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Detection of minimal residual disease in acute lymphoblastic leukemia using
immunoglobulin hypervariable region specific oligonucleotide probes
OG Jonsson, RL Kitchens, FC Scott and RG Smith
Department of Pediatrics, University of Texas Southwestern Medical Center,
Dallas 75235-8852.
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 10(6)
normal blood mononuclear cells. In contrast, the leukemic HVR3 sequences
were not detected in extracts of normal or unrelated remission 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.
Volume 76,
Issue 10,
pp. 2072-2079,
11/15/1990
Copyright © 1990 by The American Society of Hematology
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