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Disappearance of Serum Methylated p16 Indicates Longer Survival in Patients with Gastric Cancer

Purpose The aim of this study was to assess clinical correlations with postoperative alteration of p16 DNA methylation, and to clarify whether postoperative changes in the serum DNA methylation status of p16 could be used as a reliable prognostic factor for gastric cancer. Materials and Methods Fifty-three consecutive gastric adenocarcinoma patients who underwent gastric resection (Chung-Ang University Hospital, Seoul, Korea) were included. DNA methylation of p16 was evaluated by methylation-specific polymerase chain reaction using serum DNA preoperatively and at the 10th postoperative day. The correlation between changes in methylation status and patients' prognosis was analyzed. Results p16 was methylated in 79.2% of preoperative serum DNA and in 54.7% of postoperative serum DNA, respectively. Methylation in p16 disappeared more frequently in patients who underwent standard D2 lymphadenectomy compared to those who underwent modified D1+ lymphadenectomy (P=0.016). Whereas methylation of preoperative serum DNA was not correlated with survival, patients with postoperative disappearance of p16 methylation showed longer survival than those without postoperative disappearance of p16 methylation in the patients who had gastric cancer with lymph node metastasis (P=0.042). Conclusions Postoperative disappearance of p16 methylation could be an available prognostic factor for node-positive gastric cancer.


Introduction
The most effective method for treatment of gastric cancer is surgical resection including radical lymph node dissection and combined resection of the invaded organ. Improved surgical management has played an important role in treatment outcomes. 1 Many studies have indicated that surgical curability is one of the most valuable prognostic factors together with the pathologic stage. 2,3 However, patients may experience tumor recurrence after curative resection, and the recurrence pattern and survival outcomes differ among individual patients even at the same tumor stage.
Thus, metastasis may have already occurred in many gastric cancer patients at the time of surgery, presenting a limitation to assessment of the prognosis with surgical curability and pathologic stage. Strategic management of adjuvant treatment is important for patients who have the potential for cancer recurrence after curative surgery.
A variety of parameters have been studied to select the appropriate candidate for adjuvant treatment and to detect cancer recurrence earlier. DNA methylation is one such proposed prognostic factor for gastric cancer. 4,5 Epigenetic silencing of tumor-related genes by promoter hypermethylation is increasingly recognized as having an instrumental role in the development of cancers. 4 Gastric cancer is one of the tumors with a high frequency of aberrant methylation. 6,7 DNA methylation of tumor-related genes occurs in the early stages of disease 6,8,9 and increases in parallel with the multistep pathway of gastric carcinogenesis. 10 DNA methylation has also been reported to be associated with the tumor stage in gastric cancer. 11 However, DNA of cancer tissue from surgical or endoscopic biopsy can be obtained only before surgery or at the time of surgery. Thus, DNA analysis from tissue biopsy is not applicable to a continuing treatment strategy, but serum DNA methylation of tumor-related genes could be used as a method for cancer screening and surveillance for cancer recurrence. However, to date, there have been no reports of postoperative changes in methylation in gastric cancer patients.
Here we used methylation-specific polymerase chain reaction (PCR) (MSP) to evaluate p16 methylation, one of cancer-associated genes of gastric cancer, 12,13 in preoperative and postoperative serum from gastric cancer patients. Furthermore, we sought to determine the clinical correlations of the change of serum p16 methylation with the survival and prognosis of patients with gastric adenocarcinoma.   The primer sequence of p16 was based on the previous report and are listed in Table 1. 15

Statistical analysis
Statistical analyses were performed with SPSS statistical software, version 16.0 for Windows (SPSS Inc., Chicago, IL, USA).
Categorical data were analyzed by chi-square or Fisher's exact tests, and numerical data were analyzed by the Student's t test.
Correlations between the methylation status were analyzed by the   Table 2).
The relationship between clinicopathologic parameters and p16 methylation in preoperative serum DNA was evaluated. Methylation of p16 was more frequently detected in the patients with nodenegative gastric cancer than in node-positive patients (P=0.010).
Methylation in p16 disappeared more frequently in the patients who underwent standard D2 lymphadenectomy than in those who underwent modified D1+ lymphadenectomy (P=0.016) ( Table 3).
In the survival analysis for 53 patients, the disease free survival (DFS) of patients with preoperative p16 methylation was not different from that of patients without such methylation ( Fig. 2A; P=0.088).
The 3-year survival rate of patients with postoperative disappearance of p16 methylation was 94.7%, whereas the 3-year survival rate of patients without postoperative disappearance of p16 methylation was 66.9%. However, this difference was not statistically significant (Fig. 2B, P=0.238).
We further stratified the patients according to the depth of tumor (EGC or AGC) or lymph node metastasis, and compared DFS   The DNA methylation analysis in this study was conducted not from the cancer tissue but from patient's serum. Unlike tumor tissue samples, serum is usually more accessible for sampling at any time after surgery. Although many studies have suggested that tumor Values are presented as number (total number). LN = lymph node; EGC = early gastric cancer; AGC = advanced gastric cancer.  tissue DNA methylation has prognostic value, 11,12,[16][17][18] studies using serum DNA have failed to show the prognostic value of methylation in colon cancer, lung cancer, and liver cell cancer. [19][20][21][22] DNA methylation usually has a lower detection rate in serum as compared with tumor tissue. 22 Lee et al. 22  To our knowledge, this is the first report in which patients' survival has been analyzed in terms of the postoperative change in DNA methylation.
We found that the DNA methylation frequency for p16 was high in preoperative serum and had a tendency towards decreased frequency postoperatively. Thus, disappearance of methylation of p16 may be correlated with surgical curability and patient survival, making it a candidate prognostic factor. Previous studies of tumor tissue DNA indicated that methylation of p16 is a potential biomarker for early diagnosis and prognostic evaluation in gastric cancer. 17,18 Methylation of p16 gene is frequently detected in lymphatic invasive gastric cancer and correlated with chemosensitivity. 12,13 In this study, we found that p16 was useful gene for evaluation of serum DNA methylation of gastric cancer patients. were not enough to be evaluated by multivariate analysis model.
That is the limitation of this study. However, disappearance of p16 methylation was uniquely correlated with DFS of node-positive gastric cancer patients who had preoperative hypermethylation. Although the large scaled and long-term follow up study is needed to conclude our result, the survival difference according to the disappearance of p16 methylation for node-positive gastric cancer could be meaningful in itself.
In East Asia, where the incidence of gastric cancer is high, the need for a more reliable noninvasive screening test is obvious. Serum DNA methylation has been suggested as a new screening test by several reports. 22,23 However, in Korea and Japan, gastroscopy is a well established cancer screening test and gastroscopic biopsy provides high accuracy. Therefore, serum DNA methylation would be useful for postoperative surveillance and as a prognostic indicator of recurrence, rather than a screening test. Our results suggest methylation of p16 as a reliable biomarker for predicting prognosis by postoperative assessment.
In conclusion, we found that postoperative disappearance of methylation in the p16 gene in serum DNA was related to patients' survival after gastric cancer surgery. These findings suggest that the postoperative disappearance of methylation in p16 could be used as prognostic factor for node-positive gastric cancer. Additional studies are necessary to clarify the prognostic value of DNA methylation for gastric cancer patients in terms of long-term survival.