Liver fluke-associated and sporadic cholangiocarcinoma: an immunohistochemical study of bile duct, peribiliary gland and tumour cell phenotypes.

AIM: To compare cell phenotypes displayed by cholangiocarcinomas and adjacent bile duct lesions in patients from an area endemic in liver-fluke infestation and those with sporadic cholangiocarcinoma. METHODS: 65 fluke-associated and 47 sporadic cholangiocarcinomas and 6 normal livers were studied. Serial paraffin-wax sections were stained immunohistochemically with monoclonal antibodies characterising a Brunner or pyloric gland metaplasia cell phenotype (antigens D10 and 1F6), intestinal goblet cells (antigen 17NM), gastric foveolar apomucin (MUC5AC), a gastrointestinal epithelium cytokeratin (CK20) and the p53 protein. RESULTS: 60% of the 112 cholangiocarcinomas expressed antigen D10, 68% MUC5AC, 33% antigen 17NM and 20% CK20; 37% showed overexpression of p53. When present together in a cholangiocarcinoma, cancer cells expressing D10 were distinct from those displaying 17NM or MUC5AC. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinomas displayed 17NM and p53 expression. Most cases of hyperplastic and dysplastic biliary epithelium expressed D10 strongly. Pyloric gland metaplasia and peribiliary glands displayed D10 and 1F6, with peribiliary gland hyperplasia more evident in the livers with fluke-associated cholangiocarcinoma; goblet cells in intestinal metaplasia stained for 17NM. No notable association of expression between any two antigens (including p53) was found in the cancers. CONCLUSIONS: Most cases of dysplastic biliary epithelium and cholangiocarcinoma display a Brunner or pyloric gland cell phenotype and a gastric foveolar cell phenotype. The expression of D10 in hyperplastic and dysplastic epithelium and in cholangiocarcinoma is consistent with a dysplasia-carcinoma sequence. Many more fluke-associated cholangiocarcinomas than sporadic cholangiocarcinoma display an intestinal goblet cell phenotype and overexpress p53, indicating differences in the aetiopathology of the cancers in the two groups of patients.

Vertebrate phylogeny of antigen D10: identification of a conserved foregut cell lineage.

The monoclonal antibody 5HL-5D11-D10 to antigen D10 identifies a cell lineage that is restricted to certain tissues of the human foregut. We investigated the tissue distribution of antigen D10 in mammals, birds, reptiles, amphibians and fish by immunohistochemical staining. Tissue from human and each of ten other mammalian species showed staining of gastric mucous neck cells and glands of the cardia and antrum, Brunner's glands, peribiliary glands and periductal glands of the pancreas. Six of the mammalian species also expressed antigen D10 in mucosa of the larger bronchi, and five expressed it to varying degree in small bowel distal to the duodenum and in colon (three of these five species). Antigen was not detected in any of the three species of bird studied. Both reptiles and amphibians showed strong staining for antigen D10 in the gastric mucous neck cells and pyloric glands, and in a subpopulation of secretory cells in the oesophagus, with the amphibian also expressing antigen in some epithelial cells of the mouth and lung. Although absent from two species of bony fish, antigen D10 was expressed by small groups of epithelial cells of the intestine of a shark, and generally by the epithelial and connective tissue cells of the gut and gills, and hepatocytes of one species of ray. The presence of antigen D10 in different tissues and species was confirmed by both an indirect ELISA and immunoblot analysis of tissue extracts. Our observations suggest that the D10 epitope characterises a subpopulation of mucus-secreting cells, predominantly of the foregut and associated organs, which has been conserved throughout terrestrial vertebrate evolution.

Growth hormone status following treatment for Cushing's syndrome.

OBJECTIVE: Both pituitary surgery and radiotherapy for Cushing's disease can lead to growth hormone (GH) deficiency. Studies to date have, however, described the incidence of impaired GH secretion and not the incidence of severe GH deficiency following treatment of Cushing's disease. Furthermore, following cure of Cushing's disease and resolution of hypercortisolaemia, recovery of GH secretory status is seen, thus creating uncertainty as to the persistence of any documented GH deficiency. This study has two aims; to determine the incidence of severe persistent GH deficiency following treatment of Cushing's disease and to assess the time scale of any recovery of GH secretory status following surgical cure of Cushing's disease. DESIGN AND PATIENTS: The case notes of 37 patients either cured or in clinical and biochemical remission following treatment for Cushing's syndrome were reviewed to determine the incidence of severe GH deficiency. Of 34 patients with Cushing's disease, 20 were treated by pituitary surgery, and 14 with radiotherapy. Three patients with adrenal adenomas underwent unilateral adrenalectomy. MEASUREMENTS: GH secretory status was assessed by provocative testing using an insulin tolerance test (ITT, 85% of all tests), glucagon stimulation test (GST) or arginine stimulation test (AST). RESULTS: Thirty-six percent (5/14) of radiotherapy treated patients demonstrated severe GH deficiency at a mean time of 99 months following remission. Fifty-nine percent (10/17) of surgically treated patients assessed in the two years following remission demonstrated severe GH deficiency, whilst only 22% (2/9) of patients assessed beyond two years following remission demonstrated severe GH deficiency. This latter cohort is biased, with patients in whom severe GH deficiency had been demonstrated on earlier tests being over-represented. It is more accurate to estimate the incidence of persistent severe GH deficiency following surgically induced remission of Cushing's disease by incorporating data from patients in whom original testing demonstrated adequate GH reserve. Collating such data, 13% (2/15) of patients had persistent severe GH deficiency. Across all time periods five surgically treated patients demonstrated recovery of GH secretory status over a median time course of 19 months. In the surgically treated cohort, seven (35%) patients had anterior pituitary hormone deficits other than GH deficiency: 14% (2/14) of patients with normal GH secretory status at the last assessment, 83% (5/6) of patients with severe GHD at the last assessment. Of the 5 patients who demonstrated recovery of GH secretory status 40% (2) had additional anterior pituitary hormone deficits. Within the radiotherapy treated cohort 14% (2/14) of patients demonstrated additional anterior pituitary hormone deficits: 11% (1/9) of patients with normal GH secretory status and 20% (1/5) of patients with severe GH deficiency. None of the patients with adrenal adenomas treated by unilateral adrenalectomy demonstrated any abnormality of GH secretory status CONCLUSIONS: The incidence of severe persistent GH deficiency following surgically induced or radiotherapy induced remission of Cushing's disease is lower than has been suggested by previous studies, although these latter studies have assessed GH insufficiency and not severe GH deficiency. In the presence of additional pituitary hormone deficits severe GHD is common and is likely to be persistent. Recovery of GH secretory status is seen in a high proportion of patients reassessed, at a median time of 19 months following surgically induced remission of Cushing's disease. Thus, we recommend that definitive assessment of GH secretory status is delayed for at least two years following surgical cure of Cushing's disease. This has important implications for patients in whom GH replacement therapy is being considered.

Kappa-opioid receptor agonists increase locomotor activity in the monoamine-depleted rat model of parkinsonism.

Excessive glutamate transmission in the basal ganglia is a major factor in the neural mechanisms underlying parkinsonian akinesia. Activation of kappa opioid receptors causes a presynaptic reduction in glutamate release. Kappa opioid receptors are concentrated in those regions of the basal ganglia associated with increased glutamate transmission in parkinsonism. In this study, we use the alpha-methyl-p-tyrosine and reserpine-treated rat model of parkinsonism to investigate whether systemic administration of the kappa opioid agonists enadoline (CI-977) and U69,593 can alleviate the symptoms of parkinsonism either alone or in conjunction with dopamine replacement therapy. We report that, when administered alone, both enadoline and U69,593 can increase locomotion in monoamine-depleted rats. No increase in locomotor activity was seen after kappa opioid agonist administration in non-parkinsonian rats. The responses to kappa opioid agonists were blocked by co-administration of either the nonspecific opioid receptor antagonist naloxone or the selective kappa opioid receptor antagonist nor-binaltorphimine (nor-BNI). An important finding is that when enadoline and L-dopa are administered together, their anti-akinetic properties are synergistic. Thus, the doses of enadoline and L-dopa required to alleviate akinesia when administered together are lower than either administered alone. These data illustrate the importance of kappa opioid receptors in the neural mechanisms controlling voluntary movement and suggest that kappa opioid agonists may have a role as adjuncts to dopamine replacement in the management of Parkinson's disease.

Gastric mucous neck cell and intestinal goblet cell phenotypes in gastric adenocarcinoma.

AIM: To investigate the phenotype of cells comprising diffuse and intestinal-type gastric cancers using monoclonal antibodies to two antigens. One antigen (designated D10) is characteristic of gastric mucous neck cells, cardiac glands, pyloric glands, and Brunner's glands. The second antigen (designated 17NM) is specific to the mucous vacuole of intestinal goblet cells. METHODS: Thirty two gastrectomy specimens with adenocarcinoma were studied. Serial paraffin sections were stained immunohistochemically for D10 and 17NM and histochemically for acid and neutral mucins. The cancers were classified histologically as of either diffuse or intestinal type according to Lauren. RESULTS: Of 15 diffuse-type gastric carcinomas, 11 showed the majority of cancer cells staining for D10 while four were typical signet ring cell cancers staining predominantly for 17NM; five tumours displayed both phenotypes with the two phenotypes segregated in different areas of the tumours. In contrast, of 16 intestinal-type cancers, six expressed 17NM, three D10, five neither antigen, and two expressed both antigens. One indeterminate-type cancer expressed both antigens. The staining of individual cells for D10 and 17NM was mutually exclusive in both diffuse and intestinal types. In contrast to the diffuse cancers, intestinal-type cancers typically expressed either antigen only in occasional small groups of cells and individual cells. CONCLUSIONS: In disease, the gastric stem cell can assume the capacity of the duodenal stem cell for divergent differentiation into either intestinal goblet cells (for example, as in intestinal metaplasia) or Brunner's gland cells (for example, as in pyloric gland/Brunner's gland metaplasia). With neoplastic transformation, this potential for divergent differentiation is maintained and gives rise to diffuse-type cancers that display either the D10 phenotype, the 17NM phenotype, or the clonal expression of both phenotypes. In the more cell cohesive (intestinal-type) tumours, differentiation for antigen expression is poorly developed and more frequently directed towards the intestinal goblet cell phenotype.

The so-called bile duct adenoma is a peribiliary gland hamartoma.

The cell phenotype of so-called bile duct adenoma (BDA) was investigated immunohistochemically using monoclonal antibodies to two recently identified antigens (designated D10 and 1F6) extracted from human liver and cultured biliary epithelium. The acini and tubules of BDA consisted of serous and mucous cells that expressed D10 and 1F6. The intrahepatic peribiliary glands of normal liver, comprising intramural mucous glands and extramural tubuloalveolar seromucinous glands, similarly expressed D10 and 1F6 antigens. Antigen 1F6 was present in the cells forming the canals of Hering and normal bile ductules but not in interlobular and larger bile ducts. Proliferating bile ductules associated with large bile duct obstruction and alcoholic cirrhosis or the epithelia of the von Meyenberg complex and polycystic liver did not exhibit this combined profile of D10 and 1F6 expression and mucous cells. These findings suggest an origin of BDA from peribiliary glands rather than from bile ductules or ducts. Consistent with this view was our finding that 18 of the 30 BDA were spatially related to a large-calibre bile duct. Therefore, BDA, well known for its benign behavior is a small mass of disorganized but mature peribiliary gland acini and tubules within a variable amount of stroma and should properly be called a peribiliary gland hamartoma.

Phenotypic identity of gastric mucous neck cells and mucous cells of cardiac, pyloric, and Brunner's glands.

AIM: To investigate the tissue specificity of a novel monoclonal antibody raised to a tissue fraction of normal human liver and which identified certain cells of gastric and duodenal mucosa. METHODS: A total of 155 samples of various tissues obtained from 100 surgical specimens were fixed in cold ethanol-paraformaldehyde, embedded in paraffin wax, and 3 microns sections were studied by immunohistochemical and lectin staining procedures. RESULTS: Immunohistochemical staining showed a major tissue specific component which was strongly expressed by mucous neck cells of the body of the stomach, glands of the cardia and pyloric antrum, and by Brunner's glands. Staining for antigen in the periductal glands of normal major biliary and pancreatic ducts was variable and relatively weaker. It was not detected elsewhere in normal intestine or in the other normal tissues tested. Barrett's mucosa of gastric cardia type, and pyloric gland metaplasia in the gall bladder and small bowel affected with Crohn's disease stained for the antigen. The tissue distribution of the antigen was identical with that of a glycoprotein, demonstrated by an induced affinity for concanavalin A following treatment of tissue sections with periodic acid. The antigen was not sensitive to sialidase. CONCLUSIONS: The tissue component identified (designated here as antigen D10) seems to be characteristic of certain differentiated epithelial cells derived from that part of foregut giving rise to stomach, duodenum, and biliary and pancreatic ducts. The antibody will be of use in investigating pathological processes involving tissue differentiation at these sites, and in the oesophagus and intestines.

Colorectal polyps in an Australian population. A histological and immunohistochemical study.

The histology and immunohistochemistry of 896 polyps and other focal epithelial abnormalities detected macroscopically in 86 surgical resections from patients with colorectal adenocarcinoma and benign bowel disorders were studied. The lesions identified included 177 adenomas, 387 hyperplastic (metaplastic) polyps, and 202 non-neoplastic polyps designated 'focal cryptal hyperplasia'. Numbers of both neoplastic and non-neoplastic polyps were significantly increased in resections for carcinoma, with 72 per cent of all polyps in right and 10 per cent in left hemicolectomy specimens being neoplastic. Thirty per cent of adenomas were less than 2 mm in diameter and 6 per cent larger than 10 mm. Observations on polyp size, number, distribution, histological appearance, and antigenic composition suggested that focal cryptal hyperplasia evolves into the hyperplastic polyp. In doing so, there is loss of expression of a tissue specific antigen. Hyperplastic polyps were significantly larger in colons with adenoma than in those without.

Gland to gland heterogeneity in histologically normal mucosa of colon cancer patients demonstrated by monoclonal antibodies to tissue-specific antigens.

Two of three monoclonal antibodies to tissue-specific antigens of isolated colonic glands revealed gland to gland heterogeneity of antigen expression in sections of the histologically normal colonic mucosa from patients with colorectal adenocarcinoma or various nonmalignant conditions. A colon-specific goblet cell antigen (designated 3NM) was absent from rare, solitary glands randomly distributed among the otherwise strongly stained glands of distal colon. These 3NM-negative glands stained normally for the other two antigens studied and appeared morphologically and histochemically normal. They occurred in 11 of 13 cancer patients and in each of the five patients with benign conditions with median incidences of 2.2 and 0.4 per 1000 glands, respectively. Gland heterogeneity was also demonstrated in both patient groups for a cell membrane antigen (designated 6NM). In cecum and ascending colon, glands staining strongly and weakly for 6NM were found intermixed. The weakly stained glands tended to predominate in cecum and first part of ascending colon, but they were completely replaced by strongly stained glands in more distal colon. The heterogeneity shown by both 3NM and 6NM appeared due to phenotypically distinct cell clones. Our observations indicate that histologically normal colonic mucosa contains antigenically diverse gland populations.

Antigen expression in normal and neoplastic colonic mucosa: three tissue-specific antigens using monoclonal antibodies to isolated colonic glands.

Monoclonal antibodies to normal colonic glands were used to localize three tissue-specific antigens in sections of 40 hyperplastic polyps, 29 adenomas, 43 colorectal adenocarcinomas, and blocks or rolls of histologically normal large bowel mucosa. Two antibodies identified antigens (designated 3NM and 17NM) associated with the mucin vacuole of goblet cells and the third antibody identified a cell membrane antigen (designated 6NM). Antigen 3NM was absent from virtually all carcinoma cells and from all, or most, cells of hyperplastic polyps. Antigen 17NM was absent from 21 carcinomas, with some antigen-positive cells present in the others. Antigen 6NM content was unchanged compared to normal mucosa in 12 carcinomas, decreased somewhat in another 20, and either absent or markedly depleted in 11. Antigen 6NM increased in 22 hyperplastic polyps. Three cancer patients synthesized 6NM in their normal mucosa but the cellular distribution was abnormal. Antigens decreased in most adenomas, with heterogeneity of expression seen in glands and cells of some adenomas. Low concentrations of one or more antigens in normal mucosa were significantly more frequent (P less than 0.05) in patients with metastases than in those with stage A or B tumors.