Adapting K-MDS to detect KRAS-mutant ctDNA

采用 K-MDS 检测 KRAS 突变 ctDNA

• 批准号: 10408909
• 负责人: JAMES L. ABBRUZZESE
• 金额: $ 18.82万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10408909
• 关键词: Adopted; Antibiotic Resistance; Bacteria; Bar Codes; Basic Science; Biological Assay; Biopsy; Blinded; Blood; Blood Circulation; Blood specimen; Cancer Biology; Cancer Detection; Cancer Patient; Carcinogens; Cells; Clinic; Clinical; Clinical Management; Clinical Trials Design; Colonoscopy; Consent; Coupled; DNA; DNA sequencing; Detection; Discipline; Disease; Early Diagnosis; Early Intervention; Engineering; Experimental Designs; Genes; Hematopoietic Neoplasms; Human; Individual; Institutional Review Boards; KRAS2 gene; Malignant Neoplasms; Malignant neoplasm of gastrointestinal tract; Malignant neoplasm of lung; Methods; Microbiology; Molecular; Monitor; Mus; Mutation; Neoplasm Circulating Cells; Oncogenic; Oncologist; Pathologist; Patients; Performance; Point Mutation; Population; Protocols documentation; Publishing; Radiology Specialty; Recurrence; Recurrent disease; Reporting; Reproducibility; Research Personnel; Resected; Sampling; Scientist; Screening for cancer; Source; Stream; Technology; Testing; Time; Tissues; Translating; Tumor Tissue; base; biobank; cancer care; cancer cell; cancer type; clinical care; clinical development; deep sequencing; detection assay; detection limit; driver mutation; experience; extracellular vesicles; head-to-head comparison; high risk; innovation; liquid biopsy; mutant; mutational status; new technology; next generation sequencing; novel strategies; prospective; research study; resistance mutation; screening; targeted sequencing; tumor; tumor DNA

PROJECT SUMMARY/ABSTRACT DNA shed from tumors into the blood stream, termed circulating tumor DNA (ctDNA), is an easily obtained source of tumor material. As most ctDNA is identical to normal DNA, some distinguishing feature is needed to demark a cancer origin. In this regard, a fifth or more of all human cancers harbor a cancer-causing (oncogenic) point mutation in the gene KRAS. This raises the exciting possibility that sequencing for the presence of KRAS-mutant ctDNA could be used to detect many types of cancers from a simple blood draw. Indeed, the Guardant360® ctDNA-detection assay is used for just this purpose in the clinical care of cancer patients. The challenge to detecting ctDNA is that this form of DNA is found at vanishingly low levels in the blood. This limitation is borne out in our own clinical experience at Duke, where we find that the Guardant360® assay successfully detected KRAS-mutant ctDNA in only half the cases in which the patient's cancer was documented to be KRAS mutation- positive by direct sequencing of resected or biopsy tumor tissue. Thus, while Guardant360® is real-world proof that ctDNA can be used as a `liquid biopsy' in the clinic, there is clearly much room for improvement. In this regard, we adopted the Maximum Depth Sequencing (MDS) technology, originally developed in the microbiology field to detect rare antibiotic-resistance mutations in bacteria populations, to detect oncogenic mutations in KRAS. By barcoding the original KRAS template and making multiple first-strand replicates thereof, coupled with ultra-deep sequencing of these targeted DNA products, we were able to detect mutations engineered into KRAS templates at a limit of 5x10-7, which is 2,500 to 50,000 times more sensitive than the detection limit of 1x10-3 to 2x10-4 reported for the Guardant360® assay. Given this, we will combine the basic research of Dr. Counter into this KRAS-specific MDS (K-MDS) assay with the clinical and translational expertise of oncologist Dr. Abbruzzese to optimize the K-MDS assay for blood samples (aim 1) and then evaluate K-MDS to Guardant360® a prospective clinical comparison (aim 2). Completion of this study will thus provide an new technology to screen for KRAS-mutant ctDNA in the blood of cancer patients at a sensitivity orders of magnitude greater than current clinical assays, initially to monitor either recurrence of KRAS-mutant cancers or detect such cancers in high-risk patients, but more long term, in combination with screening for other hotspot mutations and using different sources of tissue, for the early detection of multiple cancer types.

项目概要/摘要 从肿瘤脱落到血流中的 DNA，称为循环肿瘤 DNA (ctDNA)，是一种容易获得的来源 肿瘤物质。由于大多数 ctDNA 与正常 DNA 相同，因此需要一些区别特征来区分 癌症起源。在这方面，五分之一或更多的人类癌症具有致癌（致癌）点 KRAS 基因突变。这提出了一个令人兴奋的可能性，即通过测序来确定 KRAS 突变体的存在 ctDNA 可用于通过简单的抽血检测多种类型的癌症。事实上，Guardant360® ctDNA 检测分析正是在癌症患者的临床护理中用于此目的。挑战 检测 ctDNA 的优点是，这种形式的 DNA 在血液中的含量极低。此限制由 根据我们在杜克大学的临床经验，我们发现 Guardant360® 检测成功检测到 在患者癌症被记录为 KRAS 突变的病例中，只有一半出现 KRAS 突变 ctDNA - 通过对切除或活检肿瘤组织进行直接测序呈阳性。因此，虽然 Guardant360® 是现实世界的证明 ctDNA可以作为临床上的“液体活检”，显然还有很大的改进空间。在这个 对此，我们采用了最初在微生物学领域发展起来的最大深度测序（MDS）技术 检测细菌群体中罕见的抗生素耐药性突变，检测细菌中的致癌突变 克拉斯。通过对原始 KRAS 模板进行条形码编码并对其进行多个第一链复制，耦合 通过对这些目标 DNA 产品进行超深度测序，我们能够检测到基因工程中的突变 KRAS 模板限制为 5x10-7，比检测限灵敏 2,500 至 50,000 倍 Guardant360® 检测报告为 1x10-3 至 2x10-4。鉴于此，我们将结合博士的基础研究。 利用肿瘤学家的临床和转化专业知识来反驳这种 KRAS 特异性 MDS (K-MDS) 检测 Abbruzzese 博士优化血液样本的 K-MDS 检测（目标 1），然后评估 K-MDS Guardant360® 前瞻性临床比较（目标 2）。因此，这项研究的完成将提供一个新的 筛查癌症患者血液中 KRAS 突变 ctDNA 的技术，灵敏度为 比目前的临床检测要大得多，最初是为了监测 KRAS 突变癌症的复发 或在高风险患者中检测此类癌症，但从长远来看，结合其他热点筛查 突变并使用不同来源的组织来早期检测多种癌症类型。