Implementation of phosphoprotein preservation technology for cancer biospecimens

描述(由申请人提供)：一个紧迫的临床目标是确定与癌症患者亚群相关的分子网络，这些患者可能对分子靶向抑制剂有单独的反应。目前的分子靶向治疗主要针对蛋白激酶和/或其磷酸化底物。因此，在肿瘤活检样本中测量这类新的磷蛋白信号通路表位对于个体化分子靶向治疗至关重要。通过福尔马林固定和石蜡包埋不能充分保存磷蛋白抗原表位，组织冷冻非常昂贵，并影响诊断的准确性。我们建议进一步开发和临床验证一种创新和变革性的技术，以保存组织磷蛋白和诊断组织形态，用于临床癌症分子图谱。应用从NIH R21资助的研究中获得的知识，我们创造了一种新的组织保存化学，它稳定了所有类别的磷蛋白，与石蜡包埋兼容，同时保持了完整的诊断组织形态，并完全保留了关键的诊断免疫组织学(IHC)抗原，包括雌激素受体、孕激素受体、HER2和Ki-67。这些IHC抗原不会被用于组织RNA保存的特殊研究固定剂保存。我们的新型非福尔马林组织防腐剂，称为生物标记和组织防腐剂(BHP)，可以无缝地引入当前社区医院的临床诊断工作流程，而不需要额外的步骤或设备。在采购时，组织可以直接浸入新的固定剂中，并加工到石蜡块中进行常规诊断，从而消除了运输或储存过程中昂贵的冷冻需求。与传统的福尔马林固定相比，必和必拓提供了实质性改进的潜力。在本申请中，我们建议在社区医院环境中对我们的新保存化学物质进行盲法临床验证，利用国际病理学家团队进行验证。该项目的目标是一步石蜡封堵稳定所有类别的细胞磷蛋白、诊断组织形态和诊断免疫组化抗原，同时保持完全诊断形态与标准福尔马林固定相同或更好。我们将在知情同意下收集新鲜的手术组织，涵盖广泛的器官和癌症组织学，以建立150例匹配的石蜡和冰冻标本的档案。我们将使用反相蛋白质微阵列(RPMA)和激光捕获显微切割(LCM)技术从提取的石蜡切片中测量100个有效的跨越膜、细胞质和细胞核的磷蛋白表位。经过诊断病理学家的客观独立验证，这项变革性的技术将准备好广泛应用于临床和研究。这项技术的采用将意味着只有一个诊断石蜡块可以用于所有类别的分子图谱，而不是目前对多个块的要求。这将提高诊断的准确性，同时大幅降低成本。 与公共健康相关：我们在收集时保存蛋白质的技术将使分析分子靶向激酶抑制剂用于个体化治疗成为可能，同时显著降低医疗成本，并提高诊断准确性。我们的新型非福尔马林组织防腐剂可以无缝地引入当前社区医院的临床诊断工作流程，而不需要额外的步骤或设备。

DESCRIPTION (provided by applicant): An urgent clinical goal is to identify molecular networks associated with subpopulations of cancer patients who may respond individually to molecular targeted inhibitors. Current molecular targeted therapeutics is directed at protein kinases and/or their phosphorylated substrates. Therefore, measurement of this new class of phosphoprotein signal pathway epitopes in tumor biopsy samples is crucial for individualizing molecular targeted therapies. Phosphoprotein antigen epitopes are not adequately preserved by formalin fixation and paraffin embedding, and freezing of tissue is very expensive and compromises diagnostic accuracy. We propose the advanced development and clinical validation of an innovative and transformative technology for preserving tissue phosphoproteins and diagnostic histomorphology for clinical cancer molecular profiling. Applying knowledge gained under an NIH R21 funded study, we created a novel tissue preservation chemistry that stabilizes all classes of phosphoproteins, is compatible with paraffin embedding, while maintaining complete diagnostic histomorphology, and fully preserving critical diagnostic immunohistology (IHC) antigens including Estrogen Receptor, Progesterone Receptor, HER2, and Ki-67. These IHC antigens are not preserved by special research fixatives used for tissue RNA preservation. Our new non-formalin tissue preservative, termed Biomarker and Histology Preservative (BHP) can be seamlessly introduced into the current community hospital clinical diagnostic workflow with no additional steps or equipment. At the time of procurement, tissue can be immersed directly in the new fixative and processed into a paraffin block for routine diagnosis, obviating the need for costly freezing during shipping or storage. BHP offers the potential for substantial improvements over conventional formalin fixation. In the present application we propose the blinded clinical validation of our novel preservation chemistry in community hospital settings, utilizing a team of international pathologists for validation. The goal of the project is one-step paraffin block stabilization of all classes of cellular phosphoproteins, diagnostic histomorphology, and diagnostic immunohistochemistry antigens, while at the same time maintaining full diagnostic morphology equivalent or superior to standard formalin fixation. We will collect fresh surgical tissue, under informed consent, covering a broad variety of organs and cancer histology to develop an archive of 150 cases of matched paraffin and frozen specimens. We will measure 100 validated phosphoprotein epitopes spanning membrane, cytoplasmic and nuclear compartments from extracted paraffin sections using Reverse Phase Protein Microarray (RPMA) and Laser Capture Microdissection (LCM) technology. Following objective independent validation by diagnostic pathologists, this transformative technology will be ready for widespread clinical and research use. Adoption of the technology would mean that only one diagnostic paraffin block could be used for all classes of molecular profiling rather than the current requirement for multiple blocks. This would increase diagnostic accuracy while substantially reducing costs. PUBLIC HEALTH RELEVANCE: Our technology for preserving proteins, at the time of collection, will enable analysis of molecular targeted kinase inhibitors for individualized therapy, while considerably reducing healthcare costs, and increasing diagnostic accuracy. Our new non-formalin tissue preservative can be seamlessly introduced into the current community hospital clinical diagnostic workflow with no additional steps or equipment.