Abstract: Trisomy 3 has been previously reported in association with T-cell lymphomas and less commonly in different types of non-Hodgkin B-cell lymphomas. Trisomy 3 has also been reported in two cases of pediatric post-transplant lymphoproliferative disorder (PTLD). We present comprehensive clinicopathologic review of two pediatric patients with cardiac and liver/intestinal allografts that developed polymorphous PTLD characterized by trisomy 3. Both patients had EBV viremia and EBV was positive in tissue by EBER in situ hybridization. Using karyotype analysis and fluorescence in situ  hybridization, we identified trisomy 3 in both patients. Both patients responded to treatment and are now free of the PTLD. Trisomy 3, an uncommon cytogenetic finding in PTLD, may be a recurrent cytogenetic if confirmed in a larger study of pediatric PTLD’s. Further clinical follow up might help stratify significance of trisomy 3 as a prognostic factor.
1. Introduction
Post-transplant lymphoproliferative disorders (PTLDs) are comprised of lymphoid or plasmacytic proliferations that develop because of immunosuppression following solid organ, bone marrow, or stem cell allotransplant 1 2 . Vast majority of PTLDs are associated with the Epstein-Barr virus (EBV) infection and constitute EBV-driven polyclonal or monoclonal B-cell or T-cell proliferations 3. There are four major subtypes of PTLDs (according to WHO 2016) – non-destructive PTLDs, monomorphic, polymorphic and classic Hodgkin lymphoma. It is important to diagnose cases as PTLD and indicate what type, because of diagnostic and prognostic implications 3. Adult patients with hepatic and cardiac allografts constitute intermediate risk to develop PTLDs (approximately 1-5%), whereas those receiving intestinal allografts have the highest risk (approximately 5%) 4. In pediatric patients incidence is much higher with most cases being EBV-positive polymorphous PTLD that develop during the first year following allograft transplantation 51. PTLDs may involve any site of the body, with lymph nodes, lungs and gastrointestinal tract being the most common sites1. Bone marrow is involved by polymorphous PTLD in about 20 percent of cases 6. Polymorphous PTLDs are composed of a heterogeneous population of lymphocytes, plasma cells and immunoblasts that efface architecture of the lymph node or extranodal tissue. Most cases of polymorphous PTLD contain numerous cells positive for EBV-encoded small RNA (EBER) 7. A microarray study series of thirty five PTLDs reported overall incidence of clonal cytogenetic abnormalities in approximately 50% of cases to include gains of included 8q24, 3q27, 2p24, 5p, 9q, 11, 12q, 14q, 17q, 18q and nonrandom losses of 17p, 1p, 4q and Xp 8. Targeted next generation sequencing studies of 50 B-cell PTLDs revealed high incidence of mutations in Lysine Methyltransferase 2D (KMT2D) and Tumor Protein p53 (TP53) genes 9. 1
Trisomy 3 has been observed in different types of lymphomas, primarily in T cell lymphomas, and less commonly in B cell Non-Hodgkin lymphomas, including extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) and mantle cell lymphoma 10. Recently, trisomy 3 has been reported in the case of an acute megakaryoblastic leukemia 11.  To date there has been one publication describing two cases of pediatric post-transplant lymphoproliferative disorder with trisomy 3 as a primary chromosomal abnormality 12. We present clinicopathologic overview of two cases of polymorphous PTLD and with trisomy 3 in pediatric patients with intestine/liver and cardiac allografts.
2. Materials and Methods
Immunophenotyping, in situ hybridization and clonality studies.  Tissue specimens were processed for histological studies using standard methods. Immunophenotyping was performed using a panel of antibodies including CD3, CD20, PAX-5, CD138, Kappa chain, Lambda chain, CD20. EBV was demonstrated using EBER in-situ hybridization on formalin fixed paraffin-embedded tissues. For the B-cell clonality studies DNA was isolated and subjected to polymerase chain reaction using primers specific to JH immunoglobulin heavy chain, followed by gel electrophoresis and fluorescent detection. For T-cell clonality studies DNA was isolated by routine laboratory procedures and subjected to the polymerase chain reaction using primers specific for the T-cell receptor gamma locus (TRG), followed by capillary electrophoresis and fluorescent scanner detection.  This technique appears to be an accurate means of detecting clonal populations of T-lymphocytes, provided the clone comprises 5% or more of the nucleated cells present in the submitted specimen.
Cytogenetic studies.  The lymph node tissues were minced and squeezed to obtain a single‐cell suspension. The cells were cultured without stimulation for 24 hours in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 15% fetal calf serum. Chromosome preparation and G‐banding analysis were performed following standard procedures. Karyotype and FISH results were described according to the International System for Human Cytogenetic Nomenclature (ISCN 2016)13.
Fluorescence in situ hybridization (FISH).  FISH was performed on the chromosome preparations using directly labeled fluorescence painting probes for the whole chromosome 3 in patient 1, and for the whole chromosomes 3 and X in patient 2. The FISH procedure was performed according to the Vysis protocol. A total of 20 metaphases were examined in each patient.
3. Results: Clinical Data and Histological, Immunophenotypic, Molecular and Cytogenetic studies
Case 1. Patient is a 16-year-old female with a history of congenital jejunoileal atresia and intestinal failure-associated liver disease who required combined orthotopic liver transplantation and intestinal transplantation at the age of 1-year-old. At the age of two patient developed refractory immune-mediated thrombocytopenic purpura, which eventually responded to splenectomy. She had a complex post-transplant course including persistent EBV viremia and a polymorphous PTLD at the age of 3-years-old, when patient presented with diffuse lymphadenopathy. At that time excisional biopsy of the supraclavicular lymph node revealed a total effacement of the nodal architecture by a polymorphous lymphoid infiltrate composed of small and large lymphoid cells, some resembling immunoblasts, as well as numerous plasma cells. Occasional atypical large cells were noted. No necrosis was identified. EBV was positive by EBER in situ hybridization in numerous lymphoid cells including the large cell component.  The findings were consistent with the EBV-positive polymorphic post-transplant lymphoproliferative disorder (PTLD) (Figure 1). 823