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

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. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-023-02613-w.


Introduction
Primary central nervous system lymphoma (PCNSL) represents a rare extranodal variant of non-Hodgkin lymphoma (NHL) with an incidence of 0.45/100,000 [49]. Per definition, the disease is restricted to the central nervous system (CNS) at diagnosis including the brain parenchyma, spinal cord, meninges and eyes [31,57].
Primary (e.g., genetic syndromes) or secondary (e.g., administration of immunosuppressants, human immunodeficiency virus (HIV) infection) immunodeficiency increases the risk of CNS lymphomagenesis and accounts for 5-10% of cases. These immunodeficiency-associated PCNSL constitute a distinct clinicopathological entity, which was recognized in the 2021 World Health Organization classification of CNS tumors [39]. Unlike disease in immunocompetent hosts, cases are typically (85-95%) Epstein-Barr virus-positive (EBV + ), suggesting an EBV-related pathogenesis, and remain associated with an inferior outcome [9,15,26,33,47]. This may reflect either the inability to tolerate high-dose methotrexate-based polychemotherapy and/or EBV-driven Leon D. Kaulen and Evgeniya Denisova have contributed equally to this article.
Extended author information available on the last page of the article 1 3 oncogenesis with peculiar molecular properties. A better pathogenetic understanding of EBV + PCNSL is hence necessary to pave the way for novel targeted therapies.
In recent years sequencing studies have helped to uncover the genetic landscape of immunocompetent, EBV − PCNSL [7,11,20,30,32,50,67]. Tumors predominantly harbor alterations in the B-cell receptor (BCR), toll-like receptor (TLR), and nuclear factor kappa B (NF-kB) signaling pathways. This includes PIM1, MYD88 L265P and CD79B Y196 hotspot mutations detected in 50-100%, 50-85% and 36-63% of cases, respectively. Genetics and tumor microenvironment of 44 EBV + PCNSL were recently assessed using targeted sequencing and digital multiplex gene expression [21]. Tumors lacked hotspot mutations typically found in EBV − disease and were characterized by elevated macrophage and immune-checkpoint gene expression in HIVnegative and low CD4 gene counts in HIV-infected individuals. While this suggested a distinct molecular landscape, alterations that characterize EBV-related CNS lymphomagenesis remained unclear precluding further molecular classification and development of targeted therapies.
In this study, a comprehensive genetic analysis of 22 EBV + PCNSL was therefore performed, which integrated clinical and pathological information with whole exome (WES) and RNA sequencing (RNASeq) data. This allowed us to uncover the protein-coding genetic landscape of EBV + PCNSL and led to the identification of alterations, that may guide targeted treatment approaches of this rare entity.

Cohort and sample collection
Retrospective case review identified 19 treatment-naïve immunocompromised adult EBV + PCNSL, collected at the Department of Neuropathology of University Hospital Heidelberg, with sufficient tissue for molecular characterization. Steroids were avoided in the two weeks before surgical diagnosis. Diagnosis was re-confirmed by a board-certified neuropathologist in all cases prior to study entry. Per histomorphological assessment, the tumor cell content exceeded 60% in all samples. Slit lamp examination and lumbar punctures were negative for ocular and cerebrospinal fluid involvement at diagnosis. Systemic disease was ruled out using fluorodeoxyglucose positron emission tomography or whole-body computed tomography as well as bone marrow biopsy. Retrospective chart review provided additional clinical and radiological data.
The discovery WES cohort included five fresh-frozen (FF) EBV + samples with matching blood (germline) controls. Given the rarity of the disease, this cohort was supplemented with analyzed genetic data from three previously reported EBV + cases that had matching blood controls (YNHH-6, LS-GD-0111, LS-GD-0106) [21,32]. The in-house WES extension cohort consisted of FF or formalin-fixed paraffin-embedded (FFPE) tissue from 11/14 EBV + samples, where matching blood controls were unavailable. Additionally, WES was carried out on two random EBV − PCNSL to confirm our pipeline detected previously established alterations. RNA sequencing (RNASeq) was performed on 19/19 EBV + specimen from our institution, including all 16 in-house cases that also underwent WES.

EBV status assessment
EBV status was evaluated with immunohistochemistry (EBV-LMP1) and Epstein-Barr encoding region (EBER) in-situ hybridization following previously described protocols [16,21]. Following previous studies, only cases where EBV was detected in more than half of the tumor cells were included in this study.

Whole exome sequencing and processing
Exome capture was performed with the Agilent SureSelect All Exon V7 kit (Agilent Technologies, Santa Clara, USA). The libraries were paired-end sequenced on an Illumina NovaSeq 6000 or NextSeq 500 (Illumina, San Diego, USA).

Alignment of reads
Mapping and preprocessing were performed in the DKFZ OTP (One Touch Pipeline) system using DKFZ Alignmen-tAndQCWorkflow version 1.2.73-202 (https:// github. com/ DKFZ-ODCF/ Align mentA ndQCW orkfl ows). Briefly, reads were mapped to the human reference genome build hs37d5 (phase II reference of the 1000 Genomes Project including decoy sequences) using the Burrows-Wheeler Aligner (BWA) version 0.7.15 mem function with all default parameters, except minimum base quality threshold which was set to zero (−T 0) [5,35]. BAM files were sorted using SAMtools version 0.1.19, and PCR duplicates were marked using Sambamba version 0.6.5 [36,63].

SNVs
Single nucleotide variants (SNVs) were detected using an in-house workflow (https:// github. com/ DKFZ-ODCF/ SNVCa lling Workfl ow) based on SAMtools/BCFtools with parameter adjustments to allow for somatic variant calling and heuristic filtering as previously described in the ICGC Pan-Cancer Analysis of Whole Genome project [65]. For paired samples, SNV calling was performed in OTP using SNVCallingWorkflow version 1.2.166-3 based on SAMtools/BCFtools version 0.1.19. For tumor samples without matched controls, SNVCallingWorkflow version 2.1.1-0 based on SAMtools/BCFtools version 1.9 was run in a nocontrol mode. No-control mode strategy included additional filters which removed common SNPs and recurrent artifacts using variant frequency information from public and local control sample pools. The local control pool contained variant frequency from 4879 whole genome (WGS) and 1198 whole exome (WES) samples analyzed with the same workflows. Variants with minor allele frequency (MAF) above 0.01 in 1000 Genomes or 0.001 in gnomAD (WGS or WES) or with a frequency above 0.01 in the local control pool (WGS or WES) were annotated as common SNPs or artifacts and removed from the downstream analysis [5]. To remove artifacts a 'confidence score' was calculated for each mutation. This score was initially set to 10 and subsequently reduced if the mutation overlapped with genomic regions that are known to be prone to artifacts. Those regions have been identified using the following UCSC genome browser tracks (http:// genome. ucsc. edu/ cgi-bin/ hgTab les [52]): GENCODE Mappability track, UCSC27 High Seq Depth track, UCSC Simple-Tandemrepeats, UCSC Repeat-Masker, DUKE-Excluded, DACBlacklist, UCSC Selfchain. High-confidence SNVs were defined as having a score of at least 8. Variants were annotated with GENCODE v19 using ANNOVAR (version 2016Feb01) [18,70]. Only somatic non-silent coding variants (i.e., nonsynonymous, stopgain, stoploss, or splicing in a vicinity of 2 bp of exon boundaries) of high confidence were selected (except for the analysis of mutational signatures, where all high confidence, including non-coding and silent, somatic variants from the capture regions were used).

INDELs
Short insertions/deletions (indels) were identified using inhouse workflow (https:// github. com/ DKFZ-ODCF/ Indel Calli ngWor kflow) based on Platypus (version 0.8.1.1) as previously described [65]. For paired samples, indel calling was performed in OTP using IndelCallingWorkflow version 2.4.1-1. For tumor samples without matched controls, Indel-CallingWorkflow version 3.1.0-0 was run in a no-control mode. As in the SNV workflow, the no-control mode strategy included additional filters based on variant frequency information from public and local control sample pools. A scoring scheme like that applied in the SNV workflow was used. Indels were annotated with GENCODE v19 using ANNOVAR and somatic high-confidence indels (minimum confidence of 8) falling into a coding sequence or splice site were selected for the analysis.

Copy number alterations (CNAs)
Copy number alterations (CNAs) and loss of heterozygosity regions of in-house samples were identified using a workflow based on the software package CNVkit version 2.1.0 with default parameter setting [61]. For tumor samples without matched controls, same-gender control samples from other patients were used as "artificial controls". Tumor cell content and ploidy were estimated using a method adapted from ACEseq (https:// aceseq. readt hedocs. io/ en/ latest/. Segments with a total copy number (TCN) at least 0.7 above the tumor ploidy were defined as gains and segments with a TCN at least 0.7 below it were defined as losses. High-level CNAs were defined as homozygous deletions and amplifications (> 2.5 × the average ploidy). CNA analysis in FFPE samples is challenging and may result in artifacts, particularly with respect to copy number losses. To address this, GISTIC2.0 workflow with default parameters (except for q = 0.1 and log2 ratio amplification/deletion threshold = 0.3/− 0.3) was used to identify regions of significantly recurring gains or losses [44]. Segmented log2 ratios (.cns) were generated by CNVkit and converted to a segmentation.seg file. This file was then used as input for the GISTIC2.0 module.

Viral integration detection
The presence of viral DNA and genomic integration in tumor samples was detected using an in-house pipeline based on VIRUSBreakend [8]. Starting with an aligned BAM file, it identifies viral reads of interest through Kraken2 taxonomic classification of all unaligned or partially aligned sequences using a custom Kraken2 database [73].

Unsupervised clustering analysis
The top 500 highly variable genes were selected for hierarchical clustering analysis. The optimal number of clusters in hierarchical clustering was selected based on the Silhouette method using R package factoextra (v1.0.7). Three clusters were obtained (i.e., EBV_1, EBV_2 and EBV_3).

Differential expression (DE) analysis
Pairwise DE analyses between three clusters (i.e., EBV_1 vs. EBV_2, EBV_2 vs. EBV_3 and EBV_1 vs. EBV_3) were performed in R package DESeq2 (v1.32.0) [40]. Genes with total counts < 10 in all samples were excluded. Genes were normalized with variance stabilizing transformation (vst) method. DE results were shrunk using apeglm. The top 50 DE genes (DEGs) in comparisons with adjusted p value < 0.05 and log2 fold change > 2 (upregulated DEGs) or log2 fold change < 2 (downregulated DEGs) were selected as features for clusters. For example, EBV_1 included the top 50 upregulated DEGs and the top 50 downregulated DEGs in EBV_1 vs. EBV_2 and EBV_1 vs. EBV_3. Expression clusters were compared with the gene read count matrix of healthy brain tissue RNA-Seq data downloaded from the Genotype-Tissue Expression (GTEx) Portal (https:// www. gtexp ortal. org/ home/). The seven samples (Supplementary Table 5) were randomly selected and included various brain areas from both genders.

Gene ontology (GO) enrichment analysis
The enriched GOs for each cluster were obtained using R package clusterProfiler (v4.0.5) with biological process Gos from the org.Hs.eg.db database. The top 10 Gos with q value < 0.05 were visualized in bar plots using ggplot2 (v3.3.5).

Validation of sequencing
Selected variants (SOCS1, NOTCH1, KMT2D, IGH-V169) were validated using Sanger Sequencing of tumor and blood control samples. For validation of copy number variants (CNVs) and RNASeq data we subjected genomic and synthesized complementary DNA to quantitative PCR, respectively. Details are provided in the supplementary.

Ethics
This study was approved by the Heidelberg University Medical Faculty institutional review board (S-128/2022). Written informed consent was obtained from all study participants or their legal guardians. The study was conducted in accordance with the Declaration of Helsinki.

Cohort characteristics
The study cohort included 8 paired and 14 unpaired treatment-naïve immunodeficiency-related EBV + PCNSL. All cases were classified as diffuse large B-cell lymphoma (DLBCL). The cell-of-origin was determined according to the Hans algorithm with most lymphomas attributed to the ABC subtype (n = 20). Clinical/pathological cohort characteristics are summarized in Supplementary Table 1.

Aberrant somatic hypermutation
Genetic landscape of EBV − PCNSL is largely shaped by aberrant somatic hypermutation (aSHM), which is driven by activation-induced cytidine deaminase (AID) enzymemediated cytosine deamination [32,50,67]. It targets the WRCY sequence motif (W = A/T; R = A/G; C = hotspot; Y = C/T) and its reverse complement, RGYW. To assess the relevance of aSHM in EBV + disease, the fraction of SNVs that overlapped with its target motifs was determined in our paired cohort (Fig. 2a) [50,67]. In addition to somatic hypermutation of immunoglobulin genes (Fig. 2b, c), SOCS1 SNVs frequently mapped to aSHM motifs (Fig. 2d) and were found within the first 2000 bases from the transcriptional start site.

Altered genes in EBV + PCNSL
Detected SNVs were filtered for relevance in tumorigenesis (Fig. 3a) using the COSMIC Gene Census database and the network of cancer genes catalogue of known and candidate cancer genes (NCG.v7). Immunoglobulin gene SNVs, which constitute a feature of healthy and neoplastic B-cells resulting from somatic hypermutation, were found in all cases. EBV + PCNSL lacked known MYD88 p.L265P and CD79B p.Y196 hotspot mutations, which were previously described to drive CNS lymphomagenesis in EBV − cases (Fig. 3b) [7,11,20,30,32,50,67]. PIM1 and TBLXR1, two frequently altered genes in EBV − PCNSL such as in our control samples, were altered in a single unpaired case only (HD-272204). Overall, except for the above-mentioned case, EBV + PCNSL harbored few alterations in the BCR and NF-kB signaling pathways.
Heterozygous germline SOCS1 variants were previously linked to severe autoimmunity with aberrant lymphoproliferation and transition to lymphomas in some cases [27]. Blood controls from this study were therefore assessed for additional SOCS1 alterations. Second hits in cases with somatic SNVs were absent. However, a germline SOCS1 variant of unknown significance (p.Q210H, rs11549428) was found in one individual with autoimmune disease-related PCNSL. This variant is found at a population frequency of 0.18% according to the 1000 Genomes Project, lies outside the SH2 domain and could also represent a benign polymorphism.
Somatic variants were detected in additional inhibitory JAK/STAT pathway genes, namely SOCS gene family

NOTCH pathway
NOTCH SNVs occurred in five EBV + PCNSL (26%). NOTCH1 variants were uncovered in three specimens (16%) including one paired case (Fig. 3a). Two of three variants localized to heterodimerization (p.R1598H) and PEST (p.H2500L) domains, respectively, that carry known hotspots for gain-of-function mutations [71]. SNVs in the heterodimerization domain were previously shown to expose the metalloprotease S2 cleavage site resulting in ligand-independent cleavage, and release of the intracellular receptor domain with subsequent pathway activation. Alterations in the PEST domain prevent ubiquitin-mediated proteasomal degradation of the activated receptor. MutationTaster prognosticated pathogenic consequences for detected NOTCH1 SNVs. Additional hits in the NOTCH pathway included NOTCH3 (n = 2, 11%) and NOTCH4 variants (n = 1, 5%). NOTCH3 p.G1422A was found in the proximity of the heterodimerization domain whereas NOTCH4 p.R1924W localized to the PEST domain suggesting gain-of-function. Of note, NOTCH and SOCS1 SNVs were mutually exclusive in EBV + PCNSL.

CNAs
A median of 6.5% (range 2.5-14.7%) of the genome were affected by CNAs (Fig. 4). CDKN2A or HLA loss, characteristic for EBV − PCNSL, were not found EBV + cases. Frequent gains were found on 11q23.3 (FOXR1, SIK3), a site known to be directly targeted for genomic aberrations by EBV protein, EBNA1 [37]. Additionally, gains included 21q22.2 (ERG) and NOTCH1. The latter were also found in samples, which lacked additional NOTCH pathway SNVs, providing an alternative mechanism of pathway activation. Losses were enriched on 5q31, the locus of STING1 critical for sensing viral DNA with subsequent induction of apoptosis [46]. Further losses were identified on 7q32 (IRF5), and 17q11 (NF1).

Expression clusters
Unsupervised hierarchical clustering revealed three expression clusters in EBV + PCNSL (Fig. 5a). As normal brain contamination was encountered in previous studies performing RNA-sequencing on PCNSL specimen [50], DEGs were compared with expression patterns from healthy brain controls deposited in GTEx. DEGs found in cluster 1 largely reflected brain tissue expression patterns. This was further corroborated by enriched gene ontology (GO) terms (Supplementary Fig. 3) and cases were excluded from further analyses. In contrast, clusters two and three shared B-cell related terms including 'regulation of B-cell activation', or 'regulation of immune effector processes' (Fig. 5b). Of note, cases with SOCS1 alterations were found within expression cluster 2, whereas tumors with NOTCH1 SNVs mapped to cluster 3.
Expression groups 2 and 3 shared robust IL1R2 and CD70 expression. Corresponding to strong expression in EBV + PCNSL, analysis of the GTEx portal database revealed CD70 overexpression in EBV-transformed B-lymphocytes (median 340.6 transcripts per million, Supplementary Fig. 4). Significantly DEGs between both clusters included stronger immunoglobulin constant gene and MAPK4 expression in cluster 2 whereas TRAF4 and SMO were among upregulated genes in group 3 ( Supplementary  Fig. 5).

CIBERSORTx analysis suggests a tolerogenic tumor microenvironment
In addition to immune checkpoints (PD-L1/L2, LAG3, TIM-3) strongly expressed IL1R2 and CD70 can inhibit immune responses and create a tolerogenic tumor microenvironment (TME) [41,64]. We found strong expression of CD163 (M2-macrophages) and FOXP3 (T-regulatory cells, Treg; Fig. 5c) across EBV + PCNSL. To further characterize the TME, deconvolution of bulk RNA sequencing data was performed using CIBERSORTx (Fig. 5d) comparing our EBV + dataset with previously published EBV − PCNSL [50]. In agreement with a tolerogenic TME significant enrichment 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 of Tregs, M2-macrophages, monocytes and mast cells were found in EBV + PCNSL.

Discussion
In this study, comprehensive genetic analyses of 22 EBV + PCNSL integrated clinical and pathological data with WES and RNASeq. This provided novel mechanistic insights into EBV-driven CNS lymphomagenesis and revealed alterations, that may serve as diagnostic markers and/or may guide targeted therapy.
Previous work suggested EBV + PCNSL lack hotspot mutations detected in EBV − PCNSL [11,32]. This was further supported by a recent study [21], that characterized 44 EBV + PCNSL FFPE samples using targeted sequencing (54 genes) and a gene expression panel (14 genes). In agreement with our WES findings, SNVs (PIM1, MYD88, CD79B) and CNVs (CDKN2A, HLA loss) characteristic of EBV − disease were absent. Corresponding to fewer alterations within their sequencing panel, WES now revealed a lower TMB in EBV + PCNSL, which parallels other EBV + lymphomas outside the nervous system [22]. However, alterations that drive EBV + PCNSL remained unclear given their study design, and top hits from this WES/RNASeq study ( The genetic landscape of NHL is largely shaped by aSHM driven by AID activity [32,50,67]. As EBV + PCNSL lacked variants in most prominent targets including PIM1, its influence on the genetic landscape was unclear. Although target genes differed, the high overlap of SNVs with aSHM motifs, however, suggested a critical role during EBV-driven CNS lymphomagenesis. This mirrors other EBV-related tumors such as Burkitt lymphoma, where the genetic landscape is largely shaped by AID activity and subsequent aSHM [24]. Underlining its pathogenetic relevance in EBV + PCNSL, like PIM1 in EBV − tumors the most frequently mutated gene in EBV + disease, SOCS1, represents a known target of aSHM and several detected SNVs clustered within its target motifs [45]. The suppressor of the cytokine signaling (SOCS) gene family including its most potent member, SOCS1, provides an inducible negative feedback loop for cytokine-triggered JAK/STAT pathway activation [69]. SOCS1 directly interacts with JAK1 via its SH2 domain, which results in ubiquitination and subsequent proteasomal degradation of JAK1 as well as direct inhibition of Janus kinase activity [38]. Most detected variants in EBV + PCNSL localized to the SH2 domain and bioinformatic scores predicted loss-of-function effects pointing to JAK/STAT pathway disinhibition. SOCS1 mutations were also documented in EBV − PCNSL albeit reported at lower frequencies [30,50]. Whether SOCS alterations are indeed enriched in EBV + disease warrants further investigation given the available case numbers. However, detection in other EBV-related lymphomas (systemic DLBCL including HIV-related disease; Hodgkin lymphoma) supports their relevance during EBV-driven lymphomagenesis [10,22,72]. Of note, JAK/STAT activation is known to drive the expression of EBV oncogenes including LMP1 via direct interaction of STAT molecules with promoter regions in EBV-related tumors [13,14]. Additionally, the pathway up-regulates immune checkpoint PD-L1, strongly expressed in EBV + PCNSL, and may hence contribute to a tolerogenic microenvironment [25]. SOCS1 variant-related disinhibition of the pathway may allow to unleash the viral oncogenic potential. Modeling of similar SOCS1 variants-including SH2 domain alterations-in EBV-transformed B-lymphocytes and HEK293 cells previously revealed excessive JAK/ STAT pathway activation upon cytokine stimulation [27]. This sensitized for treatment with the JAK1/JAK2 inhibitor ruxolitinib, which is approved for the treatment of myeloproliferative neoplasms and was shown to penetrate the blood-brain barrier in mice [28,68]. These findings provide a rationale for preclinical and early clinical assessment of JAK inhibition in EBV + PCNSL.
In addition to JAK/STAT pathway variants, mutually exclusive NOTCH SNVs were detected in EBV + PCNSL. Most alterations localized to mutational hotspots in the heterodimerization and PEST domains, which result in gainof-function in T-cell and chronic lymphocytic leukemia [53,71]. Additional NOTCH1 amplifications provide an alternative mechanism and further support genetic pathway activation in EBV + PCNSL. This matches EBV + systemic lymphomas, where activating NOTCH2 SNVs were detected [22,58], which are rare in virus-negative tumors. NOTCH activation was previously shown to maintain oncogenic viral 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 EBVrelated tumors (Mann-Whitney test; ****p < 0.0001; ***p < 0.001, **p < 0.01, *p < 0.05; ns not significant) ◂ latency transcriptional programs and prevent lytic cycles and subsequent cell death in EBV + tumors, which was reverted with NOTCH inhibitor treatment [23]. Additionally, NOTCH drives immune evasion via induction of PD-L1 and could contribute to a tolerogenic microenvironment in EBV + PCNSL [42]. Various NOTCH inhibitors are under ongoing preclinical/clinical development and evaluation in EBV + PCNSL may be warranted based on alterations identified in this study [4].
Frequent detection of variants in epigenetic regulators was in line with the previous targeted sequencing approach, which identified KMT2D/KMT2C as top hits in EBV + PCNSL although mutational frequencies were lower than in EBV − tumors [21]. However, they were among the few typical variants shared between virus-positive and -negative lymphomas. KMT2D, the most frequently altered epigenetic modifier in EBV + PCNSL, also represented the top hit in a CRISPR-Cas9 screen investigating DLBCL drivers [51]. This may suggest that epigenetic regulators are implicated in CNS lymphomagenesis irrespective of EBV status.
Distinct CNV profiles were identified in EBV + PCNSL, which overall may impair antiviral defense mechanisms and promote B-cell activation. This included 11q23 gains, the locus of SIK3, which protects cells from cytotoxic T-cell responses [59]. Of note, this locus is specifically and directly targeted for genomic aberrations by the EBV protein, EBNA1 [37]. An 11q23 gain/loss pattern was previously found in posttransplant Burkitt-lymphoma-like disease [54], whereas tumors in immunodeficient hosts from this cohort lacked respective deletions. Additionally, loss of STING1 (5q31.2) may hamper cytoplasmic viral DNA sensing and disinhibit BCR signaling [46,62,74]. 7q32 deletions cover IRF5, a known tumor suppressor in B-cell lymphoma, while individuals with NF1 loss are at increased risk for NHL [6,60].
Transcriptional profiling revealed two distinct clusters in EBV + PCNSL, that separated cases with SOCS1 and NOTCH1 SNVs. In addition to previously reported robust PD-L1 expression [11,21], both expression groups shared strong IL1R2 and CD70 expression, which may shape a tolerogenic TME. IL1R2 constitutes a decoy interleukin receptor, that abrogates interleukin-1 signaling and is known for strong expression in serum and tumor tissue in EBVrelated Hodgkin lymphoma [41]. CD70 belongs to the TNF receptor superfamily and is predominantly expressed on activated B-and T-cells. Via interaction with its receptor, CD27, it acts as an immune checkpoint that can co-stimulate B-cell activation, but also inhibit immune responses via T-cell exhaustion/apoptosis [17,64]. Overexpression in tumors was linked to more aggressive phenotypes and allowed immune evasion via a tolerogenic TME. Given the absence of expression in most healthy tissue, CD70 is evaluated as a novel immunotherapeutic target and CD70-directed antibodies as well as CAR T-cells showed promising efficacy in preclinical but also early clinical studies [17,56]. This study revealed CD70 as an interesting candidate for targeted therapy in EBV + PCNSL. Further membrane proteins that shared robust expression in EBV + tumor samples and low/absent levels in most healthy tissues included PD-L1, PD-L2, LAG3, TIM3 as well as B-cell marker CD19. As a result, these genes carry the potential for immunotherapeutic approaches, some of which have already been explored in EBV − PCNSL as well as in other malignancies [19,34]. However, immunocompromised patients were excluded from most clinical trials due to concerns for immune-related adverse events (irAE). While a growing body of evidence shows that immunotherapy can be safely administered in many immunodeficient patients and most irAE are manageable, an individual risk-benefit analysis as well as vigilant monitoring in a referral center setting are warranted [1,43,66].
In agreement with the overexpression of immune checkpoint genes, RNA-sequencing suggested a tolerogenic TME including the presence of Tregs (FOXP3) and particularly M2-macrophages (CD163). Similarly, CD163 was previously reported significantly up-regulated in EBV + PCNSL compared to EBV − counterparts [21]. Deconvolution of bulk RNASeq datasets in this study further revealed higher M2-macrophages, Treg, monocyte and mast cell fractions in EBV + PCNSL. This is in agreement with other EBV-related lymphomas (e.g. Hodgkin), where immune evasion via a tolerogenic TME contributes to virus-driven lymphomagenesis [55].
Limitations of this study include the cohort size, which reflects the rarity of EBV + PCNSL and/or the lack of tissue availability. However, sequencing studies in more prevalent EBV − disease enrolled similar or even lower case numbers [7,11,20,32,67]. Germline controls were also not available for the entire cohort, which was addressed with a rigorous filtering approach based on recurrent and previously reported alterations. Supplemented, previously described paired cases were processed using different workflows, which will have introduced batch effects. Yet high similarity to in-house paired samples including detection of recurrent SOCS1 and NOTCH alterations additionally supported our findings. Strengths of this study include the integrated WES/RNASeq approach, which led to the identification of novel variants and markers in EBV + PCNSL (e.g., SOCS1, NOTCH1, CD70) and suggested a tolerogenic TME. In addition to insights into the pathomechanism of this rare entity this provides the rational for the exploration and development of new targeted therapies. This is particularly important as many EBV + patients are unable to tolerate aggressive methotrexate-based polychemotherapy resulting in an inferior outcome. Previous functional studies together with bioinformatic scores provide a good body 1 3 of evidence for the likely functional relevance and/or targetability of identified alterations. Yet further in vitro and in vivo characterization should be considered in future studies to allow rapid translation of our findings from bench to bedside. Additionally, a future immunogenomics approach (e.g. HLA typing, neoepitope prediction with subsequent experimental validation) could allow us to identify further targets for immunotherapy.

Conclusions
The genetic landscape of EBV + PCNSL is largely shaped by aSHM although tumors lacked variants characteristic of EBV − disease. Lymphomas frequently harbored SNVs in the JAK/STAT and NOTCH signaling pathways while CNVs point to impaired antiviral defense mechanisms. Transcriptional profiling revealed two distinct expression clusters, which shared robust expression of immune checkpoint genes including CD70 and IL1R2. Correspondingly deconvolution of bulk RNASeq data suggested a tolerogenic tumor microenvironment. Findings pave the avenue for the (pre-)clinical evaluation of several targeted therapies including JAK/STAT and NOTCH inhibition as well as CD70-directed treatments.