Key Points• Programmed death ligands 1 and 2 are rearranged at a frequency of 20% in PMBCL.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. (Blood. 2014;123(13):2062-2065 IntroductionPrimary mediastinal large B-cell lymphoma (PMBCL) is an aggressive disease known to share certain genotypic and phenotypic features with classic Hodgkin lymphoma (CHL) and diffuse large B-cell lymphoma (DLBCL).1,2 However, the complete landscape of genetic alterations involved in PMBCL pathogenesis has yet to be fully elucidated.3 Among the most common chromosomal alterations in PMBCL are amplifications of chromosome 9p and translocations involving CIITA (16p13.13).4-7 These aberrations have been suggested to affect tumor-microenvironment interactions resulting in immune privilege. 7,8 Here, we demonstrate that rearrangements involving immune cell anergy-inducing programmed death ligand (PDL) 1 (CD274) and 2 (PDCD1LG2) are recurrent in and characteristic of PMBCL. Furthermore, we show such rearrangements are correlated with elevated transcript levels, and we characterize novel translocations identified using high-throughput sequencing. Study designWe studied 571 primary B-cell lymphoma samples in conjunction with 17 established B-cell-derived cell lines. Using in-house bacterial artificial chromosome probes, fluorescence in situ hybridization (FISH) or FISH combined with CD30 immunofluorescence (in the case of CHL specimens) was performed to characterize the PDL locus.7,9,10 These cases were also analyzed with Epstein-Barr virus (EBV)-encoded RNA in situ hybridization. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for CD274 and PDCD1LG2 transcript expression on a subset of cases from the FISH cohort (N 5 76). Surface PDCD1LG2 expression of the cell lines was determined via flow cytometry. To characterize the Hodgkin lymphoma cell lines found to be rearranged by FISH, 1 whole-transcriptome sequencing (RNA-seq) library (CHL-derived L-428) was reanalyzed, and 1 new wholegenome library (L-428) and 2 new RNA-seq libraries (CHL-derived L-1236 and nodular lymphocyte predominant Hodgkin lymphoma-derived DEV) were sequenced.7 This study was approved by the BC Cancer Agency ...
Introduction Primary mediastinal large B-cell lymphoma (PMBCL) is an aggressive malignancy commonly diagnosed in young adult females. In recent years, mutational and gene expression profiling has established genotypic and phenotypic similarity of PMBCL with both classical Hodgkin and diffuse large B-cell lymphoma (DLBCL). In-depth analyses of genomes and transcriptomes have highlighted several inactivating mutations (SOCS1, TP53), chromosomal amplifications (2p, 9p, Xp, Xq) and translocations (CIITA) thought to be integral in establishing and/or maintaining the PMBCL phenotype. Programmed death ligands (PDL) 1 (CD274) and 2 (PDCD1LG2), which are located on chromosome 9p24.1, are two emerging genes of interest that have been shown to be altered in PMBCL and can induce T-cell anergy by binding to the receptor, programmed death 1. Here, we describe the recurrence of chromosomal rearrangements of the PDL locus in various B-cell lymphomas and explore the association of these rearrangements with transcript levels. Methods To establish the frequency of CD274 and PDCD1LG2 aberration, we conducted fluorescence in situ hybridization (FISH) on 551 clinical samples and 20 established cell lines using in-house break-apart probes. Epstein-Barr virus encoded RNA in situ hybridization was also carried out on the clinical cohort. The clinical cases, sourced from the British Columbia Cancer Agency’s Centre for Lymphoid Cancer tissue repository, consisted of 125 PMBCLs, 216 DLBCLs, 130 primary DLBCL of the central nervous system (PCNSL), 12 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL) and 68 follicular lymphomas (FL) with diagnoses based on the WHO classification. The DLBCL cohort could be further subdivided into 134 nodal DLBCLs and 82 testicular DLBCLs (T-DLBCL). Quantitative real-time PCR (qRT-PCR) was subsequently conducted on 17 cell lines and a clinical sub-cohort of 76 samples, for which fresh-frozen material was available, to determine the effect of mutations on transcript expression. We then characterized the PDL aberrations of two clinical PMBCL cases and three cell lines (DEV, L-428, L-1236), at base pair resolution, by applying the bioinformatic tools, nFuse, deFuse and destruct to both newly produced and previously published whole genome (WGS) and whole transcriptome (RNA-seq) libraries. Results FISH revealed a PDL locus (9p24.1) break-apart frequency of 20% (25/125) in PMBCL. There were no differences in any known clinical parameters or frequency of Epstein-Barr virus positivity between positive and negative PDL break-apart cases. Break-apart frequencies in other malignancies were calculated to be 3% in DLBCL, 7% in T-DLBCL and 1% in PCNSL; no positive cases were identified in either NLPHL or FL. The proportion of break-apart positive cases was significantly higher in PMBCL as compared to the other lymphomas surveyed (P < 0.05). Further, in agreement with the published literature, we observed an amplification frequency of the PDL locus in 36% (45/125) of PMBCLs. qRT-PCR established that PDCD1LG2 transcript levels were significantly higher in cases with 9p24.1 locus rearrangements compared to copy number neutral (P = 0.0003), gain (P = 0.001) and amplified cases (P = 0.005). Likewise, CD274 transcript levels were significantly higher in rearranged cases compared to copy number neutral cases (P = 0.03). Following the analysis of WGS and RNA-seq libraries, we were able to characterize four novel fusion transcripts involving the 9p24.1 locus: PDCD1LG2-NRG1 (PMBCL clinical case), PDCD1LG2-IGHV7-81 (L-1236), CIITA-PDCD1LG2 (DEV) and KIAA1432-CLDN14 (L-428). Aberrations involving both NRG1 and CIITA have previously been implicated in breast cancer and B-cell lymphomas, respectively. We also identified a translocation in another PMBCL clinical case with breakpoints in the intergenic spaces near LRMP and CD274, though this rearrangement did not produce a fusion transcript. Conclusion Taken together, our findings show that rearrangement of the PDL locus is recurrent in PMBCL, characteristic of PMBCL and leads to overexpression of PDL transcripts. Given the well-referenced function of PDLs in repressing the anti-tumor response, these data suggest that targeting the PDL axis in a subgroup of B-cell lymphomas holds clinical promise. Disclosures: No relevant conflicts of interest to declare.
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.
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