Abstract
Background: p16INK4a 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, p16INK4a 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 (p16INK4a MS) was evaluated one day before surgery and during the follow-up period. Results: In the serum samples collected before primary resection, p16INK4a methylation was detected in 8 out of the 13 patients with same methylation in the tumor. The p16INK4a MS decreased within 2 weeks after surgery. Only two patients, who had the potential for recurrence, exhibited p16INK4a methylation in their serum. One month after surgery, in the patients with recurrence of tumor, a dramatic increase in p16INK4a MS was observed, while in the disease-free patients no methylation was seen continuously. Conclusion: p16INK4a 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 p16INK4a 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 p16INK4a 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.
Materials and Methods
Sample collection and DNA preparation. Peripheral blood samples were collected from 20 healthy donors and from 21 sporadic CRC patients who underwent surgical resection at Nagoya University Hospital, Nagoya, Japan. The CRC patient blood samples were obtained on the day before surgery and during the follow-up period at 2 weeks and 1, 3 and 6 months after surgical intervention. Tumor and corresponding normal tissue samples were obtained at the time of surgery from the 21 primary CRC and from two metastatic CRC. Written informed consent, as approved by the institutional review board, was obtained from all the patients. The tumor, normal tissue and serum samples were immediately frozen and stored at −80°C until the DNA was extracted. The samples were digested with proteinase K and the DNA was extracted.
Bisulfite modification. The DNA samples (1 μg) were denatured by NaOH and modified by sodium bisulfite as described elsewhere (10). The DNA samples were then purified using Wizard purification resin (Promega Corp, Madison, WI, USA), treated again with NaOH, precipitated with ethanol, then resuspended in water.
Quantitative methylation-specific PCR. Q-MSP was performed in an ABI sequence detection system 7000 using two amplification primers and a dual-labeled fluorogenic hybridization probe (Applied Biosystems, Foster City, CA, USA). The p16INK4a primers for RTQ-PCR have been described elsewhere (11). The sense and antisense primers and fluorogenic probe for the methylated sequence were p16INK4a MS (5’-TTATTAGAGGGTGGGGCGGATCGC-3’), p16INK4a MAS (5’-GACCCCGAACCGCGACCGTAA-3’) and p16INK4a MT [5’-(FAM)-AGTAGTATGGAGTCGGCGGCGGG-(TAMRA)-3’], respectively. The sense and antisense primers and fluorogenic probe for the unmethylated sequence were p16INK4a US (5’-TTATTAGAGGGTGGGGTGGATTGT-3’), p16INK4a UAS (5’-CAACCCCAAACCACAACCATAA-3’) and p16INK4a UT [5’-(FAM)-AGGTAGTGGGTGGTGGGGAGTAGTATGGAGμTTG-(TAMRA)-3’], respectively.
Thermocycling was performed in a final volume of 50 μl containing 5.0 μl of bisulfite-converted DNA from 180 μl of serum sample, 900 nM each of the p16 primers (sense and antisense), 300 nM of the corresponding fluorogenic probe, 5.0 μl PCR buffer, 2.5 μl dNTP mix, and 1.5 units of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The PCR amplification consisted of 50 cycles (95°C for 15 sec, 60°C for 60 sec, and 72°C for 18 sec) after an initial denaturation step (95°C for 10 min). Multiple negative water blanks were included in each analysis.
The amplification data were analyzed using the Sequence Detection System software developed by Applied Biosystems. The standard curve was established for quantifying the DNA copy number, using 9 known copy numbers of serially diluted (1 to 108 copies) plasmids containing p16INK4a methylated or unmethylated DNA. The Q-MSP assays were conducted in triplicate for each sample and the mean value (Q) was used for further calculation. The concentration (C) of p16 methylated or unmethylated DNA in the serum (genome-equivalents/μl) should be Q/180 because the 5.0 μl of bisulfite-converted DNA applied to RTQ-PCR was derived from 180 μl of serum. p16INK4a methylation score (p16INK4aMS) was defined as: p16INK4a MS (%)=CM/(CM+CU) ×100, where CM=concentration of p16INK4a methylated DNA in the serum and CU=concentration of p16INK4a unmethylated DNA in the serum.
Results
p16INK4a methylation in tumor tissues. Out of the 21 primary CRC tumor samples, 13 (62%) exhibited p16INK4a promoter hypermethylation (Table I) while no corresponding normal tissue samples exhibited p16INK4a methylation.
p16INK4a methylation in serum samples before surgery. In the corresponding serum samples collected one day before surgery, p16INK4a methylation was detected in 8 out of the 13 patients (62%) with p16INK4a methylation in the tumor DNA (Table I). The mean value of p16INK4a MS in the serum DNA was 4.2% (range 0-19.8%), with a median value of 5.0%. The p16INK4a MS according to the clinicopathological background of the patients is summarized in Table II. These data were derived from our previous study of 168 primary CRC patients (9). As a control, no methylation was found in the serum DNA from 20 healthy donors and the 8 CRC patients whose corresponding tumor DNA had no p16INK4a methylation.
p16INK4a methylation in serum samples during the follow-up period after surgery. At 2 weeks after primary resection, the CRC patients displayed a noticeable decrease in p16INK4a MS (Table I). The mean p16INK4a MS observed was 0.8% (range 0-9.1%). Only 2 patients, who had residual metastatic tumor or would have recurrence later, exhibited p16INK4a methylation in their serum DNA.
At 1 and 3 months after surgical intervention, the mean p16INK4a MS was 0.4% (range 0-6.1%) and 0.6% (range 0-6.4%). In the patients with recurrence of tumor a dramatic increase of p16INK4a MS was observed, while in the disease-free patients no methylation was seen continuously. Figures 1 and 2 show the clinical course of the patients with recurrence. In each case the p16INK4a MS in the follow-up period reflected more sensitively the recurrence status than CEA.
Discussion
In our previous studies p16INK4a methylation was detected in 60% of CRC tumor tissues (12), the same abnormal change was detected in the serum of almost 40% of primary CRC patients and of 70% of recurrent CRC patients with p16INK4a methylation in the tumor (12), the p16 methylation level in the serum of CRC patients significantly increased with the tumor stage and was closely associated with the malignancy of the carcinoma (9) and patients with high p16 methylation level in the serum had significantly worse survival rates than those with a low p16 methylation level (9).
In the present study, after surgical resection of the primary lesions, the p16INK4a methylation level in the serum DNA decreased within 2 weeks, except in those patients who had residual metastatic tumor or would have recurrence later. In the follow-up period, in the patients with recurrence of tumor a dramatic and sensitive increase in p16INK4a MS was observed, while in the disease-free patients no methylation was seen continuously.
At the time of tumor recurrence, the p16INK4a methylation level was elevated earlier than CEA in both cases. Tumor recurrence could also be detect earlier by p16INK4a methylation compared to CEA because p16INK4a methylation could be detected in the serum at an early tumor stage (TNM Stage I) whereas CEA elevation requires an advanced tumor mass (9, 10, 13). Although studies with a larger series of patients with CRC and with a longer follow-up period are required to confirm this possibility, p16INK4a MS could sensitively reflect the recurrence status and may be useful for identifying the presence of a recurrence or metastasis during the follow-up of CRC patients. p16INK4a MS may also be useful prospectively to identify high-risk individuals.
- Received January 12, 2011.
- Revision received April 11, 2011.
- Accepted April 12, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved