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TITLE OR
ABSTRACT
1 - Identification as a study -
of diagnostic accuracy
using at least one
measure of accuracy
(such as sensitivity,
specificity, predictive
values, or AUC)
ABSTRACT
2 176 Structured summary of SELDI mass spectrometry was used to
study design, methods, investigate protein expression in sera of
results, and conclusions patients with gastric cancer and gastritis
(for specific guidance, compared to normal volunteers.
see STARD for Differences in peak morphology and
Abstracts) intensity were observed in regions of 5910
Da, 5084 Da, 6640 Da and 8691 Da.
Patients with gastric cancer exhibited an
up-regulation of the 5910 Da peak and a
down-regulation of the 8691 Da peak
compared to the healthy volunteers; there
was also some bi-partitioning and tri-
partitioning at the 5084 Da peak. When
comparing the sera of these cancer patients
with those of gastritis, the former had an
up-regulation of the 5910 Da peak and a
down-regulation of the 6640 Da peak. This
is the first report showing that SELDI sera
analysis may be useful in the screening of
gastric lesions.
INTRODUCTION
3 176 Scientific & clinical Gastric cancer is a debilitating disease
background, including associated with a high mortality. Its
the intended use and successful treatment relies on an early
clinical role of the index diagnosis, but this remains a challenge
test since the progression of the malignancy is
usually silent until it reaches a more
advanced stage where the prognosis is
poor. Certainly, early detection can
drastically facilitate treatment and improve
the long-term survival of the patient
(Kodera et al., 2003; Leung and Sung,
2002; Makela et al., 2000; Yuan et al.,
1999). The sensitivity of the current single
biomarkers in tumor diagnosis is low
(usually less than 40%) and complicated by
a high return of‘false-positives’. Further,
none of the existing serum markers can be
used individually for screening for gastric
cancer (Carpelan-Holmstrom et al., 2002;
Grotzinger et al., 2001; Ohata et al., 2005;
 Pan et al., 2003; Roboz, 2005; Schneider et
al., 2005). It would be highly desirable to
have a new rapid and sensitive diagnostic
test for gastric cancer
4 176 Study objectives and to explore the possibility that a use of a
hypotheses combination of biomarkers could improve
diagnostic power. Proteomics is the large-
scale study of proteins, or the simultaneous
measurement of a large number of
expressed proteins (Conrads et al., 2004;
Petricoin et al., 2002a,b). Proteomic
profiling enables a new approach to the
discovery of biomarkers in disease
(Conrads et al., 2004; Roboz, 2005;
Simpkins et al., 2005; Solassol et al.,
2005). It has recently been shown to be
useful in identifying biomarkers for the
diagnosis of bladder, prostate (Roboz,
2005), ovary (Conrads et al., 2004;
Petricoin et al., 2002a), breast (Laronga et
al., 2003–2004; Li et al., 2005; Roboz,
2005), liver malignancies and other cancers
(Roboz, 2005). Modern proteomic
profiling involves ProteinChip technology
that sometimes utilizes an enhanced
surface laser desorption/ionization
(SELDI) time of flight mass spectrometry
system (Simpkins et al., 2005). The SELDI
assay and detection system has not been
used for the profiling of protein clusters in
gastric cancer. In the present studies,
therefore,we employed SELDI to probe for
serum proteins that may be expressed in
patients with gastric cancer.We addressed
the possibility that serum proteins critical
to the progression of gastric cancer can be
identified through the changes in
peptide/proteinmass spectral patterns.We
also investigated if such potential changes
in spectral patterns are discriminatory of
patients with gastritis compared to healthy
volunteers.
METHODS
Study design 5 - Whether data collection -
was planned before the
index test and reference
standard were performed
(prospective study) or
after (retrospective
study)
Participants 6 177 Eligibility criteria Studies were conducted using Chinese
patients diagnosed with gastric cancer
(invasive gastric adenocarcinoma, n = 45),
or gastritis (chronic superficial gastritis, n
 = 40) patients
7 - On what basis potentially Tidak dijelaskan
eligible participants were
identified (such as
symptoms, results from
previous tests, inclusion
in registry)
8 177 Where and when Studies were conducted using Chinese
potentially eligible patients diagnosed with gastric cancer
participants were (invasive gastric adenocarcinoma, n = 45),
identified (setting, or gastritis (chronic superficial gastritis, n
location and dates) = 40) patients at Tiazhou Municipal
Hospital, Zhejian, China
9 178 Whether participants Stage 1 was not blinded, and 33 samples
formed a consecutive, were randomly taken from the gastric cancer
random or convenience patient pool, 30 samples from the gastritis
series pool and 31 samples from the healthy
volunteer pool, according to verified
pathological results obtained from the
associated endoscopic biopsies. This stage
was used to establish biomarker criteria
associated with each disease and reference
biomarker data from the volunteer group.
Stage 2 of the investigation was double
blinded. Fifteen samples from the gastric
cancer pool, 10 from the gastritis pool and
11 from the volunteer pool were used. They
were randomly selected and then analyzed
based on the biomarker clusters identified in
the Stage 1. The identity of the samples was
not revealed to the investigators until the
results had been obtained and classified
Test methods 10a 177 Index test, in sufficient Sample collection Approximately 5 ml of
detail to allow replication blood was withdrawn via veinipuncture
from each patient and serum prepared and
stored at −84°C prior to analysis.
ProteinChip analysis Serum specimens
were thawed on ice, followed by
centrifugation at 20,000 rpm for 10 min
and prepared for analysis on strong anionic
exchanger (SAX2) Chips (Ciphergen
Biosystems, Inc., Fremont, USA). In brief,
SAX2-arrays were equilibrated with 150 μl
50 mM Tris–HCl, pH 8.5, containing
0.02% Triton X-100 for 5 min. The
binding/washing buffer was then rinsed
and replaced with 150 μl of fresh binding
buffer plus 20 μl of serum and incubated
on a reciprocating shaker for 20 min. The
chips were then washed with distilled
water to remove salts before allowing them
to air-dry for 15 min. 0.5 μl of saturated
sinapinic acid solution (40% v/v
acetonitrile and 0.5% v/v trifluroacetic
 acid) was added twice to each spot. After
drying, samples were analyzed using an
automated protocol in a deep-well type
protein chip reader (PBS II, Ciphergen
Biosystems, Inc., Fremont, USA) via PBS
II SELDI-MS, with a nitrogen laser (337
μm) utilizing 185 μJ as the upper energy
limit. Mass calibration was performed
using an all-in-one peptide reference
standard which contained vasopressin
(1084.2 Da), somatostatin (1637.9 Da),
bovine insulin â chain (3495.9 Da), human
insulin recombinant7 (5807.6 Da) and
hirudin (7033.6 Da) (Ciphergen
Biosystems, Fremont, CA, USA). Mass
spectrometry profiles of samples on the
SAX2-chips (Weinberger et al., 2002) were
aligned electronically and spectral peaks
were clustered and normalized for total ion
current to account for variation in
ionization efficiencies under the preset
conditions of the Biomarker Wizard
software (version 3.0, Ciphergen
Biosystems, Fremont, CA, USA). All
samples were processed in duplicate.
Protein peaks (clusters) were considered to
be differentially expressed in the sera if
there were significant differences in their
patterns and/or intensity. The non-
parametric Mann–Whitney U test or the
Wilcoxon Signed Rank Test was used to
compare statistically the potential
significant differences in the protein peaks.
A probability value less than 0.5 was
considered significant.
10b 177 Reference standard, in Sample collection Approximately 5 ml of
sufficient detail to allow blood was withdrawn via veinipuncture
replication from each patient and serum prepared and
stored at −84°C prior to analysis.
ProteinChip analysis Serum specimens
were thawed on ice, followed by
centrifugation at 20,000 rpm for 10 min
and prepared for analysis on strong anionic
exchanger (SAX2) Chips (Ciphergen
1Biosystems, Inc., Fremont, USA). In
brief, SAX2-arrays were equilibrated with
150 μl 50 mM Tris–HCl, pH 8.5,
containing 0.02% Triton X-100 for 5 min.
The binding/washing buffer was then
rinsed and replaced with 150 μl of fresh
binding buffer plus 20 μl of serum and
incubated on a reciprocating shaker for 20
min. The chips were then washed with
distilled water to remove salts before
 allowing them to air-dry for 15 min. 0.5 μl
of saturated sinapinic acid solution (40%
v/v acetonitrile and 0.5% v/v trifluroacetic
acid) was added twice to each spot. After
drying, samples were analyzed using an
automated protocol in a deep-well type
protein chip reader (PBS II, Ciphergen
Biosystems, Inc., Fremont, USA) via PBS
II SELDI-MS, with a nitrogen laser (337
μm) utilizing 185 μJ as the upper energy
limit. Mass calibration was performed
using an all-in-one peptide reference
standard which contained vasopressin
(1084.2 Da), somatostatin (1637.9 Da),
bovine insulin â chain (3495.9 Da), human
insulin recombinant (5807.6 Da) and
hirudin (7033.6 Da) (Ciphergen
Biosystems, Fremont, CA, USA). Mass
spectrometry profiles of samples on the
SAX2-chips (Weinberger et al., 2002) were
aligned electronically and spectral peaks
were clustered and normalized for total ion
current to account for variation in
ionization efficiencies under the preset
conditions of the Biomarker Wizard
software (version 3.0, Ciphergen
Biosystems, Fremont, CA, USA). All
samples were processed in duplicate.
Protein peaks (clusters) were considered to
be differentially expressed in the sera if
there were significant differences in their
patterns and/or intensity. The non-
parametric Mann–Whitney U test or the
Wilcoxon Signed Rank Test was used to
compare statistically the potential
significant differences in the protein peaks.
A probability value less than 0.5 was
considered significant.
11 178 Rationale for choosing Stage 1 was not blinded, and 33 samples
the reference standard (if were randomly taken from the gastric
alternatives exist) cancer patient pool, 30 samples from the
gastritis pool and 31 samples from the
healthy volunteer pool, according to
verified pathological results obtained from
the associated endoscopic biopsies. This
stage was used to establish biomarker
criteria associated with each disease and
reference biomarker data from the
volunteer group.
Tetapi tidak ada penjelasan mengapa
memilih biopsy.
12a Definition of and -
rationale for test
positivity cut-offs or
 result categories of the
index test, distinguishing
pre-specified from
exploratory
12b Definition of and -
rationale for test
positivity cut-offs or
result categories of the
reference standard,
distinguishing pre-
specified from
exploratory
13a 179 Whether clinical The mass spectral patterns identified in
information and Stage 1, with three special protein
reference standard results peaks,were used on the double-blinded
were available to the sera. Using these criteria, gastric cancerwas
performers/readers of the correctly identified in 14 out of 15
index test samples, gastritis was correctly identified
in 10 out of 11 patients and all 10 out of 10
normal volunteers were identified (Table
3). The biomarkers therefore had an
accuracy of 93.3%and 90.9% for the
identification of gastric cancer and
gastritis, respectively. The biomarkers had
a 100% accuracy to identify healthy
volunteers that did not have gastric cancer
or gastritis
13b Whether clinical -
information and index
test results were available
to the assessors of the
reference standard
Analysis 14 177 Methods for estimating The non-parametric Mann–Whitney U test
or comparing measures or the Wilcoxon Signed Rank Test was
of diagnostic accuracy used to compare statistically the potential
significant differences in the protein peaks.
A probability value less than 0.5 was
considered significant.
15 How indeterminate index Ada false-negatif dan false-positif pada
test or reference standard stage 1. Ada false-negatif pada stage 2,
results were handled tetapi tidak ditindaklanjuti.
16 How missing data on the Ada missing data pada stage 1, tetapi tidak
index test and reference ditindaklanjuti.
standard were handled
17 Any analyses of Tidak dijelaskan apa yang mempengaruhi
variability in diagnostic hasil pengujian.
accuracy, distinguishing
pre-specified from
exploratory
18 Intended sample size and Tidak dijelaskan
how it was determined
RESULTS
Participants 19 Flow of participants, Tidak dijelaskan
using a diagram
 20 Baseline demographic
and clinical
characteristics of
participants
21a - Distribution of severity
of disease in those with
the target condition
21b - Distribution of
alternative diagnoses in
those without the target
condition
22 - Time interval and any
clinical interventions
between index test and
reference standard
Test results 23 178- Cross tabulation of the
179 index test results (or their
distribution) by the
results of the reference
standard
24 179 Estimates of diagnostic
accuracy and their
precision (such as 95%
confidence intervals)
25 - Any adverse events from
performing the index test
or the reference standard
DISCUSSION
26 - Study limitations,
including sources of
potential bias, statistical
uncertainty, and
generalisability
27 179- Implications for practice,
180 including the intended
use and clinical role of
the index test
Tidak dijelaskan
Tidak dijelaskan, hanya menyebutkan
jumlah orang dengan gastric
adenocarcinoma dan yang sehat
Tidak dijelaskan
Tidak dijelaskan
Table 2
Table 3
The mass spectral patterns identified in
Stage 1, with three special protein
peaks,were used on the double-blinded
sera.Using these criteria, gastric cancerwas
correctly identified in 14 out of 15
samples, gastritis was correctly identified
in 10 out of 11 patients and all 10 out of 10
normal volunteers were identified (Table
3). The biomarkers therefore had an
accuracy of 93.3% and 90.9%for the
identification of gastric cancer and
gastritis, respectively. The biomarkers had
a 100% accuracy to identify healthy
volunteers that did not have gastric cancer
or gastritis (Table 4).
-
-
Recent studies have successfully
implemented the use of SELDI on sera to
identify unique proteomic patterns that
discriminate between malignant and benign
growth in the ovary (Petricoin et al.,
2002a). We decided on a similar approach
to discriminate between patients with
gastric cancer and gastritis, compared to
healthy volunteers. Our SELDI mass
spectroscopic findings were highly
 reproducible and capable of detecting
gastric tumor-specific protein profiles.
When serum profiles of each patient were
compared, they revealed similar changes in
protein peaks across different patients.

In conclusion, the importance of our

studies has been to demonstrate the
usefulness of SELDI in the discovery of
potential tumor markers in serum samples.
The comparison of protein expression
profiles from serum appears to provide an
effective approach to identifying unique
biomarkers for gastric cancer and gastritis.
Larger scale studies appear warranted to
confirm the ability of SELDI as a
proteomic screen in the clinical setting.
OTHER
INFORMATION
28 - Registration number and -
name of registry
29 - Where the full study -
protocol can be accessed
30 - Sources of funding and -
other support; role of
funders