Widespread in situ follicular neoplasia in patients who subsequently developed follicular lymphoma.

In situ follicular neoplasia (ISFN) is usually an occasional incidental finding in lymph nodes by BCL2 immunohistochemistry, and its true scale is unknown. We have identified six cases of follicular lymphoma (FL) with a history of solid neoplasm 4-16 years ago, from which ISFN was identified widely in the surgically cleared lymph nodes (LNs). Using clone-specific PCR and BaseScope in situ hybridisation with primers or probes specific to the VDJ or BCL2-IGHJ junction sequence, we confirmed the clonal identity among different ISFNs and overt-FL in each of the four cases successfully investigated. Mutation analyses of overt-FL by targeted next-generation sequencing identified multiple potential pathogenic changes involving CREBBP, EZH2, KMT2D, TNFRS14, and STAT6. Further investigations of these mutations in paired ISFNs using Fluidigm PCR and Illumina sequencing showed the presence of the FL-associated mutations in early lesions for two of the six cases investigated (CREBBP and KMT2D in one case and STAT6 in the other), with one case displaying stepwise accumulation of its observed mutations. Remarkably, there were considerable divergences in BCL2 variants among different ISFN-involved lymph nodes in all four cases successfully investigated, indicating ongoing intraclonal diversification by somatic hypermutation machinery. Our findings demonstrate widespread distribution of ISFN lesions, further implicating their dynamic nature with the neoplastic cells undergoing active trafficking and clonal evolution. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.