EBV-Negative Monomorphic B-Cell Posttransplant Lymphoproliferative Disorder with Marked Morphologic Pleomorphism and Pathogenic Mutations in ASXL1, BCOR, CDKN2A, NF1, and TP53

Hindawi Case Reports in Hematology Volume 2017, Article ID 5083463, 8 pages https://doi.org/10.1155/2017/5083463 Case Report EBV-Negative Monomorphic B-Cell Posttransplant Lymphoproliferative Disorder with Marked Morphologic Pleomorphism and Pathogenic Mutations in ASXL1, BCOR, CDKN2A, NF1, and TP53 Agata M. Bogusz Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA 19104-4283, USA Correspondence should be addressed to Agata M. Bogusz; Received 25 October 2016; Revised 18 February 2017; Accepted 19 March 2017; Published 10 April 2017 Academic Editor: Yusuke Shiozawa Copyright © 2017 Agata M. Bogusz. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Posttransplant lymphoproliferative disorders (PTLDs) are a diverse group of lymphoid or plasmacytic proliferations frequently driven by Epstein-Barr virus (EBV). EBV-negative PTLDs appear to represent a distinct entity. This report describes an unusual case of a 33-year-old woman that developed a monomorphic EBV-negative PTLD consistent with diffuse large B-cell lymphoma (DLBCL) 13 years after heart-lung transplant. Histological examination revealed marked pleomorphism of the malignant cells including nodular areas reminiscent of classical Hodgkin lymphoma (cHL) with abundant large, bizarre Hodgkin-like cells. By immunostaining, the malignant cells were immunoreactive for CD45, CD20, CD79a, PAX5, BCL6, MUM1, and p53 and negative for CD15, CD30, latent membrane protein 1 (LMP1), and EBV-encoded RNA (EBER). Flow cytometry demonstrated lambda light chain restricted CD5 and CD10 negative B-cells. Fluorescence in situ hybridization studies (FISH) were negative for cMYC, BCL2, and BCL6 rearrangements but showed deletion of TP53 and monosomy of chromosome 17. Next-generation sequencing studies (NGS) revealed numerous genetic alterations including 6 pathogenic mutations in ASXL1, BCOR, CDKN2A, NF1, and TP53(x2) genes and 30 variants of unknown significance (VOUS) in ABL1, ASXL1, ATM, BCOR, BCORL1, BRNIP3, CDH2, CDKN2A, DNMT3A, ETV6, EZH2, FBXW7, KIT, NF1, RUNX1, SETPB1, SF1, SMC1A, STAG2, TET2, TP53, and U2AF2. 1. Introduction Posttransplant lymphoproliferative disorders (PTLDs) are lymphoid and plasmacytic proliferations that arise in the setting of immunosuppression in a recipient of a solid organ transplant (SOT) or hematopoietic stem cell transplant (HSCT) [1]. PTLDs affect 1–25% of posttransplant patients, with the highest incidents for intestinal and multiorgan transplant, followed by heart and lung transplants [2]. The revised 2016 World Health Organization (WHO) categorizes PTLDs into the following categories: plasmacytic hyperplasia PTLD, infectious mononucleosis PTLD, florid follicular hyperplasia PTLD, polymorphic PTLD, monomorphic PTLD (B- and T-/NK-cell types), and classical Hodgkin (cHL) lymphoma PTLD [3]. The vast majority of PTLDs are of B-cell origin and are usually associated with Epstein-Barr virus (EBV) infection; however a significant subset are EBV-negative [1, 4, 5]. Early onset PTLDs are typically Epstein-Barr virus(EBV-) driven lymphoproliferations and may be polyclonal or oligoclonal, whereas late onset ones are typically monoclonal lymphoid malignancies that can lack EBV association. The pathogenesis of non-EBV-related PTLD may be similar to non-Hodgkin’s lymphomas (NHL) [6]. EBV-negative PTLD has been proposed to be a distinct entity and typically presents as a late complication of transplantation with a median of 50–60 months [5, 7–10]. EBV-negative PTLDs typically display monomorphic morphology [1]. Here we present a rare case of EBV-negative PTLD occurring more than a decade after solid organ transplant (SOT) and presenting with a large variety of morphologies of the malignant cells and numerous genetic alterations comprising 6 pathogenic mutations (ASXL1, BCOR, CDKN2A, NF1, and TP53x2) and 30 variants of unknown significance (VOUSs). 2 2. Materials and Methods 2.1. Histology and Immunohistochemistry. Formalin-fixed paraffin-embedded (FFPE) tissue sections were stained with hematoxylin and eosin (H&E) according to manufacturer’s instructions. Immunohistochemical staining was performed on 4 𝜇m tissue sections using an Autostainer (Leica BOND platform, Buffalo Grove, IL) according to manufacturer’s instructions. Briefly, sections were deparaffinized in xylene and graded alcohols. Detection of the antibodies was performed using a chromogenic substrate, diaminobenzene (Dako). 2.2. Molecular Analysis for Clonality. DNA was extracted from FFPE small bowel tumor tissue and analyzed for clonality as described previously [11]. Briefly, PCR amplification was performed with two sets of fluorescently labeled primers (InVivoScribe Technologies) that hybridize to a conserved V-framework, framework 2 (FR2), and framework 3 (FR3) regions and the conserved J-region of immunoglobulin heavy chain (IGH) gene. The PCR products were subsequently sizeseparated by capillary electrophoresis on a 3500xL Genetic Analyzer (Life Technologies). Data were analyzed (GeneMapper v5.0 software) and examined for peak patterns consistent with a clonal expansion. 2.3. Fluorescence In Situ Hybridization (FISH) Analysis. FISH was performed on 3 𝜇m FFPE tissue sections using the MYC break-apart probe, BCL6 break-apart probe, BCL2 breakapart probe, and TP53/NF1 probes (all from Metasystems Group, Inc.) according to the manufacturers’ instructions. Briefly, slides were deparaffinized using xylene incubation (×3), followed by ethanol wash steps (100%, 70%). The slides were treated with Dako pretreatment solution (Dako, Inc., K5799) prior to hybridization, followed by digestion with pepsin (37∘ C, 15 min). Slides were then dehydrated in ethanol (70, 85, and 100%) and dried and the FISH probes were added for incubation overnight. The next day, the slides were washed, counterstained with DAPI, manually visualized, and scored. 2.4. Gene Mutation Analysis. Mutational analysis of FFPE tissue samples was performed by the University of Pennsylvania at the Center for Personalized Diagnostics as described previously [11]. The genes sequenced were part of a custom, targeted next-generation sequencing amplicon panel testing for 68 hematologic malignancy-associated genes (ABL1, ASXL1, ATM, BCOR, BCORL1, BIRC3, BRAF, CALR, CBL, CDKN2A, CEBPA, CSF1R, CSF3R, DDX3X, DNMT3A, ETV6, EZH2, FAM5C, FBXW7, FLT3, GATA2, GNAS, HNRNPK, IDH1, IDH2, IL7R, JAK2, KIT, KLHL6, KRAS, MAP2K1, MAPK1, MIR142, MPL, MYC, MYCN, MYD88, NF1, NOTCH1, NOTCH2, NPM1, NRAS, PDGFRA, PHF6, POT1, PRPF40B, PTEN, PTPN11, RAD21, RIT1, RUNX1, SETBP1, SF1, SF3A1, SF3B1, SMC1A, SRSF2, STAG2, TBL1XR1, TET2, TP53, TPMT, U2AF1, U2AF2, WT1, XPO1, ZMYM3, and ZRSR2) (TruSeq Custom Amplicon, Illumina Inc.) based on previously described analyses [12, 13]. A custom bioinformatics pi (...truncated)