A Novel Proteomics-Based Clinical Diagnostics Technology Identifies Heterogeneity in Activated Signaling Pathways in Gastric Cancers

PurposeThe aim of this study was to utilize the proteomics-based Collaborative Enzyme Enhanced Reactive (CEER) immunoassay to investigate protein tyrosine phosphorylations as diagnostic markers in gastric cancers (GCs).
Experimental DesignProtein lysates from fresh-frozen 434 advanced stage GCs were analyzed for phosphorylation of HER1, HER2, p95HER2, HER3, cMET, IGF1R and PI3K. The pathway activation patterns were segregated based on the tumor HER2 status. Hierarchical clustering was utilized to determine pathway coactivations in GCs. Prognostic value of pathway activation patterns was determined by correlating disease-free survival times of the various GC subgroups using Kaplan-Meier survival analysis. CEER was also used to determine the presence of tyrosine phosphorylated signaling cascades in circulating tumor cells (CTCs) and ascites tumor cells (ATCs).
ResultsUtilizing a novel diagnostics immunoassay, CEER, we demonstrate the presence of p95HER2 and concomitantly activated signaling pathways in GC tumor tissues, CTCs and ATCs isolated from GC patients for the first time. p95HER2 is expressed in ∼77% of HER2(+) GCs. Approximately 54% of GCs have an activated HER1, HER2, HER3, cMET or IGF1R and demonstrate a poorer prognosis than those where these receptor tyrosine kinases (RTKs) are not activated. Hierarchical clustering of RTKs reveals co-clustering of phosphorylated HER1:cMET, HER2:HER3 and IGF1R-PI3K. Coactivation of HER1 with cMET renders GCs with a shorter disease-free survival as compared to only cMET activated GCs.
ConclusionsOur study highlights the utility of a novel companion diagnostics technology, CEER that has strong implications for drug development and therapeutic monitoring. CEER is used to provide an increased understanding of activated signaling pathways in advanced GCs that can significantly improve their clinical management through accurate patient selection for targeted therapeutics.

【研究目的】本研究旨在采用基于蛋白质组学的协作酶增强反应（Collaborative Enzyme Enhanced Reactive，CEER）免疫测定法，以蛋白质酪氨酸磷酸化作为诊断标志物，探究其在胃癌（gastric cancers, GCs）中的应用价值。【实验设计】本研究对434份新鲜冰冻的晚期胃癌组织蛋白裂解物进行分析，检测HER1、HER2、p95HER2、HER3、cMET、IGF1R及PI3K的磷酸化水平。根据肿瘤HER2状态对信号通路激活模式进行分组。采用分层聚类法明确胃癌中的信号通路共激活特征。通过Kaplan-Meier生存分析，关联不同胃癌亚组的无病生存时间，以此确定信号通路激活模式的预后价值。此外，本研究还借助CEER免疫测定法，检测循环肿瘤细胞（circulating tumor cells, CTCs）及腹水肿瘤细胞（ascites tumor cells, ATCs）中酪氨酸磷酸化信号级联反应的存在情况。【研究结果】本研究首次借助新型诊断免疫测定法CEER，在胃癌患者来源的胃癌组织、循环肿瘤细胞及腹水肿瘤细胞中，证实了p95HER2的表达及其伴随激活的信号通路。约77%的HER2阳性胃癌组织中可检测到p95HER2的表达。约54%的胃癌存在HER1、HER2、HER3、cMET或IGF1R的激活，相较于上述受体酪氨酸激酶（receptor tyrosine kinases, RTKs）未激活的胃癌患者，该类群体的预后更差。对受体酪氨酸激酶进行分层聚类分析后发现，磷酸化HER1与cMET、HER2与HER3，以及IGF1R-PI3K存在共聚类现象。相较于仅cMET激活的胃癌患者，HER1与cMET共激活的胃癌患者无病生存期更短。【研究结论】本研究证实了新型伴随诊断技术CEER的应用价值，其在药物研发与治疗监测领域具备重要意义。借助CEER可加深对晚期胃癌激活信号通路的认知，通过精准筛选适合接受靶向治疗的患者，显著改善晚期胃癌的临床诊疗效果。