[CANCER RESEARCH56, 4836-4840, November 1, 1996]

Advances in Brief

Altered Expression of hMSH2 and hMLH1 in Tumors with MicrosatelliteInstability and Genetic Alterations in Mismatch Repair Genes1

Stephen N. Thibodeau,2 Amy J. French, Patrick C. Roche, Julie M. Cunningham, David J. Tester,Noralane M. Lindor, Gabriela Moslem,3 sean M. Baker, R. Michael Liskay, Lawrence J. Burgart,Ronald Honchel,4 and Kevin C. Haffing@Department of Laboratory Medicine and Pathology (S. N. T., A. J. F., P. C. R., J. M. C., D. J. T., L J. B., R. H., K. C. H.] and Department of Medical Genetics (N. M. U, MayoClinic and Mayo Foundation, Rochester, Minnesota 55905; University of Dusseldoif@ Dusseldorf Germany (G. MI; and Department of Molecular and Medical Genetics. OregonHealth Science University, Portland, Oregon 97207 (5. M. B., R. M. LI

Abstract

To date, at least four genes involved in DNA mismatch repair (MMR)have been demonstrated to be altered in the germline of patients withhereditary nonpolyposis colon cancer: hMSH2, hMLH1, hPMSJ, andhPMS2. Additionally, loss of MMR function has been demonstrated tolead to the phenomenon ofmicrosateffite instabffity(MIN) in tumors fromthese patients. In this study, we have examined the protein expressionpattern of hMSH2 and hMLH1 by immunohistechemistry In paraffinembedded tumors from 7 patients with MIN+ sporadic cancer, 13 patients with fasnilial colorectal cancer, and 12 patients meeting the strictAmsterdam criteria for hereditary nonpolyposis colon cancer. The relationship between the expression of these two gene products, the presence

of germilne or somatic mutations, and the presence of tumor MIN wasexamined. Nineteen of the 28 tumors studied demonstrated MIN, whereasmutations in hMLHJ and hMSH2 were detected in 6 and 2 patients,respectively. Of the eight MIN+/mutation+ cases, the absence of proteinexpression was observed for the corresponding gene product in all but one

case (missense mutation in hMLHJ). However, seven MIN+/mutationcases also showed no expression ofeither hMLH1 (a 5), hMSH2 (n = 1),or both (n = 1), whereas four MIN+/mutation—cases demonstratednormal expressionfor both. Noneof the MIN—/mutation—cases (n = 9)demonstrated an altered expressionpattern for either protein. Thesedatasuggest that examination of protein expression by Immunohistochemistry

may be a rapid method for prescreening tumors for mutations in theMMR genes.

Introduction

HNPCC6 is a clinically defined autosomally dominant disorder thataccounts for approximately 6% of all cases of colon cancer (1). It ischaracterized by early age of onset, syn- and metachronous colorectalcancer, and cancers of other sites, including endometrium, stomach,small intestine, hepatobiliary tract, kidney, ureter, and ovary (1). Apedigree analysis is key in establishing the diagnosis of HNPCC. Forcomparative studies, the International Collaborative Group definedthe Amsterdam criteria for the identification of HNPCC kindreds,namely: (a) at least three relatives should have histologically verifiedcolorectal cancer, with at least two of them being first-degree relatives; (b) at least two successive generations should be affected; and

Received 7/26/96; accepted 9/18/96.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported by NIH Grant CA 68535-2.2 To whom requests for reprints should be addressed, at Laboratory Genetics/HI 970,

MayoClinic,200FirstStreetSW,Rochester,MN55905.Phone:(507)284-4696.3 G. M. was funded by Deutsche Forschungsgemeinschaft.

4 Current affiliation: Indiana University, Indianapolis, IN 46236.

5 Current affiliation: R. L. Roudebush Veterans Affairs Medical Center, Indianapolis,

IN 46236.6 The abbreviations used are: HNPCC, hereditary nonpolyposis colon cancer, MIN,

microsatellite instability.

(c) in one of the relatives, colorectal cancer should be diagnosed at

under 50 years of age (2). Although the clinical definition of thissyndrome was greatly facilitated by the establishment of the Amsterdam criteria, there remains a great deal of phenotypic variabilityamong families. Furthermore, pedigree analysis is an imprecise tool,and this imprecision impairs the reliability of studies that addressissues in FINPCC such as gene penetrance and tumor spectrum.

Recently, significant progress has been made in elucidating theunderlying molecular basis of HNPCC (3—5).This genetic disordercan now be explained, in part, by the presenceof germline mutationsin one of at least four genes that participate in DNA mismatch repair,including hMSH2, hMLHJ, hPMSJ, and hPMS2 (6—9).Mutations in/ZMSH2 and hMLHJ are thought to account for approximately 60% ofHNPCC kindreds, whereas only a few patients have had mutations inhPMSJ and hPMS2 (10). Interestingly, tumors from patients withHNPCC (1 1) and in a subgroup of patients with sporadic colon cancer(12, 13) have demonstrated a novel type of genetic instability characterized by alterations within microsatellites. Over the past threeyears, this phenomenon of MN has been reported in an ever increasing number of tumor systems (3, 4, 14). Although defective mismatchrepair is thought to be responsible for the MIN+ phenotype in tumorsfrom patients with HNPCC, the genetic defect responsible for theMIN+ phenotype in sporadic colon cancer has yet to be clearlydelineated.

The cloning and identification of DNA mismatch repair genes hasfacilitated identification of both germline and somatic mutations inindividuals with HNPCC. However, the wide spectrum of mutationsand the genetic heterogeneity involved with the disorder has beenproblematic. Furthermore, the methods available for the identificationof specific gene mutations, such as DNA sequence analysis andcoupled transcription-translation based assays, remain labor intensive.The presence of tumor MIN has offered a potential marker for theidentification of individuals who are at high risk for possessinggermline mutations in DNA mismatch repair genes. However, thepresence of tumor MIN does not necessarily define which one of thefour mismatch repair genes is involved, and more recent studies havenot detected germline or somatic mutations in a substantial number ofMIN+ sporadic colon cancer and MIN+ familial colorectal cancer(10, 15—18).An alternate method for the identification of defectivemismatch repair would be to directly examine tumor specimens forthe presence or absence of one of the DNA mismatch repair proteinsitself. Additionally, this would allow for a more direct assessment ofthe specific gene involved. In this study, we examine the proteinexpression of hMLH1 and hMSH2 in paraffin-embedded tumors frompatients with MIN+ sporadic colon cancer, patients with familialcolorectal cancer, and patients meeting the strict Amsterdam criteriafor HNPCC. The relationship between the expression of these two

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Table ISequencing, MIN, and clinicaldataCaseTissueGeneExonCodonBase

ChangesConsequenceMINstatushMLHIhMSH22F°T/N―hMLHI

(So)I 3499Del GFrameshiftPos(7/8)'@+3FT/N6MLHI(G)16590-591Del TAGAFrameshiftPos(7/8)—+4FT/NhMLHI(G)

hMSH2(G)1 321 167Del0

G —@CFrameshift Missense,Asp—*HisPos(28/32)—NA5ANhMLHJ

(G)I 3495Ins CFrameshiftPos(5/9)—+12ANhMLHI(G)8226C --vTNonsense, Arg —@stopPos(8/9)—+IFT/NhMSH2(So)14749G —@TNonsense, Glu —@stopPos(21/30)+—2ANhMSH2(G)5A —°T at 943 + 3In frame del of exon 5Pos(9/9)+—IST/NhMLHI(0)13492G —°AMissense, Ala —°ThrPos(30/34)++25T/NNegPos

(29/33)—+4ST/NNegPos(30/34)—+55T/NNegPos

(27/31)—+7ST/NNegPos(28/31)—+9FNNegPos(8/9)—+7ANNegPos

(3/6)+—1IFNNegPos(7/9)——35T/NNegPos

(28/32)++65T/NNegPos(31/34)++6FT/NNegPos(25/30)++lOFNNegPos(5/7)++l3FNNegNeg(0/9)++l4FNNegNeg(0/9)++1SFNNegNeg(0/9)++l6FNNegNeg(1/7)+NA17FNNegNA++8ANNegNeg

(0/6)+NA9ANNegNA+NAlIANNegNA++l5ANNegNeg

(0/9)++16ANNegNeg(0/5)++17ANNegNeg(0/5)++18ANNegNeg(1/9)++20ANNegNA++

EXPRESSIONOF MISMATCHREPAIRGENES

gene products, the presence of gennline mutations, and the presenceof tumor MIN was examined.

Materials and Methods

Patient Population. Thirty-two patients were identified for this study.Twelve of these fulfilled the strict Amsterdamcriteria (2) for HNPCC, 13had a family history of colon cancer, and 7 had sporadic colon cancer.Familial cases were defined as those individuals having at least one otherfamily member with colon cancer; in 9 of the 13 cases, this was afirst-degree relative. For these familial cases, the average number ofaffected (all cancers) individuals per kindred was 6, with a range of 3—16(including the proband), whereas the average number of colorectal cancerswas 3, with a range of 2—7(including the proband). When available,medical records were obtained on other affected family members to verifythe presence of cancer. In most instances, however, such records were notavailable. Tissue used for these studies included fresh frozen tumor,paraffin-embedded tumor, and peripheral blood leukocytes.

ImmunohistochemicalAnalysis. Immunoperoxidasestainingfor hMLH1in formalin-fixed, paraffin-embedded tissue sections was performed by alabeled streptavidin-biotin method. Six-pm sections were mounted on silanized slides, deparaffinized, and rehydrated through graded alcohols towater.Endogenousperoxidaseactivitywas blockedby incubationwith 0.6%H202. Sections were immersedin 10 m@isodiumcitratebuffer,pH 6.0, andsubjectedto heat-inducedantigenretrieval.Sectionswere then treatedwith10% normal goat serum for 10 mm to block nonspecific protein binding.

Mouse monoclonalantibodiesto hMLH1(clone G168-728, 1 @.tg/ml,PharMingen) were applied, and tissue sections were incubated overnight at 4°C.Aftera brief rinsing, sections were treated with biotinylated antimouse IgG for 30mm at room temperature, rinsed, and then incubated with peroxidase-labeledstreptavidin for 30 mm at room temperature. After a brief washing, sectionswere incubated with diaminobenzidine and H202 for 5 mm. Section were thenlightly counterstained with hematoxylin, dehydrated in graded alcohols,cleared in xylene, and coverslipped. For immunoperoxidase staining ofhMSH2, 6-@tmsections were deparaffinized, rehydrated, blocked, and sub

jected to antigen retrieval as described above. Mouse monoclonal antibodies(Clone FE1 I, 0.5 p.g/ml, Oncogene Science) were applied and tissue sections

were incubatedfor 20 mm at room temperature.After a brief rinsing, theCatalysed Signal Amplification system (DAKO Corp.) was used according to

the manufacturer's instructions for visualization of specific hMSH2 staining.Sections were counterstained and mounted as described for hMLHI . Thenormal staining pattern for both hMLH1 and hMSH2 was nuclear. Tumor cellsthat exhibited an absence of nuclear staining in the presence of nonneoplasticcells with nuclear staining were considered to have an abnormal pattern. CloneFEI I is a mouse monoclonal antibody generated with a carboxyl-terminalfragment of the hMSH2 protein, whereas clone 0168-728 was prepared with

full-length hMLH1 protein.DNA Extraction. DNA extractionfromfreshfrozentissue was performed

as previously described (19). Normal and tumor DNA were also obtained fromparaffin-embedded tissue. Using Toluidine Blue-stained l0-@.tm-thicksections,areas of either normal or malignant tissue (70% neoplastic cells) weremarked on the underside of the slide, and the tissue was scraped into a I .5-mlmicrocentrifuge tube. Digestion was carried out by the addition of 20 mg/mIproteinase K in a 50 mr@iTris buffer, pH 8.3 (2 @lproteinase K/l00 @lbuffer/lcm2 tissue) for 48 h at 55°C. Undigested tissue was removed by centrifugation,

and proteinase K was inactivated by boiling for 8 mm. Samples were rapidlycooled, and the DNA was stored at 4°C.

DNA Analysis. DNA sequence analysis and analysis for tumorMIN haspreviously been reported for this patient population (15). DNA sequenceanalysis was performed by the Sanger dideoxy chain termination method withthe use of the fmol sequencing kit (Promega, Madison, WI). For MIN, paired

normal and tumor DNA were analyzed with a common set of 34 microsatellitemarkers. The number used ranged from a minimum of 9 to a maximum of 34markers. All specimens were examined initially with the same group of 9markers. MIN was defined by the presence of novel fragments following PCRamplification of tumor DNA that were not present in the PCR product generated by normal DNA. Tumors were considered MIN+ if 30% or more of theloci examined demonstrated instability.

a F, familial; S. sporadic; A, Amsterdam criteria; T, fresh frozen tumor; N, normal; So, somatic mutation; 0, germline mutation; Del, deletion; Ins, insertion; Pos, positive; Neg,

negative; NA, not available or no amplification.b Tissue available for sequence analysis.C Number of markers showing instability/total number tested.

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EXPRESSiONOF MISMATCHREPAIRGENES

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Results and Discussion

In this study, we have evaluated the relationship between tumorMIN, the presence of germline or somatic mutations in hMSH2 andhMLH1, and the expression of the corresponding gene products intumor tissues. Individuals were identified for this study based oneither the presence of MIN in their tumor or the presence of a positivefamily history of colon cancer. Overall, there were 7 patients withsporadic colorectal cancer, 13 families with familial colon cancer, and12 patients with HNPCC. Results of the DNA sequence analyses andanalysis for tumor MIN in this group of patients (a subset of a largergroup) have been reported previously (15).

Nineteen (68%) of the 28 tumors included in this study demonstrated widespread MIN (Table 1). Four additional tumors could notbe evaluated due to amplification problems or the unavailability ofappropriate tissue. Four of 9 tumors from patients with HNPCC, 8 of13 tumors from patients with familial colon cancer, and all 7 tumorsfrom patients with sporadic colon cancer were MIN+.

The hMLHI and !ZMSH2 genes were sequenced in tumor andnormal colomc DNA from 12 patients and in leukocyte DNA onlyfrom the other 20 (Table 1). Changes in the hMLHJ gene weredetected in six patients (one sporadic, four familial, and one HNPCC),whereas two others showed a sequence change in /ZMSH2(one familial, one HNPCC);two weresomatic(case lF and2F),andsix weregermline alterations (Table 1). All but one of these mutations arepredicted to result in a truncated protein, and thus, these are likely tobe thecausativemutations.Theclinicalsignificanceof theone missense mutation (case lS) is, at this point, unknown. It is of significance that all of the tumors with mutations in either hMSH2 orhMLH1 were MIN+. However, not all of the MIN+ tumors had arecognizable mutation. These included six of seven patients with

sporadic colon cancer, four of eight patients with familial coloncancer, and one of four patients with HNPCC.

Protein expression for hMLHI and hMSH2 was examined in paraffin-embedded material from 32 and 28 patients, respectively (Fig.1). In all sections examined, there were nonneoplastic cells thatdemonstrated positive nuclear staining for both hMSH2 and hMLH1.However, an absence of hMLH1 expression was observed in 10tumors, an absence of hMSH2 expression in 3 tumors, and an absenceof both in 1 tumor (Table 1). In all 14 cases, altered expression ofeither hMSH2 or hMLH1 was associated with tumor MIN. None ofthe MIN—tumors (n = 9) demonstrated an altered expression patternfor either protein. Of those MIN+ tumors having mutations in eitherhMLHJ (n = 6) or hMSH2 (n = 2), the absence of one of the twomismatch repair gene products was associated with the presence of amutation in the corresponding gene in all but one case (Table 1). Inthis one discordant case (case lS), the patient had a missense mutationin exon 13 of hMLHJ and normal hMLH1 expression in the tumor. Inaddition to the MIN+/mutation+ cases that showed no detectableprotein expression with the neoplastic cells, seven MIN+/mutationcases also showed no expression of either hMLH1 (n = 5), hMSH2(n 1), or both (n = 1), whereas four MIN+/mutation— casesdemonstrated normal expression of both proteins.

These results confirm and extend those observations first made byLeach et aL (20), in which hMSH2 immunoreactivity was not detectedin colorectal tumors from a few patients with germline hMSH2mutations. Our study, however, extends these initial observations byexamining the expression pattern of both hMSH2 and hMLH1 in alarger series of patients and correlating these changes with the presence of gene mutations and tumor MIN. Our results showed that anabsence of protein expression for both hMSH2 and hMLH1 in neo

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EXPRESSIONOF MISMATCHREPAIRGENES

plastic cells was associated with the presence of widespread tumorMIN. All 14 cases lacking protein expression had MIN+ tumors,whereas all of the tumors that were MIN—(n 9) had normal proteinexpression patterns for both proteins. Additionally, the presence of agermline or somatic mutation in one of the DNA mismatch repairgenes, hMHS2 and hMLHJ, was highly associated with the presenceof both tumor MN and the absence of protein expression in thecorresponding gene product. All eight cases with either a somatic orgermline mutation had MIN+ tumors, and in all but one case, anabsence of protein expression was observed. Because none of theMIN—cases had altered protein expression, the immunohistochemical analysis of tumor tissue may be a useful screen for the detectionof defective mismatch repair. Additionally, this may be helpful for theidentification of a high-risk group of individuals having HNPCC.Other than family history, there is still a lack of useful markers forestablishing the diagnosis of HNPCC.

It is important to note that all but one of the hMSH2 and hMLHJgene alterations identified in this study are predicted to result in atruncated gene product. The lack of protein expression in the tumorfrom these cases suggests that aberrant hMSH2 and hMLH1 polypeptides or the mRNAs encoding them are unstable (20). Although theclinical significance of the one missense mutation detected is Unknown, such mutations may give rise to normal protein levels butabnonnal function. The immunohistochemical detection of protein,therefore, does not necessarily imply normal DNA mismatch repairfunction. These cases, however, would be predicted to still maintaintheir MIN+ tumor phenotype. It is also interesting to note thatindividuals with germline mutations (i.e., heterozygous) in eitherhMSH2 or hMLHJ have normal expression in nonneoplastic colonictissue and no expression in the adjacent adenocarcinoma. These datafurther support the idea that both alleles of the mismatch repair genesneed to be eliminated for abnormal function.

Not all of the tumors that lacked expression of hMSH2 or hMLH1protein had identifiable germline or somatic mutations. There wereseven such cases: four sporadic, two familial, and one HNPCC. Theseindividuals, however, may have mutations that were not detected bythe sequencing method used, such as deletions of entire exons (10) oralterations in the promoter region. For three of the seven cases, tumorDNA was notavailablefor study.The lackof proteinexpressioninthese cases, therefore, might be explained by the presence of somaticalterations in either hMSH2 or hMLH1. Alternatively, there may beother proteins or factors that are involved in the regulation or stabilization of hMSH2 or hMLH1 protein levels. Alterations of thesecomponents may influence both the amount of mismatch repair protein and subsequently MIN. It is interesting to note that of the sevenMIN+/mutation—cases that lacked protein expression, hMLH1 wasinvolved in the majority (n = 6).

Four MIN+/mutation— cases had normal expression of both geneproducts. In three of these, DNA from tumor was also available forsequence analysis, ruling out the presence of both germline andsomatic alterations. Although it is possible that missense mutations inhMSH2 or hMLH1 might have been missed, these data more likelysuggest that alterations in other mismatch repair genes, such ashPMSJ, hPMS2, or genes as yet unidentified, may be involved ingenerating the MIN+ phenotype.

The availability of antibody for both hMSH2 and hMLH1 shouldfacilitate the study of a large number of patients with familial coloncancer. Furthermore, it will be important to further study such caseswith antibody directed against hPMS1 and hPMS2. To date, thepresence of germline mutations in hPMSJ and hPMS2 has beenidentified in only a few patients (7, 10). The role of these two genesin HNPCC and MIN, therefore, remains to be clarified. The analysisof protein expression for both hPMS1 and hPMS2 by immunohisto

chemistry in MIN+ tumors and tumors from patients with familialcolon cancer should help to resolve some of these issues.

Overall, one-half of the tumors with abnormal hMLH1 or hMSH2protein expression had a mutation in the corresponding gene, and amutation in either gene resulted in abnormal expression in all but onecase. There was no case in which DNA sequence analysis implicatedone gene and immunohistochemical analysis implicated the other.Because antibody staining is more available than DNA analysis in aclinical setting, the use of immunohistochemistry appears to offer arelatively convenient and rapid method for prescreening tumors fordefects in the expression of mismatch repair genes. Ultimately, thistechnique, along with testing for tumor MIN, should help to identifythose individuals who may have germline mutations in the mismatchrepair gene complex. However, verification of these findings in amuch larger number of patients is necessary. Furthermore, it will alsobe importantto identify the underlying molecular defect in thoseMIN+ tumors that have an absence of protein expression but noapparent gene mutations.

References1. Lynch, H. T., Smyrk, T., and Lynch, J. F. Overview of natural history, pathology,

molecular genetics and management of HNPCC (Lynch syndrome). Int. i. Cancer(Pred. Oncol.), 69: 38—43,1996.

2. Vasen, H. F. A., Mecklin, i-P., Meera Khan, P., and Lynch, H. T. The InternationalCollaborative Group on Hereditary Non-polyposis Colorectal Cancer. Dis. ColonRect. 34: 424—425, 1991.

3. Honchel, R., HaIling, K. C., and Thibodeau,S. N. Genomic instability in neoplasia.Cell Biol., 6: 45—52,1995.

4. Maim, G., and Boland, C. R. Hereditary nonpolyposiscolorectal cancer: the syndrome, the genes, and historical perspectives. i. Nail. Cancer Inst., 87: 1114—1125,1995.

5. Rhyu, M. S. Molecular mechanisms underlying hereditary nonpolyposis colorectalcarcinoma. i. Nati. Cancer Inst., 88: 240—251,1996.

6. Bronner, C. E., Baker, S. M., Morrison, P. T., Warren, G., Smith, L. G., Lescoe,M. K., Kane, M., Earabino, C., Lipford, i., Lindblom, A., Tannergard, P., Bollag,R. i., Godwin, A. R., Ward, D. C., Nordenskjold, M., Fishel, R., Kolodner, R., andLiskay, R. M. Mutation in the DNA mismatch repair gene homologue hMLH1 isassociated with hereditary nonpolyposis colon cancer. Nature (Lond.), 368: 258—261,1994.

7. Nicolaides, N. C., Papadopoulos, N., Liu, B.. Wei, Y-F., Carter, K. C., Ruben, S. M.,Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D.,Venter, i. C., Dunlop, M. G., Hamilton, S. R., Petersen, G. M., de la Chapelle, A.,Vogelstein, B., and Kinzler, K. W. Mutations of two PMS homologues in hereditarynonpolyposis colon cancer. Nature (Lond.), 371: 75—80,1994.

8. Leach, F. S., Nicolaides, N. C., Papadopoulos. N., Lui, B., ien, J., Parsons, R.,Peltomäki,P., Sistonen, P., Aaltonen, L. A., Nystrom-Lahti, M., Guan, X-Y., Zhang,i., Metzler, P. S., Yu, J-W., Kao, F-T., Chen, D. I., Cerosaletti, K. M., Fournier,R. E. K., Todd, S., Lewis, T., Leach, R. i., Naylor, S. L., Weissenbach, i., Mecklin,i-K., iarvinen, H., Petersen, G. M., Hamilton, S. R., Green, i., iass, i., Watson, P.,Lynch, H. T., Trent, i. M., de la Chapelle, A., Kinzler, K. W., and Vogelstein, B.Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell, 75:1215—1225,1993.

9. Papadopoulos,N., Nicolaides,N. C., Wei, Y-F., Ruben,S. M., Carter, K. C., Rosen,C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter,i. C., Hamilton, S. R., Petersen, 0. M., Watson, P., Lynch, H. T., Peltomaki, P.,Mecklin, i-P., de la Chapelle, A., Kinzler, K. W., and Vogelstein, B. Mutation of amutL homolog in hereditary colon cancer. Science(Washington DC), 263: 1625—1629,1994.

10. Lui, B., Parsons,R., Papadopoulos,N., Nicolaides,N. C., Lynch, H. T., Watson, P.,iass. J. R., Dunlop, M., Wyllie, A., Peltomaki, P., de Ia Chapelle, A, Hamilton, S. R.,Vogelstein. B., and Kinzler, K. W. Analysis of mismatch repair genes in hereditarynon-polyposis colorectal cancer patients. Nat. Med., 2: 169—174,1996.

I I. Aaltonen, L. A., Peltomaki, P., Leach, F. S., Sistonen, P., Pylkkhnen, L., Mecklin,i-K., iBrvinen, H., Powell, S. M., ien, i., Hamilton, S. R., Peterson, G. M., Kinzler,K. W., Vogelstein, B., and de Ia Chapelle, A. Clues to the pathogenesis of familialcolorectal cancer. Science (Washington DC), 260: 812—816,1993.

12. Thibodeau, S. N., Bren, G., and Schaid, D. Microsatellite instability in cancer of theproximal colon. Science (Washington DC), 260: 816—819, 1993.

13. Ionov,Y., Peinado,M. A., Malkhosyan,S.,Shibata,D., andPerucho,M. Ubiquitoussomatic mutations in simple repeat sequences reveal a new mechanism for coloniccarcinogenesis. Nature (Lond.), 363: 558—561.1993.

14. Wooster, R., Cleton-iansen, A. M., Collins, N., Mangion, i., Cornelis, R. S., Cooper,R. S., Gusterson, B. A., Ponder, B. A. i., von Diemling, A., Wiestler, 0. D., Cornliese,C. i., Devilee, D., and Stratton, M. R. Instability of short tandem repeats (microsatellites) in human cancers. Nat.. Genet., 6: 152—156,1994.

15. Moslem, 0., Tester, D. i., Lindor, N. M., Honchel, R., Cunningham, J. M., French,A. i., Halling, K. C., Schwab, M., Goretzki, P., and Thibodeau, S. N. Microsatelliteinstability and mutation analysis of hMSH2 and hMLH1 in patients with sporadic,familial and hereditary colorectal cancer. Hum. Mol. Genet., 5: 1245—1252,1996.

4839

on July 20, 2021. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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16. Liu, B., Nicolaides,N. C., Markowitz, S.,Willson, i. K. V., Parsons,R. E., ien, i.,Papadopolous, N., Peltomaki, P., de Ia Chapelle, A., Hamilton, S. R., Kinzler, K. W.,and Vogelstein, B. Mismatch repair gene defects in sporadic colorectal cancers withmicrosatellite instability. Nat. Genet., 9: 48—55,1995.

17. Borresen, A-L., Lothe, R. A., Meling, G. I., Lystad, S., Morrison, P., Lipford, i.,Kane, M. F., Rognum, R. 0., and Kolodner, R. D. Somatic mutations in the hMSH2gene in microsatellite unstable colorectal carcinomas. Hum. Mol. Genet., 4: 2065—2072, 1995.

18. Mute, H., Noguchi, M., Perucho, M., Ushio, K., Sugihara, K., Ochiai, A., Nawata, H.,

and Hirohashi, S. Clinical implications of microsatellite instability in colorectalcancers. Cancer (Phila.), 77: 265—270,1996.

19. Cunningham, J., Lust, i. A., Schaid, D. i., Bren, 0. D., Carpenter, H. A., Rizza, E.,Kovach, i. S., and Thibodeau, S. N. Expression of p53 and lip allelic loss incolorectal carcinomas. Cancer Res., 52: 1974—1980, 1992.

20. Leach,F.S.,Polyak,K., Burrell,M., iohnson,K. A., Hill, D., Dunlop,M. G.,Wyllie,A. H.. Peltomaki, P., de la Chapelle, A., Hamilton, S. R., Kinzler, K. W., andVogelstein, B. Expression of the human mismatch repair gene hMSH2 in normal andneoplastictissues.CancerRes.,56:235—240,1996.

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1996;56:4836-4840. Cancer Res   Stephen N. Thibodeau, Amy J. French, Patrick C. Roche, et al.   Repair GenesMicrosatellite Instability and Genetic Alterations in Mismatch Altered Expression of hMSH2 and hMLH1 in Tumors with

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