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