Stephan Saikali

Comparative assessment of 5 methods (methylation-specific polymerase chain reaction, methylight, pyrosequencing, methylation-sensitive high-resolution melting, and immunohistochemistry) to analyze O6-methylguanine-DNA-methyltranferase in a series of 100 glioblastoma patients.

There is a strong need to determine the best technique for O6‐methylguanine‐DNA‐methyltranferase (MGMT) analysis, because MGMT status is currently used in clinical trials and occasionally in routine clinical practice for glioblastoma patients.

DNA methylation in glioblastoma: impact on gene expression and clinical outcome

BackgroundChanges in promoter DNA methylation pattern of genes involved in key biological pathways have been reported in glioblastoma. Genome-wide assessments of DNA methylation levels are now required to decipher the epigenetic events involved in the aggressive phenotype of glioblastoma, and to guide new treatment strategies.ResultsWe performed a whole-genome integrative analysis of methylation and gene expression profiles in 40 newly diagnosed glioblastoma patients. We also screened for associations between the level of methylation of CpG sites and overall survival in a cohort of 50 patients uniformly treated by surgery, radiotherapy and chemotherapy with concomitant and adjuvant temozolomide (STUPP protocol). The methylation analysis identified 616 CpG sites differentially methylated between glioblastoma and control brain, a quarter of which was differentially expressed in a concordant way. Thirteen of the genes with concordant CpG sites displayed an inverse correlation between promoter methylation and expression level in glioblastomas: B3GNT5, FABP7, ZNF217, BST2, OAS1, SLC13A5, GSTM5, ME1, UBXD3, TSPYL5, FAAH, C7orf13, and C3orf14. Survival analysis identified six CpG sites associated with overall survival. SOX10 promoter methylation status (two CpG sites) stratified patients similarly to MGMT status, but with a higher Area Under the Curve (0.78 vs. 0.71, p- value < 5e-04). The methylation status of the FNDC3B, TBX3, DGKI, and FSD1 promoters identified patients with MGMT-methylated tumors that did not respond to STUPP treatment (p- value < 1e-04).ConclusionsThis study provides the first genome-wide integrative analysis of DNA methylation and gene expression profiles obtained from the same GBM cohort. We also present a methylome-based survival analysis for one of the largest uniformly treated GBM cohort ever studied, for more than 27,000 CpG sites. We have identified genes whose expression may be tightly regulated by epigenetic mechanisms and markers that may guide treatment decisions.

Expression of nine tumour antigens in a series of human glioblastoma multiforme: interest of EGFRvIII, IL-13Rα2, gp100 and TRP-2 for immunotherapy

In this study, we investigated the mRNA and protein expression of nine tumour antigens in human glioblastoma multiforme with a view to their possible use in dendritic cell-based immunotherapy. Expression of ALK, EGFRvIII, GALT3, gp100, IL-13Rα2, MAGE-A3, NA17-A, TRP-2 and tyrosinase were studied by real-time RT-PCR on frozen tissues using a series of 47 tumour samples from patients with glioblastoma. Results were compared with non-neoplastic brain expression or glioblastoma samples with very low levels of expression near the limits of detection for EGFRvIII and MAGE-A3, as these latter two antigens were not detected in non-neoplastic brain. Tumour antigens showing a 5-fold increase in mRNA expression were considered as positive, and only antigens displaying an mRNA over-expression in a significant number of cases were analysed by immunohistochemistry on paraffin-embedded sections. Using real time RT-PCR, we found EGFRvIII, gp100, IL-13Rα2 and TRP-2 to be positive in 64, 38, 32 and 21% of cases, respectively. While we observed no over-expression for ALK, GALT3 and tyrosinase, 3 samples out of 47 were positive for MAGE-3 and 1 sample for NA17-A. More than 25% of tumour cells showed strong protein expression in 13, 34, 85 and 96% of GBM samples for gp100, TRP-2, EGFRvIII and IL-13Rα2, respectively. Interestingly, protein expression of at least 3 antigens was observed in 38% of cases. These results point out the importance of EGFRvIII, IL-13Rα2 and, to a less extent gp100 and TRP-2, for developing an immunotherapy strategy against glioblastoma.

A three-dimensional digital segmented and deformable brain atlas of the domestic pig

We used high-magnetic field (4.7 T) magnetic resonance imaging (MRI) to build the first high-resolution (100 microm x 150 microm x 100 microm) three-dimensional (3D) digital atlas in stereotaxic coordinates of the brain of a female domestic pig (Sus scrofa domesticus). This atlas was constructed from one hemisphere which underwent a symmetrical transformation through the midsagittal plane. Concomitant construction of a 3D histological atlas based on the same scheme facilitated control of deep brain structure delimitation and enabled cortical mapping to be achieved. The atlas contains 178 individual cerebral structures including 42 paired and 9 single deep brain structures, 5 ventricular system areas, 6 paired deep cerebellar nuclei, 12 cerebellar lobules and 28 cortical areas per hemisphere. Given the increasing importance of pig brains in medical research, this atlas should be a useful tool for intersubject normalization in anatomical imaging as well as for precisely localizing brain areas in functional MR studies or electrode implantation trials. The atlas can be freely downloaded from our institutions Website.

Integrative genome‐wide analysis reveals a robust genomic glioblastoma signature associated with copy number driving changes in gene expression

Glioblastoma multiforme shows multiple chromosomal aberrations, the impact of which on gene expression remains unclear. To investigate this relationship and to identify putative initiating genomic events, we integrated a paired copy number and gene expression survey in glioblastoma using whole human genome arrays. Loci of recurrent copy number alterations were combined with gene expression profiles obtained on the same tumor samples. We identified a set of 406 “cis‐acting DNA targeted genes” corresponding to genomic aberrations with direct copy‐number‐driving changes in gene expression, defined as genes with either significantly concordant or correlated changes in DNA copy number and expression. Functional annotation revealed that these genes participate in key processes of cancer cell biology, providing insights into the genetic mechanisms driving glioblastoma. The robustness of the gene selection was validated on an external microarray data set including 81 glioblastomas and 23 non‐neoplastic brain samples. The integration of array CGH and gene expression data highlights a robust cis‐acting DNA targeted genes signature that may be critical for glioblastoma progression, with two tumor suppressor genes PCDH9 and STARD13 that could be involved in tumor invasiveness and resistance to etoposide.

Distinct effects of human glioblastoma immunoregulatory molecules programmed cell death ligand-1 (PDL-1) and indoleamine 2,3-dioxygenase (IDO) on tumour-specific T cell functions.

Immunotherapy is a promising new treatment for patients suffering from glioma, in particular glioblastoma multiforme (GBM). However, tumour cells use different mechanisms to escape the immune responses induced by the treatment. As many other tumours, gliomas express or secrete several immunosuppressive molecules that regulate immune cell functions. In this study, we first analysed FasL, HLA-G, IDO, PDL-1 and TGF-beta1, -beta2 and -beta3 expression by transcriptomic microarray analysis in a series of 20 GBM samples and found respectively 15%, 60%, 85%, 30%, 70%, 80% and 35% of positive specimens. mRNA expression was then confirmed in 10 GBM primary cell lines and 2 immortalised cell lines U251 and U87MG. Furthermore, the protein expression of PDL-1, IDO activity and TGF-beta2 secretion were found on most of the untreated GBM primary cell lines. Remarkably, treatment with IFN-gamma increased the PDL-1 cell surface expression and the IDO activity, but reduced the TGF-beta2 secretion of GBM cell lines. We finally analysed the immunosuppressive effects of IDO, PDL-1 and TGF-beta1-3 by measuring IFN-gamma production and cell cytotoxicity activity of tumour antigen-specific T cells. PDL-1 partially affected the IFN-gamma production of antigen-specific T cells in response to GBM primary cell lines, and IDO inhibited lymphocyte proliferation induced by lectins. None of these molecules directly affected the T cell cytotoxicity function. Due to the functional role of PDL-1 and IDO molecules expressed by GBM cells, one could expect that blocking these molecules in the immunotherapy strategies would reinforce the efficiency of these treatments of GBM patients.

A 4-Gene Signature Associated with Clinical Outcome in High-Grade Gliomas

Purpose: Gene expression studies provide molecular insights improving the classification of patients with high-grade gliomas. We have developed a risk estimation strategy based on a combined analysis of gene expression data to search for robust biomarkers associated with outcome in these tumors. Experimental Design: We performed a meta-analysis using 3 publicly available malignant gliomas microarray data sets (267 patients) to define the genes related to both glioma malignancy and patient outcome. These biomarkers were used to construct a risk-score equation based on a Cox proportional hazards model on a subset of 144 patients. External validations were performed on microarray data (59 patients) and on RT-qPCR data (194 patients). The risk-score model performances (discrimination and calibration) were evaluated and compared with that of clinical risk factors, MGMT promoter methylation status, and IDH1 mutational status. Results: This interstudy cross-validation approach allowed the identification of a 4-gene signature highly correlated to survival (CHAF1B, PDLIM4, EDNRB, and HJURP), from which an optimal survival model was built (P < 0.001 in training and validation sets). Multivariate analysis showed that the 4-gene risk score was strongly and independently associated with survival (hazard ratio = 0.46; 95% CI, 0.26–0.81; P = 0.007). Performance estimations indicated that this score added beyond standard clinical parameters and beyond both the MGMT methylation status and the IDH1 mutational status in terms of discrimination (C statistics, 0.827 versus 0.835; P < 0.001). Conclusion: The 4-gene signature provides an independent risk score strongly associated with outcome of patients with high-grade gliomas. Clin Cancer Res; 17(2); 317–27. ©2011 AACR.

Human Glioblastoma Stem-Like Cells are More Sensitive to Allogeneic NK and T Cell-Mediated Killing Compared with Serum-Cultured Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most dramatic primary brain cancer with a very poor prognosis because of inevitable disease recurrence. The median overall survival is less than 1 year after diagnosis. Cancer stem cells have recently been disclosed in GBM. GBM stem‐like cells (GSCs) exhibit resistance to radio/chemotherapeutic treatments and are therefore considered to play an important role in disease recurrence. GSCs are thus appealing targets for new treatments for GBM patients. In this study, we show that GBM cells with stem cell characteristics are resistant to lysis mediated by resting natural killer (NK) cells because of the expression of MHC class I molecules. However, GSCs are killed by lectin‐activated NK cells. Furthermore, in experiments using the therapeutic antibody CetuximAb, we show that GSCs are sensitive to antibody‐mediated cytotoxicity. We confirm the sensitivity of GSC to cytotoxicity carried out by IL2‐activated NK cells and tumor‐specific T cells. More importantly, we show that GSCs are more sensitive to NK and T cell‐mediated lysis relatively to their corresponding serum‐cultured GBM cells obtained from the same initial tumor specimen. Altogether, these results demonstrate the sensitivity of GSC to immune cell cytotoxicity and, therefore, strongly suggest that GSCs are suitable target cells for immunotherapy of GBM patients.

Survival and prognostic factors in a series of adults with medulloblastomas.

OBJECT In this article, the authors report their experience in the management of adult patients with medulloblastoma at their institution to identify prognostic factors important for survival and disease control. METHODS Between 1977 and 2005, 27 patients who were >or=16 years old and had medulloblastoma were treated consecutively. There were 16 women and 11 men with a median age of 21 years (range 16-54 years). Gross-total resection was performed in 21 patients, subtotal (>or=90%) in 2, incomplete in 1, and biopsy in 3 patients. Six patients had the desmoplastic variant, and 21 patients presented with classic medulloblastoma. Staging according to the Chang classification showed 4 patients with tumors invading the brainstem (2 with Stage T3b and 2 with Stage T4), 3 patients with metastases (2 with Stage M2 and 1 with Stage M3), and 1 patient in whom the stage was unknown (Stage MX) who died 10 days postoperatively. Twenty patients were assigned to the standard-risk group and 7 to the high-risk group. All patients except the one whose status was classified as Stage MX underwent craniospinal radiotherapy at our institution. Seven patients received chemotherapy before radiotherapy. RESULTS The 5- and 10-year overall survival rates for the present study were 81 and 62%, respectively. The median overall survival time was 17.7 years. The 5- and 10-year event-free survival rates were 72 and 57%, respectively. The median event-free survival time was 17.9 years. Univariate analysis showed that survival was significantly correlated with sex (women had a better prognosis than men) and M stage (patients without metastases had a better outcome). Patient age, duration of symptoms, Karnofsky Performance Scale score at presentation, hydrocephalus, tumor location, brainstem invasion, extent of resection, histological subtype, preradiotherapy chemotherapy, risk group, and period of presentation were not significant variables. Multivariate analysis identified sex and M stage as well as the period of presentation as independent prognostic factors for overall and event-free survival times. Eleven patients suffered tumor recurrence within a median time of 4.2 years. The posterior fossa was not the most common site of recurrence, and delayed recurrence was not rare. All patients in whom the tumor recurred have died despite aggressive treatments. The median survival time after diagnosis of recurrence was 2.5 years. Questionnaires on quality of life and cognition showed high scores in favor of limited negative effects in the perception of mental and physical health after treatment. The authors observed 1 supposed second malignancy (thyroid carcinoma) and no evidence of pituitary dysfunction. CONCLUSIONS Long-term survival is possible in adults treated for medulloblastoma. Although rare, metastasis seeding at presentation is a poor prognostic factor. The possibility of delayed recurrence necessitates close follow-up of all patients. Tumor recurrences should be treated with aggressive therapies as some patients may have sustained response. Adjuvant chemotherapy should be given to high-risk patients, but its role in reducing recurrences, particularly distant ones, remains unclear in the standard-risk group.

Can Dynamic Contrast-Enhanced Magnetic Resonance Imaging Combined with Texture Analysis Differentiate Malignant Glioneuronal Tumors from Other Glioblastoma?

An interesting approach has been proposed to differentiate malignant glioneuronal tumors (MGNTs) as a subclass of the WHO grade III and IV malignant gliomas. MGNT histologically resemble any WHO grade III or IV glioma but have a different biological behavior, presenting a survival twice longer as WHO glioblastomas and a lower occurrence of metastases. However, neurofilament protein immunostaining was required for identification of MGNT. Using two complementary methods, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and texture analysis (MRI-TA) from the same acquisition process, the challenge is to in vivo identify MGNT and demonstrate that MRI postprocessing could contribute to a better typing and grading of glioblastoma. Results are obtained on a preliminary group of 19 patients a posteriori selected for a blind investigation of DCE T1-weighted and TA at 1.5 T. The optimal classification (0/11 misclassified MGNT) is obtained by combining the two methods, DCE-MRI and MRI-TA.