Adapting K-MDS to detect KRAS-mutant ctDNA

项目摘要/摘要 从肿瘤排入血液的dna称为循环肿瘤dna(Ctdna)，是一种很容易获得的来源。 肿瘤物质。由于大多数ctdna与正常dna完全相同，因此需要一些区分特征来区分。 癌症的起源。在这一点上，五分之一或更多的人类癌症存在致癌(致癌)点。 KRAS基因突变。这增加了一种令人兴奋的可能性，即对KRAS突变的存在进行测序 通过一次简单的抽血，ctDNA可以用来检测多种癌症。事实上，Guardant360® 在癌症患者的临床护理中，CTDNA检测就是为了这个目的而使用的。挑战在于 检测ctDNA是指这种形式的DNA在血液中的水平非常低。这一限制被承担了 在杜克大学我们自己的临床经验中，我们发现Guardant360®检测成功地检测到 KRAS突变ctDNA仅在患者癌症被记录为KRAS突变的病例中的一半- 对切除或活检的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 虽然ctDNA可以在临床上用作“液体活检”，但显然还有很大的改进空间。在这 在这方面，我们采用了最大深度测序(MDS)技术，该技术最初是在微生物学中开发的 现场检测细菌种群中罕见的抗药性突变，检测致癌突变 克拉斯。通过条形化原始KRAS模板并制作其多个第一链复制， 通过对这些目标DNA产物进行超深度测序，我们能够检测到基因工程产生的突变 KRAS模板的极限为5x10-7，其灵敏度是检测下限的2500到50,000倍 Guardant360®检测报告为1x10-3至2x10-4。有鉴于此，我们将结合Dr。 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 AbBruzzese博士将优化血液样本的K-MDS检测(目标1)，然后评估K-MDS以 Guardant360®一项前瞻性临床比较(目标2)。因此，完成这项研究将提供一个新的 筛查癌症患者血液中KRAS突变ctDNA的技术 比目前的临床检测更大，最初是为了监测KRAS突变癌症的复发 或在高危患者中发现此类癌症，但更长期，结合其他热点的筛查 突变和使用不同来源的组织，用于早期检测多种癌症类型。

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.