Programme Book

13 - 16 March 2017 Amsterdam, Netherlands

2nd EACR-OECI Conference

Scientific Programme CommitteeRichard Marais, Daniel Peeper (Chair),

Giorgio Stanta and Emile Voest

Making it PersonalCancer Precision Medicine

13 - 16 March 2017 • Amsterdam, Netherlands

Day 1 - Monday 13 March 2017

11.00 – 13.00 REGISTRATION Rode Hoed foyer

12.00 – 13.00 WELCOME LUNCH & OPENING OF THE TRADE EXHIBITION Rode Hoed foyer & Oosterhuiszaal

13.00 – 13.10 CONFERENCE WELCOME Oosterhuiszaal ScientificProgrammeCommittee

13.10 – 13.40 Keynote Lecture Session chair: Daniel PeeperQ&A: 13.40 – 13.55 Caroline Dive CRUK Manchester Institute (UK) “Liquid biopsies: now and next”

SESSION1:TumourEvolution SessionChair:MargaretFrame

13.55 – 14.15 Zlatko Trajanoski InnsbruckMedicalUniversity(Austria)Q&A: 14.15 – 14.25 “Immunoeditingofthecancergenomeduringtumorprogressionandcheckpointblockade”

14.25 – 14.35 Proffered Paper 1: Julia Boshuizen NKI (Netherlands)Q&A: 14.35 – 14.40 “Earlyeliminationofinvasiveandmultidrug-resistantmelanomabyanovelanti-AXL antibodydrugconjugate”

14.40 – 15.15 COFFEE BREAK Rode Hoed foyer

15.15 – 15.45 Keynote Lecture Session chair: Daniel PeeperQ&A: 15.45 – 16.00 Jeffrey Engelman NovartisInstitutesforBioMedicalResearch (USA) “Targetedtherapiesandresistance:wherearewegoing?”

SESSION2:Organoids,PDX,CTX,ODX Session Chair: Joan Seoane

16.00 – 16.20 Meritxell Huch Gurdon Institute (UK)Q&A: 16.20 – 16.30 “Liverorganoidsforthestudyofliverbiologyanddisease”

16.30 – 16.40 Proffered Paper 2: Nicholas Younger MRC Institute of Genetics & Molecular Medicine (UK)Q&A: 16.40 – 16.45 “ACombinedComputationalandOrganoid-basedGeneticScreeningStrategyforthe IdentificationofNovelDriversofIntrahepaticCholangiocarcinoma”

16.45 – 17.05 Emile Voest NKI (Netherlands)Q&A: 17.05 – 17.15 “Tailoringtreatmenttotheindividualpatientwithmetastaticcancerusingpatient-derived tumororganoids,theTUMOROIDandSENSORinitiatives”

Making it PersonalCancer Precision Medicine

17.15 – 18.30 NETWORKING RECEPTION Rode Hoed foyer and lower balcony Ahotbuffetdinnerwillbeservedforallparticipantsandexhibitorstoenjoy.Thetrade exhibitionwillbeopenatthistime,andthereisanopportunitytocontinuenetworking.

18.30 – 19.15 Keynote Lecture Sessionchair:EmileVoestQ&A: 19.15 – 19.30 Hans Clevers Hubrecht Institute (Netherlands) “Lgr5StemCell-basedorganoidsinhumandisease”

Day 2 - Tuesday 14 March 2017

08.30 – 09.30 POSTERVIEWINGLower balcony Coffee available to purchase from the bar

SESSION 3: METABOLOME TARGETS Session Chair: Maria Sibilia

09.30 – 09.50 Ayelet Erez WeizmannInstituteofScience(Israel)Q&A: 09.50 – 10.00 “Thecontributionofaminoacids’metabolismtocarcinogenesis”

10.00 – 10.20 Eyal Gottlieb TechnionIntegratedCancerCenter(Israel)Q&A: 10.20 – 10.30 “Theonco-metabolicroleandliabilitiesoftheTCAcycleincancer”

10.30 – 10.40 Proffered Paper 3: Deepti Mathur Mount Sinai (USA)Q&A: 10.40 – 10.45 “PTENinactivationcreatesvulnerabilitytoDHODHinhibition”

10.45-11.15 COFFEE BREAK Rode Hoed foyer

SESSION4:INFLUENCEOFTHEMICROENVIRONMENT SessionChair:CarolineDive

11.15 – 11.35 Margaret Frame EdinburghCRC(UK)Q&A: 11.35 – 11.45 “Cancercellpolarizationandimmuneevasionintheniche;co-regulationbythemultitasking Focal Adhesion Kinase”

11.45 – 11.55 Proffered Paper 4: Jorrit Enserink OsloUniversityHospital(Norway)Q&A: 11.55 – 12.00 “HSP90InhibitorsSelectivelyTargetFLT3-ITD-drivenAcuteMyeloidLeukemiaandBypass TKI Resistance”

12.00 – 12.20 Maria Sibilia MedicalUniversityofVienna(Austria)Q&A: 12.20 – 12.30 “Cell-specificfunctionsoftheEGFRintumordevelopment”

12.30 – 12.50 Bart Vanhaesebroeck UCL (UK)Q&A: 12.50 – 13.00 “TargetingPI3-kinasedeltainhaematologicalmalignanciesandsolidtumours”

13.00 – 13.45 LUNCH & TRADE EXHIBITION Rode Hoed foyer

13.45 – 14.15 SATELLITESYMPOSIUM1-ELITESPONSOR Lecture hall

14.15 – 15.45 POSTER DEFENCE SESSION 1 Lower balcony

SESSION 5: NEW TARGETS SessionChair:EmileVoest

15.45 – 16.05 Richard Marais CRUK Manchester Institute (UK)Q&A: 16.05 – 16.15 “Precisionmedicineformelanoma:arewethereyet?”

16.15 – 16.35 Joan Seoane Valld’HebronInstituteofOncology(VHIO)(Spain)Q&A: 16.35 – 16.45 “Tumourmicroenvironmentasatherapeutictargetinglioblastoma”

16.45 – 16.55 Proffered Paper 5: Elisabeth Chen SangerInstitute(UK)Q&A: 16.55 – 17.00 “Anapoptoticprimingassayidentifiessignaturesofapoptoticsensitivityanddrugresponse in colorectal cancer cells”

17.00 – 17.20 Lars Zender UniversityHospitalTübingen(Germany)Q&A: 17.20 – 17.30 “Title to be announced”

Day 3 - Wednesday 15 March 2017

08.30 – 09.30 POSTERVIEWINGLower balcony Coffee available to purchase from the bar

SESSION 6: COMBINATORIAL TARGETED & IMMUNO APPROACHES Session Chair: Richard Marais

09.30 – 09.40 Proffered Paper 6: Elena Galvani CRUK Manchester Institute (UK)Q&A: 09.40 – 09.45 “Tumourmutationalloaddoesnotcorrelatewithresponsetoanti-PD-1treatmentina mousemodelofmelanoma”

09.45 – 10.05 Christian Blank NKI (Netherlands)Q&A: 10.05 – 10.15 “Combiningtargetedandimmunotherapyinmelanoma–whyintermittentcombination mightbethewaytogo”

10.15 – 10.45 COFFEE BREAK Rode Hoed foyer

10.45 – 11.05 Daniel Peeper NKI (Netherlands)Q&A: 11.05 – 11.15 “Systematicgeneticperturbationstorevealtherapeuticmelanomaandimmunecell vulnerabilities”

11.15 – 11.35 Roger Lo UCLA (USA)Q&A: 11.35 – 11.45 “TherapeuticResistancetoMAPKandPD-1Targeting”

11.45 – 12.30 LUNCH & TRADE EXHIBITION Rode Hoed foyer

12.30 – 13.00 SATELLITESYMPOSIUM2-ELITESPONSOR Lecture hall

13.00 – 14.30 POSTER DEFENCE SESSION 2 Lower balcony

14.30-16.00 ROUND TABLE DISCUSSIONS Coffee served at 15:00 Thissessionenablesparticipants,speakersandexhibitorstoconverseinformallyinsmaller groups.Participantsareinvitedtoaskanyquestionstheylikeandtomovefreelybetweenthe differentgroups.

19.30-22.00 CONFERENCE DINNER Restaurant-CaféInDeWaag Pre-bookedoptionalextra

Congratulations to the winners of EACR and OECI Meeting Bursaries. Each winner received a full registration free of charge and funds of up to 500 Euros to assist with the cost of travel.

EACRandOECIMeetingBursaryAwardWinners

Day 4 - Thursday 16 March 2017

08.30 – 09.30 LASTCHANCETOVIEWPOSTERSLower balcony Coffee available to purchase from the bar

09.30 – 10.15 MEET THE EXPERT SESSION Session Chair: Daniel Peeper

Nuria Lopez-Bigas IRB Barcelona (Spain) Nuria,abiomedicalinformaticsexpert,willsharefromherpersonalperspectiveand experiences. There will be an opportunity to ask your questions.

SESSION 7: IMPLEMENTING GENOMICS INTO CLINICAL PRACTICE Session Chair: Lars Zender

10.15 – 10.35 Giorgio Stanta UniversityofTrieste(Italy)Q&A: 10.35 – 10.45 “Researchinpatients’biologicalmaterialastoday’sintegralpartofclinicalactivity”

10.45 – 11.15 COFFEE BREAK Rode Hoed foyer

11.15 – 11.20 Proffered Paper 7: João Neto NKI (Netherlands)Q&A: 11.20 – 11.30 “Quadrupleverticaltargetingofanoncogenicpathwayasatreatmentstrategytoprevent drugresistance”

11.30 – 11.50 David Solit MSKCC (USA)Q&A: 11.50 – 12.00 “Definingtheactionablegenome”

12.00 – 12.15 CLOSING SUMMARY ScientificProgrammeCommittee PosterPrizesandMeetingBursarieswillbepresentedbyGiorgioStanta

12.15 – 13.00 LUNCH AND DEPART Rode Hoed foyer

EACR winners Şeyma Aydınlık Turkey

Irina Babina UK

Elena Galvani UK

Bakho Dvali Georgia

Somaieh Hedayat UK

Tessa Paulien Sandberg Netherlands

Roy van der Meel Canada

OECI winners Milad Baradaran-Ghahfarokhi Italy

Natalia Bezdieniezhnykh Ukraine

Fabio Del Ben Italy

João Lobo Portugal

Lucia Sommerova Czech Republic

Interactive Activities at the ConferenceAn important part of the EACR Conference Series is the range of opportunities we aim to provide for participants to interact, discuss, reflect and build relationships and collaborations.

We hope you enjoy the dedicated interactive activities, which are listed below.

Networking Reception 17.15 – 18.30 Monday 13 March

A hot buffet dinner will be served prior to the evening Keynote Lecture, and this will be an ideal opportunity for participants to get to know each other and enjoy the unique venue of De Rode Hoed. De Rode Hoed’s Great Hall is the largest and oldest remaining hidden church in the Netherlands.

Poster Defence Sessions 14.15 – 15.45 Tuesday 14 March 13.00 – 14.30 Wednesday 15 March

There are two dedicated Poster Defence Sessions in the Programme. At these times, the presenters for that session are asked to stand by their posters to discuss their work with other participants and invited speakers.

Two EACR Poster Prizes worth €100 each will be awarded to the best poster presentations at the conference. The judging panel is comprised of speakers from the conference, and they will assess the top scoring abstracts based on the scientific content, the layout of the poster, and the verbal discussion. The winners will be announced during the Closing Session on Thursday.

Poster viewing 08.30 – 09.30 Tuesday 14 March 08.30 – 09.30 Wednesday 15 March 08.30 – 09.30 Thursday 16 March

Tuesday, Wednesday and Thursday will begin with an optional poster viewing slot. Participants are invited to use this time for further discussion in the poster areas, but presenters are not required to be by their posters at these sessions. Coffee and tea will be available to purchase from the bar.

Round Table Discussions 14.30 – 16.00 Wednesday 15 March

This session enables participants, speakers and exhibitors to converse informally in smaller groups. Participants are invited to ask any questions they like and to move freely between the different groups. Coffee will be served at 15:00.

Conference Dinner 19.30 – 22.00 Wednesday 15 March

The conference dinner is an excellent opportunity for participants and speakers to get to know each other in a relaxed and informal environment. The dinner will take place at the Restaurant-Cafe In de Waag, Nieuwmarkt 4, 1012 CR Amsterdam. Tickets must be purchased in advance.

Meet the Expert 09.30 – 10.15 Thursday 16 March

Invited speaker Professor Nuria Lopez-Bigas will share her personal perspective and experience as a cancer researcher from the computational genomics side. She will talk about the pancancer analysis of cancer genomes and on the interpretation of the biological and clinical relevance of tumour mutations. Professor Lopez-Bigas is a group leader at the Institute for Research in Biomedicine in Barcelona. There will be an opportunity to ask your questions.

Don’t forget to let us have your feedback about these activities in the survey we will send after the conference!

Organisation of European Cancer Institutes

www.oeci.euFor membership contact:

oeci@oeci.eu

Developing the Future in

Comprehensive CanCer Care

21-23 June 2017 Brno, Czech Republic

GENERAL ASSEMBLY SCIENTIFIC CONFERENCES

AND RELATED EVENTS

Oncology DaysORGANISATION OF EUROPEAN CANCER INSTITUTES

EUROPEAN ECONOMIC INTEREST GROUPING

3939The OECI is a non-governmental, non-profit Organisation founded in Vienna in 1979 and remodelled in 2005 into OECI-EEIG, a European Economic Interest Grouping, headquartered in Brussels. Today, the OECI counts 78 Member Institutes, which include some of the most prominent European Comprehensive Cancer Centres.The OECI aims to create a critical mass of expertise and competences, contributing to the production and dissemination of knowledge, so as to reduce fragmentation and increase competitiveness. These goals are being achieved by promoting and strengthening the concept of “comprehensiveness”, supporting quality in cancer care also through a well-structured internal organisation. OECI aims to accelerate the production and application of personalised care approaches, and to ensure equal rights to all cancer patients, with the ultimate goal of finding new and better treatments, providing more comprehensive care and improving patient quality of life, through evidence-based medicine. The increasing interest from international organisations, stakeholders & cancer community in the OECI points to the growing importance of a comprehensive cancer network of institutions, where the entire chain of cancer care provision is present.The strong alliance with the European Cancer Patient Coalition – ECPC - provides a coherent picture of today’s cancer patients’ expectations, ranging from: quality of care to information on survivorship and medical nutrition, improvement in the quality of interdisciplinary patient treatment, harmonisation in oncology healthcare practices and patient advocacy capacity to effectively tackle cancer care inequalities in Europe. Giving the crucial role to pathology departments in oncology and in lieu of the expected “influx” of new markers and diagnostics, the OECI acts in close cooperation with the European Association for Cancer Research and the European Society of Pathology, in order to better disseminate the innovation process amongst its members and abroad. The EACR-OECI training course series on “Precision Medicine” and “MolecularPathology Approach to Cancer” in Amsterdam, are just two examples of the OECI efforts to promote the innovation in cancer diagnosis and care.

O

E C

I

E E

I G

ACCREDITATION AND

DESIGNATION

CERTIFYING

COMPREHENSIVE

CANCER CARE

Organisation

of European

Cancer Institutes

Accreditation

and Designation

User Manual V. 2.0

Accreditation

and Designation U

ser Manual V. 2.0

OECI-EEIG Central Office

c/o Fondation Universitaire

11, Rue d’Egmont

B-1000, Brussels, Belgium

Phone: +32 2 512 0146

www.oeci.eu

www.selfassessment.nu

OECI Members A&D certified OECI Members in the A&D process Other OECI Members

Speaker abstracts

Keynote Lecture

Liquid Biopsies – Now and Next

Caroline Dive1

1 CRUK Manchester Institute & Manchester Centre for Cancer Biomarker Sciences, Manchester, UK

Sampling a cancer patient’s blood is easy and repeatable, less expensive than imaging and less invasive than tumour biopsy. Most cancer researchers now agree that liquid biopsies are demonstrating great potential and are beginning to inform patient management. Notably, the first FDA approved blood-based genetic test to detect gene mutation in EGFR of non-small cell lung cancer (NSCLC) patients is a companion diagnostic for the drug Tarceva (erlotinib). Landmark studies have also demonstrated the potential of ctDNA to monitor tumour evolution, to chart response to therapies and to identify possible mechanisms of therapy resistance. The development of biomarkers based on circulating miRNA (with its stability in the bloodstream) and the potential of exosomes as biomarker sources are exciting new developments in the liquid biomarkers field. The rarity and heterogeneity of circulating tumour cells (CTCs) make them a more technically demanding liquid biopsy but one that holds great promise as the technical challenges are met, allowing analysis of DNA, RNA and Proteins. CTC enumeration with CellSearch (EpCam and cytokeratin positive CTCs) in diseases with prevalent CTCs such as small cell lung cancer (SCLC) is useful for prognosis, pharmacodynamic evaluation and measurement of predictors of response. Yet phenotypic heterogeneity confounds, and marker independent technology platforms in development will better assess CTC subpopulations, including those in dynamic stages of epithelial to mesenchymal transition where EpCam has been down-regulated. The single cell revolution and parallel advances in bioinformatics approaches are enabling CTC research, and developments in single CTC isolation and genomic profiling allow insight to the biology of invasive tumour cells including cellular co-expression of cancer specific mutations. The ability to enrich blood for CTCs and develop mouse models (termed CTC derived patient explants CDX) and to culture CTCs bring added impetus to the liquid biopsy field and support drug development and, in some cases,

is amenable to real time drug testing. The holy grail of earlier detection of cancer using liquid biopsies is a high bar challenge, where efforts using ctDNA are ongoing to increase sensitivity whilst maintaining specificity. For CTCs, technology platforms such as the high definition-single cell analysis (HD-SCA) approach are needed, which eliminate an enrichment step that incurs cell losses and thus analysis every cell in the sample, with morphometric and mapped molecular analysis at high resolution.

Using small cell lung cancer as an example I will present our progress in DNA profiling of single SCLC CTCs and ctDNA, and how this approach may inform on anticipating chemotherapy response, tumour heterogeneity and, via serial sampling, on mechanisms of drug resistance. Most recently, we developed lung cancer patient CTC derived mouse models (CDX). SCLC CDX models faithfully recapitulate patient drug responses and will be useful to test novel therapeutics. CDX are generated at patient presentation and for those patients who first respond and then relapse with drug resistant disease, the relapse blood sample can be used to generate a paired CDX. Alongside single CTC profiling, the CDX approach will allow a comprehensive analysis of acquired drug resistance to chemotherapy, the discovery of new drug targets and testing of targeted therapies and drug combinations. I will also describe recent research exploring vasculogenic mimicry in SCLC as a mechanism supporting dissemination and metastasis.

Speaker abstracts

Immunoediting of the cancer genome during tumour progression and checkpoint blockade

Zlatko Trajanoski11 Medical University of Innsbruck, Innsbruck, AUSTRIA

The cancer immunoediting hypothesis postulates a dual role of the immune system: protecting the host by eliminating tumor cells, and shaping the developing tumor by editing the cancer genome. However, to what extent immunoediting is shaping the cancer genome in common malignancies is still a matter of debate. Moreover, the impact of cancer immunotherapy with checkpoint blockers on modulating immunoediting remains largely unexplored.

Here we employed a mouse model of colorectal cancer (CRC), next-generation sequencing, and computational analyses to elucidate the impact of evolutionary and immune-related forces on editing the tumor. We first carried out genomic and transcriptomic analyses of a widely-used model, MC38 cell line and show that this is a valid model for hypermutated and microsatellite-unstable CRC. Analyses of the data from longitudinal samples of wild type and immunodeficient RAG1 knockout mice transplanted with MC38 cells revealed that upregulation of checkpoint molecules and infiltration of Tregs are the major tumor escape mechanisms. Strikingly, the impact of neutral evolution on sculpting the tumor outweighed immunoediting by T cell dependent and T cell independent mechanisms in the progressing tumors. We also show that targeting the PD-1/PD-L1 pathway potentiated immunoediting and rendered tumors more homogeneous.

In summary, our study demonstrates that neutral evolution is the major force that sculpts the tumor during progression, and that checkpoint blockade effectively enforces T cell dependent immunoselective pressure in this model. The results have important implication for basic research studies on the mechanisms of resistance to checkpoint blockade and for clinical translation.

Proffered Paper 1

Early elimination of invasive and multidrug-resistant melanoma by a novel anti-AXL antibody drug conjugate

Julia Boshuizen2, Louise Koopman1, Esther Breij1, David Satijn1, Daniel Peeper2, Paul Parren1

1 Genmab, Utrecht, NETHERLANDS, 2 Netherlands Cancer Institute, Amsterdam, NETHERLANDS

The receptor tyrosine kinase AXL is overexpressed in many types of cancer, including melanoma, and is associated with EMT and increased invasiveness of tumors. AXL is also upregulated upon resistance to a variety of therapies including the oft-used BRAF and MEK inhibitors.

We have shown previously that drug resistance and upregulation of AXL occur concurrently with the downregulation of MITF. Thus, we hypothesized that targeting AXL-expressing melanoma cells may specifically eliminate invasive and drug-resistant cancer cells, while the MITF-high melanoma cells can be targeted with MAPK inhibitors.

Therefore, we developed a novel antibody drug conjugate: HuMax-AXL-ADC, consisting of an AXL antibody coupled to the microtubule-disrupting agent MMAE. We show that HuMax-AXL-ADC has strong tumor-eliminating effects in AXL-expressing melanoma cells both in vitro and in vivo, including several patient-derived melanoma xenografts. Also, we show that combining HuMax-AXL-ADC with BRAF inhibitors yields superior anti-tumor effects compared to either single agent alone. Importantly, through immunohistochemical analysis of 42 human melanoma biopsies we find rare AXL-expressing (and presumably drug-resistant) cells prior to targeted therapy, which are commonly enriched in the majority of BRAF inhibitor-resistant melanomas.

These findings suggest that drug-resistant melanoma fractions, whether present prior to therapy or upon acquisition of resistance, can be effectively targeted with HuMax-AXL-ADC. Furthermore, combining HuMax-AXL-ADC with MAPK inhibitors may prevent or delay onset of resistance to targeted therapies by specifically eliminating drug-resistant clones in heterogeneous melanomas.

Speaker abstracts

Keynote Lecture

Targeted therapies and resistance: where are we going?

Jeffrey Engelman1

1 Global Head of Oncology, Novartis Institutes for BioMedical Research, Cambridge, MA, USA

Abstract not available at the time of printing.

Liver organoids for the study of liver biology and disease

Meritxell Huch Ortega1,2,3

1 Gurdon Institute-Wellcome Trust/Cancer Research UK, Cambridge, UK, 2 Department of Physiology, Developmental Biology and Neuroscience, University of Cambridge, Cambridge, UK, 3 Wellcome Trust-Medical Research Council Stem Cell Institute, Cambridge, UK

Despite the enormous replication potential of the liver, there are currently no culture systems available that sustain hepatocyte replication in vitro. Hepatocytes can be maintained in culture for a few days. However, they lose their hepatocyte phenotype and function almost immediately, thus precluding its application for cell therapy treatments. Liver stem cells have the potential to self-renew and differentiate into functional hepatic lineages. Mouse liver stem cells can be indefinitely expanded in vitro (for >1 year), into “liver organoids”, in our liver stem cell culture system, in the absence of a mesenchymal niche. The cultured cells express ductal markers and differentiate into functional hepatocytes in vitro and in vivo. We have now further developed our culture system to study human liver stem cells and human liver disease. We describe a culture system that allows the long-term expansion of adult human liver stem cells (>3 months) from donor biopsies while maintaining their differentiation potential towards functional hepatocytes in vitro. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille Syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine and gene therapy.

Speaker abstracts

Proffered Paper 2

A Combined Computational and Organoid-based Genetic Screening Strategy for the Identification of Novel Drivers of Intrahepatic Cholangiocarcinoma

Nicholas Younger2,3, David Wilson2,3, Kevin Myant1,3, Duncan Sproul2,3, Luke Boulter2,3

1 Cancer Research UK Edinburgh Centre, Edinburgh, UK, 2 MRC Human Genetics Unit, Edinburgh, UK, 3 MRC Institute of Genetics & Molecular Medicine, Edinburgh, UK

Identifying functionally relevant oncogenic driver mutations in rare cancers is hindered by the low availability of tissue for sequencing. An exemplary cancer, with poor prognosis and broad mutational spectrum is intrahepatic cholangiocarcinoma (iCCA), where despite whole exome sequencing (WES) studies, oncogenic drivers are difficult to predict. We have developed a methodology that computationally stratifies mutations from WES data, which acts as input for a liver organoid CRISPR/Cas9-mutagenesis screen. Using this methodology we will identify functional driver mutations in iCCA.

We have pooled a number of WES data sets (N=293), and re-derived mutation calls with lenient filtering thereby capturing potential sub-clonal drivers. Using a selection of driver-prediction algorithms we have defined a list of potential oncogenes that could promote iCCA growth. When used as input for our CRISPR/Cas9-based liver organoid screen, we can identify those mutations which give rise to clones with phenotypic hallmarks of cancer, such as sustained proliferation and resistance to cell death. Mutations identified are flagged for functional validation in vivo.

Using a test data set of tumour sequences, the driver-prediction algorithms identified genes that are functionally implicated in iCCA (PBRM1, BAP1, IDH1/2), as well as candidates that are oncogenes in other cancers (KDM6B, NOTCH2, PPP6C). Moreover, we have identified genes that are not annotated as oncogenic, however could function as oncogenic modifiers. This computational discovery method provides a rich substrate for functional screening in vitro.

Using Cas9-expressing liver organoids infected with a sgRNA library, applying selection pressure and deep-sequencing, we identified genes mutated in a Cas9-dependant manner including ERBB3, CTTN, VLDLR. We also identified genes involved in pathways commonly dysregulated, including the Wnt-signalling (GSK3B, TRIB2) and Notch pathways (HES3, NCSTN). Our data shows that by combining a computational methodology with in vitro-based accelerated mutagenesis we can identify novel driver genes without the need to sequence additional tumours.

Tailoring treatment to the individual patient with metastatic cancer using patient-derived tumor organoids, the TUMOROID and SENSOR initiatives

Emile Voest1

1 NKI - Netherlands Cancer Institute, Amsterdam, NETHERLANDS

Introduction: The promise of personalized or precision medicine is seriously hampered by the lack of truly specific tests to identify patients who will respond to a specific treatment. This is largely because tumor cells contain a large number of genetic abnormalities of which relevance and function are poorly understood. In addition, the context or tissue of origin is also an important determinant of outcome. This results in over treatment in the majority of tumor types with as a consequence ineffective treatment and side effects. Furthermore, oncology is in desperate need of a test that can support early drug development. We therefore embarked on a program employing tumor organoids, 3D cultures of cancer cells from individual patients, as a potential predictive assay for drug response in the clinic.

Material and method: The TUMOROID study, is a multicenter observational cohort study for patients with metastatic colorectal or non-small cell lung cancer. The primary objective is to determine if standard of care treatment response in vitro is similar to the observed treatment response in the same patient. The SENSOR study is a mono-center intervention study in which patients with metastatic colorectal cancer are treated based on the successful outcome of a screen with experimental drugs.

Results and discussion: The TUMOROID study has now included 63 patients. Definitions of an in vitro treatment response have been established. The SENSOR study has been initiated November 2016 and has since then accrued six patients, who are currently being screened for drug sensitivity. A preliminary screen on a test panel of 20 tumor organoids has demonstrated differential drug sensitivity, where genotype-based predictions matched our in vitro observations.

Conclusion: We have established a platform that will allow us to validate the clinical value of tumor organoids as a predictive test to select patients for anti-cancer treatment.

Speaker abstracts

Keynote Lecture

Lgr5 Stem Cell-based organoids in human disease

Hans Clevers1,2

1 Hubrecht Institute, Utrecht, NETHERLANDS, 2 Princess Maxima Center for Pediatric Oncology, Utrecht, NETHERLANDS, 3 Royal Netherlands Academy of Arts and Sciences, Amsterdam, NETHERLANDS, 4 University Medical Centre Utrecht, Utrecht, NETHERLANDS

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost ‘generic’ marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear tongue and stomach epithelium.

Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so called ‘mini-guts’ in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D cultures systems have been developed for the Lgr5+ve stem cells of human stomach, liver, pancreas, prostate and kidney. Using CRISPR/Cas9 technology, genes can be efficiently modified in organoids of various origins.

The contribution of aminoacids’ metabolism to carcinogenesis

Ayelet Erez5, Narin Carmel3, Daniel Helbling2, Joo Sang-Lee3, Qin Sun1, David Dimmock2, Sandesh Nagamani1, Eytan Ruppin3, Arkaitz Carracedo4

1 Baylor College of Medicine, TX, USA, 2 Medical College Wisconsin, Wisconsin, USA, 3 University of Maryland, MD, USA, 4 University of the Basque Country, Derio, SPAIN, 5 Weizmann Institute of Science, Rehovot, ISRAEL

The UC functions in the liver to convert toxic ammonia and excess nitrogen to urea. In contrast to the established roles of the UC enzymes in ureagenesis, the UC enzyme ASS1 was found to be dysregulated in multiple different cancers. We have recently shown that ASS1 silencing leads to preferential diversion of its substrate aspartate away from the synthesis of arginine towards nucleic acid synthesis, supporting enhanced proliferation. Here we show that in addition to ASS1, most UC enzymes are deregulated in different cancers to decrease amino acid degradation and disposal and increase their utilization as carbon and nitrogen substrates mainly by the pyrimidine synthesis enzyme- CAD. We find that this shift in the nitrogen flux results from significant homology between the UC enzymes and CAD, allowing for direct substrate channeling between the two pathways. Importantly CAD activation generates nucleotide imbalance between purine and pyrimidine and increases mutagenesis in tumors. Our findings are clinically relevant as we find additional metabolic signatures generated by the changes in nitrogen flux in plasma and urine of mice and humans with cancers. Furthermore, we find worse prognosis in cancer patients in whom the UC enzymes are deregulated, CAD is highly activated and who carry this specific transversion mutation signature.

Speaker abstracts

The onco-metabolic role and liabilities of the TCA cycle in cancer

Eyal Gottlieb1

1 Technion Integrated Cancer Centre, Haifa, ISRAEL

Last century, Otto Warburg observed that cancer tissues have high rates of glycolysis in the presence of oxygen. This was attributed to defects in mitochondrial respiration, and is now considered a key hallmark of cancer. Nevertheless, convincing evidence that mitochondrial dysfunction causes metabolic switch and tumorigenesis are scarce. Recently, the identification of mutations in key metabolic enzymes with a causal link to tumorigenesis awakened interest in Warburg’s hypothesis. Cancer-associated mutations have been identified in genes coding for tricarboxylic acid (TCA) cycle and closely related enzymes.

Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are TCA cycle enzymes while SDH is also complex II of the mitochondrial respiratory chain. Despite their key housekeeping activities, the genetic loss of FH or of any of the SDH subunits is associated with susceptibility to cancers. SDH or FH inactivation leads to the accumulation of succinate or fumarate, respectively. This in turn causes the activation of hypoxia-inducible factors and the inhibition of α-ketoglutarate-dependent histone and DNA demethylases, establishing pseudohypoxic and hypermethylator phenotypes. These biochemical changes are associated with hyper-vascularization and epithelial to mesenchymal transition (EMT) resulting in increased invasiveness and motility of these tumors. However, not much is known about the metabolic mechanisms which enable the survival and proliferation of SDH- or FH-defective cells. We generated genetically engineered mouse models in which FH or SDH activity has been ablated and identified metabolic pathways dispensable in normal, but essential in TCA cycle truncated cells. Our study provides mechanistic understanding of the onco-metabolic role of the TCA cycle intermediates fumarate and succinate, and it suggests novel therapeutic approaches for treating these metabolically-atypical neoplasms.

Proffered Paper 3

PTEN inactivation creates vulnerability to DHODH inhibition

Deepti Mathur3,1, Elias Stratikopoulos3, Sait Ozturk3, Nicole Steinbach3,1, Sarah Pegno3, Sarah Schoenfeld3, Raymund Yong3, Vundavalli V Murty1, John M Asara2, Lewis Cantley4, Ramon Parsons3

1 Columbia University, New York, NY, USA, 2 Harvard Medical School, Boston, MA, USA, 3 Icahn School of Medicine at Mount Sinai, New York, NY, USA, 4 Weill Cornell Medical College, New York, NY, USA

The importance of metabolism in tumor initiation and progression is becoming increasingly clear, and metabolic changes created by oncogenic drivers could be important targets for cancer treatment. PTEN is one of the most commonly mutated tumor suppressors and is therefore also a hub for developing targeted therapy. Here, we found that PTEN regulates glutamine metabolism, DNA replication, and the DNA damage response, the integration of which creates susceptibility to inhibition of dihydroorotate dehydrogenase, a key enzyme in pyrimidine synthesis. Inhibitors of dihydroorotate dehydrogenase generated synthetic lethality in cells with PTEN inactivation, manifesting as chromosomal breaks and ultimately cell death and tumor reduction. Inhibition of dihydroorotate dehydrogenase could thus be a promising therapy for patients with PTEN mutant cancers.

The findings of this research have been accepted for publication in Cancer Discovery.

Speaker abstracts

Cancer cell polarization and immune evasion in the niche; co-regulation by the multitasking Focal Adhesion Kinase

Valerie G Brunton1, Adam Byron1, Margaret C Frame1, Tom Lund1, Robert J.B. Nibbs2, Bryan Serrels1, Alan Serrels1

1 Edinburgh Cancer Research UK Centre, University of Edinburgh, Edinburgh, UK, 2 Institute of Infection, Immunity, and Inflammation, University of Glasgow, Glasgow, UK

The behavior of tumors can be ascribed to both cell-autonomous and non cell-autonomous traits. The integrin effector protein Focal Adhesion Kinase (FAK), which is mis-regulated in cancer, is a self-regulating scaffold/ kinase that controls molecular protein complex assembly at adhesion sites and at other sub-cellular locale. Adhesion-linked complexes ‘built’ by FAK typically regulate processes associated with adhesion and actin dynamics, and consequently cancer cell polarization and invasion. However, the tumor niche is composed of multiple cell types – a mini tissue - only a proportion of which are cancer cells. It is in this context that we have now also discovered a completely new function of FAK in driving anti-tumor immune evasion. Specifically, the activity of nuclear-targeted FAK in cancer cells drives recruitment and retention of intra-tumoral regulatory T-cells (Tregs) by transcriptionally regulating chemokine and cytokine ligand-receptor networks, crucially including transcription of Ccl5 and TGFb2. In turn, these changes inhibit antigen-primed cytotoxic CD8+ T-cell activity in the tumor microenvironment, permitting survival and growth of FAK-expressing tumors. We show that immune evasion requires nuclear FAK’s catalytic activity and and small molecule FAK kinase inhibitors drive depletion of Tregs and permit CD8+ T-cell-mediated tumor clearance. It is therefore likely that FAK inhibitors may trigger both inhibition of polarization and invasion, and promote immune-mediated negative pressure on tumours, potentially providing previously unrecognized therapeutic benefits.

Proffered Paper 4

HSP90 Inhibitors Selectively Target FLT3-ITD-driven Acute Myeloid Leukemia and Bypass TKI Resistance

Beibei Zhang1, Pilar Ayuda1, Dagim Tadele1, Yngvar Floisand1, Jorrit Enserink1,2

1 Oslo University Hospital, Oslo, NORWAY, 2 University of Oslo, Oslo, NORWAY

Treatment of Acute Myeloid Leukemia (AML) has not changed significantly during the past decades. Development of new therapy is confounded by the fact that AML is a highly heterogeneous disease, and over 100 unique combinations of known or suspected oncogenic mutations have been identified. Therefore, it is likely that specific subtypes of AML require specific treatment regimens. We used a recently developed personalized medicine approach to identify compounds that may benefit specific subsets of AML patients. Briefly, we tested the sensitivity of bone marrow-derived blast cells of nearly 60 AML patients against approximately 400 anticancer drugs and several drug combinations. Clustering analysis revealed a subgroup of AML patients that mainly consisted of patients with FLT3-ITD or FLT3-TKD mutations, which have been observed in approximately 30% of AMLs and which are associated with increased risk of relapse and poor clinical outcome. Interestingly, this patient group co-clustered with a specific subset of drugs that included TKIs known to inhibit FLT3, such as quizartinib, but also Raf, MEK and mTOR inhibitors. However, among the most potent compounds in this drug cluster were HSP90 inhibitors. By unraveling the FLT3-ITD-specific proteome we discovered that FLT3-ITD, but not normal FLT3, interacts with HSP90 family members, and FLT3-ITD activity is strongly dependent on HSP90 activity. Importantly, HSP90 inhibitors sensitize FLT3-ITD+ AML cells to TKIs and bypass TKI resistance. Our study reveals a molecular basis for HSP90 addiction of FLT3-ITD-driven AML.

Speaker abstracts

Speaker abstracts

Cell-specific functions of the EGFR in tumour development

Maria Sibilia2,1

1 Comprehensive Cancer Center, Vienna, AUSTRIA, 2 Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Vienna, AUSTRIA

The epidermal growth factor receptor (EGFR) is involved in the development of several tumors and EGFR targeted inhibition is employed for the treatment of several cancer types such as colorectal and lung cancer. So far the oncogenic function of the EGFR has always been attributed to its expression in tumor cells. Recent results from our laboratory have challenged this concept, as we were able to demonstrate that in certain tumors the EGFR is oncogenic when expressed in tumor-associated myeloid cells(1). These findings have important implications for the mechanism of action of anti-EGFR cancer drugs and might provide better stratification parameters for predicting anti-EGFR therapy responses. Patients receiving anti-EGFR therapies very frequently develop a severe skin rash, which is a strong predictor for treatment response. Our lab is interested in investigating the mechanistic insight responsible for these skin pathologies and could demonstrate that EGFR signaling in skin epidermal cells regulated key factors involved in barrier function and skin inflammation(2).

(1) EGFR has a tumour-promoting role in liver macrophages during hepatocellular carcinoma formation. Lanaya H et. al. Nat Cell Biol. 2014 Oct;16(10):972-81, 1-7.

(2) Epidermal EGFR controls cutaneous host defense and prevents inflammation. Lichtenberger BM et al. Science Transl. Med. 2013 Aug 21;5(199).

Targeting PI 3-kinase delta in haematological malignancies and solid tumours

Bart Vanhaesebroeck1

1 UCL Cancer Institute, University College London, London, UK

The PI 3-kinase (PI3K) signal transduction pathway has been implicated in a variety of physiological responses, and is a therapeutic target in amongst other cancer and inflammation. Mammals have eight distinct isoforms of PI3K. In order to gain insight into the physiological roles of PI3K isoforms, we have created ‘kinase knockin’ mice that have germline inactivating point mutations in the ATP-binding site of PI3K isoforms. This knockin strategy more faithfully mimics pharmacological inhibition than the classical knock-out approach. These knockin mice have allowed us to uncover new roles for PI3K isoforms in physiology, disease and therapy.

These studies led to the discovery of the p110delta isoform of PI3K as a new drug target in inflammation, allergy, haematological malignancies and, most recently, in solid tumours. We have taken the characterization of p110delta ‘all the way’, from gene cloning, through to the development of the first mouse models, followed by the generation of p110delta inhibitors in a drug development programme with PIramed Ltd (purchased by Roche in 2008). Targeting p110delta has been the most successful clinical PI3K inhibitor development effort to date, which culminated in the approval in 2014 of the p110delta inhibitor Zydelig (Gilead Sciences Inc) for the treatment of specific blood cancers. p110delta inhibitors are also being trialled in arthritis and airway inflammation.

Our work (Nature 2014:510:407) indicates that p110delta could also be a target for immuno-stimulation in cancer, potentially widening the use of p110delta drugs from inflammation and haematological malignancies to immune-therapy of solid tumours.

We have also discovered new biological roles of oncogenic PIK3CA that will be discussed.

Speaker abstracts

Precision medicine for melanoma: are we there yet?

Richard Marais1

1 CRUK Manchester Institute, Manchester, UK

Melanoma of the skin is linked to exposure to ultraviolet radiation (UVR) and genome sequencing has revealed that there are various subtypes of this disease defined by their driver oncogenes and chromosomal aberrations. BRAF, a protein kinase of the RAS-ERK signalling pathway is mutated in about half of common cutaneous melanomas and NRAS, which activates BRAF, is mutated in a further 20% of cases. Drugs that inhibit BRAF or MEK, which is downstream of BRAF, extend both progression free and overall survival in BRAF mutant melanoma patients. However, in melanomas in which NRAS is mutated BRAF drugs are ineffective because signalling re-routes through CRAF (a kinase closely related to BRAF), and MEK drugs are only weakly effective. In parallel, significant advances have been made in the development of immunotherapies for melanoma patients, and drugs that induce the immune system to attack melanoma are effective in 20-60% of patients regardless of the driver oncogene. Critically, in some patients immunotherapies are curative. Despite these exciting results, there are still significant challenges. Most patients who receive BRAF pathway inhibitors eventually relapse due to intrinsic or acquired resistance, and we still do not know how to stratify patients for immunotherapies to improve outcomes and reduce toxicities.

We therefore need new approaches to stratify melanoma patients and so we have developed a platform of technologies to guide treatment selection in individual patients. Next generation sequencing reveals the genomic landscape of individual tumours, providing the opportunity of selecting the most appropriate drugs for an individual patient. We have developed patient-derived xenografts from over 100 patients and use these to study melanoma biology and the response of tumours to hypothesis-driven therapeutic strategies. Where tumours are inaccessible, xenografts can be developed from circulating tumour cells (CDX), allowing us to study the biology of late-stage disease. Finally, we use cell-free tumour DNA (ctDNA) in the circulation to monitor patient responses to treatments and to monitor mechanisms of resistance. We are developing clinical trials to use ctDNA to guide clinical decisions about which drugs to administer and when to switch patients’ treatments from one modality to another.

Tumour microenvironment as a therapeutic target in glioblastoma

Joan Seoane1,4

1 ICREA, Barcelona, SPAIN, 2 Universitat Autònoma de Barcelona, Barcelona, SPAIN, 3 Vall d’Hebron University Hospital, Barcelona, SPAIN, 4 Vall d’Hebron Institute of Oncology (VHIO), Barcelona, SPAIN

One the most important challenges in the treatment of cancer is its evolving genomic intratumor heterogeneity. Tumors are composed of assortments of cells with subclonal genomic alterations that evolve following Darwinian selection. Glioblastoma (GBM) is a prototypical heterogeneous tumor and still little is known about intratumor heterogeneity in brain metastasis. Most importantly not much is known about how intratumor heterogeneity impacts on the response to treatments and tumor relapse. The study of the spatial and temporal genomic architecture of GBM is essential to understand the biology and improve the treatment of this dismal disease. Through the study of primary and relapsed tumors and patient-derived xenograft models (PDX) of GBM, we are studying the genomic subclones emerging after treatment since these subclones might be enriched in genomic alterations that confer a selective advantage and resistance to treatment. In addition, we are also developing non-invasive circulating biomarkers such as the analysis of circulating tumor DNA in the cerebrospinal fluid to longitudinally monitor the evolving intratumor heterogeneity in brain tumors.

Speaker abstracts

Proffered Paper 5

An apoptotic priming assay identifies signatures of apoptotic sensitivity and drug response in colorectal cancer cells

Elisabeth Chen3,2, Andrew Barthorpe3, Patrick Bhola1, Anthony Letai1, Mathew Garnett3

1 Dana-Farber Cancer Institute, Boston, Massachusetts, USA, 2 University of Cambridge, Cambridge, UK, 3 Wellcome Trust Sanger Institute, Hinxton, UK

Avoidance of apoptosis is a hallmark of cancer. The intrinsic mitochondrial pathway of apoptosis is central to chemotherapeutic response in many cancer types. It is tightly regulated by an intricate network of BCL-2 family proteins, which have altered expression in up to 70% of tumours across various cancer types. BH3 profiling assays assessing apoptotic sensitivity in tumour cells are predictive of clinical response to chemotherapy.

Despite current efforts to modulate the activity of BCL-2 family proteins in personalised therapy, relatively little is known about the varying contributions of BCL-2 family proteins to apoptosis induction and how patient-specific molecular backgrounds affect therapeutic response.

We aim to address these questions in colorectal cancer which is the fourth most common cause of cancer death worldwide. We designed and employed a high-throughput apoptotic profiling screen across a set of 45 comprehensively annotated colorectal cancer cell lines. Our screen uses a panel of different pro-apoptotic BH3 peptides to engage and assess the varying contributions of different components of the BCL-2 family network in mediating apoptosis. In addition to creating a “baseline” profile of apoptotic sensitivity for each cell line, we also employed “dynamic” apoptotic profiling to assess the effect of commonly used colorectal cancer therapeutics on the apoptotic network.

We observe variable apoptotic sensitivity across cell lines with different genomic backgrounds. To explain different sensitivity profiles, we are performing statistical analysis on several layers of characterisations of the cell lines, including genomic annotation, gene expression and proteomic data, as well as high-throughput drug screening data. Our preliminary results indicate that not only do apoptotic sensitivities correlate with gene expression signatures, but that they may also be used to predict response to anti-cancer compounds and potential drug combinations.

Title to be announced

Lars Zender1

1 Division of Molecular Oncology of Solid Tumors, University Hospital Tübingen, GERMANY

Abstract not available at the time of printing.

Speaker abstracts

Proffered Paper 6

Tumour mutational load does not correlate with response to anti-PD-1 treatment in a mouse model of melanoma

Elena Galvani2, Piyushkumar Mundra2, Kate Hogan2, Louis Boon1, Amit Mandal2, Matthew Smith2, Jonathan Greenall2, Garry Ashton3, Nathalie Dhomen2, Richard Marais2

1 EPIRUS Biopharmaceuticals Netherlands, Utrecht, NETHERLANDS, 2 Molecular Oncology Group, Cancer Research UK Manchester Institute, Manchester, UK, 3 Research Services, Cancer Research UK Manchester Institute, Manchester, UK

Therapeutic antibodies targeting immune checkpoints, such as cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) have led to a paradigm shift in melanoma treatment, providing durable responses in patients with advanced disease. However, not all patients benefit from these therapies and in some patients significant high-grade toxicities have been observed. It is therefore important to understand the factors that contribute to response to stratify patients, both to limit toxicity and to improve patient outcomes. Amongst other factors, high mutational burden/neoantigen load and minimal intra-tumour heterogeneity have been positively associated with response to immune checkpoint blockade in melanoma and non-small cell lung cancer (NSCLC). We used chronic exposure to ultraviolet radiation (UVR) to simulate the effect of sunburn in a mouse model of BRAFV600E-driven melanoma. The tumours that developed in the UVR treated group presented with a significant increase in mutation burden compared to mice that were not treated with UVR. Approximately 70% of tumour-bearing mice did not respond to treatment with anti-PD-1 checkpoint antibodies, whereas the remaining mice either presented stable disease, or in sporadic cases tumour regression. Critically, we observed a survival benefit in the mice that presented stable disease, but there was no significant association between UVR exposure or tumour mutational load and response to anti-PD-1. Moreover, in line with clinical responses in melanoma and NSCLC patients treated with anti-CTLA-4 or anti-PD-1 respectively, clonal analysis of the murine tumours revealed an inverse association between grade of heterogeneity and response to treatment. Our data highlight the need for integrated molecular characterisation of tumours and their associated microenvironment to identify robust biomarkers of response to immune checkpoint inhibitors.

Combining targeted and immunotherapy in melanoma – why intermittent combination might be the way to go

Christian Blank1

1 NKI, Amsterdam, NETHERLANDS

Continuous combinations of targeted therapy (TT), e.g. BRAF+MEK inhibitors, with immunotherapy (IT), e.g. CTLA-4 or PD-1 blockade are currently tested in several phase1/2 trials with the aim of improving response rate and response duration. However, high toxicity rates have been observed, leaving PD-1 blockade of being currently the only possible combination partner for BRAF+MEK inhibition in BRAFV600-mutation-positive patients.

Recently we have published preclinical data, showing that short-time TT induces strong tumor T cell infiltration and is highly synergistic with PD-1 blockade. On-treatment analyses of melanoma patients treated with BRAF or BRAF+MEK inhibition indicated that long-term TT might be counterproductive, as the initial increase in T cell tumor infiltration decreases in some patients already beyond 2 weeks of TT.

This raises the question which time period of MAPK-pathway inhibition will be optimal for combination with anti-PD-1. To address this question, we have set-up the IMPemBra trial, comparing mono-treatment with PEM with combination schemes of intermittent/short-course BRAF + MEK inhibition in combination with PEM. The primary objective of this early phase trial is to explore safety, feasibility and the immune-activating capacity of the different treatment regimens.

Speaker abstracts

Systematic genetic perturbations to reveal therapeutic melanoma vulnerabilities

Daniel Peeper1

1 Netherlands Cancer Institute, Amsterdam, NETHERLANDS

For a long time, advanced-stage melanomas were refractory to the available therapeutic options, but recent developments have begun offering better perspectives for patients. The small molecule inhibitor vemurafenib, specifically targeting the mutant BRAFV600E kinase, was the first standard of personalized care for patients diagnosed with mutant BRAF metastatic melanoma. Although this compound initially reduces tumor burden dramatically, eventually most melanomas become resistant and progress on treatment. This occurs by the acquisition of additional mutations or other alterations, most of which reactivate the mitogen-activated protein kinase (MAPK) pathway. Although further suppression of BRAF-MAPK signaling by the inclusion of MEK inhibitor delays resistance, eventually most patients relapse.

The clinical outcome of late-stage melanoma patients has also greatly improved thanks to the recent availability of T cell checkpoint modulation, primarily by CTLA-4 and PD-1/PD-L1 blockade. But still, large patient groups fail to (durably) benefit from these treatments, underscoring the continuing need for developing novel therapeutic modalities.

Therefore, in spite of these new perspectives, there is a dire need to identify additional targets amenable to therapeutic intervention, possibly to be used in combination settings with tumor inhibitors alongside immune activators. We are studying (lack of) sensitivity to both tumor and immune cell treatment using patient biopsies, patient-derived xenografts (PDX) and low-passage cell lines. These systems are used for systematic function-based genetic screens to identify melanoma and immune cell factors representing pharmacologically tractable therapeutic targets. The results from these and related studies will be discussed.

Therapeutic Resistance to MAPK and PD-1 Targeting

Roger S. Lo1

1 UCLA, Los Angeles, CA, USAThe MAPK and PD-1 pathways are clinically validated therapeutic targets in advanced melanoma, and understanding innate and acquired resistance to the available agents is critical to further their survival benefits. Genomic analysis of acquired MAPKi-resistance supports deeper MAPK suppression and parallel inhibition of PI3K-AKT as clinical strategies to suppress resistance. Also, genetic alterations that drive MAPK hyper-activation and resistance can result in strong MAPKi addiction, the underlying basis of which may serve as potential drug targets. While genomic analysis fails to uncover recurrent pathway alterations in a substantial portion of melanoma with acquired MAPKi resistance, transcriptomic and CpG methylomic profiling discovered recurrent pathway dysregulations that often co-exist with resistance-causing mutations, suggesting parallel genomic and epigenomic evolution. A significant portion of melanoma also displays loss of T-cell inflammation with the acquisition of MAPKi resistance. Thus, understanding non-genomic and immune micro-environmental adaptations early during MAPKi therapy may provide insights into the origins of functional adaptations that eventually permit disease progression. Non-genomic and tumor micro-environmental adaptations early during MAPKi therapy can be captured as gene signatures that have been linked to innate aPD-1 resistance (collectively termed IPRES signatures). IPRES enrichment occurs with or without IFN signature enrichment, and vice versa. Thus, IFNhigh/IPRESlow, IFNhigh/IPREShigh, IFNlow/IPREShigh, and IFNlow/IPRESlow constitute distinct subtypes across melanoma transcriptomes. Importantly, MAPKi treatment induces temporal transitions among these subtypes, and combinations that suppress MAPKi resistance blunt transitions toward loss of T-cell inflammation. Finally, IPRES enrichment may be variable among melanoma tumors metastatic to distinct organs.

Speaker abstracts

MEET THE EXPERT SESSION

Núria Lopez-Bigas1,2

1 Institute for Research in Biomedicine, Barcelona, SPAIN, 2 University Pompeu Fabra, Barcelona, SPAIN

Nuria Lopez -Bigas is an ICREA Research Professor at the Institute for Research in Biomedicine in Barcelona and associate professor at the University Pompeu Fabra. She leads the Biomedical Genomics lab (bbglab.irbbarcelona.org), focused on the study of cancer from a genomics perspective. She is particularly interested in the identification of cancer driver mutations, genes and pathways across tumor types and in the study of their targeted opportunities.

Research in patients’ biological material as today’s integral part of clinical activity

Giorgio Stanta1

1 University of Trieste, Trieste, ITALY

Clinical research performed in patients’ biological material is today one of the most important sources for development in oncology. The extreme molecular complexity of cancer progression and the high level of heterogeneity is more and more conflicting with the application of large tumour subtypes categories in clinics. Indeed, the need for a higher level of personalization is felt today as a priority for which a higher level of molecular characterization of every cancer is necessary. This is especially related to the fact that there are many more actionable mutations in each single cancer than those we are currently using on the clinical level. In the meantime, the approval of new drugs by the big regulatory organizations is still very slow through the classical trial procedures. The possibility to develop diffused and reproducible clinical research is leading today to the new proposed approach of N-1 trials – every single patient can be a specific trial with specific clinical and molecular characteristics. Also this kind of research is anyway risky because of the low reproducibility of molecular analysis, which is generally complained of. The major obstacles to this reproducibility are related to preanalytical conditions of biological samples, standardized analytical methods, and cellular and molecular heterogeneity. There is an important effort of European organizations to try to overcome this type of obstacles by shared projects such as the new SPIDIA4P project for standardization of preanalytical conditions of biological material, and by the development of standardized analytical procedures such as liquid biopsies and clinical NGS (e.g. in the OECI organization). New approaches to overcome intratumour heterogeneity are developed within the European Society of Pathology, with the tentative drafting of new protocols of sampling and heterogeneity graduation. A new expert centre for molecular analyses in clinical tissues (ATMA-EC) has recently been set up by the European Infrastructure of Biobanking (BBMRI-ERIC) to develop further standardization of molecular methods. All these efforts are preliminary actions to have the possibility to effectively perform reproducible clinical research in patients’ biological material, and to activate large networks of N-1 trials in the future. The fact that this type of research is not for future but for today’s patients, as part of the clinical activity, is changing the perspective with the need for further bioethical considerations and the important involvement of patients’ organizations.

Speaker abstracts

Proffered Paper 7

Quadruple vertical targeting of an oncogenic pathway as a treatment strategy to prevent drug resistance

João Fernandes Neto1, Olaf van Tellingen1, René Bernards1

1 Netherlands Cancer Institute, Amsterdam, NETHERLANDS

Targeted cancer drugs elicit powerful initial responses but far less long-term benefits. The emergence of therapy resistance often involves the reactivation of the same pathway that is being blocked by the cancer drug. Our group and others have shown that inhibiting multiple nodes of the same activated signaling pathway provides longer-lasting therapeutic benefits. This “vertical” inhibition strategy is also used by microRNAs to efficiently “silence” a signaling pathway by partially inhibiting multiple nodes of the same pathway. Using this concept, we show that by combining 4 drugs, which inhibit a pathway at different nodes, using a concentration at which each drug alone does not have any significant effect on cell proliferation, resulted in complete abrogation of cell proliferation and induction of apoptosis in several cell lines of different tumor types. Also, using mouse xenograft models we have shown that this strategy induces a quick and durable tumor regression, without any associated toxicity. Further, using genome wide CRISPR/Cas9 genetic screens, we have shown that by using this concept we are able to prevent the occurrence of resistance.

Defining the actionable genome

David Solit1,2,3,4

1 Memorial Sloan Kettering Cancer Center, New York, NY, USA, 2 Geoffrey Beene Chair, 3 Director, Marie-Josée and Henry R. Kravis Center for Molecular Oncology, 4 Member, Human Oncology and Pathogenesis Program

Tumor molecular profiling is now a standard of care for many cancer types. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly-targeted therapies, demands flexible and comprehensive approaches to profile clinically significant genes across the full spectrum of cancers. This talk will review the MSKCC experience from a large-scale, prospective clinical sequencing initiative utilizing a comprehensive assay, MSK-IMPACT, through which we have compiled matched tumor and normal sequence data from a unique cohort of more than 15,000 patients with advanced cancer. Using these data, we identified clinically relevant alterations and mutational signatures that were shared among common and rare tumor types. Patients were enrolled on matched clinical trials at a rate of ~11%. Pathogenic germline mutations, some of which are predictive biomarkers of drug response, were also identified in a significantly higher proportion of patients than expected and suggest that matched germline sequencing to identify mutant alleles associated with increased heritable risk should be considered in all patients with advanced cancer. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results will be made publicly accessible through the AACR GENIE initiative.

Poster abstracts

Poster abstracts

1

The association of ShcD with ERK in acute oxidative stress in B16 melanoma cells

Samrein Ahmed2, Sara Amer2, Asha Caroline Thomas2, Mira Emad2, Sally Prigent1

1 University of Leicester, Leicester, UK, 2 University of Sharjah, Sharjah, UNITED ARAB EMIRATES

-- Abstract withheld from publication at the request of the author --

2

MITF depletion elevates expression levels of ERBB3 receptor and its cognate ligand NRG1-beta in melanoma

Tine N. Alver2, Timothy J. Lavelle2, Ane S. Longva2, Geir F. Øy2, Eivind Hovig2,1, Sigurd L. Bøe2

1 Department of Informatics, University of Oslo, Oslo, NORWAY, 2 Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, NORWAY

Loss of microphthalmia-associated transcription factor (MITF) has been associated with receptor tyrosine kinase (RTK) up-regulation and increased activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway after vemurafenib treatment in melanoma. Here, we have investigated the influence of SRY-box 10 (SOX10), forkhead box 3 (FOXD3) and MITF in the transcriptional regulation of ERBB3 and its cognate ligand neuregulin 1-beta (NRG1-beta). Our results show that both NRG1-beta and ERBB3 mRNA levels are elevated after MITF loss, by either vemurafenib or small interfering RNA (siRNA) treatment. The elevated expression levels of ERBB3 and NRG1-beta were followed by an increased activation of the PI3K-pathway, resulting in enhanced phosphorylation of AKT (S473). Together, our results suggest that MITF may play a role in the development of acquired drug resistance through NRG1-beta/ERBB3 signaling in melanoma.

3

Chloroquine improves the anticancer effects of doxorubicin in vitro and in vivo models on Mouse Mammary Tumor Ehrlich ascites

carcinoma (EAC)

Seyma Aydinlik1, Elif I. Ikitimur-Armutak2, Ayça Uvez2, Ferda Ari1, Kıvılcım Sonmez3, Osman. B. B. Esener2, F. Ustun-Alkan4, A. Aktas2, S. Umit Zeybek5, Engin Ulukaya6, Konstantinos Dimas7, Ebru Gurel-Gurevin8 1Department of Molecular Biology, Faculty of Science, Uludag University, Bursa-Turkey 2Department of Histology and Embryology, Faculty of Veterinary Medicine, Istanbul University, Istanbul-Turkey 3Department of Pathology, Faculty of Veterinary Medicine, Istanbul University, Istanbul-Turkey 4Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Istanbul University, Istanbul-Turkey 5Department of Molecular Medicine, The Institute of Experimental Medicine, Istanbul University, Istanbul-Turkey 6Department of Clinical Biochemistry, School of Medicine, Uludag University, Bursa-Turkey 7Department of Pharmacology, School of Medicine, Thessaly University, Larissa, Greece 8Department of Biology, Faculty of Science, Istanbul University, Istanbul-Turkey

Cancer still remains the most horrifying disease due to treatment failure, cause development of resistance to chemotherapeutics. Autophagy is regarded as one of the key mechanism for drug resistance and chloroquine (CQ) is an inhibitor of autophagy. Therefore, the combination of Doxorubicin (DXR) and CQ may be a realistic strategy for a new treatment modality on Ehrlich Ascites Carcinoma (EAC) cells in vivo and in vitro. EAC cells were treated with DXR (1µM) alone or in combination with CQ (8 µM). ATP assay were perfomed to determine cytotoxicity after 48h treatment. apoptosis and autophagy related proteins were also determined by Western blot analysis. Changes in survival pathway was detect by Luminex xMAP. For in vivo evaluation, 88 Balb-c mice with EAC were divided into control (n=8) and 8 experimental groups (n=10). DXR and CQ have been investigated following intraperitoneal administration of doses of 1.5 ve 3 mg/kg X 3 and 25 ve 50 mg/kg X 3 respectively. Tumor volume determined in vivo by caliper and relationship between apoptosis, proliferation and autophagy was evaluated by immunohistochemistry. According to ATP viability assay, combination of DXR and CQ caused a significant decrease in cell viability compared doxorubicin alone. Also 24h treatment of CQ and DXR combination showed increased autophagy protein levels. Cleavage of PARP1 and caspase 3, besides increase in

Poster abstracts

expression of PTEN and FAS protein indicating the enhanced apoptosis. Moreover proteins related to survival pathway were found to decrease by using Luminex xMAP. Similar to in vitro results, in vivo results showed the combination of CQ and DXR reduced tumor volume and increased apoptosis by suppressing the autophagy and proliferation. As a result combination of DXR and CQ enhances apoptosis, possibly via the inhibition of autophagy and this might be a promising therapeutic strategy in EAC cells.

This study is supported by TUBITAK (The Scientific and Technological Research Council of Turkey) with a project number of 214S609.

Ethics Committee Decision Number: 2013/65, Date: 30/05/2013.

4

Targeting drug tolerance to prevent tumour evolution and acquired resistance in FGFR2-addicted gastric cancer

Irina Babina1, Matthew Beaney1, Ros Cutts1, Eleanor Knight1, Jian Ning1, Alex Pearson1, Amanda Swain1, Nicholas Turner1

1 Institute of Cancer Research, London, UK

-- Abstract withheld from publication at the request of the author --

5

Modulation of the EMT status of tumor cells in the regulation of their sensitivity to anticancer drugs

Natalia Bezdieniezhnykh1, Ruslana Kocherga1, Yurii Kudryavets1, Oleksandra Lykhova1, Victor Zhylchuk2

1 RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, Kyiv, UKRAINE, 2 Rivne Regional Oncology Center, Rivne, UKRAINE

Heterogeneity of metastatic cancer cell population is one of the reasons why some patients are more responsive to some treatments over others; the epithelial to mesenchymal transition (EMT) of tumor cells as a key regulator of metastatic process also provides signals for increasing survival which may cause drug resistance in tumor cells. Materials and methods: cell lines from ascitic fluids of patients with cancer, methods of tissue culture, immunocytochemistry, ELISA, statistical methods.

Results: investigated the sensitivity of cells from ascitic fluids of patients with breast (n=10) and ovarian cancer (n=14) to evaluate the

response to anticancer agents, depending on the cellular composition of populations, dominated mesenchymal or epithelial cells: found increased sensitivity of cells with mesenchymal phenotype dominance to fluorouracil, whereas epithelial cells were much more sensitive to gemzar, but to DNA damaging agents (doxorubicin, cisplatin) is not proved the difference. The answer to vinca alkaloid and taxanes was different and not been associated with a particular phenotype, stress the importance of an individual approach to each patient.

Obtained permanent cell lines from ascitic fluids and was shown that cells of primary cultures characterized by the dominance expression of mesenchymal markers Vimentin and N-cadherin. After their growth in vitro was observed inhibition of EMT associated proteins and transcriptional factors simultaneously with change of cell sensitivity to anticancer drugs.

Modification of the extracellular matrix using growth on collagen or in spheroids, using the system of co-cultivation in the presence of cellular or soluble factors of microenvironment (IFN, TNF, IL-6) also significantly influenced on EMT markers expression and cells sensitivity to drugs.

Conclusion: was observed lability of phenotypic characteristics of tumor cells from ascitic fluids such as morphology, expression of EMT associated markers by modulation of specific microenvironment, that correlated with changes of their sensitivity to anticancer drugs.

6

EGFR mutation frequency in squamous cell carcinoma of the lung (SCC) in the Bulgarian population

Stoyan Bichev1, Nataliya Chilingirova2, Alexey Savov1

1 National Genetics Laboratory, Sofia, BULGARIA, 2 Specialized Hospital for Active Treatment in Oncology, Sofia, BULGARIA

Background: Molecular testing has become an important part of clinical practice since targeted therapies are routinely used in oncology, in particular in the treatment of non-small cell lung cancer (NSCLC). By squamous cell carcinoma (SCC) of the lung molecular testing is not routinely performed, since driver mutations are uncommon in this subset, but is still the main histotype in Bulgaria. The aim of this analysis is to identify the EGFR mutation frequency in SCC in order to highlight the need of mutational testing in this NSCLC subtype.

Methods: For the time period 2010-2015 our prospective study analysed 2357 samples from

Poster abstracts

patients with NSCLC, stage IIIB-IV, 926 of them with histology for SCC. DNA was extracted from paraffin-embedded (FFPE) tumor tissue using QIAamp DNA FFPE Tissue Kit® (Qiagen). The quantity and quality of extracted DNA was assessed by NanoDrop 1000 spectrophotometer (Thermo Scientific). Real-time PCR amplification by the Scorpions Amplification Refractory Mutation System (SARMS) method was used to detect mutations in the EGFR gene exons 18-21.

Results: In total, by 51 of all 926 patients (74% former or current smokers, 2/3 male, 1/3 female with median age by diagnosis 64 yo, 59% stage IV) with SCC (5,5%) mutation in EGFR was detected. Exon 21 L858R was found by 25 pts of them (49%), del19 by 17 (33,3%), T790M by 3 of them (5,9%), S768I ex21 by 3 pts (5,9%), G719X, L861Q and ins ex20 by 1 pts each (2%). All patients with activating mutations were treated according to the guidelines with first line TKI.

Conclusion: We detected activating mutations in 38 pts with SCC, which according to protocols should not be routinely tested and proved efficacy of first line TKI treatment. Our results demonstrated the need of molecular testing in certain cases with SCC NSCLC.

7

Targeting the STAT6 pathway to inhibit tumor-associated macrophages-induced tumor growth and metastasis in breast cancer

Karin Binnemars-Postma1, Ruchi Bansal1, Gert Storm1, Jai Prakash1

1 University of Twente, Enschede, NETHERLANDS

Tumor-associated macrophages (TAMs), one of the most crucial cell types in the tumor microenvironment, contribute to tumor growth and metastasis. These pro-tumoral macrophages show an M2-like phenotype whereas their counterpart M1 macrophages have been shown to inhibit tumor growth and survival. The Signal Transducer and Activator of Transcription 6 (STAT6) pathway is the downstream signaling pathway of IL-4 and IL-13, cytokines responsible for M2 differentiation. We hypothesize that inhibition of the STAT6 pathway might be an interesting strategy to inhibit TAM (M2-like macrophage) differentiation and thereby their pro-tumorigenic activities. Mouse RAW264.7 cells were differentiated into M2 type using IL-4 and IL-13 and confirmed with induction of M2-specific genes (arginase-1 and mannose receptor-1) and arginase enzyme activity. Western blot analyses showed an upregulation of pSTAT6

in M2-differentiated cells compared to the control cells. Interestingly, treatment with AS dramatically inhibited the STAT6 phosphorylation in M2 cells with increasing concentrations, as shown with Western blot analyses of pSTAT6/STAT6 and arginase activity assays. In contrast, AS did not show any inhibitory effects on M1 phenotype of RAW cells. Furthermore, we found that M2-conditioned medium strongly induced the migration of 4T1 tumor cells in vitro. Importantly, conditioned medium collected from AS-treated M2 cells did not induce these paracrine effects. In vivo, treatment with AS (20 mg/kg, i.p.) significantly attenuated the tumor growth by about >30%. Luciferase activities in different organs showed a reduction in metastasis in the AS group. This study proposes that inhibition of STAT6 pathway in TAMs is a vital therapeutic approach to reduce tumor growth and metastasis by inhibiting M2-induced pro-tumorigenic and pro-metastatic activities.

8

Gastric cancer stem cells: do they maintain the genetic alterations of driver genes identified in the primary tumor?

Tania Capeloa2,1, Claudia Giachino2, Simona Corso1, Silvia Giordano1

1 Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Turin, ITALY, 2 Department of Clinical and Biological Sciences, University of Turin, Turin, ITALY

Introduction: Gastric cancer is one of the major leading causes of cancer related deaths worldwide. The overall clinical outcome for patients with advanced gastric cancer is poor, due to the limited number of therapeutic agents currently available. Trastuzumab is the only targeted therapy recently added to the cytotoxic(s) treatments, which has anyway given unsatisfactory results. Gastric cancer stem cells (GCSCs) have been studied in recent years due to their importance for understanding tumor initiation and sensitivity to antitumor therapies. The isolation and characterization of cancer stem cells requires critical evaluation steps in order to define a cell population as “stem like”.

Aim: In the present study we aimed at isolating cancer stem cells from gastric patient-derived xenografts (xenospheres) to explore if these cells share the genetic alterations identified in the patient tumors and if they respond to targeted therapies as the original tumor.

Results: We found that two out of three xenospheres maintained the molecular alterations

Poster abstracts

identified in the primary tumor, such as MET and KRAS gene amplification. Assays such as spheroid formation, expression of specific markers, clonal self-renewal and in vivo initiating tumor ability were performed to define them as GCSCs. Viability assay showed that MET amplified xenospheres maintained the ability to respond to MET inhibitors.

Conclusion: Our data shows that GC stem like cells isolated from PDXs bear the same molecular alterations in driver genes as the original tumors a respond to therapies aimed at inhibiting driver genes. This observation is very important as it suggests that GCSCs, isolated from PDX, can be inhibited by therapies directed against driver genes identified in the whole tumor.

9

In vivo evaluation of the MDM2-p53 antagonist RO6839921 alone and in combination with temozolomide in TP53 wild-type neuroblastoma

Lindi Chen2, Fabio Pastorino1, Philip Berry2, Jennifer Bonner2, Katrina Wood2, Gareth Veal2, Mirco Ponzoni1, John Lunec2, David Newell2, Deborah Tweddle2

1 IRCCS Istituto Giannina Gaslini, Genova , ITALY, 2 Newcastle University, Newcastle upon Tyne, UK

Background: Neuroblastoma is a predominantly TP53 wt tumour which supports the use of MDM2-p53 antagonists as a novel therapy for neuroblastoma patients. This is the first study to evaluate the efficacy of RO6839921, an IV prodrug of RG7388, alone and in combination with temozolomide in TP53 wild-type orthotopic models of neuroblastoma.

Methods: Studies were conducted using a well-established orthotopic model of neuroblastoma, implanted with SHSY5Y-Luc (TP53 wt; non-MYCN amplified) and NB1691-Luc (TP53 wt; MYCN and MDM2 amplified) cells, and randomized into control, intravenous RO6839921, oral temozolomide or RO6839921 and temozolomide in combination. Tumour growth was monitored using bioluminescence imaging. Active RG7388 in plasma and tumour samples were detected using liquid chromatography–mass spectrometry, and p53 pathway activation assessed by MIC-1 ELISA assays, Western analysis and/or immunohistochemistry for p53, p21 and cleaved caspase 3.

Results: Pharmacokinetic and pharmacodynamic analysis of RO6839921 observed peak plasma levels of active RG7388 at 1h posttreatment

with maximal activation of the p53 pathway observed 3-6h posttreatment. RO6839921 had a favourable pharmacodynamic profile consistent with intermittent dosing. Assessment of anti-tumour efficacy demonstrated that RO6839921 was as effective as temozolomide and that RO6839921 in combination with temozolomide led to significantly greater tumor growth inhibition and survival than either agent given alone. RO6839921 alone and in combination with temozolomide was well tolerated.

Conclusions: These preclinical studies support the evaluation of combining RO6839921 with temozolomide in early phase clinical trials of neuroblastoma patients with wt TP53.

10

FOXM1 confers to epithelial-mesenchymal transition, stemness and chemoresistance in epithelial ovarian carcinoma cells

Wen-Tai Chiu1, Yu-Fang Huang1, Huei-Yu Tsai1, Cheng-Yang Chou1

1 National Cheng Kung University, Tainan, TAIWAN ROC

Chemoresistance to anti-cancer drugs substantially reduces survival in epithelial ovarian cancer. In this study, we showed that chemoresistance to cisplatin and paclitaxel induced the epithelial-mesenchymal transition (EMT) and a stem cell phenotype in ovarian cancer cells. Chemoresistance was associated with the downregulation of epithelial markers and the upregulation of mesenchymal markers, EMT-related transcription factors, and cancer stem cell markers, which enhanced invasion and sphere formation ability. Overexpression of FOXM1 increased cisplatin-resistance and sphere formation in cisplatin-sensitive and low FOXM1-expressing ovarian cancer cells. Conversely, depletion of FOXM1 via RNA interference reduced cisplatin resistance and sphere formation in cisplatin-resistant and high FOXM1-expressing cells. Overexpression of FOXM1 also increased the expression, nuclear accumulation, and activity of b-CATENIN in chemoresistant cells, whereas downregulation of FOXM1 suppressed these events. The combination of cisplatin and the FOXM1 inhibitor thiostrepton inhibited the expression of stem cell markers in chemoresistant cells and subcutaneous ovarian tumor growth in mouse xenografts. In an analysis of 106 ovarian cancer patients, high FOXM1 levels in tumors were associated with cancer progression and short progression-free intervals. Collectively, our findings highlight the importance of FOXM1 in chemoresistance and suggest that FOXM1

Poster abstracts

inhibitors may be useful for treatment of ovarian cancer.

11

Influences of capsaicin on normal, immortalized and cancerous cells of human mesothelium

Sabahattin Comertpay1, Fatma Gul Demirbanka1, Nagihan Helvacı1, İsmail Akyol11 Kahramanmaras Sutcu Imam University, Kahramanmaras, TURKEY

Malignant pleural mesothelioma (MPM), a type of cancer developed from the lining tissues of lung upon a prolonged exposure to asbestos and/or erionite, is a rare but a highly fatal disease with no effective treatment. In erionite-high villages of Central Anatolia, namely Cappadocia, the disease, along with another type of mesothelioma developed in the membrane of stomach-peritoneal mesothelioma, have been discovered to be responsible of 50% of the death. Capsaicin, the pungent compound produced by various species of Capsicum genus, has been shown to eliminate cancer cells through apoptosis in vitro and in vivo. In the present study, we treated commercially obtained cancerous (ATCC-5946), immortalized (Coriell Institute-AG07086) and normal cells (ZenBio-MESM012916B) of pleura with gradually increasing concentrations of synthetic capsaicin solved in ethanol, and IC50 values of the agent in 12 hours were determined via MTS Assay. As a result, we found that capsaicin in ethanol was more effective at inducing cell-death in cancerous cells (IC50: 300 µM) compared to in immortalized cells (IC50: 334 µM) and normal cells (IC50: 360 µM). Additionally, when similar experiments in 48 hours were repeated with capsaicin solved in DMSO, IC50 values were calculated as remarkably higher for the cancerous and immortalized cell lines (417 and 575 µM, respectively), indicating an increase in the amount of capsaicin required, which, overall, suggested that ethanol might be a better solvent for the therapeutic purpose of capsaicin. In conclusion, our preliminary data imply that capsaicin solved in ethanol may have the potential to be used as a drug in elimination of MPM, which might not harm normal cells in appropriate concentrations. Nevertheless, our research is still ongoing to reveal the mechanism through which the cells are eliminated by this compound.

13

Liquid biopsy platform based on single-cell

measurement of extracellular acidification rate

Eva Biscontin1, Giulia. Brisotto1, Michela Bulfoni2, Daniela Cesselli2, Alfonso. Colombatti1, Fabio Del Ben1, Wilhelm T.S. Huck3, Aigars Piruska3, Giacinto Scoles2, Agostino Steffan1, Matteo Turetta2

1 Centro di Riferimento Oncologico, Aviano, ITALY, 2 Institute of Anatomic Pathology, Udine, ITALY, 3 Radboud University, Nijmegen, NETHERLANDS

Background: The number of circulating tumor cells (CTC) in blood is correlated with the progress of metastatic cancer, and is emerging as a minimally-invasive “liquid biopsy” diagnostic tool for therapy monitoring, recurrence detection, prediction of therapy response.

In our previous work (Del Ben, Turetta et al., Angew. Chem. Int. Ed. Engl. 2016) we demonstrated a label-free technique for CTC detection based on their altered metabolism, by measuring the secretion of H+ of individual, viable tumor cells compartmentalized in microfluidically prepared picoliter-droplets. Here we present additional clinical data.

Methods: Clinical samples (2mL of whole blood collected in EDTA-tube) were lysed and CD45-depleted using Milteniy LD columns and beads. Droplets containing single cells were generated by microfluidic water-in-oil emulsification. Cells are suspended in culture medium, together with a ratiometric pH-sensitive dye (SNARF-5F). Droplets are incubated at 37C for 30 min and reinjected for fluorescent pH measurement. A triggered camera collects pictures of selected drops.

Results: According to the selected cut-off (pH=6.4) we detected significantly more events in metastatic breast (median 41/mL, range [5-3125], n=6) and lung cancer (49/mL, [28-87], n=4) vs healthy donors (3/mL, [1-9], n=7), p<0.005.

Conclusion: We observed a significant difference between patients and healthy donors, with number of events compatible with published reports on CTC. To further validate the technology, we set up a comparison study against Veridex CellSearch® and a biomarker validation trial, which are currently ongoing.

Disclosure of interest: F. Del Ben, M. Turetta, A. Piruska, W.T.S.Huck and G. Scoles filed a patent covering the described methodology and co-founded the company Cytofind Diagnostics BV to further develop the technology, aiming at improving outcomes, improving quality of life and reducing cost in oncology care.

Poster abstracts

14

Optimizing Personalized Healthcare in Gastric Cancer specimen

Archil Aladashvili3, Roland Croner1, Bakho Dvali2, Aleksandre Tsalugelashvili2, Tornike Aladashvili3, Temur Samushia2, Irine Khubua2

1 FAU-Friedrich-Alexander University, Erlangen-Nurenberg, GERMANY, 2 National Cancer Center, Tbilsi, GEORGIA, 3 Tbilisi State Medical University, Tbilsi, GEORGIA

Introduction: In our study, in order to better evaluate intratumoral heterogeneity, we employed surgical biopsy as our tumor sampling strategy, we performed an innovative pathological assessment through scoring of individual biopsies against whole biopsies from single patients.

Materials and Methods: 83 patients with multiple biopsies from different tumor areas. In Lab of TSMU were performed Immunohistochemistry (IHC) Fluorescence in situ hybridization (FISH). biomarker status including IHC and FISH staining of MET, ATM, FGFR2 and HER2 were evaluated on each biopsy. Individual scores for each biomarker were given to each biopsy. For MET IHC, MET FISH, FGFR2 FISH and HER2,

Results: The positive biopsy numbers were slightly less than the total biopsy numbers. In the 9 MET IHC positive cases, 63% of the cases showed low heterogeneity, while 34% showed medium and 3% showed high heterogeneity, significant correlation was found between MET IHC score and MET FISH results (p<0.01,κ=0.58, Fisher’s exact test) In the 29 cases with at least one ATM negative biopsy. In the 5 FGFR2 FISH positive cases, 59% of the cases showed low heterogeneity, while 32% showed medium and 9% showed high heterogeneity. In the 16 HER2 positive cases, 76% of the cases showed low heterogeneity, while 24% showed medium heterogeneity and none of the cases showed high heterogeneity.

Conclusion: Our results show a relatively low level of heterogeneity across the biomarkers analyzed in this cohort. Nevertheless, the degree of heterogeneity within other patient cohorts may be different and should be analyzed on a case-by-case basis. Furthermore, our results showed a decrease in the rate of false negative detection corresponding with an increase in the biopsy number for all biomarkers tested herein, demonstrating the benefit of multiple biopsy sampling and serving as an example of addressing intratumoral heterogeneity using statistical methods.

15

miR-34a impacts expression of key metabolic enzymes in triple-negative/Basal-like breast cancer with clinical consequences

Marie E. Engkvist1, Leonardo A. Meza-Zepeda5,4, Vessela N. Kristensen1,2, Ola Myklebost5,3, Thomas Fleischer1

1 Department of Cancer Genetics, Institute for Cancer Research, OUS Radiumhospitalet, Oslo, NORWAY, 2 Department of Clinical Molecular Biology and Laboratory Science (EpiGen), Akershus University hospital, Division of Medicine,, Lørenskog, NORWAY, 3 Department of Clinical Science, University of Bergen, Norway., Bergen, NORWAY, 4 Department of Core Facilities, Institute for Cancer Research, Oslo University Hospital, Oslo, NORWAY, 5 Department of Tumour Biology, Institute for Cancer Research, OUS Radiumhospitalet, Oslo, NORWAY

Breast cancer is the most frequent malignant disease among women in the western world, and there is a strong need to identify novel molecular targets and to develop new therapeutic agents for more personalized treatment, especially for patients with triple negative breast cancer. The miR-34a is a highly conserved microRNA found to be frequently down-regulated in breast cancer. The tumor suppressive role of miR-34a in a variety of cancers, partly as an effector molecule in p53-mediated processes, has been widely documented and promoted miR-34a as a candidate for miRNA replacement therapy.

In this study we identified a novel role of miR-34a as a regulator of the serine-glycine biosynthesis pathway, most likely through the integrated stress response axis. Our results demonstrate that re-introduction of miR-34a results in a coordinated repression of all three genes encoding the serine-glycine biosynthesis pathway (PHGDH, PSAT-1 and PSPH) and their upstream regulator ATF4 in a triple negative breast cancer cell line (MDA-MB-231). Furthermore, data from patients with basal-like tumors in the Metabric breast cancer cohort showed that the expression of these genes were negatively correlated with the expression of miR-34a, and that a higher expression of miR-34a was associated with a favorable prognosis in these patients.

In conclusion, miR-34a may regulate an important metabolic pathway in basal-like tumors, and our study supports the therapeutic potential of miR-34 in for these highly aggressive tumors.

Poster abstracts

16

Disparate responses by CD8+ T and NK cells to stimulation in the presence and absence of normal and melanoma-associated fibroblasts

Barbara Érsek4,3, Pálma Silló1, András Bencsik4, Krisztián Németh1, Zoltán Pós4,2

1 Department of Dermatology, Venereology and Dermatooncology, Semmelweis University, Budapest, HUNGARY, 2 Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, HUNGARY, 3 Office for Research Groups Attached to Universities and Other Institutions of the Hungarian Academy of Sciences, Budapest, HUNGARY, 4 SE GSI Experimental and Translational Immunomics Research Group, Budapest, HUNGARY

Cancer associated fibroblasts (CAFs) represent a highly heterogeneous population of fibroblast-like cells residing in the tumor stroma, the exact origin of which is a matter of ongoing debate. In many aspects, CAFs recapitulate key features of both mesenchymal stem cells (MSCs) having well-known immunosuppressive properties, and activated fibroblasts actively participating in matrix reorganization, a prerequisite of tumor invasion. Compared to normal fibroblasts, CAFs display high proliferation rate, show increased motility, release a skewed spectrum of extracellular matrix components and growth factors, and actively secrete both inflammatory and immunosuppressive cytokines, such as TGFβ, CXCL12, VEGF, and IL-6, into their environment.

The aim of the present study was to analyze the impact of melanoma-associated CAF (MAF)-released soluble mediators on NK and CD8+ T cells, and analyze the question whether they may support the rise of CD4+ Tregs.

MAFs and normal fibroblasts (NFs) were isolated from enzymatically disintegrated melanomas and skin biopsies of healthy donors, respectively. MAFs were identified as Melan-A-/gp100- and α-SMA+ and/or FAP+ cells by flow cytometry. Next, untouched CD8+ cytotoxic T cells and NK cells were activated by anti-CD3/CD28 or anti-CD2/NKp46, respectively, and exposed to conditioned media (CM) of NFs, MAFs, or unconditioned basal media. The response of activated CD4+/Foxp3+ Treg cells was determined in similar experiments within whole PBMC culture.

Preliminary data suggest that in the presence of MAF supernatant proliferation of CD8+ T cells is inhibited, while the opposite is true for Tregs.

It also seems that cytotoxic degranulation is more prominent in NF-CM-treated CD8+ T cells than in MAF-CM–treated cells. Moreover, both granzyme B and IFNg production of CD8+ T cells is differentially affected by MAF-CM as compared to NF-CM. Taken together, these data indicate that MAF cells are involved in the regulation of the antitumor response within the tumor microenvironment.

17

Scheduling JNK-JUN inhibition to overcome inherent and early adaptive resistance to RAF-MEK-ERK targeted therapeutics in metastatic melanoma

Petranel Ferrao3,1

1 La Trobe University, VIC, AUSTRALIA, 2 Olivia Newton-John Cancer Research Institute, VIC, AUSTRALIA, 3 The University of Melbourne, VIC, AUSTRALIA, 4 The University of South Australia, SA, AUSTRALIA, 5 The Walter and Eliza Hall Institute of Medical Research, VIC, AUSTRALIA

-- Abstract withheld from publication at the request of the author --

18

Cancer Astrocytes Have A More Conserved Molecular Status In Long Survival Glioblastoma Patients: New Emerging Cancer Players

Sara Franceschi1, Francesca Lessi1, Paolo Aretini1, Valerio Ortenzi2, Marco La Ferla1, Cristian Scatena2, Francesco G Carbone2, Riccardo Vannozzi2, Prospero Civita1, Francesco Pasqualetti2, Antonio G Naccarato2, Chiara M Mazzanti11 FPS - Pisa Science Foundation, Pisa, ITALY, 2 University Hospital of Pisa, Pisa, ITALY

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor with a median patient survival of 14.6 months. Despite decades of research and the advent of new therapies, GBM etiology and pathogenesis is still unclear and patients with GBM continue to have a very poor prognosis, almost always related to intracranial progression after surgery or radio-chemotherapy. However, 3–5% of the patients survives for more than 2 years and are referred to as long-term survivors.

The aims of this work is to identify a genetic landscape that might be associated with long

Poster abstracts

term survival and to understand the reasons why histologically identical tumors might behave less aggressively than others.

We selected 13 human GBM cases. Subjects were chosen to have same histology, similar condition and treatment. All cases had a diagnosis of GBM IV with no previous history of any brain neoplasia. Having access to samples of GBM with exceptionally long survival (>25 months), we decided to compare the mutational and gene expression profile of this group of tumors, with a group of GBM with only a few months survival (<6 months) by using NGS technology.

Gene mutational status, significantly changing between the two extreme groups of patients, revealed that the genetic constitution of less aggressive GBM is clearly more stable. Transcriptional data revealed that the functional state of cancer astrocytes in patients with long survival seems to remain closer to what is the normal astrocyte cell functionality. The more deregulated pathways are those that involve the metabolism of glutamate, an important neurotransmitter in the central nervous system. Finally combining Copy Number Variation analysis with transcriptome data we found new emerging cancer players, which confirmed at the RNA and DNA level, made them possible oncodrivers.

19

Modulation of claudin-1 expression by ∆2-TGZ in triple negative breast cancer cells is correlated to apoptosis

Marine Geoffroy3,1, Alexandra Kleinclauss3,1, Michel Boisbrun4,2, Stéphane Flament3,1, Sandra Kuntz3,1, Isabelle Grillier-Vuissoz3,1

1 CNRS, CRAN, UMR 7039, Vandœuvre-lès-Nancy, FRANCE, 2 CNRS, SRSMC, UMR 7565, Vandœuvre-lès-Nancy, FRANCE, 3 Université de Lorraine, CRAN, UMR 7039, Vandœuvre-lès-Nancy, FRANCE, 4 Université de Lorraine, SRSMC, UMR 7565, Vandœuvre-lès-Nancy, FRANCE

Breast cancer represents the second lethal malignancy across the world. Efficient targeted therapies can be used in presence of the molecular biomarkers ER, PR and HER2. Nevertheless, these therapies are ineffective on triple negative breast cancer (ER-, PR- and HER2-). Furthermore, 40% of triple negative tumors do not express claudin-1, a major constituent of tight junction. These tumors present a higher relapse incidence. A major challenge in oncology is the development of innovative therapies for these poor prognosis tumors. In this context, we study the anticancer

effects of thiazolidinediones (TZD). We develop ∆2-TGZ a compound derived from troglitazone (TGZ), which presents an improved anti-proliferative activity and a lower hepatic toxicity than the parent molecule. We showed by DNA microarray that claudin-1 is the most expressed gene in response to ∆2-TGZ treatment in MCF-7 breast cancer cells. The aim of our work is to determine if claudin-1 contributes to the pro-apoptotic effect of ∆2-TGZ. In the present study, we demonstrated that ∆2-TGZ induces apoptosis and claudin-1 protein expression in MDA-MB-231 and Hs578T “claudin 1-low” triple negative cells, but not in the non-tumorigenic human breast epithelial cell line MCF-10A. Furthermore, we showed that claudin-1 overexpression plays an important role in apoptosis in MDA-MB-231 and Hs578T cells. Concerning ∆2-TGZ-induced apoptosis, we showed that claudin-1 is involved in MDA-MB-231 cells but not in Hs578T cells suggesting a different mechanism of action. In conclusion, these data consolidate our hypothesis that claudin-1 is involved in the pro-apoptotic process in MDA-MB-231 and Hs578T cells. In fine, our study suggests that the strategy of claudin-1 re-expression could be a promising therapeutic option for the aggressive “claudin 1-low” triple negative subtype.

20

ERα36 expression: a key breast cancer risk factor?

Charlène Thiébaut1, Clémence Chamard-Jovenin1, Amand Chesnel1, Chloé Morel2, Isabelle Grillier-Vuissoz1, Alexandre Harlé1,2, Taha Boukhobza1, Jean-Louis Merlin1,2

1 CNRS-Université de Lorraine, UMR 7039, Centre de Recherches en Automatique de Nancy , Vandoeuvre lès Nancy, FRANCE, 2 Institut de Cancérologie de Lorraine, Service de Biopathologie, Nancy, FRANCE

Estrogen receptor alpha 36 (ERα36) is a variant of the canonical estrogen receptor alpha (ERα66), whose high expression level correlates with a poor survival prognosis for breast cancer patients. While our previous results indicated that a high ERα36 expression level correlates with hormone independent growth and enhanced metastatic potential (Chamard-Jovenin et al, 2015; doi: 10.1186/s12918-015-0178-7) the mechanisms of ERα36 expression onset and the consequences for the normal mammary gland are poorly documented. Therefore, we explored (i) the methylation status of ERα36 promoter in 60 mammary tumor samples and (ii) the consequences of a ERα36 overexpression

Poster abstracts

in vitro in MCF-10A normal mammary epithelial cells and in vivo in a unique model of MMTV-ERα36 transgenic mouse strain wherein the human ERα36 mRNA was specifically expressed in the mammary gland. Our data indicate a marked correlation between promoter methylation and ERα36 expression. Concomitantly, ERα36 overexpression lowered proliferation rate but enhanced migration and survival of the MCF-10A cell line. By a combination of bioinformatics and computational analyses of microarray data, we identified hierarchical gene networks, downstream of ERα36. In vivo, human ERα36 expression led to duct epithelium thinning and E-cadherin expression loss in adult but not prepubescent mice.

Here, we show that ERα36 expression could be driven, at least in part, by epigenetic mechanisms. Moreover, an enhanced expression of ERα36 is sufficient, by itself, to disrupt normal breast epithelial phenotype in vivo and in vitro through a dominant-positive effect on nongenomic estrogen signaling pathways. These results also suggest that, in the presence of endogenous estradiol, ERα36 overexpression in vivo contributes to alter mammary gland architecture which may support pre-neoplastic lesion and augment breast cancer risk.

This work was supported by ANSES (2012-2-014), INSERM (ENV201304), la Ligue contre le Cancer and CCIR-GE.

21

Human germ/stem cell-specific gene TEX19 influences cancer cell proliferation and cancer prognosis

Vicente Planells-Palop4, Ali Hazazi4, Julia Feichtinger1,5, Gerhard Thallinger1,5, Mikhlid Almutairi4,2, Lee Parry3, Jane A. Wakeman4, Ramsay J. McFarlane4

1 Computational Biotechnology and Bioinformatics Group, Institute of Molecular Biotechnology, Graz University of Technology, Graz, AUSTRIA, 2 Department of Zoology, King Saud University, Al-Ryiadh, SAUDI ARABIA, 3 European Cancer Stem Cell Research Institute, Cardiff University, Cardiff, UK, 4 North West Cancer Research Institute, School of Medical Sciences, Bangor University, North Wales, UK, 5 Omics Center Graz, BioTechMed Graz, Graz, AUSTRIA

Tumours regularly reactivate cancer germ line genes whose expression is silenced in normal healthy somatic tissue and restricted to the tissues of the testis. These genes are found to

be expressed in a wide range of tumour cells and cancer stem-like cells, so their intrinsic characteristics mark them as excellent potential cancer-specific biomarkers and a promising therapeutic target. Human TEX19 is a germ line gene that has a yet to have a functional role in oncogenesis defined. Here we demonstrate that depletion of TEX19 levels in a range of cancer cell lines in vitro and in vivo restricts cellular proliferation/self-renewal/reduces tumour volume, indicating TEX19 is required for cancer cell proliferative/self-renewal potential. We extend this to show that TEX19 has a dynamic cellular localization pattern and that it differentially regulates expression of endogenous retroviral genes and protein coding genes via different pathways in distinct cancer cell lines. TEX19 is required to drive cell proliferation in a range of cancer cell types, possibly mediated via an oncogenic transcript regulation mechanism. We also show that TEX19 expression is linked to a poor prognosis for some cancers and collectively these findings indicate that not only can TEX19 expression serve as a novel cancer biomarker, but may also offer a cancer-specific therapeutic target with broad spectrum potential.

22

A phase II organoid-based co-clinical trial identifies mechanisms of primary resistance to Regorafenib (REG) in treatment-refractory metastatic colorectal cancer (mCRC) patients

George Vlachogiannis1, Somaieh Hedayat1, Yann Jamin2, Khurum Khan1, Javier Fernández-Mateos1, Jens C Hahne1, Mihaela Rata2, Dow-Mu Koh2,1, Nina Tunariu1,1, David Collins2, Monia Bali1,2, Ian Chau1, Sheela Rao1, David Watkins1, Naureen Starling1, Nicos Fotiadis1, Ian Huntingford1, Ruwaida Begum1, Rana Isma1, Eleanor Temple1, Massimo Rugge3, Chiara Braconi4, Matteo Fassan3, Suzanne Eccles4, Simon Robinson2, David Cunningham1, Nicola Valeri11 Biomedical Research Centre at the Royal Marsden and The Institute for Cancer Research, London, UK, 2 Cancer Research UK Cancer Imaging Centre, Division of Radiotherapy and Imaging, The Institute of Cancer Research and Royal Marsden Hospital, London, UK, 3 Department of Medicine, University of Padua, Padua, ITALY, 4 Division of Cancer Therapeutics, The Institute of Cancer Research, London, UK

Background: REG demonstrated efficacy in pre-treated mCRC patients. Limited clinical benefit in unselected patient populations highlights the unmet need for better patient selection and identification

Poster abstracts

of mechanisms of action.

Methods: RAS mutant mCRC patients with biopsiable metastases were enrolled in a phase II trial (NCT03010722). Biopsies (6-12 cores) were obtained at baseline (BL), after 2 months if stable disease and at disease progression from multiple sites and used for patient-derived organoids (PDOs) and genomic/transcriptomic analyses. Dynamic contrast enhanced (DCE)-MRI was acquired in patients pre- and at day 15 post-treatment. PDOs were implanted orthotopically in the livers of NSG mice and treated with REG. Changes in tumour and fractional blood volume (fBV) in vivo were monitored by susceptibility-contrast MRI.

Results: PDOs retained genomic and transcriptomic features of matching biopsies. PDOs from BL biopsies of responder and non-responder patients (defined as RECIST 1.1 and DCE-MRI response at day 15) were successfully implanted in 97% of mice. PDOs metastasized within the liver and analysis of vascular density showed significant drop in CD-31 after REG treatment in mice from responder patients (p=0.03, n=6) and no significant changes in those from non-responders (n=10). Morphological analysis revealed a desmoplastic pattern of growth for PDOs established from responders while a pushing/replacement phenotype was observed in those from non-responder patients. Susceptibility-contrast -MRI in mice, in keeping with DCE-MRI results in responder patients, revealed a reduction in fBV in REG treated mice (p=0.04): a positive correlation was observed between fBV and CD-31 changes (r2=0.64, p=0.006) and a significant increase in necrosis was observed in treated animals (p=0.008).

Conclusion: PDOs recapitulate pathological, molecular and radiological features of matching metastases upon REG treatment. Liver xenotransplantation of PDOs is highly successful, allows the study of tumour-stroma interactions and defined vessel co-option as a mechanism of primary resistance to REG.

23

Stress suppresses autophagy and affects differentiation of bone marrow stromal cells

Zvenyslava Husak1, Michael Dworzak1

1 Children’s Cancer Research Institute, Vienna, AUSTRIA

Long-term survival of bone marrow-derived mesenchymal stromal cells (MSCs) due to

autophagy is one of their most important characteristics. Under some conditions, MSC may develop tumorigenic properties. However, these transformation-induced conditions remain largely unknown. Many studies investigate MSCs tumorigenesis under hypoxia and serum starvation. Recently we identified association between Hsp70, a main participant in cellular stress response and tumorigenesis, and CD99, which is a widely expressed surface molecule on many cells but not MSCs. Preliminary observations revealed up-regulation of both proteins in stressed long-term cultured MSCs. We hypothesized that CD99 is implicated in stress-induced mechanisms of cellular transformation in MSC. Hence, we investigated the effects of prolonged stress on MSCs and the role of CD99 and autophagy in their survival. Human telomerase reverse transcriptase (hTERT) overexpressing immortalized MSCs and primary bone marrow stromal cells were used to investigate the influence of long-term serum deprivation and hypoxia on growth and differentiation of MSCs. Cell proliferation and apoptosis were evaluated using flow cytometry, differentiation capabilities of MSCs were assessed by immunohistochemical staining followed by microscopic examination. CD99, Hsp70 expression were analyzed using flow cytometry, western blotting and reverse transcriptase polymerase chain reaction. Autophagy was explored with specific inhibitors using cell morphology examination and western blotting. We found that chronic stress factors are able to change morphology of MSCs and to inhibit spontaneous differentiation into adipocyte lineage. Furthermore, CD99 elevation and disappearance of p53 and p21 accompanied defected autophagy, which is usually associated with tumor formation. Our data show that inhibition of autophagy promoted cell detachment and modulated CD99 expression level whereas CD99 overexpression suppressed autophagy. These results provide a model for chronic stress-induced transformation of MSCs via CD99 and thus are likely of relevance for mesenchymal tumorigenesis.

24

Wild-type APC predicts poor prognosis in microsatellite-stable proximal colon cancer

Robert Jorissen15,7, Michael Christie15,8, Dmitri Mouradov15,7, Anuratha Sakthianandeswaren15,7, Shan Li15,7, Christopher Love15,7, Zheng-Zhou Xu1, Peter Molloy1, Ian Jones3, Stephen McLaughlin4, Robyn Ward10, Nicholas Hawkins13, Andrew Ruszkiewicz6, James Moore2, Antony Burgess14,7, Dana Busam9, Qi Zhao11, Robert Strausberg11,12, Lara Lipton15,5, Jayesh Desai15,5, Peter Gibbs15,5,

Poster abstracts

Oliver Sieber15,7

1 CSIRO Preventative Health Flagship, Animal, Food & Health Sciences Division, North Ryde, NSW, AUSTRALIA, 2 Department of Colorectal Surgery, Royal Adelaide Hospital, Adelaide, SA, AUSTRALIA, 3 Department of Colorectal Surgery, Royal Melbourne Hospital, Parkville, VIC, AUSTRALIA, 4 Department of Colorectal Surgery, Western Hospital, Footscray, VIC, AUSTRALIA, 5 Department of Medical Oncology, Royal Melbourne Hospital, Parkville, VIC, AUSTRALIA, 6 Department of Surgical Pathology, SA Pathology, Adelaide, SA, AUSTRALIA, 7 Faculty of Medicine, Dentistry and Health Sciences, Department of Medical Biology, University of Melbourne, Parkville, VIC, AUSTRALIA, 8 Faculty of Medicine, Dentistry and Health Sciences, Department of Surgery, University of Melbourne, Parkville, VIC, AUSTRALIA, 9 J. Craig Venter Institute, Rockville, MD, USA, 10 Lowy Cancer Research Centre, Prince of Wales Clinical School, University of New South Wales, Sydney, NSW, AUSTRALIA, 11 Ludwig Collaborative Laboratory for Cancer Biology and Therapy, Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA, 12 Ludwig Institute for Cancer Research Ltd., New York, NY, USA, 13 School of Medical Sciences, University of New South Wales, Sydney, NSW, AUSTRALIA, 14 Structural Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, AUSTRALIA, 15 Systems Biology and Personalised Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, AUSTRALIA

APC mutation (APC-mt) occurs in approximately 70% of colorectal cancer (CRC), but its relationship to prognosis is not clear. Using data from 746 CRC patients and their tumours, we observed that patients whose tumours arose from the proximal colon and were wild-type for APC and microsatellite stable (APC-wt/MSS) tumours from the proximal colon displayed worse overall and recurrence-free survival (OS, RFS) than APC-mt/MSS proximal, APC-wt/MSS distal and APC-mt/MSS distal tumours (OS HR=1.79, P=0.015; RFS HR=1.88, P=0.026). APC status did not affect outcomes in patients whose tumours exhibited microsatellite instability (MSI). For MSS/proximal colon tumours, APC mutation status was a stronger prognostic indicator than BRAF, KRAS, PIK3CA, TP53, CpG island methylator phenotype (CIMP) or chromosomal instability status (P=0.036). Using a model to predict APC mutation status from expression microarray data, we found in a validation cohort of 206 patients with proximal colon cancer, that APC-wt-like signature/MSS cases showed poorer survival than MSI or APC-mt-like signature/MSS cases (OS HR=2.50, P=0.010;

RFS HR=2.14, P=0.025). Poor prognosis APC-wt/MSS proximal tumours exhibited features of the sessile serrated neoplasia pathway (P=0.016). As APC-wt status is a marker of poor prognosis in MSS proximal colon cancer, this information is relevant for treatment decisions for colon cancer patients.

25

Development of 3D co-culture model to study prostate cancer-derived exosome effects on patient-derived fibroblast phenotype

Zane Kalnina1, Lilite Sadovska1, Baiba Brumele1, Cristina Bajo-Santos1, Kristine Sobolevska1, Elina Zandberga1, Alicia Llorente2, Aija Line1

1 Latvian Biomedical Research and Study Centre, Riga, LATVIA, 2 Oslo University Hospital, Oslo, NORWAY

Published studies suggest that, in the tumour microenvironment, cancer-cell released extracellular vesicles (EVs) can be internalised by other cancer cells as well as various types of stromal cells thus promoting cancer development and progression in vivo. Stromal cells of mesenchymal origin (fibroblasts) are a major cellular component of tumour microenvironment in most solid cancers, and activation of myofibroblast rich stroma is a step essential for cancer progression. The aim of the study was to develop an in vitro model suitable for studying uptake mechanisms and physiological effects of aggressive prostate cell-derived EVs on tumour-derived mesenchymal stromal cells.

In order to trace cancer-derived exosome (small EVs in size range of 30-100nm) uptake in stromal cells, the hormone-independent prostate cancer (PC) cell line PC3 was stably transfected with a plasmid encoding for exosomal protein CD63-GFP N-terminal fusion protein. The primary fibroblast cultures were generated from tumour tissue specimens from 3 PC patients undergoing prostatectomy (received after receiving permits from local ethics committee and a written consent). Tissues were enzymatically disintegrated and grown in standard media DMEM-F12 supplemented with 10% FBS and Primocin. The obtained tumour-derived fibroblast cultures as well as dermal fibroblasts Hs68 as a control were stained with DiD, a red fluorescent membrane dye, and seed at high density in ratios 10:1, 5:1, 1:1, 1:5 and 1:10 with PC3-GFP cells, grown as heterologous spheroids in serum-free media for 48h, and analysed by confocal microscopy and flow cytometry. Here we will report on optimal

Poster abstractsPoster abstracts

settings for the developed model and demonstrate its application for studying EV-induced changes in fibroblast phenotype in patient-derived mesenchymal stromal cells.

26

Personalised medicine approach for paediatric patients with high-risk neuroblastoma

Alvin Kamili2, Mitchell J. Lockwood2, Carol Wadham2, Shu-Oi Chow2,1, Tim W. Failes2,1, Greg M. Arndt2,1, C. Patrick Reynolds4, Murray D. Norris2, Michelle Haber2, Loretta L. Lau2,3, Toby N. Trahair2,3, Jamie I. Fletcher2

1 ACRF Drug Discovery Centre for Childhood Cancer, Lowy Cancer Research Centre, Randwick, NSW, AUSTRALIA, 2 Children’s Cancer Institute, Lowy Cancer Research Centre, Randwick, NSW, AUSTRALIA, 3 Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW, AUSTRALIA, 4 Texas Tech University Health Sciences Centre, Lubbock, Texas, USA

Background: Neuroblastoma is the most common extracranial solid tumour of children and accounts for 15% of all paediatric oncology deaths. The cure rates of children with high-risk neuroblastoma remain less than 50%, with dose limiting toxicities and the adverse effects of chemotherapy presenting major challenges. These challenges may be partially resolved by the implementation of personalized medicine approaches. In this pilot study, we recruit a limited number of patient to test personalised medicine pipeline for high-risk neuroblastoma patients. This project forms part of a larger initiative to implement personalized medicine approaches for high-risk childhood cancers in Australia, known as Zero Childhood Cancer Program.

Methods: Tumour samples were obtained from three high-risk neuroblastoma patients at the Sydney Children’s Hospital, Australia. Samples were processed for molecular profiling, tissue banking, and establishment of PDX and cell lines. Engraftment was also attempted using peripheral blood, bone marrow, or pleural fluid. We used RNA sequencing and whole genome sequencing to identify targetable molecular aberrations in individual tumours. Drug sensitivity screening was conducted using dissociated tumour cells against 165 approved oncology drugs to select drugs in the absence of targetable molecular aberration. Selected agents assessed for efficacy in the PDX models.

Results and plans: Each relapse sample

successfully engrafted, but needed 3-6 months for establishment. Each PDX models was successfully expanded for in vivo drug testing. Based on profiling analysis, one PDX model was tested against trametinib/isotretinoin combination to target NF1 mutation/loss. The other two models had no targetable molecular aberration identified, but showed high sensitivity to Venetoclax (ABT-199) and GENZ-644282, respectively. Drug testing in these PDX models are ongoing. The outcome of in vivo study may be used by oncologists to make informed decision to select patient therapies.

27

Zrf1 depletion causes an aggressive cancer phenotype and favors cell survival

Aysegul Kaymak1,2, Holger Richly1,2

1 Institute of Molecular Biology, Mainz, GERMANY, 2 Johannes Gutenberg University, Mainz, GERMANY

Zuotin-related factor 1 (Zrf1) has been identified as an epigenetic regulator of gene transcription in stem cells and cancer (Aloia L., 2015). According to the Oncomine database, Zrf1 shows a reduced expression in breast cancer. Moreover a recent study points out a potential use of auto-antibody response against Zrf1 antigen for early breast cancer detection (Dyachenko D., 2016). Based on these information, we aimed to explore Zrf1’s function in breast cancer progression in detail. After generating control and Zrf1 knockdown MCF7 cell lines, we compared main events leading to breast cancer development in those cell lines. We found that compared to control cells, Zrf1 knockdown cells display a more aggressive cancer phenotype with an increased cell motility and migration together with a less adhesive cell behavior in accordance with breast cancer patient results.

Estrogen is the major driver of breast cancer development and its biologic activities are mediated by the nuclear estrogen receptor alpha (ERα). To elucidate whether estrogen response is affected upon Zrf1 depletion, we treated control and knockdown cells either an ERα activator (17β-estradiol) or an ERα inhibitor (ICI 182,780) for 7 days. Compared to control cells, Zrf1 knockdown cells grew significantly more and stayed more viable even in the presence of ICI 182,780. Flow cytometry analysis of dual Annexin V and PI staining confirmed that Zrf1 depleted cells become less apoptotic, and desensitized to ICI 182,780 treatment. Evaluation of RT-PCR data reveals that Zrf1 knockdown cells upregulates the expression of cell survival genes including Bcl-2, Bcl-XL, Bcl-W

Poster abstractsPoster abstracts

and Survivin, whereas the expression of apoptotic genes stay same. Taken together, our data imply that Zrf1 depletion can impair the balance between apoptotic and anti-apoptotic genes, and hereby promote tumor cell survival. Therefore Zrf1 can be regarded as a potential new target to be explored for breast cancer treatment.

28

BRAFV600E Kinase Domain Duplication Identified in Therapy-Refractory Melanoma Patient-Derived Xenografts

Kristel Kemper1, Oscar Krijgsman1, Xiangjun Kong1, Paulien Cornelissen-Stijger1, Aida Shahrabi1, Daphne van der Velden1, Fleur Weeber1, Onno Bleijerveld1, Thomas Kuilman1, Roel Kluin1, Chong Sun1, Emile Voest1, Young Seok Ju3, Ton Schumacher1, Maarten Altelaar1,2, Ultan McDermott3, David Adams3, Christian Blank1, John Haanen1, Daniel Peeper1

1 Netherlands Cancer Institute, Amsterdam, NETHERLANDS, 2 University of Utrecht, Utrecht, NETHERLANDS, 3 Wellcome Trust Sanger Institute, Hinxton, UK

The therapeutic landscape of melanoma is improving rapidly. Targeted inhibitors show promising results, but drug resistance often limits durable clinical responses. There is a need for in vivo systems that allow for mechanistic drug resistance studies and (combinatorial) treatment optimization. Therefore, we established a large collection of patient-derived xenografts (PDX), derived from melanoma metastases prior to treatment with BRAF inhibitor and after resistance had occurred. The success rate was ~ 80%, yielding a total of 98 PDXs, comprising 81 BRAFV600E, 9 NRASQ61, and 8 BRAFWTNRASWT xenografts. Six matched pairs of BRAFV600E PDX models were derived from single patients both prior to treatment with the BRAF inhibitor vemurafenib and after resistance had occurred.

We have demonstrated that PDX resemble their donors at the level of biomarkers, chromosomal aberrations and RNA expression profiles. Furthermore, we show that mutations, previously identified to cause resistance to MAPK-pathway targeted therapy, are captured in PDX derived from resistant melanomas and heterogeneity is maintained.

Next, we took advantage of the limitless resource of tumour material of PDX, in contrast to patients’ biopsies. Screening in PDX cell lysates from vemurafenib-resistant melanomas for BRAF proteins with aberrant molecular weights, we identified a hitherto unknown mechanism of

resistance: a duplication of the kinase domain (encoded by exons 10-18) of BRAFV600E, which we named BRAFV600E/DK. Of clinical relevance, we demonstrate that the new pan-RAF dimerization inhibitor LY30009120, in sharp contrast to vemurafenib, effectively eliminates melanoma cells expressing BRAFV600E/DK.

Currently, we are analysing the RNA expression profiles of 95 PDX in order to determine new resistance mechanisms and study the influence of the stromal factors in these PDX on therapy response.

29

DNA methylation signature (SAM40) identifies subgroups of the Luminal A breast cancer samples with distinct survival

Thomas Fleischer1, Jovana Klajic1,3, Miriam Ragle Aure1, Riku Louhimo4, Arne V. Pladsen1, Lars Ottestad1, Nizar Touleimat5, Marko Laakso4, Ann Rita Halvorsen1, Grethe Irene Grenaker Alnæs1, Margit L. H. Ris3,7, Åslaug Helland1,8, Sampsa Hautaniemi4, Per Eystein Lønning9,10, Bjørn Naume2, Anne-Lise Børresen Dale1, Jörg Tost5, Vessela N. Kristensen1,3

1 Department of Cancer Genetics, Institute for Cancer Research, OUS Radiumhospitalet, Oslo, NORWAY, 2 Cancer Clinic, Oslo University Hospital Radiumhospitalet, Oslo, NORWAY, 3 Department of Clinical Molecular Biology and Laboratory Science (EpiGen), Akershus University hospital, Division of Medicine, Lørenskog, NORWAY, 4 Systems Biology Laboratory, Institute of Biomedicine and Genome-Scale Biology Research Program.University of Helsinki, Helsinki, FINLAND, 5 Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA – Institut de Génomique, Evry, FRANCE, 6 Department of Surgery, Akershus University Hospital, Lørenskog, NORWAY, 7 Deptartment of Breast and Endocrine Surgery, Oslo University Hospital, Ullevål, Oslo, NORWAY, 8 Department of Oncology, Oslo University Hospital, The Norwegian Radium Hospital, Oslo, NORWAY, 9 Section of Oncology, Institute of Clinical Science, University of Bergen, Bergen, NORWAY, 10 Department of Oncology, Haukeland University Hospital, Bergen, NORWAY

Breast cancer patients with Luminal A disease generally have a good prognosis, but among this patient group are patients with good prognosis that are currently overtreated with adjuvant chemotherapy, and also patients that have a bad prognosis and should be given more aggressive

Poster abstracts

treatment. There is no available method for subclassification of this patient group. Here we present a DNA methylation signature (SAM40) that segregates Luminal A patients based on prognosis, and identify one good prognosis group and one bad prognosis group. The prognostic impact of SAM40 was validated in four independent patient cohorts. Being able to subdivide the Luminal A patients may give the two-sided benefit of identifying one subgroup that may benefit from a more aggressive treatment than what is given today, and importantly, identifying a subgroup that may benefit from less treatment.

30

Detection and functional characterization of extracellular vesicles in kinase inhibitor-resistant melanoma cells

Giulia Cesi3, Demetra Philippidou3, Francois Bernardin2, Guillaume van Niel1, Yeoun-Jin Kim2, Stephanie Kreis3

1 Institut Pasteur, Paris, FRANCE, 2 Luxembourg Institute of Health, Luxembourg, LUXEMBOURG, 3 University of Luxembourg, Belval, LUXEMBOURG

Introduction: Extracellular vesicles are nano-sized structures that are released by all cell types under both physiological and pathological conditions. As extracellular vesicles can be released by “donor” cells and taken up by “recipient” cells, they can be regarded as vehicles of intercellular communication or “homing pigeons” influencing key biological functions by delivering and transporting cytokines, growth factors, proteins, mRNAs and microRNAs. Recently, extracellular vesicles have also been identified as new messengers in transferring drug resistance to still sensitive cells. In melanoma patients, drug resistance is a pressing issue. Despite the promising initial results obtained with vemurafenib and dabrafenib (BRAF kinase inhibitors) in the clinic, it soon became evident that these molecules were not able to provide durable responses, as resistance to treatment develops within months in almost all patients.

Methods: The content of the extracellular vesicles released by sensitive melanoma cells and their corresponding drug resistant cells was analysed by mass spectrometry and miRNA arrays. In addition, co-culture experiments have been performed to understand the potential involvement of extracellular vesicles in the “spreading” of drug resistance.

Results: We show that sensitive melanoma cells acquire the drug resistant phenotype if co-cultured with extracellular vesicles released by resistant

cells. Proteomic and miRNome analyses revealed distinct content profiles with a panel of proteins especially enriched in the “resistant extracellular vesicles”. Hence, potential candidates that might play a role in conferring drug resistance have been identified. Our results suggest that “resistant extracellular vesicles” have functional properties capable of making sensitive melanoma cells more resistant to BRAF inhibitors.

31

XenofilteR: separate graft from host reads in xenograft sequencing

Oscar Krijgsman1, Roel Kluin1, Thomas Kuilman1, Kristel Kemper1, Julian de Ruiter1, Vivek Iyer2, Josep V Forment2, Paulien Cornelissen-Steijger1, Iris de Rink1, Petra ter Brugge1, Young Seok Yu2, Ultan McDermott2, Jos Jonkers1, Arno Velds1, Dave J Adams2, Daniel S Peeper1

1 NKI, Amsterdam, NETHERLANDS, 2 Sanger, Hinxton, UK

Patient derived xenograft (PDX) models are increasingly used in cancer research. PDX recapitulate the 3D organization and heterogeneity of the patient tumor making it a clinically useful model to study therapy resistance, therapy responses and evolutionary dynamics but also allows for biomarker discovery. However, analysis of high-throughput sequence data of tumor material derived from these PDX is challenging. The data not only contain sequence reads that originate from the cancer cells (graft) but also sequence reads from mouse cells (host). These sequence reads have a large effect on downstream analyses which makes accurate filtering of mouse reads a mandatory step in the analysis pipeline of PDX.

We developed the open-source software tool XenofilteR to separate graft from host reads from PDX data. XenofilteR uses values readily available in every mapped sequence file (bam file) and assesses the quality of mapping for each read to both mouse and human reference genomes.

Each sequence read is assigned to either mouse or human based on the quality of mapping.

XenofilteR removes >99.9% of mouse reads from the sequence data while retaining ~99% of human sequence reads as validated on in-silico mixed mouse and human sequence data. In addition to the in-silico validation we tested XenofilteR on breast and melanoma PDX samples showing accurate detection of SNPs and somatic mutations and outperforms currently available tools.

Poster abstracts

In conclusion, XenofilteR is a fast and simple new method for accurate filtering of mouse reads from patient derived xenograft sequencing.

32

Integrin ALPHA5: a key prognostic and therapeutic target in pancreatic tumor stroma

Praneeth Reddy Kuninty3, Sanne WL De Geus2, Ruchi Bansal3, Peter Kuppen2, Alexander Vahrmeijer2, Arne Ostman1, Cornelis FM Sier2, Jai Prakash3,1

1 Cancer Centre Karolinska, Karolinska Institutet, Stockholm, SWEDEN, 2 Leiden University Medical Center, Leiden, NETHERLANDS, 3 University of Twente, Enschede, NETHERLANDS

Cancer-associated fibroblasts (CAFs) are the key cell type in the pancreatic tumor microenvironment, which induces tumor growth and metastasis. Here, we have identified integrin alpha5 (ITGA5) as a novel target overexpressed in pancreatic CAFs and studied the value of ITGA5 as a prognostic and therapeutic target.

In human patient tumor samples (n=137), 66% of the patients were positive for ITGA5 and well co-localized with -SMA, as shown with double immunostaining. Overall, clinical data analysis reveals that the overexpression of ITGA5 (log-rank p=0.022) is linked to significant poor overall survival. In vitro, activation of human primary pancreatic stellate cells (PSCs) either with recombinant TGFβ-1 or with conditioned medium obtained from Panc-1 tumor cells significantly induced ITGA5 expression in PSCs.

Importantly, knockdown of ITGA5 in PSCs using shRNA led to a dramatic reduction of ECM proteins and other fibrotic markers, analysed with human profiler gene array. Furthermore, ITGA5-KD-PSCs showed lower migration compared to control shRNA-PSCs, attributed to the loss of FAK, Rac, Cdc4 signalling. Furthermore, ITGA5-KD-PSCs had a substantial decrease in proliferation, cell adhesion and TGFβ-1 induced collagen contractility compared to control shRNA-PSCs. In vivo, co-injection of Panc-1 and PSCs in SCID mice showed a significant increase in tumor growth compared to Panc-1 tumors. Importantly, tumors formed with Panc-1 + ITGA5-KD-PSCs had no tumor growth, indicating the significance of ITGA5 in controlling PSC-induced tumor growth in vivo.

Altogether, this study reveals ITGA5 as a novel prognostic and therapeutic stromal target in the pancreatic tumor, which is in potential for clinical application.

This research was funded by Swedish Research Council.

33

Systems microscopy approaches to investigate the metabolic control of tumor cell migration

Steven Wink1, Margriet Palm1, Hans de Bont1, Xiaobing Zhang1, Joost Beltman1, Bob van de Water1, Sylvia Le Dévédec1

1 LACDR/Leiden University, Leiden, NETHERLANDS

Enhanced migratory behavior of tumor cells in metastasis is a critical hallmark of cancer progression. Cell migration is driven by dynamic changes in matrix adhesions, complexes that regulate the linkage between the actin cytoskeleton and the extracellular matrix. Aerobic glycolysis or the Warburg effect is another important hallmark of cancer. Little is known on the role of the Warburg effect in tumor cell migration. In particular the spatiotemporal relationships between energy supply and the dynamics of matrix adhesion and actin are unclear. Recently, we discovered that several enzymes of the glycolytic program are central regulators of the migratory phenotype and matrix adhesion dynamics. Therefore we hypothesize that the Warburg effect is critical to control the migratory behavior of tumor cells and hence metastasis formation. To fully understand the relationship between the Warburg effect and tumor cell migration, we aim at developing a quantitative and predictive in silico model that integrates the relationships between matrix adhesions/actin dynamics, glucose/ATP concentration/fluxes and the migratory phenotype of tumor cells. Using high-end microscopy and available genetically-encoded optical biosensors, we monitor both matrix adhesions/actin dynamics and the concentration of metabolites in real-time and in single migrating tumor cells. We established a multi-parametric image analysis pipeline to systematically quantify matrix adhesion and actin dynamics, intracellular glucose and ATP concentrations and cell migration. As a next step, the obtained data of this single-cell metabolic analysis will be integrated in an in silico model for glycolysis-driven cell migration that will be tested by genetic manipulations of the glycolytic program using siRNA approaches. Such approach will be a unique tool for the future efficient design and development of anti-metastatic drugs in relation to cancer metabolism.

Poster abstracts

34

A personalised approach for the treatment of relapsed/refractory T-cell acute lymphoblastic leukaemia (T-ALL)

Richard Lock1, Donya Moradi Manesh1, Cara Toscan1

1 Children’s Cancer Institute, UNSW Australia, Sydney, NSW, AUSTRALIA

While T-ALL accounts for only around 20% of ALL cases, relapsed T-ALL can be aggressive and often fatal, and personalised treatments are urgently required. PR-104 is a DNA alkylating pre-prodrug activated under hypoxia by one-electron reductases, but also under normoxia by the two-electron reductase aldo-keto reductase 1C3 (AKR1C3). When tested at clinically relevant doses we previously showed that PR-104 exhibited greater efficacy against paediatric T-ALL xenografts compared with B-cell precursor ALL (BCP-ALL), which was due to significantly higher AKR1C3 expression in T-ALL. However, while PR-104 was highly effective against typical T-ALL, early T-cell precursor ALL (ETP-ALL) xenografts exhibited PR-104 resistance despite expressing high levels of AKR1C3. In order to identify established drugs that could overcome PR-104 resistance in ETP-ALL we performed a high throughput screen of 96-FDA approved anticancer drugs combined with PR-104. The purine nucleoside antimetabolite clofarabine was the lead PR-104 sensitiser, and was highly synergistic when combined with PR-104 against 6/6 ALL xenografts in short-term in vitro cytotoxicity assays (mean Combination Indices 0.005-0.68). We next tested PR-104 in combination with clofarabine in vivo against an ETP-ALL xenograft (ETP-2). At a clinically achievable dose (200 mg/kg i.p. weekly x 2) PR-104 delayed the progression of ETP-2 by 22.0 days compared with vehicle control treated mice, similar to low AKR1C3-expressing BCP-ALL xenografts (median progression delay 16.6 days, n=4 xenografts), but less than typical T-ALL xenografts (49.4 days, n=4 xenografts). The combination of PR-104 and clofarabine (30 mg/kg i.p. daily x 5 x 2 weeks) was well tolerated and resulted in a progression delay of 54.4 days, which was significantly greater than either single agent (P=0.0001). These results show that the clofarabine/PR-104 combination exerts synergistic efficacy against ETP-ALL in vivo and other ALL subtypes in vitro, and warrants further consideration for the personalised treatment of relapsed/refractory T-ALL.

35

Mechanosensing in the microenvironment regulates melanoma cell phenotypes

Zsofia Miskolczi1, Emily Rowling1, Michael Smith1, Claudia Wellbrock1

1 University of Manchester, Manchester, UK

Melanoma is a cancer originating from pigment cells called melanocytes. It is the deadliest skin cancer, underlying the need to better understand the drivers of the disease. The lineage specific transcription factor, MITF is the master regulator of melanocyte development and an important regulator of genes involved in survival, migration and fate-decision. This project aims to dissect the impact of extracellular-matrix (ECM) signalling on MITF function and consequently on melanoma cell behaviour. In our study, we focused on the role of matrix stiffness, which changes during cancer progression and also differs between different metastatic sites. We found that morphology, adhesion and proliferation are all affected by the properties of the surrounding matrix. In addition, the mRNA levels of MITF and its target genes are correlated with the increasing matrix stiffness. On soft collagen gels, cells are less differentiated and less proliferative; whereas on stiffer gels, melanoma cells display a highly differentiated and proliferative phenotype. However, this phenotype is transient, and transition from a soft gel to a stiff gel causes hyper proliferation and differentiation. Because fibroblasts are a major contributor to ECM conditions, we also investigated the crosstalk between melanoma cells and fibroblasts in this context. Conditioned medium from melanoma cells increases fibroblast proliferation, makes them more contractile and alters their matrix deposition. In a reciprocal manner the remodelled matrix produced by fibroblast exposed to melanoma cells influences melanoma cell phenotypes. Our data suggest that matrix stiffness produced by a cross-talk between fibroblasts and melanoma cells could influence melanoma cell biology, which ultimately may have an impact on melanoma progression.

36

Vertical inhibition of Met by a dual decoy/antibody strategy

Chiara Modica2,1, Elisa Vigna2,1, Cristina Chiriaco1, Lara Fontani1, Paolo Maria Comoglio1, Cristina Basilico1

1 Candiolo Cancer Institute, FPO-IRCCS, Candiolo, ITALY, 2 University of Turin, Torino, ITALY

Introduction: Met oncogene, encoding the tyrosine

Poster abstracts

kinase receptor for the hepatocyte growth factor (HGF), plays a critical role in tumor progression and in resistance to targeted therapies. In a limited number of cases, a genetic lesion drives the malignant phenotype in a ligand-independent manner. In the vast majority of tumors, however, wild-type MET relies on the ligand (HGF) to sustain cell ‘scattering’, invasive growth and apoptosis protection. In this context -i.e. ligand-dependent MET activation- concomitant targeting of MET and HGF could be crucial to reach effective inhibition.

Aim: To investigate if the vertical inhibition of Met and its ligand, obtained by concomitant treatment with MvDN30 - an anti-MET antibody characterized by the property of inducing ‘shedding’, i.e. removal of MET from the cell surface and decoyMET - the soluble extracellular domain of the MET receptor, endowed with HGF sequestering ability - can neutralize the malignant properties of cancer cells fostered by HGF-dependent Met activation.

Results: To avoid antibody/decoy interaction -and subsequent neutralization- we identified a single aminoacid in the extracellular domain of MET -lysine 842- that is critical for DN30 binding, and we engineered the corresponding recombinant decoyMET. In HGF-dependent cellular models, MvDN30 antibody and decoyMETK842E used in combination cooperated in restraining invasive growth, and synergized in blocking cancer cell ‘scattering’. Moreover, the combination treatment was more effective than the single molecules to impair viability of colon cancer derived xenospheres and organoids. In a preclinical model of MET ‘expedience’, built by orthotopic transplantation of pancreatic carcinoma cells in SCID mice expressing human HGF, concomitant treatment with antibody and decoy delayed tumor growth and reduced metastatic spread.

Conclusion: The data reported indicate that vertical targeting of the MET/HGF axis results in powerful inhibition of ligand-dependent MET activation, providing proofs of concept in support of combined targeted therapy.

37

Expression of the Cocaine- and Amphetamine-Regulated Transcript (CART) recruits SWI/SNF chromatin remodelling complexes to the Estrogen Receptor

Brian Mooney2, Sudipto Das2, Rut Klinger3, Eric Conway4, Ben Doyle3, Kieran Wynne3, William M. Gallagher3, Triona Ni Chonghaile1, Adrian P. Bracken4, Darran P. O’Connor2

1 Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, EIRE, 2 Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, EIRE, 3 School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin 4, EIRE, 4 Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, EIRE

Cocaine- and amphetamine-regulated transcript (CART) peptides are involved in regulating processes such as feeding and drug reward. Previously, we demonstrated that high CART expression correlated with worse overall survival, and poor response to tamoxifen, in patients with estrogen receptor-positive (ER+), lymph node-negative breast cancer. We also demonstrated that CART can impact the transcriptional activity of ERα through the use of western blotting and qPCR, however, the impact CART expression can have on the ERα interactome remained unknown. Using stable CART-inducible cell lines, we preformed ERα-Immunoprecipitation followed by in-solution mass spectrometry to identify differentially recruited protein complexes +/- CART expression.

Mass spectrometry analysis revealed 16 proteins significantly recruited to ERα following CART expression. The vast majority of these proteins were identified as members of the SWI/SNF (BAF) chromatin remodelling complex. The identification of SMARCD1 within this complex was of particular interest to this study, as this protein has previously been reported to be a critical mediator of nuclear-hormone receptor function. In silico analysis demonstrated high expression of SMARCD1 correlates with poor overall survival (OS) (p<0.00001) and distant metastasis free survival (DMFS) (p=0.00708) in a cohort of ER+ breast cancer patients. Intriguingly, SMARCD1 expression did not correlate with poor OS or DMFS in a cohort of ER- breast cancer patients, suggesting that this negative impact on survival is potentially related to ERα.

In conclusion, we suggest that CART expression results in the recruitment of chromatin remodelling complexes to ERα in order to facilitate the regulation of ERα activity. SMARCD1 has previously been shown to be an ERα interactor, and is a known mediator of ligand-dependent ERα activity. Moving forward, we aim to modulate the expression of targets identified in our mass spectrometry study to examine whether any of these targets could be novel therapeutic targets for patients with ER+ breast cancer.

Poster abstracts

38

Investigation of MET oncogene addiction and the MET-DDR crosstalk: a phosphoproteomic approach

Eleonora Orlando1,4, Ariel Bensimon4, Michaela Medová1,2, Selina Roth1,2, Daniel Matthias Aebersold1,2, Ruedi Aebersold3,4, Yitzhak Zimmer1,2

1 Department of Clinical Research, University of Bern, Bern, SWITZERLAND, 2 Department of Radiation Oncology, Inselspital, Bern University Hospital and University of Bern, SWITZERLAND, 3 Faculty of Science, University of Zürich, SWITZERLAND, 4 Institute of Molecular Systems Biology, ETH Zürich, Zürich, SWITZERLAND

The MET receptor tyrosine kinase has acquired widespread attention in the field of cancer research since its discovery. Indeed, its role in promoting oncogenic processes, invasiveness and metastatic events has been well established. Moreover, MET inhibition (METi) was found to confer radiosensitivity to MET-addicted cell lines, potentially through the re-wiring of DNA damage response (DDR) signalling pathways. The concept of oncogene addiction postulates that, despite the array of genetic lesions that characterize cancer cells, some tumors rely on individual oncogenes for growth and survival. While accumulating data have proven the importance of this concept in therapeutic settings, the molecular pathways underlying this phenomenon remain elusive. Therefore, the major aim of this project is to investigate oncogene addiction further, particularly through the identification of crucial players involved in oncogene-mediated apoptosis and in the crosstalk with the DDR. To this purpose, MET was used as a model and further investigated by targeted proteomics based on Selected Reaction Monitoring (SRM) in order to monitor phosphorylation changes of 130 proteins in nine MET-positive cellular models upon METi, IR and the combination of the two perturbations. Remarkably, METi and IR regulate the phosphorylation status of a subset of analyzed proteins in a synergistic manner in MET-addicted cellular models. Moreover, not only crucial regulators of DDR-related processes, but also key nodes of a plethora of other cellular processes seem to be regulated by METi in MET-addicted cellular models. Finally, subsequent analysis of cellular models addicted to other oncogenes upon exposure to their respective inhibitors seems to suggest that oncogene addiction is governed by shared dominant pathways that, once disrupted, lead to radiosensitivity. In this respect, the identification of a composite phosphosignature for oncogene addiction would allow more accurate patient stratification and personalized clinical

settings, both for single agents as well as for combination therapies.

39

How to report somatic variants in molecular tumor boards

Julia Perera-Bel1, Tim Beissbarth1

1 University Medical Center Göttingen, Göttingen, GERMANY

The understanding of complex diseases, such as cancer, is becoming more comprehensive with the improvements of high-throughput technologies e.g., next-generation sequencing. However, advances in technology platforms and bioinformatic tools contrast with the scarce implementation of cancer genomics in clinical practice. One reason for this situation is that pathologists and oncologists have to face thousands of genomic alterations and unravel their clinical relevance. Accordingly, the scientific community has claimed the need of a comprehensive knowledge database as well as decision support platforms for the interpretation and reporting of genomic findings in clinical practice e.g., in molecular tumor boards.

Towards this end, we have developed a framework for reporting genomic data relying entirely on public knowledge. The method focuses on genomic alterations that predict drug response. In particular, gene-drug predictive associations are classified according the stage of development of the drug (approved, clinical trials or pre-clinical studies) and the cancer type for which the predictive association exists.

We tested the framework on the Pan-Cancer dataset from TCGA (samples from 12 cancer types). We showed that it is able to 1) accurately report the genomic variants which predict drug response; and 2) find clinically relevant findings in the majority of the patients. In addition, we assessed the clinical utility of the reports on cancer patients whose treatment was based on their genomic profiles. 10 out of 11 reports included the genomically-guided treatment option that was decided by the expert panel.

We present a method to report treatment options based on the genomic profile of the patient. It is designed as a supporting tool for all clinicians, biologists and bioinformaticians working with genomic characterization of patients in clinical routine and face complex decisions regarding treatment options.

Poster abstracts

40

Nanodelivery of Smac-mimetic peptide enhances the efficacy of doxorubicin in vivo

Dwi Priwitaningrum1, Julian Jentsch1, Sima Rahimian2, Ruchi Bansal1, Wim Hennink2, Gert Storm2, Jai Prakash1

1 University of Twente, Enschede, NETHERLANDS, 2 University of Utrecht, Utrecht, NETHERLANDS

Induction of apoptosis in cancer cells is a key challenge in suppressing tumor growth. Smac peptide (N-terminal residues of second mitochondria-derived activator of caspase), has been shown to bind to the inhibitor of apoptosis proteins (IAPs) to block their anti-apoptotic effect. Therefore, mimicking Smac-peptide to target IAPs represents a promising strategy for anti-tumor treatment. However, peptides owe many hurdles to be used as therapeutics including poor pharmacokinetics, rapid elimination, degradation, and no intracellular delivery. In the present study, we constructed a chimeric peptide containing Smac-mimetic peptide conjugated to a cell-penetrating peptide (Smac-CPP) and encapsulated it into polymeric nanoparticles to deliver it into tumor cells.

The Smac-CPP construct showed a significant inhibition of cell viability of 4T1 breast tumor cells in vitro at 20 μM by 40%. On the other hand, the scrambled construct showed no effect on the cell viability. Our Smac-CPP construct induced cell apoptosis by an increase in caspase 3/7 activity within 48 hours. After confirming the bioactivity, we encapsulated Smac-CPP in polymeric nanoparticles (Smac-NPs) for nanodelivery to tumors. In vitro, Smac-NPs showed much higher effects in killing tumor cells (40-50% at 5 μM), compared to free Smac-CPP (90%). Moreover, combining Smac-NPs with chemotherapeutics showed a synergetic effect in decreasing cell viability. Furthermore, in vivo, we examined Smac-NPs in 4T1 breast tumor model and found that Smac-NP was much better tolerated and showed higher anti-tumor effects compared to free Smac-CPP. In addition, the combination doxorubicin and Smac-NPs significantly inhibited the tumor growth compared to vehicle and Smac-NPs groups. Further histological examinations are still on-going to investigate the mechanisms for these effects.

In conclusion, we have designed a novel strategy to deliver Smac peptide using nanoparticles which seems promising to enhance the efficacy of chemotherapeutics.

41

Strategies for detecting and interpreting genomic variants in cancer patients

Carlota Rubio-Perez3, David Tamborero2, Abel Gonzalez-Perez2, Nuria Lopez-Bigas2,1

1 ICREA, Barcelona, SPAIN, 2 IRB, Barcelona, SPAIN, 3 UPF, Barcelona, SPAIN

Sequencing the tumors of cancer patients is becoming a standard tool in clinical oncology. However, the cost of sequencing the whole exome of a tumor is still high, compared to sequencing it through a gene panel, and the large number of detected variants makes difficult its interpretation. Here, I will present two tools developed in our group to solve these hurdles.

On one hand, I will present OncoPaD, to our knowledge the first tool aimed at the rational design of cancer gene panels. OncoPaD estimates the cost-effectiveness of the designed panel on a cohort of tumors and provides reports on the importance of individual mutations for tumorigenesis or therapy. OncoPaD suggests researchers relevant sets of genes to be included in the panel, based on prior knowledge or analyses indicating that their mutations either drive tumorigenesis or function as biomarkers of drug response. We demonstrate in silico that OncoPaD designed panels are more cost-effective—i.e. maximize the detection of mutations in a tumor cohort by sequencing a minimum quantity of DNA—than available cancer gene panels.

On the other hand, I will present Cancer Genome Interpreter (CGI), a tool designed to support the identification of therapeutically actionable genomic alterations in tumors. CGI first identifies validated driver events and predicts the potential effect of mutations of uncertain significance using a novel method (OncodriveMUT). Then, it identifies the alterations that are known to affect the response of the patient to anti-cancer therapies (sensitivity, resistance and toxicity) based on our expert curated database of genomic biomarkers of drug response. Our experience with two reference hospitals demonstrated that CGI is a valuable resource for allocating cancer patients to the most appropriate clinical trial and for helping in the identification of potential drug repurposing opportunities in refractory/relapsed cancer patients that have no conventional therapeutic options available.

Poster abstracts

42

Unravelling the biological mechanism of aggressive colorectal cancer with high stromal content

Tessa Paulien Sandberg2, Gabi van Pelt2, Hein Putter2, Peter Kuppen2, Rob Tollenaar2, Louis Vermeulen1, Wilma Mesker2

1 Amsterdam Medical Center, Amsterdam, NETHERLANDS, 2 Leiden University Medical Center, Leiden, NETHERLANDS

The tumor microenvironment or tumor stroma is a dominant determinant of cancer cell behaviour and disease progression. The presence of high tumor stroma content is likely associated with cancer cells acquiring pro-metastatic capacities. The tumor-stroma ratio (TSR) is a robust prognostic tool that is scored on haematoxylin and eosin (H&E) paraffin sections at the invasive front of the tumor (Huijbers et al., 2013). The importance of the tumor stroma is also emphasized in the CRC consensus molecular subtype (CMS). The CMS classification described four CRC subtypes, of which the poor-prognosis CMS4 subtype is characterized by high stromal infiltration and mesenchymal gene expression (Guinney et al., 2015). We aim to unravel the biological mechanism of tumors with high stromal content assessed based on the TSR method. We hypothesized that the TSR identifies mesenchymal tumors. First, in two CRC cohorts, tumors were scored for TSR and CMS classified using IHC as described previously (Trinh et al., 2016). The TSR and the CMS classification were independent in both cohorts (χ² test = 0.131, p = 0.717; χ² test = 1.438, p = 0.267). Secondly, we investigated the survival in one cohort of 201 patients stratified by both TSR and CMS. Both stroma-high and the mesenchymal-like CMS4 were adverse prognostic factors. We then analysed the survival by combining both methods and correcting for age, sex and TNM stage. The stroma-high and epithelial-like subgroup and the stroma-low and mesenchymal subgroup both had a 1.5 increased disease-free survival rate (HR = 1.510 [0.907 – 2.514]; HR = 1.525 [0.907 - 2.565]) while the stroma-high and CMS4 subgroup had a factor 2 increased disease-free survival rate (HR = 1.984 [1.227 – 3.207]; p = 0.050). Furthermore, gene expression analysis will be performed to compare the activated pathways between stroma-high and stroma-low tumors. The results will also be presented on the day of the symposium.

43

Integrin α11 as a key target in regulation of pancreatic tumor stromal myofibroblasts

Jonas Schnittert2, Ruchi Bansal2, Gert Storm3,2, Arne Ostman1, Jai Prakash2

1 Karolinska Institute, Stockholm, SWEDEN, 2 University of Twente, Enschede, NETHERLANDS, 3 Utrecht University, Utrecht, NETHERLANDS

The development of pancreatic ductal adenocarcinoma (PDAC) is promoted by its highly abundant tumor stroma. A major component of tumor stroma, stromal myofibroblasts or cancer-associated fibroblasts (CAFs) were previously shown to support PDAC progression by enhancing tumor cell growth, invasion and metastasis. The collagen binding transmembrane receptor integrin α11 (ITGA11) is known to be overexpressed by myofibroblasts. In this study we have for the first time stained ITGA11 in human PDAC specimens. We found that ITGA11 was highly expressed in the stromal myofibroblasts of PDAC patients, as shown by co-localization with the myofibroblast marker alpha smooth muscle actin (α-SMA). Interestingly, there was no expression in healthy human pancreas and various other tissues from human organs. Furthermore, we induced subcutaneous tumors in mice by injecting Panc-1 tumor cells or Panc-1 + pancreatic stellate cells (hPSCs) and found that ITGA11 was significantly overexpressed in stroma-rich Panc-1+hPSC tumors. The quantitative gene and protein expression of ITGA11 in subcutaneous tumors, positively correlated with the expression of the CAF markers α-SMA, Col1a1 and PDGFβR. We used human derived pancreatic stellate cells (hPSCs), precursors of pancreatic CAFs, to study the biological role of ITGA11 in CAF differentiation. Treatment of hPSCs with TGF-β or conditioned medium from Panc-1 epithelial cancer cells resulted in the significant upregulation of ITGA11 and α-SMA, Col1a1 and PDGFβR on gene and protein level. ITGA11 knockdown, mediated by siRNA, significantly inhibited hPSC differentiation and migration potential. In conclusion, this study introduces ITGA11 to be highly and specifically expressed in the tumor stroma of PDAC and explores its role in the phenotype of primary stromal myofibroblasts.

44

Inhibiting the Differentiation of Human Pancreatic Tumor Stromal Myofibroblasts Using Peptide Based Nanocomplexes as Anti-microRNA Oligonucleotide Delivery System

Poster abstracts

Jonas Schnittert2, Praneeth Kuninty2, Tomasz Bystry2, Roland Brock1, Gert Storm3,2, Jai Prakash2

1 Radboud University Medical Center, Nijmegen, NETHERLANDS, 2 University of Twente, Enschede, NETHERLANDS, 3 Utrecht University, Utrecht, NETHERLANDS

Stromal myofibroblasts, also known as cancer-associated fibroblast (CAFs), play a critical role for the tumor promoting mechanisms of the pancreatic tumor microenvironment. The main precursors of CAFs are human derived pancreatic stellate cells (hPSCs), which differentiate into CAFs during carcinogenesis. Their differentiation is controlled by microRNA (miRNA), non-protein-coding single stranded RNA molecules, which regulate mRNA expression. Inhibition of miRNA using anti-miRNA oligonucleotides (AMO) makes them an interesting class of therapeutics to challenge tumor microenvironment. However, efficient and specific delivery of miRNA into hPSCs is a major hurdle to apply them for their therapeutic application. We developed self-assembling Nanocomplexes (~40 nm) to specifically target AMO to hPSCs and inhibit their differentiation into CAFs. We designed a dimeric form of an arginine-rich cell-penetrating peptide (CPP) to form Nanocomplexes with AMO. Dimeric CPP-based Nanocomplexes (NC-2) showed ~130-fold higher uptake by hPSCs compared to monomer-based Nanocomplexes (NC-1) and superior uptake in hPSCs compared to general fibroblasts and different pancreatic tumor cells. The uptake of NC-2 into hPSCs was partly receptor-mediated. Using NC-2 for the delivery of anti-miR-199a significantly inhibited hPSC differentiation into CAFs. In addition, stroma-containing 3D tumor heterospheroids formed with Panc-1 epithelial tumor cells and hPSCs had a smaller size when hPSCs were transfected with anti-miR-199a compared to control anti-miR. Furthermore, treatment of hPSCs with anti-miR199a inhibited hPSC-induced Panc-1 tumor cell growth. In conclusion, our self-assembling Nanocomplexes show a striking structure-activity-relationship when using a dimeric CPP variant and this approach can be a valuable tool for intracellular delivery of miRNA oligonucleotides for developing novel miRNA-based therapeutics.

45

AGR2 is involved in the regulation of the epithelial phenotype by repressing EMT

Lucia Sommerova1, Eva Ondrouskova1, Roman Hrstka1

1 RECAMO, Masaryk Memorial Cancer Institute, Brno, CZECH REPUBLIC

To initiate the metastatic cascade cancer cells frequently undergo the process of epithelial-mesenchymal transition, when the epithelial cells are reprogrammed into mesenchymal-like cells exerting increased migratory potential. Enhanced expression of AGR2 was described in wide range of malignancies and AGR2 protein was shown to regulate several cancer-associated processes necessary for tumor development and progression including cellular proliferation, survival and drug resistance.

We found that induction of EMT by TGF-β was associated with decreased AGR2 expression on both mRNA and protein level. Our data indicate that downregulation of AGR2 expression results from the tight cooperation between SMAD and MAPK signalling cascades, both TGF-β inducible signalling pathway.

Moreover, induction of AGR2 in the cells with mesenchymal phenotype caused the reversion of cellular phenotype from mesenchymal to epithelial-like by the loss of mesenchymal markers and re-acquisition of epithelial one. Additionally, we observed that upregulation of EMT-related transcription factors ZEB1 and SNAI2 corresponds with decreased AGR2 level. Importantly, inhibition of AGR2 expression induced both ZEB1 and SNAI2 and facilitated their nuclear translocation, thus explaining how AGR2 depletion may contribute to the mesenchymal phenotype. Taken together our results highlight a crucial role of AGR2 in maintaining of the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.

The work was supported by the project MEYS – NPS I – LO1413 and GACR 13-00956S.

46

Generation of children-derived orthoxenograft mouse models for drug development and personalized medicine

Aroa Soriano2, Raquel Hladun3, Constantino Sábado3, Luís Gros3, Anna Llort3, Sergio López3, Gabriela Guillén3, Miguel F. Segura2, Josep Roma2, José Sánchez de Toledo3,2, Alberto Villanueva1,4, Soledad Gallego3,2

1 Chemoresistance and Predictive Factors Laboratory, Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology (ICO), Bellvitge Biomedical Research Institute (IDIBELL), Hospitalet de Llobregat, Barcelona, SPAIN, 2 Laboratory of Translational Research in Childhood and Adolescent Cancer,

Poster abstracts

Vall d’Hebron Research Institute (VHIR) - Universitat Autónoma de Barcelona (UAB), Barcelona, SPAIN, 3 Pediatric Oncology and Hematology Department, Hospital Universitari Vall d’Hebron (HUVH), Universitat Autónoma de Barcelona (UAB), Barcelona, SPAIN, 4 Xenopat S.L. Business Bioincubator Bellvitge Health Science Campus, Hospitalet de Llobregat, Barcelona , SPAIN

Paediatric cancer is a rare disease accounting for 1% of all cancers in humans and remains the leading cause of disease-related death in children over the age of 1 year in the western world. Despite the improvement in patient survival during the last 50 years, 20% of all paediatric cancer remains incurable. Paediatric research is severely limited by a lack of disease-relevant preclinical models capable to guide clinical development of paediatric drugs and to generate effective patient tailoring hypothesis. Our main objective is to create a platform based on the generation of children-derived orthotopic xenografts (CHILDOX) for testing new treatments with higher probability of success when translate to patients.

Immediately after surgery or biopsy, several tumor pieces/trucuts were transferred into Revitalazing Tumor Media and implanted into immunocompromised mice directly, without enzimatically digestion, to maintain tridimenstional structure. The small pieces of tumor were implanted in the same organ as the original tumor was. This orthotopic models are more labor-intensive than subcutaneously models but are better to predict patient tumor progression and response to treatment.

With this approach we managed to improve our engraftment rate from 12% in 2015 to 35% in 2016. We established CHILDOX mouse models of Wilms tumors (4), Rhabdomyosarcoma (1), Neuroblastoma (1), Malignant Rhabdoid tumor (1) and one lung metastases from an Osteosarcoma. Finally, we performed personalized medicine in a Rhabdomyosarcoma patient where the CHILDOX mouse model was used to test treatments based on the tumor molecular profile. This approach may be more prone to select better treatments and thus avoid the patients from being exposed to ineffective treatments.

47

Pan-RAF/Src inhibitors have broad in vitro and in vivo activity across gastro-oesophageal cancers with various MAP-kinase pathway activating aberrations

Georgia Spain2, Louise Barber2, Beatrice Griffiths2, Filipa Lopes2, Marta Gomez-Martinez2, Matthew N Davies2, Irina Babina2, Nick C Turner2,3, Adam Stewart2, Udai Banerji2,3, Richard Marais1, David Cunningham3, Caroline Springer2, Marco Gerlinger2,3

1 Cancer Research UK Manchester Institute, Manchester, UK, 2 Institute of Cancer Research, London, UK, 3 Royal Marsden Hospital, London, UK

Gastro-oesphaegeal adenocarcinomas (GOAs) frequently harbour genetic alterations that activate the Mitogen Activated Protein Kinases (MAPK) pathway. ERBB2 positive tumours are routinely treated with trastuzumab but the development of targeted therapies for molecular subgroups with other MAPK-pathway activating alterations has been hindered by frequent co-occurrence of multiple alterations within cancer cells and by intratumour heterogeneity with subclones displaying distinct MAPK activating alterations. This study investigated whether targeting of the MAPK-pathway downstream of these frequently occurring alterations with the pan-RAF/SRC inhibitor (CTC196969) can effectively inhibit GOA cell lines with different genotypes.

GOA cell lines with NRAS (MKN74), MET (MKN45), ERBB2 (NCI N87) or FGFR2 (KATOIII) amplifications and one cell line (FLO1) without known MAPK aberrations were treated with CTC196969 for 5 days. This suppressed cell growth with GI50 values of 30nM in the FGFR2 amplified cell line and of 1.2–1.5 µM in the other cell lines. In order to confirm drug activity in vivo, we established Xenograft mouse models from MKN74, MKN45 and NCI N87. Treatment with 20 mg/kg of CTC196969 halted tumour growth in all three models whereas untreated controls progressed. This was statistically significant (p<0.01).

The potential hypersensitivity of FGFR2 amplified cells to CTC196969 was further investigated in an additional GOA cell line (OCUM-2M) and in patient derived GOA spheroids (FG51). GI50 values of 380 nM and 40 nM, respectively, confirmed hypersensitivity in comparison to NRAS, MET and ERBB2 amplified lines. The molecular basis for this hypersensitivity is currently being investigated by multiplex protein phosphorylation analysis.

Taken together, our data shows in vitro and in vivo response to CTC196969 across a panel of GOA cell lines with various MAPK-pathway activating aberrations. Pan-RAF/Src inhibition could provide a novel and effective therapeutic approach for targeting heterogeneous GOAs, without the need for molecular stratification.

Poster abstracts

48

Paneth-related cells promote immune escape of colorectal cancer

Jasmin Svinka10, Ilija Crncec10, Martin Filipits10, Pornpimol Charoentong8, Markus Tschurtschenthaler2, Lukas Kenner7,6, Elisabeth Glitzner10, Maria Sibilia10, Michael Gnant4, Sigurd Lax3, Josef Thaler1, Mathias Müller5, Birgit Strobl5, Arthur Kaser2, Zlatko Trajanoski8, Gerwin Heller9, Robert Eferl10

1 Department of Internal Medicine IV, Klinikum Wels-Grieskirchen, Wels, AUSTRIA, 2 Department of Medicine, University of Cambridge, Cambridge, UK, 3 Department of Pathology, General Hospital Graz West, Graz, AUSTRIA, 4 Department of Surgery, Breast Health Center, Comprehensive Cancer Center, Medical University Vienna, Vienna, AUSTRIA, 5 Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, AUSTRIA, 6 Institute of Clinical Pathology, Medical University Vienna, Vienna, AUSTRIA, 7 Ludwig Boltzmann Institute for Cancer Research , Vienna, AUSTRIA, 8 Medical University Innsbruck, Biocenter, Division for Bioinformatics, Vienna, AUSTRIA, 9 Medical University Vienna & Comprehensive Cancer Center (CCC), Division of Oncology, Vienna, AUSTRIA, 10 Medical University Vienna & Comprehensive Cancer Center (CCC), Institute of Cancer Research, Vienna, AUSTRIA

-- Abstract withheld from publication at the request of the author --

49

The involvement of AIPL1 and NUB1 proteins in cell cycle regulation in breast cancer

Ka-Liong TAN1, Francesco Pezzella1

1 University of Oxford, Oxford, UK

Background: The interferon-induced NEDD8 ultimate buster (NUB1) protein causes degradation of neddylated and fat10ylated proteins through the ubiquitin proteasome system. Aryl Hydrocarbon Receptor Interacting Protein-Like 1 (AIPL1) interacts with NUB1 and restricts the entry of NUB1 protein into the nucleus. The mechanistic roles of AIPL1 and NUB1 protein in cancer cell cycle regulation remain unexplored.

Results: The meta-analysis of cancer databases revealed that expression transcripts of chaperones, including AIPL1, were downregulated in lung, pancreatic cancer and breast cancer in comparison

to the adjacent normal tissues. METABRIC clinical cohort highlighted that patients with low NUB1 transcripts had poor survival in the ER negative subgroup of breast cancer patients: hazard ratio (HR)=0.66, 95% confidence interval (CI)=0.5-0.87, p=0.003 and triple negative subgroup of breast cancer patients: HR=0.67, 95% CI=0.47-0.96, p=0.028. AIPL1 protein forms multimers in cancer cells. NUB1 protein moved into the nucleus in hypoxia (0.1% O2 48hrs) with final confluency at 80-90%. p21 (marker of senescence) & p27 (marker of cell cycle arrest) accumulated in NUB1-silent HCC1806 cells. It suggests that low NUB1 nuclear localisation in hypoxia cause cancer cell cycle arrest. In MDA-MB-231 cell, upon hypoxia, neddylation inhibitor (MLN4924) treated and siNUB1 transfected cells showed decreased CUL1 and further accumulated p21 & p27. The evidence suggested lower neddylated CUL1 and reduced NUB1 cooperatively stabilise p21 and p27 as the substrate of CUL1-ubiquitin ligase. In tissue microarray (TMA) study of breast cancer, compared with high cytoplasmic expression (n=73), those with low cytoplasmic expression (n=52) had worse overall survival: HR=0.432, 95% CI=0.201-0.663, p=0.002.

Conclusions: Our study investigated the cell cycle regulation role of both AIPL1 and NUB1 proteins in breast cancer. AIPL1 regulates the translocation of NUB1 into nucleus. Cell cycles are G1 phase arrested due to accumulated p21/p27 through inactive CUL1 Ub-E3 ligase.

50

Measuring Penetration and uptake of Nanoparticles in 3D spheroid model using Flow cytometry

Aleksandra Tchoryk2, Richard Argent2, Marianne Ashford1, Paul Gellert1, Snow Stolnik2, Anna Grabowska2, Martin Garnett2

1 AstraZeneca, Macclesfield, UK, 2 University of Nottingham, Nottingham , UK

An increasing number of nanodelivery systems have been reported in the literature as potential carriers of anticancer drugs; however, their penetration into the tumour is frequently affected by the complex physiology of the tumour as well as physicochemical characteristics of the nanosystems leading to their heterogeneous distribution.

In vivo models give good insight into localisation of nanosystems but are much less easy to study for penetration of nanosystems into tumour tissue

Poster abstracts

beyond the vasculature. Therefore, there is a need for a representative in vitro model and method to screen nanomedicines in the early stages of the development process.

HCT116 colorectal cancer spheroids were prepared using ultra-low attachment plates. Incubation of the spheroids with the Hoechst 33342 nuclear dye results in a fluorescent gradient which can be interrogated using FACS after spheroid disaggregation to determine nanoparticles penetration. The Hoechst dye concentration was optimised to allow discrimination between the populations of cells in the core and at the periphery of spheroids based on the degree of staining. The spheroids were incubated with the dye and nanoparticles with different characteristics, and their penetrability and distribution was assessed.

The results demonstrated that the penetration of nanoparticles were strongly influenced by their physical properties and highlight the need to control these features when designing nanomedicines.

Furthermore, the work showed that the 3D spheroid model together with FACS/Hoechst technique provides a promising in vitro screening method for assessing nanoparticles in the early stage of the development process.

51

Comprehensive profiling of signal transduction pathways and Identification of biomarkers from primary ovarian cancer using DigiWest, a novel multiplex protein profiling method

Yvonne Beiter2,4, Johanna Naskou1,4, Fridolin Treindl4, Tanja Fehm1, Christoph Sachse3, Hans Neubauer1, Markus Templin2,4

1 Heinrich-Heine University Düsseldorf, Düsseldorf, GERMANY, 2 NMI at the University of Tübingen, Reutlingen, GERMANY, 3 NMI-TT, Berlin, GERMANY, 4 University of Tübingen, Tübingen, GERMANY

The analysis of cellular signaling cascades is essential for understanding the processes that underlie drug response and drug resistance. Profiling of central signalling cascades requires the detection of protein expression and activation, which is difficult to achieve comprehensively. Our novel DigiWest technology combines the principles of Western blotting with a multiplexed Luminex bead array as a readout system. The system allows the generation of currently up to 600 of Western blot equivalents from a few micrograms of protein sample. Thereby, information on the

expression and modification of hundreds of proteins is obtained with good reproducibility and linearity in combination with a large dynamic range.

Applying the DigiWest method, we analysed 24 fresh frozen tumor specimens from relapsed vs cured ovarian cancer patients directly on the protein level. A total of 466 antibodies per sample were employed to compare activation states of signaling cascades as well as expression levels of tumor and metastasis marker proteins. The results allowed us to cluster relapsed vs cured patients via differential activation states of several signal transduction pathways, such as MAPK and NFkB signaling. Based on this, we extracted a signature of eight proteins and protein modifications whose differential expression was sufficient to clearly distinguish relapsed from cured patients, and which therefore represent promising biomarker candidates.

As such, the results of this study demonstrate the power of multiplex protein profiling in general and of our novel DigiWest in particular for identification of prognostic markers for chemotherapy, through a direct and comprehensive analysis of protein expression and protein activation states.

52

E-cadherin contributes to the proliferation of the ovarian cancer cellular aggregates by modulating integrin-independent FAK activation during the mitotic spindle assembly

Katia Rea1, Francesca Roggiani1, Francesco Raspagliesi1, Silvana Canevari1, Delia Mezzanzanica1, Antonella Tomassetti11 Fondazione IRCCS Istituto Nazionale Dei Tumori, Milan, ITALY

The disruption of E-cadherin-mediated adhesion is considered an important driver of tumor progression but contradictory data also demonstrated the involvement of E-cadherin in promoting growth- or invasion-related signaling. In epithelial ovarian cancers (EOCs), we have previously demonstrated that E-cadherin can exert a pro-tumorigenic role by contributing to PI3K/AKT activation. Recently, we have also described a novel signaling pathway whereby EGF-activated EGFR leads to the activation of CDK5, phosphorylation of FAK at serine 732, thus controlling mitotic spindle assembly. Here, we describe a new growth-promoting mechanism which requires E-cadherin expression in EOC cells. Forty percent of serous high-grade

Poster abstracts

(sHG) EOCs are diagnosed when the patients present peritoneal disseminated tumor masses associated to malignant ascites rich of EOC multicellular aggregates (MCAs). Molecular, immunohistochemical and biochemical analyses on EOC solid biopsies and MCAs, unveiled that E-cadherin was expressed in the majority of the MCAs concomitantly with phosphorylated FAK on serine 732. E-cadherin was indeed necessary for EGFR/CDK5/FAK activation in EOC cells and formed a complex with EGFR. In line with these data, 3D cultures of E-cadherin-expressing EOC cells were sensitive to growth inhibition by the CDK5 inhibitor roscovitine combined with cisplatin. Transient E-cadherin silencing reduced the growth potential of EOC cells as spheres in an Alginate 3-dimensional (3D) system and Matrigel® due to defect of the mitotic spindle formation. Finally, efforts have been devoted to investigate on the molecular mechanisms behind this pro-proliferative role of E-cadherin. In conclusion, the signaling pathways activated in E-cadherin-expressing EOC cells may represent novel targets for more effective therapeutic approach to avoid recurrence in EOC patients who present ascites rich of MCAs. Furthermore, these data represent a significant step towards untangling the role of E-cadherin present in EOC during progression.

Partially supported by Cariplo Foundation and Associazione Italiana per la Ricerca sul Cancro (AIRC).

53

3D Tumor Cultures for Personalised Diagnostics

Sander Basten1, Bram Herpers1, Leo Price1,2, Willemijn Vader2

1 OcellO B.V., Leiden, NETHERLANDS, 2 VitroScan B.V., Leiden, NETHERLANDS

Background: Genetic approaches to personalised medicine have not yet resulted in a broadly applicable method to tailor treatment for the individual patient. Drug sensitivity testing on 3D cultures of patient tumour biopsies have the potential to directly predict drug responsiveness in patients and complement genetics based approaches to support treatment decisions for the individual patient.

Our method utilises a unique automated 3D cell culture and high content screening platform which enables the sensitivity to available drugs to be determined within 2-4 weeks. Measurements of cell and tissue morphology following drug exposure report responses such as tumour cell killing, growth arrest and local invasion. We have used this approach to test standard-of-care compounds

on patient and PDX-derived tumour material from bladder, gastric, breast, colon, ovarian, cervix and lung cancer tumour tissues.

Methods and Results: After a brief recovery period, fresh and cryopreserved tumour tissues are dissociated and cultured in extracellular matrix-rich hydrogels in a 384-well format. Cultures are exposed to different combinations and doses of chemotherapeutics, small molecules, antibodies etc. After 3D imaging and analysis, morphological features are selected that describe the response to each treatment. Culture, compound exposure, imaging and analysis are all automated at the multi-384 well plate level. We present results of our PoC experiments showing in vitro drug responsiveness from cultures of fresh and cryopreserved tumour material.

Conclusion: Responsiveness to standard-of-care agents was determined in 3D cultures of biopsies and resections from several tumour types. This approach has the potential to provide predictive information of therapeutic responses in patients to guide the treating physician and patient in treatment decisions. In addition, it offers an opportunity to integrate genomics data and drug sensitivity data to identify markers that correlate with responses across a broad range of drugs and push forward personalised medicine for cancer patients.

54

Lipid nanoparticles for tumor-targeted combination treatment of prostate cancer

Roy van der Meel1,3, Sam Chen1,4, Josh Zaifman1,2, Joslyn Quick1, Raymond M. Schiffelers3, Marco A. Ciufolini2, Yuen Yi C. Tam1,4, Pieter R. Cullis1

1 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, CANADA, 2 Department of Chemistry, University of British Columbia, Vancouver, British Columbia, CANADA, 3 Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, NETHERLANDS, 4 Integrated Nanotherapeutics, Vancouver, British Columbia, CANADA

Current treatment strategies for advanced prostate cancer include androgen receptor (AR) pathway inhibition and taxane-based chemotherapy. However, the effectiveness of chemotherapy is hampered by dose-limiting adverse effects and the vast majority of tumors develop resistance mechanisms against AR inhibitors and taxane drugs. Lipid nanoparticles (LNPs) are the most clinically advanced delivery systems for chemotherapeutics and genetic

Poster abstracts

drugs such as siRNA. Long-circulating LNPs accumulate in tumors to a higher extent compared to free drugs, resulting in increased therapeutic efficacy and reduced adverse effects. We have recently developed new technology that allows the incorporation of virtually any small molecule in LNPs, raising opportunities to combine chemotherapy and gene silencing.

LNPs containing both taxane chemotherapeutics and siRNA against constitutively active AR variants (AR-V) were formulated by microfluidic rapid mixing methods. LNPs were characterized by physicochemical analysis including size and drug content. LNP stability in serum was determined by UPLC. Gene silencing efficiency of LNPs was determined by siRNA target knockdown using qPCR. Therapeutic efficacy of LNPs was determined by cell viability assays in PC cell lines expressing AR (variants) including 22Rv1, LNCaP and VCaP while the AR-negative cell line PC3 was used as control.

Physicochemical analysis indicated LNP size of 60 nm and >90% siRNA encapsulation efficiency. The incorporation of taxane chemotherapeutics was varied from 1-10 mol% without effecting the stability of the formulation in serum. LNPs containing siRNA with and without taxane drug induced >80% knockdown of AR-V in 22Rv1 cells. LNPs containing both AR-V siRNA and taxane chemotherapeutics induced greater inhibition of cell viability when compared to control formulations in 22Rv1, LNCaP and VCaP cells while no difference was observed in AR-negative PC3 cells.

LNPs containing both siRNA and chemotherapeutics are an attractive strategy for the development of effective combination treatments for advanced prostate cancer.

55

Transient tissue priming via ROCK inhibition uncouples cancer progression, sensitivity to chemotherapy and the onset of the metastatic niche in stratified patient-derived models of pancreatic cancer

Claire Vennin17,16, Venessa Chin17,16, Sean Warren17,16, Morghan Lucas17,16, David Herrmann17,16, Astrid Magenau17,16, Pauline Melenec17,16, Stacey Walters17,16, Gonzalo Del Monte-Nieto10,16, James Conway17,16, Max Nobis17,16, Rachael McCloy17,16, Richard Harvey10, Andrew Biankin1,4, Shane Grey17,16, Christopher Ormandy17,16, David Gallego-Ortega17,16, Yingxiao Wang6, Michael Samuel5, Owen Sansom4, Andrew

Burgess17,16, Thomas Cox17,16, Jennifer Morton17,16, Marina Pajic17,16

1 Australian Pancreatic Cancer Genome Initiative, Sydney, NSW, AUSTRALIA, 2 Biomedical Imaging Facility (BMIF) - Mark Wainwright Analytical Centre, Lowy Cancer Research Center, UNSW, Sydney, NSW, AUSTRALIA, 3 Cancer Diagnosis and Pathology Research Group, Kolling Institute of Medical Research and Royal North Shore Hospital, Sydney, NSW, AUSTRALIA, 4 Cancer Research UK Beatson Institute, Glasgow, UK, 5 Centre for Cancer Biology, SA Pathology & The University of South Australia and School of Medicine, University of Adelaide, Adelaide, AUSTRALIA, 6 Department of Bioengineering, Institute of Engineering in Medicine, University of California San Diego, USA, 7 Department of Dermatology Royal Prince Alfred Hospital, Camperdown, Sydney, AUSTRALIA, 8 Department of Pathology, St Vincent’s Hospital, Sydney, AUSTRALIA, 9 Department of Surgery, Royal North Shore Hospital, Sydney, NSW, AUSTRALIA, 10 Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Sydney, AUSTRALIA, 11 Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, AUSTRALIA, 12 Immune Imaging Program, The Centenary Institute, University of Sydney, NSW, AUSTRALIA, 13 Macarthur Cancer Therapy Centre, Campbelltown Hospital, Sydney, NSW, AUSTRALIA, 14 School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, AUSTRALIA, 15 School of Medicine, Western Sydney University, Sydney, NSW, AUSTRALIA, 16 St Vincent’s Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, AUSTRALIA, 17 The Garvan Institute of Medical Research & The Kinghorn Cancer Centre, Sydney, NSW, AUSTRALIA

The emerging standard-of-care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, however patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings with reciprocal feedback from micro-environmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature prior to chemotherapy can impact on tumor response. Here, we used intravital imaging to assess how transient manipulation of the tumor tissue, or ‘priming’, using the pharmaceutical Rho-kinase inhibitor Fasudil affects response to chemotherapy. Intravital FRET imaging of a CDK1 biosensor to monitor cytotoxic drugs efficacy revealed that priming improves pancreatic cancer response

Poster abstracts

to Gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress, impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we engineered an automated tool for stratification of patient samples based on an extracellular matrix signature. We demonstrate a graded response to priming in stratified patient-derived settings, highlighting that tailored fine-tuned tissue manipulation prior to chemotherapy may offer new opportunities in both primary and metastatic targeting of pancreatic cancer.

56

Enabling gene profiling in microfluidic models for precision medicine

María Virumbrales-Munoz3,1, Jose Ayuso2,4, Alodia Lacueva3,1, Sara Olivan3,1, Manuel Doblare3,1, Ignacio Ochoa3,1, Luis Fernandez3,1

1 Aragón Institute of Engineering Research (I3A), University of Zaragoza, Zaragoza, SPAIN, 2 Department of Biomedical Engineering, Wisconsin Institutes for Medical Research, University of Wisconsin, Madison, USA, 3 Group of Applied Mechanics and Bioengineering (AMB), Centro Investigacion Biomedica en Red. Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN), Zaragoza, SPAIN, 4 Medical Engineering, Morgridge Institute for Research, Madison, WI, USA

Compelling evidence over the years has demonstrated that the tumor microenvironment (TME) shapes tumor initiation, development and response to therapy. This results in a high heterogeneity within the same cancer type, and hinders the process of finding effective treatments.

In this context, microfluidics has proven a worthy sum of techniques to create comprehensive and personalized cancer in vitro 3D models reproducing the TME in a more relevant fashion than traditional in vitro setups.

Microfluidics also presents additional benefits for precision medicine, since it permits a high degree of control over the setup, combining different cell types in an orderly manner, as well as different physical and biochemical cues. Furthermore, microfluidics facilitates optical inspection and diminishes sample sizes and reagent volumes needed for each experiment. Microfluidic devices are also compatible with high-throughput approaches, which make them an interesting option for drug testing, research and development.

Hence, we developed a microfluidic tumor-slice model, which we used to model and characterize different tumor microenvironments. TME was characterized in terms of hypoxia, proliferation rates, reactive oxygen species concentration, apoptosis rate and glucose uptake. Furthermore, we carried out pharmacodynamic and drug efficiency studies in these newly-established models. Thereafter, we developed a simple enzymatic protocol to extract cells seeded in 3D from the microfluidic devices. Cells could be sorted by flow cytometry according to the expression of specific surface markers or by using different fluorescent stains. RNA was extracted for downstream quantification and gene profiling was carried out for the mentioned aspects of the tumor microenvironment.

All in all, we developed a comprehensive microfluidic model for personalized medicine capable of comprehensive reproduction of the TME, which allows characterization of tumor signatures by means of traditional benchtop methods.

57

Inhibiting glycolysis reprograms mitochondrial metabolism to fuel breast cancer cell migration

Xiaobing Zhang1, Bob van de Water1, Sylvia Le Dévédec1

1 Toxicology, LACDR, Leiden University, Leiden, NETHERLANDS

Enhanced migration of tumor cells is a critical hallmark of cancer progression. Metabolic reprogramming including the so-called “Warburg effect” is another cancer hallmark. The metabolic alterations that control cancer progression could be exploited to develop therapeutic approaches. Breast cancer (BC) is the second cause of death of women worldwide. Approximately 15-20% of breast cancers are triple negative (TNBC). Although the incidence of this BC subtype is low, TNBC has a higher rate of reoccurrence and worst outcomes. TNBC lacks expression of hormone receptors and is also described to be highly glycolytic. Targeting enhanced glycolysis in TNBC seems to be a rational therapeutic choice. A broadly used and accepted inhibitor of glycolysis is 2-deoxyglucose (2-DG) which has already been tested in animal models and in humans (e.g. clinical trial NCT00096707/ solid tumors of various cancer types). However the application of 2-DG only as an anti-cancer agent in vivo is so far inconclusive. In the caser of TNBC, our data suggest two possible explanations: i) long term effect of 2DG is

Poster abstracts

TNBC subtype dependent, and ii) 2DG treatment results in a metabolic adaption of the tumor cells resulting in emergence of resistance for tumor cell growth and switch in a more migratory phenotype in vitro and potentially also in vivo. Briefly, our preliminary results show that long term inhibition of glycolysis in TNBC cells impairs significantly cell proliferation without onset of apoptosis. In addition, those non-proliferative TNBC cells display a more mesenchymal phenotype with sustained high random cell motility. Our analysis of the cAMP-PGC1-α pathway that governs mitochondrial functions reveals that the observed metabolic adaptation to a migratory phenotype alone seems to be responsible for the metabolic adaptation upon 2DG exposure. Therefore mitochondrial respiration adaptively induced by inhibition of glycolysis can fuels tumor cell migration as efficiently as glycolysis and can also be a potential antimetastatic target.

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Tuesday 14 March 2017 13.45 – 14.15Dr Raimo Tanzi, Menarini Silicon Biosystems Chief Commercial Officer“DEPArray(TM) Copy Number Aberration CTC workflow: the future of tumor stratification by liquid biopsy”

Satellite Symposia

We are pleased to announce that Bio-Rad and Menarini Silicon Bioscience will not only be exhibiting at the conference but also inviting participants to join a Satellite Symposium. The Satellite Symposia give participants the opportunity to take part in an additional scientific session.

Wednesday 15 March 2017 12.30 – 13.00 Stephen Hague PhD, Droplet Digital PCR Specialist Europe“ddPCR Provides the Level of Sensitivity Required for Liquid Biopsy”

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