High Alpha-fetoprotein Concentrations Research Articles

Comprehensive clinicopathologic study of alpha fetoprotein-expression in a large cohort of patients with hepatocellular carcinoma.

Alpha fetoprotein (AFP) is the most widely used diagnostic and prognostic serum biomarker for hepatocellular carcinoma (HCC). Despite its wide clinical use, a systematic clinicopathologic study comparing AFP expression in HCC in situ with serum AFP concentrations has not yet been conducted. To analyze AFP expression in a large cohort of patients by immunohistochemistry, we employed a comprehensive tissue microarray with 871 different HCCs of overall 561 patients. AFP immunoreactivity was detected in only about 20% of HCC core biopsies, whereas 48.9% of the patients displayed increased serum values (>12 ng/mL). Immunostaining of whole tumor slides revealed that lack of detectable immunoreactivity in core biopsies in a subgroup of patients with elevated AFP serum concentrations is due to heterogeneous intratumoral AFP expression. Serum AFP concentrations and AFP expression in situ were moderately correlated (Spearman's rank correlation coefficient .53, P=1.2e - 13). High AFP expression detected in serum (>227.3ng/mL) or in situ predicted unfavorable prognosis and was associated with vascular invasion, higher tumor grade and macrotrabecular-massive tumor subtype. Multivariate and ROC curve analysis demonstrated that high AFP concentrations in serum is an independent prognostic parameter and represents the more robust prognostic predictor in comparison to AFP immunostaining of core biopsies. The previously published vessels encapsulating tumor clusters (VETC) pattern turned out as an additional, statistically independent prognostic parameter. AFP-positivity was associated with increased tumor cell apoptosis, but not with increased vascular densities. Additionally, AFP-positive tumors displayed increased proliferation rates, urea cycle dysregulation and signs of genomic instability, which may constitute the basis for their increased aggressiveness.

Biochemical Markers for Prediction of Hypertensive Disorders of Pregnancy.

SummaryBackgroundGestational hypertension (GH) and pre eclampsia (PE) are the most common gestational complications. Several placental biochemical markers are used to predict GH/PE, but with conflicting results.MethodsThe study aim was to estimate the biochemical markers’ ability to predict hypertensive disorders in pregnancy. On the first ultrasonographic examination, 104 healthy pregnant women were recruited. At the regular pregnancy check-ups, BMI, blood pressure, occurrence of gestational hypertension (early or late onset), preeclampsia, eclampsia and other complications were recorded. Serum concentrations (in multiples of median – MoM) of human chorionic gonadotropin (HCG) and pregnancyassociated plasma protein A (PAPPA) were measured from the 11th to 14th gestational week, while HCG, alpha feto protein (AFP), estriol and inhibin were determined between the 16th and 19th gestational week.ResultsHypertensive disorders throughout pregnancy were diagnosed in 20.2% women. Early-onset GH was registered in 7 and PE in 6 patients, while 14 had late-onset GH and 10 additional women PE. There were no significant differences (p≥0.05) in biochemical markers concentrations between women with and without GH/PE. PAPPA levels in the first and HCG in the second trimester correlated with early and late GH/PE. Moreover, higher AFP concentrations were registered in women with preeclampsia signs/symptoms. According to ROC analysis, AFP>1.05 MoM properly identified 80% of GH/PE cases. Obtained models imply that HCG, PAPPA and AFP should be used for GH/PE prediction.ConclusionsBiochemical markers HCG, PAPPA and AFP could be useful in predicting gestational hypertension and preeclampsia. However, different markers should be used for early and late onset GH/PE.

Biochemical Markers for Prediction of Hypertensive Disorders of Pregnancy

SummaryBackground: Gestational hypertension (GH) and pre eclam p - sia (PE) are the most common gestational complications. Several placental biochemical markers are used to predict GH/PE, but with conflicting results. Methods: The study aim was to estimate the biochemical markers’ ability to predict hypertensive disorders in pregnancy. On the first ultrasonographic examination, 104 healthy preg nant women were recruited. At the regular pregnancy check-ups, BMI, blood pressure, occurrence of gestational hypertension (early or late onset), preeclampsia, eclampsia and other complications were recorded. Serum concentrations (in multiples of median - MoM) of human chorionic gonadotropin (HCG) and pregnancyassociated plasma protein A (PAPPA) were measured from the 11th to 14th gestational week, while HCG, alpha feto protein (AFP), estriol and inhibin were determined between the 16th and 19th gestational week. Results: Hypertensive disorders throughout pregnancy were diagnosed in 20.2% women. Early-onset GH was registered in 7 and PE in 6 patients, while 14 had late-onset GH and 10 additional women PE. There were no significant differences (p≥0.05) in biochemical markers concentrations between women with and without GH/PE. PAPPA levels in the first and HCG in the second trimester correlated with early and late GH/PE. Moreover, higher AFP concentra tions were registered in women with preeclampsia signs/symptoms. According to ROC analysis, AFP>1.05 MoM properly identified 80% of GH/PE cases. Obtained models imply that HCG, PAPPA and AFP should be used for GH/PE prediction. Conclusion: Biochemical markers HCG, PAPPA and AFP could be useful in predicting gestational hypertension and preeclampsia. However, different markers should be used for early and late onset GH/PE.

Surgical Outcomes for the Spontaneous Rupture of Hepatocellular Carcinoma: Multicenter Analysis with a Case-Controlled Study with Propensity Score Matching

Introduction: A spontaneously ruptured hepatocellular carcinoma (HCC) has a poor prognosis; however, the true impact of a rupture on survival after hepatic resection has not been clarified. Methods: Fifty-eight patients with ruptured HCC and 1922 patients with non-ruptured HCC underwent hepatic resection between 2000 and 2013. To correct the difference in clinicopathological factors between the two groups, propensity score matching (PSM) was used at a 1:1 ratio, which resulted in a comparison of 43 patients/group. We investigated surgical outcomes in all patients with ruptured HCC and compared surgical outcomes between the two matched groups. Results: Of the 58 patients with ruptured HCC, 41 (71%) underwent transcatheter arterial embolization for hemostasis, leading to hemostasis in 40 patients (98%), while 5 underwent an emergency hepatic resection. Seven patients (13%) died postoperatively. Overall survival (OS) rate at 5/10 year after hepatic resection was 37%/34%. Emergency hepatic resection was found to be an independent risk factor for inhospital death [p = 0.026, odds ratio 12.4, 95% confidence intervals (CI) 1.3-113.2] and Child-Pugh class B for unfavorable OS [p = 0.035, hazard ratio 2.80, 95% confidence intervals (CI) 1.1-7.3] by multivariate analysis. Among all populations, incidence of in-hospital death was higher in patients with ruptured HCC than in those with non-ruptured HCC (13% vs 2%, p < 0.001). Moreover, patients with the ruptured HCC was more frequently observed in patients with high alpha-fetoprotein (AFP) concentration, large tumor, and poorly differentiated HCC than those with non-ruptured HCC, leading to unfavorable OS (5/10 year; 37%/34% vs 65%/42%, p < 0.001). After PSM, clinicopathological variables were well-balanced between the two groups. There was no significantly difference in incidence of in-hospital death (ruptured HCC 12% vs non-ruptured HCC 2%, p = 0.202), and OS rate (5/10 year; 42%/38% vs 67%/30%, p = 0.115). Conclusion: The ruptures were more frequently observed in patients with a high AFP concentration, large tumor, and poorly differentiated HCC, indicating a ruptured HCC would have tendency to associate an advanced grade of malignancy. However, PSM indicated rupture itself was not a risk for surgical outcomes. Moreover, clinicians should be careful when selecting emergency hepatic resection, and must keep in mind that prognosis of patients with Child-Pugh class B is poor.

Evaluation of MAGE‐D4 expression in hepatocellular carcinoma in Japanese patients

Though Melanoma-associated antigen (MAGE) family genes have received lots of attention as cancer-related genes and targets for immunotherapy, MAGE-D4 expression in hepatocellular carcinoma (HCC) has not yet been evaluated. MAGE-D4 mRNA expression was assayed in nine HCC cell lines and 94 HCC surgical specimens obtained from Japanese patients by quantitative real-time reverse transcription polymerase chain reaction, and the correlations between MAGE-D4 mRNA expression and clinicopathological factors were evaluated. The expression and distribution of MAGE-D4b protein were evaluated immunohistochemically. MAGE-D4 mRNA was overexpressed in five of nine HCC cell lines and 34 of 94 primary HCCs (36.2%). Median overall survival (14.8 vs. 118 months, P < 0.001) and relapse-free survival (2.7 vs. 18.3 months, P < 0.001) were significantly shorter in patients with high than with low-moderate MAGE-D4 expression. Multivariate analysis for overall survival showed that MAGE-D4 overexpression was independently prognostic for survival (hazard ratio 2.88, P = 0.009) and significantly associated with high alpha-fetoprotein concentration (P < 0.001), poor tumor differentiation (P = 0.003) and vascular invasion (P = 0.021). MAGE-D4b protein expression patterns were consistent with those of MAGE-D4 mRNA. Overexpression of MAGE-D4 may be a predictive marker of early recurrence and mortality in patients with HCC.

Improvement and Multicenter Evaluation of the Analytical Performance of an Automated Chemiluminescent Immunoassay for Alpha Fetoprotein

A new ARCHITECT® alpha fetoprotein (AFP) assay was developed to improve the linearity at the upper end of the calibration curve and to enhance other performance characteristics. In addition, this reformulation eliminated the possibility of falsely depressed samples at high AFP concentrations. The purpose of this study was to evaluate its analytical performance at multiple sites. The assay configuration, the diluent formulation, and the manufacturing process were redesigned. Analytical performance was evaluated at Abbott Laboratories, Sapporo Medical University, VU University Medical Center, and Johns Hopkins University. The limit of quantitation of the assay was 1.00-1.30 ng/mL. Total precision (%CV) across the assay range varied between 1.41 and 3.52. The assay was linear from 1.19 to 2535 ng/mL, and the range of the assay was expanded from 200 ng/mL to 2000 ng/mL. Comparison of this assay with the on-market ARCHITECT, AxSYM, ADVIA Centaur, DxI, AIA-1800, and E 170 systems yielded regression slopes of 0.91-1.08 and correlation coefficients of =0.99 for serum samples. No falsely depressed results were observed in 174 serum samples with AFP concentrations of 2018-1,196,856 ng/mL and in a spiked sample containing up to 10 mg/mL of purified AFP. The new AFP assay has improved an issue of the on-market ARCHITECT AFP assay and demonstrated excellent assay performance.

The hook effect: a need for constant vigilance

The hook effect is a well-recognized problem that can occur in assays of most tumour markers, including alpha feto-protein (AFP). We present a case of hepatoblastoma in a baby. The diagnosis was delayed as a result of unrecognized 'hooking' of a very high AFP concentration in the automated immunoassay method used. The falsely low result obtained was considered normal for the patient's age and supported the diagnosis of benign haemangioendothelioma. Liaison between clinical and laboratory staff was critical in obtaining an accurate AFP result, proceeding to liver biopsy and establishing the definitive diagnosis of hepatoblastoma. While the reasons and solutions for hook effect have been well researched and published, we believe the presence of extremely high serum AFP concentration in some hepatoblastoma patients means that the hook effect remains a problem and can generate erroneously low AFP results despite assay reformulation by manufacturers. Therefore constant vigilance by laboratory staff is still needed.

Immunoregulation of dendritic and T cells by alpha-fetoprotein in patients with hepatocellular carcinoma

Novel immunotherapeutic and other strategies are being explored for the treatment of hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) may be a target antigen for immunotherapy. Little is known, however, about the immunobiology of AFP. Therefore, the impact of AFP on dendritic cells (DC), CD4+ and CD8+ T cells was studied in detail. Immune cells from peripheral blood of 27 HCC patients were studied using FACS, ELISPOT, and proliferation assays. The in vitro generation, maturation, and T cell stimulatory capacity of DCs were not altered by AFP up to concentrations of 20 microg/ml. Higher AFP concentrations (> 20 microg/ml) resulted in phenotypic changes on DCs without impairing their capacity to stimulate CD4+ T cells. Frequencies and function of DCs and AFP specific T cells were not reduced in HCC patients independent on serum AFP levels. Finally, T lymphocytic infiltrations in the liver were not dependent on AFP serum levels. These studies clearly demonstrate that (i) DC-based immunotherapeutic approaches are a promising approach for HCC treatment and (ii) AFP-reactive T cell clones have not been deleted from the human T cell repertoire establishing AFP as a potential target for T cell based immunotherapy of HCC.

Induction of apoptosis in human hepatoma cells by alpha-fetoprotein.

We have investigated the effects of purified human alpha-fetoprotein (AFP) on the growth of the human hepatocarcinoma-cells HepG2 in culture. Cancer-derived AFP (cAFP), isolated from the culture medium of AFP-secreting HepG2 cells and embryonal AFP (eAFP), isolated from human cord serum, were used for these studies. Both AFP pre parations studied were shown to induce strong dose-dependent inhibition of HepG2 cell proliferation and complete growth arrest at high protein concentrations (more than 0.1 mg/ml). To test whether AFP may trigger an endogenous suicide program in hepatoma cells, we examined whether DNA fragmentation preceded cell death. After exposure of the cells of the high AFP dose (1.0 mg/ml), DNA fragmentation was detected as early as 2 h after treatment, and 70% of cells were apoptotic by 24 h. DNA fragmentation was shown to precede other signs of cell death for several hours. Typical morphological changes of apoptosis were observed after 4 h of exposure of cells to high AFP doses. Low concentrations of cAFP and eAFP (less than 0.1 mg/ml) failed to induce growth inhibition of HepG2 cells, rather showing a weak stimulative effect, demonstrating a biphasic AFP activity. Cell pretreatment with the transcriptional inhibitor actinomycin D had no measurable influence on AFP cytotoxicity. These findings demonstrate that protein synthesis is not required for this mechanism of cell death. The charcoal-treated ligand-free eAFP (eAFPp) had a dose-dependent growth-inhibitory activity, similar to intact protein, but slightly less intensive. The similar growth-inhibitory activities of cAFP, eAFP and eAFPp, which have a significant difference in bound-ligand content, provide evidence that the main role in cell growth regulation may be attributed to the protein moiety of the entire AFP molecule, but not to its ligands. These biologically active AFP ligands could, however, modulate AFP-growth-regulating activity. Growth factor deprivation distinctly enhanced the cytostatic activity of high AFP concentrations and also increased the mitogenic activity of low AFP levels, showing the interdependence of the growth-regulative activity of AFP and growth factors. The findings of this study demonstrated that AFP is directly introduced into the intracellular pathways of cell growth regulation and programmed cell death.

PRENATAL SCREENING FOR CONGENITAL NEPHROSIS IN EAST FINLAND: RESULTS AND IMPACT ON THE BIRTH PREVALENCE OF THE DISEASE

Congenital nephrosis of the Finnish type (CNF) is inherited as an autosomal recessive trait which maps to the long arm of chromosome 19. The disease causes massive proteinuria, and renal transplantation in early neonatal life is the only effective treatment. Prenatal diagnosis, usually in high-risk families, depends on the analysis of alpha-fetoprotein (AFP) levels in maternal serum (MS) or amniotic fluid (AF). We studied the effectiveness of MSAFP measurement as a method of screening all pregnant women for congenital nephrosis. Between 1 January 1979 and 31 December 1992, all pregnant women (N = 110,858) attending maternity care units in East Finland were offered serum AFP measurement as part of a screening programme. All patients whose MSAFP value was > or = 2.5 multiples of the median (MOM) at 15-18 weeks' gestation were given an ultrasound examination and if no morphological abnormality was found, they were then offered amniocentesis. Altogether, 105,880 pregnant women (96 per cent) in East Finland participated in the screening for CNF. A total of 47 cases of CNF were diagnosed during the study period. Elevated AFP concentrations in maternal serum and amniotic fluid (> or = 2.5 MOM) were found in all screened (44/105,880) affected pregnancies except one. The most typical feature of a CNF pregnancy was a very high AFP concentration in the amniotic fluid. As a result of the screening, the prevalence at birth of CNF decreased from 1:2600 to 1:11,086. The possibility of CNF has to be taken into account in pregnancies with a ¿false-positive' elevated AFP result (normal ultrasound and no detectable acetyl cholinesterase).

‘Idiopathic’ jaundice in Sardinian full‐term newborn infants: a multivariate study

'Idiopathic' hyperbilirubinaemia in the first 4 days of life was studied in 431 unselected healthy full-term (gestational age > or = 37 weeks) singleton Sardinian infants with birthweight > or = 2500 g. All infants were free from malformations or any disease requiring treatment other than jaundice, they were ABO and Rh compatible with their mothers and were not G6PD deficient. The serum bilirubin level was > 11.9 mg/dl (204 mumol/l) and > 14.9 mg/dl (256 mumol/l) in 37.1% and 15.3% of the study subjects. The vast majority of the infants (94%) were breast fed; no difference in the incidence of jaundice was found between breast-fed and bottle-fed infants. A logistic regression analysis indicated that high alpha-fetoprotein concentrations in cord blood, history of neonatal jaundice in previous full-term siblings, delayed first meconium passage and weight loss were associated with jaundice, defined as a serum bilirubin level > 11.9 mg/dl. These results suggest that the high rate of neonatal hyperbilirubinaemia in Sardinia is mostly related to constitutional and possibly hereditary factors.

New screening system for simultaneous determination of two marker proteins by homogeneous enzyme immunoassay.

To save time and labour in mass screening, by detecting two marker proteins on one specimen using only one test. alpha Fetoprotein and ferritin were chosen to demonstrate the principal of this system. The assay reagents were horseradish peroxidase (HRP)-labelled anti-alpha fetoprotein and HRP-labelled anti-ferritin antibodies. After the serum sample had been incubated with these reagents the substrate for HRP was added and the absorbance measured. An absorbance value below the cutoff point indicated that both parameters were within normal limits; a value above the cutoff point indicated that at least one of the two parameters was abnormally high. Fifty sera from healthy Japanese subjects were assayed by the simultaneous assay method. All samples gave absorbancy values below the cutoff point. Fifty serum samples from patients with high alpha fetoprotein concentrations (over 20 ng/ml) and 50 samples with high ferritin concentrations (over 200 ng/ml) were also assayed. The absorbancy values of all samples with high alpha fetoprotein concentrations, and all but one sample with high ferritin concentrations gave values above the cutoff point. Although this homogeneous enzyme assay method was applied to the combination of alpha fetoprotein and ferritin, it could be used in mass screening for any other combination of two markers.

Binding of 16α-[ 18F]Fluoro-17β-estradiol to alphafetoprotein in Sprague-Dawley female rats affects blood levels

To examine the relationship between blood levels of 16α-[ 18F]fluoro-17β-estradiol( 18F-ES) and serum alphafetoprotein (AFP) concentration, we undertook a study in which serum from various aged (20–33 days old) Sprague-Dawley female rats injected with 18F-ES was analyzed for both blood activity levels and AFP. There is a strong positive correlation between serum AFP concentration and 18F-ES blood levels ( r = 0.914, P < 0.001), suggesting that the binding of 18F-ES by AFP has a significant effect on blood activity levels. The AFP concentration and ultimately the AFP- 18F-ES binding is dependent on the age and weight of the rat: younger, as well as low weight rats exhibited high AFP concentrations and consequently increased 18F-ES blood activity. The rats most suitable for comparative studying of labeled estrogens are 25–28 days of age and weigh a minimum of 50–55 g. Thus, the use of the immature rat model to compare labeled estrogens requires a careful consideration of possible interference from blood binding proteins (i.e. AFP), as well as potential receptor binding competition from endogenous estrogens produced during the estrous cycle. Comparable consideration of blood binding protens (sex steroid binding protein, SBP) and endogenous estrogens must be made in human studies, as well.

A pitfall in two-site sandwich ‘one-step’ immunoassay with monoclonal antibodies for the determination of human alpha-fetoprotein

Utilizing monoclonal antibodies directed to 2 distinct antigenic determinants of the human alpha-fetoprotein (AFP), Uotila, Ruoslahti and Engvall developed the 2-site sandwich immunoassay. They found that due to the different specificities of monoclonal antibodies, 2 antigen-antibody reactions, AFP with immobilized antibody and AFP with labeled antibody, could be accomplished in a single step. We have found, however, that above a certain concentration, AFP detectable by their method decreased because the labeled antibody tended to bind with AFP that failed to react with the immobilized antibody. Consequently, 2 different AFP concentrations produced the same result, and an extremely high, still clinically expectable concentration gave a false negative result. Such an inhibition in high AFP concentrations was not observed in the conventional ‘2-step’ immunoassay within the range of concentrations tested (0.1 ng-3 mg/ml). On the basis of these observations, the 2-site ‘1-step’, immunoassay for AFP would have to be applied on multiple dilutions of the serum to avoid an erroneous interpretation of the results.

The potential of mid-trimester maternal plasma alpha-fetoprotein measurement in predicting infants of low birth weight.

Maternal plasma alpha-fetoprotein (AFP) was measured at mid-trimester in 113 women who subsequently were delivered of a singleton liveborn infant weighing less than 2.5 kg and in 113 matched controls whose infants weighed more than 2.5 kg. Plasma AFP levels were significantly higher in the subjects than in the controls, while 10 of the subjects and only 3 of the controls had values above 2.0 times the median. The higher AFP concentrations were seen both in pregnancies in which the outcome was a small premature infant and in those in which it was a small-for-dates baby.

Serum alpha-fetoprotein levels in extrahepatic biliary atresia, idiopathic neonatal hepatitis and alpha-1-antitrypsin deficiency (PiZ).

Serum alpha-fetoprotein levels were measured using a sensitive radioimmunoassay in 77 infants presenting with persistent conjugated hyperbilirubinaemia. A breed range of alpha-fetoprotein concentrations occurred in both the 23 infants with extrahepatic biliary atresia and the 35 with idiopathic neonatal hepatitis but the 13 with alpha-1-antitrypsin deficiency had uniformly low levels. High alpha-fetoprotein concentrations (above 10 000 mug/1) favoured the diagnosis of neonatal hepatitis especially in the first ten weeks of life, but the overlap between neonatal hepatitis and extrahepatic biliary atresia was large and alpha-fetoprotein determination cannot be recommended as a reliable method for distinguishing the two conditions. Serial alpha-fetoprotein values showed no consistent relationship with standard liver function tests and gave no guide to prognosis. There was an association between alpha-fetoprotein production and needle biopsy evidence of hepatic giant cell transformation. The uniformly low alpha-fetoprotein levels in alpha-1-antitrypsin deficient infants with neonatal hepatitis is a new observation and possible mechanisms for disordered glycoprotein release are discussed.

Elevated amniotic fluid alpha fetoprotein in fetal hydrocephaly

This report associates fetal hydrocephaly with high-for-dates alpha fetoprotein (AFP) values in amniotic fluid (AF) in 4 cases. All 4 hydrocephalic pregnancies showed abnormally high AF AFP concentrations (440 1800 7300 and 4750 vs. normal range of 15-340 ng/ml). Cerebrospinal fluid (CSF) was obtained from 3 fetuses. 2 contained a high AFP concentration (6400 and 148000 ng/ml); in both cases AF AFP was greatly elevated (7300 and 1800 ng/ml). In the 3rd case the moderately elevated AF AFP (440) was associated with a similar level (345 ng/ml) in fetal CSF. The total protein concentration was abnormally high in all CSF samples but no correlation existed between the total protein and AFP in either CSF or AF. AFP in maternal and fetal sera were within normal pregnancy range. The AFP level of newborn urine (23 ng/ml) was similar to previously measured normal infant urine values. The significance of high AFP on the prognosis of these infants cannot be indicated by these results because all infants died at or shortly after delivery. Prospective studies aimed at early antenatal detection of hydrocephaly by AF AFP are warranted.