【作者】 冼海兵；

【导师】 张积仁；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 1.研究背景与目的结直肠癌是全世界的第三大常见肿瘤,每年新发病例超过1000000例。在美国,结直肠癌每年约有15.4万新发病例及5.6万患者死亡,占癌症死因第2位。在我国,结直肠癌的发病率平均水平居全部恶性肿瘤的第4位,发病率每年递增4.2%,而且发病年龄明显提前,与20世纪70年代相比,在90年代城市的发病率上升了31.95%,农村上升了8.51%。2006年中国卫生统计提示大肠癌病死率位居恶性肿瘤第5位。据文献报道,肝是结直肠癌最常见的转移部位。当结直肠癌确诊时,已经有20%-25%病例发生了肝转移,而约60%的患者在其病程中会出现肝转移。治愈性根治术后再发的肝转移率为50%；结直肠癌死亡的患者肝转移率高达45%～71%。虽然手术切除、局部消融及立体定向放射外科是常用的治疗方法,但当原发灶不能切除或者肝功能不全时,内科治疗仍然是确实有效的重要治疗手段。目前,晚期结直肠癌的治疗以化疗为主,过去的40年里,5-氟尿嘧啶(5—FU)一直是治疗结直肠癌的主流药物,随后亚叶酸钙(CF)的加入及5-FU持续泵注使CRC患者的生存明显延长。20世纪90年代后期,新的细胞毒药物奥沙利铂(Oxaliplatin)、伊立替康(CPT-11)等问世,CRC的近期疗效和生存期均得到显著提高；近年来分子靶向药物贝伐单抗(Avastin)、西妥昔单抗(Cetuximab)、帕尼单抗(Panitumumab)的出现,使CRC的治疗又上了一个新的台阶。表皮生长因子受体(EGFR)是一种具有酪氨酸激酶活性的跨膜受体,目前发现该受体家族包括4个亚型：EGFR(HER1/ErbB1), HER2(ErbB2), HER3(ErbB3), HER4(ErbB4)。有超过10多种配体可以选择性与EGFR单体结合。配体与受体结合后,引起受体的寡聚化作用,激活受体酪氨酸激酶,然后使得受体细胞内部分的酪氨酸残基顺式磷酸化。受体磷酸化后构象发生改变,形成停泊位点,以便与具有SH2 (Src-homology 2)结构域和磷酸化酪氨酸结合结构域的蛋白质结合,从而触发包括丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)通路和磷脂酰肌醇-3激酶(phosphoinositide 3-kinase, PI3K)通路的信号传导级联反应,在器官形成和肿瘤生长过程中的细胞增生、抑制凋亡、血管生成和转移扩散等方面发挥着很重要的作用。表皮生长因子受体(EGFR)在结直肠癌中有过量表达,为肿瘤治疗提供了一个理想靶点。EGFR过表达与细胞恶变、肿瘤细胞增侵袭、转移、肿瘤血管生成以及凋亡抗性密切相关。西妥昔单抗(Cetuximab)是一种重组人-鼠嵌合型IgG1型单克隆抗体,对细胞膜上的EGFR的亲和力是内源性配基的5-10倍,与EGFR结合后,能阻止内源性配基与EGFR结合,切断信号的传导并阻断酪氨酸激酶的磷酸化,从而抑制细胞周期进程、诱导肿瘤细胞凋亡、减少基质金属蛋白酶和血管内皮生长因子的产生,还可引起受体的二聚化、内化和下调,抑制肿瘤浸润和转移。除了抑制EGFR发挥抗肿瘤作用外,西妥昔单抗还能激活宿主的免疫反应,通过抗体依赖的细胞毒作用(ADCC)杀伤肿瘤细胞。目前已证明用于伊立替康治疗失败的结直肠癌可提高疗效。近期多项研究均表明：在转移性结直肠癌中,K-ras野生型的患者接受西妥昔单抗联合化疗的疗效、临床获益等均显著优于K-ras突变型组,强烈提示K-ras状态与西妥昔单抗疗效相关,是独立的西妥昔单抗疗效预测指标。只有K-ras野生型患者才建议接受EGFR抑制剂如西妥昔单抗和帕尼单抗(包括单药或与化疗联合)治疗。因此,K-ras基因检测被写入2009年版《美国国立癌症综合网络(NCCN)结直肠癌临床实践指南》。也就是说,所有转移性结直肠癌患者均应检测K-ras基因状态。目前认为,大肠癌的发生是机体内因(大肠癌的遗传易感性)和外因(饮食和环境因素)共同作用的结果。大量实验证明大肠癌的发生与多个癌基因的激活或抑癌基因的失活有关。Ras基因的突变被认为在癌变过程中起启动作用并是一个早期事件。Ras基因最初是在Kirston和Hayvey大鼠肉瘤中首先发现的,转导了ras基因的鼠纤维细胞能发生恶性转化,从而确定了其癌基因的地位。Ras基因家族由K-ras、H-ras和N-ras的三个密切相关的高度同源成员组成。Ras基因定位于人类的第12号染色体上,含有4个编码外显了和1个5’端非编码外显子,共同编码含189个氨基酸组成的ras蛋白,因其相对分子质量为21×103,又称为p21蛋白。K-ras的基因最常见的激活方式为点突变,突变主要发生在1号外显子的第12位密码子上,偶也有发生于13位点及2号外显子的61位点的。K-ras的基因12位密码子野生型为甘氨酸,突变型常为半胱氨酸、缬氨酸、精氨酸三种,突变的结果导致编码的氨基酸发生改变,从而产生具有致癌活性的p21蛋白。K-ras的基因发生异常时,鸟苷三磷酸酶(GTPase)活性降低,p21GTP牢固结合而不被GTPase水解,一直处于激活状态,持续刺激细胞生长、发育、增殖,引起细胞恶变,大肠癌即可发生。另外K-ras的基因激活还可以通过扩增或过表达的方式,但发生频率较低,多数在动物实验中发现。Algar等研究认为,K-ras的基因过表达,明显降低自然杀伤细胞(NK细胞)对肿瘤的杀伤作用,是大肠癌转移,病情进展的特征。经研究认为,K-ras的基因突变是在肿瘤早期形成过程中即已发生,K-ras白勺基因突变更赋予肿瘤极大的浸润与转移倾向的可能。大肠癌K-ras基因突变率在欧美约为30%-68%,而台湾、日本人的K-ras突变率为26.5％-36％,较欧美的检出率偏低。Oliveira等发现K-ras基因的突变率在结直肠癌的淋巴结转移灶中比相应的原发灶高；而Molinari等报道K-ras基因在30例结直肠癌原发灶和相关转移灶的突变情况,有26例表达一致,一致率为86.6%；Daniele等分析99例结直肠癌原发灶和相关转移灶的K-ras基因突变情况,其一致率96.0%；Fotios等研究43例结直肠癌原发灶和相关转移灶的K-ras基因突变情况,一致率为95.0％。然而,原发灶K-ras基因突变情况可否作为对应肝转移灶突变情况的预测呢?上述国外这方面的报道的研究结果不尽相同,而目前国内缺乏相关的资料。本研究观察了75例结直肠癌肝转移的原发灶和对应肝转移灶中K-ras基因的突变情况,比较两者的一致性,为选用分子靶向药物提供新的依据。2.研究方法收集2003年1月1日至2008年5月30日佛山市第一人民医院同期或二期手术切除所得的结直肠癌及对应的肝转移灶病理标本75例。获取病理标本时,所有患者均未接受任何分子靶向药物治疗。所有标本经常规福尔马林固定、石蜡包埋,切成10μm厚的切片置于玻片上,应用QIAamp DNA FFPE Tissue Kit组织盒分别提取肠癌组织及对应的肝转移灶组织的DNA。提取的DNA经常规PCR反应扩增K-ras基因。PCR产物经2%琼脂糖凝胶电泳纯化鉴定后经过DNA测序仪进行测序。采用直接测序法分别检测原发灶组织和对应肝转移灶组织中K-ras基因(主要检测第12位及第13位密码子)的突变情况。数据结果以率表示,采用SPSS13.0统计软件进行配对χ2检验,检验水准a=0.05。3.结果3.1临床资料75例患者中,男43例(57.3%),女32例(42.7%),年龄31～74岁,中位年龄54岁。同时行肠原发灶和肝转移灶切除的64例(85.3%),异时切除的11例(14.7%)。其中结肠癌肝转移55例(73.3%),直肠癌肝转移20例(26.7%)。原发部位位于右半结肠的26例(34.7%),左半结肠29例(38.6%),直肠20例(26.7%)。伴有淋巴结转移的57例(76.0%),无淋巴结转移的18例(24.0%)。组织病理类型：低分化腺癌5例(6.7%)、中分化腺癌67例(89.3%)、黏液腺癌3例(4.0%)。3.2实验结果75例中,有3例因DNA提取质量较差,不能满足后续实验要求而出组,以下为72例的结果分析。3.2.1原发灶癌组织中K-ras基因突变的情况结直肠癌原发组织中有24例(33.3％)K-ras基因为突变型,其中结肠癌18例(75.0%),直肠癌6例(25.0%)。具体突变类型：G12C有3例(12.5％),G12D有10例(41.7%), G12V有9例(37.5%),G13D有2例(8.3％)。3.2.2肝转移灶癌组织中K-ras基因突变的情况肝转移灶癌组织中有23例(31.9％)K-ras基因为突变型。具体突变类型：G12C有4例(17.4%), G12D有11例(47.8%), G12V有7例(30.5%),G13D有1例(4.3%)。3.2.3原发灶与对应肝转移灶癌组织中K-ras基因突变情况的一致性24例原发灶K-ras基因为突变型者中,有21例(87.5%)对应的肝转移灶K-ras基因为突变型。46例(63.9%)原发灶K-ras基因为野生型者相对应的肝转移灶的K-ras基因亦为野生型。原发灶及对应肝转移灶K-ras基因表达情况的一致率为93.1%,经配对资料χ2检验无统计学差异(P=1.00),其κ系数为0.842,即表示两者的吻合度较高。经对κ系数检验,P=0.00,表明两者的吻合度有统计学意义。两者不一致的情况共5例(6.9%),其中2例原发灶K-ras基因为野生型而转移灶为突变型,3例原发灶K-ras基因为突变型而转移灶为野生型。4.结论4.1.本研究中,原发灶K-ras基因突变率为33.3%,与亚洲的报道相近；而12密码子的突变的主要类型为G12D(GGT→GAT)和G12V(GGT→GTT),与上述报道的相似。4.2.本研究中,原发灶与对应肝转移灶癌组织中K-ras基因表达情况的一致率为93.1%。因此,我们认为,对于中国人来说,在转移性结直肠癌中,原发灶和对应转移灶的K-ras基因突变是基本一致的,原发灶K-ras的状态可以指导临床用药,转移灶不一定要重取活检,或如原发灶的标本无法获取时,亦可取转移灶的病理标本进行K-ras基因状态检测。这样可以减少重复的医疗操作,减少并发症,节省时间,降低医疗成本。4.3.当然,要获得更准确的资料,需要更大样本的研究以及相关蛋白表达情况的检测和临床病例的随访分析等,我们提供的这个初步研究资料希望对进一步的研究提供可靠的参考。更多还原

【Abstract】 1. BACKGROUND & OBJECTIVESColorectal cancer (CRC) is the third most common cancer worldwide, with over a million new cases diagnosed each year. In the United States alone, an estimated 154,000 new cases will be diagnosed and over 56,000 deaths are expected from the disease every year, and CRC is the second most common cause of cancer mortality. In China, the incidence rate of CRC is the fourth among all of the cancers with an increasing rate of 4.2% per year and the population is much younger than before. Compared with that in 20th century 70s, the incidence rate increased 31.95% in cities and 8.51% in countries in 20th century 90s. In 2006 the mortality rate of CRC in china is the fifth among all of the cancers. As reported, the liver is the most frequent site of haematogenous metastatic spread of CRC. Approximately 20-25% of patients with CRC have liver metastases when diagnosed and 60% patients will develop hepatic lesions during their course of their illness. The recurrence rate is about 50% after the curative resection and approximate 45%-71% patients died from CRC had liver metastases. Although surgical resection、local ablation and radiosurgery are the common used therapies, medical therapy is the important method when the lesions cannot be removed or the hepatic insufficiency existed.Now chemotherapy is the major therapy in advanced CRC. In the past 40 years, Fluorouracil(5-FU) is the major drug in the therapy of CRC and the patient survival prolongs with Leucovorin(CF) plus 5-FU continued infusion. In 20th century 90s, the new cytotoxic drug Oxaliplatin and Irinotecan(CPT-11) improve the short-term effect and survival of CRC. In recent years the molecular targeted drug Bevacizumab、Cetuximab(C225) and Panitumumab make the new therapy of CRC.Epidermal growth factor receptor (EGFR) is a kind of tyrosine kinase activity transmembrane receptor, now found that the receptor family includes four subtypes: EGFR (HER1/ErbB1), HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4). More than 10 kinds of ligands can selectively combine with the EGFR monomer. Ligand-receptor binding has caused the role of receptor oligomerization, activated receptor tyrosine kinase, and then makes the receptor cells in parts of the tyrosine residues cis-phosphorylation. Receptor phosphorylation after the conformation change to form a point of parking spaces to work with a SH2 (Src-homology 2) domain and phosphorylated tyrosine binding domain of the protein, thereby triggering including mitogen-activated protein kinase (mitogen -activated protein kinase, MAPK) pathway and phosphatidylinositol -3 kinase (phosphoinositide 3-kinase, PI3K) pathway of signal transduction cascade. Then it plays a very important role in of organ formation and tumor growth in the process of cell proliferation, inhibiting apoptosis, angiogenesis and metastases.Epidermal growth factor receptor (EGFR) in CRC is over-expressed and it is an ideal cancer therapy target. EGFR over-expression is closely linked to the cell malignant transformation, tumor cells invasion, metastasis, tumor angiogenesis and apoptosis resistance. Cetuximab (C225) is a recombinant human-mouse chimeric IgGl monoclonal antibody, its affinity of EGFR on the cell membrane is 5-10 times to the endogenous ligand. Combined with EGFR, it can block endogenous ligand binding with EGFR, cut off the signal transduction and block the phosphorylation of tyrosine kinase, thereby inhibiting cell cycle progression,inducing apoptosis of tumor cells and reducing the matrix metalloproteinase and vascular endothelial growth factor production. It can also cause receptor dimerization, internalization and down-regulation to inhibit tumor invasion and metastasis. In addition to inhibiting EGFR, Cetuximab can also activate the host immune response by antibody-dependent cellular cytotoxicity (ADCC) to kill tumor cells. A number of recent studies show that in metastatic CRC, the efficacy and the clinical benefit of K-ras wild-type patients receiving C225 and combination with chemotherapy are significantly better than those of K-ras mutant group, strongly suggesting that K-ras status is related with efficacy of C225 and it is an independent predictor of C225 efficacy. Only K-ras wild-type patients are recommended to recieve the EGFR inhibitors treatment, such as C225 and Panitumumab(including single drug or in combination with chemotherapy). Therefore, K-ras gene detection was written in 2009 edition of "U.S. National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines for colorectal cancer." That is, K-ras gene status should be detected in patients with metastatic CRC.At present the view that the incidence of CRC is the body’s internal factors (genetic susceptibility to CRC) and exogenous (dietary and environmental factors) together results. A large number of experiments prove the occurrence of CRC is related with multiple oncogene activation or inactivation of tumor suppressor genes. Ras gene mutation is thought to play a catalytic role to carcinogenesis and is an early event. Ras gene was first discovered in Kirston and Hayvey rat sarcoma and with the transduction of the ras gene the mouse fibroblast cells could turn to malignant transformation. So it determined the status of its oncogene. Ras gene family is made up of three cancer-related genes in humans, K-ras, H-ras and N-ras, the sequences of which are highly conserved. Ras gene mapping to human chromosome No.12, contains four coding exons and a 5’non-coding exon that together code the ras protein containing 189 amino acids, and because of its relative molecular mass 21×103, also known as p21 protein. The most common way of K-ras gene activation is the point mutations which are observed mainly at, codon 12, or 13 in exon 1 and sometimes at codon 61 in exon 2. The wild-type of K-ras gene at codon 12 is glycine, while the mutant type often are cysteine, valine or arginine. These mutations result in amino acid changes, resulting in carcinogenic activity p21 protein. The guanosine triphosphatase (GTPase) activity decreased when the K-ras gene abnormalities occurred, then p21GTP with a solid combination cannot be hydrolyzed by GTPase and has been in active, continuing to stimulate cell growth, development, proliferation and causing cell malignant, so CRC may occur. Furthermore K-ras gene activation can also be observed by amplification or over-expression, but with a lower frequency, which are majority found in animal experiments. Algar et al found that the over-expression of K-ras gene significantly reduced the anti-tumor effect of natural killer cells (NK cells) and might be the features of metastasis and disease progression in CRC. The study concluded that K-ras gene mutations occurred early during the tumor formation and it has given a great tendency to the possibility of infiltration and metastasis of tumor cells.The mutation rate of K-ras gene in CRC in Europe and the United States is about 30% -68%, and in Taiwan and Japan the K-ras mutation rate is 26.5% -36.9%, lower than that in Europe and the United States. Oliveira etc have demonstrated that the frequency of K-ras mutations in lymph node metastases is higher than that in the related primary CRCs. Molinari analyzed K-ras mutational status in a small sample of 30 consecutive patients with CRC with synchronous or metachronous metastasis. The same mutational pattern between primaries and corresponding metastases was observed in 26 cases, with a total grade of concordance of 86.6%.Daniele evaluated the grade of concordance in terms of K-ras status between primaries and related metastases in 99 cases with CRC and found the concordance rate was 96.0%. Fotios analyzed K-ras mutational status between primaries and related metastases in 43 cases of CRC and found the concordance rate was 95.0%. However, can the K-ras mutational status in primaries predict that of related liver metastases in CRC? Above are some reports abroad to answer this question and there were some different conclusions. Furthermore there is little domestic-related information. This study evaluated the grade of concordance in terms of K-ras status between primaries and related liver metastases in 75 cases with CRC and provided a new basis of selection of molecular-targeted drugs.2. METHODS75 cases of CRC diagnosed in the First People’s Hospital of Foshan from January 1,2003 to May 30,2008 were enrolled in this study. These cases were all received the synchronous or metachronous surgical resection of colorectal cancer and related liver metastasis and the pathological specimens were obtained. Enrolled in this study, all of the patients were not received any molecular target drug treatment. All of the specimens were conventional formalin-fixed, paraffin-embedded, and cut into 10μm thick sections to be placed on slides, then the tissue DNA were extracted from primary tumors and related liver metastases respectively by the application of QIAamp DNA FFPE Tissue Kit Organization Box. The extracted DNA was amplified to get K-ras gene by conventional PCR. Then PCR products were purified by 2% agarose gel electrophoresis and sequenced through DNA sequencing instrument. We mainly detected K-ras codon 12 and 13 mutations in primary tumors and related liver metastases. Data results were stated by rates and analyzed by pairedχ2 test (McNemar test) with SPSS 13.0 software pack, with test level a=0.05.3. RESULTS3.1 Clinical data75 cases of CRC diagnosed in the First People’s Hospital of Foshan from January 1,2003 to May 30,2008 were enrolled. These cases were all received the synchronous or metachronous surgical resection of colorectal cancer and related liver metastasis and the pathological specimens were obtained.75 patients were composed of 43 males (57.3%) and 32 females (42.7%); median age at diagnosis,56 years (range,31-74 years).64 cases (85.3%) received synchronous surgical resection and 11 cases(14.7%) received metachronous resection. There were 55 cases (73.3%) of colon cancer with liver metastasis and 20 cases (26.7%) of rectal cancer with liver metastases.26(34.7%) were affected by right colon,29(38.6%) by left colon and 20(26.7%) by rectal cancer. Lymph node metastases were found in 57 cases (76.0%) and 18 cases (24.0%) with none. Pathological types:5 cases (6.7%) of poorly differentiated adenocarcinoma,67 cases (89.3%) of differentiated adenocarcinoma and mucinous adenocarcinoma in 3 cases (57.3%).3.2 ResultsThree pairs were excluded from the analysis because of the low quality of DNA extraction. And here is the analysis of 72 cases.3.2.1 K-ras mutational status in primary tumorsK-ras gene mutations were found in 24 cases (33.3%) in primary colorectal cancer tissues, of which 18 cases (75.0%) with colon cancer,6 cases (25.0%) with rectal cancer. Specific mutation types:G12C in 3 cases (12.5%), G12D in 10 cases (41.7%), G12V in 9 cases (37.5%) and G13D in 2 cases (8.3%).3.2.2 K-ras mutational status in liver metastasesK-ras gene mutations were found in 23 cases (31.9%) in liver metastases tissues. Specific mutation types:G12C in 4 cases (17.4%), G12D in 11 cases (47.8%), G12V in 7cases (30.5%) and G13D in 1 cases (4.3%).3.2.3 The grade of concordance in terms of K-ras status between primaries and related liver metastasesAmong 24 cases of K-ras mutation in primary tumors,21 cases (87.5%)of K-ras mutation were found in related liver metastases. And 46 cases were K-ras wild-type both in primaries and related liver metastases. The rate of concordance was 93.1%, with no significant difference (P=1.00) by McNemar test. And the value of kappa was 0.842, it means that the degree of matching between the two was high. Discordance was observed in 5(6.9%) patients. In 2 patients K-ras was wild-type in the primary tumor and mutated in the liver metastases. In 3 patients K-ras was mutated in primary tumor and wild-type in the metastatic site.4. CONCLUSION4.1 In this study, the mutation rate of K-ras gene in primary tissues was 33.3% and similar with that of Asia. And the major mutation type of codon 12 was G12D(GGT→GAT)and G12V(GGT→GTT) which was similar with the reports above.4.2 In this study, the rate of concordance in terms of K-ras status between primaries and related liver metastases was 93.1%. Therefore, we believe that to Chinese people, in metastatic colorectal cancer, K-ras mutational status between primaries and related liver metastases was concordant. K-ras status of the primary tumor can guide the clinical use of drugs and the metastases have not to be taken re-biopsy, or if the specimens of the primary tumor can not be got, we can take the pathological specimens from the metastatic site to test K-ras gene status. It will reduce duplication of medical operations, reduce complications, save time and decrease medical costs.4.3 Of course, to obtain more accurate information, it needs for researches with more samples, detection of related protein expression and analysis of clinical follow-up cases. We provide this initial research data as a reliable reference to further researches.更多还原