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Published on: September 6, 2017
Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region
O G Jonsson1, R L Kitchens, F C Scott
1Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas 75235-8852.
Insights
This study developed a novel assay using immunoglobulin heavy chain (IgH) gene rearrangements to detect minimal residual disease in acute lymphoblastic leukemia (ALL). The assay accurately identifies rare leukemic cells, offering a sensitive tool for ALL patient monitoring.
Area of Science:
- Immunology
- Molecular Biology
- Oncology
Background:
- Minimal residual disease (MRD) detection is crucial for acute lymphoblastic leukemia (ALL) treatment monitoring.
- Existing MRD assays may lack sensitivity or specificity for certain ALL subtypes.
- Immune receptor gene rearrangements offer a source of unique clonal markers.
Purpose of the Study:
- To develop a sensitive and specific assay for MRD detection in B-lineage ALL.
- To utilize the diversity of immunoglobulin heavy chain (IgH) gene rearrangements for clonal identification.
- To establish a method for detecting rare leukemic cells in patient samples.
Main Methods:
- Oligonucleotide primers targeting conserved variable (VH) and joining (JH) sequences were synthesized.
- Polymerase chain reaction (PCR) amplified the third hypervariable region (HVR3) of rearranged IgH genes from leukemic DNA.
- Sequencing and allele-specific oligonucleotide probes were used to verify and characterize amplified HVR3 sequences.
- An allele-specific amplification and hybridization procedure was developed for sensitive detection.
Main Results:
- A majority of B-lineage ALL samples yielded amplifiable HVR3 gene segments.
- Sequenced HVR3 fragments contained unique junctional sequences, serving as clonal markers.
- The developed assay detected as few as 10 leukemic cells among 10^6 normal cells.
- Leukemic HVR3 sequences were specifically detected, with no signal in normal or unrelated remission samples.
Conclusions:
- Specific IgH HVR3 sequences possess the diversity required for robust clonal markers in ALL.
- The developed assay is a sensitive and specific strategy for detecting minimal residual disease in B-lineage ALL.
- This approach holds promise for improved patient monitoring and treatment stratification in ALL.
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 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.
