Pierre Bady

Pathway Analysis of Glioblastoma Tissue after Preoperative Treatment with the EGFR Tyrosine Kinase Inhibitor Gefitinib—A Phase II Trial

Amplification of the epidermal growth factor receptor (EGFR) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we aimed at investigating the molecular effects of the tyrosine kinase inhibitor gefitinib on the EGFR signaling pathway in human glioblastoma. Twenty-two patients selected for reoperation of recurrent glioblastoma were treated within a phase II trial for 5 days with 500 mg gefitinib before surgery followed by postoperative gefitinib until recurrence. Resected glioblastoma tissues exhibited high concentrations of gefitinib (median, 4.1 μg/g), 20 times higher than respective plasma. EGFR-pathway activity was evaluated with phosphorylation-specific assays. The EGFR was efficiently dephosphorylated in treated patients as compared to a control cohort of 12 patients. However, no significant effect on 12 pathway constituents was detected. In contrast, in vitro treatment of a glioblastoma cell line, BS-153, with endogenous EGFRwt amplification and EGFRvIII expression resulted not only in dephosphorylation of the EGFR, but also of key regulators in the pathway such as AKT. Treating established xenografts of the same cell line as an in vivo model showed dephosphorylation of the EGFR without affecting downstream signal transductors, similar to the human glioblastoma. Taken together, gefitinib reaches high concentrations in the tumor tissue and efficiently dephosphorylates its target. However, regulation of downstream signal transducers in the EGFR pathway seems to be dominated by regulatory circuits independent of EGFR phosphorylation. Mol Cancer Ther; 10(6); 1102–12. ©2011 AACR.

Chromosome 7 gain and DNA hypermethylation at the HOXA10 locus are associated with expression of a stem cell related HOX-signature in glioblastoma

BackgroundHOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma.ResultsWe observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively.ConclusionsBased on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.

Phase II study of radiotherapy and temsirolimus versus radiochemotherapy with temozolomide in patients with newly diagnosed glioblastoma without MGMT promoter hypermethylation (EORTC 26082)

Purpose: EORTC 26082 assessed the activity of temsirolimus in patients with newly diagnosed glioblastoma harboring an unmethylated O6 methylguanine-DNA-methyltransferase (MGMT) promoter. Experimental Design: Patients (n = 257) fulfilling eligibility criteria underwent central MGMT testing. Patients with MGMT unmethylated glioblastoma (n = 111) were randomized 1:1 between standard chemo-radiotherapy with temozolomide or radiotherapy plus weekly temsirolimus (25 mg). Primary endpoint was overall survival at 12 months (OS12). A positive signal was considered >38 patients alive at 12 months in the per protocol population. A noncomparative reference arm of 54 patients evaluated the assumptions on OS12 in a standard-treated cohort of patients. Prespecified post hoc analyses of markers reflecting target activation were performed. Results: Both therapies were administered per protocol with a median of 13 cycles of maintenance temsirolimus. Median age was 55 and 58 years in the temsirolimus and standard arms, the WHO performance status 0 or 1 for most patients (95.5%). In the per protocol population, 38 of 54 patients treated with temsirolimus reached OS12. The actuarial 1-year survival was 72.2% [95% confidence interval (CI), 58.2–82.2] in the temozolomide arm and 69.6% (95% CI, 55.8–79.9) in the temsirolimus arm [hazard ratio (HR) 1.16; 95% CI, 0.77–1.76; P = 0.47]. In multivariable prognostic analyses of clinical and molecular factors, phosphorylation of mTORSer2448 in tumor tissue (HR 0.13; 95% CI, 0.04–0.47; P = 0.002), detected in 37.6%, was associated with benefit from temsirolimus. Conclusions: Temsirolimus was not superior to temozolomide in patients with an unmethylated MGMT promoter. Phosphorylation of mTORSer2448 in the pretreatment tumor tissue may define a subgroup benefitting from mTOR inhibition. Clin Cancer Res; 22(19); 4797–806. ©2016 AACR.

DNA fingerprinting of glioma cell lines and considerations on similarity measurements

Glioma cell lines are an important tool for research in basic and translational neuro-oncology. Documentation of their genetic identity has become a requirement for scientific journals and grant applications to exclude cross-contamination and misidentification that lead to misinterpretation of results. Here, we report the standard 16 marker short tandem repeat (STR) DNA fingerprints for a panel of 39 widely used glioma cell lines as reference. Comparison of the fingerprints among themselves and with the large DSMZ database comprising 9 marker STRs for 2278 cell lines uncovered 3 misidentified cell lines and confirmed previously known cross-contaminations. Furthermore, 2 glioma cell lines exhibited identity scores of 0.8, which is proposed as the cutoff for detecting cross-contamination. Additional characteristics, comprising lack of a B-raf mutation in one line and a similarity score of 1 with the original tumor tissue in the other, excluded a cross-contamination. Subsequent simulation procedures suggested that, when using DNA fingerprints comprising only 9 STR markers, the commonly used similarity score of 0.8 is not sufficiently stringent to unambiguously differentiate the origin. DNA fingerprints are confounded by frequent genetic alterations in cancer cell lines, particularly loss of heterozygosity, that reduce the informativeness of STR markers and, thereby, the overall power for distinction. The similarity score depends on the number of markers measured; thus, more markers or additional cell line characteristics, such as information on specific mutations, may be necessary to clarify the origin.

Cilengitide in newly diagnosed glioblastoma: biomarker expression and outcome.

Integrins αvβ3 and αvβ5 regulate angiogenesis and invasiveness in cancer, potentially by modulating activation of the transforming growth factor (TGF)-β pathway. The randomized phase III CENTRIC and phase II CORE trials explored the integrin inhibitor cilengitide in patients with newly diagnosed glioblastoma with versus without O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. These trials failed to meet their primary endpoints. Immunohistochemistry was used to assess the levels of the target integrins of cilengitide, αvβ3 and αvβ5 integrins, of αvβ8 and of their putative target, phosphorylation of SMAD2, in tumor tissues from CENTRIC (n=274) and CORE (n=224). αvβ3 and αvβ5 expression correlated well in tumor and endothelial cells, but showed little association with αvβ8 or pSMAD2 levels. In CENTRIC, there was no interaction between the biomarkers and treatment for prediction of outcome. In CORE, higher αvβ3 levels in tumor cells were associated with improved progression-free survival by central review and with improved overall survival in patients treated with cilengitide. Integrins αvβ3, αvβ5 and αvβ8 are differentially expressed in glioblastoma. Integrin levels do not correlate with the activation level of the canonical TGF-β pathway. αvβ3 integrin expression may predict benefit from integrin inhibition in patients with glioblastoma lacking MGMT promoter methylation.

Mir-21-Sox2 axis delineates glioblastoma subtypes with prognostic impact

Glioblastoma (GBM) is the most aggressive human brain tumor. Although several molecular subtypes of GBM are recognized, a robust molecular prognostic marker has yet to be identified. Here, we report that the stemness regulator Sox2 is a new, clinically important target of microRNA-21 (miR-21) in GBM, with implications for prognosis. Using the MiR-21–Sox2 regulatory axis, approximately half of all GBM tumors present in the Cancer Genome Atlas (TCGA) and in-house patient databases can be mathematically classified into high miR-21/low Sox2 (Class A) or low miR-21/high Sox2 (Class B) subtypes. This classification reflects phenotypically and molecularly distinct characteristics and is not captured by existing classifications. Supporting the distinct nature of the subtypes, gene set enrichment analysis of the TCGA dataset predicted that Class A and Class B tumors were significantly involved in immune/inflammatory response and in chromosome organization and nervous system development, respectively. Patients with Class B tumors had longer overall survival than those with Class A tumors. Analysis of both databases indicated that the Class A/Class B classification is a better predictor of patient survival than currently used parameters. Further, manipulation of MiR-21–Sox2 levels in orthotopic mouse models supported the longer survival of the Class B subtype. The MiR-21–Sox2 association was also found in mouse neural stem cells and in the mouse brain at different developmental stages, suggesting a role in normal development. Therefore, this mechanism-based classification suggests the presence of two distinct populations of GBM patients with distinguishable phenotypic characteristics and clinical outcomes. SIGNIFICANCE STATEMENT Molecular profiling-based classification of glioblastoma (GBM) into four subtypes has substantially increased our understanding of the biology of the disease and has pointed to the heterogeneous nature of GBM. However, this classification is not mechanism based and its prognostic value is limited. Here, we identify a new mechanism in GBM (the miR-21–Sox2 axis) that can classify ∼50% of patients into two subtypes with distinct molecular, radiological, and pathological characteristics. Importantly, this classification can predict patient survival better than the currently used parameters. Further, analysis of the miR-21-Sox2 relationship in mouse neural stem cells and in the mouse brain at different developmental stages indicates that miR-21 and Sox2 are predominantly expressed in mutually exclusive patterns, suggesting a role in normal neural development.

Genome-wide DNA methylation detection by MethylCap-seq and Infinium HumanMethylation450 BeadChips: an independent large-scale comparison.

Two cost-efficient genome-scale methodologies to assess DNA-methylation are MethylCap-seq and Illumina’s Infinium HumanMethylation450 BeadChips (HM450). Objective information regarding the best-suited methodology for a specific research question is scant. Therefore, we performed a large-scale evaluation on a set of 70 brain tissue samples, i.e. 65 glioblastoma and 5 non-tumoral tissues. As MethylCap-seq coverages were limited, we focused on the inherent capacity of the methodology to detect methylated loci rather than a quantitative analysis. MethylCap-seq and HM450 data were dichotomized and performances were compared using a gold standard free Bayesian modelling procedure. While conditional specificity was adequate for both approaches, conditional sensitivity was systematically higher for HM450. In addition, genome-wide characteristics were compared, revealing that HM450 probes identified substantially fewer regions compared to MethylCap-seq. Although results indicated that the latter method can detect more potentially relevant DNA-methylation, this did not translate into the discovery of more differentially methylated loci between tumours and controls compared to HM450. Our results therefore indicate that both methodologies are complementary, with a higher sensitivity for HM450 and a far larger genome-wide coverage for MethylCap-seq, but also that a more comprehensive character does not automatically imply more significant results in biomarker studies.

Ubiquitin Specific Peptidase 15 (USP15) suppresses glioblastoma cell growth via stabilization of HECTD1 E3 ligase attenuating WNT pathway activity

Expression based prediction of new genomic alterations in glioblastoma identified the de-ubiquitinase Ubiquitin Specific Peptidase 15 (USP15) as potential tumor suppressor gene associated with genomic deletions (11%). Ectopic expression of USP15 in glioblastoma cell-lines reduced colony formation and growth in soft agar, while overexpression of its functional mutant had the opposite effect. Evaluation of the protein binding network of USP15 by Mass Spectrometry in glioblastoma cells uncovered eight novel interacting proteins, including HECT Domain Containing E3 Ubiquitin Protein Ligase 1 (HECTD1), whose mouse homologue has been associated with an inhibitory effect on the WNT-pathway. USP15 de-ubiquitinated and thereby stabilized HECTD1 in glioblastoma cells, while depletion of USP15 led to decreased HECTD1 protein levels. Expression of USP15 in glioblastoma cells attenuated WNT-pathway activity, while expression of the functional mutant enhanced the activity. Modulation of HECTD1 expression pheno-copied the effects observed for USP15. In accordance, human glioblastoma display a weak but significant negative correlation between USP15 and AXIN2 expression. Taken together, the data provide evidence that USP15 attenuates the canonical WNT pathway mediated by stabilization of HECTD1, supporting a tumor suppressing role of USP15 in a subset of glioblastoma.

Abstract LB-A01: Molecular subgroup analysis of a randomized trial (EORTC 26082-22081) testing temsirolimus and radiation therapy versus chemoradiotherapy with temozolomide in patients with newly diagnosed glioblastoma without methylation of the MGMT gene promoter

Background: Preclinical data indicate activity of mammalian target of rapamycin (mTOR) inhibitors, as well as synergistic activity together with radiotherapy in glioblastoma. The aim of this non-comparative randomized phase II trial (NCT01019434) was to assess the therapeutic activity of the mTOR inhibitor temsirolimus, in patients with newly diagnosed glioblastoma with unmethylated O6 methylguanine-DNA-methyltransferase (MGMT) promoter. These patients are in particular need for new treatment options as they derive little if any benefit from the currently standard temozolomide treatment. Methods: Patients were registered and their tumors were centrally reviewed and tested for the MGMT status. Participation to the trial was offered to patients with histologically confirmed glioblastoma harboring an unmethylated MGMT promoter. Patients (n = 111) were randomized 1:1 between standard of care of radiotherapy (60 Gy; 5 times 2 Gy per week) plus concomitant and six cycles of maintenance temozolomide (TMZ/RT/→TMZ) or radiotherapy plus weekly temsirolimus at 25 mg flat dose to be continued until progression or undue toxicity. Primary endpoint was overall survival at 12 months (OS12). For a representative subgroup of 88 patients sufficient tumor tissue was available for pre-specified post hoc analyses of a panel of markers considered to be relevant for the activity of mTOR inhibition in order to identify a subgroup of patients potentially sensitive to temsirolimus. Findings: Both therapies were properly administered with a median of 13 weeks of maintenance temsirolimus or 4 cycles of TMZ. In the intention to treat population OS12 was 69.6% [95% CI (55.8-79.9) in the temsirolimus arm and 72.2% [95% CI (58.2, 82.2)] in the temozolomide arm [HR = 1.16 95% CI (0.77-1.76), p = 0.47]. In multivariable prognostic analyses of clinical and molecular factors, the phosphorylation status of mTOR [HR = 0.13, 95% CI (0.04-0.47), p = 0.002], but no clinical factor was a significant predictor in the temsirolimus arm. There was a significant interaction between treatment and phosphorylated mTOR (p-mTOR) (p = 0.047). The median overall survival in the temsirolimus group was 17.8 months (CI, 14.1-28.0) for patients with p-mTOR positive tumors and 13.11 (CI, 9.7-15.1) in the negative subgroup. In the RT/TMZ→TMZ control arm the median survival in the p-mTOR positive group was 14.0 months (CI, 9.6-19.6) and 16.5 months (CI, 9.5-18.8) in the p-mTOR negative subgroup. Interpretation: The therapeutic activity of temsirolimus is too low in the whole cohort of patients with newly diagnosed glioblastoma with an unmethylated MGMT promoter. However, the phosphorylation status of mTOR may identify a subgroup that benefits from temsirolimus, while the same treatment may be detrimental to patients with p-mTOR negative tumors. Funding: Pfizer provided an unrestricted academic grant. Molecular subgroup analysis was funded by the Swiss National Science Foundation (FN31003A-138116 to M.E.H). Previously presented in part at ASCO 2014 (abstract #2003) Citation Format: Monika E. Hegi, Thierry Gorlia, Pierre Bady, Michael Platten, Martin J. van den Bent, Martin JB Taphoorn, Jonathan Steuve, Alba A. Brandes, Marie-France Hamou, Markus Kosch, Vassilis Golfinopoulos, Mario Campone, Patrick Roth, Benoit Lhermitte, Roger Stupp, Gianfranco Pesce, Wolfgang Wick. Molecular subgroup analysis of a randomized trial (EORTC 26082-22081) testing temsirolimus and radiation therapy versus chemoradiotherapy with temozolomide in patients with newly diagnosed glioblastoma without methylation of the MGMT gene promoter. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A01.

Abstract 4031: MGMT methylation based outcome prediction is associated with two CpG regions separated by a prediction minimum centred at the initiation start site

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Promoter methylation of the repair gene MGMT is a predictive factor for benefit from alkylating agent therapy in glioblastoma patients. High through-put platforms for genome wide DNA methylation analysis have allowed establishing the methylome of large series of patient samples that has identified a glioma CpG methylator phenotype (G-CIMP). However, classification of samples as being methylated or unmethylated for a given gene is not obvious, since the relationship between CpG-methylation at a given site and the extent of the overall CpG island methylation, and the effect on gene silencing is strongly dependent on the location. For the MGMT gene 176 CpGs are interrogated on the 450K chip of which 18 are located in the CpG island associated with the promoter region. Correlation of MGMT methylation at these 18 CpG sites in 63 glioblastoma and 5 non tumoral brain tissues and methylation prediction established previously by methylation specific PCR (MSP) indicated 2 regions with highest association. The two areas were separated by a prediction minimum centered at the initiation start site. The correlation of MGMT expression with the methylation status of CpGs followed the same pattern with the strongest negative correlation reaching –0.5 at the 2 peaks. Subsequently we tested the predictive value of the 18 CpGs for outcome in these patients treated within clinical trials with temozolomide containing chemo-radiotherapy. Most intriguingly, correlation with gene expression, survival analyses and association with MSP displayed almost superposable images of CpGs, at the previously identified CpG regions! A model for MGMT methylation classification based on stepwise logistic regression was constructed with a kappa value close to 0.85. The model was validated in a published external data set of homogeneously treated patient cohort with anaplastic glioma for which methylation data and survival information was available, and for which MGMT methylation was reported as prognostic factor. The model was subsequently applied to the methylation data available from the The Cancer Genome Atlas (TCGA) glioblastoma data-set comprising 241 samples. Classification of the samples for their MGMT methylation status based on the predictor revealed that MGMT is frequently methylated in glioblastoma with G-CIMP (simulated p=0.001 chi-square, Monte-Carlo method), but is not a G-CIMP gene. Taken together, the location of methylated CpGs within the promoter is crucial for prediction of MGMT silencing and subsequent impact on outcome. Hence, for efficient mining of large scale methylation data we need models to identify regions of CpGs with a high impact on gene silencing or other features relevant for the understanding of tumor biology and effect on response to therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4031. doi:1538-7445.AM2012-4031