Ultrastructural studies of malignant cells in fluids

An ultrastructural study was performed on 104 sequential fluids in which more than eight malignant cells per ten high-power fields were found by routine light microscopy. The study included fluids associated with mesotheliomas, melanomas, lymphomas, squamous-cell carcinomas, small-cell anaplastic (oat-cell) carcinomas, and adenocarcinomas. Electron microscopic examination reliably separated lymphoid from epithelial malignancies and benign from reactive and malignant mesothelial cell proliferations. It also suggested or identified a primary site for the adenocarcinomas. Ultrastructural examination of fluids can be a valuable adjunct to routine light microscopy of cytology specimens. No false-positive diagnoses were encountered. Sampling was the most significant limitation for this technique.     

Diffuse sclerosing papillary carcinoma of the thyroid gland: immunohistochemical analysis of the local host immune response

Surgical specimens from four patients with diffuse sclerosing papillary carcinoma of thyroid were examined by electronmicroscopy. In addition, immunohistochemical examination using a panel of monoclonal and polyclonal antibodies was carried out in order to investigate the expression of HLA-DR antigen, the presence of Langerhans' cells and the phenotypic characteristics of the inflammatory infiltrate. The ultrastructural study showed that the intraglandular dissemination, typical of this tumour, was due to massive lymphatic invasion. Many Langerhans' cells were observed among tumour cells and in the lymphoid infiltrates in proximity to tumour foci. HLA-DR expression was seen on macrophages, Langerhans' cells, endothelial cells, lymphoid cells, many tumour cells and in some non-neoplastic follicles close to tumour clusters and lymphoid infiltrates. The immunohistochemical analysis of the inflammatory infiltrates showed a high proportion of B- and T-cells, and moderate numbers of plasma cells. Our results suggest that the tumour-specific immune response can give rise to an autoimmune reaction involving non-neoplastic follicles. It is suggested that this could be one of the mechanisms responsible for immunofacilitation of tumour growth.     

Ultrastructural analysis of salivary gland pleomorphic adenoma, with particular reference to myoepithelial cells

Previous ultrastructural studies of pleomorphic adenoma have presented conflicting results with regard to the role of myoepithelial cells in the histogenesis of this tumour. In the present study specimens of ten major salivary gland pleomorphic adenomas were examined ultrastructurally and a number of cell types identified. The material was subjected to quantification using the stereological method of point counting. The results showed a wide spectrum of differentiation within these tumours in which typical myoepithelial cells were rarely encountered even in situations where they are reported to occur in routine histological preparations. Cells with some myoepithelial features were more numerous but duct cells accounted for the majority of tumour cells. The ultrastructural findings correlated well with previously reported immunocytochemical data and further support certain ideas about salivary gland tumour histogenesis.     

Sections of the nipple and quadrants in mastectomy specimens for carcinoma are of limited value

AIM: To assess the value of nipple and quadrant sections in mastectomy specimens for carcinoma in detecting Paget's disease and multifocal carcinoma. METHODS: Two hundred and forty eight consecutive mastectomies performed for carcinoma were reviewed. The presence of Paget's disease of the nipple and mode of identification of any multifocal carcinoma was recorded. RESULTS: Nipple sections showed Paget's disease in eight specimens: in five the diagnosis had been made on previous biopsy and in three (1%) this was a new diagnosis. In the 220 specimens in which all four quadrants were sampled, multifocal disease was identified more often in specimens with invasive carcinoma (39 of 186; 21%) than in those with only ductal carcinoma in situ (0 of 34). In specimens with invasive carcinoma, multifocality was identified macroscopically in 20: on microscopy of tumour sections in four, on microscopic examination of quadrant sections in 11, in the nipple in three, and in both quadrant and nipple sections in one. Overall, multifocality was found on microscopic examination of quadrant or nipple sections in 15 of 220 specimens (7%). CONCLUSIONS: The low frequency of detection of multifocality or Paget's disease in nipple and quadrant sections from mastectomy specimens, combined with the fact that such findings do not affect patient management, suggest that nipple and quadrant sections should only be taken if resources permit.     

Mixed germ cell tumour of the mediastinum (seminoma, embryonal carcinoma, choriocarcinoma and teratoma). Light and electron microscopic cytology and histological investigation

A mixed germ cell tumour of the mediastinum was found in a 27-year-old subject. It had four components: seminoma, embryonal carcinoma, choriocarcinoma and teratoma. Material for light microscopy (L.M.) and ultrastructural studies were obtained with computerized tomography (CT) guided fine needle aspiration. Three histotypes were identified with ultrastructural studies while only seminoma was identified with L.M. The slides prepared for L.M. were reexamined in the light of findings with electron microscopy (E.M.) and previously unidentified cellular elements were found to be characteristic of choriocarcinoma and teratoma. Histological examination of surgical and autopsy material confirmed the cytological findings. Because of the singularity of this case, the rarity of this type of neoplasm and the difficulty in arriving at a differential diagnosis from other neoplastic and non neoplastic lesions, only the ultrastructural cytological examination allowed us to identify the mixed nature of the tumour. Such an identification is not always possible with L.M. but it is a determining factor in the prognosis and therapy.     

Adenosquamous carcinoma of the stomach: histological, histochemical and ultrastructural observations

The histochemical and ultrastructural features of an adenosquamous carcinoma of the stomach have been studied. Both the adenomatous and the squamous component of the tumour showed malignant characteristics. Elements which were considered as undifferentiated cells on light microscopy showed ultrastructural features of poorly differentiated squamous cells. No intermediate cells, sharing squamous and glandular aspects, were found by electron microscopical examination. The adenomatous component exhibited histochemical and ultrastructural features of the intestinal type of gastric carcinoma and the mucosa surrounding the tumour showed a colonic type of intestinal metaplasia. A possible histogenesis from a totipotential undifferentiated cell is considered.     

Ultrastructural and immunocytochemical study of a case of invasive cribriform breast carcinoma.

A case of recurrent invasive cribriform carcinoma of the breast mimicked the histological and mucin staining characteristics of adenoid cystic carcinoma. The diagnosis was based on negative immunocytochemical staining for laminin and ultrastructural evidence of luminal differentiation by cells lining the cystic spaces. Accurate characterisation of this type of breast tumour can be facilitated by retrospective immunocytochemical or ultrastructural examination, or both.     

Pulsed field gel electrophoresis on frozen tumour tissue sections.

The application of pulsed field gel electrophoresis (PFGE) to the molecular genetic analysis of solid tumours has been restricted by the requirement for whole single cells as a DNA source. A simple technique which allows for the direct analysis of histologically characterised solid tumour material by pulsed field gel electrophoresis was developed. Single frozen tissue sections obtained from colonic carcinoma specimens were embedded without further manipulation in molten, low melting temperature agarose. The tumour DNA contained within the agarose plug was subjected to restriction enzyme digestion and PFGE. Sufficient high molecular weight DNA is yielded by this method to obtain a hybridisation signal with a single copy probe. Histological examination of adjacent tissue sections may also be carried out, permitting correlation between molecular analysis and tumour histology.     

Acute basophilic leukemia. A clinical, morphologic, and cytogenetic study of eight cases

The authors describe eight cases of acute basophilic leukemia. In six of the eight cases, basophilic involvement was not apparent by light microscopic examination. The cases were identified on the basis of ultrastructural evidence for basophil/mast cell differentiation of the blasts with little or no differentiation into other lineages. Ultrastructural analysis revealed immature basophil granules in blasts in all eight cases and theta granules in blasts in four cases. In three cases, ultrastructural evidence of mast cell differentiation also was present, with rare cells showing evidence for both basophil and mast cell differentiation. No clinical features distinguished this group of patients from others with acute myeloid leukemia. Cytogenetically, the cases were heterogeneous. Three had a Philadelphia chromosome; none had a t(6;9). The authors conclude that ultrastructural analysis usually must be used to diagnose acute basophilic leukemia, that acute basophilic leukemia is associated frequently with the Philadelphia chromosome, and that the ultrastructural findings provide evidence for a common origin of basophils and mast cells.     

Large bowel mucosal dysplasia and carcinoma in ulcerative colitis

The clinicopathological details of eight cases of ulcerative colitis complicated by carcinoma of the colon are described. There was a total of 14 primary colonic cancers, six of which were not detected before pathological examination of the resection specimens. The reason for this may be related to atypical tumour growth patterns. Three occurred in flat mucosa, one in a mucosal plaque lesion, and another in polypoidal mucosa. The occurrence, distribution, and morphology of mucosal dysplasia were noted in both resection specimens and biopsies taken at varying stages before resection. Tumour was associated with normal and adjacent dysplastic mucosa of varying grades. The extent and grade of dysplasia were not reliable indicators of tumour differentiation or subsequent clinical outcome. Only two cancers were poorly differentiated. In five cases a total of 23 mucosal biopsies were taken, all less than 12 months before resection. Three rectal biopsies were graded positive for dysplasia and three colonic biopsies indefinite for dysplasia. The subsequent resection specimens showed both dysplastic and carcinomatous changes. Three rectal and 14 colonic biopsies were graded negative for dysplasia despite positive findings in the subsequent resection specimens. This anomaly is partly attributed to the patchy nature of dysplasia in colitic mucosa. Two cases illustrate the possibility of dysplasia pursuing a rapidly progressive course. The mucosal changes of ulcerative colitis were assessed using a recently introduced and standardised international classification.     

Ultrastructural changes of the bile secretory apparatus and bile ductule in experimental hepatitis B with neither apparent biochemical nor light-microscopic cholestasis

An ultrastructural study on the hepatic bile secretory apparatus and bile ductules was carried out using liver biopsy specimens of six chimpanzees with experimental hepatitis B. Although biochemical cholestasis was very mild or lacking and light-microscopic evidence of cholestasis was not evident, various ultrastructural changes which had been described in association with cholestasis were detected. The significance of these ultrastructural changes in relevance to cholestasis was difficult to determine, nevertheless electron-microscopic examination could possibly be the most sensitive means for the diagnosis of cholestasis. These ultrastructural changes appeared to be indifferent to the necrotizing process of liver cells in experimental hepatitis B.     

Uttrastructure of skin biopsy specimens in lysosomal storage diseases: Common sources of error in diagnosis

Common sources of error in the diagnosis of lysosomal storage diseases by ultrastructural examination of skin specimens have been identified in a series of biopsies from 72 patients. Four principal factors have emerged as leading pitfalls and sources of error in diagnosis. First, the skin biopsy technique itself may lead to alterations of normal skin ultrastructure. Second, artifacts may be produced during fixation and preparation of tissue for electron microscopy. Third, cellular organelles and structures normally present in human skin may be mistakenly interpreted as pathological. Fourth, the use of cultured skin fibroblasts for ultrastructural identification of storage material is often accompanied by artifacts induced in tissue culture and is not recommended. Recognition of these common problems may aid interpretation of the fine structure of skin abnormalities. Furthermore, when skin biopsy specimens are used as the primary source of diagnostic material, correlation of both skin ultrastructure and assay for specific lysosomal enzymes in cultured dermal fibroblasts will facilitate diagnostic accuracy.     

The relevance of ultrastructural examination in the classification of primary lung tumours

Biopsies from 50 primary lung tumours were classified according to the World Health Organisation's Histological Typing of Lung Tumours. They were also subjected to electron microscopic examination. Comparison of the diagnoses made by these separate methods showed that many poorly differentiated squamous cell carcinomas had been incorrectly classified. Agreement was good in the diagnosis of adenocarcinoma whilst ultrastructural examination of small anaplastic carcinomas disclosed a neuroendocrine tumour with a combination of squamous and glandular elements. Large cell anaplastic carcinoma proved to be a 'waste-basket' containing tumours which displayed ultrastructural characteristics of poorly differentiated squamous cell carcinoma, poorly differentiated adenocarcinoma or neuroendocrine carcinoma. Electron microscopy was also valuable in characterization of other pulmonary tumours whose identity could not be resolved at the light microscopic level. Ultrastructural examination may provide a better understanding of the histogenesis and derivation of lung tumours, as well as their behaviour and therapeutic response.     

Duodenal Ultrastructure in Patients with Chronic Renal Failure with a Comment on the Incidence of Campylobacter pylori Infection

This study was aimed at identifying ultrastructural abnormalities that may be present in the duodenum of patients with chronic renal failure who were undergoing hemodialysis and that may have an effect on the normal absorptive function of the duodenum. Duodenal biopsy specimens from ten patients and four controls were examined. Light microscopy showed gastric metaplasia in eight patients and in none of the controls. The main electron microscopic findings were related to the metaplastic changes, with abundant mucous globules and abnormal microvilli being seen in the affected columnar cells. Prominent lysosomes and wide intercellular spaces were seen in some areas. Goblet and enterochromaffin cells appeared to be normal. Some of the reported abnormal duodenal absorptive functions may be related to the ultrastructural abnormalities seen in the microvilli and the apical parts of the columnar cells. Because of the known association between gastric metaplasia and colonization of the duodenum with Campylobacter pylori, we looked for these organisms in our cases. Light microscopy demonstrated these bacteria in only one of the eight duodenal biopsy specimens with gastric metaplasia. Particles, probably representing degenerated bacteria, were seen by electron microscopy in most of the examined specimens, however, suggesting that the duodenum of these patients remains hostile to the growth of these organisms.     

ULTRASTRUCTURAL CHANGES OF THE BILE SECRETORY APPARATUS AND BILE DUCTULE IN EXPERIMENTAL HEPATITIS B WITH NEITHER APPARENT BIOCHEMICAL NOR LIGHT-MICROSCOPIC CHOLESTASIS

An ultrastructural study on the hepatic bile secretory apparatus and bile ductules was carried out using liver biopsy specimens of six chimpanzees with experimental hepatitis B. Although biochemical cholestasis was very mild or lacking and light-microscopic evidence of cholestasis was not evident, various ultrastructural changes which had been described in association with cholestasis were detected. The significance of these ultrastructural changes in relevance to cholestasis was difflcult to determine, nevertheless electron-microscopic examination could possibly be the most sensitive means for the diagnosis of cholestasis. These ultrastructural changes appeared to be indifferent to the necrotizing process of liver cells in experimental hepatitis B. ACTA PATHOL. JPN. 35 : 1343–1358, 1985.     

Intranodal and extranodal tumour growth in early metastasised non-small cell lung cancer: problems in histological diagnosis.

AIM--To question the observer reliability or agreement of reports on the intranodal and extranodal tumour growth patterns in early metastasised non-small cell lung cancer (NSCLC). METHODS--In a pilot study original histological sections of mediastinal lymph node metastases from NSCLC obtained by lymph node dissection (n = 82) or by mediastinoscopy (n = 62) were examined and classified independently by three pathologists as extranodal, intranodal, or indefinite. After clear criteria for these growth patterns had been defined sections were re-examined and recategorised one year later. Interobserver agreement was examined for both investigations. RESULTS--In the dissected lymph nodes the kappa value improved significantly from 0.52 (moderate agreement) at the first investigation to 0.72 (good agreement) at the second. In the mediastinoscopic lymph node biopsy specimens an increase in kappa value from 0.50 at the first to 0.67 at the second examination was found, although this improvement was not significant. In mediastinoscopic biopsy specimens a very high proportion of tissue samples showed indefinite tumour extension. CONCLUSION--Good reproducibility of intranodal and extranodal growth patterns in the histological examination of mediastinal lymph node metastases can be achieved, provided that pathologists use strictly defined criteria. In mediastinoscopic biopsy specimens it is often impossible to differentiate between intranodal and extranodal tumour growth.     

Comparative Ultrastructure of Needle Aspiration Biopsy and Surgical Resection Specimens of Lung Tumors

Ultrastructural examination affords conclusive evidence for classification of lung tumors. Tissue properly fixed for electron microscopy is not available in many cases, however. Ultrastructural diagnosis of resected specimens obviously follows, rather than directs, the surgical treatment. Fine-needle aspiration (FNA) of lung masses is recommended as a means to obtain lung tumor tissue for electron microscopy. Nevertheless, no comparison has been made between ultrastructural information gained from aspiration specimens and resected specimens. Electron microscopy was performed on transthoracic FNA specimens of 10 lung tumors for which surgical resection was subsequently performed. Glutaraldehyde-fixed specimens from FNA and surgical resection were prepared for electron microscopy according to routine procedures. Fixation of the FNA specimens was equivalent or superior to that of the resected specimens in 9 of the cases. Three of the FNA specimens contained necrotic as well as viable tissue. Features essential for diagnosis such as desmosomes, junctions, neurosecretory granules, intermediate filaments, glycogen, lipid, mucin, and microvilli were identifiable in both FNA and resected specimens. FNA specimens therefore yield a representative sample of the ultra-structural features of lung tumors when adequate cellular material is obtained. Use of a coaxial needle sampling technique with immediate microscopic assessment reduces the likelihood of retrieving only blood or necrotic tissue in the electron microscopy specimens.     

Is there any justification for the routine examination of bowel resection margins in colorectal adenocarcinoma?

The proximal and distal bowel resection margins of 339 specimens of colorectal adenocarcinoma were examined for the presence of tumour. In only five cases was tumour found in a resection margin. In four of these cases macroscopic examination showed that the tumour extended to the resection margin. In the fifth case the tumour was present in the pericolic fat. These results and reported data on the intramural spread of colorectal cancer suggest that examination of bowel resection margins is unnecessary unless the tumour extends to within 2 cm of the resection margin. Examination of the deep radial margins of the tumour and slides to show other prognostic indicators would be a more effective use of histopathological resources.     

Human malignant mesothelial cells: Variability of ultrastructural features in established and nude mice transplanted cell lines

The aim of this study was to determine, by transmission electron microscopy, the differentiation features of 21 human malignant mesothelioma cell lines (HMCLs) established from 13 specimens of 12 confirmed human malignant mesotheliomas, and of tumours induced in nude mice injected with 16 HMCLs. Fifty per cent of HMCLs showed typical mesothelial differentiation (long and slender microvilli, desmosomes, perinuclear intermediate filaments); 29 per cent did not show differentiation; and the remainder were poorly differentiated. Three human tumour specimens gave several different HMCLs; the cell lines obtained from a given tumour exhibited variable mesothelial differentiation. Eleven HMCLs were compared with the native tumour. Four were similar to the tumour and seven were less well differentiated, in most cases in relation to their microvilli. With six HMCLs, tumours induced in nude mice were less well differentiated than the corresponding cell lines, whereas with four HMCLs, tumours were equally or better differentiated. However, in most nude mice tumours, typical mesothelial microvilli were present. These results show that cell lines established from malignant mesothelioma may exhibit dedifferentiated features. However, while the variability in ultrastructural differentiation may result from the culture microenvironment, it could also be related to the state of differentiation, of the native tumour sample and to tumour cell heterogeneity.     

Electron microscopic assessment of smeared or imprinted specimens of solid tumours

The aim of this study was to develop a reliable method for preparation of smeared or imprinted cytology specimens for electron microscopic examination. Ten different solid tumours were studied. In each case one air-dried (Diff-Quik stained) and one alcohol-fixed (Pap stained) smear was prepared for diagnostic purposes. Simultaneously, a third smear or imprint was prepared for electron microscopy on a coverslip that was coated with poly-L-lysine and attached to a glass slide using double-sided adhesive tape. The smear or imprint was primary fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 hours. The coverslip was removed from the slide, placed in a glass petri dish and processed for electron microscopy. The smear/imprint on the coverslip was embedded in resin on a silicon embedding mould and allowed to polymerise for 8-12 hours. The coverslip was removed using liquid nitrogen and the block sectioned using standard techniques as for a monolayer. The specimens collected for electron microscopy using this technique yielded sufficient material for assessment with excellent tissue preservation producing good ultrastructural detail. Focal mechanical damage was seen in some specimens but diagnostic areas were always found within the block. As the smear/imprint can be regarded as a monolayer, the processing time can be reduced compared with solid tissue specimens. This technique ensures that well-preserved tissue is available for electron microscopy even when the sample size is very limited.