DNA/RNA Markers for Colorectal Cancer Risk in Preserved Stool Specimens: A Pilot Study

Abstract

Aims and background Exfoliated cells in human stool offer excellent opportunities to non-invasively detect molecular markers associated with colorectal tumorigenesis, and to evaluate the effects of exposures to exogenous and endogenous carcinogenic or chemopreventive substances. This pilot study investigated the feasibility of determining DNA methylation and RNA expression simultaneously in stool specimens treated with a single type of nucleic acid preservatives. Methods Stool specimens from 56 volunteers that were preserved up to a week with RNAlater were used in this study. Bisulfite sequencing was used to determine methylation at 27 CpG loci on the estrogen receptor 1 (ESR1) promoter. Taqman assay was used for quantitative reverse transcription polymerase chain reactions to measure cyclooxygenase 2 (COX2) and epidermal growth factor receptor (EGFR) mRNA expression. Subjects’ basic demographic and other selected risk factors for colorectal cancer were captured through questionnaires and correlated with the levels of these markers. Results Less than 10% of the samples failed in individual assays. Overall, 24.0% of the CpG loci on the ESR1 promoter were methylated. COX2 expression and alcohol use were positively correlated; an inverse association was present between EGFR expression and cigarette smoking; and subjects using anti-diabetic medication had higher ESR1 methylation. In addition, higher EGFR expression levels were marginally associated with history of polyps and family history of colorectal cancer. Conclusions The present study demonstrates that simultaneous analyses for DNA and RNA markers are feasible in stool samples treated with a single type of nucleotide preservatives. Among several associations observed, the association between EGFR expression and polyps deserves further investigation as a potential target for colorectal cancer screening. Larger studies are warranted to confirm some of our observations.

Noninvasive DNA methylation biomarkers in colorectal cancer: A systematic review

“…The references of the remaining 72 articles were checked manually, and two articles were further included. Finally, a total of 74 studies were eligible for this systematic review. The flowchart of study selection is shown in Figure .…”

“…Among all the 74 studies, 40 was conducted in the Western countries (including one also performed in West Asia, and one also in East Asia), 30 in East Asia, two in Iran, one in Singapore, and the remaining one unreported.…”

“…In 35 studies, samples were handled and analyzed in a blinded fashion . Methylation‐specific PCR (MSP), quantitative (qMSP) or nested MSP (nMSP) were applied in most studies, quantitative allele‐specific real‐time target and signal amplification (QuARTS) in 3 studies, Methylight or Methyl‐BEAMing (beads, emulsion, amplification and magnetics) in 9 studies, commercial methylation assay kit in 3 studies, pyrosequencing in 2 studies, Melting curve analysis in 2 studies, and methylation microarray in one study.…”

Methylated Eyes Absent 4 (EYA4) Gene Promotor in Non-neoplastic Mucosa of Ulcerative Colitis Patients with Colorectal Cancer

“…Although aberrantly methylated DNA is a very robust and chemically stable biomarker, it was beyond the scope of our aims to make direct comparisons to other classes of biomarkers for which there are promising preliminary data. 28–33 …”

Discovery of genes from feces correlated with colorectal cancer progression

“…Previously, several genes were reported as having differential expression in the feces of CRC patients (21,30). Furthermore, a number of these genes were correlated with cancer (20,21,24,(31)(32)(33)(34). The expression of the most significant of these genes must be characterized and explored in CRC cells (21,35,36).…”