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Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Mutation in the epidermal growth factor receptor (EGFR) is frequently seen in non-small cell lung cancers (NSCLCs), especially in Asian females with adenocarcinoma. The frequency of mutation and the factors associated requires to be elucidated by analyzing a large number of consecutive clinical samples. We summarized the result of the EGFR mutation analysis for 1,176 patients performed at the time of diagnosis or relapse. The PNA-LNA PCR clamp, a highly sensitive detection method for the EGFR mutation, was employed. For fresh cases a portion of samples isolated to establish the diagnosis of lung cancer was used. For cases with a relapsed disease archival tissue were tested. The variables associated with the EGFR mutation after removing the confound factors were investigated by the logistic analysis using the samples collected in our university (n = 308) where detailed information on patients were available. The frequency of the EGFR mutation and its subtypes were investigated using all samples (n = 1,176). The EGFR mutation was significantly associated with adenocarcinoma (p = 0.006) and light-smoking (p < 0.0001), but not gender. The deletions in exon 19 were more frequently associated with male gender while exon 21 deletions were with female gender (p = 0.0011). The overall frequency of the EGFR mutation was 31%. Our result suggests that the female predominance in the EGFR mutation rate is a reflection of a higher frequency of adenocarcinoma in females. The gender difference in the mutation subtypes may provide a clue for the mechanism of the occurrence of the EGFR mutation.

EGFR mutation is one of the most common genetic alterations in non-small cell lung cancers (NSCLCs).1, 2 It is more frequently seen in East Asians, females and non-smokers.3 EGFR mutation is more frequently found in adenocarcinomas than in other types of cancers.4 Since adenocarcinomas occur more frequently in females than in males, the seeming gender difference in the EGFR mutation rate may just reflect the gender difference in the rate of adenocarcinoma. The epidemiological factors associated with the EGFR mutations provide the clues to the causes that originated EGFR mutation and hence cancers. Therefore, it is important to eliminate the confounding factors and identify the factor(s) that are primarily associated with EGFR mutations. A study consisting of a large number of patients is required.

The PNA-LNA PCR clamp is a highly sensitive detection method for EGFR mutation.5, 6, 7 It detects mutations in the presence of 100 to 1000-fold background of the normal cells over the cancer cells, and thus enables one to detect the EGFR mutation from cytological specimens used to diagnose cancers in clinical practice.6 Several prospective phase II studies have shown the administration of gefitinib to the EGFR mutation-positive patients to provide a survival benefit.8, 9, 10–13 Even patients with a poor performance status have been shown to benefit from gefitinib if they are mutation-positive.14 With this information, a test for the EGFR mutation for NSCLC patients has become routine in Japan. Currently more than 10,000 NSCLC patients per year are tested for EGFR mutations by the PNA-LNA PCR clamp.

Since the development of the PNA-LNA PCR clamp in 2005, we have tested for EGFR mutations in all NSCLC patients who visited our hospital and provided informed consent. In addition, we tested for the mutation in more than 2,000 samples sent from more than 20 collaborative institutes; most of the samples were a part of a specimen isolated in order to establish the diagnosis of lung cancer. We herein summarize the results of tests and the statistical analyses for the first consecutive 1,176 patients.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Clinical samples

This study was approved by the institutional review board of each institute and performed in accordance with the Declaration of Helsinki (1995, revised in Edinburgh 2000). An aliquot of the specimens which were isolated to establish the diagnosis of NSCLC, or in the case of relapsed diseases, the archival specimens from the previous treatment were subjected to the EGFR mutation test by the PNA-LNA PCR clamp after obtaining informed consent from all patients. The period of sample collection for Saitama Medical University Hospital and Saitama Medical University International Medical Center ranged from October 2004 to February 2008, while that for the collaborative institutes differed depending on the time they joined to the study.

EGFR mutation analyses

Each sample (sputum, pleural effusion, bronchial washing, needle biopsy and paraffin embedded tissue) was divided into two parts immediately after collection at each institute. When the diagnosis of cancer was pathologically established from the first part, the other part was sent to our institute to test for the EGFR mutation by the PNA-LNA PCR clamp. The investigated gefitinib sensitive mutations included G719C, G719S, G719A, L858R, L861Q and exon 19 deletions, as well as a gefitinib resistant mutation T790M. The results were reported within 1 to 3 days after the receipt of the samples so that each institute was able to utilize the results in order to select the appropriate treatment for each patient.

Statistical analysis

Any significant differences among the categorized groups were compared using either the two-sided χ2 test or Fisher's exact test. The adjusted effects of age, sex, histology, staging and smoking history on EGFR mutation were evaluated by using a logistic regression model, and the results were described as an odd ratio with a 95% confidence interval. All analyses were performed using SPSS Statistics (SPSS version 17.0 for Windows, SPSS Inc, Chicago, IL).

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Samples

We were able to collect detailed information for the samples collected in our university. On the other hand, because of the privacy policy of the individual institutes, information was incomplete for the samples from other institutes. Therefore, we performed two different analyses. Firstly, we analyzed the samples collected in our university, and performed detailed analyses on the factors associated with EGFR mutations and their subtypes. Next, we put all samples together and calculated the frequencies of EGFR mutations and their subtypes.

Detailed analysis

We had 311 patients who visited to our university and were tested for the EGFR mutation, and 308 of them provided informative results. The source of the samples included all types of cytological and tissue specimens that are used for the diagnosis of the cancer. A total of 112 (36.4%) had EGFR mutations (Table 1). We identified the gefitinib resistant mutation T790M in 4 samples, for all of which the gefitinib sensitive mutations co-existed. All patients from whom these samples were isolated had been treated by gefitinib. Patients who were not previously treated with gefitinib did not have the T790M mutation.

Table 1. EGFR mutation identified
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We investigated the association of several variables with the EGFR mutation (Table 2). Two-sided χ2 tests revealed gender (female), histology (adenocarcinoma and adenosquamous cell carcinoma) and smoking history (less than 20 packs/year) to be significantly associated with the presence of the EGFR mutation.

Table 2. Association of each variable with the EGFR mutation
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The variables studied here may be associated with each other, and they may act as confounding factors in the analyses of other variables. For example, the smoking rate is several-fold different between males and females in Japan and thus the variables affected by smoking may show a seeming gender difference. To eliminate the effect of such confounding variables, we performed a logistic regression analysis (Table 3). The analysis revealed histology and smoking history to be significantly associated with EGFR mutation, while gender, which has often been used as a criterion for selecting a patient group populated with EGFR mutations in clinical medicine, was not significantly associated.

Table 3. Logistic regression analysis for the association with EGFR mutation (N = 289)
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In a subgroup analysis where only mutation-positive patients were studied, we noticed that exon 19 deletions were more frequently found in males, and the difference was significant according to Fisher's exact test (Figure 1). We again performed a logistic regression analysis and confirmed that exon 19 deletions were significantly associated with a male gender after removing the influence of other variables (Table 4). We concluded that not the frequency of the EGFR mutation but the subtype of it shows a gender difference.

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Figure 1. The type of EGFR mutations according to gender. The exon 19 deletions are more frequently found in males than in females (Fisher's exact test, p < 0.047).

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Table 4. 
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Frequency of the EGFR mutation

The results of the EGFR mutation analysis where samples from our university and those from other institutes were combined are shown in Figure 2. Out of 1176 samples, 1120 samples provided informative results. The EGFR mutation rate was 31%, which was very similar to the rate obtained in the detailed analysis described above.

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Figure 2. The rate of EGFR mutations. A total of 1176 samples were analyzed, of which 1120 samples provided informative results. Of the 56 uninformative samples, 52 were paraffin-embedded tissues. The number of paraffin-embedded tissue specimens examined was 344, meaning that about 1/8 of them were not suitable for the mutation analysis.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our result shows the rate of EGFR mutations to be 43% in adenocarcinoma and 12% in the other types of NSCLCs. The overall frequency of EGFR mutations was around 30% in both the samples studied in detail and in all the samples combined. We are in the field of internal medicine, as are our collaborators, and therefore advanced cancers are preferably referred to us. Our samples may better represent those from more advanced stages of NSCLCs. Instead, the frequency was found to be consistent with what determined using surgical samples3, 15, 16 which may better represent earlier stages of NSCLCs. Our results suggest that the rate of the EGFR mutation is similar irrespective of the stages of NSCLCs.

The results showing EGFR mutation to be associated with adenocarcinoma but not gender are considered to have clinical implications. Under a setting where an EGFR mutation test is not readily available, the targets for gefitinib therapy have been determined based on the patient characteristics and one of the criteria frequently employed has been adenocarcinomas which occurred in females. According to our results, there is no reason to select the patients by gender.

In contrast to the rate of the EGFR mutations, the mutation subtypes showed a gender difference. Although the exact mechanism by which each subtype occurs has not yet been elucidated, chromosomal recombination that involves DNA double strand breaks and repairs is likely to be involved in exon 19 deletions. It is well known that the meiotic recombination rate shows a clear gender difference.17 Instead, to our knowledge, there have been no reports showing a gender difference in the rate of somatic, chromosomal deletion mutations. The EGFR mutation may therefore be an interesting model to pursue the gender difference of cancers from the viewpoint of the DNA repair mechanisms.

In the current study, we reported the result of 1176 samples and found a close association between adenocarcinoma and EGFR mutations as well as the gender difference in the mutation subtypes. We also provided the frequencies of EGFR mutations in the samples that are considered to better represent the later stages of NSCLCs. Since tests for EGFR mutations are now widely performed, studies consisting of a large number of samples are now becoming realistic. Such studies will provide further valuable information on the genesis of EGFR mutations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Ms. Chieko Ono for technical assistance.

The following hospitals contributed to the study: Asahikawa Medical College (Yoshinobu Ohsaki), Dohoku National Hospital (Yuka Fujita), Fukushima Medical University (Takashi Ishida), Hokkaido Social Insurance Hospital (Toshiyuki Harada), KKR Sapporo Medical Center (Hiroshi Isobe, Testuya Kojima), Toranomon Hospital (Kunihiko Yoshimura), Kanto Medical Center NTT EC (Kazuhiro Usui), Kanagawa Cardiovascular and Respiratory Center (Takashi Ogura), Sapporo General Hospital (Shigeaki Ogura), Nippon Medical School Chiba Hokusoh Hospital (Mitsunori Hino), Hokkaido University Graduate School of Medicine (Ichiro Kinoshita), Japanese Red Cross Sendai Hospital (Makoto Miki), National Hospital Organization Hokkaido Cancer Center (Masao Harada), Sendai Medical Center (Hiroshi Miki).

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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