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Several systematic reviews and meta-analyses have summarized the association between sedentary behavior (SB) and cancer. However, the level of evidence and the potential for risk of bias remains unclear. This umbrella review summarized the current data on SB in relation to cancer incidence and mortality, with a particular emphasis on assessing the risk of bias. We searched PubMed, Web of Science and Cochrane Database for systematic reviews and meta-analyses on the association between SB and cancer incidence and mortality. We also searched for recent observational studies not yet included in existing meta-analyses. We re-calculated summary risk estimates for cancer incidence and mortality using random effects models. We included 14 meta-analyses covering 17 different cancer sites from 77 original studies. We found that high SB levels increase the risk for developing ovarian, endometrial, colon, breast, prostate, and rectal cancers, with relative risks of 1.29 (95% confidence interval (CI) = 1.08–1.56), 1.29 (95% CI = 1.16–1.45), 1.25 (95% CI = 1.16–1.33), 1.08 (95% CI = 1.04–1.11), 1.08 (95% CI = 1.00–1.17), and 1.07 (95% CI = 1.01–1.12), respectively. Also, we found an increased risk of cancer mortality of 1.18 (95% CI = 1.09–1.26). Most associations between SB and specific cancer sites were supported by a “suggestive” level of evidence. High levels of SB are associated with increased risk of several types of cancer and increased cancer mortality risk.

Background Breast cancer has been associated with activation of the WNT signaling pathway, although no driver mutations in WNT genes have been found yet. Instead, a high expression of the alternative WNT receptor ROR2 was observed, in particular in breast cancer brain metastases. However, its respective ligand and downstream signaling in this context remained unknown. Methods We modulated the expression of ROR2 in human breast cancer cells and characterized their gene and protein expression by RNA-Seq, qRT-PCR, immunoblots and reverse phase protein array (RPPA) combined with network analyses to understand the molecular basis of ROR2 signaling in breast cancer. Using co-immunoprecipitations, we verified the interaction of ROR2 with the identified ligand, WNT11. The functional consequences of WNT11/ROR2 signaling for tumor cell aggressiveness were assessed by microscopy, impedance sensing as well as viability and invasion assays. To evaluate the translational significance of our findings, we performed gene set enrichment, expression and survival analyses on human breast cancer brain metastases. Results We found ROR2 to be highly expressed in aggressive breast tumors and associated with worse metastasis-free survival. ROR2 overexpression induced a BRCAness-like phenotype in a cell-context specific manner and rendered cells resistant to PARP inhibition. High levels of ROR2 were furthermore associated with defects in cell morphology and cell-cell-contacts leading to increased tumor invasiveness. On a molecular level, ROR2 overexpression upregulated several non-canonical WNT ligands, in particular WNT11. Co-immunoprecipitation confirmed that WNT11 indeed interacts with the cysteine-rich domain of ROR2 and triggers its invasion-promoting signaling via RHO/ROCK. Knockdown of WNT11 reversed the pro-invasive phenotype and the cellular changes in ROR2-overexpressing cells. Conclusions Taken together, our study revealed a novel auto-stimulatory loop in which ROR2 triggers the expression of its own ligand, WNT11, resulting in enhanced tumor invasion associated with breast cancer metastasis.

219 HIV-negative adults ≤70 years with primary CNS lymphoma (PCNSL) were enrolled in the randomized IELSG32 trial. Enrolled patients were randomly assigned to receive methotrexate-cytarabine (arm A), or methotrexate-cytarabine-rituximab (B), or methotrexate-cytarabine-thiotepa-rituximab (MATRix; arm C). A second randomization allocated patients with responsive/stable disease to whole-brain irradiation (WBRT) or carmustine-thiotepa-conditioned autologous transplantation (ASCT). First results, after a median follow-up of 30 months, showed that MATRix significantly improves outcome, with both WBRT and ASCT being similarly effective. However, sound assessment of overall survival (OS), efficacy of salvage therapy, late complications, secondary tumors, and cognitive impairment requires longer follow-up. Herein, we report the results of this trial at a median follow-up of 88 months. As main findings, MATRix was associated with excellent long-lasting outcome, with a 7-year OS of 21%, 37%, and 56% respectively for arms A, B, and C. Notably, patients treated with MATRix and consolidation had a 7-year OS of 70%. The superiority of arm B on arm A suggests a benefit from the addition of rituximab. Comparable efficacy of WBRT and ASCT was confirmed. Salvage therapy was ineffective; benefit was recorded only in patients with late relapse re-treated with methotrexate. Eight (4%) patients developed a second cancer. Importantly, MATRix and ASCT did not result in higher non-relapse mortality or second tumors incidence. Patients who received WBRT experienced impairment in attentiveness and executive functions, whereas patients undergoing ASCT experienced improvement in these functions as well as in memory and quality of life.

Recent updates in the classification of central nervous system (CNS) tumors have increased the need for molecular testing. Assessment of multiple alterations in parallel, complex combinations of gene sequence and chromosomal changes, as well as therapy prediction by identification of actionable mutations are the major challenges. We here report on a customized next generation sequencing (NGS)-based DNA panel assay that combines diagnostic and predictive testing and -as a comprehensive approach- allows for simultaneous single nucleotide variant (SNP) / small insertion/deletion (InDel), copy number variation (CNV) and loss of heterozygosity (LOH) detection. We analyzed formalin-fixed and paraffin-embedded (FFPE) DNA from a total of 104 patients with CNS tumors. After amplicon capture-based library preparation, sequencing was performed on the relatively cost-efficient Illiumina MiniSeq platform and evaluated with freely available bioinformatical tools. 57 genes for exonic SNP/InDel calling (19 of those in intronic regions for CNV analysis), 3 chromosomal arms and 4 entire chromosomes for CNV and LOH analysis were covered. Results were extensively validated. Our approach yielded high accuracy, sensitivity and specificity. It led to refined diagnoses in a relevant number of analyzed cases, reliably enabled complex subclassifications (e.g. for medulloblastomas) and identified actionable targets for clinical use. Thus, our single-platform approach is an efficient and powerful tool to comprehensively support molecular testing in neurooncology. Future functionality is guaranteed as novel upcoming biomarkers can be easily incorporated in a modular panel design.

The macro-metastasis/organ parenchyma interface (MMPI) was previously considered an inert anatomical border which sharply separates the affected organ parenchyma from the macro-metastatic tissue. Recently, infiltrative growth of macro-metastases from various primary tumors was described in the brain, liver and lung, with significant impact on survival. Strikingly, the MMPI patterns differed between entities, so that at least nine different patterns were described. The MMPI patterns could be further classified into three major groups: displacing, epithelial and diffuse infiltrating. Additionally, macro-metastases are a source of further tumor cell dissemination in the affected organ; and these intra-organ metastatic dissemination tracks starting from the MMPI also vary depending on the anatomical structures of the colonized organ and influence disease outcome. In spite of their relevance, MMPIs and organ-specific dissemination tracks are still largely overlooked by many clinicians, pathologists and/or researchers. In this review, we aim to address this important issue and enhance our current understanding of the different MMPI patterns and dissemination tracks in the brain, liver and lung.

Summary: Melanoma is the deadliest skin cancer due to its high rate of metastasis, frequently to the brain. Brain metastases are incurable; therefore, understanding melanoma brain metastasis is of great clinical importance. We used a mouse model of spontaneous melanoma brain metastasis to study the interactions of melanomas with the brain microenvironment. We find that CXCL10 is upregulated in metastasis-associated astrocytes in mice and humans and is functionally important for the chemoattraction of melanoma cells. Moreover, CXCR3, the receptor for CXCL10, is upregulated in brain-tropic melanoma cells. Targeting melanoma expression of CXCR3 by nanoparticle-mediated siRNA delivery or by shRNA transduction inhibits melanoma cell migration and attenuates brain metastasis in vivo. These findings suggest that the instigation of pro-inflammatory signaling in astrocytes is hijacked by brain-metastasizing tumor cells to promote their metastatic capacity and that the CXCL10-CXCR3 axis may be a potential therapeutic target for the prevention of melanoma brain metastasis. : Melanoma brain metastases are incurable. Doron et al. find that astrocyte-secreted CXCL10 is functional in melanoma chemoattraction to the brain. CXCR3, the CXCL10 receptor, is upregulated in brain-seeking melanoma cells. Silencing CXCR3 expression attenuates brain metastasis, suggesting that the CXCL10-CXCR3 axis may be a therapeutic target for melanoma brain metastasis. Keywords: melanoma, brain metastasis, astrocytes, CXCL10, CXCR3, brain tropism

Brain metastases (BMs) are the most frequent malignancy of the central nervous system. Previous research suggested that some metastases show infiltrative behavior rather than sharp demarcation. We hypothesized that three magnetic resonance (MR) imaging parameters—(a) tumor size, (b) extent of peritumoral edema, and (c) presence of multiple BMs—are predictors of cellular invasion beyond the surgically identifiable tumor margins. We performed a post hoc analysis on prospectively collected data of patients with BMs. Biopsies beyond the resection margin and immunohistochemistry were performed to assess infiltration status. The three MR imaging parameters were dichotomized into diameters ≤ 30 mm (“small”) and > 30 mm (“large”), amount of peritumoral edema “extended” and “limited,” and “multiple BMs” and “single BMs,” respectively. The association between infiltration status and imaging parameters was calculated using chi-square test. Biopsy beyond the resection margin was performed in 77 patients; 49 (63.6%) had supramarginal infiltration and 28 patients (36.4%) showed no infiltration. Histological evidence of tumor infiltration was found in 25/41 patients with smaller lesions (61%) and in 24/36 with larger lesions (66.7%, p = 0.64), in 28/44 patients with limited (63.6%) and in 21/33 patients with extended edema (63.6%, p = 1.0), in 28/45 patients (62.2%) with single BM and in 21/32 patients (65.6%) with multiple BMs (p = 0.81). Based on the post hoc analysis of our prospective trial data, we could not confirm the hypothesis that infiltration of brain parenchyma beyond the glial pseudocapsule is associated with the MR imaging parameters tumor size, extent of edema, or multiplicity of metastases.

Brain metastases are more common than primary CNS tumors and confer grave prognosis on patients, as existing treatments have very limited efficacy. The tumor microenvironment has a central role in facilitating tumorigenesis and metastasis. In recent years, there has been much progress in our understanding of the functional role of the brain metastatic microenvironment. In this review, we discuss the latest advances in brain metastasis research, with special emphasis on the role of the brain microenvironment and neuroinflammation, integrating insights from comparable findings in neuropathologies and primary CNS tumors. In addition, we overview findings on the formation of a hospitable metastatic niche and point out the major gaps in knowledge toward developing new therapeutics that will cotarget the stromal compartment in an effort to improve the treatment and prevention of brain metastases.

Metabolic pathways regulate immune responses and disrupted metabolism leads to immune dysfunction and disease. Coronavirus disease 2019 (COVID-19) is driven by imbalanced immune responses, yet the role of immunometabolism in COVID-19 pathogenesis remains unclear. By investigating 87 patients with confirmed SARS-CoV-2 infection, 6 critically ill non���COVID-19 patients, and 47 uninfected controls, we found an immunometabolic dysregulation in patients with progressed COVID-19. Specifically, T cells, monocytes, and granulocytes exhibited increased mitochondrial mass, yet only T cells accumulated intracellular reactive oxygen species (ROS), were metabolically quiescent, and showed a disrupted mitochondrial architecture. During recovery, T cell ROS decreased to match the uninfected controls. Transcriptionally, T cells from severe/critical COVID-19 patients showed an induction of ROS-responsive genes as well as genes related to mitochondrial function and the basigin network. Basigin (CD147) ligands cyclophilin A and the SARS-CoV-2 spike protein triggered ROS production in T cells in vitro. In line with this, only PCR-positive patients showed increased ROS levels. Dexamethasone treatment resulted in a downregulation of ROS in vitro and T cells from dexamethasone-treated patients exhibited low ROS and basigin levels. This was reflected by changes in the transcriptional landscape. Our findings provide evidence of an immunometabolic dysregulation in COVID-19 that can be mitigated by dexamethasone treatment.

Fatigue is a common symptom in patients with rheumatoid arthritis (RA) and in patients with cancer (CA). The aim was to investigate the degree of fatigue in RA patients as compared to CA patients as well as potential influencing factors on RA-related fatigue. This was a retrospective analyses of two prospective cohort studies that used the EORTC QLQ-FA12 as a common instrument to assess fatigue. The cohort of RA patients was based on a nationwide survey in Germany. The cohort of CA patients was recruited in the context of an international validation field study. Multivariable ANCOVAs compared levels of fatigue between the two cohorts, also including various subgroup analyses. Regression analyses explored influencing factors on RA patients’ fatigue. Data of n = 705 RA patients and of n = 943 CA patients were available for analyses. RA patients reported significantly higher Physical Fatigue (mean difference = 7.0, 95% CI 4.2–9.7, p p p = 0.001). No differences in Emotional Fatigue (p = 0.678) and Interference with Daily Life (p = 0.098) were found. In RA patients, mental health and pain were associated with fatigue (p values

The diagnosis of cancer leads to high levels of emotional distress in many patients. Quality of life is an important therapeutic goal in this context. A quality-of-life guide was implemented in the oncological day clinic (ICT) at the University Hospital of Regensburg (UKR) in order to individually support outpatients, help them with their questions and needs and improve their quality of life.A screening tool is necessary for the structured assessment of quality of life/needs in the routine of tumor therapy and follow-up. As part of a mixed-methods study, focus groups with health professionals/patients were organized to specify the needs of cancer patients. On this basis, the literature was searched for questionnaires covering these needs in order to adapt an ICT-specific questionnaire and integrate it with the help of a workflow.A total of 333 individual aspects were brought up by the participants in focus groups on the needs of cancer patients in various phases of treatment/with various tumor entities. Since none of the questionnaires identified in the literature met our requirements, a new screening tool containing elements from different standardized forms and the results of the focus groups was developed and a new workflow created to integrate the questionnaire into the ICT routine.By interviewing health experts from different areas and patients with different tumor entities, the needs of cancer patients over different stages of the disease and additional possible differences between the cancer entities were identified and recorded. Through the implementation of a quality-of-life guide in the ICT, a structured assessment of the quality of life and an analysis of patient needs can take place with the help of the screening. A workflow was created to integrate screening into routine care. In addition, the questionnaire was designed in such a way that it can be used repeatedly at various points in time. In order to cover important stages in the course of therapy and to determine how patient needs change over the course, patients should be asked to complete the questionnaire several times after specified time intervals.The questionnaire is intended to assess the needs of cancer patients receiving outpatient treatment in a structured manner. Now it needs to be explored how the new screening tool and workflow interact and perform in clinical practice and how they help to improve patients' quality of life. It is also interesting to analyze which patients accept the advice offered by the quality-of-life guide and which needs are expressed most frequently.

Melanoma brain metastases (MBM) are common and associated with a particularly poor prognosis; they directly cause death in 60-70% of melanoma patients. In the past, systemic treatments have shown response rates around 5%, whole brain radiation as standard of care has achieved a median overall survival of approximately three months. Recently, the combination of immune checkpoint inhibitors and combinations of MAP-kinase inhibitors both have shown very promising response rates of up to 55% and 58%, respectively, and improved survival. However, current clinical evidence is based on multi-cohort studies only, as prospectively randomized trials have been carried out rarely in MBM, independently whether investigating systemic therapy, radiotherapy or surgical techniques. Here, an interdisciplinary expert team reviewed the outcome of prospectively conducted clinical studies in MBM, identified evidence gaps and provided recommendations for the diagnosis, treatment, outcome evaluation and monitoring of MBM patients. The recommendations refer to four distinct scenarios: patients (i) with 'brain-only' disease, (ii) with oligometastatic asymptomatic intra- and extracranial disease, (iii) with multiple asymptomatic metastases, and (iv) with multiple symptomatic MBM or leptomeningeal disease. Changes in current management recommendations comprise the use of immunotherapy - preferably combined anti-CTLA-4/PD-1-immunotherapy - in asymptomatic MBM minus/plus stereotactic radiosurgery which remains the mainstay of local brain therapy being safe and effective. Adjuvant whole-brain radiotherapy provides no clinical benefit in oligometastatic MBM. Among the systemic therapies, combined MAPK-kinase inhibition provides, in BRAFV600-mutated patients with rapidly progressing or/and symptomatic MBM, an alternative to combined immunotherapy.

C-X-C chemokine receptor 4 (CXCR4) is a transmembrane chemokine receptor involved in growth, survival, and dissemination of cancer, including aggressive B-cell lymphoma. MRI is the standard imaging technology for central nervous system (CNS) involvement of B-cell lymphoma and provides high sensitivity but moderate specificity. Therefore, novel molecular and functional imaging strategies are urgently required. Methods: In this proof-of-concept study, 11 patients with lymphoma of the CNS (8 primary and 3 secondary involvement) were imaged with the CXCR4-directed PET tracer 68Ga-pentixafor. To evaluate the predictive value of this imaging modality, treatment response, as determined by MRI, was correlated with quantification of CXCR4 expression by 68Ga-pentixafor PET in vivo before initiation of treatment in 7 of 11 patients. Results:68Ga-pentixafor PET showed excellent contrast with the surrounding brain parenchyma in all patients with active disease. Furthermore, initial CXCR4 uptake determined by PET correlated with subsequent treatment response as assessed by MRI. Conclusion:68Ga-pentixafor PET represents a novel diagnostic tool for CNS lymphoma with potential implications for theranostic approaches as well as response and risk assessment.

Background The prognosis of patients with brain tumors is widely varying. Psychooncologic need and depression are high among these patients and their family caregivers. However, the need for counselling and need for referral to psychooncology care is often underestimated. Methods We performed a single-institution cross-sectional study to evaluate psychooncologic need, depression and information need in both patients and their family caregivers. The Hornheider Screening Instrument (HSI) and the Patient Health Questionnaire (PHQ-9) were used to evaluate psychooncologic need and depression, and a study-specific questionnaire was developed to evaluate information need. Multivariable analyses were performed to detect correlations. Results A total of 444 patients and their family caregivers were approached to participate, with a survey completion rate of 35.4%. More than half of the patients and family caregivers were in need for referral to psychooncology care and 31.9% of patients suffered from clinically relevant depression. In multivariable analysis, psychooncologic need were positively associated with mild (odds ratio, OR, 7.077; 95% confidence interval, CI, 2.263–22.137; p = 0.001) or moderate to severe (OR 149.27, 95% CI 26.690–737.20; p p = 0.022). Conclusions Unmet counselling need in brain tumor patients and their family caregivers associate to high psychooncologic need and depression. Adequate information may decrease the need for referral to psychooncology care and treatment of depression in these patients. Future studies should further explore these relations to promote development of supportive structures.

B cell chronic lymphocytic leukemia (B-CLL), the most common type of leukemia in adults, is still essentially incurable despite the development of novel therapeutic strategies. This reflects the incomplete understanding of the pathophysiology of this disease. A comprehensive proteome analysis of primary human B-CLL cells and B cells from younger as well as elderly healthy donors was performed. For comparison, the chronic B cell leukemia cell line JVM-13 was also included. A principal component analysis comprising 6,945 proteins separated these four groups, placing B cells of aged-matched controls between those of young donors and B-CLL patients, while identifying JVM-13 as poorly related cells. Mass spectrometric proteomics data have been made fully accessible via ProteomeXchange with identifier PXD006570-PXD006572, PXD006576, PXD006578, and PXD006589-PXD006591. Remarkably, B cells from aged controls displayed significant regulation of proteins related to stress management in mitochondria and ROS stress such as DLAT, FIS1, and NDUFAB1, and DNA repair, including RAD9A, MGMT, and XPA. ROS levels were indeed found significantly increased in B cells but not in T cells or monocytes from aged individuals. These alterations may be relevant for tumorigenesis and were observed similarly in B-CLL cells. In B-CLL cells, some remarkable unique features like the loss of tumor suppressor molecules PNN and JARID2, the stress-related serotonin transporter SLC6A4, and high expression of ZNF207, CCDC88A, PIGR and ID3, otherwise associated with stem cell phenotype, were determined. Alterations of metabolic enzymes were another outstanding feature in comparison to normal B cells, indicating increased beta-oxidation of fatty acids and increased consumption of glutamine. Targeted metabolomics assays corroborated these results. The present findings identify a potential proteome signature for immune senescence in addition to previously unrecognized features of B-CLL cells and suggest that aging may be accompanied by cellular reprogramming functionally relevant for predisposing B cells to transform to B-CLL cells.

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia.

// Ina Hohensee 1 , Han-Ning Chuang 2, * , Astrid Grottke 3, * , Stefan Werner 1 , Alexander Schulte 4 , Stefan Horn 5 , Katrin Lamszus 4 , Kai Bartkowiak 1 , Isabell Witzel 6 , Manfred Westphal 4 , Jakob Matschke 7 , Markus Glatzel 7 , Manfred Jucker 3 , Tobias Pukrop 2, 8 , Klaus Pantel 1 , Harriet Wikman 1 1 Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, 20246, Germany 2 Department of Hematology/Oncology, University Medical Center Gottingen, 37099 Gottingen, Germany 3 Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 4 Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 5 Bone Marrow Transplantation Unit, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 6 Department of Gynecology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 7 Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg-Eppendorf, 20246 Hamburg, Germany 8 Department of Medicine III, University Medical Center Regensburg, 93053 Regensburg, Germany * Shared senior author Correspondence to: Harriet Wikman, email: h.wikman@uke.de Keywords: brain metastases, breast cancer, PTEN, microenvironment, astrocytes Received: August 04, 2016 Accepted: December 13, 2016 Published: December 20, 2016 ABSTRACT Despite improvement of therapeutic treatments for breast cancer, the development of brain metastases has become a major limitation to life expectancy for many patients. Brain metastases show very commonly alterations in EGFR and HER2 driven pathways, of which PTEN is an important regulator. Here, we analyzed PTEN expression in 111 tissue samples of breast cancer brain metastases (BCBM). Loss of PTEN was found in a substantial proportion of BCBM samples (48.6%) and was significantly associated with triple-negative breast cancer (67.5%, p = 0.001) and a shorter survival time after surgical resection of brain metastases ( p = 0.048). Overexpression of PTEN in brain-seeking MDA-MB-231 BR cells in vitro reduced activation of the AKT pathway, notably by suppression of Akt1 kinase activity. Furthermore, the migration of MDA-MB-231 BR cells in vitro was promoted by co-culturing with both astrocytes and microglial cells. Interestingly, when PTEN was overexpressed the migration was significantly inhibited. Moreover, in an ex vivo organotypic brain slice model, PTEN overexpression reduced invasion of tumor cells. This was accompanied by reduced astrocyte activation that was mediated by autocrine and paracrine activation of GM-CSF/ CSF2RA and AKT/ PTEN pathways. In conclusion, loss of PTEN is frequently detected in triple-negative BCBM patients and associated with poor prognosis. The findings of our functional studies suggest that PTEN loss promotes a feedback loop between tumor cells and glial cells, which might contribute to disease progression.

Macrophages (Mφ) are abundantly present in the tumor microenvironment and may predict outcome in solid tumors and defined lymphoma subtypes. Mφ heterogeneity, the mechanisms of their recruitment, and their differentiation into lymphoma‐promoting, alternatively activated M2‐like phenotypes are still not fully understood. Therefore, further functional studies are required to understand biological mechanisms associated with human tumor‐associated Mφ (TAM). Here, we show that the global mRNA expression and protein abundance of human Mφ differentiated in Hodgkin lymphoma (HL)‐conditioned medium (CM) differ from those of Mφ educated by conditioned media from diffuse large B‐cell lymphoma (DLBCL) cells or, classically, by macrophage colony‐stimulating factor (M‐CSF). Conditioned media from HL cells support TAM differentiation through upregulation of surface antigens such as CD40, CD163, CD206, and PD‐L1. In particular, RNA and cell surface protein expression of mannose receptor 1 (MRC1)/CD206 significantly exceed the levels induced by classical M‐CSF stimulation in M2‐like Mφ; this is regulated by interleukin 13 to a large extent. Functionally, high CD206 enhances mannose‐dependent endocytosis and uptake of type I collagen. Together with high matrix metalloprotease9 secretion, HL‐TAMs appear to be active modulators of the tumor matrix. Preclinical in ovo models show that co‐cultures of HL cells with monocytes or Mφ support dissemination of lymphoma cells via lymphatic vessels, while tumor size and vessel destruction are decreased in comparison with lymphoma‐only tumors. Immunohistology of human HL tissues reveals a fraction of cases feature large numbers of CD206‐positive cells, with high MRC1 expression being characteristic of HL‐stage IV. In summary, the lymphoma‐TAM interaction contributes to matrix‐remodeling and lymphoma cell dissemination., The study highlights the ability of Hodgkin lymphoma cells to attract and to differentiate monocytes into M2‐like Mφ and the role of IL13 in regulating CD206 but also that CD206 contributes to the remodeling of the tumor microenvironment via take up glycoconjugates as well as type‐I collagen and immunosuppression by PD‐L1 upregulation.

Diffuse large B-cell lymphoma (DLBCL) is a disease with heterogeneous outcome. Stromal signatures have been correlated to survival in DLBCL. Their use, however, is hampered by the lack of assays for formalin-fixed paraffin-embedded material (FFPE). We constructed a lymphoma-associated macrophage interaction signature (LAMIS) interrogating features of the microenvironment using a NanoString assay applicable to FFPE. The clinical impact of the signature could be validated in a cohort of 466 patients enrolled in prospective clinical trials of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL). Patients with high expression of the signature (LAMIS

More than half of all brain metastases show infiltrating rather than displacing growth at the macro-metastasis/brain parenchyma interface (MMPI), a finding associated with shorter survival. The Lymphoid Enhancer-binding Factor-1 (LEF1) is an epithelial-mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain-colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the anti-oxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma. This article is protected by copyright. All rights reserved.

Activation of AMPK by the tumor suppressor LKB1 represents an essential gatekeeping step for cells under energetic stress to prevent their growth and proliferation by inhibiting mTOR activation, until the energy supply normalizes. The LKB1/AMPK pathway is frequently downregulated in various types of cancer, thereby uncoupling tumor cell growth and proliferation from energy supply. As yet, little information is available on the role of the LKB1/AMPK pathway in tumors derived from salivary gland tissues. We performed LKB1 protein expression and AMPK and mTOR activation analyses in several salivary gland tumor types and their respective healthy control tissues using immunohistochemistry. No significant downregulation of LKB1 expression or decreased activation of AMPK or mTOR were observed in any of the salivary gland tumors tested. In contrast, we found that the salivary gland tumors exhibited an increased rather than a decreased AMPK activation. Although the PI3K/Akt pathway was found to be activated in most of the analyzed tumor samples, the unchanged robust activity of LKB1/AMPK likely prevents (over)activation of mTOR. In contrast to many other types of cancer, inactivation or downregulation of the LKB1/AMPK pathway does not substantially contribute to the pathogenesis of salivary gland tumors.

Background Wnt/β-catenin signaling is known to play an important role in colorectal cancer (CRC). Niclosamide, a salicylamide derivative used in the treatment of tapeworm infections, targets the Wnt/β-catenin pathway. The objective of this study was to investigate niclosamide as a therapeutic agent against CRC. Methods The antiproliferative effects of 1, 3, 10, and 50 μM concentrations of niclosamide on human (SW480 and SW620) and rodent (CC531) CRC cell lines were determined by MTS assay and direct cell count. The lymphoid enhancer–binding factor 1/transcription factor (LEF/TCF) reporter assay monitored the activity of Wnt signaling. Immunofluorescence staining demonstrated the expression pattern of active β-catenin. Gene expression of canonical and noncanonical Wnt signaling components was analyzed using qRT-PCR. Western blot analysis was performed with antibodies detecting nuclear localization of β-catenin and c-jun. Results Cell proliferation in CRC cell lines was blocked dose dependently after 12 and 24 h of incubation. The Wnt promoter activity of LEF/TCF significantly decreased with niclosamide concentrations of 10 and 50 μM after 12 h of incubation. Active β-catenin did not shift from the nuclear to the cytosolic pool. However, canonical target genes (met, MMP7, and cyclin D1) as well as the coactivating factor Bcl9 were downregulated, whereas the noncanonical key player c-jun was clearly activated. Conclusions Niclosamide treatment is associated with an inhibitory effect on CRC development and reduced Wnt activity. It may exert its effect by interfering with the nuclear β-catenin-Bcl9-LEF/TCF triple-complex and by upregulation of c-jun representing noncanonical Wnt/JNK signaling. Thus, our findings warrant further research into this substance as a treatment option for patients with advanced CRC.

Mutations and activation of the PI3K signaling pathway in breast cancer cells have been linked to brain metastases. However, here we describe that in some breast cancer brain metastases samples the protein expression of PI3K signaling components is restricted to the metastatic microenvironment. In contrast to the therapeutic effects of PI3K inhibition on the breast cancer cells, the reaction of the brain microenvironment is less understood. Therefore we aimed to quantify the PI3K pathway activity in breast cancer brain metastasis and investigate the effects of PI3K inhibition on the central nervous system (CNS) microenvironment. First, to systematically quantify the PI3K pathway activity in breast cancer brain metastases, we performed a prospective biomarker study using a reverse phase protein array (RPPA). The majority, namely 30 out of 48 (62.5%) brain metastatic tissues examined, revealed high PI3K signaling activity that was associated with a median overall survival (OS) of 9.41 months, while that of patients, whose brain metastases showed only moderate or low PI3K activity, amounted to only 1.93 and 6.71 months, respectively. Second, we identified PI3K as a master regulator of metastasis-promoting macrophages/microglia during CNS colonization; and treatment with buparlisib (BKM120), a pan-PI3K Class I inhibitor with a good blood-brain-barrier penetrance, reduced their metastasis-promoting features. In conclusion, PI3K signaling is active in the majority of breast cancer brain metastases. Since PI3K inhibition does not only affect the metastatic cells but also re-educates the metastasis-promoting macrophages/microglia, PI3K inhibition may hold considerable promise in the treatment of brain metastasis and the respective microenvironment.

// Laila Siam 1 , Annalen Bleckmann 2, 3 , Han-Ning Chaung 2 , Alexander Mohr 4 , Florian Klemm 2 , Alonso Barrantes-Freer 5 , Raquel Blazquez 2 , Hendrik A. Wolff 6 , Florian Luke 7 , Veit Rohde 1 , Christine Stadelmann 5 , Tobias Pukrop 2, 7 1 Department of Neurosurgery, University Medical Center, Gottingen, Germany 2 Department of Hematology and Medical Oncology, University Medical Center, Gottingen, Germany 3 Department of Medical Statistics, University Medical Center, Gottingen, Germany 4 Department of Neuroradiology, University Medical Center, Gottingen, Germany 5 Institute of Neuropathology, University Medical Center, Gottingen, Germany 6 Department of Radiotherapy and Radiation Oncology, University Medical Center, Gottingen, Germany 7 Department of Internal Medicine III, Hematology and Medical Oncology, University Hospital Regensburg, Regensburg, Germany Correspondence to: Tobias Pukrop, e-mail: tobias.pukrop@ukr.de Keywords: astrocytes, brain metastasis, glial-pseudo capsule, metastatic infiltration, organ-specific defense Received: April 11, 2015 Accepted: June 08, 2015 Published: June 17, 2015 ABSTRACT The current approach to brain metastases resection is macroscopic removal of metastasis until reaching the glial pseudo-capsule (gross total resection (GTR)). However, autopsy studies demonstrated infiltrating metastatic cells into the parenchyma at the metastasis/brain parenchyma (M/BP)-interface. Aims/Methods: To analyze the astrocyte reaction and metastatic infiltration pattern at the M/BP-interface with an organotypic brain slice coculture system. Secondly, to evaluate the significance of infiltrating metastatic tumor cells in a prospective biopsy study. Therefore, after GTR, biopsies were obtained from the brain parenchyma beyond the glial pseudo-capsule and analyzed histomorphologically. Results: The coculture revealed three types of cancer cell infiltration. Interestingly, the astrocyte reaction was significantly different in the coculture with a benign, neuroectodermal-derived cell line. In the prospective biopsy study 58/167 (34.7%) samples revealed infiltrating metastatic cells. Altogether, 25/39 patients (64.1%) had proven to exhibit infiltration in at least one biopsy specimen with significant impact on survival (OS) (3.4 HR; p = 0.009; 2-year OS was 6.6% versus 43.5%). Exceptionally, in the non-infiltrating cohort three patients were long-term survivors. Conclusions: Metastatic infiltration has a significant impact on prognosis. Secondly, the astrocyte reaction at the M/BP-interface is heterogeneous and supports our previous concept of the organ-specific defense against metastatic (organ-foreign) cells.

// Eva Rietkotter 1 , Annalen Bleckmann 1 , Michaela Bayerlova 2 , Kerstin Menck 1 , Han-Ning Chuang 1 , Britta Wenske 1 , Hila Schwartz 3 , Neta Erez 3 , Claudia Binder 1 , Uwe-Karsten Hanisch 4 , Tobias Pukrop 1, 5 1 Department of Hematology and Medical Oncology, University Medical Center, 37075 Gottingen, Germany 2 Department of Medical Statistics, University Medical Center, 37075 Gottingen, Germany 3 Department of Pathology, Sackler School of Medicine, 69978 Tel Aviv University, Israel 4 Institute of Neuropathology, University Medical Center, 37075 Gottingen, Germany 5 Department of Hematology and Medical Oncology, University Clinic Regensburg, 93053 Regensburg, Germany Correspondence to: Tobias Pukrop, e-mail: tobias.pukrop@ukr.de Keywords: anti-CSF-1, colonization, monocyte-derived cells, microglia, metastasis Received: January 5, 2015 Accepted: April 29, 2015 Published: May 12, 2015 ABSTRACT The mononuclear phagocytic system is categorized in three major groups: monocyte-derived cells (MCs), dendritic cells and resident macrophages. During breast cancer progression the colony stimulating factor 1 (CSF-1) can reprogram MCs into tumor-promoting macrophages in the primary tumor. However, the effect of CSF-1 during colonization of the brain parenchyma is largely unknown. Thus, we analyzed the outcome of anti-CSF-1 treatment on the resident macrophage population of the brain, the microglia, in comparison to MCs, alone and in different in vitro co-culture models. Our results underline the addiction of MCs to CSF-1 while surprisingly, microglia were not affected. Furthermore, in contrast to the brain, the bone marrow did not express the alternative ligand, IL-34. Yet treatment with IL-34 and co-culture with carcinoma cells partially rescued the anti-CSF-1 effects on MCs. Further, MC-induced invasion was significantly reduced by anti-CSF-1 treatment while microglia-induced invasion was reduced to a lower extend. Moreover, analysis of lung and breast cancer brain metastasis revealed significant differences of CSF-1 and CSF-1R expression. Taken together, our findings demonstrate not only differences of anti-CSF-1 treatment on MCs and microglia but also in the CSF-1 receptor and ligand expression in brain and bone marrow as well as in brain metastasis.

Central nervous system (CNS ) metastasis is not only an increasing but still a very unsatisfying clinical problem, with very few treatment options nowadays and an unmet clinical need. Additionally, the patients suffer from severe neurological symptoms. Furthermore the preclinical studies are limited and thus innovative methods are needed to study the mechanism of CNS metastasis during the final steps. Especially nowadays, the most critical step during metastasis seems to be the contention of the microenvironment of host organs with the cancer cells coming from other organs. More and more data indicate that this contention often leads to apoptosis of the pre-metastatic cells and prevents successful metastasis. However, this important step is barely understood. To further improve our knowledge about this important step of metastasis we developed a new experimental tool where we coculture an organotypic brain slice with a 3D tumor cell plug embedded in Matrigel for 3-4 days.This model especially mimics the interactions of cancer cells and glial cells at the interface of the brain parenchyma and the metastatic tissue. Therefore this coculture method with an organotypic brain slice and a tumor cell plug allows us to visualize and/or manipulate the interactions at this very important zone. Furthermore, it also allows us to use brain tissue from genetically engineered mice and/or genetically modified tumor cells to investigate genes of interest in the microenvironment or in cancer cells. Moreover this method avoids the use of a large number of animals and is especially useful in identifying the invasiveness of different tumor cell lines into the brain parenchyma as well as in studying the effect of specific treatments against brain metastasis progression during the final and most critical steps of metastasis.

Background The International Extranodal Lymphoma Study Group-32 (IELSG32) trial is an international randomised phase 2 study that addresses two key clinical questions in the treatment of patients with newly diagnosed primary CNS lymphoma. Results of the first randomisation have demonstrated that methotrexate, cytarabine, thiotepa, and rituximab (called the MATRix regimen) is the induction combination associated with significantly better outcome compared with the other induction combinations tested. Here, we report the results of the second randomisation that addresses the efficacy of myeloablative chemotherapy supported by autologous stem-cell transplantation (ASCT), as an alternative to whole-brain radiotherapy (WBRT), as consolidation after high-dose-methotrexate-based chemoimmunotherapy. Methods HIV-negative patients (aged 18–70 years) with newly diagnosed primary CNS lymphoma and an Eastern Cooperative Oncology Group performance status of 0–3 were randomly assigned to receive four courses of methotrexate 3·5 g/m2 on day 1 plus cytarabine 2 g/m2 twice daily on days 2 and 3 (group A); or the same combination plus two doses of rituximab 375 mg/m2 on days −5 and 0 (group B); or the same methotrexate–cytarabine–rituximab combination plus thiotepa 30 mg/m2 on day 4 (group C), with the three groups repeating treatment every 3 weeks. Patients with responsive or stable disease after induction treatment, with adequate autologous peripheral blood stem-cell collection, and without persistent iatrogenic side-effects, were eligible for the second randomisation between WBRT (photons of 4–10 MeV; five fractions per week; fraction size 180 cGy; started within 4 weeks from the last induction course; group D) and carmustine–thiotepa conditioned ASCT (carmustine 400 mg/m2 on day −6, and thiotepa 5 mg/kg every 12 h on days −5 and −4, followed by reinfusion of autologous peripheral blood stem cells; group E). A permuted block randomised design was adopted for both randomisations, and a computer-generated randomisation list was used within each stratum. No masking after assignment to intervention was adopted. The primary endpoint was 2-year progression-free survival, with induction group and response to induction chemotherapy as stratification parameters. Analyses were done on a modified intention-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT01011920. Findings Between Feb 19, 2010, and Aug 27, 2014, 227 patients were recruited from 53 centres in five countries. 219 of 227 enrolled patients were assessable. Of the 122 patients eligible for the second randomisation, 118 patients were randomly assigned to WBRT or ASCT (59 patients per group) and constitute the study population. WBRT and ASCT were both effective, and achieved the predetermined efficacy threshold of at least 40 progression-free survivors at 2 years among the first 52 patients in both groups D and E. There were no significant differences in 2-year progression-free survival between WBRT and ASCT: 80% (95% CI 70–90) in group D and 69% (59–79) in group E (hazard ratio 1·50, 95% CI 0·83–2·71; p=0·17). Both consolidation therapies were well tolerated. Grade 4 non-haematological toxicity was uncommon; as expected, haematological toxicity was more common in patients treated with ASCT than in those who received WBRT. Two toxic deaths (infections) were recorded, both in patients who received ASCT. Interpretation WBRT and ASCT are both feasible and effective as consolidation therapies after high-dose methotrexate-based chemoimmunotherapy in patients aged 70 years or younger with primary CNS lymphoma. The risks and implications of cognitive impairment after WBRT should be considered at the time of therapeutic decision. Funding Agenzia Italiana del Farmaco, Cancer Research UK, Oncosuisse, and Swiss National Science Foundation.

// Kerstin Menck 1,* , Florian Klemm 1,* , Julia Christina Gross 2 , Tobias Pukrop 1 , Dirk Wenzel 3 , Claudia Binder 1 1 Dept of Hematology/Oncology, University Medicine, Gottingen, Germany 2 Div. Signaling and Functional Genomics, German Cancer Research Center, Heidelberg, Germany 3 Max Planck Institute for Biophysical Chemistry, Gottingen, Germany * These authors contributed equally to this work. Correspondence: Claudia Binder, email: // Keywords : Breast cancer, exosomes, Evi, microvesicles, macrophages, Wnt 5a Received : August 27, 2013 Accepted : October 19, 2013 Published : October 21, 2013 Abstract Recently, we have shown that macrophage (MΦ)-induced invasion of breast cancer cells requires upregulation of Wnt 5a in MΦ leading to activation of β-Catenin-independent Wnt signaling in the tumor cells. However, it remained unclear, how malignant cells induce Wnt 5a in MΦ and how it is transferred back to the cancer cells. Here we identify two types of extracellular particles as essential for this intercellular interaction in both directions. Plasma membrane-derived microvesicles (MV) as well as exosomes from breast cancer cells, although biologically distinct populations, both induce Wnt 5a in MΦ. In contrast, the particle-free supernatant and vesicles from benign cells, such as platelets, have no such effect. Induction is antagonized by the Wnt inhibitor Dickkopf-1. Subsequently, Wnt 5a is shuttled via responding MΦ-MV and exosomes to the tumor cells enhancing their invasion. Wnt 5a export on both vesicle fractions depends at least partially on the cargo protein Evenness interrupted (Evi). Its knockdown leads to Wnt 5a depletion of both particle populations and reduced vesicle-mediated invasion. In conclusion, MV and exosomes are critical for MΦ-induced invasion of cancer cells since they are responsible for upregulation of MΦ-Wnt 5a as well as for its delivery to the recipient cells via a reciprocal loop. Although of different biogenesis, both populations share common features regarding function and Evi-dependent secretion of non-canonical Wnts.

Maintenance of tissue homeostasis and immune surveillance are important functions of the lymphatic vascular system. Lymphatic vessels are lined by lymphatic endothelial cells (LECs). By gene micro-array expression studies we recently compared human lymphangioma-derived LECs with umbilical vein endothelial cells (HUVECs). Here, we followed up on these studies. Besides well-known LEC markers, we observed regulation of molecules involved in immune regulation, acetylcholine degradation and platelet regulation. Moreover we identified differentially expressed WNT pathway components, which play important roles in the morphogenesis of various organs, including the blood vascular system. WNT signaling has not yet been addressed in lymphangiogenesis. We found high expression of FZD3, FZD5 and DKK2 mRNA in HUVECs, and WNT5A in LECs. The latter was verified in normal skin-derived LECs. With immunohistological methods we detected WNT5A in LECs, as well as ROR1, ROR2 and RYK in both LECs and HUVECs. In the human, mutations of WNT5A or its receptor ROR2 cause the Robinow syndrome. These patients show multiple developmental defects including the cardio-vascular system. We studied Wnt5a-knockout (ko) mouse embryos at day 18.5. We show that the number of dermal lymphatic capillaries is significantly lower in Wnt5a-null-mice. However, the mean size of individual lymphatics and the LEC number per vessel are greater. In sum, the total area covered by lymphatics and the total number of LECs are not significantly altered. The reduced number of lymphatic capillaries indicates a sprouting defect rather than a proliferation defect in the dermis of Wnt5a-ko-mice, and identifies Wnt5a as a regulator of lymphangiogenesis.

Burkitt's lymphoma (BL) is a highly malignant, aggressive non-Hodgkin's lymphoma derived from germinal center B cells. Recently, global gene expression profiling of patient samples led to a molecular definition of BL with lymphocyte enhancer-binding factor 1 (LEF1) as a signature gene. Herein, we report the expression of nucleic LEF1 in 15 of 18 patients with BL and the identification of LEF1 target genes. Germinal center B cells were devoid of detectable nuclear LEF1 expression, as were mantle cell lymphoma (0 of 5), marginal zone lymphoma (0 of 6), follicular lymphoma (0 of 12), and diffuse large B-cell lymphoma (1 of 31). Whole-genome gene expression profiling after transient knockdown of LEF1 in BL cell lines identified new LEF1 target genes; these LEF1 targets are enriched with genes associated with cancers. The expression of LEF1 and LEF1-regulated genes in primary BL suggests that LEF1 is not only aberrantly expressed but also transcriptionally active. This study supports a functionally important role for LEF1 and its target genes in BLs.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and show characteristics of skeletal muscle differentiation. The two major RMS subtypes in children are alveolar (ARMS) and embryonal RMS (ERMS). We demonstrate that approximately 50% of ARMS and ERMS overexpress the LEF1/TCF transcription factor LEF1 when compared to normal skeletal muscle and that LEF1 can restrain aggressiveness especially of ARMS cells. LEF1 knockdown experiments in cell lines reveal that depending on the cellular context, LEF1 can induce pro-apoptotic signals. LEF1 can also suppress proliferation, migration and invasiveness of RMS cells both in vitro and in vivo. Furthermore, LEF1 can induce myodifferentiation of the tumor cells. This may involve regulation of other LEF1/TCF factors i.e. TCF1, whereas β-catenin activity plays a subordinate role. Together these data suggest that LEF1 rather has tumor suppressive functions and attenuates aggressiveness in a subset of RMS.

Liver metastasis development in breast cancer patients is common and confers a poor prognosis. So far, the prognostic significance of surgical resection and clinical relevance of biomarker analysis in metastatic tissue have barely been investigated. We previously demonstrated an impact of WNT signaling in breast cancer brain metastasis. This study aimed to investigate the value of established prognostic markers and WNT signaling components in liver metastases. Overall N = 34 breast cancer liver metastases (with matched primaries in 19/34 cases) were included in this retrospective study. Primaries and metastatic samples were analyzed for their expression of the estrogen (ER) and progesterone receptor, HER-2, Ki67, and various WNT signaling-components by immunohistochemistry. Furthermore, β-catenin-dependent and -independent WNT scores were generated and analyzed for their prognostic value. Additionally, the influence of the alternative WNT receptor ROR on signaling and invasiveness was analyzed in vitro. ER positivity (HR 0.09, 95 % CI 0.01–0.56) and high Ki67 (HR 3.68, 95 % CI 1.12–12.06) in the primaries had prognostic impact. However, only Ki67 remained prognostic in the metastatic tissue (HR 2.46, 95 % CI 1.11–5.44). Additionally, the β-catenin-independent WNT score correlated with reduced overall survival only in the metastasized situation (HR 2.19, 95 % CI 1.02–4.69, p = 0.0391). This is in line with the in vitro results of the alternative WNT receptors ROR1 and ROR2, which foster invasion. In breast cancer, the value of prognostic markers established in primary tumors cannot directly be translated to metastases. Our results revealed β-catenin-independent WNT signaling to be associated with poor prognosis in patients with breast cancer liver metastasis. Electronic supplementary material The online version of this article (doi:10.1007/s10585-016-9780-3) contains supplementary material, which is available to authorized users.

Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood–brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Cancer Res; 76(15); 4359–71. ©2016 AACR.

BACKGROUND: Standard treatment for patients with primary CNS lymphoma remains to be defined. Active therapies are often associated with increased risk of haematological or neurological toxicity. In this trial, we addressed the tolerability and efficacy of adding rituximab with or without thiotepa to methotrexate-cytarabine combination therapy (the MATRix regimen), followed by a second randomisation comparing consolidation with whole-brain radiotherapy or autologous stem cell transplantation in patients with primary CNS lymphoma. We report the results of the first randomisation in this Article.METHODS: For the international randomised phase 2 International Extranodal Lymphoma Study Group-32 (IELSG32) trial, HIV-negative patients (aged 18-70 years) with newly diagnosed primary CNS lymphoma and measurable disease were enrolled from 53 cancer centres in five European countries (Denmark, Germany, Italy, Switzerland, and the UK) and randomly assigned (1:1:1) to receive four courses of methotrexate 3·5 g/m(2) on day 1 plus cytarabine 2 g/m(2) twice daily on days 2 and 3 (group A); or the same combination plus two doses of rituximab 375 mg/m(2) on days -5 and 0 (group B); or the same methotrexate-cytarabine-rituximab combination plus thiotepa 30 mg/m(2) on day 4 (group C), with the three groups repeating treatment every 3 weeks. Patients with responsive or stable disease after the first stage were then randomly allocated between whole-brain radiotherapy and autologous stem cell transplantation. A permuted blocks randomised design (block size four) was used for both randomisations, and a computer-generated randomisation list was used within each stratum to preserve allocation concealment. Randomisation was stratified by IELSG risk score (low vs intermediate vs high). No masking after assignment to intervention was used. The primary endpoint of the first randomisation was the complete remission rate, analysed by modified intention to treat. This study is registered with ClinicalTrials.gov, number NCT01011920.FINDINGS: Between Feb 19, 2010, and Aug 27, 2014, 227 eligible patients were recruited. 219 of these 227 enrolled patients were assessable. At median follow-up of 30 months (IQR 22-38), patients treated with rituximab and thiotepa had a complete remission rate of 49% (95% CI 38-60), compared with 23% (14-31) of those treated with methotrexate-cytarabine alone (hazard ratio 0·46, 95% CI 0·28-0·74) and 30% (21-42) of those treated with methotrexate-cytarabine plus rituximab (0·61, 0·40-0·94). Grade 4 haematological toxicity was more frequent in patients treated with methotrexate-cytarabine plus rituximab and thiotepa, but infective complications were similar in the three groups. The most common grade 3-4 adverse events in all three groups were neutropenia, thrombocytopenia, anaemia, and febrile neutropenia or infections. 13 (6%) patients died of toxicity.INTERPRETATION: With the limitations of a randomised phase 2 study design, the IELSG32 trial provides a high level of evidence supporting the use of MATRix combination as the new standard chemoimmunotherapy for patients aged up to 70 years with newly diagnosed primary CNS lymphoma and as the control group for future randomised trials.FUNDING: Associazione Italiana del Farmaco, Cancer Research UK, Oncosuisse, and Swiss National Foundation.

Classical Hodgkin lymphoma (cHL) has a typical clinical manifestation, with dissemination involving functionally neighboring lymph nodes. The factors involved in the spread of lymphoma cells are poorly understood. Here we show that cHL cell lines migrate with higher rates compared with non-Hodgkin lymphoma cell lines. cHL cell migration, invasion and adhesion depend on autocrine WNT signaling as revealed by the inhibition of WNT secretion with the porcupine inhibitors Wnt-C59/IWP-2, but did not affect cell proliferation. While application of recombinant WNT5A or WNT5A overexpression stimulates HL cell migration, neither WNT10A, WNT10B nor WNT16 did so. Time-lapse studies revealed an amoeboid type of cell migration modulated by WNT5A. Reduced migration distances and velocity of cHL cells, as well as altered movement patterns, were observed using porcupine inhibitor or WNT5A antagonist. Knockdown of Frizzled5 and Dishevelled3 disrupted the WNT5A-mediated RHOA activation and cell migration. Overexpression of DVL3-K435M or inhibition of ROCK (Rho-associated protein kinase) by Y-27632/H1152P disrupted cHL cell migration. In addition to these mechanistic insights into the role of WNT5A in vitro, global gene expression data revealed an increased WNT5A expression in primary HL cells in comparison with normal B-cell subsets and other lymphomas. Furthermore, the activity of both porcupine and WNT5A in cHL cells had an impact on lymphoma development in the chick chorionallantoic membrane assay. Massive bleeding of these lymphomas was significantly reduced after inhibition of WNT secretion by Wnt-C59. Therefore, a model is proposed where WNT signaling has an important role in regulating tumor-promoting processes.

Background: Programme evaluation of medical education should be multi-dimensional. While structural and organisational aspects of teaching are frequently assessed, programme evaluation tools are rarely matched to specific learning objectives. Aims: This study used one medical school's catalogue of specific learning objectives to implement and critically appraise a novel programme evaluation tool based on comparative student self-assessments. Method: Medical students enrolled in the clinical phase of the undergraduate curriculum in Gottingen were invited to self-rate their knowledge, skills and attitudes before and after each course. A newly developed formula controlling for student performance levels when entering a course was used to compute a percentage gain in knowledge, skills and attitudes. Data derived from a prospective, longitudinal intervention study on the development of electrocardiogram interpretation skills including 636 students from four consecutive cohorts were used to provide validity evidence of the new approach. Results: The novel tool appeared superior to plain mean differences and effect sizes in detecting outstanding teaching as well as shortcomings of the curriculum. In addition, it adequately reflected objectively measured performance levels and was responsive to curriculum change. Conclusions: Comparative student self-assessment is a valid tool to appraise undergraduate medical curricula at the level of specific learning objectives.

Basal cell carcinoma (BCC) is the most common skin tumor in humans. Although BCCs rarely metastasize, they can cause significant morbidity due to local aggressiveness. Approximately 20% of BCCs show signs of spontaneous regression. The understanding of molecular events mediating spontaneous regression has the potential to reduce morbidity of BCC and, potentially, other tumors, if translated into tumor therapies. We show that BCCs induced in conditional Ptchflox/floxERT2+/− knockout mice regress with time and show a more differentiated phenotype. Differentiation is accompanied by Wnt5a expression in the tumor stroma, which is first detectable at the fully developed tumor stage. Coculture experiments revealed that Wnt5a is upregulated in tumor-adjacent macrophages by soluble signals derived from BCC cells. In turn, Wnt5a induces the expression of the differentiation marker K10 in tumor cells, which is mediated by Wnt/Ca2+ signaling in a CaMKII-dependent manner. These data support a role of stromal Wnt5a in BCC differentiation and regression, which may have important implications for development of new treatment strategies for this tumor. Taken together, our results establish BCC as an easily accessible model of tumor regression. The regression of BCC despite sustained Hedgehog signaling activity seems to be mediated by tumor-stromal interactions via Wnt5a signaling. Cancer Res; 70(7); 2739–48

Bedside teaching is an important element of undergraduate medical education. However, the impact of curricular course structure on student outcome needs to be determined.This study assessed changes in fourth-year medical students' evaluations of clinical teaching sessions before and after the introduction of a new course format.The curricular structure of bedside teaching sessions in cardiology was modified without changing the amount of teaching time. Clinical teachers were instructed about the new teaching format and learning objectives. The new format implemented for adult but not paediatric cardiology sessions was piloted with 143 students in winter 2007/08. By computing effect sizes, evaluation results were compared to data obtained from 185 students before the intervention.Significant rating increases were observed for adult cardiology teaching sessions (Cohen's d = 0.66) but not paediatric cardiology sessions (d = 0.22). In addition to improving the structure and organization of the course, the intervention significantly impacted on students' perceptions of their learning outcome regarding practical skills (d = 0.69).Minimal curricular changes combined with basic faculty development measures significantly increase students' perception of learning outcome. Curricular structure needs to be considered when planning bedside teaching sessions in medical undergraduate training.

Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are known to support the establishment of a favorable tumor niche by influencing the surrounding stroma cells. Here we show that tumor-derived microvesicles (T-MV) also directly influence the tumor cells by enhancing their invasion in a both autologous and heterologous manner. Neither the respective vesicle-free supernatant nor MV from benign mammary cells mediate invasion. Uptake of T-MV is essential for the proinvasive effect. We further identify the highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) as a marker for proinvasive MV. EMMPRIN is also present at high levels on MV from metastatic breast cancer patients in vivo. Anti-EMMPRIN strategies, such as MV deglycosylation, gene knockdown, and specific blocking peptides, inhibit MV-induced invasion. Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells. In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN. peerReviewed

INTRODUCTION: WNT signaling is a complex process comprising multiple pathways: the canonical β-catenin-dependent pathway and several alternative non-canonical pathways that act in a β-catenin-independent manner. Representing these intricate signaling mechanisms through bioinformatic approaches is challenging. Nevertheless, a simplified but reliable bioinformatic WNT pathway model is needed, which can be further utilized to decipher specific WNT activation states within e.g. high-throughput data. RESULTS: In order to build such a model, we collected, parsed, and curated available WNT signaling knowledge from different pathway databases. The data were assembled to construct computationally suitable models of different WNT signaling cascades in the form of directed signaling graphs. This resulted in four networks representing canonical WNT signaling, non-canonical WNT signaling, the inhibition of canonical WNT signaling and the regulation of WNT signaling pathways, respectively. Furthermore, these networks were integrated with microarray and RNA sequencing data to gain deeper insight into the underlying biology of gene expression differences between MCF-7 and MDA-MB-231 breast cancer cell lines, representing weakly and highly invasive breast carcinomas, respectively. Differential genes up-regulated in the MDA-MB-231 compared to the MCF-7 cell line were found to display enrichment in the gene set originating from the non-canonical network. Moreover, we identified and validated differentially regulated modules representing canonical and non-canonical WNT pathway components specific for the aggressive basal-like breast cancer subtype. CONCLUSIONS: In conclusion, we demonstrated that these newly constructed WNT networks reliably reflect distinct WNT signaling processes. Using transcriptomic data, we shaped these networks into comprehensive modules of the genes implicated in the aggressive basal-like breast cancer subtype and demonstrated that non-canonical WNT signaling is important in this context. The topology of these networks can be further refined in the future by integration with complementary data such as protein-protein interactions, in order to gain greater insight into signaling processes. Open-Access-Publikationsfonds 2015 peerReviewed

Interactions between neoplastic and stromal cells contribute to tumor progression. Wnt genes, involved in cell migration and often deregulated in cancers, are attractive candidates to regulate these effects. We have recently shown that coculture of breast cancer cells with macrophages enhances invasiveness via matrix metalloproteases and TNF-α. Here we demonstrate that coculture of MCF-7 cells and macrophages leads to up-regulation of Wnt 5a in the latter. This was accompanied by activation ofAP-1/c-Junin MCF-7. Recombinant Wnt 5a mimicked the coculture effect. Wnt 5a was also detectable in tumor-associated macrophages in primary breast cancers. Experiments with agonists and antagonists of Wnt signaling revealed that a functional canonical pathway in the tumor cells was a necessary prerequisite; however, noncanonical signaling via Wnt 5a and the Jun-N-terminal kinase pathway was critical for invasiveness. It was also responsible for induction of matrix metalloprotease-7, known to release TNF-α. All these effects could be antagonized by dickkopf-1. Our results indicate that Wnt 5a is essential for macrophage-induced invasiveness, because it regulates tumor cell migration as well as proteolytic activity of the macrophages. The function of Wnt 5a as either a suppressor or promoter of malignant progression seems to be modulated by intercellular interactions. Wnt 5a detection in tumor-associated macrophages in breast cancer biopsies supports the assumption that similar events play a rolein vivo.

There is increased staining of endothelins (ET-1, -2, and -3) and receptors (ET-RA and -RB) in invasive breast tumors compared to nonneoplastic tissue, and ETs stimulate MCF-7 cell invasion in vitro. We analyzed ETstimulation of benign and transformed mammary epithelial cells, and whether expression of ETs is sufficient to induce invasiveness. In breast cancer patient serum, ET-1 was increased in those patients with lymph node metastases compared to those with no lymph node involvement; ETs, however, had no mitogenic effect on breast tumor cell lines in vitro. The benign mammary epithelial cell line, hTERT-HME1, and the poorly invasive breast tumor cell line MCF-7 secreted low levels of ET-1, while the invasive cell lines SKBR3 and MDAMB231 secreted high levels. Expression of the ETs and receptors by the cell lines broadly correlated with their in vitro invasiveness; overexpression of ETs in MCF-7 cells increased basal invasion. ET-mediated invasion involved both receptors and a calcium influx to induce a pertussis toxin-sensitive MAPK pathway. MMP-14 activity was induced via ET-RA in an autocrine manner. In contrast to transformed cells, ET stimulation or overexpression did not induce an invasive phenotype in benign cells. Benign cells do not respond to ETs, and ET expression is not sufficient to induce invasion; however, the level of ET production by tumor cells correlates with their invasiveness, and increasing expression of the ET axis promotes breast tumor cell invasion via both receptors, while MMP-14 is induced via ET-RA.

Tumor-associated macrophages may influence tumor progression, angiogenesis and invasion. To investigate mechanisms by which macrophages interact with tumor cells, we developed an in vitro coculture model. Previously we reported that coculture enhanced invasiveness of the tumor cells in a TNF-α- and matrix metalloprotease-dependent manner. In this report, we studied intracellular signaling pathways and induction of inflammatory genes in malignant cells under the influence of macrophage coculture. We report that coculture of macrophages with ovarian or breast cancer cell lines led to TNF-α-dependent activation of JNK and NF-κB pathways in tumor cells, but not in benign immortalized epithelial cells. Tumor cells with increased JNK and NF-κB activity exhibited enhanced invasiveness. Inhibition of the NF-κB pathway by TNF-α neutralizing Abs, an NF-κB inhibitor, RNAi to RelA, or overexpression of IκB inhibited tumor cell invasiveness. Blockade of JNK also significantly reduced invasiveness, but blockade of p38 MAPK or p42 MAPK had no effect. Cocultured tumor cells were screened for the expression of 22 genes associated with inflammation and invasion that also contained an AP-1 and NF-κB binding site. EMMPRIN and MIF were up-regulated in cocultured tumor cells in a JNK- and NF-κB-dependent manner. Knocking down either MIF or EMMPRIN by RNAi in the tumor cells significantly reduced tumor cell invasiveness and matrix metalloprotease activity in the coculture supernatant. We conclude that TNF-α, via NF-κB, and JNK induces MIF and EMMPRIN in macrophage to tumor cell cocultures and this leads to increased invasive capacity of the tumor cells.

Progenitor cells with differentiation capacity along multiple mesengenic lineages are attractive tools for numerous purposes in regenerative medicine. Such mesengenic progenitor cells have been isolated from adult mammalian bone marrow, and we here report placental tissue as an alternative source for these cells. By means of dissection/proteinase digestion techniques, high numbers of viable mononuclear cells were harvested from human placenta at term, and a mesenchymal cell population with characteristic expression of CD9, CD29, and CD73 was obtained in culture. The in vitro growth behavior of such placenta-derived mesengenic cells was similar to that of human bone marrow mesengenic progenitor cells. After in vitro propagation for more than three passages the cells were exclusively of maternal origin. Differentiation experiments showed differentiation potential along osteogenic, chondrogenic, adipogenic, and myogenic lineages. In conclusion, we propose human term placenta as an easily accessible, ample source of multipotent mesengenic progenitor cells.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

Introduction Various studies have identified aberrantly expressed miRNAs in breast cancer and demonstrated an association between distinct miRNAs and malignant progression as well as metastasis. Even though tumor‐associated macrophages (TAM) are known mediators of these processes, little is known regarding their miRNA expression upon education by malignant cells in vivo. Methods We profiled miRNA and mRNA expression of in vitro tumor‐educated macrophages (TEM) by indirectly co‐culturing with estrogen‐receptor‐positive (ER+) MCF‐7 breast cancer cells. The prognostic power of the resulting miRNA list was investigated in primary breast cancer datasets and compared to other signatures. Furthermore, miRNA expression levels were correlated to mRNA expression of macrophage markers and the impact on prognosis was assessed. Results Through the evaluation of the group effects between differentially‐expressed miRNAs and their target mRNAs in TEM, the power of detecting regulated miRNAs was greatly increased. The resulting list of 96 miRNAs predicts disease‐free survival (DFS) in external datasets of ER+ breast cancer patients and performs well in comparison with other miRNA signatures. Clustering with the predefined miRNA list revealed a significant difference in survival between the two resulting patient groups. Furthermore, an optimized miRNA list, based on correlations with macrophages markers, proved even more capable at identifying patient clusters significantly differing in DFS. Conclusions In vitro profiling of TEM and subsequent bioinformatic verification identified miRNAs with a high prognostic power for DFS when transferred into the clinical setting of primary breast cancer. The resulting miRNAs not only verify previously established findings but also lead to new prognostic markers. Furthermore, our data suggest that TAM contribute to the total miRNA expression profile of ER + breast cancers., Highlights miRNA and mRNA were measured in macrophages exposed to ER + breast cancer cells.Regulated miRNAs were detected by analyzing group effects of mRNA targets.The resulting miRNA list has good prognostic value for DFS in ER + breast cancer.Correlation of miRNAs to macrophage markers improved identification of clusters differing in DFS.

Basal cell carcinoma (BCC) belongs to the group of non-melanoma skin tumors and is the most common tumor in the western world. BCC arises due to mutations in the tumor suppressor gene Patched1 (Ptch). Analysis of the conditional Ptch knockout mouse model for BCC reveals that macrophages and dendritic cells (DC) of the skin play an important role in BCC growth restraining processes. This is based on the observation that a clodronate-liposome mediated depletion of these cells in the tumor-bearing skin results in significant BCC enlargement. The depletion of these cells does not modulate Ki67 or K10 expression, but is accompanied by a decrease in collagen-producing cells in the tumor stroma. Together, the data suggest that cutaneous macrophages and DC in the tumor microenvironment exert an antitumor effect on BCC. Open-Access-Publikationsfonds 2014 Open-Access-Publikationsfonds 2014 peerReviewed

// Eva Rietkotter 1 , Kerstin Menck 1 , Annalen Bleckmann 1,2 , Katja Farhat 3 , Meike Schaffrinski 1 , Matthias Schulz 1 , Uwe-Karsten Hanisch 4 , Claudia Binder 1 , Tobias Pukrop 1 1 Department of Hematology/Oncology, University Medical Center, 37099 Gottingen, Germany 2 Department of Medical Statistics, University Medical Center, 37099 Gottingen, Germany 3 Department of Cardiovascular Physiology, University Medical Center, 37099 Gottingen, Germany 4 Institute of Neuropathology, University Medical Center, 37099 Gottingen, Germany Correspondence: Tobias Pukrop, email: // Keywords : Zoledronic acid, macrophages, microglia, metastasis, tumor microenvironment Received : July 23, 2013 Accepted : August 17, 2013 Published : August 19, 2013 Abstract The bisphosphonate zoledronic acid (ZA) significantly reduces complications of bone metastasis by inhibiting resident macrophages, the osteoclasts. Recent clinical trials indicate additional anti-metastatic effects of ZA outside the bone. However, which step of metastasis is influenced and whether thisis due to directtoxicity on cancer cells or inhibition of the tumor promoting microenvironment, is unknown. In particular, tumor-associated and resident macrophages support each step of organ metastasis and could be a crucial target of ZA. Thus, we comparatively investigate the ZA effects on: i) different types of macrophages, ii) on breast cancer cells but also iii) on macrophage-induced invasion. We demonstrate that ZA concentrations reflecting the plasma level affected viability of human macrophages, murine bone marrow-derived macrophages as well as their resident brain equivalents, the microglia, while it did not influence the tested cancer cells. However, the effects on the macrophages subsequently reduced the macrophage/microglia-induced invasiveness of the cancer cells. In line with this, manipulation of microglia by ZA in organotypic brain slice cocultures reduced the tissue invasion by carcinoma cells. The characterization of human macrophages after ZA treatment revealed a phenotype/response shift, in particular after external stimulation. In conclusion, we show that therapeutic concentrations of ZA affect all types of macrophages but not the cancer cells. Thus, anti-metastatic effects of ZA are predominantly caused by modulating the microenvironment. Most importantly, our findings demonstrate that ZA reduced microglia-assisted invasion of cancer cells to the brain tissue, indicating a potential therapeutic role in the prevention of cerebral metastasis.

The metastatic colonization of the brain by carcinoma cells is still barely understood, in particular when considering interactions with the host tissue. The colonization comes with a substantial destruction of the surrounding host tissue. This leads to activation of damage responses by resident innate immune cells to protect, repair, and organize the wound healing, but may distract from tumoricidal actions. We recently demonstrated that microglia, innate immune cells of the CNS, assist carcinoma cell invasion. Here we report that this is a fatal side effect of a physiological damage response of the brain tissue. In a brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia and astrocytes comparable to that seen at the interface of human cerebral metastases. While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types. They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead. Gene expression and functional analyses revealed that the C-X-C chemokine receptor type 4 (CXCR4) and WNT signaling were involved in this process. Furthermore, CXCR4-regulated microglia were recruited to sites of brain injury in a zebrafish model and CXCR4 was expressed in human stroke patients, suggesting a conserved role in damage responses to various types of brain injuries. Together, our findings point to a detrimental misuse of the glial damage response program by carcinoma cells resistant to glia-induced apoptosis. peerReviewed

Learning outcome is an important measure for overall teaching quality and should be addressed by comprehensive evaluation tools. The authors evaluated the validity of a novel evaluation tool based on student self-assessments, which may help identify specific strengths and weaknesses of a particular course.In 2011, the authors asked 145 fourth-year students at Göttingen Medical School to self-assess their knowledge on 33 specific learning objectives in a pretest and posttest as part of a cardiorespiratory module. The authors compared performance gain calculated from self-assessments with performance gain derived from formative examinations that were closely matched to these 33 learning objectives.Eighty-three students (57.2%) completed the assessment. There was good agreement between performance gain derived from subjective data and performance gain derived from objective examinations (Pearson r=0.78; P.0001) on the group level. The association between the two measures was much weaker when data were analyzed on the individual level. Further analysis determined a quality cutoff for performance gain derived from aggregated student self-assessments. When using this cutoff, the evaluation tool was highly sensitive in identifying specific learning objectives with favorable or suboptimal objective performance gains.The tool is easy to implement, takes initial performance levels into account, and does not require extensive pre-post testing. By providing valid estimates of actual performance gain obtained during a teaching module, it may assist medical teachers in identifying strengths and weaknesses of a particular course on the level of specific learning objectives.