The Discovery and Validation of AML Prognostic Biomarkers

This study includes RNA-Seq and targeted DNA sequencing from a large collection of unsorted mononuclear cells (MNCs) and enriched viable leukemic blasts (VLBs) from bone marrow (BM) and peripheral blood (PBL) specimens collected at diagnosis from adult patients with acute myeloid leukemia (AML). Viable leukemic blasts were isolated using forward by side scatter, DAPI staining and fluorescently-labeled antibodies to CD45, CD34, CD38, and CD117. DNA and RNA were extracted using Qiagen AllPrep DNA/RNA Extraction kits. Transcriptome libraries were generated using KAPA Stranded RNA-Seq Library Preparation Kit after ribosomal RNA depletion with Roche RiboErase. Paired-end 75 bp reads were sequenced on either Illumina HiSeq 2500 (HiSeq) or NovaSeq 6000 (NovaSeq) instruments. RNA-Seq data were analyzed by the Fred Hutch Cancer Center Bioinformatics Shared Resource. STAR 2.7.1, with 2-pass mapping, was used to align reads to human reference genome GRCh37/hg19. Internal tandem duplication in FLT3 (FLT3-ITD) were identified via fragment analyses. DNA was sequenced using Illumina TruSight™ Myeloid Sequencing Panel and supplemented with in-house targeted MiSeq assays for CEBPA and NRAS exon 3 loci inadequately covered by TruSight™ platform on Illumina MiSeq instrument. Paired-end reads were first aligned to the human genome reference assembly (GRCh37/hg19) using Burrows-Wheeler Aligner (BWA, v0.7.12) and then, the resulting alignment data were processed according to the best practice of Genome Analysis Toolkit (GATK, v3.5).]]>
Inclusion Criteria: Adult (ages 18-88) patients with AML enrolled on SWOG-9031, 9333, S0106, S0112 and S0301 or in the Fred Hutch/UW Hematopoietic Diseases Repository with vials available at the time of specimen acquisitionExclusion Criteria: Patients not meeting inclusion criteria]]>