Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia 20110, USA.
Clin Cancer Res. 2012 Dec 1;18(23):6426-35. doi: 10.1158/1078-0432.CCR-12-0452. Epub 2012 Oct 8.
Targeting of the HER2 protein in human breast cancer represents a major advance in oncology but relies on measurements of total HER2 protein and not HER2 signaling network activation. We used reverse-phase protein microarrays (RPMA) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity.
Three independent study sets, comprising a total of 415 individual patient samples from flash-frozen core biopsy samples and formalin-fixed and paraffin-embedded (FFPE) surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pretreatment core biopsy samples and whole-tissue lysates from 288 FFPE samples and these results were compared with FISH and immunohistochemistry (IHC).
RPMA measurements of total HER2 were highly concordant (>90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC(+) population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC(-) tumors and, identical to that seen with FISH/IHC(+) tumors, the HER2 activation was concordant with EGF receptor (EGFR) and HER3 phosphorylation and downstream signaling endpoint activation.
Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC(-)/FISH(-)/pHER2(+) tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR.
针对人类乳腺癌中的 HER2 蛋白是肿瘤学的重大进展，但依赖于总 HER2 蛋白的测量，而不是 HER2 信号网络的激活。我们使用反相蛋白质微阵列（RPMA）来测量 HER 家族信号转导中的总 HER2 和磷酸化 HER2，以了解 HER2 的磷酸化和总水平与下游信号活性之间的相关性。
使用 RPMA 分析了由来自于 415 个个体的新鲜冷冻核心活检样本和福尔马林固定石蜡包埋（FFPE）手术和核心样本组成的三个独立研究组，共 415 个个体样本。从 127 个冷冻预处理核心活检样本的激光捕获微切割（LCM）富集肿瘤上皮和 288 个 FFPE 样本的全组织裂解物中测量了 HER 受体家族蛋白和下游信号分子的磷酸化和总水平，并将这些结果与 FISH 和免疫组织化学（IHC）进行了比较。
RPMA 对总 HER2 的测量与 FISH 和/或 IHC 数据高度一致（所有组均>90%），FISH/IHC（+）人群中 HER2 的磷酸化也是如此。HER 家族信号转导的磷酸化分析确定了一些 FISH/IHC（-）肿瘤中的 HER2 激活，与 FISH/IHC（+）肿瘤相同，HER2 激活与表皮生长因子受体（EGFR）和 HER3 磷酸化以及下游信号终点激活一致。
使用定量和敏感的功能途径激活图谱技术对大量人类乳腺癌标本的 HER2 信号进行分子分析，揭示了独立于总 HER2 水平和通过 HER3 和 EGFR 进行功能信号的 IHC（-）/FISH（-）/pHER2（+）肿瘤中的 HER2 途径激活。
