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Leukemic T-cell Precursors from T-lineage All Patients Characterized by Profound Ku80 Deficiency
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related gene expression profiles, whereby genes joined
by short branch lengths showed most similarity in
expression profile across samples (Uckun et al., 2012;
Ma et al., 2013).
In separate analyses of gene expression profiles of
lymphocyte precursors with high vs low
Ku
expression levels, we compiled the gene expression
profiles of 354 primary leukemia specimens from
newly diagnosed or relapsed ALL patients (GSE3912,
N=113; GSE18497,
N=82; GSE4698,
N=60;
GSE7440, N=99). We focused our analysis on the
genes for the regulatory cytokines/peptides (
FGFR4
,
IL10
,
IL13
,
IL4
,
IL5
,
STAT4
and
VIPR1
) comparing
samples with high versus low
Ku
expression. These
genes were identified as IK-regulated genes in human
cells (Yu et al., 2002; Umetsu and Winandy, 2009;
Mary et al., 2009; Gregory et al., 2006; Yap et al.,
2005; Dorsam and Goetzl, 2002). For each of the 4
studies, the expression values of the
IK1
target gene
transcripts common in across the Affymetrix platforms
(204579_at
(
FGFR4
),
211237_s_at
(
FGFR4
),
207433_at (
IL10
), 207844_at (
IL13
), 207538_at (
IL4
),
207539_s_at (
IL4
), 207952_at (
IL5
), 206118_at
(
STAT4
), and 205019_s_at (
VIPR1
) were standardized
relative to mean expression value across all samples in
that study (Z-scores in standard deviation units).
Expression values expressed as Z-scores were
compiled for the 4 studies and rank ordered according
to the mean expression of three highly correlated
transcripts (208642_s_at (
XRCC5
), 208643_s_at
(
XRCC5
), 200792_at (XRCC6)). Prospective power
analysis was utilized to determine Z-score cut-off for
“high”
Ku
expression and “low”
Ku
expression in the
data sets. To control for False Positive Rate (FPR) to
detect for differences in 3
Ku
transcripts out of
approximately 20 000 transcripts common across the 4
Affymetrix transcripts, we set the unadjusted P-value
at 1 × 10
-5
(FPR = 0.07). Sample size greater than 102
would provide sufficient power to detect a difference
of 1.4 standard deviation units at the 99% level.
Therefore, samples were defined as “high”
Ku
expression if their expression was greater than 0.7
standard deviations units and “low”
Ku
expression if
the sample exhibited expression level lower than 0.7
standard deviation units and intermediate expression
for the remaining samples (N=217). To determine the
differential expression of each gene transcript, T-tests
were performed for the combined Z-scores from the 4
datasets (2-sample, Unequal variance correction,
p-values<0.05 deemed significant) to compare the
expression of high
Ku
expression (N=67) versus low
Ku
expression (N=70) samples for the IK targets. We
used a one-way agglomerative hierarchical clustering
technique to organize expression patterns using the
average distance linkage method such that genes
(rows) having similar expression across patients were
grouped together (average distance metric).
Dendrograms were drawn to illustrate similar
gene-expression profiles from joining pairs of closely
related gene expression profiles, whereby genes joined
by short branch lengths showed most similarity in
expression profile across samples.
Pairwise
correlations (r; JMP Software (SAS, Cary, NC)) of the
average expression of the 3
Ku
transcripts versus each
of the 9 transcripts of IK target genes were performed
using the 354 samples compiled from the 4 studies.
The correlation co-efficient and un-adjusted P-values
(P<0.05 deemed significant) were reported to identify
IK target genes driven by
Ku
expression.
Acknowledgments
The project described was supported in part by DHHS
grants P30CA014089,
U01-CA-151837,
and
R01CA-154471 (to FMU). The content is solely the
responsibility of the authors and does not necessarily
represent the official views of the National Cancer
Institute or the National Institutes of Health. This
work was also supported in part by Children’s
Hospital Los Angeles Institutional Endowment and
Special
Funds (FMU),
2011 V-Foundation
Translational Research Award (FMU), Ronald
McDonald House Charities of Southern California
(FMU), Couples Against Leukemia Foundation
(FMU). KU70 and Ku80 cDNA were kindly provided
by Dr. Stephen P. Jackson (Wellcome Trust/Cancer
Research UK Gurdon Institute).
Authors’ Contributions
F.M.U conceived and supervised this study and wrote
the final manuscript. All authors have contributed to
the design and conduct of the research. All authors
reviewed the paper. The authors have declared no
Molecular Medical Science, Int’l Journal of