Molecular and flow cytometric analysis of the Vβ repertoire for clonality assessment in mature TCRαβ T-cell proliferations

Langerak, Anton W.; Beemd, René van den; Wolvers-Tettero, I. L. M.; Boor, Patrick P.C.; Lochem, Ellen G. van; Hooijkaas, Herbert; Dongen, Jacques J. M. van

doi:10.1182/blood.v98.1.165

Cited by 219 publications

(156 citation statements)

References 41 publications

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“…Earlier studies showed promising results by using a panel of antibodies covering over 65% of the T-cell receptor-Vb repertoire. 25,26 Langerak et al 26 showed in their study of 47 T-cell lymphomas that flow cytometry detected single Vb domain usage in 31 (66%) patients, and was non-reactive in 16 (34%) additional patients, showing a 100% concordance with PCR analysis of the T-cell receptorb gene. Subsequently, several studies validated and expanded the use of flow cytometry to assess T-cell receptor Vb in T-cell malignancies.…”

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confidence: 99%

Flow cytometric detection of peripheral blood involvement by mycosis fungoides and Sézary syndrome using T-cell receptor Vβ chain antibodies and its application in blood staging

et al. 2010

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Peripheral blood involvement has been recognized as an adverse prognostic factor in patients with mycosis fungoides and Sé zary syndrome. However, accurate identification and enumeration of the neoplastic cells in these diseases can be challenging. We assessed the clinical utility of flow cytometric immunophenotypic analysis of T-cell receptor Vb expression in 82 mycosis fungoides and 6 Sé zary syndrome patients, with an atypical T-cell immunophenotype, or abnormal CD4:CD8 ratio, identified from peripheral blood specimens of 723 patients submitted for routine mycosis fungoides/Sé zary syndrome blood staging. To improve detection sensitivity, Vb expression was analyzed on gated CD3 þ CD4 þ T cells or T cells with an aberrant immunophenotype, if present. The flow cytometric results were compared with traditional morphologic assessment (n ¼ 88) and molecular methods to assess the T-cell receptor c or b genes (n ¼ 41 tested in parallel). Flow cytometric immunophenotyping yielded a clonal Vb pattern in 60/82 mycosis fungoides and 6/6 Sé zary syndrome patients. By contrast, flow cytometric Vb was negative in all 10 healthy donors and 18 control patients, showing a specificity of 100% and concordance with molecular testing of 86%. Using flow cytometric Vb results instead of morphologic assessment, 12 patients were upstaged from B1 to B2, and 20 patients from B0 to B1 (Po0.0001). The 12 upstaged B2 patients had no morphologic evidence of involvement, but had an aggressive clinical course similar to those staged by traditional morphologic assessment (median survival 27 vs 41 months, log-rank P ¼ 0.701). In 30/44 patients with a tumor-associated Vb expression, a single Vb tube was used to monitor treatment response. In conclusion, flow cytometric Vb analysis is rapid and convenient, can assess T-cell clonality and tumor quantity simultaneously, and is useful both in initial blood staging and monitoring tumor burden during therapy in patients with mycosis fungoides or Sé zary syndrome.

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confidence: 99%

Flow cytometric detection of peripheral blood involvement by mycosis fungoides and Sézary syndrome using T-cell receptor Vβ chain antibodies and its application in blood staging

et al. 2010

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“…Furthermore, analysis in biopsies is more adapted to unambiguous identification of the malignant T-cell clone since, as mentioned previously, clonal circulating T cells are frequently observed in non-malignant conditions, leading to potential confusion between reactive and tumor cells. 17 On the basis of the promising results obtained from blood samples, [11][12][13][14][15][16] we used flow cytometry to assess the expression of 25 Vb chain members of the TCR. Indeed, T-cell clones use a single Vb domain and can thus be identified by the abnormal expansion of one Vb family or by a restricted TCR-Vb repertoire (see Materials and methods).…”

Section: Discussionmentioning

confidence: 99%

“…This technique has proved useful in detecting malignant T cells in peripheral blood samples from patients with diverse mature T-cell leukemias. [11][12][13][14][15][16] T-cell clones use the same V domain, and are thus detected by an overexpressed V family (direct identification) or by a restricted V repertoire if they are not recognized by the 25 anti-V antibodies of the panel (indirect identification). However, T-cell clone analysis in blood samples is not optimal for peripheral T-cell lymphomas, as (1) there may be no circulating lymphoma cells, (2) malignant circulating cells may not precisely reflect the clone at the site of tumor involvement and (3) when present, clonal subsets are not always malignant, as observed in infectious or autoimmune conditions, as well as in elderly people.…”

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confidence: 99%

Accurate detection of the tumor clone in peripheral T-cell lymphoma biopsies by flow cytometric analysis of TCR-Vβ repertoire

et al. 2012

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Multiparametric flow cytometry has proven to be a powerful method for detection and immunophenotypic characterization of clonal subsets, particularly in lymphoproliferative disorders of the B-cell lineage. Although in theory promising, this approach has not been comparably fulfilled in mature T-cell malignancies. Specifically, the T-cell receptor-Vb repertoire analysis in blood can provide strong evidence of clonality, particularly when a single expanded V family is detected. The purpose of this study was to determine the relevance of this approach when applied to biopsies, at the site of tumor involvement. To this end, 30 peripheral T-cell lymphoma and 94 control biopsies were prospectively studied. Vb expansions were commonly detected within CD4 þ or CD8 þ T cells (97% of peripheral T-cell lymphoma and 54% of non-peripheral T-cell lymphoma cases); thus, not differentiating malignant from reactive processes. Interestingly, we demonstrated that using a standardized evaluation, the detection of a high Vb expansion was closely associated with diagnosis of peripheral T-cell lymphoma, with remarkable specificity (98%) and sensitivity (90%). This approach also identified eight cases of peripheral T-cell lymphoma that were not detectable by other forms of immunophenotyping. Moreover, focusing Vb expression analysis to T-cell subsets with aberrant immunophenotypes, we demonstrated that the T-cell clone might be heterogeneous with regard to surface CD7 or CD10 expression (4/11 cases), providing indication on 'phenotypic plasticity'. Finally, among the wide variety of Vb families, the occurrence of a Vb17 expansion in five cases was striking. To our knowledge, this is the first report demonstrating the power of T-cell receptor-Vb repertoire analysis by flow cytometry in biopsies as a basis for peripheral T-cell lymphoma diagnosis and precise T-cell clone identification and characterization.

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“…Fresh peripheral blood was stained for VB flow cytometry analysis according to manufacturer's instructions (Beckman-Coulter, Fullerton, CA, USA) and analyzed as described previously [18,20]. Initially, 12 normal samples were characterized to define the average size and standard deviations for the VB repertoire as detected by the antibodies.…”

Section: Vb Flow Cytometrymentioning

confidence: 99%

Clonal predominance of CD8+ T cells in patients with unexplained neutropenia

Wlodarski

Nearman

Jiang

et al. 2008

Experimental Hematology

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Objective-T-cell-mediated autoimmunity may be involved in some cases of idiopathic neutropenia. We hypothesized that a precise T-cell receptor repertoire analysis may uncover cytotoxic T-cell (CTL) expansions that are less pronounced than those seen in T large granular lymphocyte leukemia (T-LGL), but are pathophysiologically analogous and thus can serve as markers of a T-cell-mediated process. Materials and Methods-Using rational algorithms for T-cell receptor analysis and in vivotracking of CTL responses previously established in our laboratory, we studied patients with unexplained chronic neutropenia (n = 20), T-LGL (n = 15), and healthy controls (n = 12). We further investigated the involvement of soluble inhibitory factors by coculture assays. To determine the level of immune activation, we studied interferon-g expression in CD8 + cells using Taqman polymerase chain reaction.Results-Fifteen expanded (immunodominant) CTL clones were detected in 12 of 20 patients. In comparison to LGL leukemia, these clones were less immunodominant, but clearly discernible from subclinical lymphoproliferations in controls. As a surrogate of cytotoxic activity, we found markedly increased production of interferon-γ in most of the neutropenia patients, irrespective of the presence of immunodominant CTL clones.Conclusions-These results suggest that, while immunodominant CTL clones are detectable in a proportion of patients only, CTL-mediated pathophysiology may be a general mechanism operating in idiopathic neutropenia. Oligogoclonal CTL expansions in chronic neutropenia may indicate an ongoing autoimmune process, while highly polarized monoclonalities in a subset of neutropenic LGL patients may represent the "extreme" end of the clonal continuum.Drugs, hereditary factors, infections, and intrinsic bone marrow diseases explain the etiology of most neutropenias. The inability to detect an immune-mediated process or other obvious cause often leads to diagnosis of chronic idiopathic neutropenia (CIN) [1][2][3][4][5][6]. Similarly, secondary autoimmune neutropenia (AIN), usually associated with collagen vascular diseases, lymphoproliferative disorders and viruses, is typically diagnosed by exclusion and thus relies on the quality of clinical and laboratory workups [6][7][8][9]. Lineage-restricted cytopenias, including neutropenia, have been associated with T-cell large granular lymphocyte leukemia It is likely that patients with AIN/CIN may belong to a heterogeneous group of diseases combining various autoimmune etiologies. Appearance of the bone marrow may reveal some clues as to the cellular targets of the autoimmune process; left shift could indicate that more mature cells are targets, while pure white cell aplasia suggests that very early myeloid precursors are affected. The similar target spectrum in AIN/CIN and T-LGL suggests that cytotoxic T-cell (CTL)-mediated autoimmunity can also operate in AIN/CIN. Consequently, detection of highly polarized CTL expansions in some cases of neutropenia patients may be consistent wi...

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Molecular and flow cytometric analysis of the Vβ repertoire for clonality assessment in mature TCRαβ T-cell proliferations

Cited by 219 publications

References 41 publications

Flow cytometric detection of peripheral blood involvement by mycosis fungoides and Sézary syndrome using T-cell receptor Vβ chain antibodies and its application in blood staging

Flow cytometric detection of peripheral blood involvement by mycosis fungoides and Sézary syndrome using T-cell receptor Vβ chain antibodies and its application in blood staging

Accurate detection of the tumor clone in peripheral T-cell lymphoma biopsies by flow cytometric analysis of TCR-Vβ repertoire

Clonal predominance of CD8+ T cells in patients with unexplained neutropenia

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