International Journal of ProteomicsVolume 2011 (2011), Article ID 628787, 13 pagesdoi:10.1155/2011/628787

Research Article

The Application of a Three-Step Proteome Analysis for Identification of New Biomarkers of Pancreatic Cancer

Mayinuer Abulaizi,1,2 Takeshi Tomonaga,1,3 Mamoru Satoh,1,2 Kazuyuki Sogawa,1,2 Kazuyuki Matsushita,1,2 Yoshio Kodera,2,4 Jurat Obul,¹ Shigetsugu Takano,⁵ Hideyuki Yoshitomi,⁵ Masaru Miyazaki,⁵ and Fumio Nomura1,2

¹Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan²Clinical Proteomics Research Center, Chiba University Hospital, Chiba 260-8677, Japan³Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka 567-0085, Japan⁴Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science, Sagamihara 252-0373, Japan⁵Department of General Surgery, Graduate School of Medicine, Chiba University, 260-8670 Chiba, Japan

Received 31 May 2011; Accepted 2 August 2011

Academic Editor: Tadashi Kondo

Copyright © 2011 Mayinuer Abulaizi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

We searched for novel tumor markers of pancreatic cancer by three-step serum proteome analysis. Twelve serum abundant proteins were depleted using immunoaffinity columns followed by fractionation by reverse-phase high-performance liquid chromatography. Proteins in each fraction were separated by two-dimensional gel electrophoresis. Then the gel was stained by Coomassie Brilliant Blue. Protein spots in which the expression levels were significantly different between cancer and normal control were identified by LC-MS/MS. One hundred and two spots were upregulated, and 84 spots were downregulated in serum samples obtained from patients with pancreatic cancers, and 58 proteins were identified by mass spectrometry. These candidate proteins were validated using western blot analysis and enzyme-linked immunosorbent assay (ELISA). As a result of these validation process, we could confirm that the serum levels of apolipoprotein A-IV, vitamin D-binding protein, plasma retinol-binding protein 4, and tetranectin were significantly decreased in patients with pancreatic cancer.

1. Introduction

Pancreatic cancer is one of the most lethal malignancies, with a 5-year survival rate of only 4-5% [1]. The major reasons for the poor prognosis may be late diagnosis and limited therapeutic options; early diagnosis of pancreatic cancer is a pressing clinical problem.

Serum levels of the conventional tumor markers including carcinoembryonic antigen (CEA) and the Lewis blood group carbohydrate antigen (CA19-9) often remain in normal range at early stages of this malignancy [2]. Therefore, search for novel biomarkers of pancreatic cancer is needed.

Recent advances in proteomic technologies have provided promising ways to discover and identify novel biomarkers in various fields of clinical medicine. Although there has been long and uncertain path from marker discovery to clinical utility [3], sophisticated technologies have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis of cancer patients [4]. Also, proteome analysis can lead to biomarkers that may be useful in the prediction of clinical response to anticancer therapy [5].

Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a representative example of a proteomics technique for the high-throughput fingerprinting of serum proteins and peptides and biomarker discovery [6]. Using this technology, we could detect and identify novel diagnostic markers for alcohol abuse [7] and also a new prognostic marker for pancreatic cancer [8].

One of the technical challenges in serum proteome analysis is that serum contains thousands of proteins and peptides that are present in a large dynamic concentration [9]. Indeed, 22 abundant proteins such as albumin, immunoglobulins, and transferring constitute up to 99% of the protein content of plasma [10]. In proteomic studies searching for low-abundance serum proteins or peptides, depletion of those abundant proteins and further fractionation of samples will be necessary.

We recently conducted a three-step proteome analysis involving removal of 12 abundant proteins and subsequent reversed-phase high-performance liquid chromatography fractionation and one-dimensional electrophoresis: we successfully identified three proteins including YKL-50 as a promising biomarker of sepsis [11].

Proteomics in pancreatic cancer research including serum or plasma biomarker search has been reviewed [12]. A three-step approach as-we used in this study has not been tried in biomarker search for pancreatic cancers before.

In this study, we applied this three-step proteome analysis to find novel biomarkers of pancreatic cancer.

2. Method

2.1. Patients Studied

Serum samples were obtained preoperatively from a total of 32 patients diagnosed with primary invasive pancreatic ductal carcinoma who had surgery at the Department of General Surgery, Chiba University Hospital. Clinical data of 32 patients are summarized in Table 1(a). Serum samples were also obtained from apparently healthy and age-matched subjects who had medical checkup at the Port-square Kashiwado Clinic, Kashiwado Memorial Foundation (Table 1(b)). All samples were frozen by liquid nitrogen and were stored at −80°C until analysis. Written informed consent was obtained from all the subjects. The ethics committee of our institute approved the protocol.

Table 1

(a) Clinical features of pancreatic cancer patients.

No
Gender
Age (years)
UICC-stage
Tumor size (mm)
TP (g/dL)
ALB (g/dL)
Che (U/L)
T-Cho (mg/dL)

1
M
66
III
35
6.7
4.3
270
220

2
M
66
III
39
6.6
4.4
359
198

3
M
79
IV
10
6.2
3.8
179
155

4
M
71
III
18
6.8
4.0
207
210

5
M
65
III
35
7.0
3.7
189
131

6
M
78
III
50
6.6
4.4
359
198

7
M
66
IIA
30
6.3
4.1
267
176

8
M
62
IV
10
5.9
3.6
171
169

9
M
38
IA
10
6.7
4.0
289
179

10
M
50
IB
30
7.3
4.6
408
203

11
M
63
IIA
18
6.6
3.9
260
138

12
M
62
IIA
38
6.0
3.4
124
149

13
M
54
IIA
24
7.0
4.5
198
162

14
M
73
IIA
25
6.7
4.1
242
176

15
F
76
IIA
26
5.0
3.3
162
95

16
M
63
IIB
15
7.1
4.2
175
163

17
M
65
IIB
32
5.8
3.5
227
113

18
M
68
IIB
80
6.8
4.0
316
217

19
M
71
IIB
24
7.5
4.1
221
144

20
M
74
IIB
27
7.0
4.3
319
178

21
F
68
IIB
27
6.5
4.0
223
177

22
M
63
IIB
26
6.9
4.3
262
219

23
F
68
IIB
27
6.1
3.5
339
163

24
M
61
IIB
30
6.2
4.0
200
176

25
F
74
IIB
40
6.6
3.8
221
170

26
M
62
IIB
60
8.3
3.5
140
155

27
F
73
IIB
35
5.9
3.2
130
169

28
F
59
IIB
18
7.2
4.4
356
130

29
M
73
IIB
50
5.7
3.3
127
135

30
F
62
IIB
25
6.8
4.1
255
293

31
F
71
III
25
6.8
4.2
294
159

32
F
78
III
50
5.9
3.3
197
182

Ave ± SD

UICC: international union against cancer, M: male, F: female. TP: total protein. ALB: albumin. Che: cholinesterase. T-Cho: total cholesterol. Ave: average. SD: standard deviation.

From number 1 to 4 were used for 2-DE, from number 1 to 8 were for first western blot, from number 9 to 32 were for second western blot.

(b) Clinical features of healthy controls.

No
Gender
Age (years)
TP (g/dL)
ALB (g/dL)
Che (U/L)
T-Cho (mg/dL)

1
M
62
7.5
4.7
401
203

2
M
61
7.7
5.1
396
269

3
M
64
7.2
4.4
284
229

4
M
73
7.6
4.7
216
203

5
M
57
8.3
5.0
430
301

6
M
57
6.7
4.8
328
253

7
M
65
7.2
4.8
375
230

8
M
64
7.2
4.6
327
296

9
M
55
6.9
4.6
300
211

10
M
71
7.1
4.7
290
267

11
F
64
8.1
5.4
293
192

12
M
71
7.2
4.5
233
213

13
M
55
7.2
4.5
365
227

14
M
68
7.4
4.4
300
255

15
F
67
7.0
4.5
279
172

16
F
71
7.0
4.5
297
220

17
M
60
6.7
4.2
284
183

18
M
61
7.0
4.7
260
198

19
M
55
6.4
3.9
304
176

20
F
70
7.6
4.7
398
225

21
M
70
7.3
4.6
257
250

22
F
67
7.4
4.5
304
194

23
M
60
7.0
4.4
338
234

24
F
77
8.1
5.0
416
274

25
F
62
7.5
4.6
284
169

26
F
64
7.2
4.5
280
208

27
M
65
7.4
4.6
264
231

28
M
61
7.8
4.3
319
239

29
M
61
7.3
4.6
309
271

30
M
66
7.5
5.0
375
278

31
F
65
7.6
4.5
339
288

32
M
73
7.1
4.4
306
211

Ave ± SD

M: male, F: female. TP: total protein. ALB: albumin. Che: cholinesterase. T-Cho: total cholesterol. Ave: average. SD: standard deviation.

From number 1 to 4 were used for 2-0 E, from number 1 to 8 were for first western blot, from number 9 to 32 were for second western blot.

2.2. A Three-Step Serum Proteome Analysis

2.2.1. Immunoaffinity Subtraction of Highly Abundant Proteins from Human Serum

Serum samples obtained from 4 patients with pancreatic cancer (Nos 1~4 in Table 1(a)) were pooled. Sera obtained from 4 age-matched healthy volunteers were also pooled (Nos 1~4 in Table 1(b)). As the first step of proteome analysis, the twelve most abundant proteins (albumin, Immunoglobulin G, transferrin, fibrinogen, Immunoglobulin A, Immunoglobulin M, apolipoprotein A-I, apolipoprotein A-II, haptoglobin, α1-acid glycoprotein and α2-macroglobulin) were removed from serum by passage through a commercially available immunoaffinity column, the ProteomeLab IgY12HC LC10 (Beckman coulter, Inc. Fullerton, CA. USA.) Ninety microliters of each pooled sample was subjected to the immunoaffinity subtraction as we previously described [11]. The combined flow-through fractions were concentrated by Vivaspin2 spin concentrators (molecular weight cutoff, 10 kDa, Vivascience, Hannover, Germany) and were stored at −80°C until use.

In addition, sera from 32 patients with pancreatic cancer and 32 healthy volunteers were used for validation. Eight healthy controls and 8 of relatively advanced cases (Nos 1~8 in Tables 1(a) and 1(b)) were chosen for first validation and 24 of them (Nos 9~32 in Tables 1(a) and 1(b)) were used for the second validation experiment.

2.3. HPLC Separation of Immunoaffinity-Subtracted Serum Samples

Immunoaffinity-subtracted serum samples prepared as described above were separated by reverse-phase HPLC in an automated HPLC system, the SHISEIDO Nanospace SI-2 (Shiseido Fine Chemicals, Tokyo, Japan) essentially as we described before [11]. A total of 40 fractions were collected at 0.5 min intervals from 19.6 to 39.6 min. Each fraction was immediately lyophilized by a centrifugal vacuum concentrator and stored at −80°C until further analysis.

2.4. Two-Dimensional Gel Electrophoresis

The IEF gels (70 mm length, Inner 2.5 mm diameter and pH ranges from 3 to 10) were prepared as previously described [13, 14]. The lyophilized samples (from fraction 6 to fraction 25) were dissolved with 15 μL sample preparation buffer and proteins were separated by two-dimensional gel electrophoresis with agarose gels in the first dimension as described by Oh-Ishi et al. [13].

2.5. In-Gel Digestion and LC-MS/MS

CBB stained 2-DE images of pooled serum samples obtained from patients with pancreatic cancer were compared with those obtained from healthy volunteers. Differentially expressed protein bands were excised from the gel and were subjected to in-gel tryptic digestion as previously reported [14]. Digested peptides were injected into a trap column: L-trap column (Chemicals Evaluation and Research Institute, Saitama, Japan) and an analytical column: Monolith column (AMR, Tokyo, Japan), which was attached to a HPLC system (Nanospace SI-2; Shiseido Fine Chemicals, Tokyo, Japan). The flow rate of a mobile phase was 1 μL/min. The solvent composition of the mobile phase was programmed to change in 35 min cycles with varying mixing ratios of solvent A (2% v/v CH₃CN and 0.1% v/v HCOOH) to solvent B (90% v/v CH₃CN and 0.1% v/v HCOOH): 5–50% B 20 min, 50–95% B 1 min, 95% B 3 min, 95–5% B 1 min, 5% B 10 min. Purified peptides were introduced from HPLC to an LTQ-XL (Thermo Scientific, CA, USA), an ion trap mass spectrometer (ITMS), via an attached Pico Tip (New Objective, MA, USA). The MS and MS/MS peptide spectra were measured in a data-dependent manner according to the manufacturer’s operating specifications. The Mascot search engine (Matrixscience, London, UK) was used to identify proteins from the mass and tandem mass spectra of peptides. Peptide mass data were matched by searching the Human International Protein Index database (IPI, July 2008, 72079 entries, European Bioinformatics Institute) using the MASCOT engine. The minimum criterion of the probability-based MASCOT/MOWSE score was set with 5% as the significant threshold level. When the candidates had SEQUEST scores lower than 100 or when the SEQEUST score was computed by using fewer than one peptides fragment, we inspected the raw MS and MS/MS spectra of peptides to judge their qualities (see Figures a–f in Supplementary Material available online at doi: 10.1155/2011/628787).

2.6. Western Blot Analysis

Western blotting was performed as we previously described [15].

Briefly, immunoaffinity-subtracted serum samples were separated on SDS-PAGE in 10–20% polyacrylamide gradient gel (DRC, Tokyo, Japan) and were transferred to polyvinylidene fluoride membranes (0.45 μm thickness, Millipore, Bedford, MA) at 10 V for overnight. The following antibodies commercially available were used as primary antibodies; mouse anti-human ApoA-IV antibody (BML Inc., Tokyo, Japan), mouse anti-human GC antibody (LifeSpan, Inc., UK), mouse monoclonal anti-human RBP4 antibody (Abnova.Com., Taipei, Taiwan) and mouse anti-human CLEC3B antibody (BioPorto, Grusbakken 8, DK-2820 Gentofte, Denmark). Antigens on the membrane were detected with enhanced chemiluminescence detection reagents (GE Healthcare). Band intensities of the western blot images were quantified by TotalLab TL12 imaging analysis software (Shimadzu Co., Ltd. Kyoto, Japan) and were presented by arbitrary units.

2.7. Other Procedures

In addition to western blotting, ELISA was conducted in some marker candidates using human ApoA-IV ELISA kit (Millipore, Missouri, USA), GC ELISA kit (immundiagnostik AG, Bensheim), and RBP4 ELISA kit; (R & D systems). Their optical density was measured at 450 nm using a microplate reader (iMark Microplate Reader S/N 10288). Serum levels of CEA and CA19-9 were determined by established commercially available kits.

2.8. Statistical Analysis

Statistical analysis was conducted using KaleidaGraph 4.0 J (Synergy Software, Reading, PA) and IBM SPSS Statistics 18 (SPSS Inc., IL, USA). Significance was defined as .

3. Results

3.1. Discovery and Identification of Differentially Expressed Proteins by a Three-Step Proteome Analysis

To discover and identify novel serum markers for pancreatic cancer, we employed a comparative three-step proteome analysis of the pooled serum samples obtained from patients with pancreatic cancer and healthy volunteers. As the first step, 12 abundant proteins were removed by immunosubtractions. The immunoaffinity-subtracted samples were separated by RP-HPLC, and 20 fractions (fractions Nos 6–25) were subjected to 2-DE. A representative example is shown in Figure 1. By comparing the 2-DE images of the proteins included in the 20 fractions, a total 186 spots were found to be differently expressed. Subsequent LC-MS/MS could identify 100 proteins. Excluding keratins, complements and trypsin, 58 proteins were selected; 37 of them were upregulated and 21 were downregulated (Tables 2(a) and 2(b)).

Table 2

(a) Proteins upregulated in pancreatic cancer.

Protein's name
Experimental
Theoretical
Score⁽¹⁾
Queries
Validation

mass (Da)
mass (Da)
matched(2)

Histidine-rich glycoprotein precursor
80000
59541
150
3
WB⁽³⁾

Plasminogen precursor
100000
90510
331
8

IGHM protein
50000
52754
105
2

TF Serotransferrin precursor
75000
77000
601
14
WB

Isoform LMW of Kininogen-1 precursor
70000
47853
242
6

F2 Prothrombin precursor (Fragment)
90000
69992
486
8

Alpha-1B-glycoprotein precursor
43000
54239
590
8
WB

Vitronectin precursor
62000
54271
581
8
WB

Hepatocyte growth factor-like protein precursor
85000
80268
284
6
WB

Plasma kallikrein precursor
90000
71323
246
8

Ceruloplasmin precursor
115000
122128
1452
49

Isoform 1 of Ficolin-3 precursor
34400
32883
58
1

Transthyretin precursor
95000
15877
4489
13

Serum amyloid P-component precursor
24000
25371
2293
12

Antithrombin III variant
61000
52658
1984
20

SERPINC1 protein
61000
29074
1147
11

Carbonic anhydrase 1
30800
28852
679
10
WB

Isoform 1 of C-reactive protein precursor
26000
25023
169
6

Apolipoprotein A-1
31600
30759
225
4

Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H3
138000
75031
529
12

Leucine-rich alpha-2-glycoprotein precursor
47000
38154
549
8

Isoform 1 of N-acetylmuramoyl-L-alanine amidase precursor
68900
67957
193
4

Xaa-Pro dipeptidase
53700
54513
144
3

Inter-alpha (globulin) inhibitor H4
130500
103261
1242
26
WB

Vitamin K-dependent protein S precursor
84000
75074
136
4
WB

Serpin peptidase inhibitor, clade D (Heparin cofactor),
72000
57034
374
9

Isoform 1 of Fibronectin precursor
200000
262442
553
14
WB

Alpha-1-antichymotrypsin precursor
62800
50566
3315
33
WB

Kallistatin precursor
59600
48511
123
2

Plastin-2
72000
70245
215
5

Corticosteroid-binding globulin precursor
62500
45112
52
1

Myosin-1
31600
222976
96
2

Isoform 1 of Serum albumin precursor
73000
69321
261
9

Cholinesterase precursor
83000
72836
50
1

AMBP protein precursor
200000
38974
68
1

Plasma protease C1 inhibitor precursor
87000
55119
1168
15

Apolipoprotein B-100 precursor
300000
515241
3982
117

⁽¹⁾MOWSE score of candidate proteins.

⁽²⁾Number of peptide fragments yielding informative MS/MS.

⁽³⁾WB: western blot.

(b) Proteins downregulated in pancreatic cancer.

Protein's name
Experimental
Theoretical
Score⁽¹⁾
Queries
Validation

mass (Da)
mass (Da)
matched(2)

Plasma retinol-binding protein precursor
19000
22995
373
8
WB⁽³⁾

Coagulation factor XII precursor
75000
67774
140
5

Tetranectin precursor
19000
22552
61
1
WB

Hyaluronan-binding protein 2 precursor
68000
62630
195
6

Vitamin D-binding protein precursor
55000
52883
284
14
WB

Hemopexin precursor
75000
51643
635
8
WB

Lumican precursor
100000
38405
120
6
WB

Isoform 1 of Gelsolin precursor
80000
85644
1360
23
WB

Afamin precursor
80000
69024
307
7

Carboxypeptidase N catalytic chain precursor
49000
52253
244
12
WB

Inter-alpha-trypsin inhibitor heavy chain H1 precursor
200000
101326
812
13
WB

Histone H4
25700
11360
59
1

JUP JUP protein
100000
81675
90
3

apolipoprotein A-IV precursor
42000
45371
2188
35
WB

Inter-alpha-trypsin inhibitor heavy chain H2 precursor
200000
106370
1636
26
WB

Pigment epithelium-derived factor precursor
50000
46313
529
11

Angiotensinogen precursor
56300
53121
986
13

SERPINF2 protein
58000
55029
75
2

Actin, cytoplasmic 1
100000
41710
208
5

Thrombospondin-1 precursor
175000
129300
152
4

Alpha-2-macroglobulin precursor
180000
163175
559
18

⁽¹⁾MOWSE score of candidate proteins.

⁽²⁾Number of peptide fragments yielding informative MS/MS data. The minimum significant threshold level of the probability-based MASCOT/MOWSE score was set at 5%.

⁽³⁾WB: western blot.

Figure 1: Comparison of 2-DE images of the same fraction of healthy volunteer control and pancreatic cancer patient sample. Electrophoresis was performed on the same gel and at the same condition. Figure 1 is an example of Coomassie blue-stained gel displaying spots from depleted and fractionated serum from control (left, , pooled) and pancreatic cancer patients (right, , pooled). (Fraction number is 10th). Double circles indicate increased spot in pancreatic cancer. Single circle indicates decreased spot in pancreatic cancer.

3.2. Validation of Marker Candidates by Western Blotting

Out of the 58 proteins listed in Tables 2(a) and 2(b), we focused on 19 proteins the alterations of which at serum level have not been studied in detail before, and also antibodies to be used for validation studies are available. Initial validation was conducted using 8 serum samples (nos. 1–8 in Table 1) obtained from relatively advanced cases with pancreatic cancer including the four cases used for the three-step analysis. Western blotting of the 19 proteins indicated in Tables 2(a) and 2(b) revealed that expression of 7 proteins were found to be significantly decreased in patients with pancreatic cancers compared with controls: they were inter-alpha trypsin inhibitor heavy chain H1 (ITIH1), hemopexin precursor (HPX), alpha-1B-glycoprotein precursor (A1BG), apolipoprotein A-IV precursor (ApoA-IV), vitamin D-binding protein precursor (GC), plasma retinol-binding protein precursor (RBP4), and tetranectin (CLEC3B).

We then conducted the second validation study to test whether differential expression of the 7 protein candidates described above is reproducible using another set of serum samples obtained from 24 patients with pancreatic cancers including cases with relatively early stages (nos. 9–32 in Table 1(a)). As shown in Figure 2(a) the expression levels of the four proteins ApoA-IV, GC, RBP4, and CLEC3B were greater in cancer patients than in controls. The differences were statistically significant assessed by densitometry Figure 2(b).

(a)

(b)

Figure 2: (a) Magnified views of 2-D gel images and western blotting analysis of ApoA-IV, GC, plasma retinol binding protein 4 RBP4, and CLEC3B in serum samples. Coomassie blue-stained 2-D gel images from pooled control and pancreatic cancer displaying the protein spots for ApoA-IV, GC, RBP4, and CLEC3B are shown in left panels (a1), (a2), (a3) and (a4). Western blotting of these four proteins are shown in the right panel (b1), (b2), (b3) and (b4). (b) Quantitation of differentially expressed serum proteins in pancreatic cancer and healthy volunteers by Western blot analysis. Intensities of each band were calculated by TotalLab TL 120 software. Closed circles indicate healthy volunteers and closed squares indicate patients with pancreatic cancer. Significance of the differences were calculated by using Wilcoxon Mann-Whitney test. Panel A: ApoA-IV levels of serum were significantly lower in the depleted sera of pancreatic cancer when compared with the depleted sera of healthy volunteers (). Panel (B, C and D) are for proteins GC, RBP4, and CLEC3B and their serum levels were likewise lower in the pancreatic cancer patients. Their values are lower than 0.0001, 0.001, and 0.009, respectively.

3.3. Validation of Marker Candidates by ELISA

ELISA kits were commercially available for GC, ApoA-IV, and RBP4. Their serum levels were determined in the 15 pairs of serum samples obtained from relatively early stages of patients used for the second validation by western blotting. Serum ApoA-IV levels of patients with pancreatic cancer (AU) were significantly lower than those in healthy volunteers (AU, ) Figure 3(a). GC levels were significantly lower in the patient group with pancreatic cancer ( UA) when compared with healthy group ( AU, ) Figure 3(b). Also, serum RBP levels in the patients ( AU) were significantly lower than in the controls (, ) Figure 3(c).

(a)

(b)

(c)

Figure 3: Quantitation of differentially expressed proteins in pancreatic cancer by ELISA. ELISA was performed using human ApoA-IV ELISA kit vitamin D binding protein ELISA kit and RBP4 ELISA kit, in serum samples obtained from 15 patients with pancreatic cancers and 15 control subjects. Analysis was performed by using Wilcoxon Mann-Whitney test. Closed circles indicate control and closed squares indicate cancer. (a): ApoA-IV levels of serum of patient with pancreatic cancer group ( AU) were lower than those in healthy group ( AU, ); (b): GC levels were significantly lower in the patient group with pancreatic cancer ( UA) when compared with healthy group ( AU, ); (c): RBP4 levels were lower in the pancreatic cancer group ( AU) than healthy group ( AU, .

3.4. Comparison of the Marker Candidates with CEA and CA19-9

Figure 4 shows the receiver-operating characteristic curve (ROC) analysis for the three marker candidates determined by ELISA and those for CEA and CA19-9.The areas under the curves for ApoA-IV, GC, RBP4, CA19-9, and CEA were 0.79, 0.72, 0.85, 0.88, 0.58, 0.88, 0.89, and 0.89, respectively. Also, AUCs of the combination assay of GC/CA19-9, ApoA-IV/CA19-9, and RBP/CA19-9 were 0.88, 0.89, and 0.89, respectively.

(a)

(b)

(c)

(d)

(e)

Figure 4: Receiver operating characteristic (ROC) curves for CA19-9, CEA, ApoA-IV, GC, RBP4 Their AUCs are described in the text.

In Tables 3(a) and 3(b), serum levels of ApoA-IV, GC and RBP4 determined by ELISA are listed together with CEA and CA19-9. There were 7 cases in which serum CA19-9 level was not elevated. Out of these 7 cases, ApoA-IV levels were below the lower reference interval value (mean SD) in 2 cases. Also, GC levels were below the lower reference interval value in one case.

Table 3

(a) Serum tumor marker levels in patients with pancreatic cancer.

Gender
Age (years)
UICC-stage
Tumor size (mm)
CA19-9 (U/mL)
CEA (ng/mL)
ApoA-IV (AU)
GC (AU)
RBP4 (AU)

M
38
IA
10
26.2
2.3
262.1
35.9
54.2

M
50
IB
30
46.5
2.3
8.5
34.3
43.9

M
63
IIA
18
157
1.1
10.2
26.7
37.0

M*
62
IIA
38
9
5
70.7
25.1
41.5

M*
54
IIA
24
11
1.4
87.5
11.3
42.5

M
73
IIA
25
15.5
3.5
203.5
13.6
49.5

F*
76
IIA
26
13.3
1.5
70.7
33.1
43.2

M
65
IIB
32
1579
3.4
352.1
24.3
36.7

M*
74
IIB
27
10.9
3.4
0
28.0
35.3

F
74
IIB
40
43
3.2
54
25.8
38.9

F
68
IIB
27
302
2.1
82.8
40.8
36.4

M
61
IIB
30
10
1.4
134.7
27.3
46.2

M
63
IIB
15
1080
—
144.6
23.9
53.3

M*
73
IIB
50
13.9
2.1
23.2
1.8
43.1

F
62
IIB
25
11.5
1.1
111.7
28.4
43.1

Ave ± SD

CA19-9: carbohydrate antigen, CEA: carcinoembryonic antigen, ApoA-IV: apolipoprotein A-IV, GC: vitamin D-binding protein, RBP4: plasma retinol binding protein 4.

The stars indicate the patient who had a normal CA19-9 level and a low ApoA-IV.

Ave: average. SD: standard deviation. AU: arbitrary unit.

(b) Serum tumor marker levels in healthy controls.

Gender
Age (years)
CA19-9 (u/mL)
CEA (ng/mL)
ApoA-IV (AU)
GC (AU)
RBP4 (AU)

M
71
33.9
2.2
157.4
29.5
47.7

M
55
0.1
4.7
243.8
35.0
48.5

M
55
6.4
1
223.0
35.3
54.3

M
55
0.1
3.5
237.1
33.0
48.1

M
60
7.7
0.8
237.1
23.9
44.1

M
60
37.4
7
214.0
39.6
48.8

M
61
7.6
4.8
277.0
16.8
49.7

F
77
7.1
1.1
294.5
24.2
43.7

M
61
7.8
1.3
80.6
33.4
55.3

F
61
6
3
210.1
45.8
56.3

F
65
2.9
2.1
65.7
61.7
53.3

M
66
5.1
0.7
156.7
33.8
44.7

F
62
11.7
1.7
122.9
33.0
50.9

F
64
8.4
1.2
222.9
38.7
50.3

F
71
24.4
2.6
184.7
30.5
56.3

Ave ± SD

CA19-9: carbohydrate antigen, CEA: carcinoembryonic antigen, ApoA-IV: apolipoprotein A-IV, GC: vitamin D-binding protien, RBP4: plasma retinol binding protein

Ave: average. SD: standard deviation. AU: arbitrary unit.

4. Discussion

The sequencing of the human genome has opened the door for comprehensive analysis of all the messenger RNA (transcriptome) and proteins (proteome). Messenger RNA concentrations, however, are not necessarily predictive of corresponding protein concentrations. Indeed, a recent report indicates that the sharing rate between cDNA microarray and proteome-based profilings is limited for the identification of candidate biomarkers in renal cell carcinoma [16]. Therefore, proteome analysis is one of the prerequisite for development of novel biomarkers. Proteomic studies in pancreatic cancers have been conducted by many research groups as reviewed [12, 17]. Hwang et al. found by using 2-DE/MS that phosphoglycerate kinase (PGK) 1, a secretable glycolytic enzyme involved in angiogenesis, is overexpressed in serum samples of pancreatic cancer patients, as compared to controls [18]. More recently, using the two-dimensional image-converted analysis of liquid chromatography and mass spectrometry (2DICAL) and a ‘‘glyco capturing’’ through concanavalin A-agarose, Ono et al. identified a novel prolyl-hydroxylation of fibrinogen alpha chain in plasma samples obtained from patients with pancreatic cancers [19].

In this study, the three-step procedure was carried out to discover novel markers of pancreatic cancer. The outline of the three-step procedures is shown in Figure 5.

Figure 5: Outline of the procedure for the three-step serum proteome analysis.

As a first step, serum samples were subjected to antibody-based immunoaffinity column that simultaneously removes 12 abundant serum proteins. The concentrated flow-through was then fractionated using reversed-phase HPLC. Proteins obtained in each HPLC fraction were further separated by 2-DE. A total of 58 differentially expressed proteins were identified. As results of initial validation by western blotting in relatively advanced cases and further validation including the less advanced cases by western blotting, the expression levels of the four proteins ApoA-IV, GC, RBP4, and CLEC3B were greater in cancer patients than in controls. Out of these four proteins, ELISA were available in apolipoprotein A-IV, retinol-binding protein precursor (RBP4), and vitamin D binding protein (GC). Serum levels of these 3 proteins were significantly lower in patients with pancreatic cancer than in healthy volunteers. In ROC analyses, the area under the curves for these three proteins was not significantly greater than that for CA19-9, but it is noteworthy that among the 4 cases of pancreatic cancers in which serum levels of both CEA and CA19-9 were within the reference intervals, at least one of ApoA-IV, RBP4, and GC was found to be decreased in 2 cases, suggesting that these candidate markers could be complementary to the conventional markers in diagnosis of pancreatic cancer.

ApoA-IV is present in human intestinal epithelial cells and is secreted as a chylomicron and VLDL apoprotein [20].

Retinol binding protein 4 (RBP4) is a 21-kDa protein synthesized in the liver and adipose tissue; its major function is to deliver retinol to tissue [21]. Fabris et al. determined serum RBP levels in patients with pancreatic cancer and found that the levels decreased concomitant with zinc and prealbumin levels [22]. Serum zinc levels were not significantly correlated with RBP4 levels in the present study (data not shown).

Vitamin D-binding protein is a plasma protein involved in vitamin D transport and other function. Although diagnostic role of this protein in pancreas cancer has not been reported yet, inhibitory role of vitamin D binding protein-macrophage activating factor (DBP-maf) in pancreatic carcinogenesis has been pointed out [23].

Tetranectin binds to kringle 4 of plasminogen, enhancing the plasminogen activation by tissue-type plasminogen activator in the presence of poly-D-lysine [24] Low serum levels of tetranectin (CLEC3B) are associated with increased risk of second-line chemoresistance in patients with ovarian cancer [25]. Also, in colorectal cancer, significantly shorter survival was found for patients with CLEC3B levels below a cut-off point of compared to patients with levels above [26].

Thus, the results of this study show that four serum proteins, apolipoprotein A-IV, vitamin D binding protein, retinol-binding protein 4, and tetranectin are significantly decreased in patients with pancreatic cancer. It was notable that these changes were observed in some patients in whom conventional tumor markers for this malignancy were not altered.

The reasons why serum levels of these proteins were decreased in pancreatic cancer patients are not clear at the moment. It is unlikely that the alterations were entirely due to malnutrition because serum levels of the 4 proteins were not significantly correlated with their serum albumin levels. It is possible that some negative mediators originated from tumor and/or the cancer-tissue microenvironments were regulating their production. It is unlikely that the alterations were due to biliary obstruction because the extent of the alterations of the four markers were not related to the extent of biliary obstruction (data not shown). Alterations of these four proteins in chronic pancreatitis as well as biliary tract diseases remain to be studied. Also, it remains to be determined whether serum levels of these four proteins are changed in other gastroenterological cancers.

Although exact mechanisms responsible for the reduction remain to be investigated, alterations of serum levels of apolipoprotein A-IV, vitamin D binding protein, tetranectin, and retinol binding protein may have complementary role in diagnosis of pancreas cancer.

Acknowledgments

The authors would like to thank Masanori Semiya for giving them helpful discussions and suggestions. Also, they thank Fumie Iida and Manami Miura for their technical supports.

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