Six-gene Assay As A New Biomarker In The Blood Of Patients With Colorectal Cancer:Establishment And Clinical Validation

Colorectal cancer(CRC)is the second most common cancer in men and the third most common cancer in women all over the world.Although long-term survival has improved over the past 30 years,at least 50%of patients with CRC will generate metastases after diagnosis.Although the oldest,CEA remains the most widely used serum marker in patients with CRC,CEA is a complex glycoprotein produced by 90%of colorectal cancers and contributes to the malignant characteristics of a tumor.Besides CEA,there are also other markers specifically in tumor tissues which are also used in circulating tumor cells(CTC)detection.CTC are tumor cells which released from the primary tumor and/or metastatic sites into the bloodstream,the conditions in the bloodstream are harsh for epithelial tumor cells,and it is likely that CTCs might undergo a strong selection process.Although CTCs are already used in numerous clinical trials,their clinical utility is still under investigation.Many issues regarding the detection and characterization of CTCs remain unknown because of the heterogeneity and complexlity of CTC.The biological relevance of ’epithelial’ CTC(that is EpCAM-positive and cytokeratin-positive cells)is evidenced by the prognostic significance of CTC captured by CellSearch in multiple tumour types.However,if at least a subset of CTCs undergoes epithelial-interstitial transformation(EMT),whereby epithelial markers are downregulated,technologies reliant on EpCAM and cytokeratin expression for CTC capture might fail to enrich an important subpopulation of cells.Thus,increasing attention has been dedicated to technology platforms that use a cocktail of tumor specific markers.In recent study in our group,we examined whether quantifying the mRNA of six CRC-related genes in the blood could improve disease assessment through detection of circulating tumor cells(CTC),and thereby improve progression prediction in relapsed CRC patients.Based on the features of CRC and CTC above,cell spiking assay and RT-PCR were performed with blood samples from healthy volunteers spiked with three CRC cell lines to generate an algorithm,herein called the Six-gene Assay,based on six genes(CEA,EpCAM,CK19,MUC1,EGFR and C-Met)for CTC detection.The CTCs of 50 relapsed CRC patients were then respectively measured by CEA Gene Assay(single-gene assay control)and Six-gene Assay in this thesis.Subsequently,receiver operating characteristic analysis of the CTC panel performance in diagnosing CRC was conducted for both assays.Moreover,the 2-year progression-free survival(PFS)of all patients was collected,and the application of CEA Gene Assay and Six-gene Assay in predicting PFS was carefully evaluated with different CTC cutoff values.Encouragingly,we successfully constructed the first multiple gene-based algorithm,named the Six-gene Assay,for CTC detection in CRC patients.Six-gene Assay was more sensitive than CEA Gene Assay;for instance,in 50 CRC patients,the positive rate of Six-gene Assay in CTC detection was 82%,whereas that of CEA Gene Assay was only 70%.Moreover,Six-gene Assay was more sensitive and accurate than CEA Gene Assay in diagnosing CRC as well as predicting the 2-year PFS of CRC patients.Statistical analysis demonstrated that CTC numbers measured by Six-gene Assay were significantly associated with 2-year PFS.This novel Six-gene Assay can be defined as fluid biopsy to improve the definition of disease status and correlates with PFS in relapsed CRC,and thus holds promise for future clinical applications.