克隆性分析技术在肝脏局灶性结节性增生诊断中的应用

Application of clonality analysis in diagnosis of focal nodular hyperplasia

目的:探讨整个肝局灶性结节性增生(FNH)病变及各结节的克隆性,以阐明其本质,同时比较其与肝细胞腺瘤(HA)鉴别的克隆性组成方法:女性肝脏标本3例共4个FNH病灶．在病变区和非病变区取组织提取基因组DNA,余标本制备石蜡切片,HE染色,应用显微切割技术分离其中3个FNH内的小结节状病变,提取基因组DNA,经甲基化敏感的HpaⅡ或Hha Ⅰ消化,巢式PCR扩增磷酸甘油酸激酶(PGK)和雄激素受体(AR)基因．应用Bst Ⅺ酶切和琼脂糖凝胶电泳显示PGK基因的单核苷酸多态性; 应用变性聚丙烯酰胺凝胶电泳显示AR基因的 CAG重复序列长度多态性．2例HA和4例HCC 作为肿瘤参照．结果:2例HA及4例HCC均为单克隆性．4个 FNH病变,直径1．5-5．3 cm,但缺乏特征性的中央星状瘢痕,结果均显示为多克隆性病变;其中的61个结节性病变中,56个呈变异肝细胞结节(NAH)形态,5个为普通再生结节．克隆性分析结果显示,56个NAH样病变中,除4个未扩增成功外,52个NAH中有21个(40．4％)显示X 染色体失活嵌合性丢失,提示为肿瘤性病变; 其中1例FNH的14个单克隆结节中,有2个与其他病变失活带型不一致,提示在同一FNH中; 存在着不同起源的NAH样病变．5个普通肝细胞增生结节及病变周围肝组织均为多克隆组成．结论:FNH是由无数个NAH构成的,其整个病变是多克隆性,但其中某些结节已经是肿瘤性增生．克隆性检测有助于其与HA的鉴别,而且所取样本必须是病变的整个切面或其大部分．

AIM： To study the clonality status of the whole lesions and each nodules of hepatic focal nodular hyper-plasia （FNH）, and simultaneously compare the clonal composition with hepatocellular adenoma （HA）. METHODS： Four FNHs from 3 women, 2 HAs and 4 HCCs were examined by clonality assays based on X-chromosome inactivation mosaicism. Nodules were microdissected from paraffin sections. Genomic DNA was isolated from each nodules, the whole lesions and surrounding liver parenchyma, pretreated with Hpa Ⅱor Hha Ⅰ, and then amplified via nested polymerase chain reaction （PCR） for phosphoglycerate kinase （PGK） and androgen receptor （AR） genes. The single nucleotide polymorphism at the PGK locus was identified by incubation with Bst XI and agarose gel electrophoresis, and the CAG repeat length polymorphism at AR locus was revealed on denaturing polyacrylamide gels and visualized by silver staining. RESULTS： The FNH lesions sized 1.5-5.3 cm in diameter, without a central stelate scar for all of them. While monoclonality was confirmed in both of 2 HAs and all of 4 HCCs examined, polyclonality was shown in all of the 4 FNHs as determined by the whole lesions, demonstrating its distinction from neoplastic lesions. A total of 61 nodules, including 56 nodules of altered hepatocytes （NAH） and 5 ordinary regenerative nodules, were microdissected from 3 of the 4 FNH lesions. The clonality analysis was successful in 52 NAHs. Loss of X-chromosome inactivation mosaicism was detected in 21 （40.4%） of them, indicating the monoclonal, neoplastic nature. In one FNH, different X-chromosomal inactivation patterns were observed in separate monoclonal nodules, revealing different clonal origins. Polyclonality was demonstrated in all of the 5 ordinary regenerative nodules and the surrounding liver parenchyma. CONCLUSION： FNH is composed of numerous NAHs. The whole lesion shows a polyclonal cell composition, but neoplastic transformation has occurred in some of the nodules. Clonality assay is useful for in the differential diagnosis of FNH from HA, and sampling the whole or larger part of the lesion is necessary.