Bcl-2 protein expression in lung cancer and close correlation with neuroendocrine differentiation.

For determination of the cellular distribution of bcl-2 expression in lung cancer and clarification of its correlation with cell neuroendocrine differentiation, Bcl-2 immunostaining was carried out on a large series of formalin-fixed, paraffin-embedded lung cancer samples, and four general neuroendocrine marker and seven peptide hormone stainings were carried out on all Bcl-2-positive squamous cell carcinomas and adenocarcinomas of the lung as well as on 8 pulmonary neuroendocrine carcinomas histologically diagnosed. In addition, 3 small cell lung cancer cell lines were studied by Western blotting. Neuroendocrine differentiation in Bcl-2-negative squamous cell carcinomas and adenocarcinomas was examined with chromogranin A and alpha-subunit of Go protein stainings. Bcl-2 protein was detected in 104/111 small cell carcinomas, 8/8 neuroendocrine carcinomas, 0/6 typical (well differentiated) carcinoids, 23/64 squamous cell carcinomas, 4/65 adenocarcinomas, and all 3 small cell lung cancer cell lines. All 8 neuroendocrine carcinomas, 11 of the Bcl-2-positive squamous cell carcinomas, and all 4 Bcl-2 positive adenocarcinomas expressed multiple neuroendocrine markers. The distributions of Bcl-2 and neuroendocrine marker immunoreactivity closely paralleled each other on consecutive sections. In squamous cell carcinomas, Bcl-2-positive cells could be roughly subdivided into those with neuroendocrine differentiation features, usually demonstrating intense Bcl-2 staining, with basaloid tumor cells usually expressing weak to moderate Bcl-2 staining. The present study clearly shows Bcl-2 protein expression to be remarkably differentially regulated according to histological types of lung cancers and to appear to quite likely be closely associated with neuroendocrine differentiation of tumor cells, indicating that bcl-2 is importantly involved in cell development and differentiation, in addition to protecting cells from apoptosis. Bcl-2 might be usable as a neuroendocrine marker in lung cancers and possibly also in neural-crest-derived tumors.     

Distribution of endothelin-like immunoreactivity and mRNA in the developing and adult human lung

Localization and characterization of endothelin-producing cells in the developing (fetal and postnatal) and adult human lung was investigated using the technics of immunocytochemistry and in situ hybridization. Immunoreactivity for endothelin was seen mainly in pulmonary endocrine cells of developing human lung. Immunoreactivity was also seen in the airway epithelium in fewer cases (about 50%) of human adults. In situ hybridization with 35S- or 32P-labeled RNA probes complementary to endothelin-1, -2, and -3, showed that endothelin mRNAs were expressed in a number of cells that were in similar sites to endocrine cells. Immunocytochemistry and in situ hybridization employed on pairs of reverse-face serial sections showed the presence of endothelin immunoreactivity and mRNAs in the same endocrine cell. Correlative studies revealed that endothelin is co-localized with general endocrine markers (synaptophysin, chromogranin, protein gene product 9.5) and regulatory peptides (e.g., gastrin-releasing peptide). The density (cells/mm2) of endocrine cells containing immunoreactivity or mRNAs was highest during fetal life and started to decline before birth, and was minimal in adults. Endothelin-like immunoreactivity and mRNAs were also expressed in endothelial cells. From these results, it is concluded that endothelin is synthesized in endocrine cells of human lung and the change of developmental expression of this peptide suggests it may play a part in growth regulation in addition to its putative vasoconstrictor role in human lung.     

IMMUNOREACTIVITY FOR HEPATOCYTE GROWTH FACTOR/SCATTER FACTOR AND ITS RECEPTOR,met, IN HUMAN LUNG CARCINOMAS AND MALIGNANT MESOTHELIOMAS

Paraffin sections from 29 lung carcinomas (28 primary and 1 metastatic) and 9 pleural malignant mesotheliomas were immunostained with antisera to human hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, met. For HGF/SF, immunoreactivity was demonstrated in all 9 mesotheliomas, 9 of 12 adenocarcinomas, and 7 of 10 squamous cell carcinomas. None of seven cases of small cell anaplastic carcinoma was positive. The adenocarcinomas frequently showed enhanced luminal staining, suggesting possible secretion of HGF/SF, and this pattern of staining was also seen occasionally in bronchial epithelium adjacent to the tumour. Stromal fibroblasts also showed immunoreactivity for HGF/SF in 6/8 cases of mesothelioma but in only 3/12 adenocarcinomas, 1/10 squamous cell carcinomas, and 1/4 small cell anaplastic carcinomas. All tumours stained for met, usually strongly. The staining was mainly cytoplasmic in nature, but some plasma membrane staining was usually evident. Adenocarcinomas showed strong luminal membrane staining, as did adjacent, histologically normal bronchial epithelium. This study demonstrates the presence of HGF/SF and met in most of the tumour types described, particularly mesotheliomas, and suggests that the HGF/SF/met signalling system may play a role in the development of these tumours, either by autocrine or by paracrine mechanisms.     

Expression of the laminin γ2 chain in different histological types of lung carcinoma. A study by immunohistochemistry and in situ hybridization

Sixty-four malignant lung tumours and 12 of their regional lymph node metastases were analysed for expression of the laminin γ2 chain by immunohistochemistry and in situ hybridization. Expression of the laminin γ2 chain was strongest in squamous cell carcinomas, followed by adenocarcinomas and large cell carcinomas. Positive cells, except for large cell carcinomas, were located at the epithelial–stromal interface of tumour clusters. An important exception was small cell lung carcinoma, with only a low level of laminin γ2 chain expression. Apart from tumour type, this may reflect the relatively scanty fibrous stroma in these tumours and supports previous observations that small cell lung carcinoma cells, contrary to other types, lack surface expression of α₆β₄ integrin, the specific laminin-5 binding receptor. In frozen sections, immunohistochemistry showed linear basement membranes around tumour clusters in squamous cell carcinomas and adenocarcinomas. This shows that carcinoma cells are capable of heavy deposition of the laminin γ2 chain around tumour clusters and suggests that a laminin γ2 chain-containing substrate may be of significance for the spread and growth of malignant tumours. Copyright © 1999 John Wiley & Sons, Ltd.     

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.     

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.     

Protein 1 and Clara cell 10-kDa protein distribution in normal and neoplastic tissues with emphasis on the respiratory system

Thirty-six different normal tissues and 13 different malignant epithelial tumours, were examined immunohistochemically for the presence of protein 1 (P1) and Clara cell 10-kDa protein (CC10). Adenocarcinomas of the lung were also examined for the expression of pulmonary surfactant apoprotein using a monoclonal antibody (PE-10). The staining results of P1 and CC10 were almost identical both in normal tissues and in malignant tumours. In normal lung, Clara cells were strongly positive for both P1 and CC10. In addition, some goblet cells and non-ciliated non-mucus cells in the upper airways were moderately positive for both proteins. In the malignant tumours, some lung cancers were positive for P1 and CC10, both of which were positive in the same tumour cells on sequential sections. In 117 lung cancers, P1 and CC10 were positive in 10.2% of adenocarcinomas, 20.5% of squamous cell carcinomas, and 12.5% of large cell carcinomas. PE-10 stained positively in 65.3% of adenocarcinomas, a frequency significantly higher than that of P1 and CC10 (P<0.01). These results suggest that P1 and CC10 are nearly identical proteins, that both are useful markers of Clara cells, and that many pulmonary adenocarcinomas express surfactant apoprotein rather than Clara cell proteins.     

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.     

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.     

Expression of the C-terminal peptide of human pro-bombesin in 361 lung endocrine tumours,a reliable marker and possible prognostic indicator for small cell carcinoma

Summary Small cell carcinoma of the lung is a highly malignant tumour. Its known biological products which include bombesin, do not allow the prediction of tumour behaviour. Molecular biology has revealed the amino acid sequence of human pro-bombesin, which consists of a signal peptide, the bioactive bombesin molecule and a C-terminal peptide. We have raised a rabbit antiserum to the first (N-terminal) 21 amino acids of the predicted C-terminal peptide. A total of 505 (361 neuroendocrine) surgically resected pulmonary tumours were evaluated for the presence of immunoreactive bombesin and C-terminal peptide. Strong immunostaining was obtained with the antiserum to the C-terminal peptide of human probombesin in 70% of the small cell carcinomas (175/250), in 63% of atypical (aggressive) carcinoids (31/49) but only in 16% of benign carcinoids (10/62). In contrast, bombesin immunostaining was focal and only moderately strong and the relative proportion of positive cases was quite evenly distributed amongst the neuroendocrine tumours: 35% of carcinoids (22/62), 22% of atypical carcinoids (11/49) and 25% of small cell carcinoma (62/250). None of the squamous, adeno, or large cell undifferentiated carcinomas were immunoreactive for bombesin or the C-terminal peptide. Radioimmunoassay and chromatography of extracts of tumours recovered from wax blocks revealed high concentrations of C-terminal peptide immunoreactivity (241±66 pmol/g of tissue) in all 12 small cell carcinomas studied, moderate concentrations in carcinoid tumours (50±7 pmol/g) and none in non-small cell carcinomas. Patients with tumours showing immunoreactivity to the C-terminal peptide of human pro-bombesin had a significantly shorter survival time than those without immunoreactive peptide (185±16.49 days, mean± SEM, and with 1128±226 days, respectivelyP> 0.02). The apparent presence of the C-terminal peptide of human pro-bombesin in higher concentrations than bombesin in the more malignant class of endocrine tumours, mainly small cell carcinomas associated with the poorest prognosis, suggests that the antiserum to this C-terminal peptide is not only a useful pathological marker but may prove to be of value in investigating the biological behaviour of small cell carcinomas and predicting the clinical course of the disease.     

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.     

Expression of epithelial and neural antigens in small cell and non small cell lung carcinoma

Seventy-one lung carcinomas from 66 different patients were stained with a panel of monoclonal antibodies. Twenty-nine were small cell lung carcinoma (SCLC), 15 adenocarcinomas, 17 squamous carcinomas and 10 large cell carcinomas. Three of the monoclonal antibodies recognize different cytokeratins, three recognize other epithelial antigens and one recognizes a neural antigen. Both formalin-fixed and cryopreserved tumours were studied using an indirect immunoperoxidase method. 23/29 SCLC reacted with all but one of the antibodies which recognize epithelial antigens. This staining was similar to that seen in non small cell lung carcinomas (NSCLC) and provides further evidence that SCLC are true epithelial tumours. All but one of the SCLC stained with the antibody recognizing a neural antigen. This antibody did not stain squamous or adenocarcinomas. However, four of the large cell carcinomas stained well with this antibody, suggesting that SCLC and some large cell carcinomas share a common pathway of differentiation. There were variations of staining seen both within and between tumours. This has obvious implications if immunotargetting with monoclonal antibodies is to be used diagnostically or therapeutically.     

Localization of the 92 kd gelatinase mRNA in squamous cell and adenocarcinomas of the lung using in situ hybridization.

We have used in situ hybridization for RNA to localize cells containing mRNA for the 92 kd gelatinase in carcinomas of the lung. We used archival material to analyze sections from 12 cases of squamous cell carcinomas of the lung including six stage I and three stage II and from three cases of adenocarcinoma of the lung. Presence of mRNA in the tissue was verified by in situ hybridization for gamma actin. The 92 kd gelatinase mRNA was found in all 12 squamous cell carcinomas tumors and was highly expressed in the tumor cells themselves. In addition, it was found in host stromal cells surrounding the tumor, but not in normal lung fibroblasts. In contrast it was not found in the adenocarcinomas of the lung or in the stroma surrounding these tumors. The mRNA for the 92 kd gelatinase was present in normal pulmonary tissue, bronchial epithelium, basal cell hyperplasia of bronchial epithelium, alveolar macrophages, and focally in bronchial mucous glands. It was not present in normal alveoli, vascular cells, cartilage, or most lymphocytes. We corroborated the presence of the mRNA for the 92 kd gelatinase by ribonuclease protection assay. The levels of mRNA for the 92 kd gelatinase in two specimens of squamous cell carcinoma were 6- to 10-fold greater than in the nonneoplastic tissue and two adenocarcinoma specimens.     

Utility of 123C3 monoclonal antibody against CD56 (NCAM) for the diagnosis of small cell carcinomas on paraffin sections

CD56 is immunohistochemically detectable in virtually all small cell carcinomas on frozen sections. The authors retrospectively tested the usefulness of the monoclonal antibody 123C3 against CD56 to differentiate pulmonary and extrapulmonary small cell carcinomas from nonneuroendocrine non—small cell carcinomas by paraffin-section immunohistochemistry after antigen retrieval. The study included 70 small cell carcinomas and 344 primary and metastatic nonneuroendocrine carcinomas of various primary sites. The staining results were compared with specific neuroendocrine markers (CD57, Chromogranin A, Synaptophysin). The monoclonal antibody 123C3 diffusely stained most small cell carcinomas with a strong membranous pattern (sensitivity: 0.99). The staining intensity was not diminished in areas with crush artifacts or after decalcification. The neuroendocrine markers had a combined sensitivity of only 0. 44 for small cell carcinomas. With regard to nonneuroendocrine carcinomas, the 123C3 antibody stained 7 of 28 ovarian carcinomas, 6 of 30 renal cell carcinomas, 2 of 10 endometrial carcinomas, two of three nonneuroendocrine large cell carcinomas of the lung, 1 of 38 adenocarcinomas, and 4 of 52 squamous cell carcinomas of the lung. Urothelial carcinomas, hepatocellular carcinomas, squamous carcinomas of the head/neck and cervix uteri, as well as adenocarcinomas of the breast, stomach, colon, pancreas, and prostate, showed no immunoreactivity for CD56. The specificities of 123C3 and the combined neuroendocrine markers for small cell carcinomas were 0. 94 and 0. 95, respectively. The authors conclude that monoclonal antibody 123C3 might be useful for the immunohistochemical differentiation of small cell carcinomas from nonneuroendocrine carcinomas on paraffin sections, especially in small and crushed biopsy specimens.     

Immunohistochemical markers of small cell carcinoma and related neuroendocrine tumours of the lung

A selected group of 263 pulmonary neuroendocrine tumours comprised 156 small cell carcinomas, five combined cell carcinomas, nine atypical carcinoid/small cell carcinomas, 32 atypical carcinoids, ten large cell/small cell carcinomas, and 51 carcinoid tumours. These were compared with a group of 109 non-small cell carcinomas, using four markers of neuroendocrine differentiation to determine differences in reactivity between the two groups and among the variants of neuroendocrine tumour. The antibodies used were neuron-specific enolase (NSE), protein gene product (PGP) 9.5, human bombesin, and the C-terminal flanking peptide of human bombesin (CTP). Most small cell carcinomas, carcinoid tumours, and atypical carcinoid variants showed immunoreactivity for both NSE and PGP 9.5 but a significant number of non-small cell carcinomas, mainly squamous cell carcinomas, were also positive (11 and 35 per cent, respectively). Bombesin was specific for neuroendocrine tumours, being demonstrable in 35 per cent carcinoids and 24 per cent small cell carcinomas, but staining was focal and often confined to scattered cells. Diffuse strongly positive immunoreactivity for CTP was seen in the majority of malignant neuroendocrine tumours, but only 12 per cent of carcinoid tumours were positive and non-small cell carcinomas were negative. CTP is therefore of potential value as a specific marker of malignant neuroendocrine tumours, particularly if the amount of biopsy material is limited and the tumour is an unusual variant, such as atypical carcinoid or large cell-small cell carcinoma.     

Apoptosis occurs independently of bcl-2 and p53 over-expression in non-small cell lung carcinoma

Certain oncogenes and tumour suppressor genes are known to modulate apoptosis. To investigate whether over-expressed bcl-2 and abnormally stabilized p53 are associated with reduced apoptosis in paraffin sections of non-small cell lung carcinoma, apoptotic, mitotic, and Ki-67 labelling indices were determined and correlated with bcl-2 and p53 immunoreactivity in 54 squamous cell carcinomas and 22 adenocarcinomas. Nineteen squamous cell carcinomas (35.2%) showed over-expression of bcl-2, but all 22 adenocarcinomas were bcl-2 negative. Thirty-seven squamous cell carcinomas (68.5%) and 13 adenocarcinomas (59.1%) showed p53 over-expression. Apoptotic tumour cells were identified among p53 positive and bcl-2 positive tumour cells. There was a significant linear correlation between apoptotic indices and mitotic indices. bcl-2 over-expression and p53 over-expression were not associated with attenuated apoptosis, or altered mitotic or Ki-67 labelling indices in either tumour type. Neither bcl-2 nor p53 was of prognostic significance. These results suggest that apoptosis in non-small cell lung carcinoma occurs independently, and is not modulated primarily by, bcl-2 or p53. It is likely that the effects on apoptosis of bcl-2 and p53 are countered by those of other oncogene products and/or additional factors that regulate apoptosis in vivo