Download Acquired Copy Number Alterations in Adult Acute Myeloid Leukemia

Microarrays
Acquired Copy Number Alterations
in Adult Acute Myeloid Leukemia Genomes
Reference: Walter MJ et al. (2009) Proc Natl Acad Sci USA 106:12950–12955
Cytogenetic analysis of acute myeloid leukemia
(AML) cells has accelerated the identification of
genes important for AML pathogenesis. As shown
in a recent scientific study, the use of an unbiased
high-resolution genomic screen yields the identification of genes altered in AML genomes not previously implicated in the disease that may be relevant
for pathogenesis.
Introduction
Acute myeloid leukemia (AML) is a heterogeneous group
of diseases, currently classified by abnormalities in bone
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marrow morphology, karyotype, acquired gene mutations,
and alterations in gene expression. Although the identification of specific gene mutations has resulted in im­proved
treatments and outcomes for some AML patients, enormous clinical heterogeneity exists and may reflect the
presence of still-undetected initiating and cooperating
mutations. Therefore, the discovery of somatic mutations
in the genomes of AML patients with normal and abnormal karyotypes will advance the understanding of the
genetics underlying AML and should lead to more specific therapies and better patient classification schemes.
Identification of mutations important for AML pathogenesis will require knowing the complete sequence of a large
number of AML genomes. To complement cytogenetic
studies and to identify genes altered in AML genomes,
Walter and co-workers performed a genome-wide copy
number analysis with paired normal and tumor DNA
obtained from adults with de novo AML.
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M6/M7
abnl
(11q23)
t(15;17)
inv(16)
t(8;21)
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Miscellaneous cytogenetics
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Log2 Ratio
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Figure 1: Copy number and UPD heatmap for 86 AML genomes. The results of
copy number and UPD (copy-neutral LOH) analysis of 86 paired tumor and normal DNA
research samples assayed on the Affymetrix Genome-Wide SNP 6.0 arrays are shown.
Each of the 86 genomes is represented by two columns, copy number as the log2 ratio
of tumor/normal DNA is shown on the left and UPD on the right. Copy number is designated by a color range from white (deletion) to red (amplification), with pink indicating
a normal copy number. The presence of UPD is shown in blue and the normal non-UPD
state in gray. The y axis represents the chromosome number, with chromosome 1 at the
top and Y on the bottom. The x axis displays samples grouped by common cytogenetic
abnormalities.
Materials and Methods
Sample processing
Genomic DNA was prepared from unfractionated bone
marrow (tumor) and normal tissue (skin biopsy) from 86
individuals with de novo AML. Eligibility criteria included
age greater than 18, more than 30% myeloblasts in the
bone marrow, and the absence of antecedent chemotherapy, radiation therapy, or history of myelodysplasia.
Research samples with two or fewer clonal cytogenetic
abnormalities were given preference for inclusion in the
study. Paired samples made it possible to distinguish
acquired copy number alterations (CNA) from inherited
copy number variants (CNV). Cases were classified in
accordance with the French-American-British (FAB) system upon diagnosis and banking of their bone marrow
specimens. The subjects included FAB M0–M7, with a
median blast count of 64% (range, 30–100%).
Genomic DNA was also prepared from unfractionated
bone marrow (tumor) and normal tissue (skin biopsy)
from an additional 38 subjects with de novo AML to
evaluate for recurrence of small copy number alterations
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Microarrays
26.0
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28.0
STAG2
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5q31.1
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7q31.31
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8q23.3
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17q11.2
18p11.3
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10-4.5
10-2.5
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Significance (q value)
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21q22.2
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MLL
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12q12.3
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UPN 831711: Chr 11
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Amplifications
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UPN 808642: Chr X
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Deletions
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Log2 Ratio
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Log2 Ratio
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UPN 269542: Chr 17
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Log2 Ratio
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Significance (q value)
Figure 2: CNAs (deletions and amplifications) include one or more genes and demonstrate significant regions of
recurrence. Log2 ratio dot plots of paired tumor and normal DNA research samples from the same individual were generated from data obtained from the Affymetrix Genome-Wide SNP 6.0 arrays (top plot of each panel) and custom NimbleGen
CGH 12 ≤135K array data (bottom plot of each panel). Solid horizontal lines indicate gene locations with selected gene
names provided. The y-axis is the log2 ratio of paired tumor/normal DNA, and the x axis represents the chromosomal
megabase position for both array platforms. (a) Deletion of a 1.9-Mb region of chromosome 17, including the NF1 gene.
(b) Deletion of a 57-kb region of chromosome X, including the STAG2 gene. (c) Partial tandem duplication of a 35.6-kb
region of MLL on chromosome 11. (d) GISTIC analysis of genomic regions of deletion. Chromosome positions are indicated along the y axis and the false-discovery rate q values on the x axis, with the significance threshold indicated by the
green line at 0.25. Deletion regions that surpass the significance threshold include chromosomes 3p14.1, 5q31.1, 7q31.31,
12p12.3, 16q22.1, 17p13.1, 17q11.2, and 18p11.31. (e) GISTIC analysis of genomic regions of amplification. Amplification
regions that surpass the significance threshold include chromosomes 8q23.2, 11q23.3, 19q13.43, and 21q22.2.
discovered in the original 86 AML genomes. All research
samples had two or fewer clonal cytogenetic abnormalities, and all specimens were labeled with a randomly generated unique number (UPN).
probes/locus). Manual review of the log2 ratio plots and
the segMNT algorithm were used for analysis of the custom arrays.
Copy number and CNA validation
DNA was quantified using UV spectroscopy and qualitatively assessed using standard agarose gel electrophoresis. For all samples, 0.5 mg DNA was digested with Nsp
and Sty enzymes, amplified, fragmented and labeled, and
hybridized to Affymetrix Genome-Wide Human SNP Array
6.0 GeneChip microarrays (906,000 SNP probes, 946,000
copy number probes).
Results and Discussion
Confirmation of CNA was performed using the independent, ultra-dense custom Roche NimbleGen CGH 12 x
135K arrays (135,000 probes per sample). Confirmation
CGH array design consisted of a dense tiling of probes
across putative CNA loci identified by SNP arrays (1,900
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Acquired CNA
In this study, Walter and co-workers identified 201
acquired CNAs in 86 AML genomes using the SNP
arrays (Figure 1). The 201 CNAs occurred in 38 of 86
AML genomes, ranged from 35 kb (34 probes) to 250 Mb
(146,524 probes) in size (median, 9.15 Mb), and involved
every chromosome at least once. There was a mean of
2.34 CNAs per AML genome (range, 0–30; median, 0),
and deletions were more common than amplifications
(1.23:1). The 201 CNAs were distributed across all FAB
subtypes, with M6 and M7 subtypes containing significantly more CNAs per genome compared with all other
subtypes (mean 21 vs. 1.4, respectively; p=0.02). Known
Microarrays
Normal kariotype
− CNA
− UPD
27% (23)
− CNA
− UPD
23% (20)
+ CNA
− UPD
10% (9)
+ CNA
− UPD
10% (8)
+ CNA
+ UPD
1% (1)
− CNA
+ UPD
5% (24 − CNA
+ UPD
1% (1)
cancer genes alone, and AML/MDS-associated genes
alone (p=0.001, p=0.02, and p<0.001, respectively). CNAs
<1 Mb (n=45) were enriched for AML/MDS-associated
genes and the combination of cancer and AML/MDS
genes, but not for cancer genes alone or all annotated
genes (p<0.001, p=0.02, p=0.16, and p=0.058, respectively). There was no enrichment for microRNA genes in
CNAs <5 Mb or <1 Mb in size.
Abnormal kariotype
+ CNA
− UPD
22% (19)
+ CNA
+ UPD
1% (1)
SNP Array Specific
CNA or UPD
Figure 3: Summary of genetic alterations in AML
genomes. Pie chart demonstrates the relative proportions of AML samples with an abnormal (red, n = 50)
and normal (white, n = 36) karyotype, with (+) and
without (-) CNA and UPD detected by SNP arrays.
(Two subjects with failed cytogenetics are included
with the normal karyotype data: UPN 295 had no CNAs
or UPD detected by SNP arrays, and UPN 327929
had 22 CNAs detected.) The number of subjects in
each group is listed in parentheses. Of 36 subjects
with a normal karyotype (including the two subjects
with failed cytogenetics), 13 (36%) had a CNA or UPD
(identified as SNP Array Specific CNA or UPD). Of 50
subjects with an abnormal karyotype, 21 (42%) had an
SNP array-specific CNA (not seen by cytogenetics) or
UPD detected by SNP arrays (identified as SNP Array
Specific CNA or UPD). Forty-three of 86 subjects (50%)
had no CNA or UPD detected by SNP arrays.
cancer- and leukemia-related genes were significantly
enriched in the CNA loci, and their mRNA expression levels were often altered, suggesting that these genes may
contribute to AML pathogenesis.
Of the 201 CNAs, 198 (99%) contained known genes, and
154 of 201 loci (77%) encompassed at least one gene
that had previously been associated with cancer-or AML/
myelodysplastic syndromes (MDS).
Of the CNAs <5 Mb (the lower limit of detection by cytogenetics), 38% (33 out of 88) contained at least one cancer- or AML/MDS-associated gene (52 total cancer- or
AML/MDS-associated genes in 88 segments), which was
significantly more than the 31 genes expected to occur in
88 size-matched segments randomly distributed across
the genome (1,000 permutations; p=0.009). CNAs <5 Mb
were also significantly enriched for all annotated genes,
The researchers identified 12 chromosomal regions (8
deletions and 4 amplifications) from the 201 CNAs that
were significantly altered in multiple AML genomes by
using the Genomic Identification of Significant Targets
in Cancer (GISTIC) algorithm (Figure 2). Most of these
regions contain at least one gene previously implicated in
cancer and/or AML/MDS.
All 12 recurrent regions displayed mRNA expression levels for the entire region that were significantly altered in
a gene dose-dependent manner, compared with samples
without CNAs (p-value range, 0.02–2.06E-16). In addition, 18 recurrent CNA regions were discovered (10
deletions and 8 amplifications, 6 of 18 loci contained
within one of the 12 GISTIC-identified loci) in the 201
CNAs that contained a cancer-or AML/MDS-associated
gene and involved at least one research sample with a
CNA <5 Mb.
There were an additional 32 recurrent CNA regions (19
deletions and 13 amplifications, 6 of 32 loci contained
within 1 of the 12 GISTIC-identified loci) that included
genes not previously implicated in cancer or AML/MDS
and involved at least one research sample with a CNA <5
Mb. The mRNA expression levels of 43 genes, located in
15 of these 32 regions (47%), were significantly altered
compared with samples without changes (p-value range,
0.049–8.37E-11).
Favorable, intermediate, and adverse cytogenetic categories at diagnosis, defined by Cancer and Leukemia Group
B (CALGB), were predictive of overall and event-free
survival in the AML subjects, as expected. However, the
total number of CNAs per subject (for all subjects or only
individuals with normal cytogenetics), identified by SNP
arrays and cytogenetics or by SNP arrays only, was not
predictive of overall or event-free survival, independent of
cytogenetic classification.
Acquired CNAs containing ≤3 genes
Of the 201 CNAs detected in the 86 genomes, 21 (10%)
encompassed ≤3 genes (all <1 Mb). Of these 21 segments,
six (29%) included known cancer-associated genes. Three
of the 21 CNAs contained no known genes but did include
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Microarrays
regions of high sequence conservation (>75%) across
chimp, rhesus, horse, dog, and mouse genomes.
To identify small (<5 Mb) recurrent CNAs involving genes
in these 21 regions, Walter et al. evaluated an additional
set of 38 independent AML research samples, and
three recurrent CNAs were detected. One included the
MYB oncogene, and another contained LOC392454 [a
pseudogene similar to proliferating cell nuclear antigen
(PCNA)].
Identification of translocations and gene
mutations
There were 23 cytogenetically defined, balanced rearrangements that occurred in >25% of metaphases in 22
subjects with noncomplex karyotypes. The researchers
detected regions of CNA at the breakpoints of subjects
with cytogenetically defined balanced rearrangements as
well as a deletion end point located at a common translocation breakpoint in the NUP98 gene on chromosome
11p15.5. However, no cytogenetic translocations were
detected in this individual.
Partial uniparental disomy
The researchers identified eight regions of UPD in seven
of 86 research samples. UPD occurred more often in cytogenetically normal AML genomes (15% vs. 3.8%; p=0.08).
All regions of UPD extended to the end of the affected
chromosome and varied in size from 11 to 95 Mb.
Twenty-four percent of AML subjects with normal cytogenetics had CNA, whereas 40% of individuals with an
abnormal karyotype had additional CNA detected by SNP
array, and several CNA regions were recurrent. The mRNA
expression levels of 57 genes were significantly altered in
27 of 50 recurrent CNA regions <5 Mb. A total of eight
uniparental disomy (UPD) segments were identified in the
86 genomes; six of eight UPD calls occurred in research
samples with a normal karyotype. Collectively, 34 of 86
AML genomes (40%) contained alterations not found with
cytogenetics, and 98% of these regions contained genes.
Of 86 genomes, 43 (50%) had no CNA or UPD at this level
of resolution.
Summary
To complement cytogenetic studies and to identify genes
altered in AML genomes, Walter and co-workers performed a genome-wide copy number analysis with paired
normal and tumor DNA obtained from 86 adult subjects with de novo AML using 1.85 million feature SNP
arrays. A total of 201 somatic CNAs were found in the
86 AML genomes (mean, 2.34 CNAs per genome), with
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FAB-System M6 and M7 genomes containing the most
changes (10-29 CNAs per genome). According to the
authors' conclusions, the use of an unbiased high-resolution genomic screen identified many genes not previously
implicated in AML that may be relevant for pathogenesis,
along with many known oncogenes and tumor suppressor
genes. Despite the ability of next-generation sequencing
platforms to accurately identify amplifications, deletions,
and translocations, a multiplatform approach (traditional
cytogenetics, FISH, SNP array, array CGH, and targeted
gene resequencing) will continue to be the most practical
approach to studying AML genomes at most institutions
for the time being. Using this multiplatform approach
(karyotype, SNP array, array CGH, and gene resequencing), Walter et al. found at least one genetic abnormality
in 95% (82 of 86) of the AML genomes, including many
known and SNP array-specific CNAs (Figure 3).
n
This article was summarized for BIOCHEMICA from
Walter MJ et al. (2009) Proc Natl Acad Sci USA 106:12950-12955.