Robert Iliev

Circulating miR-378 and miR-451 in serum are potential biomarkers for renal cell carcinoma

BackgroundThere is no standard serum biomarker used for diagnosis or early detection of recurrence for renal cell carcinoma (RCC) patients. MicroRNAs (miRNAs) are abundant and highly stable in blood serum, and have been recently described as powerful circulating biomarkers in a wide range of solid cancers. Our aim was to identify miRNA signature that can distinguish the blood serum of RCC patients and matched healthy controls and validate identified miRNAs as potential biomarkers for RCC.MethodsIn the screening phase of the study, blood serum of 15 RCC patients and 12 matched healthy controls were analyzed by use of the TaqMan Low-Density Arrays enabling parallel identification of expression levels of 667 miRNAs through qRT-PCR-based approach. In the validation phase, identified miRNAs were further evaluated on the independent group of 90 RCC patients and 35 matched healthy controls by use of individual qRT-PCR assays and statistically evaluated.ResultsWe identified 30 miRNAs differentially expressed between serum of RCC patients and healthy controls: 19 miRNAs were up-regulated and 11 miRNAs were down-regulated in RCC patients. MiR-378, miR-451 and miR-150 were further evaluated in the independent group of patients, and two of them were successfully validated: levels of miR-378 were increased (p = 0.0003, AUC = 0.71), miR-451 levels were decreased (p < 0.0001, AUC = 0.77) in serum of RCC patients. Combination of miR-378 and miR-451 enable identification of RCC serum with the sensitivity of 81%, specificity 83% and AUC = 0.86.ConclusionsCirculating miRNAs in serum are promising biomarkers in RCC.

Identification of MicroRNAs associated with early relapse after nephrectomy in renal cell carcinoma patients

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney. One of the important unmet medical needs in RCC is prognostic biomarker enabling identification of patients at high risk of relapse after nephrectomy. MicroRNAs (miRNAs) constitute a robust regulatory network with posttranscriptional regulatory efficiency for almost one‐half of human coding genes, including oncogenes and tumor suppressors. To identify potential prognostic miRNAs, we analyzed expression profiles in tumors of different prognostic groups of RCC patients. Seventy‐seven patients with clear cell RCC and detailed clinicopathological data were enrolled in a single‐center study. Global miRNA expression profiles were obtained by use of TaqMan Low Density Arrays (754 parallel quantitative reverse‐transcriptase polymerase chain reactions (qRT‐PCR) reactions). For validation of identified miRNAs individual miRNA TaqMan assays were performed in an independent group of patients. We identified tumor relapse‐signature based on the expression of 64 miRNAs differentially expressed between relapse‐free RCC patients and RCC patients who developed relapse (20 miRNAs were increased, 44 miRNAs were decreased). In the validation phase of the study, we successfully confirmed that expression levels of miR‐143, miR‐26a, miR‐145, miR‐10b, miR‐195, and miR‐126 are lower in the tumors of RCC patients who developed tumor relapse, moreover, the lowest levels of these miRNAs we observed in primary metastatic tumors. By using Kaplan–Meier analysis, we identified that miR‐127‐3p, miR‐145, and miR‐126 are significantly correlated with relapse‐free survival of nonmetastatic RCC patients. If further validated, we suggest that identified miRNAs might be used for identification of RCC patients at high risk of early relapse after nephrectomy in clinical practice.

Combination of MiR-378 and MiR-210 Serum Levels Enables Sensitive Detection of Renal Cell Carcinoma

Serum microRNAs are emerging as a clinically useful tool for early and non-invasive detection of various cancer types including renal cell carcinoma (RCC). Based on our previous results, we performed the study to analyze circulating serum miR-378 and miR-210 in patients with various histological subtypes of RCC. RNA was purified from blood serum samples of 195 RCC patients and 100 healthy controls. The levels of miR-378 and miR-210 in serum were determined absolutely using quantitative real-time PCR. Pre- and postoperative levels of both microRNAs were compared in 20 RCC patients. Significantly increased serum levels of both miR-378 and miR-210 enabled to clearly distinguish RCC patients and healthy controls with 80% sensitivity and 78% specificity if analyzed in combination (p < 0.0001), and their levels significantly decreased in the time period of three months after radical nephrectomy (p < 0.0001). Increased level of miR-378 positively correlates with disease-free survival (p = 0.036) and clinical stage (p = 0.0476). The analysis of serum miR-378 and miR-210 proved their potential to serve as powerful non-invasive diagnostic and prognostic biomarkers in RCC.

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.

Decreased expression levels of PIWIL1, PIWIL2, and PIWIL4 are associated with worse survival in renal cell carcinoma patients.

Piwi-interacting RNAs (piRNAs) are a newly discovered class of small non-coding RNAs involved in silencing of transposable elements and in sequence-specific chromatin modifications. PIWI proteins (PIWIL), which belong to the family of Argonaute genes/proteins, bind to piRNAs and function mainly in germ line cells, but more recently were described to be functional also in stem cells and cancer cells. To date, there have been four PIWI proteins discovered in humans: PIWIL1, PIWIL2, PIWIL3, and PIWIL4. Recent studies suggested that deregulated expression of PIWI proteins and selected piRNAs is common to many types of cancers. We found significantly lower expression of PIWIL1 (P<0.0001) and piR-823 (P=0.0001) in tumor tissue in comparison to paired renal parenchyma. Further, we observed a progressive decrease in PIWIL1 (P=0.0228), PIWIL2 (P=0.0015), and PIWIL4 (P=0.0028) expression levels together with increasing clinical stage. PIWIL2 (P=0.0073) and PIWIL4 (P=0.0001) expression also progressively decreased with increasing Fuhrman grade. Most importantly, low-expression levels of PIWIL1 (P=0.009), PIWIL2 (P<0.0001), and PIWIL4 (P=0.0065) were significantly associated with worse overall survival in renal cell carcinoma (RCC) patients. Our results suggest the involvement of PIWIL genes and piR-823 in RCC pathogenesis, and indicate PIWIL1, PIWIL2, and PIWIL4 as potential prognostic biomarkers in patients with RCC.

Long Noncoding RNAs in Breast Cancer: Implications for Pathogenesis, Diagnosis, and Therapy

Human genome mapping has revealed that protein-coding genes represent less than 2 % of the total genome sequence, and simultaneously more than 75 % of the genome is actively transcribed into RNA. Recent studies of the human transcriptome led to the discovery of new heterogeneous group of transcripts—noncoding RNAs. The major part of these ncRNAs consists of long noncoding RNAs (lncRNAs), which differ in size, location on genome, and biological functions. Generally, through distinct mechanisms, they affect a number of biological processes, such as modulation of protein activity, alternative splicing of mRNA, and epigenetic regulation or microRNA silencing, and play a key role in transcriptional and posttranscriptional gene expression regulation. Deregulated levels of lncRNAs were observed with a wide range of tumors, including breast cancer. Gene expression patterns of lncRNAs are able to distinguish normal and tumor tissue or even various breast cancer stages, which makes them a potential diagnostic and prognostic biomarkers or therapeutic targets.

Exercise-induced circulating microRNA changes in athletes in various training scenarios

Background The aim of the study was to compare selected extracellular miRNA levels (miR-16, miR-21, miR-93 and miR-222 with the response to 8-week-long explosive strength training (EXPL), hypertrophic strength training (HYP) and high-intensity interval training (HIIT). Methods 30 young male athletes of white European origin (mean age: 22.5 ± 4.06 years) recruited at the Faculty of Sports Studies of Masaryk University were enrolled in this study. The study participants were randomly assigned to three possible training scenarios: EXPL, HYP or HITT and participated in 8-week-long program in given arm. Blood plasma samples were collected at the baseline and at week 5 and 8 and anthropometric and physical activity parameters were measured. Pre- and post-intervention characteristics were compared and participants were further evaluated as responders (RES) or non-responders (NRES). RES/NRES status was established for the following characteristics: 300°/s right leg extension (t300), 60°/s right leg extension (t60), isometric extension (IE), vertical jump, isometric extension of the right leg and body fat percentage (BFP). Results No differences in miRNA levels were apparent between the intervention groups at baseline. No statistically significant prediction role was observed using crude univariate stepwise regression model analysis where RES/NRES status for t300, t60, IE, vertical jump and pFM was used as a dependent variable and miR-21, miR-222, miR-16 and miR-93 levels at baseline were used as independent variables. The baseline levels of miR-93 expressed an independent prediction role for responder status based on isometric extension of the right leg (beta estimate 0.76, 95% CI: -0.01; 1.53, p = 0.052). Discussion The results of the study indicate that 8-week-long explosive strength training, hypertrophic strength training and high-intensity interval training regimens are associated with significant changes in miR-16, mir-21, miR-222 and miR-93 levels compared to a baseline in athletic young men.

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 1953: MiRNA and piRNA expression profiles in renal cell carcinoma tissue detected by next generation sequencing

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 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.

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