David D. W. Twa

The pathogenesis of primary mediastinal large B-cell lymphoma (PMBCL) is incompletely understood. Recently, specific genotypic and phenotypic features have been linked to tumor cell immune escape mechanisms in PMBCL. We studied 571 B-cell lymphomas with a focus on PMBCL. Using fluorescence in situ hybridization here, we report that the programmed death ligand (PDL) locus (9p24.1) is frequently and specifically rearranged in PMBCL (20%) as compared with diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin lymphoma. Rearrangement was significantly correlated with overexpression of PDL transcripts. Utilizing high-throughput sequencing techniques, we characterized novel translocations and chimeric fusion transcripts involving PDLs at base-pair resolution. Our data suggest that recurrent genomic rearrangement events underlie an immune privilege phenotype in a subset of B-cell lymphomas.

Key Points Somatic IL4R mutations were identified in 24% of primary PMBCL cases (n = 62) and in 100% of PMBCL-derived cell lines. IL4R mutations lead to hyperphosphorylation of STAT proteins activating downstream immunoregulatory genes (CD23, CCL17).

Primary testicular diffuse large B cell lymphoma (PTL) is an aggressive malignancy that occurs in the immune-privileged anatomical site of the testis. We have previously shown that structural genomic rearrangements involving the MHC class II transactivator CIITA and programmed death ligands (PDLs) 1 and 2 are frequent across multiple B cell lymphoma entities. Specifically in PTL, we found rearrangements in the PDL locus by fluorescence in situ hybridization (FISH). However, breakpoint anatomy and rearrangement partners were undetermined, while CIITA rearrangements had not been reported previously in PTL. Here, we performed bacterial artificial chromosome capture sequencing on three archival, formalin-fixed, paraffin-embedded tissue biopsies, interrogating 20 known rearrangement hotspots in B cell lymphomas. We report novel CIITA, FOXP1 and PDL rearrangements involving IGHG4, FLJ45248, RFX3, SMARCA2 and SNX29. Moreover, we present immunohistochemistry data supporting the association between PDL rearrangements and increased protein expression. Finally, using FISH, we show that CIITA (8/82; 10%) and FOXP1 (5/74; 7%) rearrangements are recurrent in PTL. In summary, we describe rearrangement frequencies and novel rearrangement partners of the CIITA, FOXP1 and PDL loci at base-pair resolution in a rare, aggressive lymphoma. Our data suggest immune-checkpoint inhibitor therapy as a promising intervention for PTL patients harbouring PDL rearrangements.

Programmed death ligands (PDLs) are immune-regulatory molecules that are frequently affected by chromosomal alterations in B-cell lymphomas. Although PDL copy-number variations are well characterized, a detailed and comprehensive analysis of structural rearrangements (SRs) and associated phenotypic consequences is largely lacking. Here, we used oligonucleotide capture sequencing of 67 formalin-fixed paraffin-embedded tissues derived from primary B-cell lymphomas and 1 cell line to detect and characterize, at base-pair resolution, SRs of the PDL locus (9p24.1; harboring PDL1/CD274 and PDL2/PDCD1LG2). We describe 36 novel PDL SRs, including 17 intrachromosomal events (inversions, duplications, deletions) and 19 translocations involving BZRAP-AS1, CD44, GET4, IL4R, KIAA0226L, MID1, RCC1, PTPN1 and segments of the immunoglobulin loci. Moreover, analysis of the precise chromosomal breakpoints reveals 2 distinct cluster breakpoint regions (CBRs) within either CD274 (CBR1) or PDCD1LG2 (CBR2). To determine the phenotypic consequences of these SRs, we performed immunohistochemistry for CD274 and PDCD1LG2 on primary pretreatment biopsies and found that PDL SRs are significantly associated with PDL protein expression. Finally, stable ectopic expression of wild-type PDCD1LG2 and the PDCD1LG2-IGHV7-81 fusion showed, in coculture, significantly reduced T-cell activation. Taken together, our data demonstrate the complementary utility of fluorescence in situ hybridization and capture sequencing approaches and provide a classification scheme for PDL SRs with implications for future studies using PDL immune-checkpoint inhibitors in B-cell lymphomas.