Department of Biomedical Sciences and Human Oncology, University of Turin, via Santena 7, Turin, Italy.
J Pathol. 2013 Feb;229(3):390-9. doi: 10.1002/path.4074. Epub 2012 Dec 13.
A subgroup of HER2-overexpressing breast tumours co-expresses p95(HER2), a truncated HER2 receptor that retains a functional HER2 kinase domain but lacks the extracellular domain, thus impairing trastuzumab binding. We evaluated p95(HER2) expression in 99 frozen breast carcinoma samples by western blot analysis. The HER2-positive cell line BT474 treated with pervanadate or pronase was used as a positive control for p95(HER2) expression. Immunohistochemistry was performed on parallel formalin-fixed, paraffin-embedded sections of the same case series using antibodies directed against either the intra- or extra-cellular binding domain of HER2. In particular, biotinylated trastuzumab (BiotHER) was used to evaluate the binding capacity of the humanized antibody. To avoid a subjective evaluation of the score values and the percentage of immunostained cells, the slides were scanned and automatically analysed. The number of cases with HER2 overexpression (score 3+) and HER2 gene amplification was higher in the p185(HER2)-positive/p95(HER2)-positive samples than in the p185(HER2)-positive/p95(HER2)-negative group. Automated analysis confirmed a significantly higher percentage of 3+ scored cells in p95(HER2)-positive cases. Conversely, the percentage of 2+ scored cells was higher inp95(HER2)-negative cases. The status of the HER2 extracellular domain was then studied using flow cytometry on BT474 cells after pronase enzymatic digestion using trastuzumab and pertuzumab, while the presence of HER2-HER3 dimers was studied using a proximity-ligation assay. In vitro experiments showed that short-term pronase digestion of BT474 cells produced two HER2 fragments (of 95 and 150 kDa, detectable in tissue specimens as well), increased the binding affinity of trastuzumab, reduced the rate of HER2-HER3 dimers, and did not interfere with pertuzumab-binding capacity. In conclusion, the presence of p95(HER2 as detected by western blot analysis does not compromise the immunohistochemical detection of HER2. Our data suggest that a reduction of the receptor steric hindrance as induced by enzymatic shedding may facilitate the binding capacity of trastuzumab.
一种 HER2 过表达的乳腺癌亚组同时表达 p95(HER2)，这是一种截断的 HER2 受体，保留了功能性的 HER2 激酶结构域，但缺乏细胞外结构域，从而阻碍了曲妥珠单抗的结合。我们通过 Western blot 分析评估了 99 个冷冻乳腺癌样本中的 p95(HER2)表达。用过氧化物或蛋白酶处理的 HER2 阳性细胞系 BT474 被用作 p95(HER2)表达的阳性对照。对同一病例系列的平行福尔马林固定、石蜡包埋切片进行免疫组织化学染色，使用针对 HER2 的细胞内或细胞外结合域的抗体。特别是，使用生物素化曲妥珠单抗（BiotHER）来评估人源化抗体的结合能力。为了避免对评分值和免疫染色细胞百分比的主观评估，对载玻片进行了扫描和自动分析。在 p185(HER2)-阳性/p95(HER2)-阳性样本中，HER2 过表达（评分 3+）和 HER2 基因扩增的病例数高于 p185(HER2)-阳性/p95(HER2)-阴性组。自动分析证实 p95(HER2)-阳性病例中 3+评分细胞的百分比显著更高。相反，p95(HER2)-阴性病例中 2+评分细胞的百分比更高。然后使用针对 BT474 细胞的蛋白酶消化后，使用曲妥珠单抗和帕妥珠单抗通过流式细胞术研究 HER2 细胞外结构域的状态，同时使用接近连接测定法研究 HER2-HER3 二聚体的存在。体外实验表明，BT474 细胞的短期蛋白酶消化产生了两种 HER2 片段（分别为 95 和 150 kDa，在组织标本中也可检测到），增加了曲妥珠单抗的结合亲和力，降低了 HER2-HER3 二聚体的速率，并且不干扰帕妥珠单抗的结合能力。总之，Western blot 分析检测到的 p95(HER2 的存在并不影响 HER2 的免疫组织化学检测。我们的数据表明，酶切诱导的受体空间位阻减少可能有助于曲妥珠单抗的结合能力。
