Cancer Causes & Control

, Volume 23, Issue 6, pp 865–873 | Cite as

Hypomethylation of Alu repetitive elements in esophageal mucosa, and its potential contribution to the epigenetic field for cancerization

  • Yasunori Matsuda
  • Satoshi Yamashita
  • Yi-Chia Lee
  • Tohru Niwa
  • Takeichi Yoshida
  • Ken Gyobu
  • Hiroyasu Igaki
  • Ryoji Kushima
  • Shigeru Lee
  • Ming-Shiang Wu
  • Harushi Osugi
  • Shigefumi Suehiro
  • Toshikazu Ushijima
Original paper

Abstract

Background

Aberrant hypermethylation of specific genes is present in esophageal squamous cell carcinomas (ESCCs). Such hypermethylation is also present in normal-appearing esophageal mucosae of ESCC patients and is considered to contribute to the formation of a field for cancerization. On the other hand, the presence of global hypomethylation in ESCCs or in their background esophageal mucosae is unknown.

Method

We collected 184 samples of esophageal mucosae (95 normal mucosae from healthy subjects, and 89 non-cancerous background mucosae from ESCC patients) and 93 samples of ESCCs. Methylation levels of repetitive elements (Alu, LINE1) and cancer/testis antigen genes (NY-ESO-1, MAGE-C1) were measured by bisulfite pyrosequencing and quantitative methylation-specific PCR, respectively.

Results

Methylation levels of Alu, LINE1, NY-ESO-1, and MAGE-C1 were significantly lower in ESCCs than in their background and normal mucosae. Also, in the background mucosae, a significant decrease of the Alu methylation level compared with the normal mucosae was present. In ESCCs, methylation levels of the two repetitive elements and the two cancer/testis antigen genes were correlated with each other.

Conclusion

This is the first study to show the presence of global hypomethylation in ESCCs, and even in their non-cancerous background mucosae. Alu hypomethylation might reflect the severity of an epigenetic field for cancerization.

Keywords

Esophageal squamous cell carcinoma Hypomethylation Repetitive element Cancer/testis antigen Epigenetics 

Abbreviations

ALDH2

Acetaldehyde dehydrogenase 2

LINE1

Long interspersed nucleotide element 1

PCR

Polymerase chain reaction

UICC

Union for International Cancer Control

Introduction

Esophageal squamous cell carcinoma (ESCC) remains the predominant histological type of esophageal cancers in Asian countries [1, 2, 3]. It is known for its high prevalence of multiple occurrences, including synchronous and metachronous occurrence [4, 5]. As for mechanisms of multiple occurrences, it is believed that genetic/epigenetic alterations accumulate in normal-appearing tissues, forming a field for cancerization [6, 7]. We previously demonstrated that aberrant methylation of promoter CpG islands was present in non-cancerous esophageal mucosae of ESCC patients and that methylation of specific genes was associated with smoking history [8] and cancer risk [9]. Because tumor-suppressor genes, such as CDKN2A, CDH1, FHIT, and RASSF1A, are inactivated by methylation of promoter CpG islands in ESCCs [10, 11, 12, 13], it was considered that accumulation of aberrant methylation in esophageal mucosae was involved in the formation of a field for ESCCs.

In addition to methylation of promoter CpG islands, global hypomethylation is characteristic of tumor cells [14, 15]. Global hypomethylation involves hypomethylation of normally methylated repetitive elements, such as Alu and LINE1 [16], and normally methylated cancer/testis (CT) antigen genes [17, 18]. The hypomethylation of repetitive elements can serve as a surrogate marker for global DNA hypomethylation [19, 20]. “Normally methylated” genes are physiologically methylated and unexpressed in most tissues of adults, except testicular germ cells. In contrast, they are demethylated and expressed in various types of human cancers [21], and specific ones, such as NY-ESO-1 and MAGE-C1, are expected to be useful as potential targets for cancer immunotherapy [22, 23]. The presence of global hypomethylation is associated with increased rates of chromosome recombination and increased incidence of tumor formation [24, 25, 26]. Nevertheless, in ESCCs, the presence of global hypomethylation in cancer cells and its potential involvement in the field for cancerization are unknown.

In the current study, we aimed to elucidate whether or not hypomethylation is present in ESCCs, and also in non-cancerous background esophageal mucosae. To this end, we accurately quantified methylation levels of two repetitive elements, Alu and LINE1, and two CT antigen genes, NY-ESO-1 and MAGE-C1, in ESCCs, their background mucosae, and normal esophageal mucosae from healthy volunteers.

Materials and methods

Patients and tissue samples

Normal mucosae of 95 healthy volunteers (69 male and 26 female; average age = 58, median age = 59, range = 25–91) and cancerous lesions of 93 ESCC patients (85 male and 8 female; average age = 61, median age = 60, range = 43–85) before any therapeutic intervention (surgery, chemotherapy, or radiation therapy) were collected by endoscopic biopsy at the National Taiwan University Hospital, Taipei, Taiwan. In 89 of the 93 ESCC patients, we collected biopsy samples not only from cancerous lesions but also from non-cancerous background mucosae (82 male and 7 female; average age = 61, median age = 60, range = 43–85). A total of 277 samples for methylation analysis were collected. Additionally, for immunohistochemical staining, surgical specimens of primary ESCCs and their paired non-cancerous background mucosae were collected from 48 patients (41 male and 7 female; average age = 63, range = 41–83) who underwent esophagectomy without any neoadjuvant therapy at the National Cancer Center Hospital, Tokyo, Japan. Non-cancerous background mucosa was defined as the area that was at least 5 cm away from the cancerous margin and appeared normal by iodine staining. All samples were stored at −80 °C after biopsy or resection until the extraction of genomic DNA.

Informed consents and interviews for lifestyle risk factors, including cigarette smoking and alcohol intake, were obtained from all the individuals. The range of follow-up period after endoscopic examination was 461–944 days. Disease stages were classified according to the 7th edition of the TNM classification by UICC.

Bisulfite pyrosequencing for repetitive elements

Sodium bisulfite modification was performed using 1 μg of BamHI-digested genomic DNA as previously described [27]. The modified DNA was suspended in 40 μl of Tris–EDTA buffer, and an aliquot of 1 μl was used for bisulfite pyrosequencing. The CpG sites that showed most significant hypomethylation in gastric cancer [28] were selected for analysis in this study. All primers for pyrosequencing were the same as those we previously reported [28] and are listed in Supplementary Table 1. The PCR products labeled with biotin were annealed to 0.2 μM pyrosequencing primers, and pyrosequencing was carried out using the PSQ 96 Pyrosequencing System (Qiagen, CA, USA). Methylation levels were obtained using PSQ Assay Design software (Qiagen).

Quantitative methylation-specific PCR (qMSP)

Real-time MSP was performed with a primer set specific to methylated (M) or unmethylated (U) sequence by using a 2 μl aliquot of the sodium bisulfite-treated DNA, SYBR Green (Bio Whittaker Molecular Applications, MD, USA), and an iCycler Thermal Cycler (Bio-Rad Laboratories, CA, USA). MSP primers and PCR conditions are shown in Supplementary Table 2. DNA methylated by SssI methylase (New England Biolabs, MA, USA) and DNA amplified twice by a GenomiPhi DNA amplification kit (GE Healthcare Bio-Science, Buckinghamshire, England) were used as fully methylated and unmethylated control DNA, respectively. As in our previous report [8], methylation levels were calculated as the fraction of methylated molecules in the total number of DNA molecules (the number of methylated molecules + the number of unmethylated molecules).

Immunohistochemistry of CT antigens

Immunohistochemical staining of NY-ESO-1 and MAGE-C1 antigens was performed using a mouse monoclonal anti-NY-ESO-1 antibody (clone E978, Invitrogen, CA, USA) and anti-MAGE-C1 antibody (clone CT7-33, DAKO, CA, USA) as primary antibodies. Formalin-fixed and paraffin-embedded samples were sliced at 3 μm thickness, deparaffinized, and heated in 10 mM citrate buffer (pH 6.0) for 20 min (NY-ESO-1) and 5 min (MAGE-C1) at 120 °C by autoclave. After blocking with Blocking-One (Nacalai Tesque, Kyoto, Japan), the sections were incubated with a primary antibody at a concentration of 2.5 μg/ml (NY-ESO-1) or 0.21 μg/ml (MAGE-C1) at 4 °C overnight. Detection of the primary antibody was performed with the DAKO Envision Plus system at room temperature for 60 min and DAB (Wako, Osaka, Japan) as a chromogen. Slides were counterstained with hematoxylin. As a positive control, we used the normal part of a testis with intact spermatogenesis collected from an adolescent patient with seminoma.

Statistical analysis

Differences in mean methylation levels were analyzed by Student’s t test (when variances were equal) and Welch’s t test (when variances were unequal). Correlations of methylation levels among individual repetitive elements and promoter CpG islands were analyzed using Pearson’s product-moment correlation coefficients. All the analyses were performed using PASW statistics version 18.0 (SPSS Japan Inc., Tokyo, Japan), and the results were considered significant when p values <0.05 were obtained by a two-sided test.

Results

Hypomethylation of repetitive elements and CT antigen genes in ESCCs, and Alu hypomethylation in background mucosae

Methylation levels of repetitive elements were quantified by bisulfite pyrosequencing in a total of 277 samples. The mean methylation level of Alu was significantly lower in ESCCs (42.0 ± 3.8%, mean ± SD) than in non-cancerous background mucosae of cancer patients (45.7 ± 2.3%) and normal mucosae of healthy volunteers (46.5 ± 2.4%) (p < 0.001) (Fig. 1a). Notably, the mean methylation level of Alu was significantly lower even in the background mucosae than in the normal mucosae (p = 0.018). The mean methylation level of LINE1 was significantly lower in ESCCs (62.2 ± 12.1%) than in the normal and background mucosae (79.7 ± 3.9%, 78.8 ± 6.7%, respectively, p < 0.001) (Fig. 1b). Unlike Alu, significant differences between the normal and background mucosae were not observed.
Fig. 1

Methylation levels of the two repetitive elements and the two CT antigen genes in normal mucosae of healthy subjects (n = 95), non-cancerous background mucosae of cancer patients (n = 89), and ESCCs (n = 93) collected by endoscopic biopsy. Distribution of the methylation levels at a particular CpG site of Alu (a), LINE1 (b), NY-ESO-1 (c), or MAGE-C1 (d) is shown, and a horizontal line in a chart represents a mean methylation level for each group. The mean methylation levels of Alu, LINE1, NY-ESO-1, and MAGE-C1 in ESCCs were significantly lower than those in normal and background mucosae. In addition, Alu methylation levels were significantly lower in the background mucosae than in the normal mucosae

Methylation levels of CT antigen genes were quantified by real-time MSP (Fig. 1c, d). Both NY-ESO-1 and MAGE-C1 were almost fully methylated in the normal and background mucosae but demethylated in 22 (23.7%) and 23 (24.7%) samples of ESCCs, respectively, with a threshold value of 95%. The mean methylation levels of NY-ESO-1 and MAGE-C1 were significantly lower in ESCCs (89.3 ± 22.4%, 94.0 ± 14.9%, respectively) than in their background (99.6 ± 1.4%, 99.8 ± 0.6%, respectively) and normal mucosae (99.7 ± 0.9%, 99.7 ± 2.2%, respectively) (p < 0.001 each).

Correlation among methylation levels of repetitive elements, CT antigen genes, and other promoter CpG islands

We analyzed correlation of methylation levels among two repetitive elements, two CT antigen genes, and four promoter CpG islands in the background mucosae and in ESCCs (Table 1 and Supplementary Fig. 1). As promoter CpG islands hypermethylated in background mucosae, we selected four genes, HOXA9, NEFH, UCHL1, and MT1M, whose methylation levels were analyzed in our previous study (Supplementary Fig. 2) [9]. In the background mucosae, methylation levels were significantly correlated among the four genes. Also, significant negative correlations were observed between the hypomethylation of repetitive elements and hypermethylation of the four genes. In ESCCs, significant positive correlation was observed among individual repetitive elements and CT antigen genes. Correlations between Alu and LINE1 hypomethylation and between NY-ESO-1 and MAGE-C1 hypomethylation were especially stronger than the other correlations.
Table 1

Correlation among hypomethylation of individual repetitive elements, CT antigens, and hypermethylation of promoter CpG islands

 

Alu

LINE1

NY-ESO-1

MAGE-C1

HOXA9

NEFH

UCHL1

MT1M

Background mucosae

 Alu

0.063

0.152

−0.132

0.351

0.240

0.231

0.398

 LINE1

 

0.095

0.092

0.313

0.464

0.441

0.554

 NY-ESO-1

  

0.038

0.356

−0.024

0.010

0.238

 MAGE-C1

   

−0.088

−0.002

0.008

0.037

 HOXA9

    

0.341

0.632

0.490

 NEFH

     

0.561

0.544

 UCHL1

      

0.312

 MT1M

       

ESCC

 Alu

0.502

0.307

0.240

−0.162

0.093

−0.002

0.181

 LINE1

 

0.470

0.383

0.386

0.189

−0.049

0.262

 NY-ESO-1

  

0.566

−0.182

0.240

0.139

−0.019

 MAGE-C1

   

0.268

0.171

0.060

0.082

 HOXA9

    

0.057

0.133

0.178

 NEFH

     

0.268

−0.085

 UCHL1

      

0.381

 MT1M

       

Coefficient values shown in bold type denote that p value is <0.05

The lack of association between hypomethylation and exposure to risk factors, and between hypomethylation and clinicopathological findings

We first analyzed correlation between the age and degree of hypomethylation of the two repetitive elements. No significant correlation was observed in healthy volunteers (Alu; r = −0.181, p = 0.080, LINE1; r = 0.129, p = 0.214) or in ESCC patients (Alu; r = −0.085, p = 0.426, LINE1; r = 0.010, p = 0.925), showing the hypomethylation was not age-dependent. Then, in normal mucosae, background mucosae, and ESCCs, we analyzed association between exposure to risk factors and the degree of hypomethylation of the two repetitive elements and the two CT antigen genes. In any group of samples, methylation levels were not associated with history of cigarette smoking or alcohol intake (Table 2). Even when the samples were classified according to the genetic polymorphisms, whether they had an active ALDH2 allele (ALDH21/ALDH21 homozygote) or an inactive ALDH2 allele (ALDH21/ALDH22 heterozygote and ALDH22/ALDH22 homozygote), methylation levels were not associated with alcohol intake (data not shown).
Table 2

Association between hypomethylation and exposure to risk factors

Sample

N

Alu

LINE1

NY-ESO-1

MAGE-C1

Mean ± SD

p value

Mean ± SD

p value

Mean ± SD

p value

Mean ± SD

p value

Normal mucosae

 Smoking history

  (−)

53

46.3 ± 2.2

0.411

79.0 ± 3.6

0.054

99.8 ± 0.4

0.474

99.5 ± 2.9

0.316

  (+)

42

46.7 ± 2.6

 

80.6 ± 4.2

 

99.6 ± 1.3

 

99.9 ± 0.2

 

 Alcohol history

  (−)

61

46.5 ± 2.3

0.837

79.5 ± 3.8

0.517

99.8 ± 0.2

0.096

99.9 ± 0.2

0.337

  (+)

34

46.4 ± 2.6

 

80.1 ± 4.1

 

99.4 ± 1.4

 

99.3 ± 3.6

 

Background mucosae

 Smoking history

  (−)

15

45.1 ± 2.2

0.324

79.5 ± 4.9

0.650

99.5 ± 0.9

0.786

99.9 ± 0.2

0.683

  (+)

74

45.8 ± 2.3

 

78.7 ± 7.0

 

99.6 ± 1.5

 

99.8 ± 0.7

 

 Alcohol history

  (−)

11

45.6 ± 1.0

0.866

79.8 ± 5.6

0.613

99.5 ± 1.1

0.828

99.9 ± 0.2

0.623

  (+)

78

45.7 ± 2.4

 

78.7 ± 6.8

 

99.6 ± 1.4

 

99.8 ± 0.7

 

ESCC

 Smoking history

  (−)

17

42.7 ± 3.1

0.433

61.3 ± 13.0

0.734

77.4 ± 34.1

0.106

86.6 ± 26.4

0.184

  (+)

76

41.9 ± 3.9

 

62.4 ± 11.9

 

91.9 ± 18.1

 

95.7 ± 10.4

 

 Alcohol history

  (−)

12

41.7 ± 3.6

0.746

61.4 ± 15.9

0.810

85.1 ± 33.6

0.495

85.8 ± 30.8

0.316

  (+)

81

42.1 ± 3.8

 

62.3 ± 11.5

 

89.9 ± 20.4

 

95.2 ± 10.6

 
In ESCCs, we also analyzed association between methylation levels of repetitive elements or CT antigen genes and clinicopathological findings, including depth of tumor, tumor differentiation, lymph node metastasis, multiplicity of tumor, and tumor recurrence (Table 3). There was no significant association between methylation levels of repetitive elements in ESCCs and clinicopathological findings, and between methylation levels of CT antigen genes and clinicopathological findings.
Table 3

Association between hypomethylation and clinicopathological findings

Variable

N

Alu

LINE1

NY-ESO-1

MAGE-C1

Mean ± SD

p value

Mean ± SD

p value

Mean ± SD

p value

Mean ± SD

p value

Depth of tumor

 T1/T2

9

40.2 ± 3.0

0.146

64.9 ± 8.6

0.399

89.3 ± 20.7

0.894

95.2 ± 5.9

0.737

 T3/T4

66

42.1 ± 3.6

 

61.2 ± 12.5

 

90.3 ± 21.6

 

93.3 ± 17.2

 

Tumor differentiation

 Poorly

12

41.2 ± 5.0

0.463

60.1 ± 14.7

0.932

92.1 ± 16.6

0.665

98.0 ± 3.2

0.288

 Moderately/Well

33

42.3 ± 4.0

 

60.5 ± 10.8

 

88.9 ± 23.1

 

91.7 ± 20.0

 

Lymph node metastasis

 Negative

11

40.7 ± 3.4

0.208

60.6 ± 8.8

0.715

95.7 ± 12.7

0.369

97.5 ± 3.5

0.392

 Positive

65

42.1 ± 3.6

 

62.0 ± 12.6

 

89.4 ± 22.3

 

92.9 ± 17.3

 

Multiplicity of tumor

 Solitary

74

41.8 ± 3.9

0.641

61.6 ± 12.3

0.251

87.3 ± 24.5

0.240

93.0 ± 16.5

0.310

 Multiple

8

42.5 ± 3.8

 

62.0 ± 11.0

 

97.6 ± 5.6

 

99.0 ± 1.3

 

Tumor recurrence

 Negative

79

41.8 ± 3.7

0.460

62.5 ± 12.5

0.373

89.3 ± 22.2

0.609

93.4 ± 16.0

0.393

 Positive

9

42.8 ± 3.8

 

58.6 ± 10.7

 

93.2 ± 16.5

 

98.0 ± 3.3

 

Association between hypomethylation and expression of CT antigen genes

Finally, we aimed to assess association between hypomethylation and expression of CT antigen genes using immunohistochemistry. From 48 surgical specimens, we selected six ESCCs that had various degrees of hypomethylation and their paired non-cancerous background mucosae (Table 4; representative results in Fig. 2 and Supplementary Fig. 3). For NY-ESO-1, positive staining was observed in two of four ESCCs with demethylation and none of two background mucosae with demethylation. For MAGE-C1, positive staining was observed in three of five ESCCs with demethylation and none of one background mucosa with demethylation. There was no specimen that stained positive without demethylation, supporting the role of methylation status in expression regulation of the CT antigen genes.
Table 4

Association between hypomethylation and expression of CT antigen genes

Sample ID.

NY-ESO-1

MAGE-C1

Methylation level (%)

Immunohistochemistry

Methylation level (%)

Immunohistochemistry

Background mucosae

 ES-29

93.3

100.0

 ES-39

87.9

99.1

 ES-49

99.8

99.9

 ES-58

97.5

93.7

 ES-59

100.0

a

99.8

b

 ES-62

100.0

100.0

ESCC

 ES-29

12.2

+a (diffuse)

72.1

 ES-39

80.3

92.7

 ES-49

51.0

51.9

+b (hetero)

 ES-58

53.1

+a (hetero)

54.0

+ (hetero)

 ES-59

99.7

a

93.7

+ (hetero)

 ES-62

99.4

97.4

b

aMicroscopic findings are shown in Fig. 2

bMicroscopic findings are shown in Supplementary Fig. 3

Fig. 2

Representative immunohistochemical staining of NY-ESO-1 in surgical specimens. a Non-cancerous background mucosa (ES-59, methylation level = 100.0%), b ESCC with full methylation (ES-59, methylation level = 99.7%), c ESCC with partial demethylation (ES-58, methylation level = 53.1%), and d ESCC with almost complete demethylation (ES-29, methylation level = 12.2%) are presented. A scale bar represents 100 μm. Neither the background specimen nor the specimen of ESCC with full methylation had staining. Heterogeneous staining, mainly nuclear, was observed in the specimen with partial demethylation. Diffuse staining in both cytoplasm and nucleus was observed in the specimen with almost complete demethylation. The result of immunohistochemistry was consistent with methylation status of CT antigen genes

Discussion

In this study, we demonstrated that hypomethylation of Alu and LINE1 was present in ESCCs. Although the presence of global hypomethylation is considered to be a universal phenomenon across various types of cancers [14, 29, 30, 31], this is the first report on its presence in ESCCs and provides a fundamental piece of information. Also, it was striking that hypomethylation of Alu, but not LINE1, was present in non-cancerous background mucosae of ESCC patients. This strongly indicated that Alu hypomethylation is induced earlier than LINE1 hypomethylation during carcinogenesis of ESCCs. The Alu hypomethylation was considered to be involved in the epigenetic field for cancerization, and might have potential as an epigenetic marker for ESCC risk estimation.

In the background mucosae, hypomethylation of repetitive elements negatively correlated with hypermethylation of promoter CpG islands of the four genes, HOXA9, NEFH, UCHL1, and MT1M. This finding suggested a possibility that global hypomethylation and hypermethylation of some promoter CpG islands were caused by shared factors. In our study, however, while hypermethylation of these four genes is known to correlate with smoking history [8, 9], no association was found between hypomethylation and exposure to risk factors, including history of cigarette smoking and alcohol intake. It was considered that cigarette smoking and alcohol intake were more closely associated with hypermethylation of these CpG islands than with global hypomethylation.

In the stomach, we previously showed that hypomethylation of Alu, LINE1, and Satα is present and that Alu hypomethylation was more sensitive to hypomethylation due to Helicobacter pylori infection than the other repetitive elements [28]. Taken together, Alu seems to be most susceptible to hypomethylation due to exposure to carcinogenic stimuli. The different susceptibility to hypomethylation between Alu and LINE1 might be attributed to the differences in their intrinsic functions. Alu depends on the proteins encoded by LINE1 for its retrotranscription and transposition [32], whereas LINE1 is transposable autonomously [33]. Therefore, there is a possibility that LINE1 hypomethylation, which can induce its retrotranscription and transposition [34], is more strictly regulated than Alu hypomethylation.

We also showed that CT antigen genes were demethylated in a coordinated way with global hypomethylation in ESCCs and expression of these genes was consistent with methylation states of each sample by immunohistochemistry. These data suggested that CT antigen genes tended to be demethylated in ESCCs with global hypomethylation. This raises a possibility that induction of CT antigen by DNA methyltransferase inhibitors [35, 36] might lead to a more effective cancer immunotherapy targeting CT antigens.

Methylation levels of repetitive elements and CT antigen genes in ESCCs were not associated with any clinicopathological features. To exclude the possibility that a fraction of cancer cells were highly variable among the biopsy samples and made associations undetectable, we microscopically analyzed the fraction of cancer cells in biopsy samples. The fraction was 67.3 ± 6.8% (mean ± SD), and such a possibility seemed to be low. However, there still remains a possibility that use of samples with high purity, such as those prepared by laser-captured microdissection, might reveal some weak association between hypomethylation and clinicopathological findings.

In conclusion, our study demonstrated that hypomethylation of Alu and LINE1 was present in ESCCs and that Alu hypomethylation was present even in non-cancerous background mucosae of ESCC patients. It was suggested that Alu hypomethylation represents the severity of an epigenetic field for cancerization and might become an epigenetic marker for ESCC risk estimation.

Notes

Acknowledgments

This study was supported by Grants-in-Aid for the National Cancer Center Research and Development Fund, and by the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct) from the Ministry of Education, Culture, Science and Sport, Japan. Y. M. and K. G. are recipients of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10552_2012_9955_MOESM1_ESM.doc (60 kb)
Supplementary material 1 (DOC 60 kb)
10552_2012_9955_MOESM2_ESM.ppt (1.6 mb)
Supplementary material 2 (PPT 1679 kb)

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Yasunori Matsuda
    • 1
    • 2
  • Satoshi Yamashita
    • 1
  • Yi-Chia Lee
    • 3
  • Tohru Niwa
    • 1
  • Takeichi Yoshida
    • 1
  • Ken Gyobu
    • 1
  • Hiroyasu Igaki
    • 4
  • Ryoji Kushima
    • 5
  • Shigeru Lee
    • 2
  • Ming-Shiang Wu
    • 3
  • Harushi Osugi
    • 2
  • Shigefumi Suehiro
    • 6
  • Toshikazu Ushijima
    • 1
  1. 1.Division of EpigenomicsNational Cancer Center Research InstituteChuo-kuJapan
  2. 2.Department of Gastroenterological Surgery, Graduate School of MedicineOsaka City UniversityOsakaJapan
  3. 3.Department of Internal Medicine, Collage of MedicineNational Taiwan UniversityTaipeiTaiwan
  4. 4.Esophageal Surgery DivisionNational Cancer Center HospitalTokyoJapan
  5. 5.Pathology of Clinical Laboratory DivisionNational Cancer Center HospitalTokyoJapan
  6. 6.Department of Cardiovascular Surgery, Graduate School of MedicineOsaka City UniversityOsakaJapan

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