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Oncogene (2001) 20, 4416 ± 4418 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc
Frequency of ®broblast growth factor receptor 3 mutations in sporadic tumours Kathryn Sibley1, Peter Stern2 and Margaret A Knowles*,1 1
ICRF Clinical Centre, St. James's University Hospital, Leeds. LS9 7TF, UK; 2CRC Immunology Group, Paterson Institute for Cancer Research, Manchester, M20 4BX, UK
Mutations in FGFR3 have been identi®ed in several tumour types including bladder carcinoma, cervical carcinoma, and multiple myeloma. In bladder carcinoma, we recently identi®ed FGFR3 mutations in 41% of tumours, making this the most frequently mutated putative oncogene identi®ed in bladder cancer to date. We have now investigated the frequency of FGFR3 mutation in a panel of 125 tumours and 13 cell lines from various other organs. We analysed the mutation hotspots in exons 7, 10 and 15 by direct DNA sequencing, and found one mutation in exon 7 (S249C) in 1/28 (3.5%) cervical tumours. Mutations were not detected in stomach, rectum, colon, prostate, ovarian, breast, brain, or renal tumours, nor were they found in any of the cell lines included in this study. We conclude that FGFR3 is commonly mutated in bladder carcinoma and only rarely in cervical carcinoma. Several tumour types appear not to possess any mutations in FGFR3, suggesting that these mutations are important only in the development of certain types of tumour. Oncogene (2001) 20, 4416 ± 4418. Keywords: FGFR3; mutation; tumour
Germline mutations in FGFR3 are known to be the cause of several autosomal dominant skeletal dysplasias such as achondroplasia (ACH), thanatophoric dysplasia (TD) and hypochondroplasia (HCH). The mutations are invariably the result of a single base change and may arise in the extracellular ligand binding domain, the transmembrane domain, or the intracellular tyrosine kinase domain of the protein (Figure 1). All known mutations are believed to result in ligand-independent activation of the receptor (Webster and Donoghue, 1996, 1997). The severity of the skeletal disorder is dictated by the nature and position of the amino acid substitution with approximately 40% of reported mutations being lethal (PassosBueno et al., 1999). Recently, several of the lethal germline FGFR3 mutations were identi®ed at high
*Correspondence: MA Knowles; E-mail:
[email protected] Received 18 December 2000; revised 12 April 2001; accepted 12 April 2001
frequency in somatic DNA from bladder carcinoma (Capellen et al., 1999; Sibley et al., 2001) and cervical carcinoma (Cappellen et al., 1999) and at a lower frequency in multiple myeloma (Chesi et al., 1997, 1998; Richelda et al., 1997). The mutations found in sporadic tumours are located in only three of the seventeen exons which contribute to the coding sequence, namely exon 7, exon 10, and exon 15 (Table 1). This report presents a mutation analysis of these three mutation hotspots in a panel of tumours and cell lines from various organs. The panel of 125 tumours comprised 28 cervical, three stomach, ®ve rectal, 14 colon, 10 prostate, six ovarian, six breast, 27 renal cell carcinomas and 26 brain tumours (®ve glioblastoma, eight meningioma, 13 astrocytoma) (Table 2). A single base change, C to G was detected in exon 7 in 1/28 (3.6%) cervical tumours which results in the S249C mutation (Figure 2) previously reported in tumours and skeletal dysplasias. Mutations in the two adjacent amino acids resulting in R248C, known to occur in tumours, and P250R, known to occur in skeletal dysplasia (Passos-Bueno et al., 1999) were not observed. The frequency of FGFR3 mutation in cervical tumours has previously been reported to be 25% (three of 12 tumours examined) (Cappellen et al., 1999), whereas our study and two recent studies of cervical tumours (Yee et al., 2000; Wu et al., 2000) suggest that the frequency is much lower. To date, S249C is the only mutation found in cervical tumours. However, given that S248C, G372C, Y375C and K650M/E/Q arise much less frequently than S249C, it is likely that a much larger panel of tumours is required to establish whether other mutations exist in cervical carcinoma. The panel of cell lines which included four Ewing's sarcoma, one neuroblastoma, three prostate, one breast, one CML, one glioblastoma, one osteosarcoma and one transformed keratinocyte cell line, did not have mutations in FGFR3 exons 7, 10 or 15 (Table 3) whereas we previously found that 4/15 (27%) of bladder cancer cell lines possessed a mutation in FGFR3 exon 7 or exon 15 (Sibley et al., 2001). The ®nding of no mutations in FGFR3 in a panel of 26 brain tumours and 27 renal cell carcinomas, suggests that FGFR3 mutations do not contribute signi®cantly to malignancy in these tissues. We examined smaller numbers of stomach, rectum, colon,
Frequency of FGFR3 mutations in sporadic tumours K Sibley et al
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Figure 1 Coding region structure and domain organization in epithelial FGFR3. IgII ± IgIII; immunoglobulin-like domains, AB; acid box, TM; transmembrane domain, KI ± KII; kinase domains. Arrowheads denote the position of the most common mutations in exon 7, exon 10 and exon 15. Intronic primers were used to amplify exon sequence from genomic DNA (Wuchner et al., 1997). PCR reactions were carried out under standard conditions with the addition of 10% DMSO (Sigma, Dorset, UK) to all reactions. Normal human genomic DNA and no template were included as controls in all experiments. The cycle times were as follows: 10 min at 958C followed by 45 cycles of 958C for 30 s, 55 ± 658C for 30 s, 728C for 40 s and one cycle at 728C for 5 min. PCR products were cleaned using the QIAquick PCR puri®cation kit (Qiagen) according to manufacturers instructions and eluted in 50 ml ddH2O. 5 ml of clean PCR product was used in the sequencing reaction (Big Dye Terminator kit, PE Biosystems, Warrington, UK) which contained either the forward or reverse primer from the initial PCR reaction. Base pair changes were detected using sequence analysis software (PE Biosystems) and con®rmed in three independent sequencing reactions
Table 1 Summary of FGFR3 mutations in tumours Exon
Mutationa
Bladder carcinoma Bladder carcinoma and cervical carcinoma
7 7
R248C S249C
Bladder carcinoma
10
Bladder carcinoma and multiple myeloma
10
Bladder carcinoma and multiple myeloma
15
Multiple myeloma Bladder cancer
15 15
Tumour type
Reference
Capellen et al. (1999) Capellen et al. (1999) Wu et al. (2000) Sibley et al. (2000) G372G Capellen et al. (1999) Sibley et al. (2000) Y375C/Y373C Richelda et al. (1997) Chesi et al. (1997) Sibley et al. (2000) K652E/K650E Capellen et al. (1999) Sibley et al. (2000) Chesi et al. (1998) K652M/K650M Chesi et al. (1998) K652Q Sibley et al. (2000)
a
Amino acid numbering diers after exon 7 due to additional two amino acids in the FGFR3 isoform (FGFR3-IIIb) found in carcinoma samples
Table 2 Mutation analysis of FGFR3 in tumours Tumour type Cervical carcinoma Stomach Rectum Colon Prostate Ovarian Breast adenocarcinoma Brain Renal cell carcinoma
Number examined
FGFR3 mutation rate
28 3 5 14 10 6 6 26 27
1/28 (3.6%) (S249C) 0/3 0/5 0/14 0/10 0/6 0/6 0/26 0/27
Figure 2 Cervical tumour 5 (heterozygote) has a G to C transversion shown here in the reverse strand which alters the sequence of codon 249 from TCC (Ser) to TGC (Cys). Cervical tumour DNA was extracted from 10 mm sections of paranembedded tumours by proteinase K digestion as described (Brady et al., 1999). All other tumour samples obtained at surgery were either used fresh or snap frozen and stored at 7808C before use. DNA was extracted from tissue fragments using proteinase K digestion and phenol : chloroform extraction as described (Proctor et al., 1991). The three mutation hotspots in FGFR3 were also analysed in 13 cell lines: four Ewing's sarcoma cell lines, SK-ES-1 (Bloom, 1972), TTC 446, RD-ES and TC-32; one neuroblastoma cell line, IMR-32 (Tumilowicz et al., 1970); three prostate cell lines, DU 145 (Stone et al., 1978), PC-3 (Kaighn et al., 1979) and LNCaP (Horoszewicz et al., 1983); one breast adenocarcinoma cell line, SK-BR-3; one CML cell line, K652 (Lozzio and Lozzio, 1975); one glioblastoma cell line, T98G (Stein, 1979); one osteosarcoma cell line, HOS (Jones et al., 1975); and one transformed human keratinocyte cell line, SVK14 (TaylorPapadimitriou et al., 1982). All cell lines were grown under standard conditions. Genomic DNA was extracted using a DNeasy kit (Qiagen, Crawley, West Sussex, UK)
Table 3 Mutation analysis of FGFR3 in human tumour cell lines Name
Type
SK-ES-1 TTC 466 RD-ES TC-32 1MR-32 DU145 PC3 LNCaP SK-BR-3 K562
Ewing's sarcoma Ewing's sarcoma Ewing's sarcoma Ewing's sarcoma neuroblatoma prostate prostate prostate breast adenocarcinoma chronic myelogenous leukaemia glioblastoma osteosarcoma SV40 transformed keratinocyte
T98G HOS SV-K14
FGFR3 mutation status no no no no no no no no no no
mutations mutations mutations mutations mutations mutations mutations mutations mutations mutations
no no no no
mutations mutations mutations mutations
prostate, ovarian and breast tumours. These also lacked mutations in the known FGFR3 hotspots, again implying that fgfr3 is not involved at high frequency in tumour development or progression in these tissues. Our study therefore con®rms that bladder carcinomas have the highest frequency of mutations in FGFR3 Oncogene
Frequency of FGFR3 mutations in sporadic tumours K Sibley et al
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(Sibley et al., 2001) and that fgfr3 is likely to be involved in bladder tumorigenesis. It will be of great interest to examine the impact of these mutations on urothelial cell phenotype. Acknowledgements We thank the following for the generous gift of tumour DNA samples: Dr Hiroyuki Nishiyama for renal cell
carcinomas and Dr Pat Harnden for brain tumours. We also thank Dr Sue Burchill for DNA from the Ewing's sarcoma and neuroblastoma cell lines and Claire Brady for preparing DNA from cervical tumours. This work was supported by The Imperial Cancer Research Fund (K Sibley, MA Knowles) and the Cancer Research Campaign (P Stern).
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