Tumors in the lung--morphologic features and the challenge of integrating biomarker signatures into diagnostics.

In the last decade, pathologic approaches concerning diagnosis and treatment of lung carcinomas have increasingly moved towards the implementation of molecular methods into the process of decision. In this study, an overview is given referring to the variety of tumors in the lung including common primary lung neoplasms and secondary tumors, and a modus operandi is presented which integrates immunology as well as molecular pathology within the process of finding correct diagnoses. Besides the conventional and approved methods and techniques leading to appropriate treatment including so-called targeted therapies, pathologist's work meanwhile depends on both histologic and molecular results. Since molecular techniques have increasingly entered the field of routine diagnostics, challenges and possibilities have changed and are still rapidly developing. The proceeding integration of molecular-biologic investigations into the process of diagnosing has changed the nature of diagnostics and will continuously grow in the near future. Only by obtaining a proper diagnosis, the optimal treatment of a patient can be assured, whereupon the knowledge of gene mutations and/or altered protein expression is crucial. By identifying those novel molecular target structures, the therapeutic spectrum is tremendously enlarged and will finally improve the patient's prognosis by personalized targeted therapies.

Expression analysis of EML4 in normal lung tissue and non-small cell lung cancer (NSCLC) in the absence and presence of chemotherapeutics.

Despite considerable progress in the development of individualized targeted therapies of tumor diseases, identification of additional reliable target molecules is still mandatory. One of the most recent targets is microtubule-associated human EML4 generating a fusion-type oncogene with ALK demonstrating marked transforming activity in lung cancer. Since EML4 is a poorly characterized protein with regard to expression, function and regulation in human tissue, specimens of human tumor and tumor-free tissues obtained from patients with NSCLC were analyzed to determine the cellular localization. All tissue samples have been previously fixed with the novel HOPE-technique and paraffin embedded. Determination of both gene expression and protein levels of EML4 were performed using RT-PCR, in situ hybridization as well as immunohistochemistry, respectively. In human NSCLC tissue samples, possible regulation of EML4 transcription upon chemotherapy with combinations of most established cytotoxic drugs for NSCLC treatment was also studied employing the recently established ex vivo tissue culture model STST. In normal lung, both marked mRNA and protein levels of EML4 were localized in alveolar macrophages. In contrast, lung tumor tissues always showed consistent transcriptional expression in situ and by RT-PCR. Stimulation of NSCLC tissues with chemotherapeutics revealed heterogeneous effects on EML4 mRNA levels. Based on its expression patterns in both tumor-free lung and NSCLC tissues, human EML4 is likely to be closely associated with processes involved in local inflammation of the lung as well as with tumor behavior. Thus, our results suggest that EML4 may have the potential as a therapeutic target molecule in NSCLC chemotherapy.

The HOPE technique opens up a multitude of new possibilities in pathology.

Fixation of tissues with formalin results in well-preserved morphology but to a high degree leads to degradation of nucleic acids, which substantially constricts the spectrum of applicable molecular techniques. The novel HOPE-fixative with subsequent paraffin embedding, as an alternative to formalin, has been shown to result in a morphological preservation comparable to formalin-fixed, paraffin-embedded specimens. Due to a similar workflow like in formalin-fixation and paraffin embedding, the HOPE technique can be successfully established within any pathological institute. We have shown that DNA, RNA and proteins are protected in HOPE-fixed, paraffin-embedded tissues for at least eight years. Moreover, we described procedures which permit successful application of all common molecular techniques such as in situ hybridization targeting either DNA or RNA, immunohistochemistry without antigen retrieval and for formalin-refractory antigens, PCR, RT-PCR, Western blot, Northern blot, and transcription microarrays to HOPE-fixed, paraffin-embedded tissues. Furthermore, HOPE-fixed tissues can be used for the construction of tissue microarrays for enhanced high-throughput analyses on the molecular level. Using the HOPE technique as its crucial methodological base, ex vivo model systems could be established, e.g. for the simulation of early events in human infections and detection of chemotherapy resistances in human cancer. In addition to tissues, cell-culture preparations have been prepared utilizing the HOPE technique, which were then successfully applied to in situ hybridization targeting mRNA or immunocytochemistry with excellent preservation of morphological details. Taken together, the HOPE technique to date represents an alternative fixation that is, in contrary to other procedures, scientifically broadly analyzed. Therefore new possibilities are opened up especially within the rapidly growing field of molecular pathology.

Effects of crocidolite asbestos on human bronchoepithelial-dependent fibroblast stimulation in coculture: the role of IL-6 and GM-CSF.

Cocultures of human pulmonary epithelial cells (BEAS-2B) and lung fibroblasts (WISTAR-38), representing two cell types of central regulatory potential in (chronic) lung disease, were used as an in vitro model to study the role of interleukin 6 (IL-6) and of granulocyte macrophage-colony stimulating factor (GM-CSF) in early fibrogenesis. For this purpose, epithelial cells were pre-exposed to UICC crocidolite asbestos fibers or titanium dioxide (TiO2) particles for 96 h and subsequently cocultured with fibroblasts for additional 72 h. Gene expression of IL-6 or GM-CSF in both cell types as well as of alpha1 procollagens types I and III in fibroblasts was determined by RT-PCR. Synthesis of IL-6, GM-CSF or collagen I was quantified using IL-6 bioassay or ELISA tests, respectively. Both mediators were directly induced in bronchoepithelial cells by crocidolite but not by TiO2. Likewise, steady-state mRNA levels of procollagens as well as collagen synthesis were upregulated in cocultured fibroblasts. As a result of coculture, cytokine concentrations were synergistically enhanced and further increased by crocidolite in a dose-dependent manner. Suppression of cytokine induction by corresponding neutralizing antibodies consistently abrogated collagen enhancement. Direct stimulation of fibroblast monocultures with recombinant human IL-6 or GM-CSF significantly increased collagen synthesis and transcription in a dose-dependent manner. Thus, our results demonstrate that crocidolite selectively stimulated production of IL-6 and GM-CSF in bronchoepithelial cells. In epithelial-fibroblast interactions, these mediators appear to play a key role in regulating fibroblast activity, indicating a close correlation between these cytokines and the fibrogenic potential of particulates.

2,3,7,8-TCDD induces cytochrome P450 enzyme activity but not proliferation or phenotypical changes in human peripheral blood lymphocytes.

In contrast to the well documented immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in experimental animals, the impact of dioxin on the human immune system remains controversial, although adverse health effects have been reported in humans accidentally or occupationally exposed to dioxin. More recently, a dose-dependent decrease of specific subpopulations of mitogen-activated human peripheral blood lymphocytes (PBL), including helper-inducer/memory cells (CD4+CD29+) and B cells (CD20+), was reported after in vitro treatment with dioxin concentrations as low as 10(-12)-10(-14) M TCDD [1]. Therefore, the direct effects of dioxin on human PBL subpopulations have been studied in more detail, in order to assess the availability of a sensitive indicator system for human dioxin exposure. PBL from healthy volunteers were stimulated with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody (OKT3) and treated with 10(-7)-10(-14) M TCDD for 3-4 days. Cytochrome P450 (CYP1A1) enzyme induction was determined by the ethoxy-resorufin-O-deethylase (EROD) assay. Percentages of the different lymphocytes subsets, including CD2 (T cells), CD4, CD45 RA (suppressor-inducer/ virgin T cells), CD4, CD29, CD8, CD19 (B cells), as well as interleukin 2 (IL-2) receptor (CD25) and class II antigen (human leukocyte antigen HLA-DR) expression, were analyzed by flow cytometry. The proliferative activity was determined by 3H-thymidine uptake after 3-4 days of culture. In the present study, all stimulated lymphocyte cultures showed a significant increase of CYP1A1 activity at dioxin concentrations of 10(-7) and 10(-9) M. In contrast, alterations in surface antigen expression or suppression of proliferative responses did not occur in the mitogen-activated PBL over the whole concentration range of TCDD. These results clearly demonstrate that lymphoproliferation, as well as phenotypes of human PBL, are not affected by dioxin treatment and thus are not useful as sensitive biomarkers in human exposure studies.

Interactions between human bronchoepithelial cells and lung fibroblasts after ozone exposure in vitro.

Long-term exposure to ozone has been shown to cause lung fibrosis and increased collagen synthesis by fibroblasts in experimental animals. As the bronchial epithelium appears to play a major regulatory role in inflammatory processes, we investigated whether ozone induces bronchoepithelial cells in vitro to increase gene expression of procollagens and other fibrogenic mediators in human lung fibroblasts. Membrane cultures of human airway epithelial cells (BEAS-2B) in the presence or absence of lung fibroblast (HFL-1) cultures were exposed to air or 500 ppb ozone for 1 h, followed by (co-)incubation periods of 11 and 23 h. After ozone exposure of the co-cultures, there were substantial increases of steady-state mRNA levels of both alpha1 procollagens type I and III as well as TGF beta1 in the fibroblasts above the corresponding air control levels. In the absence of ozone, the presence of epithelial cells always caused significant decreases in the basal steady-state mRNA levels of both procollagens as compared to their absence. There were no significant effects of ozone on the secretion or gene expression of TGF beta2, PDGF or IL-8 in any cell type. In contrast, co-culture condition induced altered patterns of IL-8 gene expression or of PDGF production in fibroblasts and bronchoepithelial cells, respectively, both in the absence or presence of ozone. In summary, our data demonstrate that the effect of ozone on fibroblasts was mediated by epithelial cells and that mutual regulatory interactions between the different cell types occur. Thus, our co-cultivation system in vitro appears to be able to mimic the in vivo situation providing insight into the nature of cellular interactions and modulation by ozone, which may occur in the whole organism after long-term exposure.

Transforming growth factor-beta signaling leads to uPA/PAI-1 activation and metastasis: a study on human breast cancer tissues.

Metastasis represents a major problem in the treatment of patients with advanced primary breast cancer. Both Transforming Growth Factor-Beta (TGF-ß) signaling and Plasminogen Activator (PA) components, urokinase-type Plasminogen Activator (uPA) and Plasminogen Activator Inhibitor-1 (PAI-1) represent a complex network crucial for such enhanced invasiveness of tumors and imply high prognostic/predictive and promising therapeutic potential. Therefore, protein expression of specific effector molecules comprising the main parts of the TGF-ß signaling pathway were determined in HOPE-fixed human tumor tissues through IHC (Scoring) using tissue microarray (TMA) technique and correlated with respective uPA and PAI-1 levels determined earlier in the same TMAs through optimized IHC and semi-quantitative image analysis. TGF-ß signaling was active in vast majority (96 %) of the tumor samples and 88 % of all cases were significantly correlated with established metastasis markers uPA and PAI-1. In addition, TGF-ß was also closely associated with tumor size, nodal status and two steroid hormone receptors. Consistent interrelationships between TGF-ß, PA components and additional tumor characteristics underline the superiority of such more comprising data with regards to confirming TGF-ß signaling as a promising target system to inhibit metastasis in advanced breast cancer.

Optimized immunohistochemistry in combination with image analysis: a reliable alternative to quantitative ELISA determination of uPA and PAI-1 for routine risk group discrimination in breast cancer.

The determination of the invasion markers urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) has further improved the possibilities for individualized therapy of breast cancer. To date, quantitative measurement by ELISA, that needs large amounts of fresh, frozen material, is the only standardized procedure for diagnostic purposes. Therefore, the aim of this study was the establishment of a reliable alternative method based on immunohistochemistry (IHC) and image analysis requiring only small amounts of fixed tumor tissue. Protein expression of uPA and PAI-1 was analyzed in HOPE-fixed tumor samples using tissue microarrays (TMAs) and semiquantitative image analysis. The results of both methods were significantly correlated and risk assessment showed an overall concordance of 78% (83/107; high- and low-risk) and of 94% (74/79) regarding only high-risk patients. The data demonstrate that optimized IHC in combination with image analysis can provide adequate clinical significance compared to ELISA-derived determination of uPA and PAI-1.

Phosphorus-limited growth of a green alga and a blue-green alga.

The phosphorus-limited growth kinetics of the chlorophyte Scenedesmus quadricauda and the cyanophyte Synechococcus Nägeli were studied by using batch and continuous culturing techniques. The steady-state phosphate transport capability and the phosphorus storage capacity is higher in S. Nägeli than in S. quadricauda. Synechococcus Nägeli can also deplete phosphate to much lower levels than can S. quadricauda. These results, along with their morphological characteristics, were used to construct partial physiological profiles for each organism. The profiles indicate that this unicellular cyanophyte (cyanobacterium) is better suited for growth in phosphorus-limited oligotrophic niches than is this chlorophyte (green alga).

Competition between heterotrophic and autotrophic microplankton for dissolved nutrients.

When a heterotrophic (Rhodotorula rubra) and a phototrophic (Selenastrum capricornutum) plankton were grown together in dilute phosphate (Pi) continuous cultures, coexistence occurred only when the heterotroph was growthrate limited by organic carbon (C). Because of its higher affinity for Pi, and because C starvation does not affect the heterotrophic yeast's ability to transport Pi, the concentration of organic carbon indirectly controlled the biomass of the phototroph. The results support a threshold model of microbial growth.

Comparative effects of immunotoxic chemicals on in vitro proliferative responses of human and rodent lymphocytes.

In order to determine the comparability of human and rodent in vitro systems, the direct effects of various therapeutic or environmental chemicals on proliferative responses of lymphocytes of mouse, rat, and human origins were examined and analyzed by a detailed statistical approach. Four compounds of diverse structure and mechanism of action which are known to impair lymphocyte transformation, such as hydroquinone, T-2 toxin, lead nitrate, as well as the widely used immunosuppressive drug cyclosporin A, were chosen as model test substances. T cells were stimulated by phytohaemagglutinin as well as monoclonal antibodies directed at the T cell receptor/CD3 complex, while B cells were activated by the T-independent mitogens, including Staphylococcus aureus cells, Escherichia coli lipopolysaccharide, and Salmonella typhimurium mitogen with specificity for human, mouse, and rat lymphocytes, respectively. In almost all cases the chemicals altered lymphoproliferative responses in a concentration-related manner in all three species. In general, overall similarities in the relative sensitivity of lymphoblastogenesis were obtained when the human dose-response curves were compared to the rodent response curves. Frequent, statistically significant species-dependent discrepancies of the overall response curves between mice and rats were observed. Large, statistically significant differences were observed for inorganic lead, revealing obvious divergences of the effect patterns in all cases, across all species. In this case, rodent species, especially the rat, were very sensitive to immunomodulation by lead, whereas human cells were relatively resistant. It is suggested that direct interspecies comparisons of immunological effects due to chemical treatment in vitro can provide a greater understanding of the relationship between animal and human data, which will improve the confidence of extrapolation from findings in laboratory animals to human health risk.

Lack of direct immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on human peripheral blood lymphocyte subsets in vitro.

A wide variety of immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in experimental animals has been documented. In contrast, the impact of dioxin on the human immune system remains controversial, although adverse health effects have been reported in humans after occupational or accidental exposure to dioxin. Recently, Neubert et al. (1991) found that a dose-dependent decrease of peripheral blood lymphocyte (PBL) subpopulations in humans and non-human primates, including helper-inducer/memory cells (CD4+CD29+) and B cells (CD20+) occurred in pokeweed mitogen (PWM) stimulated cultures at concentrations as low as 10(-12)-10(-14) M TCDD. Therefore, the direct effects of dioxin on human PBL subpopulations have been studied, in order to determine their usefulness as sensitive biomarkers for human dioxin exposure. Lymphocyte cultures from healthy individuals were treated with 10(-7) M-10(-14) M TCDD in the absence and presence of stimulation with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody (moAb; OKT3) for 3 days. Cytochrome P450 (CYP1A1) enzyme induction, one of the best studied direct biological effects of TCDD on numerous cell types, was assayed in parallel by ethoxyresorufin-O-deethylase (EROD) activity. Percentages of the different lymphocytes subsets, including CD2 (T cells); CD4; CD45 RA (suppressor-inducer/virgin T cells); CD4 CD29; CD8; CD19 (B cells) as well as interleukin 2 (IL-2) receptor (CD25) and class II antigen (HLA-DR) expression, were analyzed by flow cytometry. DNA synthesis was determined by 3H-thymidine uptake after 3 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-specific differences in the susceptibility of potential cellular targets of human origin derived from blood and lung following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The induction of cytochrome P4501A (CYP1A1) enzyme activity is one of the best-studied direct effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds and has been shown to be a sensitive biomarker of exposure to polycyclic aromatic hydrocarbons (PAH) in different experimental animal species as well as in humans. TCDD has also been shown to modulate cytokine gene expression in human keratinocytes, including IL-1 beta, TGF-alpha and TFG-beta 2. In the present studies, the aim was to determine whether different cellular targets of human origin differed in susceptibility to TCDD as measured by CYP1A1 activity and mRNA expression, and whether cytokine gene induction/suppression correlated with TCDD susceptibility. Human airway epithelial cells, alveolar macrophages (AM), peripheral blood monocytes and lymphocytes (PBL) were exposed to 10(-10)-10(-7) mol/L TCDD. CYP1A1 enzyme activity was determined by ethoxyresorufin-O-deethylase (EROD) activity, mRNA expression of CYP1A1 was measured by semiquantitative PCR assay. The secretion and/or gene expression of specific cytokines, including IL-6, IL-8, and IL-1 beta were also examined. Overall, there was a clear correlation between TCDD-induced enzyme activity and CYP1A1 mRNA levels, which were dose-dependently increased in the bronchoepithelial cells and PBL. The human airway epithelial cells (BEAS-S6 cell line and primary cells) appeared to be the most inducible cellular target, with up to 50-fold increases at 10(-8) mol/L TCDD with an EC50 of 3 x 10(-11) mol/L TCDD. The pokeweed mitogen-activated peripheral blood lymphocytes revealed approximately 5-fold less capacity in CYP1A1 activity, with high interindividual variabilities (EC50 3 x 10(-9) mol/L TCDD). In contrast, CYP1A1 enzyme activity in both AM and purified peripheral blood monocytes, which were costimulated with LPS and/or GM-CSF, could not be detected. CYP1A1 mRNA levels, however, were detectable and only marginally enhanced in response to TCDD. The ability of all these cells to express and produce the proinflammatory cytokines IL-6 and IL-8 was neither enhanced nor impaired by TCDD. These results indicate that cell types found in human lung and peripheral blood vary in susceptibility to TCDD, with the lung epithelium being highly susceptible and the alveolar macrophage being nonsusceptible. However, expression and production of specific cytokines such as IL-6 and IL-8, which may potentiate inflammatory processes and/or work as mitogens, does not appear to be influenced by TCDD.