Genomic Profiling of Two Successive Post-Transplant Lymphoproliferative Disorders with Different Morphology in a Pediatric Lung Transplant Recipient
Authors: Rackova Marketa , Rennerova Andrea , Hubacek Petr , Mejstrikova Ester , Krskova Lenka , Sukova Martina , Kalinova Marketa , Winkowska Lucie , Campr Vit , Dousova Tereza , Sramkova Lucie , Trka Jan , Fronkova Eva 
Published: December 2024

Introduction
Post-transplant lymphoproliferative disorders (PTLD) encompass a broad spectrum of lymphoid proliferations, a consequence of externally induced immunosuppression following solid organ or hematopoietic stem cell transplantation. These disorders range from indolent proliferations to polyclonal polymorphic diseases, but they can also present as aggressive malignant lymphomas that highly resemble those observed in the immunocompetent population. Due to the shared pathogenetic mechanisms, studying PTLDs can provide valuable insights into the pathogenesis of lymphomas in general. One of the most intriguing real-life models of lymphomagenesis represent patients who develop multiple PTLDs with different morphologies, even though such cases are extremely rare.
Aims
In this study, we focused on a case of a lung transplant patient who gradually developed two PTLDs with different morphologies. Our aim was to evaluate the relationship between these two diseases and to elucidate the morphological progression.
Methods
A female EBV seronegative patient with cystic fibrosis underwent a lung transplantation from EBV seropositive donor at 16 years of age. She first developed polymorphic PTLD in the colon 11 months post-transplantation (CD20+CD30-EBV+). She was treated with reduction of immunosuppression and 6 doses of rituximab according to Ped-PTLD-2005-Pilot protocol. The clinical status of the patient improved significantly and a PET-CT scan performed after the 4th dose of rituximab showed marked regression of all the lesions. However, 15 months post-transplantation, she presented with diffuse large B-cell lymphoma (DLBCL) in the duodenum (CD20+CD30+EBER+). The patient was treated with R2 arm of NHL-BFM 2004 protocol and has achieved long-lasting remission of the disease.
DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) samples of polymorphic PTLD and DLBCL using QIAamp DNA FFPE Tissue kit (Qiagen). Immunoglobulin (IG) rearrangements were analyzed by Biomed-2 IGH FR3 Multiplex PCR. Allele-specific oligonucleotide quantitative PCR was designed based on monoclonal IGH sequence from DLBCL to backtrack the clone in PTLD and in peripheral blood (PB) samples with sensitivity of 1E^-04.
Whole exome sequencing (WES) was performed on DNA from FFPE PTLD biopsy samples and pre-transplant PB using the SureSelect XT HS2 Human All Exon V8+UTRs library preparation kit (Agilent Technologies) and NextSeq 500 instrument (Illumina). Bioinformatic analysis was performed using Varsome Clinical. Variants not present in germline with sufficient coverage and sequencing quality predicted as pathogenic or of uncertain significance were filtered out.
Results
An identical monoclonal IGHV4-39-IGHJ4 rearrangement was detected in biopsy samples of both PTLDs. This rearrangement was also detected in PB two weeks prior to the DLBCL diagnosis. PB samples (n=10) taken at previous time points before and after transplantation were all negative.
WES identified 103 somatic variants in total: 32 were present only in polymorphic PTLD, 60 only in DLBCL, and 11 were shared between the two diseases. Of the 26 variants with the highest predicted pathogenicity, only 5 were present in polymorphic PTLD and 25 in DLBCL. Notably, variants in the FAS, KMT2C, and LRP1B genes were detected in DLBCL but not in polymorphic PTLD. Variants in these genes have been previously described in pediatric PTLD-DLBCL (Salmerón-Villalobos et al., Blood, 2024) and in adult refractory DLBCL and transformed follicular lymphoma (Morin et al., Clinical Cancer Research, 2016), often occurring concurrently, suggesting synergistic effect.
Conclusions
The polymorphic PTLD and DLBCL developed from the same B-cell clone with an identical IGH rearrangement. The progression to DLBCL was most likely driven by the selection of a clone with newly acquired somatic variants in the FAS, KMT2C, and LRP1B genes. To our knowledge, only a few cases of multiple PTLDs with different morphologies in a single patient have been described in the literature, and none have been evaluated to this extent at the genomic level. This case highlights that PTLDs can provide valuable insights into the pathogenesis of NHLs in general.
Supported by the European Union - Next Generation EU - program No. LX22NPO5102, Charles University Research Centre program No. UNCE/24/MED/003 and MH CZ - DRO, Motol University Hospital, Prague, Czech Republic 00064203