Activation of the PI3K/AKT pathway correlates with prognosis in stage II colon cancer.

BACKGROUND: Patients with UICC/AJCC stage II colon cancer have a high 5-year overall survival rate after surgery. Nevertheless, a significant subgroup of patients develops tumour recurrence. Currently, there are no clinically established biomarkers available to identify this patient group. We applied reverse-phase protein arrays (RPPA) for phosphatidylinositide-3-kinase pathway activation mapping to stratify patients according to their risk of tumour recurrence after surgery. METHODS: Full-length proteins were extracted from formalin-fixed, paraffin-embedded tissue samples of 118 patients who underwent curative resection. RPPA technology was used to analyse expression and/or phosphorylation levels of six major factors of the phosphatidylinositide-3-kinase pathway. Oncogenic mutations of KRAS and BRAF, and DNA microsatellite status, currently discussed as prognostic markers, were analysed in parallel. RESULTS: Expression of phospho-AKT (HR=3.52; P=0.032), S6RP (HR=6.3; P=0.044), and phospho-4E-BP1 (HR=4.12; P=0.011) were prognostic factors for disease-free survival. None of the molecular genetic alterations were significantly associated with prognosis. CONCLUSIONS: Our data indicate that activation of the PI3K/AKT pathway evidenced on the protein level might be a valuable prognostic marker to stratify patients for their risk of tumour recurrence. Beside adjuvant chemotherapy targeting of upregulated PI3K/AKT signalling may be an attractive strategy for treatment of high-risk patients.

MiR-221/-222 differentiate prognostic groups in advanced breast cancers and influence cell invasion.

BACKGROUND: MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells. METHODS: MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT-PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222. RESULTS: In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed. CONCLUSION: This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.

A specific expression profile of heat-shock proteins and glucose-regulated proteins is associated with response to neoadjuvant chemotherapy in oesophageal adenocarcinomas.

BACKGROUND: Oesophageal adenocarcinomas often show resistances to chemotherapy (CTX), therefore, it would be of high interest to better understand the mechanisms of resistance. We examined the expression of heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) in pretherapeutic biopsies of oesophageal adenocarcinomas to assess their potential role in CTX response. METHODS: Ninety biopsies of locally advanced adenocarcinomas before platin/5-fluorouracil (FU)-based CTX were investigated by reverse phase protein arrays (RPPAs), immunohistochemistry (IHC) and quantitative RT-PCR. RESULTS: CTX response strongly correlated with survival (P=0.001). Two groups of tumours with specific protein expression patterns were identified by RPPA: Group A was characterised by low expression of HSP90, HSP27 and p-HSP27((Ser15, Ser78, Ser82)) and high expression of GRP78, GRP94, HSP70 and HSP60; Group B exhibited the inverse pattern. Tumours of Group A were more likely to respond to CTX, resulting in histopathological tumour regression (P=0.041) and post-therapeutic down-categorisation from cT3 to ypT0-T2 (P=0.040). High HSP60 protein (IHC) and mRNA expression were also associated with tumour down-categorisation (P=0.016 and P=0.004). CONCLUSION: Our findings may enhance the understanding of CTX response mechanisms, might be helpful to predict CTX response and might have translational relevance as they highlight the role of potentially targetable cellular stress proteins in the context of CTX response.

[Participation and support of clinical studies and other scientific investigations. Statement of the German Society for Pathology]. / Beteiligung und Unterstützung klinischer Studien und anderer wissenschaftlicher Untersuchungen. Stellungnahme der Deutschen Gesellschaft für Pathologie e. V.

Clinical studies and preclinical investigations are essential in order to test new therapies and diagnostics with the aim of sustained improvement in the treatment of patients. Fortunately, the number of clinical studies is continuously increasing and pathology and tissue-based research are included more often. The German Society for Pathology (DGP) and the pathologists it represents want to and can support this process and our clinical partners as best as possible as an equal partner. With our technologies and our specific expertise we can make a substantial contribution to the quality and the success of preclinical investigations, clinical studies and implementation of the results into clinical pathological diagnostics. In order to support this process the DGP has formulated a statement on the participation and support of clinical studies and other scientific investigations.

In situ quantification of HER2-protein tyrosine kinase 6 (PTK6) protein-protein complexes in paraffin sections from breast cancer tissues.

BACKGROUND: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. METHOD AND RESULTS: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6-HER2 protein-protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). CONCLUSION: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2-PTK6 complexes are of prognostic relevance.

[Analysis of signalling pathways in formalin-fixed breast cancer tissues]. / Analyse von Signalwegen in formalinfixierten Brustkrebsgeweben.

AIMS: The aim of our study was to develop and optimize methods for relative and absolute protein quantifications in formalin-fixed and paraffin-embedded (FFPE) tissues with special emphasis on HER mediated pathways in breast cancer. METHODS: Using a recently developed technology for extraction of full-length proteins from FFPE tissues, we evaluated >50 commercial antibodies for specificity using Western blots and protein microarrays. Purified HER receptor proteins were used to determine absolute protein concentrations. RESULTS: We confirmed specificity of 23 commercially available phosphospecific and non-phosphospecific antibodies using Western blots with protein extracts from cell lines and tissue extracts from breast cancer patients. Spiking known amounts of purified HER receptor proteins in HER receptor negative tissue extracts allowed us to precisely measure abundances of HER-receptors. CONCLUSIONS: Our results will provide a basis for the development of diagnostic techniques for the quantitative analysis of deregulated HER receptors and downstream signalling proteins in typical clinical tissues.

[Update on protein analysis of fixed tissues]. / Neues zur Proteinanalytik archivierter Gewebeproben.

Tissue samples have been routinely used for decades to distinguish healthy from diseased tissue in histopathological characterization. While nucleic acid-based methodologies have been successfully in use for many years, protein-based techniques, in contrast, are at a very early stage (with the exception of immunohistochemistry). One reason for this delay may be that the scientific community has long thought that formalin-fixed and paraffin embedded (FFPE) tissues are unfit for protein analysis. However, recent reports demonstrate that many protein methods that are routinely used for frozen tissues can also be applied for FFPE tissues, including Western blot, protein microarray, matrix-assisted laser desorption/ionization (MALDI) imaging and 2D gel electrophoresis. The present article provides an overview of recent developments in this field, focussing particular attention on quantitative analysis and high throughput technologies that have the potential to be integrated into the routine workflow of clinical pathology laboratories.

[Her2 testing in gastric cancer. What is different in comparison to breast cancer?]. / Her2-Diagnostik beim Magenkarzinom. Was ist anders im Vergleich zum Mammakarzinom?

Based on data from a large multicenter phase III trial (ToGA study) trastuzumab has very recently been approved by the EMEA for metastatic gastric cancer and adenocarcinoma of the gastro-esophageal junction. Only patients with tumors which over express Her2 as defined by IHC2+ and a confirmatory FISH+ result, or IHC 3+, determined by an accurate and validated assay are eligible for trastuzumab therapy. However, testing of Her2 status by immunohistochemistry (IHC) differs from breast cancer in core aspects: 1. IHC2+/3+ is scored even though membranous staining is incomplete if membrane staining is clearly detectable even at low magnification (2.5x/5x, 3+) or medium magnification (10x/20x, 2+). 2. Additionally, membrane staining at the appropriate intensity found in at least 10% of tumor cells is restricted to resection specimens. Evaluation of Her2 in situ hybridization (ISH) is similar to breast cancer with ratio values of > or =2.0 indicating Her2 gene amplification. Taking these modifications into account and defining the HER2 positive subgroup as IHC 3+ and IHC2+/FISH+, approximately 16% of gastric cancers are considered Her2 positive, affecting mainly tumor regions with intestinal (gland forming) type carcinoma. In contrast to breast cancer, up to one-third of gastric cancers show a heterogeneous Her2 status both at IHC and ISH levels which favors bright field ISH over FISH.

[MALDI imaging mass spectrometry for direct tissue analysis]. / In-situ-Proteomanalyse von Geweben: Mittels bildgebender Massenspektrometrie (MALDI Imaging).

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is a new method for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections using MALDI-TOF (time-of-flight) mass spectometry. The power of this technology lies in its capability to reliably combine protein data with specific cellular regions within the tissue. For the molecular image analysis, tissue samples are measured in a raster-like process in the mass spectrometer and for each spot a mass spectrum is obtained from molecules within the irradiated area. Software tools generate a mass spectrometry image or molecular weight-specific map of the sample at any desired molecular weight value and the presence and location of proteins and peptides are visualized in the tissue.There are three main fields of application, comprising molecular histology, screening for new biomarkers, and detection of drugs and their metabolites directly in the tissue.

Neoadjuvant continuous infusion of weekly 5-fluorouracil and escalating doses of oxaliplatin plus concurrent radiation in locally advanced oesophageal squamous cell carcinoma: results of a phase I/II trial.

Oxaliplatin and 5-fluorouracil have a significant activity in locally advanced oesophageal squamous cell cancer (OSCC). However, their optimal dosage and efficacy when combined with concurrent radiotherapy as neoadjuvant treatment are unknown. This non-randomised, phase I/II study aimed to define the maximum tolerated dose (MTD) and assessed the histopathological tumour response rate to neoadjuvant oxaliplatin in weekly escalating doses (40, 45, 50 mg m(-2)) and continuous infusional 5-fluorouracil (CI-5FU; 225 mg m(-2)) plus concurrent radiotherapy. Patients had resectable OSCC. Resection was scheduled for 4-6 weeks after chemoradiotherapy. During phase I (dose escalation; n=19), weekly oxaliplatin 45 mg m(-2) plus CI-5FU 225 mg m(-2) was established as the MTD and was the recommended dosage for phase II. Oesophageal mucositis was the dose-limiting toxicity at higher doses. During phase II, histopathological responses (<10% residual tumour cells within the specimen) were observed in 10 of 16 patients (63%; 95% confidence interval: 39-82%). Overall, 16 of the 25 patients (64%) who underwent resection had a histopathological response; tumour-free resection (R0) was achieved in 80%. Neoadjuvant weekly oxaliplatin 45 mg m(-2) plus CI-5FU 225 mg m(-2) with concurrent radiotherapy provides promising histological response rates and R0 resection rates in locally advanced OSCC.

Informatics and medicine--from molecules to populations.

OBJECTIVES: To clarify challenges and research topics for informatics in health and to describe new approaches for interdisciplinary collaboration and education. METHODS: Research challenges and possible solutions were elaborated by scientists of two universities using an interdisciplinary approach, in a series of meetings over several months. RESULTS AND CONCLUSION: In order to translate scientific results from bench to bedside and further into an evidence-based and efficient health system, intensive collaboration is needed between experts from medicine, biology, informatics, engineering, public health, as well as social and economic sciences. Research challenges can be attributed to four areas: bioinformatics and systems biology, biomedical engineering and informatics, health informatics and individual healthcare, and public health informatics. In order to bridge existing gaps between different disciplines and cultures, we suggest focusing on interdisciplinary education, taking an integrative approach and starting interdisciplinary practice at early stages of education.

[Results of reference pathology in mammography screening]. / Ergebnisse der Referenzpathologie im Mammographie-Screening.

As a measure of quality assurance in mammography screening in Germany, obligatory double reading of histopathological specimens is currently a subject of debate. Concordance rates of more than 6000 cases gathered from several reference centres were evaluated. In accordance with several international studies, overall agreement in single and double readings in German mammography screening was approximately 95%. Concordance rates were even higher for malignancies (99%). Variations are more common in the case of lesions, the biological significance of which remains unclear (flat atypia, lobular neoplasm, papilloma). This is the result of a currently unresolvable methodological rather classification problem, as seen from studies from countries with many years of experience in training and diagnostic test series. Thus, the evidence base is currently insufficient to mandate double reading of slides.

Transient elevation of messenger RNA encoding gastrin-releasing peptide, a putative pulmonary growth factor in human fetal lung.

Gastrin-releasing peptide (GRP), the mammalian homologue of the amphibian peptide bombesin, is present in pulmonary neuroendocrine cells and appears to be a growth factor for both normal and neoplastic pulmonary cells. Previously we have reported the cloning of the messenger RNAs (mRNAs) and gene that encode human GRP. We now report that GRP mRNAs are markedly elevated in human fetal lung during the canalicular phase of pulmonary development (from approximately 16 to 30 wk gestation). By RNA blot and in situ hybridization analyses, GRP mRNAs were first detectable in fetal lung at 9-10 wk, plateaued at levels 25-fold higher than in adult lungs from 16 to approximately 30 wk and then declined to near adult levels by 34 wk gestation. By contrast, GRP peptide levels remain elevated until several months after birth. Consistent with this, in situ hybridization and immunohistochemical studies showed that GRP mRNA and peptide consistently colocalized in early gestation lung but that in neonatal lung, many cells that contained GRP peptide no longer contained GRP mRNA. The transient expression of high levels of GRP mRNAs during an approximately 12-wk phase of fetal lung development suggests that the secretion of GRP or its COOH-terminal peptides from pulmonary neuroendocrine cells may play a role in normal lung development.

Comparison of automated silver enhanced in situ hybridisation (SISH) and fluorescence ISH (FISH) for the validation of HER2 gene status in breast carcinoma according to the guidelines of the American Society of Clinical Oncology and the College of American Pathologists.

HER2 is an important tumour marker in breast cancer. However, there is controversy regarding which method reliably measures HER2 status. This study evaluates the concordance between HER2 gene amplification in invasive breast cancer determined by fluorescence in situ hybridisation (FISH) and a new silver enhanced in situ hybridisation (SISH) technique. Ninety-nine cases were analysed by direct-labelled manual FISH (PathVysion(R), Abbott/Vysis) and bright field automated SISH (INFORM(R), Ventana). For comparison, all specimens were stained by immunohistochemistry (Dako-HercepTesttrade mark and Ventana-PATHWAY(R)4B5). Evaluation was performed by five pathologists following the algorithms of the manufacturers and the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Concordance was calculated and the value of kappa statistics estimated. Overall concordance between FISH and SISH was 96.0% (kappa = 0.754, 95%CI). Discrepancies were mostly seen in tumours with intra-tumoural heterogeneity of HER2 amplification. In conclusion, HER2 gene copy status can be reliably determined by SISH. The 96% concordance with FISH fulfils the ASCO/CAP requirement of greater than 95% concordance for amplified vs non-amplified cases. There was a low inter-observer variability in the interpretation of SISH, suggesting that SISH is equally reliable in determining HER2 amplification as FISH. Because SISH combines bright field microscopy with molecular analysis and full automation, it appears to be particularly suited for routine application in surgical pathology.

Analysis of the E-cadherin repressor Snail in primary human cancers.

Epithelial-mesenchymal transition (EMT), a normal developmental process, is known to play a crucial role in tumor progression. Molecules involved in this process, such as the E-cadherin repressor Snail, facilitate migration and invasion of carcinoma cells. A growing number of studies addressing the expression of Snail in clinical samples have been reported and are discussed in this review. A total of 2,112 cases from 9 different tumor types were evaluated. So far, a clear picture has emerged only in some cancer types analyzed with regard to overexpression of Snail and clinical-pathological parameters. Currently, it seems that Snail may play a role in hormone-dependent carcinomas but may be of minor importance in gastrointestinal cancers for tumor dedifferentiation and the maintenance of the invasive phenotype. It should be kept in mind, however, that the threshold for Snail activity does not have to be the same in every tumor type analyzed. The recent introduction of well-characterized novel monoclonal antibodies reacting with the short-lived nuclear Snail protein may help to establish a potential clinical usefulness for this master molecule of EMT, at least for certain types of cancer.

[Papillary adenoma of type-II pneumocytes as a rare differential diagnosis of a solitary pulmonary nodule]. / Papilläres Adenom der Typ-II-Pneumozyten als seltene Differenzialdiagnose eines pulmonalen Rundherdes.

The case of a 66-year-old, asymptomatic patient with a papillary adenoma of type-II pneumocytes is reported. Following the coincidental radiologic finding of a solitary pulmonary nodule, the diagnosis could be established in a bronchoscopically obtained endobronchial biopsy. A resection of the involved segments S8-10 on the left side was performed. Papillary adenoma of type-II pneumocytes is a rare tumor, whose origin is suspected in progenitor cells of the bronchioloalveolar epithelium with the potential to differentiate towards type-II pneumocytes and clara cells. The tumor is regarded as benign, however, a malignant potential is not excluded by some authors.

Clinical proteomics: new trends for protein microarrays.

Protein microarrays are an emerging class of nanotechnology for tracking many different proteins simultaneously. Much progress has been made for applications in basic sciences. Translation of these methods for the treatment of patients, however, is slow, because the realities in the clinic are rarely taken into account, and proteomic changes in cultured cell lines might not fully reflect human diseases due to the lack of the tissue microenvironment. In this review, we summarise current protein microarray approaches that are being developed for profiling tissues and histopathologically defined cell populations from cancer patients. We provide an overview of clinical applications for protein lysate microarrays and discuss the power of this technology for the discovery of disease markers for cancer diagnosis and individualised treatment.

Prognostic significance of expression patterns of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor in oesophageal adenocarcinoma: a tissue microarray study.

AIMS: To correlate immunohistochemical expression patterns and prognosis in oesophageal adenocarcinoma. METHODS: The expression of c-erbB-2, p53, p16INK4A, p27KIP1, cyclin D1 and epidermal growth factor receptor (EGFR) was studied in a series of 137 primarily resected oesophageal adenocarcinoma samples. The expression analysis on protein level was performed on routine paraffin wax-embedded material, with immunohistochemical staining of the samples, assembled on a tissue microarray. The results were correlated with clinicopathological features (pT, pN and G) and survival. RESULTS: 22 (16%) tumours showed an overexpression of the c-erbB-2 oncoprotein. Expression of EGFR was observed in 72 (55%) cases, accumulation of p53 in 68 (52%) cases and of cyclin D1 in 102 (77%) cases. Loss of p16INK4A expression was observed in 101 (76%) cases and low expression of p27KIP1 in 91 (71%) cases. Expression of these proteins did not correlate with tumour stage, grade, Lauren's or World Health Organization classification or lymph node status. On univariate survival analysis, more advanced tumour stage (p = 0.002), lymph node involvement (p = 0.003), high tumour grade (p = 0.017) and lack of EGFR expression (p = 0.034) were found to be associated with poorer survival. On multiple regression analysis, only tumour stage (p = 0.03) and lymph node involvement (p = 0.004) were shown to have an association with the survival of the patient. CONCLUSION: The immunohistochemical expression of c-erbB-2 oncoprotein, cylin D1, p16INK4A, p27KIP1, p53 and EGFR in most oesophageal adenocarcinomas suggests their implication in the pathogenesis of this entity. None of the molecular markers assessed, however, was of prognostic value. Identification of any marker superior to or even approaching the prognostic value of conventional histopathological markers (pT and pN) was therefore not possible.

No evidence for mutations in the alpha- and beta-catenin genes in human gastric and breast carcinomas.

Disturbed function of E-cadherin and/or of one of its anchoring proteins, the catenins, is thought to destabilize E-cadherin-mediated cell-cell adhesion, which may enhance the invasiveness of epithelial cells and thus favor carcinoma progression. Reduced expression of E-cadherin and alpha-catenin, as well as mutations in the E-cadherin gene, have been found in various carcinomas, whereas mutations in the alpha- and beta-catenin genes have been described only in carcinoma cell lines. Using reverse transcription-PCR, followed by agarose gel electrophoresis and single-strand conformational polymorphism, we examined 16 diffuse- and 5 intestinal-type gastric carcinomas, as well as 9 lobular and 2 ductal breast carcinomas, for mutations of alpha- and beta-catenin cDNA. All of the investigated tumors were analyzed previously for E-cadherin mutations. Comparing tumorous and nontumorous samples, we detected neither deletions nor aberrant single-strand conformational polymorphism patterns. At nucleotide 2220 of the alpha-catenin gene, we identified one frequent polymorphism. Our findings suggest that, in contrast to E-cadherin, mutations of alpha- and beta-catenin do not contribute to the pathogenesis or the diffuse growth patterns of gastric or breast carcinomas.