High sensitivity of chemiluminescent methodology for detection of clonal CDR3 sequences in patients with acute lymphoblastic leukemia.
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 1:1 up to 1:10(7). A sensitivity range of 1:10(3)-1:10(6) was obtained, with an average of 1:10(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.
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División de Genética, Centro de Investigación Biomédica de Occidente, IMSS, Guadalajara, Jalisco, México.
Jaloma-Cruz
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Barros-Núñez
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D000483:Alleles; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001854:Bone Marrow Cells / Q000166:cytology; D002051:Burkitt Lymphoma / Q000175:diagnosis* / Q000235:genetics*; D002869:Chromosome Aberrations; D003165:Complement System Proteins; D022801:Complementarity Determining Regions; D016384:Consensus Sequence; D003582:Cytogenetics; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004076:Digoxigenin / Q000494:pharmacology; D005434:Flow Cytometry; D015321:Gene Rearrangement; D006801:Humans; D007143:Immunoglobulin Heavy Chains; D008163:Luminescent Measurements; D011379:Prognosis; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D003165:Complement System Proteins; D004247:DNA; D004076:Digoxigenin