Detection of Mutations Associated With Colorectal Cancer in DNA From Whole-Gut Lavage Fluid

Extract

The overall 5-year survival rate for colorectal cancer is 30%-40% despite surgical intervention (1). Early diagnosis is associated with better survival (2), and the benefits of developing tests that improve early diagnosis are clear. The colorectal cancer mortality reduction from fecal occult blood testing in the general population has been well demonstrated (3); however, the poor specificity of such testing suggests the need for better strategies (4). While colonoscopy remains the most sensitive method of detecting colorectal cancer, it is costly and has associated morbidity (4). The detection of tumor-specific genetic mutations holds promise as a tool for early diagnosis and screening for colorectal cancer. Ki-ras (i.e., Kirsten-RAS) gene mutations associated with colorectal cancer have been detected in the stool of Ki-ras mutation-positive case patients (5–11), as have p53 (also known as TP53) gene mutations (12,13). The potential for the detection of premalignant lesions by use of genetic tests on stool specimens has also been recognized (10). Ki-ras mutations occur in 40%- 50% of cases of colorectal cancer (14); p53 mutations occur in 50%-75% of cases (15,16). Therefore, examination of a range of mutation sites is required to achieve an acceptable level of sensitivity for genetically based diagnostic tests (17). To our knowledge, no systematic study of an array of genes in stool or colonic washings has been reported. In several studies (5,9,11,12), persistent fecal contamination of the DNA template frequently inhibited the polymerase chain reaction (PCR), which was being used for genetic analysis, so that many samples were unsuitable for examination. Colonic washings may be more suitable for PCR analysis (5,9,11). Whole-gut lavage fluid (WGLF), the clear effluent obtained via the rectum following bowel preparation (for colonoscopy), with solutions containing polyethylene glycol (PEG) and balanced electrolytes, is suitable for protein analysis in inflammatory bowel disease (18), and well-preserved inflammatory cells can be isolated (19). Unlike inflammatory cells, colonic epithelial cells are seldom found in WGLF (19), but neoplastic cells from colorectal cancer have been identified in such samples (20–22). Since WGLF might permit noninvasive assessment of material from the entire colonic lumen, we evaluated its suitability for coordinate screening of cancer-associated mutations in the Ki-ras (codon 12) gene, the p53 (codon 248) gene, the transforming growth factor-b receptor II (TGF-b RII) gene, and the mutation cluster region of the adenomatous polyposis coli (APC) gene.