The role of immunohistochemistry in histopathological diagnostics of clinically "silent" incidentally detected adrenal masses.

BACKGROUND: The detectability of adrenal incidentalomas (incidentally found adrenal tumours) in the whole population is estimated at 0.1%; 0.42% in non-endocrine patients and at 4.3% in oncologically diagnosed ones. Even up to 16% of incidentalomas of adrenal glands can be malignant lesions. The issue of crucial importance is the histopathological differentiation between benign lesions and malignant tumours of the adrenal cortex and medulla. OBJECTIVES: To evaluate whether the immunohistochemical analysis of the expression of p53, p21, PCNA and Ki67 in the tumour's tissue can be useful in the histopathological diagnostics of adrenal incidentalomas and whether it is important for prognosis. MATERIAL AND METHODS: Our series consisted of 74 tumour samples from 164 patients operated for incidentalomas. There were 43 cortical adenomas, 11 cortical adrenocarcinomas and 20 PHEOs (including 5 malignant lesions). Using monoclonal antibodies, the expression of p53, p21, PCNA and Ki67 was evaluated. RESULTS: We found a statistically significant correlation between the expression of p53, p21, Ki67 and the differential diagnosis of adrenal cortical adenoma and adrenocortical carcinoma (for proteins: p53 p=0.010, for p21 p=0.010, for Ki67 p<0.001). The statistical significant correlation between PCNA protein and diagnosis of adrenal cortical adenoma and adrenocortical carcinoma was not found. The statistically significant correlation between p21, PCNA proteins and the diagnosis of benign and malignant PHEOs was not estimated. There was no expression of Ki67 or p53 protein above the assumed level in benign and malignant pheochromocytomas. The statistically significant correlation between p53, p21, PCNA or Ki67 and the occurrence of metastases in adrenocarcinoma and malignant PHEOs was not found.

Application of sulforaphane: histopathological study of intraportal transplanted pancreatic islets into livers of diabetic rats.

BACKGROUND: Islet cell transplantation is a promising method to restore insulin independence to patients with type 1 diabetes mellitus. A main problem in clinical islet transplantation is the fact that only a small percentage of allogeneic islet-transplanted type 1 diabetic patients can completely omit insulin injections after transplantation. One reason for the impaired survival of islet grafts is aberration of the function of islets due to toxic agents, including oxygen radicals and nitric oxide, which arise during warm or cold ischemic time. Therefore, in clinical islet transplantation, islets have been preserved with a mixture of antioxidants to reduce free radical-mediated damage of transplanted beta cells. Our aim was to examine hepatic tissue after metabolic normalization following intraportal islet transplantation after application of sulforaphane. MATERIALS AND METHODS: Islets were isolated from pancreata of WAG rats. Sulforaphane (24 mg/kg) was administered 24 hours before isolated islets were transplanted into the liver through the portal vein (1200 +/- 100 per rat). At 9 months after transplantation the animals were killed and liver tissue removed for morphological examination. RESULTS: This report indicated that the intrahepatic portal vein site was indeed an excellent locus for implantation of free pancreatic islets. The islet grafts developed rich vascularization derived from both venous and arterial sources. The islet cells maintained their structural and functional integrity after implantation. CONCLUSION: Our results showed that sulforaphane improved islet function in vivo, indicating that combination of a free radical scavenger and an antioxidant (sulforaphane) may be used to increase the effectiveness of islet transplantation.

Cytopathology of the esophagus in Transkei.

Esophageal brush biopsy capsules were used to prepare exfoliative cytologic smears of 1,000 residents of low-, intermediate-, and high-risk areas for esophageal cancer in Transkei, southern Africa. Satisfactory smears were obtained from 96.4% of the persons screened. The technique used was rapid, simple, inexpensive, safe, and effective in the determination of the prevalence of dysplasia and early carcinoma and the progression rate of dysplasia to carcinoma. In the high-risk area, precursor lesions (esophagitis and cellular atypia) were detected in 24% of adults older than 35 years, dysplastic changes in 9%, and carcinoma in 2%. Dysplastic changes were also present in 7% of young adults (25-34 yr of age) and in 1.5% of persons 15-24 years old. Precursor lesions were detected in 16% of the population group between 15 and 24 years old. Dysplastic changes and other precursor lesions were significantly less frequent in the population from the low-risk areas. A total of 14 malignant cases were detected in 8 females and 6 males. Of these, 9 were histologically confirmed squamous cell carcinomas in various stages of differentiation. The progression of dysplastic changes to carcinoma was observed in 5 of the total 14 cases. Three early cancers were successfully treated by esophagectomy. Seasonal variation occurred in the prevalence of mild cytologic changes.

Carcinogenicity of Fusarium moniliforme culture material in rats.

Two isolates of Fusarium moniliforme from corn were used in a chronic study with groups of 30 inbred male BD IX rats fed a semipurified diet that was marginally adequate nutritionally. Group 1 served as the controls and received the semipurified diet containing 5% cornmeal, group 2 received 5% of strain MRC 1069 culture material that was nontoxic to rats, and group 3 received 0.5% of strain MRC 826 culture material that was highly toxic to rats. The amount of the mutagen fusarin C detected in the culture material of strains MRC 826 and MRC 1069 was 104 and 364 mg/kg, respectively. Survival up to 2 years was good in all groups. Pathologic examination showed that many rats in group 2 had mild ductular cell hyperplasia. Almost all rats in group 3 had neoplastic nodules, gamma-glutamyltransferase-positive foci, adenofibrosis, and esophageal basal cell hyperplasia. Whereas no tumors were induced in groups 1 and 2, the 21 long-term survivors in group 3 developed 8 cholangiocarcinomas, 2 hepatocellular carcinomas, 4 carcinomas of the forestomach epithelium, and 1 esophageal papilloma. Since neoplastic lesions were confined to rats in group 3 and the diet of these rats contained much less fusarin C than that of group 2, it is highly unlikely that fusarin C was responsible for the carcinogenicity of the MRC 826 culture material. It appears that the toxicity of F. moniliforme strains may be related to their carcinogenicity, but the chemical nature of the toxic and carcinogenic metabolite(s) produced by F. moniliforme MRC 826 remains unknown.

Clinical significance of apoptotic index in non-small cell lung cancer: correlation with p53, mdm2, pRb and p21WAF1/CIP1 protein expression.

PURPOSE: The aim of this study was to assess the prognostic relevance of apoptotic index (AI), considered alone or together with expression of several proteins controlling G1 check point (p53, mdm2, pRb and p21WAF1/CIP1) in non-small cell lung cancer (NSCLC) patients. METHODS: Study group included 50 NSCLC patients who underwent curative pulmonary resection. Apoptosis was detected with the use of TUNEL technique and AI was defined as the number of apoptotic cells per 1,000 tumor cells. The expression of p53, mdm2, pRb and p21WAF1/CIP1 was assessed immunohistochemically. RESULTS: The mean and median AI calculated for all 50 patients was 14 and 9, respectively. Patients with lower (<14) and higher (> or =14) AI constituted 35 (70%) and 15 (30%) of cases, respectively. AI was not correlated with patient clinical characteristics, and expression of p53, pRb and p21WAF1/CIP1 . However, lower AI was correlated with over-expression of mdm2 protein (P=0.04). Median survival for patients with lower and higher AI was 43 months and 22 months, respectively, and 5-year survival probability-60 and 25%, respectively (P=0.03). In multivariate analysis, the only variable associated with shortened survival was AI (P=0.03, HR=2.9, 95% CI 1.95-3.86). CONCLUSIONS: These results suggest that AI correlates with mdm2 protein expression and influences survival in NSCLC.

Nesidioblastosis--a rare cause of hypoglycaemia in adults.

A case of suspected clinically hormonally active insulinoma in a 48-year-old woman is presented. Despite the lack of features, which might correspond to the insulinoma in radiological examinations, the patient was qualified for a distal subtotal pancreatectomy and then, due to persistent hyperinsulinism, for total pancreatectomy. The insulinoma was found neither in a palpable examination of the pancreas nor in the intraoperative ultrasonic examination. In a histopathological examination supplemented with immunohistochemical tests, nesidioblastosis - a rare cause of hypoglycaemia in adults - was diagnosed.

Primary sclerosing cholangitis. Requiem for biliary drainage operations?

OBJECTIVE: To review the outcome of a consecutive number of patients with primary sclerosing cholangitis (PSC) treated at one institution to define prognostic variables and determine the influence of surgery on outcome. DESIGN: Case series of patients with PSC seen in Cape Town, South Africa, between 1981 and 1991. SETTING: Tertiary referral center. PATIENTS: Thirty-six patients with PSC were studied. Diagnosis was based on cholangiographic findings of multiple strictures of the bile ducts together with compatible clinical and biochemical features. Thirty-two patients were followed up prospectively for up to 9 years. MAIN OUTCOME MEASURES: Patient outcome was defined as good (stable or slowly progressive disease) or poor (death or liver transplantation). RESULTS: During the follow-up period, seven patients with PSC died and two underwent liver transplantation. Actuarial survival at 5 years was 52%. An increased serum bilirubin concentration was the only variable at presentation that independently predicted a poor outcome. Cholangiography was unhelpful in predicting patient outcome. Six patients who developed obstructive jaundice associated with advanced liver disease underwent biliary drainage operations for surgically correctable strictures, but this did not seem to prevent progression of the disease. Two patients who progressed to end-stage liver disease went on to have liver transplantation and were alive with functioning grafts at 7 and 14 months, respectively. CONCLUSIONS: Symptomatic PSC is a progressive disorder with a poor prognosis. Our experience suggests that patients with advanced liver disease caused by PSC should be considered directly for liver transplantation rather than biliary bypass operations.

Hepatocellular carcinoma in young patients.

The study was based on the biopsy material collected in Eastern coastal region of South Africa with high incidence of primary hepatocellular carcinoma (HCC). Forty-one patients were between 9 and 25 years old of the total number of 474 cases of HCC available for examination. Liver biopsies were fixed in 10% of neutralised formalin, processed to paraffin blocks, cut and stained with hematoxylin and eosin, silver reticulin, Masson trichrome and Prussian blue stains. Representative biopsies of 21 patients younger than 25 years and 56 older than 35 years were in addition examined immunohistochemically with HBsAg antibody, endothelial marker (F VIII-related antigen) and for oncoproteins c-myc and c-erbB-2 using peroxidase-antiperoxidase method. Cirrhotic liver was evident in 41.5% of all patients and in 28% of younger than 25 years. Hemosiderosis of the liver of patients over 35 years was nearly twice as common as in younger than 25 years and showed the opposite relationship to the presence of HBsAg in liver tissue. Oncoprotein expression was also higher in tumor tissue of younger patients. These results indicate the etiological association of HCC with HBV infection, cirrhosis and possibly siderosis of the liver with HCC. Simultaneous expression of oncoproteins and HBsAg indicate the primary importance of viral infection in etiopathogenesis of HCC.

Preliminary studies on toxic effects of cyclopiazonic acid alone and in combination with aflatoxin B1 in non-human primates.

Results of experimental studies on toxicity of cyclopiazonic acid (CPA), a mycotoxin isolated from Aspergillus flavus, and toxicity of this toxin in combination with aflatoxin B1 (AFB1) are reported. Experiments were conducted in 16 vervet monkeys of both sexes. One male monkey was given 1 mg/kg per day of CPA intragastrically and the dose was doubled every third day to eventually achieve 60 mg/kg per day. Two males and 3 female monkeys were fed 20 mg/kg per day of CPA and terminated after 60 and 120 days, respectively. Two monkeys received AFB1 0.1 mg/kg per day, 2 received the same dose of AFB1 and 20 mg/kg per day of CPA. Four monkeys of both sexes served as controls. Results of experiments showed low toxicity of CPA in non-human primates with mild pathological changes in hepatocyte rough endoplasmic reticulum, small vessels and myocardium. Combined treatment with CPA and AFB1 indicated lack of a synergistic cumulative effect of both toxins. Animals treated with AFB1 only developed more advanced liver lesions and died earlier than those which also received high doses of CPA.

Pathogenesis of chronic myocarditis in mice infected with coxsackie B3 viruses.

Infection of 300 mice of the Swiss race with Coxsackie B3 viruses gave rise to chronic myocarditis and endocarditis. The virus was cultured from the heart muscle 9 days post infection. Between days 18 and 108 post infection, virtually all mice showed evidence of an active inflammatory process in the myocardium, and in one half there was proliferation of endothelial cells, and infitration and fibrosis in the endocardium. Immunomorphologic studies demonstrated the precence of antiheart antibodies in the blood serum, and Coxsackie B3 antigen and immunoglobulin deposits in the myocardium and endocardium. Highest levels of antivirus antibodies were observed 18 days post infection.

Trials of treating myocarditis in mice infected with coxsackie B3 viruses.

On the assumption of an immunologic background of chronic myocarditis after infection with Coxsackie B3 viruses, mice were treated with prednisolone or cyclophosphamide, starting 18 days after infection, for periods of three months. The favorable therapeutic effect indicates that chronic myocarditis is an immunologic disease.

Oesophageal carcinoma: cytopathology and nutritional aspects in aetiology.

This work includes results from studies on oesophageal cytopathology, dietary studies and the correlation of oesophageal cytological abnormalities and micronutrient blood levels in populations at risk for oesophageal carcinoma (OC) in remote rural areas of Southern Africa. The incidence of malignant lesions was 1.6 - 2.8%, and moderate to severe dysplastic lesions 0.5 - 1.8% in subjects aged over 35 years. Subjects 25-34 years of age showed malignant and dysplastic lesions in 0.8 and 1.3% respectively. Subjects younger than 24 years of age showed mild cytological lesions only. Early stages of OC were diagnosed in 8 patients. Three of them successfully underwent surgical intervention and are free from recurrence or metastases over a 3 year follow-up period. The incidence of mild oesophageal cytological lesions was inversely related to the frequency of the intake of green vegetables, fruit and animal proteins, and directly related to alcohol intake or tobacco smoking. A lower plasma concentration of vitamins A, E, B12 and folic acid was detected in individuals with cytological abnormalities. Of the mineral elements, only selenium, but not zinc, copper or magnesium was significantly related to the risk for OC on a regional or individual basis. An exceptionally low whole blood selenium level (58-72 ng/ml) and a relationship between its concentration and degree of cytological abnormalities were found.

Lymphoid aggregates in gastric biopsies: relationship to other mucosal lesions.

The purpose of this study is to estimate the prevalence of lymphocyte aggregates (precursor of MALT lymphomas) in gastric mucosal biopsies and to associate gastric lymphoid tissue with the age of patients, Helicobacter-associated gastritis and other gastric mucosal pathology. A consecutive series of gastric mucosal samples from 150 children and 256 adults were assessed for the presence of lymphoid aggregates as well as morphological characteristics, Helicobacter pylori status, signs of gastritis, mucosal atrophy and lymphoepithelial lesions. Fifteen selected samples with prominent lymphoid aggregates and 10 controls were examined immunohistochemically for the immunoglobulins A, G, M, lymphocytes B and T, clonality of B cell population, atypical lymphocytes and Epstein-Barr virus (EBV) antigen. There was an increase of H. pylori infection and mucosal lymphoid aggregates (MALT) rates in parallel with the increasing age of patients noted in the histological assessment of the mucosal samples. A close association of lymphoid aggregates with H. pylori infection and prominent active gastritis was found, but in adults with chronic non-active, particularly atrophic gastritis this association became weaker. No morphological and immunohistochemical signs of MALT lymphoma were present. Lymphoid aggregates in children were larger, with follicles, but less numerous and tended to be located in the intermediate and deeper parts of the gastric mucosa. Immunohistochemical studies showed an increase of IgA, IgM and lymphocytes T in the deeper part of the lamina propria in H. pylori-associated gastritis and lymphocyte T accumulation in the periphery of the lymphoid follicles. No evidence of monoclonality, CD31 positive lymphocytes or EBV antigen was detected. Lymphoid aggregates are related, but not exclusively, to H. pylori infection. Their detection rates achieve a peak in young adults with H. pylori infection. Lymphocytic aggregates are also present in chronic atrophic gastritis without H. pylori infection and may relate to autoimmune inflammatory response to other factors.

Myocarditis induced by coxsackie B3 virus in mature mice.

Forty female mice during breast-feeding were infected intraperitoneally with coxackie B3 virus. Gross and microscopic examination of the hearts of the mice 7, 20, 44 and 120 days after infection revealed myocarditis typical of the acute stage of the disease, not reported previously, and gradually increasing intensity of immunologic changes in the chronic stage.

Hepatocellular carcinoma in a rural population at risk.

This study of hepatocellular carcinoma in a homogeneous rural Transkeian population at high risk consisted of: evaluation of liver biopsies of 246 patients with hepatocellular carcinoma using routine histology and immunoperoxidase for HBV stains; collection of reliable data on alcohol consumption, blood HBsAg and ALT status in asymptomatic controls from an adequate population sample; assessment of maize intended for human consumption for contamination by carcinogenic fungi. Tumour histology of rural Blacks did not differ from those described in other studies. Cirrhotic livers were present in 45.1% and iron overload in 68% of cases. Tissue HBsAg was detected in 57% (45% of non-cirrhotic and 59% of cirrhotic livers). Asymptomatic controls showed 9.5% of HBsAg positivity, 3.5% had elevated ALT and 41.3% admitted to alcohol abuse. Maize had insignificant contamination by Aspergillus flavus and very frequent contamination by Fusarium moniloforme. This study suggests a multifactorial aetiology of hepatocellular carcinoma, with viral infection being of the most importance.

Uncommon mesenchymal tumors of the liver. Light microscopy, ultrastructure, immunohistochemistry and literature review.

This report presents the gross morphology, cytological, histological, ultrastructural and immunohistochemical features of two angiomyelolipomas of the liver. Diagnosis and classification of these tumors is difficult. There is no immunohistochemical and ultrastructural confirmation for Ito-cell pathogenesis of hepatic angiomyelolipomas.

Chronic atrophic gastritis in a population at risk for gastric carcinoma.

Gastric mucosa of 585 patients at risk for gastric carcinoma and undergoing oesophago-gastroscopy for dyspeptic symptoms was examined histologically and histochemically. Sixteen (2.7%) of the patients studied showed gastric carcinoma. The intestinal type of gastric carcinoma was the most common, present in males over 40 yrs old, and was found in 1:16. Early cancers were present in 3 patients. Chronic atrophic gastritis of the A, B and AB types was present in 32.8%. Types A and AB were noted sporadically. Males over 40 and tobacco smokers showed the prevalence of the type B (idiopathic chronic pangastritis) and incomplete intestinal metaplasia. No relationship of the mucosal precursor lesions to sulpho- and sialomucins were detected. Helicobacter pylori infestation of gastric mucosa was present in 81% of patients and relatively common in the young patients with normal gastric mucosa; therefore this finding confirms a predilection of Helicobacter pylori to the Third World.

Hepatic vascular lesions associated with malignant lymphomas.

Veno-occlusive disease is a serious complication of malignant conditions and often follows irradiation or is drug-induced. We report on the liver histology of a consecutive series of 32 cases of Hodgkin's and non-Hodgkin's lymphomas. Veno-occlusive lesions were noted in 27 biopsies prior to treatment. These patients had no apparent malignant infiltrate to the liver. The pathogenesis of these lesions is unclear.

Barrett's oesophagus: mucin composition, neuroendocrine cells, p53 protein, cellular proliferation and differentiation.

There is strong association of Barrett's oesophagus (BO) with adenocarcinoma. The sequence of events preceding malignancy appears to be reflux oesophagitis - ulceration - BO - dysplasia. One hundred and five biopsies of heterotopic columnar epithelium were stained for H&E, PAS/Alcian Blue and HID/Alcian Blue for the routine histology and neutral/acidic sialo- and sulphomucin staining. Other sections were silver impregnated by the Grimelius technique. Immunohistochemical techniques were applied for the assessment of the accumulation of p53 protein, "S" phase of the replication cell cycle using proliferating cell nuclear antigen (PCNA), marked for cell differentiation and proliferation using EGF and TGFa. 105 cases of heterotopic columnar epithelium consisted of 74 cases of BO, 25 junctional and 7 corpus mucosa. Dysplastic BO (n = 9) showed similar amount of sulphomucin and endocrine cell number when compared to non-dysplastic. PCNA study revealed a close similarity between dysplastic, indefinite for dysplasia and non-dysplastic, mucosal positive counts. Growth factors activity was significantly higher in dysplastic and indefinite than in non-dysplastic, but no such difference was found between dysplastic and indefinite for dysplasia BO. There was a significant concurrent p53 expression in dysplastic and indefinite for dysplasia BO. In conclusion, the practical utility of mucin stainings, endocrine cell count, assessment of cell proliferation and differentiation by PCNA, EGF and TGFa seems to be limited in differentiation of the dysplastic and indefinite for dysplasia BO. Altered expression of p53, particularly in combination with EGF and TGFa, may be useful in studying these lesions.

p53 gene mutants expression, cellular proliferation and differentiation in oesophageal carcinoma and non-cancerous epithelium.

An immunohistochemical study of 155 squamous cell carcinomas of the oesophagus, squamous epithelium adjacent to the tumour (n = 80), dysplastic epithelium (n = 16) and controls (n = 23) indicates that Ki67 and p53 expression is frequently present in premalignant oesophageal lesions and their related squamous cell carcinomas. Positive expression of both antibodies in apparently normal epithelium can signify early steps of malignant transformation of oesophageal epithelium and can serve in the detection of early precancerous lesions. The expression of growth factors EGF and TGF-alpha was higher in carcinomas and dysplastic lesions than in apparently normal squamous epithelium. EGF expression was unevenly distributed according to histological grade, indicating a lack of EGF immunoreactivity in poorly differentiated oesophageal carcinomas.