LINE-1 hypomethylation in gastric cancer, detected by bisulfite pyrosequencing, is associated with poor prognosis

Background Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level. In some types of human neoplasms, LINE-1 methylation level is attracting interest as a predictive marker for patient prognosis. However, the prognostic significance of LINE-1 hypomethylation in gastric cancer remains unclear. Methods Using 203 resected gastric cancer specimens, we quantified LINE-1 methylation using bisulfite-pyrosequencing technology. A Cox proportional hazards model was used to calculate the hazard ratio (HR), adjusted for the clinical and pathological variables. Results Gastric cancers showed significantly lower LINE-1 methylation levels compared to matched normal gastric mucosa (p < 0.0001; n = 74). Tumoral LINE-1 methylation range was 11.6–97.5 on a 0–100 scale (n = 203; mean 71.4, median 74.4, standard deviation 12.9). LINE-1 hypomethylation was significantly associated with shorter overall survival [log-rank p = 0.029; univariate HR 2.01, 95 % confidence interval (CI) 1.09–3.99, p = 0.023; stage-matched HR 1.88, 95 % CI 1.02–3.74, p = 0.041; multivariate HR 1.98, 95 % CI 1.04–4.04, p = 0.036]. No significant effect modification was observed by any of the covariates in survival analysis (all p interaction >0.25). Conclusions LINE-1 hypomethylation in gastric cancer is associated with shorter survival, suggesting that it has potential for use as a prognostic biomarker.


Introduction
Gastric cancer is a very common disease, the fourth most commonly diagnosed cancer and the second most common cause of cancer mortality globally [1]. Despite the developments in diagnosis and treatment technologies, the prognosis of gastric cancer patients remains poor, even for those who undergo complete resection of their carcinomas [2]. After the results of trastuzumab in patients with HER2positive gastric cancer, there is increasing interest in the development of targeted therapies in this lethal disease [3]. Importantly, epigenetic changes, including alterations in DNA methylation, are reversible, and can thus be targets for therapy or chemoprevention [4][5][6]. In addition, the identification of new prognostic or predictive molecular markers for gastric cancer could improve the risk-adapted treatment strategies and help stratify patients in future clinical trials for drugs targeting these molecular changes.
DNA methylation is a fundamental epigenetic process that modulates gene expression. Cancer cells show two types of DNA methylation alterations: global DNA hypomethylation and site-specific CpG island promoter hypermethylation [7][8][9]. Global DNA hypomethylation plays a crucial role in genomic instability, leading to cancer development and progression [10][11][12]. Because LINE-1 or the L1 retrotransposon constitutes a substantial portion (approximately 17 %) of the human genome, LINE-1 methylation levels are regarded as a surrogate marker of global DNA methylation [13]. Although LINE-1 hypomethylation is strongly associated with a poor outcome in several types of human neoplasms [14][15][16], the influence of LINE-1 hypomethylation on the prognosis of gastric cancer patients remains unclear. Given the potential relationship between LINE-1 methylation level and genomic instability, we hypothesized that LINE-1 methylation level might mark an aggressive type of gastric cancer.
In this study, to test this hypothesis, we quantified LINE-1 methylation in 203 samples of resected gastric cancers utilizing a bisulfite-polymerase chain reaction (PCR)-pyrosequencing assay, and examined the prognostic significance of LINE-1 hypomethylation in gastric cancer. Our data suggest that LINE-1 hypomethylation can have a potential role as a prognostic biomarker.

Study subjects
A total of 247 consecutive patients with gastric cancer who were undergoing resection at Kumamoto University Hospital between April 2005 and December 2009 were enrolled in this study. Nineteen patients were excluded for reasons of unavailability of adequate tissue samples. Because 22 patients received preoperative treatment, they were excluded from this study. Thus, we initially quantified LINE-1 methylation in 206 cancer specimens and obtained valid results in 203 (99 %) of the cases. Thus, a total of 203 gastric cancers were finally included in this study, and 74 cases were randomly chosen from these 203 cases to evaluate LINE-1 methylation level in normal matched mucosa. Patients were observed at 1-to 3-month intervals until death or 30 June 2011, whichever came first. Tumor staging followed the American Joint Committee on Cancer Staging Manual (7th edition) [17]. Overall survival was defined as the time between the date of the operation and the date of death. In our cohort, the 3-year overall survival rates of patients treated by gastrectomy were 91.9 % for stage I, 79.0 % for stage II, 56.8 % for stage III, and 19.5 % for stage IV. These rates are similar to those from the Japanese Gastric Cancer Association nationwide registry (94.1 % for stage I, 78.4 % for stage II, 53.2 % for stage III, and 22.4 % for stage IV), certainly supporting the absence of bias in our database. Written informed consent was obtained from each subject, and the study procedures were approved by the institutional review board. The term ''prognostic marker'' was used throughout this article according to the REMARK Guidelines [18].

DNA extraction and sodium bisulfite treatment
Hematoxylin and eosin (H&E)-stained slides of the tumors were reviewed, and areas of tumors and histologically normal gastric mucosae adjacent to tumors were marked by one pathologist (Y.B.). H&E-stained tissue sections of the largest cross-sectional slice (depending on tissue and tumor size; on average, large tumor tissue 10 lm 9 1 section) from each case were scraped off slides for DNA extraction. Genomic DNA was extracted from the tumor and normal epithelium. Genomic DNA was modified with sodium bisulfite using an EpiTect Bisulfite kit (Qiagen).
Pyrosequencing to measure the LINE-1 methylation PCR and subsequent pyrosequencing for LINE-1 were performed as previously described by Ogino et al., using the PyroMark kit (Qiagen) [14,19,20]. This assay amplifies a region of LINE-1 element (position 305-331 in accession no. X58075), which includes four CpG cites. The PCR conditions were 45 cycles of 95°C for 20 s, 50°C for 20 s, and 72°C for 20 s, followed by 72°C for 5 min. The biotinylated PCR product was purified and made singlestranded to act as a template in a pyrosequencing reaction, using the Pyrosequencing Vacuum Prep Tool (Qiagen). Pyrosequencing reactions were performed in the PyroMark Q24 System (Qiagen). The nucleotide dispensation order was ACT CAG TGT GTC AGT CAG TTA GTC TG. The non-CpG cytosine in LINE-1 repetitive sequences has been documented to be rarely methylated. Thus, complete conversion of cytosine at a non-CpG site ensured successful bisulfite conversion. The amount of C relative to the sum of the amounts of C and T at each CpG site was calculated as the percentage (i.e., 0-100). The average of the relative amounts of C in the 4 CpG sites was used as the overall LINE-1 methylation level in a given tumor (Fig. 1). In published literature, we have validated our LINE-1 methylation pyrosequencing assay; we have performed bisulfite conversion on five different DNA specimen aliquots and repeated PCR-pyrosequencing five times using four macrodissected cancers. Bisulfite-to-bisulfite (between-bisulfite treatment) standard deviation (SD) ranged from 1.4 to 2.9 (median, 2.3), and run-to-run (between-PCR pyrosequencing run) SD ranged from 0.6 to 3.3 (median, 1.2) [21]. In this study, we used ''LINE-1 methylation level'' for LINE-1 methylation as a continuous variable and ''LINE-1 hypomethylation'' for LINE-1 methylation LINE-1 methylation and prognosis in gastric cancer 481 as a categorical variable (i.e., hypomethylation vs. hypermethylation).

Statistical methods
For the statistical analyses, we used the JMP (Version 9; SAS Institute, Cary, NC, USA) and the SAS software programs (Version 9.1; SAS Institute). All p values were two sided. To compare the means, we performed the t test assuming unequal variances. For the survival analysis, the Kaplan-Meier method was used to assess the survival time distribution, and the log-rank test was used. To assess the independent effect of the LINE-1 methylation level on mortality, the tumor stage (I, II, III ? IV) was used as a stratifying (matching) variable in Cox models using the ''strata'' option in the SAS ''procphreg'' command to avoid residual confounding and overfitting.  Fig. 3). LINE-1 methylation was not significantly associated with other clinical or pathological variables. In the Kaplan-Meier analysis, LINE-1 hypomethylators (i.e., Ter2 and Ter3 cases) experienced significantly shorter overall survival (log rank p = 0.029) than those with hypermethylation (Fig. 4). In the univariate Cox regression analysis, compared to LINE-1 hypermethylated cases, LINE-1 hypomethylators experienced a significantly lower overall survival rate (HR 2.01, 95 % CI 1.09-3.99, p = 0.023) ( Table 2). In the multivariate Cox model adjusted for the clinical and pathological features, LINE-1 hypomethylation was found to be associated with a significantly lower overall survival rate (multivariate HR 1.98, 95 % CI 1.04-4.04, p = 0.036). Another independent prognostic factor was histological diffuse type (multivariate HR 1.91, 95 % CI 1.09-3.29, p = 0.023), whereas neither sex, age, nor tumor location was significantly associated with overall survival rate.

Interaction between LINE-1 hypomethylation and other variables in the survival analyses
We also examined whether the influence of LINE-1 hypomethylation on the overall survival was modified by any of the clinical and pathological variables. We did not observe a significant effect of modification by any of the covariates in survival analysis (all p interaction [0.25).
Notably, there was no significant interaction between LINE-1 methylation and tumor stage (p interaction = 0.68 for stage I, II vs. III, IV; p interaction = 0.97 for stage I vs. II-IV).

Discussion
In this study, we examined the prognostic impact of LINE-1 hypomethylation among 203 patients with resected gastric cancer. Because LINE-1 constitutes a substantial portion of the human genome, the methylation status of LINE-1 reflects global DNA methylation level [13]. We have found that LINE-1 hypomethylation (i.e., global DNA hypomethylation) in gastric cancer is associated with a poor prognosis, suggesting that LINE-1 hypomethylation may be a biomarker that can be used to identify patients who will experience an inferior outcome.  Although the prognostic factors in gastric cancer have been extensively studied [22][23][24][25], little is known regarding the prognostic value of global DNA hypomethylation. The relationship between LINE-1 hypomethylation and poor prognosis has been reported in several types of human neoplasms (e.g., prostate [26], colon [14], and ovarian [16] cancers and in chronic myeloid leukemia [27]). Our current finding in gastric cancer is in agreement with these results. On the other hand, a study of cutaneous melanoma has demonstrated that LINE-1 hypomethylation is associated with a favorable outcome [28], which did not agree with our current finding. This discrepancy might result from differences in the tumor histological type. Our data certainly support a potential role for LINE-1 hypomethylation as a prognostic biomarker for gastric cancer.
Cancer cells exhibit two types of DNA methylation alterations: global DNA hypomethylation and site-specific CpG island promoter hypermethylation [29]. It is well established that tumor suppressor genes can be silenced through promoter CpG island methylation during carcinogenesis [5,30]. In gastric cancer, a large number of genes (e.g., CDKN2A, CDK2AP2, CDH1, MGMT, RASSF1, RUNX3, and DLC1) have been shown to be suppressed by CpG island hypermethylation [31]. Of these genes, promoter hypermethylation of CDH1 [32] and MGMT [33,34] was associated with worse outcomes after surgery for gastric cancer. In contrast, the prognostic significance of global DNA hypomethylation is still unknown. To the best of our knowledge, this is the first study evaluating the relationship between LINE-1 methylation level and patient outcome in gastric cancer.
Accumulating evidence supports a crucial role of global DNA hypomethylation in tumor initiation and development: one study with a large sample collection of chronic gastritis, intestinal metaplasia, gastric adenoma, and gastric cancer demonstrated that aberrant DNA methylation occurred in early stages and tended to accumulate along the multistep gastric carcinogenesis [35]. In some types of human cancers including gastric cancer, global genomic hypomethylation has been found in the premalignant stages [36]. Nonetheless, whether global DNA hypomethylation influences cancer progression to a more advanced stage has remained uncertain. Our current finding on the relationship between LINE-1 hypomethylation and poor prognosis may support that global DNA methylation may contribute to not only initiation but also to progression of the gastric tumor.
The mechanism by which global DNA hypomethylation may confer a poor prognosis remains to be fully explored. First, genome-wide DNA hypomethylation has been shown to be associated with genomic instability [10][11][12]37], which might confer a poor prognosis. Second, the transcriptional dysregulation might be another possible mechanism, and activation of proto-oncogenes, transposable elements, or endogenous retroviruses might affect the tumor aggressiveness. Third, in addition to its role as a surrogate marker for global DNA methylation, the LINE-1 methylation status by itself likely has biological effects, because retrotransposons, such as LINE-1 elements, can provide alternative promoters [38], and contribute to noncoding RNA expression, which regulates the functions of a number of genes [39,40]. Further studies are necessary to validate our findings, as well as to elucidate Fig. 4 Kaplan-Meier curves for overall survival according to tertiles (Ter1-3) of LINE-1 methylation in gastric cancer. In panels on the right, Ter2-3 represents the hypomethylated group and Ter1 represents the hypermethylated group LINE-1 methylation and prognosis in gastric cancer 485 mechanism(s) by which LINE-1 hypomethylation affects tumor malignant behavior.
There are limitations in this study. Our cohort included relatively large numbers of patients (n = 203), but the validation cohort was missing. Our findings need to be validated in an independent dataset. In addition, epidemiological data (e.g., smoking history, alcohol drinking history, Helicobacter pylori infection) were limited.
In summary, the current study suggests that genomewide DNA hypomethylation, as measured in LINE-1, is independently associated with poor survival among patients with gastric cancer. Future studies are needed to confirm this association, as well as to examine the potential mechanism by which genome-wide DNA hypomethylation affects tumor behavior or progression.