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Clinical Trial
. 2016 Aug 31;11(8):e0161009.
doi: 10.1371/journal.pone.0161009. eCollection 2016.

Identification of IGFBP2 and IGFBP3 As Compensatory Biomarkers for CA19-9 in Early-Stage Pancreatic Cancer Using a Combination of Antibody-Based and LC-MS/MS-Based Proteomics

Toshihiro Yoneyama  1 , Sumio Ohtsuki  2   3 , Kazufumi Honda  4   3 , Makoto Kobayashi  4 , Motoki Iwasaki  5 , Yasuo Uchida  1 , Takuji Okusaka  6 , Shoji Nakamori  7 , Masashi Shimahara  8 , Takaaki Ueno  8 , Akihiko Tsuchida  9 , Naohiro Sata  10 , Tatsuya Ioka  11 , Yohichi Yasunami  12 , Tomoo Kosuge  13 , Takashi Kaneda  14 , Takao Kato  15 , Kazuhiro Yagihara  16 , Shigeyuki Fujita  17 , Wilber Huang  18 , Tesshi Yamada  4 , Masanori Tachikawa  1 , Tetsuya Terasaki  1
Affiliations
Clinical Trial

Identification of IGFBP2 and IGFBP3 As Compensatory Biomarkers for CA19-9 in Early-Stage Pancreatic Cancer Using a Combination of Antibody-Based and LC-MS/MS-Based Proteomics

Toshihiro Yoneyama et al. PLoS One. .

Abstract

Pancreatic cancer is one of the most lethal tumors, and reliable detection of early-stage pancreatic cancer and risk diseases for pancreatic cancer is essential to improve the prognosis. As 260 genes were previously reported to be upregulated in invasive ductal adenocarcinoma of pancreas (IDACP) cells, quantification of the corresponding proteins in plasma might be useful for IDACP diagnosis. Therefore, the purpose of the present study was to identify plasma biomarkers for early detection of IDACP by using two proteomics strategies: antibody-based proteomics and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. Among the 260 genes, we focused on 130 encoded proteins with known function for which antibodies were available. Twenty-three proteins showed values of the area under the curve (AUC) of more than 0.8 in receiver operating characteristic (ROC) analysis of reverse-phase protein array (RPPA) data of IDACP patients compared with healthy controls, and these proteins were selected as biomarker candidates. We then used our high-throughput selected reaction monitoring or multiple reaction monitoring (SRM/MRM) methodology, together with an automated sample preparation system, micro LC and auto analysis system, to quantify these candidate proteins in plasma from healthy controls and IDACP patients on a large scale. The results revealed that insulin-like growth factor-binding protein (IGFBP)2 and IGFBP3 have the ability to discriminate IDACP patients at an early stage from healthy controls, and IGFBP2 appeared to be increased in risk diseases of pancreatic malignancy, such as intraductal papillary mucinous neoplasms (IPMNs). Furthermore, diagnosis of IDACP using the combination of carbohydrate antigen 19-9 (CA19-9), IGFBP2 and IGFBP3 is significantly more effective than CA19-9 alone. This suggests that IGFBP2 and IGFBP3 may serve as compensatory biomarkers for CA19-9. Early diagnosis with this marker combination may improve the prognosis of IDACP patients.

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Conflict of interest statement

The authors declare no competing financial interest. One of our authors, W.H., is a CEO of Abnova and receives salary from Abnova. He also owns stock in Abnova. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Comparison of quantitative values obtained by LC-MS/MS and antibody-based absolute quantification (immunoturbidimetry and ELISA).
(A) CRP was measured by LC-MS/MS and immunoturbidimetry (n = 125). (B and C) IGFBP3 and adiponectin were measured by LC-MS/MS and ELISA (n = 125). The units of the quantitative values determined by immunoturbidimetry and ELISA were converted from gram to mol using the appropriate molecular weight based on the amino acid sequences described in uniprot/swiss-prot; CRP, 24.5 kDa; IGFBP3, 28.8 kDa; adiponectin, 24.5 kDa.
Fig 2
Fig 2. Comparison of the quantitative values obtained by LC-MS/MS and RPPAs.
C2 and IGFBP2 were measured by both LC-MS/MS and RPPAs (n = 42). U.L.D., under the limit of detection.
Fig 3
Fig 3. Dot plot showing the differences of IDACP marker candidate between healthy controls (n = 65) and early-stage IDACP (n = 38) in the early-stage set.
Each dot represents the protein level in an individual sample, and lines represent median and quartiles. N.S., no significant difference. Cont, healthy controls.
Fig 4
Fig 4. ROC curves of multivariate logistic regression.
(A) Multivariate logistic regression formulae of CA19-9+IGFBP2, 3 (AUC, 900; 95% CI, 0.837–0.962) and CA19-9 only (AUC, 836; 95% CI, 0.746–0.926) were produced from the quantitative values of the early-stage set, and ROC analysis was performed. (B) The same formulae of CA19-9+IGFBP2, 3 (AUC, 940; 95% CI, 0.903–0.976) and CA19-9 only (AUC, 0.894; 95% CI, 0.842–0.946) were applied to the quantitative values of the all-stage set, and ROC analysis was performed.
Fig 5
Fig 5. Dot plot showing the plasma levels of each IDACP biomarker candidate in controls (n = 38) and patients with stage I (n = 4), II (n = 19), III (n = 26) and IV (n = 51) in the all-stage set.
Each dot represents the protein level of an individual sample, and lines represent median and quartiles. *, p<0.05; **, p<0.01; ***, p<0.001. Healthy controls, cont.
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This study was performed as a research program in the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct, 14089014), Ministry of Education, Culture, Sports, Science and Technology of Japan (http://www.mext.go.jp). This study was also supported in part by JSPS KAKENHI Grant Numbers 14J05219, 15K15751, 23390469, and by the Industrial Technology Research Grant Program of the New Energy and the Industrial Technology Development Organization of Japan (P00041), and the Funding Program for Next Generation World-Leading Researchers (LS005) by the Cabinet Office, Government of Japan (http://www.cao.go.jp/index-e.html), and the Third-Term Comprehensive Control Research for Cancer, Research on Biological Markers for New Drug Development and Health Labour Sciences Research, and Applied Research for Innovative Treatment of Cancer grant by the Ministry of Health, Labour and Welfare of Japan, and Practical Research for Innovation Cancer Control (15ck0106101h0002), and AMED-CREST (10801082) by Japan Agency for Medical Research and Development (http://www.amed.go.jp/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. One of our authors, W.H., is a CEO of a commercial company: Abnova. The funder provided support in the form of salaries for W.H. and research materials (antibodies for RPPA), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.