肝癌肝移植患者鉴定肿瘤克隆起源的临床及病理意义的探讨

发布时间：2018-02-03 10:55

  本文关键词： 多结节肝细胞癌 弥漫型肝细胞癌 克隆起源 微卫星杂合性缺失 肝移植 病理 肿瘤复发　出处：《天津医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：【目的】探讨肝细胞癌肝移植患者鉴定肿瘤克隆起源的临床及病理意义。【方法】收集2005年08月-2010年08月于我院经肝移植治疗的符合UCSF标准的多结节型肝细胞癌(HCC)患者的病理资料、术前血清AFP水平及术后随访资料,上述资料不完整的患者予以剔除,共收集患者60例。对60例患者的各自非肿瘤组织及全部肿瘤组织的石蜡包埋块进行切片,切片数量为5张,厚10μm,常规二甲苯-乙醇法脱蜡备用。在显微切割显微镜下,精确选择脱蜡切片的肿瘤及非肿瘤组织,利用石蜡组织DNA提取试剂盒提取肿瘤及非肿瘤组织的基因组DNA。选取12个高频肝细胞癌微卫星杂合性缺失(LOH)位点,合成引物后采用SSCP-PCR法检测癌组织发生LOH的状态,根据每例患者不同微卫星位点发生LOH的情况,判定患者的肿瘤克隆起源方式。采用SPSS20.0统计软件分析患者肿瘤克隆起源方式与肝移植术后无瘤生存率、病理学特征及血清AFP水平之间的相关性。收集2004年08月-2010年11月于我院经肝移植治疗的14例弥漫型肝细胞癌(D-HCC)患者。在14例患者切除的病肝上分别于肝脏左右叶内采集肿瘤间隔≥3cm的肿瘤灶各2块和非肿瘤肝脏组织1块。各自非肿瘤组织及全部肿瘤组织的石蜡包埋块进行切片,切片数量为5张,厚10μm,常规二甲苯-乙醇法脱蜡备用。在显微切割显微镜下,精确选择脱蜡切片的肿瘤及非肿瘤组织,利用石蜡组织DNA提取试剂盒提取肿瘤及非肿瘤组织的基因组DNA。选取12个高频肝细胞癌微卫星杂合性缺失(LOH)位点,合成引物后采用SSCP-PCR法检测癌组织发生LOH的状态,根据每例患者不同微卫星位点发生LOH的情况,判定患者的肿瘤克隆起源方式。收集14例D-HCC患者术前血清AFP浓度、术后病肝病理资料、术后随访资料,总结D-HCC的临床及病理特征。【结果】60例多结节HCC患者中,男性53例,女性7例;年龄在38岁至71岁之间,平均为53岁。患者肝病背景包括乙肝肝硬化45例、丙肝肝硬化11例、酒精性肝硬化3例、隐源性肝硬化1例。有2个肿瘤结节的患者45例,有3个肿瘤结节患者15例。60例多结节HCC患者共采集肿瘤结节135个,直径0.8cm-3.8cm。微卫星LOH检测判定肿瘤克隆起源分型为IM型、IM型+MO型、MO型和不能判定分型4种类型,分别占33.33%(20/60)、8.34%(5/60)、55%(33/60)和3.33%(2/60),肿瘤克隆起源分型不能判定的2例患者剔除,剩余58例患者分为IM组、MO组和IM+MO组,分别占34.48%(20/58)、56.90%(33/58)和8.62%(5/58)。IM组、MO组和IM+MO组的3年累积无瘤生存率、镜下癌栓发生率、肿瘤低分化率(Ⅲ级-Ⅳ级)及AFP中位浓度分别为:50.00%、78.79%和40%,100%、18.18%和100%,80%、51.52%和80%,226.80μg/L(2.78μg/L-3000.00μg/L)、24.59μg/L(1.16μg/L-531.30μg/L)和122.58μg/L(16.20μg/L-1055.00μg/L)。IM组和MO组术前血清AFP水平ROC曲线下面积为0.792,其95%置信区间(CI)为0.659-0.926,最佳判定界值为122.30μg/L,灵敏度为0.750,特异度为0.818。统计显示IM组患者的3年累积无瘤生存率明显低于MO组患者(P0.05);镜下癌栓发生率和术前血清AFP浓度明显高于MO组患者(P0.05),IM组患者肿瘤病理分化程度明显低于MO组患者(P0.05),IM+MO组的无瘤生存率、肿瘤分化程度及镜下癌栓发生率均与IM组无明显差异(P0.05)。14例D-HCC患者的LOH检测结果显示,11例患者肿瘤克隆起源分型为IM,3例患者肿瘤克隆起源分型为MO和IM同时存在。14例患者术前血清AFP浓度0.53μg/L-427.04μg/L,9例患者AFP20.00μg/L,其中4例患者AFP200.00μg/L。术前影像学检查显示,患者肝脏的影像学表现类似肝硬化,伴肝脏及脾脏不同程度增大,但未发现占位性病变。肿瘤分布于全肝,直径0.1-1.0cm,数量100个,类似肝硬化结节。肿瘤病理分级为Ⅰ级-Ⅱ级,所有D-HCC均可检见镜下癌栓。肝移植术后无瘤生存时间为4.5-37.4个月,平均为13.5±6.7月。【结论】1.多结节型HCC的肿瘤克隆起源有两种主要形式:MO型与IM型;MO型在肝移植术后3年无瘤生存率明显高于IM型,而混合型(IM+MO型)预后与IM型相近。肿瘤克隆起源方式或可作为预测多结节HCC肝移植术后肿瘤复发风险的重要参考指标。2.综合肿瘤克隆起源方式、组织病理学特征及术前血清AFP水平等肿瘤学要素,有助于预测肝移植术后肿瘤复发的风险。3.D-HCC克隆起源分型以IM为主,多预示广泛的肝内转移;D-HCC发病隐匿,临床及影像学检查敏感性低,疑诊或鉴别困难时,宜借助肝脏组织穿刺活检明确诊断。
[Abstract]:[Objective] to investigate the clinical and pathological significance of liver transplantation for hepatocellular carcinoma patients with tumor identification of clonal origin. [Methods] multinodular hepatocellular carcinoma from 2005 08 months -2010 years 08 months in our hospital after liver transplantation in the treatment of the UCSF standard (HCC) and pathological data of patients, preoperative serum AFP level and operation after the follow-up data, the data is not complete with to be removed, collected a total of 60 patients. In 60 patients with their non tumor tissue and tumor tissue of paraffin embedded blocks were sliced, cut the number for 5, up to 10 mu m, conventional xylene ethanol method in dewaxing standby. Microdissection under the microscope the precise selection, dewaxed sections of tumor and non tumor tissues using tissue DNA extraction kit to extract tumor and non tumor tissue genomic DNA. from 12 hepatocellular carcinoma high frequency microsatellite loss of heterozygosity (LOH) loci, using synthetic primers after Detection of cancer tissue by SSCP-PCR LOH, according to each patient of different microsatellite loci LOH, determine the patient's tumor clone origin. By using the SPSS20.0 statistical software free survival analysis of patients with tumor clone origin and after liver transplantation, the correlation between the pathological characteristics and the level of serum AFP from 2004. 08 months -2010 year in November in our hospital after liver transplantation in the treatment of 14 cases of diffuse hepatocellular carcinoma (D-HCC) in patients with liver disease. In 14 cases the tumor resection respectively in the liver or tumor interval was 3cm acquisition leaves the 2 and 1 non tumor liver tissue. Their non tumor tissue and all the tumor tissue of paraffin embedded blocks were sliced, cut the number for 5, up to 10 mu m, conventional xylene ethanol method in dewaxing standby. Microdissection under the microscope, accurate selection of dewaxing sections of tumor and non tumor tissue, using Tissue DNA extraction kit to extract tumor and non tumor tissue genomic DNA. from 12 hepatocellular carcinoma high frequency microsatellite loss of heterozygosity (LOH) loci, detection of cancer occur LOH state was synthesized by SSCP-PCR primers, each patient according to different microsatellite loci LOH, determine the patient's tumor clonal origin. 14 D-HCC patients were collected before surgery, postoperative serum AFP concentration, liver pathological data, postoperative follow-up data, summarize the clinical and pathological features of D-HCC. [results] 60 cases of multiple nodules in HCC patients, male 53 cases, female 7 cases; age between 38 to 71 years old, average 53. Patients with liver disease including the background of 45 cases of hepatitis B, hepatitis C cirrhosis in 11 cases, 3 cases of alcoholic liver cirrhosis, 1 cases of cryptogenic cirrhosis. There were 45 cases of patients with 2 nodules, 3 nodules in 15 patients with.60 cases were multiple nodules in patients with HCC were collected and swollen 鐦ょ粨鑺，

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