A highly sensitive linear amplification based DNA methylation profiling technique for clinical cancer research

项目总结 验证可以充分利用宝贵临床标本的高灵敏度分子分析方法将 转变癌症研究和临床应用。组织活检是癌症诊断的金标准 并已成为癌症研究中临床生物制剂的主要来源。然而，其中一个最大的 基础和临床癌症研究面临的挑战是经常缺乏足够数量的肿瘤材料 从诊断程序之后用于多组学研究的活组织检查(例如，并行RNA和基于DNA的分析)， 从而限制了系统生物学的进展。因此，能够方便地采集体液样本，包括 从血浆中循环的无细胞DNA(CfDNA)，为实现高度敏感、最低限度的 侵入性、成本效益高的癌症诊断方法，并促进对高危人群和 病人监护。然而，cfDNA通常以极低的数量存在(例如，来自 几毫升血液)。癌症衍生的cfDNA预计只占已经存在的 稀有的cfDNA。为了加速和加强癌症生物学和临床应用，这款R33旨在验证 DNA甲基化的新技术，即与癌症有关的5-甲基胞嘧啶(5mC) 病理生物学，反映在患者来源的cfDNA中，并已被整合到几个FDA批准的测试中。 鉴于5mC在人类基因组中的普遍存在，它在基因调控中的作用，以及高度的化学稳定性， 在纳克或亚纳克级DNA材料中验证新的5mC技术前景看好 癌症研究中各种应用的机会一直受到可以利用的技术的限制 临床样本有限。具体地说，我们开发了基于T7-线性扩增的亚硫酸盐测序 (Labs-seq)，将专门设计的亚硫酸盐转化程序与下一代 灵敏无偏检测纳克或亚纳克级DNA中5mC的测序(NGS) 材料(例如，100加仑)。我们的初步结果证明了Labs-seq和 使用这项创新技术识别cfDNA中癌症特异性5mC变化的可行性。在这款R33中， 我们将使用库中的肿瘤组织/细胞和血浆cfdna严格验证Labs-seq技术。 来自250名不同癌症类型的患者和50名频率匹配的健康对照以及 来自200名前瞻性招募的癌症患者的纵向样本。在目标1中，我们将验证Labs-seq 从肿瘤组织/细胞的基因组DNA(GDNA)中检测癌症类型特异性的表观遗传学变化。在目标2中， 我们将验证cfDNA中的Labs-seq用于检测癌症和纵向变化。完工后 对于这个项目，我们将提供一种高度敏感、经济高效、具有变革性的表观遗传学方法。 对gDNA和cfDNA都有影响，为受技术限制的研究打开了机会。我们的 建立的多学科团队，良好的人体临床标本注释，以及良好的环境将 确保这个拟议项目的成功和未来在临床上的实施。

PROJECT SUMMARY Validating highly sensitive molecular analysis approaches that can fully exploit precious clinical specimens will transform cancer research and clinical applications. Tissue biopsy is the gold standard for cancer diagnosis and has been the primary source of clinical biospecimens in cancer research. However, one of the biggest challenges in basic and clinical cancer research is the frequent lack of sufficient amount of tumor materials from biopsy for multi-omics research (e.g., parallel RNA and DNA-based profiling) after diagnostic procedures, thus limiting progress in systems biology. As such, the ability to conveniently sample bodily fluids, including circulating cell-free DNA (cfDNA) from plasma, offers great promise for enabling highly-sensitive, minimally- invasive, cost-efficient cancer diagnostic methods, and facilitating screening of high-risk populations and patient monitoring. However, cfDNA typically exists in extremely low quantity (e.g., a few nanograms from several mL of blood). Cancer-derived cfDNA is expected to constitute an even smaller portion of the already scarce cfDNA. To accelerate and enhance cancer biology and clinical applications, this R33 aims to validate a novel technology for profiling DNA methylation, i.e., 5-methylcytosines (5mC), which contributes to cancer pathobiology, is reflected in patient-derived cfDNA, and has been integrated in several FDA-approved tests. Given the prevalence of 5mC in the human genome, its roles in gene regulation, and high chemical stability, validating a novel 5mC technology in nanogram or sub-nanogram-level DNA materials offers promising opportunities for various applications in cancer research that have been limited by technology that can utilize limited clinical samples. Specifically, we developed the T7-Linear Amplification based Bisulfite Sequencing (LABS-seq) that integrates a specially-designed bisulfite conversion procedure with the next-generation sequencing (NGS) for sensitively and unbiasedly detecting 5mC in nanogram or sub-nanogram-level DNA materials (e.g., 100 pg). Our preliminary results demonstrated the technical robustness of the LABS-seq and the feasibility of using this innovative technology to identify cancer-specific 5mC changes in cfDNA. In this R33, we will rigorously validate the LABS-seq technique using banked tumor tissues/cells and plasma cfDNA samples from 250 patients with diverse cancer types and 50 frequency-matched healthy controls as well as longitudinal samples from 200 prospectively recruited cancer patients. In Aim 1, we will validate the LABS-seq in genomic DNA (gDNA) from tumor tissues/cells to detect cancer type-specific epigenetic alterations. In Aim 2, we will validate the LABS-seq in cfDNA for the detection of cancer and longitudinal changes. Upon completion of this project, we will provide a highly sensitive, cost-efficient, transformative epigenetic approach applicable to both gDNA and cfDNA, opening up opportunities for research that have been limited by technology. Our established multidisciplinary team, well-annotated human clinical specimens, and excellent environment will ensure the success of this proposed project and future implementation in the clinic.