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MCT-11-0730
Molecular
Cancer
Therapeutics
Preclinical Development
Abstract
The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other
malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a
significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro
activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of
human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with
variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast
epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to
determine IC50 values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified
lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated
with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority
of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0G1 arrest and an
induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and
acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to
lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative
effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers
resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 197887. 2012 AACR.
Introduction
The HER (ErbB) receptor family consists of 4 type I
receptor tyrosine kinases, which include the EGF receptor (EGFR, HER1, and ErbB-1), HER2 (HER2/neu,
ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4), and their
associated ligands, including EGF, neuregulins (NRG14), TGF-a, amphiregulin, betacellulin, heparin-binding
EGF-like growth factor and epiregulin. Ligand binding
to the extracellular domain leads to homodimerization
or heterodimerization of the receptors and autophosphorylation within the intracellular domain. This
results in the activation of signaling cascades involved
in mediating cell growth and differentiation. Unlike
Authors' Afliations: 1Department of Medicine, Division of Hematology/
Oncology, Geffen School of Medicine at UCLA, Los Angeles; and 2Pzer
Global Research and Development, Pzer Inc., San Diego, California
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Richard S. Finn, Department of Medicine, Division
of Hematology/Oncology, Geffen School of Medicine at UCLA, 10833 Le
Conte Ave, 11-934 Factor Bldg, Los Angeles, CA 90095. Phone: 310-5862091; Fax: 310-586-6830; E-mail: Rnn@mednet.ucla.edu
doi: 10.1158/1535-7163.MCT-11-0730
2012 American Association for Cancer Research.
1978
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Lapatinib
Dacomitinib
N
N
H3CO
N
N
HN
O
HN
CI
F
H2O
HN
HN
O
O
O
CI
F
www.aacrjournals.org
zumab 105 mg/mL. Lapatinib-resistant BT-474 (BT-474LR) and SK-BR-3 (SK-BR-3-LR) cell lines (pools of resistant cells) were established after serial passage in the
presence of gradually increasing concentrations of lapatinib (0.17 mmol/L). These lines were established in our
laboratories, authenticated, and checked for Mycoplasma
contamination as described above.
HER2 amplification was defined as greater than 2
HER2/neu FISH signals per chromosome 17 centromere
FISH signal (22). A SpectrumOrange labeled HER2/neu
probe (ABBOTT Molecular) and SpectrumGreen labeled
chromosome 17 alpha-satellite centromere probe were
used (ABBOTT Molecular).
Proliferation assays
Cells were seeded in duplicate at 5 103 to 5 104 cells
per well in 24-well plates, and growth inhibition data was
calculated as described previously (19). Briefly, day after
plating, dacomitinib was added at 10 mmol/L and 2-fold
dilutions over 12 concentrations were carried out to generate a doseresponse curve. Control wells without the
drug were also seeded. The cells were counted on day 1
when the drug was added, as well as after 6 days when the
experiment ended. After the trypsinization cells were
placed in an Isotone solution and immediately counted
using a Coulter Z1 particle counter (Beckman Coulter,
Inc.). The suspension cultures were counted using a Coulter Vi-Cell counter (Beckman Coulter, Inc.).
Microarray analysis of cell lines
Agilent microarray analyses were developed for each
cell line as described previously (23, 24). These data are
available with GEO accession number GSE18496.
Western blots and protein quantification
To determine the effect of dacomitinib on the expression
of analyzed proteins, cells in log-phase growth were
treated with 0.1 or 1 mmol/L dacomitinib and lysates
were taken as described (19). Details about antibodies
and immunoprecipitation techniques can be found in
Supplementary Material.
Cell-cycle analysis and apoptosis studies
The effects of dacomitinib on the cell cycle were
assessed using Nim-DAPI (40 , 6-diamidino-2-phenylindole) staining (NPE Systems). The cells were plated
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Kalous et al.
Results
Dacomitinib preferentially inhibits growth of HER2amplified breast cancer cell lines in vitro
A panel of 44 human breast cancer cell lines, representing
luminal, nonluminal subtypes, and 3 immortalized breast
epithelial lines (19), was used to test the antiproliferative
effect of dacomitinib (PF-00299804). The calculated IC50
values for each cell line and its molecular classification, as
well as HER2 amplification status (by FISH) and estrogen
receptor (ER) status, were determined (Table 1 and Fig. 2).
Sensitivity was defined as IC50 < 1 mmol/L (the rationale for
selecting this cut-off is discussed below).
As a group, the HER2-amplified cell lines were most
sensitive to growth inhibition by dacomitinib (IC50 < 1
mmol/L in 14 of 16 lines; 87.5%) as compared with 5 of 28
(17.9%) of HER2-nonamplified lines (excluding immortalized lines). MDA-MB-453 and UACC-732 were the only
HER2-amplified lines resistant to the antiproliferative
effect of the compound (IC50 > 1 mmol/L). The nonluminal
lines were the most resistant to this compound. c2 (CMH)
analysis was carried out to compare the response classification (response defined as IC50 < 1 mmol/L) in the
HER2-amplified versus nonamplified groups of cell lines.
HER2-amplified lines were far more likely to be classified
as responders to dacomitinib [RR, 3.39; 95% confidence
interval (CI), 1.826.33; P < 0.0001].
ER status correlated with response when treated as a
separate, independent variable (P 0.015). However, ER
status was highly cross-correlated with HER2 status. After
controlling for HER2 status by stratified analysis, ER
status was no longer a statistically significant predictor
of response (P 0.17).
Quantitative HER2 expression associates with
response to dacomitinib
We also aimed to explore the quantitative effect of
HER2 expression on response to dacomitinib, in compar-
1980
ison with the qualitative amplified/nonamplified approach discussed above. Specifically, we wanted to determine whether a higher relative expression of HER2
mRNA is associated with a stronger response to dacomitinib, even within the HER2-amplified and nonamplified
groups. For this purpose, we ran a linear regression
analysis using log(IC50) as the response variable and
log-ratio of expression by microarray (compared with
mixed breast cancer RNA reference pool) as the predictor
variable. This analysis was carried out first on the entire
panel of lines, then separately on each group of HER2amplified and nonamplified lines. The association was
significant in all 3 analyses. (Full panel: b 1.07, SE
0.15, P < 0.0001, r 0.73; HER2 amplified: b 1.6, SE
0.57, P 0.014, r 0.6; HER2-nonamplified: b 1.57,
SE 0.6, P 0.014, r 0.44; Supplementary Fig. S1).
However, the significance of this relationship in cell
lines with higher IC50 values (resistant) is unknown. In
a separate analysis for HER2-amplified lines, we found
that the total HER2 protein levels (quantified by Western
blot) correlated with HER2 mRNA levels by microarray
(r 0.67, P 0.004). Finally, we found an association
between the HER2 protein levels and response to dacomitinib in HER2-amplified lines (b 1.99, SE 0.55, P
0.003, r 0.69; Supplementary Fig. S2).
All 4 HER family receptors were analyzed for their
relationship between mRNA expression and IC50. The
association between HER2 expression and response to
dacomitinib was by far the strongest. In addition, there
was a marginal inverse correlation between HER3 mRNA
levels and log(IC50) values (r 0.25, P 0.09). No
correlation was observed between the response to dacomitinib and EGFR mRNA (r 0.07, P 0.62) and HER4
mRNA (r 0.05, P 0.75) levels, respectively (Supplementary Fig. S3).
Biochemical effects of dacomitinib on signal
transduction
The effects of dacomitinib on HER2, EGFR, HER4, AKT,
and ERK activation were determined using Western blot
analysis of a subset of lines with variable levels of HER
receptors and sensitivities to dacomitinib.
After a 10-minute exposure to 100 nmol/L or 1 mmol/L
of dacomitinib, we observed no effect on total HER2, AKT,
and ERK in a majority of lines and a minor decrease in total
EGFR in several lines. Most of the selected lines expressed
very low baseline levels of total HER4 that mostly did not
change after the treatment with dacomitinib. Dacomitinib
caused an inhibition of HER2 phosphorylation in all lines
with detectable baseline phosphorylation (Fig. 3, densitometry in Supplementary Table S1). Dacomitinib caused
an inhibition of EGFR phosphorylation in sensitive lines,
but only 2 resistant lines (MDA-MB-453 and MDA-MB231). Dacomitinib decreased the phosphorylation of
HER4 in sensitive lines with detectable baseline phosphorylation. Dacomitinib inhibited phosphorylation of
AKT in all tested sensitive lines, but only 2 resistant lines
(HCC-70 and MDA-MB-453). Similarly, dacomitinib
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Table 1. The calculated IC50 for each cell line and its molecular classication
Cell line
IC50 SE
HER2 status
ER status
HCC-202
ZR-75-30
MDA-MB-175
HCC-2218
SUM-225
HCC-1419
184A1
MCF-10A
SK-BR-3
EFM-192A
UACC-893
BT-474
MDA-MB-361
UACC-812
HCC-1954
184B5
SUM-190
ZR-75-1
HCC-1500
MDA-MB-415
HCC-1143
HCC-1569
EFM-19
COLO-824
MDA-MB-157
T-47D
MDA-MB-468
HCC-70
HCC-1187
UACC-732
MDA-MB-134
HCC-1395
MDA-MB-453
HCC-1937
HCC-1806
BT-549
HCC-38
MDA-MB-436
BT-20
CAMA-1
CAL-51
MDA-MB-435
Hs578T
DU-4475
MCF-7
MDA-MB-231
KPL-1
<0.005
<0.005
<0.005
<0.005
0.006
0.006
0.007
0.008
0.015
0.016
0.017
0.018
0.03
0.04
0.06
0.08
0.11
0.24
0.53
0.67
0.73
0.77
1.07
1.20
1.36
1.41
1.42
1.44
1.46
1.69
1.74
1.77
2.00
2.04
2.05
2.22
2.40
2.56
2.58
2.68
2.76
2.92
2.92
3.55
3.67
4.23
4.88
n/a
n/a
n/a
n/a
0.002
0.002
0.0003
0.001
0.003
0.002
0.006
0.011
0.01
0.00
0.01
0.00
0.01
0.12
0.30
0.07
0.05
0.17
0.16
0.00
0.08
0.69
0.03
0.06
0.16
0.09
0.17
0.64
0.11
0.02
0.18
0.03
0.15
0.00
0.29
0.46
0.46
0.00
0.28
0.99
0.35
0.11
0.57
Luminal
Luminal
Luminal
Luminal
Luminal
Luminal
Immortalized
Immortalized
Luminal
Luminal
Luminal
Luminal
Luminal
Luminal
Basal
Immortalized
Luminal
Luminal
Luminal
Luminal
Basal
Post-EMT
Luminal
Basal
Post-EMT
Luminal
Basal
Basal
Basal
Luminal
Luminal
Post-EMT
Luminal
Post-EMT
Basal
Post-EMT
Basal
Post-EMT
Basal
Luminal
Post-EMT
Post-EMT
Post-EMT
Basal
Luminal
Post-EMT
Luminal
Amplied
Amplied
Normal
Amplied
Amplied
Amplied
n/a
n/a
Amplied
Amplied
Amplied
Amplied
Amplied
Amplied
Amplied
n/a
Amplied
Normal
Normal
Normal
Normal
Amplied
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Amplied
Normal
Normal
Amplied
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Positive
Positive
Positive
Positive
Negative
Positive
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Positive
Positive
Positive
Positive
Negative
Negative
Positive
Negative
Negative
Positive
Negative
Negative
Negative
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
Positive
Negative
Positive
NOTE: Included are molecular subtype and HER2 and ER status. HER2-amplied cell lines were most sensitive to the growth inhibition
effects of dacomitinib. Post-EMT, cell lines classied as representing breast cancers that had undergone an epithelial-to-mesenchymal
transition.
Abbreviation: n/a, not applicable.
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1981
Kalous et al.
10
IC50 (mol/L)
0.1
0.01
* * * *
HC
C
M ZR -2 0
DA - 7 2
-M 5-3
HC B- 0
1
C 75
SU -2 2
1
HC M-2 8
C- 2 5
14
1 1
M 84 9
CF A1
SK -1 0
EF - B A
M R
UA - 19 -3
CC 2 A
M B -89
DA T 3
-M -47
UA B- 4
C 36
HC C - 1
C- 81 2
19
1 54
SU 8 4
M B5
Z -19
HC R- 7 0
M C 5
DA - -1
-M 1 50
HC B- 0
4
C 1
HC -1 5
C- 143
1
E 56
C FM 9
M O L -1
DA O 9
-M -82
B- 4
1
M
DA T 5 7
-M -4 7
B- D
H 46
HC C C 8
-7
U C- 0
M AC 1 18
DA C 7
-M -73
H B 2
M C C -13
DA - 4
-M 1 39
HC B- 5
4
C 5
HC -1 3
C- 937
1
BT 806
M H -54
DA C 9
-M C-3
B- 8
4
B 36
CA T-2
M 0
M C ADA A 1
-M L- 5
B- 1
Hs 43 5
DU 578
-4 T
4
M
DA MC 75
-M FB- 7
2
KP 3 1
L1
0.001
Figure 2. Inhibitory concentration and cell type. Bar graph of IC50 values (mmol/L) and cell type. Cell lines are color coded by subtype: dark blue bars (stripes),
HER2 amplied; light blue, luminal; yellow, nonluminal/undergone an epithelial-to-mesenchymal transition; red, nonluminal; turquoise, immortalized. , cell
lines with IC50 < 0.005 mmol/L.
1982
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1 mol/L
MDA-MB-231
(4.23 mol/L)
100 nmol/L
MDA-MB-453
(2 mol/L)
1 mol/L
1 mol/L
HCC-70
(1.44 mol/L)
100 nmol/L
1 mol/L
100 nmol/L
MDA-MB-468
(1.42 mol/L)
1 mol/L
HCC-1143
(0.73 mol/L)
100 nmol/L
1 mol/L
HCC-1954
(0.06 mol/L)
100 nmol/L
100 nmol/L
BT-474
(0.02 mol/L)
1 mol/L
1 mol/L
EFM-192A
(0.02 mol/L)
100 nmol/L
1 mol/L
SK-BR-3
(0.02 mol/L)
100 nmol/L
1 mol/L
HCC-202
(<0.005 mol/L)
100 nmol/L
Cell line
IC50
Resistant
100 nmol/L
Sensitive
pHER2
Total HER2
pEGFR
Total EGFR
pHER4
Total HER4
pAKT
Total AKT
pERK
Total ERK
-Tubulin
Figure 3. The effects of dacomitinib on total and phosphorylated HER2, EGFR, HER4, AKT, and ERK. The effects of dacomitinib on total and phosphorylated
HER2, EGFR, HER4, AKT, and ERK were measured by Western blot as described in Materials and Methods. All cell lines were treated with 100 nmol/L
or 1 mmol/L dacomitinib for 10 minutes. Cell lines are arranged from most sensitive (low IC50; left) to least sensitive (high IC50; right). Dacomitinib had no effect
on total HER2, AKT, and ERK in a majority of lines and caused a minor decrease in total EGFR in several lines. Most of the selected lines expressed low levels of
total HER4, which remained mostly unchanged posttreatment. Dacomitinib inhibited HER2 phosphorylation in all lines with detectable baseline
phosphorylation. Dacomitinib inhibited EGFR phosphorylation in sensitive lines, but only 2 resistant lines (MDA-MB-453 and MDA-MB-231). Dacomitinib
decreased HER4 phosphorylation in sensitive lines with detectable baseline levels. Dacomitinib inhibited phosphorylation of AKT in all sensitive lines, but only
2 resistant lines (HCC-70 and MDA-MB-453). Dacomitinib caused a decrease in phosphorylation of ERK in all lines, except for resistant MDA-MB-231.
Densitometry data are available in Supplementary Table S1.
trastuzumab and that it maintains a considerable antiproliferative activity in the cell lines with acquired resistance to lapatinib.
Effects of dacomitinib on cell cycle, apoptosis, and
signal transduction in cell lines conditioned for the
acquired resistance to trastuzumab and lapatinib
We analyzed the effect of dacomitinib on cell cycle and
apoptosis in the cell lines conditioned for acquired resistance to trastuzumab (BT-474-TR, SK-BR-3-TR) and lapatinib (BT-474-LR, SK-BR-3-LR). Cells were exposed to
100 nmol/L or 1 mmol/L of dacomitinib for 48 hours (cell
cycle) and 5 days (apoptosis).
In the trastuzumab-resistant lines, dacomitinib caused a
G0G1 arrest comparable with the parental lines. In lapatinib-resistant lines, the G0G1 arrest caused by 1 mmol/L
of dacomitinib was similar to the parental lines. The effect
was less pronounced at 100 nmol/L of dacomitinib (Supplementary Fig. S5).
www.aacrjournals.org
The effect of dacomitinib on apoptosis in trastuzumabresistant lines was stronger than in the parental lines. In
lapatinib-resistant lines, dacomitinib at 100 nmol/L did
not induce apoptosis, and the effect at 1 mmol/L was less
pronounced than in the parental lines (Supplementary
Fig. S6).
In addition, the effect of dacomitinib on the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the
acquired resistant lines was analyzed by Western blots
(Supplementary Fig. S7, densitometry in Supplementary
Table S2). Dacomitinib decreased the phosphorylation of
HER2, EGFR, AKT, and ERK in trastuzumab- and lapatinib-resistant lines. The expression of baseline total and
phosphorylated HER4 was very low, making assessment
of dacomitinib effects difficult.
Discussion
Using a broad panel of 44 human breast cancer cell lines
and 3 immortalized breast epithelial lines, we have shown
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Kalous et al.
A100
90
80
70
60
% 50
40
30
20
10
0
100
90
80
70
60
50
% 40
30
20
10
0
G0/G1
G0/G1
B100
90
80
70
60
% 50
40
30
20
10
0
G2
G2
G0/G1
G2
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
G0/G1
G0/G1
G2
G2
G0/G1
1984
G2
The blockage of EGFR phosphorylation was more pronounced in HER2-amplified lines but was also present in
some resistant lines. The inhibition of HER4 phosphorylation was observed in several sensitive HER2-amplified
lines. These effects lead to the inhibition of the PI3K/AKT
signaling pathway in sensitive lines and 2 resistant lines,
as evidenced by a reduction of phosphorylated AKT. We
have also observed a loss of phosphorylated ERK in most
lines, suggesting that dacomitinib also acts by inhibiting
the RAS/MAPK pathway. These effects of dacomitinib on
HER receptors and downstream signaling pathways ultimately resulted in cell-cycle inhibition and apoptosis.
In this study, 1 mmol/L was chosen based on the distribution of IC50 values in our dataset, which has biologic
significance (i.e., there seemed to be a natural increase in
the IC50 distribution around the 1 mmol/L mark; cell lines
below this cutoff point were mostly HER2 amplified;
dacomitinib induced changes in cell cycle and apoptosis
in cell lines with IC50 values below 1 mmol/L but not
above). In addition, phase I data was recently reported
(17). In general, the maximum plasma levels of dacomitinib
in the phase I study were between 200 to 300 nmol/L and
are within the range of the 1,000 nmol/L cut-off used here.
Trastuzumab and lapatinib have an important role in
the current clinical therapy of HER2-amplified breast
cancer. Unfortunately, less than half of patients respond
to monotherapy, and though the response rates increase
when combined with chemotherapy, the responses are
often only temporary in a significant number of patients
(411, 2730). For these reasons, it is important to keep
searching for new therapies for this subset of patients.
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% Annexin-Vpositive cells
% Annexin-Vpositive cells
70
60
60
50
40
30
30
20
20
10
10
70
60
60
50
50
40
40
30
30
20
20
10
10
80
70
70
70
60
60
50
50
40
40
30
30
20
20
10
10
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70
40
80
80
50
80
% Annexin-Vpositive cells
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1985
Kalous et al.
Table 2. Comparison of dacomitinib and lapatinib responses (IC50) in parental and trastuzumab- and
lapatinib-resistant cell lines
Cell line
Dacomitinib
IC50 (mmol/L)
Lapatinib
IC50 (mmol/L)
Dacomitinib
IC50 fold change
compared with
parental line
BT-474
BT-474-TR
BT-474-LR
SK-BR-3
SK-BR-3-TR
SK-BR-3-LR
0.018 0.011
0.010a
0.031 0.020
0.015 0.003
0.005a
0.339 0.137
0.016 0.011
0.077 0.039
1.424 0.120
0.054 0.008
0.039 0.010
6.400 1.119
0.56a
1.72
0.33a
22.60
Lapatinib
IC50 fold change
compared with
parental line
4.81
89
0.72
118.52
NOTE: Growth rate decrease (fold change) in the presence or absence of 15 mg/mL of trastuzumab: BT-474 (5.00 1.05), BT-474-TR
(1.16 0.21), SK-BR-3 (1.45 0.09), SK-BR-3-TR (1.19 0.05); both resistant lines t the criteria for trastuzumab resistance (<1.2-fold
decrease in growth rate; data in part published by O'Brien and colleagues; ref. 25).
Abbreviations: TR, trastuzumab-resistant cell lines; LR, lapatinib-resistant cell lines.
a
Approximate average value from a minimum of 2 experiments.
Acknowledgments
The authors thank Veerauo Konkankit and Teodora Kolarova for their
excellent technical assistance and Dr. Habib Hamidi for reviewing the
manuscript.
Grant Support
D.J. Slamon received Department of Defense Innovator Award
W81XWH-05-1-0395. The work is also funded by a gift to D.J. Slamon by
The Wittich Family Project for Emerging Therapies in Breast Cancer at
UCLAs Jonsson Comprehensive Cancer Center.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received September 26, 2011; revised May 11, 2012; accepted June 1,
2012; published OnlineFirst July 3, 2012.
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