Validation of EGFR Protein Complexes as Molecular Diagnostics

EGFR 蛋白复合物作为分子诊断的验证

• 批准号: 10221627
• 负责人: ERIC B. HAURA
• 金额: $ 30.87万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-06 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221627
• 关键词: Biological Assay; CLIA certified; Cancer Model; Cancer Patient; Cancer cell line; Clinical; Complex; DNA Sequence Alteration; Data; Data Set; Environment; Epidermal Growth Factor Receptor; Event; Fibroblast Growth Factor Receptors; Formalin; Future; Gene Amplification; Gene Expression; Gene Mutation; Growth Factor; Hospitals; Human; In Situ; In Vitro; KRAS2 gene; Ligands; Ligation; Malignant Neoplasms; Malignant neoplasm of lung; Mass Spectrum Analysis; Measures; Mus; Mutation; Paraffin Embedding; Patients; Performance; Phosphotransferases; Publishing; Receptor Activation; Receptor Protein-Tyrosine Kinases; Resistance; Role; Sampling; Signal Transduction; Stromal Neoplasm; Technology; Testing; Therapeutic; Tissue Embedding; Tissues; Validation; Vision; Xenograft Model; base; cancer cell; cohort; growth factor receptor-bound protein 2; in vivo; kinase inhibitor; molecular diagnostics; neoplastic cell; novel strategies; patient derived xenograft model; predicting response; protein complex; protein expression; protein protein interaction; public health relevance; response; tumor; validation studies

DESCRIPTION (provided by applicant): We recently developed a new approach to characterizing activity of receptor tyrosine kinases (RTK) on tumor cells by visualizing signaling associated protein complexes using proximity ligation assay technology. This was motivated by the (i) the poor performance of many RTK expression assays, such as IHC or FISH, that measure protein or gene expression rather than signaling activity, (ii) the need to better characterize active signaling events in tumor cells driven by tumor stromal mechanisms, (iii) the realization that some patients still benefit from anti-RTK directed therapy despite their tumors lacking genomic alterations, and (iv) the need to develop assays that can better identify mechanisms of acquired resistance to kinase inhibitor therapy and thus guide clinicians about subsequent treatment. Activation of RTK through diverse mechanisms, including gene mutation, gene amplification, or overproduction of activating growth factor ligands, leads to assembly of protein complexes on RTK that facilitate downstream signal transduction. Exploiting this fact, we have developed proximity ligation assays (PLA) to annotate protein complexes reflecting an activated EGFR. PLA can be developed starting with mass spectrometry based protein-protein interaction datasets to characterize in situ EGFR complexes reflecting active signaling in cancer tissues. We have developed one such assay reflecting EGFR in complex with its adaptor protein GRB2. We show these assays reflect EGFR kinase activity both in vitro using cancer cell lines and in vivo using mouse xenograft models of lung cancer. Importantly, these assays perform well in formalin fixed paraffin embedded (FFPE) tissues enabling wide spread utility in typical hospital tumor samples. We demonstrate feasibility and utility in characterizing EGFR:GRB2 complexes in nearly 300 patient derived xenograft models of cancer. Further, our data indicate good concordance of high PLA signals across three independent cohorts of lung cancer patients numbering 350 unique cases. Our human tumor data shows high PLA signal in tumors with activating EGFR mutation as expected but we can further identify tumors with high PLA signal that lack EGFR mutation or with KRAS mutation, thus representing tumor stromal activation of EGFR. We show these assays can detect EGFR driven resistance in lung cancers driven by EML4-ALK translocations and along with other published studies indicate a role of EGFR PLA assays to detect acquired resistance to kinase inhibitors driven by EGFR signaling. Our vision is that PLA technology can be used to define signaling associated protein complexes for many RTK beyond EGFR, including MET, FGFR, and AXL, which are primary targets of emerging therapeutics and also involved in resistance. This offers the future potential to have multiple PLA assays each reflecting signaling associated protein complexes for RTK matched to therapeutics. During the UH2 aim, we will conduct analytic validation studies of our existing EGFR PLA in a CLIA-compliant environment. During the UH3 aim, we will conduct clinical validation studies by investigating the association of PLA with response to EGFR-directed therapies.

描述（由申请人提供）：我们最近开发了一种新的方法，通过使用邻位连接测定技术可视化信号相关蛋白复合物来表征肿瘤细胞上受体酪氨酸激酶（RTK）的活性。这是由于（i）许多RTK表达测定，如IHC或FISH，测量蛋白质或基因表达而不是信号传导活性的性能差，（ii）需要更好地表征由肿瘤基质机制驱动的肿瘤细胞中的活性信号传导事件，（iii）认识到尽管一些患者的肿瘤缺乏基因组改变，但他们仍然受益于抗RTK定向治疗，和（iv）需要开发能够更好地鉴定对激酶抑制剂治疗的获得性抗性的机制的测定，从而指导临床医生进行后续治疗。通过多种机制激活RTK，包括基因突变、基因扩增或活化生长因子配体的过量产生，导致蛋白质复合物在RTK上组装，从而促进下游信号转导。利用这一事实，我们已经开发了邻位连接测定（PLA）来注释反映活化EGFR的蛋白质复合物。PLA可以从基于质谱的蛋白质-蛋白质相互作用数据集开始开发，以表征反映癌组织中活性信号传导的原位EGFR复合物。我们已经开发了一种反映EGFR与其衔接蛋白GRB 2复合的检测方法。我们表明这些测定反映了EGFR激酶活性，在体外使用癌细胞系和体内使用小鼠肺癌异种移植模型。重要的是，这些测定在福尔马林固定石蜡包埋（FFPE）组织中表现良好，使得能够在典型的医院肿瘤样品中广泛应用。我们证明了在近300例患者来源的癌症异种移植模型中表征EGFR：GRB 2复合物的可行性和实用性。此外，我们的数据表明，在三个独立的肺癌患者队列中，高PLA信号具有良好的一致性，共350例独特病例。我们的人类肿瘤数据显示，如预期的那样，在具有活化EGFR突变的肿瘤中具有高PLA信号，但是我们可以进一步鉴定具有高PLA信号的缺乏EGFR突变或具有KRAS突变的肿瘤，因此代表EGFR的肿瘤基质活化。我们表明这些检测方法可以检测EML 4-ALK易位驱动的肺癌中EGFR驱动的耐药性，并且沿着其他已发表的研究表明EGFR PLA检测方法在检测EGFR信号传导驱动的激酶抑制剂获得性耐药性中的作用。我们的愿景是，PLA技术可用于定义EGFR以外的许多RTK的信号相关蛋白复合物，包括MET、FGFR和AXL，它们是新兴疗法的主要靶点，也涉及耐药性。这提供了未来具有多种PLA测定的可能性，每种测定反映与治疗剂匹配的RTK的信号传导相关蛋白质复合物。在UH 2目标期间，我们将在符合CLIA的环境中对现有EGFR PLA进行分析验证研究。在UH 3目标期间，我们将通过调查PLA与EGFR导向治疗反应的相关性来进行临床验证研究。