Discovery and Validation of Circulating Biomarkers of Colorectal Adenoma by High-Depth Small RNA Sequencing

Abstract

Purpose Colorectal cancer (CRC) is the third most common cancer worldwide, causing ~700,000 deaths each year. The majority of CRCs begin as adenomas. Definitive screening for colorectal adenomas is currently accomplished through colonoscopy but, owing largely to costs and invasiveness, is typically limited to patient groups at higher risk by virtue of age or family history. We sought to determine if blood-based small RNA markers could detect colorectal adenoma. Experimental Design We applied high-depth small RNA sequencing to plasma from a large (n=189) cohort of patients, balanced for age, sex, and ancestry. Our analytical methodology allowed for the detection of both microRNAs and other small RNA species. We replicated sequencing results by qPCR on plasma samples from an independent cohort (n=140). Results We found several small RNA species with significant associations to colorectal adenoma, including both microRNAs and non-microRNA small RNAs. These associations were robust to correction for patient covariates, including age. Among the adenoma-associated small RNAs, two, a miR-335-5p isoform and an un-annotated small RNA, validated by qPCR in an independent cohort. A classifier trained on measures of these two RNAs in the discovery cohort yields an AUC of 0.755 (0.775 with age) for adenoma detection in the independent cohort. This classifier accurately detects adenomas in patients under 50 and is robust to sex or ancestry. Conclusions Circulating small RNAs (including but not limited to miRNAs) discovered by sequencing and validated by qPCR identify patients with colorectal adenomas effectively.

Circulating microRNA/isomiRs as novel biomarkers of esophageal squamous cell carcinoma

“…Wu et al reported that expression of isomiRs in colorectal tissue differed between normal mucosa, adenoma, and adenocarcinoma [25]. Roberts et al reported that circulating small RNA, including isomiR, were associated with colorectal adenoma [26]; and Mjelle et al identified circulating miR/isomiR associated with metastasis of rectal cancer [27]. However, few studies have examined differences in miR/isomiR between cancer patients and healthy individuals.…”

TBCRC 002: a phase II, randomized, open-label trial of preoperative letrozole with or without bevacizumab in postmenopausal women with newly diagnosed stage 2/3 hormone receptor-positive and HER2-negative breast cancer

“…Raw sequencing reads were analyzed as described previously [26]. Briefly, reads were aligned to the human whole genome (hg19) requiring perfect matches.…”

“…We predicted the agreement between qPCR and sequencing data using previously described methods [26]. We evaluated the relative proportions of 3′ ends of small RNA features, predicting that those with many, equal proportioned 3′ ends would not yield concordant data between qPCR and sequencing measurements.…”

CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries

“…Adapter dimer MAD-DASH in bead cleanup samples was dramatically reduced to within 5.9× of gel extracted samples, which prompted us to further explore the degree to which overall library quality was affected and whether this level of reduction was sufficient to generate high quality libraries. We limited further in-depth differential expression analysis to changes in miRNAs and adapter dimer sequences and not other read fractions primarily because alignment of non-miRNA smRNAs to the full reference assembly can be complicated (50) and was not expected to demonstrate significantly different results.…”