BRAF V600E somatic mutation, found in all the classic HCL, does not fully explain all the distinctive morphologic, phenotypic, and immunogenetic features of HCL. Differently from other indolent B-cell neoplasms, HCL has a highly stable genomic profile. HCL is highly sensitive to purine analogs, including cladribine that has demethylating activity (Spungeon et al, 2009). We hypothesized that epigenetic changes might contribute to disease pathogenesis, and we investigated the genome-wide promoter methylation of a well characterized series of classical HCL.

Methods

DNA was extracted from circulating cells including at least 70% of neoplastic cells, of 11 BRAF V600E mutated HCL, 7 splenic marginal zone (SMZL, all negative for BRAF V600E mutation), and 29 chronic lymphocytic leukemia (CLL) and from 6 normal CD19+ B-cell (NBCs) samples. IGHV genes were mutated in all HCL and SMZL, and in 16/29 CLL cases. Samples were analyzed with the Illumina Infinium HumanMethylation27 arrays. Signal intensities and beta values were exported from Illumina Beadstudio 2.0 software. Probes interrogating CpG residues inside CpG islands (CGIs) or outside (non-CGI) were analyzed separately. Unsupervised analysis was performed using hierarchical clustering with Euclidean distance and Ward linkage. Supervised analyses were performed using a t-test on the continuous beta values, followed by the Benjamini-Hochberg multiple test correction. Probes showing q-value<0.2 were considered differentially methylated.

Results

Unsupervised clustering, with probes interrogating CGI or non-CGI CpGs, revealed 2 main clusters: one cluster contained HCL, SMZL and NBCs while the second cluster contained the CLL samples. In particular, methylation status at non-CGI CpG residues were able to reveal HCL as a separate cluster.

Supervised analysis of CGIs residues in HCL vs SMZL revealed 59 hypomethylated and 6 hypermethylated CpGs (including residues mapped in the promoters of RUNX3 and VHL) in HCL. Analysis of non-CGIs revealed 20 differentially methylated CpG residues, with only 3 more methylated in HCL. Supervised analysis of HCL vs CLL revealed a highly different profile at 467 CGI and 896 non-CGI CpGs. Such differences were independent of the IGHV mutation status of CLL. Supervised analysis of HCL vs NBCs showed higher methylation at 43 CGI CpG residues in HCL, including CpGs mapped in the promoter regions of MCM5, BLR1 (coding for CXCR5, expressed at low levels in HCL), and MGMT, and a lower methylation of 51 CpGs. HCL also showed higher methylation than NBCs at 170 non-CGI CpG residues and lower at 195 sites.

To further identify significant differences between HCL and the other entities, we classified CGI CpG residues into unmethylated (beta-values<0.3) or methylated (beta-value >0.3) and compared the frequencies of their methylation status among tumor entities. When HCL was compared to SMZL, 5 CpGs mapped in the promoter regions of VHL, GNAS, RAPGEFL1, LMX1A and TOM1L1 were more frequently methylated and 202 unmethylated. HCL differed from CLL for having 188 CpGs less and 187 more frequently methylated. Among the latter, there were CpGs in the promoters of TNFRSF10A (TRAILR1), VHL, PMAIP1 (NOXA), TP53I3, and BLR1. Finally, when compared with NBCs, HCL samples presented 48 CpGs more commonly methylated, including residues in the promoters of CDKN2B, MGMT, GNAS, LMX1A, and TOM1L1. Ninety-four probes appeared more frequently methylated in NBCs.

Conclusions

Classical HCL had a genome-wide promoter methylation profile distinct from other chronic leukemic disorders. In particular, HCL showed methylation differences from SMZL and NBCs in promoter regions of genes associated with migration/homing (CXCR5), proliferation (CDKN2B, VHL), DNA repair (MGMT), and apoptosis (NOXA, TRAILR1), which might contribute to disease pathogenesis. Our data also have clinical implications since they provide the basis to evaluate regimens including epigenetic drugs. *FB and FF are co-senior Authors.

Disclosures:

No relevant conflicts of interest to declare.

Topics:
genome, hairy-cell leukemia, methylation, splenic marginal zone b-cell lymphoma, o(6)-methylguanine-dna methyltransferase, b-cell neoplasms, chronic b-cell leukemias, chronic lymphocytic leukemia, cladribine, dna

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2013 by The American Society of Hematology
2013
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