Standardized MRD Assay for FLT3-ITD-Mutated AML Could Guide AML Therapy Decisions

In a report published in Blood Advances, researchers describe a standardized minimal residual disease (MRD) assay that can detect FLT3-ITD mutations in patients with acute myeloid leukemia (AML) in remission.

“Initial response rates to intensive chemotherapy are quite high in patients with FLT3-ITD AML and, while a substantial number of these patients will ultimately relapse if not transplanted, this is by no means uniformly seen,” explained the authors, led by Mark J. Levis, MD, PhD, of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University in Baltimore.

“The availability of a sensitive and specific FLT3-ITD mutation assay … could help guide decisions about whether patients should undergo transplantation and potentially whether FLT3 inhibitors should be administered as maintenance therapy following intensive chemotherapy or transplantation,” they wrote.

The investigators developed the assay using an improved polymerase chain reaction methodology combined with a next-generation sequencing platform to detect the presence of MRD in patients with FLT3-mutated AML that was in morphologic remission. “This MRD assay … is commercially available and accessible to any practitioner,” they noted.

In this study, investigators collected bone marrow (BM) samples from people enrolled in the phase I/II CHRYSALIS study that evaluated the FLT3 tyrosine kinase inhibitor gilteritinib in patients with relapsed or refractory AML. In the trial, 137 patients received oral gilteritinib 120 mg or 200 mg per day. Eighty patients had BM aspirates available at baseline and at least once after starting gilteritinib.

The researchers found that the linearity of the assay is “excellent” in the range of 10-2 to 10-15 (R2 = 0.98-0.99), with a reliable limit of detecting MRD to a level of 5×10-5, “although in some cases, an ITD mutation was detected at even lower levels,” they reported.

To validate the MRD assay for clinical use, the investigators analyzed BM aspirates collected from 15 patients who were in clinical remission (according to International Working Group criteria) and were negative for FLT3-ITD at the time of sample collection. BM aspirates were analyzed throughout treatment with gilteritinib.

In four patients, samples were analyzed at the time of hematologic recovery to confirm complete remission (CR) following intensive, cytarabine-based induction chemotherapy. The assay detected FLT3-ITD mutations in all cases at variant allele frequencies (VAFs) ranging from 1.35×10-5 to 3.33×10-4.

In three patients who were considered more clinically high risk, BM samples were collected while patients were in second or third remission. The assay detected mutations in all patients at VAFs ranging from 1.38×10-6 to 1.11×10-4. One patient underwent a second allogeneic hematopoietic cell transplantation (alloHCT) after the sample collection and remained in remission. The other two patients received no further therapy and died after disease relapse.

In six patients who had undergone alloHCT 24 to 48 months prior to sample collection, the FLT3-ITD MRD assay was negative. “These results suggest that the assay can be used to identify patients in whom durable remission has been achieved,” the researchers wrote.

In two patients who relapsed following alloHCT, the assay was negative for FLT3-ITD mutations, and the chimerism in both the unfractionated BM and peripheral blood T-cell compartments was 100 percent donor. The MRD assay correctly identified the length of corresponding FLT3-ITD mutation at VAFs of 1.04×10-4 to 3.67×10-3. Both patients relapsed two months after the samples were collected.

Among the 80 patients treated with gilteritinib who had BM aspirates available, the composite CR rate (defined as CR, CR with incomplete hematologic recovery, and CR with incomplete platelet recovery) was 55 percent (n=44). MRD (defined as FLT3-ITD VAF >10-4) was detected in 33 of the 44 patients.

Survival in MRD-positive patients was significantly worse than in those with a VAF of 10-4 or less, “which demonstrates that gilteritinib can induce deep molecular responses as measured by a highly sensitive MRD assay,” the researchers noted. Based on overall survival outcomes (see TABLE), the researchers defined 10-2 or less as a molecular response.

“This level also approximates the lower limit of detectability for the assay as performed at different institutions throughout the world,” they wrote.

“An apparent advantage of [FLT3-ITD mutations as an MRD target] is that each patient’s FLT3-ITD mutation is a unique length,” the researchers reported. “Detecting an FLT3-ITD mutation that is the exact sequence found in the diagnostic sample confers a distinct degree of specificity in this assay. If the FLT3-ITD mutation that was present at diagnosis is detected in a BM sample, it likely represents the disease.”

The study is limited by its small patient population. In addition, expanding the use of these types of assays in a standardized fashion across multiple institutions in several countries can be challenging, the authors noted.

The MRD assay will be prospectively tested as a secondary endpoint in an international, multicenter, pivotal trial assessing gilteritinib as maintenance therapy after alloHCT.

Astellas Pharma Global Development provided funding for the study. Invivoscribe Technologies developed and performed the FLT3-ITD MRD assay for the study.

The corresponding authors report financial support from Astellas, Novartis, Daiichi-Sankyo, Millennium/Takeda, Pfizer, Arog, Actinium, Asana BioSciences, Syros, Bristol-Myers Squibb, Celgene, Immune, and Janssen. Astellas provided editorial support.


Reference

Levis MJ, Perl AE, Altman JK, et al. A next-generation sequencing-based assay for minimal residual disease assessment in AML patients with FLT3-ITD mutations. Blood Advances. 2018 April 24.

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