Malignant Mesothelioma Epidemiology and molecular biology

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1 Malignant Mesothelioma Epidemiology and molecular biology Oluf Dimitri Røe, MD, PhD Assoc Prof. Levanger Hospital Department of Cancer Research and Molecular Medicine Norwegian University of Science and Technology (NTNU) Trondheim Norway Aalborg University Hospital, Denmark No disclosures 1 Short CV My name: Oluf Dimitri Røe Trondheim, Norway/ Mykonos, Greece 10 years GP 15 years Oncology PhD on mesothelioma Now: Consultant oncologist Postdoc NTNU Ass. Prof at Aalborg University, Aalborg, Denmark Hospital 3 1

2 Department of Cancer Research and Molecular Medicine Faculty of Medicine Norwegian University of Science and Technology Trondheim,Norway A day at the lab center Overview Asbestos Mesothelioma Epidemiology Mesothelioma Molecular Biology Biomarkers, Early Biomarkers Cancer-Biomarker HUNT - Where are we? 6 History of Asbestos One of our patients with Pleural mesothelioma A woman 42 years old The reason? She worked as a hair-dresser And used an Asbestos containing hair dryer For 10 years Asbestos (Greek, inextinguishable) a family of naturally occuring minerals that readily separate into thin fibers and are found in many parts of the world. Amiantus (Greek, untaintable, currently used in modern Greek for asbestos) was used by Pliny the Elder (23-79 AD) described that this was mined in the Montains of Arcadia, could be spun and woven to material and was resistant to fire [1]. The Greek geographer Strabo (63/64 BC-ca.24 AD) described similar use of the socalled Karystian stone from the ancient quarry in Evvia island of Greece Dioscorides (ca AD) described in his Materia Medica the use of this stone for for weaving napkins that could be cleaned and whitened by fire [1]. 2

3 History of Asbestos In Finland, asbestos was used in pottery 4500 years ago In Norway, bucket-shaped pottery tempered with crushed asbestos was used for storing and making food in the Late Roman and Migration periods (c AD) [2]. Marco Polo ( AD) also described asbestos mining in China [3]. Bucket-shaped pottery contains 20-25% clay and 65-80% asbestos Norway, AD 1. Dilek Y, Newcomb S, Geological Society of America. Meeting (2003) Geological Society of America. xii, 504 p. p. 2. Engevik A (2008) Oxford: Archaeopress. xiv, 240 p. p. 3. Rapp GR (2009) Archaeomineralogy. Berlin ; London: Springer. xv, 348 p. p. 9 Mesothelioma etiology: asbestos inhalation WHO and the IARC monograph (2012) all types of asbestos: class I carcinogens Crocidolite Amosite History of Asbestos Massive production and use started in the 20th century, due to its properties as insulation against heat, fire, corrosion and its tensile strenght. Anthrophyllite Chrysotile Asbestos was widely used for insulation of water- and combustion pipes, materials used for house construction and shipbuilding, car brakes and gaskets, even toys, jewellery, and cigarette filters, At its peak, 3000 products were registered 12 3

4 Norway Denmark World Europe Former Soviet Union Sweden AC USA AC Trends of asbestos consumption in metric tons based on Worldwide Asbestos Supply and Consumption Trends from 1900 through 2003 by Robert L. Virta Trends of asbestos consumption in metric tons based on Worldwide Asbestos Supply and Consumption Trends from 1900 through 2003 by Robert L. Virta A meso epidemic is coming in the 3 rd World mill tonn asbestos mined in countries. Russia 50% China 20% Brazil 12% Kazakhstan 10% Canada stopped

5 Metric tons Asbestos consumption in Denmark Mesothelioma incidens i North Jutland: 5/ fold incrase in 4 decades ( ) and increasing MM tilfælde År Panou V, Meristoudis C, Røe OD. 26 th European Pathology Congress, London 2014 År Asbestos exposure in North Jutland Overrepresentation of Peritoneal Mesothelioma in Women Types of asbestos exposure for females with MM 20% 20% 14% 46% DOMESTIC ENVIRONMENTAL OCCUPATIONAL UNCERTAIN Females with MM MM in pleura MM in peritoneum 20% 80% Males with MM 9% MM in pleura MM in peritoneum 91% Panou V, Omland Ø, Langhoff MD, Meristoudis C, Weinreich UM, Røe OD. 12 th International Mesothelioma Conference, Cape Town 2014 Panou V, Omland Ø, Vyberg M, Røe OD. 12 th International Mesothelioma Conference, Cape Town

6 Type of asbestos exposure and location of mesothelioma in women MPM and types of asbestos exposure MAM and types of asbestos exposure 85% 15% primary secondary 29% 71% primary secondary Panou V, Omland Ø, Meristoudis C, Hoffmann L, Røe OD. Abstract submitted to the 16 th World Conference on Lung Cancer, Denver Asbestos and Malignant Mesothelioma Asbestos and Malignant Mesothelioma Mainly by asbestos inhalation first documented by Dr. Wagner, South Africa, 1960 Both pleural and peritoneal mesothelioma is induced by asbestos inhalation Non-occupational exposures with known risk Second-hand exposure- Wife cleaning dusty clothes of husband or son, children indirectly exposed Langhoff et al Dan Med J Risk of mesothelioma is dependent on dose and duration of asbestos inhalation, but there is no lower limitone fiber can be enough Occupational exposure heavily exposed- max 10% got mesothelioma Several occupations with low exposure but still risk (plumber, carpenter, car mechanic.) Neighborhood exposure- living by an asbestos plant In Italy in Casale Monferrato (Eternit) and Bari (Fibronit) Risk- OR 10 and 5.25 (<500m from factory) respectively Barbieri et al Ann Occup Hyg 2012 Environmental exposure- asbestos or similar minerals in the soil Greece, Turkey, China, USA, Italy

7 Denmark Aalborg Eternit factory used asbestos from Cyprus The Danish Company bought and drifted the Amiantos mine in Cyprus Environmental exposure 50% of the mortality was of mesothelioma In the Karain village Genetical tendency? One village was moved! In Aalborg Path.Dept. 436 mesotheliomas from In Cyprus unknown number of mesotheliomas Cappadocia in Turkey, Erionit and Asbestos on the earth surface Other possible causes Confirmed: The contrast medium Thorotrast- alpha- emitter (not in use any more) Secondary cancer many years after thorax-peritoneal irradiation for lymphoma, testicular cancer and breast cancer. Hypothetical: SV40 virus- contaminated poliovaccine? Future challenges: Carbon nanotubes are already widely used have recently shown carcinogenic properties in vitro and in vivo The regulatory framework is not restrictive enough before letting these compounds into production The asbestos of the future? 27 Stella, Biointerphases

8 Incidence There are no reliable world-wide statistics on mesothelioma 0-6 cases / ,000-20,000 deaths / year, very unreliable figures, lack of registries USA 4000 / year UK 2200 / year Denmark 120 / year Western Australia had the highest incidence but North-Jutland in Denmark among highest in the world (5/ ) In Aalborg cases yearly Our first report linking asbestos with mesothelioma with asbestos from mainland China is now in press Zhibin Gao, Kenzo Hiroshima, Oluf D. Røe, Xing Zhang, Kenji Morinaga Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women: Analysis of 28 cases in Southeast China American Journal of Industrial Medicine, May 2015 Malignant Mesothelioma Facts Latency from asbestos exposure cancer median 40 years Diagnosis is often challenging and Patients are rarely diagnosed in an early stage Pleural Mesothelioma has 12 months median survival with pemetrexed/platinum chemotherapy 5-years overall survival 0-5% Schematic presentation of mesothelioma, the parietal and visceral pleura. Representative histology showing the most abundant cell types. Pleural plaque and mesothelioma start in the parietal pleura Parietal and visceral pleura serve different functions Parietal pleura (Mesothelial cells stain dark brown) Mesothelium Vascular endothelium Stroma with Fibrocytes, fibroblasts and collagen Fat cells Alveolar cells Mesothelioma with >80% tumour cells (brown) Visceral pleura 8

9 Mesothelioma pathophysiology Asbestos inhalation Asbestos fibers ----->Lung---->Pleura /Peritoneum ----->Cytotoxicity ----->DNA-damage--->Chronic inflammation----->mesothelioma (Latency yrs, median 40 yrs) multiple deletions eg. p16 (CDKN2A), NF2-Merlin Inactivation of p53 and prb but rarely mutation Differential gene (mrna) expression Differential DNA methylation Differential microrna expression Asbestos effects on mesothelium Clastogenic and cytotoxic in vitro Abnormal segregation at mitosis Deletions in 1p, 3p21, 6q, 9p21, 15q11-15, 22q Promoter hypermethylation at tumor suppressor loci 33 Known molecular changes in mesothelioma AP-1 transcriptional activity and NF-kB signaling pathway have been linked to mesothelial cell transformation and tumor progression. HGF and c-met are highly expressed PI3K, AKT and the downstream mtor are involved in cell growth and survival and are often found to be activated p16ink4a and p14arf are frequently inactivated and approximately 50% contain NF2 mutation Crocidolite fiber inhibits microvilli formation in the mesothelial cell Chrysotile fibers entangle In lung cancer cell division Lung cancer from asbestos exposed patient with distinct gain of 2p21(B) and loss of 2p16 (C) 9

10 Early Microarray studies-classifiers mrna expression of mesothelioma Lopez-Rios, Cancer Res 2006 Partial least square analysis (PLS) Every spot is the sum of gene expression i a biological sample related to the other samples. PV PP T 10

11 DNA repair pathways are differentially expressed CHEMOTHERAPY IRRADIATION TELOMERE MAINTENANCE DNA repair systems Repairs HR (homologous recombination) Double-strand break (DSB) NHEJ (non-homologous end joining) Single and DSB (error-prone) MMR (mismatch repair) Base mismatches BER (base excision repair) Wrong/defect bases NER (nucleotide excision repair) Wrong/defect nucleotides 41 DAMAGE RESPONSE CHEK1 DNA RAD50 ICL PCNA FEN1 DSB S/G2 HR CELL CYCLE ARREST CURCUMIN VARIOUS INHIBITORS RAD51AP1 BCCIP RAD21 RAD54L BRCA2/FANCD1 RAD18 FANCD2 TOP2A EME1 FANCA RAD50 FANCI FANCF UHRF1 UBE2V2 MMR BER/SP BER/LP NER PCNA MSH6 PCNA CCNO/Ung2 GTF2H2/p44 SHFM1 NHEJ RAD50 XRCC4 CIB1/DNA-PK RAD50 SURVIVAL NATURAL VANILIN RAD18 BORTEZOMIB 13 PROTEASOME GENES Searching for molecular resistance mechanisms in the individual patient One of our cases of Pleural mesothelioma A woman 42 years old 3 rd line Alimta Carbo 39 courses Biopsy before and after resistance >5 years later Genome-wide mrna expression 11

12 Gene expression- average signal 6/9/2015 Genes changed m ore than 2 -fold after resistance Tumour before resistance (above) and at acquired resistance (below) Change down Change up Expression before Expression after CHEK1 TYMS NQO1 CK7 TYMP SPARCL1 SPARCL1 SPARCL1 I GFBP7 I TM2 A SEPP1 FSTL1 SRPX SOX1 8 CXCL1 2 DCN LOC CRTAP OSR1 CLDN 1 CCL1 5 OGN SNAI 2 CMBL PCOLCE2 ZFP3 6 L2 ADD3 C4 orf1 8 CAV2 I L3 3 BCHE GPR1 7 PMP2 2 ECM2 KRT7 EEF1 A2 PI TX1 COMP MYOM2 SDC1 DAPL1 SYNPO2 L COL1 0 A1 MMP3 GUCY1 B2 HFE2 Gene symbols Cell cycle changes in tumour versus normal and in acquired resistance. Next-generation sequencing 12

13 Transcriptome/second generation sequencing of mesothelioma Sugarbaker et al PNAS 2007 Bueno et al PlosOne new mutations were discovered in 4 mesothelioma patients All had a different mutational profile Significant aneuploidy and numerous translocations Novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing Take home message: the deeper you search the more complex and individual changes you find Methylation and micrornas EPIGENETICS OF MESOTHELIOMA DNA methylation distinguishes mesothelioma, adenocarcinoma and normal lung and pleura Christensen et al Cancer Res 2009 microrna expression of mesothelioma 13

14 microrna origins from non-coding or junk-dna microrna facts >1800 have been discovered One microrna can regulate several hundred genes One gene can be regulated by many microrna Can act as tumor suppressors and oncogenes Can classify tumors Can show cancer risk Diagnostic/prognostic/predictive markers potential microrna are stable in body fluids and tumors and easy to measure by PCR 54 microrna in tumour can be used for differential diagnosis of mesothelioma versus other carcinomas Detection and analysis of cancer-specific microrna expression patterns Benjamin et al, J Mol Diagn November; 12(6):

15 microrna in serum for diagnosing mesothelioma microrna can be a prognostic marker of mesothelioma in serum Elevated hsa-mir-29c* was associated with a significantly longer TTP and survival time. Increased circulating mir-625-3p was found to be a potential biomarker for patients with malignant pleural mesothelioma. Kirschner et al, J Thorac Oncol Jul;7(7): Pass H I et al. Cancer Res 2010;70: by American Association for Cancer Research Genetic mesothelioma syndrome BAP1(BRCA1 associated protein 1) BAP1 is a tumor supressor gene located in the 3p21 BAP1 mutations cause a new cancer syndrome characterized by mesothelioma, uveal melanoma and melanocytic tumors BAP1 is also related to other cancers like renal cancer BAP1 provides researchers with a novel target for prevention and early detection. Carbone et al Nat Rev Cancer BAP1 mutation families and cancer In these two families (n=28): Mesothelioma (12) Uveal Melanoma (1) Skin cancer (2) Pancreatic cancer (1) Prostate cancer (2) Testa et al Nat Genetics

16 BAP1 cancer syndrome melanocytic BAP1-mutated atypical intradermal tumors (MBAITs) or epitheloid atypical Spitz tumors Carbone et al J Transl Med 2012 MOLECULAR BIOMARKERS Clinical Prognostic factors Epithelial vs non-epithelial types (biphasic and sarcomatous) Sex, female better prognosis Age, younger better prognosis But still we talk about differences in months WHAT are Molecular Biomarkers? a molecule detected in body fluids or tissues that are associated with a special process (normal or abnormal), a condition or disease (http://www.cancer.gov/) Single molecules or signatures of DNA, RNA, microrna, proteins, metabolites that may diagnose and subtype a disease prognose the natural disease path predict effect of certain treatments for this disease Ideally sensitivity and specificity higher than 80%

17 Why are diagnostic biomarkers problematic? Biomarkers for early diagnosis Tumor types and subtypes are heterogenous Clinically Histology-Morphology Molecular Genes Epigenetic Most cancers have a better prognosis by early detection Non-invasive methods based on blood analysis is an ideal method Non-invasive methods with SINGLE markers have repeatedly failed (CEA, CA125, CYFRA 19-9, mesothelin, osteopontin etc etc) BUT THERE IS HOPE Currently the only approved blood biomarker for diagnosing and monitoring mesothelioma is mesothelin (SMRP or N-ERC) Studies of biomarkers in blood samples of Mesothelioma (Panou et al, Cancer Treatment Reviews May 2015) 44 Mesothelioma Patients 228 Controls 67 Robinson et al. The Lancet

18 Current Status There are no clinically validated, non-invasive proven diagnostic markers for mesothelioma with high sensitivity and specificity Translational research urgently needed!! Studies of microrna in pre-diagnostic blood samples of Mesothelioma ZERO studies Studies only reached phase 2 of biomarker discovery and validation Our project Cancer-Biomarkers in HUNT: Biomarker discovery for early diagnosis of human mesothelioma and lung cancer. 間 皮 瘤 & 肺 癌 HUNT Research Centre HUNT Biobank Levanger, Norway microrna regulates mrna post-translationally. Cartoon from the Illumina homepage

19 Mesothelioma /9/ What is HUNT HUNT is the abbreviation of Helse-Undersøkelsen i Nord- Trøndelag (Health Survey of Nord-Trøndelag County) HUNT is a prospective Population-based biobank participants (70% of the population) Serum/ DNA collected 1998 and 2007 >400 clinical parameters registered pr. patient >500 lung cancer and 30 mesothelioma cases with blood samples 1-15 years PRIOR to clinical disease New biomarkers urgently needed Early diagnostic markers can save many lives world-wide Existing markers are not good enough or not validated for early diagnosis MicroRNA sequencing, metabolomics and proteomics of serum has not been combined and may identify unknown new markers Golden opportunity- we have the precious pre-clinical samples in the prospective HUNT biobank We identify those who got lung cancer or mesothelioma through linking to the cancer registry and identify matched samples (sex, age, smoke history, no cancer What is Cancer-Biomarker-HUNT Cancer-Biomarker HUNT: From clinical samples to biomarkers Biomarkers Clinical data Clinical collection history Physical exams Results collection Exams results Combined Clinical information and Validation Biological sample collection Serum/blood sample Tissue sample Nucleic acid extraction RNA microrna/ mrna Bioinformatics analyses Epigenetics DNA DNA Methylation Serum&samples&collected&and&accumulated&Mesothelioma& cases , ,000 25,000 20,000 15, ,000 5,

20 Techniques used in this study Is this study different? Next Generation Sequencing (NGS) -can detect 2000 micrornas Magnetic Resonance Spectrometry (MRS) Probably the first study with up-to-date, comprehensive high-throughput analysis of different classes of molecules in the same pre-diagnostic samples -can detect 40 metabolites MS Metabolomics -can detect 1500 metabolites MS Proteomics -can detect 1000 proteins Relations between microrna, proteins AND metabolites has been little studied, especially in SERUM Next-Generation sequencing (NGS) of serum microrna (Illumina) Small RNA sequencing is a powerful application, enabling the discovery and profiling of micrornas and other non-coding RNA, without prior genome annotation RNA isolation and library preparation RNA was isolated from 100 ul serum MicroRNA sequencing libraries were generated using the Illumina TruSeq small RNA Sample prep kit. Using low RNA inputs, one can profile the differential expression of known micrornas, as well as detect novel microrna targets and wide-ranging sequence variation or "isomirs" mirbase accessions Marker Small RNAs Ribosomal RNAs Illumina's small RNA sequencing methods enable accurate detection and quantification of rare small RNA sequences

21 MR based serum metabolomics Use 100microl serum Avance III 600MHz for proton Equipped with a QCI cryoprobe (for increased sensitivity) and a Bruker SampleJet (for automation) > 40 small molecular metabolites can be quantified (Chenomx) Orbitrap - MS proteomics and metabolomics MR Core Facility, NTNU: 82 Proteomic approach We correlate serum micrornas with proteomics and metabolomics Tryptic digestion of proteins, lyophilization and reconstitution for LC-MSMS Serum depletion using Mars Hu-14 (Agilent) for removal of the 14 most abundant proteins in Serum Metabolomic approach Protein precipitation using cold methanol and centrifugation Lyophilization and reconstitution for LC-MSMS Mass spectrometry for ID and/or (metabolomics) relative quantitation at PROMEC, NTNU 84 21

22 How to analyze huge amounts of data from 3-4 datasets? Comprehensive data analysis by a group of dedicated bioinformaticians (University of Crete, Prof. Tsamardinos) Using novel bioinformatics methods mine data for diagnostic signatures pathways and combinations Search for biological meaning Chemosensitivity Prediction of Tumours Based on Expression, mirna, and Proteomics Data I. Tsamardinos 1, 2, G. Bor boudakis 1, 2, E. G. Christodoulou 3, O. D. Røe 4, 5 1 Bioinformatics Laboratory, ICS-FORTH, Heraklion, Crete, Greece 2 Computer Science Department, University of Crete, Heraklion, Crete, Greece 3 Cellular Networks Group, BIOTEC TU Dresden, Dresden, Germany 4 Cancer Clinic, Levanger Hospital, Nord-Trøndelag Health Trust, Levanger, Norw ay 5 Department of Cancer Research and Molecular Medicine Norwegian University of Science and Technology (NTNU), Trondheim, Norway Conclusions I Asbestos (Greek, inextinguishable) is the term of a family of naturally occurring minerals 1960 J. C. Wagner, C. A. Sleggs and P. Marchand from South Africa linked mesothelioma to asbestos All types of asbestos are class-i carcinogens according to the WHO. Asbestos-related mesothelioma has a median lag-time from exposure to cancer of 40 years. Other etiological factors are environmental exposition to erionite, ionizing radiation and SV40 virus as a putative cofactor. Pleural mesothelioma has increasing incidence worldwide. Most mesotheliomas are of epithelial type, while the sarcomatous and biphasic types are less common Conclusions II early and combined markers Conclusions III - where are we? There were no pre-clinical sample studies for mesothelioma. The complexity of tumor biology probably calls for COMBINATION MARKERS for early diagnosis including global microrna and other non-coding RNAs metabolomics proteomics Immune markers Urgent need for large-scale, multicenter studies for mirna profiling in pre-diagnostic and general population samples and cross-validation in different populations in order to determine valid mirna signatures for timely cancer diagnosis. With a limited sample size we showed that our study design can identify potential early diagnosis biomarkers for lung cancer and mesothelioma ca 3 years prior to diagnosis. Probably detect subgroups as well Differential expression in metabolites and proteins and validation studies are ongoing

23 PUBLICATIONS ON MESOTHELIOMA 34. Zhibin Gao, Kenzo Hiroshima, Oluf D. Røe, Xing Zhang, Kenji Morinaga Asbestos textile production linked to malignant peritoneal and pleural mesothelioma in women: Analysis of 28 cases in Southeast China American Journal of Industrial Medicine, May V Panou, M Vyberg, U M Weinreich, C Meristoudis, U Falkmer, O D Røe The established and future biomarkers of malignant pleural mesothelioma Cancer Treatment Reviews, Online 8 th May Oluf D. Røe, Giulia M. Stella, Malignant pleural mesothelioma- history, controversy, and future of a man-made epidemic; review. Eur Respir Rev Mar 13. Xiao-feng Chen, Guo-qiang Ping,, Oluf Dimitri Røe, Yong-qian Shu, Ren-hua Guo, Case report: Recombinant human endostatin (endostar) decreased recurrent ascites, pleural fluid and ascitic VEGF in a case of advanced mesothelioma, J Chemother Aug 12. Oluf Dimitri Røe, Adam Szulkin, Helmut Sandeck, Endre Anderssen, Tore Amundsen, Sten Even Erlandsen, Katalin Dobra, Stein Harald Sundstrøm, Molecular Resistance Fingerprint of Pemetrexed and Platinum in a Long-Term Survivor of Mesothelioma. PLoS One European Respiratory Journal ERR Sandeck HP, Røe OD, Kjærheim K, Willén H, Larsson E. Re-evaluation of histological diagnoses of malignant mesothelioma by immunohistochemistry. Diagn Pathol Jul 4. Røe OD, Anderssen E, Sandeck H, Christensen T, Larsson E, Lundgren S. Malignant pleural mesothelioma: Genome-wide expression patterns reflecting general resistance mechanisms and a proposal of novel targets. Lung Cancer Jan 3. Oluf D. Røe, Endre Anderssen, Eli Helge, Caroline Hild Hakvaag Pettersen, Karina Standahl Olsen, Helmut Sandeck, Rune Haaverstad, Steinar Lundgren, Erik Larsson, Genome-wide profile of pleural mesothelioma versus parietal and visceral pleura: The emerging gene portrait of the mesothelioma phenotype. PLoS One Aug 2. Røe OD, Creaney J, Lundgren S, Larsson E, Sandeck H, Boffetta P, Nilsen TI, Robinson B, Kjaerheim K. Mesothelin-related predictive and prognostic factors in malignant mesothelioma: A nested case-control study. Lung Cancer Aug 1. Kjaerheim K, Røe OD, Waterboer T, Sehr P, Rizk R, Dai HY, Sandeck H, Larsson E, Anderssen A, Boffetta P, Pawlita M. Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples. Int J Cancer Jun Acknowledgements/Collaborators Norwegian University of Science and Technology (NTNU), Norway The HUNT research center Head Prof. Kristian Hveem Dept of Cancer Research and Molecular Medicine, Prof. Hans Krokan Prof Frank Skorpen Robin Mjelle, PhD student Lars Hagen, PhD Animesh Sharma, MD PhD Charalampis Giannadakis, MD Dept of Computers and Information Science Prof Pål Sætrom MRI-Cancer Group Prof Tone F Bathen Trygve Andeassen PhD Riyas Vettukatil, PhD University of Crete (UOC), Greece Dept of Computer Science Prof. Ioannis Tsamardinos Ass.Prof. Vincenzo Lagani Kostas Kerkentzes, PhD student Nanjing Medical University (NMU), China Prof Jianwei Zhou Wu Chen/Xiaofei Zhi PhD stud Aalborg University Hospital (AUH), Denmark Prof Ursula Falkmer Prof Hans E Johnsen Prof Mogens Vyberg Prof Øyvind Omland Overlege Christos Meristoudis Vasiliki Panou MD PhD stud/ Nikolaj A Jensen MD PhD stud Uppsala University (UU), Sweden Prof Erik Larsson 23

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