Down-regulation of CD95 (Fas/Apo-1) in the epithelia of adenovirus-infected appendices

 

Aims:

Adenoviral inclusions are commonly seen in appendices from infants with intussusception. They are associated with focal epithelial budding and less frequently with epithelial shedding. These morphological changes could depend on the opposing effects of adenoviral gene products on CD95-mediated apoptosis.  

Methods and results:

Appendices from intussusceptions with viral inclusions ( n  = 4) and normal appendices ( n  = 10) were studied by immunochemistry with anti-adenovirus, anti-CD95 and anti-HLA-DR antibodies. Apoptosis was studied by the TUNEL method. The mucosa of normal appendices contained no adenoviral protein. CD95 was present in all epithelial cells except Paneth cells. HLA-DR was absent in epithelial cells and apoptosis was seen only in germinal centres and in a few surface epithelial cells. The epithelium of appendices from intussusceptions contained nuclear inclusions labelled with anti-adenovirus antibody, always found in the epithelial buds. The epithelial CD95 pattern was drastically altered in adenovirus-infected appendices. CD95 was absent from the budding foci. In these foci, HLA-DR was overexpressed. There was also increased epithelial apoptosis in areas remote from those lacking CD95 antigen.  

Conclusions:

The appearance of epithelial budding or shedding in appendices from intussusception could be due to focal in situ differences in the expression of adenoviral genes.     

Loss of basement membrane components laminin and type IV collagen parallels the progression of oral epithelial neoplasia

Aims : To determine the immunohistochemical localization of basement membrane components laminin and type IV collagen in premalignant and malignant lesions of the oral epithelium.  

Methods and results

Formalin-fixed tissue sections of 12 epithelial hyperplasias with no dysplasia and 30 dysplasias, clinically diagnosed as leukoplakia and/or erythroplakia, as well as 50 invasive squamous cell carcinomas, were stained with mouse monoclonal antibodies to human laminin and type IV collagen. Statistical analysis showed that there was a linear trend for discontinuous distribution of laminin from epithelial hyperplasia to epithelial dysplasia and invasive squamous cell carcinoma ( P  < 0.001). Laminin staining showed a linear trend for discontinuity with increasing grade of dysplasia ( P  < 0.05) and was more frequently discontinuous in areas of deep tumour invasion than in central or superficial areas ( P  < 0.05). Brush-shaped thickening and reduplication of the basement membrane were also identified.  

Conclusions

Alterations in the distribution of laminin and type IV collagen in oral premalignant and malignant lesions indicate that the loss of continuity of the subepithelial basement membrane parallels the progression of the neoplastic transformation process in oral epithelium.     

Type 1 protein tyrosine kinases in benign and malignant breast lesions

Aims : To determine their significance, we examined the expression pattern of the four epidermal growth factor receptor (EGFR) family members as well as the phosphotyrosine kinase activity in breast tumour tissues.  

Methods and results

Fifty-three malignant breast tumours, four breast cancer cell lines, and 10 benign breast tumours were investigated. Fifty-three per cent (28/53) of the malignant tumours expressed EGFR protein, and the majority of these positive tumours were strongly positive. Eighty per cent (8/10) of the benign tumours also expressed EGFR protein, but all in a lower or moderate level. An association between EGFR expression and increasing malignancy grade was found in the group of infiltrating ductal carcinomas. Of the malignant tumours, 35.8% (19/53) expressed c-erbB-2 protein and 17% (9/53) c-erbB-3 protein, while no expression of c-erbB-2 and c-erbB-3 proteins was found in the benign tumours. Contrary to previous reports, we observed c-erbB-4 receptor protein to be less expressed in the malignant breast tumours. The 'normal' breast epithelial cells adjacent to the malignant tumours and the benign tumours demonstrated intensified membrane staining for c-erbB-4, while a number of the malignant tumours demonstrated a weak cytoplasmic staining or were negative. However, several malignant tumours with strong membrane staining for the c-erbB-4 protein were also found. No simple association between the expression of the four receptors and phosphotyrosine kinase activity was found.  

Conclusion

Our study has revealed a complex expression pattern of the EGFR family members in breast tumour cells. While the data about EGFR, c-erbB-2, c-erbB-3 and phosphotyrosine are largely in line with what has been reported, we found the c-erbB-4 protein expression to be decreased in the malignant tumours.     

Mammary epidermoid inclusion cysts after wide-core needle biopsies

 

Aims:

To investigate the prevalence of squamous epidermoid inclusion cysts after wide-core needle biopsy.  

Methods and results:

Epidermoid inclusion cysts were found in five of 17 surgical excisions (29%) after preliminary wide-core needle biopsies in a 7-month period. Thereafter they were not seen in 26 subsequent postwide-core surgical excisions in a period of 6 months.  

Conclusions:

The cysts appear to be an iatrogenic complication of wide-core biopsy, and need morphological recognition in order to avoid confusion with spontaneous squamous metaplasia of benign or malignant breast epithelium. Longer term implications are unknown.     

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.     

Erythrophagocytic tumour cells in melanoma and squamous cell carcinoma of the skin

 

Aims:

Erythrophagocytosis is a characteristic feature of tumour cells in malignant histiocytosis, some leukaemias, lymphomas, and also reactive histiocytes in the haemophagocytic syndrome associated with a variety of infections and neoplasms. It has also been found exceptionally in metastatic malignant epithelial cells in bone marrow and lymph nodes. We present two cases, a cutaneous malignant melanoma and an acantholytic squamous cell carcinoma, in which erythrophagocytosis by tumour cells was demonstrable by both light and electron microscopy.  

Methods and results:

The melanocytic and squamous nature of these cells was supported by the immunohistochemical detection of HMB45, S100, and NKI-C3 in the former, and cytokeratin and EMA in the latter, and at ultrastructural level by the presence of melanosomes and tonofilaments, respectively.  

Conclusions:

This is, to our knowledge, the first documented report of erythrophagocytic tumour cells in human melanomas and primary carcinomas. Biological considerations apart, this unusual feature can prove to be of value to avoid a misdiagnosis of a variety of haematopoietic malignancies.     

Hepatic stem-like cells in hepatoblastoma: expression of cytokeratin 7, albumin and oval cell associated antigens detected by OV-1 and OV-6

 

Aims:

In a recent study we described a population of small epithelial cells (SEC) in human hepatoblastoma that exhibit ultrastructural features of the oval cells of rodents. Both SEC and oval cells are immunoreactive for cytokeratin 7, a marker of biliary differentiation, and it was postulated that SEC, like oval cells, are closely related to hepatic stem cells. This study was undertaken to investigate whether SEC also exhibit immunolabelling for albumin, a marker of hepatocytic differentiation, and to determine whether other antigens typical of oval cells are detectable in hepatoblastoma.  

Methods and results:

Hepatoblastomas of various subtypes were investigated by electron microscopy, and by immunohistochemistry with the monoclonal antibodies OV-1 and OV-6, which recognize antigens associated with oval cells. Double-labelling for cytokeratin 7 and albumin was carried out by immuno-electron microscopy. OV-1 stained scattered cells in seven of 12 tumours investigated and OV-6 in nine. On immunoelectron-microscopic investigation, SEC exhibited labelling for both cytokeratin 7 and albumin.  

Conclusions:

The results demonstrate that antigens associated with oval cells are found in certain cells in hepatoblastoma. SEC, like oval cells, co-express markers for hepatocytic and biliary differentiation. The findings further support the hypothesis that SEC are closely related to the putative bipotent hepatic stem cell, which, by definition, gives rise to both hepatocytes and biliary epithelial cells.     

Adenomyoepithelioma of the skin: a case report with immunohistochemical and ultrastructural observations

 

Aims:

The histological, immunohistochemical and electron microscopic features of a primary adenomyoepithelioma of skin, a rare sweat gland tumour, are reported.  

Methods and results:

The tumour occurred on the back of a 92-year-old woman. It was composed of well-formed tubules lined by epithelial cells surrounded by clear or spindled myoepithelial cells. Immunohistochemically, the epithelial cells exhibited strong cytokeratin (CAM5.2) and weak carcinoembryonic antigen positivity. The myoepithelial cells showed diffuse positivity for smooth muscle actin and focal positivity for S100 protein. Ultrastructurally, the myoepithelial cells contained myofilaments with focal densities and hemi-desmosomes. They were limited by well-formed basal lamina. The tumour was associated with a small eccrine spiradenoma.  

Conclusion:

We predict that the tumour will behave in a benign fashion. There is no evidence of recurrence or metastasis 28 months later.     

Retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry

 

Aim:

Antigen retrieval (AR) procedures are based on the effect of heating (by either microwave or pressure cooking treatments) on routinely fixed and paraffin embedded tissues. We observed that AR procedures restore the reactivity of endogenous biotin (EB) and report on the distribution of EB following AR in a series of routinely fixed and embedded tissues.  

Methods and results:

Following pressure cooking or microwave treatments, a simple streptavidin–peroxidase staining revealed retrieved endogenous biotin (REB) in normal tissues (such as liver, kidney and adrenal cortex), in oxyphylic cells and in some tumours, especially in carcinomas of the kidney and of the adrenal cortex. In formalin-fixed (but not in alcohol-fixed) tissue sections, the heating procedures caused an intense and finely granular cytoplasmic reaction, following a routine streptavidin-conjugated peroxidase treatment. The staining was prevented by blocking of EB by a sequential avidin–biotin treatment.  

Conclusions:

Retrieval of EB reactivity can cause pitfalls in diagnostic immunohistochemistry but, alternatively, it might also constitute a useful and novel diagnostic marker.     

Immunohistochemical analysis of decalcified paraffin-embedded human bone marrow biopsies with emphasis on MHC class I and CD34 expression

 

Aims:

To identify the stromal structures and haematopoietic cell lineages in normal bone marrow. The optimal conditions were studied for the reactivity of a panel of antibodies, applicable to paraffin sections of decalcified trephine biopsies using antigen retrieval methods.  

Methods and results:

Two methods of antigen retrieval (pepsin and acid citrate buffer) were tested. For the demonstration of most antigens and for reduction of background staining, heating in acid citrate buffer was preferred. In the case of elastase and von Willebrand Factor (factor VIIIrAg) pepsin pre-treatment was optimal, whereas Ulex europaeus agglutinin (UEA-1) and α-smooth muscle actin (α-SMA) required no pre-treatment. Staining patterns obtained after 48 h EDTA decalcification and short electrolytic decalcification were identical. Both methods allowed recognition of HLA-A and HLA-B antigens, isolated CD34+ cells, mono-histiocytic cells (CD68+), myeloid cells (elastase and myeloperoxidase), erythroid cells (glycophorin C) and of megakaryocytic cells (Factor VIIIrAg). A relative simple lymphocyte-subset analysis was possible in decalcified paraffin sections allowing recognition of B-cells (CD20+) and T-cells (CD3+ and CD45RO+) in frequencies comparable to frozen sections. Suitable stromal cell staining was achieved by vimentin and desmin antibodies, whereas the bone marrow capillary network was visualized by CD34, factor VIIIrAg and UEA-1.  

Conclusions:

This immunohistochemical study indicates that all cellular components of the haematopoietic microenvironment of the bone marrow can be identified in decalcified paraffin sections using antigen retrieval methods and that the time of decalcification can be reduced to 1–1.5 h.     

Nuclear localization of human AP endonuclease 1 (HAP1/Ref-1) associates with prognosis in early operable non-small cell lung cancer (NSCLC).

The present study examined the immunohistochemical expression of human AP endonuclease 1 (HAP1/Ref-1), the major endonuclease in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA, in normal lung and lung carcinomas. Cellular expression of HAP1 was determined using a standard avidin-biotin-peroxidase complex (ABC) technique and an anti-HAP1 rabbit polyclonal antibody on paraffin-embedded tissue sections from normal lung and in 103 primary non-small cell lung carcinomas (NSCLCs). In normal lung, the staining for HAP1 was found to be both nuclear and cytoplasmic in the pneumocytes of the alveoli. Superficial ciliated cells of the bronchial epithelium presented cytoplasmic staining, while staining for the basal cells was mostly nuclear. Bronchial glandular cells demonstrated mixed nuclear and cytoplasmic staining. Lung carcinomas showed all patterns of expression for HAP1. Loss of HAP1 expression was associated with low proliferation index (p=0.01) and with squamous histology (p=0.04). In squamous carcinomas, a significant correlation was observed between positive nuclear HAP1 and negative p53 expression (p=0.03). A survival benefit was seen in patients presenting nuclear HAP1 expression and those presenting the nuclear HAP1+/p53- phenotype (p=0.01 and 0.007, respectively). It is concluded that nuclear HAP1 localization may be relevant to its role as a DNA repair protein and/or to the recently proposed role as an activator of wild-type p53, and thus to the better outcome seen in this group of patients.     

Stereological estimates of the nuclear/cytoplasmic ratio and star volume on fibreoptic biopsies are of prognostic value for survival in a preliminary study of advanced squamous cell carcinoma of the lung

 

Aims:

This study evaluated the role of morphometric and clinical parameters in establishing the prognosis of patients submitted to radiotherapy for advanced squamous cell carcinoma of the lung.  

Methods and results:

Morphometric studies were performed by point counting techniques. Forty patients were included in this study. Group 1 patients ( n  = 22) were those with survival equal to or less than 6 months; group 2 ( n  = 10) patients had a survival of 7 to 12 months; and group 3 ( n  = 8) included patients with survival greater than 12 months. To characterize these three groups of patients, models combining categorical and continuous variables were constructed by means of discriminant analysis. Weight loss, histological grade, nuclear/cytoplasmic ratio and star volume of the nuclei were selected during the backward procedure as relevant variables to characterize the three groups of patients. The overall sensitivity of the model was 90%.  

Conclusions:

Our results indicate that histopathological data may help to predict prognosis in patients with advanced squamous cell lung carcinoma, and encourage the use of morphometric procedures in histopathological analysis of this type of lung tumour.     

Expression of c-Met correlates with grade of malignancy in human astrocytic tumours: an immunohistochemical study

 

Aims:

Recent studies suggest the involvement of hepatocyte growth factor/scatter factor (HGF/SF) in glioma cell invasion and tumour progression. We investigated the distribution and rate of tumour cells that express c-Met protein, which is the cell-surface receptor for HGF/SF, in astrocytic tumours. The type of cells that express c-Met in tumour tissues was also identified.  

Methods and results:

c-Met expression was screened immunohistochemically in a total of 43 astrocytic tumours, including 14 low-grade astrocytomas (A), 13 anaplastic astrocytomas (AA) and 16 glioblastoma multiforme (GBM). c-Met reactivity was demonstrated predominantly in the cytoplasm of tumour cells. Bizarre large tumour cells tended to stain intensely. Higher c-Met expression levels (≥ 2 +, more than 25% cells were positive) were noted in 21.4% of (A) vs. 53.8% in (AA) and 87.5% in (GBM) ( P  < 0.001), indicating a clear relationship between c-Met protein staining and higher grade astrocytic tumours. Moreover, c-Met immunoreactivity was also shown in tumour microvasculature, reactive astrocytes, and neurones in the cortex infiltrated by glioma cells. In 85.7% of cases containing infiltrated cortex, neurones were positive vs. no neurones in non-neoplastic regions ( P  < 0.002).  

Conclusions:

This evidence suggests that c-Met expression in the brain could be associated with astrocytoma progression and also reactive process. Immunohistochemical determination of c-Met-expressing cell types helps to understand possible roles of c-Met in tumour tissues.     

Immunohistochemical localization of cathepsin D, proliferating cell nuclear antigen and epidermal growth factor receptor in human breast carcinoma analysed by computer image analyser: correlation with histological grade and metastatic behaviour

 

Aims:

The purpose of this study is to examine the relationship between immunohistochemical localization of cathepsin D (CD), proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGF-R) in 65 cases of breast carcinoma in Japanese women and traditional prognostic factors such as histological grade, lymph node status, mitotic rate and clinical stage, in order to possibly identify some indicator(s) that may be specifically associated with prognosis.  

Methods and results:

Serial sections of 5-μm thick were cut from the archival formalin-fixed, paraffin-embedded tissue blocks, and processed for CD, PCNA and EGF-R immunostaining. The results were analysed by computer-based image analysis system. All samples showed a positive immunoreaction for cathepsin D in both the parenchyma and stroma. However, the staining area and intensity varied from cell to cell in the parenchyma and stroma as well as among samples. Subsequently, the evaluation of immunostaining for CD was separately performed in both the parenchyma and stroma (CDpar and CDstr, respectively) and the combination of both components (CDtotal). PCNA and EGF-R showed positive immunostaining almost exclusively in the parenchymal component of the carcinoma tissue specimens. CDtotal significantly correlated with the histological grade, PCNA index (PI), mitotic rate (MR), EGF-R and lymph node metastasis. Significant correlation was also demonstrated between CDpar and the histological grade, EGF-R and lymph node metastasis, or between CDstr and MR, EGF-R and lymph node metastasis. EGF-R correlated highly with the histological grade, MR score, lymph node metastases and recurrence-free survival.  

Conclusions:

Both the CD parameters and EGF-R are valuable indicators for predicting the biological behaviour of human breast carcinoma.     

CD44 expression is up-regulated in the deep zone of osteoarthritic cartilage from human femoral heads

 

Aims:

The objective of this study was to detail the topographical and zonal distribution of the cell adhesion molecule CD44 in normal and osteoarthritic cartilage.  

Methods and results:

Immunohistochemistry utilizing well characterized anti-CD44 antibodies (clones A3D8, Bric 235, 2C5) was performed on cryostat and paraffin sections of human articular cartilage from macroscopically normal ( n  = 18) and osteoarthritic ( n  = 11) femoral heads. Samples for cryostat sections were obtained from 12 topographically different sites. Sections were divided into zones (superficial, middle, deep) and the CD44 staining scored. Chondrocytes in normal articular cartilage and cartilage from osteoarthritic femoral heads stained positive for CD44 in both cryostat and paraffin sections. Normal cartilage showed a significant decrease in CD44 staining in the deep zone as compared to the superficial zone ( P  < 0.05). However, cryostat sections of residual cartilage from osteoarthritic femoral heads showed increased CD44 staining in the deep zone as compared to normal articular cartilage. The CD44 staining showed no topographical variation in either the normal cartilage or the osteoarthritic residual cartilage.  

Conclusions:

CD44 expression displays a distinct zonal variation in normal articular cartilage which is lost in osteoarthritic cartilage due to an up-regulated expression in the deep zone. CD44 expression does not exhibit topographical variation.     

Reproducible and highly sensitive detection of the broad spectrum epithelial marker keratin 19 in routine cancer diagnosis

 

Aims:

In this study the recently developed keratin 19 antibody RCK108 is biochemically and immunohistochemically characterized. Its applicability as a keratin marker in routinely processed histological tissue specimens is assessed.  

Methods and results:

The keratin 19 antibody RCK108 antibody was tested on normal and malignant routinely formalin-fixed, paraffin-embedded tissue specimens. It stains most, although not all, glandular epithelia and showed (focal) reactivity in the basal cell compartment of stratified epithelia. It was found to react with most epithelial tumours, including adenocarcinomas, squamous cell carcinomas and endocrine tumours of various origins.  

Conclusions:

Its reproducible and highly sensitive staining characteristics make RCK108 a useful antibody to be applied as a broad epithelial marker for carcinoma detection in routinely processed paraffin sections. As such, RCK108 is a specific reagent for practically all epithelial tumours. A few types of epithelial malignancies, known not to contain keratin 19, were negative for RCK108. Therefore the antibody is also useful in some narrow differential diagnostic considerations such as cholangiocellular carcinoma (RCK108 positive) vs. hepatocellular carcinoma (RCK108 negative). Another important feature of this antibody is that it shows very little reactivity in mesenchymal tissues, or mesenchymally derived tumours, as is frequently described for other keratin antibodies. A few leiomyosarcomas showed sporadic reactivity.     

Large cell neuroendocrine carcinoma of the thymus

 

Aim:

We highlight the occurrence of an unusual neuroendocrine tumour, a large cell neuroendocrine carcinoma, arising from the thymus.  

Case details:

A 68-year-old man with a history of cigarette smoking had a large mediastinal tumour arising from the thymus removed. Two years later the tumour recurred; it was debulked surgically but the patient died 2 months later. Histological examination of both tumour specimens revealed a tumour with an endocrine pattern, composed of large pleomorphic cells with large nuclei and prominent nucleoli. The mitotic count ranged from 19 to 26 per 10 high-power fields and large tracks of coagulative tumour necrosis were present. The tumour cells were strongly positive for neuron-specific enolase (NSE), chromogranin, CAM5.2 and AE1/3, with cytoplasmic dot-like accentuation for the latter three markers. The tumour fulfilled the criteria for a diagnosis of large cell neuroendocrine carcinoma.  

Conclusions:

Large cell neuroendocrine carcinoma should be distinguished from atypical carcinoid and small cell carcinoma. It is a distinctive neuroendocrine malignancy with a prognosis between that of atypical carcinoid and small cell carcinoma, and needs to be treated aggressively.     

Synovial sarcoma of the pleura and its differentiation from other primary pleural tumours: a clinicopathological and immunohistochemical review of three cases

Aims : Synovial sarcomas are rare tumours occasionally arising in the pleural cavity, a site where their histological characteristics may be mistaken for those of malignant mesothelioma. We examined three cases of primary pleural synovial sarcoma in order to look for clinicopathological features that may help in distinguishing them from both mesotheliomas and other sarcomas that may arise in the pleura.  

Methods and results

All three patients were male, aged 42, 28 and 42, respectively, and had no known exposure to asbestos. One biphasic tumour contained neutral mucin in focal epithelial elements that also stained positively for BerEP4 and AUA1. All three tumours showed focal positivity for either keratin or EMA in the sarcomatous elements, and they also stained positively for bcl-2 protein and MIC2 gene product (CD99).  

Conclusions

Our results emphasize the importance of being aware of synovial sarcoma as a possible primary pleural malignancy, especially in a young patient with a localized mass. In addition, the presence of bcl-2 protein perhaps represents a useful marker in distinguishing synovial sarcoma, especially monophasic variants, from mesothelioma within a panel of antibodies.     

Embolization of mesothelial cells in lymphatics: the route to mesothelial inclusions in lymph nodes?

Aims : To provide evidence that lymphatic embolization is the mechanism for mesothelial inclusions in lymph nodes.  

Methods and results

A 60-year-old man with alcoholic cirrhosis and ascites had an umbilical hernia resected. The herniorrhaphy specimen contained numerous dermal and submesothelial lymphatic vessels filled by cells similar to the cells that lined the hernia sac. Most of the cells in lymphatics were submesothelial reactive cells, whose cytoplasm stained with antibodies against cytokeratins (AE1–AE3; 8,18), smooth muscle actin, vimentin, desmin and tissue polypeptide antigen (TPA). Some cells seemed to be superficial mesothelial cells, being positive with high molecular weight anticytokeratin antibody 34βE12. On ultrastructural study submesothelial cells with intermediate cytoplasmic filaments, rough endoplasmic reticulum and primitive cell junctions, and scanty superficial mesothelial cells with microvilli, tonofilaments and desmosomes were found in the lymphatics.  

Conclusions

Lymphatic dissemination of mesothelial and submesothelial cells is an uncommon and not well known phenomenon. Lymphatic dissemination is probably the route by which the mesothelial cells reach the lymphatic nodes. These cells may be mistaken for malignant cells.