Single Antibody Detection of T‐Cell Receptor αβ Clonality by Flow Cytometry Rapidly Identifies Mature T‐Cell Neoplasms and Monotypic Small CD8‐Positive Subsets of Uncertain Significance

Abstract

Background: The diagnosis of T-cell neoplasms is often challenging, due to overlapping features with reactive T-cells and limitations of currently available T-cell clonality assays. The description of an antibody specific for one of two mutually exclusive T-cell receptor (TCR) β-chain constant regions (TRBC1) provide an opportunity to facilitate the detection of clonal TCRαβ T-cells based on TRBC-restriction. Methods: Twenty patients with mature T-cell neoplasms and 44 patients without evidence of T-cell neoplasia were studied. Peripheral blood (51), bone marrow (10), and lymph node (3) specimens were evaluated by 9-color flow cytometry including TRBC1 (

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

“…Novikov et al [ 32 ] reported mean TRBC1-positive events and 95% confident intervals for normal total CD4-positive and CD8-positive T-cells in peripheral blood at 44% (36–53%) and 39% (18–61%), respectively. In patients without demonstrable T-cell neoplasia, we found a similar bimodal expression of TRBC1 on CD4-positive and CD8-positive T-cell subsets gated based on distinct immunophenotypic features, with the exception of occasional small CD8-positive T-cell subsets with a unimodal TRBC1 expression pattern [ 33 ]. Further analysis revealed that these small subsets were truly clonal based on TCR-Vβ-restriction [ 31 ] and T-cell gene rearrangement molecular studies [ 33 ], consistent with benign immunodominant clonotypes (see T-CUS section below).…”

“…In patients without demonstrable T-cell neoplasia, we found a similar bimodal expression of TRBC1 on CD4-positive and CD8-positive T-cell subsets gated based on distinct immunophenotypic features, with the exception of occasional small CD8-positive T-cell subsets with a unimodal TRBC1 expression pattern [ 33 ]. Further analysis revealed that these small subsets were truly clonal based on TCR-Vβ-restriction [ 31 ] and T-cell gene rearrangement molecular studies [ 33 ], consistent with benign immunodominant clonotypes (see T-CUS section below). In our extensive experience using TRBC1 within a single-tube comprehensive T-cell panel, we have found that T-cell neoplasms virtually always have distinct immunophenotypic features that largely separate them from background benign T-cells, but that this separation based on routine gating strategies is often imperfect and results in a very skewed rather than a purely unimodal TRBC1 expression pattern.…”

“…With the recently reported flow cytometric strategy to assess T-cell clonality using a TRBC1 antibody against one of two mutually exclusive TRBC regions [ 32 , 33 ], T-LGLL has been recognized as an excellent example to use this strategy for T-cell clonality because 1) almost all T-LGLLs show surface CD3 expression; 2) 95% of T-LGLLs show TCR αβ type, although intermittent TCR γδ type [ 53 ] and rare mixed-phenotype occur [ 54 ]. Incorporating TRBC1 into routine flow cytometric panels makes clonal T-cell identification from the reactive background T-cells possible ( Figure 3 ).…”

Sézary syndrome and mycosis fungoides: An overview, including the role of immunophenotyping

“…To further improve the specificity of FC in the diagnosis of SS (vs. reactive erythrodermic conditions), the TCR‐Vβ repertoire of CD4+ T cells showing a suspect phenotype can be analyzed using monoclonal antibodies to 24 TCR‐Vβ segments, which cover 70% of the known TCR‐Vβ repertoire: in virtually all cases, this assay will show either restriction of one of these TCR‐Vβ families or will be negative for all tested antibodies, in both cases confirming monoclonality (Gibson et al, 2016; Lima et al, 2003). However, it should be taken into account that the large number of potential Vβ chains makes the TCR‐Vβ repertoire analysis by FC challenging (and expensive) to test these markers in combination with other markers of MF/SS in routine clinical practice; interestingly, recent studies have preliminarily show the potential utility of one single antibody to confirm clonality by FC (an anti‐TCR‐Cβ1 fluorochrome‐conjugated antibody)—in addition to typical panels for identifying MF/SS cells—for diagnosis and monitoring of SS (Novikov et al, 2019; Shi et al, 2020), although the published data with this novel antibody is limited so far, and therefore more studies are needed specifically addressing its value for MF/SS diagnosis.…”

Determination of immunophenotypic aberrancies provides better assessment of peripheral blood involvement by mycosis fungoides/Sézary syndrome than quantification of CD26− or CD7− CD4+ T‐cells

“…Vβ repertoire analysis was employed in challenging cases with immunophenotypic aberrancies, in order to confirm clonality. It is noteworthy that Vβ repertoire analysis is costly, and recently an antibody specific for one of two mutually exclusive TCR β‐chain constant regions (TRBC1) has shown promise for a rapid confirmation of clonality in immunophenotypically aberrant T‐cells (Shi et al, 2019). Other antibodies with potential diagnostic utility include CD2, CD5, CD164, CD158k, CD194 (CCR4), CD279 (PD‐1), CD10, CD45RO, and CD38 (Horna et al, 2020).…”