Suitability of conventional systematic vs. MRI-guided targeted biopsy approaches to assess surgical treatment delay for radical prostatectomy.

OBJECTIVES: To assess if systematic (SBx) vs. transrectal or transperineal mpMRI-ultrasound targeted combined with systematic (TBx + SBx) biopsy confer different effects on treatment delay to radical prostatectomy measured as Gleason grade group (GGG) upgrade of prostate cancer (PCa). MATERIALS AND METHODS: We relied on a multi-institutional cohort of localized PCa patients who underwent RP in Martini-Klinik, Hamburg, or Prostate Center Northwest, Gronau, between 2014 and 2022. Analyses were restricted to PCa GGG 1-3 diagnosed at SBx (n = 4475) or TBx + SBx (n = 1282). Multivariable logistic regression modeling (MVA) predicting RP GGG upgrade of ≥ 1 was performed separately for SBx and TBx + SBx. RESULTS: Treatment delay to RP of < 90, 90-180 and 180-365 days was reported in 59%, 35% and 6.2% of SBx and in 60%, 34% and 5.9% of the TBx + SBx patients, respectively. Upgrade to GGG ≥ 4 at RP was detected in 15% of SBx patients and 0.86% of TBx patients. In MVA performed for SBx, treatment delay yielded independent predictor status (OR 1.17 95% CI 1.02-1.39, p = 0.028), whereas for TBx + SBx MVA, statistical significance was not achieved. CONCLUSION: Treatment delay remained independently associated with radical prostatectomy GGG upgrade after adjustment for clinical variables in the patients diagnosed with SBx alone, but not in those who received combined TBx + SBx. These findings can be explained through inherent misclassification rates of SBx, potentially obfuscating historical observations of natural PCa progression and potential dangers of treatment delay. Thus, mpMRI-guided combined TBx + SBx appears mandatory for prospective delay-based examinations of PCa.

Real Microgravity Influences the Cytoskeleton and Focal Adhesions in Human Breast Cancer Cells.

With the increasing number of spaceflights, it is crucial to understand the changes occurring in human cells exposed to real microgravity (r-µg) conditions. We tested the effect of r-µg on MCF-7 breast cancer cells with the objective to investigate cytoskeletal alterations and early changes in the gene expression of factors belonging to the cytoskeleton, extracellular matrix, focal adhesion, and cytokines. In the Technische Experimente unter Schwerelosigkeit (TEXUS) 54 rocket mission, we had the opportunity to conduct our experiment during 6 min of r-µg and focused on cytoskeletal alterations of MCF-7 breast cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin as well as the mCherry-tubulin fusion protein using the Fluorescence Microscopy Analysis System (FLUMIAS) for fast live-cell imaging under r-µg. Moreover, in a second mission we investigated changes in RNA transcription and morphology in breast cancer cells exposed to parabolic flight (PF) maneuvers (31st Deutsches Zentrum für Luft- und Raumfahrt (DLR) PF campaign). The MCF-7 cells showed a rearrangement of the F-actin and tubulin with holes, accumulations in the tubulin network, and the appearance of filopodia- and lamellipodia-like structures in the F-actin cytoskeleton shortly after the beginning of the r-µg period. PF maneuvers induced an early up-regulation of KRT8, RDX, TIMP1, CXCL8 mRNAs, and a down-regulation of VCL after the first parabola. E-cadherin protein was significantly reduced and is involved in cell adhesion processes, and plays a significant role in tumorigenesis. Changes in the E-cadherin protein synthesis can lead to tumor progression. Pathway analyses indicate that VCL protein has an activating effect on CDH1. In conclusion, live-cell imaging visualized similar changes as those occurring in thyroid cancer cells in r-µg. This result indicates the presence of a common mechanism of gravity perception and sensation.

Microgravity Affects Thyroid Cancer Cells during the TEXUS-53 Mission Stronger than Hypergravity.

Thyroid cancer is the most abundant tumor of the endocrine organs. Poorly differentiated thyroid cancer is still difficult to treat. Human cells exposed to long-term real (r-) and simulated (s-) microgravity (µg) revealed morphological alterations and changes in the expression profile of genes involved in several biological processes. The objective of this study was to examine the effects of short-term µg on poorly differentiated follicular thyroid cancer cells (FTC-133 cell line) resulting from 6 min of exposure to µg on a sounding rocket flight. As sounding rocket flights consist of several flight phases with different acceleration forces, rigorous control experiments are mandatory. Hypergravity (hyper-g) experiments were performed at 18g on a centrifuge in simulation of the rocket launch and s-µg was simulated by a random positioning machine (RPM). qPCR analyses of selected genes revealed no remarkable expression changes in controls as well as in hyper-g samples taken at the end of the first minute of launch. Using a centrifuge initiating 18g for 1 min, however, presented moderate gene expression changes, which were significant for COL1A1, VCL, CFL1, PTK2, IL6, CXCL8 and MMP14. We also identified a network of mutual interactions of the investigated genes and proteins by employing in-silico analyses. Lastly, µg-samples indicated that microgravity is a stronger regulator of gene expression than hyper-g.

Implications of anaemia and response to anaemia treatment on outcomes in patients with cirrhosis.

Background & Aims: Anaemia is frequently observed in patients with cirrhosis and was identified as a predictor of adverse outcomes, such as increased mortality and occurrence of acute-on-chronic liver failure. To date, the possible effects of iron supplementation on these adverse outcomes are not well described. We therefore aimed to assess the role of iron supplementation in patients with cirrhosis and its capability to improve prognosis. Methods: Laboratory diagnostics were performed in consecutive outpatients with cirrhosis admitted between July 2018 and December 2019 to the University Hospital Essen. Associations with transplant-free survival were assessed in regression models. Results: A total of 317 outpatients with cirrhosis were included, of whom 61 received a liver transplant (n = 19) or died (n = 42). In multivariate Cox regression analysis, male sex (hazard ratio [HR] = 3.33, 95% CI [1.59, 6.99], p = 0.001), model for end-stage liver disease score (HR = 1.19, 95% CI [1.11, 1.27], p <0.001) and the increase of haemoglobin levels within 6 months (ΔHb6) (HR = 0.72, 95% CI [0.63, 0.83], p <0.001) were associated with transplant-free survival. Regarding the prediction of haemoglobin increase, intake of rifaximin (beta = 0.50, SD beta = 0.19, p = 0.007) and iron supplementation (beta = 0.79, SD beta = 0.26, p = 0.003) were significant predictors in multivariate analysis. Conclusions: An increase of haemoglobin levels is associated with improvement of transplant-free survival in patients with cirrhosis. Because the prediction of haemoglobin increase significantly depends on rifaximin and iron supplementation, application of these two medications can have an important impact on the outcome of these patients. Impact and implications: Anaemia is very common in patients with cirrhosis and is known to be a predictor of negative outcomes, but little is known about the effect of iron substitution in these individuals. In our cohort, increase of haemoglobin levels improved transplant-free survival of patients with cirrhosis. The increase of haemoglobin levels was mainly induced by iron supplementation and was even stronger in the case of concomitant use of iron and rifaximin. Clinical trial registration: UME-ID-10042.

Patients with Liver Cirrhosis Show High Immunogenicity upon COVID-19 Vaccination but Develop Premature Deterioration of Antibody Titers.

SARS-CoV-2 infection is known to lead to severe morbidity and mortality in patients with liver cirrhosis. For this reason, vaccination of these patients against COVID-19 is widely recommended. However, data regarding immunogenicity in patients with liver cirrhosis is limited and even less is known about the kinetics of antibody response, as well as the optimal timing of booster immunization. We analyzed immunogenicity in 110 patients with liver cirrhosis after receiving two doses of the mRNA-based vaccine BNT162b2 following the standard protocol and compared these results to a control group consisting of 80 healthcare workers. One hundred and six patients with liver cirrhosis (96%) developed antibodies against SARS-CoV-2, compared to 79 (99%) in the control group (p = 0.400). Still, the median SARS-CoV-2 IgG titer was significantly lower in patients with liver cirrhosis compared to the control group (939 vs. 1905 BAU/mL, p = 0.0001). We also analyzed the strength of the antibody response in relation to the time between the second dose and antibody detection. Antibody titers remained relatively stable in the control group while showing a rapid and significant decrease in patients with liver cirrhosis. In conclusion, our data reveals a favorable initial outcome after vaccination with the COVID-19 vaccine BNT162b2 in cirrhotic patients but show a rapid deterioration of the antibody response after time, thereby giving a strong hint towards the importance of early booster immunization for this group of patients.

Response to olaparib in a PALB2 germline mutated prostate cancer and genetic events associated with resistance.

Prostate cancers harboring DNA repair gene alterations are particularly sensitive to PARP inhibitor treatment. We report a case of an advanced prostate cancer patient profiled within the NCT-MASTER (Molecularly Aided Stratification for Tumor Eradication Research) precision oncology program using next-generation sequencing. Comprehensive genomic and transcriptomic analysis identified a pathogenic germline PALB2 variant as well as a mutational signature associated with disturbed homologous recombination together with structural genomic rearrangements. A molecular tumor board identified a potential benefit of targeted therapy and recommended PARP inhibition and platinum-based chemotherapy. Single-agent treatment with the PARP inhibitor olaparib as well as subsequent combination with platinum-based chemotherapy resulted in disease stabilization and substantial improvement of clinical symptoms. Upon progression, we performed whole-exome and RNA sequencing of a liver metastasis, which demonstrated up-regulation of several genes characteristic for the neuroendocrine prostate cancer phenotype as well as a novel translocation resulting in an in-frame, loss-of-function fusion of RB1. We suggest that multidimensional genomic characterization of prostate cancer patients undergoing PARP inhibitor therapy will be necessary to capture and understand predictive biomarkers of PARP inhibitor sensitivity and resistance.

Rapid coupling between gravitational forces and the transcriptome in human myelomonocytic U937 cells.

The gravitational force has been constant throughout Earth's evolutionary history. Since the cell nucleus is subjected to permanent forces induced by Earth's gravity, we addressed the question, if gene expression homeostasis is constantly shaped by the gravitational force on Earth. We therefore investigated the transcriptome in force-free conditions of microgravity, determined the time frame of initial gravitational force-transduction to the transcriptome and assessed the role of cation channels. We combined a parabolic flight experiment campaign with a suborbital ballistic rocket experiment employing the human myelomonocytic cell line U937 and analyzed the whole gene transcription by microarray, using rigorous controls for exclusion of effects not related to gravitational force and cross-validation through two fully independent research campaigns. Experiments with the wide range ion channel inhibitor SKF-96365 in combination with whole transcriptome analysis were conducted to study the functional role of ion channels in the transduction of gravitational forces at an integrative level. We detected profound alterations in the transcriptome already after 20 s of microgravity or hypergravity. In microgravity, 99.43% of all initially altered transcripts adapted after 5 min. In hypergravity, 98.93% of all initially altered transcripts adapted after 75 s. Only 2.4% of all microgravity-regulated transcripts were sensitive to the cation channel inhibitor SKF-96365. Inter-platform comparison of differentially regulated transcripts revealed 57 annotated gravity-sensitive transcripts. We assume that gravitational forces are rapidly and constantly transduced into the nucleus as omnipresent condition for nuclear and chromatin structure as well as homeostasis of gene expression.

Thyroid cancer cells in space during the TEXUS-53 sounding rocket mission - The THYROID Project.

Human follicular thyroid cancer cells (FTC-133) were sent to space via a sounding rocket during the TEXUS-53 mission to determine the impact of short-term microgravity on these cells. To enable cell culture and fixation in real microgravity, an automated experiment container (EC) was constructed. In order to ensure safe cell culture, cell-chambers consisting of polycarbonate (PC) material were used. They were highly biocompatible as proved by measuring cell survival using Annexin V flow cytometry. In the follow-up experiment, FTC-133 cells were sent to space via a sounding rocket and were fixed before and after the microgravity (µg) phase with RNAlater. In addition, cells were tested for reactions on hypergravity (hyper-g) as much as 18 g to determine whether worst case acceleration during launch can have an influence on the cells. We investigated genes belonging to biological processes such as cytoskeleton, cell adhesion, tumor growth, angiogenesis and apoptosis. Pathway analyses revealed central functions of VEGFA and EGF. EGF upregulates aspartate beta-hydroxylase (ASPH) which is influencing CASP3. Hyper-g induced a significant up-regulation of TUBB1, VIM, RDX, CAV1, VEGFA and BCL2. FTC-133 cells grown in an automated EC exposed to µg revealed moderate gene expression changes indicating their survival in orbit.

Stability of gene expression in human T cells in different gravity environments is clustered in chromosomal region 11p15.4.

In the last decades, a plethora of in vitro studies with living human cells contributed a vast amount of knowledge about cellular and molecular effects of microgravity. Previous studies focused mostly on the identification of gravity-responsive genes, whereas a multi-platform analysis at an integrative level, which specifically evaluates the extent and robustness of transcriptional response to an altered gravity environment was not performed so far. Therefore, we investigated the stability of gene expression response in non-activated human Jurkat T lymphocytic cells in different gravity environments through the combination of parabolic flights with a suborbital ballistic rocket and 2D clinostat and centrifuge experiments, using strict controls for excluding all possible other factors of influence. We revealed an overall high stability of gene expression in microgravity and identified olfactory gene expression in the chromosomal region 11p15.4 as particularly robust to altered gravity. We identified that classical reference genes ABCA5, GAPDH, HPRT1, PLA2G4A, and RPL13A were stably expressed in all tested gravity conditions and platforms, while ABCA5 and GAPDH were also known to be stably expressed in U937 cells in all gravity conditions. In summary, 10-20% of all transcripts remained totally unchanged in any gravitational environment tested (between 10-4 and 9 g), 20-40% remained unchanged in microgravity (between 10-4 and 10-2 g) and 97-99% were not significantly altered in microgravity if strict exclusion criteria were applied. Therefore, we suppose a high stability of gene expression in microgravity. Comparison with other stressors suggests that microgravity alters gene expression homeostasis not stronger than other environmental factors.

Dynamic gene expression response to altered gravity in human T cells.

We investigated the dynamics of immediate and initial gene expression response to different gravitational environments in human Jurkat T lymphocytic cells and compared expression profiles to identify potential gravity-regulated genes and adaptation processes. We used the Affymetrix GeneChip® Human Transcriptome Array 2.0 containing 44,699 protein coding genes and 22,829 non-protein coding genes and performed the experiments during a parabolic flight and a suborbital ballistic rocket mission to cross-validate gravity-regulated gene expression through independent research platforms and different sets of control experiments to exclude other factors than alteration of gravity. We found that gene expression in human T cells rapidly responded to altered gravity in the time frame of 20 s and 5 min. The initial response to microgravity involved mostly regulatory RNAs. We identified three gravity-regulated genes which could be cross-validated in both completely independent experiment missions: ATP6V1A/D, a vacuolar H + -ATPase (V-ATPase) responsible for acidification during bone resorption, IGHD3-3/IGHD3-10, diversity genes of the immunoglobulin heavy-chain locus participating in V(D)J recombination, and LINC00837, a long intergenic non-protein coding RNA. Due to the extensive and rapid alteration of gene expression associated with regulatory RNAs, we conclude that human cells are equipped with a robust and efficient adaptation potential when challenged with altered gravitational environments.

Alterations of the cytoskeleton in human cells in space proved by life-cell imaging.

Microgravity induces changes in the cytoskeleton. This might have an impact on cells and organs of humans in space. Unfortunately, studies of cytoskeletal changes in microgravity reported so far are obligatorily based on the analysis of fixed cells exposed to microgravity during a parabolic flight campaign (PFC). This study focuses on the development of a compact fluorescence microscope (FLUMIAS) for fast live-cell imaging under real microgravity. It demonstrates the application of the instrument for on-board analysis of cytoskeletal changes in FTC-133 cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin during the 24(th) DLR PFC and TEXUS 52 rocket mission. Although vibration is an inevitable part of parabolic flight maneuvers, we successfully for the first time report life-cell cytoskeleton imaging during microgravity, and gene expression analysis after the 31(st) parabola showing a clear up-regulation of cytoskeletal genes. Notably, during the rocket flight the FLUMIAS microscope reveals significant alterations of the cytoskeleton related to microgravity. Our findings clearly demonstrate the applicability of the FLUMIAS microscope for life-cell imaging during microgravity, rendering it an important technological advance in live-cell imaging when dissecting protein localization.

Functional activity of plasmid DNA after entry into the atmosphere of earth investigated by a new biomarker stability assay for ballistic spaceflight experiments.

Sounding rockets represent an excellent platform for testing the influence of space conditions during the passage of Earth's atmosphere and re-entry on biological, physical and chemical experiments for astrobiological purposes. We designed a robust functionality biomarker assay to analyze the biological effects of suborbital spaceflights prevailing during ballistic rocket flights. During the TEXUS-49 rocket mission in March 2011, artificial plasmid DNA carrying a fluorescent marker (enhanced green fluorescent protein: EGFP) and an antibiotic resistance cassette (kanamycin/neomycin) was attached on different positions of rocket exterior; (i) circular every 90 degree on the outer surface concentrical of the payload, (ii) in the grooves of screw heads located in between the surface application sites, and (iii) on the surface of the bottom side of the payload. Temperature measurements showed two major peaks at 118 and 130 °C during the 780 seconds lasting flight on the inside of the recovery module, while outer gas temperatures of more than 1000 °C were estimated on the sample application locations. Directly after retrieval and return transport of the payload, the plasmid DNA samples were recovered. Subsequent analyses showed that DNA could be recovered from all application sites with a maximum of 53% in the grooves of the screw heads. We could further show that up to 35% of DNA retained its full biological function, i.e., mediating antibiotic resistance in bacteria and fluorescent marker expression in eukaryotic cells. These experiments show that our plasmid DNA biomarker assay is suitable to characterize the environmental conditions affecting DNA during an atmospheric transit and the re-entry and constitute the first report of the stability of DNA during hypervelocity atmospheric transit indicating that sounding rocket flights can be used to model the high-speed atmospheric entry of organics-laden artificial meteorites.