Elledge R M, Clark G M, Fuqua S A, Yu Y Y, Allred D C
University of Texas Health Science Center, Division of Medical Oncology, San Antonio 78284-7884.
Cancer Res. 1994 Jul 15;54(14):3752-7.
Nuclear accumulation of p53 protein is associated with a poorer clinical outcome in breast cancer patients. Heat shock protein 70 (hsp70) is a chaperone that binds to mutant p53 and consequently could regulate its accumulation or localization. The aims of this study were to determine if the prognostic significance of p53 accumulation was dependent on the type of antibody used for detection and whether hsp70 was associated with this accumulation. Node-negative breast tumors (n = 169) were examined by immunohistochemistry for nuclear p53, cytoplasmic or nuclear hsp70, and for p53 gene alteration by single-strand conformation polymorphism analysis. Frozen sections of pulverized breast tumors were stained with five p53 antibodies (240, 1801, 421, BP53-12, and CM1), a cocktail of both 240 and 1801, and the hsp70 antibody C92. Protein level was expressed as the sum of a proportion and intensity score (total 0, 2-8) with > or = 2 defined as positive staining. The cocktail 240/1801 gave the highest rate of positive staining (45%), followed by BP53-12 (35%), 1801 (27%), 240 (25%), CM1 (24%), and 421 (18%), with a high correlation between antibodies. Positive staining with each individual antibody or the cocktail was significantly associated with estrogen receptor and progesterone receptor negativity, age < 50, and high S-phase fraction. Only staining detected by the 240/1801 cocktail was associated with significantly worse overall survival; 85 versus 70% at 5 years for p53-negative compared to p53-positive tumors, respectively (P = 0.02). There was no association between nuclear or cytoplasmic hsp70 staining and accumulation of p53. Patients that were p53-negative/cytoplasmic hsp70-positive had a better overall survival than those that were p53-negative/cytoplasmic hsp70-negative. No other combination of p53 and hsp70 status could further define subsets of patients with a significantly different prognosis compared to p53 status alone. Tumors without a detectable p53 gene alteration by single-strand conformation polymorphism but with accumulated p53 protein did not have relatively increased levels of hsp70. We conclude that in node-negative breast cancer, the cocktail of two antibodies, 240/1801, resulted in the highest rate of positive staining and was most strongly associated with overall survival compared with either antibody alone or with the other individual antibodies. By immunohistochemistry, nuclear accumulation of p53 was not associated with cytoplasmic or nuclear hsp70 levels.
p53蛋白的核内积聚与乳腺癌患者较差的临床预后相关。热休克蛋白70（hsp70）是一种伴侣蛋白，可与突变型p53结合，从而调节其积聚或定位。本研究的目的是确定p53积聚的预后意义是否取决于用于检测的抗体类型，以及hsp70是否与这种积聚相关。通过免疫组织化学检测169例淋巴结阴性的乳腺肿瘤，检测核p53、细胞质或核hsp70，并通过单链构象多态性分析检测p53基因改变。将粉碎的乳腺肿瘤冰冻切片用五种p53抗体（240、1801、421、BP53 - 12和CM1）、240和1801的混合物以及hsp70抗体C92进行染色。蛋白水平以比例和强度评分的总和表示（总分0、2 - 8），≥2定义为阳性染色。240/1801混合物的阳性染色率最高（45%），其次是BP53 - 12（35%）、1801（27%）、240（25%）、CM1（24%）和421（18%），各抗体之间具有高度相关性。每种单独抗体或混合物的阳性染色与雌激素受体和孕激素受体阴性、年龄<50岁以及高S期分数显著相关。只有240/1801混合物检测到的染色与总体生存率显著较差相关；p53阴性肿瘤与p53阳性肿瘤在5年时的总体生存率分别为85%和70%（P = 0.02）。核或细胞质hsp70染色与p53的积聚之间无关联。p53阴性/细胞质hsp70阳性的患者总体生存率优于p53阴性/细胞质hsp70阴性的患者。与单独的p53状态相比，p53和hsp70状态的其他组合均不能进一步明确预后显著不同的患者亚组。通过单链构象多态性未检测到p53基因改变但p53蛋白积聚的肿瘤，其hsp70水平并未相对升高。我们得出结论，在淋巴结阴性的乳腺癌中，两种抗体的混合物240/1801导致最高的阳性染色率，与单独使用任何一种抗体或其他单个抗体相比，与总体生存率的相关性最强。通过免疫组织化学，p53的核内积聚与细胞质或核hsp70水平无关。
