The Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) is widely used to screen for hepatocellular carcinoma (HCC) in Japan and China. We developed a chemiluminescent protein microarray for determining the AFP-L3/AFP index (the ratio of AFP-L3 to total AFP, AFP-L3%) by fixing AFP-specific antibodies and Lens culinaris lectin on aldehyde-coated glass slides. Serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA) to validate the microarray. AFP-L3 was detected using Hotgen Biotech glycosyl capture spin column pretreatment technology and ELISA. When the AFP cut-off value was set to 20 ng/ml, the protein microarray displayed 89.74% sensitivity and 100% specificity for HCC diagnosis, and the ELISA displayed 87.17% sensitivity and 100% specificity. When the AFP-L3% cut-off value was set to 0.1, the protein microarray displayed 56.41% sensitivity and 100% specificity for HCC diagnosis, and the ELISA displayed 53.84% sensitivity and 100% specificity. The ROC curve for the HCC diagnosis showed that the AFP area under the ROC curve (AUC = 0.996; 95% CI: 0.986-1.005) was much higher than that of AFP-L3 (AUC = 0.857; 95% CI: 0.769-0.94) and AFP-L3% (AUC = 0.827; CI: 0.730-0.924). The microarray assay used in this study is a highly sensitive, accurate, and efficient assay for the determination of the AFP-L3%.

The level of serum glycoproteins and their glycosylation pattern change in liver diseases including hepatocellular carcinoma (HCC). Some of them, especially alpha fetoprotein (AFP), serve as useful biomarkers for HCC. The present investigation showed high level of AFP in hepatitis B cirrhosis (HBV-LC) and hepatitis C cirrhosis (HCV-LC) patients. However, increase of AFP level was not significantly high in chronic hepatitis B (HBV-CH) as determined by ELISA using monoclonal anti-human AFP (mAb-AFP). The differential expression of sialic acid linkage was observed in HBV-CH and HCV-LC by ELISA; the former bound strongly with Sambucus nigra agglutinin (SNA), which has exclusive binding specificity for NeuAcα2-6-, whereas HCV-LC reacted preferably with Maackia amurensis agglutinin (MAA) which recognizes NeuAcα2-3-. There was significantly high glycan branching in HBV-LC and HCV-LC in comparison to controls as illustrated by concanavalin A. This was further confirmed by Phaseolus vulgaris erythroagglutinin (E-PHA) and Datura stramonium agglutinin (DSA). Enhanced fucosylation of AFP was observed in HBV-LC, HCV-LC and HCC patients by ELISA using fucose binding Aleuria aurantia lectin; however, maximum binding was found in HCC. Fucosylation with α1-6 linkage was further confirmed by Lens culinaris agglutinin (LCA). From the above results it is concluded that the changes in concentration of AFP, differential expression of sialic acid, increase of glycan branching and fucosylation have a prognostic value of hepatitis and it could be possible that lectin-based assay with AFP can aid in diagnosis of hepatitis diseases besides clinical examination and routine laboratory investigation.

Updated National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed.

Published reports relevant to use of tumor markers for 4 cancer sites--liver, bladder, cervical, and gastric--were critically reviewed.

Alpha-fetoprotein (AFP) may be used in conjunction with abdominal ultrasound for early detection of hepatocellular carcinoma (HCC) in patients with chronic hepatitis or cirrhosis associated with hepatitis B or C virus infection. AFP concentrations >200 microg/L in cirrhotic patients with typical hypervascular lesions >2 cm in size are consistent with HCC. After a diagnosis of HCC, posttreatment monitoring with AFP is recommended as an adjunct to imaging, especially in the absence of measurable disease. Although several urine markers have been proposed for bladder cancer, none at present can replace routine cystoscopy and cytology in the management of patients with this malignancy. Some may, however, be used as complementary adjuncts to direct more effective use of clinical procedures. Although carcinoembryonic antigen and CA 19-9 have been proposed for use gastric cancer and squamous cell carcinoma antigen for use in cervical cancer, none of these markers can currently be recommended for routine clinical use.

Implementation of these recommendations should encourage optimal use of tumor markers for patients with liver, bladder, cervical, or gastric cancers.

Changes in N-linked glycosylation are known to occur during the development of cancer. For example, increased branching of oligosaccharides has been associated with metastasis and has been correlated to tumor progression in human cancers of the breast, colon, and melanomas. Increases in core fucosylation have also been associated with the development of hepatocellular carcinoma (HCC). To a large extent, the proteins to which these N-linked glycans are attached have been unknown. However, with the advent of sensitive glycan analysis and proteomic technologies, the ability to comprehensively identify all the fucosylated proteins in a given population is now a possibility. This method, generally referred to as targeted glycoproteomics, is shown as applied to the detection of proteins present in the fucosylated proteome of a liver cancer cell line but is generally enough to be applied in many other situations.

The serum alpha-fetoprotein (AFP) level has been used as a tumor marker for hepatoblastoma, and malignant germ cell tumors in pediatric patients. The AFP has 3 isoforms (L1, L2, L3), and the usefulness of the L3 fraction as a diagnostic marker for the adult hepatocellular carcinoma is well known. However, there are few reports dealing with various pediatric malignant tumors. In the current study, we analyzed the diagnostic value of AFP fractions for pediatric diseases, in particular, those occurring in the neoinfantile period.

From 2003 to 2006, two cases of hepatoblastoma, and 5 cases of germ cell tumor, all of which were neoinfantile, were treated in our department. In our analytical system (LiBASys), the level of the L3 fraction contains the majority of the L2 fraction. The total AFP (ng/mL) level and the L3 fraction (%) were measured to assess the usefulness of the L3 fraction as a diagnostic marker.

In all cases of hepatoblastoma and yolk sac tumor, both the total AFP and the L3 fraction were high, either before treatment or in the presence of malignant tumors. Most of the cases of neonatal immature teratoma showed a high total AFP level during the neoinfantile period, however, the L3 fraction was around 10%, and decreased after surgical treatment. Only 1 case of the immature teratoma demonstrated malignant transformation, when the patient was 8 months old. As the total AFP and the AFP-L3 fraction were proportionally elevated, the patient was treated with additional surgical resection and chemotherapy. In the case of neonatal mature teratoma, the L3 fraction was below 0.5%, even when the total AFP level was high.

Our results indicated that the level of the L3 fraction accurately confirmed the existence, or the malignant potential of hepatic tumor or germ cell tumor. The L3 fraction is useful as a tumor marker during the neoinfantile period.

The aim of the present study was to establish L3 fraction before initial treatment as a useful prognostic factor in a prospective fashion in hepatocellular carcinoma (HCC) where the alpha-fetoprotein (AFP) was very low.

From 1990 to 2004, 298 HCC patients in whom L3 could be measured were examined in the present study. Enrolled patients with HCC underwent operation, transcatheter arterial chemoembolization and percutaneous ablation therapy. The current patient status was confirmed as of the end of March 2005. L3 was determined by crossed immuno-affinoelectrophoresis when AFP was >/=30 ng/mL. It was carried out by liquid-phase binding assay system on cases where AFP < 30 ng/mL. The tentative discriminating line of L3 was set at 15%.

The HCC group included four subgroups: 110 patients with AFP concentrations 15% (high L3), was significantly lower than that in the HCC group whose L3 was Collapse

Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using alpha 1,6 fucosyltransferase gene (Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains.

Human alpha-fetoprotein (AFP) from serum of patients with cirrhosis and hepatocellular carcinoma (HCC) was separated into several bands by IEF and by erythroagglutinating phytohemagglutinin (E-PHA) affinity electrophoresis. These AFP bands were directly compared in 2-D IEF and E-PHA affinity electrophoresis. IEF of serum AFP was run in 1% agarose IEF gel with 3% Pharmalyte 4.5-5.4. After IEF, a part of the gel was stained for AFP and another part of the gel corresponding to the area of separated AFP bands was cut in 1 mm x 39 mm along the focused direction and transferred to a trough in 1% agarose gel with 0.3 mg/mL E(4)-PHA for second-dimensional affinity electrophoresis. Separated 2-D AFP spots were visualized by antibody-affinity blotting and identified by combining the systems of Johnson et al.. (Johnson, P. J., Ho, S., Cheng, P., Chan, A. et al.., Cancer 1995, 75, 1663-1668) for AFP-I-+IV and of Taketa et al.. (Taketa, K., Ichikawa, E., Taga, H., Hirai, H., Electrophoresis 1985, 6, 492-497) for AFP-P1-5. AFP-P2, the major AFP glycoform, was composed of AFP-I, AFP+I, and AFP+II; AFP-P3, a nonspecific monosialo-AFP, was composed of AFP+II; AFP-P4, HCC-specific monosialo-AFP, was composed of AFP+II, AFP+III, and AFP+IV; and malignancy-related AFP-P5 was composed of AFP+I and AFP+II. Monosialo-AFP (AFP+II) was recovered in all the glycoforms of AFP-P2, -P3, -P4, and -P5; thus, AFP-P4 is more specific to HCC than monosialylated AFP+II.

Of all protein PTMs, glycosylation is by far the most common, and is a target for proteomic research. Glycosylation plays key roles in controlling various cellular processes and the modifications of the glycan structures in diseases highlight the clinical importance of this PTM. Glycosylation analysis remains a difficult task. MS, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of glycoconjugates. This review describes methodologies based on MS for detailed characterization of glycoconjugates in complex biological samples at the sensitivity required for proteomic work.

The application of mass spectrometry to identify disease biomarkers in clinical fluids like serum using high throughput protein expression profiling continues to evolve as technology development, clinical study design, and bioinformatics improve. Previous protein expression profiling studies have offered needed insight into issues of technical reproducibility, instrument calibration, sample preparation, study design, and supervised bioinformatic data analysis. In this overview, new strategies to increase the utility of protein expression profiling for clinical biomarker assay development are discussed with an emphasis on utilizing differential lectin-based glycoprotein capture and targeted immunoassays. The carbohydrate binding specificities of different lectins offer a biological affinity approach that complements existing mass spectrometer capabilities and retains automated throughput options. Specific examples using serum samples from prostate cancer and hepatocellular carcinoma subjects are provided along with suggested experimental strategies for integration of lectin-based methods into clinical fluid expression profiling strategies. Our example workflow incorporates the necessity of early validation in biomarker discovery using an immunoaffinity-based targeted analytical approach that integrates well with upstream discovery technologies.

Some patients who are seropositive for lectin-reactive alpha-fetoprotein (AFP-L3) have intrahepatic cholangiocarcinoma (ICC). There have been no studies regarding the features of ICC patients seropositive for AFP-L3. Thus, the purpose of the present paper was to compare the features of ICC patients from the viewpoint of two different tumor markers, AFP-L3 and carbohydrate antigen (CA) 19-9.

The ICC patients who underwent hepatectomy (n = 51) were divided into three groups, and their clinicopathologic features were compared: (i) group A, seropositive for AFP-L3 >or= 15%; (ii) group B, seropositive for CA 19-9 >or= 37 U/mL; and (iii) group C, seronegative for both AFP-L3 and CA 19-9. The features of combined hepatocellular and cholangiocarcinoma (n = 11) were also studied.

Group A had a higher positivity rate for hepatitis viruses than group B (60%vs 20%, P < 0.05). More patients in group A were misdiagnosed as having hepatocellular carcinoma (HCC) at surgery (70%vs 5.7%, P < 0.001) who also had chronic liver disease (80%vs 25.7%, P < 0.01) than in group B. Seven, 10 and 11 of the 11 patients with combined hepatocellular and cholangiocarcinoma were seropositive for AFP-L3, CA 19-9 and hepatitis viruses, respectively. Ten were diagnosed as having HCC at surgery and nine had chronic liver disease.

Patients with ICC seropositive for AFP-L3 and those with combined hepatocellular and cholangiocarcinoma have features close to HCC. The present study has, for the first time, identified a subgroup of ICC patients, seropositive for AFP-L3, having features close to HCC that are very different from those of the classical ICC patients seropositive for CA 19-9.

Patients with hepatocellular carcinoma (HCC) seropositive for lectin-reactive alpha-fetoprotein (AFP-L3) have a poor prognosis. However, there have been no studies of the clinicopathologic features of patients seronegative for both AFP and des-gamma-carboxy prothrombin (DCP), and seropositive for AFP-L3 alone in comparison with other patients seropositive for AFP-L3.

Patients with primary malignant hepatic tumors seropositive for AFP-L3 who underwent hepatectomy (n = 84) were divided into four groups, and their clinicopathologic features were compared: (i) group A, seronegative for AFP <100 ng/mL and DCP <40 mAU/mL; (ii) group B, seropositive for AFP > or =100 ng/mL and seronegative for DCP; (iii) group C, seronegative for AFP and seropositive for DCP > or =40 mAU/mL; and (iv) group D, seropositive for AFP and DCP.

Among the 14 group A patients seropositive for AFP-L3 alone with low AFP concentrations, three had intrahepatic cholangiocarcinoma (ICC), one had a cholangiolocellular carcinoma, one had combined HCC and ICC, and one had undifferentiated hepatic sarcoma. Group A had a higher incidence of non-HCC tumors (P < 0.001) and tumors derived from cholangiocytes (P < 0.001) than the other three groups. They also had a high frequency of poorly differentiated tumors and sarcomatous changes, and showed a poor prognosis.

Patients with primary malignant hepatic tumors seropositive for AFP-L3 alone with low AFP concentrations have unique clinicopathologic features. Thus, we should be aware of these patients and should measure AFP-L3 levels, at least once, even in those seronegative for both AFP and DCP.

Aminotransferase levels do not always increase during acute hepatitis or during an acute flare-up of chronic hepatitis. Persistently increased levels of serum alpha-Fetoprotein in an adult with liver disease suggest not only the presence or progression of hepatocellular Carcinoma or its recurrence after hepatic resection or after other therapeutic approaches such as chemotherapy or chemoembolization, but also it suggests that there is an acute exacerbation of hepatitis or liver cirrhosis. We report here on two unusual cases of HBV- & HCV-related liver cirrhosis with acute exacerbation of hepatitis in which there was an insignificant elevation of the aminotransferase levels, but there were markedly increased alpha-Fetoprotein levels observed. The levels of alpha-Fetoprotein decreased gradually in both cases since the beginning of antiviral therapy, which implies that the increased levels were due to aggravation of the accompanying hepatitis. These cases also emphasize that using only the measurement of alpha-Fetoprotein is not sufficient for the diagnosis of hepatocellular carcinoma, and that this diagnosis also requires a more specific measurement such as AFP L3 along with the standard imaging studies.

Chronic infection with hepatitis B virus (HBV) is associated with the majority of hepatocellular carcinoma (HCC). The diagnosis of HCC is usually made in the late stages of the disease, when treatment options are limited and prognosis is poor. We therefore have developed a method of glycoproteomic analysis in an attempt to discover serum markers that can assist in the early detection of HBV-induced liver cancer. Briefly, a comparative method for analysis of oligosaccharides released from serum glycoproteins and for recovery and identification of proteins with aberrant glycosylation, as a function of cancer diagnosis, is described. The model we have used is the woodchuck (Marmota monax), which shares similarities in the glycosylation pattern associated with liver proteins in human HCC. In this report, we show that woodchucks diagnosed with HCC have dramatically higher levels of serum-associated core alpha-1,6-linked fucose, as compared with woodchucks without a diagnosis of HCC. The coupling of this methodology with 2D gel proteomics has permitted the identification of several glycoproteins with altered glycosylation as a function of cancer. One such glycoprotein, Golgi Protein 73 (GP73), was found to be elevated and hyperfucosylated in animals with HCC. Further, the study showed GP73 to be elevated in the serum of people with a diagnosis of HCC, providing a validation of our approach. The potential of this technology for biomarker discovery and the implications of increased levels of GP73 in liver cancer are discussed.

There has been no study of the clinicopathologic features of patients with hepatocellular carcinoma (HCC) who are seropositive for lectin-reactive alpha-fetoprotein (AFP-L3) alone, or seropositive for AFP-L3 and seronegative for des-gamma-carboxy prothrombin (DCP) in comparison with those who are seropositive for DCP alone. Thus, the present comparative study was performed.

The clinicopathologic features of HCC patients with either one or two tumors who underwent a hepatectomy (n = 88) were compared among the following five groups according to the seropositivity of AFP, AFP-L3 and DCP: (i) group A, seropositive for AFP above 100 ng/mL, AFP-L3 above 15% and DCP above 100 mAU/mL; (ii) group B, seropositive for AFP-L3 and seronegative for DCP below 40 mAU/mL; (iii) group C, seronegative for AFP below 20 ng/mL, AFP-L3 below 15% and seropositive for DCP; (iv) group D, seropositive for AFP and seronegative for AFP-L3 and DCP; and (v) group E, seronegative for AFP, AFP-L3 and DCP.

Group B patients showed a higher incidence of infiltrative-type HCC with an irregular margin (P < 0.05) and a higher frequency of poorly differentiated HCC (P < 0.01) compared with group C patients. Group A patients had larger tumors and more massive-type tumors than group B patients. Our HCC cases showed that advanced clinicopathologic features were demonstrated in the order of group B, group C and group D. Group A and B patients and group D and E patients showed similar characteristics.

Hepatocellular carcinoma patients who were seropositive for AFP-L3 and seronegative for DCP demonstrated clinicopathologic features of more advanced HCC compared with those who were seropositive for DCP alone.

There has been no comparative study of the clinicopathological features of HCC patients who are seropositive for alpha-fetoprotein (AFP) alone and those who are seropositive for des-gamma-carboxy prothrombin (DCP) alone. The authors, thus, performed this comparative study.

The clinicopathological features of patients with solitary hepatocellular carcinoma (HCC), who underwent a hepatectomy were compared among the four below groups according to the seropositivity of AFP and DCP: group A, seronegative for both AFP below 20 ng/mL and DCP below 40 mAU/mL; group B, seropositive for AFP above 100 ng/mL and seronegative for DCP; group C, seronegative for AFP and seropositive for DCP above 100 mAU/mL; and group D, seropositive for both AFP and DCP.

Group B patients showed a higher incidence of HCC with an indistinct margin, and a somewhat higher incidence of small HCC less than 2 cm in greatest dimension compared with group C patients. By contrast, group C patients had a higher frequency of HCC with a distinct margin compared with that of an indistinct margin, large tumors more than 3 cm compared with that of small tumors less than 2 cm, and a somewhat higher frequency of moderately to poorly differentiated HCC compared with that of well-differentiated HCC. Our HCC cases showed advanced clinicopathological features in the order of group C, group B and group A. Groups C and D patients showed similar characteristics.

Hepatocellular carcinoma patients who were seropositive for AFP alone demonstrated clinicopathological features of less advanced HCC compared with those who were seropositive for DCP alone.

Our purpose was to examine the utility of analyzing alpha-fetoprotein (AFP) microheterogeneity assessed by lectin affinity in Down's syndrome (DS) screening. Maternal sera and amniotic fluids were collected from 18 women who were carrying DS fetuses and 70 unaffected pregnancies around 16 weeks of gestation. The percentages of AFP which reacted with Lens culinaris agglutinin (AFP-L2,3) were determined by lectin affinity electrophoresis. AFP-L2,3 levels were significantly increased (P<0.0001) in both maternal serum and amniotic fluid from DS-affected versus unaffected pregnancies. The fractional areas under the receiver operating characteristic curves were 0.835 and 0.700 (P=0.106) for AFP-L3 and AFP MoM (multiples of the median) in maternal serum. No correlation was found between AFP-L3 and AFP MoM in maternal serum (r=0.006). Our data suggest that the measurement of AFP-L3 in maternal serum is a potential biochemical marker for DS.

Hepatocellular carcinoma (HCC) is increasing in many countries as a result of an increase in hepatitis C virus (HCV) infection since World War II. The epidemiology of HCC varies with the global region. There have been conflicting observations from different parts of the world concerning the frequency of HCC in patients who in the distant past had post-transfusion non-A, non-B hepatitis. The genetic basis of hepatocarcinogenesis is still poorly understood. In hepatitis B virus (HVB) associated HCC, codon 249 mutation in the p 53 gene seems more related to exposure to aflatoxin B1 than to hepatocarcinogenesis itself. HCC that occurs in children in high HBV endemic regions could be associated with germ-line mutations, but little information is available; not much is known about chemical hepatocarcinogens in the environment other than aflatoxins. The X gene of HBV seems to play an important role in HBV-associated hepatocarcinogenesis. There are preliminary observations on the molecular mechanism of HCV-associated HCC, such as HCV core protein inducing HCC in transgenic mice and the NS3 genome transforming NIH 3T3 cells. Pathological distinction between preneoplastic and very early transformed lesions still depends on classical morphology, and a more genetically oriented differential diagnosis is required. Clinical diagnosis based on modern imaging has improved greatly, but is still unsatisfactory in the differential diagnosis of preneoplastic and early transformed nodules, because the vasculature changes that occur within the nodule are not accurately discerned with the current imaging. Use of sensitive des-gamma-carboxy prothrombin (PIVKA II) assay, and lectin affinity chromatography separating HCC specific subspecies of AFP molecules with a more practical biochemical technique will further improve diagnosis. Early diagnosis and transplantation are the best treatment at the moment, but transplantation is not widely available because of the donor shortage. Despite successful resection, the remnant cirrhotic liver frequently develops new HCC lesions, seriously curtailing long-term survival. All-out efforts should be directed to the prevention of HCC, through prevention of viral hepatitis, prevention of acute hepatitis from becoming chronic, prevention of chronic hepatitis from progressing to cirrhosis, and prevention of the cirrhotic liver from developing HCC (chemoprevention). At the moment, very few such studies exist.

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-6-fucosyltransferase (alpha1-6FucT) catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycoproteins. This enzyme was purified from a human fibroblast cell line, porcine brain, a human gastric cancer cell line and human blood platelets. cDNA cloning of porcine and human alpha1-6FucT was performed from a porcine brain and gastric cancer cell cDNA libraries, respectively. Their homology is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence. No homology to other fucosyltransferases such as alpha1-2FucT, alpha1-3FucT and alpha1-4FucT was found except for a region consisting of nine amino acids. The alpha1-6FucT gene is located at chromosome 14q24.3, which is also a different location from other fucosyltransferases reported to date. The alpha1-6FucT gene is the oldest gene family in the phylogenic trees among the nine cloned fucosyltransferase genes. alpha1-6FucT is widely expressed in various rat tissues and the expression of alpha1-6FucT in the liver is enhanced during hepatocarcinogenesis of LEC rats which develop hereditary hepatitis and hepatomas. In cases of human liver diseases, alpha1-6FucT is expressed in both hepatoma tissues and their surrounding tissues with chronic liver disease, but not in the case of normal liver. Serum alpha1-6-fucosylated alpha-fetoprotein (AFP) has been employed for an early diagnosis of patients with hepatoma. The mechanisms by which alpha1-6 fucosylation of AFP occurs in the hepatoma is not due to the up-regulation of alpha1-6FucT alone. Interestingly, when the alpha1-6FucT gene is transfected into Hep3B, a human hepatoma cell line, tumor formation in the liver of nude mice after splenic injection is dramatically suppressed. In this review, we focus on alpha1-6FucT and summarize its properties, gene expression and biological significance.

It is well-known that microheterogeneity of human serum transferrin observed in alcoholics manifests as sialic acid-deficient transferrin isoforms, otherwise known as carbohydrate-deficient transferrin (CDT). A recent study demonstrated that serum CDT lacked one or both of the entire carbohydrate chains but the investigation required several troublesome procedures. The aim of the present study was to confirm the sugar chain structures of serum transferrin, and of serum CDT in particular, from patients with alcoholic liver disease (ALD) using conventional lectin affinity electrophoresis which might be useful in the clinical setting. The serum CDT obtained from ALD-patients was partially purified using an anion exchanger. Serum transferrin and the partially purified serum CDT were investigated by concanavalin A (Con A)- and Datura stramonium agglutinin (DSA)-affinity electrophoresis followed by antibody-affinity blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blotting. By Con A-affinity electrophoresis, serum CDT was separated into weakly reactive and nonreactive transferrins which showed slower electrophoretic mobilities than those from the healthy controls. Moreover, nearly all of the serum CDT was nonreactive with DSA. On SDS-PAGE, the molecular masses of serum CDT were estimated to be approximately 75 and 72 kDa, which corresponded to those of partially and completely deglycosylated transferrin obtained from the healthy controls (78 kDa), respectively. In conclusion, these results indicated that the sugar chain structures of serum CDT from patients with ALD show not merely a loss of terminal sialic acids, but also the absence of asparagine-N-linked oligosaccharides.

The aim of this study was to elucidate the usefulness of measuring the fucosylation index (FI) of alpha-fetoprotein (AFP) before the initiation of therapy as a new prognostic indicator for patients with hepatocellular carcinoma (HCC).

One hundred twelve patients with HCC who underwent transcatheter arterial embolization, chemoembolization, and/or percutaneous ethanol injection were examined in the current study. FI was determined by crossed immunoaffino-electrophoresis in the presence of Lens culinaris agglutinin.

When the tentative discriminating value of FI was set at 18%, the mean survival rate for the group whose FI was higher than 18% was significantly lower than that for the group whose FI was equal to or less than 18%, according to the generalized Wilcoxon test (P = 0.0117) and the log rank test (P = 0.0183). The survival rate for HCC patients with AFP concentrations of more than 200 ng/mL was also significantly lower than that for patients with AFP in the range of 21-200 ng/mL, according to the generalized Wilcoxon test (P = 0.0017) and the log rank test (P = 0.0018). When FI was combined with AFP concentration, a highly significant difference was observed between the group with FI >18% and AFP >200 ng/mL and another group with FI < or =18% and AFP < or =200 ng/mL, as determined by the generalized Wilcoxon test (P < 0.0001) and the log rank test (P = 0.0003). An analysis of multiple covariates in the prognostic factors with the Cox proportional hazards model showed that FI was one of the independent prognostic factors.

The current study indicates that measuring FI from sera before the initiation of treatment serves as a new prognostic factor and may improve prognostic estimates and appraisal of therapeutic outcomes for patients with HCC.

Alpha-Fetoprotein (AFP) glycoforms, defined as AFP with different chemical structures of carbohydrate, were analyzed by affinity electrophoresis with several lectins of known specificities against complex-type oligosaccharides. Serum AFP samples from cord blood on full-term delivery and from patients with hepatocellular carcinoma and extrahepatic malignancies including gastrointestinal tumors and yolk sac tumors were used. Two-dimensional lectin affinity electrophoresis and also lectin affinity chromatography coupled with lectin affinity electrophoresis were employed. More than ten AFP glycoforms were identified or characterized using the above-mentioned AFP samples. Known specificities of the lectins against complex-type oligosaccharides were refined or their additional specificities were found in this study. Lectin appeared to have specificity against carbohydrates by recognizing not only specific residues but also the whole carbohydrate molecule containing the residues, resulting in differential affinities for the lectin.

Affinity electrophoresis (AEP) is a useful technique for separation of biomolecules such as plasma proteins, enzymes, nucleic acids, lectins, receptors, and extracellular matrix proteins by specific interactions with their ligands in electric fields and for the determination of dissociation constants for those interactions. Two-dimensional affinity electrophoresis (2-D AEP), which was newly developed by a combination of isoelectric focusing with AEP, has been used for studies on immune response to haptens. Antihapten antibodies, which were induced by immunization of a mouse with the hapten-conjugated bovine serum albumin, were separated by 2-D AEP into a large number of groups of IgG spots with a few microliters of antiserum. Each group of spots showed an identical affinity for the hapten but different isoelectric points as in the case of monoclonal antibodies specific to the hapten. This enabled us to study the diversification and affinity maturation of antihapten antibodies in the course of immunization of a single mouse. Furthermore, effects of a carrier and a hapten array on the production of antihapten antibodies and the cause of charge heterogeneity of monoclonal antibodies were also examined to understand the molecular basis of the immune response in vivo.

Multimodal application of lectin affinity electrophoresis of alpha-fetoprotein (AFP) glycoforms is reviewed. Crossed affinity immunoelectrophoresis developed by Bøg-Hansen and others was extended to tandem-lectin affinity electrophoresis by tandem lining of two different lectin gels and to mixed-lectin affinity electrophoresis. By introducing an antibody-affinity blotting technique for detection of separated glycoforms of AFP, several two-dimensional combinations of lectin affinity electrophoresis became possible: two different lectins for the first and second dimension electrophoresis, lectin-gradient affinity electrophoresis, and electrophoretic separation of lectin isoforms in the first-dimension electrophoresis, followed by affinity electrophoresis against the separated lectin isoforms. Usefulness of the different modalities of lectin affinity electrophoresis for several analytical purposes has been described.

Lectin-gradient agarose gel affinity electrophoresis was developed for identification of glycoforms of glycoproteins in lectin affinity electrophoresis. Gradation of lectin was done by stacking agarose gel blocks with increasing concentrations of lectin (discontinuous system) and by keeping the plate in a moist chamber at 4 degrees C overnight (continuous system) before electrophoresis. On the visualization of separated glycoform lines, the antibody-affinity blotting was superior for low concentrations of alpha-fetoprotein. Fluoresceine isothiocyanate (FITC) labeling of whole serum proteins, enzyme activity staining for alkaline phosphatase, and prestaining for lipoproteins were also applicable for visualization of proteins at higher concentrations. The conventional Western blotting can not be recommended because of the competition between lectin and glycoproteins in binding to nitrocellulose membrane. Lectin-gradient affinity electrophoresis also had a wide application for optimization of the condition of lectin affinity electrophoresis.

To investigate a potential diagnostic use of alpha fetoprotein (alpha FP) isoform analysis by lectin affinity electrophoresis to distinguish between endodermal sinus tumours arising in the vagina in infants from those at other sites.

alpha FP in the serum of a patient with a vaginal endodermal sinus tumour was analysed for its isoforms by lectin affinity electrophoresis. The isoforms were compared with that of cord serum, sera of hepatoid adenocarcinoma of the uterus, and endodermal sinus tumour of the ovary.

The isoforms of alpha FP obtained by lectin affinity electrophoresis in the serum of the patient with vaginal endodermal sinus tumour differed from the isoforms of alpha FP in the cord serum of normal neonates, and sera of patients with hepatoid adenocarcinoma of the uterus or endodermal sinus tumour of the ovary.

Endodermal sinus tumour arising in the vagina could be distinguished from that in the ovary by the lectin affinity electrophoresis, and a potential diagnostic use of alpha FP isoform analysis by the lectin affinity electrophoresis for the detection of the endodermal sinus tumour in infants was demonstrated.

Serum or ascites alpha-fetoprotein (AFP) from patients with hepatocellular carcinoma and from a cord blood were analyzed by affinity electrophoresis with two lectins mixed in agarose gel in a combination of concanavalin A (Con A) and Lens culinaris agglutinin A (LCA-A) or of erythroagglutinating phytohemagglutinin (E-PHA) and Allomyrina dichotoma lectin (allo A). Con A- and LCA-A-reactive AFP-C2-L3 was not further retarded by mixing with either of the other lectin. It showed a mobility identical with that of AFP-C2 or AFP-L3. E-PHA- and allo A-reactive AFP-P4-A3 showed similar results. It migrated with intermediate mobilities of AFP-P4 and AFP-A3 depending on the concentrations of the two lectins mixed in the gel. The results indicate that the two mixed lectins compete with each other for the topologically different lectin-binding sites on the oligosaccharide of AFP molecule.

The oligosaccharide specificity of newly isolated Butea monosperma agglutinin (BMA) was determined by two-dimensional lectin affinity electrophoresis of alpha-fetoprotein (AFP) with concanavalin A, lentil lectin, erythroagglutinating phytohemagglutinin and Allomyrina dichotoma lectin, of which the specificities to known AFP oligosaccharides had been established. Effects of neuraminidase treatment on the reactivities of AFP to the lectins were also studied. The results indicated that BMA had the highest affinity for the exposed Gal residues of nonreducing termini of biantennary complex-type oligosaccharides, and that the affinity was reduced to zero in the following order by the presence of monosialyl residue of the Man alpha1->3 arm, monosialyl residue of the Man alpha1->6 arm, monosialyl residue of the Man alpha 1->3 arm and bisecting G1cNAc, and disialyl residues. BMA did not recognize Neu5Ac alpha2->6 and Neu5Ac alpha2->3 substitutions of Gal. These characteristics of BMA were shown to be useful in identifying malignancy-associated alteration of AFP sugar chains.

Concanavalin A (Con A) affinity electrophoresis of serum alpha-fetoprotein (AFP) can distinguish hepatocellular carcinoma (HCC) from other malignancies when the serum AFP concentration is elevated. However, Con A has not been able to distinguish HCC from benign chronic liver disease such as cirrhosis or chronic hepatitis.

The Con A affinity electrophoresis of serum AFP was analyzed in patients with a serum AFP concentration greater than 50 ng/mL by antibody affinity electrophoresis and Western blotting in an attempt to distinguish hepatocellular carcinoma from benign chronic liver disease. Before the assay, the serum AFP concentrations were adjusted between 100 ng/ml and 300 ng/ml by concentrating or diluting the samples.

Of 180 patients with HCC, 44 (24%) had a single band and 91 (51%), 35 (19%), and 10 (6%) had 2, 3, and 4 bands, respectively. All 35 patients with chronic hepatitis had a single band. All but 1 of 72 patients with cirrhosis had a single band. Multiple AFP bands on Con A affinity electrophoresis appear to be diagnostic of HCC. This method has a sensitivity of 76%, a specificity of 99%, a positive predictive value of 99%, and a negative predictive value of 71% for detecting HCC. The number of AFP bands correlated with serum AFP concentration and tumor size in patients with HCC.

This assay is useful for distinguishing HCC from benign chronic liver diseases.

Levels of two types of lectin-reactive alpha-fetoprotein (AFP), designated AFP-L3 and AFP-P4+P5, were analyzed with Lens culinaris agglutinin A and AFP-P4+P5 with erythroagglutinating phytohemagglutinin, respectively, in an attempt to determine the utility and significance of these macromolecules as early indicators of hepatocellular carcinoma during the periodic follow-up of cirrhotic patients. The subjects were 51 of 190 consecutive cirrhotic patients in whom hepatocellular carcinoma developed during a 6-year follow-up period and 21 cirrhotic patients without hepatocellular carcinoma. Serum AFP levels were of limited value to diagnose and predict hepatocellular carcinoma. The relative levels of AFP-L3 and AFP-P4+P5 in patients with hepatocellular carcinoma at the time of tumor detection were significantly higher than those in patients with cirrhosis. The sensitivity was 61%, and the specificity was 90%. Fourteen patients (48%) of 29 patients with small hepatocellular carcinomas less than 2 cm in diameter showed elevated percentage of lectin-reactive AFP. Retrospective examination of 21 patients who were positive for lectin-reactive AFP at diagnosis of hepatocellular carcinoma showed that 41% of them had already expressed lectin-reactive AFP 12 months before the direct detection of hepatocellular carcinoma by diagnostic imaging. These results lead us to conclude that the level of lectin-reactive AFP is a suitable predictive marker for the early recognition of hepatocellular carcinoma in the follow-up of patients with cirrhosis, and that measurements of the level of lectin-reactive AFP should be added to the screening methods that are now in use.

Renal cell carcinoma (RCC) producing alpha-fetoprotein (AFP) is a rare entity and merely 7 cases have been reported so far. The present case, a 71-year-old woman, showed a high serum AFP level of 204 ng/ml. The RCC of the autopsied right kidney consisted mainly of spindle-shaped or bizarre sarcomatous tumor cells. AFP was immunolocalized only in the concomitant clear cell component. Concanavalin A (Con A)-nonadsorption rate of serum AFP was 42%, which was an intermediate value between those of yolk sac tumors and metastatic liver carcinomas. Lens culinalis agglutinin (LCA)-affinity study of the patient's AFP showed an unknown peak X, which was eluted between the known peaks 2 and 3. These results suggest a certain structural alteration in carbohydrate moieties of the AFP derived from this RCC. A review of the clinicopathologic features of 8 patients with AFP-producing RCC was made to understand the pathophysiology of AFP-producing neoplasms.

Erythroagglutinating phytohemagglutinin (E-PHA)-dependent isoforms of human alpha-fetoprotein (AFP) from cord blood were analyzed for their carbohydrate structures by two-dimensional electrophoresis with E-PHA combined with extended agarose gel electrophoresis or with affinity electrophoresis with concanavalin A or Allomyrina dichtoma lectin. By means of neuraminidase and/or beta-galactosidase treatment, AFP-P2 was identified as alpha 2-->6 disialo-AFP, AFP-P3 as having biantennary structures with alpha 2-->6 monosialylated galactose of the Mannose (Man) alpha 1-->6 arm, AFP-P4 as having alpha 2-->6 monosialylated galactose of the Man alpha 1-->3 arm, and AFP-P5 as disialo-AFP with alpha 2-->3 sialylated galactose of the Man alpha 1-->6 antenna with the alpha 2-->6 sialylated galactose of the other antenna. Desialylated AFP with the terminal galactose of the Man alpha 1-->6 antenna with or without the galactose of the other arm also had a migration of AFP-P4, and other hydrolytic intermediates without the terminal galactose of the Man alpha 1-->6 arm with and without the galactose of the other antenna had mobilities of AFP-P3s and AFP-P3, respectively. Thus, the present system of two-dimensional lectin affinity electrophoreses would provide a model for the determination of the sugar chain structure of glycoproteins.

The sugar-chain structures of circulating alpha-fetoprotein in patients with hepatocellular carcinomas differ from those in patients with cirrhosis. We studied the reactivity of alpha-fetoprotein with two lectins, Lens culinaris agglutinin A and erythroagglutinating phytohemagglutinin, to monitor the evolution of hepatocellular carcinoma in patients with cirrhosis.

Among 361 patients with cirrhosis caused mainly by chronic hepatitis B or hepatitis C virus infection, 33 with base-line serum alpha-fetoprotein concentrations > or = 30 ng per milliliter or more were found to have hepatocellular carcinomas during a mean follow-up of 35 months. The lectin-reactive profiles of the alpha-fetoprotein in the serum of these 33 patients were analyzed and compared with those in the serum of 32 patients with cirrhosis who had increased base-line serum alpha-fetoprotein concentrations and were followed for at least 24 months but in whom hepatocellular carcinoma did not develop.

At the time of tumor detection, 24 (73 percent) of the 33 patients with cirrhosis and hepatocellular carcinoma had higher percentages of L. culinaris agglutinin A-reactive alpha-fetoprotein (alpha-fetoprotein L3), erythroagglutinating phytohemagglutinin-reactive alpha-fetoprotein (alpha-fetoprotein P4+P5), or both than the 32 patients with cirrhosis but no hepatocellular carcinoma. Among the 24 patients, one or both of the markers were first elevated 3 to 18 months before the hepatocellular carcinoma was detected by imaging techniques.

Measurements of the alpha-fetoprotein L3 and alpha-fetoprotein P4+P5 fractions of serum alpha-fetoprotein allow the differentiation of hepatocellular carcinoma from cirrhosis in some cases and serve as predictive markers for the development of hepatocellular carcinoma during the follow-up of patients with cirrhosis.

Diagnostic kits for determination of alpha-fetoprotein (AFP) carbohydrate chain microheterogeneity were developed using lectin affinity electrophoresis with Lens culinaris agglutinin-A (LCA-A) and erythro-agglutinating phytohemagglutinin-E4 (PHA-E4). Separated AFP bands by electrophoresis were detected with high sensitivity by antibody-affinity blotting and immunoenzymatic amplification. Densitometry was used to apportion lectin reactive AFPs. The within-run S.D. for proportions of AFP bands was below 3%. Band intensity was linearly related to AFP concentration between 2 and 200 ng/ml. Profiles of lectin reactive AFPs were compared in serum samples from 55 patients having liver diseases. The average values of lectin reactive AFPs for chronic hepatitis and liver cirrhosis patients were both below 13%, but those of hepatocellular carcinoma patients were above 25%. Correlation of data with disease states suggests that the methods can greatly facilitate the discrimination between benign and malignant liver diseases.

alpha-Fetoprotein (AFP) is widely used as a marker of hepatocellular carcinoma (HCC) for assisting diagnosis and also for screening purposes, even though its sensitivity has been decreased slightly as a result of the earlier detection of HCC by ultrasonography. Using lectin-dependent fractionation of AFP, the diagnostic sensitivity as well as the specificity of AFP can be increased compared with measurement of total AFP. Furthermore, lectin-reactive forms of AFP, AFP-L3 and AFP-P4, have been shown to serve as preclinical markers of HCC. Accordingly, AFP is still the most reliable marker of HCC in screening and monitoring high-risk patients.

The specificity and sensitivity of alpha-fetoprotein (AFP) binding to Concanavalin-A (Con-A) and Lens culinaris agglutinin (LCA) in 26 South African blacks with advanced symptomatic hepatocellular carcinoma (HCC) but only slightly raised serum AFP concentrations (20-500 ng/mL) was compared with that in patients with similar serum AFP levels from diseases that might be mistaken clinically for HCC (seven 'benign' liver disease [BLD] patients and six with metastatic liver disease [MLD] from gastrointestinal tumours). Con-A-Sepharose-4B affinity chromatography did not differentiate between the different groups: fucosylation rations for the HCC patients were 0.81 +/- 0.60, compared with 0.63 +/- 0.27 and 0.54 +/- 0.32 in patients with BLD and MLD, respectively. Electrophoresis of AFP serum and fraction in the presence or absence of Con-A and LCA showed an increase in the AFP C2 band. Rank correlation analysis of the AFP L2 and L3 bands combined could distinguish between patients with HCC and other hepatic diseases (P less than 0.05).

Principles and several modifications of lectin affinity electrophoresis are described. The results obtained using these newly developed techniques are reviewed for individual glycoproteins, the altered lectin reactivities of which have some clinical implications, showing different lectin reactivities, which occur not only on malignant transformation but also in association with inflammatory process and hormonal action.

Isoelectric focusing (IEF) of amniotic fluid alpha-fetoprotein (AFP) in thin-layer polyacrylamide gels containing 8 M urea followed by immunoblotting reveals at least nine bands, band I lying next to the cathode. Compared with 298 amniotic fluid samples from normal pregnancies, we found that the density of band V was increased in seven cases of fetal death. In 16 amniotic fluid samples from pregnancies with open neural tube defects (ONTD), band V disappeared or was markedly decreased. In seven cases with elevated AFP and positive acetylcholinesterase (AChE) due to contamination with fetal blood, no difference in pattern was observed compared with samples from normal pregnancies. It is suggested that IEF of AFP and subsequent immunoblotting are an apparently diagnostic test for ONTD and intrauterine fetal death (IUFD).

Counterflow isotachophoresis on cellulose acetate membranes of human alpha-fetoprotein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady-state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis. Smaller amounts of lectins and more diluted samples of AFP could be used in the described method.

Serum alpha-fetoprotein from 146 patients with hepatocellular carcinoma, other malignancies, and benign liver diseases, was fractionated by lectin-affinity electrophoresis coupled with our sensitive detection method of antibody-affinity blotting. Compared with chronic hepatitis and liver cirrhosis, hepatocellular carcinoma was characterized by the increase in proportions of lentil lectin A-reactive alpha-fetoprotein-L3 and erythroagglutinating phytohemagglutinin-reactive alpha-fetoprotein-P4; the yolk sac tumor was characterized by the increase of concanavalin A-nonreactive alpha-fetoprotein-C1, lentil lectin-A-weakly reactive alpha-fetoprotein-L2, erythroagglutinating phytohemagglutinin-strongly reactive alpha-fetoprotein-P5, and Allomyrina dichotoma lectin-nonreactive, slow-migrating alpha-fetoprotein-Als; and gastrointestinal tumors were characterized by alpha-fetoprotein-C1, alpha-fetoprotein-L2, alpha-fetoprotein-L3, alpha-fetoprotein-P5 and Allomyrina dichotoma-nonreactive alpha-fetoprotein-A1. By combined evaluation of alpha-fetoprotein-L3 and alpha-fetoprotein-P4, hepatocellular carcinoma was discriminated from chronic hepatitis and liver cirrhosis with a sensitivity of 97% at a specificity of 99.7%. Because the alpha-fetoprotein level of the studied cases ranged from 60-1,500,000 ng/mL (60-1,500,000 micrograms/L), mostly greater than 200 ng/mL (200 micrograms/L), additional patients with lower levels of alpha-fetoprotein [16-177 ng/mL (16-177 micrograms/L) for 16 cases of hepatocellular carcinoma with liver cirrhosis and 28-185 ng/mL (28-185 micrograms/L) for 17 cases of liver cirrhosis alone] were analyzed for alpha-fetoprotein-L3 and alpha-fetoprotein-P4. The resulting sensitivity for combined evaluation was still as high as 88% at the same high specificity of 99.7%, indicating that the simultaneous analysis of alpha-fetoprotein-L3 and alpha-fetoprotein-P4 is effective in monitoring the evolution of hepatocellular carcinoma in cirrhotic patients.

Purified alpha-fetoprotein (AFP), fucosylated AFP mixtures, and 40 sera from patients with AFP-producing hepatocellular carcinoma were analysed by monoclonal enzyme immunoassay (EIA) to distinguish fucosylated and nonfucosylated AFP molecular variants. FUC-AFP-25 was discriminated from FUC-AFP-75 by the EIA using monoclonal antibody 18H4 in the range of total AFP concentrations from 100 to 800 ng/mL. In addition, sera from 40 patients with hepatocellular carcinoma, with AFP concentrations from 100 to 1270 ng/mL and with fucosylated AFP from 0 to 100% by conventional cross immuno-affinoelectrophoresis, were also analysed by the present EIA. A statistically significant correlation was obtained between the data from the present EIA and from the conventional crossed immuno-affinoelectrophoresis in the range of fucosylated AFP more than 20% and the serum concentration of AFP more than 100 ng/mL. These results indicate that the present EIA is useful for clinical detection of hepatocellular carcinoma during the follow-up of patients with chronic liver diseases.