LEAD ARTICLE Molecular Genetics of Human Bladder Carcinomas Danielle Perucca, Pierre Szepetowski, Marie-Pierre Simon, and Patrick Gaudray
ABSTRACT: B l . d d e r cam:or corresponds t o . tumor type whose clinical b e h . v i o r is difficult to predict. A b.tter understanding (ff this i)(~th()h~gy is cxt)et:tcd t'r(m~ moh;(:ular gcmeti(:s, which bril~s tage.ther c~'togenetics and molecular biology. "l'h~rehm~. we have tried to ov~rview correl.ti(ms t)etwe,m t:hromos(;me abnormaliti~!s and the pres(!n(:c, i~ the vicilffty ()f the altered loci. ot genes /oncog(mes (rod others) that could t)e h w o l v e d in I)latlder ()ntzooetmsis and~or tlHl~t)r progressi(m. h~ (~(hliticm to ()t~cogel~e a(ztivafitm t)v /)()J/It llltttCHiolls, get~e (mq)lil](:ath)n, or deregulati(m (J( ,Aerie exl)ression, sever(d (:ytogeJleth: (is well .s m()lecular evi(len(:es l)oint t() oem~ti(: deleti(ms (existence of -tumor SUpl)ressor ?em~s"J t)eill,, iHvolved in those processes.
INTRODUCTION General Description of Bladder Tumors Transitional cell carcinomas (TCC) represent 90% human bladder cancers that are the cause of more than 10,000 deatlls annually in the United States. Epidemiologic studies have s h o wn that it is a disease of the elderly, with a strong male p r e d o m i n a n c e (3 4:1) that could be related to a long time recognized occupational exposure to chemical carcinogens (Rehn, 1895, cited in [12]). Most bladder cancers are considered to correspond to a generalized neoplastic change of the urothelium or transitional cell epithelium, w h i c h could possibly explain that it is often multifocal and recurrent. Contrary to many cancers, bladder tumors are not usually invasive at time of diagnosis. This makes bladder TCC a useful model to study tumor progression toward invasiveness, but also points to the necessity of defining good markers of prognosis in order to recognize early on the fraction of tumors that eventually will progress.
Prognostic Factors of TCC of the Bladder Histopathologic characteristics of bladder TCC have a limited predictive value with respect to tumor recurrence and/or progression. Therefore, other potential predictors of disease ev o lu ti o n have been sought out either i m m u n o l o g i cal l y with the presence or absence of ABH blood group surface antigens [1, 56] and cytokeratin 18 exDression
I"rom I,GMCII. Faculte de M6dc~cine. Av~muede Valomhrose. 06034 Nice. Franc.. Address reprint requests to: Dr. Patrick Gaudray, I.GMCH. Faculte de M(~decine, Aveml~! (h~ Valombrose, 06034 Nice, France. Received January 19, 1990: accepted January 22, 1990.
143 c |990 Elsevier Science lklblishing Co.. lnc:.
Cancer Genet Cytogene~49:143 156 {1990)
655 AvellUe ot the Americas. N e w York, NY 10010
0165-4608/90/$03.50
144
D. Perucca et al.
[29], or b i o c h e m i c a l l y with assays of the a m o u n t of e p i d e r m a l growth factor (EGFr/
C-erbB-1 [8, 49]), and of receptors for retinoic acid (hRAR [17[). Assays of the a m o u n t of DNA present in bladder tumors [5, 10, 28, 37, 51] have confirnled earlier cytc)gelletic studies that inclie:alecl that m;ar-etil)loi(I T(](; have a lesser tendenc:v to i n v a s i \ e n e s s that t u m o r s [)eing in either the hyt)()(lit)l(fi(I, the triploie], or tile llyt)ertetral)h)i(I range, 15',)1. Besides c h r ( m , ) s o m e mmd)ers, cytogeneti(:ists have looked for (:lu'c)mos o n c o g e n e s of the fibroblast g r o w t h factor (FGF) gene f a m i l y and f o u n d o n l y 3 (:ases (7%) w h e r e hst aim int-2 w e r e a m p l i f i e d at a low level [68]. In the s a m e study, 17% breast (:ar(:inomas s h o w e d amt)lification of the s a m e markers. S i m i l a r l y , T s u t s u m i et al. [69] f o u n d a 20-fold aml/lification of hst in (rely 1 out of 18 hlad(ter tumnrs. A 40-fold a m p l i f i c a t i o n of K-ras was o b s e r v e d o n c e in a series of 21 b l a d d e r TCC, and the a m p l i f i e d gene was not t r a n s f o r m i n g in an in vitro transfection assay 1211. No a m p l i f i c a t i o n of any o t h e r ras genes has e v e r b e e n d e t e c t e d either in a d v a n c e d stage t u m o r s [8, 21] or e v e n in t u m o r d e r i v e d cell lines [63]. A m o n g 31 p r i m a r y b l a d d e r tumors, Berger et al. {8] f o u n d an 8 - 1 0 - f o l d amplification of the (erbB)EGFR gene in one case of i n v a s i v e c a r c i n o m a and n o n e of the superficial t u m o r s . The o t h e r w i s e u n a l t e r e d a m p l i f i e d gene e n c o d e d an a p p a r e n t l y n o r m a l EGF-receptor. O t h e r p r o t o - o n c o g e n e s like c - m y c or c-erbB-2, w h i c h are a m p l i f i e d f r e q u e n t l y in v a r i o u s t y p e s of t u m o r s , are rarely altered in b l a d d e r TCC (Table 4). In c o n c l u s i o n , it s e e m s that e i t h e r t)rolo-oncogene a m p l i f i c a t i o n is a rare e v e n t in b l a d d e r can(:er, or p r o t o - o n c n g e n e s a m p l i f i e d in these t u m o r s have not lieen (tis(:overed yel.
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Proto-oncogene Expression Gene overexpression can be achieved by other means than amplification, lu fact, from the few detailed reports on proto-oncogenes expression, both in r i v e and in vitro, gene expression seems to be more frequently altered than gene structure in bladder TCC. In tile case of H-ras, overexpressiou has been found in the EJ/T24 cell line [13, 25, 61]. We have observed a usually h i g h - - a l t h o u g h v a r i a b l e - - l e v e l of H-ms transcripts by Northern blot analysis in a series of 40 bladder TCC, without correlating it to any clinico-histopathologic parameter (our u n p u b l i s h e d observations). On the contrary, elevated expression of c-erbB-2 (3-10-fold higher than the average in 4/30 tumors tested, as c o m p a r e d to /3-actin mRNA) seemed more prevalent in invasive tumors than in superficial ones. Using i m m u n o c h e m i c a l and ligand-binding methods, the group of Harris has shown that the related EGFR gene is expressed mostly in highgrade invasive tumors [8, 48, 49]. Abnormal expression of some proto-oncogenes is unlikely to be sufficient for oncogenesis and tumor progression as well as to be the priinary genetic defect leading to malignancy. Nevertheless, besides its potential interest in clinical evaluation, deregulation of these genes might be used to track tile molecular events responsible for it. Genetic Deletions: Possible Existence of "Tumor Suppressor Genes" One of the fields where cytogeuetics has brought a great deal to mole(:ular bioh)gy in a re(:ent past is the discovery of chromosomal deletions that are characteristic of certain tumor types. When analyzed at the mole(:ular level, such deletions have contributed to establish tile concept of tumor suppressor g e n e s - - o r "anti-oncog e n e s " - - l i k e the RB1 gene in retinoblastoma (see [35] for a review). Contrary to protooncogenes, such genes are susceptible to recessive mutations that impair their putative function in keeping the cells in a normal state. Therefore, homozygous alterations of these genes are required for expression of the inaliguant phenotype. The search for chromosomes losses has already led to the m a p p i n g of loci corresponding to predisposition for inherited cancer syndromes, e.g.: 3t) for renal carcinomas and small (:ell lung carcinoma, 5q for familial polyposis coil, 1 l p for nephroblastoma. 13q for retinoblastoma and osteosar(:oma (see [54] for references). At the cytogeneti(: level, alterations {deletions) of the short arm of c h r o m o s o m e 11 are a rather rare event in bladder TCC, and, in any case, appear to be secondary changes related--maybe--to an increased potential for invasiveuess [4, 23]. However, prompted by the recognition of a putative anti-oncogene localized on 11p that is associated with d e v e l o p m e n t of Wihns" embryonic kidney tumors [18, 38, 50, 58], Fearon et al. [19] have sought somatic losses of 11p sequences in urothelial tumors. They used DNA p o l y m o r p h i s m (RFLP) of H-ras and INS genes to detect losses of heterozygosity that could be indicative of recessive genetic changes involving 11p sequences. They observed that the frequency of loss of 11p sequences in adult b l a d d e r carcinomas a p p r o a c h e s that seen in embryonic Wilms' tumors (42% versus 56%). Although more frequent than cytogenetically visible alterations, these losses of heterozygosity on l l p do not correlate with tumor stage or histologic grade. In addition, other authors report much lower frequencies of chromosome 11p losses (8%, [53]). It is possible, as suggested by Sandberg [59], that 11p involvement in bladder TCC represents a late event in tumor progression, maybe as a consequence of a more generalized c h r o m o s o m a l instability. The relationship between 11p losses of heterozygosity and mutational activation of H-ras is an intriguing question: Do they cooperate and thus a p p e a r in the same tumors, or can deletions substitute for mutations in
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some ways and occur in tumors that do not contain an activated H-ras gene? The former possibility is supported by the chemically induced mouse skin tumors model where the loss of the normal H-ras allele seems to be involved in the progression of hemizygously H-ras-mutated tumors [55]. Concerning interstitial deletions of chrmnosome 13q, a good candidate gene to be involved is the RB1 gene that is located on 13q14. In fact, in the J82 bladder carcinoma cell line, an abe,rrant RB-1 prnte,in, has be,e,n de,te,cte,d 130l. This prote,in has lost its ability to complex to the ade,novirus E1A ont:oprote,in, and it is less stabl~,' than wiltl type plO5-Rb. However, in this case, the defe(:tiw', HB-1 protein is I)rodu(:e(I as the re,suit of a single,-point mutation rather than a (lele,tion. This situatioll is also SUl)l)(Jse,(l to take place, in retinoblastoma where gross alterations of the, 200kb I{B-1 gelie, are, not the lnost frequent, suggesting the, existence of more, subtle, ge,ne,ti(: (:banges at this level 130J. The (rely RB1 mutation found to (late' has been isolated froin a (:ell lira,' (J~2) and not directly from a tumor biopsy. It is therefore, possible, that it o(:(:urre,(I during the in vitro e,stablishment of the line,. ()nlv a svstemati(: surve,v of primary tulnor extracts for the, l)re,se,nce, of the, RB-I prote,in an(l its biologi(: inte,grity, me,asure,(I by its al)ilitv to bind EIA in vitro, e.g., will tell us about the, actual involw~,me,nt of this "anlioncogeue," product in the genesis of bla(hler TCC. CONCLUSION The recent successes in elu(:idating the molecular bases of some h u m a n pathologies (re,tinoblastonla, cystic fibrosis, D u c h e n n e muscular dystrophy} have, demonstrated the, power of molecular genetics integrating both cytogenetics and molecular biology. One of the issues addressed in this review concerned the present state, of conve,rgen(:e of these two approaches in the case of urothe,lial bladder tumors. However, as stated by Jewett 1321, "although the bladder is a relatively simple organ, its malignancy is so complex that the farther one digs for answers the more u n a n s w e r e d questions are, uncovered. It seems a never-ending process, and the, ultimate truth may reside in the p h e n o i n e n o n of cancer in general." Molecular genetics can be implicated in clinical ontology first by helping to recognize tumors subtypes. One of the striking features of bladder carcinomas is heterogeneity. In fact, although starting from a relatively simple tissue (transitional cell epithelium), it seems to represent various diseases with a lot of different outcomes. We have shown that the same heterogeneity exists at either the cytogenetic or the, molecular level, contributing to the confusing picture offered by these tumors. In addition, it is surprising to note that the prevalence of each chromosome abnormality described above, is highly variable, among different publications (Table 1). This variability could be related to some u n k n o w n bias in tumor sampling. It will probably be averaged once a lot more tumors will have, been analyzed. Hence, it has not been ye,t possible, to do genetic typing of bladder cancer. One exception might be the fraction of tumors (~15%) containing an activated H-ras gene that could correspond to a particular scheme of carcinogenesis and/or progression. Contrary to most tumors that develop from localized foci of transformed cells, bladder TCC is c o m m o n l y recurrent and multifocal, as if the entire, urotheliunl was concerned. This behavior is usually accounted for by the c o n t i n u o u s exposure of bladder to carcinogens. In order to explain that a carcinogen(s) leads to a very efficient transformation of the entire urothelium in the small fraction of the population affected by bladder cancer, one has to hypothesize either that only a minority is exposed to a particularly potent carcinogen(s) (viral?) or that there is some kind of genetic predisposition to bladder cancer. However, no such a predisposition has ever been demonstrated, maybe because, these tumors develop mostly among elderly people,. It could be interesting to screen affected i n d i v i d u a l s for germ-like alterations at the level of
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loci w h e r e d e l e t i o n s and losses of h e t e r o z y g o s i t y h a v e been e v i d e m : e d in t u m o r (:ells. On the o t h e r band, ah'eady r e c o g n i z e d b l a d d e r c a r c i n o g e n s are dive, rse, an(l at least s o m e of them, like cigarette s m o k i n g , are shared by m a n y , suggesting their role m o r e in p r o m o t i o n than in initiation. We h a v e s h o w n that a m o n g the u m : o g e n e s s t u d i e d so far, o n l y an activated H r , s has been o b s e r v e d r e p r o d u c i b l y in a subset of b l a d d e r TCC. it s h o u h l be interesting to investigate, in these tumors, lhe slate of the I~APIA w h i c h has been s h o w n to s u p p r e s s ras-modiated transforination [331. In fact, this gone is located on ( : h r o m o s o m e 1, w h i c h is the most f r e q u e n t l y altered in b l a d d e r TCC. The relation b e t w e e n o n c o g e n e a c t i v a t i o n and c h r o m o s o m a l losses has been d o c u m e n t e d in the case of genetic a l t e r a t i o n s d u r i n g c o l o r e c t a l - t u m o r d e v e l o p m e n t [74[. Here, t u m o r progression is suggested by the fact that most colorectal c a r c i n o m a s appear to arise from a d e n o m a s . K-ras m u t a t i o n s o c c u r m o s t l y in large a d e n o m a s and in c a r c i n o m a s ; a specific region of c h r o m o s o m e 18 is deleted m o r e f r e q u e n t l y in c a r c i n o m a s than in a d v a n c e d a d e n o Inas and in earlier-stage a d e n o m a s (73%, 47%, 11%, respectively): c h r o m o s o m e 17p s e q u e n c e s are u s u a l l y lost o n l y in c a r c i n o m a s (75%). Therefore, colorectal o n c o g e n e sis m i g h t r e q u i r e an initial step i n v o l v i n g inutational a c t i v a t i o n of an o n c o g e n e c o u p l e d w i t h s u b s e q u e n t losses of several genes that n o r m a l l y s u p p r e s s t u m o r i g e n e sis. S u c h a c o m p a r i s o n b e t w e e n p r e c a n c e r o u s and c a n c e r o u s lesions is not a v a i l a b l e yet in the case of b l a d d e r tumors, but it s h o u l d be e m p h a s i z e d in future studies. O n c o g e n e a c t i v a t i o n by t)oint m u t a t i o n is less f r e q u e n t in bla(tde, r t u m o r s than in colorectal (:ancer (< 15% w;rsus 4 0 % ) . T h e most (:ommon (:ytogeneti(: an(I mole(:nlar markers present in b l a d d e r t u m o r s c o r r e s p o n d to h)sses of genetic i n f o r m a t i o n (deletions, is()chromosomes, losses of heterozygosity). T h e r e f o r e , it is (:on~:eiwfl)le thai anti-on(:ogeues (or t u m o r sul)pressor genes) r e p r e s e n t I)rimarv targets of bla(hler o n c o g e n e s i s . T h e i r i n a c t i v a t i o n r e q u i r e s h o m o z y g o u s mutations, i.e., two hits, and the t i m e neede(t to a(:cumuhtte t h e m c o u l d be in part r e s p o n s i b l e for the, late at)l)earan(:e of b l a d d e r t u m o r s in the lifetime. It is to h o p e that deveh)t)in g c y t o g e n e t i c studies will h e l p to p i n p o i n t s o m e of these sites so that m o l e c u l a r bioh)gv can ta(:kle the respollsible genes. Continuous help and support from Dr. N. Ayraud is gratefully acknowledged. We thank Drs. C. Turc-Carel and A. Sandberg for critical reading of the manuscript. Such a study would not have been possible without the efficient collaboration of Drs. B. Basteris, J. Toubol, and J. Vacant who made available to us tumor and blood samples together with clinical data. Our laboratory is supported by grants from ARC, Caisse Nationale d'Assurance Maladie. FEGEFLUC, F6d6ration des Centres de Lutte Centre le Cancer, Fondation pour la Recherche M6dicale, INSERM, and Ligue Nationale Franc;aise centre le Cancer.
REFERENCES
1. Abel PD, Thorpe SJ, Williams G (1989): Blood group antigen expression in ffozeI1 sections of presenting bladder CHII(:IH':3-years prospectiw ~,follow-up of prognostic value. Br J Cancer 63:171-175. 2. Alfthan O, Tarkkanen J, GrOhn P, Heinonen E, Pyrh6nen S, Stiil~i K (1983): Tigason (etretihate) in prevention of recurrence of superficial bladder tumors. A double-blind clinical trial. Eur J Urol 9:6-9. 3. Atkin NB, Baker MC (1985): Cytogenetic study of ten (:an:ioomas of the bladder: Involvement of chromosomes 1 and 11. Can(:er Goner (]ytogenet 15:253-268. 4. Babu VR, Lutz MD, Miles BJ, Farah RN, Weiss L, Van Dyke DK (1987): Tumor behavior in transitional cell (:arcinoma of the bladder in relation to (:bromos(mml markers and histopathology. Cancer Res 47:680() 6805. 5. Badalament RA, Fair WR, Whitmore WF Jr, Melamed MR (1988): The relalive value of
15 3
M o l e c u l a r G e n e t i c s of B l a d d e r Cancer
cytomelry and ¢:ytology in Ihe managemenl of bladder cancer: Tim Memorial Sh)al>Kellering (]ant:er Center e x p e r i e n c e . Selnin tirol 6:22 3(I. 6. Barrios 1,, Mori R, (]aballin MR, Vavreda J, S u h i a s A, Egozcue ] {1986): (]onstilulional (h!l {3)(I) 14 p21 ) ill a l)atient with bhld(ler (:al'(:ill¢)ll/a. (~allcei" Gel/e{ CytogeneI 21:171 - 173. 7. Berger (]S. S a n d b e r g AA, T o d d IAI), Pct/ningIon RI), Had(tad FS. Het:ht BK, Hecht 1" ( 1986): (2womosome, s in kidney, tlreler all¢] h l a d d e r
ABSTRACT: B l . d d e r cam:or corresponds t o . tumor type whose clinical b e h . v i o r is difficult to predict. A b.tter understanding (ff this i)(~th()h~gy is cxt)et:tcd t'r(m~ moh;(:ular gcmeti(:s, which bril~s tage.ther c~'togenetics and molecular biology. "l'h~rehm~. we have tried to ov~rview correl.ti(ms t)etwe,m t:hromos(;me abnormaliti~!s and the pres(!n(:c, i~ the vicilffty ()f the altered loci. ot genes /oncog(mes (rod others) that could t)e h w o l v e d in I)latlder ()ntzooetmsis and~or tlHl~t)r progressi(m. h~ (~(hliticm to ()t~cogel~e a(ztivafitm t)v /)()J/It llltttCHiolls, get~e (mq)lil](:ath)n, or deregulati(m (J( ,Aerie exl)ression, sever(d (:ytogeJleth: (is well .s m()lecular evi(len(:es l)oint t() oem~ti(: deleti(ms (existence of -tumor SUpl)ressor ?em~s"J t)eill,, iHvolved in those processes.
INTRODUCTION General Description of Bladder Tumors Transitional cell carcinomas (TCC) represent 90% human bladder cancers that are the cause of more than 10,000 deatlls annually in the United States. Epidemiologic studies have s h o wn that it is a disease of the elderly, with a strong male p r e d o m i n a n c e (3 4:1) that could be related to a long time recognized occupational exposure to chemical carcinogens (Rehn, 1895, cited in [12]). Most bladder cancers are considered to correspond to a generalized neoplastic change of the urothelium or transitional cell epithelium, w h i c h could possibly explain that it is often multifocal and recurrent. Contrary to many cancers, bladder tumors are not usually invasive at time of diagnosis. This makes bladder TCC a useful model to study tumor progression toward invasiveness, but also points to the necessity of defining good markers of prognosis in order to recognize early on the fraction of tumors that eventually will progress.
Prognostic Factors of TCC of the Bladder Histopathologic characteristics of bladder TCC have a limited predictive value with respect to tumor recurrence and/or progression. Therefore, other potential predictors of disease ev o lu ti o n have been sought out either i m m u n o l o g i cal l y with the presence or absence of ABH blood group surface antigens [1, 56] and cytokeratin 18 exDression
I"rom I,GMCII. Faculte de M6dc~cine. Av~muede Valomhrose. 06034 Nice. Franc.. Address reprint requests to: Dr. Patrick Gaudray, I.GMCH. Faculte de M(~decine, Aveml~! (h~ Valombrose, 06034 Nice, France. Received January 19, 1990: accepted January 22, 1990.
143 c |990 Elsevier Science lklblishing Co.. lnc:.
Cancer Genet Cytogene~49:143 156 {1990)
655 AvellUe ot the Americas. N e w York, NY 10010
0165-4608/90/$03.50
144
D. Perucca et al.
[29], or b i o c h e m i c a l l y with assays of the a m o u n t of e p i d e r m a l growth factor (EGFr/
C-erbB-1 [8, 49]), and of receptors for retinoic acid (hRAR [17[). Assays of the a m o u n t of DNA present in bladder tumors [5, 10, 28, 37, 51] have confirnled earlier cytc)gelletic studies that inclie:alecl that m;ar-etil)loi(I T(](; have a lesser tendenc:v to i n v a s i \ e n e s s that t u m o r s [)eing in either the hyt)()(lit)l(fi(I, the triploie], or tile llyt)ertetral)h)i(I range, 15',)1. Besides c h r ( m , ) s o m e mmd)ers, cytogeneti(:ists have looked for (:lu'c)mos o n c o g e n e s of the fibroblast g r o w t h factor (FGF) gene f a m i l y and f o u n d o n l y 3 (:ases (7%) w h e r e hst aim int-2 w e r e a m p l i f i e d at a low level [68]. In the s a m e study, 17% breast (:ar(:inomas s h o w e d amt)lification of the s a m e markers. S i m i l a r l y , T s u t s u m i et al. [69] f o u n d a 20-fold aml/lification of hst in (rely 1 out of 18 hlad(ter tumnrs. A 40-fold a m p l i f i c a t i o n of K-ras was o b s e r v e d o n c e in a series of 21 b l a d d e r TCC, and the a m p l i f i e d gene was not t r a n s f o r m i n g in an in vitro transfection assay 1211. No a m p l i f i c a t i o n of any o t h e r ras genes has e v e r b e e n d e t e c t e d either in a d v a n c e d stage t u m o r s [8, 21] or e v e n in t u m o r d e r i v e d cell lines [63]. A m o n g 31 p r i m a r y b l a d d e r tumors, Berger et al. {8] f o u n d an 8 - 1 0 - f o l d amplification of the (erbB)EGFR gene in one case of i n v a s i v e c a r c i n o m a and n o n e of the superficial t u m o r s . The o t h e r w i s e u n a l t e r e d a m p l i f i e d gene e n c o d e d an a p p a r e n t l y n o r m a l EGF-receptor. O t h e r p r o t o - o n c o g e n e s like c - m y c or c-erbB-2, w h i c h are a m p l i f i e d f r e q u e n t l y in v a r i o u s t y p e s of t u m o r s , are rarely altered in b l a d d e r TCC (Table 4). In c o n c l u s i o n , it s e e m s that e i t h e r t)rolo-oncogene a m p l i f i c a t i o n is a rare e v e n t in b l a d d e r can(:er, or p r o t o - o n c n g e n e s a m p l i f i e d in these t u m o r s have not lieen (tis(:overed yel.
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Proto-oncogene Expression Gene overexpression can be achieved by other means than amplification, lu fact, from the few detailed reports on proto-oncogenes expression, both in r i v e and in vitro, gene expression seems to be more frequently altered than gene structure in bladder TCC. In tile case of H-ras, overexpressiou has been found in the EJ/T24 cell line [13, 25, 61]. We have observed a usually h i g h - - a l t h o u g h v a r i a b l e - - l e v e l of H-ms transcripts by Northern blot analysis in a series of 40 bladder TCC, without correlating it to any clinico-histopathologic parameter (our u n p u b l i s h e d observations). On the contrary, elevated expression of c-erbB-2 (3-10-fold higher than the average in 4/30 tumors tested, as c o m p a r e d to /3-actin mRNA) seemed more prevalent in invasive tumors than in superficial ones. Using i m m u n o c h e m i c a l and ligand-binding methods, the group of Harris has shown that the related EGFR gene is expressed mostly in highgrade invasive tumors [8, 48, 49]. Abnormal expression of some proto-oncogenes is unlikely to be sufficient for oncogenesis and tumor progression as well as to be the priinary genetic defect leading to malignancy. Nevertheless, besides its potential interest in clinical evaluation, deregulation of these genes might be used to track tile molecular events responsible for it. Genetic Deletions: Possible Existence of "Tumor Suppressor Genes" One of the fields where cytogeuetics has brought a great deal to mole(:ular bioh)gy in a re(:ent past is the discovery of chromosomal deletions that are characteristic of certain tumor types. When analyzed at the mole(:ular level, such deletions have contributed to establish tile concept of tumor suppressor g e n e s - - o r "anti-oncog e n e s " - - l i k e the RB1 gene in retinoblastoma (see [35] for a review). Contrary to protooncogenes, such genes are susceptible to recessive mutations that impair their putative function in keeping the cells in a normal state. Therefore, homozygous alterations of these genes are required for expression of the inaliguant phenotype. The search for chromosomes losses has already led to the m a p p i n g of loci corresponding to predisposition for inherited cancer syndromes, e.g.: 3t) for renal carcinomas and small (:ell lung carcinoma, 5q for familial polyposis coil, 1 l p for nephroblastoma. 13q for retinoblastoma and osteosar(:oma (see [54] for references). At the cytogeneti(: level, alterations {deletions) of the short arm of c h r o m o s o m e 11 are a rather rare event in bladder TCC, and, in any case, appear to be secondary changes related--maybe--to an increased potential for invasiveuess [4, 23]. However, prompted by the recognition of a putative anti-oncogene localized on 11p that is associated with d e v e l o p m e n t of Wihns" embryonic kidney tumors [18, 38, 50, 58], Fearon et al. [19] have sought somatic losses of 11p sequences in urothelial tumors. They used DNA p o l y m o r p h i s m (RFLP) of H-ras and INS genes to detect losses of heterozygosity that could be indicative of recessive genetic changes involving 11p sequences. They observed that the frequency of loss of 11p sequences in adult b l a d d e r carcinomas a p p r o a c h e s that seen in embryonic Wilms' tumors (42% versus 56%). Although more frequent than cytogenetically visible alterations, these losses of heterozygosity on l l p do not correlate with tumor stage or histologic grade. In addition, other authors report much lower frequencies of chromosome 11p losses (8%, [53]). It is possible, as suggested by Sandberg [59], that 11p involvement in bladder TCC represents a late event in tumor progression, maybe as a consequence of a more generalized c h r o m o s o m a l instability. The relationship between 11p losses of heterozygosity and mutational activation of H-ras is an intriguing question: Do they cooperate and thus a p p e a r in the same tumors, or can deletions substitute for mutations in
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some ways and occur in tumors that do not contain an activated H-ras gene? The former possibility is supported by the chemically induced mouse skin tumors model where the loss of the normal H-ras allele seems to be involved in the progression of hemizygously H-ras-mutated tumors [55]. Concerning interstitial deletions of chrmnosome 13q, a good candidate gene to be involved is the RB1 gene that is located on 13q14. In fact, in the J82 bladder carcinoma cell line, an abe,rrant RB-1 prnte,in, has be,e,n de,te,cte,d 130l. This prote,in has lost its ability to complex to the ade,novirus E1A ont:oprote,in, and it is less stabl~,' than wiltl type plO5-Rb. However, in this case, the defe(:tiw', HB-1 protein is I)rodu(:e(I as the re,suit of a single,-point mutation rather than a (lele,tion. This situatioll is also SUl)l)(Jse,(l to take place, in retinoblastoma where gross alterations of the, 200kb I{B-1 gelie, are, not the lnost frequent, suggesting the, existence of more, subtle, ge,ne,ti(: (:banges at this level 130J. The (rely RB1 mutation found to (late' has been isolated froin a (:ell lira,' (J~2) and not directly from a tumor biopsy. It is therefore, possible, that it o(:(:urre,(I during the in vitro e,stablishment of the line,. ()nlv a svstemati(: surve,v of primary tulnor extracts for the, l)re,se,nce, of the, RB-I prote,in an(l its biologi(: inte,grity, me,asure,(I by its al)ilitv to bind EIA in vitro, e.g., will tell us about the, actual involw~,me,nt of this "anlioncogeue," product in the genesis of bla(hler TCC. CONCLUSION The recent successes in elu(:idating the molecular bases of some h u m a n pathologies (re,tinoblastonla, cystic fibrosis, D u c h e n n e muscular dystrophy} have, demonstrated the, power of molecular genetics integrating both cytogenetics and molecular biology. One of the issues addressed in this review concerned the present state, of conve,rgen(:e of these two approaches in the case of urothe,lial bladder tumors. However, as stated by Jewett 1321, "although the bladder is a relatively simple organ, its malignancy is so complex that the farther one digs for answers the more u n a n s w e r e d questions are, uncovered. It seems a never-ending process, and the, ultimate truth may reside in the p h e n o i n e n o n of cancer in general." Molecular genetics can be implicated in clinical ontology first by helping to recognize tumors subtypes. One of the striking features of bladder carcinomas is heterogeneity. In fact, although starting from a relatively simple tissue (transitional cell epithelium), it seems to represent various diseases with a lot of different outcomes. We have shown that the same heterogeneity exists at either the cytogenetic or the, molecular level, contributing to the confusing picture offered by these tumors. In addition, it is surprising to note that the prevalence of each chromosome abnormality described above, is highly variable, among different publications (Table 1). This variability could be related to some u n k n o w n bias in tumor sampling. It will probably be averaged once a lot more tumors will have, been analyzed. Hence, it has not been ye,t possible, to do genetic typing of bladder cancer. One exception might be the fraction of tumors (~15%) containing an activated H-ras gene that could correspond to a particular scheme of carcinogenesis and/or progression. Contrary to most tumors that develop from localized foci of transformed cells, bladder TCC is c o m m o n l y recurrent and multifocal, as if the entire, urotheliunl was concerned. This behavior is usually accounted for by the c o n t i n u o u s exposure of bladder to carcinogens. In order to explain that a carcinogen(s) leads to a very efficient transformation of the entire urothelium in the small fraction of the population affected by bladder cancer, one has to hypothesize either that only a minority is exposed to a particularly potent carcinogen(s) (viral?) or that there is some kind of genetic predisposition to bladder cancer. However, no such a predisposition has ever been demonstrated, maybe because, these tumors develop mostly among elderly people,. It could be interesting to screen affected i n d i v i d u a l s for germ-like alterations at the level of
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loci w h e r e d e l e t i o n s and losses of h e t e r o z y g o s i t y h a v e been e v i d e m : e d in t u m o r (:ells. On the o t h e r band, ah'eady r e c o g n i z e d b l a d d e r c a r c i n o g e n s are dive, rse, an(l at least s o m e of them, like cigarette s m o k i n g , are shared by m a n y , suggesting their role m o r e in p r o m o t i o n than in initiation. We h a v e s h o w n that a m o n g the u m : o g e n e s s t u d i e d so far, o n l y an activated H r , s has been o b s e r v e d r e p r o d u c i b l y in a subset of b l a d d e r TCC. it s h o u h l be interesting to investigate, in these tumors, lhe slate of the I~APIA w h i c h has been s h o w n to s u p p r e s s ras-modiated transforination [331. In fact, this gone is located on ( : h r o m o s o m e 1, w h i c h is the most f r e q u e n t l y altered in b l a d d e r TCC. The relation b e t w e e n o n c o g e n e a c t i v a t i o n and c h r o m o s o m a l losses has been d o c u m e n t e d in the case of genetic a l t e r a t i o n s d u r i n g c o l o r e c t a l - t u m o r d e v e l o p m e n t [74[. Here, t u m o r progression is suggested by the fact that most colorectal c a r c i n o m a s appear to arise from a d e n o m a s . K-ras m u t a t i o n s o c c u r m o s t l y in large a d e n o m a s and in c a r c i n o m a s ; a specific region of c h r o m o s o m e 18 is deleted m o r e f r e q u e n t l y in c a r c i n o m a s than in a d v a n c e d a d e n o Inas and in earlier-stage a d e n o m a s (73%, 47%, 11%, respectively): c h r o m o s o m e 17p s e q u e n c e s are u s u a l l y lost o n l y in c a r c i n o m a s (75%). Therefore, colorectal o n c o g e n e sis m i g h t r e q u i r e an initial step i n v o l v i n g inutational a c t i v a t i o n of an o n c o g e n e c o u p l e d w i t h s u b s e q u e n t losses of several genes that n o r m a l l y s u p p r e s s t u m o r i g e n e sis. S u c h a c o m p a r i s o n b e t w e e n p r e c a n c e r o u s and c a n c e r o u s lesions is not a v a i l a b l e yet in the case of b l a d d e r tumors, but it s h o u l d be e m p h a s i z e d in future studies. O n c o g e n e a c t i v a t i o n by t)oint m u t a t i o n is less f r e q u e n t in bla(tde, r t u m o r s than in colorectal (:ancer (< 15% w;rsus 4 0 % ) . T h e most (:ommon (:ytogeneti(: an(I mole(:nlar markers present in b l a d d e r t u m o r s c o r r e s p o n d to h)sses of genetic i n f o r m a t i o n (deletions, is()chromosomes, losses of heterozygosity). T h e r e f o r e , it is (:on~:eiwfl)le thai anti-on(:ogeues (or t u m o r sul)pressor genes) r e p r e s e n t I)rimarv targets of bla(hler o n c o g e n e s i s . T h e i r i n a c t i v a t i o n r e q u i r e s h o m o z y g o u s mutations, i.e., two hits, and the t i m e neede(t to a(:cumuhtte t h e m c o u l d be in part r e s p o n s i b l e for the, late at)l)earan(:e of b l a d d e r t u m o r s in the lifetime. It is to h o p e that deveh)t)in g c y t o g e n e t i c studies will h e l p to p i n p o i n t s o m e of these sites so that m o l e c u l a r bioh)gv can ta(:kle the respollsible genes. Continuous help and support from Dr. N. Ayraud is gratefully acknowledged. We thank Drs. C. Turc-Carel and A. Sandberg for critical reading of the manuscript. Such a study would not have been possible without the efficient collaboration of Drs. B. Basteris, J. Toubol, and J. Vacant who made available to us tumor and blood samples together with clinical data. Our laboratory is supported by grants from ARC, Caisse Nationale d'Assurance Maladie. FEGEFLUC, F6d6ration des Centres de Lutte Centre le Cancer, Fondation pour la Recherche M6dicale, INSERM, and Ligue Nationale Franc;aise centre le Cancer.
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