Secretome-based identification of ULBP2 as a novel serum marker for pancreatic cancer detection.

Abstract

To discover novel markers for improving the efficacy of pancreatic cancer (PC) diagnosis, the secretome of two PC cell lines (BxPC-3 and MIA PaCa-2) was profiled. UL16 binding protein 2 (ULBP2), one of the proteins identified in the PC cell secretome, was selected for evaluation as a biomarker for PC detection because its mRNA level was also found to be significantly elevated in PC tissues.
ULBP2 expression in PC tissues from 67 patients was studied by immunohistochemistry. ULBP2 serum levels in 154 PC patients and 142 healthy controls were measured by bead-based immunoassay, and the efficacy of serum ULBP2 for PC detection was compared with the widely used serological PC marker carbohydrate antigen 19-9 (CA 19-9).
Immunohistochemical analyses revealed an elevated expression of ULPB2 in PC tissues compared with adjacent non-cancerous tissues. Meanwhile, the serum levels of ULBP2 among all PC patients (n = 154) and in early-stage cancer patients were significantly higher than those in healthy controls (p<0.0001). The combination of ULBP2 and CA 19-9 outperformed each marker alone in distinguishing PC patients from healthy individuals. Importantly, an analysis of the area under receiver operating characteristic curves showed that ULBP2 was superior to CA 19-9 in discriminating patients with early-stage PC from healthy controls.
Collectively, our results indicate that ULBP2 may represent a novel and useful serum biomarker for pancreatic cancer primary screening.

Full text

Page 1

Secretome-Based Identification of ULBP2 as a Novel
Serum Marker for Pancreatic Cancer Detection
Ya-Ting Chang1, Chih-Ching Wu2,3*, Yi-Ming Shyr4, Tse-Ching Chen5, Tsann-Long Hwang6, Ta-Sen Yeh6,
Kai-Ping Chang7, Hao-Ping Liu2, Yu-Ling Liu2, Ming-Hung Tsai1, Yu-Sun Chang2, Jau-Song Yu1,2,8*
1Graduate Institute of Biomedical Sciences, Chang Gung University, Tao-Yuan, Taiwan, 2Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan,
3Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Tao-Yuan, Taiwan, 4Divisions of General and Transplantation Surgery,
Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan, 5Department of Anatomical Pathology, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan,
6Department of Surgery, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan, 7Departments of Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital,
Tao-Yuan, Taiwan, 8Department of Cell and Molecular Biology, Chang Gung University, Tao-Yuan, Taiwan
Abstract
Background: To discover novel markers for improving the efficacy of pancreatic cancer (PC) diagnosis, the secretome of two
PC cell lines (BxPC-3 and MIA PaCa-2) was profiled. UL16 binding protein 2 (ULBP2), one of the proteins identified in the PC
cell secretome, was selected for evaluation as a biomarker for PC detection because its mRNA level was also found to be
significantly elevated in PC tissues.
Methods: ULBP2 expression in PC tissues from 67 patients was studied by immunohistochemistry. ULBP2 serum levels in
154 PC patients and 142 healthy controls were measured by bead-based immunoassay, and the efficacy of serum ULBP2 for
PC detection was compared with the widely used serological PC marker carbohydrate antigen 19-9 (CA 19-9).
Results: Immunohistochemical analyses revealed an elevated expression of ULPB2 in PC tissues compared with adjacent
non-cancerous tissues. Meanwhile, the serum levels of ULBP2 among all PC patients (n=154) and in early-stage cancer
patients were significantly higher than those in healthy controls (p,0.0001). The combination of ULBP2 and CA 19-9
outperformed each marker alone in distinguishing PC patients from healthy individuals. Importantly, an analysis of the area
under receiver operating characteristic curves showed that ULBP2 was superior to CA 19-9 in discriminating patients with
early-stage PC from healthy controls.
Conclusions: Collectively, our results indicate that ULBP2 may represent a novel and useful serum biomarker for pancreatic
cancer primary screening.
Citation: Chang Y-T, Wu C-C, Shyr Y-M, Chen T-C, Hwang T-L, et al. (2011) Secretome-Based Identification of ULBP2 as a Novel Serum Marker for Pancreatic
Cancer Detection. PLoS ONE 6(5): e20029. doi:10.1371/journal.pone.0020029
Editor: Rakesh K. Srivastava, The University of Kansas Medical Center, United States of America
Received January 21, 2011; Accepted April 10, 2011; Published May 20, 2011
Copyright: ? 2011 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the grants from the Taiwan National Science Council (NSC 96-2320-B-182-031-MY3), Department of Health (DOH-99-TD-C-
111-006), and Ministry of Education (EMRPD180241 and EMRPD180091). The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: yusong@mail.cgu.edu.tw (J-SY); luckywu@mail.cgu.edu.tw (C-CW)
Introduction
Pancreatic cancer (PC) is the fourth-leading cause of cancer-
related mortality in the United States with a 5-year survival less
than 7% [1,2]. In 2010, more than 43,000 new PC cases were
estimated to develop and 36,800 deaths were expected in the
United States [1]. In Taiwan, it is ranked tenth among cancer-
related deaths in 2008 and shows increased mortality rate in the
last decade [3]. Due to the early spread of PC and the late onset of
apparent symptoms, less than 8% of PC patients are diagnosed at
the localized stage when a surgical cure is possible [1,4,5].
Accordingly, there is an urgent need to develop improved
strategies for early detection of PC.
Current approaches for PC diagnosis are mainly based on
imaging and endoscopic methods [6], which, because of the
retroperitoneal location of the pancreas, have a limited probability
of early diagnosis [7,8]. An elevation in serum levels of
carbohydrate antigen 19-9 (CA 19-9) has been widely used for
PC detection; however, this approach is insufficient with respect to
both specificity and sensitivity [6,9,10]. In addition to CA 19-9,
more than 40 proteins have been reported as potential serological
biomarkers for PC detection [11]. Unfortunately, most either have
limited specificity and/or sensitivity, or await validation with a
large-scale cohort of specimens [11–17], despite evidence that the
use of a combinatory biomarker panel improves the accuracy of
PC diagnosis [12]. Therefore, discovery of novel and useful serum
markers could facilitate the improvement of PC diagnosis and/or
prognosis.
Recently, the secretome-based approaches have been widely
applied in the identification of potential cancer biomarkers
[18,19]. In the present study, we analyzed the secretome of two
PC cell lines, BxPC-3 and MIA PaCa-2 and evaluated one of the
identified proteins, UL16 binding protein 2 (ULBP2), as a
potential PC biomarker. Immunohistochemical staining results
PLoS ONE | www.plosone.org1 May 2011 | Volume 6 | Issue 5 | e20029

Page 2

confirmed the elevated levels of ULBP2 in PC tissues compared
with adjacent non-cancerous counterparts. Bead-based immuno-
assays further validated the elevated serum levels of ULBP2 in PC
patients versus healthy individuals. Most importantly, the
combined use of ULBP2 with CA 19-9 improved the sensitivity
and accuracy of PC early detection in this case control study,
providing a promising approach for PC diagnosis at an early stage.
Materials and Methods
Patient population and clinical specimens
Tumor specimens from 67 PC patients (median age, 64; range:
35–82) were collected in the Chang Gung Memorial Hospital
(CGMH), Lin-Kou, Taiwan. The tissue samples were collected at
surgery, evaluated by pathologists, and stored in the CGMH
Tissue Bank until use. Serum samples from 154 PC patients
(median age, 70; range, 31–87) were collected in Taipei Veterans
General Hospital, Taipei, Taiwan. Additional blood samples,
including 142 serum samples from healthy donors (median age,
58; range, 34–86), 25 plasma samples from healthy donors
(median age, 54; range, 29–79), 28 serum samples from
nasopharyngeal carcinoma (NPC) patients (median age, 46; range,
31–80), 29 serum samples from colorectal carcinoma (CRC)
patients (median age, 61; range, 30–83), and 30 plasma samples
from gastric cancer (GC) patients (median age, 66; range, 33–83),
were collected in the CGMH, Lin-Kou, Taiwan. Aliquots of these
samples were stored at 280uC until use. This research followed
the tenets of the Declaration of Helsinki and all subjects signed an
informed consent approved by Institutional Review Board of
Chang Gung Memorial Hospital or Taipei Veterans General
Hospital, Taiwan before their participation in this study and for
the use of tissue or blood samples collected before treatment.
Cell culture
The PC cell lines BxPC-3, MIA PaCa-2, PANC-1 and AsPC-1
were purchased from Bioresource Collection and Research Center
(Hsinchu, Taiwan). BxPC-3 and AsPC-1 were maintained in 10%
fetal bovine serum (FBS, Biological Industries, Israel)-supplement-
ed RPMI-1640 medium (Invitrogen, CA, USA). MIA PaCa-2 and
PANC-1 were maintained in Dulbecco’s modified Eagle’s medium
(DMEM; Invitrogen) with 10% FBS plus 2.5% horse serum
(Biological Industries) and 10% FBS, respectively. The four cell
lines were cultured at 37uC in a humidified 5% CO2environment.
Generation of secretome dataset of cancer cell lines
The methods used for preparation of cancer cell conditioned
media (CM) and secretome profiling were described previously
[20] and detailed in Materials and Methods S1.
Public domain database search for expression profiles of
selected target genes
The expression profiles of the secreted proteins in PC tissues
were searched in the National Center for Biotechnology
Information(NCBI) GeneExpression
(http://www.ncbi.nlm.nih.gov/geo/). The GSE1542 dataset was
selected for identifying candidate genes with elevated expression
levels in PC cells [21]. The GSE1542 dataset contains the gene
expression profiles of pancreatic ductal cells isolated from 24
patients with pancreatic ductal adenocarcinoma and 25 donors
with a normal pancreas [21]. The mean intensities of each gene
probe in healthy and cancerous groups were calculated to obtain
the Tumor/Normal (T/N) ratio; genes with T/N ratios $2 were
considered up-regulated candidates. Among them, those with p-
values less than 0.05 calculated by t-test were further selected as
Omnibusdatabase
the most promising candidates for elevated expression in
cancerous tissues.
Western blot analysis
Proteins in cell extracts and CM were separated by SDS-PAGE,
transferred onto PVDF membranes (Millipore, MA, USA), and
probed with antibodies against transforming growth factor-b-
induced protein ig-h3 (BIGH3) (1:1000 dilution; Santa Cruz
Biotechnology, CA, USA), ULBP2 (1:1000 dilution; R&D
Systems, MN, USA) or a-tubulin (1:10000 dilution; Millipore).
Proteins of interest were detected by incubating for 1 hour with
the appropriate horseradish peroxidase (HRP)-conjugated second-
ary antibodies (Santa Cruz Biotechnology), and visualized using an
enhanced chemiluminescence system (PerkinElmer, MA, USA).
Immunohistochemistry
The immunohistochemical staining was performed with anti-
bodies against BIGH3 (1:100 dilution; Santa Cruz Biotechnology)
and ULBP2 (1:20 dilution; R&D Systems). The staining protocols
are described in the Materials and Methods S1. Expression of
target proteins was evaluated according to the simplified H score
system, which is based on the intensity of cell staining (3, strong; 2,
moderate; 1, weak; 0, no cell staining) and the percentage of cell
staining [3, $90%; 2, 50–89%; 1, 10–49%; 0, 0–9%)]. The two
scores were multiplied and then divided by 3 to get the final score.
Positive staining was defined as a final score $0.67 [22,23].
Serum analyses
Serum BIGH3 and CA 19-9 levels were determined using the
ELISA kits from R&D Systems and Alpha Diagnostic (TX, USA),
respectively, according to the respective manufacturer’s instruc-
tions. To determine the serum ULBP2 concentration, the in-house
sandwich ELISA and bead-based immunoassay were established
in our laboratory (see Materials and Methods S1 for details).
Statistical analysis
Differences in immunohistochemical scores and serum levels of
target proteins between groups were tested using either the
Wilcoxon test or Kruskal-Wallis test. Correlations between serum
levels of target proteins were calculated using the Pearson
correlation, and immunohistochemical scores of paired samples
from the same subject were compared using paired t-tests.
Receiver operator characteristic (ROC) curves were constructed
by plotting sensitivity versus 1-specificity and areas under the
ROC curves (AUCs) were analyzed using the Hanley and McNeil
method. All tests were two-sided, and a p-value,0.05 was
considered statistically significant. All data were processed using
SPSS software version 13.0 (IL, USA).
Results
Secretome analysis of two pancreatic cancer cell lines
To discover potential serum PC biomarkers, the secreted
proteins of PC cell lines were systemically analyzed. The schematic
representation of the flowchart used is illustrated in Fig. 1A. The
proteins present in the 24 h serum-free CM of BxPC-3 and MIA
PaCa-2 cells were separated by SDS-PAGE, visualized by
Coomassie blue staining (Fig. 1B, upper panel), sliced into 40
fractions, digested individually with trypsin and analyzed by C-18
reversed-phase liquid chromatography-tandem mass spectrometry
(LC-MS/MS). The protein staining pattern of the cell lysate is also
shown in parallel to demonstrate that the secreted proteins were
enriched in the CM, and its pattern was quite different from that
generated by intracellular proteins (Fig. 1B, upper panel). The
ULBP2 as a Novel Marker of Pancreatic Cancer
PLoS ONE | www.plosone.org2 May 2011 | Volume 6 | Issue 5 | e20029

Page 3

distribution of the cytosolic protein, alpha-tubulin, was further
examined as a control. Alpha-tubulin was clearly detected in the
cell extracts, but not in the CM (Fig. 1B, lower panel), indicating
that proteins recovered from the CM was not due to cell death.
After searching with Mascot algorithm and setting a cutoff of
95.0% peptide probability and 95.0% protein probability in
Scaffold software, 1011 and 1141 proteins with multiple ($2)
peptide hits were found in the BxPC-3 and MIA PaCA-2 CM,
respectively (Supporting Tables S1 and S2), resulting in 1427 non-
redundant and 725 overlap proteins.
Generation of candidate biomarker list for PC detection
To narrow down the candidate list, expression levels of the
common 725 proteins in pancreatic ductal cells were examined in
an array-based analysis published by Ishikawa et al. [21], in which
they compared gene expression in pancreatic ductal cells derived
from 24 pancreatic ductal adenocarcinoma patients and 25 donors
with a normal pancreas (Fig. 1A). This analysis revealed that 10 of
the 725 proteins were significantly upregulated ($2-fold over-
expression, p,0.05) in pancreatic ductal carcinoma compared
with non-tumor pancreatic ductal cells (Table 1). Among them,
ceruloplasmin has been confirmed to be overexpressed in PC
tissues [24], and elevated serum level of ERO1-like protein alpha
has been reported in PC patients comparing to healthy controls
[17], indicating that the combined analysis of the secretome and
the transcriptome is a feasible strategy for discovering novel
candidate PC markers.
Overexpression of ULBP2 and BIGH3 in PC tissues
We focused our attention on ULBP2 and BIGH3 as potential
PC markers (Table 1) because another array-based analysis had
also shown that their mRNA levels were significantly increased in
PC tissues [25], and neither had yet been studied in PC. We
confirmed the presence of BIGH3 and ULBP2 in the CM
collected from four PC cell lines (BxPC-3, MIA PaCA-2, PANC-
1 and AsPC-1) by Western blot analysis (Fig. 1C). Further
Figure 1. Strategy for identification of potential PC serum biomarkers. (A), The strategy consists of cancer secretome and tissue
tranacriptome analysis. (B), The proteins (50 mg) in CM and cell extracts (CE) of the cancer cells were resolved by 10% SDS-PAGE and stained with
Coomassie blue (upper panel) or underwent immunoblot analysis with alpha-tubulin (lower panel). (C), Immunoblot analysis with anti-BIGH3 and
ULBP2 antibodies.
doi:10.1371/journal.pone.0020029.g001
ULBP2 as a Novel Marker of Pancreatic Cancer
PLoS ONE | www.plosone.org3 May 2011 | Volume 6 | Issue 5 | e20029

Page 4

immunohistochemical analyses showed positive staining for
BIGH3 and ULBP2 in 41.9% (13/31) and 100.0% (67/67),
respectively, of PC tissue sections. Inspection of each tissue
section containing both non-cancerous and cancerous ductal
tissues revealed higher expression of both proteins in the
cancerous tissues than in the non-cancerous counterparts in most
of the tissue sections examined (Fig. 2A; Supporting Figures S1
and S2). The immunohistochemical scores of both proteins in
tumor tissues were statistically higher than those in non-
cancerous counterparts: 0.5460.46 versus 0.1460.27 for BIGH3
(p,0.0001) and 2.7160.49 versus 1.8960.74 for ULBP2
(p,0.0001) (Fig. 2B). Further analyses showed that their
expression levels in tumor tissues were not statistically associated
with age, gender, histological grade, overall tumor stage, or
tumor-node-metastasis (TNM) classification of pancreatic cancer
(Supporting Tables S3 and S4).
Serum levels of BIGH3 and ULBP2 in PC patients
To evaluate the potential of BIGH3 and ULBP2 as serum PC
markers, we examined their levels in sera from PC patients
(n=154) and healthy controls (n=142). We measured serum
BIGH3 levels using a commercial ELISA kit and determined
serum ULBP2 levels using a bead-based immunoassay developed
in our laboratory that is capable of detecting the typically very low
levels of serum ULBP2 (,1 ng/mL) that could not be measured
precisely using our in-house sandwich ELISA. Our bead-based
immunoassay could accurately detect ULBP2 levels over a range
of 4.3 pg/mL to 31.9 ng/mL (Supporting Figure S3). We found
that the serum levels of ULBP2, but not BIGH3, were significantly
elevated in PC patients compared to those in the healthy controls:
1.8761.67 versus 1.2960.49 mg/mL for BIGH3 (p=0.328;
Fig. 3A) and 200.26168.6 versus 51.4664.6 pg/mL for ULBP2
(p,0.0001; Fig. 3B). Using a cutoff value of 60 pg/mL for ULBP2,
we found that the sensitivity and specificity values for cancer
detection were 83.8% and 73.9%, respectively. Serum BIGH3 and
ULBP2 levels were not statistically correlated with age, gender,
histological grade, overall tumor stage, or TNM classification of
PC in this case-control study (Supporting Table S5). These
findings indicate that ULBP2 might be a novel serum marker for
PC detection.
The efficacy of ULBP2 and CA 19-9 for PC screening
Serum levels of CA 19-9, a currently used serum PC biomarker,
were additionally assayed in the same samples. We found that
serum CA 19-9 levels were higher in PC patients than in healthy
controls (63.4624.4 versus 28.3620.2 U/mL, p,0.0001, Fig. 3C);
like ULBP2, CA 19-9 levels were not correlated with clinicopath-
ological characteristics (Supporting Table S5). Further analysis
showed that there was no correlation between the two molecules in
the patient cohort (r=0.061, p=0.453 by Pearson correlation;
Supporting Figure S4). At 40 U/mL, the cutoff value currently
applied for PC screening, the sensitivity and specificity values for
CA 19-9 were 84.4% and 74.6%, respectively. Notably, applying a
cutoff value of 60 pg/mL for ULBP2, we were able to discriminate
21 of 24 PC patients with CA 19-9 levels ,40 U/mL from healthy
individuals. In addition, 24 of 36 healthy individuals with CA 19-9
levels .40 U/mL could be further distinguished from patients
based on ULBP2 levels ,60 pg/mL.
The utility of ULBP2 and CA 19-9 as detection markers was
further tested by applying a ROC curve analysis. This analysis
demonstrated that ULBP2 (AUC=0.862; 95% confidence
interval [CI], 0.821–0.904) performed slightly better than CA
19-9 (AUC=0.856; 95% CI, 0.809–0.902) as a screening marker
(Fig. 3D). Most importantly, a logistic regression model [26]
showed that the diagnostic capacity of the combination of ULBP2
and CA 19-9 was greater than that of either marker alone
(AUC=0.910; 95% CI, 0.877–0.943; Fig. 3D). Collectively, these
results indicate that ULBP2 might be a useful serum PC marker,
especially when used together with CA 19-9.
The suitability of ULBP2 and CA 19-9 for PC early
detection
We then evaluated the suitability of ULBP2 as an early detection
marker of PC by testing serum ULBP2 levels in patients with early-
stage primary tumors (TNM-T1/T2), no lymph node metastasis
(TNM-N0), and at early overall tumor stages (stage I–II). We found
that serum ULBP2 levels were significantly higher in patients with
early-stage primary tumors (205.76184.3 pg/mL, p,0.0001,
n=34),nolymphnode metastasis
p,0.0001, n=57), andat an
(181.26158.8 pg/mL, p,0.0001, n=106) than in healthy controls
(191.66155.2 pg/mL,
overalltumorearlystage
Table 1. List of pancreatic cancer cell-secreted proteins that are overexpressed in pancreatic cancer tissue transcriptome.
Protein name (Gene symbol)
NCBI GEO GSE1542a
Probe IDb
T/N ratioc
p-valued
Alpha-soluble NSF attachment protein (NAPA)208751_at3.0160.017
Transforming growth factor-b-induced protein ig-h3 (BIGH3)201506_at2.9620.047
Ras-related protein Rab-14 (RAB14) 200928_s_at2.3000.020
UL16 binding protein 2 (ULBP2)238542_at2.2820.019
Ceruloplasmin (CP)227253_at2.2340.012
60S ribosomal protein L22 (RPL22) 238370_x_at2.0990.018
Transcriptional activator protein Pur-beta (PURB)235711_at2.0980.006
Complement C1s subcomponent (C1S) 208747_s_at2.0730.002
Annexin A11 (ANXA11)228727_at2.0050.002
ERO1-like protein alpha (ERO1L)218498_s_at2.001
,0.001
aData obtained from the NCBI GEO dataset GSE1542 (Ref. 21).
bProbe identities of Affymetrix Human Genome U133A and B arrays.
cThe expression ratios are shown as pancreatic ductal carcinoma (T) versus non-cancerous pancreatic ductal cells (N).
dThe p-values were determined using t-tests.
doi:10.1371/journal.pone.0020029.t001
ULBP2 as a Novel Marker of Pancreatic Cancer
PLoS ONE | www.plosone.org4May 2011 | Volume 6 | Issue 5 | e20029

Page 5

(51.4664.6 pg/mL; n=142; Fig. 4A). Serum CA 19-9 levels were
also elevated in the early stages of primary tumor (56.3626.9 U/
mL,
p,0.0001), lymphnode
p,0.0001), and overall tumor stage (60.7624.4 U/mL, p,0.0001)
compared to healthycontrols(28.3620.2 U/mL, n=142,) (Fig. 4B).
ROC curve analyses further showed that ULBP2 was more
appropriate than CA 19-9 for discriminating healthy controls from
patients with PC diagnosed as TNM-T1/T2 (AUC=0.854 [95%
CI, 0.778–0.930] versus AUC=0.796 [95% CI, 0.690–0.901]),
TNM-N0 (AUC=0.866[95%
AUC=0.841 [95% CI, 0.764–0.917]) or stage I/II (AUC=0.846
[95% CI, 0.798–0.895] versus AUC=0.839 [95% CI, 0.782–
0.896]; Fig. 4C). More importantly, the combination of the two
markers was better for distinguishing between healthy individuals
metastasis (62.1625.5 U/mL,
CI, 0.811–0.920]versus
and TNM-T1/T2- (AUC=0.883; 95% CI, 0.816–0.949), TNM-
N0- (AUC=0.893; 95% CI, 0.841–0.946), or stage I/II-PC patients
(AUC=0.897; 95% CI, 0.856–0.937) than either marker alone
(Fig. 4C). These results imply that ULBP2 is a potentially useful
serum marker for PC early detection, particularly in conjunction
with CA 19-9.
Blood ULBP2 levels in patients with other cancer types
To test whether ULBP2 is also a marker for other malignant
diseases, we determined ULBP2 levels in the blood of patients with
CRC, NPC, and GC. We found that serum ULBP2 levels trended
to be higher in patients suffering from NPC (65.5674.3 pg/mL,
p=0.122, n=28) compared with healthy controls (51.4664.6 pg/
mL) and were moderately, but significantly, higher in CRC
Figure 2. Elevated expression of BIGH3 and ULBP2 in PC tissues. (A), Immunohistochemical staining for BIGH3 (left panel) and ULBP2 (right
panel) in paired pericancerous adjacent non-cancerous (lower panel) and tumor (upper panel) tissues. Scale bar, 100 mm. Original magnification,
6400. (B), Box-plot analysis of the immunohistochemical staining scores in paired adjacent non-cancerous (AN) and tumor tissues. The box indicates
the 25thand 75thpercentiles of the data range; the middle line indicates the median; the dashed line shows the middle 90% distribution.
doi:10.1371/journal.pone.0020029.g002
ULBP2 as a Novel Marker of Pancreatic Cancer
PLoS ONE | www.plosone.org5 May 2011 | Volume 6 | Issue 5 | e20029

Page 6

patients (70.6673.8 pg/mL, p=0.038, n=29; Fig. 5A); plasma
levels of ULPB2 were not significantly different between healthy
individuals (86.16101.2, n=25) and GC patients (78.1679.7 pg/
mL, n=30, p=0.673; Fig. 5B). However, serum ULBP2 levels
were strikingly elevated in PC patients compared to those in CRC
(200.26168.6 versus 70.6673.8 pg/mL, p,0.0001) and NPC
(200.26168.6 versus 65.5674.3 pg/mL, p,0.0001) patients
(Fig. 5A). The observation that ULBP2 levels were unaltered or
only marginally elevated in two other gastrointestinal cancers,
CRC and GC, suggests that ULBP2 might represent a relatively
specific PC marker.
Discussion
CA 19-9 is currently regarded as the most practicable serum PC
marker [27,28]. However, because CA 19-9 is also elevated in
other gastrointestinal diseases, including pancreatitis, hepatitis,
biliary obstruction, and other gastrointestinal cancers [9,27,28], its
specificity is not reliable. Furthermore, it is not expressed in
subjects with Lewis a-b- genotype [29], and poorly differentiated
PC appears to produce less CA 19-9 than moderately or well
differentiated cancers [28], thus limiting its detection sensitivity.
Clearly, the identification of novel PC markers that circumvented
these limitations would be a welcomed development.
In this study, we used an integrated approach, combined
analyses of the PC cell secretome and transcriptome to identify
novel PC marker candidates, and provide the first evidence that
ULBP2 is a potential novel candidate serum marker for PC
diagnosis. ULBP2 was overexpressed in PC cells compared to
adjacent non-cancerous tissues (Fig. 2). Bead-based immunoassays
performed with more than 150 serum specimens from PC patients
revealed that serum ULBP2 levels not only discriminated patients
from healthy donors, but also demonstrated great potential for PC
early detection (Figs. 3 and 4). Although several potential serum
Figure 3. Serum levels of BIGH3, ULBP2, and CA 19-9 in PC patients. The serum levels of BIGH3 (A), ULBP2 (B), and CA 19-9 (C) were
measured in 154 pancreatic cancer patients (PC) and 142 healthy controls (Ctrl). Data are presented as Box plots. (D), ROC curve analyses of the ability
of BIGH3, ULBP2, CA 19-9, and the two-marker panel comprising ULBP2 and CA 19-9 to discriminate PC patients from controls.
doi:10.1371/journal.pone.0020029.g003
ULBP2 as a Novel Marker of Pancreatic Cancer
PLoS ONE | www.plosone.org6 May 2011 | Volume 6 | Issue 5 | e20029

Page 7

PC markers have been reported, very few have been shown to be
superior to CA 19-9 [30,31]. Importantly, our comparison of serum
ULBP2 with CA 19-9 in this case-control study showed that serum
ULBP2 is superior to CA 19-9 as an early diagnostic marker (Fig. 4).
This finding is especially significant in light of the fact that most PC
patients diewithinoneyearbecause they arenotdiagnoseduntil the
cancer reaches an advanced stage [1]. The diagnostic and
prognostic potential of ULBP2 in a clinical setting will need to be
verified using a larger series of serum specimens. Moreover,
additional candidates listed in Table 1 have not been studied in
detail for PC, but their expression levels could be examined in PC
tissues using Human Protein Atlas (HPA) database, which contains
the immunohistochemical (IHC) staining profiles of 10118 proteins
in a variety of cancerous and non-cancerous tissues based on 13154
antibodies (version 7.1, http://www.proteinatlas.org/) [32]. We
searched 8 proteins in the HPA database and found 5 of them were
detected in more than 50% of the PC sections examined.
Noteworthily, alpha-soluble NSF attachment protein, annexin
A11, and ERO1-like protein alpha were expressed in more than
50% of the PC sections with moderate to strong IHC staining
(Supporting Table S6). These three proteins may represent as
potential PC biomarkers that warrant further investigation.
A consensus has coalesced around the idea that effective and
accurate detection of early-stage cancer will likely rely on marker
panels that possess better specificity and sensitivity than each
marker alone [26,33]. As noted above, CA 19-9 is currently used
for PC detection, but its use as a primary screening agent is
problematic. Hence, a marker panel that combined CA 19-9 with
other useful markers, such as ULBP2, could enhance the ability to
detect and monitor cancer. Indeed, the combination of both
markers evinced an improved diagnostic efficacy compared with
the traditional approach using CA 19-9 alone, especially with
respect to the detection of early-stage PC (Fig. 4).
Although ULBP2 has been reported as a tissue and serum
prognostic marker for ovarian cancer [34] and melanoma [35],
respectively, we believe that ULBP2 still has potential as a relatively
specific and useful serum test for PC diagnosis. A number of lines of
evidence support this: (a) ULBP2 serum/plasma levels were not
significantly elevated in GC, CRC or NPC patients compared to
healthy individuals in this study; (b) Li et al. have reported that
Figure 4. Efficacy of ULBP2 and CA 19-9 for early detection of pancreatic cancer. Serum levels of ULBP2 (A) and CA 19-9 (B) in healthy
controls (Ctrl) were compared with those in patients with early-stage PC. Data are presented as Box plots. (C), ROC curve analyses of the ability of
ULBP2 (black line), CA 19-9 (dashed line), and a combination of both proteins (thick line) to discriminate early-stage PC patients from controls.
doi:10.1371/journal.pone.0020029.g004
ULBP2 as a Novel Marker of Pancreatic Cancer
PLoS ONE | www.plosone.org7 May 2011 | Volume 6 | Issue 5 | e20029

Page 8

ULBP2 was not detectable in the sera of ovarian cancer patients
[34]; (c) Paschen et al. showed that ULBP2 was not measurable in
more than 20% of tested sera from melanoma patients, particularly
in early-stage patients [35]; and (d) ULBP2, unlike CA 19-9, does
not appear to be expressed at lower levels in poorly differentiated
PC than in moderately or well differentiated cancers (Supporting
Tables S4 and S5). However, evaluation of ULBP2 as a pancreatic
cancer marker will require large-scale counter-screening, particu-
larly using serum samples from pancreatitis patients.
ULBPs and the major histocompatibility complex class I-related
chain (MIC) are cell surface ligands for NK and T cell-expressed
immunoreceptor (NKG2D) that are rarely expressed by normal
cells, but appear in a broad variety of malignancies [36,37]. These
ligands can activate natural killer (NK) and cytotoxic T cells by
binding to the NKG2D, and subsequently contribute to cell-
mediated cytolysis and clearance of cancer [38], suggesting that
tumor cells overexpressing these ligands are more susceptible to
cell-mediated immune surveillance. Indeed, MIC expression has
been described as an indicator of better prognosis in CRC patients
[39], and the release of NKG2D ligands from tumor cells has been
previously demonstrated as a novel cancer immunoevasion
mechanism [40,41]. It has recently been proposed that proteolytic
shedding of ULBP2 from tumor cells is mediated by matrix
metalloproteases (MMP), and may impair the immunogenicity of
tumor cells [41]. Via the secretome analysis in this study, several
MMPs, including MMP-1, -7, -9, -11, -13, -14, and -28 could be
detected in CM from PC cell lines (Supporting Tables S1 and S2).
Moreover, elevated expression of MMP-7, -8, -9, and -11 have
been described in PC tissues; two of these, MMP-7 and MMP-11,
are strongly associated with poor cancer prognosis [42]. These
observations collectively suggest that elevated serum ULBP2 levels
in PC patients could reflect the involvement of proteolytic cleavage
by MMPs released from the cancer itself. By activating NK and
cytotoxic T cell-mediated immunity, elimination of the ULBP2
soluble form might be an effective therapeutic strategy in PC. This
intriguing possibility warrants further investigation.
In conclusion, we herein report that ULBP2 serum levels in PC
patients are significantly higher than those in healthy controls. The
combination of ULBP2 and CA 19-9 outperformed each marker
alone in distinguishing PC patients from healthy persons. More
importantly, an analysis of the area under the ROC curve showed
that ULBP2 was superior to CA 19-9 in discriminating patients
with early-stage pancreatic cancer from healthy controls. Thus,
ULBP2 may represent a novel and useful serum biomarker for
primary screening for pancreatic cancer.
Supporting Information
Figure S1
atic cancer tissues by immunohistochemistry.
(PDF)
Detection of BIGH3 expression in 31 pancre-
Figure S2
atic cancer tissues by immunohistochemistry.
(PDF)
Detection of ULBP2 expression in 67 pancre-
Figure S3
bead-based immunoassay developed in house.
(PDF)
Standard curve of ULBP2 determined by the
Figure S4
19-9 levels.
(PDF)
Correlation of serum ULBP2 and serum CA
Table S1
conditioned medium.
(PDF)
List of proteins identified in the BxPC-3
Table S2
conditioned medium.
(PDF)
List of proteins identified in the MIA PaCa-2
Figure 5. ULBP2 levels in blood specimens from other cancers. (A), Serum ULBP2 levels in healthy controls (Ctrl) were compared to those in
patients with nasopharyngeal carcinoma (NPC, n=28) and colorectal carcinoma (CRC, n=29). (B), Plasma ULBP2 levels in controls (Ctrl, n=25) were
compared to those in gastric cancer patients (GC, n=30).
doi:10.1371/journal.pone.0020029.g005
ULBP2 as a Novel Marker of Pancreatic Cancer
PLoS ONE | www.plosone.org8 May 2011 | Volume 6 | Issue 5 | e20029

Page 9

Table S3
tures and BIGH3 expression in tissue sections from 31
pancreatic cancer patients.
(PDF)
Correlation between clinicopathological fea-
Table S4
tures and ULBP2 expression in tissue sections from 67
pancreatic cancer patients.
(PDF)
Correlation between clinicopathological fea-
Table S5
CA19-9 levels with clinicopathologic characteristics in
154 pancreatic cancer patients.
(PDF)
Correlation of serum BIGH3, ULBP2, and
Table S6
in the Human Protein Atlas (HPA) database.
(PDF)
Expression profiles of candidate PC markers
Materials and Methods S1
(PDF)
Acknowledgments
We thank for Dr. Chia-Siu Wang (Department of General Surgery,
CGMH, Chiayi, Taiwan) and Dr. Err-Cheng Chan (Department of
Medical Biotechnology and Laboratory Science, Chang Gung University,
Tao-Yuan, Taiwan) for providing blood samples.
Author Contributions
Conceived and designed the experiments: JSY CCW. Performed the
experiments: YTC YLL MHT. Analyzed the data: YTC CCW JSY.
Contributed reagents/materials/analysis tools: YMS TCC TLH TSY
KPC HPL YSC. Wrote the paper: YTC CCW JSY.
References
1. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin
60: 277–300.
2. Pliarchopoulou K, Pectasides D (2009) Pancreatic cancer: current and future
treatment strategies. Cancer Treat Rev 35: 431–436.
3. Department of Health, Executive Yuan, Taiwan (2009) Standardize death rates
from leading cancer causes of death, 1986–2008. Department of Health,
Executive Yuan, Taiwan.
4. Sohn TA, Yeo CJ, Cameron JL, Koniaris L, Kaushal S, et al. (2000) Resected
adenocarcinoma of the pancreas-616 patients: results, outcomes, and prognostic
indicators. J Gastrointest Surg 4: 567–579.
5. Winter JM, Cameron JL, Campbell KA, Arnold MA, Chang DC, et al. (2006)
1423 pancreaticoduodenectomies for pancreatic cancer: A single-institution
experience. J Gastrointest Surg 10: 1199–1210; discussion 1210–1191.
6. Katz MH, Savides TJ, Moossa AR, Bouvet M (2005) An evidence-based
approach to the diagnosis and staging of pancreatic cancer. Pancreatology 5:
576–590.
7. Oto A, Eltorky MA, Dave A, Ernst RD, Chen K, et al. (2006) Mimicks of
pancreatic malignancy in patients with chronic pancreatitis: correlation of
computed tomography imaging features with histopathologic findings. Curr
Probl Diagn Radiol 35: 199–205.
8. Yeo TP, Hruban RH, Leach SD, Wilentz RE, Sohn TA, et al. (2002) Pancreatic
cancer. Curr Probl Cancer 26: 176–275.
9. Akdogan M, Sasmaz N, Kayhan B, Biyikoglu I, Disibeyaz S, et al. (2001)
Extraordinarily elevated CA19-9 in benign conditions: a case report and review
of the literature. Tumori 87: 337–339.
10. Magnani JL, Steplewski Z, Koprowski H, Ginsburg V (1983) Identification of
the gastrointestinal and pancreatic cancer-associated antigen detected by
monoclonal antibody 19-9 in the sera of patients as a mucin. Cancer Res 43:
5489–5492.
11. Harsha HC, Kandasamy K, Ranganathan P, Rani S, Ramabadran S, et al.
(2009) A compendium of potential biomarkers of pancreatic cancer. PLoS Med
6: e1000046.
12. Chang ST, Zahn JM, Horecka J, Kunz PL, Ford JM, et al. (2009) Identification
of a biomarker panel using a multiplex proximity ligation assay improves
accuracy of pancreatic cancer diagnosis. J Transl Med 7: 105.
13. Faca VM, Song KS, Wang H, Zhang Q, Krasnoselsky AL, et al. (2008) A mouse
to human search for plasma proteome changes associated with pancreatic tumor
development. PLoS Med 5: e123.
14. Guo J, Wang W, Liao P, Lou W, Ji Y, et al. (2009) Identification of serum
biomarkers for pancreatic adenocarcinoma by proteomic analysis. Cancer Sci
100: 2292–2301.
15. Melle C, Ernst G, Escher N, Hartmann D, Schimmel B, et al. (2007) Protein
profiling of microdissected pancreas carcinoma and identification of HSP27 as a
potential serum marker. Clin Chem 53: 629–635.
16. Tian M, Cui YZ, Song GH, Zong MJ, Zhou XY, et al. (2008) Proteomic analysis
identifies MMP-9, DJ-1 and A1BG as overexpressed proteins in pancreatic juice
from pancreatic ductal adenocarcinoma patients. BMC Cancer 8: 241.
17. Yu KH, Barry CG, Austin D, Busch CM, Sangar V, et al. (2009) Stable isotope
dilution multidimensional liquid chromatography-tandem mass spectrometry
for pancreatic cancer serum biomarker discovery. J Proteome Res 8: 1565–
1576.
18. Makridakis M, Vlahou A (2010) Secretome proteomics for discovery of cancer
biomarkers. J Proteomics 73: 2291–2305.
19. Pavlou MP, Diamandis EP (2010) The cancer cell secretome: a good source for
discovering biomarkers? J Proteomics 73: 1896–1906.
20. Wu CC, Hsu CW, Chen CD, Yu CJ, Chang KP, et al. (2010) Candidate
serological biomarkers for cancer identified from the secretomes of 23 cancer cell
lines and the human protein atlas. Mol Cell Proteomics 9: 1100–1117.
21. Ishikawa M, Yoshida K, Yamashita Y, Ota J, Takada S, et al. (2005)
Experimental trial for diagnosis of pancreatic ductal carcinoma based on gene
expression profiles of pancreatic ductal cells. Cancer Sci 96: 387–393.
22. Ravn V, Rasmussen BB, Hojholt L, Barfoed M, Heiberg I, et al. (1993)
Reproducibility of subjective immunohistochemical estrogen- and progesterone
receptor determination in human endometrium. Pathol Res Pract 189:
1015–1022.
23. Wu CC, Huang YS, Lee LY, Liang Y, Tang RP, et al. (2008) Overexpression
and elevated plasma level of tumor-associated antigen 90K/Mac-2 binding
protein in colorectal carcinoma. Proteomics Clin Appl 2: 1586–1595.
24. Crnogorac-Jurcevic T, Gangeswaran R, Bhakta V, Capurso G, Lattimore S,
et al. (2005) Proteomic analysis of chronic pancreatitis and pancreatic
adenocarcinoma. Gastroenterology 129: 1454–1463.
25. Buchholz M, Braun M, Heidenblut A, Kestler HA, Kloppel G, et al. (2005)
Transcriptome analysis of microdissected pancreatic intraepithelial neoplastic
lesions. Oncogene 24: 6626–6636.
26. Mor G, Visintin I, Lai Y, Zhao H, Schwartz P, et al. (2005) Serum protein
markers for early detection of ovarian cancer. Proc Natl Acad Sci USA 102:
7677–7682.
27. Goonetilleke KS, Siriwardena AK (2007) Systematic review of carbohydrate
antigen (CA 19-9) as a biochemical marker in the diagnosis of pancreatic cancer.
Eur J Surg Oncol 33: 266–270.
28. Steinberg W (1990) The clinical utility of the CA 19-9 tumor-associated antigen.
Am J Gastroenterol 85: 350–355.
29. Tempero MA, Uchida E, Takasaki H, Burnett DA, Steplewski Z, et al. (1987)
Relationship of carbohydrate antigen 19-9 and Lewis antigens in pancreatic
cancer. Cancer Res 47: 5501–5503.
30. Hoimes CJ, Moyer MT, Saif MW (2009) Biomarkers for early detection and
screening in pancreatic cancer. Highlights from the 45th ASCO annual meeting.
Orlando, FL, USA. May 29–June 2, 2009. JOP 10: 352–356.
31. Lee MX, Saif MW (2009) Screening for early pancreatic ductal adenocarcino-
ma: an urgent call! JOP 10: 104–108.
32. Berglund L, Bjorling E, Oksvold P, Fagerberg L, Asplund A, et al. (2008) A
genecentric Human Protein Atlas for expression profiles based on antibodies.
Mol Cell Proteomics 7: 2019–2027.
33. Polanski M, Anderson NL (2007) A List of Candidate Cancer Biomarkers for
Targeted Proteomics. Biomarker Insights 2006: 1–48.
34. Li K, Mandai M, Hamanishi J, Matsumura N, Suzuki A, et al. (2009) Clinical
significance of the NKG2D ligands, MICA/B and ULBP2 in ovarian cancer:
high expression of ULBP2 is an indicator of poor prognosis. Cancer Immunol
Immunother 58: 641–652.
35. Paschen A, Sucker A, Hill B, Moll I, Zapatka M, et al. (2009) Differential clinical
significance of individual NKG2D ligands in melanoma: soluble ULBP2 as an
indicator of poor prognosis superior to S100B. Clin Cancer Res 15: 5208–5215.
36. Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, et al. (1999) Broad
tumor-associated expression and recognition by tumor-derived gamma delta T
cells of MICA and MICB. Proc Natl Acad Sci USA 96: 6879–6884.
37. Pende D, Rivera P, Marcenaro S, Chang CC, Biassoni R, et al. (2002) Major
histocompatibility complex class I-related chain A and UL16-binding protein
expression on tumor cell lines of different histotypes: analysis of tumor
susceptibility to NKG2D-dependent natural killer cell cytotoxicity. Cancer Res
62: 6178–6186.
38. Maccalli C, Nonaka D, Piris A, Pende D, Rivoltini L, et al. (2007) NKG2D-
mediated antitumor activity by tumor-infiltrating lymphocytes and antigen-
specific T-cell clones isolated from melanoma patients. Clin Cancer Res 13:
7459–7468.
39. Watson NF, Spendlove I, Madjd Z, McGilvray R, Green AR, et al. (2006)
Expression of the stress-related MHC class I chain-related protein MICA is an
ULBP2 as a Novel Marker of Pancreatic Cancer
PLoS ONE | www.plosone.org9 May 2011 | Volume 6 | Issue 5 | e20029

Page 10

indicator of good prognosis in colorectal cancer patients. Int J Cancer 118:
1445–1452.
40. Groh V, Wu J, Yee C, Spies T (2002) Tumour-derived soluble MIC ligands
impair expression of NKG2D and T-cell activation. Nature 419: 734–738.
41. Waldhauer I, Steinle A (2006) Proteolytic release of soluble UL16-binding
protein 2 from tumor cells. Cancer Res 66: 2520–2526.
42. Jones LE, Humphreys MJ, Campbell F, Neoptolemos JP, Boyd MT (2004)
Comprehensive analysis of matrix metalloproteinase and tissue inhibitor
expression in pancreatic cancer: increased expression of matrix metalloprotei-
nase-7 predicts poor survival. Clin Cancer Res 10: 2832–2845.
ULBP2 as a Novel Marker of Pancreatic Cancer
PLoS ONE | www.plosone.org10May 2011 | Volume 6 | Issue 5 | e20029