[CANCER RESEARCH 58, 5835-5841, December 15, 1998]

Clouai and Chronological Genetic Analysis of Multifocal Cancers of the Bladderand Upper Urinary Tract1

Takeshi Takahashi, Tomonori Habuchi,2 Yoshiyuki Kakehi, Kenji Mitsumori, Toshiya Akao, Toshiro Terachi, and

Osanni YoshidaDepartment of Urolog\. Graduale School of Medicine. Kyoto University. Kyoto 606-8507, Japan

ABSTRACT

Recent molecular genetic studies have suggested that multifocal urothe-

lial cancers are derived from an identical progenitor cell. However, theclonal origin of multifocal urothelial cancers of a low-grade superficial

type has not been fully defined. Using microsatellite markers, we examined genetic alterations at 20 loci on eight chromosomal arms (2q, 4p, 4q,8p, 9p, 9q, lip, and 17p) in 87 metachronous and/or synchronous multi-focal urothelial cancers, which included 84 low-grade superficial papillarytumors from 29 patients. Judging from the patterns of loss of heterozy-

gosity, microsatellite shifts, and the subchromosomal partial deletion,multifocal tumors in at least 20 (80%) of the 25 évaluablepatients wereconsidered to be derived from a single progenitor cell, although thepossibility remained that multifocal tumors in a small subset of patientsmight develop from distinct progenitor cells due to field cancerization. In13 of the 20 patients, a chronological genetic analysis was available:genetic heterogeneity was detected in 3 (23%) patients, and an apparentaccumulated pattern of genetic alterations was detected in only 1 (8%)patient. In the 20 patients with multifocal tumors of an identical clonalorigin, discordant microsatellite alterations were observed, with significantly lower frequencies on chromosome 9 compared to those on the otherchromosomes tested. The results indicate that most multifocal low-grade

superficial urothelial cancers are genetically stable despite their incidenceof frequent recurrence, and genetic divergence occurs in a subset ofpatients. This heterotopic spread and genetic divergence may occur longbefore the clinical manifestation of multiplicity from a single transformedcell. These data support the previous view that heterotopic spread oftransformed progenitor cells and genetic divergence occur after chromosome 9 alterations in most of low-grade superficial urothelial cancers.

INTRODUCTION

One of the most important features of urothelial cancers of thebladder and upper urinary tract is metachronous and/or synchronousmultifocal occurrence with high frequency (1-3). Clinically, the meta

chronous multifocality is a major problem after endoscopie treatmentof superficial urothelial cancers. Traditionally, the multifocal natureof TCC3 has been explained by the "field cancerization" hypothesis,

in which the entire uroepithelium is exposed by common carcinogenicinsults in each patient and multifocal urothelial tumors arise fromindependent clones of transformed transitional cells (4). However,detailed clinical observations have suggested that such multifocaltumors develop by the seeding (implantation) of intraluminal dispersed viable cancer cells or by intraepithelial spread (5-7). Recent

molecular genetic studies of multifocal urothelial cancers, includingthose from our laboratory, have supported the seeding or intraepithelial spread hypothesis (8-10). However, these molecular genetic

Received 7/27/98; accepted 10/14/98.The costs of publication of this article were defrayed in pan 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.

1Supported in part by Grants-in-Aid for Cancer Research from the Ministry ofEducation, Science, Sports and Culture of Japan (C0867-18I3 and B10470336) of Japan

and a grant from the Shimadzu Science Foundation.2 To whom requests for reprints should be addressed, at Department of Urology,

Graduate School of Medicine. Kyoto University. Sakyo-ku Syogoin Kawahara-cho 54,Kyoto 606-8507. Japan.

3 The abbreviations used are: TCC, transitional cell carcinoma; LOH, loss of heterozy-

gosity.

studies have focused mainly on high-grade invasive type cancers.Such high-grade invasive cancers rarely develop after previouslyresected low-grade superficial cancers, and the cases studied representa group that comprises a small minority of all urothelial cancers (1-3,8-10). Therefore, the rate of multifocal development in urothelialcancers, especially those of the low-grade superficial type, attributable

to the seeding/intraepithelial spread mechanism remains to be elucidated.

It is now widely believed that most human sporadic tumors, including TCC of the bladder and upper urinary tract and cancers of thecolon, the breast, and the respiratory tract, result from a multistepprocess of accumulation of genetic alterations. However, almost all ofthe relevant data underlying this conclusion were obtained by a seriesof single-point genetic analyses of a large number of human tumorspecimens with various pathological stages and malignancy (11-17).

A prevalent genetic alteration observed regardless of histologicalstage and grade is generally considered an early event in the tumor-

igenesis, and an alteration associated with histologically and clinicallyadvanced stage and grade is interpreted as a late event. Except for rarecases (18), because it is generally difficult to follow chronologicalgenetic alterations accompanying the growth of a tumor in humansdue to therapeutic interventions, the timing of genetic alterations hasbeen generally speculated from the cumulative genetic data obtainedby single-point analyses. If it is found that such multifocal recurrent

tumors are derived from a common transformed cell, the chronological tracing of genetic alterations in such multifocal cancers mayreveal the precise timing and role of genetic alterations in urothelialcarcinogenesis.

In this study, we tested the presence of microsatellite alterations(i.e., LOH and new alíeleor shift) in metachronous and/or synchronous multifocal low-grade superficial urothelial cancers to determine

the clonal origin of such multifocal cancers. Furthermore, we examined the chronological genetic alterations for a possible genetic divergence and the presence of genetic hierarchy in the developmentand recurrence of urothelial cancers.

MATERIALS AND METHODS

Patients and Tumor Samples. Eighty-seven topologically distinct urothe

lial tumors of the bladder, ureter, and renal pelvis in 29 patients were included

in this study. All tumors were TCC, and brief clinical and pathological data arepresented in Tables 1 and 2. Twenty-six patients had only low-grade (grade1-2 by WHO criteria) superficial papillary cancers in this study period. Three

other patients (patients 23, 5, and 35) were included in this study because theyhad at least one évaluablelow-grade superficial cancers among multifocal

tumors. At least one pair of metachronous heterotopic tumors was analyzed in21 patients. Most of the tumor specimens were obtained at the transurethralendoscopie resection. The tips of each protruding tumor were resected bycup-forceps or a resection loop and snap-frozen until DNA extraction. Anadjacent or deeper portion of the same tumor was taken for the histopatholog-

ical diagnosis. In some bladder tumors, no definite pathological staging information was obtained because the base of the tumor was electrofulgurated. Forrenal pelvic or ureteral tumors, tumor tissue was collected under direct vision,and each tumor tissue was subjected to a histopathological analysis along withDNA extraction. The location of each tumor was recorded, and cases in whichthere was a possibility that recurrent tumors might be caused by incomplete

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MOLECULAR ANALYSIS OF MULTIFOCAL UROTHELIAL CANCERS

Table 1 Clinical and genetic profiles of multiple tumors with concordant microsatellite alterations (group If

Patient391924•3634127IS273231251015TumorGroupl121234567g1*2*3»1»2»3»121212341234567«1»21*21»2*3*1»2*3»I»2»3»4»1»21»212DateSiteGradePT9P9qIIP17p4P4q2q8p1

: Multiple tumors with concordant microsatellitealterationsNov.94

B4Mar.95

B3Mar.90

BlMay.91

B2B2B2B5B5Jul.94

B2B4Iun.97

BlB2B2Nov.97

BlB2B3Jun.91

B7B2Dec.97

B2B2Nov.94

B6May.95

BlAug.96B3Oct97

B6May.97

B2Aug.97

B3OcL97

B2B2B4Nov.97

PPPMay.94

B2Iun.96

BlJun56

B2ÃŒMoSl

B2M.91

B3B4B5Jan.98

BlB2B5Jan.98

BlBlB2B2Jul.94

B4Nov.97

B2Jun.94

PJan.95

B2Mar.92

B2Afr.95

B2222111I1111111414142211221111>21>21>21>214141422112222>12>12>122222142222IbIb,aa•a•aaaNENEaaalalaaaaaaaaaaaaaaaaaaaIbIbIbaaaaaaaIba1lalaIbS.OOS.OOS.OOs,OOS.OOS.OOs,OOS.OOS.OOS.OOs,OOs,00s,OOs,00S.OOS.OOOOOOS.OOS.OO0*O*o*o*OOOO00OOOO0000OONINIOOOOOOOOOO0*o*0*OOOOOOOOo«0»00OOOOOOOO00o»0»o«0»O9O9O9o»OO00OO00OOOOOOOO00OOo*o»o«o«o«o«o«o«o*0«0«o«o«o«o*o«OO00OO0*o*0*o«o«o«o«o«o«OOOOOOOOo«0«o«o»o«o«o«o»o«o»OO00OOOOOOOOOOOOOOOO0«o«o«o«NINININININININIo«0«OOOOOO00OOOOOOOOOOOOOOOOOOOO0»o«o»o«OO00OOOO0000OOOOOOOOOO00OO00OOOOo»o*OOOOOOOOOOOOo«o»0«o»o«o»o«o«00OOOOOONININIOOOOOOOOOOOOOOo«o«0«o«o«o«OOOOO9o»0»0«O9o*o«o»OO00OO00OOOOOOOOOOOOOOOOOOOOo«o«o«o«o«o«o«o*00OOo«o*OO00OO0»o«09OOOOOOOO00OOOOOOOOOOOOOOo»o«0»o»o«o«o«o«OO00OOOOOOOONINIOOOOOOOO00OOo«o»0«o»o«o«0«o«0«o»OOOOOO00OOOOOOOOOOOOOOOO00OOOOOOOOOOOO

OO0000OO

NIOO

NIOONIOONIOONIOO

NIOONIOONIOO

OO0000OOOO00

00OOOOOOOOS,NI

C»S.NIO«NI

OONIOO00

OOOOOOOOOOOOOOOO

NIOO

NIOO

NIOO

NICO

NIOO

NIOONIOO

NIOO

OOOOOOOO

OOOOOOOO

O*000*OOO*OO

NIOO

NIOO

NIOO

NIOONIOO

NIOO

NIOO

OOOOOO00

00OOOO00

OOOOOO

" U. ureteral iunior; P. renal pelvic tumor; B, bladder tumor [the locations of the bladder tumor were as follows: Bl, trigone, B2, posterior wall; B3, right wall; B4, left wall; B5,

dome; B6, anterior wall; B7, bladder neck to prostatic urethra]; OO, retention of heterozygosity at all informative microsatellite loci on the chromosomal arm; O0. LOH at allinformative microsatellite loci on the chromosomal arm; O*, partial chromosomal deletion (LOH) of the chromosomal arm; OS and SO, distinct alíeleswere lost in each tumor sample;S, microsatellite shift (new alíele,microsatellile instability); Nl, not informative; -, same as above; NE. not examined.

The first clinical presentation of urothelial cancer.

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MOLECULAR ANALYSIS OF MULTIFOCAL UROTHELIAL CANCERS

Table 2 Clinical tint! gênent profiles of multiple Iunior* with discordant and concordant nùcro.ïalellilealterations (group 11)und multiple minors with no detected micro.ftitellilealteration (group HI)"

Patient Tumor Date Site Grade pT9p9qUp17p4p4q2q8pGroup

II: Multiple tumors with concordant and discordant microsatellitealterations21

1 May .97 Bl 2 la

2 Oct.97 B2 2 la3 - B2 2 la

4 Jan.98 B2 2>1 a

5 - B2 2>1 a

6 - B4 2>1a16

1' Jan.97 U 2 a

2 Aug.97 Bl 2 ME3 - B2 2 NE

4 - B2 2NE23

1» Iul.95 P 2 3

2 Jan.97 B4 2a5

1* Iul.94 B4 2 Ib

2 Jan.95 B3 2 a

3 Jan.98 Bl 2>3la6

1* Nov.93 U 1>2 a

2 Ian.95 B4 1,2a17

1* Iun.96 B2 1 a

2 Sep.97 B4 1a30

1 Jun.97 B2 2 a

2 Feb.98 B7 2a20

1' Jan.97 P 2 la

2 Jun.97 B3 2NE35

1' Jul.92 P 2 3

2 - B2 2 100oo

ooooooooo*o*0*

0*oo

ooo«0»o«s,O«oooo

ooo«0«oo00ooo«S.OO

S.OOs,OOS.OOS.OOS.OO0«o«

o«o«o«

o«0«o«

o«o«

o«oo

oo0«o»o»o*o»

o»NI

NI

NI

NI

NI

NI00o»

o«o»oo

oo0»o»

o«oo

oos,OOoooo

ooo»

o«oo

oo0«

00oo

oooo00o«

oocmo«oo

o»NI

NI

NIoo

oooo

oo0»ooNINIoooooo

oooooooooooo

ooooooNI

NIo«

oooooo00oo

oooooooo00oo

oooo

o»o«

oooooo

oooo

oo00ooo«

oooo

oooo

oooo

oo00ooo«

ooNI

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o«o«oo

oooooooo

ooc*o»

o«oo

oooo

oo00oooo

ooo»•oo»00

00oo

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o«o»

o»0»NI

NIoo

oo0«oooo

oooo

ooGroup

III: Multiple tumors with no detected microsatellitealteration8

1» Oct.93 B4 2>1 a

2 Jul.97 Bl 1,2a11

1 * Sep.94 B2 1,2 a

2* - U 1,2a29

1» Jul.96 B2 1a2

Nov.97 B2 1a33

1* Apr.97 B2 1 a

2 Oo97 Bl 1 a3 - B2 1 a

4 - B2 1 aoo

oooo

oooo

oooooo

oooooo

oooo

oooo

oooooo

oooooo

oooo

ooNI

NIoo

ooooooOO

00ooooOO

NIOONIoo

oooooooo

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" Boxed symbols, discordant microsatellile alterations among multifocal tumors. Group II patients are divided by a line: patients on top had mullifocal tumors that were judged to

have been derived from an identical progenitor cell; patients on bottom had tumors with undetermined clonal origin. Other symbols are as in Table I.* The first clinical presentation of urothelial cancer.

transurethral resection were omitted. Adjacent portions of each tumor specimen were routinely examined by microscopy, and tumor specimens with heavynormal cell contamination were not subjected to a DNA analysis. The tumorstage and grade were classified according to the tumor-node-metastasis system

and WHO criteria, respectively, by pathologists who were unaware of theresults of this study. Normal control DNA was obtained from peripheral bloodor from normal kidney tissue when a nephroureterectomy was performed fortumors of the upper urinary tract. Tumor and normal control DNAs were

prepared by proteinase K digestion and phenol/chloroform extraction. Onepatient was treated with prophylactic Bacillus Calmette-Guérin instillation

therapy, and 13 patients were treated with prophylactic bladder instillationchemotherapy before and during this study.

Microsatellite Analysis. We used 20 microsatellite markers to detect microsatellite alterations on eight chromosomal arms. The markers we used wereD2S206 and D2S336 on chromosome 2q; D4S404 and D4S1546 on 4p;D4S426 and D4S17ÃŒon 4q: D8S26I and D8S520 on 8p: D9S171, D9SI26,D9SI749, and D9S736 on 9p: D9S66, D9S1848, D9S1793, and GSN on 9q;DÃŒIS907and DIÃŒS922on lip; and DI7S796 and D17SII76 on 17p. Theprimer sequences were obtained from the Genome Database (available on theInternet at http://gdb www. gdb.org). None of microsatellite markers used wasconsidered to be located within or around the coding region of known genes.These eight chromosomal arms were chosen because it has been reported thatthey have a relatively frequent occurrence of LOH or chromosomal alterationin urothelial cancers (19-21). PCR was carried out in 20-/J.1 reaction volumes

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- Patient #9 -

N12345678

D17S1176

N12345678

GSNN 12345678

D9S1749 N1 2345678

D11S907

B -Patientas-

N 1 2 3 4D9S126

N 1 2 3 4D9S1749

N 1 2 3 4GSN (Chr.9)

í^V**>1' XIv_^x r\ y i/p.iip K»!

Normal tnuBíiiinal cell x~ ^ loss? ^ft,

[Cominoli progenitor eel [ i^ ^kxS-v </ V^'

N 1 2 3 4

D17S796N 1 2 3 4

D11S907

17p.8ploss

/f^~~>\ microsatelliteshift s "x

Normaltransitionalcell

N123456 N123456 N123456

D9S66 D17S1176 D2S206Common progenitor cell

orthe previous tumor 4 years before I-1

- Patient #6 - Patient #35 -

tiN 1 2

D9S1749

a.

N 1 2D9S66

N 1 2

D9S1749

N 1 2

D2S206

Fig. I. Auloradiograms of représentativepatients and possible schematic pathways of chronological genetic alteration. A, concordant micro.salellitealterations in eight multipletumors of patient 9 (group I). A complete identical microsatellite shift pattern was found at D9SI749, which suggests that these multifocal tumors were derived from an identical clonalorigin. In addition, loss of an identical alíelewas observed at GSN (9q) and Dì1S907. At DI7SÌ176.a concordant retention of heterozygosity was observed. These genetic alterationswere stable for 4 years. B. discordant microsatellite alterations in four multiple tumors with a presumable identical clonal origin in patient 16 (group II). A concordant LOH pattern(loss of an identical alíele)at [WS126 and GSN with retention of heterozygosity at D9S1749 were found, which indicates the existence of an identical subchromosomal partial deletionand suggests that these multifocal tumors were derived from a common clonal origin. At D17S1176, LOH was detected in tumors 1. 3. and 4 but not in tumor 2. At DìIS907. LOHwas found in tumors 2, 3. and 4 but not in tumor 1. The clear LOH at GSN and D9S126 indicates that each tumor specimen was not contaminated with normal cells. Thus, these fourtumors are considered to have been derived from a common clonal origin and to have acquired genetic divergence during metachronous multifocal development. C. possible schematicpathways of the chronological accumulation of genetic alterations in patient 16. It is speculated that a heterotopic spread of a transformed common progenitor cell occurred after genetic

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with 50-HX) ng of genomic DNA as the template. 1.5 mM MgCU, 2(X) m.M

each dNTP. 2 pmol of each primer. 1 unit of Taq DNA polymerase. and buffersupplied by the manufacturer (Roche Molecular Systems. Branchburg. NJ).Prior to amplification, one of each primer pair was end-labeled with[7-32P]ATP (Life Science Products. Boston. MA). The PCR protocol consistedof 28 cycles of 60 s at 95°C,60 s at 55°C,and 90 s at 72°C,followed by a finalelongation for 5 min at 72°C.Reaction products were diluted with formamide

dye. heat-denatured, and run on bl/t denaturing polyacrylamide gels. The gels

were dried and exposed to X-ray film (Fuji Film, Tokyo, Japan). Initially. LOH

was screened visually for loss of one alíeleor the presence of microsatelliteshifts, and cases with a "partial loss" or "allelic imbalance" were evaluated

further by using the public domain NIH Image program (developed at the NIHand available on the Internet at http://rsb.info.nih.gov/nih-image). The relative

decrease in the intensity with >4()7r of the signal from one tumor alíelewasscored as LOH. Each PCR experiment was performed at least twice. All tumorspecimens were tested for all 20 microsatellite loci listed above.

RESULTS

Table 1 and 2 show the summary of the microsatellite analysisresults of the 29 patients classified into three groups. Group I consisted of 16 patients with tumors with concordant genetic alterationsat all genetic markers tested (Table 1). Group II consisted of ninepatients with tumors with some discordant genetic alterations (Table2). Group III included the four patients with no detected genetic-

alterations (Table 2).In six patients (patients 3, 9, 19, 24, 26, and 34) of group I, a

complete identical microsatellite shift pattern was detected (Fig. 1A).Microsatellite shifts caused by replication error may result in a shortening or an expansion of dinucleotide repeats to any length, and thealteration provides no growth advantage for cancer cells because noneof microsatellite markers used in this study is located within or aroundthe coding region of known genes. Although the ultimate proof of theidentical microsatellite shift may require nucleotide sequencing of therepeats, each microsatellite shift pattern found on usual denaturingpolyacrylamide gels could serve as a definite genetic fingerprint (22,23). Therefore, the multifocal tumors in these six patients are considered to be derived from a common clonal origin. In the other sevenpatients (patients 1. 2. 7. 18, 27, 32, and 31) of group I, an identicalsubchromosomal partial deletions on chromosome 9, 8p, or 4 (e.g.,LOH on 9p with retention of heterozygosity on 9q or vice versa) wasfound along with concordant patterns of LOH on other chromosomes.For example, the four tumors in patient 1 had LOH at D9S126 andD9S736, with the retention of both alíelesat D9S1749. This identicalsubchromosomal partial deletion on the identical alíelein 4 tumors isevidence of an identical clonal origin in this patient. Considering thefrequencies of partial deletions on chromosomes 9, 8p, and 4, whichare reported to be below 13-20.4, and 15%, respectively (24-27), and

the findings that the identical alíelewas lost on other chromosomes,the probability of the observed alteration pattern by chance in eachpatient was roughly calculated to be <2%. [For example, in patient 7.the probability of LOH on 4q without LOH on 4p is ~0.15. The

probability of the loss of the identical alíeleon chromosomes 4q, 9q,and 1Ip is (1/2)3 = 0.125. Because each genetic alteration is presum

ably an independent event, the probability of the concordant genetic-

alterations found in two tumors in patient 7 by chance is ~0.02.] In

the remaining three patients of group I, the concordant LOH pattern isalso evidence of a common clonal origin.

Discordant genetic alterations with or without concordant alterations were observed in multifocal tumors in the nine patients ofgroup II. In one (patient 21) and two (patient 16, and 23) of the ninepatients, an identical microsatellite shift pattern and a subchromosomal partial deletion were detected, respectively, therefore providingevidence that the multifocal tumors in the three patients were derivedfrom an identical progenitor cell (Fig. l, B and Ö).In patient 5, theconcordant LOH pattern in four chromosomes indicates a commonclonal origin, whereas the allelic loss on chromosome 4 detected intumor I was not found in tumors 2 and 3. In the remaining fivepatients in group II, the discordant microsatellite shift pattern ormultiple discordant LOH patterns was found (Fig. l. Fand G). In twopatients (6 and 17), the microsatellite shift found in the first tumor wasnot detected in recurrent tumors.

Overall, excluding the 4 patients in group III with no detectedgenetic alterations. 20 (80%) of the 25 patients are considered to havea common clonal origin. In the remaining five patients of group II, wecould not determine whether or not their multiple tumors shared acommon clonal origin.

Of the 20 patients with multifocal tumors from a presumableidentical clonal origin, no discordant microsatellite alteration on 9pand/or 9q was observed in the 17 patients with chromosome 9 alterations. This finding was in contrast to the finding regarding otherchromosomes because the discordance was observed in one of eightpatients with alterations on lip, three of eight on 17p, two of seven onchromosome 4, one of three on 2q, and one of five on 8p. Overall, therate of discordance was significantly lower for chromosome 9 (0 of16, 0%) than for the other chromosomes tested (8 of 31, 26%;P = 0.038, Fisher's exact test).

In 13 (patients 3,9, 1, 2, 18, 7, 25, 10, and 15 in group I and patients21, 16, 23, and 5 in group II) of the 20 patients with multifocal tumorsof a presumable identical clonal origin, a chronological tracing ofgenetic alterations was available. Representative autoradiograms arepresented in Fig. 1, A, B, and D. In addition, possible schematic-

pathways of genetic alterations during the multifocal development ofurothelial cancers in patients 16 and 21 are presented in Fig. 1, C and£.Of the 13 patients in group I, genetic alterations were stable in 9.Genetic heterogeneity and an apparent accumulated pattern of genetic-

alterations were detected in three (patients 21, 16, and 5; 23%) andonly one (patient 23; 8%) patient, respectively.

Intravesical instillation chemotherapy was performed before orduring this study in 9 (patients 3, 9, 34, 1.2, 18. 27, 7, and 25) of 16group I patients and in 4 (patients 21, 5, 6, and 17) in 9 group IIpatients. One patient (patient 7) was treated with Bacillus Calmelte-

Giierin during this study period. The data suggest that a history ofbladder instillation chemotherapy did not influence the occurrence ofdiscordant genetic alterations among multifocal urothelial cancers.

alterations on chromosome 9 and before the clinical presentalion of Iunior I. I), discordant microsatellile alterations with a complete identical microsatellilc shift pallern in 6 multipletumors of patient 21 (group II). An identical microsatellite shift pattern was found at D9S66, which suggests lhat these tumors were derived from a common clonal origin. LOH alDÕ7SI176 delected in the tumor I was not found in the five recurrent tumors. The LOH at D2S206 in the recurrent tumors was noi detected in lumor 1. £,possible schematic pathwaysof the chronological accumulalion of genetic alterations in patient 21. A heterolopic spread of a common progenitor cell might have occurred after genetic alterations on chromosome9 (microsatellite shift) and before the clinical presentation of tumor I. This palien! also had a history of bladder lumor 4 years prior to lumor 1. F. disappearance of a microsatellileshift in patient 6. A microsatellite shift at D9S1749 in tumor I was not found in tumor 2. whereas concordant LOH was found al G5W. We could not determine whether these multipletumors were derived from a common clonal origin. G. discordant microsatellite alterations in two multiple tumors in palien! 35. Additional allelic loss was found al DVS1749 on 9p.whereas concordant LOH was found al DVS66 on 9q and distinct alíeleswere lost al D2S206. We could not determine whether these multiple tumors were derived from a commonclonal origin. Lanes N. normal: Lanes 1-8. tumors 1-8. respectively; arrows, concordant LOH pattern among multifocal tumors; arrowheads, discordant LOH pallern: *. microsatellileshift (new alíele,microsatellite instability): ///»—.9/>—,and so on, genetic alterations on the chromosomal arm indicated; üí/(.v).microsatellile shift on 9q.

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DISCUSSION

The clarification of the clonal origin of multifocal tumors of thebladder and upper urinary tract would have a significant impact on theevaluation and design of effective treatments and the prevention ofrecurrence and progression to more invasive tumors. This also holdstrue in cancers of the head and neck (28, 29), the lung (30), the ovary(31), breast (32), liver (33), and so on. There have been several studiesregarding the clonal origin of multifocal lesions of various types ofhuman cancers, using molecular genetic methods such as X-chromo-some inactivation, mutation analysis of a specific gene (e.g.,p53), andmicrosatellite analysis. In some types of cancer, the proposed mechanism of the multiplicity is contradictory among the reported studies(28, 29). Such discrepancies may be caused by the difference ingenetic markers used in each study. In this study, if we had not testedchromosome 9 markers, the multifocal tumors in four patients ofgroup II might have been misjudged regarding whether they haddistinct clonal origins. In accordance with our findings obtained usingthe patterns of LOH, subchromosomal partial deletion, and microsatellite shift as a genetic fingerprint, we propose that multifocal low-grade superficial urothelial cancers in 80% of patients or more stemfrom a clonally common progenitor cell. This number could be anunderestimation because, if a greater number of microsatellite markershad been used, specific genetic alterations identical among the tumorsmay have been detected even among the remaining five patients(patients 6, 17, 30, 20, and 35) of group II and four patients of groupIII. However, the disappearance and/or diversity of genetic alterationsfound in the five group II patients suggest that a subset of multifocalurothelial cancers might develop from distinct progenitor cells due tothe field cancerization effect.

This study provides a model for the chronological tracing ofgenetic alterations in human cancers. If multiple metachronousurothelial tumors are confirmed to be derived from a single progenitor cell in individual patients, such urothelial cancers may bea good system to evaluate sequential genetic alterations chronologically because heterotopic recurrences are very common inthese cancers. In this study, 13 patients (9 from group I and 4 fromgroup II) with multiple metachronous tumors with a possibleidentical clonal origin were followed up for periods ranging from4 months to >4 years. Interestingly, the genetic alterations detected were stable in 9 (64%) group I patients of the 13 patients. Infive patients (patients 1, 2, 9, 15, and 25), the alteration patternswere identical for S3 years. This indicates that most low-gradesuperficial papillary urothelial cancers are genetically stable andthat it is rather rare for these papillary tumors to acquire additionalgenetic alterations. It is well known that low-grade superficialtumors of the bladder rarely progress to deeply invasive tumorswith high malignancy and rarely metastasize to other organs (1-3).The phenomenon could be explained partly by the high geneticstability of low-grade superficial tumors. On the other hand, thegenetic heterogeneity detected in the multiple tumors in fourpatients of group II may provide some insights into the development and recurrence of urothelial cancers. Contrary to our expectation, an apparent accumulated pattern of genetic alteration wasfound in only one patient. Instead, genetic diversity was observedin three patients (patients 16, 21, and 5). As illustrated in Fig. 1, C(patient 16) and E (patient 21), there should have been clinicallydormant clonal spread of transitional cells with chromosome 9alterations for a relatively long time. The data suggest that theclinical chronology of evident lesions is distinct from the chronology of genetic alterations. In addition, all of the multifocal tumorsfrom the 20 patients with a possible identical clonal origin sharedan identical genetic alteration pattern of 9p and 9q, thus further

confirming the role of chromosome 9 alteration as an early eventin the urothelial carcinogenesis (10, 34-39). Presumably, the multifocal urothelial cancers acquire genetic diversity during the metachronous development after the chromosome 9 alteration (8, 10).On the other hand, it should be noted that this study focused onlow-grade superficial tumors that have been shown to have fre

quent chromosome 9 alterations, and the data presented here maybe biased for such low-grade superficial tumors with chromosome

9 alterations. It has been proposed that genetic alterations associated with another form of superficial urothelial cancer, i.e., carcinoma in situ, are different from those with papillary tumors (40,41). One study showed the relatively low frequency of 9q LOH incarcinoma in situ of the bladder (40). Because this study did notinclude carcinoma in situ patients, it would be interesting andimportant to know if such intraepithelial dormant clonal spread oftransitional cells plays an important role in formation of carcinomain situ without chromosome 9 alteration. In addition, it remains tobe determined whether carcinoma in situ is genetically more unstable than low-grade superficial papillary tumors and whether

genetic heterogeneity is more frequently present in either patient.Clinically, the efficacy of surgical treatments in individual patients

could be assessed by analyses of the microsatellite alteration pattern.For example, tumors 2-6 in patient 21 are not considered to have been

caused by a treatment failure of tumor 1, although all of these tumorsare considered to share a common clonal origin. The same conceptalso holds true in other patients of group II. In contrast, it remainsunknown whether the multiple recurrences in group I, especially inpatients 9, 2, and 1, were caused by treatment failure. Furthermore, itwould be very helpful to know whether the patients with multifocaltumors with genetic diversity are more prone to undergo progressionto biologically and clinically more malignant tumors. Interestingly, allof the three patients with high-grade or locally advanced tumors hadgenetic diversity among the multifocal tumors (patients 23, 5, and 35of group II).

Because the result of this study suggested that most multifocalsuperficial recurrent urothelial cancers are derived from a commonclonal origin and are genetically stable, the specific microsatellitealteration (LOH or shift) could be used as a genetic marker for theearly detection of recurrence in the follow-up of urothelial cancerpatients. Such strategies using the detection of microsatellite alterations from urine specimens have already been attempted by Steineret al. (42). Although our results support the validity of this approach,the disappearance of genetic alterations observed in several recurrenttumors of seven patients in this study may give a risk of false-negativeresults in such a strategy, unless an appropriate genetic alteration isused as a fingerprint.

In conclusion, we have shown that most (presumably >80% ofpatients) multifocal low-grade superficial papillary urothelial cancersstem from a common progenitor cell. Although most low-grade superficial urothelial cancers seem to be genetically stable, geneticdivergence occurs in a certain subset of patients. Genetic divergenceand heterotopic spread generally occur after chromosome 9p and 9qalterations, and in some patients, they occur long before the clinicalmanifestation of multifocal tumors.

ACKNOWLEDGMENTS

We thank the many urologists for their kind assistance in collecting samples. We also thank Itsuko Fujiwara and Tomoko Matsushita for their technical

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MOLECULAR ANALYSIS OF MULTIFOCAL UROTHELIAL CANCERS

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1998;58:5835-5841. Cancer Res   Takeshi Takahashi, Tomonori Habuchi, Yoshiyuki Kakehi, et al.   Cancers of the Bladder and Upper Urinary TractClonal and Chronological Genetic Analysis of Multifocal

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