• flat urothelial hyperplasia;
  • FGFR3;
  • LOH chromosome 9


  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References

Flat urothelial hyperplasias (FUHs) in patients with papillary bladder tumours frequently show deletions of chromosome 9, suggesting that FUH could be the first neoplastic step in the development of papillary bladder cancer. FGFR3 mutations are frequent in non-invasive papillary tumours with low risk of progression. Our aim was to investigate the frequency of FGFR3 mutations and deletions of chromosomes 9p/q and 8p/q in FUH. Thirty FUH and 9 simultaneous or consecutive tumours were detected by 5-ALA-based photodynamic cystoscopy. DNA was isolated from frozen sections and whole genome amplification was done by I-PEP-PCR, followed by LOH analysis on chromosomes 8p/q and 9p/q. FGFR3 mutations were detected by SNaPshot analysis. LOH analysis on FUH revealed deletions at 9p/q (11/30, 37%) and 8p/q (3/30, 10%). FGFR3 mutations were found in 7/30 FUH (23%). Only 2 FUH showed an FGFR3 mutation without deletions of chromosome 9. In contrast, 6 FUH revealed chromosome 9 deletions but wild type FGFR3 (p = 0.03). These results suggest that chromosome 9 deletions are the earliest genetic alterations in bladder cancer. The detection of FGFR3 mutations in FUH further supports the role of this lesion as precursor of papillary bladder cancer. © 2006 Wiley-Liss, Inc.

Flat urothelial hyperplasia (FUH), which is defined as markedly thickened mucosa without cytological atypia, is frequently found adjacent to papillary urothelial tumours. For many years, this lesion has been regarded non-neoplastic without malignant potential, although clinicopathological studies documented an association between hyperplasia and papillary neoplasia.1 Genetic analysis on FUH and concomitant papillary tumours in bladder cancer patients revealed that many hyperplasias showed the same genetic aberrations as the papillary tumour, suggesting that FUH could be the first neoplastic step in the development of urothelial carcinoma.2, 3 Chromosome 9 alterations, especially loss on 9q, are the most frequent genetic events in bladder tumours, and can be found in histologically normal urothelium.2, 4, 5, 6, 7 It has therefore been suggested that loss of 9q may be the earliest event in the initiation of bladder cancer.8, 9 Deletions at chromosome arm 8p are also frequent but predominantly associated with advanced papillary bladder tumours.10

Mutations in the Fibroblast Growth Factor Receptor 3 (FGFR3) gene, which occur in the germ line of patients with skeletal dysplasias, are present in about 50% of bladder tumours. The majority of these tumours are of low stage and grade, and the presence of an FGFR3 mutation correlates with a favourable clinical outcome; that is, significantly fewer patients show progression and disease-specific mortality.11FGFR3 mutations were also detected in 75% of urothelial papillomas and are the only genetic events reported to date in these benign lesions.12, 13 Since, mostly tumours of low stage and grade present with FGFR3 mutations, FGFR3 mutations are considered early genetic events in bladder tumorigenesis.14, 15

The aim of the present study was to analyse FUH for FGFR3 mutations and loss of heterozygosity (LOH) of chromosomes 9 and 8 to investigate these early events in preneoplastic lesions. Since hyperplasia might be the first morphologically identifiable lesion in the development of papillary bladder tumours, we hypothesised that molecular analyses of these lesions could identify early genetic alterations in bladder cancer development.

Patient material was obtained from a clinical trial assessing the photodynamic diagnosis of neoplastic urothelial lesions.16 All patients gave written informed consent for this study. Biopsies were taken during cystoscopy after instillation of 5-ALA into the bladder from both fluorescence-positive, but otherwise inconspicuous areas, and fluorescence-negative papillary tumours. The biopsies were immediately snap-frozen in the operating room. Serial frozen sections stained with haematoxylin and eosin were used for histological diagnosis. Grading was performed according to the WHO classification17 and staging according to the Union Internationale Contre le Cancer (UICC).18 FUH was diagnosed if there was a markedly thickened mucosa (at least 10 layers of cells) without any cytological atypia in serial sections. Surface umbrella cells were preserved. Cases with significant inflammatory infiltrate and edema in the adjacent stroma were excluded as well as lesions with urothelial atypia.2, 3

Microdissection and DNA isolation of the urothelial lesions was performed as described previously.3 The microdissected samples contained at least 90% urothelial cells. Normal DNA as a reference for LOH analyses was isolated from EDTA blood, or from microdissected stromal or muscle tissue from the urinary bladder.

Whole genome amplification using I-PEP-PCR was performed for all samples to obtain sufficient amounts of DNA.3, 19 For all lesions, at least 500 cells were microdissected to avoid preferential amplification of one allele in LOH analysis because of insufficient amounts of DNA.

After preamplification, exons 5–9 of the TP53 gene were directly sequenced using single exon amplification and subsequent cycle sequencing on an ABI 373 sequencer as described previously.20

Analysis of the FGFR3 gene for mutations was based on the ABI PRISM SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA), and performed as described before.21 In short, 3 regions of interest comprising 9 mutations were amplified in one multiplex polymerase chain reaction, followed by extension of primers for each mutation with a labelled dideoxynucleotide. Extended primers were separated by capillary electrophoresis, and the presence or absence of a mutation was indicated by the incorporated nucleotide.

LOH analysis was performed using 2 microsatellite markers at chromosome arm 8p (D8S1817, D8S1145), 2 markers at chromosome arm 8q (D8S587, D8S591), 2 markers at chromosome arm 9p (D9S304, pky11) and 3 at chromosome arm 9q (D9S303, D9S747, D9S1113) as described previously.22 Informative cases were scored as allelic losses when the intensity of a signal for a tumour allele had decreased to at least 50% relative to the matched normal allele. All cases of allelic loss (LOH) were confirmed at least once.

Thirty lesions diagnosed as FUH and 9 simultaneous or consecutive papillary carcinomas from 24 patients were investigated in this study (additional clinicopathological data are detailed in Table I). Good results were obtained for all lesions, however repeated LOH analyses failed in some hyperplasias (8% of the samples could not be analysed (NA), see Fig. 1).

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Figure 1. Summary of FGFR3 and TP53 mutation analysis and LOH data in hyperplasias and tumours. LOH = loss of heterozygosity; NA = not available; NI = not informative; I = informative; wt = wild type; mut = mutation.

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Table I. Histopathological and Clinical Characteristics of 24 Patients with Flat Urothelial Hyperplasia and Simultaneous or Consecutive Papillary Bladder Tumours
Case noGender/ageHyperplasiaTumour
Time pointLocalisationStage/gradeTime pointLocalisation
  1.  M, male; F, female; S, simultaneous; C, consecutive/months; LW, lateral wall; PW, posterior wall; D, bladder dome; B, bladder base; LG, low grade; HG, high grade.

1M67LW right
2M37SLW rightpTaHGSLW left
3M68SLW rightpTaLGSLW right
5F81LW left
6M62LW left
9M73LW left
10b SD
11M58LW left
12F73C4PWpT1HGC4LW left
16b SLW left
18M60B/LW left
19b SB
19c SB
20M60SLW leftpT2HGSLW left
21M62SLW leftpT1HGSLW left
22b SPW
23M81LW right
24b SB

Microsatellite analysis showed LOH in 12 of 30 hyperplasias (40%) and in 6 of 9 tumours (67%, Fig. 1). Most of the loss was observed at chromosome arm 9q (6/30 hyperplasias and 4/9 tumours), but LOH was also found on 9p (5/30 hyperplasias and 1/9 tumours) and 8p (3/30 hyperplasias and 2/9 tumours). Loss on 8q was not observed.

FGFR3 mutation analysis showed that 23% (7/30) of hyperplasias had an FGFR3 mutation (Fig. 1). Of the 9 papillary carcinomas, 2 had an FGFR3 mutation (22%, cases 3 and 7), of which one corresponded with the mutation in the concurrent hyperplasia. In the other case, the tumour was mutant and the concomitant hyperplasia wild type for FGFR3. Both tumours were of low stage and grade (Table I). None of the high grade or invasive tumours carried an FGFR3 mutation. On the other hand, 2 high-grade tumours (cases 14 and 20) showed a mutation in TP53 after analysis of all tumours (Fig. 1). This is in accordance to previous studies that demonstrated the mutual exclusiveness of FGFR3 and p53 alterations, and their role in the different molecular pathways of disease pathogenesis in bladder cancer.23, 24

When the results of LOH and FGFR3 mutation analysis for the hyperplasias were combined, 2 hyperplasia samples of 2 patients (cases 9 and 16a) displayed an FGFR3 mutation, without having deletions at chromosomes 9 or 8. These deletions, however, may be missed because of the limited set of markers we used. Alternatively, samples could be contaminated with normal tissue in spite of microdissection, and this may influence the results of LOH analysis, whereas FGFR3 mutation analysis is very robust in a large background of non-tumour DNA. Conversely, 7 hyperplasia samples of 6 patients with deletions at chromosomes 9 or 8 (cases 2, 3, 12, 14, 17, 19a and 19b) were FGFR3 wild type. Six of the 7 samples had deletions at chromosome 9. This means that 55% (6/11) of hyperplasias with LOH at chromosome 9 lacked FGFR3 mutations, while 71% (5/7) of mutated hyperplasias already had LOH (Table II, p = 0.03). This is in concordance with a previous study by Van Tilborg et al. in which we showed that FGFR3 mutations occur after loss of chromosome 9 in evolutionary genetic trees of patients with multiple tumour recurrences.25 Urothelial papillomas may also be regarded as precursors of low-grade urothelial cancer12 and the percentage of FGFR3 mutations in these lesions is high. Interestingly, so far no LOH in papillomas has been observed, although only a limited number of samples have been investigated.13 This suggests that mutations in FGFR3 may play a leading role in the formation of papillomas, whereas in this study we show that chromosome 9 deletions are more frequent in FUH. An explanation for this observation could be that FGFR3 mutations are associated with neoplasias with a papillary growth pattern, while chromosome 9 deletions might be important for all urothelial neoplasias. We previously showed that chromosome 9 deletions occur both in hyperplasias3 and in dysplasias and CIS.20

Table II. LOH at Chromosome 9 and FGFR3 Mutations in Flat Urothelial Hyperplasias
MutantWild typeTotal
LOH 9 (yes)5611
LOH 9 (no)21719

In summary, FGFR3 mutations were detected in FUHs of patients with bladder tumours, further supporting the role of this lesion as a precursor of papillary bladder carcinoma. Chromosome 9 deletions may occur earlier than FGFR3 mutations in most cases and still remain the earliest known genetic alterations in bladder cancer.


  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References

We thank M. Kerscher for excellent technical assistance.


  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. References
  • 1
    Koss LG, Tiamson EM, Robbins MA. Mapping cancerous and precancerous bladder changes. A study of the urothelium in ten surgically removed bladders. JAMA 1974; 227: 2816.
  • 2
    Hartmann A, Moser K, Kriegmair M, Hofstetter A, Hofstaedter F, Knuechel R. Frequent genetic alterations in simple urothelial hyperplasias of the bladder in patients with papillary urothelial carcinoma. Am J Pathol 1999; 154: 7217.
  • 3
    Obermann EC, Junker K, Stoehr R, Dietmaier W, Zaak D, Schubert J, Hofstaedter F, Knuechel R, Hartmann A. Frequent genetic alterations in flat urothelial hyperplasias and concomitant papillary bladder cancer as detected by CGH, LOH, and FISH analyses. J Pathol 2003; 199: 507.
  • 4
    Junker K, Boerner D, Schulze W, Utting M, Schubert J, Werner W. Analysis of genetic alterations in normal bladder urothelium. Urology 2003; 62: 11348.
  • 5
    Obermann EC, Meyer S, Hellge D, Zaak D, Filbeck T, Stoehr R, Hofstaedter F, Hartmann A, Knuechel R. Fluorescence in situ hybridization detects frequent chromosome 9 deletions and aneuploidy in histologically normal urothelium of bladder cancer patients. Oncol Rep 2004; 11: 74551.
  • 6
    Stoehr R, Zietz S, Burger M, Filbeck T, Denzinger S, Obermann EC, Hammerschmied C, Wieland WF, Knuechel R, Hartmann A. Deletions of chromosome 9 and 8p in histologically normal urothelium of patients with bladder cancer. Eur Urol 2005; 47: 5863.
  • 7
    Mazzucchelli R, Barbisan F, Stramazzotti D, Montironi R, Lopez-Beltran A, Scarpelli M. Chromosomal abnormalities in macroscopically normal urothelium in patients with bladder pT1 and pT2a urothelial carcinoma: a fluorescence in situ hybridization study and correlation with histologic features. Anal Quant Cytol Histol 2005; 27: 14351.
  • 8
    Simoneau AR, Spruck CH,III, Gonzalez-Zulueta M, Gonzalgo ML, Chan MF, Tsai YC, Dean M, Steven K, Horn T, Jones PA. Evidence for two tumor suppressor loci associated with proximal chromosome 9p to q and distal chromosome 9q in bladder cancer and the initial screening for GAS1 and PTC mutations. Cancer Res 1996; 56: 503943.
  • 9
    Reznikoff CA, Sarkar S, Julicher KP, Burger MS, Puthenveettil JA, Jarrard DF, Newton MA. Genetic alterations and biological pathways in human bladder cancer pathogenesis. Urol Oncol 2000; 5: 191203.
  • 10
    Stoehr R, Wissmann C, Suzuki H, Knuechel R, Krieg RC, Klopocki E, Dahl E, Wild P, Blaszyk H, Sauter G, Simon R, Schmitt R, et al. Deletions of chromosome 8p and loss of sFRP1 expression are progression markers of papillary bladder cancer. Lab Invest 2004; 84: 46578.
  • 11
    Van Rhijn BWG, Vis AN, Van der Kwast TH, Kirkels WJ, Radvanyi F, Ooms EC, Chopin DK, Boeve ER, Jobsis AC, Zwarthoff EC. Molecular grading of urothelial cell carcinoma with fibroblast growth factor receptor 3 and MIB-1 is superior to pathological grade for the prediction of clinical outcome. J Clin Oncol 2003; 21: 191221.
  • 12
    Van Rhijn BW, Montironi R, Zwarthoff EC, Jobsis AC, van der Kwast TH. Frequent FGFR3 mutations in urothelial papilloma. J Pathol 2002; 198: 24551.
  • 13
    Chow NH, Cairns P, Eisenberger CF, Schoenberg MP, Taylor DC, Epstein JI, Sidransky D. Papillary urothelial hyperplasia is a clonal precursor to papillary transitional cell bladder cancer. Int J Cancer 2000; 89: 5148.
  • 14
    Van Rhijn BWG, Lurkin I, Radvanyi F, Kirkels WJ, van der Kwast TH, Zwarthoff EC. The fibroblast growth factor receptor 3 (FGFR3) mutation is a strong indicator of superficial bladder cancer with low recurrence rate. Cancer Res 2001; 61: 12658.
  • 15
    Montironi R, Lopez-Beltran A, Mazzucchelli R, Bostwick DG. Classification and grading of the non-invasive urothelial neoplasms: recent advances and controversies. J Clin Pathol 2003; 56: 915.
  • 16
    Zaak D, Kriegmair M, Stepp H, Baumgartner R, Oberneder R, Schneede P, Corvin S, Frimberger D, Knuechel R, Hofstetter A. Endoscopic detection of transitional cell carcinoma with 5-aminolevulinic acid: results of 1012 fluorescence endoscopies. Urology 2001; 57: 6904.
  • 17
    EbleJN, SauterG, EpsteinJI, SesterhennIA, eds. World Health Organization Classification of Tumours. Pathology and genetics of tumours of the urinary system and male genital organs. Lyon: IARC Press, 2004.
  • 18
    SobinLH, WittekindC, eds. TNM classification of malignant tumors. New York: Wiley-Liss, 1997.
  • 19
    Dietmaier W, Hartmann A, Wallinger S, Heinmoller E, Kerner T, Endl E, Jauch KW, Hofstadter F, Ruschoff J. Multiple mutation analyses in single tumor cells with improved whole genome amplification. Am J Pathol 1999; 154: 8395.
  • 20
    Hartmann A, Schlake G, Zaak D, Hungerhuber E, Hofstetter A, Hofstaedter F, Knuechel R. Occurrence of chromosome 9 and p53 alterations in multifocal dysplasia and carcinoma in situ of human urinary bladder. Cancer Res 2002; 62: 80918.
  • 21
    Van Oers JMM, Lurkin I, van Exsel AJA, Nijsen Y, van Rhijn BWG, van der Aa MNM, Zwarthoff EC. A simple and fast method for the simultaneous detection of nine fibroblast growth factor receptor 3 mutations in bladder cancer and voided urine. Clin Cancer Res 2005; 11: 77438.
  • 22
    Hartmann A, Rosner U, Schlake G, Dietmaier W, Zaak D, Hofstaedter F, Knuechel R. Clonality and genetic divergence in multifocal low-grade superficial urothelial carcinoma as determined by chromosome 9 and p53 deletion analysis. Lab Invest 2000; 80: 70918.
  • 23
    Bakkar AA, Wallerand H, Radvanyi F, Lahaye JB, Pissard S, Lecerf L, Kouyoumdjian JC, Abbou CC, Pairon JC, Jaurand MC, Thiery JP, Chopin DK, et al. FGFR3 and TP53 gene mutations define two distinct pathways in urothelial cell carcinoma of the bladder. Cancer Res 2003; 63: 810812.
  • 24
    Van Rhijn BW, van der Kwast TH, Vis AN, Kirkels WJ, Boeve ER, Jobsis AC, Zwarthoff EC. FGFR3 and P53 characterize alternative genetic pathways in the pathogenesis of urothelial cell carcinoma. Cancer Res 2004; 64: 19114.
  • 25
    Van Tilborg AAG, de Vries A, de Bont M, Groenfeld LE, van der Kwast TH, Zwarthoff EC. Molecular evolution of multiple recurrent cancers of the bladder. Hum Mol Genet 2000; 9: 297380.