Abstract 4978: On circular RNAs in breast cancer
Authors: Smid Marcel , Weerd Vanja de , Wilting Saskia , Sieuwerts Anieta , Nik-Zainal Serena , Martens John 
Published: August 2018

Abstract
Introduction Circular RNA was already discovered many decades ago and circRNAs were long considered idiosyncrasies of the splicing machinery processing precursor mRNA to mature mRNA. A much more recent take on this phenomenon showed an unanticipated abundance of circRNA isoforms in human cells. In breast cancer some work has already been done on publicly available TCGA data, but this analysis has a big limitation since the RNA-seq data were prepared using a poly(A) selection step, thereby missing most circRNAs (which lack a poly(A) tail). Here we describe the identification of circRNAs in a large cohort of 348 primary breast tumors, using RNA-seq data obtained without using a poly(A) selection step in the generation of the sequence libraries. We developed a method that, in contrast to previous identification methods, does not rely on unmapped reads or known splice-junctions.
Methods RNA-seq data were generated, using total RNA and removing ribosomal RNA using Ribo Zero (Illumina, USA). Paired-end (75 bases) sequencing was performed on an Illumina HiSeq 2000. The resulting fastq files were mapped to GRCh38 using STAR (version 2.4.2a) and the resulting bam files were sorted and indexed. CircRNAs were identified directly on the bam file generated by STAR; assuming a circular RNA molecule is present, the sequence read that aligns over the crossing of the non-canonical junction would, on a linear reference, get two different mapped locations. The read-mate aligns somewhere in between these two locations with the head of the read-mate pointing toward the head of the junction read, as would be expected in a properly paired read. Finding additional read-pairs showing this configuration, but always with a breakpoint at the exact same location, strengthens the evidence for circular transcripts.
Results In our cohort of 348 primary breast cancer cases we identified 25,783 circRNAs having a junction over exons of a gene. Of these, 13,853 (54%) were identified in at least 2 samples, and 426 circRNAs were found in at least 150 samples. The top recurrent circRNA was CDR1, a known circRNA that functions as a miR-7 sponge. Other previously described circRNAs were also present in our list. Of the circRNAs with sufficient data, 668 (41%) showed a poor (R&lt;0.2) and 210 (13%) showed a negative correlation with the expression level of the linear gene. We validated several circRNAs by PCR and sequencing, including CNOT2 and CREBBP, the former showing prognostic value, the latter a known breast cancer driver gene. Additional analyses are ongoing and will be presented at the annual meeting.
Conclusion circRNAs are an abundant RNA species in breast cancer, several of which may just be a remnant of the splicing machinery. However, several circRNAs have characteristics of being regulated and thus may have a functional role in breast cancer. Due to their structural stability, candidate circRNAs may serve as good biomarkers for diagnosis and therapy-monitoring in liquid biopsies.
Citation Format: Marcel Smid, Vanja de Weerd, Saskia Wilting, Anieta Sieuwerts, Serena Nik-Zainal, John Martens. On circular RNAs in breast 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 4978.