Prognosefaktoren und Therapiestrategien bei Patienten mit lokal fortgeschrittenem oder metastasiertem Nierenzellkarzinom

Das Nierenzellkarzinom (NZK) stellt weltweit die dritthäufigste bösartige Erkrankung in der Urologie dar. Es werden das lokal begrenzte, das lokal fortgeschrittene und das metastasierte Krankheitsstadium unterschieden für die jeweils unterschiedliche Prognosen und Therapiestrategien bestehen. Eine Besonderheit beim lokal fortgeschrittenen NZK stellt das Tumorwachstum per continuitatem in die Vena renalis oder Vena cava inferior (VCI) dar, das bei 4-10% aller neudiagnostizierten NZK-Erkrankungen vorliegt. Trotz kurativer Therapie bei Patienten mit lokal begrenztem oder lokal fortgeschrittenem NZK erleiden zwischen 15-30% der Patienten im Verlauf ein Rezidiv oder Metastasen. Um den Patienten eine individualisierte Therapie- und Nachsorgestrategie anbieten zu können, besteht daher ein großer Bedarf an prognostischen Markern, insbesondere Biomarkern. Ziel der vorliegenden Arbeit war es, verschiedene Prognosefaktoren und die unterschiedliche Effektivität von Therapiestrategien bei Patienten mit lokal fortgeschrittenem oder metastasiertem NZK, die an der Klinik für Urologie der Charité – Universitätsmedizin Berlin behandelt wurden, zu untersuchen. In der ersten Studie dieser Arbeit wurden hierzu Patienten mit radiologischen Nachweis eines NZK und fortgeschrittenem Tumorthrombus in die VCI (Level II-IV nach der Mayo-Klassifikation) die durch eine radikale Nephrektomie mit Tumorthrombektomie behandelt wurden, untersucht. Nur das Vorliegen einer primären Metastasierung war ein statistisch signifikant unabhängiger Prädiktor für das Gesamtüberleben. Das Tumorthrombuslevel, Alter des Patienten, Komorbiditäten, OP-Technik bzw. -Dauer stellten keine statistisch signifikanten Prädiktoren dar. Eine radikale Nephrektomie mit Tumorthrombektomie stellt trotz hohen peri- und postoperativen Risikos eine anspruchsvolle, aber effektive Therapiestrategie dar. Ziel der zweiten Studie dieser Arbeit war es das Potenzial von piwi-interacting RNA (piRNA), als prognostische Biomarker für die Entwicklung eines Rezidivs bzw. für das Gesamtüberleben (OS) zu untersuchen. Es konnte gezeigt werden, dass alle drei ausgewählten piRNA in metastasiertem Tumorgewebe und Gewebe aus Knochenmetastasen verschiedene Expressionsprofile aufwiesen und statistisch signifikante Prädiktoren für OS bei Patienten mit lokal begrenzten und metastasierten NZK waren, obwohl in nicht metastasiertem Tumorgewebe alle drei piRNA im Vergleich zu Normalgewebe signifikant heruntergeregelt waren. Zusammen mit der histologischen Tumordifferenzierung war pRNA-38756 bei nicht metastasierten NZK-Patienten ein unabhängiger prognostischer Faktor für die Vorhersage des rezidivfreien Überlebens (RFS) und des OS. Weiter konnte gezeigt werden, dass piRNA als zusätzliche Biomarker konventionelle Prognosemodelle signifikant verbessern. Ziel der dritten Studie dieser Arbeit war es miRNA-Expressionsprofile aus Tumorgewebe als prädiktive Biomarker für eine primäre Resistenz einer systemischen Therapie bei Patienten mit mNZK zu identifizieren. Hierfür wurden Patienten mit mNZK eingeschlossen, die in Erstlinientherapie mit einem TKI behandelt wurden und je nach Therapieansprechen in „Responder“ und „Non-Responder“ eingeteilt wurden. Es konnte gezeigt werden, dass miR-9-5p und miR-489-3p in der Lage waren, zwischen beiden Gruppen zu differenzieren und miR-9-5p das Potenzial als prädiktiver Biomarker bzgl. des Ansprechens einer TKI-Therapie und des progressionsfreien Überlebens (PFS) unter Therapie besitzt. Ziel der vierten und fünften Studie dieser Arbeit war die Untersuchung der Effektivität einer systemischen Therapie bei metastasierten NZK-Patienten, die bereits mindestens drei Therapielinien als Sequenztherapie erhielten. Zusätzlich wurden Prädiktoren bzgl. des PFS und OS exploriert. Sowohl in Viert- als auch in Fünftlinientherapie lag die Krankheits-kontrollrate (DCR) bei 35,7% bzw. 20%. Das mediane OS von Beginn einer Viert- bzw. Fünftlinientherapie betrug 10,5 Monate bzw. 6,2 Monate mit einem PFS von 3,2 Monaten bzw. 4,1 Monaten. Sowohl in Viert- als auch in Fünftlinientherapie zeigten Patienten mitunter eine partielle Remission als bestes Therapieansprechen. Prädiktive Faktoren für ein kürzeres OS bei Patienten in Viertlinientherapie waren eine primäre Resistenz in der Erstlinientherapie, eine synchrone Metastasierung bei Diagnosestellung und ein intermediäres MSKCC-Risikoprofil

Circular RNAs in Clear Cell Renal Cell Carcinoma: Their Microarray-Based Identification, Analytical Validation, and Potential Use in a Clinico-Genomic Model to Improve Prognostic Accuracy

Circular RNAs (circRNAs) may act as novel cancer biomarkers. However, a genome-wide evaluation of circRNAs in clear cell renal cell carcinoma (ccRCC) has yet to be conducted. Therefore, the objective of this study was to identify and validate circRNAs in ccRCC tissue with a focus to evaluate their potential as prognostic biomarkers. A genome-wide identification of circRNAs in total RNA extracted from ccRCC tissue samples was performed using microarray analysis. Three relevant differentially expressed circRNAs were selected (circEGLN3, circNOX4, and circRHOBTB3), their circular nature was experimentally confirmed, and their expression-along with that of their linear counterparts-was measured in 99 malignant and 85 adjacent normal tissue samples using specifically established RT-qPCR assays. The capacity of circRNAs to discriminate between malignant and adjacent normal tissue samples and their prognostic potential (with the endpoints cancer-specific, recurrence-free, and overall survival) after surgery were estimated by C-statistics, Kaplan-Meier method, univariate and multivariate Cox regression analysis, decision curve analysis, and Akaike and Bayesian information criteria. CircEGLN3 discriminated malignant from normal tissue with 97% accuracy. We generated a prognostic for the three endpoints by multivariate Cox regression analysis that included circEGLN3, circRHOBT3 and linRHOBTB3. The predictive outcome accuracy of the clinical models based on clinicopathological factors was improved in combination with this circRNA-based signature. Bootstrapping as well as Akaike and Bayesian information criteria confirmed the statistical significance and robustness of the combined models. Limitations of this study include its retrospective nature and the lack of external validation. The study demonstrated the promising potential of circRNAs as diagnostic and particularly prognostic biomarkers in ccRCC patients

Early Continence and Extravasation After Open Retropubic Radical Prostatectomy – Interrupted vs Continuous Suturing for Vesicourethral Anastomosis

Purpose: To compare running suture (RS) and interrupted suture (IS) of vesicourethral anastomosis (VUA) during open retropubic radical prostatectomy (RRP) on early urinary continence and extravasation. Patients and methods: Single center analysis of 211 patients who underwent RRP performed by a single surgeon during 2008 to 2017 was retrospectively analyzed. For VUA, we used the standard interrupted suture technique (n=100) with a 3-0 PDS suture. The RS (n=111) was performed with 12-bite suture using 3-0 PDS. The primary endpoints were extravasation and early continence. Demographic and peri-operative data were collected and analyzed using Pearson's chi-square, t-Test and Mann-Whitney U-test. A binary logistic regression analysis was carried out to explore predictors that affected early continence after catheter removal. Results: The rates of early urinary incontinence (UI) were 7.7% vs 42.2% (p<0.001). The duration of catheterization and hospitalization was significantly shorter in the interrupted group (4 days vs 5 days, p<0.001 and 5 days vs 6 days, p<0.001). The groups did not differ significantly in body mass index or prostate volume. There were older patients and higher PSA levels in the group with RS technique. No significant difference was found in the postoperative extravasation rates between both groups (13.5% vs 12%, p=0.742). Conclusion: Running vesicourethral anastomosis increased the rate of early urinary incontinence. Both anastomosis techniques provided a similar rate of postoperative urine extravasation. VUA should only be one of the many criteria that must be considered for the preservation of urinary continence of patients after RRP

Instability of circular RNAs in clinical tissue samples impairs their reliable expression analysis using RT-qPCR: from the myth of their advantage as biomarkers to reality

Background: Circular RNAs (circRNAs) are a new class of RNAs with medical significance. Compared to that of linear mRNA transcripts, the stability of circRNAs against degradation owing to their circular structure is considered advantageous for their use as biomarkers. As systematic studies on the stability of circRNAs depending on the RNA integrity, determined as RNA integrity number (RIN), in clinical tissue samples are lacking, we have investigated this aspect in the present study under model and clinical conditions. Methods: Total RNA isolated from kidney cancer tissue and cell lines (A-498 and HEK-293) with different RIN after thermal degradation was used in model experiments. Further, RNA isolated from kidney cancer and prostate cancer tissue collected under routine surgical conditions, representing clinical samples with RIN ranging from 2 to 9, were examined. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) analysis of several circRNAs (circEGLN3, circRHOBTB3, circCSNK1G3, circRNA4, and circRNA9), their corresponding linear counterparts, tissue-specific reference genes, and three microRNAs (as controls) was performed. The quantification cycles were converted into relative quantities and normalized to the expression of specific reference genes for the corresponding tissue. The effect of RIN on the expression of different RNA entities was determined using linear regression analysis, and clinical samples were classified into two groups based on RIN greater or lesser than 6. Results: The results of model experiments and clinical sample analyses showed that all relative circRNA expression gradually decreased with reduction in RIN values. The adverse effect of RIN was partially compensated after normalizing the data and limiting the samples to only those with RIN values > 6. Conclusions: Our results suggested that circRNAs are not stable in clinical tissue samples, but are subjected to degradative processes similar to mRNAs. This has not been investigated extensively in circRNA expression studies, and hence must be considered in future for obtaining reliable circRNA expression data. This can be achieved by applying the principles commonly used in mRNA expression studies

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

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

Circular RNAs and Their Linear Transcripts as Diagnostic and Prognostic Tissue Biomarkers in Prostate Cancer after Prostatectomy in Combination with Clinicopathological Factors

As new biomarkers, circular RNAs (circRNAs) have been largely unexplored in prostate cancer (PCa). Using an integrative approach, we aimed to evaluate the potential of circRNAs and their linear transcripts (linRNAs) to act as (i) diagnostic biomarkers for differentiation between normal and tumor tissue and (ii) prognostic biomarkers for the prediction of biochemical recurrence (BCR) after radical prostatectomy. In a first step, eight circRNAs (circATXN10, circCRIM1, circCSNK1G3, circGUCY1A2, circLPP, circNEAT1, circRHOBTB3, and circSTIL) were identified as differentially expressed via a genome-wide circRNA-based microarray analysis of six PCa samples. Additional bioinformatics and literature data were applied for this selection process. In total, 115 malignant PCa and 79 adjacent normal tissue samples were examined using robust RT-qPCR assays specifically established for the circRNAs and their linear counterparts. Their diagnostic and prognostic potential was evaluated using receiver operating characteristic curves, Cox regressions, decision curve analyses, and C-statistic calculations of prognostic indices. The combination of circATXN10 and linSTIL showed a high discriminative ability between malignant and adjacent normal tissue PCa. The combination of linGUCY1A2, linNEAT1, and linSTIL proved to be the best predictive RNA-signature for BCR. The combination of this RNA signature with five established reference models based on only clinicopathological factors resulted in an improved predictive accuracy for BCR in these models. This is an encouraging study for PCa to evaluate circRNAs and their linRNAs in an integrative approach, and the results showed their clinical potential in combination with standard clinicopathological variables

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

About the influence of collagen crosslinking on laser-in-situ-keratomileusis (LASIK)

Einleitung: Die Kollagenquervernetzungs-Behandlung mit Riboflavin und UV-A-Licht (CXL) ist eine neue Behandlungsmethode, die als einziges Verfahren die Stabilität der Hornhaut erhöht und dadurch eine Progression eines Keratokonus verhindern kann. Die hierbei oft verzeichnete Rückbildung der Keratektasie reicht jedoch in der Regel nicht aus, um die damit verbundenen Brechungsfehler des Auges zu beheben. In einigen Fällen kann die Sehschärfe sogar mit formstabilen Kontaktlinsen nicht wieder hergestellt werden und es müssen operative Methoden, wie Excimer-Laserbehandlungen, in Erwägung gezogen werden. Die Gefahr der Progression der Hornhautverdünnung bis hin zur Keratolyse bleibt jedoch bestehen. Die Festigung der Cornea durch eine CXL- Behandlung vor einer Excimer-Laserbehandlung könnte diese Gefahr verringern und die Sehschärfe des Patienten entscheidend verbessern oder vollständig restituieren. Ziel dieser Arbeit war es zu untersuchen, in wie weit eine CXL-Behandlung einen Einfluss auf refraktiv-chirurgische Verfahren hat: Es sollte die Auswirkung einer CXL-Behandlung auf einen Mikrokeratomschnitt, insbesondere auf die Flapdicke, sowie die Auswirkung auf das refraktive Ergebnis einer LASIK-Behandlung untersucht werden. Material und Methoden: An frisch enukleierten Schweineaugen (n=100 je Gruppe) wurde eine CXL-Behandlung durchgeführt. Die Augen der Kontrollgruppe erhielten nur eine Epithelabrasio. Die Präparation des Flaps erfolgte mit einem Cariazzo- Pendular Mikrokeratom, die Excimer-Laser-Photoablation mit dem ESIRIS Excimer- Laser. Die Flapdickenmessung wurde durchgeführt mittels optischer Kohärenzpachymetrie und einem mechanischem Dickenmessgerät. Die Bestimmung der Refraktion erfolgte mittels Placido-basierter Videokeratographie. Ergebnisse: Die mit dem optischen Kohärenzpachymeter (OCP) gemessene mittlere Flapdicke nach einem intendierten 150μm Mikrokeratomschnitt betrug 135 ± 25μm in der Gruppe mit CXL-Behandlung und 105 ± 25μm in der Kontrollgruppe (p4D or an intended 130μm flap and refractive correction >6D, an ablation profil for higher refractive corrections has to be used

Vitamin D Metabolites in Nonmetastatic High-Risk Prostate Cancer Patients with and without Zoledronic Acid Treatment after Prostatectomy

There are limited and discrepant data on prostate cancer (PCa) and vitamin D. We investigated changes in three vitamin D3 metabolites in PCa patients after prostatectomy with zoledronic acid (ZA) treatment regarding their metastasis statuses over four years. In 32 patients from the ZEUS trial, 25(OH)D3, 24,25(OH)2D3, and 1,25(OH)2D3 were measured with liquid chromatography coupled with tandem mass spectrometry at four time points. All the patients received daily calcium and vitamin D3. Bone metastases were detected in 7 of the 17 ZA-treated patients and in 5 of the 15 controls (without ZA), without differences between the groups (p = 0.725). While 25(OH)D3 and 24,25(OH)2D3 increased significantly after the study’s start, with following constant values, the 1,25(OH)2D3 concentrations remained unchanged. ZA treatment did not change the levels of the three metabolites. 25(OH)D3 and 24,25(OH)2D3 were not associated with the development of bone metastases. In contrast, 1,25(OH)2D3 was also higher in patients with bone metastasis before the study’s start. Thus, in high-risk PCa patients after prostatectomy, 25(OH)D3, 24,25(OH)2D3, and 1,25(OH)2D3 were not affected by supportive ZA treatment or by the development of metastasis over four years, with the exception of 1,25(OH)2D3, which was constantly higher in metastatic patients. There might be potential prognostic value if the results can be confirmed