igk and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.. Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.. LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.. These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Topics: Bone Marrow; Cyclin D1; Gene Rearrangement; High-Throughput Nucleotide Sequencing; Humans; Immunoglobulin Heavy Chains; Immunoglobulin lambda-Chains; Immunoglobulins; Leukemia, Lymphocytic, Chronic, B-Cell; Limit of Detection; Multiple Myeloma; Neoplasm, Residual; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Reagent Kits, Diagnostic; Translocation, Genetic

The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of pre-B cells and prevents further development. We studied whether inhibition of BCR-ABL1 kinase activity using STI571 can relieve this differentiation block. STI571 treatment of leukemia patients induced expression of the Ig L chain-associated transcription factors IRF4 and SPIB, up-regulation of RAG1 and RAG2, Ckappa and Clambda germline transcription, and rearrangement of Ig kappa L chain (IGK) and Ig lambda L chain (IGL) genes. However, STI571-treated pre-B ALL cells expressed lambda L, but almost no kappa L chains. This could be explained by STI571-induced rearrangement of the kappa-deleting element (KDE), which can delete productively rearranged Vkappa-Jkappa joints. Amplifying double-strand breaks at recombination signal sequences within the IGK, KDE, and IGL loci revealed a coordinated sequence of rearrangement events induced by STI571: recombination of IGK gene segments was already initiated within 1 h after STI571 treatment, followed by KDE-mediated deletion of Vkappa-Jkappa joints 6 h later and, ultimately, IGL gene rearrangement after 12 h. Consistently, up-regulation of Ckappa and Clambda germline transcripts, indicating opening of IGK and IGL loci, was detected after 1 and 6 h for IGK and IGL, respectively. Continued activity of the recombination machinery induced secondary IGK gene rearrangements, which shifted preferential usage of upstream located Jkappa- to downstream Jkappa-gene segments. Thus, inhibition of BCR-ABL1 in pre-B ALL cells 1) recapitulates early B cell development, 2) directly shows that IGK, KDE, and IGL genes are rearranged in sequential order, and 3) provides a model for Ig L chain gene regulation in the human.

Topics: B-Lymphocytes; Benzamides; DNA Primers; DNA-Binding Proteins; Flow Cytometry; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Gene Rearrangement, B-Lymphocyte, Light Chain; Guanine Nucleotide Exchange Factors; Homeodomain Proteins; Humans; Imatinib Mesylate; Immunoglobulins; Nuclear Proteins; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Transcription Factors

A unique case of ALL in three monozygotic triplets diagnosed at the age of 24, 27 and 37 months is described. Archived bone marrow smears were available for molecular analysis of immunoglobulin heavy chain (IGH) and IGK genes and T-cell receptor (TCR)-delta and gamma gene rearrangements. A shared IGH rearrangement was found in triplets "A" and "B", and an identical rearrangement of TCR-delta in triplets "B" and "C". These data suggest a common, monoclonal initiation of ALL in one of these three triplets, followed by dissemination of clonal progeny to the other twins via vascular anastomoses within the single, monochorionic placenta that they shared in utero. Differences in IGH rearrangements in diagnostic samples also indicates divergent subclonal evolution of the original "pre-leukaemic" clone.

Topics: Adult; Bone Marrow; Cell Lineage; Child, Preschool; Cloning, Molecular; Female; Gene Rearrangement; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; Gene Rearrangement, T-Lymphocyte; Genes, Immunoglobulin; Genes, T-Cell Receptor delta; Genes, T-Cell Receptor gamma; Humans; Immunoglobulins; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sequence Analysis, DNA; Triplets; Twins, Monozygotic