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Introduction
T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) is a rare subtype of diffuse large B-cell lymphoma (DLBCL) characterized by a low fraction of tumor B cells and a cellular background rich in T cells and histiocytes. It has been classified as a separate entity of mature B-cell lymphoma since the fourth edition of the World Health Organization (WHO) classification of lymphoid neo- plasms.1,2 Although it has a more aggressive clinical behav- ior and distinct microenvironmental composition,3,4 THRLBCL shares several clinical and pathological features with nodular lymphocyte-predominant Hodgkin lym- phoma (NLPHL), a rare subtype of Hodgkin lymphoma. The similarities include a predominance of middle-aged male patients5 and a minority of tumor cells derived from germinal center B cells in an abundant microenviron- ment.6,7 Furthermore, a high similarity of gene expression signatures4,8 and genomic copy number changes in the microdissected tumor cells of NLPHL and THRLBCL were found.9 According to Fan et al.,10 NLPHL can be subdivided into six different histopathological patterns, which include two typical nodular growth patterns (A and B) and four histopathological variants (C-F). The NLPHL variant E, also called THRLBCL-like variant, shows particularly marked similarities with THRLBCL and can only be dis- tinguished from de novo THRLBCL by the presence of typ- ical NLPHL remnants in the same lymph node or in anoth- er lymph node simultaneously sampled. In general, histopathological NLPHL variants are associated with an advanced clinical stage and an increased relapse rate.10,11
Data on somatic gene mutations of the tumor cells of THRLBCL are still lacking. Hence, we aimed to elucidate the relationship of THRLBCL and NLPHL through a com- parison of recurrently mutated genes to obtain a more comprehensive understanding of the pathogenesis of THRLBCL.
Methods
Cases
Cases were collected based on the availability of frozen tissue at the Dr. Senckenberg Institute of Pathology, Frankfurt am Main, Germany; the Department of Pathology University Hospitals, K.U. Leuven, Belgium; the Unit of Lymphoid Malignancies, San Raffaele Scientific Institute, Milan, Italy; Tampere University Hospital and University of Tampere, Tampere, Finland; and the Department of Pathology and Laboratory Medicine and the Centre for Lymphoid Cancer, British Columbia Cancer Agency, Vancouver, Canada. The local ethics committees approved the study, and written informed consent from the donors was obtained in accordance with the Declaration of Helsinki. All cases were reviewed on a multi-head microscope by expert hematopathologists (RG, SH, MLH, and TT). Only cases meeting the diagnostic criteria of the current WHO classification for NLPHL and THRLBCL1,2 were included in the study. Coexisting NLPHL was not found in any of the THRLBCL cases. For correla- tive analysis, NLPHL cases were classified according to Fan et al.10 For this purpose, the major pattern present in the frozen sample was considered. In all cases, the initial biopsies before treatment were analyzed. All cases also presented a typical immunopheno- type of the tumor cells (CD20+, BCL6+, OCT2+, CD30–, CD15–, LMP1–). Information on the patients is provided in Table 1.
Ultra-deep targeted resequencing and identification of somatic variants
Extraction of genomic DNA from frozen tissue of cases of THRLBCL and NLPHL, ultra-deep targeted resequencing after bait-based enrichment by a custom Haloplex kit (Agilent, Santa Clara, CA, USA) and processing of sequencing reads were carried out as described previously.12 All 62 genes used for the ultra-deep targeted resequencing were selected based on mutated genes from a previous study of two clonally related composite lymphomas consisting of NLPHL and DLBCL (Online Supplementary Table S1).12 Some of these selected genes were already confirmed to be mutat- ed in primary NLPHL without transformation into DLBCL.12 The mean coverage of the 26 cases ranged between 3500 and 8500x (Online Supplementary Table S2). Non-synonymous single nucleotide variants (SNVs) were filtered for allele frequencies based on the expected tumor cell content (0.1-10%), and the pres- ence and somatic origin of selected variants were confirmed in a semi-nested polymerase chain reaction (PCR) approach followed by Sanger sequencing of the PCR products, as indicated in Online Supplementary Table S3. Details on validation of mutations using microdissected tumor cells and analysis of the somatic hypermu- tation (SHM) features of SNVs are provided in the Online Supplementary Methods section.
haematologica | 2019; 104(2)
JUNB, DUSP2, SGK1 and SOCS1 mutations in THRLBCL
Laser microdissection and Immunohistochemistry
Frozen sections (5 - 10 mm) of lymph nodes from lymphoma patients were mounted on membrane-covered slides (PALM, Zeiss, Bernried, Germany), then air-dried and fixed in acetone. Sections were stained with a mouse monoclonal anti-CD20 anti- body (clone L26, Dako, Glostrup, Denmark) in 1:200 dilution for 1 h at room temperature. Binding of the primary antibody was visualized with the aid of the Super SensitiveTM Link-Label IHC Detection System (BioGenex, Fremont, CA, USA), and counter- staining with hematoxylin was performed. For PCR analysis, 20 single tumor cells and non-tumor cells were isolated using the PALM laser capture microdissection technique (PALM MicroBeam, Zeiss, Bernried, Germany) and collected in 20 μL PCR buffer without MgCl2 (Expand High Fidelity, Roche, Grenzach, Germany) supplemented with 0.1% Triton X-100.
The immunohistochemical staining for activation-induced cyti- dine deaminase (AICDA) was performed on an independent series of 15 typical and 11 variant NLPHL as well as 12 THRLBCL with formalin-fixed paraffin-embedded tissue as previously described.13 The anti-AICDA antibody (clone EK2 5G9, Cell Signaling, Danvers, MA, USA) was applied in a dilution of 1:100.
Results
T-cell/histiocyte-rich large B-cell lymphoma shares recurrently mutated genes with nodular lymphocyte-predominant Hodgkin lymphoma
Ultra-deep targeted resequencing of the coding exons of 62 genes (Online Supplementary Table S1), which were found to be mutated in NLPHL in a previous study,12 was performed in the following three groups: THRLBCL (n=9); typical NLPHL, patterns A/B (n=6); and histopathological NLPHL variants, patterns C/D/E (n=11), yielding a total number of 26 cases. The patients’ characteristics are given in Table 1. The coding exons of selected genes were ana- lyzed for non-synonymous SNVs in order to identify mutations with a potential functional effect.
Of the 62 NLPHL-related genes, 48 (77%) were somati- cally mutated in at least one of the 26 cases (Online
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