CLINICAL NEWS

Research from ASH ’ s online peer-reviewed journal , Blood Advances

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 / 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 ( R 2 = 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

TABLE . Overall Survival ( OS ) Based on ITD VAF

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 .

– MARK J . LEVIS , MD , PhD

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 sequencingbased assay for minimal residual disease assessment in AML patients with FLT3-ITD mutations . Blood Advances . 2018 April 24 .

VAF = variant allele frequency ; NA = not achieved

62 213 ( 95 % CI 143-264 )

67 213 ( 95 % CI 144-264 )

0.003

0.002

40 ASH Clinical News May 2018