Journal of Cancer 2021, Vol.

12 5331

Ivyspring
International Publisher
Journal of Cancer
2021; 12(17): 5331-5337. doi: 10.7150/jca.59193
Research Paper

Prognostic Value of KRAS Exon 3 and Exon 4 Mutations

in Colorectal Cancer Patients
Tianan Guo1,2*, Yuchen Wu1,2*, Dan Huang2,3*, Yutong Jin4, Weiqi Sheng2,3, Sanjun Cai1,2, Xiaoyan Zhou2,3,
Xiaoli Zhu2,3, Fangqi Liu1,2, Ye Xu1,2
1. Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai, China.
2. Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China.
3. Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China.
4. Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA.

*These authors contributed equally to this study.

 Corresponding authors: Xiaoli Zhu, Fang-Qi Liu, Ye Xu, Department of Pathology, Fudan University Shanghai Cancer Center (XLZ), Department of
Colorectal Surgery, Fudan University Shanghai Cancer Center (FQL and YX). No. 270 Dong-An Road, Shanghai 200032, China. Tel: +86-021-64175590, Fax:
+86-021-64035387, E-mail: shhzxl22@[Link] (XLZ); liufq021@[Link] (FQL); yexu@[Link] (YX).

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License ([Link]
See [Link] for full terms and conditions.

Received: 2021.02.08; Accepted: 2021.05.23; Published: 2021.07.03

Abstract
Background: The clinical significance of KRAS exon 3/4 mutations in colorectal cancer (CRC) remains
unclear. We aimed to assess the prognostic value of KRAS exons 3 and 4 mutations to determine the
necessity for their testing.
Methods: KRAS mutations in exon 2/3/4 were evaluated in 1816 stage I-IV patients with colorectal
adenocarcinoma.
Results: The mutation rates of KRAS and KRAS exons 2, 3, and 4 were 49.0%, 43.0%, 1.9%, and 4.1%,
respectively. Univariate survival analysis showed that patients with exon 3 mutation had worse overall
survival (OS) compared to those with KRAS exon 2 mutation or wild-type KRAS (P = 0.044, and P =
0.001). Meanwhile, there was no difference in survival between patients with wild-type KRAS and with
exon 4 mutation (P = 0.128). In multivariate analysis, KRAS mutations in exon 3 and 2 were both
independent factors for worse OS (Exon 3, P = 0.032, HR = 1.861, 95% CI: 1.021-3.391; Exon 2, P =
0.049, HR = 1.298, 95% CI: 1.002-1.682). Among the patients with KRAS exon 2 mutations, those that had
mutations in codon 13 had significantly worse prognosis than those with wild-type KRAS (P = 0.001) or
KRAS codon 12 mutations (P = 0.003).
Conclusions: In KRAS-mutated CRC, exon 3 mutations predict the worst prognosis, while exon 4
mutations predict the best prognosis. Among KRAS exon 2 mutated patients, codon 13 mutations predict
worse prognosis than codon 12 mutations. Mutations of different KRAS exons should be analyzed
separately.
Key words: KRAS exon 3; KRAS exon 4; KRAS mutations; colorectal cancer; clinicopathologic features; prognosis

Introduction
Colorectal cancer (CRC) is the third and second are often found in exons 2, 3, and 4, with KRAS exon 2
most common malignancy in men and women mutations being the most common, accounting for
worldwide, respectively, and the fourth leading cause 81-96% of all KRAS mutations. The remaining 4-19%
of cancer-related mortality [1, 2]. KRAS is one of the of mutations are located in KRAS exons 3 and 4 [4-6].
first genes to be identified as an oncogene in CRC. Despite the lower frequency of mutations in KRAS
Detection of KRAS mutations has emerged as an exons 3 and 4, they should not be neglected given the
important assessment method for patients with CRC high prevalence of CRC.
due to its clinical value in predicting prognosis and Currently, mutations in KRAS exon 2 are
resistance to targeted therapies [3]. KRAS mutations routinely tested for metastatic CRC in most clinical

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institutions, and the identification of such mutations screened for mutations, 1949 patients were identified
has been wildly reported to be associated with poor to have mutations in KRAS, NRAS, or BRAF. We
prognosis and resistance to anti-epidermal growth excluded three patients due to a second primary
factor receptor (EGFR) therapy [3, 7, 8]. In contrast, malignant tumor (two with hepatocellular carcinoma
mutations in exons 3 and exon 4 of KRAS are not and one with lung adenocarcinoma). Further,
widely tested due to their low mutation rate. Thus, considering the effect of BRAF and NRAS mutations
the prognostic value of mutations in KRAS exon 3/4 on prognosis, we also excluded 128 patients with
remains unclear, and patients harboring KRAS infrequent mutations in BRAF (V600E or non-V600E)
mutations in exon 3 or exon 4 are usually combined or NRAS (exon 2/3/4) and 2 patients that harbored
into one group to meet the number requirements for KRAS mutations in both exons 2 and 3 or in exons 2
analysis [5, 9, 10]. Further, no consensus has been and 4. Finally, 1816 patients were included in the
reached on the clinicopathologic features and analysis (Figure 1). This study was approved by the
prognosis of patients with KRAS mutations in exon 3 Research Ethics Committee of the Fudan University
or exon 4 [11-13]. Shanghai Cancer Center in China, and all patients
Specific mutations in the KRAS gene are closely provided written informed consent.
related to the precise treatment of colorectal cancer.
We have previously analyzed the clinicopathologic Mutation analysis
features and prognostic value of KRAS, NRAS, and Mutation analysis was performed in
BRAF mutations in our cohort [14]. The present study formaldehyde fixed-paraffin embedded tissues after
aimed to assess the prognostic value of KRAS confirmation by two pathologists in hematoxylin and
mutations in exons 3 and 4 to elucidate the necessity eosin-stained slides. Genomic DNA was extracted
for their testing as well as to identify the using the QIAamp DNA Mini Kit following the
clinicopathologic characteristics of patients harboring manufacturer’s protocol (Qiagen, Valencia, CA, USA).
these mutations. DNA content was quantified using NanoDrop ND-
1000 (Nanodrop, Wilmington, DE, USA). Sequencing
Methods was performed in 1311 patients. KRAS exon 2, 3, and
4; NRAS exon 2, 3, and 4; and BRAF exon 15 were
Patients amplified using the Real-time PCR master mix
This was a retrospective study of patients who (TOYOBA, Osaka, Japan) and bidirectionally
underwent radical surgery and were pathologically sequenced via ABI 3730XL and BigDye Terminator v.
diagnosed with CRC between July 2010 and June 3.1 Cycle Sequencing kit (Applied Biosystems,
2018. Of the 18604 patients whose tumor tissues were Carlsbad, CA, USA). The positive samples were
further confirmed by three independent
experiments. For the other 505 patients,
amplification refractory mutation system
(ARMS) analysis was conducted for
mutations in KRAS exon 2 (codon 12/13),
3 (codon 59/61), and 4 (codon 117/146)
using the AmoyDx KRAS/NRAS/BRAF
Mutations Detection Kit (Amoy
Diagnostics, Xiamen, China). All
experiments were conducted as per the
manufacturer’s recommendations.
Statistical Analysis
Statistical analysis was performed
using SPSS software version 25.0 (IBM
Corporation, Armonk, NY, USA). A
two-sided P-value < 0.05 was considered
statistically significant. Chi-square tests
and Fisher’s exact tests were used to
compare the categorical variables. For
continuous variables, the Kolmogorov–
Smirnov test was performed to verify the
Figure 1. Patient inclusion flowchart. normal distribution assumptions. The

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exploratory comparison of normally distributed and extranodal tumor deposit. Factors associated with a
non-normally distributed independent groups was high KRAS exon 4 mutation rate included advanced
performed using t-tests and Mann-Whitney U tests, age, tumor type histology, and mucinous carcinoma.
respectively, for comparison between two groups and
using analysis of variance (ANOVA) for comparison Table 1. Mutations detected in the 1816 BRAF/NRAS wild-type
between more than two groups. Logistic regression patients
was used for multivariate analysis. Overall survival
Mutations Codons Amino acid alteration Number of
(OS) was defined as the time between the first surgery patients
and death from any cause. For survival analysis, Wild type 927
KRAS exon 2 Codon 12 p.G12D 174
curves were plotted using the Kaplan-Meier method mutation
and analyzed using the log-rank test. Univariate and p.G12V 121
multivariate analyses to identify prognostic p.G12S 36
p.G12C 26
biomarkers were performed using Cox proportional p.G12A 15
hazard models. p.G12R 8
Other amino acid alteration 7

Results Codon 13
Unknown amino acid alteration
P.G13D
153
184
p.G13R 3
Mutational landscape and clinicopathologic p.G13C 1
characteristics of the patients p.G13V 1
Unknown codon 51
The total KRAS mutation rate was 49.0% KRAS exon 3 Codon 61 p.Q61H 8
(889/1816), while the KRAS exon 2, 3, and 4 mutations mutation
p.Q61L 7
rates were 43.0% (780/1816), 1.9% (34/1816), and 4.1% p.Q61R 5
(75/1816), respectively. The mutation analysis results p.Q61K 2
are shown in Table 1. The patients’ clinicopathologic Codon 59 p.A59T 2
Unknown codon 10
characteristics and the results of univariate analysis KRAS exon 4 Codon 146 p.A146T 44
are shown in Table 2. Meanwhile, the results of mutation
p.A146V 4
multivariate analysis are shown in Table 3. In both p.A146N 1
univariate and multivariate analyses, a high rate of Codon 117 p.K117D 5
KRAS exon 2 mutation was associated with the p.K117N 3
p.K117R 1
following factors: female sex, advanced age, right- Codon 131 p.Q131fs 1
colon tumor, high carcinoembryonic antigen (CEA) Unknown codon 16
levels, stage IV tumor, tumor type histology, and
extranodal tumor deposit. The KRAS exon 3 mutation
rate was higher in female patients and in patients with

Table 2. Univariate analysis of the clinicopathologic features

Variables Wild type KRAS KRAS exon 2 KRAS exon 3 KRAS exon 4
N = 927 (%) Mutant P-value Mutant P-value Mutant P-value Mutant P-value
N = 889 (%) N = 780 (%) N = 34 (%) N = 75 (%)
Sex 0.001 0.003 0.020 0.379
Male 591 (63.8) 501 (56.4) 442 (56.7) 15 (44.1) 44 (58.7)
Female 336 (36.2) 388 (43.6) 338 (43.3) 19 (55.9) 31 (41.3)
Age 60.0 (22-89) 62.0 (20-91) 0.003 62.0 (20-91) 0.011 60.0 (37-90) 0.749 64.0 (34-89) 0.017
Tumor site 0.001 0.001 0.140 0.004
Rectum 430 (46.4) 429 (48.3) 373 (47.8) 12 (35.3) 44 (58.7)
Left 321 (34.6) 186 (20.9) 164 (21.0) 11 (32.4) 11 (14.7)
Right 176 (19.0) 274 (30.8) 243 (31.2) 11 (32.4) 20 (26.6)
Tumor size 4.0 (0.5-17.0) 4.2 (0.5-22.0) 0.040 4.1 (0.5-22.0) 0.089 3.9 (1.5-7.5) 0.933 4.5 (1.2-11.0) 0.014
CEA level 0.001 0.001 0.113 0.175
≤5 μg/L 520 (56.1) 411 (46.2) 361 (46.3) 14 (41.2) (48.0)
>5 μg/L 407 (43.9) 478 (53.8) 419 (53.7) 20 (58.8) 39 (52.0)
T stage 0.676 0.514 0.148 0.665
T1 31 (3.3) 25 (2.8) 21 (2.7) 4 (5.3)
T2 126 (13.6) 107 (12.0) 92 (11.8) 3 (8.8) 12 (16.0)
T3 512 (55.2) 506 (56.9) 453 (58.1) 16 (47.1) 37 (49.4)
T4 258 (27.9) 251 (28.2) 214 (27.4) 15 (44.1) 22 (29.3)
N stage 0.124 0.111 0.114 0.542
N0 399 (43.0) 351 (39.5) 306 (39.2) 10 (29.4) 35 (46.7)
N1-2 528 (57.0) 538 (60.5) 474 (60.8) 24 (70.6) 40 (53.3)

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Variables Wild type KRAS KRAS exon 2 KRAS exon 3 KRAS exon 4
N = 927 (%) Mutant P-value Mutant P-value Mutant P-value Mutant P-value
N = 889 (%) N = 780 (%) N = 34 (%) N = 75 (%)
TNM stage 0.002 0.001 0.057 0.588
I 102 (11.0) 74 (8.3) 60 (7.7) 2 (5.9) 12 (16.0)
II 235 (25.4) 206 (23.2) 180 (23.1) 7 (20.6) 19 (25.4)
III 310 (33.4) 280 (31.5) 251 (32.2) 9 (26.5) 20 (26.6)
IV 280 (30.2) 329 (37.0) 289 (37.0) 16 (47.0) 24 (32.0)
Histological 0.001 0.001 0.596 0.003
Ulcer type 662 (71.4) 565 (63.6) 495 (63.5) 27 (79.4) 43 (57.3)
Tumor type 225 (24.3) 294 (33.1) 257 (32.9) 6 (17.6) 31 (41.3)
Invasive type 40 (4.3) 30 (3.3) 28 (3.6) 1 (3.0) 1 (1.4)
Pathology 0.002 0.010 0.460 0.001
Adenocarcinoma 851 (91.8) 776 (87.3) 687 (88.1) 30 (88.2) 59 (78.7)
Mucinous 76 (8.2) 113 (12.7) 93 (11.9) 4 (11.8) 16 (21.3)
Differentiation 0.023 0.092 0.157 0.011
G3-G4 252 (27.2) 285 (32.1) 241 (30.9) 13 (38.2) 31 (41.3)
G1-G2 675 (72.8) 604 (67.9) 539 (69.1) 21 (61.8) 44 (58.7)
Lymphovascular invasion 0.811 0.990 0.447 0.095
Negative 604 (65.2) 584 (65.7) 508 (65.1) 20 (58.8) 56 (74.7)
Positive 323 (34.8) 305 (34.3) 272 (34.9) 14 (41.2) 19 (25.3)
Perineural invasion 0.164 0.097 0.358 0.237
Negative 618 (66.7) 565 (63.6) 490 (62.8) 20 (58.8) 55 (73.3)
Positive 309 (33.3) 324 (36.4) 290 (37.2) 14 (41.2) 20 (26.6)
Extranodal tumor deposit 0.011 0.013 0.024 0.878
Negative 755 (81.4) 681 (76.6) 597 (76.5) 22 (64.7) 62 (82.7)
Positive 172 (18.6) 208 (23.4) 183 (23.5) 12 (35.3) 13 (17.3)

Table 3. Multivariate analysis of the clinicopathologic features

Variables Multivariate analysis
KRAS KRAS exon 2 KRAS exon 3 KRAS exon 4
P-value OR (95% CI) P-value OR (95% CI) P-value OR (95% CI) P-value OR (95% CI)
Sex (Male) 0.002 0.728 (0.598-0.887) 0.006 0.752 (0.614-0.922) 0.035 0.473 (0.236-0.947)
Age 0.001 1.016 (1.008-1.025) 0.002 1.014 (1.005-1.022) 0.002 1.035 (1.013-1.058)
Tumor site
Rectum Ref 1 Ref 1 Ref 1
Left 0.001 0.535 (0.424-0.675) 0.001 0.545 (0.428-0.694) 0.001 0.328 (0.165-0.651)
Right 0.008 1.388 (1.088-1.773) 0.002 1.475 (1.151-1.892) 0.750 0.909 (0.505-1.637)
CEA level (>5 μg/L) 0.001 1.390 (1.140-1.695) 0.002 1.376 (1.121-1.689)
TNM stage
I Ref 1 Ref 1
II 0.160 1.310 (0.899-1.908) 0.068 1.448 (0.973-2.155)
III 0.244 1.248 (0.860-1.810) 0.066 1.443 (0.975-2.134)
IV 0.008 1.678 (1.143-2.464) 0.002 1.868 (1.246-2.801)
Histological
Ulcer type Ref 1 Ref 1 Ref 1
Tumor type 0.001 1.632 (1.307-2.037) 0.001 1.699 (1.352-2.136) 0.006 2.009 (1.219-3.311)
Invasive type 0.270 0.751 (0.452-1.248) 0.407 0.803 (0.479-1.348) 0.263 0.314 (0.041-2.385)
Pathology (Mucinous) 0.038 1.310 (1.015-1.692) 0.001 3.800 (1.974-7.313)
Extranodal tumor deposit 0.038 1.310 (1.015-1.692) 0.034 1.329 (1.022-1.729) 0.029 2.249 (1.087-4.653)

differentiation (P = 0.002, HR = 1.520, 95% CI:

Survival analysis 1.167-1.978), perineural invasion (P = 0.016, HR =
Univariate analysis revealed that a right-colon 1.385, 95% CI: 1.062-1.805), extranodal tumor deposit
tumor, larger tumor size, higher CEA levels, (P = 0.001, HR = 1.573, 95% CI: 1.198-2.066), and KRAS
neoadjuvant treatment, adjuvant treatment, stage III mutations in exon 2 (P = 0.049, HR = 1.298, 95% CI:
and stage IV tumor, palliative resection, mucinous 1.002-1.682) or 3 (P = 0.032, HR = 1.861, 95% CI:
carcinoma, poor differentiation, perineural invasion, 1.021-3.391) were independently associated with
extranodal tumor deposit, and KRAS mutations in worse OS in multivariate analyses. The survival
exon 2 or 3 were associated with shorter OS. Among curves of patients with wild-type KRAS and KRAS
these factors, larger tumor size (P = 0.001, HR = 1.073, mutations in different exons are shown in Figure 2.
95% CI: 1.028-1.120), stage IV tumor (P = 0.027, HR = Patients who harbored KRAS exon 2 mutations had
3.203, 95% CI: 1.140-9.005), palliative resection (P = poorer prognosis than those with wild-type KRAS (P
0.001, HR = 2.371, 95% CI: 1.678-3.350), poor = 0.010) and KRAS exon 4 mutations (P = 0.022).

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Meanwhile, patients with KRAS exon 3 mutations had mutations in codon 12. To our best knowledge, this is
an even worse prognosis than those with exon 2 the first study to (1) report significant differences in
mutations (P = 0.044). However, patients with KRAS prognosis between KRAS exon 3 and exon 4
exon 4 mutations survived slightly longer than mutations and (2) report worse prognosis of patients
wild-type patients, but the difference was not with KRAS codon 13 mutations compared to those
significant (P = 0.128). with mutations in codon 12. Further, our cohort of
1816 patients is the largest cohort size in which KRAS
exon 3/4 mutations have been tested.

Table 4. Univariate and multivariate analyses of the prognostic

variables for OS
Prognostic variables Univariate analysis Multivariate analysis
P- HR (95% CI) P- HR (95% CI)
value value
Sex (Male) 0.097 0.810 (0.632-1.039)
Age 0.150 0.993 (0.982-1.003)
Tumor site
Rectum Ref
Left 0.233 1.199 (0.890-1.617)
Right 0.001 1.629 (1.206-2.200)
Tumor size 0.001 1.133 (1.083-1.186) 0.001 1.073 (1.028-1.120)
Elevated CEA level 0.001 1.862 (1.439-2.408)
Neoadjuvant treatment 0.001 2.062 (1.575-2.700)
Adjuvant treatment 0.001 1.972 (1.401-2.775)
TNM stage
I Ref Ref
II 0.262 1.881 (0.624-5.668) 0.541 1.412 (0.467-4.270)
III 0.012 3.705 (1.330-10.322) 0.114 2.300 (0.818-6.469)
IV 0.001 10.058(3.735-27.084) 0.027 3.203(1.140-9.005)
Palliative resectiona 0.001 4.310 (3.347-5.552) 0.001 2.371(1.678-3.350)
Histology
Ulcer type Ref
Tumor type 0.260 0.849 (0.639-1.129)
Invasive type 0.325 1.285 (0.780-2.116)
Pathology (mucinous) 0.001 1.741 (1.261-2.404)
Differentiation (G3-G4) 0.001 2.271 (1.773-2.920) 0.002 1.520 (1.167-1.978)
Figure 2. Kaplan-Meier analysis of patients with KRAS exon 2, 3, or 4 mutations. Lymphovascular invasion 0.001 2.100 (1.639-2.691)
Perineural Invasion 0.001 2.042 (1.591-2.622) 0.016 1.385 (1.062-1.805)
Extranodal tumor deposit 0.001 2.718 (2.116-3.493) 0.001 1.573 (1.198-2.066)
KRAS exon 2 mutant 0.010 1.399 (1.084-1.806) 0.049 1.298 (1.002-1.682)
Of the 780 patients with KRAS exon 2 mutations, KRAS exon 3 mutant 0.001 2.518 (1.389-4.567) 0.032 1.861 (1.021-3.391)
540 and 189 patients had mutations in codon 12 and KRAS exon 4 mutant 0.128 0.569 (0.205-1.580) 0.141 0.472 (0.174-1.282)

13, respectively. Patients with mutations in codon 13 Cases were considered as palliative excision when primary and metastatic lesions
a)

were not both radically resected.

had worse prognosis than wild-type patients (P =
0.001; Figure 3) and patients with mutations in codon
12 (P = 0.003). Patients with KRAS codon 12 mutations Despite evidence that mutations in RAS genes,
tended to have worse prognosis than wild-type particularly KRAS, play an essential role in predicting
patients, although it was not statistically significant (P resistance to anti-EGFR therapy and worse prognosis
= 0.245). The survival of patients having codon 59/61 in CRC patients, only a few studies have investigated
and codon 117/131/146 mutations was not analyzed the clinical value of mutations in exons 3 and 4.
because of the small number of patients with these Information on the clinical relevance of KRAS
mutations. mutations is largely based on KRAS exon 2 testing.
Although KRAS exon 3/4 mutations are associated
Discussion with clinicopathologic features or patient survival, the
In this study, we retrospectively analyzed the necessity of KRAS exon 3/4 testing has not been
prognostic value of mutations in KRAS exons 2, 3, and determined to date. Thus, we conducted this single-
4. We found that patients with KRAS exon 3 center retrospective study to explore the impact of
mutations had the worst prognosis, while patients KRAS exons 3 and 4 mutations on patient survival.
with exon 4 mutations had the best prognosis. Further It has been well demonstrated that patients
analysis of codon 12 and 13 sequences in KRAS exon 2 harboring KRAS exon 2 mutations do not benefit from
mutation showed that patients harboring mutations anti-EGFR therapy. Lièvre et al. [3] found that KRAS
in codon 13 had worse prognosis than those with exon 2 mutations were associated with resistance to
cetuximab and poor prognosis. Amado et al. [8] also

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reported that KRAS exon 2 mutations influence KRAS exon 2 mutations were detected in 42.4%
response to panitumumab. Recent clinical trials also (900/2121) of their patients, while mutations in exon 3
showed that patients with KRAS exons 3 and 4 and 4 were detected in only 3.7% (19/513) and 3.3%
mutations lack response to anti-EGFR therapy. (17/513) of 513 exon 2 wild-type patients,
However, patients with KRAS mutations in exons 3 or respectively. However, in their study, no mutations in
4 were usually analyzed together as one group due to codons 59 or 117 were detected, which might explain
the small cohort size. In the PRIME study, mutations why their results differ from ours. Similarly,
in the exon 3 of KRAS were detected in 24 out of 641 Douillard et al. [5] reported mutation rates of KRAS
metastatic CRC patients, while 36 patients were mutations in exons 2, 3, and 4 of 40.1% (440/1096),
detected with mutations in exon 4. These patients, 3.7% (24/641), and 5.6% (36/641), respectively. KRAS
along with 48 patients with NRAS mutations, were codon 61 mutations were also detected in that study,
further analyzed as one group [5]. Similar situation but mutations in codon 59 were not. In an Eastern
was also seen in the CRYSTAL study, where 63 population, Guo et al. [16] evaluated 353 stage I-IV
patients harboring mutations in KRAS exons 3/4 or Chinese CRC patients, and the KRAS mutation rates
NRAS exon 2/3/4 were categorized into one group in exons 2, 3, and 4 were 42.2% (149/353), 2.3%
[10]. These findings formed the basis for international (8/353), and 8.2% (29/353), respectively. The
guidelines to recommend that all patients with differences in mutation rates might be due to the
metastatic CRC should be tested for KRAS exon 2/3/4 differences in cohort size.
mutations and that patients detected with mutations With respect to clinicopathologic features, our
in any of these exons should not be treated with study showed that KRAS mutations in exon 2/3 were
anti-EGFR therapy. However, we believe that associated with a high rate of positive extranodal
randomized, controlled trials with larger patient tumor deposit, and extranodal tumor deposit was
cohorts are needed to determine whether these known as a predictor of poor prognosis [17, 18]. This
findings are also representative for patients harboring might be related to the poor prognosis of patients
mutations in exons 3 or 4. harboring KRAS exon 2/3 mutations. No relevant
findings from other studies have been reported yet.
The prognostic value of KRAS exon 3/4
remained unclear before the current study [19-21].
Similar survival trends of patients with mutations in
KRAS exon 2/3/4 were reported by Frankel et al. [22]
in a cohort of 165 stage IV CRC patients, although the
small cohort size did not allow for robust statistical
analyses. In most studies, patients with mutations in
KRAS exon 3/4 were combined for analysis [11-13],
which was not reasonable according to our results
because mutations in these exons resulted in very
different prognosis.
The poor prognosis of patients with KRAS exon 2
mutations has been widely reported. However, the
prognostic value of mutations in codons 12/13 is
controversial. In a study of 1075 stage I-IV CRC
patients by Imamura et al. [23], survival analysis
showed that mutations in codon 12, but not codon 13,
was associated with a worse prognosis than wild-type
KRAS. Similar results were found by Margonis et al.
[24] in a study of 512 stage IV CRC patients. Passot et
al. [25] evaluated 524 stage IV CRC patients and
Figure 3. Kaplan-Meier analysis of patients with KRAS mutations in codon 12 or 13 of
reported that patients with mutations in codon 12 or
exon 2. 13 had worse prognosis than KRAS wild-type
patients. However, there was no significant difference
in the prognosis of patients with codon 12 and codon
The KRAS exon 2/3/4 mutation rates in the 13 mutations, consistent with the findings of some
current study cohort were consistent with those in other studies [13, 26]. The varying results in these
two previous studies conducted in relatively large studies could be caused by the differences in cohort
populations [5, 15]. In the study by Vaughn et al. [15], size, data analysis methods, or race.

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Acknowledgements KRAS and BRAF mutations in advanced colorectal cancer are associated with
poor prognosis but do not preclude benefit from oxaliplatin or irinotecan:
The authors would thank all the patients results from the MRC FOCUS trial. J Clin Oncol. 2009; 27: 5931–7.
included and the pathologists of the Department of 21. Taieb J, Le Malicot K, Shi Q, Penault-Llorca F, Bouché O, Tabernero J, et al.
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Center participating in this study. This work was 22. Frankel TL, Vakiani E, Nathan H DeMatteo RP, Kingham TP, Allen PJ, et al.
Mutation location on the RAS oncogene affects pathologic features and
supported by the Shanghai Committee of Science and survival after resection of colorectal liver metastases. Cancer-Am Cancer Soc.
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Technology [grant numbers 20DZ1100101, 23. Imamura Y, Morikawa T, Liao X, Lochhead P, Kuchiba A, Yamauchi M, et al.
18140903702, and 19511121202]. Specific mutations in KRAS codons 12 and 13, and patient prognosis in 1075
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Competing Interests 13 KRAS mutation predicts patterns of recurrence in patients undergoing
hepatectomy for colorectal liver metastases. Cancer-Am Cancer Soc. 2016; 122:
The authors have declared that no competing 2698–707.
interest exists. 25. Passot G, Denbo JW, Yamashita S, Kopetz SE, Chun YS, Maru D, et al. Is
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