LOSS OF HETEROZYGOSITY AT CHROMOSOME SEGMENTS 8p22 AND8p11.2-21.1 IN TRANSITIONAL-CELL CARCINOMA OF THE URINARYBLADDERChan CHOI

1,3*, Mi Hwa K IM1,3, Sang-Woo JUHNG

1,3 and Bong-Ryoul OH2,3

1Department of Pathology, Chonnam National University Medical School, Kwangju, Korea2Department of Urology, Chonnam National University Medical School, Kwangju, Korea3Research Institute of Medical Sciences, Chonnam National University, Kwangju, Korea

To identify the putative tumor-suppressor gene (TSG) in-volved in transitional-cell carcinoma (TCC) of the urinarybladder, we undertook an allelotyping analysis in 48 cases ofTCC. Relatively high percentages of allelic loss were found in2p (5 of 23, 21.7%), 8p (9 of 21, 42.9%), 9p (4 of 20, 20.0%), 12q(6 of 28, 21.4%), 15q (1 of 5, 20%; 4 of 20, 20%), 17p (7 of 26,26.9%) and 22q (6 of 23, 26.1%). On the basis of these results,fine-deletion mapping was performed on chromosome 8 in52 cases by PCR of 15 microsatellite markers. Two distinctregions of common deletion were found. A 10 cM telomericregion was located to 8p22, defined by D8S511 and D8S258. A17 cM centromeric region was located to 8p11.2-21.1, flankedby D8S298 and D8S535. The distance between the telomericand the centromeric regions of common deletion was 3 cM.Loss of heterozygosity of 8p22 was frequently observed intumors of high grade or advanced stage. Int. J. Cancer 86:501–505, 2000.© 2000 Wiley-Liss, Inc.

The development of cancer is associated with multiple geneticalterations in oncogenes and tumor-suppressor genes. Cytogeneticand molecular genetic studies suggest that such changes may beinvolved in carcinogenesis of TCC. Chromosomal analyses haveidentified frequent breakpoints in 1, 3p, 11p, 8, 13, isochromosome 5,monosomy 9 and trisomy 7. Loss of heterozygosity (LOH), suggest-ing the presence of a tumor-suppressor gene (TSG), has been reportedfor many chromosome arms in TCC, including 4p, 6p, 8p, 9p, 9q, 11p,14q, 17p and 18q (Keen and Knowles, 1994; Knowleset al., 1993,1994; Rosinet al., 1995). The results indicate that TCC undergoesmultiple genetic changes during tumor progression. However, the roleof these genetic abnormalities in the etiology and progression of thedisease remains poorly understood.

To understand the molecular mechanisms of this disease, it isnecessary to isolate the genes underlying these chromosomal ab-normalities. Toward this end, we undertook an allelotyping ofTCC. Our goal was to analyze a sufficient sample size withpolymorphic markers so that informative results could be obtainedfor all chromosome arms. We compared normal and tumor DNAfrom 48 patients using 44 markers of autosomal chromosomearms. Frequencies of LOH were highest in chromosome arms 2p,8p, 9p, 12q, 15q, 17p and 22q. On the basis of these results,fine-deletion mapping was carried out on 52 cases using 15 mic-rosatellite markers of chromosome 8. Two distinct regions ofcommon deletion were found. We also analyzed the relationshipbetween 8p22 LOH and histological grade and stage of TCC.

MATERIAL AND METHODS

Patient tissueTCC of the urinary bladder and corresponding normal tissues

were obtained from 70 patients at the Chonnam National Univer-sity Hospital (Kwangju, Korea). Allelotyping was performed in 48cases, and fine-deletion mapping of chromosome 8 was performedin 50 cases. They were graded according to WHO recommenda-tions (Mostofi et al., 1973) and staged using the TNM system(Bearhset al., 1992).

Genomic DNATumor tissues were obtained at the time of surgery and frozen at

–80°C. Sequential 5mm sections were cut for hematoxylin and

eosin staining. Hematoxylin and eosin–stained sections were ex-amined, and tumor specimens with.75% tumor cell content weresubjected to DNA isolation. Normal DNA was extracted fromperipheral blood or frozen tissue. DNA was extracted and purifiedby standard procedures.

DNA primers and PCR conditionsEach matched pair of normal and tumor DNA was subjected to

PCR analysis with 59 microsatellite markers. For allelotyping, 44microsatellite markers were used:D1S450, D1S213, D2S337,D2S172, D3S1261, D3S1311, D4S1546, D4S408, D5S630,D5S436, D6S260, D6S305, D8S261, D9S158, D10S89, D11S929,D11S912, D12S77, D12S78, D13S175, D13S260, D14S261,D14S80, D15S165, D16S407, D16S520, D17S945, D17S784,D18S54, D18S65, D19S177, D19S412, D20S120, D21S265,D22S446, D22S282(Gyapay et al., 1994), EGFR (Chi et al.,1992), D8S161 (Couch et al., 1991), IFNA (Kwiatkowski andDiaz, 1992), D20S54, D21S120(Weissenbachet al., 1992),D7S23, D10S173, FES(http://www.gdb.org). For deletion map-ping of chromosome 8, 15 microsatellite markers were used:D8S520, D8S265, D8S552, D8S549, D8S261, D8S258, D8S298,D8S283, D8S535, D8S255, D8S268, D8S509, D8S275(Gyapayetal., 1994),D8S1839(http://www.gdb.org).

PCRs were carried out as described below with the inclusion of1 [a-32P]-ATP–end-labeled primer. PCR was performed in 10mlvolumes of mixture containing 13 PCR buffer [10 mM Tris (pH8.0), 50 mM KCl, 1.5 mM MgCl2], 0.5 mM of both unlabeled andlabeled primer, 10 ng of template DNA, 0.02 units of Taq DNApolymerase and 125mM of each deoxynucleoside triphosphate.Reactions were cycled 32 times with a final elongation period of10 min at 72°C. Each cycle consisted of 1 min at 94°C, 1 min at51° to 58°C and 1 min at 72°C in a 480 thermal cycler (Perkin-Elmer Cetus, Emeryville, CA). PCR products were denatured andseparated on a 6% polyacrylamide gel containing 7 M urea for 2 hrat room temperature. After electrophoresis, the gel was fixed, driedand exposed to X-ray film for 6 to 16 hr.

LOH analysisIn cases where a particular marker was heterozygous in normal

lymphocyte DNA, LOH was assessed in the corresponding tumorDNA by 2 observers. LOH was defined as any case where the ratioof the intensity in the tumor allele was,50% of the normal alleleratio. When the results were not clear-cut, the intensity of thesignals was measured by phosphorimage analysis (ImageQuant;Molecular Dynamics, Sunnyvale, CA). The ratio of the signal

Grant sponsor: Chonnam National University; Grant number: 1996;Grant sponsor: Ministry of Education (Republic of Korea); Grant number:98-176.

*Correspondence to: Department of Pathology, Chonnam UniversityMedical School, 5 Hakdong, Dongku, Kwangju, Korea, 501-190. Fax:182-62-227-3429. E-mail: cchoi@chonnam.chonnam.ac.kr

Received 26 August 1999; Revised 12 November 1999

Int. J. Cancer:86, 501–505 (2000)© 2000 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

intensity between the 2 alleles in the tumor DNA was comparedwith that in the corresponding normal tissue DNA.

Statistical analysisThex2 test was used to evaluate the association of incidence of

8p22 LOH with histological grade and stage of TCC.

RESULTS

An average of 21 informative cases of TCC were analyzed forLOH at each of 44 microsatellite markers (Table I). Severalregions showed relatively high levels of LOH (Fig. 1). Percentagesof allelic loss in informative cases at these regions were as follows:2p, 5 of 23, 21.7%; 8p, 9 of 21, 42.9%; 9p, 4 of 20, 20.0%; 12q,6 of 28, 21.4%; 15q, 1 of 5, 20% and 4 of 20, 20%; 17p, 7 of 26,26.9%; 22q, 6 of 23, 26.1%. The relationship between LOH andclinical parameters revealed a significant trend: a higher number ofallelic losses in tumors of high grade and advanced stage.

To refine the deletion map of chromosome 8 in TCC of thebladder, 50 tumors were examined using 15 microsatellite markers(Table II). They were ordered according to Spurr and Leach(1995). Twenty-three of the 50 cases (46%) revealed LOH for 1 or

more chromosome 8 markers. Detailed analysis showed 2 distinctregions of common deletion, a telomeric region betweenD8S511and D8S258in 8p22 and a centromeric region betweenD8S298andD8S535in 8p11.2-21.1 (Fig. 2). Five cases (case numbers 25,14, 142, 209 and 31) had LOH only at 8p22, and 2 (case numbers213 and 71) had LOH at 8p11.2-21.1 only. Thirteen cases exhib-ited LOH at both 8p22 and 8p11.2-21.1, while 2 (case numbers141 and 204) showed independent deletions for 8p22 and 8p11.2-21.1. The telomeric region betweenD8S511and D8S258wasestimated to be 10 cM, and the centromeric region betweenD8S298and D8S535was estimated to be 17 cM. The distancebetween the 2 commonly deleted regions is 3cM, according toGyapayet al. (1994).

Figure 3 shows examples of PCR analysis of the LOH atD8S511, D8S549, D8S261andD8S258.Case 31 exhibited LOH atD8S549,was uninformative atD8S261andD8S258and retainedheterozygosity atD8S511.The corresponding analyses of cases142 and 25 are shown in Figure 3. Similar analyses for LOH atD8S298, D8S1839, D8S283andD8S535in cases 141, 163 and 213are illustrated in Figure 4.

When the relationship between 8p22 deletion and tumor gradeand stage was examined, a significant association was found be-tween LOH and grade (p , 0.01) and stage (p , 0.01) (Table III).

FIGURE 1 – Frequency of allelic loss on each chromosome arm.Polymorphic markers are listed in Table I. Relatively high frequencyof LOH was found in 2p, 8p, 9p, 12q, 15q, 17p and 22q.

TABLE I – INCIDENCE OF LOH IN TCC OF THE URINARY BLADDER1

Chromosomearm Locus Chromosomal

region

Cases with LOH/informative cases

(%)2

1p D1S450 1pter-qter 0/23 (0)1q D1S213 1pter-qter 3/29 (10.3)2p D2S337 2p15 5/23 (21.7)2q D2S172 2q33-37 3/20 (15.0)3p D3S1261 3p12-14.1 2/21 (9.5)3q D3S1311 3q27-qter 1/21 (4.7)4p D4S1546 4pter-qter 0/20 (0)4q D4S408 4pter-qter 2/16 (12.5)5p D5S630 5pter-qter 2/23 (8.7)5q D5S436 5pter-qter 4/23 (17.4)6p D6S260 6p23 1/20 (5.0)6q D6S305 6q25.2-27 1/21 (4.8)7p EGFR 7p12 2/23 (8.7)7q D7S23 7q31-32 0/24 (0)8p D8S261 8p22 9/21 (42.9)8q D8S161 8qter 1/20 (5.0)9p IFNA 9p22 4/20 (20.0)9q D9S158 9q34.3 1/22 (4.5)10p D10S89 10pter-11.2 2/23 (8.7)10q D10S173 10q23-25 4/25 (16.0)11p D11S929 11p14 3/19 (15.8)11q D11S912 11q23 1/20 (5.0)12p D12S77 12pter-13.2 3/26 (11.5)12q D12S78 12q14-24.3 6/28 (21.4)13q D13S175 13q11 1/23 (4.3)13q D13S260 13q32-33 4/24 (16.7)14q D14S261 14q11.1-11.2 2/23 (8.7)14q D14S80 14q12-13 1/16 (6.3)15q D15S165 15pter-qter 1/5 (20.0)15q FES 15q26.1 4/20 (20.0)16p D16S407 16p13.1 1/24 (4.2)16q D16S520 16pter-qter 1/22 (4.5)17p D17S945 17pter-qter 7/26 (26.9)17q D17S784 17pter-qter 4/22 (18.2)18p D18S54 18pter-11.2 0/21 (0)18q D18S65 18q12.2-12.3 0/23 (0)19p D19S177 19p13.3 1/14 (7.1)19q D19S412 19q13.3 3/22 (13.6)20p D20S54 20pter-qter 0/24 (0)20q D20S120 20pter-qter 2/19 (10.5)21q D21S120 21q21-22.1 2/14 (14.3)21q D21S265 21q22.1 1/16 (6.3)22q D22S446 22q11.2 2/21 (9.5)22q D22S282 22q13.2-13.3 6/23 (26.1)1A total of 48 cases examined.–2An average of 21 informative cases

of TCC of the urinary bladder analyzed per marker.

TABLE II – LOH ON CHROMOSOME 8 FOR TCCOF THE URINARY BLADDER1

Locus2 Chromosomalregion

Cases with LOH/informative cases

(%)

D8S520 8pter-qter 7/36 (19.4)D8S265 8p23-23.1 6/24 (25.0)D8S552 8p22 9/29 (31.0)D8S511 8pter-23.1 13/17 (35.1)D8S549 8pter-23.1 5/27 (18.5)D8S261 8p22 13/35 (37.1)D8S258 8p22 8/34 (23.5)D8S298 8pter-qter 6/36 (16.7)D8S1839 8pter-qter 12/39 (28.2)D8S283 8pter-qter 12/38 (31.6)D8S535 8pter-qter 7/36 (19.4)D8S255 8pter-qter 4/32 (12.5)D8S268 8p21.3-cen 3/28 (10.7)D8S509 8pter-qter 4/29 (13.8)D8S275 8q13-21.1 0/25 (0)1A total of 50 cases examined.–2Ordered according to Spurr and

Leach (1995).

502 CHOI ET AL.

DISCUSSION

By allelotyping TCC of the urinary bladder, we found rela-tively high frequencies of LOH at 2p, 8p, 9p, 12q, 15q, 17p and22q. Such high occurrences of LOH at 8p, 9p, 12q and 15q havebeen observed by other investigators (Knowleset al., 1994;Rosin et al., 1995); however, no high incidence of LOH at 2pand 22q has been reported so far. The frequency of 8p LOH washighest (42.9 %) among the chromosome arms examined. Chro-mosome 9q has been shown to be frequently affected by LOHin TCC of the bladder at 9q33-q34.1 (Keen and Knowles, 1994)and 9q32-33 (Habuchiet al.,1997). The low LOH at 9q (4.5%)in our study may result from the choice of theD9S158micro-satellite marker located at 9q34.3.

Previous studies have described 8p LOH in TCC of thebladder (Knowleset al.,1993, 1994; Ohgakiet al.,1999; Rosinet al., 1995; Takle and Knowles, 1996), colorectal carcinoma(Cunninghamet al., 1993; Farringtonet al., 1996), prostatecarcinoma (Booksteinet al., 1997; Bovaet al., 1993), breastcarcinoma (Yokotaet al., 1999), hepatocellular carcinoma(Piao et al., 1999), head-and-neck carcinoma (Liet al., 1994)and lung carcinoma (Wistubaet al., 1999). Fine-deletion map-ping of chromosome 8 using 15 polymorphic markers demon-strated 2 distinct regions of common loss at 8p. At least 2regions of loss from 8p were reported in colorectal carcinoma(Farringtonet al.,1996), TCC of the urinary bladder (Takle andKnowles, 1996) and prostatic carcinoma (Bovaet al., 1993;Trapmanet al., 1994).

D8S511andD8S258define the telomeric region (about 10 cM)of common loss in 8p22, which overlaps deleted regions frequentlyidentified in other carcinomas. Tackle and Knowles (1996) broadlydefined the commonly deleted region as delimited byD8S264andD8S133in TCC of the urinary bladder. Cunninghamet al. (1993)also identified a commonly deleted region in colorectal cancer thatis broadly defined by the markersLPL andANK1. Subsequently,this region was more strictly defined in colorectal cancer byFarringtonet al. (1996), betweenLPL and D8S298.Bova et al.(1993) found homozygous deletion of theMSR locus in a meta-static prostate carcinoma. Also of interest is the commonly deletedregion of breast carcinoma flanked byD8S511 and D8S1991(Yokota et al., 1999). This region, as well as theMSR locus, isincluded betweenD8S511andD8S258.The putative TSG of TCC

FIGURE 2 – Allelic loss patterns of TCC of the urinary bladder for chromosome 8. For each carcinoma, all informative loci are shown (opensquares, constitutional heterozygosity with LOH; solid squares, constitutional heterozygosity with no LOH; open triangles, microsatelliteinstability; blank spaces, uninformative). With the assumption that alleles in all regions between the loci exhibiting allelic loss are lost, solidlinesindicate retained regions of chromosome 8 and open spaces represent regions of allelic loss. Broken lines represent regions that are uncertainfor some loci.

FIGURE 3 – LOH at chromosome 8p22 in TCC of the urinary blad-der. A partial ideogram of chromosome 8 and the cytogenetic positionof 4 loci encompassing 8p22 are shown to the left of the diagram.Representative microsatellite analyses of normal (N) and tumor (T)DNA from cases 31, 142 and 25 were chosen to illustrate 4 informativeloci encompassing the telomeric region of common loss. Arrowheadsindicate alleles showing LOH. HET, constitutional heterozygositywith no LOH; UI, uninformative.

5038p DELETION IN URINARY BLADDER TCC

may be the same or very close to that of prostatic carcinoma. As8p22 LOH is frequently found in tumors of high grade and ad-vanced stage, the function of such a putative TSG may be associ-ated with cancer progression.

D8S298and D8S535define a centromeric region of commonloss in 8p22-p12 about 17 cM in length. Like the telomeric region,this region overlaps commonly deleted regions of other carcino-mas. In TCC of the urinary bladder, the commonly deleted regionis demarcated byD8S87andD8S259(Takle and Knowles, 1996)using the cen-D8S278-D8S535-D8S505-D8S259-D8S87-telmarker order (Spurr and Leach, 1995). In colorectal carcinoma, aregion was flanked byD8S136and D8S137(Farringtonet al.,1996), and in prostate carcinoma it was betweenD8S133andD8S87(Trapmanet al.,1994). This centromeric region of commondeletion can be divided into 2 regions: one defined byD8S136andD8S137and the other byD8S87 and D8S259.Both of theseregions are found within that denoted byD8S298andD8S535.

In summary, we identified 2 distinct regions of common loss at8p in TCC. The telomeric region (10 cM in length) is defined byD8S511andD8S258at 8p22 and the centromeric region (17 cM)by D8S298andD8S535,which maps to 8p22-p12.

ACKNOWLEDGEMENT

We thank Mr. S.-J. Jeong for proofreading.

REFERENCES

BEAHRS, O.H., HENSON, D.E., HUNTER, R.V.P. and KENNEDY, B.J. (EDS.),Manual for staging for cancer(4th ed.), American Joint Committee forCancer, pp. 195–197, Lippincott, Philadelphia (1992).

BOOKSTEIN, R., BOVA, G.S., MACGROGAN, D., LEVY, A. and ISAACS, W.B.,Tumor-suppressor genes in prostatic oncogenesis: a positional approach.Brit. J. Urol., 79 (Suppl.1), 28–36 (1997).

BOVA, G.S., CARTER, B.S., BUSSEMAKERS, M.J., EMI, M., FUJIWARA, Y.,KYPRIANOU, N., JACOBS, S.C., ROBINSON, J.C., EPSTEIN, J.I. and WALSH,P.C., Homozygous deletion and frequent allelic loss of chromosome 8p22loci in human prostate cancer.Cancer Res.,53, 3869–3873 (1993).

CHI, D.D., HING, A.V., HELMS, C., STEINBRUECK, T., MISHRA, S.K. andDONIS-KELLER, H., Two chromosome 7 dinucleotide repeat polymorphismsat gene loci epidermal growth factor receptor (EGFR) and pro alpha 2 (I)collagen (COL1A2).Hum. mol. Genet.,1, 135 (1992).

COUCH, F.J., MCCARTHY, T.V., GREGG, R.G. and HOGAN, K., Dinucleotiderepeat polymorphism at the D8S161 locus.Nucl. Acids Res.,19, 5093(1991).

CUNNINGHAM, C., DUNLOP, M.G., WYLLIE , A.H. and BIRD, C.C., Deletionmapping in colorectal cancer of a putative tumour suppressor gene in8p22-p21.3.Oncogene,8, 1391–1396 (1993).

FARRINGTON, S.M., CUNNINGHAM, C., BOYLE, S.M., WYLLIE , A.H. andDUNLOP, M.G., Detailed physical and deletion mapping of 8p with isolationof YAC clones from tumour suppressor loci involved in colorectal cancer.Oncogene,12, 1803–1808 (1996).

GYAPAY, G., MORISSETTE, J., VIGNAL, A., DIB, C., FIZAMES, C., MILLAS-SEAU, P., MARC, S., BERNARDI, G., LATHROP, M. and WEISSENBACH, J., The1993–94 Genethon human genetic linkage map.Nature (Genet.),7, 246–339 (1994).

HABUCHI, T., YOSHIDA, O. and KNOWLES, M.A., A novel candidate tumoursuppressor locus at 9q32-33 in bladder cancer: localization of the candidateregion within a single 840 kb YAC.Hum. mol. Genet.,6, 913–919 (1997).

KEEN, A.J. and KNOWLES, M.A., Definition of two regions of deletion onchromosome 9 in carcinoma of the bladder.Oncogene,9, 2083–2088(1994).

KNOWLES, M.A., ELDER, P.A., WILLIAMSON , M., CAIRNS, J.P., SHAW, M.E.and LAW, M.G., Allelotype of human bladder cancer.Cancer Res.,54,531–538 (1994).

KNOWLES, M.A., SHAW, M.E. and PROCTOR, A.J., Deletion mapping ofchromosome 8 in cancers of the urinary bladder using restriction fragmentlength polymorphisms and microsatellite polymorphisms.Oncogene,8,1357–1364 (1993).

KWIATKOWSKI, D.J. and DIAZ, M.O., Dinucleotide repeat polymorphism atthe IFNA locus (9p22).Hum. mol. Genet.,1, 658 (1992).

LI, X., LEE, N.K., YE, Y.W., WABER, P.G., SSHWEITZER, C., CHENG, Q.C.and NISEN, P.D., Allelic loss at chromosomes 3p, 8p, 13q, and 17passociated with poor prognosis in head and neck cancer.J. nat. CancerInst., 86, 1524–1529 (1994).

MOSTOFI, F.K., SOBIN, L.H. and TORLONI, H., Histological typing of uri-nary bladder tumours. International classification of tumours,19, WorldHealth Organization, Geneva (1973).

OHGAKI, K., LINDA, A., OGAWA, O., KUBOTA, Y., AKIMOTO, M. and EMI,M., Localization of tumor suppressor gene associated with distant metas-tasis of urinary bladder cancer to a 1-Mb interval on 8p22.Genes Chro-mosomes Cancer,25, 1–5 (1999).

PIAO, Z., KIM, N.G., KIM, H. and PARK, C., Deletion mapping of the short

FIGURE 4 – LOH at chromosome 8p11.2-21.1 in TCC of the urinarybladder. A partial ideogram of chromosome 8 and the cytogeneticposition of 4 loci encompassing 8p11.2-21.1 are shown to the left ofthe diagram. Representative microsatellite analyses of normal (N) andtumor (T) DNA from cases 141, 163 and 213 were chosen to illustrate4 informative loci encompassing the centromeric region of commonloss. Arrowheads indicate alleles showing LOH. HET, constitutionalheterozygosity with no LOH; UI, uninformative.

TABLE III – RELATIONSHIP BETWEEN 8P22 LOH AND TUMOR GRADEAND STAGE1

GradeStage

pTa pT1 $pT2 Total

1 1/15 (6.7) 3/11 (27.9) 0/0 (0) 4/26 (15.4)2 1/4 (25.0) 2/5 (40.0) 3/5 (60.0) 6/15 (40.0)3 0/0 (0) 1/2 (50.0) 7/7 (100) 8/9 (88.9)Total 2/19 (10.5) 6/18 (33.3) 10/12 (83.3) 18/50 (36.0)1A total of 50 cases analyzed.–Proportions of tumors showing LOH

are given in each group. Figures in brackets indicate percentage withLOH. Significant difference was found between 8p22 LOH and tumorgrade (p , 0.01) and stage (p , 0.01), determined byx2 test.

504 CHOI ET AL.

arm of chromosome 8 in hepatocellular carcinoma.Cancer Lett.,138,227–232 (1999).ROSIN, M.P., CAIRNS, P., EPSTEIN, J.I., SCHOENBERG, M.P. and SIDRANSKY,D., Partial allelotype of carcinomain situ of the human bladder.CancerRes.,55, 5213–5216 (1995).SPURR, N.K. and LEACH, R.J., Report of the second international workshopon human chromosome 8 mapping 1994.Cytogenet. Cell Genet.,68,147–164 (1995).TAKLE, L.A. and KNOWLES, M.A., Deletion mapping implicates two tumorsuppressor genes on chromosome 8p in the development of bladder cancer.Oncogene,12, 1083–1087 (1996).TRAPMAN, J., SLEDDENS, H.F., VAN DER WEIDEN, M.M., DINJENS, W.N.,KONIG, J.J., SCHRODER, F.H., ABER, P.W. and BOSMAN, F.T., Loss ofheterozygosity of chromosome 8 microsatellite loci implicates a candidate

tumor suppressor gene between the lociD8S87and D8S133in humanprostate cancer.Cancer Res.,54, 6061–6064 (1994).

WEISSENBACH, J., GYAPAY, G., DIB, C., VIGNAL, A., MORISSETTE, J., MIL-LASSEAU, P., VAYSSEIX, G. and LATHROP, M., A second-generation linkagemap of the human genome.Nature (Lond.),359,794–801 (1992).

WISTUBA, I.I., BEHRENS, C., VIRMANI , A.K., MILCHGRUB, S., SYED, S., LAM,S., MACKAY , B., MINNA, J.D. and GAZDAR, A.F., Allelic losses at chromo-some 8p21-23 are early and frequent events in the pathogenesis of lungcancer.Cancer Res.,59, 1973–1979 (1999).

YOKOTA, T., YOSHIMOTOS, M., AKIYAMA , F., SAKAMOTO, G., KASUMI, F.,NAKAMURA , Y. and EMI, M., Localization of a tumor suppressor geneassociated with the progression of human breast carcinoma within a 1-cMinterval of 8p22-p23.1.Cancer,85, 447–452 (1999).

5058p DELETION IN URINARY BLADDER TCC