**9. The use of circulating satellite MicroRNA for colorectal cancer detection**

Recent work by Mitchell & Gilad (Mitchell, et al, 2008 & Gilad, et al, 2008) has identified the presence of cancer related miRNAs in the body fluids of patients with different body organ cancers. These tumour-derived miRNAs are present in human serum or plasma in a remarkably stable form and are protected from endogenous ribonuclease activity. Given that aberrantly expressed miRNAs in CRC tissue are secreted into blood, circulating miRNAs can potentially serve as non-invasive markers for CRC detection. In 2008, Chen and colleagues used high-throughput sequencing technique and compared the miRNA expression profiles of patient with CRC and healthy controls (Chen, et al, 2008). MiRNA expression profiles of CRC and healthy controls were significantly different. However, more than 75% of the aberrantly expressed miRNAs, detected in the serum of CRC patients were also present in the serum of patients with lung cancer. A similar trend was also observed in another study where expression profiles generated from plasma of breast cancer patients were compared with colorectal cancer and other solid organ cancers (Heneghan, et al, 2010). Identification and quantification of cancer related circulating miRNAs are associated with challenges in terms of sample preparation, experimental design, and pre-analytic variation, selection of diagnostic miRNAs, data normalization and data analysis. Meyer & Kroch (Meyer, et al, 2010 & Kroh, et al, 2010) have recently addressed many of these obstacles and provided a guide for effective strategies to overcome these issues.

Preliminary studies (Ng, et al, 2009, Pu, et al, 2010 & Cheng, et al, 2011) suggest that colorectal tumour derived miRNAs are present in the circulation at detectable levels and can used as potential biomarkers for colorectal neoplasia detection. These studies used either whole plasma or total RNA extracted from a defined amount of plasma samples collected from healthy controls and diseased patients. QRT-PCR based detection systems were applied to detect selected circulating miRNAs. Selection of miRNAs was based either on results of plasma miRNA expression profiling experiments performed on relatively small cohorts of healthy and diseased patients or highly up regulated miRNAs in CRC tissue. Table 4 summarizes the sensitivity and specificity of different miRNAs investigated for their utility as biomarkers. Results of these studies are very encouraging due to the high sensitivity for detection of CRCs and adenomas. The accuracy of miRNA based detection modalities is much higher than stool based detection modalities and may be comparable with endoscopic modalities. Furthermore, the ability to detect adenomas highlights the potential role of circulating miRNAs in bowel cancer screening. Therefore, in addition to a stand alone blood test for CRC, a miRNA based blood assay can be used as a replacement of FOBT in bowel cancer screening programmes. With its higher sensitivity and specificity, it may prove cost effective and help reduce the need for unnecessary colonic investigations. Table 4 shows the comparison of sensitivity and specificity of different miRNAs for their utility as biomarkers for detection of adenocarcinoma and adenoma\*. QRT-PCR based quantification of miRNAs has been the preferred method of study in the majority of these studies.

Though the analysis of circulating miRNAs in CRC patients has identified several diagnostic miRNAs, their diagnostic accuracy is still questionable. This is due to overlapping miRNA expression with other cancers, non-cancerous conditions and variability of individual miRNA expression with stage and grade of tumour. It is possible that common carcinogenesis-related miRNAs are shared by different types of tumours and investigators


Table 4.

8 Biomarker

Preliminary studies (Ng, et al, 2009, Pu, et al, 2010 & Cheng, et al, 2011) suggest that colorectal tumour derived miRNAs are present in the circulation at detectable levels and can used as potential biomarkers for colorectal neoplasia detection. These studies used either whole plasma or total RNA extracted from a defined amount of plasma samples collected from healthy controls and diseased patients. QRT-PCR based detection systems were applied to detect selected circulating miRNAs. Selection of miRNAs was based either on results of plasma miRNA expression profiling experiments performed on relatively small cohorts of healthy and diseased patients or highly up regulated miRNAs in CRC tissue. Table 4 summarizes the sensitivity and specificity of different miRNAs investigated for their utility as biomarkers. Results of these studies are very encouraging due to the high sensitivity for detection of CRCs and adenomas. The accuracy of miRNA based detection modalities is much higher than stool based detection modalities and may be comparable with endoscopic modalities. Furthermore, the ability to detect adenomas highlights the potential role of circulating miRNAs in bowel cancer screening. Therefore, in addition to a stand alone blood test for CRC, a miRNA based blood assay can be used as a replacement of FOBT in bowel cancer screening programmes. With its higher sensitivity and specificity, it may prove cost effective and help reduce the need for unnecessary colonic investigations. Table 4 shows the comparison of sensitivity and specificity of different miRNAs for their utility as biomarkers for detection of adenocarcinoma and adenoma\*. QRT-PCR based quantification of miRNAs has been the preferred method of study in the majority of these

Though the analysis of circulating miRNAs in CRC patients has identified several diagnostic miRNAs, their diagnostic accuracy is still questionable. This is due to overlapping miRNA expression with other cancers, non-cancerous conditions and variability of individual miRNA expression with stage and grade of tumour. It is possible that common carcinogenesis-related miRNAs are shared by different types of tumours and investigators

**9. The use of circulating satellite MicroRNA for colorectal cancer detection**  Recent work by Mitchell & Gilad (Mitchell, et al, 2008 & Gilad, et al, 2008) has identified the presence of cancer related miRNAs in the body fluids of patients with different body organ cancers. These tumour-derived miRNAs are present in human serum or plasma in a remarkably stable form and are protected from endogenous ribonuclease activity. Given that aberrantly expressed miRNAs in CRC tissue are secreted into blood, circulating miRNAs can potentially serve as non-invasive markers for CRC detection. In 2008, Chen and colleagues used high-throughput sequencing technique and compared the miRNA expression profiles of patient with CRC and healthy controls (Chen, et al, 2008). MiRNA expression profiles of CRC and healthy controls were significantly different. However, more than 75% of the aberrantly expressed miRNAs, detected in the serum of CRC patients were also present in the serum of patients with lung cancer. A similar trend was also observed in another study where expression profiles generated from plasma of breast cancer patients were compared with colorectal cancer and other solid organ cancers (Heneghan, et al, 2010). Identification and quantification of cancer related circulating miRNAs are associated with challenges in terms of sample preparation, experimental design, and pre-analytic variation, selection of diagnostic miRNAs, data normalization and data analysis. Meyer & Kroch (Meyer, et al, 2010 & Kroh, et al, 2010) have recently addressed many of these obstacles and

provided a guide for effective strategies to overcome these issues.

studies.

are detecting cancer-related but not tissue specific miRNAs. Another explanation of the findings is that the detection of miRNAs released into the circulation originates in immune cells which occur as a result of a systemic immune response generated by the tumour causing abnormal proliferation of colonic cells (Dong, et al, 2011). This might also explain the finding of commonly dysregulated miRNAs in patients with CRC and Ulcerative Colitis (Pekow, et al, 2011). Furthermore, studies to date have focused on measuring the circulating levels of either single miRNAs or a subset of the known miRNAs. Due to the above reasons, a single miRNA based detection strategy would be rather ineffective whereas a CRC tissue specific expression signature generated from plasma or serum of patients with CRC and adenoma could be more informative and accurate.

The recent discovery of exosome mediated transport of cancer related miRNAs into the circulation, has shifted the focus of miRNA studies towards the isolation of tissue specific circulating exosomes and their encompassed miRNAs. Exosomes are membrane bound small vesicles (20 to 100 nm in diameter) of endocytic origin and are released by a variety of cells in both healthy and disease conditions (Théry, et al, 2002 & Keller, et al, 2006). Exosomes correspond to the internal vesicles of multivesicular bodies (MVBs) and are released in the extracellular environment upon fusion of MVBs with the plasma membrane, (Théry, et al, 2002 & Cocucci, et al, 2009). Since exosome formation includes two inward budding processes, exosomes maintain the same topological orientation as the cell, with membrane proteins on the outside and some cytosol on the inside. Exosomes contain cytoplasmic proteins, miRNAs and mRNA transcripts (Valadi, et al, 2007).

The topical orientation of exosomal membrane may help in identification of their source by using surface antigen directed antibodies e.g. anti-MHCII. One drawback of this isolation method is that unless all the exosomes contain the specific surface antigen used for the

MicroRNAs are Novel Biomarkers for Detection of Colorectal Cancer 11

Isolation method Characterisation and Validation of Exosome

(FACS)

Western Blotting

Western Blotting

Western Blotting

Western Blotting

1,200x g20 min

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

Immunobead

Transmission Electron Microscopy Immune Electron Microscopy Fluorescence-activated cell sorting

Transmission Electron Microscopy Immune Electron Microscopy

Transmission Electron Microscopy,

Transmission Electron Microscopy, Immune Electron Microscopy

400x g 20 min isolate plasma

10,000x g 30 min and filter through

anti-EpCAM coated Immunobead

anti-EpCAM antibody coated

**Isolation and Characterisation of Colorectal Cancer Cell line Exosomes** 

Differential Centrifugation

Diafiltration (5K) Ultracentrifugation Immuoaffinity

Centrifugation Diafiltration(100k) Density Gradient

Differential Centrifugation Density Gradient

**Isolation and Characterisation of Circulating Exosomes for MicroRNA Analysis**  Studies Cancer Type Isolation Method Specific Method/ Technique

> Ultracentrifugation and filtration

> Ultracentrifugation

Immunoaffinity Ultracentrifugation

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

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

Lung Cancer Immunoaffinity

Studies Colorectal

Huber, et al, 200569

Mathivanan, et

Choi, et al, 200771

van Nigel, et al, 200172

Logozzi, et al,

Rabinowits, et al, 200974

Taylor , et al, 200875

Table 5.

200973

al, 201070 Cancer Cell lines

LIM1215 Filtration,

HT29 Differential

SW403 1869col CRC28462

HT29-19A T84-

DRB1\*0401/ CIITA

Malignant Melanoma

Ovarian Cancer

#### Fig. 2.

isolation, only a fraction of the exosomes will be isolated. Circulating exosomes can also be isolated based on their size, density and surface proteins. A commonly used method of purifying exosomes involves removal of cells and debris with either a filtration process or by a series of centrifugations (differential centrifugation), followed by a final high speed centrifugation (ultracentrifugation) to pellet the exosomes. Exosomes have a specific density and can be purified by floatation in a sucrose density gradient or by sucrosedeuterium oxide (D2O) cushions. Another purification method is based on exosome size and utilizes chromatography. The size and characterisation of exosomes is performed by using transmission electron microscopy, immune-electronmicroscopy, flow cytometry and dynamic light scattering. Table 5 summarizes the exosome isolation and characterisation methods used by different groups to analyse exosomes specific to colorectal cancer cells and methods of isolation of circulating exosomes for miRNAs analysis for other cancers (Simpson, et al, 2009). There is, however, a growing need for a fast and reliable method that yields a highly purified exosome fraction.

Based on this immunoaffinity strategy, several groups have isolated exosomes from the blood of patients with different cancers and have performed miRNA expression profiles on the total RNA isolated from these purified and probably tumour specific exosomes (Taylor, et al, 2008, Logozzi, et al, 2009 & Rabinowits, et al, 2009). Patients with cancer are found to have relatively higher quantities of exosome and encompassed miRNAs in the circulation Rabinowits, et al, 2009). The analysis of miRNAs extracted from circulating exosomes in patients with ovarian cancer, has been proven to be equivalent to ovarian tissue biopsies Taylor, et al, 2008). By using a similar approach of isolation and analysis, exosomal miRNAs in colorectal cancer can be evaluated for their diagnostic accuracy and may prove a breakthrough diagnostic modality.


Table 5.

Rabinowits, et al, 200974

Taylor , et al, 200875

10 Biomarker

isolation, only a fraction of the exosomes will be isolated. Circulating exosomes can also be isolated based on their size, density and surface proteins. A commonly used method of purifying exosomes involves removal of cells and debris with either a filtration process or by a series of centrifugations (differential centrifugation), followed by a final high speed centrifugation (ultracentrifugation) to pellet the exosomes. Exosomes have a specific density and can be purified by floatation in a sucrose density gradient or by sucrosedeuterium oxide (D2O) cushions. Another purification method is based on exosome size and utilizes chromatography. The size and characterisation of exosomes is performed by using transmission electron microscopy, immune-electronmicroscopy, flow cytometry and dynamic light scattering. Table 5 summarizes the exosome isolation and characterisation methods used by different groups to analyse exosomes specific to colorectal cancer cells and methods of isolation of circulating exosomes for miRNAs analysis for other cancers (Simpson, et al, 2009). There is, however, a growing need for a fast and reliable method that

Based on this immunoaffinity strategy, several groups have isolated exosomes from the blood of patients with different cancers and have performed miRNA expression profiles on the total RNA isolated from these purified and probably tumour specific exosomes (Taylor, et al, 2008, Logozzi, et al, 2009 & Rabinowits, et al, 2009). Patients with cancer are found to have relatively higher quantities of exosome and encompassed miRNAs in the circulation Rabinowits, et al, 2009). The analysis of miRNAs extracted from circulating exosomes in patients with ovarian cancer, has been proven to be equivalent to ovarian tissue biopsies Taylor, et al, 2008). By using a similar approach of isolation and analysis, exosomal miRNAs in colorectal cancer can be evaluated for their diagnostic accuracy and may prove a

Fig. 2.

yields a highly purified exosome fraction.

breakthrough diagnostic modality.
