1994;54:3398-3406. Cancer Res   Eduardo Rodriguez, Chandrika Sreekantaiah and R. S. K. Chaganti  Genetic Changes in Epithelial Solid Neoplasia

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(CANCERRESEARCH54.3398-3406.July I. 19941

Review

Genetic Changes in Epithelial Solid Neoplasia'

Eduardo Rodriguez, Chandrika Sreekantaiah, and R. S. K. Chaganti2

Cell Biology and GeneticsProgram and the CytogeneticsService.DepartmentofPathology, Memorial Sloan-KetteringCancer Center. New York. New York 11X121

Abstract

Although chromosomal analysis ofsolld epithellal neoplasms has laggedbehind that of hematopoletic, mesenchymal, and germ cell tumors, grad

tad accumulation of data over the past 5 years enables development of a

general view. Thus, these tumors appear to be characterized by a set ofnoarandoin deletions the incidence of which varies in tumors of differenthistological types. Most tumors were studied at advanced stages; therefore, essentially no data are available on the cytogenetic characteristics ofthe earliest stages oftumorigenesis. In contrast to the status of cytogeneticdata, a large body of Information on deletions at the molecular levelassayedby the loss of heterozygosityanalysishas accumulatedover thesame period. These data have been less complete than the cytogeneticdata, although In cases such as colorectal carcinoma, genetic changes fromthe earliest to the most advanced stages have been studied in detailproviding a genetic view ofprogressloa. Equally Important is the fact thatdeletion mapping studies by the loss of heterozygosity assay directly leadto Isolation of a number of tumor suppressor genes. A comparison of thepattern of deletions Identified by chromosomal and loss of heterozygoaltyanalysis revealed, as expected, a concordance. Comparison ofthe patternsof chromosonial(and the underlyingmolecular)changesin tumorsbetween mi@jor embryological cell types demOnstrates fundamental differences In genetic mechanisms which lead to tumorigenesls.

Introduction

The cytogenetic investigation ofepithelial solid tumors is in a muchless advanced stage than that of leukemias and lymphomas, wherenonrandom or specific chromosome abnormalities define diseasetypes and predict clinical outcome (1). Carcinomas, which result fromtransformation of epithelial cells, often are examined at a late stage ofdisease when numerous and complex chromosomal changes havealready taken place. Often, multiple subclones develop and the majorclone that represents the tumor in vivo may not be the one that growsin culture performed for cytogenetic analysis. As a result, it is oftendifficult to define the primary chromosomal change associated withtumorigenic events. Additional difficulties such as low mitotic activity, lack of growth in culture, presence of large areas of necrosis,contamination with bacterial and other microbial agents, and poorchromosome morphology have contributed to the relatively slowprogress in cytogenetic analysis of epithelial tumors.

In spite of these drawbacks, the slow accumulation of cytogeneticdata on epithelial tumors over the past 5 years is beginning to revealspecific sites of the genome which participate in frequent aberrationsand therefore become candidate sites for molecular characterization.We present here a review of the current state of cytogenetic andmolecular analysis of epithelial neoplasms, identify sites of frequentaberrations, and examine their clinical status.

The data reviewed here show that the most common chromosomestructural abnormalities in epithelial solid tumors are deletions affect

ing specific chromosomal regions, suggesting that unlike sarcomas,hematopoietic, and germ cell tumors, loss of TSGs3 may be thecommon mechanism in the etiology and progression of these tumors.In contrast to the slow progress in cytogenetic analysis, there has beenan upsurge in molecular mapping of deleted sites in epithelial tumorgenomes by analysis of LOH. Because both cytogenetic and molecular analyses point to a preponderance of deletional events, we havecompared the results of the two assays to further define the sites ofnonrandom deletion and gain insights into the mechanism of origin,progression, and clinical behavior of these tumors.

Materials and Methods

Cytogenetic Data. The data for the present analysis comprised publishedkaiyotypes in primary reports and review articles up to July 1993. Of these,data up to 1991 were derived from Mitelman's catalogue of chromosomeaberrations (2). Publications subsequent to 1991 were identified in the following journals: Cancer; Cancer Genetics and Cytogenetics; Cancer Research;Oral Oncology; EuropeanJournal of Cancer, Genes. Chromosomes & Cancer, Hwnan Genetics; Modern Pathology', and International Journal of Cancer. Due to space considerationsthe full list of references from these journalsis not included here but has been deposited with the Editor and will beprovided to interested investigators upon request. Only cases of piimaiy,recurrent,andmetastatictumorsforwhichfullkaryotypesdescribedaccordingto the InternationalSystem for HumanCytogeneticNomenclature(3, 4) wereavailable were included. Data in abstracts, reports of normal karyotypes, andreports of trisomy 7 and loss of Y chromosome as solitary abnormalities intumors in male patients have been excluded. The interpretation of trisomy 7

and Y chromosome loss, occurring as solitary events, as neoplasia-relatedcytogenctic changes has been questioned by a number of investigators (5—7).

In this analysis,to be consideredas recurrent,a given aberrationor breakpoint had to be identified in at least 10% ofcases ofa given histological subsetor tumorsite. Unbalancedtranslocationswere evaluatedto identify chromosomal deletions distal to the site ofrearrangement. Breakpoints were identifiedand evaluated without regard to the type of aberration (deletion, translocation,inversion, and/or duplication) at that site.

LOHData. A summaryof thedataon LOHanalysisin epithelialtumorsup to 1991was presentedin theReportofthe Committeeon ChromosomeandGene Loss in Human Neoplasia (8). Publications subsequent to 1991 wereobtained by surveying the following journals: Acta Oncologica; AmericanJournalofHumanGenetics;AnticancerResearch;BritishJournalof Cancer,Cancer; Cancer Research; Diagnostic Molecular Pathology; European Jour

nal ofCancer; Genes, Chromosomesand Cancer; Genomics; Human Genetics;International Journal ofCancer, Japanese Journal ofCancer Research; Jour

sal of NationalCancerInstitute;Lancer,Oncogene;ProceedingsNationalAcademyofScience.For thisanalysis,to considerasrecurrent,LOH hadto beidentified in at least 30% of cases of a given histological subset or tumor site.As with the cytogenetics literature,the list of publicationsassembled fromthese journals is available from the authors and will be provided to interestedinvestigators upon request.

ResultsReceived2122194;accepted4/24/94.The costs of publication of this arnck were defrayed in pail 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 f@t.

I Supported in pwt by NIH Grants CA-05826 and CA-08748.

2 To whom requests for reprints should be addressed, at Memorial Sloan-KetteringCancer Center, 1275 York Avenue. New York, NY 10021.

Table I summarizes the frequency of recurrent cytogenetic abnormalities found in epithelial solid tumors belonging to 28 histological

3 The abbreviations used are: TSG, tumor suppressor gene; LOH. loss of heterozygosity.

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Table1 Frequencyofchronzosomalabnormalitiesin epithelialtumorsTumor

@1t@a HistologyNo.of tumors

analyzedbRecurrent abnormalitiesMostfrequent

% breakpoints'@

GENETIC QIANGES IN EP1THELIALSOLID NEOPLASIA

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Benign epitheial neoplasmsThyroidFollicularadenoma62+121919q1323+5165q11—1510t(2;3Xq12—13;p24—25)10ColorectalAdenoma53+

1338+819del(1)(j32—36)15+1415KidneyOncocytoma18—150BreastFibroadenomaI

I+ 1127l2pl218Salivary

glandAdenoma133t(3;8)(j,21—23;q12)218q1212q13—152718Malignant

epithelialneoplasmsSkinMerkellcellcarcinoma17—13

+11p11—13del(1)(j22—36)29

2918LungSmall

cell undifferentiatedadenocarcinoma

Non-small cellundifferentiatedcarcinoma

Squamous carcinoma64

59

13del(3)(j,14—24)

—13

del(1@q32—44)del(17)(pll—13)del(5@q13—33)hsrs, dminsdel(3)(jil4—23)del(15)(plO—11)del(9)(j2l—23)del(17)(pll—13)del(11)(j,11—15)del(1)(j32—36)(7)(j11—13)hsrs,dminsdel(3)(p21—26)del(17)(jill—13)del(11)(pll—13)68

452116142158545346361212173817171p32—3ó

13q12—22

lpl 1—qi17pll—2214

37

6246LiverHepatoblastoma9+20

(2@q23—25)7856larynxSquamouscarcinoma2416q22-24l4pll

lp2l—222538

3PharynxSquamous

carcinoma1111q1345TongueSquamouscarcinoma167q21—22

8q11-pllIpil‘p22llqll—1331

252525

25SalivaryglandCarcinoma42del(6Xq22—25)48Stomach-esophagusAdenocarcinoma44del(3)(jil3-25)

del(11)(j15)i(5)(plO)i(8@q10)dmitisdel(3Xqll—21)39

30181818

14l2pl2—ql218ColorectalAdenocarcinoma178del(17)(j11—13)

—18+13del(8Xpl 1—23)+xdel(1)(j32—36)—14del(5@q22—35)del(10@q22—26)70

69464438383535

29AnalcanalSquamous carcinoma8del(11@q22-25)

del(3)(j22—24)7563Kidney

PancreasWilm's

carcinoma

Non papillary carcinoma

Papillary carcinoma

Carcinoma108

110

25

27del(11)(,p13—15)

+12+18del(3)(pll—22)—14(5Xq22—35)t(35)(p13;q22)+ 17

t(X;1)(pll.2;q21)—18+20+11—12dal(8)(j2l—23)del(1)(j32—36)25

251159452211562041333026221916q11—24

1q21—24

i7pll—qll8pll—qll1q1016

14

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Table 1ContinuedTumor

siteaHistologyNo.of tumors

analyzedbRecurrent abnormalities%Mostfrequent

breakpointsc%del(17)(pll—13)15del(17@q24—25)15BladderTransitional

cellcarcinoma82—9/del(9@q11—34)241q21—3217del(11)(j11—15)

del(6)q21—25)del(3)(j14—21)del(10Xq24-26)i(5)(j10)del(13Xq14—21)22

12151311

10ipil—qil11ThyroidPapillarycarcinoma1610q21—26

11q21—255638BreastAdenocarcinoma269del(3)(j14—23)

del(1)(jil3-36)del(16@q21—24)del(6Xq21—27)dmins, hsrs24

171313401

1q23—2513ProstateAdenocarcinoma38del(10Xq24)

del(7)(q22—36)dmins50

16261q21—2518OvaryAdenocarcinoma155del(6Xq15—25)

del(11)(j11—15)del(1@q21—44)—17-xdel(1)(j31—36)del(3)(j13-23)—13del(9)(p22—24)—18dmins, hsrs29

262625241917171412

1519p1317UteruscorpusAdenocarcinoma36i(lXqlO)

del(6@q21—25)17 1411q21—2517Uterus

cervixAdenocarcinoma48?i(5)(jilO)67ipil—qillip11—1531 25

Table 2LOH analysis inepithelial tumors: summary ofpublisheddataChromosome1

23 4 56 789 10 11 13 14 1617 181922XofpqpqpqpqpqpqpqpqpqpqpqpqpqpqpqpqpqpqpqsitesParathyroid

AD―421LungSCC9994953Lung

NSCC43 3843304043 30 5040405011LungSqC703940994Liver

H531LiverHCC5547 304838 355344339Esophagus

SqC6753583Stomach-37344253535esophagus

ACColorectalAC40455967 3463337Kidney

W@F431KidneyAC70334033305BladderTCC4456 6143515BreastAC303230

404030 30 505635443012ProstateAC584050434OvarianAC3046 3030 4142 38 31383910Head

andneck691SqCCervicalAC5731403Totall7

220 180 1108 110 1 41220263080 1031030 7200410

GENETIC CHANGES IN EPITHELIAL SOLID NEOPLASIA

0 Including primary, recurrent, and metastatic tumors.b Only cases with clonal abnormality.

C Recurrent breakpoints in non recurrent aberrations.d hsrs, homogeneously staining regions.

a pj@ adenoma; AC, adenocarcinoma; H, hepatoblastoma; HCC, hepatocellular carcinoma; NPC, nonpapillary carcinoma; NSCC, non-small cell carcinoma; PC, papillary

carcinoma; SCC, small cell carcinoma; SqC, squamous carcinoma; TCC, transitional cell carcinoma; WT, Wilm's tumor.

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types (abnormalities found in >10% of cases), while Table 2 summa- Cytogeneticsrizes the deleted chromosomal regions identified by LOH analysis in17of thesehistologicalsubtypes(LOHfoundin >30%of cases). Typt@sof AberrationsandTheirOverallIncidence.A totalofTable 3 compares the deleted regions detected by both cytogenetic 1576 published karyotypes from 29 histological types fulfilled theand LOH analyses detected in >30% of cases. criteria set above. The most common recurrent cytogenetic abnormal

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Table 3 Comparison offrequent deletions by cytogenetic and LOHstudiesaChromosomal

armIplq3pSq

6q 8p 9p lOq lipllq13q14q16ql7p18qFrequencyby

cytogeneticsFrequencybyLOH7 124 1221 474

4 4 4 4 7 4

41 12 24 12 12 35 184 477 60 184 59741a

Values are percentages of tumora showing >30%deletions by cytogenetic analysis (28 histologies) versus LOH analysis (17 histologies).

GENE@flC CHANGES IN EPITHELIAL SOLID NEOPLASIA

ities exhibited by epithelial tumors as a group were deletions andmonosomies (Table 1) and many of them were shared by differentcarcinomas (Table 3). The most frequent sites of deletion were: 3p(21%); ip (7%); lip (7%); i4q (7%); and i8q (7%). However, themost frequent recurrent abnormality displayed varied histology, except for del(3p) which was encountered in 7 different histologies(small cell, non-small cell, and squamous carcinomas of lung; squamous carcinoma of esophagus; adenocarcinoma of stomach andbreast; and nonpapillary carcinoma of kidney). In 7 histological types,duplication was the most frequent recurrent abnormality noted astrisomy or isochromosome formation (follicular thyroid and colorectaladenomas, breast fibroadenoma, hepatoblastoma, papillary renal carcinoma, and cervical and endometrial adenocarcinoma). The mostcommon duplicated region was 5p (stomach adenocarcinoma, bladderand cervical carcinomas) followed by trisomies 13 (colorectal adenoma and adenocarcinoma), 12 (follicular adenoma, Wilm's tumor),and 20 (hepatoblastoma and pancreas carcinoma). Six histologies(smallcell andnon-smallcell lungcarcinomas;adenocarcinomasofstomach, prostate, and ovary) demonstrated gene amplification in theform of double minute chromosomes (dmins) and homogeneouslystaining regions. In addition, cervical carcinomas have been associated with a high frequency of dmins (9).

Chromosome Aberrations Associated with Primary Tumors. Ingeneral, there were no follow-up cytogenetic studies of progression fromdysplasia to adenoma to carcinoma to invasive carcinoma and mostcytogenetic studies were attempted on advanced stage tumors. In certainhistological subsets, specific aberrations occurring as solitary changes insome tumors presented with an overall high frequency suggesting apossible primary role in the development of that histological type oftumor. Such aberrations included del(3p) in nonpapifiary renal, breast,and lung carcinomas; del(6q) in salivary gland carcinomas; del(llp) inWihn's tumor; monosomy 9/del(9q) in transitional cell carcinomas of thebladder; t(2;3) (qi2—i3;p24—25)in fofficular thyroid adenoma; anddel(iOXq24) in prostate adenocarcinoma.

Chromosome Aberrations Associated with Progression. Certainchromosomal abnormalities correlated with metastasis. Aberrations of9p and lip were associated with metastasis of ovarian adenocarcinomas (10, 1i); and duplication of 3p, trisomy 7, and i(5p) wereassociated with a high risk for progression of transitional cell carcinoma of the bladder (12, 13). Virtually all types of tumors exhibitedabnormalities of chromosome 1, often affecting multiple bands. Inbreast cancer, overrepresentation of lq, chromosomes 7 and i i, andpresence of homogeneously staining regions or dmins were correlated

with metastasis (14).Chromosome Changes as Diagnostic Markers. No diagnostic

cytogenetic markers have thus far been identified in carcinomas; however, in most instances, careful histopathological correlations were lacking. In rare instances, cytogenetics has been shown to be of somediagnostic value. For example, in renal cell carcinomas it may be possibleto distinguish between papillary and nonpapillary types by the detectionof trisomy 17 together with trisomy or tetrasomy 7 in the former (15, 16)and del (3p) in the latter (17). Thus, in cases of kidney tumors presentingambiguous histological features, cytogenetics may help toward distinction between papifiary and nonpapillary subtypes (18).

Chromosome Changes Associated with Clinical Outcome. Therelationship between chromosomal abnormalities and prognosis ofepithelial solid tumors has received little attention. In general, tumorswith simple karyotypic abnormalities have been suggested to have alow malignant potential while tumors with extensive rearrangementswere suggested to be more aggressive (19). Prognosis is difficult topredict for some tumors by clinical and histological parameters. Forexample, in the case of ovarian carcinoma tumor stage and histological subtype are of limited prognostic value, and in transitional cellcarcinoma of the bladder histology does not predict the potential forinvasiveness and recurrence of superficial low grade tumors. However, in ovarian carcinomas, changes in ploidy and increase in thenumber of marker chromosomes have been suggested to correlatewith tumor progression, prognosis, and degree of differentiation (20,21); while in bladder carcinomas, near diploid tumors were suggestedto have a lower tendency to invasiveness and a higher level ofdifferentiation compared to triploid tumors which were suggested tobe invasive (22, 23). Invasive transitional cell carcinomas of bladderwith a propensity for recurrence and/or clinical progression alsoexhibited a high degree of karyotypic instability. However, tumorchromosome number was not a reliable prognostic indicator sincenear diploid tumors sometimes have extensive chromosomal rearrangements (24). Finally, presence of dmins has been correlated withpoor prognosis in cervical carcinoma (9).

Molecular Genetics

LOll. Overall Incidence. LOH studies confirmedthe existence ofmultiple deleted chromosomal regions in carcinomas, some of themnot previously identified by cytogenetic analysis. This may be due tosome deletions not being visible at the cytogenetic level or becausecytogenetic studies were less extensive than LOH studies for certaintumor types. For example, in the case of hepatocellular and esophageal carcinomas, virtually no cytogenetic studies have been reported,while deleted chromosomal regions have been identified by LOHstudies. On the other hand, in most LOH analyses the number oftumors studied were small and relatively few chromosomal arms weretested giving a limited, possibly biased, picture of the spectrum ofgenetic deletions. Furthermore, some tumor types were studied extensively with most of the genome scanned, while other tumor types wereanalyzed only with probes near the location of known TSGs. The mostextensively studied tumors were ovarian, colorectal, breast, hepatocellular, and lung carcinomas. Overall, I6 different carcinomas and 1adenoma have been analyzed for LOH. The chromosomal regionswhich exhibited LOH most frequently (Table 3) were: l7p (59%); 3p(47%); i3q (47%); 5q (41%); 18q (41%); 1ip (35%); 8p (24%); 1lq(18%); and 16q (18%).

The most frequent site of LOH displayed varied with histology,except for 3p, which was encountered in 4 tumor types (small celllung, kidney, and cervical adenocarcinomas and head and neck squamous carcinomas); lip, which was encountered in 3 tumor types(stomach, colorectal, and breast carcinomas); and 19p, which wasencountered in 2 tumor types (non-small cell lung and squamous lungcarcinomas).

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GENETIC CHANGES IN EPITHEUAL SOLID NEOPLASIA

Loss ofSites of TSGs. Some of the regions which showed frequentLOH included sites of previously identified TSGS such as RB1 at13q14 (25, 26), DCC at i8q21.3 (27), TP53 at l'7pi3.l (28), APC at5q2i (29), Vyrfi at lipl3 (30), NFl at l7qii.2 (31), VI-ILat 3p25—26(32), and NM23 at 17q21, the last-mentioned a candidate suppressorof tumor metastasis (33). For some of these genes, complete loss offunction of the normal allele has been shown to occur in multipletumors. Thus, mutations in the TP53 gene concomitant with LOH hasbeen reported in lung, esophageal, ovarian, pancreatic, cervical, gastric, liver, and endometrial carcinomas; Barrett's epitheium; andpremalignant lesions of the aerodigestive tract; and in advanced stagesof colorectal, bladder, hepatocellular, head and neck, breast, andprostate carcinomas (34—53).On the other hand, introduction of wildtype TP53 invariably suppressed the growth or tumorigenicity of celllines derived from cancers of many types (54—58).Similarly, mutations in the RB1 locus have been found in lung, breast, prostate, andrenal cell carcinomas and in late stage hepatocellular carcinoma (37,59—64).

The short arm of chromosome 3 may contain several tumor suppressor genes. In renal cell carcinoma, losses have been found atseveral regions: 3pi2—i4, 3p2l.3, and 3p21—pter(65—67).A familywith hereditary renal cancer and a constitutional reciprocal translocation, t(3;8)(,pi4;q22), has been described (68, 69). Mutations at thevon Hippel-Landau syndrome locus, which has been mapped to 3p25—26, predispose to renal cell carcinomas (70, 71). In lung adenocarcinomas losses have been reported at two different regions, 3p2i.3 and3pl'tl—2i.l (72, 73); in cervical carcinoma at 3pi3—2i.i (74, 75); inbreast carcinomas at 3pl3—l4.3 (76); and in head and neck squamouscarcinomas at 3pi4—25 (77).

A candidate gene for Gorlin syndrome, an autosomal dominantdisorder that predisposes to several types of cancers, among thembasal cell carcinoma of the skin and bladder carcinoma, maps to 9q31(78). Transitional cell carcinoma of the bladder has been associatedwith deletions of 9q (Tables 1 and 2).

Some deleted chromosomal regions have been related to tumorinvasion and metastasis, e.g., the cadhenn gene at i6q22.i, the product of which, normally involved in cell adhesion, has been found to bedeleted in invasive prostate carcinomas (79).

LOH Associated with Progression. Specific genetic changes havebeen identified in the multistep process of progression from dysplasiato adenoma to carcinoma by LOH analysis. In colorectal adenocarcinomas, the analysis of multistep tumongenesis was possible becauseprogression takes place through a series of well defmed molecularchanges associated with histological stages. Thus, allelic losses at Sqwere found to precede those at l'7p, which was associated with theconversion from adenoma to early carcinoma (80, 81). Both lesionswere followed by losses at i8q, 22q, and two regions of 8p (8pli.2—21.3 and 8p22—Z3.2),which were associated with invasive carcinoma(80, 82, 83). However, it should also be noted that other early geneticevents have also been identified in colorectal carcinogenesis, such ashypomethylation and mutations in the RASK protooncogene (84, 85).In contrast, in esophageal cancers, alleic losses on i7p, noted inBarrett's esophagus, preceded allelic losses on 5q, which were notedin carcinomas (86). In ovarian carcinomas, losses in 6p, Vip, and i7qwere early events, while losses in i3q and i5q were late events,associated with tumor progression (87). In transitional cell carcinomaof the bladder, loss at a 9q locus has been suggested to be a primaryevent in a subset of tumors, since 9q losses occur in superficialpapillary tumors; loss of a Sq locus has been suggested to be involvedin the transition towards invasion of lamina propria, whereas losses at17p, 1ip, 3p, 13q, 6q, and i8q were suggested to be late events(88—92).Losses at iq, ‘7p,and 7q were suggested to be involved in theprogression of gastric well-differentiated adenocarcinomas (93).

Losses at 8p, 16q, 13q, ip, and 22q were suggested to be associatedwith progression of hepatocellular carcinomas (94).

LOH studies have also identified chromosomal regions associatedwith metastasis. Thus, losses at lip, i6q, and Yip correlated withmetastasis in breast carcinoma (95—97).

LOH ChangesAssociated with ClinicalOutcome. LOH analysis hasalso identified chromosomal regions of loss which may be associatedwith clinical aggressiveness of tumors. Thus, losses affecting ip wereassociated with diminished survival from relapse and losses at 7qcorrelated with aggressive behavior in breast carcinomas (98, 99);losses at l6q and 8p2l.3—22 were associated with advanced clinicalstages and poor differentiation in hepatocellular carcinomas (100,101). The restriction fragment length polymorphism pattern of theMYCL oncogene was used as a molecular marker to predict prognosisin renal cell carcinomas and lung carcinomas (102, 103).

Gene Amplification. In addition to gene losses, amplification ofcertain genes correlated with degree of malignancy. Thus, ERBB2,RASK1, INT2, HST1, MYC, and KS4M in gastric carcinoma (104-lii) and ERBB2 and MYC in ovarian carcinoma (112—114)correlatedwith poor survival. In breast carcinomas amplification of MYC (115)and coamplification of INT2 and HSTI (116) correlated with shorterdisease-free interval and overall survival, and amplification of ERBB2(117—120) and EGFR (121) was associated with poor prognosis;however, other groups have failed to find a direct relationship betweenERBB2 amplification and prognosis in breast carcinomas (122—126).In esophageal carcinomas, coamplification of HST1 and 1N72 correlated with clinical stage and metastasis (127).

Discussion

The cytogenetic and LOH data reviewed here show that deletionsare the most prominent aberrations in epithelial solid tumors. Bothtechniques have revealed approximately the same sites of frequentdeletion (Table 3). LOH analysis was more efficient than cytogeneticanalysis for detection of losses at sites of known TSGs: i7p, 13q, 5q,lip, i8q. It is possible that LOH at other sites, such as ip, iq, 3p, 6q,8p, 9p, i0q, iiq, i4q, and i6q will be found in higher frequency whenmore appropriate probes are used and a wider array of tumor types arestudied. The important question of which changes are associated withthe earliest events in tumorigenesis cannot, however, be addressed atthe present since most data available are for tumors studied at latestages, when many genetic changes have already accumulated due tothe inherent genetic instability of tumors. Thus, the carcinomas mostextensively studied by LOH analysis generally exhibited similar multiple sites presumably due to accumulated genetic aberration. Theassociation of the evolutionary history of tumors with genetic changeshas been studied only in colon carcinoma and need to be addressed inother tumor systems. Most cases of gene amplification were found inadvanced stage tumors and therefore presumably were associated withtumor progression.

Conventional cytogenetic analysis by banding techniques oftenreveals chromosomal markers of unknown origin in tumor preparations; such chromosomal abnormalities are not completely analyzable.Recent technical advances offer enhanced resolution of chromosomalchanges in solid tumors. One such technique is in situ hybridizationusing painting probes for specific chromosomes which can identifychromosomal segments hidden in complex markers (128). Conversly,microdissection of chromosomal markers, coupled with in situ hybridization to normal metaphases, can identify their origin (129). Insitu hybridization using centromere-specific repeat DNA sequencealso can reveal chromosome ploidy changes at interphase and thistechnique can be applied to frozen, paraffin-embedded, as well asdisaggregated cells from fresh tissue (130—131).The recently devel

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GENETIC CHANGES IN EPITHELIAL SOUD NEOPLASIA

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28. Baker, S. J., Fearon, E. R., Nigro, J. M., Hamilton, S. R., Preisinger, A. C., Jessup,

oped comparative genomic hybridization technique promises rapididentification of over- and underrepresented chromosomal regions,

without attempting conventional cytogenetic analysis (132, 133).Consistent chromosomal deletions have been utilized in delineating

regions of the genome harboring TSGs and have led to the cloning ofsome of them. Generally, the critical region has been narrowed downby finding polymorphic markers flanking the deleted region in atumor. The availability of an increasingly large repertoire of polymorphic DNA markers, especially dinucleotide repeat polymorphisms(134), promises to speed the process of isolation and characterizationof other TSGs.

LOH data must be interpreted with caution, since some cases ofapparent LOH may be due to preferential duplication of chromosomesrather than true loss of alleles. Nevertheless, LOH studies usingpolymorphic markers close to candidate TSGs have led to the isolation of these genes and the resulting data have shown that manycarcinomas have deletions in these target chromosomal regions, mdicaring that in most tumors multiple suppressor genes are inactivatedduring progression.

The data discussed in this review show that epithelial neoplasmsutilize gene deletion as the predominant mechanism in their origin aswell as progression. A group of candidate TSG sites and TSGs areinvolved in these losses, some of which show specific association withhistological types (e.g., l0q and l6q in prostate cancer). Since someof the common epithelial tumors have defined environmental riskfactors (e.g., tobacco smoking in lung and head and neck cancer) therole of environmental factors as inducers of site-specific geneticlesions and site-specific instability need to be investigated. In thisregard, a new type of genetic instability associated with a replicationerror phenotype has recently been described in inherited as well as inspontaneous tumors (135—138).The role of this error in epithelialcarcinogenesis remains to be fully understood.

Finally, there appears to be a relationship between the type ofpredominant genetic lesion exhibited and the embryonal derivation ofthe precursor cell. Thus, epithelial (this review) and neurogenic (139)tumors mainly exhibit deletions implying loss of negative regulationof proliferation as the genetic event which drives their initiation whilemesenchymal (sarcomas) (140) and hematopoietic (1, 141) tumorsexhibit rearrangements (translocations) as the primary genetic events,leading to inappropriate or aberrant activation of cell growth, differentiation, or programmed death regulating genes. In contrast, germcell tumors arising in meiotic spermatocytes utilize yet another mechanism, namely obligate amplification of the entire short arm of chromosome 12, suggesting overexpression of a gene(s) on this chromosomal region as the primary genetic event (142, 143).

Acknowledgments

We thank Marilyn Evans and Paulo A. Salazar for excellent secretarialassistance.

Note Added in Proof

While this manuscript was in review, the allelotypes of head and necksquamous cell carcinoma (144) and bladder carcinoma (145) were reported.

LOH >25% was noted at ip, 3p, 3q, 4p, 4q, 6p, 6q, 7q, 8p, 8q, 9p, 9q, llq, 13q,14q, l7p, 17q, l8p, l9p, 19q, 20p, 21q, and 22q in the former and at 9p, 9q,llq, and l7p in the latter. Homozygous deletions at 9p21 in multiple solidtumor types, including breast, colon, lung, ovarian, and renal carcinomas, wereshown to involve a new gene, MTSJ (multiple tumor suppressor 1) (146).MTSJ encodes the p16 protein,which is an inhibitorofcyclin dependent kinase4 (146).Thefunctionalconsequenceof MTS1lossto tumorigenesisremainstobe determined.

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