www.fondation-medic.chAnnual Report 2014

Annual Report 2014

Consortium “Tumor-Host Interaction”supported by the MEDIC Foundation

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1. Introduction The theme ‘Tumor-host interaction’ has once again not been changed, notably in view of the strong emergence of immunotherapy, the therapeutic modality that since a few years is revolutionizing cancer treatment. Of the 12 projects presently supported by MEDIC, 11 were renewed in 2014 based upon review of a full new grant application. Due to a variety of reasons one project did not develop favorably and the research group was dissolved. The level of competition increased as not all projects could be financed at the level requested and some projects could not be financed due to lack of means in spite of positive evaluation. The interaction between the different groups in the consortium varies in intensity, new questions continue to emerge during the MEDIC day and are subsequently translated into laboratory work and bioinformatics support is shared. The external Scientific Advisory Board, already strongly involved in selecting the MEDIC prize awardee, is more actively involved in decisions on funding in view of the competitive element. All this contributes to the high quality of the research performed in the consortium. Annual reports and the annual meeting presentations provide the SAB the tools to critically follow the progress made in the studies. The web-site continues to provide updated information on the groups (see www.fondation-MEDIC.ch). Consortium members themselves can continuously update their data on the website.

Also this year the consortium members met in Lausanne to present in symposium format the progress made and the new projects under submission for funding. The one and a half day approach was appreciated as it allowed more time for discussion. The program of the 2014 meeting is presented in table 1.

Table 1 Programme of the MEDIC day 2014

Marie-Agnès ReprogrammingTie2expressingmonocytesinbreastcancertopromoteDoucey protectiveanti-tumorimmuneresponsesCurzioRuegg UnravelingmechanismsofbreastcancerdormancyGrégorySégala/ RegulatorsofestrogenreceptoralphaactivityandtheirroleinbreastcancerMarcellaBenneschChristosSotiriou Dissectingtheimpactofbreastcancerheterogeneityondiseasedissemination

andprogressionKarenWillard Theimportanceoftumorinfiltratinglymphocytedensity,subsetcompositionand organizationinbreastcancerLukasFlatz Improvedmelanomaimmunotherapybydecipheringthedifferencesof

simultaneouslyinducedTcellresponsesagainstauto-,neo-andviralantigens(MEDICprize2014lecture)

LaurencedeLeval/ MutationallandscapeofangioimmunoblasticT-celllymphomaDavidValloisMauroDelorenzi LinkingmolecularheterogeneityofcoloncancerwithclinicallyusefulsubtypesAnitaWolfer RoleofMYCintreatmentresistanceTanyaPetrova Mechanismsofresistancetoanti-angiogenictherapyincolorectalcancer

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PedroRomero HarnessingT-cellsforcancerimmunotherapyPaulWalker TheimpactofTcellandglioblastomaadaptationstohypoxia

onanti-tumourimmunityPhilippeLawalle Roleofimmunemicro-environmentinacuteleukemia:epigeneticsGhanemGhanem/ MelanogenesisinmelanomaprogressionandtherapyFabriceJourné

Productive collaborations continue to develop, feed-back of the participants is very positive so the chosen approach was continued. With the creation of the Department of Oncology in Lausanne most of the Lausanne groups are now in the Department of Oncology. The future of the consortium was again discussed with the Head of the Department. An important development was the participation of MEDIC in financing a new center for translational oncology at the University Hospital Geneva/Geneva University Medical School.

Research groups, themes and received support

Table 2 lists the research projects that are supported by the MEDIC Foundation, the project title and the total amount of annual support received.

Table 2 List of projects funded in 2014 by the MEDIC foundation

C.Ruegg Role of CYR61 in tumor progression and metastasis CHF 238’000,- A.Mariotti Elucidation of the function of Lactadherin and Sox10 in

breast cancer CHF 134’000,-

D.Picard Molecular and pharmacological investigation of the factors contributing to tamoxifen resistance of ERa-positive breast cancers

CHF 164’000,-

M.Delorenzi Linking tumor heterogeneity with clinically useful subtypes of colon cancer

CHF 122’000.-

L.de Leval Characterization of molecular biomarkers relevant to the biology, diagnosis and prognosis of peripheral T-cell lymphomas

CHF 71’000,-

T.Petrova Role and targeting of PROX1 in colon and small cell lung cancer

CHF 201'000.-

I.Stamenkovic Mechanisms that govern energy regulation in cancer stem cells *

CHF 0.-

P.Romero Role of microRNAs in CD8 T cell function CHF 126’000.- M-A.Doucey Tie-2 expressing monocytes and their ligands: appealing

targets in breast cancer angiogenesis CHF 112’000,-

A.Wolfer Resistance to MYC inhibition in breast cancer CHF 109’500.- A.Roth Comprehensive moleular characterization of colorectal

cancer liver metastases: a step toards persomalised medicine in Stage IV colorectal cancer

CHF 104’000,-

C.Sotiriou Interrogating breast cancer molecular heterogeneity € 246’000.- G.Ghanem Search for new prognosis markers, new targets for therapy

and new drug combinations in high risk melanoma € 179’000.-

Ph.Martiat Functional characterization of T cells and their regulatory subset in bone marrow and blood of acute leukemia patients. Correlations with leukemia free survival.

€ 162’000.-

* Funding on hold in view of positive balance

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It is relevant to note here that the total volume of MEDIC supported research conducted in Lausanne has grown again and in 2014 9 groups continued to profit from MEDIC support. A new project was submitted from Geneva and will be (partially) funded in 2015. Two new projects were submitted from Lausanne of which one was funded, the other remaining without support for lack of funds in spite of good reviews. One group is in the Department of Medicine of the Faculty of Sciences in Fribourg.

2. Research program

Three clusters of activities can be distinguished: general aspects of tumor biology, the pathobiology of breast cancer, pathobiology of colon cancer and cancer immunotherapy.

3.1 General aspects of tumor biology

This heading puts together research lines which address questions concerning the development and behaviour of cancer cells more in general and not necessarily limited to an organ or organ system. Two research lines fall into this category: the complex interactions between a variety of cells and molecules that make up the host response to growing tumor cells and basic aspects of cell function that are disrupted in cancer cells.

1. Role of CYR61 in tumor progression and metastasis Principal Investigator: Curzio Ruegg

In addition to the planned experiments and reported results we pursued the characterization of the mechanisms of chemotherapy induced breast cancer dormancy.

Chemotherapy is widely used to treat breast cancer patients and provides a significant survival benefit for many patients. Strikingly, however, some relapses can occur years or decades after treatment implying that treated cancers can enter a state of prolonged dormancy and at a given time resume growth. How chemotherapy-treated cancer cells are maintained in a dormant state and resume growth is unknown. We have established a model of chemotherapy-induced dormancy in immunocompetent mice recapitulating clinical observations in solid tumors. The highly aggressive, triple negative like murine cell line 4T1 was treated with chemotherapy in vivo and the surviving cells were injected in the mammary gland of BALB/c immunocompetent mice to monitor their fate. Chemotherapy induces a state of immunosurveillance that depends on CD11b+ and CD4+ cells of CD8+ T-lymphocytes. Transfer of CD11b+ myeloid derived suppressor cells (MDSC) infiltrating growing tumors from mice bearing dormant tumors prolongs dormancy, while depletion of either CD4+ or CD8+ T cells break the dormancy. By gene expression profiling we identified IRF7, an interferon-induced transcription factor regulating immune response, as critical for maintaining the state of dormancy. This model is a unique tool for the study of immune-mediated tumor dormancy in breast cancer. Manuscript in preparation.

We proceeded on two fronts to further characterize MAGI1. Firstly, we pursued to characterize its cellular function in endothelial cells (EC). We found that MAGI1 localizes at cell-cell contacts, along actin stress fibers and at focal adhesions. Laminar shear stress induces MAGI1 expression and redistribution at cell-cell contacts and MAGI1 promotes flow-induced HUVEC

Thegenerationofcancercellsfromnormalcellsinvolvestheaccumulationoftransmissiblemodificationsofthecell’sgenomeresultinginuncontrolledproliferationandsurvival.Inaddition,cuesfromtheimmediateenvironmentanddistanttissuessuchasthebonemarrow,arecriticalmediatorsoftumorinitiation,andprogression,includinginvasionandmetastasisformation.Recentevidencesuggeststhatthisinteractionbetweenthetumoranditshostalsomodulatesthetumorresponsetoanti-cancertreatments.TheRüegglaboratoryisinvestigatingselectedaspectsoftumor-hostinteractions,withemphasisoninflammation-relatedpathwaysandmechanismsmediatingmetastasisandmodulatingresponsetotherapywithpotentialfortranslationtotheclinics.

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alignment. MAGI1 induces eNOS phosphorylation and NO production in response to shear stress in a PKA dependent but Akt-, CAMKII- and AMPK-independent manner. Senescent HUVEC express high levels of MAGI1 and MAGI1 expression promotes senescence. We are now investigating the role of KLF4 in MAGI1 in response to flow. In the previous year we have generated double transgenic mice with tetracyclin-regulated MAGI1 expression in the intestine (Villin::rtTA-M2 x CMV/tet::MAGI1 and in endothelial cells (EC) (VEC::rtTA-M2 x CMV/tet::MAGI1. We have now further characterized these animals. In the Villin_rM2::tetMAGI1 line the transgene expressed in enterocytes as early as 24h after Doxo treatment. High expressing cells are rare, more frequent in small intestine compared to colon. The longer the induction, the more high expressing cells are observed and more at the villi tips compared to crypts. Transgene expression has no effect on embryonic development. Chemical carcinogenesis induced carcinoma formation in wt animals and only polyp formation in mice, thus confirming a tumor suppressive role. In VEC_tA2::tetMAGI1 (DT) line the transgene was expressed in EC in blood and lymphatic vessels of different adult organs including lung, skin, kidney and liver. Transgene expression from day E10.5 does not cause mortality or obvious vascular defects in embryonic but not adult tissues. However, FGF2 induced angiogenesis in the MG plug assays is markedly decreased in DT animals. We are currently testing the effect of MAGI1 in plaque formation. Manuscript in preparation.

We further confirmed the role of CYR61 in preventing anoikis in breast cancer cells and facilitated entry into the lung. Manuscript in preparation. We finalized the characterization of the RipTag2CYRtransgenic line. Tumors in Rip1Tag2 are significantly larger, of higher grading and more invasive compared to Rip1Tag2 mice and more angiogenic, but do not form metastases. Manuscript in preparation.

We have investigated whether fibroblasts induce colorectal cancer (CRC) motility by contact-dependent mechanisms. For this we characterized the interaction between fibroblasts and SW620 and HT29 CRC cells in 2D and 3D-coculture models in vitro. Fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independent of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF)-2. Cancer cell adhesion to fibroblasts depends on integrin αvβ5. FGFRs on CRC cells activate SRC and inhibit silencing of SRC in cancer cells but not in fibroblasts, and prevent fibroblast-mediated effects. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent CRC cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5integrin-dependent cancer cell adhesion to fibroblasts. Manuscript in revision.

Publications

Filipovic N, Ghimire K, Saveljic I, Milosevic Z, Ruegg C. Computational modeling of shear forces and experimental validation of endothelial cell responses in an orbital well shaker system. Comput Methods Biomech Biomed Engin. 2015;22:1-10 Asparuhova MB, Secondini C, Rüegg C, Chiquet-Ehrismann R. Mechanism of irradiation-induced mammary cancer metastasis: A role for SAP-dependent Mkl1 signaling. Mol Oncol. 2015;8:1510-27 Fagiani E, Bill R, Pisarsky L, Ivanek R, Rüegg C, Christofori G. An immature B cell population from peripheral blood serves as surrogate marker for monitoring tumor angiogenesis and anti-angiogenic therapy in mouse models. Angiogenesis. 2015;18:327-45. Knuchel S, Anderle P, Werfelli P, Diamantis E, Rüegg C. Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion. Oncotarget. 2015;6:14300-17.

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Ciarloni L, Hosseinian S, Monnier-Benoit S, Imaizumi N, Dorta G, Ruegg C; DGNP-COL-0310 Study Group. Discovery of a 29-gene panel in peripheral blood mononuclear cells for the detection of colorectal cancer and adenomas using high throughput real-time PCR. PLoS One. 201513;10:e0123904. Guex N, Crespo I, Bron S, Ifticene-Treboux A, Faes-Van't Hull E, Kharoubi S, Liechti R, Werffeli P, Ibberson M, Majo F, Nicolas M, Laurent J, Garg A, Zaman K, Lehr HA, Stevenson BJ, Rüegg C, Coukos G, Delaloye JF, Xenarios I, Doucey MA. Angiogenic activity of breast cancer patients' monocytes reverted by combined use of systems modeling and experimental approaches. PLoS Comput Biol. 2015;11:e1004050. D Lohri C, Schaltegger CS, VAN DEN Broek M, Wenger RH, Ruegg C, Fink D, Fehr MK, Knuth A, Zweifel M. Neutrophil expression of ICAM1, CXCR1, and VEGFR1 in patients with breast cancer before and after adjuvant chemotherapy. Anticancer Res. 2014;34:4693-9. Barras D, Lorusso G, Lhermitte B, Viertl D, Rüegg C, Widmann C. Fragment N2, a caspase-3-generated RasGAP fragment, inhibits breast cancer metastatic progression. Int J Cancer. 20141;135:242-7.

2. Mechanisms that govern energy regulation in cancer stem cells

Principal Investigator: Ivan Stamenkovic

Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSC), that have the capacity for self renewal, tumor initiation and generation of non-tumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. We have shown that the oncofetal insulin-like growth factor 2 mRNA binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), a validated in vitro model for CSC expansion. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to Complex I and Complex IV assembly. However, IMP2 is primarily known to be an RNA binding protein that is highly expressed during development, and aberrantly re-expressed in various tumour types. Its functional role may therefore be broader than regulating OXPHOS in subpopulations of cells. In the ongoing

Growthofnumerouscancertypesisbelievedtobedrivenbyasubpopulationofpoorlydifferentiatedcells,oftenreferredtoascancerstemcellsthathavethecapacityforselfrenewal,tumorinitiationandgenerationofnon-tumorigenicprogeny.Despitetheirpotentiallykeyroleintumorestablishmentandmaintenance,theenergyrequirementsofthesecellsandthemechanismsthatregulatetheirenergyproductionareunknown.Ourongoingworkisexploringthebioenergeticsofcancerstemcells,focusingmainly,butnotexclusively,onEwing’ssarcomafamilytumorsandglioblastoma.Wehaveidentifiedcancerstemcellsinglioblastomasandhavedemonstratedthatthesedependonoxydativephosphorylationfortheirenergyproductionandsurvival.Weexplorethemechanismsinvolvedinensuringoxydativephosphorylationmaintenance.

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project, we interrogated the genome-wide mRNA binding activity of Imp2 and its effects on stemness in cancer cells.

Publications

3.2 The pathobiology of breast cancer Several groups in the consortium work on breast cancer, which is the most frequently encountered type of cancer in women since 1 out of 9 will develop breast cancer and unfortunately one third of these will subsequently die from this disease. The currently used factors for predicting survival and response to treatment do not sufficiently explain why in some patients the tumors progress and in others do not or why some women respond well to therapy whereas in others the tumors continue to grow. During the last years, several prognostic predictors have been developed in breast cancer using gene expression profiling technologies. Although these predictors outperform the currently used clinico-pathologic factors, they remain suboptimal. This means that in order to get a better picture of breast cancer biology, additional elements need to be considered, such as: 1) the tumor microenvironment and 2) the disseminated and circulating tumor epithelial cells. These key elements constitute the main research axes of this group of projects. Four research projects in the consortium focus on aspects of breast cancer.

1. Interrogating breast cancer molecular heterogeneity Principal Investigator Christos Sotiriou

The initial specific aims of our research program are the following: 1) To investigate breast tumor heterogeneity by studying the clonal composition of primary tumors at different anatomic locations within a primary tumor and its relevance in breast cancer progression – focusing on multifocal breast cancers; 2) To compare the clonal composition of primary breast tumors with their respective multiple metastases in order to identify the sub clones responsible for the development of distant metastases; 3) To interrogate genetic heterogeneity of several single circulating tumor cells (CTCs) isolated from the same patient in order to identify sub clones responsible for the development of distant metastases; 4) To study the clonal relationship between CTCs and tumor cells isolated from primary tumor and matched macroscopic metastasis in order to explore if peripheral blood can be used as a “liquid biopsy” for monitoring of tumor genotype; 5) To identify novel, potentially “druggable” targets through the molecular characterization of the sub clones responsible for the development of distant metastases; 6) To validate the clinical relevance of the mutational events identified in distant metastases and CTCs studying primary tumors from a large cohort of breast cancer patients with well-annotated clinico-pathological variables and clinical outcome. In this report progress regarding the first aim is outlined.

Despitetheprogressmadeduringthelastdecadefromgeneexpression-profilingstudiesandpreliminarydataonmutationaleventsrelatedtobreasttumorigenesis,verylittleisknownregardingbreastcancerheterogeneityanditsimplicationduringdiseaseprogression,disseminationanddistantcolonization.Moreover,thereisverylittleprogressinidentifyingpotentially“druggable”mutationsthatdrivetumorprogressionanddevelopmentofdistantmetastases,whichareresponsibleforbreastcancerdeath.Weaimtoaddressthisquestionbystudyingatthegenome(wholeexome)levelbreasttumorheterogeneityattheprimarysite(tumorinitiationandprogression),circulatingtumorcells(tumordissemination)andatthemetastaticsites(tumorcolonization)usingnextgenerationsequencingtechnology.

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Patients with multifocal/multicentric breast cancer (MFBC) are defined as those with multiple lesions of invasive breast cancer in the same breast occurring at the same time (henceforth referred to only as multifocal breast cancer). These cancers are relatively frequent (approximately one-fourth of the global breast cancer population according to the most recent publications) and have been associated with a number of more aggressive features compared to unifocal tumors. Currently, based on the rationale that lesions with similar histology and grade are expected to be biologically similar, the College of American Pathologists recommends further characterizing all MFBC foci only when histology and grade differ from one another. However, recent studies question this recommendation because of well-documented intra-patient, inter-lesion discordances in estrogen receptor (ER) and HER2 expression (up to 4.4% and 10% discrepancy, respectively). In the present work, we therefore aimed to go a step further and assessed the potential genomic differences between ductal multifocal breast cancer lesions with concordant histological grade, ER, and HER2 status, which represent the majority of these cancers. To this end, we used a targeted sequencing approach specifically focusing on cancer-related genes – similar to the ones currently used by several molecular cancer profiling initiatives – on a series of 171 tumor samples from 36 clinically well-annotated multifocal breast cancers, 26 additional multifocal cancers compared to previous report. We further validated our analysis through ultra-deep re-sequencing using an alternative platform. We showed that in one-third of the patients, the lesions of multifocal breast cancers did not share any mutation despite similar histopathological features, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3 and PTEN (see figure). We further showed that heterogeneous lesions tended to be further apart in the mammary gland than the homogeneous ones.

Figure: Example of distribution of non-silent substitutions and indels for two ‘heterogeneous’ multifocal breast tumors. (Above) Heat maps representing the non-silent exonic substitutions and indels of the 2 heterogeneous multifocal lesions, which have no mutation in common between the lesions. Orange indicates the presence of an oncogenic mutation, grey, the presence of a mutation of unknown significance and white the absence of the mutation. (Below) Immunohistochemical stainings illustrating the loss of PTEN at the protein level only in the lesion of patient PD13774 carrying the PTEN mutation. This intra-patient, inter-lesion heterogeneity may have substantial therapeutic implications, especially in the context of genotype-driven trials. Indeed, although these trials are running mainly in the metastatic setting, most of them allow the identification of the mutation(s) to be performed in the primary tumor. For a subset of patients, we further characterized genome-wide structural variations to interrogate the phylogenetic relationship between the various lesions. These genome-wide data suggest that

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most multifocal breast cancers, including those without any common oncogenic mutations, are clonally related. This in turn suggests that lesions from multifocal breast cancers arise through intra-mammary spread of tumor cells. Their intrinsic invasive nature could potentially explain the worse prognostic features associated with multifocal compared to unifocal cancers. Given the prevalence of multifocal breast cancer and its currently unrecognized intra-patient, inter-lesion genomic heterogeneity, as well as the uniqueness of our research, we feel strongly that our work is innovative enough to attract interest in the medical research community and to open new paths to clinical management of this disease. Of note, we will further validate our observations in a larger number of multifocal breast cancer patients, as well as compare the intra-patient heterogeneity with the one from unifocal patients in the context of our new proposal. Publications Loibl S, von Minckwitz G, Schneeweiss A, et al. PIK3CA mutations are associated with lower rates of pathologic complete response to anti-human epidermal growth factor receptor 2 (her2) therapy in primary HER2-overexpressing breast cancer. J Clin Oncol. 2014;32:3212-20.

Rothé F, Laes JF, Lambrechts D, et al. Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer. Ann Oncol. 2014;25:1959-65.

Sonnenblick A, Fumagalli D, Sotiriou C, Piccart M. Is the differentiation into molecular subtypes of breast cancer important for staging, local and systemic therapy, and follow up? Cancer Treat Rev. 2014;40:1089-95.

Fumagalli D, Blanchet-Cohen A, Brown D, et al. Transfer of clinically relevant gene expression signatures in breast cancer: from Affymetrix microarray to Illumina RNA-Sequencing technology. BMC Genomics. 2014;15:1008.

Salgado R, Aftimos P, Sotiriou C, Desmedt C. Evolving paradigms in multifocal breast cancer. Semin Cancer Biol. 2014 [Epub ahead of print] PMID: 25066860

Tubio JM, Li Y, Ju YS, Martincorena I, et al. Mobile DNA in cancer. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes. Science. 2014;345:1251343.

Ignatiadis M, Riethdorf S, Bidard FC et al. International study on inter-reader variability for circulating tumor cells in breast cancer. Breast Cancer Res. 2014;16:R43.

2. Mechanisms of functioning of the estrogen receptor

Principal Investigator Didier Picard The project comprised of several sub-projects:

Alternate ERα-mediated responses: We have pursued our efforts to understand the molecular biology and the pathological relevance of the activation of ERα by alternate pathways, notably cAMP. While we are still optimizing the conditions ofour ChIP assays to be able to robustly determine the cistromes (i.e. genome-wide distribution) of relevant factors (ERα, CARM1, LSD1,

Estrogenreceptor(ER)playsanimportantroleinbreastcancer,bothintermsofcancerbiologyandaspredictorofresponsetotherapy:ERpositivetumorsarelikelytorespondtoanti-estrogendrugs(notablytamoxifen).Estrogenreceptorpositivebreastcancerstendtodevelopresistancefortamoxifen,however.ThePicardlaboratorystudiesthemolecularbiologyoftheestrogenreceptoragainstthisbackground.Mechanismsofresistanceareexploredaswellaspossibilitiestoovercomethisresistance.

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Hsp90) by ChIP-seq, we continue to explore the potential functional relevance of another transcription factor for the activation of two target genes, RalBP1 and NFkB2, by cAMP activated ERα. When we had investigated the sequence motifs that are associated with cAMP-induced ERα binding sites (ERBS) of breast cancer cells (data from our collaborator Wilbert Zwart at the NKI), it appeared that binding sites for the cAMP-activatable transcription factor CREB were overrepresented. This suggested that cAMP-activated CREB might collaborate with ERα at some target genes. And indeed, as previously reported, a non-phosphorylatable CREB mutant (S113A) blocks cAMP-induction of NFkB2 and RalBP1 (see 2013 report). We have now found that this CREB mutant interferes with the recruitment of Erα to the respective ERBS. If the reverse is not true, i.e. if CREB recruitment does not depend on ERα, this would indicate that CREB may function as a pioneer factor for ERα for some of its cAMP-induced actions. This also suggests that CREB and its phosphorylation by PKA might contribute to tamoxifen resistance. • LSD1 is required for cAMP-induced ERα responses: While trying to understand how cAMP activates ERα, we found the histone demethylase as an additional factor that is required. Overexpressing LSD1 boosts the response and a knock-down blunts it. Moreover, it turns out that LSD1 associates with ERα as part of a complex that also includes other components of the CoREST complex. The direct phosphorylation of LSD1 may facilitate the assembly of this complex. Intriguingly, the CoREST complex was initially discovered as a repressor complex, notably of neuronal gene expression programs. While we clearly describe a role for LSD1 and this CoREST complex as an activator of the cAMP-ERα program in breast cancer cells, it is conceivable that it contributes to promoting or maintaining a certain differentiation state of breast cancer cells.

• Authoritative review on ligand-independent pathways: On the more general subject of ligand-independent we have written a that we hope to become an authoritative source of information on the phenomena, the underlying molecular mechanisms and the biological significance. The following figure from this review illustrates the complexity of these pathways.

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• Novel ERα regulators: We have continued to follow up on two types of candidates that came out in a genetic screen for ERα regulators. We had found that components of the CORVET/HOPS complex, which is known for its role in membrane traffic, modulate ERα function. In their absence, ERα activity is augmented. Similarly, interfering with components of the ESCRT complexes, which are involved in endosomal sorting, increases ERα activity. We recently discovered that PELP1, an ERα coregulator and modulator of rapid non-genomic responses of membrane-associated ERα, counteracts the inhibitory effects of overexpressing CORVET/HOPS components. Our current working hypothesis is that we have tapped into a feedforward loop between the non-genomic and the genomic signaling branches of ERα. It is conceivable that the positive effects of rapid membrane-associated ERα signaling on ERα activity as transcription factor are modulated by membrane trafficking at several levels. We further characterized the role of the E3 ligase RNF20/40, which monoubiquitinates the histone H2B, for ERα function. We found that activation of ERα by estradiol leads to the recruitment of RNF20/40 and increased monoubiquitination of target genes. These effects seem to correlate with a reduced expression. We are currently focusing on the causal links and the relevance to breast cancer progression. • New transactivation assays: Unfortunately, there has been a setback in our efforts to develop a novel dual luciferase system based on secreted luciferases (see previous report). Without going into the details of the technical difficulties, we are currently trying to get the "standard" firefly luciferase secreted. If we can get this to work, we will have all the components that are required. • 3D cultures: As suggested in the grant application, it would be very interesting to determine whether what we know about ERα signaling and signaling crosstalk from work with breast cancer cells in plastic dishes also holds true in 3D cultures. Since we will need relatively large numbers of cells for analyses such as ChIPseq, we have realized that growing MCF7 cells in matrigel or similar natural or synthetic ECMs is not a realistic option due to prohibitively high cost. Therefore, we have begun to play around with a variety of protocols to identify conditions (with and without stromal cells) where MCF7 cells adopt a more physiological phenotype and still remain amenable to further biochemical analyses. • GPER (GPR30): It is now quite clear that this G-protein coupled receptor contributes as a membrane-bound estrogen receptor to estrogenic responses in breast cancer. Since tamoxifen works as an agonist, it also mediates some of the proliferative responses to this antiestrogen of both ERα-positive and –negative breast cancer cells. Marco Pupo, who joined my lab as a postdoc in 2014, had shown as a graduate student that GPER can also be found in the nucleus and bind chromatin in cancer-associated fibroblasts (CAFs). This suggested that GPER might contribute to breast cancer by acting in CAFs through an alternate function. We have discovered that the primary CAFs with which he had worked express a genetic variant of GPER that carries the amino acid change P16L. This in turn may affect the glycosylation of GPER at amino acids N25/N32. Indeed, the glycosylation mutant (double mutant N25/32) GPER is nuclear even in non-CAFs. We have set up a collaboration with Pierre-Yves Dietrich's service at the HUG tocharacterize the localization, polymorphism and function of GPER in CAFs from breast cancer patient. Approval by the ethical committee to get started is imminent Publications

Wang T, Echeverría PC, Picard D (2014). Overview of molecular chaperones in health and disease. In Inhibitors of molecular chaperones as therapeutic agents, T. D. Machajewski and Z. Gao, ed., Drug Discovery Series No. 37 (Cambridge: RSC Publishing), Chapter 1, pp. 1-36. Bennesch MA, Picard D. Minireview: Tipping the balance: ligand-independent activation of steroid receptors. Mol Endocrinol. 2015;29:349-63

3. New prognostic factors in breast cancer: lactadherin Principal Investigator Agnese Mariotti

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Project terminated.

4. Tie-2 expressing monocytes and their ligands: appealing targets in breast cancer angiogenesis Principal investigator Marie-Agnès Doucey

Our group pursued four lines of research: 1) Modeling of TEM responses to angiogenic and inflammatory ligands. We have combined modeling and experimental approaches to identify the treatments transitioning the highly pro-angiogenic phenotype of tumor TEM to the weak pro-angiogenic phenotype of blood TEM and vice versa. These results are highly relevant but were obtained and validated in a limited number of patients TEM (isolated from tumor and peripheral blood). Therefore, in order to strengthen our findings, we have explored breast cancer (BC) gene expression data bases to assess the contribution of these pathways on a larger number of patients and across different BC subtypes. This analysis revealed that breast cancer patients displaying high levels of CD14, PlGF and Ang-2 expression in their tumor had a significantly worse survival relative to those displaying low expression of these genes. Further, the same analysis conducted with CD14 together with PlGF or CD14 together with Ang-2 failed to show significant variations in patient survival. These results confirm that Tie-2 (and its ligand Ang-2) together with VEGFR-1 (and its ligand PlGF) pathways synergize to control TEM angiogenic activity. The results have been accepted for publication in Plos Computational Biology (Guex et al., 2014). 2) TEM are lymphangiogenic and incorporate specifically into lymphatics of human breast cancer. We have investigated TEM lymphangiogenic activity and phenotype in breast tumors using a Facs analyses, RTPC and angiogenic assays in vitro and in vivo. We were asked by the referees to provide evidence for the pro-tumoral activity of TEM. This is indeed a critical question but technically difficult to address. Currently the pro-tumoral activity of TEM from human tumor has not yet been addressed in the literature. To this end, we developed PDX models of BC in collaboration with Dr Dominique Vanhecke (project leader in the laboratory of Pr. Coukos). Primary tumor of breast cancer patients were engrafted into NSG mice. Three months later these mice developed tumors. Pairs of mice with comparable tumor size were used. One mouse was injected in the tumor with TEM isolated from autologous primary tumor whereas the control mouse was injected with buffer. Two weeks later the mice were killed and the tumors examined for their size and vascular network. Both the size and the vascular network of tumors injected

WehavefoundthatthesecretedproteinMFGE8ishighlyexpressedinbreastcancerinassociationwiththetranscriptionfactorSox10,suggestingthatSox10mayplayaroleinbreastcarcinogenesispossiblybyfunctionallyinteractingwithMFGE8.InthisprojectwehavestartedtoaddressthishypothesisbyanalyzingtheroleofSox10duringnormalmammaryglanddevelopment,andhowitsfunctionisaffectedbytransformationeventsthatwillleadtotumorigenesis.OurfinalgoalistounderstandifandhowactiveSox10cancooperatewithoncogenestopromotebreastcancer.

Tumorvascularizationisessentialfortumorgrowthandcancerprogression.Inbreastcancer,monocytesareangiogenici.e.abletoinducetumorvascularization.Inpatients,bloodcirculatingmonocytesdrasticallyincreasetheirangiogenicactivitywhenreachingthetumorsuggestingthatthetumormicroenvironmentshapestheirangiogenicactivity.Theidentificationofthetumorsignalsinducingtheangiogenicactivityofmonocyteisofparamountsignificancebecauseitrepresentstherationaleforanti-angiogenictherapiesinbreastcancer.Weaddresstheseissuesinacombinationofinsilicomodelingapproachesandexperimentalstudiesinmousemodelsofbreastcancer.

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with TEM were dramatically increased relative to control mice thus showing for the first time that tumor TEM form breast cancer patients are pro-tumoral. We are re-submitted this study to Oncoimmunology. 3) Bone marrow-derived cells are implicated as a source of lymphatic endothelial progenitors in human breast cancer We have used our in vitro cell culture model system of TEM (Guex et al., 2014) to understand the differentiation of human cord blood CD133+CD34+ progenitors into podoplanin+ cells expressing both myeloid markers (CD11b, CD14) and the canonical LEC markers. This model reflects the phenotype and the hem- and lymphangiogenic activities of TEM that we have observed in BC. We reported that these podoplanin+ cells stimulate hemangiogenesis and lymphangiogenesis through their ability to differentiate into LECs and to produce angiogenic factors. Importantly, plasma from patients with breast cancer induced differentiation of CD34+ cord blood progenitors into hemangiogenic and lymphangiogenic CD11b+ myeloid cells, whereas plasma from healthy women did not have this effect. Consistent with these findings, circulating CD11b+ cells from breast cancer patients, but not from healthy women, displayed a similar dual angiogenic activity. Taken together, our results show that marrow-derived EPCs become hemangiogenic and lymphangiogenic upon exposure to cancer plasma. These newly identified functions of bone-marrow derived EPCs are expected to influence the diagnosis and treatment of breast cancer. The results of this study were accepted in Oncoimmunology in June 2014 (Faes et al., 2014). 4) Quantification of BC angiogenic and inflammatory factors using nanobiosensors. During this project, we have built up at the CMI (EPFL Center of MacroNanotechnology) arrays of nanowire memristors made in polycrystalline silicone (Puppo et al., 2014a) and identified controlled and reproducible conditions for measurement of antibody-antigen interactions using these nanowire arrays (Puppo et al., 2014a; Puppo et al., 2014b). Most importantly, we have demonstrated that this sensor can detect antigen in a tumor microenvironment. To this end we used an anti-rabbit antibody/antigen pair that we previously used for detection in PBS thus enabling us to compare detection sensitivity levels. Further, this antigen is not expressed in humans and can therefore be spiked at a defined concentration in the tumor extract. Finally, in order to capture the complexity of the tumor microenvironment, a tumor extract was prepared by mechanical disruption of a pool of 10 distinct human breast tumor biopsies. Since the behavior of the wires in the presence of tissue extract is unpredictable, we have used FET- and memristor-based nanowires for this experiment. Our preliminary results show specific antigen detection with FET or memristor nanowires in the range of 5-20 fM. Most importantly, the wires sense the antigen in the presence of a 100 000 mass excess of non-specific protein, indicating that the sensor is extremely resistant to noise. Our current efforts are focused on the homogeneity and robustness of the signal which need to be slightly improved. Publications Faes-van’t Hull E, Bron S, Henry L, Ifticene-Treboux A, Turrini R, Coukos G, et al. Bone marrow derived cells as a source of lymphatic endothelial progenitors in human breast cancer. Oncoimmunology. 2014;3:e29080. Puppo F, Dave A, Doucey MA, Sacchetto D, Leblebici Y, De Micheli G, et al. Memristive Biosensors under Varying Humidity Conditions. IEEE Transactions on Nanobioscience. 2014a;13(1):19-30. Puppo F, Di Ventra M, De Micheli G, Carrara S. Memristive sensors for pH measure in dry conditions. Surface Science. 2014b;624:76-79.

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Van't Hull EF, Bron S, Henry L, et al. Bone marrow-derived cells are implicated as a source of lymphatic endothelial progenitors in human breast cancer. Oncoimmunology. 2014;3:e29080.

Puppo F, Dave A, Doucey MA et al. Memristive biosensors under varying humidity conditions.IEEE Trans Nanobioscience. 2014;13:19-30.

Guex N, Crespo I, Xenarios I, Bron S, Ifticene-Treboux A, Faes-van’t Hull E.,…, Xenarios I and Doucey M.-A. Angiogenic activity of breast cancer patients' monocytes reverted by combined use of systems modeling and experimental approaches Plos Comp. Biol.2015;11:e1004050. 3.3 The pathobiology of colon cancer Colon cancer is the second most frequent cancer type in the western world in females and males. In spite of the fact that it is one of the most widely studied types of cancer and that the concept of stepwise progression of molecular events has been developed based on colorectal carcinogenesis, many questions around this tumor type remain unsolved and overall about 50% of the patients with this disease cannot be cured. It is therefore not surprising that in the MEDIC consortium a significant effort is directed towards contributing answers to these questions. Two research lines address colorectal cancer.

1. Linking tumor heterogeneity with clinically useful subtypes of colon cancer Principal Investigator Mauro Delorenzi

Main results obtained

The planned generation of the RNA-seq data on paired primary and metastatic tumors got delayed: We did a pilot study and we then needed time to make a final choice of the most adapted technology. This should now be ready and data generation take place in 2015. We now hope to have first results for the next MEDIC meeting towards end of 2015. We have taken up some other previously unexpected projects, in particular a study of a colon cancer relapse risk score. We have published a report on the comparison of four prognostic risk scores in the data from the PETACC-3 clinical trial (Di Narzo et al. 2014). The main result is that while the risk scores show statistically significant prognostic classification of patients, they do not concur and this indicates a lack of understanding of the gene expression factors involved in early relapse and represent a problem for applications in the clinic

2) A new prognostic score was developed by Prof. Tim Yeatman and collaborators. It was derived combining two EMT-related signatures and has shown a strong prognostic power in their own data (collection of the Moffitt cancer center). In collaboration with the developers, we have performed tests of its prognostic value in several datasets and found a moderately stronger power

Inthisprojectthemolecularheterogeneityofcolorectalcancerisstudiedinalargeseriesofcases,withdetailedclinicalfollow-up.Thebasicaimofthisprojectistodiscovernewwaysforrecognizingtypesofcoloncancerthatdifferinthewaytheyshouldbetreated,thushelpingcliniciansandpatientstotakebetterdecisions.Decisionstobetakenareifaftersurgeryadditionalchemotherapytreatmentshouldbegiven,andifso,whichdrugshouldbeused.Informationthathelpstakingthedecisionsistwofold.Oneconcernstheprobabilitythatapatientisgoingtosufferfromametastasis;whenthisislowthebenefitofchemotherapymightnotoutweighitssideeffects.Thesecondvaluableinformationconsistsinpredictingwhichdrugismorelikelytobeeffective.Forbothquestions,thegroupingweareuncoveringcouldbeuseful.Ononehandweusegeneexpressiontofindnewsubgroups,whichreallylookdifferentfromeachother;ontheotherwesearchformarkersoftumorcharacteristicsthatareassociatedwithhowwellthepatientsurvivedwiththetreatmentreceived.Withthefirstapproachweshouldfindsubgroupsthatarerealandrobust,buttheirutilitymightbecomeclearonlylateronandbeofmoreimportanceforthechoiceofdrugthanforriskestimation.Withthesecondapproachwehopetoimprovethecurrentmethodsusedforriskestimation,buttheymighthavelimitedutilitytodecidewhatthebesttreatmentisforagivenpatient.

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than the previously tested risk scores. The new score correlates with mesenchymal expression and tend to be highest in the mesenchymal molecular subtype we and others have proposed. The score identifies a subpattern within this large set of “mesenchymal genes. This suggests that there is a “global” EMT motif associated with shorter RFS, but also finer subcomponents more relevant to high risk of early relapse. Unfortunately, we were unable to pin down the new signature to just 2-3 genes for functional characterization, though we could point to some genes (high risk markers CD109, AHNAK2, MEIS2; low risk markers CDX1, CDX2). A manuscript was submitted for publication.

miRNAs in colon cancer

3) We have continued an analysis of miRNA profiles of 645 tumors from PETACC-3 patients, 521 stage III and 124 stage II. We determined miRNAs with strong association to RFS, after adjustment with known risk factors. These contribute non-redundant information, quite from typical mRNA–based risk scores. We determined a 30 miRNAs signature for best performance. Strongest contributions are given by the miR families miR-155 and miR-16 (high expression associated with lower risk), respectively miR-616 and miR-642 (high expression associated with higher risk). The multivariable miRNA risk score performed better than selected mRNA risk scores for predicting RFS but worse for predicting survival after relapse.

Colon cancer (CC) subtypes

4) We have revised our analyses of the variation of colon cancer according to their proximal-distal position. The preliminary results were mentioned in last year's report and an updated report is now published (Missiaglia et al, 2014, copy attached).

5) We have continued the effort defining molecular subtyping in CC, now within an international collaboration of several teams coordinated by scientists at SAGE Biosystems. Almost a full year of work has been invested to consolidate a system of four consensus molecular subtypes (CMSs). The analysis was extended to the TCGA data (gene expression, mutations) and multiple other datasets, also including mutation, miRNA, DNA methylation and proteomics profiles. A manuscript was submitted for publication.

Publications Missiaglia E, Jacobs B, D'Ario G et al. Distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features. Ann Oncol. 2014;25:1995-2001. Di Narzo AF, Tejpar S, Rossi S, Yan P, Popovici V, Wirapati P, Budinska E, Xie T, Estrella H, Pavlicek A, Mao M, Martin E, Scott W, Bosman FT, Roth A, Delorenzi M. Test of four colon cancer risk-scores in formalin fixed paraffin embedded microarray gene expression data. J Natl Cancer Inst. 2014;106(10). Guinney J, Dienstmann R, Wang X, de Reyniès A, Schlicker A, Soneson C, Marisa L, Roepman P, Nyamundanda G, Angelino P, Bot BM, Morris JS, Simon IM, Gerster S, Fessler E, De Sousa E Melo F, Missiaglia E, Ramay H, Barras D, Homicsko K, Maru D, Manyam GC, Broom B, Boige V, Perez-Villamil B, Laderas T, Salazar R, Gray JW, Hanahan D, Tabernero J, Bernards R, Friend SH, Laurent-Puig P, Medema JP, Sadanandam A, Wessels L, Delorenzi M, Kopetz S, Vermeulen L, Tejpar S.The consensus molecular subtypes of colorectal cancer. Nat Med. 2015;21:1350-6. Sinicrope FA, Shi Q, Smyrk TC, Thibodeau SN, Dienstmann R, Guinney J, Bot BM, Tejpar S, Delorenzi M, Goldberg RM, Mahoney M, Sargent DJ, Alberts SR Molecular markers identify subtypes of stage III colon cancer associated with patient outcomes. Gastroenterology. 2015 ;148:88-99.

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Klingbiel D, Saridaki Z, Roth AD et al. Prognosis of stage II and III colon carcinoma treated with adjuvant 5-FU or FOLFIRI in relation to microsatellite status, results of the PETACC-3 trial. Ann Oncol. 2015;26:126-32. Schell MJ, Yang M, Missiaglia E, Delorenzi M, Soneson C, Yue B, Nebozhyn M, Loboda A, Bloom G, Yeatman TJ.A composite gene expression signature optimizes prediction of colorectal cancer metastasis and outcome. Clin Cancer Res. 2015 [Epub ahead of print]

2. The role of PROX1 in colon cancer progression and metastasis Principal Investigator Tatiana Petrova

The main goal of this project is to investigate the role of PROX1 in colon cancer metastasis. Previously, using a panel of 160 primary CRC tumors, we found that PROX1 expression is restricted to microsatellite stable cancer subtype, which has worse prognosis in comparison to microsatellite instable CRC (in collaboration with Dr. G. Marra, University of Zurich, and Dr. F. Bosman, CHUV). Furthermore, we found that PROX1 expression is increased in approx. 40% of metastatic CRC lesions in lymph nodes and liver (in collaboration with Dr. I. Letovanec, M. Gonzales and H. Bouzourene, IUP, CHUV). Using two different cell lines, we showed that PROX1 promotes the development of metastases. Moreover, PROX1 suppression after the establishment of primary tumor and metastases, further prevented the development of metastases, demonstrating that targeting PROX1 could be an important clinical goal. Analysis of the mechanism demonstrated that PROX1 acts by promoting metabolic adaptation of tumor cells, in particular by enhancing survival of cancer cells in hypoxia or nutrient-low conditions through the productive use of autophagy. During this year we addressed the questions of the mechanisms, underlying the ability of PROX1 to promote outgrowth of metastases, and potential therapeutic strategies to target PROX1+ cells in metastases. 1. In 2013, by combining the data of PROX1 ChIP-seq and gene expression profiling of colon cancer cells after PROX1 overexpression or suppression, we identified BCL2L15 and CAV1 as potential effectors of PROX1 in CRC. We further investigated the expression of BCL2L15 and CAV1 in human CRC metastases, and we demonstrated that while CAV1 is generally down regulated , BCL2L15 is restricted to Prox1- tumor cells, as expected, based on our previous findings demonstrating that PROX1 is a transcriptional repressor. We further demonstrated that suppression of BCL2L15 enhances growth of tumors, whereas BCL2L15 overexpression strongly inhibits tumor growth and metastasis. Thus, at least in part, PROX1 ability to promote tumor growth and metastasis relies on the suppression of BCL2L15 function. As BCL2L15 function is largely unknown, further studies of this protein and its role in the suppression of CRC metastases will be an important question to address in the future.

Inthisprojectoneparticularmolecularmechanismincolorectalcancerisstudied.ThisconsistsofPROX1,amoleculethathasbeenshowntobeinvolvedinintestinaladenomadevelopmentinmousemodels.TheexperimentsconductedhaveshownthatPROX1isinvolvedintheinteractionbetweenthecancercellsandthehoststromalcells.Furthermore,PROX1iscloselyassociatedwiththesignalingpathwayinvolvedinregulationofcelldifferentiationinintestinalmucosa(theWntpathway)andalsointransformationofintestinalepithelialcellsintocancercells.PresentdatasuggestthatPROX1isinvolvedinprogressiontolaterstages(e.g.metastasis)ratherthanintheinitialstagesofcolorectalcancerdevelopment.AnalysisofthemechanismdemonstratedthatPROX1actsbyenhancingsurvivalofcancercellsinhypoxiaornutrient‑lowconditionsthroughtheproductiveuseofautophagy.

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2. Signficant part of the year was dedicated to answering multiple questions of reviewers for the publication of our paper describing a key role of PROX1 in metastatic outgrowth. In particular, we further demonstrated that inhibition of authophagy has little effect on growth of primary tumors, whereas it strongly inhibits growth of metastases. These results highlighted so far unknown differences in metabolic needs of primary vs metastatic tumor cells. Importantly, they suggest a unique vulnerability of CRC metastases with high PROX1 expression to inhibition of autophagy, which may be important for the development of further treatment approaches. In addition, we demonstrated that Prox1+ cells represent a novel population of CRC stem/progenitor cells, characterised by relatively low proliferation rate, but which is important for sustaining the growth of either tumors or, in vitro, of tumor organoids. Publications Ragusa S, Cheng J, Ivanov KI, Zangger N, Ceteci F, Bernier-Latmani J, Milatos S, Joseph JM, Tercier S, Bouzourene H, Bosman FT, Letovanec I, Marra G, Gonzalez M, Cammareri P, Sansom OJ, Delorenzi M, Petrova TV. PROX1 promotes metabolic adaptation and fuels outgrowth of Wnt(high) metastatic colon cancer cells. Cell Rep. 2014;8:1957-73.

3. Comprehensive molecular characterization of liver metastasis, a step towards

personalized medicine in stage IV colorectal cancer Principal Investigators Arnaud Roth, Tom McKee

No report received.

3.4 The pathobiology of melanoma Melanogenesis in melanoma progression and therapy

Principal Investigator Ghanem Ghanem

Our 2014 work focused on two aspects : 1) Explaining the discrepancy between Tyrp1 mRNA and protein expression (50% less protein than mRNA !) that we have observed in previous studies and its impact on its prognostic value. Based on evidence from melanoma cell lines (miR-155 and Tyrp1 mRNA stability) and a validation in skin and lymph node metastases, we demonstrate a differential post-transcriptional

TheGhanemlaboratorystudiesthebiologyofmelanoma,withtheintentiontoidentitfymarkerswhichareprognostic(distinguishmelanomawithlowriskofprogressionfromhighriskmelanomas)andnewtherapiesformelanoma.ApromisingnewmarkerisTYRP1,ofwhichthebiologyisstudiedinmelanomacelllines.ApromisingnewtherapyformelanomacouldbeDasatinib.Themechanismsofactionofthisdrugareexplored,inordertoallowidentificationofpatientswhichmightbenefitfromDasatinibtreatment.

Inthisprojectadetailedcomparativeanalysisofprimarycolorectalcarcinomasandtheirlivermetastaseswillbeperformedwithafocusongeneexpressionanddigitalmorphologicanalysis.ThisprojectformspartofalargerinternationalcollaborationthatisanalyzingalsoDNAcopynumberaberrationsandmutationsonpairsofuntreatedprimaryandmetastaticsamples.ThegeneexpressionanalysisthathasalreadybeenperformedonalargegroupofstageIIandIIIcolorectalcarcinomaswillbeextendedtometastaticcarcinomas.Thequestioniswhethermolecularcharacteristicschangebetweenprimarytumorsandmetastases,whichmightexplainhowseeminglylow-riskprimarycolontumorsmanagetoformhigh-risklivermetastases.Quantitativemorphologicdatawillbeanalyzedinconjunctionwithintodatafromgeneexpressionandotheranalyses,providingadimensionofspatialresolutionallowingseparationofcancercellsfromreactivestromaandthecenterofthetumorfromtheinvasiveedge.Theintentionistodevelopsurrogateparametersallowingeasiersubtypingofcolorectalcancer.Italsoallowsintroductionofimmuneresponseparametersintosubtypingparameters.

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regulation of TYRP1 by miR-155 according to the SNPs rs683 and rs910. We could clearly identify 2 polymorphic groups (“match” and “mismatch”) having different expression levels of TYRP1 protein, but similar TYRP1 mRNA or miR-155, supporting the fact that TYRP1 protein was differentially affected by miR-155 in a SNP-dependent manner. Such polymorphisms affect TYRP1 mRNA regulation by miR-155 and subsequent translation into protein. This explains the discrepancy between the prognostic value for TYRP1 mRNA on one hand and and protein expression on the other, the latter only correlates with patient survival when stratified for the 3’UTR SNPs in the miR-155 binding site of TYRP1 mRNA. 2) Two original findings concerned the sensitivity/resistance of mutBRAF inhibitors in melanoma: (a) Despite that vemurafenib possesses a high affinity for mutBRAF, a group of melanoma cell lines with no BRAF or NRAS mutations are sensitive to this drug. We then thought that this observation can explain reported tumor regressions in 22% of patients harboring mixed type tumors containing both wild type and mutated BRAF cells. Furthermore, no correlation has been found between the presence of WTBRAF in these tumors and the non-response to the treatment. We conducted a study to uncover the mechanism underlying this unexpected sensitivity and could demonstrate that WTBRAF/WTNRAS melanoma cells with elevated cAMP levels can be intrinsically sensitive to vemurafenib, drug originally designed to inhibit mutated BRAF protein, and that, the inhibition of CRAF expression achieved by cAMP stimulators or mimetics is able to overcome the resistance to this drug. (b) The second observation motivating our work stems from the fact that in majority of melanomas, p53 protein is inactivated by a variety of recently described mechanisms and thought that this can contribute to the resistance to MAPK inhibition. First, we showed that p53 reactivation is able specifically inhibit the catalytic subunit p110α of PI3K accompanied by an increase of PTEN expression. As a p53 activator, we have chosen PRIMA-1Met for its ability to restore the active conformation of the protein whatever was the mechanism of its deactivation. We, then tested various combinations of PRIMA-1Met with vemurafenib or PI3K/AKT inhibitors in both cells with intrinsic or acquired resistance to the drug and in all cases, a synergistic effect was obtained both on the inhibition of cell survival and induction of apoptosis. These effects have been validated in a model of sensitive and resistant melanoma xenografts in nude mice without any noticeable toxicity. In conclusion, combining vemurafenib with a direct p53 activator allows to overcome and even prevent melanoma cell resistance to this drug paving the way for promising perspectives in the treatment of this disease.

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Publications Krayem M, Journe F, Wiedig M et al. Prominent role of cyclic adenosine monophosphate signalling pathway in the sensitivity of (WT)BRAF/(WT)NRAS melanoma cells to vemurafenib. Eur J Cancer. 2014;50:1310-20 Denecker G, Vandamme N, Akay O, Koludrovic D, Taminau J, Lemeire K, Gheldof A, De Craene B, Van Gele M, Brochez L, Udupi GM, Rafferty M, Balint B, Gallagher WM, Ghanem G, Huylebroeck D, Haigh J, van den Oord J, Larue L, Davidson I, Marine JC, Berx G. Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression. Cell Death Differ. 2014;21:1250-61. El Hajj P, Gilot D, Migault M, Theunis A, van Kempen LC, Salés F, Fayyad-Kazan H, Badran B, Larsimont D, Awada A, Bachelot L, Galibert MD, Ghanem G, Journe F.SNPs at miR-155 binding sites of TYRP1 explain discrepancy between mRNA and protein and refine TYRP1 prognostic value in melanoma. Br J Cancer. 2015;113:91-8. 3.5 Immunotherapy of cancer Immunotherapy of cancer has been an important research focus for several decades and remains to be so. Key questions are: why the immune system responds initially to the presence of abnormal (cancer) cells in the body but fails to eliminate these cells. Conceptually, this might be due to failure of the immune system to recognize the cancer cells as harmful, hence no longer attacking them. Alternatively, this may be due to failure of immune-competent cells to kill the cancer cells. Effective ways to reconstitute and reinforce the immune system in its efforts to eliminate cancer cells would constitute a major breakthrough in cancer research and treatment. Two groups are working in this area, one on melanoma as a model system with emphasis on the role of miRNAs in the regulation of the function of CD8+ T-cells and the other on leukemia, with similar approaches and productive interaction between the groups.

1. Role of miRNA species in regulating the immune response to melanoma Principal Investigator Pedro Romero

1 : Characterization in vivo of the role of the two major branches of mTOR signaling complexes mTORC1 and mTORC2 in memory CD8 T cell differentiation, maintenance and persistence.

Pharmacological inhibition of mTOR by rapamycin was reported to lead to increased magnitude and qualities of CD8+ memory T cell differentiation. However, in our experiments, genetic deletion of Raptor (mTORC1) impairs both the effector T cell expansion as well as the memory formation. Thus, we are now probing the mTORC1 branch using an inducible deletion system (Granzyme B-CreERT2). We have validated this system and can follow both raptor competent and raptor deleted CD8 T cells following tamoxifen administration. CD8 T cell destination will be fate mapped by YFP expression. In sharp contrast, deletion of Rictor (mTORC2) in CD8+ T

TheRomerolaboratorystudiesmechanismsdeployedbytheimmunesystemtoattackcancercells.Theultimategoalistodevelopeffectiveimmunotherapies.Inthisprojectregulationofthefunctionofaspecificsetofimmune-competentcells(CD8+T-cells)isexamined.Itwasfoundthattheirfunctionisatleastinpartregulatedthroughmicro-RNA,anewspeciesofRNAwithimportantgeneralgeneregulatoryfunctionsandpotentiallyimportantasdiagnostictoolaswellastargetfornewtherapies.Thegroupfocusesonmelanoma.miRNA155hasbeenidentifiedasparticularlypromising.

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cells does not affect their acquisition of effector functions. Intriguingly, we found that Rictor deficiency favors memory precursor effector cell (MPEC) differentiation whereas reducing at the same time the short lived effector (SLEC) differentiation. Indeed, we observed enhanced memory formation in the absence of intact mTORC2 signaling. Furthermore, we aim to investigate the role of mTORC1 and mTORC2 in the maintenance and persistence memory T cells by inducible deletion of either Raptor or Rictor in the memory phase.

2: Determination of the molecular mechanisms underlying the regulation of memory T cell differentiation by mTORC1 and mTORC2 and assessment of the interplay between mTORC1 and mTORC2 in this process.

We found increased CD127, CD62L and Bcl-2 expression in in vivo primed CD8 T cells lacking of mTORC2 signaling. Very interestingly, we found that increased Foxo1 nuclear localization in Rictor KO CD8 T cells drives the expression of Eomes and Tcf1, which promotes memory generation. We are next going to validate whether Foxo1 activity is mediating the increased memory in Rictor deficient settings by employing shRNA against Foxo1 or overexpressing a construct encoding the constitutively active Foxo1 AAA mutant.

3: Understanding how T cell metabolic profiles governed by mTOR pathway couple with CD8 memory formation.

We have collaborated with Prof. Lluis Fajas group at the Dept of Physiology, Faculty of Biology and Medicine, University of Lausanne, in this aspect. Indeed, we have shown mTORC2 deficient CD8 T cells show enhanced spare respiratory capacity (SRC), a cardinal metabolic feature of memory T cells. In line with this observation, we found that Rictor deficient CD8 T cells are highly dependent on fatty acid oxidation (FAO) compared with that of WT CD8 T cells. In this respect, we will next also explore whether Foxo1 may be involved in promoting these metabolic changes in Rictor deficient CD8 T cells.

4: Exploration of the possibility of modulating mTOR signaling and/or metabolic reprogramming to generate robust long-lived memory CD8 T cells for cancer immunotherapy.

Given the distinct metabolic features employed by mTORC2 deficient CD8 T cells, we will assess in much more depth the entire pathway related with FAO, in which we expect to find some key enzymes that could be targeted in terms of therapeutic interventions. Meanwhile, we aim to assess the mitochondrial mass, morphology and activity in the absence of mTORC1 or mTORC2.

Publications Romano E, Romero P. The therapeutic promise of disrupting the PD-1/PD-L1 immune checkpoint in cancer: unleashing the CD8 T cell mediated anti-tumor activity results in significant, unprecedented clinical efficacy in various solid tumors. J Immunother Cancer. 2015;3:15. Jebbawi F, Fayyad-Kazan H, Merimi M, Lewalle P, Verougstraete JC, Leo O, Romero P, Burny A, Badran B, Martiat P, Rouas RA microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes. J Transl Med. 2014;12:218.

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2. The role of the immune response in leukemia development Principal Investigator Philippe Martiat

Transcriptomic profiling

There is increased evidence that the immune microenvironment plays a fundamental role in the outcome of leukemia, either after high-dose chemotherapy or allogeneic stem cell transplantation. In particular, several groups have recently highlighted the role of regulatory T-cells. Known prognostic factors include leukemic burden, age, cytogenetic or specific molecular abnormalities and residual disease at the end of the induction treatment. Nonetheless, few studies had concentrated yet on a thorough investigation of the immune cellular environment as a potential independent prognostic factor. We started to investigate the entire marrow and blood T-cell compartment, which can be regarded, in the case of leukemia, as tumor-infiltrating lymphocytes. In AML and B-ALL patients, we have assessed quantitatively and qualitatively the purified T cell population, using flow cytometry and Affymetrix microarray studies, at diagnosis and in complete remission, in bone marrow and peripheral blood samples, in an effort to better correlate the role of the absolute number and percentage of the various T-cell subpopulations to the outcome of the disease in terms of relapse-free survival and overall survival, in otherwise undistinguishable leukemia as far as known prognostic factors are concerned. Unsupervised analyses revealed important significant differences between leukemic patients and healthy individuals in the gene expression profile of their T-lymphocytes that were validated by individual qPCR. We have characterized the biased polarization of patient’s T-cells (TH1, TH2, Tregs, inflammatory, activation). Moreover, our preliminary data suggest that T-cell profiling could represent an independent predictive factor of overall survival in acute leukemia.

Methylome profiling

We have also studied the methylome profiling of AML patients T lymphocytes, using the Human Methylation 450 BeadChip researchers (Illumina). AML patients (n=37; pvalue<0,01,Delta 0,15) at diagnosis, showed a significant differentially methylated CpG in the bone marrow (1699 DMCpG) and peripheral blood (1645 DMCpG) compared to healthy donors. Interestingly, our results analysis of the repartition of differentially methylated CpG showed several of them concentrated inside of the promoter, suggesting that the polarization of T-cell response during disease is controlled by epigenetic mechanisms implicating DNA methylation. Among genes known to have important role in T-cell, there is IL1RAPL2, IL21R, IL31RA, IL2RG, CD7, CD247, CD3e, CD28, CD40LG. These results also showed that DMCpG localized in the coding regions of miRNAs such as miR-16, -19, -105, -223, -450 and -500.

LiketheRomerolaboratory,theMartiat laboratorystudiesmechanismsdeployedbytheimmunesystemtoattackcancercells.Thehighincidenceofrelapseinhumancancersdemonstrates thefailureoftheimmunesystem to control cancer cells naturally. Fundamental investigations in the last decades have clearlyestablished the conceptthat crosstalk between the immune systemand tumor cells is important incancerpatientoutcome. Tumorsdevelopmanyways to escape immune surveillance and favor their owngrowth.Even when a potent immune response is demonstrated ex vivo in laboratory, tumors induce regulatory,suppressive and inhibitorymechanisms toprotect themselves from this immune response inpatients. Theinterplaybetweenthetumorandherimmunemicroenvironmenthasbeenrecentlydefinedasakeyelementin the goal to achieve a definitive cure in cancer patients. The primary objective of our project is tounderstand indetails themechanisms responsible for the lackof effective anti-tumor immune response inleukemia.Our approach is to characterize the functionality of the immune system of leukemic patients in order todefine reliable, relevant and cost effective immune signatures. These immune profiles will help us todetermine firstly which group of leukemic patient could benefit from immunotherapeutic approaches toeradicateleukemiccellsandsecondlytofollowthepatienttreatmentresponseandriskofrelapse.

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For B-ALL, preliminary analysis showed a high level of differentially DMCpG between bone marrow lymphocytes from patients and normal individuals. A preliminary comparison of patients based on age (adults vs children) showed the existence of more than 4000 DMCpG, whereas the comparison between healthy donors of the same age showed just approximately 1800 DMCpG. It is known that adult ALL patients have a poorer prognosis than children. Comparisons between different methylation profiles of TILs (patients vs healthy; BM vs PB; adults vs children; diagnosis vs remission) are carried out to identify on the one hand, different profiles of TIL in response to disease progression, risk factors and survival time, and on the other hand, the epigenetic mechanisms that are responsible for their polarization.

Circulating plasma microRNA profile in acute leukemia

Given their importance in epigenetic regulation, circulating miRNAs may serve functions other than simply being biomarkers in leukemia and their impacts in TILs remain largely unclear. Recent data showed that the population of circulating miRNAs correlates highly with immune response, suggesting that circulating miRNAs could drive anticancer effects through active immunity and might offer novel strategy for cancer therapy. We have previously identified eight plasmatic miRs differentially expressed between healthy individuals and AML patients. We described that miR-150 and miR-342 showed a decreased level of expression in AML patients at diagnosis, while it is similar to that of healthy donors when in complete remission. Few studies have concentrated yet on a thorough investigation of the immune cellular environment as a potential independent prognostic factor for B acute lymphoblastic leukemia, and little work has been conducted to study the expression profile of circulating miRNAs. Our recent results, based on 10 B-ALL patients, showed a decreased expression of miR-485-5p, -504, -198 and -330-5p in leukemic patients compared to healthy donors. The comparison between adults (poor prognosis) and children (good prognosis) revealed underexpression of miR-301, -485-5p, -92a and -223 in children patients’ plasma.

Publications

Fayyad-Kazan H, Hamade E, Rouas R et al. Downregulation of microRNA-24 and -181 parallels the upregulation of IFN-γ secreted by activated human CD4 lymphocytes. Hum Immunol. 2014;75:677-85.

Fayyad-Kazan H, Hamade E, Rouas R, Najar M, Fayyad-Kazan M, El Zein N, ElDirani R, Hussein N, Fakhry M, Al-Akoum C, Burny A, Martiat P, Badran B. Downregulation of microRNA-24 and -181 parallels the upregulation of IFN-γ secreted by activated human CD4 lymphocytes. Hum Immunol. 2014;75:677-85.

3. Molecular classification of peripheral T-cell lymphomas

Principal investigator L.de Leval

TheLevalgroupstudiesperipheralT/NKcelllymphomas(PTCLs):PTCL,nototherwisespecified(PTCL,NOS),angioImmunoblasticT-cellLymphomas(AITL)andsystemicALK-positiveandALK-negativeanaplasticlargecelllymphomas(ALCL).Thepathogenesisunderlyingmostoftheseentitiesispoorlyunderstood,andtheirboundariesareunclear.Theaimoftheprojectistousestateoftheartmolecularprofilingmethodstogainmoreinsightinmolecularcharacteristicsandusetheseformoredetailedclassification.

Ourhypothesisisthatthecombinationofoverallgeneexpressionpatternsand(epi)geneticsofalargeseriesofPTCLswillimprovetheirbiomolecularclassification.andshouldidentifyclinicaltraitsthatwillallowtherapeuticstratification.

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The aim of this project is to gain new insight into the molecular classification of the most common subtypes of peripheral T/NK cell lymphomas (PTCLs): PTCL, not otherwise specified (PTCL, NOS), angio-immunoblastic T-cell Lymphomas (AITL) and systemic ALK-positive and ALK-negative anaplastic large cell lymphomas (ALCL). The pathogenesis underlying most of these entities are poorly understood, and their boundaries are unclear. Our hypothesis is that the combination of overall gene expression patterns and (epi)genetics of a large series of PTCLs will improve their biomolecular classification and should identify clinical traits that will allow therapeutic stratificationDNMT3A mutations. The following results were obtained: 1) CD30 expression in PTCLs: correlation between immunohistochemistry and mRNA levels (Manuscript published in Blood; see attachment) 2) Characterization of TFH-like PTCL-NOS (Manuscript in preparation, published in an abstract form; see attachment) Analysis of TFH-like PTCL-NOS cases shown that relevant clinical and mutational characteristics are significantly similar to AITL compared to non-TFH-like PTCL-NOS; we also observed a significant overexpression of previously published molecular signatures (TFH, AITL, and AITL microenvironment) within the TFH-like subgroup, suggesting that AITL and TFH-like PTCL-NOS may share a common cell of origin and/or similar tumorigenic mechanisms. Supervised analysis of aCGH data were consistent with these results, showing a pattern of alterations in TFH-like PTCL-NOS intermediate between AITL and other PTCL.NOS, supporting the genetic proximity of TFH-like PTCL-NOS to AITL. Current work is on choosing genes to validate from biologically interesting gene signature enrichments. 3) Unsupervised class discovery (completion phase, manuscript in preparation) We have identified four potential subtypes within PTCLs by performing unsupervised class discovery on gene expression data (n=161), followed by integration with clinical and copy number data. The subtypes include two subclasses within AITL and two subclasses in PTCL, NOS. The two AITL subtypes are characterized by different levels of proliferation signature enrichments and different frequencies of DNMT3A mutations. One AITL group also had a higher proportion of patients with worse IPI and ECOG. Of the two PTCL-NOS subtypes, one was characterized by the high chromosomal instability, and the lowest occurrence of mutations of epigenetic modifiers TET2, DNMT3A and IDH2. The other PTCL-NOS subgroup, in contrast, was characterized by significantly lower chromosomal instability, and enrichments in cytotoxic, monocytic, and diffuse large B-cell lymphoma (DLBCL)-related signatures. These features were confirmed using an independent data set (GSE58445, n=101 after normalization, batch correction and quality control). Current work is on interpreting the signature enrichments. 4) Defining the mutational landscape of AITL and TFH-like PTCL,-NOS cases Using results from whole genome/exome sequencing results from a discovery cohort of 10 matched tumor and normal samples from AITL patients, as well as information on deregulated genes linked to the TCR pathway in sorted AITL and normal TFH cells, we selected 58 genes for targeted deep sequencing in an extended cohort of 88 patients (AITL and TFH-like PTCL-NOS) from the TENOMIC collection. We confirmed both previously reported recurrent mutations in RHOA and CD28, as well as mutations in a total of 26 genes. With the exception of TET2, none of the recurrent mutations influenced survival consistent with previous reports. Neither did these recurrent mutations (RHOA, TET2, DNMT3 and IDH2) exhibit the patterns of mutual exclusivity typically associated with driver mutations. We subsequently focused on checking for mutual exclusivity and effects on survival of mutations at the pathway level (i.e. groups of genes that are in the same pathway). At the transcriptional level, cases harboring mutations in members of the TCR activation pathway (PLCG1 and PI3K pathways) were characterized by a relative over-expression of gene sets reflective of TCR activation, and proliferation. Cases with TCR-related mutations were linked to shorter progression-free survival.

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Publications Bossard C, Dobay MP, Parrens M, Lamant L, Missiaglia E, Haioun C, Martin A, Fabiani B, Delarue R, Tournilhac O, Delorenzi M, Gaulard P, de Leval L.Immunohistochemistry as a valuable tool to assess CD30 expression in peripheral T-cell lymphomas: high correlation with mRNA levels. Blood. 2014;124:2983-6. Swerdlow SH, Jaffe ES, Brousset P, Chan JK, de Leval L, Gaulard P, Harris NL, Pileri S, Weiss LM; International Lymphoma Study Group.Cytotoxic T-cell and NK-cell lymphomas: current questions and controversies. Am J Surg Pathol. 2014;38:e60-71. Iqbal J, Wright G, Wang C, Rosenwald A, Gascoyne RD, Weisenburger DD, Greiner TC, Smith L, Guo S, Wilcox RA, Teh BT, Lim ST, Tan SY, Rimsza LM, Jaffe ES, Campo E, Martinez A, Delabie J, Braziel RM, Cook JR, Tubbs RR, Ott G, Geissinger E, Gaulard P, Piccaluga PP, Pileri SA, Au WY, Nakamura S, Seto M, Berger F, de Leval L, Connors JM, Armitage J, Vose J, Chan WC, Staudt LM; Lymphoma Leukemia Molecular Profiling Project and the International Peripheral T-cell Lymphoma Project.Gene expression signatures delineate biological and prognostic subgroups in peripheral T-cell lymphoma. Blood. 2014;123:2915-23. Gaulard P, de Leval L.Pathology of peripheral T-cell lymphomas: where do we stand? Semin Hematol. 2014;51:5-16. de Leval L, Parrens M, Le Bras F, Jais JP, Fataccioli V, Martin A, Lamant L, Delarue R, Berger F, Arbion F, Bossard C, Copin MC, Canioni D, Charlotte F, Damaj G, Dartigues P, Fabiani B, Ledoux-Pilon A, Montagne K, Molina T, Patey M, Tas P, Peoch M, Petit B, Petrella T, Picquenot JM, Rousset T, Rousselet MC, Soubeyran I, Thiebault S, Tournilhac O, Xerri L, Gisselbrecht C, Haioun C, Delsol G, Gaulard P.Angioimmunoblastic T-cell lymphoma is the most common T-cell lymphoma in two distinct French information data sets. Haematologica. 2015;100:e361-4.

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4. Outlook

The way the MEDIC foundation supported consortium ‘Tumor-host interaction’ continues to evolve confirms that high quality research can be effectively supported in an approach that is competitive in a (topographically and in terms of field of interest) limited setting. A high standard continues to be reached through auto-evaluation, internal review within the consortium and external peer review of new applications. The research program with its consortium approach continues to foster new interactions and new projects that individual groups might not have taken on. External peer review of the consortium constitutes a significant effort but remains an essential step towards a scientifically valid modus operandi. The independent external Scientific Advisory Board (prof. F.Lejeune, prof.G.Christofori, prof. H.Moch, prof. M.Mareel), evaluates the overall performance of the consortium, in participating (in part) in the annual research meeting and evaluating the annual reports submitted by the groups. The board members function as jury in the MEDIC prize applications. The trustees are satisfied with the structures developed and have confirmed their intention to continue to support the consortium - to the extent of what is financially possible - at the present level. As the Foundation did not seek a high profile but better visibility, the website serves an important purpose (www.fondation-MEDIC.ch). The site allows MEDIC member groups to remain informed as to the activities of the consortium. Important element is also the obligation of investigators supported by MEDIC to specifically mention MEDIC support in their publications. In 2014 the MEDIC foundation joined the consortium of foundations supporting the creation of a Center for Translational Oncology at the University Hospital in Geneva/Faculty of Medicine of the University of Geneva. This Center will function in close collaboration with the Department of Oncology in Lausanne, who together will provide coordinated advanced oncology care in the Lake Geneva region. IN parallel, it was decided to make the 'MEDIC prize’ for a particularly promising young clinician scientist, which was awarded for the first time in The next MEDIC prize will therefore be awarded in 2016. Awardees are beginning to seek grant support from MEDIC, which is a perfect approach towards maintaining a dynamic evolution of the consortium. Fred T Bosman Lausanne November 2015