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.

 产品说明： 近50%的晚期HER 2阳性乳腺癌患者死于该病，即使在接受精确靶向治疗后也是如此。一些最强大的精确疗法，包括具有里程碑意义的药物赫赛汀（r）（曲妥珠单抗），靶向HER 2蛋白的细胞外结构域。然而，新的研究结果表明，缺乏全长HER 2蛋白胞外结构域的HER 2蛋白亚型（称为截短的HER 2亚型）的存在使肿瘤细胞对精确治疗无反应，并与耐药性相关。因此，治疗决定将直接根据全长HER 2蛋白（曲妥珠单抗）或高水平截短亚型（小分子激酶抑制剂）的存在而做出。因此，在少量可用的乳腺活检组织中直接测量截短的HER 2亚型并具有单细胞分辨率的能力将为选择治疗和评估耐药性的潜力产生巨大的优势。遗憾的是，现有的基于免疫测定的工具无法区分微小组织活检中存在的HER 2形式（p185-erbB 2、p110-erbB 2、p95 m-、p95 c-和p95 n-erbB 2）。不存在对所有形式的HER 2蛋白具有特异性的抗体。一个跨学科的研究计划将创建和优化一套强大的微量分析工具，以直接量化的水平和身份的HER 2蛋白形式和耐药相关的信号蛋白的组织和细胞从HER 2阳性乳腺癌患者活检与斯坦福大学乳腺癌组织库。作为初步研究，将测定耐药相关蛋白的蛋白质组（mTOR、PI 3 K、HER 3、IGF-1 R、Akt、VEGF），以了解这些途径在耐药中的作用。在乳腺癌细胞系中建立性能后，在目标1中，mWB将测定新鲜冷冻和固定（FFPE）组织样本中的同种型。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，我们将比较患者结局和金标准IHC分类。在补充的Aim 2A中，研究人员将引入并优化单细胞分辨率蛋白质印迹法，以测定HER 2蛋白亚型和耐药相关信号蛋白，但现在用于数千种具有单细胞分辨率的乳腺癌细胞。蛋白质组中的细胞间异质性将包括新的HER 2阳性乳腺癌亚分类，进而评估其与患者结局的相关性。在目标2B中， 单细胞工具将被优化，以提供全长HER 2和截短亚型的细胞表面相对于核位置的亚细胞分辨率，这是目前不可能的涉及耐药性的测量。预期结果旨在消除改善BCa患者福祉的关键瓶颈。虽然HER 2亚型是这一初步努力的重点，但许多其他癌蛋白也是亚型（ER、罗恩、MDM、MYC、BAG-1、PPM 1D和FLIP），并被认为是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亚型在BCa结果中的作用以及微小组织生物标本中的治疗抗性的未决问题，否则这些生物标本将无法进行分析。