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

Abstract

T-cell clonality testing is integral to the diagnostic work-up of T-cell malignancies; however, current methods lack specificity and sensitivity, which can make the diagnostic process difficult. The recent discovery of a monoclonal antibody (mAb) specific for human TRBC1 will greatly improve the outlook for T-cell malignancy diagnostics. The anti-TRBC1 mAb can be used in flow cytometry immunophenotyping assays to provide a low-cost, robust, and highly specific test that detects clonality of immunophenotypically distinct T-cell populations. Recent studies demonstrate the clinical utility of this approach in several contexts; use of this antibody in appropriately designed flow cytometry panels improves detection of circulating disease in patients with cutaneous T-cell lymphoma, eliminates the need for molecular clonality testing in the context of large granular lymphocyte leukemia, and provides more conclusive results in the context of many other T-cell disorders. It is worth noting that the increased ability to detect discrete clonal T-cell populations means that identification of T-cell clones of uncertain clinical significance (T-CUS) will become more common. This review discusses this new antibody and describes how it defines clonal T-cells. We present and discuss assay design and summarize findings to date about the use of flow cytometry TRBC1 analysis in the field of diagnostics, including lymph node and fluid sample investigations. We also make suggestions about how to apply the assay results in clinical work-ups, including how to interpret and report findings of T-CUS. Finally, we highlight areas that we think will benefit from further research.

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

“…At the same time, we provide preliminary data on the distribution of TRBC1 + and TRBC1 − Tαβ-cells according to the specific TCRVβ family expressed by total Tαβ-cells (and their major subsets) and their maturation stage. Finally, we confirm and extend on previous observations [18,21,25,26] about the analytical sensitivity and specificity of the assay for detecting monoclonal vs. poly/oligoclonal Tαβ-cell populations, even when present at low frequencies in blood.…”

“…Despite the diagnostic value of the new TRBC1 assay here optimized and validated, it should be noted that occasionally clonal T-cell populations show dim or negative surface CD3/TCRVβ expression levels [26]. Among those T-CLPD patients with productive gene rearrangements upstream of TRBC1, expression of TRBC1 at the protein level would be also low or negative.…”

“…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.…”

“…Further molecular analysis performed here on purified suspicious Tαβ-cells populations from these otherwise healthy individuals confirmed that they systematically corresponded to clonal cells by PCR. The significance of the presence of these T-cell clones in blood remains to be elucidated, but they could be either the result of physiologically normal immune activation of T-cells [24,26], similar to the monotypic expression of kappa or lambda in B-cells that can also be seen in skewed immune responses, without additional abnormal marker expression [44], or persistent clonal (immunosenescent) cells that does not necessarily imply malignancy, in a similar way to low-count monoclonal B lymphocytosis [45].…”

“…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.…”