(CANCER RESEARCH 58. 1494-1497. April I. 1998] Clinicopathological Significance of Altered Loci of Replication Error and Microsatellite Instability-associated Mutations in Gastric Cancer1 Ming-Shiang Wu, Chung-Wei Lee, Chia-Tung Shun, Hsiu-Po Wang, Wei-Jei Lee, Jin-Chuan Sheu, and Jaw-Town Lin2 Departments of Internal Medicine ¡M-S.W., C-W. L. J-C. S.. J-T. L.j. Pathology {C-T. S.I. Emergency Medicine ¡H-P.W.]. and Surgery ¡W-J.LI. National Taiwan University Hospital. Taipei. Taiwan. Republic of China ABSTRACT Replication errors <Kl.Ksi judged by microsatellite instability and its associated mutations have been recognized as an important mechanism in tumorigenesis of gastric cancers (GCs). To gain a deeper insight into its significance, we examined the frequency of RERs using nine microsatellite markers and screened mutations in the polydeoxyadenine tract of the transforming growth factor ßtype II receptor gene (TGF-ßRII) and polydeoxyguanine tracts of insulin-like growth factor II receptor and ll.\\ genes. Twenty-four (30% ) of SO patients with (.( had RERs, of which 3, 8, and 13 had one, two, and three or more loci, respectively. In 13 tumors with RERs in three or more loci, frameshift mutations of TGF-ßRII, insulin-like growth factor II receptor, and H.\\ were identified in 12, 3, and 2, respectively. Compared with GC with none, one or two RER- positive loci as a group, GC with RERs in three or more loci showed a significantly higher frequency of antral location (12 of 13 versus 35 of 67; P= 0.01), intestinal subtype (11 of 13 versus 30 of 67; P = 0.01), and previous He lie obacter pylori infection (12 of 13 versus 41 of 67; P = 0.05) and a lower incidence of lymph node metastasis (5 of 13 versus 49 of 67; /' ~ 0.02) and tended to be in an advanced stage (12 of 13 versus 54 of 67; /•= 0.28). These data indicate that GC with multiple RERs manifest distinct Clinicopathological characteristics, and that a high frequency of frameshift mutations involving the TGF-ßRH gene may be causatively linked with tumorigenesis and progression. INTRODUCTION Tumorigenesis is a multistep process leading to accumulation of multiple genetic alterations (1). Because the rate of spontaneous mutation is low in normal cells, a mutator phenotype characterized by an increased mutation rate is required to achieve final malignant transformation (2). This notion is strongly supported by recent dem onstration of genome-wide instability of repetitive sequences (i.e., microsatellites) in HNPCC3 and in a certain portion of sporadic tumors (3, 4). Such variations in microsatellites, namely, MSI or ubiquitous somatic mutations, are generated by errors in DNA repli cation, and hence the phenotype has been named RER-positive (3-5). RER-positive colonie cancer exhibits distinct Clinicopathological characteristics and a tendency to accumulate frameshift mutations in polydeoxyadenine tract (A)H) of the TGF-ßRIIgene and polydeox yguanine tract (G)K of the IGFIIR and BAX genes (6-9). Additional information suggests that mutations of TGF-ßRIIand IGFIIR are mutually exclusive in RER-positive cancer (8). However, data con cerning frameshift mutations of TGF-ßRII,IGFIIR. and BAX genes Received 8/12/97; accepted 1/28/98. 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 (J.S.C. Section 1734 solely to indicate this fact. 1This work was supported by National Taiwan University Hospital Grant NTUH- M87103. National Science Council Grant NSC-85-2622-B002-011 and NSC87-2314- B(X)2-187, and Department of Health. Executive Yuan, Taiwan Grants DOH87-TD-1045 and DOH87-HR-525. 3 To whom requests for reprints should be addressed, at Department of Internal Medicine, National Taiwan University Hospital. 7 Chun-Shan S. Road. Taipei. Taiwan. Republic of China, 100. ' The abbreviations used are: HNPCC. hereditary nonpolyposis colorectal cancer; MSI, microsatellite instability; RF.R. replication error; TGF-ßRII. transforming growth factor ßtype II receptor: IGUIR, insulin-like growth factor II receptor; GC. gastric are scant in tumors other than colonie cancers, and their interrelation ship remains unclear, especially with respect to Clinicopathological characteristics of the tumor (10-15). GC remains a common disease with a dismal prognosis in the world (16). Both genetic and environmental factors are important in gastric tumorigenesis. MSI has been reported as a feature of HNPCC-asso- ciated GC. and a certain portion of sporadic GCs also exhibit RER phenotypes (17). However, whether the RER-positive GC can be ascribed to a specific phenotype with distinct Clinicopathological features is controversial (18). Moreover, the frequency of MSI and MSI-associated mutations vary widely, ranging from 15 to 64% for MSI (19-25) and 0 to 91% for TGF-ßRII(10-15). Although varia tions in different susceptible populations and etiological factors partly explain the discrepancy, the lack of uniformity of criteria to define MSI could also contribute to the above variability (18). For example, different altered numbers of microsatellite markers have been used to distinguish intrinsic instability from pathological genome-wide insta bility germane to cancers with a mutator phenotype. Recently, Dos Santos et al. (26) have reported that GCs with multiple (three or more) RER-positive loci have a Clinicopathological profile different from GCs with a single RER-positive locus or two RER-positive loci, of which the profile is similar to that of RER-negative GC. Others proposed further to adopt similar, more stringent criteria, such as the presence of more than one microsatellite alteration together with mutations in the target gene of TGF-ßRII to ascribe a meaningful RER phenotype (27). As a group, we have confirmed previously the frequent occurrence of an RER-positive phenotype in GC and have reported infrequent mutations of hMSH2 gene in sporadic RER-positive GC (28-30). To define the precise role of MSI in GC better, we herein extended our study to 80 patients with GC by analyzing nine microsatellite markers and screening frameshift mutations of TGF-ßRII,IGFIIR, and BAX genes. Specifically, we adopted similar stringent criteria to define the RER-positive phenotype and to elucidate the relationship among RER status, Clinicopathological features, and MSI-associated mutations. MATERIALS AND METHODS Patients and Tissues Samples. In total. 8Ãoepatients with histologically confirmed GC were included in this study. Among them, 59 patients have been reported previously (29). All 80 patients underwent surgery at the National Taiwan University Hospital without receiving previous chemotherapy or ra diation therapy. Surgical specimens were handled according to the guidelines issued by the Japanese Research Society of GC (31). All GC tissues were examined by the same pathologist (C-T. S.), who was unaware of the param eters to be investigated. These tumors were classified into 14 early GC and 66 advanced GC according to the depth of invasion (31 ). Furthermore, they were also categorized into 41 intestinal type and 39 diffuse type based on the classification of Lauren (32). Relevant demographic and Clinicopathological information for each patient was obtained from medical records. None had a family history suggestive of HNPCC. The status of Helicobacter pylori infec tion was determined by the presence of a significantly high tiler of anti-W. pylori IgG in the preoperative serum. Both tumorous and nontumorous tissues were dissected separately from each resected specimen, frozen immediately in the embedding compound 1494 Research. on December 27, 2015. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from