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.

项目总结/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点 KRAS基因突变这提出了对KRAS突变体的存在进行测序的令人兴奋的可能性 ctDNA可用于从简单的抽血中检测许多类型的癌症。事实上，Guardant 360 ® ctDNA检测测定法在癌症患者的临床护理中仅用于此目的。的挑战 检测ctDNA的另一个重要原因是，这种形式的DNA在血液中的水平极低。这一限制是由 根据我们在杜克的临床经验，我们发现Guardant 360 ®检测法成功检测到 KRAS突变ctDNA在只有一半的病例中，其中患者的癌症被记录为KRAS突变- 通过切除或活检肿瘤组织的直接测序为阳性。因此，虽然Guardant 360 ®是真实世界的证明， 虽然ctDNA可以在临床上用作“液体活组织检查”，但显然还有很大的改进余地。在这 在这方面，我们采用了最大深度测序（MDS）技术，最初是在微生物学中发展起来的。 检测细菌群体中罕见的耐药突变，检测 克拉斯。通过对原始KRAS模板进行条形码化并对其进行多个第一链复制， 通过对这些靶向DNA产物进行超深度测序，我们能够检测到基因突变， KRAS模板的检测限为5x 10 -7，灵敏度比 Guardant 360 ®测定报告了1x 10 -3至2x 10 -4。鉴于此，我们将联合收割机博士的基础研究。 利用肿瘤学家的临床和翻译专业知识对抗这种KRAS特异性MDS（K-MDS）检测 博士Abbruzzese优化血液样本的K-MDS检测（目的1），然后评价K-MDS， Guardant 360 ®前瞻性临床比较（目的2）。这项研究的完成将提供一个新的 该技术用于在癌症患者的血液中筛选KRAS突变ctDNA，灵敏度为 比目前的临床检测方法更大的幅度，最初用于监测KRAS突变型癌症的复发 或者在高风险患者中检测此类癌症，但更长期，与其他热点筛查相结合， 突变和使用不同来源的组织，用于多种癌症类型的早期检测。