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Flow cytometric identification of circulating neoplastic cells (Sezary cells) in patients with mycosis fungoides and Sezary syndrome is essential for diagnosis, staging, and prognosis. Although recent advances have improved the performance of this laboratory assay, the complex immunophenotype of Sezary cells and overlap with reactive T cells demand a high level of analytic expertise. We utilized machine learning to simplify this analysis using only 2 predefined Sezary cell-gating plots. We studied 114 samples from 59 patients with Sezary syndrome/mycosis fungoides and 66 samples from unique patients with inflammatory dermatoses. A single dimensionality reduction plot highlighted all TCR constant β chain-restricted (clonal) CD3 + /CD4 + T cells detected by expert analysis. On receiver operator curve analysis, an aberrancy scale feature computed by comparison with controls (area under the curve = 0.98) outperformed loss of CD2 (0.76), CD3 (0.83), CD7 (0.77), and CD26 (0.82) in discriminating Sezary cells from reactive CD4 + T cells. Our results closely mirrored those obtained by exhaustive expert analysis for event classification (positive percentage agreement = 100%, negative percentage agreement = 99%) and Sezary cell quantitation (regression slope = 1.003, R squared = 0.9996). We demonstrate the potential of machine learning to simplify the accurate identification of Sezary cells., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introducing a sensitive and specific peripheral blood flow cytometric assay for Sézary syndrome and mycosis fungoides (SS/MF) requires careful selection of assay design characteristics, and translation into a laboratory developed assay through development/optimization, validation, and continual quality monitoring. As outlined in a previous article in this series, the recommended design characteristics of this assay include at a minimum, evaluation of CD7, CD3, CD4, CD8, CD26, and CD45, analyzed simultaneously, requiring at least a 6 color flow cytometry system, with both quantitative and qualitative components. This article provides guidance from an international group of cytometry specialists in implementing an assay to those design specifications, outlining specific considerations, and best practices. Key points presented in detail are: (a) Pre-analytic components (reagents, specimen processing, and acquisition) must be optimized to: (i) identify and characterize an abnormal population of T-cells (qualitative component) and (ii) quantitate the abnormal population (semi/quasi-quantitative component). (b)Analytic components (instrument set-up/acquisition/analysis strategy and interpretation) must be optimized for the identification of SS/MF populations, which can vary widely in phenotype. Comparison with expert laboratories is strongly encouraged in order to establish competency. (c) Assay performance must be validated and documented through a validation plan and report, which covers both qualitative and semi/quasi-quantitative assay components (example template provided). (d) Ongoing assay-specific quality monitoring should be performed to ensure consistency., (© 2020 International Clinical Cytometry Society.)

This review discusses the definition and major categories of cutaneous T-cell lymphoma, Sézary syndrome and mycosis fungoides, and the role of immunophenotyping in their diagnosis. The following key points are raised: (a) Sézary syndrome and mycosis fungoides cells most often have a characteristic CD3+ CD4+ CD7- and/or CD26- immunophenotype. (b) This immunophenotype is not specific, but can assist in the distinction from non-neoplastic T cells and other subtypes of mature T-cell neoplasm. (c) However, small subsets of normal and reactive T-cells can have an overlapping immunophenotype, and can be distinguished by evaluating for additional changes in antigen expression., (© 2020 International Clinical Cytometry Society.)

A peripheral blood flow cytometric assay for Sézary syndrome (SS) or circulating mycosis fungoides (MF) cells must be able to reliably identify, characterize, and enumerate T-cells with an immunophenotype that differs from non-neoplastic T-cells. Although it is also important to distinguish SS and MF from other subtypes of T-cell neoplasm, this usually requires information in addition to the immunophenotype, such as clinical and morphologic features. This article outlines the approach recommended by an international group with experience and expertise in this area. The following key points are discussed: (a) At a minimum, a flow cytometric assay for SS and MF should include the following six antibodies: CD3, CD4, CD7, CD8, CD26, and CD45. (b) An analysis template must reliably detect abnormal T-cells, even when they lack staining for CD3 or CD45, or demonstrate a phenotype that is not characteristic of normal T-cells. (c) Gating strategies to identify abnormal T-cells should be based on the identification of subsets with distinctly homogenous immunophenotypic properties that are different from those expected for normal T-cells. (d) The blood concentration of abnormal cells, based on any immunophenotypic abnormalities indicative of MF or SS, should be calculated by either direct enumeration or a dual-platform method, and reported., (© 2020 International Clinical Cytometry Society.)

Background: Mycosis fungoides (MF) and Sézary Syndrome (SS) are clinically distinct cutaneous T-cell lymphomas with strikingly similar morphologic and phenotypic features. Prior studies have suggested phenotypic differences based on markers of antigen experience, suggesting a different cell of origin., Methods: Seventy-nine involved peripheral blood or bone marrow samples from 33 patients with SS and 19 patients with MF were studied by 10-color flow cytometry, including CD62L, CD45RA, CCR4, and PD-1. Gated tumor events were classified as naïve (T N ), central memory (T CM ), effector memory (T EM ), or effector memory with reacquired CD45RA (T EMRA ); based on CD62L + /CD45RA + , CD62L + /CD45RA - , CD62L - /CD45RA - , or CD62L - /CD45RA + phenotype, respectively. Sequential specimens were compared to assess for phenotypic stability., Results: The naïve/memory phenotype of the neoplastic T-cells was markedly heterogeneous, with a dominant T N , T CM , T EM , or T EMRA subset on 11 (14%), 32 (41%), 30 (38%), and 6 (8%) cases, respectively. There was no correlation between the diagnosis of MF or SS and putative cell of origin (P = 0.4). Overexpression of CCR4 and PD1 was observed in most cases, with higher intensity in SS compared to MF. The naïve/memory phenotype remained the same for 10 patients up to 273 days after the initial analysis; while on six patients, the naïve/memory phenotype was different from the original phenotype., Conclusions: Both SS and MF can have phenotypic features of any of the major naïve/memory T-cell subsets, which questions the current principle of "cell-of-origin" distinction between SS and MF. Phenotypic shifts within these subsets are common, suggesting a functional state rather than a cell-of-origin surrogate. © 2018 International Clinical Cytometry Society., (© 2018 International Clinical Cytometry Society.)

Objectives: To assess the ability of flow cytometry (FC) to detect putative neoplastic T-cell subsets on skin shave biopsy (SSB) specimens from patients with mycosis fungoides (MF) and to study the immunophenotype of skin-infiltrating tumor cells in MF., Methods: SSB specimens from patients with suspected MF were bisected and submitted for both FC and routine histopathology. Six-dimensional gating strategies were applied to identify putative neoplastic cells, independently from their expected immunophenotype., Results: Aberrant T cells were detected by FC in 18 of 33 SBB specimens, of which all had clinicomorphologic features of MF. Of the remaining 15 SSB specimens, six had clinicomorphologic features of MF and nine were diagnosed with benign inflammatory dermatoses. Unexpectedly, CD26 was aberrantly overexpressed in 11 (73%) and lost in three (20%) of 15 SSB specimens from patients with MF where this antigen was evaluated. Other detected aberrancies included CD3 dim- (13/18 [72%]), CD7 dim- (15/18 [83%]), and CD4-/CD8- (3/18 [17%])., Conclusions: FC is capable of identifying putative neoplastic cells on SSB specimens from patients with MF. Bright homogeneous CD26 expression is a common and previously undescribed immunophenotypic aberrancy on MF skin infiltrates., (Copyright© by the American Society for Clinical Pathology.)