Somatic Structural Variations in Pediatric High Hyperdiploid Acute Lymphoblastic Leukemia Revealed by Paired-End Parallel Sequencing

Abstract

Abstract 401 Introduction:High hyperdiploidy (51–67 chromosomes) is the most frequent numerical cytogenetic alteration found in pediatric B-cell precursor acute lymphoblastic leukemia (ALL), occurring in 25–30% of patients. It is characterized by nonrandom gains of chromosomes X, 4, 6, 10, 14, 17, 18, or 21. Children suffering from high hyperdiploid ALL have a good prognosis, nevertheless in 15–20% of cases the disease will recur. The mechanisms involved in the pathogenesis of primary and relapsing high hyperdiploid ALL are poorly understood. In some cases, IGH rearrangements arise in utero, indicating an early formation of pre-leukemic clones. However, the cellular origin of these pre-leukemic clones, as well as the molecular mechanism underlying the formation of high hyperdiploid cells, remains to be determined. Further genetic changes assisting in the development of ALL and recurrent disease are still unknown. Objective:By using massive parallel genome-wide next generation sequencing (Illumina/Solexa), we intended to identify specific cytogenetic structural variations (SVs) of high hyperdiploid ALL and possible clonal relationships between paired diagnostic and relapse ALL samples. Method:Paired-end sequencing libraries were generated from genomic DNA of diagnostic and relapse leukemic samples as well as germline DNA from the same patient. Libraries of two patients and one high hyperdiploid ALL cell line (MHH-CALL-2) with insert sizes of 350–400 bp were sequenced with paired end reads. Read lengths of 36 bp (Genome analyzer IIx) or 51 bp (HiSeq 2000) were sequenced, respectively. Sequencing raw data were aligned to the human reference genome hg19 (GRCh 37) by Burrows-Wheeler Aligner (BWA) and duplicate reads were removed. Copy number variants (CNVs), deletions, intrachromosomal inversions and interchromosomal translocations were analyzed by FREEC and GASV. After subtraction of germline SVs, putative leukemia-specific SVs were obtained. These were validated by PCR performed on genomic DNA. Specific breakpoints of SVs at single base resolution were identified by capillary sequencing of the PCR products. Results:Sequencing of different libraries yielded 95–279 million unique reads that mapped with both ends to the reference genome. Sequence coverages of 57–87% and fragment coverages of 4.9–12.3x were achieved (Table 1). [Display omitted] CNV profiles with 10 kb resolution were generated. A comparison of the CNVs of diagnosis and relapse ALL samples demonstrated a high degree of conformity with only few additional alterations present mainly, but not exclusively, in the relapse samples. In one of the patients, a large gain of chromosome 1q was only observed in the relapse sample (Figure 1). [Display omitted] SV analysis of all samples resulted in a total of 375 intragenic deletions, 16 intergenic inversions and 83 translocations (Table 1). PCR validation identified 2 previously unknown somatic translocations in the MHH-CALL-2 cell line concerning chromosomes 3 and 7 as well as chromosomes 15 and 18. Furthermore, 6 novel translocations present at diagnosis and relapse could be validated in patient samples. They were concerning chromosomes 3, 11, 12 and 20. One unique new relapse-specific translocation t(4;7) was identified. Conclusion:Paired-end sequencing of leukemia samples and matched non-tumor materials provides a robust tool for the discovery of genome-wide structural rearrangements. The high degree of conformity of CNVs and SVs detected in paired diagnosis/relapse samples indicate a common origin and a close relationship of the leukemic clones at diagnosis and relapse. The observation of few additional alterations in both diagnostic and relapse samples suggests the presence of different subclones at the time of diagnosis and the evolution of the relapse clone from either the diagnostic clone or a minor subclone. Disclosures:No relevant conflicts of interest to declare.