P026 | IDENTIFICATION OF PATHOLOGICAL T-CELL POPULATIONS IN T-CELL LYMPHOMAS USING TRBC1/TRBC2 FLOW CYTOMETRY ANALYSIS

Identification of a pathological T-cell population in T cell Lymphomas (T-NHL) by flow cytometry (FC) is challenging due to phenotypic overlap with reactive T-cells. Antibodies targeting the mutually exclusive isoforms of the T-cell receptor β-chain constant region, TRBC1 and TRBC2, offer a novel method for assessing monotypic restriction. Recent evidence suggests that TRBC1 <15% or >85% or a TRBC2:TRBC1 ratio <0.18 or >5.7 may indicate phenotypic TCRαβ monotypy. We evaluated a multiparameter FC panel including CD2, CD3 CD45 CD7 CD5 CD8 CD4 CD10 CD26 CD56 PD1 TCRαβ TCRγδ TRBC1 TRBC2. Following a stain-lyse-wash protocol, samples were acquired with a DXFlex cytometer (Beckman Colter). All T-cell populations, including suspected pathological clusters were analyzed for TRBC restriction. From March 2024 to April 2025, we prospectively analyzed 44 samples (25 peripheral blood [PB], 13 bone marrow [BM], and 6 lymph node biopsies [LN]). The cohort included 16 newly diagnosed T-NHL (3 T-NHL NOS, 2 AITL, 4 T-LGL leukemia, 1 T-PLL, 1 T-ALL, 2 Sézary syndrome [SS], 3 Mycosis Fungoides [MF]), 4 T-NHL cases post-therapy (2 T-NHL, 1 MF and 1 T-LGL) and 24 non-T-NHL cases (4 DLBCL, 2 HL, 1 MGUS, 1 thymoma, 1 mononucleosis, 1 lymphocytosis, 7 reactive hyperplasia, 1 solid tumor, 6 healthy controls). All 14 T-NHL with histological/morphological tissue involvement (13 at diagnosis and 1 post- therapy) displayed a monotypic pattern (TRBC1-restricted in 5/14, median ratio 0.045 range 0.002–0.13; TRBC2-restricted in 9/14, median ratio 87.9 range 19–3179). In contrast, analysis of 3 BM samples of T-NHL in complete remission and 3 PB samples of MF patients negative for presence of Sezary cells showed polytypic TRBC expression. Among the 24 non-T-NHL cases, 23 demonstrated polytypic TRBC expression (median CD4+ ratio 1.39, range 0.47-2.07; median CD8+ ratio 2.03, range 0.5–3.9). One patient with T-cell lymphocytosis showed a small double-positive CD4+ CD8+ cluster (0.18% of total leucocytes) with a high TRBC ratio (7) but a polyclonal TCR rearrangement by PCR. Notably, TCR gene rearrangement analysis was concordant with FC results in 23 available cases (12 monoclonal and 11 polyclonal). These findings support that TRBC-based FC analysis is a rapid and useful tool for identifying pathologic T-cell populations suspicious for T-NHL. However, larger studies are required to confirm its diagnostic accuracy.