Use of deep whole transcriptome sequencing in Philadelphia-positive acute lymphoblastic leukemia (ALL) to identify novel mutated genes and aberrant gene expression and alternative splicing profiles associated with disease progression

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. Methods: In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, we used a Whole Transcriptome Sequencing (Illumina/Solexa) approach to sequence the leukemia transcriptome of BCR-ABL1+ ALL cases at diagnosis and relapse. Results: This analysis generated 13.9 and 15.8 million reads from de novo and relapsed samples, respectively, detecting transcripts from 62% and 64% of human annotated genes. Reads that showed no match to the human genome reference sequence (UCSC hg18) were mapped to a dataset of all possible splice junctions created in silico and 4,334 and 3,651 primary and relapse AS were identified. The well-known alternatively spliced Ik6 of the IKZF1 gene was also detected. Finally, 2,011 and 2,103 single nucleotide variants (SNVs) were found at diagnosis and relapse respectively, about 94% of which have been already reported in the dbSNP. As potential ALL-related mutations, 124 and 114 not annotated SNVs were found at diagnosis and relapse, respectively. Of these, 43 affected both samples, while 81 and 71 resulted private variants. 12 nonsynonymous changes were found on the coding sequences of annotated genes: 1 affecting PLXNB2 on both samples, 6 affecting genes involved in metabolic processes (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46) and transport (MVP) at diagnosis and 3 affecting genes involved in cell cycle regulation (CDC2L1) and catalytic activity (CTSZ, CXorf21) at relapse. The T315I mutation in the Bcr-Abl kinase domain was also identified. All these mutations were validated by deep exon sequencing in an additional subset of 24 ALL patients and correlated with prognosis. Conclusions: Our findings demonstrated for the first time in BCR-ABL1+ positive ALL that deep sequencing is a suitable approach for identifying a wide spectrum of genetic alterations. Supported by: AIL, AIRC, FIRB 2006, European LeukemiaNet.