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Keywords:

  • epidermal growth factor receptor;
  • gene amplification;
  • stomach neoplasm;
  • survival analysis;
  • tissue array analysis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aims:  Epidermal growth factor receptor (EGFR) expression has been observed in a variety of solid tumours with the potential of new targeted therapeutic agents. The aim was to evaluate the EGFR status of gastric carcinoma (GC) using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).

Methods and results:  The EGFR status was evaluated in GC tissues from 511 patients using IHC and FISH. In addition, the clinicopathological characteristics were examined and the results were compared with the EGFR status. One hundred and forty cases (27.4%) showed EGFR overexpression by IHC. EGFR overexpression was associated with older age (P = 0.001), moderately or poorly differentiated histology (P = 0.001) and higher stage disease (P = 0.046). Sixteen cases (3.1%) showed high polysomy and 12 cases (2.3%) had gene amplification by FISH. The correlation between IHC and FISH results was statistically significant (P < 0.001). The patients with GC who had EGFR overexpression had an unfavourable prognosis and multivariate analysis showed that EGFR overexpression was a possible independent unfavourable prognostic factor.

Conclusions:  EGFR overexpression was observed in a subset of cases with GC and was associated with an unfavourable prognosis. It will be important to evaluate EGFR status to interpret future clinical trials properly using EGFR targeted agents.


Abbreviations:
CEP

chromosome enumeration probe

EGFR

epidermal growth factor receptor

FISH

fluorescence in situ hybridization

GC

gastric carcinoma

IHC

immunohistochemistry

LSI

locus specific identifier

pTNM

pathological tumour node metastasis

SSC

standard saline citrate

WHO

World Health Organization

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Gastric carcinoma (GC) is one of the common epithelial malignancies worldwide.1 Although our understanding of this disease has improved during the past decade, the prognosis for patients with advanced GC remains poor. The 5-year survival rate for patients with localized disease is approximately 60%, whereas for those with distant metastasis it is only 2%.2 Increased understanding of the molecular basis of GC may lead to improvements in patient outcome. In particular, molecular biomarkers of tumour behaviour are powerful new tools that can be used for predicting patient outcome and identifying novel targets for directed therapy.

The epidermal growth factor receptor (EGFR) gene, also called ERBB, is located at chromosomal region 7p12 and encodes a 170-kDa transmembrane tyrosine kinase receptor, which is a member of the EGFR family.3 The EGFR is activated by binding to its ligands such as epidermal growth factor or transforming growth factor-alpha, resulting in homodimerization or heterodimerization with another member of the EGFR family.4 This receptor activation is followed by phosphorylation of specific tyrosine residues within the cytoplasmic tail, stimulating the downstream signalling pathway that regulates cell proliferation, migration, adhesion, differentiation and survival.4

Gene amplification and/or protein overexpression of EGFR have been observed in a variety of solid tumours. Examples include the lung, colorectal, urinary bladder, breast, head, neck, oesophageal and gastric carcinomas.5–14 In some tumours such as non-small cell lung carcinoma and colorectal carcinoma, increased EGFR expression is associated with advanced stage and an unfavourable prognosis.5,15 The frequency of EGFR overexpression and/or amplification in GC has been variously reported to be 0–38%.12–14 However, to date no large-scale study has been performed to evaluate the frequency of EGFR overexpression and/or amplification and its association with the clinicopathological parameters and prognostic implications in patients with GC.

Recently, the development of new therapeutic agents targeting EGFR has attracted attention.16 One group is monoclonal antibodies that target the EGFR gene product and includes cetuximab, matuzumab and panitumumab. These compounds bind to the extracellular domain of the receptor, where they compete with the natural ligand binding to the receptor and thereby block activation of the receptor. Another group of agents includes low-molecular-weight tyrosine kinase inhibitors such as gefitinib, erlotinib and lapatinib. These compounds compete with adenosine triphosphate binding to the tyrosine kinase portion of the endodomain of the receptor and thereby abrogate the receptor’s catalytic activity. Both groups of compounds appear to be equally effective in blocking the downstream receptor-dependent signalling pathway. In practice, cetuximab has been approved for the treatment of advanced colorectal carcinomas refractory to irinotecan-based therapy.17,18

However, no large clinical trial on GC patients treated with cetuximab therapy has been performed to date. A response to the tyrosine kinase inhibitor gefitinib is correlated with a high EGFR gene copy number, protein expression or EGFR mutation in patients with advanced non-small cell lung carcinoma.6,19 For patients with GC, a few clinical trials have been performed but with ambiguous results. One clinical trial has found that a subpopulation of GC shows evidence of gefitinib sensitivity.20 It is likely that the alteration of EGFR in cancer cells affects sensitivity to EGFR-targeted drugs. Therefore, accurate evaluation of the EGFR protein and/or EGFR gene expression status in patients with GC is important for determining patient eligibility for new targeted therapy.

The aim of this study was to evaluate EGFR status in a large number of GC cases using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). We investigated the association between EGFR protein overexpression evaluated by IHC and EGFR gene status evaluated by FISH. In addition, we determined whether EGFR overexpression and/or high gene copy number were associated with a variety of clinicopathological features including survival.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Patient samples

Consecutively collected, surgically resected GC tissue specimens were obtained from 511 patients who underwent gastrectomy at Seoul National University Hospital between 1 January 1995 and 31 December 1995. The clinicopathological parameters such as age, gender, histological subtype, the presence of lymphatic invasion, invasion depth, the presence of lymph node or distant metastasis and pathological stage were evaluated by reviewing the medical charts and pathological records. The mean patient age was 55.4 years and 93.7% of the patients had undergone a curative resection (R0 according to the International Union Against Cancer guidelines). The clinical outcome was determined from the date of surgery until death or 31 December 2003, which resulted in a follow-up period of from 1 to 108 months (mean 68.0 months). The cases that were lost to follow-up and deaths caused by problems other than GC were regarded as censored data in the survival analysis. This study was approved by the Institutional Review Boards of Seoul National University Hospital.

Tissue array methods

Core tissue biopsy specimens (diameter 2 mm) were obtained from individual paraffin-embedded gastric tumours (donor blocks) and arranged in new recipient paraffin blocks (tissue array blocks) using a trephine apparatus (Superbiochips Laboratories, Seoul, Republic of Korea). Two separate core samples per tumour were obtained to account for tumour heterogeneity. Non-neoplastic gastric mucosa specimens were included in each of the array blocks. The tissue array blocks contained up to 60 cores, allowing for 24 array blocks from the 511 cases.

Immunohistochemistry

An EGFR pharmDx kit was used to detect EGFR expression (Dako, Carpinteria, CA, USA). According to the manufacturer’s protocol, after deparaffinization, 4-μm sections were treated with proteinase K solution for 5 min at room temperature. After peroxidase blocking for 5 min, the sections were incubated with primary antibody for 30 min at room temperature. They were then labelled with a polymer for 30 min at room temperature and reacted with diaminobenzidene tetrahydrochloride solution. EGFR immunopositivity was scored using the instructions in the EGFR pharmDx kit. Reactivity was scored as zero when there was no membranous reactivity within the tumour, and as positive when there was reactivity of the tumour cell membrane that was detected above the background level. The positive samples were classified further into 1+, 2+ and 3+ reactivity based on their intensity of reactivity. The highest intensity of reactivity of all tissue cores from the same tumour was used as the final immunohistochemical result for that tumour.21 Scores of 2+ and 3+ were classified as overexpression.14

Fluorescence in situ hybridization

Dual-colour FISH assays were performed using the locus specific identifier (LSI) EGFR SpectrumOrange/chromosome enumeration probe (CEP) 7 SpectrumGreen probe (Vysis, Downers Grove, IL, USA). Briefly, 2 μm-sectioned deparaffinized and dehydrated tissue array slides were incubated in 20% sodium bisulphate/2× standard saline citrate (2× SSC) at 75°C for 20 min. After washing in 2× SSC, the slides were treated with proteinase K at 37°C for 25 min, rinsed in 2× SSC at room temperature for 5 min and dehydrated using ethanol in a series of increasing concentrations (70%, 85%, 100%). The DNA probe set was applied onto the selected area based on the presence of tumour foci on each slide and the hybridization area was covered with a glass coverslip and sealed with rubber cement. The slides were incubated at 80°C for 10 min for co-denaturation of chromosomal and probe DNA and were then placed in a humidified chamber at 37°C for 24 h to allow hybridization to occur. After the post-hybridization washing, the slides were then counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride in antifade solution and examined under a fluorescence microscope (Olympus, Tokyo, Japan) equipped with Triple Bandpass Filter Sets (Vysis).

At least 100 tumour cell nuclei were counted per case. We then classified the tumours into six groups according to the frequency of tumour cells with specific numbers of copies of the EGFR gene and chromosome 7 centromere as previously described.6 The six groups included the following: (i) disomy (≤2 copies in ≥90% of cells); (ii) low trisomy (≤2 copies in ≥40% of cells, three copies in 10–40% of cells, ≥4 copies in <10% of cells); (iii) high trisomy (≤2 copies in ≥40% of cells, three copies in ≥40% of cells, ≥4 copies in <10% of cells); (iv) low polysomy (≥4 copies in 10–40% of cells); (v) high polysomy (≥4 copies in ≥40% of cells); and (vi) gene amplification (defined by the presence of tight EGFR gene clusters and a ratio of EGFR gene to chromosome of ≥2 or ≥15 copies of EGFR per cell in ≥10% of analysed cells). High polysomy and gene amplification were regarded as a positive FISH result.6

Statistical analysis

Differences between and among groups were compared using Fisher’s exact test or Pearson’s χ2 test for qualitative variables and Student’s t-test or analysis of variance for continuous variables. Survival curves were estimated using the Kaplan–Meier product-limit method and the significance of differences between survival curves was determined using the log rank test. Multivariate analysis was performed using Cox proportional hazards regression modelling. All statistical tests were two-sided, and statistical significance was defined as P < 0.05. All analyses were performed using the statistical package SPSS version 12.0 (SPSS Inc., Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

EGFR protein expression status and clinicopathological findings

EGFR protein expression status was determined by IHC for the 511 GC tissues (Figure 1). Of these, 139 cases (27.2%) scored 0, 232 (45.4%) scored 1+, 119 (23.3%) scored 2+ and 21 (4.1%) scored 3+. Scores of 0 and 1+ were regarded as negative for EGFR protein overexpression and scores of 2+ and 3+ as positive. Table 1 summarizes EGFR protein expression and the clinicopathological findings. EGFR overexpression was associated with an older age (P = 0.001). GC with EGFR overexpression was associated with a moderately or poorly differentiated histology by the World Health Organization (WHO) classification (P = 0.001). EGFR overexpression was correlated statistically with the presence of lymphatic invasion (P < 0.001), advanced GC (P = 0.010), presence of lymph node metastasis (P = 0.006) and higher pathological tumour node metastasis (pTNM) stage (P = 0.046). However, there was no significant correlation between EGFR overexpression and gender, Lauren classification or distant metastasis (data not shown).

image

Figure 1.  Immunohistochemical analyses of epidermal growth factor receptor (EGFR) protein expression in gastric carcinomas. A, Immunonegativity. B, 1+ reactivity intensity. C, 2+ reactivity intensity. D, 3+ reactivity intensity.

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Table 1.   Comparison of clinicopathological parameters between EGFR− and EGFR+ gastric carcinomas evaluated by immunohistochemistry
 EFGR overexpressionP-value
Negative (%) (n = 371)Positive (%) (n = 140)
  1. W/D, Well-differentiated tubular adenocarcinoma; M/D, moderately differentiated tubular adenocarcinoma; P/D, poorly differentiated tubular adenocarcinoma; SRC, signet ring cell carcinoma; AGC, advanced gastric carcinoma; EGC, early gastric carcinoma; LN, lymph node; EGFR, epidermal growth factor receptor.

Age (years)54.5 ± 13.157.8 ± 10.70.001
WHO classification0.001
 W/D38 (10.2)7 (5.0)
 M/D102 (27.5)58 (41.4)
 P/D146 (39.4)63 (45.0)
 Mucinous29 (7.8)3 (2.1)
 SRC56 (15.1)9 (6.4)
Lymphatic invasion<0.001
 Absent277 (74.7)76 (54.3)
 Present94 (25.3)64 (45.7)
Tumour invasion0.010
 AGC245 (66.0)109 (77.9)
 EGC126 (34.0)31 (22.1)
LN metastasis0.006
 Absent155 (41.8)40 (28.6)
 Present216 (58.2)100 (71.4)
pTNM stage0.046
 I167 (45.0)46 (32.9)
 II78 (21.0)29 (20.7)
 III74 (19.9)40 (28.6)
 IV52 (14.0)25 (17.9)

Comparison of EGFR status as evaluated with IHC and fish

FISH analysis was performed for all 511 cases. Of these, 293 cases (57.3%) showed disomy, 144 cases (28.2%) low trisomy, seven cases (1.4%) high trisomy, 39 cases (7.6%) low polysomy, 16 cases (3.1%) high polysomy and 12 cases (2.3%) gene amplification. Disomy, low trisomy, high trisomy and low polysomy were regarded as negative for EGFR FISH and high polysomy and gene amplification as positive (Figure 2). The results obtained showed an EGFR gene FISH+ rate of 5.5%. Only one of the 371 cases (0.27%) that was negative by IHC was positive by FISH, whereas 27 of the 140 cases (19.3%) with EGFR protein overexpression showed EGFR gene amplification or high polysomy by FISH (Table 2). The correlation between IHC and FISH results was statistically significant (P < 0.001).

image

Figure 2.  Fluorescence in situ hybridization analyses of epidermal growth factor receptor gene status in gastric carcinoma. A, Example of gene amplification. B, Example of high polysomy.

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Table 2.   EGFR protein expression and EGFR gene FISH patterns
IHCFISH patternTotal
DILTHTLPHPAMP
  1. The values are given as n (%).

  2. DI, Disomy; LT, low trisomy; HT, high trisomy; LP, low polysomy; HP, high polysomy; AMP, amplification; EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization.

096 (69.1)35 (25.2)2 (1.4)6 (4.3)0 (0.0)0 (0.0)139
1+145 (62.5)67 (28.9)2 (0.9)17 (7.3)1 (0.4)0 (0.0)232
2+49 (41.2)38 (31.9)3 (2.5)15 (12.6)12 (10.1)2 (1.7)119
3+3 (14.3)4 (19.0)0 (0.0)1 (4.8)3 (14.3)10 (47.6)21
Total293 (57.3)144 (28.2)7 (1.4)39 (7.6)16 (3.1)12 (2.3)511

Correlation between EGFR gene amplification or high polysomy and clinicopathological findings

Table 3 shows the clinicopathological differences observed between GC with EGFR gene amplification and high polysomy. GCs with EGFR high polysomy were associated with a higher pathological stage than those with EGFR amplification (P = 0.043). However, no difference was observed between GC cases with EGFR amplification and high polysomy in terms of age, gender, WHO classification or lymphatic invasion. EGFR FISH+ GCs were associated with the presence of lymphatic invasion (P < 0.001) and had a tendency for a higher disease stage, but this was not statistically significant. There were no differences noted for age, gender, WHO classification, or stage.

Table 3.   Comparison of clinicopathological parameters between EGFR FISH+ and FISH− gastric carcinomas
 EGFR FISH+EGFR FISH−3 (%) (n = 483)P-value*P-value†
AMP1 (%) (n = 12)HP2 (%) (n = 16)Total1+2 (%) (n = 28)
  1. AMP, Amplification; HP, high polysomy; W/D, well differentiated tubular adenocarcinoma; M/D, moderately differentiated tubular adenocarcinoma; P/D, poorly differentiated tubular adenocarcinoma; SRC, signet ring cell carcinoma; NS, not significant; EGFR, epidermal growth factor receptor; FISH, fluorescence in situ hybridization.

  2. *P value, 1 versus 2.

  3. P value, 1 + 2 versus 3.

Age60.3 ± 12.460.3 ± 11.960.3 ± 11.9 55.1 ± 12.6NSNS
Sex
 Male6 (50.0)12 (75.0)18 (64.3)337 (69.8)NSNS
 Female6 (50.0)4 (25.0)10 (35.7)146 (30.2)
WHO classification
 W/D0 (0.0)1 (6.3)1 (3.6)44 (9.1)NSNS
 M/D6 (50.0)3 (18.8)9 (32.1)151 (31.3)
 P/D6 (50.0)11 (68.8)17 (60.7)192 (39.8)
 Mucinous0 (0.0)1 (6.3)1 (3.6)31 (6.4)
 SRC0 (0.0)0 (0.0)0 (0.0)65 (13.5)
Lymphatic invasion
 Absent3 (25.0)8 (50.0)11 (39.3)342 (70.8)NS<0.001
 Present9 (75.0)8 (50.0)17 (60.7)141 (29.2)
pTNM stage
 I6 (50.0)1 (6.3)7 (25.0)206 (42.7)0.043NS
 II1 (8.3)6 (37.5)7 (25.0)100 (20.7)
 III4 (33.3)6 (37.5)10 (35.7)104 (21.5)
 IV1 (8.3)3 (18.8)4 (14.3)73 (15.1)

Survival analysis

The mean duration of follow-up was 68.0 months after surgery. Only four patients were lost to follow-up and this did not have a significant impact on the survival analyses. During the follow-up period, 213 of the 507 patients (42.0%) died. The overall survival rate of patients with EGFR overexpression, as determined by the log rank test, was significantly lower than the rate of those without EGFR overexpression (P = 0.0001) (Figure 3A). Patients with EGFR FISH+ GCs also had a less favourable prognosis than those with EGFR FISH− GCs (P = 0.0335) (Figure 3B). On multivariate analysis, EGFR overexpression was an independent prognostic indicator, although pTNM stage was the strongest predictive factor (Table 4). However, there were no significant differences in the multivariate analysis between the subjects with EGFR FISH+ GCs and those with EGFR FISH− GCs (data not shown).

image

Figure 3.  Survival curves using the Kaplan–Meier method by log rank test. A, Survival curves show that epidermal growth factor receptor (EGFR) immunohistochemistry (IHC)+ gastric carcinoma (broken line, n = 139) had an unfavourable prognosis compared with EGFR IHC− gastric carcinoma (solid line, n = 368) (P = 0.0001). B, Survival curves show that EGFR fluorescence in situ hybridization (FISH)+ gastric carcinoma (broken line, n = 28) had an unfavourable prognosis compared with FISH− gastric carcinoma (solid line, n = 479) (P = 0.0335).

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Table 4.   Multivariate analysis of predictive factors for survival (Cox proportional hazards model)
Prognostic factorsHazard ratio (95% CI)P-value
  1. CI, Confidence interval; EGFR, epidermal growth factor receptor.

EGFR overexpression
 Positive versus negative1.413 (1.063, 1.878)0.017
pTNM stage
 II versus I2.863 (1.815, 4.515)<0.001
 III versus I5.485 (3.577, 8.412)
 IV versus I9.899 (6.165, 15.894)
Lymphatic invasion
 Present versus absent1.383 (1.034, 1.850)0.029
Radical surgery
 R1 versus R01.425 (0.621, 3.271)0.017
 R2 versus R02.142 (1.257, 3.650)
Age
 <65 years versus ≥65 years1.495 (1.097, 2.038)0.011

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The role of growth factor-driven signalling in the pathogenesis of human malignancy has been established. Recently, due to the development of targeted agents, the EGFR gene has been highlighted in a variety of studies on human malignancies. In the present study, we evaluated the EGFR status in 511 cases of GC using IHC and FISH. We compared the results obtained using these two methods and examined the clinicopathological characteristics as well as the prognostic implications of overexpressed EGFR DNA or protein.

The frequency of EGFR overexpression in GC has been reported to vary widely.12–14 In the present study, EGFR protein overexpression was observed in 140 cases (27.4%) of GC. The possible reasons for the discrepancies in reported rates of EGFR overexpression include the use of different antibodies, the subjectivity of the pathologists’ interpretation, different scoring systems and intratumoral staining heterogeneity. In previous studies on the frequency of EGFR expression, clone 31G7 antibody (Zymed Laboratories, San Francisco, CA, USA) has been used most often. Recently, the United States Food and Drug Administration has approved the newly developed anti-EGFR antibody, the pharmDx kit antibody, for the identification of patients with colorectal cancer who are eligible for treatment with cetuximab. Here we have reported the findings of the first study on EGFR overexpression frequency in a large number of GC patients using the EGFR pharmDx kit. We suggest that use of this newer antibody may improve the discrepancies in reported rates of EGFR overexpression. Also, the findings of this study may help to identify suitable candidates for EGFR-targeted therapy.

In the current study, 28 cases (5.5%) showed gene amplification or high polysomy with FISH analysis. The FISH+ cases were correlated with IHC+ cases and this correlation was statistically significant. In cases with the ERBB2 gene, many studies have shown a correlation between FISH and IHC results in a variety of carcinomas, including GC.13,14,22 The association between EGFR overexpression and high gene copy number was first studied in non-small cell lung carcinoma.5,6 Recently, similar studies have been reported in a variety of carcinomas.7,8,11,14 However, there have been no large-scale studies on GC. Among the 140 cases with EGFR overexpression determined by IHC, only 27 cases showed gene amplification or high polysomy in our FISH analysis. Several mechanisms, other than DNA number alteration, have been proposed to explain EGFR overexpression, such as activating mutations, increasing EGFR transcription or translation causing mRNA and protein production, decreased protein destruction and overexpression of receptor ligands.23,24 Data from the current study show that DNA numerical alteration of the EGFR gene was an infrequent event. Previous reports have shown that an activating mutation of EGFR is extremely rare in GC.25,26 Therefore, the mechanisms of EGFR overexpression in GC remain unknown.

In our study, the GC patients with EGFR overexpression or a high EGFR gene copy number had an unfavourable prognosis. In non-small cell lung carcinomas, EGFR overexpression has been well documented as an unfavourable prognostic factor.5,6 In addition, patients with colorectal carcinoma and oesophageal adenocarcinoma who have EGFR expression also have an unfavourable prognosis.11,15 Multivariate analysis showed that EGFR overexpression was an independent predictor of an unfavourable prognosis. Whilst TNM stage is still the best prognostic indicator, its possible value lies in the fact that it might identify a subset of patients with tumours that would be sensitive to targeted therapy.

Patients with EGFR overexpression and/or high gene copy number have been shown to have a good response to EGFR-targeted therapy in cases with non-small cell lung carcinoma.6,19 In contrast, the relationship between EGFR overexpression and the response rate to cetuximab therapy has been controversial in colorectal carcinoma patients.18,27 Clinical trials on GC have provided minimal evidence for efficacy of anti-EGFR therapy. In a multicentre Phase II Japanese study, 13 of 75 metastatic GC patients receiving gefitinib achieved disease control.20 In contrast, a Phase II trial of erlotinib showed no response in a group of 25 unresectable or metastatic GC patients.28 Detailed information on the alteration of the EGFR gene or protein is needed to improve the assessment of response to anti-EGFR therapy.

In summary, our findings have demonstrated that a subgroup of GC cases with EGFR overexpression is associated with advanced GC, the presence of lymph node metastasis and a higher stage and lymphatic invasion. On univariate analysis, EGFR overexpression or EGFR high gene copy number was associated with an unfavourable prognosis. EGFR overexpression was confirmed to be an independent prognostic factor on multivariate analysis. Therefore, the EGFR gene and protein status should be evaluated to interpret the results of clinical trials using EGFR targeted agents.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This study was supported by a grant FG06-11-03 of 21C Frontier Functional Human Genome Project from the Ministry of Science and Technology of Korea.

References

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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