Journal of Thoracic Oncology:
Clinical Significance of Epidermal Growth Factor Receptor Gene Mutations on Treatment Outcome after First-line Cytotoxic Chemotherapy in Japanese Patients with Non-small Cell Lung Cancer
Hotta, Katsuyuki MD, PhD*; Kiura, Katsuyuki MD, PhD*; Toyooka, Shinichi MD, PhD†; Takigawa, Nagio MD, PhD*; Soh, Junichi MD, PhD†; Fujiwara, Yoshiro MD*; Tabata, Masahiro MD, PhD*; Date, Hiroshi MD, PhD†; Tanimoto, Mitsune MD, PhD*
*Department of Respiratory Medicine, Okayama University Hospital, Okayama, Japan; †Department of Cancer and Thoracic Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
This article has been selected to be presented in the 2007 annual meeting of American Society of Clinical Oncology as a part of the Scientific Program.
Disclosure: The authors declare no conflict of interest.
Address for correspondence: Katsuyuki Hotta, MD, PhD, Department of Respiratory Medicine, Okayama University Hospital, 2-5-1, Shikata-cho, Okayama, 700-8558, Japan. E-mail: email@example.com
Introduction: The relationship between the EGFR gene mutation status and clinical outcome has not fully been assessed in patients with non-small cell lung cancer (NSCLC) who received cytotoxic agents. The aim of this study was to clarify its association. We also examined whether this association could be affected by previous gefitinib treatment.
Methods: Patients with advanced or postoperative recurrent NSCLC who received both cytotoxic chemotherapy and gefitinib monotherapy in their treatment course were included in this study. An EGFR mutation was determined in exons 19 and 21 by direct sequencing.
Results: Of 194 Japanese patients with advanced or relapsed NSCLC assessable for mutation analysis, 60 received both cytotoxic chemotherapy and gefitinib monotherapy through their treatment courses. EGFR mutations significantly affected progression-free survival (PFS) in the first-line cytotoxic chemotherapy regimens in the multivariate analysis (hazard ratio for PFS = 0.422; p = 0.0422). In contrast, in 28 (47%) of the 60 patients who also received cytotoxic chemotherapy after the relapse to gefitinib monotherapy, there were no differences in PFS stratified by EGFR mutation status. The sensitivity to gefitinib was, however, correlated with EGFR mutation status, and its sensitivity was retained even in the second-line treatment setting in patients with EGFR mutations.
Conclusions: EGFR mutations were therefore significantly associated with a better PFS in the first-line cytotoxic chemotherapy regimens. However, its association was not observed in the cytotoxic regimens administered after the relapse to gefitinib monotherapy, whereas gefitinib sensitivity was associated with an EGFR mutation even in the second-line or later treatment settings.
The epidermal growth factor receptor (EGFR) is a promising target for anticancer therapy because EGFR signaling is known to be associated with proliferation and survival of cancer cells and it is frequently overexpressed in a variety of tumors including non-small cell lung cancer (NSCLC).1 Several researchers have suggested the possible association between EGFR function and sensitivity to cytotoxic chemotherapy. Dixit et al.2 demonstrated that decreased expression of the EGFR in MDA-MB-468 breast cancer cells resulted in a reduced sensitivity to cisplatin.2 It was shown that sensitivity to other cytotoxic agents including anthracyclines, vinca alkaloids, and fluorouracils could be also modified by EGFR expression level.3 As a whole, the EGFR function seems to have a certain relationship with the sensitivity to cytotoxic agents.
As a molecular oncologic alteration, somatic mutations in the tyrosine kinase domain of the EGFR gene have been recently found in NSCLC patients as gain-of-function oncogenic mutations.4 Several studies in transfected cells and human lung cancer cell lines have shown the mutant EGFR receptor to lead to a constitutive activation of the downstream signaling pathways, thus suggesting these alterations potentially confer sensitivity to EGFR tyrosine kinase inhibitors in EGFR mutant tumor cells,4–6 although no detailed mechanisms of its action have not yet been defined.
Gefitinib, EGFR tyrosine kinase inhibitor, has clinically shown promising antitumor activity in relapsed NSCLC patients of East Asian origin in phase II and III trials.7,8 The association between the EGFR gene mutation status and responsiveness to gefitinib has been extensively studied in the clinical setting, and we and other investigators demonstrated that EGFR mutant tumors yielded a high sensitivity to gefitinib as observed in preclinical studies9–11; however, the influence of EGFR mutations on the responsiveness to cytotoxic agents has not been fully investigated yet. Based on this background, we investigated whether the EGFR mutation status was associated with the efficacy of first-line cytotoxic chemotherapy in relapsed or advanced NSCLC patients.
In addition to these conventional EGFR mutations, the emergence of a second mutation at EGFR codon 790 at the time of relapse to gefitinib treatment was recently found, which is now considered to correlate with gefitinib resistance.12 The effect of cytotoxic reagent on T790M mutant tumor cell remains unknown; furthermore, the gefitinib-resistant cells induced by gefitinib treatment may acquire some features that cause higher resistance to cytotoxic reagents compared with gefitinib-naive tumor cells.13 It may indicate that gefitinib exposure might modulate the original association between EGFR mutation status and responsiveness to subsequent cytotoxic chemotherapy. We therefore also examined whether this association could be affected by the previous gefitinib treatment.
MATERIALS AND METHODS
A total of 194 patients with advanced or postoperative recurrent NSCLC who were admitted to Okayama University Hospital between January 1999 and December 2005 were assessable for EGFR mutation analysis. Of these, 60 (31.4%) patients underwent both cytotoxic chemotherapy and gefitinib monotherapy through their treatment courses. Informed consent was obtained from all patients. Tumor response was assessed as complete response (CR), partial response (PR), no change (NC), or progressive disease (PD) in accordance with the World Health Organization Criteria.14
Genomic DNA was extracted from frozen or paraffin-embedded specimens, and the mutation status of the EGFR tyrosine kinase domain (exons 19 and 21) was examined with the direct sequence method described in the previous report.11 In brief, polymerase chain reaction (PCR) amplification for extracted DNA was done using HotStarTaq DNA polymerase (Qiagen Inc., Valencia, CA) with the specific primers and PCR conditions. PCR products were incubated using ExoSAP-IT (Amersham Biosciences Corp., Piscataway, NJ) and sequenced directly using Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Corp., Foster City, CA) with ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).
The associations between the EGFR mutation status and the responses to cytotoxic agents and gefitinib were evaluated by χ2 test or Fisher’s exact test. The progression-free survival (PFS) time was defined as the time from initiation of cytotoxic chemotherapy or gefitinib treatment until the day of documented disease progression. The overall survival time was also defined as the time from initiation of first-line chemotherapy to the time of death from any cause or to the date that the patient was last known to be alive. These survival curves were constructed using the Kaplan-Meier product-limit method. Any differences between the curves were evaluated by the log-rank test. Proportional hazard regression models were applied to evaluate potential impact of confounders (covariates: EGFR mutation, gender, disease stage, histology, Brinkman index, which was calculated as follows: [average number of cigarettes consumed daily] × [number of years of smoking]), and the performance status at initiation of each chemotherapy). p values <0.05 were considered to be statistically significant.
Demographics of Patients and Treatment Regimens
The demographics of the 60 NSCLC patients are listed in Table 1. Seventeen patients (28%) had tumors harboring EGFR mutations. In-frame deletion in exon 19 was observed in six patients (35%), and the remaining 11 (65%) had a point mutation (L858R) in exon 21. As expected, the majority of the patients with mutations were female and had adenocarcinoma histology and never-smoking history.
Of the 60 patients, 54 (90%) received cytotoxic chemotherapy in the first-line treatment setting, and gefitinib monotherapy was administered in the later treatment settings. In the remaining six patients (10%) who underwent gefitinib therapy in the first-line treatment setting, cytotoxic agents were subsequently administered after the gefitinib therapy. The treatment regimens are shown in Table 2. Their types were almost comparable between patients with or without an EGFR mutation. Fifty-eight percents of the regimens in the first-line treatment were platinum-based combination chemotherapy (58%), while about two third of chemotherapy regimens administered after gefitinib treatment did not contain any platinum agents (64%).
Gefitinib was orally administered at a dose of 250 mg once daily in six (10%), 36 (60%), 15 (25%), and three (5%) patients as the first-, second-, third-, and fourth-line treatment settings among the 60 patients, respectively.
Efficacy of Cytotoxic Chemotherapy
The objective response to first-line cytotoxic chemotherapy was obtained in nine (16.7%; 95% confidence interval [CI]: 6.4%–26.9%) of the 54 patients. The median length of follow-up of the 19 surviving patients was 41.5 months (range, 1.7–90.7), and the 6-month and 1-year PFS rates and 6-month and 1-year overall survival rates in the first-line cytotoxic chemotherapy were 28.0%, 4.7%, 88.7%, and 71.5%, respectively. Regarding the efficacy of chemotherapy administered after gefitinib monotherapy in the 28 patients, the objective response rate and 6-month PFS rates were 25.0% (seven of 28 patients; 95% CI: 7.9%–42.1%) and 16.2%, respectively.
Association between EGFR Mutation Status and Efficacy of First-line Cytotoxic Chemotherapy
We investigated whether EGFR mutation status was associated with sensitivity to first-line cytotoxic chemotherapy in the 54 patients. The objective response to cytotoxic chemotherapy was not influenced by EGFR mutation status (three [21%] of 14 patients with an EGFR mutation versus six [15%] of 40 wild-type patients, respectively; p = 0.6842). In addition, neither smoking status nor types of EGFR mutations affected response to chemotherapy. However, EGFR mutations tended to yield a better PFS (log-rank test; p = 0.0535), with 6-month PFS rates of 45.8% and 21.9%, respectively (Figure 1A). This difference was also found based on the findings of a multivariate analysis (hazard ratio [HR] = 0.422, 95% CI: 0.179–0.991, p = 0.0422; Table 3). The EGFR mutation status also significantly correlated with the overall survival after initiation of first-line cytotoxic chemotherapy in both univariate (Figure 2) and multivariate analyses (HR = 0.263, 95% CI: 0.099–0.699, p = 0.0074; Table 3). Smoking status did not affect the PFS after the administration of cytotoxic chemotherapy.
Association between EGFR Mutation Status and Efficacy of Cytotoxic Chemotherapy Administered after Gefitinib Monotherapy
We next focused on the association between the EGFR mutation status and clinical outcomes after initiation of cytotoxic chemotherapy administered after gefitinib treatment. A total of 28 patients received cytotoxic chemotherapy after gefitinib monotherapy in the second-line or later treatment settings (Table 2). Of these, objective responses to cytotoxic chemotherapies after gefitinib treatment tended to be higher in patients with an EGFR mutation than in patients without any mutation (45% and 12%, respectively; p = 0.0764). There was, however, no difference in PFS between the two groups (log-rank test; p = 0.6844), with 6-month PFS rates of 23% and 13%, respectively (Figure 1B).
Efficacy of Gefitinib Monotherapy
As expected, in all 60 patients, the gefitinib sensitivity was significantly affected by EGFR mutation status with objective response rates of 76% and 16% (p < 0.0001) and 6-month PFS rates of 88% and 23% (p < 0.0001) in those with and without mutations, respectively. Even in the second-line or later treatment settings, after the administration of cytotoxic chemotherapy, a higher response rate (p < 0.0001) and better PFS (p < 0.0001) were observed in the EGFR-mutant patients than those in wild-type, with objective response rates of 92% versus 18% and 6-month PFS rates of 100% versus 22%, respectively (Figure 1C).
In this study, we found that EGFR mutation was associated with the survival time from initiation of first-line cytotoxic chemotherapy (Figures 1A and 2). In the phase III INTACT trials for advanced NSCLC patients, Bell et al.15 also demonstrated that PFS was better for EGFR mutation–positive patients than for those without mutations in the first-line treatment of platinum-based chemotherapy. A subgroup analysis of the TRIBUTE study, comparing erlotinib with placebo in combination with cytotoxic chemotherapy, also demonstrated that among the patients treated with cytotoxic chemotherapy alone, better PFS was observed in the EGFR mutation–positive patients.16 Based on these data, a certain interaction seems to exist between EGFR mutations and sensitivity to cytotoxic chemotherapy, similar to the positive association between EGFR function and responsiveness to cytotoxic chemotherapy shown in the previous studies.2,3 However, we do not know the detailed mechanism of how cytotoxic agents work differently in EGFR mutant and wild-type tumors only with our current data; therefore, future molecular analyses are needed to support its relationship.
In contrast, we also found PFS after initiation of cytotoxic chemotherapy administered after gefitinib treatment not to be affected by the EGFR mutation status (Figure 1B); this different influence of EGFR mutation status between the two periods should be simply attributable to our small sample size or the difference in types of chemotherapy regimens. However, as another explanation for this observation, a second mutation through the continuous gefitinib treatment might modulate the primarily positive association between EGFR mutation and the efficacy in cytotoxic chemotherapy. Indeed, recent reports have shown that exposure to gefitinib induced the appearance of gefitinib-resistant cells with alterations in some intercellular signal transduction pathways.13 These alterations might thus influence the cellular response to cytotoxic reagents, although the precise mechanism remains unclear. To clarify these issues, whole EGFR mutation analyses through exon 18 to 21 should thus be performed both before and after gefitinib treatment using a large cohort.
The current treatment guidelines recommend that gefitinib should be used after cytotoxic chemotherapy in unselected patients with advanced NSCLC.17 Recently, Inoue et al.18 conducted a Japanese phase II trial of first-line single-agent treatment with gefitinib for advanced NSCLC patients harboring EGFR mutations. It is interesting to note that gefitinib treatment yielded a favorable response rate of 75% and a median PFS time of 9.7 months. Contrary to these findings, our findings might support that patients who harbor EGFR mutations thus seem to still benefit from the early use of cytotoxic chemotherapy before gefitinib administration in their NSCLC treatment courses because of the significantly better PFS in the first-line cytotoxic chemotherapy in EGFR mutation–positive patients (Figure 1A). In addition, high sensitivity to gefitinib was indeed retained even in second-line or later treatment settings in patients with EGFR mutations in this study (Figure 1C). Our findings suggest that an optimal sequence of gefitinib therapy and cytotoxic chemotherapy should be extensively investigated taking EGFR mutation status into consideration, although the relevant randomized trials evaluating the optimal sequence do not stratify the patients according to the EGFR mutation status.19
As a major critical point in this study, the better PFS in the first-line cytotoxic chemotherapy in EGFR-mutation–positive patients might be essentially attributable to a favorable natural history in this genetically defined subset of NSCLC, irrespective of the efficacy of chemotherapy itself.20 Several investigators have demonstrated that classic EGFR mutations confer a favorable prognosis and that the superior survival reported for mutation-positive patients treated with tyrosine kinase inhibitors from single-arm studies,21 although majority of them were based on the retrospective analyses of highly selected and heterogeneous patients. In addition, a significantly longer survival time has been recently reported for untreated mutation-positive patients.22 Therefore, one can criticize that this superior survival reported for mutation-positive patients may not be derived as a direct result of the differential effect of therapy on the subgroup populations. However, it is also true that a better overall survival in mutation-positive NSCLC patients was not always evident.23,24 These observations still make it confusing regarding whether the EGFR mutation itself directly influences the natural history of NSCLC. Therefore, determining whether the EGFR-mutant NSCLC bears a more favorable prognosis regardless of therapy awaits further carefully designed population studies. Other limitations are that our analysis was based on a retrospective review with an unplanned analysis in a small patient cohort and that there might also be a potential bias for patient selection. Furthermore, the problems included the lack of a uniform procedure for patient follow-up. All of them might interfere with the suitability of the statistical analyses. However, if these limitations are understood, our results are hypothesis generating, which raises a critical point that needs to be evaluated in future studies.
In conclusion, we found the clinical outcome after initiation of first-line cytotoxic chemotherapy to significantly differ according to the EGFR mutation status. However, its association was not observed in the cytotoxic regimens administered after the relapse with gefitinib monotherapy, whereas gefitinib sensitivity was associated with EGFR mutations, even in the second-line or later treatment settings. Further investigations are thus warranted to confirm this observation in large-scale clinical trials.
1. 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
2. Dixit M, Yang J-L, Poirier MC, et al. Abrogation of cisplatin-induced programmed cell death in human breast cancer cells by epidermal growth factor antisense RNA. J Natl Cancer Inst
3. Dickstein BM, Wosikowski K, Bates SE. Increased resistance to cytotoxic agents in ZR75B human breast cancer cells transfected with epidermal growth factor receptor. Mol Cell Endocrinol
4. Greulich H, Chen TH, Feng W, et al. Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants. PLoS Med
5. Sordella R, Bell DW, Haber DA, Settleman J. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science
6. Tracy S, Mukohara T, Hansen M, Meyerson M, Johnson BE Janne, PA. Gefitinib induces apoptosis in the EGFRL858R non-small-cell lung cancer cell line H3255. Cancer Res
7. Fukuoka M, Yano S, Giaccone G, et al. A multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small cell lung cancer (The IDEAL 1 Trial). J Clin Oncol
8. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet
9. 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
10. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science
11. Tokumo M, Toyooka S, Kiura K, et al. Mutations and clinico-pathological features in non-small cell lung cancers. Clin Cancer Res
12. Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR mutation and resistance of non–small-cell lung cancer to gefitinib. N Engl J Med
13. Engelman JA, Mukohara T, Zejnullahu K, et al. Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer. J Clin Invest
14. Miller AB, Hoogstraten B, Staquet M, Winkler A. Reporting results of cancer treatment. Cancer
15. Bell DW, Lynch TJ, Haserlat SM, et al. Epidermal growth factor receptor mutations and gene amplification in non-small-cell lung cancer: molecular analysis of the IDEAL/INTACT gefitinib trials. J Clin Oncol
16. Eberhard DA, Johnson BE, Amler LC, et al. Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol
17. Pfister DG, Johnson DH, Azzoli CG, et al. American Society of Clinical Oncology Treatment of Unresectable Non–Small-Cell Lung Cancer Guideline: Update 2003. J Clin Oncol
18. Inoue A, Suzuki T, Fukuhara T, et al. 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
19. Nokihara H, Ohe Y, Kawaishi M, et al. A randomized phase II study of sequential carboplatin/paclitaxel (CP) and gefitinib (G) in chemotherapy-naïve patients with advanced non-small-cell lung cancer (NSCLC): preliminary results (Abstract). Proc Am Soc Clin Oncol
20. Shepherd FA, Tsao MS. Unraveling the mystery of prognostic and predictive factors in epidermal growth factor receptor therapy. J Clin Oncol
21. Takano T, Ohe Y, Sakamoto H, et al. Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non–small-cell lung cancer. J Clin Oncol
22. Sasaki H, Shimizu S, Endo K, et al. EGFR and erbB2 mutation status in Japanese lung cancer patients. Int J Cancer
23. Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst
24. Toyooka S, Soh J, Shigematsu H, Aoe M, Date H. The impact and role of EGFR gene mutation on non-small cell lung cancer. Cancer Chemother Pharmacol
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