Prognostic Analysis of E-Cadherin Gene Promoter Hypermethylation in Patients with Surgically Resected, Node-Positive, Diffuse Gastric Cancer

Home , Carcinoembryonic antigen, CDH1 (gene)

2784 Vol. 10, 2784–2789, April 15, 2004 Clinical Cancer Research

Francesco Graziano,1 Federica Arduini,2 was 35% and 67%, respectively. CDH1 promoter hyper- Annamaria Ruzzo,4 Italo Bearzi,2 methylation retained its prognostic role for disease-free sur- Bostjan Humar,5 Helen More,5 Rosarita Silva,6 vival (P < 0.001) and overall survival (P < 0.001) in multiva- 7 5 1 riate analysis. Immunohistochemistry showed a significant Pietro Muretto, Parry Guilford, Enrica Testa, association between CDH1 methylation and E-cadherin expres- 6 4 Davide Mari, Mauro Magnani, and sion (P < 0.001). Stefano Cascinu3 Conclusions: This study shows adverse prognostic effect 1Medical Oncology Unit, Hospital of Urbino, Urbino, Italy; of CDH1 promoter hypermethylation in patients with dif- 2Department of Histopathology and 3Medical Oncology, University of fuse gastric cancer. This form of cancer, and other types Ancona, Ancona, Italy; 4Institute of Biochemistry “G Fornaini,” 5 with frequent hypermethylation and silencing of critical University of Urbino, Urbino, Italy; Cancer Genetics Laboratory, tumor suppressor genes, would make appropriate targets University of Otago, Dunedin, New Zealand; 6Medical Oncology Unit, Hospital of Fabriano, Fabriano, Italy; and 7Department of for the testing of novel compounds with demethylating ac- Histopathology, Hospital of Pesaro, Pesaro, Italy tivity.

INTRODUCTION ABSTRACT CDH1 is a tumor suppressor gene located on chromosome Purpose: Recent investigations have demonstrated that 16q22.1. The mature E-cadherin protein is a transmembrane hypermethylation is a frequent mechanism for silencing tu- homodimer that is localized mainly to the adherens junctions of mor suppressor genes. This is a potentially reversible epige- epithelial cells. E-cadherin plays a fundamental role in main- netic change, and it is the target of a novel class of anticancer taining cell differentiation, polarity, and normal tissue architec- compounds with demethylating activity. Better understand- ture (1, 2). ing of the clinical implications of hypermethylation will Specific germ-line truncating mutations in CDH1 charac- allow the optimal planning of future trials with demethylat- terize the hereditary diffuse gastric cancer syndrome (3), and ing drugs. In this perspective, we investigated whether hy- somatic CDH1 mutations occur in sporadic gastric carcinomas permethylation in the CDH1 promoter region is correlated at a high frequency (4, 5). In both hereditary diffuse gastric with poor prognosis of patients with surgically resected, cancer syndrome cases and sporadic gastric carcinomas, E- node-positive, diffuse gastric cancer. cadherin immunoreactivity is frequently abolished (2). Accord- Experimental Design: Consecutive cases of diffuse gas- ing to the “two-hit” hypothesis, this is the result of an inacti- tric cancer were considered eligible for study entry. Addi- vating mutation in a CDH1 allele, and a second hit, which tional inclusion criteria were radical surgery with a minimum down-regulates the remaining wild-type allele (6). of D1 lymphadenectomy, complete follow-up information, and In mammalian DNA, through the action of methyltrans- availability of tumor specimens for methylation-specific PCR ferase enzymes, methyl groups can be added to the cytosines in and immunohistochemistry analyses. CpG dinucleotides. CpG dinucleotides occur at high density in Results: CDH1 promoter hypermethylation was found stretches of DNA termed “CpG islands” (7). About half of all in 40 of 73 cases (54%), and it was significantly associated human genes have CpG islands in their 5Ј-promoter regions, and with worse prognosis. In patients with and without hyper- in normal tissues, methylation is limited to exceptional situa- methylation, the 5-year event-free survival rate was 30% tions (embryogenesis, development, and differentiation to adult and 62%, respectively, and the 5-year overall survival rate cells; Ref. 7). Aberrant CpG island methylation and transcriptional silencing of tumor suppressor genes frequently occur in human neoplasms (7, 8). However, concomitant up-regulation of DNA methyltransferase activity is not always detected, illus- Received 9/30/03; revised 12/04/03; accepted 12/12/03. trating that the mechanisms that induce methylation in the Grant suppport: Partially supported by FIRB 2001 ϭ Prot. RBNEO01TBTR. human genome are only partially understood (8). The costs of publication of this article were defrayed in part by the Hypermethylation in CDH1 occurs in 40–80% of primary payment of page charges. This article must therefore be hereby marked human gastric carcinomas, especially in the poorly differenti- advertisement in accordance with 18 U.S.C. Section 1734 solely to ated, diffuse histotype (2, 9). It is considered as a common indicate this fact. Requests for reprints: Francesco Graziano, Oncologia Medica, Osped- inactivating second hit for CDH1 (10, 11), and the presence of ale di Urbino, Via Bonconte da Montefeltro, 61029 Urbino, Italy. hypermethylation is associated frequently with E-cadherin Phone: 39-722-301251; Fax: 39-722-301289; E-mail: [email protected]. down-regulation (9, 11–13). In recent years, several retrospec-

tive studies have found a more aggressive phenotype and poor According to the policy of participating institutions, the prognosis in gastric cancer patients with E-cadherin-negative follow-up consisted of interim history, physical examination, tumors (14–20). hematological studies, carcinoembryonic antigen levels, and Interestingly, hypermethylation is a potentially reversible diagnostic imaging (chest X-ray and abdominal ultrasonogra- epigenetic change, and, therefore, has become the target for phy) every 4 months in the first year, and every 6 months in the novel anticancer drugs (21). Several demethylating compounds second through fifth years. Patients underwent upper endoscopy have been investigated in Phase II trials in hematological and 6 months after surgery and every 12 months thereafter. Abdom- solid neoplasms over the last decades (22), and second-genera- inal and pelvic computed tomographic scan was performed for tion drugs of this family will be evaluated in the near future (23). corroborative evidence of relapse. The recurrences of gastric Early clinical trials with such agents have shown disap- carcinoma had to be proven by cytology biopsy or surgery. pointing results (22), but notably, none of these studies stratified Before study inclusion, all of the cases were reviewed by patients for the methylation status of genes involved in the two pathologists (I. B. and P. M.) for confirmation of diagnosis, critical stages of tumorigenesis and tumor progression. There- staging, and grading. The 1997 revision of the American Joint fore, better knowledge on the frequency of hypermethylation, its Committee on Cancer manual was used for the classification of involvement in the silencing of tumor suppressor genes, and the each case. The study was performed in a blind fashion, so that final influence on the natural history of the disease could lead to patient outcome was unknown to investigators performing im- the optimal use of demethylating compounds. munohistochemistry and methylation analyses. The study was According to this background and the lack of information carried out in accordance with the Institutional Review Board on the prognostic role of CDH1 promoter hypermethylation, we requirements for retrospective investigations. sought to investigate the relationship between the epigenetic Analysis of CDH1 Promoter Hypermethylation. Fif- change of CDH1 and the outcome of patients with surgically teen 5-mm thick paraffin sections of gastric tissue samples were resected, diffuse gastric cancer. used for DNA extraction. Careful microdissection of gastric carcinoma cells from representative tumor areas was performed MATERIALS AND METHODS from the slides. Genomic DNA from microdissected tissue was Human Samples and Clinicopathologic Data. In this isolated by the high pure PCR template preparation kit (Boeh- retrospective analysis, the study population consisted of consec- ringer Mannheim Corp., Indianapolis, IN; Ref. 24). Treatment utive patients with node-positive, diffuse gastric cancer who with sodium bisulfite was used to induce chemical modification underwent surgery between 1994 and 1998. None of the patients of genomic DNA and allowing identification of aberrant DNA had received preoperative or adjuvant chemotherapy. Additional methylation in CpG islands. In this procedure, the unmethylated inclusion criteria were radical gastrectomy with a minimum of cytosine nucleotides are converted to uracil, whereas methylated D1 lymphadenectomy, availability of paraffin-embedded spec- cytosine nucleotides remains unchanged. The Intergen CpGe- imens of the primary tumor, and complete follow-up informa- nome DNA modification kit (Intergen, Purchase, NY) was used tion. for the bisulfite modification procedure (24). Subsequently, the

Fig. 1 Gel electrophoresis of methylation-specific-PCR using CDH1 methylated-specific primers (A): lack of visible product in the normal mucosa (M) and presence of a product in the tumor (T); Ϫ and ϩ correspond to water and the positive control, respectively. Analysis of the same case using unmethylated-specific primers (B): a visible product is present in the normal mucosa (M) and the tumor (T); Ϫ and pl correspond to water and the DNA from peripheral lymphocytes, respectively. Experimental estimation of methylation-specific-PCR sensitivity for CDH1 promoter methylation (C). After bisulfite conversion, lymphocyte DNA with unmethylated CDH1 promoter was mixed with increasing amounts of DNA extensively methylated with SssI CpG methylase (New England Biolabs, Beverly MA). Numbers above lanes indicate the percentage of methylated DNA that is present in the mixture. U and M indicate unmethylated and methylated products, respectively. As low as 1% of methylated CDH1 promoter can be detected.

(27). For the negative control, the primary antibody was re- placed with mouse IgG. Slides with normal gastric mucosa were used as positive controls. Furthermore, positive E-cadherin staining in the adjacent noninvolved gastric mucosa served as an internal positive control. Necrotic areas and areas where the tissue had a deteriorated morphology were excluded. For the purpose of the analysis and according to the criteria used in previous studies (16, 19), the results of immunohistochemistry were classified according to the proportion of positive tumor cells. When Ͼ90% of cancer cells showed homogenous membranous E-cadherin staining, the tumor was considered positive; when 10–90% of cancer cells were positively stained, the tumor was deemed with partially reduced E-cadherin expression; tumors with Ͻ10% of positive cancer cells were considered as E-cadherin negative (Fig. 2, A and B). Statistical Methods. Statistical analyses were performed to correlate the methylation analysis with the disease-free survival and the overall survival of patients. Overall survival was measured from the date of surgery until death from any cause. Disease-free survival was measured from the date of surgery to the time of confirmed relapse or death from any cause. The Kaplan-Meier method was adopted to estimate survival curves. Differences between survival curves were studied by the log- rank test. The Cox proportional hazards model was used to assess the prognostic importance of hypermethylation adjusting for the following clinicopathological features, age, gender, tumor size and location, degree of lymph nodal involvement, tumor stage, and hypermethylation. Contingency tables were analyzed by the ␹2 test. All of the values were two-sided, and statistical significance was defined as P Ͻ 0.05.

RESULTS Fig. 2 Examples of immunohistochemistry for E-cadherin expression. The study population consisted of 73 patients with surgi- Positive homogeneous membranous staining (A) and negative staining cally resected, node-positive, diffuse gastric cancer who under- (B). went radical surgery between June 1994 and January 1998 (Table 1). During the follow-up (range, 63–108 months), 39

methylation-specific PCR (MS-PCR) was performed by adding the bisulfite-modified DNA to PCR reactions and using specific Table 1 Characteristics of the 73 patients included in the study and primers for either the methylated or the unmethylated CDH1 distribution of data in 40 methylated cases and 33 unmethylated cases promoter. Primers and conditions for the MS-PCR have been Total Unmethylated Methylated P described previously (9, 25). In vitro methylated DNA (Inter- Age (years) gen) was used as a positive control for methylation, and water Ͻ45 years 24 (33%) 15 (21%) 9 (12%) 0.04 and DNA from peripheral lymphocytes of healthy individuals Ն45 years 49 (67%) 18 (25%) 31 (42%) Sex were used as negative controls. The results of the MS-PCR were Male 40 (54%) 14 (19%) 26 (36%) NS scored when there was a clearly visible band on the gel with the Female 33 (46%) 19 (26%) 14 (19%) methylated primers (Fig. 1A) and the unmethylated primers Tumor site (Fig. 1B). Electrophoresis results were interpreted by two inde- Cardia 10 (14%) 5 (7%) 5 (7%) NS pendent investigators, and in case of discrepancy, the opinion of Noncardia 63 (86%) 28 (38%) 35 (48%) Stage grouping a third investigator was sought. All of the PCRs were repeated Stage II 30 (41%) 15 (20%) 15 (20%) NS twice, and a random sample of cases underwent external repe- Stage IIIA-B 43 (59%) 18 (25%) 25 (35%) tition of methylation analysis to ensure the reproducibility of the Outcome results (B. H., H. M., and P. G.). Disease free 34 (47%) 22 (30%) 12 (16%) 0.002 Relapsed 39 (53%) 11 (15%) 28 (39%) Immunohistochemistry. The avidin-biotin complex per- Immunohistochemistry oxidase method was used for immunostaining (26), and the Cadherin positive 30 (41%) 25 (35%) 5 (7%) Ͻ.0001 anti-E-cadherin monoclonal antibody (clone HECD-1; Zymed Cadherin reduced/ 43 (59%) 8 (10%) 35 (48%) Laboratories, San Francisco, CA) was used for detecting the negative E-cadherin protein on sections from paraffin-embedded tissue NS, not significant.

the CpG sites of the CDH1 gene is an independent prognostic factor, we performed uni- and multivariate analysis using the Cox model. Univariate analysis revealed that location and size of the tumor, degree of lymph nodal involvement, tumor stage, and hypermethylation were significant factors for both disease- free and overall survival. Multivariate analysis with factors proven to be significant in the univariate analysis confirmed that degree of lymph nodal involvement, tumor stage, and hypermethylation were independent prognostic factors for both disease-free and overall survival (Table 2). E-cadherin Immunohistochemistry. Immunohistochem- istry showed 30 cases with positive E-cadherin expression (41%), 10 cases with partially reduced E-cadherin expression (14%), and 33 cases with lack of E-cadherin expression (45%). A significant association was found between the results of immunohistochemistry and the CDH1 promoter methylation status (Table 1). Also, patients with reduced or lost E-cadherin expression showed worse event-free survival and overall survival than patients with preserved protein expression (data not shown).

DISCUSSION The results of the present investigation should be considered in a clinical perspective. Over the last 3 decades, demethylating agents for the treatment of solid tumors have been tested in Phase II studies with disappointing results. These trials showed promising response rates and durable responses in hematological malignancies, whereas low response rates and sta- bilization of disease, at most, were observed in solid neoplasms (9, 22). Studies with first-generation demethylating drugs such as azacytidine were started in the 1980s (22), when the knowledge on hypermethylation of tumor-related genes and methods for its assessment were largely unknown. Today, we know that

Fig. 3 A, disease-free survival curves of patients with and without hypermethylation in the CDH1 promoter region. B, survival curves of patients with and without hypermethylation in the CDH1 promoter Table 2 Cox multivariate analysis for disease-free and overall region. survival in the 73 patients RRa 95% CI P patients had tumor recurrence, and 35 patients had died at the Disease-free survival Tumor size (Ͻ50 mm vs. —— NS time of analysis. All of the patients had received palliative Ն50 mm) chemotherapy with fluorouracil and folinic acid (16 patients); Number of positive nodes 2.96 2.0–4.38 Ͻ0.001 fluorouracil, folinic acid, and cisplatin (25 patients); fluoroura- (Յ6 vs. Ͼ6) cil, folinic acid, cisplatin, and epirubicin (21 patients); and Tumor location —— NS (cardia vs. noncardia) fluorouracil, folinic acid, cisplatin, and mitomycin (11 patients). Tumor stage 3.24 2.35–4.46 Ͻ0.001 CDH1 promoter hypermethylation was found in 40 cases (II vs. IIIA–B) (54%), and it was not detected in the remaining 33 cases (46%). Hypermethylated vs. 2.28 1.41–3.71 Ͻ0.001 In patients aged Ͼ45 years there was a higher frequency of unmethylated hypermethylated tumors than in younger patients (Table 1). Overall survival Tumor size (Ͻ50 mm vs. —— NS A significant association was found between the methylation Ն50 mm) status in the CDH1 promoter region, and the distribution of Number of positive nodes 2.11 1.14–3.93 0.009 disease-free and relapsed patients (Table 1). (Յ6 vs. Ͼ6) In patients with and without hypermethylation, the 5-year Tumor location —— NS (cardia vs. noncardia) event-free survival rate was 30% and 62%, respectively, and the Tumor stage 2.55 1.53–4.25 0.003 5-year overall survival rate was 35% and 67%, respectively. As (II vs. IIIA–B) shown in Fig. 3, the differences between groups in both event- Hypermethylated vs. 1.94 1.23–3.06 0.004 free survival and overall survival rates were statistically signif- unmethylated icant (P Ͻ 0.001). To determine whether hypermethylation in a RR, relative risk; CI, confidence interval; NS, not significant.

hypermethylation is a relevant mechanism for silencing tumor snail; Refs. 31, 32), post-translational modification by direct or suppressor genes, but it does not occur at the same frequency in indirect phosphorylation of adherens junction components (e.g., all of the human neoplasms (7). Hematological malignancies B-catenin), or receptor tyrosine kinase-associated endocytosis may have particularly high levels of CpG island methylation and degradation of E-cadherin (33, 34). In the clinical setting, (28), whereas solid tumors show variable methylation profiles unsuccessful activity of demethylating therapies targeted to (7, 29). These data may partially explain the erratic results from CDH1 might be observed in cases with partial CDH1 promoter early clinical trials with demethylating drugs. In addition, they hypermethylation and/or silencing of E-cadherin due to other suggest that demethylating drugs may be more effective in molecular mechanisms. patients in whom hypermethylation has silenced one or more Additional concerns for interpreting results and widening critical tumor suppressor genes. applications of hypermethylation in solid tumors are related to Thus far, investigations on CDH1 promoter hypermethyla- the fact that the best method for its assessment has not been tion in gastric carcinomas have aimed at elucidating the effect of defined yet, and today, it remains a high-cost, investigational this event on tumorigenesis. In the present study, we report the assay. Under optimal experimental conditions (Fig. 1C), a sen- first evidence that CDH1 promoter hypermethylation has a sitive MS-PCR should be able to detect low numbers of meth- strong prognostic role in patients with diffuse gastric cancer. ylated tumor cells (i.e., 1–5%), which could be sufficient, after This finding is consistent with immunohistochemistry data from their selection and expansion, to influence the natural history of retrospective studies, which indicated the unfavorable outcome the disease. When used in retrospective studies, the analysis of of gastric cancer patients whose tumors showed down-regula- tumor methylation necessitates DNA isolation from archival, tion of E-cadherin expression (14–20). In the largest of these paraffin-embedded material, and even under steady MS-PCR studies involving 413 cases, Gabbert et al. (16) found that conditions a variable sensitivity of the test may occur. patients with E-cadherin-negative tumors had significantly In conclusion, the present data support the adverse prog- worse 3- and 5-year survival rates than patients with E-cadherin- nostic effect of CDH1 promoter hypermethylation in diffuse positive tumors. Our observation, together with the remarkable gastric cancer and emphasize the potential clinical relevance of frequency (40%–80%) of this epigenetic change in sporadic additional investigations in this field (35). This information can diffuse gastric carcinomas (2), suggest an attractive setting for be relevant to future studies with novel demethylating drugs and testing novel demethylating agents. for identifying high-risk gastric cancer patients who could ben- An alternative explanation for our observation of a corre- efit from adjuvant chemotherapy. The role of CDH1 hyper- lation between CDH1 methylation and survival is that CDH1 methylation in normal gastric mucosa (36), in precancerous promoter hypermethylation is a surrogate marker for CpG island gastric lesions (37, 38), in patients aged Ͼ45 years (36), and methylation in multiple tumor suppressor genes (methylator with concomitant Helicobacter pylori infection (39) may repre- phenotype), which cooperatively influence tumor behavior. sent additional fields of investigation for chemopreventive strat- However, regardless of the number and identity of any genes egies aiming at the preservation of the E-cadherin function. that may be methylated in parallel with CDH1, it is well estab- lished that E-cadherin down-regulation alone will contribute significantly to the process of tumor progression (30). ACKNOWLEDGMENTS Assessing hypermethylation in multiple genes for prognos- We thank Dr. Barry Iacopetta for helpful comments on the article. tic purposes is costly and time consuming, whereas a single determination in CDH1 in diffuse gastric carcinomas may iden- REFERENCES tify patients with poor prognosis and be relevant to the decision 1. Christofori G, Semb H. The role of the cell-adhesion molecule making in clinical practice. Immunohistochemistry for E- E-cadherin as a tumour-suppressor gene. Trends Biochem Sci 1999;24: cadherin expression may also represent a convenient prognostic 73–6. marker, and possibly, a surrogate indicator for the presence of 2. Graziano F, Humar B, Guilford P. The role of the E-cadherin gene hypermethylation in CDH1. However, the interpretation of E- (CDH1) in diffuse gastric cancer susceptibility: from the laboratory to cadherin staining may be too ambiguous and show insufficient clinical practice. Ann Oncol 2003;14:1705–13. correlation with the CDH1 methylation status (9–11) to be used 3. Guilford P, Hopkins J, Harraway J, et al. Cadherin germline muta- for this purpose. tions in familial gastric cancer. Nature 1998;392:402–5. Our combined analysis of CDH1 hypermethylation and 4. Berx G, Becker KF, Hofler H, van Roy F. Mutations of the human E-cadherin (CDH1) gene. Hum Mutat 1998;12:226–37. E-cadherin immunohistochemistry showed good correspond- 5. Becker KF, Hofler H. Frequent somatic allelic inactivation of the ence between the two methods in 40 cases, but 5 methylated E-cadherin gene in gastric carcinomas. J Natl Cancer Inst 1995;87: tumors maintained E-cadherin expression and 8 unmethylated 1082–4. cases showed reduced or negative E-cadherin expression. This 6. Knudson AG. Two genetic hits (more or less) to cancer. Nat Rev discordance has been observed in previous studies (9, 11–13). Cancer 2001;1:157–62. The few methylated cases that maintained E-cadherin immuno- 7. Santini V, Kantarjian HM, Issa JP. Changes in DNA methylation in reactivity may be explained by incomplete methylation of the neoplasia: pathophysiology and therapeutic implications. Ann Intern Med 2001;134:573–86. CDH1 promoter or the occurrence of methylation on a single 8. Herman JG, Baylin SB. Gene silencing in cancer in association with allele. The observation of reduced immunoreactivity in the promoter hypermethylation. N Engl J Med 2003;349:2042–54. absence of CDH1 hypermethylation is consistent with E- 9. Tamura G, Yin J, Wang S, et al. -Cadherin gene promoter hyper- cadherin down-regulation through other mechanisms including methylation in primary human gastric carcinomas. J Natl Cancer Inst mutations (4, 5), transcriptional repression of CDH1 (e.g., via 2000;92:569–73.

10. Grady WM, Willis J, Guilford PJ, et al. Methylation of the CDH1 25. Graff JR, Herman JG, Myohanen S, Nelkin BD, Baylin SB, Vertino promoter as the second genetic hit in hereditary diffuse gastric cancer. PM. Mapping patterns of CpG islands methylation in normal and Nat Genet 2000;26:16–7. neoplastic cells implicates both upstream and downstream regions in de 11. Machado JC, Oliveira C, Carvalho R, et al. E-cadherin gene novo methylation. J Biol Chem 1997;272:22322–9. (CDH1) promoter methylation as the second hit in sporadic diffuse 26. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase com- gastric carcinoma. Oncogene 2001;20:1525–8. plex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 12. Graziano F, Antolini ML, Mari D, Bearzi I, Silva R, Testa E, Lai V, 1981;29:577–80. Cascinu S. E-cadherin gene (CDH1) promoter hypermethylation in 27. Shimoyama Y, Hirohashi S, Hirano S, et al. Cadherin cell-adhesion gastric carcinomas. A new target for the treatment of patients with the molecules in human epithelial tissues and carcinomas. Cancer Res diffuse histotype? Proc Am Soc Clin Oncol 2003;22:263, 1055a. 1989;49:2128–33. 13. Oue N, Motoshita J, Yokozaki H, et al. Distinct promoter hyper- 28. Rush LJ, Plass C. Alterations of DNA methylation in hematologic methylation of p16INK4a, CDH1 and RAR-beta in intestinal, diffuse- malignancies. Cancer Lett 2002;185:1–12. adherent and diffuse-scattered type gastric carcinomas J Pathol 2002; 29. Paz MF, Fraga MF, Avila S, et al. systematic profile of DNA 198:55–9. methylation in human cancer cell lines. Cancer Res 2003;63:1114–21. 14. Shun CT, Wu MS, Lin JT, et al. An immunohistochemical study of 30. Guilford P. E-cadherin downregulation in cancer: fuel on the fire? E-cadherin expression with correlations to clinicopathological features Mol Med Today 1999;63:172–7. in gastric cancer. Hepatogastroenterology 1998;45:944–9. 31. Batlle, E, Sancho, E, Franci, C, et al. The transcription factor snail 15. Jawhari A, Jordan S, Poole S, Browne P, Pignatelli M, Farthing MJ. is a repressor of E-cadherin gene expression in epithelial tumour cells Abnormal immunoreactivity of the E-cadherin-catenin complex in gas- Nat Cell Biol 2000;2:84–9. tric carcinoma: relationship with patient survival. Gastroenterology 32. Cano A, Perez-Moreno MA, Rodrigo I, et al. The transcription 1997;112:46–54. factor snail controls epithelial-mesenchymal transitions by repressing 16. Gabbert HE, Mueller W, Schneiders A, et al. Prognostic value of E-cadherin expression. Nat Cell Biol 2000;2:76–83. E-cadherin expression in 413 gastric carcinomas. Int J Cancer 1996;69: 33. Kamei T, Matozaki T, Sakisaka T, et al. Coendocytosis of cadherin 184–9. and c-Met coupled to disruption of cell-cell adhesion in MDCK cells— 17. Shino Y, Watanabe A, Yamada Y, et al. Clinicopathologic evalu- regulation by Rho, Rac and Rab small G proteins. Oncogene 1999;18: ation of immunohistochemical E-cadherin expression in human gastric 6776–84. carcinomas. Cancer 1995;76:2193–201. 34. St Croix B, Sheehan C, Rak JW, Florenes VA, Slingerland JM, 18. Yonemura, Y. Ninomiya I, Kaji M, et al. Decreased E-cadherin Kerbel, RS. E-cadherin-dependent growth suppression is mediated by expression correlates with poor survival in patients with gastric cancer. the cyclin-dependent kinase inhibitor p27(KIP1). J Cell Biol 1998;142: Anal Cell Pathol 1995;8:177–90. 557–71. 19. Joo YE, Park CS, Kim HS, Choi SK, Rew JS, Kim SJ. Prognostic 35. Van Rijnsoever M, Elsaleh H, Joseph D, McCaul K, Iacopetta B. significance of E-cadherin/Catenin complex expression in gastric can- CpG islands methylator phenotype is an independent predictor of sur- cer. J Korean Med Sci 2000;15:655–66. vival benefit from 5-fluorouracil in stage III colorectal cancer. Clin Cancer Res 2003;9:2898–903. 20. Yonemura Y, Endou Y, Kimura K, et al, Sasaki T. Inverse expres- 36. Waki T, Tamura G, Tsuchiya T, Sato K, Nishizuka S, Motoyama T. sion of S100A4 and E-cadherin is associated with metastatic potential in Promoter methylation status of E-cadherin, hMLH1, and p16 genes in gastric cancer Clin Cancer Res 2000;6:4234–42. nonneoplastic gastric epithelia. Am J Pathol 2002;161:399–403. 21. Karpf AR, Jones DA. Reactivating the expression of methylation 37. To KF, Leung WK, Lee TL, et al. Promoter hypermethylation of silenced genes in human cancer. Oncogene 2002;21:5496–503. tumor-related genes in gastric intestinal metaplasia of patients with and 22. Goffin J, Eisenhauer, E. DNA. methyltransferase inhibitors-state of without gastric cancer. Int J Cancer 2002;20:623–8. the art. Ann Oncol 2002;13:1699–716. 38. Kang GH, Lee HJ, Hwang KS, Lee S, Kim JH, Kim JS. Aberrant 23. Cheng JC, Matsen CB, Gonzales FA, et al. Inhibition of DNA CpG island hypermethylation of chronic gastritis, in relation to aging, methylation and reactivation of silenced genes by zebularine. J Natl gender, intestinal metaplasia, and chronic inflammation. Am J Pathol Cancer Inst 2003;95:399–409. 2003;163:1551–6. 24. Lee TL, Leung WK, Chan MW, et al. Detection of gene promoter 39. Chan AO, Lam SK, Wong BC, et al. Promoter methylation of hypermethylation in the tumor and serum of patients with gastric car- E-cadherin gene in gastric mucosa associated with Helicobacter pylori cinoma. Clin Cancer Res 2002;8:1761–6. infection and in gastric cancer. Gut 2003;52:502–6.

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Francesco Graziano, Federica Arduini, Annamaria Ruzzo, et al.

Clin Cancer Res 2004;10:2784-2789.

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