A Glycoproteomic Approach - American Chemical Society

Study of Serum Haptoglobin and Its Glycoforms in the Diagnosis of Hepatocellular Carcinoma: A Glycoproteomic Approach Irene L. Ang,† Terence C. W. Poon,*,† Paul B. S. Lai,‡ Anthony T. C. Chan,§ Sai-Ming Ngai,| Alex Y. Hui,† Philip J. Johnson,⊥ and Joseph J. Y. Sung† Department of Medicine and Therapeutics, Department of Clinical Oncology, Department of Surgery, and Department of Biology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong SAR, Cancer Research UK Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, England Received March 22, 2006

Increased serum haptoglobin concentration and changes in its glycosylation have been reported in certain cancer types. Information for hepatocellular carcinoma (HCC) has not yet been available. In this study, we aimed to carry out a systematic analysis of serum concentrations of haptoglobin (Hp) and its glycoforms in the patients with HCC and noncancer patients only with chronic liver diseases (CLD) and to examine their clinical values. This study was divided into two major parts, (1) measurement of serum Hp concentration, and investigation of its value in the diagnosis of HCC, and (2) quantitative analysis of Hp glycoforms with alpha-2,6-sialylation and/or alpha-1,6-fucosylation by using lectin affinity purification and 2D gel electrophoresis and investigation of their relationships with tumor stage. The concentrations of serum Hp in HCC patients were significantly higher than those in noncancer patients with CLD. With the use of serum concentrations of Hp and alpha-fetoprotein, a logistic regression (LR) model was developed from the training data set and used to classify the validation cases. At a specificity of 95%, the sensitivity for HCC detection was 79%. Comparing serum concentrations of alpha-2,6sialylated Hp (S-Hp) and alpha-1,6-fucosylated Hp (F-Hp) between HCC and CLD patients suggests that purification of S-Hp and F-Hp could enrich the glycosylation variants associated with HCC. 2D gel analysis of S-Hp and F-Hp identified a total of 18 glycoforms. A unique pattern of Hp glycoforms comprising both hypersialylated fucosylated and hyposialylated fucosylated species was found in the HCC patients. Serum concentrations of these glycoproteins were significantly higher in the patients with advanced tumors, suggesting their tumor-specific nature. We have shown that serum Hp is a potential biomarker in the diagnosis of HCC. The combined use of Hp and AFP could greatly improve the diagnostic accuracy. A unique pattern of Hp glycoforms with altered sialylation and fucosylation is specific to HCC and associated tumor progression. Keywords: haptoglobin • glycoform • sialylation • fucosylation • glycosylation • hepatocellular carcinoma • diagnosis

Introduction Glycosylation is one of the major post-translational modifications of proteins. The glycosylation on individual molecules of a glycoprotein are usually similar but with some degrees of variation. The majority of serum/plasma proteins are glycosylated. Recently, studies have shown that the glycosylation machinery of cancer cells is usually altered. Such alternation leads to the production and secretion of glycoproteins with * To whom corresondence should be addressed. Dr. Terence C. W. Poon, Ph.D. Department of Medicine and Therapeutics, Prince of Wales Hospital, 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong, China; Fax: +852 26488842; Email: [email protected]. † Department of Medicine and Therapeutics. ‡ Department of Surgery. § Department of Clinical Oncology. | Department of Biology. ⊥ Cancer Research UK Institute for Cancer Studies. 10.1021/pr060109r CCC: $33.50

 2006 American Chemical Society

aberrant glycosylation, which could be identified in blood circulation and used as a biomarker in cancer diagnosis, such as glycoforms of alpha-fetoprotein.1 In the past, study of glycosylation patterns of a serum protein was mainly achieved by lectin affinity electrophoresis and immunoblotting.2 The recent advancement in proteomic technology allows for an alternative approach for quantitative analysis of glycosylation variants of a serum glycoprotein. Haptoglobin (Hp) is a tetramer composed of two alphasubunits of 9.1 kDa and two beta-subunits of 40 kDa. The carbohydrate content of Hp is found exclusively as “complex” N-linked glycans on the beta-subunit only.3 The glycans are either biantennary or triantennary, both terminating with sialic acid residue(s). Fucose is linked to the core N-acetylglucosamine residue (GlcNAc) at either the alpha-1,6 position or alpha-1,3 position.4 It is well-known that Hp is an acute phase protein. It is mainly produced by the liver and secreted into Journal of Proteome Research 2006, 5, 2691-2700

2691

Published on Web 08/23/2006

research articles the blood stream. Despite years of investigations, the physiological functions of Hp have remained enigmatic. It has been generally accepted that Hp plays an important role in hemoglobin metabolism.5 Other studies have suggested that Hp may be involved in inflammation and host defense responses to infection.6 Arterial expression of Hp has been recently discovered, suggesting its role in angiogenesis.7-10 There has been increasing evidence indicating that serum Hp may be a biomarker for cancer diagnosis, regardless of cancer type. Hp was found to be expressed in malignant ovarian epithelium,11 kidney tumor cells,7 and hepatocellular carcinoma (HCC) tissue.12 Elevation of serum concentrations of Hp and its alpha subunit has been observed in ovarian cancer and small-cell lung cancer,11,13 respectively. Changes in glycosylation of serum Hp have been observed in cancer patients. Serum concentration of fucosylated Hp has been shown to be increased in breast cancer and ovarian cancer over the past 10 years.14 Recently, a similar finding has been observed in pancreatic cancer.15 Unfortunately, information about the serum concentrations of Hp and its glycosylation variants in patients with HCC has not been available. HCC is the major primary liver cancer that carries a high mortality rate and an overall dismal prognosis. Development of HCC is strongly associated with chronic liver disease (CLD), such as chronic hepatitis B infection and alcoholic cirrhosis. Alpha-fetoprotein (AFP) is currently the best available serum marker in the diagnosis of HCC. However, AFP is also increased in some noncancer patients with CLD, leading to low diagnostic accuracy. In this study, we hypothesize that serum Hp is increased in HCC patients and can help in the diagnosis of HCC, as in the case of ovarian cancer. By undertaking a glycoproteomic approach, we aimed to measure the concentrations of Hp and its glycosylation variants in the sera from patients with HCC and noncancer patients with CLD and to examine their clinical values. As alternations in alpha-2,6sialylation and alpha-1,6-fucosylation of serum alpha-fetoprotein in HCC patients were previously reported,16,17 in this study we focused on Hp glycoforms with alpha-2,6-sialylation and/ or alpha-1,6-fucosylation.

Materials and Methods Patient Materials. With patients’ consent, pretreatment clotted blood samples were collected from patients with HCC and noncancer patients with CLD at presentation at the Joint Hepatoma Clinic at the Prince of Wales Hospital between October 1997 and December 1998. All blood samples were immediately processed, and the sera were stored at -80 °C before assay. Patients with HCC were diagnosed according to standard clinical setting as reported previously.18 All HCC cases were confirmed histologically. The cancer group consisted of 56 serum samples from patients with primary HCC at different clinical stages (TNM classification of the American Joint Committee on Cancer): stage I (n ) 12), stage II (n ) 11), stage III (n ) 27), and stage IV (n ) 6). Specimens were obtained before treatment. The median age of the HCC patients was 58 years (range, 37-82 years). All the patients were measured for serum alpha-fetoprotein (AFP) concentration with a commercial immunoassay (Dako, Glostrup, Denmark). The control group consisted of 40 serum samples from patients with CLD only. All CLD patients were followed for at least 18 months, during which the serum AFP concentration and imaging studies were assessed every 2-3 months, to exclude individuals with asymptomatic HCC. 2692

Journal of Proteome Research • Vol. 5, No. 10, 2006

Ang et al.

Measurement of Serum Hp Concentration. Concentration of serum Hp was measured by an in-house enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well microplates were coated with 100 µL of 2 µg/mL rabbit polyclonal anti-human Hp antibodies (Dako) at 4 °C for 16 h. After blocking with 0.2% BSA solution for 1 h at room temperature (RT), 1:300 000 diluted patient sera were added to the wells and incubated at 37 °C for 1 h. After washing with PBS containing 0.5% Tween20 (v/v) twice, 100 µL of 1 µg/mL digoxigenin (DIG) labeled rabbit polyclonal anti-human Hp in PBS containing 0.2% BSA (v/v) and 0.05% Tween-20 (v/v) were added to the plate and incubated at 37 °C for 1 h. After washing, 100 µL of 1 µg/mL anti-DIG peroxidase conjugate were added to the plate and incubated at 37 °C for another 1 h. After washing, 100 µL ABTS substrate solution (CalBiochem, Darmstadt, Germany) were added to the wells and incubated at RT with shaking for 20 min. The reaction was stopped with 120 µL 0.5 M H2SO4 and the optical density was read at 405 nm. All the patient samples were measured in triplicate. Samples were measured for 8 times in triplicate in a single assay for determination of the intraassay coefficient of variation (CV) and measured in triplicate in 11 separate assays for determination of the interassay CV. The intra-assay CV was 3.4%. The interassay CV was 5.3%. For the details of the standard curve and evaluation data of the in-house ELISA, please refer to the Supporting Information. Measurement of Serum Alpha-2,6-sialylated Hp (S-Hp) and Alpha-1,6-fucosylated Hp (F-Hp) Concentrations by Subtraction Assay. Serum alpha-2,6-sialylated Hp concentration was measured by combined use of a 96-well microplate coated with Sambucus Nigra (SNA) lectin (CalBiochem), which is specific to alpha-2,6-sialylated glycoproteins, and a standard ELISA for Hp. The basic principle is that the SNA lectin microplate is used to remove all the serum glycoproteins with alpha-2,6-sialylation, to allow the measurement of the serum concentration of Hp without alpha-2,6-sialylation by ELISA. Subtracting the serum concentration of Hp by the serum concentration of the Hp without alpha-2,6-sialyation will give us the serum concentration of alpha-2,6-sialylated Hp. Briefly, 100 µL of 2 µg/mL SNA lectin (CalBiochem) were coated onto a 96-well microplate in 0.2 M carbonate buffer, pH 9.6 for 16 h at 4 °C. The wells were blocked with lectin binding buffer (50 mM Tris, 150 mM NaCl, 2 mM CaCl2, 0.5% Tween-20, pH 7.2) at RT for 1 h. Patient sera diluted in lectin binding buffer were added to the lectin microplate and incubated at 4 °C for 16 h to remove the alpha-2,6-sialylated glycoproteins, including alpha-2,6-sialylated Hp. Commercial Hp calibrator (Dako) and patient sera were also diluted with lectin binding buffer containing 500 mM lactose, added to the lectin microplate, and incubated at 4 °C for 16 h for measuring total Hp concentration. After the incubation, the unbound fraction was recovered and subjected to Hp concentration measurement by the in-house ELISA, as described above. Serum alpha-2,6-sialylated Hp concentration was obtained by subtracting the Hp values in the presence of lactose by the corresponding values in the absence of lactose. Measurement of serum alpha-1,6-fucosylated Hp concentration was similar to that of serum alpha-2,6-sialylated Hp, except that the SNA lectin was replaced with lectin Lens Culinaris Agglutinin (LCA) immobilized on agarose bead (Amersham Biosciences, Uppsala, Sweden), and lactose was replaced with 200 mM methyl-D-mannopyranoside. All the patient samples were measured in triplicate. Samples were measured for 12 times in triplicate in a single assay for determination of the

Glycoproteomic Analysis of Serum Haptoglobin in HCC

intra-assay CV and measured in triplicate in 11 separate assays for determination of the interassay CV. Purification of Alpha-2,6-sialylated and Alpha-1,6-fucosylated Glycoproteins from Patient Sera by Lectin Affinity Chromatography. Patient sera were diluted in lectin binding buffer (50 mM Tris, 150 mM NaCl, 2 mM CaCl2, 0.05% Tween 20 (v/v), pH 7.2) and incubated with lectin immobilized on chromatographic beads with rotation at 4 °C for 16 h. SNA lectin was used to purify alpha-2,6-sialylated glycoproteins, whereas LCA lectin was used to purify alpha-1,6-fucosylated glycoproteins. After washing thoroughly with the lectin binding buffer, the captured glycoproteins were eluted with elution solution (500 mM lactose for SNA beads and 200 mM methylD-mannopyranoside for LCA beads). RC DC Protein Assay (BioRad, Hercules, CA) was used to monitor the protein concentrations throughout all purification steps. Quantitation of Alpha-2,6-sialylated and Alpha-1,6-fucosylated Hp Glycoforms by Combined Use of Two-Dimensional Gel Electrophoresis and Enzyme-Linked Lectin Assay. Nonlinear pH 3-10, 17 cm long IPG ReadyStrip (BioRad) was rehydrated overnight with 12.5 µg purified glycoprotein in rehydration buffer containing 8 M urea, 2% CHAPS, 50 mM dithiothreitol (DTT), 0.2% Bio-Lyte 3/10 ampholyte and 0.001% Bromophenol Blue. The first dimensional separation (IEF) was performed in PROTEAN IEF Cell (BioRad) at 20 °C, using stepwise mode to reach 8000 V and ran at 20 000 V for 1 hour. After the first dimensional separation, proteins on the strip were equilibrated with 5 mL equilibration buffer (6 M urea, 0.51 mM EDTA, 20% glycerol, 141 mM Tris-base) containing 2% SDS (w/ v) and 1% DTT (w/v) for 20 min, and then with another 5 mL equlilbration buffer containing 4% iodoacetamine (v/v) for 20 min. The strip was then transferred onto 10% Bis-Tris XT Criterion gel (BioRad) and subjected to second dimensional separation in Criterion Dodeca Cell (BioRad) at a constant voltage of 135 V for 135 min. Separated protein spots were visualized using PlusOne Silver Staining Kit (Amersham Biosciences). All the patient samples were run in duplicate. Stained gels were then digitally imaged with a densitometer GS-800 (BioRad) and analyzed using PDQuest version 7.3. The pI values of each gel image were calibrated with the pI values of known protein spots obtained from the Swiss 2D-PAGE database. The values of molecular weight (MW) of the gel image were calibrated with a commercial protein molecular weight marker (BioRad). All the protein spot intensities were normalized using sum of the second and third quartile of the spot intensities. After normalizing, total sum of intensities of all gel spots of individual gels should be equal. The concentrations of individual glycoproteins in the purified preparation were no longer reflecting the serum concentrations. Correction of the concentrations of these glycoproteins to serum concentrations was important before further analysis. Because a fixed amount of purified glycoproteins from each patient was subjected to 2D gel analysis, the normalized intensity of a spot was directly proportional to the percentage of the corresponding glycoprotein variant in the purified glycoprotein preparation. If we knew the serum concentration of the total sum of the purified glycoproteins, we could calculate back the serum concentration of individual glycoprotein variants from values of the corresponding normalized spot intensities. When two pieces of information, (1) the normalized intensity of an alpha-2,6-sialylated glycoprotein spot and (2) the serum concentration of total alpha-2,6-sialylated glycoproteins, are

research articles available, one could calculate the serum concentration of a glycoform corresponding to that glycoprotein spot by using the following equation:

After the normalization, total sum of intensities of all gel spots of each gel is a constant. Therefore, if only arbitrary unit is needed for the serum concentration of an alpha-2,6-sialylated glycoform of a glycoprotein, the equation could be simplified as following:

For calculating the serum concentration of an alpha-1,6fucosylated glycoform of a glycoprotein, the equation was as following:

Serum concentrations of all alpha-2,6-sialylated glycoproteins and of all alpha-1,6-fucosylated glycoproteins in individual patients were measured by direct enzyme-linked lectin assay (ELLA), as described previously.19 Briefly, 100 µL of 1:500 000 diluted patient sera were applied in duplicate to a 96-well microplate for direct coating at 4 °C for 16 h. Our pilot data showed that 500 000-fold dilution was needed to prevent the saturation of a microplate well. The plate was then washed and blocked with binding buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl2, 0.5% (v/v) Tween 20, pH7.2) at RT for 1 h, followed by incubation with 1 µg/mL DIG-labeled SNA or LCA lectin at 37 °C for 1 h. Unbound lectins were removed by washing with binding buffer twice before incubation with 1 µg/mL anti-DIG peroxidase conjugate at 37 °C for 1 h. Finally, 100 µL of ABTS substrate solution (CalBiochem) for SNA assay and 100 µL of BM Blue substrate solution (Roche Diagnostics, Basel, Switzerland) for LCA assay were added to the wells and incubated at RT with shaking for 20 min. The SNA assay was stopped by 0.5 M H2SO4 and the LCA assay was stopped by 1 M H2SO4. The optical density was read at 405 nm for SNA assay and read at 450 nm with reference at 690 nm for LCA assay. All the patient samples were measured in duplicate. In this assay, only arbitrary unit, but not absolute amount of all serum glycorproteins was measured. Serum concentrations of all alpha-2,6sialylated glycoproteins and of all alpha-1,6-fucosylated glycoproteins in individual patients were measured against those Journal of Proteome Research • Vol. 5, No. 10, 2006 2693

research articles of pool normal serum, undiluted preparation of which was assumed as 1 unit. The optical density was plotted against the quantity of the pool normal serum to generate a calibration curve. Protein Identification by Mass Spectrometric Analysis. Silver-stained proteins were excised from the gels and then digested with trypsin (Promega Corporation, Madison, Wisconsin, USA), as previously described.20 Mass spectrometry analyses were performed on a 4700 MALDI TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA). MALDITOF MS data of the tryptic peptide digest were searched via the ProFound search engine to obtain the protein identity by undertaking the peptide mass fingerprinting approach. Tandem MS data were subjected to MS/MS ion search via the Mascot search engine to obtain the protein sequence of a particular peptide. Construction of a Diagnostic Model. The HCC and CLD cases were randomly divided into two groups: the training group (28 HCC cases; 20 CLD cases) and independent validation group (28 HCC cases; 20 CLD cases). In the training set data, a forward stepwise method was utilized as an exploratory purpose to determine which markers were to be added or dropped from the logistic regression model. The log10 values of the serum concentrations of Hp and AFP were subjected to logistic regression to generate a model for detection of HCC. The output was the diagnostic score in the range of 0-1. During model training, the diagnostic score of a CLD patient was defined as “0”, whereas that of a HCC patient was defined as “1”. The constructed model was then evaluated on the independent validation sample set. The diagnostic score, which was computed from the model using the serum concentrations of Hp and AFP of individual cases, was used as index for classifying the HCC and CLD cases in the independent validation set. Statistical Analyses. Mann Whitney U test was performed to compare the serum concentrations of various biomarkers between the study groups. (SPSS; SPSS In., Illionis). Receiver operating characteristics (ROC) curves were constructed by calculating the sensitivities and specificities of a biomarker or the diagnostic score of a logistic regression model at different cutoff points for differentiating HCC cases from CLD cases. The area under the ROC curves (AUC) can be statistically interpreted as the probability of the test to correctly distinguish the patients with HCC from CLD. An area of “1.0” represents a perfect test; an area of “0.5” represents a worthless test.

Result Serum Total Haptoglobin, a Potential Diagnostic Marker for HCC. In the 56 patients with HCC and 40 patients with CLD, the mean serum concentration (SD) of AFP in the cancer group was 50 775 (130 417) ng/mL. As expected, it was also significantly higher than that of the control group (88 (176) ng/ mL, P < 0.001, Mann Whitney test). The mean serum concentration (SD) of total Hp in the cancer group was 902 (816) µg/ mL, which was also significantly higher than the control group (351 (348) µg/mL, P < 0.001, Mann Whitney test, Figure 1). The ratio of the mean values of HCC to CLD was 2.57. This supports our hypothesis that serum Hp concentration is increased in HCC patients. To evaluate the diagnostic value of serum Hp, ROC curve analysis was performed. The AUC of the ROC curve was 0.733 (95% CI: 0.634-0.832, P < 0.0005, Figure 2A), which was comparable to that of serum AFP (AUC ) 0.758, 95% CI: 0.662-0.853, P < 0.0005, Figure 2B). This indicated that serum 2694


Ang et al.

Figure 1. Scatter plot and box plot of serum Hp concentration measured by the in-house ELISA assay. The serum Hp concentrations of 56 patients with HCC and 40 noncancer patients with CLD were shown. The box represents the interquartile range whereas the line across the box indicates the median value. The whiskers represent the 10th and the 90th percentiles. The median value of serum Hp was higher in the patients with HCC than the patients with CLD.

Figure 2. Receiver operation characteristics (ROC) curves of the serum Hp (A), serum AFP (B) and the diagnostic model (C) in differentiating HCC cases from CLD cases. The clinical values of serum Hp and serum AFP were assessed by differentiating 56 HCC cases from 40 CLD cases. ROC curves of the serum Hp (A) and the serum AFP (B) were plotted. A trained diagnostic model comprising log10 serum Hp and log10 serum AFP was used to calculate the diagnostic score for differentiating 28 HCC cases from 20 CLD cases in the independent validation set (C).

Hp could be useful in the diagnosis of HCC. The diagnostic values were 63% sensitivity at 75% specificity. The overall accuracy was 68%. The positive and negative likelihood ratios of serum Hp were 2.5 and 0.37, respectively. The diagnostic sensitivity of serum AFP was 66% at 75% specificity. The overall accuracy was 70%. The positive and negative likelihood ratios of serum AFP were 2.6 and 0.45, respectively. Diagnostic Value of Serum Hp in Combination of AFP. To evaluate the diagnostic value of Hp in combination of the conventional marker AFP, the HCC and CLD cases were randomly divided into two groups, the training group (28 HCC cases; 20 CLD cases) and validation group (28 HCC cases; 20


research articles

Figure 3. Scatter plots and box plots of serum SNA-reactive Hp concentration (A) and serum LCA-reactive Hp concentration (B) measured by subtraction assays. The serum SNA-reactive Hp concentration and the serum LCA-reactive Hp concentration of 56 patients with HCC and 40 patients with CLD were shown. The box represents the interquartile range whereas the line across the box indicates the median value. The whiskers represent the 10th and the 90th percentiles. The median values of serum SNA-reactive Hp and serum LCA-reactive Hp were higher in the patients with HCC than the patients with CLD.

CLD cases). A multivariate logistic regression analysis was performed using the training group, indicating that Hp (P ) 0.044) and AFP (P ) 0.003) were two independent biomarkers. Both were included in the resulted diagnostic model. The odd ratios for log10 Hp and log10 AFP were 3.16 and 2.12, respectively. The logistic regression model was used to differentiate the HCC cases from the CLD cases in the independent validation set by calculating their diagnostic scores from log10 Hp and log10 AFP values. The AUC of the ROC curve of the diagnostic score was 0.898 (95% CI, 0.810-0.986, P < 0.0005, Figure 2C). At 95% specificity, the sensitivity of this diagnostic model was 79%, which was much higher than the sensitivity obtained by using conventional marker AFP (55% at 95% specificity). The overall accuracy of the diagnostic was 85%. The positive and negative likelihood ratios of the diagnostic model were 15.7 and 0.23, respectively. Increased Serum Concentrations of Alpha-2,6-sialylated Hp and Alpha-1,6-fucosylated Hp in HCC Patients. The intraassay CV and inter-assay CV were 3.2 and 4.0% for the S-Hp assay, and 14.2 and 25.1% for the F-Hp assay, respectively. For detailed data for the development of the S-Hp and F-Hp assays, please refer to the Supporting Information. The mean serum concentration (SD) of alpha-2,6-sialylated Hp (S-Hp) in the HCC group was 850 (821) µg/mL, which was significantly higher than the CLD group (290 (360) µg/mL, P < 0.001, Mann Whitney test) (Figure 3A). The HCC-CLD ratio of the mean values was 2.93. The mean serum concentration of alpha-1,6fucosylated Hp (F-Hp) in the HCC group was 155 (334) µg/ mL, which was also significantly higher than the CLD group (37 (40) µg/mL, P < 0.001, Mann Whitney test) (Figure 3B). The HCC-CLD ratio was 4.22. Therefore, HCC-CLD ratios of the serum concentrations of S-Hp and F-Hp were higher than that of serum Hp (2.57). This suggests that purification of S-Hp and F-Hp could enrich the glycosylation variants associated with HCC. Lectin Affinity Purification and 2D Gel Separation of Serum S-Hp and F-Hp Glycoforms. To analyze individual S-Hp and F-Hp glycoforms in the patients with HCC and CLD, alpha2,6-sialylated glycoproteins and alpha-1,6-fucosylated glycoproteins in individual serum samples from 20 HCC patients and 10 CLD patients were purified by lectin affinity chromatography with the use of immobilized SNA and LCA, respectively. The SNA-reactive and LCA-reactive glycoproteins were then separated and quantified by undertaking a 2D gel electrophoresis approach. Under reduced condition, each Hp

molecule was separated into two basic components, alphasubunit and beta-subunit. As glycosylation is only present on the beta-subunit, we only focused on the protein spots corresponding to Hp beta-subunit for evaluation of the distribution pattern of Hp glycoforms. Similar to the information in the Swiss-2DPAGE database,21 in the 2D gels, Hp beta-subunit molecules from both SNA- and LCA-reactive glycoprotein preparations were separated into two chains of protein spots. A total of 18 glycoforms with slight differences in molecular weight (ranging from 35 to 44kDa) and pI value (ranging from 4.6 to 5.8) were observed in the present study. The glycoforms in the upper chain were labeled from A1 to K1, whereas those in the lower chain were labeled from E2 to K2 (Figure 4). A1 is the most acidic glycoform while K2 is the most basic glycoform. All the 18 spots were excised from the gel, subjected to trypsin in-gel digestion and then analyzed by mass spectrometry. The protein identity was confirmed as beta subunit of Hp by peptide mass fingerprinting and by tandem MS ion search (Figure 5). Glycoform A1 and K1 of S-Hp were only present in 2 HCC cases and were therefore excluded from statistical analysis. Glycoform E2 and F2 of F-Hp were also excluded from analysis as their protein spots were overlapped with the spots of other proteins. Serum Profiles of S-Hp and F-Hp Glycoforms in Patients with HCC and CLD. After measuring the serum concentrations of total sialylated glycoproteins and total fucosylated glycoproteins by ELLA, serum concentrations of individual S-Hp and F-Hp glycoform were calculated from their normalized spot intensities. About the data for the development of the ELLA assays, please refer to the Supporting Information. Serum concentrations of individual S-Hp and F-Hp glycoforms were compared between the 20 HCC patients and 10 CLD patients. Figure 6A showed the distribution of the serum concentrations of individual Hp glycoforms and the HCC-CLD ratios of their mean values. Serum concentrations of most of the S-Hp and F-Hp glycoforms were significantly higher in the HCC patients (all P values < 0.05, Mann Whitney test, Figure 6A). In the case of S-Hp glycoforms, the HCC-CLD ratios showed an increasing trend toward the acidic glycoforms of the upper chain. It is worth noting that the ratios of majority of the S-Hp glycoforms are similar to the ratio of serum Hp (about 2.5), except the ratio (9.7) of the most acidic glycoform B1 (Figure 6A). As the HCC-CLD ratio of S-Hp was 2.93, our results suggest that compared to Hp, the higher ratio in the case of Journal of Proteome Research • Vol. 5, No. 10, 2006 2695

research articles

Ang et al.

Figure 4. Representative patterns of glycoforms of serum SNA-reactive Hp (A) and LCA-reactive Hp (B) separated by 2D gel electrophoresis. A total of 18 glycoforms labeled as A1 to K1 (upper chain) and E2 to K2 (lower chain) were detected in SNA-reactive Hp and LCA-reactive Hp. The calculated pI and the molecular weight (MW) of each glycoform, were labeled in the bracket (pI, MW). Both (A) and (B) are representative images derived from the HCC samples.

S-Hp might be mainly contributed by increased serum concentration of S-Hp glycoform B1 in HCC patients. In the case of F-Hp glycoforms, the HCC-CLD ratios of some glycoforms (G1, H1, I1, K1, G2, and K2) were also about 2.5. Highest ratios were also observed for F-Hp glycoforms B1, C1, J1, and I2 (Figure 6A). The mean serum concentrations of F-Hp glycoforms B1 and C1 in HCC group were as high as 14 times of those in CLD group. Compared to Hp, the higher HCC-CLD ratio of F-Hp (4.22) might be mainly caused by increased serum concentrations of F-Hp glycoforms B1, C1, J1 and I2 in HCC patients. Associations between Specific Serum Hp Glycoforms and Tumor Progression. We separated the 20 HCC patients into two subgroups with early HCC (AJCC stage I or II) and advanced HCC (AJCC stage III or IV), and compared the serum concentrations of individual Hp glycoforms. The distribution pattern of individual Hp glycoforms from patients with early HCC and advanced HCC were shown in Figure 6B. Serum concentrations of majority of the glycoforms appeared to be higher in the patients with advanced HCC. Among the S-Hp glycoforms, the acidic glycoforms B1, C1, and D1 (upper chain) were significantly different between early HCC and advanced 2696


HCC (P < 0.05, Mann Whitney test). Among the F-Hp glycoforms, the acidic glycoforms B1 and C1 and the basic glycoforms J1, H2, and I2 were significantly different between early and advanced HCC (P < 0.05, Mann Whitney test).

Discussion This is the first study clearly demonstrating that serum Hp concentrations are increased in patients with HCC. Our result strongly suggests that serum Hp is a potential diagnostic biomarker for HCC. In this study, we have also identified and examined the clinical values of HCC-specific Hp glycoforms by undertaking a glycoproteomic approach with the use of glycosylation-specifc lectins and 2D-PAGE. Specific Hp glycoforms were not only increased in the sera of HCC patients, but also positively associated with degrees of tumor progression. The aims of measuring of serum S-Hp and F-Hp were not to answer whether S-Hp and F-Hp, which were measured by the depletion approach, were potential biomarkers for HCC or not. The aims were to answer whether purification of S-Hp and F-Hp could enrich the glycosylation variants of Hp associated with HCC. Our data indicated that purification of


research articles

Figure 5. Representative protein identification result of a 2D gel spot corresponding to Hp. A MS spectrum (A) of the tryptic peptides of glycoform H1, a MS/MS spectrum (B) of one of the tryptic peptides (/), and the summary of the database search result (C) were shown.

S-Hp and F-Hp could enrich the glycosylation variants associated with HCC. This was subsequently confirmed by the observation that different HCC-specific glycosylation variants were identified among the S-Hp and F-Hp proteins in the sialylated glycoprotein and fucosylated glycoprotein preparations, respectively. During the development of the S-Hp and F-Hp assays, purified S-Hp and F-Hp (obtained by anti-Hp affinity chromatography, and subsequently by SNA and LCA lectin affinity chromatography) were used to assess the completeness of the depletion step. At the early beginning, LCA lectin coated on microplate was used to deplete the serum fucosylated glycoproteins (including F-Hp). However, after coating to microplate well, the activity of the LCA lectin was greatly reduced (data not shown). Then the LCA-immobilized on agarose beads were used to replace the LCA lectin-coated microplate, to achieve >90% depletion. Unfortunately, the use of LCA-agarose beads resulted in higher variations in the measurement (i.e., interassay CV of 25%). It was partly because it was difficult to deliver the exact amount of LCA-agarose into each assay well, leading higher variations in the background, subsequently resulting in higher measurement error. The current F-Hp assay was not good enough for clinical use; however, for an exploratory purpose, an assay with interassay CV of 25% was good enough for comparing the serum concentrations between the diseased and control subjects. The addition of one more sialic acid to the glycans will make a glycoprotein more acidic, and decrease the pI value by about 0.1 pH.22,23 Analysis of glycosylation pattern of serum Hp by 2D gel electrophoresis is advantageous because isoelectric focusing can separate Hp into a chain of glycoforms according to the number of sialic acid residue present in the glycoform. However, not all the glycoforms of serum Hp, which were observed in this study, were detectable in the previous proteomic studies. Probably some of the glycoforms might be too

low in amount for detection.11,24 In this study, using lectin affinity purification, we successfully enriched the alpha-2,6sialylated and alpha-1,6-fucosylated Hp glycoforms that were specifically associated with HCC. For both the S-Hp (i.e., alpha-2,6-sialylated Hp) and F-Hp (i.e., alpha-1,6-fucosylated Hp), alternations of acidic glycoforms (B1 and C1) were specifically observed in HCC. We speculate that the glycoforms B1 and C1 might have higher degrees of alpha-2,6-sialylation and higher degrees of alpha-1,6-fucosylation (hypersialylated fucosylated). In contrast, the basic glycoforms J1 an I2 might have lower degrees of alpha-2,6-sialylation but higher degrees of alpha-1,6-fucosylation (hyposialylated fucosylated). Only specific acidic and basic glycoforms, but not all the glycoforms of F-Hp, were able to differentiate patients with advanced HCC from early HCC, suggesting the change in the concentration of alpha-1,6-fucosylation was not a single event during HCC development. It might probably be accompanied by alternations of alpha-2,6-sialylation and other types of glycosylation, resulting in a unique pattern of HCC-specific glycoforms containing both hypersialylated fucosylated Hp and hyposialylated fucosylated Hp. Notably, our findings are consistent to the glycosylation changes on the biantennary N-linked glycan of serum AFP in HCC patients. On one hand, serum concentrations of AFP-L3, which is an AFP glycoform with alpha-1,6-fucosyaltion, are higher in the HCC patients than that in the CLD patients.25 On the other hand, serum concentrations of monosialylated AFP (msAFP), which is a AFP glycoform with reduced alpha-2,6sialylation, are also increased in the HCC patients.18 Both glycan structure analysis and mass spectrometric analysis showed that significantly amount of msAFP molecules carry alpha-1,6fucosylation.23,26 Besides using as diagnostic markers, both AFPL327 and msAFP16 have been shown to be potential prognostic markers for HCC. Journal of Proteome Research • Vol. 5, No. 10, 2006 2697

research articles

Ang et al.

Figure 6. Comparison of serum concentrations (mean ( SD) of individual Hp glycoforms between 20 HCC and 10 CLD cases (A), and between 11 advanced HCC cases and 9 early HCC cases (B). The ratios of the mean values of individual glycoforms between the two study groups were plotted. “*” indicates significant difference between the two study groups (P < 0.001, Mann Whitney test).

It is believed that these HCC-specific glycosylation changes on the biantennary N-linked glycan of serum AFP are caused 2698


by an increase in the activity of alpha-1,6-fucosyltransferase17 and a decrease in the activity of beta-galactoside alpha-2,6-

research articles


sialyltransferase in HCC tissues16. The tumor beta-galactoside alpha-2,6-sialyltransferase activity has been shown to be negatively correlated with the percentage of msAFP in the serum. Levels of other glycosyltransferases, such as N-acetylglucosaminyltransferase V,28 have also been found to be altered in the HCC tissues. In a transgenic mouse model, alternation of N-acetylglucosaminyltransferase III level in hepatic tumor led to the presence of serum Hp carrying bisecting GlcNAc, a product of N-acetylglucosaminyltransferase III,29 supporting that glycosylation pattern of serum Hp, similar to the case of AFP, can be specifically altered in the presence of liver cancer. Hp, a well-known acute phase protein, is mainly produced by hepatocytes and secreted into the blood stream. Despite years of investigations, the physiological functions of Hp have remained enigmatic. In cancer patients, serum Hp could be produced either by the tumor cells or by the normal cells of the host body in response to the presence of the tumor. In a mouse model, it was shown that plasma Hp concentration was increased after the inoculation of human stomach cancer cells, and the Hp molecules were found to originate from the mouse host.30 When studying the expression pattern of Hp in human ovarian cancer, immunohistochemical staining showed that Hp was present in malignant ovarian epithelium and stroma, but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces.11 In human kidney tumors, tumor and stromal cells were shown to produce Hp mRNA.7 Recently, we have also shown that Hp was overexpressed in human tissue of HCC associated with hepatitis C virus.12 The increase of serum Hp concentration in HCC may be contributed by both the HCC tissue and the noncancer tissue of the host body in response to the tumor. As it has been shown that alternation of activity of a glycosyltransferase in liver cancer will lead to specific alteration of structure of glycans on serum Hp,29 the Hp glycoforms (S-Hp B1, F-Hp B1, F-Hp C1, F-Hp J1, and F-Hp I2) with positive correlation with HCC stage may originate from the HCC tissue, while the other Hp glycoforms may be produced by the host body. HCC is the major primary liver cancer that carries a high mortality rate and an overall dismal prognosis. Although the prevalence demonstrates a preferential geographic distribution in China, Southeast Asia, and Sub-Saharan Africa, a rising incidence of HCC in the U.S.A. and parts of Europe has been observed in recent years.31,32 Development of HCC is strongly associated with CLD, such as chronic hepatitis B infection and alcoholic cirrhosis. Measurement of serum alpha-fetoprotein (AFP, reference range < 10 ng/mL) provides a marker for the diagnosis and management of HCC.33-35 Patients with HCC (8090%) will have concentrations above the reference range.33-35 A serum concentration of greater than 500 ng/mL is usually diagnostic of HCC. However, modestly raised concentrations of AFP (10-500 ng/mL) are also common in non-malignant CLD so that the specificity of the AFP test for HCC tends to be low.33,36 This represents a serious clinical drawback for the test because most cases of HCC arise in patients with concurrent CLD.37,38 To compensate the specificity problem, 500 ng/mL has been commonly used as a cutoff for diagnosis of HCC; however, that leads to a drop of the diagnostic sensitivity greatly to about 60%. Because the majority of the HCC arises in patients with CLD, from the practical point of view, it is important to find noninvasive biomarkers to identify the HCC cases within the CLD patients. Compared to normal healthy controls, the serum concentrations of various biomolecules change in the CLD

patients. Comparison of normal controls and HCC patients can lead to a discovery of biomarkers that are associated with the underlying chronic liver diseases instead of the HCC tumor. Therefore, compared to normal healthy controls, CLD patients will be the better controls for discovery or validation of biomarkers for HCC. Hence, instead of normal healthy subjects, CLD patients were recruited as the controls in the present study. Furthermore, to simulate the real life situation, both the CLD patients and HCC patients involved in the present study were recruited from the same Hepatoma Clinic. These CLD patients were those who were suspected with HCC but later proven to be free from cancer at the time of blood collection. By using serum Hp in combination with AFP, a diagnostic model for HCC detection with high sensitivity and specificity has been generated in this study. In the independent validation set, at diagnostic specificity of 95%, the sensitivity for detection of HCC was 79%, which was much higher than the sensitivity (55%) when using AFP alone. For HCC-specific glycoforms (SHp B1, F-Hp B1, F-Hp C1, F-Hp J1, and F-Hp I2), for the time being we cannot assess their diagnostic values, as only 20 HCC cases were subjected to Hp glycoform analysis. Development of high-throughput liquid-phase assays for these specific Hp glycoforms, such as glycosylation immunosorbent assay,18 are under taking in our laboratory for assessing their diagnostic values in future studies involving more HCC cases. As different types of cancer may have different effects on the glycosylation pattern of serum Hp, assays of HCC-specific Hp glycoforms may even allow us to differentiate HCC from other types of cancer, such as ovarian cancer. In conclusion, we have shown that serum Hp is a potential biomarker in the diagnosis of HCC. Combined use of Hp and AFP could greatly improve the diagnostic accuracy. A unique pattern of Hp glycoforms with altered sialylation and fucosylation was specific to HCC and associated tumor progression. This study also illustrates that glycoproteomics is a promising approach to discover more novel biomarkers for cancer diagnosis.

Acknowledgment. The study was supported by the RGC Earmarked Grant 4466/03M from the University Grants Committee of Hong Kong. Supporting Information Available: Experimental methods. Representative calibration curves and comparison of serum haptoglobin levels (Figures S1-3). Relative Hp concentrations (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Johnson, P. J.; Leung, N.; Cheng, P.; Welby, C.; Leung, T.; Lau, W. Y.; Ho, S. ‘Hepatoma-specific’ Alphafetoprotein may Permit Preclinical Diagnosis of Malignant Change in Patients with Chronic Liver Disease. Br. J. Cancer 1997, 75, 236-240. (2) Taketa, K.; Hirai, H. Lectin Affinity Electrophoresis of Alphafetoprotein in Cancer Diagnosis. Electrophoresis 1989, 10, 562567. (3) Dobryszycka, W. Haptoglobin: Retrospectives and Perspectives. In Acute Phase Proteins: Molecular Biology, Biochemistry, and Clinical Applications; Mackiewicz, A., Kushner, I., Baumann, H., Eds.; CRC Press: Boca Raton, 1993; pp 185-206. (4) Nilsson, B.; Lowe, M.; Osada, J.; Ashwell, G.; Zopf, D. The Carbohydrate Structure of Human Haptoglobin 1-1. In Glycoconjugates; Yamakawa, T., Osawa, T., Handa, S., Eds.; Japan Scientific Societies Press: Tokyo, 1981; pp 275-276. (5) Delanghe, J. R.; Langlois, M. R. Haptoglobin Polymorphism and Body Iron Stores. Clin. Chem. Lab. Med. 2002, 40, 212-216.

Journal of Proteome Research • Vol. 5, No. 10, 2006 2699

research articles (6) Dobryszycka, W. Biological Functions of Haptoglobin- -New Pieces to an Old Puzzle. Eur. J. Clin. Chem. Clin. Biochem. 1997, 35, 647-654. (7) Smeets, M. B.; Fontijn, J.; Kavelaars, A.; Pasterkamp, G.; de Kleijn, D. P. V. The Acute Phase Protein Haptoglobin is Locally Expressed in Arthritic and Oncological Tissues. Int. J. Exp. Pathol. 2003, 84, 69-74. (8) de Kleijn, D. P. V.; Smeets, M. B.; Kemmeren, P. P. C. W.; Lim, S. K.; Van Middelaar, B. J.; Velema, E.; Schoneveld, A.; Pasterkamp, G.; Borst, C. Acute-phase Protein Haptoglobin is a Cell Migration Factor Involved in Arterial Restructuring. FASEB J. 2002, 16, 1123-1125. (9) Smeets, M. B.; Sluijter, J. P. G.; Donners, M. M. P. C.; Velema, E.; Heeneman, S.; Pasterkamp, G.; de Kleijn, D. P. V. Increased Arterial Expression of a Glycosylated Haptoglobin Isoform After Balloon Dilation. Cardiovasc. Res. 2003, 58, 689-695. (10) Smeets, M. B.; Pasterkamp, G.; Lim, S. K.; Velema, E.; van Middelaar, B.; de Kleijn, D. P. V. Nitric Oxide Synthesis is Involved in Arterial Haptoglobin Expression After Sustained Flow Changes. FEBS Lett. 2002, 529, 221-224. (11) Ahmed, N.; Barker, G.; Oliva, K. T.; Hoffmann, P.; Riley, C.; Reeve, S.; Smith, A. I.; Kemp, B. E.; Quinn, M. A.; Rice, G. E. Proteomicbased Identification of Haptoglobin-1 Precursor as a Novel Circulating Biomarker of Ovarian Cancer. Br. J. Cancer 2004, 91, 129-140. (12) Kotaka, M.; Chen, G. G.; Lai, P. B.; Lau, W. Y.; Chan, P. K.; Leung, T. W.; Li, A. K. Analysis of Differentially Expressed Genes in Hepatocellular Carcinoma with Hepatitis C Virus by Suppression Subtractive Hybridization. Oncol. Res. 2002, 13, 161-167. (13) Bharti, A.; Ma, P. C.; Maulik, G.; Singh, R.; Khan, E.; Skarin, A. T.; Salgia, R. Haptoglobin Alpha-subunit and Hepatocyte Growth Factor can Potentially Serve as Serum Tumor Biomarkers in Small Cell Lung Cancer. Anticancer Res. 2004, 24, 1031-1038. (14) Thompson, S.; Turner, G. A. Elevated Levels of AbnormallyFucosylated Haptoglobins in Cancer Sera. Br. J. Cancer 1987, 56, 605-610. (15) Okuyama, N.; Ide, Y.; Nakano, M.; Nakagawa, T.; Yamanaka, K.; Moriwaki, K.; Murata, K.; Ohigashi, H.; Yokoyama, S.; Eguchi, H.; Ishikawa, O.; Ito, T.; Kato, M.; Kasahara, A.; Kawano, S.; Gu, J.; Taniguchi, N.; Miyoshi, E. Fucosylated Haptoglobin is a Novel Marker for Pancreatic Cancer: A Detailed Analysis of the Oligosaccharide Structure and a Possible Mechanism for Fucosylation. Int. J. Cancer 2006, 11, 2803-2808. (16) Poon, T. C. W.; Chiu, C. H. S.; Lai, P. B. S.; Mok, T. S. K.; Zee, B.; Chan, A. T. C.; Sung, J. J. Y.; Johnson, P. J. Correlation and Prognostic Significance of Beta-galactoside Alpha-2,6-sialyltransferase and Serum Monosialylated Alpha-fetoprotein in Hepatocellular Carcinoma. World J. Gastroenterol. 2005, 11, 6701-6706. (17) Noda, K.; Miyoshi, E.; Uozumi, N.; Yanagidani, S.; Ikeda, Y.; Gao, C.-X.; Suzuki, K.; Yoshihara, H.; Yoshikawa, K.; Kawano, K.; Hayashi, N.; Hori, M.; Taniguchi, N. Gene Expression of Alpha1-6 Fucosyltransferase in Human Hepatoma Tissues: A Possible Implication for Increased Fucosylation of Alpha-fetoprotein. Hepatology 1998, 28, 944-952. (18) Poon, T. C. W.; Mok, T. S. K.; Chan, A. T. C.; Chan, C. M. L.; Leong, V.; Tsui, S. H. T.; Leung, T. W. T.; Wong, H. T. M.; Ho, S. K. W.; Johnson, P. J. Quantification and Utility of Monosialylatedfetoprotein in the Diagnosis of Hepatocellular Carcinoma with Nondiagnostic Serum Total-fetoprotein. Clin. Chem. 2002, 48, 1021-1027. (19) Ching, C. K.; Rhodes, J. M. Enzyme-linked PNA Lectin Binding Assay Compared with CA19-9 and CEA Radioimmunoassay as a Diagnostic Blood Test for Pancreatic Cancer. Br. J. Cancer 1989, 59, 949-953. (20) Pang, R. T. K.; Poon, T. C. W.; Wong, N.; Lai, P. B. S.; Wong, N. L. Y.; Chan, C. M. L.; Yu, J. W. S.; Chan, A. T. C.; Sung, J. J. Y. Comparison of Protein Expression Patterns between Hepatocellular Carcinoma Cell Lines and a Hepatoblastoma Cell Line. Clin. Proteomics 2004, 1, 313-332.

2700


Ang et al. (21) Sanchez, J.-C.; Appel, R. D.; Golaz, O.; Pasquali, C.; Ravier, F.; Bairoch, A.; Hochstrasser, D. F. Inside SWISS-2DPAGE Database. Electrophoresis 1995, 16, 1131-1151. (22) Stibler, H. Carbohydrate-deficient Transferrin in Serum: A New Marker of Potentially Harmful Alcohol Consumption. Clin. Chem. 1991, 37, 2029-2037. (23) Johnson, P. J.; Poon, T. C. W.; Hjelm, N. M.; Ho, C. S.; Blake, C.; Ho, S. K. W. Structures of Disease-specific Serum Alpha-fetoprotein Isoforms. Br. J. Cancer 2000, 83, 1330-1337. (24) He, Q. Y.; Lau, G. K. K.; Zhou, Y.; Yuen, S. T.; Lin, M. C.; Kung, H. F.; Chiu, J. F. Serum Biomarkers of Hepatitis B Virus Infected Liver Inflammation: A Proteomic Study. Proteomics 2003, 3, 666674. (25) Khien, V. V.; Mao, H. V.; Chinh, T. T.; Ha, P. T.; Bang, M. H.; Lac, B. V.; Hop, T. V.; Tuan, N. A.; Don, L. V.; Taketa, K.; Satomura, S. Clinical Evaluation of Lentil Lectin-reactive Alpha-fetoproteinL3 in Histology-proven Hepatocellular Carcinoma. Int. J. Biol. Markers. 2001, 16, 105-111. (26) Johnson, P. J.; Poon, T. C.; Hjelm, N. M.; Ho, C. S.; Ho, S. K.; Welby, C.; Stevenson, D.; Patel, T.; Parekh, R.; Townsend, R. R. Glycan Composition of Serum Alpha-fetoprotein in Patients with Hepatocellular Carcinoma and Nonseminomatous Germ Cell Tumour. Br. J. Cancer 1999, 81, 1188-1195. (27) Toyoda, H.; Kumada, T.; Kiriyama, S.; Sone, Y.; Tanikawa, M.; Hisanaga, Y.; Yamaguchi, A.; Isogai, M.; Kaneoka, Y.; Washizu, J. Prognostic Significance of Simultaneous Measurement of Three Tumor Markers in Patients with Hepatocellular Carcinoma. Clin. Gastroenterol. Hepatol. 2006, 4, 111-117. (28) Yanagi, M.; Aoyagi, Y.; Suda, T.; Mita, Y.; Asakura, H. NAcetylglucosaminyltransferase V as a Possible Aid for the Evaluation of Tumor Invasiveness in Patients with Hepatocellular Carcinoma. J. Gastroenterol. Hepatol. 2001, 16, 1282-1289. (29) Ekuni, A.; Miyoshi, E.; Ko, J. H.; Noda, K.; Kitada, T.; Ihara, S.; Endo, T.; Hino, A.; Honke, K.; Taniguchi, N. A Glycomic Approach to Hepatic Tumors in N-acetylglucosaminyltransferase III (GnTIII) Ttransgenic Mice Induced by Diethylnitrosamine (DEN): Identification of Haptoglobin as a Target Molecule of GnT-III. Free Radic. Res. 2002, 36, 827-833. (30) Juan, H. F.; Chen, J. H.; Hsu, W. T.; Huang, S. C.; Chen, S. T.; Yi-Chung Lin, J.; Chang, Y. W.; Chiang, C. Y.; Wen, L. L.; Chan, D. C.; Liu, Y. C.; Chen, Y. J. Identification of Tumor-associated Plasma Biomarkers Using Proteomic Techniques: From Mouse to Human. Proteomics 2004, 4, 2766-2775. (31) El-Serag, H. B.; Mason, A. C. Rising Incidence of Hepatocellular Carcinoma in the United States. N. Engl. J. Med. 1999, 340, 745750. (32) Okuda, K. Hepatocellular Carcinoma. J. Hepatol. 2000, 32, 225237. (33) Johnson, P. J.; Portmann, B.; Williams, R. Alpha-fetoprotein Concentration Measured by Radioimmunoassay in the Diagnosing and Excluding of Hepatocellular Carcinoma. Br. Med. J. 1978, 2, 661-663. (34) Sheu, J. C.; Sung, J. L.; Chen, D. S.; Yang, P. M.; Lai, M. Y.; Lee, C. S.; Hsu, H. C.; Chuang, C. N.; Yang, P. C.; Wang, T. H.; Lin, J. T.; Lee, C. Z. Growth Rate of Asymptomatic Hepatocellular Carcinoma and its Clinical Implications. Gastroenterology 1985, 89, 259-266. (35) Nomura, F.; Ohnishi, K.; Tanabe, Y. Clinical Features and Prognosis of Hepatocellular Carcinoma with Reference to Serum Alpha-fetoprotein Levels. Cancer 1989, 64, 1700-1707. (36) Lok, A. S. F.; Lai, C. L. Alpha-fetoprotein Monitoring in Chinese Patients with Chronic Hepatitis B Virus Infection: Role in the Early Detection of Hepatocellular Carcinoma. Hepatology 1989, 9, 110-115. (37) Kew, M. C.; Popper, H. Relationship between Hepatocellular Carcinoma and Cirrhosis. Semin. Liver Dis. 1984, 4, 136-146. (38) Johnson, P. J.; Williams, R. Cirrhosis and the Aetiology of Hepatocellular Carcinoma. J. Hepatol. 1987, 4, 140-147.

PR060109R

A Glycoproteomic Approach - American Chemical Society

Recommend Documents