Abstract
Abstract 1619
Poster Board I-645
Quantitative PCR (qPCR) for fusion transcripts and overexpressed genes is a promising tool for following minimal residual disease (MRD) and detecting early relapse in patients with acute leukemia, and for identifying treatment failure in patients with chronic myeloid leukemia (CML). Its widespread use has, however, to some extent been hampered by differences in data analysis and in presentation. These issues constitute major challenges in the translation of these thoroughly pre-clinically validated assays into multicenter clinical trials. To address these problems we have designed an MRD reporting software package, in which data from the qPCR equipment can be imported, processed, and presented in a standardized fashion to generate a readily comprehensible report.
The software builds upon a database with basic patient information and data imported from qPCR runs. The software is highly flexible, and data from a variety of qPCR platforms can be accommodated (Figure 1) when exported from the equipment as tab- or comma-separated files. MRD calculations may be based on both “relative” quantification, i.e. where a diagnostic sample or a cell line is used as reference, as well as “absolute” quantification, where plasmid standards are included as controls (Figure 2). Moreover, while a series of premade report types are available, individualized report generation is also possible. Report types include graphs detailing blood and bone marrow MRD either separately or simultaneously. Moreover, two or three target genes can be displayed within the same graph.
To validate the software and provide proof of principle for applicability in multicenter studies we performed a quality control study involving analysis and reporting of multiple blinded leukemic samples by 8 laboratories. cDNA samples representing five consecutive samples from the same CML patient (BCR/ABL+), five from the same AML patient (MYH11/CBFá+, WT1+), as well as a K562 cell line sample were centrally prepared by the Aarhus laboratory. Samples were distributed to the test laboratories together with primer/probe kits from Ipsogen (Ipsogen, Marseilles, France) for the three target genes and the ABL control gene. Each laboratory performed qPCR using their in-house qPCR equipment (including ABI7500, ABI7900 (Applied Biosystems), Mx3000P (Stratagene), or LightCycler 480 (Roche)) and submitted 16 different reports as pdf files for comparison. The reports included relative quantification using the diagnostic sample as reference, relative quantification using cell line as reference, absolute quantification using plasmid standard curves, and lists of calculated MRD values and sensitivities. Excellent concordance in the quality of the reports was observed for all report types.
This software package, which has been distributed to members of the ELN MRD Workpackage, will shortly be made more widely available. The validation procedures outlined above should make this package suitable for general use in translational hematology allowing standardized reporting of MRD results (e.g. use of International Scale in CML trials) and facilitating comparison of results obtained between trial groups.
We thank Nicolas Maroc from Ipsogen for providing reagents for the QC Study and Karin Braendstrup and Lone Siig Mikkelsen for excellent technical assistance.
Grimwade:Ipsogen, Marseille, France: Membership on an entity's Board of Directors or advisory committees.
