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Highlights • Growth factor signaling causes sustained nuclear ERK1/2 activation. • The SCR and BCR/ABL inhibitor dasatinib blocks ERK1/2 and represses cell invasion. • EGF-stimulated cells may escape dasatinib inhibition of invasion through mesenchymal to amoeboid transition. • Combined inhibition of SRC and Rho-kinase signaling is necessary to completely block EGF-induced invasion., Aberrantly activated kinase signaling pathways drive invasion and dissemination in medulloblastoma (MB). A majority of tumor-promoting kinase signaling pathways feed into the mitogen-activated protein kinase (MAPK) extracellular regulated kinase (ERK1/2) pathway. The activation status of ERK1/2 during invasion of MB cells is not known and its implication in invasion control unclear. We established a synthetic kinase activation relocation sensor (SKARS) for the MAPK ERK1/2 pathway in MB cells for real-time measuring of drug response. We used 3D invasion assays and organotypic cerebellum slice culture to test drug effects in a physiologically relevant tissue environment. We found that hepatocyte growth factor (HGF), epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF) caused rapid nuclear ERK1/2 activation in MB cells, which persisted for several hours. Concomitant treatment with the BCR/ABL kinase inhibitor dasatinib completely repressed nuclear ERK1/2 activity induced by HGF and EGF but not by bFGF. Increased nuclear ERK1/2 activity correlated positively with speed of invasion. Dasatinib blocked ERK-associated invasion in the majority of cells, but we also observed fast-invading cells with low ERK1/2 activity. These ERK1/2-low, fast-moving cells displayed a rounded morphology, while ERK-high fast-moving cells displayed a mesenchymal morphology. Dasatinib effectively blocked EGF-induced proliferation while it only moderately repressed tissue invasion, indicating that a subset of cells may evade invasion repression by dasatinib through non-mesenchymal motility. Thus, growth factor-induced nuclear activation of ERK1/2 is associated with mesenchymal motility and proliferation in MB cells and can be blocked with the BCR/ABL kinase inhibitor dasatinib.

Zusammenfassung. Zystische und solide Veränderungen der Nieren sind in der Ultraschalldiagnostik häufig. Der solide Pseudotumor der Niere, der sog. Nierenparenchymzapfen, wird in bis 50 % der Patienten gefunden. Pathologisch-anatomisch handelt es sich entweder um einer Hypertrophie der Columna Bertini oder um einen ganzen Renkulus, der in den Sinus renalis hineingeruscht ist. Nierenzysten wurden in einem Sektionsgut bei 50 % der über 50-Jährigen gefunden. Die zystischen Veränderungen werden mit der Bosniak-Klassifikation in fünf Kategorien unterteilt. Diese Klassifikation ist mit CECT, CEMR und CEUS möglich. Die soliden Veränderungen werden ebenso mit diesen Verfahren beurteilt, die Unterscheidung ist hier jedoch schwieriger. Mit Messung der Echodichte im Ultraschall gelingt die Differenzierung der echoreichen Angiomyolipome von übrigen soliden Tumoren und Pseudotumoren. In der farbkodierten Doppler-Sonografie (FKDS) wird oft das klarzellige Nierenzellkarzinom mit vielen Tumorgefässen dargestellt, die übrigen Tumoren mit wenigen oder nur einzelnen Gefässen. In CEUS und TIC wird das klarzellige Nierenzellkarzinom als sehr stark perfundiert dargestellt, die Anflutung in der TIC ist oft schneller und stärker als in der umliegenden gesunden Nierenrinde. Die übrigen Tumoren sind meist schwächer perfundiert, besonders die papillären Karzinome sind deutlich schwächer als die Nierenrinde.

The composition of the plasma membrane (PM)-associated proteome of tumor cells determines cell-cell and cell-matrix interactions and the response to environmental cues. Whether the PM-associated proteome impacts the phenotype of Medulloblastoma (MB) tumor cells and how it adapts in response to growth factor cues is poorly understood. Using a spatial proteomics approach, we observed that hepatocyte growth factor (HGF)-induced activation of the receptor tyrosine kinase c-MET in MB cells changes the abundance of transmembrane and membrane-associated proteins. The depletion of MAP4K4, a pro-migratory effector kinase downstream of c-MET, leads to a specific decrease of the adhesion and immunomodulatory receptor CD155 and of components of the fast-endophilin-mediated endocytosis (FEME) machinery in the PM-associated proteome of HGF-activated MB cells. The decreased surface expression of CD155 or of the FEME effector Endophilin A1 reduces growth and invasiveness of MB tumor cells in the tissue context. These data thus describe a novel function of MAP4K4 in the control of the PM-associated proteome of tumor cells and identified two downstream effector mechanisms controlling proliferation and invasiveness of MB cells.Graphical abstractc-MET activation upon HGF stimulation induces c-MET internalization and induces downstream MAP4K4 activity. (1) MAP4K4 is required downstream of activated c-MET for the maintenance of surface presentation of CD155 in activated cells. CD155 expression is required for MB cell migration, invasion and proliferation in the tissue context. (2) MAP4K4 is required downstream of activated c-MET to maintain membrane depolarization, possibly by regulating the surface localization of several ion channels and transporters. (3) MAP4K4 is required downstream of activated c-MET cause PM-proximal localization of FEME effector CIP4, FBP17 and CIN85. The FEME effector endophilin A is necessary for MB cell migration, invasion and dissemination.

Medulloblastoma (MB) is the most frequent malignant brain tumor in children and it is subgrouped into 4 entities (SHH, WNT, Group 3, and Group 4). Molecular pathways involved in these different subgroups still are evolving and can be of clinical relevance to therapy. The YAP1-CTGF axis is known to regulate cell proliferation, differentiation, and cell death; however, its role in MB is poorly explored. We aimed to investigate the role of YAP1 gene in the MB SHH cell line DAOY and evaluate cell proliferation, doubling time and 3D spheroids invasion and its consequence on CTGF regulation. We assessed CTGF expression from 22 children with MB. Lastly, we validated our findings through in silico analysis in large cohorts dataset of patients. We observed an increased invasion rate of DAOY cells and CTGF downregulation under YAP1 knockdown (p

The metastatic potential of tumors depends in part on their response to growth factors available in the tumor microenvironment. We addressed how micro-environmental cues control tumor cell invasion in pediatric medulloblastoma (MB). We show that bFGF promotes MB tumor cell invasion through FGF receptor (FGFR) in vitro and that pharmacological blockade of FGFR represses brain tissue infiltration in vivo. TGF-ß regulates pro-migratory bFGF function in a context-dependent manner. Under low bFGF, the non-canonical TGF-ß pathway causes ROCK activation and cortical translocation of ERK1/2, which antagonizes FGFR signaling by inactivating FGFR substrate 2 (FRS2), and promotes a contractile, non-motile phenotype. Under high bFGF, negative feedback regulation of FRS2 by bFGF-induced ERK1/2 causes repression of the FGFR pathway. Under these conditions, TGF-ß counters inactivation of FRS2 and restores pro-invasive signaling [1]. These findings pinpoint coincidence detection of bFGF and TGF-ß signaling by FRS2 as a mechanism that controls tumor cell invasion and identified targeting of FRS2 as a rational strategy to abrogate aberrant FGFR signaling independent of kinase inhibition. Using computer-assisted compound discovery, we screened chemical libraries for potential small molecule inhibitors of the interaction between FGFR and FRS2. Using functional testing, we selected potential hits that block invasion comparable to current small molecule FGFR kinase inhibitors and that bind FRS2 in the relevant domain. Thus, our study provides first evidence of rationally discovered, precisely acting small molecule interaction inhibitors with the potential to effectively target pro-invasive signaling in MB and other FGFR-driven cancers. 1. Santhana Kumar K et al. Cell Rep. 2018; 23: 3798–812

Next-generation sequencing platforms are routinely used for molecular assignment due to their high impact for risk stratification and prognosis in medulloblastomas. Yet, low and middle-income countries still lack an accurate cost-effective platform to perform this allocation. TaqMan Low Density array (TLDA) assay was performed using a set of 20 genes in 92 medulloblastoma samples. The same methodology was assessed in silico using microarray data for 763 medulloblastoma samples from the GSE85217 study, which performed MB classification by a robust integrative method (Transcriptional, Methylation and cytogenetic profile). Furthermore, we validated in 11 MBs samples our proposed method by Methylation Array 450 K to assess methylation profile along with 390 MB samples (GSE109381) and copy number variations. TLDA with only 20 genes accurately assigned MB samples into WNT, SHH, Group 3 and Group 4 using Pearson distance with the average-linkage algorithm and showed concordance with molecular assignment provided by Methylation Array 450 k. Similarly, we tested this simplified set of gene signatures in 763 MB samples and we were able to recapitulate molecular assignment with an accuracy of 99.1% (SHH), 94.29% (WNT), 92.36% (Group 3) and 95.40% (Group 4), against 97.31, 97.14, 88.89 and 97.24% (respectively) with the Ward.D2 algorithm. t-SNE analysis revealed a high level of concordance (k = 4) with minor overlapping features between Group 3 and Group 4. Finally, we condensed the number of genes to 6 without significantly losing accuracy in classifying samples into SHH, WNT and non-SHH/non-WNT subgroups. Additionally, we found a relatively high frequency of WNT subgroup in our cohort, which requires further epidemiological studies. TLDA is a rapid, simple and cost-effective assay for classifying MB in low/middle income countries. A simplified method using six genes and restricting the final stratification into SHH, WNT and non-SHH/non-WNT appears to be a very interesting approach for rapid clinical decision-making. Electronic supplementary material The online version of this article (10.1186/s40478-019-0681-y) contains supplementary material, which is available to authorized users.

Introduction When laparoscopic radical prostatectomy (LRP) was introduced as a novel treatment option for prostate cancer, it had to compete with the established open techniques. The short- and intermediate-term oncologic and functional outcomes were encouraging and comparable to those with retropubic radical prostatectomy. However, the long-term oncologic safety for LRP has yet to be fully elucidated. We evaluated the long-term oncologic outcomes of an initial series of patients who had undergone LRP. Patients and Methods An initial unselected and consecutive series of 100 patients who had undergone LRP for clinically localized prostate cancer from 1999 to 2001 was identified. The pre-, intra-, and postoperative data were collected. Biochemical recurrence (BCR) was defined as a prostate-specific antigen (PSA) value of ≥ 0.2 ng/mL. The outcome measures were cancer control (CC), BCR-free survival (BCRFS), cancer-specific survival (CSS), and overall survival (OS). Results The mean patient age was 64 ± 7 years, and the mean preoperative PSA level was 9.6 ± 8.3 ng/mL. Of the 100 patients, 79 (79%) had stage pT2 and 15 (15%) had stage pT3 disease. Positive surgical margins were found in 25 patients (25%; 16.4% for pT2 and 40% for pT3). The median follow-up time was 126 months (range, 60-176 months). The 5-year CC rate was 82%. The estimated 10-year BCRFS was 83% and 80% for patients with stage pT2 and pT3 tumors, respectively. The median time to BCR was 52 months (range, 6-144 months). The estimated 10-year CSS and OS was 98% and 93%, respectively. Conclusion Our long-term follow-up data from an initial unselected patient cohort have indicated that LRP offers excellent long-term oncologic control for patients with localized prostate cancer.

Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system (CNS) tumors and greatly influence mortality and therapy-induced long-term morbidity in survivors. A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes, to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness. These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments. However, the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel, physiologically more relevant assay systems including organotypic brain slice culture. In this review, using publicly available peer-reviewed primary research and review articles, we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors. Furthermore, we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors. We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors and facilitate the development of more efficacious anti-metastatic treatments.

The molecular mechanisms contributing to distant dissemination and local recurrence of medulloblastoma, the most common malignant brain tumor in childhood, are poorly understood and no targeted anti-invasion therapies exist till date. We explored regulators and effectors of MAP4K4, a pro-invasive kinase overexpressed in MB and associated with metastatic progression in different solid malignancies. MAP4K4 is upregulated both at mRNA and protein levels in primary pediatric brain tumors compared to normal cerebellum. MAP4K4 is required for growth factor- and irradiation-induced migration and invasion of medulloblastoma cells. It furthermore promotes turnover and activation of the receptor tyrosine kinase c-Met and of the ß1 integrin adhesion receptor 1. To characterize these clinically relevant consequences and to identify druggable targets of MAP4K4 function, we profiled the interactome of MAP4K4 in starved and growth factor stimulated medulloblastoma cells. To systematically address MAP4K4 impact on receptor expression and turnover, we determined the MAP4K4-dependent surface proteome in medulloblastoma cells. We found that MAP4K4 is part of the striatin-interacting phosphatase and kinase (STRIPAK) complex and that STRIPAK component striatin 4 is controlling cell motility and invasiveness in medulloblastoma cells. Invasiveness of medulloblastoma cells is abrogated by a truncation mutant of MAP4K4 lacking the striatin 4 interaction domain. We furthermore found that MAP4K4 mediates growth factor-induced surface expression of solute carriers and immunomodulatory proteins involved in chemoresistance and immune evasion. Thus, our study identified MAP4K4 as a missing link between pro-tumorigenic growth factor signaling and tumor cell functions relevant for disease progression. It may help identifying druggable vulnerabilities in medulloblastoma cells to restrict tumor growth and dissemination. 1. Tripolitsioti, D. et al., Oncotarget9, 23220–23236 (2018).

Medulloblastoma (MB) is a paediatric cancer of the cerebellum that can develop cerebellar and leptomeningeal metastases. Local brain tissue infiltration, the underlying cause of metastasis and relapse, remains unexplored. We developed a novel approach to investigate tissue infiltration of MB using organotypic cerebellum slice culture (OCSC). We show that cellular and structural components of cerebellar tissue in OCSCs are maintained for up to 30 days ex vivo, and that OCSCs foster tumour growth and cell proliferation. Using cell-based models of sonic hedgehog (SHH) and group 3 (G3) MB, we quantified tumour growth and infiltration and determined the morphological characteristics of the infiltrating cells. We observed basal levels of dissemination occurring in both subgroups with cells migrating either individually or collectively as clusters. Collective cerebellar tissue infiltration of SHH MB cells was further enhanced by EGF but not HGF, demonstrating differential tumour cell responses to microenvironmental cues. We found G3 cells to be hyper proliferative and observed aggressive tumour expansion even in the absence of exogenous growth factors. Our study thus provides unprecedented insights into brain tissue infiltration of SHH and G3 MB cells and reveals the cellular basis of the tumour progressing functions of EGF in SHH MB.

Local tissue infiltration of Medulloblastoma (MB) tumor cells precedes metastatic disease but little is still known about intrinsic regulation of migration and invasion in these cells. We found that MAP4K4, a pro-migratory Ser/Thr kinase, is overexpressed in 30% of primary MB tumors and that increased expression is particularly associated with the frequently metastatic SHH β subtype. MAP4K4 is a driver of migration and invasion downstream of c-Met, which is transcriptionally up-regulated in SHH MB. Consistently, depletion of MAP4K4 in MB tumor cells restricts HGF-driven matrix invasion in vitro and brain tissue infiltration ex vivo. We show that these pro-migratory functions of MAP4K4 involve the activation of the integrin β-1 adhesion receptor and are associated with increased endocytic uptake. The consequent enhanced recycling of c-Met caused by MAP4K4 results in the accumulation of activated c-Met in cytosolic vesicles, which is required for sustained signaling and downstream pathway activation. The parallel increase of c-Met and MAP4K4 expression in SHH MB could predict an increased potential of these tumors to infiltrate brain tissue and cause metastatic disease. Molecular targeting of the underlying accelerated endocytosis and receptor recycling could represent a novel approach to block pro-migratory effector functions of MAP4K4 in metastatic cancers.

Medulloblastoma (MB) is a paediatric cancer of the cerebellum that can develop cerebellar and leptomeningeal metastases. Local brain tissue infiltration, the underlying cause of metastasis and relapse, remains unexplored. We developed a novel approach to investigate tissue infiltration of MB using organotypic cerebellum slice culture (OCSC). We show that cellular and structural components of cerebellar tissue in OCSCs are maintained for up to 30 days ex vivo, and that OCSCs foster tumour growth and cell proliferation. Using cell-based models of sonic hedgehog (SHH) and group 3 (G3) MB, we quantified tumour growth and infiltration and determined the morphological characteristics of the infiltrating cells. We observed basal levels of dissemination occurring in both subgroups with cells migrating either individually or collectively as clusters. Collective cerebellar tissue infiltration of SHH MB cells was further enhanced by EGF but not HGF, demonstrating differential tumour cell responses to microenvironmental cues. We found G3 cells to be hyper proliferative and observed aggressive tumour expansion even in the absence of exogenous growth factors. Our study thus provides unprecedented insights into brain tissue infiltration of SHH and G3 MB cells and reveals the cellular basis of the tumour progressing functions of EGF in SHH MB.

The search for novel targeted therapies for major human conditions such as diabetes, cardiovascular diseases and cancer is a slow and costly process. Progress is often hampered by poor drug efficaciousness in the patients, low selectivity/specificity of the compounds and cellular evasion mechanism that are rather common in anti-cancer therapies. This is particularly true also for compounds inhibiting kinases, which in theory are optimal targets thanks to their druggable enzymatic activity. Novel targeting strategies are needed to reduce side effects and treatment failure caused by non-specific drug function and target resistance, respectively. An ideal compound will repress the relevant kinase effector function, while leaving kinase functions that are not disease-relevant unaltered. To achieve function-specific inhibition, the molecular mechanism of the drug target that governs the pathological process, must be identified. The Ser/Thr kinase MAP4K4 is implicated in inflammatory and metabolic disorders and cancer progression. In this review, we describe the molecular effector functions of MAP4K4 that exert those activities and how they have been identified and characterized both in invertebrate organisms and mammals. We discuss how the modulation of the cellular cytoskeleton by MAP4K4 may be connected to pathological conditions such as aberrant angiogenesis and cancer metastasis, and we describe the molecular mechanisms that are so far known to be mechanistically involved in these processes.

Embryonal tumors of the central nervous system represent a heterogeneous group of childhood cancers with an unknown pathogenesis; diagnosis, on the basis of histological appearance alone, is controversial and patients’ response to therapy is difficult to predict. They encompass medulloblastoma, atypical teratoid/rhabdoid tumors and a group of primitive neuroectodermal tumors. All are aggressive tumors with the tendency to disseminate throughout the central nervous system. The large amount of genomic and molecular data generated over the last 5–10 years encourages optimism that new molecular targets will soon improve outcomes. Recent neurobiological studies have uncovered the key role of microRNAs (miRNAs) in embryonal tumors biology and their potential use as biomarkers is increasingly being recognized and investigated. However the successful use of microRNAs as reliable biomarkers for the detection and management of pediatric brain tumors represents a substantial challenge. This review debates the importance of miRNAs in the biology of central nervous systemembryonal tumors focusing on medulloblastoma and atypical teratoid/rhabdoid tumors and highlights the advantages as well as the limitations of their prospective application as biomarkers and candidates for molecular therapeutic targets.

Embryonal tumors of the nervous system are the leading cause of childhood cancer-related morbidity and mortality. Medulloblastoma, supratentorial primitive neuroectodermal tumors, atypical teratoid/rhabdoid tumor and neuroblastoma account for more than 20% of childhood malignancies and typify the current neural embryonal tumor model in pediatric oncology. Mechanisms driving the formation of these tumors point towards impaired differentiation of neuronal and neuron-associated cells during the development of the nervous system as an important factor. The importance of microRNAs (miRNAs) for proper embryonic cell function has been confirmed and their aberrant expressions have been linked to tumor development. The role of miRNAs in controlling essential regulators of key pathways implicated in tumor development makes their use in diagnostics a powerful tool to be used for early detection of cancer, risk assessment and prognosis, as well as for the design of innovative therapeutic strategies. In this review we focus on the significance of miRNAs involved in the biology of embryonal neural tumors, delineate their clinical significance and discuss their potential as a novel therapeutic target.

Background: microRNA-9 is a key regulator of neuronal development aberrantly expressed in brain malignancies, including medulloblastoma. The mechanisms by which microRNA-9 contributes to medulloblastoma pathogenesis remain unclear, and factors that regulate this process have not been delineated. Methods: Expression and methylation status of microRNA-9 in medulloblastoma cell lines and primary samples were analysed. The association of microRNA-9 expression with medulloblastoma patients' clinical outcome was assessed, and the impact of microRNA-9 restoration was functionally validated in medulloblastoma cells. Results: microRNA-9 expression is repressed in a large subset of MB samples compared with normal fetal cerebellum. Low microRNA-9 expression correlates significantly with the diagnosis of unfavourable histopathological variants and with poor clinical outcome. microRNA-9 silencing occurs via cancer-specific CpG island hypermethylation. HES1 was identified as a direct target of microRNA-9 in medulloblastoma, and restoration of microRNA-9 was shown to trigger cell cycle arrest, to inhibit clonal growth and to promote medulloblastoma cell differentiation. Conclusions: microRNA-9 is a methylation-silenced tumour suppressor that could be a potential candidate predictive marker for poor prognosis of medulloblastoma. Loss of microRNA-9 may confer a proliferative advantage to tumour cells, and it could possibly contribute to disease pathogenesis. Thus, re-expression of microRNA-9 may constitute a novel epigenetic regulation strategy against medulloblastoma.

UNLABELLED Study Type - Therapy (case series). Level of Evidence 4. What's known on the subject? and What does the study add? Low-risk prostate cancer is frequently diagnosed in the context of PSA screening or during a routine check-up. For those patients, to avoid possible overtreatment AS is an increasingly chosen treatment option. However, the concept of AS could possibly misclassify potentially dangerous PCa as a low-risk disease resulting in inferior cancer control outcomes. In the present study, we could demonstrate that the histopathological results of patients treated by RP in course of AS are significantly better if the selection criteria for AS are entirely fulfilled. Our findings underline the importance of a strict and precise admittance procedure for patients with early prostate cancer who are willing to undergo an AS programme. OBJECTIVE • To compare the histopathological outcomes of patients treated with radical prostatectomy (RP) after an initial active surveillance (AS) for localized, low-risk prostate cancers (PCa) among men who fulfilled the Epstein criteria at diagnosis with those who did not. PATIENTS AND METHODS • In all, 283 patients with localized PCa were initially managed at our institution with AS. • In all, ≈ 50% originated from the European Randomized Study of Screening for Prostate Cancer (ERSPC) participants from Switzerland: 75 (26.5%) patients underwent treatment during follow-up and 61 were treated with RP (21.6%). • These patients were stratified into those who did (n= 39) vs those who did not (n= 22) entirely fulfil AS inclusion criteria according to Epstein et al. at PCa diagnosis. RESULTS • Patients who did completely fulfil the AS inclusion criteria had significantly lower prostate-specific antigen (PSA)-values (4.9 vs 7.8 ng/mL; P= 0.02), a significantly lower PSA density at diagnosis (0.09 vs 0.2 ng/mL/ccm; P= 0.007) and at RP, a higher proportion of organ-confined cancers (89.7% vs 59.1%, P= 0.02) and fewer positive surgical margins (25.6% vs 40.9%). • However, the rate of favourable histopathological outcome, defined as organ-confined disease with negative surgical margins, was statistically significantly higher in the group fulfilling AS criteria (69.2% vs 40.9%; P= 0.03). CONCLUSIONS • In our AS series, 26.5% of the patients underwent definitive therapy. • Most patients treated with RP had organ-confined disease in the majority of cases, especially when the Epstein criteria were rigorously fulfilled at PCa diagnosis. • This underlines the importance of a strict and precise per protocol AS for patients with early PCa, otherwise there is a risk of missing more significant disease.

Theileria annulata is an intracellular protozoan parasite that infects B cells and macrophages of ruminants. Macrophages infected with T. annulata are de-differentiated and display tumour cell properties and a metastatic behaviour. How parasitized cells adapt their morphology, motility and invasive behaviour has not yet been addressed in detail. In this study, I investigated the regulation of host cell actin dynamics in T. annulata-transformed macrophages and how this affects host cell morphology and motility. T. annulata was found to promote the formation of filamentous-actin-rich podosome-type adhesions (PTAs) and lamellipodia, and to establish a polarized morphology of the infected cell. Characteristic for parasite-dependent host cell polarization is that infected cells display a single, persistent lamellipodium. Src kinases--in particular Hck--are required for the polar extension of this lamellipodium. Hck does so by promoting the clustered assembly of PTAs and accumulation of proteins of the Ezrin, Radixin, Moesin (ERM) family in lamellipodia. Polar accumulation of PTAs and ERM proteins correlates with focal matrix degradation underneath lamellipodia. These findings suggest that T. annulata equips its host cell with properties to adhere and invade. These properties are likely to promote the motile behaviour required for dissemination of infected cells in vivo.

Previous studies have noted an adverse relationship between implant malalignment during total knee arthroplasty (TKA) and post-operative pain. Although some evidence exists indicating that computer-assisted surgical navigation for TKA can improve the accuracy of component alignment, its impact on clinical outcomes is currently unknown. The dual goals of the present cohort/nested case-control study were to (1) compare self-reported responses to the Western Ontario-McMaster Osteoarthritis Index (WOMAC) questionnaire between computer-assisted TKA (123 patients) using the imageless PiGalileo navigation system and conventional TKA (207 patients) [cohort analysis], and (2) to investigate a potential association between malalignment and post-operative pain in 19 painful knees and 19 asymptomatic knees obtained from the cohort analysis using matched sampling [nested case-control study]. In the cohort analysis, a relevant but non-significant (P = 0.06) difference in the occurrence of chronic pain was observed between the navigated (12%) and conventional arms (20%). Median post-operative WOMAC pain score was 100 (range, 50-100) in the conventional group and 100 (range, 65-100) in the navigated group. However, the Mann-Whitney test revealed a significant difference in favor of the navigated group (P = 0.01). In the nested case-control analysis, radiological outcomes and computer tomography (CT) measurements of femoral rotation were compared between the groups. The CT rotation measurements yielded evidence of a relationship between post-operative pain and incorrect rotational alignment of the femoral component of more than 3 degrees (OR: 7; 95% CI: 1.2-42; P = .033). In conclusion, there was no clinical benefit to computer-assisted navigation; however, a statistically significant relationship was observed between incorrect rotational alignment of the femoral component and symptoms of post-operative pain following TKA.

Background To date, there is still a paucity of data in the literature on robotic-assisted laparoscopic extended pelvic lymph node dissection (RALEPLND) in patients with prostate cancer. Objective To assess the technical feasibility of RALEPLND and to present our surgical technique. Design, setting, and participants From April 2006 to March 2008, we performed RALEPLND in 99 patients prior to robotic-assisted laparoscopic radical prostatectomy. Indications for RALEPLND were a prostate-specific antigen (PSA) ≥10 ng/ml or a preoperative Gleason score ≥7. The data were evaluated retrospectively. Surgical procedure The transperitoneal approach was used in all cases. In order to gain optimal access to the common iliac bifurcation, the five trocars were placed in a more cephalad position than in patients undergoing radical prostatectomy without RALEPLND. After identification of important landmarks, the lymphatics covering the external iliac vein, the obturator lymphatic packet, and the lymphatics overlying the internal iliac artery were removed on both sides. Measurements The total lymph node yield, the frequency of lymph node metastases, and the complication rate. Results and limitations The median patient age was 64 yr (range: 45–78). The median preoperative PSA level was 7.7 ng/ml (range: 1.5–84.6). The median number of lymph nodes harvested was 19 (range: 8–53). In 16 patients (16%), we found lymph node metastasis. Complications occurred in seven patients (7%). Conclusions RALEPLND is feasible, and its lymph node yield is well in the range of open series. The robotic-assisted laparoscopic approach in itself does not seem to limit a surgeon's ability to perform a complete extended pelvic lymph node dissection.

High-throughput analysis of cancer cell dissemination and its control by extrinsic and intrinsic cellular factors is hampered by the lack of adequate and efficient analytical tools for quantifying cell motility. Oncology research would greatly benefit from such a methodology that allows to rapidly determine the motile behaviour of cancer cells under different environmental conditions, including inside three-dimensional matrices. We combined automated microscopy imaging of two- and three-dimensional cell cultures with computational image analysis into a single assay platform for studying cell dissemination in high-throughput. We have validated this new approach for medulloblastoma, a metastatic paediatric brain tumour, in combination with the activation of growth factor signalling pathways with established pro-migratory functions. The platform enabled the detection of primary tumour and patient-derived xenograft cell sensitivity to growth factor-dependent motility and dissemination and identified tumour subgroup-specific responses to selected growth factors of excellent diagnostic value.

Aim: Medulloblastoma (MB) is the most common malignant brain tumor in children. The crucial role of extracellular-microRNAs (ex-miRNAs) in cancer has been widely recognized; however, their role in MB remains unknown. This study aimed to investigate MB-driven ex-miRNAs. Methods: Microarray analysis was used to disclose the identity and quantity of key miRNAs excreted in culture-medium (CM) of 3 human MB cell lines and cerebrospinal fluid (CSF) of brain tumors (including MB) and leukemia patients. MiRNA expression was validated by quantitative reverse transcription polymerase chain reaction. Results: We have demonstrated that the 3 MB cell lines tested commonly expressed 1,083 miRNAs in their spent CM. Among them, 57 miRNAs were specific to the CM of metastasis-related cell lines which represents the aggressive group 3 and group 4 MB subtypes. A significant number (1,254) of ex-miRNAs were identified in the CSF of a MB patient. Eighty-six of these miRNAs were found to be differentially expressed in this patient's CSF compared with controls. Interestingly, 3 metastasis-associated miRNAs over-represented in CM of metastasis-related MB cell lines were found to be significantly enriched in the CSF of the MB patient. Conclusion: Although more samples are required to fully verify these results, our work provides the first evidence for the presence of a significant amount of miRNAs excreted extracellularly by MB cells and raises the possibility that, in the near future, miRNAs could be probed in CSF of MB patients and serve as novel biological markers.

How pediatric medulloblastoma (MB), a metastatic tumor of the cerebellum, infiltrates the brain tissue prior to recurrence or distant spreading is poorly understood. We hypothesized that growth factors released from the tumor microenvironment trigger tumor cell migration and brain tissue infiltration. Using a medium-throughput in vitro 3D cell migration screen and ex vivo organotypic cerebellum slice culture, we identified and validated novel potential drug targets for preventing brain tissue infiltration and growth of MB. We found that the FGFR1 ligand bFGF, which we detected in the tumor microenvironment of clinical samples and in organotypic cultures, promoted tumor cell infiltration through the adaptor protein FRS2. Interestingly, FRS2 function was antagonized by TGFβ, which induced a contractile, non-motile cell phenotype through ROCK activation and direct FRS2 repression. In the absence of TGFβ, however, bFGF-FRS2 function attenuated ROCK and enabled motility and invasiveness through Rac-PAK and ERK1/2. In contrast, at high concentrations of bFGF as observed in certain areas of MB tumor tissue, TGFβ rescued tumor cell motility impeded by a FGFR1-dependent negative feed-back and triggered invasiveness. Using organotypic cerebellum slice culture, we demonstrated that pharmacological inhibition of FGFR or genetic ablation of FRS2 prevented infiltration and proliferation of MB cells. Using a computer-assisted molecular design approach, we are currently exploring low molecular weight compounds that might selectively block FRS2 function. Together, we describe an antagonistic crosstalk between bFGF and TGFβ signaling pathways in MB that tunes the motile and invasive properties of the tumor cells for brain infiltration under varying environmental conditions. Furthermore, we identified the FRS2 adaptor protein as a novel and promising drug target in this process.

Medulloblastoma (MB), the most common pediatric malignant brain cancer, typically arises as pathological result of deregulated developmental pathways, including the NOTCH signaling cascade. Unlike the evidence supporting a role for NOTCH receptors in MB development, the pathological functions of NOTCH ligands remain largely unexplored. By examining the expression in large cohorts of MB primary tumors, and in established in vitro MB models, this research study demonstrates that MB cells bear abnormal levels of distinct NOTCH ligands. We explored the potential association between NOTCH ligands and the clinical outcome of MB patients, and investigated the rational of inhibiting NOTCH signaling by targeting specific ligands to ultimately provide therapeutic benefits in MB. The research revealed a significant over-expression of ligand JAG1 in the vast majority of MBs, and proved that JAG1 mediates pro-proliferative signals via activation of NOTCH2 receptor and induction of HES1 expression, thus representing an attractive therapeutic target. Furthermore, we could identify a clinically relevant association between ligand JAG2 and the oncogene MYC, specific for MYC-driven Group 3 MB cases. We describe for the first time a mechanistic link between the oncogene MYC and NOTCH pathway in MB, by identifying JAG2 as MYC target, and by showing that MB cells acquire induced expression of JAG2 through MYC-induced transcriptional activation. Finally, the positive correlation of MYC and JAG2 also with aggressive anaplastic tumors and highly metastatic MB stages suggested that high JAG2 expression may be useful as additional marker to identify aggressive MBs.

Theileria is an intracellular parasite that causes lymphoproliferative disorders in cattle, and infection of leucocytes induces a transformed phenotype similar to tumour cells, but the mechanisms by which the parasite induces this phenotype are not understood. Here, we show that infected B lymphocytes display constitutive phosphoinositide 3-kinase (PI3-K) activity, which appears to be necessary for proliferation, but not survival. Importantly, we demonstrate that one mechanism by which PI3-K mediates the proliferation of infected B lymphocytes is through the induction of a granulocyte-monocyte colony-stimulating factor (GM-CSF)-dependent autocrine loop. PI3-K induction of GM-CSF appears to be at the transcriptional level and, consistently, we demonstrate that PI3-K is also involved in the constitutive induction of AP-1 and NF-kappaB, which characterizes Theileria-infected leucocytes. Taken together, our results highlight a novel strategy exploited by the intracellular parasite Theileria to induce continued proliferation of its host leucocyte.

serum level in prostate cancer patients before treatment is associated with poor prognosis. Moreover, there is accumulating evidences that Gleason 7 prostate cancer shows heterogeneous behavior with Gleason 3 4 and 4 3 tumors conferring different prostate cancer specific mortality. The aim of this observational study was to characterize the aggressiveness of the prostate cancer assessed by the predominant Gleason pattern (pGP), in relation to preoperative testosterone serum concentration. METHODS: 786 patients, referred to our Department (from 03/2007 to 09/2012) for a radical prostatectomy, had preoperative TT and Bioavailable Testosterone (samplings between 7 and 10 am) serum determinations (radio-immuno assayed in a central laboratory). GS and pGP was determined in prostate biopsies and prostate tissue specimens. RESULTS: In prostate specimens, a GS7 was observed in 538 (68.4%) of the patients; 195 (24.8%) had a pGP4. Comparing the 195 patients with a pGP4 to the 343 with a pGP3, TT was lower (4.23 1.63 vs. 4.73 1.70 ng/mL, p 0.0009) and PSA higher (10.2 vs. 8.0 ng/mL, p 0.0001). Hypogonadism was more frequent (26% vs. 9%, p 0.0003). Extra prostatic extension was more frequently observed (22.5% versus 10.5%, p 0.0002). The 80 patients with TT 3.0 ng/mL were bigger ( 8kg, p 0.0001) with a BMI highier ( 2.7 kg/m, p 0.0001). They had a higher percent of GS with a pGP4: 55.0% vs. 33.0% (p 0.0002). CONCLUSIONS: The pGP in prostate specimens is of paramount importance, especially in case of a Gleason 7, to appreciate the outcomes and to choose the treatment. In cancer Gleason 7 in radical prostatectomy specimens, pGP4 is more frequent in hypogonadal patients. Preoperative testosterone should be used to improve the management of prostate cancer.

INTRODUCTION AND OBJECTIVES: The ERSPC indicates prostate biopsy at the PSA cut-off value 3.0 ng/ml. All men with a PSA below 3ng/ml at baseline are reinvited 4 years later. Whether this somewhat arbitrary interval adequately reflects the natural history of PCa is under scientific scrutiny at the moment. The current study describes the outcome of men who had an initial PSA 3.0 ng/ml in the Swiss section of the ERSPC cohort. METHODS: Our study cohort consisted of n 4332 men (55-70 years of age). Re-screening took place every 4 years. In case of PSA-values 3ng/ml, prostate biopsies were recommended. PCa cases were identified at rescreens every 4 years (screen detected PCa) or as interval cancers (through opportunistic PSA testing), which were detected through linkage with the pathology cancer registry. RESULTS: The PSA-distribution at baseline was as follows: n 587 (13.6%) ranged between 2 to 2.99ng/ml, n 1437 (33.2%)from 1 to 1.99ng/ml and n 2308 (53.3%) from 0 to 0.99ng/ml, respectively. Overall, n 342 PCa cases were identified in the study cohort (median follow-up 10.2 years). In the 8-year period after the initial screening (comprising three screening rounds), the PCa rate including interval cancers of the very low PSA-group (0-0.99ng/ml) was 1.6% (n 36). If only PCa with a Gleason Score (GS) higher than 3 3 6 were counted, the rate was 0.69% (n 16 for GS 3 4) and 0.30% (n 7 for GS 4 3), respectively. In the 1–1.99 group, during 4 years (comprising two screening rounds), 59 PCa were diagnosed including interval cancers (4.1%). Of those, only n 12 (0.83%) had a GS of 3 4 and n 5 (0.34%) had a GS of 4 3. Men with an initial PSA between 2.0-2.99ng/ml were found to have a clearly higher PCa rate during 4 years of n 89 (15.1%) of which 19 (3.2%) had a GS of 3 4 and 7 (1.2%) with a GS of 4 3. With increasing PSA ranges, only 2 (0.09%), 4 (0.28%) and 2 (0.17%) men, respectively, died from PCa. CONCLUSIONS: The incidence of clinically relevant PCa (defined by GS) and PCa specific mortality was very low during the follow-up of 10.2 years. We therefore suggest a “PSApyramid” with re-test intervals of 6-8 years at pyramid basis (0-0.99ng/ml). At center part (1.0-1.99ng/ml), a re-test should be done after 3-4 years. Men at the top level (2–2.99 ng/ml) are considered to undergo PSA testing 1–2 years. PSA-pyramid could serve as an efficient gate-keeper reassuring most of men for long time period and allowing to decrease substantially the number of PSA tests.

Objective To assess anxiety levels and health-related quality of life in partners of patients with prostate cancer (PCa) on active surveillance. Methods For low-risk PCa, active surveillance is frequently chosen as a monitoring strategy. Active surveillance has been shown to be associated with low anxiety levels and a fair health-related quality of life in patients. However, little is known about the impact on their partners. We hypothesized that the latter suffer more from PCa diagnosis than the men themselves. Therefore, between February and August 2010, 133 couples—a response rate of 46.9%—completed a written questionnaire at their individual time lags from PCa diagnosis. A Wilcoxon test was performed to assess how distress levels affected the couples' quality of life. Binary logistic regression was used to determine factors affecting distress levels. Results The mean age was 66.2 years in partners and 69.3 in men. At the time quartiles, partners had anxiety scores of 5.5, 4.6, 5.4, and 5.6. Scores in men were statistically significantly lower: 3.9 ( P = .05), 2.0 ( P P = .002), and 3.3 ( P = .02), respectively. However, the partners' scores were still well below 7 (ie, normal). Prostate-specific anxiety scores were below the clinical threshold as well: 15.5, 9.5, 6.5, and 9.0, respectively. Conclusion Active surveillance preserves an encouragingly high health-related quality of life in both men on active surveillance and their partners. Fortunately, the more adverse values of the partners are well within the normal range and thus clinically not relevant.

Purpose: To analyze rates of expression of karyopherin alpha 2 (KPNA2) in different prostate tissues and to evaluate the prognostic properties for patients with primary prostate cancer. Experimental Design: Tissue microarrays (TMA) contained 798 formalin-fixed, paraffin-embedded prostate tissue cores from two different institutes of pathology. TMAs were stained immunohistochemically for KPNA2 and NBS1. SiRNA technologies were used to inhibit KPNA2 expression in vitro, and the effect of this inhibition on cellular viability was determined. Efficiency of knockdown experiments was determined by Western blot analysis. Results: KPNA2 expression was significantly upregulated in carcinomas of the prostate, especially in metastatic and castration-resistant prostate cancer samples. Positive nuclear KPNA2 immunoreactivity was identified as a novel predictor of biochemical recurrence after radical prostatectomy (n = 348), and was independent of the well-established predictive factors preoperative PSA value, Gleason score, tumor stage, and surgical margin status. These results were validated by analyzing a second and independent prostate cancer cohort (n = 330). Further, in vitro experiments showed that the cell proliferation and viability of PC3 cells was significantly reduced when KPNA2 expression was inhibited. KPNA2 knockdown did not induce PARP cleavage as marker for apoptosis. No significantly increased sub-G1 fraction could be found by FACS analysis. Conclusions: KPNA2 is a novel independent prognostic marker for disease progression after radical prostatectomy. This allows to identify patients who need more aggressive treatment. It can moreover be speculated that patients not suited for surveillance regimens might be identified at initial biopsy by a positive KPNA2 immunohistochemistry. Clin Cancer Res; 17(5); 1111–21. ©2011 AACR.

Photoselective vaporization of the Prostate (PVP) using the 80W-Greenlight-PV(R) Laser System (Laserscope(R), San Jose, USA) has been established as a treatment option for patients suffering from obstructive symptoms caused by benign prostatic hyperplasia. However, longer operation time compared to standard trans-urethral resection of the prostate (TURP) and the high costs of the laser fibers are specific problems of this technique. In addition, many clinicians performing PVP complain about a reduced effectiveness of vaporization during treatment. Therefore, power measurement was performed during PVP using the 80W-Greenlight-PV(R) Laser System. Power output was measured at the beginning and also regularly throughout the operation. A total of 40 fibers were investigated in 35 patients. Damage to the tip of the fibers was regularly visible and increased as more energy was supplied. Additionally, in 90% of all fibers a decrease of power output was detectable during the operation. This became pronounced after the application of 200kJ, resulting in an end of lifespan (i.e. 275kJ) median power output of only 20% of the starting value. Corresponding to the clinical observations the impressive damage to the emission window was associated with a substantial decrease of power output during PVP. These observations might explain the impaired vaporization during PVP and a longer operation time compared to conventional TURP. Hence, improvements in the quality of the laser fibers are necessary to advance the efficiency of this promising technology.

Background The potassium-titanyl-phosphate (KTP) laser technique for photo-selective vaporisation of the prostate (PVP) has been regularly improved over the last decade. Nonetheless, decreasing efficiency of tissue vaporisation during the course of the operation and macroscopic alterations of the laser fibre's tip are regularly observed and seem to affect the outcome of this procedure. Objective To investigate the course of power output and to determine the type and extent of fibre deterioration during PVP. Design, Setting, and Participants Forty laser fibres were investigated during PVP in 35 consecutive patients with prostatic bladder outflow obstruction between January 2007 and August 2007 in a university hospital. Intervention All patients underwent PVP performed by three different surgeons using the 80-W KTP laser. Measurements Power output was measured at the beginning and regularly throughout PVP and throughout in vitro vaporisation without fibre–tissue contact. Microscopic documentation of the fibre tip was performed after the procedure. Results and Limitations Carbonisation and melting of the fibre tip was regularly visible and appeared to be more pronounced as more energy was applied. Additionally, 90% of the fibres showed a significant decrease of power output during PVP, resulting in an end-of-lifespan (ie, 275-kilojoule) median power output of 20% of the initial value. Final median power output after in vitro vaporisation was 83% of the starting value. The extent of the structural and functional changes might only be valid for the operative technique performed in this investigation. Conclusions Fibre deterioration caused significant reduction of power output during PVP. This finding is an explanation for the often observed decreasing efficiency of tissue ablation and may also be responsible for some of the typical drawbacks and complications of PVP. Hence, improvements in fibre quality are necessary to advance the efficiency of this technique.

Cell dissemination from an initial site of growth is a highly coordinated and controlled process that depends on cell motility. The mechanistic principles that orchestrate cell motility, namely cell shape control, traction and force generation, are highly conserved between cells of different origins. Correspondingly, the molecular mechanisms that regulate these critical aspects of migrating cells are likely functionally conserved too. Thus, cell motility deregulation of unrelated pathogenesis could be caused and maintained by similar mechanistic principles. One such motility deregulation disorder is the leukoproliferative cattle disease Tropical Theileriosis, which is caused by the intracellular, protozoan parasite Theileria annulata. T. annulata transforms its host cell and promotes the dissemination of parasite-infected cells throughout the body of the host. An analogous condition with a fundamentally different pathogenesis is metastatic cancer, where oncogenically transformed cells disseminate from the primary tumor to form distant metastases. Common to both diseases is the dissemination of motile cells from the original site. However, unlike metastatic cancer, host cell transformation by Theileria parasites can be reverted by drug treatment and cell signaling be analyzed under transformed and non-transformed conditions. We have used this reversible transformation model and investigated parasite control of host cell motile properties in the context of inflammatory signaling in Ma M. et al. [PLoS Pathog (2014) 10: e1004003]. We found that parasite infection promotes the production of the inflammatory cytokine TNFα in the host macrophage. We demonstrated that increased TNFα triggers motile and invasive properties by enhancing actin cytoskeleton remodeling and cell motility through the ser/thr kinase MAP4K4. We concluded that inflammatory conditions resulting in increased TNFα could facilitate cell dissemination by activating the actin cytoskeleton regulatory kinase MAP4K4. We discuss here the relevance of TNFα-MAP4K4 signaling for pathogen-driven cell dissemination and its potential impact on the induction of metastasis in human cancer.