Abstract 5413: MicroRNA profiling in AML
Authors: Gilligan Diana , Pandita Aakriti , Ramadas Poornima , Poudel Aarati , Saad Nibal , Anand Ankit , Basnet Alina , Middleton Frank 
Published: August 2018

Abstract
Differences in microRNA (miR) expression levels provide clues to the disruption of normal hematopoiesis that leads to the emergence of a leukemic clone. Analysis of miR expression levels may be useful to understanding normal cytogenetic acute myeloid leukemias (AMLs) and developing novel treatments for them. We collected peripheral blood samples from 10 adult patients with newly diagnosed AML, prior to induction chemotherapy, and 9 controls. Two and a half ml of whole blood was collected in Paxgene RNA tubes. miRNA was purified using standard Trizol method, followed by RNeasy mini column (Qiagen). Quality of the RNA samples was assessed using the Agilent Bioanalyzer prior to library construction using the Illumina TruSeq Small RNA Sample Prep protocol (Illumina; San Diego, CA). Multiplexed samples of RNA that exceeded quality control metrics (RIN &gt 6.0) were run on an Illumina NextSeq500 instrument at a targeted depth of 10 million reads per sample. After filtering and trimming of index and adapter sequences, whole genome alignment of the miR FASTQ reads was performed using the Homo sapiens/hg19 reference genome in the SHRiMPS aligner included in the miRNAs analysis application available in BaseSpace (Illumina), as well as the sRNA Toolbox application suite. Quantification and normalization of aligned reads to the miRBase 21 database was performed, and differential expression between AML and control groups was performed using DESeq2, NOIseq, and EdgeR algorithms. We sequenced approximately 800 miRs from each of 10 patients with AML and 9 controls. We identified 9 miRs that showed a statistically significant increase in expression in AML patients versus controls and 4 miRs that showed a statistically significant decrease in expression in AML patients versus controls, with adjusted p-value less than 0.05. Among these 13 differentially expressed miRs, only 4 were previously described in leukemia, including miR 181a-3p and 181a-2-3p, miR 409-3p, and miR 126-5p. Finding these 4 miRs differentially expressed confirmed the validity of our approach. The remaining 9 of the miRs that showed differential expression in our study have not been described in relation to AML (miR-328-3p, miR106b-3p, let-7i-5p, miR10b-5p, miR-24-3p, miR-3200-5p, miR-23a-3p, miR-323b-3p, miR-652-3p). In subset analysis, patients with NPM1 and FLT3 mutations showed lower levels of miR 181a-2-3p and higher levels of miR 10b-5p compared to patients without NPM1 and FLT3 mutations. We are continuing to accrue patients to this study and we are following them over time in order to analyze miR expression in peripheral blood as a biomarker that may predict relapse. Our approach of global sequencing of miRs as opposed to microarray analysis removes the bias regarding which miRs to assay and has demonstrated discovery of new associations of miRs with AML. Our study provides further information about the molecular changes that lead to evolution of the leukemic clone and offers new targets for development of treatments.
Citation Format: Diana Gilligan, Aakriti Pandita, Poornima Ramadas, Aarati Poudel, Nibal Saad, Ankit Anand, Alina Basnet, Frank Middleton. MicroRNA profiling in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5413.