Immunoreactivity for cadherins, HGF/SF, met, and erbB-2 in pleural malignant mesotheliomas

AIMS: To assess the immunoreactivity of malignant mesotheliomas for N- and E-cadherins, hepatocyte growth factor/scatter factor (HGF/SF) and the tyrosine kinase receptors, met and erbB-2. METHODS AND RESULTS: Pleural malignant mesotheliomas were stained using a standard indirect immunoperoxidase method applied to paraffin sections. Malignant mesotheliomas were immunoreactive for N-cadherin (26/29; 90%), met (29/29; 100%) and erbB-2 (28/29; 97%). Focal immunoreactivity was present for E-cadherin in epithelioid or mixed tumours (14/25; 56%), and for HGF/SF (9/24; 38%). CONCLUSIONS: Expression of N-cadherin supports the diagnosis of malignant mesothelioma and use of appropriate antibodies would be a useful addition to a diagnostic antibody panel. Focal staining for E-cadherin does not exclude mesothelioma. Signalling pathways mediated via met and erbB-2 may play a role in the growth and spread of malignant mesotheliomas.     

OV-TL 12/30 (keratin 7 antibody) is a marker of glandular differentiation in lung cancer

The immunoreactivity of OV-TL 12/30, a monoclonal antibody to keratin 7 was investigated on paraffin-embedded human lung cancer tissues of 61 patients. A modified AEC-immunoperoxidase method with pepsin pre-digestion was used. In normal lung tissue keratin 7 was found in bronchial and bronchiolar epithelium, pneumocytes and compound glands. Squamous metaplasia of the bronchial tree was negative. All 24 squamous cell carcinomas were negative irrespective of grade of differentiation. All differentiation grades of 20 adenocarcinomas including bronchioalveolar carcinomas were positive. Since six large cell anaplastic carcinomas did not react with keratin 7 antibody these tumours are considered to be of squamous cell rather than adenocarcinomatous origin. Small cell anaplastic carcinomas were negative in 10 of 11 cases. Our study demonstrates that this keratin 7 antibody is useful in differentiating between squamous cell carcinoma and adenocarcinoma of the lung and it may be particularly useful in making the correct diagnosis in small lung biopsy specimens.     

Secretory component in pulmonary adenocarcinoma and mesothelioma

The distribution of secretory component was examined by an immunoperoxidase method in 40 pulmonary adenocarcinomas, 11 malignant pleural mesotheliomas and areas of normal lung adjacent to the tumours. Secretory component was demonstrated in tumour cells in 25 (67%) adenocarcinomas. Its presence correlated with the degree of differentiation but was not related to tumour pattern. In the normal lung secretory component can be demonstrated in bronchial ciliated cells, bronchial gland serous cells, bronchiolar epithelium and hyperplastic alveolar epithelium. Although not usually detectable in normal mucous cells it was frequently present in mucin-producing tumours. None of the mesotheliomas examined contained secretory component and this may be an additional useful feature in the differential diagnosis between mesothelioma and adenocarcinoma.     

Immunocytochemical localization of neuron specific enolase in small cell carcinomas and carcinoid tumours of the lung

Carcinoid tumours and small cell carcinomas of the lung share many characteristics with normal neuroendocrine cells. While carcinoid tumours contain many dense-cored neurosecretory granules and are frequently argyrophil, small cell carcinomas are poorly granulated and rarely argyrophil, which casts doubt on their neuroendocrine nature. Immunostaining of the enzyme neuron specific enolase (NSE) was recently used to demonstrate the neuroendocrine components of the lung including nerves and neuroendocrine cells. We therefore used NSE immunostaining to investigate neuroendocrine differentiation in 79 lung tumours, including 18 bronchial carcinoids and 31 small cell carcinomas, and compared these results with those obtained with silver stains. Thirteen of the 18 carcinoids were reactive to silver, all other types being negative. NSE-immunoreactivity occurred in 16 carcinoids and 18 small cell carcinomas. None of the squamous cell carcinomas, large cell anaplastic carcinomas and adenocarcinomas examined showed NSE-immunoreactivity. Radioimmunoassay of extractable NSE from 10 fresh lung tumours correlated well with the immunostaining results, demonstrating large amounts in two small cell carcinomas (334 and 517 ng/mg protein) and three carcinoids (152, 908, and 1143 ng/mg protein). Values were much lower for four squamous cell carcinomas (31-44 ng/mg protein) and one large cell anaplastic carcinoma (30 ng/mg protein) and were accounted for by the presence of NSE-positive nerves and neuroendocrine cells in the surrounding lung. NSE immunostaining is a useful marker of neuroendocrine differentiation in lung tumours and should prove particularly valuable in the diagnosis of small cell anaplastic tumours and their metastases.     

Expression and localization of thrombomodulin in preneoplastic bronchial lesions and in lung cancer

Thrombomodulin (TM) is an endothelial surface glycoprotein that acts as a natural anticoagulant. It inhibits thrombin and accelerates the activation of the anticoagulant protein C. TM has been detected in dermal keratinocytes, where it is associated with terminal differentiation. It can also be detected in various types of squamous malignant neoplasms and in malignancies of endothelial and mesothelial origin, such as Kaposi's sarcoma or malignant mesothelioma, but is absent in pulmonary adenocarcinomas (AC). Seventy-two lung tumour specimens [33 squamous cell carcinomas (SQCC), 23 AC, 1 large cell carcinoma, 8 small cell lung cancers (SCLC) and 7 multidifferentiated tumours (MT)] were analysed immunohistochemically by staining with an anti-TM antibody in order to assess TM expression. All of the SQCC stained positively for TM. In contrast, only 9 AC and 4 MT and none of the SCLC showed positive anti-TM staining. Seven hyperplastic bronchial epithelial specimens and eight preneoplastic bronchial lesions (five cases of moderate dysplasia, two cases of severe dysplasia and one case of carcinoma in situ) were used as controls.Normal or hyperplastic areas of bronchial epithelium revealed no positive reaction. However, a distinct positive anti-TM staining pattern related to the degree of keratiniziation of dysplastic lesions was seen. The present results suggest that anti-TM immunostaining is a useful marker for squamous cell carcinoma in the differential diagnosis of pulmonary carcinoma, also indicating keratinocyte differentiation in dysplastic bronchial epithelium.     

Fascin expression in low-grade uterine endometrioid adenocarcinoma: correlation with microcystic, elongated and fragmented (MELF)-type alteration at the deep invasive margin

Stewart C J R, Crook M L & Manso L
(2011) Histopathology 59 , 73–80 Fascin expression in low‐grade uterine endometrioid adenocarcinoma: correlation with microcystic, elongated and fragmented (MELF)‐type alteration at the deep invasive margin Aims: The actin‐binding protein fascin appears to potentiate the migratory capacity of both normal and neoplastic cells. It has been suggested that microcystic, elongated and fragmented (MELF) glands might represent areas of active invasion within uterine low‐grade endometrioid adenocarcinomas. Therefore, fascin immunoreactivity was investigated in a series of endometrial carcinomas specifically comparing expression in conventional tumour areas, foci of MELF‐type invasion and in stromal elements. Methods and results: Fascin expression was assessed in 28 uterine endometrioid adenocarcinomas and the results compared with cytokeratin (CK) 7 expression and with tumour morphology and distribution. The conventional glandular component of most tumours showed only focal fascin reactivity (<10% cells positive), but staining was more prominent within the peripheral epithelial cells. Foci of squamous/morular type differentiation were also positive. The neoplastic epithelium in MELF‐type invasion usually showed strong fascin immunoreactivity, often contrasting with the adjacent negative or more weakly stained conventional tumour glands. There was also staining of reactive stromal cells surrounding MELF foci. Conclusions: There are distinct micro‐anatomical variations in fascin immunoreactivity within endometrial carcinoma. The localized increase in fascin expression in MELF‐type epithelium supports the proposal that MELF changes represent areas of active tumour invasion.     

Are keratin proteins a better tumor marker than epithelial membrane antigen? A comparative immunohistochemical study of various paraffin-embedded neoplasms using monoclonal and polyclonal antibodies

Epithelial membrane antigen and keratin proteins represent markers of epithelial differentiation that may be detected in routine formalin-fixed, paraffin-embedded tissues. Eighty-seven neoplasms, including 48 adenocarcinomas of various types, squamous and transitional cell carcinomas, small-cell anaplastic carcinomas, carcinoid tumors, mesotheliomas, hepatomas, melanomas (metastatic), adrenal cortical carcinomas, germ cell tumors, and extramammary Paget's disease, were assessed to determine the relative effectiveness of these antigens as tumor markers. Immunoperoxidase studies were performed using monoclonal antibodies to epithelial membrane antigen and monoclonal (combined AE1 and AE3) and polyclonal (bovine muzzle keratins) antibodies to keratin proteins. In more than half the cases (50/87%), both markers yielded comparable results. However, in 29 cases (33%), keratin proteins were clearly superior to epithelial membrane antigen as a tumor cell marker. Particular discrepancies were apparent for some gastrointestinal adenocarcinomas, squamous cell carcinomas, hepatomas (hepatocellular type), spindle cell components of mesotheliomas, and carcinoid tumors. Epithelial membrane antigen represented a better marker in eight cases (9%), mainly for small-cell anaplastic carcinomas and some renal cell and pulmonary adenocarcinomas. Adrenal cortical carcinomas, melanomas, and seminomas were nonimmunoreactive for both antigens. Epithelial membrane antigen and keratin proteins represent useful complementary markers in diagnostic surgical pathology.     

D2-40: a reliable marker in the diagnosis of pleural mesothelioma.

OBJECTIVE: Malignant mesotheliomas of the pleura, peritoneum and pericardium can easily be confused with either metastatic adenocarcinomas or reactive pleural lesions. D2-40, a monoclonal antibody used as a marker for seminomatous germ cell tumours and lymphatic endothelial cells, was recently described in mesothelial cells and type I but not type II pneumocytes. METHOD: The immunoreactivities of D2-40 in 76 lung carcinomas of different histological types (adenocarcinomas, squamous cell, small cell, and bronchioloalveolar carcinomas) were compared with those of 36 pleural epithelioid and sarcomatoid mesotheliomas and 5 specimens of chronic pleuritis. RESULTS: While all 18 analysed epithelioid mesotheliomas displayed a strong membranous immunostaining, 18 sarcomatoid mesotheliomas showed no, or a merely faint, cytoplasmic signal, comparable with fibroblasts in chronic pleuritis. Out of all analysed lung carcinomas, 49 showed no immunoreactivity for D2-40 (64%), while the other 27 (36%) showed a focal weak to moderate and only cytoplasmic signal. CONCLUSIONS: We regard D2-40 as a valid marker in the differential diagnosis of epithelioid mesotheliomas versus pulmonary adenocarcinomas. However, this marker may not properly label sarcomatoid mesotheliomas or distinguish them from reactive pleural lesions.     

P63 in pulmonary epithelium,pulmonary squamous neoplasms,and other pulmonary tumors

p63 is a p53-homologous nuclear protein that appears to play a crucial role in regulation of stem cell commitment in squamous and other epithelia. In this study, p63 expression was examined in benign lung and in neoplasms of pulmonary origin. Eighty sections from routinely fixed and processed archival bronchoscopic biopsy or lobectomy specimens were pretreated with citric acid (pH 6.0) for antigen retrieval, then incubated overnight with anti-p63 monoclonal antibody 4A4. Slides were stained using a streptavidin-biotin kit and diaminobenzidine as chromagen, and were counterstained with hematoxylin. In normal lung, p63 intensely stained nuclei of bronchial reserve cells but did not stain ciliated cells, alveolar epithelial cells, or nonepithelial cells. The lower strata of squamous metaplastic bronchial epithelium stained positively. All squamous-cell carcinomas stained positively (n = 30). In some well-differentiated carcinomas, staining was found at the periphery of tumor nests but was negative in central zones showing squamous maturation. Poorly differentiated carcinomas showed very high proportions (80% to 100%) of p63-positive nuclei. All small-cell carcinomas were p63 negative (n = 9). Staining of bronchioloalveolar carcinomas (n = 7) and adenocarcinomas (n = 23) was variable: some tumors showed no detectable staining, others showed heterogeneously positive staining. Adenosquamous carcinomas (n = 5) displayed a unique basalar staining pattern. Carcinoid tumors were almost entirely negative (n = 5). We conclude that p63 is expressed in benign bronchial stem cells, in neoplastic cells with either squamous differentiation or squamous differentiating potential, and in a subpopulation of adenocarcinomas. p63 immunostaining may also aid in some histopathologic distinctions, such as in small biopsies where the differential diagnosis is poorly differentiated squamous carcinoma versus small-cell carcinoma. A stem cell biology-based classification system for squamous carcinomas is proposed.     

Reduced expression of alpha-catenin, beta-catenin, and gamma-catenin is associated with high cell proliferative activity and poor differentiation in non-small cell lung cancer

AIMS: To investigate the expression of catenins (alpha, beta, and gamma) in non-small cell lung carcinoma (NSCLC) and its relation to clinicopathological factors and prognosis. METHODS: The expression of catenins was analysed immunohistochemically in 261 patients with resected NSCLC, diagnosed between 1978 and 1996 in eastern Finland: The cell proliferation index of the tumours was analysed by means of an image analyser. The staining results were compared with clinicopathological characteristics and survival. RESULTS: Normal catenin staining was found significantly more often in adenocarcinomas than in squamous cell carcinomas or anaplastic/large cell carcinomas. Reduced staining of alpha-catenin, beta-catenin, and gamma-catenin was related to poor differentiation of the tumour. The tumours with reduced staining of beta-catenin or gamma-catenin often had higher cell proliferation activity. Nuclear staining of beta-catenin and gamma-catenin was found in 16 (7%) and 29 (13%) cases, respectively. This nuclear staining correlated directly with increased cell proliferation and inversely with membranous staining. In survival analyses the predictors of overall and disease free survival were stage and tumour type. The expression of catenins did not affect survival. CONCLUSIONS: The expression of alpha-catenin, beta-catenin, and gamma-catenin is related to histological type and differentiation in NSCLC, although catenins have no independent prognostic value. However, this study supports the important role of the nuclear accumulation of beta-catenin and gamma-catenin in highly proliferative cells.     

Co-expression of endothelial cell and macrophage antigens in Kaposi's sarcoma cells

Mutations in the p53 tumour suppressor gene, with consequent accumulation of the p53 protein, are frequently observed in non-small cell lung cancer (NSCLC). Little is known, however, about the timing of their appearance or their maintenance through cancer progression and metastatic spread. We have examined the normal epithelium and a panel of bronchial lesions, including dysplastic, neoplastic, and metastatic leisons, for p53 immunoreactivity and for expression of proliferating cell nuclear antigen (PCNA). No p53 immunoreactivity was found in normal and hyperplastic epithelium, nor in squamous metaplastic lesions. Twenty out of 30 invasive tumours and 13 out of 17 in situ carcinomas adjacent to an invasive tumour showed p53 immunoreactivity. There was a strict correlation between the level of p53 expression in the non-invasive and the invasive components of the tumours. Five out of eight pairs of primary tumours and matching metastases expressed p53, at identical levels in both compartments. These data indicate that p53 overexpression can occur in the earliest recognized phase of NSCLC and that the alteration is maintained during progression from in situ to invasive carcinoma and metastatic spread. PCNA expression increased from early to advanced phases of NSCLC. High PCNA immunoreactivity was observed in tumours expressing high p53 levels. A significant association was observed for PCNA expression between preinvasive and invasive lesions.     

Epidermal SH-protease inhibitor in human neoplasms and their metastases

The presence of the human epidermal SH-protease inhibitor in human tumours of different types was examined using double radial immunodiffusion against specific antisera to the inhibitor. The immunoreactive protein was found to be present in all the tumours with criteria of epidermoid carcinoma identifiable microscopically. Most small cell anaplastic, fusiform, and polygonal bronchial carcinomas were also found to contain immunoreactive protein. If the primary tumour contained the inhibitor, all metastases examined were also found to contain it. The identity of the inhibitors in epidermoid carcinomas and in epidermis was confirmed by immunoelectrophoretic and gel chromatographic methods. The results suggest that the epidermal SH-protease inhibitor is a property closely associated with squamo-epithelial tissue differentation, which also appears in neoplastic squamous epithelium.     

Detection of endothelin immunoreactivity and mRNA in pulmonary tumours

Paraffin sections of 66 surgically resected lung tumours were immunostained with antisera to human endothelin-1 and to the C-terminal peptide of big endothelin. With both antisera, strong immunoreactivity was demonstrated in 11 of 15 squamous cell carcinomas and 11 of 16 adenocarcinomas. Focal immunoreactivity was seen in small cell carcinoma (2/12), large cell carcinoma (2/5), and carcinoid tumours (2/11). Four lymphomas and three sarcomas did not show endothelin immunoreactivity. Cryostat sections of 22 of the 66 tumours were hybridized with radiolabelled complementary RNA probes prepared from the 3' non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization demonstrated the presence of endothelin mRNAs in 4 of 7 squamous cell carcinomas and in 5 of 8 adenocarcinomas, in a pattern similar to that shown by immunocytochemistry. No hybridization signals were obtained from the other types of tumours. In lung tissue adjacent to the tumours, endothelin-like immunoreactivity and mRNA were detected in pulmonary endocrine cells and, in some cases, other epithelial cells, and in alveolar capillary endothelial cells. This study demonstrates the expression of endothelin in a number of pulmonary tumours and suggests a possible role for this peptide in the growth and/or differentiation of these tumours.     

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.     

Deregulated Bag-1 protein expression in human oral squamous cell carcinomas and lymph node metastases

Bag-1 is an anti-apoptotic protein that promotes metastasis in some tumour cell types. To determine whether Bag-1 expression is altered in 64 oral squamous cell carcinomas, tumour samples were compared with 17 samples of normal oral epithelium. Normal oral epithelia had pronounced nuclear staining in the basal and maturation layers and weak cytoplasmic staining that was most pronounced in the basal and suprabasal layers. Oral squamous cell carcinomas demonstrated a tendency for reduced nuclear staining intensity (p=0.036). Cytoplasmic staining intensity was not significantly different between tumour and normal tissue. However, many tumours were observed to have less of a difference between nuclear staining intensity and cytoplasmic staining intensity than normal oral epithelium. Furthermore, in lymph node metastases, cytoplasmic Bag-1 staining was stronger in 8/13 cases than in corresponding primary tumours (p=0.021). Western blotting using nine oral primary carcinoma cell lines and four normal keratinocyte cultures showed that the isoforms Bag-1s, Bag-1M, and Bag-1L were expressed in normal and malignant oral epithelial cells. Bag-1L unique sequences were shown to adopt an exclusively nuclear, and predominantly nucleolar, localization by use of transiently transfected N-terminal Bag-1L-EGFP. However, levels of Bag-1L in carcinoma cells did not differ significantly from those of normal keratinocytes. Therefore the reduced nuclear staining observed in oral squamous cell carcinomas compared with normal epithelium may reflect changes in the localization of Bag-1 isoforms, rather than decreased expression of Bag-1L. Alterations in the relative proportions of Bag-1S, Bag-1M, and Bag-1L were detected in 6/9 oral carcinoma cell lines; 5/9 oral carcinoma cell lines had a significantly greater proportion of Bag-1M than normal keratinocytes and in another cell line, Bag-1L was significantly underrepresented. Overall, the results suggest that Bag-1 deregulation plays a role in oral carcinogenesis at two different stages: during primary carcinoma development and during lymph node metastasis.     

Differential expression of c-Met, its ligand HGF/SF and HER2/neu in DCIS and adjacent normal breast tissue

AIMS: Tyrosine kinase receptors Her2/neu and c-Met play an important role in breast cancer development and progression. Our aim was to determine the expression of c-Met, its ligand hepatocyte growth factor/scatter factor (HGF/SF) and Her2/neu in ductal carcinoma in situ (DCIS) lesions of the breast (n = 39) by two different immunocytochemical techniques, classical immunohistochemistry and immunofluorescence, and to correlate their expression levels with histopathological and clinical characteristics. METHODS AND RESULTS: Both methods revealed similar c-Met staining patterns in both the in situ component and the adjacent normal tissue (P < 0.001). However, an imbalance in c-Met expression between tumour and surrounding normal tissue was correlated with high-grade DCIS (Van Nuys Grade 3). No correlation existed between Her2/neu and c-Met expression. High HGF/SF immunoreactivity was observed in 43.6% of the cases, yet the adjacent cellular stroma revealed only low levels of HGF/SF. No correlation existed between c-Met, Her2/neu or HGF/SF expression and clinicopathological factors. CONCLUSION: An imbalance in c-Met expression between tumour and surrounding normal tissue is associated with an aggressive DCIS phenotype. Moreover, c-Met and HGF/SF may contribute to tumour development by different means than those controlled by Her2/neu.