title: 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of the products of the CPD-PCR permitted the detection of a single malignant cell among 1 million polyclonal cells and superseded the constraints of prior studies that have provided a limited evaluation of family variable gene repertoire usage.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.
authors.affiliation: Department of Neurology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Greenberg
authors.forename: Y
authors.initials: Y
authors.lastname: Choi
authors.forename: M
authors.initials: M
authors.lastname: Ballow
authors.forename: T L
authors.initials: TL
authors.lastname: Du
authors.forename: P M
authors.initials: PM
authors.lastname: Ward
authors.forename: M H
authors.initials: MH
authors.lastname: Rickert
authors.forename: S
authors.initials: S
authors.lastname: Frankel
authors.forename: S H
authors.initials: SH
authors.lastname: Bernstein
authors.forename: M L
authors.initials: ML
authors.lastname: Brecher
mesh_terms: D000328:Adult; D001483:Base Sequence; D002648:Child; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000276:immunology*
chemical_list: D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region