Single-Antibody Evaluation of T-Cell Receptor β Constant Chain Monotypia by Flow Cytometry Facilitates the Diagnosis of T-Cell Large Granular Lymphocytic Leukemia

Abstract

Objectives The diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL) is challenging because of overlapping immunophenotypic features with reactive T cells and limitations of T-cell clonality assays. We studied whether adding an antibody against T-cell receptor β constant region 1 (TRBC1) to a comprehensive flow cytometry panel could facilitate the diagnosis of T-LGLL. Methods We added TRBC1 antibody to the standard T-cell and natural killer (NK) cell panel to assess T-cell clonality in 56 T-LGLLs and 34 reactive lymphocytoses. In addition, 20 chronic lymphoproliferative disorder of NK cells (CLPD-NKs) and 10 reactive NK-cell lymphocytoses were analyzed. Results Clonal T cells were detected in all available T-LGLLs by monotypic TRBC1 expression and clonal/equivocal T-cell receptor gene rearrangement (TCGR) studies, compared with only 27% of T-LGLLs by killer-cell immunoglobulin-like receptor (KIR) restriction. Overall, 85% of T-LGLLs had a blood tumor burden greater than 500 cells/µL. Thirty-four reactive cases showed polytypic TRBC1 expression, except for 5 that revealed small T-cell clones of uncertain significance. All CLPD-NKs showed expected clonal KIR expression and negative TRBC1 expression. Conclusions Addition of TRBC1 antibody to the routine flow cytometry assay could replace the TCGR molecular study and KIR flow cytometric analysis to assess clonality, simplifying the diagnosis of T-LGLL.

Emerging Role of T-cell Receptor Constant β Chain-1 (TRBC1) Expression in the Flow Cytometric Diagnosis of T-cell Malignancies

“…Incorporating TRBC1 into routine flow cytometric panels makes clonal T-cell identification from the reactive background T-cells possible (Figure 3). In our experience, all T-LGLL cases revealed monotypic TRBC1 expression, concordant with the clonal/equivocal TCR gene rearrangement results [44]. In contrast, only one-fourth of T-LGLL cases showed clonality based on the expression of restricted KIRs.…”

“…The integration of clonality assessment by TRBC1 into routine clinical practice has resulted in the frequent identification of small T-cell clones of uncertain significance (T-CUS) in patients with no clinical evidence of T-cell lymphoma [31], most likely representing dominant T immunoclones ( Figure 2). As predicted from prior reports, most T-CUS detected by TRBC1 staining has an immunophenotype reminiscent of CD8-positive T-cell large granular lymphocytes, with a clone size that only occasionally overlaps with that seen in T-cell large granular lymphocytic leukemia (T-LGLL) [44]. Interestingly, we found no association between the presence of T-CUS and clinical features typically encountered in T-LGLL (namely cytopenias, autoimmune diseases, or decreased numbers of NK cells).…”

Anti-TRBC1 Antibody-Based Flow Cytometric Detection of T-Cell Clonality: Standardization of Sample Preparation and Diagnostic Implementation

“…Several recent reports have proposed the introduction of the TRBC1-based FCM assay as a potentially useful approach to assess Tαβ-cell clonality in the diagnostic work-up of patients suspicious of T-CLPD [18][19][20][21][23][24][25][26]. Despite this, optimization of the antibody staining conditions, as well as reference ranges for normal and reactive polyclonal Tαβ-cells and their major subsets, together with the sensitivity and specificity of the TRBC1 assay for detecting clonal Tαβ-cells, remain to be fully established prior to its diagnostic routine use.…”

“…Recently, a single antibody (TRBC1-binding monoclonal antibody, clone JOVI−1) against one of the two mutually exclusive TCR β chain constant domains (TRBC1 and TRBC2) randomly selected during rearrangement of the TRB gene, has been proposed as a potential marker for rapid assessment of Tαβ-cell clonality by FCM [18]. Normal, as well as virus-specific Tαβ-cells, show an admixture of TRBC1-positive (37-51% and 36-52% of normal CD4 + and CD8 + T-cells, respectively) [18][19][20][21][22] and TRBC1-negative (presumably TRBC2 positive) T-cells (polyclonal profile in GeneScan studies), whereas monoclonal Tαβ-cells typically showed restricted (monotypic) TRBC1 expression [18][19][20][21][23][24][25]. Recent reports have further shown the potential utility of this antibody reagent for routine assessment of Tαβ-cell clonality in T-CLPD vs. normal/reactive conditions [18][19][20][21][23][24][25][26].…”

“…Normal, as well as virus-specific Tαβ-cells, show an admixture of TRBC1-positive (37-51% and 36-52% of normal CD4 + and CD8 + T-cells, respectively) [18][19][20][21][22] and TRBC1-negative (presumably TRBC2 positive) T-cells (polyclonal profile in GeneScan studies), whereas monoclonal Tαβ-cells typically showed restricted (monotypic) TRBC1 expression [18][19][20][21][23][24][25]. Recent reports have further shown the potential utility of this antibody reagent for routine assessment of Tαβ-cell clonality in T-CLPD vs. normal/reactive conditions [18][19][20][21][23][24][25][26]. Despite this, optimal standardization of the technique for routine use in diagnostic lab-oratories, and interpretation of the results based on normal reference TRBC1 + /TRBC1 − ratios and ranges for both normal and reactive Tαβ-cells (and their subsets), have not been provided.…”