Next‐generation amplicon TRB locus sequencing can overcome limitations of flow‐cytometric Vβ expression analysis and confirms clonality in all T‐cell prolymphocytic leukemia cases

Abstract

T-cell receptor (TCR) β repertoire analysis can distinguish monoclonal from polyclonal T-cell proliferations and crucially aid in the diagnosis of T-cell malignancies. TCR repertoire can be assessed either by flow cytometry (FCM), or by molecular genetic techniques. We compared the results of parallel analyses of Vβ expression by FCM and TRB rearrangements by DNA-based next-generation sequencing (NGS) in 80 diagnostic peripheral blood samples of patients with T-cell prolymphocytic leukemia (T-PLL) for (1) the diagnosis of clonality and (2) the assessment of dominant Vβ usage. FCM-based analysis of the surface expression was performed using the IOTest Beta Mark kit. The NGS-based analysis employed the multiplex Biomed-2 VB-JB primers. In all the samples, one or two clonal TRB rearrangements were detected by NGS. Although a dominant Vβ domain usage was detected by FCM in only 41/80 (51%) samples, clonality was suspected in all of them. In a total of 12 cases, the FCM missed the clone detected by NGS, despite theoretical coverage by the antibodies, the functionality of the rearrangement, and the expression of TCRαβ on the cell surface. Partly overlapping with those cases, FCM discovered predominant Vβ usage in the T-PLL population that differed from the one detected by NGS in 10 cases. Overall, the concordant NGS and FCM results were obtained on 61/80 (76%) of samples. We conclude that NGS-based TRB analysis can overcome certain limitations of FCM-based analysis by the identification of both productive and nonproductive rearrangements and by covering the whole Vβ spectrum. Currently available FCM analysis of Vβ expression lacks this breadth but has advantages, such as parallel immunophenotyping and a more accurate quantification of the Vβ usage.

Reconstruction of rearranged T‐cell receptor loci by whole genome and transcriptome sequencing gives insights into the initial steps of T‐cell prolymphocytic leukemia

“…Interestingly, both were RSS-23 (rather than, which are used by TRAV and TRDV gene segments. Taking into consideration that T-PLL cells nevertheless not only express a functional T-cell receptor but also seem to rely on that,[15][16][17] we investigated the second allele of chromosome 14 in which the TRA/TRD locus remains unaffected by the cancer-related aberration. We did not identify significant expression from any of the V(D)J gene segments of the TRD locus in the T-PLL cases.…”

“…7 The illegitimate TRA/TRD rearrangements in T-PLL prevent expression of a functional TCR alpha or delta in tumor cells from the affected allele. Taking into consideration that T-PLL cells nevertheless not only express a functional T-cell receptor but also seem to rely on that, [15][16][17] we investigated the second allele of chromosome 14 in which the TRA/TRD locus remains unaffected by the cancer-related aberration. As outlined above, we failed to detect significant expression of TRD V and J segments in the analyzed T-PLL cases.…”

A skewed distribution and increased PD-1+Vβ+CD4+/CD8+ T cells in patients with acute myeloid leukemia

“…Clonally expanded TCRαβ+ T cells were identified in patients with hematologic malignancies and cancer, and the identified tumor antigen‐specific TCRαβ gene could be used to construct TCR‐modified T cells (TCR‐T) for cancer immunotherapy . Molecular analysis, particularly using next‐generation sequencing and single‐cell sequencing, could identify clonal TCR rearrangements, confirming specifically expanded T cell clones and monitoring antigen‐specific T cell responses, which may help to design effective TCR‐based immunotherapies . However, these analyses are incapable of evaluating the functional status of clonally expanded T cells.…”

“…2,[25][26][27] Molecular analysis, particularly using next-generation sequencing and single-cell sequencing, could identify clonal TCR rearrangements, confirming specifically expanded T cell clones and monitoring antigen-specific T cell responses, which may help to design effective TCR-based immunotherapies. [28][29][30] However, these analyses are incapable of evaluating the functional status of clonally expanded T cells. Knowledge of the distribution of the TCR + subfamily in T cell subsets in patients with leukemia is lacking.…”

T-cell malignancies pathogenesis, a system hematology/oncology

“…In this type of disease, there is post-thymic monoclonal prolymphocyte, which strongly expresses CD7, CD5, and CD2, and the lack of expression of TdT and CD1a [3,4]. Malignant cells have the normal size, high ratio of nuclear-to-cytoplasm, with a round to oval shape, and a single prominent nucleolus; the cytoplasm is agranular, with blebs [1].…”