symposium advances cancer immunology immunotherapy...most young researchers that will participate in...

h t t p : / / h e l l e n i c - i m m u n o o n c o l o g y . g r / Organized by Hellenic Society of Immuno-Oncology

November 29 - December 1 . 2018 Royal Olympic Hotel Athens | Greece

Symposium Advances in

Cancer Immunology andImmunotherapy

Under the Auspices of

S y m p o s i u m A d v a n c e s i n C a n c e r I m m u n o l o g y a n d I m m u n o t h e ra p y S y m p o s i u m A d v a n c e s i n C a n c e r I m m u n o l o g y a n d I m m u n o t h e ra p y

Organizing CommitteeAthanasios KotsakisOurania Tsitsilonis

Constantin N. BaxevanisNikolaos Kentepozidis

Scientific CommitteeG. J. Adema (NL)

A. Argiris (GR)P. Bagratuni (GR)

C.N. Baxevanis (GR)T.H. Borch (DK)A. Boutis (GR)V. Bronte (IT)F. Cavallo (IT)

A. Eliopoulos (GR)W.H. Fridman (FR)V. Georgoulias (GR)

N. Giannakoulas (GR)C. Gouttefangeas (DE)

D. Heymann (FR)N. Karin (IL)

T. Katsila (GR)N. Kentepozidis (GR)

D. Kletsas (GR)A. Kotsakis (GR)

M. Koukourakis (GR)A. Koumarianou (GR)

E. Lavelle (IE)C. Le Tourneau (FR)

M. Liontos (GR)O. Mandelboim (IL)

M. Neagu (RO)R. Offringa (DE)

N. Pistamaltzian (GR)B. Seliger (DE)

O. Tsitsilonis (GR)V. Umansky (DE)P. Verginis (GR)

T.L. Whiteside (US)

Dear Colleagues,

We are delighted to welcome you to the 4th Symposium: Advances in Cancer Immunology and Immu-

notherapy, that is being held on November 29 - December 1, 2018, at the Royal Olympic Hotel, Athens,

Greece.

The meeting aims to bring together many of the leading researchers and clinicians in Europe and the US

to report on most recent advances made in cancer immunology and immunotherapy. Our main objectives

is to gather, share and exchange experiences and ideas; translate and extend this knowledge to the clinic;

promote interactions between speakers and participants and encourage stimulating discussions; provide

network opportunities and stimulate new collaborations. This year, we have introduced a new element, as

most young researchers that will participate in the poster sessions will be additionally given the opportunity

to present the highlights of their work as a short oral presentation (8 minutes each). The scientific committee

will select the three best presentations for awards.

On behalf of the Organizing and Scientific Committees, we are very pleased to have you amongst us.

We hope to enjoy the symposium,

The Organizing Committee Chairs

Athanasios Kotsakis, MD, PhD,Associate Professor of Medical Oncology,

Director of Dpt of Medical Oncology, University Hospital of Larisa,

Thessaly, Greece

Ourania Tsitsilonis, MD, PhD,Associate Professor of Immunology,

Department of Biology,National & Kapodistrian

University of Athens, Greece

Nikolaos Kentepozidis, MD, PhD,Medical Oncologist,

Head of Oncology Clinic,251 General Airforce Hospital,

Athens, Greece

Constantin N. Baxevanis, PhD,Scientific Director,

Cancer Immunology and Immunotherapy Center “Agios Savvas”

Hospital, Athens, Greece

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InfoO r g a n i z e d b y Hellenic Society of Immuno-Oncology (HeSIO) G. Theologou 5, Τ.Κ.11474,Athens,Greece www.hellenic-immunooncology.gr

U n d e r t h e A u s p i c e s o f

D a t e s November 29th-December1st.2018

W e b s i t e http://hellenic-immunooncology.eu

S y m p o s i u m V e n u e RoyalOlympicHotel,Athens28-34,AthanasiouDiakouStr.,11743,Athens,GREECETel.:+3021092.88.400Fax:+3021092.33.317E-mail:[email protected]

C e r t i f i c a t e o f A t t e n d a n c e BasedonthelatestcircularoftheNationalDrugOrganizationtheSymposiumisrequiredtouseanatten-dancetrackingsystem.Allregisteredparticipantswillreceivenamebadges,whichtheyarekindlyrequestedtowearatalltimes.Eachbadgewillhaveabarcodeformonitoringthehoursofattendance.Bytheendofthesymposiumacertificatewillbegiventothosewhohaveattendedatleast60%ofthetotalhoursoftheScientificProgram.

T h e e v e n t h a s b e e n a w a r d e d b y PanhellenicMedicalAssociation(PIS)with20CME-CPDcredits

O f f i c i a l L a n g u a g e EnglishistheofficiallanguageoftheSymposium

S y m p o s i u m S e c r e t a r i a t

Sc ien t i f i c Ι Cu l tura l Events and Pub l i ca t ions [email protected]

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Thursday,November29th,201814:45-15:00 Welcome

SESSION 1: Overcoming immune suppression Chairs: T. Whiteside (USA), O. Mandelboim (IL)

15:00-15:25 The role of myeloid-derived suppressor cells in tumor immune evasion V. Bronte (IT) 15:25-15:30 Discussion 15:30-15:55 Immune suppression in melanoma V. Umansky (DE) 15:55-16:00 Discussion 16:00-16:25 Interplay between cellular senescence and cancer D. Kletsas (GR) 16:25-16:30 Discussion

Plenary lecture Chairs: F. Cavallo (IT), P. Verginis (GR)

16:30-17:10 Tregs T. Whiteside (USA) 17:10-17:15 Discussion

17:15-17:30 Co f fee Break

SESSION 2: The role of innate immunity in cancer Chairs: V. Umansky (DE), O. Tsitsilonis (GR)

17:30-17:55 The regulatory role of chemokines in cancer progression N. Karin (IL) 17:55-18:00 Discussion 18:00-18:25 Toll-like receptors T. Bagratuni (GR) 18:25-18:30 Discussion 18:30-18:55 Regulation of tumor metastases by NK cells O. Mandelboim (IL) 18:55-1900 Discussion

Plenary lecture Chairs: G. Adema (NL), C.N. Baxevanis (GR)

19:00-19:40 Cancer stem cells in breast carcinomas F. Cavallo (IT) 19:40-19:45 Discussion

19:45 Welcome reception

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Friday,November30th,2018 SESSION 3: Monitoring of immunomodulatory mechanisms Chairs: B. Seliger (DE), O. Tsitsilonis (GR)

09:30-09:55 The emerging role of exosomes T.L. Whiteside (USA) 09:55-10:00 Discussion 10:00-10:25 Proteomics-proteogenomics T. Katsila (GR) 10:25-10:30 Discussion 10:30-10:55 Lipid metabolism and anticancer immunity G. Adema (NL) 10:55-11:00 Discussion 11:00-11:25 Immune-related adverse events during immunotherapy: the role of Tregs P. Verginis (GR) 11:25-11:30 Discussion

Plenary lecture Chairs: C.N. Baxevanis (GR), H.W. Fridman (FR)

11:30-12:10 The immunomodulatory role of microRNAs during the process of tumor immune evasion B. Seliger (DE) 12:10-12:15 Discussion

12:15-12:30 Co f fee b reak

12:30-14:10 Short oral presentations-research Chairs: O. Tsitsilonis (GR), V. Georgoulias (GR)

Ο1-1. IBTK SUPPORTS MYC-DRIVEN LYMPHOMAGENESIS IN MICE Eleonora Vecchio1, Gaetanina Golino1, Francesco Albano1, Antonio Pisano1, Enrico Iaccino1, Selena Mimmi1, Giuseppe Fiume1, Giuseppe Scala1, Ileana Quinto1

1University of Catanzaro, Experimental and Clinical Medicine, Catanzaro, Italy Ο1-2. WNT1 SILENCES CC/CXC MOTIF CHEMOKINE GENES IN DENDRITIC CELLS AND INDUCES ADAPTIVE IMMUNE RESISTANCE IN LUNG ADENOCARCINOMA Dimitra Kerdidani1,2, Panagiotis Chouvardas1,3, Ares Rocanin Arjo4, Mary Tsikitis5, George Kazamias6, Konstantinos Potaris7, Spyros Zakynthinos2, Ioannis Kalomenidis2, Vassili Soumelis4, George Kollias1,3 and Maria Tsoumakidou1

1Division of Immunology, Biomedical Sciences Research Center Alexander Fleming, Vari-Athens, Greece 21st Department of Critical Care and Pulmonary Medicine, Medical School, National and Kapodistrian University of Athens, Greece 3Department of Physiology, Medical School, National and Kapodistrian University of Athens, Greece 4Integrative Biology of Human Dendritic Cells and T Cells, Institute Curie, Paris, France 5Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Greece 6Department of Histopathology, Evaggelismos General Hospital, Athens, Greece 7Department of Thoracic Surgery, Sotiria General Hospital, Athens, Greece Ο1-3. AN IN VITRO T-CELL PRIMING ASSAY TO SELECT EPITOPES FOR THE DESIGN OF TAILORED THERAPEUTIC HEPATITIS C VIRUS VACCINES G. Koutsoumpli, N. Stasiukonyte and T. Daemen Department of Medical Microbiology, Tumor Virology and Cancer Immunotherapy, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands Ο1-4. SERUM MIRNA-BASED DISTINCT CLUSTERS DEFINE THREE GROUPS OF BREAST CANCER PATIENTS WITH DIFFERENT CLINICOPATHOLOGICAL AND IMMUNE CHARACTERISTICS Sotirios P. Fortis1, Christoforos K. Vaxevanis1, Louisa G. Mahaira1, Michael Sofopoulos2, Nectaria N. Sotiriadou2, Amalia Dinou3, Niki Arnogiannaki2, Catherine Stavropoulos-Giokas3, Dimitris Thanos4, Constantin N. Baxevanis1, Sonia A. Perez1

1 Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, Athens, Greece, 2 Pathology Department, Saint Savas Cancer Hospital, Athens, Greece, 3 Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, Greece, 4 Biomedical Research Foundation, Academy of Athens, Athens, Greece

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Ο1-5. PROGNOSTIC SIGNIFICANCE OF HLA-A*02AND HLA-A*24 INPROSTATE CANCER Savvas Stokidis1, Sotirios P. Fortis1, Amalia Dinou2, Marina I. Konstantellou1, Catherine Stavropoulos-Giokas2, Sonia A. Perez1, Constantin N. Baxevanis1

1 Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, Athens, Greece, 2 Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, Greece Ο1-6. A CELL INTRINSIC ROLE OF IL33 IN THE FUNCTIONAL STABILITY OF TREGS IN THE TUMOR MICROENVIRONMENT Aikaterini Hatzioannou1, Aggelos Banos1, Theodore Sakelaropoulos2, Constantinos Fedonidis3, Maria-Sophia Vidali4, Panagiotis Georgiadis4, Anna Henriques5, Vassiliki Koliaraki5, Aristotelis Tsirigos6, Panayiotis Verginis1

1Center of Clinical, Experimental Surgery & Translational Research, Biomedical Research Foundation Academy of Athens, Greece, 2School of Mechanical Engineering, National Technical University of Athens, Greece, 3Center of Basic Research, Biomedical Research Foundation Academy of Athens, Greece, 4Institute of Biology, Medical Chemistry & Biotechnology, National Hellenic Research Foundation, Greece, 5Department of Immunology, Biomedical Sciences Research Centre“Alexander Fleming” Vari, Greece, 6Applied Bioinformatics Laboratories, New York University School of Medicine, New York, NY, USA and Department of Pathology, New York University School of Medicine, New York, NY, USA Ο1-7. DOWN-REGULATION OF PD-L1 IN COLON CANCER CELLS SURVIVING AFTER IRRADIATION Erasmia Xanthopoulou1, Ioannis M. Koukourakis2, Achilleas Mitrakas1, Christos Kakouratos1, Alexandra Giatromanolaki3 and Michael I. Koukourakis1

1Department of Radiotherapy/Oncology, Radiobiology/Radiopathology Unit, Democritus University of Thrace, Alexandroupolis, Greece, 2National and Kapodistrian University of Athens, Greece, 3Department of Pathology, Democritus University of Thrace, Alexandroupolis, Greece Ο1-8. N-BROMOTAURINE AND STABLE DERIVATIVE OF N-BROMOTAURINE EXHIBIT THERAPEUTIC EFFECT AGAINST VARIOUS CANCERS Stella Baliou1**, Markus Nagl2, Antony M. Kyriakopoulos3 and Vassilis Zoumpourlis1* 1 Biomedical Applications Unit, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 2 Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, 3 NASCO AD Biotechnology Laboratory, Pireas, Greece Ο1-9. OVEREXPRESSION OF MIR-194 IN EPITHELIAL OVARIAN CARCINOMASIS AN INDEPENDENT MOLECULAR MARKER FOR PATIENTS’POOR TREATMENT RESPONSE ANDSURVIVAL OUTCOME Konstantina Panoutsopoulou1, Margaritis Avgeris1, Viktor Magdolen2, Andreas Scorilas1* 1 Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece, 2 Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany Ο1-10. EVALUATION OF THE LEVELS AND FUCTIONALITY OF REGULATORY T CELLS IN MULTIPLE MYELOMA Andreas Metousis1, Panagiotis Vitsos1*, Panagiotis Pothos1,2*, Ioannis Kostopoulos1, Efstathios Kastritis3, George Thyfronitis2, Nikolaos Orologas4, Evangelos Terpos3, Meletios-Athanasios Dimopoulos3, Ourania Tsitsilonis1

1Department of Biology, National and Kapodistrian University of Athens, Athens; 2Department of Biological Applications and Technology, University of Ioannina, Ioannina; 3Department of Clinical Therapeutics, “Alexandra” Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens; 4SafeBlood BioAnalytica SA, Athens, Greece (*equal contribution) Ο1-11. IDENTIFICATION OF NEW ALTERNATIVE SPLICING EVENTS RESULTING IN NOVEL SPLICE VARIANTS OF THE HUMAN L-DOPA DECARBOXYLASE (DDC) GENE IN CANCER CELLS, USING NEXT-GENERATION SEQUENCING TECHNOLOGY Michaela A. Boti, Maria Papatsirou, Panagiotis Tsiakanikas, Panagiotis G. Adamopoulos, Andreas Scorilas, Dido Vassilacopoulou, Christos K. Kontos Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece Ο1-12. ALTERNATIVE 3’-UNTRANSLATED REGIONS (3’-UTRS) IN MESSENGER RNAs OF KALLIKREIN-RELATED PEPTIDASE (KLK) FAMILY MEMBERS RESULT FROM ALTERNATIVE SPLICING OF KLK pre-mRNAs, AS REVEALED BY NEXT-GENERATION SEQUENCING Maria Morrou, Paraskevi Karousi, Panagiotis G. Adamopoulos, Panagiotis Tsiakanikas, Andreas Scorilas, Christos K. Kontos Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece Ο1-13. tRNA-DERIVED RNA FRAGMENTS (tRFs) CONSTITUTE A NOVEL CLASS OF MOLECULAR BIOMARKERS IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA: THE PARADIGM OF A NOVEL tRF PREDICTING PATIENTS’ OVERALL SURVIVAL Katerina Katsaraki1, Styliani Karagiorgou1, Panagiotis Tsiakanikas1, Panagiotis G. Adamopoulos1, Sotirios G. Papageorgiou2, Vassiliki Pappa2, Christos K. Kontos1

1Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece; 2Second Department of Internal Medicine and Research Unit, University General Hospital “Attikon”, Athens, Greece

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14:10-15:00 Lunch

SESSION 4: Special session on combinational treatment (I) Chairs: A. Kotsakis (GR), C.N. Baxevanis (GR)

15:00-15:25 Targeting strategies to enhance the effectiveness of immunotherapies E. Lavelle (IE) 15:25-15:30 Discussion 15:30-15:55 Interplay of radiation and immunotherapy M. Koukourakis (GR) 15:55-16:00 Discussion 16:00-16:25 Combination of anti-PD1/PDL1 with newer immunomodulatory agents (newer ICI, IDO, TGF-b CD40 agonists, IL-15...) C. Schultes (GR) 16:25-16:30 Discussion 16:30-16:55 Novel immune check-point inhibitors and combinations in genitourinary malignancies* R.L. Molife (UK) 16:55-17:00 Discussion *sponsored by


SESSION 5: Special session on combinational treatment (II) Chairs: A. Kotsakis (GR), E. Lavelle (IE)

17:15-17:40 ICI and targeted therapies M. Fernández (ES) 17:40-17:45 Discussion 17:45-18:10 Immunotherapy in earlier stages M. Liontos (GR) 18:10-18:15 Discussion 18:15-18:40 Adoptive cell therapy in solid tumors T.H. Borch (DK) 18:40-18:45 Discussion

Plenary lecture Chairs: V. Georgoulias (GR), D. Thanos (GR)

18:45-19:25 Precision medicine in cancer C. Le Tourneau (FR) 19:25-19:30 Discussion


Saturday,December1st,2018 SESSION 6: Biomarkers for Immunotherapy Chairs: A. Koutsopoulos (GR), N. Kentepozidis (GR)

09:30-09:55 Advances on biomarkers for immunotherapy beyond PD-L1 expression* A. Boutis (GR) 09:55-10:00 Discussion *sponsored by 10:00-10:25 Activated integrins reveal functional antigen-specific T cells C. Gouttefangeas (DE) 10:25-10:30 Discussion 10:30-10:55 Tumor-associated and non tumor associated microbiota A. Eliopoulos (GR) 10:55-11:00 Discussion


Plenary lecture Chairs: P. Foukas (GR), R. Offringa (DE)

11:30-12:10 Immune contexture as a prognostic biomarker H.W. Fridman (FR) 12:10-12:15 Discussion

12:15-13:30 Short oral presentations-clinical Chairs: O. Tsitsilonis (GR), E. Stratikos (GR)

Ο2-1. REDUCTION OF PD-L1 POSITIVE CIRCULATING TUMOR CELLS (CTCs) IN NON-SMALL CELL LUNG CANCER (NSCLC) PATIENTS, TREATED WITH NIVOLUMAB G. Kallergi1,2, E.K. Vetsika3, D. Aggouraki4, Koutsopoulos5, E. Lagoudaki5, C. Stournaras2, V. Georgoulias1, A. Kotsakis3

1 Laboratory of Τumor Cell Biology, School of Medicine, University of Crete, Heraklion, Greece, 2 Department of Biochemistry, Medical School, University of Crete, Heraklion, Crerte, Greece, 3 Medical School, University of Thessaly, Larissa, Greece, 4 Laboratory of Τranslational Oncology, School of Medicine, University of Crete, Heraklion Greece, 5 Department of pathology, Medical School, University of Crete, Greece

Ο2-2. CHARACTERIZATION OF POLYCLONAL ANTIBODIES AGAINST DIFFERENT ENVELOPE PROTEINS OF HUMAN ENDOGENOUS RETROVIRUSES IN OVARIAN CANCER Sahitya Saka1, David Díaz-Carballo1, Ali Haydar Acikelli1, Jacqueline Klein1, Gunther Wennemuth2, Andrea Tannapfel3 and Dirk Strumberg1

1 Institute for Molecular Oncology and Experimental Therapeutics. Marien Hospital Herne. Ruhr University Bochum, Medical School. Herne, Germany 2 Institute of Anatomy. University of Duisburg-Essen, Medical School. Essen, Germany 3 Institute of Pathology.Ruhr University Bochum, Medical School. Herne, Germany Ο2-3. RADIATION-ENHANCED COMPLEMENT ACTIVATION AND DOWNSTREAM C5a-C5aR1 SIGNALING MODULATE TUMORIGENIC AND PROINVASIVE RESPONSES OF GLIOBLASTOMA CELLS Stella Tzelefa1, Antigoni Stavridou1, Ioannis Kostopoulos2, Maria Georgoutsou-Spyridonos1, Georgia Terzoudi1, Georgia Sfyroera1, Ourania Tsitsilonis2, John D. Lambris3 and Dimitrios C. Mastellos1 1Division of Biodiagnostic Sciences and Technologies, INRASTES, National Center for Scientific Research ‘Demokritos’, Athens, Greece, 2Section of Animal and Human Physiology, National and Kapodistrian University of Athens, Athens, Greece, 3Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Ο2-4. JOINT EVALUATIONOF CYTOGENETICS AND IMMUNOPHENOTYPING MAY ASSIST THE PROGNOSISOF MULTIPLE MYELOMA PATIENTS Sylia Georgaki1, Paraskevi K. Micheli1, Ioannis V. Kostopoulos1, Efstathios Kastritis2, Evangelos Terpos2, Ourania E. Tsitsilonis1, Meletios A. Dimopoulos2

1Section of Animal and Human Physiology, Department of Biology & 2Department of Clinical Therapeutics, “Alexandra” Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece Ο2-5. INTESTINAL PSEUDO-OBSTRUCTION PRESENTED IN A PATIENT WITH LUNG CANCER UNDER IMMUNOTHERAPY WITH CHECK POINT INHIBITORS Dimitrios Massaras1, Georgios Fragulidis1, Eirini Pantiora1, Vasiliki Michalaki2, Elias Primetis3, Andreas Polydorou1, Georgios Polymeneas1

1 2nd Dept. of Surgery, ‘‘Aretaieio’’ Hospital, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece, 2 Dept. of Oncology, ‘‘Aretaieio’’ Hospital, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece,

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3 1st Dept. of Radiology, ‘‘Aretaieio’’ Hospital, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece O2-6. TUMOR INFILTRATING LYMPHOCYTES (TILs) IN BREAST CANCER: CORRELATIONS WITH MOLECULAR PROFILE AND GRADE Chryssoula Glava1, Argiro Mellou1, Loukia Psaridi1, Eleni Pigadioti1, Iriana Tsiakalou1, Sofokles Lanitis2, Vasilios Samaras1

1 Department of Pathology, “Korgialeneio - Benakeio”, Red Cross Hospital, Athens, Greece, 2 2nd Surgical Department and Unit of Surgical Oncology, “Korgialeneio - Benakeio”, Red Cross Hospital, Athens, Greece Ο2-7. TUMOR INFILTRATING LYMPHOCYTES (TILs) IN BLADDER CANCER: CORRELATIONS WITH RECURRENT DISEASE AND COMPARISON WITH BREAST CANCER TILs. Chryssoula Glava1, Loukis Psaridi1, Dimitrios Lepidas2, Adamantia Zizi - Serbetzoglou3

1 Department of Pathology, “Korgialeneio - Benakeio”, Red Cross Hospital, Athens, Greece, 2 Department of Urology, “Tzaneion” General Hospital of Pireaus, Greece, 3 Department of Pathology, “Tzaneion” General Hospital of Pireaus, Greece Ο2-8. A NOVEL tRNA FRAGMENT AS A MOLECULAR BIOMARKER OF POOR PROGNOSIS IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA Paraskevi Karousi1, Evdoxia Kamouza1, Marios A. Diamantopoulos1, Panagiotis Tsiakanikas1, Christos K. Kontos1, Sotirios G. Papageorgiou2, Andreas Scorilas1, Vassiliki Pappa2

1Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece; 2Second Department of Internal Medicine and Research Unit, University General Hospital “Attikon”, Athens, Greece Ο2-9. MicroRNA-15a, A NOVEL MOLECULAR BIOMARKER IN COLORECTAL CANCER, PREDICTING PATIENTS’ RELAPSE Panagiotis Tsiakanikas, Christos K. Kontos, Margaritis Avgeris, Marios A. Diamantopoulos, Andreas Scorilas Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece

13:30-14:45 Lunch b reak

SESSION 7: Immunotherapy (II) Chairs: E. Saloustros (GR), A. Koumarianou (GR)

14:45-15:10 Therapeutic advances of immunotherapy in the field of mesenchymal tumors D. Heymann (UK) 15:10-15:15 Discussion 15:15-15:40 Immunotherapy in bladder cancer* N. Pistamaltzian (GR) 15:40-15:45 Discussion *sponsored by 5:45-16:10 Immunotherapy in blood cancers N. Giannakoulas (GR) 16:10-16:15 Discussion


Plenary lecture Chairs: C. N. Baxevanis (GR), C. Gouttefangeas (DE)

16:30-17:10 Intratumoral heterogeneity detected via next generation sequencing of T cell receptors R. Offringa (DE) 17:10-17:15 Discussion


SESSION 8: Immunotherapy (III) Chairs: A. Kotsakis (GR), V. Georgoulias (GR)

17:15-17:40 Current status of immunotherapy in head and neck cancer* A. Argiris (GR) 17:40-17:45 Discussion *sponsored by 17:45-18:10 What is new in immunotherapy for melanoma M. Neagu (RO) 18:10-18:15 Discussion 18:15-19:40 Overcoming primary and acquired resistance in immune checkpoint inhibitors C.N. Baxevanis (GR) 19:40-19:45 Discussion 19:45-20:10 Management of toxicities A. Koumarianou (GR) 20:10-20:15 Discussion

20:15 Awards - Closing remarks

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AbstractsP1. MODULATION OF FOXP3+ T REGULATORY CELL FUNCTION BY EXTRACELLULAR OSTEOPONTIN

Nikolaos Paschalidis, Evangelia Kourepini, Themis Alissafi, Constantinos Fedonidis and Vily Panoutsakopoulou

Cellular Immunology Laboratory, Division of Cell Biology, Center for Basic Research I, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

Background Understanding molecular mechanisms that characterize tumor immune evasion and the interaction of cancer cells with immune cells is necessary to design new immunotherapies against cancer and optimize existing ones. Foxp3-expressing regulatory T cells (Foxp3+Tregs) are specialized T cells that infiltrate various cancers (including melanoma) and suppress immune cell activation. The molecular mechanisms responsible for Treg function in the tumor microenvironment (TME) are still poorly defined and this stands as a major hindrance for designing effective therapies. Osteopontin (Opn) is a pleiotropic cytokine produced by many types of tumor cells and is widely associated with cancer aggressiveness. Most studies on Opn have focused on its activity in effector T cells and dendritic cells, while possible effects on Foxp3+ Treg cells have been neglected. Also, while Opn is secreted by many tumors, its effect on the induction of immune tolerance in cancer cells has not been tested. Previous work from our group has demonstrated that Opn promotes immune tolerance in a mouse model of allergic airway inflammation.Our preliminary work suggested that these protective effects were partly mediated by FoxP3+ Tregs. With this study we aim to investigate the possible effects of extracellular osteopontin in Foxp3+ Tregs in the TME.

Methods To characterize the possible role of extracellular Opn in Foxp3+ Treg development and function we used assays of Foxp3 induction as well asin vitro and in vivo suppression assays.

Results and conclusions Our data show that short-term pre-treatment of activated Foxp3+ Tregs with Opn enhances their suppressive function both in vitro and in vivo. In vivo, this enhanced functionality was manifested mainly by the prevention of trafficking of effector T cell populations from draining lymph nodes to inflammatory sites in experimental multiple scle-rosis. The enhanced suppression ability of Tregs was also observed when these cells were pre-conditioned with culturing medium from B16 melanoma cells, an effect that was reduced when we depleted extracellular B16-derived osteopontin. To further study this in the context of the TME in vivo, we have generated B16 melanoma cells stably transfected with an inducible system to silence Opn expression. Furthermore, phenotypic analysis of Opn-conditioned Foxp3+ Tregs revealed increased expression of tgfb and ICOS.

Our results suggest that osteopontin can enhance Foxp3+ Treg function possibly by mediating stability and secretion of im-munomodulating factors. Results from our studies will assist in the design of more effective immunotherapies.

This work was funded by the European Research Council (ERC). This Post-doctoral research was implemented through an IKY scholarship funded by the “Strengthening Post-Academic Researchers / Researchers” Act from the OP “Human Resources Development, Education and Lifelong Learning” priority axis 6,8,9 and co-funded by the European Social Fund (ESF) and the Greek government.

P2. DEVELOPMENT OF EX-VIVO 2D & 3D CULTURE PROTOCOLS FOR THE STUDY OF MULTIPLE MY-ELOMA

Konstantinos Papadimitriou1*, Anastasia Tsopanidou1*, Ioannis Kostopoulos1, Paraskeui Micheli1, Tina Bagratuni2, Athanasios Velentzas1, Aikaterini Giannopoulou1, Nikolas Orologas3, Ioannis Trougakos1, Eustathios Kastritis2, Evangelos Terpos2, Meletios-Athanasios Dimopoulos2, Ourania Tsitsilonis1

1 Department of Biology, School of Science, and 2 Department of Clinical Therapeutics, “Alexandra”Hospital, School of Medi-cine, National and Kapodistrian University of Athens; 3SafeBloodBioanalyticaSA, Athens, Greece *equal contribution

In patients with multiple myeloma (MM) the bone marrow (BM) microenvironment supports myeloma cell growth and survival against therapeutic regiments. Our aim is to develop an ex vivo platform to assess the effectiveness of chemo-therapeutic drugs at single cell level, in an environment that simulates the BM niche using models of 2D and 3D cocultures.

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Specifically, the human MM cell line U-266, as well as primary BM stromal cells (BMSCs) obtained from MM patients’ biopsies were cocultured in 2D and 3D cultures using hydrogel (PuraMatrix, BD). Cells were plated in 96-well plates and exposed to the anthracycline doxorubicin (at concentrations 2 μΜ, 0,2 μΜ and 0,02 μΜ) for 48h. Automated analysis and evaluation of single-cell viability was performed using three platforms: A) optical microscopy of cells stained with the Try-pan Blue. Pictures were acquired using 4x objective lens (one picture/well). Data were analyzed with Nikon NIS-Elements image analysis software, counted and categorized based on TrypanBlue staining (live vs dead); B) flowcytometry analysis followingannexinV/PI staining. Live, early apoptotic and necrotic cells were detected using BD FACSDivasoftware; and C) confocal microscopy analysis after Annexin V/PI staining. Fluorescence signals of single cells were quantified with the Nikon NIS-Elements image analysis software. Our initial results indicate that using either platform, the estimation of my-eloma cell viability after doxorubicin treatment is feasible. In particular, flow cytometry results (method B) and optical microscopy results (method A) were comparable and in full concordance. Results from the 2D cultures with the use of platform B confirmed the increased resistance of U-266 to doxorubicin (20%differential viability at 2μM) after their coculture with BMSCs. Compared to methods A and B, confocal microscopy (method C) had the greatest analytical potential, as it offers the advantage of spatial (x, y, z) and quantitative analysis. Although all methods need to be standardized, our results indicate that the increase in doxorubicin concentration leads not only to increased percentages of necrotic cells but also to increased mean fluorescence intensity. The initial encouraging results of 2D and 3D MM-BMSC cultures should be further optimized and validated in comparison to standard methods evaluating cell viability.

Funding: HESMO, grant number 6954/2017

P3. ALTERNATIVE SPLICING OF THE BCL2 FAMILY MEMBERS PRODUCES NEW APOPTOSIS-RELATED PROTEIN ISOFORMS WITH DISTINCT COMBINATIONS OF BCL2-HOMOLOGY (BH) MOTIFS

Pinelopi I. Artemaki, Nikolaos V. Tsilikas, Stamatia-Maria Rapti, Panagiotis G. Adamopoulos, Andreas Scorilas, Christos K. Kontos

Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece

Background: The B-cell lymphoma-2 (BCL2) protein family consists of multiple members, best known for their role as key mediators of the intrinsic apoptotic pathway. A distinctive feature of the Bcl-2 family members is the presence of at least one out of four BCL2-homology (BH) motifs (BH1-4). The family consists of both pro- and anti-apoptotic members, com-peting for their ability to induce or suppress mitochondrial outer-membrane permeabilization (MOMP), respectively. While most of the anti-apoptotic members contain all four BH (BH1-4) motifs, the pro-apoptotic members can be further divided into multi-BH motif members and BH3-only members. Alternative splicing of BCL2 pre-mRNAs generates a wide variety of transcripts with discrete functions, a significant portion of which has been predicted via bioinformatic analysis. This study focuses on the experimental determination of the exon structures of these transcripts.

Methods: For this purpose, we conducted in-silico expressed sequence tag (EST) analysis. ESTs represent short (200-800 nucleotide) cDNA sub-sequences which, through careful pre-processing, clustering and assembling, enable the discovery of novel splice variants. Next, 44 cancer cell lines were subcultured, total RNA was isolated and poly(A) RNA was reversely transcribed into first-strand cDNA. Splice variants were amplified through nested PCR, using unique combinations of exon junction-specific PCR primers. PCR products were submitted to agarose gel electrophoresis, prior to gel extraction and Sanger sequencing.

Results and Conclusions: in silico analysis resulted in the identification of novel RNAs containing alternative combinations of previously annotated exons, new splice sites in few of these exons, and intron retentions. Some of the novel transcripts are predicted to encode new protein isoforms that lack one or more BH motifs and are hence likely to differ in their regula-tory apoptotic role, whereas other transcripts contain premature translation termination codons and are probably subjected to nonsense-mediated mRNA decay.

P4. GLYCOLYTIC ANAEROBIC METABOLISM AND PD-1+ INFILTRATING LYMPHOCYTIC (PIL) DENSITY IN NON-SMALL CELL LUNG CANCER

Alexandra Giatromanolaki1, Alison Banham2, Adrian L. Harris2, Michael I. Koukourakis1

1 Department of Pathology and Department of Radiotherapy / Oncology Medical School, Democritus University of Thrace, Alexandroupolis, Greece; Cancer Research UK, Molecular Oncology Laboratories. Radcliffe Department of Medicine, Univer-sity of Oxford, UK

Background: Intratumoral acidosis and hypoxia have been shown to suppress lymphocyte proliferation and migration, and this may be important in the efficacy of modern immunotherapy. Cancer cells are well known to exploit glycolytic pathways and anaerobic pyruvate usage for their energy demands, a result of intratumoral hypoxia or intrinsically activated molecular pathways including the HIF and Akt pathways. Acidosis-induced by lactate released into the extracellular space may be an important, still unexplored, variable defining the antitumor activity of cytotoxic T-cells.

Methods: Using immunohistochemistry for PD-1 (program death-1) protein, expressed by cytotoxic lymphocytes, we as-sessed the Tumor Infiltrating Lymphocyte (TIL) and PD-1+ lymphocytic (PIL) density in the tumor stroma of 98 non-small cell lung carcinomas (NSCLC) treated with surgery. TILs were assessed as lymphocytes stained with hematoxylin in the entire tissue section, in x40 optical fields, and the mean value was used to give a final score (TIL-score: 1 or minimal (1-10 lymphocytes/o.f.), 2 or low (10-70 lymphocytes/o.f.), 3 or medium (70-150 lymphocytes/o.f.) and, 4 or high (>150 lymphocytes/o.f.). The mean % of PD-1+ TILs (PD-1 score) was recorded, and the PIL-score was defined as the product TIL-score x PD-1-score. Three PIL scores were identified: 0 (product 0), 1 (product 1-4) and 2 (product 5-8). TIL- and PIL-scores were analyzed in parallel with the immunohistochemical expression of a large number of proteins/enzymes involved in cell metabolism (glucose and monocarboxylate transporters, glycolysis enzymes and pyruvate metabolism-related proteins).

Results and Conclusions: High % of cancer cells expressing lactate dehydrogenase LDH5, Hexokinase HXKII and mono-carboxylate transporter MCT2 were inversely related to TIL-score (p<0.04, r>0.20). High % of cancer cells expressing LDH5 or glucose transporter GLUT2 were, also, inversely associated with PIL-score (p<0.02, r>0.22). Cases with intense TIL-score and PIL-score had significantly better survival (p<0.05), while high LDH5 expression was linked with poor prognosis (p<0.02). It is concluded that markers of anaerobic cancer cell metabolism, like LDH5, HXKII or GLUT2, characterize im-munologically cold tumors. Whether LDH-targeting drugs or glycolysis inhibitors may restore immune fertility emerges as an interesting hypothesis of therapeutic relevance.

P5. OVEREXPRESSION OF THE MOLECULAR CHAPERONE CLUSTERIN (CLU) AND HEAT-SHOCK PROTEIN BETA-3 (HSPB3) mRNAs PREDICTS COLORECTAL CANCER PATIENTS’ RELAPSE

Aikaterini-Anna Liosi1, Maria-Anna Kalioraki1, Aimilia D. Sklirou2, Maria-Christina Cheimonidi2, Christos Kontos1, Andreas Scorilas1, Ioannis P. Trougakos2

1Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece; 2Department of Cell Biology and Biophysics, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece.

Background: Clusterin (CLU) is a disulfide-linked heterodimeric protein, is ubiquitously expressed and highly conserved in mammalian tissues and fluids, associated with the clearance of cellular debris and apoptosis. In humans, clusterin is encoded by the CLU gene on chromosome 8. CLU is a Golgi chaperone, which facilitates the folding of secreted proteins in an ATP-inde-pendent way. Through this function, CLU is involved in many diseases related to oxidative stress, including neurodegenerative diseases, cancers, inflammatory diseases, and aging. Heat-shock protein beta-3 (HSPB3) is a molecular chaperone protecting the proteome from uncontrolled aggregation, especially during environmental stress. In humans, HSPB3 is encoded by the HSPB3 gene on chromosome 5. A mutation in this gene is the cause of autosomal dominant distal hereditary motor neuropa-thy type 2C. However, the role of HSPB3 in colorectal cancer (CRC) has not been thoroughly investigated yet. In this study, we examined the potential prognostic value of CLU and HSPB3 mRNA expression in CRC.

Methods: For this purpose, we extracted total RNA from 167 malignant colorectal tumor specimens and 41 paired non-cancerous colorectal mucosae, and developed a sensitive and accurate SYBR-Green based real-time quantitative poly-

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merase chain reaction (qPCR) methodology to quantify CLU and HSPB3 mRNA expression in the two cohorts of specimens. For qPCR calculations, three housekeeping genes, namely glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypox-anthine phosphoribosyltransferase 1 (HPRT1), and hydroxymethylbilane synthase (HMBS) were used as reference genes, and HT-29 colorectal adenocarcinoma cells served as a calibrator. Next, extensive biostatistical analysis, including survival analysis, was performed.

Results and Conclusions: Comparison of CLU mRNA levels among 41 pairs of malignant colorectal tumors and their adjacent non-cancerous colorectal tissues revealed the remarkable downregulation of CLU mRNA expression in the ma-jority of malignant colorectal tumors. Furthermore, patients with malignant colorectal neoplasms overexpressing CLU and HSPB3 mRNAs are more prone to relapse than other CRC patients. Multivariate Cox regression analysis revealed that high mRNA expression levels of CLU and HSPB3 are independent molecular predictors of cancer recurrence in CRC patients. In conclusion, CLU mRNA expression is heavily downregulated in CRC, in contrast to the normal colon tissue specimens. Most importantly, high CLU and HSPB3 mRNA expression is associated with CRC recurrence.

O1-1. IBTK SUPPORTS MYC-DRIVEN LYMPHOMAGENESIS IN MICE

Eleonora Vecchio1, Gaetanina Golino1, Francesco Albano1, Antonio Pisano1, Enrico Iaccino1, Selena Mimmi1, Giuseppe Fiume1, Giuseppe Scala1, Ileana Quinto1

1University of Catanzaro, Experimental and Clinical Medicine, Catanzaro, Italy.

BACKGROUND: Previous studies show that IBTK is involved in cell survival. RNA interference of IBTK reduced viability of DLD-1 K-Ras-positive colorectal cancer cells. Differential methylation of the IBTK genomic region was reported for U-CLL (Unmutat-ed-Chronic Lymphocytic Leukaemia) and M-CLL (Mutated-Chronic Lymphocytic Leukaemia), suggesting a possible correlation between the IBTK expression levels and aggressiveness of disease. Recently, we provided the evidence of IBTK as a prognostic marker of CLL progression; the IBTK expression was modulated in the clinical course of disease and after first therapy. Further, the pro-survival action of IBTK was confirmed through the transcriptional inhibition of pro-apoptotic genes.

METHODS: In this study, we generated the Ibtk-/- mice by insertion of the beta-geo gene in the Ibtk locus. Ibtk-/- mice were bred with congenic E μ-myc transgenic mice, a pre-clinical mouse model of Non-Hodgkin’s Lymphoma, to generate Ibtk-/-E μ-myc mice, which were monitored for survival and tumor development. We used flow-cytometry for tumor immunophenotyping and B cell differentiation. Primary murine B cells were compared using different approaches: proliferation, cell viability and cell death assays.

RESULTS AND CONCLUSIONS: Our preliminary data provide the evidence that Ibtk gene increases survival and delays tumor onset in E μ-myc transgenic mice. The tumor phenotype of Ibtk-/-E μ-myc mice was unchanged rather than of E μ-myc trans-genic mice. Loss of Ibtk substantially reduced the number of premalignant B-lymphoid cells without affecting their proliferation rate. In particular, pre-cancerous immature B cells were reduced in bone marrow and spleen of Ibtk-/-E μ-myc compared to Ibtk+/+E μ-myc mice. The reduced number of pre-cancerous immature B cells could explain the enhanced survival and delayed tumor onset of Ibtk-/-E μ-myc, as lymphomas in this animal model generally derive from this subpopulation. In E μ-myc trans-genic mouse model of B-lymphomagenesis, the pre-cancerous state is characterized by aberrant proliferation of B-lymphoid cells, which is initially offset by pro-apoptotic action of c-Myc. Of note, resistance of pre-cancerous B cells to Myc-induced apoptosis must occur for proceeding toward malignancy.

We have previously shown that the enhanced expression of IBTK in CLL cells down regulate the expression of pro-apoptotic genes, thus counteracting apoptosis. According to this hypothesis, we found an increased spontaneous apoptosis of pre-can-cerous Ibtk-/-E μ-myc B cells ex vivo by Annexin V assay and in vitro without added cytokines as confirmed by Caspase 3/7 activity.

Since c-Myc is abnormally expressed in a great majority of human cancers, the evidence that IBTK promotes the survival of c-Myc–driven premalignant B cells could have general implications for oncogenesis. At this time, it is unclear how IBTK exactly contributes to the deregulation of apoptotic pathway, and further efforts are needed to undercover molecular partners of IBTK in cancer context. Our findings support a synergistic role of IBTK in Myc-driven B-lymphoma genesis mainly through counter-action of B cell apoptosis. In summary, this study provides the rationale for the development of novel therapeutic approaches of B-lymphoma.

O1-2. WNT1 SILENCES CC/CXC MOTIF CHEMOKINE GENES IN DENDRITIC CELLS AND INDUCES ADAP-TIVE IMMUNE RESISTANCE IN LUNG ADENOCARCINOMA

Dimitra Kerdidani1,2, Panagiotis Chouvardas1,3, Ares Rocanin Arjo4, Mary Tsikitis5, George Kazamias6, Konstantinos Potaris7, Spyros Zakynthinos2, Ioannis Kalomenidis2, Vassili Soumelis4, George Kollias1,3 and Maria Tsoumakidou1

1Division of Immunology, Biomedical Sciences Research Center Alexander Fleming, Vari-Athens, Greece. 21st Department of Critical Care and Pulmonary Medicine, Medical School, National and Kapodistrian University of Athens, Greece. 3Depart-ment of Physiology, Medical School, National and Kapodistrian University of Athens, Greece. 4Integrative Biology of Human Dendritic Cells and T Cells, Institute Curie, Paris, France. 5Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Greece. 6Department of Histopathology, Evaggelismos General Hospital, Athens, Greece. 7Department of Thoracic Surgery, Sotiria General Hospital, Athens, Greece.

Background: Lung adenocarcinoma (LUAD)-derived oncogenic Wnts increase cancer cell proliferative/stemness potential, but whether they also impact the tumor microenvironment is unknown.

Methods: We have set-up a combinatorial experimental approach that integrates primary human tumor analysis and syn-geneic tumor models (intrapulmonary engraftment of Lewis Lung Carcinoma (LLC) cells in syngeneic mice) to identify LUAD-derived WNTs that could become immunotherapeutic targets.

Results: Among all known Wnts and across all types of cancers, Wnt1 correlated strongly with tolerogenic genes in the TCGA gene expression database. In an in house cohort of LUADs, Wnt1 levels (qPCR) was inversely associated with T cell abundance (immunohistochemistry). Altering Wnt1 expression profoundly affected growth of LLC cells in syngeneic im-munocompetent mice and this was strongly dependent on conventional dendritic cells and T cells. Mechanistically, Wnt1 lead to transcriptional silencing of CC/CXC chemokines in conventional dendritic cells and T cell cross-tolerance. Wnt-target genes were up-regulated in human intratumoralconventional dendritic cells in vivo and decreased upon silencing Wnt1 ex-vivo, accompanied by enhanced T cell cytotoxicity. In our orthotopic tumor models,siWnt1-loaded nanoparticles, given as single therapy or part of combinatorial immunotherapies (dendritic cell-targeted vaccination or FLT3L), acted at both arms of the cancer-immune ecosystem to halt tumor growth.

Conclusions: Our studies show that Wnt1 enhances adaptive immune rejection of lung adenocarcinomas and highlight its potential targeting as a novel therapeutic strategy.

O1-3. AN IN VITRO T-CELL PRIMING ASSAY TO SELECT EPITOPES FOR THE DESIGN OF TAILORED THER-APEUTIC HEPATITIS C VIRUS VACCINES

G. Koutsoumpli, N. Stasiukonyte and T. Daemen

Department of Medical Microbiology, Tumor Virology and Cancer Immunotherapy, University of Groningen, University Medi-cal Center Groningen, Groningen, the Netherlands

Background: Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma and, with approximately 2 million people newly infected annually, represents a significant global health problem. Despite recent treatment advances, there are no prophylactic or therapeutic HCV vaccines available. An HCV therapeutic vaccine should induce robust, broad-spectrum T-cell responses, preferably against the non-structural proteins (nsPs) of the virus, as these proteins are the most immunogenic and conserved. We previously demonstrated that a viral vector-based vaccine encoding all nsPs of HCV, to broaden the immune response, induced lower immune responses than those vaccines encod-ing only part of the nsPs. Furthermore, considering the immunodominance observed in HCV immunity, vaccines lacking immunodominant epitopes will likely also allow broader T-cell responses. Therefore, epitope-specific vaccines might offer advantages over vaccines encoding all nsPs of HCV. Yet, the identification of epitopes that can be processed and presented by dendritic cells (DCs) is essential for the rational design of epitope (string-bead) vaccines.

Methods: We developed an in vitro T-cell priming assay using human peripheral blood mononuclear cells (PBMCs) in order to identify immunogenic HCV-specific epitopes. In brief, naïve, unfractionated PBMCs from a healthy HLA-A2+ donor, were cultured, in the presence of Fms-related tyrosine kinase 3 ligand (FLT3L), to activate DCs and subsequently were primed in vitro with irradiated cells lines expressing the NS3 protein of HCV. After a 10-day culture HCV-specific T-cell responses

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were assessed in an overnight interferon-gamma (IFN-γ) ELISPOT assay in the presence of synthetic long peptides (SLPs) spanning the whole sequence of HCV NS3 protein.

Results and Conclusions: Naïve precursors specific for 11 out of 98 SLPs tested were primed in vitro in the 10-day PB-MCs cultures. These 11 SLPs contain 9 different HLA-A2-restricted epitopes, as predicted by using a combination of three epitope prediction algorithms. Furthermore, we identified 2 peptides that were not predicted to contain immunogenic HLA class I epitopes yet showed HCV-specific responses in vitro. Additional experiments using restimulation with short synthetic peptides are ongoing to further investigate the HLA-restriction of the observed responses and validate our findings. This study demonstrates that we established an in vitro assay that enables the identification of HLA epitopes resulting from cross-presented antigens and that can cross-prime T-cells. This in vitro T-cell priming method thus enables the effective selection of immunogenic epitopes, but also less immunogenic ones, for the design of tailored therapeutic vaccines.

O1-4. SERUM MIRNA-BASED DISTINCT CLUSTERS DEFINE THREE GROUPS OF BREAST CANCER PA-TIENTS WITH DIFFERENT CLINICOPATHOLOGICAL AND IMMUNE CHARACTERISTICS

Sotirios P. Fortis1, Christoforos K. Vaxevanis1, Louisa G. Mahaira1, Michael Sofopoulos2, Nectaria N. Sotiriadou2, Amalia Dinou3, Niki Arnogiannaki2, Catherine Stavropoulos-Giokas3, Dimitris Thanos4, Constantin N. Baxevanis1, Sonia A. Perez1

1. Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, Athens, Greece, 2. Pathology Department, Saint Savas Cancer Hospital, Athens, Greece, 3. Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, Greece, 4. Biomedical Research Foundation, Academy of Athens, Athens, Greece

BACKROUND: Breast cancer (BCa) is a heterogeneous disease with different histological, prognostic and clinical aspects. There-fore, the need for identification of novel biomarkers for diagnosis, prognosis and monitoring of disease, as well as treatment outcome prediction remains at the forefront of research. The search for circulating elements, obtainable by simple peripheral blood withdrawal, which may serve as possible biomarkers, constitutes still a challenge. In the present study, we have evalu-ated the expression of 6 circulating miRNAs, (miR-16, miR-21, miR-23α, miR-146α, miR-155 and miR-181α), in operable BCa patients, with non-metastatic, invasive ductal carcinoma, not receiving neoadjuvant chemotherapy. These miRNAs, known to be involved in both tumor cell progression and immune pathways regulation, were analyzed in relation to circulating cytokines, tumor immune-cell infiltration and established prognostic clinicopathological characteristics.

METHODS: Peripheral blood from women diagnosed with invasive carcinoma (n=48), without neoadjuvant therapy, was ob-tained one day before surgery. Selected circulating miRNAs (e.g.miR-146,miR-16,miR-23,miR-21,miR-181,miR-155), crucial for the immune system, were measured inpatients’ sera and PBMCs by RT-PCR. Applying Luminex technology, we evaluated the levels of cytokines/chemokines (e.g.TGF-β1, Rantes, IL-8, IL-9,IL-10,IL-1Ra), strongly related with neoplasia and immunity. The infiltration analysis of immune subpopulations (CD4+, CD8+ and CD163+) was performed by IHC in FFPE tissues of patients with histologically-confirmed invasive carcinoma.

RESULTS: We have identified three different clusters of patients, with overall low (C1), moderate (C2) or high (C3) expression lev-els of these six circulating miRNAs, which define three distinct groups of non-metastatic BCa patients characterized by different clinicopathological (tumor size, nodal status, stage, grade, Ki67%, Her2/neu and hormone receptors expression) and immune-related characteristics (circulating chemokines/ cytokines and tumor infiltration), with possibly different clinical outcomes.

CONCLUSIONS: Our data provide the proof-of-principle to support the notion that, up- or down-regulation of the same circulat-ing miRNA may reflect different prognosis in BCa. Nonetheless, the prognostic and/or predictive potential of these three “signa-tures” needs to be further evaluated in larger cohorts of BCa patients with an, at least, 5-year clinical follow-up.

O1-5. PROGNOSTIC SIGNIFICANCE OF HLA-A*02AND HLA-A*24 INPROSTATE CANCER

Savvas Stokidis1, Sotirios P. Fortis1, Amalia Dinou2, Marina I. Konstantellou1, Catherine Stavropoulos-Giokas2, Sonia A. Perez1, Constantin N. Baxevanis1

1. Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, Athens, Greece, 2. Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, Greece

BACKROUND: The major role of the immune system in cancer development and evolution is established. Immune bio-markers for cancer prognosis and prediction, including prostate cancer (PCa), are emerging as essential tools for treat-ment, assessment and monitoring. Given the heterogeneity of PCa and the diversity of therapeutic treatments applied, intensive efforts have been recently initiated for the establishment personalized approaches also in this type of cancer. As HLA-alleles are essential for an effective antitumor response, their expression could serve as prognostic/predictive biomarkers. Studies in NSCLC and ovarian cancer have demonstrated that HLA-A*02 allele expression is correlated with poor clinical outcome. Results from our previous phase I clinical trial of PCa patients vaccinated with a HER-2/neu hybrid peptide, suggested that HLA-A*24 allele expression conferred a better clinical outcome. Based on these data we evaluate here in the prognostic relevance of both alleles in PCa.

METHODS: PCa patients (n=100) were enrolled from 06/2014-9/2018 to this combined retrospective/prospective study. Fifty of these patients were initially diagnosed with localized disease (LPCa) and 50 were already metastatic at first diagno-sis (MPCa). All patients received standard medical treatment upon diagnosis and follow-up data, either retrospective or pro-spective, were recorded. Patients were HLA-genotyped for the A locus. Time to biochemical recurrence (BCR), metastasis (M), castrate resistance (CR) and overall-survival (OS) were evaluated. To investigate the role of T-cell preexisting immunity, specific CD8+ T-cells against PSA146-151, HER2369-377 (HLA-A2-restricted), and PSA153-161, HER2780-788 (HLA-A24-restricted) pep-tides were measured. The maturation stage of these peptide-specific T cells was also defined.

RESULTS: Both, LPCa and MPCa patients expressing HLA-A*02hada worse clinical outcome than those lacking this allele, especially when compared to HLA-A*24-expressors. Interestingly, MPCaHLA-A*24+ patients had a significant, favorable, advantage in their OS.PSA-specific, circulating, CD8+ T-cells were detected at higher frequencies in HLA-A24+ than in HLA-A2+ patients. On the contrary, HER-2-specific T-cells were lower in HLA-A24 expressors. However, high frequencies of CD8+ T-cells, specific for either PSA or HER2, did not translate to differences to clinical outcome. As expected, most of the antigen-specific T-cells were terminally differentiated, although differences could be detected in the memory subsets among patients.

CONCLUSIONS: The presented data strongly support unfavorable and favorable roles for HLA-A*0201 and HLA-A*24 ex-pression, respectively, in PCa prognosis. The precise mechanisms (e.g. allelic-loss and/or immune escape or resistance) underlying this immune-based difference requires further investigation.

O1-6. A CELL INTRINSIC ROLE OF IL33 IN THE FUNCTIONAL STABILITY OF TREGS IN THE TUMOR MI-CROENVIRONMENT

Aikaterini Hatzioannou1, Aggelos Banos1, Theodore Sakelaropoulos2, Constantinos Fedonidis3, Maria-Sophia Vida-li4, Panagiotis Georgiadis4, Anna Henriques5, Vassiliki Koliaraki5, Aristotelis Tsirigos6, Panayiotis Verginis1

1Center of Clinical, Experimental Surgery & Translational Research, Biomedical Research Foundation Academy of Athens, Greece, 2School of Mechanical Engineering, National Technical University of Athens, Greece, 3Center of Basic Research, Biomedical Research Foundation Academy of Athens, Greece, 4Institute of Biology, Medical Chemistry & Biotechnology, National Hellenic Research Foundation, Greece, 5Department of Immunology, Biomedical Sciences Research Centre “Alex-ander Fleming,” Vari, Greece, 6Applied Bioinformatics Laboratories, New York University School of Medicine, New York, NY, USA and Department of Pathology, New York University School of Medicine, New York, NY, USA

Despite impressive clinical success, cancer immunotherapy remains ineffective in large proportion of patients mainly due to tumoral resistance, which is highly dependent on the abundance of Foxp3+ regulatory T cells (Tregs). In view of the improved clinical outcome upon Treg depletion in cancer patients, delineation of mechanisms that govern the immunosuppressive program of Tregs is of paramount importance. Herein, we identify a novel role of nuclear IL33 (nIL33) in the functional

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stability of Tregs in the tumor microenvironment. IL33-deficient (IL33-/-) Tregs exhibited an attenuated suppressive function in vivo empowering the anti-tumor immune responses and tumor eradication. Epigenetic and transcriptional analyses of tumor-exposed IL33-/- Tregs revealed IFNγ as a putative cause for their functional deficiency. Importantly, deletion of IFNγ in IL33-/- Tregs restored their in vivo suppressive capacity resulting in enhanced tumor growth. In a therapeutic perspective the absence of IL33 potentiated the effect of anti-CTLA4 and anti-PD1 immunotherapy in melanoma-bearing mice. Overall, our findings reveal a cell-intrinsic IL33-dependent molecular mechanism for the maintenance of Treg suppressive function in cancer with important implications in the improvement of immunotherapy protocols.

O1-7. DOWN-REGULATION OF PD-L1 IN COLON CANCER CELLS SURVIVING AFTER IRRADIATION

Erasmia Xanthopoulou1, Ioannis M. Koukourakis2, Achilleas Mitrakas1, Christos Kakouratos1, Alexandra Giatro-manolaki3 and Michael I. Koukourakis1

1Department of Radiotherapy/ Oncology, Radiobiology/Radiopathology Unit, Democritus University of Thrace, Alexandroupo-lis, Greece, 2National and Kapodistrian University of Athens, Greece, 3Department of Pathology, Democritus University of Thrace, Alexandroupolis, Greece.

Background: PD-L1 protein (CD274, B7-H1) is a key ligand of the Programmed Cell Death 1 (PD-1, CD279) protein ex-pressed by cytotoxic T-cells. Cancer cells that express PD-L1 bind the PD-1 protein on T-cells and block their cytotoxic activity, allowing cancer cell escape from immune surveillance. Radiotherapy is a principal treatment modality for rectal cancer patients. Preoperative radiotherapy induces complete responses in 30% of rectal carcinomas, while resistant rem-nant disease may be the source of post-operative loco-regional relapse and metastasis. Whether further immunotherapy may improve survival of rectal cancer patients remains obscure. The purpose of the current study was to investigate the effect of radiotherapy on the expression levels of PD-L1 protein, in the HT29 and CaCo2 colon cancer cell lines.

Materials/ methods: Cancer cells were treated with three fractions of 4Gy of radiation. After each radiation fraction, cells were allowed to recover for 15 days, before the administration of the following fraction. The new cancer cell lines (HT29-R and CaCo2-R) obtained after the 3rd fraction consisted of cancer cells resistant to radiation, simulating the clinical condition of preoperative radiotherapy. PD-L1 expression was assessed using Real-Time PCR, Western Blot analysis and Immu-nocytochemistry. Protein isolated from cell lysates from wild-type ‘WT’ and resistant ‘R’ colon cancer cell was collected for Western Blot analysis of PD-L1 (ab205921 [28-8]/Abcam and 22C3/DAKO). Also, a validated immunohistochemistry protocol with the automated Bond-max System (Leica Biosystems), was applied on cells harvested in Superfrost™ Plus Microscope Slides and fixed with acetone, to detect PD-L1 expression using primary anti-PD-L1 antibodies ((28-8/Abcam and 22C3/DAKO)). Finally, Real-Time PCR was performed to evaluate the expression of PD-L1 gene.

Results and Conclusions: Real-Time PCR showed that PD-L1 gene was repressed in resistant cell lines. Similar expres-sion patterns were observed in Western blot and Immunocytochemistry analysis. Experimental evidence suggests that exposure of cancer cells to radiation induce essential changes in their biology, transforming cells to a potent anti-tumoral vaccine. Such changes involve HLA-class-I molecule up-regulation, induction of the IFN type-I pathway and release of cytokines activating dendritic cells and T-cells. Our results suggest that, in addition to the vaccination effect, radiation down regulates PD-L1 expression in cancer cells, removing an immune check-point blockage pathway and facilitating the anti-tumor activity of PD-1+ cytotoxic T-cells.

O1-8. N-BROMOTAURINE AND STABLE DERIVATIVE OF N-BROMOTAURINE EXHIBIT THERAPEUTIC EF-FECT AGAINST VARIOUS CANCERS

Stella Baliou1**, Markus Nagl2, Antony M. Kyriakopoulos3 and Vassilis Zoumpourlis1*.1. Biomedical Applications Unit, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., 116 35 Athens, Greece, 2. Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstr. 41, 6020 Innsbruck, Austria, 3. NASCO AD Biotechnology Laboratory, 11 Sachtouri str., 18536, Pireas, Greece.

Introduction: N-Bromotaurine is a physiological molecule which arises from the neutrophil -myeloperoxidase halide sys-tem of metabolism during inflammation and constitutes the reaction product of Taurine with hypobromous acid (HOBr). N-Bromotaurine is a potent oxidant molecule which, apart from its antimicrobial ability, exerts a complex anti-inflammatory action. However, due to the poor stability of the N-bromotaurine molecule, a derivative molecule, named Βromamine T (BAT), wassubsequentlyused. In order unravel new therapeutic options, we used N-Bromotaurine and Bromamine T in both in vitro and in vivo experiments.

Methods: Cell proliferation experiments, colony formation assays, staining with CM-H2DCFDA dye, flow cytometry and western blot experiments highlighted the potential anti-cancer effect of these molecules. Α xenograft assay and an air-pouch model of inflammation induced by LPS constituted the in vivo experiments, delineating the functional significance of the molecules under study (N-Bromotaurine and BAT).

Results/Discussion: The tumor-suppressive effect of N-bromotaurine was observed in various cancer cell types, including prostate and skin cancer. In the case of skin cancer, we showed that N-bromotaurine can bypass the glucocorticoid receptor (GR)-resistance of cancer cells when used in combination with cis-platin, despite the frequently mentioned glucocorticoid unresponsiveness due to GR impairment. Cancer cell proliferation was suppressed with the use of BAT in breast, cervical, and colorectal cancer, while the anti-proliferative effect of BAT appeared to be superior to that elicited by taurine. Notably, subsequent experiments of clonogenic growth demonstrated that cancer cell colonies were eliminated following treatment with BAT. In addition, flow cytometry and western blot experiments highlighted the intrinsic apoptotic pathway used by can-cer cells following exposure to BAT. Additional experiments proved that BAT triggered oxidative burst to stimulate apopto-sis. More importantly, the in vivo experiments showed that tumor formation and distribution of immune populations in mice were impaired after treatment with BAT. Consequently, BAT appears to bean emerging anti-proliferative, anti-inflammatory, apoptotic agent in cancer cells with favorable efficacy, thus denoting its significance in cancer therapeutics.

O1-9. OVEREXPRESSION OF MIR-194 IN EPITHELIAL OVARIAN CARCINOMASIS AN INDEPENDENT MO-LECULAR MARKER FOR PATIENTS’POOR TREATMENT RESPONSE ANDSURVIVAL OUTCOME

Konstantina Panoutsopoulou1, Margaritis Avgeris1, Viktor Magdolen2, Andreas Scorilas 1*1 Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece, 2 Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany

Background: Ovarian cancer (OvCa) is the second most lethal gynecological malignancy and the third most frequent cancer in the genital system of women worldwide, while the 5-year survival is the poorest of all gynecological cancers; 38% in Europe.microRNA-194 levels have been highlighted to be dysregulated in numerous cancers. It has been demonstrated that miR-194 exerts an oncogenic role by targeting the 3’UTR of the tumor suppressor PTPN12 (Protein tyrosine phosphatase nonreceptor type 12), enhancing, thus, proliferation, migration, and invasion in ovarian cancer cells.In the present study, we aimed to inves-tigate the expression profile of miR-194 in epithelial ovarian carcinomas (EOC) and to explore its clinical value inOvCa patients’ prognosis and treatment response.

Methods: Total RNA was extracted from 103 epithelial ovarian tumors and 22 benign ovarian lesions using TRI reagent. Fol-lowing total RNA polyadenylation in 3’-end, first-strand cDNA synthesis was performed by MMLV using an oligo (dT) primer. A SYBR-Green fluorescent-based quantitative real-time PCR (qPCR) assay was developed to quantify miR-194 levels in ovarian specimens, using snoRNA RNU48 as endogenous reference control gene for normalization purposes and MDAH-2477 cells as our assay calibrator. Extensive statistical analysis has been conducted to explore the clinical utility of miR-194.

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Results and Conclusions: miR-194 expression is significantly down regulated in ovarian tumors compared to benign le-sions (p=0.002). However, Kaplan-Meier survival curves highlighted the significantly shorter disease-free survival (p=0.066) and cancer-specific survival (p=0.045) of the patients with serous ovarian carcinoma (SOC), overexpressing miR-194, following tumor resection and chemotherapy. Cox regression analysis clearly verified the higher risk for the short-term cancer-related death of the SOC patients overexpressing miR-194 (HR=1.789; 95% CI: 1.003-3.191; p=0.049), while multi-variate Cox models (HR=3.815; 95% CI: 1715-8.488; p=0.001) demonstrated that miR-194 overexpression predicts the poor response to treatment (tumor resection and first-line chemotherapy) and the adverse survival of the SOC patients indepen-dently of FIGO stage, tumor grade, residual tumor, response to chemotherapy and CA125 levels.

Conclusions: Our results highlight that miR-194 overexpression is an independent molecular cancer marker of the predic-tion of poor treatment response and survival expectancy in OvCa.

Acknowledgements: The research presented was carried out within the framework of a Stavros Niarchos Foundation grant to the National and Kapodistrian University of Athens.

O1-10. EVALUATION OF THE LEVELS AND FUCTIONALITY OF REGULATORY T CELLS IN MULTIPLE MYELOMA

Andreas Metousis1, Panagiotis Vitsos1*, Panagiotis Pothos1,2*, Ioannis Kostopoulos1, Efstathios Kastritis3, George Thyfronitis2, Nikolaos Orologas4, Evangelos Terpos3, Meletios-Athanasios Dimopoulos3, Ourania Tsitsilonis1

1Department of Biology, National and Kapodistrian University of Athens, Athens; 2Department of Biological Applications and Technology, University of Ioannina, Ioannina; 3Department of Clinical Therapeutics, “Alexandra” Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens; 4SafeBlood BioAnalytica SA, Athens, Greece (*equal contribution)

BACKROUND: Multiple myeloma (MM) is a hematologic malignancy characterized by infiltration of the bone marrow by patho-logical plasma cells. Although MM remains an incurable disease, new therapeutic protocols achieve complete remission (CR) in patients for prolonged periods. Nevertheless, many patients often relapse after treatment. Surprisingly, studies performed on regulatory T cells (Tregs), which exhibit potent immunosuppressive activity, have not yet demonstrated the correlation of Tregs with disease onset and progression. The purpose of this study was to develop a method based on Multiparameter Flow Cytometry (MFC) to study the levels and functionality of Tregs in the bone marrow and peripheral blood of MM patients, includ-ing those with detectable levels of Minimal Residual Disease (MRD+) or not (MRD-) in their bone marrow.

METHODS: In order to identify Tregs and study their functional profile, samples of both biologic materials were labeled with 12 fluorescent antibodies comprising two different 8-color panels: CD3-FITC, CD4-APC/Cy7, CD25-APC, FoxP3-PE, CD8-Per-CP/Cy5-5, Ki67-AmCyan, CD45RA-PE/Cy7, CD39-Pacific Blue, CD45RO-PerCP/Cy5-5, CTLA-4-Pacific Blue, CD127-AmCyan, HLADR-PE/Cy7. Upon recording a sufficient number of events on a BD FACS CantoII flow cytometer, data analysis was carried out using the Infinicyt software. Following a unified gating strategy, cells with immunophenotype CD3+CD4+CD25+FoxP3+ were identified as Tregs and were further analyzed, in order to evaluate the expression levels of the remaining 6 markers.

RESULTS: Tregs are numerically higher in the peripheral blood than in the bone marrow. Also, MRD- patients displayed the highest CD3+CD4+CD25hiFoxP3+ Tregs and CD3+CD4+CD25hiFoxP3+CD127lo Tregs, expressed as separate percentages on gated CD4+ T cells and total nucleated cells. The group of newly diagnosed patients was characterized by higher Treg ratio on gated CD4+ T cells in the peripheral blood rather than in the bone marrow, whereas MRD+ and MRD- patients displayed the opposite trend.

CONCLUSIONS: The results suggest that Tregs in MRD+ patients, despite not showing an increase in absolute numbers, may have a stronger immunosuppressive phenotype, therefore facilitating the escape of myeloma cells from immunosurveillance.

O1-11. IDENTIFICATION OF NEW ALTERNATIVE SPLICING EVENTS RESULTING IN NOVEL SPLICE VARI-ANTS OF THE HUMAN L-DOPA DECARBOXYLASE (DDC) GENE IN CANCER CELLS, USING NEXT-GENER-ATION SEQUENCING TECHNOLOGY

Michaela A. Boti, Maria Papatsirou, Panagiotis Tsiakanikas, Panagiotis G. Adamopoulos, Andreas Scorilas, Dido Vassilacopoulou, Christos K. Kontos


Background: L-Dopa decarboxylase (DDC) is a homodimeric pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of 3,4-dihydroxy-L-phenylalanine (L-DOPA) to dopamine. In humans, the DDC gene is located on chromo-some 7 (7p12.1) and is composed of 15 exons and 14 intervening introns. A wide repertoire of alternative splicing events within DDC mRNA has been confirmed in the past. Of note, alternative splicing within the 5’-untranslated region (5’-UTR) of DDC is re-sponsible for the production of two distinct mRNAs, characterized as neural and non-neural type, respectively. Malignant cells usually present a deregulated alternative splicing mechanism leading to the production of multiple mRNA isoforms, underlying the tremendous complexity of the cancer cell transcriptome. The aim of this study was to identify novel splice events that will support the existence of novel transcript variants of DDC in human cancer cells.

Methods: For this purpose, total RNA was extracted from 55 human cancer cell lines. Reverse transcription was performed, followed by 3’ rapid amplification of cDNA ends (3’ RACE) and nested 3’ RACE. Nested 3’ RACE products were purified and used for library construction. In the next step, Next-generation sequencing (NGS) was carried out on a platform using semiconductor sequencing technology. NGS data were analyzed by bioinformatic algorithms and in-house developed algorithms. All newly identified variants were verified using PCR and variant-specific primers.

Results and Conclusions: Analysis of the data obtained by NGS revealed novel alternative splicing events in human cancer cells. Moreover, validation via PCR of these events, using variant-specific primers unveiled novel transcripts of DDC gene. Some of them may encode new protein isoforms of DDC, whereas others may represent nonsense-mediated mRNA decay (NMD) candidates. The described workflow enables the identification of a plethora of novel transcripts that may posses a great clinical value as molecular biomarkers.

O1-12. ALTERNATIVE 3’-UNTRANSLATED REGIONS (3’-UTRS) IN MESSENGER RNAs OF KALLIKREIN-RELATED PEPTIDASE (KLK) FAMILY MEMBERS RESULT FROM ALTERNATIVE SPLICING OF KLK pre-mRNAs, AS REVEALED BY NEXT-GENERATION SEQUENCING

Maria Morrou, Paraskevi Karousi, Panagiotis G. Adamopoulos, Panagiotis Tsiakanikas, Andreas Scorilas, Christos K. Kontos


Background: Human tissue kallikreins (KLKs) belong to the S1 family of trypsin-like serine proteases and are encoded by the largest contiguous cluster of 15 protease genes, located on chromosome 19 (19q13.4). The majority of KLK pre-mRNAs appear to be alternatively spliced and consequently the human KLK genes demonstrate over 90 annotated mRNAs so far. However, deregulation of specific KLK transcripts has been noticed in multiple human malignancies, confirming their strong prognostic and/or diagnostic attributes. As a result, understanding the control of KLK expression is crucial for studying the pathogenesis of diseases and revealing their putative role as biomarkers and/or therapeutic targets. It is now known, that 3’-untranslated regions (3’-UTRs) of KLK mRNAs are targeted by specific miRNAs, regulating their expression.

Methods: In this study, we used 3’-rapid amplification of cDNA ends (3’-RACE) for the specific amplification of KLK tran-scripts and next-generation sequencing (NGS) technology to identify novel alternative KLK 3’-UTRs. For this purpose, a wide panel of human cell lines, originating from several distinct cancerous and normal tissues was used. Then, reverse transcription was carried out, using an oligo-dT–adaptor as primer. Following the cDNA synthesis, 3’-RACE was performed for the specific amplification of KLK transcripts. In detail, a forward gene-specific primer was designed to target the region of the annotated translation start codon and was used along with a universal reverse primer that annealed on the oligo-dT–adaptor primer sequence. In the next step, all 3’-RACE products were cleaned-up and 100 ng of each purified product was used for the library construction. Finally, NGS was carried out on a platform using semiconductor sequencing technology.

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NGS data were analyzed by bioinformatic algorithms and in-house developed algorithms.

Results and conclusion: Bioinformatic analysis revealed that KLK transcripts contain new alternative 3’-UTRs, support-ing the existence of novel KLK transcripts that are characterized by new alternative 3’-UTRs. Since multiple KLK genes constitute miRNA targets and miRNAs contribute to the regulation of KLKs expression in many human malignancies at the post-transcriptional level, the identification of novel KLK 3’-UTRs could be of high significance for the understanding of the miRNA-kallikrein axis of interactions.

O1-13. tRNA-DERIVED RNA FRAGMENTS (tRFs) CONSTITUTE A NOVEL CLASS OF MOLECULAR BIO-MARKERS IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA: THE PARADIGM OF A NOVEL tRF PREDICTING PATIENTS’ OVERALL SURVIVAL

Katerina Katsaraki1, Styliani Karagiorgou1, Panagiotis Tsiakanikas1, Panagiotis G. Adamopoulos1, Sotirios G. Papageorgiou2, Vassiliki Pappa2, Christos K. Kontos1

1Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece; 2Sec-ond Department of Internal Medicine and Research Unit, University General Hospital “Attikon”, Athens, Greece.

Background: Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults and still re-mains incurable. Numerous studies have identified a variety of prognostic biomarkers for CLL. Following this direction, tRFs could be promising as a new possible biomarker. tRNA-derived RNA fragments (tRFs), also known as tRNA-derived stress-induced fragments (tiRNAs), are small non-coding RNAs, having a length of 18–26 nucleotides; they do not con-stitute random degradation products, but are rather specifically cleaved from tRNAs. They are classified into three series, namely tRF-5, tRF-3, and tRF-1. tRF-5 and tRF-3 series derive from tRNA 5’ and 3’ ends, respectively, while tRF-1 series derive from the 3’ ends of pre-mature tRNA transcripts. They participate in regulation of translation and gene silencing. Furthermore, they play an important role in human diseases. In this original research study, we investigated the potential prognostic and diagnostic value of an internal fragment of the tRNA(s) bearing the Glycine anticodon CCC (itRF-34-GlyCCC) as a prognostic biomarker in CLL.

Methods: This original research study included samples collected from 69 CLL patients and 22 non-leukemic blood donors. Peripheral blood mononuclear cells (PBMCs) were positively selected after centrifugation on a Ficoll-Hypaque gradient. Most PBMCs isolated from patients’ blood samples were leukemic B cells as confirmed by cell immunophenotyping. Nu-cleic acid extraction was performed to determine the mutational status of the immunoglobulin heavy chain variable (IGHV) region. Total RNA was isolated from PBMCs and in-vitro polyadenylated using E. coli poly(A) polymerase, prior to first-strand cDNA synthesis starting from an oligo-dT–adaptor primer. Next, we developed a real-time quantitative polymerase chain reaction (qPCR) method for the quantification of particular tRFs and applied this method in all our samples. At a last step, we performed extensive biostatistical analysis, including survival analysis.

Results and Conclusions: We identified that itRF-34-GlyCCC was downregulated in PBMCs (mostly leukemic B cells) of CLL patients, compared to PBMCs of non-leukemic controls. Kaplan-Meier overall survival (OS) analysis revealed reduced OS for itRF-34-GlyCCC positive CLL patients (P=0.011). Univariate Cox regression showed that itRF-34-GlyCCC positive ex-pression is significantly associated with inferior OS of CLL patients (HR=3.75, 95%CI=1.26-11.16, P=0.018) Multivariate Cox regression analysis revealed that the prognostic value of itRF-34-GlyCCC is independent of other prognostic factors in CLL. In conclusion, itRF-34-GlyCCC appears as a promising molecular biomarker of unfavorable prognosis in CLL.

O2-1. REDUCTION OF PD-L1 POSITIVE CIRCULATING TUMOR CELLS (CTCs) IN NON-SMALL CELL LUNG CANCER (NSCLC) PATIENTS, TREATED WITH NIVOLUMAB

G. Kallergi1,2, E.K. Vetsika3, D. Aggouraki4, Koutsopoulos5, E. Lagoudaki5, C. Stournaras2, V. Georgoulias1, A. Kotsakis3

1 Laboratory of Τumor Cell Biology, School of Medicine, University of Crete, Heraklion, Greece, 2 Department of Biochemistry, Medical School, University of Crete, Heraklion, Crerte, Greece, 3 Medical School, University of Thessaly, Larissa, Greece, 4 Laboratory of Τranslational Oncology, School of Medicine, University of Crete, Heraklion Greece, 5 Department of pathology, Medical School, University of Crete, Greece

Introduction: Circulating tumor cells (CTCs) are considered as a “liquid biopsy” that allows the assessment of tumor changes over time. Tumor cells may escape from the immune system through the activation of PD-1/PD-L1 axis. Targeting these molecules with monoclonal antibodies has shown encouraging results at many types of cancers, including NSCLC. In the current study we investigated the expression of PD-1/PD-L1 molecules on the CTCs isolated from NSCLC patients treated with Nivolumab. Methods: CTCs were isolated based on their size using the ISET platform from 35 patients before treatment, after 1 cycle and 3 cycles. CTCs were detected with Giemsa staining and immunofluorescence (IF) experiments, using either pancytokeratin (A45-B/B3)(CK7)/PD-1/CD45 or (A45B/B3) (CK7)/PD-L1/CD45 combination of antibodies and analysis with the ARIOL system. Spiking experiments using the NSCLC cell lines: H460, H1299, HCC827 and SKMES in nor-mal blood were used to evaluate the detection method. Results: IF evaluation in Nivolumab-treated patients at baseline (34 evaluable samples), after the 1st cycle (13 evaluable samples) and the 3rd cycle (15 evaluable patients) of treatment showed that CTCs could be detected in 64,7% (22/34), 38% (5/13) and 73% (11/15) of patients, respectively. PD-1 (+) CTCs were detected in 50% (11/22) of CK-positive patients at baseline, in, 20% (1/5) after the 1st line and 45,5% (5/11) of CK-positive patients after the 3rd cycle. For PD-L1 the corresponding numbers were 36,4% (8/22), 20% (1/5) and 18,2%(2/11) respec-tively. The expression of PD-1 at baseline was associated with poorer OS (p=0.022) and PFS (p=0.011), while the expression of PD-L1 was associated with shorter PFS (p=0.011). Conclusion: Nivolumab reduced the number of PD-1- and PD-L1-expressing CTCs in advanced NSCLC patients and this reduction persist for PD-L1 and after the 3rd cycle. Furthermore the expression of both markers at baseline is associated with the clinical outcome.

O2-2. CHARACTERIZATION OF POLYCLONAL ANTIBODIES AGAINST DIFFERENT ENVELOPE PROTEINS OF HUMAN ENDOGENOUS RETROVIRUSES IN OVARIAN CANCER

Sahitya Saka1, David Díaz-Carballo1, Ali Haydar Acikelli1, Jacqueline Klein1, Gunther Wennemuth2, Andrea Tannapfel3, and Dirk Strumberg1

1 Institute for Molecular Oncology and Experimental Therapeutics. Marien Hospital Herne. Ruhr University Bochum, Medical School. Herne, Germany, 2 Institute of Anatomy. University of Duisburg-Essen, Medical School. Essen, Germany, 3 Institute of Pathology.Ruhr University Bochum, Medical School. Herne, Germany

Introduction: Recently, the relevance of human endogenous retroviruses (HERVs) is gaining more attention in the field of oncology. They account for about 8% of the human genome as a result of ancestral germ-cell infections in which exoviruses integrated into the host genome and vertically transmitted in a Mendelian form to the progeny. Many studies reported the over expression of HERVs in cancer of different histologies. Currently, only few commercial antibodies are available for the detection of HERV elements. So, there is an urgent need to account for antibodies against different HERVs in order to study their role in cancerogenesis as diagnostic markers.

Methods: For the generation of polyclonal antibodies, 4 different HERV envelope protein sequences HERV-Fc1, HERV-V1, HERV-H and HERV-T were obtained from NCBI data bank. Unique amino acid sequences for each were selected in such a way that they have no similarities among them and ensuring that those sequences do contain at least 3 immunogenic resi-dues. Rabbits were subjected to specific immunization schedule. Once the titers reached more than 20K dilutions, polyclonal antibodies were isolated from sera using affinity chromatography in a protein A sepharose column. Using these generated antibodies, expression of respective HERV elements were monitored in ovarian carcinoma wild type and resistant cell lines using techniques like PCR, western blots, immunocytochemistry and flow cytometry. Expression of those HERV elements was also scrutinized in normal ovarian tissue sections and in a tissue array consisting of different types and stages of ovar-ian carcinoma tissues.

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Results & Conclusions: Four unique peptides were successfully designed and antibodies were generated which induce a strong, specific and reproducible recognition of HERV-Fc1, HERV-V1, HERV-H and HERV-T envelope proteins. Those anti-bodies were successfully applied in a broad immunetechniques with reliable results. An overexpression of HERV-Fc1 and HERV-H envelope proteins was detected in both etoposide and cisplatin resistant SKOV3 ovarian carcinoma cells, indicating that both viral elements are related to acquired resistance. HERV-V1 instead is ubiquitously expressed regardless the resis-tant status of the cell lines analyzed. It was found for the very first time that the expression of those endogenous retroviral proteins is exclusively in tumors but not in normal tissues. These findings could help to elucidate the molecular biology of such viral elements. Moreover, they could represent a valuable diagnostic tools and promising therapeutic targets.

O2-3. RADIATION-ENHANCED COMPLEMENT ACTIVATION AND DOWNSTREAM C5a-C5aR1 SIGNALING MODULATE TUMORIGENIC AND PROINVASIVE RESPONSES OF GLIOBLASTOMA CELLS

Stella Tzelefa1, Antigoni Stavridou1, Ioannis Kostopoulos2, Maria Georgoutsou-Spyridonos1, Georgia Terzoudi1, Georgia Sfyroera1, Ourania Tsitsilonis2, John D. Lambris3 and Dimitrios C. Mastellos1 1Division of Biodiagnostic Sciences and Technologies, INRASTES, National Center for Scientific Research ‘Demokritos’, Ath-ens, Greece, 2Section of Animal and Human Physiology, National and Kapodistrian University of Athens, Athens, Greece, 3Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

Glioblastoma remains one of the most lethal and aggressive primary brain tumors with dismal patient prognosis, despite recent advances in therapeutic intervention. Its high recurrence rates have been partly attributed to its inherent resistance to radiotherapy. Radiotherapy has been shown to trigger intratumoral activation of complement, a key innate immune sentinel with immunoregulatory properties. Remarkably, while mainly associated with host defense against invading pathogens, accumulating evidence has provided unprecedented insight into new roles of complement in driving tumor development. Many complement components and effector pathways (e.g. C3, C5a, C5aR1) have been linked to tumorigenesis in various types of cancer, affecting tumor cell proliferation, myeloid cell-derived immunosuppression, angiogenesis and metastasis. Here, we have investigated the role of the proinflammatory complement axis C5a-C5aR1 in glioblastoma cell responses, in combination with radiotherapy. Prominent C5aR1 (CD88) expression was detected on glioblastoma cell lines using flow cytometry. Moreover, exposure of U87MG cells to radiotherapeutic regimens led to upregulation of surface C5aR1 ex-pression 24 hr post irradiation. In addition, exposure of U87MG cells to radiation led to significantly increased secretion and proteolytic cleavage of C3 into its bioactive fragments C3b/iC3B/C3c, suggesting autologous activation of tumor cell-derived complement. U87MG cell proliferation was increased in the presence of C5a and proinvasive responses of glioma cells were potentiated in a C5a-C5aR1 dependent manner, as determined by MMP zymography and cell invasion studies. Overall, our findings implicate complement activation and C5aR1 signaling as early drivers of protumorigenic responses in glioblastoma and enhance the effort of developing targeted immunotherapies that could make glioma cells more sensitive to current therapeutic strategies.

O2-4. JOINT EVALUATIONOF CYTOGENETICS AND IMMUNOPHENOTYPING MAY ASSIST THE PROGNOSI-SOF MULTIPLE MYELOMA PATIENTS

Sylia Georgaki1, Paraskevi K. Micheli1, Ioannis V. Kostopoulos1, Efstathios Kastritis2, Evangelos Terpos2, Ourania E. Tsitsilonis1, Meletios A. Dimopoulos2

1Section of Animal and Human Physiology, Department of Biology & 2Department of Clinical Therapeutics, “Alexandra” Hos-pital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

BACKROUND: Multiple Myeloma (MM) is a complex and genetically heterogenous hematological malignancy of bone mar-row (BM) plasma cells (PC’s). The sequence of genomic aberrations begins with germline events that predispose to the disease, followed by early initiating-like events, before the later acquisition of genomic aberrations that lead to disease progression. Clinical studies have shown that MM patients’ survival seems to be affected, among other factors, by the ac-cumulation of specific cytogenetic aberrations and the presence of minimal residual disease (MRD) in the BM after therapy,

which has been proven to be an independent and reliable prognostic indicator. In this study, we aimed to reveal a possible correlation between the cytogenetic profile at diagnosis and MRD status after therapy of MM patients.

METHODS: BM samples of MM patients in complete remission (CR) for ≥2 years after therapy were analyzed by next gener-ation multiparameter flow cytometry (NGF), according to the EuroFlow Consortium recommendations. Cytogenetic profiling information was acquired using fluorescent in situ hybridization (FISH) for the following aberrations: del13q, del17p, t(4;14), t(11;14), t(14;16) and add1q. GraphPad Software 7.0 was used for the statistical analysis of data.

RESULTS: Preliminary analysis on a limited number of MM patients (n=54) did not show a statistically significant correlation between MRD status and the patients’ cytogenetic background; however, we noted a trend in the accumulation of cytoge-netic aberrations in MRD+ patients. We also noticed the frequent presence of two particular aberrations, namely del13q and t(14;16),in initially MRD- MM patients that turned MRD+ at re-evaluation after 6 months, as well as in MRD+ MM patients with more than one phenotypically distinct clonal populations in their BM, although analysis of more samples is required to verify these observations.

CONCLUSIONS: Cytogenetic data analysis in conjunction with changes in the BMPC immunophenotype,and eventually also in the peripheral blood, of MM patients may provide reliable information that can help predict disease prognosis and response to treatment.

O2-5. INTESTINAL PSEUDO-OBSTRUCTION PRESENTED IN A PATIENT WITH LUNG CANCER UNDER IM-MUNOTHERAPY WITH CHECK POINT INHIBITORS

Dimitrios Massaras1, Georgios Fragulidis1, Eirini Pantiora1, Vasiliki Michalaki2, Elias Primetis3, Andreas Polydorou1, Georgios Polymeneas1

1 2nd Dept. of Surgery, ‘‘Aretaieio’’ Hospital, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece, 2 Dept. of Oncology, ‘‘Aretaieio’’ Hospital, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece, 3 1st Dept. of Radiology, ‘‘Aretaieio’’ Hospital, National and Kapodistrian University of Athens, School of Medicine, Athens, Greece

Background: Immune checkpoint inhibition therapy using monoclonal antibodies is a new therapeutic approach used in many oncology protocols with significant survival benefit for patients with several cancer types. Nivolumab is an anti-PD-1 immune checkpoint inhibitor that was recently developed for cancer immunotherapy with remarkable responses in non small cell lung cancer patients. However, immune checkpoint inhibition therapy can be associated with severe and unique immune related adverse effects as a consequence of impaired self-tolerance due to loss of T-cell inhibition. This occurs mostly via a nonselective pathway of activation of the immune system. Clinically the adverse effects can manifest as autoimmune-like/inflammatory side effects with collateral damage to various organ systems.

Methods-Results: We present an interesting case of a 62-year-old Caucasian male with recurrent lung adenocarcinoma under third-line treatment with nivolumab, who was admitted in our hospital with abdominal distension ,pain and vomiting. Radiologic findings were consistent with small bowel ileus. After conservative treatment with no improvement, the patient underwent exploratory laparotomy. Perioperative no cause of ileus was revealed. Postoperative the distension and pain persisted and considering that an adverse effect of the immune checkpoint inhibition therapy occurred, the patient received high-dose corticosteroids resulting in clinical improval.

Conclusion: Immune checkpoint inhibitors may cause various adverse effects to different organ systems and tissues. The main treatment consists of aggressive immune suppression with corticosteroids in the majority of cases. Due to the fact that clinical use of immune checkpoint inhibitors is expanding rapidly, there is an emergence of unique immune-related adverse effects in a growing patient population. Early awareness is essential in these patients in order to ensure prompt diagnosis and management.

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O2-6. TUMOR INFILTRATING LYMPHOCYTES (TILs) IN BREAST CANCER: CORRELATIONS WITH MOLECU-LAR PROFILE AND GRADE

Chryssoula Glava1, Argiro Mellou1, Loukia Psaridi1, Eleni Pigadioti1, Iriana Tsiakalou1, Sofokles Lanitis2, Vasilios Samaras1

1 Department of Pathology, “Korgialeneio – Benakeio”, Red Cross Hospital, Athens, Greece, 2 2nd Surgical Department and Unit of Surgical Oncology, “Korgialeneio – Benakeio”, Red Cross Hospital, Athens, Greece

Background Tumor infiltrating lymphocytes (TILs) are lymphocytes of the host immune system that have been observed within tumor sites, presumably migrating in order to combat the growing malignant cells. Assessment of TILs in histo-pathological specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved.

The aim of our study was to evaluate the presence of TILs in breast cancer and possible correlation of their density with Grade and molecular subtypes: overexpression of the HER2 gene (HER2-pos), presence of estrogen receptors (Luminal) and triple negative (TNBC).

Methods Stromal TILs were assessed in 100 invasive breast cancers: 50 HER2-pos and 50 without overexpression of the HER2 gene (HER2-neg), of which 30 Luminal and 20 TNBC.

Expression of estrogen receptors and overexpression of the HER2 gene in the samples examined were verified immunohis-tochemically. When necessary, HER2 gene overexpression was determined by CISH molecular method.

TILs were assessed in paraffin embedded and Hematoxylin-Eosin stained slides, according to the proposed guidelines for the assessment of TILs in solid tumors. Scores were classified in 3 groups of TILs density: Low (<10%), medium (10-49%), high (>50%), as previously for breast cancer.

Results and Conclusions Higher density of TILs was observed in TNBC and lower TILs density in Luminal tumors. There was no statistically significant difference between grade 1 and 2 cancers, while in grade 3 carcinomas the distribution of TILs was increased.

O2-7. TUMOR INFILTRATING LYMPHOCYTES (TILs) IN BLADDER CANCER: CORRELATIONS WITH RECUR-RENT DISEASE AND COMPARISON WITH BREAST CANCER TILs

Chryssoula Glava1, Loukis Psaridi1, Dimitrios Lepidas2, Adamantia Zizi - Serbetzoglou3

1 Department of Pathology, “Korgialeneio - Benakeio”, Red Cross Hospital, Athens, Greece, 2 Department of Urology, “Tza-neion” General Hospital of Pireaus, Greece, 3 Department of Pathology, “Tzaneion” General Hospital of Pireaus, Greece

BACKGROUND

Tumor infiltrating lymphocytes (TILs) are lymphocytes of the host immune system recruited into the tumour microenviron-ment in an attempt to control its growth. Assessment of TILs in histopathological specimens can provide important prog-nostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved.

The aim of our study was to evaluate whether TILs in biopsy specimens of bladder cancer was correlated with relapse and distribution of TILs at relapse and to compare distribution of TILs bladder cancer with distribution of TILs in breast cancer previously obtained by the same authors.

METHODS

TILs were assessed in 128 bladder urothelial carcinoma biopsy specimens, belonging to 95 patients at first diagnosis and in first relapse specimens of 33 of them after a median of 40 months follow-up). TILs were assessed in paraffin embedded and Hematoxylin-Eosin stained slides, according to the proposed guidelines for the assessment of TILs in solid tumors. Scores were classified in 3 groups of TILs density: Low (<10%), medium (10-49%), high (>50%), as previously for breast cancer.

RESULTS AND CONCLUSIONS

Tumor infiltrating lymphocytes were predominantly observed within cancer stroma, and only rare individual cells were observed intraepithelially. Cases without recurrent disease showed lower sTILs density than the group with recurrent dis-ease.

Both bladder and breast cancers are low immunogenic cancers, with predominance of stromal TILs rather than intraepithe-lial TILs and have similar distribution of sTILs, both globally and among grades.

O2-8. A NOVEL tRNA FRAGMENT AS A MOLECULAR BIOMARKER OF POOR PROGNOSIS IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA

Paraskevi Karousi1, Evdoxia Kamouza1, Marios A. Diamantopoulos1, Panagiotis Tsiakanikas1, Christos K. Kontos1, Sotirios G. Papageorgiou2, Andreas Scorilas1, Vassiliki Pappa2

1Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Athens, Greece; 2Sec-ond Department of Internal Medicine and Research Unit, University General Hospital “Attikon”, Athens, Greece.

Background: Chronic lymphocytic leukemia (CLL) is primarily a type of leukemia of older adults, with a median age of 71 years at the time of diagnosis. Less common, CLL sometimes affects people under the age of 40 and rarely children. Prog-nosis and survival of CLL patients depend on many factors. tRNA-derived RNA fragments (tRFs) could be promising in CLL patients’ overall survival (OS) prognosis. tRFs, also known as tRNA-derived stress-induced fragments (tiRNAs) are 16~28 nucleotide fragments derived from tRNA or pre-tRNA, which are classified by their sites of origin: tRF-5 and tRF-3 derive by cleavage from the 5’ and 3’ parts of mature tRNAs, respectively. tRF-1 originates from the 3’trailer from the pre-tRNA. i-tRF, is typically derived from the internal region of the mature tRNA. They participate in regulation of translation and gene silencing. tRFs are associated with or are causal factors for disease conditions including cancers, neurodegeneration, and metabolic disorders. This original study assesses the putative usefulness of an internal fragment of the tRNA(s) bearing the Glycine anticodon GCC (itRF-2-GlyGCC) as a prognostic biomarker in chronic CLL.

Methods: Peripheral blood mononuclear cells (PBMCs) were positively selected after centrifugation on a Ficoll-Hypaque gradient collected from 70 CLL patients and 22 non-leukemic blood donors. Cell immunophenotyping showed that most PBMCs isolated from patients’ blood samples were leukemic B cells. The mutational status of the immunoglobulin heavy chain variable (IGHV) region was specified by performing nucleic acid extraction. Total RNA was extracted from PBMCs and in-vitro polyadenylated using E. coli poly(A) polymerase, prior to first-strand cDNA synthesis starting from an oligo-dT–adaptor primer. Subsequently, a real-time quantitative polymerase chain reaction (qPCR) method was developed for the quantification of particular tRFs and applied in all samples. Extensive biostatistical analysis followed, including Kaplan-Meier survival analysis, univariate and multivariate Cox regression analysis.

Results and Conclusions: itRF-2-GlyGCC that was not differentially expressed between PBMCs of CLL patients and PBMCs of normal controls. However, Kaplan-Meier overall survival (OS) analysis showed increased OS for itRF-2-GlyGCC -negative compared to itRF-2-GlyGCC-positive CLL patients (p=0.01). Univariate Cox regression analysis for OS revealed a statisti-cally significant hazard ratio (HR) of 3.68 for itRF-2-GlyGCC-positive CLL patients (HR=3.68, (95% CI=1.29-10.53 P=0.015). Multivariate Cox regression analysis revealed that itRF-2-GlyGCC is a prognostic factor independent from Binet stage (or Rai stage), CD38 expression and IGHV mutational status. In conclusion, itRF-2-GlyGCC appears as a promising, molecular biomarker of unfavorable prognosis in CLL.

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02-9. MicroRNA-15a, A NOVEL MOLECULAR BIOMARKER IN COLORECTAL CANCER, PREDICTING PATIENTS’ RELAPSE

Panagiotis Tsiakanikas, Christos K. Kontos, Margaritis Avgeris, Marios A. Diamantopoulos, Andreas Scorilas


Background: Colorectal cancer is one of the most common gastrointestinal diseases and the second leading cause of cancer-associated deaths among adults. Many small non-coding RNAs, including microRNAs (miRNAs) are aberrantly expressed in solid tumors. miR-15a is a post-transcriptional regulator of the MYB proto-oncogene, a transcription factor essential for prolonged cancer cell proliferation and survival. In the current study, we examined the potential diagnostic and prognostic value of miR-15a expression in colorectal adenocarcinoma.

Methods: For this purpose, total RNA was extracted from 182 colorectal adenocarcinoma specimens and 86 non-cancerous colorectal mucosae. After polyadenylation of 2 μg total RNA by poly (A) polymerase and subsequent reverse transcription into first-strand cDNA using an oligo-dT–adapter primer, miR-15a expression was analyzed using an in-house developed reverse transcription quantitative real-time PCR method, based on SYBR Green chemistry. SNORD43 (RNU43) and SNORD48 (RNU48) were used as reference genes.

Results and Conclusions: miR-15a was significantly upregulated in colorectal tumors compared to non-cancerous col-orectal mucosae, while ROC analysis suggested its potential use for diagnostic purposes. Furthermore, overexpression of miR-15a predicts poor disease-free survival (DFS) and overall survival (OS). Multivariate Cox regression analysis confirmed that miR-15a overexpression is a significant predictor of poor prognosis concerning DFS in colorectal adenocarcinoma, independent of other established prognostic factors plus treatment of patients. Of note, miR-15a overexpression retains its unfavorable prognostic value in patients with T3 colorectal adenocarcinoma and in those without distant metastasis (M0). More importantly, the cumulative DFS probability of patients with early stage disease was significantly lower for those with colorectal adenocarcinoma overexpressing miR-15a. In conclusion, elevated expression of the cancer-associated miR-15a predicts poor DFS and OS of colorectal adenocarcinoma patients. The prognostic value of miR-15a expression regarding DFS is independent of clinicopathological factors currently used for colorectal adenocarcinoma prognosis.

Acknowledgments: The research presented was carried out within the framework of a Stavros Niarchos Foundation grant to the National and Kapodistrian University of Athens.

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FacultyProf. Dr. Gosse J. Adema, Chair in Molecular Immunology, Radiotherapy & OncoImmunology laboratory, Dept. of Radiation Oncology, RIMLS, Radboud University Medical Center, Nijmegen, The Netherlands

Prof. Athanassios Argiris, MD, PhD, FACP, Consultant Medical Oncologist, Hygeia Hospital/ Professor of Medi-cal Oncology, Department of Medical Oncology, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, USA

Dr. Tina Bagratuni, PhD, Post-doctoral Research Associate, Plasma Cell Dyscrasias Unit, Department of Clinical Therapeutics, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

Prof. Constantin N. Baxevanis, Scientific Director, Cancer Immunology and Immunotherapy Center “Agios Sav-vas” Hospital, Athens, Greece

Dr. Troels Holz Borch, MD, post- Doc, Center for Cancer Immune Therapy - Herlev Hospital, Herlev, Denmark

Dr. Anastasios Boutis, MD, PhD, Consultant Oncologist , Theagenio Cancer Hospital, Thessaloniki, Greece

Prof. Vincenzo Bronte, Professor of Medicine, Experimental medicine and pathophysiology, Director of Immu-nology Section, School of Medicine and Surgery, University of Verona, Verona, Italy

Prof. Federica Cavallo, Full Professor of Immunology, Department of Molecular Biotechnology and Health Sci-ences, Molecular Biotechnology Center, University of Turin, Italy

Prof. Aristides Eliopoulos, Professor of Biology , School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

Dr. Manuel Fernández Bruno, Medical Oncologist, Institute of Oncology Rosell (IOR), University Hospital Sagrat Cor, QuironSalud Group, Barcelona, Spain

Prof. Wolf H. Fridman, Professor Emeritus of Immunology, Laboratory Cancer, Immune Control and Escape, Cordeliers Research Centre, Paris, France- University Paris Descartes, Paris, France

Prof. Periklis Foukas, Associate Professor of Pathology, Attikon University Hospital, National and Kapodistrian University of Athens, Athens, Greece

Prof. Vassilis Georgoulias, Professor Emeritus of Medical Oncology, School of Medicine, University of Crete, Greece

Prof. Nikolaos Giannakoulas, MD, PhD, Assistant Professor, Internal Medicine & Hematology Dept, Faculty of Medicine, University of Thessaly, Larissa, Greece

PD Dr. Cécile Gouttefangeas, Group leader at the Institute for Cell Biology, Department of Immunology, Inter-faculty Institute for Cell Biology, University of Tübingen, Tübingen, Germany

Prof. Dominique Heymann, University Hospital Professor (Professeur des Universités -Praticien Hospitalier) - Head of Histology and Embryology Dept (Medical School, Nantes) - Head of EAL “Sarcoma Research Unit”, In-serm/University of Nantes/University of Sheffield (UK) - Head of Dept Biology-Research, Institut de Cancérologie de l’Ouest (ICO), France

Prof. Nathan Karin, PhD, Professor of Immunology, Department of Immunology, Chairman, Rappaport Inst., Rappaport Faculty of Medicine, Technion- Israel Institute of Technology, Haifa, Israel

Dr. Theodora Katsila, Senior Research Scientist-University Fellow, Department of Pharmacy, University of Pa-tras, Patra, Greece

Dr. Nikolaos Kentepozidis, MD, PhD, Medical Oncologist,Head of Oncology Clinic, 251 General Airforce Hospital, Athens, Greece

Dr. Dimitris Kletsas, Research Director, Laboratory of Cell Proliferation & Ageing Director, Institute of Biosci-ences & Applications, NCSR “Demokritos”, Athens, Greece

Prof. Michael I. Koukourakis, MD, Professor - Head Department of Radiotherapy and Oncology , Radiobiology and Radiopathology Unit , Democritus University of Thrace, Alexandroupolis - Greece

Dr. Anna Koumarianou, MD, PhD in Tumor Immunotherapies, Imperial College, Consultant in Medical Oncol-ogy, Hematology-Oncology Unit ENETs Centre of Excellence, Fourth Department of Internal Medicine, Attikon University Hospital, Haidari, Athens

Prof. Anastasios Koutsopoulos, MD, PhD, Assistant Professor of Pathology, Faculty of Medicine, University of Crete, Laboratory of Pathology, University of Heraklion, Crete, Greece

Prof. Athanasios Kotsakis, MD, PhD, Associate Professor of Medical Oncology, Director of Dpt of Medical Oncology, University Hospital of Larisa, Thessaly, Greece

Prof. Ed Lavelle, Adjuvant Research, School of Biochemistry and Immunology Trinity College, Dublin

Prof. Christoph Le Tourneau, MD, PhD, Professor of Medicine, Head of Early Phase Clinical Trials & Head and Neck Cancer Groups, Department of Medical Oncology, Institut Curie, Versailles-Saint-Quentin-en-Yvelines Uni-versity, INSERM U900 Research Unit, Paris, France

Dr. Michael Liontos, MD, PhD, Medical Oncologist, Locum Consultant at Oncology Unit, Dept of Clinical Thera-peutics, Alexandra Hospital, Athens, Greece

Prof. Ofer Mandelboim, Professor of Molecular Immunology, The Lautenberg Center for General and Tumor Immunology, The Faculty of Medicine, The Hebrew University Medical School, IMRIC, Jerusalem, Israel

Dr. Rhoda Lulama Molife, Senior Clinical Director Oncology, European Clinical Development, MSD, UK

Dr. Monica Neagu, Dr. habil. Head of Immunobiology Lab.”Victor Babes” National Institute of Pathology, Bucha-rest, Romania

Prof. Dr. Rienk Offringa, Professor, Head of Division of Molecular Oncology of Gastrointestinal Tumors (G180), Deutsches Krebsforschungszentrum - German Cancer Research Center (DKFZ), Heidelberg, Germany

Dr. Nikolaos Pistamaltzian, MD, PhD, Medical Oncologist, Consultant, Oncology Dpt “MITERA” Hospital, Athens, Greece

Prof. Emmanouil Saloustros, Assistant Professor of Oncology, Facullty of Medicine, School of Health Sciences, University of Thessaly, Greece

Dr. Christoph Schultes, PhD, Global Program Lead Oncology, Biopharma | Global R&D, Merck, Greece

Prof Dr. Barbara Seliger, Professor of Immunology, Director of the Institute for Medical Immunology, Martin-Luther-University Halle-Wittenberg, Halle/ Saale, Germany

Efstratios Stratikos, PhD, Research director INRASTES - Protein Chemistry, National Center for Scientific Re-search “Demokritos”, Athens, Greece

Dr. Dimitris Thanos, PhD, Investigator - Professor Level, President of the Scientific Board, Biomedical Re-search Foundation of the Academy of Athens, Athens, Greece

Prof. Ourania Tsitsilonis, MD, PhD, Associate Professor of Immunology, Department of Biology, National & Kapodistrian University of Athens, Greece

Prof. Viktor Umansky, PhD, Group Leader, Clinical Cooperation Unit Dermato-Oncology (G300), German Cancer Research Center (DKFZ), Foundation under Public Law, Heidelberg, Germany

Dr. Panagiotis Verginis, Investigator Assistant Professor Level - Biomedical Research Foundation of the Acad-emy of Athens, Athens, Greece

Prof. Dr. Theresa L. Whiteside, PhD, MDHC, Professor of Pathology, Immunology and Otolaryngology, UPMC Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, USA

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