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Mutation Order May Impact Clinical Outcome of Myeloproliferative Neoplasms

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NEW YORK (GenomeWeb) – Over the last several years, next-generation sequencing technology has opened windows to the genomic events that drive cancer, indicate responsiveness to targeted drugs, and predict outcomes.

But now, researchers have also found that it is not only the specific mutations, but also the order in which they are acquired, that can impact how cancer behaves.

In a study published today in the New England Journal of Medicine, researchers from the University of Cambridge found that the order in which JAK2 and TET2, two recurrent mutations in myeloproliferative neoplasms, are acquired influences disease progression and drug response.

"It is the first time in any cancer that we've been able to show that order can determine molecular and cellular characteristics of the disease," said David Kent, a co-lead author of the study and a post-doctoral researcher in Anthony Green's laboratory at the University of Cambridge.

Previous research published in the journal Haematologica in 2011 suggested that mutational order may be important, but did not demonstrate actual molecular or functional consequences that happened because of order.

Kent told GenomeWeb that the group decided to look at mutational order when they were discussing new data describing the clonal architecture of TET2 mutations. "We saw a small hint that order mattered," he said, so they reached out to clinical collaborators and designed an experiment to test their hypothesis.

Myeloproliferative neoplasms are a good disease to study because they represent early-stage tumors, Kent said. That allowed the researchers to start tracking the genetic architecture of patients' cells early in the course of the disease.

"We can see which mutation came first and ask questions about whether or not the outcomes are the same," Kent said.

Patients suspected to have a myeloproliferative neoplasm are frequently tested for JAK2 mutations, which helps diagnose some myeloproliferative neoplasms. For instance, it is present in about 95 percent of patients with the blood disorder polycythemia vera, and in about half of patients with essential thrombocythemia and idiopathic myelofibrosis. The mutation can also indicate responsiveness to a JAK2 inhibitor like ruxolitinib, which is approved for intermediate- and high-risk myelofibrosis.

Thus, the researchers first screened patients that had already been tested and were positive for JAK2 mutations. They then sequenced the exons of the TET2 gene in 246 patients positive for a JAK2 mutation, including 92 with essential thrombocythemia, 107 with polycythemia vera, and 47 with myelofibrosis. Twenty-four patients also had a TET2 mutation — seven of the essential thrombocythemia patients, 11 of the polythemia vera patients, and six of the myelofibrosis patients. From those 24 patients with both TET2 and JAK2 mutations, the researchers grew between 20 and 200 colonies per patient and sequenced them to determine clonal composition and mutation order.

The team found clones with JAK2 mutations and TET2 mutations alone in all 24 patients. In addition, they were able to do a repeat analysis of 12 patients at 1 year later and 3.7 years later, and found that the patients' subclone patterns were stable over time.

In 12 patients, TET2 mutations came first. Those patients were, on average, 12.3 years older than the 12 patients that developed the JAK2 mutation first — 71.5 years compared to 59.2 years.

In addition, the researchers found that the patients who acquired a JAK2 mutation first were more likely to develop blood clots, "which is the major thing that kills people" with myeloproliferative neoplasms, Kent said.

The team also performed exome sequencing or targeted sequencing in 23 of the 24 patients to rule out the possibility that other known mutations were contributing to the differences in disease between the two patient groups. Only four patients had known recurrent mutations in myeloid cancers.

"These data show that the effects of mutation order were not confounded by other known oncogenic mutations, but they do not exclude the possibility that additional rare drivers might also influence clinical and pathologic phenotypes," the authors wrote.

The team also studied cell colonies in vitro to determine how mutation order impacted functional differences. They found differences in the types of progenitor cells that were dominant, as well as differences in gene expression.

Specifically, they found that although JAK2 mutant cells typically demonstrate increased proliferation, when the cells acquire a TET2 mutation first, the genes associated with proliferation are no longer upregulated.

Looking more in depth at this mechanism, the team applied the JAK2 inhibitor ruxolitinib to cell colonies. As expected, they found that the drug reduced the number of JAK2 mutant cells when the JAK2 mutation was acquired first. But, interestingly, they also found that the drug had no effect on cell colonies from patients who had first acquired the TET2 mutation.

Kent said that the study helps to make the case for more in-depth genomic profiling of patients. Currently, patients suspected of having a myeloproliferative disorder are routinely screened for JAK2 mutations, which can help in diagnosis, he said. But, TET2 is rarely tested, and this study suggests that valuable information can be gleaned from a more in-depth look. In addition, he said that screening for the mutations and determining their order could be done routinely in diagnostic hematology laboratories.

Charles Swanton, a medical oncologist at the University College London Cancer Institute, also agreed that the study may have important future clinical implications. He wrote in a subsequent editorial published in the same issue of NEJM,that an "understanding of mutation order might improve the delivery of targeted therapy to attenuate early driver events that are present in the trunk of the evolutionary tree of the tumor."

Kent said additional studies would be needed to understand exactly why the mutation order impacts ruxolitinib response, but said that a possible explanation could be related to epigenetics. The TET2 mutations in the myeloproliferative neoplasms tend to be loss-of-function mutations, Kent said. And, TET2 is a known epigenetic regulator responsible for removing methylation groups from DNA, which subsequently enables genes to be transcribed. If a TET2 mutation is acquired first, one possibility is that the JAK2 gene cannot be transcribed, so a JAK2 inhibitor would be ineffective, since the JAK2 gene is not expressed, he said.

He said his group now plans to focus on "uncovering the mechanism of order" and "why order matters." In the case of TET2-first mutations, it "may lay down an epigenetic framework that JAK2 mutations are unable to change," he said.

In addition, he also hopes the work will spearhead research into other cancers. "This is the first hint that order matters, but we don't know if it matters in all cancers," he said.

Swanton added in his editorial that although "deciphering the effect of combinations of mutational events to read the evolutionary rule book of cancers may be a Herculean task," understanding cancer evolution may also "narrow the possible clinical outcomes and open avenues to limit the next evolutionary steps in cancer."

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