Mutations of BRAF and KRAS2 in the development of Barrett s adenocarcinoma

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1 (2004) 23, & 2004 Nature Publishing Group All rights reserved /04 $ Mutations of BRAF and KRAS2 in the development of Barrett s adenocarcinoma Florian Sommerer 1,6, Michael Vieth 2,6, Annett Markwarth 1, Knut Ro hrich 1, Susanne Vomschlo 1, Andrea May 3, Christian Ell 3, Manfred Stolte 4, Ulrich R Hengge 5, Christian Wittekind 1 and Andrea Tannapfel*,1 1 Institute of Pathology, University of Leipzig, Liebigstrae 26, Leipzig 04103, Germany; 2 Institute of Pathology, Otto-von-Guericke University, Magdeburg 39120, Germany; 3 Clinic of Internal Medicine II, HSK-Clinics Wiesbaden, Wiesbaden 65199, Germany; 4 Institute of Pathology, Klinikum Bayreuth, Bayreuth 95445, Germany; 5 Department of Dermatology, Heinrich-Heine-University, Düsseldorf, Germany Activation of the Raf/MEK/ERK (MAPK) signal transduction cascade byras mutations has been found in a varietyof human cancers. Mutations of BRAF provide an alternative route for activation of this signalling pathway. To determine the role of mutations in BRAF and KRAS2 in the neoplastic progression of Barrett s adenocarcinoma, we analysed both genes for common mutations. After microdissection, DNA of 19 Barrett s adenocarcinomas, 56 Barrett s intraepithelial neoplasias (n ¼ 29 low-grade intraepithelial neoplasia (LGIN) and n ¼ 27 high-grade intraepithelial neoplasia (HGIN)), 30 Barrett s mucosa without neoplasia and normal squamous, as well as gastric epithelium, were analysed for BRAF and KRAS2 mutation. Activating BRAF mutations were identified in 2/19 Barrett s adenocarcinomas (11%) and in 1/27 HGIN (4%). KRAS2 mutations were found in four out of 19 (21%) Barrett s adenocarcinomas examined and in three cases of HGIN (11%). In LGIN as well as in normal gastric or oesophageal mucosa, neither BRAF nor KRAS2 mutations were detected. All lesions with KRAS2 mutations had an intact BRAF gene. The status of mismatch-repair proteins was neither related to BRAF nor KRAS2 mutations. These data indicate that RAS or BRAF mutations are detected in about 32% of all Barrett s adenocarcinomas. We conclude that the disruption of the Raf/MEK/ERK (MAPK) kinase pathwayis a frequent but also earlyevent in the development of Barrett s adenocarcinoma. (2004) 23, ; doi: /sj.onc Keywords: BRAF; KRAS2; barrett; neoplasia Introduction *Correspondence: A Tannapfel; tana@medizin.uni-leipzig.de 6 Florian Sommerer (FS) and Michael Vieth (MV) contributed equally to the study Received 31 July 2003; revised 4 September 2003; accepted 8 September 2003 Barrett s adenocarcinoma arises from Barrett s oesophagus, in which an intestinal-type epithelium (specialized intestinal metaplasia) replaces oesophageal squamous epithelium damaged by gastro-oesophageal reflux disease. The development of cancer in Barrett s oesophagus follows a multistep pathway. Histologically, there is a progression from intestinal metaplasia (Barrett s mucosa) through low- (LGIN) and high-grade intraepithelial neoplasia (HGIN) to adenocarcinoma (Barrett et al., 1996; Spechler, 2002; Tselepis et al., 2002). To date, the exact cellular and molecular mechanisms leading to neoplastic progression in Barrett s epithelium are still not fully understood (Barrett et al., 1999; Dunn et al., 2003). Increasing evidence exists that carcinogenesis must be understood in terms of an accumulation of mutations in regulatory genes, including activation of oncogenes and inactivation or loss of the tumoursuppressor genes (Morales et al., 2002). Since the discovery of the role of RAS oncogenes in tumorigenesis, an increasing focus has been set in research of the signal transduction area (Liu et al., 2003). A key RAS effector pathway involves the kinase cascade RAF/ MEK/ERK (MEK: MAP/ERK kinase; ERK: extracellular signal-related kinase) (Mercer and Pritchard, 2003). In the quest to understand how RAS transmits extracellular growth signals, the MAP kinase (MAPK) pathway has emerged as an important route between membrane-bound RAS and the nucleus (Weber et al., 2001; Leder et al., 2002). Signalling through the MAPK cascade is transduced by GTP loading of RAS leading to the activation of RAF kinase. In mammalian cells, there are three isoforms of RAF: A-RAF, B-RAF, and C-RAF (Liao et al., 2001). Although all three of the raf isoforms share a common function with respect to MEK phosphorylation, studies have shown that these proteins might be differentially activated by oncogenic ras (Peyssonnaux and Eychene, 2001; Smalley, 2003). Recently, mutations of BRAF have been described in about 15% of all human cancers, especially in malignant melanomas, papillary thyroid cancer, as well as ovarian cancer (Davies et al., 2002; Cohen et al., 2003; Dong et al., 2003; Kimura et al., 2003; Singer et al., 2003; Soares et al., 2003). Previous studies of ras mutations in

2 Barrett s adenocarcinoma have reported varying results; mutation rates up to 88% have been described (Arber et al., 2000). In the present study, we analysed the status of the BRAF gene together with KRAS to elucidate a possible role of these genes in the carcinogenesis of Barrett s adenocarcinoma. Materials and methods Patients and tissue samples Between February 2000 and September 2001, 19 patients with well-differentiated Barrett s adenocarcinoma, 56 patients with Barrett s epithelium and intraepithelial neoplasia (n ¼ 29 with LGIN and n ¼ 27 with HGIN), and 30 patients with Barrett s mucosa without neoplasia were included in this retrospective study. The normal squamous cell epithelium as well as gastric mucosa were obtained from five patients. All patients with Barrett s neoplasia received endoscopic mucosal resection. Barrett s mucosa without neoplasia was obtained from biopsies of patients without neoplasia. The present study was in accordance with the ethical standards of the Committee on Human Experimentation of the University of Leipzig. All samples were taken during treatment procedures under therapeutical intent. Each tumour was re-evaluated with regard to typing (WHO, 2000). In all cases, H&E-stained slides were re-examined independently by three experienced GI pathologists (MV, MS, AT) without any knowledge of clinical data. In case of conflicting results of grading intraepithelial neoplasia, microscopical re-evaluation was obtained until concordance of opinion was obtained. DNA samples For each sample, the histopathological lesions of interest were first identified on routinely stained sections. Microdissection was performed as described previously (Figure 1) (Weber et al., 2003). The approximate number of cells was estimated to be at least 1000 per sample for PCR analysis. For DNA extraction, standard methods were used: after incubation with proteinase K at 371C overnight, the tissue was extracted twice in phenol and twice in chloroform, followed by ethanol precipitation. Mutation analysis All pre-pcr tissues were handled in an environment free of PCR products. All samples were coded and the investigator was blinded to all patient clinical details. Deparaffinized tissue was recovered by a 15 min incubation with xylene, followed by centrifugation for 5 min at r.p.m. two times. The tissue pellet was then washed twice in absolute ethanol, followed by two washes in phosphate-buffered saline. The pellet was incubated with 10 pellet volumes (approximately 500 ml) of lysis buffer (0.32 M sucrose, 10 mm Tris-HCl, 1% (v/v) Triton X-100) and 0.2 volumes of proteinase K (final concentration 400 mg/ml) for 2 3 days at 371C. DNA was phenol chloroform extracted and precipitated in ethanol using conventional techniques. The resulting DNA pellet was resuspended in 50 ml TEbuffer, ph 7.4, (10 mm Tris-HCl ph 7.4, 1 mm EDTA ph 8.0). DNA samples were stored at 201C. Using these matched samples (tumour and normal squamous epithelium from the same patient), screening for exon 2 18 BRAF mutations was performed using an ABI PRISM 3100 Genetic Analyser. PCR primers were designed to amplify the exon plus at least 50 bp of the flanking intronic sequence, according to previously published protocols. The primers were adopted from those published in the literature to omit analysing the BRAF pseudogene (Davies et al., 2002; Naoki et al., 2002; Yuen et al., 2002). The resulting traces were analysed to identify samples that produced a shift in peak migration relative to the matched normal control from the same individual or a standard normal control, indicating the presence of a putative sequence variation. The first exon of KRAS2 was amplified by PCR using primers designed to avoid amplification of the KRAS2 pseudogene. The primers used were 5 0 -ATTATAAGG- CCTGCTGAAAATG-ACTGA-3 0 (upstream primer) and ATATGCATATTAAAACAAGATTTACCT-CTA-3 0 (downstream primer), giving a 155 bp product. Amplification was performed using a touchdown PCR technique (Tannapfel et al., 2003; Weber et al., 2003) from 63 to 531C over 10 cycles, followed by 30 cycles at 94, 53, and 721C. PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced using dye primer cycle sequencing and AmpliTaq polymerase FS on an Applied Biosystems DNA sequencer (ABI PRISM 3100; Applied Biosystems-Perkin-Elmer/Cetus, Norwalk). 555 Figure 1 Microdissection of the Barrett s mucosa and adenocarcinoma compartments. Microdissection of the Barrett s mucosa with HGIN (a c) and Barrett s adenocarcinoma (pt1m) (d f). The outlined areas (b, e (green arrows)) were microdissected (c, f) by the laser system (Palm s MicrobeamSystem) and catapulted as described in Materials and methods

3 556 As a positive control for RAS mutation analysis, DNA from colon carcinoma cell lines SW480 (Clontech, Palo Alto, CA, USA) and HCT116 (American Type Culture Collection, ATCC, Rockville, MD, USA) with known KRAS2 mutations at codon 12 (GTT) and codon 13 (GAC), respectively, were used. Negative controls, without DNA, were run as controls for contamination. If a mutation was detected, it was confirmed by amplification and sequencing of a fresh DNA sample using the upstream primer. Any sequences, which proved difficult to read, were reamplified and resequenced. Immunohistochemistry of active MAPK and MLH1/MSH2 The immunohistochemical analysis was performed as described recently (Tannapfel et al., 2003; Weber et al., 2003). In all cases, the tumour and non-neoplastic epithelium were examined. For the immunohistochemical visualization of ERK and active ERK, we used the antibodies anti-p44/42 MAPK and antiphospho-p44/42 MAPK (Anti-ACTIVE R MAPK Ab; Promega R, Madison, WI, USA). Antiphospho-p44/42 MAPK specifically recognizes the dually phosphorylated, active form of MAPK (also known as p44/erk1 and p42/erk2) enzymes. The working dilution was 1 : 200. To analyse the MSI status of our tumours, immunohistochemistry of the mismatch repair proteins hmlh1 and hmsh2 was performed according to previously published standardized protocols (Rüschoff et al., 1998; Stahl, 2000) on formalin-fixed, paraffin-embedded specimens, using the standard avidin biotin method (Tannapfel et al., 2003; Weber et al., 2003) with the following antibodies: MLH1 (Clone G , dilution 1 : 100, BD Pharmingen R,Germany) and MSH2 (Clone GB12, dilution 1 : 10, -Calbiochem R, USA). The positive controls included were sections known to express the investigated antibodies. The negative controls were obtained by omitting the primary antibodies. For the estimation of nuclear hmsh2 and hmsh2 protein expression, the sections were subjected to microwave epitope retrieval. DAKO R TechMate 500 slide-processing equipment was used for all immunohistochemical procedures. BRAF gene alterations Genomic DNA from Barrett s adenocarcinoma and precursor lesions was analysed for BRAF gene mutations. Somatic BRAF mutations were found in 2/19 Barrett s adenocarcinomas (11%) and in 1/27 (4%) of HGIN. All mutations were within exons 11 and 15 (Table 1), with a predominant nucleotide change (Figure 2). The mutation of HGIN was found in exon 11 (G1403T; resulting in the substitution of glycine to valine at 468). The two mutations of Barrett s adenocarcinomas were T1796A (resulting in the substitution of valine 599 by glutamate), a previously documented hotspot. In all three cases, the normal epithelium of the same patient exhibits wild-type BRAF. Samples with LGIN, Barrett s mucosa, and normal gastric and squamous cell epithelium were wildtype as well. We failed to detect a significant correlation between the mutation status of BRAF, grade or other histopathological factors (vascular density, multiplicity, desmoplastic reaction, grade of peritumourous inflammation) in Barrett s neoplasia. KRAS2 status PCR amplification and DNA sequencing enabled the detection of heterozygous mutations in 4/19 Barrett s adenocarcinomas (21%). Two patients had a mutation of codon 12 and two of codon 13. In HGIN, three Results Figure 2 Mutation analysis of the BRAF gene. Electropherograms of the DNA sequences of normal mucosa and Barrett s adenocarcinoma of patient no. 8 Table 1 BRAF and KRAS2 alterations in Barrett s mucosa, intraepithelial neoplasia and adenocarcinoma KRAS2 Nucleotide change No of points BRAF mutation Codon 12 Codon 13 Barrett mucosa 30 0/30 0/30 0/30 Intraepithelial neoplasia (IN) Low grade 29 0/29 0/29 0/29 High grade 27 1/27 2/27 1/27 GGT-GAT GGC-TGC GGT-GCT Adenocarcinoma (pt1m) 19 2/19 2/19 2/19 GGT-GTT GGC-TGC GGT-TGT GGC-GAC

4 KRAS2 mutations were found (11%): two in codon 12, and one in codon 13. No patient had multiple mutations (Table 1). In normal Barrett s mucosa without intraepithelial neoplasia and in the corresponding normal squamous and gastric epithelium, KRAS2 was wildtype in all cases. We failed to observe an association between KRAS2 mutation pattern and histopathological variables. We failed to detect a simultaneous BRAF and RAS mutation within the same tumour specimen. However, the number of mutations identified in either gene is too small to evaluate a genetic interaction. Immunohistochemistry Active, that is, phosphorylated ERK was observed in 14/19 (74%) Barrett s adenocarcinomas with a nearly homogeneous intratumoural expression (Figure 3b, d, f), while unphosphorylated ERK showed baseline levels (Figure 3a, c, e). The active protein was also observed in the 12/27 HGIN (44%) and in 5/29 (17%) samples of LGIN and 5/30 (17%) of Barrett s mucosa without neoplasia. Especially, globlet cells and regenerative immature cells were positive for phosphorylated ERK. In the normal squamous cell epithelium as well as gastric mucosa, unphosphorylated ERK was the predominant detected protein. In general, active ERK immunostaining was more intense in areas with inflammation and also in lesions with intraepithelial neoplasia, as compared to unphosphorylated ERK (Figure 3). To analyse the status of MSI, immunohistological assessment of MLH1 and MSH2 was performed in all Figure 3 Immunostaining of the anti-p44/42 MAPK (ERK) (a, c, e) and antiphospho-p44/42 MAPK MAPK (active ERK) (b, d, f). (a, c, e) Weak perinuclear and slight nuclear ERK staining (red/brown reaction product) in Barrett s mucosa (a), HGIN and Barrett s adenocarcinoma (original magnifications: 20). (b, d, f) Weak to absent antiphospho-erk staining in Barrett s mucosa. Strong staining of anti-erk in HGIN and Barrett s adenocarcinomas (original magnification: 20; insets: 60) cases. The normal staining pattern for MLH1 and MSH2 is nuclear, and nuclei in stromal cells were used as internal positive controls. Some cases showed weak cytoplasmic staining for MLH1, but only the nuclear staining was recorded. We could detect both proteins in all cases of Barrett mucosa as well as LGIN. In HGIN, immunostaining for both proteins occurred in 26 cases. In one patient with HGIN and two patients with Barrett s adenocarcinoma, we failed to detect a specific staining for MLH1 and MSH2. These patients had neither BRAF nor KRAS2 mutations. Discussion In the study presented, we examined the frequency of mutations of BRAF and KRAS2 in Barrett s adenocarcinoma and precursor lesions. Both, BRAF and KRAS2 are members of the RAS-RAF-MEK-ERK-MAP kinase pathway, which mediates cellular response to growth signals (Hakimi et al., 2002). Whereas KRAS2 was examined in a variety of human malignancies (for a review, see Mercer and Pritchard, 2003), this is the first study of the mutational status of BRAF in Barrett s neoplasia. In contrast to melanoma (Davies et al., 2002; Pollock et al., 2003), colon (Rajagopalan et al., 2002), (low-grade serous) ovarian (Singer et al., 2003), and thyroid cancer (Kimura et al., 2003), we detected BRAF mutation only in a small number of Barrett s adenocarcinomas and precursor lesions. The predominant BRAF mutations occurred at nucleotide 1796, leading to a T to A change at exon 15 of the BRAF gene. It has been shown, previously, that this mutation in the activation segment, leading to a conversion of valine 599 to glutamic acid, is a hotspot for BRAF mutation in human cancer (Davies et al., 2002; Yuen et al., 2002). This mutation results in the insertion of a negatively charged residue adjacent to a site of regulatory phosphorylation at T598, which may mimic regulatory phosphorylation, thus leading to constitutive activation of BRAF independent of RAS (Satyamoorthy et al., 2003). According to our results, KRAS2 and BRAF mutations never occur simultaneously in Barrett s adenocarcinoma or HGIN. Since oncogenic KRAS2 activates wild-type BRAF, but mutated BRAF does not require KRAS2 for growth induction (Davies et al., 2002; Hakimi et al., 2002), a simultaneous BRAF and KRAS2 mutation in the same tumour may be redundant. Both KRAS2 and BRAF influence the same effector pathway (promoting cell survival by activating the MAPK pathway); a simultaneous double mutation may not confer a selection advantage for a single tumour cell. To prove this hypothesis, further studies are necessary, especially to look for RAS and BRAF alterations in preneoplastic lesions or early tumour stages. In the present study, we show that most Barrett s adenocarcinomas signal through a constitutively activated Erk pathway. One may speculate that, for Barrett s carcinoma, neither RAS nor BRAF mutations may be a prerequisite for the activation of Ras-RAF- 557

5 558 MEK-Erk pathway. Alternative ways of activation of ERK have recently been detected. The inhibition of Rac1 or Cdc42 signalling led to MAPK Erk activation via a pathway involving PI(3)K, Akt, Raf, and MEK (Aguirre-Ghiso et al., 2003). Interestingly, Cdc42 was implicated in the activation of p38, which, in relation to the activation status of Erk, determined whether a tumour cell was actively proliferating (high Erk/p38 ratio) or was dormant (low Erk/p38 ratio) (Aguirre- Ghiso et al., 2003). Although the prevalence of BRAF mutations in Barrett s adenocarcinomas is relatively low compared to colon, thyroid, low-grade ovarian carcinomas or melanomas, the inhibition of the RAS-RAF- MEK-ERK-MAP kinase pathway may be a new diagnostic or even therapeutic strategy in Barrett s adenocarcinomas (Fitzgerald, 2003). The successful application of inhibitors of activated kinases was reported for STI571, an inhibitor of the BCR-ABL kinase, which is activated in chronic myeloid disorders or gastrointestinal stroma tumours, and may facilitate a new therapeutic approach for Barrett s adenocarcinomas and their precursor lesions. If BRAF inhibitors are found to be effective in these tumours, they should also be considered for (adjuvant) treatment of BRAF-mutant Barrett s adenocarcinomas and precursor lesions, especially HGIN. Acknowledgements This paper was supported by the Bundesministerium fu r Bildung und Forschung (BMB þ F), Interdisciplinary Centre for Clinical Research (IZKF) at the University of Leipzig (01KS9504/1, project D01). References Aguirre-Ghiso JA, Estrada Y, Liu D and Ossowski L. (2003). Cancer Res., 63, Arber N, Shapira I, Ratan J, Stern B, Hibshoosh H, Moshkowitz M, Gammon M, Fabian I and Halpern Z. (2000). Gastroenterology, 118, Barrett MT, Sanchez CA, Galipeau PC, Neshat K, Emond M and Reid BJ. (1996)., 13, Barrett MT, Sanchez CA, Prevo LJ, Wong DJ, Galipeau PC, Paulson TG, Rabinovitch PS and Reid BJ. (1999). Nat. Genet., 22, Cohen Y, Xing M, Mambo E, Guo Z, Wu G, Trink B, Beller U, Westra WH, Ladenson PW and Sidransky D. (2003). J. Natl. Cancer Inst., 95, Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard- Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR and Futreal PA. (2002). Nature, 417, Dong J, Phelps RG, Qiao R, Yao S, Benard O, Ronai Z and Aaronson SA. (2003). Cancer Res., 63, Dunn JR, Risk JM, Langan JE, Marlee D, Ellis A, Campbell F, Watson AJ and Field JK. (2003)., 22, Fitzgerald RC. (2003). Gut, 52, Hakimi MA, Bochar DA, Chenoweth J, Lane WS, Mandel G and Shiekhattar R. (2002). Proc. Natl. Acad. Sci. USA, 99, Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE and Fagin JA. (2003). Cancer Res., 63, Leder A, Lebel M, Zhou F, Fontaine K, Bishop A and Leder P. (2002)., 21, Liao Y, Satoh T, Gao X, Jin TG, Hu CD and Kataoka T. (2001). J. Biol. Chem., 276, Mercer KEand Pritchard CA. (2003). Biochim. Biophys. Acta, 1653, Morales CP, Souza RF and Spechler SJ. (2002). Lancet, 360, Naoki K, Chen TH, Richards WG, Sugarbaker DJ and Meyerson M. (2002). Cancer Res., 62, Peyssonnaux C and Eychene A. (2001). Biol. Cell, 93, Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins C, Moses TY, Hostetter G, Wagner U, Kakareka J, Salem G, Pohida T, Heenan P, Duray P, Kallioniemi O, Hayward NK, Trent JM and Meltzer PS. (2003). Nat. Genet., 33, (Epub 2002 Nov 25). Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B and Velculescu VE. (2002). Nature, 29, 934. Ru schoff J, Dietmaier W, Bocker T, Wallinger S, Kullmann F, Beham A and Hofstadter F. (1998). Pathologe, 19, Satyamoorthy K, Li G, Gerrero MR, Brose MS, Volpe P, Weber BL, Van Belle P, Elder DE and Herlyn M. (2003). Cancer Res., 63, Singer G, Oldt III R, Cohen Y, Wang BG, Sidransky D, Kurman RJ and Shih IeM. (2003). J. Natl. Cancer Inst., 95, Smalley KS. (2003). Int. J. Cancer, 104, Soares P, Trovisco V, Rocha AS, Lima J, Castro P, Preto A, Maximo V, Botelho T, Seruca R and Sobrinho-Simoes M. (2003)., 22, Spechler SJ. (2002). N. Engl. J. Med., 346, Stahl J. (2000). Adv. Anat. Pathol., 7, Tannapfel A, Sommerer F, Benicke M, Katalinic A, Uhlmann D, Witzigmann H, Hauss J and Wittekind C. (2003). Gut, 52, Tselepis C, Perry I, Dawson C, Hardy R, Darnton SJ, McConkey C, Stuart RC, Wright N, Harrison R and Jankowski JA. (2002)., 21, Weber A, Langhanki L, Sommerer F, Markwarth A, Wittekind C and Tannapfel A. (2003)., 22, Weber CK, Slupsky JR, Kalmes HA and Rapp UR. (2001). Cancer Res., 61, WHO (2000). Pathology and Genetics. Hamilton SR, Aaltonen LA (eds) IARC Press: Lyon, pp Yuen ST, Davies H, Chan TL, Ho JW, Bignell GR, Cox C, Stephens P, Edkins S, Tsui WW, Chan AS, Futreal PA, Stratton MR, Wooster R and Leung SY. (2002). Cancer Res., 62,