Iris Halbwedl

Protein expression profiles in adenocarcinomas and squamous cell carcinomas of the lung generated using tissue microarrays.

With the appearance of defect‐targeted therapies, the definition of tumour protein expression profiles has gained increasing importance. Two lung carcinoma tissue microarrays, one including 75 primary adenocarcinomas (ACs) and the other comprising 67 primary squamous cell carcinomas (SQCCs), were generated in the present study. On both arrays, each tumour was represented by an average of five cores. In addition, one punch of normal lung parenchyma adjacent to each tumour was included in the array. Immunohistochemical expression of 86 proteins was evaluated and the results were analysed by non‐parametric tests, hierarchical clustering, and principal component analysis. In both tumour entities, parenchyma and tumours were clearly separated by hierarchical clustering. By the same statistical approach, it was possible to distinguish ACs from SQCCs with 98% accuracy and to distinguish parenchyma adjacent to ACs from that adjacent to SQCCs with 96% accuracy. It was also possible to separate ACs into three groups that significantly differed in survival. Cathepsin E and hsp105 were identified as previously unknown predictors of survival in lung AC. In summary, this study has shown that protein profiles are feasible tools for anticipating biological behaviour. Copyright

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.

Bronchiolar columnar cell dysplasia—genetic analysis of a novel preneoplastic lesion of peripheral lung

Atypical adenomatous hyperplasia is the only known precursor lesion of lung adenocarcinomas (ACs) so far. Here, we describe a new dysplastic lesion in the bronchioles of peripheral lung for which we propose the name bronchiolar columnar cell dysplasia (BCCD). Eight of fourteen BCCDs were successfully analyzed by means of comparative genomic hybridization (CGH). The average number of chromosomal aberrations was 2.6 in BCCD and 14.7 in concomitant AC. Among the aberrations were losses of 3p, 9, 13, 14 and gains of 1q, 17, 19q and 20q. Summarizing our data from morphological and genetic analysis, we conclude that BCCD is a preneoplasia of the bronchiolar epithelium and most probably represents an additional precursor lesion of lung adenocarcinomas.

BAP1 Protein is a Progression Factor in Malignant Pleural Mesothelioma

Human malignant pleural mesothelioma (MPM) is an aggressive cancer due to former asbestos exposure with little knowledge about prognostic factors of outcome and resistance to conventional therapy. BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene that is frequently lost in MPM. Germline mutations of BAP1 predispose to several different tumors including malignant mesothelioma. Our study aimed to clarify if asbestos exposure has an influence on BAP1 expression and if BAP1 expression could be used as a prognostic factor of outcome. An immunohistochemical staining for BAP1 was performed on 123 MPM tissue samples and the expression levels have been correlated with asbestos exposure and overall survival time. BAP1 expression was not associated with asbestos exposure but we detected a significant effect of BAP1 expression on overall survival time - the higher the BAP1 expression (non-mutated BAP1), the shorter the overall survival. BAP1 mutation has been linked to non-asbestos induced familial mesotheliomas, which usually belong to the long survivor group and BAP1 is most probably functioning differently than in sporadic cases. Further investigations need to be performed to characterize the BAP1 mutations and to identify the BAP1 downstream targets in MPM.

The VEGF-system in primary pulmonary angiosarcomas and haemangioendotheliomas: New potential therapeutic targets?

Malignant epitheloid vascular tumors (epitheloid haemangioendotheliomas and angiosarcomas) of the lung are very rare lesions often posing difficulties in diagnosis. Due to their rare incidence no standardized therapy regimen is established. Surgical resection of the tumor is the mainstay of treatment, but in many cases, especially due to the multifocality of the tumor, negative resection margins cannot be achieved. A blockade of members of the vascular endothelial growth factor (VEGF) system either by antibodies for their ligands or by kinase inhibitors has been increasingly used for the therapy of solid tumors. The aim of our study was to highlight the main distinguishing morphological factors between the two entities for diagnostic purposes. Next, we investigated several factors of the VEGF-signalling pathway as well as Tie 2 in eight primary pulmonary epitheloid haemangioendotheliomas and ten primary pulmonary epitheloid angiosarcomas by means of immunohistochemistry using commercially available antibodies against VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR-2, VEGFR-3 and endothelium specific kinase Tie2. The observed positivity for the factors of the VEGF-signalling pathway points towards a possible new therapeutic option in the therapy of pulmonary vascular tumors by a blockade of the ligands or their receptors.

Analysis of chromosome-11 aberrations in pulmonary and gastrointestinal carcinoids: an array comparative genomic hybridization-based study

Carcinoid tumors are a heterogeneous group of neoplasms arising from the diffuse neuroendocrine system. Pulmonary as well as gastrointestinal carcinoids can be separated into those with low malignant and intermediate malignant potential. DNA losses of chromosome arm 11q are commonly seen in pulmonary neuroendocrine tumors. Conflicting results have been published comparing atypical with typical lung carcinoids with respect to imbalances of chromosome 11. In the present study, a DNA microarray with genomic clones mapped to chromosome 11 was created, and array comparative genomic hybridization (CGH) with DNA derived from formalin-fixed, paraffin-embedded tissue was performed. We investigated 4 typical and 12 atypical carcinoids of the lung and, for comparison, 9 gastrointestinal carcinoids and 6 endocrine pancreatic tumors. We have shown that formalin-fixed, paraffin-derived DNA can be successfully used for array CGH. Alterations of 11q were rarely detected not only in typical carcinoids of the lung but also in gastrointestinal carcinoids. Atypical lung carcinoids that comprised extensive DNA losses also presented retained fragments in between these deleted regions. The array CGH data were consistent with the data of a previously published classical CGH study and were additionally confirmed using fluorescence in situ hybridization in the present investigation.

Positioning of necrotic lobular intraepithelial neoplasias (LIN, grade3) within the sequence of breast carcinoma progression.

Lobular intraepithelial neoplasia Grade 3 (LIN3) is a recently recognized variant of intraepithelial lobular neoplasia (LIN) of the breast composed of either uniform, generally small cells with massive lobular distension, pleomorphic cells, signet‐ring cells, or any cell type with necrosis. In contrast to classic forms of LIN, there is no consensus on therapeutic strategies for LIN3. In part this is due to the paucity of molecular data that could assist in defining the relationship of LIN3 to classic LIN and carcinomas. In this study we have employed array comparative genomic hybridization to determine the patterns of chromosomal aberrations in nine LIN3 lesions. By comparison to array CGH data of 13 classic LIN lesions, we demonstrate that classic LIN and LIN3 share several recurrent changes, in particular gains of 1q and losses of 16q. Both aberrations are known to appear early in tumorigenesis and to be associated with good prognosis. However, apart from this overlap, there were a number of karyotypic features that were observed exclusively in LIN3. Clearly, this lesion was characterized by a significantly higher number of DNA copy number changes (9 vs. 31 on average), a considerable complexity of chromosomal rearrangements with more than 16 breakpoints in one chromosome and overlapping high copy amplifications encompassing a number of known oncogenes. Our data suggest that, at the genetic level, LIN3 represents a highly advanced lesion with considerable resemblance to carcinomas and, therefore, might represent the transition state from an intraepithelial neoplasm to breast carcinoma.

Is high-grade adenomatous hyperplasia an early bronchioloalveolar adenocarcinoma?

Atypical adenomatous hyperplasia (AAH) is a probable forerunner of bronchioloalveolar carcinoma (BAC) and pulmonary adenocarcinoma (AC) of mixed type. The present study analysed four low‐grade AAHs, 13 high‐grade AAHs, two BACs, nine mixed ACs, and one squamous cell carcinoma derived from 13 patients using comparative genomic hybridization. The average number of chromosomal aberrations was 1.2 in low‐grade AAH, 9.6 in high‐grade AAH, and 12.5 in AC. A high degree of overlap of genetic changes was found in high‐grade AAH, BAC, and AC within individual patients. The high number of aberrations and the degree of shared aberrations found in high‐grade AAH and AC raises questions about the separation of these two entities. In addition, in view of the monoclonal origin of multiple foci within the same patient, AAH may not be a precursor of AC in some cases, but rather may represent intraepithelial spread. Copyright

Reduced Folate Carrier and Folylpolyglutamate Synthetase, but not Thymidylate Synthase Predict Survival in Pemetrexed-Treated Patients Suffering from Malignant Pleural Mesothelioma

Background: Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment. Methods: Samples from 63 patients were tested using immunohistochemistry (IHC) and quantitative polymerase chain reaction(qPCR) for expression levels of TS, GARFT, AICARFT, RFC, and FPGS. Clinical data were evaluated to determine associations between efficacy of pemetrexed and enzyme expression levels. Evaluation of expression levels was done through TaqMan-based qPCR, and IHC was evaluated semiquantitatively by using the H-score. Results: qPCR analysis showed no difference in expression pattern of GARFT and AICARFT. IHC analysis revealed a heterogeneous staining pattern for all the enzymes. No significant association was found between TS expression and survival or objective response of the tumors after pemetrexed treatment. FPGS (p = 0.0111) and RFC (p = 0.0088) mRNA expression levels were strongly associated with overall survival in these patients. Conclusions: Our results reveal that in pemetrexed-treated MPMs TS expression levels have no influence on patient outcome. Furthermore, GARFT and AICARFT were homogenously expressed in the patient samples. Folate uptake mechanisms by RFC and activation by FPGS were associated with clinical benefit from pemetrexed treatment.