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人类成人白血病。由于CLL被认为无法用标准疗法治愈，所以通常在患者出现侵袭性疾病的症状体征时才开始治疗。然而，患者的临床病程是异质性的。一些患者病情进展相对较快，需要早期治疗，并在诊断后几年内死于疾病。另一方面，大多数患者最初有一个不痛的过程，不需要治疗诊断后很长一段时间；然而，这类患者中有很大一部分最终进展到较晚期的临床阶段，通常死于该病。在诊断时，约5-20%的CLL患者可出现p53、SF3B1、Notch1基因的互斥性改变，几位研究人员已分别显示这些基因与不良预后和治疗耐药性有显著相关性。p53、Notch1和SF3B1的突变可能在发病时不存在，但可能在病程中出现。总的来说，发生新的高风险遗传病变的可能性很大（10年约25%），随着时间的推移，高风险遗传病变的获得也会影响

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.