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

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 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 repair (NER) and chain crosslink repair (7). Overexpression of ERCC1 can rapidly repair damaged DNA arrest at the G 2 /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. 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 4.29x10 -3 , and RRM1 had a standard mean of 11.37x10 -3 (Fig. 1C).

Specimens
Statistical analysis. The data were analyzed using the statistical software SPSS (version 19.0; IBM SPSS, Armonk, NY, USA) using the χ 2 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.     (Table IV).

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