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NGS Bests Flow Cytometry for MRD-Based Prediction of ALL Relapse, Survival, ASH Study Suggests

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NEW YORK – Research presented at the American Society for Hematology's annual meeting suggested that using next-generation sequencing to detect residual disease in acute lymphoblastic leukemia (ALL) patients after treatment can better gauge their risk of relapse and overall survival than multiparameter flow cytometry.

Although flow cytometry is commonly used to assess residual disease in leukemia patients, some patients deemed to be negative for measurable residual disease (MRD-negative) at remission by this method later relapse. Researchers led by Nicholas Short, an assistant professor in the department of leukemia at The University of Texas MD Anderson Cancer Center, wanted to determine if using a more sensitive test to measure residual disease, like next-generation sequencing, would benefit ALL patients.

In this study, researchers retrospectively analyzed data from 67 previously untreated Philadelphia chromosome-negative ALL patients who had received either a standard-intensity chemotherapy regimen or lower-intensity regimen. The researchers tested 109 bone marrow samples provided by these patients by both flow cytometry and NGS to determine MRD status.

The higher sensitivity of NGS is able to detect residual leukemia that would normally be missed by flow cytometry, said Short during a presentation at the meeting.

"Current MRD assays are far from perfect, in part due to limited sensitivity," Short said. "For example, when MRD is assessed by standard multiparameter flow cytometry, many patients with apparent MRD-negativity still relapse. Highly sensitive NGS assays have been developed that detect dominant rearrangements within the B-cell and T-cell receptor genes at a sensitivity that is two logs deeper than standard flow cytometry."

The researchers in this study used the ClonoSEQ assay developed by Adaptive Biotechnologies, which was the first NGS test approved by the US Food and Drug Administration in 2018 for measuring residual disease in ALL patients. Prior to that test being approved, flow cytometry and PCR-based assays were the standard and could measure residual disease down to 1 in 10,000 or 1 in 100,000 cells. The NGS assays, however, can measure residual disease down to 1 in 1 million cells.

In the analysis by Short and colleagues, of the 84 samples that were MRD-negative by flow cytometry, the NGS test found 38 percent of those were MRD-positive, meaning leukemia remained in these samples that was below the level of detection from flow cytometry.

Overall, when samples across patient subgroups were tested with NGS, they were less likely to achieve MRD-negative status than when tested with flow cytometry. The researchers broke up the study cohort into two subgroups by the type of frontline chemotherapy patients received, either a standard chemotherapy-based regimen (involving cyclophosphamide/vincristine/doxorubicin/dexamethasone), called hyper-CVAD, or a lower-intensity version of the regimen, called hyper-CVD.

At the time of remission, 61 percent of patients who were treated with hyper-CVAD were deemed MRD-negative by flow cytometry, but only 32 percent in this subgroup were MRD-negative by NGS. Patients who received the lower intensity hyper-CVD regimen saw slightly better results by both methods, with 74 percent reaching MRD-negative status by flow cytometry and 46 percent by NGS.

Short noted that this data suggests further study is needed "to evaluate the feasibility of de-intensifying therapy in those patients who rapidly achieve MRD-negativity."

Because NGS was able to detect more residual disease, the researchers concluded that achieving MRD-negativity from NGS was a better predictor of durable remission than MRD-negative results from flow cytometry. The number of patients who relapsed within five years after achieving MRD-negative status was higher among patients tested with flow cytometry (27 percent) than those with NGS (8 percent). Only one of the 14 ALL patients who were deemed MRD-negative by NGS later relapsed.

NGS was also a better predictor of overall survival among these patients. All of the patients who were MRD-negative by NGS survived for five years, compared to 67 percent of those who were MRD-negative by flow cytometry but MRD-positive by NGS.

Short suggested that providers might use a combination of the two tests to gather insights on patients' outcomes, while noting that the NGS test is limited by necessitating bone marrow samples. He said that further research is needed to determine if peripheral blood samples could also be used for NGS, which would make it easier to perform these tests more frequently.

"It will always be somewhat complementary, even with the more sensitive flow assay, to use two different types of MRD assays that are looking at slightly different things, for example, using flow [cytometry] for other specific markers and an NGS panel looking at these rearrangements," Short said. "I suspect there might be added benefit to the NGS, but of course those studies will have to be performed."