ABSTRACT
Introduction and Aim: T-cell lymphomas (TCL) are a rare group of non-Hodgkin lymphomas characterized by heterogeneity, challenging diagnostics, and poor prognosis. Diagnosis relies on a combination of clinical, morphological, immunophenotypic, and molecular features, with a particular challenge being the distinction between neoplastic and reactive T-cell proliferations. The TRBC1 antibody has emerged as a promising tool for clonality assessment by flow cytometry (FC). The aim of this study was to immunophenotypically characterize neoplastic and reactive populations and to develop a reliable FC method for determining T-cell clonality.
Hypotheses: In our study, we aimed to test the following hypotheses: 1) The most common immunophenotypes of angioimmunoblastic T-cell lymphomas and peripheral T-cell lymphomas, not otherwise specified, will differ from each other in at least one of the observed T-cell markers. 2) In more than 60% of T-cell lymphoma cases, compared to reactive T lymphocytes, we will detect differences in the degree of expression in at least two different T-cell markers. 3) Determination of T-cell clonality by flow cytometric detection of the constant region 1 of the T-cell receptor beta (β) chain will be a highly specific and sensitive method, enabling us to distinguish between reactive and lymphomatous T-cell populations.
Materials and Methods: In the first part of the study, we analyzed 251 TCL samples using multiparametric FC to evaluate a broad panel of T-cell markers. In the second part, clonality was assessed in 275 samples with TRBC1 and the results were compared with PCR. In addition to manual evaluation, we also applied the Phenograph and t-SNE algorithms.
Results: Aberrant T-cell populations were detected in 96% of TCL samples. The most frequent alterations included decreased expression of CD3, CD7, CD8, and TCRαβ, and increased expression of CD10, CD279, and HLA-DR. TCL subtypes were distinguished by the expression of key markers. In ALCL, we observed strong CD30 expression, while PTCL and nTFHL showed differences in CD3, CD10, CD26, CD45RO, and CD279 expression. Monoclonal T lymphocytes were identified with the TRBC1 method in 94.5% of TCL cases, with high concordance with PCR. Algorithm-based analysis revealed monotopic T-cell populations in 7.3% of cases that were missed by manual evaluation, confirming the higher sensitivity of the algorithmic approach. In addition, we identified T-cell clones of uncertain significance, mostly CD8 positive, which were most frequently observed in patients with B-cell lymphomas.
Conclusions: FC is a reliable method for detecting aberrant T-cell populations. The TRBC1-based clonality assessment method achieved high sensitivity and specificity and proved useful in the diagnostics of TCL.
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