Limited utility of qPCR-based detection of tumor-specific circulating mRNAs in whole blood from clear cell renal cell carcinoma patients

BACKGROUND: RNA sequencing data is providing abundant information about the levels of dysregulation of genes in various tumors. These data, as well as data based on older microarray technologies have enabled the identification of many genes which are upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched normal tissue. Here we use RNA sequencing data in order to construct a panel of highly overexpressed genes in ccRCC so as to evaluate their RNA levels in whole blood and determine any diagnostic potential of these levels for renal cell carcinoma patients. METHODS: A bioinformatics analysis with Python was performed using TCGA, GEO and other databases to identify genes which are upregulated in ccRCC while being absent in the blood of healthy individuals. Quantitative Real Time PCR (RT-qPCR) was subsequently used to measure the levels of candidate genes in whole blood (PAX gene) of 16 ccRCC patients versus 11 healthy individuals. PCR results were processed in qBase and GraphPadPrism and statistics was done with Mann-Whitney U test. RESULTS: While most analyzed genes were either undetectable or did not show any dysregulated expression, two genes, CDK18 and CCND1, were paradoxically downregulated in the blood of ccRCC patients compared to healthy controls. Furthermore, LOX showed a tendency towards upregulation in metastatic ccRCC samples compared to non-metastatic. CONCLUSIONS: This analysis illustrates the difficulty of detecting tumor regulated genes in blood and the possible influence of interference from expression in blood cells even for genes conditionally absent in normal blood. Testing in plasma samples indicated that tumor specific mRNAs were not detectable. While CDK18, CCND1 and LOX mRNAs might carry biomarker potential, this would require validation in an independent, larger patient cohort

Cooperative Effect of miR-141-3p and miR-145-5p in the Regulation of Targets in Clear Cell Renal Cell Carcinoma

Background Due to the poor prognosis for advanced renal cell carcinoma (RCC), there is an urgent need for new therapeutic targets and for prognostic markers to identify high risk tumors. MicroRNAs (miRNAs) are frequently dysregulated in tumors, play a crucial role during carcinogenesis and therefore might be promising new biomarkers. In previous studies, we identified miR-141-3p and miR-145-5p to be downregulated in clear cell RCC (ccRCC). Our objective was to investigate the functional association of these miRNAs, focusing on the cooperative regulation of new specific targets and their role in ccRCC progression. Methods The effect of miR-141-3p and miR-145-5p on cell migration was examined by overexpression in 786-O cells. New targets of both miRNAs were identified by miRWalk, validated in 786-O and ACHN cells and additionally characterized in ccRCC tissue on mRNA and protein level. Results In functional analysis, a tumor suppressive effect of miR-141-3p and miR-145-5p by decreasing migration and invasion of RCC cells could be shown. Furthermore, co-overexpression of the miRNAs seemed to result in an increased inhibition of cell migration. Both miRNAs were recognized as post-transcriptional regulators of the targets EAPP, HS6ST2, LOX, TGFB2 and VRK2. Additionally, they showed a cooperative effect again as demonstrated by a significantly increased inhibition of HS6ST2 and LOX expression after simultaneous overexpression of both miRNAs. In ccRCC tissue, LOX mRNA expression was strongly increased compared to normal tissue, allowing also to distinguish between non-metastatic and already metastasized primary tumors. Finally, in subsequent tissue microarray analysis LOX protein expression showed a prognostic relevance for the overall survival of ccRCC patients. Conclusion These results illustrate a jointly strengthening effect of the dysregulated miR-141-3p and miR-145-5p in various tumor associated processes. Focusing on the cooperative effect of miRNAs provides new opportunities for the development of therapeutic strategies and offers novel prognostic and diagnostic capabilities

Piwi-interacting RNAs as novel prognostic markers in clear cell renal cell carcinomas

Background Piwi-interacting RNAs (piRNAs) are small RNAs of 27–30 nucleotides mapping to transposons or clustering in repeat genomic regions. Preliminary studies suggest an important role in cancerogenesis. This study is the first one investigating their prognostic impact in clear cell renal cell cancer (ccRCC) patients. Methods Three piRNAs (piR-30924, piR-57125, and piR-38756) selected on the basis of initial piRNA microarray analyses were determined using RT-qPCR in non-metastatic (n = 76) and metastatic (n = 30) ccRCC tissue at the time of nephrectomy in comparison to normal renal tissue (n = 77) and tissue from distant ccRCC metastases (n = 13). Primary clinical end points were recurrence-free and overall survival. Results piR-57125 showed lower expression in metastatic than in non-metastatic tumors, whereas the expression of piR-30924 and piR-38756 increased in metastatic tumors. The higher expression of piR-30924 and piR-38756 as well as the lower expression of piR-57125 in metastatic primary tumors were significantly associated with tumor recurrence and overall survival. Multivariate Cox regression analyses revealed both piR-30924 and piR-57125 as independent prognostic predictors. This impact was even more pronounced in non-metastatic patients. Conclusions This study demonstrates that the expression levels of these piRNAs in primary non-metastatic and metastatic ccRCC tissue can serve as potential prognostic biomarkers in combination with clinicopathological factors

High-pressure calorimetry: Thermophysical properties of gases and polymers

During the last decades polymers were important materials for technological progress of our western consumption society. The importance of advanced polymer materials is still increasing and polymers are not only used for packages anymore, but for high-value products such as high-voltage cable insulations, solar cell coatings or medical applications. Due to that, the necessity for optimized and energy efficient processes including a proper waste treatment is increasing. In 2015 worldwide plastic productions were at 322 million tons, mostly common polymers are Polypropylene (PP) and polyethylene (PE) with a content of 48.5%. [1] Some of the production processes, for example low-density polyethylene (LDPE), are performed at high pressures and temperatures of up to 3000 bar and 300 °C. To optimize respective processes, simulation-models have been used more commonly during the last years. Computational models are a comparatively cheap and safe alternative in comparison to actual changes in the process and can be used to test new concepts or recipes. Beside kinetics, simulation-models are based on thermophysical properties such as density or heat capacity. Correlations of these parameters are applied to describe the heat balance of the process and therefore influence the conversion during a simulation-run. To describe a process as detailed as possible, correlations of applied components like monomers or polymers have to rely on exact substance data. Although the temperature dependence of different properties is described well by currently applied correlations, a pressure dependence is mainly achieved by extrapolating fragmentary datasets. These data-sets were partly determined at atmospheric pressure and have not been evaluated further since their publication during the second half of the last century, which makes an application at the demanding polymerization process conditions rather imprecise.Consequently, for an improvement of such simulation-models new and exact substance data at the respective process conditions are required. In this work high-pressure calorimetry has been used for this purpose. Regular calorimetric devices, such as the DSC, have been applied to determine thermophysical properties at atmospheric pressure, but they are not suited for measurements at higher pressures. Such conditions require precise sensor technologies combined with rather massive mountings to withstand the extreme conditions. One device, which merges these properties is the transitiometer. This special Tian-Calvet calorimeter can withstand pressures up to 4000 bar without a decrease in precision. In a measurement one parameter (temperature, pressure or volume) is kept constant, the second one is varied with time and the third is measured as a function of the first two. Applying a pressure program in the transitiometer, expansion coefficients of different gases and polymers could be calculated when evaluating the measured difference heat fluxes. Coming from a temperature and pressure dependence of expansion coefficients, heat capacities could be determined in both dependencies as well. While testing different experimental setups, a pressure- and temperature dependence of densities of polymers could be calculated as well. In heat capacities of carbon dioxide, ethane or ethene a maximum in the temperature dependence in all measured pressures could be observed, which cannot be seen in methane or nitrogen. Beside a temperature-dependent maximum, a pressure dependent maximum is indicated in those molecules as well. Those maxima might be first experimental evidence of results regarding different molecular-dynamic simulations. Here, phenomena in the super-critical area of a phase diagram so called “Widom-Line” and/or “Frenkel-Line” that indicate a “pseudo” phase transition are predicted. That phenomena divide assumed homogeneous phase of the supercritical area, in a gas-like and a liquid-like region. [2] Next steps will be to validate measured data sets and to compare different types of polymers, including homo- and co-polymers. Additionally, it will be necessary to investigate not only pure substances such as polymers or gases, but to examine excess- and solubility effects between different molecules. First indications of such effects have already been observed since melting points of polymers change with pressure and different pressure media. Such effects might also influence already determined heat capacities or expansion coefficients. Another important task in the future will be the investigation of peroxide kinetics in dependence of pressure, temperature and solvent viscosity. [1] PlasticsEurope, 2016, http://www.plasticseurope.org/documents/document/20161014113313-plastics_the_facts_2016_final_version.pdf [2] T. Bryk and T. Scopigno, J. Phys. Chem. Lett., 2017, 8 (20), pp 4995–5001

miR-9-5p in Nephrectomy Specimens is a Potential Predictor of Primary Resistance to First-Line Treatment with Tyrosine Kinase Inhibitors in Patients with Metastatic Renal Cell Carcinoma

Approximately 20-30% of patients with metastatic renal cell carcinoma (mRCC) in first-line treatment with tyrosine kinase inhibitors (TKIs) do not respond due to primary resistance to this drug. At present, suitable robust biomarkers for prediction of a response are not available. Therefore, the aim of this study was to evaluate a panel of microRNAs (miRNAs) in nephrectomy specimens for use as predictive biomarkers for TKI resistance. Archived formalin-fixed, paraffin embedded nephrectomy samples from 60 mRCC patients treated with first-line TKIs (sunitinib, n = 51; pazopanib, n = 6; sorafenib, n = 3) were categorized into responders and non-responders. Using the standard Response Evaluation Criteria in Solid Tumors, patients with progressive disease within 3 months after the start of treatment with TKI were considered as non-responders and those patients with stable disease and complete or partial response under the TKI treatment for at least 6 months as responders. Based on a miRNA microarray expression profile in the two stratified groups of patients, seven differentially expressed miRNAs were validated using droplet digital reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) assays in the two groups. Receiver operating characteristic curve analysis and binary logistic regression of response prediction were performed. MiR-9-5p and miR-489-3p were able to discriminate between the two groups. MiR-9-5p, as the most significant miRNA, improved the correct prediction of primary resistance against TKIs in comparison to that of conventional clinicopathological variables. The results of the decision curve analyses, Kaplan-Meier analyses and Cox regression analyses confirmed the potential of miR-9-5p in the prediction of response to TKIs and the prediction of progression-free survival after the initiation of TKI treatment

Use of quantitative T2 mapping for the assessment of renal cell carcinomas: first results

Background: Correct staging and grading of patients with clear cell renal cell carcinoma (cRCC) is of clinical relevance for the prediction of operability and for individualized patient management. As partial or radial resection with postoperative tumor grading currently remain the methods of choice for the classification of cRCC, non-invasive preoperative alternatives to differentiate lower grade from higher grade cRCC would be beneficial. Methods: This institutional-review-board approved cross-sectional study included twenty-seven patients (8 women, mean age ± SD, 61.3 ± 14.2) with histopathologically confirmed cRCC, graded according to the International Society of Urological Pathology (ISUP). A native, balanced steady-state free precession T2 mapping sequence (TrueFISP) was performed at 1.5 T. Quantitative T2 values were measured with circular 2D ROIs in the solid tumor portion and also in the normal renal parenchyma (cortex and medulla). To estimate the optimal cut-off T2 value for identifying lower grade cRCC, a Receiver Operating Characteristic Curve (ROC) analysis was performed and sensitivity and specificity were calculated. Students’ t-tests were used to evaluate the differences in mean values for continuous variables, while intergroup differences were tested for significance with two-tailed Mann-Whitney-U tests. Results: There were significant differences between the T2 values for lower grade (ISUP 1–2) and higher grade (ISUP 3–4) cRCC (p < 0.001), with higher T2 values for lower grade cRCC compared to higher grade cRCC. The sensitivity and specificity for the differentiation of lower grade from higher grade tumors were 83.3% (95% CI: 0.59–0.96) and 88.9% (95% CI: 0.52–1.00), respectively, using a threshold value of ≥110 ms. Intraobserver/interobserver agreement for T2 measurements was excellent/substantial. Conclusions: Native T2 mapping based on a balanced steady-state free precession MR sequence might support an image-based distinction between lower and higher grade cRCC in a two-tier-system and could be a helpful addition to multiparametric imaging

Identifying Customer Values of B2C-Fintech Services in the Area of Personal Financial Management

Banks face the challenge of providing value that consumers are comfortable paying for. Although customer value is essential for fintech services, scant research exclusively focuses on technological advantages. This paper applies the theValue-Focused-Thinking approach to identify which values fintech services regarding personal financial management (PFM) can create for customers. Through 24 qualitative interviews, we identify 14 fundamental objectives and 15means objectives, which represent the potential customer values of PFM services(PFMS). The relationships of the identified objectives are illustrated in aMeans-Objective-Network. We prioritized the identified values through a quantitative online survey with 167 potential customers. The results provide insights into the characteristics that PFMSs should have to achieve the highest possible value for customers. Customers see PFMS as valuable if they deem the service trustworthy and give them control over their finances. This paper provides an early exploratory research contribution about the customer values of PFMSs

Measurement of Spin Correlation Parameters ANN_{NN}, ASS_{SS}, and A_SL{SL} at 2.1 GeV in Proton-Proton Elastic Scattering

At the Cooler Synchrotron COSY/J\"ulich spin correlation parameters in elastic proton-proton (pp) scattering have been measured with a 2.11 GeV polarized proton beam and a polarized hydrogen atomic beam target. We report results for A$_{NN}$, A$_{SS}$, and A_${SL}$ for c.m. scattering angles between 30$^o$ and 90$^o$. Our data on A$_{SS}$ -- the first measurement of this observable above 800 MeV -- clearly disagrees with predictions of available of pp scattering phase shift solutions while A$_{NN}$ and A_${SL}$ are reproduced reasonably well. We show that in the direct reconstruction of the scattering amplitudes from the body of available pp elastic scattering data at 2.1 GeV the number of possible solutions is considerably reduced.Comment: 4 pages, 4 figure

Rock characteristics and reservoir properties of Upper Carboniferous (Stephanian A–B) tight siliciclastic rocks from the Saar–Nahe basin (SW Germany)

The environmental consequences of mine flooding in the Saar hard coal district, post-mining re-use concepts in the course of the energy transition, and the potential of coalbed methane production require an understanding of subsurface rock properties on the microscale. In this study, mineralogy, microtexture, microstructure, porosity, permeability, and geochemistry of an Upper Carboniferous (Stephanian A–B) drill core recovered in the Saar–Nahe basin are quantified. Based on these data, the diagenetic history and reservoir quality are analyzed regarding mine flooding and coalbed methane potential. The feldspar-poor and igneous rock fragment-free siliciclastic rock succession shows multiple fining upward sequences deposited in a fluvial environment during the pre-volcanic syn-rift phase of the Variscan intramontane Saar–Nahe basin. Intercalated small-scale coarsening upward sequences are related to the floodplain where near-surface soft-sediment deformation and paleosol formation took place. Porosity (< 7%) of the tight siliciclastic rocks is mainly controlled by an interplay of authigenic microporous kaolinite, dissolution porosity, and quartz cement, whereas permeability (< 0.05 mD) shows no systematic variation with petrography. During burial, quartz cements preserved porosity by stabilizing the granular framework against mechanical compaction, while phyllosilicates were ductilely deformed reducing reservoir quality. Relative phyllosilicates and quartz contents and mean grain size are reliably inferred from SiO$_2$/Al$_2$O$_3$ ratios (1.8–28.8), Ba (0.0108–0.0653 wt%), Rb (0.0024–0.0181 wt%), and Sr (0.0013–0.0086 wt%) concentrations measured with a portable x-ray fluorescence analyzer. Regarding coalbed methane production and mine flooding, sealing of cleats and heterogeneous subsurface rock properties due to dynamically changing depositional settings during the Late Carboniferous need to be considered

Morphing Attack Detection using Laplace operator based features

The vulnerability of facial recognition systems through morphing attacks is a known problem. Since the first publication about this vulnerability of facial recognition systems, a variety of morphing attack detection methods have been presented, promising an automated detection of such fraudulent attacks. In this work, a new approach is presented attempting to distinguish bona fide from morphed images based on information about the edges in the image extracted by the Laplace operator. It can be demonstrated that the features employed contain information that can contribute to the detection of morphed face images