Whole stromal fibroblast signature is linked to specific chemokine and immune infiltration patterns and to improved survival in NSCLC.

Cancer associated fibroblasts (CAF) are known to orchestrate multiple components of the tumor microenvironment, whereas the influence of the whole stromal-fibroblast compartment is less understood. Here, an extended stromal fibroblast signature was investigated to define its impact on immune cell infiltration. The lung cancer adenocarcinoma (LUAD) data set of the cancer genome atlas (TCGA) was used to test whole stroma signatures and cancer-associated fibroblast signatures for their impact on prognosis. 3D cell cultures of the NSCLC cancer cell line A549 together with the fibroblast cell line SV80 were used in combination with infiltrating peripheral blood mononuclear cells (PBMC) for in-vitro investigations. Immune cell infiltration was assessed via flow cytometry, chemokines were analyzed by immunoassays and RNA microarrays. Results were confirmed in specimens from NSCLC patients by flow cytometry or immunohistochemistry as well as in the TCGA data set. The TCGA analyses correlated the whole stromal-fibroblast signature with an improved outcome, whereas no effect was found for the CAF signatures. In 3D microtumors, the presence of fibroblasts induced infiltration of B cells and CD69+CD4+ T cells, which was linked to an increased expression of CCL13 and CXCL16. The stroma/lymphocyte interaction was confirmed in NSCLC patients, as stroma-rich tumors displayed an elevated B cell count and survival in the local cohort and the TCGA data set. A whole stromal fibroblast signature was associated with an improved clinical outcome in lung adenocarcinoma and in vitro and in vivo experiments suggest that this signature increases B and T cell recruitment via induction of chemokines.

A drug library screen identifies Carbenoxolone as novel FOXO inhibitor that overcomes FOXO3-mediated chemoprotection in high-stage neuroblastoma.

The transcription factor FOXO3 has been associated in different tumor entities with hallmarks of cancer, including metastasis, tumor angiogenesis, maintenance of tumor-initiating stem cells, and drug resistance. In neuroblastoma (NB), we recently demonstrated that nuclear FOXO3 promotes tumor angiogenesis in vivo and chemoresistance in vitro. Hence, inhibiting the transcriptional activity of FOXO3 is a promising therapeutic strategy. However, as no FOXO3 inhibitor is clinically available to date, we used a medium-throughput fluorescence polarization assay (FPA) screening in a drug-repositioning approach to identify compounds that bind to the FOXO3-DNA-binding-domain (DBD). Carbenoxolone (CBX), a glycyrrhetinic acid derivative, was identified as a potential FOXO3-inhibitory compound that binds to the FOXO3-DBD with a binding affinity of 19 µM. Specific interaction of CBX with the FOXO3-DBD was validated by fluorescence-based electrophoretic mobility shift assay (FAM-EMSA). CBX inhibits the transcriptional activity of FOXO3 target genes, as determined by chromatin immunoprecipitation (ChIP), DEPP-, and BIM promoter reporter assays, and real-time RT-PCR analyses. In high-stage NB cells with functional TP53, FOXO3 triggers the expression of SESN3, which increases chemoprotection and cell survival. Importantly, FOXO3 inhibition by CBX treatment at pharmacologically relevant concentrations efficiently repressed FOXO3-mediated SESN3 expression and clonogenic survival and sensitized high-stage NB cells to chemotherapy in a 2D and 3D culture model. Thus, CBX might be a promising novel candidate for the treatment of therapy-resistant high-stage NB and other "FOXO-resistant" cancers.

Treatment According to a Comprehensive Molecular Profiling Can Lead to a Better Outcome in Heavily Pretreated Patients With Metastatic Cancer: Data of a Pooled Analysis.

PURPOSE: Improvements in systemic treatment have led to a prolongation of survival and quality of life in patients with metastatic tumors in recent years. However, despite this improved standard of care, it is expected that the progression-free survival (PFS) for patients with refractory cancers will continue to decline over subsequent therapy lines. In those patients, studies and meta-analyses showed that treatment based on multiplatform molecular profiling (MMP) of tumor tissue may derive a clinical benefit. The aim of this study was to analyze if molecular-based therapy may prolong PFS compared with the PFS of the immediately prior therapy. METHODS: We pooled clinical data of 140 patients treated within 3 recently conducted pilot studies and included an additional 21 patients who were treated within the ongoing ONCO-T-PROFILE program. The PFS of the molecular-based treatment was compared with the PFS of the previous therapy using Kaplan-Meier curves. RESULTS: In heavily pretreated cancer patients, the PFS could be significantly improved using molecular-based treatment options (120.0 vs. 89.5 days). More than 50% of patients showed a clinical benefit from MMP-guided therapy as defined by a PFS ratio of 1.3 or greater. CONCLUSIONS: We conclude that pretreated cancer patients can benefit from incorporation of molecular profiling, as demonstrated by not only an increase of the PFS ratio but also PFS. Further randomized trials in specific tumor subtypes may help establish specific patient populations who might benefit most from MMP guidance.

Aviscumine, a recombinant ribosomal inhibitor, increases the antitumor activity of natural killer cells.

Aviscumine, a recombinant lectin I, has been identified as an immunomodulatory agent within a new class of ribotoxic stress-inducing anticancer substances that have demonstrated efficacy in phase I/II trials. The aim of the present study was to elucidate the presumed effect of aviscumine on enhancing human natural killer (NK) cell antitumor cytotoxicity. To measure the effect of aviscumine on human NK cell cytotoxicity, chromium-51-release assays against K-562 cells were performed with isolated NK cells from the whole blood of 34 healthy volunteers. Two effector-to-target cell ratios (12.5:1 and 25:1) were used by two independent investigators with a focus on the concentration-dependent effect (0.5 vs. 1 ng/ml aviscumine), reproducibility (first vs. second investigator) and the specificity of the effect by comparison to a heat-inactivated aliquot and interleukin 2 (IL-2) stimulation (10 ng/ml). The mediation of the effect via degranulation was demonstrated by flow cytometric analyses of CD107α expression. Statistics were performed with SPSS using Student's t-tests for normally distributed data. Aviscumine induced a significant and reproducible, concentration-dependent increase in NK cell cytotoxicity (n=22; P<0.01 for both concentrations and ratios), which was also demonstrated when administered in combination with IL-2 (n=12; 12.5:1 ratio, P<0.001; 25:1 ratio, P=0.025) and when compared with the heat-inactivated aliquots (n=12; 12.5:1, P=0.004; 25:1 ratio, P=0.007). The mediation of its effect via interferon γ degranulation was demonstrated by significantly enhanced CD107α expression (n=7; P=0.005). Taken together, the results indicate that aviscumine induced an increase in NK cell anticancer cytotoxicity. These results highlight its clinical potential as an immunostimulatory agent, particularly with regard to combined use with chemotherapeutics or immune checkpoint inhibitors. However, further studies are required.

The influence of stromal cells and tumor-microenvironment-derived cytokines and chemokines on CD3+CD8+ tumor infiltrating lymphocyte subpopulations.

The tumor microenvironment has been identified as a major mediator of immunological processes in solid tumors. In particular, tumor-associated fibroblasts are known to interact with tumor infiltrating immune cells. We describe the influence of fibroblasts and tumor-microenvironment-derived cytokines on the infiltration capacity of CD3+CD8+ cytotoxic T lymphocyte subpopulations using a multicellular 3D co-culture system. 3D tumor microtissues were cultivated using a hanging drop system. Human A549 and Calu-6 cancer cell lines were incubated alone or together with the human fibroblast cell line SV80 for 10 d to form microtissues. On day 10, peripheral blood mononuclear cells (PBMC) were added with or without cytokine stimulation for 24 h. Infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of the microtissues and the infiltration of the PBMCs were analyzed by immunohistochemistry, and endogenous cytokine and chemokine expression was analyzed with a multi-cytokine immunoassay. Secretion of chemokines is increased in microtissues consisting of cancer cells and fibroblasts. PBMC infiltrate the whole spheroid in cancer cell monocultures, whereas in co-cultures of cancer cells and fibroblasts, PBMCs are rather localized at the margin. Activated CD69+ and CD49d+ T lymphocytes show an increased microtissue infiltration in the presence of fibroblasts. We demonstrate that the stromal component of cancer microtissues significantly influences immune cell infiltration. The presence of fibroblasts in cancer microtissues induces a shift of T lymphocyte infiltration toward activated T lymphocytes.

Development of a 3D angiogenesis model to study tumour - endothelial cell interactions and the effects of anti-angiogenic drugs.

The tumour microenvironment and tumour angiogenesis play a critical role in the development and therapy of many cancers, but in vitro models reflecting these circumstances are rare. In this study, we describe the development of a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines (A549 and Colo699) in combination with a fibroblast cell line (SV 80) and two different endothelial cell lines in a hanging drop technology. Endothelial cells aggregated either in small colonies in Colo699 containing microtissues or in tube like structures mainly in the stromal compartment of microtissues containing A549. An up-regulation of hypoxia and vimentin, ASMA and a downregulation of E-cadherin were observed in co- and tri-cultures compared to monocultures. Furthermore, a morphological alteration of A549 tumour cells resembling "signet ring cells" was observed in tri-cultures. The secretion of proangiogenic growth factors like vascular endothelial growth factor (VEGF) was measured in supernatants. Inhibition of these proangiogenic factors by using antiangiogenic drugs (bevacizumab and nindetanib) led to a significant decrease in migration of endothelial cells into microtissues. We demonstrate that our method is a promising tool for the generation of multicellular tumour microtissues and reflects in vivo conditions closer than 2D cell culture.

Implementing liquid biopsies into clinical decision making for cancer immunotherapy.

During the last decade, novel immunotherapeutic strategies, in particular antibodies directed against immune checkpoint inhibitors, have revolutionized the treatment of different malignancies leading to an improved survival of patients. Identification of immune-related biomarkers for diagnosis, prognosis, monitoring of immune responses and selection of patients for specific cancer immunotherapies is urgently required and therefore areas of intensive research. Easily accessible samples in particular liquid biopsies (body fluids), such as blood, saliva or urine, are preferred for serial tumor biopsies.Although monitoring of immune and tumor responses prior, during and post immunotherapy has led to significant advances of patients' outcome, valid and stable prognostic biomarkers are still missing. This might be due to the limited capacity of the technologies employed, reproducibility of results as well as assay stability and validation of results. Therefore solid approaches to assess immune regulation and modulation as well as to follow up the nature of the tumor in liquid biopsies are urgently required to discover valuable and relevant biomarkers including sample preparation, timing of the collection and the type of liquid samples. This article summarizes our knowledge of the well-known liquid material in a new context as liquid biopsy and focuses on collection and assay requirements for the analysis and the technical developments that allow the implementation of different high-throughput assays to detect alterations at the genetic and immunologic level, which could be used for monitoring treatment efficiency, acquired therapy resistance mechanisms and the prognostic value of the liquid biopsies.

3D-cultivation of NSCLC cell lines induce gene expression alterations of key cancer-associated pathways and mimic in-vivo conditions.

This work evaluated gene expression differences between a hanging-drop 3D NSCLC model and 2D cell cultures and their in-vivo relevance by comparison to patient-derived data from The Cancer Genome Atlas. Gene expression of 2D and 3D cultures for Colo699 and A549 were assessed using Affymetrix HuGene 1.0 ST gene chips. Biostatistical analyses tested for reproducibility, comparability and significant differences in gene expression profiles between cell lines, experiments and culture methods. The analyses revealed a high interassay correlation within specific culture systems proving a high validity. 979 genes were altered in A549 and 1106 in Colo699 cells due to 3D cultivation. The overlap of changed genes between the cell lines was small (149), but the involved pathways in the reactome and GO- analyses showed a high overlap with DNA methylation, cell cycle, SIRT1, PKN1 pathway, DNA repair and oxidative stress as well known cancer-associated representatives. Additional specific GSEA-analyses revealed changes in immunologic and endothelial cell proliferation pathways, whereas hypoxic, EMT and angiogenic pathways were downregulated. Gene enrichment analyses showed 3D-induced gene up-regulations in the cell lines 38 to be represented in in-vivo samples of NSCLC patients using data of The Cancer Genome Atlas. Thus, our 3D NSCLC model might provide a tool for early drug development and investigation of microenvironment-associated mechanisms. However, this work also highlights the need for further individualization and model adaption to address remaining challenges.

Evaluation of assays for drug efficacy in a three-dimensional model of the lung.

BACKGROUND: The focus of the outlined work is the establishment of a three-dimensional lung model for various drug-screening applications. METHODS: The non-small cell lung cancer (NSCLC) cell line Colo699 was cultivated as monolayer (2D) on plates for 5 days or as microtissues (3D) using a hanging-drop system for 5 and 10 days. Cells and microtissues were treated with afatinib (10-80 µM), cisplatin (100-800 µM) or vinorelbine (25-200 µM) for 24 or 48 hours (h). Cell proliferation and viability were analysed by intra-cellular adenosine triphosphate (ATP) and lactate dehydrogenase release (LDH) assays, annexin V/propidium iodide (PI) staining, and cell cycle determination. Microtissue morphology and size, as well as cell death were evaluated via phase contrast microscopy. RESULTS: Our results demonstrate the valid determination of viability and cell death using established assays in the 3D system for drug testing. The comparison of ATP, LDH and cytometry data showed moderate (0.40) to very strong (0.99) correlations. Thereby, we observed partially significant differences in drug efficacy between microtissues and 2D cultures dependent from the applied treatment and read-out method. Altogether, microtissues developed resistance to cisplatin and vinorelbine; but remained more vulnerable to afatinib. These findings were confirmed with microscopy. CONCLUSION: In summary, we established an NSCLC 3D test system with multiple assays compatible for drug-testing applications of substances with different mechanisms of action. In addition, our data support the usage of microtissues as more accurate tools for drug-efficacy testing with the possibility of long-term cultivation and treatment.

The impact of metformin and salinomycin on transforming growth factor ß-induced epithelial-to-mesenchymal transition in non-small cell lung cancer cell lines.

The epithelial-to-mesenchymal transition (EMT) is highly involved in the development of metastases. EMT transforms epithelial carcinoma cells into mesenchymal-like cells, characterized by increased cell migration and invasiveness. Transforming growth factor ß (TGFß) appears to be crucial in this process. Metformin and salinomycin have demonstrated an EMT inhibitory effect. The current experiments indicate that these substances specifically inhibit TGFß-induced EMT in non-small cell lung cancer (NSCLC) cell lines. The NSCLC cell lines A549 and HCC4006 were stimulated with TGFß for 48 h to induce EMT. Metformin or salinomycin was added simultaneously with TGFß to inhibit TGFß-induced EMT. Western blot analyses of E-cadherin and vimentin were performed to detect changes in EMT marker expression, and a wound healing assay was conducted to determine the potential effects on cell migration. The effects of the two drugs on cell viability were also investigated using MTS tetrazolium dye assays. The results revealed that cells undergoing EMT by application of TGFß exhibited a downregulation of E-cadherin and an upregulation of vimentin protein expression on western blot analyses, and an increased capacity for cell migration. Simultaneous application of TGFß and metformin specifically inhibited EMT and increased E-cadherin expression. At the higher dose tested, salinomycin also inhibited EMT, despite an increase in vimentin expression in the two cell lines. Furthermore, metformin and salinomycin, at the two concentrations tested, inhibited cell migration. These findings demonstrate that metformin and salinomycin are able to block EMT and inhibit EMT-induced cell migration. Thus, these two substances are novel EMT inhibiting drugs that have the potential to specifically control EMT and metastatic spread in NSCLC.

Infiltration of lymphocyte subpopulations into cancer microtissues as a tool for the exploration of immunomodulatory agents and biomarkers.

INTRODUCTION: The interaction between the immune system and malignant diseases is a proven key target for cancer therapy. We describe an innovative 3D cell culture system comprising both immune and cancer cells to evaluate their interaction and immune cell infiltration to provide an innovative in vitro screening of immunomodulatory agents and biomarkers. METHODS: 3D tumor microtissues were cultivated using a hanging drops system. Human non-small-cell lung cancer cell lines were incubated for 7 days to form microtissues. On day 5, peripheral blood mononuclear cells (PBMC) were added with or without interleukin-2 (IL-2) for 24 or 48h. Viability of cancer cells and the infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of tumor cells and PBMC and the infiltration of the PBMC into the tumor microtissues were analyzed by immunohistochemistry. Quantification of infiltration was measured by applying the TissueFAXS system. RESULTS: Immunohistochemistry revealed PBMC infiltration in all cell lines which increased under IL-2 stimulation. Analysis of infiltrating populations showed both lymphocyte subpopulations and monocytes within the tumor microtissues. In all three co-cultures, CD3+CD8+ and CD3+CD8+CD45R0+CD28+ lymphocytes were increased with IL-2, whereas CD3+CD8+CD45R0-CD28+ PBMCs were decreased with and without IL-2 stimulation. CONCLUSION: In summary, we present a novel cell culture system to study the interaction between cancer cells and immune cells in 3-dimensional microtissues. In addition, we report for the first time an in vitro infiltration assay based on 3D microtissues. This model has the potential to provide a tool for ex-vivo drug testing and biomarker screening of immunomodulatory agents.

Novel technologies and emerging biomarkers for personalized cancer immunotherapy.

The culmination of over a century's work to understand the role of the immune system in tumor control has led to the recent advances in cancer immunotherapies that have resulted in durable clinical responses in patients with a variety of malignancies. Cancer immunotherapies are rapidly changing traditional treatment paradigms and expanding the therapeutic landscape for cancer patients. However, despite the current success of these therapies, not all patients respond to immunotherapy and even those that do often experience toxicities. Thus, there is a growing need to identify predictive and prognostic biomarkers that enhance our understanding of the mechanisms underlying the complex interactions between the immune system and cancer. Therefore, the Society for Immunotherapy of Cancer (SITC) reconvened an Immune Biomarkers Task Force to review state of the art technologies, identify current hurdlers, and make recommendations for the field. As a product of this task force, Working Group 2 (WG2), consisting of international experts from academia and industry, assembled to identify and discuss promising technologies for biomarker discovery and validation. Thus, this WG2 consensus paper will focus on the current status of emerging biomarkers for immune checkpoint blockade therapy and discuss novel technologies as well as high dimensional data analysis platforms that will be pivotal for future biomarker research. In addition, this paper will include a brief overview of the current challenges with recommendations for future biomarker discovery.

Treatment of patients with refractory metastatic cancer according to molecular profiling on tumor tissue in the clinical routine: an interim-analysis of the ONCO-T-PROFILE project.

INTRODUCTION: Patients with refractory metastatic cancer have been shown to benefit from molecular profiling of tumor tissue. The ONCO-T-PROFILE project was launched in March 2014 at the Innsbruck Medical University. Within 2 years our project aims to recruit 110 patients with stage IV cancer refractory to standard therapy. Our data presented here are based on an interim-analysis. METHODS: Tumor tissue specimens were submitted for molecular profiling to the certified laboratory (Caris Life Science, USA). Druggable tumor targets were selected based on biomarker status to agents with potential clinical benefit. Clinical benefit was defined as a PFS ratio (=PFS upon treatment according to the molecular profile/ PFS upon the last prior therapy) ≥ 1.3. RESULTS: As of April 2015, tumors from 50 patients have been molecularly profiled and one or more targets were detectable in 48 specimens (98%). So far, 19 (38%) patients have been treated according to their molecular tumor profile. To date, 8 (42%) patients have reached a PFS ratio of ≥ 1.3. CONCLUSIONS: We could show that molecular profiling is feasible in the clinical routine. A proportion of patients might benefit from an individualized treatment approach based on molecular profiling. As a result, we will proceed to enroll patients in ONCO-T-PROFILE.

Creating a unique, multi-stakeholder Paediatric Oncology Platform to improve drug development for children and adolescents with cancer.

Seven years after the launch of the European Paediatric Medicine Regulation, limited progress in paediatric oncology drug development remains a major concern amongst stakeholders - academics, industry, regulatory authorities, parents, patients and caregivers. Restricted increases in early phase paediatric oncology trials, legal requirements and regulatory pressure to propose early Paediatric Investigation Plans (PIPs), missed opportunities to explore new drugs potentially relevant for paediatric malignancies, lack of innovative trial designs and no new incentives to develop drugs against specific paediatric targets are some unmet needs. Better access to new anti-cancer drugs for paediatric clinical studies and improved collaboration between stakeholders are essential. The Cancer Drug Development Forum (CDDF), previously Biotherapy Development Association (BDA), with Innovative Therapy for Children with Cancer Consortium (ITCC), European Society for Paediatric Oncology (SIOPE) and European Network for Cancer Research in Children and Adolescents (ENCCA) has created a unique Paediatric Oncology Platform, involving multiple stakeholders and the European Union (EU) Commission, with an urgent remit to improve paediatric oncology drug development. The Paediatric Oncology Platform proposes to recommend immediate changes in the implementation of the Regulation and set the framework for its 2017 revision; initiatives to incentivise drug development against specific paediatric oncology targets, and repositioning of drugs not developed in adults. Underpinning these changes is a strategy for mechanism of action and biology driven selection and prioritisation of potential paediatric indications rather than the current process based on adult cancer indications. Pre-competitive research and drug prioritisation, early portfolio evaluation, cross-industry cooperation and multi-compound/sponsor trials are being explored, from which guidance for innovative trial designs will be provided.

Development of an innovative 3D cell culture system to study tumour--stroma interactions in non-small cell lung cancer cells.

INTRODUCTION: We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. METHODS: Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC. RESULTS: Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. CONCLUSION: We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers.

Increase in antibody-dependent cellular cytotoxicity (ADCC) in a patient with advanced colorectal carcinoma carrying a KRAS mutation under lenalidomide therapy.

The failure of EGFR inhibitors in colorectal tumors with KRAS mutations requires the development of alternative treatment strategies for this patient subgroup. Among the hallmarks of cancer the disturbed immunosurveillance and cancer immune evasion have become emerging targets for cancer therapy. Due to their pleiotropic functions immunomodulatory drugs (IMiDs) are interesting agents for combination therapies in solid tumors. However, their possible contribution and a way of monitoring their biological effects have yet to be revealed. In a heavily pretreated patient with advanced colorectal cancer carrying mutations in APC and KRAS genes, we show an early metabolic response and enhanced NK cell activity to monotherapy with lenalidomide. After subsequent lenalidomide/cetuximab combination treatment, the patient had progressive disease. At the same time a reduced performance status, complicated by febrile neutropenia, occurred, as well as a slight increase in metabolic activity. Concordantly NK cell activity dropped back to baseline. Thus, laboratory measurements and metabolic response assessment correlated with clinical conditions. This case report describes the feasibility and potential of a functional assessment of patient derived immune competent cells in combination with functional imaging for the detection of a biological response.

Cancer classification using the Immunoscore: a worldwide task force.

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the 'Immunoscore' into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Value of infliximab (Remicade®) in patients with low-risk myelodysplastic syndrome: final results of a randomized phase II trial (EORTC trial 06023) of the EORTC Leukemia Group.

Tumor-necrosis factor alpha activity has been correlated to ineffective erythropoiesis in lower risk myelodysplastic syndromes. Infliximab (Remicade(®)) is an anti-tumor necrosis factor alpha chimeric antibody that is used in the treatment of patients with rheumatoid arthritis or Crohn's disease. Forty-six patients with myelodysplastic syndromes and a relatively low risk of developing acute leukemia were included in a randomized phase II study assessing the therapeutic activity of two dosages of infliximab administration (3 mg/kg vs. 5 mg/kg). The primary end point was the response rate. Responses were observed in 3 of 22 patients (13.1%) randomized to the 3 mg/kg arm, versus 0 of 21 patients randomized in the 5 mg/kg arm. According to the statistical design of the current study, neither of the two infliximab dose schedules tested showed sufficient activity as a single agent in this cohort of unselected patients with early myelodysplastic syndrome.

The preclinical and clinical activity of aviscumine: a potential anticancer drug.

Extracts from the European mistletoe plant Viscumalbum have been studied for decades for their direct and indirect anticancer activity. Therefore, scientists were interested in identifying the active compound (mistletoe lectin) in these extracts and making it available as a highly purified molecule for drug development. Recombinant mistletoe lectin (INN: aviscumine) was produced in Escherichiacoli. It has been shown to have immunomodulatory and cytotoxic activity in invitro and in animal models and can target tumour cells. Clinical phase I studies also demonstrated immunomodulatory activity, which appears to have a positive effect on disease stabilisation. This review explores the current knowledge base for aviscumine's mechanism of action, efficacy and side-effects in both preclinical studies and clinical trials, and it considers aviscumine's potential as a cancer therapy.

Preventive therapy for breast cancer: a consensus statement.

In March, 2010, a group of breast cancer experts met to develop a consensus statement on breast cancer prevention, with a focus on medical and therapeutic interventions. We present the conclusions in this Review. First we agreed that the term chemoprevention is inappropriate and suggested that the term preventive therapy better represents this feature of management. Two selective oestrogen-receptor modulators--tamoxifen and raloxifene--are so far the only medical options approved by the US Food and Drug Administration for preventive therapy. Of these tamoxifen has greater efficacy and can be used in premenopausal women, but raloxifene has fewer side-effects. Two newer drugs in this class, lasofoxifene and arzoxifene, also show efficacy and possibly a better overall risk-benefit profile, but need further assessment. Aromatase inhibitors might be more efficacious, and results of prevention trials are eagerly awaited. Newer agents, notably bisphosphonates and metformin, have shown promise in observational studies and need to be assessed in randomised prevention trials. Other agents, such as aspirin, other non-steroidal anti-inflammatory drugs, COX-2 inhibitors, retinoids, rexinoids, and dietary components have limited effects or are in the early phases of investigation. New contralateral tumours in women with breast cancer might be generally useful as a model for prevention, as has been seen for tamoxifen. If valid such a model would facilitate the design of simpler, cheaper, and better-focused trials for assessing new agents.