Abstract P5-03-13: Circular RNAs express heterogeneously across different breast cancer subtypes and correlate with invasive disease-free survival
Authors: Li James L. , Yoshimatsu Toshio F. , McClellan Julian C. , Zhao Fangyuan , Zheng Yonglan , Olopade Olufunmilayo I. , Huo Dezheng 
Published: March 2023

Abstract
Background: Circular RNAs (circRNAs) are a large class of RNAs derived from back splicing and subsequent circularization of precursor mRNAs. Due to their circular structures, circRNAs are protected from exonuclease-induced degradation and are thereby comparatively more stable than linear RNAs. circRNAs have been implicated in the progression of multiple types of cancers but few studies have systematically examined how circRNAs associate with different subtypes of breast cancer and prognosis. Methods: We conducted a nested case-control study of prospectively ascertained participants in the Chicago Multiethnic Breast Cancer Cohort (ChiMEC), in which patients without recurrence were matched with patients with recurrence on time to recurrence, age of diagnosis, tumor stage, and clinical subtype. We performed whole exome capture RNA sequencing on tumor samples that passed quality control. We then used CIRIquant to identify circRNAs by aligning back-splice junction (BSJ) reads to pseudo-circular reference sequences and retained circRNAs that had a BSJ read count of 2 or greater in at least 5 patients. After normalization using the trimmed mean of M-values method, we used edgeR to model circRNA expression via a negative binomial distribution and performed differential expression (DE) analyses of circRNAs by ER and HER2 status, as well as between Black (self-reported) and White patients. Furthermore, we conducted survival analyses for each circRNA using Cox proportional hazards models to assess how the expression of each circRNA associated with survival outcomes of invasive disease-free survival (IDFS) and overall survival, while adjusting for age at diagnosis, stage, and HER2 status. Results: A total of 123 of 126 sequenced patients were included in the analysis, including 56 Black patients, 59 White patients, and 8 patients from other racial groups. The mean age of diagnosis was 51.9 years of age (SD 13.2) with 68% ER+, 48% PR+, and 30% HER2+ patients. We identified 16,927 high-confidence circRNAs. In the crude DE analysis, we found 489 circRNAs differentially expressed between patients with ER+ compared to patients with ER- tumors and 33 circRNAs between HER2+ vs. HER2- at a false discovery rate of 0.05. After adjusting for race and grade, we discovered 187 circRNAs differentially expressed by ER status and 38 by HER2 status. In the DE analysis by race, we found 88 circRNAs that were differentially expressed between Blacks and Whites. After adjusting for grade, ER, PR, and HER2 status, 14 circRNAs remained significantly different between racial groups. After a median of follow up of 8 years, 41 patients died, 41 patients had invasive recurrent diseases, and 2 patients had second primary breast cancers, for a total of 57 events in the IDFS analysis. Because of the matching study design to limit the impact of known prognostic factors, none of known prognostic factors (stage, ER, PR, HER2, grade, and race) were statistically associated with IDFS. In the survival analyses, we discovered two circRNAs (hsa-GSK3B_0001 and hsa-CMPK1_0006) that met the Bonferroni threshold for significance for their associations with IDFS but did not detect any circRNAs that were significantly associated with overall survival after correction for multiple testing. Discussion: This preliminary study demonstrates that multiple candidate circRNAs were differentially expressed between BC subtypes and racial groups, and several circRNAs were associated with IDFS. Future studies are warranted to validate our findings and cement the portability of these circRNAs as prognostic biomarkers across populations.
Citation Format: James L. Li, Toshio F. Yoshimatsu, Julian C. McClellan, Fangyuan Zhao, Yonglan Zheng, Olufunmilayo I. Olopade, Dezheng Huo. Circular RNAs express heterogeneously across different breast cancer subtypes and correlate with invasive disease-free survival [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-13.