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Genetic landscape of plasmablastic lymphoma
Introduction
Plasmablastic lymphoma (PBL) is an aggressive lymphoid neoplasm characterized by a diffuse proliferation of large neoplastic cells with an immunoblastic or plasmablastic morphology but expressing a plasma cell phenotype includ- ing positivity for BLIMP1/PRDM1, XBP1, CD138, CD38, Vs38c and MUM1/IRF4 and negativity or weak positivity for CD45 and markers of mature B cells such as CD20 and PAX5. CD79a is positive in approximately 50-85% of the cases.1,2 This tumor was initially described as a subtype of diffuse large B-cell lymphoma (DLBCL) present in the oral cavity of patients positive for human immunodeficiency virus (HIV). Subsequent studies expanded the spectrum of presentation of this lymphoma to other extranodal sites and immunodeficient conditions such as iatrogenic immuno- suppression associated with post-transplant therapy or chronic treatments for autoimmune diseases and aging.1,3,4
Epstein-Barr virus (EBV) is known to be an important ele- ment in the development of PBL, especially in HIV-positive patients, and can be detected in 60-75% of PBL cases by in situ hybridization.1,4,5 The role of EBV infection has been related to the anti-apoptotic effect in B cells through several mechanisms related to EBV antigens.4 Around 50% of PBL carry immunoglobulin (IG)/MYC rearrangements, a hall- mark of Burkitt lymphoma (BL). This rearrangement seems to be observed more frequently in EBV-positive than in EBV-negative PBL (74% vs. 43%).4,6,7
Few studies have investigated the genetic landscape of PBL. Copy number profiling revealed that recurrent gains of 1p36, 1p36-p34, 1q21-q23, 7q11, 11q12-q13 and 22q12-q13 are common features of PBL.8 More recently a 34-gene tar- geted next-generation sequencing study highlighted PRDM1 and STAT3 as key genes in the pathogenesis of PBL in addition to MYC.9 Furthemore, during the preparation of this manuscript, a genomic analysis of HIV-positive PBL patients from South Africa identified recurrent mutations in the JAK–STAT and RAS-MAPK signaling pathways and recurrent copy number alterations including large chromo- somal gains of 1q and chr7 and focal gains in 6p22.1, 1q21.3 and 11p13 but the possible differential representation of these alterations in relationship to EBV infection could not be defined.10 A gene expression profiling study has shown that, compared to DLBCL, PBL downregulates B-cell recep- tor signaling genes including transcriptionally activated tar- gets of nuclear factor κB (NFκB) signaling and upregulation of MYB and MYC target genes.11
Some PBL share morphological and immunophenotypic features with other aggressive lymphoid neoplasias such as BL, plasmablastic transformation of plasma cell myeloma (PCM) and some variants of DLBCL with immunoblastic features and/or activated B-cell like (ABC) cell of origin.2,12,13 Of note, plasmablastic PCM has been associated with MYC translocations and worse prognosis,4,14 which adds difficulty to the already challenging differential diagnosis. The man- agement of patients with PBL is still not standardized and most patients are treated with cyclophosphamide, doxoru- bicin, vincristine, and prednisone (CHOP) or CHOP-like regimens. However, responses are relatively poor with a median overall survival of about 1 year.1,3,15 More intensive regimens, such as infusional EPOCH, Hyper-CVAD, or CODOX-M/IVAC, have also been used without improving the prognosis.16 On the other hand, bortezomib, a proteo- some inhibitor used in the treatment of PCM, has shown potential efficacy in PBL with promising early results.17
A better understanding of the genetic profiling of PBL may substantiate its distinction from other entities and may contribute to the design of novel therapeutic strategies. We performed a high-resolution genetic analysis of PBL to dis- cover the hallmarks of this disease which may enhance greater diagnostic accuracy and provide insight into the clinical behavior of this lymphoma.
Methods
Case selection and DNA/RNA extraction
Thirty-four cases with a consensus diagnosis of PBL according to the World Health Organization (WHO) classification1 were obtained from the archives of the Hospital Clínic of Barcelona (Barcelona, Spain), the National Institutes of Health National Cancer Institute (Bethesda, USA) and the Department of Clinical Pathology, Robert-Bosch-Krankenhaus (Stuttgart, Germany). The cases were reviewed by the Leukemia and Lymphoma Molecular Profiling Project (LLMPP) pathology panel. Only cases with more than 50% neoplastic cells were included.
DNA and RNA from formalin-fixed paraffin-embedded material were extracted simultaneously using a Qiagen AllPrep DNA/RNA FFPE kit with Deparaffinization Solution (Qiagen Inc.), according to manufacturer's instructions. This study was approved by our Institutional Review Board. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki.
Immunohistochemistry and fluorescence in situ hybridization
Immunohistochemical studies were performed using standard protocols. (Online Supplementary Table S1). Fluorescence in situ hybridization (FISH) analyses for the detection of BCL2, BCL6, MYC and IGH translocations were performed using standard tech- niques and commercial Dual Color Break-Apart Rearrangement Probes (Vysis, Abbott Molecular, Wiesbaden, Germany).
Copy number analysis
Copy number alterations were examined in 33 PBL samples using an Oncoscan FFPE Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to standard protocols. Gains and losses and copy number neutral loss of heterozygosity (CNN- LOH) regions were evaluated using Nexus Biodiscovery 9.0 soft- ware (Biodiscovery, Hawthorne, CA, USA). The proportion of tumor cells or cancer cell fraction carrying each copy number alter- ation (CCFCNA) was estimated from the B-allele frequency and cor- rected for the tumor cell content or purity of the sample obtained from ASCAT (Online Supplementary Methods).18 Copy number alter- ations were considered clonal if their CCFCNA was ≥85%.19 Previously published data from 35 BL, 41 PCM and 49 ABC- DLBCL were used for comparisons.20–23
Mutational analysis
Twenty-seven PBL were examined for the mutational status of 94 B-cell lymphoma-related genes (Online Supplementary Table S2) using a SureSelectXT Target Enrichment System Capture NGS strategy library (Agilent Technologies, Santa Clara, CA, USA) before sequencing with MiSeq equipment (Illumina, San Diego, CA, USA). The bioinformatic pipeline included a filtering process excluding intronic, synonymous and single nucleotide polymor- phic variants and a selection of driver mutations with potential functional effect (Online Supplementary Methods, Online Supplementary Figure S1). The cancer cell fraction carrying each spe- cific mutation (CCFmut) was calculated as previously described.18 As applied to copy number alterations, mutations were classified
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