Genetic Characterization and Clinical Impact of Loss of MHC Class I and II Expression in De Novo Diffuse Large B-Cell Lymphoma

Introduction: Immune recognition by T cells requires the presentation of processed peptides in association with major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules. Among the tumor immune escape mechanisms described to date, alterations in the expression of MHC molecules play a crucial role in the development of diffuse large B-cell lymphoma (DLBCL). Loss of MHC-I or -II expression in DLBCL can occur through multiple mechanisms, including deletion of the MHC-I and -II locus, loss of function mutations of B2M, and genetic alterations of CIITA ; however, the role of additional genetic and epigenetic mechanisms remains largely unknown. With regard to clinical significance, a number of studies have reported that low MHC-II expression on tumor cells is associated with shorter survival in DLBCL, while the prognostic effect of MHC-I expression has not been systematically evaluated in a large, homogeneously treated cohort. In addition, although the frequency of loss of MHC-II expression differs between ABC- and GCB-DLBCL cell of origin (COO) subtypes, the prognostic impact remains ill-defined. Here, we conducted a comprehensive phenotypic and genetic analysis in a population-based DLBCL cohort to investigate the genetic background and clinical relevance of loss of MHC-I and -II expression.

Method: We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL and uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analysis was performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 57 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of MHC-I and -II was performed on tissue microarrays (n=332). COO was assigned by gene expression (Lymph2Cx assay) in 323 cases (183 GCB, 104 ABC and 36 unclassifiable).

Results: We found the following MHC-I and -II staining patterns:membrane-positive (n=188 (57%) and n=233 (72%), respectively), cytoplasmic only-positive / membrane-negative (n=86 (26%) and 31 (10%), respectively) and negative staining for both membrane and cytoplasmic (n=55 (17%) and n=59 (18%), respectively). Overall, membranous MHC-I and -II expression was not detected in 141/329 (43%) and 90/323 (28%) cases, respectively. While the frequency of loss of MHC-I expression was not different between COO subtypes, loss of MHC-II expression occurred more often in ABC-DLBCL (36% vs 22%, p=0.026). This is consistent with previous observations showing that MHC-II expression is reduced during plasmacytic differentiation and in ABC-DLBCL. Loss of MHC-I expression was associated with B2M mutation and deletion as well as with HLA-C deletions (all p <0.001, Fisher's exact test). Interestingly, mutations of EZH2 and GNA13 were detected more frequently in cases that had loss of MHC-I expression (p <0.001 and p=0.004, respectively). Moreover, mutations of CIITA and RFXAP (components of the MHC-II enhanceosome), and mutations of PRDM1 and TNFAIP3, ABC-DLBCL-specific mutations, were significantly enriched in MHC-II negative cases. Of note, mutations of B2M and EZH2 were significantly associated with loss of MHC-II expression, as well (p=0.001 and p=0.016, respectively). The associations between EZH2 mutation and MHC-I and -II expression were also observed when the analysis was restricted to GCB-DLBCL (both p <0.0001).

With a median follow up of 6.5 years for living patients, the loss of MHC-I expression was not associated with survival in the entire DLBCL cohort, as well as within each COO subtype. Although there was also no significant survival difference according to the MHC-II expression status in the entire DLBCL cohort, loss of MHC-II expression was associated with an inferior 5y-time to progression (TTP, 59% vs 79%, p=0.007) in the GCB subtype. Interestingly, in the ABC subtype, loss of MHC-II expression was associated with a superior 5y-TTP (64% vs 40%, p=0.03).

Conclusion: Our approach using next generation sequencing and high resolution SNP arrays uncovered the genetic background, as well as the clinical impact of loss of MHC-I and-II expression in a uniformly R-CHOP-treated large population-based cohort of DLBCL patients. Our results warrant further studies investigating a potential mechanism of EZH2 mutations regulating MHC-I and -II expression in DLBCL.