Ann Surg Oncol DOI 10.1245/s10434-014-3826-z

ORIGINAL ARTICLE – COLORECTAL CANCER

KRAS Mutation is Associated with Worse Prognosis in Stage III or High-risk Stage II Colon Cancer Patients Treated with Adjuvant FOLFOX Dae-Won Lee, MD1, Kyung Ju Kim, MD2, Sae-Won Han, MD, PhD1,3, Hyun Jung Lee, MD1, Ye Young Rhee, MD2, Jeong Mo Bae, MD2, Nam-Yun Cho, MS2, Kyung-Hun Lee, MD1, Tae-Yong Kim, MD1, Do-Youn Oh, MD, PhD1,3, Seock-Ah Im, MD, PhD1,3, Yung-Jue Bang, MD, PhD1,3, Seung-Yong Jeong, MD, PhD4, Kyu Joo Park, MD, PhD4, Jae-Gahb Park, MD, PhD4, Gyeong Hoon Kang, MD, PhD2, and Tae-You Kim, MD, PhD1,3,5 1

Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea; 2Department of Pathology, Seoul National University College of Medicine, Seoul, Korea; 3Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea; 4Department of Surgery, Seoul National University Hospital, Seoul, Korea; 5Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Korea

ABSTRACT Background. Although KRAS mutation has a predictive role in stage IV colorectal cancer (CRC) patients treated with anti-EGFR therapy, there have been controversies in the prognostic impact of KRAS mutation in stage II or III disease. The purpose of this study was to assess the prognostic impact of KRAS and BRAF mutation in patients treated with adjuvant 5-fluorouracil/leucovorin/oxaliplatin (FOLFOX). Methods. KRAS exon 2 and BRAF codon 600 were analyzed in patients with stage II and III CRC who underwent curative resection followed by adjuvant FOLFOX. Clinicopathologic features and disease-free survival (DFS) were compared. Results. Among a total of 437 patients, mutational data of KRAS and BRAF were available in 388 and 433 patients, respectively. KRAS mutation (codon 12 and 13) and BRAF

Dae-Won Lee and Kyung Ju Kim contributed equally to this article, and both should be considered first author. Ó Society of Surgical Oncology 2014 First Received: 11 March 2014 S.-W. Han, MD, PhD e-mail: [email protected] G. H. Kang, MD, PhD e-mail: [email protected]

V600E mutation was found in 26.5 and 3.7 % of patients. DFS was significantly worse in the KRAS mutant patients compared to KRAS wild type patients (3-year DFS 79 and 92 %, p = 0.006). Multivariate analysis revealed KRAS mutation as an independent negative prognostic factor for DFS (adjusted hazard ratio 2.30, 95 % confidence interval 1.23–4.32). Among the various subtypes of KRAS mutation, G13D (3-year DFS 76 %, p = 0.008) was significantly associated with poor DFS, while G12D was not associated with prognosis (3-year DFS 86 %, p = 0.61). There was no association between BRAF mutation and DFS. Conclusions. KRAS mutation has an adverse prognostic impact on stage II or III CRC treated with adjuvant FOLFOX.

KRAS mutation is found in 35–40 % of patients with colorectal cancer (CRC), which are point mutations that occur mostly in codons 12 and 13 of exon 2.1 KRAS mutation is a well-established biomarker predicting lack of benefit from anti-EGFR monoclonal antibodies in stage IV CRC.2,3 Therefore, it is recommended to test KRAS mutational status in every patient with stage IV CRC to guide treatment decisions.4 In contrast, the value of testing KRAS mutation in an earlier stage of the disease has been controversial. Pooled analyses of earlier studies of KRAS mutation in CRC have shown that the mutation is associated with increased risks of recurrence and death.5,6 However, the impact of KRAS mutation on recurrence has been inconsistent in the adjuvant clinical trials evaluating

D. -W. Lee et al.

5-fluorouracil (5-FU)-based treatments or addition of irinotecan to 5-FU/leucovorin.7,8 BRAF is a serine/threonine kinase that regulates cellular signaling pathway downstream of KRAS.9 BRAF mutation and KRAS mutations are usually mutually exclusive, and BRAF mutation is associated with poor response to antiEGFR therapy in metastatic CRC.3 BRAF mutation is associated with distinct clinicopathologic features such as female sex, proximal location, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI).10–12 Although BRAF mutation is associated with adverse clinical outcome in metastatic CRC, there have been controversies in prognostic role of BRAF mutation in other cancer stage.8,13–17 Although KRAS mutation is routinely tested in patients with stage IV CRC as a result of its predictive role in antiEGFR therapy, the clinical value of testing KRAS mutation in patients with stage II or III disease is unclear. Moreover, only limited data are available regarding the association between KRAS and BRAF mutation and treatment outcome in CRC patients treated with adjuvant 5-FU, leucovorin, and oxaliplatin (FOLFOX), which is the current standard care in patients with stage III disease.18 Therefore, we analyzed the KRAS and BRAF mutations in patients treated with adjuvant FOLFOX chemotherapy.

MATERIAL AND METHODS Patients and Treatment Among the stage II or III CRC patient cohort (n = 521) reported previously, only patients who had KRAS (n = 388) or BRAF (n = 433) mutation data were included in this study.19 As previously described, patients with stage III or high-risk stage II CRC who had completed at least 6 cycles of adjuvant FOLFOX chemotherapy after complete resection of the tumor between April 2005 and December 2011 were included. Other eligibility criteria included age over 18 years and adenocarcinoma histology. High-risk stage II was defined if the patient had any of the following: T4 lesion, obstruction or perforation, lymphovascular invasion, perineural invasion, or poorly differentiated histology.20 Patients with upper rectal cancer were included if the patient did not receive pre- or postoperative radiation. Exclusion criteria were the following: previous chemotherapy for CRC, previous radiotherapy for CRC, signet ring cell histology, distant metastasis, and history of other malignancy within 5 years. Patients received FOLFOX chemotherapy as either a FOLFOX-4 or modified FOLFOX-6 regimen.19 None of the patients received anti-EGFR or anti-VEGF treatment as an adjunct to FOLFOX. Adjuvant chemotherapy was planned for a total of 12 cycles. Patients were assessed every 2 weeks

during chemotherapy treatment, and then at least every 6 months for 5 years. The postchemotherapy period assessment included medical history, physical examination, measurement of the carcinoembryonic antigen level, chest computed tomography, and abdominal computed tomography. All patients underwent their operation and subsequent adjuvant chemotherapy at Seoul National University Hospital (SNUH), and eligible patients were identified from the SNUH electronic medical record. The study protocol was reviewed and approved by the SNUH institutional review board. Analysis of KRAS and BRAF DNA was extracted from formalin-fixed, paraffinembedded tumor specimens. Areas in which tumor cells were most dense were delineated by light microscopy of tissue slides. The corresponding areas were marked on 10 serial unstained tissue slides; the marked areas were then manually scraped from the glass slides and collected in microtubes containing lysis buffer solution (50 mM Tris, 1 mM EDTA, pH 8.0, and 1 % Tween-20) and proteinase K (3 mg/ml). The samples were incubated at 55 °C for up to 48 h. After centrifugation, the supernatants were transferred into a newly labeled microtube. The samples were then placed into a 95 °C heat block for 10 min to inactivate the proteinase K. After extraction of the genomic DNA, KRAS exon 2 was amplified by hemi-nested PCR (with rTaq DNA Polymerase, Takara, Kyoto, Japan) with the following primer set: forward primer (50 -ACTGAATATAAACTTGT GGTAGTTGGCCCT-30 ), reverse primer 1 (50 -TAATATG TCGACTAAAACAAGATTTACCTC-30 ), and reverse primer 2 (50 -TCAAAGAATGGTCCTGGACC-30 ). The first PCR reaction had a 20 lL volume and contained: forward primer and reverse primer 1 with a concentration of 400 nM, 19 rTaq PCR buffer, 3 mM MgCl2, 0.625 U of rTaq polymerase, 400 lM of each deoxynucleotide, and 100 ng of genomic DNA. The PCR reaction ran with the following program: 95 °C initial denaturation, 25 cycles of amplification (denaturation at 94 °C for 30 s, annealing at 55 °C for 40 s, and extension at 30 s at 72 °C for primer extension), and a final extension step at 72 °C for 10 min. One microliter of the first PCR product was used for the second PCR, which was conducted in a 25 lL volume containing 19 PCR buffer (16.6 mM (NH4)2SO4; 67 mM Tris, pH 8.8; 6.7 mM MgCl2; and 10 mM b-mercaptoethanol), dNTP (each 1 mM), and primers (0.4 lM each of forward primer and reverse primer 2). Amplifications were carried out in a thermal cycler for 35 cycles (30 s at 95 °C, 40 s at 57 °C, and 30 s at 72 °C) and were given a final 10-min extension at 72 °C. Five microliters of PCR products was treated with 1.2 U of shrimp alkaline phosphatase and 6 U of exonuclease in a final

KRAS Mutation and Adjuvant FOLFOX

volume of 10 lL at 37 °C for 15 min and then heat-inactivated at 80 °C for 15 min. The purified PCR products were sequenced with BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and analyzed with a 3730 ABI capillary electrophoresis system (Applied Biosystems, Foster City). All somatic mutations found were further validated by a new independent amplification and sequencing. BRAF mutations at codon 600 (V600E) were analyzed by a real-time PCR-based allelic discrimination method, as previously described.21

TABLE 1 Patient characteristics Characteristic

No. of patients (n = 437)

Patients with KRAS status (n = 388) Wild type

Mutation

437

285 (73.5 %)

103 (26.5 %)

Median (range)

60 (25–81)

60 (29–78)

59 (36–78)

C65 year

134 (30.7 %)

89 (31.2 %)

29 (28.2 %)

Male

268 (61.3 %)

174 (61.1 %)

62 (60.2 %)

Female

169 (38.7 %)

111 (38.9 %)

41 (39.8 %)

Proximal

144 (33.0 %)

90 (31.5 %)

39 (37.9 %)

Distal

293 (67.0 %)

195 (68.4 %)

64 (62.1 %)

T1

10 (2.3 %)

7 (2.5 %)

0 (0.0 %)

T2

34 (7.8 %)

26 (9.1 %)

5 (4.9 %)

T3

332 (76.0 %)

216 (75.8 %)

80 (77.7 %)

T4

61 (14.0 %)

36 (12.6 %)

18 (17.5 %)

Frequency

p

Age, year 0.56

Sex

Microsatellite Analysis The microsatellite status of each tumor was determined by evaluating five microsatellite markers (D2S123, D5S346, D17S250, BAT25, and BAT26). Either forward or reverse primer for each marker was labeled with fluorescence, and PCR products were electrophoresed and analyzed. We classified MSI status as follows: high (MSIH; instability at 2 or more microsatellite markers), low (MSI-L; instability at 1 marker), or MSS (no instability). Only MSI-H was regarded as having MSI, and MSI-L was grouped with MSS.22 Statistical Analysis The primary objective of this study was to investigate the effect of KRAS mutation and BRAF mutation status on the treatment outcome (disease-free survival, DFS) of CRC patients treated with adjuvant FOLFOX chemotherapy. We chose DFS as the primary objective because 3-year DFS has been shown to have good correlation with 5-year overall survival in colon cancer.23 Secondary objectives were to investigate whether individual subtypes of KRAS mutations have different effects and to investigate the potential interaction between KRAS and BRAF mutations and clinicopathologic characteristics on treatment outcome. The clinical database was last updated in March 2013. DFS was calculated from the date of operation to the first date of documented recurrence or the date of death from any cause. Data from patients who were free of recurrence were censored at the date of the last follow-up visit for DFS. Categorical variables were compared by the chi-square test or Fisher’s exact test. DFS was calculated by the Kaplan– Meier method, and comparisons were made by the log-rank tests. Hazard ratios were calculated by the Cox proportional hazard model, and baseline characteristics were adjusted by using forward stepwise model including covariates that have a prognostic role: age, sex, T stage, N stage, tumor location, mucinous histology, angiolymphatic invasion, venous invasion, perineural invasion, and MSI status. Twosided p values of less than 0.05 were considered statistically

0.88

Location 0.25

T stage 0.15

N stage N0

65 (14.9 %)

46 (16.1 %)

15 (14.6 %)

N1

256 (58.6 %)

166 (58.2 %)

64 (62.1 %)

N2

116 (26.5 %)

73 (25.6 %)

24 (23.3 %)

MAC

20 (4.6 %)

10 (3.5 %)

6 (5.8 %)

Non-MAC

417 (95.4 %)

275 (96.5 %)

97 (94.2 %)

0.80

Histology 0.31

Angiolymphatic invasion Present

185 (42.3 %)

122 (42.8 %)

41 (39.8 %)

Absent

252 (57.7 %)

163 (57.2 %)

62 (60.2 %)

Present

43 (9.8 %)

27 (9.5 %)

12 (11.7 %)

Absent

394 (90.2 %)

258 (90.5 %)

91 (88.3 %)

0.60

Venous invasion 0.53

Perineural invasion Present

95 (21.7 %)

59 (20.7 %)

25 (24.3 %)

Absent

342 (90.2 %)

226 (79.3 %)

78 (75.7 %)

Mutation

16 (3.7 %)

12 (4.3 %)

1 (1.0 %)

Wild type

417 (95.4 %)

270 (95.7 %)

101 (99.0 %)

0.45

BRAF (n = 433) 0.20

Microsatellite status (n = 433) MSS/MSI-L

399 (91.3 %)

259 (91.5 %)

96 (94.1 %)

MSI-H

34 (7.8 %)

24 (8.5 %)

6 (5.9 %)

0.40

MAC mucinous adenocarcinoma, MSS microsatellite stable, MSI-L microsatellite instability-low, MSI-H microsatellite instability-high

D. -W. Lee et al.

significant. Statistical analysis was performed by SPSS software for Windows, version 18.0 (IBM, Armonk, NY, USA). RESULTS Patient Characteristics A total of 437 patients with mutation data of either KRAS or BRAF were included in the present study. Baseline characteristics are summarized in Table 1. Tumor location was cecum in 17, ascending colon in 96, transverse in 31, descending in 28, sigmoid in 236, and rectum in 29 patients. Collectively, 144 patients had tumor in proximal (from cecum to transverse colon) location and 293 patients had tumor in distal location. Tumor stage was stage II in 65 patients (IIA in 42, IIB in 19, and IIC in 4) and III in 372 patients (IIIA in 37, IIIB in 241, and IIIC in 94). All stage II patients had high-risk features. MSI-H was demonstrated in 7.8 % of tumors. According to the inclusion criteria, all patients received at least 6 cycles of chemotherapy, and 89.4 % of patients completed the planned 12 cycles of chemotherapy. Ninety percent of KRAS wild type patients and 88 % of KRAS mutation patients finished 12 cycles of chemotherapy (p = 0.68). Clinicopathologic Characteristics of KRAS or BRAF Mutations One hundred three patients (26.5 %) of 388 patients had the KRAS mutation in either codon 12 or 13. Seventy-one patients had the mutation in codon 12, 31 in codon 13, and 1 in both codon 12 and 13. One patient having concomitant codon 12 (G12D) and 13 (G13D) mutation was excluded from analysis of subtypes. For KRAS codon 12 mutations, we identified 41 patients (39.8 %) with c.35G [ A (p.G12D, codon 12 GGT [ GAT), 17 patients (16.5 %) with c.35G [ T (p.G12 V, codon 12 GGT [ GTT), 5 patients (4.9 %) with c.34G [ T (p.G12C, codon 12 GGT [ TGT), 5 patients (4.9 %) with c.34G [ A (p.G12S, codon 12 GGT [ AGT), and 3 patients (2.9 %) with c.35G [ C (p.G12A, codon 12 GGT [ GCT). In codon 13, all 31 patients (30.1 %) had c.38G [ A (p.G13D, codon 13 GGC [ GAC). Baseline characteristics including age, sex, tumor location, tumor stage, and tumor histology were similar between KRAS mutation and KRAS wild type. Proximal CRC had a higher percentage of KRAS codon 13 mutation compared to distal cancer (13.2 % in proximal location vs. 5.4 % in distal location, p = 0.030). Female patients had similar incidence of KRAS codon 13 mutation regardless of tumor location (9.2 % in proximal location vs. 7.0 % in distal location, p = 0.36); however, male patients had higher percentage of

KRAS codon 13 mutation in a proximal location (16.9 % in proximal location vs. 4.7 % in distal location, p = 0.003). Wild type and codon 12 mutations were equally distributed, regardless of tumor location and patient sex. Other baseline characteristics were similar between codon 13 mutation, codon 12 mutation, and wild type. Among 433 patients, we detected BRAF mutation V600E in 16 patients (3.7 %). Tumors with BRAF mutation were more frequently found in proximal location compared to BRAF wild type (75.0 vs. 31.2 %, p = 0.001); otherwise no significant difference were found when comparing mutant versus wild type. Impact of KRAS and BRAF Mutation on DFS After a median follow-up of 39 months, the 3-year DFS of the entire cohort was 88.2 %. DFS was significantly worse in the KRAS mutation compared to KRAS wild type (3-year DFS 79 and 92 %, respectively; p = 0.006) (Fig. 1a). There was no difference in DFS between BRAF mutation and BRAF wild type patients (3-year DFS 93 and 88 %, respectively; p = 0.43) (Fig. 1b). There was no difference in pattern of recurrence (local recurrence vs. distant metastasis) according to KRAS mutation status. In addition to KRAS mutation, the following clinicopathologic features were associated with poor treatment outcome: T4 stage, N2 stage, angiolymphatic invasion, venous invasion, perineural invasion, and mucinous histology (Table 2). Multivariate analysis using the Cox proportional hazard model revealed that KRAS mutation was an independent negative prognostic factor (Table 3). KRAS mutation was associated with a significantly higher risk of recurrence (adjusted hazard ratio 2.3, 95 % confidence interval 1.23–4.32) compared to KRAS wild type. We next assessed whether the detrimental effect of KRAS mutation was different according to clinicopathological factors (Fig. 2). There was no interaction between KRAS mutation and clinicopathologic characteristics. The effect of KRAS mutation was consistent between the subgroups analyzed. In the analysis of individual subtypes of KRAS mutation, G13D (3-year DFS 76 %, p = 0.008 vs. wild type) and G12C (3-year DFS 33 %, p = 0.002 vs. wild type) were associated with poor DFS compared to wild type (3-year DFS 92 %) (Fig. 3). In contrast, G12D showed comparable DFS with wild type (3-year DFS 86 %, p = 0.61 vs. wild type). DISCUSSION In the present homogenous cohort of stage III or highrisk stage II Korean patients treated with adjuvant FOLFOX, we observed that KRAS mutation was associated with a higher risk of recurrence. The detrimental effect of KRAS

KRAS Mutation and Adjuvant FOLFOX

B 100

Disease-free survival (percentage)

Disease-free survival (percentage)

A

80

60 KRAS WT (N = 285) KRAS MT (N = 103)

40

p = 0.006 20

100

80

60 BRAF WT (N = 417) BRAF MT (N = 16)

40

p = 0.43 20

0

0 0

12

24

36

48

60

72

84

0

12

24

Time (months)

36

48

60

72

84

Time (months)

FIG. 1 Kaplan–Meier curves of disease-free survival according to mutation status of KRAS (a) and BRAF (b). WT wild type, MT mutation TABLE 2 Univariate analysis of DFS Characteristic Sex Age Location Stage T stage N stage Angiolymphatic invasion Venous invasion Perineural invasion Histology KRAS BRAF Microsatellite status

Variable

TABLE 3 Multivariate analysis of disease-free survival 3-year DFS, %

Male

87.8

Female

88.7

p

Characteristic 0.52

Histology

C65 year

89.1 87.7

Proximal

90.2

Distal

87.2

II III

89.8 87.9

0.39

KRAS

0.038

HR hazard ratio, adenocarcinoma

T1–3

89.3 83.9

N0–1

91.2

N2

80.6

Present

80.0

Absent

94.0

Present

78.7

Absent

89.2

Present

80.1

Absent

90.1

MAC

68.1

Non-MAC

89.2

Adjusted HR (95 % CI) p

MAC

4.76 (1.64–13.8)

0.004

Non-MAC 1

\65 year

T4

Variable

0.68 0.23

Angiolymphatic invasion N stage

Present

2.95 (1.50–5.80)

Absent

1

N2

1.99 (1.06–3.74)

N0-1

1

Mutation 2.30 (1.23–4.32) Wild type 1 CI

confidence

interval,

MAC

0.002 0.031 0.009

mucinous

0.001 \0.001 0.017 0.004 0.001

Mutation

78.7

WT

92.1

0.006

Mutation WT

92.9 87.8

0.43

MSS/MSI-L

87.3

0.096

MSI-H

96.9

DFS disease-free survival, MSS microsatellite stable, MSI-L microsatellite instability-low, MSI-H microsatellite instability-high, MAC mucinous adenocarcinoma, WT wild type

mutation on DFS was similar throughout the clinicopathologic subgroups analyzed. However, we could not evaluate MSI status and mucinous histology in the

interaction model as a result of the small number of patients with these characteristics. (Among 103 KRAS mutation patients, only 6 had MSI-H and 6 had mucinous histology.) We also found that proximal CRC had higher percentage of KRAS codon 13 mutation compared to distal CRC. Recent data show that KRAS-mutated cancer is more frequently located in the proximal colon.24 The association between KRAS or BRAF mutation and worse DFS were reported in North Central Cancer Treatment Group N0147 trial, in which stage III colon cancer patients received FOLFOX with or without cetuximab.25 The adverse effect of KRAS mutation is in line with our study. However, we could not find a significant difference in DFS according to BRAF mutation. This may be due to the lower incidence of BRAF mutation in the study population. The prevalence of BRAF mutation, MSI-H, and CIMP-H in CRC is lower in Eastern compared to Western populations.12,26 The detrimental effects of KRAS mutation on recurrence have also been observed in the Quick and Simple and Reliable (QUASAR) trial, which evaluated 5-FU-based adjuvant treatment.16 In contrast, there was no prognostic value of

D. -W. Lee et al. FIG. 2 Forest plot demonstrating the risk of recurrence by KRAS mutation stratified by clinicopathological factors. Hazard ratio stratified by nodal stage was adjusted by histology and angiolymphatic invasion. All other hazard ratios were adjusted by histology, angiolymphatic invasion and nodal stage. HR hazard ratio, CI confidence interval

N

HR (95% Cl)

Interaction p-value

Age < 65

270

2.09 (0.99 – 4.43)

0.75

Age ≥ 65

118

2.83 (0.98 – 8.20)

Male

236

1.88 (0.85 – 4.16)

Female

152

2.95 (1.06 – 8.22)

Proximal

129

4.78 (1.36 – 16.82)

Distal

259

178 (0.85 – 3.74)

T1-3

334

2.01 (1.00 – 4.05)

T4

54

4.61 (1.09 – 19.43)

N0-1

291

1.77 (0.74 – 4.20)

N2

97

2.92 (1.18 – 7.22)

Total

388

2.30 (1.23 – 4.32)

Disease-free survival (percentage)

0.1

1

0.31

0.23

0.36

0.31

10

100

N

3 year DFS (%)

p-vaue vs. WT

Wild type

285

92.1

-

G12D

41

86.2

0.61

G13D

31

75.5

0.008

G12V

17

82.5

0.18

G12C

5

33.3

0.002

G12S

5

100

0.49

G12A

3

50

0.28

Subtype

80

60

40

20

0 0

12

24

36

48

60

72

84

Time (months)

FIG. 3 Kaplan–Meier curves of disease-free survival according to KRAS subtypes. DFS disease-free survival, WT wild type

KRAS mutation in the Pan-European Trials Adjuvant Colon Cancer (PETACC)-3 trial and the Cancer and Leukemia Group B (CALGB) 89803 trial, which were randomized trials to test the addition of irinotecan to 5-FU/leucovorin.8,27 KRAS mutation did not show a prognostic role in the National Surgical Adjuvant Breast and Bowel Project (NSABP) clinical trials C-07 and -08, in which patients treated with bolus 5-FU/leucovorin plus oxaliplatin (FLOX) and FOLFOX were included.28 Different prognostic effects between individual subtypes of KRAS mutation have been investigated in previous studies. In line with the present study, KRAS G13D mutation was associated with a higher risk of death in a population-based cohort.29 Moreover, analysis of KRAS status in the chemotherapy-alone arms of the CRYSTAL

and OPUS trials revealed that G13D mutation is associated with worse outcomes.30 Different biological consequence in G13D mutation is also suggested by the fact that only G13D mutation is sensitive to anti-EGFR monoclonal antibodies, whereas other subtypes are associated with lack of response.30,31 However, other studies have shown the importance of G12V mutation. The Kirsten ras in CRC collaborative group II study (RASCAL II) showed a negative prognostic value of KRAS G12V mutation.6 G12V mutation was also associated with higher CRC-specific mortality in the cohort study by Imamura et al.32 Both KRAS codon 12 and 13 mutations were associated with shorter DFS in patients receiving adjuvant FOLFOX alone or combined with cetuximab (N0147).33 In contrast, we could not find a significant difference in DFS between

KRAS Mutation and Adjuvant FOLFOX

G12V mutation (82.5 %) and wild type (92.1 %). This may be due to the small number of patients with G12V mutation or the effect of adjuvant FOLFOX chemotherapy. The implication of the less frequent subtypes should be investigated in a larger cohort of patients or in a pooled analysis. The limitation of our study is that we had relatively short duration of follow-up and thus could not evaluate overall survival. However, the median follow-up duration had passed 3 years; DFS at that point has been demonstrated to have good correlation with 5-year overall survival in colon cancer.23 In addition, limited sensitivity of direct sequencing used for KRAS testing could have resulted in the relatively lower frequency of mutation. Selection bias of the retrospective design may have also contributed to lower incidence of KRAS and BRAF mutation. Last, the sample size was insufficient to test the meaning of infrequent KRAS subtypes and BRAF mutation. In conclusion, KRAS mutation was independently associated with a higher risk of recurrence in colorectal patients treated with adjuvant FOLFOX. Different subtypes of KRAS mutation had different prognostic implication. Further validation of these findings and elucidation of underlying mechanism of the differences between the subtypes are warranted in the future.

8.

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12. 13.

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16. ACKNOWLEDGMENT Supported in part by Grants from Priority Research Centers Program (2009-0093820) and Basic Science Research Program (NRF-2013R1A1A2058552) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology and the National R&D Program for Cancer Control (0720540), Ministry for Health, Welfare & Family Affairs, Republic of Korea. DISCLOSURE

17.

18.

The authors declare no conflict of interest. 19.

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KRAS mutation is associated with worse prognosis in stage III or high-risk stage II colon cancer patients treated with adjuvant FOLFOX.

Although KRAS mutation has a predictive role in stage IV colorectal cancer (CRC) patients treated with anti-EGFR therapy, there have been controversie...
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