Colorectal cancer (CRC) is one of the most common

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1 Microsatellite Instability in Colorectal Carcinoma The Comparison of Immunohistochemistry and Molecular Biology Suggests a Role for hmlh6 Immunostaining Valérie Rigau, MD; Nicole Sebbagh, BS; Sylviane Olschwang, MD, PhD; François Paraf, MD, PhD; Najat Mourra, MD; Yann Parc, MD; Jean-François Flejou, MD, PhD Context. Microsatellite instability (MSI) due to defective mismatch repair (MMR) genes has been reported in the majority of colorectal tumors from patients with hereditary nonpolyposis colorectal cancer syndrome and in 1% to 15% of sporadic colorectal cancers. The identification of cancers associated with MSI requires classical molecular testing as the gold standard. Objective. The aim of this study was to evaluate the role of immunohistochemistry with antibodies directed against 4 MMR proteins as a screening tool for carcinomas with MSI. Methods. In this study, 24 formalin-fixed, paraffinembedded colorectal carcinomas were examined for MMR protein expression (, hmsh6, and hpms2) and analyzed for MSI (MSI-H indicates at least 2 of 6 markers affected). These results were correlated with histopathologic parameters. Results. Immunohistochemical analysis revealed that loss of expression of at least 1 protein was present in 17% of cases. One hundred percent of carcinomas that showed high instability (MSI-H) showed loss of expression of, or hmsh6. Loss of expression of 2 proteins was present in 5.4% of MSI-H cases, with only 2 combinations, namely, hmlh1/hpms2 and hmsh2/ hmsh6. Isolated loss of hmsh6 expression was present in 2 MSI-H cases. Conclusions. These findings confirm that examination of MMR protein expression by immunohistochemistry is a simple method to diagnose colorectal cancer with MSI. Our data suggest that the study of hmsh6 may be useful, in addition to hmlh1 and hmsh2. Moreover, immunohistochemistry could represent a screening method with which to direct research on the mutations of MMR genes observed in hereditary nonpolyposis colorectal cancer syndrome. (Arch Pathol Lab Med. ;127:64 7) Colorectal cancer (CRC) is one of the most common forms of cancer in western countries. In most cases, these cancers are seen sporadically, but 5% to 1% of cases are associated with a primary genetic factor. Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common syndrome. It is secondary to an inherited mutation in one of the D mismatch repair (MMR) genes (, hmsh6, and hpms2). 1,2 The phenomenon of microsatellite instability (MSI) results from inactivation, mutational and/or epigenetic, of MMR genes, mostly hmlh1 and hmsh2. 3 It is a characteristic molecular finding in most cases of HNPCC and is also observed in a subgroup (1% 15%) of sporadic CRCs. 4 It has been shown that MSI tumors, either hereditary or sporadic, have particular morphologic features 5 11 Accepted for publication January,. From the Departments of Pathology (Drs Rigau, Mourra, and Flejou, and Ms Sebbagh), Surgery (Drs Olschwang and Parc), AP-HP, Hôpital Saint-Antoine, Paris, France; Centre d Etude du Polymorphisme Humain (CEPH) and Institut National de la Santé et de la Recherche Médicale (INSERM) U434, Paris, France (Drs Rigau, Olschwang, Parc, and Flejou, and Ms Sebbagh); and the Department of Pathology, Hôpital Dupuytren, Limoges, France (Dr Paraf). Reprints: Jean-François Flejou, MD, PhD, Service d Anatomie Pathologique, Hôpital Saint-Antoine, AP-HP 184, rue du Faubourg Saint- Antoine, 75571, Paris cedex 12, France ( jean-francois.flejou@ sat.ap-hop-paris.fr). and, most importantly, have an improved survival rate relative to microsatellite-stable (MSS) tumors of similar stage, 12,13 and may have distinct sensitivity to the action of certain commonly used chemotherapeutic agents. Therefore, it appears that recognition of the microsatellite phenotype is becoming important in the management of patients with CRC. Determination of tumor microsatellite status by polymerase chain reaction has been suggested as the best method to recognize MSI-positive CRC. 15 However, the demonstration by immunohistochemistry of loss of expression of MMR proteins has been proposed as an alternative method to recognize these tumors. 5,,11,16 32 In most studies, only hmlh1 and hmsh2 proteins were studied, although rare cases of HNPCC are secondary to mutations in other MMR genes. Moreover, the expression of other MMR proteins has not been studied extensively in sporadic CRC. The aim of this study was to compare the expression of 4 MMR proteins (, hmsh6, and hpms2) to the MSI status determined by polymerase chain reaction in a series of 24 CRCs. MATERIALS AND METHODS Samples Resection specimens were obtained from 24 patients who underwent surgical resection of CRC at Saint-Antoine Hospital (Paris, France) from 18 to 1. Site of tumors was determined as 64 Arch Pathol Lab Med Vol 127, June Microsatellite Instability in Colorectal Carcinoma Rigau et al

2 Table 1. Antibodies Used for Immunohistochemical Study Antigen Antibody Clone Dilution Time of Incubation Source hmlh1 hmsh2 hmsh6 hpms2 G FE :7 1: 1: 1:2 1h 3 min 3 min 3 min Pharmingen, San Diego, Calif Calbiochem, Cambridge, Mass Becton Dickinson, Franklin Lakes, NJ Oncogene Research Products, Cambridge, Mass rectum, left colon (distal to the splenic flexure), or right colon. Family cancer history was obtained from the clinical charts and was used to identify those families that met either the Amsterdam criteria or extended Bethesda criteria for HNPCC. 33,34 Histopathologic Review of Tumors For all tumors, all hematoxylin-phloxine-safranin stained slides were reviewed independently by 2 observers (V.R. and J.F.F.). The following variables were assessed: tumor grade (well, moderately, or poorly differentiated), extent of mucin production (mucinous cancers were those containing more than 5% of extracellular mucin), tumor growth pattern (expanding or infiltrating), 35 presence of a Crohn-like inflammatory infiltrate (defined as intense lymphoid reaction with numerous lymphoid aggregates with frequent germinal centers), 36 and intraepithelial lymphocytes (classified as conspicuous when more than 3 were present per 1 high-power fields).,37 The tumors were classified in the TNM, Dukes, Astler-Coller, and Jass 38 staging systems. A specific medullary type of cancer was recorded, characterized by uniform, poorly differentiated tumor cells arranged in solid sheets with a prominent intratumoral lymphocytic infiltrate. 3 Presence of residual adenomatous tissue around the cancer and of distant colonic polyps (hyperplastic, adenomatous, or serrated) was noted. Immunohistochemical Analysis One block of formalin-fixed, paraffin-embedded adenocarcinoma tissue was selected in each case. In all cases, this block comprised an area of normal colon mucosa adjacent to the tumor. Four-micrometer sections were affixed to Superfrost Plus slides (CML, Nemours, France) and dried overnight at 37 C. After dewaxing and rehydration in distilled water, endogenous peroxidase activity was quenched by incubating the slides in 3% hydrogen peroxide in methanol for (2 15 minutes). Sections were subject to heat antigen retrieval by autoclaving in citrate buffer (ph 6.) for 15 minutes at 75 W and 15 minutes at 15 W. Details of primary antibodies for MMR proteins, hmsh6, and hpms2 are summarized in Table 1. All incubations were performed at room temperature. The following methods were used: avidin-biotin complex method for hmlh1, indirect immunoperoxidase for hmsh2 and hmsh6, and supersensitive detection kit method (BioGenex, San Ramon, Calif) for hpms2. Immunohistochemical reactions were performed with the Optimax Plus system (BioGenex). Color was developed with aminoethyl-carbazole (Vector, Burlingame, Calif) as the chromogen. The sections were washed in running tap water and lightly counterstained in Mayer hematoxylin. The results were scored without knowledge of the MSI results. Loss of expression was recorded when nuclear staining was absent in all malignant cells, but preserved in normal epithelial and stromal cells. Two observers (V.R. and J.F.F.) assessed all cases independently. The few cases with discrepant scoring were reevaluated jointly on a second occasion, and agreement was reached in all cases. Microsatellite Instability Testing The same paraffin block was used for D extraction as for immunohistochemistry. D was extracted from both normal and malignant tissue embedded in paraffin. Selected areas of malignant tissue comprised more than 7% of malignant cells in all cases. Five to six 2- m sections were microdissected and placed into microcentrifuge tubes. D was extracted with the Qiamp tissue kit (Qiagen, Santa Clarita, Calif). The fluorescent multiplex microsatellite assay was used as previously described with a panel of the following 6 markers: D2S1, D5S346, D17S25, TGFbRII, BAT25, and BAT Tumors were considered MSI-H (high) if at least 2 markers showed instability and MSI-L (low) if only 1 marker was unstable. Statistical Analysis Qualitative data were compared with the 2 test using Yates correction when appropriate. Quantitative data were expressed as the mean SEM and compared by the Mann-Whitney test. A P value less than.5 was considered to indicate statistical significance. RESULTS Clinical and Pathologic Characteristics The patients ranged in age from 26 to years. One hundred twenty patients were women and 84 were men. Six patients fulfilled the Amsterdam criteria, and 3 met the extended Bethesda criteria for HNPCC syndrome. The cancers were located in the proximal colon (%), the distal colon (%), and the rectum (48%). The Astler-Coller stages were as follows: A, 11%; B1, 17%; B2, 28%; C1, 3%; C2, 3%; and D, 11%. Sixty-eight percent of tumors were well differentiated, 21% moderately differentiated, and 11% poorly differentiated. Fourteen percent of cases were mucinous, and 5% were of the medullary type. Frequency of MSI Thirty-one tumors (15%) were MSI-H, (11%) MSI-L, and 15 (74%) MSS. In MSI-H tumors, all 6 microsatellite markers showed instability in a majority of tumors. BAT26 was unstable in all 31 MSI-H tumors, and BAT25 was unstable in 25 of the 31 MSI-H tumors. Frequency of Loss of MMR Protein Thirty-five tumors (17%) lost the expression of 1 (13 cases) or 2 (22 cases) proteins. The following proteins were lost: hmlh1 alone (7 cases), hpms2 alone (4 cases), hmlh1 and hpms2 (15 cases) (Figure 1), hmsh6 alone (2 cases), and hmsh2 and hmsh6 (7 cases) (Figure 2). A comparison of microsatellite testing and immunohistochemistry results is presented in Table 2. All cases with loss of expression were MSI-H, except the 4 cases showing isolated loss of hpms2, which were MSS. There was no loss of expression of any protein in the MSI-L cases nor in the remaining 6 MSS cases. Therefore, when hmlh1, hmsh2, and hmsh6 expression was considered, there was a % sensitivity and specificity of immunohistochemistry for the MSI-H phenotype. The specificity, sensitivity, positive predictive value, and negative predictive value of immunohistochemistry for MSI are presented in Table 3, along with data from other published series. Relationship Between MMR Protein, HNPCC Clinical Criteria, and Mutation in MMR Genes Among the patients who fulfilled the Amsterdam or Bethesda clinical criteria for HNPCC syndrome, 4 had an Arch Pathol Lab Med Vol 127, June Microsatellite Instability in Colorectal Carcinoma Rigau et al 65

3 Figure 1. Well-differentiated colorectal carcinoma with high microsatellite instability (A), showing loss of expression of hmlh1 (B) and hpms2 (C), and normal expression of hmsh2 (D) and hmsh6 (E) (A, hematoxylinphloxine-safranin, original magnification 2; B through E, immunoperoxidase, original magnification 4). hmlh1 germline mutation with loss of hmlh1 expression, 4 had an hmsh2 germline mutation with loss of expression of hmsh2 and hmsh6 proteins, and 1 had an hmsh6 germline mutation with loss of expression of hmsh6 protein (mutation data not shown). In one 36-year-old patient with loss of both hmsh2 and hmsh6 and in one 5-year-old patient with loss of hmsh6, no family history was obtained, and in one 33- year-old patient with loss of hmsh2 and hmsh6, there was a familial history of CRC insufficient to diagnose HNPCC. Relationship Between MMR Protein and Characteristics of the Tumors The relationship between loss of expression of MMR proteins and clinical and pathologic characteristics of the tumors is presented in Table 2. Tumors with a loss of hmlh1 expression occurred in older patients (P.1) and those with loss of expression of hmsh2 occurred in younger patients (P.1) than those with no loss of expression of either hmlh1 or hmsh2. Female sex was correlated with loss of hmlh1 (P.1). Proximal location of the tumor was associated with loss of hmlh1 (P.1) and hmsh2 (P.1). Poor differentiation was associated with loss of hmlh1 (P.1). Mucinous and medullary types were more frequent in tumors that were hmlh1 negative (P.1) and hmsh2 negative (P.1). Tumors showing loss of hmlh1 more often had expanding margins (P.2) and lower stages in the Astler-Coller system (P.1). There was no relationship between the expression of hmlh1 and hmsh2 and increased number of intratumorous lymphocytes, Crohnlike reaction, presence of adenomatous residue, T and N stage, or grade in Jass classification. COMMENT In this study, we compared tumor MSI testing by polymerase chain reaction versus tumor immunohistochemistry for assessing the competence of the MMR mechanism of CRCs. In our study, MSI-H tumors were seen in 15.2% of the cases. This rate is slightly higher than percentages previously reported in most studies.,17,1 This high rate could be partially explained by the presence of patients belonging to an HNPCC family in our population, as in most studies results were considered separately for HNPCC and sporadic MSI-positive CRCs. In our series, 11% of cases had an MSI-L phenotype. As in all reported series, MSI-L CRCs showed no loss of expression of MMR proteins. 7,16,17,32 Although some authors have proposed that the MSI-L phenotype reflects a distinctive pathway of development of CRC, 4 this theory is very much debated in the literature. 41 We do not furnish arguments in favor of this hypothesis, as MSI-L cases were similar to MSS cases 6 Arch Pathol Lab Med Vol 127, June Microsatellite Instability in Colorectal Carcinoma Rigau et al

4 Figure 2. Well-differentiated colorectal carcinoma with high microsatellite instability (A), showing normal expression of hmlh1 (B) and hpms2 (C), and loss of expression of hmsh2 (D) and hmsh6 (E) (A, hematoxylinphloxine-safranin, original magnification 2; B through E, immunoperoxidase, original magnification 4). in regard to all clinicopathologic characteristics of the tumors. Our immunohistochemical analysis of MMR enzymes provided a highly specific and sensitive approach to the detection of MSI-H tumors. The performance of immunohistochemistry in comparison with molecular MSI testing is shown in Table 3, which presents the sensitivities, specificities, positive predictive values, and negative predictive values reported in previously published series of CRC and in the present series. As in most other series, we observed a predominance of loss of hmlh1 protein in MSI-H tumors, probably due to hypermethylation of the promoter of this gene in sporadic MSI-H cancers. 42 In most published studies, only 2 antibodies were used, directed against hmlh1 and hmsh2 proteins (Table 3). These 2 antibodies allowed a diagnosis of MSI with a high specificity (5% %) in almost all series, and usually with a high sensitivity, although in some studies this sensitivity was between 7% and 8%, with a relatively high number of false negatives.,1,21,26 When considering the results of 16 series representing 344 cases as a whole, the following performances of immunohistochemistry were obtained: sensitivity, 2.4%; specificity,.6%; positive predictive value, 8.5%; and negative predictive value, 7.8% (Table 3). Even if molecular MSI testing remains the current gold standard for assessing tumor D mismatch pair competency, all these studies confirm that hmlh1 and hmsh2 immunohistochemistry is a sensitive, rapid, and cost-effective method. Previous studies suggested that one explanation for false-negative cases obtained by immunohistochemistry might be the presence of mutations in still unidentified genes or in other MMR genes. 7,17 It seems therefore of interest to study the expression of other MMR proteins, such as hmsh6 and hpms2, that have been shown to be responsible for rare cases of HNPCC syndrome. We found cases that did not express hmsh6 protein, of which 7 cases also showed loss of hmsh2 expression and represented the entire group of tumors lacking hmsh2 expression. This association between hmsh2 and hmsh6 expression has already been reported by others. 11,32,43 We observed 2 cases with isolated loss of expression of hmsh6 protein, both of which were MSI-H. One patient had an HNPCC syndrome with a germline mutation of the hmsh6 gene, and the second patient was 5 years old. Although we did not have a family history in the latter case, this result might suggest that this patient also had an HNPCC syndrome, as hmsh6 is only rarely implicated in the development of MSI in sporadic CRCs. 32 In our series, these 2 cases lacking hmsh6 expression represented % of MSI-H tumors. Therefore, it seems interesting to study the expression of hmsh6 by immunohistochemistry when a clinical or morphological suspicion of MSI exists, especially when an HNPCC syndrome is sus- Arch Pathol Lab Med Vol 127, June Microsatellite Instability in Colorectal Carcinoma Rigau et al 67

5 Table 2. Characteristics of Colorectal Adenocarcinomas and Results of Immunohistochemistry for Mismatch Repair Proteins in 24 Patients (All Numerical Results Are Expressed as Percentages Except the Mean Age in Years)* MSI status, % MSI-H MSI-L MSS Age, y (mean SD) Male sex, % Site, % Proximal Distal Rectum Differentiation, % Well-differentiated Moderately differentiated Poorly differentiated Histologic type, % Not otherwise specified Mucinous Medullary Expanding margins, % Increased intraepithelial lymphocytes, % Crohn-like reaction, % Adenomatous residue, % Astler-Coller stage, % A B1 B2 C1 C2 D pt stage, % T1 T2 T3 T4 pn stage, % N N1 N2 Jass stage, % I II III IV Lack of hmlh1 (n 22) Lack of hmsh2 (n 7) Lack of hmlh1 and/or hmsh2 and/or hmsh6 (n 31) Normal hmlh1, hmsh2 and hmsh6 (n 173) * MSI indicates microsatellite instability; MSI-H, at least 2 markers showed instability; MSI-L, only 1 marker showed instability; and MSS, microsatellite stable. Statistical comparison with tumors showing no loss of expression: P.1, P.1, and P pected, and when there is no loss of either hmlh1 or hmsh2 expression, although the MSI status of hmsh6- related tumors is still a matter of debate. 3,32,44 We also observed a close association between hmlh1 and hpms2 expression, as 15 of 22 cases with loss of hmlh1 also lacked hpms2, and 15 of 1 cases with loss of hpms2 also lacked hmlh1. This relationship between hmlh1 and hpms2 has been reported previously and is considered as secondary to the degradation of hpms2 in the absence of its binding partner, hmlh1. 11,32 We observed 4 cases with isolated loss of hpms2, but these 4 tumors were all MSS. It appears, therefore, that there is no need to include hpms2 in the panel of antibodies to be used when looking for MSI by immunohistochemistry. Although our unselected series mostly included sporadic CRCs, some cases developed in patients with an HNPCC syndrome. In our study, all cases identified by clinical criteria (Amsterdam or Bethesda) showed loss of expression of at least 1 MMR protein. However, it has been demonstrated recently that when a larger number of cases are studied, there is a significant proportion of cancer in HNPCC syndrome with germ-line mutation of hmlh1 or 68 Arch Pathol Lab Med Vol 127, June Microsatellite Instability in Colorectal Carcinoma Rigau et al

6 Table 3. Reference, y Thibodeau et al, 16 1 Dietmaier et al, 5 17 Cawkwell et al, 17 1 Marcus et al, 18 1 Chaves et al, 1 2 Cawkwell et al, 2 2 Dieumegard et al, 21 2 Jass, 22 2 Iino et al, 24 2 Ward et al, 21 Young et al, Stone et al, Salahshor et al, Chiaravelli et al, Lindor et al, 22 Lanza et al, Plaschke et al, Present series Total of 16 series Results of Immunohistochemistry of Mismatch Repair (MMR) Proteins to Assess Microsatellite Instability in Previously Documented Series and in the Present Series* No. of Cases MMR Proteins Studied by Immunohistochemistry, hmsh6, hpms2, hmsh6, hpms2, hmsh6, hpms2... Sensitivity, % Specificity, % Positive Predictive Value, % Negative Predictive Value, % * indicates not available. Results calculated with hmlh1 and hmsh2,, and hmsh6, and, hmsh6, and hpms2 immunostainings. All series were included except Young et al 11 and Salahshor et al. 26 hmsh2 that remains positive on immunohistochemistry for these 2 proteins. 45 These apparent discrepancies between molecular and immunohistochemical results may be due to the synthesis of mutant proteins that are detected by immunohistochemistry, but that are not functionally active. If these results are confirmed in other series, they may indicate that immunohistochemistry is unlikely to be a primary test for detecting the presence of germline mutations in HNPCC cases. However, as immunohistochemistry is a simple and cheap technique, it can be used to indicate which gene has to be studied for mutation, in case the corresponding protein is not expressed in the tumor. 46 In sporadic cancers, false-negative immunohistochemical results are unlikely to occur, because in most MSI-H tumors the promoter for the MMR gene is turned off by hypermethylation, and then no protein is produced. Immunohistochemistry could therefore be a useful screening method to detect MSI-H cases, which may need different chemotherapy. In our series, we observed the same clinical and morphologic characteristics of MSI-H CRCs that have been described previously: frequent loss of hmlh1 expression in elderly women, excess of mucinous and medullary types, poorly differentiated cancers, and circumscribed borders. 6,,11 On the contrary, none of these features was related to the MSI-L phenotype. However, although some features are relatively specific for the MSI-H phenotype, they cannot be used as the only detection test, owing to their low sensitivity. 6,1 Moreover, we did not confirm the relationship between the MSI-H phenotype and other features previously described in the literature, such as a Crohn-like lymphoid response and an increased number of intraepithelial lymphocytes. 6, 11,37 These discrepancies may be at least partially due to the relatively subjective diagnosis of these features, with criteria variable in different studies. For the intraepithelial lymphocyte count, it may also be explained by the count that we performed on hematoxylin-eosin stained sections, contrary to most studies with a count made by immunohistochemistry for T lymphocytes. 1,37 It seems therefore necessary to include an immunohistochemical study of MMR proteins when the MSI phenotype has to be determined in a large series of patients. In conclusion, our findings confirm that examination of MMR protein expression by immunohistochemistry is a simple method for diagnosing CRC with MSI. Our data suggest that the study of hmsh6 may be useful, in addition to hmlh1 and hmsh2. Moreover, immunohistochemistry could represent a screening method to direct the research of mutation of MMR genes observed in HNPCC syndrome, and to detect MSI-H sporadic CRCs that may need different chemotherapy. The authors acknowledge the Fonds de Recherche de la Société Nationale Française de Gastroentérologie (Tours, France), the Fonds de Recherche de la Société Française de Pathologie (Paris, France), and the Association Charles Debray (Chevigny Saint- Sauveur, France). Dr Rigau was supported by the Association pour la Recherche sur le Cancer (Villejuif, France). References 1. Lynch HT, De La Chapelle A. Genetic susceptibility to non-polyposis colorectal cancer. J Med Genet. 1;36: Rüschoff J, Bocker T, Schlegel J, Stumm G, Hofstaedter F. Microsatellite instability: new aspects in the carcinogenesis of colorectal carcinoma. Virchows Arch. 15;426: Ionov Y, Peinado M, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature. 13;363: Arch Pathol Lab Med Vol 127, June Microsatellite Instability in Colorectal Carcinoma Rigau et al 6

7 4. Fujiwara T, Stolker JM, Watanabe T, et al. Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences. Am J Pathol. 18;153: Dietmaier W, Wallinger S, Bocker T, Kullmann F, Fishel R, Rüschoff J. Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression. Cancer Res. 17;57: Jass JR, Do KA, Simms LA, et al. Morphology of sporadic colorectal cancer with D replication errors. Gut. 18;42: Nicholl ID, Dunlop MG. Molecular markers of prognosis in colorectal cancer. J Natl Cancer Inst. 1;1: Purdie CA, Piris J. Histopathological grade, mucinous differentiation and D ploidy in relation to prognosis in colorectal carcinoma. Histopathology. 2;36: Ward R, Meagher A, Tomlinson I, et al. Microsatellite instability and the clinicopathological features of sporadic colorectal cancer. Gut. 21;48: Alexander J, Watanabe T, Wu T-T, Rashid A, Li S, Hamilton SR. Histopathological identification of colon cancer with microsatellite instability. Am J Pathol. 21;158: Young J, Simms LA, Biden KG, et al. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol. 21;15: Gryfe R, Kim H, Hsieh ETK, et al. Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med. 2;342: Wright CM, Dent OF, Barker M, et al. Prognostic significance of extensive microsatellite instability in sporadic clinicopathological stage C colorectal cancer. Br J Surg. 2;87: Elsaleh H, Joseph D, Grieu F, Zeps N, Spry N, Iacopetta B. Association of tumour site and sex with survival benefit from adjuvant chemotherapy in colorectal cancer. Lancet. 2;355: Boland CR, Thibodeau SN, Hamilton SR, et al. A national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 18;58: Thibodeau SN, French AJ, Roche PC, et al. Altered expression of hmsh2 and hmlh1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes. Cancer Res. 1;56: Cawkwell L, Gray S, Murgatroyd H, et al. Choice of management strategy for colorectal cancer based on a diagnostic immunohistochemical test for defective mismatch repair. Gut. 1;45: Marcus VA, Madlensky L, Gryfe R, et al. Immunohistochemistry for hmlh1 and hmsh2: a practical test for D mismatch repair- deficient tumors. Am J Surg Pathol. 1;: Chaves P, Cruz C, Lage P, et al. Immunohistochemical detection of mismatch repair gene proteins as a useful tool for the identification of colorectal carcinoma with the mutator phenotype. J Pathol. 2;11: Cawkwell L, Sutherland F, Murgatroyd H, et al. Defective hmsh2/hmlh1 protein expression is seen infrequently in ulcerative colitis associated colorectal cancers. Gut. 2;46: Dieumegard B, Grandjouan S, Sabourin JC, et al. Extensive molecular screening for hereditary colorectal cancer. Br J Cancer. 2;82: Jass JR. hmlh1 and hmsh2 immunostaining in colorectal cancer. Gut. 2;47: Debniak T, Kurzawski G, Gorski B, Kladny J, Domagala W, Lubinski J. Value of pedigree, clinical data, immunohistochemistry and microsatellite instability analyses in reducing the cost of determining hmlh1 and hms2 gene mutations in patients with colorectal cancer. Eur J Cancer. 2;36: Iino H, Simms L, Young J, et al. D microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer. Gut. 2;47: Stone JG, Robertson D, Houlston RS. Immunohistochemistry for MSH2 and MHL1: a method for identifying mismatch repair deficient colorectal cancer. J Clin Pathol. 21;54: Salahshor S, Koelble K, Rubio C, Lindblom A. Microsatellite instability and hmlh1 and hmsh2 expression analysis in familial and sporadic colorectal cancer. Lab Invest. 21;81: Chiaravalli AM, Furlan D, Facco C, et al. Immunohistochemical pattern of hmsh2/hmlh1 in familial and sporadic colorectal, gastric, endometrial and ovarian carcinomas with instability in microsatellite sequences. Virchows Arch. 21;438: Perrin J, Gouvernet J, Parriaux D, et al. MSH2 and MLH1 immunodetection and the prognosis of colon cancer. Int J Oncol. 21;1: Lindor NM, Burgart LJ, Leontovich O, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol. 22;2: De La Chapelle A. Microsatellite instability phenotype of tumors: genotyping or immunohistochemistry? The jury is still out. J Clin Oncol. 22;2: Lanza G, Gafa R, Maestri I, Santini A, Matteuzzi M, Cavazzini L. Immunohistochemical pattern of MLH1/MSH2 expression is related to clinical and pathological features in colorectal adenocarcinomas with microsatellite instability. Mod Pathol. 22;15: Plaschke J, Kruger S, Pistorius S, Theissig F, Saeger HD, Schackert HK. Involvement of hmsh6 in the development of hereditary and sporadic colorectal cancer revealed by immunostaining is based on germline mutations, but rarely on somatic inactivation. Int J Cancer. 22;7: Vasen HFA, Watson P, Meklin J-P, Lynch HT, and the International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative Group on HNPCC. Gastroenterology. 1;116: Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al. A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines. J Natl Cancer Inst. 17;8: Jass JR, Ajioka Y, Allen JP, et al. Assessment of invasive growth pattern and lymphocytic infiltration in colorectal cancer. Histopathology. 1;28: Graham DM, Appelman HD. Crohn s-like lymphoid reaction and colorectal carcinoma: a potential histologic prognosticator. Mod Pathol. 1;3: Michael-Robinson JM, Biemer-Huttmann A, Purdie DM, et al. Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status. Gut. 21;48: Jass JR, Love SB, Northover JM. A new prognostic classification of rectal cancer. Lancet. 187;1: Hamilton SR, Aaltonen LA. Pathology and genetics: tumours of the digestive system. In: World Health Organization Classification of Tumours. Lyon, France: IARC Press; 2: Iino H, Jass JR, Simms LA, et al. D microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol. 1;52: Tomlinson I, Halford S, Aaltonen L, Hawkins N, Ward R. Does MSI-low exist? J Pathol. 22;17: Kane MF, Loda M, Gaida GM, et al. Methylation of the hmlh1 promoter correlates with lack of expression of hmlh1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 17;57: Schweizer P, Moisio AL, Kuismanen SA, et al. Lack of MSH2 and MSH6 characterizes endometrial but not colon carcinomas in hereditary nonpolyposis colorectal cancer. Cancer Res. 21;61: Parc YR, Halling KC, Wang L, et al. hmsh6 alterations in patients with microsatellite instability-low colorectal cancer. Cancer Res. 2;6: Wahlberg SS, Schmeits J, Thomas G, et al. Evaluation of microsatellite instability and immunohistochemistry for the prediction of germ-line MSH2 and MLH1 mutations in hereditary nonpolyposis colon cancer families. Cancer Res. 22;62: Paraf F, Gilquin M, Longy M, et al. MLH1 and MSH2 protein immunohistochemistry is useful for detection of hereditary non-polyposis colorectal cancer in young patients. Histopathology. 21;3: Arch Pathol Lab Med Vol 127, June Microsatellite Instability in Colorectal Carcinoma Rigau et al