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Supplementary Materials
Radiotherapy
The gross tumor volume (GTV) was defined as all macroscopically
identifiable tumor determined by CT scan, EUS, esophagogram, and PET
scan. The clinical target volume (CTV) was defined as 4 cm proximal and distal
as well as 0.5 cm lateral beyond GTV. The CTV included the supraclavicular
nodes for upper-third primaries and celiac nodes for distal-third primaries. The
planning target volume (PTV) was defined as the CTV plus 0.5-cm margins. A
0.5-cm margin around the spinal cord was added for the planning organ-at-risk
volume. Inverse intensity-modulated radiation therapy (IMRT) plans were
generated using a commercial treatment planning system (Pinnavle3, v9.0,
ADAC Laboratories, Milpitas, CA). The CTV was irradiated with a total dose of
40 Gy at 2 Gy per fraction. The treatment was delivered with 6- and/or 10-MV
photons using 3–5 beam fields (median 4 beams). The typical beam
orientation was similar to the conventional four-beam arrangement with
anteroposterior, posteroanterior, and two posterior oblique fields. The goals of
inverse IMRT planning were to ensure that ≧95% of the PTV received the
prescribed dose while keeping the exposure of normal structures within
normally acceptable tolerances.
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Proximity ligation assay (PLA)
Probe pairs were made for each of the 15 protein markers by attaching
goat or rabbit antibodies specific for each protein to complementary
oligonucleotides using a Solulink Antibody-Oligonucleotide All-In-One
Conjugation Kit.
These probes were divided into two panels. Each of the
individual probes and the probe panels were validated in a pilot study using
either anti-goat or anti-rabbit antibodies as a pseudo-antigen. Multiplex PLA
was performed on 158 frozen serum samples. Samples were thawed and
mixed in a 1:1 ratio with buffer (Olink AB) and incubated for 10 minutes at room
temperature. Two μL of either of the probe panels was mixed with 2 μL of the
buffered serum sample and incubated for 2 hours at 37°C to allow the probes
to bind to the analytes. Ligation was achieved by incubating 120 μL of reaction
mixture with the 4 μL probed samples for 15 minutes at 30°C to dilute and
separate any free probes. To stop the ligation, 2 μL of uracil-DNA excision mix
(Epicentre) was added and incubated for 15 minutes at room temperature.
Preamplification of bar-coded amplicons required mixing 25 μL of stopped
ligation reaction mixture with 25 μL of pooled PCR mix (Platinum Taq kit,
Invitrogen). After 13 cycles at 95°C for 30 seconds and a 4-minute extension at
60°C, the preamplification products were diluted 10-fold in TE. For each of the
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15 proteins assayed, a separate qPCR reaction was run in a 384-well plate
with 2 μL of diluted preamplication product sample, 5 μL of iTaq mix (iTaq
SYBR Green Supermix with ROX, Bio-Rad), 2 μL qPCR primer mix, and 1 μL
water. Real-time qPCR was performed with a sample volume of 10 μL per well
for 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. To ensure
standardization of values for each biomarker investigated, all 158 samples
were simultaneously probed and evaluated on a single 384-well plate with a
PBS-BSA blank well.
Cycle threshold (Ct) values resulting from qPCR were converted into
estimated number of starting amplicons, or PLA units, by calculating 10(-0.301 ×
Ct+11.439)
as previously reported 10.
Enzyme-linked immunosorbent assay (ELISA)
The serum biomarker concentration was measured with a commercially
available human protein sandwich enzyme immunoassay kit with two mouse
monoclonal antihuman antibodies (R&D Systems, Inc., Minneapolis, MN,
USA). All serum samples from patients and standards were incubated in
microplate wells coated with the first mouse monoclonal anti-human biomarker
antibody. After washing, a second antihuman biomarker antibody labeled with
peroxidase (HRP) was added for subsequent incubation. The reaction
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between HRP and substrate (hydrogen peroxide and tetramethylbenzidine)
resulted in color development and the intensities were measured with a
microplate reader at an absorbance of 450 nm. Concentrations of serum
biomarkers were determined against a standard curve.
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Supplementary Table F factor and the significance (p value) by two-way
analysis of variance (ANOVA) for the association of 15 serum biomarkers'
pre-chemoradiotherapy (CCRT), post-CCRT, and pre-/post-CCRT changes of
proximity ligation assay (PLA) data with pathological response.
Biomarker
Time factor (Pre-
Response factor
Interaction of Time
versus
(CR versus
factor and Response
Post-CCRT)
microscopic
factor
F factor (p value)
residual, versus
macroscopic
residual)
Axl
3.0 (0.09)
0.43 (0.65)
0.66 (0.52)
BMP2/4
2.37 (0.13)
0.40 (0.67)
0.40 (0.67)
EGFR
3.53 (0.06)
0.53 (0.59)
0.81 (0.45)
FAM84B
9.37 (0.003)
0.85 (0.43)
0.80 (0.45)
Gas6
1.44 (0.23)
0.39 (0.68)
0.99 (0.38)
Her2
4.26 (0.04)
0.39 (0.68)
0.35 (0.71)
IGFBP3
0.13 (0.72)
0.69 (0.51)
0.32 (0.73)
MMP1
0.18 (0.67)
0.70 (0.50)
2.55 (0.09)
MMP13
2.68 (0.11)
0.20 (0.82)
1.06 (0.35)
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OPN
0.15 (0.70)
2.15 (0.12)
0.14 (0.87)
PDGFR-α
0.001 (0.97)
0.70 (0.50)
0.89 (0.42)
Periostin
10.26 (0.002)
1.95 (0.15)
0.62 (0.54)
SPARC
0.07 (0.79)
2.72 (0.07)
1.21 (0.30)
TGF-β1
9.7 (0.003)
5.07 (0.009)
0.97 (0.38)
VEGF-A
2.09 (0.15)
0.65 (0.53)
1.98 (0.15)