Circulating RNA in Kidney Cancer: What We Know and What We Still Suppose.

Renal cancer represents the 7th most common tumor worldwide, affecting 400,000 people annually. This malignancy, which is the third most frequent cancer among urological diseases, displays a completely different prognosis if the tumor is detected in the early stages or advance phases. Unfortunately, more than 50% of renal cancers are discovered incidentally, with a consistent percentage of cases where the tumor remains clinically silent till the metastatic process is established. In day-to-day clinical practice, no available predictive biomarkers exist, and the existent imaging diagnostic techniques harbor several gaps in terms of diagnosis and prognosis. In the last decade, many efforts have been reported to detect new predictive molecular biomarkers using liquid biopsies, which are less invasive in comparison to renal biopsy. However, until now, there has been no clear evidence that a liquid biopsy biomarker could be relevant to the creation of a precise and tailored medical management in these oncological patients, even though circulating RNA biomarkers remain among the most promising. Given the idea that liquid biopsies will play a future key role in the management of these patients, in the present review, we summarize the current state of circulating RNA (miRNA, lncRNAs, and circRNAs) as possible biomarkers of renal cancer presence and aggressiveness in patients.

Urine miRNA signature as a potential non-invasive diagnostic and prognostic biomarker in cervical cancer.

MicroRNAs as cancer biomarkers in serum, plasma, and other body fluids are often used but analysis of miRNA in urine is limited. We investigated the expression of selected miRNAs in the paired urine, serum, cervical scrape, and tumor tissue specimens from the women with cervical precancer and cancer with a view to identify if urine miRNAs could be used as reliable non-invasive biomarkers for an early diagnosis and prognosis of cervical cancer. Expression of three oncomiRs (miR-21, miR-199a, and miR-155-5p) and three tumor suppressors (miR-34a, miR-145, and miR-218) as selected by database search in cervical pre-cancer, cancer, and normal controls including cervical cancer cell lines were analyzed using qRT-PCR. The expression of miRNAs was correlated with various clinicopathological parameters, including HPV infection and survival outcome. We observed a significant overexpression of the oncomiRs and the downregulation of tumor suppressor miRNAs. A combination of miR-145-5p, miR-218-5p, and miR-34a-5p in urine yielded 100% sensitivity and 92.8% specificity in distinguishing precancer and cancer patients from healthy controls and it well correlates with those of serum and tumor tissues. The expression of miR-34a-5p and miR-218-5p were found to be independent prognostic factors for the overall survival of cervical cancer patients. We conclude that the evaluation of the above specific miRNA expression in non-invasive urine samples may serve as a reliable biomarker for early detection and prognosis of cervical cancer.

Combining urinary DNA methylation and cell-free microRNA biomarkers for improved monitoring of prostate cancer patients on active surveillance.

PURPOSE: Prostate cancer (CaP) patients with low-grade tumors are enrolled in active surveillance (AS) programs and monitored with digital rectal exams (DREs), prostate-specific antigen (PSA) tests, and periodic invasive biopsies. Patients are "reclassified" with higher-risk disease if they show signs of disease progression. However, AS patients who will reclassify cannot be easily identified upfront and suffer morbidities associated with biopsy. Biomarkers derived from noninvasively obtained specimens such as serum or urine samples are promising alternatives to monitor patients with clinically insignificant cancer. Previously, we have characterized and validated a urinary DNA methylation panel and a serum miRNA panel for the prediction of patient reclassification in 2 independent AS cohorts. In this exploratory study, we have investigated cell-free miRNAs in the urinary supernatant combined with urinary DNA methylation markers to form an integrative panel for prediction of AS patient reclassification. METHODS: Post-DRE urine was collected from 103 CaP patients on active surveillance. Urinary sediment DNA methylation levels of selected genes were previously analyzed using qPCR-based MethyLight assay. Using qRT-PCR, we analyzed the urinary supernatants for relative quantities of 10 miRNAs previously shown to be associated with AS reclassification. Logistic regression and Receiver Operating Characteristics curve analyses were performed to assess the predictive ability of miRNAs and DNA methylation biomarkers. RESULTS: We identified a 3-marker panel, consisting of miR-24, miR-30c and CRIP3 methylation, that was significant for prediction of patient reclassification (Odds ratio = 2.166, 95% confidence interval = 1.22-3.847) with a negative predictive value of 90.9%. Our 3-marker panel also demonstrated additive value to PSA for prediction of patient reclassification (c-statistic = 0.717, ROC bootstrapped 1000 iteration P = 0.041). CONCLUSION: A urinary integrated panel of methylation and miRNA markers is a promising approach to identify AS patients at risk for reclassification. Our 3-marker panel, with its high negative predictive value, would be beneficial to identify and preclude AS patients with truly indolent cancer and to personalize monitoring strategies for AS patients.

Urinary cell-free microRNA biomarker could discriminate bladder cancer from benign hematuria.

The most common symptom of bladder cancer (BC) is hematuria. However, not all patients with hematuria are diagnosed with BC. Here, we explored a novel method to discriminate BC from hematuria under nonmalignant conditions by measuring differences in urinary cell-free microRNA (miRNA) expression between patients with BC and those with hematuria. A multicenter study was performed using 543 urine samples obtained from the National Biobank of Korea, including 326 BC, 174 hematuria and 43 pyuria without cancer. The urinary miR-6124 to miR-4511 ratio was considerably higher in BC than in hematuria or pyuria, and enabled the discrimination of BC from patients with hematuria at a sensitivity of >90% (p < 0.001). Conclusively, the proposed noninvasive diagnostic tool based on the expression ratio of urinary cell-free miR-6124 to miR-4511 can reduce unnecessary cystoscopies in patients with hematuria undergoing evaluation for BC, with a minimal loss in sensitivity for detecting cancer.

Cell-Free Urinary MicroRNAs Expression in Small-Scale Experiments.

Cell-free microRNAs (miRNAs) have become one of the novel promising diagnostic and prognostic biomarkers for various diseases recently. Blood serum and plasma along with urine are the most common sources of clinically well, almost noninvasively available samples containing various types of miRNAs. Here, we present a protocol for a small-scale study investigating expression of several candidate miRNAs. Small-scale experiments may be worth investigating in cases where no information is available on miRNAs expression in particular diseases, for validation of previously published miRNAs with promising diagnostic potential, particularly in situations where follow-up study is aimed at validating miRNAs coming from array or NGS experiments, or where funding for these large-scale experiments is not available.Using urine miRNAs expression as the novel diagnostic tools is challenging and currently this approach is still in its infancy. Therefore, various methods may result in different conclusions depending on clinical sample sets and differences among methods used for the miRNAs isolation and quantitation. In this protocol, we present the method evaluated in the study focused on cell-free urinary miRNAs in ovarian and endometrial cancers. We recommend using stabilization tubes for the urine collection, as this step may be necessary to stop activity of RNases. Further, routine real-time PCR methods are described. We demonstrate that assessment of urinary miRNAs expression may reveal as a feasible method to explore the potential for finding novel diagnostic and prognostic markers.

Cell-free microRNA expression signatures in urine serve as novel noninvasive biomarkers for diagnosis and recurrence prediction of bladder cancer.

Urinary microRNAs (miRNAs) are potential biomarkers for the noninvasive diagnosis of bladder cancer (BC). In this study, we aimed to develop a urinary miRNAs panel for diagnosing and predicting recurrence of BC. Genome-wide miRNAs analysis by deep sequencing followed by two phases of quantitative real-time PCR assays were performed on urine supernatant of 276 BC patients and 276 controls. We identified a seven-miRNA panel (miR-7-5p, miR-22-3p, miR-29a-3p, miR-126-5p, miR-200a-3p, miR-375, and miR-423-5p) that provided high diagnostic accuracy of BC with an AUC of 0.923 and 0.916 in training and validation set, respectively. The corresponding AUCs of this panel for Ta, T1 and T2-T4 were 0.864, 0.930 and 0.978, significantly higher than those of urine cytology, which were 0.531, 0.628 and 0.724, respectively (all p < 0.05). Moreover, Kaplan-Meier analysis showed that nonmuscle-invasive BC (NMIBC) patients with high miR-22-3p and low miR-200a-3p level had worse recurrence-free survival (RFS) (p = 0.002 and 0.040, respectively). Multivariate Cox regression analysis revealed that miR-22-3p and miR-200a-3p were independently associated with RFS of NMIBC (p = 0.024 and 0.008, respectively). In conclusion, our results suggested that urinary miRNAs may have considerable clinical value in diagnosis and recurrence prediction of BC.

Detection of urinary cell-free miR-210 as a potential tool of liquid biopsy for clear cell renal cell carcinoma.

BACKGROUND: Recent findings show that cell-free miRNAs are stable in biological fluid. Urine provides an alternative to blood serum or plasma as a potential source of tumor biomarkers. MiR-210 is proven to be overexpressed in patients with a clear cell renal cell carcinoma (ccRCC). The purpose of this pilot study was to examine the urinary cell-free miR-210 as a potential tool of liquid biopsy for ccRCC. METHODS: Overall, 75 patients with a ccRCC and 45 control subjects without a cancer were included in this study. Urine samples were centrifuged twice and the cell-free urine supernanants were stored in -80°C until use. A total of 350µl cell-free urine was used for the extraction of total RNA. The expression levels of these miRNAs were performed by using quantitative RT-PCR. RESULTS: The level of urinary cell-free miR-210 was significantly higher in patients with ccRCC than in control subjects (P<0.001). The urinary cell-free miR-210 yielded the areas under the receiver operating characteristics curve of 0.76 in discriminating the patients with ccRCC from the control subjects with a sensitivity of 57.8% and a specificity of 80.0%. Moreover, the expression level of urinary cell-free miRNA-210 was significantly decreased in the patients a week after surgery (P<0.001). CONCLUSION: Urinary cell-free miR-210 may be used as a potential tool of liquid biopsy for ccRCC diagnosis. Further studies are needed to confirm our results.