Publications by authors named "Daniela Lötsch"

25 Publications

  • Page 1 of 1

Heme Biosynthesis mRNA Expression Signature: Towards a Novel Prognostic Biomarker in Patients with Diffusely Infiltrating Gliomas.

Cancers (Basel) 2021 Feb 7;13(4). Epub 2021 Feb 7.

Department of Neurosurgery, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria.

Diffusely infiltrating gliomas are characterized by a variable clinical course, and thus novel prognostic biomarkers are needed. The heme biosynthesis cycle constitutes a fundamental metabolic pathway and might play a crucial role in glioma biology. The aim of this study was thus to investigate the role of the heme biosynthesis mRNA expression signature on prognosis in a large glioma patient cohort. Glioma patients with available sequencing data on heme biosynthesis expression were retrieved from The Cancer Genome Atlas (TCGA). In each patient, the heme biosynthesis mRNA expression signature was calculated and categorized into low, medium, and high expression subgroups. Differences in progression-free and overall survival between these subgroups were investigated including a multivariate analysis correcting for WHO grade, tumor subtype, and patient age and sex. In a total of 693 patients, progression-free and overall survival showed a strictly monotonical decrease with increasing mRNA expression signature subgroups. In detail, median overall survival was 134.2 months in the low, 79.9 months in the intermediate, and 16.5 months in the high mRNA expression signature subgroups, respectively. The impact of mRNA expression signature on progression-free and overall survival was independent of the other analyzed prognostic factors. Our data indicate that the heme biosynthesis mRNA expression signature might serve as an additional novel prognostic marker in patients with diffusely infiltrating gliomas to optimize postoperative management.
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http://dx.doi.org/10.3390/cancers13040662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916021PMC
February 2021

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

NPJ Vaccines 2020 Jan 16;5(1). Epub 2020 Jan 16.

Activartis Biotech GmbH, Vienna, Austria.

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.
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http://dx.doi.org/10.1038/s41541-019-0149-xDOI Listing
January 2020

TCGA mRNA Expression Analysis of the Heme Biosynthesis Pathway in Diffusely Infiltrating Gliomas: A Comparison of Typically 5-ALA Fluorescent and Non-Fluorescent Gliomas.

Cancers (Basel) 2020 Jul 24;12(8). Epub 2020 Jul 24.

Department of Neurosurgery, Medical University Vienna, 1090 Vienna, Austria.

5-Aminolevulinic acid (5-ALA) is a fluorescent dye that after metabolization to Protoporphyrin IX (PpIX) by the heme biosynthesis pathway typically leads to visible fluorescence in WHO grade IV but not grade II gliomas. The exact mechanism for high PpIX levels in WHO grade IV gliomas and low PpIX levels in WHO grade II gliomas is not fully clarified. To detect relevant changes in mRNA expression, we performed an in-silico analysis of WHO grade II and IV glioma sequencing datasets provided by The Cancer Genome Atlas (TCGA) to investigate mRNA expression levels of relevant heme biosynthesis genes: Solute Carrier Family 15 Member 1 and 2 (SLC15A1 and SLC15A2), Aminolevulinate-Dehydratase (ALAD), Hydroxymethylbilane-Synthase (HMBS), Uroporphyrinogen-III-Synthase (UROS), Uroporphyrinogen-Decarboxylase (UROD), Coproporphyrinogen-Oxidase (CPOX), Protoporphyrinogen-Oxidase (PPOX), ATP-binding Cassette Subfamily B Member 6 (ABCB6)/G Member 2 (ABCG2) and Ferrochelatase (FECH). Altogether, 258 WHO grade II and 166 WHO grade IV samples were investigated. The mRNA expression levels showed significant differences in 8 of 11 examined genes between WHO grade II and IV gliomas. Significant differences in mRNA expression included increases of HMBS, UROD, FECH and PPOX as well as decreases of SLC15A2, ALAD, UROS and ABCB6 in WHO IV gliomas. Since the majority of changes was found in directions that might actually impair PpIX accumulation in WHO grade IV gliomas, additional studies are needed to analyze the corresponding factors of the heme biosynthesis also on protein level.
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http://dx.doi.org/10.3390/cancers12082043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466145PMC
July 2020

Single-Cell RNA-Seq Reveals Cellular Hierarchies and Impaired Developmental Trajectories in Pediatric Ependymoma.

Cancer Cell 2020 07;38(1):44-59.e9

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.

Ependymoma is a heterogeneous entity of central nervous system tumors with well-established molecular groups. Here, we apply single-cell RNA sequencing to analyze ependymomas across molecular groups and anatomic locations to investigate their intratumoral heterogeneity and developmental origins. Ependymomas are composed of a cellular hierarchy initiating from undifferentiated populations, which undergo impaired differentiation toward three lineages of neuronal-glial fate specification. While prognostically favorable groups of ependymoma predominantly harbor differentiated cells, aggressive groups are enriched for undifferentiated cell populations. The delineated transcriptomic signatures correlate with patient survival and define molecular dependencies for targeted treatment approaches. Taken together, our analyses reveal a developmental hierarchy underlying ependymomas relevant to biological and clinical behavior.
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http://dx.doi.org/10.1016/j.ccell.2020.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479515PMC
July 2020

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

NPJ Vaccines 2020 16;5. Epub 2020 Jan 16.

Activartis Biotech GmbH, Vienna, Austria.

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 ( = 36) with microRNA sequencing plus RT-qPCR ( = 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.
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http://dx.doi.org/10.1038/s41541-019-0149-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965118PMC
January 2020

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

Acta Neuropathol Commun 2019 08 7;7(1):128. Epub 2019 Aug 7.

Comprehensive Cancer Center-Central Nervous System Tumors Unit, Medical University of Vienna, Spitalgasse 23, BT86/E 01, 1090, Vienna, Austria.

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 BRAF and TERT promoter mutations in our clinical data (n = 8) in addition to published datasets (n = 103) and established a BRAF-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 BRAF-mutated HGG as compared to BRAF-mutated LGG. In vitro, BRAF/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 BRAF-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 BRAF 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.
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http://dx.doi.org/10.1186/s40478-019-0775-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685154PMC
August 2019

Resolving medulloblastoma cellular architecture by single-cell genomics.

Nature 2019 08 24;572(7767):74-79. Epub 2019 Jul 24.

Tumor Initiation and Maintenance Program, NCI-Designated Cancer Center, Sanford Burnham Prebys Medical Research Discovery Institute, La Jolla, CA, USA.

Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.
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http://dx.doi.org/10.1038/s41586-019-1434-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754173PMC
August 2019

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.

Cytotherapy 2019 06 8;21(6):643-658. Epub 2019 Apr 8.

Activartis Biotech GmbH, Vienna, Austria.

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.
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http://dx.doi.org/10.1016/j.jcyt.2019.03.002DOI Listing
June 2019

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

J Cell Mol Med 2019 01 22;23(1):281-292. Epub 2018 Nov 22.

Department of Tumor Immunology, St. Anna Kinderkrebsforschung Children's Cancer Research Institute, Vienna, Austria.

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.
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http://dx.doi.org/10.1111/jcmm.13927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307809PMC
January 2019

TERT promoter mutations are associated with poor prognosis and cell immortalization in meningioma.

Neuro Oncol 2018 11;20(12):1584-1593

Institute of Cancer Research and Comprehensive Cancer Center, Department of Medicine I, Medical University Vienna, Vienna, Austria.

Background: Meningiomas are mostly benign tumors tending to progress to higher-grade lesions. Mutations in the telomerase reverse transcriptase (TERT) gene promoter are comparably rare in meningioma, but were recently suggested to predict risk of recurrence and progression. Here we have analyzed a cohort of World Health Organization grades I-III meningiomas regarding the impact of TERT promoter mutations on patient prognosis and in vitro cell propagation feasibility.

Methods: From 110 meningioma patients, 128 tissue samples were analyzed for the TERT promoter mutations C228T and C250T by direct sequencing. Of the 128 samples, 121 were tested for cell propagation in vitro. Telomerase activity, TERT mRNA expression, and telomere lengths were investigated by telomeric repeat amplification protocol assay, reverse transcription PCR, and quantitative PCR, respectively. Impact of the E-twenty-six (ETS) transcription factor inhibitor YK-4-279 on cell viability and TERT promoter activity was analyzed.

Results: TERT promoter mutations were found in 5.5% of all samples analyzed and were associated with a significantly upregulated telomerase activity and TERT mRNA expression (P < 0.0001 both). Regarding telomere lengths, no significant difference between the TERT promoter wild-type and mutated subgroups was detected. Patients with TERT promoter mutated tumors exhibited significantly shorter overall survival (P = 0.0006; 53.8 vs 115.6 mo). The presence of TERT promoter mutations but not telomerase activity or TERT mRNA expression predicted indefinite cell growth in vitro. TERT promoter mutated meningioma cells were hypersensitive against the ETS transcription factor inhibitor YK-4-279, inducing a distinct downregulation of TERT promoter activity.

Conclusion: TERT promoter mutations drive meningioma aggressiveness, resulting in reduced patient survival, but might also open novel therapeutic options for progressive disease.
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http://dx.doi.org/10.1093/neuonc/noy104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231195PMC
November 2018

CD44 drives aggressiveness and chemoresistance of a metastatic human osteosarcoma xenograft model.

Oncotarget 2017 Dec 9;8(69):114095-114108. Epub 2017 Dec 9.

Department of Orthopaedics, Medical University Vienna, 1090 Vienna, Austria.

Background: Osteosarcoma is the most common primary malignant bone tumor with a 5 year survival rate of up to 70%. However, patients with metastatic disease have still a very poor prognosis. Osteosarcoma metastasis models are essential to develop novel treatment strategies for advanced disease.

Methods: Based on a serial transplantation approach, we have established a U-2 OS osteosarcoma xenograft model with increased metastatic potential and compared it to other metastatic osteosarcoma models from international sources. Subclones with differing invasive potential were compared for genomic gains and losses as well as gene expression changes by several bioinformatic approaches. Based on the acquired results, the effects of a shRNA-mediated mRNA knockdown on migration, invasion and chemosensitivity were evaluated.

Results: The gene was part of an amplified region at chromosome 11p found in both U-2 OS subclones with enhanced metastatic potential but not in parental U-2 OS cells, corresponding with distinct CD44 overexpression. Accordingly, shRNA-mediated knockdown significantly attenuated osteosarcoma cell migration, invasion, and viability especially in the metastatic subclones of U-2 OS and Saos-2 cells. Metastatic subclones generally were hypersensitive against the integrin inhibitor cilengitide paralleled by alterations in integrin expression pattern following knock-down. Additionally, attenuation of CD44 expression sensitized these cell models against osteosarcoma chemotherapy with doxorubicin but not methotrexate and cisplatin.

Conclusions: The osteosarcoma xenograft models with increased metastatic potential developed in this study can be useful for identification of mechanisms driving metastasis and resistance towards clinically used and novel therapeutic regimens.
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http://dx.doi.org/10.18632/oncotarget.23125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768389PMC
December 2017

Telomerase activation in posterior fossa group A ependymomas is associated with dismal prognosis and chromosome 1q gain.

Neuro Oncol 2017 Sep;19(9):1183-1194

Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Austria; Comprehensive Cancer Center, Medical University of Vienna, Austria; Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Austria; Neuromed Campus, Kepler University Hospital, Linz, Austria; Institute of Neurology, Medical University of Vienna, Austria; Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, Germany; Department of Pediatric Oncology, Hematology and Immunology, University Hospital Heidelberg, Germany; German Cancer Consortium (DKTK), Heidelberg, Germany; Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany; Department of Neuropathology, University Hospital Heidelberg, Germany; Division of Molecular Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany; Department of Neurosurgery, Medical University of Vienna, Austria; Department of Radiotherapy, Medical University of Vienna, Austria.

Background: Ependymomas account for up to 10% of childhood CNS tumors and have a high rate of tumor recurrence despite gross total resection. Recently, classification into molecular ependymoma subgroups has been established, but the mechanisms underlying the aggressiveness of certain subtypes remain widely enigmatic. The aim of this study was to dissect the clinical and biological role of telomerase reactivation, a frequent mechanism of cancer cells to evade cellular senescence, in pediatric ependymoma.

Methods: We determined telomerase enzymatic activity, hTERT mRNA expression, promoter methylation, and the rs2853669 single nucleotide polymorphism located in the hTERT promoter in a well-characterized cohort of pediatric intracranial ependymomas.

Results: In posterior fossa ependymoma group A (PF-EPN-A) tumors, telomerase activity varied and was significantly associated with dismal overall survival, whereas telomerase reactivation was present in all supratentorial RelA fusion-positive (ST-EPN-RELA) ependymomas. In silico analysis of methylation patterns showed that only these two subgroups harbor hypermethylated hTERT promoters suggesting telomerase reactivation via epigenetic mechanisms. Furthermore, chromosome 1q gain, a well-known negative prognostic factor, was strongly associated with telomerase reactivation in PF-EPN-A. Additional in silico analyses of gene expression data confirmed this finding and further showed enrichment of the E-twenty-six factor, Myc, and E2F target genes in 1q gained ependymomas. Additionally, 1q gained tumors showed elevated expression of ETV3, an E-twenty-six factor gene located on chromosome 1q.

Conclusion: Taken together we describe a subgroup-specific impact of telomerase reactivation on disease progression in pediatric ependymoma and provide preliminary evidence for the involved molecular mechanisms.
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http://dx.doi.org/10.1093/neuonc/nox027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570194PMC
September 2017

Podoplanin expression in primary brain tumors induces platelet aggregation and increases risk of venous thromboembolism.

Blood 2017 Mar 10;129(13):1831-1839. Epub 2017 Jan 10.

Clinical Division of Hematology and Hemostaseology, Department of Medicine I, Medical University of Vienna, Vienna, Austria.

Venous thromboembolism (VTE) is common in patients with brain tumors, and underlying mechanisms are unclear. We hypothesized that podoplanin, a sialomucin-like glycoprotein, increases the risk of VTE in primary brain tumors via its ability to induce platelet aggregation. Immunohistochemical staining against podoplanin and intratumoral platelet aggregates was performed in brain tumor specimens of 213 patients (mostly high-grade gliomas [89%]) included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study of patients with newly diagnosed cancer or progressive disease aimed at identifying patients at risk of VTE. Platelet aggregation in response to primary human glioblastoma cells was investigated in vitro. During 2-year follow-up, 29 (13.6%) patients developed VTE. One-hundred fifty-one tumor specimens stained positive for podoplanin (33 high expression, 47 medium expression, 71 low expression). Patients with podoplanin-positive tumors had lower peripheral blood platelet counts ( < .001) and higher D-dimer levels ( < .001). Podoplanin staining intensity was associated with increasing levels of intravascular platelet aggregates in tumor specimens ( < .001). High podoplanin expression was associated with an increased risk of VTE (hazard ratio for high vs no podoplanin expression: 5.71; 95% confidence interval, 1.52-21.26; 010), independent of age, sex, and tumor type. Podoplanin-positive primary glioblastoma cells induced aggregation of human platelets in vitro, which could be abrogated by an antipodoplanin antibody. In conclusion, high podoplanin expression in primary brain tumors induces platelet aggregation, correlates with hypercoagulability, and is associated with increased risk of VTE. Our data indicate novel insights into the pathogenesis of VTE in primary brain tumors.
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http://dx.doi.org/10.1182/blood-2016-06-720714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823234PMC
March 2017

Acquired nintedanib resistance in FGFR1-driven small cell lung cancer: role of endothelin-A receptor-activated ABCB1 expression.

Oncotarget 2016 Aug;7(31):50161-50179

Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Austria.

Genomically amplified fibroblast growth factor receptor 1 (FGFR1) is an oncogenic driver in defined lung cancer subgroups and predicts sensibility against FGFR1 inhibitors in this patient cohort. The FGFR inhibitor nintedanib has recently been approved for treatment of lung adenocarcinoma and is currently evaluated for small cell lung cancer (SCLC). However, tumor recurrence due to development of nintedanib resistance might occur. Hence, we aimed at characterizing the molecular mechanisms underlying acquired nintedanib resistance in FGFR1-driven lung cancer. Chronic nintedanib exposure of the FGFR1-driven SCLC cell line DMS114 (DMS114/NIN) but not of two NSCLC cell lines induced massive overexpression of the multidrug-resistance transporter ABCB1. Indeed, we proved nintedanib to be both substrate and modulator of ABCB1-mediated efflux. Importantly, the oncogenic FGFR1 signaling axis remained active in DMS114/NIN cells while bioinformatic analyses suggested hyperactivation of the endothelin-A receptor (ETAR) signaling axis. Indeed, ETAR inhibition resensitized DMS114/NIN cells against nintedanib by downregulation of ABCB1 expression. PKC and downstream NFκB were identified as major downstream players in ETAR-mediated ABCB1 hyperactivation. Summarizing, ABCB1 needs to be considered as a factor underlying nintedanib resistance. Combination approaches with ETAR antagonists or switching to non-ABCB1 substrate FGFR inhibitors represent innovative strategies to manage nintedanib resistance in lung cancer.
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http://dx.doi.org/10.18632/oncotarget.10324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226575PMC
August 2016

Another step toward DNA selective targeting: Ni(II) and Cu(II) complexes of a Schiff base ligand able to bind gene promoter G-quadruplexes.

Dalton Trans 2016 05;45(18):7758-67

Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Viale delle Scienze, Edificio 17, 90128 Palermo, Italy.

DNA G-rich sequences are able to form four-stranded structures organized in stacked guanine tetrads. These structures, called G-quadruplexes, were found to have an important role in the regulation of oncogenes expression and became, for such a reason, appealing targets for anticancer drugs. Aiming at finding selective G-quadruplex binders, we have designed, synthesized and characterized a new water soluble Salen-like Schiff base ligand and its Ni(II) and Cu(II) metal complexes. UV-Vis, circular dichroism and FRET measurements indicated that the nickel complex can stabilize oncogene promoter G-quadruplexes with high selectivity, presenting no interactions with duplex DNA at all. The same compound exhibited dose-dependent cytotoxic activity in MCF-7 breast cancer cells when combined with lipofectamine as lipophilic carrier.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5048337PMC
http://dx.doi.org/10.1039/c6dt00648eDOI Listing
May 2016

New Brain Tumor Entities Emerge from Molecular Classification of CNS-PNETs.

Cell 2016 Feb;164(5):1060-1072

Department of Radiological, Oncological and Anatomic-Pathological Sciences, Sapienza University of Rome, 00185 Rome, Italy.

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.
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http://dx.doi.org/10.1016/j.cell.2016.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5139621PMC
February 2016

EGFR is not a major driver for osteosarcoma cell growth in vitro but contributes to starvation and chemotherapy resistance.

J Exp Clin Cancer Res 2015 Nov 2;34:134. Epub 2015 Nov 2.

Institute of Cancer Research and Comprehensive Cancer Center, Department of Medicine I, Medical University Vienna, Borschkegasse 8a, 1090, Vienna, Austria.

Background: Enhanced signalling via the epidermal growth factor receptor (EGFR) is a hallmark of multiple human carcinomas. However, in recent years data have accumulated that EGFR might also be hyperactivated in human sarcomas. Aim of this study was to investigate the influence of EGFR inhibition on cell viability and its interaction with chemotherapy response in osteosarcoma cell lines.

Methods: We have investigated a panel of human osteosarcoma cell lines regarding EGFR expression and downstream signalling. To test its potential applicability as therapeutic target, inhibition of EGFR by gefitinib was combined with osteosarcoma chemotherapeutics and cell viability, migration, and cell death assays were performed.

Results: Osteosarcoma cells expressed distinctly differing levels of functional EGFR reaching in some cases high amounts. Functionality of EGFR in osteosarcoma cells was proven by EGF-mediated activation of both MAPK and PI3K/AKT pathway (determined by phosphorylation of ERK1/2, AKT, S6, and GSK3β). The EGFR-specific inhibitor gefitinib blocked EGF-mediated downstream signal activation. At standard in vitro culture conditions, clinically achievable gefitinib doses demonstrated only limited cytotoxic activity, however, significantly reduced long-term colony formation and cell migration. In contrast, under serum-starvation conditions active gefitinib doses were distinctly reduced while EGF promoted starvation survival. Importantly, gefitinib significantly supported the anti-osteosarcoma activities of doxorubicin and methotrexate regarding cell survival and migratory potential.

Conclusion: Our data suggest that EGFR is not a major driver for osteosarcoma cell growth but contributes to starvation- and chemotherapy-induced stress survival. Consequently, combination approaches including EGFR inhibitors should be evaluated for treatment of high-grade osteosarcoma patients.
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http://dx.doi.org/10.1186/s13046-015-0251-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4630894PMC
November 2015

Prognostic quality of activating TERT promoter mutations in glioblastoma: interaction with the rs2853669 polymorphism and patient age at diagnosis.

Neuro Oncol 2015 Sep 13;17(9):1231-40. Epub 2015 Feb 13.

Department of Neurosurgery, Wagner-Jauregg Hospital, Linz, Austria (S.S.-K., M.L., A.O.); Institute of Pathology and Neuropathology, Wagner-Jauregg Hospital, Linz, Austria (S.W.); Department of Internal Medicine, Wagner-Jauregg Hospital, Linz, Austria (J.P.); Institute of Cancer Research and Comprehensive Cancer Center-Central Nervous System Tumors Unit, Department of Medicine I, Medical University of Vienna, Vienna, Austria (D.L., C.P., T.M., W.B.); Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria (B.G.); Laboratory of Molecular Biology and Tumorcytogenetics, Hospital Barmherzige Schwestern, Linz, Austria (G.W.).

Background: Expression of the telomerase reverse transcriptase (TERT) might be altered by activating mutations of the rs2853669 polymorphism within the promoter region. Here we investigate the impact of these genomic alterations on telomerase activation and dissect their prognostic potential in glioblastoma (GBM).

Methods: The respective TERT promoter region was sequenced in 126 GBM tissues and compared with clinical parameters and glioma biomarkers MGMT promoter methylation and IDH1 mutation. TERT mRNA expression, telomerase activity, and telomere lengths were determined by reverse transcriptase PCR, TRAP assay, and real-time PCR, respectively.

Results: Seventy-three percent of GBM patients harbored TERT promoter mutations associated with enhanced telomerase activity and TERT mRNA expression but reduced telomere lengths (P < .001 for all). Patients with mutated tumors exhibited significantly shorter overall survival in the entire cohort (11.5 vs 23.1 months; P < .0001) and in the primary GBM patient subgroup lacking IDH1 mutations (n = 120; P = .0084). This prognostic impact was confined to younger patients (aged <65 years), while the negative prognostic power of enhanced age at diagnosis was limited to those patients lacking TERT promoter mutations. Presence of the common single nucleotide polymorphism rs2853669, disrupting an endogenous Ets2 transcription factor-binding site, was associated with improved survival exclusively in patients with a wild-type TERT promoter. On the contrary, the shortest mean overall survival was detected in those patients harboring both an activating TERT promoter mutation and homozygous rs2853669 alleles.

Conclusion: In summary, TERT promoter mutations are powerful prognosticators for worse course of disease in human GBM patients but their prognostic value is influenced by the rs2853669 polymorphism and age at diagnosis.
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http://dx.doi.org/10.1093/neuonc/nov010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588753PMC
September 2015

Major vault protein supports glioblastoma survival and migration by upregulating the EGFR/PI3K signalling axis.

Oncotarget 2013 Nov;4(11):1904-18

Institute of Cancer Research, Department of Medicine I.

Despite their ubiquitous expression and high conservation during evolution, precise cellular functions of vault ribonucleoparticles, mainly built of multiple major vault protein (MVP) copies, are still enigmatic. With regard to cancer, vaults were shown to be upregulated during drug resistance development as well as malignant transformation and progression. Such in a previous study we demonstrated that human astrocytic brain tumours including glioblastoma are generally high in vault levels while MVP expression in normal brain is comparably low. However a direct contribution to the malignant phenotype in general and that of glioblastoma in particular has not been established so far. Thus we address the questions whether MVP itself has a pro-tumorigenic function in glioblastoma. Based on a large tissue collection, we re-confirm strong MVP expression in gliomas as compared to healthy brain. Further, the impact of MVP on human glioblastoma aggressiveness was analysed by using gene transfection, siRNA knock-down and dominant-negative genetic approaches. Our results demonstrate that MVP/vaults significantly support migratory and invasive competence as well as starvation resistance of glioma cells in vitro and in vivo. The enhanced aggressiveness was based on MVP-mediated stabilization of the epidermal growth factor receptor (EGFR)/phosphatidyl-inositol-3-kinase (PI3K) signalling axis. Consequently, MVP overexpression resulted in enhanced growth and brain invasion in human glioblastoma xenograft models. Our study demonstrates, for the first time, that vaults have a tumour-promoting potential by stabilizing EGFR/PI3K-mediated migration and survival pathways in human glioblastoma.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875758PMC
http://dx.doi.org/10.18632/oncotarget.1264DOI Listing
November 2013

Prognostic significance of telomerase-associated parameters in glioblastoma: effect of patient age.

Neuro Oncol 2013 Apr 7;15(4):423-32. Epub 2013 Feb 7.

Institute of Cancer Research, Department of Medicine I, Medical University Vienna, Borschkegasse 8a, 1090 Vienna, Austria.

Background: Glioblastoma multiforme (GBM) is a heterogeneous, highly aggressive primary brain tumor with strongly variable patient survival. Because reliable prognostic biomarkers are lacking, we investigated the relation between telomerase-associated parameters and the disease course.

Methods: Telomerase-associated parameters were determined in 100 GBM tissues and associated with clinical characteristics and overall survival. Expressions of telomere length, telomerase activity (TA), and human telomerase reverse transcriptase (hTERT) were analyzed by quantitative PCR, telomeric repeat amplification protocol assay, and reverse transcriptase-PCR, respectively. Mutation status of isocitrate dehydrogenase (IDH)1 was determined by direct sequencing, and O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation by methylation-specific PCR.

Results: Of 100 GBM tissues, 61 were positive for both hTERT mRNA and TA, with a highly significant correlation between both parameters (linear regression, P < .0001). Telomere length determination revealed a significant difference between the hTERT/TA-positive and -negative subgroups, with markedly longer telomeres in the hTERT/TA-negative cohort (unpaired Student's t-test, P = .0001). Accordingly, significantly shorter telomeres were detected in GBM tissues derived from older patients (>60 y at diagnosis, P < .0001). While no association of telomere parameters with MGMT promoter status was found, all tumors with IDH1 mutation (6/100) were negative for both hTERT expression and TA and harbored significantly longer telomeres. Patients with tumors lacking hTERT expression/TA showed a significant survival benefit (Kaplan-Meier test, both P < .01), which, however, was based exclusively on the younger patient subgroup (≤60 y, both P < .005; >60 y, both ns).

Conclusions: Telomerase activation is not an independent prognostic parameter in GBM but predicts aggressive tumor behavior solely in a younger patient cohort.
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http://dx.doi.org/10.1093/neuonc/nos329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607268PMC
April 2013

Aggressiveness of human melanoma xenograft models is promoted by aneuploidy-driven gene expression deregulation.

Oncotarget 2012 Apr;3(4):399-413

Laboratory of Toxicology, Faculty of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium.

Melanoma is a devastating skin cancer characterized by distinct biological subtypes. Besides frequent mutations in growth- and survival-promoting genes like BRAF and NRAS, melanomas additionally harbor complex non-random genomic alterations. Using an integrative approach, we have analysed genomic and gene expression changes in human melanoma cell lines (N=32) derived from primary tumors and various metastatic sites and investigated the relation to local growth aggressiveness as xenografts in immuno-compromised mice (N=22). Although the vast majority >90% of melanoma models harbored mutations in either BRAF or NRAS, significant differences in subcutaneous growth aggressiveness became obvious. Unsupervised clustering revealed that genomic alterations rather than gene expression data reflected this aggressive phenotype, while no association with histology, stage or metastatic site of the original melanoma was found. Genomic clustering allowed separation of melanoma models into two subgroups with differing local growth aggressiveness in vivo. Regarding genes expressed at significantly altered levels between these subgroups, a surprising correlation with the respective gene doses (>85% accordance) was found. Genes deregulated at the DNA and mRNA level included well-known cancer genes partly already linked to melanoma (RAS genes, PTEN, AURKA, MAPK inhibitors Sprouty/Spred), but also novel candidates like SIPA1 (a Rap1GAP). Pathway mining further supported deregulation of Rap1 signaling in the aggressive subgroup e.g. by additional repression of two Rap1GEFs. Accordingly, siRNA-mediated down-regulation of SIPA1 exerted significant effects on clonogenicity, adherence and migration in aggressive melanoma models. Together our data suggest that an aneuploidy-driven gene expression deregulation drives local aggressiveness in human melanoma.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380575PMC
http://dx.doi.org/10.18632/oncotarget.473DOI Listing
April 2012

Trabectedin has promising antineoplastic activity in high-grade meningioma.

Cancer 2012 Oct 5;118(20):5038-49. Epub 2012 Mar 5.

Department of Medicine I, Medical University of Vienna, Vienna, Austria; Comprehensive Cancer Center-Central Nervous System Tumors Unit, Medical University of Vienna, Vienna, Austria.

Background: Meningiomas are common intracranial tumors arising from the meninges and usually are benign. However, a few meningiomas have aggressive behavior and, for such patients, effective treatment options are needed. Trabectedin is a novel, marine-derived, antineoplastic agent that has been approved and is used routinely as therapy for advanced soft tissue sarcoma and ovarian cancer.

Methods: The authors investigated the in vitro effects of trabectedin alone and in combination with hydroxyurea, cisplatin, and doxorubicin in primary cell cultures of benign (n = 9), atypical (n = 6), and anaplastic (n = 4) meningiomas using chemosensitivity assays (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT]), Western blot analysis, cell cycle analysis, and immunofluorescent staining.

Results: Strong antimeningioma activity of trabectedin was observed and was characterized by distinct cell cycle arrest, down-regulation of multiple cyclins, deregulated expression of cell death-regulatory genes, and massive apoptosis induction. Cytotoxic activity was especially intense in higher grade meningiomas with a half-maximal inhibitory concentration <10 nM. Combination with trabectedin synergistically enhanced the antimeningioma activity of hydroxyurea but also enhanced the activity of doxorubicin and cisplatin. On the basis of these findings, trabectedin was given to 1 patient who had heavily pretreated, anaplastic meningioma, and a favorable response was observed with radiologic disease stabilization, marked reductions in brain edema and requirement for corticosteroids, and improvement of clinical symptoms. However, treatment had to be discontinued after 5 cycles because of adverse drug effects.

Conclusions: The current results indicated that trabectedin may represent a promising new therapeutic option for patients with aggressive meningioma and should be evaluated in prospective clinical studies.
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http://dx.doi.org/10.1002/cncr.27460DOI Listing
October 2012

The major vault protein mediates resistance to epidermal growth factor receptor inhibition in human hepatoma cells.

Cancer Lett 2012 Jun 17;319(2):164-172. Epub 2012 Jan 17.

Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria. Electronic address:

To better understand the response of HCC to EGFR inhibition, we analyzed factors connected to the resistance of HCC cells against gefitinib. Sensitive HCC3 cells co-expressed EGFR and ErbB3 but lacked kinase-domain mutations in EGFR. Interestingly, expression of MVP was restricted to resistant cell lines, whereas ABCB1 and ABCC1 showed no association with gefitinib resistance. Moreover, ectopic MVP expression in HCC3 cells decreased gefitinib sensitivity, increased AKT phosphorylation and reduced the expression of inflammatory pathway-associated genes, whereas silencing of MVP in Hep3B and HepG2 cells increased sensitivity. These findings suggest MVP as a novel player in resistance against EGFR inhibition.
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http://dx.doi.org/10.1016/j.canlet.2012.01.002DOI Listing
June 2012

Response of experimental malignant melanoma models to the pan-Aurora kinase inhibitor VE-465.

Exp Dermatol 2010 Dec;19(12):1040-7

Institute of Cancer Research, Clinics for Medicine I, Medical University Vienna, Vienna, Austria.

Aurora kinases represent promising novel cancer therapy targets. Genomic analyses of human cutaneous melanoma (CMM) models (N = 51, low passage) by classical and/or array CGH revealed frequent gains at chromosome 20q (65%, amplifications in 45%) repeatedly including the Aurora A gene locus. Accordingly, the majority of CMM cell cultures overexpressed Aurora A when compared to proliferating non-malignant cells. Moreover, CMM cells even when arrested in G1/S cell cycle phase contained readily detectable levels of Aurora A indicating incomplete degradation during mitosis. Already at low concentrations (10-100 nm), long-term (7-10 days) application of the pan-Aurora kinase inhibitor VE-465 completely prevented colony formation in all CMM models tested. In contrast, blockade of cell survival/proliferation and DNA synthesis as well as the induction of apoptosis by VE-465 distinctly differed in short-term experiments (up to 72 h exposure). Both cell cycle arrest and DNA synthesis blockade depended on the level of VE-465-mediated p53/p21 activation while p53/p21 unresponsiveness led to repetitive endoreduplication (>8n DNA content). In contrast, apoptosis induction by VE-465 and Aurora A siRNA did not correlate with p53/p21 responsiveness and DNA synthesis blockade. Moreover, application of the Aurora B-specific inhibitor ZM447439 and siRNA was less efficient to induce CMM cell death proofing that apoptosis induction by VE-465 depended predominantly on Aurora A targeting. In combination experiments with chemotherapeutic agents, VE-465 acted additive to antagonistic when applied concomitantly but in several cases even synergistic when applied consecutively. In summary, we suggest that the Aurora A kinase might represent a promising target of well-designed novel antimelanoma strategies.
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http://dx.doi.org/10.1111/j.1600-0625.2010.01182.xDOI Listing
December 2010

O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients.

Neuro Oncol 2010 Jan 15;12(1):28-36. Epub 2009 Oct 15.

Department of Neurosurgery, Wagner-Jauregg Hospital, Wagner Jauregg Weg 15, 4020 Linz, Austria.

O(6)-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (chi(2) test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76-17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45-2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.
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http://dx.doi.org/10.1093/neuonc/nop003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2940563PMC
January 2010