Mutations of the BRAF Gene in Squamous Cell Carcinoma of the Head and Neck

Mutations of the BRAF gene in squamous cell carcinoma of the head and neck

Anette Weber1, Larissa Langhanki2, Florian Sommerer2, Annett Markwarth2, Christian Wittekind2 and Andrea Tannapfel*,2

1Department of Otorhinolaryngology, Head and Neck Surgery, University of Leipzig; Liebigstrabe 18a; 04103 Leipzig, Germany; 2Institute of Pathology, University of Leipzig, Liebigstrabe 26, 04103 Leipzig, Germany

The RAF/MEK/ERK (MAPK) signal transduction cas- Signalling through the MAPK cascade is transduced cade is an important mediator of a number of cellular by GTP loading of RAS leading to the activation of fates including growth, proliferation and survival. The RAF kinase. In mammalian cells, there are three BRAF gene, one of the human isoforms of RAF,is isoforms of RAF: A-RAF, B-RAF and C-RAF (Mikula activated by oncogenic RAS, leading to cooperative et al., 2001; Liao et al., 2001). Although all three of the effects in cells responding to growth factor signals. This RAF isoforms share a common function with respect to study was performed to elucidate a possible function of MEK phosphorylation, studies have shown that these BRAF in squamous cell carcinoma of the head and neck proteins might be differentially activated by oncogenic (HNSCC). Mutations of BRAF and KRAS2 were RAS (Peyssonnaux and Eychene, 2001). In contrast to evaluated in 89 HNSCC and corresponding normal many other tumours of different lineage, oncogenic mucosa by direct DNA sequencing analyses after micro- RAS mutations are rarely found within head and neck dissection. The results obtained were correlated with squamous cell carcinoma (HNSCC) (Xu et al., 1998; histopathological variables. Activating BRAF missense Hao and Rowinsky, 2002; Hoa et al., 2002). Recently, it mutations were identified in 3/89 HNSCC (3%). KRAS2 was described, that wild-type levels of KRAS2 protein mutations were found in five out of 89 (6%) HNSCC expression is a major determinant of proliferation of examined. There were no mutations of KRAS2 and BRAF HNSCC cells and suggest that amplification of non- in non-neoplastic mucosa. We failed to observe a mutated KRAS2 in HNSCC contributes to tumour correlation between BRAF or KRAS2 mutations and growth (Hoa et al., 2002). histopathological factors. Our data indicate that BRAF Recently, mutations of BRAF have been described in gene mutations are relatively rare events in HNSCC. about 15% of all human cancers, especially in malignant Although uncommon, BRAF mutations may identify a melanomas (Davies et al., 2002). In the present study, subset of patients with HNSCC sensitive to targeted we analysed the status of the BRAF gene together with therapy. KRAS2 to elucidate a possible role of these genes in the Oncogene (2003) 22, 4757–4759. doi:10.1038/sj.onc.1206705 carcinogenesis of HNSCC. Genomic DNA from HNSCC and corresponding Keywords: Squamous cell carcinoma of the head and normal epithelium was analysed for BRAF gene muta- neck; BRAF; KRAS2 tions. Somatic BRAF mutations were found in 3/89 HNSCC (3%) (Table 1). All mutations were within exons 11 and 15 (Table 1), with a predominant nucleotide change (Figure 1). Two mutations were Since the discovery of the role of RAS oncogenes in T1796A (resulting in the substitution of valine 599 by tumorigenesis, we have witnessed an explosion of glutamate), a previously documented hot spot. The third research in the signal transduction area (Hagemann mutation was found in exon 11 (G1403T; resulting in the and Rapp, 1999; Fitzgerald et al., 2000). A key RAS substitution of glycine to alanine at 468). In all three effector pathway involves the kinase cascade cases the normal epithelium of the same patient exhibits RAF/MEK/ERK (MEK: MAP/ERK kinase; ERK: wildtype BRAF. All 89 patients had a wild-type BRAF extracellular signal related kinase). In the quest to status in the corresponding normal squamous epithe- understand how RAS transmits extracellular growth lium. signals, the MAP kinase (MAPK) pathway has emerged We failed to detect a significant correlation between as an important route between membrane-bound RAS the mutation status of BRAF, tumour stage or grade or and the nucleus (Weber et al., 2001; Hakimi et al., 2002, other histopathological factors (tumour size, vascular Leder et al., 2002; Gire and Wynford-Thomas, 2000). invasion, multiplicity, desmoplastic reaction). Heterozygous mutations of KRAS2 were found in *Correspondence: A Tannapfel; E-mail: [email protected] 5/89 HNSCC (6%). Four patients had a mutation of Received 27 January 2003; revised 24 March 2003; accepted 24 March codon 12 and one of codon 13. No patient had multiple 2003 mutations (Table 1). In the corresponding normal Mutated BRAF in HNSCC A Weber et al 4758 Table 1 BRAF and KRAS2 mutation in HNSCC Pat. No. Location Stage Grade Nucleotide Amino-acid substitution

BRAF 6 Pharynx III 1 1796 T-A 599 Valine-Glutamate 19 Oropharynx IV 3 1403 G-T 468 Glycine-Alanine 56 Hypopharynx I 2 1796 T-A 599 Valine-Glutamate

KRAS Codon 12: GGT (wild type) 12 Hypopharynx III 2 GGT-GTT Glycine-Valine 28 Oropharynx I 1 GGT-TGT Glycine-Cysteine 51 Floor of mouth II 3 GGT-GCT Glycine-Alanine 84 Hypopharynx IV 2 GGT-TGT Glycine-Cysteine Codon 13: GGC (wild type) 71 Larynx III 2 GGC-TGC Glycine-Cysteine

Figure 1 DNA sequencing was performed on tissue samples of 89 patients with HNSCC after microdissection and DNA extraction as described before (Pindborg et al., 1997; Shanmugaratnam, 1991; Tannapfel et al., 2001). The tumours were located in the oropharynx (33 cases), followed by larynx (25 cases), hypopharynx (18 cases) and floor of mouth (13 cases). According to UICC (2002), staging of HNSCC was as follows: stage l n ¼ 17, stage ll n ¼ 24, stage lll n ¼ 21 and stage lV n ¼ 27 tumours. All patients were informed of special examination of tumour samples which was in accordance with the ethical standards of the Committee on Human Experimentation of the University of Leipzig. PCR primers were designed to amplify the exon plus at least 50bp of flanking intronic sequence according to previously published protocols for BRAF sequencing. The primers were adopted from those published in the literature to omit analysing the BRAF pseudogene (Davies et al., 2002; Yuen et al., 2002; Naoki et al., 2002). The first exon of KRAS2 was amplified by PCR using primers described recently (Tannapfel et al., 2000; Tannapfel and Weber, 2002).

squamous epithelium, KRAS2 was wild type in all cases. cancer (Rajagopalan et al., 2002), we detected BRAF We failed to observe an association between KRAS2 mutation only in a small number of HNSCC. Two out mutation pattern and histopathological variables. of three BRAF mutations occurred at nucleotide 1796, We failed to detect a simultaneous BRAF and KRAS2 leading to a T to A change at exon 15 of the BRAF gene. mutation within the same tumour specimen. However, It has been shown previously that this mutation in the the number of mutations identified in either gene is too activation segment, leading to a conversion of valine 599 small to evaluate a genetic interaction. to glutamic acid, is a hot spot for BRAF mutation in Both KRAS2 and BRAF are members of the RAS- human cancer (Davies et al., 2002; Yuen et al., 2002; RAF-MEK-ERK-MAP kinase pathway, which med- Brase et al., 2002). As a result of this mutation, a iates cellular response to growth signals (Hakimi et al., negatively charged residue is inserted adjacent to a 2002). Whereas was examined in a variety of human regulatory phosphorylation site, mimicking phosphor- malignancies (Garcia et al., 2001), this is the first study ylation and thus leading to BRAF activation indepen- of the mutational status of BRAFand KRAS2 in dent of KRAS2 mutation (Davies et al., 2002). HNSCC in a large series of patients. In its initial report, Consistent with the proposed autonomous nature of Davies et al. (2002) examined 19 HNSCC and failed to this mutation in the RAS-RAF-MEK-ERK-MAP detect specific mutations. In contrast to melanoma kinase signalling pathway, mutation of KRAS2 is not (Davies et al., 2002; Pollock et al., 2003) or colon required and hence not observed in any of the tumours

Oncogene Mutated BRAF in HNSCC A Weber et al 4759 carrying V599E, as it was shown previously by Davies hypothesis, further studies are necessary, especially in et al. (2002). preneoplastic lesions or early tumour stages. According to our results, KRAS2 and BRAF mutations never occur simultaneously in HNSCC. Since oncogenic KRAS2 activates wild-type BRAF, but Abbreviations mutated BRAF does not require KRAS2 for growth HNSCC, head and neck squamous cell carcinoma; MAP, mitogen-activated protein; ERK, extracellular signal-regulated induction (Davies et al., 2002; Hakimi et al., 2002), a kinase. simultaneous BRAF and KRAS2 mutation in the same tumour may be redundant. Both KRAS2 and BRAF Acknowledgements inﬂuence the same effector pathway (promoting cell This paper was supported by the Bundesministerium fu¨ r survival by activating the MAPK pathway); a simulta- Bildung und Forschung (BMB þ F), Interdisciplinary Centre neous ‘double mutation’ may not confer a selection for Clinical Research (IZKF) at the University of Leipzig advantage for a single tumour cell. To prove this (01KS9504/1, Project D01).

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