Integrated genetic analyses of immunodeficiency-associated Epstein-Barr virus- (EBV) positive primary CNS lymphomas

Abstract

Immunodeficiency-associated primary CNS lymphoma (PCNSL) represents a distinct clinicopathological entity, which is typically Epstein-Barr virus-positive (EBV⁺) and carries an inferior prognosis. Genetic alterations that characterize EBV-related CNS lymphomagenesis remain unclear precluding molecular classification and targeted therapies. In this study, a comprehensive genetic analysis of 22 EBV⁺ PCNSL, therefore, integrated clinical and pathological information with exome and RNA sequencing (RNASeq) data. EBV⁺ PCNSL with germline controls carried a median of 55 protein-coding single nucleotide variants (SNVs; range 24-217) and 2 insertions/deletions (range 0-22). Genetic landscape was largely shaped by aberrant somatic hypermutation with a median of 41.01% (range 31.79-53.49%) of SNVs mapping to its target motifs. Tumors lacked established SNVs (MYD88, CD79B, PIM1) and copy number variants (CDKN2A, HLA loss) driving EBV^- PCNSL. Instead, EBV⁺ PCNSL were characterized by SOCS1 mutations (26%), predicted to disinhibit JAK/STAT signaling, and mutually exclusive gain-of-function NOTCH pathway SNVs (26%). Copy number gains were enriched on 11q23.3, a locus directly targeted for chromosomal aberrations by EBV, that includes SIK3 known to protect from cytotoxic T-cell responses. Losses covered 5q31.2 (STING), critical for sensing viral DNA, and 17q11 (NF1). Unsupervised clustering of RNASeq data revealed two distinct transcriptional groups, that shared strong expression of CD70 and IL1R2, previously linked to tolerogenic tumor microenvironments. Correspondingly, deconvolution of bulk RNASeq data revealed elevated M2-macrophage, T-regulatory cell, mast cell and monocyte fractions in EBV⁺ PCNSL. In addition to novel insights into the pathobiology of EBV⁺ PCNSL, the data provide the rationale for the exploration of targeted therapies including JAK-, NOTCH- and CD70-directed approaches.

Keywords: Epstein-Barr virus; Exome sequencing; Genetics; Immunodeficiency; Non-Hodgkin lymphoma; Primary CNS lymphoma; RNA sequencing.

Fig. 1

Single nucleotide variant and insertion/deletion counts. a Functional exonic single nucleotide variants (SNVs, blue) and insertions/deletions (INDELs, yellow) counts are displayed. Paired EBV⁺ PCNSL carried a median of 55 protein-coding SNVs (range 24–217) and 2 INDELs (range 0–22). b Tumor mutational burden (TMB: variants/megabase (Mb)) for various malignancies deposited in the cancer genome atlas (TCGA) are shown and contrasted to EBV⁺ PCNSL from this study and EBV⁻ counterparts from the ICGC MMML-Seq consortium. TMB in EBV⁺ PCNSL was lower than in systemic lymphomas (DLBCL) and EBV⁻ PCNSL. c Base substitutions were conserved across cases and indicate a C > T predominance in agreement with the deamination of cytosines. d EBV⁺ PCNSL carried significantly (Mann–Whitney test; ***p < 0.001; **p < 0.01) fewer non-synonymous exonic SNV and INDELs (e) than EBV⁻ PCNSL from the ICGC MMML-Seq consortium

Fig. 2

Aberrant somatic hypermutation EBV⁺ PCNSL. a SNV fractions that overlapped with aberrant somatic hypermutation target motifs WRCY (yellow) and RGYW (blue) were determined (W = A/T; R = A/G; C or G = hotspot; Y = C/T). A median 41.01% of SNVs localized to either motif, suggesting a critical role for aSHM in the pathobiology of EBV⁺ PCNSL. b, c, d Genes with alterations within the first 2000 (K = 1000) bases from the transcriptional start site, that frequently mapped to aSHM motifs, are shown. Positions of detected variants are indicated with symbol shapes denoting relation to target motifs. Targets in EBV⁺ PCNSL included immunoglobulin genes (b, c), but also SOCS1 (d)

Fig. 3

Single nucleotide variants in EBV⁺ PCNSL. a Filtered genes with functional exonic SNVs and INDELs are shown. Colors indicate the type of alteration. SOCS1 was the most frequently (26%, n = 5) altered gene in EBV⁺ PCNSL. Mutually exclusive NOTCH variants were detected in 26% (n = 5) of tumors. b Frequencies of selected alterations are contrasted to EBV⁻ PCNSL (blue) from the ICGC-MMML-Seq dataset. EBV⁺ cases (red) mostly lacked MYD88, CD79B, and PIM1 variants characteristic of virus-negative disease (blue). Frequencies were compared with Fisher Exact tests and significance levels are displayed (****p < 0.0001; ns = not significant). c Normalized single base substitution Alexandrov-COSMIC (AC) mutational signatures in EBV⁺ PCNSL are shown. Colors indicate the signature type and proportions reflect related SNVs counts/sample. Signatures associated with spontaneous deamination of cytosines (AC1), and defective DNA mismatch repair (AC20, AC21) were frequently found. d The SOCS1 gene and its domains are displayed (KIR, kinase inhibitory region; ESS, extended SH2 subdomain; SH2, Src2 homology domain; SOCS box) and detected alterations are indicated. Most (n = 10/13) variants clustered to the SH2 domain, which mediates binding and subsequent inhibition of JAK1. e Crystal structure of the SOCS1 SH2 domain (red) in complex with the JAK1 kinase domain (green) obtained from the RCSB protein data bank (accession number 6C7Y). Positions of altered amino acids (blue) in EBV⁺ PCNSL are indicated within the complex

Fig. 4

Copy number alterations in EBV⁺ PCNSL. Chromoplot summarizes copy number variants (CNVs) of in-house EBV⁺ PCNSL. Chromosomes are displayed on the x-axis. The G-score, which integrates frequency across samples and the magnitude of CNVs, is shown on the y-axis. Gains (red) and losses (blue) are indicated. Lymphomas carried 11q23, a site known to be specifically targeted for genomic aberrations by EBV, and 21q22 gains. Losses covered 5q31, the locus of STING1 necessary for sensing of viral cytoplasmic DNA, 7q32 and 17q11

Fig. 5

Expression clusters and tumor microenvironment in EBV⁺ PCNSL. a Heatmap shows scaled expression levels of feature genes from three EBV⁺ PCNSL expression groups, identified with unsupervised hierarchical clustering analysis, across samples and healthy brain controls from the GTEx database. Cluster 1 (EBV_1) showed high overlap with GTEx controls in line with healthy brain contamination and cases were removed from further analyses. Clusters 2 (EBV_2) and 3 (EBV_3) separated lymphomas with SOCS1 and NOTCH1 SNVs and displayed robust IL1R2 and CD70 expression. b Top 10 enriched gene ontology (GO) terms for feature genes from these clusters are shown. GOs were ordered with rising q values from top to bottom and number (N) of cluster genes overlapping with respective GO terms are indicated along the x-axis. GOs largely reflected B-cell-related terms. c Heatmap indicates robust expression of immune checkpoint genes and markers of a tolerogenic tumor microenvironment (TME) across EBV⁺ PCNSL. d Correspondingly, deconvolution of bulk RNASeq data (CIBERSORTx) from EBV⁺ PCNSL and EBV⁻ cases from the ICGC-MMML-Seq dataset revealed a tolerogenic TME with enrichment of T-regulatory cell, M2-macrophage, monocyte, and mast cell fractions in EBV-related tumors (Mann–Whitney test; ****p < 0.0001; ***p < 0.001, **p < 0.01, *p < 0.05; ns not significant)