【作者】 孙伟；

【导师】 郝继辉；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2014， 硕士

【摘要】 目的近年来胰腺癌的发病率在国内外均呈明显上升趋势,且预后极差,其五年生存率低于5%。即使术后辅助化疗的病人,5年生存期仍只有10%～30%。以吉西他滨为主的化疗方案是胰腺癌化疗的标准方案。而吉西他滨耐药仍然是延长患者总生存期(overall survival, OS)和无病生存期(disease free survival,DFS)的重要阻碍。因此,预测胰腺癌术后患者的化疗耐药性,从而选择最佳的化疗方案,实现更好的个体化治疗,对提高胰腺癌患者的生存具有重大意义。吉西他滨是一种很强的亲水性药物,不能以被动扩散的方式穿过细胞膜,而主要通过人平衡型核苷转运体1(human equilibrative nucleoside transporter-1, hENT-1)进入胞浆。在胞浆中,吉西他滨在被脱氧胞苷激酶(deoxycytidine kinase,DCK)磷酸化成双氟脱氧胞苷一磷酸(difluorodeoxycytdine monophosphate, dFdCMP),随后,DCK将dFdCMP转换成其活性代谢物,即双氟脱氧胞苷二磷酸(difluorodeoxycytidine diphosphate, dFdCDP)及双氟脱氧胞苷三磷酸(difluorodeoxycytidine triphosphate, dFdCTP)。dFdCDP和dFdCTP通过抑制核苷酸还原酶(ribonucleotide reductase, RR)来阻碍三磷酸脱氧核苷酸(deoxynucleotide triphosphates)的生成,而三磷酸脱氧核苷酸是合成DNA的必需材料。RR由其大亚基核苷酸还原酶M1(ribonucleoside reductase subunit M1,RRM1)和小亚基核苷酸还原酶M2(ribonucleoside reductase subunit M2RRM2)组成。近年来,国外关于吉西他滨敏感性的研究逐渐增多,hENT-1, DCK, RRM1和RRM2等几种吉西他滨代谢相关的基因也各有报道,但关于这几种基因能否预测胰腺癌患者吉西他滨辅助化疗的敏感性,结论尚有争议,不能对临床产生较大的指导意义。并且到目前为止,还没有关于中国胰腺癌人群的大宗报道。本课题拟回顾性分析我院2005.1月到2011.6月期间收治的胰腺癌术后患者,其hENT-1, DCK, RRM1和RRM2表达与吉西他滨辅助化疗敏感性的关系,为其应用于临床提供证据。方法一、病人的筛选及病例资料的收集和总结我们收集了从2005.1月到2011.6月我院收治的所有病理确诊为胰腺导管腺癌的行肿瘤切除术病人的临床资料。剔除接受新辅助化疗、放化疗、非吉西他滨化疗、R2切除的病人及术后1月内死亡、随访信息不全的病例,共筛选出102例病人,其中45例术后未行化疗,为非化疗组(对照组),57例术后行至少3个周期的吉西他滨或GEMOX(吉西他滨+奥沙利铂)方案的化疗,为化疗组(实验组)。两组性别,年龄,手术方式,Rx(R0或R1)切除,临床分期,病理分级,肿瘤大小等临床指标相匹配,无统计学差异(P>0.05)。设立对照组,以期排除hENT-1, DCK, RRM1, RRM2基因的表达本身即为胰腺癌术后患者的独立预后因素的可能,进而证明其仅在吉西他滨辅助化疗的患者中发挥作用。二：免疫组化及结果评级我们收集了此102例病人的福尔马林固定石蜡包埋的肿瘤组织蜡块,HE染色的切片用来确认病理类型为胰腺导管腺癌。组织蜡块切成小于4um厚度的连续切片,做hENT-1, DCK, RRM1, RRM2的蛋白免疫组化。并由本人与本院病理科程润芬医生分别评级。结合双方的组化评级结果,评分不一致的请本院病理科孙燕副主任医师评定。三：QRT-PCR及结果计算102例胰腺癌病人中可收集到53例病人的新鲜冰冻肿瘤组织,其中化疗组24人,非化疗组29人。两组性别,年龄,手术方式,Rx(R0或R1)切除,临床分期,病理分级,肿瘤大小等临床指标相匹配,无统计学差异(P>0.05)。我们采用RNA提取试剂盒从这53例病人的新鲜冰冻肿瘤组织中提取全RNA,反转录并用QRT-PCR验证hENT-1, DCK, RRM1, RRM2在mRNA水平表达与吉西他滨化疗敏感性的关系。四：结果分析在化疗组和非化疗组中分别分析hENT-1, DCK, RRM1, RRM2四个基因在蛋白组化水平及mRNA水平与患者OS和DFS的关系。结果一：蛋白水平1.患者总生存期(OS)分析非化疗组中,RRM1蛋白表达为患者OS的独立相关因素。RRM1蛋白高表达组生存期较高,这与相关文献报道一致。化疗组中,RRM2, hENT-1的蛋白表达是患者OS的独立相关因素。RRM2低表达组、hENT-1高表达组分别拥有较长的OS。2.患者无瘤生存期(DFS)分析非化疗组中,RRM1的蛋白表达为患者DFS的独立相关因素。RRM1蛋白高表达组的患者DFS较长。化疗组中,RRM2, hENT-1的蛋白表达为患者DFS的独立相关因素。RRM2低表达组、hENT-1高表达组分别拥有较长的DFS。二：mRNA水平1.患者总生存期(OS)分析两组中,hENT-1, DCK, RRM1, RRM2的mRNA表达均非患者OS的独立相关因素。化疗组中,hENT-1mRNA高表达组的生存期较长,但达不到统计学差异(P=0.091)。2.患者无瘤生存期(DFS)分析非化疗组中,hENT-1, DCK, RRM1, RRM2的mRNA表达均非患者DFS的独立相关因素。化疗组中,]hENT-1mRNA的表达为患者DFS的独立相关因素。结论RRM2, hENT-1的蛋白水平的表达是吉西他滨辅助化疗患者DFS和OS的独立相关因素,RRM2低表达组、hENT-1高表达组分别拥有较长的DFS和OS。在mRNA水平,仅hENT-1为吉西他滨辅助化疗患者DFS的独立相关因素。RRM2, hENT-1免疫组化染色的方法,操作简单,灵敏度高,重复性强,有望成为预测胰腺癌患者术后吉西他滨辅助化疗敏感性的重要方法。更多还原

【Abstract】 ObjectivePancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with five-year survival rates of less than5%.Although patients with postoperative adjuvant chemotherapy have relatively better prognosis, most of them suffer from local recurrence or metastasis that occurs after treatment, with five-year survival rates of10%to30%. Efforts to improve survival in patients with resectable pancreatic adenocarcinoma (PA) have focused on adjuvant therapy. For more than a decade, gemcitabine (Gemzar(?), Eli Lilly and Company, IN, USA) remains the first-line drug for adjuvant therapy. The resistance to gemcitabine is still the important obstacle to prolong patients’overall survival and disease-free survival. Therefore, it is of great significance for improving the survival of pancreatic cancer patients to predict the sensitivity of gemcitabine adjuvant chemotherapy and choose the best chemotherapy regimen for better individualized treatment.Gemcitabine is strongly hydrophilic, and its uptake into human cells is mainly mediated by human equilibrative nucleoside transporter-1(hENT-1), rather than by passive diffusion through hydrophobic cellular membranes. In the cytoplasm, gemcitabine is phosphorylated by deoxycytidine kinase (DCK) to its mononucleotide in the rate-limiting step. Subsequent nucleotide kinases convert gemcitabine monophosphate to its active metabolites, that is, gemcitabine diphosphate and gemcitabine triphosphate. Gemcitabine diphosphate and gemcitabine triphosphate inhibit ribonucleotide reductase, comprising ribonucleoside reductase rubunit M1(RRM1) and ribonucleoside reductase rubunit M2(RRM2), which block the production of the deoxynucleotide triphosphates that are necessary for DNA synthesis.Several preclinical studies suggest that the sensitivity of gemcitabine depends on molecular parameters, such as HENT-1, DCK, RRM1, and RRM2. An increasing number of clinical studies of these factors at both mRNA and protein levels have been reported in predicting the efficacy of gemcitabine-based chemotherapy in resected PDAC. However, except studies on HENT-1, the results were controversial, and data on Asian patients are rare. Thus, a formal evaluation of these factors and their relationships to disease progression and prognosis of PDAC is still needed before they can be applied in the prediction of gemcitabine chemosensitivity in clinical practice. This study characterized the in situ expression of hENTl, DCK, RRM1, and RRM2at both protein and mRNA levels to evaluate their use as prognostic biomarkers for resected pancreatic cancer patients that have received gemcitabine-based adjuvant chemotherapy (AC) and to explore the clinical feasibility.Methods1. Patient CohortWe retrospectively enrolled all consecutive patients with histologically confirmed diagnosis of PDAC by operation and were treated at Tianjin Cancer Hospital, Tianjin Medical University in this study between January2005and June2011. Patients with at least one of the following conditions were excluded:(1) patients who received neoadjuvant chemotherapy, chemoradiotherapy, or non-gemcitabine-based chemotherapy;(2) patients with macroscopically incomplete resection;(3) patients who died within one month after the operation or because of non-cancer reasons;(4) patients who were lost during the follow up period; and (5) patients whose tumor samples were unavailable. Patient demographics and clinical characteristics including sex, age, maximal tumor size, operation method, Rx resection, tumor stage according to the Union for International Cancer Control, TNM classification, histological grade, and lymph node metastasis were collected. Patients were examined each two weeks for tumor markers and by abdominal ultrasound, and every two months by computed tomography (CT). The follow up ended on June30,2013. This study was approved by the appropriate institutional review boards.2. ImmunohistochemistryA series of4μm thick, formalin-fixed, and paraffin-embedded (FFPE) sections were prepared for each specimen. Tissue sections stained with hematoxylin and eosin (HE) were histologically evaluated for verification of diagnosis and eligibility for immunohistochemistry (IHC) analysis.HE and IHC slides were independently graded by pathologists Runfen Cheng and Yan Sun, who were blinded to patient outcomes. Discordant cases were assessed by a third pathologist, and a consensus was reached.Membranous and cytoplasmic staining for hENTl was regarded as positive. Cytoplasmic staining for DCK was considered positive. Cytoplasmic staining for RRM1and RRM2was regarded as positive. Immunoreactivity was scored semi-quantitatively according to the percentage of positive tumor cells. Intensity of tumor cell staining was scored as0=negative;1=low;2=medium;3=high, which was based on the relative intensities to staining of the lymphocytes in the same section, as previously reported. The extent of staining was scored as0=0%to5%stained;1=5%to25%stained;2=26%to50%stained; and3=51%to100%stained. The final score was determined by multiplying the scores of intensity with the extent of staining, in the range0-9.3. mRNA Extraction and Real-time QRT-PCRFresh tumor tissue samples were stored at-150℃in the refrigerator immediately after resection until used for mRNA extraction. The mRNA was extracted by using the TriPure Isolation Reagent (Roche) according to the manufacturer’s instructions. Integraty of the mRNA was guaranteed by a28S/18S ratio between1.8and2.0, and a RNA Integrity Number (RIN) value higher than five. Purity of the mRNA was tested by the A260/A280ratio, and an A260/A280ratio greater than1.8is considered as an acceptable indicator of qualified mRNA.4. Statistical AnalysesX2tests were used to analyze the categorical variables between the chemotherapy group and the non-chemotherapy group. OS was studied by the Kaplan-Meier method analysis. Protein and mRNA expression levels were split into high-and low-level groups, respectively, by using recursive descent partition analysis (RPA). Univariate survival curve differences were tested for significance with a log-rank test. A Cox proportional hazards regression model was used to assess the prognostic significance of the parameters obtained in association. HR<1indicates a benefit in OS or disease-free survival (DFS). Analyses were performed by using the Statistical Package for the Social Sciences, SPSS18.0for Windows (SPSS Inc., Chicago, IL).A p-value of<0.05indicated statistically significant difference.Results1. Immunohistochemical EvaluationRRM1levels showed significant association with the OS of the non-AC patients. By comparison, RRM2and hENT-1levels affected the OS of the GEM-treated patients. In the non-AC group, patients with high protein levels of RRM1showed longer median OS time. In the GEM group, patients with low levels of RRM2or high levels of hENT-1had a relatively longer overall survival time.RRM1were associated with the DFS of the non-AC group. RRM2and hENT-1levels were associated with the DFS of the GEM group. Longer median DFS was observed in patients with higher levels of RRM1in the non-AC group and patients with low levels of RRM2or high levels of hENT-1in the GEM group.Patients with both low levels of RRM2and high levels of hENT-1had relatively much longer median OS and DFS.2. QRT-PCR EvaluationIn the non-AC group, none of the four gene mRNA levels was correlated with the OS or DFS. In the GEM group, hENT-1levels were independently associated with the DFS, but not with the OS.ConclusionImmunohistochemical staining of FFPE tumor sections for hENT-1and RRM2protein levels, especially the combination of the two protein levels, may be superior to qRT-PCR as a method for gemcitabine sensitivity prediction.更多还原