Profile of immunoglobulin heavy chain variable gene repertoires and highly selective detection of malignant clonotypes in acute lymphoblastic leukemia

Abstract

The predominant B cell immunoglobulin heavy chain variable gene (IgH-V) usage and the uniquely rearranged, clonotype-specific variable-diversity-joining region gene (VDJ) sequences were identified in patients with B cell acute lymphoblastic leukemia (B-ALL) using a novel DNA-based gene amplification strategy, The approach allows a thorough and sensitive determination of the number of clonal leukemic IgH rearrangements and their precise V gene usage, This strategy may be applied in the detection of minimal residual disease, in surveillance after induction of disease-free states, and in analyzing the effectiveness of purging autologous bone marrow of malignant clones, An initial primary polymerase chain reaction (PCR), directed by an IgH-J generic primer and a complement of family-specific IgH-V primers, defined the major B cell IgH-V gene usage, Use of an IgH-J generic primer supplanted the use of a constant region primer anchor and thus eliminated the need to target mRNA by the traditional RNA reverse transcription-PCR amplification method, Monoclonality of rearranged VDJ bands was further substantiated by high resolution denaturant gel electrophoretic analysis, The predominant amplified bands were subcloned and sequenced, By sequencing through VDJ juxtaposed regions, that is, the third complementarity-determining region, clonotype-specific primers were developed and used in a secondary clonotype primer-directed PCR (CPD-PCR) to detect, with extreme sensitivity and specificity, a unique B cell clone, Analysis of the products of the CPD-PCR permitted the detection of a single malignant cell among 1 million polyclonal cells and superseded the constraints of prior studies that have provided a limited evaluation of family variable gene repertoire usage, Leukemic clonal rearrangements were detected in 100% of the eight cases of pediatric and two cases of adult B-ALL studied, Two or more clonal IgH-VDJ amplified sequences were observed in 50% of the B-ALL bone marrows analyzed. In two cases, clonotype-specific oligodeoxynucleotide primers, derived from B-ALL VDJ sequences, directed the secondary CPD-PCR, and disease activity was monitored after chemotherapy and allogeneic bone marrow transplantation.