Multimodal laboratory tests for angioimmunoblastic T-cell lymphoma using multicolor flow cytometry and polymerase chain reaction-based assays

We developed multimodal tests to support the diagnosis of angioimmunoblastic T-cell lymphoma (AITL), for which the cell of origin was previously determined to be follicular helper T-cells in the germinal center and RHOAG17V has been reported as a highly recurrent mutation. In this study, we subjected clinical specimens from 5 patients with AITL to multicolor flow cytometry (FCM) and polymerase chain reaction (PCR)-based assays. We found that lymphoma cells in the 5 cases were invariably characterized by diminished or loss of CD3 expression compared with non-neoplastic T-cells. CD10 was expressed in lymph node specimens in 4 cases; however, the proportion of CD10+ neoplastic T-cells varied. Multicolor FCM using a 10-color antibody panel detected neoplastic T-cell populations in all cases, comprising 2 to 41% within each clinical specimen. We next developed three PCR-based assays for detection of the RHOAG17V mutation, and demonstrated that allele-specific (AS)-PCR was capable of detecting as low as 0.1% RHOAG17V mutant DNA. Using this sensitive AS-PCR, the mutation was found in 4 cases, whereas one was negative. We propose the sequential diagnostic use of multicolor FCM for immunophenotypic and quantitative assessment of lymphoma cells and AS-PCR for the detection of RHOAG17V mutation.