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1.  Critical roles of specimen type and temperature before and during fixation in the detection of phosphoproteins in breast cancer tissues 
The most efficient approach for therapy selection to inhibit the deregulated kinases in cancer tissues is to measure their phosphorylation status prior to the treatment. The aim of our study was to evaluate the influence of pre-analytical parameters (cold ischemia time, temperature before and during tissue fixation, and sample type) on the levels of proteins and phosphoproteins in breast cancer tissues, focusing on the PI3 kinase/AKT pathway. The BALB-neuT mouse breast cancer model expressing HER2 and pAKT proteins and human biopsy and resection specimens were analyzed. By using quantitative reverse phase protein arrays (RPPA), 9 proteins and 16 phosphoproteins relevant to breast cancer biology were assessed. Cold temperatures before and during fixation resulted in a marked improvement in the preservation of the reactivity of biological markers (eg, ER, HER2) in general and, specifically, pHER2 and pAKT. Some phosphoproteins, eg, pHER2 and pAKT, were more sensitive to prolonged cold ischemia times than others (eg, pS6RP and pSTAT5). By comparing the phosphoprotein levels in core needle biopsies with those in resection specimens, we found a marked decrease in many phosphoproteins in the latter. Cold conditions can improve the preservation of proteins and phosphoproteins in breast cancer tissues. Biopsies≤1 mm in size are the preferred sample type for assessing the activity of deregulated kinases for personalized cancer treatments because the phosphoprotein levels are better preserved compared with resection specimens. Each potential new (phospho)protein biomarker should be tested for its sensitivity to pre-analytical processing prior to the development of a diagnostic assay.
PMCID: PMC4421866  PMID: 25730369
2.  Heat Shock Protein 90 (HSP90) and Her2 in Adenocarcinomas of the Esophagus 
Cancers  2014;6(3):1382-1393.
Her2 overexpression and amplification can be found in a significant subset of esophageal adenocarcinomas. The activity of Her2 has been shown to be modulated by molecular chaperones such as HSP90. We analyzed expression/amplification data for HSP90 and Her2 on 127 primary resected esophageal adenocarcinomas in order to evaluate a possible relationship between these two molecules. HSP90 expression determined by immunohistochemistry was observed in various levels. Thirty nine (39) tumors (30.7%) were classified as Her2-positive according to their immunoreactivity and amplification status. There was a significant correlation between HSP90 expression and Her2-status (p = 0.008). This could also be demonstrated by quantitative protein expression analysis with reverse phase protein arrays (r = 0.9; p < 0.001). Her2-status was associated withpT-category (p = 0.041), lymph node metastases (p = 0.049) and tumor differentiation (p = 0.036) with a higher percentage of cases with negative Her2 status in lower tumor stagesA negative Her2-status was also associated with better survival in univariate and multivariate analysis (p = 0.001 and p = 0.014). For HSP90, no associations between clinical and pathological parameters were found. The observed association between HSP90 expression and Her2 suggests a co-regulation of these molecules in at least a subset of esophageal adenocarcinomas. Anti-HSP90 drugs, which recently have been introduced in cancer treatment, may also be an option for these tumors by targeting HSP90 alone or in combination with Her2.
PMCID: PMC4190546  PMID: 24978439
HSP90; Her2; esophageal adenocarcinoma; immunohistochemistry; RPPA; in situ hybridization
3.  Variation in Cell Signaling Protein Expression May Introduce Sampling Bias in Primary Epithelial Ovarian Cancer 
PLoS ONE  2013;8(10):e77825.
Although the expression of cell signaling proteins is used as prognostic and predictive biomarker, variability of protein levels within tumors is not well studied. We assessed intratumoral heterogeneity of protein expression within primary ovarian cancer. Full-length proteins were extracted from 88 formalin-fixed and paraffin-embedded tissue samples of 13 primary high-grade serous ovarian carcinomas with 5–9 samples each. In addition, 14 samples of normal fallopian tube epithelium served as reference. Quantitative reverse phase protein arrays were used to analyze the expression of 36 cell signaling proteins including HER2, EGFR, PI3K/Akt, and angiogenic pathways as well as 15 activated (phosphorylated) proteins. We found considerable intratumoral heterogeneity in the expression of proteins with a mean coefficient of variation of 25% (range 17–53%). The extent of intratumoral heterogeneity differed between proteins (p<0.005). Interestingly, there were no significant differences in the extent of heterogeneity between phosphorylated and non-phosphorylated proteins. In comparison, we assessed the variation of protein levels amongst tumors from different patients, which revealed a similar mean coefficient of variation of 21% (range 12–48%). Based on hierarchical clustering, samples from the same patient clustered more closely together compared to samples from different patients. However, a clear separation of tumor versus normal tissue by clustering was only achieved when mean expression values of all individual samples per tumor were analyzed. While differential expression of some proteins was detected independently of the sampling method used, the majority of proteins only demonstrated differential expression when mean expression values of multiple samples per tumor were analyzed. Our data indicate that assessment of established and novel cell signaling proteins as diagnostic or prognostic markers may require sampling of serous ovarian cancers at several distinct locations to avoid sampling bias.
PMCID: PMC3810127  PMID: 24204986
4.  The PI3K/Akt/mTOR Pathway Is Implicated in the Premature Senescence of Primary Human Endothelial Cells Exposed to Chronic Radiation 
PLoS ONE  2013;8(8):e70024.
The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.
PMCID: PMC3731291  PMID: 23936371
5.  The PAXgene® Tissue System Preserves Phosphoproteins in Human Tissue Specimens and Enables Comprehensive Protein Biomarker Research 
PLoS ONE  2013;8(3):e60638.
Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology.
PMCID: PMC3612043  PMID: 23555997
6.  Evidence of Prognostic Relevant Expression Profiles of Heat-Shock Proteins and Glucose-Regulated Proteins in Oesophageal Adenocarcinomas 
PLoS ONE  2012;7(7):e41420.
A high percentage of oesophageal adenocarcinomas show an aggressive clinical behaviour with a significant resistance to chemotherapy. Heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) are molecular chaperones that play an important role in tumour biology. Recently, novel therapeutic approaches targeting HSP90/GRP94 have been introduced for treating cancer. We performed a comprehensive investigation of HSP and GRP expression including HSP27, phosphorylated (p)-HSP27(Ser15), p-HSP27(Ser78), p-HSP27(Ser82), HSP60, HSP70, HSP90, GRP78 and GRP94 in 92 primary resected oesophageal adenocarcinomas by using reverse phase protein arrays (RPPA), immunohistochemistry (IHC) and real-time quantitative RT-PCR (qPCR). Results were correlated with pathologic features and survival. HSP/GRP protein and mRNA expression was detected in all tumours at various levels. Unsupervised hierarchical clustering showed two distinct groups of tumours with specific protein expression patterns: The hallmark of the first group was a high expression of p-HSP27(Ser15, Ser78, Ser82) and low expression of GRP78, GRP94 and HSP60. The second group showed the inverse pattern with low p-HSP27 and high GRP78, GRP94 and HSP60 expression. The clinical outcome for patients from the first group was significantly improved compared to patients from the second group, both in univariate analysis (p = 0.015) and multivariate analysis (p = 0.029). Interestingly, these two groups could not be distinguished by immunohistochemistry or qPCR analysis. In summary, two distinct and prognostic relevant HSP/GRP protein expression patterns in adenocarcinomas of the oesophagus were detected by RPPA. Our approach may be helpful for identifying candidates for specific HSP/GRP-targeted therapies.
PMCID: PMC3404067  PMID: 22911792
7.  Common Protein Biomarkers Assessed by Reverse Phase Protein Arrays Show Considerable Intratumoral Heterogeneity in Breast Cancer Tissues 
PLoS ONE  2012;7(7):e40285.
Proteins are used as prognostic and predictive biomarkers in breast cancer. However, the variability of protein expression within the same tumor is not well studied. The aim of this study was to assess intratumoral heterogeneity in protein expression levels by reverse-phase-protein-arrays (RPPA) (i) within primary breast cancers and (ii) between axillary lymph node metastases from the same patient. Protein was extracted from 106 paraffin-embedded samples from 15 large (≥3 cm) primary invasive breast cancers, including different zones within the primary tumor (peripheral, intermediate, central) as well as 2–5 axillary lymph node metastases in 8 cases. Expression of 35 proteins including 15 phosphorylated proteins representing the HER2, EGFR, and uPA/PAI-1 signaling pathways was assessed using reverse-phase-protein-arrays. All 35 proteins showed considerable intratumoral heterogeneity within primary breast cancers with a mean coefficient of variation (CV) of 31% (range 22–43%). There were no significant differences between phosphorylated (CV 32%) and non-phosphorylated proteins (CV 31%) and in the extent of intratumoral heterogeneity within a defined tumor zone (CV 28%, range18–38%) or between different tumor zones (CV 24%, range 17–38%). Lymph node metastases from the same patient showed a similar heterogeneity in protein expression (CV 27%, range 18–34%). In comparison, the variation amongst different patients was higher in primary tumors (CV 51%, range 29–98%) and lymph node metastases (CV 65%, range 40–146%). Several proteins showed significant differential expression between different tumor stages, grades, histological subtypes and hormone receptor status. Commonly used protein biomarkers of breast cancer, including proteins from HER2, uPA/PAI-1 and EGFR signaling pathways showed higher than previously reported intratumoral heterogeneity of expression levels both within primary breast cancers and between lymph node metastases from the same patient. Assessment of proteins as diagnostic or prognostic markers may require tumor sampling in several distinct locations to avoid sampling bias.
PMCID: PMC3390380  PMID: 22792263
8.  uPA and PAI-1-Related Signaling Pathways Differ between Primary Breast Cancers and Lymph Node Metastases12 
Translational Oncology  2012;5(2):98-104.
The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell-related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-β, ILK, pGSK3-β, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-β, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-β). Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node-negative breast cancer tissues.
PMCID: PMC3323931  PMID: 22496926
9.  The HOPE fixation technique - a promising alternative to common prostate cancer biobanking approaches 
BMC Cancer  2011;11:511.
The availability of well-annotated prostate tissue samples through biobanks is key for research. Whereas fresh-frozen tissue is well suited for a broad spectrum of molecular analyses, its storage and handling is complex and cost-intensive. Formalin-fixed paraffin-embedded specimens (FFPE) are easy to handle and economic to store, but their applicability for molecular methods is restricted. The recently introduced Hepes-glutamic acid-buffer mediated Organic solvent Protection Effect (HOPE) is a promising alternative, which might have the potential to unite the benefits of FFPE and fresh-frozen specimen. Aim of the study was to compare HOPE-fixed, FFPE and fresh-frozen bio-specimens for their accessibility for diagnostic and research purposes.
10 prostate cancer samples were each preserved with HOPE, formalin, and liquid nitrogen and studied with in-situ and molecular methods. Samples were H&E stained, and assessed by immunohistochemistry (i.e. PSA, GOLPH2, p63) and FISH (i.e. ERG rearrangement). We assessed DNA integrity by PCR, using control genes ranging from 100 to 600 bp amplicon size. RNA integrity was assessed through qRT-PCR on three housekeeping genes (TBP, GAPDH, β-actin). Protein expression was analysed by performing western blot analysis using GOLPH2 and PSA antibodies.
Of the HOPE samples, morphologic quality of H&E sections, immunohistochemical staining, and the FISH assay was at least equal to FFPE tissue, and significantly better than the fresh-frozen specimens. DNA, RNA, and protein analysis of HOPE samples provided similar results as compared to fresh-frozen specimens. As expected, FFPE-samples were inferior for most of the molecular analyses.
This is the first study, comparatively assessing the suitability of these fixation methods for diagnostic and research utilization. Overall, HOPE-fixed bio-specimens combine the benefits of FFPE- and fresh-frozen samples. Results of this study have the potential to expand on contemporary prostate tissue biobanking approaches and can serve as a model for other organs and tumors.
PMCID: PMC3248383  PMID: 22151117
HOPE technique; HOPE fixation; Prostate cancer
10.  Histological Assessment of PAXgene Tissue Fixation and Stabilization Reagents 
PLoS ONE  2011;6(11):e27704.
Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.
PMCID: PMC3218013  PMID: 22110732
11.  Discovery of New Molecular Subtypes in Oesophageal Adenocarcinoma 
PLoS ONE  2011;6(9):e23985.
A large number of patients suffering from oesophageal adenocarcinomas do not respond to conventional chemotherapy; therefore, it is necessary to identify new predictive biomarkers and patient signatures to improve patient outcomes and therapy selections. We analysed 87 formalin-fixed and paraffin-embedded (FFPE) oesophageal adenocarcinoma tissue samples with a reverse phase protein array (RPPA) to examine the expression of 17 cancer-related signalling molecules. Protein expression levels were analysed by unsupervised hierarchical clustering and correlated with clinicopathological parameters and overall patient survival. Proteomic analyses revealed a new, very promising molecular subtype of oesophageal adenocarcinoma patients characterised by low levels of the HSP27 family proteins and high expression of those of the HER family with positive lymph nodes, distant metastases and short overall survival. After confirmation in other independent studies, our results could be the foundation for the development of a Her2-targeted treatment option for this new patient subgroup of oesophageal adenocarcinoma.
PMCID: PMC3179464  PMID: 21966358
12.  Correction: Successful Protein Extraction from Over-Fixed and Long-Term Stored Formalin-Fixed Tissues 
PLoS ONE  2011;6(4):10.1371/annotation/a42e114f-a708-4423-8a3e-a1d8919b9b60.
PMCID: PMC3084773
13.  Successful Protein Extraction from Over-Fixed and Long-Term Stored Formalin-Fixed Tissues 
PLoS ONE  2011;6(1):e16353.
One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease.
PMCID: PMC3031559  PMID: 21305021
14.  Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs 
The Journal of Cell Biology  2009;184(3):399-408.
Epithelial–mesenchymal transition (EMT) is required for mesodermal differentiation during development. The zinc-finger transcription factor, Snail1, can trigger EMT and is sufficient to transcriptionally reprogram epithelial cells toward a mesenchymal phenotype during neoplasia and fibrosis. Whether Snail1 also regulates the behavior of terminally differentiated mesenchymal cells remains unexplored. Using a Snai1 conditional knockout model, we now identify Snail1 as a regulator of normal mesenchymal cell function. Snail1 expression in normal fibroblasts can be induced by agonists known to promote proliferation and invasion in vivo. When challenged within a tissue-like, three-dimensional extracellular matrix, Snail1-deficient fibroblasts exhibit global alterations in gene expression, which include defects in membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invasive activity. Snail1-deficient fibroblasts explanted atop the live chick chorioallantoic membrane lack tissue-invasive potential and fail to induce angiogenesis. These findings establish key functions for the EMT regulator Snail1 after terminal differentiation of mesenchymal cells.
PMCID: PMC2646556  PMID: 19188491
15.  Noninvasive Visualization of the Activated αvβ3 Integrin in Cancer Patients by Positron Emission Tomography and [18F]Galacto-RGD 
PLoS Medicine  2005;2(3):e70.
The integrin αvβ3 plays an important role in angiogenesis and tumor cell metastasis, and is currently being evaluated as a target for new therapeutic approaches. Several techniques are being studied to enable noninvasive determination of αvβ3 expression. We developed [18F]Galacto-RGD, a 18F-labeled glycosylated αvβ3 antagonist, allowing monitoring of αvβ3 expression with positron emission tomography (PET).
Methods and Findings
Here we show by quantitative analysis of images resulting from a small-animal PET scanner that uptake of [18F]Galacto-RGD in the tumor correlates with αvβ3 expression subsequently determined by Western blot analyses. Moreover, using the A431 human squamous cell carcinoma model we demonstrate that this approach is sensitive enough to visualize αvβ3 expression resulting exclusively from the tumor vasculature. Most important, this study shows, that [18F]Galacto-RGD with PET enables noninvasive quantitative assessment of the αvβ3 expression pattern on tumor and endothelial cells in patients with malignant tumors.
Molecular imaging with [18F]Galacto-RGD and PET can provide important information for planning and monitoring anti-angiogenic therapies targeting the αvβ3 integrins and can reveal the involvement and role of this integrin in metastatic and angiogenic processes in various diseases.
Visualising integrins with PET scanning can show angiogenesis in tumors and also be used to monitor anti-angiogenic therapy
PMCID: PMC1069665  PMID: 15783258

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