Rescuing Nucleic Acids from Formalin Damage in Cancer Specimens

总结 我们提出的研究旨在开发催化分子和方法，大大提高催化剂的性能。 从福尔马林固定的组织标本中恢复生物分子信息。福尔马林（甲醛） 组织的处理被普遍用于数亿次活检和手术的准备中 世界各地的标本。不幸的是，甲醛形成许多加合物和交联物， 生物分子，强烈抑制获得序列和定量的能力， 这些样本中的RNA和DNA，这些信息对于诊断和 癌症的治疗。RNA/DNA提取的标准的基于加热的方法仅去除一小部分RNA/DNA。 加合物，它们是苛刻的，在这个过程中破坏核酸。 在我们的第一阶段工作中，我们证明了催化方法的使用可以大大提高 从DNA和RNA碱基中除去甲醛加合物。重要的是，我们能够开发出 催化协议在实际福尔马林固定的样本中成功发挥作用。可扩增RNA 回收量高达25倍以上的世界领先的商业协议， 前所未有的改善。 这一合作第二阶段研究的目标是走向实际发展， 用于多种组织类型的临床重要应用的技术。我们将进一步 优化我们的催化方案，用于DNA恢复和最小化损伤，用于下一个- 代测序（NGS）。我们将测试新的催化剂结构是否更有效 比早期的例子，我们将研究是否最佳的RNA回收结果改善 通过NGS和微阵列分析表示基因表达和microRNA表达。最后， 我们将测试在肺肿瘤针活检中的应用，这可能难以通过电流分析。 由于材料量少， 这项研究计划是创新的，因为它代表了使用催化剂的第一个概念， 逆转生物分子的甲醛加合物，因为新的第二代催化剂和协议 都计划好了我们提出的工作是有意义的，因为FFPE方法是世界范围内的通用方法 用于在癌症诊断期间存储组织样本的标准。现有约4亿 标本可以受益于工作的成功，数以百万计的新患者可以积极 每年受影响。我们希望我们的催化方法将成为新的实践标准的一部分 用于分子诊断和治疗。

SUMMARY Our proposed research is aimed at developing catalytic molecules and methods that greatly improve the recovery of biomolecular information from formalin-fixed tissue specimens. Formalin (formaldehyde) treatment of tissue is universally used in preparation of hundreds of millions of biopsy and surgery specimens worldwide. Unfortunately, the formaldehyde forms many adducts and crosslinks with the biomolecules in the specimens, strongly inhibiting the ability to obtain sequences and quantification of RNAs and DNAs from such specimens, information that is increasingly essential to diagnosis and treatment of cancer. Standard heating-based methods of RNA/DNA extraction remove only a fraction of adducts, and they are harsh, damaging the nucleic acids in the process. In our phase I work we demonstrated that the use of catalytic methods could greatly enhance the removal of formaldehyde adducts from DNA and RNA bases. Importantly, we were able to develop catalytic protocols that function successfully with actual formalin-fixed specimens. Amplifiable RNA was recovered in amounts as much as 25-fold greater than a world-leading commercial protocol, an unprecedented magnitude of improvement. The goals of this collaborative phase II research are to move to practical development of this technology for application in clinically important applications with multiple tissue types. We will further optimize our catalytic protocols for DNA recovery and minimization of damage, for application in next- generation sequencing (NGS). We will test whether new catalyst structures can be yet more effective than early examples, and we will examine whether optimal RNA recovery results in improved representation of gene expression and microRNA expression by NGS and microarray analysis. Finally, we will test application in lung tumor needle biopsies, which can be difficult to analyze by current methods because of the small amount of material present. This research program is innovative because it represents the first concept of using catalysts to reverse formaldehyde adducts of biomolecules, and because new, 2nd-generation catalysts and protocols are planned. The work we propose is significant because FFPE methodology is the universal worldwide standard for storing tissue specimens during cancer diagnosis. Approximately 400 million existing specimens could benefit from success of the work, and millions of new patients could be positively affected annually. We expect that our catalytic methods will become part of the new standard of practice for molecular diagnosis and therapy.