[Mass spectrometry-applications in pathology]. / Massenspektrometrie ­ Anwendungsmöglichkeiten in der Pathologie.

BACKGROUND: Currently, more complex and extensive diagnostic pathology work-up of sometimes only limited sample material is necessary to ensure optimal patient treatment. This often includes genomic analyses. However, dynamic changes within an organism or tumor can be better reflected at the protein level. Therefore, proteomic technologies would seem to be the solution. OBJECTIVES: To evaluate the application of different proteomic techniques to analyze body fluids and tissue samples with regards to implementation in diagnostics. MATERIALS AND METHODS: All studies utilized mass spectrometry-based methods in order to achieve differentiation of a number of different patient groups in various diseases. RESULTS: Whereas classical proteomic methods are particularly suitable for analyzing serum samples in order to diagnose bladder cancer or chronic hepatitis C, tissue analyses would require prior tissue lyses, thus erasing possible information to be obtained from histology. Imaging mass spectrometry offers a solution as it allows for the analysis of an intact tissue section. Possible applications and the added benefit of this method could be shown using various examples of tumors (prostate cancer, Hodgkin's lymphoma, cervical cancer, and different types of adenocarcinomas). CONCLUSIONS: Mass spectrometry-based technologies allow diagnostic confirmation with high sensitivity and specificity. In comparison to routine diagnostic approaches, results can be achieved faster, using less sample material, and with comparable accuracy.

[Histopathological research laboratories in translational research : Conception and integration into the infrastructure of pathological institutes]. / Histopathologische Forschungslabors in der translationalen Forschung : Konzeption sowie Integration in die Infrastruktur pathologischer Institute.

A systematic review of histopathology from experimental animal systems is an essential part of up-to-date biomedical research. Pathologists at university hospitals are especially and increasingly challenged by these specialized and time-consuming duties. This article presents and analyzes a new laboratory structure of comparative experimental pathology-jointly lead by veterinary and human pathologists-which might solve this problem. The focus is on the establishment and full integration of this laboratory structure into a local, regional, and nationwide biomedical research cluster. A detailed comparison with an established structure of routine histopathology laboratories discusses merits and benefits as well as disadvantages.

CXCR4: A Potential Marker for Inflammatory Activity in Abdominal Aortic Aneurysm Wall.

OBJECTIVES: The aim of the study was to evaluate the potential role of chemokine receptor CXCR4 and its ligand CXCL12 in the pathogenesis of abdominal aortic aneurysm (AAA). METHODS: AAA tissue specimens were obtained from the anterior or lateral aneurysm sac of patients (n = 32, 26 males, 6 females; 66.8 ± 11.2 years, diameter 64.4 ± 17.0 mm), who underwent elective open surgical repair. Twelve non-aneurysmal aortic specimens from transplant donors served as controls. Expression analysis of CXCR4 and CXCL12 at mRNA and protein level was determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Immunohistochemical staining of corresponding histological sections for CD3 (T-cells), CD20 (B-cells), and CD68 (macrophages) was performed to determine the cellular localization of CXCR4 and CXCL12. Data were analyzed with SPSS 20.0 using Mann-Whitney U test and Spearman's rank correlation coefficient. RESULTS: Gene expression of CXCR4 and CXCL12 was 9.6 and 4.6 fold higher in AAA than in non-aneurysmal aorta (p = .0004 and p < .0001, respectively). Likewise, the protein level of CXCR4 was increased 3.2 fold in AAA wall compared with non-aneurysmal aortic tissue (p < .0001), although CXCL12 could not be detected. Immunohistochemical analysis revealed that CXCR4 was expressed in B and T lymphocytes and macrophages, and CXCL12 was observed only in plasma cells. CONCLUSIONS: This study confirmed the over expression of CXCR4 in human AAA tissue. CXCR4 was detected both at the mRNA and the protein level and by immunohistochemistry, especially in inflammatory cells. In contrast, CXCL12 expression was observed only at the mRNA level, with the exception of plasma cells. The exact role of CXCR4/CXCL12 in AAA has to be further elucidated.

MALDI MS imaging as a powerful tool for investigating synovial tissue.

OBJECTIVE: To identify and image protein biomarker candidates in the synovial tissue of patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). METHODS: A novel matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) technique was applied to the analysis of synovial tissue. Patients were classified according to the American College of Rheumatology (ACR) criteria for RA. Frozen sections were stained to obtain morphological data. Serial sections were desiccated, and spotted with matrix for MALDI analysis. Ions generated by laser irradiation of the tissue were separated in time, based on their m/z ratio, and were subsequently detected. IMS was used in a 'profiling' mode to detect discrete spots for rapid evaluation of proteomic patterns in various tissue compartments. Photomicrographs of the stained tissue images were reviewed by a pathologist. Areas of interest (10 discrete areas/compartment) were marked digitally and the histology-annotated images were merged to form a photomicrograph of the section taken before the MALDI measurement. Pixel coordinates of these areas were transferred to a robotic spotter, the matrix was spotted, and the coordinates of the spots were transferred to a mass spectrometer for spectral acquisition. The data generated were then subjected to biocomputation analysis to reveal the biomarker candidates. RESULTS: Several peaks (m/z) consistent in mass with calgranulins, defensins, and thymosins were detected and their distribution in various synovial compartments (synovial lining and sublining layer) was demonstrated. CONCLUSION: MALDI IMS is a powerful tool for the rapid detection of numerous proteins (in situ proteomics) and was applied here for the analysis of the distribution of proteins in synovial tissue sections.

[Current knowledge in molecular pathology of urothelial cancer]. / Stand des Wissens zur molekularen Pathologie des Urothelkarzinoms.

Results of molecular pathology have supported changes in the 2004 WHO classification of urothelial cancer. Since then new molecular data such as the distribution pattern of the fibroblast growth factor receptor 3 (FGFR3) has further supported the principle of low and high grade entities of urothelial carcinoma. Animal experiments with knockout mice and conditional knockout systems reveal important parallels to humans and results emphasize the cellular context as a trigger for malignancy. One special feature of the urothelium is its high protection of the urothelial cells by members of the retinoblastoma gene family, efficiently inhibiting invasion even in the presence of p53 mutations. In search of the tumor stem cell phenotype the basal cell phenotype is the focus of attention providing a high clonogenic potential. At the same time detailed analysis of the distribution of mutations in the mitochondrial genome within the urothelium will help to gain insight into the spreading of normal cell or tumor cell clones. The overall data in urological oncology provide evidence that diagnostic and prognostic tools for urothelial cancer can only be reached with multiparametric approaches.

[Status of the availability and use of next generation sequencing (NGS) in bladder cancer-a questionnaire within the uropathology working group]. / Status der Verfügbarkeit und Anwendung von "next generation sequencing" (NGS) beim Harnblasenkarzinom ­ eine Umfrage in der Arbeitsgemeinschaft Uropathologie.

BACKGROUND: Technical advancement and availability of high-throughput analysis has advanced molecular subtyping of most cancers. Thus, new possibilities for precision oncology have emerged. AIM: Therefore, we aimed to collect data regarding availability and use of next generation sequencing (NGS) for urothelial cancer within the uropathology working group of the German Society of Pathology. METHODS: We collected data by questionnaires and additionally asked for sequencing results of bladder cancers in the participating institutions. RESULTS: A total of 13 university-affiliated institutes of pathology took part in the survey. All university institutes offer NGS-based molecular panel diagnostics and provide panels covering between 15 and 170 genes. Altogether, only 20 bladder cancers were sequenced in routine diagnostics and for 10 cancers potential targeted treatment options were available. DISCUSSION: So far, despite availability of NGS diagnostics at university institutes of pathology, only few bladder cancer samples have been sequenced. Based on current data from the molecular subtyping of bladder cancers, we recommend a step-by-step protocol with basic immunohistochemistry analysis and subsequent subtype-dependent analyses, e.g., alterations of the fibroblast growth factor receptors (FGFR) or comprehensive gene panel analyses.

Protein profiling of non-malignant and malignant ascites by SELDI-TOF MS: proof of principle.

Ascites is a common clinical symptom in liver cirrhosis, inflammatory disorders of the abdomen and a major late manifestation of metastatic malignancies. Standard cytopathological techniques and immunocytochemistry have specificities and sensitivities of approximately 95 and 60%, respectively for the presence of tumor cells. Development of faster and more accurate screening methods would be of great clinical utility. In this work we examined differential analysis of the unbound proteins in the supernatant of ascites fluid by Protein-Chip SELDI mass spectrometry. There were 21 tumor cell-positive and 34 tumor cell-negative samples. We used principal component analysis coupled with linear regression applied to the mass spectra of the samples to distinguish between the sample groups. Two sample sets for statistical analysis were created after randomization, a training set with 37 samples and a validation set with 18 samples resulting in a specificity of 93% and a sensitivity of 83% on the training set. The validation set yielded a specificity and sensitivity of 75%. This study suggests that SELDI-TOF mass spectrometry appears to have great potential as a surrogate diagnostic tool to evaluate effusion specimens.

[Precancerous lesions of the urothelium. From Feulgen staining to single cell CGH]. / Präkanzerosen des Urothels. Von der Feulgen-Färbung bis zur Einzelzell-CGH.

Feulgen staining represents a staining method to quantitatively document the DNA content of a nucleus. Thus it is an excellent and straightforward method to reflect the irregular increase in DNA content of a malignant cell as a sign of genetic instability. Genetic instability of the tumour cell is the key feature of the 2004 WHO classification of bladder tumours, in which flat and papillary neoplasia are grouped into low- and high-grade lesions. "High grade" represents the tumor with genetic instability and consequently a higher likelihood of progression. Concomitant distinct genetic aberrations other than the numeric ones are increasingly identified as discriminators and help group the entities. The current status of genetic investigations, especially those in precancerous lesions, will be outlined in this review in the context of morphology (histology and cytology) as well as clinical situation.

The Importance of Histology and Pathology in Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) has become a valuable tool in cancer research. Even more, due to its capability to directly link molecular changes with histology, it holds the prospect to revolutionize tissue-based diagnostics. In order to learn to walk before running, however, information obtained through classical histology should not be neglected but rather used to its full capacity and integrated with mass spectrometry data to lead to a superior molecular histology synthesis. In order to achieve this, pathomorphological analyses have to be integrated into MSI analyses right from the beginning to avoid errors and pitfalls of MSI application possibly leading to incorrect or imprecise study outcomes. Such errors can be caused by different sample or tissue inherent factors or through factors in sample preparation. Future studies should, therefore, aim for a comprehensive incorporation of histology and pathology characteristics to ensure the generation of high-quality data in MSI to exploit its full capacity in tissue-based basic and translational research.

Opposing role of Notch1 and Notch2 in a Kras(G12D)-driven murine non-small cell lung cancer model.

Lung cancer is the leading cause of cancer-related deaths worldwide. Recently, we have shown that Notch1 inhibition resulted in substantial cell death of non-small cell lung cancer (NSCLC) cells in vitro. New compounds targeting Notch signal transduction have been developed and are now being tested in clinical trials. However, the tumorigenic role of individual Notch receptors in vivo remains largely unclear. Using a Kras(G12D)-driven endogenous NSCLC mouse model, we analyzed the effect of conditional Notch1 and Notch2 receptor deletion on NSCLC tumorigenesis. Notch1 deficiency led to a reduced early tumor formation and lower activity of MAPK compared with the controls. Unexpectedly, Notch2 deletion resulted in a dramatically increased carcinogenesis and increased MAPK activity. These mice died significantly earlier due to rapidly growing tumor burden. We found that Notch1 regulates Ras/MAPK pathway via HES1-induced repression of the DUSP1 promoter encoding a phosphatase specifically suppressing pERK1/2. Interestingly, Notch1 but not Notch2 ablation leads to decreased HES1 and DUSP1 expression. However, Notch2-depleted tumors showed an appreciable increase in ß-catenin expression, a known activator of HES1 and important lung cancer oncogene. Characteristically for ß-catenin upregulation, we found that the majority of Notch2-deficient tumors revealed an undifferentiated phenotype as determined by their morphology, E-Cadherin and TTF1 expression levels. In addition, these carcinomas showed aggressive growth patterns with bronchus invasion and obstruction. Together, we show that Notch2 mediates differentiation and has tumor suppressor functions during lung carcinogenesis, whereas Notch1 promotes tumor initiation and progression. These data are further supported by immunohistochemical analysis of human NSCLC samples showing loss or downregulation of Notch2 compared with normal lung tissue. In conclusion, this is the first study characterizing the in vivo functions of Notch1 and Notch2 in Kras(G12D)-driven NSCLC tumorigenesis. These data highlight the clinical importance of a thorough understanding of Notch signaling especially with regard to Notch-targeted therapies.

Sticky platelet syndrome: an underrecognized cause of graft dysfunction and thromboembolic complications in renal transplant recipients.

Sticky platelet syndrome (SPS) leads to hyperaggregabilty of platelets in response to physiologic stimuli. In this report we describe three patients with clinical symptoms of SPS after renal transplantation. The first patient developed an infarction of her transplant kidney with additional, subsequent renal microinfarctions. The second patient suffered multiple strokes and deep vein thrombosis with episodes of pulmonary embolism and ischemic bowel disease due to colonic microinfarctions. The third patient experienced a long episode of unexplained respiratory and graft dysfunction immediately after transplantation until therapy for SPS was initiated, at which point symptoms resolved quickly. Kidney transplant recipients with SPS may be at increased risk of developing thrombosis, given that most immunosuppressive drugs are known to induce either endothelial cell damage or augment platelet aggregation. All patients awaiting renal transplantation should be screened for a history of thrombosis and, if appropriate, tested for SPS. Affected patients should receive dose-adjusted acetylsalicylic acid.

Analyzing effects of photodynamic therapy with 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) in urothelial cells using reverse phase protein arrays.

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) is clinically established approach for a number of defined applications. However, in order to optimize the therapeutic benefits of PDT, the specific mode of cell destruction should be better defined. Apoptosis is favored over necrosis for clinical practice as the latter causes more side-effects. In the present study, we analyse PDT-induced cell death and its correlation to various PDT parameters (different doses applied, time after PDT treatment) in vitro using reverse phase protein arrays. Human urothelial cell lines with varying degrees of differentiation (UROtsa, RT4, RT112, J82) were subjected to in vitro-PDT using increasing doses of irradiation. In addition, positive controls for apoptosis, necrosis and un-/specific cellular damage were included. Cells were harvested over a specified time course, lysed and arrayed onto nitrocellulose-covered glass slides. The arrays were analyzed for expression of apoptosis-related proteins by immunohistochemistry. Analysis of caspase-3 and -9 expression, the activation of HIF-1alpha, Bcl2, Cox2 and the phosphorylation of AKT reveals signal activation due to a PDT-stimulus in correlation with the positive controls. Data were analyzed by unsupervised hierarchical clustering and depicted as a heat map revealing cell-specific patterns of pathway stimulation. Higher differentiated phenotypes showed a more distinct signal response in general and a higher apoptotic response in detail. Lower differentiated cell lines lost pathway regulation capabilities according to their state of dedifferentiation. Reverse phase protein arrays are a promising technique for signal pathway profiling: they exceed the range of traditional western blots by sensitivity, high-throughput capability, minimal sample consumption and easy quantification of results obtained.

The histone H1(0) contains multiple sequence elements for nuclear targeting.

We have investigated the nuclear transport of the replacement histone H1(0) and have searched for its nuclear localization sequence (NLS). The lysine-rich H1(0) histone differs from the other H1 histones with respect to its mode of expression and to the processing of the respective mRNA. Using the digitonin-permeabilized cell import assay we demonstrate that H1(0) is transported into the nucleus in an energy- and temperature-dependent manner. In competition experiments we show that the transport of H1(0) from the cytoplasm into the nucleus is competed by the SV40 T-antigen-NLS-peptide coupled to HSA, an established substrate of the importin pathway. In transfection studies we have expressed in HeLa cells a series of plasmid constructs containing different fragments of the coding region of the H1(0) histone gene that were fused to the beta-galactosidase gene, and we have determined the subcellular localization of each fusion protein. The results show that H1(0) contains multiple transport-competent sequence elements that can function as NLS and that H1(0) meets the requirements for a transport into the nucleus by an importin-dependent pathway.

Replacement of invariant bZip residues within the basic region of the yeast transcriptional activator GCN4 can change its DNA binding specificity.

Two residues are invariant in all bZip basic regions: asparagine -18 and arginine -10 (we define the first leucine of the leucine zipper of GCN4 as +1). X-ray structures of two specific GCN4-DNA complexes (Ellenberger et al., Cell, 71, 1223-1237, 1992; König & Richmond, J. Mol. Biol., 233, 139-154, 1993) demonstrate the involvement of both residues in specific base pair recognition. We replaced either asparagine -18 or arginine -10 with all other amino acids and tested the DNA binding properties of the resulting mutant peptides by gel mobility shift assays. Peptides with histidine -18 or tyrosine -10 bind with changed specificities to variants of the ATF/CREB site 5'A4T3G2A1C0*G0'T1'C2'A3'T4'3' with symmetric exchanges in positions 2/2' or 0/0', respectively. The double mutant with histidine -18 and tyrosine -10 combines the features of the parental single mutants and binds specifically to the respective double exchange target. Furthermore, the tyrosine -10 mutant clearly prefers the palindrome 5'ATGATATCAT3' over the corresponding pseudo-palindrome 5'ATGATTCA-T3', whereas the lysine -10 mutant binds better to the pseudo-palindromic AP1 site 5'ATGACTCAT3' than to the palindromic ATF/CREB site. Thus, although invariant within natural bZip proteins, asparagine -18 or arginine -10 can be functionally replaced by other amino acids, and their replacement can lead to new DNA binding specificities.

Phorbol esters and cytokines regulate the expression of the NEMO-related protein, a molecule involved in a NF-kappa B-independent pathway.

The NF-kappaB signaling pathway plays a crucial role in the immune, inflammatory, and apoptotic responses. Recently, we identified the NF-kappaB Essential Modulator (NEMO) as an essential component of this pathway. NEMO is a structural and regulatory subunit of the high molecular kinase complex (IKK) responsible for the phosphorylation of NF-kappaB inhibitors. Data base searching led to the isolation of a cDNA encoding a protein we called NRP (NEMO-related protein), which shows a strong homology to NEMO. Here we show that NRP is present in a novel high molecular weight complex, that contains none of the known members of the IKK complex. Consistently, we could not observe any effect of NRP on NF-kappaB signaling. Nonetheless, we could demonstrate that treatment with phorbol esters induces NRP phosphorylation and decreases its half-life. This phosphorylation event could only be inhibited by K-252a and stauroporin. We also show that de novo expression of NRP can be induced by interferon and tumor necrosis factor alpha and that these two stimuli have a synergistic effect on NRP expression. In addition, we observed that endogenous NRP is associated with the Golgi apparatus. Analogous to NEMO, we find that NRP is associated in a complex with two kinases, suggesting that NRP could play a similar role in another signaling pathway.

The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1.

Import of proteins into the nucleus proceeds through nuclear pore complexes and is largely mediated by nuclear transport receptors of the importin beta family that use direct RanGTP-binding to regulate the interaction with their cargoes. We investigated nuclear import of the linker histone H1 and found that two receptors, importin beta (Impbeta) and importin 7 (Imp7, RanBP7), play a critical role in this process. Individually, the two import receptors bind H1 weakly, but binding is strong for the Impbeta/Imp7 heterodimer. Consistent with this, import of H1 into nuclei of permeabilized mammalian cells requires exogenous Impbeta together with Imp7. Import by the Imp7/Impbeta heterodimer is strictly Ran dependent, the Ran-requiring step most likely being the disassembly of the cargo-receptor complex following translocation into the nucleus. Disassembly is brought about by direct binding of RanGTP to Impbeta and Imp7, whereby the two Ran-binding sites act synergistically. However, whereas an Impbeta/RanGTP interaction appears essential for H1 import, Ran-binding to Imp7 is dispensable. Thus, Imp7 can function in two modes. Its Ran-binding site is essential when operating as an autonomous import receptor, i.e. independently of Impbeta. Within the Impbeta/Imp7 heterodimer, however, Imp7 plays a more passive role than Impbeta and resembles an import adapter.