Identification of novel serum biomarkers of hepatocellular carcinoma using glycomic analysis

Abstract

The altered N-glycosylation of glycoproteins has been suggested to play an important role in the behavior of malignant cells. Using glycomics technology, we attempted to determine the specific and detailed N-glycan profile for hepatocellular carcinoma (HCC) and investigate the prognostic capabilities. From 1999 to 2011, 369 patients underwent primary curative hepatectomy in our facility and were followed up for a median of 60.7 months. As normal controls, 26 living Japanese related liver transplantation donors were selected not infected by hepatitis B and C virus. Their mean age was 40.0 and 15 (57.7%) were male. We used a glycoblotting method to purify N-glycans from preoperative blood samples from this cohort (10 lL serum) which were then identified and quantified using mass spectrometry (MS). Correlations between the N-glycan levels and the clinicopathologic characteristics and outcomes for these patients were evaluated. Our analysis of the relative areas of all the sugar peaks identified by MS, totaling 67 N-glycans, revealed that a proportion had higher relative areas in the HCC cases compared with the normal controls. Fourteen of these molecules had an area under the curve of greater than 0.80. Analysis of the correlation between these 14 N-glycans and surgical outcomes by univariate and multivariate analysis identified G2890 (m/z value, 2890.052) as a significant recurrence factor and G3560 (m/z value, 3560.295) as a significant prognostic factor. G2890 and G3560 were found to be strongly correlated with tumor number, size, and vascular invasion. Conclusion: Quantitative glycoblotting based on whole serum N-glycan profiling is an effective approach to screening for new biomarkers. The G2890 and G3560 N-glycans determined by tumor glycomics appear to be promising biomarkers for malignant behavior in HCCs. (HEPATOLOGY 2013;57:2314-2325 H epatocellular carcinoma (HCC) is a common and fatal malignancy with a worldwide occurrence.1 Liver resection has shown the highest level of control among the local treatments for HCC and is associated with a good survival rate.2,3 However, the recurrence rates for HCC are still high even when a curative hepatectomy is performed. 4 Many factors associated with the prognosis and recurrence of HCC have now been reported. Vascular invasion of the portal vein and/or hepatic vein and tumor differentiation are important factors affecting survival and recurrence in HCC cases after a hepatectomy. 5,6 However, microvascular invasion and differentiation can only be detected by pathological examination just after a hepatectomy, and cannot be diagnosed preoperatively, and thus cannot be identified preoperatively either. Hence, the serum biomarkers alpha-fetoprotein (AFP) and protein induced by vitamin K absence-II (PIVKA-II) are used as prognostic markers 7,8 and also as surrogate markers for microvascular invasion and tumor differentiation.9,10 AFP is associated with grade differentiation, 11 whereas PIVKA-II is related to vascularAbbreviations: AFP, alpha-fetoprotein; AFP-L3, lens cul...

A comprehensive glycome profiling of Huntington's disease transgenic mice

“…In this study, we demonstrated the importance of the total glycomics of HD transgenic (R6/2) and WT mice (BCF1) using the glycoblotting method [35][36][37][38][39][40][41]47,56]. This technique enabled us to efficiently estimate the structure and quantify the total glycomes derived from the brain and serum glycoproteins and glycosphingolipids.…”

“…These include the study of conserved oligomeric Golgi (COG)-deficient patients and the increasing prevalence age and agerelated diseases identified via glycoform characterization of serum, plasma and CSF by using ESI-MS, affinity chromatography, and 2-DE followed by MS/MALDI-MS/DNA sequencer adapted-fluorophore assisted carbohydrate electrophoresis (DSA-FACE) and HILIC-HPLC [13]. Moreover, glycosylation changes in non-invasive bodily fluids, inter alia, serum, urine, plasma glycomes and on individual glycoproteins have been identified as promising biomarkers for the detection of prostate cancer [28], hepatocarcinoma cancer (HCC) and cirrhosis of the liver [35,36], pancreatic cancer [37], ulcerative colitis [38], and bladder cancer [39]. Measuring the degree of alteration of specific glycans in serum/plasma or whole blood has helped to provide a novel, noninvasive method for determining prognosis in cases of cancer and the existence of specific altered glyco-phenotypes, which might represent risk factors for the sequela of certain diseases [40][41][42][43][44].…”

“…The purpose of this enrichment process was to assess changes in the N-, O-, and GSL-glycan expression levels of PTMs in various human diseases when compared with those of normal controls. This new operational method has been applied to large scale structural and functional analyses of protein glycosylation [48][49][50][51], diseases related to glycan biomarker exploration [36][37][38][39]52], and to reveal the trends and complex patterns of glycans in diverse taxonomic groups of different bird species [53]. It seems likely that MS-based glycome analysis is one of the rapid and sensitive methods to carry out large-scale analyses for the discovery of disease-relevant biomarkers [54].…”

C1GALT1 Enhances Proliferation of Hepatocellular Carcinoma Cells via Modulating MET Glycosylation and Dimerization

“…For instance, expression of N-acetylglucosaminyltransferase-III and -V is increased in hepatocellular carcinoma (9,10). An N-glycan profiling study identified novel N-glycan structures in serum as prognostic markers of hepatocellular carcinoma (11). In addition, a-1,6-fucosyltransferase can generate fucosylated a-fetoprotein (AFP), which provided a more accurate diagnosis of hepatocellular carcinoma from chronic liver diseases (12,13).…”

Glycomic Analysis of Cancer

“…Serum glycomic studies to discover novel glycan cancer biomarkers have been highlighted (Adamczyk et al 2012 ). Numerous glycan tumor marker candidates for various cancers, including liver (Kaji et al 2013 ;Kamiyama et al 2013 ;Wu et al 2012 ;Tang et al 2010 ;Goldman et al 2009 ;Comunale et al 2009 ;Tanabe et al 2008 ;Ressom et al 2008 ), ovary (Biskup et al 2013 ;Hua et al 2013 ;Alley et al 2012 ;Abbott et al 2010 ;Leiserowitz et al 2008 ), pancreas (Li et al 2009 ;Okuyama et al 2006), breast (de Leoz et al 2011Alley et al 2010 ;Zeng et al 2010 ;Storr et al 2008 ;Abd Hamid et al 2008 ;Kyselova et al 2008 ;Kirmiz et al 2007 ), prostate , lung (Arnold et al 2011 ), colorectum (Zhao et al 2012 ), bile duct (Silsirivanit et al 2011 ), and esophagus ) cancers, have been reported. Most of these glycomic analyses have been carried out by newly developed high-throughput platform technologies, such as mass spectrometry-based methods and lectin-based methods, which have enabled the effi cient analysis of large cohorts of samples.…”