Abstract 1299: Circular RNA expression is abundant and correlated to aggressiveness in early-stage bladder cancer
Authors: Okholm Trine Line H. , Nielsen Morten M. , Hamilton Mark P. , Christensen Lise-Lotte , Vang Søren , Hedegaard Jakob , Hansen Thomas B. , Kjems Jørgen , Dyrskjøt Lars , Pedersen Jakob S. 
Published: August 2018

Abstract
Background: Due to their stability, specificity, and accessibility, circular RNAs (circRNAs) may represent an attractive new class of biomarkers in early-stage bladder cancer (BC) and possess regulatory functions.
Experimental procedures: We characterize circRNA transcripts using whole transcriptome RNA-Seq data from 457 non-muscle-invasive bladder cancer (NMIBC) samples (348 Ta and 109 T1). We identify backsplice-spanning reads by using a modified version of the find_circ pipeline with an increased filtering stringency on both anchor sequences. Same pipeline is used to identify circRNAs in publicly available tissue samples obtained from ENCODE (n = 113) and in locally generated RNA-Seq data from unfractionated BC cell lines (n = 8) as well as from the nucleic and cytoplasmic fractions of three BC cell lines.
Results and limitations: Here, we identify more than 15,000 unique circRNAs supported by at least two reads in at least two different samples. We show that a set of highly expressed circRNAs have conserved core splice sites, are likely to be surrounded by inverted homologous Alu repeats, and are enriched with Synonymous Constraint Elements as well as microRNA target sites. Moreover, we identify 113 abundant circRNAs that are differentially expressed between high and low-risk tumor subtypes. Analysis of progression-free survival reveals 13 circRNAs that are associated with BC progression independently of the linear transcript and parent gene. We point to circHIPK3 and circCDYL as important candidates because they possess strong clinical and biological associations. The progression-free survival analyses reveal a significantly lower risk of progression for patients with high circHIPK3 and circCDYL expression levels compared to patients with low levels. Correspondingly, both circRNA candidates are found at higher levels in non-malignant BC cell lines than metastatic BC cell lines. We are currently conducting knockdown and overexpression studies in BC cell lines to reveal their biological role. Future studies should address whether circRNAs that correlate with BC progression are present in urine and plasma samples, and importantly, validation in independent cohorts should be performed in order to confirm their clinical relevance.
Conclusions: We demonstrate that abundant circRNAs possess key biological characteristics, distinguishing them from low-expressed circRNAs and non-circularized exons, and suggest that circRNAs might serve as a new class of prognostic biomarkers in NMIBC.
Citation Format: Trine Line H. Okholm, Morten M. Nielsen, Mark P. Hamilton, Lise-Lotte Christensen, Søren Vang, Jakob Hedegaard, Thomas B. Hansen, Jørgen Kjems, Lars Dyrskjøt, Jakob S. Pedersen. Circular RNA expression is abundant and correlated to aggressiveness in early-stage bladder cancer [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 1299.