evolution of genomic abnormalities in relapsed acute lymphoblastic leukemia

102 Pages·2008·2.9 MB·English

www.sciencemag.org/cgi/content/full/322/5906/1377/DC1

Supporting Online Material for

Genomic Analysis of the Clonal Origins of Relapsed Acute Lymphoblastic
Leukemia
Charles G. Mullighan, Letha A. Phillips, Xiaoping Su, Jing Ma, Christopher B. Miller,
Sheila A. Shurtleff, James R. Downing*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 28 November 2008, Science 322, 1377 (2008)
DOI: 10.1126/science.1164266

This PDF file includes:

Materials and Methods
SOM Text
Figs. S1 to S15
Tables S1 to S21
References
SUPPLEMENTARY METHODS
Cases and samples studied
Sixty one cases of relapsed acute lymphoblastic leukemia treated at St Jude Children’s Research
Hospital from 1988-2007 with cryopreserved peripheral blood or bone marrow samples obtained
at both diagnosis and relapse were identified. The cohort comprised 47 B-progenitor (six high
hyperdiploid, one TCF3-PBX1, five ETV6-RUNX1, five MLL-rearranged, four BCR-ABL1, and
26 with low hyperdiploidy, pseudodiploid or miscellaneous/normal karyotypes) and 14 T-lineage
ALL cases (Supplementary Table 1). The study fulfilled requirements of the Declaration of
Helsinki, and was approved by the St Jude Children’s Research Hospital Institutional Review
Board.
Sample preparation and DNA extraction.
Samples with at least 80% blasts (as assessed by morphologic examination or
immunophenotyping at the time of cryopreservation) were thawed, pelleted and DNA extracted
without further processing. Diagnostic or relapse samples with less than 80% blasts were flow
sorted to at least 90% blast purity prior to DNA extraction using FACSVantage SE (with DiVa
option) flow cytometers (BD Biosciences, San Jose, CA) and fluorescein isothiocyanate labelled
CD45, allophycocyanin labelled CD33, allophycocyanin labelled CD7 (Invitrogen (CALTAG),
Carlsbad, CA) and phycoerythrin labelled CD3, CD5, CD13 CD19 antibodies (BD Biosciences).
DNA was extracted from leukemic blasts using the DNA blood mini kit (Qiagen, Valencia, CA)
or phenol/chloroform. DNA quality and integrity was assessed using a Nanodrop ND-1000
spectrophotometer (Wilmington, DE) or by agarose gel electrophoresis.
Single nucleotide polymorphism microarray analysis
500ng of sample DNA was digested with NspI and StyI restriction enzymes and processed
according to the manufacturer’s instructions for Affymetrix 250k Nsp and Sty arrays (14
diagnosis and relapse pairs) or Affymetrix SNP 6.0 arrays (47 pairs). For the 250k mapping
arrays, SNP genotype calls were generated using the DM algorithm in GTYPE v 4.0
(Affymetrix). For 6.0 arrays, SNP genotype calls were generated using the birdseed v1 or v2
algorithms in Genotyping console v1 or v2.0 (Affymetrix). Genotyping using the birdseed
algorithm was performed using at least 44 arrays in each analysis.
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Array normalization and copy number inference was performed according to a previously
published workflow (1, 2). Extraction and summarization of SNP/CNV probe hybridization
intensities from CEL files was performed in dChip (3) using the PM/MM (250k arrays) or PM-
only (6.0 arrays) model-based expression algorithms. Un-normalized data was exported from
dChip and normalization performed using a “reference normalization” algorithm that uses only
probes from chromosomes known or predicted to be diploid (based on SNP genotype frequency
data) to guide array normalization. This approach normalizes each array independently without
“borrowing” information across arrays, and results in more accurate downstream copy number
inferences than alternative approaches (ref. (1) and SB Pounds et al., submitted). Normalized
data were then viewed in dChip and regions of abnormal copy number identified
computationally by circular binary segmentation (CBS) (4). CBS results were filtered to use only
segments of at least 5 markers (SNP and/or CNV) with segment copy number means of >2.29 or
<1.69 for downstream analysis. Notably, signals of both SNP and copy number variant probes on
the 6.0 arrays were processed, normalized, and used for copy number inference simultaneously,
thus maximizing the resolution of the arrays. SNP array CEL, CHP and TXT file data are
available from the authors upon request.
For samples with matched germline array data, segmentation was performed pairwise, with each
diagnostic or relapse sample compared directly to the corresponding germline sample. For
samples without a matched germline sample, segmentation was performed using a pool of
germline samples run in the same SNP array batch (typically 7-12 germline samples in each
batch). To distinguish somatic from inherited copy number variants, each identified region of
copy number alteration was compared to a local database of over 300 reference samples, and
public databases of copy number polymorphisms (http://projects.tcag.ca/variation/ and
http://www.genome.ucsc.edu/). Copy number abnormalities in unpaired samples similar or
matching those previously described or observed in our local repository were deemed inherited
and excluded from subsequent analysis.
To systematically identify copy number abnormalities acquired at relapse, we first identified all
segments of CNA at diagnosis by comparing each diagnosis sample to the corresponding
germline or pool of unpaired germline samples, and then identified all segments of CNA in
relapse samples similarly. We then subtracted curated diagnosis segmentation data from curated
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relapse segmentation data. As confirmation, circular binary segmentation was also performed
pairwise by comparing the relapse sample directly to the corresponding diagnostic sample. Final
lesion listings and tallies exclude DNA gains and losses arising from B- and T- cell antigen
receptor gene rearrangements at 2p11.2 (IGK@), 7p14.1 (TRG@), 7q34 (TRB@), 14q11.2
(TRA@), 14q32.33 (IGH@) and 22q11.22 (IGL@).
In order to perform unsupervised hierarchical clustering of copy number abnormalities, CBS
segmentation data was filtered as described above, and then manually curated to exclude
inherited copy number variants and segments arising from experimental artifact (e.g. inter-batch
effects). To ensure data from the SNP 250k arrays (the annotations for which are based on the
hg17 genome build) were comparable to data from the SNP 6.0 arrays (based on the hg18
genome build), genome coordinates of the segments from the SNP 6.0 arrays were converted to
the corresponding hg17 genome coordinates using the command line liftOver tool available from
UCSC genome browser (http://hgdownload.cse.ucsc.edu/admin/exe/). Curated CBS segments
were then re-extrapolated into a genome-wide matrix of 615,000 probe-set features for all
diagnosis and relapse cases. To reduce data set complexity and permit clustering, autosomal
copy number data was binned into 500Kb sliding windows with 50% overlap between windows,
resulting in a matrix of 10,746 rows (500Kb copy number bins) and 122 columns (61 diagnosis-
recipient pairs). When deletions and gains were present in the same window, deletion was given
precedence. Clustering was performed using the Pearson correlation similarity and Ward linkage
clustering algorithm in GeneMaths XT v 2.1 (Applied Maths, Austin, TX).
Pathway analysis of CNAs acquired at relapse
Enrichment of genes involved by CNAs acquired at relapse in biological pathways was assessed
using Fisher’s exact test. The pathways examined included the 147 canonical pathways defined
by Ingenuity Pathway analysis (Ingenuity Systems, Redwood City, CA) and a B-lymphoid
developmental pathway consisting of transcriptional regulators of normal lymphoid development
(comprising IL7RA, SPI1, TCF3, IKZF1, IKZF2, IKZF3, EBF1, PAX5, LEF1, IRF4, IRF8,
BCL11A, SOX4, STAT3, STAT5A, STAT5B, BLNK, RAG1, RAG2, MEF2C, CD79A, GAPBA).
Relapse-CNAs were mapped to the genomic locations of each gene, including a window of 20kb
proximal and distal to each gene.
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To identify pathways most frequently affected by the relapse-CNAs, Fisher’s exact test was
performed for the genes involved by CNAs for each case using a contingency table as follows:
Genes involved by relapse Genes not involved by relapse
CNAs for each case CNAs for each case
Pathway X Number of genes in a given Number of genes in pathway
pathway X involved by CNAs X not involved by CNAs at
at relapse relapse
Non-Pathway X Number of genes not in Number of genes not in
pathway X, but involved by pathway X and not involved
CNAs at relapse by CNAs at relapse

Significantly enriched pathways for each case were identified, and the false discovery rate
estimated using the method of Benjamini and Hochberg (5). Pathways below an FDR threshold
of 30% were reported. The number of cases showing significant enrichment for each pathway
was used to rank the pathways.
Genomic quantitative real-time PCR
Confirmation of copy number abnormalities using genomic quantitative real-time PCR (gqPCR)
was performed for PAX5 and IKZF1 deletions as previously described. (1, 2). gqPCR for
deletions involving ADD3, C20orf94, CDKN2A/B, the IKZF1 promoter region, IKZF2, IKZF3,
and MKKS was performed using primers and probes detailed in Supplementary Table 2.
Genomic PCR for lesion backtracking
Lesion-specific qualitative genomic PCR assays were designed by tiling primers outward from
the genomic locations of SNP array probes defining the minimal regions of deletion at 500-1500
bp intervals to beyond the next non-deleted probes up- and downstream of the deletion. Primers
were designed using Primer3 (6), and a matrix of PCR reactions using all combinations of
forward and reverse PCR primers used to amplify the region of genomic deletion. The genomic
deletion breakpoints were defined by direct sequencing of PCR products. PCR was performed
using 25-50ng genomic DNA in 25 l reaction volumes using the Advantage 2 PCR Kit
(Clontech, Mountain View, CA), Mastercycler PCR machines (Eppendorf) and the following
thermal cycling conditions: 5 cycles of 94°C for 30 sec and 72°C 3 min, followed by 5 cycles of
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94°C for 30 sec, 70°C for 30 sec and 72°C 3 min, followed by 25 cycles of 94°C for 30 sec,
68°C for 30 sec and 72°C 3 min. Sequences of primers used for lesion-specific PCR are shown
in Supplementary Table 3.

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Supplementary Table 1. ALL samples examined by SNP array analysis

*Published in refs (1, 2). 6.0, Affymetrix SNP 6.0 arrays; 500K, Affymetrix 250k Sty and Nsp
arrays. TCF3-PBX1 cases are referred to as E2A-PBX1, and ETV6-RUNX1 as TEL-AML1 for
consistency with sample naming in our previous publications.
SNP
Previously array
Diagnosis ID published?* Remission sample ID data Relapse ID
Hyperdip50-SNP-#27 Yes Hyperdip50-SNP-#27GL 6.0 Hyperdip50-SNP-#27R
Hyperdip50-SNP-#51 No 6.0 Hyperdip50-SNP-#51R
Hyperdip50-SNP-#52 No 6.0 Hyperdip50-SNP-#52R
Hyperdip50-SNP-#53 No Hyperdip50-SNP-#53GL 6.0 Hyperdip50-SNP-#53R
Hyperdip50-SNP-#54 No Hyperdip50-SNP-#54GL 6.0 Hyperdip50-SNP-#54R
Hyperdip50-SNP-#55 No 6.0 Hyperdip50-SNP-#55R

E2A-PBX1-SNP-#12 Yes E2A-PBX1-SNP-#12GL 6.0 E2A-PBX1-SNP-#12R
TEL-AML1-SNP-#10 Yes TEL-AML1-SNP-#10GL 500K TEL-AML1-SNP-#10R
TEL-AML1-SNP-#44 Yes TEL-AML1-SNP-#44GL 500K TEL-AML1-SNP-#44R
TEL-AML1-SNP-#48 Yes TEL-AML1-SNP-#48GL 6.0 TEL-AML1-SNP-#48R
TEL-AML1-SNP-#49 No TEL-AML1-SNP-#49GL 6.0 TEL-AML1-SNP-#49R
TEL-AML1-SNP-#50 No 6.0 TEL-AML1-SNP-#50R
MLL-SNP-#6 Yes MLL-SNP-#6GL 500K MLL-SNP-#6R
MLL-SNP-#8 Yes MLL-SNP-#8GL 500K MLL-SNP-#8R
MLL-SNP-#23 Yes MLL-SNP-#23GL 6.0 MLL-SNP-#23R
MLL-SNP-#24 No MLL-SNP-#24GL 6.0 MLL-SNP-#24R
MLL-SNP-#25 No MLL-SNP-#25GL 6.0 MLL-SNP-#25R
BCR-ABL-SNP-#11 Yes 6.0 BCR-ABL-SNP-#11R
BCR-ABL-SNP-#16 Yes BCR-ABL-SNP-#16GL 6.0 BCR-ABL-SNP-#16R
BCR-ABL-SNP-#15 Yes BCR-ABL-SNP-#15GL 6.0 BCR-ABL-SNP-#15R
BCR-ABL-SNP-#18 Yes BCR-ABL-SNP-#18GL 6.0 BCR-ABL-SNP-#18R
Hyperdip47-50-SNP-#4 Yes Hyperdip47-50-SNP-#4GL 6.0 Hyperdip47-50-SNP-#4R
Hyperdip47-50-SNP-#24 Yes 500K Hyperdip47-50-SNP-#24R
Pseudodip-SNP-#2 Yes Pseudodip-SNP-#2GL 500K Pseudodip-SNP-#2R
Pseudodip-SNP-#7 Yes Pseudodip-SNP-#7GL 500K Pseudodip-SNP-#7R
Pseudodip-SNP-#18 Yes Pseudodip-SNP-#18GL 6.0 Pseudodip-SNP-#18R
Pseudodip-SNP-#23 Yes 500K Pseudodip-SNP-#23R
Other-SNP-#12 Yes Other-SNP-#12GL 500K Other-SNP-#12R
Other-SNP-#13 Yes Other-SNP-#13GL 500K Other-SNP-#13R
Other-SNP-#21 Yes Other-SNP-#21GL 6.0 Other-SNP-#21R
Other-SNP-#27 No 6.0 Other-SNP-#27R
Other-SNP-#28 No 6.0 Other-SNP-#28R
Other-SNP-#29 No 6.0 Other-SNP-#29R
Other-SNP-#30 No Other-SNP-#30GL 6.0 Other-SNP-#30R
Other-SNP-#31 No 6.0 Other-SNP-#31R
Other-SNP-#32 No Other-SNP-#32GL 6.0 Other-SNP-#32R
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SNP
Previously array
Diagnosis ID published?* Remission sample ID data Relapse ID
Other-SNP-#33 No Other-SNP-#33GL 6.0 Other-SNP-#33R
Other-SNP-#34 No Other-SNP-#34GL 6.0 Other-SNP-#34R
Other-SNP-#35 No Other-SNP-#35GL 32 Other-SNP-#35R
Other-SNP-#36 No Other-SNP-#36GL 6.0 Other-SNP-#36R
Other-SNP-#37 No Other-SNP-#37GL 6.0 Other-SNP-#37R
Other-SNP-#38 No Other-SNP-#38GL 6.0 Other-SNP-#38R
Other-SNP-#40 No Other-SNP-#40GL 6.0 Other-SNP-#40R
Other-SNP-#41 No Other-SNP-#41GL 6.0 Other-SNP-#41R
Other-SNP-#42 No Other-SNP-#42GL 6.0 Other-SNP-#42R
Other-SNP-#43 No Other-SNP-#43GL 32 Other-SNP-#43R
Other-SNP-#39 No Other-SNP-#39GL 6.0 Other-SNP-#39R
T-ALL-SNP-#4 Yes T-ALL-SNP-#4GL 500K T-ALL-SNP-#4R
T-ALL-SNP-#31 Yes T-ALL-SNP-#31GL 500K T-ALL-SNP-#31R
T-ALL-SNP-#34 Yes T-ALL-SNP-#34GL 500K T-ALL-SNP-#34R
T-ALL-SNP-#37 Yes T-ALL-SNP-#37GL 500K T-ALL-SNP-#37R
T-ALL-SNP-#36 Yes T-ALL-SNP-#36GL 6.0 T-ALL-SNP-#36R
T-ALL-SNP-#43 Yes T-ALL-SNP-#43GL 6.0 T-ALL-SNP-#43R
T-ALL-SNP-#46 Yes T-ALL-SNP-#46GL 6.0 T-ALL-SNP-#46R
T-ALL-SNP-#51 No 6.0 T-ALL-SNP-#51R
T-ALL-SNP-#52 No 6.0 T-ALL-SNP-#52R
T-ALL-SNP-#53 No T-ALL-SNP-#53GL 6.0 T-ALL-SNP-#53R
T-ALL-SNP-#54 No T-ALL-SNP-#54GL 6.0 T-ALL-SNP-#54R
T-ALL-SNP-#55 No T-ALL-SNP-#55GL 6.0 T-ALL-SNP-#55R
T-ALL-SNP-#57 No T-ALL-SNP-#57GL 6.0 T-ALL-SNP-#57R
T-ALL-SNP-#58 No T-ALL-SNP-#58GL 6.0 T-ALL-SNP-#58R

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Supplementary Table 2. Genomic quantitative PCR primers

Quantitative PCR primers were designed using Primer Express 3.0 (Applied Biosystems, Foster
City, CA). Primers for CDKN2B exon 1 reaction 1 (C745-747) and exon 1 reaction 2 (C748-750)
are alternative primer sets that are both located in exon 1 of CDKN2B. Primers for CDKN2A
exon 2 reaction 1 (C769-771) are located in intron 1 immediately upstream of exon 2. Primers
for CDKN2A exon 2 reaction 2 are located in intron 2 downstream of exon 2. F, forward primer;
FAM, 6-carboxyfluorescein; MGB, minor groove binder; P, probe; R, reverse primer.
Primer Description Sequence (5' → 3')
C745 CDKN2B exon 1 F1 GACGACGGGAGGGTAATGAA
C746 CDKN2B exon 1 R1 GGCGCACTCTCTCCTTCCT
C747 CDKN2B exon 1 P1 FAM-AGCCCAGGTCTCC-MGB
C748 CDKN2B exon 1 F2 CGTGGGAAAGAAGGGAAGAGT
C749 CDKN2B exon 1 R2 GCGGCCCGGATAATCC
C750 CDKN2B exon 1 P2 FAM-TCGTTAAGTTTACGGCCAAC-MGB
C754 CDKN2B exon 2 F2 GCACTGGGTGAAAACTTTGCA
C755 CDKN2B exon 2 R2 CCAGAGAGAGCAGAGTGGTCAGA
C756 CDKN2B exon 2 P2 FAM-TAGGTGTTTCTTTAAATGGC-MGB
C757 CDKN2A exon 1 beta F1 CCGCTTCCTAGAAGACCAGGTA
C758 CDKN2A exon 1 beta R1 AACAAAACAAGTGCCGAATGC
C759 CDKN2A exon 1 beta P1 FAM-AAAGGCCCTCGAAAAG-MGB
C763 CDKN2A exon 1 alpha F1 CCAACGCACCGAATAGTTACG
C764 CDKN2A exon 1 alpha R1 TTCCAATTCCCCTGCAAACTT
C765 CDKN2A exon 1 alpha P1 FAM-CCGATCCAGGTGGGTAG-MGB
C769 CDKN2A exon 2 F1 AAGAATGGATAGAGAACTCAAGAAGGA
C770 CDKN2A exon 2 R1 CCCAGGTGTCTAATTACCCCTACA
C771 CDKN2A exon 2 P1 FAM-ATTGGAAACTGGAAGCAA-MGB
C772 CDKN2A exon 2 F2 ATTGGAAACTGGAAGCAA
C773 CDKN2A exon 2 R2 GCTCTCAGGGTACAAATTCTCAGAT
C774 CDKN2A exon 2 P2 FAM-CTCAGGTGAGGACTGAT-MGB
C933 IKZF1 promoter F GATTTCCGCCAGGCTCAAG
C934 IKZF1 promoter R CAATCAGCACACGGGACAAG

C935 IKZF1 promoter P FAM-TTGGCCTCTTGGTTTC-MGB

C1035 C20orf94 exon 2 F TCAGCCCATCTTTTTCCTGTATAAT

C1036 C20orf94 exon 2 R TGCCATGGCTTATTGATTCCA
C1037 C20orf94 exon 2 P FAM-TTTCTTAAAGGTCTGTAGTTACT-MGB
C1038 MKKS exon 2 F CATGCAGTGCCGTCTGACA
C1039 MKKS exon 2 R GATGTGAACGTCTGATTCTGATGAT
C1040 MKKS exon 2 P FAM-TGAGCTAAGAAAAAAC-MGB
C1054 IKZF3 (Aiolos) exon 1 F CCGCCCTTTGGAAAGTGATA
C1055 IKZF3 (Aiolos) exon 1 R CCCCCATGTCTGACAACTCTTTA
C1056 IKZF3 (Aiolos) exon 1 P FAM-CTTCTTCGCAATTTT-MGB
C1057 ADD3 intron 1 F TGTGACTCCAAGGCTCTGAAAA
C1058 ADD3 intron 1 R GCAGACTTCAGTGAGAGCAAACTG
C1059 ADD3 intron 1 P FAM-TGTGTGGCAATATTAATGT-MGB
C1060 IKZF2 (Helios) 5’ F GGGCCCCTCGACACTTG
C1061 IKZF2 (Helios) 5’ R AGGGTGTTCCTCTTCCACTATCC
C1062 IKZF2 (Helios) 5’ P FAM-CGAAAAAGAGTTTAAGAAAC-MGB
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Supplementary Table 3. Primers used for backtracking of deletions identified at relapse on
SNP array analysis.

*Case Other-SNP-#28. **Case Other-SNP-#29. ***Case Other-SNP-#30
Primer Description Sequence (5' → 3')
C1002 ADD3 F ggggagtgagaggttcctgcgctta
C1008 ADD3 R cgcgcccggctaaatgacagttttt
C874 C20orf94 F gataattcacccggcatttccccatc
C872 C20orf94 R atgcccccgaggcctctctacaaact
C966 IKZF2 (Helios) F tggaaaggccactggtcaatgttgtt
C972 IKZF2 (Helios) R ctgtccattgggtgtgcttgaggtg
C952 IKZF3 (Aiolos) F ggggtttcacaggtgtggaattgga
C955 IKZF3 (Aiolos) R aggttgaggcggcaagtaagccttg
C1083 ETV6 F* gcacctggccctgctatgactttca
C1088 ETV6 R* tcaaggcatccagaagtgaaccagga
C1138 ETV6 F** catggtctggacatggctgaggaaa
C1140 ETV6 R** tccccacgcattctctctccatcat
C1074 ETV6 F*** cccaccgttttgatgggcttatggt
C1078 ETV6 R*** gaccagccctggcattctggttgta

12