Abstract
Loss of expression of the MHCII in a subset of DLBCL cases studied by the Leukemia and Lymphoma Molecular Profiling Project (LLMPP) has been previously associated with extremely poor prognosis. Large genetic deletions of the MHC II loci were not seen in these cases. Furthermore, gene expression profiling analysis demonstrated that the MHCII gene expression was coordinated and most likely suppressed through altered transcription. We therefore investigated the possibility of small deletions or genetic mutations of 2 key transcriptional regulators of MHCII, CIITA and RFX, as possible causes of decreased MHCII expression in DLBCL. These transcription factors were chosen because mutations in the coding region of these proteins have been shown to cause the rare genetic disease, bare lymphocyte syndrome, in which MHCII expression is lost. We designed primers to amplify all coding exons of CIITA and RFX (a multimer containing RFX5, RFXB, and RFXAP), including internal splicing regions, in a minimal number of amplifications. DNA samples were amplified by 6 multiplex PCRs of genomic DNA, then the products sequenced with separate sequencing primers and compared to NCBI curated sequences. DLBCL DNA samples for which gene expression profiling data on MHCII expression was available, were obtained from the LLMPP research group. 23 of these samples were from the lowest 10% de novo untreated average MHCII expressers, 4 from non-de novo samples expressing MHCII in the same range, 4 from low MHCII expressers in the lowest 10–25% range, and 15 were primary mediastinal B cell lymphoma (PMBL) samples. The PMBL subset of DLBCL expresses MHCII at a lower range than other DLBCL. A number of other MHCII positive and negative lymphoma samples and cell lines were also sequenced. Although various SNPs and silent changes were noted, there were few small point mutations, deletions, or splicing mutations in the low MHCII DLBCL expressers that would explain loss of MHCII expression. In RJ2.2.5, an MHCII negative Burkitts lymphoma cell line derived from Raji, which is known to have only one CIITA allele expressing RNA lacking exons 11 and 12, the genomic deletion was sequenced. One of the lowest 10% MHCII expressing LLMPP samples had an insertional duplication that caused a frameshift in the C-terminus of all copies of the CIITA gene. Another tumor sample showing functional mutations was an MHCII negative T cell lymphoma (non-LLMPP), which had nonsense mutations in both RFXAP and RFX5, all copies. In conclusion, critical deletions or mutations were not common in the studied samples. These results confirmed previous data implying loss of MHCII expression in DLBCL was most likely due to altered transcriptional regulation, and indicate that this unfortunate circumstance may frequently be amenable to therapeutic intervention to upregulate the MHCII pathway.
Gain-of-function IKBKB mutation causes human combined immune deficiency
