Page 146 - Haematologica Vol. 107 - September 2022

ARTICLE - FL3B and simultaneous DLBCL
K. Koch et al.
necessity of treatment strategies that may differ from those for FL 1, 2 and 3A.7 In a large retrospective study FL3B seemed to be associated with a favorable outcome.8 How- ever, following the current definition of the WHO classifica- tion FL3B harboring areas of diffuse growth fulfilling the criteria of DLBCL are rather rare and probably underrepre- sented5,9 or even excluded from analysis in more recent studies on the clinical features of FL3B.6,8 It is, therefore, unclear whether histological transition of follicular to diffuse growth reflects a clinically relevant transformation at all. Lack of knowledge regarding the biological and clinical sig- nificance of transformation of FL3B may however create a considerable clinical problem since FL3B occurs in patients who are often young - a subgroup of patients who warrant careful consideration of therapy intensity with respect to long-term toxicity.10
As FL3B is one of the rarest grades of FL very few mol- ecular studies have been conducted and most of them fo- cused on the distinction of FL3B from other grades and DLBCL.11-14 In the current study we aimed to understand the molecular and clinical features of FL3B showing tran- sition into DLBCL in order to provide a definition of “trans- formation” in FL3B guiding clinical decision-making.
Methods
Case selection, histological evaluation and immunohistochemistry
Cases with a diagnosis of FL3B with or without a DLBCL component diagnosed between 2012 and 2016 at the Hematopathology Section and Lymph Node Registry of the University Hospital Schleswig-Holstein, (Kiel, Germany) were identified in the files and re-evaluated with regard to diagnosis and growth patterns as well as suitability for molecular analysis. In total, 51 specimens with a histologi- cal picture of FL3B were identified, two of which were ex- cluded later as they turned out to have an IRF4 break and these are considered a separate entity by the WHO clas- sification.1 Staining for CD20 (clone L26, DAKO), CD10 (clone 56C6, Novocastra), Mum1 (clone MUM1P, Dako), Bcl6 (clone BL6.02, DCS) and Bcl2 (clone 100/D5, DBS) was evaluated semiquantitatively (negative, <25% of lymphoma cells positive, <50% positive, <75% positive, >75% positive) and proliferative rate was determined according to Ki67 (clone SP6, Neomarkers) in steps of 10% by visual inspec- tion. Immunohistochemical classification of the cell of ori- gin of DLBCL components was performed applying the Hans classifier.15 Areas of defined growth patterns were selected for tissue microarray construction in 47 cases with sufficient material. Meshworks of follicular dendritic cells were evaluated in tissue microarrays for each growth pattern semiquantitatively by CD21 staining (0 = absence of meshworks, 1 = markedly reduced covering <50% of fol-
licles, 2 = slightly reduced covering >50% of follicles and 3 = intact covering whole area of follicle; clone 2G9, No- vocastra). Punches with a tissue microarray needle (1 mm diameter) from each area were also used for nucleic acid extraction. To prevent cross-contamination of DNA/RNA analytes the tissue microarray needle was punched three to five times in an empty paraffin block between each punching of a patient’s specimen.
The study was conducted in accordance with the recom- mendations of the ethics board of the Medical Faculty, University of Kiel (D447/10) for the use of archival tissue specimens. Informed consent was obtained from patients treated in the PETAL trial (see below).
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) was performed on 5 mm slides from the tissue microarrays, allowing sep- arate analysis of different growth patterns. Probes for IRF4, BCL2, BCL6 and MYC were obtained from ZytoVision (Bre- merhaven, Germany) and applied as previously described.16
DNA and RNA extraction
Cases for molecular genetic analyses were chosen when the tissue quality was assumed to be sufficient for nucleic acid extraction. Simultaneous DNA and RNA extraction was done using the AllPrep DNA/RNA FFPE Kit (Qiagen) ac- cording to the manufacturer´s instructions. For each pa- tient different growth patterns were extracted separately from tissue microarray needle biopsies taken from the re- spective areas (2-3 biopsies per specimen).
Mutation analyses
Mutation analyses were done on DNA isolated from the for- malin-fixed paraffin-embedded material of the diagnostic biopsy using an AmpliSeq Custom DNA Panel covering genes or mutational hotspots known to be recurrently mu- tated in aggressive B-cell lymphomas (Online Supplemen- tary Table S1). Libraries were prepared according to the manufacturer’s instructions and sequencing was performed on a MiSeq instrument using V3 sequencing chemistry. FASTQ data were analyzed using JSI SeqNext software (JSI Medical Systems GmbH, Ettenheim, Germany). Formalin- fixed paraffin-embedded tissue from reactive lymph nodes from eight healthy individuals (a cancer infiltration was ex- cluded by expert hematopathologists) was used as control tissue, and sequence alterations identified in those samples were subtracted from those of the lymphoma patients. Fur- thermore, common single nucleotide polymorphisms with ≥1% variant allele frequency were excluded. Potential pro- tein changing alterations with at least 15% variant allele fraction were analyzed in more detail using the ENSEMBL variant effect predictor (http://www.ensembl.org/Homo_sa- piens/Tools/VEP). However, as the library preparation pro- cess contains polymerase chain reaction amplification
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