Immunohistochemical study of lung adenocarcinoma using monoclonal antibody for 60-kilodalton antigen in type II pneumocytes and nonciliated bronchiolar epithelial cells. Comparison with two antisurfactant apoprotein antibodies.

Monoclonal antibody KP16D3 was produced by immunizing mice with monkey bronchoalveolar lavage. KP16D3 revealed the immunohistochemical reactivity in the cytoplasm of some nonciliated bronchiolar epithelial cells and type II pneumocytes and thereby recognized specifically a protein with an apparent molecular weight of 60 kD with the use of Western blotting and immunoaffinity column chromatography followed by SDS-PAGE. Examination of 76 primary and 4 metastatic lung carcinomas in primary lung carcinoma KP16D3 showed immunohistochemical positivity only to mucin-nonproducing papillary adenocarcinoma (27/28) and bronchioloalveolar carcinoma (2/2), except for one case of large cell carcinoma. All other primary lung carcinomas such as squamous cell carcinoma, acinar adenocarcinoma, and small cell carcinoma had negative results. From these findings, KP16D3 seems to be an effective immunohistochemical marker of mucin-nonproducing papillary adenocarcinoma and bronchioloalveolar carcinoma of the lung and it appears to be useful to investigate both the histogenesis and functional expression of primary lung adenocarcinoma.

Sheep bronchoalveolar carcinoma: tissue associated protein complex (TAPC) in normal lung tissue and in the tumor differed quantitatively.

Sheep lungs experimentally and naturally affected by bronchoalveolar carcinoma were washed out exhaustively of soluble components by phosphate-buffered saline, pH 7.4 (PBS), followed by glycine buffer, pH 2.8 (GB), and then again by 1M KCl followed by PBS. The tissue matrix (TM) of the tumor-free region and the tumor-affected tissue were analysed separately by sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis. Normal lung tissues obtained from normal sheep served as controls. Several protein fractions and fragments, identified in both the normal and the tumorous lung, have the molecular weight (MW) of 130,000-228,000, as compared with the major soluble tissue associated protein having MW of 70,000. Coomassie blue staining used in the SDS polyacrylamide system and alkaline phosphatase immunoreaction used in the Enzyme Linked Immunosorbant Assay (ELISA) showed tenfold increased concentration of the TAPC in the TM of the tumor tissue and in the blood of tumor-affected animals, respectively. Total concentration of the TAPC in the serum of tumor-affected animals was higher than in the normal. Immunofluorescent antibody test (IAT) detected the TAPC in the cytoplasm of tumor as well as in normal lung cells, and the study suggested that the TAPC reaches the peripheral blood during tissue destruction occurring at the tumor site, as observed by light and electron microscopy (LM and EM). The concentration of each of the TAPC fractions was higher in the tumor-affected sheep lung as compared with normal sheep lung. Antibodies prepared against the TAPC fractions were toxic to sheep lung cells in tissue culture. Tumor cells were more susceptible.

Nuclear DNA content in squamous cell carcinoma, small cell carcinoma, and bronchiolo-alveolar carcinoma of the lung.

Nuclear DNA content in individual, morphologically identified tumor cells from 33 squamous lung carcinomas, 20 small cell lung carcinomas, and 10 bronchiolo-alveolar carcinomas were analyzed by means of cytophotometry on Feulgen-stained histologic and cytologic specimens. Twenty-eight of the squamous cell carcinomas and 17 of the small cell carcinomas had high and scattered DNA values, indicative of high malignancy potentials. None of the bronchiolo-alveolar carcinomas showed such high DNA values. These results are in line with clinical experience that squamous cell and small cell carcinoma are associated with rapid progression and death in patients, whereas bronchiolo-alveolar carcinomas have a more indolent course.

Immunohistochemical study of basement membrane antigens in bronchioloalveolar carcinoma.

Bronchioloalveolar carcinoma (BAC), not yet completely defined as a biologic entity, has recently been classified into two different types. Immunohistochemical investigations, aimed at characterizing basement membrane (BM) behavior in the two types of BAC, revealed different distribution patterns. The first (Type I BAC) showed a linear staining for laminin and Type IV collagen similar to normal lung. Fibronectin was widely present in the septal interstitium and patchily distributed along the BM. The second (Type II BAC) showed a variable reaction for Type IV collagen and fibronectin, whereas laminin was absent or appeared as short, interrupted tracts around the epithelial neoplastic population, similar to conventional adenocarcinoma of the lung. These results suggest that only Type I BAC shows structural characteristics different from those of conventional adenocarcinoma of the lung.

Detection of human lung epithelial cell growth factors produced by a lung carcinoma cell line: use in culture of primary solid lung tumors.

Serum-free medium conditioned for 72 h by a human bronchioloalveolar carcinoma of lung, A549-1, stimulated the colony formation of normal human bronchial epithelial cells, newly cultured cells from human solid lung tumors, and established human lung tumor cell lines, including A549-1 cells themselves. This activity was concentration dependent and was stable to acid. Growth factors in A549-1 conditioned medium (CM) supported culture of solid lung tumors; primary cell cultures were obtained from nine of 10 solid lung tumors of non-small cell origin and from one small cell tumor using A549-1 CM. In addition, three cell lines have been established to date from these primary cultures. Gel filtration of concentrated A549-1 CM on Biogel P-10 separated the growth promoting activity into four regions of apparent Mr 70,000, 12,000, 8,000, and 6,000, and two broad regions of apparent Mr 3000-5000. All but the 12,000 Mr fraction contained activity which competed for specific binding of epidermal growth factor (EGF) to A431 cell membranes. CM was superior to both EGF and TGF alpha in stimulating growth of normal and neoplastic lung cells. EGF also was inhibitory to tumor cells while TGF alpha stimulated both normal and tumor cell growth. TGF beta was also found in CM but inhibited normal and neoplastic lung epithelial cell growth. Of other substances tested, ILGF-I stimulated colony formation. The results suggest that autocrine factors may be important in non-small cell lung tumor cell growth and that differences in response to EGF and TGF alpha may provide the basis for selective culturing of normal and neoplastic lung epithelial cells.

Ultrastructural and immunohistochemical studies of bronchiolo-alveolar carcinoma.

A detailed ultrastructural study was made of seven cases of bronchiolo-alveolar carcinoma, and the findings were correlated with histochemical and immunohistochemical data. By electron microscopic examination all seven tumors displayed glandular differentiation, manifested by the presence of microvilli and intercellular junctions, with or without mucin production. Variable proportions of tumor cells retained ultrastructural characteristics of alveolar type II cells and Clara cells. In addition, some tumor cells revealed desmosomes and tonofilaments consistent with squamous differentiation. Immunohistochemical evaluation was carried out using a peroxidase-antiperoxidase technique and specific antibodies against surfactant high molecular weight glycoproteins, keratin proteins, IgA + secretory piece, carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), and alpha-fetoprotein (AFP). Four tumors with type II cell-like differentiation stained with anti-surfactant glycoprotein sera. All seven tumors stained focally with anti-keratin and IgA + anti-secretory piece antibodies, and diffusely with CEA. These tumors failed to stain with antisera against HCG and AFP. It is concluded that bronciolo-alveolar carcinomas are primarily composed of cells with alveolar and bronchiolar cell differentiation. Adequate criteria were established for ultrastructural identification of tumor cells with differentiation to type II alveolar cell or Clara cell. Moreover, the findings of this study indicate that the surfactant glycoprotein marker, when present in a given tumor either diffusely or focally, is diagnostic of bronchiolo-alveolar carcinoma.

Keratin proteins in human lung carcinomas. Combined use of morphology, keratin immunocytochemistry, and keratin immunoprecipitation.

Light-microscopic immunocytochemistry and electron microscopy demonstrated that adenocarcinomas (AC) and squamous cell (epidermoid) carcinomas (SCCs) of human lung contained keratin proteins in the form of tonofilament bundles. However, moderately differentiated (md) SCCs contained abundant keratin, whereas poorly differentiated (pd) SCCs and all ACs contained lesser amounts. Lung tumors with the diagnosis of AC or SCC, as defined by WHO criteria, were also analyzed by immunoprecipitation techniques for the presence of keratin proteins. Regardless of the degree of tumor differentiation, SCCs contained a 44 kd keratin which was lacking in ACs. Interestingly, normal bronchial epithelium also contained the same 44 kd keratin. In addition, as SCCs became more differentiated, they exhibited even greater differences in the profile of synthesized keratins. Specifically, the relative abundance of the intermediate-sized keratins (57 and 59 kd) was increased in the md SCCs. Although keratin protein patterns appear to be a valuable adjunct in distinguishing AC from SCC, their usefulness as a diagnostic tool will require survey of a larger number of poorly differentiated tumors.

Characterization of a glycoprotein isolated from a continuous tumor-cell line of human lung carcinoma.

A glycoprotein with a molecular weight of 62 000 has been isolated from a tumor-cell line, A549, and purified to homogeneity by gel chromatography. The glycoprotein contained sialic acid, galactose, mannose, N-acetylglucosamine and a relatively high amount of glutamic acid and proline. The data indicated that the overall composition of this glycoprotein was different from that of the glycoprotein of Mr 62 000 isolated from lung lavage of patients with alveolar proteinosis. The glycoprotein did not react with the antiserum raised against glycoprotein of Mr 62 000 isolated from lung lavage of patients with alveolar proteinosis.

Biochemical characterization of pulmonary washings of patients with alveolar proteinosis, interstitial pneumonitis and alveolar cell carcinoma.

We have studied the biochemical compositions of fifteen pulmonary washings from seven patients with alveolar proteinosis, and two washings from two patients with interstitial pneumonitis and two from two patients with alveolar cell carcinoma. The pulmonary washing was separated into the supernatant and precipitate fractions by a brief centrifugation. Analytical results revealed that the pulmonary washings from patients with alveolar proteinosis contained much more protein and lipids as well as a higher percentage of phospholipid than did the pulmonary washings from other patients. With regards to alveolar proteinosis, the precipitate fraction, i.e., water-insoluble material, contained lipids as the major component, the majority of which was dipalmitoyl phosphatidylcholine. Protein in the sedimental material was small in amount, but was composed of proteins mainly of molecular weights of 62,000, 36,000, 28,000 and 15,000 as measured by SDS-gel electrophoresis. On the other hand, the supernatant fraction, i.e., water-soluble material, was composed predominantly of serum proteins, with the lipid content being lower than those in the precipitate fraction. These analytical findings support the idea that materials normally existing in the alveoli are excessively accumulated as alveolar-filling materials in alveolar proteinosis. It was also noted that there were marked differences in the lipid profiles between pulmonary washings from patients with alveolar proteinosis, and those from patients with other diseases, indicating that the biochemical composition of pulmonary washings tends to reflect the nature of an underlying disease. From these findings, the cause of the alveolar-filling materials found in alveolar proteinosis was discussed.

Immunohistological analysis of surfactant-apoprotein in the bronchiolo-alveolar carcinoma.

Fifty-five cases of bronchiolo-alveolar carcinoma were examined immunohistochemically using mono-specific antisurfactant apoprotein IgG obtained from a rabbit immunized with monkey surfactant preparations. Formalin-fixed and paraffin-embedded lung tissues were stained by an immunoperoxidase method. Antibody stained the normal and hyperplastic alveolar type II pneumocytes, but did not stain bronchial epithelium or other lung cells. In tumor tissue, 26 (47.3%) of 55 cases were positively stained in the cytoplasm, and 15 showed reaction products in both the cytoplasm and intranuclear regions. By electron microscopy osmiophilic lamellar bodies and microvilli on the free surface, thought to be characteristic of type II pneumocytes, were seen in tumor cells (2 cases). In the five cases, the nuclei contained branching tubular inclusions. The results of this study support the idea that certain bronchiolo-alveolar carcinomas originate from type II pneumocytes, the intranuclear inclusions may represent an abnormal proliferation of nuclear membranes containing surfactant-apoprotein.

Detection of tissue CEA-like substance as an aid in the differential diagnosis of malignant mesothelioma.

Sections of various adenocarcinomas and malignant mesotheliomas were tested for carcinoembryonic antigen (CEA) localized in tissues by the immunoperoxidase technique; epithelial mucin was demonstrated with the PAS technique. While CEA and mucin were found in many adenocarcinomas, both were absent in the 43 cases of malignant mesothelioma we investigated. In the problem of distinguishing between adenocarcinoma and mesothelioma, the CEA-test in combination with conventional strains for mucin is a useful technique and clearly identifies most adenocarcinomas. A dual negative result for CEA and mucin, although not proving that a given lesion is a mesothelioma, adds considerable support to this histological diagnosis.

Intravascular bronchiolo-alveolar tumor (IVBAT): A low-grade sclerosing epithelioid angiosarcoma of lung.

Intravascular bronchiolo-alveolar tumor (IVBAT) is a rare and highly distinctive pulmonary tumor of disputed cellular nature. Both epithelial and endothelial differentiation of this neoplasm have been suggested. We have studied multiple nodules of IVBATs from three patients by light and electron microscopy and by immunohistochemical methods for Factor VIII-related antigen (FVIII RAG). Our light and ultrastructural studies are in essential agreement with the previous suggestion of the endothelial nature of the neoplasm and our demonstration of the presence of FVIII RAG in many of the tumor cells offers new evidence strongly supportive of their endothelial differentiation. We believe that IVBAT and a group of extrapulmonary tumors described as epithelioid hemangioendothelioma and endovascular papillary angioendothelioma are similar biologically indolent neoplasms of epithelioid and dendritic endothelial cells characterized by stromal sclerosis, intravascular spread, a low incidence of metastases and slow clinical evolution. Thus, we regard IVBAT as a low-grade sclerosing angiosarcoma of the lung.

Purification and partial characterization of high-molecular-weight forms of ectopic calcitonin from a human bronchial carcinoma cell line.

Studies on the high-molecular-weight immunoreactive calcitonin produced ectopically in culture by an epidermoid bronchial carcinoma cell line are reported. In cell-exposed medium, the principal component has a molecular weight of 40000 and molecules of mol.wts. 13000 and 10000 also occur. Only a trace amount of material co-eluting with 35000-mol.wt. human calcitonin is detectable. None of the calcitonins show cross-reactivity with anti-corticotropin serum. The 40000-mol.wt. immunoreactive calcitonin is readily proteolysed to the 13000- and 10000-mol.wt. components, but the 10000-mol.wt. component behaves as a comparatively stable 'core' molecule. By using immunoprecipitation and high-pressure liquid chromatography (h.p.l.c.), it is possible to prepare radiochemically homogeneous 10000-mol.wt. immunoreactive calcitonin from cells grown in the presence of individual 35S- or 3H-labelled amino acids. Peptide mapping of enzymic digests of this material by h.p.l.c. shows that it contains peptides in common with synthetic human calcitonin.

[Intracellular and extracellular acid mucopolysaccharides in human bronchus carcinoma and lung metastases - prognostic parameters?]. / Infiltratzellen und extrazelluläre saure Mukopolysaccharide in menschlichen Bronchuskarzinomen und Lungenmetastasen - prognostische Parameter?

Local cellular and extracellular (acid mucopolysaccharide) reactions in human lung cancers and metastases - prognostic parameters? The role of immunologic reactions in human malignant tumors is still unsettled. In the work presented, 72 primary lung cancers and 17 pulmonary metastases of 8 different organs of origin were studied with histochemical techniques for cellular infiltrates (eosinophils and macrophages) and extracellular acid mucopolysaccharides (AMPS). The results showed a positive correlation between eosinophils and macrophages and a negative correlation between cellular reactions and the deposition of AMPS. The clinical course of radically operable patients with primary lung cancer 2 to 3 1/2 years after operation showed that patients with the reactive pattern "strong local eosinophilia, no AMPS deposition" had the best prognosis with 12 of 14 patients free of tumor. Of the patients with the same tumor stage, but lacking local eosinophilia, more than half died from the tumor or had generalized metastases within the same period. The most frequent pattern in patients with pulmonary metastases was "no local eosinophilia, strong AMPS reaction". These observations suggest that cellular reactions in radically operable primary lung cancer express immunologic responses. These are often absent in metastases.

Phospholipid composition and ultrastructure of A549 cells and other cultured pulmonary epithelial cells of presumed type II cell origin.

In order to assess the usefulness of A549, L-2, and AK-D cell lines as model systems for alveolar type II cells, we compared their phospholipid composition to that of fibroblasts grown under similar conditions. The percentage of disaturated phosphatidylcholine and phosphatidylglycerol, key phospholipids of purified surface-active material, was the same in epithelial cells and fibroblasts. When A549 cells were maintained in serum-free media for two days, ultrastructural examination showed an increase in cytoplasmic lamellar inclusions but there was no change in the percentage of disaturated phosphatidylcholine or phosphatidylglycerol. Because the lipid content of these cultured cells was very different from that of freshly isolated rat type II cells, we conclude that their suitability as model cell systems for type II cells is questionable.