不同位点显微切割对宫颈鳞癌及其上皮内瘤变克隆性分析的影响

The effect of microdissections of different sites on clonality analysis of cervical squamous cell carcinoma and its intraepithelial neoplasia

目的探讨同一病例不同位点组织显微切割方法对宫颈鳞状细胞癌(squamous cell carcinoma,SCC)及其宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)克隆性检测结果的影响和意义。方法随机选取病理存档宫颈SCC 22例,CIN 48例,采用单位点或多位点显微切割方法,分别切取病变上皮组织及其相应的间质,抽提DNA,用限制性内切酶HpaⅡ酶切,聚合酶链反应(PCR)扩增人类X染色体上的雄激素受体基因第一外显子,比较酶切前后的结果,判断病变的克隆性特征。结果纯合子8例,杂合子62例,杂合率88.6%。CIN组单克隆性病变明显高于正常对照组,单位点显微切割病变的单克隆率为CINⅠ5/20(25%)、CINⅡ/Ⅲ16/22(72.7%)和宫颈SCC17/20(85%);多位点显微切割病变的单克隆率为CINⅠ3/20(15%)、CINⅡ/Ⅲ13/22(59%)和宫颈SCC12/20(60%),其检测结果差别有统计学意义(P<0.05)。多点微切割的病变中,10例呈现不同的X染色体失活类型。结论单位点显微切割的方法检测宫颈病变单克隆性比多位点显微切割更为准确,对判断CIN的肿瘤属性有意义。多位点显微切割对研究SCC发生的单中心或多中心起源有意义。本组结果提示大部分SCC起源于单个位点的单克隆改变,而部分病例为多位点的单克隆发生。

Objective To study the effect and the significance of different microdissection sites of the same case on the clonality analysis of cervical squamos cell carcinoma（SCC） and cervical intraepithelial neoplasia（CIN）.Methods X chromosome inactivation pattern was assessed in 22 cases of cervical SCC and 48 cases CIN.Dysplasia cells of CIN and cervical SCC were microdissected from the routine sections stained with hematoxylin and eosin.Stroma components were also isolated from the same sample for parallel control.Genomic DNA was extracted by routine methods, and the exonl of AR gene located on X chromosome was amplified by means of polymerase chain reaction and the results of polyacrylamide eleetrophoresis were compared before and after digesting with Hpa Ⅱ. Synchronous lesions were analyzed respectively by two single lesions and mixed lesions. Resuits 62 cases were proved to be heterogeneous（heterogeneous proportion 88.6 % ） in spite of 8 homogeneous cases; Monoclonality proportion of CIN was significantly higher than that of normal cervix, 25% CIN Ⅰ ,72. 7 % CIN Ⅱ/Ⅲ and 85 % cervical SCC showed monoclonality by analyzing with single site. However, 15 % CIN Ⅰ ,59% CIN Ⅱ/Ⅲ and 60% cervical SCC showed monoelonality and 10 cases of them were revealed dissimilar pattern of monoclonality by analysis with multiple sites of the lesions. There was a statistic difference between them （P 〈 0.05）. Conclusion It is more precise to analyze the monoclonality of CIN and to evaluate its neoplastic property by using single site microdissection rather than multiple sites microdisseetion. But it is significant to study the origin site of cervical squamous cell carcinogenesis by analyzing the X chromosome inactivation pattern of two or more sites. Most of the cervical squamous cell carcinoma is monoclonal, originated from single foci and some of them may be multicentricaUy originated from multiple monoclonal cells.