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WashU Team IDs Recurrent, Outcome-Related Mutations in AML

By Andrea Anderson

NEW YORK (GenomeWeb News) – The presence or absence of mutations in the DNA methyltransferase gene DNMT3A can help identify a sub-group of acute myeloid leukemia patients prone to poor outcomes, according to a paper appearing online last night in the New England Journal of Medicine.

By sequencing the genome of a relapse tumor from an AML patient whose primary tumor and matched sample had been sequenced previously, Washington University researchers identified a frameshift mutation in DNMT3A — a gene that they subsequently found to be mutated in more than one-fifth of AML tumors tested.

Individuals with DNMT3A mutations typically had worse outcomes and shorter survival times than those who didn't, they found, suggesting DNMT3A status may help sub-stratify AML tumors that would otherwise be grouped together.

"These mutations in DNMT3A seem to be enriched in cytogenetically normal — these sort of intermediate risk — people," senior author Richard Wilson, co-director of the Washington University Genome Center, told GenomeWeb Daily News. "And it allows us, really for the first time, to have a really good marker for whether or not they should be further classified as high risk or low risk."

So far, Wilson and his Washington University team have sequenced matched tumor-normal samples for about 50 individuals with AML.

Whole-genome analyses and other studies of AML indicate that recurrent mutations affecting the isocitrate dehydrogenase genes IDH1 and IDH2 are linked to poor AML outcomes, the authors explained.

But past research also hints at a possible role for DNA methyltransferase enzyme-coding genes such as DNMT1, DNMT3A, and DNMT3B in AML, they added. And compounds that inhibit DNA methyltransferase are sometimes used to treat AML, with varying success.

For the current study, the researchers focused on DNMT3A, a gene that appears to be mutated in 11 of the 38 cytogenetically normal AML genomes they've sequenced — including the genome of a relapse tumor from the same woman whose primary tumor and matched skin sample were sequenced in the first published cancer genome sequencing paper.

"What we're trying to do is to weave together some of these key genes that appear to be mutated in leukemia, along with the epigenetic landscape, and really try to get a handle on how this all fits together in the AML picture," Wilson said.

Using the Illumina Genome Analyzer II, the team generated more than 116 billion bases of paired-end sequence data on DNA from the relapse tumor, covering 99.6 percent of the diploid relapse tumor genome at an average depth of about 30 times.

In the process, they uncovered six mutations not originally detected in the primary tumor, Wilson explained. Of these, three were subsequently identified in the primary tumor sample, while three appear to be relapse tumor-specific, he said. The relapse sample also contained a translocation not present in the primary tumor.

Among the newly identified mutations found in both tumor genomes was a single base, frameshift-causing deletion in DNMT3A.

When the researchers used PCR amplification and Sanger sequencing to assess the exons of DNMT3A and DNMT3L — a gene coding for a protein that heterodimerizes with DNMT3A and DNMT3B gene products — in a panel of AML tumors, they found that 62 of 281 samples contained DNMT3A mutations.

These included 18 missense mutations in DNMT3A, as well as frameshift, nonsense, and splice-site mutations, and a 1.5 million base deletion affecting DNMT3A and eight neighboring genes.

Tumors harboring DNMT3A mutations also appear to be more likely to contain mutations IDH1, FLT3, and NPM1 gene mutations, they noted.

Such changes were particularly common in tumors from AML patients classified as "intermediate-risk" based on their cytogenetic profiles, the team found. Of the 166 individuals in this category, 56 had tumors with DNMT3A mutations.

In contrast, none of the 79 AML patients classified as low risk had mutations in DNMT3A, NPM1, IDH1, or IDH2.

The findings are promising, Wilson explained, because many AML patients have tumors that are cytogenetically normal, making them difficult to classify. "That group contains good outcomes and poor outcomes, but there's no way to differentiate between the two genetically or genomically," he said.

Moreover, the team reported, DNMT3A mutations in the AML tumors tested tended to correspond to AML outcomes. Whereas individuals whose tumors did not contain DNMT3A mutations had a median survival time of almost three-and-a-half years, the DNMT3A mutation group had a median survival time of just over a year.

"This finding strongly suggests that DNMT3A mutations are probably relevant to the pathogenesis of AML," the researchers wrote.

Based on their results so far, the team plans to not only continue investigating DNMT3A in AML, but also other methylation and epigenetic-related genes, Wilson noted. In addition, they are optimistic about the possibility of using DNMT3A mutation status to help classify AML patients and guide treatment.

"This is a new marker that one could use to sort of determine how aggressive treatment might need to be," Wilson said, noting that the results will need to be validated in even larger sets of AML samples.

The new results underscore the value of sequencing cancer genomes to find clinically relevant and biologically informative genetic changes, according to University of California at San Francisco researcher Kevin Shannon and Dana Farber Cancer Institute researcher Scott Armstrong, who co-authored an editorial appearing in the same issue of NEJM.

"These findings have implications for clinical investigators, for researchers working to characterize the molecular mechanisms that contribute to leukemic growth, and more broadly, for illumination mechanisms of epigenetic reprogramming in cancer cells," Shannon and Armstrong wrote.

Still, the pair cautioned that "more research is required before these results can be directly translated into clinical practice."

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