Immunohistochemical Pattern of MLH1/MSH2 Expression Is Related to Clinical and Pathological Features in Colorectal Adenocarcinomas with Microsatellite Instability Giovanni Lanza, M.D., Roberta Gafà, M.D., Iva Maestri, M.Sc., Alessandra Santini, M.D., Maurizio Matteuzzi, M.Sc., Luigi Cavazzini, M.D. Department of Experimental and Diagnostic Medicine, Section of Anatomic Pathology, University of Ferrara (GL, RG, IM, MM, LC); and Division of Clinical Oncology, St. Anna Hospital (AS), Ferrara, Italy cinomas more frequently died of disease than did Detection of colorectal carcinomas with high- patients with MLH1/MSH2-positive and MSH2- frequency microsatellite instability (MSI-H) is clin- negative MSI-H tumors, but the difference was not ically important for several reasons. Recent studies statistically significant. The results of the present suggested that immunohistochemical analysis of investigation strongly indicate that immunohisto- MLH1 and MSH2 expression is a rapid and accurate chemical analysis of MLH1 and MSH2 expression is method for identifying large bowel tumors of the a practical and reliable method for the routine de- MSI-H phenotype. In this study, we evaluated by tection of the vast majority of MSI-H large bowel immunohistochemistry MLH1 and MSH2 protein adenocarcinomas. Our data also point out that expression in 132 MSI-H, 23 MSI-L (low-frequency MSI-H MLH1/MSH2-positive colorectal carcinomas MSI), and 150 microsatellite stable (MSS) colorectal are characterized by distinctive pathological adenocarcinomas. Loss of MLH1 or MSH2 expres- features. sion was detected in 120 (90.9%) MSI-H carcinomas, whereas all MSI-L and MSS tumors showed normal KEY WORDS: Colorectal carcinoma, DNA mismatch expression of both proteins. Lack of MLH1 nuclear repair, Hereditary nonpolyposis colorectal cancer, staining was observed much more often than ab- Immunohistochemistry, Microsatellite instability, sence of MSH2 nuclear staining (106 and 14 cases, MLH1 gene, MSH2 gene, Pathology, Prognosis. respectively). Among MSI-H carcinomas, MLH1/ Mod Pathol 2002;15(7):741–749 MSH2 pattern of expression was significantly re- lated to several clinical and pathological variables. High-frequency microsatellite instability (MSI-H) In particular, MSI-H MLH1/MSH2-positive carcino- occurs in most colorectal adenocarcinomas from mas were more often located in the distal colon, patients with hereditary nonpolyposis colorectal were more frequently classified as ordinary adeno- cancer (HNPCC) and in about 15% of sporadic carcinomas, and were more likely to be well or large bowel cancers. MSI-H tumors develop moderately differentiated, p53 positive, and <7cm through a mutational pathway different from that in diameter than were MLH1-negative and MSH2- operating in common colorectal adenocarcino- negative carcinomas. In addition, MLH1-negative mas (1, 2) and display distinctive pathological carcinomas were less common among patients with features, such as proximal location, poor differ- hereditary nonpolyposis colorectal cancer (HNPCC) entiation, frequent mucinous or medullary/un- or suspected HNPCC and in the group of patients differentiated histotype, and intense peritumoral aged <50 years. Patients with MLH1-negative car- and intratumoral lymphocytic infiltration (3–5). Besides identifying HNPCC gene carriers (6–8), Copyright © 2002 by The United States and Canadian Academy of detection of MSI-H large bowel adenocarcinomas Pathology, Inc. VOL. 15, NO. 7, P. 741, 2002 Printed in the U.S.A. is clinically important for several reasons. Pa- Date of acceptance: April 1, 2002. tients with MSI-H cancers demonstrated a better This work was supported by grants from MURST and Fondazione Cassa di Risparmio di Ferrara. clinical outcome than did patients with common Address reprint requests to: Giovanni Lanza, M.D., Dipartimento di Me- large bowel adenocarcinomas, and recent studies dicina Sperimentale e Diagnostica, Sezione di Anatomia Istologia e Cito- logia Patologica, Università di Ferrara, Via Fossato di Mortara 64/b, 44100 performed on very large series of patients showed Ferrara, Italy; e-mail: [email protected]; fax: 39-0532-248021. that the survival advantage conferred by the DOI: 10.1097/01.MP.0000018979.68686.B2 MSI-H phenotype is independent of tumor stage 741 and other clinical and pathological variables (5, with a predominant solid growth pattern (Ն70% 9 –12). In addition, clinical evidence suggests that of the tumor area) and mild or moderate nuclear MSI-H colorectal carcinomas may be particularly pleomorphism were classified as medullary ade- sensitive to 5-fluorouracil– based adjuvant che- nocarcinomas (29). Pattern of growth, lympho- motherapy (13, 14). Finally, patients with MSI-H cytic infiltration at the advancing tumor margin, tumors are thought to be at increased risk of and extramural vein invasion were evaluated ac- developing metachronous large bowel carcino- cording to the criteria of Jass et al. (30). Peritu- mas and therefore to require long-term colono- moral Crohn’s-like lymphoid reaction was as- scopic follow-up (15). sessed according to Graham and Appelman (31) MSI-H is determined by defects in the DNA and classified as conspicuous (intense) and in- mismatch repair (MMR) system (16, 17). In conspicuous (5, 29). HNPCC, MSI-H is caused by germline mutations Detailed clinical follow-up data were available of one of the MMR genes, in most cases the MLH1 for all patients operated on before December or MSH2 gene, with somatic inactivation of the 1994. Personal and family histories of colorectal remaining wild-type allele (17). In sporadic carcinoma and other cancers were obtained by MSI-H large bowel carcinomas, mutations of chart review and in most cases by interview of MMR genes have been detected at a very low patients and/or family members. Whenever pos- frequency, and MLH1 gene inactivation by pro- sible, pathological review of HNPCC-related tu- moter hypermethylation is the major mechanism mors of all family members was performed. How- leading to the mutator phenotype (18–20). Ge- ever, for the large majority of patients, only data netic or epigenetic inactivation of MLH1 and concerning first-degree relatives were accessible, MSH2 genes is generally associated with loss of and no attempt to construct expanded family immunohistochemical expression of the corre- trees was done. sponding protein (18, 20, 21). Recent studies sug- gest that immunohistochemical analysis of MLH1 and MSH2 gene product expression may allow Microsatellite Analysis one to specifically identify MSI-H colorectal ade- Representative samples of tumor and corre- nocarcinomas (15, 22–26). Therefore, immuno- sponding normal mucosa were obtained from fresh histochemistry for MLH1 and MSH2 seems to surgical specimens, frozen in liquid nitrogen, and represent a rapid and accurate method for the stored at Ϫ80° C. DNA was extracted by a standard routine detection of these tumors. phenol–chloroform procedure. Before DNA extrac- In the present investigation, we evaluated MLH1 tion, from each tumor sample, a frozen section was and MSH2 expression in a large number of MSI-H, cut and stained with hematoxylin and eosin to ver- MSI-L (low-frequency MSI), and microsatellite sta- ify by microscopic examination the presence of ad- ble (MSS) colorectal adenocarcinomas. The rela- equate neoplastic material (Ն60–70% of tumor tionship between the immunohistochemical pat- cells). tern of MLH1/MSH2 expression and the clinical In the majority of cases (n ϭ 614), MSI was and pathological features of MSI-H tumors was also evaluated at six microsatellite loci (D18S58, analyzed. D18S61, D17S855, D17S786, BAT26, and BAT40) using a fluorescence-based polymerase chain re- action (PCR) method, as reported elsewhere (5, MATERIALS AND METHODS 29). Analysis of the PCR products was performed with an automated laser-activated fluorescent The study included 724 colorectal adenocarcino- DNA sequencer (ALF; Pharmacia Biotech, Co- mas surgically resected in the years 1988–1994 and logno Monzese, Italy) using the Fragment Man- 1999–2000. To obtain a very large number of MSI-H ager (FM1.1) software (Pharmacia Biotech). More tumors, a nearly equal number of carcinomas of the recently, in the last 110 tumors, MSI was ana- proximal (caecum, ascending, and transverse co- lyzed with the Bethesda panel of five microsatel- lon; n ϭ 338) and distal colon (left colon and rec- lite markers (BAT26, BAT25, D2S123, D5S346, tum; n ϭ 386) were examined. Availability of frozen D17S250) (32) and a fluorescence-based multi- tissue samples suitable for genetic analyses was the plex PCR method, using the HNPCC Microsatel- only criterion for selection. Patients with familial lite Instability Test kit (Boehringer Mannheim, adenomatous polyposis or inflammatory bowel dis- Mannheim, Germany) according to manufactur- ease were excluded. er’s instructions. PCR products were run in an Tumors were staged according to the TNM ABI PRISM 377 DNA sequencer (Perkin-Elmer Ap- staging system (27). Tumor type and grade of plied Biosystems Division, Foster City, CA) and differentiation were determined by the criteria of analyzed by the GeneScan 3.1 software (Perkin- the World Health Organization (28). Carcinomas Elmer) (Fig. 1). 742 Modern Pathology (clone FE11; Oncogene Research Products, Cam- bridge, MA), both diluted 1:100. An UltraVision streptavidin—biotin peroxidase detection kit (TP- 060-HL; Lab Vision Corporation, Fremont, CA) was employed as the secondary detection system, and the peroxidase reaction was developed using diamino-