Rare Circulating MicroRNAs as Biomarkers of Colorectal Neoplasia Scott V. Adams 1 *, Polly A. Newcomb 1 , Andrea N. Burnett-Hartman 1,2 , Michelle A. Wurscher 1 , Margaret Mandelson 3 , Melissa P. Upton 3 , Lee-Ching Zhu 4 , John D. Potter 1,2,5 , Karen W. Makar 1 1 Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 2 School of Public Health, University of Washington, Seattle, Washington, United States of America, 3 School of Medicine, University of Washington, Seattle, Washington, United States of America, 4 Anatomic Pathology, Group Health Cooperative, Seattle, Washington, United States of America, 5 Centre for Public Health Research, Massey University, Wellington, New Zealand Abstract Background: MicroRNAs (miRNAs) are regulatory RNAs, stable in circulation, and implicated in colorectal cancer (CRC) etiology and progression. Therefore they are promising as early detection biomarkers of colorectal neoplasia. However, many circulating miRNAs are highly expressed in blood cells, and therefore may not be specific to colorectal neoplasia. Methods: We selected 7 miRNA candidates with previously reported elevated expression in adenoma tissue but low expression in blood cells (‘‘rare’’ miRNAs), 2 previously proposed as adenoma biomarkers, and 3 implicated in CRC. We conducted a colonoscopy-based case-control study including 48 polyp-free controls, 43 advanced adenomas, 73 non- advanced adenomas, and 8 CRC cases. miRNAs from plasma were quantified by qRT-PCR. Correlations between miRNA expression levels, adjusted for age and sex, were assessed. We used polytomous logistic regression to estimate odds ratios (ORs) and 95% confidence intervals quantifying the association between expression levels of miRNAs and case groups. We also conducted nonparametric receiver operating characteristic (ROC) analyses and estimated area under the curve (AUC). Results: miRNAs with high expression levels were statistically significantly correlated with one another. No miRNAs were significantly associated with non-advanced or advanced adenomas. Strong (ORs .5) and significant associations with CRC were observed for 6 miRNA candidates, with corresponding AUCs significantly .0.5. Conclusions: These candidate miRNAs, assayed by qRT-PCR, are probably unsuitable as blood-based adenoma biomarkers. Strong associations between miRNAs and CRC were observed, but primarily with miRNAs highly expressed in blood cells. These results suggest that rare miRNAs will require new detection methods to serve as circulating biomarkers of adenomas. Citation: Adams SV, Newcomb PA, Burnett-Hartman AN, Wurscher MA, Mandelson M, et al. (2014) Rare Circulating MicroRNAs as Biomarkers of Colorectal Neoplasia. PLoS ONE 9(10): e108668. doi:10.1371/journal.pone.0108668 Editor: Alfons Navarro, University of Barcelona, Spain Received April 18, 2014; Accepted August 9, 2014; Published October 6, 2014 Copyright: ß 2014 Adams et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that, for approved reasons, some access restrictions apply to the data underlying the findings. Data are from the Screening Marker Study, a collaboration of the Fred Hutchinson Cancer Research Center and Group Health. Data are available for researchers meeting criteria for access to confidential data and pending review by FHCRC Institutional Review Board. Contact Dr. Polly Newcomb pnewcomb@fhcrc.org. Funding: This research was primarily supported by a Listwin Family Foundation and Gregory Fund Pilot Award. Additional support was provided by US National Institutes of Health (NIH)/National Cancer Institute grants P01 CA074184 (PAN, JDP), R03 CA165153 (KWM), K05 CA152715 (PAN), and NIH/National Center for Advancing Translational Sciences KL2 TR00042 (ABH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: sadams@fhcrc.org Introduction MicroRNAs (miRNAs) are short, non-coding regulatory RNAs that play critical roles in cell-fate determination and in controlling gene regulation [1]. In addition to regulating expression within cells, cell-derived miRNAs are stable in circulation and have been implicated in the etiology of many cancers including colorectal cancer (CRC) [2–4]. Thus, miRNAs are a promising class of potential cancer biomarkers that could augment current CRC screening and surveillance methods [5]. Nonetheless, candidate circulating miRNAs identified for use as potential biomarkers of CRC have also been implicated in the etiology of many cancers as well as other chronic diseases [3,6,7]. In addition, most of the previously identified candidate circulating miRNA biomarkers of CRC are also highly expressed in blood cells [8]. This may confound their associations with CRC because small contaminants or variations in blood cell counts could overwhelm disease-specific changes in these circulating miRNAs. Because current early detection practices can reduce the risk of developing CRC by identifying and removing adenomas, as well as identifying early stage invasive cancers, a potential biomarker to replace or supplement current CRC screening practices must detect premalignant and invasive lesions. Thus, while published studies of circulating miRNAs as CRC biomarkers have generally focused on invasive carcinoma [5], an increasing number of studies have included colorectal adenomas, recognized precursors of CRC [9–15]. PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e108668