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

Abstract

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.

Quantitative flow cytometric identification of aberrant T cell clusters in erythrodermic cutaneous T cell lymphoma. Implications for staging and prognosis

“…We demonstrate that a 6D gating strategy effectively overcomes individual immunophenotypical variations and provides additional parameters to visually separate presumed benign from suspected malignant CD4 T cells. While T cell receptor-β analysis by FC has been proposed as an accurate approach to identify SCs,18 19 we found immunophenotypical analysis to be sufficient for SC identification when appropriate antibody combinations and gating strategies were applied.…”

Flow Cytometric Identification of Immunophenotypically Aberrant T-Cell Clusters on Skin Shave Biopsy Specimens From Patients With Mycosis Fungoides

“…In peripheral blood, Feng et al 33 previously showed that the immunophenotypically aberrant T-cell subsets in patients with advanced CTCL correspond to lymphoid cells harboring a clonal T-cell receptor Vβ chain usage, supporting the use of immunophenotypic analysis for the identification of neoplastic cells. On skin biopsy specimens from patients with MF, Clark et al 34 showed the presence of T-cell receptor Vβ-restricted clonal T-cell subsets exhibiting high light scatter by FC.…”

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

“…36 Aberrant expression of T-cell markers by malignant T cells further allows for improved gating for Vb clonality analysis, leading to increase detection sensitivity and specificity. 12,37 In this series of 30 peripheral T-cell lymphomas, a wide variety of Vb families was involved (14 out of 25 specific and 5 out of panel families). They were observed at low frequencies, except Vb17 that appeared to be expanded in 5/30 cases (Figure 3).…”

“…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).…”

“…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.…”