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Team Tracks Down Epigenomic Heterogeneity in Lymphomas

NEW YORK (GenomeWeb News) – Epigenetic programs tend to become increasingly out of whack in B-cells as lymphoma develops, progresses, and becomes more aggressive, a new study suggests.

As they reported online last night in PLoS Genetics, researchers from the University of Colorado, Weill Cornell Medical College, the Dana-Farber Cancer Institute, and elsewhere used custom arrays and methylation assays to compare the DNA methylation profiles in normal B-cell samples and samples from the two most common subtypes of non-Hodgkin lymphoma.

The analyses uncovered altered cytosine methylation marks in the lymphoma genome. These patterns varied not only within and between the two lymphoma types, but also in ways related to chromosomal and gene contexts. In general, though, the team saw more and more cytosines with intermediate methylation as lymphomas became increasingly advanced and aggressive.

"Epigenetic heterogeneity is initiated in normal germinal center B-cells, increases markedly with disease aggressiveness, and is associated with unfavorable clinical outcome," Weill Cornell Medical College hematologist and oncology researcher Ari Melnick and Dana-Farber biostatistics and computational biology researcher Franziska Michor, the study's co-senior authors, and their colleagues explained.

The team was prompted to look at epigenetic features in lymphomas based, in part, based on the realization that lymphoma subtypes such as follicular lymphoma and diffuse large B-cell lymphoma sometimes show similar genetic glitches despite manifesting themselves differently in the clinic.

Past research published by members of the same group supported the notion that epigenetics could contribute to such differences. In 2010, for instance, researchers detected DNA methylation and gene expression patterns that seem to split lymphomas in the diffuse large B-cell lymphoma, or DLBCL, subtype into additional molecular sub-groups.

Similarly, a study by Melnick, Michor, and colleagues in the journal Blood in 2011 indicated that specific DNA methylation dynamics are at play during the normal development and differentiation of B-cells.

For the current study, researchers delved more deeply into epigenetic profiles in some typical B-cell populations and in samples from the follicular lymphoma and DLBCL subtypes of non-Hodgkin lymphoma.

Both follicular lymphoma and DLBCL arise from mature B-cells, the authors explained. But whereas follicular lymphomas tend to be diagnosed at a fairly early stage, before they've gone on to become more aggressive, DLBCL cases more often involve high-grade tumors with a spectrum of gene expression patterns.

"While it is known that [follicular lymphomas] accumulate new mutations as they progress, the underlying cause of the different phenotype of de novo [follicular lymphoma] and DLBCL, which share many of the same mutant alleles, remains unclear."

By interrogating methylation marks within these lymphoma cell types relative to normal naïve and germinal center B-cells, the team was able at to start unraveling the interplay between methylation at cytosine nucleotides, lymphoma development, and disease features or aggressiveness.

The study relied on analyses with custom Nimblegen arrays as well as an assay called "HpaII tiny fragment enrichment by ligation-mediated PCR," or HELP, that involves chopping up DNA with restriction enzymes targeting bases with different sequence and methylation contexts.

Using such approaches, the team profiled methylation patterns in 18 normal naïve or germinal center B-cell samples, eight follicular lymphoma samples, and 57 DLBCL samples, including 39 samples from a form of BLBCL called germinal center B-like DLBCL and 18 activated B-like DLBCL samples.

Together, results from these and other analyses indicated that lymphomas were more apt to have intermediate levels of methylation within gene promoter regions in the genome, researchers reported. In contrast, the corresponding sites in typical B-cells tended to be predominantly methylated or unmethylated.

The lymphoma-related methylation heterogeneity tended to be more pronounced within the more aggressive DLBCL samples than they were in the follicular lymphomas, the study authors explained, noting that "[e]ach subtype is characterized by a different extent of epigenetic heterogeneity, which likely reflects different mechanisms of lymphomagenesis."

Given the more advanced nature of many DLBCL cases, the investigators speculated that the type of epigenetic variability picked up in those samples could be related to disease aggressiveness and patient outcomes — a prediction supported by their preliminary assessments of patient lymphoma samples.

The group's follow-up experiments suggested that the prevalence of sites with intermediate or aberrant methylation in lymphoma samples varied depending on everything from transcriptional activity in the region to genomic features such as chromosomal locale, gene density, and the like.

Unusual methylation profiles sometimes seems to spread from one gene to the next, too, the team explained, though a transcriptional repressor called CTCF apparently insulate some regions of the genome from one another, curbing the extent to which that occurs.

Based on the patterns found so far, those involved in the study suggested that "focal aberrant hyper- and hypo-methylation via target-specific recruitment of master regulators and non-specific spreading of aberrant methylation drives the generation of epigenetic abnormalities in follicular lymphoma and DLBCL."