**10. The use of stool MicroRNAs for detection of colorectal neoplasia**

Ultracentrifugation

Immunoaffinity Ultracentrifugation

Lung Cancer Immunoaffinity

Ovarian Cancer

Colonic epithelium is the most dynamic cell population of the human organism. Highly differentiated colonocytes are continuously shed into the colon of healthy individuals and

10,000x g 30 min and filter through

anti-EpCAM coated Immunobead

anti-EpCAM antibody coated

0.22um filter 1,00,000x g 60 min

Immunobead

MicroRNAs are Novel Biomarkers for Detection of Colorectal Cancer 13

adenoma and carcinoma from healthy controls (Link, et al, 2010). The detection of miRNAs in stool specimens requires efficient protocols for stool preparation, stool miRNA extraction and quantitative analysis (Ahmed, et al, 2009). The utility of stool miRNAs as a biomarker is still in its infancy; further studies of stool miRNA are needed on larger cohorts to validate its

In summary, systemic and faecal miRNAs can accurately correlate with disease status and can potentially be used for colorectal cancer detection and screening. Detection of colorectal cancer based on miRNA expression analysis requires extensive pre analytical considerations for sample selection & processing, isolation of miRNAs, the method of expression analysis, selection of endogenous controls for normalisation and data analysis. Studies performed so far have shown great promise for miRNA based detection of colorectal carcinoma and adenoma. There is, however, a further need to develop and evaluate miRNA based assays

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patients with CRC (Brittan, et al, 2004 & Loktionov, et al, 2007) . It is presumed that exfoliated colonocytes from healthy colon and neoplastic lesions carry important genetic and epigenetic information that could be utilized for subsequent testing, such as the detection of mutant genes or dysregulated mRNAs, proteins and miRNAs (Loktionov, et al, 2009). It is proposed that even small neoplastic loci can alter colonic cell exfoliation rate and may lead to early detection of these lesions (Loktionov, et al, 2007). The effectiveness of an exfoliated colonocyte based detection system requires an efficient isolation of colonocytes while minimizing the amount of background faecal debris. In order to achieve maximum retrieval of colonocytes, strategies that have been employed include density gradient centrifugation and/or immunoaffinity on either homogenized stool samples or scrapings from the stool surface (Loktionov, et al, 2007). However, cell yields are generally very low, often with conspicuous background debris, which makes cell identification difficult and time consuming (Deuter, et al, 1995). Consequently, such preparations would be unsuitable for high-throughput population screening programs (White, et al, 2009). Furthermore, colonocytes shed from a proximal colonic region travel a longer distance and are more exposed to cytolytic agents, thus making them less likely to be preserved and sampled. If this does prove to be a common problem, stool miRNA markers for right-sided CRC will be less effective. There is evidence, from the work of Koga and Colleagues (Koga, et al, 2010) that this is indeed the case. In this study immunomagnetic beads were conjugated with EpCAM monoclonal antibody to isolate colonocytes from stool. Despite the selection of two highly up regulated miRNAs in CRC cells, the sensitivity of detection was approximately 70% as shown in table 6. However, the detection rate for left sided colonic and rectal tumour was significantly higher, suggesting the potential utility of exfoliated colonocytes based miRNA assay as an alternative to flexible sigmoidoscopy. It is well established that profound deregulation of apoptosis is a characteristic feature of cancer. As a result of apoptosis, tumour specific proteins and genetic information i.e. DNA, RNA and miRNA are released into the lumen of colon (Ahlquist, et al, 2010). Stool environment is much more complex and hostile than plasma, and human RNA are rapidly degraded and only constitute <1% of total stool RNA (Ahlquist, et al, 2010). In contrast with the fast degradation of mRNA, human miRNAs are packed in micro vesicles and are well protected from degradation. The available data indicates that stool miRNA analysis can distinguish


Table 6.

adenoma and carcinoma from healthy controls (Link, et al, 2010). The detection of miRNAs in stool specimens requires efficient protocols for stool preparation, stool miRNA extraction and quantitative analysis (Ahmed, et al, 2009). The utility of stool miRNAs as a biomarker is still in its infancy; further studies of stool miRNA are needed on larger cohorts to validate its diagnostic accuracy.

In summary, systemic and faecal miRNAs can accurately correlate with disease status and can potentially be used for colorectal cancer detection and screening. Detection of colorectal cancer based on miRNA expression analysis requires extensive pre analytical considerations for sample selection & processing, isolation of miRNAs, the method of expression analysis, selection of endogenous controls for normalisation and data analysis. Studies performed so far have shown great promise for miRNA based detection of colorectal carcinoma and adenoma. There is, however, a further need to develop and evaluate miRNA based assays before their clinical application.
