Detection of Tumor Markers Using Stools with RT-PCR

ABSTRACT

PURPOSE: There is a need for sensitive, specific diagnostic and prognostic molecular markers that can monitor the early patterns of gene expression in non-invasive exfoliated colonocytes shed in stools. It has been estimated that approximately 10(10) normal adult colonic epithelial cells, each having a lifespan of 3~4 days, are shed from the lower two-thirds of colon crypts daily; thus, the development of a screening test using colonocytes is an realistic goal. Due to the characteristics of stools, few studies have been conducted on RNA based detection methods. Herein, a mass RNA analysis, using stools in colorectal cancer, is reported. METHODS: The study included 15 colorectal cancer patients, and 15 control patients without neoplastic disease. RNA was isolated from routinely collected stool samples using a modified method. The expression levels of survivin, livin, Akt-1, caveolin, histone deacetylase (HDAC)1, matrix metalloproteinases (MMP)-2, MMP-7, MMP-9, MMP-12, hepatoma derived growth factor (HDGF), peptideYY, hypoxia-inducible factor (HIF)-1, epidermal growth factor receptor (EGFR), N-cadherin, catenin-beta, inducible nitric oxide synthase (iNOS), ring-3, enolase-1beta, insulin-like growth factor binding protein (IGFBP)-2, tissue inhibitors of metalloproteinase (TIMP)-1 and EphB2 were determined by reverse transcriptase- polymerase chain reactions (RT-PCR). RESULTS: The rates of expression of fecal survivin, livin and Akt-1 assays for colorectal cancer were all 93%; whereas, those of the fecal caveolin, HDAC1, MMP-2, HDGF and peptideYY assays for colorectal cancer were 13, 6, 20, 6 and 6%, respectively. The remaining 13 assays did not show any expression in either the colorectal or normal groups. The expression levels of survivin, livin and Akt-1 were higher in the colorectal cancer than normal group in a semiquantitative analysis (P < 0.05). CONCLUSIONS: These fecal survivin, livin and Akt-1 assays had both high expression rate and levels (colorectal cancer as distinguished from normal group) for detecting colorectal cancer; although, a larger study will be necessary to assess the expression rates and levels.