Abstract 4878: Sensitive and specific DNA and RNA sequencing techniques for detecting minimal residual disease
Authors: Crowgey Erin L. , Mahajan Nitin , Wong Wing H. , Kolb Edward A. , Druley Todd 
Published: July 2017

The goal of this study was to assess the clinical applicability of molecular genetic techniques in the study of minimal residual disease (MRD) in pediatric acute myeloid leukemia (AML) via DNA and RNA-based methods. Currently, multi-parametric flow cytometry (MPFC) for surface immunophenotypes is the gold standard for MRD with a limit of detection between 0.001-0.002. However, approximately one-third of children with no detectable MRD by MPFC after first induction still relapse and suffer inferior outcomes. One potential reason is that a majority of refractory or recurrent AML clones will present with altered immunophenotypes compared to diagnostic specimens. In contrast, DNA mutations present at diagnosis will remain in refractory disease and therefore, a gene-based MRD platform surveying many genes could enable targeted, gene-specific therapy.

Recently, the heterogeneous and unique genomic landscape of pediatric AML was characterized in the TARGET project and highlights the potential for leveraging companion molecular screens in the analysis of AML MRD. Contrary to adult AML, pediatric AML is not primarily characterized by single nucleotide variants (SNVs), but rather by complex structural variants (SV) that are difficult to detect using standard next generation sequencing techniques and analysis pipelines. The need to detect these complex SV and SNVs, all of which are at low allelic ratios (AR) at the remission state, demands assays that are capable of analyzing both DNA and RNA molecules at levels of detection comparable to MPFC.

As proof of principal, we leveraged a unique capture technique (ArcherDx; CoreAML) to detect an important SV in pediatric AML, the internal tandem duplication in Fms related tyrosine Kinase (FLT3-ITD). Via a serial dilution of cells with known FLT3-ITD allelic ratio (MV4-11 cells), to determine a limit of detection, we were able to detect the mutation as low as 0.001. Next, we bench-marked the technique using diagnostic and relapse samples, and successfully detected FLT3-ITD at AR &lt;0.01.

To complement the DNA-based approach, we compared two error-corrected (EC) RNA assays (a Druley lab developed protocol and the ArcherDx; HemeV2 panel) to further assay SVs that cause novel gene fusions and aberrant exon usage, which are not detectable via DNA assays. Compared to existing cytogenetic and RNA-seq data, this new platform was capable of detecting known and novel cryptic translocations.

Finally, we integrated the results from the FLT3-ITD assay and the RNA assay with our custom error-corrected sequencing data to create a novel bioinformatics workflow for assessing the biological implications of MRD clones detected. Collectively, our data support that EC sequencing, at both the DNA and RNA level, enable accurate detection of low allelic variants that could be used for improved clinical MRD diagnostics, prognostication and therapeutic selection.

Citation Format: Erin L. Crowgey, Nitin Mahajan, Wing H. Wong, Edward A. Kolb, Todd Druley. Sensitive and specific DNA and RNA sequencing techniques for detecting minimal residual disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4878. doi:10.1158/1538-7445.AM2017-4878