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

项目摘要 验证能够充分利用珍贵临床标本的高灵敏度分子分析方法， 改变癌症研究和临床应用。组织活检是癌症诊断的金标准 并且是癌症研究中临床生物样本的主要来源。然而，最大的 基础和临床癌症研究的挑战是经常缺乏足够量的肿瘤材料 从用于多组学研究的活组织检查（例如，平行的基于RNA和DNA的分析）在诊断程序之后， 从而限制了系统生物学的进展。因此，能够方便地对体液进行采样，包括 来自血浆的循环无细胞DNA（cfDNA），为实现高灵敏度、最低限度地 侵入性的、具有成本效益的癌症诊断方法，并促进高危人群的筛查， 病人监护然而，cfDNA通常以极低的量存在（例如，几毫微克 几毫升血）。预计癌症来源的cfDNA将构成已经存在的cfDNA的甚至更小的部分。 缺乏cfDNA。为了加速和增强癌症生物学和临床应用，R33旨在验证 用于分析DNA甲基化的新技术，即，5-甲基胞嘧啶（5 mC），这有助于癌症 病理生物学上的差异反映在患者来源的cfDNA中，并且已经整合在几种FDA批准的测试中。 考虑到5 mC在人类基因组中的普遍性、其在基因调控中的作用以及高化学稳定性， 在纳克或亚纳克级别的DNA材料中验证一种新的5 mC技术， 癌症研究中的各种应用的机会，这些机会受到可以利用的技术的限制， 有限的临床样本。具体来说，我们开发了基于T7-线性扩增的亚硫酸氢盐测序技术， （LABS-seq），将专门设计的亚硫酸氢盐转化程序与下一代 用于灵敏且无偏地检测纳克或亚纳克水平DNA中的5 mC的测序（NGS） 材料（例如，100 pg）。我们的初步结果证明了LABS-seq的技术稳健性， 使用这种创新技术鉴定cfDNA中癌症特异性5 mC变化的可行性。在这款R33中， 我们将使用库存的肿瘤组织/细胞和血浆cfDNA严格验证LABS-seq技术 来自250名不同癌症类型患者和50名频率匹配的健康对照的样本， 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 在来自肿瘤组织/细胞的基因组DNA（gDNA）中检测癌症类型特异性表观遗传改变。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。完成后 在这个项目中，我们将提供一种高度敏感、具有成本效益、变革性的表观遗传方法， gDNA和cfDNA，为受技术限制的研究开辟了机会。我们 已建立的多学科团队、注释良好的人类临床标本和优良的环境将 确保该拟议项目的成功和未来在诊所的实施。

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.