Extracellular vesicle microRNA as liquid biopsy biomarkers in early stage colorectal cancer patients

Abstract

New Zealand has one of the highest rates of colorectal cancer (CRC) in the world. Current strategies to diagnose CRC, including population level stool-based screening, are flawed due to poor sensitivity in early disease and limited participation. Patients have expressed a preference for a blood-based test, or ‘liquid biopsy’, if it were available. A liquid biopsy that has parity with, or outperforms current stool based screening would likely lead to earlier diagnosis of CRC and improved patient outcomes. Extracellular Vesicles (EVs) are produced in abundance by CRC cells, their contents also reflect their parent cell of origin. microRNA (miRNA) are small non-coding RNA molecules, which are dysregulated in CRC cells compared to normal cells. Identifying EV miRNA in the bloodstream of early stage CRC patients that are sufficiently different from healthy patients may enable identification of CRC from a liquid biopsy. Our study examined four miRNAs, miR-19a, miR-23a, miR-183 and miR-1246, in both tumour tissue and plasma EVs that have previously been reported as dysregulated in CRC.

miRNA expression of the candidate miRNAs was in examined in CRC tumour tissue vs normal colonic mucosa, and in plasma EVs of stage I and II CRC patients vs healthy controls, by RT-qPCR. Changes in expression in tissue were assessed to establish if they would translate to circulating EVs. Furthermore, any differences between the miRNA expression in plasma EVs were examined to assess for a capacity to differentiate early stage CRC patients from healthy controls and be useful as a liquid biopsy biomarker in CRC.

From our results, miR-1246 and miR-183 were significantly overexpressed in tumour tissue compared to normal colonic mucosa. However, neither miRNA was consistently expressed in the circulating EVs. In the plasma analysis, miR-19a was significantly downregulated in EVs of CRC patients. It demonstrated significant differences even when stage I patients alone were compared against controls and it also had superior sensitivity to CEA in our cohort. This appears to be the first study to document a significant decrease in miR-19 in the EVs of early stage CRC patients. From our results, significant variance from the current literature was seen. Some possible factors for this variance may include differences in methodology and control selection.

Although, plasma EV miRNA display great promise as liquid biopsy biomarkers, there are a number of challenges in identifying a marker specific for CRC. Standardisation of methodology may help identify candidate miRNAs that offer the greatest potential. Finding a liquid biopsy biomarker for CRC that is equally effective, or outperforms, current faecal-based methods would likely increase uptake in screening. The subsequent effects would include decreased healthcare costs due to earlier diagnoses of CRC with improved patient outcomes for the growing millions of people who will suffer from this disease.