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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 < 0,01) compared to healthy controls, thereof 64 highly up-regulated and 12 down-regulated. These miRNAs showed specificity for UCUB even when compared to other examined cohorts (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). Set of 12 miRNAs with highest specificity and expression level was validated in training phase of study. Based on training phase results the panel of 3 miRNAs was profiled. Validation phase confirmed diagnostic potential and ability of this urine miRNA-based panel to differentiate between UCUB patients and controls with high sensitivity and specificity (AUC = 0,857, sensitivity = 82%, specificity = 80%). 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.

MicroRNAs (miRNAs) are the class of small non-coding RNAs, about 21-25 nucleotides in length, that play an important role in regulation of transcription. They affect gene expression at post-transcriptional levels through binding to complementary mRNAs and mediate their degradation in RISC complex. Piwi-interacting RNAs (piRNAs) is newly discovered class of non-coding RNA. 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. It was repeatedly shown that piRNAs are present also in somatic cells and are dysregulated in kidney, bladder, gastric, breast, pancreatic and liver cancers. According to small non-coding RNA databases there are about 2600 mature miRNAs and more than 24 000 piRNAs in humans. There were extensive studies aiming to discover miRNAs and to analyze their functions. However, there are only few published studies of miRNA and piRNA profiles in renal cell carcinoma (RCC) using next generation sequencing (NGS) technology. In our study we used the tumor tissue and the paired adjacent non-tumor renal parenchyma of 12 patients (8 males and 4 females) with RCC treated in Masaryk Memorial Cancer Institute (Brno, Czech Republic). RNA was isolated with mirVana™ miRNA Isolation Kit. For preparing RNA library was used TruSeq Small RNA Sample Preparation Kit from Illumina and then the miSeq sequencing technology was employed to detect small RNAs. In our 12 paired samples of tumor tissue and the paired adjacent non-tumor renal parenchyma we detected 283 miRNAs with over 1 read in at least 7 samples. Expression levels of 55 miRNAs were significantly different expressed (p < 0.05) in tumor tissue and adjacent non-tumor parenchyma. Among miRNAs with the most significantly altered expression (p < 0.01) were for example, miR-129, miR-138, miR-142, miR-149, miR-154, miR-155, miR-200b, miR-210, miR-218, miR-340, miR-584, miR-885, miR-891a, miR-1270, miR-3690 and miR-7641. After analyzing piRNA sequences we found 440 piRNAs with over 1 read in at least 7 samples. From these piRNAs were 38 piRNAs significantly deregulated (p In our pilot study we found altered expression patterns of miRNAs and piRNAs in tumor tissue of RCC and paired adjacent non-tumor renal parenchyma. For the first time, we have described piRNAs expression profile in RCC tissue by NGS approach. 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: Robert Iliev, Petra Faltejskova-Vychytilova, Zuzana Ozanova, Silvia Rybecka, Jaroslav Juracek, Jitka Mlcochova, Lenka Radova, Michal Stanik, Jan Dolezel, Michal Fedorko, Dalibor Pacik, Ondrej Slaby. MiRNA and piRNA expression profiles in renal cell carcinoma tissue detected by next generation sequencing. [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 1953.

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 Juráček, Hana Mlčochová, Michal Staník, Barbora Peltanová, Robert Iliev, Táňa Macháčková, Jitka Mlčochová, Renata Héžová, 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

Introduction: Colorectal cancer (CRC) is one of the most common types of cancers worldwide and it is the third leading cause of cancer-related death. Therefore, several efforts have been made to identify not only new diagnostic, prognostic and predictive biomarkers, but also therapeutic targets. One of the promising approaches is the characterization of the tumors using microRNAs (miRNAs). Patients and Methods: We have analyzed expression profiles of 667 miRNAs in 8 patients diagnosed for CRC and 8 paired adjacent non-tumoral tissues using TaqMan Low Density miRNA arrays. We have found miR-215 to be one of the most deregulated miRNAs, therefore its expression was further validated on independent cohort of 250 paired samples and correlated with clinicopathological features of the patients. Consequently, involvement of this miRNA in CRC pathogenesis was investigated using numerous in vitro and in vivo assays. Results: By use of miRNA expression profiling we have identified miR-215 to be highly down-regulated in tumor tissue of CRC patients (P < 0,0001). Subsequent validation on independent cohort of 250 paired samples of tumor tissues and adjacent non-tumor tissues confirmed this result, moreover, correlation between low expression of this miRNA and clinical stage (P < 0.0001) and lymph node metastasis (P < 0.0001) has been observed. In addition, significantly reduced levels of miR-215 have been detected in metastatic tissue (P < 0.0001) compared to primary tumor tissue and low expression of miR-215 was associated with shorter disease free survival (P = 0.0403). Subsequently, in vitro analyses have been performed using stable colorectal cancer cell lines in which the level of miR-215 was increased using transient or stable transfection. Higher levels of miR-215 lead to the cell cycle arrest in G1 phase of HCT-116+/+ cells (P = 0.01) and in G2/M phase of DLD-1 cells (P = 0.05), HCT-116-/- cells (P < 0.001) and HT-29 cells (P = 0.01), increased apoptosis of HCT-116+/+ cells (P < 0.001), reduced migration of DLD-1 cells (P < 0.001) and HCT-116+/+ cells (P < 0.001) and decreased proliferation activity of all analyzed cell lines (P < 0.001). Using qRT-PCR we have validated several predicted targets of miR-215 including XIAP, CD164, ALCAM or HOXB9. Finally, we have established human tumor xenografts in immunodeficient mice. We have observed that HCT-116+/+ transduced with vector containing precursor of miR-215 were associated with the slower growth of the tumors in vivo (N = 5; P = 0.04). Conclusions: The reduced expression of miR-215 has been observed in several cancers, therefore we presume that this miRNA functions as an important tumor suppressor. The results of our study indicate that miR-215 could serve as a new diagnostic and prognostic biomarker as well as potential therapeutic target for the treatment of CRC patients. The work has been supported by IGA MZCR NT13549-4/2012, NT13860-4/2012, NT13547-4/2012 and NT13514-4/2012. Citation Format: Petra Vychytilova-Faltejskova, Jana Merhautova, Jitka Mlcochova, Lenka Radova, Robert Iliev, Marek Svoboda, Rostislav Vyzula, Ondrej Slaby. MiR-215 is a tumor suppressor in colorectal cancer in vitro and in vivo. [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 3116. doi:10.1158/1538-7445.AM2015-3116

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