Identification of a novel serum biomarker for pancreatic cancer, C4b-binding protein α-chain (C4BPA) by quantitative proteomic analysis using tandem mass tags

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease due to the lack of specific early diagnostic markers. To improve the outcomes, proteomic approaches are being developed for the discovery of novel biomarkers of PDAC.

Methods: Using tandem mass tag labelling and LC-MS/MS, we performed comparative analyses of pre- and postoperative sera from PDAC patients to identify specific serum biomarkers for PDAC. In validation studies, we evaluated the discriminatory power of candidate proteins.

Results: Among the 302 proteins analysed, 20 were identified as potential biomarkers, with C4b-binding protein α-chain (C4BPA) and polymeric immunoglobulin receptor (PIGR) being selected for further analysis. The sera levels of C4BPA and PIGR were significantly higher in the preoperative PDAC patients than in the postoperative ones (P<0.008, P<0.036, respectively). In addition, serum C4BPA levels, but not PIGR, in patients with PDAC were significantly higher than those in healthy controls as well as in patients with pancreatitis and other malignancies including biliary tract cancers (BTC) (P<0.001). The respective area under the receiver operator characteristics (ROC) curve (AUC) was 0.860 for C4BPA, 0.846 for CA19-9 and 0.930 for the combination of C4BPA and CA19-9 in PDAC vs non-cancer individuals. The respective AUC was 0.912 for C4BPA, 0.737 for CA19-9 in Stages I and II of PDAC, 0.854 for C4BPA and 0.264 for CA19-9 in PDAC vs BTC.

Conclusions: We have demonstrated that C4BPA is a novel serum biomarker for detecting early stage PDAC, as well as for distinguishing PDAC from other gastroenterological cancers. Further analysis in large cohort studies will warrant C4BPA as a promising biomarker of PDAC in clinical use.

Figure 1

Identification of candidate proteins using TMT labelling and LC-MS/MS analyses. (A) TMT six-plex approach. A total of six serum samples (three pairs of sera from pre- and postoperative PDAC patients) were differentially labelled with TMT, pooled, and subjected to analysis using LC-MS/MS. (B) A: Pre- and postoperative sera of three PDAC patients were analysed by LC-MS/MS using an LTQ Orbitrap XL mass spectrometer. Proteomic analysis revealed 302 proteins with unique peptide sequences. B: Twenty proteins had serum levels increased by twofold in at least one preoperative patient compared with the postoperative levels. C: The serum levels of two candidate proteins, C4BPA and PIGR were elevated in all three preoperative patients by greater than twofold compared with those in postoperative patients.

Figure 2

The serum C4BPA and PIGR levels are increased in PDAC patients. (A) Comparison of serum C4BPA levels in pre- and postoperative sera of PDAC patients (P=0.008; Mann–Whitney U-test). Samples of test set are indicated as red lines. (B) Comparison of serum PIGR levels in pre- and postoperative sera of PDAC patients (P=0.036; Mann–Whitney U-test). Samples of test set are indicated as red lines. (C) Comparison of serum C4BPA levels in healthy volunteers (HVs) and patients with pancreatitis (PT) and PDAC using ELISA. The serum C4BPA levels in PDAC patients were significantly greater than in the HVs and PT patients (PDAC vs HVs: P<0.001, PDAC vs PT: P<0.001; Mann–Whitney U-test). (D) Comparison of serum PIGR levels in HVs, PT and PDAC using ELISA. The serum PIGR levels in PDAC patients were not significantly higher than in PT patients but in HVs (P<0.001; Mann–Whitney U-test).

Figure 3

C4BPA excels CA19-9 and CEA in the early detection of PDAC. (A) The ROC analyses were performed for the serum levels of C4BPA, CA19-9 and CEA between PDAC and non-cancer (HVs and PT) populations. The respective AUC was 0.860 for C4BPA, 0.846 for CA19-9, 0.765 for CEA, and 0.930 for the combination of C4BPA and CA19-9. (B) The ROC analyses were performed for the serum levels of C4BPA, CA19-9, and CEA between the early stage (stages I and II) of PDAC patients and non-cancer (HVs and PT) populations. The respective AUC was 0.912 for C4BPA, 0.737 for CA19-9, and 0.868 for CEA.

Figure 4

C4BPA is a potentially new specific serum biomarker for PDAC. (A) Serum C4BPA levels in HVs, and in patients with PT, PDAC and other gastroenterological cancers. Serum C4BPA levels in patients with PDAC were significantly higher than those in HVs and in patients with PT and other gastroenterological cancers (P<0.001; Mann–Whitney U-test). (B) The ROC analyses were performed for the serum levels of C4BPA and CA19-9 between patients with PDAC and biliary tract cancers (BTC). The respective AUC was 0.854 for C4BPA and 0.264 for CA19-9.