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Cancer Epidemiology Biomarkers & Prevention Vol. 14, 2987-2989, December 2005
© 2005 American Association for Cancer Research

Short Communication

The Angiotensin I–Converting Enzyme Gene Insertion/Deletion Polymorphism Is Linked to Early Gastric Cancer

Matthias P.A. Ebert1,2, Uwe Lendeckel3, Sabine Westphal4, Jutta Dierkes4, Jürgen Glas6, Christian Folwaczny6, Albert Roessner5, Manfred Stolte7, Peter Malfertheiner2 and Christoph Röcken5

1 Fred Hutchinson Cancer Center, Public Health Sciences, Seattle, Washington; 2 Department of Gastroenterology, Hepatology and Infectious Diseases; Institutes of 3 Experimental Internal Medicine, 4 Clinical Chemistry, and 5 Pathology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany; 6 Medizinische Poliklinik der Ludwig-Maximilians Universität, Munich, Germany; and 7 Institute of Pathology, Klinikum Bayreuth, Bayreuth, Germany

Requests for reprints: Matthias Ebert, Department of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke University Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany. Phone: 49-391-672-1520; Fax: 49-391-672-90054; E-mail: matthias.ebert{at}medizin.uni-magdeburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The insertion/deletion polymorphism of the angiotensin I–converting enzyme (ACE) gene has recently been linked to the pathogenesis and progression of human cancers. Using genomic DNA from 88 patients with early gastric cancer confined either to mucosa (pT1a) or submucosa (pT1b), we assessed the insertion (I) and deletion (D) polymorphism by PCR analysis and compared it with a large noncancer control population (n = 145). In the noncancer control group, the II genotype was observed in 33 (23%) individuals, whereas the ID and DD genotypes were found in 72 (50%) and 40 (27%) individuals, respectively. Interestingly, in the cancer group, we found the II genotype in six (7%) patients and the ID genotype in 46 (52%) individuals, whereas the DD genotype was observed in 36 (41%) individuals (P = 0.0034). Accordingly, the odds ratio for the II genotype was 0.20 [95% confidence interval (95% CI), 0.08-0.54; P = 0.009] and 0.55 for the ID/II genotype (95% CI, 0.31-0.96; P = 0.044) using the high-activity genotype DD as the reference category. No correlation was found among tumor type, tumor stage, the presence of Helicobacter pylori, and the ACE genotype. Our study provides further evidence that the ACE insertion/deletion gene polymorphism may be linked to the development of early gastric cancer. (Cancer Epidemiol Biomarkers Prev 2005;14(12):2987–9)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Gastric cancer is the second most common cancer worldwide, and mortality ranks only second to lung cancer (1, 2). Most cancers are diagnosed in advanced stages, in which curative resection is impossible. This leads to poor survival rates for the vast majority of gastric cancer patients (1, 2). Localized gastric cancers of stage I are diagnosed in <10% of the cases and ~10% to 20% of T1 cancers already present with lymph node metastases, which is the most important prognostic factor in this malignancy (3). Recently, the angiotensin I–converting enzyme (ACE), which is a cell surface zinc metallopeptidase, has been linked to cancer development (4, 5). A polymorphism of the ACE gene, comprising an insertion (I) or deletion (D) of a 287-bp DNA fragment in intron 16, leads to variances in ACE expression and activity in blood and tissues of affected individuals (5, 6). Previous studies have linked this polymorphism to various malignancies (4, 5, 7, 8), and recently, we also reported an increased number of lymph node metastases in individuals with gastric cancer harboring the DD genotype, indicating that this polymorphism may also be linked to advanced gastric cancer (9). The role of this polymorphism in the early stages of gastric cancer is thus far unknown.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Samples
Samples from 88 patients with noncardia cancer were obtained by gastric resection (10). Tissue samples were fixed in 10% neutralized formalin and embedded in paraffin for histologic assessment. Forty-one of these cancers were confined to the mucosa (pT1a), whereas 47 reached the submucosa (pT1b). Regional lymph node metastases were observed in five cases; however, in none of the cases, distant metastases were found. Tumors were classified according to the Lauren classification (11). By histologic assessment, Helicobacter pylori was identified in 77 of 88 patients. Blood samples were also obtained from a further set of 145 healthy control patients without gastric cancer as previously reported and described below (9). All cancer patients and healthy controls were of Caucasian ethnicity. The healthy controls were recruited for an epidemiologic study assessing the role of gene polymorphisms on general health and nutritional status during 2001. Inclusion criteria were no history of neoplasia at any site, no history of gastrointestinal disease or surgery, and no history of cardiovascular disease. The healthy controls were examined by a physician: weight and height were recorded, blood pressure was measured, and a venous blood sample was taken. They filled in a questionnaire on lifestyle habits, drug use, and medical history. The general characteristics of the controls are presented in Table 1. General medical characteristics of this group of individuals were as follows (mean ± SD): body mass index (kg/m2), 26.2 ± 3.7; systolic blood pressure (mm Hg), 140 ± 20; diastolic blood pressure (mm Hg), 82 ± 11. This study was carried out in accordance with the guidelines of the Ethics Committee of the University of Magdeburg, and all data were encoded to ensure patient protection.


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  		Table 1. Patient characteristics

 
DNA Analysis
Genomic DNA from control patients was extracted from peripheral blood mononuclear cells using the QIAamp DNA blood kit (Qiagen, Chatsworth, CA). In the cancer patients, genomic DNA was extracted from paraffin-embedded tissue sections, which were histologically confirmed to be devoid of any infiltrating cancer cells using the E.Z.N.A. Tissue DNA Mini kit (EQLAB Biotechnologie GmbH, Erlangen, Germany; ref. 9). The ACE genotype was then determined by PCR as described recently with some modifications (9). A typical 50-µL reaction mixture consisted of 25 µL HotStarTaq Master Mix (Qiagen), 100 ng of genomic DNA, 250 pmol primer, and 5% (v/v) DMSO. An initial 15-minute denaturation at 95°C was followed by 40 cycles of 1 minute at 64°C, 1 minute at 72°C, and 0.6 minute at 94°C. Amplified ACE gene fragments were separated on 1.6 % agarose gels and visualized by ethidium bromide staining. D or I alleles were identified by the presence of 190- or 490-bp fragments, respectively, when ACE-1, 5'-CTGGAGACCACTCCCATCCTTTCT-3' was used together with ACE-2, 5'-GATGTGGCCATCACATTCGTCAGAT-3'. DNA isolated from paraffin-embedded tissue samples often exhibits a small average size. This could result in false-negative results, especially in case of the larger I allele. Therefore, the presence or absence of this allele was confirmed by an additional PCR using the ACE-1 primer together with ACE-2.2 (5'-AGCTACTTGGGAGGCTGAGG-3'), which gives a 188-bp PCR fragment that is indicative for the presence of the I allele (9, 12).

Statistical Analysis
Statistical analysis was done using the {chi}2 test and the Fisher's exact test. Continuous variables were compared by means of the unpaired Student's t test. For determining the strength of the gene-cancer association, we also determined the odds ratio, the corresponding 95% confidence interval, and the Ps, using the high-activity genotype DD as the reference category. P < 0.05 was considered significant. The Hardy-Weinberg equilibrium was assessed in the control group by the {chi}2 test (13).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The clinical characteristics of the gastric cancer and noncancer control populations are given in Table 1. Overall, we found no significant difference in the distribution of gender and mean age between the two groups. In the control group, the II genotype was observed in 33 (23%) individuals, whereas the ID and DD genotypes were observed in 72 (50%) and 40 (27%) individuals, respectively. The distribution of the ACE genotypes in the control group did not differ from the distribution predicted by the Hardy-Weinberg equilibrium (Table 2). In the cancer group, we found the II genotype in only six (7%) patients and the ID genotype in 46 (52%), whereas the DD genotype was present in 36 (41%). The DD genotype was significantly more frequent in gastric cancer patients compared with the noncancer group (P = 0.0034). To assess the strength of the gene-cancer association, we also determined the odds ratio and the corresponding 95% confidence interval using the high-activity genotype DD as the reference category. Accordingly, the odds ratio for the II genotype was 0.20 (95% confidence interval, 0.08-0.54; P = 0.009) and 0.55 for the ID/II genotype (95% confidence interval, 0.31-0.96; P = 0.044) compared with the high-activity genotype DD (Table 3). Next, we compared the ACE genotype with various clinicopathologic variables of the cancer patients as detailed in Table 2. However, univariate analyses showed that the ACE genotype did not correlate with the type of gastric cancer, according to the Lauren classification, the T category (pT1a versus pT1b), the presence of lymph node metastases, or H. pylori infection (Table 2).


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  		Table 2. Distribution of the ACE gene polymorphism in cancer and control patients

 

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  		Table 3. Risk assessment of ACE genotypes in gastric cancer patients

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Identifying early gastric cancers and screening of high-risk individuals are the most viable options in the effort to improve cancer diagnosis and prognosis (1, 2). The recent introduction of chromoendoscopy has improved the identification of early gastric cancer restricted to either mucosa or submucosa, which are accessible to endoscopic resection (14). However, most patients are still diagnosed in advanced stages with lymph node or distant metastases prohibiting curative resection. Therefore, the biological mechanisms underlying gastric carcinogenesis and the initial stages of cancer progression as well the molecular basis for increased cancer risk need to be further elucidated.

The pathogenesis and progression of gastric cancer is a multifactorial process, which includes increased cell proliferation, dedifferentiation, angiogenesis, and degradation of the extracellular matrix. Recently, ACE has been implicated in cancer biology (4, 5). Angiotensin II, the major effector of the renin-angiotensin system, acts as a potent vasoconstrictor and raises blood pressure; however, it also modulates tumor cell proliferation, migration, angiogenesis, and metastasis formation (15). Accordingly, a polymorphism of the ACE gene, which leads to increased activity of ACE in blood and tissues among individuals harboring the DD genotype, has been associated with various cancers, including prostate and breast cancer (7, 8). Recently, we also showed a significant association among the ACE polymorphism, lymph node metastases, and the Unio Internationale Contra Cancrum tumor stage in patients with gastric cancer (9). However, the role of this gene polymorphism in the early phases of gastric carcinogenesis remained unknown. There is mounting evidence that the genetic background of early-stage gastric cancers differs considerably from advanced gastric cancers (1, 10). Therefore, we studied the distribution of the ACE genotypes in a well-characterized set of 88 patients with gastric cancers confined either to the mucosa (pT1a) or submucosa (pT1b) and compared the distribution with a set of healthy controls. Although the genotype distribution in the control group was comparable with other studies done in Germany (16, 17), the DD genotype was significantly more frequent in this cancer group compared with a noncancer population. All other clinicopathologic variables proved to be independent of the ACE genotype.

Although in a previous investigation we were unable to find any association between T category and ACE genotype, probably because only 15 patients of the previous series had suffered from pT1 tumors (9), we found a significant association with pT1 tumors in this study. The small sample size and the statistical significance based on the sample sizes limit further interpretation of our findings. In addition, some of the samples used for the genetic analysis were derived from gastric tissue. Thus, the genotype of the cases may also reflect loss of heterozygosity, although the genotype distribution indicates that loss of heterozygosity may be unlikely. Nonetheless, the former and present observations point to a role of the renin-angiotensin system for the pathogenesis and biology of gastric cancer. In addition, the renin-angiotensin system and ACE may exert different effects in early and advanced gastric cancer. In the early phases, ACE may promote carcinogenesis, whereas in later stages, it contributes to nodal spread. In this respect, it is interesting to note that ACE was expressed in intestinal metaplasia in 28 of 29 (97%) cases (9). In contrast to our previous study, we did not find an association between ACE genotype and N category. However, lymph node metastases were only observed in five patients, which were too few to be informative. Nonetheless, three of them harbored the DD genotype.

Although recent studies have shown the direct involvement of ACE in the biology and progression of human cancers (7-9), the increased frequency of the DD genotype in early gastric cancers may also have another reason. Thus, individuals with the DD genotype also carry an increased risk of developing hypertension (18), atherosclerotic cardiovascular complications (19), and diabetic nephropathy (20) due to increased ACE activity. Interestingly, several epidemiologic studies indicate that gastric cancer incidence may be increased in patients with diabetes (21). At this point, the direct biological mechanism of enhanced ACE activity in patients with the DD genotype with regard to gastric cancer development is not known. ACE may exert direct tumor promoting functions, such as promoting angiogenesis, cell proliferation, and tumor growth (7-9); however, this effect may also be related to indirect actions, such as its proinflammatory role (15) or the development of other nonmalignant conditions that may carry an increased cancer risk (21).

In conclusion, individuals with the DD genotype may carry an increased risk of developing gastric cancer. Thus, this study further supports previous observations indicating that ACE may play a role in the pathogenesis and progression of gastric cancer.


   Footnotes
 
Grant support: Deutsche Forschungsgemeinschaft Heisenberg-Programme grant Eb 187/5-1 and Land Sachsen-Anhalt (M. Ebert).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/20/05; revised 9/12/05; accepted 9/22/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  7. Medeiros R, Vasconcelos A, Costa S, et al. Linkage of angiotensin I-converting enzyme gene insertion/deletion polymorphism to the progression of human prostate cancer. J Pathol 2004;202:330–5.[CrossRef][Medline]
  8. Haiman CA, Henderson SO, Bretsky P, Kolonel LN, Henderson BE. Genetic variation in angiotensin I-converting enzyme (ACE) and breast cancer risk: the multiethnic cohort. Cancer Res 2003;63:6984–7.[Abstract/Free Full Text]
  9. Rocken C, Lendeckel U, Dierkes J, et al. The number of lymph node metastases in gastric cancer correlates with the angiotensin I-converting enzyme gene insertion/deletion polymorphism. Clin Cancer Res 2005;11:2526–30.[Abstract/Free Full Text]
  10. Glas J, Torok HP, Schneider A, et al. Allele 2 of the interleukin-1 receptor antagonist gene is associated with early gastric cancer. J Clin Oncol 2004;22:4746–52.[Abstract/Free Full Text]
  11. Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification. Acta Pathol Microbiol Scand 1965;64:31–49.[Medline]
  12. Yoshida H, Mitarai T, Kawamura T, et al. Role of the deletion of polymorphism of the angiotensin converting enzyme gene in the progression and therapeutic responsiveness of IgA nephropathy. J Clin Invest 1995;96:2162–9.[Medline]
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  16. Klemm T, Mittrach-Schorin S, Neumann S, et al. No association between the angiotensin-converting-enzyme gene insertion/deletion polymorphism and the occurrence of macroangiopathy in patients with diabetes mellitus type 2. Horm Metab Res 2003;35:43–7.[Medline]
  17. von Depka M, Czwalinna A, Wermes C, et al. The deletion polymorphism in the angiotensin-converting enzyme gene is a moderate risk factor for venous thromboembolism. Thromb Haemost 2003;89:847–52.[Medline]
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