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类型 基础研究 预答辩日期 2017-12-15
开始(开题)日期 2013-06-25 论文结束日期 2017-09-26
地点 东南大学医学院基一楼会议室 论文选题来源 非立项    论文字数 6 (万字)
题目 肺腺癌的全基因组表达谱分析和Polo样激酶1表达的临床病理研究
主题词 肺腺癌,全基因组表达谱,PLK1,临床病理特征,预后
摘要 背景: 驱动基因突变如EGFR和KRAS突变,MET扩增以及ALK、RET、ROS1融合基因等,在肺腺癌的发病和个体化治疗中发挥关键作用。但仍有大约30%的肺腺癌未发现明确的驱动基因突变,大约10-30%的KRAS突变型肺腺癌缺乏有效的靶向治疗手段。深入研究这部分肺腺癌发病的分子机制并寻找潜在的治疗靶点仍然是目前最重要课题之一。 肿瘤研究领域高通量的基因组表达谱分析技术正在应用于肿瘤高危人群筛查、早期诊断、分子学分型、精准治疗、监控和预测肿瘤复发、疗效评估以及预后判断等多个方面。应用高通量技术分析不同分子改变肺腺癌(EGFR突变、KRAS突变和三阴性肺腺癌)基因组表达差异将为新的潜在治疗靶点的寻找提供实验依据。 Polo样激酶1在人类多种恶性肿瘤中高表达并且是预后不良的指标。多项研究证实PLK1是治疗肿瘤的理想分子靶点,但迄今PLK1表达在肺腺癌中的研究报道不多,是否可以作为肺腺癌潜在的治疗靶点也不清楚。本研究通过观察PLK1表达与肺腺癌临床病理特征、分子改变和预后的关系,以及对肺腺癌细胞株增殖、周期分布、凋亡和侵袭能力的影响,探讨PLK1在肺腺癌中过表达的意义及作为个体化治疗分子靶点的临床价值。 方法: 1. 应用Nimblegen全基因组表达谱芯片技术分析7例肺腺癌,包括2例EGFR突变型肺腺癌、2例KRAS突变型肺腺癌和3例三阴性肺腺癌(EGFR-/KRAS-/EML4-ALK-, triple negative)的全基因组表达谱,Gene Ontology(GO)分析信号通路和基因表达差异。 2. 应用免疫组化法检测266例I-IV期肺腺癌中PLK1、TTF1、p53蛋白的表达,分别应用直接基因测序技术、免疫组化VENTANA法和FISH技术检测EGFR 18-21号外显子和KRAS 12/13号密码子突变以及EML4-ALK融合基因。分析PLK1、TTF1、p53蛋白表达与肺腺癌临床病理特征、EGFR和KRAS突变以及EML4-ALK融合的关系。采用Kaplan-Meier法进行单因素生存分析,采用Cox比例风险回归法进行多因素分析。 3. 为了观察PLK1特异性小分子抑制剂Poloxin对人肺腺癌细胞株A549(KRAS突变,TP53野生型)、H1975(EGFR突变,TP53突变型)、H1563(三阴性,TP53野生型)细胞生物学性状的影响,分别应用MTT法、流式细胞仪、Transwell试验观察Poloxin对不同人肺腺癌细胞株增殖能力、细胞周期分布、凋亡以及侵袭能力的影响,应用Real-time PCR 和Westernblot方法检测PLK1 mRNA 和蛋白的表达。 结果: 1. 肺腺癌中存在显著异常的细胞信号通路基因表达,包括糖类代谢、缺氧诱导因子、细胞黏附分子、病毒癌基因和黏蛋白合成等。本研究共确认976个基因呈显著高表达,813个基因呈显著低表达。参与肺腺癌发病和进展的45个关键基因中包括著名的驱动基因EGFR、ERBB2、KRAS、MET等和重要的抑癌基因CDKN2A、ID1、RB1和TP53等。EGFR突变型肺腺癌中显著异常表达的基因有EGFR、MYCL1、NKX2-1、SP-B/C、DUSP4等。 KRAS突变型肺腺癌异常表达的基因有KRAS、PLK1、CEACAM5、DDR1、LKB1和TP53等。三阴性肺腺癌显著异常表达基因有AHNAK2、CYP24A1、EGFR、KRTAP5-8、MMP7、PLK1等。PLK1在KRAS突变型和三阴性肺腺癌均呈显著高表达。 2. 62%(165/266)的肺腺癌呈PLK1蛋白过表达。PLK1过表达与吸烟史(P<0.05)、转移/复发(P<0.01)以及高临床分期(III-IV期)(P<0.05)显著相关。在不同组织学类型的肺腺癌中,PLK1过表达常见于胶样腺癌(87.5%)、浸润性黏液腺癌(86.7%)和实体型腺癌(81.1%),在其他类型的肺腺癌中表达较低(P<0.01)。 PLK1过表达在泌黏液型腺癌(73.1%)中发生率显著高于非泌黏液型腺癌(57.5%)(P<0.05)。 3. PLK1过表达与KRAS突变、p53表达存在显著相关性(P<0.01),与TTF1表达、EGFR突变和EML4-ALK融合基因无显著相关性(P>0.05)。79%(98/124)的三阴性肺腺癌呈PLK1过表达,与非三阴性肺腺癌比较存在显著性差异(P<0.01)。 4. PLK1过表达的患者总生存率(OS)显著低于PLK1不/低表达的患者(P<0.01),三阴性肺腺癌中,PLK1过表达者的总生存率(OS)也显著低于PLK1不/低表达者(P<0.05)。多因素分析显示临床分期是肺腺癌最重要的预后因素(HR=2.874, P<0.001),PLK1过表达可作为肺腺癌的独立预后因素(HR=2.235, P<0.01)。 5. PLK1小分子抑制剂Poloxin对不同分子改变的肺腺癌细胞株的生物学性状均产生显著影响,其中对A549和H1563的影响较H1975显著。表现为细胞增殖能力的显著降低(P<0.01),周期分布G2期细胞比率(P<0.05)和凋亡指数(P<0.05)的显著增加,侵袭能力的显著降低(P<0.05)以及PLK1蛋白表达水平的显著降低(P<0.01)。 结论: 1. 肺腺癌存在显著的基因组表达谱异常,EGFR突变型、KRAS突变型与三阴性肺腺癌的基因组表达谱存在明显的差异,该研究发现为进一步探讨不同分子改变的肺腺癌的发病机制,以及筛查个体化治疗靶点奠定了实验基础。 2. PLK1过表达的肺腺癌具有明显的临床病理特征:常见于吸烟患者,组织学特征常表现为富于细胞内/外黏液的腺癌,多为高临床分期(III-IV期),常表达p53,常见于KRAS突变和三阴性肺腺癌,预后较差。PLK1过表达在部分肺腺癌的发病和进展过程中可能发挥重要作用,对于PLK1过表达的患者临床应密切随访并建议采取更积极的治疗措施。 3. 体外实验显示PLK1特异性抑制剂Poloxin对肺腺癌细胞株有明显的抑制作用,但不同细胞株的敏感性存在明显差异,进一步证实PLK1可以作为KRAS突变和三阴性肺腺癌潜在的个体化治疗靶点。
英文题目 Global gene expression profiling analysis and clinicopathological study of Polo-like kinase 1 expression in lung adenocarcinoma
英文主题词 lung adecnocarcinoma,global gene expression profiling,PLK1,clinicopathological characteristics, prognosis
英文摘要 Background: Driver mutations such as EGFR and KRAS mutation, MET amplification, and ALK, RET, ROS1 translocations, play a key role in tumorigenesis and personalized therapy of lung adenocarcinoma (ADC). However, about 30% lung ADCs have not been found the known oncogenic driver mutations. Moreover, there was about 10-30% lung ADCs harbored KRAS mutation, which the clinical benefits of KRAS-targeting therapy have not been defined yet. So the exploration of molecular pathogenesis and potential personalized therapeutic targets in these ADCs is still one of the most important topics and should be further studied. In the field of cancer research,high-throughputt gene expression profiling was applied in many aspects, including the screening of those tumor susceptible people, early diagnosis, the classification of molecular phenotype, targeted therapy, Monitoring and predicting tumor recurrence, therapeutic effect evaluation and prediction of prognosis. The global gene expression profiling was screened by high throughput genome scanning in EGFR mution ADCs, KRAS mutation ADCs and triple negative ADCs, which will provide experimental bases for searching new potential therapeutic targets. Numerous studies have confirmed Polo-like kinase 1 (PLK1) is highly expressed in a broad spectrum of cancers and its overexpression often correlates with poor prognosis. PLK1 could play an ideal personalized therapeutic target of tumors. However, the study about PLK1 expression in lung ADC is very rare and whether or not PLK1 as a potential therapeutic target remains unknown so far. In the present study, the correlation with PLK1 overexprssion and the clinicopathological characteristics, molecular genetic alterations and patient prognosis was surveyed and the impact of PLK1 overexprssion on cell proliferation, cell cycle, apoptosis and tumor invasion was observed. The present study further demonstrated the clinic significance of PLK1 overexprssion and the value as a potentially personalized therapeutic target of lung ADC. METHODS 1. The global gene expression profiling was screened by Nimblegen DNA array based whole genome scanning in 7 lung ADC cases (2 cases of EGFR mution ADC, 2 cases of KRAS mutation ADC and 3 cases of triple negative ADC). The key cell signaling pathways and differentially Gene Ontology (GO) term-based genes were identified using clustering and discriminant analysis. 2. The expressions of PLK1, TTF1 and p53 protein in 266 patients with stage I-IV lung ADC were detected by immunohistochemistry (IHC). Mutations of EGFR 18-21 exons, KRAS 12/13 codons and EML4-ALK fusion were detected by directive gene sequencing,VENTANA immunohistochemical staining and fluorescence in situ hybridization (FISH), respectively. The correlations with PLK1, TTF1 and p53 expression and the clinicopathological characteristics, the mutation of EGFR and KRAS, and EML4-ALK fusion were further analyzed. Kaplan-Meier curves were constructed to analyze patient survival and Cox multivariate analyses were performed to identify independent factors indicating an unfavorable prognosis. 3. For the purpose of observing the influences of small molecular PLK1 inhibitor, Poloxin, on the biological properties of human lung ADC cell A549 (KRAS mutation, wild type p53), H1975 (EGFR mutation, p53 mutation) and H1563 (triple negative, wild type p53). MTT assay was performed to evaluate cell proliferation, and flow cytometry was performed to evaluate cell cycle and apoptosis, and transwell test was conducted to observe tumor cell invasion. Moreover, Real-time PCR and Westernblot were applied to analyze the expression of PLK1 mRNA and protein. Results: 1. The significant difference in key cell signaling pathways was observed between lung ADCs and normal lung tissues using clustering and discriminant analysis, including fructose and mannose metabolism, HIF-1 signaling pathway, cell adhesion molecules, viral carcinogenesis, mucin type O-Glycan biosynthesis and other cell signaling pathways. There were 976 genes exhibiting significantly higher expression and 813 genes showing significantly lower expression using GO term-based genes analysis. Forty-five key genes involving the pathogenesis and evolution were identified, and some of which were crucial driver genes including EGFR, ERBB2, KRAS, MET, CDKN2A, ID1, RB1 and TP53. Some important genes involving lung ADC harbored EGFR mutation were identified such as EGFR, MYCL1, NKX2-1, SP-B/C, DUSP4, and KRAS, PLK1, CEACAM5, DDR1, LKB1 and TP53 involving lung ADC harbored KRAS mutation as well as AHNAK2, CYP24A1, EGFR, KRTAP5-8, MMP7 and PLK1 involving triple-negative lung ADC. Both lung ADC harbored KRAS mutation and triple-negative tumor showed significantly higher PLK1 expression. 2. PLK1 overexpression was exhibited in 62% (165/266) cases of lung ADC. There was positive correlation with PLK1 overexprssion and smoking history (P<0.05), metastesis/recurrence (P<0.01), and higher stage (stage III-IV) (P<0.05). According to different histological subtypes, PLK1 overexpression was more frequently found in colloid ADCs (87.5%), invasive mucinous ADCs (86.7%) and solid ADCs (81.1%), yet relatively uncommon in other ADC subtypes (P<0.01). PLK1 overexprssion was more common in mucin-product ADCs than in non mucin-product ADCs (P<0.05). 3. PLK1 overexpression significiantly correlate with KRAS mutation (P<0.01), p53 protein expression (P<0.01), other than TTF1 expression, EGFR mutation or EML4-ALK fusion (P>0.05). 79% (98/124) triple negative ADCs showed PLK1 overexpression. There was significant difference of PLK1 overexpression between triple negative and non-triple negative ADCs (P<0.01). 4. There was significant difference with respect to overall survival between lung ADCs with PLK1 overexpression and no or weak expression (P<0.01), as well as in the triple negative ADCs (P<0.05). Cox multivariate analysis indicated that stage was the most important determinant of overall survival (HR=2.874, P<0.001), and PLk1 overexpression was acted as an independent prognostic factor (HR=2.235, P<0.01). 5. Poloxin influenced significantly on the biological properties of human lung ADC cell lines with different molecular alterations. The effect was more obvious on A549 and H1563 as compared with H1975 cell line. The results showed that the ability of cell proliferation (P<0.01) and invasion (P<0.05) dramatically declined, the ratio of G2 cells (P<0.05) and the apoptotic index (P<0.05) significantly increased, and the level of PLK1 protein expression (P<0.01) sharply inhibited in vitro test. Conclusion: 1. There were amount of abnormally expressed genes in lung ADCs. The significant difference in gene expression profiling was confirmed among lung ADCs hanbored EGFR mutation, KRAS mutation and triple negative lung ADCs. These important founds laid a foundation on further study of pathogenesis and screening the potentially personalized therapeutic target of lung ADCs 2. Lung ADCs with PLK1 overexpression exhibited some definitely clinicopathological characteristics, such as smoking, ADCs with intra-/ extra-cellular mucin product, higher stage (stage III-IV), p53 expression, KRAS mutation and triple negative pattern. The cases of lung ADC with PLK1 overexpression presented unfavorable prognosis. PLK1 overexpression maybe plays an important role in pathogenesis and evolution of lung ADCs and acts as a predictor of patein’s strengthening treatment and follow-up. 4. In vitro tests of the study showed that Poloxin influenced significantly on the biological properties of human lung ADC cell lines, but sensitivity of cell lines with different molecular alterations was significant different. The results preliminarily indicated that PLK1 overexpression could act as a potentially personalized therapeutic target of lung ADCs hanbored KRAS mutation and triple negative lung ADCs.
学术讨论
主办单位时间地点报告人报告主题
东南大学附属中大医院肿瘤科 2011年5月11日 东南大学附属中大医院肿瘤科会议室 徐艳 信号基因体细胞突变检测对非小细胞肺癌分子靶向治疗的意义
东南大学病理和病理生理学系 2012年9月12日 基一楼203 徐艳 肺活检标本中免疫组化对低分化非小细胞癌分类诊断的作用
东南大学病理和病理生理学系 2013年4月17日 基一楼203 徐艳 Cribriform adenocarcinoma of the lung: a distinctive morphologic subtype of lung adenocarcinoma
南京军区南京总医院病理科 2013年10月14日 南京军区南京总医院病理科会议室 徐艳 肺腺癌术前芯针活检所致活检部位的改变—判断间质浸润潜在的诊断陷阱
东南大学肾脏病研究所 2013年10月18日 综合楼 沈振涛 生物医药领域专利申请与撰写的特殊要求
东南大学病理和病理生理学系 2013年10月23日 基一楼203 丁粉干 Methods in Mammalian Autophagy Research
东南大学病理和病理生理学系 2013年12月03日 基一楼203 丁粉干 金纳米粒对缺氧肾小管上皮细胞的损伤作用及其机制研究
南京军区南京总医院病理科 2012年6月4日 南京军区南京总医院病理科会议室 刘标 中国肺癌EGFR基因检测现状和规范化要求
     
学术会议
会议名称时间地点本人报告本人报告题目
中华医学会第十九次病理学术会议暨第三届全国病理学术年会 2013年11月8日 广州
中华医学会第二十次病理学术会议暨第四届全国病理学术年会 2014年11月15日 重庆 筛状为主型和寡粘液实体型Ⅰ 期肺腺癌的预后研究
2014年中华医学会病理学分会胸部疾病学组成立大会及辽宁省第25届学术年会 2014年7月19日 长春 肺小细胞肺癌(NSCLC)免疫组化诊断专家共识
首届全国淋巴增生性疾病病理读片会 2012年9月14日 成都
     
代表作
论文名称
Circulating tumor cell detection A direct comparison between negative and unbiased enrichment inlung
肺腺癌中PLK1的异常表达及其临床病理意义
Ⅰ期肺腺癌中肿瘤经肺泡腔隙扩散的临床病理观察及预后意义
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
王亚平 正高 教授 博导 南京大学医学院医学遗传学
石群立 正高 教授 主任医师 硕导 中国人民解放军南京总医院病理科
陈平圣 正高 教授 博导 东南大学医学院病理与病理生理学系
余卫平 正高 教授 博导 东南大学医学院病理与病理生理学系
张海军 副高 副研究员 博导 东南大学附属中大医院肿瘤科精准治疗中心
      
答辩秘书信息
姓名职称工作单位备注
丁粉干 其他 博士 住院医师 东南大学附属中大医院