Inactivating Gene Alterations of MHC Class II Transactivator CIITA Are Recurrent in Primary Mediastinal B Cell Lymphoma and Hodgkin Lymphoma

Escape from immune surveillance has been proposed to play an important role in cancer and in particular in the pathogenesis of a subgroup of lymphomas. Using massively parallel sequencing, we have recently identified recurrent rearrangements of the CIITA gene in classical Hodgkin lymphoma (HL) and primary mediastinal B cell lymphoma (PMBCL) (Steidl et al, Nature 2011). Specifically, a gene fusion in the HL cell line KM-H2, involving the genes CIITA and FLJ27352, suppressed expression of CIITA-regulated MHC class II genes in a dominant-negative manner. Therefore, we sought to further investigate if gene alterations targeting the CIITA locus are recurrent somatic events in HL and PMBCL that lead to loss of gene function and thereby reduce immunogenicity of the malignant cells. PATIENTS AND METHODS: To characterize CIITA gene alterations, we comprehensively studied the cell lines DEV (nodular lymphocyte predominance HL-derived) and KARPAS1106P (PMBCL-derived) by whole-transcriptome paired-end sequencing (RNA-seq) and single nucleotide polymorphism arrays (Affymetrix SNP 6.0). To identify single nucleotide mutations in the CIITA coding sequence we studied additional 7 mediastinal biopsy specimens of PMBCL by RNA-seq. Fluorescence in-situ hybridization (FISH) was used to determine and validate structural and copy number alterations of the CIITA locus. 23 samples of PMBCL, 3 PMBCL-derived cell lines and 9 HL cell lines were studied for mutations and small deletions in CIITA intron 1 by long-range PCR and direct sequencing. CIITA and HLA-DR expression levels were determined by quantitative reverse transcriptase PCR. RESULTS: We identified chromosomal rearrangements affecting both alleles of CIITA in DEV cells resulting in expression of CIITA-PDL2, CIITA-SOCS1 and SOCS1-CIITA fusion transcripts. In KARPAS1106P cells chromosome 16 deletions result in the loss of one entire CIITA allele and partial loss of the other allele. As a consequence, in both cell lines wildtype CIITA and HLA-DR expression was undetectable. Genomic breakpoints within the CIITA rearrangements, together with previously identified translocations, defined a 1.6Kb breakpoint cluster region in CIITA Intron 1. Further analysis of this breakpoint cluster region revealed small intronic deletions in 10 of 23 (43%) PMBCL cases while no such deletions were detected in 18 diffuse large B cell lymphoma and 15 reactive lymph node samples. Furthermore, sequencing revealed multiple deletions ranging from 1–1948 bp and a high incidence of single nucleotide mutations in the breakpoint cluster region of the deleted alleles. The base pair changes were enriched for C to T and G to A transitions over other transitions and transversions, indicative of a somatic hypermutation process. We found significantly lower CIITA expression in cases with CIITA rearrangement and/or small intronic deletions compared to cases without CIITA alterations (p=0.044). RNAseq revealed three cases with non-synonymous coding-sequence mutations including a truncating mutation in exon 4. These changes were somatic in 3 cases with available matching normal DNA. DISCUSSION:CIITA is the target of multiple genomic hits including chromosomal translocation, deletion and coding sequence mutations in HL and PMBCL. In two HL and PMBCL cell lines this leads to complete gene inactivation and loss of HLA class II expression. These data characterize CIITA alterations as a recurrent underlying genomic event of the previously described immunophenotype of reduced HLA class II expression in a subset of PMBCL cases. Our data show that an immune escape mechanism utilized by tumor cells can be directly linked to somatically acquired alterations in cancer genomes.

Siebert:Abbott/Vysis: .