PD-L1 expression in tumor and inflammatory cells is associated with favorable tumor features and favorable prognosis in muscle-invasive urothelial carcinoma of the bladder not treated by immune checkpoint inhibitors.

BACKGROUND: A high level of PD-L1 expression is the most relevant predictive parameter for response to immune checkpoint inhibitor (CPI) therapy in urinary bladder cancer. Existing data on the relationship between PD-L1 expression and the natural course of disease are controversial and sparse. METHODS: To expand our understanding of the relationship between PD-L1 expression and parameters of cancer aggressiveness, PD-L1 was analyzed on tissue microarrays containing 2710 urothelial bladder carcinomas including 512 patients with follow-up data who underwent radical cystectomy and follow-up therapies in the pre-immune checkpoint inhibitor therapy era. RESULTS: Tumor cell positivity in ≥10% of cells were seen in 513 (20%) and an immune cell positivity occurred in 872 (34%) of 2566 interpretable cancers. PD-L1 positivity in tumor cells increased from pTaG2 low grade (0.9% positive) to pTaG3 high grade (4.1%; p = 0.0255) and was even higher in muscle-invasive (pT2-4) carcinomas (29.3%; p < 0.0001). However, within pT2-4 carcinomas, PD-L1 positivity was linked to low pT stage (p = 0.0028), pN0 (p < 0.0001), L0 status (p = 0.0005), and a better prognosis within 512 patients with cystectomy who never received CPIs (p = 0.0073 for tumor cells and p = 0.0086 for inflammatory cells). PD-L1 staining in inflammatory cells was significantly linked to PD-L1 staining in tumor cells (p < 0.0001) and both were linked to a positive p53 immunostaining (p < 0.0001). CONCLUSION: It cannot be fully excluded that the strong statistical link between PD-L1 status and favorable histological tumor features as well as better prognosis could influence the outcome of studies evaluating CPIs in muscle-invasive urothelial carcinoma.

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.

Circular RNAs: a new class of biomarkers as a rising interest in laboratory medicine.

Circular RNAs (circRNAs) are a distinct family of RNAs derived from the non-regular process of alternative splicing. CircRNAs have recently gained interest in transcriptome research due to their potential regulatory functions during gene expression. CircRNAs can act as microRNA sponges and affect transcription through their complex involvement in regular transcriptional processes. Some early studies also suggested significant roles for circRNAs in human diseases, especially cancer, as biomarkers and potential clinical targets. Therefore, there is a great need for laboratory scientists to translate these findings into clinical tools to advance testing for human diseases. To facilitate a better understanding of the promise of circRNAs, we focus this review on selected basic aspects of circRNA research, specifically biogenesis, function, analytical issues regarding identification and validation and examples of expression data in relation to human diseases. We further emphasize the unique challenges facing laboratory medicine with regard to circRNA research, particularly in the development of robust assays for circRNA detection in different body fluids and the need to collaborate with clinicians in the design of clinical studies.

Loss of Upk1a and Upk1b expression is linked to stage progression in urothelial carcinoma of the bladder.

BACKGROUND: Uroplakin-1a (Upk1a) and uroplakin-1b (Upk1b) have recently been identified as diagnostic markers for the distinction of urothelial carcinomas from other solid tumor entities. Both proteins play an important role in the stabilization and strengthening of epithelial cells that line the bladder. METHODS: To evaluate the prognostic role of uroplakin expression in urothelial carcinomas, more than 2700 urothelial neoplasms were analyzed in a tissue microarray format by immunohistochemistry. To further assess the diagnostic role of uroplakin immunohistochemistry, results were compared with preexisting GATA3 data. RESULT: The fraction of Upk1a/Upk1b positive cases decreased slightly from pTaG2 low-grade (88% positive for Upk1a/87% positive for Upk1b) and pTaG2 high-grade (92%/89%) to pTaG3 (83%/88%; p > 0.05) and was lower in muscle-invasive (pT2-4) carcinomas (42%/64%; p < 0.0001/p < 0.0001 for pTa vs. pT2-4). Within pT2-4 carcinomas, high expression of Upk1a and Upk1b was linked to nodal metastasis and lymphatic vessel infiltration (p < 0.05) but unrelated to patient outcome. There were significant associations between Upk1a, Upk1b and GATA3 immunostaining (p < 0.0001 each), but 11% of GATA3 negative cancers were Upk1a/b positive and 8% of Upk1a/b negative cancers were GATA3 positive. Absence of GATA3/Upk1a/b staining was significantly linked to poor patient survival in the subgroup of 126 pT4 carcinomas (p = 0.0004) but not in pT2 and pT3 cancers. CONCLUSIONS: In summary, the results of our study demonstrate that Upk1a and/or Upk1b immunohistochemistry can complement GATA3 for the distinction of urothelial carcinomas. Furthermore, a progressive loss of Upk1a/b expression during stage progression and a prognostic role of the combination GATA3/Upk1a/Upk1b in pT4 carcinomas is evident.

CEA (CEACAM5) expression is common in muscle-invasive urothelial carcinoma of the bladder but unrelated to the disease course.

Objectives: Carcinoembryonic antigen (CEA) is a cell surface glycoprotein that represents a promising therapeutic target. Serum measurement of shedded CEA can be utilized for monitoring of cancer patients. Material and Methods: To evaluate the potential clinical significance of CEA expression in urothelial bladder neoplasms, CEA was analysed by immunohistochemistry in more than 2500 urothelial bladder carcinomas in a tissue microarray format. Results: CEA staining was largely absent in normal urothelial cells but was observed in 30.4% of urothelial bladder carcinomas including 406 (16.7%) with weak, 140 (5.8%) with moderate, and 192 (7.9%) with strong staining. CEA positivity occurred in 10.9% of 411 pTaG2 low-grade, 32.0% of 178 pTaG2 high-grade, and 43.0% of 93 pTaG3 tumours (p < 0.0001). In 1335 pT2-4 carcinomas, CEA positivity (34.1%) was lower than in pTaG3 tumours. Within pT2-4 carcinomas, CEA staining was unrelated to pT, pN, grade, L-status, V-status, overall survival, recurrence free survival, and cancer specific survival (p > 0.25). Conclusion: CEA increases markedly with grade progression in pTa tumours, and expression occurs in a significant fraction of pT2-4 urothelial bladder carcinomas. The high rate of CEA positivity in pT2-4 carcinomas offers the opportunity of using CEA serum measurement for monitoring the clinical course of these cancers. Moreover, CEA positive urothelial carcinomas are candidates for a treatment by targeted anti-CEA drugs.

[Precision oncology options in urological cancers]. / Zielgerichtete Therapieoptionen in der Uroonkologie.

Advancements in the molecular genetic understanding of urological tumors have enabled the identification of numerous new therapeutic targets. Based on routinely applicable tumor sequencing, individual treatment decisions have been introduced in the context of precision oncology. This work provides an overview of the latest targeted tumor therapies in the treatment of prostate cancer, urothelial carcinoma, and renal cell carcinoma. Current studies on the administration of FGFR-inhibitors ("fibroblast growth factor receptor") in metastatic urothelial carcinoma show a high tumor response in patients with selected FGFR alterations. PARP-inhibitors ("Poly-[ADP-Ribose-]Polymerase") are routinely used in the treatment of metastatic prostate cancer. Patients with a BRCA mutation ("BReast CAncer gene") show high radiological response rates. Moreover, we discuss the latest results of the combination of PARP inhibitors with novel androgen receptor pathway inhibitors. In metastatic prostate cancer, there are numerous ongoing studies evaluating the promising drug targets PI3K/AKT/mTOR ("Phosphatidylinositol-3-Kinase")/AKT/mTOR ("mammalian target of rapamycine") and VEGF signaling pathways ("vascular endothelial growth factor"). A HIF-2a inhibitor ("hypoxia inducible factor") offers a promising new therapeutic option for metastatic renal cell carcinoma. Overall, molecular diagnostics to determine the right therapy for the right patient subgroup at the right time is important for uro-oncological precision medicine.

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.

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.