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Journal of Clinical Oncology, Vol 22, No 22 (November 15), 2004: pp. 4584-4594 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.02.154 Colorectal Cancer With Mutation in BRAF, KRAS, and Wild-Type With Respect to Both Oncogenes Showing Different Patterns of DNA MethylationFrom the Departments of Gastroenterological Surgery and Surgical Oncology and Pathological Research, Okayama University Graduate School of Medicine and Dentistry, Okayama; Department of Statistics, Radiation Effects Research Foundation, Hiroshima, Japan; Conjoint Gastroenterology Laboratory, Royal Brisbane and Women's Hospital Research Foundation and Queensland Institute of Medical Research, Brisbane, Australia; and Department of Pathology, McGill University, Montreal, Quebec, Canada Address reprint requests to Nagahide Matsubara, MD, Department of Gastroenterological Surgery and Surgical Oncology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan; e-mail: nagamb{at}cc.okayama-u.ac.jp
PURPOSE: BRAF mutations are common in sporadic colorectal cancers (CRCs) with a DNA mismatch repair (MMR) deficiency that results from promoter methylation of hMLH1, whereas KRAS mutations are common in MMR proficient CRCs associated with promoter methylation of MGMT. The aim of this study was to further investigate the link between genetic alterations in the RAS/RAF/ERK pathway and an underlying epigenetic disorder. PATIENTS AND METHODS: Activating mutations of BRAF and KRAS were identified and correlated with promoter methylation of 11 loci, including MINT1, MINT2, MINT31, CACNA1G, p16INK4a, p14ARF, COX2, DAPK, MGMT, and the two regions in hMLH1 in 468 CRCs and matched normal mucosa. RESULTS: BRAF V599E mutations were identified in 21 (9%) of 234 CRCs, and KRAS mutations were identified in 72 (31%) of 234 CRCs. Mutations in BRAF and KRAS were never found in the same tumor. CRCs with BRAF mutations showed high-level promoter methylation in multiple loci, with a mean number of methylated loci of 7.2 (95% CI, 6.6 to 7.9) among 11 loci examined (P < .0001). Tumors with KRAS mutations showed low-level promoter methylation, and CRCs with neither mutation showed a weak association with promoter methylation, with an average number of methylated loci of 1.8 (95% CI, 1.5 to 2.1) and 1.0 (95% CI, 0.79 to 1.3), respectively. CONCLUSION: In CRC, the methylation status of multiple promoters can be predicted through knowledge of BRAF and, to a lesser extent, KRAS activating mutations, indicating that these mutations are closely associated with different patterns of DNA hypermethylation. These changes may be important events in colorectal tumorigenesis.
There has been considerable progress in understanding of the genetic and epigenetic events occurring during colorectal carcinogenesis. Mechanisms that maintain genomic fidelity are targets for inactivation during carcinogenesis, a well-documented example being the DNA mismatch repair (MMR) system. The hallmark of defective MMR is microsatellite instability (MSI). Hereditary nonpolyposis colorectal cancer syndrome–related cancers, caused by germline mutation of an MMR gene, are characterized by high-level MSI (MSI-H), whereas 10% to 15% of sporadic colorectal cancers (CRCs) show MSI-H as a result of epigenetic silencing of an MMR gene, hMLH1.1,2 The RAS/RAF/ERK pathway mediates the cellular response to extracellular signals that regulate cell proliferation, differentiation, and apoptosis.3 Mutation of the RAS proto-oncogene commonly occurs in various human cancers, including 95% of pancreatic cancers and up to 50% of large bowel adenocarcinomas.4-7 KRAS mutation occurs in hot spots mainly of codon 12 and 13.8 Recent studies have shown that the BRAF gene, one of three human isoforms of RAF, is activated by oncogenic RAS, leading to cooperative effects in cells responding to growth factor signals.3 BRAF somatic mutation presents in 66% of malignant melanoma and approximately 10% of CRCs. A hotspot for BRAF mutation is conversion of varine 599 to glutamic acid (V599E) and accounts for 80% of the BRAF mutations in CRC. This hot spot is suggested to be biologically distinct from other infrequent BRAF mutations, because the cancer cells having V599E mutation can grow without functional RAS,9 and thus BRAF V599E mutation has not been found in CRCs with KRAS mutation.10,11 BRAF mutations were initially identified in CRCs with MMR deficiency.10 Subsequently, BRAF mutation has been shown to be associated with the epigenetic silencing of hMLH1, but not with germline mutation of MMR genes.12 In contrast, KRAS mutation is associated with epigenetic silencing of O6-methylguanine-DNA methyltransferase (MGMT), which is known to encode a DNA repair protein that removes potentially carcinogenic and cytotoxic alkyl adducts from the O6 position of guanine.13-15 In this study, we examined BRAF codon 599 mutation as well as KRAS codon 12 and 13 mutations by our newly designed enriched polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis and highlighted the association between KRAS or BRAF mutations versus multiple epigenetic alterations, including the silencing of hMLH1 in a total of 234 CRC samples.
Tissue Samples Tumor tissues and samples of the corresponding normal mucosa were obtained from 234 patients with CRC who had undergone curative surgery at Okayama University and Okayama Saisekai Hospitals between 1994 and 2002 and the Royal Brisbane Hospitals in Brisbane, Queensland, Australia, between 1989 and 2002. Ethical approval was obtained, and we obtained informed consent in writing from all patients. Tumor staging was based on Dukes' classification. Samples of both tumor and normal mucosal tissue were stored at –80°C, and DNA was extracted by the standard procedure involving digestion with proteinase K and phenol-chloroform extraction.
Detection of BRAF Codon 599 and KRAS Codon 12 and 13 Mutations
To detect KRAS codon 12 and 13 mutations, we modified the artificial RFLP/PCR approach used by previous investigators.16 The primers we used (K12&13F and Kwt-R) were designed to introduce a MvaI restriction site for codon 12 and a BglI restriction site for codon 13 of the wild allele (Table 1). Using these primers, first-stage PCR amplification was performed. PCR was as follows: 95°C for 11 minutes and 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and finally 5 minutes at 72°C. Aliquots (5 µL) of the first PCR product were then digested with 10 units of MvaI (Takara Co, Kyoto, Japan) and 8 units of BglI (Takara Co) at 37°C for 3 hours, respectively; next, 1-µL aliquots of these MvaI and BglI digests were used in a second-stage PCR for mutations in codons 12 and 13, respectively. The PCR for codon 12 was performed with the K12&13F and 12mt-R primers, whereas the PCR for codon 13 was performed with the K12&13F and 13mt-R primers (Table 1). The second-stage PCR conditions were as described above. Aliquots (25 µL) of the KRAS codon 12 and 13 products obtained after second-stage PCR were then digested with either MvaI (for codon 12) or BglI (for codon 13) at 37°C for more than 6 hours. This method of RFLP analysis for KRAS codon12 led to the generation of 69-bp, 29-bp, and 22-bp fragments after MvaI digestion if there was no KRAS codon 12 mutation present, and of 98-bp and 22-bp fragments if KRAS codon 12 mutation was present. A similar approach for codon 13 mutations led to the generation of 74-bp, 32-bp, and 14-bp fragments by BglI in the case of a wild-type codon 13, as opposed to 106-bp and 14-bp fragments in the case of codon 13 mutations. All products were visualized on 3% agarose gels stained with ethidium bromide. If the first PCR step does not seem to be necessary, it may be possible to omit it without losing sensitivity in the detection of BRAF codon 599 and KRAS codon 12 and 13 mutations. We confirmed the nature of each of the mutations that we detected by RFLP analysis by sequencing the appropriate DNA samples in a Hitachi Autosequencer SQ-5500E (Hitachi Inc, Tokyo, Japan) used in accordance with the manufacturer's instructions.
Bisulfite Modification and Detection of Methylation Status of Multiple Loci
Methylation Analysis for hMLH1 and MGMT The methylation status of the MGMT promoter was examined using the primer set shown in Table 1. This enabled us to detect two discrete regions of MGMT promoter in a single-step PCR reaction, for which we used a final volume of 25 µL containing 10 x buffer, 1.5 mmol/L of MgCl2, 0.4 µmol/L of NS-S and NS-AS primers, 0.2 µmol/L of MS-S and MS-AS primers, 0.1 mmol/L of dNTP, and 0.65 units of Taq polymerase (Ampli-TaqGold). The methylation status of the MGMT promoter was determined as follows: methylated if a 100-bp and/or 137-bp fragment could be detected; unmethylated if only a single 405-bp fragment was present. Our MSSP method has an advantage over the conventional methylation-specific PCR because each individual assay includes a reaction for an internal control. In other words, a pair of primers hybridized to the non-CpG island sequences generate a band that represents an internal control and thereby assures the reliability of the assay. For this reason, MSSP does not require an individual reaction with unmethylated specific primers.
Immunohistochemical Analysis for hMLH1
Microsatellite Analysis
Statistical Analysis
PCR/RFLP Analysis and Sequencing Results of BRAF and KRAS Mutations Using our designed enriched RFLP analyses, we detected BRAF V599E mutations in 21 (9%) and KRAS mutations in 72 (31%) of the 234 CRCs that we examined (Fig. 1 and 2). The spectrum of BRAF/KRAS mutations is shown in Table 2. All of the BRAF mutations were T to A transversions at bp position 1,796 (V599E); of the 72 KRAS mutations detected, 51 (71%) were G to A transitions, 17 (24%) were G to T transversions, and seven (10%) were G to C transversions.
As noted by previous investigators,10,11 there did not seem to be a single CRC in our 234-CRC study that had mutation in both BRAF and KRAS. We therefore decided to divide our collection of 234 into the following subgroups for further investigation: CRCs with BRAF mutations (BRAF-mt), CRCs with KRAS mutations (KRAS-mt), and CRCs that were not mutant in either gene (wild type; Wt). These three groups and associated clinicopathologic features, including MSI status, are shown in Table 3. Sixteen (76%) of the 21 CRCs with BRAF-mt CRCs proved to be MSI-H, whereas most of the CRCs in the other two subgroups (KRAS-mt and Wt) were MSI-L/MSS. Most patients with CRCs harboring BRAF mutations (76%) or KRAS mutations (64%) were older than 65 years. Only 45% of patients in whom CRCs were Wt with respect to both BRAF and KRAS were older than 65 years. With respect to sex, CRCs with BRAF-mt were more frequent in female than male patients (Table 3). The proximal colon was the site of predilection for tumors with BRAF mutation (76%), less obviously for CRCs with KRAS mutation (44%), and least for tumors that were Wt for both genes (23%; Table 3). In terms of histologic grade, BRAF-mt was more frequent in poorly differentiated or mucinous (57%) CRCs than in moderately (33%) or well differentiated (10%) CRCs. Tumor stage showed no relation with mutation status.
Comparisons of the Methylation Status of the Two Distinct hMLH1 Promoter Regions in CRCs With Their hMLH1 Protein Expression Levels We assessed the methylation status of both 5' region and 3' regions of the hMLH1 promoter using an MSSP (Fig 3a, 3b, and 3c). One key feature of this method is that its validity does not depend on the availability of unmethylated controls. Methylation of the 5' region of the hMLH1 promoter was observed in MSI-H and non-MSI-H tumors as well as in normal mucosa, whereas methylation of its 3' region was only ever detected in MSI-H tumors and was correlated with the hMLH1 protein expression detected by immunohistochemical staining (Table 4 and Fig 3d and 3e).
Association Between the Methylation Status of Multiple Loci and the Presence of BRAF or KRAS Mutations in 234 CRCs We assessed the methylation status of nine loci, including the MINT1, MINT2, MINT31, CACNA1G, p16INK4a, p14ARF, COX2, DAPK, and MGMT, as well as of the 5' and 3' regions of the hMLH1 promoter region, and assessed the results on the basis of whether the CRCs in question harbored BRAF or KRAS mutations (Fig 4 and Table 5). Methylation of the hMLH1-5'region, hMLH1-3' region, MINT1, MINT2, MINT31, CACNA1G, p16INK4a, and p14ARF was much more common (P < .0001) in the subgroup we have described as BRAF-mt CRCs. Methylation of hMLH1-3'region (extensive methylation in both 5' and 3' regions) was almost exclusively observed in the BRAF-mt group (BRAF-mt, 76%; KARS-mt, 0%; Wt, 1%; P < .0001). In comparison to the Wt group, KRAS mutations were statistically correlated with the methylation of MINT2, p16INK4a, and p14ARF similar to BRAF mutations. However, methylation of MGMT was only associated with the KRAS-mt group (P = .007). Promoter methylation of COX2 and DAPK showed no correlation with any of the groups. We provide statistical results in Table 5 for 3 x 2 contingency tables, which establish the fact that differences exist among BRAF mutants, KRAS mutants, and tumors that are Wt with respect to both genes. The particular differences as indicated above are evident from examination of the proportions in the table, and these differences were confirmed as statistically significant by individual two-group comparisons using 2 x 2 tables.
The frequency of methylation among 11 promoters of three groups was determined (Table 6; Fig 5). Among 234 CRCs, 93 CRC samples (40%) displayed either KRAS or BRAF mutations. Twenty-one CRCs with BRAF-mt showed methylation at one or more loci, and the average number of methylated loci was 7.2 (SE, 0.31; 95% CI, 6.6 to 7.9 loci). Seventy-two tumors with KRAS-mt had 1.8 methylated loci (SE, 0.17; 95% CI, 1.5 to 2.1 loci), and 141 tumors with neither KRAS nor BRAF mutations contained 1.0 methylated loci (SE, 0.12; 95% CI, 0.79 to 1.3 loci). Thus there were significant differences in the average numbers of methylated loci that were detected in the BRAF-mt, KRAS-mt, and Wt subgroups of CRCs (Wilcoxon/Kruskal-Wallis' test, P < .0001).
This is the first report correlating KRAS and BRAF mutational status with epigenetic alterations of multiple loci and genes. Among the genes studied for epigenetic alterations, we focused particularly on the precise promoter methylation status and protein expression of hMLH1, an important MMR gene, in relation to RAS/RAF mutations. We investigated the hot-spot KRAS mutations in codons 12 and 13 and hot-spot BRAF mutation in codon 599 using a newly designed enriched PCR/RFLP analysis, and all the detected mutations were confirmed by sequencing. KRAS codon 12 and 13 mutations cover 90% of the entire KRAS mutation spectrum in CRC.25-27 BRAF V599E mutation accounts for 80% of the activating BRAF mutations in CRC and was thought to activate a BRAF protein by mimicking phosphorylation of T598 and S601.9,10,28 No mutation of BRAF except for V599E upregulates the RAS/RAF/ERK pathway independent of KRAS mutation. Thus our analysis of a total of 234 CRCs and normal counterpart tissue samples is sufficient to study the frequency of KRAS and BRAF activating mutations. In our study, BRAF mutation was strongly associated with the high-level methylation status of multiple loci, including MINT1, MINT2, MINT31, CACNA1G, p16INK4a, and p14ARF, in addition to methylation in 5' and 3' regions of hMLH1. The average number of methylated loci examined per tumors with BRAF mutations was 7.2 loci (95% CI, 6.6 to 7.9 loci). Sixteen (94%) of 17 CRCs with methylation in the hMLH1 3' region (resulting in MMR deficiency) showed BRAF mutations, whereas BRAF mutations were also detected in a small percentage of non–MSI-H CRCs displaying no methylation in the hMLH1 3' region (MMR proficient). These results suggest that mutation in BRAF is not simply explained by the result of defective MMR. Wang et al12 suggested that BRAF mutations might arise as a consequence of the inactivation of another gene or genes not involved in DNA MMR, because CRCs with epigenetic inactivation of hMLH1 also exhibit promoter hypermethylation at other loci. Previous studies reported that non-MSI CRCs, which exhibit a high frequency of CpG island methylator phenotype, showed high frequency of KRAS mutation.29,30 However, in our analysis, the average number of methylated loci per tumor with KRAS mutation was 1.8 loci (95% CI, 1.4 to 2.2 loci), which was much less than in CRCs with BRAF mutation. The subset of CRCs with MGMT methylation also showed frequent KRAS mutation, consistent with previous reports.13,14 Inactivation of MGMT by promoter hypermethylation was theoretically associated with the presence of G:C to A:T transitions in KRAS and p53 in human CRCs.13,31 In our study, among the 72 CRC patients with KRAS mutation, codon 12 mutations were identified in 60 patients, and 39 (65%) of them were G to A transitions. Conversely, mutation in KRAS codon 13 was detected in 15 patients, and 14 (93%) of them were G to A transition. The majority of KRAS mutations were G to A transitions. However, the hot-spot activating mutation in BRAF V599E was a transversion at bp position 1,796. Accordingly, it is conceivable that hypermethylation of MGMT can explain the majority of KRAS mutations but not of BRAF mutations. By contrast, the average number of methylated loci in CRCs with neither BRAF nor KRAS mutations was only 1.0 (95% CI, 0.79 to 1.3 loci), which was statistically less than that of the KRAS-mt group. Consistent with the previous studies, upstream promoter of hMLH1 (hMLH1-5'region in this study) showed methylation in both MSI-H and non–MSI-H CRCs,1,23 and methylation at the hMLH1-5'region itself is not sufficient for the silencing of the gene. On the other hand, methylation in the hMLH1-3' region, thus extensive methylation in both 5' and 3' regions, was exclusively identified in MSI-H CRCs and was correlated with the loss of expression of hMLH1 protein. In our analysis, 16 tumors harboring methylation at hMLH1-3' region showed both loss of expression of hMLH1 and BRAF mutations. However, five CRCs without methylation in hMLH1-3' region (MMR proficient) also exhibited BRAF mutations. Four (80%) of five such tumors showed frequent methylation in multiple loci, but one tumor had single (hMLH1-5') methylation. Dissimilar to the hMLH1-5'region, methylation of hMLH1-3'region was almost exclusively identified in CRCs with BRAF-mt and was never identified in those with KRAS-mt. The close relation between BRAF mutation and methylation of multiple genes observed in our study may indicate that BRAF mutation predisposes to hypermethylation, that hypermethylation and silencing of an unknown gene or genes leads to BRAF mutation or simply that a synergy exists between BRAF mutation and DNA methylation. Further research is required to determine the mechanistic basis of the association. The mechanisms leading to MGMT methylation may be different from those causing methylation of the majority of gene promoters, such as 3'-hMLH1, because methylation of MGMT was not related to the methylation status of other multiple loci. In conclusion, our data elucidate the clear differences in epigenetic alterations among CRCs with BRAF mutations, those with KRAS mutations, and those that were Wt with respect to both genes. Approximately 40% of CRCs examined have genetic alteration in the RAS/RAF/ERK pathway; roughly 10% with BRAF and 30% with KRAS mutations. Mutations in both genes may be caused by or associated with at least two distinct epigenetic mechanisms causing promoter methylation of multiple cancer-related genes. Moreover, we can anticipate the methylation level of multiple promoters by the presence of BRAF and KRAS activating mutations. It remains to be seen whether a dual classification of CRC based on both mutation and epigenetic alterations has clinical utility.
The authors indicated no potential conflicts of interest.
Supported in part by a grant-in aid from the Japanese Ministry of Education, Science, Sports and Culture of Japan (grant Nos. 12671227, 11671237, 11671240, 14031227). Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
40% of the markers displayed MSI, respectively. Tumors displaying no MSI with any of the microsatellite markers that we tested were classified as MSS. 
2 test where appropriate. The association among the average number of methylated loci of three CRC subsets was analyzed using a Wilcoxon/Kruskal-Wallis's test. All reported P values are two-sided, and a P value less than .05 was considered statistically significant. 
), mutation in codon 599; (*), mismatch base in artificial forward primer.
), promoter methylation; (
), promoter unmethylation.
