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Study Describes DNA Methylation-based CLL Subtypes

NEW YORK (GenomeWeb News) – A team from Spain and the US has used a combination of whole-genome bisulfite sequencing and array-based analyses to begin characterizing epigenetic patterns in healthy populations of white blood cells known as B cells and in B cells affected by chronic lymphocytic leukemia.

As they reported online yesterday in Nature Genetics, the researchers identified DNA methylation profiles specific to each of the two main CLL subtypes, particularly across gene body and enhancer regions of the genome — patterns that seem to stem from methylation marks in the original B-cell populations that gave rise to the cancers.

The DNA methylation data also led to a previously undescribed clinical subtype of CLL that appears to have intermediate treatment outcomes compared with tumors in the other two methylation-based subgroups, prompting the study's authors to suggest that "differential methylation in the gene body may have functional and clinical implications in leukemogenesis."

The two subtypes that have been traditionally used to classify CLL are based on the presence or absence of excessive mutation to a gene called IGHV, the researchers explained.

CLL tumors that contain hyper-mutated versions of the immunoglobulin heavy chain variable region-coding gene are known as M-CLLs. And survival outcomes for M-CLL cases are generally better than those for patients with the other subtype, called U-CLL, which shows little to no somatic IGHV mutation.

The distinct IGHV mutation profiles within each CLL subtype appears to be related to the stage of B cell differentiation at which cancer develops, senior author José Martín-Subero, from the University of Barcelona, and his colleagues noted, though there is debate over which B cell populations underlie the cancers.

For their part, the team reasoned that DNA methylation profiling might provide new information not only about CLL biology, but also related to the stages of differentiation associated with CLL development in each IGHV mutation-defined subtype.

The researchers started by doing whole-genome bisulfite sequencing of one M-CLL sample and one U-CLL sample at single-base resolution using the Illumina HiSeq 2000. They also did similar sequencing-based DNA methylation analyses on three kinds of mature B cells, all obtained from the same healthy individual.

The three normal B cell types — naïve B cells, class-switched memory B cells, and non-class-switched memory B cells — represent progressive stages of B-cell differentiation. By comparing the methylation profiles within each group of B cells, the researchers were able to get a glimpse at the epigenetic shifts that accompany this process.

In particular, they reported, DNA methylation tends to drop off extensively during the transition from naïve B cells to class-switched memory B cells, whereas relatively few DNA methylation differences turned up between the class-switched memory B cell and the non-class-switched memory B cell samples.

By folding in array-based methylation data on normal B cell samples from healthy donors and on 139 CLL samples, the team delved deeper into the epigenetic profiles that distinguish both the healthy B cell populations and the CLL subtypes.

The bisulfite sequencing and array-based approaches each uncovered differentially methylated regions between the M-CLL and U-CLL subtypes, for instance. Comparisons with the healthy B cell samples indicated that U-CLL samples are generally more similar to B cells from earlier stages of differentiation, such as naïve B cells. On the other had most M-CLL samples looked far more like mature B cells.

Even so, the data revealed methylation shifts between the leukemias and the B cell populations they resembled. Nearly 3.8 million cytosine and guanine nucleotide-rich regions, or CpGs, sported differentially methylated sites in U-CLL tumors compared with naïve B cells, the researchers reported, with many of these sites showing a dip in methylation in the cancer.

A comparison of M-CLL tumors with class-switched memory B cells also found CpGs containing differentially methylated regions, though the extent of the methylation changes in that cancer subtype seemed to be less pronounced.

Additional analyses also uncovered a set of CLLs with DNA methylation profiles falling between these typical U-CLL and M-CLL groups.

Many of the tumors within this third group had been classified as M-CLLs, but showed lower-than-usual levels of IGHV mutation, the team explained, hinting that they had arisen from yet another B cell population.

Such distinctions appear to have prognostic importance, too, the researchers found. For example, cancers with methylation profiles most like mature B cells — including many of the M-CLL samples — had the best treatment outcomes, they noted. Around one-fifth of patients with that form of CLL had to be treated again within a decade of their original treatment.

That figure jumped to 43 percent and 100 percent, respectively, for patients with intermediate methylation profiles and with naïve B cell-like methylation profiles, according to their analysis.

"These data suggest that the putative cell of origin of the CLL identified by the DNA methylation imprint is a relevant factor affecting the clinical outcome of this disease," the study authors argued. "This finding may suggest a new approach to the classification and management of CLL in patients."