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: 10436863
• 负责人: ERIC B. HAURA
• 金额: $ 13.86万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10436863
• 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; inhibitor therapy; 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来表征受体酪氨酸激酶（RTK）在肿瘤细胞上的活性，通过使用近距离连接测定技术可视化信号相关蛋白复合物。这是由于(i)许多RTK表达分析，如IHC或FISH，测量蛋白质或基因表达而不是信号活性，表现不佳，（ii）需要更好地表征肿瘤细胞中由肿瘤基质机制驱动的活性信号事件，（iii）认识到一些患者尽管肿瘤缺乏基因组改变，但仍然受益于抗RTK定向治疗。（iv）需要开发能够更好地识别对激酶抑制剂治疗获得性耐药机制的检测方法，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、基因扩增或激活生长因子配体的过量生产，导致RTK上的蛋白质复合物组装，促进下游信号转导。利用这一事实，我们开发了接近连接法（PLA）来注释反映激活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，以表征反映癌症组织中活性信号的原位EGFR复合物。我们已经开发了一种这样的检测方法，可以反映EGFR及其适配蛋白GRB2的复合物。我们表明，这些检测在体外使用癌细胞系和体内使用肺癌小鼠异种移植模型中都反映了EGFR激酶活性。重要的是，这些检测在福尔马林固定石蜡包埋（FFPE）组织中表现良好，从而在典型的医院肿瘤样本中广泛应用。我们证明了在近300例患者来源的异种肿瘤移植模型中表征EGFR:GRB2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350例肺癌患者队列中，高PLA信号具有良好的一致性。我们的人类肿瘤数据显示，正如预期的那样，在激活EGFR突变的肿瘤中存在高PLA信号，但我们可以进一步识别缺乏EGFR突变或KRAS突变的高PLA信号肿瘤，从而代表肿瘤间质激活EGFR。我们表明，这些检测方法可以检测由EML4-ALK易位驱动的肺癌中EGFR驱动的耐药性，并与其他已发表的研究一起表明，EGFR PLA检测方法可以检测由EGFR信号驱动的对激酶抑制剂的获得性耐药性。我们的愿景是，PLA技术可用于定义除EGFR之外的许多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，也与耐药性有关。这提供了未来多种PLA检测的潜力，每种检测都反映与治疗方法匹配的RTK信号相关蛋白复合物。在UH2目标期间，我们将在符合clia的环境中对我们现有的EGFR PLA进行分析验证研究。在UH3目标期间，我们将通过调查PLA与egfr导向治疗反应的关系进行临床验证研究。