Patient-Tailored Analysis of Minimal Residual Disease in Acute Myeloid Leukemia Using Next Generation Sequencing

Background and aim : The importance of sensitive minimal residual disease (MRD) analysis for determination of response to treatment in acute myeloid leukemia (AML) is becoming increasingly evident. Routinely, this analysis is performed using multiparameter flow cytometry, and in select cases with fusion transcripts using reverse transcription polymerase chain reaction. The drawback with flow cytometry is that it is associated with false negativity due to immunophenotypic shifts during treatment and in pending relapse. In addition, leukemia immunophenotypes often overlap with the normal regenerating bone marrow cell populations. Therefore, other means of identifying remaining leukemic cells are warranted. Leukemic cells in AML are characterized by somatic mutations in recurrently mutated genes as well as in random genes, in most cases as single nucleotide variations (SNVs). We have previously reported that leukemia-specific mutations can be readily identified at the time of diagnosis of AML using exome sequencing of high purity sorted leukemic cells and lymphocytes. The aim here was to show that leukemia-specific mutations identified with exome sequencing at diagnosis can serve as markers for MRD, quantified with targeted deep sequencing, during follow-up. Method: Seventeen cases of AML, age 2-71 years old, were included in the study. Leukemic cells and lymphocytes were sorted using fluorescence activated cell sorting (FACS), from blood or bone marrow at diagnosis of AML. Exome sequencing of sorted cell populations was performed on the Illumina platform. Variant calling was performed with Mutect for SNVs and with Strelka and Varscan for short insertions/deletions. The data was subjected to an in-house statistical algorithm to identify variants present in all leukemic cells and thus suitable for MRD analysis. For targeted deep sequencing, the Truseq-library system was used for in-house PCR and sequencing on the Illumina Miseq platform (2x150 bp). The acquired reads were stitched using PEAR, aligned to the human reference genome and the resulting alignments were analyzed with in-house scripts with respect to specific SNVs and NPM1 insertion. Results: Exome sequencing of the paired leukemia/lymphocyte samples identified 240 leukemia-specific SNVs (14 (0-29) per case (median, range) and 22 small insertions and deletions (1 (0-5) per case). The most common type of mutation was, as expected, substitution of cytosine to thymine (CaT). The number of leukemia specific SNVs correlated with age (r=0.76, p Conclusions: Exome sequencing of high purity sorted leukemic cells and lymphocytes at the time of diagnosis of AML can identify leukemia-specific mutations suitable for MRD analysis. With targeted deep sequencing of leukemia-specific SNVs identified in this manner, leukemic cell burden can be estimated with high sensitivity during follow-up. The method could be used for patient-tailored MRD analysis in AML. Disclosures No relevant conflicts of interest to declare.