Next-generation sequencing for MRD was more sensitive and accurate than flow cytometry and B-cell aplasia monitoring.
DNA sequencing-based detection of residual disease between three and twelve months accurately identified all patients who would eventually relapse in pediatric and young adult patients with acute lymphoblastic leukemia (ALL) treated with tisagenlecleucel (Kymriah), whereas other methods were less predictive.
Tisagenlecleucel is a chimeric antigen receptor T-cell (CAR-T) therapy—a form of therapy in which patients’ own T cells are taken, reprogrammed to specifically target a protein called CD19 found on the surface of cancer cells, and then reintroduced to the patient to combat ALL and lymphoma. According to Pulsipher, about 80% of ALL patients treated with tisagenlecleucel achieve complete remission.
However, over half of patients who achieve remission ultimately have relapse and require additional treatment, such as a bone marrow transplant. Accurately forecasting relapse may enable patients in need of a transplant to initiate the process prior to the illness recurrence.
Since the CD19 receptor is expressed on normal B cells as well, therapy results in B-cell aplasia—a patient’s B cells being depleted.
“Our current recommendation to centers giving tisagenlecleucel is to follow B cells in the blood monthly, using a standard test, as a way to predict patients at higher risk of relapse,” says Stephan Grupp, senior author of the study.
“Monitoring B-cell aplasia is not ideal because it only picks up part of the relapse risk.”
He noticed that recurrence can occasionally occur in the absence of B-cell recovery, in the form of tumor cells that lack CD19 and hence are unable to be targeted by functional CAR-T cells.
“A key goal for our patients and families is not only to define which patients are at increased relapse risk and need further therapy, but also to identify patients heading toward longer-term benefit who might not need further therapies such as transplant,” Grupp said.
While liquid biopsies of the blood or bone marrow have grown in popularity, these procedures have not been directly compared. Pulsipher and colleagues examined the predictive value of flow cytometry—a technique that examines proteins on the cell surface—and next-generation DNA sequencing minimal residual disease (NGS-MRD) monitoring using blood and bone marrow samples from the ELIANA and ENSIGN phase II clinical trials at one, three, six, nine, and twelve months after tisagenlecleucel infusion. The presence of CD9, CD10, CD13, CD19, CD20, CD22, CD33, CD34, CD38, CD45, CD58, CD66c, and CD123 was determined using flow cytometry. For NGS, the IgH, IgK, and IgL gene sequences were examined for rearrangements and translocations.
While flow cytometry could detect around one cancer cell per 10,000 blood cells, the researchers discovered that NGS-MRD was significantly more sensitive, detecting one cancer cell per 1 to 10 million blood cells, depending on the amount of cells in the sample. This resulted in a 131 percent increase in the number of positive samples discovered with NGS-MRD versus flow cytometry.
NGS-MRD analysis of bone marrow samples was more accurate than flow cytometry in predicting relapse. At three or six months after infusion, 100% of patients with detectable disease DNA suffered relapse or progression to another therapy, with the exception of one patient who was lost to follow-up. Additionally, NGS-MRD detection was able to identify patients at risk of relapse far in advance of relapse. Those with the lowest levels of NGS-MRD positivity relapsed a median of 168 days following the positive test, and the assay detected 100% of relapses. In comparison, flow cytometry was positive 52 days before relapse and missed 50% of relapses.
These findings indicate that more sensitive NGS assessments detect disease with enough lead time prior to recurrence to allow for repeat sampling and/or coordination of therapeutic interventions, according to Pulsipher.
NGS-MRD was also more accurate at predicting relapse than B-cell aplasia. B-cell recovery was not predictive of recurrence three months after therapy, whereas patients with a positive NGS sample had a 12-fold increased risk of recurrence.
Pulsipher proposed that NGS-MRD be used in conjunction with, rather than in place of, B-cell aplasia monitoring. He noted that for certain individuals, 28 days may not be enough time for CAR-T cells to eradicate all underlying illness, which means they may test positive at one month but negative at three months. Thus, at the one-month time point, the predictive value of NGS-MRD was decreased, representing a 4.87-fold greater risk for positive patients. Early after treatment, B-cell recovery exhibited a stronger predictive value, with positive individuals having a 3.33-fold increased chance of recurrence, which Pulsipher highlighted was insufficient on its own due to the method’s inability to identify tumor cells missing the CD19 receptor.
Pulsipher and colleagues concluded that patients who develop B-cell aplasia after six months of therapy or who present with NGS-MRD-positive illness in the bone marrow during the first year following therapy should get extra treatment to prevent relapse.
Pulsipher stated that his team is now conducting a prospective clinical trial to investigate the possibility of intervening prior to a patient experiencing a recurrence using NGS-MRD results.
“This is the first paper to show an approach that identifies markers of relapse that are very specific, allowing clinicians to add additional therapy prior to relapse that will prevent it,” adds Pulsipher.
“To date there have been no reports systematically exploring the use of molecular MRD assessment following therapy with tisagenlecleucel. There is utility in the data Pulsipher et al. provide, comparing [flow cytometry] with NGS-MRD beyond the already-established increased sensitivity of the latter, but importantly, also exploring the optimal threshold for positivity and lead time to relapse from a positive result to obtain the best biomarker of relapse risk.”
They further write: “Whist it is clear from these data that NGS-MRD has the potential to be a powerful predictor of relapse post infusion of tisagenlecleucel, to take these data forward, the wider applicability of this approach in a multi-centre prospective validation is needed.”
The study’s limitations include the study’s small sample size (just under 400), particularly from peripheral blood, and the fact that samples were accessible for a limited number of time points (one, three, six, nine, and 12 months).
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