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UV and chemical crosslinking mass spectrometry for the analysis of protein-nucleic acid interactions
No full textProtein-nucleic acid interactions are a key part of essential cellular processes and their disturbance often results in the development of diseases. Crosslinking mass spectrometry (XL-MS)-based approaches have become increasingly popular for the proteome-wide discovery of nucleic acid-binding proteins. The basic principle of the methodology relies on covalent attachment of proteins and binding nucleic acids by crosslinking, which makes the heteroconjugates amenable to MS analysis. Building up on comprehensive nucleic acid-binding protein inventories, there is an increasing demand for higher resolution analyses identifying the interaction sites at the level of crosslinked amino acids. Amino acid-resolved XL-MS data is of significant value for integration in structural modelling approaches as distance restrains, and for the discovery of novel nucleic acid-binding sequence and structure motifs. At the outset of the research detailed in this doctoral thesis, amino acid-resolved XL-MS could not be applied comprehensively in native cellular environments, due to low crosslink yields and the absence of specialized software solutions. In this work, we focused on i) the optimization of crosslinking conditions comparing conventional UV-light induced crosslinking with chemical crosslinking reagents; ii) crosslinked peptide-(oligo)nucleotide enrichment; iii) identification of crosslinked (oligo)nucleotide-adducts; iv) the development of a specialized software solution for crosslinked peptide-(oligo)nucleotide database search; and v) the adaptation of a labelling-based quantitative MS approach. In the published manuscript by Stützer et al., which constitutes the first part of this thesis, an optimized UV-light based XL-MS workflow was established, leading to the identification of numerous crosslinked amino acids within eukaryotic nucleosomes and HeLa nuclei. With the aim to increase XL-MS efficiency and sensitivity, we established chemical crosslinking XL-MS, which is presented in the second, yet unpublished manuscript by Welp and colleagues. We compared the results obtained by chemical XL-MS with those from UV XL-MS, and applied it to Escherichia coli cells, which delivered a comprehensive crosslink site inventory. We further present the database search tool, NuXL, specialized on the identification of peptide-(oligo)nucleotide crosslinks and providing crosslink localisation on the level of single amino acids. Building upon these studies, we conducted a preliminary quantitative amino acid-resolved XL-MS experiment using tandem mass tag (TMT)-labelling in combination with our established workflow. This thesis provides a valuable contribution to the application toolkit for the investigation of protein-nucleic acid complexes and contains exciting protein-DNA and protein-RNA datasets from human cell nuclei and E. coli cells.2025-04-0
Measurement of the Branching Ratio using the hadronic Full Event Interpretation with Belle II Data
No full textThe Belle II detector is situated at the asymmetric collider SuperKEKB at KEK in Tsukuba in Japan, which operates with an energy slightly above the mass of the Y(4S) resonance. As a result, pairs of B mesons are predominantly produced. Due to the clean experimental environment, various measurements to test the Standard Model and precisely determine its parameters can be performed. This is e.g. the element of the CKM matrix among others, where one of the leading systematic uncertainties arises due to decays of the nature .
This thesis presents the first measurement of the branching ratio using the data set collected with the Belle II detector between 2019 and 2022 and corresponds to about of integrated luminosity at the Y(4S) resonance. To reconstruct the "other" B meson, a multivariate analysis tool called "Full Event Interpretation" is used, which reconstructs a variety of different hadronic decay modes. For the reconstruction of the signal side B meson, a light charged lepton is combined with a meson that is reconstructed from various and combinations. In contrast to previously performed analyses, this analysis also takes the into account in the reconstruction of the meson.
A selection of events is applied to obtain a sample enriched in decays and a binned maximum likelihood fit is performed to determine the branching ratio for the different decay channels. To account for effects in the simulation that are currently not well understood, the branching ratio is normalised to the branching ratio of the much more frequent decay .2024-07-2
Investigation of active mechanical properties in the cytoplasm of living cells
No full textOver the past decades, the study of intracellular active mechanical properties has experienced
a growth in interest. This is mainly caused by recognizing that vital cellular
functions rely on the intricate interplay between the viscoelastic mechanical properties
of the cytoplasm and the active force generated by the consumption of metabolic energy.
However, quantifying these properties poses significant challenges due to the complexity
of the physical quantities and the elaborate and low-throughput experimental methods
required for their investigation. In this work, we propose two techniques to meet these
challenges. Firstly, we introduce a mechanical fingerprint that reduces the complexity
of intracellular active mechanical properties to a set of six parameters. Demonstrated
initially on HeLa cells, the fingerprint accurately captured changes in mechanics upon
disrupting cytoskeletal components, showcasing its capability to represent intracellular
dynamics concisely. The investigation was then expanded to 7 diverse cell types, where
their unique fingerprint could identify individual cell types.
Further correlational analysis led to the introduction of a three-dimensional phase space
comprised of resistance, activity, and fluidity. Here, the positions in phase space correlated
with expected cell functions, underlining the fingerprint’s capability of accelerating
the investigation of such relations. Secondly, we introduced the Mean Back Relaxation
(MBR) as a novel statistical tool to determine the breaking of detailed balance in confined
systems. This approach was first established in a well-controlled model system, mimicking
the intracellular space. It was then applied to living cells, where we could observe
surprising relations between the MBR and intracellular activity. Strikingly, by deploying
this relation, we determined the mechanical properties of MDCK cells by purely passive
observations. We thus present an alternative approach for the quantification of intracellular
active mechanical properties. This will drastically reduce experimental complexity
and increase the experimental throughput.2024-07-1
Investigation of Cells and Cellular Components with X-Rays in Liquid Sample Environments
No full textRöntgenstrahlung besitzt aufgrund ihrer kurzen Wellenlänge eine hohe räumliche Auflösung und eine hohe Durchdringungsfähigkeit für weiche kondensierte Materie. Daher eignet sich Röntgenstrahlung zur Untersuchung von Strukturen und Strukturänderungen in in-vitro Systemen sowie in intakten biologischen Zellen und Geweben. Für solche Untersuchungen müssen jedoch geeignete Probenumgebungen entwickelt und verwendet werden, um Effekte von Oberflächen oder der Probenvorbereitung zu vermeiden. Solche Probenumgebungen müssen sowohl mit der Probe als auch mit der Röntgenstrahlung kompatibel sein.
In dieser Arbeit verwenden wir Röntgenkleinwinkelstreuung (SAXS) und Fluoreszenzmikroskopie, um das Assemblierungsverhalten des Muskelproteins Desmin zu Filamenten in 2 mM MOPS-Puffer und bei verschiedenen KCl Konzentrationen zu untersuchen. Menschliches und Maus-Desmin zeigen das gleiche Assemblierungsverhalten, wobei die optimale Salzkonzentration für die Bildung von Filamenten bei 40-50 mM KCl liegt. Der entsprechende Gyrationsradius beträgt etwa 5 nm. In Mischungen aus menschlichem Desmin-Wildtyp und weniger als 50% der Mutante R406W werden Filamente beobachtet; oberhalb von 70% R406W ist keine Filamentbildung zu erkennen.
Zur Untersuchung von intakten Zellen in kontinuierlichem Fluss, stellen wir einen röntgenkompatiblen mikrofluidischen Chip vor, der aus PDMS und einer Kapillare besteht und der als Probenzufuhrsystem und Messumgebung dient. Zum Nachweis des Konzepts werden mit diesem Gerät chemisch fixierte rote Rinderblutzellen (RBC) mittels SAXS untersucht. Der Vergleich der Messergebnisse mit Daten von etablierten statischen Messmethoden zeigt eine große Übereinstimmung und damit die Verlässlichkeit des Aufbaus zur Messung von Zellen in kontinuierlichem Fluss. Mithilfe einer Anpassung eines Hard-Sphere-Modells mit abgeschirmten Coulomb-Wechselwirkungen and die Daten wird außerdem der Radius des Proteins Hämoglobin innerhalb der RBCs bestimmt.
Um einzelne Zellen zu untersuchen, kombinieren wir Röntgen-Phasenkontrast-Bildgebung mit einem röntgenkompatiblen optischen Stretcher (OS) und einer mikrofluidischen Probenzuführung. Mit diesem Aufbau können einzelne Zellen in Lösung räumlich fixiert werden und gleichzeitig mit dem Röntgenstrahl gemessen werden. Diese Methode testen wir erfolgreich an lebenden sowie an fixierten und gefärbten NIH3T3-Mausfibroblasten. Obwohl das Färbeverfahren für einzelne Zellen weiter verbessert werden muss, erhöht es die Sichtbarkeit der Zellkonturen und teilweise der Zellkerne. Eine Strahlungsenergie von 9.9 keV ist für diese Messungen einer Strahlungsenergie von 13.8 keV vorzuziehen. Durch Rotation der Zelle im OS während der Messung wird auch Tomographie ermöglicht. Mithilfe von BaSO4 Markern, einem algebraischen 3D-Rekonstruktionsschema und einer speziellen mathematischen Beschreibung der Bewegung der Zellen im optischen Stretcher konnten wir eine 3D Rekonstruktion berechnen.
Prinzipiell können in zukünftigen Experimenten mit dem PDMS-Kapillarchip und dem OS auch Kräfte auf die Zellen ausgeübt und deren Effekte auf die Zelle untersucht werden. Insgesamt zeigen unsere Experimente die erfolgreiche Kombination von verschiedenen röntgenbasierten Untersuchungsmethoden und Probenumgebungen. Dies erlaubt die Untersuchung von Proteinkonformationen in chemischen Umgebungen, Zellen in kontinuierlichem Fluss oder einzelnen Zellen ohne die Notwendigkeit Oberflächen zu verwenden und ohne aufwendige Probenvorbereitung.Due to their short wavelength, X-rays offer a high spatial resolution and a high ability to penetrate soft condensed matter. Therefore, X-rays are well-suited for investigating structures and structural changes in in-vitro systems as well as in intact biological cells and tissues. For such investigations, suitable sample environments need to be developed and used to avoid effects from surfaces or sample preparation. The applied sample environments need be compatible with both the sample under investigation and the X-ray radiation.
In this thesis, we use small-angle X-ray scattering (SAXS) and fluorescence microscopy to investigate the assembly behavior of the muscle protein desmin into filaments in 2 mM MOPS buffer and at different KCl concentrations. Human and mouse desmin show the same assembly behavior with an optimal salt concentration of 40-50 mM KCl. The corresponding radius of gyration is approximately 5 nm. Filaments are observed in mixtures of human wild-type desmin and less than 50% of its mutant R406W; no filament formation is found above 70% R406W.
To study intact cells in continuous flow, we present an X-ray compatible microfluidic chip consisting of PDMS and a capillary, which serves as both a sample delivery system and a measurement environment. As a proof-of-concept, we investigate chemically fixed bovine red blood cells (RBCs) with this device and SAXS. The comparison of the measurement results with data from established static measurement methods shows a high level of agreement and thus the reliability of the set-up for measuring cells in continuous flow. In addition, the radius of the protein hemoglobin within the RBCs is determined by fitting a hard-sphere model with screened Coulomb interactions to the data.
To examine individual cells in solution with X-rays, we combine X-ray phase-contrast imaging with an X-ray compatible optical stretcher (OS) and a microfluidic sample delivery system. With this setup, individual cells can be trapped in solution and simultaneously measured with the X-ray beam. We successfully test this method on living as well as fixed and stained NIH3T3 mouse fibroblasts. Despite the need for further improvement of the staining procedure for single cells, it increases the visibility of the cell outlines and partially of the cell nuclei. A photon energy of 9.9 keV should be preferred to a photon energy of 13.8 keV for these measurements. Furthermore, rotating the cell in the OS during the acquisition enables tomography. With the help of BaSO4 markers, an algebraic 3D reconstruction scheme, and a dedicated mathematical description of the movement of the cells in the optical stretcher, we are able to calculate a 3D reconstruction.
In principle, future experiments with the PDMS-capillary chip and the OS can also be used to exert forces and investigate their effects on the cell. The presented experiments show how different X-ray-based investigation methods and sample environments can be successfully combined to investigate protein conformations in chemical environments, cells in continuous flow or individual cells without the need to use surfaces or complex sample preparations.2025-04-1
Development of an in-silico framework for the engineering and evaluation of future optogenetic cochlear implants
No full textHearing loss is a prevalent global challenge with profound cognitive, psychological, social, and economic implications. Traditional cochlear implants (CIs)—neuroprosthetic devices to partially restore hearing—stimulate the spiral-ganglion neurons (SGNs) with electrical pulses via an electrode array at a high stimulation rate. The wide spread of current and channel interactions lead to a poor understanding of speech and limited appreciation of music. Optogenetic cochlear implants (oCIs) represent a promising alternative, leveraging light-sensitive proteins (opsins) to activate the SGNs with spatially-confined light pulses. While several pre-clinical studies have shown promising results for improved spectral resolution, the question of potential improvements in speech perception remains elusive. This dissertation presents an exploration of the engineering and evaluation of the oCIs through a computational framework development, integrating models of sound processing, spatial spread in a human cochlea, and neuron stimulation. The modular structure of the framework allowed to simulate electrical and optical stimulation for an audio dataset in a fairly comparable manner. The spike patterns were compared to the sound spectrograph using a similarity measure based on the structural similarity index. The results demonstrate the superior spectral resolution of oCIs and offer an approach to evaluate the impact of lower stimulation rates that could arise from opsin kinetics and/or energy budget. The model indicates that the increased number of spectral channels (64 instead of 20) can compensate for a lower optical stimulation rate. Further, the oCIs are predicted to provide better hearing than the electrical CIs in noisy environments. By employing computational modelling approaches, this dissertation attempts to guide and accelerate the development and translation of oCIs.2025-05-1
Einfluss von chronischer THC-Gabe auf den zerebralen Glukosemetabolismus in weiblichen C57BL/6-Mäusen
No full textCannabis is the most widely consumed recreational drug in the world and is also becoming increasingly relevant in the medical sector with promising pharmacological effects.
THC interacts with the endocannabinoid system, which plays a crucial role in brain development, learning, memory, anxiety, behavior, neuroprotection, and plasticity, particularly through the CB1 receptor. Studies show that cannabis use can be associated with increased risks of mental health disorders and cognitive impairments.
This study aims to assess the impact of chronic THC consumption on cerebral metabolism in healthy female C57BL/6 mice using 18F-FDG-PET imaging. Mice were divided into two age groups: " young" (seven months) and "old" (twelve months). Both groups received high-dose THC (20 mg/kg) intraperitoneally over the course of 42 days. A third group, "young abstinent THC," received THC at three months old followed by a two-month abstinence. With this third group, behavioral experiments were conducted to detect persistent cerebral effects post-abstinence and before PET-Imaging at the age of 7 months.
Imaging results showed significant reductions in brain activity across all examined regions in the "young" mice after chronic THC exposure. The "young abstinent THC" group exhibited the lowest cerebral glucose uptake, indicating persistent effects despite abstinence. Behavioral tests of the third group suggested compensatory mechanisms, as no learning, anxiety, or motor deficits were observed. In contrast, "old" mice showed no significant changes except in the amygdala, suggesting age-dependent neuroadaptation and tolerance.
Overall, this study demonstrates, that chronic THC exposure significantly affects the brains metabolism of young mice. These effects persist despite prolonged abstinence, without affecting the behaviour of the mice.2025-07-2
Wie viel Aussage hat DRIL? Bedeutung der inneren Netzhautschichten bei Patienten mit retinalen Gefäßverschlüssen
Get PDFPurpose: To examine the interrater and intrarater reliability of qualitatively and quantitatively assessed disorganization of retinal inner layers (DRIL) and disorganization of retinal outer layers (DROL) by multiple raters. Subjectively assessing these surrogate biomarkers can be challenging in daily routine, despite the high resolution of spectral-domain (SD) OCT scans.
Design: Retrospective trial.
Participants: Three hundred six pooled SD OCT scans of 34 patients treated for macular edema caused by retinal vein occlusion (RVO) between January 2016 and December 2017.
Methods: SD OCT scans were assessed by 6 raters regarding presence of cystoid macular edema, subretinal fluid (SRF), vitreoretinal traction, and epiretinal membrane and extent of DRIL and DROL.
Main outcome measures: Interrater and intrarater reliability were calculated applying κ statistics for qualitative assessment regarding each pathologic feature's presence in all evaluated OCT scans, and for quantified horizontal DRIL and DROL extent within each OCT cross-section.
Results: Cystoid macular edema and SRF assessments revealed excellent inter- and intrarater reliability with almost perfect strength of agreement, whereas subjective DRIL and DROL evaluations yielded low κ statistics with slight to moderate strength of agreement. Furthermore, the presence of SRF remarkably compromised the reliability of DROL detection.
Conclusions: Our data highlight the limited subjective assessibility of DRIL and DROL, underscoring the need for automated image analysis to improve the reliability of OCT biomarkers for clinical studies and daily practice.2024-09-0
Systematische Zeitpunkt- und ortsabhängige Untersuchung der Legionellenkontamination im Trinkwassersystem der Universitätsmedizin Göttingen
Get PDFWarmwasserleitungssysteme bieten dem Süßwasserbakterium Legionella optimale Lebensbedingungen. Die Trinkwasserverordnung regelt eine Untersuchungspflicht auf Legionellen. Kommt es zu einer Überschreitung des technischen Maßnahmewertes, müssen entsprechende Maßnahmen ergriffen werden. Diese Dissertation hatte das Ziel, den Einfluss verschiedener Faktoren auf die Legionellenkonzentration und somit die Robustheit dieser Messgröße darzustellen. Dazu wurden Saisonalität, Temperatur und der Einfluss des Verbraucherverhaltens im Tagesverlauf untersucht. Es konnte eine signifikante negative Korrelation zwischen den Legionellenkonzentrationen und der Wassertemperatur gezeigt werden. Dahingegen ergab sich kein eindeutig signifikanter Unterschied hinsichtlich der Legionellenkonzentrationen im Vergleich der verschiedenen Jahreszeiten. Eine Abhängig- keit ist aber unter veränderten Beprobungsbedingungen durchaus denkbar. Dies zeigt, dass das gehäufte Auftreten von Legionellosen in den Sommermonaten nicht zwangsläufig im Zusammenhang mit höheren Legionellenkonzentrationen im Trinkwasser steht. Auch ergaben die Untersuchungsergebnisse keinen Hinweis auf signifikante Unterschiede hinsichtlich des Verbraucherverhaltens im Tagesverlauf und damit der Durchflussmengen. Dass die Legionellenkonzentrationen über das Jahr gesehen an den verschiedenen Probenahmestellen stark variieren, jedoch kein eindeutiger Trend im jahreszeitlichen Verlauf erkennbar ist, bestätigt aus der Literatur bekannte Resultate, nach denen Legionellenanalysen häufig nur Momentaufnahmen sind, die möglicherweise von anderen Faktoren als der Jahreszeit beeinflusst werden. Des Weiteren konnte in nur 42% der untersuchten Proben die am häufigsten als pathogen beschriebene Serogruppe 1 nachgewiesen werden. Für die restlichen Proben mit anderen Serogruppen von geringerem pathogenen Potenzial stehen folglich die durchgeführten Interventionen in einem schlechteren Aufwand-Nutzen- Verhältnis. Die Ergebnisse dieser Arbeit erlauben es nicht, klimatische Perioden festzule- gen, in denen die Überwachung intensiviert oder gelockert werden könnte. Auch wäre es fahrlässig, vereinzelt niedrige oder hohe Werte einer Probenahmestelle zur Vorhersage eines verminderten oder erhöhten Gesundheitsrisikos oder gar für Rückschlüsse auf ein gesamtes System heranzuziehen. Aufgrund der Individualität jedes einzelnen wasserfüh- renden Leitungssystems und der Vielzahl teils bisher nicht verstandener Einflussfaktoren auf eine Legionellenkontamination erscheint die Übertragung auf ein anderes System ausgeschlossen bzw. sollte nur mit äußerster Vorsicht in Erwägung gezogen werden.Hot water pipe systems offer the freshwater bacteria Legionella optimal living conditions. The Drinking Water Ordinance stipulates that testing for Legionella is mandatory. If the technical action value is exceeded, appropriate measures must be taken. The aim of this dissertation was to show the influence of various factors on the Legionella concentration and thus the robustness of this measurement value. To this end, seasonality, temperature and the influence of consumer behavior over the course of the day were examined. A significant negative correlation was shown between the Legionella concentrations and the water temperature. On the other hand, there was no clearly significant difference in the Legionella concentrations when comparing the different seasons. However, a dependency is certainly conceivable under changed sampling conditions. This shows that the increased occurrence of Legionellosis in the summer months is not necessarily related to higher Legionella concentrations in drinking water. The test results also showed no indication of significant differences in consumer behavior over the course of the day and thus in flow rates. The fact that Legionella concentrations vary greatly at the various sampling points over the course of the year, but that no clear seasonal trend can be identified, confirms results from the literature that Legionella analyses are often only snapshots that may be influenced by factors other than the season. Furthermore, serogroup 1, which is most frequently described as pathogenic, was detected in only 42% of the samples examined. For the remaining samples with other serogroups of lower pathogenic potential, the interventions carried out therefore have a poorer cost-benefit ratio. The results of this study do not allow us to determine climatic periods in which monitoring could be intensified or relaxed. It would also be negligent to use isolated low or high values from a sampling point to predict a reduced or increased health risk or even to draw conclusions about an entire system. Due to the individuality of each water-carrying pipe system and the multitude of factors that influence Legionella contamination, some of which are not yet understood, transmission to another system appears to be impossible or should only be considered with extreme caution.2024-09-1
Characterization of the Phosphomimetic Mutant Y39E of a-Synuclein
Get PDFα-Synuclein (aSyn) has long been identified as a key factor in the pathogenesis of Parkinson's Disease (PD), with familial mutations in the SNCA gene contributing to the aberrant aggregation of aSyn. Its aggregation results in the formation of Lewy Bodies, a characteristic hallmark of PD, ultimately leading to neurodegeneration and cell loss. While mutations have been extensively studied, post-translational modifications (PTMs), particularly phosphorylation, have emerged as crucial players in aSyn's physiological function and pathological aggregation. The kinase c-abl is known to phosphorylate aSyn predominantly at Tyrosine 39 (Y39), a modification often observed in advanced PD stages associated with elevated c-abl levels. Given the challenges in purifying phosphorylated proteins, phosphomimetic mutants, in this case Y39E, serves to simulate phosphorylation effects.
This study extensively characterizes the Y39E aSyn mutant across various aspects, including monomeric structure and dynamics, membrane binding, in vitro and in vivo aggregation properties, dopaminergic neurodegeneration, and toxicity. Employing a multidisciplinary approach, biophysical methods such as nuclear magnet resonance (NMR), circular dichroism (CD) spectroscopy, Thioflavin-T (ThT) fluorescence measurements, and size exclusion chromatography (SEC) were combined with biological methods including molecular cloning, protein expression, and in vivo investigations using cell-based assays and the animal model C. elegans.
Structural analysis revealed that the Y39E monomer closely resembled the wild-type (WT) monomeric structure, with no significant differences in backbone dynamics nor in the hydrodynamic radius. However, slightly higher R2 relaxation rates and minor changes in transient long-range interactions were observed in the Y39E variant. The overall membrane affinity remained equal between WT and Y39E, although the NAC region of Y39E exhibited reduced interaction. Notably, during the aggregation process, Y39E incorporated fewer monomers and displayed a prolonged lag phase with concentration-dependent kinetics. In cellular and animal models, the Y39E mutant demonstrated fewer cellular inclusions and smaller aggregates, respectively. Compared to the WT species, dopaminergic neurodegeneration was significantly elevated when expressing Y39E aSyn in C. elegans, impacting the nematode's behavior, while mitochondrial pathways were ruled out as a cause of toxicity.
Additionally, data acquired on pY39 aSyn by other research groups aligned closely with results obtained in this study, indicating the Y39E mutant as a valuable tool for probing phosphorylation at this specific site, and confirming that pY39 can be mimicked by the Y39E mutant. Furthermore, the significance of pY39 is underscored by evidence of various research groups, showing that inhibiting phosphorylation effectively hinders disease progression and diminishes pathology in animal models.
In summary, this work characterizes de Y39E mutant of aSyn. The structure and dynamics of the Y39E aSyn monomer closely resembled those of the WT species. However, differences were observed in the aggregation profiles between WT and Y39E aSyn, and dopaminergic neurodegeneration was found to be elevated for the Y39E mutant. Furthermore, this work highlights the crucial role of PTMs in aSyn pathology, showing that mutations, such as Y39E emerge as valuable instruments for mimicking PTMs, thereby simplifying the research process.2024-08-1
Totale neoadjuvante Therapie des Rektumkarzinoms – Sicherheit, Durchführbarkeit und Wirksamkeit – (Langzeit-) Ergebnisse eines monozentrischen Patientenkollektivs
Get PDFMit der vorliegenden Arbeit sollte die Sicherheit, Durchführbarkeit und Wirksamkeit einer totalen neoadjuvanten Therapie (TNT) für Patienten mit lokal fortgeschrittenen Rektumkarzinomen (LARC; cUICC-Stadien II - IV; mittleres und unteres Rektum; Tumorunterrand < 12 cm ab Anokutanlinie) geprüft werden. Die Patienten (n = 62) waren im Rahmen der TransValid-KFO179/GRCSG-B-Phase- I/II-Studie (WHO-UTN-U1111-1132-0235; EudraCT-Nr.: 2011-004228-37) und der vorausgegangenen Proof-of-concept-Phase erstmals in Deutschland mit einer TNT, d. h. mit einer präoperativen 5-Fluorouracil-basierten (5-FU) Chemoradiotherapie (präopCRT) + Oxaliplatin (OX), nachfolgender FOLFOX-Konsolidierungschemotherapie (kCTx; 3 Gaben) und einer qualitätskontrollierten totalen mesorektalen Exzision (TME) behandelt worden. Das Ziel hatte darin bestanden, bei einer akzeptablen Rate an TNT-assoziierten Toxizitäten (CTC-AE-Klassifika- tion), eine höhere Rate an pathologisch-bestätigten Komplettremissionen (pCR), eine höhere CTx-Adhärenz und Überlebensvorteile vs. der Standard-Multimodaltherapie (MMT; präopCRT → TME → adjuv. (ad) CTx) zu erreichen.
Die TNT erwies sich als eine nebenwirkungsarme, hoch-effektive und zeitlich effiziente MMT mit einer hohen Adhärenz, insbesondere zur kCTx. In 96,7 % war die kCTx vollständig ± Dosisreduktionen durchgeführt worden vs. einer adCTx- Einleitung in 35 %. Die hohe Therapieeffizienz zeigte sich u. a. darin, dass ein beim Staging als prognostisch ungünstig eingeschätztes LARC (Nachweis eines N1- / N2-, eines cT3- / cT4- Status und / oder positiven zirkumferentiellen Resektionsrandes im MRT) durch das TNT-induzierte Downsizing / -staging in ein prognostisch vorteilhaftes Tumorstadium überführt werden konnte.
Im Vergleich zu den wichtigsten (internationalen) MMT-Studien war das eigene TNT-Regime mit seiner auf 3 Applikationen verkürzten kCTx der Standard-MMT im 5-Jahres-DFS (~ 70 %) und in der Rate von Fernmetastasen (22,5 %) mindestens gleichwertig. Zudem konnten für verschiedene Prädiktoren (wie z. B. für den nach TNT ermittelten ypUICC-Status ≤ II) in uni- und multivariablen Analysen deutliche Vorteile in Bezug auf das DFS, OS, CSS und auf die Rate an Fernmetastasen gezeigt werden.The aim of the present thesis was to evaluate safety, feasibility and efficacy of a total neoadjuvant therapy (TNT) for patients with locally advanced rectal cancer (LARC; cUICC stages II - IV; mid and lower rectum; lower tumor margin < 12 cm from the anal verge). All patients (n = 62) were treated as part of the TransValid- KFO179/GRCSG-B-phase-I/II-trial (EudraCT no.: 2011-004228-37; WHO-UTN- U1111-1132-0235;) and the preceding proof-of-concept phase for the first time in Germany with TNT, i. e. with a preoperative 5-fluorouracil-based (5-FU) chemoradiotherapy (preopCRT) + oxaliplatin (OX), followed by a FOLFOX consolidation chemotherapy (kCTx; 3 courses) and a quality-controlled total mesorectal excision (TME). The aim of the TNT was to achieve a higher rate of pathologically confirmed complete remissions (pCR), a higher CTx adherence and survival benefits with an acceptable rate of TNT-associated toxicities (CTC-AE classification) compared to standard multimodal therapy (MMT; preopCRT → TME → adjuvant (ad) CTx).
TNT proved to be a highly effective and time efficient MMT with low rates of adverse events and high adherence, especially to kCTx. In 96.7 %, kCTx was performed completely ± dose reductions vs. adCTx initiation in 35 %) in standard MMT. The high efficacy of TNT was demonstrated, for example, by the fact that a LARC assessed as prognostically unfavorable at initial diagnosis due to lymph node metastases, the cT status or a positive circumferential resection margin (in MRI) could be converted into a prognostically more favorable malignoma situation by downsizing / -staging with TNT.
Despite the kCTx being shortened to 3 single applications, TNT was not inferior to standard MMT in terms of the 5-year DFS (~ 70 %) and the rate of distant metastases (22.5 %). In addition, univariable and multivariable analyses showed considerable advantages for various predictors (such as status ypUICC ≤ II) regarding DFS, OS and CSS as well as the rate of distant metastases.2024-10-0