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Study Suggests Presence of AML Mutations at Remission Heralds Relapse Risk

NEW YORK (GenomeWeb) – The proportion of bone marrow cells harboring leukemia-related mutations during acute myeloid leukemia remission can provide a window into relapse risk, according to a study published online today in the Journal of the American Medical Association.

Researchers from Washington University, St. Jude Children's Research Hospital, and elsewhere did whole-genome and/or exome sequencing on tumor samples taken at diagnosis from 71 individuals with AML. For 25 of these individuals and another 25 new AML cases, they also compared diagnostic and remission mutation patterns using bone marrow samples collected roughly a month after successful induction chemotherapy treatment.

While mutation patterns in tumor samples at diagnosis did not provide much insight into patient outcomes, the team did see far shorter event-free survival and overall survival times in individuals who still had leukemia-related mutations in at least 5 percent of their bone marrow cells at remission.

"When you look at day 30 [at remission], for the patients where you find a lot of leukemia-associated mutations, they're clearly at an increased risk for relapse," first author Jeffery Klco told GenomeWeb.

"I suspect that, in time, clinicians are going to start looking at this and making preemptive decisions," he noted. "Even if the patient is in morphologic remission, if we're finding a lot of these variants, we know that something bad is eventually going to happen."

Klco was a pathology and immunology researcher at Washington University when the study was done. He is now based at St. Jude Children's Research Hospital.

About half of AML patients who reach remission through induction chemotherapy go on to develop relapsed disease, the researchers noted. Though hematopoietic stem cell transplantation can cure some individuals with AML, its use is currently limited by its potential side effects and high cost, prompting interest in finding ways to target it to AML patients most prone to relapse.

"[H]igh-risk patients need aggressive, potentially curative therapy with a stem-cell transplant when they are in remission, early in the course of the disease," senior author Timothy Ley, an oncology researcher at Washington University, said in a statement.

"However, we don't want to transplant patients who are unlikely to relapse following conventional chemotherapy because the transplant procedure is expensive and carries a significant risk of severe side effects and even death," Ley explained.

At the moment, age and tumor cytogenetic abnormalities — which are present in about half of cases — still provide the biggest clues to AML patient outcomes.

In an effort to find new molecular markers of relapse risk, Klco, Ley, and colleagues did whole-genome or exome sequencing on 71 individuals with AML who were treated with induction therapy at Washington University and Barnes-Jewish Hospital's Siteman Cancer Center.

Clinical information and follow-up data was available for the group, which included patients with long remission times that exceeded a year, individuals with refractory disease within six months or less, and patients who remained in remission for between six months to a year.

From recurrent mutations detected in diagnostic tumor samples from 58 individuals whose tumors were tested by whole-genome sequencing, the researchers narrowed in on sites to test by deep sequencing across the tumor set. For a subset of the samples, they also assessed microRNA expression profiles, transcriptome and microRNA sequence patterns, and/or genome-wide methylation marks.

Similar to past studies of AML, the team found that tumors at diagnosis tended to contain mutations affecting genes such as FLT3, NPM1, and DNMT3A. Even so, it found little in the way of prognostic information by looking at the diagnostic tumors alone.

"Comprehensive sequencing at the time of diagnosis adds a little bit, but not a ton to help predict how patients are going to do," Klco said. Consequently, the group turned its attention to samples taken at morphologic remission, searching for other genomic insights into relapse risk.

"We decided to do the official test to see if you could use deep sequencing on these remission samples to see if you could actually detect residual mutations in patients who were in remission by morphologic standards and then what that actually meant for the patient in terms of outcome," he said.

Depending on the quality of the sample available, the investigators used either targeted sequencing or exome sequencing to assess genetic patterns in post-treatment bone marrow samples collected about 30 days after diagnosis for 25 individuals from the original cohort and 25 additional cases.

All of the individuals were deemed to be in remission based on standard bone marrow morphology analyses and 32 individuals had AML cases classified as intermediate risk based on tumor cytogenetics.

As it turned out, the team did see persistent mutations in a subset of the patients' tumors, particularly for mutations falling in genes such as DNMT3A, IDH1, and IDH2. And the proportion of bone marrow cells containing the AML-related glitches was associated with the patients' eventual relapse patterns.

For the 24 individuals with leukemia-linked mutations in 5 percent or more of their bone marrow cells during remission, the team saw survival times of 10.5 months and event-free survival times of just six months.

In contrast, individuals whose remission samples were more or less mutation-free — with "cleared" disease — had overall survival times exceeding three-and-a-half years, on average, with average event-free survival times approaching 18 months.

To distinguish between leukemia-related alterations and age-related mutations in the remission samples, the researchers carefully scrutinized the diagnostic tumor samples to identify genetic glitches that they could confidently attribute to the patient's leukemia. By focusing at read counts at these sites, they were then able to see how many of the mutations stuck around at remission.

"We knew what positions we were looking for and looked at the read counts at those positions," Klco said. "In other studies that are ongoing, we're looking for mutations that we couldn't really appreciate at day zero and looking in the day-30 sample."

Klco noted that he and his colleagues are conducting related follow-up research both at Washington University and at St. Jude, including studies to look at the possibility of finding similar prognostic hints from remission samples for pediatric leukemia cases.

In an accompanying editorial slated to appear in the same issue of JAMA, Memorial Sloan Kettering Cancer Center researchers Friederike Pastore and Ross Levine said findings from the study "provide critical insights into the role of molecular monitoring in AML and into the dynamics of genetic mutations during AML treatment."

"The next steps should involve development of assays that can be used to enumerate [minimal residual disease] in the clinic, performance studies to enumerate how best to use MRD monitoring in clinical care, and formulation of therapeutic regimens to target MRD and eradicate mutant clones to improve outcomes for patients with AML," Pastore and Levine concluded.