Immunophenotypic Analysis of the TCR-Vβ Repertoire in 98 Persistent Expansions of CD3+/TCR-αβ+ Large Granular Lymphocytes

Abstract

At present, a major challenge in the initial diagnosis of leukemia of large granular lymphocytes (LGLs) is to establish the clonal nature of the expanded population. In the present study we have analyzed by flow cytometry immunophenotyping the TCR-Vbeta repertoire of 98 consecutive cases of persistent expansions of CD4(+) or CD8(+bright) CD3(+)/TCR-alphabeta(+) LGLs and compared the results with those obtained in molecular studies of TCR-beta gene rearrangements. Fifty-eight cases were considered to be monoclonal in molecular studies whereas in the remaining 40 cases there was no evidence for monoclonality (11 cases were considered oligoclonal and 29 polyclonal). The TCR-Vbeta repertoire was biased to the preferential use of one or more TCR-Vbeta families in 96% of cases, a total of 124 TCR-Vbeta expansions being diagnosed: one TCR-Vbeta expansion in 71 cases and two or more TCR-Vbeta expansions in 23 cases. The highest TCR-Vbeta expansion observed in each case was higher among monoclonal (74 +/- 19%) as compared to nonmonoclonal cases (24 +/- 14%) (P = 0.001), as did the fraction of LGLs that exhibited a TCR-Vbeta-restricted pattern (86 +/- 16% and 42 +/- 23%, respectively; P = 0.0001); by contrast, the proportion of cases displaying more than one TCR-Vbeta expansion was higher in the latter group: 7% versus 48%, respectively (P = 0.001). Results obtained in oligoclonal cases were intermediate between those obtained in polyclonal and monoclonal cases and similar results were observed for CD4(+) as for CD8(+bright) T-cell expansions. TCR-Vbeta families expressed in CD8(+bright) T-cell-LGL proliferations showed a pattern of distribution that mimics the frequency at which the individual TCR-Vbeta families are represented in normal peripheral blood T cells. Assuming that a given proliferation of LGLs is monoclonal whenever there is an expansion of a given TCR-Vbeta family of at least 40% of the total CD4(+) or CD8(+bright) T-cell compartment, we were able to predict clonality with a sensitivity of 93% and a specificity of 80%. By increasing the cut-off value to 60%, sensitivity and specificity were of 81% and 100%. In summary, our results suggest that flow cytometry immunophenotypic analysis of the TCR-Vbeta repertoire is a powerful screening tool for the assessment of T-cell clonality in persistent expansions of TCR-alphabeta(+) LGLs.

An approach to diagnosis of T‐cell lymphoproliferative disorders by flow cytometry

“…In contrast to B-cell lymphoid proliferations, in which flow cytometric analysis of immunoglobulins for light chain restriction can determine clonality, neoplasia cannot be determined easily by flow cytometry analysis of T-cell or NK cell proliferations. This may change, however, with the advent of the development of antibodies to the family of T-cell antigen receptor beta chains (31,32). Large panels of monoclonal antibodies to the TCR-V␤ repertoire can detect expansion of one or more TCR-V␤ families and can predict clonality in some instances (32).…”

“…This may change, however, with the advent of the development of antibodies to the family of T-cell antigen receptor beta chains (31,32). Large panels of monoclonal antibodies to the TCR-V␤ repertoire can detect expansion of one or more TCR-V␤ families and can predict clonality in some instances (32). However, the large panel is unwieldy.…”

Abnormal NK Cell Lymphocytosis Detected after Splenectomy: Association with Repeated Infections, Relapsing Neutropenia, and Persistent Polyclonal B-Cell Proliferation

“…It has been suggested that LGL proliferations arise as a consequence of chronic immune stimulation [9][10][11].…”

“…of these cases [2,12,13]. Unlike evaluation of T-cell LGL proliferations, in which clonality can be assessed with molecular analysis of the TCR genes and TCR variable region repertoire studies [9], assessment of clonality remains a challenge in NK cell disorders. Thus demonstration of an aberrant immunophenotype may be of a great help in the detection of clonal expansion of NK cells.…”

T-Cell Large Granular Lymphocyte Leukemia and Related Disorders

“…Monoclonal antibodies are available for more than 80% of the variable region families of the β chain (Vβ). TCR Vβ antibody studies can be used as an initial screening method for the detection of unbalanced use of β chain gene families [25,26].…”