Effects Of Expression Of ERCC1,TS And TUBB3 On Chemotherapy Sensitivity And Clinical Outcome Of Gastric Cancer

Background and Objective:Gastric cancer (GC) is one of the most common malignant tumors, GC remains the fourth most common cancer and the second most frequent cause of cancer death worldwide. Our country belongs to the area of high morbidity of gastric cancer,42% of the world morbidity and new cases of gastric cancer patients showed a trend of younger, its mortality rate ranks second only to lung cancer and liver cancer. Due to include China, of the most countries in the world have yet to carry out the routine screening of gastric cancer, gastric cancer onset occult early often had typical symptoms. In most cases often only in the late diagnosed, simple operation of the poor efficacy, postoperative 5-year survival rate of only about 20%, serious harm to people’s life and health. GC occurrence and development is a multi-stage multi-factor and multi-steps together complex process involving multiple oncogene activation and anti oncogene inactivation, but the molecular mechanism of gastric cancer has not yet been elucidated. The results of the present study show that H.pylori infection, diet, smoking and host genetic susceptibility is an important factor in the occurrence of gastric cancer.The current study showed that the difference in the efficacy of chemotherapy between individuals can not be explained by the patient’s age, sex, type of tissue, growth site, organ function, and so on. The main factor influencing the efficacy and toxicity of chemotherapy is drug genomics. Pharmacogenomics is a new subject developed on the basis of pharmacogenetics. As early as 1950s, it was discovered that different genetic backgrounds would lead to differences in drug response. Due to the different sensitivity of chemotherapy in patients with gastric cancer, so that the real benefit in patients with chemotherapy is not much, if the choice is inappropriate, but make some patients with chemotherapy is not sensitive to delay treatment, increase the pain and treatment costs. Therefore, chemotherapy in patients with advanced gastric cancer susceptibility biomarker screening, prediction of chemotherapy sensitivity in patients can effectively avoid the risk, make the patients to maximize the benefit.Platinum compound forms the platinum-DNA adducts in the DNA, which causes inter-cross-linking and induces bulky distortion of DNA which subsequently inhibits the DNA replication. These DNA adducts are reported to be involved in clinical outcome and response to chemotherapy for cancers. Nucleotide excision repair (NER) is responsible for repairing platinum-DNA adducts and is involved in removing and restoring a segment of DNA containing a bulky adduct by cooperating with about 20 enzymes. DNA repair may not only prevent mutagenesis but also retard molecular events related to progression of cancers. Excision repair cross-complementing 1 (ERCC1) is one key gene during the NER pathway, and its altered expression has been associated with resistance to platinum compounds.The antitumor effect of fluoropyrimidine mainly stems from its competitive inhibition of thymidylate synthase (TS). TS is the rate-limiting enzyme in the pyrimidine nucleotide synthetic pathway for DNA and is a key target of fluoropyrimidine agents, such as 5-FU, capecitabine, and S-1.TS holds promise as a prognostic biomarker because of its role as the molecular target of 5-FU, a commonly used chemotherapeutic agent in many cancer. TS is a rate-limiting enzyme in the phosphorylation of 5-d UMP to 5-dTMP. The 5-dTMP under normal conditions is converted to 5-dTTP which is incorporated into DNA during its routine synthesis. In the absence of TS DNA synthesis is inhibited and this results in cell death. Increased expression of TS in the tumor cells enables them to overcome 5-FU-induced nuclear cytotoxicity and promotes DNA synthesis and repair of tumor cells.Tubulin-binding agents are an important class of compounds in the field of anti-neoplastic chemotherapy,with broad activity in both solid tumors and hematological malignancies.Intracellular tubulin divided into α、β two subtypes is an important component of the cytoskeleton, the main role is anti microtubule drug target.Class Ⅲ β-tubulin(TUBB3) has been shown to play a role in chemotherapy resistance in various cancer types.These agents block cell division by inhibiting the mitotic spindle. Taxanes (paclitaxel and docetaxel) promote the polymerization of purified tubulin in vitro at high concentrations and enhance the fraction of polymerized tubulin in cells. Thus, they have been referred to as microtubule-stabilizing agents.Several mechanisms have been reported to be involved in resistance to tubulin-binding agents. Many preclinical studies have shown that high levels of TUBB3 expression are associated.with taxane resistance in various human cancer cell lines, including lung, ovary, prostate, breast, and pancreas.In 2012 the National Comprehensive Cancer Network(NCCN) non small cell lung cancer guidelines have been pointed out, detection of ERCC1, RRM1 expression level can improve the efficiency and the survival rate of the patients before received chemotherapy, but in gastric cancer and other tumors has not yet been pointed out. However, nearly years published studies that ERCC1 can not prediction with platinum based chemotherapy efficacy in lung cancer, suggesting that clinical study of chemotherapy molecular marker detection and guiding chemotherapy is controversial and chemotherapy related molecular mark in clinical studies is still a great challenge. At present, domestic and foreign experts on whether biomarkers can predict the prognosis of patients with advanced gastric cancer and chemotherapy efficacy related research is relatively small, and the current research is in a retrospective study. Our research group in clinical collected cases of gastric cancer and to explore the correlation between gastric cancer in ERCC1, TS and TUBB3 expression and clinical pathological characteristics and analysis of they and sensitivity to chemotherapy and prognosis in and through the experiments in vitro confirmed that individualized treatment for stomach cancer target object detection to provide clinical and experimental basis, standardizing clinical treatment, efficient use of medical resources service for the patient has important significance.Materials and Methods:Part 1 Expression and correlation of ERCC1, TS and TUBB3 in gastric cancer1.This study reviewed the cases of 118 patients with primary GC in patients between December 2008 and December 2011 at Shaoyang Central Hospital. There were 2 cases were excluded because of immunohistochemical(IHC) staining does not satisfiable, a total of 116 patients completed all the tests available for evaluation. There are 23 cases of stage Ⅱ,66 cases of stage III, and 27 cases of stage IV, according to AJCC stage.62 cases were male,54 cases were female,20 cases were high differentiation,35 cases were median differentiation and 61 cases were poorly differentiated.69 cases of lymph node metastasis, and without metastasis in 47 cases. Collected data included the patient’s age, sex, admission number, pathological type, ECOG score, lymph node metastasis, tumor size and clinical staging. All tissue samples of gastric cancer by 10% formalin fixed and conventional dehydration, paraffin embedding, a part of samples for IHC; another part for branch liquid phase chip technology. Analysis of the relationship between the expression of ERCC 1, TS and TUBB3 in gastric carcinoma and the clinical pathological characteristics and correlation of ERCC1, TS and TUBB3 at the same time.2.1mmunohistochemical analysis:Formalin-fixed, paraffin-embedded samples of tumor tissues from each patient were reacted with anti-ERCC1, TS and TUBB3 monoclonal antibodies was employed to determine the tumor expression levels of its protein. IHC staining was quantified by a single pathologist (from Shaoyang Central Hospital), who was blinded to outcomes, using a previously described, validated method for scoring IHC specimens on the basis of the percentage and intensity of cellular staining. All IHC scoring was subsequently reviewed and confirmed by a second, senior pathologist (from Nanfang Hospital), who also was blinded to patient outcomes. Overall scores were assigned on a 0 to 4 scale, and patients were dichotomized into a high-expression group (scores>2) and a low-expression group (scores ≤2).Part 2 Study on the expression of ERCC 1, TS and TUBB3 in advanced gastric cancer and the effect of chemotherapy and prognosis1.This study reviewed the cases of 67 patients with advanced GC in patients between December 2008 and December 2011 at Shaoyang Central Hospital. ERCC1,TS and TUBB3 expression were determined using immunohistochemistry. Relationships between expressions of ERCC 1,TS and TUBB3 protein with platinum, fluorouracil and taxanes chemotherapy response, progression-free survival(PFS) and overall survival(OS) were analyzed.2.Treatment and evaluation:Patients, included in the study, were treated with chemotherapy following regimen. Fluorouracil-based regimen included capecitabine at a dose of 1,000-1,250mg/m2 or S-1 40mg/m2 (oral administration after breakfast and after dinner) and 5-fluorouracil 1,000 mg/m2 IV continuous infusion over 24 hours daily on days 1 and 3,cycled every 21 days, capecitabine/S-1 was administered twice daily from D1 to D14, repeated every 3 weeks. Paclitaxel/docetaxel-based regimen included paclitaxel at a dose of 150-175mg/m2 or docetaxel at a dose of 75mg/m2 on day 1.Platinum-based regimen included cisplatin at a dose of 75mg/m2 on days 1-3 or or oxaliplatin at a dose of 130mg/m2 on day 1, repeated every 3 weeks. After the second cycle of chemotherapy, response was reevaluated based on CT imaging, gastroscope results according to RECIST (version 1.1) criteria. Maintenance continued until disease progression or appearance of intolerable side effects. When the chemotherapy was finished, follow-up was conducted every month by telephone up to death or the end of study. We also recorded the overall survival (OS) and progression-free survival (PFS). The OS was defined from the start of therapy to the date of death, and PFS was defined from the start of therapy to the date of progression or death without progression. The last follow-up time was December 2014.Part 3 Relationship between ERCC1, TS and TUBB3 in gastric cancer and sensitivity of chemotherapeutic drugs in vitroThis study reviewed the fresh specimens of 48 patients with gastric cancer after surgical operation between October 2013 and September 2014 at Shaoyang Central Hospital and Nanfang Hospital. There are 18 cases of stage II,28 cases of stage III, and 2 cases of stage IV, according to AJCC stage.28 cases were male,20 cases were female,8 cases were high differentiation,10 cases were median differentiation and 30 cases were poorly differentiated.27 cases of lymph node metastasis, and without metastasis in 21 cases. All fresh specimens of gastric cancer were collection and each cancer tissue samples were divided into two parts, a part of samples of the fixed, paraffin embedding,make-up the paraffin sections and IHC detection ERCC1,TS and TUBB3 expression in there organizations; another part used for the original generation of cultured cells,single cell suspension and planting in 96-well plates,the cells for drug sensitive test,and then determined by ATP-tumor chemosensitivity assay were used to detect cell proliferation activity of cancer cells resistant index calculation. Analyzing the relationships between expressions of ERCC1,TS and TUBB3 protein with cisplatin,5-fluorouracil and paclitaxel chemotherapy responseResults:Part 11. ERCC1, TS and TUBB3 protein in the high, medium and low degree of differentiation of gastric cancer tissues have different degrees of expression in 116 cases. ERCC1 protein expression analyses from paraffin tumor tissues were successfully performed. The immunostaining patterns showed that ERCC1 was mainly localized to the cytoplasm and nucleus. Of all the archival specimens, 50.86%(59/116) showed low-level expression of ERCC1 protein,49.14%(57/116) showed high-level expression, respectively. TS was mainly localized to the cytoplasm and nucleus. Of all the archival specimens,48.28%(56/116) showed low-level expression of TS protein,51.72%(60/116) showed high-level expression, respectively. TUBB3 was mainly localized to the nucleus. Of all the archival specimens, 55.17%(64/116) showed low-level expression of TUBB3 protein,44.83%(52/116) showed high-level expression, respectively.2. The level of ERCC1, TS and TUBB3 protein and mRNA expression were not related with the patient’s sex, age, ECOG score, differentiation, clinical stage and lymphatic metastasis with the method of IHC and branch liquid phase chip technology(P>0.05). The high-level expression of both ERCC1, TS and TUBB3 was 21 cases(18.10%), low-level expression of both ERCC1, TS and TUBB3 was 15 cases(12.93%), ERCC1 and TS high-level expression were 31.03%(36/116), low-level expression were25.00%(29/116), ERCC1、TUBB3 high-level expression were 26.72%(31/116), low-level expression were 19.83%(23/116), TS and TUBB3 high-level expression were 37.07%(43/116), low-level expression were 25.86%(30/116). The protein expression of TS had correlation with TUBB3 in gastric cancer(χ2=5.690, P=0.017), but the rest had no obvious correlation(P>0.05).Part 21.57 cases of 60 cases of gastric cancer patient to obtain valid data, among the 57 cases treated with platinum-based regimen, treatment response was as follows: complete response (CR) in 2 patient, partial response(PR) in 26 patients, stable disease (SD) in 17 patients, and progressive disease (PD) in 12 patients. Overall response rate (CR+PR) was 49.12%(28/57). The chemotherapy response rates in low and high expression of ERCC1 were 66.67%(20/30) and 29.63%(8/27), respectively (χ2=7.8, P=0.005). Among the 52 valid cases treated with fluorouracil-based regimen, treatment response was as follows:CR in 1 patient, PR in 20 patients, SD in 18 patients, and PD in 13 patients. Overall response rate (CR+PR) was 40.38%(21/52). The chemotherapy response rates in low and high expression of TS were 63.46%(16/27) and 36.54%(5/25), respectively (χ2=8.310, P=0.004). Among the 46 valid cases treated with Paclitaxel/docetaxel-based regimen, treatment response was as follows:CR in 1 patient, PR in 17 patients, SD in 22 patients, and PD in 6 patients. Overall response rate (CR+PR) was 39.13%(18/46). The chemotherapy response rates in low and high expression of TUBB3 were 54.17%(13/24) and 22.73%(5/22), respectively (χ2=4.736, P=0.029).2.The patients with low ERCC1 expression had a longer median PFS and OS than those with high ERCC1 expression (PFS 6.0 vs 3.1 m,P=0.005; OS 12.9 vs 7.8m, P=0.002). The patients with low TS expression had a longer median PFS and OS than those with high TS expression (PFS 7.8 vs 3.1m, P=0.007; OS 13.3 vs 8.3m, P=0.003).The patients with low TUBB3 expression had a longer median PFS and OS than those with high TUBB3 expression (PFS 5.9 vs 3.9 m, P=0.032; OS 116 vs 7.9m, P=0.001).3.univariate analysis and multivariate Cox regression analysis showed that both the expression of ERCC1, TS and TUBB3 were independent prognostic factors in gastric cancer.Part 31.Primitive cells of gastric cancer cell in vitro medicine sensitive experiment results show 45 cases of 48 cases of gastric cancer cell to obtain valid data, of which 21 cases of cisplatin-resistant,24 cases of sensitive, sensitive rate was 53.33%; 22 cases of 5-fluorouracil-resistant,23 cases of sensitive, sensitive rate was 51.11%; 24 cases of paclitaxel-resistant,21 cases of sensitive, sensitive rate was 46.67%%, according to the IC90 and IC50 to determine its drug sensitivity in vitro, is almost the same with the clinical use of chemotherapeutics sensitive rate.2.Among the 45 cases of gastric cancer tissues, ERCC1 high expression in 21 cases (46.67%), low expression in 24 cases (53.33%). Combined with susceptibility results obtained before in vitro, there were 7 cases of drug sensitivity in 21 cases of ERCC1 high expression group,17 cases of drug sensitivity in 24 cases of ERCC1 low expression group. ERCC1 high expression group was obviously lower than the low expression group to sensitivity cisplatin, and the differences were significant statistically (P< 0.05).TS high expression in 19 cases (42.22%), low expression in 26 cases (57.78%), there were 5 cases of drug sensitivity in 19 cases of TS high expression group,18 cases of drug sensitivity in 26 cases of TS low expression group.TS high expression group was obviously lower than the low expression group to sensitivity 5-fluorouracil(P<0.01);TUBB3 high expression in 22 cases (48.89%), low expression in 23 cases (51.11%), there were 5 cases of drug sensitivity in 22 cases of TUBB3 high expression group,16 cases of drug sensitivity in 23 cases of TUBB3 low expression group.TUBB3 high expression group was obviously lower than the low expression group to sensitivity paclitaxel(P<0.01).Conclusion:1.ERCC1, TS and TUBB3 protein have different degrees of expression in gastric cancer tissues, the level of ERCC1, TS and TUBB3 protein and mRNA expression were not related with the patient’s clinical feature, the protein expression of TS had correlation with TUBB3 in gastric cancer.2.The present study demonstrates that ERCC1,TS and TUBB3 expression of advanced gastric cancer is associated with chemotherapy response and prognosis, low expression group were better than high expression group.3.The expression of ERCC1,TS and TUBB3 in gastric cancer cell were associated with drug resistance, low expression group were lower than high expression group.4.ERCC1、TS and TUBB3 could be assessment of the chemotherapy sensitivity and clinical outcome indicators in gastric cancer.