Seminars in Surgical Oncology 8260-266 (1992)

Genetics of Transitional Cell Carcinoma ERIC A. KLEIN, MD, AND R.S.K. CHACANTI, P h o From the Section of Urologic Oncology, Department of Urology, Cleveland Clinic Foundation, Cleveland, Ohio (€.A.K.) and Laboratory of Cancer Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York (R.S.K.C.)

Several models of genetic events which define the origin and progression of human tumors have been elucidated over the last several years. These models suggest that the study of tumors at the level of both the chromosome and the gene can be useful in elucidating molecular events in tumor progression and in determining the biologic behavior of individual tumors. The genetics of transitional cell carcinomas are reviewed with emphasis on potential mechanisms of tumorigenicity and the clinical utility of genetic markers. 0 1992 WiIey-Liss, Inc. KEY WORDS:genetics, chromosomes, karyotypes, transitional cell carcinoma INTRODUCTION The analysis of human tumors over the last two decades has yielded significant insight into the genetic events which are important in human malignancy. This research has produced a large body of information cataloging in detail the karyotypic abnormalities observed in both hematologic and solid tumors [l]. This information has suggested that tumor origin and progression is comprised of a series of specific genetic events which take place in a target cell and its descendents. The identification of these genetic events has also led to definition of the molecular events which occur as a result of chromosomal changes and which may alter a cell’s capacity for growth and differentiation [2]. These observations have suggested that chromosomal and gene abnormalities characterize virtually all tumors, that the abnormalities are nonrandom and may show specificity to a histologic subtype, and that tumor progression is accompanied by additional or new abnormalities which occur in defined patterns. This article will review several general models of genetic mechanisms of tumorigenicity and apply them to cytogenetic and molecular biologic observations about transitional cell carcinoma. THE HUMAN CHROMOSOME COMPLEMENT Normal human somatic cells contain 46 chromosomes which can be arranged into seven groups comprising chromosomes of similar length and morphology (Fig. 1). The introduction of chromosomal staining techniques during the 1970s allowed identifi0 1992 Wiley-Liss, Inc.

cation of chromosomal regions which exhibit characteristic differences in staining called bands. These techniques make it possible not only to recognize individual chromosomes but also to map their subregions precisely. Figure 1 shows a metaphase spread from a cultured human blood lymphocyte stained to reveal one type of banding (Q-banding). The chromosomes are arranged as homologous pairs, an arrangement termed the karyotype. A formal system of nomenclature has been developed that is used to describe chromosomes, their regions, and their alterations [3]. By convention, the two regions of a chromosome divided by the centromere are recognized as two arms. Each arm is divided into regions and each region further subdivided into bands. The letters p, q, and c designate short arm, long arm, and centromere, respectively, of the chromosome. In a karyotype, the p arm always is above the centromere and the q arm always below. Chromosomes in Cancer Cells Tumors are characterized by groups of cells called clones which share common numerical and/ or structural chromosomal abnormalities (Fig. 2). A cytogenetic clone is defined by detection of at least three cells with an identical numerical abnormality or two cells with an identical structural abnormality in a population of tumor cells. Numerical abnormalities involve Address reprint requests to Dr. Eric A. Klein, M.D., Head, Section of Urologic Oncology, Department of Urology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195.

Genetics of TCC 261

Fig, 1. Normal karyotype derived from peripheral blood Iymphocyte of a healthy male. Chromosomes are arranged by groups and chromosome number in homologous pairs. Horizontal arrows denote position of centromeres; oblique arrows denote p arms of various chromosomes.

gains and losses of chromosomes. Gain of an entire chromosome is termed trisomy, and loss of an entire chromosome, monosomy. Cells with chromosome numbers in excess of 46 are termed hyperdiploid, while those with less than 46 chromosomes are termed hypodiploid. A cell containing three copies of the haploid genome (69 chromosomes) is called triploid and one with four copies (92 chromosomes) tetraploid. Structural abnormalities result from breakage and reunion of broken ends of chromosomes forming translocations (t), inversion (inv), deletions (del), duplications (dup), or isochromosomes (i). Translocations involve breakage and exchange of broken segments between nonhomologous chromosomes. Inversions involve breakage and reattachment of the broken segment within the chromosome in an inverted orientation. Deletions and duplications involve loss or gain of segments of the chromosome. Isochromosomes occur

when both arms are derived from one of the two arms of a normal chromosome by breakage at the centromere; hence the two arms of an isochromosome are genetically identical. Another type of chromosomal abnormality seen in some cancer cells is amplification of a chromosomal region which contains extra copies of a specific gene. The amplified region may be located at the normal chromosomal position of the gene or at an unrelated chromosomal site. Amplified genes may appear as a homogeneously stained region (HSR) within a chromosome or as tiny extrachromosomal elements in the cytoplasm called double minute (DM) chromosomes. When studied with appropriate techniques, chromosome changes can be seen to characterize virtually all neoplasms. Such changes generally affect chromosomes nonrandomly and may be specific for a histologic subtype or etiologic factor. Tumor progression is accompanied by karyotypic evolution which in some tumors follows defined patterns. Elucidation of such karyotypic evolution lead to the concept that some changes are primary (related to tumor origin) and others are secondary (related to tumor evolution) in tumor development. The karyotypic abnormalities of primary or secondary changes include the entire spectrum of aberrations (e.g., gain, loss, deletion, duplication, translocation, and inversion) described above. The tendency for instability in tumor genomes occurs early in tumor progression and accounts for variation in tumor evolution. The analysis of this genetic instability has significant implications in understanding of the mechanisms of neoplastic transformation. Any nonrandom chromosome translocation can be considered potentially capable of leading to tumor formation if perturbation of genes relevant to cell transformation, proliferation, or lineage-sepcific function are situated at the breakpoints. Gene amplification may result in overproduction of the product of a gene involved in malignant proliferation or in conferring resistance to antineoplastic drugs. The major contribution to the understanding of cellular genetics of cancer during the 1980s was the discovery that certain classes of genes, in particular cellular oncogenes (cellular DNA sequences homologous to transforming sequences of RNA tumor viruses), and genes that encode molecules such as growth factors and their receptors map at the breaksites of nonrandom rearrangement in cancer cells [4].Molecular studies of structure and function of several of these genes has suggested several mechanisms of genetic carcinogenesis. Broadly stated, normal cellular homeostasis results from the balance of growth-promoting and growth-inhibitory effects [ 5 ] . At the most basic level cancer is a disorder of growth resulting from the

262 Klein and Chaganti

Fig. 2. A: Karyotype of a noninvasive papillary transitional cell carcinoma containing 47 chromosomes. This tumor is characterized by loss of one chromosome 2 and extra copies of chromosomes 20 and 22 (arrows). The monosomy 2 seen in this tumor is consistant with molecular observations of deletions of chromosome 2p in some TCC. (Reproduced from [2] with permission of W.B. Saunders Company.) B: Karotype of tumor cell from a noninvasive carcinoma in situ of the bladder. In addition to 46 normal chromosomes there are two abnormal chromosomes present (arrows) one of which is a duplication of 3p and one of which is an unidentified marker chromosome.

loss of the normal constraints on cellular proliferation and differentiation. At the genetic level, cellular homeostasis results from the balanced effects of genes which enhance and those which inhibit growth. Cancer can result from mutations in either growth-enhancing or growth-inhibitory genes. Oncogenes typically enhance cellular growth when mutations cause them to be activated or “turned on.” Genes which inhibit cellular growth have been termed anti-oncogenes, or tumor suppressor genes, because mutations which inactivate them allow uncontrolled cellular growth or tumor development. Genetic changes in tumor cells can be recognized at the level of the chromosome or at the level of the gene. Chromosomal changes are identified by preparation of tumor karyotypes after growth of the tumor in short-term tissue culture. Changes in indi-

vidual genes are studied by extracting intact DNA or RNA in fresh tumors. Changes in gene structure (translocations, rearrangements, amplification, and point mutations) can be detected by digestion of DNA with (restriction) enzymes of known specificity and hybridization with homologous DNA (“probes”) of known sequence by a technique called Southern hybridization and blotting [6]. Alterations in the expression of individual genes can be detected by quantitative and qualitative assay of messenger RNA using similar probes by the technique of Northern blotting [6]. Both techniques have yielded useful information on the nature and specificity of genetic changes important in identifying tumor histology, biological markers of tumor progression, insight into cellular mechanisms of growth control, and markers useful for predicting prognosis.

Genetics of TCC 263

Models of Genetic Carinogenesis n

Several examples of genetic observations in nonurologic tumors will serve to illustrate the current level of understanding of tumor genetics and how they may be useful in understanding tumor biology and clinical tumor behavior. The first specific chromosomal change identified in a human tumor was the Philadelphia chromosome, a translocation between chromosomes 9 and 22. This chromosomal abnormality was detected in 1960 by light microscopy of chromosomes obtained by growth of peripheral blood lymphocytes from patients with chronic myelogenous leukemia (CML) [7].Detailed study of this system has yielded much useful biologic and clinical information and has served as a model for the study of other tumors. For example, the Ph’ chromosome is present in 85% of patients with CML; those patients with CML who lack Ph’ are usually older and have a better prognosis [ 11. The chronic phase of CML is characterized by the addition of other chromosomal abnormalities in addition to Ph’, including extra copies of chromosome 8, formation of isochromosome 17q, additional copies of Ph ’, and loss of the Y chromosome. The addition of these abnormalities can be seen several months or weeks prior to the onset of blast crisis and some success has been achieved in defining prognostic subgroups based on the specific markers observed at a given time. The molecular analysis of the Ph ’ chromosome has yielded insight into specific cellular growth mechanisms. The translocation between chromosomes 9 and 22 results in the movement of an oncogene called c-abl from chromosome 9 to chromosome 22 (Fig. 3). The abl gene fuses with a gene called bcr on chromosome 22. The result is the expression of a new “fusion” gene which produces a protein not present in normal cells. This protein has been shown to contain the ability to transform normal cells into neoplastic ones in vitro and is thought to represent a very early event in CML [8,9]. A similar activating mutation occurs in acute lymphocytic leukemia (ALL), which also may contain the Ph’. Here, the abl-bcr fusion occurs at a different site in the bcr gene and results in a different fusion protein. Recent work has shown that the abl-bcr protein formed in ALL is even more potent in producing neoplastic transformation than that seen in CML, paralleling the more aggressive clinical course of ALL [lo]. Another useful tumor model has come from the study of retinoblastoma (RB). This tumor occurs in two forms, familial and sporadic. Patients with sporadic RB have no family history of RB and are usually affected by solitary tumors in one retina. Individuals with familial RB typically have a parent with RB and

U U

9

22

ABL mRNA

BCR mRNA

9q-

22q+

BCR-ABL FUSION mRNA

Fig. 3. Schematic diagram of genetic events in chronic myelogenous leukemia. Reciprocal translocation between chromosomes 9 and 22 results in fusion of the BCR and abl oncogenes. This results in a novel messenger RNA and cellular protein not present in normal cells. A similar event in acute lymphocytic leukemia with a different breakpoint in the BCR gene results in a cellular protein with more potent transforming capacity.

other relatives affected by an autosomal dominant mode of inheritance. Familial tumors are identical histologically to sporadic tumors but typically occur at an earlier age and affect both retina with multiple tumors. The differences in the epidemiology of familial and sporadic RB and the observed loss of genetic material from chromosome 13 in karyotypes in familial RB generated the hypothesis that loss of a tumorsuppressor gene allows RB to form [Ill. In sporadic tumors it is hypothesized that both copies of the RB gene in all cells are normal, and that a single cell must undergo two mutations which inactivate or delete both copies of the gene; consequently sporadic tumors arise in a single cell and are unifocal and unilateral (Fig. 4). In familal tumors one of these mutations is inherited in all cells (including both retina) and only a single additional mutation is necessary for tumors to form. Because many cells are at risk familial RB occurs multifocally and bilaterally (Fig. 4). The molecular technique of restriction fragment length polymorphism analysis by Southern blotting has allowed the identification of children whose DNA already contains the inherited mutation. This has permitted efforts aimed at the early detection of individuals at risk for RB before retinal tumors have become clinically apparent and opened the possibility for vision-sparing treatment. Thus the study of RB has led to insight into the cellular and genetic mechanisms by which tumors can form (i.e., by mutations in genes which normally inhibit cell growth) and created new diagnostic tools which have significant clinical relevance. Recent studies have identified loss of specific genetic loci which may contain tumor suppressors genes in many human

FAMILIAL

Fig. 4. Schematic representation of loss of a tumor suppressor gene leading to retinoblastoma. In sporadic RB, two somatic mutations in the same cell are necessary for tumor formation. In familial RB, the first mutation is inherited in every cell, putting each cell at risk for tumor formation. Only a single somatic mutation corresponding to the second mutation in sporadic RB is necessary for tumor formation. RB + , normal tumor suppressor gene; RB - , inactivated or deleted suppressor gene.

malignancies and raise the spector of similar advances in the understanding of biologic mechanisms and earlier diagnosis for these tumors as well [5]. The third genetic model of tumors comes from the study of colorectal cancers using a variety of genetic techniques. These studies have defined a series of genetic events which appear to cocur in each stage of the progression of normal colonic mucosa to dysplasia, low and high grade adenomas, and frank cancers [ 121 (Fig. 5). Like CML and RB, these studies have yielded some insight into,the biologic mechanisms by which colorectal tumors originate and grow. When applied clinically these observations may serve as an adjunct to help define in an objective way the stage in the natural history of an adenoma (instead of relying solely on histologic appearance) and the degree of risk of progression to cancer. This may in turn help decide the need for adjunctive treatment in the form of surgical or medical therapy designed to prevent further tumor formation. GENETIC EVENTS IN TRANSITIONAL CELL CARCINOMAS Karyotypic Abnormalities Detailed chromosomal banding studies have been reported in about 100 transitional cell cancers (TCC) of the bladder. These studies have identified abnormalities of chromosomes 1, 3, 5, 7, 9, and 11 as occurring with increased frequency in TCC [14-231. The

Fig. 5. Hypothesized chain of genetic events associated with the origin and progression of colorectal cancers. Modified and reproduced from [I31 with permission of Cold Spring Harbor Laboratory, Copyright 1989.

observed abnormalities of chromosome 1 have mirrored those seen in other malignancies and do not appear to be specific to TCC [14-161. Additions to the short arm of chromosome 3 (3p+) in the region of 3p14-3p26 have been observed in several tumors. (Figs. 2, 3) [15,17]. The nature of the additional chromosomtil segment has not yet been identified in these tumors. Duplication of the short arm of chromosome 5, forming an isochromosome [i(Sp)], has also been seen in several tumors [16,18]. This marker has also been observed in ureteral TCC [19]. The occurrence of i(5p) in both invasive and noninvasive tumors suggests its formation may be an early event in the pathogenesis of TCC. One extra copy of chromosome 7 (trisomy 7) has been observed in many tumors in conjunction with other abnormalities [15]. In addition trisomy 7 has been observed as the only chromosomal abnormality in three TCC of the bladder and two of the ureter, strongly suggesting that addition of this chromosome may be an early and specific event in all TCC [ 15,16,21,221. Partial or complete losses of chromosome 9 are commonly seen and have been described as the only chromosomal abnormality in one noninvasive papillary tumor, suggesting this as another early chromosomal change [I 5,16,20,21]. Deletions of the short arm of chromosome 11 (1 l p - ) have been described in conjunction with various anomalies of other chromosomes and is usually seen in higher grade and invasive tumors [ 14-1 71. In addition to trisomy 7 and monosomy 9, two other solitary chromosomal changes have been identified in TCC. Two low grade noninvasive TCC characterized by deletion of band 22 on chromosome 2 1[de1(21q22)] have been described [21,22]. One case

Genetics of TCC 265

of del (10q24) has also been described as the sole ab- (21q22) suggests that loss of the ets oncogene, which normality in a noninvasive grade 2 papillary TCC [20]. is located at 21q22, may be important in the pathogenWhile the identification of these solitary changes sug- esis of TCC. At present, there are no good models of gest an important early role in the pathogenesis of alterations in oncogene structure or expression that TCC, the actual genetic events which may occur as the account for origin or progression of TCC. Similarly, result of these changes is unknown. The order in which there are no identified oncogene markers of clinical the primary and secondary genetic abnormalities are utility in the management of bladder tumors, acquired and a pathogenetic sequence of events in Genetic Predictors of Tumor Recurrence and tumor progression is not yet possible for TCC. An Progression insufficient number of cases has also yet to be identified to say with certainty that these changes are speSeveral studies have addressed a common and difcific genetic markers for TCC whose identification ficult question in the clinical management of superfimay substantiate histologic diagnoses or identify tu- cial bladder tumors-is there a way to identify which mors of equivocal histology with certainty. low or intermediate grade noninvasive tumors are likely to recur and/or progress in stage and gain the Molecular Studies potential for metastases? These studies have demonTwo studies have reported the loss of genetic mate- strated several trends in the answer to this question. In rial from several chromosomes as detected by South- one study of 42 superficial (Ta) grade 1 or 2 papillary ern blotting. The first study confirmed the finding of TCC, only 50% of diploid or near-diploid tumors reloss of part of the short arm of chromosome 11 seen curred, while 100% of the hyperdiploid tumors did so in karyotypes of other tumors in 5 of 12 TCC [24]. One [29]. Similarly, several studies have shown that superhigh grade invasive tumor also contained deletions of ficial tumors with marker chromosomes (any abnorchromosome 2p and 14q; a grade 2 noninvasive tumor mal chromosome) or aneuploidy recur more fresimilarly contained a deletion of chromosome 2p. The quently and carry a higher risk of death than those second study identified frequent deletions of 1l p and without marker chromosomes. In one study Ta tu14q in both superficial and invasive TCC [25]. In addi- mors without markers had a 10% recurrence rate and tion deletions of 17q, 9q, and 6p were seen without a 5% risk of death from bladder cancer; Ta tumors specificity for stage or grade. Several metastatic le- with markers recurred in 90% with a similar death sions were also studied and showed the same deletions rate, and T1 (submucosally invasive) tumors with as the corresponding primary tumor [25]. Together markers recurred in 80% with a 50% death rate [30]. these observations suggest loss of genetic material (im- Longer follow-up (up to 17 years) of a similar group plying loss of tumor suppressor genes) from chromo- of patients revealed an 86% ultimate recurrence risk some 1 l p and 14q occur early in the genesis of TCC for superficial noninvasive tumors with marker and are retained as tumors progress to muscle inva- chromosomes vs. a 50% risk for tumors without marksion. Losses from other chromosomes (17q, 9q, and ers. Risk of death from bladder cancer also was related 6p) may represent randomly acquired losses during to the presence of markers, with rates of 33% vs. 9% tumorigenesis or may dictate other aspects of tumor in the groups with and without markers, respectively. behavior. The lack of a clear pattern of secondary Submucosally invasive tumors, all with markers, had genetic changes in association with stage or grade in a 100% recurrence rate and a 61% death rate [31]. these studies limits the interpretation of their signifi- While these studies appear promising in prognosticating the risk of tumor progression they are all limited cance. Little is known about which oncogenes may be im- by the fact that none of the genetic indices (degree of portant in TCC. Early work focused on the rus family aneuploidy or marker chromosomes) occur indepenof oncogenes because of the identification of mutated dently of stage or grade. Specifically, low grade turus genes in some TCC which were capable of trans- mors tend to have diploid or near-diploid chromoforming mouse fibroblasts [26]. However, recent some numbers, with an increasing trend to aneuploidy screening of a larger number of TCC with a very sensi- with higher grade [17,29-321. Similarly, aneuploidy tive technique (polymerase chain reaction) has failed increases with increasing stage from Ta to invasive to identify a significant number of mutations in ras tumors [ 17,20,30,33]. Marker chromosomes (any abgenes in bladder tumors [27]. One recent study has normal chromosome) also increase in incidence paraldetected high levels of expression of the epidermal lel to grade and stage [29,32]. Do genetic markers have any value independent of growth factor receptor gene in TCC, suggesting a potential mechanism for enhanced tumor growth by re- currently used (histologic) criteria? One study has ceptor overexpression [28]. The observation of del shown that hyperdiploid tumors of any stage tend to

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recur sooner than diploid tumors, with 10% of diploid as compared to 54% of the hyperdiploid tumors recurring within 18 months of initial diagnosis 1291. Another study showed that noninvasive or superficially invasive TCC with either 3p duplication or 1Ip deletion, or both, were associated with a shorter time to recurrence (3.8 vs. 12 months) and higher risk of progression to muscle invasiveness [ 171. Whether these markers will ultimately prove useful clinically will require study of more tumors. CONCLUSION It is clear from the examples cited that genetic analysis of tumors have enhanced the understanding of genetic mechanisms of tumor origin and progression. At present there is limited data in TCC suggesting that gains (chromosomes 5p, 7, and 3p) and losses (chromosomes 9 and l l p ) of specific chromosomal segments are important in the genesis of TCC. It also appears that loss of certain tumor suppressor genes from chromosome 11 and 14 may be important in the biologic progression of papillary noninvasive tumors to those with aggressive clinical behavior. The lack of clear association of other genetic markers with the behavior (stage and grade) of TCC limits interpretation of their significance. Descriptive observations in TCC of tumor behavior based on aneuploidy and the presence of marker chromosomes agree with general notions of tumor progression being associated with progressively abnormal genetic complements within tumor cells but are of limited clinical utilitv at uresent. REFERENCES 1. Chaganti RSK, Klein EA: Cytogenetic basis for molecular

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