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Correlation between microsatellite instability and metachronous disease recurrence after endoscopic mucosal resection in patients with early stage gastric carcinoma

2001; Wiley; Volume: 91; Issue: 2 Linguagem: Inglês

10.1002/1097-0142(20010115)91

1097-0142

Akira Kawamura, Kyoichi Adachi, Shunji Ishihara, Tomoko Katsube, Toshiharu Takashima, Mika Yuki, Kazutoshi Amano, Ryo Fukuda, Yukimasa Yamashita, Yoshikazu Kinoshita,

Gastrointestinal Tumor Research and Treatment

CancerVolume 91, Issue 2 p. 339-345 Original ArticleFree Access Correlation between microsatellite instability and metachronous disease recurrence after endoscopic mucosal resection in patients with early stage gastric carcinoma Akira Kawamura M.D., Corresponding Author Akira Kawamura M.D. kadachi@shimane-med.ac.jp Department of Internal Medicine II, Shimane Medical University, Shimane, Japan Fax: +81-853-20-2187Department of Internal Medicine II, Shimane Medical University, 89-1 Enya-cho, Izumo-shi, Shimane 693-8501, Japan===Search for more papers by this authorKyoichi Adachi M.D., Kyoichi Adachi M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorShunji Ishihara M.D., Shunji Ishihara M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorTomoko Katsube M.D., Tomoko Katsube M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorToshiharu Takashima M.D., Toshiharu Takashima M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorMika Yuki M.D., Mika Yuki M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorKazutoshi Amano M.D., Kazutoshi Amano M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorRyo Fukuda M.D., Ryo Fukuda M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorYukimasa Yamashita M.D., Yukimasa Yamashita M.D. Department of Internal Medicine, Miki City Hospital, Hyougo, JapanSearch for more papers by this authorYoshikazu Kinoshita M.D., Yoshikazu Kinoshita M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this author Akira Kawamura M.D., Corresponding Author Akira Kawamura M.D. kadachi@shimane-med.ac.jp Department of Internal Medicine II, Shimane Medical University, Shimane, Japan Fax: +81-853-20-2187Department of Internal Medicine II, Shimane Medical University, 89-1 Enya-cho, Izumo-shi, Shimane 693-8501, Japan===Search for more papers by this authorKyoichi Adachi M.D., Kyoichi Adachi M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorShunji Ishihara M.D., Shunji Ishihara M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorTomoko Katsube M.D., Tomoko Katsube M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorToshiharu Takashima M.D., Toshiharu Takashima M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorMika Yuki M.D., Mika Yuki M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorKazutoshi Amano M.D., Kazutoshi Amano M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorRyo Fukuda M.D., Ryo Fukuda M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this authorYukimasa Yamashita M.D., Yukimasa Yamashita M.D. Department of Internal Medicine, Miki City Hospital, Hyougo, JapanSearch for more papers by this authorYoshikazu Kinoshita M.D., Yoshikazu Kinoshita M.D. Department of Internal Medicine II, Shimane Medical University, Shimane, JapanSearch for more papers by this author First published: 23 January 2001 https://doi.org/10.1002/1097-0142(20010115)91:2 3.0.CO;2-2Citations: 11 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract BACKGROUND Since endoscopic treatment was first evaluated and established as a treatment for patients with early stage gastric carcinoma, metachronous disease recurrence at other sites in the stomach after endoscopic treatment has become a major problem. METHODS A retrospective case–control study was conducted on 10 patients with metachronous recurrence of gastric carcinoma after undergoing successful endoscopic mucosal resection (EMR) therapy for early stage gastric carcinoma and on 14 patients without recurrence. Gastric mucosal tissues obtained during the initial EMR were dissected, and DNA samples from the tumor tissue and surrounding nonneoplastic mucosa were extracted separately. Microsatellite instability (MSI) was tested in five microsatellite markers (D2S137, D3S1067, TP53, TGFβRII, and BAX). The authors also looked for K-ras codon 12 point mutations in the tumor tissues. In addition, immunohistochemical staining was done to test for the presence of proliferating cell nuclear antigen (PCNA), p53, hMSH2, and hMLH1 in the mucosal tissues. Finally, the correlation between the presence or absence of metachronous recurrence and the characteristics of the primary tumor (MSI, K-ras, p53, etc.) were investigated. RESULTS Three of 10 patients with recurrent disease showed MSI in more than two microsatellite markers among 3–5 investigated site (MSI-H), whereas none of the patients with nonrecurrent disease did so. There was no significant correlation between metachronous recurrence after EMR and immunohistochemical staining reactions, including those for PCNA, p53, hMSH2, and hMLH1. None of the patients showed K-ras mutations. CONCLUSIONS Thirty percent of patients with recurrent disease showed MSI-H, whereas none of the patients with nonrecurrent disease did so. Cancer 2001;91:339–45. © 2001 American Cancer Society. Since endoscopic treatment was first established to treat patients with early stage gastric carcinoma and has become widely used, metachronous disease recurrence at other sites in the stomach after endoscopic treatment has become a major problem.1-8 If it was possible to predict disease recurrence in patients at the time of initial treatment, we could follow different standards for endoscopic follow-up programs, and this would reduce medical costs. In our previous study, we showed that metachronous recurrence frequently was observed in patients with multiple synchronous lesions at the time of initial treatment.9 Recent advances in molecular biology have revealed the many genetic abnormalities in malignancies of various organs. For example, microsatellite instability (MSI), which is well known to be caused by the germline mutation of mismatch repair (MMR) genes in patients with hereditary nonpolyposis colorectal carcinoma (HNPCC), was shown to be present in 20–30% of patients with sporadic gastric carcinoma in surgically resected specimens.10-16 Furthermore, MSI was observed more frequently in patients with simultaneous multiple gastric tumors than in those with a single tumor.17, 18 In this study, we examined the correlation between metachronous disease recurrence and proliferative ability and several genetic abnormalities in the initial endoscopically treated lesions. The objective was to find a way to identify those patients with gastric tumors that are likely to recur after endoscopic treatment. MATERIALS AND METHODS Patients One hundred fifty-four patients with early stage gastric carcinoma were treated endoscopically at our clinic and carried out a regular endoscopic follow-up (1 month, 3 months, and 6 months and 1–8 years after the initial treatment) between 1990 and 1998. Initial screening endoscopy was performed using an electric video endoscope (GIF XQ230, Q200, or Q230; Olympus Optical Co. Ltd., Tokyo, Japan). Before endoscopic treatment, reexaminations were performed by one of five expert endoscopists (A.K., K.A., S.I., T.K., or K.A.) using indigocarmine dye spraying to detect the possible presence of other synchronous gastric lesions. Because of the possibility of microscopic synchronous tumors that may have been missed initially, we intended to regard any lesion detected within one year after initial treatment as a synchronous lesion. All synchronous lesions, however, were detected before treatment, and no cancerous lesion was detected within 1 year after the treatment in our series. Metachronous recurrence was defined as the appearance of a new gastric tumor at a different site in the stomach distant from the primary lesion at least 1 year after the initial treatment. During the follow-up period, we found 10 patients with metachronous recurrence in the stomach distant from the primary lesion. The period from the initial treatment to the detection of the new lesion ranged from 13 months to 51 months (average, 27.8 months). Table 1 shows that we selected 14 patients with nonrecurrent disease who were age- and gender-matched with the patients who had recurrent disease. The follow-up for patients with nonrecurrent disease ranged from 24 months to 61 months (average, 44.7 months). None of the patients investigated met the Amsterdam criteria for HNPCC19 according to the family histories that were collected from a review of medical records. Written, informed consent was obtained from all of the patients. Table 1. Characteristics and Follow-Up Period of Patients with Early Gastric Carcinoma Treated Endoscopically by an Endoscopic Mucosal Resection Technique Characteristic Recurrent diseaseaa In patients with recurrent disease, follow-up means the interval between initial treatment and the day the new malignancy was detected. Data are expressed as mean ± standard deviation. Nonrecurrent disease Age (yrs) 73.9 ± 9.2 68.5 ± 5.9 Gender (male:female) 5:5 9:5 Follow-up (months) 27.8 ± 8.1 44.7 ± 12.0 a In patients with recurrent disease, follow-up means the interval between initial treatment and the day the new malignancy was detected. Data are expressed as mean ± standard deviation. Immunohistochemical Staining The EMR method allows us to obtain gastric carcinoma tissue with the surrounding normal mucosa and part of the submucosa.3, 7 Tissues from the initial EMR specimens were fixed in 10% buffered formalin and embedded in paraffin. Immunohistochemical staining for the proliferating cell nuclear antigen (PCNA), p53, hMSH2 and hMLH1 in formalin fixed, paraffin embedded tissue sections was performed using the avidin-biotin complex method. Briefly, 3-μm sections were mounted on silanized slides, deparaffinized, and rehydrated through graded alcohol to water. Endogenous peroxidase activity was blocked by incubation with 3% H2O2. Sections were immersed in 10 mM sodium citrate buffer, pH 6.0, and subjected to heat-induced antigen retrieval. Sections were then treated with 10% normal goat serum for 30 minutes to block nonspecific protein binding. After antigen retrieval, the slides were incubated with one of four antibodies: anti-human PCNA antibody (PC10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), antihuman p53 antibody (clone DO-7; DAKO, Glostrup, Denmark), anti-human hMSH2 antibody (clone FE 11; Oncogene Research Products, Cambridge, MA), or antihuman hMLH1 antibody (clone G 168-728; Pharmingen, San Diego, CA). Clone FE11 is a mouse monoclonal antibody generated with a carboxy-terminal fragment of hMSH2 protein, whereas clone G168-728 was raised with full-length hMLH1 protein.20 The slides were then incubated with the secondary antibody, biotinylated antimouse immunoglobulin, for 30 minutes before being exposed to the avidin-biotin complex. The specimens were then incubated with a solution of diaminobenzidine tetrahydrochloride and hydrogen peroxide for 5–10 minutes. The extent of PCNA positivity in each tissue was evaluated by determining the percentage of positive nuclei in at least 500 malignant cells. At least 500 cells were counted from each tumor sample, and sections were graded positive for p53 expression if ≥5% of the tumor cells showed nuclear staining and negative if <5% of the tumor cell nuclei were stained.21 The normal pattern for both hMSH2 and hMLH1 was for the nucli to be positive. Tumor cells that showed negative nuclear staining with hMSH2 or hMLH1 antibody were considered to have an abnormal pattern. Genomic DNA Extraction Serial 10-μm sections of tumor tissues and nonneoplastic areas were stained with hematoxylin and eosin and carefully dissected under a microscope. Tumor tissues were selected in the area where all the epithelial cells were malignant. Nonneoplastic parts included nonneoplastic mucosa, mucosal muscular layer, and submucosa without tumor cells. DNA was extracted using the proteinase K and sodium dodecyl sulfate method. Tissues were incubated overnight at 52 °C in lytic solution, and DNA was extracted with phenol-chloroform, as described previously. Detection of the K-ras Codon 12 Point Mutation For detecting the K-ras codon 12 point mutation, we used the polymerase chain reaction dependent preferential homoduplex formation assay (PCR-PHFA) according to the method reported previously by Oka et al.22, 23 The PCR-PHFA kit was supplied by Wakunaga Pharmaceutical Co. Ltd. (Hiroshima, Japan). MSI and Loss of Heterozygosity Analysis We used the primer pairs for three microsatellite markers (D2S137, D3S1067, and TP53). All of these markers were dinucleotide repeats. The nucleotide sequences of these primers have been described previously.24 One of the paired primers was labeled with two different fluorescent dyes (i.e., rox or fam), which made it possible to detect only one strand of amplified DNA. The fluorescent-labeled primers were purchased from the Takara Shuzo Co. Ltd. (Tokyo, Japan). The conditions for fluorescent PCR were the same for all the loci examined and consisted of 35 cycles at 95 °C for 30 seconds for denaturing, at 55 °C for 30 seconds for annealing, and at 72 °C for 30 seconds for extension. PCR was performed in a 50-μL solution (10 mM Tris-HCL, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 200 μM deoxynucleotide triphosphate; 0.5 units of Taq polymerase [Takara Shuzo Co. Ltd.]; 25–50 ng genomic DNA, and 10 pmol of each primer) using a GeneAmp PCR System 9600 thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). The fluorescent PCR products were separated using 6% urea-polyacrylamide denaturing gels in a model 373A automated fluorescent DNA sequencer (Applied Biosystems, Foster City, CA). The GeneScan Analysis program (Applied Biosystems) was used for the assessment of MSI. In this study, tumors with a positive MSI in more than two microsatellite regions among three to five analyzed sites (D2S137, D3S1067, TP53, TGFβRII, and BAX) were defined as high frequency MSI (MSI-H), whereas Boland et al.25 classified tumors with more than two microsatellite regions in five sites as MSI-H. Amplification of the Target Genes (TGFβRII and BAX) Direct sequencing of the target genes (TGFβRII and BAX) was done in 4 tumors with positive MSI and 10 tumors with negative MSI in all of the microsatellite regions investigated in this study. The primers used for TGFβRII and BAX were synthesized from nucleotide positions containing (A) 10 for TGFβRII and (G) 8 for BAX. The sequences of these primers were as follows; TGFβRII; 5′-TAGAGACAGTTTGCATGAC-3′ (sense) and 5′-ATGAAGAAAGTCTCACCAGG-3′ (antisense); BAX: 5′-TTGTGGCACAGATTTGAGGA-3′ (sense) and 5′-GGGTCCAGGGCCAGCTCGGG-3′ (antisense). Statistical Analysis Statistical analysis was performed with SPSS software (version 6.1; SPSS, Inc., Chicago, IL) for Macintosh (Apple Computers, Cupertino, CA) using chi-square tests and t tests (if the data followed a normal distribution) or Mann–Whitney U tests (if the data did not follow a normal distribution); differences at P < 0.05 were considered statistically significant. RESULTS All data in this study are summarized in Table 2. The median score of the PCNA labeling index, which indicates the proliferative ability of a tumor,26, 27 was essentially the same for patients with recurrent (48%) and nonrecurrent (51%) disease. The number of patients with positive p53 nuclear immunohistochemical staining, which is the result of possible mutation of the p53 gene, was 4 of 10 patients with recurrent disease and 10 of 14 patients with nonrecurrent disease. The K-ras codon 12 point mutation was not detected in any of the patients investigated. Table 2. Molecular and Immunohistochemical Analysis of Patients with Early Gastric Carcinoma Patient Age (yrs) Gender Follow-up period (mo) PCNAaa PCNA labeling index. p53bb Presence (+) or absence (−) of overexpression of immunohistochemical staining for p53. K-rascc Presence (+) or absence (−) of the K-ras codon 12 point mutation. D2S137 D3S1067 TP53 TGFβRII (A) 10ff −, Wild type; +, 1-base pair deletion. BAX (G) 8ff −, Wild type; +, 1-base pair deletion. HMSH2gg +, Equal or more expression of immunohistochemical staining for hMLH1 or hMSH2 in tumor tissue compared with normal tissue. hMLH1gg +, Equal or more expression of immunohistochemical staining for hMLH1 or hMSH2 in tumor tissue compared with normal tissue. MSIdd Presence (+) or absence (−) of MSI. LOHee Presence (+) or absence (−) of LOH. MSI LOH MSI LOH Recurrent disease 1 75 F 51 26 − − + Homo + − + Homo − NI + + 2 84 F 46 34 − − − Homo − − − − − − + + 3 86 F 21 41 − − + − + + + − + + + − 4 66 M 29 55 + − − + + + + − − NI + + 5 82 M 17 56 + − − − − Homo − Homo − − + + 6 70 M 15 51 + − − − − − − Homo − − + + 7 63 F 32 48 − − − Homo − − − Homo NE NE + + 8 61 M 36 50 − − NI NI − Homo NI NI NE NE + + 9 70 F 13 48 − − NI NI − Homo − − NE NE + + 10 82 M 18 42 + − − Homo − − − − NE NE + + Nonrecurrent disease 11 70 F 46 23 + − − − − − − + NI − + + 12 74 F 61 59 + − − − − − − + NI − + + 13 71 F 56 52 + − − − − − − + − − + + 14 75 M 43 47 + − − Homo − Homo − + − − + + 15 75 M 42 54 − − − − − − − − − − + + 16 62 F 59 45 + − − − − − − + NE NE + + 17 72 M 24 53 + − − + + − − + − − + − 18 71 M 39 58 − − − − − − − − NE NE + + 19 64 F 33 46 − − − Homo − − − − NE NE + + 20 76 M 42 45 + − − Homo − − − + NE NE + + 21 66 M 37 50 + − − − − − − − − − + + 22 64 M 29 50 − − − Homo − − − − − − + + 23 59 M 55 53 + − − Homo − Homo − + NE NE + + 24 60 M 60 57 + − − Homo − − − − NE NE + + PCNA: proliferating cell nuclear antigen; TGF: transforming growth factor; MSI: microsatellite instability; LOH: loss of heterozygosity; F: female; M: male; Homo: homozygosity; NI: not informative; NE: not examined. a PCNA labeling index. b Presence (+) or absence (−) of overexpression of immunohistochemical staining for p53. c Presence (+) or absence (−) of the K-ras codon 12 point mutation. d Presence (+) or absence (−) of MSI. e Presence (+) or absence (−) of LOH. f −, Wild type; +, 1-base pair deletion. g +, Equal or more expression of immunohistochemical staining for hMLH1 or hMSH2 in tumor tissue compared with normal tissue. The numbers of patients with positive MSI in the D2S137, D3S1067, and TP53 regions were 2, 3, and 3 of 10 patients with recurrent disease, respectively, and 0, 1, and 0 of 14 patients with nonrecurrent disease, respectively. The numbers of patients with positive MSI for any microsatellite regions were three patients with recurrent disease and one patient with nonrecurrent disease. The number of patients with positive MSI in more than two microsatellite markers (MSI-H) was three among those with recurrent disease. Conversely, there were no patients with positive MSI in more than two microsatellite markers among those with nonrecurrent disease (P = 0.028). A single base deletion was found in 10 tracts of the TGFβRII gene and in 8 tracts of the BAX gene in only 1 of 14 patients investigated. This patient had recurrent gastric carcinoma with positive MSI in three microsatellite regions investigated. No negative hMSH2 nuclear immunohistochemical staining was found in any of the patients investigated. Only two patients with MSI, one with recurrent disease and one with nonrecurrent diseases, showed negative hMLH1 nuclear immunohistochemical staining. Therefore, hypermethylation of the promoter region in the hMSH2 and hMLH1 genes followed by negative transcription and translation of hMSH2 and hMLH128-32 may not have been responsible for the positive MSI observed in the other two patients. The numbers of patients with positive loss of heterozygosity (LOH) in the D2S137, D3S1067, and TP53 regions in informative cases were 1 of 4 (25%), 2 of 7 (28.6%), and 0 of 5, respectively, among the patients with recurrent disease. Among the patients with nonrecurrent disease, the numbers with positive LOH in the D2S137, D3S1067, and TP53 regions in informative cases were 1 of 8 (12.5%), 0 of 12, and 8 of 14 (57.1%), respectively. In TP53, the frequency of positive LOH in patients with nonrecurrent disease was significantly greater than in patients with recurrent disease (P = 0.026). DISCUSSION Endoscopic treatment for patients with early stage gastric carcinoma has been widely used for curative therapy, and its safety and effectiveness have been confirmed.1-8 Although the indications for endoscopic treatment are limited to small, well differentiated adenocarcinomas without submucosal invasion and ulceration, the prognosis of treated patients is good and is nearly equal to that of surgically treated patients.6-8 One of the most serious problems, however, is the recurrence of gastric carcinoma in other parts of the gastric mucosa, distant from the initial site.9 This probably is because endoscopic treatment excises only gastric carcinoma tissue without influencing other areas of gastric mucosa that may have a high potential for developing new tumor cells. The frequency of metachronous recurrence after endoscopic treatment is reported to be 2.5–11%.9 In addition, our previous study revealed that metachronous recurrence frequently is detected in patients with synchronous multiple lesions at the time of initial treatment.9 However, to our knowledge, the proliferative ability and genomic abnormalities of tumors were not examined with a view to predicting future disease recurrence after endoscopic treatment for patients with early stage gastric carcinoma. Recent advances in immunohistochemical staining and molecular biologic techniques have revealed that several factors contributing to the proliferative ability and genomic abnormalities of cancer malignancies in various organs can be correlated with disease stage and patient prognosis. Indeed, PCNA is known to be related to the cellular proliferation and malignant potential of tumors.26, 27 It also has been reported that there was a strong association between p53 tumor status and patient survival after receiving a diagnosis of gastric carcinoma. The 5-year survival of patients with p53-expressing tumors is poorer than that of patients with non-p53-expressing tumors.33 Furthermore, the K-ras codon 12 point mutation, which is observed frequently in pancreatic and colorectal tumors, has been reported in some differentiated types of gastric tumors.34-36 In view of this, we investigated the relation of these factors and metachronous recurrence after endoscopic treatment in patients with early stage gastric carcinoma. The PCNA labeling index, positive immunostaining for p53, and the mutation of K-ras codon 12 in initially treated lesions did not differ between patients with recurrent and nonrecurrent disease. These results suggest that there is no difference in proliferative ability and carcinogenic pathway through the K-ras mutation between patients with recurrent and nonrecurrent disease and that these markers are not useful for predicting future metachronous disease recurrence. MSI, which is well known to be caused by the germline mutation of MMR genes in patients with HNPCC, was found in 20–30% of sporadic gastric carcinoma tissues in surgically resected specimens.10-16 In addition, MSI was observed more often in patients with of simultaneous, multiple gastric tumors than in those with a single tumor.17, 18 LOH, which reflects the loss of one of two alleles, also is observed in gastric carcinoma.37 This study showed that MSI and LOH differed significantly in patients with recurrent and nonrecurrent disease. MSI was observed more frequently in patients with recurrent disease than in those with nonrecurrent disease. Furthermore, MSI-H was observed only in patients with recurrent disease. For the TP53 region, positive MSI was observed only in patients with recurrent disease, whereas positive LOH was observed only in patients with nonrecurrent disease. These results suggest that there may be different pathways of carcinogenesis in recurrent and nonrecurrent tumors and that the detection of MSI and LOH is useful for predicting future metachronous recurrence of gastric carcinoma after endoscopic treatment. We also performed direct sequencing of TGFβRII and BAX, which are known to be functional target genes of MSI.38-42 TGFβRII is reported to be linked to growth inhibition of epithelial cells, and BAX has been linked to apoptosis in epithelial cells. It is reported that patients with gastric tumors with MSI have a relatively good prognosis, whereas those with gastric tumors that have MSI and a concomitant BAX mutation have a poor prognosis.43 Single base deletion of TGFβRII and BAX was found only in one patient with recurrent disease with high MSI in this study. Therefore, loss of growth inhibition and/or apoptosis of epithelial cells may have been responsible for carcinogenesis and recurrence in this patient. Recently, hypermethylation of the promoter region in DNA MMR genes, such as hMSH2 and hMLH1, followed by negative transcription and translation of hMSH2 and hMLH1 was found to be responsible for the positive MSI in sporadic tumors.28-32 Because all of the patients investigated in this study did not meet the Amsterdam criteria for HNPCC,19 we examined the existence of hMSH2 and hMLH1 protein by immunohistochemical staining. Only two patients showed negative hMLH1 nuclear immunohistochemical staining. This result suggested that hypermethylation of the promoter region in the hMSH2 and hMLH1 genes may not be a major cause of MSI in the gastric tumors investigated. Therefore, some novel pathway other than germline mutation and hypermethylation of hMSH2 and hMLH1 may have been responsible for MSI in these tumors. 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