Letter to the Editor
Analysis of fibroblast growth factor receptor 3 G697C mutation in oral squamous cell carcinomas
Article first published online: 16 OCT 2006
Copyright © 2006 Wiley-Liss, Inc.
International Journal of Cancer
Volume 120, Issue 9, pages 2058–2059, 1 May 2007
How to Cite
Aubertin, J., Tourpin, S., Janot, F., Ahomadegbe, J.-C. and Radvanyi, F. (2007), Analysis of fibroblast growth factor receptor 3 G697C mutation in oral squamous cell carcinomas. Int. J. Cancer, 120: 2058–2059. doi: 10.1002/ijc.22285
- Issue published online: 28 FEB 2007
- Article first published online: 16 OCT 2006
- Manuscript Accepted: 27 JUL 2006
- Manuscript Received: 14 JUN 2006
Zhang et al. recently reported an activating mutation of the receptor tyrosine kinase fibroblast growth factor receptor 3 in 62% of 71 oral squamous cell carcinomas (OSCC).1 More than 300,000 new cases of OSCC are diagnosed annually, worldwide. This aggressive epithelial cancer is associated with severe morbidity and a 5-year survival rate of less than 50%, despite advances in surgical treatment, radiotherapy and chemotherapy.2, 3 Indeed the high rates of morbidity and mortality associated with this devastating disease have not improved in decades. Agents for the molecular targeting of OSCC treatment are urgently needed.2 The discovery of an activating mutation in a receptor tyrosine kinase (RTK) may represent a major advance towards the identification of new therapies, as RTKs have recently been successfully used as targets for cancer treatment.4, 5, 6 FGFRs are involved in cell growth, differentiation and migration and plays important roles in embryogenesis and tissue homeostasis.7, 8 FGFR3 exists in 2 mutually exclusive isoforms: FGFR3b, which is the main form expressed in epithelial cells and FGFR3c, the main form expressed in chondrocytes.9 Activating somatic mutations in exons 7, 10 and 15 of FGFR3 have been reported in several types of cancer: multiple myeloma, bladder and cervical carcinomas. Such mutations are very frequent in bladder carcinoma (about 50% of all cases), and rarer in multiple myeloma and cervical carcinomas.10, 11, 12 No such mutations have been found in OSCC.1, 13
Zhang et al. previously reported that FGFR3b is expressed in cells derived from OSCC.14 In their recent publication, these authors reported that 62% (44 of 71) of OSCC contain the same somatic missense mutation at codon 697 of the FGFR3 gene (697 FGFR3c numbering, 699 FGFR3b numbering): a GGC(Gly) to TGC(Cys) mutation. The G697C amino-acid substitution results in the presence of a cysteine residue in the cytoplasmic kinase domain. Zhang et al. reported that this replacement of a glycine by cysteine might cause the ligand-independent dimerization of FGFR3 molecules. They also reported higher levels of autophosphorylation for the mutated, recombinant expressed kinase domain in vitro and differences in the cellular distribution of the G697C form and wild-type FGFR3 in vivo.1 This is the first time that a G697C amino-acid substitution has been reported in FGFR3. The observations that FGFR3 is expressed in OSCC-derived cell lines, that the G697C amino-acid substitution is present in 62% of OSCC and the activating nature of this mutation strongly suggest that FGFR3 may play an important role in oral tumorigenesis.
We screened a set of OSCC for G697C mutations by 2 different methods—sequencing and a simple enzymatic test—to confirm the existence of this mutation in a different patient population. We analyzed tumors from a series of consecutive, unselected patients undergoing primary OSCC surgery. Pathological findings were reviewed in each case to confirm the diagnosis of OSCC. Tumor materials were snap-frozen in liquid nitrogen. We cut 5 μm sections of frozen tissue, which we stained with hematoxylin and eosin, as a means of checking tissue quality. Only samples with more than 60% tumor cells were selected. DNA was isolated from ten 80 μm sections, by standard procedures. A 183-base pair polymerase chain reaction (PCR) product containing codon 697 was generated using the following primers: 5′-GACAGCCTGACCTCACCT-3′ and 5′-CCAGGGATGCCACTCACA-3′. A single band of the appropriate size was obtained on agarose gel electrophoresis, using the Multiplex PCR Kit and Q solution (QIAGEN, Germany) according to the manufacturer's instructions, with Tm = 58°C. The PCR product was subjected to restriction endonuclease digestion with HpyCH4 V. The recognition site of HpyCH4 V, TGCA, is present twice in the amplified product, and complete digestion therefore generates a 129 bp fragment, a 29 bp fragment and a 25 bp fragment. In the case of the G697C mutation (due to a G2089T mutation, FGFR3c numbering), an additional restriction site is created, resulting in 4 fragments when the PCR product is digested (25 bp, 29 bp, 63 bp and 66 bp). All of the 39 OSCC examined were found to have the wild-type sequence on complete digestion. In no case were additional bands with an approximate size of 66 bp or 63 bp observed. We can, therefore, conclude that these tumors contained only the wild-type sequence (Fig. 1). For confirmation of these results, we selected 16 of the PCR products randomly and subjected them to direct bidirectional sequence analysis. In all cases, the wild-type FGFR3 sequence was obtained. Thus, the FGFR3 G697C amino-acid substitution is probably extremely rare in this population of OSCC patients.
It is difficult to account for the discrepancy between our data and those of Zhang et al. The absence of the FGFR3 G697C mutation in our OSCC patients may reflect differences in the molecular genetics of OSCC from the population studied here (urban north-eastern France) and the unspecified, but probably Japanese population studied by Zhang et al., because of to any number of ethnic, socioeconomic, lifestyle or environmental factors affecting oral tumorigenesis in the 2 patient populations. Nevertheless, our data are consistent with the conclusion that FGFR3 G679C is unlikely to be a common oncogenic mutation in OSCC generally.
Johannes Aubertin, Sophie Tourpin, François Janot, Jean-Charles Ahomadegbe, François Radvanyi.