Multiplexed isoform quantification in HER2-positive breast cancer

HER2 阳性乳腺癌的多重亚型定量

• 批准号: 9390766
• 负责人: Amy Elizabeth Herr
• 金额: $ 34.78万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9390766
• 关键词: Address; Aftercare; Antibodies; Attention; Biological Assay; Biology; Biometry; Biopsy; Breast; Breast Cancer Cell; Breast Cancer Patient; Breast Cancer Treatment; Breast Cancer cell line; Breast biopsy; Buffers; Cell Nucleus; Cell surface; Cells; Classification; Clinical; Clinical Oncology; Collection; Complex; Cytolysis; Disease; Drug resistance; ERBB2 gene; ERBB3 gene; Engineering; Extracellular Domain; FRAP1 gene; Flow Cytometry; Freezing; Gel; Gold; Her2/erbb2/neu Staining Method; HercepTest; Heterogeneity; Human; Immunoassay; Immunohistochemistry; Knowledge; Length; Liquid substance; Location; Malignant Neoplasms; Measurement; Measures; Membrane Proteins; Microfluidics; Minor; Molecular; Monitor; Nuclear; Oncoproteins; Outcome; PPM1D gene; Pathology; Pathway interactions; Patient-Focused Outcomes; Performance; Personal Satisfaction; Pharmaceutical Preparations; Pilot Projects; Population; Precision therapeutics; Progression-Free Survivals; Protein Isoforms; Proteins; Reproducibility; Research; Research Personnel; Resistance; Resolution; Role; Selection for Treatments; Signal Transduction; Signaling Protein; Specificity; Surface; Testing; Therapeutic; Tissue Banks; Tissue Sample; Tissues; Translations; Trastuzumab; Vascular Endothelial Growth Factors; Western Blotting; base; cancer cell; cancer classification; design; experience; improved; kinase inhibitor; laser capture microdissection; malignant breast neoplasm; neoplastic cell; novel; novel therapeutics; overexpression; programs; protein biomarkers; public health relevance; resistance mechanism; small molecule; targeted cancer therapy; targeted treatment; therapy resistant; tool; tumor; tumor heterogeneity; tumor progression; ultra high resolution; ward

 DESCRIPTION: Nearly 50% of advanced HER2-positive breast cancer patients succumb to the disease, even after treatment with precision targeted therapies. Some of the most powerful precision therapies, including the landmark drug Herceptin(r) (trastuzumab), target the extracellular domain of the HER2 protein. Yet, new findings suggest that the presence of HER2 protein isoforms that lack the extracellular domain of the full-length HER2 protein (called truncated HER2 isoforms) make tumor cells non-responsive to precision therapies and correlate with drug resistance. Consequently, treatment decisions will be directly informed by the presence of the full-length HER2 protein (trastuzumab) or high levels of truncated isoforms (small molecule kinase inhibitors). Consequently, the ability to directly measure the truncated HER2 isoforms in sparingly available breast biopsy tissues and with single-cell resolution would yield a tremendous advantage for selecting treatment and assessing the potential for drug resistance. Regrettably, existing immunoassay based tools cannot distinguish the forms of HER2 (p185-erbB2, p110-erbB2, p95m-, p95c-, and p95n-erbB2) present in minute tissue biopsies. No antibodies specific to all forms of the HER2 protein exist. A transdisciplinary research program will create and optimize a suite of powerful microanalytical tools to directly quantify the levels and identities of HER2 protein forms and resistance-related signaling proteins in tissues and cells from HER2-positive breast cancer patient biopsies with the Stanford Breast Cancer Tissue Bank. A Protein Panel of drug-resistance related proteins will be assayed (mTOR, PI3K, HER3, IGF-1R, Akt, VEGF) as a pilot study to understand the role of these pathways in drug resistance. After establishing performance in breast cancer cell lines, in Aim 1 the mWB will assay the isoforms in both fresh-frozen and fixed (FFPE) tissue samples. We will assess differences in signaling and expression between leading edge and interior tumor regions. Further, we will compare with patient outcomes and gold-standard IHC classification. In a complementary Aim 2A, the investigators will introduce and optimize a single-cell resolution western blot to assay the HER2 protein isoforms and drug resistance-related signaling proteins but now for thousands of breast cancer cells with single-cell resolution. Cell-to-cell heterogeneity in the Protein Panel will comprise new HER2-positive breast cancer sub-classifications, which will in turn be assessed for correlation to patient outcomes. In Aim 2B, the single-cell tool will be optimized to provide sub-cellular resolution on the cell surface versus nuclear location of full-length HER2 and a truncated isoform, a currently impossible measurement implicated in drug resistance. Anticipated outcomes seek to eliminate a critical bottleneck to improving the well-being of BCa patients. While HER2 isoforms are a focus of this initial effort, numerous other oncoproteins are isoforms (ER, RON, MDM, MYC, BAG-1, PPM1D, and FLIP) and acknowledged contributors to drug resistance and cancer progression. The unique analytical approaches developed will address open questions regarding the role of isoforms in BCa outcomes and therapy resistance in miniscule tissue biospecimens that are otherwise out of analytical reach.

 描述：即使在针对精确的靶向疗法治疗后，近50％的晚期HER2阳性乳腺癌患者也屈服于该疾病。一些最强大的精确疗法，包括具有里程碑意义的药物Herceptin（R）（Trastuzumab），靶向HER2蛋白的细胞外结构域。然而，新发现表明，缺乏全长HER2蛋白（称为截短的HER2同工型）的HER2蛋白同工型的存在使肿瘤细胞无反应地对精度治疗，并且与药物抗性相关。因此，将通过全长HER2蛋白（曲妥珠单抗）或高水平的截短同工型（小分子激酶抑制剂）直接告知治疗决策。因此，在很少有可用的乳房活检组织和单细胞分辨率中直接测量截短的HER2同工型的能力将对选择治疗和评估耐药性的潜力产生巨大优势。遗憾的是，现有的基于免疫测定的工具无法区分Minute组织活检中存在的HER2（P185-ERBB2，P110-ERBB2，P95M-，P95C-，P95C-和P95N-ERBB2）的形式。没有针对所有形式的HER2蛋白的特异性抗体。跨学科研究计划将创建并优化一套强大的微分析工具，以直接量化HER2阳性乳腺癌患者活检中与斯坦福大学乳腺癌组织的组织和细胞中HER2蛋白形式的水平和身份。将分配一个与药物抗性相关蛋白质的蛋白质面板（MTOR，PI3K，HER3，IGF-1R，AKT，VEGF），以了解这些途径在耐药性中的作用。在在乳腺癌细胞系中建立性能后，在AIM 1中，MWB将在新鲜冻干和固定（FFPE）组织样品中主张同工型。我们将评估前缘和内部肿瘤区域之间的信号传导和表达差异。此外，我们将与患者结局和金标准IHC分类进行比较。在一个完整的目标2a中，研究人员将引入和优化单细胞分辨率蛋白质印迹，以主张HER2蛋白同工型和耐药性相关的信号传导蛋白，但现在用于成千上万的具有单细胞分辨率的乳腺癌细胞。蛋白质面板中细胞到细胞的异质性将完成新的HER2阳性乳腺癌亚分类，而这些乳腺癌又将对患者结局的相关性进行评估。在AIM 2B中 单细胞工具将被优化，以在全长HER2的细胞表面与核位置以及截短的同工型的核位置提供亚细胞分辨率，这是耐药性中暗示的目前不可能的测量。预期的结果试图消除关键的瓶颈，以改善BCA患者的福祉。尽管HER2同工型是这项初步努力的重点，但许多其他癌蛋白是同工型（ER，RON，MDM，MYC，BAG-1，PPM1D和FLIP），并确认了耐药性和癌症进展的贡献者。开发的独特分析方法将解决有关同工型在BCA结局中的作用和在微小组织生物质量中的耐药性作用的问题，这些问题否则是无法分析的。