【作者】 郝萍；

【导师】 陈正堂；

【作者基本信息】 第三军医大学 ， 肿瘤学， 2009， 博士

【摘要】 在我国,肺癌的发病率和死亡率均居恶性肿瘤首位,而非小细胞肺癌(non-small cell lung cancer NSCLC)占所有肺癌患者80%左右,其中65%～70%的NSCLC就诊时已处于晚期。因此,化疗在Ⅲ/Ⅳ期NSCLC患者综合治疗中占重要地位。自80年代以来,随着第三代化疗新药异长春花碱、吉西他滨及紫杉类等药物的开发和利用,以铂类为基础联合第三代化疗新药的方案已成为NSCLC标准化疗(Standard Chemotherapy SC)方案,但是化疗反应率(Response Rate RR)和中位生存时间(Median Survival Time MST)仍然很低。由于遗传的异质性,同一种病理类型和分期的NSCLC对同一种化疗方案敏感性差异很大。因此在2004美国肿瘤临床学会(American Society of Clinical Oncology ASCO)年会上提出了个体化疗(tailor chemotherapy TC)概念并成为研究热点。TC是根据癌症病人的遗传学特点使用对其最佳的化疗药物,以提高化疗敏感性,提高RR及MST,同时将化疗的副作用降至最低。由于条件限制体外肿瘤细胞药物敏感性试验较少开展,而且体外药敏结果与体内药物敏感性差异很大,表明肿瘤细胞的内在的化疗敏感性有显著不同。DNA损伤修复系统作为机体抵抗各种损伤的分子基础,对于维护基因组的稳定与完整起着至关重要的作用;然而对于通过损伤DNA杀死癌细胞的放、化疗,这一过程无疑削弱了治疗效果。因此能够提示化疗药物敏感性及预后的分子标记物在TC方案制定中将发挥重要作用。切除修复交叉互补基因1(Excision repair cross-complementation group 1 ERCC1)、p53是与肺癌化疗耐药关系密切的两个重要基因,在化疗前对NSCLC组织进行p53、ERCC1基因检测可以使化疗药物的应用更个体化,更具科学性和合理性。本研究在征得患者知情同意和符合医学伦理学原则的前提下,检测49例NSCLC手术患者组织、外周血淋巴细胞(peripheral blood lymphocyte,PBL)化疗敏感性分子标记物ERCC1、p53蛋白表达,术前血清p53抗体水平,证实NSCLC癌组织与患者PBL中ERCC1、p53表达呈显著正相关;检测74例晚期NSCLC患者PBL中ERCC1、p53表达,对ERCC1、p53与含铂方案(TP、NP或GP)化疗敏感性的关系进行了研究,证实ERCC1,p53高表达与NSCLC患者顺铂类化疗药物的耐药显著相关。本研究为TC药物选择提供参考,以期提高化疗敏感性,提高MST及RR。研究目的1.检测p53、ERCC1在NSCLC癌组织、癌旁组织、和外周血淋巴细胞(peripheral blood lymphocytes,PBL)的表达,探讨p53、ERCC1在癌组织和PBL中表达的相关性;2.探讨III、IV期NSCLC患者PBL中p53、ERCC1的表达与铂类SC方案化疗敏感性关系;3.探讨NSCLC p53蛋白表达与血清p53抗体的相关性。研究内容和方法1. p53、ERCC1在NSCLC癌组织表达及其临床意义:采用免疫荧光化学方法检测49例NSCLC癌组织和PBL中p53、ERCC1蛋白表达,分析p53、ERCC1表达与NSCLC临床病理因素的关系,探讨NSCLC癌组织和PBL中p53、ERCC1表达的相关性,为进一步以PBL内p53、ERCC1表达情况为依据制定TC方案提供临床病理依据。2. PBL中ERCC1、p53的表达与Ⅲ/Ⅳ期NSCLC患者含铂类标准化疗方案敏感性的关系:采用免疫荧光细胞化学染色方法检测74例晚期NSCLC患者PBL中p53、ERCC1蛋白表达。给予患者2～4疗程铂类为基础的SC化疗,对病人化疗后的总生存期(OS)、肿瘤进展时间(TTP)、化疗疗效及一年生存率进行评价。3. NSCLC患者血清p53抗体检测及其与化疗敏感性相关性研究:采用免疫荧光化学染色方法检测NSCLC患者ERCC1、p53蛋白表达, ELISA检测术前血清p53抗体浓度,术后给予患者铂类为基础的SC化疗,对近期疗效进行评价。研究结果1. p53、ERCC1在NSCLC癌组织表达及其临床意义:p53、ERCC1主要呈细胞核染色,在细胞核内均匀分布,一些肿瘤细胞的胞质中也有表达,集中于核膜周围。2.癌组织、癌旁组织、正常肺组织、NSCLC的PBL和健康人的PBL的ERCC1阳性率分别为55.10%、26.53%、10.2%、51.10%和10%,p53阳性率分别为63.26%、30.61%、8.186、59.18%和5%, NSCLC患者癌组织和PBL的ERCC1、p53表达阳性率均高于癌旁组织、正常肺组织、健康人PBL(P<0.01)。癌组织及PBL中ERCC1、p53表达率与临床分期和淋巴结转移相关(P<0.05)。3. NSCLC癌组织与患者PBL中ERCC1、p53表达呈显著正相关(x2=14.8596 p<0.01;x2=21.291, p<0.01)。4. ERCC1、p53的表达与Ⅲ/Ⅳ期NSCLC患者含铂类标准化疗方案敏感性的关系:根据ERCC1,p53免疫荧光标记结果将NSCLC患者分为4组:EP组(阳性组:ERCC1及p53均高表达组)37例、N组(阴性组:ERCC1及p53均低表达组)29例、E组(仅ERCC1高表达组)4例、P组(仅p53高表达组)4例。4组患者的OS分别为(月)10.65±4.91、14.17±6.63、11.00±7.5、11.50±5.75。TTP分别为(月)7.78±4.59、12.41±7.16、8.25±7.75、10.75±6.12,组间差异有统计学意义(P<0.05),两组间两两比较结果示N组与其它各组间OS及TTP差异均有统计意义(P<0.01),而其它3组间OS及TTP差异没有统计学意义(P>0.05);N组化疗后RR为65.52%,显著高于EP组35.13%,而其它3组间比较则没有统计意义(P>0.05)。提示:ERCC1和p53均低表达的NSCLC患者,可以更好的从含铂类药物化疗方案中受益。5. p53蛋白在NSCLC高分化、中分化和低分化患者癌组织中的阳性表达率分别为:37.50%、53.84%和90.00%,p53阳性表达率与肿瘤分化程度显著相关(P=0.004);p53抗体在NSCLC高分化、中分化和低分化患者血清中的阳性率分别为43.75%、30.77%和75.00%,血清p53抗体与肿瘤分化程度相关(P=0.03)。49例NSCLC患者癌组织p53蛋白、血清p53抗体均阳性20例,均阴性12例,癌组织p53蛋白阳性而血清p53抗体阴性2例,癌组织p53蛋白阴性而血清p53抗体阳性5例。NSCLC患者癌组织p53蛋白与血清p53抗体表达相关(χ2=4.446 P=0.035)。结论1、NSCLC癌组织p53、ERCC1表达与肿瘤分化程度、TNM分期以及有无淋巴结转移相关。2、检测NSCLC患者PBL的p53、ERCC1表达可间接反映NSCLC组织的p53、ERCC1表达状况。3、PBL中p53、ERCC1表达情况与含铂化疗方案敏感性显著相关。4、PBL中p53、ERCC1可作为含铂化疗方案化疗敏感性及预后分子标记物,二者均低表达的NSCLC患者为最适含铂案TC靶向人群。5、p53蛋白、血清p53抗体阳性NSCLC患者,肿瘤分化程度低、恶性程度高。6、癌组织p53蛋白和血清p53抗体联合检测将有助于NSCLC的诊断及判断生物学行为和患者的预后。更多还原

【Abstract】 Morbidity and mortality of lung cancer occupied the first place of malignant tumors in our country, with 80% of nonsmall-cell lung cancer (NSCLC) in lung cancer. Unfortunately 65-70% of NSCLC was already in advanced stage when be diagnosed. Therefore chemotherapy plays a vital role in the combined therapy of stage III/IV NSCLC. Since 1980s, accompanying the pharmaprojects and utilization of the third generation agents, such as vinorelbine, gemcitabine, and Japanese yew branchlet, the third generation combined chemotherapy based on platinum became the standard chemotherapy profile. However, the reaction rate (RR) and meso-survive time of chemotherapy remained unsatisfactory. Because of the genetic heterogeneity, even the NSCLC patients with the same kind of pathological phenotype and stage can behave much differently to the same chemotherapy. Hence the tailor chemotherapy (TC) was presented in the 2004 American Society of Clinical Oncology (ASCO), and was prevailed. TC means to take out the most optimal chemotherapy agents based on its genetic characteristics, in order to improve the chemical sensitivity, improve the RR and MST, and to decrease the side effects. According to the conditions, there are seldom studies on the chemosensitivity of extracorporeal tumor cells. There would be much different between the extracorporeal and corporeal chemosensitivity. DNA damage-repair system is the molecular basis for the body to resist the damage, which is important to maintain the stabilization and integrity of the gene groups. Radiotherapy or chemotherapy would induce the damage to DNA, which would consequently weaken the therapeutic efficacy. Molecular markers which can hint the chemosensitivity and prognosis seem to play important roles in TC profiles. P53 and ERCC1 gene are two genes with tight relationship to chemoresistance of lung cancer. To detect the p53 and ERCC1 genes of NSCLC tissues before the chemotherapy can individualize the chemical agents, to be more scientific and rational. This study was approved by the patients and the medical ethics committee. In this study, ERCC1 and p53 protein expressions, pre-operational p53 antigbody of the carcinoma tissue and peripheral blood lymphocyte (PBL) in 49 patients with NSCLC were detected, and the correlation of ERCC1 and p53 expressions were confirmed between those in the NSCLC tissue and in PBL. ERCC1 and p53 expressions of PBL in 74 patients with NSCLC were detected, to evaluate the relationship between the ERCC1, p53 expressions and chemosensitivity to project with TP, NP or GP. Correlation between the hyper-expressions of ERCC1, p53 and chemoresistance to platinum in NSCLC was confirmed. This study can provide a reference for the choice of TC agents, to improve the chemosensitivity, MST and RR.Objects1. To detect the impression of p53, ERCC1 in the carcinoma tissue, peri-carcinoma tissue and PBL of NSCLC patients, to study the correlations of p53 and ERCC1 expressions between carcinoma tissue and PBL.2. To study the relationship between p53, ERCC1 expression of PBL in NSCLC and the SC project with platinum.3. To study the correlation of the p53 expression between in peri-carcinoma tissues and in serum.Methods1. Expressions of p53 and ERCC1 in peri-carcinoma tissues in NSCLC and their clinical values: To detect the expressions of p53 and ERCC1 protein in peri-carcinoma tissues and PBL in NSCLC with immonofluorescence chemistry, and to evaluate their relationship with pathological characteristics, to provide a clinical pathological basis for the TC profiles.2. Relationship between p53, ERCC1 expression of PBL in NSCLC and the SC project with platinum: To dye the p53 and ERCC1 protein in the NSCLC with immunofluorescence cytochemical staining. After 2-4 courses of SC chemotherapy based on platinum, the total overall survival (OS), time to progression (TTP) chemical efficacy and one-year survival of the patients were evaluated.3. Correlation between the p53 antibody, p53 expression and the chemosensitivity of NSCLC: To detect the expression of ERCC1 and p53 protein in peri-carcinoma tissue of NSCLC with immunofluorescence chemistry, to detect the serum p53 antibody concentration, and to perform the SC chemotherapy based on platinum, so as to estimate the near future efficacy.Results1. p53 and ERCC1 expression in the peri-carcinoma tissues of NSCLC and their clinical values: p53 and ERCC1 were mostly nuclear-dyed. They homogeneously distributed in the nuclei, and also plasmatically distributed along the nuclear membrane.2. p53 positive rate of carcinoma tissue, peri-carcinoma tissue, normal lung tissue, PBL of NSCLC, normal PBL were respectively 63.26%、30.61%、8.186、59.18% and 5%. ERCC1 positive rates were 55.10%、26.53%、10.2%、51.10%和10%. P53 and ERCC1 expressions of carcinoma tissue and PBL in NSCLC significantly higher that those of peri-carcinoma tissue, normal lung tissue and normal PBL (P<0.01). the p53 and ERCC1 expressions in the peri-carcinoma tissue and PBL correlated with the clinical stages and lymphonode metastasis (p<0.05).3. p53 and ERCC1 expressions in peri-carcinoma tissue of NSCLC correlated with those in PBL (x2=14.8596 p<0.01;x2=21.291, p<0.01).4. Relationship between ERCC1, p53 expressions and the chemosensitivity of III/IV stage NSCLC to chemotherapy based on platinum: NSCLC patients were divided into 4 groups based on the ERCC1 and p53 positive immunoinfluence markers: EP group (both ERCC1 and p53 highly-expressed) 37 cases; N group (both ERCC1 and p53 lowly-expressed) 29 cases; E group (only ERCC1 highly-expressed) 4 cases; P group (only p53 highly-expressed). The OS in the 4 groups were respectively (months) 10.65±4.91,14.17±6.63,11±7.5 and 11.5±5.75. The TTP in the 4 groups were respectively (months) 7.78±4.59、12.41±7.16、8.25±7.75、10.75±6.12. Both OS and TPP were statistically significant among the groups (P<0.05). OS and TPP in N group were significantly different against the other 3 groups (P<0.01), and no significant difference between the other 3 groups (P>0.05). post-chemotherapy RR in N group was 65.52%, significantly increased against that of EP group (35.13% increase), and there were no significant difference among the other 3 groups (P>0.05). This hints those NSCLC patients with low ERCC1 and p53 expressions can benefit more from the chemotherapy based on platinum.5. Positive expressions of p53 protein in carcinoma tissue with well, moderate and poor differentiation NSCLC were respectively 37.50%、53.84% and 90.00%. p53 positive expression correlated with the tumor differentiation (P=0.04). p53 antibody in NSCLC serum with well, moderate and poor differentiation were 43.75%、30.77% and 75.00%. Serum p53 antibody correlated with the tumor differentiation (P=0.03). In 49 cases, 20 cases were both positive of p53 in NSCLC carcinoma tissue and serum, 12 cases were both negative. 2 cases were positive in carcinoma tissue and negative in serum, 5 cases were negative in carcinoma tissue and positive in serum. There is correlation between the carcinoma tissue p53 protein and the serum p53 antibody (χ2=4.446).Conclusions1. p53 and ERCC1 expressions in NSCLC tissue correlate with the tumor differentiation stage, TNM stage and lymphonode metastasis.2. p53 and ERCC1 expressions in PBL of NSCLC can reflect the p53 and ERCC1 expressions in NSCLC tissue.3. p53 and ERCC1 expressions in PBL correlated with the chemosensitivity based on platinum.4. p53 and ERCC1 expressions in PBL can be used as markers for chemosensitivity and prognosis based on platinum. NSCLC patients with low expressions of p53 and ERCC1 are most suitable for the TC project with platinum.5. Positive expressions of p53 protein in carcinoma tissue or serum p53 antibody hint a low tumor differentiation stage and high malignance.6. Combine test of the carcinoma tissue p53 protein and the serum p53 antibody can be valuable for the diagnose and prognosis of the NSCLC.更多还原