Global Identification of Genomic Structural Variants In Childhood ETV6/RUNX1 (TEL/AML1) Acute Lymphoblastic Leukemias by Mate-Pair Massively Parallel Sequencing.

Chimeric fusion genes generated by chromosomal translocations are highly prevalent in childhood acute lymphoblastic leukemia (ALL) and mainly represent early, prenatal events. The t(12;21)(p13;q22) translocation generates the ETV6/RUNX1 fusion gene and is the most frequent gene recombination in childhood B-lineage ALL, occurring in approximately 25% of the cases. It is associated with favorable prognosis even though a substantial proportion of the cases relapse. The fusion gene itself initiates the pre-leukemic process but additional genetic hits are needed to trigger a full blown leukemia. Being one of the best-characterized childhood leukemias, both nature and mechanisms of the events that cooperate with the chimeric protein are still poorly understood. Furthermore, studies addressing the genetic origin of relapse demonstrated a clonal relationship between relapse and diagnostic sample, assuming the existence of an ancestral, pre-leukemic clone.

Second-generation sequencing of both ends of huge numbers of DNA fragments allows comprehensive characterization of patterns of somatic rearrangements on an unprecedented, high-resolution level. By using the Illumina mate-pair massively parallel sequencing technology and intra-individual side-by-side comparison of leukemic and normal germline DNA, we aim to elucidate the cooperating genetic events in leukemogenesis in ETV6/RUNX1-ALLs as well as the clonal relationship between relapse and diagnostic sample.

We investigated diagnostic and relapse samples as well as non-leukemic germline material from one pediatric patient, diagnosed with ETV6/RUNX1-ALL in Germany. Mate-pair genomic sequencing libraries with an insert size of approximately 2-kb were constructed and paired-end sequence reads of 36-bp each were generated on the Illumina Genome Analyzer IIx from randomly created ~500-bp DNA fragments. Data were filtered and aligned to the human reference genome (GRCh37) using BWA. Reads considered PCR duplicates were removed and detection as well as clustering of structural variants (translocations, deletions, inversions) was subsequently carried out with GASV. Discordantly mapping read pairs defined potential structural variations and cluster sizes of at least 4 uniquely and correctly mapping read pairs were included in further analyses. In order to confirm breakpoints and resolve them to base-pair level, areas of putative chromosomal rearrangements were amplified from genomic DNA of tumor and matched normal sample and were conventionally sequenced.

An average of ~73,000,000 read pairs were generated for each sample and after alignment, the whole genome was sequenced with a mean fragment coverage of 18.8X. A substantial variation in prevalence of structural variants could be detected between paired diagnostic and relapse samples. Within the diagnostic sample we could in total observe 739 deletions, 66 inversions and 107 translocations while the relapse sample exhibited 26 deletions, 14 inversions and 240 translocations. In a first analysis we focused on translocations, presuming the high impact of chromosomal rearrangements on leukemogenesis. Subtracting translocations being of germline origin, 73 translocations at diagnosis and 207 translocations in relapse could be detected, of which 183 (74%) were identified being intragenic. Remarkably, both samples shared only 16 translocations (6%), while 57 (22%) uniquely appear in the diagnostic sample and 191 (72%) could only be observed in the relapse sample. Intragenic, shared translocations in diagnostic and relapse samples include the t(12;24) PDE3A/RN18S1, the t(2;17) PID1/UNC45B as well as the t(1;6) HFM1/EYA4 fusion gene products. Detection of the known ETV6/RUNX1 translocation in diagnostic and relapse sample as well as confirmation of selected breakpoints via Sanger sequencing validated our methodological approach.

Mate-pair sequencing of leukemic samples in comparison to germline material provides a powerful tool to identify genome-wide chromosomal structural variations and will allow analysis of clonality between diagnostic and relapse samples. The low percentage of shared translocations gives a first hint probably objecting the thesis of a common pre-leukemic clone, but will only be elucidated by analysis of further patient samples.

No relevant conflicts of interest to declare.