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Detection of genomic rearrangements in archival lymphoma tissues using targeted capture sequencing Chong, Lauren Camille


The B-cell lymphomas are a heterogeneous group of disease entities arising from mature B lymphocytes and are characterized by frequent genomic rearrangements. Recurrent rearrangements involving the MHC class II transactivator CIITA and the programmed death 1 ligands PDL1 and PDL2 have been shown to contribute to an immune privilege phenotype in multiple B-cell lymphomas, with implications for novel therapeutic approaches. However, the landscape of fusion partners for these genes has not been well characterized and methods that utilize formalin-fixed paraffin-embedded (FFPE) tumour samples for breakpoint discovery have not been explored. We selected 68 B-cell lymphoma patients with known CIITA and PDL1/2 rearrangement status determined by fluorescence in situ hybridization (FISH) break-apart assays. DNA surrounding the CIITA and PDL1/2 loci was captured from FFPE tumour libraries using a hybridization-based target enrichment assay and sequenced on an Illumina HiSeq 2500. Multiple structural variant (SV) detection tools were used in an ensemble approach to generate SV predictions. We identified 35 novel translocation partners and observed translocation cluster breakpoint regions (CBRs) in CIITA, PDL1, PDL2 and the SOCS1 tumour suppressor gene downstream of CIITA. Recurrent intrachromosomal deletions, inversions and duplications were also identified in each region. Immunohistochemistry (IHC) analysis of PD-L1 and PD-L2 surface expression demonstrated that CBR translocations and a subset of intra-chromosomal rearrangements are significantly associated with increased protein expression of the respective ligand. In conjunction with published reports this suggests that distinct rearrangement types have variable functional consequences. We also report many SVs below the detection resolution of FISH, suggesting the value of a combined approach integrating FISH, capture sequencing and IHC data for characterizing genomic rearrangements in lymphomas. This study confirms the utility of a targeted sequencing approach for detecting structural variation in FFPE lymphoma tissues. Future capture designs interrogating the full set of recurrently rearranged lymphoma genes are being explored with the aim of designing a comprehensive, high-throughput and clinically relevant assay for routine profiling of rearrangement status to guide clinical decision making.

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