Observational Study Open Access
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 28, 2015; 21(4): 1275-1283
Published online Jan 28, 2015. doi: 10.3748/wjg.v21.i4.1275
Prognostic value of KRAS and BRAF mutations in curatively resected colorectal cancer
Shigenori Kadowaki, Akira Ooki, Kensei Yamaguchi, Division of Gastroenterology, Saitama Cancer Center, Saitama 362-0806, Japan
Miho Kakuta, Shuhei Takahashi, Akemi Takahashi, Yoshiko Arai, Kiwamu Akagi, Division of Molecular Diagnosis and Cancer Prevention, Saitama Cancer Center, Saitama 362-0806, Japan
Yoji Nishimura, Toshimasa Yatsuoka, Division of Gastroenterological Surgery, Saitama Cancer Center, Saitama 362-0806, Japan
Keitaro Matsuo, Department of Preventive Medicine, Kyushu University Faculty of Medical Sciences, Fukuoka 812-8582, Japan
Shigenori Kadowaki, Kei Muro, Department of Clinical Oncology, Aichi Cancer Center Hospital, Aichi 464-8681, Japan
Author contributions: Kadowaki S, Matsuo K and Akagi K designed the study, analyzed the data, interpreted the results and wrote the paper; Kakuta M, Takahashi S, Takahashi A, Arai Y and Akagi K carried out all the laboratory experiments; Kadowaki S, Kakuta M, Ooki A, Nishimura Y, Yatsuoka T and Akagi K collected the data; Yamaguchi K and Muro K supervised this study; and all authors have read and approved the manuscript.
Supported by Japanese Ministry of Health, Labor and Welfare.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Kiwamu Akagi, MD, PhD, Division of Molecular Diagnosis and Cancer Prevention, Saitama Cancer Center, 818 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan. akagi@cancer-c.pref.saitama.jp
Telephone: +81-48-7221111 Fax: +81-48-7235197
Received: June 19, 2014
Peer-review started: June 20, 2014
First decision: July 21, 2014
Revised: September 9, 2014
Accepted: October 14, 2014
Article in press: October 15, 2014
Published online: January 28, 2015

Abstract

AIM: To investigate the prognostic role of KRAS and BRAF mutations after adjustment for microsatellite instability (MSI) status in Japanese colorectal cancer (CRC) population.

METHODS: We assessed KRAS and BRAF mutations and MSI status in 813 Japanese patients with curatively resected, stage I-III CRC and examined associations of these mutations with disease-free survival (DFS) and overall survival (OS) using uni- and multivariate Cox proportional hazards models.

RESULTS: KRAS and BRAF mutations were detected in 312 (38%) of 812 and 40 (5%) of 811 tumors, respectively. KRAS mutations occurred more frequently in females than in males (P = 0.02), while the presence of BRAF mutations was significantly associated with the female gender (P = 0.006), proximal tumor location (P < 0.001), mucinous or poorly differentiated histology (P < 0.001), and MSI-high tumors (P < 0.001). After adjusting for relevant variables, including MSI status, KRAS mutations were associated with poorer DFS (HR = 1.35; 95%CI: 1.03-1.75) and OS (HR = 1.46; 95%CI: 1.09-1.97). BRAF mutations were poor prognostic factors for DFS (HR = 2.20; 95%CI: 1.19-4.06) and OS (HR = 2.30; 95%CI: 1.15-4.71). Neither the BRAF by MSI interaction test nor the KRAS by MSI interaction test yielded statistically significant results for DFS and OS.

CONCLUSION: KRAS and BRAF mutations are associated with inferior survival, independent of MSI status, in Japanese patients with curatively resected CRC.

Key Words: Colorectal cancer, KRAS, BRAF, Microsatellite instability, Prognostic factor

Core tip: Although KRAS and BRAF mutations play a critical role in colorectal cancer development, little is known regarding the prognostic role of these genetic alterations after adjustment for microsatellite instability status in Asian populations. To the authors’ knowledge, the current study is the first large-scale study to clarify the impact of KRAS and BRAF mutations on the survival outcomes of colorectal cancer in Asian populations. We found that KRAS and BRAF mutations were separately associated with inferior disease-free survival and overall survival, independent of microsatellite instability status, in patients with curatively resected colorectal cancer.



INTRODUCTION

Colorectal cancer (CRC) develops through diverse mechanisms such as chromosomal instability (CIN), microsatellite instability (MSI), and epigenetic DNA promoter methylation [CpG island methylator phenotype (CIMP)][1]. CIMP and MSI-high (MSI-H) phenotypes are closely associated. Most sporadic MSI-H tumors develop through CIMP-associated methylation of MLH1, and BRAF mutations occur frequently in both phenotypes[2,3]. KRAS mutations mainly occur in CIN and are partly associated with intermediate CIMP epigenotype[4]. KRAS and BRAF mutations are mutually exclusive; both cause RAS/RAF/MAPK signaling pathway upregulation and are crucial in CRC development.

KRAS encodes a guanosine triphosphate/guanosine diphosphate binding protein; KRAS mutations are observed in approximately 30%-40% CRCs[5-8]. KRAS mutations are well known as predictive markers of resistance to epidermal growth factor receptor-targeted antibodies in metastatic CRC, but their prognostic value remains controversial. Some studies have shown that KRAS mutations are associated with poorer survival in CRC[8,9], while others found no association[6,7].

BRAF encodes a serine/threonine protein kinase, a downstream effector of the KRAS protein. Activating BRAF mutations occur in approximately 4%-20% CRCs[6,10-14], with the vast majority being the V600E hotspot mutation. Although some previous studies have shown that BRAF mutations confer poorer prognosis in CRC[10-12], others have not[6,13], probably because of associations with favorable MSI-H CRC prognosis[15-17].

Although genetic background and geographical factors may influence mutation frequency and prognosis, most reports are from Western countries; less data are available regarding the prognostic role of KRAS and BRAF mutations in Asian populations. Two independent studies from Taiwan and Japan have been published recently. However, both had a small sample size and heterogeneous cohorts including metastatic disease; the study from Taiwan did not examine MSI status[14,18]. Hence, a large homogenous cohort with MSI status is essential for assessing the prognostic value of various clinical or molecular variables in CRC. Here, we clarified associations of KRAS and BRAF mutations and MSI status with survival outcomes in a larger Japanese cohort of patients with curatively resected CRC.

MATERIALS AND METHODS
Patients and tissue samples

A total of 813 consecutive stage I-III CRC patients undergoing curative resection at Saitama Cancer Center between July 1999 and May 2006 were included. Written informed consent was obtained from all patients. Patients with the following conditions were excluded: (1) history of radiotherapy or chemotherapy preoperatively; (2) inflammatory bowel disease; or (3) history of familial adenomatous polyposis. Pathological staging was performed according to the tumor, node, and metastasis (TNM) classification system (6th edition)[19]. CRCs were typically divided into 3 types: rectum, distal colon (splenic flexure and descending and sigmoid colon), and proximal colon (cecum and ascending and transverse colon). Adjuvant chemotherapy was administered to 40% (129/322) and 76% (232/307) of stage II and III CRC patients, respectively. Among 361 patients treated with adjuvant chemotherapy, only 10 patients received combination chemotherapy with 5-fluorouracil, leucovorin, and oxaliplatin, while remaining were treated with single-agent fluoropyrimidines. Patients were followed-up until death or February 2012, whichever came first. We obtained approval from the Ethics Committee of Saitama Cancer Center.

Genomic DNA extraction and KRAS and BRAF mutation analysis

Primary CRCs and paired healthy colorectal mucosa obtained perioperatively were immediately frozen at -80 °C until analysis. Genomic DNA was extracted from fresh frozen specimens using the standard phenol-chloroform extraction method. Exons 2 and 3 of KRAS were examined for mutations by denaturing gradient gel electrophoresis, as described previously[20]. The BRAF V600E mutation was detected using PCR and restriction enzyme digestion, as described previously[21].

MSI analysis

MSI analysis was performed using fluorescence-based PCR, as described previously[22]. Five Bethesda markers BAT25, BAT26, D5S346, D2S123, and D17S250 were used to classify tumor MSI status. MSI status was graded as MSI-H with 2 or more unstable markers, MSI-low (MSI-L) with only 1 unstable marker, and microsatellite-stable (MSS) with no unstable marker. MSI-positive markers were re-examined at least twice to confirm the result.

Statistical analysis

The aim of this study was to evaluate the impact of KRAS/BRAF mutations on prognosis in patients with resected CRC. Prognosis was evaluated according to 2 measures: overall survival (OS) and disease-free survival (DFS). OS was defined as the interval from the date of resection until death due to any cause or until the censor date of February 1, 2012. DFS was defined as the time from the date of resection to tumor recurrence, occurrence of a new primary colorectal tumor, or death due to any cause. Survival probability was estimated using the Kaplan-Meier method and compared using the log-rank test. Cox proportional hazards models were used to estimate uni- and multivariate adjusted hazard ratios for DFS and OS according to mutation status. Factors for which the multivariate models were adjusted are age (≥ 65 vs < 65), gender (male vs female), tumor stage (III vs II vs I), adjuvant chemotherapy (Yes vs No), and status of MSI and BRAF or KRAS mutations (Yes vs No). To further evaluate the potential heterogeneity of the impact of KRAS and BRAF mutations according to MSI status and other covariates [age (≥ 65 vs < 65), gender (male vs female), tumor location (distal/rectum vs proximal), and stage (III vs I/II)], we tested the models that included interaction terms, cross-products of gene mutation status, and another variable of interest in a multivariate Cox model. The likelihood ratio test was performed to determine the significance of the results.

Clinicopathological factor distribution according to gene mutation status was assessed using the χ2 or Fisher’s exact tests for categorical variables, when appropriate, and Student’s t-test for continuous variables. All statistical analyses were performed using Dr. SPSS II software (SPSS Japan Inc., Tokyo, Japan); 2-sided P < 0.05 was considered statistically significant.

RESULTS
Clinicopathological characteristics of KRAS and BRAF mutant tumors

Patient characteristics according to KRAS or BRAF status are summarized in Table 1. MSI status was determined in all cases, whereas mutation status was not determined in 1 case for KRAS and 2 for BRAF. KRAS or BRAF mutations were detected in 38% (312/812) and 5% (40/811) of cases, respectively. Only 1 patient harbored KRAS and BRAF mutations. KRAS mutations were more frequent in females than in males (43% vs 35%, P = 0.02). BRAF mutations were significantly more frequent in females than in males (7% vs 3%, P = 0.006), proximal than in distal or rectal tumors (13% vs 1% vs 2%, P < 0.001), mucinous or poorly differentiated tumors than in moderately or well-differentiated tumors (17% vs 4%, P < 0.001), and MSI-H tumors than in MSS/MSI-L tumors (36% vs 2%, P < 0.001).

Table 1 Patient characteristics according to BRAF or KRAS status n (%).
CharacteristicsKRAS status
BRAF status
Wild-type n = 500Mutant n = 312P valueWild-type n = 771Mutant n = 40P value
Age (yr)0.110.40
mean ± SD63.5 ± 10.364.7 ± 10.363.9 ± 10.365.4 ± 11.6
Gender0.020.006
Male308 (62)166 (53)459 (60)15 (38)
Female192 (38)146 (47)312 (40)25 (63)
Tumor location0.37< 0.001
Proximal134 (27)98 (31)201 (26)31 (78)
Distal213 (43)125 (40)332 (43)5 (13)
Rectum153 (31)89 (29)238 (31)4 (10)
Histological grade0.24< 0.001
Well/moderate472 (94)288 (92)728 (94)31 (78)
Poor/mucinous28 (6)24 (8)43 (6)9 (23)
T stage0.120.89
152 (10)31 (10)79 (10)4 (10)
2106 (21)46 (15)145 (19)7 (18)
3286 (57)200 (64)462 (60)23 (58)
456 (11)35 (11)85 (11)6 (15)
LN metastasis0.180.96
Yes180 (36)127 (41)292 (38)15 (38)
No320 (64)185 (59)479 (62)25 (63)
TNM stage0.090.92
I125 (25)58 (19)173 (22)10 (25)
II195 (39)127 (41)306 (40)15 (38)
III180 (36)127 (41)292 (38)15 (38)
Adjuvant chemotherapy0.440.57
Yes217 (43)144 (46)344 (45)16 (40)
No283 (57)168 (54)427 (55)24 (60)
MSI status0.33< 0.001
MSS/MSI-L455 (91)290 (93)728 (94)16 (40)
MSI-H45 (9)22 (7)43 (6)24 (60)
Survival analysis

The median follow-up time was 87.7 mo (range: 13-148 mo). Based on univariate Cox proportional hazard analysis results (Table 2), greater age (≥ 65), male gender, advanced TNM stage, and presence of KRAS mutations were significantly associated with poor prognosis for DFS and OS. For KRAS mutant vs KRAS wild-type tumors, 5-year DFS was 71% vs 77% (log-rank P = 0.02; Figure 1A); 5-year OS was 80% vs 84%, respectively (log-rank P = 0.01; Figure 1B). Presence of BRAF mutations was not significantly associated with poorer DFS and OS in the entire cohort. For BRAF mutant vs wild-type tumors, 5-year DFS was 70% vs 75% (log-rank P = 0.23; Figure 1C); 5-year OS was 77% vs 83% (log-rank P = 0.11; Figure 1D), respectively.

Figure 1
Figure 1 Kaplan-Meier curves for disease-free survival and overall survival according to KRAS or BRAF status. A: Disease-free survival (DFS) according to KRAS status; B: Overall survival (OS) according to KRAS status; C: DFS according to BRAF status; D: OS according to BRAF status.
Table 2 Univariate prognostic analysis of disease-free survival and overall survival.
CharacteristicsDisease-free survival
Overall survival
HR95%CIHR95%CI
Age (yr)
< 651Reference1Reference
≥ 651.731.35-2.282.211.64-2.98
Gender
Female1Reference1Reference
Male1.571.20-2.061.571.16-2.13
Tumor location
Proximal1Reference1Reference
Distal0.920.67-1.250.90.64-1.26
Rectum1.170.85-1.620.970.67-1.40
Histological grade
Well/moderate1Reference1Reference
Poor/mucinous1.530.97-2.421.430.84-2.42
AJCC stage
I1Reference1Reference
II2.61.61-4.192.261.36-3.75
III4.682.95-7.423.492.14-5.70
Adjuvant chemotherapy
No1Reference1Reference
Yes1.240.96-1.601.291.10-1.51
MSI
MSS/MSI-L1Reference1Reference
MSI-H0.710.42-1.200.920.54-1.59
KRAS
Wild-type1Reference1Reference
Mutant1.351.04-1.741.441.08-1.92
BRAF
Wild-type1Reference1Reference
Mutant1.380.82-2.321.570.90-2.76

In multivariate analysis, adjusting for potential prognostic variables, KRAS retained its prognostic impact on DFS (HR = 1.35; 95%CI: 1.03-1.75) and OS (HR = 1.46; 95%CI: 1.09-1.97; Table 3). Presence of BRAF mutations was significantly associated with poorer DFS (HR = 2.20; 95%CI: 1.19-4.06) and OS (HR = 2.30; 95%CI: 1.15-4.71) after adjustment (Table 3).

Table 3 Prognostic effects of microsatellite instability, KRAS, and BRAF status in Cox proportional models.
Disease-free survival1
Overall survival1
HR (95%CI)P valueHR (95%CI)P value
MSI
MSS/MSI-L1 (reference)0.141 (reference)0.53
MSI-H0.64 (0.35-1.16)0.81 (0.42-1.56)
KRAS
Wild-type1 (reference)0.031 (reference)0.01
Mutant1.35 (1.03-1.75)1.46 (1.09-1.97)
BRAF
Wild-type1 (reference)0.011 (reference)0.02
Mutant2.20 (1.19-4.06)2.30 (1.15-4.71)
Survival analysis stratified by MSI status

Given the potential prognostic effect of MSI status, we evaluated interactions of KRAS or BRAF mutations with MSI status (Table 4). The effect of KRAS mutations on DFS and OS was limited to patients with MSS/MSI-L tumors (HR = 1.37; 95%CI: 1.05-1.80; HR = 1.49; 95%CI: 1.10-2.02, respectively); however, the KRAS by MSI interaction test was not significant (P = 0.95 and 0.70, respectively). BRAF mutations were significantly associated with reduced OS (HR = 2.74; 95%CI: 1.19-6.30) in MSS/MSI-L, but not MSI-H, tumors. However, the BRAF by MSI interaction test did not reach statistical significance (P = 0.44).

Table 4 Prognostic Effects of KRAS and BRAF mutations according to microsatellite instability status.
KRAS
BRAF
HR (95%CI)P valueHR (95%CI)P value
DFS1
MSS/MSI-L1.37 (1.05-1.80)0.952.06 (0.96-4.43)0.91
MSI-H1.34 (0.34-5.24)2.46 (0.49-12.4)
OS1
MSS/MSI-L1.49 (1.10-2.02)0.702.74 (1.19-6.30)0.44
MSI-H1.39 (0.33-5.78)1.18 (0.23-6.02)
Survival analysis stratified by other potential variables

We also analyzed the prognostic value of KRAS and BRAF mutations for OS across strata of other potential prognostic factors (Figure 2). The prognostic effect of KRAS mutations appeared to be consistent across potential variables, and interactions between KRAS status and these factors were not significant. In contrast, BRAF mutations were significantly associated with poor OS in stage III, but not stage I-II, disease. Interactions between BRAF status and TNM stage showed suggestive statistical significance (P = 0.10).

Figure 2
Figure 2 Stratified analysis of KRAS or BRAF status and overall survival. Loge [adjusted hazard ratio (HR)] and 95%CI for BRAF and KRAS mutant tumors (vs wild-type tumors) in various strata are shown. A: KRAS mutant tumors; B: BRAF mutant tumors.
DISCUSSION

To our knowledge, this is the largest study to assess the prognostic value of KRAS and BRAF mutations for survival outcomes in CRC patients in Asian populations. Tumor specimens were prospectively collected from patients with curatively resected CRC (stage I-III); KRAS and BRAF mutations and MSI status were analyzed using a consistent methodology at a single institution. KRAS and BRAF mutations were associated with poor prognosis, independent of MSI status.

Many studies have examined associations of KRAS mutations with various clinical features, with no consistent results[5-8]. KRAS mutations were more frequent in females; however, these mutations were not associated with any other clinical variable. Similarly, Watanabe et al[5] found relationships of KRAS mutations with the female gender and older age. In contrast, the Kirsten Ras Colorectal Cancer Collaborative Group study (RASCAL) demonstrated that KRAS mutations were associated with histological grade but no other variables[8]. In analysis of the PETACC-3 trial, Roth et al[6] reported associations of KRAS mutations with histological grade and tumor location but not gender. Such inconsistencies may be attributed to differences in the distribution of age, race, stage, or other factors among subject groups.

Currently, no convincing evidence exists that KRAS mutations are independent prognostic factors in CRC. In a Taiwanese study by Liou et al[14], KRAS mutations were not associated with inferior OS; however, the magnitude of multivariate HR (HR = 1.61; 95%CI: 0.91-2.84) was of the same order as that in the present study. A study from Japan revealed that the prognostic impact of KRAS mutations on recurrence-free survival was limited in patients with stage II CRC, and the association of KRAS mutations with OS was not observed[18]. Both studies had a small sample size and heterogeneous cohorts, including stage IV disease. In the large homogeneous cohort in this study, we found significant association of KRAS mutations with inferior DFS and OS. Because we previously found no difference in survival outcomes among different KRAS mutations, including those in exons 2, 3, and 4[23], prognostic analyses of specific codons for these mutations were not performed in the present study. Similarly, the RASCAL study indicated that KRAS mutations resulted in overall poorer prognosis[8], whereas subsequent analysis (RASCAL II) showed that only the glycine to valine substitution in codon 12 (G12V) was associated with poor prognosis in patients with Dukes’ C disease[24]. Furthermore, recent randomized phase III trial results supported KRAS mutations as prognostic factors; 3-year DFS ranged from 72% to 75% across treatments for KRAS wild-type tumors, with 65% to 67% for KRAS mutant tumors[25]. In contrast, in the PETACC-3 trial, no association was found between KRAS mutations and poorer relapse-free survival or OS[6]. Although further research of the prognostic effect of KRAS mutations is needed, the influence of these mutations seems to be mild across previously reported studies.

The frequency of BRAF mutations (5%) and MSI-H (8%) in our cohort was lower than that in Western populations (BRAF: 8%-20%, MSI-H: 11%-17%)[6,9,11-13,15,16] and comparable with that in Asian populations (BRAF: 4%-7%, MSI-H: 6%-12%)[14,18,26]. Generally, BRAF mutations and MSI-H are frequently observed in females, proximal tumors, and poorly differentiated tumors. In a systematic review including 9885 CRC patients, a BRAF mutation was associated with a proximal tumor location, poor differentiation, and female sex[27]. Consistent with this observation, BRAF mutations were more frequent in proximal tumors, poorly differentiated tumors, and females. Previous Western cohorts showed more patients with proximal and poorly differentiated tumors compared with Asian cohorts, including the current cohort. Thus, the discrepancy in BRAF mutations and MSI-H status between Western and Asian populations may be attributed to the different distribution of patients’ characteristics such as gender, tumor location, histological grade, or racial and/or environmental differences.

Most previous studies found associations of BRAF mutations with poorer survival[6,10-12]. In meta-analysis of 26 independent studies (11773 patients), BRAF mutations increased the risk of mortality in CRC patients (HR = 2.25; 95%CI: 1.82-2.83)[28]. However, this evidence is mainly based on studies in Western populations; little is known regarding the prognostic role of BRAF mutations in Asian populations. In a Taiwanese study[14], BRAF mutations were associated with reduced OS, but MSI status was not estimated. In a Japanese study, Nakanishi et al[18] found no such association because of the insufficient number of patients with BRAF mutations. In the present study with larger sample size and homogeneous cohorts, we found associations of BRAF mutations with poorer DFS and OS in CRC patients with stage I-III disease, with the same order of magnitude of HR for OS as in the above meta-analysis. The prognostic effect of BRAF mutations on survival seems to be even stronger than that of KRAS mutations.

In contrast to previous reports[6,9,15-17], our analysis did not show that patients with MSI-H tumors exhibited better survival than those with MSS/MSI-L tumors. However, the number of patients with MSI-H tumors was too small to draw meaningful conclusions regarding the prognostic effect of MSI status. Therefore, additional larger studies are needed to clarify the prognostic impact of MSI status. Inconsistent results were reported regarding the prognostic effect of BRAF mutations according to MSI status[6,10,13]. Samowitz et al[10] found associations of BRAF mutations with poor survival in MSS/MSI-L, but not MSI-H tumors. Meanwhile, French et al[13] reported associations of BRAF mutations with poor survival in MSI-H tumors. In our analysis, associations of BRAF mutations with reduced OS were limited in MSS/MSI-L tumors. However, the BRAF by MSI interaction test was not significant; statistical power was considerably limited due to the small number of patients with MSI-H and BRAF mutant tumors. Larger studies are needed to clarify the modifying effect on the relation between BRAF mutations and survival outcome according to MSI status. Advantages of this study include comprehensive analysis of molecular markers using consistent methodology at a single institution, large sample size, and homogeneous cohort of Japanese patients. These results suggest that constitutive activation of the RAS/RAF/MAPK signaling pathway may be closely associated with clinical prognosis in CRC. Prognostic effects of KRAS and BRAF mutations seem to be consistent across most strata of clinical variables, while the adverse effect of BRAF mutations on OS may be attenuated in stage I-II CRC patients, with marginal statistical significance. The interaction of BRAF mutations with tumor stage warrants further research.

In conclusion, we found that Japanese CRC patients with KRAS or BRAF mutations have poorer survival, independent of MSI status. Additional investigations are warranted to clarify the interaction between these mutations and potential relevant factors, such as MSI status and tumor stage.

COMMENTS
Background

KRAS and BRAF mutations occur in 30%-40% and 4%-20% of colorectal cancers (CRCs), respectively. Microsatellite instability (MSI) is characterized by inactivation of the DNA mismatch repair system and is observed in 5%-15% of CRCs. MSI-high tumors are less likely to metastasize compared with the other phenotypes and have favorable survival outcomes. KRAS mutations are well known as predictive markers of resistance to epidermal growth factor receptor-targeted antibodies, and BRAF mutations are of current interest as a therapeutic target in metastatic CRCs. However, their prognostic value remains controversial for patients with curatively resected CRCs.

Research frontiers

Most previous studies investigating the prognostic role of KRAS and BRAF mutations in CRCs are from Western countries. Genetic background and geographical factors may influence mutation frequency and prognosis; however, few data are available regarding the prognostic role of these genetic alterations in Asian populations. Thus, clinical implications will be obtained by assessing the prognostic value of these mutations in a large cohort of CRCs in Japan, after adjustment for MSI status.

Innovations and breakthroughs

This study is the first large-scale study to demonstrate the prognostic impact of KRAS and BRAF mutations in Asian populations. After adjustment for relevant factors, including MSI, KRAS and BRAF mutations were independently associated with inferior disease-free survival and overall survival in patients with curatively resected CRCs. These findings will offer new insight into prognostic role of KRAS and BRAF mutations in CRCs.

Applications

BRAF and KRAS mutations may be useful as molecular markers for stratification of the clinical prognosis of curatively resected CRCs. Further investigation on whether the prognostic impact of KRAS and BRAF mutations could be modified by MSI status may provide more precise stratification of clinical outcomes in CRC.

Terminology

The protein product of the KRAS gene is a guanosine triphosphate/guanosine diphosphate-binding protein, and KRAS mutations play a key role in the development of various malignancies, including lung cancer, pancreatic cancer, and CRC. The protein product of the BRAF gene, a protein called B-Raf, is a serine/threonine protein kinase serving as downstream effector of the KRAS protein. BRAF mutations are involved in the development of many malignancies, e.g., malignant melanoma, papillary thyroid cancer, and CRC.

Peer review

This is well written and illustrated paper. The authors investigate the prognostic role of KRAS and BRAF mutations after adjustment for MSI status. And they demonstrated that KRAS and BRAF mutations are associated with inferior survival, independent of MSI status in Asian colorectal cancer population. As the authors mentioned, in contrast to previous reports, their analysis did not show that patients with MSI-high tumors exhibited better survival than those with microsatellite-stable/MSI-low tumors.

Footnotes

P- Reviewer: Paoluzi OA, Sakakura C, Tajika M, Wang JY S- Editor: Ma YJ L- Editor: A E- Editor: Wang CH

References
1.  Markowitz SD, Bertagnolli MM. Molecular origins of cancer: Molecular basis of colorectal cancer. N Engl J Med. 2009;361:2449-2460.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1274]  [Cited by in F6Publishing: 1304]  [Article Influence: 86.9]  [Reference Citation Analysis (2)]
2.  Wang L, Cunningham JM, Winters JL, Guenther JC, French AJ, Boardman LA, Burgart LJ, McDonnell SK, Schaid DJ, Thibodeau SN. BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair. Cancer Res. 2003;63:5209-5212.  [PubMed]  [DOI]  [Cited in This Article: ]
3.  Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, Kang GH, Widschwendter M, Weener D, Buchanan D. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38:787-793.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1423]  [Cited by in F6Publishing: 1418]  [Article Influence: 78.8]  [Reference Citation Analysis (0)]
4.  Yagi K, Akagi K, Hayashi H, Nagae G, Tsuji S, Isagawa T, Midorikawa Y, Nishimura Y, Sakamoto H, Seto Y. Three DNA methylation epigenotypes in human colorectal cancer. Clin Cancer Res. 2010;16:21-33.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 172]  [Cited by in F6Publishing: 182]  [Article Influence: 12.1]  [Reference Citation Analysis (0)]
5.  Watanabe T, Yoshino T, Uetake H, Yamazaki K, Ishiguro M, Kurokawa T, Saijo N, Ohashi Y, Sugihara K. KRAS mutational status in Japanese patients with colorectal cancer: results from a nationwide, multicenter, cross-sectional study. Jpn J Clin Oncol. 2013;43:706-712.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 28]  [Cited by in F6Publishing: 31]  [Article Influence: 2.8]  [Reference Citation Analysis (0)]
6.  Roth AD, Tejpar S, Delorenzi M, Yan P, Fiocca R, Klingbiel D, Dietrich D, Biesmans B, Bodoky G, Barone C. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol. 2010;28:466-474.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 801]  [Cited by in F6Publishing: 876]  [Article Influence: 58.4]  [Reference Citation Analysis (0)]
7.  Samowitz WS, Curtin K, Schaffer D, Robertson M, Leppert M, Slattery ML. Relationship of Ki-ras mutations in colon cancers to tumor location, stage, and survival: a population-based study. Cancer Epidemiol Biomarkers Prev. 2000;9:1193-1197.  [PubMed]  [DOI]  [Cited in This Article: ]
8.  Andreyev HJ, Norman AR, Cunningham D, Oates JR, Clarke PA. Kirsten ras mutations in patients with colorectal cancer: the multicenter “RASCAL” study. J Natl Cancer Inst. 1998;90:675-684.  [PubMed]  [DOI]  [Cited in This Article: ]
9.  Hutchins G, Southward K, Handley K, Magill L, Beaumont C, Stahlschmidt J, Richman S, Chambers P, Seymour M, Kerr D. Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. J Clin Oncol. 2011;29:1261-1270.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 473]  [Cited by in F6Publishing: 486]  [Article Influence: 37.4]  [Reference Citation Analysis (0)]
10.  Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA, Wolff RK, Slattery ML. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res. 2005;65:6063-6069.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 564]  [Cited by in F6Publishing: 577]  [Article Influence: 30.4]  [Reference Citation Analysis (0)]
11.  Fariña-Sarasqueta A, van Lijnschoten G, Moerland E, Creemers GJ, Lemmens VE, Rutten HJ, van den Brule AJ. The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients. Ann Oncol. 2010;21:2396-2402.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 177]  [Cited by in F6Publishing: 198]  [Article Influence: 14.1]  [Reference Citation Analysis (0)]
12.  Ogino S, Shima K, Meyerhardt JA, McCleary NJ, Ng K, Hollis D, Saltz LB, Mayer RJ, Schaefer P, Whittom R. Predictive and prognostic roles of BRAF mutation in stage III colon cancer: results from intergroup trial CALGB 89803. Clin Cancer Res. 2012;18:890-900.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 215]  [Cited by in F6Publishing: 219]  [Article Influence: 18.3]  [Reference Citation Analysis (0)]
13.  French AJ, Sargent DJ, Burgart LJ, Foster NR, Kabat BF, Goldberg R, Shepherd L, Windschitl HE, Thibodeau SN. Prognostic significance of defective mismatch repair and BRAF V600E in patients with colon cancer. Clin Cancer Res. 2008;14:3408-3415.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 174]  [Cited by in F6Publishing: 198]  [Article Influence: 12.4]  [Reference Citation Analysis (0)]
14.  Liou JM, Wu MS, Shun CT, Chiu HM, Chen MJ, Chen CC, Wang HP, Lin JT, Liang JT. Mutations in BRAF correlate with poor survival of colorectal cancers in Chinese population. Int J Colorectal Dis. 2011;26:1387-1395.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 26]  [Cited by in F6Publishing: 36]  [Article Influence: 2.8]  [Reference Citation Analysis (0)]
15.  Sinicrope FA, Foster NR, Thibodeau SN, Marsoni S, Monges G, Labianca R, Kim GP, Yothers G, Allegra C, Moore MJ. DNA mismatch repair status and colon cancer recurrence and survival in clinical trials of 5-fluorouracil-based adjuvant therapy. J Natl Cancer Inst. 2011;103:863-875.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 366]  [Cited by in F6Publishing: 383]  [Article Influence: 29.5]  [Reference Citation Analysis (0)]
16.  Ribic CM, Sargent DJ, Moore MJ, Thibodeau SN, French AJ, Goldberg RM, Hamilton SR, Laurent-Puig P, Gryfe R, Shepherd LE. Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med. 2003;349:247-257.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1585]  [Cited by in F6Publishing: 1554]  [Article Influence: 74.0]  [Reference Citation Analysis (0)]
17.  Popat S, Hubner R, Houlston RS. Systematic review of microsatellite instability and colorectal cancer prognosis. J Clin Oncol. 2005;23:609-618.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 1267]  [Cited by in F6Publishing: 1270]  [Article Influence: 66.8]  [Reference Citation Analysis (1)]
18.  Nakanishi R, Harada J, Tuul M, Zhao Y, Ando K, Saeki H, Oki E, Ohga T, Kitao H, Kakeji Y. Prognostic relevance of KRAS and BRAF mutations in Japanese patients with colorectal cancer. Int J Clin Oncol. 2013;18:1042-1048.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 26]  [Cited by in F6Publishing: 33]  [Article Influence: 2.8]  [Reference Citation Analysis (0)]
19.  Sobin LH, Wittekind C.  TNM Classification of Malignant Tumors, 6th edition. New York: Wiley-Liss 2002; .  [PubMed]  [DOI]  [Cited in This Article: ]
20.  Akagi K, Uchibori R, Yamaguchi K, Kurosawa K, Tanaka Y, Kozu T. Characterization of a novel oncogenic K-ras mutation in colon cancer. Biochem Biophys Res Commun. 2007;352:728-732.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 37]  [Cited by in F6Publishing: 37]  [Article Influence: 2.1]  [Reference Citation Analysis (0)]
21.  Asaka S, Arai Y, Nishimura Y, Yamaguchi K, Ishikubo T, Yatsuoka T, Tanaka Y, Akagi K. Microsatellite instability-low colorectal cancer acquires a KRAS mutation during the progression from Dukes’ A to Dukes’ B. Carcinogenesis. 2009;30:494-499.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 53]  [Cited by in F6Publishing: 53]  [Article Influence: 3.5]  [Reference Citation Analysis (0)]
22.  Ishikubo T, Nishimura Y, Yamaguchi K, Khansuwan U, Arai Y, Kobayashi T, Ohkura Y, Hashiguchi Y, Tanaka Y, Akagi K. The clinical features of rectal cancers with high-frequency microsatellite instability (MSI-H) in Japanese males. Cancer Lett. 2004;216:55-62.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 36]  [Cited by in F6Publishing: 39]  [Article Influence: 2.0]  [Reference Citation Analysis (0)]
23.  Ogura T, Kakuta M, Yatsuoka T, Nishimura Y, Sakamoto H, Yamaguchi K, Tanabe M, Tanaka Y, Akagi K. Clinicopathological characteristics and prognostic impact of colorectal cancers with NRAS mutations. Oncol Rep. 2014;32:50-56.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 25]  [Cited by in F6Publishing: 28]  [Article Influence: 2.8]  [Reference Citation Analysis (0)]
24.  Andreyev HJ, Norman AR, Cunningham D, Oates J, Dix BR, Iacopetta BJ, Young J, Walsh T, Ward R, Hawkins N. Kirsten ras mutations in patients with colorectal cancer: the ‘RASCAL II’ study. Br J Cancer. 2001;85:692-696.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 632]  [Cited by in F6Publishing: 649]  [Article Influence: 28.2]  [Reference Citation Analysis (0)]
25.  Alberts SR, Sargent DJ, Nair S, Mahoney MR, Mooney M, Thibodeau SN, Smyrk TC, Sinicrope FA, Chan E, Gill S. Effect of oxaliplatin, fluorouracil, and leucovorin with or without cetuximab on survival among patients with resected stage III colon cancer: a randomized trial. JAMA. 2012;307:1383-1393.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 308]  [Cited by in F6Publishing: 339]  [Article Influence: 28.3]  [Reference Citation Analysis (0)]
26.  Lin CH, Lin JK, Chang SC, Chang YH, Chang HM, Liu JH, Li LH, Chen YT, Tsai SF, Chen WS. Molecular profile and copy number analysis of sporadic colorectal cancer in Taiwan. J Biomed Sci. 2011;18:36.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 17]  [Cited by in F6Publishing: 21]  [Article Influence: 1.6]  [Reference Citation Analysis (0)]
27.  Clancy C, Burke JP, Kalady MF, Coffey JC. BRAF mutation is associated with distinct clinicopathological characteristics in colorectal cancer: a systematic review and meta-analysis. Colorectal Dis. 2013;15:e711-e718.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 80]  [Cited by in F6Publishing: 88]  [Article Influence: 8.0]  [Reference Citation Analysis (0)]
28.  Safaee Ardekani G, Jafarnejad SM, Tan L, Saeedi A, Li G. The prognostic value of BRAF mutation in colorectal cancer and melanoma: a systematic review and meta-analysis. PLoS One. 2012;7:e47054.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 146]  [Cited by in F6Publishing: 164]  [Article Influence: 13.7]  [Reference Citation Analysis (0)]