A New and Simple TRG Multiplex PCR Assay for Assessment of T-cell Clonality: A Comparative Study from the EuroClonality Consortium

• Main Authors: Marine Armand, Coralie Derrieux, Kheira Beldjord, Tamara Wabeke, Dido Lenze, Elke Boone, Monika Bruggemann, Paul A.S. Evans, Paula Gameiro, Michael Hummel, Patrick Villarese, Patricia J.T.A. Groenen, Anton W. Langerak, Elizabeth A. Macintyre, Frederic Davi
• Format: Article
• Language: English
• Published: Wolters Kluwer 2019-06-01
• Series: HemaSphere
• Online Access: http://journals.lww.com/10.1097/HS9.0000000000000255

Abstract. T-cell Receptor Gamma (TRG) rearrangements are commonly used to detect clonal lymphoproliferations in hematopathology, since they are rearranged in virtually all T lymphocytes and have a relatively limited recombinatorial repertoire, which reduces the risk of false negative results, at the cost of potential false positivity. We developed an initial one-tube, 2-fluorochrome EuroClonality TRG PCR multiplex (TRG-1T-2F) which was compared to the original 2-tube, 2-fluorochrome EuroClonality/BIOMED-2 TRG PCR (TRG-2T-2F) and a commercial Invivoscribe one-tube, one-fluorochrome kit (IVS-1T-1F) on a series of 239 samples, including both T-cell malignancies and reactive cases. This initial assay yielded discrepant results between the 10 participating EuroClonality laboratories when using 2 fluorochromes, leading to adoption of a final single color EuroClonality strategy (TRG-1T-1F). Compared to TRG-2T-2F, both TRG-1T-1F and IVS-1T-1F demonstrated easier interpretation and a lower risk of false positive from minor peaks in dispersed repertoires. Both generate smaller fragments and as such are likely to be better adapted to analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples. Their differential performance was mainly explained by (i) superposition of biallelic rearrangements with IVS-1T-1F, due to more extensive overlapping of the repertoires and (ii) intentional omission of the TRGJP primer in TRG-1T-1F, in order to avoid the potential risk of confusion of consensus TRG V9-JP normal rearrangements with a pathological clone.