MicroRNAs as molecular markers for colon cancer Diagnostic screening in stool & blood

Abstract

Screening for colon cancer (CC) allows for diagnosis of the early stage for malignancy and potentially reduces disease mortality as the cancer could be cured at disease earliest stages. Early detection could be desirable if accurate, practical and cost effective diagnostic measures for this cancer are available. Mortality and morbidity from colon cancer represent a major health problem involving a malignant disease that is theoretically preventable through screening. Current screening methods (e.g., the immunological fecal occult blood test, FOBTi, obtained from patients' medical records) either lacks sensitivity and requires dietary restriction, which impedes compliance and use; are costly (e.g., colonoscopy), which decreases compliance; or could lead to mortality. In comparison to the FOBT test, a noninvasive sensitive screen for which there is no requirement for dietary restriction would be a more convenient test. Colorectal cancer (CRC) is the only cancer for which screening by colonoscopy is recommended. Although colonoscopy is a reliable screening tool, its invasive nature, accompanying abdominal pain, potential complications and high cost have hampered the application of this screening method worldwide.A novel screening approach using the stable miRNA molecules, which are relatively nondegradable when extracted from noninvasive stool and semi-invasive blood samples by currently available commercial kits and manipulated thereafter, would be preferable to a transcriptomic messenger (m)RNA-, a mutation DNA-, an epigenetic-or a proteomic-based test. The approach utilizes reverse transcriptase (RT), followed by a modified quantitative real-time polymerase chain reaction (qPCR). Although exosomal RNA would not be measured, using a restricted extraction of total RNA from stool or blood, then a parallel test could also be carried out on RNA obtained from stool or plasma samples, and appropriate corrections for exsosomal loss can be made to obtain accurate and quantitative results. Eventually, a chip would be developed to facilitate diagnosis, as has been carried out for the quantification of genetically modified organisms (GMOs) in foods. The gold standard to which the molecular miRNA test is compared is colonoscopy. If performance criteria are met, as detailed herein, then a miRNA test in human stool or blood samples based on high throughput automated technologies and quantitative expression measurements --commonly used in the diagnostic clinical laboratory--would eventually be advanced to the clinical setting, which will make a vivid impact on the prevention of colon cancer.Correspondence to: Farid E. Ahmed, GEM

Expression of specific microRNAs in tissue and plasma in colorectal cancer

“…Screening is based on several methods with varied advantages and drawbacks and different economic impact. Existing screening modalities to diagnose CRC are most often impractical due to invasiveness and cost (e.g., colonoscopy) or insufficient diagnostic accuracy (e.g., fecal-based occult blood tests) [ 5 , 6 ].…”

Importance of Micrornas in Human Cancer Development: A Molecular Analytical Approach

“…We have shown that we can routinely and systematically be able to extract a high quality small total RNA containing microRNAs from a small number of Laser Capture Micro Dissected (LCM) cells from tissue [134], co-lonocytes isolated from human stool, or circulating blood using commercially-available kits (RNeasy isolation Kit®) from Qiagen, Valencia, CA, USA, followed by another kit from Qiagen "The "Sensiscript RT Kit". We can then quantify these microRNAs using various quantification technologies [135][136][137][138][139][140].…”

“…Global microarray expression studies [147,148], have shown similarity in expression between stool, plasma and tissue [147]. Microarray studies in stool samples ob-tained from fifteen individuals (three controls, and three each with TNM stage 0-1, stage 2, stage 3, and stage 4 colon cancer) showed 202 preferentially expressed microRNA genes that were either increased (141 mi-croRNAs), or decreased (61 microRNAs) in expression [135].…”

Quantification of MicroRNAs for the Diagnostic Screening of Colon Cancer in Human Stool by Absolute Digital(d)PCR*

“…Two studies have shown that a single miRNA act as a rheostat to fine tune the expression of hundreds of proteins [12,13]. Hence, for CRC screening, miRNA markers are much more comprehensive and preferable to a DNA-, epigenetic-, mRNA-or a protein-based marker [14][15][16][17][18]. An added advantage for the use of the stable, nondegradable miRNAs by PCR expression, by chip-based methods, is its being automatable, making them much more economical and more easily acceptable by laboratory personnel performing these assays [4].…”

“…Unlike screening for large numbers of messenger (m)RNA [1], a modest number of miRNAs is used to differentiate cancer from normal [35], and unlike mRNA, miRNAs in stool remain largely intact and stable for detection [36], therefore, leading to conclude that miRNA molecules are better markers to use for developing a reliable noninvasive diagnostic marker screen for colon cancer [14][15][16][17][18], since: a) the presence of the bacterium Escherichia coli does not hinder detection of miRNA by a sensitive technique such as dPCR [36], and b) the miRNA expression patterns are the same in primary tumor, or diseased tissue, as in stool samples [35][36][37].…”