Antineuron specific enolase staining reactions in sarcomas and carcinomas: its lack of neuroendocrine specificity.

A commercially available polyclonal antiserum (Dakopatts) raised against bovine neuron specific enolase (NSE) was reacted with 197 sarcomas, 32 carcinomas, 11 carcinoid tumours and 20 malignant melanomas to assess its specificity for neuroendocrine tumours. All the tumours had been fixed in formalin and embedded in paraffin. Positive tumour cells were found in two of 11 squamous cell carcinomas, one of 11 adenocarcinomas, 10 of 10 oat cell carcinomas, 11 of 11 carcinoid tumours, 16 of 20 malignant melanomas, four of seven clear cell sarcomas, nine of 25 leiomyosarcomas, four of 22 rhabdomyosarcomas, one of seven angiosarcomas and one of 20 synovial sarcomas.     

Intracellular keratins in normal and pathological bronchial mucosa

Summary The distribution of intracellular keratins was investigated in normal bronchial epithelium and in several morphologically distinct forms of respiratory tract carcinomas. This study was performed with two different experimentally produced antisera against normal human stratum corneum keratin and against keratin protein of MW 67000 dalton, using indirect immunofluorescence and immunoperoxidase methods on tissue sections and cell suspensions.In normal bronchial epithelium, the basal cells were strongly labelled by both antisera. The ciliated columnar cells appeared devoid of cytokeratins in tissue sections but were strongly labelled with both antisera in cell suspensions. The goblet cells remained negative in every case. In squamous metaplasia of the bronchus, all epithelial cells were unevenly stained with both antisera.Among tumours, only the squamous cell carcinomas were strongly labelled by both antisera. Primary lung adenocarcinoma appeared weakly positive, whereas metastatic lung carcinomas, undifferentiated lung carcinomas, oat cell tumours, carcinoid tumours were negative.The immunocytochemical determination of keratins appears to be of value in the study of normal and abnormal epithelial differentiation, in the diagnosis of poorly differentiated carcinomas and in their distinction from metastatic tumours of the lung.     

Immunohistochemical examination of lung cancers using monoclonal antibodies reacting with sialosylated Lewisx and sialosylated Lewisa

Summary In order to explore the relationship between the expression of cancer-associated glycolipids such as sialylated Le^x (SLEX) and sialylated Le^a (SLEA) and the histological subtypes of lung cancers, 30 cases of small cell carcinoma (SCC) and 47 cases of non-small cell carcinoma (non-SCC) were examined immunohistochemically using monoclonal antibodies reacting with SLEX and SLEA. The forty-seven cases of non-SCC included 20 cases of adenocarcinoma, 20 of squamous cell carcinoma and 7 of large cell carcinoma. Tumour cells of most non-SCCs expressed SLEX and SLEA. In adenocarcinomas, the number of tumour cells having SLEX and SLEA was more than that of squamous cell carcinomas, large cell carcinomas and SCC. In SCC, 14 of the 30 cases were found to be positive for both antigens. Although the cancer cells of 11 cases of 17 intermediate cell type SCC had both antigens, the cells of only 3 of 13 oat cell tumours expressed SLEX and SLEA. The present study shows that SLEX and SLEA are useful markers for lung adenocarcinomas, that most cases of intermediate cell type of SCCs have characteristics similar to non-SCC but that many oat cell tumours lack them.     

Anaplastic small cell (oat cell) carcinoma of the tonsils: report of two cases

Two cases of anaplastic small cell (oat cell) carcinoma of the tonsils are presented. In the first, cervical metastases preceded the manifestation of the primary tumour by 2 years. In case 2 the tonsillar carcinoma was accompanied by a bronchial tumour of the same histological type and by cervical and axillary metastases. Positive Grimelius stain, positive immunohistochemical staining for chromogranin A and neurone-specific enolase and the presence, in case 1, of membrane-bound granules indicate that these tumours display many similarities with neuroendocrine carcinomas even if they originate from pluripotential ductal cells of tonsillar minor salivary glands and not from Kulchitsky-like cells.     

Primary oat cell (neuroendocrine) carcinoma of the breast

Summary Four cases of oat cell (neuroendocrine) carcinoma of the breast are reported. Three patients died within 15 months of the diagnosis and the fourth patient is alive after 44 months. Histochemical, ultrastructural and mRNA markers of endocrine differentiation were present in three cases. These tumours show histological similarities to breast metastases of bronchial oat cell carcinoma, but a distinguishing feature is the presence of in situ ductal lesions. It appears that the breast is a further site which has to be added to the long list of extrapulmonary oat cell carcinomas.     

Gastrin releasing peptide in human neuroendocrine tumours

Neuroendocrine tumours of the lung and gut are known to possess bombesin-like immunoreactivity. The recent observation that gastrin releasing peptide (GRP), a 27 amino acid peptide isolated from the porcine intestine, may be the mammalian analogue of bombesin led us to look for this peptide in a variety of human neoplasms. Formalin-fixed tissues from 85 tumours were examined by the immunoperoxidase technique, using specific antisera to the GRP molecule (1-27) and the GRP fragment (1-16). Intense cytoplasmic GRP immunoreactivity was seen in thyroid medullary carcinomas (3/3), carcinoids of lung, pancreas, and intestine (22/36), and paragangliomas (2/3). Less frequent staining was present in pulmonary small cell (oat cell) carcinomas (1/8) and pituitary adenomas (1/6). Complete absence of immunoreactivity was observed in three phaeochromocytomas, five Merkel cell tumours, six neuroblastomas and 15 non-neuroendocrine tumours. Normal neuroendocrine cells of the thyroid (C-cells) and bronchial mucosa (Kulchitsky cells) exhibited GRP immunoreactivity; nerve fibres from all sites failed to demonstrate staining for GRP. In each positive case, the pattern of staining for GRP (1-27) and GRP (1-16) was identical, although the GRP (1-16) immunostaining was weaker. These findings indicate that bombesin immunoreactivity in human neuroendocrine cells and tumours is attributable to GRP-like molecules and that GRP is a useful marker of neuroendocrine differentiation in many tumours.     

PRIMARY MIXED CARCINOMAS OF THE BRONCHUS

Primary mixed carcinomas of the bronchus are found to occur more frequently than previously reported. Forty-two cases of primary mixed carcinomas of the bronchus were seen at the London Chest Hospital from 1966 to 1969. During this time, 3853 patients were admitted to the hospital and among them 733 had bronchial carcinomas. In 27 instances, the tumour histologically was squamous and adenocarcinomatous in type. Of the other 15; squamous and oat cell characters, and undifferentiated carcinoma of large polygonal cell and adenocarcinomatous characters were present in five instances; adenocarcinoma and oat cell, and undifferentiated carcinoma of large polygonal cell and oat cell characters were present in two instances; and only one had mixed alveolar and oat cell carcinomatous pattern. A study of the pathological changes in these mixed bronchial carcinomas revealed little differences, if any, from a primary bronchial carcinoma.
The histogenesis of these mixed carcinomas remains uncertain. The most plausible view is that they arise by divergent differentiation from the descendants of the original multipotent cancer cells. ACTA PATH. JAP. 22:555–564, 1972.     

p53 expression in oat and non-oat small cell lung carcinomas: correlations with proliferating cell nuclear antigen.

AIMS--To investigate the immunohistochemical expression of p53 protein in small cell lung carcinoma (SCLC), comparing it with that in non-small cell carcinoma (NSCLC); and to evaluate the correlation between p53 and proliferating cell nuclear antigen (PCNA) expression as well as between p53 and PCNA expression and survival. METHODS--Paraffin wax embedded tissues from 61 cases of primary lung carcinoma were stained for p53 protein and PCNA using the monoclonal antibodies 1801 and PC-10, respectively, in a standard avidin-biotin immunoperoxidase method. RESULTS--Of the 61 lung carcinomas 35 (57%) were positive for p53 (range 1% to 90%). Ninety percent of the non-oat SCLC contained positive cells and the labelling index (LI) was significantly higher than that of the oat SCLC (p < 0.001). SLCC also displayed a higher p53 LI than NSCLC (p < 0.01); no difference was found between squamous carcinoma, adenocarcinoma, and oat cell carcinoma. A p53 LI of greater than 1% tended to be associated with nodal metastases (p < 0.05), and p53 expression in node positive tumours as well as in oat cell carcinomas was indicative of poorer survival (p < 0.01 and p < 0.1, respectively). A p53 Li of greater than 60% was a negative prognostic factor in non-oat SCLC (p < 0.05). PCNA LI was highest in non-oat SCLC and lowest in NSCLC; oat cell carcinomas had a mean LI intermediate between NSCLC and non-oat SCLC (NSCLC v oat cell carcinoma p < 0.05 and oat cell v non-oat cell carcinomas p < 0.01). A PCNA LI was not correlated with nodal metastases or survival, but there was a significant positive correlation between PCNA LI and p53 LI (rs = 0.484, p < 0.001). CONCLUSIONS--p53 and PCNA expression differ substantially among the various types of lung carcinomas. Substantial differences were also found between oat and non-oat types of SCLC, indicating that SCLC is heterogeneous as far as proliferation rate and altered p53 expression is concerned. p53 seems to be of some prognostic value. The relation between PCNA and p53 expression indicates that the PCNA gene is slightly upregulated by p53.     

Acinar cell neoplasms of the exocrine pancreas.

The pathological features of 12 acinar cell neoplasms of the pancreas are described; these comprise 11 carcinomas, of which seven were pure acinar cell growths and four were mixed acinar and ductal carcinomas, and one adenoma. These tumors occurred in a series of 105 during the period 162-75. Thrombotic endocarditis developed in three out the 11 carcinoma cases. The distinctive histological features of these neoplasms and the means of differentiating them from anaplastic carcinomas and certain other carcinomas, for example, islet cell carcinoma, oat cell carcinomas, and carcinoid tumours, are discussed. The poor prognosis of pancreatic cancers is emphasized, and reasons are put forward for believing that future epidemiological studies may need to take account of the histological types of pancreatic carcinoma.     

An analysis of the sensitivity and specificity of the cytokeratin marker CAM 5.2 for epithelial tumours. Results of a study of 203 sarcomas, 50 carcinomas and 28 malignant melanomas

Two hundred and three sarcomas, 40 carcinomas, 10 carcinomas with spindle cell features, 27 malignant melanomas and one spindle cell melanoma were examined using CAM 5.2, a monoclonal antibody to cytokeratin. This antibody which was prepared against colorectal carcinoma cells and which identifies low molecular weight intermediate filament cytokeratin proteins is suitable for use in formalin fixed, paraffin embedded material. Seventeen of the 203 sarcomas showed positive staining. These included 15/21 synovial sarcomas, 1/5 epithelioid sarcomas and 1/18 malignant neural tumours. Five carcinosarcomas showed positive staining of their epithelial components but negative staining of their spindle cell components; three out of four pure spindle cell carcinomas stained positively; a metastasis from a spindle cell renal carcinoma was negative. A spindle cell thymoma also stained positively. Thirty-seven of the 40 carcinomas stained positively; the three negative carcinomas were a squamous cell carcinoma, a renal cell carcinoma and an oat cell carcinoma. All malignant melanomas were negative. These results are compared with those of other workers and the sensitivity and specificity of CAM 5.2 as an epithelial marker is assessed.     

Monoclonal antibody JC1: new reagent for studying cell proliferation.

AIM: To characterise a newly developed mouse monoclonal antibody JC1 which recognises a nuclear antigen present in proliferating cells in normal tissues and neoplastic lesions, and which is absent in resting cells. METHODS: The methodology was established using a representative range of frozen sections from normal tissues and from certain tumours which were immunostained with antibodies Ki67 and JC1. The molecular weight of the antigen recognised by JC1 was obtained by western blot analysis and this was compared with that of Ki67. IM-9 cell lysates containing Ki67 derived plasmids were also tested with JC1 antibody. RESULTS: Biochemical investigation indicated that the antigen recognised by JC1 gives two molecular weight bands of 212 and 123 kilodaltons, which is distinct from the well characterised anti-proliferation monoclonal antibody Ki67 (395-345 kilodaltons). In addition recombinant Ki67 protein is not recognised by JC1. Immunohistological reactivity was seen in areas known to contain numerous proliferating cells such as lymphoid germinal centres, splenic white matter, cortical thymocytes and undifferentiated spermatogonia. In tumours many cells from adenocarcinomas, oat cell carcinomas, squamous cell carcinomas of lung, and seminomas were labelled by JC1 with a distribution and proportion similar to that seen with Ki67. In normal tissues the only apparent difference was in testis where JC1 stained a considerably greater number of cells than Ki67. In all cases studied the new antibody showed nuclear reactivity only. JC1 did not show any cytoplasmic crossreactivity with squamous cells as is frequently seen with Ki67. CONCLUSION: Antibody JC1, which recognises a nuclear antigen present in proliferating cells, should provide a useful adjunct to Ki67 as a marker of proliferation especially in those cases such as squamous cell carcinomas where a Ki67 index cannot be determined.     

CD68 reactivity of non-macrophage derived tumours in cytological specimens.

AIMS--To investigate the presence of the macrophage associated antigen CD68 in non-haematopoietic tumours. METHODS--Cytological specimens from non-macrophage derived tumours were stained using the alkaline phosphatase anti-alkaline phosphatase immunocytochemical method (APAAP) and three monoclonal anti-CD68 antibodies, Y1/82A, EBM11, and KP1. RESULTS--Reactivity of malignant cells with one or more of the antibodies was seen in 11 out of 40 adenocarcinomas and in one of seven poorly differentiated carcinomas; other neoplasms, including 10 cases of squamous carcinoma, three of malignant melanoma, and four of oat cell carcinoma were negative. Monoclonal antibody KP1 gave the strongest staining and reacted with the highest proportion of neoplastic cells. CONCLUSIONS--CD68 is expressed in a proportion of epithelial tumours although the labelling is usually less intense than in macrophages. Anti-CD68 antibodies should therefore be used as part of a panel in the diagnosis of poorly differentiated neoplasms in cytological material.     

Distribution of the Ca (Oxford) antigen in lung neoplasms and non-neoplastic lung tissues.

The Ca (Oxford) antigen was originally isolated from a malignant neoplasm and with few exceptions was reported to discriminate between malignant and non-malignant neoplasms or normal tissues. Using the Ca 1 antibody we have studied the Ca distribution in 54 lung neoplasms and adjacent non-neoplastic lung tissue. Staining of tumours was very focal and the proportion of positive cells varied from about 50% for adenocarcinomas to less than 1% for oat cell carcinomas, which were often negative. Focal cytoplasmic staining can be seen in all neoplasms, whereas membrane staining is mainly seen in their areas of glandular and squamous differentiation. We found consistently strong membrane staining of alveolar type II pneumocytes in non-neoplastic lung. This staining may be useful in differentiating type II cells from alveolar macrophages which only occasionally showed granular cytoplasmic staining, probably due to phagocytosed Ca. Mucin from tumours and bronchi did not stain but there was consistent staining of alveolar serous exudate suggesting extracellular location of Ca.     

Ultrastructural and immunocytochemical definition of component neoplasms in an unusual gastrooesophageal collision tumour

An unusual collision tumour (concrescence of two neighbouring independent neoplasms) is reported. One tumour was a small cell undifferentiated (oat cell) carcinoma of the lower oesophagus and the other was a gastro-oesophageal adenocarcinoma. There was little intermingling of the two patterns. The adenocarcinoma stained strongly positive for mucin and carcinoembryonic antigen (CEA), but the small cell carcinoma was negative for both and also argyrophil-negative; both were negative for neurone-specific enolase and common leukocyte antigen. Ultrastructural study showed extra-cellular glandular lumina lined by cells with apical microvilli and junctional complexes in the adenocarcinoma; primitive cells without tonofilaments or dense-core granules, and joined by rudimentary desmosomes were seen in the small cell carcinoma. Collision carcinomas may result from a carcinogenic stimulus affecting two neighbouring regions of mucosa or may simply be the chance apposition of two unrelated tumours.     

Ultrastructural and biochemical analysis of "undifferentiated" pulmonary carcinomas.

Seven cases of "undifferentiated" pulmonary carcinoma were studied ultrastructurally; five were of the typical oat cell variety and the remaining two consisted of larger cells. In three of the former and both of the latter cases neurosecretory-like granules were demonstrated. Biochemical analysis of tumor tissue extracts revealed 5-hydroxy-3-indoleacetic acid, vanilylmandelic acid, and catecholamine activity in all instances. No hormonal syndrome or metabolic abnormality was detected in any of the patients. The concomitant morphologic demonstration of neurosecretory-like granules and the presence of 5-hydroxy-3-indoleacetic acid, vanilylmandelic acid, and catecholamines in neoplastic tissue would provide further evidence that these tumors may indeed arise from bronchial endocrine cells and could therefore be classified within the group of neuroendocrine carcinomas. Also it seems apparent that these neuroendocrine bronchial carcinomas may include tumors consisting of cells somewhat larger than the typical oat cell. The observation of 5-hydroxy-3-indoleacetic acid, vanilylmandelic acid, and catecholamine activity in two oat cell carcinomas in which neurosecretory granules could not be demonstrated poses an interesting problem whose solution may only derive from further studies.     

Criteria for the diagnosis of primary endocrine carcinoma of the skin (Merkel cell carcinoma). A histological, immunohistochemical and ultrastructural study of 13 cases

Thirteen cases of primary endocrine carcinoma of the skin (Merkel cell carcinoma) were reviewed with the aim of defining the morphological, immunohistochemical and ultrastructural criteria for diagnosis. The tumour cells were characterized by their scanty cytoplasm, generally small uniform nuclei with finely dispersed chromatin and multiple small nucleoli. Nuclear shapes varied from round to spindle, with larger and pleomorphic forms predominating in 2 tumours. A striking feature seen in 12 tumours was the occurrence of a "ball-in-mitt" pattern represented by 1 or 2 crescentic tumour cells closely wrapped around an oval cell. Staining for neuron-specific enolase was the most consistent marker of the tumour and the characteristic juxtanuclear globular staining for keratin and cytokeratin and the occasional coexpression of neurofilament set this tumour apart from other cutaneous neoplasms, in particular, metastatic carcinoid tumours and oat cell carcinoma from the lung. The fine structural features of note were striking paranuclear or juxtanuclear whorls of intermediate filaments, seen in 7 cases, the presence of variable numbers of membrane-bound dense core granules of 80-150 nm diameter in all cases and cytoplasmic spinous or microvillous projections containing microfilaments in 4 cases. Less consistent characteristics of primary endocrine carcinomas of the skin included cell moulding, argyrophilia and immunohistochemical staining for ACTH, VIP and calcitonin. The high frequency of vessel invasion in this series is in keeping with the high rate of local recurrence, lymph node metastases and visceral dissemination reported. The distinction from other similar appearing tumours in the skin is discussed.     

Oat cell carcinoma of the larynx. A study of six new cases

Clinicopathological details of six oat cell carcinomas of the larynx are presented. Five patients were male and heavy smokers. Only two patients had lymph node metastases at diagnosis. Only one tumour was unequivocally Grimelius stain positive. Immunostaining for neurone-specific enolase was negative in all cases. Neurosecretory granules were found in two tumours studied electron microscopically. Three tumours had areas of squamous carcinoma. Laryngectomy was the treatment of choice. Two patients are alive without evidence of disease 21 and 25 months after diagnosis.     

Oat cell carcinoma of the bronchus and the carcinoid syndrome.

A man with high urinary levels of 5-hydroxyindole acetic acid had an oat cell carcinoma of the bronchus. The patient had symptoms of the carcinoid syndrome and at autopsy was found to have evidence of carcinoid heart disease. This report supports the hypothesis that bronchial carcinoids and oat cell carcinomas are derived from argentaffin (Kulchitsky)-type cells of the lung.     

Pulmonary oat cell carcinomas. Expression of plasma membrane antigen correlated with presence of cytoplasmic neurosecretory granules.

A plasma membrane antigen highly associated with pulmonary oat cell carcinoma has recently been identified. Expression of this surface antigen was now examined in routine surgical pathology material from 32 primary lung tumors by immunostaining. The staining patterns of 17 tumors diagnosed as oat cell carcinoma by light microscopy were compared to those of 10 epidermoid carcinomas, four carcinoid tumors, and one lymphocytic lymphoma. In addition, when available, the oat cell carcinoma group was also studied by electron microscopy for demonstration of cytoplasmic neurosecretory granules. Twelve of the 17 oat cell carcinomas and one of the epidermoid carcinomas expressed the antigen. In this group of small cell tumors of the lung, aggrement was found between the expression of this immunochemical marker and the presence of neurosecretory granules in the cytoplasm of the tumors. Demonstration of this surface antigen in routine surgical pathologic material may be a useful aid in the diagnosis of these tumors. The failure of a group of tumors, which satisfied the light microscopic criteria for oat cell carcinoma to stain for the antigen, demonstrates heterogeneity within the oat cell carcinoma group.     

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.