Monitoring Leukemic Mutations Using Ultrasensitive IBSAFE Digital Droplet Analysis Predicts Relapse of Acute Myeloid Leukemia

Introduction

Optimal management of patients with acute myeloid leukemia (AML) depends on accurate monitoring of minimal residual disease (MRD) after treatment. The presence of MRD predicts outcome, with implications for risk stratification and treatment decisions. Therefore, improvements in MRD determination are desirable. Current MRD methods include multicolor flow cytometry (MFC), real-time quantitative polymerase chain reaction (QPCR), and more recently next generation sequencing (NGS) and droplet digital PCR (ddPCR). MFC can be applied on most patients, but suffers from limited sensitivity (rarely below 0.1% leukemic cells). QPCR is more sensitive but can only be applied on leukemias carrying the targeted mutation. NGS can track several mutations simultaneously but with limited sensitivity. We recently developed the method IBSAFE, which utilizes compartmentalized ddPCR micelles with a limit of detection of 0.001% mutant allele frequency (MAF). Given that even patients with negative MFC-MRD results (<0.1%) often have poor prognosis, we set out to analyse relapsing AML patients using IBSAFE-ddPCR to investigate if this method would be helpful for predicting the relapses.

Materials and Methods

In this pilot study, 10 relapsing AML patients were retrospectively tested for MRD with IBSAFE, a patent-pending method, in bone marrow aspirates taken at 3 to 13 (average 8) follow-up time points between 145 and 2607 days after diagnosis. First, the mutational profile of each leukemia was determined at diagnosis and relapse by whole-exome sequencing and targeted AML gene panel sequencing. Skin fibroblasts were used as germline controls.Based on the identified mutations, candidates for MRD monitoring were selected with priority towards recurrent mutations and mutations present at both diagnosis and relapse. IBSAFE assays were developed for selected mutations and used to analyse the diagnostic and follow-up samples.

Results

NGS of AML samples revealed between 10 and 36 somatic mutations at diagnosis (average 18) in each patient of which 0 to 7 (average 4) were known recurrent AML mutations including NPM1 , DMNT3A and TET2 . For a total of 66 selected mutations, IBSAFE assays were designed and validated, representing between 5 to 9 mutations (average 7) for each patient. The concordance between MAF by IBSAFE and NGS was excellent, corroborating the precision of our IBSAFE method. For MRD monitoring, molecular evidence of MRD/relapse was apparent prior to clinical relapse in all 10 patients, and three different patterns of emerging and disappearing mutations could be discerned. In some patients (pattern one; n=5), it was possible to monitor two or more separate clones, all of which reappeared at relapse. In these, some mutations were undetectable at certain time points, whereas others (e.g. TET2 ) were present at all time points at low levels, possibly representing preleukemic clones. In other patients (pattern two; n=4), most monitored mutations were undetectable (<0.001% MAF) at certain time points. Interestingly, in this second group, the emerging relapsing leukemia carried only some of the mutations monitored, demonstrating clonal heterogeneity with selection of one relapsing clone, which often showed additional new mutations. In one patient (pattern three; n=1) there was no distinct decrease before stem cell transplantation for several of the mutations (including DNMT3A and TET2 ) despite morphological remission and negative MFC-MRD, demonstrating the preleukemic nature of the regenerating hematopoiesis. For 21 samples (affecting 9 out of 10 patients), mutations were detected at MAF between 0.1% and 0.001%.

Conclusions

We demonstrate that our IBSAFE method can track early recurrence of leukemic clones. The advantage as compared to other MRD methods is the high sensitivity down to 0.001% MAF and simplified workflow to follow several mutations and track different emerging clones. One drawback is that our method does not allow tracking of acquired new mutations after treatment. However, developed IBSAFE assays can quickly be applied on follow-up samples and easily utilized for other patients carrying the same mutation. Of course, it may still be necessary to design some new assays for each patient, given the mutational heterogeneity of AML. In conclusion, these pilot results demonstrate the power of the IBSAFE method with possible applications in clinical routine AML-MRD analyses.