Genomic profiling of post-transplant lymphoproliferative disorders using cell-free DNA

Abstract

Diagnosing post-transplant lymphoproliferative disorder (PTLD) is challenging and often requires invasive procedures. Analyses of cell-free DNA (cfDNA) isolated from plasma is minimally invasive and highly effective for genomic profiling of tumors. We studied the feasibility of using cfDNA to profile PTLD and explore its potential to serve as a screening tool. We included seventeen patients with monomorphic PTLD after solid organ transplantation in this multi-center observational cohort study. We used low-coverage whole genome sequencing (lcWGS) to detect copy number variations (CNVs) and targeted next-generation sequencing (NGS) to identify Epstein-Barr virus (EBV) DNA load and somatic single nucleotide variants (SNVs) in cfDNA from plasma. Seven out of seventeen (41%) patients had EBV-positive tumors, and 13/17 (76%) had stage IV disease. Nine out of seventeen (56%) patients showed CNVs in cfDNA, with more CNVs in EBV-negative cases. Recurrent gains were detected for 3q, 11q, and 18q. Recurrent losses were observed at 6q. The fraction of EBV reads in cfDNA from EBV-positive patients was 3-log higher compared to controls and EBV-negative patients. 289 SNVs were identified, with a median of 19 per sample. SNV burden correlated significantly with lactate dehydrogenase levels. Similar SNV burdens were observed in EBV-negative and EBV-positive PTLD. The most commonly mutated genes were TP53 and KMT2D (41%), followed by SPEN, TET2 (35%), and ARID1A, IGLL5, and PIM1 (29%), indicating DNA damage response, epigenetic regulation, and B-cell signaling/NFkB pathways as drivers of PTLD. Overall, CNVs were more prevalent in EBV-negative lymphoma, while no difference was observed in the number of SNVs. Our data indicated the potential of analyzing cfDNA as a tool for PTLD screening and response monitoring.

Keywords: Cell-free DNA; Copy number variation; Epstein–Barr virus; Genomic profiling; Liquid biopsy; Low-coverage whole genome sequencing; Next generation sequencing; Post-transplant lymphoproliferative disorder; Single nucleotide variants.

Fig. 1

Copy number aberrations found in cell-free DNA from PTLD patients. A Overview of copy number variations (CNVs) for individual PTLD patients, grouped by Epstein–Barr virus (EBV) status and sorted on fraction of genome altered (FGA). Chromosomal regions with gains are indicated in red and losses in blue. B A significant correlation between estimated tumor fraction (ETF) and lactate dehydrogenase (LDH) was observed. Dashed line represents cut-off value at 248 U/L discriminating clinically elevated LDH from normal LDH value. C Correlation between ETF and metabolic tumor volume (MTV). In panels B and C, the grey areas around the regression lines represent 95% CI and the Spearman coefficient is indicated with ρ. D FGA in PTLD patients categorized by Epstein–Barr virus (EBV) status, as determined by EBER-ISH, shows that EBV-negative patients have a higher FGA, although insignificant according to Wilcoxon Signed Rank test. Only PTLD samples with CNVs (FGA > 0) are shown in B–D

Fig. 2

Single nucleotide variants in genes found in cfDNA of PTLD patients. A Waterfall plot displaying types of mutations in each plasma sample for each gene. Top- and right-side bar plots show total number of mutations found in a sample (tumor mutation burden, TMB) or a gene, respectively. Genes are sorted based on this number. Samples are grouped by Epstein Bar virus (EBV) status. EBV status, Ann-Arbor staging, Estimated tumor fraction (ETF) and fraction of genome altered (FGA) information is shown below. Pathway information per gene is indicated by color, left of the gene names. B A significant correlation between SNV load and lactate dehydrogenase (LDH) was observed. Dashed line represents cut-off value at 248 U/L discriminating clinically elevated LDH from normal LDH value. C Correlation between SNV load and metabolic tumor volume (MTV). In panels B and C, the grey areas around the regression lines represent the 95% CI and the Spearman coefficient is indicated with ρ. D The total number of SNVs per sample is shown, grouped by EBV status. EBV-negative samples show a slightly higher mean, though this difference is insignificant as tested by Wilcoxon Signed Rank test