10.1245/s10434-006-9111-z
Annals of Surgical Oncology 14:1229-1236 (2007)
© 2007 Society of Surgical Oncology
Role of the BRAF Mutations in the Microsatellite Instability Genetic Pathway in Sporadic Colorectal Cancer
M. L. Maestro1,
M. Vidaurreta1,
M. T. Sanz-Casla1,
S. Rafael1,
S. Veganzones1,
A. Martínez2,
C. Aguilera2,
M. D. Herranz2,
J. Cerdán3 and
M. Arroyo1
1 Department of Clinical Analysis, Hospital Clínico San Carlos, Madrid, Spain
2 Department of Clinical Analysis, Hospital Reina Sofía, Cordoba, Spain
3 Department of General Surgery III, Hospital Clínico San Carlos, c/Martín Lagos s/n, 28040, Madrid, Spain
Correspondence: Address correspondence and reprint requests to: Ma Luisa de las Casas; E-mail: mmaestro.hcsc{at}salud.madrid.org
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ABSTRACT
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Background and Aims: Between 10 and 15% of all cases of colorectal cancer are the result of microsatellite instability (MSI) in the genetic pathway due to an alteration in the DNA repair genes. Tumors with high MSI are characterized by a better prognosis. The BRAF oncogene has been linked to the MSI pathway in tumorogenesis. The objective of this study was to determine whether alterations in BRAF are related to MSI and whether they can result in differences in survival rates.
Methods: The study cohort comprised 351 patients diagnosed with sporadic colorectal cancer. MSI was determined in accordance with the National Cancer Institute’s (NCI) recommendations by means of PCR and sequence analyses. Mutational analysis of the BRAF gene was performed by real-time PCR and subsequent sequencing of the altered samples. The methylation pattern of the hMLH1 gene was determined using methylation-specific PCR analyses of bisulfite-treated DNA and the results confirmed by sequencing.
Results: Of the patients tested, 6.9% showed high MSI and 3.7% showed a BRAF gene mutation. hMLH1 methylation was observed in 67.2% of the patients with MSI and/or the BRAF alteration. The BRAF mutation was related to the MSI genetic pathway (P < 0.0001) and with hMLH1 methylation. In the analysis of overall survival only MSI had an independent prognostic value for the risk of death. Patients with the BRAF mutation showed a higher risk of death, although this association was found not to be statistically significant.
Conclusions: There is a subgroup of carcinomas which develop via the MSI pathway that carry an alteration of the BRAF gene. This alteration confers a poorer outcome on these patients within the total group of patients with MSI who have a better prognosis. This hypothesis should be further investigated in a larger study population due to the low incidence of BRAF mutations in colorectal cancer.
Key Words: hMLH1 methylation • MSI • BRAF • Colorectal cancer • Prognosis
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INTRODUCTION
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Colorectal cancer (CRC) is one of the tumors that has been better characterized with respect to genetic mechanisms involved in its development. However, this means that it is important to know whether these genetic alterations identify patients who will have a worse prognosis. Two different genetic pathways have been described in tumorogenesis. The most frequent pathway is the chromosomal instability pathway characterized by alterations in oncogenes and suppressor genes, with the first step being the alteration of the APC gene.1 In 10–15% of all cases of CRC the microsatellite instability (MSI) pathway is involved, which results from alterations in the DNA repair genes (MMR) responsible for the repair of errors that occur during DNA replication. Two genetic pathways give rise to two clinical phenotypes of colorectal tumors that differ in the characteristics of the tumor and, probably, in their outcome. A high-frequency microsatellite instability (MSI-H) phenotype is associated with small insertions and deletions in repetitive sequences (microsatellites).2 MSI-H tumors are characterized by the diploid state, right-sided location, mucinous histology and poor differentiation, and this phenotype has been associated with a better prognosis.2–5
Raf proteins participate in the Ras/Raf/MEK/ ERK signaling pathway, which mediates the response of the cell to growth signals. Recently, mutations in the BRAF gene in CRC have been described as an alternative mechanism to the alteration of KRAS in this pathway.6,7 Alterations in the BRAF gene are described as being associated with the MSI pathway of colorectal tumorogenesis. The V599E mutation, which is located on exon 15 of the BRAF gene, is the most frequently identified mutation in CRC tumors with MMR defects.7 This mutation constitutively activates BRAF, independently of Ras. Koinumka et al. reported an association between BRAF mutations and promoter methylation of the hMLH1 repair gene, that latter of which has been found to be altered in 80% of the cases of MSI sporadic CRC.8
The aim of this study was to determine whether these alterations in the BRAF gene are related to the MSI genetic pathway in colorectal tumorogenesis and whether the presence of these alterations leads to differences in the survival of CRC patients.
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PATIENTS AND METHODS
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Study Population
The study cohort comprised 351 patients undergoing surgery consecutively for colorectal cancer at the Hospital Clínico San Carlos in Madrid (Spain) between March 1995 and April 2003. This was a prospective cohort study. All the patients were operated on by the same surgeon who performed radical oncological surgery based on the location of the tumor. The surgery was defined as curative when there was no evidence of macroscopic residual tumor after resection. Using this criterion, the surgeon performed a curative resection on 290 patients (82.6%) and resected the primary tumor in 61 patients (17.4%) as palliative treatment. Patients with metacronic carcinoma, familial polyposis, a familial predisposition for hereditary nonpolyposis colon cancer (HNPCC) and inflammatory bowel disease were excluded from the study. None of the patients had received neoadjuvant treatment. Informed consent was obtained from each patient, and the project was approved by the clinical research and ethics committee of this hospital. The follow-up was performed according to the protocol designed by the authors. The tumors were staged according to Duke’s classification. Proximal tumors were defined as occurring in the cecum through to the transverse colon; tumors in the splenic flexure, descending and sigmoid colon were defined as being distal. Of the study cohort, 51.3% were subjected to adjuvant chemotherapy with 5-fluorouracil (5-FU).
Tumoral and non-tumoral tissue samples were obtained during the surgical procedure itself and immediately immersed in liquid nitrogen for storage in a freezer at –80°C. The specimens were then independently examined by two pathologists, who confirmed the samples had over 80% tumor cells.
To extract DNA, we first incubated tumoral and non-tumoral samples overnight at 50°C in a lysis buffer (10 mM Tris HCl, 1 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate, 500 µg/ml proteinase K); we then isolated the DNA using a phenol:chloroform method and precipitated it with ethanol.
MSI Determination
The DNA was amplified in a 25-µl final volume containing 100 ng of DNA, 200 µM dNTP, 1–2 mM MgCl2, 350 nM of each primer and 2 U Taq polymerase. The PCR conditions were as follows: 25–35 cycles [95°C for 30 s, 50–58°C for 30 s (depending on the marker) and 72°C for 30 s]. The primers used were BAT25, BAT26, D17S250, D5S346 and D2S123 in accordance with the NCI criteria.9 Each pair of primers was tagged with a fluorochrome for later analysis (TIB MOLBIOL, Roche, Germany).
The sizes of the amplified products were determined using an ABI 310 PRISM sequencer and analysed using Genescan and Genotyper software (Applied BioSystems, Foster City, Calif).
BRAF Gene Analysis
The analysis of the BRAF gene was performed using the LightCycler (Roche). The PCR analyses were performed in the hybridization probe mode. The primers for exon 15 used were BRAF-F (5'-CTACTgTTTTCCTTTACTTACTACAC-3') and BRAF-R (5'-gACCTTCAATgACTTTCTAgTAAC-3'), and the hybridization probes that hybridizes inner to primers were BRAF-Red (5'-Red- TCACC-TATTTTTACTgTgAggTCTTCATg-3') and BRAF-fluo (5'– AgATTTCACTgTAgCTAgACCAAA-fluo –3') (designed over genebank sequence no. AC006344.2). The PCR was performed with 5 µl of DNA in 20-µl volumes in glass capillaries. After completing the amplification process the samples were denatured and then slowly heated. The decline in fluorescence was monitored, and melting curves were constructed automatically by LightCycler software (Roche Molecular Biochemicals, Indianapolis, Ind.). These curves were converted to melting peaks by plotting the negative derivative of the fluorescence against temperature. The expected peaks were at 61 °C for the 599 wild-type and 53°C for V599E. When the real-time PCR showed a mutation the sample was sequenced in an ABI PRISM 3100 Genetic Analyzer.
Analysis of Methylation Status of the hMLH1 Gene
Analysis of the methylation status of the hMLH1 promoter region was performed by Na-bisulfite treatment and single-strand conformation polymorphism (SSCP). The primers used for the PCR analyses were hMLH1-F (5'-ACGTAGACGTT-TTATTAGGGTCGC) and hMLH1-R (5'- CCTCA TCGTAACTACCCGCG) for methylated DNA and hMLH1-F (5'-TTTTGATGTAGATGTTTTATTA GGGTTGT) and hMLH1-R (5'-ACCACCTCATC ATAACTACCCACA) for unmethylated DNA. When the bands showed mobility shifts in the SSCP analysis, they were cut from the gels and sequenced in an ABI PRISM 3100 Genetic Analyzer.
Statistical Analysis
Qualitative variables were provided with their corresponding frequency distributions. Quantitative variables were expressed as their mean, standard deviation (SE) and range. Age was recoded into two groups according to median age (71 years). Associations between qualitative variables were evaluated using the 
2 test or Fisher’s exact test when 25% of the expected frequencies fell below 5. The overall survival (OS) was estimated by the Kaplan-Meier method and compared among the groups using Breslow’s exact test. The event in OS was defined as deaths occurring as a consequence of the tumor, censuring live patients and those dying of another cause. OS was calculated as the time elapsed from the date of surgery until death or last follow-up. The data was fitted to Cox’s proportional risks regression model. The hazard ratio (HR) is given with a 95% confidence interval (95% CI). The variables included were sex, age, Duke’s stage, tumor site, differentiation grade, histological type, adjuvant chemotherapy, MSI and BRAF. In each comparison, the null hypothesis was rejected when the type I error was less than 0.05. All statistical tests were performed using SSPS ver. 11.5 software (SPSS, Chicago, Ill.).
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RESULTS
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The study cohort comprised 351 patients, 52.1% of whom were male and 47.9% female. The mean patient age was 69.6 ± 11.3 years (range: 30–95 years). Patients younger than 71 years represented 48.4% of the study population. The clinicopathological variables are show in Table 1
. With respect to location, 59.8% of the tumors were located in colon (105 in proximal colon and 105 in distal) and 40.2% (141 patients) were located in the rectum. The smaller portion (7.1%) was mucinous adenocarcinomas and the majority (92.9%) adenocarcinomas. In 54 patients it was not possible to determine the differentiation grade. The MSI study was performed in 350 patients and the determination of mutation of the BRAF gene in 324 patients. The MSI analysis revealed that 6.9% of the tumors (24 patients) were MSI-high (MSI-H), 10.2% (36 patients) were MSI-low (MSI-L) and 82.9% (290 patients) showed no change (MSS). Mutations of the BRAF gene were observed in 3.7% of the tumors (12 cases), all of which were V599E. The promoter methylation pattern of the hMLH1 gene was studied in 61 patients (in all patients who presented MSI-L or MSI-H and/or had BRAF mutations). Full methylation of the gene was observed in tumors from 30 patients (49.2%), partial methylation in tumors from 11 patients (18%) while there was an absence of methylation in tumors from 20 patients (32.8%). Tumors from 41 patients (67.2%) showed methylation (full or partial).
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TABLE 1. Clinicopathological variables of the 351 patients with colorectal carcinoma. Relationship between BRAF alteration and MSIa and these variables
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MSI and alterations in the BRAF gene were related to the site of the tumor. MSI-H was more frequent in colon tumors than the ones located in the rectum (P = 0.008) and BRAF mutations were only found in colon tumors and not seen in rectal tumors (P = 0.002). MSI was related to differentiation grade, with undifferentiated tumors being frequently associated with MSI-H phenotype (P = 0.03) (Table 1
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The relationship between promoter methylation and clinicopathological variables is summarized in Table 2. Full methylation of the promoter occurred more frequently in proximal tumors (P = 0.002). All of the mucinous tumors showed full methylation (P = 0.01).
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TABLE 2. Clinicopathological variables of the 61 patients analyzed for hMLH1 methylation. Relationship between hMLH1 methylation and these variables
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The relationship between mutations in the BRAF gene and MSI are shown in Table 3. Of the 12 patients who presented BRAF mutations, nine also had MSI (P < 0.0001).
Of those patients with MSS tumors, only two patients had tumors which showed simultaneously hMLH1 methylation and BRAF mutations, and in both cases only partial methylation was observed. A total of 83.4% of the patients with MSI-H also showed methylation of hMLH1 (66.7% full methylation and 16.7% partial); 54.3% of the patients with MSI-L showed methylation of hMLH1 (Table 4).
All of the patients with the BRAF mutation showed methylation of hMLH1 (Table 5).
Overall Survival
The median follow-up period was 43 months (3 years); the interquartile range was 27–63 months. The OS at 43 months was 68.9%. All survival analyses refer to this median follow-up period. During follow-up, 130 patients died; 102 as a consequence of neoplasia. One patient was lost to follow-up.
Univariate analysis of OS is shown in Table 6. According to the stratified analysis of OS, in women with MSI-H survival rate was 92% and in men with MSI-H 64%, which was significant (P = 0.04).
In patients with adenocarcinomas and MSI-H the OS was 87% and in mucinous tumors 75% (P = 0.03). Colon carcinomas with MSI-H had an OS of 79% (67% in proximal colon and 100% in distal) and in rectum tumors the OS was 100% (P = 0.02).
According to the stratified analysis of hMLH1 methylation, in Dukes’ stage D the OS of the patients with full methylation was 80% and with partial or no methylation, 0% (P = 0.009).
In the analysis adjusted by stage no differences in OS were observed between the group of patients who were treated and the group that were not treated.
The stratified analyses of the OS for the genetic alterations studied are shown in Tables 7 and 8.
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TABLE 8. Overall survival (OS) analysis of MSI stratified by hMLH1 methylation in 351 colorectal carcinoma patients
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Tumor stage (P < 0.001) and MSI (P = 0.036) showed independent prognostic value in the multivariate analysis. The HR of patients with MSI-L was 0.72 (95% CI = 0.29–1.8) and with MSI-H was 0.33 (95% CI = 0.12–0.92). Patients with BRAF mutations showed a 62% higher risk of dying, independent of MSI, although this relationship was not significant (P = 0.38) (Table 9).
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DISCUSSION
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In our study a mutation of the BRAF gene was observed in 3.7% of the CRC tumors; all were of the V599E type, which is in accordance with prior results in this type of carcinoma.7 This alteration was related to the MSI genetic pathway: 75% of the carcinomas with BRAF alterations showed MSI (P < 0.0001).
Some authors have suggested that alterations in this gene may provide an alternative mechanism to the KRAS mutations in the CRC tumorogenesis.6,7
We determined promoter methylation in hMLH1 in tumors with MSI because this is the most frequently modified repair gene in this pathway. This genetic alteration was also determined in tumors with BRAF mutations according to the criteria of Koinuma et al. who in a previous report associated alterations in this gene with the methylation of hMLH1.6 In our study 83.3% of the MSI-H tumors showed full or partial promoter methylation. As well, in two patients with MSS and the BRAF mutation, partial methylation was observed, thereby confirming the fact that hMLH1 is frequently altered in MSI tumors. Miyakura et al. also observed hMLH1 hypermethylation in 34.8% of the MSS tumors; in all cases this took the form of partial methylation and protein expression was maintained.10
In 100% of the carcinomas with BRAF mutations methylation of hMLH1 occurred. Samowitz et al. have recently speculated about the fact that MSI colorectal tumors that develop from hyperplasic polyps frequently show BRAF mutations and the methylator phenotype (CIMP), including the methylation of hMLH1. According to Domingo et al. the inactivation of hMLH1 by methylation is related to the activation of BRAF, suggesting that specific modulations in the Ras/Raf system could occur, depending on the hMLH1 methylation status in CRC.11 However, Wang et al. suggested that BRAF mutations are not the result of defects in the repair genes because these mutations rarely occur in tumors with defects in the MMR in the germinal line and, additionally, MSS tumors which show alterations of this gene exist.12
Promoter methylation is a characteristic of carcinomas situated in the proximal colon and, according to Miyakura et al. it is the key factor to understanding why MSI tumors are more frequent in this location.10 In our study, the genetic alterations studied were more frequent in proximal tumors, and it seems clear that rectal tumors do not develop through the MSI genetic pathway with alterations in the BRAF gene. Furthermore, as has been described previously, we observed that MSI is related to undifferentiated tumors and mucinous histology. This fact coincides with the association of full methylation of hMLH1 with proximal tumors and the mucinous component. These results are consistent with those published by the Kazama group which show that mucinous histology is related to proximal tumors, with the phenotype MSI-H and with the methylation of hMLH1.13
It is important to know whether there are genetic alterations that differentiate sub-groups in terms of the agressivity of the tumor in those tumors that develop through the MSI pathway and show better prognosis. As has been described previously,14 tumors which develop through the MSI genetic pathway are less aggressive. In our study of the genetic parameters examined, only MSI showed a prognostic value independent of the various classic prognostic factors. In patients with MSI the presence of hMLH1 methylation did not differentiate sub-groups in terms of overall survival.
At the beginning of this study we hoped that the result would reveal a strong relationship between MSI and BRAF mutations so that the latter genetic alteration would lead to a better prognosis for these patients. However, we found instead that the mutation of the BRAF gene does not present a significant relationship with prognosis due to the small size of the population with this genetic alteration. Contrary to what we had expected to see, a poorer survival was observed in patients with this mutation. In the stratified analysis of MSI, we observed a tendency for a worse prognosis in patients with the BRAF mutation – in both patients with MSS and in those with MSI . However, it was not possible to confirm this tendency due to the small sample size of each of the sub-groups. Samowitz et al. found a worse prognosis in patients with MSS tumors and BRAF mutations, but the same was not found in those with MSI tumors, for whom the prognosis was excellent independent of the genetic alteration in the BRAF gene.5 We propose that it is not the mutation itself which confers a poor prognosis but rather that the mutation has different effects depending of the type of genetic pathway in which it is produced.
We conclude that there are tumors which develop by the MSI genetic pathway through the methylation of hMLH1 repair genes that show a better prognosis than MSS tumors. Furthermore, there is a subgroup of carcinomas within this pathway which show alterations in the BRAF gene, and this leads them to a worse outcome within the better prognosis which MSI would confer. This hypothesis will have to be confirmed in a study with a larger sample size due to the low frequency of mutations in the BRAF gene in CCR.
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FOOTNOTES
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Grant support: This study was supported by grant no. PI030514 from the "Fondo de Investigaciones Sanitarias", Spain
Received for publication May 19, 2006.
Accepted for publication May 23, 2006.
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ABSTRACT
INTRODUCTION
