Journal of Cancer Research and Clinical Oncology

, 135:313

EGFR R497K polymorphism is a favorable prognostic factor for advanced lung cancer

Authors

    • Department of Surgery IINagoya City University Medical School
  • Katsuhiro Okuda
    • Department of Surgery IINagoya City University Medical School
  • Shigeki Shimizu
    • Department of Pathology IINagoya City University Medical School
  • Minoru Takada
    • Department of Internal MedicineNational Hospital Organization, Kinki-chuo Chest Medical Center
  • Masaaki Kawahara
    • Department of Internal MedicineNational Hospital Organization, Kinki-chuo Chest Medical Center
  • Naoto Kitahara
    • Department of SurgeryNational Hospital Organization, Kinki-chuo Chest Medical Center
  • Meinoshin Okumura
    • Department of SurgeryNational Hospital Organization, Kinki-chuo Chest Medical Center
  • Akihide Matsumura
    • Department of SurgeryNational Hospital Organization, Kinki-chuo Chest Medical Center
  • Keiji Iuchi
    • Department of SurgeryNational Hospital Organization, Kinki-chuo Chest Medical Center
  • Tomoya Kawaguchi
    • Department of Internal MedicineNational Hospital Organization, Kinki-chuo Chest Medical Center
  • Akihito Kubo
    • Department of Internal MedicineNational Hospital Organization, Kinki-chuo Chest Medical Center
  • Osamu Kawano
    • Department of Surgery IINagoya City University Medical School
  • Haruhiro Yukiue
    • Department of Surgery IINagoya City University Medical School
  • Motoki Yano
    • Department of Surgery IINagoya City University Medical School
  • Yoshitaka Fujii
    • Department of Surgery IINagoya City University Medical School
Original Paper

DOI: 10.1007/s00432-008-0464-5

Cite this article as:
Sasaki, H., Okuda, K., Shimizu, S. et al. J Cancer Res Clin Oncol (2009) 135: 313. doi:10.1007/s00432-008-0464-5

Abstract

Introduction

It has been reported that the R497K polymorphism of the epidermal growth factor receptor (EGFR) gene has attenuated functions in ligand binding, tyrosine kinase activation, and growth stimulation. On other hand, EGFR gene mutations at kinase domain in non-small cell lung cancer (NSCLC) have been examined for their ability to predict sensitivity to gefitinib or erlotinib.

Materials and methods

We investigated the EGFR mutations and/or R497K polymorphism statuses in 225 surgically treated NSCLC cases. 192 adenocarcinoma cases were included. The presence or absence of EGFR polymorphism of exon 13 was analyzed by PCR–RFLP method.

Results

EGFR mutations at kinase domain were found from 95 of 225 lung cancer patients. In 86.2% of patients, homo- or heterozygous Lys497 allele was present. No correlation existed between R497K EGFR genotype and clinico-pathological features, such as gender, smoking status, and pathological subtypes.

Conclusions

EGFR mutation status was not correlated with R497KEGFR genotype of lung cancers. In node-negative patients, R497KEGFR genotype was not correlated with disease outcome. In node-positive patients, however, R497K EGFR was significantly associated with better overall survival. This association was attributable to neo-adjuvant or adjuvant chemotherapy. In 46 total gefitinib treated NSCLC patients, the prognosis was not different between the EGFR wild type (GG) patients and AG+AA patients. R497KEGFR polymorphism might be associated with favorable prognosis of advanced lung cancers and correlated with chemosensitivity.

Keywords

EGFRLung cancerPolymorphismR497K

Introduction

Lung cancer is a major cause of death from malignant diseases, due to its high incidence, malignant behavior, and lack of major advancements in treatment strategy (Ginsberg et al. 1993). There are much accumulated evidences that epidermal growth factor receptor (EGFR) and its family member are strongly implicated in the development and progression of numerous human tumors, including lung cancer (Nicolson et al. 2001; Onn et al. 2004). The EGFR tyrosine kinase inhibitor, gefitinib, was approved in Japan for the treatment of non-small cell lung cancer (NSCLC) since 2002. In 2004, two reports have shown that EGFR mutation statuses at tyrosine kinase (TK) domain in NSCLC patients were correlated with the clinico-pathological features related to good response to gefitinib (Paez et al. 2004; Lynch et al. 2004). EGFR mutations in lung cancer have been correlated with clinical response to gefitinib therapy in vivo and in vitro (Paez et al. 2004; Lynch et al. 2004; Pao et al. 2004). Genomic profiling of the EGFR signaling is also helpful in identifying lung cancer patients who are at risk of tumor recurrence and those who are more likely to benefit from chemoradiation therapy. For example, the NSCLC patients with more than 35 (CA)n repeats in EGFR intron 1 polymorphism had a significantly longer overall survival than the patients with the 35 or fewer (CA)n alleles, who received radiation (RT; 50.4 Gy) or RT concurrent with chemotherapy (CT; four cycles of cisplatin plus etoposide) (Dubey et al. 2006; Keller et al. 2000). EGFR intron 1 and −216G/T polymorphisms influenced clinical outcomes in gefitinib-treated NSCLC patients (Liu et al. 2008). A polymorphic variant EGFR arising from a single nucleotide change (G–A) leading to an arginine (Arg) to lysine (Lys) substitution in codon 497 (R497K) in the extracellular domain of EGFR has been identified (Moriai et al. 1994). This polymorphism alone or in combination with another polymorphism in the same gene is associated with a lower recurrence of tumor in rectal cancer patients treated with chemoradiation (Zhang et al. 2005). To determine this EGFR polymorphism status and correlation with clinico-pathological features in Japanese lung carcinoma, we investigated EGFR gene status by PCR–RELP method and direct sequencings. The findings were compared to the clinico-pathologic features of lung cancer.

Materials and methods

Patients and samples

The study group included 206 lung cancer patients who had undergone surgery at the Department of Surgery II, Nagoya City University Medical School between 1997 and 2005. Fifty eight patients were treated with platinum-based neo-adjuvant or adjuvant chemotherapy. Twenty seven patients were treated with gefitinib for their recurrence of lung cancer after they had undergone surgery. We have also investigated EGFR R497K status for 19 NSCLC patients who had treated with gefitinib for their recurrence of lung cancer after undergone surgery at the National Hospital Organization, Kinki-chuo Chest Medical Center. The lung tumors were classified according to the general rule for clinical and pathological record of lung cancer in Japan, as well as WHO classification. All tumor samples were immediately frozen and stored at −80°C until assayed.

The clinical and pathological characteristics of the 225 lung cancer patients were as follows; 132 (58.6%) were male and 93 were female. One hundred and ninety two were diagnosed as adenocarcinoma, and 33 were diagnosed as other types of carcinoma (20 squamous cell carcinomas, eight adenosquamous carcinomas and five large cell carcinomas). One hundred and twenty five (55.6%) were smoker (current smoker or ever smoker) and 100 were non-smoker. Written informed consent was obtained from the patients, and the institutional ethics committee of the Nagoya City University approved the study.

PCR assays for EGFR polymorphism

Genomic DNA was extracted using Wizard SV Genomic DNA purification Systems (Promega) according to the manufacturers’ instructions. EGFR mutation statuses at kinase domain were investigated using TaqMan PCR assay (Applied Biosystems). The sequences of 13 allele-specific TaqMan MGB probes and primer sets used in the TaqMan PCR assay were already shown (Endo et al. 2005). The results of TaqMan PCR assays were already reported. The R497K EGFR (G–A) polymorphism was examined by PCR–RELP method as described previously (Zhang et al. 2005; Wang et al. 2007). Briefly, the PCR reactions were performed using LA-Taq kit (Takara Bio Inc, Shiga, Japan) in a 50 μl reaction volume. The primer sequences for EGFR gene at exon 13 were as follows: the forward primer, 5′-TGCTGTGACCCACTCTGTCT-3′ and the reverse primer, 5′-CCAGAAGGTTGCACTTGTCC-3′. The cycling conditions were as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles at 94°C for 60 s, 59°C for 60 s, 72°C for 60 s. The products were purified by Qiagen PCR purification kit (Qiagen, Valencia, CA), and then digested by BstN1 restriction enzyme (New England Biolabs) at 60°C for 16 h. These samples were separated on 4% ethidium bromide-stained agarose gels. In some cases, direct sequencing were performed and analyzed by BLAST and chromatograms by manual review.

Statistical analysis

Statistical analyses were done using the Mann–Whitney U test for unpaired samples and Wilcoxon’s singed rank test for paired samples. Linear relationships between variables were determined by means of simple linear regression. Correlation coefficients were determined by rank correlation using Spearman’s test and χ2 test. The overall survival of lung cancer patients was examined by the Kaplan–Meier methods, and differences were examined by the Log-rank test. All analysis was done using the Stat-View software package (Abacus Concepts Inc. Berkeley, CA), and was considered significant when the p value was less than 0.05.

Results

EGFR gene mutation status

Of 225 patients, in exon 19, 51 patients had the deletion type mutation. In exon 18 or exon 21, 39 patients had the missense point mutations (1 G719S, 3 G719C, 34 L858R and 1 L861Q). Five patients had exon 20 insertion mutations (Sasaki et al. 2007). Of these 95 patients, 34 were male and 61 were female. Sixty seven were non-smokers and 28 were smokers. Ninety two patients had adenocarcinoma and three had adenosquamous cell carcinoma. Thus EGFR mutation statuses at exon 18–21 were significantly correlated with gender (p < 0.0001), tobacco-smoking (p < 0.0001), and pathological subtypes (adenocarcinoma vs. non-adenocarcinoma, p < 0.0001). Of 206 patients from Nagoya City University, 97 (51.5%) were stage I. There was a higher EGFR mutation in stage I (51/97, 28.4%) than in stage II–IV (33/89, 19.7%, p = 0.0235).

EGFR polymorphism at exon 13

Using the PCR–RFLP assay, a sequence difference in exon 13 (R497K) was found in tumors that defined in the EGFR gene. Example of the EGFR gene analyzed by PCR–RELP method was shown in Fig. 1. Same codon 497 polymorphism of EGFR was found in both DNAs isolated from several lung cancer samples and adjacent peripheral blood samples. Several samples were also confirmed by direct sequencing (Fig. 2). Of 225 patients, 194 patients had the EGFR polymorphism (80 AA and 114 GA), 117 were male and 77 were female, 110 were non-smokers and 84 were smoker, and 166 patients had adenocarcinoma and 28 had other types of lung cancers. The R497K polymorphism did not correlate with gender (p = 0.2410), smoking status (p = 0.4387), pathological subtypes (p = 0.7865), and EGFR-TK mutation status of lung cancer (p = 0.5566) (Table 1). Major components of adenocarcinomas with R497K were as follows; acinar 58.3%, solid 25.0%, and papillary 12.5%. Major components of adenocarcinomas with wild type (Lys/Lys) were as follows; acinar 40.0%, papillary 40.0%, and solid 20.0%. Thus polymorphism status did not correlated with the major components of adenocarcinomas. No significant association between R497K EGFR genotype and patient outcome was seen for the 206 patients from Nagoya City University (p = 0.1121). Pathological stages (p < 0.0001) but not gender (p = 0.0696) was a prognostic factor. In node-negative patients, 119 (28 were dead) were R497 KEGFR and 14 (three were dead) were wild type EGFR. Thus EGFR genotype was not correlated with disease outcome (Log-rank test p = 0.8882) (Fig. 3). In node-positive patients, however, 59 (33 were dead) were R497K EGFR and 14 (12 were dead) were wild type. Thus R497K EGFR was significantly associated with better overall survival (Log-rank test, p = 0.0072) (Fig. 4). In this cohort, pathological stage (stage II, n = 17 vs. stage III–IV, n = 56, p = 0.2932) or gender (male, n = 41 vs. female, n = 32, p = 0.7957) was not a prognostic factor. Multi-variate analysis showed that R497K status was a prognostic factor (p = 0.0104, relative risk 2.4, 1.229–4.689). We also compared associations between EGFR polymorphism status and patient outcome who were treated with platinum-based adjuvant or neo-adjuvant chemotherapy who had undergone surgery. The overall survival of 58 lung cancer patients with follow-up through March 1, 2008 was studied in reference to the EGFR polymorphism status. Ten were wild type (eight were dead) and 48 were R497K (23 were dead). The prognosis was significantly worse in EGFR wild type than in EGFR R497K polymorphism (p = 0.0038) (Fig. 5). In this cohort, pathological stages (stage I, n = 11, stage II, n = 14, stage III–IV, n = 33, p = 0.0445) but not gender (male, n = 42 vs. female, n = 16, p = 0.9103) was a prognostic factor. However, multi-variate analysis showed none of them was a prognostic factor.
https://static-content.springer.com/image/art%3A10.1007%2Fs00432-008-0464-5/MediaObjects/432_2008_464_Fig1_HTML.gif
Fig. 1

Representative PCR–RELP patterns of different EGFR codon 497 status. PCR products after being digested by BstN1 were separated by agarose gel electrophoresis

https://static-content.springer.com/image/art%3A10.1007%2Fs00432-008-0464-5/MediaObjects/432_2008_464_Fig2_HTML.gif
Fig. 2

The sequence results of EGFR exon 13. Left upper wild type (GG). Right upper heterozygous change (GA). Left lower homozygous change (AA)

Table 1

Clinico-pathological data of 225 lung cancer patients

Factors

EGFR

p-value

GG

GA+AA

Patients

Patients

Mean age (years)

63.2 ± 10.3

63.4 ± 10.0

62.0 ± 12.0

0.6685

Gender

 Male

 

15 (48.4%)

117 (60.3%)

0.2410

 Female

 

16 (51.6%)

77 (49.7%)

Smoking

 Non-smoker

 

16 (51.6%)

84 (43.3%)

0.4387

 Smoker

 

15 (48.4%)

110 (56.8%)

Pathological subtype

 Adeno

 

26 (83.9%)

166 (85.6%)

0.7865

 Others

 

5 (16.1%)

28 (14.4%)

EGFR mutation

 Positive

 

14 (45.2%)

81 (41.8%)

0.5566

 Negative

 

17 (54.8%)

113 (58.2%)

Age

 ≤60

 

12 (38.7%)

72 (39.1%)

>0.9999

 >60

 

19 (61.3%)

112 (60.8%)

Pathological stages

 I

 

10 (35.7%)

96 (53.9%)

0.1073

 II

 

4 (14.3%)

29 (16.3%)

 III–IV

 

14 (50.0%)

53 (29.8%)

Lymph node metastasis

 

 Negative

 

14 (50.0%)

118 (66.3%)

0.1366

 Positive

 

14 (50.0%)

60 (33.7%)

*EGFR epidermal growth factor receptor, Smoker current smoker or ever smoker, Adeno adenocarcinoma

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Fig. 3

The overall survival of node-negative lung cancer patients was studied in reference to the EGFR (R497K) status. There was no difference of survival between the patient with EGFR wild type (GG) (n = 14, 3 were dead) and the patient with R497K EGFR (GA or AA) (n = 119, 28 were dead) (Log-rank test, p = 0.8882)

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Fig. 4

The overall survival of node-positive lung cancer patients was studied in reference to the EGFR (R497K) status. The patients with EGFR wild type (GG) (n = 14, 12 were dead, median follow up = 21.7 months) had significantly worse prognosis than the patients with R497K EGFR (GA or AA) (n = 59, 33 were dead, median follow up = 42.7 months) (Log-rank test, p = 0.0072) (relative risk 2.4, 1.229–4.689)

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Fig. 5

The overall survival of adjuvant or neo-adjuvant chemo-untreated lung cancer patients was studied in reference to the EGFR (R497K) status. The patients with EGFR wild type (GG) (n = 10, 8 were dead, median follow up = 23.7 months) had significantly worse prognosis than the patients with R497K EGFR (GA or AA) (n = 48, 23 were dead, median follow up = 55.1 months) (Log-rank test, p = 0.0038)

Relationship between clinical courses of lung cancer patients treated with gefitinib and EGFR

The overall survival of gefitinib treated lung cancer patients from Nagoya City University, with follow-up through March 1, 2008, was studied in reference to the EGFR polymorphism status. Of 206 patients from Nagoya City University, 27 were treated with gefitinib therapy. Total 46 gefitinib treated patients were investigated the R497K polymorphism statuses. In this analysis, 38 patients had EGFR polymorphism (AG or GG). The prognosis after gefitinib therapy was not significantly different between EGFR wild type patients (GG, 5/8 were dead) and EGFR polymorphism patients (AG+GG; 28/38 were dead) (p = 0.3100) (Fig. 6).
https://static-content.springer.com/image/art%3A10.1007%2Fs00432-008-0464-5/MediaObjects/432_2008_464_Fig6_HTML.gif
Fig. 6

The overall survival of 46 gefitinib untreated lung cancer patients was studied in reference to the EGFR (R497K) status. There was no difference of survival between the patients with EGFR wild type (GG) (n = 8, 5 were dead) and the patients with R497K EGFR (GA or AA) (n = 38, 28 were dead) (Log-rank test, p = 0.3100)

Discussion

In the present study, we showed that the R497 polymorphism of EGFR in node-positive lung cancer patients who received curative surgery might account for a longer overall survival. Moreover, this polymorphism was shown to correlate with a better prognosis after platinum-based adjuvant treatment. Although the underlying mechanisms remain unclear, an attenuated ligand interaction and consequential signal transduction might be the main reason for the subopitimal function of this receptor variant (Moriai et al. 1994).

The quantification of certain intratumoral molecules involved in the targeting or metabolism of specific chemotherapeutic agents may be valuable in predicting their efficacies or toxicities in cancer patients. For example, patients with a higher intratumoral level of excision repair cross complementation group 1 (ERCC1), an enzyme involved in nucleotide excision repair, may have a higher resistance to cisplatin-based adjuvant therapy in NSCLC (Olaussen et al. 2006). Moreover, NSCLC patients with a higher class III beta tubulin may have a higher resistance to taxane chemotherapy (Dumontet et al. 2005).

In this report, the R497K EGFR SNP(exon 13) is not associated with somatic EGFR-TK mutation. Approximately 563 EGFR-SNPs have been identified in human genome according to the National Cancer for Biotechnology information database. However, there are few studies examining associations between EGFR SNPs and human disease (Shintani et al. 1999; Kang et al. 2005; Fukushima et al. 2006; Zhang et al. 2006; Wang et al. 2007; Liu et al. 2008). In this study, we detected a polymorphism in exon 13 of the EGFR-extracellular domain, which changed amino acid Arg (R) to Lys (K), and the K allele seems to decrease the activity of EGFR (Moriai et al. 1994). Previous reports suggested that EGFR R497K polymorphism was weakly associated with gefitinib response (Liu et al. 2007). However, in our Japanese cohort, EGFR R497K was not associated with response to gefitinib. Although the survival curve of R497K showed higher than EGFR wild type (G/G) in our data, the larger number would help to determine the correlation between the R497K polymorphism and gefitinib sensitivity.

Previous report showed that patients with 497 Arg/Arg genotype tended to have a higher risk of local recurrence in chemo-treated rectal cancer patients (Zhang et al. 2005; Brandt et al. 2006). The patients with Arg/Arg genotype showed the highest risk of disease-specificity mortality and none of the patients with the Lys/Lys genotype died throughout the follow-up period of head and neck cancer treated with chemoradiation (Bandres et al. 2007). The mechanism through which the variant human EGFR R497K may account for lower local failures after chemotherapy is unknown (Zhang et al. 2005). A study with Chinese hamster ovary cells, the variant EGFR 497K cell line, showed an attenuated growth response to EGF and transforming growth factor-α, and a reduced induction of the proto-oncogenes fos, jun, and myc (Moriai et al. 1994). It was suggested that the amino acid substitution in the extracellular domain might modulate ligand binding and transmembrane signaling to the intracellular domain (Zhang et al. 2005). Thus, variant EGFR receptor may be less efficient in the recruitment of intracellular substrates and/or cause downstream activation of alternative signaling pathways with decreased proto-oncogene induction or growth stimulation, affecting chemosensitivity. Shintani et al. (1999) demonstrated that another EGFR-SNP at position 2073 was correlated with truncated EGFR transcription, which might interfere with EGFR three-dimensional structure and EGFR expression.

In summary, R497 polymorphism of EGFR in node-positive lung cancer patients had a better overall survival. R497KEGFR polymorphism might be associated with favorable prognosis of advanced lung cancers.

Acknowledgments

The authors would like to thank Mrs. Emi Sugiyama for his excellent technical assistances. This work was supported by AstraZeneca Research Grant 2004, Grand-in-Aid for Research in Nagoya City University (2006), and Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science (JSPS) (Nos, 19390367, 18390381,18659407).

Conflict of interest statement

None declared.

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© Springer-Verlag 2008