Classical Hodgkin lymphoma (cHL) is an atypical germinal center derived B-cell lymphoma (gcdBCL) that has lost its B-cell identity. 1 Some reports have suggested that epigenetic modifications are involved in this cHL-associated B-cell reprogramming. 2, 3 Here, aiming at investigating DNA methylation patterns on a more genome-wide level, we have performed 27K BeadArray analysis4 in five cHL cell lines. Owing to the low numbers of tumor cells in primary cHL biopsies (B1%), our microarray analyses were restricted to cHL cell lines, which represent widely used models to study this lymphoma. The results from cHL cell lines were compared with those of 20 previously analysed gcdBCL diagnosed as Burkitt lymphomas and diffuse large B-cell lymphomas5 and three normal B-cell (NBC) populations6 (for details see Supplementary Information). Results of the BeadArray analysis were exemplary validated by bisulfite pyrosequencing (Supplementary Figure S2). After filtering sex chromosome-specific and low-quality CpGs (outlined in the Supplementary Information), a total of 24312 autosomal CpGs corresponding to 13088 genes entered the statistical analyses. We initially classified these CpGs according to their methylation status in NBC. We identified 16 446 CpGs unmethylated in NBC (mean β-values o0. 25 and all individual β-values below 0.5), 2781 CpGs methylated in NBC (mean β-values> 0.75 and all individual β-values above 0.5) and 5085 CpGs partially methylated in NBC (mean β-values between 0.25 and 0.75). As we aimed at identifying genes clearly hyper-or hypomethylated in lymphomas as compared with NBCs, this third group was not used for further analyses. We then identified CpGs showing differential methylation between B-cell lymphoma, that is, joint cHL and gcdBCL, as compared with NBCs. Applying the criteria detailed in the Supplementary Information, we observed a total of 383 CpGs (329 genes) commonly hypermethylated in cHL, gcdBCL cell lines and primary gcdBCL as compared with NBC (Figure 1a). From now on, this group of genes will be called hypermethylated in cHL/gcdBCL (Hmet-cHL/gcdBCL). These genes were mostly enriched for gene ontology (GO) terms associated with development and morphogenesis (Po0. 001), regulation of the canonical Wnt receptor signaling pathway (Po0. 001) and regulation of adenylate cyclase activity (Po0. 001)(Table 1, Supplementary Figure S5 and Supplementary Table S3). As the main goal of the present study was to characterize the specific changes in the methylome of cHL as compared with gcdBCL, we analyzed cHL vs NBC and gcdBCL, and gcdBCL vs NBC and cHL. By this approach, we identified a total of 247 CpGs (209 genes) that were exclusively hypermethylated in cHL but not in gcdBCL or controls (from now on called Hmet-cHL)(Figure 1b) and 12 CpGs (9 genes) that were hypermethylated only in gcdBCL but not in cHL or controls (Figure 1c). Genes Hmet-cHL were enriched for immune system functions like positive regulation of B-cell activation (Po0. 001) and regulation of T-cell differentiation (Po0. 001)(Table 1, Supplementary Figure S6 and Supplementary Table S4). Also, if the GO analysis is restricted to those genes silenced de novo in cHL (that is, expressed in NBCs and silenced in cHL), the B-cell program was also identified (Supplementary Figure S7 and Supplementary Table S5). These findings indicate that genes Hmet-cHL target the B-cell program.

Lymphoma-associated hypomethylation was a much less common event. Comparing lymphomas (that is, joint cHL and gcdBCL) to NBC, only 10 CpGs (10 genes) were identified as hypomethylated in the …