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High Frequency of Epidermal Growth Factor Receptor Mutations with Complex Patterns in Non–Small Cell Lung Cancers Related to Gefitinib Responsiveness in Taiwan

¹ Division of Molecular and Genomic Medicine, National Health Research Institutes, Departments ² Pathology, ³ Cardio-Thoracic Surgery, ⁴ Hematology and Oncology, and ⁵ Thoracic Medicine, Chang-Gung Memorial Hospital, and ⁶ Institute of Genetics and Genome Research Center, National Yang-Ming University, Taipei, Taiwan

	ABSTRACT

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Purpose: Epidermal growth factor receptor (EGFR) mutations related to gefitinib responsiveness in non–small cell lung cancer have been found recently. Detection of EGFR mutations has become an important issue for therapeutic decision-making in non–small cell lung cancer.

Experimental Design: Mutational analysis of the kinase domain of EGFR coding sequence was done on 101 fresh frozen tumor tissues from patients without prior gefitinib treatment and 16 paraffin-embedded tumor tissues from patients treated with gefitinib. Detection of phosphorylated EGFR by immunoblot was also done on frozen tumor tissues.

Results: The 101 non–small cell lung cancer tumor specimens include 69 adenocarcinomas, 24 squamous cell carcinomas, and 8 other types of non–small cell lung cancers. Mutation(s) in the kinase domain (exon 18 to exon 21) of the EGFR gene were identified in 39 patients. All of the mutations occurred in adenocarcinoma, except one that was in an adenosquamous carcinoma. The mutation rate in adenocarcinoma was 55% (38 of 69). For the 16 patients treated with gefitinib, 7 of the 9 responders had EGFR mutations, and only 1 of the 7 nonresponders had mutations, which included a nonsense mutation. The mutations seem to be complex in that altogether 23 different mutations were observed, and 9 tumors carried 2 mutations.

Conclusions: Data from our study would predict a higher gefitinib response rate in lung adenocarcinoma patients in Chinese and, possibly, other East Asian populations. The tight association with adenocarcinoma and the high frequency of mutations raise the possibility that EGFR mutations play an important role in the tumorigenesis of adenocarcinoma of lung, especially in East Asians.

	INTRODUCTION

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Epidermal growth factor receptor (EGFR) is a M_r 170,000 transmembrane glycoprotein. Binding of ligands to the extracellular domain results in activation of the tyrosine kinase activity of the receptor. Activation of the downstream pathways of EGFR leads to cell proliferation, differentiation, migration/motility, adhesion, protection from apoptosis, enhanced survival, and gene transcription (1 , 2) . Overactivity of these signaling pathways can lead to abnormal growth control and cellular transformation. Thus, deregulated expression of EGFR through mutation, amplification, or deregulation is linked with malignancy (1 , 2) . In recent years, inhibition of activated protein kinases with small molecule drugs or antibodies has become an effective approach to cancer therapy (3, 4, 5) . Because EGFR is highly expressed in 88 to 99% of non–small cell lung cancers (6, 7, 8, 9) , and most chemotherapy regimens seem to have limited efficacy with disappointing survival results for non–small cell lung cancer (10, 11, 12) , EGFR-tyrosine kinase has become a particularly promising drug target for treating non–small cell lung cancer. Among these, gefitinib is an orally active, targeted, small-molecule drug with evidence of antitumor activity in a range of cancers including non–small cell lung cancer, as shown by Phase I clinical trials (13 , 14) . One interesting observation about this new drug is the significant variability in the response rate. Although only 10 to 19% of patients with chemotherapy-refractory advanced non–small cell lung cancer showed a response to this new drug in large multi-institutional Phase II trials, a subgroup of patients had a dramatically rapid and profound response with only mild side effects (15 , 16) . Additionally, a significantly higher response rate was noted in Japanese patients than in a predominantly European-derived population (27.5% versus 10.4%; refs. 15 , 16 ). In Taiwan, a high response rate for gefitinib for pretreated, advanced non–small-cell lung cancers has also been noted (26 to 36%; refs. 17 , 18 ), which was similar to that of the Japanese patients. The response rate in chemonaive advanced non–small cell lung cancer patients was even higher (56.5%; ref. 19 ). In addition to the difference between ethnic backgrounds, several clinical characteristics were also noted to be associated with higher response rate, i.e., adenocarcinoma, female gender, and nonsmoking history (16 , 20 , 21) . EGFR expression detected by immunohistochemistry, however, is not correlated with the clinical response to gefitinib (22) . Thus, the search for an effective predictor of the response to gefitinib and its molecular mechanism has become an important issue.

Recently, two research groups have discovered that EGFR mutations correlated with the clinical responsiveness to gefitinib in non–small cell lung cancer (23 , 24) . All of these mutations were within exons 18 through 21 of the kinase domain of EGFR. These mutations led to increased tyrosine kinase activity and conferred susceptibility to gefitinib. All of these mutations, except one, occurred in adenocarcinomas. The study by Paez et al. (24) also reported a much higher mutation rate in Japanese patients than in those from the United States (32% versus 3% for adenocarcinoma) and highest of all in Japanese women with adenocarcinoma (57%). These findings are highly correlated with the clinical characteristics known to be associated with a higher response rate to gefitinib (15 , 16) .

We have conducted mutational analysis of the EGFR gene from exons 18 to 21 in a series of 101 surgically resected non–small cell lung cancers with no prior chemotherapy or gefitinib treatment. We observed a high mutation rate of EGFR in the regions that have been reported to be associated with responsiveness to gefitinib. We also identified a range of new mutations that have not been reported previously. Additional mutational analysis of 16 patients with adenocarcinoma of lung treated with gefitinib also revealed mutations of EGFR in most of the responders.

	MATERIALS AND METHODS

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Fresh Frozen Tumor Tissues from Patients without Prior Gefitinib Treatment
One hundred and one cases of fresh frozen tumor tissues from surgically resected lung specimens together with their adjacent non-neoplastic lung tissues and the peripheral blood samples of the patients were collected from Chang-Gung Memorial Hospital between 2002 and 2004. Informed consent from each patient was obtained before the operation. All of the tumor and non-neoplastic lung tissues were snapped frozen and stored at –80°C within 30 minutes after resection. None of these patients had received gefitinib treatment before the surgery.

For each specimen, frozen sections were done and stained with H&E first to exam the tumor proportion of the tissue. Only tumors with >50% of tumor component were sent forward for DNA extraction and sequence analysis. Immunoblot analysis was also carried out in most of the fresh tumor specimens to check whether there was autophosphorylation of EGFR. Complete clinical data including smoking history, clinical stage, pathological diagnosis, and follow-up status were obtained from medical records and reviewed by the physicians.

Formalin-Fixed Paraffin-Embedded Tumor Tissues from Patients Treated with Gefitinib
Tumor tissues obtained before the patients received any systemic treatment were collected retrospectively from patients treated with gefitinib. All of these tumors were formalin-fixed paraffin-embedded tissues from the Department of Pathology (Chang-Gung Memorial Hospital, Taipei, Taiwan). To minimize non-neoplastic tissue contamination, the tumor portion was selected and marked on the H&E stained tissue section slide first by a pathologist (Shiu-Feng Huang). Only the tumor portion was dissected from the unstained tissue section slides and sent for DNA extraction. The latter was done with DEXPAT (Takara Biomedical, Shiga, Japan) according to the manufacturer’s instructions.

Nucleotide Sequence Analysis
For mutational analysis of the kinase domain of EGFR coding sequence, exon 18, 19, 20, and 21 were amplified with four pairs of primers, specific to the flanking sequences of individual exon, and PCR amplicons were subjected to direct sequencing (the PCR primers and amplification procedures are shown in supplemental text and supplemental Tables 1 and 2 for frozen and paraffin-embedded tissue, respectively). Forward and reverse sequencing reactions were done with the same primers for PCR amplification and ABI BigDye Terminator kit v3.1 (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions. Sequencing reactions were electrophoresed on an ABI3700 genetic analyzer. Sequence variations were determined by using Seqscape software (Applied Biosystems) with the EGFR reference sequence (NM_005228.3, National Center for Biotechnology Information). All of the sequence variations were confirmed by multiple, independent PCR amplifications and repeated sequencing reactions.

Immunoblot Analysis
Tumor and adjacent non-neoplastic tissues were homogenized in lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% (w/v) SDS, 1 mmol/L dithiothreitol, 5 µg/mL leupeptine, 3 µg/mL aprotinin, 0.5 mmol/L phenylmethylsulfonylfluoride, and 1 mmol/L Na₃VO₄] with the MagNA Lyser Green Beads kit (Roche Applied Science, Mannheim, Germany). The extracts were centrifuged at 14,000 g to remove the tissue debris. One hundred micrograms of extracted protein were resolved by 8% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and then subjected to immunoblot analyses with an antiphosphorylated EGFR monoclonal antibody (Chemicon, Temecula, CA) according to the manufacturer’s instructions. The expression level of EGFR was determined by an anti-EGFR antibody (Cell Signaling Technology, Inc., Beverly, MA), and an equal loading of extract was confirmed by immunoblotting with an anti-ß–actin antibody (AC-15, Sigma, St. Louis, MO).

	RESULTS

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Fresh Frozen Tumor Tissues from Patients without Prior Gefitinib Treatment
The 101 non–small cell lung cancer tumor specimens include 69 adenocarcinomas, 24 squamous cell carcinomas, and 8 other types of non–small cell lung cancers. Fifty-six were male patients and 45 were female. Among the 69 adenocarcinomas, 33 were male patients and 36 were female. Mutation(s) in the kinase domain (exon 18 to exon 21) of the EGFR gene were identified in 39 tumor tissues. The overall mutation rate was 38.6% (39 of 101). All of the mutations occurred in adenocarcinoma, except for one in an adenosquamous carcinoma. The mutation rate in adenocarcinoma was 55% (38 of 69). Among them, 18 patients were male and 20 were female. The mutation rate in male patients with adenocarcinoma was 54.5% (18 of 33) and 55.5% (20 of 36) in female patients. The pathology slides of the 101 cases were reviewed by one pathologist at Chang-Gung Memorial Hospital (Shiu-Feng Huang). All of the adenocarcinomas with EGFR mutations were well to moderately differentiated. There were only 3 pure bronchioloalveolar carcinomas among the 69 adenocarcinomas, but none of them had EGFR mutation. Sixteen of the 24 patients with squamous cell carcinoma had a history of smoking for 30 to 60 years (smoking rate 66.6%). Twelve of the 69 patients with adenocarcinoma had a history of smoking for 20 to 50 years (smoking rate 17.4%). Among the 38 cases of adenocarcinoma with EGFR mutation, there were 5 patients with a smoking history (smoking rate 13.1%). Thus, although the smoking rate in the patients with adenocarcinoma was lower than for squamous cell carcinoma, there was no significant difference in the smoking rate between adenocarcinoma patients with or without EGFR mutations (P = 0.304). The clinical characteristics and the mutation patterns of these 39 patients are summarized in Table 1 .

View larger version (42K): [in this window] [in a new window]   Fig. 1. Mutations in the EGFR gene in non–small cell lung cancer. A. Five patterns of in-frame deletion in exon 19. B. The nucleotide sequence with heterozygous in-frame deletion (double peaks). Tracings in both sense and antisense directions are shown to highlight the two breakpoints of deletion; the wild-type nucleotide sequence is in capital letters, and the mutant sequence is in lowercase letters. The 5' breakpoint of the delE746_751insA mutation is preceded by an A to C substitution that does not alter the encoded amino acid. C. Homozygous missense mutation (one peak) at nucleotide 2819 from T to G in exon 21. D. A combination of two heterozygous missense mutations (two peaks) at nucleotide 2743 (T to G) and 2750 (A to T) in exon 21. E. Heterozygous missense mutation (two peaks) at nucleotide 2372 (A to C) in exon 18. F. Homozygous missense mutation (one peak) at nucleotide 2372 (A to G) in exon 18.

In exon 18, four missense mutations (E709A, E709G, G719S, and G719C) were found, and each occurred in 1 tumor (Fig. 1, E and F) . All four patients (Case 29, 30, 38, and 39) with a mutation in exon 18 had another mutation in exon 20 (S768I) or exon 21 (L858R).

In exon 20, 2 missense mutations and two in-frame duplications were found. The S768I mutation was found in 2 tumors (Case 38 and 39). The V769M mutation occurred in 1 tumor (Case 10). This tumor also had an in-frame deletion in exon 19. The two types of duplication patterns are shown in Fig. 2A . Case 36 had a duplication of 12 bp from nucleotide 2530–5 to 2536 (Fig. 2B) . The duplicated sequence spanned an intron-exon junction. Because this duplication involved the splice acceptor site of intron 19, reverse-transcriptase-PCR of the tumor RNA followed by sequencing was done. The result showed that the first splice acceptor site was used for the mutant form. The consequence was insertion of 4 amino acids (EAFQ) between codon 761 and 762 (Fig. 2C) . Case 37 had a duplication of 9 bp at nucleotide 2549 to 2557, which resulted in insertion of 3 amino acids (SVD).

View larger version (43K): [in this window] [in a new window]   Fig. 2. A. Two types of duplication mutation in exon 20. Case 36 had a heterozygous in-frame duplication of 12 bp from nucleotide 2530–5 to 2536. Case 37 had a duplication of 9 bp at nucleotide 2549 to 2557. The duplicated sequence of Case 36 spanned an intron-exon junction. Intronic sequence is in lowercase letters, and exonic sequence in capital letters. B. Sequence tracing of genomic DNA of Case 36. The duplication was confirmed in both strands. C. Sequence tracing of cDNA of Case 36. The nucleotide sequence showed splicing at the first acceptor site and an insertion of 4 amino acids (shown in red) between codon 761 and 762.

We have additionally analyzed the entire exonic sequences of the EGFR gene in all 39 tumors with mutations and 38 other non–small cell lung cancers (31 adenocarcinomas, 4 squamous cell carcinoma, and 3 adenosquamous carcinoma) without mutations in the tyrosine kinase domain among our 101 patients. We found no additional mutation.

Formalin-Fixed Paraffin-Embedded Tumor Tissue from Patients Treated with Gefitinib
To ascertain the EGFR mutations within the tyrosine kinase domain are associated with responsiveness to gefitinib in Taiwanese lung cancer patients, we also did mutational study in tumors from 16 patients who had received gefitinib treatment as a single agent at Chang-Gung Memorial Hospital. A significant clinical response was defined according to the response evaluation criteria for global trial in solid tumors (25) . Among them, 6 patients showed partial response to gefitinib, 2 patients had clinical benefit, 1 patient had serologic response, and 7 patients had no response. In total, 9 patients were considered as responders, and 7 patients were nonresponders. Mutations of EGFR were found in 7 of the 9 responders. The 2 responders with no mutations were the 2 patients with clinical benefit. Only 1 of the 7 nonresponders had mutations. The difference of the mutation rates between the responders and nonresponders is statistically significant (P = 0.041, by ²/Fisher’s exact tests). The clinical features and the results of mutational study are shown in Table 2 .

A diagram showing all of the mutations and the amino acid sequence of the sequenced regions of EGFR discovered from the current study are shown in Fig. 3 .

View larger version (27K): [in this window] [in a new window]   Fig. 3. Distribution of the mutations in the kinase domain (exons 18 to 21) of the EGFR gene. Exons are shown as box, and mutation types are indicated by different colored arrows. Solid arrows represent mutations found in fresh frozen tumor tissue of 101 patients not treated with gefitinib. Open arrows represent mutations found in paraffin-embedded tumor tissue from 16 patients treated with gefitinib. Each arrow represents a single mutation event. The amino acid sequence of each mutation is shown below the box. For those mutations found in responders to gefitinib, amino acid sequences are shown in red. *, These 2 mutations occurred in 1 nonresponder.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Immunoblot analysis of phosphorylated EGFR and EGFR in lung cancers. (ß-actin serves as a control). A. Six tumors with EGFR mutation and increased phosphorylated EGFR expression in T compared with its N are shown. The number in this figure is the same as the case number in Table 1 . B. Expression levels of EGFR and phosphorylated EGFR of 6 tumors without EGFR mutation are shown. Start from right, the first three pairs showed lower phosphorylated EGFR-expression levels in T compared with N. The fourth pair show equal phosphorylated EGFR expression between T and N. The last two pairs show higher level of phosphorylated EGFR in T compared with N. The numbers in this figure are the serial numbers of those specimens. (T, tumor tissue; N, adjacent non-neoplastic lung tissue)

	DISCUSSION

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We have taken a resequencing approach to detect directly any sequence variation in the exons and exon-intron junctions of the genomic region that encodes the kinase domain of EGFR. In total, 18 different mutations were observed in the 39 patients, and 12 of them have not been reported previously. The mutation spectrum covers deletion, duplication, and single-nucleotide substitution. Most of the mutations were within exons 19 or 21, which is a finding similar to the two previous reports (23 , 24) . All of the deletions were observed in exon 19, but we also identified known and new single-nucleotide substitutions in exon 18 and 21. In addition, we discovered new mutations (missense mutations and duplication) in exon 20. In nearly all of the cases, the mutation was acquired in the tumor tissue, as we did not detect mutation in the leukocyte of the same blood sample of the individual. Moreover, the mutations seem to be complex in that 7 tumors carried 2 mutations and that a homozygous mutational status was detected in 4 tumors. Among them, one (Case 30) had homozygous point mutations in both exon 18 and exon 21. These findings would suggest that the cancer cells might be accumulating multiple molecular events in the EGFR gene in these tumor specimens.

Recently, activating mutations in the EGFR gene have been reported to underline the response to chemotherapy with a kinase inhibitor, gefitinib. A much higher mutation rate was found in Japanese patients with adenocarcinoma. Our study revealed a high mutation rate in EGFR gene similar to that seen in Japanese patients. This result is highly consistent with an ethnical difference in the responsive rate to gefitinib as shown by clinical trials carried out in the United States, Japan, and Taiwan (15 , 19) . To ascertain the EGFR mutations within the tyrosine kinase domain are associated with responsiveness to gefitinib in Taiwanese lung cancer patients, we also did mutational analysis on tumors of 16 patients who had received gefitinib treatment. There were 9 responders and 7 nonresponders. All of the mutations in the tyrosine kinase domain except two were found in the responders. Among the 8 mutations found in 7 of the 9 responders, 5 were also found in our 101 fresh frozen tumor series and had been reported to be associated with gefitinib responsiveness by others (23 , 24) . There were 3 new mutations (del747_A750InsP, A839T, and K846R) found in the responders. Interestingly, we also found double mutations in 1 responder (both mutations have been reported to be associated with gefitinib responsiveness) and in 1 nonresponder. In the latter, a nonsense mutation (W731Stop) in exon 19 and 1 missense mutation (H773R) in exon 20 were found. Because the stop codon could have resulted in protein truncation and loss of EGFR function, it is understandable why this patient did not respond to gefitinib treatment.

Female patients with adenocarcinoma have been reported to be associated with a higher gefitinib response rate, especially in Japanese patients (16 , 21) . However, in our analysis of the 101 patients, we did not see significant difference in the EGFR mutation rate between male (18 of 33, 54.5%) and female patients (20 of 36, 55.5%) with adenocarcinoma, and the mutation patterns are also similar between the two groups. Of note, the report by Miller et al. (20) also found no significant difference in the response rate between male and female patients in their study of 139 patients. It remains to be determined whether gender might play a role in determining gefitinib treatment response in patients with EGFR mutation. We also found no significant difference in the smoking rate between adenocarcinoma patients with or without mutation.

By immunoblot analysis, we observed EGFR activation, as evidenced by increased EGFR phosphorylation in 26 of the 39 tumors (66.6%) with EGFR mutations. This result suggests that the EGFR mutations in these tumors may enhance their kinase activity. All of the new mutations identified in this study showed EGFR phosphorylation in at least one case, except Case 34, which was unique and had two missense mutations close to each other in exon 21. The functional change caused by these 2 mutations remains to be determined. For the 38 tumors without EGFR mutations, there were only 36.8% of tumors that had increased level of phosphorylated EGFR. As a whole, the EGFR kinase domain mutations seem to have a different effect on kinase activation. The functional significance of this finding requires additional investigation.

We have analyzed the entire exonic sequences of the EGFR gene in all of the 39 tumors with mutations and 38 other non–small cell lung cancers (31 adenocarcinomas, 4 squamous cell carcinoma, and 3 adenosquamous carcinoma) without mutations in the tyrosine kinase domain among our 101 patients. We found no additional mutation. Thus, the EGFR mutations in lung cancer seem to cluster in exons 18 to 21 in a predictable pattern. In the report of Lynch et al. (23) , they searched for the EGFR mutations in exon 19 and 21 in primary tumors from breast, colon, kidney, pancreas, brain, and a panel of 108 cancer-derived cell lines, and the result was totally negative. We also did not detect any mutation in exons 18 to 21 of EGFR in 30 pairs of tumor and nontumor liver tissues of hepatocellular carcinomas (data not shown). The tight association with adenocarcinoma and the recurrent pattern of EGFR mutations raise the possibility that these mutations play an important role in the tumorigenesis of adenocarcinoma of lung, especially in East Asians.

Finally, although the previous EGFR studies have revealed that the clinical response to gefitinib is associated with specific mutations in the EGFR kinase domain, and the finding is supportive of treatment selection based on molecular classification, it is still possible that not all of the patients with EGFR mutation will respond to gefitinib. The nonsense mutation found in 1 of the nonresponders in our series is a good example. Given the wide spectrum and complexity of EGFR mutations shown by this study, it will require additional analysis to investigate whether the new EGRF mutations discovered by this study also confer a similar beneficial effect with the gefitinib treatment. Nevertheless, data from our study would suggest that there should be a higher gefitinib response rate in lung adenocarcinoma patients in Chinese and, possibly, other East Asian populations.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. Bae-Li Hsi, Jeng-Chang Chen, and Zee-Feng Chang for their valuable suggestions and Drs. Tsang-Wu Liu, Chong-Chi Huang, and Ying-Hung Tsai for their help in collecting the information of gefitinib usage in Taiwan.

	FOOTNOTES

 
Grant support: Grants from the National Science Council (NSC92-2314-B-182A-124 and NHRI92A1-NSCCA16-5) and Chang-Gung Medical Research Funds (BMRP680).

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.

Note: Supplementary data for this article can be found at Clinical Cancer Research Online at http://clincancerres.aacrjournals.org.

Requests for reprints: Shih-Feng Tsai, Division of Molecular and Genomic Medicine, National Health Research Institutes, 128 Yen-Chiu-Yuan Road, Section 2, Taipei 115, Taiwan. Phone: 886-2-26534401, extension 6120; Fax: 886-2-27890484; E-mail: petsai{at}nhri.org.tw

Received 6/30/04; revised 9/26/04; accepted 10/14/04.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Olayioye MA, Neve RM, Lane HA, Hynes NE. The erbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000;19:3159-67.[CrossRef][Medline]
2. Pinkas-Kramarski R, Soussan L, Waterman H, et al Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J 1996;15:2452-67.[Medline]
3. Demetri GD, von Mehren M, Blanke CD, et al Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 2002;347:472-80.[Abstract/Free Full Text]
4. Druker BJ, Sawyers CL, Kantarjian H, et al Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001;344:1038-42.[Abstract/Free Full Text]
5. Slamon DJ, Leyland-Jones B, Shak S, et al Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783-92.[Abstract/Free Full Text]
6. Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001;37:S9-15.
7. Rusch V, Baselga J, Cordon-Cardo C, et al Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Res 1993;53:2379-85.[Abstract/Free Full Text]
8. Fontanini G, De Laurentiis M, Vignati S, et al Evaluation of epidermal growth factor-related growth factors and receptors of neoangiogenesis in completely resected stage I-IIIa non-small-cell lung cancer: amphiregulin and microvessel count are independent prognostic indicators of survival. Clin Cancer Res 1998;4:241-49.[Abstract]
9. Lai WW, Chen FF, Wu MH, et al Immunohistochemical analysis of epidermal growth factor receptor family members in stage I non-small cell lung cancer. Ann Thorac Surg 2001;72:1868-76.[Abstract/Free Full Text]
10. Breathnach OS, Freidlin B, Conley B, et al Twenty-two years of phase III trials for patients with advanced non-small-cell lung cancer: sobering results. J Clin Oncol 2001;19:1734-42.[Abstract/Free Full Text]
11. Kelly K, Crowley J, Bunn PA, Jr, et al Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine plus cisplatin in the treatment of patients with advanced non–small-cell lung cancer: a Southwest Oncology Group trial. J Clin Oncol 2001;19:3210-8.[Abstract/Free Full Text]
12. Schiller JH, Harrington D, Belani CP, et al Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 2002;346:92-8.[Abstract/Free Full Text]
13. Nakagawa K, Tamura T, Negoro S, et al Phase I pharmacokinetic trial of the selective oral epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (‘Iressa’, ZD1839) in Japanese patients with solid malignant tumors. Ann Oncol 2003;14:922-30.[Abstract/Free Full Text]
14. Herbst RS, Maddox AM, Rothenberg ML, et al Selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 is generally well-tolerated and has activity in non-small-cell lung cancer and other solid tumors: results of a phase I trial. J Clin Oncol 2002;20:3815-25.[Abstract/Free Full Text]
15. Fukuoka M, Yano S, Giaccone G, et al Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer. J Clin Oncol 2003;21:2237-46.[Abstract/Free Full Text]
16. Kris MG, Natale RB, Herbst RS, et al Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. J Am Med Assoc 2003;290:2149-58.[Abstract/Free Full Text]
17. Tsai CM, Chiu CH, Chen YM, Perng RP. Gefitinib in patients with advanced non-small cell lung cancer: relationship between drug toxicity and tumor response. Thoracic Medicine 2003;18:S6:93
18. Wu MF, Fahn HJ, Wu TC, et al Experience of gefitinib (Iressa) for previously treated patients with advanced non-small cell lung cancer. Thoracic Medicine 2003;18:S6:203
19. Chen KC, Chang GC, Yang TY. A comparison of gefitinib monotherapy and chemotherapy with cisplatin and gemcitabine in chemonaive patients with advanced non-small cell lung cancer: A case control study. Thoracic Medicine 2003;18:S6:218
20. Miller VA, Kris MG, Shah N, et al Bronchioloalveolar pathologic subtype and smoking history predict sensitivity to gefitinib in advanced non-small-cell lung cancer. J Clin Oncol 2004;22:1103-9.[Abstract/Free Full Text]
21. Janne PA, Gurubhagavatula S, Yeap BY, et al Outcomes of patients with advanced non-small cell lung cancer treated with gefitinib (ZD1839, "Iressa") on an expanded access study. Lung Cancer 2004;44:221-30.[CrossRef][Medline]
22. Bailey R, Kris M, Wolf M, et al Gefitinib (‘Iressa’,ZD1839) monotherapy for pretreated advanced non-small cell lung cancer in IDEAL 1 and 2: tumor response is not clinically relevantly predictable from tumor EGFR membrane staining alone. Lung Cancer 2004;41:S2:71
23. Lynch TJ, Bell DW, Sordella R, et al Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129-39.[Abstract/Free Full Text]
24. Paez JG, Jänne PA, Lee JC, et al EGFR Mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science (Wash DC) 2004;304:1497-500.[Abstract/Free Full Text]
25. Therasse P, Arbuck SG, Eisenhauer EA, et al New guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst (Bethesda) 2000;92:205-16.[Abstract/Free Full Text]

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				J. Subramanian and R. Govindan Lung Cancer in Never Smokers: A Review J. Clin. Oncol., February 10, 2007; 25(5): 561 - 570. [Abstract] [Full Text] [PDF]

				L. V. Sequist, D. W. Bell, T. J. Lynch, and D. A. Haber Molecular Predictors of Response to Epidermal Growth Factor Receptor Antagonists in Non-Small-Cell Lung Cancer J. Clin. Oncol., February 10, 2007; 25(5): 587 - 595. [Abstract] [Full Text] [PDF]

				M. Shrader, M. S. Pino, G. Brown, P. Black, L. Adam, M. Bar-Eli, C. P.N. Dinney, and D. J. McConkey Molecular correlates of gefitinib responsiveness in human bladder cancer cells Mol. Cancer Ther., January 1, 2007; 6(1): 277 - 285. [Abstract] [Full Text] [PDF]

				L. V. Sequist, V. A. Joshi, P. A. Janne, A. Muzikansky, P. Fidias, M. Meyerson, D. A. Haber, R. Kucherlapati, B. E. Johnson, and T. J. Lynch Response to treatment and survival of patients with non-small cell lung cancer undergoing somatic EGFR mutation testing. Oncologist, January 1, 2007; 12(1): 90 - 98. [Abstract] [Full Text] [PDF]

				R. Rosell, M. Taron, N. Reguart, D. Isla, and T. Moran Epidermal Growth Factor Receptor Activation: How Exon 19 and 21 Mutations Changed Our Understanding of the Pathway Clin. Cancer Res., December 15, 2006; 12(24): 7222 - 7231. [Abstract] [Full Text] [PDF]

				G. J. Riely, K. A. Politi, V. A. Miller, and W. Pao Update on Epidermal Growth Factor Receptor Mutations in Non-Small Cell Lung Cancer Clin. Cancer Res., December 15, 2006; 12(24): 7232 - 7241. [Abstract] [Full Text] [PDF]

				B. A. Helfrich, D. Raben, M. Varella-Garcia, D. Gustafson, D. C. Chan, L. Bemis, C. Coldren, A. Baron, C. Zeng, W. A. Franklin, et al. Antitumor Activity of the Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor Gefitinib (ZD1839, Iressa) in Non-Small Cell Lung Cancer Cell Lines Correlates with Gene Copy Number and EGFR Mutations but not EGFR Protein Levels Clin. Cancer Res., December 1, 2006; 12(23): 7117 - 7125. [Abstract] [Full Text] [PDF]

				K. D. Carey, A. J. Garton, M. S. Romero, J. Kahler, S. Thomson, S. Ross, F. Park, J. D. Haley, N. Gibson, and M. X. Sliwkowski Kinetic Analysis of Epidermal Growth Factor Receptor Somatic Mutant Proteins Shows Increased Sensitivity to the Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Erlotinib Cancer Res., August 15, 2006; 66(16): 8163 - 8171. [Abstract] [Full Text] [PDF]

				W.-C. Chou, S.-F. Huang, K.-Y. Yeh, H.-M. Wang, M.-Y. Liu, J.-J. Hsieh, Y.-C. Cheung, and J. W.-C. Chang Different Responses to Gefitinib in Lung Adenocarcinoma Coexpressing Mutant- and Wild-Type Epidermal Growth Factor Receptor Genes Jpn. J. Clin. Oncol., August 1, 2006; 36(8): 523 - 526. [Abstract] [Full Text] [PDF]

				A. Inoue, T. Suzuki, T. Fukuhara, M. Maemondo, Y. Kimura, N. Morikawa, H. Watanabe, Y. Saijo, and T. Nukiwa Prospective Phase II Study of Gefitinib for Chemotherapy-Naive Patients With Advanced Non-Small-Cell Lung Cancer With Epidermal Growth Factor Receptor Gene Mutations J. Clin. Oncol., July 20, 2006; 24(21): 3340 - 3346. [Abstract] [Full Text] [PDF]

				P. A. Janne Gefitinib for Epidermal Growth Factor Receptor Mutant Lung Cancers: Searching for a Weapon of Mass Destruction J. Clin. Oncol., July 20, 2006; 24(21): 3319 - 3321. [Full Text] [PDF]

				E. L. Kwak, J. Jankowski, S. P. Thayer, G. Y. Lauwers, B. W. Brannigan, P. L. Harris, R. A. Okimoto, S. M. Haserlat, D. R. Driscoll, D. Ferry, et al. Epidermal growth factor receptor kinase domain mutations in esophageal and pancreatic adenocarcinomas. Clin. Cancer Res., July 15, 2006; 12(14): 4283 - 4287. [Abstract] [Full Text] [PDF]

				R. K. Thomas, B. Weir, and M. Meyerson Genomic approaches to lung cancer. Clin. Cancer Res., July 15, 2006; 12(14): 4384s - 4391s. [Abstract] [Full Text] [PDF]

				L. V. Sequist, V. A. Joshi, P. A. Janne, D. W. Bell, P. Fidias, N. I. Lindeman, D. N. Louis, J. C. Lee, E. J. Mark, J. Longtine, et al. Epidermal growth factor receptor mutation testing in the care of lung cancer patients. Clin. Cancer Res., July 15, 2006; 12(14): 4403s - 4408s. [Abstract] [Full Text] [PDF]

				P. A. Janne and B. E. Johnson Effect of epidermal growth factor receptor tyrosine kinase domain mutations on the outcome of patients with non-small cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors. Clin. Cancer Res., July 15, 2006; 12(14): 4416s - 4420s. [Abstract] [Full Text] [PDF]

				Y. Yatabe, T. Hida, Y. Horio, T. Kosaka, T. Takahashi, and T. Mitsudomi A Rapid, Sensitive Assay to Detect EGFR Mutation in Small Biopsy Specimens from Lung Cancer J. Mol. Diagn., July 1, 2006; 8(3): 335 - 341. [Abstract] [Full Text] [PDF]

				M. Sonobe, T. Manabe, H. Wada, and F. Tanaka Lung Adenocarcinoma Harboring Mutations in the ERBB2 Kinase Domain J. Mol. Diagn., July 1, 2006; 8(3): 351 - 356. [Abstract] [Full Text] [PDF]

				K.-H. Lee, S.-W. Han, P. G. Hwang, D.-Y. Oh, D.-W. Kim, D. H. Chung, S.-A. Im, T.-Y. Kim, D. S. Heo, and Y.-J. Bang Epidermal Growth Factor Receptor Mutations and Response to Chemotherapy in Patients with Non-Small-Cell Lung Cancer. Jpn. J. Clin. Oncol., June 1, 2006; 36(6): 344 - 350. [Abstract] [Full Text] [PDF]