Charakterisierung und molekulare Mechanismen Therapie-induzierter Seneszenz durch epigenetisch aktive Therapeutika

Ziel dieser Promotionsarbeit ist die Identifizierung und Charakterisierung epigenetischer Wirkmechanismen einer Therapie-induzierten Seneszenz in Tumorzellen. Um mögliche intrazelluläre, Seneszenz-induzierende Signalwege aufzudecken, sollten potentielle Überschneidungen der Wirkmechanismen der primär unterschiedlich wirkenden Substanzen 5-aza-dC und des Topoisomerase II-Inhibitors Doxorubicin auf Ebene der Zellkultur in den Hepatomzelllinien HepG2 und Hep3B phänotypisch untersucht werden. Der erste Schwerpunkt der Promotionsarbeit lag in einer vergleichenden Betrachtung des miRNoms beider Substanzen. 5-aza-dC als DNMTi reguliert je nach verwendeter Methode und miRBase-Version 16,3% bzw. 12,8% aller miRNAs und somit deutlich mehr und zusätzlich stärker als Doxorubicin mit 3,2 %. Es konnte gezeigt werden, dass miR-494 und miR-369-5p die einzig gemeinsam regulierten miRNAs darstellen. miR-494 war zudem in der Lage, Effekte der Chemotherapeutika bezüglich der Proliferationshemmung und der SA-ßGal-Modulation nachzuahmen. Eine AntagomiR-494-Transfektion führte jedoch nicht zur Aufhebung der 5-aza-dC- und Doxorubicin-vermittelten Effekte, so dass die Therapie-induzierte Seneszenz dieser beiden Substanzen nicht ausschließlich auf eine Modulation von miR-494 zurückgeführt werden konnte. Der zweite Schwerpunkt der Promotionsarbeit lag auf der Untersuchung des Einflusses von HDAC4 in der Wirkungsweise von 5-aza-dC und Doxorubicin, da beide Chemotherapeutika zu einer Abnahme des Proteingehalts von HDAC4 führten. Beide Substanzen können somit über eine HDAC4-Regulation epigenetische Mechanismen für eine transkriptionelle Aktivität regulieren, die nicht ihrem primären Wirkmechanismus entsprechen. Neben einer miR-1 vermittelten Abnahme des Proteingehaltes von HDAC4, ahmte ebenfalls ein spezifischer siRNA-vermittelter HDAC4-Knockdown die Proliferationshemmung und die SA-ßGal-Modulation von 5-aza-dC und Doxorubicin in HepG2-Zellen nach. Eine HDAC4-Überexpression führte zudem zu einer Abnahme der von 5-aza-dC- und Doxorubicin-induzierten SA-ßGal-Färbung als Surrogat-Parameter einer Therapie-induzierten Seneszenz. Es ergeben sich Hinweise darauf, dass die HDAC4-Regulation maßgeblich an der Seneszenzinduktion durch 5-aza-dC und Doxorubicin beteiligt sein könnte. Dass HDAC4 hierfür ein wesentlicher Faktor sein dürfte, kann durch dessen pleiotrope Wirkungsweise erklärt werden. Eine Überprüfung eines HDAC4-Knockdowns weiterer p53-kompetenter und –nicht-kompetenter Tumorzelllinien in vitro und in vivo sollte als Ausblick zur weiteren Bestätigung dieser Beobachtung durchgeführt werden. Im Gegensatz zu den bisher klinisch eingesetzten HDACi, welche meist mehrere HDACs inhibieren und als Pan-HDACi gelten, hätte ein selektiver HDAC4-Knockdown bzw. -Inhibierung den Vorteil eines potentiell geringeren Nebenwirkungsprofils und nach unseren Daten das Potential einer gezielten Auslösung einer Therapie-induzierten Seneszenz. Zusammenfassend konnte in dieser Promotionsarbeit die Modulation der Faktoren miR-494, miR-1 und HDAC4 als molekulare Mechanismen einer Therapie-induzierten Seneszenz identifiziert werden. Inwieweit eine gezielte Modulation dieser Zielstrukturen für therapeutische Zwecke ausgenutzt werden kann, bedarf einer über den Rahmen dieser Promotionsarbeit hinausgehenden Überprüfung

Retrospective longitudinal assessment of optic nerve sheath diameter in patients with malignant glioma

Abstract Introduction Glioblastoma (GBM) is a tumor with rapid growth and a possible relationship to elevated intracranial pressure (ICP). High ICP may not always be associated with clinical signs. A non‐invasive technique for assessment of ICP is measuring the optic nerve sheath diameter (ONSD). Identifying patients who need immediate intervention is of importance in neuro‐oncological care. The goal of this study is to assess the available magnetic resonance imaging (MRI) of patients with GBM with respect to pre‐ and postoperative ONSD. Methods and Materials Retrospective data analysis was performed on all patients operated for GBM at a tertiary care center between 2010 and 2020. Two pre and one postoperative MRI had to be available. Clinical data and ONSD at multiple time points were analyzed and correlated, as well as preoperative volumetrics. Results Sixty‐seven patients met the inclusion criteria. Clinical signs of elevated ICP were seen in 25.4% (n = 17), while significant perifocal edema was present in 67.2% (n = 45) of patients. Clinical signs of preoperatively elevated ICP were associated with significantly elevated ONSD at diagnosis (p < 0.001) as well as preoperative tumor volume (p < 0.001). Significant perifocal edema at the time of diagnosis was associated with elevated ONSD (p = 0.029) and higher tumor volume (p = 0.003). In patients with significant edema, ONSD increased significantly between preoperative MRIs (p = 0.003/005). In patients with clinical signs of raised ICP, ONSD also increased, whereas it was stable in asymptomatic patients (yes: 5.01+/−4.17 to 5.83+/−0.55 mm, p = 0.010, no: 5.17+/−0.46 mm to 5.38+/−0.41 mm, p = 0.81). A significant increase of ONSD from diagnosis to preoperative MRI and a significant decrease until 3 months postoperatively were observed (p < 0.001). Conclusions ONSD might help identify high ICP in patients with GBM. In this first‐of‐its kind study, we observed a significant increase of ONSD preoperatively, likely associated with edema. Postoperatively, ONSD decreased significantly until 3 months after surgery and increased again at 12 months. Further prospective data collection is warranted

Transarterial Chemoembolization of Hepatocellular Carcinoma Using Radiopaque Drug-Eluting Embolics: Impact of Embolic Density and Residual Tumor Perfusion on Tumor Recurrence and Survival

Purpose!#!To evaluate the value of dual-phase parenchymal blood volume (PBV) C-arm mounted cone-beam-CT (CBCT) to enable assessment of radiopaque, doxorubicin-loaded drug-eluting embolics (rDEE) based on the visual degree of embolization, embolic density and residual tumor perfusion as early predictors for tumor recurrence after transarterial chemoembolization (TACE) of hepatocellular carcinoma (HCC).!##!Material and methods!#!Thirty patients (50 HCCs) were prospectively enrolled, underwent cross-sectional imaging before and after TACE using 100-300 µm rDEE and had regular follow-up examinations. Directly before and after the TACE procedure, PBV-CBCT was acquired. The response was evaluated and compared to visual degree of embolization (DE) and embolic density (ED) of rDEE deposits, as well as the presence of residual tumor perfusion (RTP) derived from PBV-CBCT. Outcome was assessed by mid-term tumor response applying mRECIST and patient survival after 12 months.!##!Results!#!RTP was detected in 16 HCCs and correlated negatively with DE (p = .03*) and ED (p = .0009*). The absence of RTP significantly improved lesion-based mid-term response rates regarding complete response (CR, 30/34 (88%) vs 2/16 (12.5%), p = .0002*), lesion-based complete response rate was 75% (21/28) for DE ≥ 50% vs. 50% (11/22) for DE &amp;lt; 50% (p =  .08) and 82% (27/33) for ED ≥ 2 vs. 29% for ED &amp;lt; 2 (5/17), p =  .005*). Thirteen patients were treated with re-TACE within 12 months, 11 of which had shown RTP. 12-month survival rate was 93%.!##!Conclusion!#!Residual tumor perfusions as assessed by PBV-CBCT during rDEE-TACE proved to be the best parameter to predict mid-term response. 'Level of Evidence: Level 3'

Detection of spinal long fiber tract degeneration in HSP: Improved diffusion tensor imaging

Spinal diffusion tensor imaging (sDTI) is still a challenging technique for selectively evaluating anatomical areas like the pyramidal tracts (PT), dorsal columns (DC), and anterior horns (AH) in clinical routine and for reliably quantifying white matter anisotropy and diffusivity. In neurodegenerative diseases, the value of sDTI is promising but not yet well understood. The objective of this prospective, single-center study was to evaluate the long fiber tract degeneration within the spinal cord in normal aging (n = 125) and to prove its applicability in pathologic conditions as in patients with molecular genetically confirmed hereditary spastic paraplegias (HSP; n = 40), a prototypical disease of the first motor neuron and in some genetic variants with affection of the dorsal columns. An optimized monopolar Stejskal-Tanner sequence for high-resolution, axial sDTI of the cervical spinal cord at 3.0 T with advanced standardized evaluation methods was developed for a robust DTI value estimation of PT, DC, and AH in both groups. After sDTI measurement at C2, an automatic motion correction and an advanced semi-automatic ROI-based, standardized evaluation of white matter anisotropy and diffusivity was performed to obtain regional diffusivity measures for PT, DC, and AH. Reliable and stable sDTI values were acquired in a healthy population without significant decline between age 20 and 65. Reference values for PT, DC, and AH for fractional anisotropy (FA), mean diffusivity (MD), and radial diffusivity (RD) were established. In HSP patients, the decline of the long spinal fiber tracts could be demonstrated by diffusivity abnormalities in the pyramidal tracts with significantly reduced PT(FA) (p < 0.001), elevated PT(RD) (p = 0.002) and reduced PT(MD) (p = 0.003) compared to healthy controls. Furthermore, FA was significantly reduced in DC(FA) (p < 0.001) with no differences in AH. In a genetically homogeneous subgroup of SPG4 patients (n = 12) with affection of the dorsal columns, DC(RD) significantly correlated with the overall disease severity as measured by the Spastic Paraplegia Rating Scale (SPRS) (r = − 0.713, p = 0.009). With the most extensive sDTI study in vivo to date, we showed that axial sDTI combined with motion correction and advanced data post-processing strategies enables robust measurements and is ready to use, allowing recognition and quantification of disease- and age-related changes of the PT, DC, and AH. These results may also encourage the usage of sDTI in other neurodegenerative diseases with spinal cord involvement to explore its capability as selective biomarkers