Microsatellite instability in human solid tumors

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Microsatellite instability in human solid tumors A genome-wide instability has been found in almost all analyzed malignant tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC), and in a subgroup of sporadic (noninherited) cancers of the same type.This mutator phenotype was initially seen as novel alleles at microsatellite loci (a family of repetitive DNA sequences) and was shown to be caused by mutations in the highly conserved mismatch repair genes. Mutations have been found in each of four of these human genes: hMSH2, h/W-/I, hPMS7 and hPMS2, in the germline of HNPCC patients and in their tumors, as well as in sporadic tumors.These recent discoveries provide new molecular diagnostic tools for the detection of patients at high risk of developing carcinomas of the large bowel and other HNPCC-related tumors. Ongoing international research is progressively solving many of the unanswered questions at the genotypic and phenotypic levels of this newly identified mechanism in carcinogenesis. THE genetic changes (mutations) that are known to cause or contribute to neoplastic growth occur within genes belonging to two major gene classes, the proto-oncogenes and the tumor suppressor genes. The proteins encoded by these genes perform opposing functions during normal homeostasis; the proto-oncogenes stimulate cell proliferation and the tumor suppressors inhibit cell proliferation. The balance is altered if a proto-oncogene is activated to an oncogene, or if a tumor suppressor gene is inactivated, either of which can result in abnormal growth stimulation. During the past three years, evidence has accumulated showing that a third class of genes involved in DNA repair (the mismatch repair genes) can contribute to tumor growth if their normal DNA sequence is mutated. Several cancers are thought to arise through a stepwise accumulation of mutations. One of the best-characterized malignancies at the genetic level is carcinoma of the colon and rectum. Most carcinomas of the large bowel arise from adenomas and, in parallel with the phenotypic adenoma-carcinoma progression, a stepwise acquisition Copyright

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of genetic changes occurs’ (Fig. 1). According to the somatic mutation theory of cancer, FAP HNPCC these changes are causative events in tumoriGermline mutations \ / hMSH2, hMLH1, genesis. The genetic model for colorectal hPMS1, hPMS2 carcinogenesis put forward by Fearon and Vogelstein’ summarizes the genetic changes that preferentially occur at the various phenotypic stages. The sequence of events Normal includes deletions of chromosome arm 5q, Somatic mutations in Somatic mutations in which can inactivatethe tumor suppressor gene oncogenes and/or mismatch repair genes for familial adenomatous polyposis (AK), tumor suppressor genes 1. and/or mutations of the proto-oncogene K-RAS (which encodes ~21” and maps to Hyperptasia chromosome arm 12~) as some of the early events. Deletions of chromosome arm 18q, which result in inactivation of the tumor supMicrosatellite pressor gene DCC (deleted in colorectal instability APC/MCC p cancer), occur later and are frequently found KRAS in large adenomas and in carcinomas. Another N Ad late event, the inactivation of the tumor suppressor gene TP53 (which encodes tumor villous component -protein 53 and maps to chromosome arm DCC m17p), marks the transformation to malignant J TP53 growth. hMSH2 Some individuals are predisposed to canhMLH7 Others cer as the result of germline mutations; as IN Ca many as 20% of adenomatous polyps and carcinomas of the colon and rectum are estimated to occur in familial aggregates’, including several well-known familial syn+ Others dromes, some of which follow an autosomaldominant inheritance pattern. One of these, the hereditary non-polyposis colorectal cancer syndrome (HNPCC), is presumed to account for l-5% of all colorectal cancers. A gene co-segregating with this disease Figure 1. Gene mutations contributing to colorectal tumorigenesis. Abbreviations: FAP, familial adenowas mapped in 1993 to chromosome band matous polyposis; HNPCC, hereditaty non-polyposis colorectal cancer. Mismatch repair genes are coded 2p15-p16 by linkage analysis in families in red and oncogenes and tumor suppressor genes in blue. with HNPCC selected according to strict clinical criteria3. Later that year, this HNPCC L gene was isolated”x5.Initially, it was assumed that the HNPCCgenemappingto 2~15~16 wasa tumorsuppressor the genome.Sometimesa distinctionis madebetween2 bp repeat gene.Thus, accordingto the two-hit model for inactivation of a microsatellitesand 3-5 bp short tandemrepeats.The dinucleotide tumor suppressor genewith a recessiveaction at the cellular level, repeatsare estimatedto be the most frequently occurring microboth parentalalleleswouldbe expectedto be inactive6.If one allele satellites,with an incidenceof at least1 every 100 000 bp. Locusis carrying a mutation,the second,wild-type alleleis expectedto be specificfragmentscan be amplified by PCR, by using the short inactivatedasa resultof a larger deletion,at leastin sometumors. sequences flanking the repeatsasprimers.This locusspecificity,and Such deletionscan be detectedin tumor DNA by analyzing DNA the fact that the two parentalallelesare often of different lengths markersin the candidateregion,and comparingthe result with the becauseof a variablenumberof repeatsbetweenthem,makesthese patient’s normal genotype for those markers.However, the 2p polymorphicmarkerssuitablefor a wide rangeof analyses.Sincethe microsatellitemarkersanalyzeddid not reveal lossof one parental initial reportsof microsatelliteinstability in colorectalcarcinomas, allele in neoplastictissue;instead,additionalnew alleleswere fre- suchmarkersmappingto all humanchromosomes have beenused quently seenat the analyzedloci (microsatelliteinstability), suggest- to analyzenumeroushumantumorsfrom varioustissues(Tables1 ing an erroneousreplicationprocess7-9. In this way, colorectalcar- and2). cinogenesis wastied to a novelmechanism (Fig. 1).

Apt\

Microsatellites Microsatellitesare shortnucleotidesequences [2-5 basepairs(bp)] that areusuallyrepeated15-30 timesanddistributedthroughoutthe 62

/

Mismatch repair genes:from bacteria to humans This genome-widemutation pattern at short repetitive DNA sequencesresembleda mutationalfingerprint found in Escherichia coli andwasknown to be causedby failure of the mismatchrepair

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system, mutHLS, in this organism (Fig. 2). Mismatch repair differs from nucleotide and base excision repair in that the components of the system recognize normal nucleotides that are either mispaired or unpaired, rather than recognizing abnormal nucleotides”. Mismatched nucleotides arise in the DNA by: misincorporation by DNA polymerase during replication; physical damage to existing nucleotides; or as the result of forming heteroduplex intermediates during genetic recombination. Although the most-studied and best-understood mismatch repair pathway remains mutHLS in E. coli, certain components of this system are strongly conserved throughout evolution (Fig. 3) and have facilitated the identification and characterization of yeast and human mismatch repair genes. The Saccharomyces cerevisiae genes MSHl, MSH2 and MLHl, which are homologous to mutS and mutL in E. coli, were cloned by using a degenerate PCR approach”. Using the same methodology, Kolodner and co-workers cloned the first human mismatch repair gene, hMSH2, in 1993, which was published simultaneously by the groups of Vogelstein and de la Chapelle, who used a positional cloning approach4B5. During the past three years, five components of the human mismatch-repair system have been isolated4B5.“m*4. These are: hMSH2 (human mutS homolog 2), hMLH1 (human mutL homolog 1), hPMSl

and hPMS2 (human homologs of mutL that, if inactivated in Saccharomyces cerevisiae, cause a high frequency of post-meiotic segregation) and GTBP (G/T-binding protein; a human mutS homolog). There is a third human mutS homolog, hMSH3, but its role in mismatch repair is so far poorly understood”.

Mismatch

repair and HNPCC

Two lines of evidence suggested that hMSH2 was one of the HNPCC genes. First, it mapped to the same chromosomal position as the markers linked to the disease. Second, phenotypic instability - a characteristic of mismatch repair defects - was found in tumors from these patients. Conclusive evidence that hMSH2 was the diseasecausing gene in at least some of the HNPCC families came when germhne mutations were identified in affected family members4x5.Later, it was shown that mutations in hMSH2 were also responsible for the Muir-Torre syndrome’, a variant of HNPCC characterized by cutaneous tumor manifestations in addition to the tumor types characteristic of the HNPCC syndrome. Current data suggest that hMSH2 is one of the major HNPCC genes, causing 3040% of all cases455,‘5, but three other components of the human mismatch repair system have also been cloned and shown to be mutated in the germline of HNPCC patients.The mostimportantof thesegenes, hMLHl, mapsto chromosomearm 3p and accountsfor an additional30% of the germTable 1. Microsatellite instability in sporadCcand familial cokwectal line mutationsin HNPCC families”~‘2~‘5~‘6. The two hPMSgenes,whichmapto chromotumors” somearms 2q (hPMS1) and 7p (hPMS2), Tumortype No. of tumors %OftUmOtSWith No.ofboi fhf. have been found to be mutated in the germlineof a few HNPCCpatients’3x’5. 8nalyzsd m~rosatesite im 8nal*

Sporadic tumors 33 46 70

3 0 IO

7 4 13

17 21 38

46 137 90 49 230 108 80 158

13 12 26 16 16 6.5 20 12

7 4 7 7 5 8 4

7 8 9 17 20 21 22 23

36 20

22 20

8 43

22 41

Adenomas

14 (HNPCC)

57

7

17

Carcinomas

12 (HNPCC) 29 (HNPCC) 4 (HNPCC) 13 (Lynch I and II) 31 (early onset)” 9 (Muir-Ton@

75 86 100 31 56 67

7 7 9 7 4 4

7 17

Adenomas

Carcinomas

M&stases(liver) Familial tumors

fbbfeviatione: HNPCC, here&q non-plyposis colorectei cancer. bathe minimum number of microe~tellite !wi that were enatyeed in all tumors. ‘Patients younger than 35 years.

18 20 23 49

Microsatellite tumors

instability

in solid

Hereditary versussporadic cancer Phenotypicinstability,believedto be caused by mutationsof mismatchrepair genes,is morefrequentin tumorsfrom familial cases thanin sporadictumorsof the sametype, as illustratedfor colorectaltumorsin Table 1. Colorectal cancer is emphasizedin this review for two reasons.First, it is the most commonmalignancy(in primary aswell as metachronictumors) in HNPCC families, even amongthose individuals with extracolonic tumors,suchasendometrial,gastric, pancreatic and ovarian cancers. Second, carcinomasof the large bowel have been more extensively studiedfor microsatellite instability; 75100% of HNPCC carcinomas exhibit the instabilityphenotype7”7~‘8, implying that screening with microsatellitemarkers couldbe usedasa molecular-diagnostic tool. If a family fulfills the clinical criteria for HNPCC, linkage analysis of the family and/orinstability studiesof tumorscould be performedto indicatethe mostlikely target (mismatchrepair gene) for germlinemutations.This will reducethe amountof work in the searchfor the disease-associated mutation. The coding sequences for the two 63

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major HNPCC genes, hMsH2 and hMLHI, are divided into 16 and 19 exons, respectively. Table 2. Microsatellite Instability in human solid tumors” No mutational ‘hot spots’ have been found in these genes,unlike the 77’53 suppressor gene”. mmor typeb No. of tumors % of tumors with Ref(s)c Therefore it will be necessary to screen the microsstellits instability whole gene, preferably with a combination of Tumor types &uWerktk of the HNPCC tumor spectrum different methods, to ensure that all possible Color6ctal adenomas 149 5.5 d mutations are detected. It can, for various reasons, be difficult to obtain linkage data in Colorectal carcinomas 898 15 d families; however, microsatellite analysis of 22 27 Endometrium 18 tumors is almost always feasible because 36 17 30 archival material (such as formalin-fixed paraffin-embedded sections) can now be used Stomach 57 39 26 for such analysis. If no instability is detectedin 34 32 27,29 a tumor from an HNPCC patient, it is unlikely 52 31 28 that either RMsH2 or /zMLHl is mutated. Ovary 19 16 26 Previous studies of sporadic colorectal 10 31 20 carcinomas (n = 898) have shown that a Pancreas 9 67 26 subgroup of 15% exhibit microsatellite instability (Table 1)7-9~‘7~20-23. Whether or not this Tumor types not chsrsctertstk of the NNPCC tumor spectrum instability in sporadic colorectal tumors is 2 31 Brain 54 caused by mutations in the same genes as in Breast 26 4 26 the familial cases is not yet clear, but mutated 0 20 84 mismatch repair genes have already been found 104 11 31 in some sporadic microsatellite-unstable tuBladder 200 3 35 mors2’~*5. Tables 1 and 2 summarize the 41 36 61 instability frequencies observed in sporadic tumors, some of which occur nonrandomly 15 26 Cervix 13 in the HNPCC syndrome. The differences in 89 8 50 microsatellite instability frequencies detected 2 27 Lung NSCLC 87 among tumors of the same type in different 55 29 33 studies could be explained by: (1) a variation 45 32 in the number of loci analyzed; (2) the definiLung SCLC 33 37 0 33 tion of instability used (how large a percentage of the analyzed loci needs to be detected Prostate 68 20 51 as unstable); (3) which markers and type of 11 31 Soft tissue 18 markers (di-, tri- or tetranucleotide repeats) were analyzed; and (4) the fact that different 0 20 TestiS 86 materials were examined. These parameters 29 21 34 might also, at least in part, account for the variable instability frequencies found among *Abbrevi8tion8: HNPCC, heredllaly non-pdyposis coforectal cancel; NSCLC, non-sm8lk8ll lung cancer; SCLC, M8lfdifferent tumor types. It is also unlikely that cell lung c8ncer. all of the previously analyzed cases (Table 2) ?he different organs for mdignant tumors em indffte~ Lwdgn and malignant tumore are inckided only for colorectum. cSeleti8d references. were truly sporadic, because very few studies have documented the sporadic nature dSumm8riz8d from T8bl8 1, excludiig the familial c8se8. using information obtained by questionnaires and/or from cancer registries. Even when known HNPCC families are excluded from analyses, other familial and early-onset cases can unknowingly be numerous alleles often observed in the HNPCC tumor types (Fig. 4a, b; included in the series analyzed, and thereby result in an overesti- R.A. Lothe, unpublished). mation of the instability frequency among ‘sporadic’ cases. Among sporadic tumors, other than colorectal cancers, that are The number of microsatellite loci analyzed determines the part of the HNPCC tumor spectrum (gastric, endometrial, pancreatic ,frequency of instabilky and ovarian cancers), a significant subgroup exhibits the instability In lung carcinomas, microsatellite instability is reported at frequenphenotypezG3’, and the pattern is comparable to that found in colo- cies of 045%, thus illustrating the effect of the confounding factors rectal carcinomas (Fig. 4a). Sporadic tumors of types not characteris- mentioned above. Merlo et al.‘* reported instability in 15/33 (45%) tic to the HNPCC tumor spectrum generally show microsatellite small-cell lung carcinomas at one or more of 34 analyzed loci, The instability at lower frequencies (Table 2), and the pattern is often percentage of altered loci within each tumor in this study varied from characterized by one or only a few new alleles, in contrast to the 4% to 44%, suggesting that, in general, the ratio of the number of

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altered loci to the number of tested loci is less than the value typically seen in extremely unstable colorectal cancers (~50% affected loci). This implies different underlying causes of the phenotypic changes in lung cancer compared with colorectal cancer. Among the 15 unstable tumors reported by these investigators3’, 8 were affected at more than 10% of the loci examined. By contrast, Adachi et ~1.~~analyzed 11 loci in 37 comparable lung tumors, and found no microsatellite instability. According to the report by Merlo et aL3*,instability is expected to occur at 10% of the loci in 24% of the tumors, suggesting that 9 tumors in the report by Adachi et ~1.~~ shouldhaveshownnewallelesat onelocus. Although the numberof analyzedloci alone doesnot explain the

a

Methyl group

i

GTTC -Old -

Mismatched

b

CTAG bases

strand New strand

MutS

Glossary APC - Adenomatous polyposis coli, the tumor suppressor gene that, if mutated in the germline, causes familial adenomatous polyposis. APC is located on chromosome arm 5q.

MutL

c

/XC - Deleted in colorectal cancer; a tumor suppressor gene that is frequently inactivated in colorectal adenomas and carcinomas. DCC is located on chromosome arm 18q. Degenerate PCR approach -A cloning strategy in which primers are designed to represent all possible coding sequences for a selected peptide region. Such degenerate primers were designed to target the protein sequences in the conserved regions of the bacterial mismatch repair components and used to identify the human homologs.

MutS I

UvrD helicase ssDNA exonuclease

K-RAS- A human proto-oncogene originally identified in the Kirsten strain of murine sarcoma virus. K-RAS is located on chromosome arm 12~.

I

MCC- Mutated in colorectal cancer, a tumor suppressor gene that is frequently deleted in colorectal tumors. MCC is located on chromosome arm 5q.

DNA polymerase

III

Microsatellites - Site-specific DNA markers scattered throughout the genome and consisting of short nucleotide sequences (25 bp) repeated several times and flanked by unique sequences.

I

Microsatellite instability - The formation of new alleles in tumor DNA as compared with the two parental alleles in the normal DNA of the patient. Mismatch repair - A DNA repair system highly conserved from bacteria to humans. Mismatch repair occurs by excision of a DNA sequence from the strand containing an incorrect paired nucleotide, followed by resynthesis of a new strand. Mutator phenotype - An instability at several microsatellite loci mapping to different chromosomes, and caused by mutation in a mismatch repair gene. Somatic mutation theory of cancer -Acquired mutations in somatic cells that are the essential pathogenetic events in tumorigenesis. (Theodor Boveri hypothesised in 1914 in his book Zur Frage der Entsfehung maligner Tumoren that chromosome abnormalities were the cellular changes causing the transition from normal to malignant proliferation.)

Dam methylation

-

cTiG -

Figure 2. A simplified view of mismatch repair in Escheticbia co/i. (a)Mismatched base pairs that are formed during bacterial DNA replication (new strand) are repaired using the methylated parental (old) strand as a template, thereby reducing misincorporation-induced errors. (b) MutS recognizes and binds to the mismatched base pair. (c) MutL binds to the mismatch complex and, together with MutS, activates the MutH endonuclease, which recognizes and nicks the unmethylated strand. (d) Part of the DNA strand, including the mismatched base, is removed by the helicase UvrD and digested by a single-strand exonuclease, leaving a gap to be (e) filled by DNA polymerase Ill. Once the error has been corrected, (f) methylation of the replicated strand by Dam (methylase) makes the strands indistinguishable to the repair system.

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extreme differences between these two reports, it probably contributes significantly. Differences in the number of analyzed markers at leastpartly accountfor the differentresults obtainedin other studiesof microsatellite instability in non-small-celllung cancers (NSCLC)“7x33.

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mutH

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mutS

J\

Saccharomyces cerevisiae

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/I MLHl

MSHl

V-L MSH2

MSH3

MSH4

J\ \ J\ \ Homo hPMS1’ hPMS2+ hMLH1’ hMSH2’ GTBP hMSH3 Diferent types of microsatellite loci are sapiens altered in different tumor types Breastcancersandtesticularcancersexhibit Figure 3. Relationship between mismatch repair genes among different species. Genes that are known to microsatelliteinstability at tri- and tetrabe mutated in the germline of hereditary non-polyposis colorectal cancer (HNPCC) patients are marked nucleotiderepeatsbut, in contrastto colorectal with an asterisk. The arrows indicate the most closely related gene relatives”. cancers,are rarely affected at dinucleotide loci’“,3’X”4. This observationimpliesthat the underlying causeof the instability in colorectal cancerdiffers from that in theseother diseases, and argues Microsatellite instability during tumor progression againstthe involvementof hMSH2 and hMLHl in sporadicbreast The hypothesisthat genomic instability at microsatellitesoccurs and testicularcancers.Of sevenanalyzeddinucleotiderepeatloci, early in tumorigenesis fits well with the observationof suchalternone showednovel allelesin 84 breastcarcinomastested”. Similar ationsin precancerous lesions.Severalsuchlesionscan exhibit this resultswerereportedby Woosteret aL31, who analyzed 104breast phenotype,including: benign tumors and dysplasiasof the large carcinomas,of which 1 wasalteredat a dinucleotidelocusand 10 at bowe1’7.38*39 and stomach’*;Barrett’smetaplasiaof the esophagus4’; tri- or tetranucleotiderepeatloci. The two reportsstudyingmalegerm-celltumorsarein full agreementwith each other, because novel allelesin five of the six microsatellitea b unstabletumorsreportedby Huddartet ~1.~~ were found in tri- and tetranucleotide repeat Gastric Colon Endometrial Cervical Breast MPNST loci, whereas only dinucleotides wereanalyzed carcinoma carcinoma carcinoma carcinoma carcinoma in the secondstudy”. 8887

Locus-specificinstability? Someresults indicate that locus- or areaspecificinstability occursin somecancers. Gonzalez-Zulueta et ~1.~’ haveanalyzeda large numberof transitionalcell carcinomas of the bladder(n = 200),andfoundthat microsatellite instability is rare (3%). Five of the seven analyzedloci mapto chromosomearm 9p. By contrast, Orlow et ~1.~~reported 14/61 (23%)bladdertumorswith expansions or rearrangements of the locusD9S54(9~). This specificlocuswasnot includedin the analysis by Gonzalez-Zuluetaet aL3’Takentogether, thesereportssuggesta locus-specificinstability in bladder carcinomasrather than a genome-wide occurrence of thisphenomenon, because the latterimpliesinstabilityat several 9p loci, aswell asat loci on other chromosomes. Area-specificmicrosatelliteinstability is suggested by onereporton malegermcell tumorsthat reportedinstability restrictedto chromosome bandlq4243 (Ref. 37). Becausemutationsin hMSH2andhMLH1 causea genome-wide tendencyto instability at microsatelliteloci, any locus-specificinstability,if it is confirmedby additionalstudies, shouldpresumablybe soughtin changes of genesotherthanthese. 66

178787

88255 NT

NT

5s404 178787

17S787

8S259

N T

N T

N T

9s171

N T

Figure 4. Novel alleles exhibited at microsatellite loci in human solid tumors. (a) Tumor types associated with hereditary non-polyposis colorectal cancer (HNPCC). (b) Tumor types not associated with HNPCC. Incorporation of radioactive nucleotides in PCR amplification of microsatellite markers, followed by electrophoretic size separation of the DNA fragments and autoradiography, have been used to visualize the alleles for each sample. Instability at several dinucleotide repeat loci in different solid tumors (T) is indicated by arrows. The normal parental alleles (N) are shown in DNA from corresponding peripheral blood. The type of tumor and the chromosome map position of the locus analyzed are indicated above each N and T pair. Microsatellite instability is shown in (a) sporadic tumors associated with or (b) not associated with the HNPCC tumor spectrum. MPNST, malignant peripheral nerve sheath tumor.

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and benign thyroid tumors (M. Sobrinho-Simoes, pets. commun.). cancers with microsatellite instabilitya. Similar studies were done in The fact that malignant tumors reveal instability at more loci than two other HNPCC-associated tumor types, gastric and endometrial do precancerous lesions of the same organ supports the view that carcinomas”‘. The RI1 mutations were also found in microsatellitethese changes accumulate as tumors gro~‘~,*~,“~.In colorectal tumori- unstable gastric carcinomas, but not in endometrial carcinomas, suggenesis, adenomas show fewer new alleles than do carcinomas, and gesting a different route for the genesis of these tumors. the microsatellite instability frequency increases with adenoma size38. Clinicopathological characteristics of patients with Advanced colorectal tumors also reveal this type of instability”~“. microsatellite-unstable tumors Although the frequency of these is comparable to that found in early Patients with gastric, endometrial or colorectal carcinomas that show primary carcinomas, the pattern and the restricted number of affected microsatellite instability share several clinicopathological features. loci resemble those reports for adenomas”.4’. A higher frequency of First, a predilection for a specific site in the respective organ has instability has been shown in metastatic NSCLC than in primary been shown for carcinomas of the stomach (in the antrum)29 and NSCLC (55% versus 12%y3, but 75% of the unstable metastatic colon (in the proximal portion)‘,” and both tumor types are often tumors showed alterations at only a single locus. These results, and poorly differentiated adenocarcinomas8~‘0’26. Second, the presence of the prolonged survival of patients with either colorectal cancer or lymphoid-cell infiltration into the tumors and a relatively good outgastric cancer whose tumors show microsatellite instability9~20~29,come has been reported for the subgroup of patients with microsatelfavor the hypothesis that the mutator phenotype does not provide lite-unstable tumors of all three types9.20,‘9.30. Thus, it is tempting to tumor cells with a greater likelihood to metastasize. speculate that the relatively good prognosis of these patients could result from a strong immune response to proteins encoded by mutated The underlying mechanisms for development of cancer genes.

microsatellite-unstable

tumors

A germline mutation of a mismatch repair gene has been found in most analyzed HNPCC patients, but additional somatic mutations are necessary for tumor development. One question that has been dealt with in various types of studies is whether or not inactivation of the remaining wild-type allele is one of these necessary somatic events. Mutations of both alleles of a mismatch repair gene were initially reported in a few HNPCC tumors5,“-13. A later study from de la Chapelle’s group reported loss of the normal hMLHI allele in 35% (6/17) of colorectal tumors from HNPCC patients with known hMLH1 germline mutations4’. In sporadic microsatellite-unstable tumors, inactivation of both alleles of hMSH2 or hMLH1 has also been reported, as well as an hMSH2 heterozygous mutant with a possible dominant effect24’5. Biochemical analyses revealed that on the cellular level one mismatch repair gene mutation (heterozygous condition), as opposed to homozygous inactivation, does not severely affect mismatch repair43, and animal studies have shown that mice completely deficient in hMSH2 function (M,SH2-‘- mice) lead to predisposition for cance?. In summary, these observations suggest a shared requirement between mismatch repair genes and tumor suppressor genes for homozygous inactivation on the cellular level. The second important (and virtually unanswered) question is how does the defective mismatch repair contribute to tumorigenesis? One alternative is that this defect promotes mutations in oncogenes or tumor suppressor genes known to be of pathogenetic importance to the tumor type in question, such as K-US, APC and TP53 in the colorectal system. Recent data suggest that this might occur7-9.However, the frequency of alterations within these genes is lower in microsatellite-unstable tumors compared with tumors that do not share this phenotype819,in accordance with the observations that microsatellite-unstable tumors are usually not accompanied by gross chromosomal rearrangements, as shown by flow cytometry*‘, image cytometry3’, chromosome analysesj5 or studies of loss of heterozygosity’. The other alternative suggests that defective mismatch repair might promote instability in novel genes, and perhaps preferentially in those associated with microsatellites. This has indeed been shown for the gene encoding the transforming growth factor l3receptor @II), found to be mutated in a polyadenine tract in 90% of colorectal

Concluding remarks The genome-wide microsatellite instability phenotype does not itself provide tumor cells with a proliferative advantage, let alone the ability for malignant transformation. Nor do mutations in defective mutator genes, known to cause the phenotypic instability, necessarily provide a neoplastic growth advantage. Instead, the genotypic and/or phenotypic instability might increase the likelihood of additional mutations in other mutator genes and in even more directly cancerrelevant gene6*. These target cancer genes are not necessarily the oncogenes and suppressor genes known to be mutated in equivalent microsatellite-stable tumors, as suggested by the inverse correlation between mutations in K-RAS or TP53 and microsatellite instability in colorectal carcinomas8~9.Other genes encoding proteins involved in cell growth, senescence or apoptosis could be targets for mutations, and the RZZgene has already been found to be mutated in microsatellite-unstable gastrointestinal cancers46.47.

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Acknowledgements. Heim for critically Cancer Society.

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I thank my colleagues Anne-Lise Borresen-Dale and Sverre reading the manuscript. I am a Senior Fellow of the Norwegian

References 1 Fearon. E.R. and Vogelstein, B. (1990) A genetic model for colorectal genesis, Cell 61.759-767

tumori-

2 Marra. Cl. and Boland. R. (1995) Hereditary nonpolyposis colorectal cancer: the syndrome, the genes, and historical perspectives, J. Narl. Cancer Inst. 87. 1114-1125 3 Peltomaki. P. ef al. (1993) Genetic mapping of a locus predisposing to human colorectal cancer, Science 260,810-812 4 Fishel. R. et al. (1993) The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer, Cell 75,1027-1038 5 Leach, ES. et al. (1993) Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer, Cell 75, 1215-1225 6 Knudson, A.G. (1971) Mutation and cancer. Statistical study of retinohlastoma, Proc. Nat!. Acad. Sci. U. S. A. 68, 82@823 7 Aaltonen, L.A. et al. (1993) Clues to the pathogenesis of familial colorectal cancer, Science 260, X12-816 8 Ionov. Y. et al. (1993) Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis, Nature 363. 55X-561 9 Thibodeau, S.N., Bren, G. and Schaid, D. (1993) Microsatellite instability in cancer of the proximal colon, Science 260,816-819 10 Fishel, R. and Kolodner, R.D. (1995) Identification of mismatch repair genes and their role in the development of cancer, Curr Upin. Gene?. Dev. 5,3X3-395 11 Bronner, C.E. et al. (1994) Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer, Nature 368,258-261 12 Papadopoulos. N. er al. (1994) Mutation of a mutL homolog in hereditary colon cancer, Science 263.1625-1629 13 Nicolaides. N.C. et al. (1994) Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 371,75-80 14 Palombo, E et al. (1995) GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells, Science 268, 1912-1914 15 Liu, B. er al. (1996) Analysis of mismatch repair genes in hereditary nonpolyposis colorectal cancer patients, Nat. Med. 2, 169-174 16 Tannergard, P. et al. (1995) Mutation screening in the hMLHl gene in Swedish hereditary nonpolyposis colon cancer families, Cancer Res. 55,6092-6096 17 Aaltonen, L.A. et al. (1994) Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients, Cancer Res. 54, 1645-1648 18 Lindblom, A., Tanner&d. I?, Werelius, B. and Nordenskjold. M. (1993) Genetic mapping of a second locus predisposing to hereditary non-polyposis colon cancer, Nat. Genet. 5,279-282 19 Nigro, J.M. er al. (1989) Mutations in the p53 gene occur in diverse human tumour types. Nature 342, 705-708 20 Lothe, R.A. et al. (1993) Genomic instability in colorectaI cancer: relationship to clinicopathological variables and family history, Cancer Res. 53, 5849-5852 21 Young, J. et a[. (1993) Genomic instability occurs in colorectal carcinomas but not in adenomas, Hum. Mutat. 2,351-354 22 Ishimaru, G. et al. (1995) Microsatellite instability in primary and metastatic colorectal cancers. ht. J. Cancer 64.153-157 23 Liu, B. et al. (1995) Genetic instability occurs in the majority of young patients with colorectal cancer, Nat Med. 1,348-352 24 Borresen, A-L. et al. (1995) Somatic mutations in the hMSH2 gene in microsatellite unstable colorectal carcinomas, Hum. Mol. Genet. 4,2065-2072

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FEBRUARY

1997

25 Liu, B. et al. (1995) Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability, Nuf. Genet. 9.48-55 26 Han, H-J. et al. (1993) Genetic instability in pancreatic cancer and poorly differentiated type of gastric cancer, Cancer Res. 53. 5087-5089 27 Peltom&i, P. et al. (1993) Microsatellite instability is associated with tumors that characterize the hereditary non-polyposis colorectal carcinoma syndrome, Cancer Rex 53,5853-5X55 28 Rhyu, M-G., Park, W-S. and Meltzer, S.J. (1994) Microsatellite instability occurs frequently in human gastric carcinoma. Oncogene 9,29-32 29 Seruca, R. et al. (1995) Sporadic gastric carcinomas with microsatellite instability display a particular clinicopathologic profile, ht. J. Cancer 64,32-36 30 Risinger, J.I. er al. (1993) Genetic instability of microsatellites in endometrial carcinoma, Cancer Res. 53.510&5103 31 Wooster. R. et al. (1994) Instability of short tandem repeats (microsatellites) in human cancers, Nat. Gem. 6,152-156 32 Merlo, A. et al. (1994) Frequent microsatellite instability in primary small cell lung cancer, Cancer Rex 54,2098-2101 33 Ada& J-I. et al. (1995) Microsatelhte instability in primary and metastatic lung carcinomas, Genes Chromosomes Cancer 14,301-306 34 Huddart, R.A.. Wooster, R., Horwich, A. and Cooper, C.S. (1995) Microsatellite instability in human testicular germ cell turnouts, Brit. 1. Cancer 72. 642-645 35 Gonzalez-Zulueta, M. et al. (1993) Microsatellite instability in bladder cancer, Cancer Res. 53,5620-5623 36 Orlow, I. ef al. (1994) Chromosome 9 allelic losses and microsatellite alterations in human bladder tumors, Cancer Res. 54,2848-285 1 37 Murty, V.V.V.S. et al. (1994) Replication error-type genetic instability at lq42-43 in human male germ cell tumors, Cancer Rex 54.39X3-3985 38 Lothe, R.A. et al. (1995) Deletion of lp loci and microsatellite instability in colorectal polyps, Genes Chromosomes Cancer 14,1X2-188 39 Suzuki. H. et al. (1994) Microsatellite instability in ulcerative colitis-associated colorectal dysplasias and cancers, Cancer Res. 54,48414844 40 Meltzer, S.J. et al. (1994) Microsatellite instability occurs frequently and in both diploid and aneuploid cell populations of Barrett%-associated esophageal adenocarcinomas, Cancer Res. 54.3379-3382 41 Thorstensen, L. ef al. (1996) Alleletype profiles of local recurrences and distant metastases from colorectal-cancer patients , Int. J. Cancer 69,452+$56 42 Hemminki, A. et al. (1994) Loss of the wild-type MLHl gene is a feature of hereditary nonpolyposis colorectal cancer, Nat. Gene?. 8,405410 43 Parsons, R. et al. (1993) Hypermutability and mismatch repair deficiency in RRR+ tumour cells, Cell 75.1227-1236 44 Reitmair, A.H. et al. (1995) MSH2 deficient mice are viable and susceptible to lymphoid turnouts, Nat. Genet. 11,64-70 45 Remvikos, Y. et al. (1995) DNA-repeat instability is associated with colorectal cancers presenting minimal chromosome rearrangements, Genes Chromosomes Cancer 12.272-276 46 Parsons, R. et al. (1995) Microsatellite instability and mutations of the transforming growth factor b type II receptor gene in colorectal cancer, Cancer Res. 55.5548-5550 47 Myeroff, L.L. er al. (1995) A transforming growth factor b receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability. Cancer Rex 55,5545-5547 48 Loeb, L.A. (1994) Microsatellite instability: marker of a mutator phenotype in cancer, Cancer Res. 54,5059-5063 49 Honchel. R. et al. (1994) Microsatellite instability in Muir-Torre syndrome, Cancer Res. 54,1159-1163 50 Larson. A.A. er al. (1996) Analysis of replication error (RER’) phenotypes in cervical carcinoma, Cancer Res. 56, 1426-1431 51 Egawa, S. ef al. (1995) Genomic instability of microsatellite repeats in prostate cancer: relationship

to clinicopathological

variables,

Cancer Res. 55,2418-2421