Jaroslav Juracek

Dynamic changes in microRNA expression profiles reflect progression of Barrett’s esophagus to esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) is highly aggressive malignancy that frequently develops from Barretts esophagus (BE), a premalignant pathologic change occurring in the lower end of the esophagus. MicroRNAs (miRNAs) are small, non-coding RNAs that function as posttranscriptional regulators of gene expression and were repeatedly proved to play key roles in pathogenesis of BE as well as EAC. In our study, we used Affymetrix GeneChip miRNA arrays to obtain miRNA expression profiles in total of 119 tissue samples [24 normal esophageal mucosa (EM), 60 BE and 35 EAC]. We identified a number of miRNAs, that showed altered expression progressively in sequence EM, BE and EAC, including for instance miR-21, miR-25, miR-194 and miR-196a with increasing levels (P < 0.0015) and miR-203, miR-205, miR-210 and miR-378 with decreasing levels (P < 0.0001). The subsequent analysis revealed four diagnostic miRNA signatures enabling to distinguish EM and BE [12 miRNAs, area under curve (AUC) = 0.971], EM and EAC (13 miRNAs, AUC = 1.0), BE without and BE with dysplasia (21 miRNAs, AUC = 0.856) and BE without dysplastic changes and BE with dysplasia together with EAC (2 miRNAs, AUC = 0.886). We suggest that miRNA expression profiling expands current knowledge in molecular pathology of Barretts-based carcinogenesis and enables identification of molecular biomarkers for early detection of BE dysplasia and progression to EAC.

MicroRNAs in the pathogenesis of renal cell carcinoma and their diagnostic and prognostic utility as cancer biomarkers.

Purpose To provide information about the role of microRNAs in the pathogenesis of renal cell carcinoma (RCC) and their diagnostic and prognostic utility as cancer biomarkers. Methods A literature search was performed in the PubMed and Web of Science databases using the keywords “renal cancer/renal cell carcinoma/kidney cancer” and “miR*/miRNA*/microRNA*”. Articles dealing with the role of miRNAs in the pathogenesis of RCC, diagnostic miRNAs and prognostic miRNAs were separated. Results MiRNAs act both as oncogenes and tumor suppressors. They regulate apoptosis, cell growth, migration, invasion, proliferation, colony formation and angiogenesis through target proteins involved in several signaling pathways, and they are involved in key pathogenetic mechanisms such as hypoxia (HIF/VHL dependent) and epithelial-to-mesenchymal transition. Differentially expressed miRNAs can discriminate either tumor tissue from healthy renal tissue or different RCC subtypes. Circulating miRNAs are promissing as diagnostic biomarkers of RCC. Information about urinary miRNAs associated with RCC is sparse. Detection of a relapse is another implication of diagnostic miRNAs. The expression profiles of several miRNAs correlate with the prognosis of RCC patients. Comparison between primary tumor tissue and metastasis may help identify high-risk primary tumors. Finally, response to target therapy can be estimated thanks to differences in miRNA expression in tissue and serum of therapy-resistant versus therapy-sensitive patients. Conclusions Our understanding of the role of microRNAs in RCC pathogenesis has been increasing dramatically. Identification and validation of their gene targets may have direct impact on developing microRNA-based anticancer therapy. Several microRNAs can serve as diagnostic and prognostic biomarkers.

Detection of let-7 miRNAs in urine supernatant as potential diagnostic approach in non-metastatic clear-cell renal cell carcinoma

Introduction Urinary microRNAs (miRNAs) are emerging as a clinically useful tool for early and non-invasive detection of various types of cancer. The aim of this study was to evaluate whether let-7 family miRNAs differ in their urinary concentrations between renal cell carcinoma (RCC) cases and healthy controls. Materials and methods In the case-control study, 69 non-metastatic clear-cell RCC patients and 36 gender/age-matched healthy controls were prospectively enrolled. Total RNA was purified from cell-free supernatant of the 105 first morning urine specimens. Let-7 family miRNAs were determined in cell-free supernatant using quantitative miRNA real-time reverse-transcription PCR and absolute quantification approach. Results Concentrations of all let-7 miRNAs (let-7a, let-7b, let-7c, let-7d, let-7e and let-7g) were significantly higher in urine samples obtained from RCC patients compared to healthy controls (P < 0.001; P < 0.001; P = 0.005; P = 0.006; P = 0.015 and P = 0.002, respectively). Subsequent ROC analysis has shown that let-7a concentration possesses good ability to differentiate between cases and controls with area under curve being 0.8307 (sensitivity 71%, specificity 81%). Conclusions We have shown that let-7 miRNAs are abundant in the urine samples of patients with clear-cell RCC, and out of six let-7 family members, let-7a outperforms the others and presents promising non-invasive biomarker for the detection of RCC.

Genome-wide identification of urinary cell-free microRNAs for non-invasive detection of bladder cancer

Urinary microRNAs (miRNAs) are emerging as clinically useful tool for early and non‐invasive detection of various types of cancer including bladder cancer (BCA). In this study, 205 patients with BCA and 99 healthy controls were prospectively enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays (2578 miRNAs) and candidate miRNAs further validated in independent cohorts using qRT‐PCR. Whole‐genome profiling identified 76 miRNAs with significantly different concentrations in urine of BCA compared to controls (P < 0.01). In the training and independent validation phase of the study, miR‐31‐5p, miR‐93‐5p and miR‐191‐5p were confirmed to have significantly higher levels in urine of patients with BCA in comparison with controls (P < 0.01). We further established 2‐miRNA‐based urinary DxScore (miR‐93‐5p, miR‐31‐5p) enabling sensitive BCA detection with AUC being 0.84 and 0.81 in the training and validation phase, respectively. Moreover, DxScore significantly differed in the various histopathological subgroups of BCA and decreased post‐operatively. In conclusion, we identified and independently validated cell‐free urinary miRNAs as promising biomarkers enabling non‐invasive detection of BCA.

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.

Abstract 521: Dynamic measurements of circulating microRNAs reflect different biological effects of radiofrequency ablation and transarterial chemoembolisation in liver cancer patients

Introduction: The majority of primary or metastatic liver tumors are unresectable (because of tumor size, location, poor performance status or multifocality), therefore other therapeutic modalities as radiofrequency ablation (RFA) and transarterial chemoembolization (TACE) are applied. RFA is a localized thermal treatment technique designed to produce tumor destruction by heating tumor tissue, while TACE combines cytotoxic effect of particle based tumor ischemia and locoregional chemotherapy. Both methods cause characteristic changes in liver tissue (inflammation, hypoxia, elevated temperature, tissue destruction) accompanied by targeted systemic secretion of microRNA into the bloodstream. Since RFA and TACE differ in the dynamics with which they affects the tumor tissue, we aimed to investigate whether the expression level of circulating microRNAs related to hypoxia (miR-21 and miR-210), liver injury (miR-122) and epithelial-mesenchymal transition (miR-200a) could reflect such changes. Material and methods: This study consisted of 14 patients diagnosed with primary hepatocellular carcinoma (HCC) (median age 73; TACE) and 20 patients diagnosed with liver metastases of colorectal cancer (median age 63; 17 patients - RFA, 3 patients - TACE). RFA was performed using the rf/mw generator (AngioDynamics). For TACE drug eluting beads (Biocompatibles Ltd.) loaded with irinotecan for mCRC patients and doxorubicin for HCC patients were used. The concentrations of miRNA were determined for all patients in series of blood plasma from 4 time points (before intervention, immediately after intervention, 24 hours after intervention, 1 week after intervention) using miRNA-specific TaqMan assays and qRT-PCR method. Results: In RFA cases we observed significant increase of investigated miRNA concentrations immediately after intervention (miR-122, FC = 15, P = 0.0002; miR-200a, FC = 1.9, P = 0.015). In TACE we observed delayed increase in circulating miRNA concentrations at time point 24 hours after intervention (miR-21, FC = 10.4, P Conclusions: Our preliminary data indicates potential usage of circulating miRNAs for monitoring of the systemic effects of RFA and TACE therapy and their ability to reflect efficacy of intervention procedures. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 16-31314A, and 15-34678A. Citation Format: Jaroslav Juracek, Tomas Andrasina, Barbora Cechova, Petra Vesela, Jan Zavadil, Tana Machackova, Jiri Sana, Marek Vecera, Natalia Gablo, Marek Svoboda, Nahum Goldberg, Ondrej Slaby. Dynamic measurements of circulating microRNAs reflect different biological effects of radiofrequency ablation and transarterial chemoembolisation in liver cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 521.

Abstract 3445: ZFAS1 is upregulated in GBM tissue and affects viability and migration of GBM cells in vitro

Introduction: Glioblastoma multiforme (GBM) is the most frequent primary brain tumor of astrocytic origin characterized by very poor prognosis. Despite conventional therapeutic protocol the prognosis of GBM patients is very poor with median of overall survival ranging between 12 and 15 months from diagnosis. Therefore, many financial charges and lot of effort is spent in research of new therapeutic approaches that could prolong the survival of GBM patients. Long non-coding RNAs (lncRNAs) are a relatively new class of noncoding gene regulators playing critical roles in tumor biology, including GBM. From this perspective, lncRNAs seem to be promising therapeutic targets in GBM patients. Material and Methods: We performed next-generation sequencing analysis of fresh-frozen histopatologically confirmed 45 GBM tissues and 5 non-tumor brain tissues obtained from non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient before the treatment. rRNA depletion and cDNA library preparation were performed with GeneRead rRNA Depletion Kit (Qiagen) and NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), respectively. Sequencing was held using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). Statistical analysis evaluated 24 087 protein-coding and 8 414 non-coding RNAs and their sequential variants with non-zerou RPKM (Reads Per Kilobase of transcript per Million mapped reads) at least in one sample. We used CLC genomic workbench for the alignment and target counts. Targeted regulation of ZFAS1 level have been carried out by the transient transfection of specific siRNA in GBM stable cell lines (A172, T98G, U87MG, U251). Viability and migration were analyzed in vitro using MTT and scratch wound healing assay, respectively. Results: Statistical analysis has revealed 274 (P Note: This abstract was not presented at the meeting. Citation Format: Jiri Sana, Marek Vecera, Romana Butova, Jaroslav Juracek, Tana Machackova, Parwez Ahmad, Natalia Anna Gablo, Kamila Souckova, Leos Kren, Radim Lipina, Martin Smrcka, Ondrej Slaby. ZFAS1 is upregulated in GBM tissue and affects viability and migration of GBM cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3445. doi:10.1158/1538-7445.AM2017-3445

Abstract 3425: MiR-215-5p is a tumor suppressor in colorectal cancer targeting EGFR ligand epiregulin and its transcriptional inducer HOXB9

Growing evidence suggests that microRNAs (miRNAs) are involved in the development and progression of colorectal cancer (CRC). In the present study, deregulation and functioning of tumor suppressive miR-215-5p was evaluated in CRC. In total, 448 tumor tissues and 325 paired adjacent healthy tissues have been used for miRNA expression analyses. We proved that miR-215-5p is significantly down-regulated in tumor tissues compared to non-tumor adjacent tissues and its decreased levels correlate with the presence of lymph node metastases, tumor stage, and shorter overall survival in CRC patients. To identify specific cellular processes affected by ectopic expression of miR-215-5p, a series of in vitro experiments have been performed using transient transfection of miR-215-5p mimics into four CRC cell lines. Increased levels of miR-215-5p significantly reduced proliferation, clonogenicity, and migration of CRC cells, lead to cell cycle arrest in G2/M phase and p53-dependent induction of apoptosis. The ability of miR-215-5p to inhibit tumor growth was confirmed in vivo by use of NSG mice model and stable cell line over-expressing miR-215-5p. Finally, we proved epiregulin and HOXB9 to be the direct targets of miR-215-5p by luciferase assay and western blot analyses. Since epiregulin is EGFR ligand and HOXB9, is its transcriptional inducer, we suggest, that the main molecular link between miR-215-5p and CRC cells phenotypes presents the EGFR signaling pathway, which is one of the canonical pathogenic pathways in CRC. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S. Citation Format: Petra Faltejskova-Vychytilova, Jana Merhautova, Pablo Conesa-Zamora, Katerina Slaba, Tana Machackova, Marek Svoboda, Marek Vecera, Jitka Mlcochova, Jaroslav Juracek, Jiri Sana, Parwez Ahmad, Natalia Gablo, Ondrej Slaby. MiR-215-5p is a tumor suppressor in colorectal cancer targeting EGFR ligand epiregulin and its transcriptional inducer HOXB9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3425. doi:10.1158/1538-7445.AM2017-3425

Abstract 5451: Panel of urinary cell-free microRNAs in detection of urinary bladder cancer

Bladder cancer is the most common cancer of the urinary tract. More than 90% of bladder cancers are urothelial carcinoma, which are divided into non-muscle-invasive and muscle-invasive forms. Non-muscle-invasive tumors frequently recur (50-70%) and can also progress to invasion form (10-15%). These patients are monitored by cystoscopy and may have multiple resections over many years. Improved monitoring method is needed, ideally via urine analysis, which could reduce the morbidity and costs associated with long follow up. Currently there are no molecular biomarkers which could diagnose or accurately predict disease progression. We aimed to develop a clinically applicable, specific and sensitive panel of urine microRNAs enabling detect bladder cancer and predict risk of progression to muscle-invasive form.Within the exploratory phase of study we have analyzed expression profiles of 1733 miRNAs in urine supernatant of 16 bladder cancer patients (6 muscle invasive, 5 high-grade muscle non-invasive, 5 low-grade muscle non-invasive), 17 controls, 10 RCC patients and 4 urinary tract infections (UTI) using Affymetrix miRNA microarrays. Diagnostic and prognostic potential of selected microRNAs was further validated on independent samples in training phase (50 bladder cancer patients, 15 controls) and validation phase (100 bladder cancer patients, 55 controls, 45 renal cancer patients) using specific TaqMan assays and qRT-PCR method.Global expression profiling identified set of 76 miRNAs able distinguish bladder cancer patients from healthy controls (P Citation Format: Jaroslav Juracek, Tana Machackova, Marek Vecera, Kamila Souckova, Jiri Sana, Parwez Ahmad, Natalia Anna Gablo, Ondrej Slaby, Michal Stanik, Jan Dolezel. Panel of urinary cell-free microRNAs in detection of urinary bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2017-5451

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.