Structural Variations Involving Programmed Death Ligands in B-Cell and T-Cell Lymphomas

Immune checkpoint blockade using anti-PD-1 or anti-PD-L1 antibodies is a highly promising therapy that can induce a durable anti-tumor response and a long-term remission in many patients with multiple cancer types. In particular, the excellent efficacy of anti-PD-1 antibody has been reported in advanced cases with classical Hodgkin lymphoma (cHL), of which high frequency of genetic lesions involving PD-L1 and/or PD-L2 somatic alterations is a defining feature, suggesting a close link between the relevant genetic lesions and the efficacy of anti-PD-1/PD-L1 therapy. In addition to cHL, several subtypes of B-cell lymphomas are shown to have structural variations (SVs) involving PD-1 ligands, such as gene amplification and chromosomal translocation causing promoter replacement. Moreover, recently we reported unique SVs disrupting the 3′-untranslated region (UTR) of PD-L1 in a diversity of cancers, including adult T-cell leukemia/lymphoma (ATL) and diffuse large B-cell lymphoma (DLBCL). However, the comprehensive landscape of PD-L1 and PD-L2 alterations in non-Hodgkin lymphomas has not been fully elucidated. Therefore, in this study, we interrogated PD-L1 and PD-L2 genetic aberrations and characterized their features in a variety of non-Hodgkin lymphomas.

To do this, lymphoma-derived DNA was captured for the entire region of PD-L1 and PD-L2 genes including their exons, introns, and 3′- and 5′-untranslated regions (UTRs) and subjected to high-throughput DNA sequencing. More than 300 samples from different lymphoma subtypes were analyzed, including DLBCL, follicular lymphoma, mantle cell lymphoma, MALT lymphoma, primary mediastinal B-cell lymphoma, peripheral T-cell lymphoma-not otherwise specified, and cutaneous T-cell lymphoma. We also analyzed publicly available sequencing data as well as our own data for lymphomas, which included Burkitt and angioimmunoblastic T-cell lymphomas as well.

PD-L1/PD-L2-involving SVs were most frequently observed in PMBCL, accounting for 26.3% of the cases, but widely observed in various B- and T-cell lymphomas at varying but generally low frequencies. However, in contrast to PD-L1-involving SVs, which were found in both B- and T-cell lymphomas, PD-L2-involving SVs were exclusively seen in B-cell lymphomas. Depending on samples, different SV types were observed, including deletion, inversion, tandem duplication, and translocation, but most of SVs resulted in a truncation of the 3'-UTR of the PD-L1 or PD-L2 genes. Unlike previous reports, we rarely found those SVs that translocate PD-L1/PD-L2 to an ectopic regulatory element. Of particular interest were those cases in which multiple, independent SVs that converged to PD-L1 and PD-L2, were observed in a single tumor sample, underscoring the importance of PD-L1 and PD-L2 SVs in clonal selection and expansion of these tumors

Given that PD-L1-involving SVs are detected not only in aggressive lymphomas but also in a variety of solid cancers, we hypothesized that PD-L2 genetic alterations are also present in other human cancers. However, no PD-L2-involving SVs were identified among > 10,000 cancer samples from 32 tumor panels, for which RNA sequencing data were available from the Cancer Genome Atlas (TCGA). These results suggest that PD-L1 is affected in a broad spectrum of human malignancies, whereas PD-L2 SVs are a characteristic alteration of B-cell lymphomas, which is consistent with their expression patterns.

Based on these findings, we assessed whether disruption of PD-L2 3'-UTR also induces PD-L2 overexpression as seen for that of PD-L1 3'-UTR. When introduced in T2 human B and T lymphoblast hybrid cell line using the CRISPR/Cas9 system, SVs involving an almost entire PD-L2 3'-UTR sequence actually induced a significant elevation of PD-L2 expression, confirming the relevance of 3'-UTR in the regulation of PD-L2 expression.

Taken together, our findings clarified the entire picture of PD-L1/PD-L2-involving SVs ligands in B- and T-cell lymphomas. Detection of these SVs might help the identification of patients with non-Hodgkin lymphomas who potentially benefit from PD-1/PD-L1 blockade therapy.