We report incidence and deep molecular characteristics of lineage switch in 182 pediatric patients affected by B-cell precursor acute lymphoblastic leukemia (BCP-ALL), who were treated with blinatumomab. We documented six cases of lineage switch that occurred after or during blinatumomab exposure. Therefore, lineage conversion was found in 17.4% of all resistance cases (4/27) and 3.2% of relapses (2/63). Half of patients switched completely from BCP-ALL to CD19-negative acute myeloid leukemia, others retained CD19-positive B-blasts and acquired an additional CD19-negative blast population: myeloid or unclassifiable. Five patients had KMT2A gene rearrangements; one had TCF3::ZNF384 translocation. The presented cases showed consistency of gene rearrangements and fusion transcripts across initially diagnosed leukemia and lineage switch. In two of six patients, the clonal architecture assessed by IG/TR gene rearrangements was stable, while in others, loss of clones or gain of new clones was noted. KMT2A-r patients demonstrated very few additional mutations, while in the TCF3::ZNF384 case, lineage switch was accompanied by a large set of additional mutations. The immunophenotype of an existing leukemia sometimes changes via different mechanisms and with different additional molecular changes. Careful investigation of all BM compartments together with all molecular –minimal residual disease studies can lead to reliable identification of lineage switch.
Summary
Rearrangements of T‐ and B‐cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high‐throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high‐throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V‐ and J‐segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T‐lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.
High-throughput sequencing of adaptive immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful TCR/BCR repertoire reconstruction and analysis methods have been developed in the past decade. However, detecting and correcting the discrepancy between real and experimentally observed lymphocyte clone frequencies is still challenging. Here we discovered a hallmark anomaly in the ratio between read count and clone count-based frequencies of non-functional clonotypes in multiplex PCR-based immune repertoires. Calculating this anomaly, we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on the OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: Immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5' RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches and compared with a biological spike-in-based method for PCR bias evaluation. The developed approach can increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.
High-throughput sequencing of immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful methods for TCR/BCR repertoire reconstruction and analysis have been developed in the past decade. However, detection and correction of the discrepancy between real and experimentally observed lymphocyte clone frequencies are still challenging. Here we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on the OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: Immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5′ RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches. The developed tool can be used to increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.
The distribution of polymorphic variants of innate immunity genes TLR9 (+2848G>A) and DEFB1(-20G>A; -44C>G; -52G>A) was evaluated in long-living individuals. No significant differences were found in the distribution of genotypes and alleles of the TLR9 gene. The following features were revealed: increase in the frequency of AA and GG genotypes and decrease in the frequency of the AG genotype of the DEFB1(-20G>A) gene; increase in the frequency of the CC genotype and C allele and decrease in the frequency of CG and GG genotypes and G allele of the DEFB1(44C>G) gene; and increase in the frequency of AA and AG genotypes and A allele and decrease in the frequency of the GG genotypes and G allele of the DEFB1(-52G>A) gene. Genotypes and alleles of the DEFB1 gene found in long-living individuals can be considered as the factors that increase the probability of longevity and favorable course of age-related diseases.
T-cell receptor (TCR) diversity is generated by VDJ recombination. The classical course of TCR beta (TRB) chain production starts with D and J segment recombination and finishes with subsequent recombination between the resulting DJ junction and V segment. In this study, we performed deep sequencing of poorly explored incomplete TRBD1 to TRBD2 rearrangements in T-cell genomic DNA. Here we reconstructed full human TRB DD rearrangements repertoires for the first time. We validated its authenticity by detecting excision circles with RSS (recombination signal sequence) junctions. The found rearrangements generated in compliance with the classical 12/23 rule are common for humans, rats, and mice, and contain typical VDJ footprints: random nucleotide deletions and insertions. Detected bimodal distribution of DD junctions indicates two active recombination sites producing long and short rearrangements. Unlike long DD rearrangements, the short ones have unusual origin resulting from non-canonical intrachromosomal RSSs junctions formation. Identified DD rearrangement leads to the deletion of J1 and C1 segments and creates a diverse hybrid D segment that can further recombine with J2 and V segments. However, in most complete TRB genes, the traces of DD junctions are blurred by cutting in DDJ and VDDJ rearrangements which made this stage of VDJ recombination mostly hidden in the final completely rearranged TRB gene.
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