High sensitivity of chemiluminescent methodology for detection of clonal CDR3 sequences in patients with acute lymphoblastic leukemia.

ABSTRACT

Detection of minimal residual disease (MRD) in patients with B-cell acute lymphoblastic leukemia (B-ALL) has been achieved using several radioactive labelling methodologies; however, limited information exists about the use of chemiluminescent labelling. Although many malignant disorders are related to cytogenetic alterations, there is not a consistent chromosomal translocation that could serve as a tumour marker for the monitoring of MRD. ALL are derived from B-lymphocytes in 80% of cases. In the early stages of their maturation, the immunoglobulin heavy chain genes (IgH) undergo rearrangements among their V, D, and J segments, giving rise to the Complementary Determining Regions (CDR). Among these, CDR3 is considered unique for each lymphocyte and used as a tumour-specific marker in B-ALL patients. In this study, the CDR3 was labelled with digoxigenin and used as a patient-specific probe to test its sensitivity for further detection of MRD. Fourteen pretreatment samples of bone marrow (BM) or peripheral blood (PB) from B-ALL patients were included. Tumour-specific probes were designed from each clonal product by elimination of the consensus sequences. Ten digoxigenin-labelled probes were hybridized with a mixture of their respective clonal DNA and the polyclonal product from a normal healthy donor, in serial dilutions from 11 up to 110(7). A sensitivity range of 110(3)-110(6) was obtained, with an average of 110(5). Crossed tests performed in four patients, showed right probe specificity in all cases. We propose that the design of allele-specific probes with chemiluminescent labelling, represents a reliable, sure and sensitive alternative methodology for MRD detection in patients with B-cell lymphoproliferative disorders.