Supplemental Material
1. Supplemental Methods
Leukemic Cell Isolation and FACS
GFP+ leukemic tissues were dissected under fluorescent microscopy into Zebrafish
Kidney Stromal media (Stachura et al 2009). Single cell suspensions were made by
repetitive pipetting, filtering through SmallParts 35 micron filters (Miramar, FL, USA), and
final passage through 70 micron Filcon filters (Becton Dickinson, San Jose, CA, USA)
immediately prior to flow cytometric sorting. Fluorescence-activated cell sorting (FACS)
was performed as described previously (Frazer et al 2009) using a BD FACSVantage
instrument (Becton Dickinson). GFP intensity, forward- and side-scatter were used for
gating. Cells were collected into Zebrafish Kidney Stromal media.
Serial Transplantation
Tumors were dissected and cells prepared as above. GFP+ cells were FACS-purified,
diluted in Zebrafish Kidney Stromal media and cell concentrations confirmed by
hemocytometer counting. Using a
137
Cesium source, strain-matched, non-isogenic host
fish were irradiated with 25 Gy, and intra-peritoneally injected 2 days later with 1x 105 of
FAC-sorted GFP+ cells in injection volumes of 10 ul. Recipients were monitored by serial
fluorescence microscopy to follow engraftment and disease progression.
Array Comparative Genomic Hybridizations and Imaging
After FACS collection, genomic DNA was extracted from purified GFP+ cells as well as
individual-matched tail tissue using a DNeasy Blood & Tissue Kit (Qiagen Inc., Valencia,
CA). DNA from tail tissue was used as a reference (i.e. cancerous test vs. noncancerous control) to determine CNVs using a self-self hybridization to reduce potential
inter-individual effects. After heat denaturation, genomic DNA (500 ng) from the test and
reference sample was labeled with Cy5-dCTP or Cy3-dCTP by random priming using
the BioPrime Labeling Kit (Invitrogen, Carlsbad, CA), in which fluorescently labeled
nucleotides are incorporated during genomic DNA replication. Unincorporated
nucleotides were removed with Amicon Ultra 0.5 mL 30K filters (Millipore) prior to
determination of DNA concentration and dye incorporation. For array hybridizations, 3.5
μg of labeled tumor DNA was co-precipitated with 3.5 μg of reference DNA at a total
volume of 79 μl; 25 μl of herring sperm DNA, 26 μl of blocking agent, and 130 μl of
hybridization solution were combined and denatured at 95°C for 3 minutes. Samples
were then pre-hybridized at 37°C for 30 minutes before being placed on custom
NimbleGen or Agilent zebrafish arrays. Arrays were placed in individual hybridization
chambers and hybridized for 40 hours at 65°C.
After hybridization, cover slips were removed by placing slides into Oligo aCGH
wash buffer I. Slides were then transferred to a second wash solution of Oligo aCGH
wash buffer I at room temperature and washed for 3 minutes. After the first wash, slides
were transferred to a preheated container of Oligo aCGH wash buffer II at 37°C for 1
minute. Slides were then gently removed from the solution to prevent streaking and
placed directly into the scanner for image processing.
A two-color scan of the arrays was conducted using an Agilent G2565CA
Microarray Scanner System with SureScan High Resolution Technology (Agilent
Technologies, Inc., Santa Clara, CA) at a 3 μm resolution. Images were analyzed using
Agilent Feature Extraction software which normalizes Cy3 and Cy5 fluorescence
intensities across the array and quantifies each DNA probe for color intensities and
background intensity to produce Cy3/Cy5 log2 ratios.
Array analysis
Copy-number analysis using the Rank Segmentation algorithm and group comparisons
were performed employing the BioDiscovery Nexus Copy Number 5.0 software
(BioDiscovery, El Segundo CA, USA). Copy-number analysis was done using a
significance threshold (p- value) of 1x10-6 and a minimum of 3 affected adjacent probes.
Thresholds for gains and losses were set such that aneuploidies were detectable, and
deletions in the T cell receptor regions were visible in at least 90% of samples. For
zebrafish analyses, Nexus-produced CNA calls were refined by removing regions of
“self-self” hybridization, regions where >3Mb was amplified or deleted, and areas where
CNAs were also present in normal zebrafish T lymphocytes.
Sample
hlk 1
srk 3
srk 3
otg 1
hlk 4
hlk 4
hlk 4
hlk 4
mMyc
Event
trisomy
trisomy
4 MB amplification
trisomy
trisomy
trisomy
trisomy
5 MB amplification
trisomy
Boundaries
chr 6
chr 8
chr23:0-4,075,864
chr 23
chr 6
chr 21
chr 23
chr1:19,044,570-24,073,766
chr 19
A gene list was created first by identifying genes altered in all CNAs from both the
NimbleGen and Agilent platforms, and then cross-referencing to Zv6 and Zv8,
respectively, to determine current official zebrafish gene names. Each zebrafish gene
was then cross-referenced to the NCBI human genome database 36.1 and/or sequencesearched by BLAST to determine homologous human genes. Zebrafish genes with
homology to multiple human genes were only counted once. The human gene list was
then used to analyze human T-ALL datasets by using the “Query” function of Nexus 5.
Human Datasets
Two human T-ALL datasets were used. The first dataset includes 47 patients treated on
Children’s Oncology Group study P9404 or Dana-Farber Cancer Institute study 00-01
clinical trials. Among these, 25 achieved event free survival, 9 showed induction failure,
and 13 eventually relapsed (Gutierrez et al 2010a, Gutierrez et al 2010b). The second
dataset contained paired primary and relapse samples from 14 T-ALL patients treated at
St Jude Children’s Research Hospital between 1993 and 2005 (Mullighan et al 2007). All
14 patients relapsed, and aCGH data was available from both their primary and relapsed
T-ALL samples. Eight of these patients also had non-malignant germline DNA analyzed
by aCGH, and any genes with CNAs in these germline samples were excluded from
study, as they represent germline CNVs and not somatically-acquired CNAs.
2. Supplemental Tables
Supplemental Table 1. CNAs Observed in Zebrafish and Human T-ALLs
17 Zebrafish T-ALLs
Total CNAs
Amplifications
Deletions
176
22.0 (4-68)
117
14.6 (1-54)
210,165
59
7.4 (2-14)
72,810
664
73.8 (28-143)
218
24.2 (7-81)
189,639
446
49.6 (22-105)
143,276
840
49.4
335
19.7
196,808
505
29.7
135,044
Total CNAs
Amplifications
Deletions
Agilent 244K (n=47)
average (range)
average size (bp)
509
17.8 (9-35)
130
3.4 (0-11)
2,661,949
379
14.4 (9-25)
4,108,918
Affymetrix 500K (n=8) or snp6 (n=20)
average (range)
average size (bp)
1069
35.3 (3-90)
380
11.6 (0-37)
1,759,019
689
25.1 (3-72)
1,914,300
1578
24.2
510
6.4
1,989,178
1068
18.3
2,693,102
Nimblgen (n=8)
average (range)
average size (bp)
Agilent (n=9)
average (range)
average size (bp)
Summary (n=17)
average
average size (bp)
75 Human T-ALLs
Summary (n=75)
average
average size (bp)
Supplemental Table 2. Zebrafish Primary and Passaged T-ALL CNAs
Primary T-ALLs (n=3)
Passaged T-ALLs (n=3)
Total # of CNAs
Average # of CNAs (range)
154
51.3 (31-89)
186
62.0 (28-119)
Primary-only CNAs (% of total)
Average # of CNAs (range)
69 (45%)
23.0 (15-34)
n/a
n/a
n/a
n/a
101 (54%)
33.7 (17-64)
85 (55%)
avg. 28.3 (11-55)
85 (46%)
avg. 28.3 (11-55)
Passaged-only CNAs (% of total)
Average # of CNAs (range)
Shared Primary and Passaged CNAs
Average # of CNAs (range)
Supplemental Table 3. Algorithm for Identifying D. rerio CNA Genes with Human Homologues
Algorithm Processing Step
D. rerio T-ALL CNA Genes
NimbleGen platform (8 samples)
311
(+)
Agilent platform (9 samples)
759
(=)
# of Genes in >1 CNA on either platform
1070
(-)
# of Genes present in CNAs on both platforms
127
(=)
Total # of unique CNA genes
943
(-)
Genes lacking human homologues
Genes not located on human genome build 36.1
15
2
(=)
D. rerio CNA genes with human homologues
926
(-)
Genes with human germline CNVs
33
(=)
Analyzable homologous gene pairs
893
Supplemental Table 4. Recurrent CNA genes in D. rerio and Human T-ALLs
Shared and recurrent CNA genes in zebrafish (n=14) and human (n=61) primary T-ALLs
D. rerio
gene
col15a1
tgfbr1b
lrp6
zgc:158374
osr2
zgc:66488
pus7
matn4
zgc:65779
zgc:77727
# of D. rerio T-ALLs
with gene in CNA
5 (35.7%)
5
4 (28.6%)
4
4
4
4
3 (21.4%)
3
3
Human
gene
COL15A1
TGFBR1
LRP6
MANSC1
OSR2
FBXO43
PUS7
MATN2
RNF170
TOR1A
# of human T-ALLs
(n=61) with gene in CNA
(inc. LOH)
# of human EFS T-ALLs
(n=25) with gene in CNA
(inc. LOH)
# of human poor-outcome primary
T-ALLs (n=36) with gene in CNA
(inc. LOH)
5 (6)
5 (6)
6 (7)
6 (7)
6 (6)
6 (6)
4 (4)
4 (4)
4 (6)
4 (4)
4 (4)
4 (4)
3 (3)
3 (3)
2 (2)
2 (2)
2 (2)
2 (2)
2 (2)
3 (3)
1 (2)
1 (2)
3 (4)
3 (4)
4 (4)
4 (4)
2 (2)
2 (2)
2 (4)
1 (1)
Shared and recurrent CNA genes of passaged zebrafish (n=3) and poorest-outcome human (n=23) T-ALLs
D. rerio
gene
zgc:153606
zgc:158222
zgc:114085
flt4
ckmt1
ckmt1
zgc:112384
katnb1
zgc:113984
# of D. rerio T-ALLs
with gene in CNA
2 (66.7%)
2
2
2
2
2
2
2
2
Human
gene
C7orf60
AHCYL2
ERGIC1
FLT4
CKMT1A
CKMT1B
WBP4
KATNB1
HIST2H3C
# of human T-ALLs
(n=75) with gene in CNA
(inc. LOH)
# of human EFS T-ALLs
(n=25) with gene in CNA
(inc. LOH)
# of human poor-outcome T-ALLs
(n=50) with gene in CNA
(inc. LOH)
7 (7)
6 (7)
6 (7)
6 (7)
0 (5)
0 (5)
2 (2)
3 (3)
0 (1)
1 (1)
1 (1)
1 (1)
1 (1)
0 (0)
0 (0)
0 (0)
1 (1)
0 (0)
6 (6)
5 (6)
5 (6)
5 (6)
0 (5)
0 (5)
2 (2)
2 (2)
0 (1)
3. Supplemental Figure Legends
Supplemental Figure 1. Distribution of Zebrafish T-ALLs Analyzed by aCGH
Genomic DNA from 17 zebrafish T-ALLs was studied by aCGH. Cancers from 4 D. rerio
genetic models were used: hlk (n=6), otg (n=4), srk (n=6), and mMyc (n=1). Neoplasias
consisted of 14 primary T-ALLs (hlk n=5, otg n=3, srk n=5, mMyc n=1) and 3 seriallytransplanted T-ALLs (hlk passage #7, otg passage #4, and srk passage #3).
Supplemental Figure 2. CNAs are Abundant in Primary Zebrafish T-ALLs
(A) Genomic representations of 11 primary zebrafish T-ALLs (3 other primary T-ALLs
are shown in Supp. Figure 4) and pooled normal T cells from 25 wild-type fish. Nonmalignant tissue from each fish was also tested by aCGH for signal normalization and to
prevent spurious detection of germline copy number variations (CNVs) as somaticallyacquired CNAs. T cells were normalized to pooled non-T cell DNA from the same group
of 25 fish. Two arrays were used: NimbleGen 385K (top 8 samples) or Agilent 418K
(bottom 3 samples and 6 samples in Supp. Figure 4). Red lines denote deletions; green
lines signify amplifications. TCR rearrangement-associated deletions on chromosomes 2
and 17 are marked by black arrows on the normal T cell genome, and were also seen in
T-ALL samples. Seven trisomies in 5 T-ALLs are highlighted by asterisks. Graphics were
created using Nexus5 Copy Number software. (B) Genomic representations of all 14
primary T-ALLs, now with aCGH data pooled and distributed by chromosome. Samples
analyzed with NimbleGen arrays (n=8) are at top; 6 T-ALLs tested using the Agilent
platform are shown at bottom (3 passaged T-ALLs are shown in Supp. Figure 4). TCR
deletions are denoted by red arrowheads. Red and green lines again correspond to
deleted and amplified loci. Lines with greater amplitude represent recurrent CNAs in
multiple samples.
Supplemental Figure 3. CNAs in Zebrafish and Human T-ALLs
Zebrafish (n=17) and human (n=75) T-ALL DNA was hybridized to genomic arrays [for
zebrafish: NimbleGen 385K (n=8) or Agilent 418K (n=9); for humans: Agilent 244K
(n=47), Affymetrix 500K (n=8), or Affymetrix snp6 (n=20)]. aCGH results were analyzed
using Nexus5 Copy Number software. CNAs, amplifications, and deletions per sample
are plotted by platform. (a) In zebrafish, 840 total CNAs were detected. Black bars depict
mean (NimbleGen 22.0, Agilent 73.8, Total 49.4). Grey whiskers represent standard
error of the mean. A total of 335 amplifications (19.7/sample) were seen, with 14.6 gains
per sample detected by the NimbleGen array and 24.2 gains/sample detected using the
Agilent array. Deletions were also prevalent (n=505; 29.7 per sample), with 7.4/sample
and 49.6/sample on NimbleGen and Agilent arrays, respectively. (b) In humans, 1578
total CNAs were detected. Black bars depict mean (Agilent 17.8, Affymetrix 35.3, Total
24.2). Genomic gains (n=510; 6.4 per sample) and losses (n=1068; 18.3 per sample)
were also common. Agilent platforms detected 3.4 amplifications/sample and 14.4
deletions/sample. Affymetrix arrays identified 11.6 gains/T-ALL and 25.1 losses/T-ALL.
Supplemental Figure 4. Genomes of Primary and Passaged D. rerio T-ALLs
Genomic depictions of 3 primary zebrafish T-ALLs paired with the same cancer after
several transplant iterations (srk 3rd passage; hlk 7th passage; otg 4th passage). CNAs in
pooled normal T cells, including T cell receptor deletions (arrowheads) are shown at
bottom. Samples were normalized to non-malignant/non-T cell tissue from matched fish.
In all 3 pairs, most CNAs are retained in the passaged T-ALL, demonstrating its clonal
relationship to the original cancer. Other figure details are as in Supp. Figure 2a.
Supplemental Figure 5. Examples of Primary and Passaged Zebrafish T-ALLs
(A) D. rerio lines used in these studies carry an lck::EGFP transgene (Langenau et al
2004), rendering their T cells GFP+. At left, wild type fish is shown. The thymus is GFP+,
located posterior and dorsal to the eye. Three panels at right show progression of T cell
malignancy in one fish over a 4 week time course. Disease begins as a focal tumor in
the thymic region, grows locally, and spreads throughout the fish, including the marrow
and peripheral blood (Frazer et al 2009). (B) Fish from 2 iterative transplantation series
are shown. In upper left panel, a primary T-ALL (i.e., donor fish) is shown. This fish was
sacrificed, dissected, and GFP+ cells captured by FACS. Some primary T-ALL cells were
used to prepare DNA for aCGH. Other GFP+ cells were intra-peritoneally injected into
irradiated recipients as described in the methods. In the 1st round host, GFP+ cells can
be seen at the injection site by day 10 (d10), throughout the abdomen by d20, are widely
disseminated by d25, and host death occurred on d35. In later transplant rounds, this
same sequence of events follows an accelerated course, as shown in d3, d12, and d17
images on the bottom row. Survival curves depicting results from >100 recipients
transplanted with otg, srk, or hlk T-ALLs [Figure 1a and our previous studies (Frazer et al
2009, Rudner et al 2010)] demonstrate the consistency of this experimental finding.
Supplemental Figure 6. Expression of Recurrent CNA Genes in Human T-ALL
Top 2 rows show expression levels for 2 recurring CNA genes from primary T-ALLs.
Each box signifies an individual human primary T-ALL case (n=40). Transcript levels are
represented as a heat map, with red indicating high expression. OSR2 was deleted in
4/14 (28.6%) zebrafish primary T-ALLs and seen in CNAs from 6/61 (9.8%) human
primary T-ALLs. In 40 human primary cases, 38 samples (95%) had low OSR2 mRNA
levels. This suggests OSR2 repression may be common in T-ALL, occurring by either
genomic deletion or other mechanisms. For TGFBR1 [deleted in 5/14 (35.7%) of D. rerio
primary T-ALLs and found in CNAs from 6/61 (9.8%) human primary T-ALLs], transcript
levels were low in 30 cases (75%), intermediate in 7 cases (17.5%), and high in only 3
cases (7.5%). No appreciable skewing is seen between good outcome (n=21) and poor
outcome (n=19) cases, as expected for CNA gene candidates identified from primary
samples. Bottom row shows expression levels for C7orf60, a gene amplified in 2/3
passaged zebrafish T-ALLs. C7orf60 CNAs were present in 6/50 poor outcome human
T-ALLs, but only 1/25 good outcome cases. Consistent with CNA findings, C7orf60
mRNA was low in good outcome cases (1/21 with intermediate-to-high level), but high in
several poor outcome cases (2/6 induction failures and 4/13 cases destined to relapse).
The 5 samples with highest C7orf60 levels all derive from the poor outcome group.
Survival was also poorer in cases with C7orf60 rearrangements (Figure 2c). Expression
data is from publicly-accessible sources (Gutierrez et al 2010a, Gutierrez et al 2010b).
References for Supplemental Methods, Tables, and Figures
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