[Show abstract][Hide abstract] ABSTRACT: Background:
Temozolomide (TMZ) is an oral DNA-alkylating agent used for treating patients with glioblastoma. However, therapeutic benefits of TMZ can be compromised by the expression of O6-methylguanine methyltransferase (MGMT) in tumor tissue. Here we used MGMT-expressing glioblastoma stem cells (GSC) lines as a model for investigating the molecular mechanism underlying TMZ resistance, while aiming to explore a new treatment strategy designed to possibly overcome resistance to the clinically relevant dose of TMZ (35 μM).
MGMT-expressing GSC cultures are resistant to TMZ, and IC50 (half maximal inhibitory concentration) is estimated at around 500 μM. Clonogenic GSC surviving 500 μM TMZ (GSC-500 μM TMZ), were isolated. Molecular signatures were identified via comparative analysis of expression microarray against parental GSC (GSC-parental). The recombinant protein of top downregulated signature was used as a single agent or in combination with TMZ, for evaluating therapeutic effects of treatment of GSC.
The molecular signatures characterized an activation of protective stress responses in GSC-500 μM TMZ, mainly including biotransformation/detoxification of xenobiotics, blocked endoplasmic reticulum stress-mediated apoptosis, epithelial-to-mesenchymal transition (EMT), and inhibited growth/differentiation. Bone morphogenetic protein 7 (BMP7) was identified as the top down-regulated gene in GSC-500 μM TMZ. Although augmenting BMP7 signaling in GSC by exogenous BMP7 treatment did not effectively stop GSC growth, it markedly sensitized both GSC-500 μM TMZ and GSC-parental to 35 μM TMZ treatment, leading to loss of self-renewal and migration capacity. BMP7 treatment induced senescence of GSC cultures and suppressed mRNA expression of CD133, MGMT, and ATP-binding cassette drug efflux transporters (ABCB1, ABCG2), as well as reconfigured transcriptional profiles in GSC by downregulating genes associated with EMT/migration/invasion, stemness, inflammation/immune response, and cell proliferation/tumorigenesis. BMP7 treatment significantly prolonged survival time of animals intracranially inoculated with GSC when compared to those untreated or treated with TMZ alone (p = 0.0017), whereas combination of two agents further extended animal survival compared to BMP7 alone (p = 0.0489).
These data support the view that reduced endogenous BMP7 expression/signaling in GSC may contribute to maintained stemness, EMT, and chemoresistant phenotype, suggesting that BMP7 treatment may provide a novel strategy in combination with TMZ for an effective treatment of glioblastoma exhibiting unmethylated MGMT.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma stem cells (GSC) co-exhibiting a tumor-initiating capacity and a radio-chemoresistant phenotype, are a compelling cell model for explaining tumor recurrence. We have previously characterized patient-derived, treatment-resistant GSC clones (TRGC) that survived radiochemotherapy. Compared to glucose-dependent, treatment-sensitive GSC clones (TSGC), TRGC exhibited reduced glucose dependence that favor the fatty acid oxidation pathway as their energy source. Using comparative genome-wide transcriptome analysis, a series of defense signatures associated with TRGC survival were identified and verified by siRNA-based gene knockdown experiments that led to loss of cell integrity. In this study, we investigate the prognostic value of defense signatures in glioblastoma (GBM) patients using gene expression analysis with Probeset Analyzer (131 GBM) and The Cancer Genome Atlas (TCGA) data, and protein expression with a tissue microarray (50 GBM), yielding the first TRGC-derived prognostic biomarkers for GBM patients. Ribosomal protein S11 (RPS11), RPS20, individually and together, consistently predicted poor survival of newly diagnosed primary GBM tumors when overexpressed at the RNA or protein level [RPS11: Hazard Ratio (HR) = 11.5, p<0.001; RPS20: HR = 4.5, p = 0.03; RPS11+RPS20: HR = 17.99, p = 0.001]. The prognostic significance of RPS11 and RPS20 was further supported by whole tissue section RPS11 immunostaining (27 GBM; HR = 4.05, p = 0.01) and TCGA gene expression data (578 primary GBM; RPS11: HR = 1.19, p = 0.06; RPS20: HR = 1.25, p = 0.02; RPS11+RPS20: HR = 1.43, p = 0.01). Moreover, tumors that exhibited unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) or wild-type isocitrate dehydrogenase 1 (IDH1) were associated with higher RPS11 expression levels [corr (IDH1, RPS11) = 0.64, p = 0.03); [corr (MGMT, RPS11) = 0.52, p = 0.04]. These data indicate that increased expression of RPS11 and RPS20 predicts shorter patient survival. The study also suggests that TRGC are clinically relevant cells that represent resistant tumorigenic clones from patient tumors and that their properties, at least in part, are reflected in poor-prognosis GBM. The screening of TRGC signatures may represent a novel alternative strategy for identifying new prognostic biomarkers.
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive tumor growth are not well understood. Here, through integrated epigenome and transcriptome analyses of cell lines, genotyped clinical samples, and TCGA data, we show that EGFR mutations remodel the activated enhancer landscape of GBM, promoting tumorigenesis through a SOX9 and FOXG1-dependent transcriptional regulatory network in vitro and in vivo. The most common EGFR mutation, EGFRvIII, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in a SOX9, FOXG1-dependent manner. These results identify the role of transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy.
[Show abstract][Hide abstract] ABSTRACT: Background:
Immunotherapy is an ideal treatment modality to specifically target the diffusely infiltrative tumor cells of malignant gliomas while sparing the normal brain parenchyma. However, progress in the development of these therapies for glioblastoma has been slow due to the lack of immunogenic antigen targets that are expressed uniformly and selectively by gliomas.
We utilized human glioblastoma cell cultures to induce expression of New York-esophageal squamous cell carcinoma (NY-ESO-1) following in vitro treatment with the demethylating agent decitabine. We then investigated the phenotype of lymphocytes specific for NY-ESO-1 using flow cytometry analysis and cytotoxicity against cells treated with decitabine using the xCelligence real-time cytotoxicity assay. Finally, we examined the in vivo application of this immune therapy using an intracranially implanted xenograft model for in situ T cell trafficking, survival, and tissue studies.
Our studies showed that treatment of intracranial glioma-bearing mice with decitabine reliably and consistently induced the expression of an immunogenic tumor-rejection antigen, NY-ESO-1, specifically in glioma cells and not in normal brain tissue. The upregulation of NY-ESO-1 by intracranial gliomas was associated with the migration of adoptively transferred NY-ESO-1-specific lymphocytes along white matter tracts to these tumors in the brain. Similarly, NY-ESO-1-specific adoptive T cell therapy demonstrated antitumor activity after decitabine treatment and conferred a highly significant survival benefit to mice bearing established intracranial human glioma xenografts. Transfer of NY-ESO-1-specific T cells systemically was superior to intracranial administration and resulted in significantly extended and long-term survival of animals.
These results reveal an innovative, clinically feasible strategy for the treatment of glioblastoma.
[Show abstract][Hide abstract] ABSTRACT: Lymphocytic hypophysitis (LH) is a poorly understood autoimmune disorder of the pituitary gland. Symptoms include headache, pituitary dysfunction, visual disturbances and neurological deficits. The diagnosis can be made based on clinical and biochemical findings but for atypical presentations, no circulatory diagnostic biomarkers exist and pituitary biopsy is necessary for diagnosis.
We used high-resolution human leukocyte antigen (HLA) screening assays to investigate for a relationship between specific HLA markers and lymphocytic hypophysitis.
This was a retrospective analysis Setting: The study was conducted at a tertiary referral center.
Fifteen patients with sporadic LH and 4 melanoma patients who developed hypophysitis following administration of CTLA4 antibodies and one patient with sarcoid-associated hypophysitis were evaluated.
Clinical data including endocrine function, were assessed and HLA typing performed in all 20 patients with hypophysitis, 50 control patients with other sellar abnormalities and 4 CTLA4 antibody-treated patients without hypophysitis.
Two major histocompatibility class II HLA markers, DQ8 and DR53, were found in 13/15 (87%) and 12/15 (80.0%) patients with sporadic lymphocytic hypophysitis respectively. In contrast, none of 4 patients who developed hypophysitis after administration of the CTLA4 antibodies exhibited the HLA-DQ8 marker and only 1/ 4(25%) exhibited the HLA-DR53 marker. In a parallel group of 50 control subjects with sellar masses and 4 CTLA4-Ab treated patients who did not develop evidence of pituitary failure, the candidate HLA subtypes were found in ∼20% for DQ8 and ∼48% for DR53 respectively.
& Relevance: The HLA markers, DQ8 and DR53, were found to be commonly present in patients with LH. The odds ratio of a patient with LH expressing the HLA-DQ8 marker is 23.1-fold higher compared to a patient with another sellar mass. HLA-DQ8 testing may assist in diagnosis and avoid unnecessary biopsy in atypical LH cases.
Preview · Article · Aug 2015 · The Journal of Clinical Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Latino Americans are a rapidly growing ethnic group in the United States. The characteristics of glioblastoma in this population is poorly studied. We have evaluated the data of 47,540 glioblastoma patients from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute. This SEER data from 1973-2000 includes up to 13 cancer registries. For 2001 to 2011, the data has improved geographic coverage with 18 registries encompassing 28% of the U.S. population.
Latinos have a lower incidence of GBM than non-Latino Whites. Gender distribution is similar. The total SEER data show that Latinos present slightly younger and have a higher incidence of giant cell glioblastoma and gliosarcoma than non-Latino Whites. Despite higher rates of radiation therapy, the one year survival rate (34.7%) for non-Latino White populations is less than for Latinos (39.0%, p <0.001). Subset analyses (2001-2011) of all the above parameters show similar results except for gliosarcoma incidence. A literature search does not identify MGMT or IDH1 data regarding Latino Americans.
We have assessed 2 prognostic markers in 30 Latino glioblastoma patients. MGMT methylation is present in 24% and IDH1 mutation is found in 12.5%. Our preliminary data suggests that Latinos may have a greater incidence of MGMT unmethylated tumors. Younger age may possibly contribute to improved survival in Latinos but the underlying molecular basis is unresolved.
Preview · Article · Aug 2015 · The Canadian journal of neurological sciences. Le journal canadien des sciences neurologiques
[Show abstract][Hide abstract] ABSTRACT: Cancer cells adapt their signaling in response to nutrient availability. To uncover the mechanisms regulating this process and its functional consequences, we interrogated cell lines, mouse tumor models, and clinical samples of glioblastoma (GBM), the highly lethal brain cancer. We discovered that glucose or acetate is required for epidermal growth factor receptor vIII (EGFRvIII), the most common growth factor receptor mutation in GBM, to activate mechanistic target of rapamycin complex 2 (mTORC2) and promote tumor growth. Glucose or acetate promoted growth factor receptor signaling through acetyl-CoA-dependent acetylation of Rictor, a core component of the mTORC2 signaling complex. Remarkably, in the presence of elevated glucose levels, Rictor acetylation is maintained to form an autoactivation loop of mTORC2 even when the upstream components of the growth factor receptor signaling pathway are no longer active, thus rendering GBMs resistant to EGFR-, PI3K (phosphoinositide 3-kinase)-, or AKT (v-akt murine thymoma viral oncogene homolog)-targeted therapies. These results demonstrate that elevated nutrient levels can drive resistance to targeted cancer treatments and nominate mTORC2 as a central node for integrating growth factor signaling with nutrient availability in GBM.
Full-text · Article · Jul 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Diffusion magnetic resonance imaging (MRI) and 6-[(18)F]fluoro-L-dopa ([(18)F]FDOPA) positron emission tomography (PET) are used to interrogate malignant tumor microenvironment. It remains unclear whether there is a relationship between [(18)F]FDOPA uptake, diffusion MRI estimates of apparent diffusion coefficient (ADC), and mitotic activity in the context of recurrent malignant gliomas, where the tumor may be confounded by the effects of therapy. The purpose of the current study is to determine whether there is a correlation between these imaging techniques and mitotic activity in malignant gliomas.
We retrospectively examined 29 patients with recurrent malignant gliomas who underwent structural MRI, diffusion MRI, and [(18)F]FDOPA PET prior to surgical resection. Qualitative associations were noted, and quantitative voxel-wise and median measurement correlations between [(18)F]FDOPA PET, ADC, and mitotic index were performed.
Areas of high [(18)F]FDOPA uptake exhibited low ADC and areas of hyperintensity T2/fluid-attenuated inversion recovery (FLAIR) with low [(18)F]FDOPA uptake exhibited high ADC. There was a significant inverse voxel-wise correlation between [(18)F]FDOPA and ADC for all patients. Median [(18)F]FDOPA uptake and median ADC also showed a significant inverse correlation. Median [(18)F]FDOPA uptake was positively correlated, and median ADC was inversely correlated with mitotic index from resected tumor tissue.
A significant association may exist between [(18)F]FDOPA uptake, diffusion MRI, and mitotic activity in recurrent malignant gliomas.
No preview · Article · Dec 2014 · Molecular Imaging & Biology
[Show abstract][Hide abstract] ABSTRACT: Background
Ventriculoperitoneal shunt obstruction remains a major problem in pediatric neurosurgery. We analyzed the tissue reaction to ventriculoperitoneal shunts and compared the histology versus time elapsed to shunt failure.
85 ventricular catheter tissues samples obtained from 71 patients were reviewed along with time elapsed to shunt revision. Pathology reports of all tissue samples were divided into three categories: inflammatory based on the presence of lymphocytes, macrophages, and microglial cells; reactive based on the presence of fibro-connective tissue, reactive astrocytes, and Rosenthal fibers; and normal brain tissue based on presence of choroid plexus. These categories were then grouped according to time elapsed to shunt revision. Group I had those shunts revised < 6 months, group II included shunts revised between 6 months to 3 years, while group III had shunts revised after more than 3 years.
The incidence of inflammatory type of histology was 44% (16/36) in group I, 22% (6/27) in group II, and 18% (4/22) in group III. The reactive histology was 42% (15/36) in group I, 67% (18/27) in group II, and 77% (17/22) in group III. There was a clear noted difference of incidence between inflammatory versus reactive histology between early shunt failure compared to late shunt failure. Incidence of normal brain tissue remained high in group I with 8%, 11% in group II, and none in group III.
Early shunt obstruction arises from pathologies different from those causing late shunt obstructions.
No preview · Article · Dec 2014 · Clinical Neurology and Neurosurgery
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: To ascertain the radiologic response and serial volumetric changes in patients with glioblastoma multiforme (GBM)
treated with dendritic cell (DC) vaccine using contrast enhanced (CE) T1-weighted subtraction maps. METHODS: MR images were
acquired for fourteen patients with newly diagnosed or recurrent GBM. Genetic analyses and intratumoral histopathology were
performed on all but one patient. Changes in CE tumor volumes were assessed with CE T1-weighted subtraction maps generated
via voxel-wise subtraction of Gaussian-normalized signal intensities T1-weighted and CE T1-weighted images. Response criterion
was determined as 25% reduction in the T1 subtraction CE tumor volume with respect to pre-vaccination baseline. Response stratification
was tested as a predictor of PFS using log-rank univariate analysis. RESULTS: Four patients responded to the treatment within
the first three months of treatment. Ten patients did not respond within the same period of time. One patient exhibited a
growth pattern consistent with pseudo-progression between 100 and 300 days after vaccination. A significant difference in
tumor growth rate (P = 0.0064) months between responders and non-responders was found within the first three months via linear
regression. A preference for unfavorable response in MGMT unmethylated, EGFR amplified, and EGFRVIII mutated tumors, and favorable
response in IDH1 mutant tumors was observed. A significantly longer PFS was found both from the opening date of the trial
for each patient (Log-rank, P = 0.0149) and from the date of first vaccination (Log-rank, P = 0.0440) for responders versus
non-responders. CONCLUSION: The clinical utility of T1 subtraction maps for tumor visualization and burden evaluation was
shown. Response outcomes to DC vaccine therapy become apparent within the first three months of following treatment. Mischaracterization
of pseudo-progression in the context of DC vaccine therapy for malignant glioma may not be a major concern. Genetic analyses
and histopathological considerations may be a component in assessing treatment response to immunotherapy in malignant gliomas.
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Persistent bacterial infection prolongs hospitalizations, leading to increased healthcare costs. Treatment of these infections costs several billion dollars annually. Biofilm production is one mechanism by which bacteria become resistant. With the help of biofilms, bacteria withstand the host immune response and are much less susceptible to antibiotics. Currently, there is interest in the use of laser-generated shockwaves (LGS) to delaminate biofilm from infected wound surfaces; however, the safety of such an approach has not yet been established. Of particular concern are the thermal and mechanical effects of the shockwave treatment on the epidermis and the underlying collagen structure of the dermis. The present study is a preliminary investigation of the effect of LGS on freshly harvested ex vivo porcine skin tissue samples.
Materials and methods:
Tissue samples for investigation were harvested immediately post-mortem and treated with LGS within 30 minutes. Previous studies have shown that laser fluences between 100 and 500 mJ/pulse are capable of delaminating biofilms off a variety of surfaces, thus our preliminary investigation focused on this range of laser energy. For each sample, LGS were produced via laser irradiation of a thin layer (0.5 µm) of titanium sandwiched between a 50 and 100 µm thick layer of water glass and a 0.1 mm thick sheet of Mylar. The rapid thermal expansion of the irradiated titanium film generates a transient compressive wave that is coupled through a liquid layer to the surface of the ex vivo pigskin sample. Shocked samples were immediately fixed in formalin and prepared for histological analysis. A blinded pathologist evaluated and scored each section on the basis of its overall appearance (O) and presence of linear/slit-like spaces roughly parallel to the surface of the skin (S). The scores were given on a scale of 0-3.
The present investigation revealed no visible difference between the tissue sections of the control sample and those that were subjected to laser-generated shockwaves. There was no relationship between the scores received by the samples and the energy with which they were shocked.
Preliminary investigation into the safety of the LGS treatment for biofilm delamination appears promising. Additional investigation will continue on ex vivo porcine samples, followed by an in vivo animal trial to better understand the physiological response to LGS treatment.
No preview · Article · Oct 2014 · Lasers in Surgery and Medicine
[Show abstract][Hide abstract] ABSTRACT: Purpose:
IDH1/2-mutant gliomas harbor a distinct glioma-CpG island methylation phenotype (G-CIMP) that may promote the initiation and progression of secondary pathway gliomas by silencing tumor-suppressive genes. The potential role of tumor-suppressive microRNAs (miRNA; miR) in this process is not understood.
To identify potential tumor-suppressive miRNA hypermethylated in glioma, the methylation profiles of IDH1/2(WT) gliomas (n = 11) and IDH1(MUT) glioma (n = 20) were compared by using massively parallel reduced representation bisulfite sequencing (RRBS). The methylation status of selected miRNA was validated by using targeted bisulfite sequencing (BiSEQ) in a large cohort of glioma tissue samples including 219 IDH1(WT) and 72 IDH1/2(MUT) samples. The expression of selected miRNAs was determined by using the TaqMan qPCR. Functional analyses of miR148a were conducted and target genes were identified.
We identify miR148a as a novel, G-CIMP-associated miRNA whose methylation is tightly correlated with IDH1 mutation and associated with improved survival in patients with malignant glioma. We confirm that downregulation of miR148a can occur via DNA methylation. We demonstrate that IDH1 mutation provides a mechanism of miR148a methylation and downregulation, and that restoration of miR148a reduced tumorigenic properties of glioma cells, possibly by targeting DNMT1.
We identify miR148a as a novel G-CIMP-associated miRNA, and provide results suggesting that miR148a restoration may have therapeutic implications.
Full-text · Article · Sep 2014 · Clinical Cancer Research