Oncology 2014;87:7–20
DOI: 10.1159/000360989
Received: May 17, 2013
Accepted after revision: February 21, 2014
Published online: June 24, 2014

© 2014 S. Karger AG, Basel
0030–2414/14/0871–0007$39.50/0
www.karger.com/ocl

Clinical Study

Phase II Trial of Cetuximab plus Irinotecan
for Oxaliplatin- and Irinotecan-Based
Chemotherapy-Refractory Patients with
Advanced and/or Metastatic Colorectal
Cancer: Evaluation of Efficacy and Safety
Based on KRAS Mutation Status (T-CORE0801)
Hiroshi Soeda a, b Hideki Shimodaira a, b Makio Gamoh f Hideaki Ando g
Hideki Isobe h Takeshi Suto i Shin Takahashi a, b Yuichi Kakudo a, b
Kenji Amagai j Takahiro Mori c Mika Watanabe d Takuhiro Yamaguchi e
Shunsuke Kato a, b Chikashi Ishioka a–ca
Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku
University, b Department of Clinical Oncology, c Cancer Center and d Department of
Pathology, Tohoku University Hospital, and e Division of Biostatistics, Tohoku University
Graduate School of Medicine, Sendai, f Department of Clinical Oncology, South Miyagi
Medical Center, Ogawara, g Department of Surgery, Nakadori General Hospital, Akita,hDepartment of Surgery, Yamagata University Hospital, and i Department of Surgery,Yamagata Prefectural Central Hospital, Yamagata, and j Division of Gastroenterology and
Gastrointestinal Oncology, Ibaraki Prefectural Central Hospital and Cancer Center, Ibaraki,Japan

Key Words
Cetuximab · Colorectal carcinoma · Epidermal growth factor receptor · KRAS
Abstract
Background: Mutations in the KRAS gene have been identified as negative predictors of response to anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapy by patients with metastatic colorectal cancer (mCRC). However, it has been based on the study ofmainly Caucasian mCRC patients. This prospective study investigated the relationship between the mutation status of EGFR-related genes including KRAS and the response rate (RR)
to cetuximab plus irinotecan therapy in Japanese mCRC patients. Methods: Samples takenfrom 43 chemotherapy-refractory mCRC patients who had undergone cetuximab plus irino-

Chikashi Ishioka, MD, PhD
Department of Clinical Oncology
Institute of Development, Aging and Cancer, Tohoku University
4-1, Seiryo-machi, Aobaku, Sendai 980-8575 (Japan)
E-Mail chikashi @ idac.tohoku.ac.jp

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Hiroshi Soeda and Hideki Shimodaira contributed equally to this work.

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Oncology 2014;87:7–20
DOI: 10.1159/000360989© 2014 S. Karger AG, Baselwww.karger.com/ocl

Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy Resistancetecan therapy at 11 medical centers in Japan were subjected to direct DNA sequencing todetermine the KRAS, BRAF, PIK3CA, NRAS, and AKT1 mutation status. The clinical outcome
after the treatment was evaluated for each mutation status. Results: KRAS mutations weredetected in 31.7% of 41 eligible patients. The RR to cetuximab plus irinotecan therapy was
found to be 17.9 and 0% in the KRAS wild-type and mutant subgroups, respectively. Conclusion: Despite the identification of a lower-than-expected RR to treatment by the KRAS wildtype subgroup, KRAS mutation status appears to be a useful predictive marker of responseto cetuximab plus irinotecan therapy in Japanese mCRC patients.
© 2014 S. Karger AG, Basel

Anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) therapy has
been established as the standard therapy for advanced colorectal cancer (CRC). However,retrospective examination of tumors from metastatic colorectal cancer (mCRC) patients who
had undergone first and subsequent lines of anti-EGFR mAb therapy has revealed that tumors
with KRAS mutation are resistant to cetuximab or panitumumab therapy [1–8]. Based onthese findings, KRAS mutations have been identified as negative predictors of response toanti-EGFR mAb therapy. However, the identification of KRAS mutations as negative markers
was confirmed by the findings of prospective studies of the effectiveness of panitumumab intreating Caucasian mCRC patients, while few prospective studies have examined its effectiveness in Asian populations [6]. Moreover, a clear ethnic difference in the frequency of EGFR
mutations, a critical predictor of response to gefitinib in patients with non-small cell lungcancer, has been found between Caucasian and Asian populations, with EGFR mutations identified in approximately 30–60% of Asian patients with non-small cell lung cancer compared
to approximately 10–20% of Caucasian patients [9]. Despite this finding, differences between
Caucasian and Asian mCRC patients in the prevalence of mutations of KRAS and other EGFRdownstream genes, including BRAF, PIK3CA, NRAS, and AKT1, have not been fully evaluated.
In fact, more than half of the patients with KRAS wild-type tumors have been found to be
primarily resistant to anti-EGFR antibodies, and the results of several studies suggest that
BRAF and PIK3CA mutations abrogate the efficacy of anti-EGFR mAb therapy [10, 11].
Mutation of the BRAF gene, which occurs in approximately only 5–10% of patients withwild-type KRAS, has also been shown to be both a prognostic factor and predictive factor ofcetuximab response [11–13]. As such, the clinical significance of BRAF mutations cannot be
simply evaluated. As the BRAF gene encodes a serine-threonine kinase function downstreamof the KRAS gene, constitutively active BRAF mutations are mutually exclusive of KRAS mutations. Moreover, mutation of the PIK3CA gene, which encodes the catalytic subunit p110α of
PI3K, occurs in approximately 15–20% of CRC patients. Because tumor-derived mutant PI3K
stimulates the AKT pathway and promotes cell growth in several cancers, including CRC,PIK3CA mutations have also been associated with poor prognosis and significantly impaired
response to anti-EGFR mAb therapy in mCRC patients [14, 15]. In addition, recent contradictory evidence indicates no strong rationale for using the presence of PIK3CA mutations asa single predictive marker of cetuximab response in chemotherapy-refractory mCRC [16]. A
large-scale European study reported that the identification of KRAS, BRAF, NRAS, and PIK3CA
mutation status improved the prediction of response to anti-EGFR mAb therapy [17]. Anotherstudy found that mutation of AKT1, a downstream gene in the PI3K/AKT pathway, occurs in
6% of mCRC patients [18]. E17K, which is a somatic hot-spot mutation in the AKT1 gene,
results in the pathological localization of AKT1 to the plasma membrane, and the constitutive
activation of the downstream signal transduction.

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Introduction

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Oncology 2014;87:7–20
DOI: 10.1159/000360989© 2014 S. Karger AG, Baselwww.karger.com/ocl

Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy Resistance

Although the mutation status of KRAS and that of other EGFR-downstream genes should
be validated as a predictive marker of anti-EGFR mAb therapy in Asian populations by aprospective study, no well-designed studies have been attempted so far. To fill this researchgap, this prospective study evaluated the relationship between KRAS mutation status and theseveral clinical outcomes, such as response rate (RR) to cetuximab-based therapy, progressionfree survival (PFS), overall survival (OS) after cetuximab-based therapy in Japanese mCRC
patients who had failed to respond to prior chemotherapies consisting of irinotecan, oxaliplatin, and fluoropyrimidine treatment and for whom no other standard anticancer therapy
had been available. To contribute to the optimization of the selection of patients most likely
to benefit from anti-EGFR mAb therapy, the association between BRAF V600E and PIK3CA
mutations in exons 9 and 20; NRAS mutations in codons 12, 13, and 61, and AKT1 E17K mutations and clinical outcome was investigated.
Materials and Methods

Study Design
Before enrollment, the tumor specimens collected from all patients had been confirmed to exhibit EGFR
expression in more than 1% of malignant cells, as determined by immunohistochemistry with the Dako EGFR
PharmDx kit (DakoCytomation, Glostrup, Denmark), and all patients confirmed not to have undergone
previous anti-EGFR mAb therapy. After enrollment, patients were intravenously administered cetuximab at
a standard dosage of 400 mg/m2 over 2 h on day 1 of treatment, followed by 250 mg/m2 over 1 h on 1 day
per week, and intravenously administered irinotecan at two standard dosages which were approved for CRC
treatment in Japan based on the phase II study (150 mg/m2 every 2 weeks or 100 mg/m2 weekly for 3 consecutive weeks, followed by 1 week of rest) [19]. Patients were evaluated for tumor response or progression
every 8 weeks by means of radiologic imaging. If severe skin toxicity (more than grade 3) happened, cetuximab
treatment was postponed until the skin toxicity went down to less than grade 2 and was reduced to 200, 150,
and 100 mg/m2 in each event. If a severe hematological (neutropenia more than grade 4, thrombocytopenia
more than grade 2) or nonhematological adverse event (more than grade 3) was observed, irinotecan
treatment was postponed until these events recovered and was reduced to 80, 60 and 50% of doses in each
event. Cetuximab-based treatment was continued until disease progression or unacceptable toxicity occurred.
Even though KRAS mutant patients would be expected to show a lower RR in Japanese as well as Caucasian
populations, we did not plan an interim analysis or two-stage design because the study period was limited
from the approval of cetuximab treatment to that of KRAS gene analysis in Japan. Only in this period were
cetuximab treatments for KRAS mutant patients allowed ethically, since cetuximab treatments were
performed without KRAS analyses in clinical practice.

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Patients
Enrollment of eligible patients began in October 2008 and concluded in May 2010, which is almost
equivalent to the period from just after the approval of cetuximab until just before the approval of the KRAS
examination to predict the efficacy of cetuximab-based therapy in CRC in Japan. Inclusion criteria were: (1)age 18 years or older, (2) presence of histologically confirmed EGFR-positive CRC, (3) presence of unresectable primary or metastatic tumor and presence of measurable lesions based on the Response Evaluation
Criteria in Solid Tumors (RECIST) ver. 1.0 criteria, (4) Eastern Cooperative Oncology Group (ECOG) performance status score of ≤2, (5) adequate hematological function (neutrophil count ≥1,500/μl, platelet count≥100,000/μl, and hemoglobin count ≥8.0 g/dl), (6) adequate hepatic function (aspartate aminotransferase
and alanine aminotransferase levels ≤100 IU/μl and bilirubin level ≤2.0 mg/dl), (7) adequate renal function(serum creatinine level <1.5 mg/dl), (8) treatment with more than 2 rounds of oxaliplatin- or irinotecanbased chemotherapies, (9) elapsed time of the appropriate interval from a previous treatment (4 weeks fromradiotherapy, 2 weeks from surgical intervention with some organ resection, 2 weeks from chemotherapy,
and 4 weeks from another form of treatment provided during a clinical trial), and (10) a prognosis of morethan 2 months. The protocol was approved by the Ethics Committee of Tohoku University School of Medicine
and all patients provided written informed consent.

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Oncology 2014;87:7–20
DOI: 10.1159/000360989© 2014 S. Karger AG, Baselwww.karger.com/ocl

Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy Resistance

Tumor Collection and Processing
Formalin-fixed paraffin-embedded samples of tumor tissue from archival specimens that had beencollected at the time of diagnosis were stored at Tohoku University Hospital. The extent of KRAS, BRAF,PIK3CA, NRAS, and AKT1 mutation was assayed at the Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University. All patients’ samples were screened for KRAS mutations incodons 12, 13, and 61; BRAF V600E and PIK3CA mutations in exons 9 and 20; NRAS mutations in codons 12,
13, and 61; AKT1 mutations in codon 17, and PIK3R1 mutations in exon 16. All available tissue samples werecategorized into either a KRAS mutant or a KRAS wild-type subgroup depending on the results.
Nucleotide Sequence Analysis
Mutation analysis was performed by extraction of genomic DNA from macro-dissected formalin-fixedparaffin-embedded tissue slides or sections. DNA was extracted using the QIAamp DNA formalin-fixedparaffin-embedded tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The
appropriate sites of each gene were amplified using nested primer sets under specified cycle and temperature conditions and analyzed by 1.0% agarose gel electrophoresis. Reactions were performed using theautomated CEQ2000XL DNA analysis system (Beckman Coulter, Fullerton, Calif., USA) and including theappropriate positive and negative controls for each gene to be analyzed. To minimize bias, the researchers
who performed the mutation analyses remained blinded to the clinical outcomes.
Statistical Analysis
Statistical analysis of categorical variables was performed using the χ2 test and RR was defined according
to RECIST ver. 1.0 criteria. An independent review committee assessed response and the time to progression
during the study according to RECIST criteria and categorized patients as either responders who had achieved
complete response (CR) or partial response (PR) or as nonresponders who showed stable disease (SD) or
progressive disease (PD). To determine the sample size, the expected RR was set at 30%, the RR thresholdat 10%, the significance level for the results of one-sided testing at 5%, and the power at 80% for the KRAS
wild-type subgroup. After estimating the proportion of patients in each KRAS subgroup (wild-type andmutant) and the number of patients in the KRAS wild-type subgroup (approximately 24), it was determinedthat the sample should consist of at least 40 patients.
To evaluate the RR, the primary endpoint, the study began by testing the hypothesis that the RR of the
KRAS wild-type subgroup is equal or less than 10% assuming the efficacy of cetuximab to be similar between
Japanese and Caucasian populations. If a significant treatment effect (p ≤ 0.05) as measured by RR was identified for the KRAS wild-type subgroup, then the KRAS mutant subgroup would be analyzed similarly. PFS was
defined as the time from the beginning of chemotherapy until the first objective evidence of diseaseprogression or death from any cause and was determined using the Kaplan-Meier method. The survival
curves developed from the results of the determination of PFS of each subgroup were compared using the
log-rank test. The level of statistical significance was set at 0.05 for the results of one-sided testing for RR andthe results of two-sided testing for other variables.

Patient Characteristics
The clinical characteristics of the patients are listed in table 1. As can be observed, of the
43 patients who had undergone cetuximab-based treatment, 28 were categorized as ECOG
performance status 0 and 15 as ECOG performance status 1. All patients had failed to respondto prior chemotherapy, including irinotecan, oxaliplatin, and 5-fluorouracil treatment, andnone had previously been treated with anti-EGFR mAb therapy. The 41 patients who had
undergone oxaliplatin-based therapy were treated only by the FOLFOX regimen (infusion andbolus of 5-fluorouracil plus oxaliplatin). Treatment of the 42 patients who had undergone
irinotecan-based therapies was based on the FOLFIRI regimen (infusion and bolus of 5-fluorouracil with irinotecan) for 34 patients, S-1 plus irinotecan for 6 patients and irinotecan
monotherapy for 2 patients. Bevacizumab therapy had been administered as first-line,
second-line, or both lines of treatment to 26 patients.

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Results

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Oncology 2014;87:7–20© 2014 S. Karger AG, Baselwww.karger.com/ocl

DOI: 10.1159/000360989

Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy Resistance

Table 1. Patient characteristics

Total number of patientsMedian age (range)
GenderMale
Female
ECOG performance status0
1
Prior chemotherapy for advanced disease
FOLFOXFOLFIRI/IRIS/irinotecan
Bevacizumab
Primary tumor
Cecum
Ascending colon
Transverse colon
Descending colon
Sigmoid colon
RectumMetastatic sites
Lung
Liver
Intra-abdominal lymph nodes
Peritoneum
Bone
OthersAll

KRAS mutant

KRAS wild-type

43
68 (50 – 82)

13
67 (50 – 81)

30
68 (52 – 82)

27
16

7
6

20
10

28
15

10
3

18
12

41
34/6/226

13
11/2/0828
23/4/2
18

1
9
4
2
9
18

0
3
0
1
4
5

1
6
4
1
5
13

30
28
12
5
2
8

10
11
2
2
0
3

20
17
10
3
2
5

FOLFOX = 5-Fluorouracil, leucovorin, oxaliplatin; FOLFIRI = 5-fluorouracil, leucovorin, irinotecan; IRIS =irinotecan, S-1.

Concerning the sites of metastases, lung metastases were found in 30 patients (69.8%),
followed by liver metastases found in 28 (65.1%), intra-abdominal lymph node metastases
found in 12 (27.9%), and peritoneum metastases found in 5 (11.6%) patients. No significant
differences were seen regarding clinical characteristics between the KRAS wild-type and
KRAS mutant subgroups.

Mutation Analysis of KRAS, BRAF, PIK3CA, NRAS and AKT1
Table 3 provides a list of the mutations detected by direct sequencing. The common KRAS
mutations in codons 12 and 13 and rarer mutations in codon 61 were analyzed to increase
the sensitivity of mutation detection. Of the 13 tumors (30.2%) that harbored KRAS muta-

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Toxicity
Table 2 provides a summary of the toxicity data. As can be observed, grade 3 or 4 neutropenia was identified in 12 patients (29.3%) and grade 3 or 4 anemia in 4 (9.8%). Althoughskin toxicity, including acneiform rash, dry skin and pruritus, was observed in the majority ofpatients (82.9%), grade 3 or 4 skin toxicity was observed in only 2 patients (4.9%). Other
conditions for which grade 3 or 4 toxicity was identified were diarrhea (4.9%), stomatitis(2.4%), and hypomagnesemia (4.9%). No significant differences were found between the
toxicity profiles of the KRAS wild-type and KRAS mutant subgroups.

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DOI: 10.1159/000360989

Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy Resistance

Table 2. Toxicity profile in 43 mCRC patients
Event

All (n = 41)

Leukopenia
Neutropenia
AnemiaThrombocytopenia
Diarrhea
Skin toxicity
HFS
Stomatitis
NauseaVomiting
Fatigue
Anorexia
Hypomagnesia

KRAS mutant (n = 13)

KRAS wild-type (n = 28)

grade 1-4

grade 3-4

grade 1-4

grade 3-4

grade 1-4

grade 3-4

21 (51.2, 35.1 – 67.1)
20 (48.8, 32.9 – 64.9)
36 (87.8, 73.8 – 95.9)
13 (31.7, 18.1 – 48.1)
23 (56.1, 39.8 – 71.5)
34 (82.9, 67.9 – 92.9)
11 (26.8, 14.2 – 42.9)
14 (34.1, 20.1 – 50.6)
19 (46.3, 30.7 – 62.6)
4 (9.8, 2.7 – 23.1)
22 (53.7, 37.4 – 69.3)
26 (63.4, 46.9 – 77.9)
19 (46.3, 30.7 – 62.6)

9 (22.0, 10.6 – 37.6)
12 (29.3, 16.1 – 45.5)
4 (9.8, 2.7 – 23.1)
2 (4.9, 0.6 – 16.5)
2 (4.9, 0.6 – 16.5)
2 (4.9, 0.6 – 16.5)
1 (2.4, 0.06 – 12.9)
1 (2.4, 0.06 – 12.9)
2 (4.9, 0.6 – 16.5)
0 (0.0, 0.0 – 8.6)
3 (7.3, 1.54 – 19.9)
6 (14.6, 5.57 – 29.2)
2 (4.9, 0.6 – 16.5)

5 (38.5, 13.9 – 68.4)
5 (38.5, 13.9 – 68.4)
11 (84.6, 54.6 – 98.1)
5 (38.5, 13.9 – 68.4)
6 (46.2, 19.2 – 74.9)
12 (92.3, 64.0 – 99.8)
2 (15.4, 1.92 – 45.4)
6 (46.2, 19.2 – 74.9 )
7 (53.8, 25.1 – 80.8)
0 (0.0, 0.0 – 24.7)
9 (69.2, 38.6 – 90.9)9 (69.2, 38.6 – 90.9)
7 (53.8, 25.1 – 80.8)

3 (23.1, 5.0 – 53.8)
4 (30.8, 9.1 – 61.4)
1 (7.7, 0.2 – 36.0)
0 (0.0, 0.0 – 24.7)
1 (7.7, 0.2 – 36.0)
0 (0.0, 0.0 – 24.7)
0 (0.0, 0.0 – 24.7)
0 (0.0, 0.0 – 24.7)
1 (7.7, 0.2 – 36.0)
0 (0.0, 0.0 – 24.7)
0 (0.0, 0.0 – 24.7)
1 (7.7, 0.2 – 36.0)
1 (7.7, 0.2 – 36.0)

16 (57.1, 37.2 – 75.5)
15 (53.6, 33.9 – 72.5)
25 (89.3, 71.8 – 97.7)
8 (28.6, 13.2 – 48.7)
17 (60.7, 40.6 – 78.5)
22 (78.6, 59.1 – 91.7)
9 (32.1, 15.9 – 52.4)
8 (28.6, 13.2 – 48.7)
12 (42.9, 24.5 – 62.8)
4 (14.3, 4.0 – 32.7)
13 (46.4, 27.5 – 66.1)
17 (60.7, 40.6 – 78.5)
12 (42.9, 24.5 – 62.8)

6 (21.4, 8.3 – 41.0)
8 (28.6, 13.2 – 48.7)
3 (10.7, 2.27 – 28.2)
2 (7.1, 0.88 – 23.5)
1 (3.6, 0.09 – 18.4)
2 (7.1, 0.88 – 28.2)
1 (3.6, 0.09 – 18.4)
1 (3.6, 0.09 – 18.4)
1 (3.6, 0.09 – 18.4)
0 (0.0, 0.0 – 12.3)
3 (10.7, 2.27 – 28.2)
5 (17.9, 6.1 – 36.9)
1 (3.6, 0.09 – 18.4)

Figures in parentheses are percentages and 95% confidence intervals. HFS = Hand-foot syndrome.

Table 3. KRAS, BRAF andPIK3CA mutation frequencies(n = 43)

Gene

Codon

Nucleotide
substitution

Amino acid Number
substitution

KRAS

12

G12D
G12V
G13D

3 (7.0)7 (16.2)
3 (7.0)13 (30.2)

13

GGT→GATGGT→GTTGGC→GAC

BRAF

600

GTG→GAG

V600E

3 (7.0)3 (7.0)PIK3CA

542
545

GAA→AAA
GAG→GGG
CAT→CGT
CAT→TATCAT→CTT

E542K
E545G
H1047R
H1047YH1047L

1 (2.3)2 (4.7)
1 (2.3)1 (2.3)1 (2.3)6 (14.0)

1,047
NRAS

12

GGT→GAT

G12D

2 (4.7)2 (4.7)AKT1

17GAG→AAG

E17K

2 (4.7)2 (4.7)

Figures in parentheses are percentages.

Cetuximab Efficacy
The RR of the KRAS wild-type subgroup was found to be 17.9% (exact 90% confidence
interval = 7.3–33.9%). As no significant treatment effect was obtained for the KRAS mutant
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tions in codons 12 and 13, none harbored mutations in codon 61. Of the 13 detected mutations in codons 12 and 13, the most frequent mutation was G12V (16.3%), followed by G13D
(7.0%), and G12D (7.0%). Three tumors (7.0%) harbored BRAF mutations in codon 600(V600E). Six tumors (14.0%) had PIK3CA mutations in exon 9 (E542K and E545G) and in exon
20 (H1047R, H1047Y, and H1047L). Two tumors showed NRAS mutations in codon 12. Twotumors harbored AKT1 E17K mutations. Analysis of the findings indicated that KRAS mutations are mutually exclusive of BRAF and NRAS mutations (fig. 1). Two of the 13 KRAS mutant
tumors harbored PIK3CA exon 20 mutations but not exon 9 mutations. One of the 3 BRAF
mutant tumors showed AKT1 mutations. None of the tumors harbored PIK3R1 mutations.

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DOI: 10.1159/000360989

KRAS only, n = 11 (25.6%)
Wild-type, n = 20
(46.5%)

Fig. 1. Prevalence of mutation in
EGFR-related genes. Numbers refer to the numbers of tumors harboring single or double mutationsin the indicated genes.

KRAS and PIK3CA,n = 2 (4.7%)
PIK3CA only, n = 4 (9.3%)
BRAF only, n = 2 (4.7%)
BRAF and AKT1, n = 1 (2.3%)

NRAS only, n = 2 (4.7%)

AKT1 only, n = 1 (2.3%)

Color version available online

Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy Resistance

Table 4. Response to cetuximab according to the presence or absence of gene mutation in the 41 patientsTumor
response

KRAS

Genetic status of KRAS and BRAF

Genetic status of KRAS, BRAF,PIK3CA, NRAS and AKT1

All
patients

mutantwild-typemutant of any
genes

wild-type of
all genes

mutant ofany genes

wild-type of
all genes

Total

13

28

15

26

22

19

41

CR
PR
SD
PD
NE

0 (0)
0 (0)
2 (15.4)
9 (69.2)
2 (15.4)0 (0)5 (17.9)9 (32.1)
12 (42.9)
2 (7.1)

0 (0)
0 (0)
2 (12.5)
11 (78.5)
2 (14.3)0 (0)5 (19.2)
9 (34.6)
10 (38.5)
2 (7.7)

0 (0)
0 (0)
5 (22.7)
13 (59.1)
4 (18.2)0 (0)5 (26.3)
6 (31.6)
8 (42.1)
0 (0)

0
5
11
21
4

RR
90% CI

0.0
0.0 – 20.6

17.9a
7.3 – 33.9

0.0
0.0 – 18.1

19.2
7.9 – 36.3

0.0
0.0 – 12.7

26.3
11.0 – 47.6

12.2
4.9 – 23.9

DCR
90% CI

15.4
2.8 – 41.0

50.0
33.3 – 66.7

13.3
2.4 – 36.3

39.6
36.2 – 70.8

22.7
9.4 – 42.0

57.9
36.8 – 77.0

39.0
27.6 – 55.4

mPFS,
months
95% CI
p valueb

1.6

3.7

1.6

5.2

1.8

5.2

2.5

1.3 – 2.6
0.00392.1 – 5.8

1.5 – 2.20.0008

2.1 – 6.2

1.5 – 2.6
0.0042

2.5 – 6.5

1.8 – 5.2

7.5

10.3

7.4

11.8

7.4

11.8

8.47

3.6 – 8.9
0.02

5.9 – 13.7

3.6 – 8.5
0.0026

6.0 – 14.4

4.3 – 9.1
0.24

6.0 – 14.4

6.0 – 11.6

mOS,
months
95% CI
p valuebsubgroup to identify an RR of 0% (90% confidence interval = 0.0–20.6%). As can be observedin table 4, which shows the RR and median PFS (mPFS) according to the presence or absenceof gene mutations, the KRAS mutant subgroup was found to have a lower RR than the KRAS
wild-type subgroup. Combined analysis of KRAS, BRAF, PIK3CA, NRAS, and AKT1 mutation
status indicated that none of the 23 patients with mutations in any of these 5 genes, whose
objective RR was 0.0%, had responded to treatment, whereas 5 of the 18 patients with nomutations, whose objective RR was 27.8%, had responded, indicating that KRAS, BRAF,PIK3CA, NRAS, and AKT1 mutations were associated with a lack of response. Combined

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Figures in parentheses are percentages. DCR = Disease control rate.
aOne-sided p = 0.083 (z test); the null hypothesis (the true RR of the KRAS wild-type group was more than 0.1) was tested.
b log-rank test.

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DOI: 10.1159/000360989

Genetic status Median
Wild-type KRAS 3.7 m
Mutant KRAS
1.6 m

60
40
20
0

p = 0.0039
0

50

100

a

250

300

60
40
20

50

100

150

200

250

60
40
20
0

50

100

150

Time (days)

100

200

200

250

300

400

500

600

MedianGenetic status
Wild-type KRAS and BRAF 11.8 m
Mutant KRAS and BRAF
7.4 m

60
40
20
p = 0.0026
0

100

200

300

400

500

600

Time (days)
MedianGenetic status
Wild-type KRAS, BRAF, 11.8 m
PIK3CA, NRAS and AKT1
Mutant KRAS, BRAF,PIK3CA, NRAS and AKT1 7.4 m

100
80
60
40
20
0

f

300

Time (days)0

p = 0.004200dMedianGenetic status
Wild-type KRAS, BRAF,
5.2 m
PIK3CA, NRAS and AKT1
Mutant KRAS, BRAF,PIK3CA, NRAS and AKT1 1.8 m

80

p = 0.02080300Time (days)
100

20

100

p = 0.0013
0

40

b

Overall survival (%)

Progression-free survival (%)

80cProgression-free survival (%)

200

60

0

MedianGenetic status
Wild-type KRAS and BRAF 5.2 m
Mutant KRAS and BRAF
1.6 m

100

e

150

Time (days)

080
Overall survival (%)

80

Genetic status Median
Wild-type KRAS 10.3 m
Mutant KRAS
7.5 m

100

Overall survival (%)

Progression-free survival (%)

100

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Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy Resistancep = 0.24
0

100

200

300

400

500

600

Time (days)

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Fig. 2. Kaplan-Meier analysis of cumulative PFS and OS based on KRAS, BRAF and PIK3CA mutational status
in mCRC patients treated with cetuximab. PFS (a) and OS (b) of patients with wild-type KRAS versus mutant
KRAS. PFS (c) and OS (d) of patients with wild-type KRAS or BRAF versus mutant KRAS or BRAF. PFS (e) andOS (f) of patients with wild-type KRAS, BRAF, PIK3CA, NRAS, or AKT1 versus mutant KRAS, BRAF, PIK3CA,
NRAS, or AKT1. m = Months.

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100
Change from baseline (%)

80
60

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Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy Resistance

PR
SD
PD

40
20
0
–20
–40
–60
–80

KRAS
BRAF
PIK3CANRAS
AKT1KRAS G13DPIK3CA exon 9

PIK3CA exon 20

analysis of KRAS, BRAF, PIK3CA, NRAS, and AKT1 mutations improved RR (26.3%) of thesubgroups expected to show response compared to the analysis of KRAS mutations alone(17.9%). Whereas the combined analysis of KRAS, BRAF, PIK3CA, NRAS, and AKT1 mutations
did not reduce the disease control rate (22.7%) of the subgroup expected to have no responsecompared to the analysis of KRAS mutations alone (15.4%), the combined analysis of KRAS
and BRAF mutations did reduce it (12.5%).
As can be seen in figure 2a and b, the mPFS and median overall survival (mOS) of the KRAS
wild-type subgroup were found to be significantly longer than those of the KRAS mutant
subgroup (mPFS = 3.7 vs. 1.6 months, respectively, p = 0.0039; mOS = 10.3 vs. 7.5 months,
respectively, p = 0.02). However, as shown in figure 2c and d, the difference in mPFS and mOS
between both the KRAS and BRAF wild-type subgroups and the mutant subgroups was foundto be greater (mPFS = 5.2 vs. 1.6 months, respectively, p = 0.0013; mOS = 11.8 vs. 7.4 months,
respectively, p = 0.0026). The difference in mPFS and mOS between the subgroup with all 5
wild-type genes (KRAS, BRAF, PIK3CA, NRAS, and AKT1) and the subgroup with mutations in
any one of the 5 genes was found to be less significant (mPFS = 5.2 vs. 1.8 months, respectively, p = 0.0042; mOS = 11.8 vs. 7.4 months, p = 0.24; fig. 2e, f).
The analysis of a waterfall plot of the best response in target lesions and mutation statusindicates a similar tendency to RR and mPFS analysis. As shown in figure 3, almost all patientswith KRAS, BRAF, and/or PIK3CA mutations failed to respond to cetuximab-based treatment.
Whereas 23% (3/13) of the patients in the KRAS mutant subgroup experienced tumor
reduction, 57.7% (15/26) of the patients in the KRAS wild-type subgroup showed tumor
reduction, including patients with PR, SD, and PD. In contrast, 28% (5/18) of patients in the
KRAS, BRAF or PIK3CA mutant subgroup and 72% (13/19) of patients in any of the wild-typesubgroup experienced tumor reduction. These results indicate the clinical relevance of theanalysis of mutation status of these genes in predicting the efficacy of cetuximab-based
treatment in patients with mCRC.

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Fig. 3. Waterfall plots showing maximal reduction in size of target lesions based on analysis of KRAS, BRAF,PIK3CA, NRAS, and AKT1 mutational status in mCRC patients treated with cetuximab.

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Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy Resistance

The RR of the KRAS wild-type subgroup in this study (17.9%) was found to be lower thanwould be expected according to the results of previous studies. Several studies have shownthat the RR of KRAS wild-type patients to cetuximab therapy with or without chemotherapy
in second-line or later lines of treatment was higher (27–41%) [2, 3, 5, 8, 20, 21]. One European
study found that the RR of the KRAS wild-type subgroup to cetuximab plus irinotecan in thirdline therapy was 37% [22]. Whereas the RR of all patients in this study was 12.2%, the RR ofthe 206 patients in the third-line subgroup in the BOND study, who had undergone cetuximab
plus irinotecan therapy, was 22.2% [23]. The lower RR in this study might have arisen frombias in several steps, such as during KRAS testing, patient sampling, and/or evaluation ofefficacy. Specifically, while this study performed direct sequencing to determine KRAS status,
several other studies used the Scorpion-Arms method, which is more sensitive to detectmutations. However, as the frequency and spectrum of KRAS mutation in this study weresimilar to those of previous studies, the sensitivity of KRAS examination was not likely to havediffered greatly between this and previous studies [24, 25]. Retrospective analysis of the
KRAS status of 43 other chemorefractory patients who had undergone cetuximab treatment
in our previous study that we had conducted using the same method as that used in thepresent study revealed that the RR of the KRAS wild-type subgroup had been higher than thatof the patients examined in the present study (22%) [26]. Using the single-base primer
extension method, the direct sequencing results were validated in this previous study. Specifically, the characteristics of the patients in both studies were similar except the total mutation
rates of the BRAF, PIK3CA, NRAS, and AKT1, which were higher in the present study (23% in
the present study vs. 9% in the previous study). One explanation for this difference may be ahigh rate of tumors with mutations in the EGFR pathway in the present study’s cohort, whichis associated with a putative poor prognosis or nonresponse to cetuximab-based therapy.
However, this explanation is not completely acceptable, since this mutation rate was not
significantly higher than that identified in studies of Caucasian patients [17]. In addition to
our previous study, a South Korean study found that the RR of Korean patients with refractory
KRAS wild-type CRC to cetuximab plus irinotecan treatment was similar (33%) to that ofcomparable Caucasian patients, indicating that the existence of ethnic differences in responseto cetuximab therapy is unlikely [20].
Exploratory analysis of the study data confirmed that KRAS mutation is a negative
predictive marker of cetuximab efficacy in Japanese as well as Caucasian patients. The RR, theprimary endpoint of this study, was higher and the mPFS and mOS were significantly longer
in the KRAS wild-type subgroup compared to the KRAS mutant subgroup. The rate of KRAS
mutation identified in this study (30.2%) was consistent with that reported by previous
studies of various populations (30–40%), and the spectrum of the KRAS mutation was very
similar to that reported by previous studies of Caucasian populations [24, 25]. All of the 3common KRAS mutations examined in the RASCAL and RASCAL II studies – the G12V, G13D,
and G12D mutations – were frequently detected in this study. Based on these findings, theKRAS mutation, in terms of both frequency and the nature of the mutation spectrum, wasconcluded not to differ significantly between Japanese and Caucasian populations. Recently,
the KRAS G13D mutation has been shown to be associated with better outcome after cetuximab
treatment compared to that associated with other mutations [27]. However, the 3 patientswith KRAS G13D-mutated tumors examined in this study had no better response to cetuximabbased therapy than had patients with other mutations (fig. 3). The sample size was too small
to evaluate the relationship between the efficacy of cetuximab-based therapy and the KRAS
G13D mutation.

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Discussion

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In this study, 42.9% of patients, even those in the KRAS wild-type subgroup, were foundto show PD after cetuximab treatment. Several studies have revealed that analysis of mutation
status of EGFR-related downstream genes, such as BRAF, PIK3CA, and NRAS, improves the
specificity of prediction of cetuximab efficacy [17]. In addition to analysis of mutation statusof these 3 genes, this study analyzed that of AKT1 and PIK3R1, for which mutations have been
identified in between 6 [18] and 8.3% [28] of CRC patients and found to result in the activation of the AKT pathway. However, the prevalence of mutations in these genes in thepatients of this study was found to be low (BRAF = 7.0%, PIK3CA = 14.0%, NRAS = 4.7%,
AKT1 = 4.7%, and PIK3R1 = 0%), indicating that these mutations can hardly be evaluated as
independent predictors. Therefore, future studies with larger sample sizes are required toanalyze the clinical significance of these mutations as predictive markers for the efficacy ofcetuximab. To obtain suggestive evidence, we evaluated which combinations of these genes
produce the most significant differences in the clinical outcome of cetuximab treatment
between the wild-type and mutant subgroups. The most significant difference in RR was
obtained by combined analysis of all 5 genes. However, the more significant differences indisease control rate, PFS and OS were obtained by combined analysis of only KRAS and BRAF.
These findings indicate that the combined analysis of KRAS and BRAF genes clearly contributes
to detecting the responders to cetuximab treatment by evaluating the various clinicaloutcomes but that the contribution of additional analyses of other EGFR-related genes is
uncertain. Therefore, in the assessment of predictive markers, careful evaluation of the resultsof combined analysis of EGFR-related genes must be performed to prevent erroneously identifying patients who may benefit from cetuximab therapy as nonresponders.
In this study, analysis of BRAF mutations was found to contribute to the identification of
additional nonresponders, as only 2 (7.0%) patients with BRAF mutations were found to have
PD. BRAF mutations were observed at a rate comparable to that found in Caucasian populations (5–10%) and mutually exclusive of KRAS mutation, as previously shown [29]. Although
BRAF mutations have been shown to be negative predictive markers of response to anti-EGFR
mAb therapy, they have also been found to be strong prognostic factors [11]. Moreover,recent studies have indicated that even patients with BRAF mutations may benefit fromcetuximab treatment, having found better clinical outcomes for cetuximab-treated compared
to non-cetuximab-treated patients with BRAF mutations, although the difference in outcome
between these 2 groups did not reach a level of statistical significance [30, 31]. While theresults of the present study reveal that the analysis of BRAF mutation status contributes tobetter identification of responders to cetuximab treatment, the possibility that patients witha poor prognosis will be erroneously identified as nonresponders, regardless of resistance tocetuximab, cannot be ruled out in a one-arm phase II study. To evaluate the benefit ofcetuximab in BRAF mutant patients, large-scale randomized studies are required. A recent
study found that treatment with EGFR inhibitors, including cetuximab, has a synergistic effectwith treatment with BRAF inhibitors in BRAF mutant CRC patients. This indicates that BRAF
inhibitor monotherapy achieves only a limited effect because it causes rapid feedback activation of EGFR [32]; moreover, it shows that genetic analysis of BRAF status will become more
useful if combination therapies inhibiting both BRAF and EGFR are developed in the future.
Of the 2 AKT1 mutant patients showing PD in this study, 1 patient was found to have an
overlapping BRAF mutation. As such, only the findings for 1 of these patients can be considered
representative of the impact of AKT1 mutation on cetuximab efficacy. Nevertheless, it indicates that hot-spot somatic mutations such as AKT1 E17K, which is reported in various
tumors, including breast, colorectal and ovarian cancers, may be a novel predictive marker ofcetuximab efficacy. Even though AKT1 E17K has been shown to be oncogenic in both in vitro
and in vivo analysis, it has not been found to be associated with the efficacy of anti-EGFR mAb
treatment [18]. Previous studies have found no association between phosphorylated AKT

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level and clinical outcome after anti-EGFR mAb treatment [33]. In contrast to the reportobserving absence of tumors with AKT1 mutations in Asian populations, 2 patients (7.0%)
were found to have AKT1 mutations in this study of Japanese patients [34].
The prevalence of PIK3CA mutations in the patients of this study (14.0%) was similar tothat in studies of Caucasian populations (10–20%). The clinical relevance of PIK3CA mutations in the prediction of response to anti-EGFR therapy remains controversial. Among the 5
PIK3CA mutant patients examined in this study, only 2 patients with exon 9 mutations wereavailable for evaluation of clinical outcome. Both PIK3CA mutant patients showed SD,
suggesting that PIK3CA analysis did not contribute to improving the specificity of theprediction of response to cetuximab. This finding is consistent with a recent large European
study that found that PIK3CA mutations in exon 20 but not exon 9 are associated with resistance to anti-EGFR mAb therapy [17]. Nevertheless, the spectrum of PIK3CA mutation could
differ by ethnicity; both PIK3CA mutations detected in KRAS mutant tumors were located inexon 20, which contrasts with a previous report saying that PIK3CA exon 9 mutations arefound more frequently in KRAS mutant tumors than exon 20 mutations. Unfortunately, nodata were collected in the present study that could provide more information regarding exon
20 mutations. Of the 5 detected mutations, E542K and H1047R are among the 3 most frequent
mutations (E542K, E545K, and H1047R), whereas E545G, H1047Y, and H1047L are relatively
rare mutations [17]. Especially, E545G was detected in 2 patients of this study in contrast to
the reports saying that E545G is quite rare in Caucasian populations [16].
NRAS mutation, which was detected in 2 patients, has been associated with poor responseto cetuximab treatment [17]. However, NRAS analysis did not clearly contribute to an
improvement of the sensitivity of the prediction of treatment response, with the 2 NRAS
mutant patients showing SD and PD. These 2 NRAS mutations were found in codon 12,
whereas NRAS mutations are more commonly seen in codon 61 in Caucasian populations,suggesting ethnic differences may have led to the differences in the sensitivity of prediction.
NRAS mutations were mutually exclusive of KRAS mutations, as shown previously. Unfortunately, no data regarding KRAS codon 61 and PIK3R1 mutations could be collected because
no patients with these mutations were identified in this study.
This is a prospective study to evaluate the relationship between BRAF, PIK3CA, and KRAS
mutation status and response to cetuximab therapy in Japanese patients with mCRC. Despite
the study’s focus on mutation status, almost 42.1% of the patients for whom no KRAS, BRAF,PIK3CA, NRAS, or AKT1 mutations were detected were found not to have responded tocetuximab therapy and to have suffered tumor progression. This finding suggests that otheras yet unidentified biomarkers are important determinants of response, including the tumor
suppressor PTEN protein, a negative regulator of PI3-kinase-initiated signaling for which lossof expression has been associated with lack of response to cetuximab [33, 35].
In conclusion, the results of this study prospectively confirmed that cetuximab-based
treatment is effective and well tolerated in both Japanese and Caucasian mCRC patients with
KRAS wild-type mutations who have failed to respond to prior chemotherapy consisting of
irinotecan, oxaliplatin, and fluoropyrimidine administration. The results also indicate theclinical relevance of analysis of not only KRAS mutation status in predicting the efficacy ofcetuximab-based treatment in Asian patients with mCRC but also BRAF mutation status in
improving identification of patients most likely to benefit from anti-EGFR mAb therapy.
Nevertheless, as the maximum RR to cetuximab-based treatment was found to be only 26.6%,even with genetic analysis, they also indicate the need for the development of new diagnostics
to enable the identification of anti-EGFR mAb-sensitive mCRC patients without any known
mutations to improve the cost-effectiveness of providing anti-EGFR mAb therapy.

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Soeda et al.: Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and IrinotecanBased Chemotherapy ResistanceAcknowledgements
This study was supported by the Tohoku Clinical Oncology Research and Education Society (T-CORE),
a nonprofit organization. We thank all the patients and hospitals that participated in this study as well as
Hiroyoshi Suzuki of Sendai Medical Center, Yayoi Takahashi of Tohoku University Hospital, Noriyuki Iwama
of Sendai Kosei Hospital, and all the pathologists and technicians in the participating hospitals who prepared
the samples. We also thank the members of the Data and Safety Monitoring Committees in this trial, Yasuo
Saijo, Kenichi Ishizawa, and Akira Inoue.

Disclosure Statement
Chikashi Ishioka received a research grant from the Chugai and Novartis pharmaceutical companies.

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