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  • Korshunov, Andrey  (12)
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  • 1
    In: Neuro-Oncology, 2018, Vol. 20(suppl2), pp.i182-i182
    Description: Precise diagnosis and robust detection of actionable alterations is required for individualized treatments. The Pediatric Targeted Therapy (PTT) 2.0 program aims at improvement of diagnostic accuracy and detection of targetable alterations by extended molecular diagnostics. The impact of these analyses on clinical management is being evaluated. Pediatric patients with relapsed or progressive tumors after treatment according to standard protocols are included, independent of the histological diagnosis. Formalin fixed paraffin embedded material and a blood sample for germline correction are requested. The methods employed are DNA methylation array, customized targeted gene panel sequencing (130 genes), RNA and Sanger sequencing in selected cases, and immunohistochemistry (IHC) of selected markers. A questionnaire-based follow-up is used to determine the clinical impact of the analysis. We have included n=111 cases from 22.02.2017.-31.12.2017, analysis was completed for n=83 cases (75%) at the time of abstract submission. The most common entities were brain tumors (n=56/83, 67%). DNA methylation array alone allowed diagnostic classification in n=45/83 cases (54.2%) and n=34/56 brain tumor cases (60,7%), respectively. Actionable targets as detected by copy number calculation, gene panel sequencing, RNA sequencing and IHC were found in n=47/83 cases (56.6%). Pathogenic germline alterations with clinical relevance were identified in n=7/83 cases (8.4%) and were confirmed by Sanger sequencing. Follow-up analyses are ongoing. In conclusion, combination of next-generation diagnostics such as methylation arrays and targeted sequencing in addition to selected IHC markers added robust information concerning diagnosis and targetable alterations. The impact on clinical decision-making and on outcome is currently being evaluated.
    Keywords: Medicine;
    ISSN: 1522-8517
    E-ISSN: 1523-5866
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  • 2
    Language: English
    In: Ecker, J., I. Oehme, R. Mazitschek, A. Korshunov, M. Kool, T. Hielscher, J. Kiss, et al. 2015. “Targeting class I histone deacetylase 2 in MYC amplified group 3 medulloblastoma.” Acta Neuropathologica Communications 3 (1): 22. doi:10.1186/s40478-015-0201-7. http://dx.doi.org/10.1186/s40478-015-0201-7.
    Description: Introduction: Medulloblastoma (MB) is the most frequent malignant brain tumor in children. Four subgroups with distinct genetic, epigenetic and clinical characteristics have been identified. Survival remains particularly poor in patients with Group 3 tumors harbouring a MYC amplification. We herein explore the molecular mechanisms and translational implications of class I histone deacetylase (HDAC) inhibition in MYC driven MBs. Material and Methods Expression of HDACs in primary MB subgroups was compared to normal brain tissue. A panel of MB cell lines, including Group 3 MYC amplified cell lines, were used as model systems. Cells were treated with HDAC inhibitors (HDACi) selectively targeting class I or IIa HDACs. Depletion of HDAC2 was performed. Intracellular HDAC activity, cellular viability, metabolic activity, caspase activity, cell cycle progression, RNA and protein expression were analyzed. Results: HDAC2 was found to be overexpressed in MB subgroups with poor prognosis (SHH, Group 3 and Group 4) compared to normal brain and the WNT subgroup. Inhibition of the enzymatic activity of the class I HDACs reduced metabolic activity, cell number, and viability in contrast to inhibition of class IIa HDACs. Increased sensitivity to HDACi was specifically observed in MYC amplified cells. Depletion of HDAC2 increased H4 acetylation and induced cell death. Simulation of clinical pharmacokinetics showed time-dependent on target activity that correlated with binding kinetics of HDACi compounds. Conclusions: We conclude that HDAC2 is a valid drug target in patients with MYC amplified MB. HDACi should cover HDAC2 in their inhibitory profile and timing and dosing regimen in clinical trials should take binding kinetics of compounds into consideration. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0201-7) contains supplementary material, which is available to authorized users.
    Keywords: Medulloblastoma ; Hdac ; Hdac Inhibitor ; Hdac2 ; Myc
    ISSN: 2051-5960
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  • 3
    In: Nature, 2018
    Description: Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.
    Keywords: DNA Methylation ; Central Nervous System Neoplasms -- Diagnosis;
    ISSN: 0028-0836
    E-ISSN: 1476-4687
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  • 4
    In: Neuro-Oncology, 2017, Vol. 19(12), pp.1607-1617
    Description: BackgroundEmbryonal tumor with multilayered rosettes (ETMR) is a rare and aggressive embryonal brain tumor that solely occurs in infants and young children and has only recently been recognized as a separate brain tumor entity in the World Health Organization classification for CNS tumors. Patients have a very dismal prognosis with a median survival of 12 months upon diagnosis despite aggressive treatment. The aim of this study was to develop novel treatment regimens in a preclinical drug screen in order to inform potentially more active clinical trial protocols. MethodsWe have carried out an in vitro and in vivo drug screen using the ETMR cell line BT183 and its xenograft model. Furthermore, we have generated the first patient-derived xenograft (PDX) model for ETMR and evaluated our top drug candidates in an in vitro drug screen using this model. ResultsBT183 cells are very sensitive to the topoisomerase inhibitors topotecan and doxorubicin, to the epigenetic agents decitabine and panobinostat, to actinomycin D, and to targeted drugs such as the polo-like kinase 1 (PLK1) inhibitor volasertib, the aurora kinase A inhibitor alisertib, and the mammalian target of rapamycin (mTOR) inhibitor MLN0128. In xenograft mice, monotherapy with topotecan, volasertib, and actinomycin D led to a temporary response in tumor growth and a significant increase in survival. Finally, using multi-agent treatment regimens of topotecan or doxorubicin combined with methotrexate and vincristine, the response in tumor growth and survival was further increased compared with mice receiving single treatments. ConclusionsWe have identified several promising candidates for combination therapies in future clinical trials for ETMR patients.
    Keywords: Actinomycin D ; Brain Tumor ; Etmr ; Topotecan ; Volasertib
    ISSN: 1522-8517
    E-ISSN: 1523-5866
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  • 5
    In: Neuro-Oncology, 2017, Vol. 19(suppl6), pp.vi200-vi200
    Description: Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. This single-pathway disease exhibits aberrant activation of the MAPK pathway driving the tumor into oncogene-induced senescence (OIS). OIS is proposed to be the source of the unstable however benign growth observed in PA patients. Senescence is thought to be regulated by the senescence-associated secretory phenotype (SASP) which comprises a variety of cytokines, growth factors and proteases. Markers of senescence have been detected in PA, but the functional relevance of the SASP and its relation to OIS in PA is unknown. The first patient-derived PA cell culture model DKFZ-BT66 was utilized for the characterization of OIS and the role of the SASP in PA. The model allows experimental switching between senescent and proliferating states by modulation of the p53/RB pathway. Both conditions were analyzed by gene-expression profiling (GEP), Western Blot, real-time qPCR, ELISA, cell counts and viability by automated trypan blue exclusion staining. A significant increase of the SASP could be detected by GEP in the OIS state of the PA cell line. Moreover, the OIS expression signature was associated with improved progression free survival in a cohort of n=112 PA patients. Upregulation of IL-6 and IL-1B, two representative SASP factors, could be demonstrated on mRNA and protein level in DKFZ-BT66 during OIS. Both cytokine receptors are expressed and activation of the respective pathways was confirmed. Activation of the IL-1 pathway led to decreased growth of proliferating PA cells. Overall, the novel primary PA tumor model provides functional evidence of the presence of OIS in PA and exhibits increased activity of the SASP during the senescent state. In order to find an explanation for the clinically observed spontaneous on/off growth behavior, current studies aim to investigate the disruption of the OIS-characteristic growth arrest by inhibition of inflammatory signaling pathways.
    Keywords: Medicine;
    ISSN: 1522-8517
    E-ISSN: 1523-5866
    Source: Oxford University Press
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  • 6
    In: Neuro-Oncology, 2018, Vol. 20(suppl2), pp.i106-i106
    Description: The main challenge in the clinical management of pilocytic astrocytoma (PA) is its unpredictable growth behavior. PA cells are driven into oncogene-induced senescence (OIS) by aberrant MAPK activation. In other senescence models OIS was shown to be regulated and maintained by the senescence-associated secretory phenotype (SASP). In this study, the first patient-derived cell culture model DKFZ-BT66 was used to show presence of the SASP in PA and to analyze its impact on OIS. This model allows for shifting between proliferation and senescence via doxycycline-inducible inhibition of the OIS-relevant p53/RB pathway. Both states were studied using gene-expression profiling (GEP), Western blot, qPCR, ELISA, and automated trypan blue exclusion staining. The GEP shows significant upregulation of SASP factors during OIS in DKFZ-BT66 cells. Conditioned medium of senescent DKFZ-BT66 cells is sufficient to induce growth arrest of proliferating DKFZ-BT66 cells. Upregulation of the SASP factors IL-1B and IL-6 was validated on mRNA and protein levels and both pathways are active during OIS. Stimulation of the IL-1 pathway reduces growth of proliferating DKFZ-BT66 cells. While pharmacological inhibition of single cytokines is not sufficient to overcome growth arrest during OIS, treatment with the anti-inflammatory drug dexamethasone induces regrowth of senescent cells. Overall, the primary PA model provides evidence of the presence of OIS in PA and exhibits increased activity of the SASP during senescence. Our data suggest that the SASP has an important impact on the growth regulation of senescent PA cells. Alteration of SASP factors may result in spontaneous regrowth of senescent PA tumors.
    Keywords: Medicine;
    ISSN: 1522-8517
    E-ISSN: 1523-5866
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  • 7
    In: Brain Pathology, July 2016, Vol.26(4), pp.506-516
    Description: The “pediatric targeted therapy” (PTT) program aims to identify the presence and activity of druggable targets and evaluate the clinical benefit of a personalized treatment approach in relapsed or progressive tumors on an individual basis. 10 markers (HDAC2, HR23B, p‐AKT, p‐ERK, p‐S6, p‐EGFR, PDGFR‐alpha/beta, p53 and BRAFV600E) were analyzed by immunohistochemistry. Pediatric patients with tumors independent of the histological diagnosis, with relapse or progression after treatment according to standard protocols were included.  = 61/145 (42%) cases were eligible for analysis between 2009 and 2013, the most common entities being brain tumors. Immunohistochemical stainings were evaluated by the ‐Score (0–300). In 93% of the cases potentially actionable targets were identified. The expressed or activated pathways were histone deacetylase (HDACs; 83.0% of cases positive), EGFR (87.2%), PDGFR (75.9%), p53 (50.0%), MAPK/ERK (43.3%) and PI3K/mTOR (36.1%). Follow‐up revealed partial or full implementation of PTT results in treatment decision‐making in 41% of the cases. Prolonged disease stabilization responses in single cases were noticed, however, response rates did not differ from cases treated with other modalities. Further studies evaluating the feasibility and clinical benefit of personalized diagnostic approaches using paraffin material are warranted.
    Keywords: Brain Tumors ; Pediatric Oncology ; Personalized Medicine ; Targeted Therapy ; Relapsed Childhood Tumors ; Predictive Markers
    ISSN: 1015-6305
    E-ISSN: 1750-3639
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  • 8
    Language: English
    In: Acta Neuropathologica, 2016, Vol.131(6), pp.903-910
    Description: With the number of prognostic and predictive genetic markers in neuro-oncology steadily growing, the need for comprehensive molecular analysis of neuropathology samples has vastly increased. We therefore developed a customized enrichment/hybrid-capture-based next-generation sequencing (NGS) gene panel comprising the entire coding and selected intronic and promoter regions of 130 genes recurrently altered in brain tumors, allowing for the detection of single nucleotide variations, fusions, and copy number aberrations. Optimization of probe design, library generation and sequencing conditions on 150 samples resulted in a 5-workday routine workflow from the formalin-fixed paraffin-embedded sample to neuropathological report. This protocol was applied to 79 retrospective cases with established molecular aberrations for validation and 71 prospective cases for discovery of potential therapeutic targets. Concordance of NGS compared to established, single biomarker methods was 98.0 %, with discrepancies resulting from one case where a TERT promoter mutation was not called by NGS and three ATRX mutations not being detected by Sanger sequencing. Importantly, in samples with low tumor cell content, NGS was able to identify mutant alleles that were not detectable by traditional methods. Information derived from NGS data identified potential targets for experimental therapy in 37/47 (79 %) glioblastomas, 9/10 (90 %) pilocytic astrocytomas, and 5/14 (36 %) medulloblastomas in the prospective target discovery cohort. In conclusion, we present the settings for high-throughput, adaptive next-generation sequencing in routine neuropathology diagnostics. Such an approach will likely become highly valuable in the near future for treatment decision making, as more therapeutic targets emerge and genetic information enters the classification of brain tumors.
    Keywords: Medicine & Public Health ; Pathology ; Neurosciences ; Medicine;
    ISSN: 0001-6322
    E-ISSN: 1432-0533
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  • 9
    Language: English
    In: Oncotarget, 14 February 2017, Vol.8(7), pp.11460-11479
    Description: Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo, preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro. Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.
    Keywords: Kiaa1549:Braf-Fusion ; Mapk-Inhibitors ; Oncogene-Induced Senescence (Ois) ; Pediatric Low Grade Glioma ; Pilocytic Astrocytoma ; Astrocytoma ; Brain Neoplasms ; Cell Culture Techniques ; Cell Line, Tumor ; Cellular Senescence -- Physiology
    E-ISSN: 1949-2553
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  • 10
    Language: English
    In: Clinical cancer research : an official journal of the American Association for Cancer Research, 15 March 2019, Vol.25(6), pp.1851-1866
    Description: Pilocytic astrocytoma is the most common childhood brain tumor, characterized by constitutive MAPK activation. MAPK signaling induces oncogene-induced senescence (OIS), which may cause unpredictable growth behavior of pilocytic astrocytomas. The senescence-associated secretory phenotype (SASP) has been shown to regulate OIS, but its role in pilocytic astrocytoma remains unknown. The patient-derived pilocytic astrocytoma cell culture model, DKFZ-BT66, was used to demonstrate presence of the SASP and analyze its impact on OIS in pilocytic astrocytoma. The model allows for doxycycline-inducible switching between proliferation and OIS. Both states were studied using gene expression profiling (GEP), Western blot, ELISA, and cell viability testing. Primary pilocytic astrocytoma tumors were analyzed by GEP and multiplex assay. SASP factors were upregulated in primary human and murine pilocytic astrocytoma and during OIS in DKFZ-BT66 cells. Conditioned medium induced growth arrest of proliferating pilocytic astrocytoma cells. The SASP factors IL1B and IL6 were upregulated in primary pilocytic astrocytoma, and both pathways were regulated during OIS in DKFZ-BT66. Stimulation with rIL1B but not rIL6 reduced growth of DKFZ-BT66 cells and induced the SASP. Anti-inflammatory treatment with dexamethasone induced regrowth of senescent cells and inhibited the SASP. Senescent DKFZ-BT66 cells responded to senolytic BCL2 inhibitors. High and SASP expression in pilocytic astrocytoma tumors was associated with favorable progression-free survival. We provide evidence for the SASP regulating OIS in pediatric pilocytic astrocytoma, with IL1B as a relevant mediator. SASP expression could enable prediction of progression in patients with pilocytic astrocytoma. Further investigation of the SASP driving the unpredictable growth of pilocytic astrocytomas, and its possible therapeutic application, is warranted.
    ISSN: 1078-0432
    E-ISSN: 15573265
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