An evaluation of whole gut lavage fluid for the detection of colorectal cancer using molecular techniques

• Main Author: Potter, Mark Adrian
• Published: University of Edinburgh 2000
• Subjects: 616.994
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.660707

DNA was extracted from WGLF obtained pre-operatively from 40 patients undergoing cancer resections. This was analyzed for mutations in 4 genes commonly altered in CRC. Analysis consisted of a PCR/restriction enzyme enrichment strategy for Ki-ras and p53 mutations, while APC and Transforming growth factor beta receptor II (TGFb RII) mutations were analyzed by non-enriched single strand conformational polymorphism (SSCP). There were 26 mutations in 18/40 primary tumours (45%); 7 Ki-ras, 2 p53, 2 TGFb RII and 15 APC. In the WGLF 2/7 Ki-ras mutations; 2/2 p53 mutations; 0/15 APC mutations; 0/2 TGFb RII mutations were detected. PCR inhibition was encountered and mutations were only detected with the enriched techniques. To assess the potential of detecting telomerase activity in cancer detection. WGLF from 6 patients was examined using the telomerase repeat amplification protocol (TRAP). Analysis of WGLF samples indicated that polyethylene glycol (PEG) at high concentrations inhibited the PCR portion of the TRAP assay. Dilution of the WGLF sample to give PEG concentrations that allowed PCR, still did not yield positive TRAP assay results. This suggests dilution of telomerase to concentrations below the sensitivity of the assay, however, spiking diluted WGLF samples with 10³ cells from telomerase positive cells still yielded negative results. Identical APC mutations in synchronous CRC would strongly support the notion of mosaicism. In a related line of investigation to identify potential research uses for the molecular analysis of WGLF, I initially examined synchronous colorectal cancers for APC mutations. In 12 cases the mutation cluster region of APC was examined. No identical APC mutations were identified. However, a replication error phenotype was identified in 20% of cases, suggesting an underlying mismatch repair deficiency might predispose to synchronous cancers. While DNA extracted from WGLF is suitable for PCR analysis, many samples are lost due to contamination or insufficient DNA. WGLF genetic diagnosis requires selective mutation enrichment, other methods used for mutation detection are too insensitive.