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R.J. Leeman-Neill et al.
ated PBL have highlighted the prognostic significance of disease stage and Epstein-Barr virus (EBV) status2-4 and genomic analyses have revealed frequent MYC rearrange- ments, heterogeneous chromosome/DNA copy number abnormalities,5-7 variable transcriptional and microRNA pro- files2,8-10 and, recently, recurrent mutations in JAK/STAT, MAPK and NOTCH pathway genes.7,11
PBL occurring after solid organ transplantation (PT-PBL) is an uncommon type of post-transplant lymphoproliferative disorder (PTLD), accounting for 6-7% of PTLD and consti- tuting a minor fraction (5-14%) of all PBL.2-4,12,13 Only limited data regarding the pathological and molecular features of PT-PBL have been reported.2-4,12,14
In order to clarify the pathogenetic bases of PT-PBL, we performed morphological, immunophenotypic and molec- ular analyses, including targeted genomic sequencing, of a series of PT-PBL, comprising de novo PBL, both primary and recurrent tumors, and those preceded by other types of PTLD.
Methods
Case selection
We searched our departmental database for cases of PTLD diag- nosed over the past 18 years (2002-2019) to select those fulfilling morphological and immunophenotypic features of PBL according to the current World Health Organization classification.1 Other types of PTLD preceding PT-PBL were also identified. Clinical and laboratory data were retrieved from electronic health records. This study was performed according to the principles of the Declaration of Helsinki and a protocol approved by the Institutional Review Board of Columbia University.
Morphology and immunohistochemistry
Formalin-fixed, paraffin-embedded (FFPE) tissue sections were stained with hematoxylin and eosin for cytomorphological evalu- ation and semi-quantitative assessment of the percentage of mature plasma cells. Immunohistochemistry/in situ hybridization was performed to analyze expression of B- and plasma-cell anti- gens, the cellular microenvironment, a variety of biomarkers and the EBV status/latency profiles (see Online Supplementary Methods).
Immunoglobulin heavy chain (IGH) gene rearrangement analysis
Polymerase chain reaction analysis for immunoglobulin heavy chain (IGH) gene rearrangement was performed on DNA extracted from fresh or FFPE tissue using the BIOMED-2 primers, as described previously.15
Cytogenetic analysis
G-band karyotyping was performed on metaphase preparations obtained after unstimulated overnight culture. Fluorescence in situ hybridization (FISH) was performed on metaphase spreads or FFPE sections using TP53/CEP 17 and MYC/IGH/CEP8 probes (Abbott Molecular, Des Plaines, IL, USA) using standard methods. Two hundred cells per hybridization were evaluated. For interphase FISH analysis, the cut-off was 1% for IGH/MYC and 4% for TP53/CEP 17 alterations.
Targeted genomic sequencing
DNA was extracted from tumors and matched non-tumor tissue for sequencing a panel of 465 cancer-associated genes, as described previously16 (Online Supplementary Methods). Microsatellite instabil- ity (MSI) was also analyzed (Online Supplementary Methods).
Results
Clinical characteristics
We analyzed 18 samples from 11 patients (8 males, 3 females; median age 61 years; range, 12-76 years) with PT- PBL, accounting for 11/177 (6%) of ‘destructive’B-cell PTLD and 11/98 (11%) of monomorphic B-cell PTLD diagnosed at our institution during the study period. PT-PBL occurred in recipients of heart (4/11, 36%), kidney (3/11, 27%), lung (3/11, 27%) and combined liver/kidney (1/11, 9%) allo- grafts at a median of 9.6 years after transplantation (range, 0.6-11.9 years). The intestines were the most common sites of disease (6/11, 55%). Three patients had recurrent PBL and in three patients the PBL was preceded by another type of PTLD. Staging marrow biopsies, performed in seven patients, including three of five with EBV– PBL, showed no evidence of PBL/PTLD. Therapy and outcome data are summarized in Table 1 and details are provided in the Online Supplementary Data. Serum protein electrophoresis revealed low-level monoclonal paraproteins in four of seven (57%) patients with available results; none had lytic bone lesions on imaging. Results of pertinent laboratory tests and imaging studies are listed in Online Supplementary Table S2.
Morphologic and immunophenotypic features
All PT-PBL showed diffuse infiltrates of large immunoblastic or plasmablastic cells (Figure 1). A minor component of small, more mature plasma cells (plasmacytic differentiation), comprising 10-20% of the neoplastic infil- trate, was seen in five (45%) cases (Figure 1A, Table 2). Some PBL had numerous tingible body macrophages, imparting a ‘starry sky’ appearance (Figure 1B) or multinu- cleated/anaplastic cells (Figure 1C). Foci of necrosis were observed in six of 11 (55%) cases.
Details of the immunophenotypes of all cases are listed in Table 2 and flow cytometry results in Online Supplementary Table S3. Representative cases are illustrated in Figure 2. All PBL expressed MUM1/IRF4, nine of 11 (82%) were CD138+ and subsets showed B-cell antigen, CD10, CD56, PD-1 or PD-L1 expression. IgG, IgA or IgM was expressed by five (45%), two (18%), and two (18%) of the 11 cases. All evaluable PBL were positive for EMA and negative for Cyclin D1, CD117, HHV8 and ALK. Variable CD30 positiv- ity was noted in seven of the 11 (64%) cases. The Ki-67 pro- liferation index ranged from 20 to >90% (median 90%). MYC expression ranged from <10% to 90% (median 45%), with six of ten (60%) cases showing ≥40% MYC expres- sion. Two of the latter cases expressed BCL2 in ≥50% of cells (‘double expressors’). P53 overexpression was observed in five of ten (50%) cases. The immunoprofiles and/or proportions of cells expressing certain antigens dif- fered in some PBL on recurrence. A mild to moderate infil- trate of reactive PD-1+ lymphocytes was observed in all ten cases evaluated and five of ten (50%) had PD-L1+ macrophages admixed.
Three PBL were preceded by other forms of PTLD; nodal EBV+ monomorphic PTLD (plasmacytoma) (case 5), duode- nal EBV+ P-PTLD (case 8), and intestinal EBV– monomorphic PTLD (diffuse large B-cell lymphoma, DLBCL) (case 10).
Epstein-Barr virus status and latency profiles
The neoplastic cells were positive for EBER in six of 11 (55%) PBL, with four of these six (67%) showing latency II and one case each displaying latency 0/I and latency III pro- files. Four EBV+ PBL and two EBV– PBL occurred in patients
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