Scott Ian S, Morris Lesley S, Bird Kate, Davies R Justin, Vowler Sarah L, Rushbrook Simon M, Marshall Aileen E, Laskey Ronald A, Miller Richard, Arends Mark J, Coleman Nicholas
MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 2XZ, UK.
J Pathol. 2003 Oct;201(2):187-97. doi: 10.1002/path.1444.
An immunohistochemical method for assessing cell-cycle phase distribution in colorectal resection specimens would enable phase data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in colorectal cancer. In contrast to flow cytometry, an immunohistochemical method would also allow the phase distribution to be examined within morphologically heterogeneous regions of neoplasms. Paraffin sections of normal colon (n = 25), colonic adenoma (n = 15), and colonic adenocarcinoma (n = 30) were analysed by immunohistochemistry using antibodies against markers of cell-cycle entry, Mcm-2 and Ki67, and putative markers of the cell-cycle phase, cyclins D1 and E (putative markers of G1 phase), cyclin A (S phase), cytoplasmic cyclin B1 (G2 phase), and phosphohistone H3 (M phase). The phase specificity of each marker was assessed by examining the degree of co-expression of adjacent phase markers using double-antibody fluorescence confocal microscopy and by comparison with flow cytometric analysis performed on adjacent tissue sections. The S-phase specificity of detectable cyclin A was also assessed in combination with in situ DNA replication using fluorescence confocal microscopy. All cells expressing phase markers co-expressed Mcm-2. Adjacent phase markers were not significantly co-expressed, confirming the relative specificity of these markers in tissue sections of colon. Cell-cycle phase distribution, calculated by immunohistochemistry, compared well with phase analyses obtained by flow cytometry. No cells expressed cyclin A in the absence of active DNA replication. The S-phase labelling index, as defined by detectable cyclin A expression, showed a positive correlation with the Mcm-2 labelling index and increased in the progression from normal colon to adenocarcinoma. In conclusion, a combination of these cell-cycle phase markers can be used to calculate the distribution of cells throughout each phase of the cell cycle in colorectal tissue sections. Detectable cyclin A can be used as a surrogate marker of S phase and may be of value in predicting prognosis and response to adjuvant therapy.
一种用于评估结直肠癌切除标本中细胞周期阶段分布的免疫组织化学方法，将使阶段数据能够纳入诊断算法，以估计结直肠癌的预后和对辅助化疗的反应。与流式细胞术不同，免疫组织化学方法还能在肿瘤形态学异质性区域内检查阶段分布。使用针对细胞周期进入标记物Mcm-2和Ki67以及细胞周期阶段推定标记物细胞周期蛋白D1和E（G1期推定标记物）、细胞周期蛋白A（S期）、细胞质细胞周期蛋白B1（G2期）和磷酸化组蛋白H3（M期）的抗体，通过免疫组织化学分析正常结肠（n = 25）、结肠腺瘤（n = 15）和结肠腺癌（n = 30）的石蜡切片。通过使用双抗体荧光共聚焦显微镜检查相邻阶段标记物的共表达程度，并与在相邻组织切片上进行的流式细胞术分析相比较，评估每个标记物的阶段特异性。还结合使用荧光共聚焦显微镜的原位DNA复制评估可检测到的细胞周期蛋白A的S期特异性。所有表达阶段标记物的细胞均共表达Mcm-2。相邻阶段标记物未显著共表达，证实了这些标记物在结肠组织切片中的相对特异性。通过免疫组织化学计算的细胞周期阶段分布与通过流式细胞术获得的阶段分析结果良好相符。在没有活跃DNA复制的情况下，没有细胞表达细胞周期蛋白A。由可检测到的细胞周期蛋白A表达定义的S期标记指数与Mcm-2标记指数呈正相关，并且在从正常结肠到腺癌的进展过程中增加。总之，这些细胞周期阶段标记物的组合可用于计算结直肠组织切片中细胞在细胞周期各阶段的分布。可检测到的细胞周期蛋白A可作为S期的替代标记物，可能在预测预后和辅助治疗反应方面具有价值。
