Global Genomic Characterization of Acute Lymphoblastic Leukemia

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A key goal in cancer research is to identify the total complement of genetic and epigenetic alterations that contribute to tumorigenesis. We are currently witnessing the rapid evolution and convergence of multiple genome-wide platforms that are making this goal a reality. Leading this effort are studies of the molecular lesions that underlie pediatric acute lymphoblastic leukemia (ALL). The recent application of microarray-based analyses of DNA copy number abnormalities (CNAs) in pediatric ALL, complemented by transcriptional profiling, resequencing and epigenetic approaches, has identified a high frequency of common genetic alterations in both B-progenitor and T-lineage ALL. These approaches have identified abnormalities in key pathways, including lymphoid differentiation, cell cycle regulation, tumor suppression, and drug responsiveness. Moreover, the nature and frequency of CNAs differ markedly among ALL genetic subtypes. In this article, we review the key findings from the published data on genome-wide analyses of ALL and highlight some of the technical aspects of data generation and analysis that must be carefully controlled to obtain optimal results.

Section snippets

Microarray Analysis of DNA Copy Number Alterations in Acute Leukemia

Several microarray platforms are available for the analysis of DNA copy number abnormalities (CNAs) and LOH in cancer, each with its advantages and disadvantages. The basic method involves the hybridization of a labeled sample of test DNA to microarrays that carry thousands to millions of probes, each of which is specific for a different region of the genome. The hybridization strategy may be single color (in the case of single-nucleotide polymorphism [SNP] microarrays) or dual color (in the

SNP Array Studies in ALL

The first reported SNP array study of ALL examined 10 pediatric cases using 10K SNP arrays (incorporating approximately 11,000 markers; Affymetrix, Santa Clara, CA) and detected LOH in eight cases.38 A region of chromosome 9p harboring the CDKN2A/CDKN2B tumor-suppressor locus was most frequently involved, but the low resolution of the arrays (100-200 kb between markers) prevented precise delineation of the regions involved, and the copy number status at each region was not reported.

We performed

Mutations of Genes Regulating B-Lymphoid Development in ALL

Our study identified more than 50 recurring regions of CNA in ALL (Table 2). Most were focal (minimal common region of CNA <1 Mb) and many involved genes with known or putative roles on leukemogenesis and cancer. The most striking novel finding was that genes encoding regulators of normal lymphoid development were mutated in over 40% of cases of B-progenitor ALL. The most frequent target was the lymphoid transcription factor PAX5, which harbored deletions or focal amplifications in almost 30%

Genomic Determinants of Lineage and Disease Progression in BCR-ABL1 Leukemia

In our initial ALL SNP array analysis, abnormalities of PAX5, IKZF1, and CDKN2A/B appeared to be frequent in pediatric BCR-ABL1 ALL; however, the number of cases examined was small (N = 9). Thus, we extended our existing studies to an expanded cohort of pediatric ALL (N = 304), including 21 pediatric and 22 adult BCR-ABL1 cases, and 23 cases of BCR-ABL1–positive chronic myeloid leukemia (CML), that included samples obtained at various stages of disease progression, including 24 chronic phase

Mutations in Other Cellular Pathways in B-ALL

We also identified CNAs targeting other genes regulating cellular growth, differentiation and drug responsiveness in B-ALL. In addition to previously identified deletions involving the CDKN2A/CDKN2B and ETV6 genes, we found deletions of tumor suppressors (RB1, PTEN, NF1, FHIT), the histone gene complex at 6p22, the Arf-induced antiproliferative gene and regulator of apoptosis BTG1,57, 58, 59 the glucocorticoid and mineralocorticoid receptors NR3C1 and NR3C2,60, 61ATM, the IL3RA and CSF2RA

Mechanism of Deletion in ALL

Long-range PCR mapping and sequencing of the genomic breakpoints at sites of recurring CNAs in B-ALL has provided important insights into the mechanism responsible for the CNAs in ALL. By sequencing across the genomic regions of the breakpoints of the most common IKZF1 deletion (exons 3-6) in BCR-ABL1 B-ALL cases, we found that the proximal and distal breakpoints were extremely conserved, varying by only a few nucleotides, and had a pattern suggestive of aberrant activity of the recombinase

Role of CNAs in B-ALL Leukemogenesis

An important question is whether the identified CNAs play a mechanistic role in leukemogenesis (driver mutation), or represent background genetic “noise” (passenger mutation). Several observations suggest that the CNAs are biologically important. The average number of CNAs per ALL case is low, suggesting that this disease is not characterized by inherent genomic instability. Secondly, the majority of identified CNAs target small regions of the genome, frequently containing only a single gene in

Genome-Wide Profiling of T-Lineage ALL

One of the first CNA studies in T-ALL used BAC array comparative genomic hybridization (array-CGH) and identified high-level amplification resulting in fusion of the NUP214 and ABL1 genes present on extrachromosomal episomes. The fusion of NUP214 to ABL1 results in constitutive activation of the kinase,73 and NUP214-ABL1–positive cell lines are responsive to tyrosine kinase inhibition, suggesting that these agents may be therapeutically useful in this type of leukemia. Using SNP, BAC, or

Copy-Neutral LOH (Acquired Uniparental Disomy) in ALL

The identification of copy-neutral LOH (CN-LOH) in tumor cells can indicate duplication of a gene within the region of LOH that has been altered by point mutations or epigenetic modification. Consequently accurate detection of CN-LOH is important to identify genes for subsequent mutation studies. For example, CN-LOH accompanying acquisition of homozygous mutations of FLT3 and CEBPA has been described in acute myeloid leukemia.81 We and others have reported recurring CN-LOH in ALL, most commonly

Technical Aspects of Copy Number Profiling Studies in Leukemia

The results described above have transformed our understanding of the genetic basis of ALL, but our ability to accurately and comprehensively detect all regions of copy number alteration and LOH in cancer is critically dependent on the resolution and genomic coverage of the platform used, the selection of appropriate reference samples, and appropriate array normalization and careful CNA/LOH inference.

Other Approaches to Identify Genomic Abnormalities in ALL

Genome-wide techniques to characterize epigenetic changes,88 sequence mutations, and noncoding RNA expression are evolving rapidly, but thus far they have not been widely applied to ALL. Most investigations have focused on limited panels of individual genes. As with CNA/LOH analysis, it is likely that these techniques will provide insights into the pathogenesis of ALL. Several microarray platforms are available for studying epigenetic changes and microRNA expression, but as with gene expression

Summary

The advent of high-resolution genome-wide platforms to detect CNAs and regions of LOH, coupled with methods to accurately and quantitatively assess the total transcriptome of a leukemia cell, has provided critical new insights into the pathogenesis of pediatric ALL. These data are defining key pathways required for cellular transformation and are highlighting combinations of lesions that appear to influence the response of the leukemia to our present therapeutic approaches. Over the next

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    Supported in part by grants from the Haematology Society of Australasia, the Royal Australasian College of Physicians, the National Health and Medical Research Council (Australia), and the American Lebanese and Syrian Associated Charities of St Jude Children's Research Hospital.

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