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.