Quantification of Minimal Residual Disease (MRD) is a general concern in oncology since this parameter is likely to give valuable information to clinicians to customize chemotherapeutic treatments and to anticipate possible relapses. An automated system will be developed for the quantification of MRD by using real-time PCR to quantify cancer cells in a background of non pathologic DNA. The system will be derived from an existing high-throughput sample handling system (developed by Meldrum and team) named Acapella. Acapella has a pipelined serial architecture which makes it possible to develop an adaptive PCR control algorithm ensuring a level of sensitivity and accuracy for the quantification results specified by the clinician. In the R21 phase of the project, a critical component of the system is the real-time thermocycler. It will provide DNA quantification results of greater precision than what is currently possible with commercial instruments such as the ABI PRISM 7700 Sequence Detector by taking advantage of a high performance custom fluorescence analyzer and sophisticated data analysis methods. The fluorescence analyzer will have a signal to noise ratio and a dynamic range of at least one order of magnitude larger than the optics used on commercial systems. This unique feature will permit precise measurements of the amplification kinetics during at least five cycles in the exponential phase of the reaction. The amplification yield will he derived from these data using statistical estimators customized to meet the requirements of real-time PCR data analysis. The sample DNA content will be derived from the amplification yield and the calibrated fluorescence measurements of the reaction kinetics. This new approach will make it possible to run series of real-time PCRs with more flexibility than would be possible if the assay was based on standard reactions required to have the same amplification yield as the clinical samples. PCR conditions will be adapted online without concerns about possible differences of amplification rate. The assay control algorithm will adapt the reaction DNA content, the primer selection, and the number of PCRs to meet the clinician requirements for particular patient DNA. During the R21 phase of the project a prototype of the real-time thermocycler will be developed to demonstrate the optic performance and the possibility to estimate the PCR amplification rates with 5% accuracy. A conceptual design of the fully integrated process from patient blood sample to MRD quantification data will be completed to allow assessment of expected performance, cost, and risks associated with the development of the fully engineered system. The development of the various hardware and software components along with their integration into the automated system will take place during the R33 phase of the project. Performance of the system will he evaluated on real biological samples provided by the UW Department of Laboratory Medicine. Results returned by the system will he compared with results of t(14; 18) PCR performed in this department with an ABI PRISM 7700 for the diagnosis of patients suffering from follicular lymphomas.
