Proc. Natl. Acad. Sci. USAVol. 92, pp. 2854-2858, March 1995Medical Sciences

Comprehensive allelotyping of human renal cell carcinomas usingmicrosatellite DNA probesCATHERINE A. THRASH-BINGHAM*, RICHARD E. GREENBERGt, SHARON HOWARD*, ALAN BRUZEL*,MARILYN BREMER*, ALEX GOLL*, HERNANDO SALAZARt, JEROME J. FREED*, AND KENNETH D. TARTOF*t*Institute for Cancer Research and tDivision of Medical Science, Fox Chase Cancer Center, Philadelphia, PA 19111

Communicated by Alfred G. Knudson, Jr., Fox Chase Cancer Center, Philadelphia, PA, December 19, 1994

ABSTRACT The von Hippel-Lindau locus on chromo-some 3p is a tumor suppressor gene known to be involved innonpapillary renal cell carcinoma. A previous loss of het-erozygosity (LOH) study aimed at determining the allelotypeof kidney tumors has indicated that in addition to 3p, chro-mosome arms 5q, 6q, 10q, llq, 17p, and i9p may also harbortumor suppressor genes. However, cytogenetic studies revealthat chromosomes 3p, 6q, 8p, 9pq, and 14q most frequentlyundergo karyotypic changes in renal tumors. To resolve thesedifferences, a collection of microsatellite DNA probes has beenused to scan forLOH so that 90%o of individual tumor genomeswere rendered informative for allele loss. The assay is capableof detecting quantitative genomic alterations in tumor cells aswell. We find that LOH is most frequent for chromosome arm3p. However, in no tumor is 3p exclusively affected. LOH for6q, 8p, 9pq, and 14q is also distinctly elevated for bothnonpapillary as well as papillary tumors and suggest thatmany of the tumor suppressor loci involved may be commonto the etiology of both forms of kidney cancer.

Renal cell carcinoma (RCC) is the most common malignancyof the adult kidney. The vast majority of these may bebroadly assigned to either of two histologic classes: nonpap-illary and papillary. Nonpapillary disease is about 6 timesmore prevalent than the papillary form. Over the last severalyears, cytogenetic and molecular analyses of RCC havedemonstrated that alterations of chromosome 3p are spe-cifically associated with nonpapillary carcinomas (1-5). Thevon Hippel-Lindau (VHL) disease gene that predisposes torenal tumors and maps to 3p25-3p26 (6) is the most likelycandidate for the nonpapillary renal tumor suppressor locuson chromosome 3p. Indeed, loss of heterozygosity (LOH) for3p has been reported to occur in 60-90% of sporadic andhereditary renal tumors (4, 7, 8), and VHL itself is mutatedin at least 57% of those that exhibit 3p loss (6-8).However, VHL mutations alone may not be sufficient for the

development of RCC. Cytogenetic observations indicate thatseveral chromosomes, in addition to 3p, are routinely lost orrearranged in these tumors (9). Also, chromosome 3p is notcytogenetically aberrant in papillary renal cancers. Lastly,RCC typically arises in adults rather than in childhood, evenamong individuals who inherit a mutant VHL gene. Thisobservation suggests that the mutation of other genes inaddition to VHL is required for malignant tumor growth.We have sought to define the allelotype of nonpapillary and

papillary tumors in order to identify those chromosome armsthat may harbor tumor suppressor genes involved in RCC. Therationale for this approach is that since tumor suppressor genesregulate cell proliferation in a negative manner, both alleles ofsuch a gene must be mutated or lost for malignant growth toensue. While the first allele may be mutated in any of severalways, the second allele is typically inactivated as a consequence

of either a point mutation, deletion, or chromosome loss or iseliminated by mitotic recombination. Point mutations andsubmicroscopic deletions do not result in LOH. Hence, thisapproach necessarily underestimates the extent to which aparticular chromosome arm may actually contain a tumorsuppressor gene. However, large deletions, chromosome loss,and mitotic recombination, which together comprise abouthalf of the second events leading to inactivation of a tumorsuppressor gene, do result in LOH (4, 7, 10). Mitotic recom-bination may be detected only when the exchange event takesplace between a polymorphic allele and the centromere.Therefore, to capture instances of both mitotic recombinationand chromosome loss leading to LOH, it is only necessary touse highly polymorphic markers located at the ends of eachchromosome arm.For this purpose we have assayed the length variation of

DNA tandem repeat (C-A), sequence called "CA microsat-ellites" located at or near the ends of all 41 chromosome armsfor which such markers are currently available (11). Unlikeprevious genome scans for LOH that depended on restrictionfragment length polymorphisms (RFLPs), the amplification ofmicrosatellites by PCR provides a uniform method of assaythat is rapid, requires very small amounts of DNA, and iscapable of rendering about 90% of chromosome arms in agiven tumor DNA sample informative for LOH. Because themicrosatellite assay is -3 times more informative than RFLPs,it is now possible to obtain a nearly complete description ofLOH status in individual tumors. The utility, as well as thepower, of this approach is general and may also be applied toother types of tumors.

MATERIALS AND METHODSEstablishment of Renal Tumor Cell Lines. Tumor tissue

specimens were obtained from patients undergoing surgeryat the American Oncologic Hospital of the Fox ChaseCancer Center. The tissue was minced into 1-mm pieces andincubated at 37°C for several hours in divalent cation-freesaline solution containing 0.25% trypsin. Cells released intothe supernatant were pelleted and then resuspended in 10 mlof IIA growth medium (12) supplemented with 15% (vol/vol) fetal bovine serum, 10 ng of epidermal growth factor(Sigma) per ml, and in some instances, 100 gg of ciprofloxa-cin per ml. The resuspended cells were initially cultured ata high density to promote tumor cell attachment and toinhibit the growth of fibroblasts. When fibroblasts persisted,cultures were transferred to a growth medium lacking fetalbovine serum to arrest fibroblast growth. When tumor cellsdid not grow well, 2% serum was added. The epithelialnature of the resulting cultures was confirmed by morphol-

Abbreviations: RCC, renal cell carcinoma; LOH, loss of heterozygos-ity; VHL, von Hippel-Lindau disease; RFLP, restriction fragment

i length polymorphism; FISH, fluorescence in situ hybridization.ITo whom reprint requests should be addressed.

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Proc. NatL Acad Sci USA 92 (1995) 2855

ogy and, in most cases, by a positive immunochemicalstaining for the presence of cytokeratin.

Establishment of Lymphoblastoid Cell Lines. Peripheralblood lymphocytes from each patient were recovered fromwhole blood by centrifugation over Histopaque 1077 (Sigma).Lymphocytes were resuspended in Hepes-buffered RPMI1640 medium (GIBCO) supplemented with 1 mM L-glu-tamine; 20% fetal bovine serum (Intergen, Purchase, NY); and50 units of penicillin, 50 ,g of streptomycin, and 100 ,ug ofkanamycin sulfate per ml. Cultures were initiated by theaddition of phytohemagglutinin P (Sigma) to a final concen-tration of 2 jig/ml and Epstein-Barr virus isolate B95-8 as a1:10 volume of filtered supernatant from marmoset B-cell lineGM 7404. Cultures were incubated at 37°C for 1 week at whichtime half of the supernatant medium was replaced withsupplemented RPMI 1640 medium. Transformed lympho-blasts were subcultured for eventual harvest.DNA Isolation and Loss of Heterozygosity Analyses. DNA

was prepared from lymphocyte and tumor cell cultures usingstandard procedures (13, 14). CA repeat microsatellites (Table1) were amplified from 20 ng of genomic DNA by using 200 ngof the appropriate primer pair in lx PCR buffer (Perkin-Elmer) with 20 ,uM each of dGTP, dATP, dTTP; 2 ALM dCTP;0.1 ,uM [a-32P]dCTP at 800 Ci/mmol; and 0.5 unit ofTaq DNApolymerase (Perkin-Elmer) in a total reaction volume of 25 AlI.Amplifications were performed with an initial denaturation at94°C for 2 hr, followed by 25 cycles of 94°C for 30 min, 60°C(55°C for D9S166 and D11S992) for 30 min, and 72°C for 30min. At the end of the last cycle, samples were incubated at72°C for 2 hr to allow for complete elongation of the amplifiedfragments. The reaction products were separated on 5% LongRanger acrylamide gels (AT Biochem, Malvern, PA).

Fluorescence in Situ Hybridization (FISH). Metaphasechromosomes were obtained from renal tumor cell cultures.Cells in exponential growth phase were synchronized bytreatment with 5-bromodeoxyuridine and processed for FISHas described (15, 16). Biotin-labeled chromosome-specificpainting probes (Oncor) were hybridized to chromosomespreads using the supplier's recommended conditions.

RESULTSLOH Analysis of Renal Tumors. Kidney tumor tissue was

obtained from 28 RCC patients, and a portion of each wasprepared for in vitro cell culture as described in Methods andMaterials. Of 23 tumors that grew in culture, 12 cell linesproliferated for 2-10 passages, whereas 11 others continued togrow for >10 passages. Tumor DNA was prepared from thesecultured cells, and in five additional cases it was extracteddirectly from tissue samples. It was expected that tumor cellline DNA would be relatively free of contaminating normalcell alleles, since the culture conditions stringently selectedagainst the growth of fibroblasts and lymphocytes typicallypresent in tumor tissues. Normal DNA was obtained fromEpstein-Barr virus-transformed lymphoblastoid cell lines forall but four patients; normal kidney tissue was used in the lattercases.LOH was assayed byPCR with primer pairs that flank highly

polymorphic CA microsatellites. We selected 70 primer pairsfrom the Genethon collection (11) that are located at or nearthe ends of each chromosome arm as listed in Table 1.Typically, three different loci were amplified in the samereaction tube by selecting primer pairs whose products did notoverlap in size, so that as many as 144 loci could be analyzedon a single 48-lane sequencing gel.The majority of tumor DNAs analyzed here were derived

from cultured cell lines. Typical LOH data obtained from suchsamples are shown in Fig. 1A. Allelic losses at the D3S1307locus on chromosome 3p were noted in cell lines RCC8,RCC13, and RCC14. In each, one of the two alleles present in

Table 1. PCR primer pairs for determining LOH

PCR product size,HSA* Locus amplified bplplq2p2q3p

D1S243, DIS228D1S251D2S207, D2S131D2525D3S1263, D3S1307, D3S1297

3q D3S13144p D4S4314q D4S426, D4S4155p D5S392Sq D5S4296p D6S3096q D6S281, D6S311, D6S278

7p D7S5177q D7S5508p D8S264, D8S265, D8S560

8q9p9qloplOqllpllq12p12q13q14qlSq16p

16q17p17q18p18q19p19q20p20q21q

D8S272, D8S284D9S16, D9S166D9S164DlOS249, DlOS191D10S212, DlOS190DlIS922D11S968D12S94, D12S77D12S97, D12S86D13S173, D13S174D14S65, D14S81D15S20, D15S127D16S404, D16S414, D16S403

D16S413, D16S402D17S849, D75796D17S784D18S59D18S58, D18S61D19S216D19S210, D19S224D20S117D205O10D21S267, D21S265, D21S263

22q D22S280, D22S282Xp DXS1060, DXS993Xq DXSIOOI

142-170,117-129249-271144-156, 229-24788-100231-249,237-251,

219-233144-170246-270177-191,172-20283-117160-186254-272203-219,230-276,

125-139239-257177-200121-145, 208-231,

131-156192-239,243-273227-247, 233-261187-199118-134, 124-152189-201,203-21988-138137-151183-211, 163-193265-279,124-160166-178, 171-189125-149,175-209150-174,114-147117-137,152-161,

134-152131-149,161-187251-261, 144-174226-238148-164144-160,157-183179-191165-177, 240-262150-182194-218175-203, 244-258,

175-201208-220,144-164134-150, 292-312197-215

*HSA (Homo sapiens) is the prefix for designating a particular humanchromosome.

normal DNA was absent in the tumor. Overexposure of theautoradiogram revealed no trace of the missing allele, indi-cating little or no contamination by normal cells. RCC11 andRCC15 were were not informative for this particular micro-satellite marker, and RCC9 and RCC10 retained both alleles.To determine the extent to which DNA from normal cells

might obscure the ability to detect LOH directly in tumortissue, a reconstruction experiment using cell culture-derivedDNA was performed. RCC32 and RCC14 each contain asingle allele of the D8S264 locus, 141 nt and 129 nt, respec-tively. DNA from these tumors was mixed in ratios (RCC32/RCC14) of 1/100, 1/10, and 1/5 and amplified by PCR. Theresulting products of these reactions are illustrated in Fig. 1B.Although the 141-nt allele was not detected when present at1%, it was visualized as a faint band when present at 10% or20%. Fig. 1 C illustrates a typical case of allele loss in a tumortissue sample when using the D8S560 locus. RCC17 tumor lost

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2856 Medical Sciences: Thrash-Bingham et al.

A 8 9 10 11 13 14 15NT NT NT NT NT NT NT

| ~~~~.247B

,. \CJ<

C 8 14 17NT NT N T

-148

-141 -142

-129 ^

FIG. 1. LOH in renal tumor DNA using highly polymorphic CAmicrosatellites. In all panels, the size in nt of one or more CA-richstrands is indicated. The GT strands migrate slower than the CAstrands under these electrophoresis conditions. The faint 2-nt ladderof products migrating faster than the prominent CA and GT bands issometimes observed and is an artifact produced by the Taq poly-merase. (A) PCR amplification of the 3p locus D3S1307 from normal(lanes N) and tumor (lanes T) cell line DNA obtained from sevennonpapillary renal cell carcinomas as indicated. (B) DNAs fromRCC14 and RCC32, each containing a different allele of the D8S264locus, were mixed in ratios of 1/100, 1/10, and 1/5 and then amplifiedby PCR. (C) The 8q locus D8S560 was amplified from normal (lanesN) and tumor (lanes T) tissue DNA of a papillary renal cell carcinoma(RCC17). For comparison, cell line DNAs from RCC8 and RCC14 arealso illustrated.

one of the alleles found in normal DNA. For comparison, twotumor cell line DNAs, RCC8 and RCC14, that lost heterozy-gosity for this locus are also shown. Clearly, even in the caseof tumor tissue samples, LOH could be readily determinedwhen the amount of contaminating normal cells was low.The results ofLOH analyses for each of the 41 chromosome

arms in 28 renal tumors are summarized in Table 2. Exami-nation of one or two microsatellites on each chromosome armwas sufficient to render, on average, 25 of the 28 tumorsinformative for any given autosomal arm and 10 of 11 tumorsfrom female patients informative for either arm of the Xchromosome. Most chromosomes show little or no allelic loss.In fact, no losses were detected on 12 of the 41 chromosomearms examined, and another 22 arms showed loss in only oneor two tumors. This indicates an extremely low background ofallelic loss. Only six autosome arms (3p, 6q, 8p, 9p, 9q, and 14q)exhibited elevated levels of LOH.

Consistent with previous findings (2-5, 17, 18), losses onchromosome 3p are restricted to nonpapillary tumors (Table2). Nine of the 20 nonpapillary tumors studied lost one or moreCA repeat loci on 3p, whereas none of the six papillary tumorswas similarly affected. This is close to the 50% value for LOHof 3p in nonpapillary RCCs previously reported by others (4,7). However, allelic losses on 6q, 8p, 9p, 9q, and 14q (18%,18%, 21%, 15%, and 21%, respectively) arose in both papillaryand nonpapillary tumors. The involvement of these chromo-somes in RCC has also been observed in prior cytogeneticstudies. Karyotype analyses of nonpapillary renal carcinomasindicate that chromosomes 6q, 8p, 9pq, and 14q were lost in14%, 22%, 14%, and 30-50% of tumors, respectively (9). It isnot clear whether the loss of chromosome 9 in these primarycultures and the LOH for 9pq described here is a consequenceof cell culture (19) or reflects the involvement of a tumorsuppressor gene. As a whole, the LOH data presented in Table2 are in excellent agreement with both the qualitative as wellas quantitative aspects of published cytogenetic observations.A summary of our LOH analysis with respect to each

individual tumor is provided in Table 3. Nearly 90% of the

Proc. NatL Acad ScL USA 92 (1995)

Table 2. Loss of heterozygosity in renal tumors sorted accordingto chromosome

NonpapillaryHSA* RCC (22)

lplq2p2q3p3q4p4q5pSq6p6q7p7q8p8q9p9qlop10qllpllq12p12q13q14q15q16p16q17p17q18p18ql9p19q20p20q21q22qXpXq

1/220/200/211/189/221/171/191/200/191/191/184/220/210/184/221/213/193/200/211/191/191/170/200/191/205/221/210/220/211/211/200/210/221/170/190/210/161/190/221/82/7

PapillaryRCC (6)

0/60/40/60/60/60/50/40/50/50/31/41/60/60/51/60/62/51/60/50/51/61/51/50/60/51/60/60/60/60/60/61/61/60/30/60/50/62/60/60/30/2

LOH/Informative(% LOH)1/28 (3.6)0/24 (0.0)1/27 (3.7)1/24 (4.2)9/28 (32)1/22 (4.5)1/23 (4.4)1/25 (4.0)0/24 (0.0)1/22 (4.5)2/22 (9.1)5/28 (18)0/27 (0.0)0/23 (0.0)5/28 (18)1/27 (3.7)5/24 (21)4/26 (15)0/26 (0.0)1/24 (4.2)2/25 (8.0)2/22 (9.1)1/25 (4.0)0/25 (0.0)1/25 (4.0)6/28 (21)1/27 (3.7)0/28 (0.0)0/27 (0.0)1/27 (3.7)1/26 (3.8)1/27 (3.7)1/28 (3.6)1/20 (5.0)0/25 (0.0)0/26 (0.0)0/22 (0.0)3/25 (12)0/28 (0.0)1/11 (9.1)2/9 (22)

*HSA (Homo sapiens) is the prefix for designating a particular humanchromosome.

chromosome arms examined were informative in each patient;a mean of only 3.8 chromosome arms was uninformative in anygiven tumor. The largest number of noninformative chromo-some arms was 8, and this occurred in two individuals (RCC6and RCC34); on the other hand, RCC41 was informative forall chromosomes. LOH was observed in 17 of the 28 tumorsexamined; 11 tumors revealed no LOH for any of the infor-mative chromosomes tested. LOH was detected an average of2.7 chromosome arms per tumor in the 23 cultured cell lines,and 0.8 chromosome arms per tumor in the five tissue-derivedDNAs. However, it is difficult to determine if this differenceis meaningful, since RCC8 substantially contributes to theapparent excess LOH in the cultured material and since thenumber of tumor tissue samples is small.

It is interesting to note that loss of chromosome arm 3p wasnever observed as the sole aberration in any tumor (P < 0.025,by G test of independence). LOH of 3p usually occurred inassociation with losses of 6q, 8p, 9pq, and/or 14q. It is likelythat the loss of 3p reflects the role of the VHL gene in RCC.However, the relationship of 3p loss with that of these otherchromosomes strongly suggests that VHL mutations may be

Proc. NatL Acad ScL USA 92 (1995) 2857

Table 3. LOH in 28 individual renal carcinomas

Pathology Patient Loss of heterozygosity Intensity differences* Not informative

Nonpapillary RCC5 4p 6pRCC7t - 4p 8pq 10q 17pq 2q 7p 13q l9pqRCC8 2q 3pq 5q 8p 9pq 13q lq 2p

14q 15q 17p 19p 21qRCC9t 3p 9p l9pRCC10 - 3q 12qRCCllt 3pq 6q 5p llq 13q 18p 19q 20qRCC13 3p 6q 5q 7q 10p llq 12pRCC14 3p 8p 5q 6p 9q llpq 16qRCC15 3p 6q 2p 7p 13q 14q 2q 4q 7q llq 21qRCC21 Xpq 9p l9pRCC22 3p 4pq 14q 2p 12p 15q 16pq lq 20pqRCC25 5q llpRCC26 14q 7pq 2q 3q 4p 5p 10q 12qRCC28 3p 8p 16p 3q 7q 9q 17q 19q 20qRCC29 lp 3p 8pq 9pq 14q 5p 7pq 4q 10q 17q 21qRCC32 3p 6pq 10p 17q 7q 12p 19pRCC33 3p 6q 10q 14q lpq 4p 5q 7q 18p 3q 20qRCC34 6q 3q 10q llq 12q 19p 20q 21q XqRCC35 2q 5qRCC41 XqRCC42 - 5p 6p 8q llpRCC48 9pq llpq 17q lpq 6q 15q 4p 6p 20q

Papillary RCC6t 4pq 5pq 10q llq 19p XqRCC17t 8p 9pq 12p lq 5q 10p i9pRCC23 llq 6p l9pRCC36 6pq 18pq 21q 2pq 3pq 16pq 17pq 4p 5qRCC37 21q 16pq 17pq lq 3q 7q 9p 13q 20pRCC38 9p llpq 14q 2p 3pq 8q lOpq 16pq 17q 18q 6p 12p

tPatients from which tumor tissue was used as the source of DNA. All other tumor DNAs were extracted from cell lines.*Alleles that showed intensity differences were assayed twice to confirm the results except in those cases where multiple alleles onthe same chromosome arm were all similarly affected.

necessary but are not sufficient for the development and/orprogression of nonpapillary RCC.LOH for chromosome arms 6q, 8p, 9pq, and 14q were also

detected in papillary tumors. In addition, two of six papillarytumors showed LOH for chromosome 21q, and in one of these(RCC37), 21q was the only informative chromosome to displayLOH. Although LOH for 21q also arose in one nonpapillarytumor (RCC8), the fact that two of six papillary tumors showedLOH for 21q suggests the involvement of a gene on thischromosome arm in papillary RCC. However, since the num-ber of papillary tumors studied here is small, additionalspecimens need to be analyzed.Changes in Chromosome Ploidy as Detected by the PCR

Assay of Microsatellites. In the course of screening tumors forLOH, we noticed that PCR amplification of particular micro-satellite sequences in some tumors reproducibly yielded frag-ments that differed in intensity, where a band representing oneallele was conspicuously darker than the other, relative to thatof normal DNA. A typical example of such allelic imbalancesat the D16S414 locus is illustrated in Fig. 2. The bandsrepresenting smaller fragments amplified from tumorDNA ofRCC22 (153 nt) and RCC37 (155 nt) were always more intensethan those representing the larger fragments. In RCC36, andto a lesser extent in RCC38, it is the bands representing thelarger-161 nt and 159 nt, respectively-that are more intense.In contrast, bands representing the two alleles amplified fromnormal DNA are of similar intensity in each of these patients.RCC36, RCC37, and RCC38 tumor DNAs are free of detect-able amounts of normal DNA as indicated by the completeallelic losses (listed as LOH in Table 3) observed by PCR.Therefore, the faint allelic bands seen in the tumor DNAs inFig. 2 are not due to contaminating normal sequences. How-ever, it is possible that such intensity differences could reflectthe gain or loss of alleles in some, but not all, tumor cells.

Chromosome arms for which allelic intensity differences wereobserved are listed in Table 3. The most frequent alterationsoccurred for human chromosomes 16 and 17, in five tumors each.Loci on both the short and long arms of chromosome 16 wereaffected in three of the six papillary tumors and in one nonpap-illary tumor (Table 3). Fragments amplified from the p arm ofchromosome 16 exhibited allelic imbalances in RCC28 but the qarm gave an electrophoretic pattern equivalent to that of normalDNA. Intensity differences for alleles on chromosomes 3 and 7were detected in four different tumors each. It isworth noting thattrisomy of chromosome 7 is a condition that occurs frequently inboth normal and malignant cells (20, 21). Apparent intensitydifferences were also observed in three tumor tissue samples(RCC7, RCC9, and RCC11) that showed no LOH. However, the-50% reduction in intensity of one allele in these samples couldbe due to LOH that is obscured by substantial contaminationfrom normal cells. Since it is not possible to determine preciselythe extent towhich normal cells maybe present,we have classifiedthese tumor tissue patterns as imbalances rather than LOH.The intensity variations described here may be the result of

chromosome gains or could be due to nonclonal chromosomelosses. It is possible to distinguish between these possibilitiesby FISH of tumor cells. As an example, metaphase chromo-somes from RCC38 tumor cells were hybridized with a chro-mosome 16 painting probe. A typical metaphase spread dem-onstrating trisomy 16 in a tumor cell is shown in Fig. 3.Metaphase spreads of RCC38 reveal that these cells have amean of 67 chromosomes and three or five copies of HSA16that leads to the unbalanced dosage of alleles observed here.While we have not examined every tumor by FISH to

confirm the inferred chromosome aneuploidy detected byPCR, similar chromosome gains have been observed by others.For example, gains of chromosomes 7, 16, and 17 have beenreported in 75%, 62%, and 80% of papillary tumors, respec-

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2858 Medical Sciences: Thrash-Bingham et al.

22 36 37 38

N T N T N T N T

- 159157

FIG. 2. Intensity differences in PCR products amplified fromnormal/renal tumor DNA pairs. The D16S414 locus was amplifiedfrom normal (lanes N) and tumor (lanes T) DNA of four patients, andthe reaction products were separated on an acrylamide gel. The sizes

in nucleotides of the two CA-rich strands in RCC38 are indicated.

tively (9). Our results indicate an imbalance in 50% of papillarytumors for both chromosomes 16 and 17; however, we observeno intensity difference of chromosome 7, although this doesoccur in nonpapillary tumor cells (20%). Despite this differ-ence, the similarities between our data and previous cytoge-netic observations suggest that genome scans by the PCR assayof microsatellite loci may reveal those chromosomes that are

aneuploid in tumor cells.

DISCUSSIONThe methods described here permit a rapid and complete scanof a tumor genome for loss of heterozygosity. Unlike previousRFLP procedures, the markers used here are highly informa-tive, typically located at the ends of chromosomes and rely ona uniform means of assay. Since both LOH and allele imbal-ance information can be obtained by this unified approach, itmay have application for diagnostic and prognostic purposes.The LOH data for nonpapillary tumors reported here are

quantitatively and qualitatively in very close agreement withprevious cytogenetic studies that describe monosomy for chro-mosome arms 3p, 6q, 8p, 9pq, and 14q as the major karyotypicchanges in renal tumors (9). A previously reported partialgenome scan using RFLP markers noted elevated levels ofLOH for 3p, 5q, 6q, 10q, llq, 17p, and l9p in nonpapillarytumors (4). However, neither our study nor previous cytoge-netic data (9) indicate the regular loss of chromosomes 10q,llq, 17p, or l9p. Nevertheless, it is possible that such dispar-

FIG. 3. FISH of RCC38 tumor cells with a chromosome 16-specificpainting probe. The metaphase spread depicted here contains 58chromosomes, 3 of which are human chromosome 16.

ities may reflect differences in methods, the patient population(Caucasian vs. Japanese), or some environmental factor af-fecting the pathway of events leading to renal cancer.

It is of considerable interest that 3p, the chromosome mostfrequently exhibiting LOH, was never observed as the onlychromosome arm displaying allele loss in nonpapillary tumors.LOH for 3p occurred in combination with either 6p, 8p, 9pq,or 14q. These results, together with the fact that renal cancerusually occurs in adulthood, even in individuals that inherit amutant VIL gene, suggest that mutations of tumor suppressorgenes on these other chromosomes are necessary for malignantrenal disease to arise. This conclusion is reinforced further byour observation that, except for 3p, the patterns of allele lossin papillary and nonpapillary renal carcinomas are very sim-ilar. In both cases, chromosomes 6q, 8p, 9pq, and 14q areaffected. Precisely what genes are involved and the pathwaysthey regulate are unknown, though they are likely to be ofconsiderable significance in the etiology of malignant renaldisease.

We thank Robert Muhlhauser and C. Glenn Miller for oligonu-cleotide synthesis, Brendan Bingham for statistical analysis, JaneBishop for her support, and Al Knudson for his encouragement. Thisresearch was supported by grants from the Betz Foundation, LucilleW. Markey Foundation, Council for Tobacco Research (CTR2901to K.D.T.), U.S. Public Health Service (CCSG CA-06927), and anappropriation from the Commonwealth of Pennsylvania.

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