1 Hospital Doce de Octubre, Madrid; 2 Xeral Cies de Vigo, Vigo; 3 Catalan Institute of Oncology, Badalona; 4 Clinica Sagrado Corazon, Sevilla; 5 Hospital General Yague, Burgos; 6 Hospital La Princesa, Madrid; 7 Catalan Institute of Oncology, Girona; 8 Hospital de Leon, Leon; 9 Hospital General de Valencia, Valencia; 10 Hospital Arnau de Vilanova, Valencia; 11 Catalan Insitute of Oncology, Bellvitge; 12 Hospital Clinico San Carlos, Madrid; 13 Hospital General de Asturias, Oviedo; 14 Hospital Alcoy, Alicante; 15 Autonomous University of Madrid, Madrid, Spain
* Correspondence to: Dr R. Rosell, MD, Chief, Medical Oncology Service, Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Ctra Canyet, s/n, 08916 Badalona, Barcelona, Spain. Tel: +34-93-497-89-25; Fax: +34-93-497-89-50; Email: rrosell{at}ns.hugtip.scs.es
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Abstract |
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Patients and methods:: Eighty-three Spanish advanced NSCLC patients who had progressed after chemotherapy, were treated with compassionate use of gefitinib. Patients were selected on the basis of available tumor tissue. Tumor genomic DNA was retrieved from paraffin-embedded tissue obtained by laser capture microdissection. EGFR mutations in exons 19 and 21 were examined by direct sequencing.
Results:: EGFR mutations were found in 10 of 83 (12%) of patients. All mutations were found in adenocarcinomas, more frequently in females (P=0.007) and non-smokers (P=0.01). Response was observed in 60% of patients with mutations and 8.8% of patients with wild-type EGFR (P=0.001). Time to progression for patients with mutations was 12.3 months, compared with 3.6 months for patients with wild-type EGFR (P=0.002). Median survival was 13 months for patients with mutations and 4.9 months for those with wild-type EGFR (P=0.02).
Conclusions:: EGFR TK mutational analysis is a novel predictive test for selecting lung adenocarcinoma patients for targeted therapy with EGFR TK inhibitors.
Key words: EGFR, gefitinib, mutations, NSCLC, predictive markers
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Introduction |
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TKs are central regulators of signaling pathways that control differentiation, transcription, cell cycle progression, apoptosis, motility and invasion. Mutations in several genes have been identified in key regions of the TK domain in several cancers [5], paving the way for the identification of biomarkers for gefitinib sensitivity. The hypothesis that mutations in the EGFR TK domain may play a role in non-small-cell lung cancer (NSCLC) is supported by several lines of evidence. In three studies in NSCLC [6
8
], mutations were identified in the EGFR TK domain, with the majority clustering within exons 19 and 21. Mutations were either in-frame deletions or amino acid substitutions clustered around the ATP binding pocket. Missense mutations changing leucine 858 to arginine (L858R) and leucine 861 to glutamine (L861Q), as well as multiple deletions clustered in the region spanning codons 746 to 759, were found [6
8
]. In a very small number of patients, other mutations have been found in exons 18 and 20, some of which were identified in patients who did not respond to gefitinib. Duplication mutations in exon 20 have been described in Taiwanese patients [9
]. Another recent study [10
] examined 38 patients, of whom 21 were treated with gefitinib; a point mutation was found in exon 18 in only one case, while 19 cases had a deletion in exon 19, and 18 cases had a point mutation in exon 21. No mutations were found in exon 20. EGFR TK mutations represent bona fide somatic mutations in NSCLC and have not been identified in other primary tumors such as breast, colon, kidney, pancreas and brain, or in 108 cancer cell lines [6
]. Mutations were found more frequently in women, adenocarcinomas and Japanese patients [7
]. However, EGFR mutations were found in 11 of 96 (12%) primary NSCLCs resected from untreated patients, none of whom were East Asians [8
]. Fourteen of 182 (8%) primary lung cancer patients in the United States harbored EGFR mutations [6
8
]. The NSCLC H3255 cell line, harboring the L858R mutation, was 50-fold more sensitive to gefitinib than other adenocarcinoma cell lines containing wild-type EGFR TK [7
]. Accumulated data of the three studies [6
8
] show that 25 of 31 (81%) tumors from patients having partial responses or marked clinical improvement while taking gefitinib or erlotinib contained mutations in the EGFR TK domain. In contrast, none of 29 specimens from patients refractory to gefitinib or erlotinib had such mutations.
The frequency of EGFR mutations across different populations and the robustness of the correlation between the mutations and clinical benefit [11] prompted us to examine the presence of EGFR TK mutations in exons 19 and 21 and their association with clinical outcome in chemoresistant advanced NSCLC patients who received gefitinib in a compassionate-use program in Spain.
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Patients and methods |
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Laboratory methods
Pure tumor genomic DNA was derived from paraffin-embedded tissue obtained by laser capture microdissection (Palm, Oberlensheim, Germany). For isolation of DNA from deparaffinated, microdissected tissue, the material was incubated with proteinase K, and DNA was extracted with phenol-chloroform and ethanol precipitation. Primers and cycling conditions for PCR amplification and direct sequencing for exons 19 and 21 of EGFR (Gen Bank accession number: X00558) are shown in Table 1. Sequencing was performed using forward and reverse primers with the ABI Prism 3100 DNA Analyzer (Perkin-Elmer, Applied Biosystems). Electropherograms were analyzed for the presence of mutations using Seqscape v2.1.1 software in combination with Factura to mark heterozygous positions. The NSCLC cell line (PC9) derived from an adenocarcinomas [12] was also examined using the same methods.
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Age differences were analyzed using the MannWhitney U-test. Normality of the distribution of continuous variables was assessed with the KolmogorovSmirnov test. To identify relevant parameters of influence, a multivariable logistic regression model was used, and the fit of the models was evaluated with the HosmerLemeshow likelihood ratio test. The Wald test was used to test the statistical significance of each variable in the model. Survival and time to progression curves were drawn with the KaplanMeier product limit method. All reported P values are two-sided; P <0.05 was considered statistically significant. SPSS software version 11.5 (SPSS Inc, Chicago, IL) was used for all analyses.
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Results |
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Discussion |
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The 60% objective response rate observed in our study among NSCLC tumors harboring EGFR mutations is somewhat lower than that previously described [68
]. This disparity may be due to the retrospective nature of our study, where patients were included based on the availability of tumor tissue. In addition, the limitations imposed by the use of computed tomography to measure tumor size, especially in non-spherical lesions for which one-dimensional measurements are subject to inaccuracy [17
], may have led to discrepancies. Nevertheless, a highly significant difference in response rate (P=0.001) and survival (P=0.02) was observed between patients whose tumors contained EGFR TK mutations and those harboring wild-type EGFR.
There is a need for markers that can predict the efficacy of chemotherapy, and genetic changes in metastases in different organs may be a contributing factor in second-line chemotherapy failure. Objective responses after second-line treatment with docetaxel do not necessarily translate into longer time to progression [18]; median time to progression with either docetaxel or pemetrexed was only 2.9 months in previously-treated NSCLC patients [19
]. Even in first-line chemotherapy, median time to progression is rather short: 4.2 months in a randomized phase III trial comparing four different platinum-based doublets [20
]. In the present study, the first to examine the relation between EGFR TK mutational status and time to progression in gefitinib-treated stage IV NSCLC chemotherapy failures, patients with EGFR mutations had a remarkable 12.3-month time to progression.
The presence of EGFR TK mutations clearly identifies a subset of NSCLC patients with oncogene addiction [3, 16
] who will respond dramatically to EGFR TK inhibitors such as gefitinib or erlotinib [6
8
]. The frequency of EGFR TK mutations in our Spanish NSCLC cohort is clinically meaningful, particularly in adenocarcinomas, and represents a new paragon in lung cancer management. EGFR TK mutational analysis constitutes a novel predictive test for selecting NSCLC patients for upfront treatment with EGFR TK inhibitors in preference to chemotherapy [21
]. Based on accumulated evidence and the results of our study, the Spanish Lung Cancer Group is initiating a new trial of first-line gefitinib in stage IV NSCLC patients carrying EGFR TK mutations.
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Acknowledgements |
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Received for publication December 20, 2004. Revision received February 16, 2005. Accepted for publication February 17, 2005.
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References |
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