CLL Mutation Detection and Monitoring from Blood using AC Electrokinetics

描述（由申请人提供）：慢性淋巴细胞白血病（CLL）是西方世界最常见的成人白血病。由于CLL被认为不能用标准疗法治愈，因此通常在患者出现侵袭性疾病症状的体征时开始治疗。然而，患者的临床过程是异质性的。一些患者进展相对较快，需要早期治疗，并在诊断后几年内死于疾病。另一方面，大多数患者最初具有惰性过程，并且在诊断后不需要长时间的治疗;然而，大部分这样的患者最终进展到更晚期的临床阶段，并且通常死于该疾病。p53、SF 3B 1、Notch 1基因的相互排斥存在的改变可以在诊断时发生在约5-20%的CLL患者中，并且这些基因已经被几个研究者单独显示与不良预后和治疗抗性具有显著相关性。p53、Notch 1和SF 3B 1突变在发病时可能不存在，但在病程中可能出现。总的来说，发生新的高危遗传病变的概率很大（10年时约为25%），随着时间的推移，高危遗传病变的发生会影响 以独立于其他随时间变化的因素（如患者年龄和疾病阶段）的改变的方式生存。鉴于越来越多的新目标代理，CLL的管理将被修改，早期干预也可能成为一种选择。在这种不断变化的情况下[8，9]，人们对使用预后标志物的兴趣越来越大，这些标志物可以指导疾病早期的患者管理。检测突变的标准程序需要：DNA提取，然后进行靶向PCR、凝胶电泳和纯化，然后进行常规DNA桑格测序。将患者特异性序列与相应的种系参考序列进行比较。最耗时和劳动密集的步骤仍然是DNA提取和无细胞循环DNA（CFC DNA）的AC电动（ACE）分离代表了缩短该步骤而不增加成本的有吸引力的方式。该项目将侧重于临床前方面，即使用专有的原型ACE设备从CLL患者血浆中分离和分析CFC DNA。I期提案的具体目标是：1 -使用ACE芯片分离CFC DNA，并在12名患者中进行VH分析。2 -证明ACE分离样品中的肿瘤DNA。3 -定量通过ACE分离的总CFC DNA和肿瘤CFC DNA的水平。未来的第二阶段提案将构建用于商业化的分析系统，测试和关联新鲜CLL血液与冷冻血浆，并结合基于芯片上定量荧光的PCR机制，以允许直接在芯片上测定突变水平。

DESCRIPTION (provided by applicant): Chronic lymphocytic leukemia (CLL) is the most common human adult leukemia in the Western world. Because CLL is not considered curable with standard therapy, treatment generally is initiated when the patient develops signs of symptoms of aggressive disease. However, the clinical course of patients is heterogeneous. Some patients progress relatively rapidly, require early therapy, and succumb to the disease within a few years of diagnosis. On the other hand, most patients initially have an indolent course and do not require therapy for long periods after diagnosis; however a large proportion of such patients ultimately progress to a more advanced clinical stage and generally die from the disease. The mutually exclusive presence of alterations of the p53, SF3B1, Notch1 genes can occur in ~5-20% of CLL patients at diagnosis and these genes have individually shown to have significant correlations with poor prognosis and treatment resistance by several investigators. Mutations in p53, Notch1 and SF3B1 can be absent at presentation but may emerge during disease course. Overall, the probability of developing new high-risk genetic lesions is substantial (~25% at 10- years), and the acquisition of high-risk genetic lesions over time affects survival in a manner that is independent of modifications of other time-varying factors, such as patient age and disease stage. Given the growing number of newly targeted agents, the management of CLL will conceivably be revised, and early intervention may also become an option. In this changing scenario [8, 9] there is increasing interest in the use of prognostic markers that may guide management of patients since the early phases of the disease. The standard procedure to detect mutations requires: DNA extraction followed by targeted PCR, gel electrophoresis and purification, followed by conventional DNA Sanger sequencing. Patient specific sequences are compared to the corresponding germline reference sequences. The most time consuming and labor intense step remains DNA extraction and AC Electrokinetic (ACE) separation of Cell-free circulating DNA (CFC DNA) represents an attractive way to shorten this step without increasing the cost. This project will focus on the pre-clinical aspect o using a proprietary prototype ACE device to isolate and analyze CFC DNA from CLL patient plasma. The specific aims for the Phase I proposal are: 1 - Isolate CFC DNA using ACE Chip and perform VH analysis in 12 patients. 2 - Demonstrate tumor DNA in ACE isolated samples. 3 - Quantify level of total and tumor CFC DNA isolated by ACE. A future Phase II proposal will construct an analytical system for commercialization, test and correlate fresh CLL blood vs. frozen plasma, and incorporate an on-chip quantitative fluorescence based PCR mechanism to allow for assaying for mutation levels directly on-chip.