Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action.
Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action.
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DOI：
10.1186/bcr2866
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发表时间：
2011-04-15
期刊：
影响因子：
--
通讯作者：
Winslow J
中科院分区：
文献类型：
--
作者：
DeFazio-Eli L;Strommen K;Dao-Pick T;Parry G;Goodman L;Winslow J
配体结合的和磷酸化的ErbB/HER异二聚体是该受体家族的有效信号形式，并且这些活性受体的定量测量可以预测患者对靶向治疗的反应。使用VeraTag™技术，我们开发并表征了定量测定法，其测量肿瘤细胞系裂解物和福尔马林固定石蜡包埋（FFPE）肿瘤切片中活化HER受体的表皮生长因子（EGF）依赖性增加。我们证明了该试验定量测量2C 4、厄洛替尼和拉帕替尼处理后细胞系中活化HER 1和HER 2受体水平变化的能力。我们利用这些检测方法来确定43例HER 2阳性乳腺肿瘤中活化的HER 1、HER 2和HER 1-HER 2异二聚体的患病率和分布。使用VeraTag™技术开发了FFPE和细胞裂解物形式的活化HER 1和HER 2受体的测定，该技术需要抗体对的接近以用于荧光标记标签的光依赖性释放，然后进行基于毛细管电泳的定量。通过裂解物和FFPE VeraTag™测定法测量的配体依赖性和非依赖性HER 1-HER 2异二聚体水平与肿瘤细胞系中的HER 1和HER 2表达水平呈趋势，这通过免疫共沉淀得到证实。EGF依赖性HER 1-HER 2异二聚体的形成被HER 2靶向单克隆抗体2C 4抑制，并被HER 1酪氨酸激酶抑制剂（TKI）厄洛替尼稳定。拉帕替尼和厄洛替尼可抑制EGF依赖性HER 1和HER 2磷酸化。此外，我们观察到，根据药物作用机制和HER受体的相对水平，显性受体信号传导模式可能在HER 1-HER 1和HER 1-HER 2之间转换。在同时表达HER 1和HER 2的FFPE乳腺肿瘤中，根据检测方法，在25 - 50%的肿瘤中检测到HER 1-HER 2异二聚体。在对43例HER 2阳性乳腺肿瘤的分析中，活化的磷酸化HER 1-HER 2异二聚体水平与HER 1或HER 2水平相关。VeraTag™裂解物测定可用作了解靶向HER家族抑制剂在临床前环境中的作用机制的工具，而活化HER受体的VeraTag™ FFPE测定结合肿瘤样本中的总HER 2测量（HERmark®）可更准确地预测对HER 1和HER 2靶向治疗的临床反应。
Ligand-bound and phosphorylated ErbB/HER heterodimers are potent signaling forms of this receptor family, and quantitative measurements of these active receptors may be predictive of patient response to targeted therapies. Using VeraTag™ technology, we developed and characterized quantitative assays measuring epidermal growth factor (EGF)-dependent increases in activated HER receptors in tumor cell line lysates and formalin-fixed, paraffin-embedded (FFPE) tumor sections. We demonstrated the ability of the assays to quantitatively measure changes in activated HER1 and HER2 receptor levels in cell lines following treatment with 2C4, erlotinib, and lapatinib. We utilized these assays to determine the prevalence and distribution of activated HER1, HER2, and HER1-HER2 heterodimers in 43 HER2-positive breast tumors. Assays for activated HER1 and HER2 receptors in FFPE and cell lysate formats were developed using VeraTag™ technology, which requires the proximity of an antibody pair for light-dependent release of a fluorescently labeled tag, followed by capillary electrophoresis-based quantitation. Ligand-dependent and independent HER1-HER2 heterodimer levels measured by lysate and FFPE VeraTag™ assays trended with HER1 and HER2 expression levels in tumor cell lines, which was confirmed by co-immunoprecipitation. The formation of EGF-dependent HER1-HER2 heterodimers were inhibited by the HER2-targeted monoclonal antibody 2C4 and stabilized by the HER1 tyrosine kinase inhibitor (TKI) erlotinib. EGF-dependent HER1 and HER2 phosphorylation was inhibited by lapatinib and erlotinib. Further, we observed that dominant receptor signaling patterns may switch between HER1-HER1 and HER1-HER2, depending on drug mechanism of action and relative levels of HER receptors. In FFPE breast tumors that expressed both HER1 and HER2, HER1-HER2 heterodimers were detected in 25 to 50% of tumors, depending on detection method. The levels of activated phospho-HER1-HER2 heterodimers correlated with HER1 or HER2 levels in an analysis of 43 HER2-positive breast tumors. VeraTag™ lysate assays can be used as a tool for understanding the mechanism of action of targeted HER-family inhibitors in the preclinical setting, while VeraTag™ FFPE assays of activated HER receptors combined with total HER2 measurements (HERmark®) in tumor samples may provide a more accurate prediction of clinical response to both HER1 and HER2 targeted therapies.
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影响因子：
4.8
作者：
Gan, Hui K.;Walker, Francesca;Johns, Terrance G.
通讯作者：
Johns, Terrance G.
影响因子：
11.4
作者：
GREENFIELD, C;HILES, I;MCDONALD, N
通讯作者：
MCDONALD, N
影响因子：
4.8
作者：
Arteaga, CL;Ramsey, TT;Guyer, CA
通讯作者：
Guyer, CA
影响因子：
50.3
作者：
Agus, DB;Akita, RW;Sliwkowski, MX
通讯作者：
Sliwkowski, MX
影响因子：
11.5
作者：
Arteaga, Carlos L.;O'Neill, Anne;Davidson, Nancy E.
通讯作者：
Davidson, Nancy E.