【作者】 李丽；

【导师】 周庚寅； Anders Zetterberg；

【作者基本信息】 山东大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 【研究背景】乳腺癌目前已经成为全球女性发病率最高的恶性肿瘤,严重影响着女性的生命和健康。乳腺癌是一种多基因参与的复杂疾病,参与调控细胞生长、分化与凋亡的一系列染色体异常导致了肿瘤的发生。更好的认识其基因改变能为患者提供更为精确的诊断和合理的治疗。染色体不稳定性是指癌细胞较之于正常细胞,在细胞分裂时丧失和(或)获得整条染色体或染色体片段频率的升高,可以具体的分为数目改变和结构改变。染色体不稳定是恶性肿瘤的最基本特征,几乎所有人类肿瘤都存在染色体不稳定,对肿瘤恶性生物学行为的判断具有重要价值。过去大批基因表达图谱分析发现了一系列的乳腺癌分子异常和一些对临床诊断治疗有用的基因表达亚型。但是由于分辨率的不足,比较基因组杂交(comparative genomic hybridization,CGH)等常用方法确定基因组的DNA拷贝数的能力受到限制。最新的细胞分子遗传学技术进展为我们提供了高分辨率检测细胞染色体不稳定性的一个平台。采用代表性寡核苷酸芯片分析(representationaloligonucleotide microarray analysis,ROMA)和定量多基因荧光原位杂交(quantitative multi-gene FISH,QM-FISH)使得对肿瘤基因组进行高分辨率分析成为可能。ROMA是迄今为止敏感性和特异性最强的微阵列杂交技术。QM-FISH技术可在同一细胞核内检测多个不同基因,并且可用于石蜡包埋组织。使用ROMA技术,可以寻找有意义的染色体异常,确定研究靶向,而使用QM-FISH技术则能检测细胞内具体位点的拷贝数改变(copy number alterations,CNAs),反映单个细胞的基因改变,对有意义的基因进行深入研究。两种技术结合,可以发现更广泛的基因扩增、缺失和易位。p53基因是迄今发现与人类肿瘤相关性最高的基因,但并非所有的恶性肿瘤都存在p53突变。MDM2(murine double minute 2)和MDMx是是两种重要的p53负性调节因子。MDM2与MDMx扩增或过表达频发于原发性乳腺癌,其中大部分肿瘤表达野生型P53蛋白。本研究分为两部分。第一部分在前期对原发性乳腺癌ROMA研究的基础上,采用QM-FISH对人乳腺癌染色体改变进行高分辨率检测,筛选出一组特异性分子标记物,用于系统的鉴定不同患者的分子异常,并且检测同一肿瘤导管内癌与浸润性导管癌之间的分子表达差异,研究特定分子异常在乳腺癌发生发展中的作用机制。第二部分研究MDMx-MDM2-p53在在乳腺癌发生中的作用机制。该研究将提高我们对乳腺癌发生发展过程中遗传学改变的认识,为乳腺癌病理诊断、预后评估以及制定个体化治疗方案提供理论基础。【研究方法】收集山东大学齐鲁医院2005-2006年乳腺癌34例,所有病例均为原发性乳腺癌,同时包含原位癌和浸润癌。分别取所有石蜡标本肿瘤原位癌与浸润癌组织制作组织芯片。图像细胞学分析肿瘤细胞DNA倍体。根据ROMA检测结果,筛选在乳腺癌中扩增或缺失比较频繁的30个基因,使用UCSC(University ofCalifornia,Santa Cruz)genome browser选择设计探针,Qiagen(?)质粒纯化试剂盒分离提取BAC克隆,切口平移法对分离纯化的探针进行荧光素标记,QM-FISH法检测30个基因的拷贝数改变,同一视野照相记录。使用激光扫描共聚焦显微镜进行图像采集,Axio Vision分析软件进行图像分析。并比较30个基因在导管内癌与浸润性导管癌中的拷贝数差异,以及与临床病理指标之间的关系。FISH检测34例原发性乳腺癌p53,MDMx与MDM2的基因拷贝数,免疫组织化学技术检测P53与MDM2蛋白表达情况,分析三种基因之间的相互作用以及与临床病理指标之间的联系。【实验结果】1.34例乳腺癌中,11例为二倍体肿瘤即D-tumor(32%),23例为非整倍体肿瘤即A-tumor(68%)。2.QM-FISH检测34例原发性乳腺癌中30个候选基因的拷贝数,所有病例均发生了两个或两个以上的基因改变,平均每例乳腺癌6.5个基因发生数目增益或缺失。3.CCNE2,C-erbB2,IGF1R,CKS1a,c-myc,CCND1扩增和chk1,p53,Rb1,CDH1,chk2,Nek9缺失均见于25%以上的病例,其中MDMx扩增(59%)最为常见。4.34例肿瘤浸润癌成分共发生222个基因异常事件,原位癌成分共检测到194个基因异常事件,差异无显著性,部分基因拷贝数仅在少数病例的导管内癌和浸润癌中基因有差别,无显著差异。5.在检测的30个基因中,我们发现了多种基因异常存在相关性。包括CCND1扩增与chk1缺失呈正相关(P＜0.05);c-myc扩增与C-erbB2扩增、CCNE2扩增、LZST1缺失正相关(P＜0.05),其中c-myc、LZST1与CCNE2基因改变相互之间存在相关关系(p＜0.01),p53缺失与MDMx扩增呈负相关(P＜0.05)。6.CNAs在组织学分级高(Ⅱ级与Ⅲ级)的肿瘤、非整倍体肿瘤和腋窝淋巴结转移阳性肿瘤更为常见(P＜0.05),并且不同的临床病理亚型有不同的CNAs。7.34例原发性乳腺癌中,16例存在p53杂合性缺失,20例存在MDMx扩增,MDM2扩增仅有1例,但27例显示MDM2蛋白过度表达。8.MDM2过表达或者p53功能异常见于33例肿瘤,但二者不同时出现(P＜0.01);31例肿瘤有MDM4扩增与P53功能异常,二者呈负相关(P＜0.05)。【结论】1.多种基因异常(基因扩增、缺失)参与了乳腺癌的发病与进展。2.MDMx,CKS1a,CCNE2,IGF1R,C-erbB2,c-myc,CCND1扩增与chk1,chk2,Rb1,p53,CDH1缺失是原发性乳腺癌常见的染色体异常,可视为乳腺癌相关基因,对于乳腺癌早期诊断和预后判断具有重要意义。3.原位癌和浸润癌基因拷贝数改变基本一致,即与浸润癌有同等程度的基因组不稳定性,是一种遗传学上处于进展期的病变。4.CNAs在体积较大的肿瘤、非整倍体肿瘤和腋窝淋巴结转移阳性肿瘤更为常见,且不同临床病理亚型的乳腺癌有不同的CNAs。5.乳腺癌中出现多种细胞周期调控基因扩增/缺失,细胞周期失控与恶性肿瘤发生密切相关。6.乳腺癌的发生发展是一个多基因参与的过程,诸多基因相互作用、相互影响,单一基因改变不能完满解释这一过程,进行多个基因之间相互作用的研究,能更好地从分子水平上阐述它们之间的联系和肿瘤发生的机制,大样本分析对于深入研究乳腺癌多基因异常之间的联系有帮助。7.MDMx扩增与p53缺失是原发性乳腺癌中频发事件,而MDM2则为低发事件。8.原发性乳腺癌中MDM2过表达/MDMx扩增与p53异常相互排斥,高水平MDM2与MDMx导致p53通路中断。9.MDMx通过MDM2非依赖性途径抑制p53功能、发挥致癌作用,联合应用MDM2与MDM4抑制剂能有效治疗表达野生型p53的乳腺癌患者。更多还原

【Abstract】 [Background]Breast cancer is a leading cause of cancer-related morbidity and mortality among women worldwide. As cancers evolve, their genomes undergo many alterations, including point mutations, rearrangements, deletions, and amplifications (often including growth control genes, anti-apoptotic genes and cell cycle checkpoint genes). An understanding of these changes will allow the design of more rational therapies and, by providing precise diagnostic criteria, allow fitting the correct therapy to each patient according to need.Chromosomal instability refers to the rate with which whole chromosomes or large portions thereof are gained or lost in cancers. The accumulation of chromosomal instability is characteristic of all carcinomas, including breast cancer. Chromosomal instability has been proposed to play an important role in cancer by accelerating the accumulation of genetic changes responsible for cancer cell evolution.More recently, molecular profiling methods have been used to identify clinically-relevant tumor features. Expression profiling has been very effective at revealing phenotypic subtypes of breast cancer and clinically useful diagnostic patterns of gene expression in tumors. But microarray studies have not had sufficient resolution to detect copy number. Recent technological advances have provided platforms that allow genomic amplifications and deletions to be analyzed in association studies. The combination of the two newly developed techniques ROMA (representational oligonucleotide microarray analysis) and QM-FISH(quantitative multi-gene fluorescent in situ hybridization) allow high resolution genomic analysis of specific deletions and amplifications. ROMA is a high sensitive and specific microarray technique and measures copy number values across the genome averaged over the population of a tumor. QM-FISH is a cytological technique that measures copy number values in individual cells. It has recently been shown that the QM-FISH technique works on formaldehyde fixed tumour cells in paraffin blocks. QM-FISH is therefore a very suitable technique for large-scale retrospective clinical studies. In summary ROMA is the technique to identify interesting chromosomal regions and genes, but QM-FISH is the technique of choice to quantify copy number changes of these genes or chromosomal regions in clinical tumour samples. These two powerful tools open the possibility for high resolution genome wide analysis and a detailed study of chromosomal instabilities.The p53 gene is often referred as the gene most related to tumours. The p53 gene is mutated in many, but not all human malignancies.MDM2 and MDM4 are the key negative regulators of p53 function in vivo. MDM2 and MDMx overexpression have been observed in a subset of human tumors, some of which retain wild-type p53.The thesis composed of two parts. The specific aim of the first study was scanning a panel of specific molecular markers for identifying the genetic aberration of individual patients, defining the distinct genetic subtypes of breast cancer and comparison of patterns of chromosomal alterations in DCIS(ductal carcinoma in situ) and their concurrent invasive breast cancer. High resolution genome analysis in primary breast cancer was performed by QM-FISH based on the previous ROMA analysis. Furthermore, we explored associations between CNAs and clinicopathological parameters in breast cancer. The second study was focused on evaluation of abnormalities of p53, MDMx and MDM2 genes and the relationships between each other. Here we assessed the genetic instability at p53, MDMx and MDM2 using FISH protocol and detected the expression status of P53 and MDM2 proteins by immunohistochemistry in 34 archived primary breast cancers. This study may provide a basis for improved patient prognostication, as well as a starting point to define important genes contributing to breast cancer development and progression.[Methods]The first part was based on thirty-four primary invasive breast carcinoma samples with foci DCIS diagnosed at the Department of Pathology, Qilu Hospital of Shandong University. The part of the tissue that contains normal ducts or lobules, DCIS and invasive carcinoma was cored and placed in the recipient block forming the array. Frequently amplified and deleted loci were chosen based on the previous ROMA data and CGH data. Hybridization probes for FISH were created from bacterial artificial chromosomes (BAC) selected using the UCSC Genome Browser. QM-FISH for 30 genes were performed on 4μm slides. Evaluation of signals was carried out in an epifluorescence microscope. Selected cells were photographed in a Zeiss Axioplan 2 microscope equipped with an Axio Cam MRM CCD camera and Axio Vision software. The difference of patterns of chromosomal alterations in DCIS and invasive breast cancer and the correlation between FISH data and clinicopathological parameters were evaluated.In the second study, to evaluate the abnormalities of p53, MDMx and MDM2 genes and the relationship between each other, we assessed the genetic instability at p53, MDMx and MDM2 loci using FISH protocol and detected the expression status of p53 and MDM2 by immunohistochemistry in 34 archived primary breast cancers. The interaction of three genes and their correlation with clinicopathological parameters were also assessed.[Results]1. Overall, among the 34 breast cancers,11 were D-tumors(32%) and 23 were A-tumors (68%). 2. All cases had at least two chromosomal aberrations out of 30 loci. The mean number of chromosomal alterations was 6.5.3. Gains of CCNE2, C-erbB2, IGF1R,CKS1a,c-myc,CCND1 and loss of chkl,p53, Rbl,CDHl,chk2, Nek9 were observed in more than 25% of the analyzed cases .Gain of MDMx is the most frequently affected chromosomal region in breast cancer.4. The invasive component of individuals breast carcinoma totally presented 222 CNAs by QM-FISH, however the DCIS component presented 194 CNAs (P>0.05). All the pairs showed a strong similarity between the DCIS and IDC with few differences.5. There are significant association between CCND1 amplification and chk1 deletion (P <0.05), c-myc amplification and LZST1 deletion (P<0.01), MAPK3 amplification and JARID2 deletion (P<0.001), WTAP amplification and TPTE deletion(P <0.001), c-myc amplification and CCNE2 amplification(P <0.001).Inverse relationship between LOH (loss of heterozygosity) of p53 and MDMx amplification was found(P <0.05).6. The frequencies of occurrence of the gene copy number gains/losses in larger tumors(>2cm), A-tumors, and node positive tumors were higher than in smaller tumors, D-tumors and node negative tumors (P<0.05) .7. Allelic loss of p53 was detected in 47% (16/34) of the cases and amplification of MDMx was found in 20 cases (59%), while MDM2 amplification was identified only in one of these samples. But 27 out of 34 cases (79%) showed MDM2 overexpression indicating MDM2 amplification is an uncommon event in primary breast cancer.8. Most tumors contained either p53 dysfunction or MDM2 alteration, but not both. This distribution was significant (P < 0.01). Significant inverse correlation between MDM4 amplification and p53 expression was also observed (P < 0.05).[Conclusions]1. Breast cancer develops multiple chromosomal alteration (including gene amplifications and deletions) as they evolve. 2. Frequently affected gene aberrations include loss of chk1, chk2, Rb1, Nek9, p53 and CDH1, and gains of MDMx,CKS1a,CCNE2, IGF1R,C-erbB2,c-myc and CCND1. The identified CNAs may provide a basis for improved patient prognostication, as well as a starting point to define important genes to further our understanding of the pathobiology of breast cancer.3. Levels of genomic instability are equivalent in DCIS lesions and advanced invasive rumors. DCIS lesions have the same extent of genomic instability as the synchronous invasive carcinomas; thus supporting the notion that invasive carcinomas evolve from or in parallel with DCIS.4. The frequencies of occurrence of the gene copy number gains/losses in larger tumors(>2cm), aneuploid tumors, and node positive tumors were higher than in smaller tumors,diploid tumors and node negative tumors (p<0.05) . Distinct spectra of CNAs underlie the different clinicopathological subtypes of breast cancer.5. Several genes undergo amplifications or deletions in tumorgenensis of breast cancer. Uncontrlled cell cycle is crucial for tumorigenesis6. Human solid tumors commonly develop multiple genetic abnormalities. Single gene alteration can not explain tumorgenensis. Study on interaction of multigenes involved in tumor development and tumor progression would be helpful to understand the mechanism of tumorgenesis.7. Overexpression of MDM2 or MDMx and p53 mutations in primary breast cancer are mutually exclusive events and combined use of MDM2 and MDM4 antagonists should be considered in the treatment of breast cancer expressing wild-type p53.更多还原