Simony J, Pujol J L, Radal M, Ursule E, Michel F B, Pujol H
Centre Régional de Lutte contre le Cancer, Montpellier, France.
Cancer Res. 1990 Jul 15;50(14):4382-7.
Ploidy and growth fraction were analyzed by means of a computer-assisted image processor in surgically resected non-small cell lung cancer (NSCLC). This study was done in order (a) to evaluate the distribution of anti-Ki-67 immunostaining and (b) to correlate this distribution to ploidy status and pTNM stage of NSCLC. Thirty-two patients underwent a surgical resection for primary NSCLC following complete staging. Indirect immunoperoxidase reactions of monoclonal antibody Ki-67 were done on frozen tissue sections. Integrated optical density and index of stained nuclear surface were calculated by means of a computer-assisted image processor in 120 fields of each preparation in order to quantify the Ki-67 immunostaining. DNA content was determined by means of cytometry of Feulgen-stained cytological prints. The ploidy status was defined for each tumor by DNA index, percentage of hypodiploid cells, and type of DNA content histogram (near diploid, hyperdiploid, hypodiploid, and multiploid). Reproducibility of immunostaining quantitative analysis was demonstrated by iterative measurements of the same slide. Intratumoral heterogeneity of Ki-67 immunostaining induced integrated optical density variation assessed on six nonconsecutive tissue sections from at least two regions of the same tumor. This intratumoral variability was 15 times lower than integrated optical density variability between tumors. The Ki-67 immunostaining varied significantly according to the DNA content histogram type (P less than 0.05, Kruskal-Wallis test); most of the specimens with high Ki-67 immunostaining were multiploid or hypodiploid. Moreover, Ki-67 immunostaining correlated to the percentage of hypodiploid cells. Ki-67 immunostaining and ploidy status did not vary significantly according to the tumor-nodes-metastasis stage. We conclude that (a) quantitative analysis of Ki-67 immunostaining is a reliable evaluation of growth fraction in NSCLC if a large number of fields are analyzed to take into account intratumoral variability, (b) hypodiploidy and multiploidy are frequent abnormalities of DNA content, (c) Ki-67 immunostaining is significantly higher in hypodiploid and multiploid tumors. Thus, determination of growth fraction and ploidy in surgically resected NSCLC specimens may be considered as complementary prognostic parameters independent of the stage of the disease.
采用计算机辅助图像处理器对手术切除的非小细胞肺癌（NSCLC）的倍性和生长分数进行分析。开展本研究的目的是：（a）评估抗Ki-67免疫染色的分布情况；（b）将该分布情况与NSCLC的倍性状态和pTNM分期相关联。32例患者在完成全面分期后接受了原发性NSCLC的手术切除。对冷冻组织切片进行单克隆抗体Ki-67的间接免疫过氧化物酶反应。通过计算机辅助图像处理器在每份标本的120个视野中计算积分光密度和染色核表面积指数，以量化Ki-67免疫染色。通过对Feulgen染色的细胞学印片进行细胞计数法测定DNA含量。根据DNA指数、亚二倍体细胞百分比和DNA含量直方图类型（近二倍体、超二倍体、亚二倍体和多倍体）确定每个肿瘤的倍性状态。通过对同一张玻片进行重复测量证明了免疫染色定量分析的可重复性。在来自同一肿瘤至少两个区域的6个非连续组织切片上评估Ki-67免疫染色诱导的积分光密度变化所反映的肿瘤内异质性。这种肿瘤内变异性比肿瘤之间的积分光密度变异性低15倍。Ki-67免疫染色根据DNA含量直方图类型有显著差异（P<0.05，Kruskal-Wallis检验）；大多数Ki-67免疫染色高的标本为多倍体或亚二倍体。此外，Ki-67免疫染色与亚二倍体细胞百分比相关。Ki-67免疫染色和倍性状态根据肿瘤-淋巴结-转移分期无显著差异。我们得出结论：（a）如果分析大量视野以考虑肿瘤内变异性，Ki-67免疫染色的定量分析是评估NSCLC生长分数的可靠方法；（b）亚二倍体和多倍体是常见的DNA含量异常；（c）亚二倍体和多倍体肿瘤中的Ki-67免疫染色显著更高。因此，手术切除的NSCLC标本中生长分数和倍性的测定可被视为独立于疾病分期的补充性预后参数。
