Integrated Analysis of Whole-Exome Sequencing and Micrornas Expression in Blast Crisis Transformation of Chronic Myeloid Leukemia
Authors: Magistroni Vera , Sharma Nitesh , Piazza Rocco , Harb Jason G , Spinelli Roberta , Pirola Alessandra , Ellis Justin , Dong-Wook Kim , Perrotti Danilo , Gambacorti-Passerini Carlo 
Published: October 2019

Abstract
Abstract 3727

Objective.
The molecular events leading to the evolution of BCR/ABL+ chronic myeloid leukemia (CML) from the chronic phase (CP) to the advanced phase (blast crisis-BP) are poorly understood. The aggressiveness of BP and the poor over-all survival of BP patients needs deep investigation of the biological basis of blastic transformation. We here present the results obtained from the analysis of paired (CP)/(BP) CML samples from patients that underwent progression after standard therapy. The access to matched CP/BP samples will render this data highly valuable.

Methods.
We performed whole-exome sequencing analysis using high-throughput technologies (Illumina Genome Analyzer IIx) from genomic DNA of four paired samples. The cross-match between BP and CP exomes was performed with dedicated in-house C#software. Gross chromosomal rearrangements were evaluated using CEQer(Comparative-Exonic-Quantification-Analyzer) software from whole-exome sequencing data. We also evaluated microRNA(miRNA) differential expression extracting RNA from five paired samples, using Nanostring nCounter miRNA expression assay. Differentially-expressed miRNAs with a p-value<0.001 were considered significant. Putative targets of significantly deregulated miRNAs were generated using miRGen software analysis(http://www.diana.pcbi.upenn.edu/miRGen.html).

Results.
By comparing exome-sequences of four paired CP (used as a control) and BP samples we found a total of 8 single nucleotide somatic mutations. Among the 8 variants identified, 4 of them ranked >1 in the GenRanker cancer scoring system (http://cbio.mskcc.org/tcga-generanker). We show here that, unexpectedly, blast crisis samples have a limited number of acquired mutations compared to chronic phases (average=2 mutations/patient) with patient number two and four displaying the lowest and the higher frequencies, respectively (patient n.2=0 mutations, patient n.4=4 mutations). CEQer analysis of whole exome data showed that 3/4 patients present gross chromosomal rearrangements of at least 2 chromosomes (bulky alteration of chromosome7 present in 2/4 patients); critical regulators of cell cycle control (e.g. CDKN2A and p53) have also been shown to be deleted in patient number 1 and 4, respectively. The individual mutations and rearrangements identified will be presented at the meeting.
Differential expression of miRNAs showed that miR-106a, miR-17, miR-20a and miR-20b were significantly down-regulated while miR-148a was significantly up-regulated in all the blast crisis compared to chronic phase samples. In-silico analysis of the putative deregulated targets revealed a strong enrichment of genes involved in molecular mechanisms of cancer, with the 20% of these genes involved in cell cycle regulation. The integrated analysis of these informative data will help to understand the molecular mechanisms responsible for blast crisis progression.

Disclosures:
No relevant conflicts of interest to declare.