肝癌患者来源的移植瘤模型构建、特征描述及基于此模型的索拉菲尼耐药标志物研究

发布时间：2018-06-06 17:38

本文选题：肝细胞癌 + 患者来源的移植瘤模型　；参考：《第二军医大学》2016年博士论文

【摘要】：背景:虽然近几年外科及其他治疗手段的不断进步,但是肝癌患者总体生存率依旧不理想,原因在于肝癌患者发病隐匿,早期诊断困难,多数患者在确诊时已为晚期,失去手术机会;即使行手术治疗,术后仍有较高的复发率,且复发后缺乏有效治疗手段。因此,寻找对晚期肝癌和复发性肝癌切实有效的治疗方法,对延长肝癌患者生存有重要意义。分子靶向药在肿瘤治疗中的应用越来越多,索拉菲尼也被证实在肝癌中有效,提示分子靶向药物可能是治疗肝癌的新方法。但是目前进入肝癌临床试验的靶向药物中,除了索拉菲尼以外,其余全部被证实在总体人群中无效。即使是索拉菲尼,在临床应用中同样有约50%的患者无效,但是我们无法在用药之前判断索拉菲尼的疗效。因此,肝癌缺乏有效的分子靶向药物和用来指导靶向药物治疗的生物标志物或者分子分型。患者来源的移植瘤模型(Patient-derived xenograft models,PDXs)是目前被认为最接近临床患者的药物筛选模型。PDX模型保持了所来源肿瘤的生物学特征,并且PDX对药物的疗效评价与药物临床实际效果较为一致,逐渐地被应用于药物研发、敏感标志物筛选等研究中。但是肝癌PDX模型和其对临床患者肿瘤的代表性报道相对较少,其应用价值值得探讨。索拉菲尼作为唯一应用于肝癌临床治疗的药物,在整体人群中仅有约50%患者有效,而另一半患者则对索拉菲尼不敏感,使得索拉菲尼的整体疗效欠佳,并使得一半患者承受药物不良反应带来的痛苦和巨大的经济负担。而在索拉菲尼耐药研究中,受限于体内外肿瘤模型的限制,如体外模型多,体内模型少,模式动物多,人源化的模型少,使得索拉菲尼耐药机制和标志物仍不清楚,亟待进一步研究。方法:本研究首先通过新鲜肿瘤组织移植方法构建了大规模的肝癌PDX模型队列,并对比PDX模型与对应来源肿瘤组织的生物学特征,通过全外显子组测序、SNP芯片、表达谱芯片等技术,描绘了PDX模型的分子遗传学特征,并与现有肝癌测序数据对比,来研究PDX模型与来源患者肿瘤组织的生物学一致性。同时对建模成功与未建成功患者的临床资料进行对比分析,分析建模过程本身的应用价值。并且根据PDX模型遗传学特征进行可能用于肝癌治疗的靶向药物整理和初步筛选。通过联合模型对索拉菲尼的敏感性与表达谱数据,寻找与索拉菲尼药物敏感性相关的生物标志物,并通过体内外实验验证。结果:本研究共建成肝细胞癌PDX模型242例、肝内胆管癌PDX模型71例,建模成功率分别为30.2%和33.8%,平均建成时间为90(65,130)天和110(80,150)天。PDX建模成功所需的时间短于相应患者的无瘤生存期。PDX模型保持了所来源肿瘤组织的病理学特点,并且在肿瘤标志物甲胎蛋白(alpha fetoprotein,AFP)的表达上与患者一致。建模成功在肝细胞癌患者中与患者年龄50、HBe Ag阳性、肿瘤直径5cm、AFP20U/L、Edmondson GradeⅢⅣ级、微血管侵犯、大血管侵犯、子灶、包膜不完整、早期复发等恶性表型相关,并且预示患者较差的无瘤生存期及总体生存期。同样建模成功在肝内胆管癌中与CA19-939U/L、早期复发等指标相关,也预示肝内胆管癌较差的临床预后。肝癌患者常见的基因突变中,除少数基因的变异未在PDX模型中检测出,绝大多数均可在PDX模型中找到。在PDX模型中肝癌常见突变基因的突变率分别为TP53(67.62%)、TP53(67.6%)、TTN(93.3%)、CTNNB1(11.4%)、JAK1(6.7%)等;常见拷贝数扩增ARNT(20.5%)、MTDH(16.2%)、CCND1(12.4%)、FGF19(11.4%)和MET(16.1%)等;拷贝数缺失RB1(11.4%)、SMAD4(18.1%)、WRN(15.2%)、ARID1A(7.6%)、CDKN2A(12.4%)和PTEN(5.7%)等。索拉菲尼在PDX模型中的有效率约为40%,与临床试验的结果相符。对索拉菲尼耐药的PDX模型中细胞周期、WNT信号通路、IFNa/b信号通路异常,联合应用细胞周期的抑制剂LEE011及IFNa均可提高索拉菲尼抗肿瘤效果。DKK1在耐药组中高表达,并且在体外诱导耐药细胞系中高表达,同时在PDX模型中与肿瘤较差的无进展生存期相关,且在连续用药过程中上调。干扰DKK1后可影响细胞对索拉菲尼的敏感性。临床上,用药一年内死亡的患者血清DKK1水平高于用药一年以上的患者。结论:1.肝癌PDX模型保持了患者肿瘤组织的生物学特点,肝癌常见的基因突变均可在PDX模型队列中找到对应模型,PDX模型建成时间早于肿瘤的复发时间,PDX建成功意味着患者较差的临床预后,需进行密切随访及积极干预。2.索拉菲尼耐药过程中细胞周期、WNT信号通过发挥重要作用,联合应用这些信号通路抑制剂可增强索拉菲尼疗效。DKK1是索拉菲尼耐药的潜在标志物,并且干扰DKK1可增强肿瘤对索拉菲尼的敏感性。
[Abstract]:Background: Despite the continuous progress of surgery and other treatments in recent years, the overall survival rate of the patients with liver cancer is still not ideal. The reason lies in the concealment of the patients with liver cancer and the difficulty of early diagnosis. Most patients are late at the time of diagnosis and lose the chance of operation. Even if the operation is performed, there is still a high recurrence rate after the operation, and the recurrence is lack after the recurrence. Therefore, it is of great significance to search for the effective treatment of advanced liver cancer and recurrent liver cancer, which is of great significance for prolonging the survival of the patients with liver cancer. More and more applications of molecular targeting drugs are used in the treatment of cancer. Sola Feeney has also been proved to be effective in liver cancer, suggesting that molecular targeting drugs may be a new method for the treatment of liver cancer. At present, all the target drugs that enter the clinical trial of liver cancer, except Sola Feeney, are all proved to be ineffective in the general population. Even Sola Feeney, about 50% of the patients are ineffective in clinical application, but we can not judge the effect of Sola Feeney before using the drug. Therefore, the liver cancer lacks effective molecular targeting drugs. And the biomarkers or molecular types used to guide targeted drug therapy. The Patient-derived xenograft models (PDXs) is the drug screening model that is currently considered the closest to the clinical patients. The.PDX model of the drug screening model maintains the biological characteristics of the cancer, and the evaluation of the efficacy of PDX on the drug and the presence of the drug. The actual effect of the bed is more consistent and is gradually used in the research of drug development and sensitive marker screening. However, the PDX model of liver cancer and its representative reports on the clinical patients are relatively less, and its application value is worth discussing. As the only drug that should be used for the clinical treatment of liver cancer, only about 50% of the whole population are affected by Sola Feeney. The other half of the patients were insensitive to Sola Feeney, which made the overall efficacy of Sola Feeney poor and made half of the patients suffering from the pain and enormous economic burden of adverse drug reactions. In Sola Feeney's drug resistance study, the restriction on the tumor model in the body and outside the body, such as in vitro models, less models in the body, and pattern movement, was limited. The mechanisms and markers of Sola Feeney resistance are still unclear. It is not clear that the drug resistance mechanism and markers are still unclear. Methods: first of all, a large PDX model of liver cancer was constructed by the method of fresh tumor tissue transplantation, and the biological characteristics of the PDX model and the corresponding source of the tumor tissue were compared, and the whole exons were sequenced. SNP chip, expression spectrum chip and other techniques, describe the molecular genetic characteristics of the PDX model, and compare with the existing liver cancer sequencing data to study the biological consistency of the PDX model and the cancer tissue from the source of the patients. The clinical data of the successful modeling and the unsuccessful patients are compared and analyzed, and the application value of the modeling process itself is analyzed. And according to the PDX model genetic characteristics to carry out the targeted drug arrangement and preliminary screening for the treatment of liver cancer. Through the combined model of Sola Feeney's sensitivity and expression data, look for biomarkers related to the sensitivity of Sola Feeney drug, and verify it through the test in vivo and in vitro. Results: this study was built into hepatocellular carcinoma PDX In 242 cases and 71 cases of intrahepatic cholangiocarcinoma PDX model, the successful rate of modeling was 30.2% and 33.8%, the average construction time was 90 (65130) days and 110 (80150) days. The time needed for the success of.PDX modeling was shorter than that of the corresponding patient's tumor free survival.PDX model, which kept the pathological features of the tumor tissue, and the tumor marker alpha fetoprotein (alph). The expression of a fetoprotein, AFP) was consistent with the patient. The modeling was successful in patients with hepatocellular carcinoma with age 50, HBe Ag positive, tumor diameter 5cm, AFP20U/L, Edmondson Grade III IV, microvascular invasion, large vascular invasion, subfoci, incomplete capsule, early recurrence and other malignant phenotype related, and predict the patient's poor tumor free survival and the prognosis. The same modeling success is associated with CA19-939U/L and early recurrence in intrahepatic cholangiocarcinoma. It also indicates the poor clinical prognosis of intrahepatic cholangiocarcinoma. Most of the common gene mutations in the liver cancer patients are found in the PDX model except that a few genes are not detected in the PDX model. In the PDX model, the liver cancer is common. The mutation rates of the mutant genes were TP53 (67.62%), TP53 (67.6%), TTN (93.3%), CTNNB1 (11.4%), JAK1 (6.7%), common copy number amplification ARNT (20.5%), MTDH (16.2%), CCND1 (12.4%), FGF19 (11.4%) and MET (16.1%), SMAD4 (18.1%), 15.2%, 15.2%, 15.2%, 16.1%, etc. The effective rate in the model is about 40%, which is consistent with the results of clinical trials. The cell cycle, WNT signaling pathway, IFNa/b signaling pathway in the Sola Feeney resistant PDX model, the combination of LEE011 and IFNa, the combined use of cell cycle inhibitors, LEE011 and IFNa, can increase the high expression of sorafeni's anti-tumor effect in the drug resistant group, and induce drug resistance in vitro. High expression in the cell line was associated with a poor progression free survival in the PDX model and increased during continuous use. Interfering with DKK1 could affect the sensitivity of the cell to Sola Feeney. In clinical, the serum DKK1 level of patients who died within one year was higher than that of patients who had been used for more than one year. Conclusion: 1. the PDX model of liver cancer has been maintained. The biological characteristics of the tumor tissues of the patients, the common gene mutation of the liver cancer can be found in the PDX model queue. The time of the PDX model is earlier than the time of the tumor recurrence. The success of PDX means that the patient has poor clinical prognosis. It is necessary to follow up closely and actively intervene in the cell cycle during the process of.2. Sola Feeney resistance, and the WNT signal is connected. The combined use of these signaling pathways can enhance the efficacy of the Sola Feeney effect.DKK1 as a potential marker for Sola Feeney resistance, and interfering with DKK1 can enhance the sensitivity of the tumor to Sola Feeney.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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