Hypermethylation Is A Key Feature of the Transition of Multiple Myeloma to Plasma Cell Leukemia

Abstract 535

PCL is associated with a failure of the plasma cells to retain their normal homing patterns to the bone marrow, is difficult to treat and developing targeted treatments based on its pathogenesis would be a step forward. PCL lies at the end of the multistep pathway from normal to malignant plasma cells and little is known about the genetic mechanisms mediating the final stages of this pathway. The methylation status of genes in myeloma can change as the malignancy progresses and as such identifying genes deregulated by methylation that mediate the progression of MM to PCL may offer epigenetically relevant therapeutic targets. We have previously analyzed 181 samples including B cell, normal plasma cells, MGUS and MM samples for methylation differences using the Illumina Infinium humanmethylation27 array, which interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. Here we have added 32 PCL samples to our dataset in order to further investigate the methylation changes that occur on the transition from MM to PCL. Data are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Data were analyzed in GenomeStudio using the methylation module (Illumina). Further analyses were performed using R and the LIMMA package. Differential methylation between samples was identified using an empirical Bayes moderated t-test and the resulting p-values were adjusted using the Benjamini and Hochberg method. P-values below 0.05 were considered significant. We have shown genome-wide hypomethylation at the transition from MGUS to MM with gene-specific hypermethylation of tumor suppressor genes. The methylation patterns within myeloma samples correlate with translocations or hyperdiploidy, where the latter are split into 2 distinct clusters with a significant difference in overall survival, p=0.03. However, the t(4;14) subgroup had the most distinct methylation profile with hypermethylation of genes specific to the t(4;14) subgroup including CD79A, FAM49A and SOCS2 which correlate with loss of expression in this subgroup. Herein, we analyzed presentation MM (n=161) and PCL (n=32) samples for methylation changes which may contribute to expression and phenotypic changes. When comparing all MM and PCL samples, irrespective of cytogenetic subgroup, we find hypermethylation of the genome in PCL samples. Upon clustering we find that PCL samples segregate within the translocation groups with which they belong i.e. t(11;14) PCL samples cluster with t(11;14) myeloma samples. However, when MM and PCL samples from the same cytogenetic group are compared there is exclusive hypermethylation of genes occurring as the disease progresses. Although t(4;14) myeloma samples have considerable more methylation than t(11;14) samples, the increase in methylation from MM to PCL is consistent in both groups, resulting in t(4;14) PCL samples with increased methylation over t(11;14) PCL samples. The genes which are hypermethylated in the transition of MM to PCL can be classified as either tumor suppressor genes, genes involved in cell-cell signaling, or as cell adhesion molecules. The further analysis of these genes will allow us to identify genes which are down-regulated through methylation and mediate the progression of MM to PCL allowing the clone to become independent of the bone marrow microenvironment. In addition, these analyses will enable us to identify targets which may be sensitive to modulation by epigenetic therapies in vivo. In conclusion, we have demonstrated that methylation changes play a significant part in differentiating the various cytogenetic subgroups of MM and in mediating the transition to PCL. Hypermethylation affects genes and pathways important in retaining plasma cells in the bone marrow as well as in their growth factor independent growth in the absence of stromal cell support.