Association between epidermal growth factor receptor mutations and the expression of excision repair cross-complementing protein 1 and ribonucleotide reductase subunit M1 mRNA in patients with non-small cell lung cancer

  • Authors:
    • Chun‑Wei Xu
    • Gang Wang
    • Wu‑Long Wang
    • Wen‑Bin Gao
    • Chuan‑Jun Han
    • Jing‑Shan  Gao
    • Yang Li
    • Lin Wang
    • Li‑Ying Zhang
    • Yu‑Ping Zhang
    • Yu‑Wang Tian
    • Jin‑Nv Fang
  • View Affiliations

  • Published online on: January 21, 2015     https://doi.org/10.3892/etm.2015.2196
  • Pages: 880-884
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

The present study aimed to investigate the association between epidermal growth factor receptor (EGFR) gene mutations and excision repair cross‑complementing protein 1 (ERCC1) and ribonucleotide reductase subunit M1 (RRM1) mRNA expression in non‑small cell lung cancer (NSCLC) tissue. The quantitative polymerase chain reaction was used to detect EGFR mutations, and ERCC1 and RRM1 mRNA expression in 257 cases of NSCLC. In the NSCLC samples the EGFR mutation rate was 49.03% (126/257). The rate was higher in females and non‑smoking patients (P<0.05). High expression of ERCC1 mRNA was observed in 47.47% of the samples (122/257), while a high RRM1 mRNA expression was observed in 61.87% of the samples (159/257). In comparison with patients with NSCLC without EGFR mutations, patients with EGFR mutations had significantly lower levels of ERCC1 mRNA expression (P<0.05); however, EGFR mutations and expression levels of RRM1 mRNA were not correlated in NSCLC tissues (P>0.05). In addition, ERCC1 mRNA expression was not correlated with the expression levels of RRM1 mRNA (P>0.05). In conclusion, patients with NSCLC with EGFR mutations tend to have a low expression of ERCC1 mRNA and may potentially benefit from platinum‑based chemotherapy.

Introduction

Globally, lung cancer has the highest rates of morbidity and mortality of all malignancies (1). Non-small cell lung cancer (NSCLC) accounts for 80–85% of lung cancer cases worldwide (2).

Human epidermal growth factor receptor (EGFR) belongs to the type I receptor family. This family has four cognate family members, including EGFR (HER1), HER2, HER3 and HER4, which mediate the following signal transduction pathways: Ras-Raf-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase-mitogen-activated protein kinase, phospholipase C-γ, phosphatidylinositol-3-kinase/phosphoinositide-dependent kinase 1 and Janus kinase/signal transducers and activators of transcription (3). These receptors regulate cell proliferation, differentiation and apoptosis (4). EGFR mutations in patients with NSCLC occur in the intracellular tyrosine kinase (TK) region above the first four exons (18 to 21). A total of 30 different types of mutations have been identified in the TK region (5), the most common occurring in exons 19 and 21, which account for ~85% of all the mutations (6).

Excision repair cross-complementing gene 1 (ERCC1) is the key gene in two DNA repair pathways: nucleotide excision repair (NER) and chain crosslink repair (7). Overexpression of ERCC1 can rapidly repair damaged DNA arrest at the G2/M phase and cause cells to be resistant to platinum (8). Ribonucleotide reductase subunit M1 (RRM1) is involved in DNA synthesis and repair (9). Results from the Iressa® Pan-Asia Study (10) clinical trial indicated that, in Asian populations, patients with EGFR mutations were more responsive to chemotherapy than patients with wild-type EGFR. Patients with lung cancer with low expression of ERCC1 and RRM1 are more responsive to gemcitabine- and platinum-based chemotherapy, respectively (11,12). Patients with NSCLC are generally treated with chemotherapy drugs, including cisplatin and gemcitabine. In the present study, 257 patients with stages I-IV NSCLC from multiple hospitals were analyzed for the presence of EGFR mutations and expression levels of ERCC1 and RRM1 mRNA. The data were statistically analyzed to determine significant correlations between EGFR mutations and expression of these two chemotherapy resistance genes in patients with NSCLC. These data may prove useful in further identifying more effective individualized treatment plans for patients with EGFR mutations, particularly for patients with small-molecule EGFR-tyrosine kinase inhibitor (EGFR-TKI) primary or secondary resistance.

Materials and methods

Specimens

For the detection of EGFR mutations, as well as ERCC1 and RRM1 mRNA expression levels, paraffin tissue specimens were collected from 257 patients from the General Military Hospital of Beijing PLA (Beijing, China; 103 cases), the Affiliated Zhongshan Hospital of Dalian University (Dalian, China; 58 cases) and the People’s Hospital of Weifang (Weifang, China; 96 cases). The patients had undergone surgery between 2004 and 2013; the pathological diagnosis was adenocarcinoma, and patients had not received preoperative chemotherapy, radiotherapy or biological immunotherapy. All protocols in the present study were approved by the Human Clinical and Research Ethics Committees of the General Military Hospital of Beijing PLA, the Affiliated Zhongshan Hospital of Dalian University (Dalian, China) and the People’s Hospital of Weifang (Weifang, China). Written informed consent was obtained from all of the patients.

Reagents and instruments

The DNA and RNA extraction kits were purchased from Qiagen (Hilden, Germany). The human EGFR mutation qualitative detection kit and the tumor-related gene expression relative quantification detection kit (ERCC1 and RRM1) were obtained from Amoy Diagnostics Co., Ltd. (Xiamen, China). The B-500 instrument to measure nucleic acid protein concentrations was purchased from Shanghai Chong Meng Biotechnology Co. Ltd. (Shanghai, China) and the ABI 7500 quantitative polymerase chain reaction (qPCR) instrument was purchased from Applied Biosystems® (Life Technologies, Foster City, CA, USA).

qPCR to detect EGFR mutations in NSCLC tissues

Between four and eight 4-μm-thick paraffin tissue sections were obtained and dewaxed. The genomic RNA extraction kit was used to extract RNA from the tissue samples according to the manufacturer’s instructions. A spectrophotometer was used to determine the purity and concentration of the extracted RNA, which was used as a template to synthesize the corresponding DNA. The human EGFR mutation qualitative detection kit, which contains 29 different fusion mutant primers and probes to amplify EGFR exons 18, 19, 20 and 21, was used (ADx-EG09; Amoy Diagnostics Co., Ltd.); the DNA was amplified in an ABI 7500 qPCR instrument (Fig. 1A and B). The qPCR cycling conditions were set as follows: 95°C for 5 min, follwed by 45 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec.

qPCR to detect the expression of ERCC1 and RRM1 mRNA in NSCLC tissue

RNA was extracted from 4-μm-thick paraffin tissue sections in accordance with the aforementioned methods. The tumor-associated gene expression detection kit for ERCC1 (ADx-ER01) and RRM1 (ADx-RR01) (Amoy Diagnostics Co., Ltd.) was used to determine the mRNA expression level of these genes using the absolute quantitative method; β-actin was used as the reference gene. ERCC1 had a standard mean of 4.29×10−3, and RRM1 had a standard mean of 11.37×10−3 (Fig. 1C).

Statistical analysis

The data were analyzed using the statistical software SPSS (version 19.0; IBM SPSS, Armonk, NY, USA) using the χ2 and Fisher’s exact test with a test level α=0.05. The P-value was set to bilateral distribution and P<0.05 was considered to indicate a statistically significant difference.

Results

Association between EGFR mutations, expression of ERCC1 and RRM1 mRNA, and patient clinical characteristics

Of the 257 cases of NSCLC, EGFR mutations were present in 126 cases (49.03%). The EGFR mutation rate was higher in non-smoking patients (92/158, 58.23%; P<0.001); however, the EGFR mutation rate was not associated with the age, tumor size, lymph-node metastasis or clinical stage of the patient. High expression of ERCC1 mRNA was observed in 122/257 cases (47.47%). ERCC1 mRNA expression levels were not associated with the gender, age, smoking status, tumor size, lymph-node metastasis or clinical stage of the patient. High expression of RRM1 mRNA was observed in 159/257 cases (61.87%). RRM1 mRNA expression levels were not associated with the gender, age, smoking status, tumor size, lymph-node metastasis or clinical stage of the patient (Table I).

Table I

Association between EGFR mutations, ERCC1 and RRM1 mRNA expression, and patient clinical characteristics.

Table I

Association between EGFR mutations, ERCC1 and RRM1 mRNA expression, and patient clinical characteristics.

EGFRERCC1RRM1



Clinical featuresMutant (n)Wild-type (n)P-valueHigh (n)Low (n)P-valueHigh (n)Low (n)P-value
Gender<0.0010.8980.088
 Male528665739246
 Female744557626752
Age, years0.2870.9430.714
 ≥59686262687951
 <59586960678047
Smoking history<0.0010.1990.129
 Yes346552476732
 No926670889266
Tumor diameter, cm0.2820.6870.292
 ≥5566760637251
 <5706462728747
Lymph node metastasis0.9030.4700.188
 Yes515447587035
 No757775778963
Clinical stage0.2030.3390.577
 I504240525933
 II+III+IV7689828310065

[i] EGFR, epidermal growth factor receptor; ERCC1, excision repair cross-complementing protein 1; RRM1, ribonucleotide reductase subunit M1.

Association between EGFR mutations and expression levels of ERCC1 mRNA

Of the 126 patients with NSCLC with an EGFR mutation, 79 (62.70%) showed low expression of ERCC1. Of the 131 patients with NSCLC with the wild-type EGFR gene, 56 (42.75%) had low expression of ERCC1 mRNA. These data indicate that patients with NSCLC with an EGFR mutation had significantly lower expression of ERCC1 mRNA (P<0.05) (Table II).

Table II

Association between EGFR mutations and the expression level of ERCC1 mRNA.

Table II

Association between EGFR mutations and the expression level of ERCC1 mRNA.

ERCC1 mRNA expression

EGFRHigh (n)Low (n)Total (n)
Mutant4779126
Wild-type7556131
Total (n)122135257

[i] EGFR, epidermal growth factor receptor; ERCC1, excision repair cross-complementing protein 1.

Association between EGFR mutations and the expression level of RRM1 mRNA

Of the 126 patients with NSCLC with an EGFR gene mutation, 51 (40.48%) had low expression of RRM1 mRNA, while 47 out of the 131 patients (35.88%) with the wild-type EGFR gene had low expression of RRM1 mRNA. These data indicate that there was no correlation between EGFR and RRM1 mutations in patients with NSCLC (P>0.05) (Table III).

Table III

Association between EGFR mutations and the expression level of RRM1 mRNA.

Table III

Association between EGFR mutations and the expression level of RRM1 mRNA.

RRM1 mRNA expression

EGFRHigh (n)Low (n)Total (n)
Mutant7551126
Wild-type8447131
Total (n)15998257

[i] EGFR, epidermal growth factor receptor; RRM1, ribonucleotide reductase subunit M1.

Association between the expression levels of ERCC1 and RRM1 mRNA

Of the 122 patients with NSCLC with high expression of ERCC1 mRNA, 72 cases (59.02%) had high expression of RRM1 mRNA. Of the 135 patients with NSCLC with low expression of ERCC1 mRNA, 48 cases (35.56%) had low expression of RRM1 mRNA. A total of 120 cases had low or high expression of both ERCC1 and RRM1 mRNA, and 137 cases had contrasting expression levels of ERCC1 and RRM1 mRNA. These data indicate that the expression of ERCC1 and RRM1 mRNA was not significantly correlated (χ2=0.800, P=0.371) in NSCLC tissue (Table IV).

Table IV

Association between the expression levels of ERCC1 and RRM1 mRNA.

Table IV

Association between the expression levels of ERCC1 and RRM1 mRNA.

RRM1 mRNA expression

ERCC1 mRNA expressionHigh (n)Low (n)Total (n)
High7250122
Low8748135
Total (n)15998257

[i] ERCC1, excision repair cross-complementing protein 1; RRM1, ribonucleotide reductase subunit M1.

Discussion

EGFR mutations and the responsiveness of NSCLC to the molecular targeted drugs gefitinib (trade name, Iressa) and erlotinib (trade name, Tarceva®) have a close association (13,14). Small-molecule TKIs have been shown to have a high efficiency in patients with an exon 19 deletion in the EGFR gene (15); however, patients with NSCLC with EGFR mutations in exon 20 are resistant to drug treatment with TKIs (16). Other studies have reported that the NER complexes prognosis is good but not suitable for receiving platinum-based chemotherapy (17,18).

The results of the present study showed that the EGFR mutation rate was 48.03% (126/257) in patients with stages I-IV NSCLC, which is consistent with previously reported data (14,19). A higher percentage of patients with NSCLC (47.47%; 122/257) showed high mRNA expression levels of ERCC1 compared with data from a previous study (20), while the percentage of patients with NSCLC with a high RRM1 mRNA expression level (61.87%; 159/257) was consistent with data presented in a previous study (21). The expression levels of ERCC1 and RRM1 mRNA were not associated with the gender, age, smoking status, tumor size, lymph node metastasis, pathological staging or other clinical characteristics of the patient.

The current study found that the mutational status of EGFR was associated with ERCC1 mRNA expression levels in patients with NSCLC; patients with EGFR mutations had a significantly lower expression of ERCC1 mRNA (P<0.05). EGFR mutations did not, however, significantly correlate with RRM1 expression levels (P>0.05) in patients with NSCLC. A previous study has shown that EGFR mutations in patients with NSCLC were correlated with expression levels of ERCC1 (P<0.001). Furthermore, EGFR mutations in the adenocarcinoma subgroup were correlated with ERCC1 expression levels (P=0.001) (22). It has also been shown that NER enzymes in cells can lead to cell damage, resulting in genomic instability and an increased mutation rate (23). Cancer cells with low expression of ERCC1 have a decreased ability to repair DNA damage, an increase in the number of EGFR mutations and increased sensitivity to platinum-based chemotherapy, which may be why patients with NSCLC with EGFR mutations have a higher response rate to chemotherapy.

The present study also found that, in NSCLC tissues, mRNA expression levels of ERCC1 and RRM1 were not correlated (P>0.05), which is inconsistent with data presented by Reynolds et al (24). The association between the expression of ERCC1 and RRM1 mRNA remains controversial and warrants further research. At present, numerous studies have confirmed that therapy can be selected based on patient expression levels of ERCC1 and RRM1, and this can be extended to patients with NSCLC (25,26).

In conclusion, the current study has demonstrated that patients with EGFR mutations tend to have lower expression levels of ERCC1 mRNA. We hypothesize that patients with EGFR mutations may be more responsive to cisplatin-based chemotherapy, although the molecular mechanisms require further study. No difference in the expression levels of RRM1 mRNA was observed between patients with NSCLC with EGFR mutations and those without mutations. In the present study, we have determined the first-line chemotherapy for tumors involving platinum and gemcitabine drug resistance genes and EGFR mutations but not the microtubule drug resistance gene TUBB3 or the thymidylate synthase resistance gene TYMS. Future studies, therefore, are likely to focus on EGFR, TUBB3 and TYMS mutations to better identify effective individualized treatment plans, particularly individualized treatment plans for EGFR-TKI (e.g. imatinib) primary or secondary resistance observed in certain patients.

Acknowledgements

This study was supported by the Wu Jieping Medical Foundation Clinical Research Special Project Fund (320.6750.1360).

References

1 

Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar : PubMed/NCBI

2 

Sher T, Dy GK and Adjei AA: Small cell lung cancer. Mayo Clin Proc. 83:355–367. 2008. View Article : Google Scholar : PubMed/NCBI

3 

Cao C, Lu S, Sowa A, et al: Priming with EGFR tyrosine kinase inhibitor and EGF sensitizes ovarian cancer cells to respond to chemotherapeutical drugs. Cancer Lett. 266:249–262. 2008. View Article : Google Scholar : PubMed/NCBI

4 

Herbst RS: Review of epidermal growth factor receptor biology. Int J Radiat Oncol Biol Phys. 59(2 Suppl): 21–26. 2004. View Article : Google Scholar : PubMed/NCBI

5 

Sharma SV, Bell DW, Settleman J and Haber DA: Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 7:169–181. 2007. View Article : Google Scholar : PubMed/NCBI

6 

Chan SK, Gullick WJ and Hill ME: Mutations of the epidermal growth factor receptor in non-small cell lung cancer - search and destroy. Eur J Cancer. 42:17–23. 2006. View Article : Google Scholar

7 

Simon GR, Ismail-Khan R and Bepler G: Nuclear excision repair-based personalized therapy for non-small cell lung cancer: from hypothesis to reality. Int J Biochem Cell Biol. 39:1318–1328. 2007. View Article : Google Scholar : PubMed/NCBI

8 

Rosell R, Lord RV, Taron M and Reguart N: DNA repair and cisplatin resistance in non-small-cell lung cancer. Lung Cancer. 38:217–227. 2002. View Article : Google Scholar : PubMed/NCBI

9 

Su C, Zhou S, Zhang L, et al: ERCC1, RRM1 and BRCA1 mRNA expression levels and clinical outcome of advanced non-small cell lung cancer. Med Oncol. 28:1411–1417. 2011. View Article : Google Scholar

10 

Mok TS, Wu YL, Thongprasert S, et al: Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 361:947–957. 2009. View Article : Google Scholar : PubMed/NCBI

11 

Olaussen KA, Dunant A, Fouret P, et al: DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med. 355:983–991. 2006. View Article : Google Scholar : PubMed/NCBI

12 

Bepler G, Kusmartseva I, Sharma S, et al: RRM1 modulated in vitro and in vivo efficacy of gemcitabine and platinum in non-small-cell lung cancer. J Clin Oncol. 24:4731–4737. 2006. View Article : Google Scholar : PubMed/NCBI

13 

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. 350:2129–2139. 2004. View Article : Google Scholar : PubMed/NCBI

14 

Paez JG, Jänne PA, Lee JC, et al: EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 304:1497–1500. 2004. View Article : Google Scholar : PubMed/NCBI

15 

Riely GJ, Pao W, Pham D, et al: Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clin Cancer Res. 12:839–844. 2006. View Article : Google Scholar : PubMed/NCBI

16 

Kobayashi S, Boggon TJ, Dayaram T, et al: EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. 352:786–792. 2005. View Article : Google Scholar : PubMed/NCBI

17 

Ceppi P, Volante M, Novello S, et al: ERCC1 and RRM1 gene expressions but not EGFR are predictive of shorter survival in advanced non-small-cell lung cancer treated with cisplatin and gemcitabine. Ann Oncol. 17:1818–1825. 2006. View Article : Google Scholar : PubMed/NCBI

18 

Cobo M, Isla D, Massuti B, et al: Customizing cisplatin based on quantitative excision repair cross-complementing 1 mRNA expression: a phase III trial in non-small-cell lung cancer. J Clin Oncol. 25:2747–2754. 2007. View Article : Google Scholar : PubMed/NCBI

19 

Pao W, Miller V, Zakowski M, et al: EGF receptor gene mutations are common in lung cancers from ‘never smokers’ and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA. 101:13306–13311. 2004. View Article : Google Scholar

20 

Deng QH, Qiu Y, Mo MC, et al: EGFR gene copy number, ERCC1 and BRCA1 protein expression and their relationship in non-small cell lung cancer. Zhonghua Zhong Liu Za Zhi. 33:508–512. 2011.(In Chinese). PubMed/NCBI

21 

Lin XY, Chen Y and Chen ZZ: Expression of ERCC1 and RRM1 in non-small cell lung cancer (NSCLC) and clinical prognosis. Fujian Yike Daxue Xuebao. 45:10–14. 2011.(In Chinese).

22 

Gandara DR, Grimminger P, Mack PC, et al: Association of epidermal growth factor receptor activating mutations with low ERCC1 gene expression in non-small cell lung cancer. J Thorac Oncol. 5:1933–1938. 2010. View Article : Google Scholar : PubMed/NCBI

23 

Mitsudomi T, Morita S, Yatabe Y, et al: West Japan Oncology Group: Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 11:121–128. 2010. View Article : Google Scholar

24 

Reynolds C, Obasaju C, Schell MJ, et al: Randomized phase III trial of gemcitabine-based chemotherapy with in situ RRM1 and ERCC1 protein levels for response prediction in non-small-cell lung cancer. J Clin Oncol. 27:5808–5815. 2009. View Article : Google Scholar : PubMed/NCBI

25 

Simon GR, Schell MJ, Begum M, et al: Preliminary indication of survival benefit from ERCC1 and RRM1-tailored chemotherapy in patients with advanced nonsmall cell lung cancer: evidence from an individual patient analysis. Cancer. 118:2525–2531. 2012. View Article : Google Scholar :

26 

Hu YY, Zhang DB, Dong YG, et al: The protein expression and the significance of RRM1 and ERCC1 in non-small cell lung cancer. Shandong Yi Yao. 52:1–3. 2012.(In Chinese).

Related Articles

Journal Cover

March-2015
Volume 9 Issue 3

Print ISSN: 1792-0981
Online ISSN:1792-1015

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Xu CW, Wang G, Wang WL, Gao WB, Han CJ, Gao JS, Li Y, Wang L, Zhang LY, Zhang YP, Zhang YP, et al: Association between epidermal growth factor receptor mutations and the expression of excision repair cross-complementing protein 1 and ribonucleotide reductase subunit M1 mRNA in patients with non-small cell lung cancer. Exp Ther Med 9: 880-884, 2015
APA
Xu, C., Wang, G., Wang, W., Gao, W., Han, C., Gao, J. ... Fang, J. (2015). Association between epidermal growth factor receptor mutations and the expression of excision repair cross-complementing protein 1 and ribonucleotide reductase subunit M1 mRNA in patients with non-small cell lung cancer. Experimental and Therapeutic Medicine, 9, 880-884. https://doi.org/10.3892/etm.2015.2196
MLA
Xu, C., Wang, G., Wang, W., Gao, W., Han, C., Gao, J., Li, Y., Wang, L., Zhang, L., Zhang, Y., Tian, Y., Fang, J."Association between epidermal growth factor receptor mutations and the expression of excision repair cross-complementing protein 1 and ribonucleotide reductase subunit M1 mRNA in patients with non-small cell lung cancer". Experimental and Therapeutic Medicine 9.3 (2015): 880-884.
Chicago
Xu, C., Wang, G., Wang, W., Gao, W., Han, C., Gao, J., Li, Y., Wang, L., Zhang, L., Zhang, Y., Tian, Y., Fang, J."Association between epidermal growth factor receptor mutations and the expression of excision repair cross-complementing protein 1 and ribonucleotide reductase subunit M1 mRNA in patients with non-small cell lung cancer". Experimental and Therapeutic Medicine 9, no. 3 (2015): 880-884. https://doi.org/10.3892/etm.2015.2196