1 Department of Pulmonary Medicine, 2 Department of Pathology, 3 Department of Radiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
* Correspondence to: Dr X.-T. Zhang, Department of Pulmonary Medicine, Peking Union Medical College Hospital and Chinese Academy of Medical Sciences, Beijing 100730, China. Tel: +86-010-84251629; Fax: +86-010-84251629; Email: ajxt{at}public3.bta.met.cn
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Abstract |
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Methods: A total of 98 patients who had failed at least one platinum-based regimen received gefitinib 250 mg once daily. The mutation analysis of the EGFR kinase domain was performed for 30 patients using paraffin-embedded tumor tissue.
Results: The response rate was 31.6% and the disease control rate was 67.3%. Objective response was correlated with adenocarcinoma, female gender and non-smokers. Median progress free survival (PFS) was 7.0 months, median overall survival (OS) was 12.0 months and 1-year survival was 53.1%. The median PFS and OS were improved among patients with adenocarcinoma, gefitinib responders and non-smokers. Active gene mutation was detected in 12 patients. Mutation rates were higher among gefitinib responders, non-smokers, patients with adenocarcinoma and female patients. OS was longer for patients with gene mutation than for patients without mutation.
Conclusion: Gefitinib demonstrated significant antitumor activity with a favorable toxicity profile for pretreated Chinese patients with advanced NSCLC. The active mutation of the EGFR kinase domain was strongly associated with response to gefitinib and prolonged overall survival.
Key words: EGFR mutation, gefitinib, non-small-cell lung cancer, p-EGFR, predictive marker, targeted therapy
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Introduction |
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It has been clearly demonstrated that aberrant activity of epidermal growth factor receptor (EGFR) signaling plays a key role in the development of tumor cell growth in a variety of common solid tumors, including NSCLCs [4]. Once activated, tyrosine kinase can promote autophosphorylation of protein tyrosine residue, thereby switching on intracellular signal transmission. Inhibition of tyrosine kinase phosphorylation by agents designed to target these specific molecular process appears to induce substantial therapeutic benefits [5
].
Gefitinib (IressaTM, ZD1839) is an orally active and highly effective EGFR tyrosine kinase selective inhibitor that blocks signal transduction pathways implicated in cancer growth [6]. Phase I studies showed that gefitinib was generally well tolerated, with evidence of tumor regression in patients with NSCLC [7
, 8
]. Two large phase II studies indicated clinically meaningful antitumor activity in previously treated patients with advanced NSCLC [9
, 10
]. Although EGFR is commonly expressed in NSCLC, there was no evidence for a consistent relationship between EGFR expression levels and response to gefitinib [11
]. Recently, two studies have shown that a gain-of-function somatic mutation of EGFR accounts for the response of some patients with NSCLC to gefitinib [12
, 13
]. The efficacy of gefitinib has been explored extensively in the West and in Japan. We aimed to assess the antitumor activity of gefitinib as well as the relationship between response and EGFR mutation in a series of Chinese patients in Peking Union Medical College Hospital.
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Patients and methods |
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Drug administration
One tablet of gefitinib (250 mg) was taken once daily at about the same time. Patients continued the course uninterrupted until disease progression, intolerable toxicity or withdrawal of consent. All drugs were supplied by AstraZeneca through the Extensive Access Program and local trail.
No systemic anticancer treatment was permitted during the study, except for palliative radiotherapy in patients with isolated symptomatic bone metastases provided that drug administration was not interrupted for >14 days.
Assessment of response and toxicity
Baseline evaluation included medical history and physical examination, electrocardiogram, chest radiography, thorax CT scan and ultrasonography of the upper abdomen. Laboratory investigations included complete blood counts, urinalysis and renal and liver function tests. Performance status was evaluated according to the Eastern Cooperative Oncology Group (ECOG) criteria. MRI of the brain and radionuclide bone scans were performed only if metastatic disease was suspected according to the clinical manifestations of each patient. Patients were re-evaluated using the same method as at the baseline at the end of the first and third months of therapy, and then every 3 months. Objective tumor response and its duration were assessed according to the RECIST criteria [14], and all responses had to be confirmed
28 days after the initial assessment of response. Progress-free survival (PFS) was calculated from the date of start of therapy to the date of disease progression or last follow-up. Overall survival (OS) was assessed from the first dose of gefitinib to the date of death for any reason. Patients alive at the data cut-off were censored at the last date that the patient was known to be alive. All patients were evaluated in an intent-to-treat analysis. All adverse events were recorded and graded according to National Cancer Institute Common Toxicity Criteria, Version 2.0 [15
].
EGFR gene mutation analysis
Tumor tissues obtained before any systemic treatment were collected retrospectively from patients treated with gefitinib. All tumor tissues were formalin-fixed and embedded in paraffin. DNA was extracted from the tumor sections with DEXPAT (Takara Biomedical, Shiga, Japan) according to the manufacturer's instruction.
For mutational analysis of the kinase domain of EGFR coding sequence, exons 18, 19, 20 and 21 were amplified with four pairs of primers. Forward and reverse sequencing reactions were performed with the same primers for PCR amplification and ABI BigDye Terminator kit v3.1 (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Sequencing reactions were electrophoresed on an ABI3700 genetic analyzer. Sequence variations were determined using Seqscape software (Applied Biosystems) with the EGFR reference sequence (NM-005228.3, National Center for Biotechnology Information, Hyattsville, MD). All the sequence variations were confirmed by multiple independent PCR amplifications and repeated sequencing reactions.
Immunoactivity analysis
Phosphorylation EGFR (p-EGFR) status was evaluated by immunohistochemical staining using Moab phospho-EGFr Tyr1068 (Cell Signaling, Beverly, MA) according to the manufacturer's instruction. The slides were evaluated by two independent pathologists. The result of membrane and cytoplasma immunoreactivity was scored based on the percentage of stained cells and staining intensity according to a three-stage grading system: 0, no tumor cells were stained; 1+, <50% of the tumor cells were stained or weakly stained; 2+, >50% cells were stained vividly. The negative group includes tissues with score 0, and the positive group includes tissues with score 1+ to 2+.
Statistical analysis
Logistical regression test models were used to identify baseline factors (gender, smoking history, physical status, histology, TNM stage and prior chemotherapy) that might independently predict tumor response. Median PFS and OS were calculated using the KaplanMeier method. The log-rank test was used to detect differences in PFS and OS between strata. The gene mutation rate was compared between patients with and without objective tumor response using Pearson's 2-test or Fisher's exact test.
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Results |
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Mutations of EGFR were detected in 12 tumors. The clinical features and the results of mutational study are listed in Table 6. Four were in-frame deletions in exon 19 (delE746-A750, delL747-P753 insS). Eight mutations were in exon 21. All were the 2819TG, which resulted in an L858 change. Representative cases are shown in Figure 1.
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Discussion |
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In the present study, the response rate was 31.6% and the disease control rate was 67.3%, both of which are higher than in the phase II trials described above but similar to results obtained in Japan. A total of 102 Japanese patients with advanced lung cancer were evaluated in IDEAL1. Similar to our result, the RR for Japanese patients was higher than that of non-Japanese patients (27.5% compared with 10.45%; OR 3.72; P=0023). However, the difference in RR was imputed to bias of the baseline predictive factors of the patients (i.e. gender, PS and pathology) rather than ethnicity [9, 20
]. In our study, logistic regression analysis showed that histology, gender and smoking history were prognostic factors associated with an objective tumor response. Physical status, pre-chemotherapy and TNM stage were not predictive factors for tumor response. The data were similar to the two phase II trials.
The present study demonstrated that a daily oral dose of 250 mg of gefitinib was well tolerated in Chinese patients. Similar to prior clinical trials evaluating the toxicity of gefitinib, the most frequently reported adverse events were acne-like rash and grade 1 and 2 diarrhea. Severe adverse events were rare. It has been reported that 291 (1.7%) of 17 500 patients treated with ZD1839 in Japan had developed suspected interstitial pneumonia or acute lung injury in contrast with a lower incidence worldwide [21]. We paid more attention to this severe adverse event in our study because of the similarity in ethnicity between Chinese and Japanese, but we did not observe any evidence of interstitial pneumonia.
The most recent study showed that EGFR gene mutations were correlated with response to gefitinib in NSCLC. All these mutations were within exons 1821 of the kinase domain of EGFR. The mutations were most frequently found in patients who responded to gefitinib, patients with adenocarcinoma, patients who were non-smokers and female patients [12, 13
]. In the present study, a total of 12 mutations were detected, with four in-frame deletion mutations in exon 19 and eight missense mutations in exon 21. The mutation rate was significantly higher for gefitinib responders, non-smokers, female patients and patients with adenocarcinoma than for gefitinib non-responders, male patients, former smokers and patients with non-adenocarcinoma. These data agreed with the above studies and clinical data. The previous study showed that the gene mutation rate of Japanese (26%) was much higher than that of non-Japanese. The mutation rate of Japanese patients with adenocarcinoma was 46% [12
]. That may partially explain the different response rates of Japanese and Caucasians. The therapeutic efficacy for Chinese patients in the present study was close to that for Japanese which suggests that the EGFR gene mutation rate would be higher in the Chinese population because of the ethnic similarity.
EGFR is frequently expressed in NSCLC, and EGFR activation after ligand binding is believed to be its most important role in clinical settings. The relationships of EGFR expression and EGFR activation status to the sensitivity of EGFR inhibitors have been studied in a few retrospective series. The predictive association of EGFR and p-EGFR with tumor sensitivity has not been verified, but the activation of the downstream signaling molecule (p-Akt) seems to be highly sensitive to gefitinib in NSCLC [2225
]. In the present study, patients with positive p-EGFR expression had a higher response rate (P=0.013) and a tendency to have longer median PFS and OS (P-values of 0.0805 and 0.0526, respectively). The relationships between p-EGFR activation and activation of the downstream signaling pathways, as well as the relationship of activation status about EGFR and downstream signaling pathways to the response of gefitinib need further investigation.
In the present study, EGFR phosphorylation was detected by immunohistochemical analysis in six out of 12 tumors with EGFR mutation in contrast with four out of 19 tumors without EGFR mutation. The data agreed with the results obtained by Huang et al. [26]. In their study, increased EGFR phosphorylation was detected by immunoblot in 26 out of 39 tumors with EGFR mutation compared with 14 out of 38 tumors without mutation. In general, the EGFR kinase domain mutations seem to have a different effect on kinase activation. The functional significance of mutation on phosphorylation requires additional investigation.
Although previous studies have shown that the EGFR kinase mutations are correlated with clinical response, it is possible that not all patients with EGFR mutation respond to gefitinib. In the present study, four patients with disease control also had EGFR gene mutation, which has not been documented in other studies. All except one of these four patients had histological evidence of adenocarcinoma and OS was >10 months (follow-up 1025 months). Our data suggest that even patients with gene mutation who failed to show objective tumor response could probably also benefit from gefitinib treatment. In general, OS for patients with mutation was significant longer than that for patients without mutation. Further study of the antitumor mechanism of gefitinib may provide better selection criteria for patients. We should note that all mutations were performed with tissues at diagnosis. Could this gene status represent that after exposure to gefitinib? Does cytotoxic therapy affect the mutation status? At present, we lack information on the incidence of mutation after cytotoxic therapy. Further investigations are needed to determine the true incidence in untreated and treated tumors.
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Conclusion |
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Received for publication March 9, 2005. Revision received May 1, 2005. Accepted for publication May 3, 2005.
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References |
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