BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency

(2003) 22, 9192 9196 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency Carla Oliveira 1, Mafalda Pinto 1, Alex Duval 2, Caroline Brennetot 2, Enric Domingo 3, Eloi Espı n 3, Manel Armengol 3, Hiroyuki Yamamoto 4, Richard Hamelin 2, Raquel Seruca 1 and Simo Schwartz Jr*,3 ONCOGENOMICS 1 Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto 4200-465, Portugal; 2 INSERM U434 CEPH, Paris 75010, France; 3 Molecular Pathology Program, Centre d Investigacions en Bioquímica i Biologia Molecular (CIBBIM), Passeig Vall d Hebron 119-129, Barcelona 08035, Spain; 4 First Department of Internal Medicine, Sapporo Medical University, S.1, W.16, Chuo-ku, Sapporo 060-8543, Japan Genes from the RAF family are Ras-regulated kinases involved in growth cellular responses. Recently,a V599E hotspot mutation within the BRAF gene was reported in a high percentage of colorectal tumors and significantly associated to defective mismatch repair (MMR). Additionally,BRAF mutations were described only in K-Rasnegative colon carcinomas,suggesting that BRAF/K-Ras activating mutations might be alternative genetic events in colon cancer. We have addressed to what extent the tumorigenic-positive selection exerted by BRAF mutations seen in colorectal MMR-deficient tumors was also involved in the tumorigenesis of gastric cancer. Accordingly,BRAF mutations were detected in 34% (25/74) of colorectal MMR-deficient tumors and in 5% (7/142) of MMR-proficient colorectal cases (P ¼ 0.0001). All mutations found in the MSI cases corresponded to the previously reported hotspot V599E. Two D593K and a K600E additional mutations were also detected in three MSS cases. However,only one mutation of BRAF was found within 124 MSS gastric tumors and none in 37 MSI gastric tumors,clearly suggesting that BRAF mutations are not involved in gastric tumorigenesis. Nonetheless,a high incidence of mutations of K-Ras was found within the MSI gastric group of tumors (P ¼ 0.0005),suggesting that the activation of K-Ras-dependent pathways contributes to the tumorigenesis of gastric cancers with MMR deficiency. Accordingly,our results show evidences that BRAF mutations characterize colon but not gastric tumors with MMR deficiency and are not involved in the tumorigenesis of gastric cancer of the mutator phenotype pathway. (2003) 22, 9192 9196. doi:10.1038/sj.onc.1207061 Keywords: genomic instability; BRAF; K-Ras; DNA mismatch repair; mutator phenotype; gastrointestinal cancer *Correspondence: S Schwartz; E-mail: sschwartz@vhebron.net Received 15 April 2003; revised 30 July 2003; accepted 31 July 2003 Introduction Approximately 15% of sporadic colorectal and gastric tumors show microsatellite instability due to defects in their DNA mismatch repair (MMR) system. In both tumor types, this molecular phenotype is associated with a particular clinicopathological behavior, and characterized by an underlying genomic instability and a specific profile of target gene mutations (Aaltonen et al., 1993; Ionov et al., 1993; Thibodeau et al., 1993; Perucho, 1996). Moreover, several evidences have shown that stomach and colon tumors with microsatellite instability share the same target genes of the mutator phenotype, with similar mutational incidences (Yamamoto et al., 1997, 1999; Schwartz et al., 1999; Duval and Hamelin, 2002). Recently, a high incidence of activating mutations in BRAF, a gene from the Rasregulated kinase-encoding RAF family, has been found in colorectal tumors and associated to MMR-deficient cases (Davies et al., 2002; Rajagopalan et al., 2002; Yuen et al., 2002). Further, BRAF data showed a mutational hotspot in nucleotide 1796 within exon 15, accounting for a T:A transversion mutation and a valine to glutamic acid substitution, which is the most frequent somatic substitution ever identified in MMR-deficient colon cancers. In addition, these mutations were inversely associated to K-Ras mutations, reinforcing the idea that colorectal tumors with defective MMR progress through the same Ras/Raf/MAPK pathway than MMR-proficient tumors (Rajagopalan et al., 2002). However, it is not clear if colon and gastric tumors of the mutator phenotype share the alterations of the Ras/RAF/MAPK genes found in MMR-deficient colon tumors, nor if K-Ras and BRAF genes are also alternative genetic events in gastric cancer. Here, we show evidences to support that K-Ras but not BRAF mutations contribute to the tumorigenesis of MMR-deficient gastric cancer and therefore that K-Ras and BRAF mutations are not alternative genetic events in gastric cancer. Further, we also conclude that although MMR-deficient gastrointestinal tumors share the same mutational profile of the target genes of the mutator phenotype, they differ concerning

oncogenic mutations in genes from the Ras/RAF/ MAPK pathway. Results and discussion It has been recently shown that the V599E mutation of BRAF, the most common BRAF mutation found within colorectal tumors, has a transforming and oncogenic activity in NIH3T3 cells 138 times over wild-type BRAF (Davies et al., 2002). The presence of BRAF mutations in a high percentage of colorectal MMR-deficient carcinomas suggests its tumorigenic involvement within these tumors, and also its capability to induce tumor cell clonal expansions. Further, the absence of concomitant K-Ras and BRAF mutations in these tumors has suggested that both are alternative genetic events in colorectal tumorigenesis, and also that alterations within the Ras/RAF/MAPK pathway characterize MMR-proficient and -deficient colorectal tumors accordingly (Rajagopalan et al., 2002). In agreement with these findings, BRAF and K-Ras mutations were, respectively, detected in 34% (25/74) and 18% (11/60) of tumors from our collection of MSI colorectal cases (Figures 1 and 2). Further, all BRAF mutations detected corresponded to the V599E hotspot substitution reported by Davies et al. (2002) and were clearly associated to the presence of MMR deficiency (P ¼ 0.0001). In fact, only 5% (7/142) of colorectal MSS tumors showed BRAF mutations, from which four (57%) were V599E, one K600E and two D593K (Figures 1 and 2). As expected, the analysis of K-Ras showed higher mutational frequencies in colorectal MSS 9193 Figure 1 Mutation analysis of BRAF and K-Ras in colon and gastric carcinoma cases. SSCP/HA from BRAF are shown. DGGE analysis of K-Ras is also shown. N, normal tissue counterpart; T, tumor. BRAF mutations and wild-type sequence (normal) are indicated on top and abnormal bands pointed by arrows. (a) A representative SSCP/HA analysis of V599E BRAF mutations within MSI colorectal tumors. The corresponding mutated sequence is also shown at the right side. (b) A representative SSCP/HA analysis of BRAF mutations in MSS colorectal tumors. Two additional D593K and one K600E mutations were detected in the HA analysis and also by automatic sequencing (right). (c) SSCP/HA analysis of BRAF in MSS (left panel) and MSI (right panel) gastric carcinomas. Sequencing analysis of the single V599E BRAF mutation found in an MSS gastric tumor is also shown (right side). (d) Representative DGGE analysis of K-Ras in MSI gastric tumors. HAs corresponding to G12D and G13D mutations were found in several MSI tumor cases (arrows). A normal case is shown on the left. Two G12V mutations were also detected by automatic sequencing. Sequences are also shown. Additional V599E BRAF mutations were also detected in our collection of gastrointestinal tumor cell lines, including three MSI colon cell lines (Co115, RKO and LS411), two MSS colon cell lines (WIDR and HT29) and one MSI gastric cell line (St2957). No mutations were detected in other cell lines from the colon, including nine MSI (LS174 T, LoVo, TC71, HCT15, HCT116, TC7, SW48, HCT8 and KM12) and 19 MSS (GLY, EB, Isreco1, LS513, CBS, FET, V9P, ALA, FRI, LS1034, Isreco2, Colo320, Isreco3, SW480, SW1116, Colo205, SW620, CaCo2 and T84), neither in the MSI gastric cell line SNU1 nor in the MSS gastric cell lines GTL16, SNU16, KatoIII, MKN28, N87, TMK1, MKN1, MKN74, HGT1, AGS, MKN45, GP220, GP202 and L195

9194 Figure 2 Comparative analysis of the mutation frequencies of BRAF and K-Ras in MSS and MSI colon and gastric tumors. The upper part of the figure represents the mutation frequency of BRAF mutations in colon (light bars) and gastric (dark bars) in MSS and MSI tumors. The lower part of the figure represents the mutation frequency of K-Ras mutations in colon (light bars) and gastric (dark bars) in MSS and MSI tumors (38%, 28/73) tumors than in MSI (18%, 11/60) colorectal cases (P ¼ 0.01) (Figure 2). Mutations in both genes were also detected in a variety of colorectal MSS and MSI tumor cell lines (data not shown see legend of Figure 1). In the series of MSI (n ¼ 60) and MSS (n ¼ 73) colon tumors that were analysed for both genes, 50% of MSI and 41% of MSS cases show activating mutations of K-Ras or V599E BRAF mutations or both (Figure 3). These results clearly link the activation of the Ras/RAF/MAPK pathway in colorectal tumorigenesis to the oncogenic activation of K-Ras or BRAF genes (Figure 3). Even though K-Ras and BRAF mutations have been described as alternative events in these tumors (Rajagopalan et al., 2002), we found two concomitant mutations in two Japanese cases of our collection (Figure 3). These results might suggest either a possible synergistic contribution of both genes to the tumorigenic clonal expansion of these cases or a heterogeneous clonal population of cells within these tumors. Further analysis also revealed that the colorectal Japanese cases (n ¼ 22) from our collection showed higher levels of BRAF mutations (11/22, 50%) than tumors from European origin (14/52, 27%) (P ¼ 0.05), even though similar levels of K-Ras mutations were detected in both groups (data not shown). These results might suggest a modulation of the tumorigenic contribution from the Ras/RAF/MAPK pathway activation by the genetic ethnical background. Further, because stomach tumors of the mutator phenotype share similar target genes than colorectal tumors (Yamamoto et al., 1997, 1999; Schwartz et al., 1999; Duval and Hamelin, 2002), we did investigate whether BRAF mutations are also involved in gastric MMR-deficient cancer. However, no significant mutations were found in our collection of stomach primary tumors, including 37 MSI gastric primary tumors (Figures 1 and 2). In fact, we only detected a V599E mutation in an MSS tumor (1/124, 0.8%) within our series. These data led us to the conclusion that BRAF mutations are not involved in gastric tumorigenesis. However, as we have recently reported (Brennetot et al., in press), a high incidence of K-Ras mutations was found in tumors from the MSI group (10/36, 28%) of our gastric tumor collection whereas no mutations were detected in any of the MSS cases (P ¼ 0.0005), clearly suggesting that the activation of K-Ras contributes to the tumorigenesis of gastric cancer of the mutator phenotype, but not to MSS gastric cancer (Figures 2 and 3). Further, these data also support that K-Ras and BRAF mutations are not alternative events in gastric cancer. Further, although MMR-deficient colon and gastric tumors show a similar spectrum of mutations regarding the typical target genes of the mutator phenotype, they differ in the oncogenic mutational incidences found in genes from the Ras/RAF/MAPK pathway (Figure 4), raising the question of whether alternative K-Ras-depending pathways, other than the classical Figure 3 Comparative analysis of the percentage of cases harboring activating K-Ras and/or V599E BRAF mutations in colon and gastric MSS and MSI tumors. The upper bar represents the percentage of MSS and MSI colon carcinoma cases harboring activating K-Ras (light gray) and V599E BRAF (dark gray) or both (black). The lower bar represents the percentage of MSS gastric carcinoma cases harboring the V599E BRAF (dark gray) and the percentage of MSI gastric carcinoma cases harboring activating K-Ras (light gray). Cases without activating K-Ras and/or V599E BRAF, including K600E and D593K BRAF, are represented in white

tumor tissue or from macrodissected areas with at least 50% of tumor cells. Our collection of tumors and cell lines was analysed for microsatellite instability, using the mononucleotide repeats BAT-26 and BAT-25, and also a panel of dinucleotide repeat sequences, as previously described (Hoang et al., 1997; Boland et al., 1998; Oliveira et al., 1998; Yamamoto et al., 2001). We gather for this study 124 tumors and cell lines with MMR deficiency (74 sporadic colon carcinomas, including 22 cases from Japan, and also 37 sporadic gastric carcinomas, 12 colon cancer cell lines and one gastric cancer cell line), and 302 MMR-proficient tumors and cell lines (142 sporadic colon carcinomas, 124 sporadic gastric carcinomas, 21 colon cancer cell lines and 15 gastric cancer cell lines). All tumors and cell lines classified as MSI showed additional mutations in several target genes of the mutator phenotype, including hmsh3, hmsh6, BAX and TGFbRII (not shown). 9195 Figure 4 Mutation frequencies observed at BRAF, K-Ras and at coding repeat sequences in MSI sporadic colorectal (dark gray) and MSI gastric tumors (light gray). *Significant differences found between MSI colon and gastric tumors Ras/RAF/MAPK, might also be activated during gastric MMR-deficient tumorigenesis. Future research will have to answer to this possibility. Materials and methods Tissue samples and microsatellite instability analysis Tumors were obtained from the Hospital of S. Joa o (Porto, Portugal), the Saint-Antoine Hospital (Paris, France), the Sapporo Medical University (Sapporo, Japan) and from the Centre d Investigacions en Bioquimica i Biologia Molecular Vall d Hebron (CIBBIM) (Barcelona, Spain). Sample collection was carried out in accordance with previously established ethical protocols. Collected tumors were immediately frozen in liquid nitrogen for further analysis. Human cancer cell lines were obtained from different sources, including the American Type Culture Collection and the Japanese Cancer Research Resources Bank in Osaka, Japan. Gastric cell lines GP202, GP220 and L195 were established at the IPATIMUP. Some of the cell lines were previously characterized for a number of genetic alterations (Gayet et al., 2001). A family history was obtained for every carcinoma case. None of the patients included in the present study had a family history suggestive of hereditary nonpolyposis colorectal cancer. Hematoxylin- and eosin-stained sections were used to classify the tumors and allowed their macrodissection. High molecular weight DNA was isolated using standard methods from total sections of the tumors whenever tumor cells occupied more than 50% of BRAF and K-Ras mutation screening Mutational analysis of BRAF was performed by singlestranded conformation polymorphism and heteroduplex analysis (SSCP/HA). The fragment encompassing exon 15 was amplified by PCR in 376 carcinoma samples and 48 cell lines. Primer sequences and PCR conditions were based on those reported previously (Davies et al., 2002). Genomic DNA (25 100 ng) was amplified by PCR using the following cycling conditions: 30 s at 941C, 30 s at 601C and 45 s at 721C for 35 cycles. Reaction products were diluted with denaturing buffer (formamide with 0.025% xylene cyanol and 0.025% bromophenol blue) and heated up to 991C for 10 min before being loaded onto 0.8 mutation detection enhancement gels (MDE Flowgen, Rockland, ME, USA), run at 81C for 12 18 h and stained with silver nitrate. Selected bands were recovered from the gels and submitted to PCR reamplification with the original primer sets. Reamplified products were purified and sequenced on an ABI Prism 377 automatic sequencer (Perkin-Elmer, Foster City, CA, USA) using the ABI Prism Dye Terminator Cycle Sequencing Kit (Perkin- Elmer). All detected mutations were confirmed in a second independent PCR. K-Ras mutations were screened in 133 colorectal tumors (73 MSS and 60 MSI) and in 77 gastric carcinomas (42 MSS and 35 MSI). Mutational analysis of the K-Ras gene was performed by denaturing gradient gel electrophoresis (DGGE) as described (Gayet et al., 2001). Acknowledgements This work was supported by Grant FIS 01/1350 from the Spanish Fondo de Investigaciones Sanitarias and Fundac a o para a Cieˆ ncia e a Tecnologia, Portugal (Project: POCTI/ 35374/CBO/2000 and POCTI/CBO/40820/2001). 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