Carbonic anhydrase-related protein VIII increases invasiveness of non-small cell lung adenocarcinoma

Carbonic anhydrase-related protein VIII (CA-RP VIII) is believed to be an oncofetal antigen and is overexpressed in colorectal and non-small cell lung cancer. However, the pathobiological properties of CA-RP VIII in lung cancer remain unclear. In the present study, we examined ultrastructural changes caused by exogenous CA-RP VIII expression in a well-differentiated lung adenocarcinoma cell line, PC-9. Many vacuoles lined by cilia, sometimes large vacuoles pushing the nuclei to one side, were found in the cytoplasm of CA-RP VIII-expressing PC-9 cells, but not in control PC-9 cells. Moreover, signet-ring cells containing abundant intracytoplasmic mucin were often found among CA-RP VIII-expressing PC-9 cells, but rarely among control PC-9 cells. We subsequently examined CA-RP VIII expression in atypical adenomatous hyperplasia and early-stage lung adenocarcinoma (Stage Ia). Significant expression of CA-RP VIII was observed in invasive lung adenocarcinoma but not in noninvasive adenocarcinoma. Interestingly, CA-RP VIII was strongly expressed in signet-ring cell cancer and invasive mucinous adenocarcinoma components. CA-RP VIII also appeared to enhance the invasiveness of PC-9 cells in Matrigel invasion assay. The present findings suggest that CA-RP VIII expression in lung adenocarcinoma is related to cancer cell invasion.     

Developmental expression of carbonic anhydrase-related proteins VIII,X, and XI in the human brain

Three cDNA homologues of carbonic anhydrase with unknown biological functions have been reported: carbonic anhydrase-related proteins (CA-RP) VIII, X, and XI. In the present study, we produced monoclonal antibodies to these CA-RPs and studied their regional and cellular distributions in the human adult and fetal brains by immunohistochemical analysis. In the adult brain, CA-RP VIII was expressed in the neural cell body spreading to most parts of the brain. CA-RP X was expressed in the myelin sheath and its expression was shown in the cytoplasm of cultured tumor cells by immunocytochemical analysis. CA-RP XI was expressed in the neural cell body, neurites, and astrocytes in relatively limited regions of the brain. In the fetal brain, CA-RP VIII and XI were expressed in the neuroprogenitor cells in the subventricular zone as early as the 84th day of gestation and subsequently detected in the neural cells migrating to the cortex. CA-RP X first appeared in the neural cells in the cortex at the 141st day. In the choroid plexus, the epithelial cells gave CA-RP VIII and XI expressions in both adult and fetal brains.From the findings in the present study on the distribution and the developmental expression of CA-RP VIII, X, and XI in the human brain we suggest that these CA-RPs play roles in various biological process of the CNS.     

High-level expression of CD109 is frequently detected in lung squamous cell carcinomas

CD109 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein, which is a member of the alpha2-macroglobulin/C3, C4, C5 family of thioester-containing proteins. It has been reported that CD109 is expressed in a subset of hematopoietic cells, endothelial cells and several kinds of human tumors. Herein it is reported that the CD109 protein is preferentially expressed in lung squamous cell carcinomas compared with other types of lung carcinoma including adenocarcinomas, large cell carcinomas and small cell carcinomas. Immunohistochemical staining of surgically resected lung specimens using an anti-CD109 antibody detected CD109 expression in basal cells of bronchial and bronchiolar epithelia and myoepithelial cells of bronchial secretary glands, but not in bronchial and bronchiolar apical epithelial cells and alveolar epithelial cells. Furthermore, the CD109 immunoreactivity was observed in squamous cell carcinomas at a high frequency compared with other types of lung carcinoma. Although the detailed function of CD109 protein is unclear, these results suggest that CD109 expression may play a role in the development of lung squamous cell carcinoma.     

Overexpression of carbonic anhydrase-related protein VIII in human colorectal cancer

Carbonic anhydrase-related protein (CA-RP) VIII, which is a member of the CA gene family, has been shown to have no catalytic CA activity and its biological function is still unknown. Recently, overexpression of CAs IX and XII has been reported in certain types of malignancy. To investigate a potential role for CA-RP VIII in human colorectal epithelial carcinogenesis, colorectal tissue specimens from surgically resected adenocarcinomas (n = 60) and endoscopically polypectomized adenomas (n = 13) were analysed by immunohistochemistry using monoclonal antibodies to CA-RP VIII and Ki-67 antigen. Less than 5% of epithelial cells in normal colonic mucosae (n = 73) were CA-RP VIII-positive and these were localized to the deep part of the cryptal epithelium. Increased expression of CA-RP VIII was observed in 78% of colorectal carcinomas. An intense signal was frequently observed at the tumour invasion front and its distribution was completely different from that of Ki-67 antigen. Colorectal adenomas also showed significant immunopositivity for CA-RP VIII, but its expression level was much lower than in adenocarcinomas. These findings suggest that CA-RP VIII plays a role in the process of invasion in colorectal cancer.     

Laminin and type VII collagen distribution in different types of human lung carcinoma: correlation with expression of keratins 14, 16, 17 and 18

The expression patterns of basement membrane components and keratin intermediate filament proteins were studied in normal human bronchial epithelium and 56 lung carcinomas using monoclonal antibodies to laminin, type VII collagen and the individual keratins 14, 16, 17 and 18. In normal lung, laminin and type VII collagen were present between the epithelium and the lamina propria of bronchi and bronchioles. Keratin 14 was expressed in the basal cells, keratin 17 in the basal and some suprabasal cells and keratin 18 in the columnar cells of the bronchi and bronchioles. Keratin 16 was not present in normal bronchial epithelium. Laminin was found in all subtypes of lung carcinoma, but type VII collagen was present only in squamous cell carcinomas, where it showed a reduction in expression with decreasing differentiation. Type VII collagen was not identified in adenocarcinomas, small cell carcinomas or carcinoids. Antibodies to basal cell keratins 14 and 17 also displayed positivity only in squamous cell carcinomas, although no correlation with the degree of differentiation could be observed. Keratin 16 appeared to be a marker of the squamous phenotype, rather than of hyperproliferation. The keratin 18 marker for columnar epithelial cells showed a reaction pattern opposite to that of the basal cell keratins, being extensively present in adenocarcinomas, small cell carcinomas and carcinoids, with less expression in squamous cell carcinomas. This study shows a correlation between the presence of type VII collagen and the basal cell keratins 14 and 17, and a negative correlation between these components and keratin 18. These findings are likely to be useful in identifying lung cancer subtypes.     

Adenosquamous carcinoma of the lung diagnosed by cytology?: A diagnostic dilemma

Adenosquamous cell carcinomas of the lung are rare tumours and are associated with a poor prognosis compared to other non-small cell carcinomas. We report a case of a solitary lung carcinoma evaluated by bronchial brush and lavage cytology, bronchial biopsy and pleural fluid cytology. Cytological assessment of the pleural fluid demonstrated non-small cell carcinoma and immunohistochemical staining confirmed a metastatic lung adenocarcinoma. The bronchial brush and lavage specimens, however, demonstrated the cytomorphological features of squamous cell carcinoma, which was confirmed by the bronchial biopsy. The finding of a mixed squamous and glandular component predicts a poor prognosis for this patient. The identification of a squamous component with the non-small cell carcinoma is important as this excludes the patient from anti-VEGF monoclonal antibody treatment due to the increased risk of haemorrhage.     

SEL1L expression in non-small cell lung cancer

SEL1L gene product plays a role in cell transformation and tumor progression in human breast, pancreas, esophageal, and prostate cancer. SEL1L expression was evaluated in a series of 76 surgically resected non-small cell lung carcinomas to investigate its clinical significance. SEL1L is scarcely detectable in normal lung, whereas in the initial stages of cell transformation, it becomes consistently expressed with evident staining in bronchial squamous metaplasia and in associated dysplastic changes. SEL1L immunoreactivity can be detected both in the cytoplasm and less commonly in the nuclei; the subcellular location correlates with tumor histotype, with cytoplasmic immunoreactivity being most prevalent in squamous cell carcinomas (P = .0005) and nuclear immunoreactivity being associated with adenocarcinomas (P = .02). Nuclear import and export signals are present in the SEL1L coding sequence, justifying the different subcellular location of the protein. SEL1L immunoreactivity was inversely correlated with tumor grade (P = .05); when considering only the adenocarcinomas, a stronger association was found (P = .006). SEL1L messenger RNA and protein evaluation in lung cancer cell lines confirmed the expression of the gene and the dual subcellular location of the protein in lung tumors. The data here reported suggest that, in non-small cell lung carcinoma, SEL1L may be an indicator of cell transformation, thus having important biologic and clinical implications.     

Neurotrophins and neurotrophin receptors in human lung cancer.

The expression of neurotrophins (NTs) and related high- and low-affinity receptors was studied in surgical samples of histologically diagnosed human tumors of the lower respiratory tract. The experiment was conducted with 30 non-small cell lung cancer specimens and in eight small cell lung cancer specimens by Western blot analysis and immunohistochemistry to assess expression and distribution of NT and NT receptor proteins in tissues examined. Immunoblots of homogenates from human tumors displayed binding of anti-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and NT-3 antibodies as well as of anti-tyrosine-specific protein kinase (Trk) A, TrkB, and TrkC receptor antibodies, with similar migration characteristics than those displayed by human beta-NGF and proteins from rat brain. A specific immunoreactivity for NTs and NT receptors was demonstrated in vessel walls, stromal fibroblasts, immune cells, and sometimes within neoplastic cell bodies. Approximately 33% of bronchioloalveolar carcinomas exhibited a strong membrane NGF and TrkA immunoreactivity, whereas 46% adenocarcinomas expressed an intense TrkA immunoreactivity but a weak immunostaining for NGF within tumor cells. Moreover, squamous cell carcinomas developed an intense TrkA immunoreactivity only within stroma surrounding neoplastic cells. A faint BDNF and TrkB immunoreactivity was documented in adenocarcinomas, squamous cell carcinomas, and small cell lung cancers. NT-3 and its corresponding TrkC receptor were found in a small number of squamous cell carcinomas within large-size tumor cells. No expression of low-affinity p75 receptor protein was found in tumor cells. The detection of NTs and NT receptor proteins in tumors of the lower respiratory tract suggests that NTs may be involved in controlling growth and differentiation of human lung cancer and/or influencing tumor behavior.     

Oncogene expressions detected by in situ hybridization of squamous metaplasia, dysplasia and primary lung cancer in human

In order to elucidate the dynamic changes of oncogene expression in the sequential cascade of squamous metaplasia, dysplasia, and squamous cell carcinoma of the bronchial epithelium, hybridization in situ was employed with a biotinylated oncogene probe. The expression of c-myc was localized exclusively in nuclei. While normal bronchial epithelium revealed no discernible clumps of c-myc grains, except occasional grains less than 3 per cell, squamous metaplasia showed increased number of grains and a few clusters of c-myc grains. In dysplasia, c-myc expression was more intensive than in squamous metaplasia. Approximately, 1/3 to 2/3 of tumor cell populations of squamous cell carcinomas of the lung revealed tremendously increased c-myc expression. In addition clumpy grains of c-myc in squamous cell carcinoma appeared more frequently than in squamous metaplasia or dysplasia. The c-myc expression was found to vary between different samples and within each cancer, and not all cancer cells expressed c-myc. These data indicate that c-myc oncogene plays it's role on reprogramming for growth control of cell populations particularly in multistage carcinogenesis and progression of lung cancer. These dynamic alterations of c-myc expression suggest that neoplastic transformation may occur conceivably at the dysplastic phase eventually resulting in carcinoma in situ. This means, in turn, squamous dysplasia is a putative precancerous lesion of the human lung.     

Immunohistochemical expression of aminopeptidase N (CD13) in human lung squamous cell carcinomas, with special reference to Bestatin adjuvant therapy

Bestatin, a specific inhibitor of aminopeptidase N (CD13), has been reported to prolong survival time in patients with completely resected stage I lung squamous cell carcinoma. Considering the antitumor mechanism of Bestatin, it is interesting to know whether CD13 is expressed in human lung squamous cell carcinoma. The immunohistochemical expression of CD13 was examined in human lung carcinoma and the question of whether CD13 was immunohistochemically expressed in the interstitial tissue was investigated, mainly in the fibroblasts and blood vessels, surrounding the tumor nests of various kinds of non-small cell lung cancers, especially of squamous cell carcinomas. In Japanese squamous cell carcinoma of the lung, 38 (61.3%) out of 62 cancers were positively stained in the same manner on immunohistochemistry for CD13. The area of interstitial tissue positively stained for CD13 varied depending on the case. To confirm the cell nature of the interstitial tissue with CD13 positivity, double immunohistochemistry using CD34 and alpha-smooth muscle actin was performed. Double immunohistochemistry showed that the majority of CD13-positive cells were slender fibroblastic cells around the blood vessels and some endothelial cells.     

hnRNP B1 expression in benign and malignant lung disease

Evidence is accumulating to suggest that hnRNP B1 expression may be a useful tool in the early diagnosis of lung cancer. This study examined the immunohistochemical expression of hnRNP B1 in archived sections of resected lung cancers and compared the patterns of expression with those seen in similar archived sections of non-neoplastic lung. Particular attention was paid to the expression of hnRNP B1 in the benign bronchial cells in both cases, to establish if overexpression of this protein in respiratory epithelial cells is specific for malignancy. Nineteen cases of different types of non-small cell carcinoma were examined (eight squamous cell, six adenocarcinomas, two carcinosarcomas, two undifferentiated large cell carcinomas, and one mucoepidermoid carcinoma) and compared with sections from 16 open lung biopsies (three cases of cryptogenic fibrosing alveolitis, two cases of sarcoidosis, two cases of organizing pneumonia, and one case each of tuberculosis, extrinsic allergic alveolitis, non-specific interstitial pneumonitis, pneumocystis pneumonia, aspergilloma, respiratory bronchiolitis-interstitial lung disease, mineral dust disease, Sj?gren's syndrome and systemic sclerosis vascular variant). All the tumours showed positive staining, with the vast majority, 16/19 (84%), showing strong diffuse nuclear staining. The background cells of these cases showed positive staining in alveolar macrophages, lymph node germinal centres, bronchial mucous glands, and bronchial epithelial cells. No significant difference was seen in the percentage of positive bronchial epithelial cells in bronchi adjacent to the tumour compared with the resection margins. In the benign lung cases, positive bronchial epithelial cells were seen in a small percentage, 3/16 (18%), of cases, but the majority of cases showed no or very focal staining. The levels of expression between benign epithelial cells of malignant cases, compared with benign, showed a significant difference when the staining was assessed in percentage of positive nuclei (p = 0.001, Fisher's exact test). The results confirm that hnRNP B1 is widely expressed in a range of lung carcinomas; that expression is seen in benign bronchial epithelial cells and inflammatory cells; and that expression in background bronchial epithelial cells appears to be higher in malignant than in benign lung disease. It is feasible that this biomarker may be of use in the detection of early lung cancer, provided that levels of expression can be accurately quantified.     

Immunohistochemical analysis of calcitonin and calcitonin gene-related peptide in human lung

Calcitonin and calcitonin gene-related peptide (CGRP) have been localized immunohistochemically in neuroendocrine cells of normal and diseased human lungs. Cells immunoreactive for calcitonin and CGRP first appeared in immature bronchi in the 27th gestational week. Thereafter, both peptides were found in the same bronchial neuroendocrine cells throughout fetal and neonatal life. In adult lungs with or without neuroendocrine cell hyperplasia, only calcitonin was present. In 17 of 18 (94%) pulmonary tumorlets, variable numbers of calcitonin-positive cells were identified. A few CGRP immunoreactive cells, as a subset of calcitonin-containing cells, were found in only three (17%) lesions. Of 37 bronchial carcinoids, calcitonin was detected in 14 and CGRP was detected in 16 (38% and 43%, respectively), and both peptides were predominantly localized in the same cells. Ten of 45 (22%) small cell lung carcinomas were calcitonin-immunoreactive. CGRP was noted in only one (2%) of these tumors, and both peptides coexisted in single cells. These findings indicate that the patterns of calcitonin/CGRP expression in hyperplastic bronchial neuroendocrine cells, pulmonary tumorlets, and, to some extent, small cell lung carcinomas are similar to those of normal adult lungs. On the other hand, calcitonin/CGRP expression in bronchial carcinoids is similar to that of late fetal and neonatal lungs.     

Peripheral airway cell marker expression in non-small cell lung carcinoma. Association with distinct clinicopathologic features.

Paraffin sections of 247 primary and metastatic non-small cell lung carcinomas, the corresponding non-neoplastic lungs, and 75 other specimens were examined by immunohistochemical procedures using a panel of antibodies against the specific products of peripheral airway cells: the major surfactant-associated protein and 10-kD Clara cell protein. Non-small cell lung carcinoma tumors most frequently positive for either peripheral airway cell marker were adenocarcinomas (41%), especially those with papillolepidic growth pattern (56%), followed by large cell carcinomas (25%), other adenocarcinomas (22%), and squamous cell carcinomas (16%). Immunoreactivity was mainly focal and the expression of the two peripheral airway cell markers was discordant. The incidence of marker expression was similar in metastatic and primary non-small cell lung carcinoma. Other organs and their tumors were negative, with few exceptions. Non-small cell lung carcinomas positive for peripheral airway cell markers were associated with younger age and less-intense smoking, and surfactant-associated protein reactivity was more common in women than in men. Peripheral airway cell markers were independent prognostic factors for survival and delayed development of metastases in patients with less-advanced disease. It is concluded that surfactant-associated protein and 10-kD Clara cell protein are specific markers for non-small cell lung carcinoma and peripheral airway cell differentiation and provide useful tools to study the pathogenesis, biology, and prognosis of non-small cell lung carcinoma.     

Prognostic significance of anti-apoptosis proteins survivin and bcl-2 in non-small cell lung carcinomas: a clinicopathologic study of 102 cases.

Inhibitors of apoptosis, including bcl-2 and survivin (a novel gene encoding a unique apoptosis inhibitor), regulate cell proliferation by promoting cell survival. Although survivin has been detected in several human cancers, its prognostic significance and relationship to bcl-2 are not well characterized in lung cancer. Tissue sections from 102 non-small cell lung carcinomas (NSCLC) were immunostained using antibodies against survivin and bcl-2. Staining results were correlated with prognostic variables. Immunoreactivity for survivin and bcl-2 was observed in 53% and 21% of NSCLCs, respectively. Fifty-two percent of the 50 squamous cell carcinomas and 54% of the 52 adenocarcinomas expressed survivin. Survivin positivity correlated with tumor stage in squamous cell carcinoma. On univariate analysis, survivin expression correlated with decreased patient survival in NSCLC and in the subset of squamous cell carcinomas, but not in adenocarcinomas. On multivariate analysis, survivin was an independent predictor, along with distant metastasis and large tumor size. Eighteen percent of squamous cell carcinomas and 24% of adenocarcinomas expressed bcl-2. On univariate analysis, bcl-2 expression correlated with increased patient survival in NSCLC and in the subset of squamous cell carcinomas. An inverse correlation between the expression of survivin and bcl-2 was noted. Survivin immunoreactivity is an independent predictor of shortened survival in NSCLC, while bcl-2 protein expression correlated with prolonged patient survival. These findings indicate an inverse relationship between survivin and bcl-2 expression and suggest that these two inhibitors of apoptosis function through different pathways in the regulation of tumorigenesis in NSCLC.     

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

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

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.