【作者】 高东伟；

【导师】 刘奇；

【作者基本信息】 山东大学 ， 肿瘤学， 2010， 博士

【摘要】 一.研究背景和研究目的转移是导致癌症患者死亡的根本原因,目前尚无有效的措施可以阻止转移。阐明癌症转移的机制是干预或控制肿瘤转移的基础,目前对于肿瘤转移认识有不同的学说,但是这些学说尚不能解释全部肿瘤转移的现象。现代生物技术和纳米制药技术的进步为发现新的抗肿瘤转移的靶点和实现药物商品化提供了可能,但是现有对肿瘤转移的认识是建立在细胞和动物实验基础上,很少有开展原发肿瘤和转移肿瘤的配对比较研究的。研究同一病人原发癌和转移癌为加深认识肿瘤转移的机制、发现新的药物靶点提供了可能。已有文献开展的对原发癌和转移癌的研究则主要集中在乳腺癌和结肠癌,而对肺癌报道的文献仅占少部分,并且主要集中在EGFR和其调节通路上。肺癌是世界上发病率最高的恶性肿瘤之一,就诊时70%左右的患者已处于中晚期,即使手术切除后患者2年内也多死于复发或转移,研究肺癌转移有望解决肺癌目前治疗困境。乳腺癌转移机制研究比较深入,现在已经发现一些关键的基因有可能成为新的抗转移的靶点,由于这些关键基因和分子在癌症中普遍发挥重要作用,这为研究肺癌转移机制提供了研究基础。分子靶向治疗是目前癌症治疗的组成部分,但是分子靶向治疗的疗效与其分子表达状态有关,比较肺癌原发灶和转移灶分子状态的异同性可以为临床医生在选择分子靶向治疗时提供参考意见。肺癌肺内转移和肺多原发癌一直是困扰临床医生的主要难题,由于多原发肺癌和肺内转移预后不同,比较原发和转移首先是确定的肺癌转移灶,因此区分肺内原发和转移是进行肺癌原发灶和转移灶比较研究的基础。一些关键分子蛋白的表达与肺癌的转移和预后密切相关,癌基因EGFR已经作为分子靶点用于治疗肺癌,血管生成因子VEGF抑制剂正在进行临床试验,MET、HIF-1 a、TGF-β1是癌症潜在的治疗靶点。但是对于这些基因的研究在肺癌主要集中在原发肿瘤的研究,在肺癌转移中发挥怎样的作用还没有文献报告。p53作为肺癌的关键抑癌基因,其突变和多态性和肺癌易感性密切相关,有研究者通过p53基因突变鉴别肺内原发和转移,其理论基础是建立在p53基因突变发生在肿瘤转移之前,转移和原发灶有共同的克隆来源和分子基础,一致的为肺内转移,不一致的为肺内原发,但是其他研究者在乳腺癌和结肠癌研究p53基因突变在原发灶和转移灶存在一定的差异。这就使得需要寻找更好的鉴别肺内原发和转移的工具。单核苷酸多态性(SNP)是第三代基因多态性,是更好地反映个体遗传差异的分子标记,在肿瘤研究中有广泛用途,p53的72密码子SNP与肺癌个体易感性已有相关研究,本课题意在比较原发肺癌和其转移癌的p53的72密码子SNP状态并且探讨作为鉴别肺内原发和肺内转移的可能性。因此,本课题研究目的是：1.比较肺癌原发和转移的肿瘤干细胞标记物CD133、血管生成因子VEGF、缺氧诱导因子HIF-α、在器官选择性转移发挥重要作用的TGF-β1、参与肿瘤微环境的肿瘤坏死因子TNF-α、抑癌基因p53、间质上皮转化因子MET和参与肿瘤特异性转移的COX2的蛋白的表达,为认识肺癌转移机制、丰富和发展肿瘤转移学说、预测肺癌转移、发现可能的抗肺癌转移的靶点奠定研究基础。2.通过p53的72密码子SNP鉴别肺内原发和肺内转移,研究p53的72密码子的多态性作为鉴别肺癌肺内原发和转移的工具的可能性。二.临床资料和方法在病理库中收集2002年-2009年期间在山东省立医院住院的原发肺癌和转移肺癌均切除的石蜡标本,共有11例为确认的肺癌原发和转移,3例其转移灶从现有标准不能确认是肺内原发和转移,其中1例肺癌原发灶有2个石蜡标本。共收集29个石蜡标本进行下列实验：1.常规HE染色：确认肺癌原发和转移来自同一标本2.SP方法免疫组化实验步骤按照说明书进行,并设阳性对照和阴性对照。3.提取石蜡标本中的DNA采用凯杰生物技术有限公司石蜡标本提取试剂盒,严格按照说明书操作。4.设计p53的72密码子的多态性引物,提取的DNA样本进行聚合酶连反应扩增,反应产物进行限制酶内切反应,图像仪下观察p53的72密码子基因型。5.蛋白表达强度在原发癌和转移癌的比较采用Marginal Homogeneity统计检验。三.结果1.临床资料：14例临床标本中11例明确诊断为转移的病例,肿瘤分级均为中低分化,9例分级相同(81.8%),其余2例患者,原发癌为中分化和低分化,转移癌仅为中分化。3例不能确定转移的患者,均为肺癌术后再次发现肺占位。2.免疫组化结果VEGF:11对组织标本中,5例样本低表达(占22.7%),14个样本中度表达(63.6%),3例高度表达(13.6%)。原发癌与转移癌表达一致9例,不一致2例,转移较原发表达增强。一致性比例为81.8%。VEGF在肺癌原发肿瘤和转移瘤表达差异未达到统计学意义,P值=0.157>0.05。不能确认转移的3例中有2例一致,1例不一致。COX2：22个样本9个低表达(40.9%),9个样本中度表达(40.9%),4个样本高度表达(18.2%)。比较原发癌和转移癌的表达情况,11个病例10例原发癌和转移癌表达一致,不一致的1例,一致性占90.9%,肺癌原发肿瘤和转移瘤表达差异未达到统计学意义P值=0.317>0.05。3例不能确认转移的2例一致,1例不一致。MET：13个样本强阳性(59.1%),6个样本呈中度表达(27.2%),低表达的为3个样本(13.6%)。9例表达一致(占81.8%),2例表达不一致(占18.2%),主要发生在肺癌脑转移,脑转移癌相对原发癌表达增强。两者比较,差异未达到统计学意义,P值=0.157>0.05。3例不能确定转移的,2例一致,1例不一致。HIF-1α：肿瘤细胞浆和核均可见到HIF-1α表达,4个低度表达(18.2%),15个中度表达(68.2%),高表达的3例(13.6%)。11例病例中,2例原发癌和转移癌表达情况不一致,转移癌HIF-1α较原发癌表达增强,9例表达一致,一致性占81.8%。两者比较,差异未达到统计学意义P值=0.180>0.05。3例不能确定转移的,原发和转移表达一致。CD133：CD133与病理类型有关,在腺癌中不表达,4例鳞癌及含有鳞癌成分的标本有少数细胞阳性表达,1例腺鳞癌中原发灶阳性细胞约2%,但是脑转移灶腺癌未发现表达。而另一例含有部分腺鳞癌成分的,其皮肤转移灶也为腺癌,均未见表达。考虑CD133可能表达与病理类型有关两者。样本阳性数目太少未进行统计学比较。3例不能确定转移的7个标本中5个腺癌标本未见表达,2个鳞癌标本有表达。TGF-β1：在肺癌原发灶和转移灶表达较弱,13个样本肿瘤无表达,9个样本虽呈阳性,但肿瘤细胞普遍染色较弱。而在间质中17个标本可见间质细胞阳性表达较高,并可排除非特异性染色,5个标本间质表达细胞较少,阳性细胞主要是间质炎症细胞和成纤维细胞。。对于肺癌肿瘤细胞而言,1例表达差异,原发肿瘤细胞表达为阴性,转移瘤为阳性。以肿瘤间质而言,3例在转移表达增强。TGF-β1可能与肿瘤间质和促进转移有关。实验时假设TGF-β1主要在肿瘤细胞表达,实际发现肿瘤细胞中表达病例较少,因此两者未进行统计学比较。3例不能确定的转移肿瘤2例不一致,1例表达一致TNF-α：在肿瘤细胞和间质炎症细胞中均有表达,22个样本中有2个标本原发癌和转移癌为阴性,6个标本低表达(27.2%),11个标本中度表达(50%),3个样本高表达(13.6%)。在肺癌原发灶和转移灶,表达一致的为10例(90.9%),1例表达有差异,原发癌呈高表达,脑转移癌阳性表达下降,为中度表达,差异未达到统计学意义P值=0.317>0.05。3例不能确定转移的表达一致。p53：p53蛋白在肿瘤细胞核和浆中均有表达,9个样本低表达(40.9%),9个样本中度表达(40.9%),4个样本高表达(18.2%),一致有8例(72.7%),不一致3例(27.3%),差异未达到统计学意义,P值=0.564＞0.05。3例不能确定转移的2例患者一致,1例患者不一致。3.DNA提取结果3个病例未能从对应的石蜡标本中提取出有效的DNA。11例23标本中DNA质量在lOug-15ug之间,纯度经测定符合PCR反应标准4. p53codon 72 SNP:9例确认为转移的8例一致,不一致的1例原发肿瘤为基因型纯合子,脑转移肿瘤为杂合子。另外3例不能确认是否转移的,原发肿瘤一致的基因型一致,通过对照同一病例不同标本和原发转移SNP的状态确定这3例为转移瘤。5.14例原发和转移的功能蛋白表达COX2不一致的2例,P值=0.157；MET不一致3例,P值=0.564；VEGF不一致的3例,P值=0.083；、HIF-1α不一致的2例,P值=0.180、TNFα不一致的1例,P值=0.317；p53表达不一致4例,P值=0.317,差异均未达到显著性统计学意义,均大于0.05。四.结论1.肺癌原发灶和转移灶在病理分级方面存在一些差异,这种差异主要见于原发癌具有多种分化程度的,而转移癌只有一种分化程度,没有观察到肿瘤演进过程中转移分化程度降低,提示转移癌和原发癌有共同的克隆来源。肺癌原发和转移在功能蛋白表达具有一定的差异性,但无明显统计学差异,原发癌和转移癌具有较高的一致性,提示转移癌保持了原发癌的关键生物学特征。2.CD133是否可以作为肺癌干细胞的标记还需要进一步研究,VEGF、COX2原发灶和转移灶表达具有高度的相似性,在应用贝伐单抗和赛来昔布治疗肺癌时如果不能获得转移癌组织可以通过原发癌的表达预测其抑制剂的疗效。MET、HIF-1α是肺癌抗转移潜在的有效靶点,TGF-β1和TNFα在肿瘤间质和肿瘤细胞均有表达,是否可以作为抗转移的靶点需要进一步研究。3.p53的密码子72的SNP是区分原发肺癌和肺内转移的有效工具,原发肺癌和肺内转移以及其他部位的转移灶存在高度一致性,反映原发肺癌和转移癌在遗传背景上高度一致,但仍然有1例不一致,提示p53在转移过程中可能发生了突变。4.转移癌和原发癌在细胞形态和分级、功能蛋白表达和p53的72密码子多态性的高度相似性,提示转移癌和原发癌有共同的克隆来源,转移癌保持了原发癌的关键生物学特征。而功能蛋白表达及p53 condon72 SNP在转移和原发存在的微小差异可能是由于癌细胞DNA基因组的不稳定性,基因表达从复制到转录会经历一系列变化,可能是导致变异的主要原因。更多还原

【Abstract】 Background and AimsMetastasis is the major cause of death in cancer patients, and there are currently no therapeutic agents available to prevent this disease.It would be useful to clarify the mechanism of metastasis for prediction or prevention of metastasis. Several model hypotheses try to explain the mechanism of metastasis, but there are some limitations in them. Modern molecular technologies and nanotechnology offer the possibilities of discovering new biomarkers for prevention metastasis and the feasibility of producing drugs on these new biomarkers. Current studies on the mechanism of metastasis are mostly based on cells in ex vivo experiments and animal models design, which are often artifical. Few studies focused on comparsion of primary cancer and their metastasis in a same patient. However, it is very important to further develop the theory of metastasis mechanism and discovering new target drugs.The papers that have been reported on comparison of primary cancer and matched metastasis maily focused on breast cancer and colon cancer. Few data have been published on primary lung cancer and their metastases.Lung cancer is one of the most common malignant tumors in the world, especially in China. About 70% of lung cancer patients are in late stages at the time of primary diagnose. Even if these patients who had been completely removed primary tumors, most of them would develop metastasis. Researchers should devote themselves to study lung cancer metastasis to resolve the puzzle of lung cancer treatment. Some key genes play important roles in many cancers. The mechanism of breast cancer had been clear clarified in some extent. The molecules that exert their effects on breast cancer metastasis maybe play the same role in lung cancer metastasis. Therefore, it may be helpful for research of lung cancer metastasis. Molecular target therapy is becoming available in treatment of lung cancer. Howerver, the efficiency of target therapy is related to their molecules status. Now the molecule status is mainly based on primary lung cancer tissue. Few data reveals whether changes in lung cancer metastasis tissues on expressions of these key genes. Some genes have been reported that their expressions are related to prognosis of lung cancer,such as VEGF, MET、HIF-1α、TGF-β1. These molecules inhibitors are in clinical trial or becoming possible target therapies in lung cancer. But we don’t know whether expression on metastasis tissue. Doctors are often puzzled by diagnosed of pulmonary metastasis or double primay lung cancer in some patients. Reseachers hold the idea that the gene background between parimay cancer and metastasis are identical while it is heterogeneity among multiprimary lung cancer. Based on this, p53 mutation that often occur in lung cancer, had been used to discriminate pulmonary metasis from multiprimary lung cancer. However, there is heterogeneity of p53 mutation between breast cancer and matched metastasis. It is urgent to search for a more powerful tool to discriminate metastasis or primary cancer. Single nucleotide polymorphisms is the most advanced of polymorphisms, which may reflect heterogeneity of genetic background of individual. P53 72 codon poly-morphisms is reported to induce susceptibility of lung cancer. We hypothsized P53 72 codon polymorphisms can discriminate pulmonary metastasis from second primary lung cancer.Therefore, there are two major goals in this paper. One is to evaluate the similarities and differences on proteins expressions of CD133(Lung cancer stem cell marker), COX2, VEGF, MET, HIF-la, TNFa, TGF-betal and p53 between primary lung cancer and their metastases. The other is to discriminate pulmonary metastasis from multiprimary lung cancer through detection of p53 codon 72 SNP under RFSCP.Patients and Methods14 patients had been removed primay lung cancer tissues and metastasis tissues between 2002 and 2009.11 patients are diagnosed of metastasis.3 cases aren’t certain according to current criteria on diagnosed of pulmonary metastasis. Their paired paraffin sections were collected to conduct the following procedure. 1. Paraffin sections were cut into microsections to conduct routine HE staining and SP immunohistochemistry.2.DNA were extracted from archived, paraffin-embedded sections under the guidelines of QiAGEN kits. The purification of DNA were examined on a special mechanism.3. PCR of p53 condon 72 was proceed on the valid DNA of samples.The productions were proceed to the method of restriction fragment length polymor-phism. The gene type were identified under a laser photo instrument.4. Non-parameter method were used to compare proteins expression insentity between paried tissues through Marginal Homogeneity test. P value<0.05 of two sides test were considered statistically significant.Results1. Clinical data Tumor grades are identical in 9 cases of 11 cases. Tumor grades of the other two cases were G1 and G2 in primary lung cancer but were only G2 in metastasis. The three cases who cann’t be clearly diagnosed of metastasis showed masses again in pulmonary.2. Immunohistochemical ResultsVEGF:In the 11 paried primary and metastasis tissues, low expression was in 5 sections(22.7%), moderate expression in 14 sections(63.6%), high expression in 3 sections(13.6%). The expression scores of primary lung cancer were consistent with that of metastasis in 9 cases(81.8%). The differences don’t show significantly statistical (P value=0.157>0.05).There are two cases who showed more intensity in metastases of lung and brain than in primary lung cancder. In the three cases who cann’t be clearly diagnosed of metastasis, there are two cases concordance and one inconcordance.COX-2:In the 11 paried primary and metastasis tissues, the low, moderate and high expressions were respectively 9 sections(40.9%),9sections(40.9%),4 sections (18.2%). There is one cases incordance(9.1%). The differences don’t show significantly statistical (P value=0.317>0.05). In the three cases who cann’t be clearly diagnosed of metastasis, there are two cases concordance and one inconcordance. MET: In the 11 paried primary and metastasis tissues, the low, moderate and high expressions were respectively 3 samples (13.6%),6 samples (27.2%),13 samples(59.1%). There are two cases incordance(9.1%). The differences don’t show significantly statistical (P value=0.157>0.05). In the three cases who cann’t be clearly diagnosed of metastasis, there are two cases concordance and one inconcordance.HIF-la:In the 11 paried primary and metastasis tissues, the low, moderate and high expressions were respectively 4 samples (18.2%),15 samples(68.2%),3 samples (13.6%). There are two cases incordance(18.2%). The differences don’t show significantly statistical (P value=0.180>0.05). In the three cases who cann’t be clearly diagnosed of metastasis, they are concordance.CD 133:CD 133 showed membrane positive in only squamous tissues. The percent of positive cell was very low. CD 133 expression may be relation to the histological type.TGF-β1:The intensity was weak in tumor cell but strong in stromal cell. The positive cells are inflammations cells and fibroblast. TGF-β1 showed stronger in metastatis stromal than primay stromal, which maybe play a role in interaction of stromal and metastasis.TNFa: In the 11 paried primary and metastasis tissues, there is one case who show negative in both primary and metastasis lung cancer, the low, moderate and high expressions were respectively 6 samples (27.2%),11 samples(50%),3 samples (13.6%). There is one cases incordance(9.1%). The differences don’t show significantly statistical (P value=0.317>0.05). In the three cases who cann’t be clearly diagnosed of metastasis, they are concordance.P53:In the 11 paried primary and metastasis tissues, the low, moderate and high expressions were respectively 9 samples(40.9%),9 samples(40.9%),4 samples (18.2 %). There are three cases incordance(27.2%). The differences don’t show significantly statistical (P value=0.564>0.05). In the three cases who cann’t be clearly diagnosed of metastasis, there are one cases concordance and one inconcordance.3. The results of DNA extraction Three cases fail to extract poor DNA. The quality and purity of DNA in the other 11 cases can meet the demand of PCR.4. SNP of p53 condon 72:In the 9 cases who diganosed of primay and metastatis lung cancer, there is one cases inconcordance in gene type. The other three cases were all identical. Taking into account of positive control and metastatic control, they are paired primay and metastatis tissues.5. The proteins expressions in 14 cases. There are respectively inconcordance in 2 cases of COX2 expression,3 cases expression of MET,3 cases expression of VEGF,2 cases expression of HIF-la,1 cases of TNF-a,4cases of p53. Their P valles are more than 0.05.Conclusions:There are high concordance in selected protein expression and p53 SNP status between primay lung cancer and matched metastatis, suggest there are common clonal origin between them. However, there are still minor differences between them. The VEGF, COX2 expression in metastasis maybe depend on primay lung cancer tissue when their inhibitors were used in lung cancer. The Met, HIF-1αwere potential targets in prevent metastasis.P53 condon 72 polymorphism was a useful tool to discriminate pulmonary metastasis from multiprimay lung cancer.Although there is minor difference, high concordance of gene type prevails in between primay and metastasis. The variations exist between primay and metastasis, which may be caused by genome DNA instability of cancer cells which developed in the process of metastasis.更多还原