Standardized Approach for Microsatellite Instability Detection in Gastric Carcinomas EVA MUSULE ´ N, MD, PHD, VI ´ CTOR MORENO, MD, PHD, GERMAN REYES, MD, PHD, FRANCISCO JAVIER SANCHO, MD, PHD, MIGUEL ANGEL PEINADO, PHD, MANEL ESTELLER, MD, PHD, JAMES G. HERMAN, MD, PHD, NEUS COMBALIA, MD, MERCE ` REY, MD, AND GABRIEL CAPELLA ´ , MD, PHD Microsatellite instability (MSI) defines a specific type of genetic instability. Although consensus diagnostic criteria for MSI definition in colorectal cancer have been established, their utility in other tumor types remain to be proven. Previously we developed a mathematical model for MSI definition in colorectal cancer. The aim of this study was to establish diagnostic criteria for MSI evaluation in human gastric cancer. We designed an algorithm for the efficient character- ization of MSI and used it to analyze data on 7 microsatellite markers in 35 gastric carcinomas. Theoretical models considering 1, 2, or 3 populations were tested against the data collected. Also, hypermeth- ylation of hMLH1 gene promoter and hMLH1 protein expression were studied. The observed frequencies of MSI in our series of samples best fit a 2-population model: stable and unstable, defined by instability in 2 or more of a minimum of 7 markers analyzed. MSI was observed in 29% of the tumors. Misclassification rate was <4% when any 7 loci were analyzed. MSI() tumors inversely associated with p53 protein overexpression. A good correlation between hMLH1 status (either protein or promoter hypermethylation) and MSI clas- sification was observed. We have developed a simple, sensitive, and specific approach to assess the presence of MSI in gastric cancer that may have clinical applications. HUM PATHOL 35:335-342. © 2004 Elsevier Inc. All rights reserved. Key words: gastric cancer, microsatellite instability, hMLH1. Abbreviations: CI, confidence interval; CRC, colorectal cancer; HNPCC, hereditary nonpolyposis colorectal cancer; LOH, loss of heterozygosity; MSI, microsatellite instability; PCR, polymerase chain reaction; ICH, immunohistochemistry. A subset of gastrointestinal carcinomas (mainly colorectal and gastric) and other types of human tu- mors exhibit ubiquitous microsatellite instability (MSI). This discrete type of genomic instability is character- ized by small deletions or insertions within short-tan- dem repeats in tumor DNA when compared with the corresponding DNA from normal tissue. 1-3 MSI is a symptom of defective DNA repair and characterizes a distinctive tumor phenotype known as the microsatel- lite mutator phenotype. 4,5 This phenotype is present in 7% to 20% of human colorectal cancers (CRCs), cor- relates with specific tumor characteristics, and is strongly associated with hereditary nonpolyposis colo- rectal cancer (HNPCC), 2,6 which is caused by germline mutations in mismatch repair genes. In other tumor types, namely endometrial and gastric, a significant incidence of MSI has been reported. In gastric tumors, this type of instability has been detected in 14% to 47% of cases 7,8 and is associated with specific tumor fea- tures, 9-16 including low rate of p53 mutations, 7,10,14,16-21 and better prognosis. 9,13,16,20 The ubiquitous nature of MSI and the intrinsic somatic instability of microsatellite sequences 22,23 have led to the development of heterogeneous approaches to define the presence of MSI in colorectal and gastric tumors. Consensus has been achieved in defining MSI in colorectal cancer. 5 Despite this, however, some con- troversy remains about the existence of only 2 tumor categories, MSI(+) and MSI(-), and whether MSI(+) tumors should be divided into those exhibiting low and high instability. 24 Recently, we developed a mutational population model for determining the presence and degree of MSI in CRC. 25 The presence of 2 or more altered microsatellites out of 4 to 6 evaluated enabled the classification of most MSI(+) cases. The CRC con- sensus report evidenced the need to more precisely define the MSI phenotype in tumors other than CRCs. Moreover, a more accurate definition will also allow evaluation of its potential clinical usefulness. The aim of the present study was to establish criteria to define MSI in gastric tumors using a mathematical algorithm. MATERIALS AND METHODS Patients Thirty-five patients harboring gastric carcinomas surgi- cally resected at the Hospital de Sant Pau i de la Santa Creu between 1991 and 1997 were prospectively included. Inclu- sion criteria were (1) resected primary adenocarcinomas, (2) fresh paired normal mucosal tumor samples obtained within From the Servei de Patologia, UDIAT-CD, Corporacio ´ Sanitaria Parc Taulı ´, Sabadell, Spain; Laboratori de Recerca Translacional, Institut Catala ` d’Oncologia, L’Hospitalet de Llobregat, Spain; Hos- pital Clı ´nica Kennedy, Guayaquil, Ecuador; Servei d’Anatomia Pato- lo ` gica, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; Centre d’Oncologia Molecular, Institut de Recerca Oncolo ` gica, L’Hospitalet de Llobregat, Spain; Johns Hopkins Oncology Center, Baltimore, MD; and Cancer Epigenetics Laboratory, Molecular Pathology Pro- gram, Centro Nacional de Investigaciones Oncolo ´ gicas, Majada- honda, Spain. Accepted for publication October 28, 2003. Supported by grants from Comisio ´ n Interministerial de Ciencia y Tecnologia (CICYT SAF00/81 and FIS 01/1264). Address correspondence and reprint requests to Gabriel Capella ´ MD, PHD, Laboratori de Recerca Translacional, Institut Catala ` d’Oncologia, Av. Gran Via s/n Km 2.7, 08907 L’Hospitalet de Llo- bregat, Spain. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2003.10.021 335