Clinicopathological characteristics and prognostic impact of colorectal cancers with NRAS mutations
- Authors:
- Published online on: May 6, 2014 https://doi.org/10.3892/or.2014.3165
- Pages: 50-56
Abstract
Introduction
The epidermal growth factor receptor (EGFR) is one of the most important molecular targets for advanced colorectal cancer. Activation of this transmembrane receptor tyrosine kinase stimulates signaling pathways supporting cell proliferation, adhesion, migration, evasion of apoptosis, angiogenesis and survival (1–3). Oncogenic signaling pathways downstream of EGFR, including RAS/Raf/MAPK and PI3K/PTEN/Akt pathways, are important mechanisms of tumor progression.
Activating mutations in the RAS oncogene family are present in ~30% of all human cancers. RAS genes encode highly homologous proteins: KRAS, NRAS and HRAS (4). Mutations in the KRAS gene are frequency reported in various human neoplasms, including pancreatic cancer, biliary tract cancer and lung adenocarcinoma (4–6). Cancer types with a high rate of NRAS mutations include myeloid leukemia and cutaneous melanoma, whereas HRAS mutations are typical of bladder and cervical cancers (4,7,8). In colorectal cancer (CRC), rates of KRAS, NRAS and HRAS mutation are 30–42%, 2.2–5% and 0–0.8%, respectively (9–14). Relatively low rates of NRAS and HRAS mutations in CRC remain unexplained.
Anti-EGFR antibody therapy exhibits antitumor effects by inhibiting multiple EGFR signaling pathways, including RAS/RAF/MAPK and PI3K/PTEN/AKT pathways. Clinical trials have demonstrated that anti-EGFR monoclonal antibodies (i.e., cetuximab or panitumumab) are largely ineffective for metastatic CRC patients when tumors harbor mutations in the codon 12 or 13 of KRAS exon 2 (15–20). These mutations cause constitutive activation of the RAS/RAF/MAPK pathway, regardless of EGFR inhibition. Therefore, KRAS exon 2 mutations are recognized as predictive markers of anti-EGFR therapy resistance for metastatic CRC patients. Accordingly, these clinical trials routinely exclude CRC patients harboring KRAS exon 2 mutations.
Recent studies suggest that other activating mutations in KRAS or NRAS, in addition to KRAS exon 2, confer resistance to anti-EGFR therapy (9,11,13,21). Since KRAS and NRAS mutations tend to be mutually exclusive, they may be present in approximately half of metastatic CRC patients (9–14). Therefore, personalized cancer therapy should be tailored to the KRAS and NRAS mutation profile of each patient to improve treatment outcomes.
Previous studies have evaluated the clinicopathological features and prognostic influence of KRAS or BRAF mutations in colorectal cancer. The prognostic value of KRAS mutations in CRC remains controversial (22–24). In contrast, BRAF mutations are associated with proximal colon tumor location, poor differentiation, mucinous component and microsatellite instability. Patients with BRAF-mutated tumors revealed lower survival rates compared with wild-type tumors, particularly those with BRAF-mutated and microsatellite-stable CRC (22,25–27). On the other hand, clinicopathological characteristics, molecular features, and the prognostic value of the NRAS mutation remain largely unknown (10,12). To date, analyses of NRAS mutations in colorectal cancer were performed as part of a subset analysis of clinical studies for treatment of metastatic CRC with anti-EGFR antibodies, and few studies have described NRAS mutations in the early stage of CRC. Irahara et al (12) associated NRAS mutations with left-sided cancers in females, but the data did not reach statistical significance since NRAS mutations were only detected in 5 (2.2%) of the 225 cases. Therefore, the prognostic role and clinical characteristics of NRAS mutations should be clarified using large tissue samples to guide future clinical studies on the predictive impact of the NRAS gene.
The present study used 1,304 consecutive samples of stage 0-IV CRC to investigate the impact of mutations in NRAS exon 2 and 3, in addition to KRAS and BRAF. We evaluated the relationship between NRAS mutations and other clinicopathological or molecular features, including KRAS and BRAF mutations, microsatellite instability (MSI) status and patient survival.
Materials and methods
Patients and tissue samples
The present study was conducted on 1,304 consecutive primary CRC patients at the Saitama Cancer Center from July 1999 to July 2008. Information on clinical data, including age at diagnosis, gender, tumor size, histological differentiation, tumor location, International Union against Cancer (UICC) stage and prognosis were collected from medical records. Tissue samples were surgically excised after obtaining informed consent from each patient. All tumor tissues were paired with normal colorectal tissues and immediately stored at −80°C. The present study was approved by the Ethics Committee of the Saitama Cancer Center.
Mutation analysis of KRAS, BRAF and NRAS
Genomic DNA from each sample was extracted by standard SDS-proteinase K procedure, followed by ethanol precipitation. All tumor samples were tested for KRAS exon 2, 3 and 4; BRAF exon 15 (codon 600); NRAS exon 2 and 3; and MSI status.
KRAS mutations in exon 2 and 3 were detected by denaturing gradient gel electrophoresis (DGGE), and BRAF mutations in exon 15 by PCR-restriction fragment length polymorphism (RFLP), as previously described (28,29).
High resolution melting (HRM) analysis was used to identify mutations in NRAS exon 2 and 3 and in KRAS exon 4 using a Rotor-Gene Q (Qiagen, Hilden, Germany). Primer sets for NRAS were as follows: exon 2, 5′-GGTTTCCAACAGGT TCTTGC-3′ (forward) and 5′-CACTGGGCCTCACCTCTA TG-3′ (reverse); exon 3, 5′-CACACCCCCAGGATTCTTAC-3′ (forward) and 5′-TGGCAAATACACAGAGGAAGC-3′ (reverse). The primer set for KRAS exon 4 was as follows: 5′-GCCTTCTAGAACAGTAGACAC-3′ (forward) and 5′-GA CATAACAGTTATGATTTTGCAGA-3′ (reverse). The reaction mixture contained 7 μl of 2× LightCycler 480 High Resolution Melting Master Reaction Mix (Roche Diagnostics, Mannheim, Germany) with 0.21 μM of each forward and reverse primer, 3.2 mM MgCl2, 20 ng purified genomic DNA, and water to a total volume of 14 μl. PCR cycling and melting conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of 10 sec at 95°C, 20 sec at 57°C, and 10 sec at 72°C. One heteroduplex cycle was performed at 95°C for 1 min and 40°C for 1 min, followed by melting from 72°C to 95°C with 10 acquisitions per °C. HRM data were analyzed using the Rotor-Gene Q software ver.2.0.2.4.
The DNA sequence of NRAS exon 2 and 3 mutations was determined by HRM using primers particularly designed for HRM. Amplified products were labeled with GenomeLab™DTCS Quick Start kit (Beckman Coulter Inc., Fullerton, CA, USA) according to the manufacturer’s instructions and sequenced using the GenomeLab™ GeXP Genetic Analysis System (Beckman Coulter). Sequencing was performed in both directions, and sequence analysis was performed using the GenomeLab Genetic Analysis System v10.2 (Beckman Coulter).
Analysis of microsatellite status
The MSI status was determined using Bethesda markers: BAT25, BAT26, D5S346, D2S123 and D17S250. PCR and subsequent analyses were performed as previously described (30). CRC samples showing instability in two or more markers were defined as microsatellite instability-high (MSI-H), and the ones with none or one marker as microsatellite stable (MSS).
Statistical analysis
Possible associations between each mutation and clinicopathological parameters of CRC were assessed by the Chi-square or Fisher’s exact test for categorical variables and Mann-Whitney U or Kruskal-Wallis test for continuous variables. Overall survival (OS) time was calculated from the date of surgery to the date of death by any cause or censored at the last follow-up visit. Cox proportional hazards analysis was used to estimate clinicopathological- and biomarker-specific survival hazard ratios (HRs) and 95% confidence intervals (CIs). A multivariable model stratification by UICC stage was performed. All P-values were calculated from two-sided test, and P-values <0.05 were considered statistically significant. All statistical analyses were performed with the SPSS Statistics v.20 (SPSS, Inc., Chicago, IL, USA).
Results
Patient characteristics
All 1,304 patients enrolled in the present study were diagnosed with either CRC stage 0 (n=48), stage I (n=248), stage II (n=407), stage III (n=384) or stage IV (n=217) (Table I). Three hundred and seventy-nine cancers were from the proximal colon (cecum to transverse colon), 544 from the distal colon (descending colon to sigmoid colon) and 381 from the rectum. The median follow-up period was 5.6 years (interquartile range, 4.1–7.8 years), during which there were 435 deaths (33%).
Frequency of KRAS, NRAS and BRAF mutations
All 1,304 CRC cases were examined for mutations in KRAS (exon 2, 3 and 4), NRAS (exon 2 and 3) and BRAF (exon 15), as well as MSI status and clinicopathological factors (Table I). KRAS mutations were detected in 42.4% (n=553), NRAS in 2.7% (n=35) and BRAF in 4.5% (n=59) of patients. MSI-H was detected in 5.5% (n=72) of cases. Table II presents changes in the nucleotides and corresponding amino acids detected in NRAS, with p.G12D (codon 12), p.G13R (codon 13) and p.Q61K (codon 61) as the most frequently noted mutations.
Mapping associations between molecular markers revealed that 3 patients had both KRAS and NRAS mutations, whereas 1 patient had both KRAS and BRAF mutations (Fig. 1). KRAS/NRAS mutation combinations were as follows: p.G12D/p.V9A, pG12V/p.R68S and p.G57T/p.G13D. In contrast, NRAS and BRAF mutations were mutually exclusive. Regarding the MRI status, 28 patients with KRAS mutations also had MSI-H tumors compared with 23 patients with BRAF mutations. None of the patients with NRAS mutations had MSI-H tumors.
BRAF mutations were significantly more frequent in MSI-H than in MSS tumors (P<0.001), whereas no significant association was observed between MSI status and KRAS or NRAS mutations.
Frequency of KRAS and NRAS mutations in each exon
In the KRAS gene, most mutations were located in exon 2, with 495 of 1,304 cases (38.0%), whereas exon 3 or 4 mutations were detected in 26 (2.0%) and 32 (2.5%) cases, respectively (Table III). In the NRAS gene, 20 (1.5%) mutations were identified in exon 2 and 15 (1.2%) mutations in exon 3.
Impact of KRAS, NRAS and BRAF mutation status on clinicopathological and molecular characteristics of the colorectal cancer patients
CRC patients were categorized into three groups on the basis of KRAS, NRAS and BRAF mutations, and they were compared in terms of gender, age, colorectal tumor location, tumor maximum size, histological differentiation, mucinous component, depth of tumor invasion, UICC stage, extramural venous invasion and MSI status (Table IV). BRAF-mutated tumors were more frequently associated with mucinous component tumors (KRAS, P=0.003; NRAS, P=0.002), poorly differentiated tumors (KRAS, P<0.001; NRAS, P=0.013), female gender (KRAS, P=0.022) and MSI-H (KRAS and NRAS, P<0.001). NRAS-mutated tumors were more frequently located in the distal colorectum compared with KRAS- or BRAF-mutated tumors (P=0.015 and P<0.001, respectively). Compared with triple wild-type tumors (KRAS, NRAS and BRAF wild-type), KRAS- and BRAF-mutated tumors were more commonly noted in the proximal colon (P<0.001 and P<0.001, respectively), whereas no significant difference was observed between NRAS-mutated tumors and triple wild-type tumors (P=0.201).
Mutation rates of KRAS, NRAS and BRAF for each UICC stage are presented in Fig. 2. KRAS mutations were detected at similar frequencies in stage 0–I to IV. NRAS mutations tended to occur more frequently in stage IV cancers than in stage 0–III cancers compared with KRAS mutations (P=0.061).
Impact of KRAS, NRAS and BRAF mutations on CRC patient survival
Univariate analysis was conducted in regards to age, gender, tumor location, stage, histological subtype, mucinous component, extramural venous invasion, MSI status, KRAS, NRAS and BRAF mutations (Table V). Patients with KRAS and BRAF mutations had significantly worse survival compared with wild-type cases [HR=1.25; 95% confidence interval (CI) 1.03–1.52; P=0.027 and HR=1.73; 95% CI, 1.15–2.60; P=0.009, respectively]. Four other variables were significantly associated with poor prognosis, namely age ≥65 years (HR=1.39; 95% CI, 1.15–1.69; P=0.001), UICC stage (stage II: HR=2.33; stage III: HR=3.58; stage IV: HR=14.14; P<0.001 respectively), histological subtype (HR=1.82; 95% CI, 1.31–2.52; P<0.001), and extramural venous invasion (HR=3.28; 95% CI, 2.50–4.30; P<0.001). The only predictor of good prognosis was female gender (HR=0.73; 95% CI, 0.60–0.89; P=0.002). In multivariable analysis, KRAS and BRAF mutations were associated with significantly higher mortality rates when stratified according to UICC staging (HR=1.44; 95% CI, 1.18–1.79; P<0.001 and HR=2.09; 95% CI, 1.36–3.28; P=0.001, respectively). Notably, NRAS-mutated tumors demonstrated a trend towards favorable prognosis (HR=0.53; 95% CI, 0.27–1.03; P=0.059).
Discussion
The present study investigated clinicopathological and prognostic features of KRAS, NRAS and BRAF mutations in tumors from 1,304 consecutive CRC patients. An important finding was that patients undergoing CRC tumor resection at all stages were targeted by all these mutations. In addition, to the best of our knowledge, this is the first large study to present statistically significant comparisons between these three categories of RAS/RAF mutations in CRC patients.
NRAS mutations were observed in 35 (2.7%) of the 1,304 patients, of which 20 (1.5%) patients revealed a mutation in exon 2 and others (1.2%) in exon 3. These data are consistent with previous studies that reported NRAS mutations in 2.2–5.0% of CRC tumors, with approximately equal frequency in exon 2 and 3 (9,10,12,29). Moreover, we showed that NRAS mutations are detected in early stages of CRC and tend to occur more frequently in stage IV cancers than in stage 0–III cancers. Therefore, NRAS mutations appear to be acquired at early and advanced stages of CRC (31). Nonetheless, the tendency of higher NRAS mutation rates in stage IV CRC should be ascertained in a larger scale study. The frequency of KRAS mutations was also compatible with previous studies, but the frequency of BRAF mutations (4.5%) declined below 7.4–10.1% of the values previously reported (10,13,14). On the other hand, Yokota et al (32) reported an incidence of 4.7% for BRAF mutations (15/319 patients) in Japanese CRC patients. Such agreement with our Japanese study suggests that racial or environmental factors may affect the frequency of BRAF mutations. The strong overlap between BRAF mutation and MSI-H status that we detected suggests that the low frequency in the BRAF mutation is affected by the low MSI-H frequency reported among Asians compared with Westerners (33).
In the present study, 3 cases of 1,304 had mutations in both KRAS and NRAS, which is inconsistent with previous reports of mutual exclusivity. Notably, all three tumors presented rare mutations (NRAS p.V9A, NRAS p.R68S and KRAS p.G57T), whereas these tumors had common mutations (KRAS p.G12D, KRAS p.G12V and NRAS p.G13D). The oncogenic activity of these minor mutations is unknown, except for KRAS p.G57T (28). Therefore, KRAS and NRAS mutation detection only in major mutation lesion may have missed these rare mutations.
Colorectal tumors with NRAS mutations were found more frequently in the distal colon and rectum compared with tumors with KRAS or BRAF mutations, while the distribution of BRAF-mutated tumors was consistent with previous studies reporting that BRAF-mutated tumors are primarily located in the proximal colon (22,32,34). It was proposed that cellular transformation and mutations occur more frequently in the proximal colon due to close contact of epithelial cells with stimulating bowel content (34). However, this theory does not explain the higher frequency of NRAS-mutated tumors in the distal colon and rectum compared with those with KRAS or BRAF mutations. Elucidating factors responsible for distinct locations of KRAS- and NRAS-mutated tumors may be crucial to our understanding of NRAS mutations in CRC patients.
The prognosis of advanced CRC patients carrying NRAS mutations has been reported in the form of subset analysis of clinical studies using anti-EGFR drugs such as cetuximab and panitumumab (9,13,14). Although there is currently no consistent view regarding the efficacy of anti-EGFR drugs for CRC patients with NRAS mutations, several reports consider this form of therapy inappropriate for such CRC patients. However, the fundamental malignant potential of the NRAS mutation should be considered to determine the efficacy of anti-EGFR drugs. To the best of our knowledge, this is the first study to compare patient survival in NRAS-mutated and triple wild-type CRCs. Numerous studies report an association between BRAF mutations and poor clinical outcome (15,22,23,27,32,35). Although the prognostic value of the KRAS mutation is still controversial, several studies suggest a poor prognosis (24,35,36). Since NRAS is a RAS family member, we expected NRAS-mutated CRC patients to have a poor prognosis compared with those without RAS mutations. However, multivariate analysis showed a tendency toward a better prognosis for NRAS-mutated CRC patients. Therefore, the present study suggests that CRC cases with NRAS mutations exhibit different characteristics than CRC cases with KRAS mutations.
Recent studies report that common KRAS exon 2 mutations targeting the RAS/RAF/MAPK pathway are currently used to determine patient eligibility for anti-EGFR monoclonal antibody therapy (19,37,38). Minor mutations (KRAS exon 3 and 4, NRAS, BRAF) are expected to become the next predictive biomarkers. In fact, a recent clinical trial on anti-EGFR monoclonal antibodies excluded patients with the KRAS exon 2 mutation and those with these less frequent mutations (39). In the present study, the incidence of KRAS exon 2 mutations was 38%, whereas the combined mutation rate for KRAS exon 3 and 4, NRAS and BRAF was 49.3%. For stage IV CRC patients subjected to anti-EGFR monoclonal antibody therapy, the total mutation rate increased from 37.3 to 51.6%. If the validity of excluding CRC patients with these minor RAS mutations from anti-EGFR monoclonal antibody therapy is verified by future trials, more than 50% of CRC patients would greatly benefit from personalized medicine for enhanced efficacy and a better prognosis.
In conclusion, the present study demonstrated that KRAS- and NRAS-mutated CRC tumors exhibit distinct characteristics and distributions along the colorectum. Future molecular biology studies should address the significance of these differences between NRAS- and KRAS-mutated CRC and confirm possible positive prognoses associated with NRAS mutations.
Acknowledgements
We would like to thank Akemi Takahashi, Mina Yamada and Syuhei Takahashi for their excellent technical assistance. The present study was supported by the Japanese Ministry of Health, Labour and Welfare.
References
|
Scaltriti M and Baselga J: The epidermal growth factor receptor pathway: a model for targeted therapy. Clin Cancer Res. 12:5268–5272. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Mendelsohn J and Baselga J: Epidermal growth factor receptor targeting in cancer. Semin Oncol. 33:369–385. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Fang JY and Richardson BC: The MAPK signalling pathways and colorectal cancer. Lancet Oncol. 6:322–327. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Schubbert S, Shannon K and Bollag G: Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer. 7:295–308. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Ginesta MM, Mora J, Mayor R, et al: Genetic and epigenetic markers in the evaluation of pancreatic masses. J Clin Pathol. 66:192–197. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Mascaux C, Iannino N, Martin B, et al: The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer. 92:131–139. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Kohlmann A, Grossmann V, Klein HU, et al: Next-generation sequencing technology reveals a characteristic pattern of molecular mutations in 72.8% of chronic myelomonocytic leukemia by detecting frequent alterations in TET2, CBL, RAS, and RUNX1. J Clin Oncol. 28:3858–3865. 2010.PubMed/NCBI | |
|
Uhara H, Ashida A, Koga H, et al: NRAS mutations in primary and metastatic melanomas of Japanese patients. Int J Clin Oncol. June 6–2013.(Epub ahead of print). | |
|
De Roock W, Claes B, Bernasconi D, et al: Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis. Lancet Oncol. 11:753–762. 2010. | |
|
Vaughn CP, Zobell SD, Furtado LV, Baker CL and Samowitz WS: Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer. Genes Chromosomes Cancer. 50:307–312. 2011.PubMed/NCBI | |
|
Loupakis F, Ruzzo A, Cremolini C, et al: KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. Br J Cancer. 101:715–721. 2009. View Article : Google Scholar | |
|
Irahara N, Baba Y, Nosho K, et al: NRAS mutations are rare in colorectal cancer. Diagn Mol Pathol. 19:157–163. 2010. View Article : Google Scholar | |
|
Seymour MT, Brown SR, Middleton G, et al: Panitumumab and irinotecan versus irinotecan alone for patients with KRAS wild-type, fluorouracil-resistant advanced colorectal cancer (PICCOLO): a prospectively stratified randomised trial. Lancet Oncol. 14:749–759. 2013.PubMed/NCBI | |
|
Smith CG, Fisher D, Claes B, et al: Somatic profiling of the epidermal growth factor receptor pathway in tumors from patients with advanced colorectal cancer treated with chemotherapy ± cetuximab. Clin Cancer Res. 19:4104–4113. 2013.PubMed/NCBI | |
|
Farina-Sarasqueta A, van Lijnschoten G, Moerland E, et al: The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients. Ann Oncol. 21:2396–2402. 2010. View Article : Google Scholar | |
|
Van Cutsem E, Kohne CH, Hitre E, et al: Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med. 360:1408–1417. 2009.PubMed/NCBI | |
|
Bokemeyer C, Bondarenko I, Makhson A, et al: Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. J Clin Oncol. 27:663–671. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Karapetis CS, Khambata-Ford S, Jonker DJ, et al: K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl J Med. 359:1757–1765. 2008. View Article : Google Scholar | |
|
Douillard JY, Siena S, Cassidy J, et al: Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study. J Clin Oncol. 28:4697–4705. 2010. View Article : Google Scholar | |
|
Amado RG, Wolf M, Peeters M, et al: Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol. 26:1626–1634. 2008.PubMed/NCBI | |
|
Douillard JY, Oliner KS, Siena S, et al: Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 369:1023–1034. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Samowitz WS, Sweeney C, Herrick J, et al: Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res. 65:6063–6069. 2005.PubMed/NCBI | |
|
Roth AD, Tejpar S, Delorenzi M, et al: 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. 28:466–474. 2010. | |
|
Imamura Y, Morikawa T, Liao X, et al: Specific mutations in KRAS codons 12 and 13, and patient prognosis in 1075 BRAF wild-type colorectal cancers. Clin Cancer Res. 18:4753–4763. 2012.PubMed/NCBI | |
|
Lochhead P, Kuchiba A, Imamura Y, et al: Microsatellite instability and BRAF mutation testing in colorectal cancer prognostication. J Natl Cancer Inst. 105:1151–1156. 2013.PubMed/NCBI | |
|
Gavin PG, Colangelo LH, Fumagalli D, et al: Mutation profiling and microsatellite instability in stage II and III colon cancer: an assessment of their prognostic and oxaliplatin predictive value. Clin Cancer Res. 18:6531–6541. 2012. View Article : Google Scholar | |
|
Ogino S, Shima K, Meyerhardt JA, et al: Predictive and prognostic roles of BRAF mutation in stage III colon cancer: results from intergroup trial CALGB 89803. Clin Cancer Res. 18:890–900. 2012.PubMed/NCBI | |
|
Akagi K, Uchibori R, Yamaguchi K, Kurosawa K, Tanaka Y and Kozu T: Characterization of a novel oncogenic K-ras mutation in colon cancer. Biochem Biophys Res Commun. 352:728–732. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Asaka S, Arai Y, Nishimura Y, et al: Microsatellite instability-low colorectal cancer acquires a KRAS mutation during the progression from Dukes’ A to Dukes’ B. Carcinogenesis. 30:494–499. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Ishikubo T, Nishimura Y, Yamaguchi K, et al: The clinical features of rectal cancers with high-frequency microsatellite instability (MSI-H) in Japanese males. Cancer Lett. 216:55–62. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Andreyev HJ, Norman AR, Cunningham D, Oates JR and Clarke PA: Kirsten ras mutations in patients with colorectal cancer: the multicenter ‘RASCAL’ study. J Natl Cancer Inst. 90:675–684. 1998. | |
|
Yokota T, Ura T, Shibata N, et al: BRAF mutation is a powerful prognostic factor in advanced and recurrent colorectal cancer. Br J Cancer. 104:856–862. 2011. View Article : Google Scholar | |
|
Oh JR, Kim DW, Lee HS, et al: Microsatellite instability testing in Korean patients with colorectal cancer. Fam Cancer. 11:459–466. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Yamauchi M, Morikawa T, Kuchiba A, et al: Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal versus distal colorectum. Gut. 61:847–854. 2012. View Article : Google Scholar | |
|
Richman SD, Seymour MT, Chambers P, et al: KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. J Clin Oncol. 27:5931–5937. 2009. View Article : Google Scholar | |
|
Phipps AI, Buchanan DD, Makar KW, et al: KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumour markers. Br J Cancer. 108:1757–1764. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Tol J, Koopman M, Cats A, et al: Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer. N Engl J Med. 360:563–572. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Bokemeyer C, Bondarenko I, Hartmann JT, et al: Efficacy according to biomarker status of cetuximab plus FOLFOX-4 as first-line treatment for metastatic colorectal cancer: the OPUS study. Ann Oncol. 22:1535–1546. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Fornaro L, Lonardi S, Masi G, et al: FOLFOXIRI in combination with panitumumab as first-line treatment in quadruple wild-type (KRAS, NRAS, HRAS, BRAF) metastatic colorectal cancer patients: a phase II trial by the Gruppo Oncologico Nord Ovest (GONO). Ann Oncol. 24:2062–2067. 2013. View Article : Google Scholar : PubMed/NCBI |
