T-cell receptor (TR) diversity of the variable domains is generated by recombination of both the alpha (TRA) and beta (TRB) chains. The textbook process of TRB chain production starts with TRBD and TRBJ gene rearrangement, followed by the rearrangement of a TRBV gene to the partially rearranged D-J gene. Unsuccessful V-D-J TRB rearrangements lead to apoptosis of the cell. Here, we performed deep sequencing of the poorly explored pool of partial TRBD1-TRBD2 rearrangements in T-cell genomic DNA. We reconstructed full repertoires of human partial TRBD1-TRBD2 rearrangements using novel sequencing and validated them by detecting V-D-J recombination-specific byproducts: excision circles containing the recombination signal (RS) joint 5’D2-RS – 3’D1-RS. Identified rearrangements were in compliance with the classical 12/23 rule, common for humans, rats, and mice and contained typical V-D-J recombination footprints. Interestingly, we detected a bimodal distribution of D-D junctions indicating two active recombination sites producing long and short D-D rearrangements. Long TRB D-D rearrangements with two D-regions are coding joints D1-D2 remaining classically on the chromosome. The short TRB D-D rearrangements with no D-region are signal joints, the coding joint D1-D2 being excised from the chromosome. They both contribute to the TRB V-(D)-J combinatorial diversity. Indeed, short D-D rearrangements may be followed by direct V-J2 recombination. Long D-D rearrangements may recombine further with J2 and V genes forming partial D1-D2-J2 and then complete V-D1-D2-J2 rearrangement. Productive TRB V-D1-D2-J2 chains are present and expressed in thousands of clones of human antigen-experienced memory T cells proving their capacity for antigen recognition and actual participation in the immune response.

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 are 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.

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. We reconstructed full repertoires of human incomplete TRB DD rearrangements and validated its authenticity by detecting excision circles with RSS (recombination signal sequence) junctions for the first time. The identified rearrangements generated in compliance with the classical 12/23 rule are common for humans, rats, and mice and contain typical VDJ recombination footprints. Detected bimodal distribution of DD junctions indicates two active recombination sites producing long and short DD rearrangements. Unlike long DD rearrangements, the short ones have unusual origin resulting from non-canonical intrachromosomal RSSs’ junctions formation. Identified DD rearrangements lead to deleting J1 and C1 segments and creating diverse hybrid D segments, which recombine further with J2 and V segments. Resulting functional TRB VDDJ rearrangements are present in the memory T-cells subset proving its participation in antigen recognition.

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.

Background. Genomic rearrangements of T and B cell receptor genes (BCR and TCR) have become the gold standard marker for clonality assessment and minimal residual disease monitoring (MRD) in acute lymphoblastic leukemia (ALL). B-ALL can bear both TCR and BCR rearrangements, whereas T-ALL contains mainly TCR rearrangements. The limitation on the number of potential markers decreases T-ALL cases suitable for MRD monitoring and its reliability. We discovered a missed stage of TRB locus VDJ-recombination during which rearrangement between D1 and D2 segments occurs. Here we examined this new type of rearrangement in pediatric T-ALL as a potential clonal marker for MRD diagnostics for the first time.

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 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.

Introduction Clonality assessment using IG/TCR rearrangements is an essential tool for clonal evolution analysis and minimal residual disease monitoring in acute lymphoblastic leukemia (ALL). Here we present a new multiplex method for ALL clonal structures analysis at the single-cell level. Material and Methods: DNA from bone marrow (BM) samples of 20 T-ALL and 60 B-ALL patients was used for initial rearrangements detection by targeted high-throughput sequencing. The nuclei from all 80 BM samples were extracted, measured, pooled together evenly and sorted by FACS in 96-tube plate 75 nuclei per tube. The detection of initially identified IG/TCR was performed in all nuclei aliquots. Results: The presence of all patient-specific IG/TCR was detected and analyzed for each sample. The clonal structure was resolved by pairing of clonal and subclonal rearrangements in aliquots. Discussion: The developed method is a useful tool for single-cell level clonality analysis and can be easily implemented in routine ALL diagnostics. The work is supported by RSF grant 18-14-00244, RFBR grants 20-015-00462 and 18-29-09132 and Charity foundation Podari Zhizn.

Introduction While immunotherapy is highly effective in leukemia, one of the main obstacles to MRD monitoring in ALL patients are CD19- relapses that impede FCM. Detection of clonal rearrangements of Ig/TCR genes is used as a routine method for MRD monitoring along with FCM, and unlike latter is applicable in all immunotherapy patients. Materials and methods: We tested 210 bone marrow samples from 35 patients diagnosed with refractory or relapsed B-ALL aged 1-21 years, receiving immunotherapy. Initial detection of clonal rearrangements was carried out by 8 multiplex PCRs of Ig and TCR loci followed by NGS. MRD detection included NGS of previously detected rearrangements in post-treatment samples and quantitative analysis. Results: In our cohort CD19- relapses were observed twice as often as CD19+ relapses. Both CD19- and CD19+ relapses were detected timely or at least 1 month earlier by NGS than by any other method, including FCM. Conclusions: High-throughput sequencing in patients undergoing immunotherapy is necessary for an adequate assessment of MRD level. The study is supported by RFBR grants 20-015-00462, 18-315-20038 and 18-29-09132 and Charity foundation Podari Zhizn

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.

Relapses are still the major cause of poor outcome in therapy of T-lineage acute lymphoblastic leukemia (T-ALL) in children. Over 30% cases of relapse in T-ALL are accompanied by changing of the main clones. At the present time the most reliable markers for analysis of clonal structure of T-ALL are rearrangements of T-cell receptors (TCR) genes: TCR beta, gamma and delta. The purpose of this study is identification and characterization a new type of clonal markers based on previously unanalyzed in ALL rearrangements of TCR alpha locus.

Being the major cause of relapse in leukemia, minimal residual disease (MRD) is considered to be the strongest prognostic factor, allowing to evaluate the efficiency of a treatment, and to make a decision on the subsequent therapy. Detection of MRD by Next generation sequencing (NGS) is proven to be one of the most efficient and sensitive techniques. Detection of clonal rearrangements of immunoglobulin (Ig) and T-cell receptor (TCR) genes is widely used for clonality assessment and for MRD monitoring along with RT-PCR of fusion transcripts, and unlike the latter one, is applicable in most cases of lymphoblastic malignancies.

Diagnostics of minimal residual disease (MRD) is the most powerful prognostic tool in acute lymphoblastic leukemia. MRD testing allows to determine risk of relapse and guides treatment decision. Here we describe a new method for MRD detection based on targeted high-throughput sequencing (HTS) of rearranged immunoglobulin genes specific for leukemic clones.

A new quantitative method for minimal residual disease monitoring based on next generation dequencing and digital PCR principal, A new quantitative method for minimal residual disease monitoring based on next generation dequencing and digital PCR principal

Monitoring of minimal residual disease (MRD) proved to be a valuable tool for predicting relapse in patients with acute lymphoblastic leukemia (ALL). However, the universal use of MRD monitoring in routine clinical practice is limited because current MRD assays have some methodological difficulties, high price and ambiguity of assay results interpretation. Here we describe next generation sequencing based system for the detection of clonal rearrangements in immunoglobulin genes loci. We performed testing of the system on 17 initial bone marrow samples from B-ALL patients. We revealed 1 to 6 characteristic immunoglobulin genes rearrangements in each of 16 samples. These results are in a good accordance with the results obtained by traditional BIOMED-2 assay. Further improvement of the reported system will provide highly reliable and sensitive technique for MRD monitoring.