Visscher D W, Sarkar F H, Shimoyama R K, Crissman J D
Department of Pathology, Harper Hospital, Detroit, MI 48201, USA.
Diagn Mol Pathol. 1996 Sep;5(3):187-93. doi: 10.1097/00019606-199609000-00007.
We performed p53 immunostaining in 82 invasive breast carcinomas by using two commercially available antibodies, one of which (DO7) was employed in formalin-fixed paraffin-embedded sections. The other antibody (PAb1801) was evaluated in corresponding acetone-fixed cryostat sections. A greater percent of cases were immunostained with DO7 compared to PAb1801 (52% vs 33%); however, the staining was more often heterogeneous (6-50% cells positive) or focal (< or = 5% cells positive) with DO7 (9% vs 31%). To investigate the genetic relevance of p53 immunostaining, single-strand conformational polymorphism (SSCP) analysis and DNA sequencing were performed on exons 2-11 by using archival tissue samples of 18 cases that were selected on the basis of certain immunostaining patterns. Two (33%) of six tumors with negative staining for DO7 had gene sequence mutations; however, one of these mutations was a base-pair deletion that caused a reading-frame shift and the other was a base-pair insertion that resulted in a stop codon. Both of these tumors exhibited immunostaining with PAb1801, although it was weak and cytoplasmic in one case. Conversely, three (30%) of 10 tumors showing immunoreactivity in 6-100% of cells with both reagents lacked a gene sequence mutation. Of the remaining seven tumors that were positive by SSCP, six contained a point mutation resulting in a base-pair substitution. Despite repeat analyses, one of the cases positive by SSCP failed to demonstrate a mutation in the sequenced exons. Four (80%) of five cases with heterogeneous DO7 immunoreactivity (that is, 6-50% of nuclei positive) were positive for gene sequence mutation. Neither of two cases showing focal DO7 nuclear staining in < 5% of tumor cells contained a mutation in the sequenced exons, and neither of these cases was strongly positive with PAb1801. Staining for either antibody was significantly associated with adverse outcome, as determined by disease recurrence at 52 months median follow-up (DO7, p = 0.01; and PAb1801 p = 0.002, chi-squared test). We conclude that a variety of factors may account for discrepancies when immunohistology is used to evaluate p53 status. These include fixation artifacts, differing epitope specificities of monoclonal reagents, presence of immunohistologically "silent" mutations and, possibly, aberrant overexpression of wild-type protein.
我们使用两种市售抗体对82例浸润性乳腺癌进行了p53免疫染色，其中一种抗体（DO7）用于福尔马林固定石蜡包埋切片，另一种抗体（PAb1801）用于相应的丙酮固定冰冻切片。与PAb1801相比，DO7免疫染色的病例百分比更高（52%对33%）；然而，DO7染色更常呈异质性（6 - 50%细胞阳性）或局灶性（≤5%细胞阳性）（9%对31%）。为了研究p53免疫染色的遗传学相关性，我们对18例根据特定免疫染色模式选择的存档组织样本的外显子2 - 11进行了单链构象多态性（SSCP）分析和DNA测序。6例DO7染色阴性的肿瘤中有2例（33%）存在基因序列突变；然而，其中一个突变是导致阅读框移位的碱基对缺失，另一个是导致终止密码子的碱基对插入。这两个肿瘤均显示对PAb1801有免疫染色，尽管其中1例较弱且为细胞质染色。相反，10例肿瘤中有3例（30%）在两种试剂检测下6 - 100%的细胞显示免疫反应性，但缺乏基因序列突变。在其余7例SSCP阳性的肿瘤中，6例含有导致碱基对替换的点突变。尽管进行了重复分析，但1例SSCP阳性的病例在测序的外显子中未显示突变。5例DO7免疫反应性异质性（即6 - 50%细胞核阳性）的病例中有4例（80%）基因序列突变阳性。2例肿瘤细胞中<5%显示DO7核局灶性染色的病例在测序的外显子中均未发生突变，且这2例病例对PAb1801均无强阳性反应。中位随访52个月时，根据疾病复发情况判断，两种抗体的染色均与不良预后显著相关（DO7，p = 0.01；PAb1801，p = 0.002，卡方检验）。我们得出结论，当使用免疫组织化学评估p53状态时，多种因素可能导致差异。这些因素包括固定假象、单克隆试剂不同的表位特异性、免疫组织学“沉默”突变的存在以及可能的野生型蛋白异常过表达。
