RNA Bead-Based Assays to Detect Tumour Gene Amplifications in Blood-Derived Exosomes

Extracellular vesicles provide a tool to measure tumour characteristics in blood, at various stages of cancer development.  We have extensively used multiplex RNA bead-based assays (Quantigene assays Thermo Fisher Scientific) to detect gene amplifications in colorectal cancer tissues in multiple sites within a primary tumour, showing intra-tumour heterogeneity and in matched lymph nodes and metastatic lesions. These studies provided the selection of a gene amplification panel to study tumour-derived exosomes in blood from various stages of colorectal cancer. The selected panel includes TPX2, TOP2A, CCND3 and MYC. Of interest, TPX2 is overexpressed in 86% of colorectal cancer (CRC) samples when compared to normal colon epithelium, 50% of lung metastatic lesions show the highest fold increase of TOP2A when compared to the primary CRC tissue, CCND3 was exclusively overexpressed in lug metastasis, and MYC is consistently gained in metastasis, irrelative to site of metastatic lesion. To ensure that the methodology used is sensitive enough to measure these amplifications in patient blood-derived exosomes, we used colorectal cancer cell lines and generated exosomes following serum starvation. The mean fluorescence intensity (xMAP® Technology, Luminex) of RNA representing the amplified genes, were measured for exosomes and respective matched originator cells. Correlation analysis show an coefficient of determination, r2 of 0.95, showing that MFI magnitude derived from exosomes reflect the RNA amount from originator cells, and that gene amplification in originator cells can be measured using RNA quantification in exosomes. This led us to isolate exosomes from patients’ plasma and run a 40-plex RNA bead-based assay to measure gene amplification in tumour-derived extracellular vesicles. The 40-plex assay included 18 epithelial-metastatic transition and metastasis associated gene amplifications.  TPX2, MET and TOP2A were detected in the exosomes and matched tissue samples from the same patient. This shows the versatility of the Quantigene® assay (Thermo Fisher Scientific) and the sensitivity of the xMAP® Technology (Luminex) to detect tumour characteristics in blood. This augers well to develop protocols for early detection of disease, and early detection of metastatic disease during therapy or tumour recurrence.

Learning Objectives:

1. Assess the use of multiplex assays to measure panels of biomarkers simultaneously provides a solution to the complexity of cellular biology.

2. Examine how RNA bead-based assays provide the sensitivity required to measure mRNA in exosomes that reflect gene amplification/s in originator cellular models. 

3. Interpret how optimising methodologies through the integration of technologies, enhance end-point measurement sensitivity for tumor-derived exosome detection in patients.