Med Oncol (2015) 32:117 DOI 10.1007/s12032-015-0571-5
ORIGINAL PAPER
Relationship between LINE-1 hypomethylation and Helicobacter pylori infection in gastric mucosae Keisuke Kosumi1 • Yoshifumi Baba1 • Takatsugu Ishimoto1 • Kazuto Harada1 • Keisuke Miyake1 • Daisuke Izumi1 • Ryuma Tokunaga1 • Asuka Murata1 • Kojiro Eto1 • Hidetaka Sugihara1 • Hironobu Shigaki1 • Shiro Iwagami1 • Yasuo Sakamoto1 • Yuji Miyamoto1 • Naoya Yoshida1 • Masayuki Watanabe2 • Yoshihiro Komohara3 • Motohiro Takeya3 • Hideo Baba1
Received: 9 March 2015 / Accepted: 13 March 2015 / Published online: 18 March 2015 Ó Springer Science+Business Media New York 2015
Abstract The DNA methylation alterations occurring in human cancers have two types: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Recently, to assess global DNA methylation, long interspersed nucleotide element 1 (LINE-1) retrotransposons, constituting a substantial portion of the human genome, attracts much attention. The aim of the current study was to clarify the significance of LINE-1 methylation level for epigenetic field defects and the relationships among LINE-1 methylation level in gastric mucosae, clinical and pathological features, including infection by Helicobacter pylori (H. pylori), a bacterium implicated in gastric cancer. By bisulfite-PCR pyrosequencing, we quantified the LINE-1 methylation levels in noncancerous gastric mucosae and cancer tissues from 87 gastric cancer patients, in gastric mucosae from 17 autopsied individuals without gastric cancers and in 20 gastric fresh frozen samples from non-gastric cancer patients. LINE-1 methylation in the noncancerous gastric mucosae of gastric cancer patients was significantly higher than in cancer
tissues (P = 0.0006), but significantly lower than in the gastric mucosae of the autopsied individuals (P = 0.026), suggesting the formation of epigenetic field defect in noncancerous gastric mucosae. Moreover, LINE-1 hypomethylation of noncancerous gastric mucosae in gastric cancer patients significantly correlated with H. pylori infection (P = 0.037). We prospectively confirmed the similar result in 20 non-gastric cancer patients (P = 0.010). LINE-1 hypomethylation of gastric mucosae significantly correlated with H. pylori infection, supporting the potential of LINE-1 methylation level as a surrogate marker of epigenetic field defects for gastric cancer cancerization, particularly induced by H. pylori. Keywords Infection of Helicobacter pylori LINE-1 Hypomethylation Gastric mucosae Epigenetic field defect Abbreviations H. pylori Helicobacter pylori LINE-1 Long interspersed nucleotide element-1
Electronic supplementary material The online version of this article (doi:10.1007/s12032-015-0571-5) contains supplementary material, which is available to authorized users. & Hideo Baba [email protected] 1
Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan
2
Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan
3
Department of Cell Pathology, Graduate School of Medical Science, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan
Introduction DNA methylation alteration is a fundamental epigenetic process that modulates gene expression, and two patterns of altered DNA methylation are recognized in cancer cells: global DNA hypomethylation and site-specific CpG island promoter hypermethylation [1–3]. Of these, global DNA hypomethylation is important in human cancers [4], being associated with various pathways of multistep tumorigenesis. As global DNA hypomethylation appears to play
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an important role in genomic instability leading to cancer development [5–7], early detection of such alteration may signify an increased risk of cancer development [8]. A large proportion (approximately 17 %) of the human genome comprises long interspersed nucleotide element-1 (LINE-1 or L1) retrotransposons. Consequently, LINE-1 methylation level is regarded as a surrogate marker of global DNA methylation [9]. Moreover, LINE-1 methylation levels can now be measured at high throughput and low cost using pyrosequencing technology [10–12]. Indeed, we have reported that LINE-1 hypomethylation has emerged as a promising prognostic or predictive biomarker in esophageal cancer and gastric cancer [13, 14]. Gastric cancer is the fourth most commonly diagnosed cancer and the second most common cause of global cancer mortality [15]. Despite improved diagnostic and treatment technologies, the prognosis of gastric cancer patients remains poor, even for those undergoing complete resection of their carcinomas [16]. Helicobacter pylori (H. pylori), a Gram-negative bacterium that colonizes the human stomach plays a well-recognized role in the pathogenesis of gastritis, ulcers and gastric cancer [17–19]. H. pylori may facilitate gastric cancer by altering the DNA methylation levels [20– 22], and DNA methylation is altered in the gastric mucosae. The aim of the current study was to investigate the relationships between LINE-1 methylation levels in the gastric mucosae, clinical and pathological features, including H. pylori infection, smoking history, drinking history and age. First, we sampled noncancerous gastric mucosae and cancer tissues from gastric cancer patients, and gastric mucosae from autopsied individuals without gastric cancer, and quantified their LINE-1 methylation levels by bisulfite-PCR pyrosequencing assay. Second, we prospectively examined the relationship between LINE-1 methylation levels and H. pylori infection in gastric mucosae from non-gastric cancer patients. Our data support a role for LINE-1 methylation level as a surrogate marker of epigenetic field defects for gastric cancer cancerization, induced by H. pylori infection.
Materials and methods Study subjects Informed consent was obtained from all individual participants included in the study, and the study procedures were approved by the institutional review board in Kumamoto University. This study enrolled 247 consecutive gastric cancer patients who underwent resection at Kumamoto University Hospital between April 2005 and December 2009. Among these, we randomly sampled 87 cases, evaluated the LINE-1 methylation levels of their cancer tissues and matched
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noncancerous gastric mucosae. In addition, we randomly selected 46 cases from the 87 cases, and H. pylori infection was verified under microscopic observation by the pathologist, due to high sensitivity and high specificity (Fig. 1a) [23]. Smoking consumption was evaluated by using Brinkman index, i.e., the number of cigarettes smoked per day 9 the number of years the person has smoked. Daily alcohol consumption was expressed in grams of ethanol per day calculated by using a standard conversion for alcoholic beverages. Beer was assumed to be 5 % ethanol (v/v); wine, 12 %; sake, 16 %; shochu, 25 %; and whiskey, 40 %. These data were prospectively collected by clinical staff at the initial visit. Gastric mucosae from 17 autopsied individuals who had died of causes other than gastric cancer were also investigated. These individuals included 15 male and two female patients, aged 56–87 years (71.0 ± 7.8 years) at the time of autopsy. The causes of death were malignant diseases excepting gastric cancer (nine patients), pneumonitis (one patient), arrhythmia (one patient), cirrhosis of the liver (one patient), familial adenomatous polyposis (one patient), AL amyloidosis (one patient), diffuse alveolar damage (one patient), infectious endocarditis (one patient) and aortic dissection (one patient). We prospectively collected 20 gastric fresh frozen samples obtained from patients who underwent gastric resection for esophageal cancer in 2014. These patients were verified as having or not having an H. pylori infection using a urease test. Tumor staging complied with the American Joint Committee on Cancer Staging Manual (7th edition) [24]. In this article, the term ‘‘prognostic marker’’ is used as specified in the REMARK Guidelines [25]. DNA extraction and sodium bisulfite treatment Hematoxylin and eosin (H&E)-stained slides of the tumors were reviewed, and areas of tumors or histologically noncancerous gastric mucosae adjacent to the tumors were marked by two pathologic investigators (Y.K. & Y.B.) (Fig. 1b). In each case, H&E-stained tissue sections of the largest cross-sectional slice (in terms of tissue and tumor size; on average, a large tumor tissue section measured 10 lm 9 1) were scraped off the slides and subjected to DNA extraction. Genomic DNA was extracted from both tumor and noncancerous mucosae by using a QIAamp DNA FFPE Tissue Kit or a QIAamp DNA Mini Kit (Qiagen) and modified with sodium bisulfite using an EpiTect Bisulfite kit (Qiagen). Pyrosequencing measurement of LINE-1 methylation PCR and subsequent pyrosequencing of LINE-1 were performed as previously described by Ogino et al., using
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the PyroMark kit (Qiagen) [26–28]. This assay amplifies a region of the LINE-1 element (positions 305–331 in accession no. X58075) containing four CpG sites. At each CpG site, the amount of C relative to the combined C and T content was calculated as a percentage (0–100 %). The average relative C content among the four CpG sites was assumed as the overall LINE-1 methylation level in a given tissue (Fig. 1c). Our LINE-1 methylation pyrosequencing assay was validated in our previously published work; briefly, we subjected aliquots of five different DNA specimens from four macrodissected cancers to bisulfite conversion and five rounds of PCR pyrosequencing. The standard deviation (SD) of the bisulfite to bisulfite (between bisulfite treatments) ranged from 1.4 to 2.9 (median 2.3), whereas that of the run to run (between PCR pyrosequencing runs) ranged from 0.6 to 3.3 (median 1.5) [29]. Statistical methods The results were statistically analyzed using the JMP software, version 10 (SAS Institute, Cary, NC). All P values were two-sided. The means were compared by the student’s t test with unequal variances or by analysis of variance for variables with more than two categories. Correlation between LINE-1 methylation level and age was evaluated by the Pearson correlation coefficient.
Results Pyrosequencing of LINE-1 methylation level We quantified the LINE-1 methylation levels in noncancerous gastric mucosae and gastric cancer tissues extracted from 87 gastric cancer patients and in gastric mucosae from 17 autopsied individuals without gastric cancer (Fig. 1a, b). Quantification was performed by bisulfite-PCR pyrosequencing assay (Fig. 1c). Fig. 1 Study subjects and methods (macrodissection and pyrosequencing assay). a Flow chart of the study population. b Macrodissection from noncancerous gastric mucosa and cancer tissue in a gastric cancer patient. c Pyrosequencing assay for measuring the LINE-1 methylation level. Percentage (%) is the proportion of C at each CpG site after bisulfite conversion; methylation level of each CpG site was estimated by the proportion of C (%). Overall LINE-1 methylation level was calculated as the average of proportions of C (%) at the four CpG sites. The level of one gastric mucosa was 75.8 %. The arrows indicate no residual C at the non-CpG site, ensuring complete bisulfite conversion
LINE-1 methylation levels of noncancerous gastric mucosae and cancer tissues in gastric cancer patients The LINE-1 methylation level of 87 noncancerous gastric mucosae was distributed as follows: mean 76.9, median 78.2, SD 8.3 and range 48.3–94.8. The level in matched gastric cancer tissues was distributed as: mean 71.4,
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For further analysis, we randomly selected 46 cases from 87 gastric cancer patients and identified the infection of H. pylori in the tissues of them by using a high sensitivity
and high specificity microscope (Fig. 1a) [23]. First, we analyzed the correlation between the LINE-1 methylation level of noncancerous gastric mucosae as a continuous variable and various clinical and pathological features in all of 46 samples (Table 1). A significant correlation was found between the LINE-1 methylation levels of noncancerous gastric mucosae and gastric cancer tissues. However, the LINE-1 methylation level in noncancerous gastric mucosae was unrelated to all of the clinical and pathological features, even tumor stage. We then evaluated the relationships between the LINE1 methylation level of noncancerous gastric mucosae and H. pylori infection. Interestingly, the LINE-1 hypomethylation of noncancerous gastric mucosae was strongly correlated with H. pylori infection in gastric cancer patients (P = 0.037, Fig. 3a). We also examined the relationships between the LINE-1 methylation level of noncancerous gastric mucosae and smoking history, drinking history and age, but LINE-1 methylation level was unrelated to all three of these environmental variables (P = 0.76, P = 0.21, P = 0.72, respectively, Fig. 3b–d). In addition, we analyzed the correlation between the LINE-1 methylation level of noncancerous gastric mucosae, Brinkman index, amount of alcohol and age as a continuous variable. However, the LINE-1 methylation level in noncancerous gastric mucosae was unrelated to all of them (P = 0.91, P = 0.17, P = 0.67, respectively, Figure S1a–c).
Fig. 2 LINE-1 methylation levels in noncancerous gastric mucosae and cancer tissues. a The distribution of the LINE-1 methylation levels in noncancerous gastric mucosae and cancer tissues sampled from 87 gastric cancer patients. b LINE-1 methylation levels in gastric mucosae sampled from autopsied individuals and in
noncancerous gastric mucosae and cancer tissues sampled from gastric cancer patients. LINE-1 methylation in the noncancerous gastric mucosae of gastric cancer patients was significantly higher than in cancer tissues (P = 0.0006), but significantly lower than in the gastric mucosae of the autopsied individuals (P = 0.026)
median 73.6, SD 12.1 and range 28.0–97.5 (Fig. 2a). The LINE-1 methylation level was significantly higher in the noncancerous gastric mucosae than in the matched cancer tissues (P = 0.0006, Fig. 2b). LINE-1 methylation levels of gastric mucosae in autopsied individuals and of noncancerous gastric mucosae in gastric cancer patients The LINE-1 methylation level of gastric mucosae extracted from the 17 autopsied individuals was distributed as follows: mean 81.6, median 82, SD 4.04 and range 75.3–90.8. Interestingly, the LINE-1 methylation level was significantly lower in the noncancerous gastric mucosae of gastric cancer patients than in the gastric mucosae of autopsied individuals (P = 0.026; see Fig. 2b). This finding suggests that LINE-1 hypomethylation (i.e., global DNA hypomethylation) in histologically noncancerous gastric mucosae might manifest from an epigenetic field defect in gastric cancer cases. Relationship between LINE-1 hypomethylation of noncancerous gastric mucosae, clinical and pathological features in gastric cancer patients
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Med Oncol (2015) 32:117 Table 1 LINE-1 methylation level as a continuous variable and its associations with clinical and pathological features in gastric cancer patients
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Clinical and pathological features
N (%)
LINE-1 methylation level of noncancerous gastric mucosae (mean, %)
All cases
46
79.2
P value
Age (years) \65
12 (26.1)
79.8
C65
34 (73.9)
79.1
0.72
Sex Male
29 (63.0)
80.5
17 (37.0)
77.0
1
41 (89.1)
79.5
2
4 (8.7)
76.5
3
1 (2.2)
79.9
Female Number of tumors
0.067
0.67
Tumor size (mm) \30
21 (45.7)
78.8
C30
25 (54.3)
79.6
U
9 (19.6)
79.0
M
18 (39.1)
81.2
L
19 (41.3)
77.5
Ant
13 (28.3)
77.8
Gre
4 (8.7)
82.6
Post Less
13 (28.3) 16 (34.7)
80.1 78.8
tub
29 (63.0)
79.4
por/sig/muc
16 (34.8)
78.5
MALT
1 (2.2)
85.8
I
34 (73.9)
79.1
II
7 (15.2)
80.0
III
4 (8.7)
80.7
IV
1 (2.2)
73.8
0.64
Tumor location 1 0.21
Tumor location 2 0.57
Histological type 0.53
Tumor stage
These data suggest that LINE-1 methylation level is a potential surrogate marker of epigenetic field defects in H. pylori-induced gastric cancer development.
gastric mucosae from (P = 0.010, Fig. 4b).
Prospective study to analyze the relationship between LINE-1 hypomethylation and H. pylori infection in gastric mucosae from non-gastric cancer patients
Discussion
Using gastric mucosae from patients without gastric cancer and the H. pylori urease test, we tested the hypothesis that LINE-1 hypomethylation level is associated with H. pylori infection in gastric mucosae from non-gastric cancer patients (Fig. 4a). As a result, the LINE-1 hypomethylation was significantly correlated with H. pylori infection in
0.79
non-gastric
cancer
patients
In the current study, we examined whether LINE-1 hypomethylation was a viable epigenetic marker of gastric mucosae induced by H. pylori infection. Because LINE-1 retrotransposons constitute a substantial portion (approximately 17 %) of the human genome, the level of LINE-1 methylation is considered as a surrogate marker of global DNA methylation [9]. Using bisulfite-PCR pyrosequencing assay, we quantified the LINE-1 methylation levels in the noncancerous gastric mucosae and cancer
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Fig. 3 Relationships between the LINE-1 methylation level of noncancerous gastric mucosae and several environmental factors. LINE-1 hypomethylation of noncancerous gastric mucosae in gastric
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cancer patients significantly correlated with H. pylori infection (P = 0.037, a), but was independent of smoking (b), drinking (c) and age (d) (P = 0.76, P = 0.21, P = 0.72, respectively, b–d)
Fig. 4 Prospective study to analyze the relationship between LINE-1 hypomethylation and H. pylori infection in gastric mucosae from non-gastric cancer patients. a Study design of the prospective study. b LINE-1 methylation levels in gastric mucosae sampled from nongastric cancer patients. LINE-1 hypomethylation significantly correlated with H. pylori infection in gastric mucosae from non-gastric cancer patients (P = 0.010)
tissues of gastric cancer patients and in gastric mucosae of autopsied individuals without gastric cancer. LINE-1 methylation levels were significantly lower in the
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noncancerous gastric mucosae of the cancer patients than in the gastric mucosae of the autopsied individuals. In addition, LINE-1 hypomethylation in the noncancerous
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gastric mucosae of gastric cancer patients was significantly associated with H. pylori infection, but was independent of smoking history, drinking history and age. Importantly, we also conducted the prospective study to examine the relationship between LINE-1 methylation level and H. pylori infection in gastric mucosae from non-gastric cancer patients and confirmed the significant correlation between them. The prospective data are more beneficial, and our data support that LINE-1 methylation level is a surrogate marker of epigenetic field defects in the gastric mucosa, particularly those induced by H. pylori infection. Global DNA hypomethylation appears to play an important role in genomic instability leading to cancer development [5–7]. In addition, the premalignant stages of certain cancers, including gastric cancer, are characterized by global DNA hypomethylation [30]. Aberrant DNA methylation occurs in the early stages and tends to accumulate along the multistep pathway of gastric carcinogenesis [31]. Therefore, DNA methylation has been emphasized as a useful marker of past exposure to carcinogens and future risk of cancer development [8, 32]. In the current study, our findings of reduced LINE-1 methylation in the gastric mucosae of non-gastric cancer patients suggest that precancerous lesions of depleted DNA methylation develop in the mucosae of these patients. Because measurement of LINE-1 methylation by pyrosequencing technology is a cost-effective and high-throughput method to assess global DNA methylation status, our findings may have considerable clinical implications [10– 12, 33]. H. pylori is a widely recognized pathogenic inducer of gastritis, ulcers and gastric cancer [17–19]. H. pylori infection has also been implicated in DNA methylation alterations, site-specific CpG island promoter hypermethylation and global DNA hypomethylation, the precursors of gastric cancer [20–22]. Interestingly, we demonstrated that LINE-1 methylation levels were lower in the noncancerous gastric mucosae of gastric cancer patients infected with H. pylori than in those of their uninfected counterparts. In addition, we prospectively confirmed the significant correlation between LINE-1 hypomethylation and H. pylori infection in gastric mucosae from non-gastric cancer patients for the first time. These findings suggested that infection of H. pylori might contribute to LINE-1 hypomethylation by some mechanisms. LINE-1 hypomethylation appears to occur during the transition from intestinal metaplasia to gastric adenoma, not during the transition from gastric adenoma to gastric cancer [34]. Since LINE-1 methylation level is regarded as a surrogate marker of global DNA methylation [9], our finding is consistent with these experimental results. Some mechanisms by which one pattern of altered DNA methylation, site-specific CgG island promoter hypermethylation, may correlate with gastric cancer have been
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identified. Infection by H. pylori induces the methylation of specific genes, and a cancerization field might emerge from accumulated aberrant methylation in the gastric mucosae [35]. FHL1 methylation has been associated with H. pylori infection, and FHL1 silencing might contribute to forming an epigenetic cancerization field [36]. Similarly, methylation silencing of miRNA genes contributes to the formation of epigenetic field defects in gastric cancers [37]. Among these, methylated miR-34b/c and RASGRF1 have been identified as useful biomarkers for identifying individuals at high risk of gastric cancer [38–40]. However, whether H. pylori influences the methylation of miR34b/c and RASGRF1 remains unclear. In the current study, we confirmed the significant correlation between LINE-1 hypomethylation, i.e., global DNA hypomethylation, and infection of H. pylori in noncancerous mucosae from gastric cancer patients and in gastric mucosae from non-gastric cancer patients. However, the mechanism by which global DNA hypomethylation induced by H. pylori correlates with human carcinogenesis remains undiscovered. Possibly, as occurs in esophageal squamous cell carcinoma, global DNA hypomethylation facilitates aggressive tumor behavior through genomic gains of oncogenes such as CDK6 [41]. Further studies are needed to confirm our findings and to examine the biological mechanisms by which global DNA hypomethylation induced by H. pylori infection contributes to epigenetic field defects and carcinogenesis. Carcinogenesis is induced by multiple factors, and the risk factors of gastric cancer have been fully explored. Various indicators such as life expectancy, education attainment and income are combined into a risk measure called the human development index (HDI). According to a recent global population study, increased incidence of gastric cancer is linked to higher HDIs coupled with increased per capita consumption of alcohol and tobacco [42]. In gastric cancer patients, smoking and age might lower the methylation levels of insulin-like growth factor 2 (IGF2) in blood leukocytes [43]. A possible relationship has been identified between LINE-1 methylation levels in blood leukocytes and smoking and drinking [44]. However, our current study found no effect of smoking, drinking or age on LINE-1 hypomethylation in the noncancerous mucosae of gastric cancer patients. An existing relationship might have been obscured by our small sample size; therefore, further studies are necessary to validate our findings. In summary, the current study suggests that LINE-1 hypomethylation is a phenomenon of the gastric cancer epigenetic field defect. Furthermore, LINE-1 hypomethylation is significantly associated with H. pylori infection, supporting a role for LINE-1 methylation level as a surrogate marker for the epigenetic field defect, particularly that induced by H. pylori infection. These findings require
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confirmation in future studies. We must also elucidate the mechanisms by which H. pylori infection induces aberrant DNA methylation in normal gastric mucosae. Conflict of interest
16.
None. 17.
References 18. 1. Rodriguez-Paredes M, Esteller M. Cancer epigenetics reaches mainstream oncology. Nat Med. 2011;17(3):330–9. doi:10.1038/ nm.2305. 2. Taby R, Issa JP. Cancer epigenetics. CA Cancer J Clin. 2010; 60(6):376–92. doi:10.3322/caac.20085. 3. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128(4):683–92. doi:10.1016/j.cell.2007.01.029. 4. Ehrlich M. DNA hypomethylation in cancer cells. Epigenomics. 2009;1(2):239–59. doi:10.2217/epi.09.33. 5. Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW, et al. Induction of tumors in mice by genomic hypomethylation. Science. 2003;300(5618):489–92. doi:10.1126/ science.1083558. 6. Holm TM, Jackson-Grusby L, Brambrink T, Yamada Y, Rideout WM 3rd, Jaenisch R. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell. 2005;8(4):275–85. doi:10.1016/j.ccr.2005.09.007. 7. Suzuki K, Suzuki I, Leodolter A, Alonso S, Horiuchi S, Yamashita K, et al. Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage. Cancer Cell. 2006;9(3):199–207. doi:10.1016/j.ccr.2006.02.016. 8. Ushijima T. Epigenetic field for cancerization. J Biochem Mol Biol. 2007;40(2):142–50. 9. Cordaux R, Batzer MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10(10):691–703. doi:10.1038/nrg2640. 10. Ogino S, Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, et al. LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Int J Cancer. 2008;122(12):2767–73. doi:10. 1002/ijc.23470. 11. Irahara N, Nosho K, Baba Y, Shima K, Lindeman NI, Hazra A, et al. Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells. J Mol Diagn. 2010;12(2):177–83. doi:10. 2353/jmoldx.2010.090106. 12. Ahn JB, Chung WB, Maeda O, Shin SJ, Kim HS, Chung HC, et al. DNA methylation predicts recurrence from resected stage III proximal colon cancer. Cancer. 2011;117(9):1847–54. doi:10. 1002/cncr.25737. 13. Iwagami S, Baba Y, Watanabe M, Shigaki H, Miyake K, Ishimoto T, et al. LINE-1 hypomethylation is associated with a poor prognosis among patients with curatively resected esophageal squamous cell carcinoma. Ann Surg. 2013;257(3):449–55. doi:10.1097/SLA.0b013e31826d8602. 14. Shigaki H, Baba Y, Watanabe M, Murata A, Iwagami S, Miyake K, et al. LINE-1 hypomethylation in gastric cancer, detected by bisulfite pyrosequencing, is associated with poor prognosis. Gastric Cancer. 2013;16(4):480–7. doi:10.1007/s10120-0120209-7. 15. Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic
123
19. 20.
21.
22.
23. 24.
25.
26.
27.
28.
29.
30. 31.
32.
33.
regions of the world. J Clin Oncol. 2006;24(14):2137–50. doi:10. 1200/JCO.2005.05.2308. Macdonald JS, Smalley SR, Benedetti J, Hundahl SA, Estes NC, Stemmermann GN, et al. Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N Engl J Med. 2001;345(10): 725–30. doi:10.1056/NEJMoa010187. Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M, et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med. 2001;345(11):784–9. doi:10.1056/NEJMoa001999. Gerhard M, Rad R, Prinz C, Naumann M. Pathogenesis of Helicobacter pylori infection. Helicobacter. 2002;7(Suppl 1):17–23. Marshall BJ. Helicobacter pylori. Am J Gastroenterol. 1994;89(8 Suppl):S116–28. Maekita T, Nakazawa K, Mihara M, Nakajima T, Yanaoka K, Iguchi M, et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk. Clin Cancer Res. 2006;12(3 Pt 1):989–95. doi:10.1158/1078-0432.CCR-05-2096. Compare D, Rocco A, Liguori E, D’Armiento FP, Persico G, Masone S, et al. Global DNA hypomethylation is an early event in Helicobacter pylori-related gastric carcinogenesis. J Clin Pathol. 2011;64(8):677–82. doi:10.1136/jcp.2010.087858. Park SY, Yoo EJ, Cho NY, Kim N, Kang GH. Comparison of CpG island hypermethylation and repetitive DNA hypomethylation in premalignant stages of gastric cancer, stratified for Helicobacter pylori infection. J Pathol. 2009;219(4):410–6. doi:10. 1002/path.2596. McColl KE. Clinical practice. Helicobacter pylori infection. N Engl J Med. 2010;362(17):1597–604. doi:10.1056/NEJMcp1001110. Washington K. 7th edition of the AJCC cancer staging manual: stomach. Ann Surg Oncol. 2010;17(12):3077–9. doi:10.1245/ s10434-010-1362-z. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM, et al. Reporting recommendations for tumor marker prognostic studies (REMARK). J Natl Cancer Inst. 2005;97(16):1180–4. doi:10.1093/jnci/dji237. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Chan AT, Schernhammer ES, et al. A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer. J Natl Cancer Inst. 2008;100(23):1734–8. doi:10.1093/jnci/djn359. Baba Y, Huttenhower C, Nosho K, Tanaka N, Shima K, Hazra A, et al. Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors. Mol Cancer. 2010;9:125. doi:10.1186/1476-4598-9-125. Baba Y, Nosho K, Shima K, Huttenhower C, Tanaka N, Hazra A, et al. Hypomethylation of the IGF2 DMR in colorectal tumors, detected by bisulfite pyrosequencing, is associated with poor prognosis. Gastroenterology. 2010;139(6):1855–64. doi:10.1053/ j.gastro.2010.07.050. Iwagami S, Baba Y, Watanabe M, Shigaki H, Miyake K, Ida S, et al. Pyrosequencing assay to measure LINE-1 methylation level in esophageal squamous cell carcinoma. Ann Surg Oncol. 2012;19(8):2726–32. doi:10.1245/s10434-011-2176-3. Jang BG, Kim WH. Molecular pathology of gastric carcinoma. Pathobiology. 2011;78(6):302–10. doi:10.1159/000321703. Kang GH, Lee S, Kim JS, Jung HY. Profile of aberrant CpG island methylation along multistep gastric carcinogenesis. Lab Invest. 2003;83(4):519–26. Nakajima T, Enomoto S, Ushijima T. DNA methylation: a marker for carcinogen exposure and cancer risk. Environ Health Prev Med. 2008;13(1):8–15. doi:10.1007/s12199-007-0005-x. Zhang FF, Cardarelli R, Carroll J, Fulda KG, Kaur M, Gonzalez K, et al. Significant differences in global genomic DNA
Med Oncol (2015) 32:117
34.
35.
36.
37.
38.
methylation by gender and race/ethnicity in peripheral blood. Epigenetics. 2011;6(5):623–9. Bae JM, Shin SH, Kwon HJ, Park SY, Kook MC, Kim YW, et al. ALU and LINE-1 hypomethylations in multistep gastric carcinogenesis and their prognostic implications. Int J Cancer. 2012;131(6):1323–31. doi:10.1002/ijc.27369. Ushijima T, Hattori N. Molecular pathways: involvement of Helicobacter pylori-triggered inflammation in the formation of an epigenetic field defect, and its usefulness as cancer risk and exposure markers. Clin Cancer Res. 2012;18(4):923–9. doi:10. 1158/1078-0432.CCR-11-2011. Asada K, Ando T, Niwa T, Nanjo S, Watanabe N, Okochi-Takada E, et al. FHL1 on chromosome X is a single-hit gastrointestinal tumor-suppressor gene and contributes to the formation of an epigenetic field defect. Oncogene. 2013;32(17):2140–9. doi:10.1038/ onc.2012.228. Ando T, Yoshida T, Enomoto S, Asada K, Tatematsu M, Ichinose M, et al. DNA methylation of microRNA genes in gastric mucosae of gastric cancer patients: its possible involvement in the formation of epigenetic field defect. Int J Cancer. 2009;124(10): 2367–74. doi:10.1002/ijc.24219. Suzuki H, Yamamoto E, Nojima M, Kai M, Yamano HO, Yoshikawa K, et al. Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect. Carcinogenesis. 2010;31(12):2066–73. doi:10.1093/carcin/bgq203.
Page 9 of 9 117 39. Yamamoto E, Suzuki H, Maruyama R, Shinomura Y. Developing technologies for epigenomic analysis and clinical application of molecular diagnosis. Rinsho Byori. 2012;60(7):637–43. 40. Takamaru H, Yamamoto E, Suzuki H, Nojima M, Maruyama R, Yamano HO, et al. Aberrant methylation of RASGRF1 is associated with an epigenetic field defect and increased risk of gastric cancer. Cancer Prev Res (Phila). 2012;5(10):1203–12. doi:10. 1158/1940-6207.CAPR-12-0056. 41. Baba Y, Watanabe M, Murata A, Shigaki H, Miyake K, Ishimoto T, et al. LINE-1 hypomethylation, DNA copy number alterations, and CDK6 amplification in esophageal squamous cell carcinoma. Clin Cancer Res. 2014;. doi:10.1158/1078-0432.CCR-13-1645. 42. Ferrari F, Reis MA. Study of risk factors for gastric cancer by populational databases analysis. World J Gastroenterol. 2013;19(48):9383–91. doi:10.3748/wjg.v19.i48.9383. 43. Yuasa Y, Nagasaki H, Oze I, Akiyama Y, Yoshida S, Shitara K, et al. Insulin-like growth factor 2 hypomethylation of blood leukocyte DNA is associated with gastric cancer risk. Int J Cancer. 2012;131(11):2596–603. doi:10.1002/ijc.27554. 44. Hou L, Wang H, Sartori S, Gawron A, Lissowska J, Bollati V, et al. Blood leukocyte DNA hypomethylation and gastric cancer risk in a high-risk Polish population. Int J Cancer. 2010;127(8):1866–74. doi:10.1002/ijc.25190.
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ORIGINAL PAPER
Relationship between LINE-1 hypomethylation and Helicobacter pylori infection in gastric mucosae Keisuke Kosumi1 • Yoshifumi Baba1 • Takatsugu Ishimoto1 • Kazuto Harada1 • Keisuke Miyake1 • Daisuke Izumi1 • Ryuma Tokunaga1 • Asuka Murata1 • Kojiro Eto1 • Hidetaka Sugihara1 • Hironobu Shigaki1 • Shiro Iwagami1 • Yasuo Sakamoto1 • Yuji Miyamoto1 • Naoya Yoshida1 • Masayuki Watanabe2 • Yoshihiro Komohara3 • Motohiro Takeya3 • Hideo Baba1
Received: 9 March 2015 / Accepted: 13 March 2015 / Published online: 18 March 2015 Ó Springer Science+Business Media New York 2015
Abstract The DNA methylation alterations occurring in human cancers have two types: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Recently, to assess global DNA methylation, long interspersed nucleotide element 1 (LINE-1) retrotransposons, constituting a substantial portion of the human genome, attracts much attention. The aim of the current study was to clarify the significance of LINE-1 methylation level for epigenetic field defects and the relationships among LINE-1 methylation level in gastric mucosae, clinical and pathological features, including infection by Helicobacter pylori (H. pylori), a bacterium implicated in gastric cancer. By bisulfite-PCR pyrosequencing, we quantified the LINE-1 methylation levels in noncancerous gastric mucosae and cancer tissues from 87 gastric cancer patients, in gastric mucosae from 17 autopsied individuals without gastric cancers and in 20 gastric fresh frozen samples from non-gastric cancer patients. LINE-1 methylation in the noncancerous gastric mucosae of gastric cancer patients was significantly higher than in cancer
tissues (P = 0.0006), but significantly lower than in the gastric mucosae of the autopsied individuals (P = 0.026), suggesting the formation of epigenetic field defect in noncancerous gastric mucosae. Moreover, LINE-1 hypomethylation of noncancerous gastric mucosae in gastric cancer patients significantly correlated with H. pylori infection (P = 0.037). We prospectively confirmed the similar result in 20 non-gastric cancer patients (P = 0.010). LINE-1 hypomethylation of gastric mucosae significantly correlated with H. pylori infection, supporting the potential of LINE-1 methylation level as a surrogate marker of epigenetic field defects for gastric cancer cancerization, particularly induced by H. pylori. Keywords Infection of Helicobacter pylori LINE-1 Hypomethylation Gastric mucosae Epigenetic field defect Abbreviations H. pylori Helicobacter pylori LINE-1 Long interspersed nucleotide element-1
Electronic supplementary material The online version of this article (doi:10.1007/s12032-015-0571-5) contains supplementary material, which is available to authorized users. & Hideo Baba [email protected] 1
Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan
2
Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan
3
Department of Cell Pathology, Graduate School of Medical Science, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan
Introduction DNA methylation alteration is a fundamental epigenetic process that modulates gene expression, and two patterns of altered DNA methylation are recognized in cancer cells: global DNA hypomethylation and site-specific CpG island promoter hypermethylation [1–3]. Of these, global DNA hypomethylation is important in human cancers [4], being associated with various pathways of multistep tumorigenesis. As global DNA hypomethylation appears to play
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an important role in genomic instability leading to cancer development [5–7], early detection of such alteration may signify an increased risk of cancer development [8]. A large proportion (approximately 17 %) of the human genome comprises long interspersed nucleotide element-1 (LINE-1 or L1) retrotransposons. Consequently, LINE-1 methylation level is regarded as a surrogate marker of global DNA methylation [9]. Moreover, LINE-1 methylation levels can now be measured at high throughput and low cost using pyrosequencing technology [10–12]. Indeed, we have reported that LINE-1 hypomethylation has emerged as a promising prognostic or predictive biomarker in esophageal cancer and gastric cancer [13, 14]. Gastric cancer is the fourth most commonly diagnosed cancer and the second most common cause of global cancer mortality [15]. Despite improved diagnostic and treatment technologies, the prognosis of gastric cancer patients remains poor, even for those undergoing complete resection of their carcinomas [16]. Helicobacter pylori (H. pylori), a Gram-negative bacterium that colonizes the human stomach plays a well-recognized role in the pathogenesis of gastritis, ulcers and gastric cancer [17–19]. H. pylori may facilitate gastric cancer by altering the DNA methylation levels [20– 22], and DNA methylation is altered in the gastric mucosae. The aim of the current study was to investigate the relationships between LINE-1 methylation levels in the gastric mucosae, clinical and pathological features, including H. pylori infection, smoking history, drinking history and age. First, we sampled noncancerous gastric mucosae and cancer tissues from gastric cancer patients, and gastric mucosae from autopsied individuals without gastric cancer, and quantified their LINE-1 methylation levels by bisulfite-PCR pyrosequencing assay. Second, we prospectively examined the relationship between LINE-1 methylation levels and H. pylori infection in gastric mucosae from non-gastric cancer patients. Our data support a role for LINE-1 methylation level as a surrogate marker of epigenetic field defects for gastric cancer cancerization, induced by H. pylori infection.
Materials and methods Study subjects Informed consent was obtained from all individual participants included in the study, and the study procedures were approved by the institutional review board in Kumamoto University. This study enrolled 247 consecutive gastric cancer patients who underwent resection at Kumamoto University Hospital between April 2005 and December 2009. Among these, we randomly sampled 87 cases, evaluated the LINE-1 methylation levels of their cancer tissues and matched
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noncancerous gastric mucosae. In addition, we randomly selected 46 cases from the 87 cases, and H. pylori infection was verified under microscopic observation by the pathologist, due to high sensitivity and high specificity (Fig. 1a) [23]. Smoking consumption was evaluated by using Brinkman index, i.e., the number of cigarettes smoked per day 9 the number of years the person has smoked. Daily alcohol consumption was expressed in grams of ethanol per day calculated by using a standard conversion for alcoholic beverages. Beer was assumed to be 5 % ethanol (v/v); wine, 12 %; sake, 16 %; shochu, 25 %; and whiskey, 40 %. These data were prospectively collected by clinical staff at the initial visit. Gastric mucosae from 17 autopsied individuals who had died of causes other than gastric cancer were also investigated. These individuals included 15 male and two female patients, aged 56–87 years (71.0 ± 7.8 years) at the time of autopsy. The causes of death were malignant diseases excepting gastric cancer (nine patients), pneumonitis (one patient), arrhythmia (one patient), cirrhosis of the liver (one patient), familial adenomatous polyposis (one patient), AL amyloidosis (one patient), diffuse alveolar damage (one patient), infectious endocarditis (one patient) and aortic dissection (one patient). We prospectively collected 20 gastric fresh frozen samples obtained from patients who underwent gastric resection for esophageal cancer in 2014. These patients were verified as having or not having an H. pylori infection using a urease test. Tumor staging complied with the American Joint Committee on Cancer Staging Manual (7th edition) [24]. In this article, the term ‘‘prognostic marker’’ is used as specified in the REMARK Guidelines [25]. DNA extraction and sodium bisulfite treatment Hematoxylin and eosin (H&E)-stained slides of the tumors were reviewed, and areas of tumors or histologically noncancerous gastric mucosae adjacent to the tumors were marked by two pathologic investigators (Y.K. & Y.B.) (Fig. 1b). In each case, H&E-stained tissue sections of the largest cross-sectional slice (in terms of tissue and tumor size; on average, a large tumor tissue section measured 10 lm 9 1) were scraped off the slides and subjected to DNA extraction. Genomic DNA was extracted from both tumor and noncancerous mucosae by using a QIAamp DNA FFPE Tissue Kit or a QIAamp DNA Mini Kit (Qiagen) and modified with sodium bisulfite using an EpiTect Bisulfite kit (Qiagen). Pyrosequencing measurement of LINE-1 methylation PCR and subsequent pyrosequencing of LINE-1 were performed as previously described by Ogino et al., using
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the PyroMark kit (Qiagen) [26–28]. This assay amplifies a region of the LINE-1 element (positions 305–331 in accession no. X58075) containing four CpG sites. At each CpG site, the amount of C relative to the combined C and T content was calculated as a percentage (0–100 %). The average relative C content among the four CpG sites was assumed as the overall LINE-1 methylation level in a given tissue (Fig. 1c). Our LINE-1 methylation pyrosequencing assay was validated in our previously published work; briefly, we subjected aliquots of five different DNA specimens from four macrodissected cancers to bisulfite conversion and five rounds of PCR pyrosequencing. The standard deviation (SD) of the bisulfite to bisulfite (between bisulfite treatments) ranged from 1.4 to 2.9 (median 2.3), whereas that of the run to run (between PCR pyrosequencing runs) ranged from 0.6 to 3.3 (median 1.5) [29]. Statistical methods The results were statistically analyzed using the JMP software, version 10 (SAS Institute, Cary, NC). All P values were two-sided. The means were compared by the student’s t test with unequal variances or by analysis of variance for variables with more than two categories. Correlation between LINE-1 methylation level and age was evaluated by the Pearson correlation coefficient.
Results Pyrosequencing of LINE-1 methylation level We quantified the LINE-1 methylation levels in noncancerous gastric mucosae and gastric cancer tissues extracted from 87 gastric cancer patients and in gastric mucosae from 17 autopsied individuals without gastric cancer (Fig. 1a, b). Quantification was performed by bisulfite-PCR pyrosequencing assay (Fig. 1c). Fig. 1 Study subjects and methods (macrodissection and pyrosequencing assay). a Flow chart of the study population. b Macrodissection from noncancerous gastric mucosa and cancer tissue in a gastric cancer patient. c Pyrosequencing assay for measuring the LINE-1 methylation level. Percentage (%) is the proportion of C at each CpG site after bisulfite conversion; methylation level of each CpG site was estimated by the proportion of C (%). Overall LINE-1 methylation level was calculated as the average of proportions of C (%) at the four CpG sites. The level of one gastric mucosa was 75.8 %. The arrows indicate no residual C at the non-CpG site, ensuring complete bisulfite conversion
LINE-1 methylation levels of noncancerous gastric mucosae and cancer tissues in gastric cancer patients The LINE-1 methylation level of 87 noncancerous gastric mucosae was distributed as follows: mean 76.9, median 78.2, SD 8.3 and range 48.3–94.8. The level in matched gastric cancer tissues was distributed as: mean 71.4,
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For further analysis, we randomly selected 46 cases from 87 gastric cancer patients and identified the infection of H. pylori in the tissues of them by using a high sensitivity
and high specificity microscope (Fig. 1a) [23]. First, we analyzed the correlation between the LINE-1 methylation level of noncancerous gastric mucosae as a continuous variable and various clinical and pathological features in all of 46 samples (Table 1). A significant correlation was found between the LINE-1 methylation levels of noncancerous gastric mucosae and gastric cancer tissues. However, the LINE-1 methylation level in noncancerous gastric mucosae was unrelated to all of the clinical and pathological features, even tumor stage. We then evaluated the relationships between the LINE1 methylation level of noncancerous gastric mucosae and H. pylori infection. Interestingly, the LINE-1 hypomethylation of noncancerous gastric mucosae was strongly correlated with H. pylori infection in gastric cancer patients (P = 0.037, Fig. 3a). We also examined the relationships between the LINE-1 methylation level of noncancerous gastric mucosae and smoking history, drinking history and age, but LINE-1 methylation level was unrelated to all three of these environmental variables (P = 0.76, P = 0.21, P = 0.72, respectively, Fig. 3b–d). In addition, we analyzed the correlation between the LINE-1 methylation level of noncancerous gastric mucosae, Brinkman index, amount of alcohol and age as a continuous variable. However, the LINE-1 methylation level in noncancerous gastric mucosae was unrelated to all of them (P = 0.91, P = 0.17, P = 0.67, respectively, Figure S1a–c).
Fig. 2 LINE-1 methylation levels in noncancerous gastric mucosae and cancer tissues. a The distribution of the LINE-1 methylation levels in noncancerous gastric mucosae and cancer tissues sampled from 87 gastric cancer patients. b LINE-1 methylation levels in gastric mucosae sampled from autopsied individuals and in
noncancerous gastric mucosae and cancer tissues sampled from gastric cancer patients. LINE-1 methylation in the noncancerous gastric mucosae of gastric cancer patients was significantly higher than in cancer tissues (P = 0.0006), but significantly lower than in the gastric mucosae of the autopsied individuals (P = 0.026)
median 73.6, SD 12.1 and range 28.0–97.5 (Fig. 2a). The LINE-1 methylation level was significantly higher in the noncancerous gastric mucosae than in the matched cancer tissues (P = 0.0006, Fig. 2b). LINE-1 methylation levels of gastric mucosae in autopsied individuals and of noncancerous gastric mucosae in gastric cancer patients The LINE-1 methylation level of gastric mucosae extracted from the 17 autopsied individuals was distributed as follows: mean 81.6, median 82, SD 4.04 and range 75.3–90.8. Interestingly, the LINE-1 methylation level was significantly lower in the noncancerous gastric mucosae of gastric cancer patients than in the gastric mucosae of autopsied individuals (P = 0.026; see Fig. 2b). This finding suggests that LINE-1 hypomethylation (i.e., global DNA hypomethylation) in histologically noncancerous gastric mucosae might manifest from an epigenetic field defect in gastric cancer cases. Relationship between LINE-1 hypomethylation of noncancerous gastric mucosae, clinical and pathological features in gastric cancer patients
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Med Oncol (2015) 32:117 Table 1 LINE-1 methylation level as a continuous variable and its associations with clinical and pathological features in gastric cancer patients
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Clinical and pathological features
N (%)
LINE-1 methylation level of noncancerous gastric mucosae (mean, %)
All cases
46
79.2
P value
Age (years) \65
12 (26.1)
79.8
C65
34 (73.9)
79.1
0.72
Sex Male
29 (63.0)
80.5
17 (37.0)
77.0
1
41 (89.1)
79.5
2
4 (8.7)
76.5
3
1 (2.2)
79.9
Female Number of tumors
0.067
0.67
Tumor size (mm) \30
21 (45.7)
78.8
C30
25 (54.3)
79.6
U
9 (19.6)
79.0
M
18 (39.1)
81.2
L
19 (41.3)
77.5
Ant
13 (28.3)
77.8
Gre
4 (8.7)
82.6
Post Less
13 (28.3) 16 (34.7)
80.1 78.8
tub
29 (63.0)
79.4
por/sig/muc
16 (34.8)
78.5
MALT
1 (2.2)
85.8
I
34 (73.9)
79.1
II
7 (15.2)
80.0
III
4 (8.7)
80.7
IV
1 (2.2)
73.8
0.64
Tumor location 1 0.21
Tumor location 2 0.57
Histological type 0.53
Tumor stage
These data suggest that LINE-1 methylation level is a potential surrogate marker of epigenetic field defects in H. pylori-induced gastric cancer development.
gastric mucosae from (P = 0.010, Fig. 4b).
Prospective study to analyze the relationship between LINE-1 hypomethylation and H. pylori infection in gastric mucosae from non-gastric cancer patients
Discussion
Using gastric mucosae from patients without gastric cancer and the H. pylori urease test, we tested the hypothesis that LINE-1 hypomethylation level is associated with H. pylori infection in gastric mucosae from non-gastric cancer patients (Fig. 4a). As a result, the LINE-1 hypomethylation was significantly correlated with H. pylori infection in
0.79
non-gastric
cancer
patients
In the current study, we examined whether LINE-1 hypomethylation was a viable epigenetic marker of gastric mucosae induced by H. pylori infection. Because LINE-1 retrotransposons constitute a substantial portion (approximately 17 %) of the human genome, the level of LINE-1 methylation is considered as a surrogate marker of global DNA methylation [9]. Using bisulfite-PCR pyrosequencing assay, we quantified the LINE-1 methylation levels in the noncancerous gastric mucosae and cancer
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Fig. 3 Relationships between the LINE-1 methylation level of noncancerous gastric mucosae and several environmental factors. LINE-1 hypomethylation of noncancerous gastric mucosae in gastric
Med Oncol (2015) 32:117
cancer patients significantly correlated with H. pylori infection (P = 0.037, a), but was independent of smoking (b), drinking (c) and age (d) (P = 0.76, P = 0.21, P = 0.72, respectively, b–d)
Fig. 4 Prospective study to analyze the relationship between LINE-1 hypomethylation and H. pylori infection in gastric mucosae from non-gastric cancer patients. a Study design of the prospective study. b LINE-1 methylation levels in gastric mucosae sampled from nongastric cancer patients. LINE-1 hypomethylation significantly correlated with H. pylori infection in gastric mucosae from non-gastric cancer patients (P = 0.010)
tissues of gastric cancer patients and in gastric mucosae of autopsied individuals without gastric cancer. LINE-1 methylation levels were significantly lower in the
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noncancerous gastric mucosae of the cancer patients than in the gastric mucosae of the autopsied individuals. In addition, LINE-1 hypomethylation in the noncancerous
Med Oncol (2015) 32:117
gastric mucosae of gastric cancer patients was significantly associated with H. pylori infection, but was independent of smoking history, drinking history and age. Importantly, we also conducted the prospective study to examine the relationship between LINE-1 methylation level and H. pylori infection in gastric mucosae from non-gastric cancer patients and confirmed the significant correlation between them. The prospective data are more beneficial, and our data support that LINE-1 methylation level is a surrogate marker of epigenetic field defects in the gastric mucosa, particularly those induced by H. pylori infection. Global DNA hypomethylation appears to play an important role in genomic instability leading to cancer development [5–7]. In addition, the premalignant stages of certain cancers, including gastric cancer, are characterized by global DNA hypomethylation [30]. Aberrant DNA methylation occurs in the early stages and tends to accumulate along the multistep pathway of gastric carcinogenesis [31]. Therefore, DNA methylation has been emphasized as a useful marker of past exposure to carcinogens and future risk of cancer development [8, 32]. In the current study, our findings of reduced LINE-1 methylation in the gastric mucosae of non-gastric cancer patients suggest that precancerous lesions of depleted DNA methylation develop in the mucosae of these patients. Because measurement of LINE-1 methylation by pyrosequencing technology is a cost-effective and high-throughput method to assess global DNA methylation status, our findings may have considerable clinical implications [10– 12, 33]. H. pylori is a widely recognized pathogenic inducer of gastritis, ulcers and gastric cancer [17–19]. H. pylori infection has also been implicated in DNA methylation alterations, site-specific CpG island promoter hypermethylation and global DNA hypomethylation, the precursors of gastric cancer [20–22]. Interestingly, we demonstrated that LINE-1 methylation levels were lower in the noncancerous gastric mucosae of gastric cancer patients infected with H. pylori than in those of their uninfected counterparts. In addition, we prospectively confirmed the significant correlation between LINE-1 hypomethylation and H. pylori infection in gastric mucosae from non-gastric cancer patients for the first time. These findings suggested that infection of H. pylori might contribute to LINE-1 hypomethylation by some mechanisms. LINE-1 hypomethylation appears to occur during the transition from intestinal metaplasia to gastric adenoma, not during the transition from gastric adenoma to gastric cancer [34]. Since LINE-1 methylation level is regarded as a surrogate marker of global DNA methylation [9], our finding is consistent with these experimental results. Some mechanisms by which one pattern of altered DNA methylation, site-specific CgG island promoter hypermethylation, may correlate with gastric cancer have been
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identified. Infection by H. pylori induces the methylation of specific genes, and a cancerization field might emerge from accumulated aberrant methylation in the gastric mucosae [35]. FHL1 methylation has been associated with H. pylori infection, and FHL1 silencing might contribute to forming an epigenetic cancerization field [36]. Similarly, methylation silencing of miRNA genes contributes to the formation of epigenetic field defects in gastric cancers [37]. Among these, methylated miR-34b/c and RASGRF1 have been identified as useful biomarkers for identifying individuals at high risk of gastric cancer [38–40]. However, whether H. pylori influences the methylation of miR34b/c and RASGRF1 remains unclear. In the current study, we confirmed the significant correlation between LINE-1 hypomethylation, i.e., global DNA hypomethylation, and infection of H. pylori in noncancerous mucosae from gastric cancer patients and in gastric mucosae from non-gastric cancer patients. However, the mechanism by which global DNA hypomethylation induced by H. pylori correlates with human carcinogenesis remains undiscovered. Possibly, as occurs in esophageal squamous cell carcinoma, global DNA hypomethylation facilitates aggressive tumor behavior through genomic gains of oncogenes such as CDK6 [41]. Further studies are needed to confirm our findings and to examine the biological mechanisms by which global DNA hypomethylation induced by H. pylori infection contributes to epigenetic field defects and carcinogenesis. Carcinogenesis is induced by multiple factors, and the risk factors of gastric cancer have been fully explored. Various indicators such as life expectancy, education attainment and income are combined into a risk measure called the human development index (HDI). According to a recent global population study, increased incidence of gastric cancer is linked to higher HDIs coupled with increased per capita consumption of alcohol and tobacco [42]. In gastric cancer patients, smoking and age might lower the methylation levels of insulin-like growth factor 2 (IGF2) in blood leukocytes [43]. A possible relationship has been identified between LINE-1 methylation levels in blood leukocytes and smoking and drinking [44]. However, our current study found no effect of smoking, drinking or age on LINE-1 hypomethylation in the noncancerous mucosae of gastric cancer patients. An existing relationship might have been obscured by our small sample size; therefore, further studies are necessary to validate our findings. In summary, the current study suggests that LINE-1 hypomethylation is a phenomenon of the gastric cancer epigenetic field defect. Furthermore, LINE-1 hypomethylation is significantly associated with H. pylori infection, supporting a role for LINE-1 methylation level as a surrogate marker for the epigenetic field defect, particularly that induced by H. pylori infection. These findings require
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confirmation in future studies. We must also elucidate the mechanisms by which H. pylori infection induces aberrant DNA methylation in normal gastric mucosae. Conflict of interest
16.
None. 17.
References 18. 1. Rodriguez-Paredes M, Esteller M. Cancer epigenetics reaches mainstream oncology. Nat Med. 2011;17(3):330–9. doi:10.1038/ nm.2305. 2. Taby R, Issa JP. Cancer epigenetics. CA Cancer J Clin. 2010; 60(6):376–92. doi:10.3322/caac.20085. 3. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128(4):683–92. doi:10.1016/j.cell.2007.01.029. 4. Ehrlich M. DNA hypomethylation in cancer cells. Epigenomics. 2009;1(2):239–59. doi:10.2217/epi.09.33. 5. Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW, et al. Induction of tumors in mice by genomic hypomethylation. Science. 2003;300(5618):489–92. doi:10.1126/ science.1083558. 6. Holm TM, Jackson-Grusby L, Brambrink T, Yamada Y, Rideout WM 3rd, Jaenisch R. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell. 2005;8(4):275–85. doi:10.1016/j.ccr.2005.09.007. 7. Suzuki K, Suzuki I, Leodolter A, Alonso S, Horiuchi S, Yamashita K, et al. Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage. Cancer Cell. 2006;9(3):199–207. doi:10.1016/j.ccr.2006.02.016. 8. Ushijima T. Epigenetic field for cancerization. J Biochem Mol Biol. 2007;40(2):142–50. 9. Cordaux R, Batzer MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10(10):691–703. doi:10.1038/nrg2640. 10. Ogino S, Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, et al. LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Int J Cancer. 2008;122(12):2767–73. doi:10. 1002/ijc.23470. 11. Irahara N, Nosho K, Baba Y, Shima K, Lindeman NI, Hazra A, et al. Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells. J Mol Diagn. 2010;12(2):177–83. doi:10. 2353/jmoldx.2010.090106. 12. Ahn JB, Chung WB, Maeda O, Shin SJ, Kim HS, Chung HC, et al. DNA methylation predicts recurrence from resected stage III proximal colon cancer. Cancer. 2011;117(9):1847–54. doi:10. 1002/cncr.25737. 13. Iwagami S, Baba Y, Watanabe M, Shigaki H, Miyake K, Ishimoto T, et al. LINE-1 hypomethylation is associated with a poor prognosis among patients with curatively resected esophageal squamous cell carcinoma. Ann Surg. 2013;257(3):449–55. doi:10.1097/SLA.0b013e31826d8602. 14. Shigaki H, Baba Y, Watanabe M, Murata A, Iwagami S, Miyake K, et al. LINE-1 hypomethylation in gastric cancer, detected by bisulfite pyrosequencing, is associated with poor prognosis. Gastric Cancer. 2013;16(4):480–7. doi:10.1007/s10120-0120209-7. 15. Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic
123
19. 20.
21.
22.
23. 24.
25.
26.
27.
28.
29.
30. 31.
32.
33.
regions of the world. J Clin Oncol. 2006;24(14):2137–50. doi:10. 1200/JCO.2005.05.2308. Macdonald JS, Smalley SR, Benedetti J, Hundahl SA, Estes NC, Stemmermann GN, et al. Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N Engl J Med. 2001;345(10): 725–30. doi:10.1056/NEJMoa010187. Uemura N, Okamoto S, Yamamoto S, Matsumura N, Yamaguchi S, Yamakido M, et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med. 2001;345(11):784–9. doi:10.1056/NEJMoa001999. Gerhard M, Rad R, Prinz C, Naumann M. Pathogenesis of Helicobacter pylori infection. Helicobacter. 2002;7(Suppl 1):17–23. Marshall BJ. Helicobacter pylori. Am J Gastroenterol. 1994;89(8 Suppl):S116–28. Maekita T, Nakazawa K, Mihara M, Nakajima T, Yanaoka K, Iguchi M, et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk. Clin Cancer Res. 2006;12(3 Pt 1):989–95. doi:10.1158/1078-0432.CCR-05-2096. Compare D, Rocco A, Liguori E, D’Armiento FP, Persico G, Masone S, et al. Global DNA hypomethylation is an early event in Helicobacter pylori-related gastric carcinogenesis. J Clin Pathol. 2011;64(8):677–82. doi:10.1136/jcp.2010.087858. Park SY, Yoo EJ, Cho NY, Kim N, Kang GH. Comparison of CpG island hypermethylation and repetitive DNA hypomethylation in premalignant stages of gastric cancer, stratified for Helicobacter pylori infection. J Pathol. 2009;219(4):410–6. doi:10. 1002/path.2596. McColl KE. Clinical practice. Helicobacter pylori infection. N Engl J Med. 2010;362(17):1597–604. doi:10.1056/NEJMcp1001110. Washington K. 7th edition of the AJCC cancer staging manual: stomach. Ann Surg Oncol. 2010;17(12):3077–9. doi:10.1245/ s10434-010-1362-z. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM, et al. Reporting recommendations for tumor marker prognostic studies (REMARK). J Natl Cancer Inst. 2005;97(16):1180–4. doi:10.1093/jnci/dji237. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Chan AT, Schernhammer ES, et al. A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer. J Natl Cancer Inst. 2008;100(23):1734–8. doi:10.1093/jnci/djn359. Baba Y, Huttenhower C, Nosho K, Tanaka N, Shima K, Hazra A, et al. Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors. Mol Cancer. 2010;9:125. doi:10.1186/1476-4598-9-125. Baba Y, Nosho K, Shima K, Huttenhower C, Tanaka N, Hazra A, et al. Hypomethylation of the IGF2 DMR in colorectal tumors, detected by bisulfite pyrosequencing, is associated with poor prognosis. Gastroenterology. 2010;139(6):1855–64. doi:10.1053/ j.gastro.2010.07.050. Iwagami S, Baba Y, Watanabe M, Shigaki H, Miyake K, Ida S, et al. Pyrosequencing assay to measure LINE-1 methylation level in esophageal squamous cell carcinoma. Ann Surg Oncol. 2012;19(8):2726–32. doi:10.1245/s10434-011-2176-3. Jang BG, Kim WH. Molecular pathology of gastric carcinoma. Pathobiology. 2011;78(6):302–10. doi:10.1159/000321703. Kang GH, Lee S, Kim JS, Jung HY. Profile of aberrant CpG island methylation along multistep gastric carcinogenesis. Lab Invest. 2003;83(4):519–26. Nakajima T, Enomoto S, Ushijima T. DNA methylation: a marker for carcinogen exposure and cancer risk. Environ Health Prev Med. 2008;13(1):8–15. doi:10.1007/s12199-007-0005-x. Zhang FF, Cardarelli R, Carroll J, Fulda KG, Kaur M, Gonzalez K, et al. Significant differences in global genomic DNA
Med Oncol (2015) 32:117
34.
35.
36.
37.
38.
methylation by gender and race/ethnicity in peripheral blood. Epigenetics. 2011;6(5):623–9. Bae JM, Shin SH, Kwon HJ, Park SY, Kook MC, Kim YW, et al. ALU and LINE-1 hypomethylations in multistep gastric carcinogenesis and their prognostic implications. Int J Cancer. 2012;131(6):1323–31. doi:10.1002/ijc.27369. Ushijima T, Hattori N. Molecular pathways: involvement of Helicobacter pylori-triggered inflammation in the formation of an epigenetic field defect, and its usefulness as cancer risk and exposure markers. Clin Cancer Res. 2012;18(4):923–9. doi:10. 1158/1078-0432.CCR-11-2011. Asada K, Ando T, Niwa T, Nanjo S, Watanabe N, Okochi-Takada E, et al. FHL1 on chromosome X is a single-hit gastrointestinal tumor-suppressor gene and contributes to the formation of an epigenetic field defect. Oncogene. 2013;32(17):2140–9. doi:10.1038/ onc.2012.228. Ando T, Yoshida T, Enomoto S, Asada K, Tatematsu M, Ichinose M, et al. DNA methylation of microRNA genes in gastric mucosae of gastric cancer patients: its possible involvement in the formation of epigenetic field defect. Int J Cancer. 2009;124(10): 2367–74. doi:10.1002/ijc.24219. Suzuki H, Yamamoto E, Nojima M, Kai M, Yamano HO, Yoshikawa K, et al. Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect. Carcinogenesis. 2010;31(12):2066–73. doi:10.1093/carcin/bgq203.
Page 9 of 9 117 39. Yamamoto E, Suzuki H, Maruyama R, Shinomura Y. Developing technologies for epigenomic analysis and clinical application of molecular diagnosis. Rinsho Byori. 2012;60(7):637–43. 40. Takamaru H, Yamamoto E, Suzuki H, Nojima M, Maruyama R, Yamano HO, et al. Aberrant methylation of RASGRF1 is associated with an epigenetic field defect and increased risk of gastric cancer. Cancer Prev Res (Phila). 2012;5(10):1203–12. doi:10. 1158/1940-6207.CAPR-12-0056. 41. Baba Y, Watanabe M, Murata A, Shigaki H, Miyake K, Ishimoto T, et al. LINE-1 hypomethylation, DNA copy number alterations, and CDK6 amplification in esophageal squamous cell carcinoma. Clin Cancer Res. 2014;. doi:10.1158/1078-0432.CCR-13-1645. 42. Ferrari F, Reis MA. Study of risk factors for gastric cancer by populational databases analysis. World J Gastroenterol. 2013;19(48):9383–91. doi:10.3748/wjg.v19.i48.9383. 43. Yuasa Y, Nagasaki H, Oze I, Akiyama Y, Yoshida S, Shitara K, et al. Insulin-like growth factor 2 hypomethylation of blood leukocyte DNA is associated with gastric cancer risk. Int J Cancer. 2012;131(11):2596–603. doi:10.1002/ijc.27554. 44. Hou L, Wang H, Sartori S, Gawron A, Lissowska J, Bollati V, et al. Blood leukocyte DNA hypomethylation and gastric cancer risk in a high-risk Polish population. Int J Cancer. 2010;127(8):1866–74. doi:10.1002/ijc.25190.
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