Primary hepatic carcinoid tumor presenting as Cushing's syndrome.

Hepatic carcinoid tumors are very uncommon; most are clinically non-functional and very few present with the symptoms of carcinoid syndrome. ACTH-producing carcinoid tumors most commonly originate in the lung or thymus and present insidiously with bronchospasm and/or chest mass. Occasionally, ectopic ACTH syndromes have been reported in association with pancreatic islet cell tumors, medullary thyroid cancer, pheochromocytoma, small-cell lung carcinoma, and rarely, ovarian and prostate tumors. We report here a patient with an ectopic ACTH-secreting primary hepatic carcinoid tumor who presented with cushingoid appearance, profound proximal muscle weakness, severe lower extremity edema, and markedly elevated urinary free cortisol. ACTH levels were in the low normal range. A solitary vascular hepatic lesion was found on magnetic resonance imaging, which was isodense with the surrounding liver on octreotide scan and photopenic on an 18-fluorodeoxyglucose (18FDG)-positron emission tomography (PET) scan. Following surgical resection of the hepatic tumor, histopathology confirmed an ACTH-secreting neuroendocrine tumor (NET), the patient had complete resolution of hypercortisolemic symptoms and remains in remission, now 4 yr after hepatic tumor resection. This case reports the first ACTH-secreting primary hepatic NET presenting as ectopic Cushing's syndrome. Interesting aspects of this case include the presence of a pituitary incidentaloma, the low normal ACTH, and photopenia on 18FDG-PET imaging.

Wide expression of the human erythrocyte glucose transporter Glut1 in human cancers.

Glucose uptake has been found to be increased in cancer cells. Previous work has shown increased expression of the human erythrocyte glucose transporter (Glut1) mRNA in some human cancers, indicating that Glut1 may play a significant role in glucose uptake by these tumors. The distribution of Glut1 protein in normal and malignant human tissues is still largely unknown. Using immunohistochemistry, we found that Glut1 is largely undetectable in normal epithelial tissues and benign epithelial tumors but is expressed in a significant proportion of a variety of human carcinomas. We hypothesize that Glut1 expression by human carcinomas indicates an increased glucose uptake, and probably increased utilization of energy, which may correlate with an aggressive behavior. The biological significance of Glut1 expression needs to be determined.

Overexpression of the human erythrocyte glucose transporter occurs as a late event in human colorectal carcinogenesis and is associated with an increased incidence of lymph node metastases.

Energy metabolism of human colon cancer in vivo relies predominantly on glucose. Although studies have revealed increased expression of Glut1 mRNA in colon cancer, Glut1 protein (Glut1) expression in the large intestine and its significance are still unknown. The objective of this work was to determine whether Glut1 is present in human colorectal neoplasms and whether that presence is of biological significance. Formalin-fixed, paraffin-embedded tissue sections of 53 colonic adenocarcinomas, 82 adenomas, 46 hyperplastic polyps, and 38 normal colon samples were immunostained with the anti-Glut1 antibody MYM. The localization was carried out using the avidin-biotin immunoperoxidase technique. No Glut1 immunoreactivity was present in normal colonic mucosa or in hyperplastic polyps, whereas 8 (10%) of 82 adenomas showed such immunoreactivity. The frequency of Glut1 expression in adenomas increased with villous morphology and with the size of the adenoma. Forty-four (83%) of 53 colorectal adenocarcinomas expressed Glut1, and, of these, tumors in which >50% of the cancer cells expressed Glut1 had a significantly higher incidence of metastasis to the lymph nodes (P = 0.0001). It is concluded that (a) Glut1 is expressed as a late event in the carcinogenesis process in human colorectal cancer, and (b) expression of Glut1 in a high proportion of cancer cells is associated with a high incidence of lymph node metastases.

Human erythrocyte glucose transporter (Glut1) is immunohistochemically detected as a late event during malignant progression in Barrett's metaplasia.

We have previously demonstrated that the human erythrocyte glucose transporter (Glut1) is expressed in adenocarcinoma arising in Barrett's metaplasia (BM). We have also shown that Glut1 is expressed as a late event during colorectal carcinogenesis. The aim of this work was to determine the chronology of Glut1 expression during the neoplastic progression in Barrett's metaplasia. Formalin-fixed, paraffin-embedded tissue sections of 251 biopsies from 97 patients with BM were immunostained with the anti-Glut1 antibody MYM, after microwave-aided antigen retrieval, using the standard avidin-biotin complex immunoperoxidase technique. Dysplasia was graded as negative (ND), low grade (LGD)/indefinite or high grade (HGD). None of the 181 biopsies with ND (0%) or 51 biopsies with LGD (0%) showed Glut1 immunoreactivity. More importantly, although 0 of 6 biopsies with HGD (0%) expressed Glut1, 9 of 13 biopsies with adenocarcinoma (69%) were Glut1 positive (P = 0.0108, Fisher's exact test). Our results indicate that Glut1 is expressed as a late event during the neoplastic progression in BM. Prospective studies are needed to determine the clinical utility of Glut1 immunoreactivity as a marker of carcinoma in patients with BM.

A restaining method to restore fluorescence in faded preparations of tissues treated with the indirect immunofluorescence technique.

We report a restainin method for restoring fluorescence in paraffin-embedded tissue sections previously treated with the indirect immunofluorescence technique. Antisera to gastrin and group II pepsinogens were used. Fluorescence was restored in completely faded sections retrived from storage files, as well as in sections that had faded partially either with exposure to fluorescence microscope illumination or after counterstaining with hematoxylin and eosin.

The endocrine cells of the digestive tract. General concepts and historic perspective.

The chromaffin cells of the digestive mucosa were discovered by Heidenhain in 1870, long before the hormone concept was formulated. Following the discovery of the first hormone, secretin, by Bayliss and Starling in 1902, the study of the cells producing such messengers acquired new impetus. After Masson, in 1914, showed that the digestive chromaffin cells were argentaffin and suggested their endocrine nature, a series of technological advances led to the gradual characterization of diverse cell types, their classification, and the discovery of their function. Histochemistry, including silver impregnations, fluorescence microscopy, and aniline stains, exposed the complexity of the digestive endocrine cells. Electron microscopy provided structural markers for their characterization and the basis for the formulation of the first universally accepted classifications. Light- and electron-microscopic immunocytochemistry contributed to the understanding of the function of many of these cells, thus opening the way for modern classifications. Many unsolved problems still remain. These include the existence of cells without defined function, of chemical messengers without known cell of origin, and the presence of multiple messengers in some cell types. Answers are expected to emerge from further application of immunocytochemistry and from the introduction of modern approaches, such as in-situ hybridization histochemistry.

Diagnosing primary and metastatic renal cell carcinoma: the use of the monoclonal antibody 'Renal Cell Carcinoma Marker'.

The diagnosis of primary or metastatic renal cell carcinoma (RCC) can be difficult, especially in small biopsies, because of the wide variety of histologic appearances and clinical presentations that RCC can assume. An immunomarker specific for RCC is currently not available. We tested the relevant diagnostic use of the Renal Cell Carcinoma Marker (RCC Ma), a monoclonal antibody, against a normal human proximal tubular brush border antigen. Immunostaining using RCC Ma and the avidin-biotin-peroxidase complex technique was performed on archival tissues from primary and metastatic tumors of renal or nonrenal origin. A total of 122 of 153 primary RCCs (79.7%) were positive [clear cell (84%), papillary (96%), chromophobe (45%), sarcomatoid (25%), and collecting duct (0%)], with > or =10% of tumor cells stained in 93% of cases. None of the 64 primary renal tumors other than RCC, including 15 oncocytomas, was positive. Fifteen of 146 (10.2%) nonrenal primary tumors were positive (5 of 17 breast tumors, 8 of 8 parathyroid adenomas, and 2 of 7 embryonal carcinomas). Forty-two of 63 (67%) metastatic RCCs were positive with > or =10% of cells being stained in 83% of them. Two of 108 (2%) metastases from tumors other than RCCs were positive, both of which were metastatic breast carcinomas; however, only 10% (2 of 19) of metastatic breast carcinomas were positive. RCC Ma is an excellent marker for primary RCC, which should facilitate its diagnosis in a small biopsy. Although RCC Ma remains highly specific (98%) for metastatic RCC, a negative result may not rule out metastatic RCC because of a rather low sensitivity and a focal staining pattern in some of the positive cases. RCC Ma may also facilitate the differential diagnosis between oncocytoma and other types of RCC when they are composed mostly of eosinophilic cells.

Immunocytochemical localization of a granuliberin-like peptide in Rana pipiens brain.

The histamine-releasing peptide "granuliberin", originally isolated from the skin of Rana rugosa, was localized by immunofluorescence within nerve cell bodies and fibers in the brain of Rana pipiens. The granuliberin-positive neurons were characterized ultrastructurally by electron microscopic observation of ultrathin sections stained either with immunoperoxidase or with conventional stains. Granuliberin-positive nerve cell bodies were seen throughout the hypothalamus, from the suprachiasmatic area rostrally to the full length of the periinfundibular grey matter caudally. Similarly positive nerve fibers were localized in the hypothalamus radiating upwards to the optic vesicles in the midbrain, extending through the preoptic area into the subpallium in the forebrain, and throughout the white matter surrounding the floor and lateral walls of the fourth ventricle in the brainstem. The granuliberin-positive nerve cells showed the presence of variable numbers of small cytoplasmic neurosecretory granules, possessing an electron dense elongated core, and measuring 250-350 nm in their largest diameter. The functional significance of a granuliberin-like peptide in neurons of the frog brain is not known.

Human colonic substance P-producing cells are a separate population from the serotonin-producing enterochromaffin cells.

Human colonic mucosa was immunostained with antibodies against substance P to identify the endocrine cells containing this peptide in the mucosal glands. Dual immunohistochemical and histochemical studies were also carried out to determine whether these cells are enterochromaffin cells and contain serotonin as claimed in the literature. The results obtained indicate that the normal human colonic substance P-producing cells are not argentaffin cells, nor do they contain serotonin. In addition, they are also negative both for several silver and for other techniques commonly used to identify digestive endocrine cells. They are positive, however, with the argyrophilic technique of Churukian-Schenk. It is concluded, therefore, that the substance P-producing cells of the human colonic mucosa are not a subpopulation of the enterochromaffin cells, but constitute a distinct and independent cell type.

Simultaneous visualization of two antigens in the same tissue section by combining immunoperoxidase with immunofluorescence techniques.

A simple method was developed whereby immunoperoxidase and immunofluorescence techniques were applied in consecutive steps to demonstrate the presence of two antigens in the same tissue section. This method was applied in three model, two antigens were shown: a) each (gastrin and pepsinogen II) inside one of two different cell types (gastrin (G) and antral peptic cells), b) each (kappa or gamma light chains) inside different cells of the same type (plasma cells); also, both (kamma and gamma light chains) inside the same cell (Reed-Sternberg cell), and c) both (pepsinogen I and II) inside the same cell (chief cell of oxyntic glands). The results could be viewed and photographed either simultaneously, when the antigens were in different cells, or sequentially, when the antigens were in the same cells.

Immunohistochemical localization of relaxin in human prostate.

We identified relaxin in human male prostate by use of an anti-human relaxin analogue polyclonal antibody and the avidin-biotin-immunoperoxidase method. The antibody was obtained by immunizing a rabbit with a synthetic human relaxin analogue which has 95% sequence homology with native human relaxin. Human prostate tissues incubated with the anti-human relaxin analogue exhibited positive immunostaining up to an antibody dilution of 1:3200. Inhibition of immunostaining with this antibody by excess relaxin analogue demonstrated specificity of the antibody. The exact role of relaxin in human male reproductive physiology remains to be fully elucidated.

Immunocytochemical localization of epidermal growth factor in mouse kidney.

Epidermal growth factor (EGF) was originally isolated from mouse submandibular glands (SMG). However, SMG removal failed to lower circulating EGF, and large amounts of EGF have been found in mouse urine. In addition, the presence of pre-pro-EGF mRNA in mouse kidney has recently been reported by others. Kidneys may therefore represent an alternate source of EGF. In the present study, we investigated the immunocytochemical localization of EGF in mouse kidney. Male and female adult Swiss Webster mice were fixed by perfusion with 4% paraformaldehyde or Zamboni's fixative, the kidneys were frozen, and serial sections were obtained. Rabbit EGF antiserum was used for the primary incubation and the avidin-biotin complex immunoperoxidase procedure was utilized for immunostaining. EGF was immunolocalized in the apical portion of the cells lining the thick ascending limb of Henle (TALH) and the distal convoluted tubule (DCT). The macula densa, in contrast, lacked EGF immunoreactivity. No sex differences were observed in the distribution pattern or intensity of immunostaining. Infusion of EGF into sheep renal artery has been reported to induce changes in urine flow and ionic composition. Immunolocalization of EGF in the TALH and DCT documented here supports a regulatory role for EGF in the function of the mouse distal nephron.

Gastric mucosal atrophy: interobserver consistency using new criteria for classification and grading.

BACKGROUND AND AIMS: Considerable difficulties persist amongst pathologists in agreeing on the presence and severity of gastric atrophy. An international group of pathologists pursued the following aims: (i) to generate an acceptable definition and a simple reproducible classification of gastric atrophy; and (ii) to develop guidelines for the recognition of atrophy useful for increasing agreement among observers. METHODS: After redefining atrophy as the 'loss of appropriate glands' and examining histological samples from different gastric compartments, three categories were identified: (i) negative; (ii) indefinite; (iii) atrophy, with and without intestinalization. Atrophy was graded on a three-level scale. Interobserver reproducibility of the classification was tested by kappa statistics (general and weighted) in a series of 48 cases. RESULTS: The medians of the general agreement and weighted kappa values were 0.78 and 0.73, respectively. The weighted kappa coefficients, obtained by cross-tabulating the evaluation of each pathologist against all others, were, with only one exception, > 0.4 (moderate to excellent agreement). CONCLUSIONS: By using the definition of atrophy as the loss of appropriate glands and distinguishing the two main morphological entities of metaplastic and non-metaplastic types, a high level of agreement was achieved by a group of gastrointestinal pathologists trained in different cultural contexts.

Gastrointestinal neuroendocrine cell proliferations.

The gastrointestinal neuroendocrine cell proliferations are comprised of a few hyperplasias and various neoplasias. The better characterized hyperplasias include G-cell hyperplasia, either primary or secondary, enterochromaffin-like (ECL)-cell hyperplasias, generally secondary to hypergastrinemia, and EC-cell hyperplasias. The neoplasias include carcinoid tumors, demonstrating low malignancy and divided into foregut, midgut, and hindgut varieties, poorly differentiated neuroendocrine carcinomas resembling their pulmonary counterparts the "oat cell" carcinomas both in histological pattern and in their highly malignant behavior mixed endo-exocrine tumors, which in turn can be divided into composite tumors formed by a population of endocrine cells and a population of exocrine cells, and amphicrine tumors formed by a uniform population of cells with a mixture of endocrine and exocrine phenotypic traits. Although some of these mixed tumors show a degree of malignancy intermediate between the classical carcinoid and an adenocarcinoma, more information must be gathered to establish firm prognostic parameters for these relatively new entities.

Bile duct carcinoma associated with multiple von Meyenburg complexes in the liver.

A 61-year-old white woman was found to have disseminated abdominal carcinoma at autopsy. The primary site proved to be a bile duct carcinoma arising from ductal epithelium of multiple von Meyenburg complexes of the liver. No other neoplastic lesion was found elsewhere after careful, gross, microscopic examination. This case is thought to represent malignant transformation of the hamartomatous growth, previously reported only exceptionally.

Decreased expression of Fas (CD95/APO1) associated with goblet cell metaplasia in Barrett's esophagus.

Fas ligand (FasL) has been shown to induce apoptosis in cells expressing its receptor Fas. We have recently shown that Fas ligand is overexpressed in all cases of Barrett's metaplasia (BM) with dysplasia and esophageal adenocarcinomas, and in a few cases of BM negative with dysplasia. The aim of this work was to determine the status of Fas expression in BM with and without dysplasia or carcinoma. Formalin-fixed and paraffin-embedded tissue sections from esophageal biopsies and esophagectomy specimens with BM, with and without dysplasia and carcinoma, were immunostained for Fas and FasL using the immunoperoxidase technique. The percentage of positive cells in each case was evaluated and compared with the degree of dysplasia. When Fas expression was assessed in glands with goblet cell metaplasia, Fas immunoreactivity was either undetected or present in less than 10% of the cells in 85% of the cases, and only 1 (4%) of the 28 cases examined showed Fas immunoreactivity in more than 25% of the cells. When we compared Fas expression in goblet cell-containing glands with glands of gastric cardia phenotype, we found that in the 26 cases of BM with or without dysplasia Fas was completely undetectable in goblet cell-containing glands in 15 (58%) of the cases but was undetectable in only 3 (12%) of the glands with gastric cardia phenotype (P = .002). Fas is usually undetectable or is expressed at a low level in BM with or without dysplasia or carcinoma. Fas expression in goblet cell-containing glands is less frequent than in glands with gastric cardia phenotype in the same specimens. BM with dysplasia or carcinoma overexpress FasL, so decreased Fas expression may protect BM with dysplasia and carcinoma from self-destruction while allowing them to evade immune surveillance.

Metastatic breast disease 40 years after the initial diagnosis.

This is the case of a patient with metastatic disease diagnosed 40 years after a radical mastectomy which was followed by radiation treatment for breast cancer. The patient had nonspecific symptoms for 3 years, and a lymph node biopsy revealed the underlying cause to be recurrent breast cancer. Excision of the largest metastases combined with chemotherapy resulted in a further 3-year remission.

Immunohistochemical studies in endocrine carcinoma of the skin.

Twenty-two endocrine carcinomas of the skin were examined immunohistochemically for the presence of several polypeptides and serotonin. Eight tumors contained one or more polypeptides. Occasional cells containing calcitonin were found in several tumors. Rare cells with substance P, somatostatin, and ACTH were present in only one tumor each. None of the patients presented a clinical syndrome associated with any of these polypeptides. Met-enkephalin and serotonin were negative in all tumors. The lack of demonstration of met-enkephalin in these tumors makes their relationship to the Merkel cell uncertain.

Duodenal gangliocytic paraganglioma. Clinicopathologic and immunocytochemical study of 11 cases.

The clinicopathologic features of 11 cases (8 in men) of duodenal gangliocytic paraganglioma are presented. The patients averaged 56 years of age; none showed evidence of phakomatosis. Ten tumors occurred in the second portion of the duodenum, and one arose in the third portion. All tumors were polypoid, and half presented with gastrointestinal bleeding. The neoplasms were composed of paraganglioma and carcinoid-like elements, neurons, and Schwann as well as sustentacular cells. All tumors behaved in a benign fashion after local resection or snare polypectomy; long-term follow-up (1-25 years; mean, 8.3 years) showed no recurrence in any case. Immunocytochemical examination demonstrated the presence of somatostatin, serotonin, and human pancreatic polypeptide within endocrine cells and neurons.

Tissue-specific expression of kallikrein family transgenes in mice and rats.

To define the regulatory strategy for the transcriptional control of the kallikrein multigene family, we analyzed the expression of several kallikrein/SV40 T-antigen (TAg) fusion genes in transgenic mice and rats. Kallikrein family members are normally expressed at a high level in the submandibular gland and are expressed in a wide range of tissues that vary among individual family members. A total of 1.7 kb of proximal 5'-flanking DNA from the tissue kallikrein gene (rKlk1) was sufficient to confer much of the correct tissue-specific pattern on a TAg reporter gene. TAg mRNA was detectable in tissues that normally express rKlk1 and TAg-induced tumors arose in brain and pancreas. However, absolute levels of transgene mRNA were very low relative to the expression of the normal endogenous tissue kallikrein gene. In particular, expression in the salivary glands, normally very high for endogenous rKlk1, was either low or absent. An intact rKlk1 transgene with extensive flanking DNA (4.5 kb 5' and 4.7 kb 3') and complete intragenic (4 kb) sequences was expressed similarly to the fusion transgene, demonstrating that regulatory elements necessary for comprehensively correct expression are not contained within these additional gene regions. Two additional kallikrein/SV40 fusion transgenes were derived from other family members, one from the rKlk2 gene, which encodes tonin, and another from the rKlk8 gene, which encodes a prostate kallikrein. Whereas the endogenous rKlk2 and rKlk8 genes normally are expressed at high levels in rat salivary glands, they were not expressed in the salivary glands as transgenes. The results for these transgenes of three different family members indicate that control elements that direct the particular nonsalivary gland expression pattern characteristic of each family member may be present within the proximal 5'-flanking region of each gene, whereas regulatory sequences necessary for normal levels of expression in these tissues and for maximal salivary gland expression are not. We propose that the gene-associated regulatory sequences are complemented by a dominant control region that imposes salivary gland expression on the extended kallikrein family locus.