A Novel Predictor Tool of Biochemical Recurrence after Radical Prostatectomy Based on a Five-MicroRNA Tissue Signature

Within five to ten years after radical prostatectomy (RP), approximately 15-34% of prostate cancer (PCa) patients experience biochemical recurrence (BCR), which is defined as recurrence of serum levels of prostate-specific antigen >0.2 µg/L, indicating probable cancer recurrence. Models using clinicopathological variables for predicting this risk for patients lack accuracy. There is hope that new molecular biomarkers, like microRNAs (miRNAs), could be potential candidates to improve risk prediction. Therefore, we evaluated the BCR prognostic capability of 20 miRNAs, which were selected by a systematic literature review. MiRNA expressions were measured in formalin-fixed, paraffin-embedded (FFPE) tissue RP samples of 206 PCa patients by RT-qPCR. Univariate and multivariate Cox regression analyses were performed, to assess the independent prognostic potential of miRNAs. Internal validation was performed, using bootstrapping and the split-sample method. Five miRNAs (miR-30c-5p/31-5p/141-3p/148a-3p/miR-221-3p) were finally validated as independent prognostic biomarkers. Their prognostic ability and accuracy were evaluated using C-statistics of the obtained prognostic indices in the Cox regression, time-dependent receiver-operating characteristics, and decision curve analyses. Models of miRNAs, combined with relevant clinicopathological factors, were built. The five-miRNA-panel outperformed clinically established BCR scoring systems, while their combination significantly improved predictive power, based on clinicopathological factors alone. We conclude that this miRNA-based-predictor panel will be worth to be including in future 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

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

COVID-19 vaccines in patients with cancer:immunogenicity, efficacy and safety

Patients with cancer have a higher risk of severe coronavirus disease (COVID-19) and associated mortality than the general population. Owing to this increased risk, patients with cancer have been prioritized for COVID-19 vaccination globally, for both primary and booster vaccinations. However, given that these patients were not included in the pivotal clinical trials, considerable uncertainty remains regarding vaccine efficacy, and the extent of humoral and cellular immune responses in these patients, as well as the risks of vaccine-related adverse events. In this Review, we summarize the current knowledge generated in studies conducted since COVID-19 vaccines first became available. We also highlight critical points that might affect vaccine efficacy in patients with cancer in the future

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

Mice with renal-specific alterations of stem cell-associated signaling develop symptoms of chronic kidney disease but surprisingly no tumors

Previously, we found that Wnt and Notch signaling govern stem cells of clear cell kidney cancer (ccRCC) in patients. To mimic stem cell responses in the normal kidney in vitro in a marker-unbiased fashion, we have established tubular organoids (tubuloids) from total single adult mouse kidney epithelial cells in Matrigel and serum-free conditions. Deep proteomic and phosphoproteomic analyses revealed that tubuloids resembled renewal of adult kidney tubular epithelia, since tubuloid cells displayed activity of Wnt and Notch signaling, long-term proliferation and expression of markers of proximal and distal nephron lineages. In our wish to model stem cell-derived human ccRCC, we have generated two types of genetic double kidney mutants in mice: Wnt-β-catenin-GOF together with Notch-GOF and Wnt-β-catenin-GOF together with a most common alteration in ccRCC, Vhl-LOF. An inducible Pax8-rtTA-LC1-Cre was used to drive recombination specifically in adult kidney epithelial cells. We confirmed mutagenesis of β-catenin, Notch and Vhl alleles on DNA, protein and mRNA target gene levels. Surprisingly, we observed symptoms of chronic kidney disease (CKD) in mutant mice, but no increased proliferation and tumorigenesis. Thus, the responses of kidney stem cells in the tubuloid and genetic systems produced different phenotypes, i.e. enhanced renewal versus CKD

Radium-223 for primary bone metastases in patients with hormone-sensitive prostate cancer after radical prostatectomy

Radium-223 dichloride (Ra-223) is the first bone-targeting agent showing improvement in overall survival in patients with castration-resistant prostate cancer (CRPC) and bone metastases. We aimed to assess feasibility of Ra-223 treatment in patients with metastatic hormone-sensitive prostate cancer (mHSPC). Ten patients with primary bone metastases received Ra-223 following radical prostatectomy (RP). Changes in alkaline phosphatase (ALP) and prostate-specific antigen (PSA) were recorded, while pain intensity was evaluated using the self-reporting Brief Pain Inventory (BPI) questionnaire. Bone scintigraphy (BS) was performed to assess treatment response. Seven patients completed six cycles of Ra-223. Discontinuation was due to leuko-and lymphopenia, progressive lymph node metastasis or newly diagnosed liver metastasis. Treatment-related adverse events occurred in three patients and included leuko-and lymphopenia, fatigue, abdominal discomfort and nausea. Overall, a median decrease of 28% in ALP and a median decrease of 83% in PSA were noted at follow-up. However, PSA progressed in five patients at follow-up. Improvement of pain was observed in all patients (median decrease of 36% after 3 cycles and of 40% at the end of therapy). On BS, three patients showed remission, four had stable disease, and one showed progressive disease at follow-up. Our results suggest that Ra-223 for primary bone metastases in patients with mHSPC after RP is feasible and alleviates pain. ALP, rather than PSA, may be a good marker for assessing treatment response. Ra-223 could therefore be taken into consideration as part of a multimodal approach for carefully selected patients with advanced prostate cancer

Outcome after PSMA PET/CT based radiotherapy in patients with biochemical persistence or recurrence after radical prostatectomy

Background: PSMA PET/CT visualises prostate cancer residual disease or recurrence at lower PSA levels compared to conventional imaging and results in a change of treatment in a remarkable high number of patients. Radiotherapy with dose escalation to the former prostate bed has been associated with improved biochemical recurrence-free survival. Thus, it can be hypothesised that PSMA PET/CT-based radiotherapy might improve the prognosis of these patients. Methods: One hundred twenty-nine patients underwent PSMA PET/CT due to biochemical persistence (52%) or recurrence (48%) after radical prostatectomy without evidence of distant metastases (February 2014-May 2017) and received PSMA PET/CT-based radiotherapy. Biochemical recurrence free survival (PSA <= 0.2 ng/ml) was defined as the study endpoint. Results: Patients with biochemical persistence were significantly more often high-risk patients with significantly shorter time interval before PSMA PET/CT than patients with biochemical recurrence. Patients with biochemical recurrence had significantly more often no evidence of disease or local recurrence only in PSMA PET/CT, whereas patients with biochemical persistence had significantly more often lymph node involvement. Seventy-three patients were started on antiandrogen therapy prior to radiotherapy due to macroscopic disease in PSMA PET/CT. Cumulatively, 70 (66-70.6) Gy was delivered to local macroscopic tumor, 66 (63-66) Gy to the prostate fossa, 61.6 (53.2-66) Gy to PET-positive lymph nodes and 50.4 (45-52.3) Gy to lymphatic pathways. Median PSA after radiotherapy was 0.07 ng/ml with 74% of patients having a PSA <= 0.1 ng/ml. After a median follow-up of 20 months, median PSA was 0.07 ng/ml with ongoing antiandrogen therapy in 30 patients. PET-positive patients without antiandrogen therapy at last follow-up (45 patients) had a median PSA of 0.05 ng/ml with 89% of all patients, 94% of patients with biochemical recurrence and 82% of patients with biochemical persistence having a PSA <= 0.2 ng/ml. Post-radiotherapy PSA <= 0.1 ng/ml and biochemical recurrence vs. persistence were significantly associated with a PSA <= 0.2 ng/ml at last follow-up. Conclusions: PSMA PET/CT-based radiotherapy is an effective local salvage treatment option with significant PSA response in patients with biochemical recurrence or persistence after radical prostatectomy leading to deferral of long-term ADT or systemic therapy