INTEGRATION OF T‐CELL CLONALITY SCREENING USING TRBC‐1 IN LYMPHOMA SUSPECT SAMPLES BY FLOW CYTOMETRY

Abstract

Background: The diagnosis of T-cell non-Hodgkin lymphomas (NHL) is challenging. Many cases of T-NHL have normal immunophenotypes, and currently available T-cell clonality assays have limitations, including suboptimal sensitivity. The development of a monoclonal antibody (mAb) specific for T-cell receptor β constant region 1 (TRBC1) provides an alternative to discriminate clonal T cells. The aim of this study was to evaluate the diagnostic potential of an anti-TRBC1 mAb for the identification of T-NHL. Materials and Methods: We performed a cross-sectional diagnostic analytic study of samples tested for lymphoma. All samples sent for lymphoma screening were first evaluated using the standard Euroflow Lymphoma Screening Tube (LST), to which a second additional custom-designed T-cell clonality assessment tube was added. LST tube included CD45/CD3/CD4/CD5/CD8/CD19/ CD20/CD38/TRCγδ/CD56 and the second CD45/TRBC1/CD2/CD7/CD4/TRCγδ/CD3. The results of TRBC1 expression in patient samples were compared to the normal expression of TRBC1 in 10 normal samples of peripheral blood (PB). Flow cytometry reports were compared with morphological and molecular tests. Clinical data were obtained from the electronic database. This study was approved by the institutional IRB and Clinical Ethics Committee. Results: 69 samples were evaluated. 10 healthy controls, and 59 patient samples. Overall, individuals had a median of 52 years (interquartile range [IRQ], 35–70 years) and 53.6% were female. Normal TRBC1 expression was assessed in 10 normal PB samples. Within the T-cell population (CD45+/CD3+), CD4+ cells expressed (±SD) TRBC1 in 42% (4.2), while CD8+ cells in 38% (4.7). This resulted in a TRBC1+/- ratio of 0.72 ± 0.13 for CD4+ and 0.61 ± 0.13 for CD8+ T cells. Cut-off percentages in the CD4+ cells were from 29.4% to 54.6%, and from 23.9 to 52.1% in CD8+ T-cells. Cut-off ratios in CD4+ T cells were from 0.33 to 1.1, and in CD8+ T cells between 0.22 and 1.0. Using predefined normal cut-off values, 18 of 59 (30.5%) samples showed a restricted (monotypic) expression of TRBC1. 6/18 were monotypic positive (33.3%) and 12/18 were monotypic negative (66.6%). A final diagnosis of a T-NHL was confirmed by IHC in 15 of the 18 cases (83.3%). A molecular TCR gene rearrangement assay was available in 3/15 cases, which confirmed clonality. The 3 discordant cases were 2 samples from an EBV+ primary effusion lymphoma (PEL) and 1 of lung adenocarcinoma. There were no cases of a diagnosis of a T-NHL by morphology / IHC with normal TRBC1 expression. Non-neoplastic patient samples behaved between predefined TRBC1 cut-off values. Conclusions: Expression of TRBC1 provides a robust method for T- cell clonality assessment, with very high sensitivity and good correlation with complementary methods. TRBC1 can be integrated into routine lymphoma screening strategies via flow cytometry to improve T-NHL diagnosis capacities. The research was funded by: Fondecyt 11200805 Clínica Alemana de Santiago ID1025 Keyword: diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.