Improved diagnostics targeting c-MET in non-small cell lung cancer: expression, amplification and activation?

BACKGROUND: Several c-MET targeting inhibitory molecules have already shown promising results in the treatment of patients with Non-small Cell Lung Cancer (NSCLC). Combination of EGFR- and c-MET-specific molecules may overcome EGFR tyrosine kinase inhibitor (TKI) resistance. The aim of this study was to allow for the identification of patients who might benefit from TKI treatments targeting MET and to narrow in on the diagnostic assessment of MET. METHODS: 222 tumor tissues of patients with NSCLC were analyzed concerning c-MET expression and activation in terms of phosphorylation (Y1234/1235 and Y1349) using a microarray format employing immunohistochemistry (IHC). Furthermore, protein expression and MET activation was correlated with the amplification status by Fluorescence in Situ Hybridization (FISH). RESULTS: Correlation was observed between phosphorylation of c-MET at Y1234/1235 and Y1349 (spearman correlation coefficient rs = 0.41; p < 0.0001). No significant correlation was shown between MET expression and phosphorylation (p > 0.05). c-MET gene amplification was detected in eight of 214 patients (3.7%). No significant association was observed between c-MET amplification, c-MET protein expression and phosphorylation. CONCLUSION: Our data indicate, that neither expression of c-MET nor the gene amplification status might be the best way to select patients for MET targeting therapies, since no correlation with the activation status of MET was observed. We propose to take into account analyzing the phosphorylation status of MET by IHC to select patients for MET targeting therapies. Signaling of the receptor and the activation of downstream molecules might be more crucial for the benefit of therapeutics targeting MET receptor tyrosine kinases than expression levels alone.

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.

Pulmonary haptoglobin and CD163 are functional immunoregulatory elements in the human lung.

BACKGROUND: The acute-phase protein haptoglobin (Hp) and its receptor CD163 serve as immunomodulators and possess anti-inflammatory besides antioxidant functions. OBJECTIVES: To further understand the role of the recently described pulmonary Hp (pHp) and its receptor CD163 in case of inflammation and infection, pHp and CD163 were investigated on mRNA and protein level to gain insight into the cellular events taking place upon stimulation with the inflammatory mediators LPS, Pam3, cytokine IL-6 and dexamethasone, and upon infection with respiratory pathogens (Haemophilus influenzae, Streptococcuspneumoniae and Chlamydia pneumoniae) by use of a human ex vivo tissue culture model and cell cultures of A549 and alveolar epithelial cells type II. In addition, pHp and CD163 expression in COPD and sarcoidosis was assessed. METHODS: We conducted experiments using 942 ex vivo cultured lung samples applying immunohistochemistry, immunocytochemistry, in situ hybridization, immunofluorescence, real-time PCR, RT-PCR, slot and Western immunoblot analyses with tissue lysates and culture supernatants as well as ELISA and cytometric bead array analyses. RESULTS: This study describes for the first time the expression, regulation and secretion of pHp and its receptor CD163 in the human lung. The release of soluble mediators from A549 cell line and human monocyte-derived macrophages was observed indicating that Hp differentially activates the release of soluble mediators and major chemoattractants. CONCLUSIONS: The findings indicate a native function of pHp and CD163 as functional pulmonary defense elements due to local expression, regulation and secretion during lung infection and as part of the inflammatory immune response of the respiratory system.

Generation and evaluation of a monoclonal antibody, designated MAdL, as a new specific marker for adenocarcinomas of the lung.

BACKGROUND: Different therapy regimens in non-small-cell lung cancer (NSCLC) are of rising clinical importance, and therefore a clear-cut subdifferentiation is mandatory. The common immunohistochemical markers available today are well applicable for subdifferentiation, but a fraction of indistinct cases still remains, demanding upgrades of the panel by new markers. METHODS: We report here the generation and evaluation of a new monoclonal antibody carrying the MAdL designation, which was raised against primary isolated human alveolar epithelial cells type 2. RESULTS: Upon screening, one clone (MAdL) was identified as a marker for alveolar epithelial cell type II, alveolar macrophages and adenocarcinomas of the lung. In a large-scale study, this antibody, with an optimised staining procedure for formalin-fixed tissues, was then evaluated together with the established markers thyroid transcription factor-1, surfactant protein-A, pro-surfactant protein-B and napsin A in a series of 362 lung cancer specimens. The MAdL displays a high specificity (>99%) for adenocarcinomas of the lung, together with a sensitivity of 76.5%, and is capable of delivering independent additional diagnostic information to the established markers. CONCLUSION: We conclude that MAdL is a new specific marker for adenocarcinomas of the lung, which helps to clarify subdifferentiation in a considerable portion of NSCLCs.

Pathology on the edge of interdisciplinarity. A historical epitome.

Pathology is a bridging discipline that involves both basic and clinical biomedical sciences. In this context, it includes both descriptive and mechanistic approaches, with the final goals of further understanding the anatomic and functional changes and underlying molecular events involved in disease-related processes. Pathology studies mainly comprise macroscopic and microscopic examinations, and involve the visual recognition of different patterns in cells and tissues. In time elapsed to this end, it has adopted a dazzling array of methods and techniques from the most varied fields of natural and engineering sciences in order to optimize tissue analysis, which will be summarized in this article.

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.

Malignant and non-malignant lung tissue areas are differentially populated by natural killer cells and regulatory T cells in non-small cell lung cancer.

Even though the lung represents a special immune compartment with the capacity of a high inflammatory response, ineffective anti-tumour immunity is common in lung-associated malignancies. We asked whether a differential composition of the immune cell infiltrate in malignant (MLTAs) and non-malignant lung tissue areas (N-MLTAs) exists and might potentially contribute to this effect. We performed a comparative analysis of immune cells residing in MLTAs and N-MLTAs of non-small cell lung cancer (NSCLC) patients. To this end, we used immunophenotyping and functional analyses on directly isolated immune cells and tissue arrays on archived paraffin-embedded specimens. A strong T cell infiltration was prominent in both tissue compartments whereas CD4(+)CD25(+)CD127(-) T regulatory cells were present in MLTAs only. Nonetheless, concurrent functional ex vivo T cell analyses revealed no significant difference between T cells of MLTA and N-MLTA, suggesting that tumour-infiltrating T cells were not functionally impaired. Interestingly, T cell infiltration was less pronounced in specimens with a high neutrophilic infiltrate. NK cell infiltration was strikingly heterogenous between MLTA and N-MLTA. While NK cells were almost absent in the malignant tissue regions, non-malignant counterparts were selectively populated by NK cells and those NK cells showed strong cytotoxic activity ex vivo. We report that malignant and non-malignant tissue areas in NSCLC are selectively infiltrated by certain immune cell types with NK cells being displaced from the tumour tissue. These phenomena have important implications for tumour immunology of NSCLC and should be considered for the development of future immunologic intervention therapies.

Secondary bronchial botryomycosis due to foreign body aspiration.

Botryomycosis is recognised mainly as a visceral disorder with rare cases of pulmonary manifestation. The most frequent cause of pulmonary Botryomycosis is aspiration of a foreign body which induces bacteria to group together instead of spreading out forming conglomerates resembling the granules of Actinomyces. Here we report on the clinical and pathologic findings of a 38-year-old patient without any further predisposing factors. It should be mentioned that the disease was cured following the extraction of a foreign body without the need for any surgery or antibiotic therapy. Factors influencing the course of the disease are discussed below.

Cytological diagnosis of malignant mesothelioma--improvement by additional analysis of hyaluronic acid in pleural effusions.

Cytology allows the diagnosis of malignant mesothelioma (MM) from effusions with high specificity but low sensitivity. Conversely, elevated levels of hyaluronic acid (HA) in effusions are sensitive indicators of MM, although specificity is insufficient. We studied whether the cytological diagnosis of MM could be improved by HA analysis. HA was analysed in patients with histologically confirmed MM (n=162), adenocarcinoma or other malignant tumours (n=100) and in 90 patients with benign pleural diseases. In 77 out of 162 effusions, all, and in 33 some, cytological criteria of MM were satisfied. The cut-off value of HA showing maximum diagnostic reliability (86%) regarding MM was 30 mg/l (sensitivity 87%, specificity 86%). A HA value of 100 mg/l yielded 39 and 98%, respectively. Seventy three out of 77 patients with cytological findings indicative of MM showed HA levels greater than 30 mg/l as well as 27 of 33 patients with suspicious lesions. These 100 patients were correctly recognised as having MM. The addition of HA analysis to cytology, requiring all or some criteria of MM as positive, increased sensitivity for MM from 48 to 71-91%, whereas specificity only slightly decreased to 94-96%. We conclude that the combined cytological and HA analysis of pleural effusions had the potential to improve the diagnosis of MM.

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.

Characterisation of guinea pig precision-cut lung slices: comparison with human tissues.

Precision-cut lung slices (PCLS) allow comparison of the airway responses of different species under identical experimental conditions. The aim of this study was to establish and characterise PCLS from guinea pigs (GPs) and to compare them with human PCLS. GP PCLS were prepared according to previously published procedures with the exception that the agarose solution and the initial incubation medium contained isoproterenol to avoid post mortem airway contraction. The median effective concentrations (EC50, expressed as nM) for agonist-induced bronchoconstriction in GP and human PCLS, respectively, were: leukotriene D4 (1.8, 5.0); thromboxane (16, 1.3); serotonin (69, unresponsive); histamine (217, 2,170); and methacholine (231, 234). Allergen-induced bronchoconstriction of passively sensitised PCLS was attenuated by histamine or thromboxane-prostanoid receptor antagonists and was almost completely prevented by their combination with leukotriene receptor antagonists. Airways pre-contracted with methacholine were relaxed by the beta-agonist salbutamol or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Simultaneous studies of airways and vessels are possible with, for example, EC50 values for endothelin-1 of 37 nM (pulmonary arteries), 10 nM (pulmonary veins) and 9.6 nM (airway). When compared with previous findings in rat and mouse, these data show that guinea pig lungs are a more appropriate model for human airway pharmacology than lungs from rats or mice.

Real-time PCR assay for improved detection of Mycobacterium tuberculosis complex in paraffin-embedded tissues.

OBJECTIVE: To test the usefulness of a commercially available real-time polymerase chain reaction (PCR) kit for the detection of Mycobacterium tuberculosis complex (MTBC) in formalin-fixed, paraffin-embedded tissues. RESULTS: The examination of 24 specimens of patients with a final diagnosis of TB shows that the real-time PCR assay exhibits a higher sensitivity (66.7%) for the detection of MTBC DNA than an alternative in-house IS6110 PCR (33.3%), whereas staining detected acid-fast bacilli in only two cases (8.3%). CONCLUSION: The real-time PCR assay provides a highly sensitive and specific means for the detection of MTBC DNA in histopathological specimens.

Phenotype and genotype associations of lung carcinoma with atypical adenomatoid hyperplasia, squamous cell dysplasia, and chromosome alterations in non-neoplastic bronchial mucosa.

The frequency of preneoplastic lesions of the lung and bronchial mucosa as well as potential genotype alterations in spatial relationship to pulmonary malignancies still need intensive investigations in order to understand the occurrence and manifestation of lung cancer in detail. To investigate the contemporary manifestation of lung cancer precursor lesions, peripheral (non-neoplastic) lung parenchyma and bronchial mucosa of operated lung carcinomas were analyzed at distinct distances (1, 2, 3, and 4 cm) from the tumor boundary for pre-neoplastic lesions--atypical adenomatoid hyperplasia (AAH) and squamous cell dysplasia (SCD), in 150 surgical specimens. Short-term tissue cultures of additional 55 primary and secondary lung tumors and their surrounding non-neoplastic bronchial mucosa were performed at the same distances in order to search for chromosome alterations, i.e. genotype aberrations. In phenotype observations, atypical adenomatoid hyperplasia was noted in 19/150 (13%) cases, and squamous cell dysplasia in 46/150 (31%) cases. The degree of cellular atypia decreased with increasing distance from the tumor boundary in both AAH and SCM. AAH was observed more frequently in adenocarcinomas, SCQ more frequently in squamous cell carcinomas. In genotype observations, the average number of abnormal metaphases measured 4.5/10 high power fields (HPF) in primary lung carcinomas, and only 2/10 in metastases. Data indicate that the so-called preneoplastic lesions in the lung are not completely tumor-precursor lesions, but, in addition, induced by the tumor itself.

Fatal outcome of a hyperinfection syndrome despite successful eradication of Strongyloides with subcutaneous ivermectin.

We report the case of a 77-year-old man who developed a Strongyloides hyperinfection syndrome following immunosuppressive therapy more than 60 years after he moved away from an area endemic for Strongyloides stercoralis. Successful eradication of the nematode was achieved with an off label subcutaneous formulation of ivermectin. However, the patient subsequently died from acute respiratory distress syndrome (ARDS). Despite a high wormload in the stool and sputum of the patient and delayed infection control measures in the hospital, testing of the medical staff revealed a very low risk of Strongyloides transmission among healthcare workers.

Improved detection of mycobacterial DNA by PCR in formalin-fixed, paraffin-embedded tissues using thin sections.

PCR is a unique methodology allowing for the sensitive detection of mycobacterial DNA-sequences in cases in which no fresh material can be obtained for classic analyses. Despite the limitations of this technique, for example the less satisfactory quality of DNA from paraffin-embedded specimens and the high effort necessary to control contamination, PCR still represents a useful additional tool for routine diagnostic examinations of mycobacterial infections. Fragmentation of the DNA extracted from formalin-fixed, paraffin-embedded samples on the one hand and the rigid cell wall of mycobacteria on the other hand are obstacles to detecting the DNA of these microorganisms by PCR. Here, we describe a simple mechanical procedure that allows us to improve the detection of mycobacterial DNA with the use of thin (1 microm) sections instead of thicker sections. This could be explained by a gentle, mechanical opening of the acid fast mycobacterial cell wall. Thus, even the application of heat/cold shock treatments is not necessary. This inexpensive fast procedure can also be used for the detection of other infectious agents.