Of note, in multiple attempts, we were unable to amplify the region with COBRA or bisulfite sequencing primers in the WAC3CD5 cell line, even though they were successful in the other two cell lines and main samples. This study demonstrates that CLL is usually affected by CpG island methylation in some genes that segregate with CD38 expression levels, while most others show comparable methylation patterns across all levels. The CpG island methylation in certain functional gene groups and pathway-associated genes that are known to be deregulated in CLL provides additional insights into the CLL methylome and epigenetic contribution to RCBTB1 cellular dysfunction. It will now be useful to investigate the effectiveness of epigenetic therapeutic reversal of these alterations to develop effective treatments for the disease. mutational status, more recent studies suggest that each parameter is usually independently prognostic, but with considerable overlap [6,7]. Expression of CD38 is usually tightly regulated in normal B-cell ontogeny, with low expression in resting B cells and higher expression in stimulated B cells . Both CD38 and B-cell receptor (BCR) signaling are altered in, and segregate with, clinical subsets of CLL patients, but the reasons for this are unclear. Based on our previous studies suggesting variance of genome methylation in small B-cell lymphomas, including CLL , we hypothesized that these modifications might relate to CD38 expression and the biological behavior of these patient groups, or conversely, methylation may be a functional attribute of the disease in general. We now present data from discovery-based DNA methylation studies of CLL patients with a range of CD38 expression and demonstrate mainly similarities, but a few differences, in the methylation status of specific genes related to CD38 expression levels. The genes affected across all CD38 levels were functionally classified into groups including ion and solute transport, and pathways such as WNT that are known to be deregulated in CLL, thus suggesting an important epigenetic underpinning of cellular dysfunction. Those segregating with CD38 levels will require further research to define their potential role(s) in differential clinical behaviors. Nevertheless, with the ongoing and future clinical trials using epigenetic modifiers, it becomes important to understand the CLL epigenome and how demethylating brokers, histone modifiers and other novel agents impact the underlying biological behavior and clinical outcomes. Patients & methods Samples Blood samples were obtained from patients following diagnostic evaluation, and before any treatment, at the Ellis Fischel Malignancy Center in Columbia (MO, USA), the Holden Malignancy Center in Iowa City (IA, USA) and the Mayo Medical center in Rochester (MN, USA) NK-252 in compliance with local Institutional Review Table requirements. DNA was isolated using the QIAmp DNA Blood Minikit (Qiagen, CA, USA). The samples (n = 38) experienced levels of CD38 expression around the CLL cells varying from 1 to 92% by circulation cytometry , and all contained more than 60% (range 60C96) neoplastic cells as determined by CD19/CD5/CD23 expression (data not shown). The percentage of CD38 expression was adjusted for NK-252 CD19 expression and used as a variable in the clustering analyses. NK-252 Genomic DNA (Promega, WI, USA) was used as an unmethylated normal control. In addition, CD19+ nonmalignant B cells were also used as a normal control, as well as CD19+ B cells (Invitrogen, CA, USA). The source in both cases is usually from peripheral blood, and for the genes tested, there is no difference in methylation. Cell culture & pharmacological treatments Three CLL cell lines with differing levels of CD38 expression by circulation cytometry (not shown) were included: WAC3CD5 (4.7%, CD38), MEC1 (69.5%, CD38) and MEC2 (96.6%, CD38). These were managed in RPMI 1640 media as previously reported . Included in this study were three CLL cell lines with differing levels of CD38 expression: WAC3CD5 (4.73%, CD38), MEC-1 (50.5%, CD38) and MEC-2 (6.6%, CD38). MEC-1 was initially obtained 3 years after diagnosis from peripheral blood lymphocytes (PBLs) of a 58-year-old Caucasian patient with CLL. A year later, a second cell line (MEC-2) was obtained from PBLs of the same patient. Analysis of IgVH showed that these cell lines have not undergone somatic hypermutation, but they differ in expression of CD23 and FMC7. The WAC3CD5 line was induced by cytokines and infected with EpsteinCBarr virus. For gene reactivation experiments, cells were cultured in the presence of a combination of a demethylating agent (5-aza-2-deoxycytidine [5-Aza]) and/or a histone deacetylase (HDAC).