Integrated Analysis of Whole-Exome Sequencing and Micrornas Expression in Blast Crisis Transformation of Chronic Myeloid Leukemia

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.

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).

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.

No relevant conflicts of interest to declare.