Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme.

BACKGROUND: Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). METHODS: RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. RESULTS: Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. CONCLUSION: Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.

Implementation of a molecular tumor board at a regional level to improve access to targeted therapy.

BACKGROUND: With the development of precision oncology, Molecular Tumor Boards (MTB) are developing in many institutions. However, the implementation of MTB in routine clinical practice has still not been thoroughly studied. MATERIAL AND METHODS: Since the first drugs approved for targeted therapies, patient tumor samples were centralized to genomic testing platforms. In our institution, all tumor samples have been analyzed since 2014 by Next Generation Sequencing (NGS). In 2015, we established a regional MTB to discuss patient cases with 1 or more alterations identified by NGS, in genes different from those related to drug approval. We conducted a retrospective comparative analysis to study whether our MTB increased the prescriptions of Molecular Targeted Therapies (MTT) and the inclusions of patients in clinical trials with MTT, in comparison with patients with available NGS data but no MTB discussion. RESULTS: In 2014, 86 patients had UGA, but the results were not available to clinicians and not discussed in MTB. During the years 2015 and 2016, 113 patients with an UGA (unreferenced genomic alteration) were discussed in MTB. No patients with an UGA were included in 2014 in a clinical trial, versus 2 (2%) in 2015-2016. 13 patients with an UGA (12%) were treated in 2015-2016 with a MTT whereas in 2014, no patient (p = 0.001). CONCLUSIONS: In this retrospective analysis, we showed that the association of large-scale genomic testing and MTB was feasible, and could increase the prescription of MTT. However, in routine clinical practice, the majority of patients with UGA still do not have access to MTT.

Correction to: Neuro-radiological characteristics of adult diffuse grade II and III insular gliomas classified according to WHO 2016.

In the initial, online publication, the authors' given names were captured as family names and vice versa. The names are correctly shown here. The original article has been corrected.

Neuro-radiological characteristics of adult diffuse grade II and III insular gliomas classified according to WHO 2016.

INTRODUCTION: The phenotypic heterogeneity of diffuse gliomas is still inconsistently explained by known molecular abnormalities. Here, we report the molecular and radiological features of diffuse grade WHO II and III gliomas involving the insula and its potential impact on prognosis. METHODS: Clinical, pathological, molecular and neuro-radiological features of 43 consecutive patients who underwent a surgical resection between 2006 and 2013 for a grade II and III gliomas involving the insula was retrospectively analyzed. RESULTS: Median age was 44.4 years. Eight patients had oligodendrogliomas, IDH mutant (IDHmut) and 1p/19q-codeleted (6 grade II, 2 grade III). Twenty-eight patients had diffuse astrocytomas, IDHmut (22 grade II and 6 grade III) and seven patients had grade II diffuse astrocytomas, IDHwt (A-IDHwt). Vimentin staining was exclusively recorded in tumor cells from A-IDHwt (p = 0.001). Mean cerebral blood volume (CBV) (p = 0.018), maximal value of CBV (p = 0.017) and ratio of the corrected CBV (p = 0.022) were lower for A-IDHwt. Volumetric segmentation of ADC allowed the identification of the tumor cores, which were smaller in A-IDHwt (p < 0.001). The tumor occurrences of A-IDHwt were exclusively located into the temporo-insular region. Median progression-free survival (PFS) and overall survival (OS) were 50.9 months (95% CI: 26.7-75.0) and 80.9 months (60.1-101.6). By multivariate analysis, A-IDHwt (p = 0.009; p = 0.019), 7p gain and 10q loss (p = 0.009; p = 0.016) and vimentin positive staining (p = 0.011; p = 0.029) were associated with poor PFS and OS respectively. CONCLUSIONS: Insular low-grade A-IDHwt presented with poor prognosis despite a smaller tumor core and no evidence of increased perfusion on MR imaging.

Integration of transcriptome and proteome profiles in glioblastoma: looking for the missing link.

BACKGROUND: Glioblastoma (GB) is the most common and aggressive tumor of the brain. Genotype-based approaches and independent analyses of the transcriptome or the proteome have led to progress in understanding the underlying biology of GB. Joint transcriptome and proteome profiling may reveal new biological insights, and identify pathogenic mechanisms or therapeutic targets for GB therapy. We present a comparison of transcriptome and proteome data from five GB biopsies (TZ) vs their corresponding peritumoral brain zone (PBZ). Omic analyses were performed using RNA microarray chips and the isotope-coded protein label method (ICPL). RESULTS: As described in other cancers, we found a poor correlation between transcriptome and proteome data in GB. We observed only two commonly deregulated mRNAs/proteins (neurofilament light polypeptide and synapsin 1) and 12 altered biological processes; they are related to cell communication, synaptic transmission and nervous system processes. This poor correlation may be a consequence of the techniques used to produce the omic profiles, the intrinsic properties of mRNA and proteins and/or of cancer- or GB-specific phenomena. Of interest, the analysis of the transcription factor binding sites present upstream from the open reading frames of all altered proteins identified by ICPL method shows a common binding site for the topoisomerase I and p53-binding protein TOPORS. Its expression was observed in 7/11 TZ samples and not in PBZ. Some findings suggest that TOPORS may function as a tumor suppressor; its implication in gliomagenesis should be examined in future studies. CONCLUSIONS: In this study, we showed a low correlation between transcriptome and proteome data for GB samples as described in other cancer tissues. We observed that NEFL, SYN1 and 12 biological processes were deregulated in both the transcriptome and proteome data. It will be important to analyze more specifically these processes and these two proteins to allow the identification of new theranostic markers or potential therapeutic targets for GB.

The expression of EMX2 lead to cell cycle arrest in glioblastoma cell line.

BACKGROUND: Glioblastoma (GB) is a highly invasive primary brain tumor that nearly always systematically recurs at the site of resection despite aggressive radio-chemotherapy. Previously, we reported a gene expression signature related to tumor infiltration. Within this signature, the EMX2 gene encodes a homeodomain transcription factor that we found was down regulated in glioblastoma. As EMX2 is reported to play a role in carcinogenesis, we investigated the impact of EMX2 overexpression in glioma-related cell lines. METHODS: For that purpose, we constructed tetracycline-inducible EMX2 expression lines. Transfected cell phenotypes (proliferation, cell death and cell cycle) were assessed in time-course experiments. RESULTS: Restoration of EMX2 expression in U87 glioblastoma cells significantly inhibited cell proliferation. This inhibition was reversible after EMX2 removal from cells. EMX2-induced proliferative inhibition was very likely due to cell cycle arrest in G1/S transition and was not accompanied by signs of cell death. CONCLUSION: Our results suggest that EMX2 may constitute a putative therapeutic target for GB treatment. Further studies are required to decipher the gene networks and transduction signals involved in EMX2's effect on cell proliferation.

Independent association of PD-L1 expression with noninactivated VHL clear cell renal cell carcinoma-A finding with therapeutic potential.

Clear cell renal cell carcinoma (ccRCC) is an aggressive tumor that is characterized in most cases by inactivation of the tumor suppressor gene VHL. The VHL/HIF/VEGF pathway thus plays a major role in angiogenesis and is currently targeted by anti-angiogenic therapy. The emergence of resistance is leading to the use of targeted immunotherapy against immune checkpoint PD1/PDL1 that restores antitumor immune response. The correlation between VHL status and PD-L1 expression has been little investigated. In this study, we retrospectively reviewed 98 consecutive cases of ccRCC and correlated PD-L1 expression by immunohistochemistry (IHC) with clinical data (up to 10-year follow-up), pathological criteria, VEGF, PAR-3, CAIX and PD-1 expressions by IHC and complete VHL status (deletion, mutation and promoter hypermethylation). PD-L1 expression was observed in 69 ccRCC (70.4%) and the corresponding patients had a worse prognosis, with a median specific survival of 52 months (p = 0.03). PD-L1 expression was significantly associated with poor prognostic factors such as a higher ISUP nucleolar grade (p = 0.01), metastases at diagnosis (p = 0.01), a sarcomatoid component (p = 0.04), overexpression of VEGF (p = 0.006), and cytoplasmic PAR-3 expression (p = 0.01). PD-L1 expression was also associated with dense PD-1 expression (p = 0.007) and with ccRCC with 0 or 1 alteration(s) (non-inactivated VHL tumors; p = 0.007) that remained significant after multivariate analysis (p = 0.004 and p = 0.024, respectively). Interestingly, all wild-type VHL tumors (no VHL gene alteration, 11.2%) expressed PD-L1. In this study, we found PD-L1 expression to be associated with noninactivated VHL tumors and in particular wild-type VHL ccRCC, which may benefit from therapies inhibiting PD-L1/PD-1.

Routine molecular profiling of patients with advanced non-small-cell lung cancer: results of a 1-year nationwide programme of the French Cooperative Thoracic Intergroup (IFCT).

BACKGROUND: The molecular profiling of patients with advanced non-small-cell lung cancer (NSCLC) for known oncogenic drivers is recommended during routine care. Nationally, however, the feasibility and effects on outcomes of this policy are unknown. We aimed to assess the characteristics, molecular profiles, and clinical outcomes of patients who were screened during a 1-year period by a nationwide programme funded by the French National Cancer Institute. METHODS: This study included patients with advanced NSCLC, who were routinely screened for EGFR mutations, ALK rearrangements, as well as HER2 (ERBB2), KRAS, BRAF, and PIK3CA mutations by 28 certified regional genetics centres in France. Patients were assessed consecutively during a 1-year period from April, 2012, to April, 2013. We measured the frequency of molecular alterations in the six routinely screened genes, the turnaround time in obtaining molecular results, and patients' clinical outcomes. This study is registered with ClinicalTrials.gov, number NCT01700582. FINDINGS: 18,679 molecular analyses of 17,664 patients with NSCLC were done (of patients with known data, median age was 64·5 years [range 18-98], 65% were men, 81% were smokers or former smokers, and 76% had adenocarcinoma). The median interval between the initiation of analysis and provision of the written report was 11 days (IQR 7-16). A genetic alteration was recorded in about 50% of the analyses; EGFR mutations were reported in 1947 (11%) of 17,706 analyses for which data were available, HER2 mutations in 98 (1%) of 11,723, KRAS mutations in 4894 (29%) of 17,001, BRAF mutations in 262 (2%) of 13,906, and PIK3CA mutations in 252 (2%) of 10,678; ALK rearrangements were reported in 388 (5%) of 8134 analyses. The median duration of follow-up at the time of analysis was 24·9 months (95% CI 24·8-25·0). The presence of a genetic alteration affected first-line treatment for 4176 (51%) of 8147 patients and was associated with a significant improvement in the proportion of patients achieving an overall response in first-line treatment (37% [95% CI 34·7-38·2] for presence of a genetic alteration vs 33% [29·5-35·6] for absence of a genetic alteration; p=0·03) and in second-line treatment (17% [15·0-18·8] vs 9% [6·7-11·9]; p<0·0001). Presence of a genetic alteration was also associated with improved first-line progression-free survival (10·0 months [95% CI 9·2-10·7] vs 7·1 months [6·1-7·9]; p<0·0001) and overall survival (16·5 months [15·0-18·3] vs 11·8 months [10·1-13·5]; p<0·0001) compared with absence of a genetic alteration. INTERPRETATION: Routine nationwide molecular profiling of patients with advanced NSCLC is feasible. The frequency of genetic alterations, acceptable turnaround times in obtaining analysis results, and the clinical advantage provided by detection of a genetic alteration suggest that this policy provides a clinical benefit. FUNDING: French National Cancer Institute (INCa).

EGFR tyrosine kinase inhibitors versus chemotherapy in EGFR wild-type pre-treated advanced nonsmall cell lung cancer in daily practice.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are approved for second-line treatment of EGFR wild-type (EGFR-wt) nonsmall cell lung cancer (NSCLC). However, results from randomised trials performed to compare EGFR-TKIs with chemotherapy in this population did not show any survival benefit. In the era of immunotherapy, many drugs are approved for second-line treatment of EGFR-wt NSCLC and there is a need to reassess the role of EGFR-TKIs in this setting.The Biomarkers France study is a large nationwide cohort of NSCLC patients tested for EGFR mutations. We used this database to collect clinical, biological, treatment and outcome data on EGFR-wt patients who received second-line treatment with either EGFR-TKIs or chemotherapy.Among 1278 patients, 868 received chemotherapy and 410 received an EGFR-TKI. Median overall survival and progression-free survival were longer with chemotherapy than with an EGFR-TKI. Overall survival was 8.38 versus 4.99 months, respectively (hazard ratio 0.70, 95% CI 0.59-0.83; p<0.0001) and progression-free survival was 4.30 versus 2.83 months, respectively (hazard ratio 0.66, 95% CI 0.57-0.77; p<0.0001).This study is helpful to guide a multiline treatment strategy for EGFR-wt NSCLC patients. Immunotherapy is approved for second-line treatment. For third-line treatment, chemotherapy results in longer overall survival and progression-free survival, and should be preferred to EGFR-TKIs.

Genome-wide association study identifies TF as a significant modifier gene of iron metabolism in HFE hemochromatosis.

BACKGROUND & AIMS: Hereditary hemochromatosis (HH) is the most common form of genetic iron loading disease. It is mainly related to the homozygous C282Y/C282Y mutation in the HFE gene that is, however, a necessary but not a sufficient condition to develop clinical and even biochemical HH. This suggests that modifier genes are likely involved in the expressivity of the disease. Our aim was to identify such modifier genes. METHODS: We performed a genome-wide association study (GWAS) using DNA collected from 474 unrelated C282Y homozygotes. Associations were examined for both quantitative iron burden indices and clinical outcomes with 534,213 single nucleotide polymorphisms (SNP) genotypes, with replication analyses in an independent sample of 748 C282Y homozygotes from four different European centres. RESULTS: One SNP met genome-wide statistical significance for association with transferrin concentration (rs3811647, GWAS p value of 7×10(-9) and replication p value of 5×10(-13)). This SNP, located within intron 11 of the TF gene, had a pleiotropic effect on serum iron (GWAS p value of 4.9×10(-6) and replication p value of 3.2×10(-6)). Both serum transferrin and iron levels were associated with serum ferritin levels, amount of iron removed and global clinical stage (p<0.01). Serum iron levels were also associated with fibrosis stage (p<0.0001). CONCLUSIONS: This GWAS, the largest one performed so far in unselected HFE-associated HH (HFE-HH) patients, identified the rs3811647 polymorphism in the TF gene as the only SNP significantly associated with iron metabolism through serum transferrin and iron levels. Because these two outcomes were clearly associated with the biochemical and clinical expression of the disease, an indirect link between the rs3811647 polymorphism and the phenotypic presentation of HFE-HH is likely.

Identification of two glioblastoma-associated stromal cell subtypes with different carcinogenic properties in histologically normal surgical margins.

Glioblastoma (GB) is a highly infiltrative tumor recurring within a few centimeters of the resection cavity in 85 % of cases, even in cases of complete tumor resection and adjuvant chemo/radiotherapy. We recently isolated GB-associated stromal cells (GASCs) from the GB peritumoral zone, with phenotypic and functional properties similar to those of the cancer-associated fibroblasts present in the stroma of carcinomas. In particular, GASCs promote blood vessel development and have tumor-promoting effects on glioma cells in vitro and in vivo. In this study, we characterized these cells further, by analyzing the transcriptome and methylome of 14 GASC and five control stromal cell preparations derived from non-GB peripheral brain tissues. We identified two subtypes of GASCs in surgical margins in GB patients: GASC-A and GASC-B. GASC-B promoted the development of tumors and endothelium, whereas GASC-A did not. A difference in DNA methylation may underlie these two subtypes. We identified various proteins as being produced in the procarcinogenic GASC-B. Some of these proteins may serve as prognostic factors for GB and/or targets for anti-glioma treatment. In conclusion, in this era of personalized therapy, the status of GASCs in GB-free surgical margins should be taken into account, to improve treatment and the prevention of recurrence.

Characterizing the peritumoral brain zone in glioblastoma: a multidisciplinary analysis.

Glioblastoma (GB) is the most frequent and aggressive type of primary brain tumor. Recurrences are mostly located at the margin of the resection cavity in the peritumoral brain zone (PBZ). Although it is widely believed that infiltrative tumor cells in this zone are responsible for GB recurrence, few studies have examined this zone. In this study, we analyzed PBZ left after surgery with a variety of techniques including radiology, histopathology, flow cytometry, genomic, transcriptomic, proteomic, and primary cell cultures. The resulting PBZ profiles were compared with those of the GB tumor zone and normal brain samples to identify characteristics specific to the PBZ. We found that tumor cell infiltration detected by standard histological analysis was present in almost one third of PBZ taken from an area that was considered normal both on standard MRI and by the neurosurgeon under an operating microscope. The panel of techniques used in this study show that the PBZ, similar to the tumor zone itself, is characterized by substantial inter-patient heterogeneity, which makes it difficult to identify representative markers. Nevertheless, we identified specific alterations in the PBZ such as the presence of selected tumor clones and stromal cells with tumorigenic and angiogenic properties. The study of GB-PBZ is a growing field of interest and this region needs to be characterized further. This will facilitate the development of new, targeted therapies for patients with GB and the development of approaches to refine the per-operative evaluation of the PBZ to optimize the surgical resection of the tumor.

Outcome-based determination of optimal pyrosequencing assay for MGMT methylation detection in glioblastoma patients.

The methylation of O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter is a key biological marker in clinical neuro-oncology. Nevertheless, there is no consensus concerning the best technique for its assessment. In a recent study comparing five methods to analyze MGMT status, we found that the best prediction of survival was obtained with a pyrosequencing (PSQ) test assessing methylation of 5 CpGs (CpGs 74-78). In the present study we extended our PSQ analysis to 16 CpGs (CpGs 74-89) identified as critical for transcriptional control of the gene. The predictive value of the methylation levels at each CpG, as well as the mean methylation levels of selected sets of consecutive CpGs was tested in a cohort of 89 de novo glioblastoma patients who had received standard of care treatment (Stupp protocol). Using an optimal risk cut-off, each CpG or combination of CpGs, was associated with overall survival (OS) and progression free survival. The best predictive models for OS after stratification on performance score and age were obtained with CpG 89, CpG 84 and mean methylation of CpG 84-88 (Hazard ratio (HR), 0.31; p < 0.0001). The improvement compared to the predictive value of the test analyzing average methylation of CpG 74-78 (HR, 0.32; p < 0.0001) was however marginal. We recommend to test CpGs 74-78 when analyzing MGMT methylation status by PSQ because a commercial kit that has successfully been used in several studies is available, allowing reproducible and comparable results from one laboratory to another.

IRE1 endoribonuclease signaling promotes myeloid cell infiltration in glioblastoma.

BACKGROUND: Intrinsic or environmental stresses trigger the accumulation of improperly folded proteins in the endoplasmic reticulum (ER), leading to ER stress. To cope with this, cells have evolved an adaptive mechanism named the unfolded protein response (UPR) which is hijacked by tumor cells to develop malignant features. Glioblastoma (GB), the most aggressive and lethal primary brain tumor, relies on UPR to sustain growth. We recently showed that IRE1 alpha (referred to IRE1 hereafter), 1 of the UPR transducers, promotes GB invasion, angiogenesis, and infiltration by macrophage. Hence, high tumor IRE1 activity in tumor cells predicts a worse outcome. Herein, we characterized the IRE1-dependent signaling that shapes the immune microenvironment toward monocytes/macrophages and neutrophils. METHODS: We used human and mouse cellular models in which IRE1 was genetically or pharmacologically invalidated and which were tested in vivo. Publicly available datasets from GB patients were also analyzed to confirm our findings. RESULTS: We showed that IRE1 signaling, through both the transcription factor XBP1s and the regulated IRE1-dependent decay controls the expression of the ubiquitin-conjugating E2 enzyme UBE2D3. In turn, UBE2D3 activates the NFκB pathway, resulting in chemokine production and myeloid infiltration in tumors. CONCLUSIONS: Our work identifies a novel IRE1/UBE2D3 proinflammatory axis that plays an instrumental role in GB immune regulation.

Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

Isolation of a new cell population in the glioblastoma microenvironment.

Glioblastoma (GB) is a highly infiltrative tumor recurring in 90% of cases within a few centimeters of the resection cavity, even in cases of complete tumor resection and adjuvant chemo/radiotherapy. This observation highlights the importance of understanding this special zone of brain tissue surrounding the tumor. It is becoming clear that the nonneoplastic stromal compartment of most solid cancers plays an active role in tumor proliferation, invasion, and metastasis. Very little information, other than that concerning angiogenesis and immune cells, has been collected for stromal cells from GB. As part of a translational research program, we have isolated a new stromal cell population surrounding GB by computer-guided stereotaxic biopsies and primary culture. We named these cells GB-associated stromal cells (GASCs). GASCs are diploid, do not display the genomic alterations typical of GB cells, and have phenotypic and functional properties in common with the cancer-associated fibroblasts (CAFs) described in the stroma of carcinomas. In particular, GASCs express markers associated with CAFs such as fibroblast surface protein, alpha-smooth muscle actin (α-SMA), and platelet-derived growth factor receptor-beta (PDGFRß). Furthermore, GASCs have a molecular expression profile different from that of control stromal cells derived from non-GB peripheral brain tissues. GASCs were also found to have tumor-promoting effects on glioma cells in vitro and in vivo. The isolation of GASCs in a tumor of neuroepithelial origin was unexpected, and further studies are required to determine their potential as a target for antiglioma treatment.

A Novel Pseudogene Methylation Signature to Predict Temozolomide Outcome in Non-G-CIMP Glioblastomas.

Objective: Alterations in the methylation state of pseudogenes may serve as clinically useful biomarkers of glioblastomas (GBMs) that do not have glioma-CpG island methylator phenotype (G-CIMP). Methods: Non-G-CIMP GBM datasets were included for evaluation, and a RISK-score signature was determined from the methylation state of pseudogene loci. Both bioinformatic and experimental analyses were performed for biological validation. Results: By integrating clinical information with DNA methylation microarray data, we screened a panel of eight CpGs from discovery cohorts of non-G-CIMP GBMs. Each CpG could accurately and independently predict the prognosis of patients under a treatment regime that combined radiotherapy (RT) and temozolomide (TMZ). The 8-CpG signature appeared to show opposite prognostic correlations between patients treated with RT/TMZ and those treated with RT monotherapy. The analyses further indicated that this signature had predictive value for TMZ efficacy because different survival benefits between RT/TMZ and RT therapies were observed in each risk subgroup. The incorporation of other risk factors, such as age and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, with our pseudogene methylation signature could provide precise risk classification. In vitro experimental data revealed that two locus-specific pseudogenes (ZNF767P and CLEC4GP1) may modulate TMZ resistance via distinct mechanisms in GBM cells. Conclusion: The biologically and clinically relevant RISK-score signature, based on pseudogene methylation loci, may offer information for predicting TMZ responses of non-G-CIMP GBMs, that is independent from, but complementary to, MGMT-based approaches.

Aberrant DNA methylation impacts HOX genes expression in bone marrow mesenchymal stromal cells of myelodysplastic syndromes and de novo acute myeloid leukemia.

DNA methylation, a major biological process regulating the transcription, contributes to the pathophysiology of hematologic malignancies, and hypomethylating agents are commonly used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). In these diseases, bone marrow mesenchymal stromal cells (MSCs) play a key supportive role through the production of various signals and interactions. The DNA methylation status of MSCs, likely to reflect their functionality, might be relevant to understand their contribution to the pathophysiology of these diseases. Consequently, the aim of our study was to analyze the modifications of DNA methylation profiles of MSCs induced by MDS or AML. MSCs from MDS/AML patients were characterized via 5-methylcytosine quantification, gene expression profiles of key regulators of DNA methylation, identification of differentially methylated regions (DMRs) by methylome array, and quantification of DMR-coupled genes expression. MDS and AML-MSCs displayed global hypomethylation and under-expression of DNMT1 and UHRF1. Methylome analysis revealed aberrant methylation profiles in all MDS and in a subgroup of AML-MSCs. This aberrant methylation was preferentially found in the sequence of homeobox genes, especially from the HOX family (HOXA1, HOXA4, HOXA5, HOXA9, HOXA10, HOXA11, HOXB5, HOXC4, and HOXC6), and impacted on their expression. These results highlight modifications of DNA methylation in MDS/AML-MSCs, both at global and focal levels dysregulating the expression of HOX genes well known for their involvement in leukemogenesis. Such DNA methylation in MSCs could be the consequence of the malignant disease or could participate in its development through defective functionality or exosomal transfer of HOX transcription factors from MSCs to hematopoietic cells.

Differential analysis of glioblastoma multiforme proteome by a 2D-DIGE approach.

BACKGROUND: Genomics, transcriptomics and proteomics of glioblastoma multiforme (GBM) have recently emerged as possible tools to discover therapeutic targets and biomarkers for new therapies including immunotherapy. It is well known that macroscopically complete surgical excision, radiotherapy and chemotherapy have therapeutic limitations to improve survival in these patients. In this study, we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify proteins that may serve as brain tumor antigens in new therapeutic assays. Five samples of patients presenting a GBM and five samples of microscopically normal brain tissues derived from brain epileptic surgery specimen were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis) with an internal pool sample on each gel. Five gels were matched and compared with DIA (Difference In-gel Analysis) software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained. RESULTS: From 51 protein-spots significantly up-regulated in GBM samples, mass spectrometry (MS) identified twenty-two proteins. The differential expression of a selected protein set was first validated by western-blotting, then tested on large cohorts of GBM specimens and non-tumor tissues, using immunohistochemistry and real-time RT-PCR. CONCLUSIONS: Our results confirmed the importance of previously described proteins in glioma pathology and their potential usefulness as biological markers but also revealed some new interesting targets for future therapies.