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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.

Background Sleep apnoea and type 2 diabetes (T2D) have been linked to malignancy. The aim of the present study was to evaluate the association between sleep apnoea and incidence of malignancy in patients with T2D. Methods The DIACORE (DIAbetes COhoRtE) study is a prospective, population-based cohort study in T2D patients. In the sleep disordered breathing substudy, the apnoea–hypopnoea index (AHI), oxygen desaturation index (ODI) and percentage of night-time spent with a peripheral oxygen saturation of, In patients with type 2 diabetes, sleep apnoea is not associated with the incidence of malignancy. However, stratified analysis shows that sleep apnoea is associated with incident malignancy in females, but not in males. https://bit.ly/37RAK8V

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 (LAMIShigh) had shorter EFS, PFS, and OS. Multivariate analyses revealed independence from IPI factors in EFS (HR 1.7, 95% CI 1.2–2.4, p-value = 0.001), PFS (HR 1.8, 95% CI 1.2–2.5, p-value = 0.001) and OS (HR 1.8, 95% CI 1.3–2.7, p-value = 0.001). Multivariate analyses adjusted for the IPI factors showed the signature to be independent from COO, MYC rearrangements and double expresser status (DE). LAMIShigh and simultaneous DE status characterized a patient subgroup with dismal prognosis and early relapse. Our data underline the importance of the microenvironment in prognosis. Combined analysis of stromal features, the IPI and DE may provide a new rationale for targeted therapy.

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.

Objective: Developing an integrative approach to early treatment response classification using survival modeling and bioinformatics with various biomarkers for early assessment of filgrastim (granulocyte colony stimulating factor) treatment effects in amyotrophic lateral sclerosis (ALS) patients. Filgrastim, a hematopoietic growth factor with excellent safety, routinely applied in oncology and stem cell mobilization, had shown preliminary efficacy in ALS. Methods: We conducted individualized long-term filgrastim treatment in 36 ALS patients. The PRO-ACT database, with outcome data from 23 international clinical ALS trials, served as historical control and mathematical reference for survival modeling. Imaging data as well as cytokine and cellular data from stem cell analysis were processed as biomarkers in a non-linear principal component analysis (NLPCA) to identify individual response. Results: Cox proportional hazard and matched-pair analyses revealed a significant survival benefit for filgrastim-treated patients over PRO-ACT comparators. We generated a model for survival estimation based on patients in the PRO-ACT database and then applied the model to filgrastim-treated patients. Model-identified filgrastim responders displayed less functional decline and impressively longer survival than non-responders. Multimodal biomarkers were then analyzed by PCA in the context of model-defined treatment response, allowing identification of subsequent treatment response as early as within 3 months of therapy. Strong treatment response with a median survival of 3.8 years after start of therapy was associated with younger age, increased hematopoietic stem cell mobilization, less aggressive inflammatory cytokine plasma profiles, and preserved pattern of fractional anisotropy as determined by magnetic resonance diffusion tensor imaging (DTI-MRI). Conclusion: Long-term filgrastim is safe, is well-tolerated, and has significant positive effects on disease progression and survival in a small cohort of ALS patients. Developing and applying a model-based biomarker response classification allows use of multimodal biomarker patterns in full potential. This can identify strong individual treatment responders (here: filgrastim) at a very early stage of therapy and may pave the way to an effective individualized treatment option.

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

Breast cancer has been associated with activation of the WNT signaling pathway, although the underlying molecular mechanisms are still unclear. Here, we found the WNT receptor ROR2 to be highly expressed in aggressive breast tumors and associated with worse metastasis-free survival. In order to understand the molecular basis of these observations, we overexpressed ROR2 in human breast cancer cell lines, inducing a BRCAness-like phenotype and rendering them resistant to PARP inhibition. High levels of ROR2 were associated with defects in cell morphology and cell-cell-contacts leading to increased tumor invasiveness. Using gene expression analysis we demonstrated an upregulation of several non-canonical WNT ligands in ROR2-overexpressing breast cancer cells, in particular WNT11. Co-immunoprecipitation confirmed that WNT11 is indeed a novel ligand for ROR2 that interacts with its cysteine-rich domain and triggers the invasion-promoting signaling via RHO/ROCK. Knockdown of WNT11 reversed the pro-invasive phenotype and the cellular changes in ROR2-overexpressing cells. Taken together, our studies 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.

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.

Brain and leptomeningeal metastasis (LMM) of non-small cell lung cancer is still associated with poor prognosis. Moreover, the current diagnostic standard for LMM often yields false negative results and the scientific progress in this field is still unsatisfying. We present a case of a 71-year old patient with an isolated LMM. While standard diagnostics could only diagnose a cancer of unknown primary, the use of [68Ga]-Pentixafor-PET/CT (CXCR4-PET/CT, a radiotracer targeting CXCR4) and a liquid biopsy of the cerebrospinal fluid revealed the primary NSCLC. The detection of L858R-EGFR, a common driver mutation in NSCLC, enabled us to treat the patient with Afatinib and monitor treatment using [68Ga]-Pentixafor PET/CT. To estimate the impact of CXCR4 signaling and its ligands in NSCLC brain metastasis we looked at their expression and correlation with EGFR mutations in a primary and brain metastasis data set and investigated the previously described binding of extracellular ubiquitin to CXCR4. In conclusion, we describe a novel approach to improve diagnostics towards LMM and underline the impact of the CXCL12/CXCR4 axis in brain metastasis in a subset of NSCLC patients. We cannot confirm a correlation of CXCR4 expression with EGFR mutations or the binding of extracellular ubiquitin as previously reported.

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.

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: In glioma surgery, 5-aminolevulinic acid (5-ALA) fluorescence reflects tumor infiltration, and fluorescence-assisted resection correlates with higher removal rates and improved progression-free survival. Recent studies report that a sizable proportion of brain metastases exhibit peritumoral infiltration on the cellular level. There is little information regarding whether 5-ALA is useful to guide surgery in the peritumoral zone in metastases. The aim of this study was to assess histologically whether 5-ALA fluorescence accurately reflects metastatic brain infiltration. Methods and Materials: Fluorescence-assisted tumor resection was performed in 27 patients with brain metastases. Patients received 20 mg/kg 5-ALA 3 hours before anesthesia. After resection, biopsy specimens of the surrounding parenchyma were analyzed for 5-ALA fluorescence and histologic evidence of infiltrating tumor cells. The correlation between 5-ALA positivity and immunohistochemical evidence of tumor in the peritumoral zone was also assessed. Results: Of 27 metastases, 23 (85%) were 5-ALA positive. For qualitative tissue analysis, 110 of 125 samples were collected. Metastatic infiltration was present in 49 samples with faint or red fluorescence; 33 samples without fluorescence were tumor-free. The presence of metastatic infiltration correlated with fluorescence (P < 0.001). Tumor infiltration correlated with fluorescence (blue fluorescence 0.09% ± 0.04% and red or faint fluorescence 3.26%; P = 0.003). Conclusions: Infiltration of surrounding brain tissue is a common finding in brain metastases in selected primary tumors. 5-ALA fluorescence correlates with tumor cell infiltration and might guide more radical resection. Key words: 5-ALA, Brain metastasis, Brain tumor, Fluorescence-guided surgery, Histology, Tumor infiltration

Introduction Central nervous system lymphoma (CNSL) is a rare neoplasia and arises as primary (PCNSL) or secondary CNSL (SCNSL) and most commonly occurs as diffuse large B-cell lymphoma. Although prognosis of this disease has significantly improved due to advantages in diagnostic procedures and intensification of treatment over the last decade there remain major challenges in the clinical management. Magnetic resonance imaging (MRI) - as the standard imaging modality - has difficulties to discriminate CNSL from other brain-derived tumors or metastasis. Moreover, prognostic scores in CNSL lack the ability to reliable define patients with high relapse risk 1. Therefore, novel strategies to facilitate diagnosis and to select patients that profit from intense treatment protocols are urgently needed. The C-X-C chemokine receptor 4 (CXCR4) is a transmembrane chemokine receptor with pivotal roles in cell homing and is often overexpressed in hematologic malignancies. CXCR4-directed positron emission tomography (PET) imaging with the tracer [68Ga]Pentixafor has been proven to be a suitable in vivo imaging modality for CXCR4 expression in lymphoid malignancies 2. To evaluate the feasibility of CXCR4-directed PET and the prognostic value of this imaging modality this retrospective proof-of-concept study evaluated [68Ga]Pentixafor PET imaging in patients with CNSL. Methods 11 patients with lymphoma of the CNS (n=8 PCNSL, n=3 SCNSL) were imaged with the CXCR4-directed PET tracer [68Ga]Pentixafor in this retrospective proof-of-concept study after signing inform consent. Lymphoma tissue was assessed for CXCR4 expression ex vivo by immunohistochemistry. The prognostic value of CXCR4-directed PET imaging was evaluated in a computed analysis in 7 patients with follow-up MRI. Treatment response calculated as treatment efficiency η by sequential MRI was correlated with [68Ga]Pentixafor PET derived parameters at diagnosis such as volume of PET positive lymphoma lesion V(PET), maximal PET uptake value within the lesion max(PET) or integrated uptake values over the lymphoma lesion ∫(PET). Analysis was performed in a lesion- and patient-based manner. Results [68Ga]Pentixafor PET imaging was positive in all patients with active disease (10/11 patients) with excellent contrast characteristics to the surrounding brain parenchyma. PET positive lesions correlated well with lymphoma lesions in MRI-T1c sequences. The surrounding edema depicted in MRI-FLAIR sequences was evaluated as PET negative (Figure 1). Semi-quantitative analysis revealed maximum standard uptake values of [68Ga]Pentixafor-derived PET from 4.2 to 23.3 within the lesions with a high tumor-to-background-ratio ranging from 13.2 to 83.0. The computed analysis revealed that CXCR4-directed PET parameters at diagnosis given by max(PET) and ∫(PET) significantly correlated with treatment response in the lesion-based as well as in the patient-based analysis. Specifically, ∫(PET) was the most significant prognostic factor in the present study (Figure 2 C, D, F). On the other hand tumor volume at diagnosis measured either by MRI or by [68Ga]Pentixafor PET did not denote a predictive marker for treatment response (Figure 2 A, B, E). Conclusion/Outlook CXCR4-directed PET imaging represents a novel imaging modality for CNSL. Due to its excellent contrast properties it might help to facilitate diagnosis and refine response assessment. Moreover, owing the predictive value for treatment response, CXCR4-directed PET could serve as a biomarker for the selection of patients profiting from intense treatment protocols. Furthermore, the feasibility of endoradiotherapeutic approaches targeting CXCR4 with Pentixather - the therapeutic twin of the imaging peptide Pentixafor - has been shown in hematologic malignancies and could be incorporated in the treatment of CNSL. References 1. Han CH, Batchelor TT. Diagnosis and management of primary central nervous system lymphoma. Cancer. 2017;123(22):4314-4324. 2.Kircher M, Herhaus P, Schottelius M, et al. CXCR4-directed theranostics in oncology and inflammation. Ann Nucl Med. 2018;32(8):503-511. Disclosures Wester: Scintomics: Other: Spouse CEO of Company; CXCR4-targeted radiopharmaceuticals: Other: Inventor; Scintomics GmbH, Germany: Other: Shareholder. Bassermann:Celgene: Consultancy, Research Funding.

Continuous updates add novel molecular markers to the WHO classification of CNS tumors. Additionally, molecularly informed trials may provide the basis for the introduction of additional predictive biomarkers. All these developments will prospectively require molecular testing to a yet unknown extent and still pose challenges to many neuropathology labs. Some of the relevant alterations (e.g. C11orf95-RELA) are hard to assess or complex subclassifications (e.g. for medulloblastomas) are required. Some labs rely on a two-tiered approach: i) methylation arrays for tumor classification and ii) targeted NGS panel sequencing for identifying actionable mutations. However, high initial investment and annual costs for running two platforms in parallel hinder a fast expansion of the new techniques in the breadth of neuropathology. We decided to focus on the sole use of the relatively cost-efficient Mini-Seq NGS platform (Illumina). First, we designed a DNA panel (FFPE, HaloPlex, Agilent; no Covaris required) that is suited for detection of DNA mutations, copy number alterations and chromosomal alterations (459 kbp, 58 genes, 4082 SNPs, 98.83% coverage). We successfully used this platform to support tumor classification in astrocytomas, oligodendrogliomas, meningiomas and medulloblastomas and identified actionable targets of clinical use. We then designed a RNA panel (FFPE, Sure Select, Agilent) that detects gene fusions and covers a broad range of alterations particularly relevant to pediatric tumors (e.g. pediatric glioblastomas, ependymomas, CNS PNETs) (148 kbp, coding sequence of 31 genes, 99.88% coverage). With these two panels we cover the most relevant molecular alterations in the field of neurooncology. The cost-efficient and flexible single platform approach enables state-of-the-art molecular diagnostics in virtually every neuropathology lab in a quality-controlled manner. Functionality is guaranteed for future revisions of the WHO classification as novel biomarkers can be easily included in an adapted panel design.

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.

To evaluate whether tumour-derived microvesicles (T-MV), originating from the plasma membrane, represent suitable cancer biomarkers, we isolated MV from peripheral blood samples of cancer patients with locally advanced and/or metastatic solid tumours (n = 330, including 79 head & neck cancers, 74 lung cancers, 41 breast cancers, 28 colorectal cancers and 108 with other cancer forms) and controls (n = 103). Whole MV preparations were characterised using flow cytometry. While MV carrying the tumour-associated proteins MUC1, EGFR and EpCAM were found to be enhanced in a tumour-subtype-specific way in patients’ blood, expression of the matrix metalloproteinase inducer EMMPRIN was increased independent of tumour type. Higher levels of EMMPRIN+-MV correlated significantly with poor overall survival, whereas the other markers were prognostic only in specific tumour subgroups. By combining all four tumour-associated antigens, cancer patients were separated from healthy controls with an AUC of up to 0.85. Ex vivo, whole MV preparations from cancer patients, in contrast to those of controls, induced a tumour-supporting phenotype in macrophages and increased tumour cell invasion, which was dependent on the highly glycosylated isoform of EMMPRIN. In conclusion, the detection of T-MV in whole blood, even in minor amounts, is feasible with standard techniques, proves functionally relevant and correlates with clinical outcome.

// 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.

The putative protein tyrosine kinase (PTK) inhibitor tyrphostin AG126 has proven beneficial in various models of inflammatory disease. Yet molecular targets and cellular mechanisms remained enigmatic. We demonstrate here that AG126 treatment has beneficial effects in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. AG126 alleviates the clinical symptoms, diminishes encephalitogenic Th17 differentiation, reduces inflammatory CNS infiltration as well as microglia activation and attenuates myelin damage. We show that AG126 directly inhibits Bruton's tyrosine kinase (BTK), a PTK associated with B cell receptor and Toll-like receptor (TLR) signaling. However, BTK inhibition cannot account for the entire activity spectrum. Effects on TLR-induced proinflammatory cytokine expression in microglia involve AG126 hydrolysis and conversion of its dinitrile side chain to malononitrile (MN). Notably, while liberated MN can subsequently mediate critical AG126 features, full protection in EAE still requires delivery of intact AG126. Its anti-inflammatory potential and especially interference with TLR signaling thus rely on a dual mechanism encompassing BTK and a novel MN-sensitive target. Both principles bear great potential for the therapeutic management of disturbed innate and adaptive immune functions.

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.

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.

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.

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

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

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.

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.

Introduction: IELSG32 is an international randomized phase II trial with 2 key clinical questions in pts with PCNSL. The first question focused on optimal induction therapy, and results of the 1st randomization have demonstrated that MATRix combination (methotrexate (MTX), cytarabine (ARAC), thiotepa, rituximab (R)) is associated with significantly better outcome [Ferreri AJ, et al. Lancet Haematol 2016]. The second question addresses the efficacy and neurotolerability of ASCT, as an alternative to WBRT as consolidation. Herein, we report the results of the 2nd randomization (NCT01011920). Methods: HIV-neg pts 18-70 ys and ECOG PS ≤3 with new histology-proven PCNSL and measurable disease were randomly assigned to receive 4 courses of MTX-ARAC (arm A); or R-MTX-ARAC (arm B); or MATRix (arm C). ASC were collected after 2nd c. Pts with responsive or stable disease were further randomized between WBRT 36 ± 9 Gy (arm D) and BCNU-thiotepa conditioned/ASCT (arm E). Stratification parameters were induction arm and response . Primary endpoint of the 2ndrandomization was 2-year PFS. To demonstrate a 2-yr PFS improvement from 65% (P0) to 85% (P1), 52 pts/arm (one-sided; α 5%; β 95%) were required. Consolidation arm would be considered effective if ≥40 pts were progression-free survivors at 2 ys. Analyses were performed on an intention-to-treat (ITT) basis. Effects of consolidation treatments on neurocognitive functions and quality of life (QoL) were assessed with the IPCG tests panel and EORTC-QLQ at baseline, after treatment and every 6 months afterwards. Results: 227 pts were enrolled in 52 centers of 5 countries; 219 assessable pts were referred to 1st randomization (arm A 75; B 69; C 75). 167 pts had responsive or stable disease after induction: 49 pts were excluded from 2nd randomization (poor mobilization, poor condition or patients' refusal); 118 pts were thus randomized (59 pts/arm) and constitute the study population (median age 58 ys; range 18-70). 15 (13%) pts had high IELSG risk, 3 pts had ocular disease and 20 pts had meningeal disease; with the exception of a higher male prevalence in arm D, there were no differences in clinical presentation between two arms; There were 6 protocol violations: 4 pts randomly allocated to arm D were treated with ASCT and 2 pts randomly allocated to arm E were treated with WBRT; 5 pts (D 2; E 3) refused consolidation. Therefore, per-protocol (PP) groups consisted of 55 pts treated with WBRT and 58 with ASCT. Both consolidation therapies were well tolerated. Grade 4 toxicity was uncommon (≤5% of pts); as expected, hematological toxicity was more common in arm E (neutropenia 5% vs. 71%; p=0.00001 - thrombocytopenia 2% vs. 72%; p=0.00001). There were 2 toxic deaths (infections), both in arm E. Neuropsychological tests showed a significant impairment of attention/executive functions and a non-significant trend to impaired memory among pts treated with WBRT, whereas pts treated with ASCT exhibited improved functions. Both consolidation therapies were associated with significant improvement in language and QoL. Both WBRT and ASCT were active and resulted in significant improvement in CR rate: from 54% after induction to 95% (95%CI: 90-100) after consolidation in arm D, and from 53% to 93% (95%CI: 87-99) in arm E. After a median follow-up of 40 months (range 24-76), there were 20 events (16 relapses, 2 PDs, 2 off-therapy deaths) in arm D, and 25 events (18 relapses, 2 PDs, 2 toxic deaths, 3 off-therapy deaths) in arm E. Importantly, WBRT and ASCT were both effective, with a number of progression-free survivors at 2 ys equal to the pre-determined efficacy threshold: 40 among both the first 52 arm-D and 52 arm-E pts. There were no significant differences in PFS between WBRT and ASCT; on ITT basis, the 2-yr PFS was 80 ± 5% after WBRT and 70 ± 6% after ASCT; per protocol, the 2-yr PFS was 76 ± 6% and 75 ± 6%, respectively. In multivariate analysis, IELSG risk group, number of lesions and induction arm were independently associated with PFS; gender, CSF cytology status and consolidation arm were not. Forty-two pts randomized to arm D and 37 randomized to arm E are alive, with 2-year OS of 85 ± 5% and 71 ± 6% (p=0.12), respectively. Conclusions: WBRT and ASCT are both feasible, active and effective as consolidation therapies after high-dose-MTX-based chemoimmunotherapy in pts ≤70 ys with PCNSL. Potential impairment of specific neurocognitive functions after WBRT should be considered at the time of therapeutic decision. Disclosures Ferreri: Sandoz: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Research Funding, Speakers Bureau; Italfarmaco: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adienne: Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees. Fox:Adienne: Honoraria, Research Funding; Takeda: Honoraria, Other: travel funding; Gilead: Honoraria, Other: travel funding; Janssen: Honoraria, Other: travel funding; AbbVie: Consultancy. Röth:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Johnson:Celldex Therapeutics: Research Funding. Linton:Takeda: Research Funding. Hess:Roche: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Roche, CTI, Pfizer, Celgene: Research Funding; Celgene: Honoraria; Pfizer: Honoraria. Stilgenbauer:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau. Rummel:Roche Pharma AG: Other: Personal fees, Research Funding; Mundipharma GmbH: Other: Personal fees, Research Funding. Illerhaus:Riemser: Honoraria; Amgen: Honoraria.

// 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

Melanoma frequently metastasizes to the brain, resulting in dismal survival. A major obstacle for characterizing mechanisms of brain metastasis is the lack of tractable pre-clinical models that mimic the multi-stage process of brain metastasis. We established a novel model of spontaneous melanoma brain metastasis in immunocompetent mice. Aggressive local tumors are formed following orthotopic injection, and 3-4 months after surgical excision of the primary tumor, 50% of the mice develop brain macrometastases. By utilizing a unique ex-vivo modeling system, we detected brain micrometastases in 50-60% of the mice and quantified the metastatic load. Moreover, we show that the detection of melanoma transcripts in cerebrospinal fluid (CSF) is correlated with brain metastases. Interestingly, preliminary findings suggest that the melanoma-derived transcripts are found in exosomes. Astrocytes are glial cells that maintain brain homeostasis, and constitute a central part of the brain microenvironment. Reactive astrogliosis is the primary response of astrocytes to brain insult. While much data have accumulated on the contribution of astrogliosis to neurodegenerative diseases, the functional role of astrogliosis in promoting melanoma brain metastases is unknown. Utilizing our model we demonstrate early changes in the microenvironment and show that micrometastases are associated with activation of astrocytes and with hyperpermeability of the blood-brain-barrier. In-vitro, paracrine signaling by melanoma cells activated astrocytes to up-regulate a ‘wound healing program’ including Cxcl10 and Serpins. Reciprocally, astrocytes facilitated the invasion of melanoma cells and were necessary for primary outgrowth in 3D co-cultures. Finally, we show that intracranial co-injection of astrocytes with melanoma cells resulted in a 9 fold increase of tumor volume, and that the same gene-signature was up-regulated in astrocytes in-vivo. Studying spontaneous melanoma brain metastasis in a clinically relevant setting enables in-depth understating of the metastatic microenviromet which is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Citation Format: Hila Schwartz, Eran Blacher, Malak Amer, Nir Livneh, Anat Klein, Dikla Ben-Shushan, Shelly Soffer, Reuven Stein, Ronit Satchi-Fainaro, Viktor Umansky, Tobias Pukrop, Neta Erez. Melanoma brain metastasis is facilitated by instigation of reactive astrogliosis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C26.

With the improvement of therapeutic options for the treatment of breast cancer, the development of brain metastases has become a major limitation to life expectancy in many patients. Loss of Phosphatase and tensin homolog (PTEN) is correlated with occurrence of breast cancer brain metastases, but its functional role in metastases formation remains unclear. Here, we first assessed the clinical role of PTEN expression in brain metastases, by performing immunohistochemical analyses on a TMA consisting of 132 breast cancer brain metastases samples. Loss of PTEN protein expression was found in 48.6% of brain metastases, and significantly associated with the triple-negative breast cancer subtype (67.5%, p = 0.01). Loss of PTEN was furthermore significantly associated with shorter overall survival (p = 0.048). We then investigated the biological role of PTEN in breast cancer brain metastasis formation by overexpressing PTEN in the triple-negative brain metastatic breast cancer cell cline MDA-MB-231 BR. Overexpression of PTEN was shown to reduce the AKT pathway activation especially by reducing the Akt1 kinase activity. Coculture experiments with glia cells (astrocytes and microglia) showed that astrocytes can inhibit cell proliferation of the tumor cells in a PTEN dependent manner. Both astrocytes and microglia promote migration of MDA-MB-231 BR cells in a PTEN-dependent manner in vitro. Using an organotypic brain slice model as a representation of the brain tumor microenvironment, the PTEN-dependent invasion of tumor cells could be confirmed. Furthermore, PTEN overexpression reduced the astrocyte activation. Coculture experiments with glia cells showed PTEN-dependent, differential cytokine expression. We could identify an endocrine and paracrine regulation between tumor cells and astrocytes, where GM-CSF, CSF2RA, EGF and PTEN play an important role. In conclusion, loss of PTEN is associated with poor prognosis and is an important mediator of a “vicious circle” mediated by the cross talk between tumor cells and glia cells. Citation Format: Harriet Wikman, Ina Hohensee, Han-Ning Chuang, Astrid Grottke, Stefan Werner, Alexander Schulte, Jakob Matschke, Markus Glatzel, Stefan Horn, Manfred Jücker, Tobias Pukrop, Klaus Pantel. PTEN is an important mediator of tumor and glia cell crosstalk in breast cancer brain metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1571.

An essential function of the transcription factors LEF1/TCF4 in cerebral metastases of lung adenocarcinomas has been described in mouse models, suggesting a WNT/β-catenin effect as potential mechanism. Their role in humans is still unclear, thus we analyzed LEF1, TCF4, β-catenin, and early stage prognostic markers in 25 adenocarcinoma brain metastases using immunohistochemistry (IHC). IHC revealed nuclear TCF4 in all adenocarcinoma samples, whereas only 36 % depicted nuclear LEF1 and nuclear β-catenin signals. Samples with nuclear LEF1 as well as high TCF4 (++++) expression were associated with a shorter survival (p = 0.01, HR = 6.68), while nuclear β-catenin had no significant impact on prognosis and did not significantly correlate with nuclear LEF1. High proliferation index Ki67 was associated with shorter survival in late-stage disease (p = 0.03, HR 3.27). Additionally, we generated a LEF1/TCF4 as well as an AXIN2 signature, the latter as representative of WNT/β-catenin activity, following a bioinformatics approach with a gene expression dataset of cerebral metastases in lung adenocarcinoma. To analyze the prognostic relevance in primary lung adenocarcinomas, we applied both signatures to a microarray dataset of 58 primary lung adenocarcinomas. Only the LEF1/TCF4 signature was able to separate clusters with impact on survival (p = 0.01, HR = 0.32). These clusters displayed diverging enrichment patterns of the cell cycle pathway. In conclusion, our data show that LEF1/TCF4, but not β-catenin, have prognostic relevance in primary and cerebrally metastasized human lung adenocarcinomas. In contrast to the previous in vivo findings, these results indicate that LEF1/TCF4 act independently of β-catenin in this setting. Electronic supplementary material The online version of this article (doi:10.1007/s10585-012-9552-7) contains supplementary material, which is available to authorized users.

A considerable percentage of rectal cancers are resistant to standard preoperative chemoradiotherapy. Because patients with a priori-resistant tumors do not benefit from multimodal treatment, understanding and overcoming this resistance remains of utmost clinical importance. We recently reported overexpression of the Wnt transcription factor TCF4, also known as TCF7L2, in rectal cancers that were resistant to 5-fluorouracil-based chemoradiotherapy. Because Wnt signaling has not been associated with treatment response, we aimed to investigate whether TCF4 mediates chemoradioresistance. RNA interference-mediated silencing of TCF4 was employed in three colorectal cancer (CRC) cell lines, and sensitivity to (chemo-) radiotherapy was assessed using a standard colony formation assay. Silencing of TCF4 caused a significant sensitization of CRC cells to clinically relevant doses of X-rays. This effect was restricted to tumor cells with high T cell factor (TCF) reporter activity, presumably in a β-catenin-independent manner. Radiosensitization was the consequence of (i) a transcriptional deregulation of Wnt/TCF4 target genes, (ii) a silencing-induced G(2)/M phase arrest, (iii) an impaired ability to adequately halt cell cycle progression after radiation and (iv) a compromised DNA double strand break repair as assessed by γH2AX staining. Taken together, our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance. Moreover, they suggest that TCF4 is a promising molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy.

A role of WNT signaling for primary breast cancers of the basal-like subtype and as a predictor of brain metastasis has been described. However, a responsible WNT ligand has not been identified. To further clarify this question, we comparatively investigated 22 human breast cancer brain metastases as well as the highly invasive human breast cancer cell line MDA-MB-231 and the weakly motile MCF-7 as models for the basal-like and the luminal A subtype. WNT5A and B were found overexpressed in MDA-MB-231 cells as compared with MCF-7. This corresponded to reduction of MDA-MB-231 invasiveness by WNT inhibitors, whereas MCF-7 invasion was enhanced by recombinant WNT5B and abolished by WNT and Jun-N-terminal kinase antagonists. Expression and subcellular distribution of β-catenin remained uninfluenced. Consistently, β-catenin was not localized in the nuclei of brain metastases while there was strong nuclear c-Jun staining. Similar to MDA-MB-231, metastases showed expression of WNT5A/B and the alternative WNT receptors ROR1 and 2. These findings were validated using external gene expression datasets (Gene Expression Omnibus) of different breast cancer subtypes and brain metastases. Hierarchical cluster analysis yielded a close relation between basal-like cancers and brain metastases. Gene set enrichment analyses confirmed WNT pathway enrichment not only in basal-like primaries but also in cerebral metastases of all subtypes. In conclusion, WNT signaling seems highly relevant for basal-like and other subtypes of breast cancers metastasizing into the brain. β-catenin-independent WNT signaling, presumably via ROR1-2, plays a major role in this context.

B-cell chronic lymphocytic leukemia (B-CLL) is phenotypically characterized by cell surface co-expression of CD19, CD20, CD5, and CD23. However, the concomitant presence of other antigens distinctive of a particular leukocyte subset, e.g. T cells, is an unusual finding in B-CLL. In the present report, a case of B-CLL with aberrant expression of the T-cell-associated antigen CD8 is described. Flow cytometric analysis of the patient's cells demonstrated lack of CD38 expression, and cytogenetic analysis by FISH did not show any abnormalities of chromosomes 17p, 11q, and 12. Furthermore, the B-CLL cells expressed only low levels of the tyrosine kinase ZAP-70, which was in agreement with the mutated status of the immunoglobulin heavy-chain variable-region gene (IgVH). In summary, the determined profile of prognostic markers together with the highly stable and indolent disease in the described patient suggest a good prognosis of B-CLL with aberrant CD8 expression.

Background: The clinical response of locally advanced rectal cancers to preoperative chemoradiotherapy is very heterogeneous. We previously identified TCF4, the key downstream effector of the Wnt/β-catenin signaling pathway, as over-expressed in tumors that were resistant to chemoradiotherapy. The aim of this study was to explore the potential functional relevance of Wnt/ß-catenin signaling for mediating treatment resistance. Methods: Using RNA interference, TCF4 and β-catenin were inhibited in the colorectal cancer cell lines SW837 and SW480. Subsequently, these cell lines were irradiated with varying doses of X-rays. In addition, normal retinal epithelial cells (RPE) were stimulated with Wnt-3a for 28 hours, and also irradiated. Wnt/β-catenin activity was assessed using the TOPFLASH/FOPFLASH reporter assay, and induction of Axin2 was confirmed by Western blotting. Results: Protein levels of both TCF4 and β-catenin were considerably reduced following RNAi, accompanied by a decreased TOP/FOP reporter activity. Importantly, silencing of both proteins led to a pronounced radiosensitization in SW837 and SW480 cells. Exogenous stimulation of canonical Wnt signaling in RPE cells using Wnt-3a resulted in an increased Axin2 expression and an increased TOP/FOP reporter activity. Importantly, however, this activation of canonical Wnt signaling was associated with significant increase in resistance to radiation therapy. Conclusion: TCF4 was found to be over-expressed in resistant rectal carcinomas, and its RNAi-mediated silencing caused a significant radiosensitization. To determine whether this effect was dependent on canonical Wnt signaling, we inhibited ß-catenin, another key factor of this pathway and binding partner of TCF4, and observed a similar sensitization to radiation therapy. Follow-up experiments show that external stimulation of canonical Wnt signaling confers radiation resistance in normal cells. These data suggest that targeting canonical Wnt signaling may represent a potential therapeutic strategy for sensitizing tumor cells to radiation. In vivo studies are ongoing to validate these results. Citation Format: Georg Emons, Janneke Möller, Melanie Spitzner, Emil Kendziorra, Jochen Gaedcke, Margret Rave-Fränk, Tobias Pukrop, Michael Ghadimi, Thomas Ried, Marian Grade. Canonical Wnt signaling mediates resistance of colorectal cancer cells to radiation therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1588. doi:10.1158/1538-7445.AM2013-1588

Background: We recently demonstrated that the Wnt transcription factor TCF4 is over-expressed in rectal cancers that were resistant to preoperative chemoradiotherapy. Because the association between Wnt signaling and chemoradioresistance is a novel finding, we aimed to investigate whether this over-expression is functionally relevant. Methods: Three colorectal cancer cell lines were transfected with two shRNA-vectors targeting TCF4. Stable single-cell clone (SCC) populations were established, and selected clones were irradiated at 0, 1, 2, 4, 6 and 8 Gy. γH2AX staining was used to evaluate DNA double strand repair after irradiation, and changes in cell cycle distribution were analyzed before and after radiation. Finally, β-catenin activity was inhibited using both siRNAs and the small molecule inhibitor XAV939. Results: Silencing of TCF4 led to a significant radiosensitization in SW837 and SW480, whereas there was no effect in HT-29. Well-fitting, we observed significantly more γH2AX foci 24 hrs after radiation in SW837 SCCs compared to HT-29 SCCs, pointing to an impaired DNA damage repair in SW837. Furthermore, in SW837 SCCs, but not in HT-29 SCCs, we noticed a change in the cell cycle distribution towards radiosensitive cell cycle phases before radiation, and compromised cell cycle control after radiation. Finally, both siRNA- and small molecule-mediated inhibition of β-catenin activity did not influence the sensitivity to chemoradiotherapy. Conclusion: Our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance in a β-catenin-independent manner. Moreover, they suggest that TCF4 is a potential molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5729. doi:1538-7445.AM2012-5729

Background: The clinical response of locally advanced rectal cancers to preoperative chemoradiotherapy is very heterogeneous. We recently profiled a series of responsive and resistant rectal carcinomas using gene expression microarrays and identified TCF7L2, the main downstream effector of the Wnt/β-catenin pathway, as over-expressed in the resistant tumors. The aim of this study was to explore the relevance of TCF7L2 for resistance to radiation and to study the underlining mechanisms. Methods: We transfected three microsatellite-stable colorectal cancer cell lines (SW837, SW480 and HT-29) with two different shRNA-vectors targeting TCF7L2 and a non-silencing. Next, we established single cell clone (SCC) populations, and after confirming the reduction of TCF7L2 protein levels by Western blotting, we irradiated the SCCs at 0, 1, 2, 4, 6 and 8 Gy and calculated survival fractions. To gain functional insight, we chose SW837 and HT-29 for further experiments. Using γH2AX staining we evaluated DNA double strand repair after irradiation at 2 Gy and analyzed cell cycle changes before and after radiation. Furthermore, we studied Wnt/β-catenin pathway activity using the TOPFLASH/FOPFLASH reporter assay. Results: RNAi-mediated silencing of TCF7L2 led to a pronounced reduction in TCF7L2 protein levels. In radiation experiments we observed a highly significant radiosensitization in SW837 and SW480 SCCs, whereas no effect was observed in HT-29 SCCs. Well-fitting, we observed significantly more γH2AX foci 24h after radiation in SW837 SCCs compared to HT-29 SCCs, pointing to impaired DNA damage repair in the SCCs from SW837. Furthermore, in SW837 SCCs but not in HT-29 SCCs we noticed a change in the cell cycle distribution towards radiosensitive cell cycle phases before radiation and compromised cell cycle control after radiation. Finally, using the TOPFLASH/FOPFLASH reporter assay we found SW837 wild-type cells to show high Wnt/β-catenin pathway activity, while HT-29 wild-type cells only showed very low activity. Conclusion: TCF7L2 was found to be over-expressed in resistant rectal carcinomas, and its RNAi-mediated silencing caused a significant radiosensitization, mediated by DNA damage repair deficiencies and impaired cell cycle control. Interestingly, radiosensitization effetcts were only observed in cell lines showing Wnt/β-catenin pathway activity. Thus, we have uncovered a novel role of Wnt/β-catenin signaling for mediating resistance to multimodal cancer therapy. Moreover, these data suggest that TCF7L2 is a potential molecular target for sensitizing Wnt/β-catenin-dependent tumor cells to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2011-2490

Background: The standard treatment for locally advanced rectal carcinomas consists of preoperative chemoradiotherapy (CT/RT), followed by radical surgery. However, the clinical response to this treatment strategy is very heterogeneous. To determine the molecular basis of disparate response, we recently profiled a series of responsive and resistant rectal cancers using gene expression microarrays and identified a set of differentially expressed genes. One gene that was significantly over-expressed in the resistant tumors was TCF7L2 (also known as TCF4), the main downstream effector of the Wnt signaling pathway. The aim of this study was to understand its role in treatment response by testing the hypothesis that RNAi-mediated silencing of TCF7L2 sensitizes tumor cells to radiation therapy. Methods: The colorectal cancer cell line SW480, which is relatively resistant to radiation, was transfected with two different shRNA-vectors targeting TCF7L2, and a non-silencing control. Subsequently, stable single cell clones were established, and knockdown efficiency was analyzed by Western blot analysis. For each vector, individual clones were irradiated at 2, 4, 6 and 8 Gy. Colonies were counted after 10 days to calculate the respective surviving fractions. Results: TCF7L2 protein levels were reduced to ∼ 34% for shRNA_1, and ∼ 29% for shRNA_2. This led to a ∼ 1.6-fold growth inhibition (plating efficiency) compared to the non-silencing control. Importantly, RNAi-mediated silencing of TCF7L2 significantly reduced colony-formation after radiation (dose reduction factors of ∼ 1.5 and ∼ 2.0 at 37% survival for shRNA_1 and shRNA_2, respectively). These results were validated in a second cell line (HT29). In addition, we analyzed global gene expression profiles after RNAi to analyze the global transcriptomic changes induced by silencing TCF7L2. Conclusion: TCF7L2 is over-expressed in rectal carcinomas resistant to chemoradiotherapy. RNAi-mediated silencing of this gene caused a significant growth inhibition and, importantly, radiosensitization of colorectal cancer cells. Thus, TCF7L2 might represent a potential molecular target to sensitize a priori resistant tumor cells. Additionally, these data provide first evidence that the Wnt signaling pathway might not only be central for the development of colorectal cancers, but also for the resistance to therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 487.