Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes

Olafur G. Jonsson, Richard L. Kitchens, Frederick C. Scott, R. Graham Smith

Research output: Contribution to journalArticle

81 Citations (Scopus)

Abstract

To develop a sensitive and specific assay for minimal residual disease in acute lymphoblastic leukemia (ALL), we exploited the enormous diversity of genomic sequences created by immune receptor gene rearrangements. To isolate clone-specific sequences, we first synthesized oligonucleotides that match conserved variable (VH) and joining (JH) sequences flanking the third hypervariable region (HVR3) in the rearranged immunoglobulin heavy chain (IgH) locus. In polymerase chain reactions (PCR), these primers were then used to amplify the intervening HVR3 segments from leukemic DNA samples. Of 12 B-lineage ALLs studied, ten generated one or more fragments of the size expected for HVR3 gene segments. Thus, this single pair of amplimers was sufficient to isolate HVR3 sequences from a majority of acute lymphoblastic leukemias. To verify that the amplified fragments originated from HVR3 alleles and to assess their diversity, we sequenced 7 PCR products derived from 6 leukemias. In addition to elements of recognized D segments, each of the 7 fragments contained novel VH-D and D-JH junctional sequences, including N nucleotides, not known to be present in the germline. Each sequence was unique, and allele-specific oligonucleotide probes hybridized only to HVR3 segments from which the probes were derived. Therefore, as anticipated, these HVR3 segments appeared to possess the diversity required to serve as clonal markers for leukemic populations. To demonstrate that these amplified HVR3 alleles could serve as the basis for a sensitive and specific assay to detect rare leukemic cells, we analyzed in detail one pre-B leukemia that had rearranged 2 IgH alleles. The HVR3 sequences were shown to be linked to rearranged JH-containing restriction fragments in digests of genomic DNA, establishing their origin in the leukemic cells. We synthesized oligonucleotides corresponding to the unique junctional sequences in the HVR3 segments. Using these novel amplimers in an allele-specific amplification and hybridization procedure, we showed that this assay can detect 10 leukemic cells in a background of 10B normal blood mononuclear cells. In contrast, the leukemic HVR3 sequences were not detected in extracts of normal or unrelated remissison leukemic leukocytes. We conclude that the assay for specific IgH HVR3 sequences is a realistic strategy for detection of minimal residual disease in B-lineage ALL.

Original languageEnglish (US)
Pages (from-to)2072-2079
Number of pages8
JournalBlood
Volume76
Issue number10
StatePublished - Nov 15 1990

Fingerprint

Complementarity Determining Regions
Oligonucleotide Probes
Residual Neoplasm
Precursor Cell Lymphoblastic Leukemia-Lymphoma
Immunoglobulin Heavy Chains
Assays
Alleles
Polymerase chain reaction
Oligonucleotides
Genes
Leukemia
DNA
Polymerase Chain Reaction
Reaction products
Joining
Gene Rearrangement
Amplification
Blood
Nucleotides
Blood Cells

ASJC Scopus subject areas

  • Hematology

Cite this

Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes. / Jonsson, Olafur G.; Kitchens, Richard L.; Scott, Frederick C.; Smith, R. Graham.

In: Blood, Vol. 76, No. 10, 15.11.1990, p. 2072-2079.

Research output: Contribution to journalArticle

Jonsson, Olafur G. ; Kitchens, Richard L. ; Scott, Frederick C. ; Smith, R. Graham. / Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes. In: Blood. 1990 ; Vol. 76, No. 10. pp. 2072-2079.
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