Genome-Wide Analysis of Genetic Alterations In Acute Myeloid Leukaemia (Massively parallel, high-throughput, paired-end DNA sequencing and genotyping of an AML genome).

Acute Myeloid Leukaemia (AML) has a worldwide incidence of approximately 3.5 per 100,000 population per year with most cases occurring in adults. Survival at 12 months is less than 30% and at 5 years is less than 10% (NCI, 2010). AML is a complex disease that demonstrates marked heterogeneity morphologically, cytogenetically and molecularly. The largest cytogenetic subgroup of AML has normal cytogenetics and accounts for approximately 45% of de novo AML cases. Furthermore, the response to treatment and survival outcomes of cytogenetically normal AML (cn-AML) is remarkably heterogeneous.

– To undertake whole-genome and exome sequence analysis of a cn-AML case at diagnosis and at relapse to identify subtle, potentially oncogenic mutations at the molecular level that may initiate AML and those that may be responsible for drug resistance or relapse.

i. –Comparison of the AML genomes at diagnosis, remission and relapse to identify genomic regions with changes in copy number and loss of heterozygosity, using single nucleotide polymorphism (SNP) microarray analysis. ii. Identify chromosomal rearrangements such as small insertions, duplications, inversions and deletions, (“indels”) and translocations that may contribute to AML initiation or relapse, using massively parallel, ultra-high throughput (“next generation”), paired-end sequencing of total genomic DNA at low depth. iii. Identify DNA point mutations potentially affecting protein function that may contribute to AML initiation or relapse by using next generation sequencing at high depth of the captured exomes (putative coding regions) of the AML and control genomes.

The PwC (Price-Waterhouse-Coopers) Leukaemia and Lymphoma Tissue Bank, a joint initiative of the Australasian Leukaemia and Lymphoma Group and the Leukaemia Foundation, provided samples from an AML patient with normal cytogenetics and a blast count at diagnosis of 70% and at relapse of 85%. Genomic DNA extracted from autologous mesenchymal stem/stromal cells (MSCs) was used to represent non-leukaemic, germline, control DNA. Primary cell culture of MSCs from cryopreserved bone marrow aspirate cells (50 × 10⁶) of our test patient at remission was achieved with standard tissue culture methods. High molecular weight DNA was extracted from the patient's MSCs and marrow cells at diagnosis, remission and relapse samples. Sonication (Covaris) and libraries appropriate for paired-end high-throughput sequencing (Illumina Genome Analyzer II instrument) were prepared from gel purified DNA fragments approximately 200 bp in size.

Primary cell culture of MSCs from bone marrow aspirates proved to be a robust source of germline genomic DNA with several advantages over skin. Firstly, tissue banked samples, will not include biopsies of normal skin. Furthermore, skin can be contaminated by circulating leukaemic cells, which is problematic with low-depth genomic sequencing. The homogenous immunophenotype of the cultured MSCs indicate their purity. Preliminary SNP microarray analysis identified a large region of uniparental disomy (copy number neutral loss of heterozygosity) involving most of chromosome 13q, which was not identified by standard cytogenetic analysis. Low-pass or shallow paired-end genomic DNA sequencing has generated the following outputs. MSC genome: 10.7 Gb (3.6X haploid genome coverage) resulting from 152 × 10⁶ paired sequence reads, AML_diagnosis genome: 21.6 Gb (7.2X) resulting from 142 × 10⁶ paired sequence reads, AML_relapse genome: 26.8 Gb (8.9X) resulting from 177 × 10⁶ paired sequence reads. The capture baits representing the human exome span 26,225,870 bp (approximately 0.9% of haploid human genome). Exome capture libraries were prepared from the three sources of DNA (MSC, AML_diagnosis and AML_relapse) and sequenced using a single lane for each library. The outputs for all three were very similar, approximately 23 × 10⁶ paired sequence reads, representing 1.3 Gb (49X haploid exome coverage). The reads for whole genome or exome capture were of high quality and more than 98% could be unambiguously aligned to the human reference genome in the correct orientation and interval distance. Here we will present MSC enrichment results and sequencing output results, quality control and preliminary analysis of genomic alterations and exonic mutations.

No relevant conflicts of interest to declare.