Purpose: In this phase Ib/II study, we aimed to assess the safety and efficacy of afatinib plus nimotuzumab (N) in advanced non–small cell lung cancer (NSCLC) patients with acquired resistance to gefitinib or erlotinib.

Experimental Design: In phase Ib stage, patients received afatinib (40 mg or 30 mg once daily) plus nimotuzumab (100 mg or 200 mg once weekly) for 28-day cycles to determine the recommended phase II dose (RPIID). The safety and efficacy of RPIID dose was evaluated in phase II stage.

Results: In total, 50 patients were enrolled (13 to phase Ib and 37 to phase II). In the first dose-finding cohort (afatinib 40 mg plus nimotuzumab 100 mg), one patient experienced dose-limiting toxicity (DLT) of grade 3 diarrhea and in the subsequent cohort (afatinib 40 mg plus nimotuzumab 200 mg), two DLTs (grade 3 diarrhea and grade 3 neutropenia) occurred in 2 of 6 patients. Accordingly, RPIID was determined as afatinib 40 mg plus nimotuzumab 100 mg. In 44 patients treated with RPIID, 7 (16%) patients had grade 3 toxicities; skin rash (7%), diarrhea (5%), acne (2%), and fatigue (2%). The overall response rate was 23% and the median duration of response was 4.3 months (range, 0.7–16.2 months). The median progression-free survival and overall survival were 4.0 months [95% confidence interval (CI), 2.3–5.7 months] and 11.7 months (95% CI, 9.4–14.0 months), respectively.

Conclusions: Combination treatment of afatinib and nimotuzumab demonstrated an acceptable safety profile and encouraging antitumor activity in advanced NSCLC patients with acquired resistance to gefitinib or erlotinib. Larger phase III trial is warranted to confirm its efficacy and safety. Clin Cancer Res; 22(9); 2139–45. ©2015 AACR.

Translational Relevance

Acquired resistance after initial clinical benefit inevitably occurs in patients with EGFR-mutant non–small cell lung cancer (NSCLC), usually within 1 year of starting gefitinib or erlotinib. Afatinib is a potent irreversible ErbB family blocker and nimotuzumab is a humanized anti-EGFR monoclonal antibody. We aim to assess the safety and efficacy of dual inhibition of EGFR with afatinib and nimotuzumab in kinase inhibitor–resistant EGFR-mutant NSCLC. Combination of afatinib and nimotuzumab showed acceptable safety profiles and encouraging antitumor activity in patients with acquired resistance to gefitinib or erlotinib.

Non–small cell lung cancer (NSCLC) is one of the leading causes of deaths from cancer worldwide (1, 2). Randomized phase 3 studies have shown better progression-free survival (PFS) and responses with gefitinib or erlotinib than platinum-based chemotherapy for NSCLC harboring EGFR mutations (3–8). However, most of NSCLC patients treated with EGFR tyrosine kinase inhibitors (TKI) eventually develop acquired resistance, necessitating alternative treatment strategies (9, 10). Approximately half of patients have a secondary EGFR mutation in exon 20 (T790M), which is proposed to cause resistance by interfering with binding of TKIs or by increasing the affinity for ATP (11, 12).

New approaches for treatment beyond progression were explored in preclinical studies on dual targeting of the EGFR (13, 14). Combined treatment with EGFR TKIs and anti-EGFR monoclonal antibodies (mAbs) resulted in superior antitumor activity than either agent given alone in cancer cell lines (14). Afatinib is an oral, irreversible ErbB family blocker that selectively and potently blocks signaling from all relevant ErbB family receptors (ErbB1, ErbB2, and ErbB4; refs. 15, 16). Although afatinib showed clinical efficacy as single agent in the TKIs-naïve setting (17), the response rates in acquired resistance setting were below 10% (18). In xenograft models of TKI-resistant tumors harboring T790M mutation, dual inhibition of EGFR with afatinib plus cetuximab induced encouraging tumor shrinkage (19). This combination regimen has demonstrated an augmented response rate of 29% for TKI-resistant EGFR-mutant NSCLC patients in a recent clinical trial (20). Notwithstanding promising efficacy of this combination therapy for heavily pretreated patients, grade 3 adverse events, mainly skin rash and gastrointestinal toxicities occurred in nearly half of the patient population, making it difficult to adopt its regimen for wider clinical use.

Nimotuzumab is a humanized IgG1 mAb against EGFR, which binds to the extracellular domain III of EGFR with a moderate affinity (21). Several phase I and II trials with nimotuzumab have mainly focused on head and neck cancer and brain malignancies, and showed antitumor activity while minimizing skin toxicity compared with other anti-EGFR drugs (22–24). Bebb and colleagues and Choi and colleagues confirmed the minimal toxicity of nimotuzumab in combination with thoracic radiation, and also demonstrated favorable results compared with historical controls in a NSCLC (25–27). Therefore, we hypothesized that dual blockade of EGFR with afatinib and nimotuzumab demonstrated robust clinical activity with a manageable safety profile.

Patient population

Eligible patients were at least 20 years old and had a diagnosis of stage IIIB/IV NSCLC harboring activating EGFR mutation (exon 19deletion or L858R) who had progressed on gefitinib or erlotinib. Patients with unknown genotype of EGFR, were included if disease progression was present after at least 6 months of treatment with gefitinib or erlotinib. All patients had measurable disease by Response Evaluation Criteria in Solid Tumors (RECIST version 1.1), and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1. Patients with prior afatinib or nimotuzumab therapy, uncontrolled or symptomatic central nervous system metastasis, active pulmonary fibrosis, uncontrolled heart disease, and inadequate hematologic, hepatic, or renal function were excluded.

All patients provided written informed consent before study enrollment. The study was approved by the Institutional review boards of the Samsung Medical Center and conducted in accordance with the Declaration of Helsinki and the Good Clinical Practice guidelines.

Study design and treatment

This is a phase Ib/II, open-label, single-arm study comprising of two stages: dose-escalation and dose-expansion (Fig. 1). The phase Ib followed a standard 3 + 3 dose-escalation design to determine the maximum tolerated dose (MTD) and the recommended phase II dose (RPIID) of combination therapy of afatinib and nimotuzumab. Treatment consisted of oral afatinib at two dose levels (30 mg or 40 mg per day) and intravenous infusion of nimotuzumab at two dose levels (100 mg or 200 mg weekly) until disease progression or unacceptable toxicity. Each treatment cycle was defined as 28 days regardless of omitted doses.

Dose-limiting toxicities (DLT) were defined as follows: grade 2 left heart failure; grade 2 diarrhea refractory to antidiarrheal medication for 7 days or grade 3 to 4 diarrhea refractory to antidiarrheal medication for 2 days; grade 3 to 4 rash; other grade 3 to 4 nonhematologic toxicities; or treatment-related death. Toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0). The dose level I started with 40 mg of afatinib plus 100 mg of nimotuzumab and was subsequently escalated to dose level II (afatinib 40 mg + nimotuzumab 200 mg). If ≥2 of 3 subjects experienced DLT in the initial cohort, deescalated dose (afatinib 30 mg + nimotuzumab 100 mg) would be considered. In the absence of DLTs among initial 3 patients, dose-escalation would proceed according to treatment protocol. If one DLT is observed in the initial cohort, then3 additional patients would be treated at dose level I. The MTD was determined as the highest dose where no more than 1 of 6 patients develop DLT. The RPIID was defined as the MTD or the higher dose level of afatinib (40 mg) and nimotuzumab (200 mg) if MTD is not reached. Intrapatient dose escalation was not permitted.

In phase II stage, all patients received a fixed dose of afatinib plus nimotuzumab as determined by dose-finding phase Ib stage. The primary objectives of the phase II stage were overall response rate (ORR) and occurrence of adverse events. The secondary objectives included PFS and overall survival (OS).

For patients who experienced severe toxicities during treatment, dose modification was permitted for the subsequent cycle.

Assessments

At baseline, all patients provided medical history and underwent physical examination. Laboratory evaluations and CT scans of the chest and of other known metastatic regions were performed within 2 weeks of the first dose of study treatment. EGFR (exon 18–21) mutations were analyzed by directional sequencing or the peptide nucleic acid-locked nucleic acid PCR clamp method especially for detection of T790 M (28, 29). Tumor response was evaluated according to the response evaluation criteria in solid tumors (RECIST version 1.1) after 4 weeks of treatment (1 cycle) and every 8 weeks (2 cycles) thereafter.

Statistical analysis

The sample size of the phase II stage was calculated using the Simon two-stage minimax design. Predicated on the results of afatinib under a Named Patient Use(30), we presumed H0 to be less than 20% of the ORR and H1 to be more than 40%, yielding a sample size of 33 with 80% power and a two-sided significance level set as α = 0.1. If more than 5 of 18 patients in stage I showed treatment response, then the study would proceed to stage II. If more than 10 of the total 33 patients showed treatment response, H0 would be rejected. Assuming a 10% dropout, a total of 37 patients were required for this study.

For analysis, we included all patients who received at least one dose of study drug. For efficacy analysis, we included all patients enrolled in this study. Separate analysis of efficacy was performed for each subgroup, including phase Ib stage, phase II stage, and patients who were treated with RPIID of afatinib and nimotuzumab. Survival estimates were calculated according to Kaplan–Meier method. All analyses were performed using SPSS 20.0 (IBM SPSS Statistics, IBM Corp.) and P value of < 0.05 was considered statistically significant. This study was registered at ClinicalTrials.gov, number NCT 01861223.

Patient characteristics

Between April 2013 and October 2014, a total of 50 patients were enrolled in two stages of the study; 13 patients for phase Ib and 37 patients for phase II. Table 1 summarizes baseline patient characteristics. Median age was 55 years, 62% were female, and 98% had an ECOG performance status of 1. The majority of patients were never-smokers (60%) with adenocarcinoma (96%), and all patients had stage IV disease at screening. Seventy percent of patients have been previously treated with at least three chemotherapies. All patients have received prior erlotinib (68%) or gefitinib (32%) and 80% patients showed partial response (PR) to prior EGFR TKI. Of 48 patients with known EGFR mutation status prior to gefitinib or erlotinib therapy, exon 19 deletion was the most common, accounting for 48%, followed by L858R mutation at 30%. In 27 (54%) of the 50 patients, rebiopsy was performed at the time of acquired resistance to gefitinib or erlotinib; 18 patients (67%) were T790 M-positive and 9 patients (33%) were T790M-negative.

At the data cutoff for this report (May 5, 2015), 49 patients discontinued study treatment due to the following reasons: progressive disease (42 patients, 82%), adverse events (5 patients, 10%), or other reasons (2 patients, 4%). The median number of cycles was 4 (range, 1–19) and the median follow-up duration for all 50 patients was 10.1 months (range, 2.5–23.9 months).

Determination of MTD and safety assessment

In phase Ib, 1 of 3 patients who were treated with dose level I experienced DLT (grade 3 diarrhea), which prompted enrollment of 3 additional patients resulting in only one DLT of 6 patients. At dose level II, 2 of 6 patients developed DLTs (grade 3 diarrhea and grade 3 neutropenia), leading to MTD. On the basis of these results, RPIID was defined as 40 mg of afatinib plus 100 mg of nimotuzumab. In stage I, 3 of 11 (> 27%) patients showed treatment response, the study would proceed to stage II. In phase II stage, 37 patients were treated with this regimen.

Table 2 shows treatment-related toxicity profiles which occurred in > 10% of total patients. There were no grade 4 or 5 toxicities. The most common adverse events of any grade for all patients included diarrhea (88%), mucositis (74%), rash (58%), anorexia (52%), and paronychia (46%). Among 44 patients treated with RPIID, 7 (16%) patients experienced grade 3 toxicities such as diarrhea (5%), rash (7%), acne (2%), scalp eruption (2%), fatigue (2%), and dyspnea (2%).

Treatment-related adverse events led to dose reduction in 18 (36%) patients, including 6 (46%) of 13 in the phase Ib stage and 12 (32%) of 37 in the phase II stage. Three patients in phase Ib stage discontinued therapy due to grade 3 diarrhea. Two patients in phase II stage withdrew from the study because of grade 2 skin rash and grade 3 diarrhea.

Antitumor activity

Of total 44 patient treated with RPIID, 43 patients were evaluable for tumor response (Table 3). The ORRs were observed in 10 (23%) of 43 patients, all of which were PRs. DCR were noted in 36 (84%) of 43 patients including PR in 10 (23%) and SD in 26 (61%). Of 35 patients harboring activating EGFR mutation and treated with RPIID, 9 (26%) had a confirmed ORR; ORR was 30% for exon 19 deletion and 20% for L858R mutation (Supplementary Table S1). There was a trend toward improved ORR in patients with exon 19 deletion relative to L858R mutation. Median interval between initial EGFR TKI and afatinib plus nimotuzumab was 7 months (range, 0–30). There was a trend toward improved ORR with respect to the interval of EGFR TKI “holiday,” although comparisons between groups (≥ 7 months vs. < 7 months) were not statistically significant (36% vs. 10%, P = 0.069). When we compared the ORR according to the number of prior treatment regimens (< 3 vs. ≥ 3), no significant differences were observed (P = 0.440). The maximum percent change in radiographic assessment of tumor target lesions is shown in Fig. 2. The PFS and OS for 44 patients who received the RPIID dose are shown in Fig. 3. The median duration of response was 4.3 months (range, 0.7–16.2 months) and the median PFS and OS were 4.0 months (95% CI, 2.3–5.7 months) and 11.7 months (95% CI, 9.4–14.0 months), respectively (Fig. 3).

Twenty-seven patients underwent rebiopsy after progression with gefitinib or erlotinib whose genomic aberrations were available for subgroup analysis. Patients were categorized according to the T790M mutation status (T790M-positive; n = 18 vs. T790M-negative; n = 9). ORR and median PFS for this subgroup patients were as follows: ORR, 18% T790M-positive vs. 33% T790M-negative, P = 0.628; median PFS, 3.7 months T790M-positive vs. 2.8 months T790M-negative, P = 0.720.

The development of acquired resistance to EGFR TKIs have galvanized research efforts in identifying effective alternative treatment strategies for EGFR-mutant NSCLC patients. The high frequency of EGFR T790M mutation in acquired resistance signified the critical role of continued signaling through EGFR in survival of EGFR-mutant lung cancer (31, 32). Dual EGFR blockade using an EGFR TKI coupled with an antibody to EGFR has been explored as a promising approach for acquired resistance (13, 14, 19, 20). To our knowledge, this is the first prospective study of combination therapy with afatinib plus nimotuzumab in patients with acquired resistance to gefitinib or erlotinib.

Toxicity is a critical aspect of treating patients with dual anti-EGFR inhibition given the potential overlapping side effects, especially skin rash and diarrhea. In this study, the RPIID was determined as afatinib 40 mg daily and nimotuzumab 100 mg weekly. For patients who were treated with RPIID, grade 3 toxicities were found in 16% of patients with low incidences of skin rash and diarrhea (7% and 5%, respectively), which were more favorable the previous study of afatinib plus cetuximab combination (20) where grade 3 or 4 treatment-related adverse events were noted in 46% of patients with the most common grade 3 events of rash (20%) and diarrhea (6%). According the toxicity profiles of afatinib (40 mg per day), rash and diarrhea occurred in 76% of patients, with 4% grade 3 rash and 7% grade 3 diarrhea (33). On the basis of these data, nimotuzumab seems to have no additive toxicities such as skin rash and diarrhea when combined with afatinib. Prior data also reported that severe adverse events such as skin rash or diarrhea commonly associated with cetuximab and panitumumab remain extremely rare with nimotuzumab in other solid tumors (34–36). Fewer side effects of nimotuzumab compared with other anti-EGFR mAbs may be attributed to unique binding affinity in the range of 10−8 to 10−9 mol/L to maximize tumor cell targeting while minimizing normal cell toxicity (37, 38). Also, recent experimental observations suggest that in contrast to other anti-EGFR antibodies, bivalent binding property of nimotuzumab contributes to the differences in toxicity profiles (38, 39). We noted that one patient experienced DLT with grade 3 neutropenia in cycle 1 at dose level II without any symptom. On the basis of the toxicity profile of nimotuzumab, prior systemic chemotherapy and radiotherapy to pelvic bone could have influenced on bone marrow capacity of the patient.

Although afatinib as single agent demonstrated high antitumor activity in preclinical model in both sensitizing EGFR mutants and resistant cells harboring T790M, the ORR with afatinib alone for NSCLC patients who progressed during prior treatment with gefitinib or erlotinib was disappointing with only 7% to 8% (18, 40). However, a novel treatment strategy with dual blockade of EGFR with afatinib and cetuximab has increased the response rate to 29% with an improvement in median PFS of 4.7 months (20). In the current study evaluating afatinib 40 mg plus nimotuzumab 100 mg, 23% achieved partial response and 84% showed disease control with median duration of response of 4.3 months. Collectively, these results provide additional evidence that dual blockade of EGFR are more effective than afatinib alone in NSCLC patients who progressed on first-generation EGFR TKI. Furthermore, the survival benefit of this combination (4.0 months for median PFS and 11.7 months for median OS) is particularly meaningful given that the majority of patients were heavily pretreated. Although treatment with higher dose level (afatinib 40 mg plus nimotuzumab 200 mg) seems to have higher response rates, the clinical relevance is limited because of the small sample size. To increase the clinical benefit of this regimen, the more defined patient selection strategy should be investigated in the future.

It is noteworthy that subgroup analysis according to the EGFR mutation subtype showed numerically higher response in patients with exon 19 deletion. In the current study, patients with longer interval duration between prior EGFR TKI and afatinib plus nimotuzumab showed better ORR, although no statistically significant finding (36% vs. 10%, P = 0.069). Several retrospective studies regarding gefitinib readministration have shown that a prolonged gefitinib-free interval was a predictive factor for a favorable clinical result (41, 42), suggesting that cytotoxic chemotherapy modify the heterogeneous tissue distribution in sensitive or resistant cells.

It still remains unknown whether dual blockade of EGFR is effective irrespective of T790M mutation status in patients with acquired resistance to gefitinib or erlotinib. According to the study by Janjigian and colleagues, there was no significant difference in ORR between patients harboring T790M-positive and T790M-negative tumors (32% vs. 25%; P = 0.341; ref. 20). In the current study, although the response rate was numerically higher in T790M-negative patients, it should be cautious to make conclusion due to limitation of study population. However, given that no alternative therapies exist for EGFR TKI resistance patients without T790M mutation, this combination therapy may be a viable strategy. Recently, third-generation EGFR TKI, AZD9291 and rociletinib demonstrated robust and durable response in both T790M-positive and -negative patients (43, 44). Moreover, Meador and colleagues reported that secondary resistance to afatinib plus cetuximab can be overcome by AZD9291 (45). However, given the heterogeneity of T790M-negative EGFR TKI-resistant tumor, dual targeting of EGFR with afatinib plus nimotuzumab still has therapeutic option for patients who developed resistance to EGFR TKIs.

In conclusion, combination therapy of afatinib and nimotuzumab was effective and tolerable in advanced NSCLC patients with acquired resistance to gefitinib or erlotinib. A large trial assessing clinical impact of afatinib plus nimotuzumab combination in conjunction with accurate molecular profiling of genomic aberrations is warranted to identify and guide optimal treatment strategy in EGFR-mutant patients resistant to gefitinib or erlotinib.

K. Park is a consultant/advisory board member for Boehringer Ingelheim. No potential conflicts of interest were disclosed by the other authors.

Conception and design: J.Y. Lee, H.S. Kim, K.H. Yoo, K. Park, M.-J. Ahn

Development of methodology: J.Y. Lee, J.-M. Sun, H.S. Kim, K.H. Yoo, K. Park, M.-J. Ahn

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.Y. Lee, J.-M. Sun, H.S. Kim, K.H. Yoo, B.M. Ku, J. Koh, Y.-H. Bae, S.-H. Lee, J.S. Ahn, K. Park, M.-J. Ahn

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.Y. Lee, J.-M. Sun, H.S. Kim, K.H. Yoo, J. Koh, J.S. Ahn, K. Park, M.-J. Ahn

Writing, review, and/or revision of the manuscript: J.Y. Lee, J.-M. Sun, S.H. Lim, H.S. Kim, K.H. Yoo, B.M. Ku, S.-H. Lee, J.S. Ahn, K. Park, M.-J. Ahn

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.Y. Lee, J.-M. Sun, H.S. Kim, K.H. Yoo, K.S. Jung, K. Park, M.-J. Ahn

Study supervision: J.Y. Lee, H.S. Kim, K.H. Yoo, K.S. Jung, K. Park, M.-J. Ahn, H.-N. Song

This study was supported in part by Boehringer Ingelheim and by the Samsung Biomedical Research Institute Grant (GE1-B3-081-1).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Jemal
A
,
Bray
F
,
Center
MM
,
Ferlay
J
,
Ward
E
,
Forman
D
. 
Global cancer statistics
.
CA Cancer J Clin
2011
;
61
:
69
90
.
2.
Jung
KW
,
Park
S
,
Kong
HJ
,
Won
YJ
,
Lee
JY
,
Park
EC
, et al
Cancer statistics in Korea: incidence, mortality, survival, and prevalence in 2008
.
Cancer Res Treat
2011
;
43
:
1
11
.
3.
Mok
TS
,
Wu
YL
,
Thongprasert
S
,
Yang
CH
,
Chu
DT
,
Saijo
N
, et al
Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma
.
N Engl J Med
2009
;
361
:
947
57
.
4.
Mitsudomi
T
,
Morita
S
,
Yatabe
Y
,
Negoro
S
,
Okamoto
I
,
Tsurutani
J
, et al
Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial
.
Lancet Oncol
2010
;
11
:
121
8
.
5.
Maemondo
M
,
Inoue
A
,
Kobayashi
K
,
Sugawara
S
,
Oizumi
S
,
Isobe
H
, et al
Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR
.
N Engl J Med
2010
;
362
:
2380
8
.
6.
Zhou
C
,
Wu
YL
,
Chen
G
,
Feng
J
,
Liu
XQ
,
Wang
C
, et al
Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study
.
Lancet Oncol
2011
;
12
:
735
42
.
7.
Rosell
R
,
Carcereny
E
,
Gervais
R
,
Vergnenegre
A
,
Massuti
B
,
Felip
E
, et al
Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial
.
Lancet Oncol
2012
;
13
:
239
46
.
8.
Han
JY
,
Park
K
,
Kim
SW
,
Lee
DH
,
Kim
HY
,
Kim
HT
, et al
First-SIGNAL: first-line single-agent iressa versus gemcitabine and cisplatin trial in never-smokers with adenocarcinoma of the lung
.
J Clin Oncol
2012
;
30
:
1122
8
.
9.
Kosaka
T
,
Yatabe
Y
,
Endoh
H
,
Yoshida
K
,
Hida
T
,
Tsuboi
M
, et al
Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib
.
Clin Cancer Res
2006
;
12
:
5764
9
.
10.
Ohashi
K
,
Maruvka
YE
,
Michor
F
,
Pao
W
. 
Epidermal growth factor receptor tyrosine kinase inhibitor-resistant disease
.
J Clin Oncol
2013
;
31
:
1070
80
.
11.
Pao
W
,
Miller
VA
,
Politi
KA
,
Riely
GJ
,
Somwar
R
,
Zakowski
MF
, et al
Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain
.
PLoS Med
2005
;
2
:
e73
.
12.
Yun
CH
,
Mengwasser
KE
,
Toms
AV
,
Woo
MS
,
Greulich
H
,
Wong
KK
, et al
The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP
.
Proc Natl Acad Sci U S A
2008
;
105
:
2070
5
.
13.
Huang
S
,
Armstrong
EA
,
Benavente
S
,
Chinnaiyan
P
,
Harari
PM
. 
Dual-agent molecular targeting of the epidermal growth factor receptor (EGFR): combining anti-EGFR antibody with tyrosine kinase inhibitor
.
Cancer Res
2004
;
64
:
5355
62
.
14.
Matar
P
,
Rojo
F
,
Cassia
R
,
Moreno-Bueno
G
,
Di Cosimo
S
,
Tabernero
J
, et al
Combined epidermal growth factor receptor targeting with the tyrosine kinase inhibitor gefitinib (ZD1839) and the monoclonal antibody cetuximab (IMC-C225): superiority over single-agent receptor targeting
.
Clin Cancer Res
2004
;
10
:
6487
501
.
15.
Li
D
,
Ambrogio
L
,
Shimamura
T
,
Kubo
S
,
Takahashi
M
,
Chirieac
LR
, et al
BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models
.
Oncogene
2008
;
27
:
4702
11
.
16.
Solca
F
,
Dahl
G
,
Zoephel
A
,
Bader
G
,
Sanderson
M
,
Klein
C
, et al
Target binding properties and cellular activity of afatinib (BIBW 2992), an irreversible ErbB family blocker
.
J Pharmacol Exp Ther
2012
;
343
:
342
50
.
17.
Yang
JC
,
Wu
YL
,
Schuler
M
,
Sebastian
M
,
Popat
S
,
Yamamoto
N
, et al
Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-Lung 3 and LUX-Lung 6): analysis of overall survival data from two randomised, phase 3 trials
.
Lancet Oncol
2015
;
16
:
141
51
.
18.
Miller
VA
,
Hirsh
V
,
Cadranel
J
,
Chen
YM
,
Park
K
,
Kim
SW
, et al
Afatinib versus placebo for patients with advanced, metastatic non-small-cell lung cancer after failure of erlotinib, gefitinib, or both, and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial
.
Lancet Oncol
2012
;
13
:
528
38
.
19.
Regales
L
,
Gong
Y
,
Shen
R
,
de Stanchina
E
,
Vivanco
I
,
Goel
A
, et al
Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer
.
J Clin Invest
2009
;
119
:
3000
10
.
20.
Janjigian
YY
,
Smit
EF
,
Groen
HJ
,
Horn
L
,
Gettinger
S
,
Camidge
DR
, et al
Dual inhibition of EGFR with afatinib and cetuximab in kinase inhibitor-resistant EGFR-mutant lung cancer with and without T790M mutations
.
Cancer Discov
2014
;
4
:
1036
45
.
21.
Talavera
A
,
Friemann
R
,
Gomez-Puerta
S
,
Martinez-Fleites
C
,
Garrido
G
,
Rabasa
A
, et al
Nimotuzumab, an antitumor antibody that targets the epidermal growth factor receptor, blocks ligand binding while permitting the active receptor conformation
.
Cancer Res
2009
;
69
:
5851
9
.
22.
Crombet
T
,
Osorio
M
,
Cruz
T
,
Roca
C
,
del Castillo
R
,
Mon
R
, et al
Use of the humanized anti-epidermal growth factor receptor monoclonal antibody h-R3 in combination with radiotherapy in the treatment of locally advanced head and neck cancer patients
.
J Clin Oncol
2004
;
22
:
1646
54
.
23.
Ramos
TC
,
Figueredo
J
,
Catala
M
,
Gonzalez
S
,
Selva
JC
,
Cruz
TM
, et al
Treatment of high-grade glioma patients with the humanized anti-epidermal growth factor receptor (EGFR) antibody h-R3: report from a phase I/II trial
.
Cancer Biol Ther
2006
;
5
:
375
9
.
24.
Rodriguez
MO
,
Rivero
TC
,
del Castillo Bahi
R
,
Muchuli
CR
,
Bilbao
MA
,
Vinageras
EN
, et al
Nimotuzumab plus radiotherapy for unresectable squamous-cell carcinoma of the head and neck
.
Cancer Biol Ther
2010
;
9
:
343
9
.
25.
Bebb
G
,
Smith
C
,
Rorke
S
,
Boland
W
,
Nicacio
L
,
Sukhoo
R
, et al
Phase I clinical trial of the anti-EGFR monoclonal antibody nimotuzumab with concurrent external thoracic radiotherapy in Canadian patients diagnosed with stage IIb, III or IV non-small cell lung cancer unsuitable for radical therapy
.
Cancer Chemother Pharmacol
2011
;
67
:
837
45
.
26.
Choi
HJ
,
Sohn
JH
,
Lee
CG
,
Shim
HS
,
Lee
IJ
,
Yang
WI
, et al
A phase I study of nimotuzumab in combination with radiotherapy in stages IIB-IV non-small cell lung cancer unsuitable for radical therapy: Korean results
.
Lung Cancer
2011
;
71
:
55
9
.
27.
Fairchild
A
,
Harris
K
,
Barnes
E
,
Wong
R
,
Lutz
S
,
Bezjak
A
, et al
Palliative thoracic radiotherapy for lung cancer: a systematic review
.
J Clin Oncol
2008
;
26
:
4001
11
.
28.
Choi
YL
,
Sun
JM
,
Cho
J
,
Rampal
S
,
Han
J
,
Parasuraman
B
, et al
EGFR mutation testing in patients with advanced non-small cell lung cancer: a comprehensive evaluation of real-world practice in an East Asian tertiary hospital
.
PLoS One
2013
;
8
:
e56011
.
29.
Nagai
Y
,
Miyazawa
H
, Huqun,
Tanaka
T
,
Udagawa
K
,
Kato
M
, et al
Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp
.
Cancer Res
2005
;
65
:
7276
82
.
30.
Khan
F
,
Ottensmeier
C
,
Popat
S
,
Dua
D
,
Dorey
N
,
Ellis
S
, et al
Afatinib use in non-small cell lung cancer previously sensitive to epidermal growth factor receptor inhibitors: the United Kingdom Named Patient Programme
.
Eur J Cancer
2014
;
50
:
1717
21
.
31.
Balak
MN
,
Gong
Y
,
Riely
GJ
,
Somwar
R
,
Li
AR
,
Zakowski
MF
, et al
Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors
.
Clin Cancer Res
2006
;
12
:
6494
501
.
32.
Bean
J
,
Riely
GJ
,
Balak
M
,
Marks
JL
,
Ladanyi
M
,
Miller
VA
, et al
Acquired resistance to epidermal growth factor receptor kinase inhibitors associated with a novel T854A mutation in a patient with EGFR-mutant lung adenocarcinoma
.
Clin Cancer Res
2008
;
14
:
7519
25
.
33.
Genova
C
,
Rijavec
E
,
Barletta
G
,
Burrafato
G
,
Biello
F
,
Dal Bello
MG
, et al
Afatinib for the treatment of advanced non-small-cell lung cancer
.
Expert Opin Pharmacother
2014
;
15
:
889
903
.
34.
Reddy
BK
,
Lokesh
V
,
Vidyasagar
MS
,
Shenoy
K
,
Babu
KG
,
Shenoy
A
, et al
Nimotuzumab provides survival benefit to patients with inoperable advanced squamous cell carcinoma of the head and neck: a randomized, open-label, phase IIb, 5-year study in Indian patients
.
Oral Oncol
2014
;
50
:
498
505
.
35.
Agero
AL
,
Dusza
SW
,
Benvenuto-Andrade
C
,
Busam
KJ
,
Myskowski
P
,
Halpern
AC
. 
Dermatologic side effects associated with the epidermal growth factor receptor inhibitors
.
J Am Acad Dermatol
2006
;
55
:
657
70
.
36.
Giusti
RM
,
Cohen
MH
,
Keegan
P
,
Pazdur
R
. 
FDA review of a panitumumab (Vectibix) clinical trial for first-line treatment of metastatic colorectal cancer
.
Oncologist
2009
;
14
:
284
90
.
37.
Boland
WK
,
Bebb
G
. 
Nimotuzumab: a novel anti-EGFR monoclonal antibody that retains anti-EGFR activity while minimizing skin toxicity
.
Expert Opin Biol Ther
2009
;
9
:
1199
206
.
38.
Ramakrishnan
MS
,
Eswaraiah
A
,
Crombet
T
,
Piedra
P
,
Saurez
G
,
Iyer
H
, et al
Nimotuzumab, a promising therapeutic monoclonal for treatment of tumors of epithelial origin
.
MAbs
2009
;
1
:
41
8
.
39.
Garrido
G
,
Tikhomirov
IA
,
Rabasa
A
,
Yang
E
,
Gracia
E
,
Iznaga
N
, et al
Bivalent binding by intermediate affinity of nimotuzumab: a contribution to explain antibody clinical profile
.
Cancer Biol Ther
2011
;
11
:
373
82
.
40.
Katakami
N
,
Atagi
S
,
Goto
K
,
Hida
T
,
Horai
T
,
Inoue
A
, et al
LUX-Lung 4: a phase II trial of afatinib in patients with advanced non-small-cell lung cancer who progressed during prior treatment with erlotinib, gefitinib, or both
.
J Clin Oncol
2013
;
31
:
3335
41
.
41.
Tomizawa
Y
,
Fujita
Y
,
Tamura
A
,
Shirai
M
,
Shibata
S
,
Kawabata
T
, et al
Effect of gefitinib re-challenge to initial gefitinib responder with non-small cell lung cancer followed by chemotherapy
.
Lung Cancer
2010
;
68
:
269
72
.
42.
Yokouchi
H
,
Yamazaki
K
,
Kinoshita
I
,
Konishi
J
,
Asahina
H
,
Sukoh
N
, et al
Clinical benefit of readministration of gefitinib for initial gefitinib-responders with non-small cell lung cancer
.
BMC Cancer
2007
;
7
:
51
.
43.
Janne
PA
,
Yang
JC
,
Kim
DW
,
Planchard
D
,
Ohe
Y
,
Ramalingam
SS
, et al
AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer
.
N Engl J Med
2015
;
372
:
1689
99
.
44.
Sequist
LV
,
Soria
JC
,
Goldman
JW
,
Wakelee
HA
,
Gadgeel
SM
,
Varga
A
, et al
Rociletinib in EGFR-mutated non-small-cell lung cancer
.
N Engl J Med
2015
;
372
:
1700
9
.
45.
Meador
CB
,
Jin
H
,
de Stanchina
E
,
Nebhan
CA
,
Pirazzoli
V
,
Wang
L
, et al
Optimizing the sequence of anti-EGFR-targeted therapy in EGFR-mutant lung cancer
.
Mol Cancer Ther
2015
;
14
:
542
52
.