Ultrastructural immunostaining of infiltrating ductal breast carcinomas with the monoclonal antibody H: a comparative study with cytokeratin 8

The monoclonal antibody H (mAbH) detects an epitope consisting of an O-linked N -acetyl glucosamine (O-GlcNAc) and neighboring amino acids. This epitope has been found by using extracts from the MCF-7 human breast carcinoma cell line in immunoblotting experiments, on cytokeratin 8 (CK8) and 5 other polypeptides. In the present study, a double immunogold method was applied for the colocalization of CK8 and mAbH epitope on epoxy thin sections in 18 cases of infiltrating ductal breast carcinomas (IDBC) and in 6 cases of fibroadenomas, to study the accurate subcellular distribution of CK8 in breast cancer cells, as compared to the 5 polypeptides, recognized by mAbH. Furthermore, a detailed quantitative evaluation of the double immunolocalization over the cellular compartments of cancer cells was undertaken with the aid of a computerized image analysis system and the results were assessed statistically. The distribution pattern of CK8 and the mAbH epitope in the neoplastic mammary epithelial cells was similar in IDBC as compared to fibroadenomas, while the gold labeling intensity of these epitopes differed over the cellular compartments between malignant and benign biopsies. The results reveal the significance of the role of CK8 and O-GlcNAc glycosylation in the biology of the neoplastic mammary cells in vivo, determining their malignant potential.     

Expression of bcl-2, Bax and Fas in oxyphil cells of Hashimoto thyroiditis

Immunoreactivity for bcl-2, Bax and Fas was analysed in 16 cases with Hashimoto thyroiditis. Bcl-2-expression was constantly seen in regular thyrocytes and in the mantle-zone of lymphofollicular infiltrates. However, thyrocytes in the vicinity of lymphoid infiltrates and, especially, mitochondria-rich oxyphil cells exhibited reduced staining or none at all for bcl-2. Bax was found to be weakly reactive or negative in normal thyrocytes and was not up-regulated in bcl-2-deficient epithelial cells. In contrast, expression of Fas was markedly increased both in typical thyrocytes and in oxyphil cells within areas of lymphocytic infiltration. In conclusion, focal lack of bcl-2 expression together with up-regulation of Fas is a constant feature of Hashimoto thyroiditis. The reaction pattern of oxyphil cells is identical to that of affected typical thyrocytes without proliferation of mitochondria. Loss of bcl-2 with up-regulation of Fas is therefore likely to precede oncocytic change. Whether these alterations are involved in the process of oncocytic transformation remains to be clarified, however. Received: 25 June 1999 / Accepted: 14 January 1999     

Hypoxia upregulates the expression of the O-linked N-acetylglucosamine containing epitope H in human ependymal cells

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by mouse IgM monoclonal antibody H (mabH). Epitope H is present in several types of cells and in several polypeptides outside the CNS. Previous results have shown that in the adult human brains, epitope H is confined mostly to a minority of fibrous astrocytes, and it is greatly upregulated in the reactive astrocytes. Post-translational modification with O-GlcNAc occurs on many proteins involved in several cell processes, such as cell cycle progression, apoptosis, proteasome degradation pathways, and modulation of cellular function in response to nutrition and stress. Hypoxia is one of the major causes of cellular stress. Therefore, in this study, we used the mAbH and the indirect immunoperoxidase method to investigate the expression of epitope H in ependymal cells in brains of persons who died with signs of hypoxic encephalopathy. The results of the present study showed that practically all ependymal cells showed cytoplasmic staining for epitope H in supranuclear cytoplasm in the brain of two premature neonates and in ten infants who died with signs of hypoxic encephalopathy. However, the overwhelming majority of ependymal cells of the nine human embryos taken from legal abortions, ranging from 26 days until 13 weeks of gestational age, and of the ten infants’ brains without any sign of hypoxic encephalopathy remained negative. Only occasionally did the ependymal cells show weak cytoplasmic staining in some foci. In addition, the reactive astrocytes in the hypoxic brains showed strong cytoplasmic staining, confirming previous results.     

Nontyrosine crystalloids in salivary gland lesions: report of seven cases with fine-needle aspiration cytology and follow-up surgical pathology

We report a series of seven patients who underwent fine-needle aspiration (FNA) for clinically apparent parotid gland lesions. In all seven cases, numerous to abundant polyhedral, multifaceted (nontyrosine) crystalloids were noted in the background of scanty cellular specimens composed predominantly of oncocytic cells. Subsequent surgical excision showed that three of the seven glands revealed sialolithiasis and sialadenitis without evidence of neoplasia. The histology of the remaining four cases consisted of two Warthin's tumor, one oncocytic papillary cystadenoma, and one cellular benign mixed tumor. In all seven cases the nontyrosine crystalloids were found in highest concentrations in cystic spaces lined with oncocytic metaplastic cells. We conclude that nontyrosine cystalloids can be associated with both neoplastic and nonneoplastic salivary gland disease, and they may be a product of oncocytic cell secretion. Diagn. Cytopathol. 2000;22:167-171.     

Multifocal nodular oncocytic hyperplasia of bilateral parotid glands: A case report with a histological variant of clear cells

A case of multifocal nodular oncocytic hyperplasia (MNOH) of the bilateral parotid gland is presented. An 80-year-old woman was admitted to hospital because of painless swellings in bilateral parotid regions. Histologically, the nodular lesion had incomplete capsules and engulfed the surrounding parotid gland at the periphery. The lesions were mostly composed of clear cells, while the peripheries of the lesions had typical oncocytic cells with abundant fine granules. The histological existence of the clear cell component in the lesions led to misdiagnoses of other clear cell neoplasms. However, this case had multiple nodules in bilateral glands. No evidence of malignant histological findings was found. Moreover, the clear cells, as well as the oncocytic cells, were demonstrated to have mitochondria and glycogen in their cytoplasm using special staining. Based on these findings, the diagnosis of this case was MNOH in the parotid gland. We also discuss the differential diagnosis for clear cell lesions.     

Detection of damage to the mitochondrial genome in the oncocytic cells of Warthin's tumour

Warthin's tumour of the salivary glands is composed of oncocytic cells containing excessive numbers of mitochondria which show frequent structural abnormalities and reduced metabolic function. Recent evidence of a strong association between cigarette smoking and the occurrence of Warthin's tumour prompted this study, to look for evidence of damage to mitochondrial DNA (mtDNA) that could be the result of an increase in oxidative stress; two-colour fluorescence in situ hybridization (FISH) was developed to show the distribution of mitochondria with deleted mtDNA in paraffin wax-embedded material. Approximately 10% of mtDNA bears the 'common' 4977 bp deletion. Using the polymerase chain reaction (PCR), the 4977 bp deletion was further quantified, in Warthin's tumour and age-matched normal parotid control tissue. Whilst the deletion was present in all parotid tissue, its presence was significantly higher in oncocytic tumour cells. In a small number of controls, there was a trend towards higher concentrations of the deletion in smokers.     

Prominent oncocytic metaplasia in pleomorphic adenoma: A potential diagnostic pitfall

Pleomorphic adenoma (PA) is the most common salivary gland tumor. The cytological features of PA are well recognized, and its diagnosis is straightforward in most cases. Some metaplastic changes in PA are well known; however, occurrence of oncocytic metaplasia in PA is very rare. In this report, we describe the first cytological case of prominent oncocytic metaplasia in PA identified based on immunocytochemical analysis. We report the case of a 62‐year‐old Japanese female who presented with swelling of the left neck region. A fine‐needle aspiration cytologic examination was performed followed by surgical resection. The Papanicolaou smear revealed the presence of discohesive neoplastic cells in a myxoid background. These neoplastic cells had a relatively rich, granular cytoplasm, and round nuclei with moderate pleomorphism. Initial cytodiagnosis revealed carcinoma ex PA (CXPA). Immunocytochemical analysis showed that abundant mitochondria were present in the cytoplasm of these neoplastic cells. Histopathological examination of the resected tumor demonstrated proliferation of oncocytic neoplastic cells within a myxoid material and the presence of conventional PA components. A final diagnosis of prominent oncocytic metaplasia in PA was made. Oncocytic metaplasia showed nuclear atypia and pleomorphism; therefore, CXPA, which presents with severe nuclear atypia and necrotic background, must be differentiated from oncocytic metaplastic PA. Recognition of oncocytic metaplasia in PA is important for correct diagnosis.     

Identification of oncocytic lesions of salivary glands by anti-mitochondrial immunohistochemistry

 

Aims:

To evaluate the immunohistochemistry using an anti-mitochondria antibody in the investigation of various oncocytic lesions of the salivary glands.  

Methods and results:

Ten cases of adenolymphoma (Warthin's tumour) and one case each of benign oncocytoma and oncocytic carcinoma of the salivary glands were examined. Normal salivary glands were also tested. They were investigated immunohistochemically using mouse monoclonal antibody against human mitochondria. In normal salivary glands, epithelial cells of the striated ducts showed a thick linear immunoreactivity, which corresponded well to the intracytoplasmic distribution pattern of mitochondria. In addition, a small number of swollen epithelial cells showing an intense, finely granular immunoreactivity in the cytoplasm were scattered in the ductal system and acini ('oncocytic metaplasia'). Almost all neoplastic cells involved in adenolymphoma, benign oncocytoma, and oncocytic carcinoma showed an intense, finely granular immunoreactivity in the cytoplasm.  

Conclusions:

Immunohistochemistry using the anti-mitochondria antibody proved to be a highly sensitive and specific method for light microscopic identification of mitochondria and superior to routine H & E or PTAH stain especially in the detection of isolated oncocytic cells.     

Oncocytic adrenocortical carcinoma: a morphologic, immunohistochemical and ultrastructural study of four cases.

We present the clinical, histologic, immunohistochemical, and ultrastructural findings of four cases of non-functioning oncocytic adrenocortical carcinomas. The patients' ages ranged from 39 to 71 years. There was no sex predilection. Large yellow-tan tumors (8.5 to 17.0 cm), well demarcated from the adjacent kidney, were seen with a thin rim of normal adrenal gland along one edge. One tumor invaded the inferior vena cava and extended up to the level of the right atrium, and another metastasized to bone. The other two tumors had similar morphologic features and therefore were considered carcinomas. Histologic sections of all four cases showed a diffuse proliferation of polygonal neoplastic cells with large nuclei containing prominent nucleoli and abundant granular and eosinophilic cytoplasm. Occasional mononuclear and binucleated giant cells were noted in one case. There were rare mitotic figures (less than one per 10 high power fields). All tumors were immunoreactive for cytokeratins (AE1/AE3 and CAM5.2). Inhibin was focally expressed by one tumor and its bone metastasis. Ultrastructurally, the cytoplasm of the neoplastic cells was packed with innumerable mitochondria. Cytologic atypia or mitotic rate cannot reliably predict the biologic behavior of oncocytic adrenocortical neoplasms. Large tumor size (4/4), extracapsular extension (3/4), blood vessel invasion (2/4), necrosis (4/4), and metastasis (1/4) are features of malignancy for oncocytic adrenocortical carcinomas. The treatment of these tumors is complete surgical excision.     

Random cytochrome-C-oxidase deficiency of oxyphil cell nodules in the parathyroid gland. A mitochondrial cytopathy related to cell ageing?

Cytochrome-c-oxidase (complex IV) was histochemically studied in oncocytic adenoma (n = 10) and carcinoma of the thyroid gland (n = 3), cystadenolymphomas and oncocytic adenomas of the major salivary glands (n = 9), oncocytic neoplasia of the kidney (n = 1) and in 21 parathyroid glands with primary hyperparathyroidism and adenomatous proliferation (n = 17) and secondary hyperparathyroidism with hyperplasia (n = 4). Only in the parathyroids defects of cytochrome-c-oxidase were found being expressed in all 4 glands with hyperplasia (14 defects) and in 5 of the 17 adenomas (11 defects). All defects were confined to foci with oxyphil cell differentiation, the defect areas varying from 0.09 to 21.10 sq mm in hyperplastic glands and from 0.11 to 13.88 sq mm in adenomas, the size of the oxyphil foci varying from 0.12 sq mm-105.38 sq mm. However, not every oxyphil nodule of a gland was devoid of cytochrome-c-oxidase activity. Of 6 predominantly oxyphil adenomas, 4 showed no defects. No defects were observed either in 2 adenomas without oxyphil cells. Further enzymes of the respiratory chain, succinate dehydrogenase (complex II) and ATP synthetase, (complex V) were devoid of defects. In parathyroids with hyperplasia and oxyphil areas, defects of cytochrome-c-oxidase occurred significantly more often and tended to be larger than in adenomas, statistical analysis revealing a significant correlation between the occurrence of defects and the number of oxyphil foci but not with the total oxyphil area.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure of sialadenoma papilliferum

Sialadenoma papilliferum is a rare tumor of salivary gland origin and has been reported in the parotid and minor salivary glands of the oral cavity. This tumor is morphologically similar to the papillary syringoadenoma of the sweat gland. We report the clinical and morphologic features of five cases and review the literature. Ultrastructural examination of case 1 revealed the predominant cell type to be an oncocytic cell. These cells contained numerous mitochondria, exhibited parallel filaments within the cell cytoplasm, and were attached by desmosomes. The neoplastic cells appear to exhibit characteristic features of various cell types of the salivary gland duct apparatus.     

Cystic lesions of the salivary glands: cytologic features in fine-needle aspiration biopsies

A variety of neoplastic and nonneoplastic lesions of the salivary glands have a predominantly cystic architecture. Fine-needle aspirates of these lesions yield watery or mucoid material, frequently of low cellularity. Such aspirates may be obtained from mucus retention cysts, lymphoepithelial cysts, cystadenomas, Warthin's tumors, cystic pleomorphic adenomas, low-grade mucoepidermoid carcinomas, cystadenocarcinomas, and examples of polycystic disease of the parotid gland. The cellular component within the fluid obtained from these lesions may be exceedingly scant or absent, making cytologic diagnosis difficult and, at times, impossible. We studied a series of 56 cystic lesions of the salivary glands, including 38 Warthin's tumors, 6 benign cysts, 2 lymphoepithelial cysts, 5 low-grade mucoepidermoid carcinomas, 1 cystic pleomorphic adenoma, 2 cystadenomas, and 2 cystadenocarcinomas. Careful attention to the cellular elements present often allowed definitive cytologic diagnosis, with an overall accuracy rate of 84%. The presence of atypical squamous metaplasia in oncocytic lesions was a significant cause of false-positive diagnoses of carcinoma (4 cases, 7%). Aspirates of low-grade mucoepidermoid carcinoma may contain no epithelial cells and result in false-negative diagnoses (1 case, 2%).     

Metaplastic lesions of the extrahepatic bile ducts: a morphologic and immunohistochemical study.

Although metaplastic changes can occur in the extrahepatic bile ducts, a detailed morphologic study of these lesions has not been done. We examined the bile duct mucosa in 42 pancreaticoduodenectomy specimens, 32 with neoplastic lesions and ten with inflammatory lesions of the extrahepatic bile ducts, to assess the prevalence and type of metaplastic lesions. For comparison, the common bile ducts from 10 autopsy cases were reviewed. Twenty of the 42 total cases (48%), 13 of the 32 neoplastic cases (40%), and 7 of the 10 inflammatory cases (70%) had metaplastic changes. Pyloric gland metaplasia was the most common type (16/20 cases; 80%), whereas intestinal metaplasia was seen in 1/20 cases (5%). A combination of pyloric gland and intestinal metaplasia occurred in 2/20 cases (10%), and squamous metaplasia plus the above-mentioned two types of metaplasia was seen in 1/20 cases (5%). None of the normal common bile ducts obtained from ten autopsies had metaplastic changes. Endocrine cells were identified in nine (56%) of 17 metaplastic lesions. In contrast, endocrine cells within the intramural glands were seen in only 2 of the 10 normal common bile ducts. Although a significant proportion of carcinomas (6/13 cases) was in close proximity to areas of metaplasia, we were unable to find dysplastic foci within the metaplastic glands or the metaplastic surface epithelium. Reactive atypical cells involved the surface biliary epithelium and intramural glands and were associated with inflammation and metaplastic changes. The presence of goblet, mucinous, squamous, and reactive atypical cells in association with hyperplasia of intramural glands in frozen sections or small biopsy specimens may be mistaken for malignancy; hence, recognition of these lesions is of diagnostic importance.     

Lymphoid proliferations and lymphomas associated with gastric metaplasia, dysplasia, and carcinoma.

Gastric carcinomas are invariably accompanied by lymphoid proliferations. We studied their features in 22 resected gastric carcinomas in which the lymphoid proliferations ranged from reactive lymphoid follicles to mucosa-associated lymphoid tissue (MALT) lymphomas. In most cases, the collections of lymphocytes were abundant, which is remarkable considering the lack of lymphoid tissue in the normal stomach. They were not haphazardly located but in direct contact with the metaplastic, dysplastic, and neoplastic epithelial cells, in positions suggestive of defense barriers. They consisted of newly formed lymphoid follicles with reactive germinal centers sometimes high up in the superficial mucosa, collections of plasma cells beneath the surface epithelium, and large aggregates of B cells above and below the muscularis mucosae as well as abundant T cells. The latter, both CD4+ and CD8+, were seen within metaplastic epithelial cells as well as within carcinomatous glands that were partially destroyed, resembling apparent neoplastic lympho-epithelial lesions (LEL). In three cases, the B cells infiltrating the gastric muscular layers represented MALT-lymphomas adjacent to gastric carcinomas, as confirmed by polymerase chain reaction (PCR) analysis in two cases. In a case of lymphoepithelioma-like carcinoma, the excessive lymphoid cells were predominantly of T-CD8+ type. In this case, EBV identified by EBV-encoded RNA and latent membrane protein was present in large amounts. Helicobacter pylori was seen in only six cases in areas of chronic gastritis that were distant from carcinoma. H. pylori was not present in the areas of metaplasia, dysplasia, or carcinoma. It appears that the lymphoid proliferations accompanying these gastric changes do not arise in response to the pathogenic agent H. pylori, which caused the persistent infection leading to them yet is no longer present, but rather in response to the existence of the abnormal epithelial cells. Thus the lymphoid proliferations consistently associated with gastric metaplasia, dysplasia, and neoplasia may be regarded as immune reactions to the long-term cellular changes triggered by the initial chronic gastritis. On rare occasions, the exaggerated lymphoid proliferations may reach the end of the spectrum, resulting in MALT lymphomas coexistent with gastric carcinomas.     

An immunohistochemical study of the tissue distribution of the breast cyst fluid protein, zinc alpha2 glycoprotein

Zinc alpha 2 glycoprotein is one of the proteins present in breast cyst fluids, being found at levels 30-50 times its plasma concentration. Using an immunoperoxidase technique the distribution of this glycoprotein has been studied in a range of non-mammary tissues and carcinomas, as well as in normal, benign and malignant breast specimens. The breast cyst fluid protein was detected in all apocrine cells of skin and in the apocrine metaplastic epithelium lining of breast cysts. A progression was apparent from normal to hyperplastic breast in the number of cells reacting, particularly of cystically dilated acini, to a final consistent staining of apocrine-lined cysts. Zinc alpha 2 glycoprotein was demonstrated in 16 of 33 invasive carcinomas, 15 of which were eosinophilic on haematoxylin and eosin staining, and in one of three non-invasive carcinomas. No staining was apparent in other non-mammary tissues and carcinomas apart from weak reactivity of serous cells of the parotid gland. Zinc alpha 2 glycoprotein is, therefore, a reliable immunohistochemical marker of apocrine cell differentiation.     

Oncocytic carcinoma of the breast: frequency, morphology and follow-up

Oncocytic breast carcinomas are tumors composed of no fewer than 70% of oncocytic cells (World Health Organization). The purpose of this study was to determine the frequency, morphologic, immunohistochemical, and clinical features of invasive oncocytic carcinoma in a large series. Twenty-eight cases of putative oncocytic breast carcinoma (selected cases group) and 76 consecutive cases of invasive breast carcinoma (consecutive cases group) were analyzed. Immunohistochemistry for mitochondria, gross cystic disease fluid protein 15, chromogranin, estrogen receptor, progesterone receptor, androgen receptor, HER2/Neu, cytokeratin 7, cytokeratin 14, epithelial membrane antigen, and differentiation cluster 68 was performed. Score for mitochondria was based on intensity and percentage of immunopositive cells. Classes were as follows: (1) oncocytic carcinoma: at least 70%, 3+; (2) mitochondrion-rich carcinoma: 50% to 70%, 3+, or more than 50%, 2+; and (3) all the other cases were referred to as invasive breast carcinoma. Ultrastructural examination was available for 6 cases of oncocytic carcinoma. Morphologic and immunohistochemical features of the 3 groups were compared using Fisher exact test (P < .05). For overall survival analysis, Kaplan-Maier curves were compared using log-rank and Wilcoxon tests (P < .05). Our results suggest that oncocytic breast carcinoma is a morphologic entity with distinctive histologic and ultrastructural features. Mitochondrion-rich carcinomas are histologically similar to oncocytic carcinomas and constitute 19.7% of all invasive carcinomas, indicating that cytoplasmic eosinophilia in breast cancer cells is often due to accumulation of mitochondria. Oncocytic carcinomas and mitochondrion-rich carcinomas are more often grade III tumors and show human epidermal growth factor receptor 2 overexpression. Clinical features and overall survival of oncocytic carcinomas are not distinctive because they are similar to those of the other cases when matched for grade and stage.     

Immunoreactivity of p53, Ki-67, and Bcl-2 in oncocytic adenomas and carcinomas of the thyroid gland.

To analyze relevant factors of neoplastic transformation in oncocytic neoplasms of the thyroid, expression of p53, Ki-67, and bcl-2 has been studied in oncocytic carcinomas (n = 17) and compared with results obtained in oncocytic adenomas (n = 20). P53 protein accumulation was found immunohistochemically in 75% of the oncocytic adenomas (15 of 20) and 88% of the oncocytic carcinomas (15 of 17). Eight of 17 of the carcinomas (47%), but only 3 of the 20 adenomas (15%), showed nuclear p53 accumulation in more than 10% of the cells, mostly in a focal pattern. Ki-67 expression also differed significantly between adenomas and carcinomas. The median of Ki-67-positive cells was 12/10 high-power fields (HPF) for adenomas and 76/10 HPF for carcinomas (P < .001). Furthermore, metastatic carcinomas had a significantly higher Ki-67 positivity than nonmetastasized carcinomas (164/10 HPF v 42/10 HPF, P < .05). Bcl-2 immunohistochemistry showed a constantly positive reaction in normal thyroid tissue. In contrast, bcl-2 protein was not detected in most of the adenomas (70%) and carcinomas (76%). In conclusion, p53 protein and Ki-67 is more prevalent in oncocytic carcinomas than in oncocytic adenomas of the thyroid, indicating that these factors may be involved in the progression of oncocytic neoplasms in the thyroid. In contrast, loss of bcl-2 appears to be an early event in the formation of oncocytic neoplasms of the thyroid. Its importance for malignant transformation is, however, unclear.     

Metastatic atypical meningioma to the parotid gland – A cytopathological correlation

The tumors involving the parotid gland are mainly primary, with metastatic lesions comprising only 5% of malignant salivary gland neoplasms. We are presenting a rare case of metastatic meningioma to the parotid in a 51-year-old male with a past medical history of recurrent atypical meningioma involving the frontal lobe. For the past 1.5 years, routine imaging showed parotid lesions with interval growth including a 2.0 cm dominant tender preauricular parotid mass. The chronicity and the number of lesions made malignancy unlikely. The differential diagnosis included non-specific lesions such as intraparotid lymph nodes and benign neoplasms. Fine needle aspiration of the parotid mass was performed to show loosely cohesive fragments and singly scattered neoplastic cells with mild nuclear pleomorphism and oncocytic cytoplasm. The main cytomorphologic differential diagnosis included oncocytic and myoepithelial-rich tumors. The neoplastic cells were immunoreactive to p63, calponin and SSTR2A and were negative for cytokeratins, progesterone receptor, S100, DOG-1, EMA, synaptophysin, and chromogranin. The cytology slides and the parotid gland mass resection were compared to the previous meningioma resection specimen which showed a similar morphology of the oncocytic tumor cells in some areas. The overall morphologic and immunohistochemical findings of the parotid tumor were consistent with metastatic meningioma. Extracranial metastases from intracranial tumors are extremely rare. Meningiomas arise from the dura matter, constitute 15% of primary brain tumors, and metastasize at an estimated rate of 0.1%. Despite how uncommon metastatic meningioma is, our case emphasizes the critical role of clinical history when evaluating parotid gland lesions.