NEXT GENERATION TRANSCRIPTOME RESEQUENCING IDENTIFIES NOVEL POINT MUTATIONS, GENE EXPRESSION AND ALTERNATIVE SPLICING PROFILES IN BCR-ABL1 POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

Background: Although the pathogenesis of BCR-ABL1+ ALL is mainly related to the expression of BCR-ABL1, additional genetic lesions are supposed to be involved in its development and progression. Aim: In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, the leukemia transcriptome of a BCR-ABL1+ ALL patient at diagnosis and relapse was sequenced using a Whole Transcriptome Sequencing (RNA-Seq) approach. The selected cases had previously been profiled by high-resolution SNP and gene expression arrays and candidate gene resequencing. Methods: Poly(A) RNA from blast cells was used to prepare cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained sequence reads were mapped to the human genome reference sequence (UCSC hg18) to identify single nucleotide variants (SNVs). Reads that showed no match were mapped to a dataset of all possible splice junctions created in silico to identify AS events. The number of reads corresponding to RNA from known exons was also estimated and a normalized measure of gene expression level (RPKM) was computed. Results: RNA-seq analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, most of which successfully mapped to the reference sequence of the human genome. With the exclusion of the T315I mutation in the BCR-ABL kinase domain, seven novel missense mutations were detected after applying stringent criteria to reduce the SNV discovery false positive rate and validating novel substitutions with genomic DNA Sanger sequencing: 4 were exclusively found in the primary ALL sample and affected genes involved in metabolic processes (DPEP1, ZC3H12D, TMEM46) or transport (MVP); 3 missense mutations specific to the relapse sample affected genes involved in cell cycle regulation (CDC2L1) and catalytic activity (CTSZ, CXorf21). Differences in mutational patterns suggest that the leukemia clone from which relapsed cells have been developed was not the predominant one at diagnosis and that relapse specific variants were mutations probably acquired during Ph+ ALL progression. Moreover, 4,334 and 3,651 primary ALL and relapse isoforms with at least one AS event were identified. An average of 1.5 and 1.3 AS per isoform was estimated. The well-known alternatively spliced IKZF1 gene was also detected. Finally, a detailed gene expression profile was obtained indicating that more than 60% of annotated human genes were transcribed in leukemia cells in both diagnosis and relapse phases. Approximately 23% of genes were up-regulated at relapse with respect to diagnosis. Many of these genes affect cell cycle progression (AURORA A, SURVIVIN, PLK1, CDK1, Cyclin A, Cyclin B), suggesting that the loss of cell cycle control and the subsequent increased proliferation play a role in disease progression. Conversely, only 9% of active genes in both samples were down-regulated at relapse with respect to diagnosis. Conclusions: Discovery of novel missense mutations, as well as exhaustive alternative splicing and gene expression profiles were achieved for the first time for a BCR-ABL1+ positive ALL demonstrating that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations. Supported by AIL, AIRC, FIRB 2006, European LeukemiaNet, GIMEMA ONLUS.