SEVERAL FUSION TRANSCRIPTS ARE DETECTED BY NEXT GENERATION PAIRED END TRANSCRIPTOMIC RE-SEQUENCING APPROACH IN ADULT BCR-ABL1-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

Introduction. Mutations, chromosomal rearrangements and associated gene fusions resulting from inversions, interstitial deletion or translocations represent recurrent findings in leukemia. Nowadays high-throughput “Next Generation Sequencing” technologies and novel developed algorithmic methods allow a unified deep analysis of such alterations with a single base resolution. Taking advantage from these tools, we performed an RNA-Seq (whole transcriptome shotgun sequencing) approach using the Illumina/Solexa platform to define in a single procedure and at high sensitivity the full repertoire of leukemia-related mutations and fusion genes in 3 adult BCR-ABL1+ ALL cases treated with tyrosine kinase inhibitors. Methods. Patient median age was 55.3 years (range, 40-70); no additional chromosome abnormalities were detected except for one case with del(9)(p13p22). All the selected cases had previously been profiled by high resolution SNP (Affymetrix SNP6.0) and gene expression (Affymetrix Human Exon 1ST Array) arrays, as well as candidate gene re-sequencing (IKZF1, PAX5, JAK2, CDKN2A, CDKN2B, IDH1, IDH2), lacking missense point mutations. Two cases harbored the deletion of IKZF1, and in one case PAX5 and CDKN2A/B losses were also found. Poly(A) RNA from blast cells was used to prepare Illumina cDNA libraries according to the manufacturer’s recommendations. Sequencing by synthesis was performed on an Illumina Genome Analyzer IIx platform, with standard sequencing kits and nucleotide incorporation cycles, generating 75 base pairs (bp) paired end sequence reads. Results. A total of 57, 51 and 9 million reads were obtained from the 3 samples and high quality sequence reads were mapped to the reference sequence of the human genome (UCSC hg19) using the Maq software, finding out 58,205, 48,913 and 136,937 putative new single nucleotide variants (SNVs) in the CDS/EXON regions not reported in the dbSNP build 130. Of these, 874 distributed on 290 genes, affected both samples. Thereafter, we analyzed RNA-seq data by deFuse (McPherson A et al. PLoS Computational Biology May 2011), a novel computational method for fusion discovery between a gene and an intergenic region. This software uses clusters of discordant paired-end alignments to inform a split read alignment analysis for finding fusion boundaries. First of all, we evaluated the ability of deFuse to rediscovery the BCR-ABL1 gene fusion, then we focused on the most intriguing fusions such as the PBXIP1- PMVK (pre-B-cell leukemia homeobox interacting protein 1- phosphomevalonate kinase) gene fusion. All new gene fusions are under evaluation in a bigger ALL cohort. Conclusions. This study provided a comprehensive overview of a BCR-ABL1+ ALL transcriptome, identifying novel mutations and gene fusions involved in Ph+ ALL. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna- Ravenna, FIRB 2006, Ateneo RFO grants, PIO project, Programma di Ricerca Regione–Università 2007–2009.