Next-Generation Sequencing-Based T-Cell Receptor Gene Rearrangement Analysis in Nodal T Follicular Helper Cell Lymphoma, a Comparison with the EuroClonality/BIOMED-2 Assay.

Nodal T follicular helper cell lymphoma (nTFHL) can be difficult to diagnose because it often shows features of immune dysregulation and can have a low number of neoplastic T cells in involved lymph nodes. The analysis of T-cell receptor (TR) gene rearrangements by next-generation sequencing (NGS) and the conventional EuroClonality/BIOMED-2 were performed to compare their performance on this challenging diagnosis. DNA was extracted from 32 formalin-fixed, paraffin-embedded nTFHL samples from two pathology archives. NGS amplicon-based analysis of TRBV-TRBD-TRBJ, TRBD-TRBJ, and TRGV-TRGJ rearrangements was performed using the two-step PCR protocols developed by EuroClonality. The nucleotide sequences were analyzed for abundance, clonotype, and functionality. Both the NGS-based and the conventional clonality assays resulted in a high detection of clonality (97% and 94%, respectively), including both monoclonal and biclonal cases. There was an overrepresentation of TRBV20-1 and TRBV19 gene use that was in line with the frequent use of these genes in T cells of reactive lymph nodes and tonsils. The NGS-based approach detected two or more clonal targets in all clonal samples, whereas the conventional assay detected a single (isolated) dominant rearrangement in three cases. Hence, NGS enables more reliable detection of even small clones, as is frequent in nTFHL. NGS-based TR rearrangement analysis provides the abundance, the sequences, clonotypes, and productivity of the clonal rearrangements and thus stresses the need for novel guidelines for interpretation.