An immuno-electron microscopic study on intra and extracellular localization of alpha-fetoprotein in human hepatocellular carcinoma

Intra and extracellular localization of alpha-fetoprotein (AFP) has been studied by an indirect peroxidase labeled antibody method using 12 cases of human hepatocellular carcinoma (HCC). With light microscopic observation, positive immuno-staining for AFP was observed in 6 out of 12 cases and demonstrated as granular or diffuse deposits in the cytoplasm of neoplastic hepatocytes. In electron microscopic studies, 8 cases showed the positive immuno-staining for AFP in the neoplastic hepatocytes. Intracellular antigen was well circumscribed within certain cell organelles with the positive immuno-staining for AFP being observed in perinuclear space, cisternae of rough endoplasmic reticulum (RER), Golgi complexes, and secretory vesicles. In addition, the positive immuno-staining for AFP was observed in bile canaliculus-like space in most cases with increased levels serum AFP and in some cases which showed normal levels of serum AFP. Furthermore, the positive immuno-staining for AFP was also observed in intercellular, Disse's-like and sinusoid-like spaces, and micropinocytotic and lysosome-like vesicles in the endothelial cells in a few cases which showed excessively high value of serum AFP.     

Localization of ferritin in human liver diseases studied by immuno-histochemical and immuno-electron microscopic procedures

Localization of ferritin using a pre-embedding diffusion technique and an indirect localization sequence has been made in 34 cases of human liver under normal and several pathological conditions. With light microscopic observation, positive immuno-staining for ferritin was demonstrated as diffuse deposits in the hepatocytes and Kupffer cells. Intensity of the positive immuno-staining for ferritin in these cells appeared to roughly coincide with serum ferritin levels of each patient, but showed no disease specificity, although hepatoma cells contained weak deposits or were negative from immuno-staining for ferritin. With electron microscopic studies, intracellular antigen was well preserved in the hepatocytes and Kupffer cells in most cases with the positive immuno-staining for ferritin being observed in cytosol and a few cisternae of rough endoplasmic reticulum. Content of the positive immuno-staining for ferritin differed considerably from one case to another and one cell to another even in the same case. There was no immuno-staining for ferritin in hemosiderin pigment, lysosome, most of rough endoplasmic reticulum, Golgi complexes, and nucleus in both cells.     

LOCALIZATION OF FERRITIN IN HUMAN LIVER DISEASES STUDIED BY IMMUNO-HISTOCHEMICAL AND IMMUNO-ELECTRON MICROSCOPIC PROCEDURES

Localization of ferritin using a pre-embedding diffusion technique and an indirect localization sequence has been made in 34 cases of human liver under normal and several pathological conditions. With light microscopic observation, positive immuno-staining for ferritin was demonstrated as diffuse deposits in the hepatocytes and Kupffer cells. Intensity of the positive immuno-staining for ferritin in these cells appeared to roughly coincide with serum ferritin levels of each patient, but showed no disease specificity, although hepatoma cells contained weak deposits or were negative from immuno-staining for ferritin. With electron microscopic studies, intracellular antigen was well preserved in the hepatocytes and Kupffer cells in most cases with the positive immuno-staining for ferritin being observed in cytosol and a few cisternae of rough endoplasmic reticulum. Content of the positive immuno-staining for ferritin differed considerably from one case to another and one cell to another even in the same case. There was no immuno-staining for ferritin in hemosiderin pigment, lysosome, most of rough endoplasmic reticulum, Golgi complexes, and nucleus in both cells.     

大鼠肝大部切除与D-氨基半乳糖肝中毒后肝细胞增殖及甲胎蛋白和白蛋白免疫细胞化学研究

大鼠肝大部切除后和D-氨基半乳糖肝中毒后第1～7天,逐日取肝组织及血液样本,观察两种类型损伤后肝再生中肝细胞的增殖分化,以及血清甲胎蛋白(AFP)与白蛋白(ALB)浓度变化及其免疫细胞化学变化。结果表明:1.肝大部切除后肝细胞分裂高峰(1.5％)出现于术后第2天,半乳糖肝中毒组的高峰在第3天,且峰值仅为前者的一半(0.8％)。2.半乳糖肝中毒后,门管区和小叶周边带出现许多小型细胞,而大部切除后的肝内无此种小型细胞。3.AFP与ALB免疫细胞化学观察表明,两种损伤后肝再生中均有AFP阳性肝细胞,于第2～3天增多,第4天后渐少。部分小型细胞呈AFP阳性。电镜下见粗面内质网、滑面内质网、高尔基复合体及核周隙等处显示AFP阳性。ALB阳性细胞第1～3天较少,于第4天起渐增多。4.血清AFP含量于第1天开始升高,第4天达高峰,此后下降,ALB含量变化恰与AFP相反,第3天下降至最低点,此后回升。     

A human vitelline component in embryonal carcinoma of the testis

The correlation between increased serum alpha-fetoprotein (AFP) and a human vitelline component of 5 testicular embryonal carcinomas (1 was one histological type, 4 were more than one histological type) with no histological features specific for yolk sac tumor (YST) or endodermal sinus tumor (EST) was presented. In all 5 cases, distinct cells simulating the human yolk sac endodermal cell (HYSEC) with eosinophilic-granular or clear-vacuolated cytoplasm were found. These cells contained PAS positive, diastase resistant eosinophilic hyaline globules (EHG) which were positive for AFP. In the embryoid body, the same cells with positive AFP as the HYSEC were also seen. Electron microscopic investigation of these cells revealed basement membrane-like materials which were observed in organoid structure of YST. Positive AFP in these cells showed no binding to Concanavalin A (Con A), which was similar to AFP in YST. It was suggested that testicular embryonal carcinoma with higher serum AFP showing no histological features specific for YST had a vitelline component simulating the HYSEC and produced an increased serum AFP. Immunohistochemical staining for AFP of embryonal carcinoma with higher serum AFP is useful for detecting a human vitelline component and is important to guess the survival for the patient.     

A HUMAN VITELLINE COMPONENT IN EMBRYONAL CARCINOMA OF THE TESTIS

The correlation between increased serum alpha-fetoprotein (AFP) and a human vitelline component of 5 testicular embryonal carcinomas (1 was one histological type, 4 were more than one histological type) with no histological features specific for yolk sac tumor (YST) or endodermal sinus tumor (EST) was presented. In all 5 cases, distinct cells simulating the human yolk sac endodermal cell (HYSEC) with eosinophilic-granular or clear-vacuolated cytoplasm were found. These cells contained PAS positive, diastase resistant eosinophilic hyaline globules (EHG) which were positive for AFP. In the embryoid body, the same cells with positive AFP as the HYSEC were also seen. Electron microscopic investigation of these cells revealed basement membrane-like materials which were observed in organoid structure of YST. Positive AFP in these cells showed no binding to Concanavalin A (Con A), which was similar to AFP in YST. It was suggested that testicular embryonal carcinoma with higher serum AFP showing no histological features specific for YST had a vitelline component simulating the HYSEC and produced an increased serum AFP. Immunohistochemical staining for AFP of embryonal carcinoma with higher serum AFP is useful for detecting a human vitelline component and is important to guess the survival for the patient.     

Immunocytochemical identification of human chorionic gonadotropin- and alpha-fetoprotein-producing cells of hepatoblastoma associated with precocious puberty

Summary A one-year-five-month-old boy with hepatoblastoma producing both human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP) is presented. Histologically, the primary tumor was mainly composed of well differentiated hepatoblastoma cells, with minor areas of poorly differentiated cells. Immunoperoxidase staining of the tumor for hCG and AFP showed that a few well differentiated, fetal type cells and multinucleated giant cells were positive for hCG, and AFP was never stained in the same cells. In areas where cells were poorly differentiated, positive reactions for either hCG or AFP were not observed. Electron microscopic studies revealed focal aggregates of cytoplasmic cored vesicles in some tumor cells, similar to secretory granules.     

Experimental Hepatocarcinogenesis in Rat and Cellular Detection of Alpha1-fetoprotein by Use of Peroxidase Conjugates

Hepatomas were induced in rats by feeding low doses (6 mg/kg) or high doses (20 mg/kg) of N-nitrosomorpholine (NNM) for 12 and 6 weeks respectively. Necroses of adult hepatocytes and proliferation of oval-shaped cells occurred only at high doses of NNM. In parallel, early reappearance of alpha₁-fetoprotein (AFP) in sera was observed in rats which were fed high doses of NNM. At this time, AFP was detected in oval-shaped cells by means of immunoperoxidase staining techniques. Both NNM feeding schedules (low and high doses) resulted in development of hepatomas. At this stage of hepatocarcinogenesis, AFP staining nodules were seen concomitantly with non AFP staining nodules in the same animal. AFP staining cells were populations of distinct neoplastic hepatocytes with a certain degree of retrodifferentiation. Pulse-chase experiments have shown the highly proliferative character of carcinoma cells from which the AFP staining population was the most proliferating one.     

The Dynamics of Serum Alpha-fetoprotein in the Course of Testicular Teratoma

A retrospective study was carried out on a total of 279 cases of teratoma, out of which 135 were AFP positive, and 123 cases of seminoma. The AFP patterns were established by serial serum AFP assays carried out by radioimmunoassay at appropriately spaced time intervals. The following patterns could be distinguished: (1) Serum AFP levels normal throughout the whole course of the disease; (2) AFP levels consistently elevated irrespective of treatment; (3) normal levels for varying periods of time, then rising consistently; (4) elevated levels on presentation falling to normal for varying periods of time; (5) fluctuating with treatment, occasionally dropping to normal levels. Sequential AFP assays in patients with teratoma following orchidectomy, without evidence of residual tumour, suggest a half-life of circulating AFP of approximately five days. A study was made to correlate any subsequent recurrence or absence of AFP elevation with the actual level of the patient's T½, compared with that of a normal T½ of five days. Appropriate formulas can be used to determine the patient's serum AFP T½ or to calculate the expected AFP level assuming a standard T½ of five days. The results suggest that a prolonged T½ is usually associated with recurrent elevations of serum AFP.     

实验性肝癌癌组织的发生和甲胎蛋白表达

目的：研究实验性肝癌的组织发生过程中甲胎蛋白的表达,旨在为肝癌诊断奠定形态学基础。方法：Ｗｉｓｔａｒ大白鼠喂０．０４％３‘－Ｍｅ－ＤＡＢ不同日程处死,３６周处互完,观察动力形态变化和免疫组化ＡＦＰ表达。结果：实验早期卵圆细胞增生,该细胞是多向分化的干细胞。它进一步分化成过渡细胞,胚胎性小肝细胞,后者可呈不典型增生,它们散在或群集,这些细胞ＡＦＰ强阳性,群集不典型增生的小肝细胞形态上不同于肝细胞增生     

ras癌基因产物P21与肿瘤胚胎蛋白AFP、CEA在人肝癌中表达的对比研究

应用同一组织连续石蜡切片免疫组化染色技术，检测了ＡＦＰ、ＣＥＡ与Ｐ２１￣（ｒａｓ）在人肝细胞癌及其癌旁肝组织中的免疫定位关系，并探讨了ＡＦＰ与Ｐ２１￣（ｒａｓ）在肝癌辅助诊断中的意义。结果显示（１）ＡＦＰ及Ｐ２１￣（ｒａｓ）较ＣＥＡ与肝细胞癌的关系更为密切，半数以上肝癌可同时表达ＡＦＰ与Ｐ２１￣（ｒａｓ）两种抗原，且二者在组织中的阳性分布极相似；（２）癌组织ＡＦＰ的阳性率显著高于癌旁，而Ｐ２１￣（ｒａｓ）与之相反。由此可见，ＡＦＰ作为肝细胞癌相对特异的标志物用于肝癌的辅助诊断优于Ｐ２１￣（ｒａｓ）；癌旁同时表达ＡＦＰ与Ｐ２１￣（ｒａｓ）的肝细胞很可能是已经启动的、尚未产生表型转化的、已具备肿瘤性增殖能力的癌前细胞群。     

甲胎蛋白异质体L3对低浓度甲胎蛋白肝癌诊断的临床意义

目的探讨在低浓度甲胎蛋白（AFP）肝病患者中,甲胎蛋白异质体L3（AFP-L3）的百分含量（AFP—L3％）对肝癌的早期诊断和疗效评估的临床意义。方法收集245例血清低AFP含量（5～40ng／m1）的肝病患者样本（其中肝硬化患者100例、肝癌患者145例）,检测AFP—L3％,并对其中100例肝硬化患者和20例肝癌术后患者分别进行3个月和12个月的随访。结果以AFP—L3％≥10％作为阳性判断标准,100例肝硬化患者中阳性为23例,经3个月随访后其中8例诊断为肝癌;145份肝癌患者血清AFP-L3％阳性率为46．2％（67／145）。低浓度AFP肝癌组的AFP—L3％水平显著高于低AFP肝硬化组（t=7．318,P=0．001〈0．01）;20例肝癌患者术后有5例AFP．L3％仍为阳性,其在12个月内的生存率为0,而术后AFP—L3％阴性患者生存概率为15／15。结论AFP—L3％在低浓度AFP肝病患者中对肝癌的早期诊断和术后疗效评估都具有一定的临床意义。     

Studies on the mechanism of elevation of serum PIVKA-II levels in alcoholic liver cirrhosis

We measured serum PIVKA-II concentrations in 18 patients with alcoholic liver cirrhosis. Alcoholic liver disease was diagnosed by the history of ethanol intake of more than 900 ml/day for over 10 years. Liver cirrhosis was diagnosed histologically. Infections with hepatitis B and C viruses were ruled out by assaying serum virus markers. No tumor was detected in liver by ultrasonography and computed tomography during observation period. None of the patients studied were positive for alpafetoprotein (AFP). Eight out of 18 (44.4%) patients with alcoholic liver cirrhosis showed elevated serum PIVKA-II levels. In contrast, only eight out of 93 (8.6%) patients with nonalcholic liver cirrhosis had elevated serum PIVKA-II levels. PIVKA-II is well known as a tumor marker of hepatocellular carcinoma (HCC). The rates of positive PIVKA-II found in alcoholic liver cirrhosis approached its rates in HCC. However, the time course for the elevation of serum PIVKA-II levels was different each other in alcoholic liver cirrhosis and HCC. In HCC, serum PIVKA-II "levels" continued to elevate until therapy. In contrast, its elevation was transient and its levels returned to baseline in alcoholic liver cirrhosis. The values of ALT (GPT), gamma-GTP, and ALP correlated poorly with serum PIVKA-II levels in patients with alcoholic liver cirrhosis. To investigate the mechanism by which elevation of serum PIVKA-II levels in patients with alcoholic liver cirrhosis occurred, we studied the effect of vitamin K on production of PIVKA-II and AFP by hepatocytes. Hepatocytes(Alexander PLC/PRF/F cell line) were cultured in the presence of various concentrations of vitamin K (Kaytwo, Eisai, Tokyo). Vitamin K had no effect on AFP production. In contrast, PIVKA-II production was inhibited by addition of vitamin K in a dose dependent manner. Moreover, elevation of serum PIVKA-II levels in patients with alcoholic liver cirrhosis was suppressed by administration of vitamin K (Kaytwo) to these patients. Taken together, these results suggest that vitamin K may have a role in the mechanism of PIVKA-II elevation in sera of these patients. Then, we measured serum concentrations of vitamin K(PK, MK-4, MK-7) in these patients. There was no correlation observed between vitamin K and PIVKA-II in these patients. This result suggests that elevation of serum PIVKA-II in these patients may not be due to vitamin K deficiency. One question not answered here is how serum PIVKA-II levels in these patients are suppressed by treatment with vitamin K (Kaytwo). More detailed analysis of the mechanism of elevation of serum PIVKA-II levels in patients with alcoholic liver cirrhosis is needed.     

Immunohistological Studies of CEA, AFP and CPALP in Gastric Cancer

In studying 150 cases of gastric cancer, 93 (62%) were found to be CEA-positive. Among them, 5 cases had concurrent elevation of serum AFP over 200 ng/ml and appearance of serum CPALP. Further, there were another 2 cases simultaneously positive in serum CPALP. All these 7 cases were of pathologically well-differentiated adenocarcinoma with liver metastases.
Immunohistologically, CEA was observed on the surface of cancer cells as well as in glandular lumens of cancer tissues, forming a centroglandular pattern, while CPALP was detected in cytoplasm of cancer cells generally but absent in glandular lumens, and no formation of centroglandular pattern was observed. In contrast, most of the cancer cells were found AFP-negative except for some cancer cells with abundant chromatin in nucleus and acidophilic cytoplasm, in which AFP was detected in cytoplasm. Furthermore, AFP was also scarcely observed in cytoplasm of some normal liver transient cells scattered close to metastatic cancer lesions, suggesting that in gastric cancer, occurrences of CEA, AFP and CPALP originate from cancer cells while mild amounts of AFP are also being synthesized in some normal liver transient cells scattered close to metastatic cancer lesions.     

Serum alpha-foetoprotein as a marker for endodermal sinus tumour (yolk sac tumour) or a vitelline component of "teratocarcinoma".

The correlation between serum alpha-foetoprotein (AFP) and the clinical pathological finding of 24 germ cell tumours arising from the testes (14 cases), the ovaries (3 cases), the mediastinum (3 cases), the retroperitoneal region (2 cases), and the sacrococcygeal region (2 cases) are presented. Irrespective of marked differences in age and sex of the patients, primary site of the tumours and clinical outcome, the 24 cases constituted a homogeneous group in fundamental histological patterns and in AFP synthesis. In all cases of endodermal sinus tumour or teratocarcinomas with a distinct vitelline component an increased serum AFP concentrations was found in the pre-operative serum samples. AFP was also demonstrated in the tumour tissue by quantitative determination of AFP in tumour homogenate (5 cases) and, by immunofluorescence technique, positive staining of the cells lining the endodermal sinuses and of the hyaline globules was found (3 cases). In 12 germ cell tumours without vitelline components in the tumour tissue sections, a normal AFP concentration below 20 mug/1 was found in preoperative serum samples.     

Hepatocytic differentiation in retiform Sertoli-Leydig cell tumors: distinguishing a heterologous element from Leydig cells.

Sertoli-Leydig cell tumors (SLCT) of the ovary are rare sex cord-stromal neoplasms. A minority of SLCT are characterized by a pattern resembling that of the rete ovarii and frequently have a range of homologous and heterologous tissues. Approximately 20 cases of SLCT have been reported to have elevation of serum alpha-fetoprotein (AFP) levels, or tissue immunoreactivity for AFP, a protein usually associated with germ cell neoplasms, especially yolk sac tumor. We identified hepatocytic differentiation in five cases of retiform SLCT (RSLCT), and confirmed immunohistochemically that these cells are hepatocytes rather than Leydig cells. Hepatocytes are positive for keratins (AE1/3 and Cam 5.2), AFP, and ferritin, negative for vimentin, and show weak to moderate staining for inhibin. Leydig cells are negative for keratins, positive for vimentin, and intensely positive for inhibin. Immunohistochemistry is needed to distinguish hepatocytic differentiation from Leydig cells with certainty. Including the cases in this report, hepatocytic differentiation has been associated with a retiform pattern in SLCT in 14 of 25 cases (56%). The association of these two patterns appears to be characteristic of a relatively primitive sex cord-stromal neoplasm.     

Diagnostic and Follow-up Studies on CEA, AFP and ALP4 Isoenzyme in the Sera of Gynecological Malignancies

Simultaneous determination of CEA, AFP and ALP₄ was made in patients with various gynecological malignancies. CEA was positive in 65%, AFP was positive in 1.8% of 55 cases with cervical carcinoma stage 0—-II. ALP₄ was positive in one patient in these stages. In the group of cervical carcinoma stage III, stage IV and recurrence, CEA and AFP were positive in 100% and in 8.6% of 35 cases respectively. ALP₄ was positive in 14.3%. High levels and/or progressively increasing CEA with positive ALP₄ were found to be significant in poor prognostic patients in whom ALP₄ was not always found positive. Out of 17 cases with endometrial carcinoma, CEA was positive in 12 patients. AFP and ALP₄ were each positive in one case. Of 30 cases with ovarian carcinoma, CEA was positive in 70%, AFP in 23% and ALP₄ in 14%. Increasing CEA and/or increasing AFP appeared to be correlated with poor prognosis. In the group of 5 vulvar carcinoma, only CEA was positive in 80% of the patients. Of 19 cases of choriocarcinoma, CEA was positive in 11%, AFP was positive in 5.3% and ALP₄ was positive in 5.6%. There existed reverse-correlation between serum CEA and AFP in some cases.     

Oncofetal protein glypican-3 in testicular germ-cell tumor

The expression of an oncofetal protein, the glypican-3 (GPC3), was immunohistochemically evaluated in a wide variety of primary testicular germ-cell tumors (GCTs) in comparison with other markers, alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG)-beta, and OCT3/4. Eighty-nine cases of GCT including 22 cases of mixed GCT were evaluated with reference to each tumor component. GPC3 expression was observed in neoplastic cells of yolk-sac tumor (YST) (25/25), teratoma (2/10), components of syncytiotrophoblastic giant cells (STGCs) (10/14), and choriocarcinoma (1/3), but none in intratubular germ-cell neoplasias, unclassified type (0/33), seminomas (0/61), or embryonal carcinoma (0/19). All cases of YST showed diffuse labeling of neoplastic cells in cytoplasmic and membranous patterns, and the positive area of GPC3 was much larger than that of AFP. Glandular structures in teratomas showed GPC3 immunostaining as well as AFP. Although the number of GPC3-positive cells was smaller in STGC components and choriocarcinoma, there was no diffusion artifact in GPC3 immunostaining, as was frequently encountered in hCG-beta staining. Thus, GPC3 is a unique oncofetal protein, which is useful as an immunohistochemical marker for GCT differentiated to extraembryonic tissue, especially YST.     

Immunohistochemical and Ultrastructural Analysis of Bronchopulmonary Neuroendocrine Neoplasms. I. Carcinoids

Twenty-five strictly defined bronchopulmonary carcinoids were studied by light microscopic immunohistochemistry by the peroxidase technique for NSE (neuronspecific enolase), serotonin, and a broad spectrum of neuropeptides. Eighteen cases were also studied by electron microscopy.

Twenty-three of the twenty-five cases showed immunostaining for NSE; 24 cases displayed immuno-staining for at least two of the hormones tested for; the single case that failed to show hormonal immuno-reactivity was however positive for NSE and had granules by electron microscopy. Serotonin was the most frequently demonstrated hormone followed by bombesin, vasoactive intestinal peptide, gastrin, leuenkephalin, alphamelanocyte stimulating hormone, somatostatin, substance P, and calcitonin. In several cases, adjacent-step sections stained for different hormones strongly indicated immunoreactivity for more than one hormone in single neoplastic cells.

By electron microscopy, all 18 cases studied showed generally abundant neurosecretory granules, which, however, displayed considerable heterogeneity in their size, shape, and density. Twelve of these eighteen cases displayed evidence of squamous differentiation and 10 showed characteristic exocrine lumina.

The capability of single neuroendocrine tumors and single neuroendocrine tumor cells to produce more than one immunoreactive hormone is hereby amply confirmed; these broad capabilities are certainly reflected in the heterogeneous granule populations seen by electron microscopy. The synchronous presence of squamous and exocrine features in bronchopulmonary carcinoids indicates that they too are capable of multidirectional differentiation, which should not detract from their being regarded basically as well-differentiated neuroendocrine neoplasms. The clinical significance of strictly defining bronchial carcinoids is underscored by the fact that of 25 cases followed for 2-13 years, only one developed metastases 9 years postoperatively.     

Distribution of albumin- and/or alpha-fetoprotein-positive cells in hepatocellular carcinoma

Albumin (ALB)-positive cells were identified by an immunoperoxidase technique in 52 of 53 autopsy cases and in all of 13 surgical cases of hepatocellular carcinoma (HCC). The distribution pattern of ALB-positive cells could be classified into three groups: diffuse, localized, and sparse. The diffuse type was the most common pattern and was usually seen in well or moderately differentiated HCC, showing a trabecular growth pattern. The localized or sparse patterns were more frequent in poorly differentiated HCC showing a compact growth pattern. alpha-Fetoprotein (AFP)-positive cells were detected in 37 of the 53 autopsy cases of HCC and 11 of the 13 surgical cases. The number of AFP-positive cells and the intensity of the immunoperoxidase reaction were roughly proportional to serum AFP levels in most cases. In most regions of HCC, there seemed to be an inverse relationship between the number of ALB- and AFP-positive cells, suggesting tht most HCC cells synthesized only one of the two antigens studied. ALB-positive hepatocytes were found in all of the normal or cirrhotic livers examined and in the tumor-free regions of the HCC-containing livers. In contrast, AFP was not detected in nonneoplastic hepatocytes.