Chromosome 8P Deletions Are Associated with Invasive Tumor Growth in Urinary Bladder Cancer

American Journal ofPathology, Vol. 151, No. 3, September 1997 Copynight © American Societyfor Investigative Pathology

Chromosome 8p Deletions Are Associated with Invasive Tumor Growth in Urinary Bladder Cancer

Urs Wagner,* Lukas Bubendorf,* a third locus telomeric to 8p23.1.912 Previous studies Thomas C. Gasser,t Holger Moch,* using molecular analysis and comparative genomic hy- Jean-Philippe Gorog,* Jan Richter,* bridization (CGH) have found 8p deletions in more than Michael J. Mihatsch,* Frederick M. Waldman, 25% of bladder tumors.57' 13 Chromosome 8 mapping by and Guido Sauter* Takle et al13 revealed common areas of deletion span- ning from 8p11.2 to 8p12 and from 8p21.1-8pter. In From the Institute ofPathology* and Urologic Clinics,t University another study, we found a strong association between ofBasel, Basel, Switzerland, and the Division ofMolecular chromosome 8q gains and histological phenotype, sug- Cytometry,* Department ofLaboratory Medicine, University of gesting that a gain of chromosome 8q material might also California, San Francisco, San Francisco, California contribute to bladder cancer progression.14 To better understand the role of chromosome 8 alterations in bladder cancer 87 tumor specimens were ex- Alterations of chromosome 8, including deletions of amined by dual labeling fluorescence in situ hybridization (FISH). aims were to and 8p, occur frequently in many tumors. In this study, Specific explore prevalence fluorescence in situ hybridization was used to study extent of chromosome 8p losses in bladder cancer and to the relationship between 8p deletions, 8q gains, and determine the relationship of chromosome 8p losses with tumor phenotype in bladder cancer. Celis from 87 tumors 8q gains and histopathologic phenotype. were examined by dual-labeling fluorescence in situ hybridization with a centromere 8 probe (pJM12) and P1 probes for 8p22, 8p12, 8q12, and 8q24. Both 8p22 Materials and Methods deletions and 8q24 gains were strongly associated Tumor Material with tumor phenotype. There was a marked difference in 8p22 deletions between noninvasive (pTa) Seventy-two formalin-fixed, paraffin-embedded bladder tumors (3/33) and minimally invasive (pTl) tumors tumors were randomly selected from the archives of the (8/19; P = 0.005) whereas there was no significant Institute of Pathology, University of Basel. Nuclei of for- difference between pTl and muscle-invasive (pT2-4) malin-fixed tissue blocks were dissociated and dropped tumors (19/35; P = 0.3926). Six tumors with 8p22 onto slides as previously described.15 Additionally, im- deletion were examined at 8p12. Three of these tu- print preparations were made immediately after surgery mors showed no 8p12 deletion, narrowing down the from 15 unfixed bladder tumor samples and stored at site of a putative tumor suppressor gene distal to -20°C. All hematoxylin and eosin slides of each case 8pl2. In one other case, there was a marked increase were reviewed by a single pathologist (G. Sauter). Tumor in 8p12 copy number (>40 per cell; amplification), stage and grade were defined according to UICC and suggesting the presence of an oncogene involved in World Health Organization classifications.16,17 Particu- bladder cancer at 8p12. The marked difference in larly stringent criteria were applied for the diagnosis of 8p22 deletions between noninvasive (pTa) and mini- stage pTl, which is defined by a tumor invasion of the mally invasive (pTl) tumors is consistent with a role suburothelial stroma but not of the muscular bladder wall. of a putative tumor suppressor gene on 8p for devel- An unequivocal stromal invasion was required to prevent opment of invasive tumor phenotype. (AmJ Pathol overstaging of pTa tumors. To minimize the risk of under- 1997, 151:753-759) staging muscle-invasive tumors (pT2-T4), the presence of tumor-free fragments of the muscular bladder wall in Recent cytogenetic and molecular studies have shown that deletions involving the short arm of chromosome 8 Supported by Krebsforschung Schweiz, Schweizerische Krebsliga (SKL are frequent in numerous tumors including prostate,1 137-7-1995), Krebsliga Beider Basel, and Thurgauer Krebsliga (G. Sau- lung,2 colon,3,4 and bladder cancer.5 It has been sug- ter, T. C. Gasser, and M. J. Mihatsch) and National Institutes of Health gested that 8p may be the site of at least one tumor grant CA47537 (F. M. Waldman). suppressor gene.5'6 Mapping studies have proposed Accepted for publication June 18, 1997. several different loci as sites of putative tumor suppressor Address reprint requests to Dr. G. Sauter, Institut fOr Pathologie, Uni- genes, 9 including an area between 8p21.3 and versitatskliniken, Kantonsspital Basel, Schonbeinstrasse 40, CH-4003 8p22,231011 another region centromeric to 8p21,4 and Basel, Switzerland. 753 754 Wagner et al AJP September 1997, Vol. 151, No. 3 the primary biopsy, and the absence of muscle invasion were counted for 100 nuclei. To avoid misinterpretation in a subsequent transurethral resection performed within due to insufficient hybridization efficiency, cells were 8 weeks after the first biopsy was required. Muscle inva- scored only when at least one bright P1 and one bright sion in a follow-up biopsy after less than 8 weeks was centromere signal were present. Two P1 signals were considered to indicate a sampling error at the initial bi- counted as one signal when they were situated very close opsy. Because of the limitations of transurethral biopsies to each other (O0.5 p.m) to avoid misinterpretation due to in accurately determining the depth of invasion of higher- sister chromatids of cells in S or G2M phase. The defini- stage bladder cancer, all tumors showing muscle inva- tions used for deletions and gains of loci were based on sion were categorized into one group (pT2-T4). Only the results obtained in normal urothelial cells from 10 tumors for which histological staging was unequivocal patients without a bladder cancer history (mean + 3 SD). were included in the study.

DNA Probes for FISH Statistics A chromosome 8 centromere probe (pJM12) was used x2 tests were used to compare the frequency of genomic in combination with P1 probes mapping to 8p22 alterations between tumors of different grades and (RMC08POO5, LPL, Flpter 0.140), 8p12 (RMC08PO03, stages and to evaluate the relationship between 8p de- D8S549, Flpter 0.307), 8q12 (RMC08PO02, D8S285, letions and 8q gains. Flpter 0.420), and 8q24 (RMC08POO1, c-myc, Flpter 0.845) to determine gains and deletions of chromosome 8 sequences. All probes were kindly provided by Dr. Joe Gray, Resource for Molecular Cytogenetics, University of Results California, San Francisco. The centromere 8 probe was labeled with biotin-14-dATP and P1 probes were labeled Pathology with digoxigenin-1 1-dUTP by nick translation using stan- Eighty-seven primary bladder tumors (72 paraffin-disso- dard protocols. ciated and 15 fresh touch preparations) were success- fully analyzed by FISH (17 additional dissociated cases Fluorescence in Situ Hybridization had insufficient hybridization intensity and were excluded). Thirty-three tumors were confined to the bladder In all hybridizations, a chromosome 8 centromere probe mucosa (pTa), nineteen showed invasion of the lamina (pJM12) was simultaneously used with a locus-specific propria (pTl), and thirty-five were muscle invasive (pT2- P1 probe to define relative gains and losses. FISH was as T4). Fourteen tumors were classified as grade 1, thirty- previously described.18 Briefly, cells on slides were de- nine were grade 2, and thirty-four were grade 3. natured in 70% formamide/2X SSC (1X SSC is 0.15 mol/L NaCI, 0.015 mol/L sodium Citrate), pH 7, at 75°C for 2.5 minutes. After dehydration in graded ethanol, samples were treated with proteinase K (Sigma Chemical Co., St. Normal Controls Louis, MO; 2.0 ,ug/ml for formalin-fixed cells and 0.2 Ten tissue samples (three unfixed bladder washes and ,ug/ml for unfixed specimens) in PBS (pH 7.0) for 7 min- dissociated nuclei from seven formalin-fixed biopsies) utes at 37°C, followed by ethanol dehydration. The hy- containing normal urothelial cells from patients without a bridization mixture was denatured for 5 minutes at 750C. bladder cancer history were used as normal controls. All A 10-,ul aliquot of hybridization mixture consisted of 10 ng normal controls showed an equal number of centromere of P1 probe, 30 ng of centromere 8 probe, and 10 ng of and P1 signals in .85% of cells. The fraction of cells with unlabeled, sonicated (200- to 500-bp) human placental fewer P1 signals than centromere signals ranged from 0 DNA (Sigma) in 50% formamide, 10% dextran sulfate, to 14% (7.5 ± 5.2, mean ± SD). These cells were attrib- and 2X SSC (pH 7). Re-annealing of hybridization mixture uted to a decreased hybridization efficiency of the small was allowed for 30 minutes at 37°C. Hybridization was P1 probes as compared with the much larger centromere overnight at 37°C. Lymphocyte metaphase spreads were probes. The fraction of cells with more P1 signals than used to assure probe specificity. The slides were washed centromere signals (considered to represent cells in S or in 50% formamide/2X SSC, pH 7, at 450 C. Immunohis- G2 phase with both sister chromatids visible) was gen- tochemical probe detection using Texas Red avidin (Vec- erally lower than the fraction of cells with deletion and tor Laboratories, Burlingame, CA) and FITC-conjugated ranged from 0 to 10% (4.5 ± 3.1, mean ± SD). Our cutoff sheep anti-digoxigenin (Vector) was as described.18 Nu- levels for definition of deletions and gains on a tumor clei were counterstained with 0.07 ptg/ml 4.5-diamino-2- basis were based on these results (mean number of phenyl-Indole (DAPI) in antifade solution. aberrant cells found in normal tissues + 3 SD). More than 25% of cells with fewer P1 signals than centromere 8 Scoring of FISH Signals signals were required to define a tumor as deleted for a specific locus. More than 15% of cells with more P1 Criteria for scoring of cells were as described.18 Copy signals than centromere 8 signals were required to define numbers of both centromere- and locus-specific signals a tumor as having a gain at a particular locus. 8p Deletions in Bladder Cancer 755 A/P Septembfller 1997 I'ol.lf SiNf 3

A 8p22 Deletion 100- (0- FISH with a P1 probe for 8p22 and a centromere repeat 80- probe for chromosome 8 was used to define 8p22 dele- 70- tions. The percentages of cells with an 8p22 deletion are shown in Figure 1A for all cases. Twenty-six of thirty 90 - tumors meeting our definition for 8p22 deletion had at ,,,40-.- had minor 30i least 70% deleted cells. Four additional tumors populations of deleted cells (between 27 and 36% deleted cells), but were still above the cutoff. Cells of a representative tumor with an 8p22 deletion are shown in Figure 2A. Tumor cells with an 8p22 deletion generally B had an increased centromere 8 copy number. The largest deleted tumor cell population having the same com- 9(1 (). bination of centromere 8 and 8p22 signals (predominant 70- tumor cell population) is shown in Table 1 for all 30 6(tO tumors with an 8p22 deletion. There were no tumors with 5()) 8p22 gains. 40 - 8p22 deletions were strongly associated with invasive (4) tumor phenotype (Figure 3). 8p22 deletions were found in 20-20- only 3 of 33 noninvasive pTa tumors (9%) but in 8 of 19 pTl carcinomas (42%; P = 0.005 for pTa versus pT1). 04 f- There was a much smaller difference in 8p22 deletions Figure 1 )of c'ls w ith c hiruiio(isomiii 8 .alteritionlsX iaio.niosx ot celix tumors carcinomas xWitih XI (ITCluCInS (8- ilHIMrs cxiiincdi: A) and clX gatins (-- tumrs c,xim- between pTl and muscle-invasive ited; B ,r >11()Xxh i iic, irlpsi-I-p-csciiits (nL tuLIn()l 'li'ii Ctitot'l t'()IxS tott (deletion in 19 of 35 pT2-4 tumors (54%); P = 0.3926). ciit'inlitimls()I it'ciluRtmis (A) ()I (.ja.ins (B) 0 n tit.1iciOIi1\'i l 11i S'ho)e.'nlS The comparison of 8p22 deletions with histological grade 1aI )I'I/.( )nt.ll lillt'S. revealed no difference between grade 1 tumors (deletion in 3 of 14 tumors) and grade 2 tumors (7 of 39 deleted) but a sharp increase in deletion frequency in grade 3

Figure 2.iFSHcletection of chroimooxsone 8 .hrrtiliitonx 1Sl1 x ithi prohxs fto- cenitronile-c 8 (piN18X) are in recl! intl I1 prohex for Xp22 (A) 8cl2 i (B), and 8pl2 (C) air- in gretn D)API CoUntirstIining,; miagnificaltion, X 1()() A: 8p22 tliletion, giii1p cif clix xxith tvxi centromiiiiri 8 signasix and one 8p22 signal. B: 8cl2l gain ccll xxitih tli-ce centromire si8ignals and sxevn 8q24 signailsx C: Xpl2 aniplification. cells with 3 centi-onitere 8 signails and miore than 20 8pl2 signals. Noti thue arIrange nt of tic XSpI2 signails in a clistinct Cixstrc indiicl tix of . holintlgeuneoILisI Ist(4alini l eoion 756 Wagner et al AJP September 1997, Vol. 151, No. 3

Table 1. Predominant Populations in Tumors with 8p22 TumwNowAc Deletions and 8q24 Gains 1 2 3 4 5 6 7 9 10 11 12 13 CoUls Piobu(fllr) 22 tp (0.140) p atL, El_ EEE3ED3E3PE-0EENN-E Centromere 8/8p22 Centromere 8/8q24

Predominant population n Predominant population n w t C - 3- m - - - - | g ~~~~~~~~~~~~~~~~8q12{0&42Q) .2.E1as 47 4/2 1 1 2/4 5 2/1 7 3/4 4 26.1 3/1 3 3/5 2 .2. 4/3 3 4/5 2 Others* 6 Otherst 9 _qu 2 1 a 2 2 a 2 2 a a 3 2 2 3 3 1 2 3 3 1 1 2 2

Thirty tumors showed an 8p22 deletion, and twenty-two tumors *ddd -g RNPECE - E showed an 8q24 gain. Results are expressed as the copy number of N chromosome 8 centromere (numerator) and 8p22/8q24 (denominator) Figure 4. Chromosome 8 mapping with probes for 8p22, 8p12, 8q12, and in the predominant population of tumor nuclei. 8q24. n = percentage of cells with deletion (on 8p) or gains (on 8q). See *The predominant populations in other tumors with 8p22 deletions text for definitions. **This tumor showed 3 to 4 centromere 8 signals and were 3/2, 4/1, 5/2, 5/3, 6/2, and 8/2 in one tumor each. more than 40 8pl2 signals per nucleus (Figcire 2C) tThe predominant populations in other tumors with 8q24 gains were 2/3, 2/5, 4/6, 4/7, 4/10, 5/6, 5/7, 5/8, and 6/11 in one tumor each. 8q24 gains were significantly more frequent in tumors tumors (20 of 34 deleted; P = 0.0003 for grade 2 versus with an 8p22 deletion that in tumors without deletion. grade 3). 8q24 gains were detectable in 14 of 26 tumors with an 8p22 deletion (54%) but in only 8 of 50 tumors without 8p22 deletion (16%; P = 0.0006). The relationship be- 8q24 Gains tween 8p22 deletions and 8q24 gains was still significant when the analysis was restricted to invasive tumors A probe for 8q24 was used together with the centromere (pTl-4) where both alterations were most prevalent. 8 probe in 76 tumors to detect 8q24 gains. The fractions 8q24 gains were found in 13 of 23 pTl-4 tumors with in of cells with 8q24 gain for all tumors are shown Figure 8p22 deletion (57%) but in only 6 of 22 pTl-4 tumors 1 B. The fraction of cells with an 8q24 gain was between without 8p22 deletion (27%; P = 0.0006). 15 and 30% in 6, between 31 and 60% in 2, and greater than 60% in 14 tumors. Cells of a representative tumor with an 8q24 gain are shown in Figure 2B. The largest Chromosome 8 Mapping abnormal tumor cell population having the same combination of centromere 8 and 8q24 signals (predominant To better define the size of chromosome 8 aberrations, a tumor cell population) of all 22 tumors with an 8q24 gain subset of 13 tumors was examined using additional P1 is shown in Table 1. Most tumors with an 8q24 gain had probes for 8p12 and 8q12 (Figure 4). This analysis an increased centromere 8 copy number. 8q24 deletions showed that three of eight tumors with an 8p22 deletion were not found. had retained 8p12 (tumors 3, 7, and 8). This narrows The frequency of 8q24 gains increased with advanced down the site of a putative bladder cancer tumor sup- tumor stage and high grade (Figure 3). 8q24 gains were pressor gene distal to 8p12. Unexpectedly, another tu- found in 3 of 31 pTa (10%), 5 of 19 pTl (26%; P = 0.1193 mor with an 8p22 deletion (tumor 1) showed a marked for pTa versus pTl), and 14 of 26 pT2-4 tumors (54%; increase of 8p12 copy number (>40 per cell) as com- P = 0.0648 for pTl versus pT2-4). 8q24 gains were most pared with the centromere 8 copy number (three or four frequently found in grade 3 tumors (14 of 29, 48%). They per cell), indicating a high-level amplification at 8p12 were significantly less frequent in grade 2 tumors (7 of 35, (Figure 2C). The arrangement of amplified signals in dis- 19%; P = 0.0165 for grade 3 versus grade 2) whereas tinct clusters suggested an intrachromosomal amplifica- there was only a minor difference in 8q24 gains between tion (homogeneously staining region) in this tumor. Both grade 2 and grade 1 tumors (1 of 12, 8%; P = 0.3534 for 8p12 amplification and 8p21-pter deletion was also con- grade 1 versus grade 2). firmed by CGH in this tumor (data not shown). An 8q24 gain was present in four of these tumors. Two tumors (tumors 2 and 9) had a gain of both 8q12 and 8q24, which is consistent with a gain of the entire q arm. Whereas tumor 9 had retained 8p, tumor 2 had a con- comitant deletion of both 8p12 and 8p22, thus suggesting the presence of an isochromosome 8q in this case. The remaining two tumors with an 8q24 gain (tumors 3 and 10) showed a normal copy number at the 8q12 locus, indicating that partial chromosome 8q gains can occur.

N 33 31 19 20 35 26 14 12 39 36 34 29 pTa pTI pT2-4 Grade I Grade 2 Grade 3 Discussion Figure 3. Prevalence of 8p22 deletions and 8q24 gains in bladder cancer. The frequencies of both 8p22 deletions and 8q24 gains increase significantly with In this study, dual-labeling FISH using centromere advanced tumor stage and high grade. probes together with locus-specific probes for 8p and 8q 8p Deletions in Bladder Cancer 757 AJP September 1997, Vol. 151, No. 3 was applied to detect subregional chromosome 8 alter- Previous mapping studies have suggested at least ations. In previous studies using different probes, we three different putative tumor suppressor gene loci on have shown that this approach yields a high agreement 8p.1-4,8-12 However, the results of several studies sug- with detection of allelic loss by restriction fragment length gest that the same gene(s) on 8p may be involved in polymorphism analysis.1819 The close similarity in the multiple tumor types as some minimally deleted regions prevalence of 8p22 deletions seen by FISH in this study were identical in different tumors. This is, for example, and by restriction fragment length polymorphism in a true for 8p21.3-8p22, which was the minimal deleted previous study5 was therefore expected. Knowles et al5 region in lung, liver, and colon cancer.2,3 10 11 Other stud- found 8p loss of heterozygosity (LOH) in 12% of pTa ies have suggested that deletions of some loci may be (versus 10% by FISH in this study) and in 45% of pT2-4 more tumor type specific, such as 8p1 1 .1-8p21 for ma- bladder carcinomas (54% by FISH). The striking differ- lignant fibrous histiocytoma.8 In bladder cancer, a recent ence between noninvasive (pTa) and muscle-invasive study has found two separate candidate regions span- tumors (pT2-4) in both studies suggests a role of a tumor ning from 8p11.2 to 8p12 and from 8p21.1-8pter.13 Our suppressor gene on 8p for bladder cancer progression. mapping results locate the minimally deleted region dis- This assumption is also consistent with the comparatively tal to 8p12, which is consistent with the results of Takle et low degree of heterogeneity found for 8p22 deletions in al.13 Candidate tumor suppressor genes on 8p include our patients. Although there were some tumors with fewer the monomorphic and the polymorphic forms of 8p22 signals than centromere 8 signals in small sub- arylamine N-acetyl transferase mapped to 8p21.3- populations ranging from 25 to 40% of cells, it is notable 23.1.22,23 It has been shown that smokers and individuals that the vast majority of tumors with an 8p22 deletion with industrial exposure to arylamines having a slow acet- contained more than 80% deleted cells. A clearly higher ylation phenotype of their arylamine N-acetyl-transferase degree of heterogeneity had been found in previous FISH are at an increased risk of developing bladder cancer.24 studies where we investigated erbB-2 gene amplifica- Knowles et al6 interpreted an association between al- tion.15'20 The high proportion of cells with 8p22 deletion in lelic imbalances of 8p and 8q as due to a loss of the tumors carrying this aberration might suggest that these entire chromosome 8. The results of this study confirm a cells have a strong proliferation advantage resulting in a significant association between genomic alterations on quick overgrowth of cell populations having 8p deletions. 8p and 8q. It shows, however, that 8p losses are asso- A major difficulty in the histological interpretation of ciated with 8q gains but not with 8q deletions. Recent transurethral bladder tumor biopsies is the finding of studies have indicated that the combination of 8p dele- stromal invasion for the diagnosis of stage pT1. Studies tions and 8q gains is also frequent in other tumors.25 have shown that the interobserver variability in the dis- Such a combination could best be explained by a single tinction between stage pTa and pTl is high between event affecting chromosome 8 that would simultaneously pathologists21 and that a thorough review of pT1 tumors lead to both 8q gain and 8p loss, for example, formation can result in downstaging of pTl tumors to pTa in as of an isochromosome 8q. It is likely that isochromosome many as 29% of cases.21 Special emphasis was there- 8q is present in some tumors with 8p deletions and 8q fore put on the diagnosis of stage pTl in this study. Using gains as isochromosome 8q has been described in cy- stringent criteria for a pT1 diagnosis, a striking difference togenetic analyses of bladder tumors,26 and our map in 8p deletions was found between stage pTa and pTl ping results are also consistent with isochromosome 8q (P = 0.005). Depending on what model is assumed for in one case (tumor 2). Either two independent events or a development of invasive bladder cancer, this result may more complex mechanism may be responsible for the have different implications. If it holds true that minimally combination of a terminal 8p22 deletion and a terminal invasive papillary tumors (pT1) do usually develop from 8q24 gain in tumor 3 or the combination of an 8p12 low-grade pTa tumors, the higher frequency of 8p22 amplification and an 8p22 deletion found in tumor 1. deletions in pT1 than in pTa tumors would suggest that The biological significance of low-level 8q gains is 8p22 deletions occur at the transition from noninvasive to unclear. Given their association with 8p deletions, 8q minimally invasive bladder cancer. This would raise the gains could represent side effects of a complex alteration possibility that inactivation of the putative tumor suppres- of chromosome 8 leading to the much more relevant 8p sor gene on 8p could play a role for the development of loss. In that case, cells containing 8q gains could be invasive tumor growth. Alternatively, it is possible that selected for due to a growth advantage exerted by the minimally invasive tumors do not develop from pTa tu- inactivation of the putative tumor suppressor gene on 8p mors but arise de novo in most if not all cases. In that whereas 8q gains by themselves might have little biolog- case, our result would imply that inactivation of a putative ical significance. Although it cannot be excluded that tumor suppressor gene on 8p may be part of a genetic tumors having retained 8p12 and 8p22 may still carry pathway leading to a bladder cancer subtype with a relevant deletions on 8p, the finding of a number of particularly high risk of developing invasive growth po- tumors having 8q gains in the absence of 8p deletions tential. The higher prevalence of 8p deletions in our pTl argues for a biological significance of 8q gains indepen- tumors (8 of 19) as compared with the study of Knowles dent of 8p deletions. The stronger increase in 8q24 gains et al (9 of 36), where no significant difference between from pT1 to pT2-4 than from pTa to pT1 could suggest pTa and pT1 had been found, could be explained by that 8q gains occur later in bladder cancer progression varying interpretations of stromal invasion by different than 8p deletions. One possible explanation for frequent pathologists.5 21 8q24 gains is an overexpression of at least one gene on 758 Wagner et al AJP September 1997, Vol. 151, No. 3

8q conferring a growth advantage to cells having 8q Storz, Ms. Sandy DeVries, and the staff of the Institute of overrepresentation. In a previous study, we found no Pathology, University of Basel, for their excellent techni- association between 8q24 gains and c-myc (located at cal support. 8q24) protein overexpression as detected by immunohistochemistry.14 Given the comparatively low sensitivity of immunohistochemistry for subtle differences in protein References expression, this result cannot totally exclude a subtle but relevant c-myc overexpression caused by 8q gains. The 1. Bova GS, Carter BS, Bussemakers MJ, Emi M, Fujiwara Y, Kyprianou of not including the c-myc N, Jacobs SC, Robinson JC, Epstein JI, Walsh PC, Isaacs W: Ho- finding several 8q24 gains, mozygous deletion and frequent allelic loss of chromosome 8p22 loci locus in a previous study applying CGH, suggests that a in human prostate cancer. Cancer-Res 1993, 53:3869-3873 gene proximal to c-myc could be involved in at least 2. Ohata H, Emi M, Fujiwara Y, Higashino K, Nakagawa K, Futagami R, some of these tumors.7 8q21 is a candidate site for Tsuchiya E, Nakamura Y: Deletion mapping of the short arm of chro- another 8q oncogene as a small amplification unit has mosome 8 in non-small cell lung carcinoma. Genes Chromosomes & Cancer 1993, 7:85-88 been detected at this locus by CGH.7 3. Cunningham C, Dunlop MG, Wyllie AH, Bird CC: Deletion mapping in Another putative oncogene with potential relevance for colorectal cancer of a putative tumour suppressor gene in 8p22- bladder cancer might be located at 8p12 where amplifi- p21.3. Oncogene 1993, 8:1391-1396 cation was found in 1 of 13 tumors examined for this 4. Fujiwara Y, Emi M, Ohata H, Kato Y, Nakajima T, Mori T, Nakamura Y: locus. As previous studies have found 8p12 amplifica- Evidence for the presence of two tumor suppressor genes on chromosome 8p for colorectal carcinoma. Cancer Res 1993, 53:1172- tions in breast carcinomas,27 this region has already 1174 been postulated to carry an unknown oncogene. The 5. Knowles MA, Shaw ME, Proctor AJ: Deletion mapping of chromosome potential significance of this putative 8p12 oncogene for 8 in cancers of the urinary bladder using restriction fragment length bladder cancer is further supported by the finding of an polymorphisms and microsatellite polymorphisms. Oncogene 1993, 8:1357-1364 additional tumor with an 8p12 amplification by CGH in 6. Knowles MA, Elder PA, Williamson M, Cairns JP, Shaw ME, Law MG: another series of 8 pT1 bladder carcinomas (unpublished Allelotype of human bladder cancer. Cancer Res 1994, 54:531-538 data) and a previous cytogenetic study reporting a ho- 7. Kallioniemi A, Kallioniemi 0, Citro G, Sauter G, DeVries S, mogeneously staining region at 8p12.28 Candidate onco- Kerschmann R, Carroll P, Waldman F: Identification of novel DNA on the for a amplifications and deletions in primary bladder cancer by compara- genes 8p include heregulin gene coding tive genomic hybridization. Genes Chromes & Cancer 1995, 12:213- ligand for the erbB-2 oncogene and fibroblast growth 219 factor 1. In breast cancer, fibroblast growth factor recep- 8. Chang M, Tsuchiya K, Batchelor R, Rabinovitch P, Kulander B, Hag- tor 1 was found overexpressed in 7 of 10 breast carcino- gitt R, Burmer G: Deletion mapping of chromosome 8p in colorectal mas with 8p12 amplification in one study,29 whereas carcinoma and dysplasia arising in ulcerative colitis, prostatic carcinoma, and malignant fibrous histiocytomas. Am J Pathol 1994, 144: heregulin expression was not related to 8p12 amplifica- 1-6 tion in another series.30 The expression of both genes in 9. Yaremko M, Wasylyshyn M, Paulus K, Michelassi F, Westbrook C: bladder cancer remains to be studied. The occurrence of Deletion mapping reveals two regions of chromosome 8 allele loss in circumscribed amplification units at 8p12 is also impor- colorectal carcinomas. Genes Chromosomes & Cancer 1994,10:1-6 tant for the of LOH results in this 10. Emi M, Fujiwara Y, Ohata H, Tsuda H, Hirohashi S, Koike M, Miyaki M, interpretation region. Monden M, Nakamura Y: Allelic loss at chromosome band 8p21.3- Small amplification units could significantly hamper posi- p22 is associated with progression of hepatocellular carcinoma. tional cloning studies. It is likely that all amplified copies Genes Chromosomes & Cancer 1993, 7:152-157 in such a case are derived from one allele leading to the 11. Fujiwara Y, Ohata H, Emi M, Okui K, Koyama K, Tsuchiya E, Nakajima finding of an allelic imbalance. An 8p12 amplification T, Monden M, Mori T, Kurimasa A, Oshimura M, Nakamura Y: A 3-Mb physical map of the chromosome region 8p21.3-p22, including a could therefore easily mimic a small interstitial deletion in 600-kb region commonly deleted in human hepatocellular carcinoma, this region in LOH studies. colorectal cancer, and non-small cell lung cancer. Genes Chromo- In summary, the results of this study show that 8p22 somes & Cancer 1994, 10:7-14 deletions are frequent in bladder cancer and are strongly 12. van der Bosch K, Becker I, Savelyeva L, Bruderlein S, Schlag P, of at Schwab M: Deletions in the short arm of chromosome 8 are present associated with invasive phenotype. Inactivation in up to 90% of human colorectal cancer cell lines. Genes Chromo- least one tumor suppressor gene located between 8p12 somes & Cancer 1992, 5:91-95 (D8S549) and 8pter might be linked to the development 13. Takle LA, Knowles MA: Deletion mapping implicates two tumor sup- of invasive tumor growth. Future studies are needed not pressor genes on chromosome 8p in the development of bladder only to further localize the 8p tumor suppressor gene but cancer. Oncogene 1996, 12:1083-1087 14. Sauter G, Carroll P, Moch H, Kallioniemi A, Kurschmann R, Narayam also to clarify whether the identification of 8p deletions in P, Mihatsch M, Waldman F: Increased c-myc gene copy number in noninvasive bladder tumors may indicate impending tu- advanced bladder cancer. Am J Pathol 1995, 146:1131-1139 mor progression and therefore advocate a more aggres- 15. Sauter G, Moch H, Moore D, Carroll P, Kerschmann R, Chew K, sive treatment in these patients. Mihatsch M, Gudat F, Waldman F: Heterogeneity of erbB-2 gene amplification in bladder cancer. Cancer Res 1993, 53:2199-2203 16. UICC: TNM Classification of malignant tumours. Berlin, Springer, 1992 Acknowledgments 17. Mostofi F: Histological typing of urinary bladder tumors. Geneva, World Health Organization, 1973 FISH probes were generously supplied by the University 18. Sauter G, Deng G, Moch H, Kerschmann R, Matsumura K, DeVries S, George T, Fuentes J, Carroll P, Mihatsch M, Waldman F: Physical of California, San Francisco, Resource for Molecular Cy- deletion of the p53 gene in bladder cancer: detection by fluores- togenetics (Dr. Joe W. Gray, Director). The authors thank cence in situ hybridization. Am J Pathol 1994, 144:756-766 Ms. Hedvika Novotny, Ms. Carole Egenter, Ms. Martina 19. Matsumara K, Kallioniemi 0, Kallioniemi A, Chen L, Smith H, Pinkel D, 8p Deletions in Bladder Cancer 759 AJP September 1997, Vol. 151, No. 3

Gray J, Waldman F: Deletion of chromosome 17p loci in breast rescence in situ hybridization on chromosome 8 in prostate cancer. cancer cells detected by fluorescence in situ hybridization. Cancer Genes Chromosomes & Cancer 1994, 11:153-162 Res 1992, 52:3474-3477 26. Mitelman F: Catalog of chromosome aberrations in cancer. New York, 20. Sauter G, Moch H, Gasser T, Mihatsch M, Waldman F: Heterogeneity Wiley-Liss, 1994 of chromosome 17 and erbB-2 gene copy number in primary and 27. Adnane J, Gaudray P, Dionne C, Crumley G, Jave M, Schlessinger J, metastatic bladder cancer. Cytometry 1995, 21:40-46 Jeanteur P, Birnbaum D, Theillet C: BEK and FLG, two receptors for 21. Abel PD, Henderson D, Bennett MK, Hall RR, Williams G: Differing members of the FGF family, are amplified in subsets of human breast interpretations by pathologists of the pT category and grade of tran- cancers. Oncogene 1991, 6:659-663 sitional cell cancer of the bladder. Br J Urol 1988, 62:339-342 28. Gibas Z, Prout G, Pontes J, Conolly J, Sandberg A: A possible 22. Hickman D, Risch A, Buckle V, Spurr N, Jeremiah S, McCarthy A, Sim specific chromosome change in transitional cell carcinoma of the E: Chromosomal localization of human genes for arylamine N-acetyl- transferase. Biochem J 1994, 297:441-445 bladder. Cancer Genet Cytogenet 1986, 29:229-238 23. Spurr N: Report of the second international workshop on chromo- 29. Jacquemier J, Adelaide J, Parc P, Penault Llorca F, Planche J, some 8 mapping 1994. Cytogenet Cell Genet 1995, 68:147-164 deLapeyriere 0, Birnbaum D: Expression of the FGFR1 gene in 24. Risch A, Wallace D, Bathers S, Sim E: Slow-N-acetylation genotype is human breast-carcinoma cells. Int J Cancer 1994, 59:373-378 a susceptibility factor in occupational and smoking related bladder 30. Adelaide J, Penault Llorca F, Dib A, Yarden Y, Jacquemier J, Birn- cancer. Hum Mol Genet 1995, 4:231-236 baum D: The heregulin gene can be included in the 8p12 amplifica- 25. Cher ML, MacGrogan D, Bookstein R, Brown JA, Jenkins RB, Jensen tion unit in human breast cancer. Genes Chromosomes & Cancer RH: Comparative genomic hybridization, allelic imbalance, and fluo- 1994, 11:66-69