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metavectum Recklinghausen Modern Moleculare Diagnostic in Oncology Aims of Molecular Oncology Early Diagnosis of Tumors Detection of disseminated Tumor-Cells and Micrometastasis Deciding of therapeutic Aims und Therapy Resistance Monitoring of Therapy Individualisised Therapy Tumorprogression by genetic Alterations Dissemination/Metastasis Cancer is caused by genetic Alteration cancer cells obtain Unkontrolled Growth Signal unnormal features Unresponsive against growth inhibitors Loss of Apoptosis Angiogenesis Metastasis creation Unlimited Replication Genetic Changes in Tumor-Cells Genes for Growth Factors Erb-b2, EGFR, c-Kit, PDGFR, K-ras Replications facilitating Genes Telomerase, c-myc Angiogenesis facilitating Genes VEGF Apoptosis facilitating Genes Bcl-2, p53 Circulating Tumor-cells (CTC) Risk-Factor for recurrance and metastasis. indicate risk for micro-metastasis. Easier indentification of patients profiting from adjuvant therapy. Suggested further reading: 1. Cristofanilli M. et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004 Aug 19;351(8):781-91. 2. Allard WJ et al.: Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases Clin Cancer Res. 2004 Oct 15;10(20):6897-904. Molekular Charakterisation of CTCs Isolation of mRNA from CTCs Measuring different Gene-Expression Quantitative real-time PCR of molekular Tumor-Marker (e.g.): • Cytokeratine • PSA (Prostata), G250 (kidney), AFP (liver) • Growth / Cell-cycle-Regulating Gene (c-myc, erbb2, Telomerase) Effect of Soy on Gene-Expression disseminated Tumor-Cells Five Patients per group suffering from metastastastic Prostate-, Ovaryand Breast -Ca received Chemo + fermented soy Clinical response in all patients Question: Can molecular factors predict clinical outcome of CaTherapy by fermented Soy ? Clincial : Bestimmung der modulierende Wirkung von Soja auf die Gen-Expression vor und nach 100 Tagen Einnahme des SojaExtraktes Funktion der Östrogenrezeptoren (ER) in der Zelle ER-alpha a a ERE DNA-Doppelstrang Zellteilungsaktivität wird gesteigert (Wucherung) ER-beta ß ß ERE DNA-Doppelstrang Zellteilungsaktivität wird verringert Expression der Östrogenrezeptoren (ER) alpha und beta Normale Zellen Brust / Ovar Prostata Tumorzellen mit Soja-Extrakt Tumorzellen unbehandelt konstantes Verhältnis Anstieg Absinken Anstieg ß Absinken a ß ER-beta ER-alpha ER-beta a ER-alpha ß ER-beta a ER-alpha Estrogen-receptor beta is induced by Haelan 951 Haelan increases ER2 expression in circulating tumor cells of P-CA patients (n=5) relative gene expression 600 vor H nach H 500 400 300 200 100 0 cases Modulation of estrogen-receptor expression by Haelan 951 relative gene expression Circulating Tumor Cells (CTCs) of prostate cancer patients were analysed for expression of ER-alpha and ER-beta 600 500 400 300 200 100 0 vor H nach H ER 1 vor H nach H ER 2 -> Expression of ER-beta was significantly induced by Haelan 951 Modulation of estrogen-receptor expression by Haelan 951 Mamma-Ca. patients n= 5 all shown breast cancer patients are ER-beta positive + relative Genexpression Estrogenreceptor ER-beta Estrogenreceptor-beta (ER-beta) Expression = No Haelan951 = Haelan951 application 33,9 Healthy persones without Haelan951 with Haelan951 6,2 Healthy persones n=7 Mamma-Ca. patients n=5 Relative Genexpression Mamma-Ca. Patients Estrogen-receptor beta is induced by Haelan 951 Haelan increases ER2 expression in circulating tumor cells of ovarian cancer patients (n=5) relative gene expression 100 vor H nach H 50 0 cases Modulation of estrogen-receptor expression by Haelan 951 Circulating Tumor Cells (CTCs) of ovarian cancer patients were analysed for expression of ER-alpha and ER-beta Induction of ER1 and ER2 by Haelan relative gene expression 200 100 0 vor H nach H ER 1 vor H nach H ER 2 -> Expression of ER-beta was significantly induced by Haelan 951 Stoffwechselweg der Apoptose Duales Signal von c-myc: • Der apoptotische Stoffwechselweg wird blockiert durch bcl2 / bax und c-myc fördert Proliferation • Nach Verlust des bcl2 Überlebenssignals, fördert c-myc die Apoptose proliferation mitogens c- myc apoptosis blocking Bcl2 NF - kB Bax (survival signals) Apoptotic conditioning induced by Healan 951 CTCs of prostate cancer patients showed an increased gene expression ratio of apoptosis-triggering genes bax/bcl2 after Haelan951-therapy Healan induces pro-apoptotic condition in circulating tumor cells of P-CA patients vor H nach H bax/bcl2 ratio 70 60 50 10 5 0 cases Apoptotic conditioning induced by Healan 951 Mamma-Ca. patients without Haelan951 n= 5 Haelan951 application 100 days relative Genexpression BAX/BCL2 no significant change 1,2 1,1 Apoptotic conditioning induced by Healan 951 Ovarian-Ca. patients n=6 relative gene expression 10 5 0 vor H Bax/Bcl2 nach H Bax/Bcl2 Apoptotic conditioning induced by Healan 951 relative gene expression Haelan increases BAX expression in circulating tumor cells of ovarian cancer patients (n=5) 30 vor H nach H 20 10 0 cases The p53 Signaling Pathway Bax block MDM2 p53 Bcl-2 Caspases block Cell cycle inhibitor Cyclin D1 CDK4 Cyclin E CDK2 Apoptosis p21 (Ovarian Ca.) block Cell Cycle Progression p21 Involvement in Cell Cycle p53 p21 CIP1 Cell cycle inhibitor Mitosis block Cyclin A/B CDK1 CDK2 M-phase entry G1 DNA-Synthesis M G2 S Chromosomes Duplication Cellcycle Inhibitor P21 is induced by Haelan951 Prostata-Ca. patients n=5 Haelan951 (100) Days) without Haelan951 56,3 relative Genexpression 37,8 12,4 7,1 1,9 0,5 0,9 cases 1,0 0,6 0,9 Cellcycle Inhibitor P21 is induced by Haelan951 without Haelan951 Haelan951 (100 days) Mamma-Ca. patients n=5 332,5 211,9 7,36 4,5 0,11 1,11 cases 2,28 scale unit progressive 15,0 14,3 relative Genexpression 15,8 Cellcycle Inhibitor P21 is induced by Haelan951 Mamma-Ca. patients without Haelan951 n= 5 Haelan951 application 100 days relative Genexpression p21 CIP increased 2,0 13,1 Cellcycle Inhibitor P21 is induced by Haelan951 Haelan increases P21expression in circulating tumor cells of ovarian cancer patients (n=5) relative gene expression 150 vor H nach H 100 50 0 cases Schlussfolgerungen • Alle Patienten zeigten auf die Gabe fermentierten Soja-Extraktes klinischen Response • Fermentiertes Soja-Extrakt fördert den pro-apoptotischen Zustand der Prostata-Karzinomzellen durch Herunterregulation von Bcl2. Dies dürfte die Zellen sensitiver machen für eine Chemotherapie und somit für eine Kombinationstherapie mit konventionellen Chemotherapeutika sprechen. • Zugabe von fermentiertem Soja induziert die Expression des antiproliferativen ER-beta bei Prostata- und Ovar-Ca Patienten. • Fermentiertes Soja-Extrakt induziert die Transkription des p21, welches zu einem Stillstand des Zellzyklus führt und damit das Krebswachstum unterdrückt, sowohl bei Prostata-, Ovar- als auch bei Mamma-Ca Patienten. metavectum Interaction The classical view of biological organization is to consider the flow of information from the genome to the transcriptome, to the proteome and then the metabolome. The metabolome can be used to monitor changes in the genome or to measure the effects of downregulation or upregulation of a specific gene transcript. Invluence of Haelan951 to VEGF Expression Haelan951 (100 Days) without Haelan951 37,5 Vascular Endothelial Growth Factor 1,46 Prostata-Ca. patients n=5 0,97 0,97 Mamma-Ca. patients n=5 relative Genexpression No alteration