1. VTE and Cancer Cancer, Thrombosis, and the Biology of Malignancy Scientific Foundations for the Role of Low-Molecular-Weight Heparin Frederick R. Rickles, MD Professor of Medicine, Pediatrics, Pharmacology and Physiology The George Washington University Washington, DC Frederick R. Rickles, MD Professor of Medicine, Pediatrics, Pharmacology and Physiology The George Washington University Washington, DC Clotting, Cancer, and Controversies

2. VTE and Cancer (1801–1867) Cancer and Venous Thromboembolism The Legacy of Armand Trousseau

3. VTE and Cancer Professor Armand Trousseau Lectures in Clinical Medicine “ I have always been struck with the frequency with which cancerous patients are affected with painful oedema of the superior or inferior extremities….” New Syndenham Society – 1865

4. VTE and Cancer Professor Armand Trousseau More Observations About Cancer and Thrombosis “In other cases, in which the absence of appreciable tumour made me hesitate as to the nature of the disease of the stomach, my doubts were removed, and I knew the disease to be cancerous when phlegmasia alba dolens appeared in one of the limbs.” Lectures in Clinical Medicine, 1865 Lectures in Clinical Medicine, 1865

5. VTE and Cancer Trousseau’s Syndrome Ironically, Trousseau died of gastric carcinoma six months after writing to his student, Peter, on January 1st, 1867: “I am lost... the phlebitis that has just appeared tonight leaves me no doubt as to the nature of my illness”

6. VTE and Cancer Trousseau’s Syndrome ► Occult cancer in patients with idiopathic venous thromboembolism ► Thrombophlebitis in patients with cancer

7. VTE and Cancer Silver In: The Hematologist - modified from Blom et. al. JAMA 2005;293:715 Population-based case-control (MEGA) study N=3220 consecutive patients with 1 st VTE vs. n=2131 control subjects CA patients = OR 7x VTE risk vs. non- CA patients Population-based case-control (MEGA) study N=3220 consecutive patients with 1 st VTE vs. n=2131 control subjects CA patients = OR 7x VTE risk vs. non- CA patients Effect of Malignancy on Risk of Venous Thromboembolism (VTE) 0 10 20 30 40 50 Hematological Lung Gastrointestinal Breast Distant metastases 0 to 3 months 3 to 12 months 1 to 3 years 5 to 10 years > 15 years Adjusted odds ratio Type of cancer Time since cancer diagnosis 28 22.2 20.3 4.9 19.8 53.5 14.3 2.6 1.1 3.6

8. VTE and Cancer Cancer, Mortality, and VTE Epidemiology and Risk ► Patients with cancer have a 4- to 6-fold increased risk for VTE vs. non-cancer patients ► Patients with cancer have a 3-fold increased risk for recurrence of VTE vs. non-cancer patients ► Cancer patients undergoing surgery have a 2-fold increased risk for postoperative VTE ► Death rate from cancer is four-fold higher if patient has concurrent VTE ► VTE 2 nd most common cause of death in ambulatory cancer patients (tied with infection) Heit et al Arch Int Med 2000;160:809-815 and 2002;162:1245-1248; Prandoni et al Blood 2002;100:3484-3488; White et al Thromb Haemost 2003;90:446-455; Sorensen et al New Engl J Med 2000;343:1846-1850); Levitan et al Medicine 1999;78:285-291; Khorana et al Heit et al Arch Int Med 2000;160:809-815 and 2002;162:1245-1248; Prandoni et al Blood 2002;100:3484-3488; White et al Thromb Haemost 2003;90:446-455; Sorensen et al New Engl J Med 2000;343:1846-1850); Levitan et al Medicine 1999;78:285-291; Khorana et al J Thromb Haemost 2007;5:632-4

9. VTE and Cancer Mechanisms of Cancer-Induced Thrombosis: The Interface 1.Pathogenesis? 2.Biological significance? 3.Potential importance for cancer therapy?

10. VTE and Cancer “There appears in the cachexiae…a particular condition of the blood that predisposes it to spontaneous coagulation.” Lectures in Clinical Medicine, 1865 Trousseau’s Observations (continued)

11. VTE and Cancer Copyright ©2007 American Society of Hematology. Copyright restrictions may apply. Varki, A. Blood 2007;110:1723-1729 Multiple Mechanisms in Trousseau's Syndrome Tissue Factor microparticles

12. VTE and Cancer Fibrinolytic activities : t-PA, u-PA, u-PAR, PAI-1, PAI-2 Procoagulant Activities FIBRIN Endothelial cells IL-1, TNF-  VEGF Tumor cellsMonocyte PMN leukocyte Activation of coagulation Platelets Angiogenesis, Basement matrix degradation Falanga and Rickles, New Oncology: Thrombosis, 2005; Hematology, 2007 Interface of Biology and Cancer

13. VTE and Cancer Pathogenesis of Thrombosis in Cancer – A Modification of Virchow’s Triad 1.Stasis l Prolonged bed rest l Extrinsic compression of blood vessels by tumor 2.Vascular Injury l Direct invasion by tumor l Prolonged use of central venous catheters l Endothelial damage by chemotherapy drugs l Effect of tumor cytokines on vascular endothelium 3.Hypercoagulability l Tumor-associated procoagulants and cytokines (tissue factor, CP, TNF , IL-1 , VEGF, etc.) l Impaired endothelial cell defense mechanisms (APC resistance; deficiencies of AT, Protein C and S) l Enhanced selectin/integrin-mediated, adhesive interactions between tumor cells,vascular endothelial cells, platelets and host macrophages

14. VTE and Cancer Mechanisms of Cancer-Induced Thrombosis: Clot and Cancer Interface 1.Pathogenesis? 2.Biological significance? 3.Potential importance for cancer therapy?

15. VTE and Cancer Activation of Blood Coagulation in Cancer Biological Significance? ► Epiphenomenon? Is this a generic secondary event where thrombosis is an incidental finding Is this a generic secondary event where thrombosis is an incidental finding or, is clotting activation... or, is clotting activation... ► A Primary Event? Linked to malignant transformation Linked to malignant transformation

16. VTE and Cancer TF VEGF Angiogenesis Endothelial cells IL-8 Blood Coagulation Activation FIBRIN PAR-2 Angiogenesis FVII/FVIIa THROMBIN Tumor Cell TF Falanga and Rickles, New Oncology:Thrombosis, 2005;1:9-16 Interface of Clotting Activation and Tumor Biology

17. VTE and Cancer Coagulation Cascade and Tumor Biology TFThrombin Clotting- dependent Clotting- independent Clotting- dependent Fibrin Clotting- independent PARs Fernandez, Patierno and Rickles. Sem Hem Thromb 2004;30:31; Ruf. J Thromb Haemost 2007;5:1584 VIIa Xa Angiogenesis, Tumor Growth and Metastasis

18. VTE and Cancer Regulation of Vascular Endothelial Growth Factor Production and Angiogenesis by the Cytoplasmic Tail of Tissue Factor 1.TF regulates VEGF expression in human cancer cell lines 2.Human cancer cells with increased TF are more angiogenic (and, therefore, more “metastatic’) in vivo due to high VEGF production Abe et al Proc Nat Acad Sci 1999;96:8663-8668; Ruf et al Nature Med Abe et al Proc Nat Acad Sci 1999;96:8663-8668; Ruf et al Nature Med 2004;10:502-509

19. VTE and Cancer 3.The cytoplasmic tail of TF, which contains three serine residues, appears to play a role in regulating VEGF expression in human cancer cells, perhaps by mediating signal transduction 4. Data consistent with new mechanism(s) by which TF signals VEGF synthesis in human cancer cells may provide insight into the relationship between clotting and cancer Abe et al Proc Nat Acad Sci 1999;96:8663-8668; Ruf et.al. Nature Med 2004;10:502-509 Regulation of Vascular Endothelial Growth Factor Production and Angiogenesis by the Cytoplasmic Tail of Tissue Factor

20. VTE and Cancer Activation of Blood Coagulation in Cancer and Malignant Transformation ► Epiphenomenon vs. Linked to Malignant Transformation? 1.MET oncogene induction produces DIC in human liver carcinoma (Boccaccio lab) (Boccaccio et al 2005;434:396-400) (Boccaccio et al Nature 2005;434:396-400) 2.Pten loss and EGFR amplification produce TF activation and pseudopalisading necrosis through JunD/Activator Protein-1 in human glioblastoma (Bratt lab) (Rong et al 2005;65:1406-1413; 2009;69:2540-9) (Rong et al Ca Res 2005;65:1406-1413; Ca Res 2009;69:2540-9) 3.K-ras oncogene, p53 inactivation and TF induction in human colorectal carcinoma; TF and angiogenesis regulation in epithelial tumors by EGFR (ErbB1) – relationship to EMTs (Rak lab) (Yu et al 2005;105:1734-1741; Milson et al 2008;68:10068-76) (Yu et al Blood 2005;105:1734-1741; Milson et al Ca Res 2008;68:10068-76)

21. VTE and Cancer ► MET encodes a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF)  l Drives physiological cellular program of “invasive growth” (tissue morphogenesis, angiogenesis and repair) l Aberrant execution (e.g. hypoxia-induced transcription) is associated with neoplastic transformation, invasion, and metastasis Boccaccio et al 2005;434:396-400 Boccaccio et al Nature 2005;434:396-400 “1. MET Oncogene Drives a Genetic Programme Linking Cancer to Haemostasis” Activation of Blood Coagulation in Cancer: Malignant Transformation Activation of Blood Coagulation in Cancer: Malignant Transformation

22. VTE and Cancer ► Mouse model of Trousseau’s Syndrome l Targeted activated human MET to the mouse liver with lentiviral vector and liver-specific promoter  slowly, progressive hepatocarcinogenesis l Preceded and accompanied by a thrombo- hemorrhagic syndrome l Thrombosis in tail vein occurrs early and is followed by fatal internal hemorrhage l Syndrome characterized by  d-dimer and PT and  platelet count (DIC) “MET Oncogene Drives a Genetic Programme Linking Cancer to Haemostasis”

23. VTE and Cancer Blood Coagulation Parameters in Mice Transduced with the MET Oncogene Transgene Parameter Parameter Time after Transduction (days) Time after Transduction (days) 0 30 90 0 30 90GFP_________MET Platelets (x10 3 ) D-dimer (µg/ml) PT (s) ________________ Platelets (x10 3 ) D-dimer (µg/ml) PT (s) 968 656 800 <0.05 <0.05 <0.05 12.4 11.6 11.4 _______________________________ 974 350 150 <0.05 0.11 0.22 12.9 11.8 25.1

24. VTE and Cancer ► Mouse model of Trousseau’s Syndrome l Genome-wide expression profiling of hepatocytes expressing MET - upregulation of PAI-1 and COX- 2 genes with 2-3x  circulating protein levels l Using either XR5118 (PAI-1 inhibitor) or Rofecoxib (Vioxx; COX-2 inhibitor) resulted in inhibition of clinical and laboratory evidence for DIC in mice “MET Oncogene Drives a Genetic Programme Linking Cancer to Haemostasis”

25. VTE and Cancer Activation of Blood Coagulation in Cancer: Malignant Transformation 2. “Pten and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation By Glioblastoma” ► Pten = tumor suppressor with lipid and protein phosphatase activity ► Loss or inactivation of Pten (70-80% of glioblastomas) leads to Akt activation and upregulation of Ras/MEK/ERK signaling cascade Rong et al Ca Res 2005;65:1406-1413

26. VTE and Cancer ► Glioblastomas characterized histologically by “pseudopalisading necrosis” ► Thought to be wave of tumor cells migrating away from a central hypoxic zone, perhaps created by thrombosis ► Pseudopalisading cells produce VEGF and IL-8 and drive angiogenesis and rapid tumor growth ► TF expressed by >90% of grade 3 and 4 malignant astrocytomas (but only 10% of grades 1 and 2) “Pten and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation By Glioblastoma”

27. VTE and Cancer Results: 1.Hypoxia and PTEN loss  TF (mRNA, Ag and procoagulant activity); partially reversed with induction of PTEN 2.Both Akt and Ras pathways modulated TF in sequentially transformed astrocytes. 3.Ex vivo data:  TF (by IH-chemical staining) in pseudopalisades of # 7 human glioblastoma specimens “Pten and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation By Glioblastoma”

28. VTE and Cancer Both Akt and Ras Pathways Modulate TF Expression By Transformed Astrocytes N = Normoxia H = Hypoxia H = Hypoxia Similar data for EGFR – upregulation of TF via JunD/ AP-1 transcription (CA Res 2009;69:2540-9)

29. VTE and Cancer Pseudopalisading necrosis Vascular Endothelium H&E TF IHC “Pten and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation By Glioblastoma”

30. VTE and Cancer Activation of Blood Coagulation in Cancer: Malignant Transformation Activation of Blood Coagulation in Cancer: Malignant Transformation 3. “Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells: Implications For Tumor Progression And Angiogenesis” 3. “Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells: Implications For Tumor Progression And Angiogenesis” ► Activation of K-ras oncogene and inactivation of p53 tumor suppressor  TF expression in human colorectal cancer cells ► Transforming events dependent on MEK/MAPK and PI3K ► Cell-associated and MP-associated TF activity linked to genetic status of cancer cells ► TF siRNA reduced cell surface TF expression, tumor growth and angiogenesis ► TF may be required for K-ras-driven phenotype Yu et al 2005;105:1734-41 Yu et al Blood 2005;105:1734-41

31. VTE and Cancer “Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells: Implications For Tumor Progression And Angiogenesis” TF expression in cancer cells parallels genetic tumor progression with an impact of K-ras and p53 status Activation of Blood Coagulation in Cancer: Malignant Transformation Activation of Blood Coagulation in Cancer: Malignant Transformation Mean Channel TF Flourescence TF Activity (U/10 6 cells) del/+mut/+mut/+ +/++/+del/del

32. VTE and Cancer “Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells: Implications For Tumor Progression And Angiogenesis” Effect of TF si mRNA on tumor growth in vitro and in vivo Activation of Blood Coagulation in Cancer: Malignant Transformation Activation of Blood Coagulation in Cancer: Malignant Transformation

33. VTE and Cancer Effect of TF si mRNA on new vessel formation in colon cancer “Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells” “Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells” %VWF-Positive Area

34. VTE and Cancer “Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells: Implications For Tumor Progression And Angiogenesis” Matrigel Assay: (D) HCT 116; (E) SI-3 cells – vWF immunohistology Activation of Blood Coagulation in Cancer: Malignant Transformation Activation of Blood Coagulation in Cancer: Malignant Transformation Similar amplification of TF with upregulated VEGF induced by mutated EGFR in glioblastoma and lung cancer cells, accompanied by epithelial-to-mesenchymal transition (EMT) Milsom et al 2008;68:10068-76 Milsom et al CA Res 2008;68:10068-76

35. VTE and Cancer Kalluri and Kansaki Nature 2008;452:543 (  21 nucleotides)* * Kleinman et al Nature 2008;452:591 2008;452:591 Class Effect of siRNA for Angiogenesis Inhibition via Toll-Like Receptior 3 (TLR 3)

36. VTE and Cancer Mechanisms of Cancer-Induced Thrombosis: Implications 1.Pathogenesis? 2.Biological significance? 3.Potential importance for cancer therapy?

37. VTE and Cancer Activation of Blood Coagulation in Cancer: Malignant Transformation ► Q: What do all of these experiments in mice have to do with real patients with cancer? ► A: They suggest two things: ● Tumor cell-derived, TF-rich microparticles (MPs) may be important as a predictive test for VTE ● All patients with oncogene-driven cancer may need prophylactic anticoagulation

38. VTE and Cancer ► Retrospective study ► Immunohistologic (IH) and microarray data on expression of TF and VEGF, as well as microvascular density (MVD) in: l Normal pancreas (10) l Pre-malignant pancreatic lesions: Intraductal papillary mucinous neoplasms (IPMN; 70)Intraductal papillary mucinous neoplasms (IPMN; 70) Pancreatic intrepithelial neoplasia (PanIN; 40)Pancreatic intrepithelial neoplasia (PanIN; 40) l Resected or metastatic pancreatic adenoca (130) ► Survival ► VTE Rate Tissue Factor Expression, Angiogenesis, and Thrombosis in Human Pancreatic Cancer Khorana et al Clin Cancer Res 2007;13:2870

39. VTE and Cancer Immunohistologic Correlation of TF with the Expression of Other Angiogenesis Variables in Resected Pancreatic Cancer High TF Low TFP High TF Low TFP expression expression ____________________________________________________ VEGF expression l Negative 13 41 <0.0001 l Positive 53 15 Microvessel density l V6 per tissue core 27 33 0.047 l >6 per tissue core 39 23 l Median 8 6 0.01 ---------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------- Khorana et.al. Clin CA Res 2007:13:2870

40. VTE and Cancer Symptomatic VTE in Pancreatic Cancer Khorana et al Clin CA Res 2007;13:2872 5/19; 26.3% 1/22; 4.5%

41. VTE and Cancer Median Survival of #122 Resected Pancreatic Cancer Patients months 17.9 12.6 P = 0.16 (HR 2.06; 0.74-5.7) Khorana et al Clin CA Res 2007;13:2872

42. VTE and Cancer 1. Does activation of blood coagulation affect the biology of cancer positively or negatively? 2. Can we treat tumors more effectively using coagulation protein targets? 3. Can anticoagulation alter the biology of cancer? Cancer and Thrombosis: Year 2009 State-of-the-Science Update Cancer and Thrombosis: Year 2009 State-of-the-Science Update Key Questions Key Questions

43. VTE and Cancer 1. Epidemiologic evidence is suggestive that VTE is a bad prognostic sign in cancer 2. Experimental evidence is supportive of the use of antithrombotic strategies for both prevention of thrombosis and inhibition of tumor growth 3. Results of recent, randomized clinical trials of LMWHs in cancer patients indicate superiority to oral agents in preventing recurrent VTE, as well as increasing survival (not due to prevention of VTE) Cancer and Thrombosis: Year 2009 State-of-the-Science Update Tentative Answers

44. VTE and Cancer LMWH and Prolongation of Cancer Survival Mechanistic Explanations VTE Coagulation Proteases Direct Heparin Other

45. VTE and Cancer Heparins and Tumour Biology Multiple Potential Modes of Action Angiogenesis Apoptosis Heparanase Adhesion

46. VTE and Cancer Ex Vivo Angiogenesis: Embryonic Chick Aortic Rings Control Aortic Ring: Day 510U/ml Dalteparin-Treated Aortic Ring: Day 5 Fernandez, Patierno and Rickles. Proc AACR 2003;44:698 (Abstr. #3055)

47. VTE and Cancer Effects of Low-Molecular Weight Heparin on Lung Cancer Cell Apoptosis P<0.05 Chen et al Cancer Invest 2008;26:718-24 G1 arrest G1 arrest decrease in decrease in S phase S phase 3-fold  in p21 WAF1 3-fold  in p21 WAF1 and p27 KIP1 (p <0.01) and p27 KIP1 (p <0.01) reversed apoptosis reversed apoptosis and G1 arrest with and G1 arrest with p21 or p27 siRNA p21 or p27 siRNA

48. VTE and Cancer 0 100 200 300 400 500 VEGFFGF-2 TNF-  * * * * * * Cytokine+enoxaparin +dalteparin+UFH § § § Control * * * Marchetti et al. Thromb Res 2008;121:637-645 Heparins Inhibit Cytokine–Induced Capillary Tube Formation § = p<0.05 vs control, * = p<0.05 vs cytokine Tube Length (mm/cm 2 )

49. VTE and Cancer LMWH and VEGF Antisense Oligonucleotides Inhibit Growth and Metastasis of 3LL Tumors in Mice ► 40 mice with Lewis Lung Cancer (3LL) ► Rx qod x 15 with: ● Control (saline) ● VEGF antisense oligos (ASODN) ● VEGF mismatch sense oligo (MSODN) ● LMWH (dalteparin) ● LMWH + ASODN ► RESULTS: Growth Inhibit * Lung Mets * ● ASODN47%38% ● LMWH27%38% ● Combined59%25% * P < 0.05 Zhang YH et al Chinese Med J 2006;86:749-52

50. VTE and Cancer Inhibition of Binding of Selectins to Human Colon Carcinoma by Heparins Stevenson et al Clin Ca Res 2005;11:7003-11

51. VTE and Cancer Heparin Inhibition of B16 Melanoma Lung Metastasis in Mice Stevenson et al Clin Ca Res 2005;11:7003-11

52. VTE and Cancer Coagulation Cascade and Tumor Biology TFThrombin Clotting- dependent Clotting- independent Clotting- dependent Fibrin Clotting- independent PARs Fernandez, Patierno and Rickles. Sem Hem Thromb 2004;30:31; Ruf. J Thromb Haemost 2007; 5:1584 VIIa Xa Angiogenesis, Tumor Growth and Metastasis ? LMWHs (e.g. dalteparin); Non-anticoagulant heparins