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Study objectives: The prevalence of epidermal growth factor receptor (EGFR) mutations in gefitinib-naive lung cancer patients is higher in adenocarcinomas, in women, and in Japanese. To further investigate the prevalence of EGFR mutations in relation to ethnic and geographic factors, we evaluated EGFR mutations in a series of Taiwanese patients with primary lung adenocarcinomas who had never been treated with gefitinib.

Design and methods: We retrospectively studied 35 primary lung adenocarcinoma samples for mutations in the tyrosine kinase domain of EGFR; exons 18, 19, and 21 were analyzed by nested polymerase chain reaction and automated sequencing. Clinicopathologic information was obtained from patient records and pathology reports. Correlation between EGFR mutations and patient characteristics, including sex, smoking history, and pathologic subtypes, were evaluated by using the [chi square] test and logistic regression analysis.

Results: Heterozygous EGFR mutations were detected in 17 of 35 patients (48%). Missense mutations in exon 21 (13 of 17 patients, 76%) were the most frequent mutations detected. EGFR mutations were more frequent in women (13 of 18 patients [72%]) than in men (4 of 17 patients [23%]; p = 0.004), more frequent in nonsmokers (14 of 21 patients [66%]) than in current smokers (3 of 14 patients [21%]; p = 0.009), and when any degree of bronchioloalveolar carcinoma (BAC) was present (14 of 21 patients [66%]) compared with pure adenocarcinoma (3 of 14 patients [21%]; p = 0.009). Logistic regression analysis demonstrated that female gender (odds ratio [OR], 10.913; 95% confidence interval [CI], 1.778 to 66.97; p = 0.01) and BAC, including adenocarcinomas with any bronchioloalveolar features (OR, 9.708; 95% CI, 1.464 to 64.393; p = 0.019), were significantly associated with EGFR mutations.

Conclusions: In our series, female sex and bronchioloalveolar pathologic subtype predicted the presence of EGFR mutations in lung adenocarcinomas, and the high frequency of EGFR mutations supports the hypothesis that genetic backgrounds and/or environmental factors may affect the pathogenesis of certain lung cancers.

Key words: bronchioloalveolar carcinoma; epidermal growth factor receptor; female; mutations; non-small cell lung cancer

Abbreviations: BAC = bronchioloalveolar carcinoma; EGFR = epidermal growth factor receptor; NSCLC = non-small cell lung cancer; OR = odds ratio; PCR = polymerase chain reaction; TKI = tyrosine kinase inhibitor

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Lung cancer is the leading cause of cancer death in the world. (1) Non-small cell lung cancer (NSCLC) accounts for approximately 80% of cases of lung cancer. Chemotherapy for advanced NSCLC provides only a modest benefit but considerable toxicity. (2) Epidermal growth factor receptor (EGFR) is overexpressed in 40 to 80% of NSCLC patients and is therefore an important target of interest for therapy in this disease. Gefitinib, a synthetic anilinoquinazoline that specifically inhibits EGFR tyrosine kinase, has been used to treat advanced, chemotherapy-refractory NSCLC, yielding clinical responses of 9 to 19%. (3,4) However, EGFR expression does not correlate with either tumor response or symptom improvement in trials of gefitinib monotherapy. (5,6)

Recently, the molecular mechanisms underlying clinical responsiveness to gefitinib in NSCLC have been shown to correlate with somatic mutations in the EGFR gene, which result in increased sensitivity to inhibition of growth by gefitinib. (7,8) In previous studies, (7,8) the reported prevalence of EGFR mutations in gefitinib-naive patients is 8 to 13.4%. All such EGFR mutations were found to cluster in exons 18, 19, and 21. EGFR mutations have been found more frequently in adenocarcinomas (21%), in women (20%), and in Japanese patients (26%). Japanese women with adenocarcinoma have had the highest prevalence of these mutations (57%). There is a striking difference in the frequency of EGFR mutations between Japanese and US patients (26% vs 2%). To further explore possible ethnic and geographic factors related to the prevalence of EGFR mutations, we evaluated a series of 35 Taiwanese patients from a single institution who had primary lung adenocarcinoma never treated with gefitinib.

MATERIALS AND METHODS

Patients

The records of 35 patients with primary lung adenocarcinoma that had never been treated with gefitinib were randomly retrieved from the files of the Department of Pathology, Mackay Memorial Hospital, Taipei, over the period between August 30, 1996, and June 1, 2004. Clinicopathologic information was obtained from the medical records and pathology reports.

Analysis of EGFR Mutations

All specimens used for EGFR mutations analysis were surgically resected tumors, and only lung cancer cells were used for analysis. DNA was isolated from formalin-fixed, paraffin-embedded tumors according to previously described procedures. (9) In brief, representative paraffin blocks were cut in 8-[micro]m sections using a clean disposable microtome blade for each block. To ensure representative sampling, excess tissue was trimmed before sectioning. The first and the last sections from each ribbon were subjected to light microscopic examination after routine hematoxylin-eosin staining. Because the interface between the cancer cells and the normal tissues was generously trimmed, both the first and the last sections indeed showed that the selected specimens contained nearly uniform cell populations. However, the possibility of trivial contamination could not be totally excluded.

The paraffin sections were transferred directly into polymerase chain reaction (PCR) tubes and incubated in 300 [micro]L xylene at 25[degrees]C for 5 min, pelleted at 12,000 g for 5 min, resuspended in 300 [micro]L absolute alcohol at room temperature, spun down, and lyophilized. The pellets were then processed using the Puregene DNA isolation kit (Gentra; Minneapolis, MN) according to the instructions of the manufacturer, which included proteinase K (300 [micro]g/mL) digestion overnight at 55[degrees]C. The final extracts were dissolved in Tris-ethylenediaminetetra-acetic acid buffer and kept at 4[degrees]C for later use.

A total of six pairs of oligonucleotide primers were used to amplify exons 18, 19, and 21 of EGFR by nested PCR according to previously described procedures. (10) The PCR products were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and ABI Prism 377 Genetic Analyzer (PE Applied Biosystems; Foster City, CA). Experiments involving recombinant DNA were performed in accordance with National Institutes of Health Guidelines for Research Involving Recombinant DNA molecules.

Statistical Analysis

We looked for associations between EGFR mutations and various patient characteristics, including sex, smoking history, and pathologic subtypes. For statistical analysis, the [chi square] test was conducted to analyze associations between mutations and the different variables. Odds ratios (ORs) were estimated using logistic regression with EGFR mutations as the dependent variable. Statistical analysis was carried out using a commercially available computer program (SPSS version 10.0; SPSS; Chicago, IL). Significance was defined at p < 0.05; all analyses were two sided.

RESULTS

Patients

The patients included 17 men and 18 women (median age, 64 years; range, 42 to 83 years) [Table 1]. Thirteen men and 1 woman were current smokers, and all other patients were never-smokers. Histologically, there were 22 adenocarcinomas (14 pure adenocarcinomas, and 8 adenocarcinomas with a bronchioloalveolar component) and 13 bronchioloalveolar carcinomas (BACs).

EGFR Mutations

Heterozygous EGFR mutations were detected in 17 of the 35 patients (48%) [Table 2]. Three tumors had in-frame deletions within exon 19, resulting in the loss of codons 745 through 747 (delK745-L747) in one tumor, and loss of codons 746 through 750 (delE746-A750) in two tumors. Nine tumors had missense mutations with amino acid substitutions within exon 21: leucine to arginine at codon 858 (L858R). Another two tumors were found to have double EGFR mutations involving exon 18. One tumor had two missense mutations within exon 18: glutamic acid to lysine at codon 709 (E709K) and glycine to alanine at codon 719 (G719A). The other tumor had one missense mutation in exon 18 leading to the substitution of methionine for threonine at codon 725 (T725M) and another in exon 21, leucine to arginine substitution at codon 858 (L858R).

Clinicopathologic Correlation With EGFR Mutations

EGFR mutations were more frequent in women (13 of 18 patients [72%]) than in men (4 of 17 patients [23%]) [p = 0.004], more frequent in never-smokers (14 of 21 patients [66%]) than in current smokers (3 of 14 patients [21%]) [p = 0.009], and when any degree of BAC was present (14 of 21 patients [66%]), compared with pure adenocarcinoma (3 of 14 patients [21%]) [p = 0.009]. When adenocarcinoma with a bronchioloalveolar component was included in the adenocarcinoma group, no statistically significant difference between the two pathologic subtypes was found (p = 0.631). Logistic regression analysis demonstrated that female gender (OR, 10.913; 95% confidence interval [CI], 1.778 to 66.97; p = 0.01) and BAC, including adenocarcinomas with any bronchioloalveolar features (OR, 9.708; 95% CI, 1.464 to 64.393; p = 0.019), were significantly associated with EGFR mutations.

DISCUSSION

In the present study of gefitinib-naive Taiwanese patients with lung adenocarcinoma, the frequency of EGFR mutations was 48%. This is comparable to the prevalence in Japanese patients with lung adenocarcinoma (32%), but much higher that for US patients (range, 3 to 9%). (7,8) Our findings provide further evidence that EGFR mutations, and presumably the molecular pathogenesis of NSCLC, vary by ethnicity and geography. Investigation of EGFR mutations studies in Japanese or Taiwanese immigrants to Western countries may shed further light on this issue.

It is worth noting that adenocarcinoma is the most frequent histologic type in women, and BAG is also more common in women. (11) In addition, adenocarcinoma and BAC are more common among nonsmokers of either sex. These baseline demographic factors, including female gender, adenocarcinoma histology that includes the BAC subtype, and nonsmoking, have been found to be significant prognostic factors for response to gefitinib. (12,13) Moreover, these demographic characteristics have also been associated with EGFR mutations in previous reports. (7,8) In the present study, female gender, never-smokers, and adenocarcinoma with any bronchioloalveolar component correlated significantly with EGFR mutations. This is consistent with previous Studies (7,8) showing that EGFR mutations are more common in women and adenocarcinoma. Overall, our findings suggest that adenocarcinoma with any bronchioloalveolar component in a female never-smoker may be a unique pathologic subtype and may perhaps have a distinctive pathogenesis related to mutations of EGFR.

BAC is uncommon and accounts for < 3% of all invasive lung malignancies. (14) In the 1999 World Health Organization/International Association for the Study of Lung Cancer classification of lung tumors, BAC is defined as an adenocarcinoma of the lung that grows in a lepidic fashion along the alveolar septa without invasive growth. (15) BAC is distinguished from pure adenocarcinoma by four major histologic features that must be present in at least 75% of the tumor. (16) However, in contrast to the World Health Organization/International Association for the Study of Lung Cancer classification, Lynch et al (7) regarded an adenocarcinoma with any element of BAC as BAC; most of their EGFR mutations occurred in BAC. However, EGFR mutations were detected in only one case of BAC not treated with gefitinib in the series of Paez et al. (8) In our study, the frequency of EGFR mutations was significantly higher when any element of BAG was present but not when we adhered to the strict definition of BAC. Since the BAC pathologic subtype, regardless of how much there was, correlated significantly with EGFR mutations, it appears that any BAC features in NSCLC should be carefully identified, since it may influence treatment, given the greater responsiveness of this type of tumor to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib.

We identified two classes of somatic EGFR mutations in lung adenocarcinomas that are consistent with the findings of two previous studies. (7,8) The first class included missense mutations with amino acid substitutions in exon 18 (the p-loop of the kinase domain) or in exon 21 (the activation loop of the kinase domain). The second class involved in-frame deletions within exon 19 that change the structure and spatial orientation of the catalytically important [alpha]C helix of the kinase domain. In-frame deletions within exon 19 are the most frequent mutations (60 to 68%) found by the two previous studies, (7,8) and were found to affect different amino acid residues in the Japanese and US patients. In contrast to their findings, missense mutations in exon 21 (13 of 17 patients [76%]) were the most frequent mutations detected in our study in Taiwanese patients. Whether these differences in EGFR mutational type are related to genetic background or environmental factors is currently not clear. The relationship between specific EGFR mutations and clinical response to an EGFR-TKI is also unknown. Precedents found with imatinib suggest that lung cancers with different EGFR mutations may respond differently to EGFR-TKI. (17) Further studies directed at clinical outcome are needed to clarify this issue.

Interestingly, we detected two heterozygous double EGFR mutations in two of our patients. One patient had stage IV adenocarcinoma with mixed histologic subtypes, and the other patient had stage IB BAC. Double mutations in a single gene, especially those related to human cancer, are rare (18) and have never been reported in EGFR. A second new mutation in c-KIT in a GI stromal tumor resulting in acquired resistance to imatinib was reported. (19) However, the clinical significance of these two de novo double EGFR missense mutations we found is unknown, since these two patients had a very diverse clinical course. As neither had ever been treated with gefitinib, the mutations could not have been related to that particular treatment. The double EGFR mutations were entirely within the tyrosine kinase domain in both patients.

Our study focused on surgically resected tumor specimens rather than biopsy specimens. Therefore, sufficient cancer cells were investigated in our study. It is possible that the findings derived from surgically resected specimens may not be able to represent the whole clinical spectrum of NSCLC. However, the results of our study are still comparable to and corroborative of previous reports. Another limitation of our study is that only three exons of EGFR were studied. The frequency of EGFR mutations may increase if more exons were sequenced, and this is supported by the findings of two de novo double EGFR mutations in our series.

CONCLUSIONS

In conclusion, female sex and bronchioloalveolar pathologic subtype predicted EGFR mutations in our series. The high frequency of EGFR mutations in lung adenocarcinomas observed in this study, particularly involving exon 21, suggests that genetic background or environmental factors or both may be involved in the pathogenesis of lung cancer. This may also have implications for the treatment response to EGFR-TKI in different ethnic groups. Investigation of Japanese or Taiwanese immigrant populations to the West may help clarify the role of environmental factors.

Table 1--Patient Characteristics and Frequency of
EGFR Mutations

                                      Patients With Mutations,
        Variables             No.             No. (%)

Age, yr
  Median                      64
  Range                      42-83
Sex
  Male                        17              4 (23)
  Female                      18             13 (72)
Smoking history
  Current smoker              14              3 (21)
  Never-smoker                21             14 (66)
Pathology
  Adenocarcinoma              14              3 (21)
  Adenocarcinoma with BAC      8              7 (87)
    component
  BAC                         13              7 (53)
Stage
  I                           15              7 (46)
  II                           3              2 (66)
  IIIA                         6              4 (66)
  IIIB                         2              1 (50)
  IV                           9              3 (33)
Mutations                                    17 (48)
  Exon 18                                     2 (11)
  Exon 19                                     3 (17)
  Exon 21                                    13 * (76)

* Including double mutations involved exon 21.

Table 2--EGFR Mutations in 17 Patients With Non-Small Cell Lung
Cancer *

Patient No.   Sex      Age     Pathology      Smoking        Exon

 1            Female   72    Adeno              No      Exon 19
 2            Female   70    Adeno plus BAC     No      Exon 19
 3            Female   52    Adeno plus BAC     No      Exon 19
 4            Female   55    Adeno plus BAC     No      Exon 21
 5            Male     74    Adeno plus BAC     Yes     Exon 21
 6            Female   64    Adeno plus BAC     No      Exon 21
 7            Female   79    Adeno              No      Exon 21
 8            Female   71    Adeno plus BAC     No      Exon 21
 9            Female   65    Adeno              No      Exon 21
10            Female   68    BAC                No      Exon 21
11            Female   62    BAC                No      Exon 21
12            Female   77    BAC                No      Exon 21
13            Male     62    BAC                Yes     Exon 21
14            Male     72    BAC                No      Exon 21
15            Female   68    BAC                No      Exon 21
16            Male     42    Adeno plus BAC     Yes     Double exon 18
17            Female   57    BAC                No      Exons 18 and 21

Patient No.   Effect of Mutation

 1               delK745-L747
 2               delE746-A750
 3               delE746-A750
 4                   L858R
 5                   L858R
 6                   L858R
 7                   L858R
 8                   L858R
 9                   L858R
10                   L858R
11                   L858R
12                   L858R
13                   L858R
14                   L858R
15                   L858R
16               E709K, G719A
17               T725M, L858R

* Adeno = adenocarcinoma.

ACKNOWLEDGMENT: We thank Dr. Mary Jeanne Buttrey, Consulting Physician, Department of Internal Medicine, Mackay Memorial Hospital, for review and revision of the article.

* From the Department of Internal Medicine (Drs. Hsieh, Lim, and Huang), Division of Hematology and Oncology (Dr. Kuo), Division of Chest Medicine, and the Department of Pathology (Dr. Tzen), Mackay Memorial Hospital, Taipei, Taiwan.

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This study was supported in part by grants from AstraZeneca. Manuscript received October 14, 2004; revision accepted December 3, 2004.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml).

Correspondence to: Ming-Jer Huang, MD, Division of Hematology and Oncology, Department of Internal Medicine, Mackay Memorial Hospital 156, Min-Sheng West Rd, Taipei 104, Taiwan; e-mail: huanghhh@ms23.hinet.net

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