FGFR3 mutational status and protein expression in patients with bladder cancer in a Jordanian population

Cancer Epidemiology xxx (2010) xxx–xxx

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CANEP-175; No. of Pages 9

FGFR3 mutational status and protein expression in patients with bladdercancer in a Jordanian population

Khaldon Bodoor a,*, Abdulhameed Ghabkari a,b, Ziad Jaradat a, Asem AlKhateeb a, Saied Jaradat a,b,Mohammed A. Al-Ghazo c, Ismail Matalka d, Hisham Musleh d, Yazan Haddad a,b

a Department of Applied Biology, Jordan University of Science and Technology, Irbid, Jordanb Princess Haya Biotechnology Center, Jordan University of Science and Technology, Irbid, Jordanc Department of Urology, Jordan University of Science and Technology, Irbid, Jordand Department of Pathology and Laboratory Medicine, Jordan University of Science and Technology, Irbid, Jordan

A R T I C L E I N F O

Article history:

Accepted 8 May 2010

Keywords:

Bladder cancer

FGFR3

Prognosis

A B S T R A C T

Bladder cancer accounts for nearly 5% of all newly diagnosed cancers in Jordan, with a much higher

frequency in males. Recent studies have shown that activating mutations in FGFR3 are the most common

findings in non-invasive low grade bladder tumors. In this study, we, retrospectively, investigated a

cohort of 121 bladder cancer patients with various grades and stages of the tumor for molecular changes

in FGFR3. Overexpression of FGFR3 was observed in 49%, 34%, 15%, and 2% of pTa, pT1, pT2, and pT3 cases,

respectively. Further, FGFR3 expression was positive in 45%, 26%, and 30% of G1, G2 and G3 cases,

respectively. Mutational analysis of exons 7, 10 and 15 of FGFR3 identified four previously reported

mutations, namely R248C (n = 4; 10%), S249C (n = 23; 59%), Y375C (n = 7; 18%), G382R (n = 4; 10%), and

one novel mutation, G382E (n = 1; 3%). Our results indicate that both mutations and overexpression of

FGFR3 are correlated together, and are more prevalent in early stage (pTa and pT1) and low grade (G1

and G2) bladder tumors. Survival analysis showed no contribution of changes in FGFR3 on the patient’s

survival. Multivariate Cox proportional hazards model analysis of overall survival for the following

variables: age, gender, stage and grade of tumor, and FGFR3 (expression and mutation) revealed that age,

stage and grade of tumor are independent predictors of overall survival in patients with bladder cancer.

Our work is the first to address the molecular status of FGFR3 in Jordanian patients with bladder cancer,

and provides further support for FGFR3 as a key player in the initiation of bladder tumors.

� 2010 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Cancer EpidemiologyThe International Journal of Cancer Epidemiology, Detection, and Prevention

journal homepage: www.cancerepidemiology.net

1. Introduction

Worldwide, 250,000–300,000 new cases of bladder cancer arediagnosed every year claiming the lives of more than 120,000patients [1]. Bladder cancer is the fifth most common cancer inJordanian males accounting for more than 4% of new cancer casesdiagnosed each year [2]. Studies show that four times as manycases of bladder cancer occur in males as in females whileapproximately 90% of patients diagnosed with bladder cancer areover the age of 60 [3,4]. Transitional cell carcinoma (TCC) is themost common type of bladder cancer accounting for more than

* Corresponding author at: Department of Pharmacology and Cancer Biology,

LSRC room C112, Duke University Medical Center, P.O. Box 3813, Durham, NC

27710, USA. Tel.: +1 919 613 8750; fax: +1 919 668 0977.

E-mail addresses: [email protected], [email protected]

(K. Bodoor), [email protected] (A. Ghabkari), [email protected] (Z. Jaradat),

[email protected] (A. AlKhateeb), [email protected] (S. Jaradat),

[email protected] (M.A. Al-Ghazo), [email protected] (I. Matalka),

[email protected] (H. Musleh), [email protected] (Y. Haddad).

Please cite this article in press as: Bodoor K, et al. FGFR3 mutational sJordanian population. Cancer Epidemiology (2010), doi:10.1016/j.ca

1877-7821/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.canep.2010.05.003

90% of the cases [5]. Bladder cancer is staged according to thedegree of tumor invasion into the bladder wall [6,7]. At the time ofdiagnosis, 70–80% of tumors are non-invasive being restricted tothe inner epithelial lining (pTa, CIS) and/or the lamina propria(pT1) [7]. However, invasive tumors are usually presented withpenetration into the muscle (stage T2), the perivesical layerbeyond the muscle (stage T3) and, rarely, involving local nodesand/or distant organs (stage T4) [8]. Non-invasive tumors areusually well-differentiated (low grade; G1) whereas invasivetumors are poorly differentiated (high grade; G2 or G3) [8]. Non-invasive bladder tumors are generally treated by transurethralresection of tumor (TURT) however; 50–70% of patients willdevelop recurrences within 5 years [9]. Patients with muscle-invasive urothelial tumors are commonly treated with cystectomyand are associated with a poor prognosis [5,10,11].

Recent studies have suggested non-invasive low grade tumorsand invasive high grade tumors arise through distinct molecularpathways [12,13]. Alterations on chromosome 9 and activatingFGFR3 mutations have been described as the most commonfindings in non-invasive low grade tumors whereas, p53 mutations

tatus and protein expression in patients with bladder cancer in anep.2010.05.003

mailto:[email protected]










http://dx.doi.org/10.1016/j.canep.2010.05.003

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K. Bodoor et al. / Cancer Epidemiology xxx (2010) xxx–xxx2

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and loss of heterozygosity (LOH) on chromosome 17 are morecommon in invasive high grade tumors [14–16]. FGFR3 belongs toa class of structurally related receptor tyrosine kinases (FGFR1–4)which binds to fibroblast growth factors (FGFs) with differentaffinities with FGFs 1, 2, 4, 8 and 9 being the most relevant [17,18].The 4.4-kb cDNA of FGFR3 contains an open reading frame of 2520nucleotides encompassing 19 exons, and encoding an 840 aminoacids protein. FGFR3 consists of three glycosylated extracellularimmunoglobulin-like ligand binding domains, a single transmem-brane domain, and two cytoplasmic tyrosine kinase domains[17,19]. Alternative mRNA splicing between exons 8 and 9generates two isoforms with variable ligand specificity; FGFR3band FGFR3c. FGFR3b is mainly expressed in epithelial cells whereasFGFR3c is mainly found in chondrocytes [20].

FGFR3 is involved in a variety of biological processes includingcell proliferation, differentiation, migration, angiogenesis, andapoptosis. Cellular functions of FGFR3 are activated by ligand-induced dimerization which in turn leads to transphosphorylationof key tyrosine residues in the kinase domain of the receptor [20].Phosphorylated tyrosines act as docking sites for the recruitmentof signaling molecules and activation of downstream pathways[21].

Four main signal transduction pathways have been implicatedin mediating FGFR3 functions including the MAP kinase, STAT1,PI3K-AKT, and PLCg [22–24]. It is well documented that activatingmutations in FGFR3 are found in the germline in several autosomaldominant human skeletal disorders [25,26]. Surprisingly, recentstudies identified identical mutations in FGFR3b, exons 7, 10 and15, in patients with non-invasive low grade bladder tumors [27–30]. Multiple myeloma, colon cancer and cervical cancer are the

Table 1Clinical and histopathological characteristics.

Clinical characteristic Stage

pTa pT1

Number 63 30

Grade

G1 (well differentiated) 48 6

G2 (moderately differentiated) 12 17

G3 (poorly differentiated) 3 7

Sex

Male 56 28

Female 7 2

Age (years)

Mean 60.6 65

Range 24–83 42–81

Smoking

Yes 49 22

No 14 8

Tumor size

�3 cm 32 19

<3 cm 31 11

Tumor number

Solitary 19 10

Multiple 31 12

Missing 13 8

Treatment

TURT 47 17

Radical cystectomy 16 13

Recurrence

No 9 11

One or more 54 19

Follow-up (months)

Median 30 24

Range 11–72 10–52


only other types of cancer with FGFR3 mutations [31,32]. Multiplestudies have been published describing, in detail, FGFR3 muta-tional spectrum and frequencies [27–30]. However, few studieshave examined the correlation between mutation status, level ofexpression of the protein and their association with the clinical andhistopathological variables of patients [28,33]. Additionally, ourwork is the first to address the molecular status of FGFR3 inJordanian patients.

In this study, we analyzed a cohort of 121 bladder cancerpatients with various grades and stages of the tumor. We examinedthe mutation and expression profiles of FGFR3 and theircorresponding frequencies and attempted to correlate thesemolecular changes to a number of clinical and pathologicalvariables to better identify markers associated with favorableprognosis.

2. Methods

2.1. Patients and tumor samples

One hundred and thirty patients diagnosed with bladder cancerwere randomly selected in a retrospective analysis (2004–2007) ofthe pathological records in the Department of Pathology at theKing Abdullah University Hospital. Tumors were staged accordingto the American Joint Committee on Cancer and graded accordingto the WHO 1973 classification system [34]. The 1973 system wasused instead of the WHO 2004 classification system because of thefact that this system has been implemented in our hospital formore than two decades, it is clinically approved, reproducible, andprovides more consistency when results have to be compared to

Total

pT2 pT3

24 4 121

0 0 54

3 0 32

21 4 35

20 2 106

4 2 15

61.8 59.8

41–71 47–71

18 3 92

6 1 29

15 3 69

9 1 52

11 1 41

6 1 50

7 2 30

11 1 76

13 3 45

15 4 39

9 0 82

.5 24.5 28.5

2–39 21–61



K. Bodoor et al. / Cancer Epidemiology xxx (2010) xxx–xxx 3

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the ones from different hospitals in the region. Re-staging TURTwas performed for cases of pTa and pT1 high grade tumors. Onehundred and twenty-nine cases were classified as transitional cellcarcinoma (TCC) and only one case was classified as squamous cellcarcinoma (SCC) which was not further investigated. Completeclinical records were available for 121 cases and those wereselected for further analysis. Approval for this work was obtainedfrom the Faculty of Medicine Research Ethics Committee at theJordan University of Science and Technology. Patients’ mainclinical features are listed in Table 1.

2.2. Immunohistochemical studies

Immunohistochemical studies were performed on formalin-fixed paraffin sections (3-mm thickness) using the avidin–biotin–peroxidase complex method. Immunostaining was performed usingan automated immunostainer (XL, LEICA, Germany) according to themanufacturer’s protocols. Sections were incubated with a monoclo-nal antibody against FGFR3 (Clone B9, Santa Cruz, CA, USA) for 1 h atroom temperature and detected with a biotinylated secondaryantibody and 3,3-diaminobenzidine (DAB). Slides were brieflycounterstained with Mayer’s hematoxyline and examined by lightmicroscopy (OLYMPUS). At least 1000 tumor cells were scored semi-quantitatively in well-preserved areas of each slide. Evaluation ofslides was carried out independently by pathologists I.M. and H.M.,whom were blinded for any knowledge regarding patient’sclinicopathogical data. Cytoplasmic and/or cell membranous stain-ing of FGFR3 was scored negative (<5% indicating absent or weakstaining) or positive (+: 5–70% moderate staining; ++:>70% intensestaining). The expression of FGFR3 was also analyzed in normalindividuals and patients diagnosed with other urological disorders.

2.3. DNA extraction and mutational analysis

Five micrometers serial sections of paraffin-embedded tissueswere deparafinized and DNA extracted using the DNeasy tissue kit(Qiagen GmbH, Hilden, Germany). Exons 7, 10, and 15 of FGFR3 wereselected for mutational analysis. Primers for the amplification ofthese exons were previously described [35]. PCR reactions weredone in a 50 ml volume using 2 ml tumor DNA (�40 ng), 2 ml of5 pmol of each primer, 25 ml PCR master mix (GoTaq1 Green mastermix, Promega, USA) and nuclease-free water was added to attain afinal volume of 50 ml. PCR conditions were as follows: 95 8C for7 min, followed by 40 cycles of 95 8C for 1 min, 62 8C for 1.5 min(exon 15 of FGFR3), 61 8C for 1.5 min (exons 7 and 10 of FGFR3) and72 8C for 1 min, and a final extension step of 72 8C for 7 min. PCRproducts were fractionated by electrophoresis and visualized withethidium bromide. Purification of PCR products was carried out atthe Macrogen Sequencing Labs in Seoul, South Korea. Sequencing ofpurified PCR products (using both forward and reverse PCR primers)was carried out using the bigDye terminator V1.1 cycle sequencingkit (Applied Biosystems). Mutational analysis was done using theChromasPro software 1.34 (Technelysium, Australia).

2.4. Statistical analysis

Pearson’s x2 test of independence was used to analyze therelationship between categorical variables (stage and grade oftumor, FGFR3 expression and FGFR3 mutation). Overall survival(OS) and disease-free survival (DFS) curves were constructed usingthe Kaplan–Meier method. Accordingly, OS was defined as theperiod from the time of diagnosis to death from any cause or lastcontact. DFS was defined as the period from the time of diagnosis todeath from any cause or recurrence of the disease or last contact.The log rank test was used to evaluate the survival curves. Coxproportional hazard regression model was carried out to deter-


mine the multivariable effect of the different prognostic variables.A forward stepwise likelihood ratio method was used for choosingthe model. The Rho test and scaled Schoenfeld residuals plot wereused to test for the assumption of proportionality, whereas theMartingale residual plot was used to test for the assumption oflinearity of continuous variable according to previously describedmethods [36,37]. A value of p � 0.05 was considered statisticallysignificant. All data were analyzed using the Statistical Package forthe Social Sciences program (SPSS for Windows 16.0, SPSS Inc.,USA). Validation of Cox regression analysis and test of assumptionswere confirmed using R language (R version 2.6.1, The R foundationfor Statistical Computing, Austria).

3. Results

3.1. Clinical data

A retrospective analysis of the Department of Pathology recordsat the King Abdullah University Hospital carried out between theyears 2004 and 2007 identified 130 cases of bladder cancer.Complete clinical records were available for 121 cases and thosewere selected for further investigation. The main clinical andhistopathological features are presented in Table 1. In brief, 106patients were males and 15 were females. The median age ofdiagnosis was 63 years with a range of 24–83. Clinical follow-up ofpatients was available for up to 72 months. Therapeuticapproaches included both transurethral resection of the tumor(76 patients) and radical cystectomy (45 patients). Eighty-twopatients experienced multiple recurrences and this was mostevident in patients presented with early stages of the tumor. 93(77%) cases were early stage tumors (pTa and pT1) and 28 (23%)cases were late stage tumors (pT2 and pT3). Furthermore, 54(44.6%) cases were of G1 grade, 32 (26.4%) cases were of G2 grade,and 35 (29%) cases were of G3 grade.

3.2. FGFR3 protein expression

The expression of FGFR3 was investigated by immunohis-tochemistry. FGFR3 expression (both cytoplasmic and membra-nous) was observed in 47 (38.9%) cases of bladder cancer as shownin Fig. 1a and b. In total, positive expression of FGFR3 was observedin 49% of pTa cases, 34% of pT1 cases, 15% of pT2 cases, and 2% ofpT3 cases (Fig. 1c). Furthermore, FGFR3 expression was positive in45% of G1 cases, 26% of G2 cases, and 30% of G3 cases (Fig. 1d). Eventhough a large number of positive FGFR3 expression was seen incases of early stage and low grade tumors, the correlation betweenFGFR3 expression and the stage and grade of the tumors was notconsidered statically significant (Pearson x2 = 1.6, p = 0.2; Pearsonx2 = 0.03, p = 0.9, respectively).

3.3. FGFR3 mutations

DNA was extracted from paraffin-embedded tissues of all 121cases of bladder cancer and exons 7, 10 and 15 of FGFR3b wereamplified and subjected for direct sequencing. Out of the total 121cases analyzed we detected FGFR3 mutations in 39 cases. FGFR3

mutational analysis identified four previously reported mutations,namely R248C, S249C, Y375C, G382R and one novel mutation,G382E. The frequencies of mutations were as follows: S249C(n = 23; 59%), Y375C (n = 7; 18%), R248C (n = 4; 10%), G382R (n = 4;10%) and G382E (n = 1; 3%) as shown in Table 2. The pointmutations identified in this study (R248C and S249C; Y375C andG382R) are located in the extracellular domain (exon 7) andtransmembrane domain (exon 10) of the protein, respectively.Sequencing of exon 15 did not detect the previously reportedmutations (K652Q and K652E) in our group of patients. The



Fig. 1. Immunohistochemical staining of FGFR3 and corresponding frequencies. (a) Negative staining of FGFR3. (b) An example of tumor cells from a pT1G2 case showing

cytoplasmic and/or membranous staining of FGFR3. (c) Frequencies of FGFR3 expression according to the stage. (d) Frequencies of FGFR3 expression according to grade of the

tumor. Positive expression of FGFR3 is predominant in early stage and low grade tumors.


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correlation between mutation status and stage/grade was alsoanalyzed. FGFR3 mutations were found to be more prevalent in lowstage bladder tumors and this association was statisticallysignificant (p < 0.001).

3.4. G382E is a novel mutation in the TM domain of FGFR3

Mutational analysis of exon 10 of the FGFR3 gene resulted in theidentification of three point mutations Y375C, G382R and G382E.Y375C, G382R are reported in patients with various types ofskeletal disorders and also in patients with bladder cancer,whereas the G382E mutation was not previously reported. Fig. 2shows a heterozygous G-to-A transition resulting in a glycine-to-glutamic acid change at codon 382 of exon 10. The absence of this

Table 2FGFR3 mutations identified in 121 cases of bladder cancer.

Nucleotide position Mutation Exon Frequency % of total

mutations

746 C>G S249C 7 23 59

742 C>T R248C 7 4 10

1124 A>G Y375C 10 7 18

1144 G>A G382R 10 4 10

1145 G>A G382E 10 1 3


mutation in 100 healthy individuals suggests that it is not apolymorphism (data not shown). This mutation was present inonly one male patient of 63 years old, at the time of diagnosis, andpresented with a pT1G2 bladder tumor with no other major clinicaldisorders. The patient was subjected to TURT and experiencedmultiple recurrences of the tumor in the years 2003–2005. Thepatient died at the age of 65. Molecular analysis revealed anintense (++) cytoplasmic/membranous staining of FGFR3, whereas;no nuclear p53 staining was seen (data not shown). G382E was theonly mutation identified in this patient in the case of FGFR3 (exons7, 10 and 15). In addition, we did not detect any mutations in exons4, 5, 6, 7 and 8 of p53 (data not shown).

3.5. Survival analysis

We investigated the overall survival (OS) and disease-freesurvival (DFS) of all 121 cases of bladder cancer included in thepresent study. Variables analyzed were tumor stage and grade,FGFR3 expression levels and FGFR3 mutational status. Meansurvival times for patients with early stage tumors (pTa and pT1)and late stage tumors (pT2 and pT3) were 60.6 and 40.6 months,respectively. Patients with early stage tumors had a significantlybetter OS compared to patients with late stage tumors (log rankx2 = 10.7, p = 0.001, Fig. 3a). Mean survival times for patients with



Fig. 2. G382E is a novel FGFR3 mutation in bladder cancer. A heterozygous G1145A

transition resulting in a glycine-to-glutamic acid change at codon 382 of exon 10.


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low grade tumors (G1 and G2) and high grade tumors (G3) were60.3 and 43.5 months, respectively. Patients with low gradetumors had a significantly better OS compared to patients withhigh grade tumors (log rank x2 = 4.7, p = 0.03, Fig. 3b). Mean DFStimes for patients with early stage tumors and late stage tumorswere 14.1 and 28.0 months, respectively. Patients with early stagetumors had a worse DFS compared to patients with late stages (logrank x2 = 7.8, p = 0.005, Fig. 3c). Mean OS times for patients withlow grade tumors and high grade tumors were 14.7 and 23.9months, respectively. Patients with low grade tumors had asignificantly worse DFS compared to patients with high gradetumors (log rank x2 = 3.9, p = 0.05, Fig. 3d). Mean survival times forpatients with positive and negative FGFR3 expression were 53.6and 57.2 months, respectively. We did not see any differences in OSbetween the two groups (log rank x2 = 0.06, p = 0.8, Fig. 4a). Meansurvival times for patients with wild-type and mutant FGFR3 were56.4 and 54.0 months, respectively. Similarly, we did not see anydifferences in OS between the two groups (log rank x2 = 0.1, p = 0.8,Fig. 4b). Mean DFS times for patients with positive and negativeFGFR3 were 15.0 and 18.7 months, respectively. Similarly, we didnot see any differences in DFS between the two groups (log rankx2 = 2.3, p = 0.1, Fig. 4c). Mean DFS times for patients with wild-type and mutant FGFR3 were 18.1 and 15.2 months, respectively.Similarly, we did not see any differences in DFS between the twogroups (log rank x2 = 0.8, p = 0.4, Fig. 4d).


Multivariate Cox proportional hazards model analysis of overallsurvival for the following variables: age, gender, stage and grade oftumor, and FGFR3 (expression and mutation) revealed that age,stage and grade of tumor are independent predictors of OS inpatients with bladder cancer (Table 3). Forward stepwise modelingindicated that age and stage are dependent predictors of OS (�2 loglikelihood = 206.5, overall x2 = 17.5, p = 0.0002). On the otherhand, multivariate analysis of disease-free survival for theaforementioned variables revealed that stage was the onlyindependent predictor of DFS (Table 3). Forward stepwisemodeling indicated that stage and gender are dependentpredictors of DFS (�2 log likelihood = 782.2, overall x2 = 10.5,p = 0.005). Considerable violations of the model’s assumptionswere found in OS for gender and FGFR3 mutation; hence thesevariables were subsequently removed from multivariate OSanalysis (Rho test, Table 3).

4. Discussion

Recent studies have reported that low stage and low gradebladder tumors are associated with FGFR3 mutations andalterations on chromosome 9 whereas high grade invasive tumorsare associated with mutations in p53 and loss of heterozygosity onchromosome 17 [14–16]. Germline mutations in FGFR3 areresponsible for a number of autosomal dominant human skeletaldisorders (reviewed by L’Hote and Knowles [22]). Recent studieshave identified identical mutations in exons 7, 10 and 15 of FGFR3in patients with non-invasive bladder tumors. Several reports haveinvestigated the mutational spectrum and frequency of FGFR3 inpatients with bladder cancer [27–30]. However, few studies haveexamined the correlation between mutation status, level ofexpression of the protein and their association with the clinicaland histopathological variables of patients [28,33].

4.1. Clinical characteristics of patients

All, except for one, were classified as transitional cell carcinoma.Our results indicate that Jordanian patients with bladder cancerare diagnosed with the disease in the late 50s (i.e., median age is61). Pathological grading and staging indicate that pTa and pT1comprised 77% of all cases analyzed whereas pT2 and pT3comprised 23% of the cases. Further, 44.6% of all cases were G1,26.4% were G2 and 29% were G3 bladder tumors.

4.2. FGFR3 expression

Out of 121 patients diagnosed with bladder cancer, 47 (38.9%)cases exhibited high overexpression of the protein. This wasevident by increased cytoplasmic and/or membranous stainingpattern. No significant correlation between FGFR3 expression andstage of the tumor (p = 0.2) or grade of the tumor (p = 0.9) was seen.Similarly, Matsumoto et al. [38] who investigated a similar groupof patients were not able to find such correlation. However, anumber of other studies have reported a significant correlationbetween FGFR3 expression and the stage/grade of the tumor[28,39,40]. This discrepancy could be explained by a number ofreasons including the selection of the patients (low stage/grade vs.all stages/grades), sample size, choice of the antibody used,differences in IHC protocols, or simply reflects the heterogeneousnature of bladder tumors as described by Tomlinson et al. [28].

4.3. FGFR3 mutational analysis

In the present study, the frequency of mutations was as follows:S249C (59%), R248C (10%), Y375C (18%), G382R (10%) and G382E(3%). The K652E and K652Q were not detected in the present study



Fig. 3. Kaplan–Meier curves of the stage and grade of all bladder cancer cases analyzed. (a) Overall survival according to the stage of the tumor. (b) Overall survival according

to the grade of the tumor. (c) Disease-free survival according to the stage of the tumor. (d) Disease-free survival according to the grade of the tumor.

Fig. 4. Kaplan–Meier curves of the FGFR3 expression and mutations of all bladder cancer cases analyzed. (a) Overall survival according to the FGFR3 expression. (b) Overall

survival according to the FGFR3 mutations. (c) Disease-free survival according to the FGFR3 expression. (d) Disease-free survival according to FGFR3 mutations.


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Please cite this article in press as: Bodoor K, et al. FGFR3 mutational status and protein expression in patients with bladder cancer in aJordanian population. Cancer Epidemiology (2010), doi:10.1016/j.canep.2010.05.003


Table 3Multivariate Cox regression model analysis.

Variable Rho test Univariate Multivariate

77-value p-value Hazard ratio (CI 95%) p-value

Overall survival

Age 0.09 0.02 1.06 (1.02–1.11) 0

Gender 0.02a N/A

Stage 0.8 0 0.23 (0.10–0.52) 0

Grade 0.5 0.03

FGFR3 expression 0.2 0.8

FGFR3 mutation 0.03a N/A

Disease-free survival

Age 0.8 0.08

Gender 0.8 0.2 1.85 (1.00–3.40) 0.05

Stage 0.5 0.01 2.2 (1.30–3.73) 0

Grade 1 0.06

FGFR3 expression 0.3 0.1

FGFR3 mutation 0.3 0.4

aA significant Rho test suggests violation of the proportional hazard assumption; hence the variables were removed from the analysis. Graphical methods (Schoenfeld and

Martingale plots) were also used to assess the proportionality and linearity assumptions (data not shown).

Fig. 5. A hypothetical structural model of mutant FGFR3 TM domain created using

Swiss-Pdb Viewer v4.0.1. This model was built on the basis of the structural

homology to ErbB-2/neu (Pdb Id: 2JWA) TM domain. (a) Y375C mutation which

results in the formation of a putative disulfide bridge (shown in yellow) with the

corresponding cysteine at the opposite helix. (b) G382R mutation which results in

the formation of putative hydrogen bonds (shown as red dashes) with the

backbones of Y375 and A376 at the opposite helix. (c) G382E mutation which

results in the formation of putative hydrogen bonds (shown as red dashes) with the

backbone and sidechain of S380 at the opposite helix. The helix of the TM domain is

shown as a green ribbon. Amino acid backbones and sidechains are shown as sticks

(carbon is in white, nitrogen is in blue, oxygen is in pink, and sulfur is in yellow).

(For interpretation of the references to color in this figure legend, the reader is

referred to the web version of the article.)


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and previous reports indicate that mutations in the kinase domainof FGFR3 have very low frequencies [27,28,41]. Overall, thefrequencies of cases presented with mutations according to thestage and grade of the tumor were as follows: pTa (36.5%), pT1(33.3%), pT2 (20.8%) and pT3 (25%); G1 (22%), G2 (41%) and G3(40%). Out of the 39 cases with FGFR3 mutations, 31 of those wereof pTa and pT1, an 80% frequency. The frequencies of FGFR3

mutations reported in this study are higher than those reported fora group of low stage patients with bladder cancer from theNetherlands (67%), UK (58%) and Spain (50%) ([41,28,27],respectively). S249C was the most common mutation identified(59%) which is within the range of frequencies (48–69%) reportedin previous studies [27–29,42,43]. The mutational frequency ofR248C (10%) and Y375C (18%) was also similar to those reported inother studies; (7–17%) and (19–24%), respectively [27,28,42]. Thefrequency of G382R (10%) was much higher than those reported byother groups [27,28]. The relative differences between our resultsand those reported in the literature could be explained by anumber of reasons including the ethnicity of the population and itsassociated lifestyles/risk factors, and the use of direct sequencingvs. single-strand conformation polymorphism (SSCP) followed bysequencing.

Previous studies indicated a strong correlation between FGFR3

mutations and the stage/grade of the tumor [27–29,44]. In thepresent study, we were able to find a significant correlation(p < 0.001) between the stage of the tumor and FGFR3 mutations.However, in contrast to other studies, we could not find anycorrelation between the grade of the tumor and FGFR3 mutations.

Several mechanisms have been suggested to explain howactivating FGFR3 mutations affect its signaling function. In the caseof S249C and R248C introducing cysteines to the extracellulardomain are thought to either increase ligand binding affinity orresult in receptor activation in the absence of ligand binding. Inboth cases, this would facilitate dimerization of the receptorthrough the formation of disulfide bridges, which in turn wouldresult in the phosphorylation of tyrosines in the kinase domain andactivation of downstream signaling pathways. In support of thishypothesis, the expression of FGFR3b–S249C was shown to inducethe transformation of NIH-3T3 cells [20].

According to the structural organization of FGFR3b, amino acids378–398 represent the potential transmembrane (TM) domain ofthe protein. The TM domain plays an active role in FGFR3 signalingpathways as evident by the number of mutations mapped to thisdomain including G372C, S373C, Y375C, G377C, G382R, G384D,and A393E [22]. In the case of Y375C, located in the vicinity of the


TM domain, introducing a cysteine at this position is expected tostabilize the dimer and activate the kinase domain as shown in thehypothetical model in Fig. 5a. The oncogenic ability of thismutation is supported by a study in which a bladder carcinoma cellline expressing FGFR3b–Y375C lost its transforming ability upontreatment with an FGFR kinase inhibitor and/or FGFR3 shRNA[20,45]. Glycine382 is within proximity to the proposed motifinvolved in mediating TM dimerization (i.e., residues Leu379,Val383, Phe386, and Ile389). These amino acids are highlyconserved in the TM domain of receptor tyrosine kinases [46].Substituting glycine for arginine, G382R, is expected to stabilizethe dimer through the formation of putative hydrogen bonds withthe backbones of tyrosine375 and alanine376 as shown in Fig. 5b.This model assumes correct positioning of the helices in the TMdimer so that the kinase domain could be fully activated. Perviouswork by You et al. [47] have shown that this mutation is notexpected to change the dimerization energetics of the FGFR3 TMdomain nor does it destabilizes the dimer.

Our hypothesis is supported by the work of Webster andDonoghue [48] which demonstrates that arginine mediated-hydrogen bonding increased the level of FGFR3 phosphorylationand that of a hybrid neu/FGFR3 receptor. G382E has not beenreported previously. Substituting glycine for glutamic acid at thisposition is also expected to stabilize the dimer through the




G Model


formation of putative hydrogen bonds with the backbone andsidechain of serine380 as shown in Fig. 5c. Support for this comesfrom studies of the oncogenic neu mutant [46,49,50], in which thesubstitution of valine for glutamic acid at position 664 (equivalentto glycine382 of FGFR3) has been shown to activate the kinasedomain of the receptor and enhance its transforming capacity.Furthermore, polarized FTIR and NMR spectroscopy studiesdemonstrated that the sidechain of E664 is protonated andinvolved in the formation of hydrogen bonds with the backboneof G665 in the adjacent helix of the neu dimer [51,52]. The A393Eand the G384D FGFR3 mutations are also expected to stabilize TMdimerization through the formation of putative hydrogen bonds[53]. We suggest that glycine382 of FGFR3 could be substituted bya number of amino acids that have the ability to form hydrogenbonds (i.e., glutamic acid, glutamine, aspartic acid and tyrosine)which results in TM dimer stabilization and constitutive, ligand-independent, activation of the receptor [54]. Mutations affectingthe kinase domain of FGFR3, K652Q, and K652E are thought toresult in ligand-independent activation of the receptor by favoringthe active conformation of regulatory loop of the kinase domain[55]. FGFR3 could also be activated by mutation-independentmechanisms including delayed turnover of activated receptors andsubsequent increase in signaling [56].

Together, we identified 47 (38.9%) cases with overexpression ofFGFR3 and 39 (32.2%) cases with FGFR3 mutations. 27 (69.2%) ofthose had a combined FGFR3 overexpression and mutationswhereas, 12 cases had a mutation in FGFR3 with no signs ofoverexpression of the protein. In bladder cancer, it is conceivablethat the overexpression of FGFR3 could arise through a mechanismthat does not involve activating mutations in the gene [28,40].Similarly, mutations in the gene could not always result inincreased expression of FGFR3 as reported by Matsumoto et al.[38]. We believe that investigating the expression levels andmutational status of FGFR3, in addition to other genes, is required ifwe are to get a better picture of the molecular changes associatedwith early stage/low grade bladder tumors. Furthermore, identi-fying the molecular consequences of FGFR3 mutations is animportant first step in understanding the role of FGFR3 signaling inbladder cancer initiation.

4.4. Survival analysis

Several reports indicate that patients with bladder cancer ofearly stage and low grade tumors usually have a favorableprognosis [29,57–59]. Similarly, our results indicate that patientswith early stage (pTa and pT1) and low grade (G1 and G2) bladdertumors had a better OS when compared to patients with late stage(pT2 and pT3) and high grade (G3) of the tumor (Fig. 3). Patientswith bladder cancer presented with non-invasive tumors arecommonly treated with transurethral resection of bladder,however; the majority of patients will develop recurrences within5 years [9]. When we analyzed our cohort in terms of disease-freesurvival, defined as the first recurrence or death from any cause, wewere able to show that patients with early stage (p = 0.005) andlow grade (p = 0.05) of the disease are more susceptible torecurrences of the tumor when compared to patients with latestage and high grade tumors. We were not able to find significantdifferences in susceptibility to recurrence between cases of earlystage (pTa vs. pT1: p = 0.2) and low grade tumors (G1 vs. G2:p = 0.7). Similar findings were reported by other groups[40,57,58,60].

Further, we were not able to find any significant correlationbetween levels of FGFR3 expression and survival (both overall anddisease-free). In addition, we did not find any correlation betweenFGFR3 mutations and survival (both overall and disease-free). Bothfindings were previously reported [27,40,59,60]. We also used the


multivariate Cox regression model to determine the prognosticvalue of the different variables included in this study (age, gender,stage and grade of the tumor, FGFR3 expression and FGFR3

mutations) in order to determine the ones with favorableprognosis for patients. Our results indicate that combining thestage of the tumor with the age provides a better prognosticindicator of OS whereas, combining the stage of the tumor withgender provides a better prognostic indicator of DFS for patients asshown in Table 3. Unfortunately, the combination of stage of thetumor with gender can be missed by bias of sample size, or the useof conditional stepwise Cox regression analysis. Nevertheless,these results as a whole indicate the role of clinical andpathological characteristics in predicting the outcome of patientswith bladder cancer.

We have to point out that in our cohort of patients we noticed aclear distinction in terms of survival rates when compared tosimilar groups from the United States and Europe. Jordanianpatients diagnosed with pTa and pT1 usually die from the diseasewithin 3 years whereas same stage groups from the US have anaverage 5-year relative survival of 75–82% while in Europe patientshave an average 5-year relative survival of 68% [1].

Trying to explain these differences, patients’ medical recordspoint to a number of reasons including, among others, the highpercentage of smokers in our cohort (more than 50%), the lowsocioeconomic status of the majority of patients and the possibleexposure to a variety of environmental factors (i.e., dyes, textile-related products, exposure to aromatic compounds). Furthermore,patients diagnosed with pTa and pT1 are also presented with othercomplications including diabetes and hypertension which arepresumed to accelerate the progression of the disease. In addition,few patients were noncompliant with the therapeutic regimen.

In conclusion, our work is the first in Jordan to address themolecular changes of FGFR3 in bladder cancer patients and adds tothe relatively small number of studies addressing the prognosticvalue of both protein expression and mutational status of FGFR3.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgments

We are grateful to Dr. Khaled Bodoor and Dr. Waleed Gharaibehfor critical analysis of the manuscript. This work was supported bythe Arab Science and Technology Foundation grant number(BT062290) 41/2007.

References

[1] Garcia M, Jemal A, Ward EM, Center MM, Hao Y, Siegel RL, et al. Global cancerfacts & figures. Atlanta 2007.

[2] Tarawneh M, Nimri O. Cancer incidence in Jordan. Amman: Jordan CancerRegistry, 2007.

[3] Begum G, Dunn JA, Bryan RT, Bathers S, Wallace DM. Socio-economic depri-vation and survival in bladder cancer. BJU Int 2004;94:539–43.

[4] Kirkali Z, Chan T, Manoharan M, Algaba F, Busch C, Cheng L, et al. Bladder cancer:epidemiology, staging and grading, and diagnosis. Urology 2005;66:4–34.

[5] Al-Sukhun S, Hussain M. Molecular biology of transitional cell carcinoma. CritRev Oncol Hematol 2003;47:181–93.

[6] Sobin LH, Wittekind C. TNM classification of malignant tumors. New York:Wiley–Liss, 1997.

[7] Reuter VE. The pathology of bladder cancer. Urology 2006;67:11–7.[8] MacVicar AD. Bladder cancer staging. BJU Int 2000;86(Suppl. 1):111–22.[9] Sanchez-Carbayo M. Recent advances in bladder cancer diagnostics. Clin

Biochem 2004;37:562–71.[10] Simerville JA, Maxted WC, Pahira JJ. Urinalysis: a comprehensive review. Am

Fam Phys 2005;71:1153–62.[11] Cote RJ, Datar RH. Therapeutic approaches to bladder cancer: identifying

targets and mechanisms. Crit Rev Oncol Hematol 2003;46(Suppl. 1):S67–83.[12] Spiess PE, Czerniak B. Dual-track pathway of bladder carcinogenesis: practical

implications. Arch Pathol Lab Med 2006;130:844–52.




G Model


[13] van Rhijn BW, van der Kwast TH, Vis AN, Kirkels WJ, Boeve ER, Jobsis AC, et al.FGFR3 and P53 characterize alternative genetic pathways in the pathogenesisof urothelial cell carcinoma. Cancer Res 2004;64:1911–4.

[14] van Tilborg AA, de Vries A, de Bont M, Groenfeld LE, Zwarthoff EC. The randomdevelopment of LOH on chromosome 9q in superficial bladder cancers. J Pathol2002;198:352–8.

[15] Hirao S, Hirao T, Marsit CJ, Hirao Y, Schned A, Devi-Ashok T, et al. Loss ofheterozygosity on chromosome 9q and p53 alterations in human bladdercancer. Cancer 2005;104:1918–23.

[16] van Oers JM, Adam C, Denzinger S, Stoehr R, Bertz S, Zaak D, et al. Chromosome9 deletions are more frequent than FGFR3 mutations in flat urothelial hyper-plasias of the bladder. Int J Cancer 2006;119:1212–5.

[17] Johnson DE, Williams LT. Structural and functional diversity in the FGFreceptor multigene family. Adv Cancer Res 1993;60:1–41.

[18] Eswarakumar VP, Lax I, Schlessinger J. Cellular signaling by fibroblast growthfactor receptors. Cytokine Growth Factor Rev 2005;16:139–49.

[19] Wuchner C, Hilbert K, Zabel B, Winterpacht A. Human fibroblast growth factorreceptor 3 gene (FGFR3): genomic sequence and primer set information forgene analysis. Hum Genet 1997;100:215–9.

[20] Bernard-Pierrot I, Brams A, Dunois-Larde C, Caillault A, Diez de Medina SG,Cappellen D, et al. Oncogenic properties of the mutated forms of fibroblastgrowth factor receptor 3b. Carcinogenesis 2006;27:740–7.

[21] Hart KC, Robertson SC, Donoghue DJ. Identification of tyrosine residues inconstitutively activated fibroblast growth factor receptor 3 involved in mito-genesis, Stat activation, and phosphatidylinositol 3-kinase activation. Mol BiolCell 2001;12:931–42.

[22] L’Hote CG, Knowles MA. Cell responses to FGFR3 signalling: growth, differen-tiation and apoptosis. Exp Cell Res 2005;304:417–31.

[23] Powers CJ, McLeskey SW, Wellstein A. Fibroblast growth factors, their recep-tors and signaling. Endocr Relat Cancer 2000;7:165–97.

[24] Horton WA, Garofalo S, Lunstrum GP. FGFR3 signaling in achondroplasia: areview. Cells Mater 1998;8:83–7.

[25] d’Avis PY, Robertson SC, Meyer AN, Bardwell WM, Webster MK, Donoghue DJ.Constitutive activation of fibroblast growth factor receptor 3 by mutationsresponsible for the lethal skeletal dysplasia thanatophoric dysplasia type I.Cell Growth Differ 1998;9:71–8.

[26] Vajo Z, Francomano CA, Wilkin DJ. The molecular and genetic basis of fibro-blast growth factor receptor 3 disorders: the achondroplasia family of skeletaldysplasias, Muenke craniosynostosis, and Crouzon syndrome with acanthosisnigricans. Endocr Rev 2000;21:23–39.

[27] Hernandez S, Lopez-Knowles E, Lloreta J, Kogevinas M, Amoros A, Tardon A,et al. Prospective study of FGFR3 mutations as a prognostic factor in non-muscle invasive urothelial bladder carcinomas. J Clin Oncol 2006;24:3664–71.

[28] Tomlinson DC, Baldo O, Harnden P, Knowles MA. FGFR3 protein expressionand its relationship to mutation status and prognostic variables in bladdercancer. J Pathol 2007;213:91–8.

[29] van Oers JM, Zwarthoff EC, Rehman I, Azzouzi AR, Cussenot O, Meuth M, et al.FGFR3 mutations indicate better survival in invasive upper urinary tract andbladder tumours. Eur Urol 2009;55(3):650–8.

[30] Zieger K, Dyrskjot L, Wiuf C, Jensen JL, Andersen CL, Jensen KM, et al. Role ofactivating fibroblast growth factor receptor 3 mutations in the development ofbladder tumors. Clin Cancer Res 2005;11:7709–19.

[31] Ronchetti D, Greco A, Compasso S, Colombo G, Dell’Era P, Otsuki T, et al.Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14):comparative analysis of Y373C, K650E and the novel G384D mutations.Oncogene 2001;20:3553–62.

[32] Cappellen D, De Oliveira C, Ricol D, de Medina S, Bourdin J, Sastre-Garau X,et al. Frequent activating mutations of FGFR3 in human bladder and cervixcarcinomas. Nat Genet 1999;23:18–20.

[33] Lindgren D, Liedberg F, Andersson A, Chebil G, Gudjonsson S, Borg A, et al.Molecular characterization of early-stage bladder carcinomas by expressionprofiles, FGFR3 mutation status, and loss of 9q. Oncogene 2006;25:2685–96.

[34] Bostwick D, Mikuz G. Urothelial papillary (exophytic) neoplasms. VirchowsArch 2002;441:109–16.

[35] Bakkar AA, Wallerand H, Radvanyi F, Lahaye JB, Pissard S, Lecerf L, et al. FGFR3and TP53 gene mutations define two distinct pathways in urothelial cellcarcinoma of the bladder. Cancer Res 2003;63:8108–12.

[36] Fox J, An R. S-PLUS companion to applied regression. Thousand Oaks: Sage, 2002.[37] Landau S, Everitt BS. A handbook of statistical analysis using SPSS. New York:

Chapman & Hall/CRC, 2004.


[38] Matsumoto M, Ohtsuki Y, Ochii K, Seike Y, Iseda N, Sasaki T, et al. Fibroblastgrowth factor receptor 3 protein expression in urothelial carcinoma of theurinary bladder, exhibiting no association with low-grade and/or non-invasivelesions. Oncol Rep 2004;12:967–71.

[39] Gomez-Roman JJ, Saenz P, Molina M, Cuevas Gonzalez J, Escuredo K, Santa CruzS, et al. Fibroblast growth factor receptor 3 is overexpressed in urinary tractcarcinomas and modulates the neoplastic cell growth. Clin Cancer Res2005;11:459–65.

[40] Mhawech-Fauceglia P, Cheney RT, Fischer G, Beck A, Herrmann FR. FGFR3 andp53 protein expressions in patients with pTa and pT1 urothelial bladdercancer. Eur J Surg Oncol 2006;32:231–7.

[41] van Rhijn BW, Vis AN, van der Kwast TH, Kirkels WJ, Radvanyi F, Ooms EC, et al.Molecular grading of urothelial cell carcinoma with fibroblast growth factorreceptor 3 and MIB-1 is superior to pathologic grade for the prediction ofclinical outcome. J Clin Oncol 2003;21:1912–21.

[42] Billerey C, Chopin D, Aubriot-Lorton MH, Ricol D, Gil Diez de Medina S, VanRhijn B, et al. Frequent FGFR3 mutations in papillary non-invasive bladder(pTa) tumors. Am J Pathol 2001;158:1955–9.

[43] van Rhijn BW, van Tilborg AA, Lurkin I, Bonaventure J, de Vries A, Thiery JP,et al. Novel fibroblast growth factor receptor 3 (FGFR3) mutations in bladdercancer previously identified in non-lethal skeletal disorders. Eur J Hum Genet2002;10:819–24.

[44] van Rhijn BW, Lurkin I, Radvanyi F, Kirkels WJ, van der Kwast TH, Zwarthoff EC.The fibroblast growth factor receptor 3 (FGFR3) mutation is a strong indicatorof superficial bladder cancer with low recurrence rate. Cancer Res 2001;61:1265–8.

[45] Tomlinson DC, Hurst CD, Knowles MA. Knockdown by shRNA identifies S249Cmutant FGFR3 as a potential therapeutic target in bladder cancer. Oncogene2007;26:5889–99.

[46] Sternberg MJ, Gullick WJ. A sequence motif in the transmembrane region ofgrowth factor receptors with tyrosine kinase activity mediates dimerization.Protein Eng 1990;3:245–8.

[47] You M, Li E, Hristova K. The achondroplasia mutation does not alter thedimerization energetics of the fibroblast growth factor receptor 3 transmem-brane domain. Biochemistry 2006;45:5551–6.

[48] Webster MK, Donoghue DJ. Constitutive activation of fibroblast growth factorreceptor 3 by the transmembrane domain point mutation found in achondro-plasia. EMBO J 1996;15:520–7.

[49] Sternberg MJ, Gullick WJ. Neu receptor dimerization. Nature 1989;339:587.[50] Li E, Hristova K. Role of receptor tyrosine kinase transmembrane domains in

cell signaling and human pathologies. Biochemistry 2006;45:6241–51.[51] Smith SO, Smith CS, Bormann BJ. Strong hydrogen bonding interactions

involving a buried glutamic acid in the transmembrane sequence of theneu/erbB-2 receptor. Nat Struct Biol 1996;3:252–8.

[52] Smith SO, Smith C, Shekar S, Peersen O, Ziliox M, Aimoto S. Transmembraneinteractions in the activation of the neu receptor tyrosine kinase. Biochemistry2002;41:9321–32.

[53] He L, Hristova K. Pathogenic activation of receptor tyrosine kinases in mam-malian membranes. J Mol Biol 2008;384:1130–42.

[54] Bargmann CI, Weinberg RA. Oncogenic activation of the neu-encoded receptorprotein by point mutation and deletion. EMBO J 1988;7:2043–52.

[55] Gibbs L, Legeai-Mallet L. FGFR3 intracellular mutations induce tyrosine phos-phorylation in the Golgi and defective glycosylation. Biochim Biophys Acta2007;1773:502–12.

[56] Monsonego-Ornan E, Adar R, Feferman T, Segev O, Yayon A. The transmem-brane mutation G380R in fibroblast growth factor receptor 3 uncouplesligand-mediated receptor activation from down-regulation. Mol Cell Biol2000;20:516–22.

[57] Zieger K, Wolf H, Olsen PR, Hojgaard K. Long-term survival of patients withbladder tumours: the significance of risk factors. Br J Urol 1998;82:667–72.

[58] Kurth KH, Denis L, Bouffioux C, Sylvester R, Debruyne FM, Pavone-Macaluso M,et al. Factors affecting recurrence and progression in superficial bladdertumours. Eur J Cancer 1995;31A:1840–6.

[59] Eltze E, Wild PJ, Wulfing C, Zwarthoff EC, Burger M, Stoehr R, et al. Expressionof the endothelin axis in noninvasive and superficially invasive bladdercancer: relation to clinicopathologic and molecular prognostic parameters.Eur Urol 2009;56(5):837–47.

[60] Lamy A, Gobet F, Laurent M, Blanchard F, Varin C, Moulin C, et al. Molecularprofiling of bladder tumors based on the detection of FGFR3 and TP53 muta-tions. J Urol 2006;176:2686–9.



FGFR3 mutational status and protein expression in patients with bladder cancer in a Jordanian population

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