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Clin Cancer Res. Author manuscript; available in PMC Feb 1, 2013.
Published in final edited form as:
PMCID: PMC3271172
NIHMSID: NIHMS342824
Predictive and Prognostic Roles of BRAF Mutation in Stage III Colon Cancer: Results from Intergroup Trial CALGB 89803
Shuji Ogino,1,2 Kaori Shima,1 Jeffrey A. Meyerhardt,1 Nadine J. McCleary,1 Kimmie Ng,1 Donna Hollis,3 Leonard B. Saltz,4 Robert J. Mayer,1 Paul Schaefer,5 Renaud Whittom,6 Alexander Hantel,7 Al B. Benson, III,8 Donna Spiegelman,9,10 Richard M. Goldberg,11 Monica M. Bertagnolli,12 and Charles S. Fuchs1,10
1Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA; supported by CA32291, CA118553
2Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA
3CALGB Statistical Center, Duke University Medical Center, Durham, NC; supported by CA33601
4Memorial Sloan-Kettering Cancer Center, New York, NY; supported by CA77651
5Toledo Community Hospital Oncology Program, Toledo, OH; NCCTG, supported by CA35415
6Hôpital du Sacré-Coeur de Montréal; NCIC, supported by grant CO15
7Loyola University Stritch School of Medicine, Maywood, IL; SWOG, supported by CA38926, CA32101, CA46282
8Northwestern University, Chicago, IL; supported by CA23318
9Departments of Biostatistics and Epidemiology, Harvard School of Public Health, Boston, MA
10Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA
11University of North Carolina at Chapel Hill, Chapel Hill, NC; supported by CA47559
12Department of Surgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA
Correspondence to: Shuji Ogino, MD, PhD, MS(Epidemiology), Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, 450 Brookline Ave., Room JF-215C, Boston, MA 02215, USA, Tel: 617-632-1972; Fax: 617-582-8558, shuji_ogino/at/dfci.harvard.edu
Purpose
Alterations in the RAS-RAF-MAP2K (MEK)-MAPK signaling pathway are major drivers in colon and rectal carcinogenesis. In colorectal cancer, BRAF mutation is associated with microsatellite instability (MSI), and typically predicts inferior prognosis. We examined the effect of BRAF mutation on survival and treatment efficacy in patients with stage III colon cancer.
Methods
We assessed status of BRAF c.1799T>A (p.V600E) mutation and MSI in 506 stage III colon cancer patients enrolled in a randomized adjuvant chemotherapy trial [5-fluorouracil and leucovorin (FU/LV) vs. irinotecan (CPT11), FU and LV (IFL); CALGB 89803]. Cox proportional hazards model was used to assess the prognostic role of BRAF mutation, adjusting for clinical features, adjuvant chemotherapy arm and MSI status.
Results
Compared to 431 BRAF-wild-type patients, 75 BRAF-mutated patients experienced significantly worse overall survival [OS; log-rank p=0.015; multivariate hazard ratio (HR)=1.66; 95% confidence interval (CI), 1.05-2.63]. By assessing combined status of BRAF and MSI, it appeared that BRAF-mutated MSS (microsatellite stable) tumor was an unfavorable subtype, while BRAF-wild-type MSI-high tumor was a favorable subtype, and BRAF-mutated MSI-high tumor and BRAF-wild-type MSS tumor were intermediate subtypes. Among patients with BRAF-mutated tumors, a non-significant trend toward improved OS was observed for IFL vs. FU/LV arm (multivariate HR=0.52; 95% CI, 0.25-1.10). Among patients with BRAF-wild-type cancer, IFL conferred no suggestion of benefit beyond FU/LV alone (multivariate HR=1.02; 95% CI, 0.72-1.46).
Conclusions
BRAF mutation is associated with inferior survival in stage III colon cancer. Additional studies are necessary to assess whether there is any predictive role of BRAF mutation for irinotecan-based therapy.
Keywords: colorectal cancer, RAS, biomarker, prognosis, response, resistance
BRAF is a part of the RAS-RAF-MAP2K (MEK)-MAPK signaling pathway. BRAF mutations are observed in 10-20% of colon cancers in population-based studies (1-9). In colon cancer, BRAF mutation is associated with proximal tumor location and microsatellite instability (MSI) (1, 3, 10-13), and with significantly worse patient survival in most (1, 6, 14-22), though not all studies (2). In contrast, MSI-high colon cancers have been associated with a significantly improved survival (1, 2, 6, 16, 23), and several studies have suggested the prognostic impact of BRAF mutation status may vary according to the concurrent presence or absence of MSI-high (1, 14, 21). Thus, investigation of the prognostic impact of BRAF mutation or MSI in colon cancer may be most informative when these markers are simultaneously assessed.
The predictive role of BRAF mutation in colon cancer remains less clear. Few studies have examined the impact of BRAF mutation on the efficacy of available chemotherapy regimens (24, 25). A recent analysis of stage III colon cancer patients enrolled in a randomized trial comparing 5-fluorouracil (5-FU) and leucovorin (FU/LV) to irinotecan (CPT11), 5-FU and leucovorin (IFL) (CALGB 89803) suggested that, among patients with MSI-high cancer, IFL conferred a superior disease-free survival when compared to FU/LV (23). In light of the association between BRAF mutation and MSI, we hypothesized that BRAF mutation in colon cancer may similarly influence the efficacy of irinotecan-based chemotherapy in this setting.
We therefore examined prognostic and predictive roles of BRAF mutation among stage III colon cancer patients enrolled in this National Cancer Institute (NCI)-sponsored randomized clinical trial comparing postoperative adjuvant FU/LV to IFL (CALGB 89803) (26). Since data on pathologic stage, performance status, post-operative treatment, follow-up and tumor molecular features such as KRAS and MSI status were carefully recorded in this trial, the simultaneous impact of disease characteristics and the use of adjuvant therapy could be assessed to control for potential confounding. Moreover, the simultaneous impact of BRAF mutational status and MSI on patient outcome could be explored.
Study population
Patients in this study were participants in the National Cancer Institute (NCI)-sponsored Cancer and Leukemia Group B (CALGB) adjuvant therapy trial for stage III colon cancer comparing therapy with the weekly Roswell Park regimen of 5-FU and leucovorin (FU/LV) to weekly bolus regimen of irinotecan, 5-FU, and leucovorin (IFL) (CALGB 89803) (26). Between April 1999 and May 2001, 1,264 patients were enrolled on the treatment trial. Patients in the treatment trial (and thus this companion study) were eligible if they underwent a complete surgical resection of the primary tumor within 56 days prior to study entry, and had regional lymph node metastases (stage III colon cancer) but no evidence of distant metastases. Moreover, patients were required to have a baseline Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 (ambulatory) and have adequate bone marrow, renal and hepatic function. Data on family history of colorectal cancer in first-degree relatives were obtained by questionnaire at diagnosis (26). The current analysis was limited to 506 patients for whom archived formalin-fixed paraffin-embedded tumor tissue and BRAF sequencing data were available. All patients signed informed consent, approved by each site’s institutional review board.
We compared baseline characteristics of the patients who were included in this study (with available BRAF data, N=506) with those who were excluded from this study due to unavailability of tissue data (N=758). We did not detect any significant or substantial difference between these two groups in terms of age, sex, body mass index (BMI), family history, tumor location, pT stage, pN stage, performance status, bowel perforation, bowel obstruction or treatment arm (all p>0.08). In addition, recurrence-free and disease-free survival did not significantly differ in subjects with available BRAF data as compared to those without BRAF data (multivariate HR=0.96; 95% CI, 0.79-1.18; and multivariate HR=0.95; 95% CI, 0.78-1.15, respectively).
As part of the quality assurance program of the CALGB, members of the Audit Committee visit all participating institutions at least once every three years to review source documents. The auditors verify compliance with federal regulations and protocol requirements, including those pertaining to eligibility, treatment, adverse events, tumor response, and outcome in a sample of protocols at each institution. Such on-site review of medical records was performed for a subgroup of 328 patients (26%) of the 1264 patients included in this study.
Definitions of study endpoints
The study endpoints were; (1) recurrence-free survival (RFS), defined as the time from the study enrollment to tumor recurrence or occurrence of a new primary colon tumor; (2) disease-free survival (DFS), defined as time from the study enrollment to tumor recurrence, occurrence of a new primary colon tumor, or death from any cause; and (3) overall survival (OS), defined as the time from the study enrollment to death from any cause. For RFS, patients who died without known tumor recurrence were censored at last documented evaluation by a treating provider.
DNA extraction from tumor, BRAF and KRAS sequencing, and MSI, MLH1 and MSH2 analyses
Tumor molecular analyses were performed blinded to patient and outcome data. DNA was extracted from paraffin-embedded colon cancer tissue (27). We marked tumor areas on H&E slide, and dissected tumor tissue by a sterile needle. PCR and Pyrosequencing spanning BRAF codon 600 (28), and KRAS codons 12 and 13 were performed as previously described (27) in the laboratory at the Dana-Farber Cancer Institute. Our previous study (27) has shown that Pyrosequencing assay is more sensitive than Sanger sequencing (29), and can detect approximately 5-10% of mutant allele among a mixture of mutant and normal alleles. Microsatellite instability (MSI) was assessed by PCR for 10 markers, and MLH1 and MSH2 expression was examined by immunohistochemistry as previously described (23). Tumors with instability in ≥50% of the loci were classified as MSI-high, and those with instability in 0-40% of the loci as microsatellite stable (MSS), and the concordance between MSI testing and immunohistochemistry for MLH1 or MSH2 loss was 97% (23). For 28 cases without PCR MSI results, those with loss of MLH1 or MSH2 were classified as MSI-high, and those with intact expression of MLH1 and MSH2 as MSS. All tumor tissue analyses were performed completely blinded to data patient identity, clinical and outcome data.
Statistical analyses
The goal of this correlative study was to determine whether tumor BRAF mutation status was associated with clinical outcome for patients with stage III colon cancer. Patient registration and clinical data collection were managed by the CALGB Statistical Center, and analyses were conducted collaboratively between the CALGB Statistical Center and Dana-Farber Cancer Institute. All analyses were based on the study database frozen on November 9, 2009, except for the tumor BRAF data. All analyses used SAS version 9.2 (SAS Institute, Cary, NC) and all p values were two-sided.
The Kaplan-Meier method was used to estimate the distribution of survival time according to BRAF status, and the log-rank test was used to compare survival between subgroups. We used the multivariable Cox proportional hazards model to estimate survival hazard ratio (HR) by tumor BRAF status. The following variables were considered in the multivariable analysis: age at study entry (continuous), sex, baseline body mass index (BMI; ≥30 vs. <30 kg/m2), family history of colorectal cancer in first-degree relatives (present vs. absent), baseline performance status (0 vs. 1-2), presence of bowel perforation or obstruction at time of surgery, treatment arm, tumor location (proximal vs. distal), pT stage (pT1-2 vs. pT3 vs. pT4 vs. unknown), pN stage (pN1 vs. pN2), KRAS (wild-type vs. codon 12 mutation vs. codon 13 mutation), and MSI status (high vs. MSS). A backward stepwise elimination with a threshold of p=0.20 was conducted to select covariates in the final model. pT stage was used as a stratifying variable using the strata option in the SAS “proc phreg” command. No collinearity was evident among the variables studied. Although KRAS and BRAF mutations were almost mutually exclusive (Table 1) and KRAS mutation overall did not influence outcome in this dataset (30), we included KRAS codon 12 and 13 mutations separately in the model, to examine codon-specific effects of KRAS mutation. The proportionality of hazards assumption was assessed using standard survival plots and by evaluating a time-dependent variable, which was the cross-product of BRAF and survival time (p=0.011 for RFS; p=0.22 for DFS; p=0.26 for OS). Data were missing on family history in 1% of patients, tumor location in 1% of patients, pN stage in 0.6% of patients, perforation status in 1.8% of patients, obstruction status in 0.6% of patients, and MSI status in 0.2% of patients; those were included in a majority category in multivariable Cox models to maximize the efficiency of multivariable analyses. To assess the potential differential effect of treatment arm according to BRAF status (or combined BRAF and MSI status), we performed a single multivariate Cox regression analysis, in which we could estimate the effect of treatment arm simultaneously in two strata of BRAF status (or in four strata of combined BRAF and MSI status) using a re-parameterization of the interaction term(s) (3). Interaction was also assessed by including the cross product of BRAF and another variable of interest (without data-missing cases) in a multivariate model, using the Wald test.
Table 1
Table 1
Baseline characteristics according to BRAF mutational status in stage III colon cancer
BRAF mutation in stage III colon cancer
Study participants were drawn from a multi-center study of post-operative adjuvant chemotherapy in stage III colon cancer patients who underwent a curative-intent surgical resection (CALGB 89803 protocol) (26). We included 506 cases in the current study based on availability of tumor tissue for BRAF sequencing, which detected c.1799T>A (p.V600E) mutation in 75 (15%) patients. This BRAF mutation frequency is comparable to data in the previous large population-based studies in the U.S. (1, 16). Table 1 summarizes baseline characteristics according to BRAF mutation status. BRAF mutation was significantly associated with female sex, older age, proximal tumor location, microsatellite instability (MSI)-high, and wild-type KRAS (all p<0.0045; a p value for significance was adjusted to p=0.0045 by Bonferroni correction).
Prognostic role of BRAF mutation
With median follow-up of 7.6 years among survivors, there were 183 events for recurrence-free survival (RFS) analysis, 202 events for disease-free survival (DFS) analysis, and 160 events for overall survival (OS) analysis. In a Kaplan-Meier analysis (Figure 1), BRAF-mutated cases experienced a non-significant trend towards inferior RFS and DFS. For BRAF-mutated vs. wild-type cases, 5-year RFS was 60% vs. 65%, and 5-year DFS was 55% vs. 64%, respectively. BRAF mutation was associated a statistically significant reduction in OS (5-year OS: 63% in BRAF-mutant vs. 75% in BRAF-wild-type; log-rank p=0.015).
Figure 1
Figure 1
Figure 1
BRAF mutation and clinical outcome in colon cancer.
In multivariate Cox regression analysis, we examined the prognostic association of BRAF mutation adjusting for other predictors of patient survival (Table 2). Compared to BRAF-wild-type cases, BRAF-mutated cases experienced a significantly worse OS [multivariate hazard ratio (HR)=1.66; 95% confidence interval (CI), 1.05-2.63], adjusting for other factors including MSI and KRAS mutational status. For RFS and DFS analyses, trends were similar in direction, but not statistically significant.
Table 2
Table 2
BRAF c.1799T<A (p.V600E), KRAS and MSI status and clinical outcome in stage III colon cancer
We also examined the associations of MSI and KRAS mutation with patient outcome. Although MSI-high tumors were independently associated with an improved OS [multivariate hazard ratio (HR)=0.61; 95% CI, 0.38-0.97], adjusting for other factors including BRAF and KRAS mutational status, KRAS mutations in either codon 12 or codon 13 were not associated with patient outcome.
Combined BRAF and MSI status, and prognosis
We further categorized patients according to both BRAF and MSI status to assess the joint effect on patient outcome (Table 3). Compared to patients whose tumors were both BRAF-wild-type and MSS (microsatellite stable), those with BRAF-mutated and MSS tumors experienced a trend towards an inferior OS (multivariate HR=1.61; 95% CI, 0.96-2.69). In contrast, compared to BRAF-wild-type MSS patients, those with BRAF-wild-type MSI-high tumors demonstrated consistent trends toward superior RFS, DFS, and OS. Finally, patients with BRAF-mutated MSI-high cancers experienced no significant difference in outcome when compared to BRAF-wild-type MSS patients [multivariate hazard ratio (HR)=1.02; 95% CI, 0.54-1.93], suggesting opposing prognostic effects of BRAF mutation and MSI-high.
Table 3
Table 3
Combined BRAF mutation and MSI status and clinical outcome in stage III colon cancer
Predictive role of BRAF mutation for irinotecan-based therapy
We assessed the prognostic role of BRAF mutation within each treatment arm and the effect of treatment according to BRAF status. Among patients treated with FU/LV, the presence of BRAF mutation was associated with a significantly reduced DFS and OS (multivariate OS HR=2.43; 95% CI, 1.34-4.40) when compared BRAF wild-type tumors (Table 4). In contrast, among subjects treated with IFL, BRAF mutation was not significantly associated with patient outcome (multivariate OS HR=1.24; 95% CI, 0.67-2.31; vs. BRAF-wild-type).
Table 4
Table 4
Stage III colon cancer and clinical outcome according to treatment arm and BRAF mutation status
Among patients with BRAF-mutated tumors, we observed a non-significant trend toward improved RFS, DFS, and OS for subjects treated with IFL when compared with FU/LV (Table 4); however, statistical power was limited and results should be interpreted with caution. Among patients with BRAF wild-type cancer, IFL was associated with no benefit when compared to FU/LV alone.
In a Kaplan-Meier analysis by treatment arm and BRAF status (Figure 1), BRAF-mutated cases treated with FU/LV experienced a significantly worse OS compared to BRAF-mutated cases treated with IFL or to BRAF-wild-type cases in either treatment arm (log-rank p=0.030).
Predictive role of combined BRAF and MSI subtyping for irinotecan-based therapy
We examined the predictive role of combined BRAF and MSI status on adjuvant treatment efficacy (Table 5). Among subjects with either BRAF-wild-type MSS tumors or BRAF-mutated MSI-high tumors, IFL was not associated with any improvement in patient outcome. Although statistical power was limited, among patients with either BRAF-wild-type MSI-high tumors or BRAF-mutated MSS tumors, IFL appeared to confer a consistent trend toward improved RFS, DFS, and OS when compared to FU/LV-treated subjects. In contrast, there appeared to be no appreciable benefit of IFL (compared to FU/LV) among BRAF-mutated MSI-high or BRAF-wild-type MSS patients.
Table 5
Table 5
Effect of treatment arm on stage III colon cancer outcome, according to combined BRAF and MSI status
We also performed analyses for response to IFL (vs. FU/LV) according to MSI status (Supplementary Table 1, Supplementary Figure 1) in the current dataset. There might be a possible beneficial effect of IFL in MSI-high patients, similar to the previous analysis in the CALGB 89803 trial (23).
Finally, we examined treatment effects according to status of BRAF mutation and MLH1 and MSH2 by immunohistochemistry (IHC) with available IHC data. There were 4 cases with MSH2 loss, and all those 4 cases were BRAF-wild-type and likely Lynch syndrome cases. There were 37 cases of MLH1 loss. Among those 37 cases, 17 cases were BRAF-wild-type and included Lynch syndrome cases. Among the 4 cases with MSH2 loss, 2 cases received IFL with no RFS, DFS or OS event (follow-time, 8.1 and 8.5 years). Among the other 2 cases with MSH2 loss in the FU/LV arm, one case experienced a RFS/DFS/OS event at 3.5 years, and the other case was censored at 6.7 years. We analyzed the effects of IFL (vs. FU/LV) in the 17 cases with MLH1 loss and wild-type BRAF, and multivariate HR (with 95% CI) for IFL treatment (vs. FU/LV) was 0.11 (0.011-1.08) for RFS; 0.11 (0.011-1.07) for DFS; 0.33 (0.021-5.40) for OS. These data were suggestive of good response of Lynch syndrome cases to IFL (vs. FU/LV), although statistical power was limited.
In this study of patients with stage III colon cancer participating in the randomized trial comparing post-operative IFL to FU/LV, somatic mutations in BRAF were associated with a statistically significant reduction in OS, with a non-significant trend toward an inferior RFS and DFS. These results persisted in multivariate analyses that adjusted for other predictors for patient outcome, supporting BRAF mutation as an independent prognostic marker in colon cancer. Furthermore, combined BRAF and MSI subtyping analysis suggests that BRAF-mutated MSS tumor is an unfavorable subtype, while BRAF-wild-type MSI-high tumor is a favorable subtype, and BRAF-mutated MSI-high and BRAF-wild-type MSS tumors are intermediate subtypes (Figure 1G). The independent, opposing prognostic effects of BRAF mutation and MSI observed in the current study is also consistent with several previous studies (6, 16-20, 22).
Interestingly, the prognostic association of BRAF mutation appeared to be somewhat attenuated among patients treated with IFL, whereas BRAF mutation was associated with a significant increase in mortality among subjects treated with FU/LV. Among patients with BRAF-mutated colon cancer, IFL might be associated with a non-significant trend toward improved RFS, DFS, and OS compared to FU/LV, whereas there was no apparent benefit by IFL among BRAF wild-type cases. However, statistical power was quite limited and caution must be taken to interpret the results. Additional studies are needed to examine the predictive role of BRAF mutation in colon cancer.
Although a number of studies (31-34) have assessed potential predictive roles of various genetic or tumor biomarkers for irinotecan therapy [e.g., APTX expression (31), ABCB1 polymorphism (32), EGFR and ERCC1 mRNA expression (33)], none of these markers has yet been proven to be clinically useful. A previous analysis of patients in this clinical trial suggested that MSI-high might predict an improved patient outcome for treatment with IFL relative to FU/LV (23), although this finding was not observed in a concurrent trial conducted in Europe (35). Possibly, mismatch repair deficiency may cause DNA repair gene mutations, inhibit the DNA repair process for double strand breaks induced by irinotecan, and thereby potentiate tumor cell death (23).
Analysis of interactions between host factors (e.g., therapy) and tumor markers is increasingly important in cancer research (36, 37). A few previous studies have examined the influence of BRAF status on the effect of chemotherapy in colon cancer (24, 25). In the largest previous analysis, the QUASAR trial (25) observed no predictive role of BRAF mutation for 5-FU-based chemotherapy in stage II colorectal cancer. The MRC FOCUS trial (24) observed greater treatment effects with 5-FU plus oxaliplatin (vs. 5-FU alone) in advanced colorectal cancers with BRAF mutations, compared to smaller effects of 5-FU plus oxaliplatin (vs. 5-FU alone) in BRAF-wild-type cases. In contrast, there was a greater treatment effects on progression-free survival with 5-FU plus irinotecan (vs. 5-FU alone) in advanced colorcetal cancer with wild-type BRAF, compared to smaller effects on progression-free survival with 5-FU plus irinotecan (vs. 5-FU alone); however, there was no significant interaction between BRAF mutation and any of the treatment comparisons (24). Additional studies are necessary to assess efficacy of various treatment regimens in stage III or IV colorectal cancers. A number of studies have assessed a predictive role of BRAF status in targeted therapy against EGFR in stage IV colorectal cancer (38-41); BRAF mutation may have a predictive role for anti-EGFR therapy in monotherapy or in chemorefractory patients, but its predictive role for other settings remains to be fully determined.
Evidence suggests increased sensitivity of cells with defective mismatch repair to irinotecan (42, 43), and improved response of Lynch syndrome MSI-high cancers to 5-FU-based chemotherapy (44). On the other hand, mechanisms underlying the apparent improved outcome for patients with BRAF-mutated colon cancers treated with irinotecan remain speculative. BRAF mutation in colon cancer has been associated with high-level global DNA methylation (45) as well as widespread gene promoter methylation termed the CpG island methylator phenotype (CIMP)-high (46-49). A recent laboratory analysis found that increasing levels of DNA methylation substantially increased sensitivity of cancer cells to camptothecin whereas widespread hypomethylation induced resistance to camptothecin (50). Thus, responsiveness of BRAF-mutated cells to irinotecan may reflect increased DNA methylation associated with BRAF mutation. Confirmation of our observations and elucidation of the exact mechanisms underlying potential responsiveness of BRAF-mutated cells to irinotecan await future studies.
There are several advantages in evaluating prognostic and predictive roles of molecular biomarkers in this NCI-sponsored clinical trial of adjuvant chemotherapy. All patients had stage III colon cancer, reducing the impact of heterogeneity by disease stage. Moreover, treatment and follow-up care were all standardized within the clinical trial, and the date and nature of recurrence were prospectively recorded. In addition, detailed information on other prognostic variables was routinely collected at study entry.
We recognize that patients who enroll in randomized trials may differ from the population-at-large. To participate, patients must meet eligibility criteria, be selected as an appropriate candidate, and be motivated to participate. In addition, patients were particularly selected for this study on the basis of availability of colon cancer tissue specimens. Nonetheless, demographic data of the patients in this study did not suggest considerable selection bias. Moreover, because the study included patients from both community and academic centers across North America, our findings should reflect the general population of stage III patients in North America. In addition, although data on BRAF mutational status were available on a subset of patients enrolled in the trial, baseline characteristics and patient survival did not substantially differ for patients with and without available archived tumor tissue in this trial. Finally, since BRAF status was not available on all patients, statistical power was attenuated. As such, confirmation of our findings is clearly needed.
In conclusion, we found that BRAF mutation was associated with an inferior prognosis in stage III colon cancer patients, supporting tumor BRAF mutation as an independent prognostic biomarker in colon cancer. Although BRAF mutation in stage III colon cancer may possibly predict improved response to irinotecan-based chemotherapy, the predictive role of BRAF mutation testing remains uncertain at this time, and additional trial studies are needed.
Statement of Translational Relevance
BRAF mutation is associated with microsatellite instability (MSI) in colon cancer. Thus, the prognostic role of BRAF mutation or MSI in colon cancer can only be properly assessed when these markers are simultaneously determined. We examined BRAF mutation status in stages III colon cancer patients who enrolled in a phase III trial CALGB 89803, which randomized patients to either a combination of irinotecan, 5-fluorouracil, and leucovorin (IFL) or 5-fluorouracil, and leucovorin (FU/LV). We found that BRAF mutation was independently associated with inferior overall survival. We also observed a non-significant trend toward an improved overall survival of patients randomized to IFL (vs. FU/LV) among BRAF-mutated patients, but not among BRAF-wild-type patients. Our findings provide important data on the prognostic role of BRAF mutation. Whether BRAF status has any predictive role for irinotecan-based chemotherapy needs to be examined by additional studies.
Supplementary Material
Supplementary Figure
Supplementary Figure legend
Supplementary Table
Acknowledgements
We would like to thank the CALGB Pathology Coordinating Office at the Ohio State University for banking and preparing the materials for the study. The following institutions participated in this study:
Baptist Cancer Institute CCOP, Memphis, TN–Lee S. Schwartzberg, M.D., supported by CA71323
Christiana Care Health Services, Inc. CCOP, Wilmington, DE–Stephen Grubbs, M.D., supported by CA45418
Dana-Farber Cancer Institute, Boston, MA–Eric P. Winer, M.D., supported by CA32291
Dartmouth Medical School – Norris Cotton Cancer Center, Lebanon, NH–Marc S. Ernstoff, M.D., supported by CA04326
Duke University Medical Center, Durham, NC–Jeffrey Crawford, M.D., supported by CA47577
Georgetown University Medical Center, Washington, DC–Minetta C. Liu, M.D., supported by CA77597
Cancer Centers of the Carolinas, Greenville, SC–Jeffrey K. Giguere, M.D, supported by CA29165
Hematology-Oncology Associates of Central New York CCOP, Syracuse, NY–Jeffrey Kirshner, M.D., supported by CA45389
Long Island Jewish Medical Center, Lake Success, NY–Kanti R. Rai, M.D., supported by CA11028
Massachusetts General Hospital, Boston, MA–Jeffrey W. Clark, M.D., supported by CA12449
Memorial Sloan-Kettering Cancer Center, New York, NY–Clifford A. Hudis, M.D., supported by CA77651
Missouri Baptist Medical Center, St. Louis, MO–Alan P. Lyss, M.D., supported by CA114558-02
Mount Sinai Medical Center, Miami, FL–Rogerio C. Lilenbaum, M.D., supported by CA45564
Mount Sinai School of Medicine, New York, NY–Lewis R. Silverman, M.D., supported by CA04457
Nevada Cancer Research Foundation CCOP, Las Vegas, NV–John A. Ellerton, M.D., supported by CA35421
North Shore-Long Island Jewish Health System, New Hyde Park, NY – Daniel Budman, MD, supported by CA35279
Rhode Island Hospital, Providence, RI–William Sikov, M.D., supported by CA08025
Roswell Park Cancer Institute, Buffalo, NY–Ellis Levine, M.D., supported by CA02599
Southeast Cancer Control Consortium Inc. CCOP, Goldsboro, NC–James N. Atkins, M.D., supported by CA45808
State University of New York Upstate Medical University, Syracuse, NY–Stephen L. Graziano, M.D., supported by CA21060
The Ohio State University Medical Center, Columbus, OH–Clara D Bloomfield, M.D., supported by CA77658
University of California at San Diego, San Diego, CA–Barbara A. Parker, M.D., supported by CA11789
University of California at San Francisco, San Francisco, CA–Alan P. Venook, M.D., supported by CA60138
University of Chicago, Chicago, IL –Gini Fleming, M.D., supported by CA41287
University of Illinois MBCCOP, Chicago, IL–Lawrence E. Feldman, M.D., supported by CA74811
University of Iowa, Iowa City, IA–Daniel A. Vaena, M.D., supported by CA47642
University of Maryland Greenebaum Cancer Center, Baltimore, MD–Martin Edelman, M.D., supported by CA31983
University of Massachusetts Medical School, Worcester, MA–William V. Walsh, M.D., supported by CA37135
University of Minnesota, Minneapolis, MN–Bruce A Peterson, M.D., supported by CA16450
University of Missouri/Ellis Fischel Cancer Center, Columbia, MO–Michael C Perry, M.D., supported by CA12046
University of Nebraska Medical Center, Omaha, NE–Anne Kessinger, M.D., supported by CA77298
University of North Carolina at Chapel Hill, Chapel Hill, NC–Thomas C. Shea, M.D., supported by CA47559
University of Tennessee Memphis, Memphis, TN–Harvey B. Niell, M.D., supported by CA47555
University of Vermont, Burlington, VT–Hyman B. Muss, M.D., supported by CA77406
Wake Forest University School of Medicine, Winston-Salem, NC–David D Hurd, M.D., supported by CA03927
Walter Reed Army Medical Center, Washington, DC–Thomas Reid, M.D., supported by CA26806
Washington University School of Medicine, St. Louis, MO–Nancy Bartlett, M.D., supported by CA77440
Weill Medical College of Cornell University, New York, NY–John Leonard, M.D., supported by CA07968
Funding: The research for CALGB 89803 was supported, in part, by grants from the National Cancer Institute (CA31946) to the Cancer and Leukemia Group B (currently, Alliance for Clinical Trials in Oncology) (Monica M. Bertagnolli, MD, Chairperson) and to the CALGB Statistical Center (Stephen George, PhD, CA33601), as well as support from Pharmacia & Upjohn Company, now Pfizer Oncology. Each author was supported as listed with affiliations. S.O., J.A.M. and C.S.F. were supported in part by R01 awards from the National Cancer Institute (R01 CA151993 to S.O.; R01 CA149222 to J.A.M.; R01 CA118553 to C.S.F.), and K.N. was supported in part by K07 award from the National Cancer Institute (K07 CA148894). S.O., J.A.M., K.N. and C.S.F. were supported in part by the SPORE grant (P50 CA127003). The sponsors did not participate in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.
Abbreviations
AJCCAmerican Joint Committee on Cancer
CALGBCancer and Leukemia Group B
CIconfidence interval
DFSdisease-free survival
5-FU5-fluorouracil
FU/LV5-fluorouracil and leucovorin
HRhazard ratio
IFLirinotecan, 5-fluorouracil and leucovorin
MSImicrosatellite instability
MSSmicrosatellite stable
NCINational Cancer Institute
OSoverall survival
RFSrecurrence-free survival

Footnotes
Disclosure of potential conflicts of interest:
L.B.S.; consultant to Genomic Health, Genzyme, Asuragen.
R.W.; honorarium from speakers bureau, Hoffmann-La Roche; consultant to Eli-Lilly, Amgen, Novartis, Pfizer, Boehringer Ingelheim.
A.H.; member of Foundation Medicine Advisory Board
A.B.B.; research funding from Pfizer, Imclone, Bristol Myer Squibb, Amgen, Sanofi Aventis; scientific advisor for Pfizer, Imclone, Bristol Myer Squibb, Amgen, Sanofi Aventis.
R.M.G.; research funding from Abbot, Amgen, Bayer, Pfizer, Genentech, Myriad, Sanofi-Aventis; consultant to Genentech, Amgen, Genomic Health, Jennerex, Sanofi-Aventis.
C.S.F.: consultant to Sanofi-Aventis, Pfizer, Genentech, Roche, Bristol Myers Squibb, Amgen.
No other conflicts of interest exist.
1. Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA, et al. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res. 2005;65:6063–9. [PubMed]
2. Barault L, Charon-Barra C, Jooste V, de la Vega MF, Martin L, Roignot P, et al. Hypermethylator phenotype in sporadic colon cancer: study on a population-based series of 582 cases. Cancer Res. 2008;68:8541–6. [PubMed]
3. Nosho K, Irahara N, Shima K, Kure S, Kirkner GJ, Schernhammer ES, et al. Comprehensive biostatistical analysis of CpG island methylator phenotype in colorectal cancer using a large population-based sample. PLoS ONE. 2008;3:e3698. [PMC free article] [PubMed]
4. English DR, Young JP, Simpson JA, Jenkins MA, Southey MC, Walsh MD, et al. Ethnicity and risk for colorectal cancers showing somatic BRAF V600E mutation or CpG island methylator phenotype. Cancer Epidemiol Biomarkers Prev. 2008;17:1774–80. [PubMed]
5. Rozek LS, Herron CM, Greenson JK, Moreno V, Capella G, Rennert G, et al. Smoking, gender, and ethnicity predict somatic BRAF mutations in colorectal cancer. Cancer Epidemiol Biomarkers Prev. 2010;19:838–43. [PMC free article] [PubMed]
6. Dahlin AM, Palmqvist R, Henriksson ML, Jacobsson M, Eklof V, Rutegard J, et al. The Role of the CpG Island Methylator Phenotype in Colorectal Cancer Prognosis Depends on Microsatellite Instability Screening Status. Clin Cancer Res. 2010;16:1845–55. [PubMed]
7. Naguib A, Mitrou PN, Gay LJ, Cooke JC, Luben RN, Ball RY, et al. Dietary, lifestyle and clinicopathological factors associated with BRAF and K-ras mutations arising in distinct subsets of colorectal cancers in the EPIC Norfolk study. BMC Cancer. 2010;10:99. [PMC free article] [PubMed]
8. Limsui D, Vierkant RA, Tillmans LS, Wang AH, Weisenberger DJ, Laird PW, et al. Cigarette Smoking and Colorectal Cancer Risk by Molecularly Defined Subtypes. J Natl Cancer Inst. 2010;102:1012–22. [PMC free article] [PubMed]
9. Hughes LA, Simons CC, van den Brandt PA, Goldbohm RA, de Goeij AF, de Bruine AP, et al. Body size, physical activity and risk of colorectal cancer with or without the CpG island methylator phenotype (CIMP) PLoS One. 2011;6:e18571. [PMC free article] [PubMed]
10. Yamauchi M, Morikawa T, Kuchiba A, Imamura Y, Qian ZR, Nishihara R, et al. Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal vs. distal colorectum. Gut. 2011 [PMC free article] [PubMed]
11. Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet. 2006;38:787–93. [PubMed]
12. Nagasaka T, Koi M, Kloor M, Gebert J, Vilkin A, Nishida N, et al. Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer. Gastroenterology. 2008;134:1950–60. 60 e1. [PMC free article] [PubMed]
13. Suehiro Y, Wong CW, Chirieac LR, Kondo Y, Shen L, Webb CR, et al. Epigenetic-Genetic Interactions in the APC/WNT, RAS/RAF, and P53 Pathways in Colorectal Carcinoma. Clin Cancer Res. 2008;14:2560–9. [PMC free article] [PubMed]
14. French AJ, Sargent DJ, Burgart LJ, Foster NR, Kabat BF, Goldberg R, et al. Prognostic Significance of Defective Mismatch Repair and BRAF V600E in Patients with Colon Cancer. Clin Cancer Res. 2008;14:3408–15. [PMC free article] [PubMed]
15. Zlobec I, Bihl MP, Schwarb H, Terracciano L, Lugli A. Clinicopathological and protein characterization of BRAF- and K-RAS-mutated colorectal cancer and implications for prognosis. Int J Cancer. 2010;127:367–80. [PubMed]
16. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Meyerhardt JA, Loda M, et al. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. 2009;58:90–6. [PMC free article] [PubMed]
17. Roth AD, Tejpar S, Delorenzi M, Yan P, Fiocca R, Klingbiel D, 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. 2010;28:466–74. [PubMed]
18. Kim JH, Shin SH, Kwon HJ, Cho NY, Kang GH. Prognostic implications of CpG island hypermethylator phenotype in colorectal cancers. Virchow Arch. 2009;455:485–94. [PubMed]
19. Souglakos J, Philips J, Wang R, Marwah S, Silver M, Tzardi M, et al. Prognostic and predictive value of common mutations for treatment response and survival in patients with metastatic colorectal cancer. Br J Cancer. 2009;101:465–72. [PMC free article] [PubMed]
20. Farina-Sarasqueta A, van Lijnschoten G, Moerland E, Creemers GJ, Lemmens VE, Rutten HJ, et al. The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients. Ann Oncol. 2010;21:2396–402. [PubMed]
21. Saridaki Z, Papadatos-Pastos D, Tzardi M, Mavroudis D, Bairaktari E, Arvanity H, et al. BRAF mutations, microsatellite instability status and cyclin D1 expression predict metastatic colorectal patients’ outcome. Br J Cancer. 2010;102:1762–8. [PMC free article] [PubMed]
22. Kalady MF, Sanchez JA, Manilich E, Hammel J, Casey G, Church JM. Divergent oncogenic changes influence survival differences between colon and rectal adenocarcinomas. Dis Colon Rectum. 2009;52:1039–45. [PubMed]
23. Bertagnolli MM, Niedzwiecki D, Compton CC, Hahn HP, Hall M, Damas B, et al. Microsatellite Instability Predicts Improves Response to Adjuvant Therapy With Irinotecan, Fluorouracil, and Leucovorin in Stage III Colon Cancer: Cancer and Leukemia Group B Protocol 89803. J Clin Oncol. 2009;27:1814–21. [PMC free article] [PubMed]
24. Richman SD, Seymour MT, Chambers P, Elliott F, Daly CL, Meade AM, 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. 2009;27:5931–7. [PubMed]
25. Hutchins G, Southward K, Handley K, Magill L, Beaumont C, Stahlschmidt J, et al. Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. J Clin Oncol. 2011;29:1261–70. [PubMed]
26. Saltz LB, Niedzwiecki D, Hollis D, Goldberg RM, Hantel A, Thomas JP, et al. Irinotecan fluorouracil plus leucovorin is not superior to fluorouracil plus leucovorin alone as adjuvant treatment for stage III colon cancer: results of CALGB 89803. J Clin Oncol. 2007;25:3456–61. [PubMed]
27. Ogino S, Kawasaki T, Brahmandam M, Yan L, Cantor M, Namgyal C, et al. Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn. 2005;7:413–21. [PubMed]
28. Ogino S, Kawasaki T, Kirkner GJ, Loda M, Fuchs CS. CpG island methylator phenotype-low (CIMP-low) in colorectal cancer: possible associations with male sex and KRAS mutations. J Mol Diagn. 2006;8:582–8. [PubMed]
29. Ogino S, Meyerhardt JA, Cantor M, Brahmandam M, Clark JW, Namgyal C, et al. Molecular alterations in tumors and response to combination chemotherapy with gefitinib for advanced colorectal cancer. Clin Cancer Res. 2005;11:6650–6. [PubMed]
30. Ogino S, Meyerhardt JA, Irahara N, Niedzwiecki D, Hollis D, Saltz LB, et al. KRAS mutation in stage III colon cancer and clinical outcome following intergroup trial CALGB 89803. Clin Cancer Res. 2009;15:7322–9. [PMC free article] [PubMed]
31. Dopeso H, Mateo-Lozano S, Elez E, Landolfi S, Ramos Pascual FJ, Hernandez-Losa J, et al. Aprataxin tumor levels predict response of colorectal cancer patients to irinotecan-based treatment. Clin Cancer Res. 2010;16:2375–82. [PubMed]
32. Glimelius B, Garmo H, Berglund A, Fredriksson LA, Berglund M, Kohnke H, et al. Prediction of irinotecan and 5-fluorouracil toxicity and response in patients with advanced colorectal cancer. Pharmacogenomics J. 2010:1–11. [PMC free article] [PubMed]
33. Vallbohmer D, Iqbal S, Yang DY, Rhodes KE, Zhang W, Gordon M, et al. Molecular determinants of irinotecan efficacy. Int J Cancer. 2006;119:2435–42. [PubMed]
34. Ma LC, Kuo CC, Liu JF, Chen LT, Chang JY. Transcriptional repression of O6-methylguanine DNA methyltransferase gene rendering cells hypersensitive to N,N’-bis(2-chloroethyl)-N-nitrosurea in camptothecin-resistant cells. Mol Pharmacol. 2008;74:517–26. [PubMed]
35. Tejpar S, Bosman F, Delorenzi M, Fiocca R, Yan P, Klingbiel D, et al. Microsatellite instability (MSI) in stage II and III colon cancer treated with 5FU-LV or 5FU-LV and irinotecan (PETACC 3-EORTC 40993-SAKK 60/00 trial) J Clin Oncol. 2009;27 abstract 4001. [PubMed]
36. Ogino S, Galon J, Fuchs CS, Dranoff G. Cancer immunology-analysis of host and tumor factors for personalized medicine. Nat Rev Clin Oncol. 2011 in press (doi:10.1038/nrclinonc.2011.122) [PMC free article] [PubMed]
37. Ogino S, Chan AT, Fuchs CS, Giovannucci E. Molecular pathological epidemiology of colorectal neoplasia: an emerging transdisciplinary and interdisciplinary field. Gut. 2011;60:397–411. [PMC free article] [PubMed]
38. Dasari A, Messersmith WA. New strategies in colorectal cancer: biomarkers of response to epidermal growth factor receptor monoclonal antibodies and potential therapeutic targets in phosphoinositide 3-kinase and mitogen-activated protein kinase pathways. Clin Cancer Res. 2010;16:3811–8. [PubMed]
39. Tejpar S, Bertagnolli M, Bosman F, Lenz HJ, Garraway L, Waldman F, et al. Prognostic and predictive biomarkers in resected colon cancer: current status and future perspectives for integrating genomics into biomarker discovery. Oncologist. 2010;15:390–404. [PMC free article] [PubMed]
40. Lievre A, Blons H, Laurent-Puig P. Oncogenic mutations as predictive factors in colorectal cancer. Oncogene. 2010;29:3033–43. [PubMed]
41. De Roock W, Vriendt VD, Normanno N, Ciardiello F, Tejpar S. KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer. Lancet Oncol. 2011;12:594–603. [PubMed]
42. Rodriguez R, Hansen LT, Phear G, Scorah J, Spang-Thomsen M, Cox A, et al. Thymidine selectively enhances growth suppressive effects of camptothecin/irinotecan in MSI+ cells and tumors containing a mutation of MRE11. Clin Cancer Res. 2008;14:5476–83. [PubMed]
43. Vilar E, Scaltriti M, Balmana J, Saura C, Guzman M, Arribas J, et al. Microsatellite instability due to hMLH1 deficiency is associated with increased cytotoxicity to irinotecan in human colorectal cancer cell lines. Br J Cancer. 2008;99:1607–12. [PMC free article] [PubMed]
44. Sinicrope FA, Foster NR, Thibodeau SN, Marsoni S, Monges G, Labianca R, et al. DNA mismatch repair status and colon cancer recurrence and survival in clinical trials of 5-fluorouracil-based adjuvant therapy. J Natl Cancer Inst. 2011;103:863–75. [PMC free article] [PubMed]
45. Ogino S, Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, et al. LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG methylator phenotype in colorectal cancer. Int J Cancer. 2008;122:2767–73. [PMC free article] [PubMed]
46. Tanaka N, Huttenhower C, Nosho K, Baba Y, Shima K, Quackenbush J, et al. Novel Application of Structural Equation Modeling to Correlation Structure Analysis of CpG Island Methylation in Colorectal Cancer. Am J Pathol. 2010;177:2731–40. [PubMed]
47. Curtin K, Slattery ML, Samowitz WS. CpG island methylation in colorectal cancer: past, present and future. Pathology Research International. 2011;2011:902674. [PMC free article] [PubMed]
48. Lao VV, Grady WM. Epigenetics and colorectal cancer. Nat Rev Gastroenterol Hepatol. 2011 [PMC free article] [PubMed]
49. Hughes LA, Khalid-de Bakker CA, Smits KM, van den Brandt PA, Jonkers D, Ahuja N, et al. The CpG island methylator phenotype in colorectal cancer: Progress and problems. Biochim Biophys Acta. 2011
50. Orta ML, Mateos S, Cortes F. DNA demethylation protects from cleavable complex stabilization and DNA strand breakage induced by the topoisomerase type I inhibitor camptothecin. Mutagenesis. 2009;24:237–44. [PubMed]