Ito Yasushi, Kobayashi Susumu, Taniguchi Tetsushi, Kainuma Osamu, Hara Tsuyosi, Ochiai Takenori
Department of Surgery, Shimizu Kosei General Hospital, Chiba, Japan.
Int J Oncol. 2002 Jun;20(6):1263-8.
Fecal occult blood testing is widely used in the clinical screening of colorectal tumors. However, this method has so frequent false-positive results that more accurate screening-strategy should be established. Although the molecular screening using K-ras gene mutation in stools has been attempted to improve the results, the low rate of DNA extraction from stools leaves this measurement under utility value. In this study, we investigated whether or not our applied DNA extraction method from stools could produce enough DNA for the molecular screening of colorectal tumors by K-ras gene mutations in stools. We applied cetyltrimethylammonium bromide (CTAB) solution to improve human DNA extraction from stools and a mutant-allele-sensitive amplification (MASA) method to detect K-ras mutation within codon 12. We were able to confirm the stool DNA by identifying K-ras fragments in all the 20 patients. Tissue K-ras mutation was identified in 4 (2 cancers and 2 adenomas) of 20 patients. Stool K-ras mutations were found in 6 patients, 3 tissue K-ras mutation positive patients (2 cancers and an adenoma) and 3 tissue K-ras mutation negative patients. These results indicate that it is possible to extract enough DNA from human stool samples of all patients with colorectal tumors for K-ras mutation studies. K-ras mutations are more frequently detected in stools than in resected colorectal tumors. This study indicates that K-ras mutation screening in stools for colorectal cancer may include not only a primary colorectal cancer but also precancerous lesions in all parts of a gastrointestinal tract.
粪便潜血检测广泛应用于结直肠肿瘤的临床筛查。然而，该方法假阳性结果频繁，因此应建立更准确的筛查策略。尽管已尝试通过检测粪便中K-ras基因突变进行分子筛查以改善结果，但粪便DNA提取率低，使得该检测实用价值有限。在本研究中，我们调查了所应用的粪便DNA提取方法能否为通过检测粪便中K-ras基因突变进行结直肠肿瘤分子筛查提供足够的DNA。我们应用十六烷基三甲基溴化铵（CTAB）溶液改进从粪便中提取人DNA的方法，并采用突变等位基因敏感扩增（MASA）法检测第12密码子内的K-ras突变。我们在所有20例患者中均通过鉴定K-ras片段证实了粪便DNA的存在。20例患者中有4例（2例癌症和2例腺瘤）检测到组织K-ras突变。6例患者粪便中检测到K-ras突变，其中3例组织K-ras突变阳性患者（2例癌症和1例腺瘤），3例组织K-ras突变阴性患者。这些结果表明，从所有结直肠肿瘤患者的粪便样本中提取足够的DNA用于K-ras突变研究是可行的。粪便中检测到K-ras突变的频率高于切除的结直肠肿瘤。本研究表明，结直肠癌粪便K-ras突变筛查可能不仅包括原发性结直肠癌，还包括胃肠道各部位的癌前病变。
