【作者】 蔡毅然；

【导师】 苏勤；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的：肝细胞腺瘤(HCA)和局灶性结节性增生(FNH)是发生于结构正常或基本正常肝脏的良性病变。已经知道前者是一种肿瘤性病变,具有特征性的分子病理学改变,而后者的性质及发病机制尚未被阐明。本研究选取多个与HCC发生有关联的微卫星标志,以期能够揭示FNH和HCA中的染色体等位基因杂合性丢失(LOH)在肝肿瘤发生、发展中的作用。方法：选取5例经典型FNH,9例HCA和18例分化好的(Edmondson I或Ⅱ级)肝细胞肝癌(HCC),通过聚合酶链式反应(PCR)扩增57个微卫星位点,应用变性聚丙烯酰胺凝胶电泳显示LOH。通过免疫组织化学方法对9例肝腺瘤检测β-catenin蛋白表达。结果：18例HCC均为乙型肝炎病毒感染,其中一例同时合并丙型肝炎病毒感染。所有HCC标本中均可检测LOH,单一病例发生LOH的微卫星均数为4个(0-12)。57个检测的位点中,33个(58%)被累及,6个在这些HCC标本中呈现高频(≥30%)LOH,位于6q、8p、11p、16q和17p。9例HCA中,2例伴有不典型增生(即小细胞性改变,SCC),β-catenin免疫组织化学检测均显示胞膜阳性,未见明确胞浆和胞核阳性。9例HCA均显示有等位基因失衡,累及16个(28%)位点,其中8个为高频位点,位于11p、13q和17p。单个HCA病变发生LOH位点均数为3个(1-5)。5例FNH中均未检测到SCC改变,LOH检测均为阴性。然而,这些FNH标本中都可以见到多个透明细胞为主型的变异肝细胞病灶(FAH)和结节(NAH),后一种病变构成FNH中多数体积较大(直径>1 mm)的小结节。选取NAH较多且明显的1例FNH病变,应用显微切割分离了25个NAH,其中13个病变多数微卫星位点扩增成功。这些NAH病变均显示出LOH,共累及21个(37%)位点,其中6个为高频位点,位于8p、11p、13q和17p。单个病变发生LOH的位点均数为4个(1-6)。不同NAH病变之间LOH组型也不相同,显示是互相独立的病变个体。NAH、HCA和HCC的LOH位点分布不同,频率最高的位点分别是D8S298(70%)、D11S1301 (75%)和D6S1008 (50%)。这三种病变中,NAH中发生LOH的位点累及染色体区域与HCA基本相似。然而,前者中8p发生LOH的频度高于后者；NAH与HCA在16q改变中也不相同,前者累及7个位点中的5个,后者仅累及1个。结论：分化较好的HCC中存在广泛的等位基因失平衡,高频率LOH位点定位于染色体6q、8p、11p、16q和17p；HCA中也存在LOH,但分布较为局限,高频位点位于11p、13q和17p。FNH中未检测到LOH,与其多克隆特征一致,但组织学检查发现广泛存在FAH和NAH。对显微切割的不同NAH微卫星位点检测显示,所有小结节均存在LOH,提示他们都已经属于肿瘤性病变；NAH中LOH高频位点位于8p、11p、13q和17p,与HCA相似。来自同一FNH的不同NAH病变具有不同的LOH组型,显示它们相互独立。这组数据提示,经典FNH中小结节状的组织学改变实际上反映了多个NAH的成簇发生。更多还原

【Abstract】 Objective:Hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH) are benign focal lesions occuring in normal or nearly normal liver. HCA has been found to be neoplastic, characterized by alterations in a few molecular pathways. The clonality status of FNH remains to be clarified, and its pathogenesis is largely unknown. In the current study, a comprehensive observation was carried out on FNH and HCA for loss of heterrozygosity (LOH) using 57 microsatellite markers, where allelic imbalances had been associated to HCC development. Possible roles of the genetic alterations in hepatotocarcinogenesis were evaluatedMethods:5 classical FNH lesions,9 HCAs and 18 well-differentiated HCCs (Edmondson grade I or II) were used in this study. A total number of 57 microsatellite markers were examined. The sequences were amplified through polymerase-chain reaction, and LOH were visualized on denatured polyacrylamide gels by silver staining. Expression of P-catenin was assessd in 9 HCAs by immunohistochemistry.Results:All of the 18 HCCs were found in livers with chroni hepatitis B and cirrhosis, with coinfection of hepatitis C virus found in one of the cases. LOH was demonstrated in all of the HCCs examined. Numbers of loci revealed in each case ranged from 0 to 12, with their average being 4. Among the 57 loci,33 (58%) showed allelic imbalances in HCCs, with highly frequent (≥30%) LOH detected in 6 loci located on 6q,8p, 11p,16q and 17p.Liver cell dysplasia (small-cell change, SCC) was observed in 2 out of 9 HCAs examined. A memebranous, rather than cytoplasmic or nuclear, immunoreactivity was demonstrated in all of the HCA. LOH was also detected in these lesions. Numbers of loci with LOH in each HCA lesion ranged from 1 to 5, with their average being 3. Sixteen loci (28%) were affected, with 8, on 11p,13q and 17p, showing highly frequent imbalances.LOH was not found in the 5 FNH lesions as detected as a whole. However, foci of altered hepatocytes (FAH) and nodules of altered hepatocytes (NAH) composed mainly of clear cells were identified in these lesions. NAH were found to occupy majority of the larger nodules, sizing>1 mm in diameter, within FNH. NAH were microdissected from an FNH lesion showing many well-circumscribed larger nodules. A total number of 25, NAH were obtained, and amplification was successful for most of the 57 loci in 13 of them. Numbers of loci ranged from one to 6, with their average being 4. Twenty-one loci (37%) were affected, with 6, located on 8p, 11p,13q and 17p, showing highly frequent LOH.NAH, HCA and HCC were found to be different in their LOH profiles, with LOH detected in highest frequencies at the loci D8S298 (70%), D11S1301 (75%) and D6S1008 (50%), respectively. Distribution pattern of LOH in NAH was largely similar to that in HCA. However,8p was affected more frequently in the former than in the latter lesion. In addition, they were also shown to be different in the 16q alterations, with 5 of the 7 loci affected in NAH and only one in HCA.Conclusions:Allelic imbalances were found in well-differentiated HCC, with highly frequent LOH detected on chromosomal arms 6q,8p, 11p,16q and 17p. LOH was also identified in HCA, but it affected fewer loci than in HCC samples, detected in high frequencies at the loci on 11p, 13q and 17p. No LOH was found in whole FNH lesions, which is in agreement with its polyclonality as demonstrated previously. However, frequent occurrences of FAH and NAH were demonstrated within the lesions. The microsatellite sequences from the microdissected NAH were also examined. LOH was detected in all of the lesions at lest at one locus, indicating their monoclonalality. Chromosomal arms 8p, 11p,13q and 17p were affected in NAH in high frequencies, the distribution pattern being largely similar to HCA. Different LOH profiles were found in different NAH, indicating that they developed independently. These data hint that the nodular pattern characterized by classical FNH may reflect the clustered occurrence within the lesion.更多还原