Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas

Karin Fransén1,4, Maria Klintenäs1, Anna Österström1,2, Jan Dimberg2, Hans-Jürg Monstein3 and Peter Söderkvist1

1 Division of Cell Biology, Floor 9, Department of Biomedicine and Surgery, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden, 2 Department of Natural Science and Biomedicine, University College of Health Sciences, Jönköping, Sweden and 3 Molecular Biology Laboratory, Strategic Development-LMÖ/IBK, University Hospital, Linköping, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1–2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.

Abbreviations: APC, adenomatous polyposis coli; CRC, colorectal cancer; DCC, deleted in colorectal cancer; ERK-1/-2, extracellular regulated kinases; MAPK, mitogen-activated protein kinase; MAPKKs, MAP kinase kinases; MAPKKKs, MAP kinase kinase kinases; MCR, mutation cluster region; MDE, mutation detection enhancement; MEK-1/-2, Map or Erk kinases; MSI, microsatellite instability; SSCA, single strand conformation analysis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Colorectal cancer (CRC) is one of the four most prevalent cancer types in the world and affects almost exclusively elderly people (1). CRC develops through a multi-step process, which is characterized by the inactivation of tumor suppressor genes and activation of protooncogenes by mutations and/or allelic loss or mismatch repair deficiency (2). A model sequence of six to seven crucial genetic alterations from normal intestinal epithelium to the development of metastatic colorectal carcinomas has been suggested (24). Some of the earliest events involve genetic alterations of the APC/ß-catenin signaling pathway and the mutational activation of the protooncogene KRAS (2,3), which are frequently found altered in colorectal polyps. KRAS belongs, together with HRAS and NRAS, to a family of GTPases which when activated induce a cascade of mitogen-activated protein kinases (MAPKs) and transfer signals from the cell membrane via the cytoplasm into the nucleus (5). The ras gene products activate proteins in the Raf family, which consists of the ARAF, BRAF and RAF-1 members (6), and are considered as MAP kinase kinase kinases (MAPKKKs), due to their ability to phosphorylate and activate the MAP kinase kinases (MAPKKs) MEK-1 and MEK-2. The MAPKKs subsequently phosphorylate the MAPKs ERK-1 and ERK-2, which transmits the signal to the nucleus and binds to different transcription factors (6,7). Recently, mutations in the MAPKKK BRAF gene were found in colorectal cancer cell lines, xenografts and in primary tumors (810), which emphasize the importance of the MAPK pathway in the development of human cancers. In the present investigation we have studied mutational activation of four members of the MAPK pathway, the KRAS, ARAF, BRAF and RAF-1 genes, and their interplay in 130 primary CRC tumors of varying stages. In addition, the relation between alterations in the MAPK pathway and other prevalent genetic alterations in CRC, i.e. the p53 and adenomatous polyposis coli (APC)/ß-catenin pathways, microsatellite instability (MSI) and some clinico-pathological data have been evaluated. The identification of specific mutated target genes in tumor tissue is crucial for the understanding of cell growth regulatory pathways in human cancers.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
Tissue collection
The samples were obtained from 130 patients (68 females and 60 males from Southeastern Sweden; clinical information was missing for 2 cases) undergoing surgical resection for primary colorectal adenocarcinomas diagnosed at the Department of Surgery, Ryhov County Hospital, Jönköping, Sweden. One part of the tumor was saved for routine pathological evaluation by an experienced pathologist and stored at the Department of Pathology, Ryhov County Hospital. The other part of the tumor and a corresponding normal blood sample from each patient was immediately frozen at -70°C until the extraction of nucleic acids.

The age of the patients ranged from 25 to 93 years (median age 74 years). The stage of the tumor was described according to Duke's staging system: 17 of the tumors were in Duke's stage A, 58 in B, 42 in C and 7 in D. Five adenomas were also included in the study. 73 tumors were located in the colon (caecum 12; ascending colon 24; transverse colon 4; descending colon 2; sigmoid colon 31) and 55 in the rectum. Seventy-six of the tumors displayed an ulcerative phenotype, 36 were polypoid and 5 showed a mixed ulcerative and polypoid phenotype (13 of the tumors were not classified). Smoking data was available for 51 of the patients: 25 were smokers and 26 non-smokers. The study protocol was approved by the research ethics committee at the Faculty of Health Sciences, Linköping, Sweden (no. 98113).

RNA extraction and RT–PCR
RNA was extracted with a RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the supplier's recommendations from 15–30 mg intestinal tumor tissue from three different patients. RNA was also extracted from the corresponding normal tissue from the same patient, surgically resected 10 cm from the tumor. The RNA was on-column DNase treated with a RNase-free DNase Set (Qiagen) according to the manufacturer's protocol. Equal amounts of total RNA were used for cDNA synthesis, which was performed with Superscript IITM RT (Invitrogen, Stockholm, Sweden) and random primers (Invitrogen) with RNase-free utilities. The samples were stored at -70°C until used.

Aliquots of 1 µl of tumor or corresponding normal cDNA were amplified in 20 µl multiplex PCR reactions containing 75 mM Tris–HCl (pH 9.0), 20 mM (NH4)2SO4, 0.01% Tween 20, 2 mM MgCl2, 200 µM each dGTP, dATP, dTTP and dCTP, 1 µM each ARAF, BRAF or RAF-1 primer and 0.1 µM each housekeeping gene primer (Table I), 0.5 U Taq DNA polymerase (Invitrogen). The PCR temperature profile was 1 cycle of 94°C for 2.5 min, followed by 35 cycles of 94°C for 1 min, annealing temperature for 1 min (Table I) and 1 min at 72°C, with final extension at 72°C for 7 min. The primers targeted to regions with as little homology as possible between the three RAF genes and at least one of the primers in each set should cover exon/exon boundaries. After amplification, the PCR products were separated on 1.5% agarose gels, stained with ethidium bromide and visualized in UV light.


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Table I. Primer sequences and annealing temperatures used for amplification of the human ARAF, BRAF and RAF-1 genes from cDNA and for amplification of the human ARAF, BRAF, RAF-1, KRAS, ß-catenin and p53 genes from genomic DNA

 
DNA isolation
Genomic DNA was isolated from the tumor and the normal blood samples with a Wizard® Genomic DNA Purification Kit according to the supplier's recommendations (Promega, Madison, WI).

PCR amplification of genomic DNA
Aliquots of 20 ng of genomic tumor or corresponding normal genomic DNA were amplified in 20 µl PCR reactions as described above (except for the housekeeping gene). Intronic primers were used for amplification of the ARAF, BRAF, RAF-1, KRAS and p53 genes, covering exons 10 and 13 of the ARAF gene, 11 and 15 of the BRAF gene, 10 and 14 of the RAF-1 gene, 1 and 2 of the KRAS gene and 5–8 of the p53 gene (Table I). Exonic primers were used to amplify the hot-spot codons in exon 3 of the ß-catenin gene and the overlapping PCR fragments of the mutation cluster region (MCR) in exon 15 (codons 832–892 and 1242–1584) of the APC gene. The PCR temperature profile and agarose gel detection was performed as described above.

Mutation analysis
An aliquot of 1 µl of the primary PCR product was reamplified for single strand confirmation analysis (SSCA) in a similar reaction mixture to that described above, with the addition of 1 µCi [32P]dATP per sample (3000 Ci/mmol; Amersham Biosciences, Piscataway, NJ) for 8 cycles. Samples of 2 µl of the labeled PCR products were diluted with 6 µl of 0.2% SDS, 20 mM EDTA and 12 µl of loading buffer (98% formamide, 9.8 mM EDTA, pH 8.0, 0.098% bromphenol blue and xylene cyanol FF), heated to 95°C for 5 min and applied to a non-denaturing 6% polyacrylamide gel containing 10% glycerol and a 0.5x MDETM gel (Mutation Detection Enhancement; BMA Products, Rockland, ME). Electrophoresis was performed at 4–6 W constant power at room temperature for 12–17 h. The gels were transferred to a 3MM filter paper and exposed to X-ray film at -70°C for ~4–17 h with intensifying screens. Shifted fragments were cut out from the gel, eluted in water, reamplified and purified with GFXTM PCR DNA and a Gel Band Purification Kit (Amersham Biosciences) for DNA sequencing analysis. All mutations identified were confirmed by a second mutational analysis of the original DNA sample.

DNA sequencing
Mobility shifted, reamplified and purified samples were sequenced with a ThermoSequenase Radioabeled Termination Cycle Sequencing Kit (USB Corp., Cleveland, OH) and 33P-labeled ddNTPs (Amersham Biosciences) according to the supplier's protocol. The samples were diluted with 4 µl of denaturing loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol FF) and electroporesed in 6% denaturing polyacrylamide gels containing 7 M urea for 1–2 h at 70 W and 50°C. The gels were dried and autoradiography was performed at room temperature for ~48–72 h.

MSI analysis
MSI analysis was performed in a similar fashion to that described in Jansson et al. (13). In brief, 20 ng of tumor and paired normal DNA from each patient was amplified with primers for the microsatellite marker BAT-26 (14), for 35 cycles at 57°C annealing temperature, and labeled with 1 µCi [32P]dATP per sample for 8 cycles as described above. The fragments were mixed with denaturing loading buffer and separated on a sequencing gel (see above), transferred to a filter paper and autoradiography was performed. MSI was visually estimated by two independent observers and MSI was confirmed in a separate experiment.

Statistical analysis
Fishers exact test, the Mann–Whitney U-test and {chi}2 analysis were performed with the SPSS 11.0.0 statistical software package (SPSS Inc., Chicago, IL). The results were considered significant at the P < 0.05 level.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
ARAF, BRAF and RAF-1 expression in colorectal tissue
In this study we performed mRNA expression and mutation analyses of genes in the MAPK pathway. To study whether the three MAPKKKs were expressed in colorectal tissue, we performed RT–PCR of ARAF, BRAF and RAF-1 in normal and tumor tissue from three patients. Since the three genes were found to be expressed in colorectal tissue (data not shown), we selected all of the MAPKKK genes for mutation analysis.

Mutation screening of the MAPK pathway
Mutation analysis was performed for the known hot-spot regions of the BRAF and KRAS genes in 130 primary CRC tumors. The results were correlated with clinical and mutational data of the APC/ß-catenin and p53 pathways (Tables II and III) and MSI (Table II). We also studied the corresponding regions of ARAF and RAF-1, similar to exons 11 and 15 in the BRAF gene (Table IV). SSCA combined with direct DNA sequencing analysis revealed somatic mutations in the MAPK pathway in 50.7% of the primary CRC tumors. The KRAS and BRAF genes exhibited missense mutations in 52/130 (40%) and 15/130 (11.5%) of the tumors, respectively.


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Table II. Mutations in the BRAF gene compared with mutational and clinical data in human primary colorectal cancer

 

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Table III. Mutations in the KRAS gene compared with mutational and clinical data in human primary colorectal cancer

 

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Table IV. Germline polymorphisms in the ARAF and RAF-1 genes in primary colorectal cancer

 
In detail, three different mutation types were found in the BRAF gene in primary CRC tissue. The most frequently found mutation in the hot-spot regions in the BRAF gene was a T -> A mutation in codon 599 (Table II), resulting in a valine (V) to glutamic acid (E) amino acid shift. Interestingly, we also identified one novel and to our knowledge previously undescribed mutation in CRC at codon 600, the AAA -> AAC (K600N) mutation. We also identified one previously detected mutation (10) in codon 593, the GAT -> GGT (D593G) mutation (Figure 1), which was found in an adenoma of the sigmoid colon. All of the BRAF mutations found in the present study were somatic and therefore most likely contributors to tumor progression, since paired normal blood DNA from the patient displayed the wild-type sequence (Figure 1). Earlier reports (810) have found a low frequency of mutations in exon 11 of the BRAF gene in CRC. However, in the present study no genetic alterations could be identified in exon 11.



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Fig. 1. Mutations in the BRAF gene in human primary colorectal cancer tumors showing the three samples with different mutation types. Arrows indicate the mutations. T, tumor; N, normal. (A) Single strand conformation analysis (SSCA). (B) Direct sequencing.

 
Mutation analysis of the ARAF and RAF-1 genes revealed only rare germline polymorphisms; one tumor displayed a silent exonic genetic variant and five additional germline intronic polymorphisms were found (Table IV).

As expected, a high frequency of mutations was found in the KRAS gene (Table III). The majority of the mutations were located in exon 1 and only one mutation was found in exon 2. Codon 12 harbored the highest number of genetic alterations with a frequency of 43/52 (83%). Codons 13 and 61 displayed mutations in 8/52 (15%) and 1/52 (2%), respectively. The most frequent nucleotide substitution was a G -> T transversion that changed glycine to valine and occurred in 14/43 (32%) of the KRAS mutated tumors.

BRAF/KRAS mutations and MSI
In total, 12/130 (9%) of all the tumors were microsatellite unstable for the BAT-26 marker. Six of 15 tumors (40%) with BRAF mutations were microsatellite unstable, compared with six of 115 (5%) tumors without BRAF mutations displaying MSI (P < 0.001, Pearson {chi}2 test). All of the six microsatellite unstable tumors with BRAF mutations displayed the V599E mutation and no MSI was found in tumors with BRAF mutations in the other codons.

Consequently, a significantly low incidence of MSI in KRAS mutated tumors was also observed (P = 0.027, Fisher's exact test) and in only one single case out of 52 KRAS mutations was MSI found. The KRAS mutation in this microsatellite unstable tumor was found in codon 12, yielding a G -> D amino acid shift.

BRAF/KRAS mutations and correlation to clinical data
The BRAF mutations were also subjected to statistical analysis with respect to available clinical data for CRC tumors. The majority of the BRAF mutated tumors were located in the colon (87%) rather than the rectum (13%, P = 0.014, Fisher's exact test). However, no such correlation between tumor localization and KRAS mutations was evident (P = 0.45, Pearson {chi}2). Eight BRAF mutated tumors arose from the right side of the colon and three from the left side, but no significant difference could be found (P = 0.137, Pearson {chi}2). Previously, an association was reported between mutations in codon 12 of the KRAS gene and polypoid tumor growth in CRC (15), but no such correlation could be observed in our study. Furthermore, no correlation with other clinico-pathological features could be found between mutations in KRAS or BRAF and age, gender, Duke's stage or smoking data (Mann–Whitney U-test or {chi}2 test).

Mutation screening of the APC, ß-catenin and p53 genes
Mutation screening of the APC and ß-catenin genes revealed mutations in 38/130 (29%) and 1/130 (0.8%) CRC tumors, respectively. The majority of the APC mutations were deletions or insertions yielding a truncated protein. Mutations in the p53 gene were found in 54/130 (42%) of the tumors and the majority of the mutations were missense mutations. Detailed results from the mutation screening of the APC, ß-catenin and p53 genes are available upon request.

Correlation between genetic alterations
A negative correlation between mutations in two cancer genes may reflect a similar mechanism for tumor-specific activation of that pathway. In agreement with earlier studies (8,9), statistical analysis showed that mutations in the KRAS and BRAF genes were mutually exclusive and in no case in the present study could such mutations be observed simultaneously.

Furthermore, we analyzed the BRAF/KRAS mutation data in relation to mutations in the p53 and APC/ß-catenin pathways. Ten of fifteen (60%) of the tumors with BRAF mutations did not display mutations in any of the investigated hot-spot regions of the APC/ß-catenin or p53 genes. The corresponding number of tumors with KRAS mutations without mutations in these pathways was 20/52 (38.5%). In total, 29/130 (22.3%) tumors did not display mutations in either of the genes investigated in the MAPK, APC/ß-catenin or p53 pathways, which suggests tumorigenic activation through other mechanisms or genes than those studied in the present investigation. No significant patterns of KRAS and/or BRAF mutations related to genetic alterations in the APC/ß-catenin and p53 genes were evident (Fisher's exact test and {chi}2 test). The APC/ß-catenin/p53 mutational data were also tested for correlations with each other, but no significant results were found, which indicates more complex regulatory mechanisms in the large network of genetic alterations in these pathways.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
The mapping of mutation patterns in tumor suppressor genes and oncogenes in human tumors is a powerful instrument to determine important regulatory pathways in the development of tumors in vivo. In this study we have investigated mutational activation of the ARAF, BRAF, RAF-1 and KRAS genes of the MAPK pathway in relation to certain clinical data and mutations in major CRC disease genes in 130 primary CRC tumors. Activating mutations in the MAPK pathway were found in approximately half of the CRC tumors investigated. Recent studies have identified genetic alterations in the kinase domain of the BRAF gene in human CRC (810). In the present study BRAF gene mutations were found in 15/130 (11.5%) primary CRC, which is consistent with Davies et al. (8) and Rajagopalan et al. (9). However, only 5% of the tumors in the study performed by Yuen et al. (10) displayed mutations in the BRAF gene, although these authors used the same method as in a previous study (8). A statistical evaluation reveals a significant difference (P < 0.028, {chi}2 test) between the study made by Yuen et al. (10) and the present study. This may indicate different population-specific sensitivities for BRAF mutations or different environmental exposures, but such a hypothesis will obviously have to be confirmed by future studies. Only rare germline polymorphisms were found in the ARAF and RAF-1 genes. The majority of such polymorphisms were intronic, which indicates a less important or no role for these mutations in the development of CRC.

In the present study all of the mutations found were located in exon 15 of the BRAF gene, between codons 593 and 600. All of the mutations found in the BRAF gene were somatic and were not found in corresponding normal leukocyte DNA, which supports an active role for these BRAF mutations in tumor progression. In good agreement with Rajagopalan et al. (9) and Yuen et al. (10), 83% of the mutations were the T -> A transversion found in codon 599, which indicates a crucial role for this amino acid in BRAF gene activation. Several of the mutations identified by Davies et al. (8) displayed an increased kinase activity in vitro and the V599E mutated variant showed a 10-fold increased activity compared with wild-type BRAF activity in COS cells (8). The amino acid valine has been conserved throughout evolution and is located close to the conserved T598 and S601, which are required for the kinase activity of BRAF (16). These observations might be indicative of the importance of this amino acid. We also found one novel and one previously identified (10) mutation in exon 15. Codon 600 has earlier been reported to display the K600E mutation (9). In the present study we found K600N and D593G mutations in the BRAF gene, which both are located in an evolutionarily conserved region of the RAF genes (8).

Furthermore, mutations in BRAF exon 11 were earlier identified in only 1/33 and 3/333 CRC tumors, xenografts or cell lines (8,9). Previously, Yuen et al. (10) found 2/215 exon 11 mutants in colorectal tumors. In all cancer types in total, only a limited frequency of exon 11 mutations have been reported so far in human tumors (810,17,18), targeting either the phosphorylation sites for Akt or the glycine-rich domain, responsible for orientation of the ATP molecule (8). Exon 11 mutations therefore seem to be less frequent than exon 15 mutations, which may indicate a different activating mechanism for mutations in exon 11, engaging a different intracellular signaling pathway.

Mutations in the KRAS gene were found in 40% of the tumors in codons 12, 13 and 61, which are responsible for the transforming potential of the KRAS oncogene (19), which is consistent with earlier reports (20,21). The ras gene products are located upstream of the three RAF genes in the MAPK signaling cascade and binds to the conserved region I in the BRAF and RAF-1 gene products (22). After activation of the BRAF and RAF-1 gene products, the conserved regions II and III are needed for interaction with the downstream MEK-1 and MEK-2 proteins (22). BRAF is the strongest activator of MEK-1 and MEK-2 compared with RAF-1 (23). The role of ARAF in activating MEK-1 and MEK-2 seems to be of limited importance (24), which is also supported by the present study, in which mutational activation of ARAF could not be seen. In agreement with Davies et al. (8) and Rajagopalan et al. (9), none of the KRAS mutated tumors contained mutations in the BRAF gene or vice versa. Papin et al. (22) showed that KRAS and BRAF are located in the same regulatory pathway and that a mutation in either gene gives a selective proliferative advantage.

We also found a special preference for BRAF mutations in the colonic part compared with rectal tumors. Interestingly, an earlier study (20) found more KRAS mutations in rectal compared with colonic tumors. However, this distribution pattern could not be confirmed in our study. Yuen et al. (10) found a higher preference for BRAF mutations in the earlier Duke's tumor stages. However, this trend is neither supported by us nor by the study made by Rajagopalan et al. (9). Yuen et al. (10) also found a slight, but non-significant, preference for BRAF mutated tumors from the left colon, but we could see an opposite, but non-significant, pattern in our study.

In the present study we also found a significant association between BRAF mutations and MSI analyzed using the BAT-26 marker, which has been shown to detect nearly 100% of microsatellite unstable tumors (14,25). Our results therefore support the study made by Rajagopalan et al. (9), who also observed a correlation between BRAF mutations and MSI. However, Yuen et al. (10) could not confirm these results. A significantly lower incidence of MSI in KRAS mutated tumors was consequently found in the present study, which was earlier also shown by Jass et al. (26). These differences between the present study and the investigation made by Yuen et al. (10) may therefore depend on different lifestyle factors between Asian and Caucasian populations, such as diet, alcohol consumption and smoking.

We also tried to correlate mutations in the MAPK pathway with other genetic alterations in CRC, p53, APC and ß-catenin mutations, but no significant correlations could be found. However, the frequency of APC and ß-catenin mutations was lower than expected from other reports (27,28). This may partly be due to the analysis strategy, where the MCR of the APC gene and the GSK3ß phosphorylation sites of ß-catenin only were screened for mutations. However, according to the APC mutation database, this region covers the majority of APC mutations found in sporadic CRC. Twenty-two percent of the tumors did not reveal any mutation in the genes included in this study, which suggests that other genes of the studied pathways or other pathways and genes than those indicated in the Kinzler–Vogelstein model (2) of CRC may be involved.

Collectively, these results therefore imply increased importance of the MAPK pathway in CRC development, especially via mutational activation of the KRAS and BRAF genes.


    Note added in proof
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 
While the present study was being considered for publication, a study by Wang et al. ‘BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair’, was published (29).


    Notes
 
4 To whom correspondence should be addressed. Email: karfr{at}ibk.liu.se Back


    Acknowledgments
 
With the greatest appreciation we kindly thank Dr Anders Hugander (County Hospital Ryhov, Jönköping) for collection of the tumor material. We also thank Anita Öst and Annette Molbaek for their excellent assistance with partial mutation analysis of the p53 gene, Dr Mats Fredriksson for statistical consultation and Dr Jon Jonasson for critical reading of the manuscript. This study was supported by grants from the National Swedish Cancer Society, the Regional Cancer Society of Östergötland and via the Graduate School in Biomedical Research in Linköping by the Foundation of Strategical Research.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Note added in proof
 References
 

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Received August 20, 2003; revised November 19, 2003; accepted November 30, 2003.