Publications by authors named "Katrin Lamszus"

108 Publications

Integrated Molecular-Morphologic Meningioma Classification: A Multicenter Retrospective Analysis, Retrospectively and Prospectively Validated.

J Clin Oncol 2021 Oct 7:JCO2100784. Epub 2021 Oct 7.

Department of Neurosurgery, NYU Langone Hospital, New York, NY.

Purpose: Meningiomas are the most frequent primary intracranial tumors. Patient outcome varies widely from benign to highly aggressive, ultimately fatal courses. Reliable identification of risk of progression for individual patients is of pivotal importance. However, only biomarkers for highly aggressive tumors are established ( and ), whereas no molecularly based stratification exists for the broad spectrum of patients with low- and intermediate-risk meningioma.

Methods: DNA methylation data and copy-number information were generated for 3,031 meningiomas (2,868 patients), and mutation data for 858 samples. DNA methylation subgroups, copy-number variations (CNVs), mutations, and WHO grading were analyzed. Prediction power for outcome was assessed in a retrospective cohort of 514 patients, validated on a retrospective cohort of 184, and on a prospective cohort of 287 multicenter cases.

Results: Both CNV- and methylation family-based subgrouping independently resulted in increased prediction accuracy of risk of recurrence compared with the WHO classification (c-indexes WHO 2016, CNV, and methylation family 0.699, 0.706, and 0.721, respectively). Merging all risk stratification approaches into an integrated molecular-morphologic score resulted in further substantial increase in accuracy (c-index 0.744). This integrated score consistently provided superior accuracy in all three cohorts, significantly outperforming WHO grading (c-index difference = .005). Besides the overall stratification advantage, the integrated score separates more precisely for risk of progression at the diagnostically challenging interface of WHO grade 1 and grade 2 tumors (hazard ratio 4.34 [2.48-7.57] and 3.34 [1.28-8.72] retrospective and prospective validation cohorts, respectively).

Conclusion: Merging these layers of histologic and molecular data into an integrated, three-tiered score significantly improves the precision in meningioma stratification. Implementation into diagnostic routine informs clinical decision making for patients with meningioma on the basis of robust outcome prediction.
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http://dx.doi.org/10.1200/JCO.21.00784DOI Listing
October 2021

CD74 and CD44 Expression on CTCs in Cancer Patients with Brain Metastasis.

Int J Mol Sci 2021 Jun 29;22(13). Epub 2021 Jun 29.

Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.
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http://dx.doi.org/10.3390/ijms22136993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268634PMC
June 2021

Cannabidiol converts NFκB into a tumor suppressor in glioblastoma with defined antioxidative properties.

Neuro Oncol 2021 Apr 17. Epub 2021 Apr 17.

Neurosurgical Research, Department of Neurosurgery, University Hospital, LMU Munich, Germany.

Background: The transcription factor NFκB drives neoplastic progression of many cancers including primary brain tumors (glioblastoma; GBM). Precise therapeutic modulation of NFκB activity can suppress central oncogenic signalling pathways in GBM, but clinically applicable compounds to achieve this goal have remained elusive.

Methods: In a pharmacogenomics study with a panel of transgenic glioma cells we observed that NFκB can be converted into a tumor suppressor by the non-psychotropic cannabinoid Cannabidiol (CBD). Subsequently, we investigated the anti-tumor effects of CBD, which is used as an anticonvulsive drug (Epidiolex) in pediatric neurology, in a larger set of human primary GBM stem-like cells (hGSC). For this study we performed pharmacological assays, gene expression profiling, biochemical and cell-biological experiments. We validated our findings using orthotopic in vivo models and bioinformatics analysis of human GBM-datasets.

Results: We found that CBD promotes DNA binding of the NFκB subunit RELA and simultaneously prevents RELA-phosphorylation on serine-311, a key residue which permits genetic transactivation. Strikingly, sustained DNA binding by RELA lacking phospho-serine 311 was found to mediate hGSC cytotoxicity. Widespread sensitivity to CBD was observed in a cohort of hGSC defined by low levels of reactive oxygen-species (ROS), while high ROS-content in other tumors blocked CBD induced hGSC death. Consequently, ROS levels served as predictive biomarker for CBD-sensitive tumors.

Conclusions: This evidence demonstrates how a clinically approved drug can convert NFκB into a tumor suppressor and suggests a promising repurposing option for GBM-therapy.
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http://dx.doi.org/10.1093/neuonc/noab095DOI Listing
April 2021

Genome-wide methylation profiling of glioblastoma cell-derived extracellular vesicle DNA allows tumor classification.

Neuro Oncol 2021 07;23(7):1087-1099

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Genome-wide DNA methylation profiling has recently been developed into a tool that allows tumor classification in central nervous system tumors. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioblastoma cell-derived EVs reflects genome-wide tumor methylation and mutational profiles and allows noninvasive tumor subtype classification.

Methods: DNA was isolated from EVs secreted by glioblastoma cells as well as from matching cultured cells and tumors. EV-DNA was localized and quantified by direct stochastic optical reconstruction microscopy. Methylation and copy number profiling was performed using 850k arrays. Mutations were identified by targeted gene panel sequencing. Proteins were differentially quantified by mass spectrometric proteomics.

Results: Genome-wide methylation profiling of glioblastoma-derived EVs correctly identified the methylation class of the parental cells and original tumors, including the MGMT promoter methylation status. Tumor-specific mutations and copy number variations (CNV) were detected in EV-DNA with high accuracy. Different EV isolation techniques did not affect the methylation profiling and CNV results. DNA was present inside EVs and on the EV surface. Proteome analysis did not allow specific tumor identification or classification but identified tumor-associated proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids.

Conclusions: This study provides proof of principle that EV-DNA reflects the genome-wide methylation, CNV, and mutational status of glioblastoma cells and enables their molecular classification.
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http://dx.doi.org/10.1093/neuonc/noab012DOI Listing
July 2021

Intrathecal and systemic alterations of L-arginine metabolism in patients after intracerebral hemorrhage.

J Cereb Blood Flow Metab 2021 08 18;41(8):1964-1977. Epub 2021 Jan 18.

Institute of Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Alterations in the concentration of nitric oxide (NO) and L-arginine metabolites have been associated with the pathophysiology of different vascular diseases. Here, we describe striking changes in L-arginine metabolism after hemorrhagic stroke. Blood and cerebrospinal fluid (CSF) samples of patients with intracerebral hemorrhage (ICH) and/or intraventricular hemorrhage were collected over a ten-day period. Liquid chromatography-tandem mass spectrometry was used to quantify key substrates and products of L-arginine metabolizing enzymes as well as asymmetric (ADMA) and symmetric dimethylarginine (SDMA). Changes in the plasma were limited to early reductions in L-ornithine, L-lysine, and L-citrulline concentrations. Intrathecally, we observed signs of early NO synthase (NOS) upregulation followed by a decrease back to baseline accompanied by a rise in the level of its endogenous NOS-inhibitor ADMA. SDMA demonstrated increased levels throughout the observation period. For arginase, a pattern of persistently elevated activity was measured and arginine:glycine amidinotransferase (AGAT) appeared to be reduced in its activity at later time points. An early reduction in CSF L-arginine concentration was an independent risk factor for poor outcome. Together, these findings further elucidate pathophysiological mechanisms after ICH potentially involved in secondary brain injury and may reveal novel therapeutic targets.
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http://dx.doi.org/10.1177/0271678X20983216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8327100PMC
August 2021

The genetic landscape of choroid plexus tumors in children and adults.

Neuro Oncol 2021 04;23(4):650-660

Institute of Neuropathology, University Hospital Münster, Münster, Germany.

Background: Choroid plexus tumors (CPTs) are intraventricular brain tumors predominantly arising in children but also affecting adults. In most cases, driver mutations have not been identified, although there are reports of frequent chromosome-wide copy-number alterations and TP53 mutations, especially in choroid plexus carcinomas (CPCs).

Methods: DNA methylation profiling and RNA-sequencing was performed in a series of 47 CPTs. Samples comprised 35 choroid plexus papillomas (CPPs), 6 atypical choroid plexus papillomas (aCPPs) and 6 CPCs plus three recurrences thereof. Targeted TP53 and TERT promotor sequencing was performed in all samples. Whole exome sequencing (WES) and linked-read whole genome sequencing (WGS) was performed in 25 and 4 samples, respectively.

Results: Tumors comprised the molecular subgroups "pediatric A" (N=11), "pediatric B" (N=12) and "adult" (N=27). Copy-number alterations mainly represented whole-chromosomal alterations with subgroup-specific enrichments (gains of Chr1, 2 and 21q in "pediatric B" and gains of Chr5 and 9 and loss of Chr21q in "adult"). RNA sequencing yielded a novel CCDC47-PRKCA fusion transcript in one adult choroid plexus papilloma patient with aggressive clinical course; an underlying Chr17 inversion was demonstrated by linked-read WGS. WES and targeted sequencing showed TP53 mutations in 7/47 CPTs (15%), five of which were children. On the contrary, TERT promoter mutations were encountered in 7/28 adult patients (25%) and associated with shorter progression-free survival (log-rank test, p=0.015).

Conclusion: Pediatric CPTs lack recurrent driver alterations except for TP53, whereas CPTs in adults show TERT promoter mutations or a novel CCDC47-PRKCA gene fusion, being associated with a more unfavorable clinical course.
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http://dx.doi.org/10.1093/neuonc/noaa267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041331PMC
April 2021

Discovery of Targetable Genetic Alterations in NSCLC Patients with Different Metastatic Patterns Using a MassARRAY-Based Circulating Tumor DNA Assay.

Cells 2020 10 22;9(11). Epub 2020 Oct 22.

Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Circulating tumor DNA (ctDNA) has shown great promise as a minimally invasive liquid biopsy for personalized cancer diagnostics especially among metastatic patients. Here, we used a novel sensitive assay to detect clinically relevant mutations in ctDNA in blood plasma from metastatic non-small cell lung cancer (NSCLC) patients, including patients with a limited oligo-brain metastatic disease. We analyzed 66 plasma samples from 56 metastatic NSCLC patients for 74 hotspot mutations in five genes commonly mutated in NSCLC using a novel MassARRAY-based lung cancer panel with a turnaround time of only 3 days. Mutations in plasma DNA could be detected in 28 out of 56 patients (50.0%), with a variant allele frequency (VAF) ranging between 0.1% and 5.0%. Mutations were detected in 50.0% of patients with oligo-brain metastatic disease, although the median VAF was lower (0.4%) compared to multi-brain metastatic patients (0.9%) and patients with extra-cranial metastatic progression (1.2%). We observed an overall concordance of 86.4% ( = 38/44) for status between plasma and tissue. The MassARRAY technology can detect clinically relevant mutations in plasma DNA from metastatic NSCLC patients including patients with limited, oligo-brain metastatic disease.
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http://dx.doi.org/10.3390/cells9112337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7690267PMC
October 2020

Highlights of the inaugural ten - the launch of Neuro-Oncology Advances.

Neurooncol Adv 2019 May-Dec;1(1):vdz016. Epub 2019 Aug 28.

Division of Neurosurgery, University Health Network and MacFeeters-Hamilton Neuro-Oncology Program, Princess Margaret Hospital, University of Toronto, Toronto, Canada.

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http://dx.doi.org/10.1093/noajnl/vdz016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212904PMC
August 2019

Glioma escape signature and clonal development under immune pressure.

J Clin Invest 2020 10;130(10):5257-5271

Department of Neurosurgery and.

Immunotherapeutic strategies are increasingly important in neuro-oncology, and the elucidation of escape mechanisms that lead to treatment resistance is crucial. We investigated the impact of immune pressure on the clonal dynamics and immune escape signature by comparing glioma growth in immunocompetent versus immunodeficient mice. Glioma-bearing WT and Pd-1-/- mice survived significantly longer than immunodeficient Pfp-/- Rag2-/- mice. While tumors in Pfp-/- Rag2-/- mice were highly polyclonal, immunoedited tumors in WT and Pd-1-/- mice displayed reduced clonality with emergence of immune escape clones. Tumor cells in WT mice were distinguished by an IFN-γ-mediated response signature with upregulation of genes involved in immunosuppression. Tumor-infiltrating stromal cells, which include macrophages/microglia, contributed even more strongly to the immunosuppressive signature than the actual tumor cells. The identified murine immune escape signature was reflected in human patients and correlated with poor survival. In conclusion, immune pressure profoundly shapes the clonal composition and gene regulation in malignant gliomas.
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http://dx.doi.org/10.1172/JCI138760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524465PMC
October 2020

High-Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies.

Cytometry A 2020 06 16;97(6):602-609. Epub 2020 May 16.

Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.

Extracellular vesicles (EVs) are released from basically all cells. Over the last decade, small EVs (sEVs; 50-150 nm) have gained enormous attention in diagnostics and therapy. However, methodological limitations coupled to the lack of EV standards leave many questions in this quickly evolving field unresolved. Recently, by using enhanced green fluorescent protein (eGFP)-labeled sEVs as biological reference material, we systematically optimized imaging flow cytometry for single sEV analysis. Furthermore, we showed that sEVs stained with different fluorescent antibodies can be analyzed in a multiparametric manner. However, many parameters potentially affecting the sEV staining procedure still require further evaluation and optimization. Here, we present a concise, systematic evaluation of the impact of the incubation temperature (4°C, room temperature and 37°C) during sEV antibody staining on the outcome of experiments involving the staining of EVs with fluorescence-conjugated antibodies. We provide evidence that both the staining intensity and the sample recovery can vary depending on the incubation temperature applied, and that observed differences are less pronounced following prolonged incubation times. In addition, this study can serve as an application-specific example of parameter evaluation in EV flow cytometry. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.24034DOI Listing
June 2020

FASN Is a Biomarker Enriched in Malignant Glioma-Derived Extracellular Vesicles.

Int J Mol Sci 2020 Mar 12;21(6). Epub 2020 Mar 12.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Extracellular vesicles (EVs) are known for their important role in cancer progression and hold considerable potential as a source for tumor biomarkers. However, purification of tumor-specific EVs from patient plasma is still an urgent unmet need due to contamination by normal host cell-derived EVs, that results in compromised analytical sensitivity. Here we identified fatty acid synthase (FASN), a key lipogenic enzyme which is highly expressed in malignant glioma cells, to be elevated in CD63- and CD81-positive EVs in glioma patient plasma samples, opening vital opportunities to sort brain tumor-specific EVs.
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http://dx.doi.org/10.3390/ijms21061931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139767PMC
March 2020

Molecular profiling of an osseous metastasis in glioblastoma during checkpoint inhibition: potential mechanisms of immune escape.

Acta Neuropathol Commun 2020 03 9;8(1):28. Epub 2020 Mar 9.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany.

Peripheral metastases of glioblastoma (GBM) are very rare despite the ability of GBM cells to pass through the blood-brain barrier and be disseminated through the peripheral blood. Here, we describe a detailed genetic and immunological characterization of a GBM metastasis in the skeleton, which occurred during anti-PD-1 immune checkpoint therapy. We performed whole genome sequencing (WGS) and 850 K methylation profiling of the primary and recurrent intracranial GBM as well as one of the bone metastases. Copy number alterations (CNA) and mutational profiles were compared to known genomic alterations in the TCGA data base. In addition, immunophenotyping of the peripheral blood was performed. The patient who was primarily diagnosed with IDH-wildtype GBM. After the resection of the first recurrence, progressive intracranial re-growth was again detected, and chemotherapy was replaced by PD-1 checkpoint inhibition, which led to a complete intracranial remission. Two months later MR-imaging revealed multiple osseous lesions. Biopsy confirmed the GBM origin of the skeleton metastases. Immunophenotyping reflected the effective activation of a peripheral T-cell response, with, however, increase of regulatory T cells during disease progression. WGS sequencing demonstrated distinct genomic alterations of the GBM metastasis, with gains along chromosomes 3 and 9 and losses along chromosome 4, 10, and 11. Mutational analysis showed mutations in potentially immunologically relevant regions. Additionally, we correlated tumour-infiltrating lymphocyte and microglia presence to the occurrence of circulating tumour cells (CTCs) in a larger cohort and found a decreased infiltration of cytotoxic T cells in patients positive for CTCs. This study exemplifies that the tumour microenvironment may dictate the response to immune checkpoint therapy. In addition, our study highlights the fact that despite an effective control of intracranial GBM, certain tumour clones have the ability to evade the tumour-specific T-cell response and cause progression even outside of the CNS.
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http://dx.doi.org/10.1186/s40478-020-00906-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063778PMC
March 2020

ALCAM contributes to brain metastasis formation in non-small-cell lung cancer through interaction with the vascular endothelium.

Neuro Oncol 2020 07;22(7):955-966

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Brain metastasis (BM) in non-small-cell lung cancer (NSCLC) has a very poor prognosis. Recent studies have demonstrated the importance of cell adhesion molecules in tumor metastasis. The aim of our study was to investigate the role of activated leukocyte cell adhesion molecule (ALCAM) in BM formation in NSCLC.

Methods: Immunohistochemical analysis was performed on 143 NSCLC primary tumors and BM. A correlation between clinicopathological parameters and survival was developed. Biological properties of ALCAM were assessed in vitro by gene ablation using CRISPR/Cas9 technology in the NCI-H460 NSCLC cell line and in vivo by intracranial and intracardial cell injection of NCI-H460 cells in NMRI-Foxn1nu/nu mice.

Results: ALCAM expression was significantly upregulated in NSCLC brain metastasis (P = 0.023) with a de novo expression of ALCAM in 31.2% of BM. Moderate/strong ALCAM expression in both primary NSCLC and brain metastasis was associated with shortened survival. Functional analysis of an ALCAM knock-out (KO) cell line showed a significantly decreased cell adhesion capacity to human brain endothelial cells by 38% (P = 0.045). In vivo studies showed significantly lower tumor cell dissemination in mice injected with ALCAM-KO cells in both mouse models, and both the number and size of BM were significantly diminished in ALCAM depleted tumors.

Conclusions: Our findings suggest that elevated levels of ALCAM expression promote BM formation in NSCLC through increased tumor cell dissemination and interaction with the brain endothelial cells. Therefore, ALCAM could be targeted to reduce the occurrence of BM.

Key Points: 1. ALCAM expression associates with poor prognosis and brain metastasis in NSCLC.2. ALCAM mediates interaction of NSCLC tumor cells with brain vascular endothelium.3. ALCAM might represent a novel preventive target to reduce the occurrence of BM in NSCLC.
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http://dx.doi.org/10.1093/neuonc/noaa028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339886PMC
July 2020

Local Intracerebral Immunomodulation Using Interleukin-Expressing Mesenchymal Stem Cells in Glioblastoma.

Clin Cancer Res 2020 06 27;26(11):2626-2639. Epub 2020 Jan 27.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Purpose: Mesenchymal stem cells (MSCs) show an inherent brain tumor tropism that can be exploited for targeted delivery of therapeutic genes to invasive glioma. We assessed whether a motile MSC-based local immunomodulation is able to overcome the immunosuppressive glioblastoma microenvironment and to induce an antitumor immune response.

Experimental Design: We genetically modified MSCs to coexpress high levels of IL12 and IL7 (MSC, Apceth-301). Therapeutic efficacy was assessed in two immunocompetent orthotopic C57BL/6 glioma models using GL261 and CT2A. Immunomodulatory effects were assessed by multicolor flow cytometry to profile immune activation and exhaustion of tumor-infiltrating immune cells. Diversity of the tumor-specific immune response as analyzed using T-cell receptor sequencing.

Results: Intratumoral administration of MSC induced significant tumor growth inhibition and remission of established intracranial tumors, as demonstrated by MR imaging. Notably, up to 50% of treated mice survived long-term. Rechallenging of survivors confirmed long-lasting tumor immunity. Local treatment with MSC was well tolerated and led to a significant inversion of the CD4/CD8 T-cell ratio with an intricate, predominantly CD8 effector T-cell-mediated antitumor response. T-cell receptor sequencing demonstrated an increased diversity of TILs in MSC-treated mice, indicating a broader tumor-specific immune response with subsequent oligoclonal specification during generation of long-term immunity.

Conclusions: Local MSC-based immunomodulation is able to efficiently alter the immunosuppressive microenvironment in glioblastoma. The long-lasting therapeutic effects warrant a rapid clinical translation of this concept and have led to planning of a phase I/II study of apceth-301 in recurrent glioblastoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0803DOI Listing
June 2020

Immune Characterization in Aneurysmal Subarachnoid Hemorrhage Reveals Distinct Monocytic Activation and Chemokine Patterns.

Transl Stroke Res 2020 12 19;11(6):1348-1361. Epub 2019 Dec 19.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany.

The pathophysiology of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) is incompletely understood. Intrathecal activation of inflammatory immune cells is suspected to play a major role for the induction of DCI. The aim of this study is to identify immune cell subsets and mediators involved in the pathogenesis of DCI. We prospectively collected blood and CSF from 25 patients with aSAH at early and late time points. We performed multicolor flow cytometry of peripheral blood and CSF, analyzing immune cell activation and pro-inflammatory cyto- and chemokines. In addition to the primary immune analysis, we retrospectively analyzed immune cell dynamics in the CSF of all our SAH patients. Our results show an increased monocyte infiltration secondary to aneurysm rupture in patients with DCI. Infiltrating monocytes are defined by a non-classical (CD14 CD16) phenotype at early stages. The infiltration is most likely triggered by the intrathecal immune activation. Here, high levels of pro-inflammatory chemokines, such as CXCL1, CXCL9, CXCL10, and CXCL11, are detected. The intrathecal cellular activation profile of monocytes was defined by upregulation of CD163 and CD86 on monocytes and a presumable later differentiation into antigen-presenting plasmacytoid dendritic cells (pDCs) and hemosiderophages. Peripheral immune activation was reflected by CD69 upregulation on T cells. Analysis of DCI prevalence, Hunt and Hess grade, and clinical outcome correlated with the degree of immune activation. We demonstrate that monocytes and T cells are activated intrathecally after aSAH and mediate a local inflammatory response which is presumably driven by chemokines. Our data shows that the distinct pattern of immune activation correlates with the prevalence of DCI, indicating a pathophysiological connection to the incidence of vasospasm.
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http://dx.doi.org/10.1007/s12975-019-00764-1DOI Listing
December 2020

Immune Characterization in Aneurysmal Subarachnoid Hemorrhage Reveals Distinct Monocytic Activation and Chemokine Patterns.

Transl Stroke Res 2020 12 19;11(6):1348-1361. Epub 2019 Dec 19.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany.

The pathophysiology of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) is incompletely understood. Intrathecal activation of inflammatory immune cells is suspected to play a major role for the induction of DCI. The aim of this study is to identify immune cell subsets and mediators involved in the pathogenesis of DCI. We prospectively collected blood and CSF from 25 patients with aSAH at early and late time points. We performed multicolor flow cytometry of peripheral blood and CSF, analyzing immune cell activation and pro-inflammatory cyto- and chemokines. In addition to the primary immune analysis, we retrospectively analyzed immune cell dynamics in the CSF of all our SAH patients. Our results show an increased monocyte infiltration secondary to aneurysm rupture in patients with DCI. Infiltrating monocytes are defined by a non-classical (CD14 CD16) phenotype at early stages. The infiltration is most likely triggered by the intrathecal immune activation. Here, high levels of pro-inflammatory chemokines, such as CXCL1, CXCL9, CXCL10, and CXCL11, are detected. The intrathecal cellular activation profile of monocytes was defined by upregulation of CD163 and CD86 on monocytes and a presumable later differentiation into antigen-presenting plasmacytoid dendritic cells (pDCs) and hemosiderophages. Peripheral immune activation was reflected by CD69 upregulation on T cells. Analysis of DCI prevalence, Hunt and Hess grade, and clinical outcome correlated with the degree of immune activation. We demonstrate that monocytes and T cells are activated intrathecally after aSAH and mediate a local inflammatory response which is presumably driven by chemokines. Our data shows that the distinct pattern of immune activation correlates with the prevalence of DCI, indicating a pathophysiological connection to the incidence of vasospasm.
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http://dx.doi.org/10.1007/s12975-019-00764-1DOI Listing
December 2020

T cell receptor next-generation sequencing reveals cancer-associated repertoire metrics and reconstitution after chemotherapy in patients with hematological and solid tumors.

Oncoimmunology 2019;8(11):e1644110. Epub 2019 Jul 25.

Department of Internal Medicine IV, Oncology/Hematology, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany.

The dynamics of immunoaging and the onset of immunoparesis in healthy individuals and cancer patients has been controversially discussed. Moreover, the role of chemotherapy on T cell regeneration needs further elucidation in light of novel immunotherapies that have become standard of care for many elderly cancer patients. We used next-generation immunosequencing to study T cell receptor (TCR) repertoire metrics on 346 blood samples from healthy individuals and cancer patients producing a dataset with around 8.8 million TCR reads. This analysis showed that decline of T cell diversity and increase in T cell clonality is a continuous process beginning in healthy individuals over 40 years of age. Untreated patients with both hematological and solid tumors showed blood TCR repertoires with significantly lower diversity and higher clonality as compared to healthy individuals across all decades. Loss in T cell diversity was essentially driven by a loss in richness in aging healthy individuals, while in cancer patients a loss in repertoire evenness was an additional contributing factor. Interestingly, chemotherapy did not impair the regeneration of blood TCR repertoire diversity to pre-treatment age-specific levels. Surprisingly, even patients over the age of 70 years receiving highly T cell toxic therapies reestablished their pre-treatment T cell diversity suggesting rebound thymic activity rather than recovery of T cell counts by peripheral expansion only. Taken together, these data suggest that human TCR repertoire metrics gradually deteriorate in the aging individual, but age-specific TCR metrics are restored after T cell depleting therapy even in elderly cancer patients.
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http://dx.doi.org/10.1080/2162402X.2019.1644110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791461PMC
July 2019

Clonality of circulating tumor cells in breast cancer brain metastasis patients.

Breast Cancer Res 2019 09 3;21(1):101. Epub 2019 Sep 3.

Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: The incidence of brain metastases in breast cancer (BCBM) patients is increasing. These patients have a very poor prognosis, and therefore, identification of blood-based biomarkers, such as circulating tumor cells (CTCs), and understanding the genomic heterogeneity could help to personalize treatment options.

Methods: Both EpCAM-dependent (CellSearch® System) and EpCAM-independent Ficoll-based density centrifugation methods were used to detect CTCs from 57 BCBM patients. DNA from individual CTCs and corresponding primary tumors and brain metastases were analyzed by next-generation sequencing (NGS) in order to evaluate copy number aberrations and single nucleotide variations (SNVs).

Results: CTCs were detected after EpCAM-dependent enrichment in 47.7% of the patients (≥ 5 CTCs/7.5 ml blood in 20.5%). The CTC count was associated with ERBB2 status (p = 0.029) of the primary tumor as well as with the prevalence of bone metastases (p = 0.021). EpCAM-independent enrichment revealed CTCs in 32.6% of the patients, especially among triple-negative breast cancer (TNBC) patients (70.0%). A positive CTC status after enrichment of either method was significantly associated with decreased overall survival time (p < 0.05). Combining the results of both enrichment methods, 63.6% of the patients were classified as CTC positive. In three patients, the matched tumor tissue and single CTCs were analyzed by NGS showing chromosomal aberrations with a high genomic clonality and mutations in pathways potentially important in brain metastasis formation.

Conclusion: The detection of CTCs, regardless of the enrichment method, is of prognostic relevance in BCBM patients and in combination with molecular analysis of CTCs can help defining patients with higher risk of early relapse and suitability for targeted treatment.
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http://dx.doi.org/10.1186/s13058-019-1184-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6720990PMC
September 2019

Immunologic Profiling of Mutational and Transcriptional Subgroups in Pediatric and Adult High-Grade Gliomas.

Cancer Immunol Res 2019 Sep 2;7(9):1401-1411. Epub 2019 Jul 2.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Immunologic treatment strategies are under investigation for high-grade gliomas. Determining relevant immunologic pathways is required for invigorating a tumor-specific immune response. We therefore investigated the immunologic phenotypes within different subgroups of high-grade gliomas, with a focus on rare genetic subgroups of pediatric and adolescent patients to identify potentially targetable mechanisms. We gathered published gene-expression data from 1,135 high-grade glioma patients and applied a machine-learning technique to determine their transcriptional (mesenchymal, classic, neural, and proneural) and mutational [K27, G34, IDH, and wild type (WT)] subtypes. Gene signatures of infiltrating immune cells and functional immune pathways were evaluated in correlation to histologic diagnosis, age, and transcriptional and mutational subgroups. Our analysis identified four distinct microenvironmental signatures of immune cell infiltration (immune 1-4), which can be stratified into vascular, monocytic/stromal, monocytic/T-cell-, and antigen-presenting cell (APC)/natural killer (NK) cell/T-cell-dominated immune clusters. Immune cell expression profiles correlated with transcriptional and mutational subgroups but were independent of age and histologic diagnosis. By including functional pathways and correlating the expression of immunostimulatory and -inhibitory receptor-ligand interactions, we were able to define the immunologic microenvironment and identify possible immunologic subtypes associated with poor prognosis. In addition, comparison of overall survival with the immunologic landscape and with checkpoint molecules revealed correlations within the transcriptional and mutational subgroups, highlighting the potential application of PD-1/PD-L1 checkpoint inhibition in K27-mutated tumors. Our study shows that transcriptional and mutational subgroups are characterized by distinct immunologic tumor microenvironments, demonstrating the immunologic heterogeneity within high-grade gliomas and suggesting an immune-specific stratification for upcoming immunotherapy trials.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0939DOI Listing
September 2019

Cytotoxic T Cells and their Activation Status are Independent Prognostic Markers in Meningiomas.

Clin Cancer Res 2019 09 21;25(17):5260-5270. Epub 2019 Jun 21.

Division of Experimental Neurosurgery, Department of Neurosurgery, University Hospital of Heidelberg, Heidelberg, Germany.

Purpose: Clinically aggressive meningiomas (MGMs) are rare but treatment-resistant tumors in need for more effective therapies. Because tumor-infiltrating T lymphocytes (TILs) are essential for successful immunotherapy, we assessed TIL numbers and their activation status in primary (p-) and recurrent (r-) meningiomas and their impact on survival.

Experimental Design: Presence of TILs was analyzed in 202 clinically well-annotated cases ( = 123 pMGMs and = 79 rMGMs) focusing on higher-grade meningiomas [ = 97 World Health Organization (WHO) °II, = 62 WHO°III]. TILs were quantified by a semiautomated analysis on whole-tissue sections stained by multicolor immunofluorescence for CD3, CD8, FOXP3, and programmed cell death protein 1 (PD-1).

Results: Median T-cell infiltration accounted for 0.59% TILs per total cell count. Although there were no significant WHO°-dependent changes regarding helper (CD3CD8FOXP3) and cytotoxic (CD3CD8FOXP3) TILs in pMGMs, higher number of cytotoxic TILs were associated with an improved progression-free survival (PFS) independent of prognostic confounders. rMGMs were characterized by lower numbers of TILs in general, helper, and cytotoxic TILs. The additional analysis of their activation status revealed that a proportion of PD-1CD8 TILs within the TIL population was significantly decreased with higher WHO grade and in rMGMs. Furthermore, lower proportions of PD-1CD8 TILs were associated with inferior PFS in multivariate analyses, arguing for PD-1 as activation rather than exhaustion marker.

Conclusions: We identified higher numbers of CD3CD8FOXP3 TILs and proportions of PD-1-expressing CD3CD8FOXP3 TILs as novel biomarkers for better survival. These findings might facilitate the selection of patients who may benefit from immunotherapy and argue in favor of an intervention in primary rather than recurrent tumors.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-0389DOI Listing
September 2019

EGFR and HER3 expression in circulating tumor cells and tumor tissue from non-small cell lung cancer patients.

Sci Rep 2019 05 15;9(1):7406. Epub 2019 May 15.

Department of Tumour Biology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.

Although clinically relevant, the detection rates of EpCAM positive CTCs in non-small cell lung cancer (NSCLC) are surprisingly low. To find new clinically informative markers for CTC detection in NSCLC, the expression of EGFR and HER3 was first analyzed in NSCLC tissue (n = 148). A positive EGFR and HER3 staining was observed in 52.3% and 82.7% of the primary tumors, and in 62.7% and 91.2% of brain metastases, respectively. Only 3.0% of the brain metastases samples were negative for both HER3 and EGFR proteins, indicating that the majority of metastases express these ERBB proteins, which were therefore chosen for CTC enrichment using magnetic cell-separation. Enrichment based on either EGFR or HER3 detected CTCs in 37.8% of the patients, while the combination of EGFR/HER3 enrichment with the EpCAM-based CellSearch technique detected a significantly higher number of 66.7% CTC-positive patients (Cohen's kappa = -0.280) which underlines the existence of different CTC subpopulations in NSCLC. The malignant origin of keratin-positive/CD45-negative CTC clusters and single CTCs detected after EGFR/HER3 based enrichment was documented by the detection of NSCLC-associated mutations. In conclusion, EGFR and HER3 expression in metastasized NSCLC patients have considerable value for CTC isolation plus multiple markers can provide a novel liquid biopsy approach.
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http://dx.doi.org/10.1038/s41598-019-43678-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520391PMC
May 2019

Mass Spectrometric Lipid Profiles of Picosecond Infrared Laser-Generated Tissue Aerosols Discriminate Different Brain Tissues.

Lasers Surg Med 2020 03 8;52(3):228-234. Epub 2019 May 8.

Department of Mass Spectrometric Proteomics, Institute for Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.

Background And Objectives: A picosecond infrared laser (PIRL) has recently been demonstrated to cut biological tissue without scar formation based on the minimal destructive action on the surrounding cells. During cutting with PIRL, the irradiated tissue is ablated by a cold vaporization process termed desorption by impulsive vibrational excitation. In the resulting aerosol, all molecules are dissolved in small droplets and even labile biomolecules like proteins remain intact after ablation. It is hypothesized that these properties enable the PIRL in combination with mass spectrometry as an intelligent laser scalpel for guided surgery. In this study, it was tested if PIRL-generated tissue aerosols are applicable for direct analysis with mass spectrometry, and if the acquired mass spectra can be used to discriminate different brain areas.

Materials And Methods: Brain tissues were irradiated with PIRL. The aerosols were collected and directly infused into a mass spectrometer via electrospray ionization without any sample preparation or lipid extraction.

Results: The laser produced clear cuts with no marks of burning. Lipids from five different classes were identified in the mass spectra of all samples. By principal component analysis the different brain areas were clearly distinguishable from each other.

Conclusions: The results demonstrate the potential for real-time analysis of lipids with a PIRL-based laser scalpel, coupled to a mass spectrometer, for the discrimination of tissues during surgeries. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/lsm.23096DOI Listing
March 2020

Identification of KIF11 As a Novel Target in Meningioma.

Cancers (Basel) 2019 Apr 15;11(4). Epub 2019 Apr 15.

Division of Experimental Neurosurgery, Department of Neurosurgery, University of Heidelberg, INF 400, D-69120 Heidelberg, Germany.

Kinesins play an important role in many physiological functions including intracellular vesicle transport and mitosis. The emerging role of kinesins in different cancers led us to investigate the expression and functional role of kinesins in meningioma. Therefore, we re-analyzed our previous microarray dataset of benign, atypical, and anaplastic meningiomas ( = 62) and got evidence for differential expression of five kinesins (KIFC1, KIF4A, KIF11, KIF14 and KIF20A). Further validation in an extended study sample ( = 208) revealed a significant upregulation of these genes in WHO°I to °III meningiomas (WHO°I = 61, WHO°II = 88, and WHO°III = 59), which was most pronounced in clinically more aggressive tumors of the same WHO grade. Immunohistochemical staining confirmed a WHO grade-associated upregulated protein expression in meningioma tissues. Furthermore, high mRNA expression levels of KIFC1, KIF11, KIF14 and KIF20A were associated with shorter progression-free survival. On a functional level, knockdown of kinesins in Ben-Men-1 cells and in the newly established anaplastic meningioma cell line NCH93 resulted in a significantly inhibited tumor cell proliferation upon siRNA-mediated downregulation of KIF11 in both cell lines by up to 95% and 71%, respectively. Taken together, in this study we were able to identify the prognostic and functional role of several kinesin family members of which KIF11 exhibits the most promising properties as a novel prognostic marker and therapeutic target, which may offer new treatment options for aggressive meningiomas.
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http://dx.doi.org/10.3390/cancers11040545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521001PMC
April 2019

Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours.

J Extracell Vesicles 2019 21;8(1):1588555. Epub 2019 Mar 21.

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Cells release heterogeneous nano-sized vesicles either as exosomes, being derived from endosomal compartments, or through budding from the plasma membrane as so-called microvesicles, commonly referred to as extracellular vesicles (EVs). EVs are known for their important roles in mammalian physiology and disease pathogenesis and provide a potential biomarker source in cancer patients. EVs are generally often analysed in bulk using Western blotting or by bead-based flow-cytometry or, with limited parameters, through nanoparticle tracking analysis. Due to their small size, single EV analysis is technically highly challenging. Here we demonstrate imaging flow cytometry (IFCM) to be a robust, multiparametric technique that allows analysis of single EVs and the discrimination of distinct EV subpopulations. We used IFCM to analyse the tetraspanin (CD9, CD63, CD81) surface profiles on EVs from human and murine cell cultures as well as plasma samples. The presence of EV subpopulations with specific tetraspanin profiles suggests that EV-mediated cellular responses are tightly regulated and dependent on cell environment. We further demonstrate that EVs with double positive tetraspanin expression (CD63/CD81) are enriched in cancer cell lines and patient plasma samples. In addition, we used IFCM to detect tumour-specific GFP-labelled EVs in the blood of mice bearing syngeneic intracerebral gliomas, indicating that this technique allows unprecedented disease modelling. In summary, our study highlights the heterogeneous and adaptable nature of EVs according to their marker profile and demonstrates that IFCM facilitates multiparametric phenotyping of EVs not only but also in patient plasma at a single EV level, with the potential for future functional studies and clinically relevant applications. EDTA = ethylenediamine tetraacetic acid.
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http://dx.doi.org/10.1080/20013078.2019.1588555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442086PMC
March 2019

Evolutionary Trajectories of IDH Glioblastomas Reveal a Common Path of Early Tumorigenesis Instigated Years ahead of Initial Diagnosis.

Cancer Cell 2019 04 21;35(4):692-704.e12. Epub 2019 Mar 21.

Division of Molecular Genetics, German Cancer Research Consortium (DKTK), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; Heidelberg Center for Personalized Oncology, DKFZ-HIPO, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Electronic address:

We studied how intratumoral genetic heterogeneity shapes tumor growth and therapy response for isocitrate dehydrogenase (IDH)-wild-type glioblastoma, a rapidly regrowing tumor. We inferred the evolutionary trajectories of matched pairs of primary and relapsed tumors based on deep whole-genome-sequencing data. This analysis suggests both a distant origin of de novo glioblastoma, up to 7 years before diagnosis, and a common path of early tumorigenesis, with one or more of chromosome 7 gain, 9p loss, or 10 loss, at tumor initiation. TERT promoter mutations often occurred later as a prerequisite for rapid growth. In contrast to this common early path, relapsed tumors acquired no stereotypical pattern of mutations and typically regrew from oligoclonal origins, suggesting sparse selective pressure by therapeutic measures.
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http://dx.doi.org/10.1016/j.ccell.2019.02.007DOI Listing
April 2019

Frequency of Circulating Tumor Cells (CTC) in Patients with Brain Metastases: Implications as a Risk Assessment Marker in Oligo-Metastatic Disease.

Cancers (Basel) 2018 Dec 19;10(12). Epub 2018 Dec 19.

Department of Tumor Biology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany.

Forty percent of non-small cell lung cancer (NSCLC) patients develop brain metastases, resulting in a dismal prognosis. However, patients in an oligo-metastatic brain disease setting seem to have better outcomes. Here, we investigate the possibility of using circulating tumor cells (CTCs) as biomarkers to differentiate oligo-metastatic patients for better risk assessment. Using the CellSearch system, few CTCs were detected among NSCLC patients with brain metastases ( = 52, 12.5% ≥ two and 8.9% ≥ five CTC/7.5 mL blood) and especially oligo-metastatic brain patients ( = 34, 5.9%, and 2.9%). Still, thresholds of both ≥ two and ≥ five CTCs were independent prognostic indicators for shorter overall survival time among all of the NSCLC patients ( = 90, two CTC ≥ HR: 1.629, = 0.024, 95% CI: 1.137⁻6.465 and five CTC ≥ HR: 2.846, = 0.0304, CI: 1.104⁻7.339), as well as among patients with brain metastases (two CTC ≥ HR: 4.694, = 0.004, CI: 1.650⁻13.354, and five CTC ≥ HR: 4.963, = 0.003, CI: 1.752⁻14.061). Also, oligo-brain NSCLC metastatic patients with CTCs had a very poor prognosis ( = 0.019). Similarly, in other tumor entities, only 9.6% of patients with brain metastases ( = 52) had detectable CTCs. Our data indicate that although patients with brain metastases more seldom harbor CTCs, they are still predictive for overall survival, and CTCs might be a useful biomarker to identify oligo-metastatic NSCLC patients who might benefit from a more intense therapy.
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http://dx.doi.org/10.3390/cancers10120527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315958PMC
December 2018

The secreted glycolytic enzyme GPI/AMF stimulates glioblastoma cell migration and invasion in an autocrine fashion but can have anti-proliferative effects.

Neuro Oncol 2018 11;20(12):1594-1605

Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: Aerobic glycolysis confers several advantages to tumor cells, including shunting of metabolites into anabolic pathways. In glioblastoma cells, hypoxia induces a flux shift from the pentose phosphate pathway toward glycolysis and a switch from proliferation to migration. The mechanistic link between glycolysis and migration is poorly understood. Since glucose-6-phosphate isomerase (GPI) is identical to the secreted cytokine autocrine motility factor (AMF), we investigated whether GPI/AMF regulates glioblastoma cell invasion.

Methods: The expression and hypoxic regulation of GPI/AMF and its receptor AMFR were analyzed in glioblastoma tissue and cell lines. Functional effects were studied in vitro and in xenograft models.

Results: High GPI/AMF expression in glioblastomas was found to be associated with a worse patient prognosis, and levels were highest in hypoxic pseudopalisades. Hypoxia upregulated both GPI/AMF and AMFR expression as well as GPI/AMF secretion in vitro. GPI/AMF stimulated cell migration in an autocrine fashion, and GPI/AMF expression was upregulated in migratory cells but reduced in rapidly proliferating cells. Knockdown or inhibition of GPI/AMF reduced glioblastoma cell migration but in part stimulated proliferation. In a highly invasive orthotopic glioblastoma model, GPI/AMF knockdown reduced tumor cell invasion but did not prolong survival. In a highly proliferative model, knockdown tumors were even larger and more proliferative than controls; however, perivascular invasion, provoked by simultaneous bevacizumab treatment, was reduced.

Conclusions: GPI/AMF is a potent motogen for glioblastoma cells, explaining in part the association between glycolysis and migration. Targeting GPI/AMF is, however, problematic, since beneficial anti-invasive effects may be outweighed by unintended mitogenic effects.

Key Points: 1.Increased glycolysis is linked with increased cell migration and invasion in glioblastoma cells. 2.The glycolysis enzyme GPI/AMF may serve as a target for antimetabolic and anti-invasive therapy. 3.Despite reducing tumor invasion, GPI/AMF targeting may have unwanted growth stimulatory effects.
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http://dx.doi.org/10.1093/neuonc/noy117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231211PMC
November 2018
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