A phase II study of bevacizumab and erlotinib after radiation and temozolomide in MGMT unmethylated GBM patients.

Survival for glioblastoma (GBM) patients with an unmethyated MGMT promoter in their tumor is generally worse than methylated MGMT tumors, as temozolomide (TMZ) response is limited. How to better treat patients with unmethylated MGMT is unknown. We performed a trial combining erlotinib and bevacizumab in unmethylated GBM patients after completion of radiation (RT) and TMZ. GBM patients with an unmethylated MGMT promoter were trial eligible. Patient received standard RT (60 Gy) and TMZ (75 mg/m2 × 6 weeks) after surgical resection of their tumor. After completion of RT they started erlotinib 150 mg daily and bevacizumab 10 mg/kg every 2 weeks until progression. Imaging evaluations occurred every 8 weeks. The primary endpoint was overall survival. Of the 48 unmethylated patients enrolled, 46 were evaluable (29 men and 17 women); median age was 55.5 years (29-75) and median KPS was 90 (70-100). All patients completed RT with TMZ. The median number of cycles (1 cycle was 4 weeks) was 8 (2-47). Forty-one patients either progressed or died with a median progression free survival of 9.2 months. At a follow up of 33 months the median overall survival was 13.2 months. There were no unexpected toxicities and most observed toxicities were categorized as CTC grade 1 or 2. The combination of erlotinib and bevacizumab is tolerable but did not meet our primary endpoint of increasing survival. Importantly, more trials are needed to find better therapies for GBM patients with an unmethylated MGMT promoter.

Two phase II trials of temozolomide with interferon-alpha2b (pegylated and non-pegylated) in patients with recurrent glioblastoma multiforme.

BACKGROUND: Because of the poor outcomes for patients with recurrent glioblastoma multiforme (GBM), and some laboratory and clinical evidence of efficacy using interferon in GBM, we assessed the toxicity and efficacy of temozolomide (TMZ) combined with either short-acting (IFN) or long-acting (pegylated) interferon alpha2b (PEG) in two single-arm phase II studies, and compared the results to 6-month progression-free survival (PFS-6) data from historical controls. METHODS: Two single-arm phase II studies were carried out in adults with GBM. Patients were treated with the standard regimen of TMZ (150-200 mg m(-2) per day x 5 days every month) combined with either 4 million units per m(2) subcutaneously (SQ) three times weekly of IFN or 0.5 microg kg(-1) SQ weekly of PEG. Physical exams and imaging evaluations were carried out every 8 weeks. RESULTS: On the IFN study, 34 adults (74% men) were enrolled, and 29 adults (55% men) on the PEG study; median Karnofsky performance status was 80 and 90 for the IFN and PEG studies, respectively. Grade 3 or 4 toxicities were common, leucopoenia and thrombocytopoenia occurring in 35-38% and 18-21% of patients, respectively. Grade 3 or 4 fatigue occurred in 18% of patients on both studies. Lymphopoenia was infrequent. PFS-6 was 31% for 29 evaluable patients in the IFN study and 38% for 26 evaluable patients in the PEG study. CONCLUSION: In recurrent GBM patients, both studies of standard dose TMZ with either IFN or PEG showed improved efficacy when compared to historical controls, or reports using TMZ alone. Even though the TMZ+PEG study met criteria for further study, the results of both of these studies must be considered in light of the standard of care (TMZ plus radiotherapy) for newly diagnosed GBM, which has evolved since the inception of these studies. Despite the results of the current studies being eclipsed by the new GBM standard of care, these results can still inform the development of newer approaches for GBM, either in an earlier, upfront setting, or by extrapolation of the results and consideration of the use of PEG or IFN in conjunction with other antiglioma strategies.

A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

Tumor recurrence in glioblastoma (GBM) is, in part, attributed to increased epithelial-to-mesenchymal transition (EMT) and enhanced tumor cell dissemination in adjacent brain parenchyma after ionizing radiation (IR). EMT is associated with aggressive behavior, increased stem-like characteristics and treatment resistance in malignancies; however, the underlying signaling mechanisms that regulate EMT are poorly understood. We identified grade-dependent p21-activated kinases 4 (PAK4) upregulation in gliomas and further determined its role in mesenchymal transition and radioresistance. IR treatment significantly elevated expression and nuclear localization of PAK4 in correlation with induction of reactive oxygen species (ROS) and mesenchymal transition in GBM cells. Stable PAK4 overexpression promoted mesenchymal transition by elevating EMT marker expression in these cells. Of note, transcription factor-DNA-binding arrays and chromatin immunoprecipitation experiments identified the formation of a novel nuclear PAK4/PPARγ complex which was recruited to the promoter of Nox1, a peroxisome proliferator-activated receptor gamma (PPARγ) target gene. In addition, IR further elevated PAK4/PPARγ complex co-recruitment to Nox1 promoter, and increased Nox1 expression and ROS levels associated with mesenchymal transition in these cells. Conversely, specific PAK4 downregulation decreased PPARγ-mediated Nox1 expression and suppressed EMT in IR-treated cells. In vivo orthotopic tumor experiments showed inhibition of growth and suppression of IR-induced PPARγ and Nox1 expression by PAK4 downregulation in tumors. Our results provide the first evidence of a novel role for PAK4 in IR-induced EMT and suggest potential therapeutic efficacy of targeting PAK4 to overcome radioresistance in gliomas.

Fenretinide activates caspases and induces apoptosis in gliomas.

The synthetic retinoid fenretinide (N-[4-hydroxyphenyl] retinamide or 4HPR) has been shown to not only inhibit cell growth but also to induce apoptosis in a variety of malignant cell lines. It is being tested presently for its potential as a chemopreventive agent against several cancers. A related retinoid, 13-cis-retinoic acid (cRA), has been shown to have activity against gliomas in vitro as well as in a recent clinical study. The present study aimed at assessing the activity of fenretinide against glioma cells in vitro and comparing it with that of cRA at pharmacologically relevant doses. We hypothesized that the ability of fenretinide to induce apoptosis would make it more potent against gliomas than cRA. Four glioma cell lines (D54, U251, U87MG, and EFC-2) were treated with fenretinide (1-100 microM) and showed dose- and time-dependent induction of cell death. At pharmacologically relevant doses, fenretinide was more active against glioma cells than cRA because of its ability to induce apoptosis. Flow cytometric studies using D54 cells demonstrated no significant changes in the cell cycle distribution compared with untreated control, but a sub-G1 fraction consistent with apoptosis was detected. Terminal deoxynucleotidyl transferase-mediated nick end labeling assay indicated that the apoptotic fraction was cell cycle nonspecific. Fenretinide treatment resulted in cleavage of poly ADP-ribose polymerase, indicating an activation of the caspase 3. Immunofluorescence studies using the nuclear stain 4',6-diamidine-2'-phenylindole dihydrochloride showed nuclear condensation and an apoptotic morphology. Hence, this study demonstrates that, at clinically relevant doses, fenretinide is a potent inducer of apoptosis in gliomas acting via the caspase pathway. We also show that at clinically achievable doses, fenretinide has more activity against gliomas than comparable doses of cRA. The favorable side effect profile seen in previous clinical studies and the in vitro activity against gliomas demonstrated in this study suggest that fenretinide could be a promising therapeutic agent against gliomas.

Patterns of expression of Rb and p16 in astrocytic gliomas, and correlation with survival.

The retinoblastoma pathway is a key cell cycle regulatory complex that controls the passage of cells through the G1 checkpoint and is a frequent target of genetic alterations in gliomas. In this study, we examined the expression of Rb and p16 in 170 primary astrocytic gliomas by immunohistochemical techniques, and correlated the expression with overall survival to determine their prognostic value as immunomarkers. There were 130 patients with glioblastoma multiforme (GBM) and 40 with anaplastic astrocytoma (AA). Alterations in the levels of Rb or p16 expression were seen in the majority (>90%) of the gliomas studied. The expression of Rb was completely absent or low in 47.5% of the GBM and 67.5% of the AA. The remainder of the tumors was immunopositive for Rb to varying degrees. Immunoreactivity for p16 was absent in 56% of the GBM and 77.5% of the AA. Kaplan-Meier survival plots (log-rank test) and Cox proportional hazards regression analysis, adjusted for age and histology, showed that neither Rb nor p16 expression independently predicted survival. The results of our study suggest that although genetic alterations of Rb and p16 are common in gliomas, immunohistochemical analysis of these markers correlates poorly with prognosis.

Adenovirus-mediated p16 transfer to glioma cells induces G1 arrest and protects from paclitaxel and topotecan: implications for therapy.

Malignant gliomas are highly resistant to chemotherapy, in part because of the blood-brain barrier, which restricts the delivery of chemotherapy to certain areas of tumor and their cellular heterogeneity, which leads to the selection and propagation of resistant clones. However, the molecular basis of the drug resistance is poorly understood. In this study, we examined the effect of the cell cycle-inhibitory protein p16 on the chemosensitivity of human glioma cells. Treatment of the p16-null glioma cells, U-251 MG and D-54 MG, with paclitaxel and topotecan, resulted in cell death within 4 days. However, overexpression of exogenous wild-type p16 protein using an adenovirus vector resulted in G1 arrest of glioma cells and resistance to the anticancer effect of paclitaxel or topotecan. Specifically, the p16-expressing cells showed a 30-fold increase in the ID50 of topotecan and a more than 40-fold increase in the ID50 of paclitaxel. These observations indicate that overexpression of molecules that control cell-cycle progression may be partially responsible for causing the resistance of glioma cells to cytocidal drugs.

Induction of apoptosis by N-(4-hydroxyphenyl)retinamide in glioma cells.

N-(4-hydroxyphenyl)retinamide (fenretinide) is a synthetic retinoid with anticancer properties. We investigated the effects of fenretinide on the growth of glioma cells. Four glioma cell lines (C6, 9L, Med3 and U87) were treated with fenretinide. Cell viability and independent growth was determined by MTS assay and soft agar assay, respectively. The induction of apoptosis was evaluated by microscopic examination, flow cytometric DNA content analysis, and in situ TdT methods. Fenretinide markedly reduced cell viability of all the glioma cell lines examined at a range of concentrations from 1 to 10 microM. In all cell lines examined, fenretinide also induced morphological changes consistent with apoptosis, including cellular shrinkage, chromatin condensation, and nuclear fragmentation. Flow cytometric analysis also revealed an apoptotic pattern of the DNA content, and in situ detection of apoptosis showed increased incorporation of digoxigenin-nucleotide triphosphate in fenretinide-treated glioma cells. These findings indicate that fenretinide inhibits the growth of glioma cells via the induction of apoptosis, suggesting potential clinical use of fenretinide for treatment of glioma patients.

Epigenetic regulation of CD133/PROM1 expression in glioma stem cells by Sp1/myc and promoter methylation.

Tumor stem cells, postulated to be the source cells for malignancies, have been identified in several cancers using cell-surface expression of markers including CD133, a pentaspan membrane protein. CD133+ve cells form neurospheres, exhibit self-renewal and differentiation, and are tumorigenic. However, despite its association with stem cells, a causal relationship of CD133 to tumorigenesis remains to be defined. Hypothesizing that specific epigenetic and transcription factors implicated in driving the stem cell state may concurrently regulate CD133 expression in stem cells, we analyzed the structure and regulation of CD133 promoter in glioma stem cells and glioma cell lines. Initially, a minimal promoter region was identified by analyzing the activity of CD133 promoter-driven luciferase-expressing 5'-and 3'-deletion-constructs upstream of the transcription start site. This region contained a CpG island that was hypermethylated in CD133-ve glioma stem cells (GSC) and glioma cells but unmethylated in CD133+ve ones. Of several predicted TF-binding sites in this region, the role of tandem Sp1 (-242 and -221) and two Myc (-541 and -25)-binding sites were examined. Overexpression of Sp1 or Myc increased CD133 minimal promoter-driven luciferase activity and CD133 levels in GSC and in glioma cell line. Mithramycin, a Sp1 inhibitor, decreased minimal promoter activity and downregulated CD133 levels in GSC. Gel-shift assays demonstrated direct binding of Sp1 to their predicted sites that was competitively inhibited by oligonucleotide-binding-site sequences and supershifted by anti-Sp1 confirming the interaction. Sp1 and Myc-antibody chromatin immunoprecipitation (ChIP) analysis in GSC showed enrichment of regions with Sp1 and Myc-binding sites. In CD133-ve cells, ChIP analysis showed binding of the methyl-DNA-binding proteins, MBD1, MBD2 and MeCP2 to the methylated CpG island and repression of transcription. These results demonstrate that Sp1 and Myc regulate CD133 transcription in GSC and that promoter methylation and methyl-DNA-binding proteins cause repression of CD133 by excluding transcription-factor binding.

Efficacy of adenovirally expressed soluble TRAIL in human glioma organotypic slice culture and glioma xenografts.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in malignant cells, including gliomas, and is currently in anticancer clinical trials. However, the full-length and tagged forms of TRAIL, unlike the untagged ligand (soluble TRAIL (sTRAIL)), exhibits toxicity against normal cells. Here, we report the generation and testing of an adenovirus (AdsTRAIL) that expresses untagged sTRAIL in an intracranial xenograft model and a human glioma organotypic slice culture model. AdsTRAIL efficiently induced apoptosis in glioma cell lines, including those resistant to sTRAIL, but not in normal human astrocytes (NHAs). It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays. Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity. Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

Phase II study of carboplatin and erlotinib (Tarceva, OSI-774) in patients with recurrent glioblastoma.

Targeting the epidermal growth factor receptor (EGFR) may be effective in a subset of glioblastoma patients. This phase II study assessed the clinical activity of erlotinib plus carboplatin and to determine molecular predictors of response. The primary endpoint was progression free survival (PFS). Patients with recurrent glioblastoma with no more than two prior relapses received carboplatin intravenously on day 1 of every 28-day cycle (target AUC of 6 mg x ml/min). Daily erlotinib at 150 mg/day was dose escalated to 200 mg/day, as tolerated. Clinical and MRI assessments were made every 4 and 8 weeks, respectively. Tumor tissue was evaluated for EGFR, AKT and phosphatase and tensin homolog (PTEN) status. One partial response (PR) was observed out of 43 assessable patients. Twenty patients (47%) had stable disease (SD) for an average of 12 weeks. Median PFS was 9 weeks. The 6-month PFS rate was 14%. Median overall survival (OS) was 30 weeks. This regimen was well tolerated with grade 3/4 toxicities of fatigue, leukopenia, thrombocytopenia and rash requiring dose reductions. A recursive partitioning analysis (RPA) predicted that patients with KPS >or=90 treated with more than 1 prior regimen had the highest OS. No correlation was observed between EGFR, Akt or PTEN expression and either PFS or OS. Carboplatin plus erlotinib is well tolerated but has modest activity in unselected patients. Future trials should be stratified based on optimal molecular or clinical characteristics.

Combination chemotherapy with 13-cis-retinoic acid and celecoxib in the treatment of glioblastoma multiforme.

In a phase II clinical trial, we sought to determine if combining celecoxib with 13-cis-retinoic acid (13-cRA, Accutane) was efficacious in the treatment of recurrent (progressive) glioblastoma multiforme (GBM). In parallel, we also sought to determine to what extent the outcomes from this clinical trial correlated with the findings from studies utilizing two murine intracerebral GBM models, U87MG and U251HF, to determine the predictive value of these murine models. In the clinical trial, 25 patients were studied at recurrence. Stable disease, which occurred in 44% of the patients, was the best response. The median progression-free survival (PFS) was 8 weeks, with a PFS at 6 months of only 19%. For the patients with stable disease, the median PFS was 24 weeks. The toxicity profile was unremarkable. The modest effect on PFS seen in this study agreed with the recent findings of another study, which showed a 19% PFS at 6 months in patients treated with 13-cRA alone. Thus, the combination of 13-cRA with celecoxib is not more effective than 13-cRA in the treatment of progressive GBM. In the murine model study, we found that long-term dosing with 13-cRA or celecoxib alone or in combination did not increase survival in animals with U87MG tumors but modestly increased survival in animals with U251HF tumors. There was no evidence of synergism between the two drugs. From this, we concluded that the animal studies generally predicted that the two agents would have only a modest effect alone and no additive effect when given in combination to patients.

TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation.

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.

Brain metastases: biology and the role of the brain microenvironment.

Metastatic lesions constitute the most frequently occurring malignancy in the brain, and their detection portends a grim prognosis. Efforts to treat these lesions have failed partly because the biologic processes that govern their development are poorly understood. In recent years, it has become evident that metastases occur as a result of a multistep process involving a rigorous natural selection of cells in the primary tumor that bear molecular and biologic characteristics permitting brain metastasis. In addition, recent studies have uncovered the importance of the brain microenvironment and its contribution to the metastatic process. The development of targeted therapies against brain metastases demands a better understanding of these molecular processes and the factors that influence them. This review examines the interplay between tumor cells and host brain tissue in the context of our current understanding of the role of various molecules involved in the metastatic process.

Antiangiogenesis -- therapeutic strategies and clinical implications for brain tumors.

The poor prognosis of patients with malignant brain tumors in spite of aggressive multimodality therapy has led to the search for novel therapeutic strategies. Among the targets for such treatment approaches, tumor angiogenesis has captured the attention of not only the medical field but also of the lay public because of its conceptual departure from traditional methods of cancer therapy. Angiogenesis and vascular proliferation are particularly important in the growth and progression of malignant gliomas and are used as indicators of the degree of malignancy. Recent studies have helped us gain a better understanding of the molecular mediators of this process. It is now evident that after the initial formation of malignancy the continued growth of a glioma is critically dependent on its angiogenic potential. Hence, several approaches to control angiogenesis are being developed and tested. In the present review, we examine some of these approaches from a therapeutic perspective and summarize the findings from early human trials of such agents.

New frontiers in therapy of malignant gliomas.

The prognosis of patients with malignant gliomas remains dismal despite the development of a multidisciplinary approach to their treatment. There is a strong need for novel therapeutic approaches that can make a definite impact in the clinical course of these tumours. Although there have been several advances in diagnostic modalities, surgical techniques and cytotoxic therapies, the development of newer therapies has been hampered by the limited understanding of the factors that determine the biological nature of gliomas. However, inroads are now being made into the understanding of the genetic make-up, biological behaviour and therapeutic response of these tumours, which are expected to pave the way for new modes of treatment. In this article, we review the advances made in the identification of potential targets for glioma therapy and the recent clinical trials utilising biological therapies and newer cytotoxic agents.

Carpal tunnel syndrome associated with interleukin-2 therapy.

BACKGROUND: Interleukin (IL-2) has been extensively used in our institution in the treatment of cancer and has protean neurologic side effects. Carpal tunnel syndrome developing in patients receiving IL-2 appears to have a good prognosis and may spare the patient unneeded investigation. METHODS: A retrospective evaluation was undertaken for all patients using our institution's Patient Studies database. The patients were examined and their charts reviewed. RESULTS: We found eight patients with renal cell carcinoma who developed carpal tunnel syndrome (CTS) during treatment with IL-2, fluorouracil (5-FU), and alpha-interferon (alpha-IFN). The symptoms were bilateral in five patients and all patients improved with cessation of therapy. Three patients had recurrent symptoms with subsequent courses of therapy. Symptoms occurred during or shortly after IL-2 infusion and resolved after therapy was completed with conservative management. The number of courses given did not seem to correlate with development of symptoms. Neurophysiologic studies demonstrated conduction velocity slowing without evidence of acute denervation. CONCLUSIONS: IL-2 can produce focal entrapment of the median nerve at the wrist, which reverses with drug withdrawal. IL-2 mediates the inflammatory response and can cause interstitial edema that likely causes CTS to develop in predisposed patients undergoing treatment.