Phase 1 lead-in to a phase 2 factorial study of temozolomide plus memantine, mefloquine, and metformin as postradiation adjuvant therapy for newly diagnosed glioblastoma.

BACKGROUND: Repurposed memantine, mefloquine, and metformin have putative anticancer activity. The objective of this phase 1 study was to determine the maximum tolerated doses (MTDs) of combinations of these agents with temozolomide (TMZ). METHODS: Adults with newly diagnosed glioblastoma who completed chemoradiation were eligible. The patients were assigned to receive doublet, triplet, or quadruplet therapy with TMZ combined with mefloquine, memantine, and/or metformin. Dose-limiting toxicities (DLTs) were determined, using a 3 + 3 study design. RESULTS: Of 85 enrolled patients, 4 did not complete cycle 1 (the DLT observation period) for nontoxicity reasons, and 81 were evaluable for DLT. The MTDs for doublet therapy were memantine 20 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 850 mg twice daily. For triplet therapy, the MTDs were memantine 10 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 850 mg twice daily. For quadruplet therapy, the MTDs were memantine 10 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 500 mg twice daily. DLTs included dizziness (memantine) and gastrointestinal effects (metformin). Lymphopenia was the most common adverse event (66%). From study entry, the median survival was 21 months, and the 2-year survival rate was 43%. CONCLUSIONS: Memantine, mefloquine, and metformin can be combined safely with TMZ in patients with newly diagnosed glioblastoma.

A randomized phase II trial of standard dose bevacizumab versus low dose bevacizumab plus lomustine (CCNU) in adults with recurrent glioblastoma.

Antiangiogenic therapy can rapidly reduce vascular permeability and cerebral edema but high doses of bevacizumab may induce selective pressure to promote resistance. This trial evaluated the efficacy of low dose bevacizumab in combination with lomustine (CCNU) compared to standard dose bevacizumab in patients with recurrent glioblastoma. Patients (N = 71) with recurrent glioblastoma who previously received radiation and temozolomide were randomly assigned 1:1 to receive bevacizumab monotherapy (10 mg/kg) or low dose bevacizumab (5 mg/kg) in combination with lomustine (90 mg/m(2)). The primary end point was progression-free survival (PFS) based on a blinded, independent radiographic assessment of post-contrast T1-weighted and non-contrast T2/FLAIR weighted magnetic resonance imaging (MRI) using RANO criteria. For 69 evaluable patients, median PFS was not significantly longer in the low dose bevacizumab + lomustine arm (4.34 months, CI 2.96-8.34) compared to the bevacizumab alone arm (4.11 months, CI 2.69-5.55, p = 0.19). In patients with first recurrence, there was a trend towards longer median PFS time in the low dose bevacizumab + lomustine arm (4.96 months, CI 4.17-13.44) compared to the bevacizumab alone arm (3.22 months CI 2.5-6.01, p = 0.08). The combination of low dose bevacizumab plus lomustine was not superior to standard dose bevacizumab in patients with recurrent glioblastoma. Although the study was not designed to exclusively evaluate patients at first recurrence, a strong trend towards improved PFS was seen in that subgroup for the combination of low dose bevacizumab plus lomustine. Further studies are needed to better identify such subgroups that may most benefit from the combination treatment.

Integrated chromosome 19 transcriptomic and proteomic data sets derived from glioma cancer stem-cell lines.

One subproject within the global Chromosome 19 Consortium is to define chromosome 19 gene and protein expression in glioma-derived cancer stem cells (GSCs). Chromosome 19 is notoriously linked to glioma by 1p/19q codeletions, and clinical tests are established to detect that specific aberration. GSCs are tumor-initiating cells and are hypothesized to provide a repository of cells in tumors that can self-replicate and be refractory to radiation and chemotherapeutic agents developed for the treatment of tumors. In this pilot study, we performed RNA-Seq, label-free quantitative protein measurements in six GSC lines, and targeted transcriptomic analysis using a chromosome 19-specific microarray in an additional six GSC lines. The data have been deposited to the ProteomeXchange with identifier PXD000563. Here we present insights into differences in GSC gene and protein expression, including the identification of proteins listed as having no or low evidence at the protein level in the Human Protein Atlas, as correlated to chromosome 19 and GSC subtype. Furthermore, the upregulation of proteins downstream of adenovirus-associated viral integration site 1 (AAVS1) in GSC11 in response to oncolytic adenovirus treatment was demonstrated. Taken together, our results may indicate new roles for chromosome 19, beyond the 1p/19q codeletion, in the future of personalized medicine for glioma patients.

Survival outcome of early versus delayed bevacizumab treatment in patients with recurrent glioblastoma.

Bevacizumab (BEV) is widely used for treatment of patients with recurrent glioblastoma. It is not known if there are differences in outcome between early versus delayed BEV treatment of recurrent glioblastoma. We examined the relationship between the time of starting BEV treatment and outcomes in patients with recurrent glioblastoma. In this retrospective chart review, we identified patients with recurrent glioblastoma diagnosed between 2005 and 2011 who were treated with BEV alone or BEV-containing regimens. Data was analyzed to determine overall survival (OS) from time of diagnosis and progression free survival (PFS) from time of starting BEV. A total of 298 patients were identified, 112 patients received early BEV, 133 patients received delayed BEV, and 53 patients were excluded because they either progressed within 3 months of radiation or received BEV at the time of diagnosis. There was no significant difference in PFS between patients that received early BEV and those that received delayed BEV (5.2 vs. 4.3 months, p = 0.2). Patients treated with delayed BEV had longer OS when compared to those treated with early BEV (25.9 vs. 20.8 months, p = 0.005). In patients with recurrent glioblastoma, there was no significant difference in PFS from the time of starting BEV between early and delayed BEV. Although patients treated with delayed BEV seemed to have longer OS, a conclusion regarding OS outcome requires further prospective trials. These results may indicate that delaying treatment with BEV is not detrimental for survival of patients with recurrent glioblastoma.

Chromosome 19 annotations with disease speciation: a first report from the Global Research Consortium.

A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented ( http://www.c-hpp.org ). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC-MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks.

Phase 2 trial of irinotecan and thalidomide in adults with recurrent anaplastic glioma.

BACKGROUND: Therapeutic options for patients with anaplastic gliomas (AGs) are limited despite better insights into glioma biology. The authors previously reported improved outcome in patients with recurrent glioblastoma treated with thalidomide and irinotecan compared with historical controls. Here, results of the AG arm of the study are reported, using this drug combination. METHODS: Adults with recurrent AG previously treated with radiation therapy, with Karnofsky performance score ≥70, adequate organ function and not on enzyme-inducing anticonvulsants were enrolled. Treatment was in 6-week cycles with irinotecan at 125 mg/m(2) weekly for 4 weeks followed by 2 weeks off, and thalidomide at 100 mg daily increased to 400 mg/day as tolerated. The primary endpoint was progression-free survival rate at 6 months (PFS-6), and the secondary endpoints were overall survival (OS) and response rate (RR). RESULTS: In 39 eligible patients, PFS-6 for the intent-to-treat population was 36% (95% confidence interval [CI] = 21%, 53%), median PFS was 13 weeks (95% CI = 6%, 28%) and RR was 10%(95% CI = 3%, 24%). Radiological findings included 2 complete and 2 partial responses and 17 stable disease. Median OS from study registration was 62 weeks, (95% CI = 51, 144). Treatment-related toxicities (grade 3 or higher) included neutropenia, diarrhea, nausea, and fatigue; 6 patients experienced venous thromboembolism. Four deaths were attributable to treatment-related toxicities: 1 from pulmonary embolism, 2 from colitis, and 1 from urosepsis. CONCLUSIONS: The combination of thalidomide and irinotecan did not achieve sufficient efficacy to warrant further investigation against AG, although a subset of patients experienced prolonged PFS/OS. A trial of the more potent thalidomide analogue, lenalidomide, in combination with irinotecan against AG is currently ongoing.

Activity of temozolomide and bevacizumab in the treatment of locally advanced, recurrent, and metastatic hemangiopericytoma and malignant solitary fibrous tumor.

BACKGROUND: Hemangiopericytomas and malignant solitary fibrous tumors (HPC/SFT) are rare, closely related sarcomas with unpredictable behavior that respond infrequently to chemotherapy. An optimal systemic treatment strategy for advanced HPC/SFT has not yet been identified. METHODS: We retrospectively analyzed the records of 14 patients with histopathologically confirmed HPC/SFT who were treated at The University of Texas MD Anderson Cancer Center between May 2005 and June 2007. All patients were treated with temozolomide 150 mg/m(2) orally on days 1-7 and days 15-21 and bevacizumab 5 mg/kg intravenously on days 8 and 22, repeated at 28-day intervals. Computed tomography assessment of tumor size and density (Choi criteria) was used to determine the best response to therapy. The Kaplan-Meier method was used to estimate progression-free survival. RESULTS: The median follow-up period was 34 months. Eleven patients (79%) achieved a Choi partial response, with a median time to response of 2.5 months. Two patients (14%) had stable disease as the best response, and 1 patient (7%) had Choi progressive disease as the best response. The estimated median progression-free survival was 9.7 months, with a 6-month progression-free rate of 78.6%. The most frequently observed toxic effect was myelosuppression. CONCLUSION: Combination therapy with temozolomide and bevacizumab is a generally well-tolerated and clinically beneficial regimen for HPC/SFT patients. Additional investigation in a controlled, prospective trial is warranted.

Combination of 6-thioguanine, capecitabine, and celecoxib with temozolomide or lomustine for recurrent high-grade glioma.

We evaluated the efficacy of temozolomide (TMZ) or lomustine (CCNU) in combination with 6-thioguanine, capecitabine, and celecoxib for the treatment of recurrent high-grade glioma. Forty-three patients with recurrent glioblastoma and 31 patients with recurrent anaplastic glioma (AG) were enrolled in this open-label, non-comparative study. Patients previously treated with TMZ received CCNU while all others received TMZ; all patients received 6-thioguanine, capecitabine, and celecoxib. Endpoints were 12-month progression-free survival (PFS) for patients with AG, 6-month PFS for patients with glioblastoma, duration of PFS, and MRI-based objective response rates. Results from the TMZ and CCNU treatment arms were combined in the final analysis because there was no statistically significant difference between them. Thirty-eight patients with glioblastoma were treated with the lomustine-based regimen, and five received the TMZ-based regimen. For the 43 glioblastoma patients, the objective response rate was 12 and 33% had stable disease; the 6-month PFS was 14% and median overall survival 32 weeks. For the 31 AG patients, the combined objective response rate was 26 and 42% had stable disease; the 12 month PFS was 44%. Treatment was reasonably well tolerated with hematological toxicity common and more frequent with CCNU than TMZ. The combination therapy with 6-thioguanine, capecitabine and celecoxib plus CCNU or TMZ does not appear to be more effective than other alkylating agent schedules for patients with recurrent glioblastoma. The combination, however, is promising for patients with recurrent high-grade AG.

Glycomic and transcriptomic response of GSC11 glioblastoma stem cells to STAT3 phosphorylation inhibition and serum-induced differentiation.

A glioblastoma stem cell (GSC) line, GSC11, grows as neurospheres in serum-free media supplemented with EGF (epidermal growth factor) and bFGF (basic fibroblast growth factor), and, if implanted in nude mice brains, will recapitulate high-grade glial tumors. Treatment with a STAT3 (signal transducer and activator of transcription 3) phosphorylation inhibitor (WP1193) or 10% FBS (fetal bovine serum) both led to a decrease in expression of the stem cell marker CD133 in GSC11 cells, but differed in phenotype changes. Altered glycolipid profiles were associated with some differentially expressed glycogenes. In serum treated cells, an overall increase in glycosphingolipids may be due to increased expression of ST6GALNAC2, a sialyltransferase. Serum treated cells express more phosphatidylcholine (PC), short chain sphingomyelin (SM) and unsaturated long chain phosphatidylinositol (PI). Decrease of a few glycosphingolipids in the STAT3 phosphorylation inhibited cells may be linked to decreased transcripts of ST6GALNAC2 and UGCGL2, a glucosylceramide synthase. A rare 3-sulfoglucuronylparagloboside carrying HNK1 (human natural killer-1) epitope was found expressed in the GSC11 and the phenotypically differentiated cells. Its up-regulation correlates with increased transcripts of a HNK1 biosynthesis gene, B3GAT2 after serum treatment. Taken together with a quantitative phosphoproteomic study of the same GSC line (C. L. Nilsson, et al. J. Proteome Res. 2010, 9, 430-443), this report represents the most complete systems biology study of cancer stem cell (CSC) differentiation to date. The synergies derived by the combination of glycomic, transcriptomic and phosphoproteomic data may aid our understanding of intracellular and cell-surface events associated with CSC differentiation.

Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network: an initial study in GSC11 glioblastoma stem cells.

Initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called cancer stem cells (CSCs). CSCs may provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of tumors. STAT3 is a key transcription factor associated with regulation of multiple stem cell types. Recently, a novel autocrine loop (IL-6/STAT3/HIF1alpha) has been observed in multiple tumor types (pancreatic, prostate, lung, and colon). The objective of this study was to probe perturbations of this loop in a glioblastoma cancer stem cell line (GSC11) derived from a human tumor by use of a JAK2/STAT3 phosphorylation inhibitor (WP1193), IL-6 stimulation, and hypoxia. A quantitative phosphoproteomic approach that employed phosphoprotein enrichment, chemical tagging with isobaric tags, phosphopeptide enrichment, and tandem mass spectrometry in a high-resolution instrument was applied. A total of 3414 proteins were identified in this study. A rapid Western blotting technique (<1 h) was used to confirm alterations in key protein expression and phosphorylation levels observed in the mass spectrometric experiments. About 10% of the phosphoproteins were linked to the IL-6 pathway, and the majority of remaining proteins could be assigned to other interlinked networks. By multiple comparisons between the sample conditions, we observed expected changes and gained novel insights into the contribution of each factor to the IL6/STAT3/HIF1alpha autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.

Polar lipid remodeling and increased sulfatide expression are associated with the glioma therapeutic candidates, wild type p53 elevation and the topoisomerase-1 inhibitor, irinotecan.

We report changes in gene and polar lipid expression induced by adenovirus-delivered wild-type (wt) p53 gene and chemotherapy of U87 MG glioblastoma cells, a treatment known to trigger apoptosis and cell cycle arrest. Sulfatides (sulfonated glycolipids) were most highly modulated by wild-type p53 treatment; however, no changes were observed in expression levels of mRNA for genes involved in sulfatide metabolism, indicating post-transcriptional control of sulfatide synthesis. Modulation of the aglycones of GD1 and GM1b was observed in wild-type p53-treated cells. The treatment also leads to an increase in phospholipids such as phosphatidyl inositols, phosphatidyl serines, phosphatidyl glycerols, and phosphatidyl ethanolamines, especially hydroxylated phospholipids. These dramatic changes in the composition of cellular glycolipids in response to p53 gene expression and cytotoxic chemotherapy treatment indicate the large role that they play in cell signaling. The use of the human glioma cell line U87 appears to be an excellent model system both in tissue culture and in intracranial murine xenograft models to further characterize the role of sulfatides in modulating glioma responsivity to therapeutic agents.

Biomarkers of disease: cerebrospinal fluid vascular endothelial growth factor (VEGF) and stromal cell derived factor (SDF)-1 levels in patients with neoplastic meningitis (NM) due to breast cancer, lung cancer and melanoma.

BACKGROUND: Breast cancer, lung cancer and melanoma metastasize to the meninges in 5-15% of patients. The identification of specific biomarkers of disease may allow for earlier diagnosis and treatment. Preclinical evidence suggests the possible relevance of SDF-1 and VEGF in the homing and neoangiogenesis of metastases. We chose to measure these molecules in the cerebrospinal fluid (CSF) of melanoma, breast, and lung cancer patients being evaluated for neoplastic meningitis (NM). MATERIALS AND METHODS: We collected CSF from patients with these cancers who were being evaluated for possible NM. CSF was assayed for SDF-1 and VEGF levels using Enzyme-linked Immunosorbent Assay (ELISA) assays. RESULTS: CSF samples from 89 patients met criteria for analysis, including 41 with breast cancer, 35 with lung cancer and 13 with melanoma. Twenty-five percent (22/89) of all samples were positive for malignant cells; 8/41 (20%) from breast cancer, 10/35 (29%) from lung cancer and 4/13 (31%) from melanoma. CSF VEGF levels were available from 83 patients, and were elevated (>20 pg/ml) in 15/22 (68%) of patients with positive CSF cytology and normal (<20 pg/ml) in 59/61 (97%) of patients with negative CSF cytology. The two patients with negative CSF cytology who also had elevated CSF VEGF levels had MRI evidence of NM. CSF SDF-1 levels were available from 81 patients, and were elevated (>950 pg/ml) in 11/18 (61%) of patients with positive CSF cytology and normal (<950 pg/ml) in 57/63 (90%) of patients with negative CSF cytology. CONCLUSIONS: Elevated CSF levels of VEGF are sensitive and highly specific for the diagnosis of NM from breast cancer, lung cancer and melanoma, and may serve as a useful biomarker of NM in high risk patients. CSF SDF-1 levels add little to the diagnostic information provided by CSF VEGF. Evaluation of CSF VEGF levels as a trigger for early treatment in high risk breast cancer, lung cancer and melanoma patients at risk for NM, is warranted.

VEGF Trap induces antiglioma effect at different stages of disease.

Pathological angiogenesis is a hallmark of cancer, specifically of glioblastomas, the most malignant and common primary brain tumor. Vascular endothelial growth factor (VEGF) is the key protein in the regulation of the hypervascular phenotype of primary malignant brain tumors. In this study, we tested VEGF Trap, a soluble decoy receptor for VEGF, in an intracranial glioma model. VEGF Trap was administered in short or prolonged schedules to animals bearing human gliomas at different stages of disease. Of importance, VEGF Trap treatment was efficacious in both initial and advanced phases of tumor development by significantly increasing overall survival. Furthermore, this effect was enhanced in animals treated with more prolonged regimens. In addition, we observed the emergence of a VEGF Trap-resistant phenotype characterized by tumor growth and increased invasiveness. Our results suggest that VEGF Trap will be effective in treating both patients with recurrent or progressive resectable glioblastoma and patients that have undergone extensive initial surgery. Finally, our results indicate that the clinical success of VEGF Trap may depend on a prolonged treatment in combined therapy aiming to simultaneously inhibit angiogenesis and tumor invasion.

Phase II trial of irinotecan and thalidomide in adults with recurrent glioblastoma multiforme.

This phase II study aimed at determining the efficacy and safety of irinotecan combined with thalidomide in adults with recurrent glioblastoma multiforme (GBM) not taking enzyme-inducing anticonvulsants (EIACs). Adult patients (> or =18 years) with recurrent GBM with up to three relapses following surgery and radiation therapy were eligible for this trial. The primary end point was rate of progression-free survival at 6 months (PFS-6); secondary end points were response rate, overall survival, and toxicity. Patients were treated in 6-week cycles with 125 mg/m(2) irinotecan weekly for 4 weeks followed by 2 weeks off treatment and 100 mg of thalidomide daily increased as tolerated to 400 mg/day. Of 32 evaluable patients, 8 (25%) were alive and progression free at 6 months. The median PFS was 13 weeks. One patient experienced a complete response, one a partial response, and 19 stable disease. Median overall survival time from entry into the study was 36 weeks, and the 1-year survival rate was 34%. Adverse events (grade 3 or 4) included diarrhea, abdominal cramps, lymphopenia, neutropenia, and fatigue. Two of the four deaths that occurred were possibly due to treatment-related toxicity. The combination of irinotecan, a cytotoxic agent, and thalidomide, an antiangiogenic agent, shows promising activity against recurrent GBM in patients not receiving EIACs and warrants further study. The results also provide support for similar strategies using combination therapies with newer targeted antiangiogenic agents to generate effective therapies against malignant gliomas.

A multicenter phase II trial of intrathecal topotecan in patients with meningeal malignancies.

To determine the therapeutic efficacy (13-week and 26-week CNS progression-free survival [PFS], response rate, and overall survival) and safety of intraventricular (IVent) topotecan in patients with neoplastic meningitis (NM), we conducted a phase II, open-label, nonrandomized, single-arm trial of IVent topotecan in patients with NM using 400 mug of topotecan IVent twice weekly for 6 weeks, followed by evaluation with imaging, cerebrospinal fluid (CSF), and physical examinations. In the absence of disease progression, patients were then treated with IVent topotecan weekly for 6 weeks, twice monthly for 4 months, and monthly thereafter. Sixty-two patients (23 males and 39 females) were enrolled from April 2001 through March 2006. Median age and KPS at enrollment were 56 (range 5-83) and 80 (range 60-100), respectively. Primary cancers included breast (19), lung (13), CNS (14), and others (16). Forty patients (65%) completed the 6-week induction period, among whom 13 (21%) had CSF clearance of malignant cells. Kaplan-Meier estimates of PFS at 13 and 26 weeks were 30% (95% confidence interval [CI], 20%-45%) and 19% (95% CI, 11%-34%). Overall median survival (50 deaths) was 15 weeks (95% CI, 13-24 weeks). The most common side effect was chemical meningitis in 32% of patients (5% grade 3); 32% experienced no drug side effects. IVent topotecan is well tolerated, but provides no added benefit over other IVent therapies. Because of its modest side effect profile, combining IVent topotecan with other IVent or systemic interventions should be considered.

Effect of bevacizumab on radiation necrosis of the brain.

PURPOSE: Because blocking vascular endothelial growth factor (VEGF) from reaching leaky capillaries is a logical strategy for the treatment of radiation necrosis, we reasoned that bevacizumab might be an effective treatment of radiation necrosis. PATIENTS AND METHODS: Fifteen patients with malignant brain tumors were treated with bevacizumab or bevacizumab combination for their tumor on either a 5 mg/kg/2-week or 7.5 mg/kg/3-week schedule. Radiation necrosis was diagnosed in 8 of these patients on the basis of magnetic resonance imaging (MRI) and biopsy. MRI studies were obtained before treatment and at 6-week to 8-week intervals. RESULTS: Of the 8 patients with radiation necrosis, posttreatment MRI performed an average of 8.1 weeks after the start of bevacizumab therapy showed a reduction in all 8 patients in both the MRI fluid-attenuated inversion-recovery (FLAIR) abnormalities and T1-weighted post-Gd-contrast abnormalities. The average area change in the T1-weighted post-Gd-contrast abnormalities was 48% (+/-22 SD), and the average change in the FLAIR images was 60% (+/-18 SD). The average reduction in daily dexamethasone requirements was 8.6 mg (+/-3.6). CONCLUSION: Bevacizumab, alone and in combination with other agents, can reduce radiation necrosis by decreasing capillary leakage and the associated brain edema. Our findings will need to be confirmed in a randomized trial to determine the optimal duration of treatment.

Combination therapy of inhibitors of epidermal growth factor receptor/vascular endothelial growth factor receptor 2 (AEE788) and the mammalian target of rapamycin (RAD001) offers improved glioblastoma tumor growth inhibition.

Malignant gliomas are highly lethal tumors that display striking genetic heterogeneity. Novel therapies that inhibit a single molecular target may slow tumor progression, but tumors are likely not dependent on a signal transduction pathway. Rather, malignant gliomas exhibit sustained mitogenesis and cell growth mediated in part through the effects of receptor tyrosine kinases and the mammalian target of rapamycin (mTOR). AEE788 is a novel orally active tyrosine kinase inhibitor that decreases the kinase activity associated with the epidermal growth factor receptor and, at higher concentrations, the vascular endothelial growth factor receptor 2 (kinase domain region). RAD001 (everolimus) is an orally available mTOR inhibitor structurally related to rapamycin. We hypothesized that combined inhibition of upstream epidermal growth factor receptor and kinase domain region receptors with AEE788 and inhibition of the downstream mTOR pathway with RAD001 would result in increased efficacy against gliomas compared with single-agent therapy. In vitro experiments showed that the combination of AEE788 and RAD001 resulted in increased rates of cell cycle arrest and apoptosis and reduced proliferation more than either agent alone. Combined AEE788 and RAD001 given orally to athymic mice bearing established human malignant glioma tumor xenografts resulted in greater tumor growth inhibition and greater increases in median survival than monotherapy. These studies suggest that simultaneous inhibition of growth factor receptor and mTOR pathways offer increased benefit in glioma therapy.

PD-L1 expression and prognostic impact in glioblastoma.

BACKGROUND: Therapeutic targeting of the immune checkpoints cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) and PD-1/PD-L1 has demonstrated tumor regression in clinical trials, and phase 2 trials are ongoing in glioblastoma (GBM). Previous reports have suggested that responses are more frequent in patients with tumors that express PD-L1; however, this has been disputed. At issue is the validation of PD-L1 biomarker assays and prognostic impact. METHODS: Using immunohistochemical analysis, we measured the incidence of PD-L1 expression in 94 patients with GBM. We categorized our results according to the total number of PD-L1-expressing cells within the GBMs and then validated this finding in ex vivo GBM flow cytometry with further analysis of the T cell populations. We then evaluated the association between PD-L1 expression and median survival time using the protein expression datasets and mRNA from The Cancer Genome Atlas. RESULTS: The median percentage of PD-L1-expressing cells in GBM by cell surface staining is 2.77% (range: 0%-86.6%; n = 92), which is similar to the percentage found by ex vivo flow cytometry. The majority of GBM patients (61%) had tumors with at least 1% or more PD-L1-positive cells, and 38% had at least 5% or greater PD-L1 expression. PD-L1 is commonly expressed on the GBM-infiltrating T cells. Expression of both PD-L1 and PD-1 are negative prognosticators for GBM outcome. CONCLUSIONS: The incidence of PD-L1 expression in GBM patients is frequent but is confined to a minority subpopulation, similar to other malignancies that have been profiled for PD-L1 expression. Higher expression of PD-L1 is correlated with worse outcome.

A novel E1A-E1B mutant adenovirus induces glioma regression in vivo.

Malignant gliomas are the most frequently occurring primary brain tumors and are resistant to conventional therapy. Conditionally replicating adenoviruses are a novel strategy in glioma treatment. Clinical trials using E1B mutant adenoviruses have been reported recently and E1A mutant replication-competent adenoviruses are in advanced preclinical testing. Here we constructed a novel replication-selective adenovirus (CB1) incorporating a double deletion of a 24 bp Rb-binding region in the E1a gene, and a 903 bp deleted region in the E1b gene that abrogates the expression of a p53-binding E1B-55 kDa protein. CB1 exerted a potent anticancer effect in vitro in U-251 MG, U-373 MG, and D-54 MG human glioma cell lines, as assessed by qualitative and quantitative viability assays. Replication analyses demonstrated that CB1 replicates in vitro in human glioma cells. Importantly, CB1 acquired a highly attenuated replicative phenotype in both serum-starved and proliferating normal human astrocytes. In vivo experiments using intracranially implanted D-54 MG glioma xenografts in nude mice showed that a single dose of CB1 (1.5 x 10(8) PFU/tumor) significantly improved survival. Immunohistochemical analyses of expressed adenoviral proteins confirmed adenoviral replication within the tumors. The CB1 oncolytic adenovirus induces a potent antiglioma effect and could ultimately demonstrate clinical relevance and therapeutic utility.

Phase II study of fenretinide (NSC 374551) in adults with recurrent malignant gliomas: A North American Brain Tumor Consortium study.

PURPOSE: Fenretinide induces apoptosis in malignant gliomas in vitro. This two-stage phase II trial was conducted to determine the efficacy of fenretinide in adults with recurrent malignant gliomas. PATIENTS AND METHODS: Twenty-two patients with anaplastic gliomas (AG) and 23 patients with glioblastoma (GBM) whose tumors had recurred after radiotherapy and no more than two chemotherapy regimens were enrolled. Fenretinide was given orally on days 1 to 7 and 22 to 28 in 6-week cycles in doses of 600 or 900 mg/m(2) bid. RESULTS: Six of 21 (29%) patients in the AG arm and two of 23 (9%) patients in the GBM arm had stable disease at 6 months. One patient with AG treated at 900 mg/m(2) bid dosage had a partial radiologic response. Median progression-free survival (PFS) was 6 weeks for the AG arm and 6 weeks for the GBM arm. PFS at 6 months was 10% for the AG arm and 0% for the GBM arm. Grade 1 or 2 fatigue, dryness of skin, anemia, and hypoalbuminemia were the most frequent toxicities reported. The trial was closed after the first stage because of the inadequate activity at the fenretinide doses used. The first-administration mean plasma C(max) for fenretinide was 832 +/- 360 ng/mL at the 600 mg/m(2) bid dosage and 1,213 +/- 261 ng/mL at the 900 mg/m(2) bid dosage. CONCLUSION: Fenretinide was inactive against recurrent malignant gliomas at the dosage used in this trial. However, additional studies using higher doses of the agent are warranted based on the tolerability of the agent and the potential for activity of a higher fenretinide dosage, as suggested in this trial.