Stool DNA Methylation Analysis Of SFRP1 Gene Promoter In The Diagnosis Of Colorectal Cancer

BackgroundThe morbidity of colorectal cancer (CRC) has increased so fast, which is a major cause of malignant tumor related death in both male and female patients. The cost of treating CRC is raised at the same time, especially for advanced CRC. As a curable disease, the outcome of the patient depends on the key that the stage of tumor: earlier stage means better outcome. So the CRC screening of the average risk people is of great social and economic significance. Among the frequently used screening methods, such as colonoscopy, fecal occult blood testing (FOBT), no one can satisfied us completely, because of poor sensitivity and specificity, or lack of patients compliance, or the high cost. Along with the development of gene technology, detecting DNA alterations in stool DNA, as a promising noninvasive screening method for CRC, is recommended. And some pilot studies have found that detection of epigenetic alterations in stool DNA, such as DNA methylation, can get better outcome than that of detection of genetic alteration, such as mutation. Secreted frizzled-related protein 1 (sFRP1) is a newly discovered tumor suppressor gene. The sFRP1 protein acts as an antagonist of Wnt signaling. And the continuous activation of Wnt pathway plays a very important role in the carcinogenesis and progression of CRC. Mutations had rarely been found in sFRP1 gene, but the methylation at the CpG islands of its promoter is frequently present, and the methylation changes correlated with the down-regulation of its expression, which occurs frequently in CRC. Because of this characteristic of sFRP1 gene, it is a promising biomarker in stool DNA for the detection of CRC.ObjectiveTo study the use of stool DNA methylation analysis of sFRP1 gene promoter in diagnosing CRC and the relationship between sFRP1 gene promoter hypermethylation and the clinicopathological characteristics of CRC patients. We also evaluated the stool DNA isolation kit in isolating and purifying stool DNA at the same time.MethodsStool from patients suffering from colorectal cancer, colorectal polyps or normal people, who were admitted to Chang Hai Hospital in the second department of general surgery during June 2007 to Feburary 2008, were collected. Clinical characteristics of these patients and medical record reports were reviewed. After stool DNA isolated by the stool DNA isolation kit, MS-PCR (methylation-specific PCR) was applied to analyze the promoter hypermethylation of sFRP1 gene in the stool DNA.ResultsStool DNA were isolated from stool samples of 55 patients and at last 45 samples passed all the testing procedures, including 21 patients with CRC and 15 patients with colorectal polyps and 9 patients without colorectal neoplasia. The patients without colorectal neoplasia were included in control group. The sFRP1 gene promoter hypermethylation in CRC and colorectal polyps were 66.7% (14/21) and 46.7% (7/15) respectively. There was no promoter hypermethylation in the 9 patients without colorectal neoplasia. The difference in hypermethylation status of the sFRP1 gene promoter between the CRC and the control groups was statistically significant (P<0.001). There was also a significant difference between the colorectal polyps and the control groups (P<0.05).But there was no significant difference between the CRC and the colorectal polyps groups. This method had a sensitivity of 66.7% and specificity of 70.8% for detection CRC. If the precancerous lesions included (colorectal polyps characterized by size >1cm, villous histology, and dysplasia), the sensitivity was 65.5% and the specificity was 87.5%.The promoter hypermethylation of sFRP1 gene didn't correlate with age, gender, tumor site, lymphatic metastasis and TNM stage.ConclusionsThe stool DNA isolation kit can provide purified stool DNA, but the quantity of stool DNA mainly depended on the sample itself. The stool DNA sFRP1 gene promoter hypermethylation test can provide high sensitivity for detection CRC, especially for the early lesions, but the specificity is low. It can not distinguish carcinoma from adenoma very well. The sFRP1 gene promoter hypermethylation was not associated with the clinicopathological characteristics of the CRC patients, including age, gender, tumor site, lymphatic metastasis and TNM stage. The detection of sFRP1 gene promoter hypermethylation in stool DNA is a potential noninvasive screening method for CRC and deserves further research.