gene in MGMT-positive cells altered glioma cell responses to cilengitide alone or to cilengitide in combination with temozolomide. These data suggest that the beneficial clinical effects derived from cilengitide in vivo may arise from altered perfusion, which promotes temozolomide delivery to glioma cells.

In glioma patients, differentiation between tumor progression (TP) and treatment-related changes (TRCs) remains challenging. Difficulties in classifying imaging alterations may result in a delay or an unnecessary discontinuation of treatment. PET using O-(2-18 F-fluoroethyl)-L-tyrosine ( 18 F-FET) has been shown to be a useful tool for detecting TP and TRCs. Methods: We retrospectively evaluated 127 consecutive patients with World Health Organization grade II-IV glioma who underwent 18 F-FET PET imaging to distinguish between TP and TRCs. 18 F-FET PET findings were verified by neuropathology (40 patients) or clinicoradiologic follow-up (87 patients). Maximum tumor-to-brain ratios (TBR max ) of 18 F-FET uptake and the slope of the time-activity curves (20-50 min after injection) were determined. The diagnostic accuracy of 18 F-FET PET parameters was evaluated by receiver-operating-characteristic analysis and χ 2 testing. The prognostic value of 18 F-FET PET was estimated using the Kaplan-Meier method. Results: TP was diagnosed in 94 patients (74%) and TRCs in 33 (26%). For differentiating TP from TRCs, receiver-operating-characteristic analysis yielded an optimal 18 F-FET TBR max cutoff of 1.95 (sensitivity, 70%; specificity, 71%; accuracy, 70%; area under the curve, 0.75 ± 0.05). The highest accuracy was achieved by a combination of TBR max and slope (sensitivity, 86%; specificity, 67%; accuracy, 81%). However, accuracy was poorer when tumors harbored isocitrate dehydrogenase (IDH) mutations (91% in IDH-wild-type tumors, 67% in IDH-mutant tumors, P , 0.001). 18 F-FET PET results correlated with overall survival (P , 0.001). Conclusion: In our neurooncology department, the diagnostic performance of 18 F-FET PET was convincing but slightly inferior to that of previous reports.

The mechanism by which Raf-1 antagonizes Rok-α to promote migration and tumorigenesis is revealed.

The identification of high-risk patients with thyroid cancer and the preoperative differentiation between follicular adenoma and carcinoma remain clinically challenging. Our study was conducted to analyze whether the quantification of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator receptor (u-PAR) and transcription factor binding to the u-PAR promoter improve prognostic predictability and differential diagnosis of thyroid tumors. Tumor/normal tissue was collected from 69 prospectively followed patients with thyroid carcinomas (papillary, medullary, follicular and anaplastic, PTC, MTC, FTC and ATC) or follicular adenomas. U-PAR, MMP-1, MMP-7 and MMP-9 amounts were determined by ELISA, and transcription factor binding was determined by electrophoretic mobility shift assay. Binding of transcription factors to the u-PAR promoter was observed, but not associated with u-PAR expression. Carcinomas except MTC expressed significantly more u-PAR/MMPs than adenomas/normal tissues, this being associated with advanced pT- or M-stages. MMP-1 and MMP-9 were significantly higher in follicular carcinomas than in adenomas. In carcinomas, high u-PAR-gene expression correlated significantly with high MMP-9, the latter being associated with MMP-7 in normal tissues. Poor survival in differentiated tumors was associated in trend (p = 0.07); poor survival of all patients (p = 0.043) and especially of patients with carcinomas of follicular origin (including ATC), but not medullary carcinomas, were significantly associated with high u-PAR-protein (p = 0.015). Quantification of u-PAR is of prognostic relevance in thyroid carcinomas of non-c-cell origin, and u-PAR in part may be regulated nontranscriptionally in thyroid cancers. This is the first study to suggest MMP-1/-9 as significant differentiation markers between follicular adenoma and follicular carcinoma.

The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival.