genomic alterations and instabilities in renal cell ... · renal cell carcinomas indicatesthat...

ICANCER RESEARCH55. 6189-6195. December 5. 9951

composed of more than one cell type and may exhibit two differentpauerns of growth as well (e.g., nonpapillary with areas of focalpapillary growth). To understand the etiology of these diseases it isnecessary to discover the complete set of tumor suppressor genes thedysfunction of which allows kidney cancer to develop.

It has been recognized for some time that alterations of the shortarm of chromosome 3 are associated specifically with nonpapillaryRCC and are not usually observed in papillary tumors (2â€”6).The vonHippel-Lindau (VHL) disease gene that predisposes to renal tumorsand maps to distal 3p is the most likely candidate for a nonpapillaryrenal tumor suppressor locus (7). LOH for 3p has been reported tooccur in 60â€”90%of sporadic and hereditary renal tumors (5, 8, 9), andVHL is mutated in at least 57% of those that exhibit 3p loss (7â€”9).TheVHL protein functions to negatively regulate the transcription dongation factor, elongin B/C ( 10, 11).

The fact that VHL appears to be the most commonly mutated genein nonpapillary RCC indicates that it affects an early step in thepathway of events that ultimately lead to cancer of the kidney.However, several lines of evidence indicate that mutations in VHLalone may not be sufficient to produce malignant disease: (a) cytogenetic observations indicate that several chromosomes, in addition to3p, are routinely lost or rearranged in these tumors (12); (b) as statedabove, chromosome 3p is not cytogenetically aberrant in papillaryrenal cancers; and (c) RCC typically arises in adults rather than inchildhood, even among individuals who inherit a mutated VHL gene.These observations suggest that the mutation of other genes, in addition to VHL, is required for malignant tumor growth.

More compelling evidence for the hypothesis that VHL alone is notsufficient to cause RCC comes from allelotyping studies. Recently,we have carried out a genome scan for LOH at CA microsatellitesequences in both nonpapillary and papillary tumors (13). Theseexperiments demonstrated that 45% of nonpapillary tumors showedLOH for 3p, but in no case was this loss the sole aberration. Rather,LOH for 3p occurs in association with allele losses on one or morechromosome arms that include 6q, 8p, and l4q. This observationsuggests that 3p (VHL) loss may be necessary, but not sufficient, forthe development and/or progression of nonpapillary RCC.

The LOH study reported here has been expanded to include 33RCCs as well as data on chromosome imbalance and CA microsatellite instability for each tumor. This has allowed us to examine theassociation of specific chromosome losses with histopathology, dinical stage, allelic imbalance, and microsatellite stability. The experiments described below consider the relationships among these factorsfor malignant renal disease and contribute to a more integrated pictureof renal tumor etiology and pathogenesis.

MATERIALS AND METHODS

Establishment of Paired Lymphoblastoid and Tumor Cell Lines.Tumor tissue and whole blood samples were obtained from patients who hadundergone surgery at the American Oncological Hospital. The preparation ofcell cultures from renal tumors and the derivation of cell lines from thesetumors have been described elsewhere (13). Peripheral blood lymphocyteswere recovered from whole blood by centrifugation over Histopaque 1077(Sigma Chemical Co.) and transformed with Epstein-Barr Virus. The resulting

ABSTRACT

A comprehensive genome scan for loss of heterozygosity (LOH) in 33renal cell carcinomas Indicates that mutations of tumor suppressor geneson several different chromosomes are required for malignant transformation in this disease. In the case of nonpapillary renal carcinomas chromosomes3p, 6q, Sp, 9pq, and 14qexhibit elevatedlevelsof LOH. Although3p is the most frequently lost chromosome arm, in no case is 3p observedas the sole allelic loss because it always occurs in conjunction with the lossof either 6q, 8p, or 14q. This result indicates that the mutation of a tumorsuppressor geneon 3p, most likelyvonHlppel-Lindaudisease(VHL),maybe necessary but is not sufficient for the development of nonpapillary

renal cell carcinoma.In papillary renal tumors, LOH is observed most often for chromo

somes 6pq, 9p, llq, 14q, and 21q. This suggests that tumor suppressorgenes located on chromosomes6q, 9pq, and 14q may be involvedIn thedevelopment and/or progression of both nonpapillary and papillary renalcell carcinomas. However, LOH in papillary tumors appears to be especially elevated for llq and 21q and reduced for 3p and 8p indicating thatthere are also tumor suppressor genes specific to each form of the disease.

There is no correlation between stage ofdisease and the extent of LOH,loss of a particular chromosome, or the number of chromosomes thatshow allele imbalance. Early and late stage tumors may exhibit eitherextensive LOll or no apparent allele loss; similarly, allelic imbalances areobserved in both early and late stage renal cell carcinomas. This suggeststhat a gene (or genes) regulating mitotic chromosome stability may bemutated in some renal tumors. Preliminary evidence points to an association between genome instability and LOH of 14q.

Finally, a distinct type of microsateilite instability has been detected in21% of renal cell carcinomas and occurs at a frequency of 4.4 x 10@/locus. The most common mutation Is a 2-bp insertion in a CA repeat. Thisalteration is not restricted to a particular histopathology or clinical stage,and it is not associated with allelic loss of a specific chromosome. Thefrequency of this event is similar to that which occurs spontaneously ingermline microsatellite loci and is probably not the result of a defect in amismatch repair gene. It is possible that this type of microsatellite instability is general and may occur in most, if not all, carcinomas.

INTRODUCTION

RCC3 is the most common malignancy of the adult kidney and isthe eighth and eleventh most frequent cancer among males andfemales, respectively. In general, such tumors may be broadly assigned to one of two histological patterns of growth, nonpapillary orpapillary. These may be further subdivided according to cell type(clear cell, granular cell, mixed clear and granular cell, and morerarely, cystic or sarcomatoid; Ref. 1). Some renal tumors may be

Received 8/I 0/95; accepted 10/20/95.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 Supported by grants from the Bets Foundation, Lucille P. Markey Charitable Trust,

Council for Tobacco Research (CTR29OI, K. D. T.), Public Health Service (CancerCenter Support Grant CA-O6927), and an appropriation from the Commonwealth ofPennsylvania.

2 To whom requests for reprints should be addressed, at Institute for Cancer Research,

Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 191 Il. Phone:(215)728-2473;Fax:(215)728-3574.

3 The abbreviations used are: RCC, renal cell carcinoma; LOH, loss of heterozygosity;

VHL, von Hippel-Lindau disease.

6189

Genomic Alterations and Instabilities in Renal Cell Carcinomas and TheirRelationship to Tumor Pathology'

Catherine A. Thrash-Bingham, Hernando Salazar, Jerome J. Freed, Richard E. Greenberg, and Kenneth D. Tartof2

Institute for Cancer Research (C. A. T-B., J. J. F., K. D. TI, Division of Medical Science IR. E. G.J, Fox Chase Cancer Center, Philadelphia, Pennsylvania 191 11, and ReadingHospital, Department of Pathology. West Reading. Pennsylvania 19612 [H. S.]

on April 10, 2017. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Table 1Molecular pathology of 33RCCsPathologyPatientâ€•StageGradeLOHIntensity

differencesNotinformativeNonpapillary

Clear cellRCC9 RCC10â€•RCCI3â€•RCC14RCC2IRCC22RCC26RCC28RCC29Â°RCC33RCC34RCC35RCC42RCC59Vf1N@M0

/T20M1IITr,N@M0ITr,N@M@Vr1N@M@JlITr2N@,M@Il!F,N@M@IITr2N@M@lVrr,N1@M1IV/TSbN,MIIIIJTsbN@Mo

â€˜r3bNO@O

â€œ@3bNO@'{)IIITliaN@M()II

IIIIIIIIIIIIIIIIIllIIIIII3p

6q3p 8pXpq3p 4pq 14q14q3p 8plp 3p 8pq 9pq l4q3P 6@ilOq l4q3p

7p

Sq

Iq 2p 8p I2p l5q l6p7pq

Sp 7pqlpq 4p Sq 7q 17p9p

l9p3q I2q7q lOp I lq I2pSq 6p 9q I Ipq 16q9p l9p

2Opq2q 3q 4p 5p l0q 12q3q 7q 9q I7q 19q 2Oq4q IOq I7q3q 20q3q lOq I lq l2q 19p 2OqXq2q Sq5p 6p 8q I Ip 2Oplq 2p SptipMixedRCC5

RCC7RCC8â€•

RCCIIRCCI5RCC32RCC48â€•RCCS7IV!TiaN@Mi

IV/TSbNIMIIIIITIbNIM()

lV/T3aN@MiIITr,N@,M,@IIfT,N@M,@Il!r,N@M@II1F,N@M,@III

IIIII

IIIIIIIIIII2q

3pq Sq 8p 9pq l3q 14q15ql7pl9p2lq

3p 6q3p 6pq9pq I lpq l7qâ€˜4p

8pq

3pq6q7p l3q I4q17q

lpq 6q l4qâ€˜4P

6P2q â€˜lpl3q l9pq2p

Sp Ilq 13q I8p l9q2Oq2q 4q 7q Ilq7q l2p 19p4p 6p20qGranular

cellRCCS5IlT@,N@M@IIpq 2p 6pq lOpq l7pq2q Spq20qPapillaryRCC6

RCCI7RCC23RCC36RCC37â€•RCC38

RCC47RCC6IIlfT2N@M0

llTr,N@M,@IITF2N1@M@lITF,N@M@IITr2N@M0IlTr2N(@M0

IV/T1N2M1lV/TSbNIMII

IIIIIIIIII

IVIII8p

9pq l2p

6pq l8pq 2lq2lq9p I lpq l4q

3pq 4p lOp llq l4ql'lp 22qlp 6pq 9p 1lpq l3q I4q

l8pq 21q 22qllq

2pq 3pq l6pq 17ql6pq l7pq2p 3pq 8q lOpq l6pq I7q18q19q21q

Sp â€˜lplOq l6pq l7p4pq

Spq IOq 1lq 19p XqSq lOp l9ptip l9pâ€˜(pSq3q 7q 9p l3q 2Optip l2p

lq 2q 4q tip 7q 19p3q Sq 7pq20pCystic

a Patients with micrRCC2S

IlfF2N@M0RCC4I IVr,N@M@

osatellite instability.I

IIXqSqI lp

GENOMIC CHANGES IN RCC

cell lines served as a source of normal genome DNA for the correspondingtumor from each patient.

Histopathology of Renal Tumors. Renal tumors were staged by using thetwo principle systems for categorizing this disease (1): (a) classifying tumorsI-IV, depending on the extent of local disease and the presence of metastasis(stage I, confined to the kidney; stage II, perirenal fat involvement; stage III,lymphatic involvement; and stage IV, distant metastases); (b) the tumor-nodemetastasis system that describes separately the local extent of the tumor, nodalinvolvement, and distant metastasis and is used by the International UnionAgainst Cancer (14).

DNA Isolation and the Detection of LOH by PCR Assay of Microsatellites. DNA was prepared from lymphocyte and tumor cell cultures usingstandard procedures (15). DNA from tumor tissue samples was prepared bydigestion with 2% SDS-proteinase K, followed by extraction with phenol (13).Conditions for the PCR amplification and analysis of microsatellites have beendescribed elsewhere (13). Five additional CA repeat loci, D1S197, D1S249,D8S505, D21S1252, and D21S1253 were selected from the MicrosatelliteMapcatalog (Genethon) and included in the studies described here.

Sequencing DNA Microsatellites. DNA from normalcells and from thecorresponding tumor that displayed an altered microsatellite length was amplified in a 25 @xlreaction volume containing 20 ng of DNA, 200 ng of primerpair, 50 p@mdNTPs, and 1 unit AmpliTaq polymerase (Perkin-Elmer) in 1XPCR buffer (Perkin Elmer Cetus). Amplification began with an initial denaturation at 94Â°Cfor 2', followed by 35 cycles of denaturation at 94Â°Cfor 30S and annealing at 60Â°C for 15 s. Samples were then incubated at 72Â°C for 2'

to permit complete elongation of the synthesized fragments. PCR productswere ligated into the plasmid vector pCRII (Invitrogen) using the supplier'srecommended procedure, except that bacterial strain DH5ct was used as thetransformation host. Bacteria containing inserts were identified by colonyhybridization using a 32P-labeled (CA)25 oligonucleotide as a probe (16). Thenormal and mutant alleles were identified from among these clones by PCRamplification of DNA from each colony, followed by electrophoresis of theresulting products on a sequencing gel. The appropriate plasmid DNA was

prepared by an alkaline lysis procedure and the insert was sequenced usingSequenase version 2 (United States Biochemical) with 17 and SP6 primers.

Statistical Analysis. Data were analyzed by the G test of independence for2 X 2 contingency tables. Williams' correction was applied to the G value.

Results with P < 0.05 were deemed statistically significant.

RESULTS

LOH and TumorPathology.Highlypolymorphicmicrosatelliteloci located at or near the ends of each chromosome arm wereamplified by PCR to determine the LOH allelotypes of 33 RCCs.Terminally positioned probes were chosen because they most readilydetect allele losses that may arise by either chromosome loss ormitotic recombination. Seventy-four CA repeat loci were used to scaneach tumor genome so that all 41 chromosome arms (except the Y andthose bearing a nucleolus organizer) would be informative in mosttumors.

The allelotype and pathology of these renal tumors are presented inTable 1. In six cases (RCC6, RCC7, RCC9, RCC1 1, RCC17, andRCC55)tumortissuewas used as the sourceof DNA;in the remaining 27 cases, DNA was obtained from cultured tumor cells. Onaverage, 9 1% of chromosome arms have been rendered informative ineach tumor. The largest number of noninformative chromosome armsin any tumor was eight, and this occurred only once (RCC6); on theother hand, RCC4I and RCC57 were informative for all chromosomes. Thirteen of the 33 tumors examined revealed no LOH for anyof the informative chromosomes tested. LOH was detected in anaverage of 2.9 chromosome arms/tumor in the 27 cultured cell linesand in 2.2 chromosome arms/tumor in the six tissue-derived DNAs,indicating that in vitro cell culture has little, if any, impact on alleleloss.

6190



Table 2 Statistical analysis of LOH associations in 33 RCCs described in TableIType

of LOHG valuePvalueSingle

chromosome loss3p not as sole loss6q not as sole loss8p not as sole loss9pq not as sole lossI4q not as sole lossI

2.0357.5985.0296.2804.060<0.005

<0.01<0.025<0.025

<0.05Pairwise

chromosome losses3p lost with 6q3p lost with 8p3p lost with 9pq3p lostwith14q6q lost with 8p6q lost with 9pq6q lost with l4q8p lost with 9pq8p lost with 14q9pq lost with 14q2.590

5.6610.0284.5022.2340.0830.0794.5950.6325.551<0.025

<0.05

<0.05

<0.025


cytogenetic observations in which monosomy for chromosomes 6p,8p, 9p, and l4q have been reported (12). The common involvement ofat least chromosome arms 6q, 9p, and l4q suggests the genetic basesof both papillary and nonpapillary carcinomas are likely to be similar.However, present LOH evidence also suggests that tumor suppressorgenes on chromosomes 1lq and 2lq may be involved specifically inpapillary RCC.

Two cystic and one granular tumor were also examined in thisstudy. The cystic tumors showed few gross alterations; only onechromosome arm, Xq, was affected in one cystic tumor, and no allelicimbalances were seen. One granular renal carcinoma exhibited LOHfor several chromosome arms. In this case, 6pq was the only chromosome lost in common with other RCCs.

Relationship between LOH, Allelic Imbalance, and Stage ofDisease. All 33 renal tumors have been analyzed for a number ofcharacteristics that might correlate LOH with stage of disease. Wefind no obvious association of LOH or allelic imbalance with tumorsize, nodal involvement, metastasis, or nuclear grade. However, astriking pattern does emerge from a comparison of clinical stage withthe general property of genomic instability, as reflected by either thedegree of LOH or chromosome imbalance.

The renal tumors studied here may be divided into four stages ofdisease as indicated in Table I . All tumors have been grouped by stageand the extent of LOH: those that exhibit no LOH, those that lose onlyone chromosome arm, and those that lose two or more chromosomearms (Table 3). Of the three stage I tumors, one retained heterozygosity for all informative loci, and two lost heterozygosity for twochromosome arms each. Four stage II tumors exhibited no LOH, threelost only one chromosome arm, and ten lost heterozygosity for two ormore chromosome arms. Interestingly, four stage III and four stage IVcarcinomas showed no evidence of LOH, and only one stage Ill andfour stage IV cancers showed LOH for two or more chromosomes.These results demonstrate that there are late stage tumors that displaylittle if any LOH and early stage cells that exhibit considerable alleleloss.

A similar comparisonof disease stage with allelic imbalancesispresented in Table 4. The distribution of tumor genomes showingimbalances in each disease stage closely parallels the data obtained forLOH. Of the 33 tumors, 16 show no evidenceof allelic imbalance,whereas 14 tumors exhibit allelic imbalances for 2 or more chromosome arms. There is no correlation between the extent of allelicimbalance and the stage of disease.

Clearly, stage II renal carcinomas displaying considerable LOH andchromosome imbalance cannot give rise to stage III and IV tumorswith no apparent LOH or aneuploidy (Tables 3 and 4). We suggest,therefore, that there are two different mechanisms underlying theprogression of kidney cancer: one that proceeds from an early eventinvolving overt chromosome loss or gain, and another that mayinvolve point mutations or small deletions that are not detectable bythe methods used here.

These data also suggest that LOH for l4q is associated with anelevated level of allele loss. Of the eight cases with allele loss on l4q,seven occur in RCCs where an average of 7.6 chromosome arms arelost/tumor and one has l4q loss as the sole event. A similar association occurs between LOH l4q and allelic imbalance. In the 33 renaltumors examined, a total of 64 chromosome arms exhibit allelicimbalance, and 35 of these occur in tumor cells that also display LOHfor 14q. Stated another way, a mean of 4.4 chromosome arms exhibitallelic imbalance in tumors with LOH of 14q, whereas only 1.2 armsshow allelic imbalance when 14q is retained. These results point to thepresence of a gene on 14q that controls chromosome stability.

CA MicrosateffiteInstability.In the courseof scanningrenaltumor genomes for LOH, a distinct pattern of the RER+ phenotype

6191

LOHof chromosome3p is almostexclusivelyrestrictedto nonpapillary tumors (Table I ). This is consistent with previous findings (3â€”6,13, 17, 18) and most likely reflects mutation or inactivation of theVHL gene. In one papillary carcinoma (RCC47), loss of an entirechromosome 3 occurred in a late stage tumor in which six otherchromosome arms were also lost. It is possible that in this case thedeletion of 3pq is adventitious and a consequence of gross alterationof the genome. Despite the striking association of LOH for 3p innonpapillary RCCs, there are no specific allele losses that distinguishclear cell, granular, or mixed clear/granular nonpapillary tumors.

Of the 20 tumors in which LOH was observed (Table 1), only 3 losta single chromosome arm (RCC26 lost l4q, RCC37 lost 21q, andRCC4I lost Xq). In most cases, two or more different chromosomesare lost concomitantly. To examine the pattern of chromosome losses,each of the most frequently lost chromosome arms (3p, 6q, 8p, 9pq,and l4q) was tested for loss as the sole event and for pairwisecoincident loss with each other. Two important conclusions emergefrom this analysis, the results of which are presented in Table 2: (a)LOH for any chromosomearm does not usually occur as the soleevent; (b) there is a significant association for loss of 3p with 8p, 3pwith l4q, 8p with 9pq, and 9pq with l4q. These results provideevidence that the loss of multiple tumor suppressor genes in preferredcombinations may be required for the initiation and progression ofRCC.

Table I also indicates those tumor genomes in which allelic imbalances have been observed. An allele imbalance is detected as arelative intensity difference between two alleles in tumor versusnormal DNA. As noted elsewhere, these most likely reflect chromosomal aneuploidy ( 13). The chromosome imbalances reported hereoccur at frequencies similar to, yet in some ways different from, thosedescribed in previous cytogenetic studies (12). For example, gains ofHSA I6 and HSA 17 have been reported in 62 and 80% of papillarytumors, respectively (12). Our results indicate an imbalance of 50%for both HSA16 and HSA17.

LOH events in 23 nonpapillary tumors for each chromosome armare graphically displayed in Fig. 1A. On average, the background levelof allelic loss is extremely low, <5%. In fact, 13 chromosome armsexhibit no apparent allele loss, and another 18 have been lost onlyonce or twice in all 23 tumors. In total, six autosome arms thatinclude, 3p, 6q, 8p, 9pq, and 14q exhibit elevated levels of LOH(>15%).

A similar representation of chromosome arms exhibiting LOH ineight papillary RCCs is illustrated in Fig. lB. Because fewer tumorshave been examined, the background of allele loss is higher (12â€”14%). For papillary tumors, chromosomes 6pq, 9p, llq, l4q, and 2lqexhibit elevated levels of LOH. These results concur, in part, with



40

35

@20.

@15@

@ 1; .@@

1 2 3 4 5 6 7 8 9 10111213141516171819202122 X

CHROMOSOMEARM

@LJLL ill iL I@1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

CHROMOSOMEARM

As indicated in Table 5, mutations in 11 CA repeats were detectedin 7 (21%) of the 33 RCCs examined here. Microsatellite instabilityoccurred in six nonpapillary tumors representing all three histopathological types, as well as in one papillary tumor. There is no apparentcorrelation of RER+ with the extent of LOH or the stage of disease.Nine of the 11 mutations are 2 nucleotide insertions, one is a 4

No. ofinfonnative chromosomearmswithStageLOH012II02II4310III401IV404

Table 4 Tumor stage aad number of chromosome arms displaying allelicin 33 renaltumorsinthalancesNo.

of chromosome arms withallelicStageimbalances0

12I2

01II737IIIS

00IV206


A

Fig. I. Chromosome arms exhibiting LOH in 23 nonpapillary (A) and eight papillary (B) RCCs. LI, p arms;@,qarmsofeachchromosomeexamined.B

Table 3 Tumor stage and number of chromosome arms displaying LOH in 33renal tumors

was observed. Typically, only 2 nucleotide increases in a CA repeatare observed as illustrated in Fig. 2, where the results for two differenttumors are displayed. In both RCC8 and RCC13, a 2-nucleotideincrease is observed for the larger allele of the D6S311 locus. Thealtered alleles are present in DNA derived from both cultured tumorcells and from primary tumor tissue but are not present in normallymphocytes.

6192

45

40

= 350._J 30

@ 25

@200

@ 15

I-1

* 05




DNA mismatch repair gene hMLHJ is located at 3p2l, and mutationof this locus is known to cause replication errors in microsatellites (20,21). However, six of the seven instances of microsatellite instabilityarose in nonpapillary tumors where LOH for 3p is common and mayrelate to mutation of VHL rather than hMLHI. Moreover, LOH for 3p,6q, and 9pq occurs in tumors that show no evidence of RER +.

It is possible that the CA repeat instability observed here simplyreflects spontaneous events that become detectable in tumors by virtueof the clonal expansion of a single malignant cell. The averagemutation rate of a dinucleotide repeat measured as a new alleleappearing among the pedigrees of Centre d' Etude du PolymorphismeHumain reference families is 1.2 X l0@, and the vast majority ofthese appear as increases of one repeat unit length and, to a lesserextent, decreases of a similar amount (22). Because the frequency andthe spectrum of CA repeat mutations observed in renal tumors is verysimilar to those that arise spontaneously in the human germline, it islikely that they too are spontaneous and not a defect in a DNA repairgene. The same mutations are not detected in the transformed lymphocytes used as the source of normal control DNA because thesecells are not derived from the clonal expansion of a single cell.

DISCUSSION

The LOH studies presented here indicate a multigenic etiology forRCC that involves the mutation of several putative tumor suppressorgenes located on six different chromosome arms. LOH for 3p isspecifically associated with nonpapillary tumors and probably servesto uncover a recessive mutation in the VHL gene. However, because3p is not lost alone in any tumor, it is likely that other loci arenecessary for malignant transformation. Allele losses on 6q, 8p, 9pq,and l4q frequently occur in nonpapillary kidney tumors and maycontain tumor suppressor genes that are also required for tumordevelopment. All of these chromosomes, except for 3p and 8p, appearto be involved in papillary disease as well. In addition, LOH for 1lqand 2lq may be specific to papillary RCC. Consistent with thismultigenic origin is the fact that RCC is a late onset disease.

Other than the association of LOH for 3p and 8p with nonpapillarytumors and LOH for llq and 21q with papillary tumors it has not beenpossible to correlate a specific allele loss with clear or mixed histopathologies. This suggests that the histopathological state of a renaltumor is not the result of mutations in a specific gene, but rather, mayreflect the state of gene expression in the target tumor cell at theinception of malignant transformation.

Those chromosome arms that most frequently exhibit LOH inkidney tumors are also lost in other tumors. LOH for 6q has beenreported for carcinoma of the breast (23) and malignant mesotheliomaof the lung (24). A high frequency of allele loss on 8p occurs inprostate (25) and colon tumors (26), whereas LOH for 14q has beenobserved in neuroblastoma (27). It has been thought that LOH onchromosome 9p may reflect mutation of the cyclin-dependent kinase4, located at 9p2l, and referred to as p16 (CDKN2/p16). p16 plays animportant role in the control of the cell cycle and has been shownrecently to be mutated or deleted in a wide variety of cancers including S of 9 (56%) renal tumors (28). However, it is possible that thereexists another tumor suppressor gene in the region because LOH on9p has been detected in cases where CDKN2/p16 is unaffected.

Finally, it is of interest that mutation of the tuberous sclerosis 2(TS2) gene, known to be located at l6pl33 in humans, causes renaltumors in the Eker rat (29, 30). Yet, l6p shows no evidence of LOHin any of the renal tumors studied here. However, it is possible that thethis gene could be important in the more rare chromophobe renaltumors, which are histologically similar to those observed in the Ekerrat.

Fig. 2. Microsatellite instability in RCCs. Typical 2 nucleotide (ut) insertion mutationsin CA repeats are present in tumor DNAs obtained from RCC8 and RCCI3. Microsatelliteloci D6S31 I and D7S550 were amplified from DNA of normal cells (lymphocytes),cultured tumors cell (Tu.Cell), and primary tumor tissue (Tu-Tis).

6193

RCC8 RCC13D6S311 D7S550

\@@c)Q@dc:@1@p6Ã§s:@cÃ§)@

I--250nt@_234nt

nucleotide insertion, and one is a 2 nucleotide deletion. The D12S97and D16S403 microsatellite loci are mutated in two tumors each,otherwise, different loci are affected in each carcinoma. Four of themutant microsatellite repeats were sequenced and found to contain theexpected 2 nucleotide CA insertion.

It is of interest to note that there is very little, if any, evidence of thenormal microsatellite allele in tumor DNA displaying the RER+phenotype (Fig. 2; RCC8). This is the case even when several different loci are affected in a given tumor genome and suggests that thesemultiple mutational events may have arisen very early, perhaps in apremalignant cell. This conclusion is further supported by the following observation regarding RCC55. This tumor had not only the granular RCC reported here but also an oncocytoma in the same kidney.The granular RCC and the oncocytoma share a common microsatellitemutation at the D12S97 locus; however, the spectra of chromosomearms displaying LOH were entirely different.4 This indicates that anearly mutation may have produced a clone of cells with a mutantmicrosatellite and that different cells in the clone gave rise to the twotumors.

The mutations detected here are similar to the type II class ofmicrosatellite instability that has been described for a subset ofcolorectal carcinomas. The type II pattern consists of small changesof Â±2 nucleotides in the tandem array of CA repeats and is distinguished from the more frequently occurring type I alternations thatinvolve increases or decreases of >4 nucleotides (19). Both type I andtype II forms of instability are found in colon tumors. However, wehave observed only type II mutations in kidney cancers.

More than 2475 microsatellite loci have been examined, and only11 mutations have been detected. Thus, the mutation rate in kidneytumors is low, approximately 4.4 X l0@ mutations/dinucleotiderepeat locus. As the data of Table 5 indicate, we observed fourpatients with a single mutant microsatellite, two patients with twomutations, and one patient with three altered CA repeats. These datado not depart significantly from a Poisson distribution that predictsthat 8, 1, and 0 tumors would harbor 1, 2, or 3 microsatellite mutations, respectively.

It is not clear if LOH of a specific chromosome is associated withRER+. LOH for chromosomes 3p, 6q, or 9pq occurred in three

different tumors displaying microsatellite instability (Table 5). The

4 C. Thrash-Binghamand K. D. Tartof. unpublisheddata.

Jâ€”248nt



Table 5Microsatellite instabilityinRCCsPatientPathologyChromosome

armswith LOHStageMicrosatellite locusObserved changeSequencedmutationRCCIO

RCCI3Clearcell

Clear cell3p 6qIV IID12S97 D7S550DI6S403D22S280+2ntâ€•

+2 nt+2 nt+2 nt+CA

+CAÃ·CARCC29Clear

cellIp 3p 8pq 9pq l4qIVD3S1314+2nti-CARCC55Granularlpq

2p 6pq lOpq17pqIID12S97â€”2

ntRCC8Mixed2q

3pq Sq 8p 9pq13q 14q lSq lip19p 21qIllD6S311

D165403+2

nt

+2ntRCC48Mixed9pq

llpq 17qIID19S224+2ntRCC37Papillary21qIIDIOSI9O

D9SJ65Ã·2nt

+4nta

nt, nucleotide.


The genome scan reported here confirms and extends previouskaryotypic and partial genome LOH studies that found no associationof tumor stage or pathological grade with chromosome or allele loss(3 1â€”33).Neither our results nor those just cited support the conclusionthat there is a significant correlation between tumor stage and the ratioof chromosome arms showing LOH to the number of informativearms (5). An inspection of these latter data parallel the results reportedhere, namely, that there are early stage tumors (I and II) that exhibitconsiderable LOH and late stage tumors (III and IV) that reveal liuleorno LOH.

There are two different, but possibly related, aspects of genomestability that are relevant to renal carcinomas. One concerns chromosome arm instability and the other is microsatellite instability. Withregard to the former, as noted in Table 1, approximately one-fourth (9of 33) of RCC genomes exhibit remarkable stability. They show noevidence of LOH or chromosome imbalance. An additional 25% (7 of33) of the tumors show LOH or allelic imbalance for only one or twochromosome arms. The remaining tumor genomes demonstrate eitherextensive LOH or gross imbalance or both. However, the degree ofgenome instability does not appear to be diagnostic of clinical stagebecause highly stable and unstable genomes may be found in early orlate stage tumors. Obviously, early stage tumors displaying considerable LOH and chromosome imbalance cannot give rise to late stagedisease with no apparent LOH or aneuploidy. This suggests that thereare two different pathways underlying the development of renalcancer, one that proceeds by overt chromosome loss or gain and onethat does not. Mutations in DNA repair genes are known to causechromosome instability in both meiotic and mitotic cells of Drosophila (34â€”36). It is possible that mutation or inactivation of similar

genes in human tumor cells may be responsible for those cases inwhich extensive LOH occurs. Chromosome 14q may harbor such agene because tumors displaying LOH for 14q lose on average fourtimes as many chromosome arms as those that retain l4q.

The microsatellite instability in RCC observed here is similar to thetype LI RER+ phenotype that has been reported in several othertumors. At present, no gene defect has been found to be responsiblefor this effect, and we have found no evidence for LOH of a particularchromosome arm in cells expressing the type II RER+ phenotype inrenaltumors.

Although microsatellite instability has been observed previously inkidney tumors (37), there are notable differences between these observations and the data presented here. Although Uchida et a!. (37) didnot provide values for the length change in mutant microsatellites, itmay be inferred from the data that both type I and type II RER+changes are present. In addition, they suggest that microsatellite

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instability may be more common in nonclear, high-grade, and latestage tumors. In contrast, we observe only type II alterations and noassociation of tumor stage, grade, or histopathology with the expression of RER+ . The reasons for these differences are uncertain, butthey may reflect an interesting difference in the etiology of renaltumors in Japanese and Caucasian populations.

We have observed that type II RER+ microsatellite instabilities inrenal tumors arise at a frequency similar to that of naturally occurringspontaneous mutation in CA repeats. Moreover, the mutations in CArepeats appear to be early clonal events because there is little, if any,evidence of the presence of the normal allele in the tumor cell DNA.This is the case even in those tumors that contain two and threedifferent altered repeats. For these reasons, we suggest that the type IIRER+ events arise as a natural consequence of spontaneous mutational events that may otherwise occur in normal cells but are specifically observed in tumors because of their clonal nature.

ACKNOWLEDGMENTS

We thank A. Knudson for his encouragement and B. Bingham for commentson statistical analyses.

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1995;55:6189-6195. Cancer Res Catherine A. Thrash-Bingham, Hernando Salazar, Jerome J. Freed, et al. and Their Relationship to Tumor PathologyGenomic Alterations and Instabilities in Renal Cell Carcinomas

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