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ACGH Detektiert Genomische Alterationen Des Intrahepatischen

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ACGH Detektiert Genomische Alterationen Des Intrahepatischen Aus der Klinik für Allgemein-, Viszeral- und Kinderchirurgie der Heinrich-Heine-Universität Düsseldorf Direktor: Univ.-Prof. Dr. med. Wolfram T. Knoefel ACGH detektiert genomische Alterationen des intrahepatischen Cholangiokarzinoms sowie der korrespondierenden Lymphknotenmetastasen und identifiziert eine Subklasse mit signifikanter Korrelation zum Überleben. Dissertation zur Erlangung des Grades eines Doktors der Medizin der Medizinischen Fakultät der Heinrich-Heine- Universität Düsseldorf vorgelegt von Ruben Jansen 2018 Als Inauguraldissertation gedruckt mit der Genehmigung der Medizinischen Fakultät der Heinrich-Heine-Universität Düsseldorf gez.: Dekanin/Dekan: Univ.-Prof. Dr. med. Wolfram T. Knoefel Referentin/Referent: PD Dr. med. Anja Lachenmayer Korreferentin/Korreferent: Prof. Dr. med. Nikolas Stoecklein Meinen Eltern Teile dieser Arbeit wurden veröffentlicht: Jansen R, Moehlendick B, Bartenhagen C, Toth C, Lehwald N, Stoecklein NH, et al. ACGH detects distinct genomic alterations of primary intrahepatic cholangiocarcinomas and matched lymph node metastases and identifies a poor prognosis subclass. Sci Rep. 2018;8(1):10637. doi: 10.1038/s41598-018-28941-6. PubMed PMID: 30006612; PubMed Central PMCID: PMCPMC6045619 [1] I Zusammenfassung Das intrahepatische cholangiozelluläre Karzinom (ICC) ist der zweithäufigste maligne primäre Lebertumor mit einer weltweit zunehmenden Inzidenz [2, 3]. Obwohl das Vorkommen von Lymphknotenmetastasen (LKM) bereits als negativer Prädikator für das Gesamtüberleben identifiziert werden konnte, sind die den Primärtumoren und LKM zugrundeliegenden genetischen Alterationen noch weitestgehend unverstanden [4]. In dieser Arbeit wurden mittels der komparativen genomischen Hybridisierung auf DNA-Microarrays (aCGH) 37 primäre intrahepatische ICCs und 14 korrespondierende LKM analysiert. Insgesamt wurden 60 Patienten mit ICC in diese Studie eingeschlossen, um relevante histo-pathologische und klinische Daten zu analysieren und mit den Ergebnissen zu korrelieren. Die aCGH Analyse aller Proben bestätigte die bereits bekannten Kopienzahlalterationen (Copy Number Alterations, CNAs) für das ICC und identifizierte zudem bisher nicht beschriebene Zugewinne auf Chromosom 19q. N1 Primärtumore zeigten insgesamt mehr CNAs und exklusive Zugewinne auf den Chromosomen 3p, 4q, 5p und 13q sowie Verlusten auf 17p und 20p im Vergleich zu den N0 Primärtumoren. In der gepaarten Analyse hatte ein Großteil der LKM (86%) die gleichen CNAs wie der korrespondierende Primärtumor, jedoch zeigten 79% der LKM auch isolierte Zugewinne auf (12q14 (36%); 1p13, 2p23, 7p22, 7q11, 11q12, 13q13 und 14q12 (>20%)). Die Clusteranalyse der Primärtumore konnte 2 unterschiedliche Gruppen identifizieren. Cluster B zeichnete sich durch eine hohe Anzahl an CNAs aus und zeigte eine signifikante Korrelation zu einer schlechten Tumordifferenzierung (G3/4) sowie ein signifikant schlechteres Überleben. Die Mehrzahl der Primärtumore clusterte bei der Analyse aller Tumore aufgrund der großen Ähnlichkeit der CNAs mit ihren korrespondierenden LKM ohne signifikante Unterschiede zwischen den untersuchten Paaren. TP53 und KRAS Mutationen konnten in 19% bzw. 6% der N1 Primärtumore und LKM detektiert werden. Gene, die in den neuen Zugewinnen und Verlusten der N1 Primärtumore und der LKM lagen, waren in wichtige Karzinom-assoziierte Signalwege involviert. Rauchen, Carbohydrat-Antigen 19-9 (CA 19-9) Serumlevel, Tumorstadium, mikrovaskuläre- und Perineuralscheideninvasion und R1-Resektion waren signifikant mit dem Lymphknotenstatus korreliert, während der Lymphknotenstatus, die R1- Resektion, eine schlechte Tumordifferenzierung (G3/4) signifikant mit einem schlechten Überleben korrelierten. Die aCGH Analyse der ICC Primärtumore und LKM identifizierte eindeutige genetische Unterschiede zwischen N0 und N1 Primärtumoren, wohingegen die N1 Primärtumore und ihre korrespondierenden LKM eine große Ähnlichkeit mit jedoch einigen exklusiven Zugewinnen in den Metastasen aufwiesen. Eine neue Subklasse an Tumoren zeigte eine signifikante Korrelation zu schlechter Tumordifferenzierung und schlechterem Überleben. Die in den alterierten Regionen lokalisierten Gene könnten möglicherweise für den Prozess der Metastasierung und der Tumorprogression ursächlich sein und stellen somit potentielle Zielstrukturen für eine zielgerichtete Krebstherapie dar. II Summary Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver malignancy with increasing incidence worldwide [2, 3]. While the occurrence of lymph node metastases (LNM) has been identified as a prognostic factor [4], genetic alterations in metastases and matched primary tumors have not been analyzed yet. Whole genome array comparative genomic hybridization (aCGH) was performed in 37 primary ICCs and 14 matched LNM. Sixty patients were studied for correlation of clinical and histopathological data. ACGH of all primary tumors confirmed known copy number alterations (CNAs) in ICC and identified to date undescribed gains in 19q. N1 tumors had more CNAs and harbored exclusive gains (3p, 4q, 5p, 13q) and losses (17p and 20p) compared to N0 tumors. LNM shared most of the CNAs with their matched primary tumor (86%), but 79% acquired new isolated gains (12q14 (36%); 1p13, 2p23, 7p22, 7q11, 11q12, 13q13 and 14q12 (>20%)). Unsupervised clustering revealed 2 significantly different groups. Cluster B was characterized by a high amount of the CNAs and was significantly associated to poor differentiation and worse survival. The majority of the primary tumors clustered with their matched LNM when analyzed together due to the high similarity of the CNAs. TP53 and KRAS occurred in 19% of tumors and in 6% of metastases. Pathway analyses revealed that genes located in the altered regions of N1 tumors and LNM were involved in cancer- associated pathways. Several clinical factors like advanced tumor stage, microvascular/perineural invasion, R1-resection, smoking and high carbohydrat-antigen 19-9 (CA-19-9) level were significantly correlated to the occurrence of LNM, while N1-status, R1- resection, and poor tumor differentiation were significantly correlated to survival. ACGH identified clear differences between N0 and N1 tumors, while N1 tumors and their LNM displayed great similarity with exclusive gains in the metastases. A novel cluster with a high amount of CNAs and poor tumor differentiation was significantly correlated to poor survival. Genes located in the altered regions are most likely involved in important cancer associated signaling pathways and might represent potential therapeutic targets for anti-tumoral therapy. III Abkürzungsverzeichnis AASLD Amerikanische Gesellschaft für Studien bei Lebererkrankungen ACG Amerikanische Gesellschaft für Gastroenterologie aCGH Array comparative genomic hybridization ADM-2 Aberration detection method 2 AFP Alpha Fetoprotein AP Alkalische Phosphatase Bcl-2 B-cell lymphoma 2 Bp Baasenpaare BRAF V-Raf murine sarcoma viral oncogene homolog B BRCA1 Breast Cancer 1 CA 19-9 Carbohydrat-Antigen 19-9 CA-125 Carbohydrat-Antigen 125 CAND1 Cullin Associated And Neddylation Dissociated 1 CCC Cholangiocellular Carcinoma (Cholangiozelluläres Karzinom) CD10 Cluster of differentiation 10 CDK6 Cyclin Dependent Kinase 6 CDKN2A cyclin-dependent kinase Inhibitor 2A CEA Carcinoembryonales Antigen CK Cytokeratin c-Met Tyrosine-protein kinase Met CNA Kopienzahlalterationen COX-2 Cyclooxygenase-2 CSNK2A1 Casein Kinase 2 Alpha 1 CT Computertopographie CTC Circulating tumor cell DNA Deoxyribonucleic acid DP1 Transcription Factor Dp-1 DTC Disseminated tumor cell ECC Extrahepatisches/Distales Cholangiokarzinom EGFR Epidermal Growth Factor Receptor ERBB3 Erb-B2 Rezeptor Tyrosinkinase 3 FFPE Formaldehyde-fixed paraffin-embedded FGFR2 Fibroblast growth factor receptor 2 FISH Fluoreszenz in situ Hybridisierung FTL1 Fms Related Tyrosine Kinase 1 GHR Growth Hormone Receptor GNS Glucosamine (N-Acetyl)-6-Sulfatase GSTP Glutathione S-Transferase Pi 1 HBV Hepatitis B Virus HCC Hepatozelluläres Karzinom HCV Hepatitis C Virus HE Hämatoxylin-Eosin HepPar-1 Hepatozyten-assozierten monoklonalen Antikörper HI Heterogenitätsindex HIV Humane Immundefizienz-Virus HMGA2 High Mobility Group AT-Hook 2 ICC Intrahepatisches Cholangiokarzinom IDH1 Isocitrat Dehydrogenase 1 IDH2 Isocitrat Dehydrogenase 2 IgG4 Immunglobulin G4 IL-6 Interleukin-6 INR International Normalized Ratio JAK- JAK = Januskinase, STAT = Signal Transducers and Activators of STAT Transcription KRAS V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog LIFR Leukemia Inhibitory Factor Receptor Alpha LNM Lymph node metastasis LKM Lymphknotenmetastase LTX Lebertransplantation MAPK Mitogen-Activated Protein Kinase 1 MDM2 Mouse double minute 2 homolog MRT Magnetresonanztomographie MSKCC Memorial Sloan-Kettering Cancer Center mTOR Mechanistic Target of Rapamycin NF1 Neurofibromin 1 NF-κB Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells NOS Stickstoffmonoxid-Synthase NRAS Neuroblastom RAS viral Onkogen homolog PCC Perihiläre Cholangiokarzinom PCR Polymerase chain reaction PDT Photodynamische Therapie PIK3CA Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha PLCB1 Phospholipase C Beta 1 PLCB4 Phospholipase C Beta 4 PPAR Peroxisome proliferator-activated receptors PSC Primär sklerosierende Cholangitis PT Primärtumor PTCH1 Patched 1 PTEN Phosphatase And Tensin Homolog RASSF1A Ras Association Domain Family Member 1 RKI Robert Koch Institut STAT3 Signal transducer and activator of transcription 3 TACE Transarterielle Chemoembolisation
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