the molecular pathology of urological malignancies

, . 183: 380–387 (1997)

REVIEW ARTICLE

THE MOLECULAR PATHOLOGY OF UROLOGICALMALIGNANCIES

. *, . .

School of Surgical Sciences, Medical School, Framlington Place, University of Newcastle upon Tyne, NE2 4HH, U.K.

SUMMARY

Urological malignancies kill over 16 000 people annually in England and Wales. There have been exciting recent developments in ourunderstanding of the molecular pathogenesis of these diseases, although many questions remain unanswered. Three separate genes (WT1,WT2, andWT3) have been implicated in Wilms’ tumour development. Patients with von Hippel–Lindau (VHL) syndrome develop renalcell carcinoma and it has been shown that VHL protein inhibits elongin, a cellular transcription factor which controls RNA elongation.Use of molecular markers to identify superficial bladder tumours likely to progress to muscle invasive disease has met with some success.Increased epidermal growth factor receptor (EGFR) and p53 expression, and decreased E-cadherin expression all correlate with tumourprogression. Tumours in patients with carcinoma in situ have distinct molecular features. Androgen ablation delays disease progressionin men with prostate cancer, but relapse is inevitable. Research has been directed towards elucidating the mechanisms by which prostatecancer ‘escapes’ hormonal control. Mutations in the androgen receptor have been identified. It is apparent that locally produced growthfactors mediate androgen-dependent processes and these too have been implicated in prostate carcinogenesis. ? 1997 John Wiley &Sons, Ltd.

J. Pathol. 183: 380–387, 1997.No. of Figures: 0. No. of Tables: 0. No. of References: 98.

KEY WORDS—Wilms’ tumour; von Hippel–Lindau; renal cell carcinoma; bladder cancer; prostate cancer

INTRODUCTION

The management of patients with malignancies affect-ing the urinary tract constitutes a large portion of theworkload of the urologist. In 1995, over 2600 peopledied from malignant neoplasms of the kidney, 4800 frombladder cancer, and 8800 men from prostate cancer inEngland and Wales alone.1 Prostate cancer is now thesecond commonest cause of cancer death in the West2and receives increasing publicity in the media as ‘malehealth’ issues become less of a taboo subject. In recentyears, there have been rapid advances in our under-standing of the molecular bases of these diseases. Thisreview aims to highlight those advances which possessthe greatest potential clinical relevance in terms ofdiagnosis, prognosis and therapy. Malignancies of thekidney, bladder, and prostate will be discussed.

KIDNEY

Wilms’ tumourThere are two forms of Wilms’ tumour, familial and

sporadic, and the study of this contributed towards thedevelopment of the two-hit model of malignant trans-formation postulated by Knudson and Strong.3 Wilms’

tumour arises from the mesenchymal stem cells of thekidney and accounts for 8 per cent of all childhoodmalignancies (incidence 1 in 10 000 live births).4 Patientsusually present between the ages of 2 and 5 years andwith improvements in treatment, the 5-year survival nowapproaches 90 per cent. Wilms’ tumour has been exten-sively investigated in terms of genetic abnormalitiesbecause of the known (albeit low) familial incidence.The proposed mechanism of carcinogenesis in Wilms’

tumour is complex, as three separate genes (WT1,WT2,and WT3) are now known to be involved. Loss ofheterozygosity (LOH) on the short arm of chromosome11 was seen in 5/7 cases of sporadic Wilms’ tumour5 andtheWT1 gene was subsequently mapped to 11p13.6 TheWT1 gene is a tumour suppressor gene and a recentstudy by Hewitt et al. has shown that WT1 suppressestranscription of bcl-2 and c-myc, proto-oncogenesinvolved in the regulation of cellular proliferation andapoptosis.7 Overexpression of bcl-2, which occurs inWilms’ tumour and renal cell carcinoma,8 results insuppression of apoptosis. WT1 shares 60 per centhomology with the early growth response gene 1 (Egr1)9and the WT1 protein product binds a DNA sequencesimilar to the target binding site of EGR1.10 Egr1regulates cell growth and differentiation. Sequenceanalysis of cDNA clones of the WT1 gene reveals aprotein with features commonly found in transcriptionfactors—the protein has four zinc fingers and a prolineand serine-rich NH2-terminus.6 Egr1 transcriptionis stimulated as a result of growth factor-receptor

*Correspondence to: Trevor J. Dorkin, School of Surgical Sciences,Medical School, Framlington Place, University of Newcastle uponTyne, NE2 4HH, U.K.

CCC 0022–3417/97/120380–08 $17.50 Received 16 May 1997? 1997 John Wiley & Sons, Ltd. Accepted 9 June 1997

interactions and it is postulated that WT1 behaves asan antagonist to Egr1 by binding to the sites of targetgenes which would otherwise be bound by the EGRproduct.11It has also been suggested that one mode of action of

WT1 is the down-regulation of the insulin-like growthfactor receptor type 1 (IGFR-1) gene.11 WT1-negativecells were stably transfected with a WT1 expressionvector, which resulted in a decrease in IGFR-1 promoteractivity and IGFR-1 mRNA expression with decreasedcellular proliferation. IGF-2, a mitogen, is over-expressed in Wilms’ tumour, and the WT1 product hasalso been shown to inhibit IGF-2 transcription.12 It ispossible that an autocrine growth loop is facilitated byIGF-2 and its interaction with IGFR-1. WT1 maytherefore exert its effect via four distinct pathways:down-regulation of IGFR-1; binding of genes targetedby EGR1; IGF-2 suppression; and bcl-2 suppression.Reeve et al. observed that in 5/21 cases of sporadic

Wilms’ tumour, deletions at 11p15 were more commonthan at 11p13.13 Further evidence implicating theinvolvement of a second tumour suppressor gene waspublished by Koufos et al., who noted LOH at 11p15.5only in three Wilms’ tumours.14 The suppressor role ofthis gene, WT2, was confirmed by experiments in whichchromosome constructs with 11p13 (but not 11p15)deletions introduced into a Wilms’ tumour cell line G401were able to inhibit malignant growth in nude mice.15The control construct (with p15 deletion) had no sucheffect. The WT2 locus has recently been localized distalto D11S998 on 11p15.5.16There are studies of families predisposed to Wilms’

tumour which have revealed no chromosome 11 abnor-malities, suggesting that a third gene must be involved intumourigenesis.17 Austruy et al. recently analysed 28patients for chromosome 12 and 16 abnormalities,having observed such abnormalities in sporadic Wilms’tumour; 25 per cent had LOH for 16q and 18 per centhad duplication of chromosome 12.18 In one tumour,16q LOH was the only abnormality, suggesting that thismay be an early event in tumorigenesis.It is hoped that with an increased knowledge of the

cytogenetic abnormalities found in Wilms’ tumour,more informed genetic counselling will be available toaffected couples.

Renal cell carcinoma

Renal cell carcinoma (RCC) accounts for 90 per centof renal neoplasms in the adult. A small number (1 percent) are familial and are seen in patients with the vonHippel–Lindau (VHL) syndrome. These individualsdevelop multiple benign and malignant tumours ofvarious organs (cysts and carcinomas in the kidney,adrenal carcinoma, pancreatic and liver cysts, retinalcysts, and cerebellar and spinal haemangioblastomas).They are more likely to develop renal neoplasms at anearlier age than sporadic cases. Indeed, they have a 70per cent risk of developing RCC by the age of 60 years.19The VHL disease gene (a tumour suppressor gene) hasbeen mapped to chromosome 3p25–p2620,21 and encodesa protein of 213 amino acids.

Elongin, a cellular transcription factor, is the target ofthe VHL protein. It activates transcription elongationby RNA polymerase II and is composed of three sub-units (A, B, and C). In vitro studies have revealed thatthe VHL protein binds the B and C subunits, resultingin the inhibition of elongin action (and hence RNAtranscription).22 Evidence suggests that the A subunit isthe active component in transcription and the B and Csubunits act as regulators.23 Mutant VHL gene productsbind elongin less effectively and this has been demon-strated in vivo as well as in vitro.24 RNA transcriptionregulation will therefore be unhinged with subsequentuncontrolled cellular proliferation. The VHL genealso alters the transcription of vascular endothelialgrowth factor (VEGF), an important regulator ofangiogenesis.Hereditary RCCs are also seen in tuberous sclerosis.

Tuberous sclerosis genes (TSC1, TSC2) have beenmapped to chromosomes 9q34 and 16p13.3, respect-ively.25,26 These patients develop RCC at a younger agethan patients with sporadic tumours, although it wouldappear that they have a better prognosis. However, thismay simply be due to the fact that RCCs in tuberoussclerosis patients are discovered incidentally during theinvestigation of other renal pathology associated withthis condition (e.g., angiomyolipomas).VHL gene abnormalities may play a role in sporadic

renal cell tumorigenesis. However, other genetic abnor-malities have been observed, although further putativetumour suppressor genes still remain to be character-ized. Deletions have been frequently identified at3p13–14 and 3p21.27 3p deletions or mutations areconsistent findings, having been identified in 51/58 (88per cent) of patients in one study,28 but these abnormali-ties do not occur alone, as one would expect with thecomplex multistep nature of genetic aberrations seen insolid tumours. Thrash-Bingham et al. analysed 33tumours and found LOH on 6q, 8p, 9pq, and 14q, aswell as on 3p.29 Cytogenetic analysis of a renal carci-noma cell line, KTCTL-26A, performed by Hogemannet al. identified numerous chromosome abnormalities(on 2, 3, 5, 7, 9, 13, 18, 21, 22, and Y), the mostprevalent being incomplete 3p deletions and trisomy ofchromosome 7.30 Further analysis revealed increasedexpression of pro-transforming growth factor á(TGFá) and epidermal growth factor receptor (EGFR)mRNA. It has been demonstrated in other studies thatthe EGFR, which binds both EGF and TGFá, isoverexpressed in RCC.31 Most malignant cells express-ing the EGFR also produce TGFá, thereby completingan autocrine loop favouring tumour growth. IncreasedEGFR protein content correlated significantly withhigh tumour grade and the development of metastaticdisease.32Leukaemia inhibitory factor (LIF) is an inflammatory

cytokine secreted by RCCs which also acts in anautocrine/paracrine fashion, stimulating tumourgrowth.33 It may also be responsible for the cachexiasyndrome associated with renal malignancy. Basic fibro-blast growth factor (FGF-2) is expressed by both normalkidney and malignant tissue, and antibodies directedagainst FGF-2 inhibit tumour growth.34

381THE MOLECULAR PATHOLOGY OF UROLOGICAL MALIGNANCIES

? 1997 John Wiley & Sons, Ltd. , . 183: 380–387 (1997)

BLADDER

The majority (60–70 per cent) of bladder tumours aresuperficial at presentation and can be adequately treatedby transurethral resection. The majority of thesetumours will recur, but more importantly 10–20 per centwill progress to invasive disease with a poorer prognosis.The challenge to the urologist and pathologist is toidentify means by which the behaviour of an individualtumour can be predicted so that the appropriate thera-peutic measures can be instigated. Research at themolecular level has been directed towards this goal.One of the potential markers for bladder tumour

progression is the EGFR, encoded by the gene c-erbB-1.This is a transmembrane tyrosine kinase receptor whichbinds both EGF and TGFá. Immunohistochemicalstudies have shown a significant correlation betweenEGFR expression and both tumour stage and grade.35Following this observation, a prospective study wascarried out to see whether EGFR status could predictoutcome. EGFR expression at diagnosis was associatedwith tumour progression (P<0·0001).36 A similar studyincorporating more patients (n=212) was reported byour group in 1995;37 this confirmed EGFR positivity tobe an independent prognostic indicator of survival andtumour progression. In the high-risk group of patientswith pT1G3 disease (n=20), EGFR expression was 80per cent sensitive and 93 per cent specific in predictingprogression to muscle invasive disease. If these findingswere to be confirmed in a larger series, EGFR statuswould provide useful information for advising patientswith T1G3 disease to undergo early radical cystectomy.It is also apparent that it is TGFá, and not EGF, whichbinds to the EGFR in this biological system and it hasbeen demonstrated recently that there is a correlationbetween TGFá and EGFR in bladder cancer (with lowlevels of EGF).38 It seems likely that as in RCC, TGFáis acting as an autocrine or paracrine growth factor inthis situation and further research is needed to delineateits mode of action more clearly.Another potential marker for tumour progression is

p53. The gene encoding this protein is located on theshort arm of chromosome 17 (17p13.1). LOH on 17pand p53 mutations, usually resulting in an abnormal,non-functional protein with a prolonged half-life (which,unlike its normal counterpart, can be detected immuno-histochemically) occur more frequently in muscle inva-sive tumours and tumours of poor histological grade.Olumi et al. demonstrated that none of ten low-gradeand 20 of 31 high-grade tumours exhibited homozy-gosity for 17p,39 demonstrating that loss of the normalp53 tumour suppressor gene correlates with the develop-ment of high-grade tumours. Spruck et al. detected p53gene mutations in 1/36 (3 per cent) Ta tumours and25/49 (51 per cent) muscle invasive tumours.40 p53abnormalities would appear to be critical in tumourprogression and indeed several studies suggest that p53is also an independent prognostic factor for superficialtransitional cell carcinoma (TCC) of the bladder. Serthet al., analysing a group of 69 patients, showed that12/14 (85·7 per cent) patients who had more than 20 percent of tumour cells staining positively for p53 had

progression to muscle invasive disease, compared withonly 1/55 (1·8 per cent) with less than 20 per cent tumourcell positivity.41 Sarkis et al. observed similar findings intheir series of 43 patients with T1 bladder carcinoma.42These patients had a median follow-up of nearly 10years. Patients with greater than 20 per cent p53 posi-tivity had an approximately 11-fold increased risk ofdisease progression based on multivariate analysis; p53staining was an independent prognostic factor for dis-ease progression. Indeed, in both studies, p53 positivitywas the only independent factor predicting diseaseprogression; other non-predictive factors included age,tumour grade, vascular invasion, and carcinoma in situ).Other studies, however, have refuted the suggestion

that p53 accumulation in superficial TCC is related totumour progression. Gardiner et al. used three anti-bodies (including PAb1801 used in the aforementionedstudies) for immunostaining and found no correlationbetween p53 positivity and disease progression in T1disease (n=28).43 A similar conclusion was reached byLipponen, who performed a multivariate analysis on 212patients with bladder cancer.44 p53 protein was detectedusing the CM1 antibody in this study. p53 also does notcorrelate with prognosis in those patients with invasivedisease.45 The overall picture therefore remains confusedat present.Unlike 17p LOH, chromosome 9 deletions are an

early event in bladder tumorigenesis, being found inmany pTa and pT1 tumours and being independent ofboth tumour grade and stage.46 In one series, 72/116 (62per cent) tumours had chromosome 9 deletions.47 Inanother series, 40/70 (57 per cent) newly diagnosed casesof TCC of the bladder demonstrated LOH at one ormore loci on 9q.48 It has been postulated that thisdeletion is the initiating event in bladder carcinogenesis.Research has been directed towards identifying a puta-tive tumour suppressor gene in the region of thisdeletion (9p21–9q22).One tumour suppressor locus which has been exten-

sively investigated is 9p21. Homozygous deletions of thisregion have been identified in both cultured cell lines andprimary tumours.49 A candidate tumour suppressorgene for this locus is p16. p16 protein is integral to cellcycle regulation, acting as an inhibitor of cyclin depen-dent kinases 4 and 6, which normally form complexeswith cyclin D. Williamson et al. examined 140 bladdertumours for p16 aberrations. All of 13 tumours with9p21 deletions, 18 of 31 (58 per cent) tumours withchromosome 9 LOH, and 9 of 91 tumours (10 per cent)with no LOH for chromosome 9 had homozygousdeletions of p16. Their conclusion was that p16 is thetarget tumour suppressor gene at 9p21.50 There are,however, reports that are not consistent with this con-clusion. Cairns et al. found that only 1 of 15 (6·7 percent) tumours with 9p LOH had a p16 defect51 andsimilar results were obtained by Okajima et al.52 It hasbeen recognized, however, that detection of homozygousdeletions using the polymerase chain reaction (PCR) canbe difficult and different methodologies may account forsuch disparate results.The Rb1 gene product is essential for cell cycle con-

trol. When dephosphorylated, it binds and inactivates

382 T. J. DORKIN ET AL.


transcription factors necessary for cell cycle progression.These factors are released on Rb1 phosphorylation,which is the end product of cyclin-dependent kinaseactivation. The Rb1 locus is on 13q and in one study,28/94 (29 per cent) specimens showed LOH with asignificant correlation to high tumour grade and stage.53Chromosome 8 deletions have also been identified inbladder cancer.54

Carcinoma in situ

Superficial papillary carcinomas will occasionallyprogress to muscle invasive disease, but progression ismore likely in patients with carcinoma in situ (CIS). Thisobservation highlights the possibility of two separatemolecular pathways in bladder cancer. Evidence for thishas been published by Spruck et al., who found that 1/36(2·8 per cent) Ta tumours and 15/23 (65 per cent) CIScases (P<0·001) contained p53 mutations.40 A low fre-quency of chromosome 9 LOH (3/24) was seen in theCIS specimens. Rosin et al. detected a much higherfrequency (77 per cent) of chromosome 9 LOH.55 Over-all, papillary tumours and CIS behave differently both atthe molecular and at the clinical levels, and it may bethat they should be regarded as separate entities.

Angiogenesis

Tumour survival is dependent on adequate neo-vascularization and therefore angiogenic factors areimportant in carcinogenesis. Indeed, it has been shownthat vascular density, as a measure of angiogenesis, is anindependent predictor of tumour recurrence and overallsurvival.56 Several members of the fibroblast growthfactor (FGF) family stimulate new vessel formationboth physiologically (e.g., tissue response to injury) andpathologically (e.g., neoplasia). Elevated levels ofFGF-2 have been found in the urine of patients withbladder cancer.57 Unfortunately, similar levels of FGF-2were also detected in the urine of patients with benignprostatic hyperplasia, so it is unlikely that urinaryFGF-2 measurements will be sufficiently specific toprovide useful information on disease status in bladdercancer. The same investigators have examined midkine(MK) expression in bladder cancer. Like FGFs, MK is aheparin-binding angiogenic factor which was alsoexpressed at higher levels in bladder cancer. With limiteddata (n=22), they found that MK expression wasassociated with a worse prognosis in invasive disease.58

Cell adhesion molecules

Deregulation of cell–cell adhesion facilitates tumourinvasion and dissemination. In bladder cancer, this maycontribute to the generation of multifocal disease, seenin approximately 30 per cent cases at presentation.There are four major families of cell surface adhesionreceptors (cadherins, immunoglobulin superfamily,selectins, and integrins). E-cadherin, expressed by epi-thelial cells, has been the most widely studied. DecreasedE-cadherin expression has been reported in muscle in-vasive bladder tumours.59 It is also associated with an

increased risk of progression in patients with superficialdisease.60 A soluble form of E-cadherin can be detectedin the serum of patients using conventional ELISAmethods. Soluble E-cadherin is elevated in patients withbladder cancer and these levels correlate with poorhistological grade and multifocal tumours at presenta-tion, as well as the likelihood of tumour recurrence atthe first check cystoscopy (3 months).61

PROSTATE

Over 50 per cent of men presenting to the urologistwith clinically apparent prostate cancer have metastaticdisease.62 Most are therefore initially treated withandrogen deprivation therapy, which results in a symp-tomatic improvement in approximately 70 per cent, butthis response is only transient because of the appearanceof hormone refractory phenotypes. Research is directedtowards elucidating the mechanisms which cause pros-tate cancer to become unresponsive to anti-androgentherapy.The prostate requires androgens for maintenance of

normal glandular structure and function. Androgens(testosterone and dihydrotestosterone) interact withnuclear receptor proteins. Upon ligand binding, there isa conformational change in the androgen receptor (AR),which exposes DNA binding sites. The activated ligand–receptor complex is then capable of binding specificDNA sequences called androgen response elements,hence controlling the transcription of specific targetgenes (i.e., the ligand–receptor complex is a transcrip-tion factor).63 One such androgen response element islocated within the promoter region of the prostate-specific antigen gene.64 Abnormalities in this biologicalsystem could account for the eventual androgenindependence of prostate tumours.It has been demonstrated that AR mRNA expression

is absent in the androgen-independent prostate carci-noma cell lines PC3 and DU145; this is not the case withthe LNCaP cell line, which is androgen-sensitive.65 TheAR concentration is also significantly higher in well-differentiated tumours than in moderately and poorlydifferentiated tumours.66 However, the picture is farfrom clear, as Habib et al. showed that AR contentcorrelated with higher tumour stage and Gleason scoresin 13 patients with prostate carcinoma.67 Similar resultswere found by van der Kwast et al.68 Absence of ARprevents the apoptosis that is induced in normal prostatefollowing androgen withdrawal.69,70Abnormalities of the AR itself have also been docu-

mented in prostate cancer. The LNCaP cell line carries asingle point mutation in codon 868, substituting alaninefor threonine.71 This mutation (an A to G mutation)does not alter the AR’s ability to interact with testoster-one and dihydrotestosterone and, in fact, results in anincreased affinity of the receptor for progesterone andoestradiol.72 In addition, the mutant AR binds theanti-androgens hydroxy-flutamide and cyproteroneacetate, but such binding results in paradoxicallyincreased transcriptional activity and growth stimula-tion. This same mutation has been identified in 6/24



(25 per cent) cases of advanced prostate cancer in oneseries.73 This mutation may therefore be important inthe development of hormone refractory disease. It is ofdirect clinical relevance, because prescribing standardpharmacological anti-androgen therapy in the formof flutamide to patients with this mutation may bedetrimental to their treatment. Other AR gene muta-tions have been delineated, including a guanine-to-adenine mutation in codon 73074 and the sametransition in codon 715. This latter mutation results inan increased ability of progesterone and the adrenalandrogens to enhance androgen-responsive transcrip-tional activity.75 It would appear that several mutantAR phenotypes, rather than becoming non-functional asone might expect, actually acquire increased functionalcapacity.Another possibility to account for the development of

hormone refractory disease is a change in the responseof target genes to AR binding. In one experiment, cellsof the PC3 cell line were transfected with a vectorcarrying AR cDNA. Addition of dihydrotestosteroneactually resulted in a 50 per cent decrease in growth ratecompared with the controls.76 The precise mechanism toexplain this has yet to be established.Tissue recombination experiments using normal wild-

type urogenital sinus mesenchyme/stroma and epi-thelium lacking functional AR have shown that theAR-negative epithelium is still able to undergoandrogen-dependent differentiation, albeit in a smallfraction of experiments (5 per cent).77 This is becausemany such androgen-dependent processes are actuallymediated by local paracrine growth factors secretedby prostatic stroma. These growth factors have beenimplicated in prostate cancer, because their increasedexpression or activity would explain how tumours couldproliferate in the absence of androgens. EGF is pro-duced by both benign and malignant prostate tissue andis androgen-regulated. EGFR mRNA is expressed athigh levels in the androgen-independent cell linesDU145 and PC3, and at lower levels by the androgen-dependent LNCaP cell line.78,79 TGFá, a ligand for theEGFR, is produced by the PC3 cell lines, suggesting anautocrine role for this growth factor in malignancy.80Immunohistochemical analysis has shown that poorlydifferentiated tumours express TGFá at higher levels.81As with bladder cancer, there may be a transition in thepredominant ligand from EGF to TGFá.82 Evidence forthis has been published recently by Glynne-Jones et al.,who found a highly significant (P<0·0001) correlationbetween EGFR and TGFá.83Members of the FGF family have also been identified

in prostate cancer. FGF-2 (basic FGF) is produced bythe DU145 and PC3 cell lines.84 In the rat Dunningmalignant epithelial cells, FGF-3 and FGF-5 mRNA areexpressed.85 FGF-8 mRNA (or androgen-inducedgrowth factor) has been shown to be overexpressed bythe malignant prostatic epithelium and the increasedlevel of expression correlates with poor histologicalgrade.86 Experimental evidence has demonstrated thatFGF-8 isoforms have different transformation proper-ties in NIH3T3 cells in vitro and in vivo,87 one of themtransforming these cells so that they are highly tumori-

genic. There are four distinct genes encoding the tyrosinekinase FGF receptors. However, splice variants of thereceptors exist, which adds to the complexity of possibleligand–receptor interactions. Indeed, in an experimentby Yan et al., a switch in the FGFR-2 from the IIIbisoform (which binds FGF-7 with high affinity) to theIIIc isoform (which binds FGF-2 and not FGF-7) wasidentified in the progression of Dunning epithelialcell tumours from an androgen-dependent to anandrogen-independent state.85Another complex system involves insulin-like growth

factors (IGFs), of which there are three: IGF-I, IGF-II,and insulin. In addition, there are two receptors, type Iand type II, as well as at least six IGF-binding proteins.IGF-II is produced by the stroma of the normal prostateand stimulates growth of both stroma and epithelium,its effects being mediated primarily by the IGF type Ireceptor.88 In the prostate cancer cell lines DU145 andPC3, however, IGF-I mRNA is overexpressed89 andblockage of the type I receptor causes reduced prolifer-ation, suggesting yet another functional autocrine loopallowing androgen-independent growth.90 Prostate-specific antigen (PSA) is a serine protease that cleavesIGF-I from IGFBP3.Unlike the above growth factors, transforming

growth factor â (TGFâ) is a growth inhibitor of normalprostate cells.91 In vitro studies on human prostatecancer cells have shown, however, that this inhibition islost, which may be important in malignant progres-sion.91 Indeed, overexpression of TGFâ in a rat prostatecancer model led to the development of a more aggres-sive tumour with increased metastasis, compared withcontrols.92 Obviously, the interaction of these growthfactors and their receptors is complex and it is importantnot to view each in isolation. To illustrate this, Mortonand Barrack examined rat prostate cancer cells whichare resistant to the inhibitory effects of TGFâ in vivo butnot in vitro. They found that the inhibitory effect in vitrowas abolished in the presence of FGF-2 and greatlyreduced in the presence of EGF.93Androgen withdrawal causes prostate regression

by means of apoptosis or programmed cell death.69However, androgen-insensitive tumour cells retain theirability to undergo apoptosis when exposed to chemo-therapeutic agents.94 This is associated with a concomi-tant rise in TGFâ and testosterone-repressed prostatemessage 2 (TRPM-2) gene expression. bcl-2 is a proteinwhich prevents apoptosis and which has been implicatedin malignancy, allowing malignant cells to ‘escape’ suchnormal homeostatic mechanisms. Increased expressionof bcl-2 has been demonstrated in prostate cancer. Inone study, 9/9 hormone refractory prostate tumoursand/or metastases expressed bcl-2.95 Bubendorf et al.found that bcl-2 expression was more frequent in higher-grade tumours (31 per cent pT3 vs. 5 per cent pT2;P=0·001) and that it predicted disease progression.96 Inan elegant study by Raffo et al., androgen-dependentLNCaP cells were transfected with the bcl-2 proto-oncogene. In vitro studies showed that the transfectedcolonies were resistant to apoptotic stimuli and in vivostudies using nude mice demonstrated that these trans-fected cells developed larger tumours than controls and



only these tumours were resistant to the effects ofandrogen withdrawal (surgical castration).97 The apop-totic index (AI) can be measured on haematoxylin andeosin-stained sections. In one study of 28 radical pros-tatectomy specimens followed up for 5–9 years, patientswith a low AI (defined as less than the median value ofall 28 AI scores) had significantly lower rates of diseaseprogression (7 per cent vs. 50 per cent; P<0·007) thanpatients with a high AI.98

CONCLUSIONS

Our knowledge of the molecular pathological pro-cesses involved in urological malignancies is increasingrapidly. At a purely scientific level, it is important thatwe have a complete understanding of molecular biologyin health and disease. It is only with this knowledge thatapplications can be devised to aid clinicians seeking toimprove the management of these malignancies. Unfor-tunately, in terms of routine urology practice, suchapplications have yet to come to fruition, although thepotential is apparent. For example, EGFR and p53status in bladder cancer do offer prognostic informationwith regard to the likelihood of tumour progression,which is of particular importance in patients with T1G3disease. It may be that patients who have EGFR,p53-positive T1G3 disease should be advised to undergoradical cystectomy in preference to a trial of BCGimmunotherapy. Coordinated multicentre prospectivetrials are urgently required.Another potential difficulty lies in the deployment of

molecular techniques in a standard DGH pathologydepartment. Immunohistochemical studies are carriedout routinely and it is quite feasible that apoptoticindices can be assessed, should larger trials confirm theirusefulness. However, routine PCR-based assays arecomplex and prone to error due to contamination, evenin the hands of the expert molecular scientist. Welook forward to finding answers to these questions andchallenges.

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