Hannelore Kothmaier

Sarcomatoid carcinomas of the lung—are these histogenetically heterogeneous tumors?

Sarcomatoid carcinomas (SC) of the lung are a heterogeneous group of nonsmall cell lung carcinomas (NSCLC) containing a sarcoma or sarcoma-like component. SC may represent an epithelial neoplasm undergoing divergent tissue differentiation originating from a single clone. Epithelial–mesenchymal transition (EMT) best describes the origin of the spindle and giant cells. We aimed to define chromosomal aberrations within the subgroups of SC and if EMT does play a role in SC. Twenty-two SC were investigated by chromosomal comparative genomic hybridization (CGH). Immunohistochemical staining was performed with antibodies for E-cadherin, Vimentin, c-Fos, c-Jun, Snail, TGFβ1, Notch1, β-catenin, Glycogen synthase kinase 3β (GSK3β), and Fascin. Gains occurred more frequently than losses (70.5 vs 29.5%). The shortest regions of overlap were gains on chromosomes 8q and 7 followed by 1q, 3q, and 19, supporting the common origin of the different subtypes of SC. The immunohistochemical staining suggests that the sarcomatoid components of SC might have undergone EMT, not triggered by the signaling pathways Notch1, Snail, and TGFβ1, but probably initiated by an upregulation of c-Jun and a consecutive overexpression of Vimentin and Fascin. The Wnt-pathway was not deregulated because combined membrane and cytoplasmic reactivity for β-catenin and GSK3β was observed.

EGFR and PDGFR differentially promote growth in malignant epithelioid mesothelioma of short and long term survivors.

Background: Malignant pleural mesothelioma (MPM) is an asbestos related tumour difficult to detect early and treat effectively. Asbestos causes genetic modifications and cell signalling events that favour the resistance of MPM to apoptosis and chemotherapy. Only a small number of patients, approximately 10%, survive more than 3 years. The aim of our study was to assess possible differences within signalling pathways between short term survivors (survival <3 years; STS) and long term survivors (survival >3 years; LTS) of MPM. Methods: 37 antibodies detecting proteins engaged in cell signalling pathways, enforcing proliferation, antiapoptosis, angiogenesis and other cellular activities were investigated by tissue microarray (TMA) technology. Results: Epidermal growth factor receptor (EGFR) was expressed stronger in LTS whereas platelet derived growth factor receptor (PDGFR) signalling was more abundant in STS. Expression of TIE2/Tek, a receptor for tyrosine kinases involved in angiogenesis, was differentially regulated via PDGFR and thus is more important in STS. Antiapoptosis was upregulated in STS by signal transducer and activator of transcription 1 (STAT1)–survivin and related molecules, but not in LTS. Our study provides novel insights into the regulatory mechanisms of signalling pathways in MPM, which differentially promote tumour growth in LTS and STS. Conclusion: We have demonstrated that small scale proteomics can be carried out by powerful linkage of TMA, immunohistochemistry and statistical methods to identify proteins which might be relevant targets for therapeutic intervention.

Comparison of formalin-free tissue fixatives: a proteomic study testing their application for routine pathology and research.

CONTEXT Formalin-fixed, paraffin-embedded tissue is the routine processing method for diagnostics practiced in pathology departments worldwide. OBJECTIVE To determine the potential value of non-cross-linking, formalin-free tissue fixation for diagnostics in pathology and proteomic investigations. DESIGN We tested 3 commercially available, formalin-free tissue fixatives-FineFIX, RCL2, and HOPE-in lung cancer specimens from 10 patients. The fixatives were evaluated for their effects on tissue morphology, protein recovery, and immunoreactivity for a selected panel of proteins differently expressed in lung cancer, using immunohistochemistry and Western blotting. RESULTS Tumor-cell analysis with hematoxylin-eosin worked equally well for all tested fixatives when compared with the standard formalin-fixed, paraffin-embedded procedure. Movat pentachrome stains showed comparable results for the different matrices and cellular proteins analyzed. The RCL2 (P = .01) and HOPE fixatives (P = .03) improved protein recovery when compared with formalin-fixed, paraffin-embedded or frozen tissues. Our data clearly show that the fixatives evaluated influenced immunoreactivity to matched, formalin-fixed, paraffin-embedded lung cancer tissue. In particular, membrane-bound proteins, such as epidermal growth factor receptor EGFR, can be detected more efficiently by immunohistochemistry and Western blotting. CONCLUSION We have demonstrated that formalin-free fixatives have the potential in routine pathology and research to replace formalin in histomorphology and protein preservation.

Signal transducer and activator of transcription 1 (STAT1) acts like an oncogene in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is increasing in Europe and the prognosis remains poor. We compared epithelioid MPM in short and long survivors, and identified signal transducer and activator of transcription 1 (STAT1) as probably being responsible for antiapoptotic signaling and chemoresistance. Six mesothelioma cell lines were evaluated by Western Blot. We also analyzed 16 epithelioid MPM tissue samples for the phosphorylation status of STAT1 and the expression of its negative regulator, the suppressor of cytokine signaling 1 (SOCS1). Formalin-fixed and paraffin-embedded tissue specimens were evaluated by protein-lysate microarray and immunohistochemistry. We found STAT1 to be highly expressed and STAT3 downregulated in MPM cell lines. The expression of STAT1 phosphorylated on tyrosine 701 (Y701) was increased by interferon-gamma (IFN-γ) treatment, whereas SOCS1 was not expressed. The expression of STAT1 phosphorylated on serine 727 (S727) was not detected in mesothelioma cell lines and was not stimulated by IFN-γ. STAT1 was phosphorylated on tyrosine 701 and serine 727 in MPM tissue samples. The expression of pSTAT1-Y701 was increased compared to pSTAT1-S727. SOCS1 was again not detectable. STAT1 is upregulated in MPM, and its action may be prolonged by a loss of the negative regulator SOCS1. STAT1 might, therefore, be a target for therapeutic intervention, with the intention to restore apoptotic mechanisms and sensitivity to chemotherapy. However, other regulatory mechanisms need to be investigated to clarify if lack of expression of SOCS1 is the only reason for sustained STAT1 expression in MPM.

Comparative Genomic Hybridisation (CGH)

CGH is a technique used to identify unbalanced chromosomal aberrations in solid tumors. It provides a global overview of chromosomal gains and losses. Two variants are used in research and diagnostics, chromosomal CGH and array CGH. The major difference is within the detection system. In chromosomal CGH preparations of normal test chromosomes serve as the reference, on which tumor and normal DNA is competitively hybridized, whereas in array CGH either bacterial artificial clones (BAC) clones or oligonucleotides are used. Whereas in chromosomal CGH the resolution of detected aberrations is in the 10 MB range, the resolution of array CGH ranges from 1 MB to 100 bp.