Performance of biomarkers SMRP, CA125, and CYFRA 21-1 as potential tumor markers for malignant mesothelioma and lung cancer in a cohort of workers formerly exposed to asbestos.

The aim of the study is to examine the cancer-predictive values of SMRP (soluble mesothelin-related peptides), CA125, and CYFRA21-1 as potential tumor markers for lung cancer and malignant mesothelioma in a cohort of workers formerly exposed to asbestos. A voluntary surveillance program has been established for German workers with former asbestos exposure. A subgroup of 626 subjects with a mean age of 63 years (range 53-70 years) at baseline was enrolled in an extended health examination program with high-resolution computer tomography (HRCT) of the chest and blood drawing between 1993 and 1997. Serum concentrations of SMRP, CA125, and CYFRA21-1 were measured in archived serum samples in 2005 and 2006. A mortality follow-up was conducted through 2007. So far, 12 cases with lung cancer and 20 cases with malignant mesothelioma have been observed in this cohort. The average time between sample collection and diagnosis was 4.7 years. Analyzed biomarkers showed low sensitivities (5-25%) and positive predictive values (4-30%) for both cancer sites. Marker combinations resulted in sensitivities between 5 and 50% and positive predictive values ranging from 3 to 14%. Even in those cases, where biomarker concentrations were available within 36 months before diagnosis, no trend for increasing biomarker levels was observed. The analyzed tumor markers were characterized by high specificities, but low sensitivities. SMRP, CA125, and CYFRA21-1 alone or in combination were less suitable to serve as predictors for the diagnosis of lung cancer or malignant mesothelioma. However, a prospective study with annual sampling might reveal a better predictive value of these markers.

Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas.

Nineteen specimens from primary human malignant mesotheliomas obtained from 19 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 by combined RFLP-PCR/SSCP analysis. In addition, all tumours were screened for deletions and point mutations in the tumour suppressor genes p53, p16INK4a (CDKN2A) and p14ARF (exon-1beta) by combined multiplex-PCR/SSCP analysis. No mutations were found in N-ras, p53 and CDK4. Three tumours displayed homozygous deletion (co-deletion of exons 1, 2 and 3) of p16INK4a. One of them displayed additional homozygous deletion of p14ARF (exon-1beta). Two silent point mutations and 2 polymorphisms were found in p16INK4a in 3 tumours. Our preliminary data indicate that disarrangement of the Rb1 pathway may be involved in mesothelioma formation.

Detection of MDM2-proto-oncogene in paraffin embedded human bronchial epithelium.

Recently a new proto-oncogene, the murine double-minute 2 (MDM2), has been described. MDM2 becomes oncogenic due to amplification and overexpression. Among other proto-oncogenes MDM2 becomes interesting since MDM2 protein can associate with both mutant and wild type p53 tumor suppressor gene products and thus inhibit p53-mediated transactivation of other genes. Loss of p53 tumor suppressor function is the most frequently observed alteration in human tumors. Immunohistochemical studies investigating the quantity of MDM2 protein in human sarcomas revealed an overexpression in 30% of the specimens. Here we describe the successful use of a monoclonal antibody (IF2) for the detection of MDM2 protein in paraffin-embedded tissue from human lung biopsies. 18 out of 44 specimens (41%), predominantly mucosal epithelial and glandular epithelial cells, stained positive for MDM2. No significant difference was observed between non-cancerogenic cells adjacent to tumor cells and those specimens without any tumor cells but altered by inflammatory processes. In general, the staining pattern was restricted not to the nuclei, but to selected subnuclear compartments, probably representing the golgi apparatus or the endoplasmatic reticulum. Our data support the hypothesis that in addition to its nuclear function of forming a complex with p53, MDM2 may also be secreted and thus have a transcellular effect.

Pathology of small-cell lung cancer.

The morphological differentiation between small-cell and non-small-cell lung cancer has great prognostic and therapeutic significance for the patient. Malignant lung tumors are now classified according to the new 1999 WHO/IASLC classification of lung and pleural tumors. The variant of heterogeneously differentiated "combined small-cell carcinoma" can be distinguished from classical small-cell carcinoma, whereas the subtype of "intermediate cell carcinoma" is no longer used. Together with "large-cell neuroendocrine carcinomas" and typical or atypical carcinoid tumors, small-cell lung cancers are currently histogenetically categorized as neuroendocrine lung tumors. In contrast to large-cell neuroendocrine carcinoma, the immunohistochemical demonstration of neuroendocrine differentiation is not a prerequisite for the diagnosis of small-cell lung cancer. Although electron-microscopical, immunohistochemical, and molecular-biological findings have considerably increased our understanding of the pathogenesis and progression of malignant lung tumors, routine pathological-anatomical diagnostics are still decisively based on light-microscopical evaluation of tissue samples.

P53 accumulation and proliferating-cell nuclear antigen expression in human lung cancer.

Mutations in the p53 gene are currently the commonest genetic alterations in human malignant tumors, including non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). Alterations of the protein induced by gene mutations enables the mutant protein to become more stable, resulting in the accumulation of P53 in quantities detectable by immunohistochemistry. Although previous studies document the accumulation of P53 in lung cancer, there is little information regarding the usual frequency of accumulation based on a comprehensive number of lung tumors. A total of 328 paraffin-embedded lung carcinoma specimens were analyzed for P53 accumulation and for the expression of the proliferating-cell nuclear antigen (PCNA) by standard immunohistochemistry. Among 49 SCLC, 35% were positive for p53 and 51% were positive for PCNA. Out of 279 NSCLC, 43% showed a positive P53 immunoreaction and 72% displayed detectable amounts of PCNA. In squamous-cell carcinomas a statistically significant increased accumulation of P53 was found compared to adenocarcinomas (P = 0.001). Among the 233 PCNA-positive tumors the relative number of P53-positive specimens was higher compared to the total number of tumors. Since immunohistochemical investigations should contribute to the improvement of the clinical diagnosis and treatment or give information on the prognosis, we conclude from our results that it seems to be legitimate to assess the P53 status exclusively in the specimens positive for PCNA. Immunohistochemical investigations under consideration of the PCNA status yielded good and fast recognition of p53 mutations leading to intracellular P53 protein accumulation.

p53 tumour-suppressor gene in non-small-cell lung cancer with neoadjuvant therapy.

In a phase II study for optimizing therapeutic management of locally advanced non-small-cell lung cancer the prognostic and therapeutic relevance of the p53 status was investigated. Biopsy or mediastinoscopy samples, collected prior to neoadjuvant chemoradiotherapy and corresponding resection specimens, were analysed immunohistochemically (CM1 antiserum) for p53 accumulation and molecular biologically (polymerase chain reaction/single-strand conformation polymorphism) for p53 mutations. The results were correlated to the response to therapy (regression grade) and to the survival times. p53 accumulation was found in 41.7% (prior to neoadjuvant therapy) and in 40.0% (after surgery) of the tumours. p53 mutation was demonstrated in 45.4% (prior to neoadjuvant therapy) and in 46.4% (after surgery) of the investigated tumours. Neither before nor after therapy was any correlation to the survival times or to the response to therapy seen in the collective analysed. Thus, such investigations are not suitable for identifying patients with locally advanced non-small-cell lung cancer who might benefit, to different extents, from neoadjuvant therapy.

Hepatocyte growth factor/scatter factor and its receptor c-Met are overexpressed and associated with an increased microvessel density in malignant pleural mesothelioma.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates cell proliferation, motility and invasiveness via its receptor c-Met during embryogenesis and repair processes. It induces angiogenesis, promoting endothelial cell migration and capillary-tube formation in vivo. Co-expression of HGF/SF and c-Met receptor results in enhanced tumour growth, invasiveness and a mesenchymal-epithelial transition in some experimental tumours. Since mesothelioma cells have been reported to express c-Met receptor and to migrate in response to HGF/SF, we investigated human malignant pleural mesotheliomas for the demonstration of possible co-expression of the growth factor and its receptor. The microvessel density of the tumours was also analysed in order to assess the influence of HGF/SF expression on tumour angiogenesis. Thirty-nine paraffin-embedded specimens of malignant pleural mesotheliomas were immunostained by anti-HGF/SF and anti-c-Met antibodies and semiquantitatively evaluated. c-Met mRNA expression was visualised in ten tumour samples by a fluorescent in situ hybridisation method. Microvessel density was calculated by counting microvessels with a high-power field (200x) on von-Willebrand-factor-stained slides. We found an increased production of HGF/SF in 33/39 tumours and a corresponding overexpression of c-Met receptor in 29/39 specimens. The FISH method detected increased transcription of c-Met mRNA in malignant cells and in neighbouring vascular endothelial cells. HGF/SF-positive mesotheliomas had significantly higher microvessel densities compared to their HGF/SF-negative counterparts. The observed co-expression of HGF/SF and c-Met in malignant pleural mesotheliomas suggests a possible self-stimulation (autocrine loop) of tumour cells. On the basis of the significantly higher microvessel density values of malignant mesotheliomas overexpressing HGF/SF, we postulate, that HGF/SF may be an additional relevant factor in tumour angiogenesis in malignant pleural mesotheliomas.

Expression of type IV collagenase correlates with the expression of vascular endothelial growth factor in primary non-small cell lung cancer.

Tumor growth and metastasis are angiogenesis-dependent processes initiated and regulated by a number of cytokines. Vascular endothelial growth factor (VEGF) is a potent angiogenic protein with a selective mitogenic effect on vascular endothelial cells, known to be involved in physiological (embryogenesis) and pathophysiological (rheumatoid arthritis, tumor) angiogenesis. An increased expression of matrix metalloproteinase type IV collagenase has been reported in invading endothelial cells in vitro and in malignant cells, degrading structures of the basement membranes in various human malignancies. In the present study we investigated the expression of the genes for type IV collagenase and vascular endothelial growth factor (VEGF) in 40 cases of primary non-small-cell lung cancer (NSCLC). Specimens were immunostained by an antibody directed against VEGF and mRNA transcripts of VEGF and type IV collagenase were localized by non-radioactive in situ hybridization. VEGF mRNA was detected in 33 neoplasms, while in 23 cases transcripts of the type IV collagenase gene were visualized by digoxigenin-labeled cDNA probes. Transcripts of both mRNAs were detected in malignant cells. Furthermore, anti-VEGF immunostaining was present in newly formed microvessels close to the atypical cells, and mRNA of type IV collagenase was present in stromal cells adjacent to the tumor. A statistically significant correlation was found between the expression of type IV collagenase and VEGF (P = 0.0061). These data suggest a double role for type IV collagenase in the metastatic process of NSCLC: (1) facilitating the invasion of tumor cells by the proteolytic cleavage of the basement membrane and (2) similarly supporting the endothelial cell invasion essential for tumor angiogenesis. Furthermore, our findings sustain the hypothesis that metastatic spread and angiogenesis are associated with a clonal expansion of highly angiogenic and invasive tumor cell clones.

German uranium miner study--historical background and available histopathological material.

Mining activities in the former German Democratic Republic were documented as early as 1168 in the ore mountains (Erzgebirge) of Saxony. Silver, bismuth, cobalt, nickel and tungsten were mined from then up to the end of the 19th century. After the Second World War, the Soviet Occupation Authorities reopened the old silver mines in Saxony to mine uranium for the Soviet nuclear industry. About 400, 000 workers produced a total of 220,000 tons of uranium during the years 1946 to 1990. After the reunification of Germany, the archive of the Institute of Pathology of the mining area was opened for research. It contains protocols of 28,975 autopsy cases and about 400,000 slides collected from 1957 to 1992, about 66,000 tissue blocks, and 238 whole lungs. From the autopsy cases, 17,466 could be identified as workers of the uranium mining company. The remainder of the cases were in the population of the mining area. A comparison of the frequencies of malignancies of male workers older than 15 years with those of the population of the mining area for the years 1957 to 1989 demonstrates a significantly higher percentage of lung cancer among the uranium miners. There was no significant difference for other solid cancers and leukemias.

German uranium miner study--pathological and molecular genetic findings. German Uranium Miner Study, Research Group Pathology.

Uranium miners of the former Wismut company in Germany form the largest cohort of workers exposed to (222)Rn and dust in the world. The German Uranium Miner Study, Research Group Pathology, is evaluating the central pathology archive of the Wismut company. The main tasks of our study are pathological-anatomical and molecular genetic investigations of 28,975 autopsy cases and the evaluation of mining pollutants in the lungs by neutron activation analysis. As part of an observer agreement study, lung tumors are classified according to the WHO/IASLC classification and nontumorigenic lung disorders are registered. Lung tumors were analyzed for the presence of a proposed radon-specific mutation in the TP53 gene (formerly known as p53). Interim results are: (a) In the years 1957 to 1965, a high rate (69%) of small cell carcinomas was found which had declined to 34% by 1990. (b) The percentage of the deceased who suffered from silicosis is not higher in the group of lung tumors than in other tumor groups or the nontumor group. (c) The hypothesis of a radon-characteristic hotspot mutation in the TP53 tumor suppressor gene is not supported by our investigations. (d) Neutron activation analysis demonstrates that uranium, arsenic, chromium, cobalt and antimony can be found in tissue samples from the miners even when they had stopped working more than 20 years before death.

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

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

p53 accumulation in polynuclear-giant-cells.

Accumulation of p53 has been reported in nearly all malignant human tumours. Macrophage derived giant cells of sarcoid granulomas in human lung tissue also show intense staining for p53 while normal alveolar macrophages remain unstained. Since sarcoid giant cells are not considered to be either pre-neoplastic nor to exhibit p53 gene mutations, two different physiological functions of p53 may be illustrated. Alveolar macrophages were isolated from rat lungs and cultured in vitro. Accumulation of p53 was observed by indirect immunohistochemistry after application of polyclonal rabbit serum directed against murine p53 (CM5). Antiproliferating cell nuclear antigen (PCNA) antibodies were used to study DNA synthesis. Most of the multinucleated giant cells derived from macrophages accumulated p53 in the cytoplasm, while only few nuclei were stained. PCNA was found in most giant cells nuclei. However, PCNA positivity was visible in few mononucleated macrophages. Isolated alveolar macrophages in vitro clearly divide and since nuclear division is a late event in the cell cycle, p53 may be involved in G1/S-control and in other cell-cycle-checkpoints between mitosis and cytokinesis.

Expression and localization of vascular endothelial growth factor and its receptor flt in pulmonary sarcoidosis.

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine, which has recently been reported to enhance the activation and migration of monocytes through the flt receptor in vitro, which are key events in granuloma formation of granulomatous disorders and in sarcoidosis. Since activated macrophages and monocytes are known to be involved in sarcoid granuloma formation in sarcoidosis, we investigated the expression of VEGF and its receptor flt in 33 paraffin-embedded lung tissue biopsies of patients with pulmonary sarcoidosis. VEGF-mRNA was localized by nonradioactive in situ hybridization, VEGF and flt expression were visualized immunohistochemically. We found an increased transcription and protein production of VEGF and an overexpression of flt in activated alveolar macrophages, in epitheloid cells, and in multinuclear giant cells of pulmonary sarcoid granulomas.

Expression of vascular endothelial growth factor in diffuse malignant pleural mesothelioma.

Angiogenesis is an important part of normal and pathological processes, including tumour growth, metastasis, inflammation and wound healing. VEGF is the best known angiogenic factor, implicated in tumour-associated microvascular hyperpermeability and carcinogenesis. We investigated 103 malignant pleural mesotheliomas, analysing the expression of vascular endothelial growth factor using immunohistochemistry and in situ hybridization. The grade of microvessel density was assessed with the aid of anti-factor-VIII antibodies. An increased expression of VEGF was found in biphasic and epithelioid mesotheliomas, correlating in a statistically significant manner (P<0.042). In situ hybridization confirmed the specificity of VEGF mRNA expression. There was a robust correlation between VEGF expression and increased microvessel density (P<0.001), and positive mesotheliomas had significantly higher microvessel densities than negative specimens. There was also a significant correlation between microvessel density and histological pattern. As growth pattern tended towards biphasic and sarcomatoid mesotheliomas the density of micovessels decreased (P<0.05).

Detection of transforming growth factor beta 1 mRNA in cerebrospinal fluid cells of patients with meningitis by non-radioactive in situ hybridization.

Meningitis is a serious disease mostly caused by viral or bacterial infections. In complicated cases it may lead to brain damage and death. The infection and cell damage result in a cellular and immunological response. Following this, a high secretion of cytokines can be expected. Cytokines, especially tumour necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1), promote the inflammatory reactions in the subarachnoid space. Transforming growth factor beta 1 (TGF-beta 1) has antagonistic effects on TNF-alpha and IL-1-mediated processes. Therefore, it suppresses inflammatory reactions. To observe the expression of TGF-beta 1 in transcellular signalling in the inflammatory processes of meningitis, we investigated TGF-beta 1 mRNA in cells in the cerebrospinal fluid of three patients with meningitis by non-radioactive in situ hybridization. All patients fulfilled the usual clinical criteria of meningitis. In one case Neisseria menigitidis could be identified as the pathogenic agent. In the remainder, no agent could be isolated. In all cytological preparations of the cerebrospinal fluid of these patients a high level of TGF-beta 1 mRNA was detectable in the cell populations. It was possible to distinguish between the different cell types of the cerebrospinal fluid and to attach the mRNA expression to them. On the one hand, this makes it possible to investigate pathogenesis and defence mechanisms in bacterial and aseptic meningitis on a cellular level; on the other hand, it may open new perspectives in the control of disease development, prognosis, diagnosis and supporting therapy.

Mutational analysis of the PTEN/MMAC1 tumour suppressor gene in primary human malignant mesotheliomas.

Eighteen primary human malignant mesotheliomas obtained from 18 patients were screened for point mutations and microdeletions/insertions in all exons of the tumour suppressor gene PTEN/MMAC1 by SSCP analysis. No mutation could be found. Our preliminary data indicate that disarrangements of PTEN/MMAC1 are at least not frequently involved in mesothelioma formation.

Localization of collagen types I, II, and III mRNAs in human heterotopic ossification by non-radioactive in situ hybridization.

Heterotopic ossification is a metabolically active process which shares several properties of orthotopic bone formation and, therefore, represents an excellent model for the investigation of matrix components. A novel tool for studying the ossifying process at the level of transcription is the technique of non-radioactive in situ hybridization. Using digoxigenin labeled cDNA probes we investigated the distribution patterns of types I, II and III collagen mRNAs in heterotopic ossification of pressure sores of paraplegic patients. The three collagen mRNAs exhibited substantially divergent distribution patterns. Type I (alpha 1) collagen mRNA was predominantly detectable in preosteoblasts, prechondroblasts and chondrocytes of the ossification zone. Type II (alpha 1) collagen mRNA was nearly exclusively found in cells of the chondrogenic lineage. Type III (alpha 1) collagen mRNA was detectable at low levels in soft tissue, but was strongly expressed by prechondroblasts and chondrocytes of heterotopic cartilage. Our in situ hybridization experiments provide evidence that chondrogenic cells in heterotopic ossification show a phenotypic alteration in collagen type expression. These results indicate that chondrocytes of heterotopic cartilage show a co-expression of types I (alpha 1), II (alpha 1) and III (alpha 1) collagen mRNAs.

Localization of elastase and tumor necrosis factor alpha mRNA by non-radioactive in situ hybridization in cultures of alveolar macrophages.

Digoxigenin is a new tool for labeling probes which can be detected with the help of specific antibodies in the cell by indirect or direct immunostaining. In contrast to the biotin-reaction, the advantage of digoxigenin is that it does not appear in animal or human cells in nature. In comparison to radioactive labeling methods it is favorable in terms of short exposure time and precise localization of signals in the cell. In this paper we describe the localization of elastase and tumor necrosis factor alpha (TNF alpha) mRNA by non-radioactive in situ hybridization of rat alveolar macrophages in cell culture after stimulation with welder steam dusts. Using digoxigenin labeled probes the determination of specific mRNA's expression and their precise localization in the cytoplasm of the cell could be achieved within one day.

Biological durability and oxidative potential of man-made vitreous fibres as compared to crocidolite asbestos fibres.

In this study we investigated relationships between redox properties and biodurability of crocidolite asbestos fibres and three different man-made vitreous fibres (MMVF): traditional stone wool fibres (MMVF 21), glass fibres (MMVF 11) and refractory ceramic fibres (RCF). Each fibre type was incubated up to 22 weeks in four different incubation media: gamble solution (GS) pH 5.0 and pH 7.4, representing blood plasma without proteins, and surfactant-like solution (SLS) pH 5.0 and pH 7.4. During incubation time aliquots of incubation mixtures were removed and analysed in a biochemical model reaction, mimicking activated phagocytes. In addition, changes of fibre morphology and chemical composition were examined using SEM- and EDX-technology. In the presence of crocidolite asbestos fibres and MMVF 21 the formation of OH*-radicals according to the Haber-Weiss sequence could be demonstrated, whereas MMVF 11 and RCF showed no reactivity. Crocidolite asbestos fibres exhibited a significant higher activity compared with the stone wool fibres at the onset of incubation. The oxidative capacities of these fibre types were shown to depend on both specific surface area and iron content. The oxidative potentials of crocidolite asbestos fibres as well as MMVF 21 were not constant during incubation over several weeks in each incubation medium. The reactivities showed sinoidal curves including reactivities much higher than those at the onset of incubation time. These irregular changes of oxidative capacity may be explained by changes of the redox state of fibre surface-complexed iron. Furthermore our results showed clear differences between incubation of fibres in GS and SLS, respectively, indicating that phospholipids play an important part in fibre dissolution behaviour and oxidative reactivity. In conclusion we suggest, that biodurability testing procedures should not exclusively concentrate on dissolution rates of fibres. They should include fibre characteristics concerning known pathogenic mechanisms to evaluate the real toxic potential of the fibre type looking at. Secondly we suggest, that phospholipids should be constituents of incubation liquids used for standardised fibre biodurability test procedures thus representing more realistic incubation conditions.