[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.

[Staging of liver fibrosis in biliary atresia : Comparison of Chevallier and Ishak score as well as automated evaluation]. / Staging der Leberfibrose bei Gallengangatresie : Vergleich von Chevallier- und Ishak-Score sowie automatisierter Auswertung.

BACKGROUND: Biliary atresia (BA) is a rare disease of the newborn, resulting in liver cirrhosis due to obliterative cholangiopathy. Liver biopsies are commonly performed in order to confirm the diagnosis and in order to stage fibrosis. OBJECTIVES: The present study intended to analyze two established scores for evaluating liver fibrosis focusing on the interobserver variability as well as the prognostic reliability towards the time of liver transplantation. MATERIALS AND METHODS: Liver biopsies of BA patients between 2012 and 2015 were evaluated retrospectively by two pathologists at the Hannover Medical School (MHH) and the RWTH Aachen University Hospital. Fibrosis was measured using Ishak and Chevallier scores. Furthermore, a computerized automatically algorithm-based analyzation (ABAA) was performed. Results were evaluated towards the time point of liver transplantation and hepatoportoenterostomy (HPE). RESULTS: Overall, 34 liver biopsies were analyzed. The Ishak score showed a remarkable interobserver variability (ΚW = 0.68) while the Chevallier score was proven to have a poor interobserver variability (Fleiss' Κappa = -0.01). However, both scores were correlated positively, as was the ABAA (p < 0.001). Regarding prognostic reliability, ROC analyses of the Ishak score revealed the best validity towards an early liver transplantation within 12 months (AUC 0.813, p = 0.011). In addition, an increased Ishak score ≥4 reduced the survival time with the native liver (hazard ratio 6.6 [95% CI 1.9-23.3]). CONCLUSIONS: The Ishak score was revealed to have the best interobserver variability as well as prognostic validity towards an early liver transplantation in BA patients. Due to its easy applicability, the Ishak score was proven superior in comparison to the Chevallier score and ABAA. Therefore, use of the Ishak score is recommended in daily clinical routine for analyzing liver biopsies in BA patients.

[Adult autopsies during the past decade in Germany : Data from two university hospitals]. / Erwachsenenobduktionen im letzten Jahrzehnt in Deutschland : Daten zweier Universitätskliniken.

BACKGROUND: The clinical autopsy is the ultimate medical service for a patient and plays a crucial role in the education of physicians and other medical personnel, as well as in the context of quality control. Nevertheless, the number of autopsies is constantly decreasing. Numerous factors, such as the personal attitude of relatives and also clarification of relatives, as well as the increasing application of imaging methods while the patient is still alive, play a central role in this decline. OBJECTIVE: This study aimed to demonstrate the development of autopsy services over the past decade in two university hospitals in Germany and therefore to underline the importance of this investigation procedure in pathology. MATERIAL AND METHODS: Autopsy reports between the years 2005 and 2014 from 2 university institutes of pathology were analyzed regarding a diverse dataset, including age and sex of the deceased as well as the clinical and pathological causes of death. RESULTS: The data showed that the number of autopsies has continuously decreased over the past decade; however, the distribution of characteristics of the deceased remained relatively stable. In this cohort the clinically assumed cause of death differed from the pathological cause of death in 6% of the autopsies. Frequently occurring discrepant diagnoses were cardiac tamponade, aortic dissection and endocarditis/myocarditis. DISCUSSION: Our results show that, despite significant improvements in imaging methods, findings do not yield more accurate results than does autopsy. This underscores once again the need to encourage the performance of this final medical act on patients.

Growth factor-functionalized silk membranes support wound healing in vitro.

Chronic wounds represent a serious problem in daily medical routine requiring improved wound care. Silk of the domesticated silkworm (Bombyx mori) has been used to form a variety of biomaterials for medical applications. We genetically engineered B. mori to produce silk functionalized with growth factors to promote wound healing in vitro. In this study FGF-, EGF-, KGF-, PDGF- or VEGF-functionalized silk membranes were compared to native B. mori silk membranes without growth factors for their ability to support wound healing in vitro. All silk membranes were cytocompatible and supported macrophage secretion of neutrophil recruiting factor CXCL1 and monocyte chemoattractant protein 1 (MCP-1). VEGF-functionalized silk significantly outperformed other growth factor-functionalized silk membranes, but not native silk in angiogenesis assays. In addition, EGF- and VEGF-functionalized silk membranes slightly enhanced macrophage adhesion compared to silk without growth factors. In wound healing assays in vitro (reduction of wound lesion), dermal equivalents showed a higher wound healing capacity when covered with EGF-, FGF- or VEGF-functionalized silk membranes compared to native, KGF- or PDGF-functionalized silk membranes. Keratinocyte migration and growth is overstimulated by KGF- and VEGF-functionalized silk membranes. In conclusion, growth factor-functionalized silk membranes prepared from genetically engineered silk worm glands are promising wound dressings for future wound healing therapies.

Mesenchymal stem cells can be recruited to wounded tissue via hepatocyte growth factor-loaded biomaterials.

Mesenchymal stem cells (MSC) are precursor cells of mesodermal tissue and, because of their trophic phenotype, they are known to play beneficial roles in wound healing. In addition, various tissue engineering strategies are based on MSC/biomaterial constructs. As the isolation and expansion of MSCs is a long-term process, a major goal is to develop an endogenous stem cell recruitment system that circumvents all ex vivo steps generally used for tissue engineering. Therefore collagen and silk fibroin were loaded with hepatocyte growth factor (HGF), a chemoattractant for MSCs. Collagen was mixed with HGF during polymerization, while silk fibroin and HGF were produced as fusion proteins by transgenic silkworms. To demonstrate release of active HGF, enzyme-linked immunosorbent assay, in vitro migration assays and animal studies were performed to demonstrate MSC migration in vivo, followed by detailed examinations of the immunological effects of the biomaterials. Hepatocyte growth factor was released burst-like, both from silk fibroin and collagen during the first 8 h and gradually for up to 168 h in vitro. Directed migration in vitro was demonstrated when MSCs were exposed to HGF. In vivo, HGF-loaded collagen and silk fibroin were tolerated as subcutaneous implants. In addition, it was proved that endogenous MSCs were recruited from the local environment. These results show for the first time recruitment of endogenous MSCs to HGF-loaded collagen (fast degradable) and silk fibroin scaffolds (long-term degradable) in vitro and in vivo. This knowledge could be applied to make off-the-shelf, readily available constructs for use in patients with chronic wound or burns. Copyright © 2016 John Wiley & Sons, Ltd.

[How do metastases of urological tumors develop?]. / Wie entwickeln sich Metastasen urologischer Tumoren?

This overview mainly focuses on the topic of epithelial tumors (carcinomas) because urological tumors are generally of this type. The importance of the topic is reflected by the fact that patients rarely die of the primary tumor, but the majority die of metastases that cause life-threatening situations. More recent findings show that treatment decisions should be based on the metastasis site and less on the tumor's tissue of origin. Given the progression of clinical oncology toward individualized medicine, a better understanding of the biology of metastases is therefore acute and includes some important challenges.

Hypoxia induces the expression of transketolase-like 1 in human colorectal cancer.

BACKGROUND AND AIMS: Transketolase-like (TKTL) 1 is one of the key enzymes for anaerobic sugar degradation even in the presence of oxygen (aerobic glycolysis). Transketolase-dependent reactions supply malignant tumors with ribose and NADPH. Therefore, TKTL1 activity could be crucial for tumor proliferation and survival. The aim of the study was to evaluate the expression of TKTL1 in colorectal cancer (CRC) and its regulation under hypoxic conditions. METHODS: We studied TKTL1 mRNA and protein expression in CRC cell lines and human CRC biopsies by quantitative real-time PCR, Western blotting and immunohistochemistry. Regulation of TKTL1 under oxygen depletion was analyzed by cultivating cells either in a three-dimensional spheroid model or in a hypoxia incubator chamber. RESULTS: TKTL1 mRNA was heterogeneously expressed in monolayers of cells with high levels in HT-29 and SW480. TKTL1 protein was also clearly detectable in HT-29 and SW480. Hypoxia-inducible factor (HIF)-1α protein expression correlated with TKTL1 protein expression in SW480 spheroids over time. On the one hand, induction of hypoxia in T84 spheroids did not induce TKTL1; on the other hand, hypoxia by incubation at 1% O2 in a hypoxia incubator chamber clearly showed an upregulation of TKTL1. In 50% of CRC patients, TKTL1 protein expression was upregulated in tumor compared to non-tumor tissue. The immunohistochemical staining of TKTL1 in CRC patient samples resulted in 14 positive and 30 negative samples. CONCLUSIONS: TKTL1 expression correlated with HIF-1α protein expression and was induced upon hypoxic conditions which could facilitate energy supply to tumors under these circumstances.

[Tumors of Vater's ampulla]. / Tumoren der Papilla Vateri.

Tumors of Vater's ampulla are generally uncommon. In this location intestinal type adenomas are frequently found, followed by noninvasive papillary neoplasms of the pancreaticobiliary type and neuroendocrine tumors (carcinoids). Carcinomas of Vater's ampulla represent about 0.5% of all gastrointestinal malignancies. Intestinal type adenocarcinoma is the most common malignant epithelial tumor followed by the pancreaticobiliary type adenocarcinoma. Highly malignant neuroendocrine carcinomas of Vater's ampulla are very uncommon. Carcinomas of the ampullary region can be sporadic or a component of several disease syndromes. Designation of large carcinomas as tumors with an ampullary or extra-ampullary origin can be difficult but is of relevance for a TNM conform classification. Helpful in the decision are the relationship between the tumor centre and Vater's ampulla, the existence of premalignant lesions in the ampullary epithelium as well as histology and immunostaining of the tumor.

Nuclear transport receptor karyopherin-α2 promotes malignant breast cancer phenotypes in vitro.

Tumorigenesis and tumor progression are associated with dysfunction of the nuclear transport machinery at the level of import and export receptors (karyopherins). Recent studies have shown that the nuclear import factor karyopherin-α2 (KPNA2) is a novel prognostic marker for poor prognosis in human breast cancer. Based on the well-defined hallmarks of cancer progression, we performed a detailed in vitro characterization of the phenotypic effects caused by KPNA2 overexpression and KPNA2 silencing in benign and malignant human breast cells. KPNA2 overexpression clearly increased proliferation of MCF7 tumor cells and further led to a reduction of cell-matrix adhesion in benign MCF10A cells, whereas cell migration was significantly increased (P<0.0001) in both tumor models. Remarkably, these individual effects of KPNA2 overexpression on proliferation, cell-matrix adhesion and migration resulted in an increased colony spreading of benign MCF10A breast cells and malignant MCF7 tumor cells (P<0.001), which is a hallmark of cancer progression. Conversely, RNA interference-mediated KPNA2 silencing caused a complete inhibition of MCF7 tumor cell proliferation and migration (P<0.0001). In addition, in these experiments apoptosis was increased (P<0.05) and formation of tumor cell colonies was reduced (P<0.01). Thus, KPNA2 overexpression provoked increased aggressiveness of malignant MCF7 breast tumor cells and induced a shift in benign MCF10A breast cells toward a malignant breast cancer phenotype. In conclusion, we demonstrate for the first time in experimental tumor models that forced KPNA2 expression drives malignant features relevant for breast cancer progression, while its silencing is required for the remission of those progressive phenotypes. This study gives clear evidence that KPNA2 acts as a novel oncogenic factor in human breast cancer, in vitro.

[Mesenchymal stem cells and their interaction with biomaterials: potential applications in tissue engineering]. / Mesenchymale Stammzellen und ihre Interaktionen mit Biomaterialien: Anwendungsmöglichkeiten im Tissue-Engineering.

INTRODUCTION: Mesenchymal stem cells (MSC) are an important cell type for regenerative medicine and tissue engineering. They are involved in tissue regeneration by means of: (a) differentiation into specialised mesodermal cells and (b) their biosynthetic activity that is both immunomodulatory and trophic. In recent studies we analysed MSC in contact with different biomaterials to identify suitable combinations for tissue engineering. METHODS: A biomaterial test platform was established to analyse cell adhesion, viability, proliferation, cytotoxicity according to ISO 10993-5, apoptosis and differentiation to adipocytes and osteoblasts on a variety of polymers (degradable biopolymers, degradable synthetic polymers, non-degradable synthetic polymers, shape memory polymers, and ceramics). RESULTS: Using this platform, biomaterials which support MSC growth by maintaining their stem cell characteristics and support the differentiation of MSC towards mature osteoblasts were identified. Furthermore, we showed that MSC possess fibrinolytic capacities and perform extracellular matrix remodelling. CONCLUSION: The data support the theory that MSC are involved in tissue regeneration both via their differentiation capacity and their trophic characteristics. We identified different MSC/biomaterial combinations which are suitable for stem cell-based bone tissue engineering.

[Bladder cancer update: what was new at the 2010 annual congress of the German Association of Urology in Düsseldorf?]. / Blasenkarzinom - Update : Was gab es neues auf dem Jahreskongress 2010 der Deutschen Gesellschaft für Urologie in Düsseldorf?

One of the principal objects of the scientific research network "German Bladder Cancer Network" is to consolidate research activities on bladder cancer. An overview about directions of current projects on this research topic was given at the annual meeting of the German Association of Urology in Düsseldorf from September 22 to 25 September 2010. As representatives of the"German Bladder Cancer Network" we summarize and comment on some of the most interesting projects on bladder cancer presented at this meeting. A special focus will be on current developments in the field of uropathology and on different aspects in preclinical research on bladder cancer.

[Characterisation of DNA methylation biomarkers for bladder cancer]. / Charakterisierung von DNA-Methylierungs-Biomarkern für das Harnblasenkarzinom.

Despite considerable advances in recent years in our understanding of the genetic changes occurring in urinary bladder cancer, similar progress in the field of epigenetics has hitherto been lacking. Increasingly, however, focus has shifted in the direction of aberrant DNA methylation as a result of recent studies showing the direct impact of such promoter hypermethylation on the loss of tumor suppressor gene expression and function, therefore potentially affecting tumor genesis and progression. The purpose of this study is the identification and characterization of new DNA methylation markers in urinary bladder cancer, with the expectation that these markers could then be incorporated in a multi-gene panel for clinical use in early cancer detection. In addition, better understanding of the signalling pathways involved will undoubtedly impact the development of new treatment strategies. Potential candidate genes, including the Wnt antagonist SFRP5 among others, will be validated by different epigenetic techniques using invasive and superficial urothelial cell lines as well as tumor and urine samples from bladder cancer patients.

[Mesenchymal stem cells for bone tissue engineering]. / Mesenchymale Stammzellen für das "tissue engineering" des Knochens.

Human mesenchymal stem cells (MSC) represent an attractive option for cell replacement strategies (tissue engineering, TE). TE applications require stability of a stem cell/biomaterial-hybrid via cell migration, matrix-remodelling and differentiation. We focus on these mechanisms in organotypic culture systems for bone TE using MSC from the umbilical cord (UC-MSC) and from bone marrow (BM-MSC). For the organotypic differentiation of MSC into functional osteoblasts, MSC were embedded in a collagenous matrix and subjected to osteogenic differentiation. Under these culture conditions, UC-MSC exceeded BM-MSC in the expression and synthesis of extracellular matrix (ECM) proteins, while BM-MSC show enhanced osteogenic gene upregulation. In both cell types the biosynthetic activity was accompanied by the ultrastructural appearance of hydroxyapatite/calcium crystals. Following secretion of matrix metalloproteinases, both MSC types migrated into and colonised the collagenous matrix causing matrix strengthening and contraction. In conclusion, MSC promise a broad therapeutical application for a variety of connective tissues requiring ECM synthesis and remodelling.

Intermediate filament dynamics and breast cancer: aberrant promoter methylation of the Synemin gene is associated with early tumor relapse.

Synemin (SYNM) is a type IV intermediate filament that has recently been shown to interact with the LIM domain protein zyxin, thereby possibly modulating cell adhesion and cell motility. Owing to this multiplicity of potential functions relevant to cancer development, we initiated a study to decipher SYNM expression and regulation in benign human breast tissue and breast cancer. Dot blot array analysis showed significant SYNM mRNA downregulation in 86% (n=100, P<0.001) of breast cancers compared with their normal tissue counterparts, a result that was confirmed by real-time PCR analysis (n=36, P<0.0001). Immunohistochemistry analysis showed abundant SYNM protein expression in healthy myoepithelial breast cells, whereas SYNM expression loss was evident in 57% (n=37, P<0.001) of breast cancer specimens. Next, we analyzed methylation of the SYNM promoter to clarify whether the SYNM gene can be silenced by epigenetic means. Indeed, methylation-specific PCR analysis showed tumor-specific SYNM promoter methylation in 27% (n=195) of breast cancers. As expected, SYNM promoter methylation was tightly associated (P<0.0001) with SYNM expression loss. In-depth analysis of the SYNM promoter by pyrosequencing showed extensive CpG methylation of DNA elements supposed to regulate gene transcription. Demethylating treatment of SYNM methylated breast cancer cell lines with 5-aza-2-deoxycytidine clearly reestablished the SYNM expression. Statistical analysis of the patient cohort showed a close association between SYNM promoter methylation and unfavorable recurrence-free survival (hazard ratio=2.941, P=0.0282). Furthermore, SYNM methylation positively correlated with lymph node metastases (P=0.0177) and advanced tumor grade (P=0.0275), suggesting that SYNM methylation is associated with aggressive forms of breast cancer. This is the first study on the epigenetic regulation of the SYNM gene in a cancer entity. We provide first hints that SYNM could represent a novel putative breast tumor suppressor gene that is prone to epigenetic silencing. SYNM promoter methylation may become a useful predictive biomarker to stratify breast cancer patients' risk for tumor relapse.

Endogenous myoglobin in human breast cancer is a hallmark of luminal cancer phenotype.

BACKGROUND: We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. METHODS: Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-alpha (ERalpha)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERalpha or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. RESULTS: Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2alpha and CAIX, but not to HIF-1alpha or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERalpha or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. CONCLUSION: We conclude that in breast cancer, Mb is co-expressed with ERalpha and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours.

Different proliferative and survival capacity of CLL-cells in a newly established in vitro model for pseudofollicles.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B-lymphocytes that manifests in a variety of clinical courses. The accumulation of CLL-cells is primarily caused by defective apoptosis; however, a higher proliferative capacity has also been found to correlate with poorer prognostic factors. Proliferating CLL-cells are confined to specialized structures called pseudofollicles, which contain CLL-cells, T-lymphocytes, and stromal cells. We established an in vitro model for pseudofollicles to characterize the behavior of CLL-cells in relation to clinical courses with different outcomes. Only CLL-cells from progressive clinical cases were inducible to proliferate by a combination of soluble CD40L/IL-2/IL-10 in co-culture with stromal cells. Proliferating CLL-cells showed a higher and more extensive expression of antigens, which are important in T-B-cell interactions such as CD40, MHC II, and adhesion molecules. IL-4 increased interferon regulatory factor-4 expression and induced a specific immunophenotype, which may imply plasmacytic differentiation. Furthermore, it was shown that co-cultured stromal cells protected CLL-cells from apoptosis. CLL-cells from clinically indolent cases had a far worse survival rate in medium than the cells from poor prognostic cases. Thus, we can assume that not only a different resistance to apoptosis, but also proliferation contributes to the progression of CLL resulting in bone marrow failure with thrombocytopenia and anemia.

[Urine-based tumour diagnostics for bladder cancer: effects of the new histopathological classification--food for thought]. / Urinbasierte Tumordiagnostik des Harnblasenkarzinoms: Auswirkungen der neuen histopathologischen Klassifikation--ein Denkanstoss.

The new WHO classification of bladder cancer was published in 2004 and consequently cannot be regarded as very recent. However, it is still timely since it picks up considerations affecting other schemes of tumour classification as well. Genetic results are included in the context of morphology, and at the same time a high inter- and intra-observer agreement is striven for as a matter of high quality patient care. The WHO classification of 2004 does not include cytological diagnosis. Thinking about and considering tumour tissue diagnosis, the style of cytological diagnoses is also affected. For tissue diagnoses, low- and high-grade tumours are differentiated from benign lesions including reactive changes. The element of this classification which has to be transferred to cytology is especially the unequivocal diagnosis of high-grade lesions. The low-grade lesion, correlating with tissue of well-differentiated papillary tumours and dysplasias, mostly cannot be distinguished cytologically with certainty from a broad spectrum of non-malignant lesions (papillomas, reactive urothelial detachment in urolithiasis patients, cytology specimen from vigorously irrigated bladders). For the latter group our aim should be to establish an additional diagnostic tool of high quality driven by clinical questions (e.g. potential of tumour progression).

[Virilizing adrenal ganglioneuroma : A rare differential diagnosis in testosterone secreting adrenal tumours]. / Virilisierendes Ganglioneurom der Nebenniere : Eine seltene Differenzialdiagnose testosteronproduzierender Nebennierentumoren.

Testosterone secreting tumours of the adrenal glands are usually adrenal carcinomas or adenomas. Here we report the rare case of an adrenal ganglioneuroma with ectopic Leydig cells, a so-called virilizing adrenal ganglioneuroma. Clinically it is characterized by symptoms of virilization, histologically by the occurrence of a population of eosinophilic cells. In the absence of crystalloids of Reinke this cell population can be identified as Leydig cells based on positive immunohistochemical staining of inhibin and calretinin.

Long-term survival and bipotent terminal differentiation of human mesenchymal stem cells (hMSC) in combination with a commercially available three-dimensional collagen scaffold.

Researchers working in the field of tissue engineering ideally combine autologous cells and biocompatible scaffolds to replace defect tissues/organs. Due to their differentiation capacity, mesenchym-derived stem cells, such as human mesenchymal stem cells (hMSC), are a promising autologous cell source for the treatment of human diseases. As natural precursors for mesenchymal tissues, hMSC are particularly suitable for bone, cartilage, and adipose tissue replacement. In this study a detailed histological and ultrastructural analysis of long-term cultured and terminally differentiated hMSC on 3D collagen scaffolds was performed. Standardized 2D differentiation protocols for hMSC into adipocytes and osteoblasts were adapted for long-term 3D in vitro cultures in porous collagen matrices. After a 50-day culture period, large numbers of mature adipocytes and osteoblasts were clearly identifiable within the scaffolds. The adipocytes exhibited membrane free lipid vacuoles. The osteoblasts were arranged in close association with hydroxyapatite crystals, which were deposited on the surrounding fibers. The collagen matrix was remodeled and adopted a contracted and curved form. Human MSC survive long-term culture within these scaffolds and could be terminally differentiated into adipocytes and osteoblasts. Thus, the combination of hMSC and this particular collagen scaffold is a possible candidate for bone and adipose tissue replacement strategies.

[Novel prognostic marker in invasive breast cancer. ITIH5 expression is abrogated by aberrant promoter methylation]. / Neuer Prognosemarker beim invasiven Mammakarzinom. ITIH5-Expression wird über aberrante Promotormethylierung inaktiviert.

We have recently characterized ITIH5 as a new extracellular matrix protein that exhibits clear expression loss in a variety of human tumour entities, including breast cancer. The aim of the present study was to decipher the molecular cause of ITIH5 expression loss in breast cancer and to learn more about the possible role of this molecule in cancer diseases. ITIH5 protein expression was found to be strongly reduced in 42% of invasive breast carcinomas-interestingly, with significant association with poor patient outcome. ITIH5 promoter methylation was frequently detected in breast cell lines and in primary carcinomas (40%), and it was functionally correlated with loss of ITIH5 mRNA expression. Moreover, ITIH5 promoter methylation was also significantly associated with poor clinical patient outcome and also with the occurrence of lymph node and distant metastases. In conclusion, we propose that ITIH5 may represent a novel metastasis repressor in human breast cancer. Both ITIH5 protein expression and ITIH5 promoter methylation may serve as prognostic biomarkers, thereby helping improve clinical patient outcome.