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. 2021 Feb;21(2):127.
doi: 10.3892/ol.2020.12388. Epub 2020 Dec 17.

Fucosylated C4b-binding protein α-chain, a novel serum biomarker that predicts lymph node metastasis in pancreatic ductal adenocarcinoma

Kazuyuki Sogawa  1 , Sakino Yamanaka  1 , Shigetsugu Takano  2 , Kosuke Sasaki  2 , Yoji Miyahara  2 , Katsunori Furukawa  2 , Tsukasa Takayashiki  2 , Satoshi Kuboki  2 , Hirotaka Takizawa  3 , Fumio Nomura  4 , Masayuki Ohtsuka  2
Affiliations

Fucosylated C4b-binding protein α-chain, a novel serum biomarker that predicts lymph node metastasis in pancreatic ductal adenocarcinoma

Kazuyuki Sogawa et al. Oncol Lett. 2021 Feb.

Abstract

C4b-binding protein α-chain (C4BPA) was previously identified as a novel serum biomarker for pancreatic ductal adenocarcinoma (PDAC). To apply this biomarker for clinical diagnosis, a lectin ELISA was established to measure serum fucosylated (Fuc)-C4BPA levels in 45 patients with PDAC, 20 patients with chronic pancreatitis (CP) and 50 healthy volunteers (HVs) in one training and three validation sets. The lecithin ELISA developed in the current study exhibited satisfactory within-run (2.6-6.7%) and between-day (1.8-3.6%) coefficient of variations. Serum Fuc-C4BPA levels in patients with PDAC (0.54±0.27 AU/ml) was significantly higher than that in HVs (0.21±0.06 AU/ml; P<0.0001) and patients with CP (0.25±0.03 AU/ml; P<0.0001). Additionally, serum Fuc-C4BPA levels in preoperative patients were significantly decreased compared with postoperative patient sera (P<0.0003). The receiver operating characteristic (ROC) curve analyses revealed that the area under the curve (AUC) of Fuc-C4BPA (0.985) was higher than that of carbohydrate antigen (CA)19-9 (0.843), carcinoembryonic antigen (0.548) and total C4BPA (0.875) (P<0.001). To analyze the clinical significance of Fuc-C4BPA, the ability of Fuc-C4BPA to predict lymph node metastasis was compared with that of CA19-9. The AUC of serum Fuc-C4BPA levels (0.703) was significantly higher than that of serum CA19-9 levels (0.500) in patients with PDAC (P<0.001). The current study established a novel lectin ELISA for measuring serum Fuc-C4BPA levels. Thus, Fuc-C4BPA has potential clinical applications owing to its high diagnostic value in PDAC.

Keywords: fucosylated C4-binding protein α-chain; lectin enzyme-linked immunosorbent assay; lymph node metastasis; pancreatic ductal adenocarcinoma; serum biomarker.

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Figures

Figure 1.
Figure 1.
Establishment of lens culinaris agglutinin-lectin ELISA for the detection of fucosylated C4BPA. (A) Western blot analysis. Immunoreactive bands were observed at the predicted molecular weight of C4BPA (67 kDa) upon incubation of recombinant human C4BPA protein and the serum sample from a patient with PDAC with anti-C4BPA polyclonal antibodies. Lane 1, internal references. The molecular weights of the upper and lower bands are 75 and 50 kDa, respectively. Lane 2, recombinant human C4BPA protein. Lane 3, serum sample from a patient with PDAC. (B) Schematic diagram of the antibody-lectin sandwich assay. Immobilized anti-C4BPA antibody captures C4BPA in the serum. Fucosylation of C4BPA is detected using a biotinylated lectin, followed by incubation with horseradish peroxidase-conjugated streptavidin. (C) Standard curves of Fuc-C4BPA following ELISA. Correlation between absorbance values and concentration of Fuc-C4BPA. Four concentrations of Fuc-C4BPA were examined using ELISA. (D) Linearity of the ELISA results, which fitted into the following linear equation: y=1.0009× (r2=0.9988; P<0.0001). C4BPA, C4b-binding protein α-chain; PDAC, pancreatic ductal adenocarcinoma; HRP, horseradish peroxidase; GlcNac, N-Acetylglucosamine; Fuc, fucosylated; LCA, lens culinaris agglutinin.
Figure 2.
Figure 2.
Serum Fuc-C4BPA levels are upregulated in patients with PDAC. (A) Comparison of serum Fuc-C4BPA levels between HVs, patients with CP and patients with PDAC using ELISA. Serum Fuc-C4BPA levels in patients with PDAC were significantly higher than those in HVs and patients with CP. (B) Comparison of serum Fuc-C4BPA levels before and after surgery in patients with PDAC. Fuc-C4BPA, fucosylated C4b-binding protein α-chain; PDAC, pancreatic ductal adenocarcinoma; HV, healthy volunteers; CP, chronic pancreatitis.
Figure 3.
Figure 3.
Serum levels of total C4BPA and Fuc-C4BPA are upregulated in patients with PDAC. (A) Comparison of total serum C4BPA levels among HVs, patients with CP and patients with PDAC using ELISA. Total serum C4BPA levels in patients with PDAC were significantly higher than those in HVs and patients with CP. HVs and patients with CP did not exhibit significant differences in total serum C4BPA levels. (B) Comparison of Fuc-C4BPA serum levels between HVs, patients with CP and patients with PDAC using ELISA. The serum Fuc-C4BPA levels in patients with PDAC were significantly higher than those in HVs and patients with CP. (C) Comparison of serum levels of total C4BPA and Fuc-C4BPA. Serum Fuc-C4BPA levels were not correlated with total serum C4BPA levels in patients with PDAC. (D) Fuc-C4BPA has higher PDAC diagnostic accuracy than total C4BPA, CA19-9 and carcinoembryonic antigen (CEA). The receiver operating characteristic curve analyses of the serum levels of Fuc-C4BPA, total C4BPA, CA19-9 and CEA between patients with PDAC, HVs and patients with CP. PDAC, pancreatic ductal adenocarcinoma; C4BPA, C4b-binding protein α-chain; Fuc, fucosylated; HVs, healthy volunteers; CP, chronic pancreatitis; CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; AUC, area under the curve.
Figure 4.
Figure 4.
Confirmation of upregulated serum Fuc-C4BPA levels in patients with PDAC in the validation set 3. (A) Comparison of serum Fuc-C4BPA levels among HVs, patients with CP and patients with PDAC using ELISA. Serum Fuc-C4BPA levels in patients with PDAC were significantly higher than those in HVs and patients with CP. (B) Fuc-C4BPA has increased PDAC diagnostic accuracy compared with CA19-9. The receiving operating characteristic curve analyses of Fuc-C4BPA and CA19-9 serum levels in patients with PDAC, HVs and patients with CP. (C) The ability of Fuc-C4BPA to identify CA19-9-negative cases among patients with PDAC. The percentage of the Fuc-C4BPA-positive cases (75%) was significantly higher than that of duke pancreatic monoclonal antigen type 2-positive cases (18.8%) among CA 19-9-negative patients with PDAC. Fuc-C4BPA, fucosylated C4b-binding protein α-chain; PDAC, pancreatic ductal adenocarcinoma; HVs, healthy volunteers; CP, chronic pancreatitis; DUPAN-2, duke pancreatic monoclonal antigen type 2; AUC, area under the curve.
Figure 5.
Figure 5.
Correlation between serum Fuc-C4BPA levels and lymph node metastasis in patients with pancreatic ductal adenocarcinoma. (A) Patients with lymph node metastasis exhibited significantly higher serum Fuc-C4BPA levels than patients without lymph node metastasis in the validation set 3. (B) The area under the curve of serum Fuc-C4BPA levels (0.703) was significantly higher than that of serum carbohydrate antigen 19-9 levels (0.500), which indicated the ability of Fc-C4BPA to predict lymph node metastasis. Fuc-C4BPA, fucosylated C4b-binding protein α-chain; pN+, patients with lymph node metastasis; PN-, patients without lymph node metastasis; LN, lymph node; AUC, area under the curve; CA19-9, carbon antigen 19-9.
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