Association of MGMT Promoter and Enhancer Methylation with Genetic Variants, Clinical Parameters, and Demographic Characteristics in Glioblastoma.

The response of glioblastoma (GBM) patients to the alkylating agent temozolomide (TMZ) vitally depends on the expression level of the repair protein O6-methylguanine-DNA methyltransferase (MGMT). Since MGMT is strongly regulated by promoter methylation, the methylation status of the MGMT promoter has emerged as a prognostic and predictive biomarker for GBM patients. By determining the methylation levels of the four enhancers located within or close to the MGMT gene, we recently found that enhancer methylation contributes to MGMT regulation. In this study, we investigated if methylation of the four enhancers is associated with SNP rs16906252, TERT promoter mutations C228T and C250T, TERT SNP rs2853669, proliferation index Ki-67, overall survival (OS), age, and sex of the patients. In general, associations with genetic variants, clinical parameters, and demographic characteristics were caused by a complex interplay of multiple CpGs in the MGMT promoter and of multiple CpGs in enhancer regions. The observed associations for intragenic enhancer 4, located in intron 2 of MGMT, differed from associations observed for the three intergenic enhancers. Some findings were restricted to subgroups of samples with either methylated or unmethylated MGMT promoters, underpinning the relevance of the MGMT promoter status in GBMs.

Association between MGMT Enhancer Methylation and MGMT Promoter Methylation, MGMT Protein Expression, and Overall Survival in Glioblastoma.

The repair protein O6-methylguanine-DNA methyltransferase (MGMT) is regulated epigenetically, mainly by the methylation of the MGMT promoter. MGMT promoter methylation status has emerged as a prognostic and predictive biomarker for patients with newly diagnosed glioblastoma (GBM). However, a strong negative correlation between MGMT promoter methylation and MGMT protein expression cannot be applied as a rule for all GBM patients. In order to investigate if the DNA methylation status of MGMT enhancers is associated with MGMT promoter methylation, MGMT expression, and the overall survival (OS) of GBM patients, we established assays based on high-resolution melting analysis and pyrosequencing for one intragenic and three intergenic MGMT enhancers. For CpGs in an enhancer located 560 kb upstream of the MGMT promoter, we found a significant negative correlation between the methylation status and MGMT protein levels of GBM samples expressing MGMT. The methylation status of CpGs in the intragenic enhancer (hs696) was strongly negatively correlated with MGMT promoter methylation and was significantly higher in MGMT-expressing GBM samples than in MGMT-non-expressing GBM samples. Moreover, low methylation of CpGs 01-03 and CpGs 09-13 was associated with the longer OS of the GBM patients. Our findings indicate an association between MGMT enhancer methylation and MGMT promoter methylation, MGMT protein expression, and/or OS.

A whole-genome scan for Artemisinin cytotoxicity reveals a novel therapy for human brain tumors.

The natural compound Artemisinin is the most widely used antimalarial drug worldwide. Based on its cytotoxicity, it is also used for anticancer therapy. Artemisinin and its derivates are endoperoxides that damage proteins in eukaryotic cells; their definite mechanism of action and host cell targets, however, have remained largely elusive. Using yeast and haploid stem cell screening, we demonstrate that a single cellular pathway, namely porphyrin (heme) biosynthesis, is required for the cytotoxicity of Artemisinins. Genetic or pharmacological modulation of porphyrin production is sufficient to alter its cytotoxicity in eukaryotic cells. Using multiple model systems of human brain tumor development, such as cerebral glioblastoma organoids, and patient-derived tumor spheroids, we sensitize cancer cells to dihydroartemisinin using the clinically approved porphyrin enhancer and surgical fluorescence marker 5-aminolevulinic acid, 5-ALA. A combination treatment of Artemisinins and 5-ALA markedly and specifically killed brain tumor cells in all model systems tested, including orthotopic patient-derived xenografts in vivo. These data uncover the critical molecular pathway for Artemisinin cytotoxicity and a sensitization strategy to treat different brain tumors, including drug-resistant human glioblastomas.

Surgeon experience in glioblastoma surgery of the elderly-a multicenter, retrospective cohort study.

PURPOSE: To assess the impact of individual surgeon experience on overall survival (OS), extent of resection (EOR) and surgery-related morbidity in elderly patients with glioblastoma (GBM), we performed a retrospective case-by-case analysis. METHODS: GBM patients aged ≥ 65 years who underwent tumor resection at two academic centers were analyzed. The experience of each neurosurgeon was quantified in three ways: (1) total number of previously performed glioma surgeries (lifetime experience); (2) number of surgeries performed in the previous five years (medium-term experience) and (3) in the last two years (short-term experience). Surgeon experience data was correlated with survival (OS) and surrogate parameters for surgical quality (EOR, morbidity). RESULTS: 198 GBM patients (median age 73.0 years, median preoperative KPS 80, IDH-wildtype status 96.5%) were included. Median OS was 10.0 months (95% CI 8.0-12.0); median EOR was 89.4%. Surgery-related morbidity affected 19.7% patients. No correlations of lifetime surgeon experience with OS (P = .693), EOR (P = .693), and surgery-related morbidity (P = .435) were identified. Adjuvant therapy was associated with improved OS (P < .001); patients with surgery-related morbidity were less likely to receive adjuvant treatment (P = .002). In multivariable testing, adjuvant therapy (P < .001; HR = 0.064, 95%CI 0.028-0.144) remained the only significant predictor for improved OS. CONCLUSION: Less experienced neurosurgeons achieve similar surgical results and outcome in elderly GBM patients within the setting of academic teaching hospitals. Adjuvant treatment and avoidance of surgery-related morbidity are crucial for generating a treatment benefit for this cohort.

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

Implementation of TERT promoter mutations improve prognostication of the WHO classification in meningioma.

TERT promoter mutations have been associated with increased risk of recurrence in meningioma cohorts, thus a potential biomarker for aggressive phenotypes. A main purpose of refining tumour classification is better predictions on the patient level. We compiled data from previous published cohorts to investigate patient-level predictions of recurrence based on TERTp-mut status. Implementation of TERTp-mut into the WHO grading led to better patient prognostication by improved prediction of recurrence. Our results support implementation of TERTp-mut into diagnostics and classification of meningiomas.

Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the MGMT Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set.

Due to its cost-efficiency, high resolution melting (HRM) analysis plays an important role in genotyping of candidate single nucleotide polymorphisms (SNPs). Studies indicate that HRM analysis is not only suitable for genotyping individual SNPs, but also allows genotyping of multiple SNPs in one and the same amplicon, although with limited discrimination power. By targeting the three C>T SNPs rs527559815, rs547832288, and rs16906252, located in the promoter of the O6-methylguanine-DNA methyltransferase (MGMT) gene within a distance of 45 bp, we investigated whether the discrimination power can be increased by coupling HRM analysis with pyrosequencing (PSQ). After optimizing polymerase chain reaction (PCR) conditions, PCR products subjected to HRM analysis could directly be used for PSQ. By analyzing oligodeoxynucleotide controls, representing the 36 theoretically possible variant combinations for diploid human cells (8 triple-homozygous, 12 double-homozygous, 12 double-heterozygous and 4 triple-heterozygous combinations), 34 out of the 36 variant combinations could be genotyped unambiguously by combined analysis of HRM and PSQ data, compared to 22 variant combinations by HRM analysis and 16 variant combinations by PSQ. Our approach was successfully applied to genotype stable cell lines of different origin, primary human tumor cell lines from glioma patients, and breast tissue samples.

Targeting fibroblast growth factor receptors to combat aggressive ependymoma.

Ependymomas (EPN) are central nervous system tumors comprising both aggressive and more benign molecular subtypes. However, therapy of the high-risk subtypes posterior fossa group A (PF-A) and supratentorial RELA-fusion positive (ST-RELA) is limited to gross total resection and radiotherapy, as effective systemic treatment concepts are still lacking. We have recently described fibroblast growth factor receptors 1 and 3 (FGFR1/FGFR3) as oncogenic drivers of EPN. However, the underlying molecular mechanisms and their potential as therapeutic targets have not yet been investigated in detail. Making use of transcriptomic data across 467 EPN tissues, we found that FGFR1 and FGFR3 were both widely expressed across all molecular groups. FGFR3 mRNA levels were enriched in ST-RELA showing the highest expression among EPN as well as other brain tumors. We further identified high expression levels of fibroblast growth factor 1 and 2 (FGF1, FGF2) across all EPN subtypes while FGF9 was elevated in ST-EPN. Interrogation of our EPN single-cell RNA-sequencing data revealed that FGFR3 was further enriched in cycling and progenitor-like cell populations. Corroboratively, we found FGFR3 to be predominantly expressed in radial glia cells in both mouse embryonal and human brain datasets. Moreover, we detected alternative splicing of the FGFR1/3-IIIc variant, which is known to enhance ligand affinity and FGFR signaling. Dominant-negative interruption of FGFR1/3 activation in PF-A and ST-RELA cell models demonstrated inhibition of key oncogenic pathways leading to reduced cell growth and stem cell characteristics. To explore the feasibility of therapeutically targeting FGFR, we tested a panel of FGFR inhibitors in 12 patient-derived EPN cell models revealing sensitivity in the low-micromolar to nano-molar range. Finally, we gain the first clinical evidence for the activity of the FGFR inhibitor nintedanib in the treatment of a patient with recurrent ST-RELA. Together, these preclinical and clinical data suggest FGFR inhibition as a novel and feasible approach to combat aggressive EPN.

p53 Loss Mediates Hypersensitivity to ETS Transcription Factor Inhibition Based on PARylation-Mediated Cell Death Induction.

The small-molecule E26 transformation-specific (ETS) factor inhibitor YK-4-279 was developed for therapy of ETS/EWS fusion-driven Ewing's sarcoma. Here we aimed to identify molecular factors underlying YK-4-279 responsiveness in ETS fusion-negative cancers. Cell viability screenings that deletion of P53 induced hypersensitization against YK-4-279 especially in the BRAFV600E-mutated colon cancer model RKO. This effect was comparably minor in the BRAF wild-type HCT116 colon cancer model. Out of all ETS transcription factor family members, especially ETS1 overexpression at mRNA and protein level was induced by deletion of P53 specifically under BRAF-mutated conditions. Exposure to YK-4-279 reverted ETS1 upregulation induced by P53 knock-out in RKO cells. Despite upregulation of p53 by YK-4-279 itself in RKOp53 wild-type cells, YK-4-279-mediated hyperphosphorylation of histone histone H2A.x was distinctly more pronounced in the P53 knock-out background. YK-4-279-induced cell death in RKOp53-knock-out cells involved hyperPARylation of PARP1, translocation of the apoptosis-inducible factor AIF into nuclei, and induction of mitochondrial membrane depolarization, all hallmarks of parthanatos. Accordingly, pharmacological PARP as well as BRAFV600E inhibition showed antagonistic activity with YK-4-279 especially in the P53 knock-out background. Taken together, we identified ETS factor inhibition as a promising strategy for the treatment of notoriously therapy-resistant p53-null solid tumours with activating MAPK mutations.

Evaluation of an Assay for MGMT Gene Promoter Methylation in Glioblastoma Samples.

BACKGROUND/AIM: To compare the GeneXpert® O6-methylguanine DNA methyltransferase (MGMT) methylation prototype (GX MGMT) assay with pyrosequencing in glioblastomas. MATERIALS AND METHODS: The MGMT methylation status was retrospectively assessed in formalin-fixed paraffin embedded (FFPE) tumor blocks from 262 glioblastoma patients obtained from three independent cohorts using either a standard of care pyrosequencing laboratory developed test or the GX MGMT assay. RESULTS: The concordance rate was 92.1% (58/63) for Oregon Health and Science University (OSHU) samples, 91.7% (88/96) for Medical University of Vienna (MUV) samples, and 82.5% (85/103) for Kepler University Hospital (KUH) samples. Patients with MGMT promoter hypermethylation assessed by pyrosequencing or the GX MGMT test had a significantly longer overall survival compared to patients without hypermethylation (HR=0.43, 95%CI=0.26-0.72, p=0.001 and HR=0.51, 95%CI=0.31-0.84, p=0.008, respectively). CONCLUSION: Standardized, simplified, and on-demand testing of MGMT promoter methylation by the GX MGMT assay is feasible.

Telomerase Reverse Transcriptase Promoter Mutations Identify a Genomically Defined and Highly Aggressive Human Pleural Mesothelioma Subgroup.

PURPOSE: Human malignant pleural mesothelioma (MPM) is characterized by dismal prognosis. Consequently, dissection of molecular mechanisms driving malignancy is of key importance. Here we investigate whether activating mutations in the telomerase reverse transcriptase (TERT) gene promoter are present in MPM and associated with disease progression, cell immortalization, and genomic alteration patterns. EXPERIMENTAL DESIGN: TERT promoters were sequenced in 182 MPM samples and compared with clinicopathologic characteristics. Surgical specimens from 45 patients with MPM were tested for in vitro immortalization. The respective MPM cell models (N = 22) were analyzed by array comparative genomic hybridization, gene expression profiling, exome sequencing as well as TRAP, telomere length, and luciferase promoter assays. RESULTS: TERT promoter mutations were detected in 19 of 182 (10.4%) MPM cases and significantly associated with advanced disease and nonepithelioid histology. Mutations independently predicted shorter overall survival in both histologic MPM subtypes. Moreover, 9 of 9 (100%) mutated but only 13 of 36 (36.1%) wild-type samples formed immortalized cell lines. TERT promoter mutations were associated with enforced promoter activity and TERT mRNA expression, while neither telomerase activity nor telomere lengths were significantly altered. TERT promoter-mutated MPM cases exhibited distinctly reduced chromosomal alterations and specific mutation patterns. While BAP1 mutations/deletions were exclusive with TERT promoter mutations, homozygous deletions at the RBFOX1 and the GSTT1 loci were clearly enriched in mutated cases. CONCLUSIONS: TERT promoter mutations independently predict a dismal course of disease in human MPM. The altered genomic aberration pattern indicates that TERT promoter mutations identify a novel, highly aggressive MPM subtype presumably based on a specific malignant transformation process.

Ferritin in glioblastoma.

Elevated levels of serum ferritin (SF) are observed in several types of cancer; however, little is known on the association between ferritin and glioma, the most frequent type of human primary brain tumour. Here we report that GBM patients show significantly increased pre-surgical SF levels (i.e. ferritinaemia) within the SF reference range and a marked ferritin immunoreactivity of resected tumour tissue. Our findings account for an indirect association between ferritin synthesis in glioma-tissue and altered SF levels, which limits the clinical value of SF as a tumour marker in glioma. Importantly, we show for the first time that GBM-derived glioma cells release ferritin in vitro, which exerts an apoptosis-stimulating activity. Albeit the pathophysiologic context of apoptosis induction by a tumour-derived ferritin remains to be defined, our findings account for a distinct growth-regulatory role of these ferritin species in tumour biology.

Poor prognosis associated with TERT gene alterations in meningioma is independent of the WHO classification: an individual patient data meta-analysis.

BACKGROUND: TERT gene alterations (TERT-alt) have been linked to increased risk of recurrence in meningiomas, whereas the association to mortality largely remain incompletely investigated. As incongruence between clinical course and WHO grade exists, reliable biomarkers have been sought. METHODS: We applied the Preferred Reporting Items for Systematic Review and Meta-Analyses of individual participant data Statement. We compiled data from eight studies and allocated patients to TERT-alt (n=59) or TERT promoter wild-type (TERTp-wt; n=618). We compared the two groups stratified for WHO grades as: incidence rates, survival probabilities and cumulative recurrences. We estimated the effects of WHO grade, age at diagnosis and sex as HRs. RESULTS: TERT-alt occurred in 4.7%, 7.9% and 15.4% of WHO-I/WHO-II/WHO-III meningiomas, respectively. The median recurrence-free survival was 14 months for all TERT-alt patients versus 101 months for all TERTp-wt patients. The HR for TERT-alt was 3.74 in reference to TERTp-wt. For all TERT-alt patients versus all TERTp-wt patients, the median overall survival was 58 months and 160 months, respectively. The HR for TERT-alt was 2.77 compared with TERTp-wt. TERT-alt affected prognosis independent of WHO grades. Particularly, the recurrence rate was 4.8 times higher in WHO-I/-II TERT-alt patients compared with WHO-III TERTp-wt patients. The mortality rate was 2.7 times higher in the WHO-I and WHO-II TERT-alt patients compared with WHO-III TERTp-wt patients. CONCLUSIONS: TERT-alt is an important biomarker for significantly higher risk of recurrence and death in meningiomas. TERT-alt should be managed and surveilled aggressively. We propose that TERT-alt analysis should be implemented as a routine diagnostic test in meningioma and integrated into the WHO classification. TRIAL REGISTRATION NUMBER: PROSPERO: CRD42018110566.

Combined proteomics/miRNomics of dendritic cell immunotherapy-treated glioblastoma patients as a screening for survival-associated factors.

Glioblastoma is the most prevalent and aggressive brain cancer. With a median overall survival of ~15-20 months under standard therapy, novel treatment approaches are desperately needed. A recent phase II clinical trial with a personalized immunotherapy based on tumor lysate-charged dendritic cell (DC) vaccination, however, failed to prolong survival. Here, we investigated tumor tissue from trial patients to explore glioblastoma survival-related factors. We followed an innovative approach of combining mass spectrometry-based quantitative proteomics (n = 36) with microRNA sequencing plus RT-qPCR (n = 38). Protein quantification identified, e.g., huntingtin interacting protein 1 (HIP1), retinol-binding protein 1 (RBP1), ferritin heavy chain (FTH1) and focal adhesion kinase 2 (FAK2) as factor candidates correlated with a dismal prognosis. MicroRNA analysis identified miR-216b, miR-216a, miR-708 and let-7i as molecules potentially associated with favorable tissue characteristics as they were enriched in patients with a comparably longer survival. To illustrate the utility of integrated miRNomics and proteomics findings, focal adhesion was studied further as one example for a pathway of potential general interest. Taken together, we here mapped possible drivers of glioblastoma outcome under immunotherapy in one of the largest DC vaccination tissue analysis cohorts so far-demonstrating usefulness and feasibility of combined proteomics/miRNomics approaches. Future research should investigate agents that sensitize glioblastoma to (immuno)therapy-potentially building on insights generated here.

Risks and Benefits of Glioblastoma Resection in Older Adults: A Retrospective Austrian Multicenter Study.

OBJECTIVE: To assess the prognostic profile, clinical outcome, treatment-associated morbidity, and treatment burden of elderly patients with glioblastoma (GBM) undergoing microsurgical tumor resection as part of contemporary treatment algorithms. METHODS: We retrospectively identified patients with GBM ≥65 years of age who were treated by resection at 2 neuro-oncology centers. Survival was assessed by Kaplan-Meier analyses; log-rank tests identified prognostic factors. RESULTS: The study population included 160 patients (mean age, 73.1 ± 5.1 years), and the median contrast-enhancing tumor volume was 31.0 cm3. Biomarker analyses revealed O(6)-methylguanine-DNA methyltransferase-promoter methylation in 62.7% and wild-type isocitrate dehydrogenase in 97.5% of tumors. The median extent of resection (EOR) was 92.3%, surgical complications were noted in 10.0% of patients, and the median postoperative hospitalization period was 8 days. Most patients (60.0%) received adjuvant radio-/chemotherapy. The overall treatment-associated morbidity was 30.6%. The median progression-free and overall survival were 5.4 months (95% confidence interval [CI], 4.6-6.4 months) and 10.0 months (95% CI, 7.9-11.7 months). The strongest predictors for favorable outcome were patient age ≤73.0 years (P = 0.0083), preoperative Karnofsky Performance Status Scale score ≥80% (P = 0.0179), postoperative modified Rankin Scale score ≤1 (P < 0.0001), adjuvant treatment (P < 0.0001), and no treatment-associated morbidity (P = 0.0478). Increased EOR did not correlate with survival (P = 0.5046), but correlated significantly with treatment-associated morbidity (P = 0.0031). CONCLUSIONS: Clinical outcome for elderly patients with GBM remains limited. Nonetheless, the observed treatment-associated morbidity and treatment burden were moderate in the patients, and patient age and performance status remained the strongest predictors for survival. The risks and benefits of tumor resection in the age of biomarker-adjusted treatment concepts require further prospective evaluation.

Long interspersed element-1 ribonucleoprotein particles protect telomeric ends in alternative lengthening of telomeres dependent cells.

Malignant cells ensure telomere maintenance by the alternative lengthening of telomeres (ALT) in the absence of telomerase activity (TA). The retrotransposons "long interspersed nuclear element-1" (LINE-1, L1) are expressed in malignant cells and are primarily known to contribute to complex karyotypes. Here we demonstrate that LINE-1 ribonucleoprotein particles (L1-RNPs) expression is significantly higher in ALT+- versus in TA+-human glioma. Analyzing a role of L1-RNP in ALT, we show that L1-RNPs bind to telomeric repeat containing RNA (TERRA), which is critical for telomere stabilization and which is overexpressed in ALT+ cells. In turn, L1-RNP knockdown (KD) abrogated the nuclear retention of TERRA, resulted in increased telomeric DNA damage, decreased cell growth and reduced expression of ALT characteristics such as c-circles and PML-bodies. L1-RNP KD also decreased the expression of Shelterin- and the ALT-regulating protein Topoisomerase IIIα (TopoIIIα) indicating a more general role of L1-RNPs in supporting telomeric integrity in ALT. Our findings suggest an impact of L1-RNP on telomere stability in ALT+ dependent tumor cells. As L1-RNPs are rarely expressed in normal adult human tissue those elements might serve as a novel target for tumor ablative therapy.

TERT expression is susceptible to BRAF and ETS-factor inhibition in BRAFV600E/TERT promoter double-mutated glioma.

The BRAF gene and the TERT promoter are among the most frequently altered genomic loci in low-grade (LGG) and high-grade-glioma (HGG), respectively. The coexistence of BRAF and TERT promoter aberrations characterizes a subset of aggressive glioma. Therefore, we investigated interactions between those alterations in malignant glioma. We analyzed co-occurrence of BRAFV600E and TERT promoter mutations in our clinical data (n = 8) in addition to published datasets (n = 103) and established a BRAFV600E-positive glioma cell panel (n = 9) for in vitro analyses. We investigated altered gene expression, signaling events and TERT promoter activity upon BRAF- and E-twenty-six (ETS)-factor inhibition by qRT-PCR, chromatin immunoprecipitation (ChIP), Western blots and luciferase reporter assays. TERT promoter mutations were significantly enriched in BRAFV600E-mutated HGG as compared to BRAFV600E-mutated LGG. In vitro, BRAFV600E/TERT promoter double-mutant glioma cells showed exceptional sensitivity towards BRAF-targeting agents. Remarkably, BRAF-inhibition attenuated TERT expression and TERT promoter activity exclusively in double-mutant models, while TERT expression was undetectable in BRAFV600E-only cells. Various ETS-factors were broadly expressed, however, only ETS1 expression and phosphorylation were consistently downregulated following BRAF-inhibition. Knock-down experiments and ChIP corroborated the notion of a functional role for ETS1 and, accordingly, all double-mutant tumor cells were highly sensitive towards the ETS-factor inhibitor YK-4-279. In conclusion, our data suggest that concomitant BRAFV600E and TERT promoter mutations synergistically support cancer cell proliferation and immortalization. ETS1 links these two driver alterations functionally and may represent a promising therapeutic target in this aggressive glioma subgroup.

Spheroid glioblastoma culture conditions as antigen source for dendritic cell-based immunotherapy: spheroid proteins are survival-relevant targets but can impair immunogenic interferon γ production.

BACKGROUND: Glioblastoma is the most aggressive type of brain cancer. Dendritic cell (DC)-based immunotherapy against glioblastoma depends on the effectiveness of loaded antigens. Sphere-inducing culture conditions are being studied by many as a potential antigen source. Here, we investigated two different in vitro conditions (spheroid culture versus adherent culture) in relation to DC immunotherapy: (1) We studied the specific spheroid-culture proteome and assessed the clinical importance of spheroid proteins. (2) We evaluated the immunogenicity of spheroid lysate - both compared to adherent conditions. METHODS: We used seven spheroid culture systems, three of them patient-derived. Stemness-related markers were studied in those three via immunofluorescence. Spheroid-specific protein expression was measured via quantitative proteomics. The Cancer Genome Atlas (TCGA) survival data was used to investigate the clinical impact of spheroid proteins. Immunogenicity of spheroid versus adherent cell lysate was explored in autologous ELISPOT systems (DCs and T cells from the three patients). RESULTS: (1) The differential proteome of spheroid versus adherent glioblastoma culture conditions could successfully be established. The top 10 identified spheroid-specific proteins were associated with significantly decreased overall survival (TCGA MIT/Harvard cohort; n = 350, P = 0.014). (2) In exploratory experiments, immunogenicity of spheroid lysate vis-á-vis interferon (IFN)γ production was lower than that of adherent cell lysate (IFNγ ELISPOT; P = 0.034). CONCLUSIONS: Spheroid culture proteins seem to represent survival-relevant targets, supporting the use of spheroid culture conditions as an antigen source for DC immunotherapy. However, immunogenicity enhancement should be considered for future research. Transferability of our findings in terms of clinical impact and regarding different spheroid-generation techniques needs further validation.

Gliomasphere marker combinatorics: multidimensional flow cytometry detects CD44+/CD133+/ITGA6+/CD36+ signature.

Glioblastoma is the most dangerous brain cancer. One reason for glioblastoma's aggressiveness are glioblastoma stem-like cells. To target them, a number of markers have been proposed (CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6). A comprehensive study of co-expression patterns of them has, however, not been performed so far. Here, we mapped the multidimensional co-expression profile of these stemness-associated molecules. Gliomaspheres - an established model of glioblastoma stem-like cells - were used. Seven different gliomasphere systems were subjected to multicolor flow cytometry measuring the nine markers CD133, CD44, CD15, A2B5, CD36, CXCR4, IL6R, L1CAM, and ITGA6 all simultaneously based on a novel 9-marker multicolor panel developed for this study. The viSNE dimensionality reduction algorithm was applied for analysis. All gliomaspheres were found to express at least five different glioblastoma stem-like cell markers. Multi-dimensional analysis showed that all studied gliomaspheres consistently harbored a cell population positive for the molecular signature CD44+/CD133+/ITGA6+/CD36+. Glioblastoma patients with an enrichment of this combination had a significantly worse survival outcome when analyzing the two largest available The Cancer Genome Atlas datasets (MIT/Harvard Affymetrix: P = 0.0015, University of North Carolina Agilent: P = 0.0322). In sum, we detected a previously unknown marker combination - demonstrating feasibility, usefulness, and importance of high-dimensional gliomasphere marker combinatorics.