[CANCER RESEARCH 61, 3139 –3144, April 1, 2001] Genetic and Clinical Features of Human Pancreatic Ductal Adenocarcinomas with Widespread Microsatellite Instability 1 Hiroyuki Yamamoto, 2 Fumio Itoh, Hideaki Nakamura, Hiroshi Fukushima, Shigeru Sasaki, Manuel Perucho, and Kohzoh Imai First Department of Internal Medicine [H. Y., F. I., H. N., H. F., S. S., K. I.], Sapporo Medical University, Sapporo 060-8543, Japan, and The Burnham Institute, La Jolla, California 92037 [M. P.] ABSTRACT The incidences of microsatellite instability (MSI) and underlying DNA mismatch repair (MMR) defects in pancreatic carcinogenesis have not been well established. We analyzed 100 sporadic and 3 hereditary pan- creatic ductal adenocarcinomas for MSI, and high-frequency MSI (MSI-H) and low-frequency MSI (MSI-L) tumors were further analyzed for frameshift mutations of possible target genes and for promoter meth- ylation and mutation of DNA MMR genes, including hMLH1, hMSH2, hMSH3, and hMSH6 genes. Among the 100 sporadic tumors, 13 (13%) were MSI-H, 13 (13%) were MSI-L, and 74 (74%) were microsatellite stable (MSS) tumors. All of the three hereditary tumors from hereditary nonpolyposis colorectal cancer (HNPCC) patients were MSI-H. MSI-H tumors were significantly associated with poor differentiation and the presence of wild-type K-RAS and p53 genes. Patients with MSI-H tumors had a significantly longer overall survival time than did those with MSI-L or MSS tumors (P  0.0057). Frameshift mutations of hMSH3, hMLH3, BRCA-2, TGF- type II receptor, and BAX genes were detected in MSI-H tumors. Hypermethylation of the hMLH1 promoter was observed in 6 (46%) of the 13 sporadic MSI-H tumors but not in any of the 3 hereditary MSI-H tumors or 13 MSI-L tumors. All of the 3 HNPCC cases had germ-line hMLH1 mutation accompanied by loss of heterogeneity or other mutation in the tumor. Our results suggest that pancreatic carcinomas with MSI-H represent a distinctive oncogenic pathway because they ex- hibit peculiar clinical, pathological, and molecular characteristics. Our results also suggest the principal involvement of epigenetic or genetic inactivation of the hMLH1 gene in the pathogenesis of pancreatic carci- noma with MSI-H. INTRODUCTION A novel type of genetic instability characterized by length alter- ations within simple repeated sequences, termed MSI, 3 occurs in the majority of HNPCC and in a subset of sporadic cancers (1–5). This phenotype is also known as a replication error or microsatellite mutator phenotype. Recent studies have revealed that there are two types of MSI. Tumors with widespread or high-frequency MSI (MSI-H; for colorectal cancers, 30 – 40% of markers showing insta- bility) and MSS tumors exhibit fundamental differences in clinical, pathological, and molecular characteristics. On the other hand, tumors with MSI-L are indistinguishable in genotype (other than sporadic microsatellite length alterations) and phenotype from microsatellite stable tumors (6 – 8). A confusing factor in this field has been the overestimation of rare microsatellite length alterations as being MSI caused by DNA MMR defects. Thus far, there has been no experi- mental evidence supporting the notion that MSI-L is caused by defective DNA MMR (8). Pancreatic adenocarcinoma is included in the broad spectrum of the HNPCC syndrome, but the incidences of MSI (MSI-H and MSI-L) and underlying MMR defects have not been well established. There have been few studies on MSI in pancreatic carcinomas (9 –11). The reported frequencies of MSI vary from as low as 0% (0/27) to as high as 75% (6/8; unstable at least at 1 of the 12 loci tested, of which 4 cases were unstable at multiple loci). Recent studies have used strin- gent criteria and have distinguished MSI-H and MSI-L in pancreatic carcinomas. Ouyang et al. (12) reported MSI (40% or more instabil- ity) in 9 (15%) of 60 pancreatic cancers. Goggins et al. (13) reported MSI-H (two or more unstable loci of six loci examined) in 4% of 82 xenografted pancreatic carcinomas. Venkatasubbarao et al. (14) re- ported MSI-H (2 or more unstable loci of 6 loci examined) in 4 (29%) of 14 pancreatic adenocarcinomas. In contrast, Ghimenti et al. (15) detected MSI-L but not MSI-H in 66.7% of 21 pancreatic cancers. Wilentz et al. (16) recently reported 1 MSI in 13 medullary carcino- mas but none in 5 nonmedullary carcinomas. Gastrointestinal cancers with MSI-H and those without MSI-H have been shown to exhibit fundamental differences in clinical, path- ological, and molecular characteristics. In this regard, it is of interest that pancreatic carcinoma with MSI-H has been shown to exhibit characteristic features such as a wild-type K-RAS gene and a medul- lary phenotype characterized by poor differentiation, an expanding invasion, and syncytical growth (13, 16). Gastrointestinal cancers with MSI-H have a high rate of slippage-induced frameshift mutations in target genes such as TGFRII (17), hMSH3 (18), hMSH6 (18), and BAX (19) and high frequencies of aberrant DNA hypermethylation of tumor suppressor genes, including the hMLH1 gene (20 –24). As is the case of TGFRII, target genes could be differentially mutated in MSI-H tumors from different primary sites. However, target genes of frameshift mutations have not been characterized in pancreatic carci- noma with MSI-H. Although hypermethylation of the hMLH1 pro- moter has been reported in two of four MSI pancreatic carcinomas (25), the number of cases is too small to draw conclusions regarding the mechanism underlying the MSI-H phenotype in this carcinoma. In an attempt to address these issues, we analyzed 100 sporadic and 3 hereditary pancreatic ductal adenocarcinomas for MSI status, and we subsequently analyzed both MSI-H and MSI-L tumors for pro- moter methylation and mutation of DNA MMR genes and frameshift mutations of mononucleotide repeat sequences within possible target genes, including hMSH3 (18), hMSH6 (18), hMBD4 (26), hMLH3 (27), BRCA1 (28), BRCA2 (28), BLOOM (29), TGFRII (17), IGFIIR (30), PTEN (31), RIZ (32), APC (33), BAX (19), FAS (34), APAF-1 (34), BCL-10 (34), CDX2 (35), CHK1 (36), CASPASE-5 (37), and TCF-4 (38) genes. PATIENTS AND METHODS Patients and Tissue Samples. Seventy fresh paired surgical specimens of sporadic pancreatic carcinoma and adjacent nontumorous tissue and 30 paraf- fin-embedded sporadic tumor specimens were obtained from Japanese patients who had undergone surgical resection at our university hospital and related Received 9/8/00; accepted 1/29/01. 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. 1 Supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture and from the Ministry of Health and Welfare, Japan. 2 To whom requests for reprints should be addressed, at First Department of Internal Medicine, Sapporo Medical University, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan. Phone: 81-11-611-2111; Fax: 81-11-613-1141; E-mail: h-yama@sapmed.ac.jp. 3 The abbreviations used are: MSI, microsatellite instability; HNPCC, hereditary nonpolyposis colorectal cancers; MSI-H, high-frequency MSI; MSS, microsatellite stable; MSI-L, low-frequency MSI; MMR, mismatch repair; TGFRII, TGF- type II receptor; IGFIIR, insulin-like growth factor II receptor; SSCP, single-strand conformation poly- morphism; LOH, loss of heterozygosity; NCI, National Cancer Institute. 3139