High frequency of epidermal growth factor receptor mutations in non small cell lung cancers in vietnam

JOURNAL OF MEDICAL RESEARCH HIGH FREQUENCY OF EPIDERMAL GROWTH FACTOR RECEPTOR MUTATIONS IN NON - SMALL CELL LUNG CANCERS IN VIETNAM Nguyen Ha Minh1, Tran Huy Thinh1,2, Tran Van Khanh2 Dept of Biochemistry, Hanoi Medical University Center for Gene & Protein Research, Hanoi Medical University Mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene impact a patient’s response to targeted therapy in non - small cell lung cancer (NSCLC) Detection of these mutations plays an important role in therapeutic decision - making in NSCLC Methods: 181 paraffin embedded tumor tissues from grade IIIB/IV NSCLC patients were screened for mutations in exons 18 to 21 of the EGFR gene, using both the direct sequencing and the Scorpion ARMS method Mutations in the kinase domain (exon 18 to exon 21) of the EGFR gene were identified in 106 of the 181 NSCLC specimens (58.6%) The following mutations were the two common mutation types found: in-frame deletions in exon 19 (found in 48.1% of the patients in our study with EGFR mutations) and single missense mutations L858R in exon 21 (found in 40.6% of the patients in our study with EGFR mutations) Two tumors carried double mutations and four tumors showed new mutations The EGFR TK mutations were more frequent in never smokers than ever smokers (74.4% versus 44.2%), in females than males (75.0% versus 45.5%) and in adenocarcinoma NSCLC versus other histologic subtypes (65.9% versus 20.7%) In conclusion: EGFR TK mutations can be found in many patients with advanced NSCLC in Vietnam Direct sequencing and the Scorpion ARMS method could be combined to screen for these mutations Keywords: Epidermal growth factor receptor, Non-small cell lung cancer, EGFR mutation I INTRODUCTION Despite improvements in diagnostic and therapeutic approaches to treatment, lung cancer remains the major cause of cancerrelated deaths worldwide [1] Non - small cell lung cancer (NSCLC), the major form of lung cancer, is classified into three histologic subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma NSCLC is also characterized by the accumulation of multiple genetic alterations, including those that result in the activation of oncogenes and the inactivation of tumor suppressor genes [2; 3] Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor Activation of the downstream pathways of EGFR leads to cell proliferation, differentiation, migration and adhesion, protection from apoptosis, enhanced survival, and gene transcription [4] Targeting the EGFR gene is an appealing strategy for the treatment of NSCLC, as the EGFR gene has been found to be expressed, sometimes strongly, in NSCLC tumors [5] Researchers have found that EGFR mutations are strong determinants of tumor response to EGFR Corresponding author: Tran Van Khanh, Center for Gene and Protein Research, Hanoi Medical University E-mail: tranvankhanh@hmu.edu.vn Received: 02 November 2016 Accepted: 10 December 2016 JMR 105 E1 (7) - 2016 tyrosine kinase (EGFR TK) inhibitors [5 – 8] These mutations cluster in a wide spectrum from exon 18 to exon 21 within the EGFR gene Numerous mutations, including deletion, 27 JOURNAL OF MEDICAL RESEARCH insertion, substitution ers are used in a fluorescence - based way for mutations, are classified into EGFR TK the specific detection of PCR products [11] inhibitor-sensitizing or - resistant mutations Scorpion primers can be used in combination The two most common NSCLC-associated with the Amplified Refractory Mutation System EGFR mutations are the 15 - bp nucleotide (ARMS) to enable the detection of single - in-frame deletion in exon 19 (E746_A750del) base mutations and to enhance specificity [11; and the point mutation that replaces leucine 12] The Scorpion - ARMS method is highly with arginine at codon 858 in exon 21 (L858R) sensitive and fast when it comes to the [5; 9] Previous research has shown that rates detection of known mutations [13] This study of duplication EGFR TK mutations and among NSCLC patients varies according to patients’ ethnic backgrounds Asian NSCLC patients have been found to carry higher numbers of mutations than those in Europe and the United States In addition, several clinical characteristics have been found to be associated with overall better clinical response, i.e adenocarcinoma, female gender and a history of non - smoking [7; 10] sought to investigate the occurrence of the EGFR TK mutation in Vietnamese NSCLC patients by simultaneously employing the direct sequencing and the Scorpion ARMS methods We also sought to clarify the relationship between EGFR mutation status and the clinical characteristics of NSCLC in Vietnamese patients II SUBJECTS AND METHODS As efforts to identify clinically relevant mutations intensify, mutation testing of tumors will become more routine Direct sequencing is the classic method for the detection of Subjects Tumor tissues and DNA extraction In total, 181 tumor tissue biopsies were genomic mutations and is still widely em- taken from the Departments of Pathology in ployed to uncover ‘‘new’’ mutations However, five hospitals across Northern Vietnam: the this technique has several drawbacks in the National Cancer Hospital, the Hanoi Cancer clinical setting, where the focus is on the Hospital, detection of known recurrent mutations that Hospital and the Hanoi Medical University have clinical relevance Direct sequencing Hospital These tissue biopsies were formalin- involves multiple steps, so several days are fixed and paraffin - embedded Patient data required to obtain a result after tissue acquisi- was collected from medical records, and tion More importantly, the sensitivity of direct included: smoking status, age, pathological sequencing is suboptimal for clinical tumor diagnosis, and clinical stage This data was samples Mutant DNA needs to comprise over reviewed by oncologists at the hospitals where 25% of the total DNA to be easily detected tumor tissue biopsies were obtained Bach Mai Hospital, Huu Nghi Thus, mutation detection by direct sequencing All samples underwent a haematoxylin and could lead to ‘‘false - negative’’ results An- eosin pathology review to confirm the diagno- other method to detect genomic mutations is sis of NSCLC and the presence of tumor in the the Scorpion - ARMS method Scorpion prim- samples The NSCLC samples were macro- 28 JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH dissected by scraping only the tumor area that µL of primer mix and 0.5 µL of Taq poly- had been selected by a pathologist Genomic merase Real-time PCR was carried out using DNA was extracted and purified from 20μm Stratagen MX 3000P (Life Technologies, thin sections using the phenol/Chloroform Thermo Fisher Scientific Inc.) under the follow- method [14] The concentration and purity of ing conditions: initial denaturation at 95oC for the extracted DNA were determined by Nano- minutes, 40 cycles of 95oC for 30 seconds, Drop ND - 1000 (NanoDrop Technologies, and finally 60 seconds at 60oC with fluores- Rockland, DE) The extracted DNA was cence reading (set to FAM (fluorescein) that o stocked at -20 C until use Methods allows optical excitation at 480 nm and measurement at 520 nm) at the end of each cycle The cycle threshold (Ct) was defined as the PCR amplification and direct sequencing cycle at the highest peak of the second derivative curve, which represented the point of Exons 18 to 21 of the EGFR gene were maximum curvature of the growth curve The amplified using four specific pairs of primers, ∆Ct value was calculated as ∆Ct = sample Ct and the PCR amplicons were subjected to – control Ct, after ensuring that the sample direct sequencing (the sequences of the and control Ct values were from the same primers and the amplification procedures are sample The ∆Ct value was then compared shown in Supplemental Tables and 2) with the cut-off value of the mutation, which Sequencing reactions were done with the was obtained from the rQiagen’s instructions same primers for PCR amplification and ABI A positive result was defined as a ∆Ct value BigDye Terminator kit v3.1 (Applied Biosys- less than or equal to the cut-off value tems, Foster City, CA), according to the manu- Statistical analysis facturer’s instructions Sequencing reactions were electrophoresed on an ABI3700 genetic Chi-square tests and Fisher’s exact tests analyzer Sequence variations were determi- (when there were fewer than five expected ned using Seqscape software (Applied Biosys- counts in the contingency table) were used to tems) with the EGFR reference sequence assess the relationship between the presence (NM_005228.3, National Center for Biotechno- of EGFR mutations in patients with NSCLC logy Information) and other patient characteristics, including age, gender, tumor histology and smoking Scorpion ARMS analysis status All statistical tests were two sided and An EGFR Scorpion Kit (Qiagen Manches- a p value of less than 05 was considered sta- ter Ltd., UK), which combines the ARMS and tistically significant Statistical analyses were Scorpion methods to detect 29 EGFR TK mu- done using the SPSS software package ver- tations in real-time PCR reactions, was used sion 21.0 to conduct the Scorpion-ARMS analysis The scorpion primers were designed and synthe- Research ethics sized by Qiagen All reactions were done in 25 This research was approved by the ethics -µL volumes using µL of template DNA, 19.5 committee of Hanoi Medical University, decree JMR 105 E1 (7) - 2016 29 JOURNAL OF MEDICAL RESEARCH No.161 - HMUIRB, signed February 15th, 2014 III RESULTS Extracted DNA from paraffin - embedded tissues 181 patients were enrolled between January 02, 2012 and December 30, 2013 Tumor tissues were collected from all participants before the initiation of gefitinib or erlotinib treatment Genomic DNA was extracted in all 181 samples at a range of purity from 1.7 to 2.0 and a concentration from 1,213 to 4,380 ng/µL The relationship between EGFR mutations and patients’ clinical characteristics The 181 NSCLC tumor specimens included 80 women showed EGFR mutations (75.0%) versus 46 out of 101 men (45.5%) f (p < 0001) When the subgroup of adenocarcinomas was analysed according to gender, women continued to show a higher mutation rate (56 out of 70 women (80.0%) versus 40 out of 82 men (48.8%) (p < 0001) Smoking status was also closely associated with EGFR mutation rate Among patients with EGFR mutations (n = 106), the rate of never smokers was 1.7 times higher than the rate for ever smokers 64 out of 86 never smokers showed EGFR mutations (74.4%) versus 42 out of 95 ever smokers (44.2%) (p < 0001) This difference was also observed when smoking status was studied in patients with adenocarcinomas 57 out of 75 never smokers 152 adenocarcinomas (84.0%), 21 squamous with cell carcinomas (11.6%), and large cell car- mutations (76.0%) versus 39 out of 77 ever cinomas (4.4%) 101 patients were male pa- smokers tients (55.8%) and 80 were female patients (p < 0001)) adenocarcinomas with displayed adenocarcinoma EGFR (50.6%) (44.2%) The median age of all patients was However, no significant difference in the 62 years old (range from 36 to 82 years old), rate of mutation was found with regards to with 61.3% of participants (111 of 181) being age For patients over 65 years old, 44 out of under 65 years old In terms of smoking his- 70 tory, 47.5% patients (86 of 181) were never (62.9%), and for those patients under 65 years smokers old, 62 out of 111 displayed EGFR mutations patients displayed EGFR mutations Mutations in the kinase domain (exon 18 to (55.9%) (p= 352) We also found no signifi- exon 21) of the EGFR gene were identified in cant difference in the rate of mutations with 106 tumor tissues, giving an overall mutation regards to age among patients with adenocar- rate of 58.6% (106 of 181) Adenocarcinomas cinomas Moreover, 39 out of 56 patients over showed mutations more often than any other 65 years old with adenocarcinoma displayed histology Moreover, 100 out of 152 adenocar- EGFR mutations (69.6%) versus 57 out of 96 cinomas (65.9%) showed mutations, versus patients under 65 years old with adenocarci- six out of 29 samples (20.7%) for all other noma (59.4%) (p= 206) Patient characteris- samples (p = 004) When all histologies were tics and EGFR mutation rates are summarized analysed together, women also showed a in table The relationship between EGFR significantly higher frequency of EGFR muta- mutation status and patient characteristics are tions compared with men Moreover, 60 out of graphed in figure 30 JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH Table Characteristics of patients according to EGFR mutation status Screened (n = 181) Characteristics With EGFR mutation With no EGFR mutation n1 % n2 % n3 % 101 55.8 46 45.5 55 54.5 50 44.2 60 75.0 20 25.0 Gender Male Female Age Median (yr) range 62 (36 - 82) 63 (36 - 81) 61 (40 - 82) < 65 yrs 111 61.3 62 55.9 49 44.1 >= 65 yrs 70 38.6 44 62.9 26 37.1 Never 86 47.5 62 72.1 24 27.9 Ever 95 52.5 44 46.3 51 53.7 152 84.0 100 65.9 56 34.1 Squamous 21 11.6 23.8 14 76.2 Large cell 4.4 12.5 87.5 Smoking status Histology subtypes Adeno Figure EGFR mutation status and patient characteristics JMR 105 E1 (7) - 2016 31 JOURNAL OF MEDICAL RESEARCH EGFR mutation spectrum of direct sequencing compared to Scorpion ARMS Exon 18 to exon 21 of the EGFR gene were screened in all 181 tumors Mutations were found in all four exons, but mainly clustered in exons 19 and 21 (Fig 2) In exon 19, the only mutation found was a 15 - nucleotide deletion from nucleotides 2481 to 2495, which resulted in elimination of codons 746 to 750 This type of in - frame deletion, called an LREA deletion, was found in 51 of 106 tumors with EGFR mutations (48.1%) tide deletion, spanning from nucleotide 2499 to 2521 (c.2499_2521del mutation); and one tumor carried a 3-nucleotide ACA insertion at the position between nucleotide 2554 and 2555, leading to a threonine insertion into the position between valine (V851) and lysine (K852) (V851_K852insT mutation) (Fig 3) In exon 18, the two forms of mutation seen were missense mutation G719S (c.2155G>A) and the small deletion c.2137delA Each of these mutations was found once In exon 20, three missense mutations and one small deletion were found A concurrent S768I and In exon 21, two types of missense muta- V769L mutation was found in one tumor The tions were observed A c.2819T > G mutation T790M mutation occurred in two tumors, one of which had an L858R mutation in exon 21 resulted in a change to L858R in 43 tumors L861Q, which occurred in tumors In addi- Deletion of two adenines at the position of nucleotide 2373 caused the non-in-frame tion, one tumor contained a new 23 - nucleo- deletion c.2373_2374delAA (Fig 3) (40.6%) The other missense mutation was Figure Incidence of EGFR mutations in 181 NSCLC patients Although most tumors had a single mutation in the kinase domain of the EGFR gene, a combination of two types of mutation were seen in two patients, one of which contained the T790M (exon 20) and L858R (exon 21) mutations and the other of which carried the S768I and V769L mutations (both in exon 20) 32 JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH The EGFR mutation frequency detected by Direct sequencing, however, discovered direct sequencing and the Scorpion ARMS four novel or unknown mutations from four methods were 44.2% (80 out of 181 patients) tumors, including c.2137delA in exon 18, and 55.8% (101 out of 181 patients), respec- c.2373_2374delAA tively The Scorpion ARMS method found c.2499_2521del23 more tumors containing LREA deletion muta- c.2554/2555insACA (V851_K852insT) in exon tions and single missense mutations than the 21 In addition, the direct sequencing method direct sequencing method Moreover, the detected Scorpion - ARMS method found 51 tumors missense mutations, while the Scorpion- containing LREA deletion mutations, versus ARMS found only three Thus, the combina- 45 tumors with LREA deletion mutations found tion of the two methods elevated the overall by the direct sequencing method; the Scorpion mutation rate of this study to 58.6% (106 out -ARMS method also discovered 49 tumors of 181 patients) The frequency and the mo- with single missense mutations, compared lecular features of EGFR mutations detected with 29 tumors with single missense mutations by direct sequencing and the Scorpion ARMS discovered via direct sequencing method are shown in table four in in exon exon mutations in 21 two 20, and double- Figure Novel mutations in the EGFR gene in non - small cell lung cancer JMR 105 E1 (7) - 2016 33 JOURNAL OF MEDICAL RESEARCH (A) A deletion of one adenine at the position of nucleotide 2137 in exon 18 caused a c.2137delA mutation (B) A deletion of two adenines at the position between nucleotides 2373 and 2374 in exon 20 led to a c.2373_2374delAA mutation (C) An insertion of three nucleotides, Adenine - Cytosine - Adenine, at the position between nucleotides 2554 and 2555 created a c.2554/2555insACA mutation This is an in - frame insertion mutation with amino acid change V851_K852insT (D) A deletion of 23 nucleotides at the position between nucleotides 2499 and 2521 in exon 19 caused a c.2499_2521del23 mutation The mutations in (A), (B) and (D) resulted in alterations in encoded amino acids The arrow shows the position of the mutation Table Molecular features of 106 lung cancer patients with EGFR mutations EGFR mutation Mutation No of patients detected Mutation Scor- Site, nucleotide change Amino acid change Site, nucleotide Amino acid change change Sequencing pion Both ARMS Exon 18, 2137delA ? 1 Exon 18, 2155G > A G719S 1 DelE746_A750 45 51 55 1 1 1 1 Exon 19, 2235_2249del15 Exon 20, 2303G > T S768I Exon 20, 2369C > T T790M Exon 20, 2369C > T T790M Exon 20, 2305G > T Exon 21, 2819T > G V769L L858R Exon 20, 2373_2374delAA ? 1 Exon 21, 2499_2521del23 ? 1 V851_K852ins T 1 Exon 21, 2573T > G L858R 25 43 43 Exon 21, 2582T > A L861Q 4 Total 80 101 106 Exon 21, 2554/2555insACA 34 JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH IV DISCUSSION other ethnic groups [17 – 22] Since we only The current study reports the results of a molecular analysis of the EGFR gene in 181 Vietnamese NSCLC patients before the initiation of gefitinib or erlotinib therapy Among the 181 patients, 106 patients (58.6%) recruited Vietnamese patients for this study, the mutation rate here was both higher than that reported in studies with non - Asian patients and also differed from frequencies reported in studies conducted in other Asian countries were found to carry EGFR mutations in their NSCLC tumors The rate of EGFR mutations found in this study was much higher than that previously reported for Caucasian NSCLC patients [7; 15; 16] but only somewhat higher than that observed in East Asian patients The frequency of EGFR mutations was reported to be 39% in Japan, 34.6% in Korea, 38.6% in Taiwan and 57.4% in Thailand [17 - 20] One possible explanation for the higher mutation rate in this study lies in the distribution of histological subgroups While the expected proportion of adenocarcinomas in routine studies is between 50% to 70% depending on literature, 84% of the participants in this study had adenocarcinoma In a manner consistent with previous reports, the EGFR mutation was tightly associated with this pathologic subtype Since the adenocarcinomas included in this study were more likely to exhibit an EGFR mutation adenocarconima (100 tumors, out of 152 65.9%), the The method that we used to determine the EGFR mutation rate is also an important factor impacting our results In this study, the rate of mutations in tumor DNA detected by the Scorpion-ARMS was compared with that determined by the direct sequencing method, which is the current standard DNA from tumor samples always consisted of a mixture of mutant DNA and wild - type DNA because the EGFR mutation status was heterogeneous in all samples and the complete removal of normal cells, such as normal epithelial cells and inflammatory cells, from tumor specimens is difficult Since parallel tumor tissue investigations were done on all specimens using both direct sequencing and the Scorpion - ARMS method - a recognized advantage in the present study - the maximum number of EGFR mutations were detected (106 cases in total) This is in contrast to the 101 cases that would have been detected using Scorpion - ARMS alone, or the 80 cases that would have been frequency of the EGFR mutation may have detected using direct sequencing alone Elli- been overestimated Similarly high mutation son et al reported an overall EGFR mutation rates have been observed in previous studies rate of 12.6%, higher than the single Scorpion that ARMS method or sequencing (8.4% and have recruited patients with adenocarcinoma [21; 22] 7.9%, respectively) [23] We can speculate Another factor with great influence on the that the high sensitivity and specificity of the EGFR mutation rate was the geographical EGFR Scorpion kit allowed us to detect more origin of patients included in this study For mutations and to confirm uncertain results that quite some time, it has been known that East we obtained from direct sequencing alone Asian patients with NSCLC have a higher inci- Figure shows an example of how the dence of EGFR mutations compared with Scorpion ARMS method was used to confirm JMR 105 E1 (7) - 2016 35 JOURNAL OF MEDICAL RESEARCH uncertain results Although Shi et al, in the with EGFR mutations, 46.3%) found in this PIONEER study, reported a high frequency of study are comparable with rates found in of EGFR mutations in seven Asian countries numerous previous studies [19; 21; 23; 25] using the Scorpion-ARMS method, they did The LREA mutation in exon 19 occurred at a not discover any new or unknown mutations higher rate than the L858R mutation in exon [22] In this study, four novel mutations 21, corresponding to what past research has (deletion and insertion mutations) and one shown [5] We also discovered new mutations, V769L mutation were determined by using including deletions in exons 18, 20 and 21 and direct sequencing alone This is the first time an insertion in exon 21 (listed in Table 2) in Vietnam that an EGFR mutation analysis These genetic changes have not been reported was carried out by both direct sequencing and in the literature Thus, their roles for EGFR the Scorpion-ARMS method One limitation of tyrosine kinase inhibitory therapy are unclear the EGFR Scorpion kit is that while it is able to detect mutations targeted by the designed Scorpion primers, it misses those EGFR mutations that are not at these unique sites but are instead clustered around the ATPbinding site in exon 18, 19, 20 and 21 [5 – 9] In addition, the likelihood of discovering an EGFR mutation depends on various technical factors such as the tissue extraction method, the qualification of the tumor tissue, and the DNA purification procedure Moreover, the new mutations that we found in this study are complex Two tumors carried double mutations, including both T790M + L858R and S768I+V769L This finding would suggest that the cancer cells in these patients' tumor specimens might be accumulating multiple molecular mutations in the EGFR gene In terms of the impact of EGFR mutations on the responsiveness of tyrosine kinase inhibitors, sensitizing mutations were more frequent than resistant mutations in this study (99 sensitizing In this study, we found that NSCLC pa- mutations out of 106 tumors, 93.4%, versus tients with EGFR mutations were more likely resistant mutations out of 106, 2.8%), which is to be non - smokers, women, and/or have similar to what has been found in previous tumors of the adenocarcinoma subtype These studies [5; 8] Two of three tumors containing associations by resistant mutations carried double mutations numerous previous studies [7, 24] The high T790M, S768I and V769L were reported as mutation rates in these subgroups suggest resistant mutations, while L858R is classified have been confirmed that it is possible to screen for potential NSCLC patients who are likely to carry EGFR mutation(s) Such screening could play an important role in selecting patients for targeted therapeutic decision-making in NSCLC as a sensitizing mutation Although EGFR double mutations have been reported in previous studies, where the frequency has varied from 13% to 17% [19; 26], they have not previously been studied in Vietnamese Independent of the total rate of mutations, NSCLC patients [22] T790M + L858R is a the frequency of EGFR mutations in exon 19 complex double mutation that leads to a (51 out of 106 patients with EGFR mutations, combination of sensitization and resistance In 48.1%) and exon 21 (49 out of 106 patients a study by Tam et al., the in vitro gefitinib 36 JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH response of EGFR T790M+L858R mutants Sekido Y, Fong KM, Minna JD (1998) was investigated and compared with other Progress in understanding the molecular single and double mutants The T790M + pathogenesis of human lung cancer Biochim L858R Biophys Acta, 1378, 21 - 59 double mutants showed strong resistance to gefitinib treatment because they Olayioye MA, Neve RM, Lane HA et al increased the ATP affinity of the EGFR kinase (2000) The erbB signaling network: receptor domain at a 5-fold higher rate than the L858R heterodimerization in development and can- single mutants [26] This should be further cer EMBO J, 19, 3159 - 67 investigated among Vietnamese patients In conclusion, EGFR tyrosine Lynch TJ, Bell DW, Sordella R et al kinase (2004) Activating mutations in the epidermal domain mutations were found with high growth frequency in Vietnamese NSCLC patients responsiveness of nonsmall-cell lung cancer These mutations included deletion, insertion to gefitinib N Engl J Med, 350, 2129 - 2139 and missense mutations, ranging from exon factor receptor underlying Pao W, Miller VA (2005) Epidermal 18 to exon 21 of the EGFR gene New growth mutations and double mutations were also molecule kinase inhibitors, and non-small-cell detected, thanks to the combination of the lung cancer: current knowledge and future direct sequencing and the Scorpion ARMS directions J Clin Oncol, 23(11), 2556 - 2568 methods These findings support We also found that among the patients in our Vietnamese receptor mutations, small- patient selection based on molecular classification study, factor women with adenocarcinoma had the highest frequency of tumors with an activating EGFR mutation Shigematsu H, Lin L, Takahashi T et al (2005) Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers J Natl Cancer Inst, 97, 339 - 346 Paez JG, Janne PA, Lee JC et al Acknowledgements We thank the patients and their families for their voluntary involvement in this study This (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy Science, 304,1497 - 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