C082 | T-CELL RECEPTOR-VΒ REPERTOIRE FLOW CYTOMETRY ANALYSIS SHOWED ABSOLUTE CONCORDANCE WITH MOLECULAR ANALYSIS OF TCR GENE REARRANGEMENT IN THE DIAGNOSIS OF T-CELL LYMPHOPROLIFERATIVE DISORDER: OBSERVATIONAL STUDY FROM A SINGLE CENTER EXPERIENCE.

Background: Clonality assessment is critical to differentiate between reactive and pathological T- cell lymphocytosis, and it usually relies on molecular analysis of T-Cell Receptor (TCR) gene rearrangement, which is considered the gold standard assay. However, multiparametric flow cytometry (MFC) represents a valuable tool to pinpoint potential clonality in T-cell proliferation, especially through the analysis of the TCR-Vβ repertoire. A restricted expansion of a single out of the 24 evaluable Vβ families, or the presence of a pathological “clonogram-off” pattern, strongly suggest the presence of a clonal T-cell population. Nevertheless, the concordance between MFC TCR-Vβ repertoire and molecular TCR gene rearrangement analysis to assess T-cell clonality has been poorly investigated. Methods and Objective: We conducted a monocentric, retrospective observational study, collecting data from 307 patients (pts) referred to our center for peripheral blood T-cell lymphocytosis between 2003 and 2024. The aim was to evaluate the diagnostic accuracy of MFC TCR-Vβ repertoire analysis in detecting T-cell clonality by comparing the results with molecular analysis of TCR gene rearrangement. Results: MFC TCR-Vβ repertoire analysis was performed in 270 patients (87.9%), while molecular analysis of TCR gene rearrangement was available for 252 patients (82%). In clonally restricted cases, MFC TCR-Vβ repertoire analysis revealed the expansion of a single Vβ family in 67.5% of cases, while clonality in the remaining 32.5% was inferred based on a pathological “clonogram-off” pattern. Among the 24 TCR-Vβ families, Vβ13.1 was the most frequently expanded (26 pts; 15.3%), followed by Vβ8 and Vβ2 (17 pts each; 10%), Vβ17 (11 pts; 6.5%), and Vβ1 (10 pts; 5.9%). The most common rearrangement detected by PCR was Vγ2-8Jγ1.3 (110 pts; 43.6%), followed by Vγ9Jγ1.3 (61 pts, 24.2%), Vγ2-8Jγ 1.1 (52 pts; 20.6%), Vγ10Jγ1.3 (42 pts; 16.7%), and Vγ10-11Jγ1.3 (26 pts; 10.3%). For 215 (70%) patients both MFC TCR-Vβ repertoire analysis and molecular analysis of TCR gene rearrangement were available, showing an absolute concordance (215 out of 215 cases, 100%) between the two methods. Conclusion: MFC TCR-Vβ repertoire analysis represents a rapid, sensitive, affordable, and reliable tool to infer clonality in T-cell lymphocytosis. It is an effective first-line diagnostic approach, and should be included into the routine laboratory work-up of MFC analysis for peripheral T lymphocytosis.