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A. Vogelsberg et al.
BCL2 and effectively abrogates negative selection in the germinal center (GC), leading to prolonged survival in the GC environment.4-6 Another category of aggressive BCL harboring BCL2 (18q21) translocations are high-grade B- cell lymphomas (HGBL) with an additional MYC rearrangement, so-called double-hit (DH) or triple-hit (TH) lymphomas, when also carrying BCL6 transloca- tions.3 In addition to de novo presentation, both DLBCL and HGBL can arise from indolent BCL, most commonly FL.3 Transformation of FL into an aggressive lymphoma occurs in 2-3% of patients per year and usually results in a GCB phenotype.3,7
The t(14;18)(q32;q21) occurs during early B-cell devel- opment and is considered a founding alteration.4 However, this translocation alone is insufficient to cause the development of FL, as t(14;18)+ B cells can be identi- fied at low frequencies in the peripheral blood of about half of otherwise healthy adults over the age of 50.3,8 Distinct clones of these t(14;18)-carrying cells, termed fol- licular lymphoma like B cells (FLLC), have been shown to persist and even expand over years without progressing to manifest FL in most individuals.3,9 The risk for progres- sion depends on the clone size, rather than on the number of different t(14;18)+ clones.10 The earliest identifiable tis- sue-based precursor of FL is in situ follicular neoplasia (ISFN), defined as colonization of GC by a monoclonal population of t(14;18)+ B cells in otherwise reactive lym- phoid tissues.3,11 Although by definition the normal lym- phoid architecture is not altered, ISFN can be identified immunohistochemically by virtue of its strong staining for BCL2 and CD10 and a low proliferation index.11 ISFN can occur syn- or metachronously with FL, as well as with other BCL, but can also be found in individuals without history of lymphoma.12,13 The risk of progression seems to be low.12 Although ISFN is considered a precur- sor lesion, it already demonstrates secondary genetic alterations typically associated with manifest FL, espe- cially affecting chromatin modifier genes such as CREBBP, and less frequently KMT2D and EZH2, known as important factors in FL pathogenesis.14-16 Importantly, both ISFN and FLLC also exhibit persistent expression of activation-induced cytidine deaminase (AID), which cat- alyzes class switch recombination and somatic hypermu- tation (SHM).17,18 AID activity is responsible for the intra- clonal heterogeneity of the re-arranged immunoglobulin heavy chain (IGH) genes and the acquisition of novel N- glycosylation sites in the IG variable regions.18,19 Novel glycosylation motifs are a feature frequently observed in FL, but less so in normal B cells or other BCL, and are thought to enable the interaction with mannose-binding lectins, eliminating the need for conventional B-cell recep- tor signaling through antigen binding.18,20 Furthermore, AID activity is believed to be an important driver for the genetic evolution of FL, leading to increased genomic instability and the accumulation of additional aberra- tions.21,22
Given the well-known role of ISFN as clonally related premalignant FL precursor and the frequent occurrence of the t(14;18) translocation in both de novo and second- ary DLBCL and HGBL,3,14,23,24 we aimed to identify syn- or metachronous ISFN in patients with DLBCL and HGBL, and used these paired samples to investigate the clonal relationship, clonal evolution and underlying genetic changes driving progression from ISFN to aggressive BCL.
Methods
Sample selection
Suitable cases were identified by searching the archives of the Institutes of Pathology of Tuebingen University Hospital and the Robert-Bosch-Krankenhaus (Stuttgart, Germany) for patients with a diagnosis of DLBCL or HGBL, with or without antecedent FL, for which reactive lymphoid tissue from any time point was available, and staining the lymphoid tissues for BCL2 to identify ISFN (as detailed in the Online Supplementary Appendix). The ISFN of case 8 has already been included in pre- vious studies.14,15 An additional case was provided by the Hospital Universitario Fundación Jiménez Díaz (Madrid, Spain). All diagnoses were made according to the criteria of the 2017 World Health Organization classification and reviewed by two experienced hematopathologists (LQ-M and FF).3 This study was approved by the Ethics Committee of the University of Tuebingen (096/2016/B02).
Microdissection, immunohistochemistry and fluorescence in situ hybridization
Microdissection of ISFN samples was performed on 5 mm hematoxylin and eosin stained formalin-fixed, paraffin-embed- ded (FFPE) sections with an Axiovert 200M microscope (Zeiss, Oberkochen, Germany) and the P.A.L.M. system (Palm@Robo software 3.0; Zeiss). Fluorescence in situ hybridization (FISH) was performed on FFPE sections using Vysis LSI BCL2, BCL6 and MYC Dual Color Break Apart Rearrangement Probes (Abbott Molecular, Wiesbaden, Germany). For additional infor- mation, including DNA extraction and immunohistochemistry, see the Online Supplementary Appendix.
Polymerase chain reaction and sequencing of the t(14;18) breakpoint region
The t(14;18) breakpoint regions were amplified by poly- merase chain reaction (PCR) and sequenced using major break- point, minor cluster and intermediate cluster region primers together with a joining region consensus primer as previously described, as well as the IdentiClone BCL2/JH Translocation Assay (Invivoscribe, San Diego, CA, USA) (Online Supplementary Appendix).
Clonality analysis and immunoglobulin sequencing
Detection of monoclonal IGH and IGκ light chain (IGK) gene rearrangements was performed using BIOMED-2 primers as pre- viously described.25 Next-generation sequencing (NGS) of IGH genes was accomplished with the LymphoTrack Dx IGH Assay – PGM (Invivoscribe) on the Ion Torrent Personal Genome Machine (PGM; Thermo Fisher Scientific, Waltham, MA, USA). Data were analyzed with the LymphoTrack Dx Software – PGM (Invivoscribe). For a description of the IG sequence analysis and the construction of phylogenetic trees to illustrate the clonal evolution of the IGH, see the Online Supplementary Appendix.
Targeted next-generation sequencing analysis
Samples were subjected to NGS on the Ion Torrent PGM using AmpliSeq Custom Panels created with the Ion AmpliSeq Designer (Thermo Fisher Scientific). The panels target recurrent mutations of FL and DLBCL, covering 95.21% of the coding sequence of BCL2, BCL6, BTG1/2, CARD11, CD79B, CREBBP, EP300, FOXO1, GNA13, HIST1H1B-E, IGLL5, KMT2D, IRF4, MEF2B, PIM1, PRDM1, TBL1XR1, TNFAIP3, and TNFRSF14 as well as exons 2-5 of MYD88 and the Y646 EZH2 hotspot (Online Supplementary Table S1). In addition, all aggressive BCL were analyzed with the Ion Ampliseq TP53 Panel (Thermo Fisher
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haematologica | 2021; 106(10)