Frequent structural variations involving programmed death ligands in Epstein-Barr virus-associated lymphomas

Abstract

Viral infection induces potent cellular immunity and activated intracellular signaling, which may dictate the driver events involved in immune escape and clonal selection of virus-associated cancers, including Epstein-Barr virus (EBV)-positive lymphomas. Here, we thoroughly interrogated PD-L1/PD-L2-involving somatic aberrations in 384 samples from various lymphoma subtypes using high-throughput sequencing, particularly focusing on virus-associated lymphomas. A high frequency of PD-L1/PD-L2-involving genetic aberrations was observed in EBV-positive lymphomas [33 (22%) of 148 cases], including extranodal NK/T-cell lymphoma (ENKTL, 23%), aggressive NK-cell leukemia (57%), systemic EBV-positive T-cell lymphoproliferative disorder (17%) as well as EBV-positive diffuse large B-cell lymphoma (DLBCL, 19%) and peripheral T-cell lymphoma-not otherwise specified (15%). Predominantly causing a truncation of the 3'-untranslated region, these alterations represented the most prevalent somatic lesions in ENKTL. By contrast, the frequency was much lower in EBV-negative lymphomas regardless of histology type [12 (5%) of 236 cases]. Besides PD-L1/PD-L2 alterations, EBV-positive DLBCL exhibited a genetic profile distinct from EBV-negative one, characterized by frequent TET2 and DNMT3A mutations and the paucity of CD79B, MYD88, CDKN2A, and FAS alterations. Our findings illustrate unique genetic features of EBV-associated lymphomas, also suggesting a potential role of detecting PD-L1/PD-L2-involving lesions for these lymphomas to be effectively targeted by immune checkpoint blockade.

Fig. 1

Genetic alterations involving PD-1 ligands in various subtypes of lymphomas. a Frequency of genetic alterations involving PD-L1 and/or PD-L2 in each lymphoma subtype. Type of alterations is indicated by color. Cases harboring both SV and focal CNA affecting PD-L1 and/or PD-L2 are combined into the corresponding SV group. Multiple represents cases harboring both PD-L1 and PD-L2 SVs. DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; MALT, mucosa-associated lymphoid tissue lymphoma; MCL, mantle cell lymphoma; PMBCL, primary mediastinal B-cell lymphoma; PTCL-NOS, peripheral T-cell lymphoma-not otherwise specified; ENKTL, extranodal NK/T-cell lymphoma; ANKL, aggressive NK-cell leukemia; EBV T-LPD, Systemic EBV-positive T-cell lymphoproliferative disorder; Amp, amplification. b, c Different types of SVs affecting PD-L1 (b) and PD-L2 (c) are shown by indicated colors

Fig. 2

CRISPR/Cas9-mediated 3′-UTR disruption induces PD-L2 overexpression. a Proportion of PD-L1 and PD-L2 SVs in B-NHL and T/NK-NHL as well as other tumors. b PD-L1 and PD-L2 mRNA expression in normal human B, CD4⁺ T, CD8⁺ T, and NK cells. Expression microarray data were obtained from HemaExplorer [25]. c Positions of targeting sgRNAs used for CRISPR/Cas9-mediated deletions and inversions of PD-L1 and PD-L2 3′-UTR. d, e Representative plots (d) and frequencies (e) of PD-L1⁺ (top) or PD-L2⁺ (bottom) cells in green fluorescent protein (GFP)⁺ fraction by flow cytometry in HEK293T (e, left) or T2 (d, e, right) cells transfected with Cas9 and no, single, or pairwise sgRNAs targeting PD-L1 or PD-L2 3′-UTR (n = 3 biological replicates). Data represent means ± s.d. *P < 0.05, Welch’s t-test

Fig. 3

ENKTL harbors frequent PD-L1 SVs, leading to its overexpression. a FISH analysis showing PD-L1 break-apart (middle) and cluster formation (right) in ENKTL cells. Design of the break-apart assay using BAC probes recognizing the 5′-part (green) and 3′-part (red) of PD-L1 loci is shown. Amp, amplification. b PD-L1 IHC (top and middle) and EBER-ISH (bottom) of ENKTL cases with or without PD-L1 genetic alterations. Antibodies specifically detecting N-terminal (top) and C-terminal (middle) domains of PD-L1 were used. c Percentage of Ki-67⁺ cells in tumor cell fraction in ENKTL cases with or without PD-L1 genetic alterations. Each dot represents a single case. **P < 0.005, Brunner–Munzel test. A summary of the results is shown in Table 1. d Frequently altered genes identified by whole-exome sequencing for 25 ENKTL cases [28]. Type of somatic alterations is indicated by color. e Hierarchy of somatic mutations and PD-L1 SVs is shown with their allele frequencies in 8 ENKTL cases analyzed by whole-exome sequencing. Driver mutations are shown in green, and PD-L1 SVs are shown in red

Fig. 4

PD-L1/PD-L2 genetic alterations associated with EBV infection. a Proportion of EBV DNA reads to total mapped reads according to lymphoma subtype (offset, 0.0001). Each dot represents a single case. Red line indicates the cut-off value (0.00015%) for EBV positivity. Type of genetic alterations involving PD-1 ligands is indicated by color. Multiple represents cases harboring both PD-L1 and PD-L2 SVs. b–d Frequency of genetic alterations involving PD-L1/PD-L2 according to EBV status in DLBCL (b), PTCL-NOS (c), and stomach adenocarcinoma (d). Fisher’s exact test is used for DLBCL and PTCL-NOS, and Cochran–Armitage trend test is applied to stomach adenocarcinoma. EBV high group corresponds to the EBV-positive group in the TCGA classification of stomach adenocarcinoma [38]

Fig. 5

Genetic differences between EBV-negative and -positive DLBCLs. a Frequency and type of somatic alterations identified by targeted-capture sequencing for lymphoma-associated genes in 48 EBV-negative and 27 EBV-positive DLBCL cases. Type of alterations is indicated by color. BCR, B-cell receptor; GCB, germinal center B-cell. *P < 0.05, **P < 0.01, Fisher’s exact test. b Distribution of somatic mutations encoded in MYD88 (NM_002468), CD79B (NM_000626), TET2 (NM_001127208), and FAS (NM_000043) detected in 48 EBV-negative and 27 EBV-positive DLBCL cases. The protein domains were obtained from the NCBI Gene database (https://www.ncbi.nlm.nih.gov/gene/) and the UniProt database (https://www.uniprot.org/)