High-Throughput Sequencing Detects Minimal Residual Disease in Acute T Lymphoblastic Leukemia

Abstract

High-throughput sequencing (HTS) of lymphoid receptor genes is an emerging technology that can comprehensively assess the diversity of the immune system. Here, we applied HTS to the diagnosis of T-lineage acute lymphoblastic leukemia/lymphoma. Using 43 paired patient samples, we then assessed minimal residual disease (MRD) at day 29 after treatment. The variable regions of TCRB and TCRG were sequenced using an Illumina HiSeq platform after performance of multiplexed polymerase chain reaction, which targeted all potential V-J rearrangement combinations. Pretreatment samples were used to define clonal T cell receptor (TCR) complementarity-determining region 3 (CDR3) sequences, and paired posttreatment samples were evaluated for MRD. Abnormal T lymphoblast identification by multiparametric flow cytometry was concurrently performed for comparison. We found that TCRB and TCRG HTS not only identified clonality at diagnosis in most cases (31 of 43 for TCRB and 27 of 43 for TCRG) but also detected subsequent MRD. As expected, HTS of TCRB and TCRG identified MRD that was not detected by flow cytometry in a subset of cases (25 of 35 HTS compared with 13 of 35, respectively), which highlights the potential of this technology to define lower detection thresholds for MRD that could affect clinical treatment decisions. Thus, next-generation sequencing of lymphoid receptor gene repertoire may improve clinical diagnosis and subsequent MRD monitoring of lymphoproliferative disorders.

Using synthetic templates to design an unbiased multiplex PCR assay

“…These data were used to estimate the frequency of blast cells and CD7 þ cells (T cells) in each sample. As previously described, using TCRG sequencing we identified one or two dominant CDR3 sequences in the pre-treatment sample (which are presumed to be the TCRG rearrangements in the malignant clone) and determined MRD status based on the presence of those sequences in the post-treatment sample 9 . We compared the frequency of the top cancer clone in each patient to the results obtained by mpFC.…”

“…In extreme cases, such bias can result in undetectable levels of specific under-amplifying target templates. A bias-free assay is critical for studies aiming to quantitatively measure the frequency of specific immune receptor rearrangements, such as minimal residual disease (MRD) monitoring in leukaemia [9][10][11][12] , following exposure-specific immune repertoires over time 13,14 and research to study basic B-and T-cell biology 15,16 .…”

Prognostic and therapeutic implications of minimal residual disease at the time of transplantation in acute leukemia

“…13,46,[95][96][97] As the sensitivity, specificity and timing of MRD assays is critical, the development of standardized methods of MRD detection and analysis is an important step toward optimized use of this tool, and efforts in that direction are under way. 11,13,16,98,99 All transplant centers should be encouraged to participate in the development of standardized tools and adopt them as soon as they are available.…”

Immunoglobulin transcript sequence and somatic hypermutation computation from unselected RNA-seq reads in chronic lymphocytic leukemia

“…Others have previously identified clonal IGH and TCR transcripts from massively parallel sequencing data (5,(26)(27)(28) via targeted or enriched sequencing with ad hoc experiments for this specific purpose, and specifically for the detection of MRD. Early techniques required patient-specific amplification, whereas latter methods have been refined to work with consensus primers; at least one group has described the use of degenerate IGH targeted primers (28).…”