Towards the introduction of the ‘Immunoscore’ in the classification of malignant tumours

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named ‘Immunoscore’ has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune). © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Inhibition of Caveolin-1 Restores Myeloid Cell Function in Human Glioblastoma

Background: Gliomas are the most common primary brain tumor in both children and adults. The prognosis for glioblastoma (GBM), the most common type of malignant glioma, has remained dismal, with median survival a little over one year despite maximal therapy with surgery, chemotherapy, and radiation. Although immunotherapy has become increasingly successful against many systemic tumors, clinical efficacy against brain tumors has been limited. One reason for this is an incomplete understanding of the local immunologic tumor microenvironment, particularly the function of large numbers of infiltrating myeloid derived cells. Monocytes/microglia are myeloid derived immunomodulatory cells, and they represent the predominant infiltrating immune cell population in gliomas. Our group has previously demonstrated using complementary in vitro and in vivo approaches that GBM tumor cells polarize tumor-associated myeloid cells (TAMs) and suppress their immunostimulatory function. Methods and Results: To better understand the mechanisms responsible for this immunosuppression, we used gene expression profiling of stimulated monocytes in the presence or absence of GBM tumor cells. Our analysis identified caveolin-1 (CAV1), a plasma membrane molecule with pleiotropic functions, as significantly up-regulated in monocytes in the presence of GBMs. We validated these findings ex vivo by confirming up-regulation of CAV1 in TAMs isolated from GBMs immediately after surgical resection. Finally, we demonstrate that siRNA inhibition of CAV1 restores myeloid cell function, as measured by TNF-alpha secretion, in the presence of GBMs. Conclusions: Restoration of TAM function through pharmacologic blockage of CAV1 may facilitate more successful immunotherapeutic strategies directed against a variety of solid human tumors infiltrated by TAMs

Modeling of miRNA and Drug Action in the EGFR Signaling Pathway

MicroRNAs have gained significant interest due to their widespread occurrence and diverse functions as regulatory molecules, which are essential for cell division, growth, development and apoptosis in eukaryotes. The epidermal growth factor receptor (EGFR) signaling pathway is one of the best investigated cellular signaling pathways regulating important cellular processes and its deregulation is associated with severe diseases, such as cancer. In this study, we introduce a systems biological model of the EGFR signaling pathway integrating validated miRNA-target information according to diverse studies, in order to demonstrate essential roles of miRNA within this pathway. The model consists of 1241 reactions and contains 241 miRNAs. We analyze the impact of 100 specific miRNA inhibitors (anit-miRNAs) on this pathway and propose that the embedded miRNA-network can help to identify new drug targets of the EGFR signaling pathway and thereby support the development of new therapeutic strategies against cancer

BIRC3 but Not BIRC2 mRNA Expression in Chronic Lymphocytic Leukemia Correlates with Disease Progression and Mediates Decreased Fludarabine Sensitivity in Vitro

Abstract Abstract 563 Introduction The molecular characterization of chronic lymphocytic leukemia (CLL) with whole genome sequencing has recently begun to foster a novel understanding of the disease, identifying pathways of importance in CLL biology. One of the target genes that was identified by this approach was BIRC3 (Rossi et. al. Blood 2012). BIRC3 (cIAP2) and its close homolog BIRC2 (cIAP1), are members of the IAP (inhibitior of apoptosis) protein family, that unlike other IAPs, do not directly interfere with apoptosis but are key components of the canonical and non-canonical NFKB signalling pathways. As such they play an important role in B-cell differentiation, survival and proliferation. In the murine system BIRC2 and BIRC3 were shown to be redundant (Gardam et. al. Blood 2011). Their combined loss rendered murine B-cells independent of some survival factors and led to uncontrolled B-cell accumulation. In CLL, Rossi et. al. reported recurrent mutations in BIRC3 that were rare at diagnosis (&lt;5%), but associated with increased non-canonical NFKB signalling, worse prognosis and fludarabine refractory disease, independent of p53. We aimed to determine if BIRC3 mutations might be a flag for an importance of the pathway in a larger context of CLL biology and wanted to know whether BIRC3 expression levels are important for clinical outcome. Materials and Methods Peripheral blood MCs of 63 fully characterized CLL patients were collected. mRNA levels of BIRC2 and BIRC3 were analysed using Real Time PCR and normalized to 18S as housekeeping control. Cutoffs to detect different time to treatment intervals were determined by ROC analyses. In vitro culture of CLL cells was performed under standard culture conditions in presence or absence of accessory cells (NIH 3T3 fibroblasts +/− CD40L transgene) in order to abrogate spontaneous apoptosis or achieve CLL cell activation for 3 days. siRNA transfections were performed with non-targeting siRNA (NTC), BIRC2 or BIRC3 siRNA using lipofection as recommended by the manufacturer (Qiagen). Results BIRC3 and BIRC2 mRNA levels were compared with established risk factors and clinical course. We detected significantly downregulated BIRC3 expression levels (P &lt; 0.001) in CLL with unmutated (N = 24) as compared to CLL with a mutated B-cell receptor status (N = 33) and in CLL of advanced RAI stages (N = 19, P &lt; 0.01). Low expression of BIRC3 mRNA was furthermore significantly correlated to decreased time to first treatment (P &lt; 0.001, Fig 1A), which highlights the prognostic impact of BIRC3 mRNA levels independent of potential BIRC3 mutations. Since our test population was previously untreated and cut-offs for prediction were close to the median, this effect was presumably independent of the occurrence of BIRC3 mutations, that should be rare in such a population. To clarify this, mutation analysis is currently ongoing. Despite the proposed functional redundancy, the mRNA levels of the homologue BIRC2 did not correlate with prognostic factors or time to treatment (Fig 1B) giving first hints, that in the context of CLL BIRC3 may be more important as compared to its homolog, which is also reflected in the dominance of BIRC3 mRNA levels as compared to those of BIRC2 (N = 63, P &lt; 0.0001). To further address the question of functional redundancy we performed siRNA mediated downregulation of either BIRC2 or BIRC3. We found that BIRC3 but not BIRC2 siRNA mediated downregulation significantly inhibited fludarabine induced cell death of CLL cells in vitro (N = 6, Fig 1C). Discussion and Conclusion Here we demonstrate for the first time that in addition to the reported effect of mutations in BIRC3, mRNA levels were related to negative prognostic factors and predictive for shorter time to treatment. We further provided evidence that BIRC3 and BIRC2 potentially have non-redundant roles indicated by their different behavior, opposite clinical parameters and different outcomes in response to siRNA mediated downregulation in vitro. Importantly and consistent with a potential selection for mutations, downregulation of BIRC3, but not BIRC2, increased in vitro resistance of CLL cells against fludarabine, the backbone of CLL therapy. Considering the accumulation of BIRC3 mutations in fludarabine refractory CLL (Rossi et. al. Blood 2012), BIRC3 might serve as a novel biomarker, that together with aberrations in p53, could characterize CLL patients with suboptimal therapy success and inferior outcome. Disclosures: No relevant conflicts of interest to declare. </jats:sec