| Background and objective Lung cancer is one of the most common malignancy. Platinum is the essential drugs for the treatment of non-small cell lung cancer。However, chemotherapy is failure to some of patients. The mechanism of Platinum-resistant tumor cells is very complicated. Individual differences in DNA damage repair capacity are one of the most important factors to determine the efficacy of platinum treatment in advanced non-small cell lung cancer patients. So far, the main mechanisms of DNA damage excision repair in cells are: Nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). NER and BER are the most important mechanisms. XRCC1 is a key member and play important role in BER. It is involved in cisplatin or carboplatin induced DNA damage repair process. In DNA repair pathway, NER recognizes DNA damage and related to the rate-limited removal step. ERCC1 is the most important functional gene in this step. The functional activity of the ERCC1 is corrected to the the NER repair activity. The expression levels of ERCC1 can impact the efficacy of platinum drugs. Ribonucleotide reductase (RR) is the only the rate-limited enzyme in DNA synthesis pathway, by which the ribose nucleoside diphosphate (NDP) generates to the corresponded deoxyribose nucleotides (including Purine and pyrimidine oligodeoxynucleotides). RR is an essential protein for DNA synthesis, repair and one of the key enzymes in cell survival. RRM1 is adjusted RR subunit and the tumor suppressor gene. XRCC1, ERCC1 remove damaged DNA fragment, the vacancy left in the DNA chain will be filled by ribonucleotides, which are supplied by the RRM1. The expression of XRCC1, ERCC1 and RRM1 gene is corrected with DNA repair capacity and may affect the susceptibility of non-small cell lung cancer, and affect the sensitivity of chemotherapy. Recent years, many studies are focusing on this topic. The aims of this study are to detect the protein expression level of XRCC1, ERCC1, RRM1 genes in tumors and adjacent tissues among advanced lung cancer (NSCLC) patients, by SP immunohistochemical detection method, to identify the predictive value of chemotherapy. Meanwhile, we study the polymorphisms in these genes in patients with advanced NSCLC by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) method, further explore the association between genetic variation and predictive value chemotherapy. According the efficacy of platinum treatment in advanced non-small cell lung cancer patients, combining the information of the protein expression level of XRCC1, ERCC1, RRM1 genes and genotypies of these genes'polymorphisms studied, to obtain the knowledge of the susceptibility of non-small cell lung cancer and the sensitivity of platinum chemotherapy, we could eventually targete the genotypies of these three DNA repair genes and provide the basis for predictability of the susceptibility for lung cancer and and the sensitivity to platinum treatment, to achieve individualized cancer treatment.Methods (1) Samples collection: Blood samples were collected between January 2009 ~ June 2010, from the patients (n= 54) were diagnosed by histological and confirmed by imaging studies in advanced NSCLC with measurable lesions and initially treated in Ningxia Medical University Hospital, and 50 healthy subjects. PCR-RFLP and sequencing technique were used to detect cases and the control group XRCC1, ERCC1, RRM1 gene polymorphisms. Fifty-four patients with advanced NSCLC patients received at least four cycles of platinum-containing chemotherapy.(2) In the case group, 38 patients, their cancer tissue samples were collected and also 24 patients, the adjacent normal lung tissue samples were collected. SP immunohistochemical method was used to detect ERCC1, XRCC1, RRM1 expression of three proteins. (3) Efficacy analysis of chemotherapy: Advanced NSCLC patients were received platinum-based chemotherapy shown effective results, after 2 cycles of chemotherapy by using original proposal. If the treatment was invalid, changing treatment proposal and continue the 2 cycles of treatment and re-evaluated. Finally, we analyze the association between the expression level of ERCC1, XRCC1, RRM1 proteins and their gene polymorphism with efficacy of platinum-based chemotherapy in advanced NSCLC patients.Results 1,(1) XRCC1 in NSCLC, the positive parts of the tumor cells located in the nucleus, cell membrane and cytoplasm no coloring, adjacent normal lung tissue and no expression or low expression in patients with NSCLC, 38 patients with positive expression of XRCC1 was 63.2% (24 / 38), 24 cases of adjacent normal lung tissue expression was 33.3%(8/24), significantly different from the two groups (P <0.05); ERCC1 in NSCLC, the positive rate was 57.9% (22/38), expression site is located in the nucleus, adjacent normal lung tissue and no expression or low expression, positive expression rate was 29.2%(7/24), significantly different from the two groups (P<0.05);RRM1 in NSCLC, the positive expression area in the cytoplasm and (or) cell membrane, the positive rate was 60.5% (23/38), adjacent normal tissue had no expression or low expression, positive expression was 29.2%(7/24) in group also statistically significant (P<0.05). (2) XRCC1 and ERCC1, RRM1 protein expression was no significant correlation (r values were 0.233,0.053; P values were greater than 0.05),ERCC1 and RRM1 expression of two proteins positively correlated (r=0.838, P<0.05). (3) 38 patients, chemotherapy in 20 cases, the effective rate was 52.6% (20/38), of which: 20 cases of chemotherapy in patients with positive expression of XRCC1 was 45.0% (9/20), 18 cases of patients with chemotherapy is not valid XRCC1 expression was positive in 83.3% (15/18), with a significant difference between the two (P<0.05);20 cases of chemotherapy in patients with ERCC1 expression was 40.0% (8/20), 18 cases of ineffective chemotherapy in patients with ERCC1 expression was positive in 77.8% (14/18), there are significant differences between the two (P<0.05 ); 20 cases of chemotherapy in patients with positive expression of RRM1 was 40.0% (8/20), 18 cases of ineffective chemotherapy in patients with positive expression of RRM1 was 83.3% (15/18), also has significant difference between the two (P<0.05). 2, (1) in patients with advanced NSCLC gene XRCC1 exon 6 codon 194 codon194 There are three genotypes: Arg/Arg, Arg/Trp and Trp/rp; ERCC1 exon 4 codon 118 codon118 total of three genotypes: C/C, C/T and T/T; RRM37AC two genotypes were detected: C/C and C/A, not detected in A/A genotype.XRCC1 gene to the wild genotype Arg/Arg as the reference, XRCC1 gene to the wild genotype Arg/Arg as the reference, carrying Arg/Trp and Trp/Trp and Arg/Trp patients were increased relative risk of lung cancer (OR values were 3.344,3.071;95%CI values were 1.099~10.179,1.154~8.175). ERCC1 in the wild genotype C/C as a reference, carrying C/T+T/T and C/T genotype relative risk of lung cancer were significantly increased (OR values were 2.680, 2.756;95%CI values were 1.080～6.64,81.016~7.476). RRM1 in the wild genotype C/C as a reference, to carry C/A, although the relative risk of lung cancer increased (OR=2.177,95% CI=0.934~5.704), but not statistically significant(P >0.05). (2) in the XRCC1 gene, carrying the Arg/Arg, Arg/Trp, Trp/Trp genotype of the efficiency of chemotherapy patients was 36.1% (13/36), 71.4% (10/14), 75.0% (3/4). Arg/Arg and Arg/Trp genotype groups in patients with chemotherapy statistically significant difference between the efficient (χ~2=5.062, P=0.024); Arg/ Arg and Trp/Trp genotype groups between the efficiency of chemotherapy in patients no significant difference (χ~2=0.938,P=0.333);Carrying Trp allele (Arg/Trp+Trp/Trp) in the chemotherapy group was 72.2%, significantly higher than the Arg/Arg genotype (χ~2=6.268,P=0.012), is carrying Arg/Arg genotype 3 times (OR=3.453, 95% CI=1.361~8.764). In the ERCC1 gene, carrying C/C, C/T, T/T genotype in patients with chemotherapy, response rates were 32.4% (11/34), 75.0% (12/16), 75.0% (3/4).C/C and C/T genotype groups in patients with chemotherapy statistically significant difference between the efficient (χ~2=7.966, P=0.005); C/C and T/T genotype groups between the efficiency of chemotherapy in patients no significant difference (χ~2=1.265, P=0.261); carrying T allele (C/T+T/T) chemotherapy was 75.0%, significantly higher than C/C wild-type genome (χ~2=9.174, P=0.002), the latter 4 times (OR=4.11,95% CI=1.622~10.418). In the RRM1 gene, carrying C/C, C/A genotype in patients with chemotherapy response rate was 46.9% (15/32), 50.0% (11/22),Between the two groups was not significant (χ~2 =0.051, P=0.821).Conclusion 1. XRCC1, ERCC1, RRM1 is highly expressed in advanced NSCLC, but low expression or no expression in adjacent normal lung tissue. It prompted that there is abnormal DNA repair in advanced NSCLC.2. In advanced NSCLC, XRCC1 and ERCC1, RRM1 no correlation between protein expression, but expression of ERCC1 and RRM1 high correlation exists between, suggesting that ERCC1 and RRM1 protein on the development of advanced NSCLC, may play a synergistic role. 3. XRCC1, ERCC1 and RRM1 expression and platinum resistance to chemotherapy of advanced NSCLC, which may be predicted in patients with advanced NSCLC platinum drug sensitivity of biological markers for the realization of individual clinical therapy. 4. In patients with advanced NSCLC in the XRCC1, ERCC1 genotype and T allele carriers of the proportion of Trp increased, suggesting that in advanced NSCLC plays an important role. 5. XRCC1 genotypes carrying Trp allele, ERCC1 genotypes carrying T allele in patients with advanced NSCLC platinum drug sensitivity, suggesting that in patients before chemotherapy XRCC1, ERCC1 genotyping, to a certain extent with advanced NSCLC to predict the sensitivity to platinum drugs. |
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