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OHSU Team Uses Sequencing to ID Drug Targets, Dx Markers for Two Types of Leukemia

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Researchers at Oregon Health and Science University have uncovered diagnostic markers and druggable driver mutations in chronic neutrophilic leukemia and atypical chronic myeloid leukemia.

The mutations indicated two different drugs that have approval for other leukemias, and already one CNL patient has been treated and showed clinical response to the drug. The researchers are now in the process of designing a clinical trial to test its efficacy in patients with CNL and atypical CML.

In a study published this month in the New England Journal of Medicine, the team sequenced the coding regions of 1,862 genes, including all the kinase genes, phosphatases, non-kinase growth factor or cytokine receptors, and selected adapter genes in 27 patients with either CNL or atypical CML.

They found mutations to the CSF3R gene in 16 of those 27 patients, or 59 percent. Looking across other types of leukemia, they found that the gene was very rarely mutated, indicating it could be a diagnostic marker.

"These two diseases have been challenging for oncologists and pathologists to diagnose because they [are] largely diagnosed on exclusionary criteria," explained Jeffrey Tyner, senior author of the study and assistant professor at the Knight Cancer Institute at OHSU. "Now we have this mutation that is very enriched" and could provide a molecular marker.

Additionally, he said, the mutations are indicative of treatment.

The team used a targeted sequencing strategy, looking at kinase genes and other regions that often contain driver mutations in cancer.

Tyner said that the team chose to use this strategy because at the time it was more feasible from a cost perspective. Additionally, kinase genes are frequently mutated in cancer and are also good candidates for targeted therapies.

Going forward, he said the team would move to more comprehensive sequencing, either exome or whole-genome. However, he said they will likely still take a tiered approach to analysis, prioritizing the kinase genes. "The things that are most interesting, still often fall within that gene set," he said.

Conducting sequencing on the Illumina HiSeq 2000, the researchers found that mutations were significantly enriched in the CSF3R gene. Additionally, they identified two classes of mutations. The most common types of mutations were single base substitutions in the extracellular domain of the gene. The other class was mutations that caused protein truncation.

The team also conducted a series of functional studies testing a panel of small interfering RNAs and small-molecule kinase inhibitors on patients' cells, and candidate oncogenes were validated with in vitro assays.

The functional studies found that the two different types of CSF3R mutations had different drug-sensitivity profiles. The mutations to the extracellular domain of the gene were sensitive to JAK inhibitors, while the truncating mutations were not sensitive to JAK inhibitors, but were sensitive to kinases inhibitors targeting the SRC family and TNK2 genes.

There are currently approved drugs in both classes of inhibitors, including a multikinase inhibitor dasatinib, which is marketed by Bristol-Myers Squibb as Sprycel; and the JAK inhibitor ruxolitinib, which is marketed by Incyte Pharmaceuticals and Novartis as Jafaki and Jakavi, respectively.

The drugs are not currently approved for the treatment of CNL or atypical CML, but they are approved for other related indications. Dasatinib is approved for both chronic phase chronic myeloid leukemia and a rarer subtype known as Philadelphia chromosome positive CP-CML, while ruxolitinib is approved for intermediate or high-risk myelofibrosis.

In the study, one of the patients with a mutation in the extracellular domain of CSF3R was treated with ruxolitinib and showed "marked clinical improvement."

"A lot of drugs out there are being underutilized because we don't have enough information on the genetics of different types of cancer," said Tyner.

The team is now in the process of designing a prospective clinical trial for ruxolitinib to test its efficacy in CNL and atypical CML, which he said would likely start enrolling patients within the next year.

The team is continuing to follow up with the 40 percent of patients who did not have one of the two types of CSF3R mutations.

Julia Maxson, lead author of the study and a postdoc at the Knight Cancer Institute at OHSU, said that the team has already done whole-exome sequencing on a subset of those patients and is currently analyzing the results. They plan to first focus on related pathways to the CSF3R gene.

There's "every reason to believe that these other patients have similar mutations that will activate related pathways or even the same pathway, so could be treated either with the same drugs or similar drugs with slightly different targets," Tyner said.

Tyner said that there were no clinical differences between the group of patients that had a CSF3R mutation and the group that did not.

However, one interesting finding that still needs to be validated because it was not statistically significant is that the CSF3R mutations appeared to be more common in patients with CNL. Close to 90 percent of those patients had the mutation, compared to 60 percent of the entire patient cohort, Tyner said. "It could be a defining mutation in CNL and may be present at a little lower [frequency] in atypical CML," he said, "but we didn't have enough patients to say whether that was a true difference."

Moving forward, Maxson said the group is continuing to do similar studies for other cancer types, including acute myeloid leukemia.

"There's a much larger population of patients," said Tyner, so unlike CNL and atypical CML, he anticipates there will be a much "more diverse spectrum of gene targets."

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