Monitoring Minimal Residual Disease in Acute Myeloid Leukemia Using Genomic or cfDNA with MyMRD®, a Targeted NGS Panel

Zhiyi Xie, Lisa Chamberlain, Andrew Carson, Veronika Atkinson, Valerie McClain, James Sprague, Ogeen Kiya, Peng Xia, Wenli Huang, Bradley Patay, Martin Blankfard and Jeffrey E Miller


Acute myeloid leukemia (AML) is a genetically and phenotypically heterogeneous disorder. Precision therapies for AML have been developed that target specific driver mutations. The efficacies of these therapies are variable, making it critical to determine successful therapies prior to patient relapse. For patients achieving a first complete remission, minimum residual disease (MRD) is an important prognostic factor, as MRD may provide a powerful and timely tool to evaluate therapeutic efficacy. There is a growing demand that new and promising drugs are approved as quickly as possible and accelerated approvals will require biomarker surrogate endpoints, such as MRD, rather than long-term survival endpoints.

We have developed a sensitive NGS gene panel (MyMRD®), which identifies pathogenic variants in AML. The MyMRD panel targets single nucleotide variants (SNVs), insertions and deletions (indels) in coding exons of hotspots of 21 genes (ASXL1 BRAF CALR CEBPA CSF3R DNMT3A FLT3 IDH1 IDH2 JAK2 KIT KRAS MPL NPM1 NRAS PTPN11 RUNX1 SF3B1 SRSF2 TP53 ZRSR2), and structural variants of potential genomic breakpoint hotspots within 3 somatic gene fusion partners (CBFB-MYH11 KMT2A RUNX1-RUNX1T1). This 23 gene targeted panel can identify driver mutations that cause relapse in >90% of all AML patients, as well as common drivers in other myeloid neoplasms and myelodysplasic syndromes.

MyMRD panel validation included determining the limit of blank (LoB), limit of detection (LoD), and linearity. Validation samples were generated using known variant containing DNA from cell lines and clinical samples diluted into NA12878 ("Genome in a Bottle") DNA. The average background, crossover, and carryover rates were determined to be 0.017%, 0.023%, and 0.016%, respectively. The LoB was determined to be 0.13% from calculating each of expected variants using the 95th percentile of all negative samples. Overall, we established an LoD of 0.5% for >95% of the targeted SNV and indel sites in the assay with lower LoDs for specific mutations of interest, such as 0.17% for FLT3 TKD and 0.34% for NPM1. The LoD for structure variants was determined to be 1.8%. The assay shows strong linearity with R2 = 0.969 - 0.994 of 11 selected targets in the entire range (0.17% - 50%) of variant allele frequencies (VAFs) tested. Linearity and LoD determined from clinical samples containing SNVs and indels were consistent with conclusions obtained from contrived cell line samples.

Cell-free DNA (cfDNA) isolated from plasma is becoming a readily available source ofcirculating tumor DNA for testing. A similar molecular profile has been observed from cfDNA to that of bone-marrow tumor cells from multiple myeloma patients. Therefore, we investigated whether the MyMRD assay, developed for genomic DNA analysis, could be applied to cfDNA to assess variants at a level comparable to testing of genomic DNA. To overcome the limitation of cfDNA availability, DNA fragments with size similar to cfDNA (140-170bp), were generated from contrived cell line DNA to assess the LoD and linearity of assay. The cfDNA assay also showed strong linearity (R2=0.975 - 0.998) in the range of VAFs (0.1- 20%) tested. With cfDNA input of 25ng, the LoD was established to be 0.5% for targeted SNV sites, and 1% for targeted indel sites. The MyMRD cfDNA assay detected variants at the same LoD for SNVs and a slightly higher LoD for indels.

Additional clinical samples were tested with the MyMRD assay. Variants detected were verified with capillary electrophoresis and/or amplicon-based NGS assays. The results showed 100% concordance with the MyMRD panel assay.

The MyMRD gene panel is a sensitive and reliable assay that provides monitoring of MRD using genomic DNA and cfDNA. The assay is shown to detect clinically important driver variants and to have excellent linearity and LoD for targeted sites. The assay provides an accurate method for detecting mutations in multiple targets in patients and can be used to stratify patients for therapy and clinical trials, and may provide the sensitivity necessary to serve as a surrogate endpoint assay for targeted therapies in AML.

Disclosures Xie: Invivoscribe Inc.: Employment. Chamberlain: Invivoscribe Inc.: Employment. Carson: Invivoscribe Inc.: Employment. Atkinson: Invivoscribe Inc.: Employment. McClain: Invivoscribe Inc.: Employment. Sprague: Invivoscribe, Inc.: Employment. Kiya: Invivoscribe Inc.: Employment. Xia: Invivoscribe Inc.: Employment. Huang: Invivoscribe Inc.: Employment. Patay: Invivoscribe Inc.: Consultancy, Equity Ownership, Patents & Royalties. Blankfard: Invivoscribe Inc.: Employment. Miller: Invivoscribe, Inc.: Employment, Equity Ownership.

  • * Asterisk with author names denotes non-ASH members.