TY - JOUR A1 - Dirks, Johannes A1 - Haase, Gabriele A1 - Cantaert, Tineke A1 - Frey, Lea A1 - Klaas, Moritz A1 - Rickert, Christian H. A1 - Girschick, Hermann A1 - Meffre, Eric A1 - Morbach, Henner T1 - A novel AICDA splice-site mutation in two siblings with HIGM2 permits somatic hypermutation but abrogates mutational targeting JF - Journal of Clinical Immunology N2 - Hyper-IgM syndrome type 2 (HIGM2) is a B cell intrinsic primary immunodeficiency caused by mutations in AICDA encoding activation-induced cytidine deaminase (AID) which impair immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM). Whereas autosomal-recessive AID-deficiency (AR-AID) affects both CSR and SHM, the autosomal-dominant form (AD-AID) due to C-terminal heterozygous variants completely abolishes CSR but only partially affects SHM. AR-AID patients display enhanced germinal center (GC) reactions and autoimmune manifestations, which are not present in AD-AID, suggesting that SHM but not CSR regulates GC reactions and peripheral B cell tolerance. Herein, we describe two siblings with HIGM2 due to a novel homozygous AICDA mutation (c.428-1G > T) which disrupts the splice acceptor site of exon 4 and results in the sole expression of a truncated AID variant that lacks 10 highly conserved amino acids encoded by exon 4 (AID-ΔE4a). AID-ΔE4a patients suffered from defective CSR and enhanced GC reactions and were therefore indistinguishable from other AR-AID patients. However, the AID-ΔE4a variant only partially affected SHM as observed in AD-AID patients. In addition, AID-ΔE4a but not AD-AID patients revealed impaired targeting of mutational hotspot motives and distorted mutational patterns. Hence, qualitative defects in AID function and altered SHM rather than global decreased SHM activity may account for the disease phenotype in these patients. KW - hyper-IgM syndrome type 2 (HIGM2) KW - AICDA KW - AID-ΔE4a KW - AD-AID KW - mutational targeting KW - somatic hypermutation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324253 VL - 42 IS - 4 ER - TY - JOUR A1 - Bohnert, Simone A1 - Trella, Stefanie A1 - Preiß, Ulrich A1 - Heinsen, Helmut A1 - Bohnert, Michael A1 - Zwirner, Johann A1 - Tremblay, Marie-Ève A1 - Monoranu, Camelia-Maria A1 - Ondruschka, Benjamin T1 - Density of TMEM119-positive microglial cells in postmortem cerebrospinal fluid as a surrogate marker for assessing complex neuropathological processes in the CNS JF - International Journal of Legal Medicine N2 - Routine coronal paraffin-sections through the dorsal frontal and parieto-occipital cortex of a total of sixty cases with divergent causes of death were immunohistochemically (IHC) stained with an antibody against TMEM119. Samples of cerebrospinal fluid (CSF) of the same cases were collected by suboccipital needle-puncture, subjected to centrifugation and processed as cytospin preparations stained with TMEM119. Both, cytospin preparations and sections were subjected to computer-assisted density measurements. The density of microglial TMEM119-positive cortical profiles correlated with that of cytospin results and with the density of TMEM119-positive microglial profiles in the medullary layer. There was no statistically significant correlation between the density of medullary TMEM119-positive profiles and the cytospin data. Cortical microglial cells were primarily encountered in supragranular layers I, II, and IIIa and in infragranular layers V and VI, the region of U-fibers and in circumscribed foci or spread in a diffuse manner and high density over the white matter. We have evidence that cortical microglia directly migrate into CSF without using the glympathic pathway. Microglia in the medullary layer shows a strong affinity to the adventitia of deep vessels in the myelin layer. Selected rapidly fatal cases including myocardial infarcts and drowning let us conclude that microglia in cortex and myelin layer can react rapidly and its reaction and migration is subject to pre-existing external and internal factors. Cytospin preparations proved to be a simple tool to analyze and assess complex changes in the CNS after rapid fatal damage. There is no statistically significant correlation between cytospin and postmortem interval. Therefore, the quantitative analyses of postmortem cytospins obviously reflect the neuropathology of the complete central nervous system. Cytospins provide forensic pathologists a rather simple and easy to perform method for the global assessment of CNS affliction. KW - cerebrospinal fluid KW - forensic neuropathology KW - forensic neurotraumatology KW - immunohistochemistry KW - immunocytochemistry KW - biomarker Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325009 VL - 136 IS - 6 ER - TY - JOUR A1 - Zamò, Alberto A1 - Gerhard-Hartmann, Elena A1 - Ott, German A1 - Anagnostopoulos, Ioannis A1 - Scott, David W. A1 - Rosenwald, Andreas A1 - Rauert-Wunderlich, Hilka T1 - Routine application of the Lymph2Cx assay for the subclassification of aggressive B-cell lymphoma: report of a prospective real-world series JF - Virchows Archiv N2 - The subclassification of diffuse large B-cell lymphoma (DLBCL) into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes has become mandatory in the 2017 update of the WHO classification of lymphoid neoplasms and will continue to be used in the WHO 5\(^{th}\) edition. The RNA-based Lymph2Cx assay has been validated as a reliable surrogate of high-throughput gene expression profiling assays for distinguishing between GCB and ABC DLBCL and provides reliable results from formalin-fixed, paraffin-embedded (FFPE) material. This test has been previously used in clinical trials, but experience from real-world routine application is rare. We routinely applied the Lymph2Cx assay to day-to-day diagnostics on a series of 147 aggressive B-cell lymphoma cases and correlated our results with the immunohistochemical subclassification using the Hans algorithm and fluorescence in situ hybridization findings using break-apart probes for MYC, BCL2, and BCL6. The routine use of the Lymph2Cx assay had a high technical success rate (94.6%) with a low rate of failure due to poor material and/or RNA quality. The Lymph2Cx assay was discordant with the Hans algorithm in 18% (23 of 128 cases). Discordant cases were mainly classified as GCB by the Hans algorithm and as ABC by Lymph2Cx (n = 11, 8.6%). Only 5 cases (3.9%) were classified as non-GCB by the Hans algorithm and as GCB by Lymph2Cx. Additionally, 5.5% of cases (n = 7) were left unclassified by Lymph2Cx, whereas they were defined as GCB (n = 4) or non-GCB (n = 3) by the Hans algorithm. Our data support the routine applicability of the Lymph2Cx assay. KW - diffuse large B-cell lymphoma KW - Hans algorithm KW - Lymph2Cx assay KW - cell of origin Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324686 VL - 481 IS - 6 ER - TY - JOUR A1 - Gschmack, Eva A1 - Monoranu, Camelia-Maria A1 - Marouf, Hecham A1 - Meyer, Sarah A1 - Lessel, Lena A1 - Idris, Raja A1 - Berg, Daniela A1 - Maetzler, Walter A1 - Steigerwald, Frank A1 - Volkmann, Jens A1 - Gerlach, Manfred A1 - Riederer, Peter A1 - Koutsilieri, Eleni A1 - Scheller, Carsten T1 - Plasma autoantibodies to glial fibrillary acidic protein (GFAP) react with brain areas according to Braak staging of Parkinson’s disease JF - Journal of Neural Transmission N2 - Idiopathic Parkinson’s disease (PD) is characterized by a progredient degeneration of the brain, starting at deep subcortical areas such as the dorsal motor nucleus of the glossopharyngeal and vagal nerves (DM) (stage 1), followed by the coeruleus–subcoeruleus complex; (stage 2), the substantia nigra (SN) (stage 3), the anteromedial temporal mesocortex (MC) (stage 4), high-order sensory association areas and prefrontal fields (HC) (stage 5) and finally first-order sensory association areas, premotor areas, as well as primary sensory and motor field (FC) (stage 6). Autoimmunity might play a role in PD pathogenesis. Here we analyzed whether anti-brain autoantibodies differentially recognize different human brain areas and identified autoantigens that correlate with the above-described dissemination of PD pathology in the brain. Brain tissue was obtained from deceased individuals with no history of neurological or psychiatric disease and no neuropathological abnormalities. Tissue homogenates from different brain regions (DM, SN, MC, HC, FC) were subjected to SDS-PAGE and Western blot. Blots were incubated with plasma samples from 30 PD patients and 30 control subjects and stained with anti-IgG antibodies to detect anti-brain autoantibodies. Signals were quantified. Prominent autoantigens were identified by 2D-gel-coupled mass spectrometry sequencing. Anti-brain autoantibodies are frequent and occur both in healthy controls and individuals with PD. Glial fibrillary acidic protein (GFAP) was identified as a prominent autoantigen recognized in all plasma samples. GFAP immunoreactivity was highest in DM areas and lowest in FC areas with no significant differences in anti-GFAP autoantibody titers between healthy controls and individuals with PD. The anti-GFAP autoimmunoreactivity of different brain areas correlates with the dissemination of histopathological neurodegeneration in PD. We hypothesize that GFAP autoantibodies are physiological but might be involved as a cofactor in PD pathogenesis secondary to a leakage of the blood–brain barrier. KW - Parkinson KW - GFAP KW - autoantibodies KW - Braak Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325161 VL - 129 IS - 5-6 ER - TY - THES A1 - Haßler, Markus Sebastian T1 - NFATc3 in der akuten GvHD T1 - NFATc3 in acute GvHD N2 - Bei Leukämien, Lymphomen und dem Multiplen Myelom stellt die allogene hämatopoetische Stammzelltransplantation (allo-HCT) oft die letzte kurative Therapieoption dar. Spender-T-Zellen (v.a. CD8+-T-Zellen), die im Transplantat enthalten sind, erkennen nach Chemo-/Strahlentherapie verbliebene Reste des entarteten Empfängergewebes, eradizieren dieses und verhindern somit ein Tumorrezidiv (Graft-versus-Leukämie Reaktion/GvL). Häufig attackieren Spender-T-Zellen (v.a. CD4+-Th1-Zellen) aber auch nicht-malignes Gewebe (z.B. Haut, Leber und Darm), was bis zum Tod des Patienten führen kann (Graft-versus-Host Disease/GvHD). Calcineurin-Inhibitoren wie Cyclosporin A (CsA) und Tacrolimus, die oft schon prophylaktisch verabreicht werden, verhindern über eine unselektive Inhibition aller Mitglieder der NFAT-Transkriptionsfaktorfamilie (Nuclear factor of activated T-cells) die Aktivierung der Spender-T-Zellen. Es folgt eine klinische Besserung der GvHD-Symptomatik, während jedoch der GvL-Effekt ebenfalls supprimiert wird. Bisherige Untersuchungen unserer Arbeitsgruppe am Mausmodell hatten gezeigt, dass die selektive Inhibition eines NFAT-Familienmitgliedes (NFATc1 oder NFATc2) in den Donor-T-Zellen zu einer signifikanten Besserung der aGvHD bei jedoch erhaltener GvL führt. Es wurde nun der Einfluss des dritten, in Lymphozyten exprimierten NFAT-Mitglieds NFATc3 im Kontext der aGvHD untersucht. Zur Basisanalyse der neu kreierten Nfatc3fl/fl.Cd4cre- und Nfatc1fl/fl.Nfatc3fl/fl.Cd4cre-Mauslinien erfolgten durchflusszytometrische und Western-Blot-Analysen. Anschließend wurden In-vivo-Untersuchungen unter Verwendung eines etablierten major-mismatch-aGvHD-Modells (H-2b→H-2d) durchgeführt. Es konnte gezeigt werden, dass durch eine NFATc3- (+/- NFATc1-) Defizienz direkt ex vivo die CD4+/CD8+-Ratio durch Abnahme der CD4+- hin zu den CD8+-T-Zellen verschoben wird. Auch zeigte sich in den entsprechenden Genotypen eine Abnahme der naiven- und dafür vice versa eine Zunahme der Effektor-T-Zellen. In den wiederholt durchgeführten aGvHD-Versuchen zeigte sich in vivo als Korrelat der (ebenfalls erneut nachgewiesenen) Abnahme des CD4+/CD8+-Quotienten in den Zielorganen eine geringere Expansion der NFAT-defizienten als der wildtypischen T-Zellen. Leider spiegelte sich dies nicht in dem clinical score zur Quantifizierung der aGvHD-Symptomatik wider. Auch das Körpergewicht der Versuchsgruppe nahm rapide ab. Ursächlich hierfür ist – als Korrelat zur direkt ex vivo nachgewiesenen Aktivierungsneigung – ein vermehrter Th1-Shift der NFATc3 (+/-NFATc1-) defizienten T-Zellen. Eine Inhibierung von NFATc3 – im Gegensatz zu NFATc1 und NFATc2 – ist demzufolge kein sinnvoller Ansatzpunkt für eine mögliche, zielgerichtetere aGvHD-Therapie. Der positive Effekt der reduzierten Proliferationsneigung der NFATc3-defizienten Lymphozyten wird durch deren vermehrte Aktivierungsneigung mit erhöhter Sekretion von pro-inflammatorischen Zytokinen zunichte gemacht. N2 - In malignant diseases such as multiple myeloma, leukemia and lymphoma the allogenic hematopoietic stem cell transplantation (allo-HCT) often represents the final curative treatment option. Donor T cells (esp. CD8+ T cells) within the graft recognize and eradicate tumor cells which have remained after chemo- and radiotherapy. This graft-versus-leukemia (GvL) effect can prevent tumor relapses. However, donor T cells (esp. CD4+ Th1 cells) often attack non-malignant tissue (e.g. skin, liver, colon) with potentially life-threatening consequences for the host. (Graft-versus-host disease = GvHD). To prevent the development of aGvHD, calcineurin-inhibitors (CNI) like cyclosporin A (CsA) and tacrolimus are often administered prophylactically. By means of an unselective suppression of the nuclear factor of activated T cells (NFAT) transcription factors, both drugs inhibit the activation of donor T cells. While leading to a clinical improvement of the GvHD-symptoms, coevally, the GvL effect is also suppressed. Previous research of our study group showed that a selective inhibition of one NFAT family member (NFATc1 oder NFATc2) in donor T cells leads to a significant decline of aGvHD symptoms while maintaining GvL. We have now analysed the iκluence of NFATc3, the third NFAT member expressed in lymphocytes, in context of aGvHD. Initially we analysed the new created Nfatc3fl/fl.Cd4cre- and Nfatc1fl/fl.Nfatc3fl/fl.Cd4cre-mouse strains by western blot and flow cytometry. Subsequently, these were followed by in vivo studies, using an already established major-mismatch-aGvHD-model (H-2b→H-2d). It could be shown that directly ex vivo a NFATc3- (+/- NFATc1-) deficiency leads to a reduction in the CD4+/CD8+ ratio. This is mainly caused by a diminution of CD4+ T cell population, while the CD8+ population remains unaffected. Furthermore, a lower number of naive but an increased number of effector T cells has been observed. This effect (which was also present in the aGvHD-experiments) correlated in vivo with a decreased expansion of NFAT-deficient T cells – compared to wild type T cells – in target organs. Unfortunately, the expected clinical improvement could not be demonstrated. The clinical score which objectifies the aGvHD-symptoms, as well as the body weight of the mice in the experimental group declined rapidly, comparable with or even worse than in the control group due to an increased Th1-shift of the NFATc3- (+/- NFATc1) deficient T cells. This also correlates with the increased effector function which has been observed in the previous ex vivo experiments. In conclusion an inhibition of NFATc3 – in contrast to NFATc1 or NFATc2 – is not a useful target point for a more specific aGvHD-therapy. The positive effect of a reduced proliferation in NFATc3-deficient lymphocytes is over-compensated by their augmented activation. KW - Transplantat-Wirt-Reaktion KW - Gvhd KW - Transplantatabstoßung KW - NFAT KW - NFATc3 KW - GvL KW - Stammzelltransplantation KW - allogenic stem cell transplantation Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323681 ER - TY - THES A1 - Jaklin, Tamara T1 - Nachweis von PD-1 und PD-L1 in Plattenepithelkarzinomen des Larynx und Hypopharynx T1 - Detection of PD-1 and PD-L1 in squamous cell carcinomas of the larynx and hypopharynx N2 - In der modernen Tumortherapie sind Checkpoint-Inhibitoren ein fester Bestandteil. Die Oberflächenproteine PD-L1 und PD-1 stellen die Angriffspunkte dieser spezifischen Therapie dar. Die Datenlage hinsichtlich PD-L1 in HNSCC ist sehr heterogen. Diese Arbeit beschäftigte sich daher mit der Expression von PD-L1 und PD-1 in einem Kollektiv von 118 Plattenepithelkarzinomen in Larynx und Hypopharynx und einer prognostischen Aussagekraft hinsichtlich mehrerer histopathologischer und epidemiologischer Faktoren. Außerdem wurde ein möglicher Zusammenhang zwischen der Expression von PD-L1, PD-1 und CD5 als T-Zell-Marker in besagtem Kollektiv untersucht. Die IHC-Färbungen wurden lichtmikroskopisch an tissue micro arrays untersucht. Für die Auswertung von PD-L1 wurde der bereits etablierte Cologne-Score verwendet, welcher zum einen zunächst erweitert und anschließend zwecks einer fundierten statistischen Auswertung ergänzend modifiziert wurde. Für CD5 und PD-1 wurden eigene Cut-Off-Werte generiert. 48 % der Fälle waren PD-L1+, die Spannweite im Literaturvergleich schwankt zwischen 30 – 90 %. PD-1+ waren insgesamt 31% der Fälle, auch hier zeigen sich deutliche Abweichungen zu den vorliegenden Publikationen. Hinsichtlich der prognostischen Aussagekraft konnte ein signifikanter Zusammenhang zwischen dem T-Stadium und der PD-L1-Expression aufgezeigt werden. Ob dies Einfluss auf mögliche Behandlungsstrategien hat, bleibt Gegenstand weiterer Forschung. Auch im Literaturvergleich finden sich wiederholt signifikante prognostische Zusammenhänge, jedoch beziehen sich diese auf differente Faktoren. Ursächlich dafür sind aller Wahrscheinlichkeit nach Diskrepanzen in der PD-L1-Expression sowie deren Schwankungen durch äußere Einflüsse und nicht standardisierte Testverfahren. Es zeigten sich weiterhin Korrelationen zwischen den Markern, welche sich abschließend nicht alle gänzlich herleiten lassen. Zusammenfassend könnten einheitliche Testverfahren die Datenlage zu PD-L1 und PD-1 homogenisieren, auch mögliche Vortherapien sollten dementsprechend berücksichtigt werden. Allerdings erscheint die prognostische Aussagekraft von PD-L1 und auch von PD-1 insgesamt aufgrund der inkonstanten Expression hochgradig eingeschränkt, sodass sich in Zukunft vermehrt auf andere Marker konzentriert werden sollte. N2 - Checkpoint inhibitors are an essential part of modern tumor therapy. The surface proteins PD-L1 and PD-1 are the targets of this specific therapy. But the data situation regarding PD-L1 in HNSCC is very heterogeneous. This study therefore dealt with the expression of PD-L1 and PD-1 in a collective of 118 squamous cell carcinomas in the larynx and hypopharynx and a prognostic significance with regard to several histopathological and epidemiological factors. Furthermore, a possible link between the expression of PD-L1, PD-1 and CD5 as a T cell marker has been studied. The IHC stains were examined by light microscopy on tissue micro arrays. The already established Cologne score was used for the evaluation of PD-L1. Initially this score was expanded and additionally has been modified for a substantiated statistical analysis. Separate cut-off values were generated for CD5 and PD-1. 48 % of the cases were PD-L1+, literature shows a range between 30 – 90 %. PD-1+ were a total of 31% of the cases, there were distinctly discrepancies from the existing publications too. With regard to the prognostic significance, a significant correlation between the T-stage and PD-L1 expression could be shown. Whether this has an impact on possible treatment strategies remains the subject of further research. Significant prognostic correlations are also repeatedly found in the literature comparison, but these refer to different factors. In all likelihood, this is due to discrepancies in PD-L1 expression and its fluctuations due to external influences and non-standardized test procedures. There were also correlations between the used markers, not all of them could be fully explained. In summary, common test procedures could homogenize the data on PD-L1 and PD-1, furthermore possible previous therapies should also be considered. However, the prognostic significance of PD-L1 and also of PD-1 is highly limited due to the inconstant expression, so that in the future more focus should be placed on other markers. KW - Plattenepithelkarzinom KW - Hals-Nasen-Ohren-Heilkunde KW - Pathologie KW - Immun-Checkpoint KW - Larynxkarzinom KW - Hypopharynxkarzinom KW - Checkpoint-Inhibitor KW - PD-L1 KW - PD-1 Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313019 ER - TY - THES A1 - Marquardt, André T1 - Machine-Learning-Based Identification of Tumor Entities, Tumor Subgroups, and Therapy Options T1 - Bestimmung von Tumorentitäten, Tumorsubgruppen und Therapieoptionen basierend auf maschinellem Lernen N2 - Molecular genetic analyses, such as mutation analyses, are becoming increasingly important in the tumor field, especially in the context of therapy stratification. The identification of the underlying tumor entity is crucial, but can sometimes be difficult, for example in the case of metastases or the so-called Cancer of Unknown Primary (CUP) syndrome. In recent years, methylome and transcriptome utilizing machine learning (ML) approaches have been developed to enable fast and reliable tumor and tumor subtype identification. However, so far only methylome analysis have become widely used in routine diagnostics. The present work addresses the utility of publicly available RNA-sequencing data to determine the underlying tumor entity, possible subgroups, and potential therapy options. Identification of these by ML - in particular random forest (RF) models - was the first task. The results with test accuracies of up to 99% provided new, previously unknown insights into the trained models and the corresponding entity prediction. Reducing the input data to the top 100 mRNA transcripts resulted in a minimal loss of prediction quality and could potentially enable application in clinical or real-world settings. By introducing the ratios of these top 100 genes to each other as a new database for RF models, a novel method was developed enabling the use of trained RF models on data from other sources. Further analysis of the transcriptomic differences of metastatic samples by visual clustering showed that there were no differences specific for the site of metastasis. Similarly, no distinct clusters were detectable when investigating primary tumors and metastases of cutaneous skin melanoma (SKCM). Subsequently, more than half of the validation datasets had a prediction accuracy of at least 80%, with many datasets even achieving a prediction accuracy of – or close to – 100%. To investigate the applicability of the used methods for subgroup identification, the TCGA-KIPAN dataset, consisting of the three major kidney cancer subgroups, was used. The results revealed a new, previously unknown subgroup consisting of all histopathological groups with clinically relevant characteristics, such as significantly different survival. Based on significant differences in gene expression, potential therapeutic options of the identified subgroup could be proposed. Concludingly, in exploring the potential applicability of RNA-sequencing data as a basis for therapy prediction, it was shown that this type of data is suitable to predict entities as well as subgroups with high accuracy. Clinical relevance was also demonstrated for a novel subgroup in renal cell carcinoma. The reduction of the number of genes required for entity prediction to 100 genes, enables panel sequencing and thus demonstrates potential applicability in a real-life setting. N2 - Molekulargenetische Analysen, wie z. B. Mutationsanalysen, gewinnen im Tumorbereich zunehmend an Bedeutung, insbesondere im Zusammenhang mit der Therapiestratifizierung. Die Identifizierung der zugrundeliegenden Tumorentität ist von entscheidender Bedeutung, kann sich aber manchmal als schwierig erweisen, beispielsweise im Falle von Metastasen oder dem sogenannten Cancer of Unknown Primary (CUP)-Syndrom. In den letzten Jahren wurden Methylom- und Transkriptom-Ansätze mit Hilfe des maschinellen Lernens (ML) entwickelt, die eine schnelle und zuverlässige Identifizierung von Tumoren und Tumorsubtypen ermöglichen. Bislang werden jedoch nur Methylomanalysen in der Routinediagnostik eingesetzt. Die vorliegende Arbeit befasst sich mit dem Nutzen öffentlich zugänglicher RNA-Sequenzierungsdaten zur Bestimmung der zugrunde liegenden Tumorentität, möglicher Untergruppen und potenzieller Therapieoptionen. Die Identifizierung dieser durch ML - insbesondere Random-Forest (RF)-Modelle - war die erste Aufgabe. Die Ergebnisse mit Testgenauigkeiten von bis zu 99 % lieferten neue, bisher unbekannte Erkenntnisse über die trainierten Modelle und die entsprechende Entitätsvorhersage. Die Reduktion der Eingabedaten auf die 100 wichtigsten mRNA-Transkripte führte zu einem minimalen Verlust an Vorhersagequalität und könnte eine Anwendung in klinischen oder realen Umgebungen ermöglichen. Durch die Einführung des Verhältnisses dieser Top 100 Gene zueinander als neue Datenbasis für RF-Modelle wurde eine neuartige Methode entwickelt, die die Verwendung trainierter RF-Modelle auf Daten aus anderen Quellen ermöglicht. Eine weitere Analyse der transkriptomischen Unterschiede von metastatischen Proben durch visuelles Clustering zeigte, dass es keine für den Ort der Metastasierung spezifischen Unterschiede gab. Auch bei der Untersuchung von Primärtumoren und Metastasen des kutanen Hautmelanoms (SKCM) konnten keine unterschiedlichen Cluster festgestellt werden. Mehr als die Hälfte der Validierungsdatensätze wiesen eine Vorhersagegenauigkeit von mindestens 80% auf, wobei viele Datensätze sogar eine Vorhersagegenauigkeit von 100% oder nahezu 100% erreichten. Um die Anwendbarkeit der verwendeten Methoden zur Identifizierung von Untergruppen zu untersuchen, wurde der TCGA-KIPAN-Datensatz verwendet, welcher die drei wichtigsten Nierenkrebs-Untergruppen umfasst. Die Ergebnisse enthüllten eine neue, bisher unbekannte Untergruppe, die aus allen histopathologischen Gruppen mit klinisch relevanten Merkmalen, wie z. B. einer signifikant unterschiedlichen Überlebenszeit, besteht. Auf der Grundlage signifikanter Unterschiede in der Genexpression konnten potenzielle therapeutische Optionen für die identifizierte Untergruppe vorgeschlagen werden. Zusammenfassend lässt sich sagen, dass bei der Untersuchung der potenziellen Anwendbarkeit von RNA-Sequenzierungsdaten als Grundlage für die Therapievorhersage gezeigt werden konnte, dass diese Art von Daten geeignet ist, sowohl Entitäten als auch Untergruppen mit hoher Genauigkeit vorherzusagen. Die klinische Relevanz wurde auch für eine neue Untergruppe beim Nierenzellkarzinom demonstriert. Die Verringerung der für die Entitätsvorhersage erforderlichen Anzahl von Genen auf 100 Gene ermöglicht die Sequenzierung von Panels und zeigt somit die potenzielle Anwendbarkeit in der Praxis. KW - Maschinelles Lernen KW - Krebs KW - Tumor KW - Sequenzdaten KW - Random Forest KW - Vorhersage KW - RNA-Sequenzierung KW - Prognose Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-329548 ER - TY - JOUR A1 - Stock, Benjamin A1 - Möckel, Sigrid A1 - Zander, Christine A1 - Heinsen, Helmut A1 - Bohnert, Simone A1 - Bohnert, Michael T1 - „Black esophagus“ – zwei Obduktionsfälle mit infektiöser Beteiligung JF - Rechtsmedizin N2 - „Black esophagus“ oder „akute Ösophagusnekrose“ (AÖN) ist eine seltene Erkrankung, die sich makroskopisch durch eine zirkumferente Schwarzverfärbung der Ösophagusmukosa mit abruptem Ende am gastroösophagealen Übergang auszeichnet. Die genaue Pathogenese ist unbekannt; es werden multifaktorielle Einflüsse wie z. B. Säurereflux, Ischämie und verringerte Schutzmechanismen der Mukosa als mögliche Ursachen diskutiert. Vorgestellt werden 2 Obduktionsfälle, die typische Befunde einer AÖN aufwiesen. Zusätzlich hatten Fall 1 eine Candida-Infektion und Fall 2 eine Appendizitis, sodass eine infektiöse Genese in beiden Fällen eine Rolle gespielt haben könnte. N2 - Black esophagus, also known as acute esophageal necrosis, is a rare disease characterized by a circumferential black discoloration of the esophageal mucosa with an abrupt stop at the gastroesophageal junction. The exact pathogenesis is unknown, but multifactorial influences, such as acid reflux, ischemia and reduced protective mechanisms of the mucosa are discussed as possible causes. Two autopsy cases are presented with typical signs of a black esophagus. The first case showed an infection with Candida albicans, the second one died of appendicitis, so in both cases an infectious genesis might have played a role. T2 - Black esophagus—Two autopsy cases with infectious involvement KW - Histopathologie KW - Blinddarmentzündung KW - Speiseröhre KW - Nekrose KW - Schleimhaut KW - Candida KW - Akute Ösophagusnekrose KW - Schleimhaut-Ulzera KW - Appendizitis KW - acute esophageal necrosis KW - histopathology KW - mucosal ulcers KW - appendicitis KW - Candida Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-325022 SN - 0937-9819 VL - 33 IS - 3 ER - TY - JOUR A1 - Löb, Sanja A1 - Linsmeier, Eva A1 - Herbert, Saskia-Laureen A1 - Schlaiß, Tanja A1 - Kiesel, Matthias A1 - Wischhusen, Jörg A1 - Salmen, Jessica A1 - Kranke, Peter A1 - Quenzer, Anne A1 - Kurz, Florian A1 - Weiss, Claire A1 - Gerhard-Hartmann, Elena A1 - Wöckel, Achim A1 - Diessner, Joachim T1 - Prognostic effect of HER2 evolution from primary breast cancer to breast cancer metastases JF - Journal of Cancer Research and Clinical Oncology N2 - Purpose Therapeutic options for breast cancer (BC) treatment are constantly evolving. The Human Epidermal Growth Factor 2 (HER2)-low BC entity is a new subgroup, representing about 55% of all BC patients. New antibody–drug conjugates demonstrated promising results for this BC subgroup. Currently, there is limited information about the conversion of HER2 subtypes between primary tumor and recurrent disease. Methods This retrospective study included women with BC at the University Medical Centre Wuerzburg from 1998 to 2021. Data were retrieved from patients' records. HER2 evolution from primary diagnosis to the first relapse and the development of secondary metastases was investigated. Results In the HR-positive subgroup without HER2 overexpression, HER2-low expression in primary BC was 56.7 vs. 14.6% in the triple-negative subgroup (p < 0.000). In the cohort of the first relapse, HER2-low represented 64.1% of HR-positive vs. 48.2% of the triple-negative cohort (p = 0.03). In patients with secondary metastases, HER2-low was 75.6% vs. 50% in the triple negative subgroup (p = 0.10). The subgroup of HER2-positive breast cancer patients numerically increased in the course of disease; the HER2-negative overall cohort decreased. A loss of HER2 expression from primary BC to the first relapse correlated with a better OS (p = 0.018). No clinicopathological or therapeutic features could be identified as potential risk factors for HER2 conversion. Conclusion HER2 expression is rising during the progression of BC disease. In view of upcoming therapeutical options, the re-analysis of newly developed metastasis will become increasingly important. KW - breast cancer KW - HER2 conversion KW - HER2-low KW - trastuzumab deruxtecan KW - HER2 targeted therapy KW - trastuzumab Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324068 VL - 149 IS - 8 ER - TY - THES A1 - Maier, Claudia T1 - Untersuchungen zu neuen potenziellen N-Glykosylierungsmotiven in t(14;18)-positiven und t(14;18)-negativen follikulären Lymphomen T1 - Investigations about novel potential N-glycosylation sites in t(14;18)-positive and t(14;18)-negative follicular lymphoma N2 - In der vorliegenden Arbeit wurde das Vorkommen neu erworbener N-Glykosylierungsmotive in t(14;18)-positiven und -negativen FL der lokalisierten (FL I/II) und fortgeschrittenen Stadien (FL III/IV), sowie zum Zeitpunkt der Primärdiagnose und des Rezidivs untersucht. Dabei wurde der jeweilige Haupttumorklon mit Hilfe von „Next Generation Sequencing“ und unter Verwendung des „LymphoTrack® Assays“ in einer Serie von 68 kryoasservierten FL identifiziert 36 t(14;18)-negative und 32 t(14;18)-positive FL. Die Frequenz neu erworbener N-Glykosylierungsmotive unterschied sich signifikant zwischen t(14;18)-positiven und -negativen PD/R-FL III/IV, während man zwischen t(14;18)-positiven und -negativen PD/R-FL I/II keinen Unterschied beobachten konnte. Des Weiteren zeigten t(14;18)-negative PD/R-FL I-IV im Vergleich zu t(14;18)-positiven PD/R-FL I-IV signifikant häufiger einen Zugewinn neuer N-Glykosylierungsmotive in der FR3 Region des BCL2 Gens, sowie eine vermehrte Nutzung des IGHV4-34 Keimbahngens. Interessanterweise beschränkte sich die Nutzung des IGHV4-34 Gens auf PD-FL und konnte in R-FL nicht nachgewiesen werden. Da sowohl das Vorkommen neu erworbener N-Glykosylierungsmotive in FR3 als auch die Nutzung von IGHV4-34 im Zusammenhang mit Autoimmunerkrankungen beschrieben wurden, deuten unsere Ergebnisse darauf hin, dass die Subgruppe der t(14;18)-negativen FL im pathologischen Prozess der Onkogenese mehr auf die Stimulation durch (Auto)-Antigene als durch die Stimulation des B-Zell Rezeptors mit Lektinen (DC-SIGN) angewiesen sein könnte. N2 - This study investigated the occurrence of newly acquired n-glycosylation motifs (NANGS) in a cohort of t(14;18)-positive and t(14;18)-negative FL, including early (I/II) and advanced (III/IV) stage treatment-naive and relapsed tumors. The clonotype was determined by using a next generation sequencing approach in a series of 68 FL with fresh frozen material [36 t(14;18) positive and 32 t(14;18) negative]. The frequency of NANGS differed considerably between t(14;18)-positive and t(14;18)-negative FL stage III/IV, but no difference was observed among t(14;18)-positive and t(14;18)-negative FL stage I/II. The introduction of NANGS in all t(14;18)- negative clinical subgroups occurred significantly more often in the FR3 region. Moreover, t(14;18)-negative treatment-naive FL, specifically those with NANGS, showed a strong bias for IGHV4-34 usage compared with t(14;18)-positive treatment-naive cases with NANGS; IGHV4-34 usage was never recorded in relapsed FL. In conclusion, subgroups of t(14;18)- negative FL might use different mechanisms of B-cell receptor stimulation compared with the lectin-mediated binding described in t(14;18)-positive FL, including responsiveness to autoantigens as indicated by biased IGHV4-34 usage and strong NANGS enrichment in FR3. KW - Glykosylierung KW - Non-Hodgkin-Lymphom KW - B-Zell-Lymphom KW - N-Glykosylierungsmotive KW - N-glycosylation sites KW - Follikuläres Lymphom KW - follicular lymphoma KW - t(14;18)-negative follikuläre Lymphome KW - t(14;18)-negative follicular lymphoma Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-330261 ER - TY - JOUR A1 - Schulmeyer, Carla E. A1 - Fasching, Peter A. A1 - Häberle, Lothar A1 - Meyer, Julia A1 - Schneider, Michael A1 - Wachter, David A1 - Ruebner, Matthias A1 - Pöschke, Patrik A1 - Beckmann, Matthias W. A1 - Hartmann, Arndt A1 - Erber, Ramona A1 - Gass, Paul T1 - Expression of the immunohistochemical markers CK5, CD117, and EGFR in molecular subtypes of breast cancer correlated with prognosis JF - Diagnostics N2 - Molecular-based subclassifications of breast cancer are important for identifying treatment options and stratifying the prognosis in breast cancer. This study aimed to assess the prognosis relative to disease-free survival (DFS) and overall survival (OS) in patients with triple-negative breast cancer (TNBC) and other subtypes, using a biomarker panel including cytokeratin 5 (CK5), cluster of differentiation 117 (CD117), and epidermal growth factor receptor (EGFR). This cohort–case study included histologically confirmed breast carcinomas as cohort arm. From a total of 894 patients, 572 patients with early breast cancer, sufficient clinical data, and archived tumor tissue were included. Using the immunohistochemical markers CK5, CD117, and EGFR, two subgroups were formed: one with all three biomarkers negative (TBN) and one with at least one of those three biomarkers positive (non-TBN). There were significant differences between the two biomarker subgroups (TBN versus non-TBN) in TNBC for DFS (p = 0.04) and OS (p = 0.02), with higher survival rates (DFS and OS) in the non-TBN subgroup. In this study, we found the non-TBN subgroup of TNBC lesions with at least one positive biomarker of CK5, CD117, and/or EGFR, to be associated with longer DFS and OS. KW - early breast cancer KW - therapy KW - prognosis KW - CK5 KW - CD117 KW - EGFR KW - triple-negative breast cancer Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304987 SN - 2075-4418 VL - 13 IS - 3 ER - TY - JOUR A1 - Stefanakis, Mona A1 - Bassler, Miriam C. A1 - Walczuch, Tobias R. A1 - Gerhard-Hartmann, Elena A1 - Youssef, Almoatazbellah A1 - Scherzad, Agmal A1 - Stöth, Manuel Bernd A1 - Ostertag, Edwin A1 - Hagen, Rudolf A1 - Steinke, Maria R. A1 - Hackenberg, Stephan A1 - Brecht, Marc A1 - Meyer, Till Jasper T1 - The impact of tissue preparation on salivary gland tumors investigated by Fourier-transform infrared microspectroscopy JF - Journal of Clinical Medicine N2 - Due to the wide variety of benign and malignant salivary gland tumors, classification and malignant behavior determination based on histomorphological criteria can be difficult and sometimes impossible. Spectroscopical procedures can acquire molecular biological information without destroying the tissue within the measurement processes. Since several tissue preparation procedures exist, our study investigated the impact of these preparations on the chemical composition of healthy and tumorous salivary gland tissue by Fourier-transform infrared (FTIR) microspectroscopy. Sequential tissue cross-sections were prepared from native, formalin-fixed and formalin-fixed paraffin-embedded (FFPE) tissue and analyzed. The FFPE cross-sections were dewaxed and remeasured. By using principal component analysis (PCA) combined with a discriminant analysis (DA), robust models for the distinction of sample preparations were built individually for each parotid tissue type. As a result, the PCA-DA model evaluation showed a high similarity between native and formalin-fixed tissues based on their chemical composition. Thus, formalin-fixed tissues are highly representative of the native samples and facilitate a transfer from scientific laboratory analysis into the clinical routine due to their robust nature. Furthermore, the dewaxing of the cross-sections entails the loss of molecular information. Our study successfully demonstrated how FTIR microspectroscopy can be used as a powerful tool within existing clinical workflows. KW - formalin KW - fixation KW - tissue preparation KW - salivary gland neoplasia KW - FTIR spectroscopy KW - principal component analysis KW - discriminant analysis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304887 SN - 2077-0383 VL - 12 IS - 2 ER - TY - THES A1 - Majumder, Snigdha T1 - Selective inhibition of NFAT in mouse and human T cells by CRISPR/Cas9 to ameliorate acute Graft-versus-Host Disease while preserving Graft-versus-Leukemia effect T1 - Selektive Hemmung von NFAT in murinen und humanen T-Zellen durch CRISPR/Cas9 zur Linderung der akuten Graft-versus-Host-Erkrankung bei gleichzeitigem Erhalt des Graft-versus-Leukemia-Effekts N2 - Allogenic hematopoietic stem cell transplantation (allo-HCT) is a curative therapy for the treatment of malignant and non-malignant bone marrow diseases. The major complication of this treatment is a highly inflammatory reaction known as Graft-versus-Host Disease (GvHD). Cyclosporin A (CsA) and tacrolimus are used to treat GvHD which limits inflammation but also interferes with the anticipated Graft-versus-Leukemia (GvL) effect. These drugs repress conventional T cells (Tcon) along with regulatory T cells (Treg), which are important for both limiting GvHD and supporting GvL. Both of these drugs inhibit calcineurin (CN), which dephosphorylates and activates the nuclear factor of activated T-cells (NFAT) family of transcription factors. Here, we make use of our Cd4cre.Cas9+ mice and developed a highly efficient non-viral CRISPR/Cas9 gene editing method by gRNA-only nucleofection. Utilizing this technique, we demonstrated that unstimulated mouse T cells upon NFATc1 or NFATc2 ablation ameliorated GvHD in a major mismatch mouse model. However, in vitro pre-stimulated mouse T cells could not achieve long-term protection from GvHD upon NFAT single-deficiency. This highlights the necessity of gene editing and transferring unstimulated human T cells during allo-HCT. Indeed, we established a highly efficient ribonucleoprotein (RNP)-mediated CRISPR/Cas9 gene editing for NFATC1 and/or NFATC2 in pre-stimulated as well as unstimulated primary human T cells. In contrast to mouse T cells, not NFATC1 but NFATC2 deficiency in human T cells predominantly affected proinflammatory cytokine production. However, either NFAT single-knockout kept cytotoxicity of human CD3+ T cells untouched against tumor cells in vitro. Furthermore, mouse and human Treg were unaffected upon the loss of a single NFAT member. Lastly, NFATC1 or NFATC2-deficient anti-CD19 CAR T cells, generated with our non-viral ‘one-step nucleofection’ method validated our observations in mouse and human T cells. Proinflammatory cytokine production was majorly dependent on NFATC2 expression, whereas, in vitro cytotoxicity against CD19+ tumor cells was undisturbed in the absence of either of the NFAT members. Our findings emphasize that NFAT single-deficiency in donor T cells is superior to CN-inhibitors as therapy during allo-HCT to prevent GvHD while preserving GvL in patients. N2 - Die allogene hämatopoetische Stammzelltransplantation (allo-HCT) ist eine kurative Therapie zur Behandlung bösartiger und nicht bösartiger Knochenmarkerkrankungen. Die Hauptkomplikation dieser Behandlung ist eine hochgradige Entzündungsreaktion, die als Graft-versus-Host-Disease (GvHD) bekannt ist. Zur Behandlung der GvHD werden Cyclosporin A (CsA) und Tacrolimus eingesetzt, die die Entzündung eindämmen, aber auch den gewünschten Graft-versus-Leukämie-Effekt (GvL) beeinträchtigen. Diese Medikamente unterdrücken sowohl konventionelle T-Zellen (Tcon) als auch regulatorische T-Zellen (Treg), die sowohl für die Begrenzung der GvHD, als auch für die Unterstützung der GvL wichtig sind. Beide Medikamente hemmen Calcineurin (CN), das die Transkriptionsfaktoren der Familie der Nuclear Factor of Activated T-Cells (NFAT) dephosphoryliert und aktiviert. Hier nutzten wir unsere Cd4cre.Cas9+-Mäuse und entwickelten eine hocheffiziente, nicht-virale CRISPR/Cas9-Geneditierungsmethode mittels reiner gRNA-Nukleofektion. Mithilfe dieser Technik konnten wir zeigen, dass unstimulierte T-Zellen der Maus nach Ablation von NFATc1 oder NFATc2 die GvHD in einem Major-Mismatch-Mausmodell mildern. In vitro vorstimulierte T-Zellen von Mäusen konnten jedoch keinen langfristigen Schutz vor GvHD bei NFAT-Einzeldefizienz erreichen. Dies unterstreicht die Notwendigkeit der Gen-Editierung und des Transfers unstimulierter menschlicher T-Zellen während einer allo-HCT. In der Tat konnten wir ein hocheffizientes Ribonukleoprotein (RNP)-vermitteltes CRISPR/Cas9 gene-editing für NFATC1 und/oder NFATC2 nicht nur in vorstimulierten, sondern auch in unstimulierten primären menschlichen T-Zellen etablieren. Im Gegensatz zu T-Zellen von Mäusen wirkte sich der Mangel an NFATC2, nicht aber so sehr an NFATC1, in menschlichen T-Zellen überwiegend auf die Produktion proinflammatorischer Zytokine aus. Bei beiden NFAT-Single-Knockouts blieb jedoch die Zytotoxizität menschlicher CD3+ T-Zellen gegen Tumorzellen in vitro unangetastet. Darüber hinaus wurden die Treg von Maus und Mensch durch den Verlust eines einzelnen NFAT-Mitglieds nicht beeinträchtigt. Schließlich bestätigten NFATC1- oder NFATC2-defiziente Anti-CD19-CAR-T-Zellen, die mit unserer nicht-viralen "Ein-Schritt-Nukleofektionsmethode" erzeugt wurden, unsere Beobachtungen zu T-Zellen von Maus und Mensch. Die Produktion proinflammatorischer Zytokine hing hauptsächlich von der NFATC2-Expression ab, während die In-vitro-Zytotoxizität gegen CD19+-Tumorzellen in Abwesenheit eines der beiden NFAT-Mitglieder ungestört war. Unsere Ergebnisse unterstreichen, dass der Mangel eines einzelnen NFAT-Mitglieds in Spender-T-Zellen einer Therapie mit CN-Inhibitoren während einer allo-HCT überlegen ist. Hier könnten wir eine GvHD verhindern und gleichzeitig den GvL-Effekt in allo-HCT-Patienten erhalten. KW - Allogenic hematopoietic stem cell transplantation KW - CRISPR/Cas9 KW - Graft-versus-host-disease KW - Graft-versus-leukemia KW - allografts KW - CRISPR/Cas-Methode Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-293256 ER - TY - JOUR A1 - Marquardt, André A1 - Hartrampf, Philipp A1 - Kollmannsberger, Philip A1 - Solimando, Antonio G. A1 - Meierjohann, Svenja A1 - Kübler, Hubert A1 - Bargou, Ralf A1 - Schilling, Bastian A1 - Serfling, Sebastian E. A1 - Buck, Andreas A1 - Werner, Rudolf A. A1 - Lapa, Constantin A1 - Krebs, Markus T1 - Predicting microenvironment in CXCR4- and FAP-positive solid tumors — a pan-cancer machine learning workflow for theranostic target structures JF - Cancers N2 - (1) Background: C-X-C Motif Chemokine Receptor 4 (CXCR4) and Fibroblast Activation Protein Alpha (FAP) are promising theranostic targets. However, it is unclear whether CXCR4 and FAP positivity mark distinct microenvironments, especially in solid tumors. (2) Methods: Using Random Forest (RF) analysis, we searched for entity-independent mRNA and microRNA signatures related to CXCR4 and FAP overexpression in our pan-cancer cohort from The Cancer Genome Atlas (TCGA) database — representing n = 9242 specimens from 29 tumor entities. CXCR4- and FAP-positive samples were assessed via StringDB cluster analysis, EnrichR, Metascape, and Gene Set Enrichment Analysis (GSEA). Findings were validated via correlation analyses in n = 1541 tumor samples. TIMER2.0 analyzed the association of CXCR4 / FAP expression and infiltration levels of immune-related cells. (3) Results: We identified entity-independent CXCR4 and FAP gene signatures representative for the majority of solid cancers. While CXCR4 positivity marked an immune-related microenvironment, FAP overexpression highlighted an angiogenesis-associated niche. TIMER2.0 analysis confirmed characteristic infiltration levels of CD8+ cells for CXCR4-positive tumors and endothelial cells for FAP-positive tumors. (4) Conclusions: CXCR4- and FAP-directed PET imaging could provide a non-invasive decision aid for entity-agnostic treatment of microenvironment in solid malignancies. Moreover, this machine learning workflow can easily be transferred towards other theranostic targets. KW - machine learning KW - tumor microenvironment KW - immune infiltration KW - angiogenesis KW - mRNA KW - miRNA KW - transcriptome Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-305036 SN - 2072-6694 VL - 15 IS - 2 ER - TY - JOUR A1 - Feldheim, Jonas A1 - Kessler, Almuth F. A1 - Feldheim, Julia J. A1 - Schmitt, Dominik A1 - Oster, Christoph A1 - Lazaridis, Lazaros A1 - Glas, Martin A1 - Ernestus, Ralf-Ingo A1 - Monoranu, Camelia M. A1 - Löhr, Mario A1 - Hagemann, Carsten T1 - BRMS1 in gliomas — an expression analysis JF - Cancers N2 - The metastatic suppressor BRMS1 interacts with critical steps of the metastatic cascade in many cancer entities. As gliomas rarely metastasize, BRMS1 has mainly been neglected in glioma research. However, its interaction partners, such as NFκB, VEGF, or MMPs, are old acquaintances in neurooncology. The steps regulated by BRMS1, such as invasion, migration, and apoptosis, are commonly dysregulated in gliomas. Therefore, BRMS1 shows potential as a regulator of glioma behavior. By bioinformatic analysis, in addition to our cohort of 118 specimens, we determined BRMS1 mRNA and protein expression as well as its correlation with the clinical course in astrocytomas IDH mutant, CNS WHO grade 2/3, and glioblastoma IDH wild-type, CNS WHO grade 4. Interestingly, we found BRMS1 protein expression to be significantly decreased in the aforementioned gliomas, while BRMS1 mRNA appeared to be overexpressed throughout. This dysregulation was independent of patients’ characteristics or survival. The protein and mRNA expression differences cannot be finally explained at this stage. However, they suggest a post-transcriptional dysregulation that has been previously described in other cancer entities. Our analyses present the first data on BRMS1 expression in gliomas that can provide a starting point for further investigations. KW - glioblastoma KW - metastasis KW - suppressor KW - behavior KW - mRNA KW - protein Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-319225 SN - 2072-6694 VL - 15 IS - 11 ER - TY - JOUR A1 - Nickl, Vera A1 - Eck, Juliana A1 - Goedert, Nicolas A1 - Hübner, Julian A1 - Nerreter, Thomas A1 - Hagemann, Carsten A1 - Ernestus, Ralf-Ingo A1 - Schulz, Tim A1 - Nickl, Robert Carl A1 - Keßler, Almuth Friederike A1 - Löhr, Mario A1 - Rosenwald, Andreas A1 - Breun, Maria A1 - Monoranu, Camelia Maria T1 - Characterization and optimization of the tumor microenvironment in patient-derived organotypic slices and organoid models of glioblastoma JF - Cancers N2 - While glioblastoma (GBM) is still challenging to treat, novel immunotherapeutic approaches have shown promising effects in preclinical settings. However, their clinical breakthrough is hampered by complex interactions of GBM with the tumor microenvironment (TME). Here, we present an analysis of TME composition in a patient-derived organoid model (PDO) as well as in organotypic slice cultures (OSC). To obtain a more realistic model for immunotherapeutic testing, we introduce an enhanced PDO model. We manufactured PDOs and OSCs from fresh tissue of GBM patients and analyzed the TME. Enhanced PDOs (ePDOs) were obtained via co-culture with PBMCs (peripheral blood mononuclear cells) and compared to normal PDOs (nPDOs) and PT (primary tissue). At first, we showed that TME was not sustained in PDOs after a short time of culture. In contrast, TME was largely maintained in OSCs. Unfortunately, OSCs can only be cultured for up to 9 days. Thus, we enhanced the TME in PDOs by co-culturing PDOs and PBMCs from healthy donors. These cellular TME patterns could be preserved until day 21. The ePDO approach could mirror the interaction of GBM, TME and immunotherapeutic agents and may consequently represent a realistic model for individual immunotherapeutic drug testing in the future. KW - glioblastoma KW - organoids KW - slice culture KW - tumormicroenvironment Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-319249 SN - 2072-6694 VL - 15 IS - 10 ER - TY - JOUR A1 - Böck, Julia A1 - Maurus, Katja A1 - Gerhard-Hartmann, Elena A1 - Brändlein, Stephanie A1 - Kurz, Katrin S. A1 - Ott, German A1 - Anagnostopoulos, Ioannis A1 - Rosenwald, Andreas A1 - Zamò, Alberto T1 - Targeted panel sequencing in the routine diagnosis of mature T- and NK-cell lymphomas BT - report of 128 cases from two German reference centers JF - Frontiers in Oncology N2 - Diagnosing any of the more than 30 types of T-cell lymphomas is considered a challenging task for many pathologists and currently requires morphological expertise as well as the integration of clinical data, immunophenotype, flow cytometry and clonality analyses. Even considering all available information, some margin of doubt might remain using the current diagnostic procedures. In recent times, the genetic landscape of most T-cell lymphomas has been elucidated, showing a number of diagnostically relevant mutations. In addition, recent data indicate that some of these genetic alterations might bear prognostic and predictive value. Extensive genetic analyses, such as whole exome or large panel sequencing are still expensive and time consuming, therefore limiting their application in routine diagnostic. We therefore devoted our effort to develop a lean approach for genetic analysis of T-cell lymphomas, focusing on maximum efficiency rather than exhaustively covering all possible targets. Here we report the results generated with our small amplicon-based panel that could be used routinely on paraffin-embedded and even decalcified samples, on a single sample basis in parallel with other NGS-panels used in our routine diagnostic lab, in a relatively short time and with limited costs. We tested 128 available samples from two German reference centers as part of our routine work up (among which 116 T-cell lymphomas), which is the largest routine diagnostic series reported to date. Our results showed that this assay had a very high rate of technical success (97%) and could detect mutations in the majority (79%) of tested T-cell lymphoma samples. KW - T-cell lymphoma KW - panel-sequencing KW - NGS KW - diagnostics KW - mutation KW - FFPE Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-326478 SN - 2234-943X VL - 13 ER - TY - JOUR A1 - Kreß, Julia Katharina Charlotte A1 - Jessen, Christina A1 - Hufnagel, Anita A1 - Schmitz, Werner A1 - Da Xavier Silva, Thamara Nishida A1 - Ferreira Dos Santos, Ancély A1 - Mosteo, Laura A1 - Goding, Colin R. A1 - Friedmann Angeli, José Pedro A1 - Meierjohann, Svenja T1 - The integrated stress response effector ATF4 is an obligatory metabolic activator of NRF2 JF - Cell Reports N2 - Highlights • The integrated stress response leads to a general ATF4-dependent activation of NRF2 • ATF4 causes a CHAC1-dependent GSH depletion, resulting in NRF2 stabilization • An elevation of NRF2 transcript levels fosters this effect • NRF2 supports the ISR/ATF4 pathway by improving cystine and antioxidant supply Summary The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease. KW - NRF2 KW - ATF4 KW - integrated stress response KW - CHAC1 KW - melanoma KW - SLC7A11 KW - GSH Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350312 VL - 42 IS - 7 ER - TY - JOUR A1 - Bassler, Miriam C. A1 - Knoblich, Mona A1 - Gerhard-Hartmann, Elena A1 - Mukherjee, Ashutosh A1 - Youssef, Almoatazbellah A1 - Hagen, Rudolf A1 - Haug, Lukas A1 - Goncalves, Miguel A1 - Scherzad, Agmal A1 - Stöth, Manuel A1 - Ostertag, Edwin A1 - Steinke, Maria A1 - Brecht, Marc A1 - Hackenberg, Stephan A1 - Meyer, Till Jasper T1 - Differentiation of salivary gland and salivary gland tumor tissue via Raman imaging combined with multivariate data analysis JF - Diagnostics N2 - Salivary gland tumors (SGTs) are a relevant, highly diverse subgroup of head and neck tumors whose entity determination can be difficult. Confocal Raman imaging in combination with multivariate data analysis may possibly support their correct classification. For the analysis of the translational potential of Raman imaging in SGT determination, a multi-stage evaluation process is necessary. By measuring a sample set of Warthin tumor, pleomorphic adenoma and non-tumor salivary gland tissue, Raman data were obtained and a thorough Raman band analysis was performed. This evaluation revealed highly overlapping Raman patterns with only minor spectral differences. Consequently, a principal component analysis (PCA) was calculated and further combined with a discriminant analysis (DA) to enable the best possible distinction. The PCA-DA model was characterized by accuracy, sensitivity, selectivity and precision values above 90% and validated by predicting model-unknown Raman spectra, of which 93% were classified correctly. Thus, we state our PCA-DA to be suitable for parotid tumor and non-salivary salivary gland tissue discrimination and prediction. For evaluation of the translational potential, further validation steps are necessary. KW - salivary gland tumor KW - confocal Raman imaging KW - principal component analysis KW - discriminant analysis KW - multivariate data analysis KW - molecular diagnostics Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-355558 SN - 2075-4418 VL - 14 IS - 1 ER - TY - JOUR A1 - Madrahimov, Nodir A1 - Mutsenko, Vitalii A1 - Natanov, Ruslan A1 - Radaković, Dejan A1 - Klapproth, André A1 - Hassan, Mohamed A1 - Rosenfeldt, Mathias A1 - Kleefeldt, Florian A1 - Aleksic, Ivan A1 - Ergün, Süleyman A1 - Otto, Christoph A1 - Leyh, Rainer G. A1 - Bening, Constanze T1 - Multiorgan recovery in a cadaver body using mild hypothermic ECMO treatment in a murine model JF - Intensive Care Medicine Experimental N2 - Background Transplant candidates on the waiting list are increasingly challenged by the lack of organs. Most of the organs can only be kept viable within very limited timeframes (e.g., mere 4–6 h for heart and lungs exposed to refrigeration temperatures ex vivo). Donation after circulatory death (DCD) using extracorporeal membrane oxygenation (ECMO) can significantly enlarge the donor pool, organ yield per donor, and shelf life. Nevertheless, clinical attempts to recover organs for transplantation after uncontrolled DCD are extremely complex and hardly reproducible. Therefore, as a preliminary strategy to fulfill this task, experimental protocols using feasible animal models are highly warranted. The primary aim of the study was to develop a model of ECMO-based cadaver organ recovery in mice. Our model mimics uncontrolled organ donation after an “out-of-hospital” sudden unexpected death with subsequent “in-hospital” cadaver management post-mortem. The secondary aim was to assess blood gas parameters, cardiac activity as well as overall organ state. The study protocol included post-mortem heparin–streptokinase administration 10 min after confirmed death induced by cervical dislocation under full anesthesia. After cannulation, veno-arterial ECMO (V–A ECMO) was started 1 h after death and continued for 2 h under mild hypothermic conditions followed by organ harvest. Pressure- and flow-controlled oxygenated blood-based reperfusion of a cadaver body was accompanied by blood gas analysis (BGA), electrocardiography, and histological evaluation of ischemia–reperfusion injury. For the first time, we designed and implemented, a not yet reported, miniaturized murine hemodialysis circuit for the treatment of severe hyperkalemia and metabolic acidosis post-mortem. Results BGA parameters confirmed profound ischemia typical for cadavers and incompatible with normal physiology, including extremely low blood pH, profound negative base excess, and enormously high levels of lactate. Two hours after ECMO implantation, blood pH values of a cadaver body restored from < 6.5 to 7.3 ± 0.05, pCO2 was lowered from > 130 to 41.7 ± 10.5 mmHg, sO2, base excess, and HCO3 were all elevated from below detection thresholds to 99.5 ± 0.6%, − 4 ± 6.2 and 22.0 ± 6.0 mmol/L, respectively (Student T test, p < 0.05). A substantial decrease in hyperlactatemia (from > 20 to 10.5 ± 1.7 mmol/L) and hyperkalemia (from > 9 to 6.9 ± 1.0 mmol/L) was observed when hemodialysis was implemented. On balance, the first signs of regained heart activity appeared on average 10 min after ECMO initiation without cardioplegia or any inotropic and vasopressor support. This was followed by restoration of myocardial contractility with a heart rate of up to 200 beats per minute (bpm) as detected by an electrocardiogram (ECG). Histological examinations revealed no evidence of heart injury 3 h post-mortem, whereas shock-specific morphological changes relevant to acute death and consequent cardiac/circulatory arrest were observed in the lungs, liver, and kidney of both control and ECMO-treated cadaver mice. Conclusions Thus, our model represents a promising approach to facilitate studying perspectives of cadaveric multiorgan recovery for transplantation. Moreover, it opens new possibilities for cadaver organ treatment to extend and potentiate donation and, hence, contribute to solving the organ shortage dilemma. KW - extracorporeal membrane oxygenation KW - cadaver multiorgan preservation KW - mild hypothermia KW - post-mortem heart recovery Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357381 VL - 11 ER - TY - JOUR A1 - Hung, Sophia A1 - Kasperkowitz, Amelie A1 - Kurz, Florian A1 - Dreher, Liane A1 - Diessner, Joachim A1 - Ibrahim, Eslam S. A1 - Schwarz, Stefan A1 - Ohlsen, Knut A1 - Hertlein, Tobias T1 - Next-generation humanized NSG-SGM3 mice are highly susceptible to Staphylococcus aureus infection JF - Frontiers in Immunology N2 - Humanized hemato-lymphoid system mice, or humanized mice, emerged in recent years as a promising model to study the course of infection of human-adapted or human-specific pathogens. Though Staphylococcus aureus infects and colonizes a variety of species, it has nonetheless become one of the most successful human pathogens of our time with a wide armory of human-adapted virulence factors. Humanized mice showed increased vulnerability to S. aureus compared to wild type mice in a variety of clinically relevant disease models. Most of these studies employed humanized NSG (NOD-scid IL2Rgnull) mice which are widely used in the scientific community, but show poor human myeloid cell reconstitution. Since this immune cell compartment plays a decisive role in the defense of the human immune system against S. aureus, we asked whether next-generation humanized mice, like NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF) with improved myeloid reconstitution, would prove to be more resistant to infection. To our surprise, we found the contrary when we infected humanized NSG-SGM3 (huSGM3) mice with S. aureus: although they had stronger human immune cell engraftment than humanized NSG mice, particularly in the myeloid compartment, they displayed even more pronounced vulnerability to S. aureus infection. HuSGM3 mice had overall higher numbers of human T cells, B cells, neutrophils and monocytes in the blood and the spleen. This was accompanied by elevated levels of pro-inflammatory human cytokines in the blood of huSGM3 mice. We further identified that the impaired survival of huSGM3 mice was not linked to higher bacterial burden nor to differences in the murine immune cell repertoire. Conversely, we could demonstrate a correlation of the rate of humanization and the severity of infection. Collectively, this study suggests a detrimental effect of the human immune system in humanized mice upon encounter with S. aureus which might help to guide future therapy approaches and analysis of virulence mechanisms. KW - humanized mice KW - Staphylococcus aureus KW - MRSA KW - NSG KW - NSG-SGM3 KW - staphylococcal abscess KW - Staphylococcus aureus immune response KW - humanized hemato-lymphoid mice Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-306966 VL - 14 ER - TY - JOUR A1 - Murti, Krisna A1 - Fender, Hendrik A1 - Glatzle, Carolin A1 - Wismer, Rhoda A1 - Sampere-Birlanga, Salvador A1 - Wild, Vanessa A1 - Muhammad, Khalid A1 - Rosenwald, Andreas A1 - Serfling, Edgar A1 - Avots, Andris T1 - Calcineurin-independent NFATc1 signaling is essential for survival of Burkitt lymphoma cells JF - Frontiers in Oncology N2 - In Burkitt lymphoma (BL), a tumor of germinal center B cells, the pro-apoptotic properties of MYC are controlled by tonic B cell receptor (BCR) signals. Since BL cells do not exhibit constitutive NF-κB activity, we hypothesized that anti-apoptotic NFATc1 proteins provide a major transcriptional survival signal in BL. Here we show that post-transcriptional mechanisms are responsible for the calcineurin (CN) independent constitutive nuclear over-expression of NFATc1 in BL and Eµ-MYC – induced B cell lymphomas (BCL). Conditional inactivation of the Nfatc1 gene in B cells of Eµ-MYC mice leads to apoptosis of BCL cells in vivo and ex vivo. Inhibition of BCR/SYK/BTK/PI3K signals in BL cells results in cytosolic re-location of NFATc1 and apoptosis. Therefore, NFATc1 activity is an integrated part of tonic BCR signaling and an alternative target for therapeutic intervention in BL. KW - apoptosis KW - Burkitt lymphoma KW - cyclosporin A KW - nuclear localization KW - NFATc1 KW - activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) KW - B cell receptor (BCR) KW - Burkitt lymphoma (BL) Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323103 VL - 13 ER - TY - JOUR A1 - Lisowski, Dominik A1 - Hartrampf, Philipp E. A1 - Hasenauer, Natalie A1 - Nickl, Vera A1 - Monoranu, Camelia-Maria A1 - Tamihardja, Jörg T1 - Complete loss of E-cadherin expression in a rare case of metastatic malignant meningioma: a case report JF - BMC Neurology N2 - Background Hematogenous tumor spread of malignant meningiomas occurs very rarely but is associated with very poor prognosis. Case presentation We report an unusual case of a patient with a malignant meningioma who developed multiple metastases in bones, lungs and liver after initial complete resection of the primary tumor. After partial hepatic resection, specimens were histologically analyzed, and a complete loss of E-cadherin adhesion molecules was found. No oncogenic target mutations were found. The patient received a combination of conventional radiotherapy and peptide receptor radionuclide therapy (PRRT). Due to aggressive tumor behavior and rapid spread of metastases, the patient deceased after initiation of treatment. Conclusions E-cadherin downregulation is associated with a higher probability of tumor invasion and distant metastasis formation in malignant meningioma. Up to now, the efficacy of systemic therapy, including PRRT, is very limited in malignant meningioma patients. KW - beta-catenin KW - E-cadherin KW - meningioma KW - peptide receptor radionuclide therapy (PRRT) KW - radiotherapy Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357996 VL - 23 ER - TY - JOUR A1 - Seal, Rishav A1 - Schwab, Lara S. U. A1 - Chiarolla, Cristina M. A1 - Hundhausen, Nadine A1 - Klose, Georg Heinrich A1 - Reu-Hofer, Simone A1 - Rosenwald, Andreas A1 - Wiest, Johannes A1 - Berberich-Siebelt, Friederike T1 - Delayed and limited administration of the JAKinib tofacitinib mitigates chronic DSS-induced colitis JF - Frontiers in Immunology N2 - In inflammatory bowel disease, dysregulated T cells express pro-inflammatory cytokines. Using a chronic azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis model resembling ulcerative colitis, we evaluated whether and when treatment with the Janus kinase (JAK) inhibitor tofacitinib could be curative. Comparing the treatment with two and three cycles of tofacitinib medication in drinking water – intermittently with DSS induction – revealed that two cycles were not only sufficient but also superior over the 3-x regimen. The two cycles of the 2-x protocol paralleled the second and third cycles of the longer protocol. T cells were less able to express interferon gamma (IFN-γ) and the serum levels of IFN-γ, interleukin (IL)-2, IL-6, IL-17, and tumor necrosis factor (TNF) were significantly reduced in sera, while those of IL-10 and IL-22 increased under the 2-x protocol. Likewise, the frequency and effector phenotype of regulatory T cells (Tregs) increased. This was accompanied by normal weight gain, controlled clinical scores, and restored stool consistency. The general and histologic appearance of the colons revealed healing and tissue intactness. Importantly, two phases of tofacitinib medication completely prevented AOM-incited pseudopolyps and the hyper-proliferation of epithelia, which was in contrast to the 3-x regimen. This implies that the initial IBD-induced cytokine expression is not necessarily harmful as long as inflammatory signaling can later be suppressed and that time-restricted treatment allows for anti-inflammatory and tissue-healing cytokine activities. KW - anti-inflammatory cytokines KW - AOM/DSS KW - pro-inflammatory cytokines KW - effector Treg (eTreg) KW - chronic IBD model KW - JAK inhibitor KW - tofacitinib KW - treatment regimens Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-317815 VL - 14 ER - TY - JOUR A1 - Breun, Maria A1 - Flock, Katharina A1 - Feldheim, Jonas A1 - Nattmann, Anja A1 - Monoranu, Camelia M. A1 - Herrmann, Pia A1 - Ernestus, Ralf-Ingo A1 - Löhr, Mario A1 - Hagemann, Carsten A1 - Stein, Ulrike T1 - Metastasis associated in colorectal cancer 1 (MACC1) mRNA expression is enhanced in sporadic vestibular schwannoma and correlates to deafness JF - Cancers N2 - Vestibular schwannoma (VS) are benign cranial nerve sheath tumors of the vestibulocochlear nerve. Their incidence is mostly sporadic, but they can also be associated with NF2-related schwannomatosis (NF2), a hereditary tumor syndrome. Metastasis associated in colon cancer 1 (MACC1) is known to contribute to angiogenesis, cell growth, invasiveness, cell motility and metastasis of solid malignant cancers. In addition, MACC1 may be associated with nonsyndromic hearing impairment. Therefore, we evaluated whether MACC1 may be involved in the pathogenesis of VS. Sporadic VS, recurrent sporadic VS, NF2-associated VS, recurrent NF2-associated VS and healthy vestibular nerves were analyzed for MACC1 mRNA and protein expression by quantitative polymerase chain reaction and immunohistochemistry. MACC1 expression levels were correlated with the patients’ clinical course and symptoms. MACC1 mRNA expression was significantly higher in sporadic VS compared to NF2-associated VS (p < 0.001). The latter expressed similar MACC1 concentrations as healthy vestibular nerves. Recurrent tumors resembled the MACC1 expression of the primary tumors. MACC1 mRNA expression was significantly correlated with deafness in sporadic VS patients (p = 0.034). Therefore, MACC1 might be a new molecular marker involved in VS pathogenesis. KW - vestibular schwannoma KW - metastasis associated in colorectal cancer 1 (MACC1) KW - pathogenesis KW - deafness KW - NF2-related schwannomatosis (NF2) KW - mRNA expression Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-362543 SN - 2072-6694 VL - 15 IS - 16 ER - TY - JOUR A1 - Meinert, Madlen A1 - Jessen, Christina A1 - Hufnagel, Anita A1 - Kreß, Julia Katharina Charlotte A1 - Burnworth, Mychal A1 - Däubler, Theo A1 - Gallasch, Till A1 - Da Xavier Silva, Thamara Nishida A1 - Dos Santos, Ancély Ferreira A1 - Ade, Carsten Patrick A1 - Schmitz, Werner A1 - Kneitz, Susanne A1 - Friedmann Angeli, José Pedro A1 - Meierjohann, Svenja T1 - Thiol starvation triggers melanoma state switching in an ATF4 and NRF2-dependent manner JF - Redox Biology N2 - The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; Braf\(^{CA}\); Pten\(^{lox/+}\) melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2. KW - thiol starvation KW - ATF4 KW - NRF2 KW - melanoma Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350328 VL - 70 ER -