Molecular and flow cytometric analysis of the Vβ repertoire for clonality assessment in mature TCRαβ t-cell proliferations

Clonality assessment through Southern blot (SB) analysis of TCRB genes or polymerase chain reaction (PCR) analysis of TCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)-PCR analysis of Vβ-Cβ transcripts and flow cytometry with a Vβ antibody panel covering more than 65% of Vβ domains was validated using 28 SB-defined clonal T-cell receptor (TCR)αβ⁺ T-ALL samples and T-cell lines. Next, the diagnostic applicability of the Vβ RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal Vβ-Cβ RT-PCR products were detected in all 47 samples, whereas single Vβ domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of Vβ monoclonal antibody reactivity that was confirmed by molecular data showing the usage of Vβ gene segments not covered by the applied Vβ monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the "clonal" character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant Vβ-Cβ products and single Vβ domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigen-activated cytotoxic T cells. It is concluded that molecular Vβ analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper Vβ antibody studies can be used as a powerful screening method for the detection of single Vβ domain expression, followed by molecular studies in patients with more than 20% single Vβ domain expression or large suspect T-cell populations (more than 50%-60%) without Vβ reactivity.