Search

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors with a strong hereditary background and a large genetic heterogeneity. Identification of the underlying genetic cause is crucial for the management of patients and their families as it aids differentiation between hereditary and sporadic cases. To improve diagnostics and clinical management we tailored an enrichment based comprehensive multi-gene next generation sequencing panel applicable to both analyses of tumor tissue and blood samples. We applied this panel to tumor samples and compared its performance to our current routine diagnostic approach. Routine diagnostic sequencing of 11 PPGL susceptibility genes was applied to blood samples of 65 unselected PPGL patients at a single center in Dresden, Germany. Predisposing germline mutations were identified in 19 (29.2%) patients. Analyses of 28 PPGL tumor tissues using the dedicated PPGL panel revealed pathogenic or likely pathogenic variants in known PPGL susceptibility genes in 21 (75%) cases, including mutations in IDH2, ATRX and HRAS. These mutations suggest sporadic tumor development. Our results imply a diagnostic benefit from extended molecular tumor testing of PPGLs and consequent improvement of patient management. The approach is promising for determination of prognostic biomarkers that support therapeutic decision-making. Acknowledgments: We thank the patients and their families who have made this research possible. We want to thank JacquesW. Lenders for his support. We further thank Alexander Krüger, Lydia Rossow and Franziska Stübner for technical support as well as Katharina Langton and Uwe Siemon for their assistance in patient administration. Sí

Metastases are the main reason for cancer-related deaths. Initiation of metastases, where newly seeded tumor cells expand into colonies, presents a tremendous bottleneck to metastasis formation. Despite its importance, a quantitative description of metastasis initiation and its clinical implications is lacking. Here, we set theoretical grounds for the metastatic bottleneck with a simple stochastic model. The model assumes that the proliferation-to-death rate ratio for the initiating metastatic cells increases when they are surrounded by more of their kind. For a total of 159,191 patients across 13 cancer types, we found that a single cell has an extremely low median probability of successful seeding of the order of 10−8. With increasing colony size, a sharp transition from very unlikely to very likely successful metastasis initiation occurs. The median metastatic bottleneck, defined as the critical colony size that marks this transition, was between 10 and 21 cells. We derived the probability of metastasis occurrence and patient outcome based on primary tumor size at diagnosis and tumor type. The model predicts that the efficacy of patient treatment depends on the primary tumor size but even more so on the severity of the metastatic bottleneck, which is estimated to largely vary between patients. We find that medical interventions aiming at tightening the bottleneck, such as immunotherapy, can be much more efficient than therapies that decrease overall tumor burden, such as chemotherapy. ISSN:1553-734X ISSN:1553-7358

Astrocytomas are primary human brain tumors including diffuse or anaplastic astrocytomas that develop towards secondary glioblastomas over time. However, only little is known about molecular alterations that drive this progression. We measured multi-omics profiles of patient-matched astrocytoma pairs of initial and recurrent tumors from 22 patients to identify molecular alterations associated with tumor progression. Gene copy number profiles formed three major subcluters, but more than half of the patient-matched astrocytoma pairs differed in their gene copy number profiles like astrocytomas from different patients. Chromosome 10 deletions were not observed for diffuse astrocytomas, but occurred in corresponding recurrent tumors. Gene expression profiles formed three other major subclusters and patient-matched expression profiles were much more heterogeneous than their copy number profiles. Still, recurrent tumors showed a strong tendency to switch to the mesenchymal subtype. The direct progression of diffuse astrocytomas to secondary glioblastomas showed the largest number of transcriptional changes. Astrocytoma progression groups were further distinguished by signaling pathway expression signatures affecting cell division, interaction and differentiation. As expected, IDH1 was most frequently mutated closely followed by TP53, but also MUC4 involved in the regulation of apoptosis and proliferation was frequently mutated. Astrocytoma progression groups differed in their mutation frequencies of these three genes. Overall, patient-matched astrocytomas can differ substantially within and between patients, but still molecular signatures associated with the progression to secondary glioblastomas exist and should be analyzed for their potential clinical relevance in future studies.

Oligodendrogliomas are primary human brain tumors with a characteristic 1p/19q co-deletion of important prognostic relevance, but little is known about the pathology of this chromosomal mutation. We developed a network-based approach to identify novel cancer gene candidates in the region of the 1p/19q co-deletion. Gene regulatory networks were learned from gene expression and copy number data of 178 oligodendrogliomas and further used to quantify putative impacts of differentially expressed genes of the 1p/19q region on cancer-relevant pathways. We predicted 8 genes with strong impact on signaling pathways and 14 genes with strong impact on metabolic pathways widespread across the region of the 1p/19 co-deletion. Many of these candidates (e.g. ELTD1, SDHB, SEPW1, SLC17A7, SZRD1, THAP3, ZBTB17) are likely to push, whereas others (e.g. CAP1, HBXIP, KLK6, PARK7, PTAFR) might counteract oligodendroglioma development. For example, ELTD1, a functionally validated glioblastoma oncogene located on 1p, was overexpressed. Further, the known glioblastoma tumor suppressor SLC17A7 located on 19q was underexpressed. Moreover, known epigenetic alterations triggered by mutated SDHB in paragangliomas suggest that underexpressed SDHB in oligodendrogliomas may support and possibly enhance the epigenetic reprogramming induced by the IDH-mutation. We further analyzed rarely observed deletions and duplications of chromosomal arms within oligodendroglioma subcohorts identifying putative oncogenes and tumor suppressors that possibly influence the development of oligodendroglioma subgroups. Our in-depth computational study contributes to a better understanding of the pathology of the 1p/19q co-deletion and other chromosomal arm mutations. This might open opportunities for functional validations and new therapeutic strategies. Electronic supplementary material The online version of this article (10.1186/s40478-018-0544-y) contains supplementary material, which is available to authorized users.

Figure S1. Comparison of normalized coverage of targeted capture-based NGS to multiplex PCR-based NGS applied to FFPE tumor samples. A: Normalized coverage (y-axis) of targeted capture-based and multiplex PCR-based NGS of FFPE samples from patient 1 to 13 (Additional file 3: Table S2, P01 to P13) at single-base resolution along all concatenated BRCA1/2 targets (x-axis). Samples P01 to P13 are color-coded in blue. B: Normalized coverage (y-axis) of targeted capture-based and multiplex PCR-based NGS of FFPE samples from patient P14-P15 (Additional file 3: Table S2, P14 to P15) of FFPE samples from patient P14-P15 (Additional file 3: Table S2, P14 to P15) of FFPE samples from patient P14-P15 (Additional file 3: Table S2, P14 to P15) of FFPE samples from patient P14-P15 (Additional file 3: Table S2, P14 to P15) at single-base resolution along all concatenated BRCA1/2 targets (x-axis). Samples are color-coded in red and represent highly fragmented DNA. Exemplary, randomly chosen capture target dropouts and amplicon dropouts are marked by a red arrow. The vertical green line indicates the end of BRCA2 targets (target number is increasing from left to right which corresponds to five to three prime orientation of the gene) and the start of BRCA1 targets (target number is decreasing from left to right which corresponds to five to three prime orientation of the gene). The horizontal green line displays normalized coverage of 1.0. All target exons are separated by gray dotted vertical lines. Selected exons are marked. (PDF 2100 kb)

Although intellectual disability is one of the major indications for genetic counselling, there are no homogenous diagnostic algorithms for molecular testing. While whole exome sequencing is increasingly applied, we questioned whether analyzing a partial exome, enriched for genes associated with Mendelian disorders, might be a valid alternative approach that yields similar detection rates but requires less sequencing capacities. Within this context 106 patients with different intellectual disability forms were analyzed for mutations in 4.813 genes after pre-exclusion of copy number variations by array-CGH. Subsequent variant interpretation was performed in accordance with the ACMG guidelines. By this, a molecular diagnosis was established in 34% of cases and candidate mutations were identified in additional 24% of patients. Detection rates of causative mutations were above 30%, regardless of further symptoms, except for patients with seizures (23%). We did not detect an advantage from partial exome sequencing for patients with severe intellectual disability (36%) as compared to those with mild intellectual disability (44%). Specific clinical diagnoses pre-existed for 20 patients. Of these, 5 could be confirmed and an additional 6 cases could be solved, but showed mutations in other genes than initially suspected. In conclusion partial exome sequencing solved >30% of intellectual disability cases, which is similar to published rates obtained by whole exome sequencing. The approach therefore proved to be a valid alternative to whole exome sequencing for molecular diagnostics in this cohort. The method proved equally suitable for both syndromic and non-syndromic intellectual disability forms of all severity grades.

Objective: Multifocal glioblastoma, i.e. multiple synchronous lesions in one patient, are considered to be a good model to elucidate the pathogenesis and progression of malignant gliomas. Our aim was to perform a molecular characterization of a small set of this tumor entity. Methods: Tumor tissue[for full text, please go to the a.m. URL], 69. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Mexikanischen und Kolumbianischen Gesellschaft für Neurochirurgie

Texts S1â S4 and Figures S1â S7. (PDF 1800 kb)

Intrinsic and acquired resistances are major obstacles in cancer therapy. Genetic characterization is commonly used to identify predictive or prognostic biomarker signatures and potential cancer targets in samples from therapy-naïve patients. By far less common are such investigations to identify specific, predictive and/or prognostic gene signatures in patients or cancer cells refractory to a specific molecular-targeted intervention. This, however, might have a great value to foster the development of tailored, personalized cancer therapy. Based on our identification of a differential radiosensitization by single and combined β1 integrin (AIIB2) and EGFR (Cetuximab) targeting in more physiological, three-dimensional head and neck squamous cell carcinoma (HNSCC) cell cultures, we performed comparative whole exome sequencing, phosphoproteome analyses and RNAi knockdown screens in responder and non-responder cell lines. We found a higher rate of gene mutations with putative protein-changing characteristics in non-responders and different mutational profiles of responders and non-responders. These profiles allow stratification of HNSCC patients and identification of potential targets to address treatment resistance. Consecutively, pharmacological inhibition of mTOR and KEAP1 effectively diminished non-responder insusceptibility to β1 integrin and EGFR targeting for radiosensitization. Our data pinpoint the added value of genetic biomarker identification after selection for cancer subgroup responsiveness to targeted therapies.

Comparison of revealed subtypes to subtypes revealed for histologically classified oligoastrocytomas and astrocytomas. (PDF 2365 kb)

We report a novel CASP9 germline mutation that may increase susceptibility to the development of brain tumors. We identified this mutation in a family in which three brain tumors had developed within three generations, including two anaplastic astrocytomas occurring in cousins. The cousins were diagnosed at similar ages (29 and 31 years), and their tumors showed similar histological features. Genetic analysis revealed somatic IDH1 and TP53 mutations in both tumors. However, no germline TP53 mutations were detected, despite the fact that this family fulfills the criteria of Li-Fraumeni-like syndrome. Whole exome sequencing revealed a germline stop-gain mutation (R65X) in the CASP9 gene, which encodes caspase-9, a key molecule for the p53-dependent mitochondrial death pathway. This mutation was also detected in DNA extracted from blood samples from the two siblings who were each a parent of one of the affected cousins. Caspase-9 immunohistochemistry showed the absence of caspase-9 immunoreactivity in the anaplastic astrocytomas and normal brain tissues of the cousins. These observations suggest that CASP9 germline mutations may have played a role at least in part to the susceptibility of development of gliomas in this Li-Fraumeni-like family lacking a TP53 germline mutation.

Precision oncology implies the ability to predict which patients will likely respond to specific cancer therapies based on increasingly accurate, high-resolution molecular diagnostics as well as the functional and mechanistic understanding of individual tumors. While molecular stratification of patients can be achieved through different means, a promising approach is next-generation sequencing of tumor DNA and RNA, which can reveal genomic alterations that have immediate clinical implications. Furthermore, certain genetic alterations are shared across multiple histologic entities, raising the fundamental question of whether tumors should be treated by molecular profile and not tissue of origin. We here describe MASTER (Molecularly Aided Stratification for Tumor Eradication Research), a clinically applicable platform for prospective, biology-driven stratification of younger adults with advanced-stage cancer across all histologies and patients with rare tumors. We illustrate how a standardized workflow for selection and consenting of patients, sample processing, whole-exome/genome and RNA sequencing, bioinformatic analysis, rigorous validation of potentially actionable findings, and data evaluation by a dedicated molecular tumor board enables categorization of patients into different intervention baskets and formulation of evidence-based recommendations for clinical management. Critical next steps will be to increase the number of patients that can be offered comprehensive molecular analysis through collaborations and partnering, to explore ways in which additional technologies can aid in patient stratification and individualization of treatment, to stimulate clinically guided exploratory research projects, and to gradually move away from assessing the therapeutic activity of targeted interventions on a case-by-case basis toward controlled clinical trials of genomics-guided treatments.

LGCA transition dynamics. LGCA transition dynamics D : = R ∘ A $\mathcal {D}:=\mathcal {R} \, \circ \, \mathcal {A}$ in a von Neumann neighbourhood N r $\mathcal {N}_{\boldsymbol {r}}$ around node r (gray) followed by translocation T i $\mathcal {T}_{i}$ . (a) During reorientation R $\mathcal {R}$ cells are stochastically redistributed within nodes according to a probability function P. The highest probability is assigned to a resulting node configuration η ′(r)with post-reorientation momentum J:=J(η ′,a ″)(r)parallel to the pre-reorientation local adhesivity gradient G:=G(η,a ′)(r)of the neighbourhood excluding r [Fig. 1b]. (b) In our model, the newly introduced adhesivity update operator A $\mathcal {A}$ couples the time scales of the LGCA and ODE models. The graph shows an example for the analytical solution y r (t)[Eq. (1)] of the underlying ODE [Eq. 1] according to which A $\mathcal {A}$ decreases the adhesive states a i (r,k)of all cells as a i (r,k)>a i (r,k+τ) ∀ k; in this example there is no adhesion heterogeneity. The update from time-step k = t to k+τ = t+τ shown here is labelled red in the graph. After reorientation, cells are moved by the translocation operator T i $\mathcal {T}_{i}$ (see Additional file 1 for details). Note that all nodes have only one rest channel. Notation as in Fig. 1. (PDF 28 kb)

Item does not contain fulltext The tremendous advances over the past two decades in both clinical genetics and biochemical testing of chromaffin cell tumours have led to new considerations about how these aspects of laboratory medicine can be integrated to improve diagnosis and management of affected patients. With germline mutations in 15 genes now identified to be responsible for over a third of all cases of phaeochromocytomas and paragangliomas, these tumours are recognised to have one of the richest hereditary backgrounds among all neoplasms. Depending on the mutation, tumours show distinct differences in metabolic pathways that relate to or even directly impact clinical presentation. At the same time, there has been improved understanding about how catecholamines are synthesised, stored, secreted and metabolised by chromaffin cell tumours. Although the tumours may not always secrete catecholamines it has become clear that almost all continuously produce and metabolise catecholamines. This has not only fuelled changes in laboratory medicine, but has also assisted in recognition of genotype-biochemical phenotype relationships important for diagnostics and clinical care. In particular, differences in catecholamine and energy pathway metabolomes can guide genetic testing, assist with test interpretation and provide predictions about the nature, behaviour and imaging characteristics of the tumours. Conversely, results of genetic testing are important for guiding how routine biochemical testing should be employed and interpreted in surveillance programmes for at-risk patients. In these ways there are emerging needs for modern laboratory medicine to seamlessly integrate biochemical and genetic testing into the diagnosis and management of patients with chromaffin cell tumours. 01 april 2017

Model details. Mathematical formalism and full model description. (PDF 225 kb)

BACKGROUND A major hallmark of glioblastoma (GBM) is its invasive capacity, contributing to its aggressive behaviour. Invasive cells cannot be easily removed by surgery or irradiation and eventually result in lethal recurrence. A better understanding of the invasion process and the key molecular players underlying the invasive potential of GBM may lead to the identification of new therapeutic targets for GBM patients. MATERIAL AND METHODS To identify candidate genes responsible for invasion, a genome-wide shRNA screen was performed in patient-derived GBM cultures. The most promising candidate was validated in in vitro invasion assays, ex vivo brain slice cultures and in vivo orthotopic xenografts in mice. Gene knockdown in invasive GBM cells was compared with overexpression in non-invasive cells. RNAseq of knockdown cells, along with the generation of deletion constructs were applied to uncover the mechanisms regulating invasion. RESULTS A zinc-finger domain containing protein was identified as an invasion essential candidate gene. Knockdown of this gene confirmed a strong impact on invasion in highly invasive GBM cells. In contrast, gene overexpression switched non-invasive GBM cells to an invasive phenotype. Deletion of one or both zinc-finger motifs decreased invasion indicating that both are essential for regulating invasion. Mutation of the nuclear localisation signal resulted in retention of the protein in the cytoplasm and loss of the invasion phenotype demonstrating that the protein activity is required in the nucleus. Gene expression analyses revealed that invasion-related genes are significantly regulated by the candidate gene once it is localized in the nucleus. CONCLUSION We identified a zinc-finger containing protein as a novel driver of GBM invasion, presumably through transcription factor activity resulting in the induction of an invasive transcriptional program. This protein and its downstream pathway may represent novel promising targets to overcome invasive capacities in GBM.

Objective: The mitotic Aurora B kinase is overexpressed in various cancers and several inhibitors for Aurora B are currently under clinical assessments. Yet, the precise mode of the anti-tumoral action of Aurora B inhibitors and the possible impact of p53 on treatment remained unclear. In this study,[for full text, please go to the a.m. URL], 67. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), 1. Joint Meeting mit der Koreanischen Gesellschaft für Neurochirurgie (KNS)

Radiotherapy is a mainstay of curative prostate cancer treatment, but risks of recurrence after treatment remain significant in locally advanced disease. Given that tumor relapse can be attributed to a population of cancer stem cells (CSC) that survives radiotherapy, analysis of this cell population might illuminate tactics to personalize treatment. However, this direction remains challenging given the plastic nature of prostate cancers following treatment. We show here that irradiating prostate cancer cells stimulates a durable upregulation of stem cell markers that epigenetically reprogram these cells. In both tumorigenic and radioresistant cell populations, a phenotypic switch occurred during a course of radiotherapy that was associated with stable genetic and epigenetic changes. Specifically, we found that irradiation triggered histone H3 methylation at the promoter of the CSC marker aldehyde dehydrogenase 1A1 (ALDH1A1), stimulating its gene transcription. Inhibiting this methylation event triggered apoptosis, promoted radiosensitization, and hindered tumorigenicity of radioresistant prostate cancer cells. Overall, our results suggest that epigenetic therapies may restore the cytotoxic effects of irradiation in radioresistant CSC populations.

Background Astrocytomas are the most common primary brain tumors distinguished into four histological grades. Molecular analyses of individual astrocytoma grades have revealed detailed insights into genetic, transcriptomic and epigenetic alterations. This provides an excellent basis to identify similarities and differences between astrocytoma grades. Methods We utilized public omics data of all four astrocytoma grades focusing on pilocytic astrocytomas (PA I), diffuse astrocytomas (AS II), anaplastic astrocytomas (AS III) and glioblastomas (GBM IV) to identify similarities and differences using well-established bioinformatics and systems biology approaches. We further validated the expression and localization of Ang2 involved in angiogenesis using immunohistochemistry. Results Our analyses show similarities and differences between astrocytoma grades at the level of individual genes, signaling pathways and regulatory networks. We identified many differentially expressed genes that were either exclusively observed in a specific astrocytoma grade or commonly affected in specific subsets of astrocytoma grades in comparison to normal brain. Further, the number of differentially expressed genes generally increased with the astrocytoma grade with one major exception. The cytokine receptor pathway showed nearly the same number of differentially expressed genes in PA I and GBM IV and was further characterized by a significant overlap of commonly altered genes and an exclusive enrichment of overexpressed cancer genes in GBM IV. Additional analyses revealed a strong exclusive overexpression of CX3CL1 (fractalkine) and its receptor CX3CR1 in PA I possibly contributing to the absence of invasive growth. We further found that PA I was significantly associated with the mesenchymal subtype typically observed for very aggressive GBM IV. Expression of endothelial and mesenchymal markers (ANGPT2, CHI3L1) indicated a stronger contribution of the micro-environment to the manifestation of the mesenchymal subtype than the tumor biology itself. We further inferred a transcriptional regulatory network associated with specific expression differences distinguishing PA I from AS II, AS III and GBM IV. Major central transcriptional regulators were involved in brain development, cell cycle control, proliferation, apoptosis, chromatin remodeling or DNA methylation. Many of these regulators showed directly underlying DNA methylation changes in PA I or gene copy number mutations in AS II, AS III and GBM IV. Conclusions This computational study characterizes similarities and differences between all four astrocytoma grades confirming known and revealing novel insights into astrocytoma biology. Our findings represent a valuable resource for future computational and experimental studies. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1939-9) contains supplementary material, which is available to authorized users.

Contains supporting Texts S1â S5 and supporting Figures S1â S9. Text S1: Processing of Rembrandt gene copy number data. Text S2: Lasso-based regulatory network inference. Text S3: Endothelial cells express Ang2 in PA I and GBM IV. Text S4: Network validation based on independent glioma cohorts. Text S5: Comparison of motif search-based and gene expression-based TF-target links. Figure S1: Age distribution of PA I, AS II, AS III and GBM IV patients. Figure S2: Functional categorization of differentially expressed genes. Figure S3: Verhaak classification results of two independent PA I cohorts. Figure S4: Ang2 immunohistochemistry. Figure S5: Associations of PA I, AS II, AS III and GBM IV with hypermethylator subtype. Figure S6: Differential expression in individual signaling pathways. Figure S7: Gene expression-based regulatory network validation. Figure S8: Overlap of network-based TF-target interactions and motif-based TF-binding sites. Figure S9: TF-TF interaction network. (PDF 1249 kb)

Objective: Carmustine wafers (Gliadel™) are the only available local therapy for glioma treatment and are frequently used for recurrent glioblastomas. Although the therapeutic effect has been shown for primary and recurrent glioblastomas in phase II and III studies, respectively; there are only[for full text, please go to the a.m. URL], 61. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC) im Rahmen der Neurowoche 2010

Metastases are the main reason for cancer-related deaths. Initiation of metastases, where newly seeded tumor cells expand into colonies, presents a tremendous bottleneck to metastasis formation. Despite its importance, a quantitative description of metastasis initiation and its clinical implications is lacking. Here, we set theoretical grounds for the metastatic bottleneck with a simple stochastic model. The model assumes that the proliferation-to-death rate ratio for the initiating metastatic cells increases when they are surrounded by more of their kind. For a total of 159,191 patients across 13 cancer types, we found that a single cell has an extremely low median probability of successful seeding of the order of 10−8. With increasing colony size, a sharp transition from very unlikely to very likely successful metastasis initiation occurs. The median metastatic bottleneck, defined as the critical colony size that marks this transition, was between 10 and 21 cells. We derived the probability of metastasis occurrence and patient outcome based on primary tumor size at diagnosis and tumor type. The model predicts that the efficacy of patient treatment depends on the primary tumor size but even more so on the severity of the metastatic bottleneck, which is estimated to largely vary between patients. We find that medical interventions aiming at tightening the bottleneck, such as immunotherapy, can be much more efficient than therapies that decrease overall tumor burden, such as chemotherapy., PLoS Computational Biology, 16 (10), ISSN:1553-734X, ISSN:1553-7358

Glioblastomas are the most common malignant brain tumors in adults. Despite resection of the tumor, combined radio- and chemotherapy and new-targeted therapy approaches, the average life expectancy is only 15 months with a 3-year survival rate of less than 5%. Invasive growth of tumor cells into the surrounding brain tissue complicates treatment and causes recurrence. Imaging techniques such as magnetic resonance imaging (MRI) are conventionally used for diagnosis and for monitoring after surgery in order to detect tumor lesions. However, next to tumor progression, contrast enhancement could also result from pseudoprogression or radiation necrosis. Similarly, next to therapy response, a reduction of contrast enhancement could also mimic a success in therapy (pseudoresponse). Furthermore, the use of targeted therapies requires the molecular detection of biomarkers, which is often performed on tissue biopsies. During therapy, resistance mechanisms to therapy may develop, which have a significant impact on the success of the therapy. Repeated biopsies are needed to distinguish tumor progress from therapy-associated tissue changes and simultaneously detect therapy resistance. Often they are not feasible due to the high risk for the patient. Currently, there are methods to improve monitoring in glioblastoma patients. Besides improved imaging techniques, liquid biopsies are a promising method to detect tumor progression. At the same time, liquid biopsies also allow molecular characterization of the lesions. A key advantage over tissue biopsies is that they are minimally invasive, making them well suited for longitudinal tumor monitoring. For clinical application of liquid biopsies in the form of blood and CSF, components such as circulating tumor cells, circulating tumor DNA, extracellular vesicles, or tumor-educated platelets are used, with the former two probably being the most common tumor markers at present. In this study, a highly sensitive next generation sequencing-based method was established to detect tumor mutations in plasma and CSF of glioblastoma patients. To this end, crucial sample preparation steps were initially optimized. Therefore, four isolation kits extracting cell-free DNA from plasma were compared and the best-performing kit was chosen for the analysis of patient material. Furthermore, a multi-gene next generation sequencing panel (cfDNA-GBM panel) was designed specifically for use in liquid biopsies from glioblastoma patients and tested on a reference standard for cell-free DNA with mutations of different allele frequencies. The newly designed cfDNA-GBM panel, which uses hybrid-capture based enrichment, was compared to a primer-extension based panel. Due to the more consistent coverage of target regions, superior sensitivity, and better clinical applicability due to its smaller size, the cfDNA-GBM panel was used for subsequent clinical investigations. In both enrichments, unique molecular barcodes were used to label all original fragments and thus identify and eliminate errors in subsequent data analysis that occur during amplification in library preparation This increases the sensitivity of the method. During the establishment of the methodology, the use of molecular barcodes clarified the limitation of sequence information due to the small input amount of cell-free DNA. For tumor tissue analysis, an already established larger panel was adapted and extended based on the results of a proof of concept study. After the establishment of the sensitive method, clinical samples of glioblastoma patients were analyzed. Isolation and analysis of cell-free DNA from plasma and CSF resulted in highly variable amounts of cell-free DNA with characteristic oligonucleosomal fragment lengths. Furthermore, no difference in the amount or fragmentation of cell-free DNA from pre- or post-surgery plasma was determined. To increase the amount of cell-free DNA for sequencing, the amount of blood samples was increased from 10 ml (5 patients) to 50 ml (9 patients). Detection of circulating tumor DNA in plasma and CSF from glioblastoma patients was performed using two strategies. In a first approach, somatic tumor mutations were detected in genomic DNA from tumor tissue, which were searched for in the sequence data of liquid biopsies in a second step. In this approach, an increased detection rate (in 56% of patients compared to 25% in 10 ml blood samples) of circulating tumor DNA was detected in blood samples with a volume of 50 ml. Almost all tumor mutations lying in the target region of the smaller cfDNA-GBM panel could be detected in CSF (92%) with similar allele frequencies to those detected in the tumor. In a second strategy, somatic variants were detected in plasma and CSF without prior knowledge of variants in tumor tissue. Interestingly, three variants were detected in the CSF of one patient, which occurred with an allele frequency over 5% in the CSF, while they were not detectable in the analyzed tumor tissue. These variants may be indicators for tumor heterogeneity that was not accessible by analyzing sections of tumor tissue. Finally, mutation-specific probes were designed for three tumor-somatic variants detected in the patients' plasma to validate them by digital PCR. A pair of probes for a mutation in PTEN was successfully established to robustly detect minimal allele frequencies down to 0.1% but could not validate the mutation in cell-free DNA from plasma of the respective patient. Overall, it was shown that analysis of tumor somatic mutations using the cfDNA-GBM sequencing panel designed and established in this study is limited in the analysis of cell-free DNA from plasma of patients with glioblastomas, whereas the detection of circulating tumor DNA from CSF is promising.