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.
This project is directed at the development of a point-of-care Chronic Lymphocytic Leukemia (CLL) mutation detection and monitoring system that will allow rapid, cost effective therapy monitoring for all Americans afflicted with CLL. The system does this by rapidly isolating cell- free circulating DNA in blood, a cancer biomarker, using a prototyp AC Electrokinetic device. Further development of this device will create a new point-of-care CLL monitoring device that will lead to better quality healthcare for all Americans with CLL as well as lowering costs associated with treatment.