Andreas-Claudius Hoffmann

Proteomic Differences Between Hepatocellular Carcinoma and Nontumorous Liver Tissue Investigated by a Combined Gel-based and Label-free Quantitative Proteomics Study

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.

A Three-Gene Signature for Outcome in Soft Tissue Sarcoma

Purpose: Finding markers or gene sets that would further classify patients into different risk categories and thus allow more individually adapted multimodality treatment regimens in soft tissue sarcomas is necessary. In this study, we investigated the prognostic values of hypoxia-inducible factor 1a (HIF1a), heparin-binding epidermal growth factor–like growth factor (HB-EGF), vascular endothelial growth factor (VEGF), and other angiogenesis-related gene expressions, as well as their interrelationships. Experimental Design: Formalin-fixed paraffin-embedded tissue samples were obtained from 45 patients with soft tissue sarcoma (median age 57 years, range 16–85 years). After laser capture microdissection direct quantitative real-time reverse transcription-PCR (TaqMan) assays were done in triplicates to determine HIF1a, HB-EGF, VEGF, and other gene expression levels. Results: Multivariate Cox regression analysis revealed significant independent associations of HB-EGF, HIF1a, and VEGF-C gene expression to the overall survival (P < 0.0001). A combined factor of these three genes showed a relative risk for shorter survival of 5.5, more than twice higher as in an increasing International Union against Cancer Stage. Receiver operating characteristic curve analysis showed a significant sensitivity of 73% and specificity of 82% of this factor for the diagnosis of short (<3 years) versus long (3-9 years) survival (P = 0.0002). VEGF-A showed significant gender differences in the association to survival. Conclusions: Measuring HIF1a, HB-EGF, and VEGF-C expression may contribute to a better understanding of the prognosis of patients with soft tissue sarcoma and may even play a crucial role for the distribution of patients to multimodal therapeutic regimens. Prospective studies investigating the response to different adjuvant or palliative therapies seem to be warranted. (Clin Cancer Res 2009;15(16):5191–8)

A practical data processing workflow for multi-OMICS projects☆

Multi-OMICS approaches aim on the integration of quantitative data obtained for different biological molecules in order to understand their interrelation and the functioning of larger systems. This paper deals with several data integration and data processing issues that frequently occur within this context. To this end, the data processing workflow within the PROFILE project is presented, a multi-OMICS project that aims on identification of novel biomarkers and the development of new therapeutic targets for seven important liver diseases. Furthermore, a software called CrossPlatformCommander is sketched, which facilitates several steps of the proposed workflow in a semi-automatic manner. Application of the software is presented for the detection of novel biomarkers, their ranking and annotation with existing knowledge using the example of corresponding Transcriptomics and Proteomics data sets obtained from patients suffering from hepatocellular carcinoma. Additionally, a linear regression analysis of Transcriptomics vs. Proteomics data is presented and its performance assessed. It was shown, that for capturing profound relations between Transcriptomics and Proteomics data, a simple linear regression analysis is not sufficient and implementation and evaluation of alternative statistical approaches are needed. Additionally, the integration of multivariate variable selection and classification approaches is intended for further development of the software. Although this paper focuses only on the combination of data obtained from quantitative Proteomics and Transcriptomics experiments, several approaches and data integration steps are also applicable for other OMICS technologies. Keeping specific restrictions in mind the suggested workflow (or at least parts of it) may be used as a template for similar projects that make use of different high throughput techniques. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

High Expression of Heparanase is Significantly Associated with Dedifferentiation and Lymph Node Metastasis in Patients with Pancreatic Ductal Adenocarcinomas and Correlated to PDGFA and Via HIF1a to HB-EGF and bFGF

BackgroundPancreatic cancer still has one of the worst prognoses of all cancers with a 5-year survival rate of 5%, making it necessary to find markers or gene sets that would further classify patients into different risk categories and thus allow more individually adapted multimodality treatment regimens. Especially heparanase (HPSE) has recently been discussed as a key factor in pancreatic cancer.Materials and MethodsParaffin-embedded tissue samples were obtained from 41 patients with pancreatic adenocarcinoma who were scheduled for primary surgical resection. Direct quantitative real-time reverse transcriptase polymerase chain reaction (TaqMan™) assays were performed in triplicates to determine HPSE, hypoxia inducible factor-1 alpha (HIF1a), platelet-derived growth factor alpha (PDGFA), heparin-binding EGF-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF) gene expression levels.ResultsHPSE was significantly correlated to PDGFA (p = 0.04) and HIF1a (p = 0.04). The correlation of HIF1a to bFGF and HB-EGF was significant (p = 0.04, p = 0.02). Stepwise multiple linear regression models showed a significant independent association of HPSE with lymph node metastasis (p = 0.025) and with dedifferentiation (p = 0.042).ConclusionsHeparanase seems to be significantly associated with lymph node metastasis (p = 0.025) as well as dedifferentiation (p = 0.042). We assume that HPSE plays a crucial role for the aggressiveness of pancreatic cancer. Larger studies including more patients seem to be warranted.

Analysis of disease-associated protein expression using quantitative proteomics—fibulin-5 is expressed in association with hepatic fibrosis.

Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n = 77). Finally, we performed a targeted proteomics approach, multiple reaction monitoring (MRM), to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM), and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.

Lack of prognostic significance of serum DNA methylation of DAPK, MGMT, and GSTPI in patients with non‐small cell lung cancer

To further improve the screening, diagnosis and therapy of patients with non‐small cell lung cancer (NSCLC) additional diagnostic tools are desperately warranted. Aim of this study was to investigate the potential of the DNA methylation of DAPK, MGMT, and GSTPI in serum of patients with NSCLC as a prognostic molecular marker in this disease.

Preoperative survivin mRNA detection in peripheral blood is an independent predictor of outcome in esophageal carcinoma.

AIMS Survivin (SVV) mRNA expression levels in peripheral blood of patients with gastrointestinal malignancies change significantly during the course of treatment. We wanted to scrutinize these findings in patients with esophageal carcinoma and furthermore evaluate whether the detection of mRNA and the change in detecting ability have an association with overall survival. MATERIALS & METHODS Whole blood was drawn 1 day pre- and 10 days post-operatively from 62 patients with esophageal carcinoma. Tumor cells were enriched from whole blood by density-gradient centrifugation prior to extraction of total cellular RNA and subsequent direct quantitative reverse transcriptase-PCR assays. RESULTS SVV was detectable in 48 out of 62 patients (77%). Stepwise multivariate Cox linear regression models demonstrated a significant and independent association of measured SVV with overall survival (6.6 exp[b]; 95% CI: 1.97-22.12; p = 0.002). Increased SVV levels after the operation were linked to shorter overall survival (p = 0.04). CONCLUSION Preoperative SVV expression levels appear to be associated with overall survival in patients with esophageal cancers. Increasing levels could potentially indicate a higher risk for shorter overall survival and therefore demand adapted treatment modalities.

Identification of Novel Biomarker Candidates for the Immunohistochemical Diagnosis of Cholangiocellular Carcinoma

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.

Detection of Circulating Tumor Cell Subpopulations in Patients with Head and Neck Squamous Cell Carcinoma (HNSCC)

Background Since image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples. Methods 20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed. Results We were able to detect cells with epithelial properties like CK+/N-cadherin−/CD45− and CK+/CD133−/CD45− as well as cells with mesenchymal features such as N-cadherin+/CK−/CD45− and cells with both characteristics like N-cadherin+/CK+/CD45−. We also observed cells showing stem cell-like features like CD133+/CK−/CD45− and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45−. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1). Conclusions This is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.

High-risk PCI in acute coronary syndromes with Impella LP 2.5 device support

OBJECTIVES To evaluate feasibility, safety, efficacy as well as acute and short-term outcome of hemodynamically supported percutaneous coronary intervention (PCI) by a percutaneous, catheter-based left ventricular assist device (LVAD) (Impella LP 2.5, Abiomed Europe GmbH, Aachen, Germany) in a high-risk patient population with acute coronary syndrome. BACKGROUND Although hemodynamic support by intraaortic balloon pump favorably affects myocardial oxygen supply and demand, it has modest effects on cardiac output, providing passive support only. In contrast, the Impella LP 2.5 microaxial pump, which is placed within the left ventricular outflow tract and actively ejects blood into the ascending aorta, might offer additional hemodynamic support and thereby procedural safety during PCI. METHODS Thirty-eight consecutive high-risk patients (mean age, 69.7 ± 10.3 years, logistic EuroSCORE, 22.4 ± 14.9%) with unstable angina pectoris or non-ST-segment elevation myocardial infarction and severe three-vessel-disease were included in the study. Clinical and laboratory examinations were performed at baseline as well as at 6, 24 and 48 h after the procedure and 30 days after discharge. RESULTS Device insertion and explantation was feasible in all patients without vascular complications and continuous hemodynamic stability was obtained during PCI. PCI was uneventfully performed in all but one patient for technical reasons. One non procedure-related death occurred 7 days after the intervention, accounting for a total 30-day mortality of 2.86%. Other major cardiac or cerebrovascular events did not occur. CONCLUSIONS LVAD support using a percutaneous microaxial flow pump is a promising and safe approach for high-risk PCI providing good short-term results.