How standardization of the pre-analytical phase of both research and diagnostic biomaterials can increase reproducibility of biomedical research and diagnostics.

Comparison of published biomedical studies shows that a large proportion are irreproducible, causing severe damage to society and creating an image of wasted investments. These observations are of course damaging to the biomedical research field, which is currently full of future promise. Precision medicine and disease prevention are successful, but are progressing slowly due to irreproducible study results. Although standardization is mentioned as a possible solution, it is not always clear how this could decrease or prevent irreproducible results in biomedical studies. In this article more insight is given into what quality, norms, standardization, certification, accreditation and optimized infrastructure can accomplish to reveal causes of irreproducibility and increase reproducibility when collecting biomaterials. CEN and ISO standards for the sample pre-analytical phase are currently being developed with the support of the SPIDIA4P project, and their role in increasing reproducibility in both biomedical research and diagnostics is demonstrated. In particular, it is described how standardized methods and quality assurance documentation can be exploited as tools for: 1) recognition and rejection of 'not fit for purpose' samples on the basis of detailed sample metadata, and 2) identification of methods that contribute to irreproducibility which can be adapted or replaced.

[Preanalytics and biobanking : Influence of preanalytical factors on tissue sample quality]. / Präanalytik und Biobanking : Einfluss präanalytischer Faktoren auf die Gewebeprobenqualität.

Access to well-characterized human biosamples is one of the most important prerequisites for modern biomedical research. Biobanks play a decisive role here, as they provide corresponding biosamples for planned analyses. Many interfering factors influencing the quality of biosamples have to be taken into account. In addition to logistical, ethical, and data protection aspects, preanalytical variables in the context of sample acquisition, storage, and processing should be mentioned in particular. In this paper, therefore, the most important preanalytical influencing factors are presented systematically and an overview of current national and international activities for the standardized recording of these factors is provided with the goal of being able to better understand their influence on results and to minimize them in the near future.

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.

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.

UPA and PAI-1 analysis from fixed tissues - new perspectives for a known set of predictive markers.

The urokinase-type plasminogen activator (uPA) and its main inhibitor PAI-1 play key roles in tumor-associated processes such as the degradation of the extracellular matrix (ECM), tissue remodeling, cell adhesion and migration. Elevated expression of both molecules is known to correlate with negative outcomes in node negative breast cancer. To date, these molecules are the only prognostic markers to have reached the highest level of evidence (LOE I) in multi-centered clinical trials for prognosis of node negative breast cancer. Unfortunately, the clinical utility of these molecules as markers is limited by the use of enzyme-linked immunoassay (ELISA) tests for their detection. The ELISA relies on the use of fresh or frozen tissue, which are rarely available in routine clinical settings. In this review article, we provide an overview of the clinical relevance of uPA and PAI-1 and present alternative methods for their detection. Common uPA and PAI-1 detection methods discussed in literature include RT-PCR-based assays and classical immunohistochemistry approaches. In recent years, attempts have been made to isolate and analyze proteins of formalin fixed, paraffin embedded (FFPE) tissues. These new methods are of special interest because up to now neither RT-PCR nor immunohistochemistry are recommended for the detection of uPA and PAI-1. Here, we present an approach for the analysis of uPA and PAI-1 directly from FFPE tissues that may eventually overcome the limitations of current assays and make the use of both markers widely available for routine prognosis and therapy decisions for breast cancer patients.

[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.

[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.

Deciphering signaling pathways in clinical tissues for personalized medicine using protein microarrays.

The current transition in cancer therapy from general treatment approaches, based mainly on chemotherapy and radiotherapy, to more directed approaches that aim to inhibit specific molecular targets has brought about new challenges for pathology. In the past, classical assignment of pathology consisted of tumor diagnosis and staging for further therapy decisions; nowadays, pathologists are asked to predict possible therapeutic results by detecting and quantifying therapeutic targets in tumors such as the human epidermal growth factor receptor 2 (HER2). The best approach to analyze such molecular targets is to provide a tumor-specific protein expression profile prior to therapy. To further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow fast, precise, cheap, and simultaneous analysis of many network components while requiring only a small amount of clinical material. Reverse phase protein microarray (RPPA) is a promising technology that meets these requirements while enabling quantitative measurement of proteins. Recently, methods for the extraction of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues have become available. In this article, we demonstrate how the use of RPPA to analyze signaling pathways from FFPE tissues may improve quantification of therapeutic targets and diagnostic markers in the near future.

Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks.

In general, it is believed that the extraction of proteins from formalin-fixed paraffin embedded samples is not feasible. However, recently a new technique was developed, presenting the extraction of non-degraded, full length proteins from formalin fixed tissues, usable for western blotting and protein arrays. In the study presented here, we applied this technique to punch biopsies of formalin fixed tissues embedded in paraffin to reduce heterogeneity of the tissue represented in sections, and to ensure analysing mainly defined cellular material. Successful extraction was achieved even from very small samples (0.7 mm(3)). Additionally, we were able to detect highly glycosylated proteins and protein modification, such as phosphorylation. Interestingly, with this technique it is feasible to extract high quality proteins from 14 year old samples. In summary, the new technique makes a great pool of material now usable for molecular analysis with high throughput tools.

The E-cadherin repressor Snail is associated with lower overall survival of ovarian cancer patients.

Epithelial ovarian cancer is the leading cause of death among female genital malignancies. Reduced expression of the cell adhesion molecule E-cadherin was previously shown to be associated with adverse prognostic features. The role of the E-cadherin repressor Snail in ovarian cancer progression remains to be elucidated. We analysed formalin-fixed and paraffin-embedded specimens of 48 primary ovarian tumours and corresponding metastases for expression of E-cadherin and Snail by immunohistochemistry. We found a significant correlation between E-cadherin expression in primary cancers and their corresponding metastases (P<0.001). This correlation was found for Snail expression as well (P<0.001). There was a significant (P=0.008) association of reduced E-cadherin expression in primary ovarian cancer with shorter overall survival. Similarly, Snail expression in corresponding metastases (P=0.047) was associated with reduced overall survival of the patients. Additionally, the group of patients showing reduced E-cadherin and increased Snail immunoreactivity in primary tumours and corresponding metastases, respectively, had a significantly higher risk of death (P=0.002 and 0.022, respectively) when compared to the patient group with the reference expression profile E-cadherin positive and Snail negative. Taken together, the results of our study show that the E-cadherin repressor Snail is associated with lower overall survival of ovarian cancer patients.

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.

Slug is overexpressed in gastric carcinomas and may act synergistically with SIP1 and Snail in the down-regulation of E-cadherin.

Epithelial-mesenchymal transition (EMT) involving down-regulation of E-cadherin is known to play an important role in tumour progression. The aim of our study was to investigate the mRNA expression of two EMT regulators-Slug and E12/E47-in primary human gastric carcinomas and to compare this with the expression of E-cadherin and other EMT regulators (Snail, Twist, and SIP1). We studied a series of 59 gastric carcinomas by real-time quantitative RT-PCR in formalin-fixed and paraffin-embedded tissues. Thirty-four cases (58%) showed Slug up-regulation in the tumour; reduced or negative expression of E-cadherin was present in 24 of these (71%, p<0.0001). Twenty-one cases (36%) showed E12/E47 up-regulation that was not significantly associated with E-cadherin down-regulation (p=0.5734). Slug up-regulation accompanied by E-cadherin down-regulation correlated with the presence of distant metastases (p=0.0029) and with advanced pTNM stages (p=0.0424). A statistically significant association was found between Slug up-regulation and the expression of SIP1 in intestinal (p=0.0014) and Snail in diffuse (p=0.0067) carcinomas. We present the first study integrating the analysis of several EMT regulators in primary gastric carcinomas and conclude that Slug up-regulation is associated with E-cadherin down-regulation in diffuse and intestinal-type gastric carcinoma, and that this effect could be complemented by the presence of other EMT regulators.

Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis.

Owing to its cross-linking effects, it is currently believed that formalin fixation of routinely processed tissues in the clinic prevents protein extraction and profiling. The aim of our study was to develop a robust, fast, standardized, and easy to use technique for the solubilization of non-degraded, full length, and immunoreactive proteins from formalin-fixed tissues for western blot and protein microarray analysis. Sections of routinely processed formalin-fixed and paraffin-embedded tissues of various origin were analysed. After deparaffination, tissues were manually dissected from the slides and transferred into an optimized protein extraction buffer system. Proteins were solubilized and subsequently analysed by western blot and reverse phase protein microarrays. We succeeded in isolating non-degraded, soluble, and immunoreactive proteins from routinely processed formalin-fixed tissues. We were able to detect membrane, cytoplasmic and nuclear proteins at the expected molecular weight. No differences were found in the protein yield and protein abundances between fresh frozen and formalin-fixed tissues. Using western blots and reverse phase protein microarrays, the receptor tyrosine kinase HER2, an important protein target for antibody based cancer treatment, was reliably measured in formalin-fixed breast cancer biopsy samples when compared with measurement by immunohistochemistry and fluorescence in situ hybridization; remarkably, immunohistochemically equivocal cases (score 2+) can be categorized according to HER2 protein abundance. Our new clinically orientated multiplexed protein measurement system may be generally applicable to determine the relative abundances of known disease-related proteins in small amounts of routinely processed formalin-fixed tissue samples for research and diagnosis. This technique may also be used to identify, characterize, and validate known and new protein markers in a variety of human diseases.

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.

The antibody 2B4 directed against the Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) detects MAGE-4: implications for studies on the EBV association of human cancers.

We have previously developed two monoclonal antibodies against the Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1), designated 1H4 and 2B4. Both detect EBNA1 by in situ staining in established EBV-positive tumours, e.g. Hodgkin's lymphoma and nasopharyngeal carcinoma. An association of EBV with other tumours, notably breast carcinomas, has been reported but remains controversial. Using the antibody 2B4, a nuclear protein has been detected in breast carcinomas that were EBV-negative by other methods, suggesting cross-reactivity with a cellular protein. Furthermore, an association of EBV with various other carcinomas has been reported on the basis of 2B4 immunohistochemistry. Here we show that 2B4 also binds to MAGE-4, a cancer testis antigen expressed in a variety of tumour cells, including breast carcinoma, seminoma and EBV-negative cases of Hodgkin's lymphoma. We conclude that the 2B4 antibody is not suitable for the detection of EBV infection but that additional techniques, particularly in situ hybridization for the detection of the EBV-encoded RNAs (EBERs), should be employed to confirm the presence of EBV. Our results add to the evidence indicating that breast cancer is not an EBV-associated disease.

[Molecular carcinogenesis of the upper gastrointestinal tract]. / Molekulare Karzinogenese des oberen Gastrointestinaltraktes.

Carcinomas of the upper gastrointestinal tract have been intensively studied for decades in order to identify markers for a) design of simple blood tests to detect presence or recurrence of the disease, b) prediction of therapy response, c) identification of molecular targets for novel therapies. These aims have not yet been fully reached by analysing single genes. However, some genes, including E-cadherin that is altered in sporadic and familial cases of gastric cancer and interleukin 1-beta whose polymorphisms together with Helicobacter pylori infection are associated with an increased risk for gastric cancer, may have the potential for clinical use. In this review we summarize current data for the single gene approach and provide an overview for recent results from cDNA microarray studies.

Gastric adenocarcinoma: pathomorphology and molecular pathology.

Two types of gastric adenocarcinoma can be distinguished histopathologically: the diffuse and the intestinal type. Molecular pathology supports this theory by showing differences in the genetic pathways of both tumor types. In addition to known pathomorphological factors of prognosis, e.g., depth of tumor infiltration, number of lymph node metastases and resection margins, a few genes have been suggested to have prognostic impact in gastric carcinoma. Clinically relevant molecules whose expression or structure is altered include the plasminogen activator (uPA) and its inhibitor PAI-1 (plasminogen activator inhibitor type 1), the cell cycle regulator cyclin E, epidermal growth factor (EGF), the apoptosis inhibitor bcl-2, the cell adhesion molecule E-cadherin, and the multifunctional protein beta-catenin. Gene amplification and protein overexpression of the growth factor receptors c-erbB-2 and K-sam may be prognostic factors for intestinal-type and diffuse-type gastric cancer, respectively. In addition, genetic instability is commonly seen. There has long been evidence for a genetic predisposition to gastric cancer by epidemiological studies and case reports. Very recently, germ line mutations of E-cadherin have been identified that are responsible for a dominantly inherited form of diffuse-type gastric cancer and could be used to identify individuals that are at high risk.

Highly specific tumor binding of a 213Bi-labeled monoclonal antibody against mutant E-cadherin suggests its usefulness for locoregional alpha-radioimmunotherapy of diffuse-type gastric cancer.

A monoclonal antibody (E-cadherin delta 9-1) directed against a characteristic E-cadherin mutation (in-frame deletion of exon 9), found in diffuse-type gastric cancer but not in any normal tissue, was conjugated with the high linear energy transfer alpha-emitter 213Bi and tested for its binding specificity in s.c. and i.p. nude mice tumor models. After intratumoral application in s.c. tumors expressing mutant E-cadherin, the 213Bi-labeled antibody was specifically retained at the injection site as shown by autoradiography. After injection into the peritoneal cavity, uptake in small i.p. tumor nodules expressing mutant E-cadherin was 17-fold higher than in tumor nodules expressing wild-type E-cadherin (62% injected dose/g versus 3.7% injected dose/g). 78% of the total activity in the ascites fluid was bound to free tumor cells expressing mutant E-cadherin, whereas in control cells, binding was only 18%. The selective binding of the 213Bi-labeled, mutation-specific monoclonal antibody E-cadherin delta 9-1 suggests that it will be successful for alpha-radioimmunotherapy of disseminated tumors after locoregional application.

Single amino acid substitutions in conserved extracellular domains of E-cadherin differ in their functional consequences.

The calcium-dependent homophilic cell adhesion molecule E-cadherin typically connects epithelial cells. The extracellular portion of the mature transmembrane protein consists of five homologous domains. The four sequences linking these domains contain the structural amino acid motif DXXD that is thought to be involved in direct calcium binding. In gastric cancer patients mutations affecting this motif between the second and third domain are frequently seen. In order to determine the functional significance of similar sequence alterations with regard to their location, we analyzed single amino acid substitutions changing the DXXD motif to DXXA in each linker region according to a mutation found in gastric cancer (D370A). The cDNA sequences coding for DQND, DVLD and DVND were changed (D257A, D479A, D590A, respectively) and stably expressed in E-cadherin negative MDA-MB-435S mammary carcinoma cells. We found that the D257A and D370A mutations result in abnormal protein localization, changes in the actin cytoskeleton, markedly reduced homophilic cell adhesion, and altered cell morphology. Unexpectedly, the tumor-associated D370A mutation but not the D257A mutation induced increased cell motility. The D479A mutation only had slight functional consequences whereas cells expressing the D590A mutant did not differ from cells expressing the wild-type molecule. Although the putative calcium binding motif DXXD is located at repetitive positions in the extracellular portion of E-cadherin, our results indicate that it has different functions depending on the location. Remarkably, tumor cells select for mutations in the most critical domains resulting both in loss of function (decreased cell adhesion) and in gain of function (increased cell motility). Since multiple DXXD motifs are typically seen in other cadherins, our structure-function study is relevant for this gene family in general.

Mutant cell surface receptors as targets for individualized cancer diagnosis and therapy.

The catalogue of gene alterations in human cancer is growing rapidly. Alterations in specific genes that play important roles in diverse cellular functions such as cell adhesion, signal transduction, differentiation, development or DNA-repair have been identified. Cancer-associated mutant cell surface molecules are very attractive candidates to target tumor cells because they offer the possibility of minimizing toxic effects to non-tumor cells. The cell adhesion molecule E-cadherin has been shown to play a major role in determining which of the two subtypes of gastric cancer, diffuse or intestinal type, develops. E-cadherin gene mutations typically affect the extracellular portion of the homophilic receptor and are frequently found in patients with diffuse-type tumors. Cancer-specific monoclonal antibodies against the E-cadherin mutational hot spot region are now available. In cell culture and in animal studies we have shown that mutation-specific antibodies exclusively target cells expressing abnormal E-cadherin. Those cells expressing the normal protein were not affected, demonstrating the specificity of our approach. After linking to toxins, drugs or radiolabeled mutation-specific antibodies could serve as very specific agents to treat small tumor deposits. Patients for this novel individualized cancer therapy can be identified within a day using routine immunohistochemistry of biopsies.