Glycoproteomic Discovery of Serological Biomarker Candidates for HCV/HBV Infection-Associated Liver Fibrosis and Hepatocellular Carcinoma

We previously proposed a high-throughput strategy to discover serological biomarker candidates of cancer. This strategy focuses on a series of candidate glycoproteins that are specifically expressed in the original tissues (cells) of the target cancer and that carry glycan structures associated with carcinogenesis [Narimatsu, H., et al. <i>FEBS J.</i> <b>2010</b>, <i>277</i>(1), 95–105]. Here, we examined the effectiveness of our strategy in identifying biomarkers to assess progression of liver fibrosis and for the early detection of hepatocellular carcinoma (HCC). On the basis of the results of lectin array analyses in culture media of hepatoma cell lines, we captured glycopeptides carrying AAL-ligands (fucosylated glycans) or DSA-ligands (branched glycans) from digests of culture media proteins and sera from HCC patients with a background of liver cirrhosis (LC). Glycoproteins were identified by the IGOT-LC–MS method. In all, 21 candidates were selected from 744 AAL-bound glycoproteins for further verification according to (i) their abundance in serum, (ii) their specific expression in liver, and (iii) the availability of antibodies to the glycoproteins. All selected candidates showed enhancement of AAL-reactivity in sera of HCC patients compared with that of healthy volunteers (HV). These results indicate that our glycoproteomic strategy is effective for identifying multiple glyco-biomarker candidates in a high-throughput manner

Relationship between post-treatment WFA⁺-M2BP values and HCC development.

<p>The distribution of WFA⁺-M2BP values was plotted. The dashed line indicates the 2.0 COI for WFA⁺-M2BP. The 16 patients who developed HCC were stratified according to the duration from SVR to HCC development in 5-year increments. Each time point is designated by a distinct symbol as indicated. 222 patients did not develop HCC, and 209 of these 222 patients (94.1%) were in the post-treatment WFA⁺-M2BP ≤ 2.0 COI group (white circles). During the follow-up period, 5 of 18 patients (27.8%) developed HCC in the post-treatment WFA⁺-M2BP > 2.0 COI group, which was significantly higher than the rate in the post-treatment WFA⁺-M2BP ≤ 2.0 COI group (5.0%, <i>P</i> < 0.001). In the post-treatment WFA⁺-M2BP > 2.0 COI group, 4 of 5 cases developed HCC within 5 years after IFN treatment (black circles).</p

Cumulative incidence of hepatocellular carcinoma (HCC) according to post-treatment WFA⁺-M2BP values.

<p>Cumulative incidences of HCC according to post-treatment WFA⁺-M2BP values were analyzed using the Kaplan-Meier method. The black solid and dotted lines indicate the stratified post-treatment WFA⁺-M2BP values with a COI ≤ 2.0 and a COI > 2.0, respectively. The incidence rate differed significantly between the two groups (<i>P</i> < 0.0001 by the log-rank test). The numbers of patients at risk at each time point are shown below the graphs.</p

Chronological changes in the WFA⁺-M2BP and AFP values at pre-treatment, post-treatment, the time of HCC development, and the last visit of the 238 patients with sustained virological response.

<p>Dots represent the median serum WFA⁺-M2BP values at each time point, and the error bar represents the interquartile range. Diamonds represent the median serum AFP values at each time point, and the error bar represents the interquartile range. (A): Patients who developed HCC (n = 16). WFA⁺-M2BP values were decreased at post-treatment and increased at HCC development. And AFP values were decreased at post-treatment and increased at HCC development. (B): Patients who did not developed HCC (n = 222). WFA⁺-M2BP values were decreased at post-treatment and last clinical visit. And AFP values were decreased at post-treatment and last clinical visit.</p

Characteristics of Patients Enrolled in the Present Study.

<p>Data are given as the medians with ranges.</p><p>*Results are expressed as the means ± standard deviation. Unless otherwise indicated, data were collected at pre-treatment (before administration of IFN therapy; pre-Tx). Several biochemical measurements were made at both pre-Tx and post-treatment (24 weeks after completion of IFN therapy; post-Tx).</p><p>Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; γ-GTP, γ-glutamyl transpeptidase; HbA1c, glycated hemoglobin; BMI, body mass index; AFP, α-fetoprotein; HCV, hepatitis C virus; PEG-IFN, pegylated interferon; RBV, ribavirin.</p><p>Characteristics of Patients Enrolled in the Present Study.</p

Cumulative incidence of hepatocellular carcinoma (HCC) according to post-treatment WFA⁺-M2BP values, stratified by age.

<p>(A): Age ≤ 60 years (n = 165). (B): Age > 60 years (n = 73). Cumulative incidences of HCC according to post-treatment WFA⁺-M2BP values were analyzed using the Kaplan-Meier method. The black solid and dotted lines indicate the stratified post-treatment WFA⁺-M2BP values with a COI ≤ 2.0 and a COI > 2.0, respectively. The incidence rate differed significantly between the two groups (<i>P</i> < 0.0001 by the log-rank test). The numbers of patients at risk at each time point are shown below the graphs.</p

Cumulative incidence of hepatocellular carcinoma (HCC) according to post-treatment WFA⁺-M2BP values, stratified by stage of fibrosis.

<p>(A): F1/2 (n = 172). (B): F3/4 (n = 66). Cumulative incidences of HCC according to post-treatment WFA⁺-M2BP values were analyzed using the Kaplan-Meier method. The black solid and dotted lines indicate the stratified post-treatment WFA⁺-M2BP values with a COI ≤ 2.0 and a COI > 2.0, respectively. There were no significant differences in HCC incidence with F1/2 group (<i>P</i> = 0.09 by the log-rank test). On the other hand, the incidence rate differed significantly with F3/4 group (<i>P</i> < 0.01 by the log-rank test). The numbers of patients at risk at each time point are shown below the graphs.</p

Factors Associated with Hepatocellular Carcinoma.

<p>Hazard ratios for the development of hepatocellular carcinoma were calculated by Cox proportional hazards analysis.</p><p>Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; γ-GTP, γ-glutamyl transpeptidase; HbA1c, glycated hemoglobin; BMI, body mass index; AFP, α-fetoprotein; HCV, hepatitis C virus; WFA⁺-M2BP, <i>Wisteria floribunda</i> agglutinin-positive human Mac-2 binding protein.</p><p>Factors Associated with Hepatocellular Carcinoma.</p