Comprehensive Molecular Characterization of Monomorphic Post-Transplant Lymphoproliferative Disorder (PTLD)
Authors: Koff Jean L. , Balakrishnan Kumudha , Churnetski Michael , Lee Michelle J. , Kurilovich Anna , Paponova Svetlana , Nesmelov Alexander , Zornikova Ksenia , Zemskiy Pavel , Bagaev Alexander , Kotlov Nikita , Flowers Christopher R. 
Published: December 2024

Introduction. Post-transplant lymphoproliferative disorder (PTLD) is a rare life-threatening complication that occurs in the setting of iatrogenic immunosuppression after solid organ or hematopoietic stem cell transplantation, often with associated Epstein-Barr virus (EBV) infection/reactivation. EBV+ PTLD typically occurs earlier in the post-transplant period than EBV-cases, which more often present as monomorphic PTLD that morphologically resembles de novo lymphomas. Histologically heterogeneous, the most common subtype of monomorphic PTLD is diffuse large B-cell lymphoma (DLBCL), but comprehensive characterization of molecular changes and lymphoma microenvironment (LME) in PTLD-DLBCL lags far behind that of immunocompetent (IC) DLBCL. To help bridge this knowledge gap, we assessed genetic abnormalities and LME in a cohort of PTLD-DLBCLs with detailed clinical data available, with comparisons between EBV- and EBV+ cases, as well as between PTLD- and IC-DLBCL.
Methods. Whole-exome (WES) and RNA sequencing analysis (RNA-seq) were performed in formalin-fixed, paraffin-embedded tissues from 13 patients with PTLD-DLBCL; all biopsies occurred prior to treatment initiation. Based on WES, genomic rearrangements were detected using a customized GRIDSS-based pipeline (Cameron et al., Genome Biol., 2021). Somatic mutations were calculated using a supervised machine learning approach. LME types were estimated as described in Kotlov et al. (Cancer Discov, 2021). Cell deconvolution using the Kassandra algorithm (Zaitsev et al., 2022) predicted cell percentages in the LME from bulk RNA-seq data. Cell-of-origin (COO) and double-hit signature (DHITsig) (Ennishi et al., J Clin Oncol., 2019 ) status was estimated as previously described (Kotlov et al., Cancer Discov., 2021) to determine DLBCL subgroups. EBV positivity was derived either from clinical testing (i.e., EBER in situ hybridization in tumor biopsy) or by evidence of viral transcripts detected by our next-generation sequencing. IC-DLBCL (n = 58) were previously collected, sequenced and analyzed in the same way by BostonGene.
Results. A cohort of 13 patients who developed PTLD in 5-238 months (median = 63) after solid organ transplantation was analyzed. WES data of tumor and matched-normal were available for 8 patients, and WES of tumor-only for 2 patients. RNA-seq analysis (RNA-seq) was performed for 13 patients (6 EBV+ and 7 EBV-). Compared to patients with EBV- PTLD, EBV+ PTLD patients demonstrated shorter median PTLD-free survival (24 vs. 86 months, P=0.2), and shorter median overall survival (51 vs.75 months, P=0.2).
The most commonly mutated genes in the PTLD cohort included KMT2D (n = 7), CSMD3 (n = 6), and MYC (n = 3). In 2 cases, translocations involving BCL2 (n = 1) and MYC (n = 1) were identified, but no DHITsig+ cases were detected. In terms of COO subtype, PTLD was classified as activated B cell-like in the majority of cases (n = 7), with germinal center B cell-like subtype in n = 3.
Based on cell deconvolution from RNA-seq, EBV+ cases had a higher percentage of CD8+ cells and M2 macrophages (FDR-adjusted P=0.037, Mann-Whitney U test) compared to EBV- cases. EBV+ PTLDs also tended to have a higher fraction of monocytes (raw P=0.019, Mann-Whitney U test) and higher values of gene signatures associated with major histocompatibility complex class I (MHCI, raw P=0.019), natural killer cells (raw P=0.0095) and checkpoint inhibitors (raw P=0.0095). These findings suggest that pro-tumor components are relatively more prevalent in EBV+ PTLD samples. However, both EBV-negative and -positive samples (six and four patients with RNA-seq data, respectively) exhibited mostly mesenchymal LME (60% in whole cohort, 67% and 50% in EBV- and EBV+, respectively).
While cell composition and cell-specific gene signatures were generally comparable between PTLD and IC-DLBCL samples, plasma B cell fractions tended to be higher in PTLD (raw P=0.0228). Moreover, EBV- PTLD was more similar to IC-DLBCL than EBV+ PTLD in the proportion of macrophages, T cells and expression of checkpoint inhibitors.
Conclusion. We present evidence that LME composition differs by EBV status in DLBCL-PTLD. While EBV- PTLDs largely resembled de novo DLBCL, EBV+ PTLDs were enriched for LME elements associated with a pro-tumor effect. Additional findings linking molecular changes with LME features and clinical outcomes will be presented at the meeting.