Previously Unknown Stage of TCR Beta VDJ Recombination As a Novel Rich Source of Markers for Clonality Assessment and MRD Monitoring in T-ALL
Authors: Komkov Alexander Y , Smirnova Anastasia O , Miroshnichenkova Anna M , Popov Alexander M , Karachunskiy Alexander I , Olshanskaya Yulia V , Maschan Michael A , Mamedov Ilgar Z 
Published: November 2021

Abstract
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.
Methods. Identification of TRB D1-D2 rearrangements was performed using targeted high-throughput sequencing on the Illumina MiSeq instrument [1]. Genomic libraries for sequencing were obtained using PCR with intronic primers flanking the TRBD1 and TRBD2 genes. Genomic DNA for the libraries was extracted from T-ALL patients' bone marrow and peripheral blood of healthy volunteers. DNA from normal CD4+ and CD8+ T-cells was used as a positive control. DNA from normal B-cells and the RMS cell line was used as a negative control. Extraction of TRB D1-D2 rearrangements from sequencing data was performed using modified MiXCR software. Post-analysis was performed using VDJtools software.
Results. This study analyzed a cohort of 144 children aged 2-16 years with T-ALL and 12 healthy individuals aged 25-40 years. We identified TRB D1-D2 rearrangements in all analyzed samples except negative controls (B-cells and RMS cell line). The dominant TRB D1-D2 clone in all normal samples occupied 25-30% and had zero length of the D1-D2 junction. The clonal size of the second clone in the top was 3-8%. Based on this observation, we set a 30% cut-off for zero-length D1-D2 junction rearrangements and a 10% cut-off for others to identify abnormally proliferated T-ALL specific clones.
We identified 221 different leukemia-related D1-D2 rearrangements in 124 T-ALL samples in total. We intersected them with those in normal samples to test the uniqueness of leukemia-related rearrangements as potential markers. 89% of analyzed rearrangements were specific for leukemia samples, so they can be used for tracking leukemic cells. 11% of rearrangements were present in at least one normal sample and can be used for clonality analysis but are useless for MRD monitoring due to its relatively high generation probability. Zero-length D-D junction was detected as a major clonal marker (25-100%) in 42 T-ALL samples. Nevertheless, 23 out of these 42 samples contained additional unique rearrangements useful for MRD. 29 out of the other 82 samples included one major rearrangement (clonal rate 80-90%), six samples had two major rearrangements with equal clonal rates (30-50%), and the rest of the samples contained 3-5 rearrangements representing oligoclonality.
The length of the D-D junction among detected rearrangements in both leukemic and normal samples has a bimodal distribution which is probably linked to two different levels of TdT or exonuclease activity during D1-D2 recombination. Additionally, we observed a strong correlation (Mann-Whitney-U-test, p-value &lt; 0.001) between the presence of TRB D1-D2 rearrangements in T-ALL samples and the CD117 negative immunophenotype of leukemic cells.
Conclusions. Obtained results show that TRB D1-D2 rearrangements are a common feature of normal and malignant T-cells. Unique leukemia-related TRB D1-D2 rearrangements can be detected in 73% of pediatric T-ALL cases. It can be routinely detected by PCR with subsequent NGS and can be easily integrated into an existing multiplex system for T-ALL clonality and MRD analysis.
References.  Komkov et al. High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid malignancies. British Journal of Haematology. 2020 Mar. 188(5): 723-731.
Acknowledgment. This work was supported by Russian Science Foundation grant № 20-75-10091 to AK.

Disclosures
No relevant conflicts of interest to declare.