Novel Genomic Structural Variations in Relapsed Childhood ETV6/RUNX1 (TEL/AML1) Acute Lymphoblastic Leukemias Identified by Next Generation Sequencing

The chimeric fusion gene ETV6/RUNX1 generated by the interchromosomal translocation t(12;21) presents the most frequent chromosomal aberration in childhood acute lymphoblastic leukemia (ALL), occurring in approximately 25% of all patients. This ALL subtype is associated with an overall favorable prognosis, nevertheless 10–20% of children will suffer from relapse. ETV6/RUNX1-positive preleukemic clones arise already in utero, but require additional cooperating oncogenic lesions for the development of overt leukemia. The nature of the assisting genetic alterations and the mechanisms driving the development of leukemia and recurrent disease are still not well understood.

We applied state-of-the-art whole genome and whole exome next-generation-sequencing to comprehensively analyze the assisting oncogenic alterations in pediatric patients with initial and/or recurrent ETV6/RUNX1+ ALL (primary disease n=11, recurrent disease n=7). Matched sample sets taken at initial diagnosis, remission and relapse were compared. Mate pair and/or paired end sequencing was carried out for whole genome analysis with inserts spanning 2 kb or 500 kb, respectively. Constructed libraries were sequenced from both ends with 36- or 50-bp reads on a Genome Analyzer IIx or a HiSeq 2000 (Illumina/Solexa), respectively. Reads were aligned against the human reference genome (GRCh37) using BWA. Duplicate reads were removed. Unique reads with high mapping quality (q>35) served as input for GASV which detected translocations and inversions based on the mapping coordinates, insert sizes and read orientation. Variations covered by at least three reads in the tumor sample and not detected in the remission sample or the Database of Genomic Variants were reported. For detection of copy number variations, the program FREEC carried out coverage normalization, computation of copy number ratios between paired leukemia and remission samples (with up to 10 kb resolution) and subsequent segmentation. A subset of six selected patients was further investigated by targeted enrichment of whole exomic regions employing SeqCap EZ libraries (Roche) and 100 bp single read next-generation-sequencing on a HighSeq 2000. Mutations were called using GATK and further processed by an in-house bioinformatic pipeline. Putative somatically acquired mutations were validated by PCR, Sanger sequencing and FISH analysis.

Genomes were sequenced to at least 13× physical coverage (mate pair) and 6.7× sequence coverage (paired-end). Exome sequencing achieved a minimum of 25× sequence coverage. In silico we detected 155 tumor-specific intragenic translocations. On average each tumor harbored 9 acquired translocations. With the exception of one case (13 translocations at diagnosis, 9 at relapse), the number of translocations was higher in relapse than in the matched diagnosis sample (additional 3 translocations on average). Ongoing validation studies confirmed the defining ETV6/RUNX1 translocation t(12;21) and identified 13 novel translocations. The genes affected are involved in essential signaling pathways, such as cytokine signaling (LIFR), calcium signaling (RCAN2), insulin and anti-apoptotic signaling (PHIP). Interestingly, also a factor essential for pre-mRNA splicing (IBP160) and genes encoding regulatory RNAs (miRNAs, lincRNAs and RNAs involved in splicing) were rearranged. A validated intragenic deletion of 836 bp leading to a frameshift and premature stop affected a calcium ion sensor of the ferlin protein family. Recurrent deletions in 9 of 11 cases (82%) ranging from 5 to 200 kb were detected in the immunoglobulin lambda variable gene cluster (IGLV) on chromosome 22q11. Some of the deletions were extending into the pre-B lymphocyte 1 gene (VPREB1) locus. In silico the probabilty of illegitimate RAG-mediated recombination at the breakpoint sites was determined by evaluation of RIC scores. RIC scores indicated that aberrant V(D)J rearrangements involving cryptic recombination sequence signals had caused the deletions on chromosome 22q11.

We present somatic mutations that are promising novel candicate genes (e.g. LIFR, RCAN2, PHIP, IBP160) for cooperating secondary mutations in ETV6/RUNX1+ ALL and discuss their impact on the molecular pathology of primary and recurrent disease.

No relevant conflicts of interest to declare.