High throughput transcriptome sequencing of pediatric relapsed acute lymphoblastic leukemia (ALL).

Abstract

9521 Background: Relapsed ALL carries a very poor prognosis despite intensive therapy, indicating the need for new insights into disease mechanisms. We have previously used global gene expression profiling and copy number analysis in diagnosis/relapse pairs to better understand the biologic mechanisms leading to relapse. To create an integrated genomic profile we have now focused on high throughput RNA sequencing to detect mutations, insertions, deletions and fusion transcripts. Methods: To date we have sequenced 4 pediatric matched diagnosis/relapse pairs (i.e. 8 marrow samples) from B-precursor ALL patients. RNA sequencing was performed using the Illumina GAIIx Analyzer. Each sample was sequenced in 7 lanes using single-read 54 base pair sequencing to cover 52% of genes at 5X and 34% of genes at 20X. BWA (v0.5.5) and Samtools (v1.7) were used to align the reads to the human genome and predict single nucleotide variants (SNVs) and insertion/deletions (indels) across the genome. Known single nucleotide polymorphisms (SNPs) were filtered out by comparison to diagnostic samples and through use of dbSNP (r130) and 1,000 Genomes Project databases. We have focused our initial analysis on relapse specific lesions shared by samples presumably indicating selection for common chemoresistance pathways. Results: We found 232 variants that were relapse specific and shared among all 4 patients. Sixty-seven of the variants were within exons: 14 deletions, 6 indels, 34 insertions and 13 SNVs. There were 2,454 variants that were relapse specific and shared among 3 of 4 patients. Of these, 766 variants were within exons: 144 deletions, 69 indels, 344 insertions and 209 SNVs. There were 16,100 relapse specific variants found in 2 of 4 patients. Of these, 5,184 were within exons: 1,058 deletions, 300 indels, 2,146 insertions and 1,680 SNVs. Validation of a subset of these variants in a larger cohort of patients using Taqman assays and Sanger sequencing is underway. Conclusions: RNA sequencing in relapsed pediatric ALL reveals many novel relapse specific variants that are shared among patients. Furthur analysis of these variants will be necessary to determine their functional significance and potential therapeutic relevance. No significant financial relationships to disclose.