Changes in the Glycosylation of Kininogen and the Development of a Kininogen-Based Algorithm for the Early Detection of HCC

Abstract

Background: Hepatocellular carcinoma (HCC) has the greatest increase in mortality among all solids tumors in the United States related to low rates of early tumor detection. Development of noninvasive biomarkers for the early detection of HCC may reduce HCC-related mortality.Methods: We have developed an algorithm that combines routinely observed clinical values into a single equation that in a study of >3,000 patients from 5 independent sites improved detection of HCC as compared with the currently used biomarker, alpha-fetoprotein (AFP), by 4% to 20%. However, this algorithm had limited benefit in those with AFP <20 ng/mL. To that end, we have developed a secondary algorithm that incorporates a marker, fucosylated kininogen, to improve the detection of HCC, especially in those with AFP <20 ng/mL and early-stage disease.Results: The ability to detect early-stage AFP-negative (AFP <20 ng/mL) HCC increased from 0% (AFP alone) to 89% (for the new algorithm). Glycan analysis revealed that kininogen has several glycan modifications that have been associated with HCC, but often not with specific proteins, including increased levels of core and outer-arm fucosylation and increased branching.Conclusions: An algorithm combining fucosylated kininogen, AFP, and clinical characteristics is highly accurate for early HCC detection.Impact: Our biomarker algorithm could significantly improve early HCC detection and curative treatment eligibility in patients with cirrhosis. Cancer Epidemiol Biomarkers Prev; 26(5); 795-803. ©2017 AACR.

Conflict of interest statement

Conflict of Interest: M. Wang, MA. Comunale, T.M. Block and A. Mehta have filed a patent regarding the markers in this manuscript (through Drexel University). They have been licensed to Glycotest, Inc. A. Mehta, T.M. Block and C. Swindell have an ownership stake in Glycotest, Inc. T.M Block receives a sponsored research grant from Arbutus bioPharma and is a member of the Advisory board for ContraVir Pharmaceuticals Inc. A. Singal acts as consultant for Instadiagnostics, Bayer, Wako Diagnostics, Eisai Co., Ltd., EMD Serano and Abbott. A. Singal has also received an Honoraria from Bayer.

©2017 American Association for Cancer Research.

Figures

Figure 1

AUROC for AFP, the DA or the DA Plus (DA+) algorithm in A) All patients; B) Patients with early stage HCC; C) patients with AFP

Figure 2. Characterization of low molecular weight kininogen glycosylation

A) Normal phase UPLC analysis of low molecular weight kininogen following sequential exoglycosidase digestion. B) Lectin blotting of low molecular weight kininogen. The left panel is stained with colloidal Coomassie brilliant blue (red channel) and the right panel (green channel) is a lectin blot with the AAL lectin. Proteins are Bovine serum albumin (BSA, as negative control, human IgG as a positive control) and low molecular weight kininogen (LMWK). C) Lectin-FLISA of captured kininogen. Supernatant from Hep G2 cells served as the positive control.

Figure 3. Glycopeptide analysis of tryptic glycopeptide 290–300 highlighting the presence of branched and fucosylated glycan

For glycan structures, blue squares represent N-acetylglucosamine monosaccharides (GlcNAc); green circles represent mannose residues; yellow circles represent galactose residues and red triangles represent fucose residues.

Figure 4. Glycopeptide analysis of kininogen tryptic glycopeptide 290–300 containing site N294

The median, 25%, 75% and standard deviation are shown for each fucosylated glycan observed. Results are from an analysis of 10 individual cirrhotic and 20 individual HCC patients. For structures, A2G2F1, biantennary N-glycan glycan with a single fucose residue; A3G3F1, tri-antennary N-glycan glycan with a single fucose residue; A3G3F2, tri-antennary N-glycan glycan with two fucose residues; A4G4F1, tetra-antennary N-glycan glycan with a single fucose residue; A4G4F2, tetra-antennary N-glycan glycan with two fucose residues.