Identification Of Leukemia-Specific Mutations For Detection Of Minimal Residual Disease In Acute Myeloid Leukemia Using Cell Sorting and Whole Exome Sequencing

The introduction of next generation sequencing techniques into the field of leukemia research has revealed that acute myeloid leukemia (AML) is characterized by a limited number of somatic mutations, in most cases single nucleotide variations (SNVs). In addition to providing insight into the pathogenesis of AML, this information can potentially be used for detection of small amounts of leukemic cells in follow-up samples (minimal residual disease, MRD). The aim of this study was to identify leukemia-specific mutations in AML cells that can serve as leukemia-specific MRD-markers.

Identification of leukemia-specific mutations was performed using whole exome sequencing of DNA from sorted leukemic cells and comparison with sorted lymphocytes from the same individual. Cells were obtained from 8 cases of AML, age 30-71 years old, from blood samples taken at the time of diagnosis of AML. Cell sorting was carried out by fluorescence activated cell sorter (FACS), where leukemic cells were defined by their FSC and SSC properties and expression of CD45, CD34, CD117, and HLA-DR. Lymphocytes were sorted based on FSC, SSC and CD45 expression. Purity of cell populations were >98% for leukemic cells and >99% for lymphocytes (with undetectable amounts of leukemic cells). Exome sequencing of sorted cell populations was performed on the Illumina platform with HiScanSQ yielding around 4^10⁷reads per sample. Data were quality assessed by FastQC, aligned to the reference human genome, processed for PCR duplicate removal, variant calling with Genome Analysis Toolkit (GATK) package, annotation of variants with ANNOVAR, and verification in Integrative Genomic Viewer. SNVs and short insertions or deletions present in the dbSNP database were excluded and the resulting SNVs and short insertions and deletions with minimum coverage of 10 were compared between leukemic cells and lymphocytes from the same individual. Leukemia-specific heterozygous mutations were defined as present in >40% of the reads in the leukemic cell sample and present in none of the reads from the corresponding lymphocyte sample.

By using these rather strict criteria at least three leukemia-specific SNVs were found in each AML case. Leukemia-specific SNVs (with coverage spanning between 10 and 250) were detected in recurrently mutated genes but also in genes not previously reported to be mutated in AML, e.g. CNNM4, GLYAT, NCKAP1L, PPBP, and PRB1. In the case of previously reported recurrently mutated genes in AML, at least one SNV was found in most AML cases. SNVs in recurrently mutated genes were found to be leukemia-specific in most cases, but in some cases, including PRPF40B, ETV6, and EZH2, SNVs were present in a heterozygous pattern in both leukemic cells and in lymphocytes, indicating that they are germ-line mutations. Genes with leukemia-specific insertions or deletions included NPM1, STAG2, RUNX1, and BCOR. The finding of the insertion in NPM1 in two cases was confirmed by detection of the insertion with conventional fragment analysis used in our clinical laboratory. When the same data analysis was used on exome sequencing data of neutrophilic cells and lymphocytes sorted from normal control samples (n=2), no SNVs or short insertions or deletions were found to differ between these two cell populations.

Our results show that by using exome sequencing on sorted cell populations with high purity, leukemia-specific mutations can be identified in AML samples already at diagnosis without the need for additional sampling of normal material or access to remission samples. Information on leukemia-specific mutations at diagnosis could provide a basis for detection of MRD in follow-up samples, either by polymerase chain reaction or targeted deep sequencing.