p16INK4a Methylation in Serum as a Follow-up Marker for Recurrence of Colorectal Cancer

Background: p16 INK4a methylation present in the tumors of colorectal cancer (CRC) patients can be detected in their serum using quantitative methylation-specific PCR (Q-MSP). To investigate the possibility that this technique could be applied to the monitoring for cancer recurrence in CRC patients, p16 INK4a methylation in the serum of CRC patients during their follow-up period was evaluated. Materials and Methods: Using Q-MSP on serum samples from 21 CRC patients undergoing surgery for primary CRC, the p16INK4a methylation score (p16 INK4a MS) was evaluated one day before surgery and during the follow-up period. Results: In the serum samples collected before primary resection, p16 INK4a methylation was detected in 8 out of the 13 patients with same methylation in the tumor. The p16 INK4a MS decreased within 2 weeks after surgery. Only two patients, who had the potential for recurrence, exhibited p16 INK4a methylation in their serum. One month after surgery, in the patients with recurrence of tumor, a dramatic increase in p16 INK4a MS was observed, while in the disease-free patients no methylation was seen continuously. Conclusion: p16 INK4a MS could sensitively reflect the recurrence status and may be useful for identifying the presence of recurrence during the follow-up of CRC patients. Colorectal cancer (CRC) is one of the most common and fatal carcinomas in the world (1). The earlier detection of recurrence would help to reduce deaths from this disease. Although much progress has been made in the identification and characterization of the genetic changes in CRC, there are few reports of the detection of recurrence using molecular biological techniques. Previous studies have proposed that tumor DNA is released into the circulation and is present in plasma and serum (2-5). The precise mechanism of the release of DNA into the bloodstream remains to be proven, however the major sources of circulating DNA were suggested to be apoptotic and necrotic cancer cells (5-7). Accordingly, it is possible to detect tumor-specific DNA, such as genetic or epigenetic alterations identified in the primary tumor DNA, in the serum of patients with various cancers (8). We previously proved that p16 INK4a promoter methylation, present in the tumors of CRC patients, can be detected in the serum of those same patients using quantitative methylation- specific PCR (Q-MSP) (9), which indicated that this approach could potentially be useful for screening and monitoring the disease. To investigate the possibility that this technique could be applied to the monitoring of cancer recurrence during the follow-up of CRC patients, the p16 INK4a methylation level in the serum of patients with CRC collected before surgery and during the follow-up period was examined using Q-MSP. Since CEA in the serum has been used as the orthodox tumor maker for monitoring CRC occurrence, CEA was also measured in this study.