BACKGROUND: For the purpose of early detection of carcinomas, detection of p16 hypermethylation by methylation-specific polymerase chain reaction (MSP) in the serum of many kinds of malignancies has been introduced. An attempt to increase the sensitivity of this assay is reported for colorectal cancer. MATERIALS AND METHODS: DNA samples were subjected to limiting dilution before being divided into ten smaller samples. Subsequently, hemi-nested MSP was performed on those ten samples. The limiting dilution-MSP (LD-MSP) provided a 10-fold increase in sensitivity of the detection of methylated DNA compared with conventional MSP. RESULTS: Of 44 colorectal cancer patients, 30 (68%) exhibited abnormal promoter methylation of p16 in their serum DNA by LD-MSP, while 13 (30%) exhibited it by conventional MSP. As a control, the serum DNA of 50 patients with colorectal carcinomas whose corresponding tumor DNA had no methylation in the p16 promoter was screened for aberrant methylation. No methylation was found in the serum DNA of this control group by LD-MSP or conventional MSP. CONCLUSION: The high sensitivity of LD-MSP makes it possible to detect smaller amounts of tumor DNA in the serum than conventional MSP. This technique also has great specificity and no abnormal methylation in serum has yet been observed if the corresponding tumor does not exhibit methylation. This observation supports the idea that LD-MSP could be applied in clinical use for the detection of tumor DNA in serum.
BACKGROUND: For the purpose of early detection of carcinomas, detection of p16 hypermethylation by methylation-specific polymerase chain reaction (MSP) in the serum of many kinds of malignancies has been introduced. An attempt to increase the sensitivity of this assay is reported for colorectal cancer. MATERIALS AND METHODS: DNA samples were subjected to limiting dilution before being divided into ten smaller samples. Subsequently, hemi-nested MSP was performed on those ten samples. The limiting dilution-MSP (LD-MSP) provided a 10-fold increase in sensitivity of the detection of methylated DNA compared with conventional MSP. RESULTS: Of 44 colorectal cancerpatients, 30 (68%) exhibited abnormal promoter methylation of p16 in their serum DNA by LD-MSP, while 13 (30%) exhibited it by conventional MSP. As a control, the serum DNA of 50 patients with colorectal carcinomas whose corresponding tumor DNA had no methylation in the p16 promoter was screened for aberrant methylation. No methylation was found in the serum DNA of this control group by LD-MSP or conventional MSP. CONCLUSION: The high sensitivity of LD-MSP makes it possible to detect smaller amounts of tumor DNA in the serum than conventional MSP. This technique also has great specificity and no abnormal methylation in serum has yet been observed if the corresponding tumor does not exhibit methylation. This observation supports the idea that LD-MSP could be applied in clinical use for the detection of tumor DNA in serum.