Analysis of serum alpha-fetoprotein (AFP) and AFP-L3 levels by protein microarray.

Objectives This study aimed to examine a simple, effective, time-saving, and low-cost protein microarray method for detecting serum alpha-fetoprotein (AFP) and AFP-L3 levels. Methods Serum samples from patients with hepatocellular carcinoma (HCC) (n = 33) and control subjects (n = 39) were collected and evaluated for the presence of AFP using a novel protein microarray. Glycoprotein (including AFP-L3) was enriched from crude samples by a Hotgen Biotech glycosyl capture spin column and then detected by protein microarray. An electrochemiluminescence immunoassay (ECLIA) was used to validate the measured values. Results Neither AFP levels lower than 20 ng/mL in the HCC group nor AFP levels higher than 20 ng/mL in the control group were found when tested by the ECLIA and protein microarray. The kappa test showed good consistency in the diagnostic performance of measuring serum AFP levels and the percentage of AFP-L3 in total AFP by the ECLIA and protein microarray. Protein microarray had advantages of smaller sample size required, low cost, and convenience compared with the ECLIA. Conclusion The protein microarray assay that was developed in the present study shows potential as an economic and convenient technique for detecting AFP and AFP-L3 levels in serum samples from patients with HCC.

[The killing effects of cytotoxic T lymphocytes stimulated by specific hAFP-derived peptides on hepatocellular cancer cells].

OBJECTIVES: To compare the in vitro killing effects of cytotoxic T lymphocytes (CTLs) induced by dendritic cells(DCs) that modified with 4 different specific hAFP-derived peptides. METHODS: DCs derived from normal human peripheral monocytes were activated by GM-CSF and IL-4. MTT assay was applied to analyse the proliferation activities of the DCs. To induce the specific CTLs, DCs modified with four immunodominant epitopes from alpha fetoprotein (AFP) were cocultured with MNC derived CD8+ lymphocytes. The acquired specific CTLs were cocultured with SMMC-7721 cells at different dilutions, and the killing effects were detected by flow-cytometry and Non-Radioactive Cytotoxity kit. RESULTS: The CTLs stimulated by DCs modified with the four immunodominant epitopes from alpha fetoprotein (AFP) had specific killing activities on the SMMC-7721 cells. And statistical differences of the killing effects existed between the hAFP(158-166) (FMNKFIYEI) group and the other three groups. CONCLUSION: CTLs induced by the DCs modified with the four immunodominant peptides had significant killing effects on the hepatocellular cancer cells in vitro. The hAFP(158-166) (FMNKFIYEI) peptide had a relatively higher efficiency in inducing DCs and stimulating specific CTLs.

Hierarchical role of fetuin-A and acidic serum proteins in the formation and stabilization of calcium phosphate particles.

The serum protein fetuin-A is a potent systemic inhibitor of soft tissue calcification. Fetuin-A is highly effective in the formation and stabilization of protein-mineral colloids, referred to as calciprotein particles (CPPs). These particles ripen in vitro in a two-step process, indicated by a morphological conversion from spheres to larger prolate ellipsoids. Using a combined light scattering and electron microscopic imaging approach we determined that the second-stage particles resulted from a highly anisotropic outgrowth of the first-stage particles. Electron microscopy of ascites fluid from a patient with calcifying peritonitis revealed particles reminiscent of secondary CPPs. Thus, CPPs form in the body and undergo the two-step ripening at least in pathological conditions. Unlike in vitro generated CPPs, ascites-derived CPPs contained little fetuin-A but large amounts of albumin. This prompted us to study the role of fetuin-A combined with other serum proteins in CPP formation. Fetuin-A was indispensable for primary CPP formation. Albumin and acidic proteins in general greatly enhanced the fetuin-A triggered formation of secondary CPPs and, thus, substituted substantial amounts of fetuin-A without loss of inhibition of calcium phosphate precipitation. Thus, direct mineral deposition from solute in the body is unlikely even at low fetuin-A serum levels as long as sufficient bulk acidic protein is available. Collectively fetuin-A and other acidic bulk plasma proteins may be considered as mineral chaperones mediating the stabilization, safe transport, and clearance in the body of calcium and phosphate as colloidal complexes, thus, preventing ectopic calcification.

Relationship between Lens culinaris agglutinin-reactive alpha-fetoprotein and pathologic features of hepatocellular carcinoma.

AIM: We investigated pathological features of Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3)-positive hepatocellular carcinoma (HCC) in order to seek a pathological basis of poor prognosis of HCC patients with elevated AFP-L3. METHODS: A total of 111 patients with HCC < or =5 cm in diameter who underwent hepatic resection were studied. Serum AFP-L3 concentration was measured within a month prior to surgery by lectin-affinity electrophoresis coupled with antibody-affinity blotting, and expressed as AFP-L3 percentage of total AFP. AFP-L3 of 10% or higher was judged to be positive. Pathologic features of resected HCC specimens were evaluated and classified concerning growth pattern (expansive or infiltrative growth), capsule formation, capsule infiltration, septal formation, portal vein invasion, hepatic vein invasion, bile duct invasion, and intrahepatic metastasis. These macroscopic and microscopic findings were compared between AFP-L3-positive and negative HCC specimens. RESULTS: Thirty-three (29.7%) were positive for AFP-L3. The prevalence of HCC with infiltrative growth, with capsule infiltration, with septum formation, with portal vein invasion, and with hepatic vein invasion was significantly higher in AFP-L3-positive group (P=0.0121, 0.0290, 0.0442, 0.0314, and 0.0433, respectively). These pathologic features reportedly indicate the progression of the tumor. CONCLUSIONS: AFP-L3-positive HCC had several pathologic features of progressed state of HCC, which accounted for the AFP-L3 as an indicator of poor prognosis of HCC.

Lectin-reactive alpha-fetoprotein as a marker for testicular tumor activity.

BACKGROUND: Lens culinaris agglutin (LCA)-affinity electrophoresis resolves serum alpha-fetoprotein (AFP) into three isoforms, AFP-L1, -L2 and -L3. The ratio of AFP-L3 to total AFP (AFP-L3%) is frequently high in hepatocellular carcinoma (HCC) patients, and thus, it is widely used for early diagnosis of HCC. In the present study, we used the subfraction profile of LCA-binding AFP to diagnose and monitor testicular tumor activity. METHODS: Serum samples were collected from 21 testicular tumor patients, and the LCA-reactive fractions were determined by LCA-affinity electrophoresis coupled with antibody-affinity blotting. The histological diagnosis was non-seminomatous germ cell tumor (NSGCT) in 15 patients and pure seminoma in six patients. RESULTS: Serum AFP levels were abnormally elevated (>20 ng/mL) in 10 of 15 NSGCT patients. One NSGCT patient and two seminoma patients showed borderline AFP levels between 10 and 20 ng/mL. LCA-reactive AFP was detected in all 11 NSGCT patients with serum AFP levels above 10 ng/mL, but not in the two seminoma patients with serum AFP levels above 10 ng/mL. In testicular tumor patients, the broad band of AFP-L2 could not be completely separated from AFP-L3. The mean ratio of AFP-L3 plus AFP-L2 (AFP-L2 + 3%) was as high as 94% (range 80-99%) in these patients. Serial determinations of LCA-reactive fractions were performed in eight of the 11 LCA-reactive AFP-positive patients. They included five patients who received chemotherapy, and three patients who underwent orchiectomy for stage I NSGCT. In three of eight patients, LCA-reactive AFP was detected even after normalization of total AFP levels. All three patients relapsed, with elevation of serum AFP within several months. CONCLUSION: Determination of LCA-reactive AFP might be a useful marker for testicular tumor activity in patients with lower AFP levels.

Endodermal sinus tumor of the ovary: review of 10 cases.

Endodermal sinus tumors are rare but highly malignant neoplasms of children and young adults. In all, only about 200 cases have been reported in the literature. Ten patients with endodermal sinus tumor of the ovary who were treated at Jackson Memorial Hospital from 1977 to 1989 were retrospectively reviewed. The median age of these patients was 24.7 years. The most common presenting symptom was abdominal pain, occurring in all our patients. Nine of the 10 patients received combination chemotherapy; all but one survived and are currently well. The patient who received no chemotherapy died. Eight of the ten patients are alive and well. The roles of alpha-fetoprotein monitoring, second-look laparotomy, and conservative surgery are discussed. The need for aggressive combination chemotherapy for this tumor is confirmed by this study.

Hepatoid adenocarcinoma of the renal pelvis producing alpha-fetoprotein of hepatic type and bile pigment.

A right renal pelvic mass in a 72-year-old man was resected. The histologic appearance of the tumor was a mixture of tubular adenocarcinoma cells and hepatoid neoplastic cells, and there was a resemblance to hepatoid adenocarcinoma. The intraoperative level of serum alpha-fetoprotein (AFP) was calculated to be 2246 ng/ml, and the postoperative level ranges from 183.6 to 285.6 ng/ml. Lectin binding assays showed that the serum AFP was the hepatic carcinoma type. In a hepatoid portion, an iron-negative, brown to green pigment was positive for bile. Alpha-fetoprotein was immunohistochemically evident in the neoplastic cells. In addition to the hepatic differentiation, the tumor had differentiated into intestinal absorptive or pancreatobiliary tract cells, as deduced from the frequent presence of spicular bodies, a unique light microscopic feature equivalent to microvilli with an actin core. The hepatoid adenocarcinoma is a distinct type of AFP-producing carcinoma present in the organs with epithelium of endodermal origin. Hepatoid adenocarcinoma in the renal pelvis may arise from a metaplasia of neoplastic mesonephric cells into endodermal cells.

Lectin affinity electrophoresis of alpha-fetoprotein in cancer diagnosis.

Lectin affinity electrophoresis of serum alpha-fetoprotein (AFP) was carried out on samples obtained from patients with benign and malignant diseases and on cord blood, and separated AFP bands were detected by antibody-affinity blotting. The following major bands were identified by determination of kinetic constants: AFP-C1 and -C2 with concanavalin A, AFP-L1, -L2 and -L3 with Lens culinaris agglutinin A, AFP-P1, -P2, -P3, -P4 and -P5 with erythroagglutinating phytohemagglutinin, and AFP-A1, -A2 and -A3 with Allomyrina dichotoma lectin. AFP bands with the lowest number had either low or no affinity and those with higher numbers had higher affinities for respective lectins. AFP from cord blood and chronic liver disease was characterized by the predominance of AFP-C2, AFP-L1, AFP-P2 and AFP-A3. Hepatocellular carcinoma was differentiated from the benign liver disease by increased proportions of AFP-L3 and AFP-P4. Extrahepatic tumors had additional increases of AFP-C1, AFP-L2, AFP-P5 and AFP-A1 (or slow-migrating AFP-Als, particularly in yolk sac tumor).

[Early determination of the effect of preoperative TAE by observation of serum AFP changes in patients with hepatocellular carcinoma].

Serum Alpha-fetoprotein (AFP) was assessed several times after preoperative TAE on 18 patients with hepatocellular carcinoma, and postoperatively the resected specimens were examined histopathologically. Changes in AFP level after TAE showed five typical patterns: AFP continued to rise in spite of treatment (non-effective type); AFP shows regular and equal increases and decreases (undulating type); AFP showed an increase after initially decreasing (increasing after decreasing type); AFP decreased gradually to normal levels (gradually decreasing type); and AFP decreased along a half-value period line to normal levels (half-value period type). These AFP patterns were intimately related to necrosis rate of tumor and other factors such as tumor size and existence of daughter tumors. Even when the AFP level decreased to 1/2 one week after TAE, the tumor showed only about 70% necrosis. Only when the AFP level fell into 1/4 of the original level after one week we could expect complete necrosis. Immuno-histological findings revealed AFP-producing cells in carcinoma but not in necrotic cells. It seems therefore, that the immuno-histological technique is uneffective in determining the viability of tumor cells.

Distinct molecular species of human alpha-fetoprotein due to differential affinities to lectins.

Resolution of human alpha-fetoprotein (AFP) into four distinct molecular species was demonstrated by a combination of two affinity chromatographies with crossed-immuno-affino-electrophoresis (CIAE) using concanavalin A (Con A) and Lens culinaris hemagglutinin (LcH)-A and LcH-B as affinity media. Of the four AFPs, AFP1 had no affinity for Con A, LcH-A, or LcH-B; AFP2 showed a high affinity for Con A, a low affinity for LcH-A, and an intermediate affinity for LcH-B (or a low affinity, depending on the lot of LcH-B preparations used); AFP3 revealed strong affinities for all of the three lectins; and AFP4, a trace component of hepatoma AFP in the present study, showed no interaction with Con A, but a definite interaction with LcH-A or LcH-B. These results were based on the determination of dissociation constants (Kd) of AFP-lectin complex by CIAE on isolated preparations of the three major hepatoma AFPs. These AFPs had identical electrophoretic mobilities of 0.86-0.87 (relative to human albumin) in the absence of lectins. The calculated mobilities of AFP2 and AFP3 were both reduced to 0.50-0.58 by saturation with lectins, but these two AFPs were clearly separated by 1 mg/ml LcH-A or LcH-B because of their large differences in Kd.

Four subfractions of amniotic fluid AFP identified by lentil lectin crossed-line affinity immunoelectrophoresis.

The subfraction of AFP was examined in 11 amniotic fluids at 43-121 days of gestation by lentil lectin crossed-line affinity immunoelectrophoresis. AFP in the amniotic fluid could be separated into four subfractions (type A, B, C, and D) according to reactivity with lentil lectin. Types A and C seem to be produced by the fetal liver, and types B and D by the yolk sac. However a part of type B may be synthesized in the fetal liver.