Hana Mlčochová

Urine microRNAs as potential noninvasive biomarkers in urologic cancers

Micro-ribonucleic acids (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. The ability of miRNAs to inhibit translation of oncogenes and tumor suppressors implies that they may be involved in carcinogenesis. Our review focuses on the potential of urinary miRNAs to serve as biomarkers of urologic cancers. We discuss in detail the recent knowledge about the origin of urinary miRNAs, their stability, quality control, and their utility as a potential new class of biomarkers in urologic cancer. Finally, we summarize the studies focusing on detection and characterization of urinary miRNAs as potential biomarkers in bladder, prostate, and kidney cancers.

Epithelial-mesenchymal transition-associated microRNA/mRNA signature is linked to metastasis and prognosis in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinomas (ccRCCs) are genetically heterogeneous tumors presenting diverse clinical courses. Epithelial-mesenchymal transition (EMT) is a crucial process involved in initiation of metastatic cascade. The aim of our study was to identify an integrated miRNA/mRNA signature associated with metastasis and prognosis in ccRCC through targeted approach based on analysis of miRNAs/mRNAs associated with EMT. A cohort of 230 ccRCC was included in our study and further divided into discovery, training and validation cohorts. EMT markers were evaluated in ccRCC tumor samples, which were grouped accordingly to EMT status. By use of large-scale miRNA/mRNA expression profiling, we identified miRNA/mRNA with significantly different expression in EMT-positive tumors and selected 41 miRNAs/mRNAs for training phase of the study to evaluate their diagnostic and prognostic potential. Fifteen miRNAs/mRNAs were analyzed in the validation phase, where all evaluated miRNA/mRNA candidates were confirmed to be significantly deregulated in tumor tissue. Some of them significantly differed in metastatic tumors, correlated with clinical stage, with Fuhrman grade and with overall survival. Further, we established an EMT-based stage-independent prognostic scoring system enabling identification of ccRCC patients at high-risk of cancer-related death. Finally, we confirmed involvement of miR-429 in EMT regulation in RCC cells in vitro.

Urinary MicroRNAs as a New Class of Noninvasive Biomarkers in Oncology, Nephrology, and Cardiology

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression. In the last decade, number of evidences showing miRNAs contribution to the regulation of apoptosis, cellular proliferation, differentiation, and other important cellular processes is constantly growing. Specific miRNA expression signatures have been identified in variety of human cancers as well as pathologies of cardiovascular and urinary systems. Our chapter focuses on the potential of urinary miRNAs to serve as biomarkers in uro-oncology, nephrology, and cardiology. We discuss in detail recent knowledge about the origin of urinary miRNAs, their stability, quality control, and their utility as a potential new class of biomarkers in medicine. Finally, we summarize the studies focusing on detection and characterization of urinary miRNAs as potential biomarkers in urologic cancers, nephrology, and cardiology.

Abstract 1946: Urinary cell-free microRNA panel in detection of urothelial carcinoma of the urinary bladder

Urothelial carcinoma of the urinary bladder (UCUB) is the most common malignancy of the urinary system. Although about 80% of cases is a non-muscle invasive form of UCUB a high rate of local recurrence and progression to invasive form is observed. Early-stage tumors have very good prognosis, but current diagnostic methods (cystoscopy and urine cytology) suffer from low sensitivity. This reflects a large number of relapse, which occurs in almost 70% of superficial UCUB. This has led to the development of multiple molecular urinary biomarkers, but none are sufficiently robust to enter clinical practice. In this study we aimed to develop a clinically applicable, specific and sensitive panel of urine microRNAs enabling early detection of UCUB and prediction of risk of progression to muscle-invasive form. In the first phase of study we have analyzed expression profiles of 1733 miRNAs in urine supernatant of 16 UCUB patients (6 invasive, 5 high-grade non-invasive, 5 low-grade non-invasive), 17 controls, 10 RCC patients and 4 urinary tract infections (UTI) using Affymetrix miRNA microarrays. MicroRNAs able distinguish between UCUB and control groups were further validated using specific TaqMan assays and qRT-PCR method on independent cohort of 100 UCUB patients, 40 controls and 25 RCC patients (training phase - 40 UCUB, 15 controls, 10 RCC; validation phase - 60 UCUB, 25 controls, 15 RCC, 20 UTI). Global expression profiling revealed set of 76 miRNAs significantly differentially expressed in urine of UCUB patients (P Our data have shown that urinary microRNAs could serve as sensitive and specific biomarkers of UCUB and could be useful tool to increase sensitivity of standard cytological examination and to decrease high costs for long-term follow-up of UCUB patients. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A and 15-34678A. All rights reserved. Citation Format: Jaroslav Juracek, Barbora Peltanova, Hana Mlcochova, Michal Stanik, Tana Machackova, Michal Fedorko, Robert Iliev, Jitka Mlcochova, Jiri Sana, Zuzana Ozanova, Jan Dolezel, Ondrej Slaby. Urinary cell-free microRNA panel in detection of urothelial carcinoma of the urinary bladder. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1946.

Abstract 3979: Urinary cell-free microRNAs as potential biomarkers of urothelial carcinoma of the urinary bladder

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Urothelial carcinoma of the urinary bladder (UCUB) is the most common malignancy of the urinary system. UCUB is divided into muscle invasive and non-muscle invasive bladder cancer (superficial), which represents approximately 80% of cases. Despite the relatively high degree of superficial tumors is UCUB associated with high local recurrence rate and approximately 20% of non-muscle invasive tumors progress to invasive form. Although cystoscopy remains a fundamental investigative tool in the detection and surveillance of urothelial bladder cancer, carcinoma in situ (CIS) or small papillary tumors can be with this method easily missed. This has led to the development of newer technologies and several molecular urinary tests, but currently there is no sensitive biomarker enabling early detection of relapse, which occurs in almost 70% of cases of superficial UCUB or biomarker with ability to predict the risk of progression of non-invasive to invasive form of UCUB. Such requirements could fit with diagnostic approach based on the detection of microRNAs (miRNAs) in urine, where have already showed remarkably high stability and good analytical properties. Patients and methods: Using Affymetrix miRNA microarrays we have analyzed expression profiles of 1733 miRNAs in urine supernatant of 16 UCUB patients (6 invasive, 5 high-grade non-invasive, 5 low-grade non-invasive), 17 controls, 10 RCC patients and 4 urinary tract infections (UTI). Ability of selected miRNAs to identify UCUB from urine was confirmed in the validation phase based on independent cohort of 80 UCUB patients using qRT-PCR method. Results: Global expression profiling revealed set of 76 miRNAs significantly differentially expressed in urine of UCUB patients (P < 0,01) compared to healthy controls, thereof 64 highly up-regulated and 12 down-regulated. These miRNAs were specific for UCUB also when compared to other examined cohorts of patients (RCC, UTI). Moreover 23 miRNAs were able distinguish invasive and non-invasive forms of UCUB (P < 0,01) and 18 miRNAs high-grade and low-grad non-invasive (p < 0,01). Subsequent validation on larger independent cohort of UCUB patients lead to definition of urinary miRNA panel enabling sensitive and highly specific diagnosis of UCUB from urine. Conclusion: Our data have shown that urinary miRNAs could serve as sensitive and specific biomarkers of UCUB and after further independent validations could be useful tool to increase sensitivity of standard cytological examination potentially decreasing high costs for long-term follow-up of UCUB patients. This work has been supported by IGA MZCR: NT/13860-4/2012. Citation Format: Jaroslav Juracek, Hana Mlcochova, Michal Stanik, Barbora Peltanova, Robert Iliev, Taňa Machackova, Jitka Mlcochova, Renata Hežova, Jan Doležel, Ondřej Slabý. Urinary cell-free microRNAs as potential biomarkers of urothelial carcinoma of the urinary bladder. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3979. doi:10.1158/1538-7445.AM2015-3979

Abstract 237: Piwi genes and tissue/serum piR-651 are related to clinicopathologic features of renal cell carcinoma

Introduction: Piwi-interacting RNAs (piRNAs) consists a newly discovered class of non-coding RNA. PiRNAs were first identified in the germ cells of various animal species. They bind to Piwi proteins which are a subfamily of Argonaut proteins. In humans were identified four Ago genes and four Piwi genes. Human Piwi genes include PIWIL1, PIWIL2, PIWIL3 and PIWIL4. Recent studies suggest that deregulated expression of Piwi proteins is common to many types of tumors and also correlate with clinicopathologic features and worse prognosis in patients with breast, cervical, ovarian, colorectal and other cancers. PiRNAs are short single-stranded RNAs with 26-31 nucleotides in length. They are involved in silencing of transposable elements and it is assumed that also participate in sequence-specific chromatin modifications. The differential expression of piRNAs was found in gastric and breast cancer. In our pilot study we analyzed expression of Piwi genes and piR-651, which was previously found to be deregulated in various types of tumors. Patients and Methods: In our study, we have used the tumor tissue and the paired renal parenchyma tissue of 56 patients with renal cell carcinoma (RCC). From the tissue was isolated total RNA and by RT-qPCR were examined the expression of Piwi genes and piR-651. For the analysis of circulating piR-651, blood serum samples of 75 patients with RCC and 75 age, gender-matched healthy donors were used. We have compared expression levels of the studied genes in tumor and non-tumor tissues, serum samples, and also correlated them with clinicopathologic features of RCC patients (stage, grade, RFS, OS). Results: We found a significant down-regulation of PIWIL1 (p Conclusion: Accordingly to our pilot data expression of Piwi genes is altered in tumor tissue and is correlated to selected clinicopathologic features of RCC. We also suggest the potential of piR-651 in blood serum as novel non-invasive diagnostic biomarker in RCC. Acknowledgments: IGA MZCR No: NT/13860-4/2012, NT/13549-4/2012, NT/13547-4/2012, NT/13514-4/2012 Citation Format: Robert Iliev, Petra Vychytilova-Faltejskova, Jaroslav Juracek, Hana Mlcochova, Michal Stanik, Jan Dolezel, Michal Fedorko, Dalibor Pacik, Marek Svoboda, Ondrej Slaby. Piwi genes and tissue/serum piR-651 are related to clinicopathologic features of renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2015-237