VTE and Cancer Cancer, Thrombosis, and the Biology of Malignancy Scientific Foundations for the Role...

VTE and Cancer

Cancer, Thrombosis, and the Cancer, Thrombosis, and the Biology of Malignancy Biology of Malignancy

Scientific Foundations for the Role ofScientific Foundations for the Role ofLow-Molecular-Weight HeparinLow-Molecular-Weight Heparin

Cancer, Thrombosis, and the Cancer, Thrombosis, and the Biology of Malignancy Biology of Malignancy

Scientific Foundations for the Role ofScientific Foundations for the Role ofLow-Molecular-Weight HeparinLow-Molecular-Weight Heparin

Frederick R. Rickles, MDFrederick R. Rickles, MDProfessor of Medicine, Pediatrics, Professor of Medicine, Pediatrics,

Pharmacology and PhysiologyPharmacology and PhysiologyThe George Washington UniversityThe George Washington University

Washington, DCWashington, DC

Frederick R. Rickles, MDFrederick R. Rickles, MDProfessor of Medicine, Pediatrics, Professor of Medicine, Pediatrics,

Pharmacology and PhysiologyPharmacology and PhysiologyThe George Washington UniversityThe George Washington University

Washington, DCWashington, DC

Clotting, Cancer, and ControversiesClotting, Cancer, and Controversies

VTE and Cancer

(1801–1867)(1801–1867)

Cancer and Venous ThromboembolismCancer and Venous ThromboembolismThe Legacy of The Legacy of Armand TrousseauArmand Trousseau

VTE and Cancer

Professor Armand TrousseauProfessor Armand TrousseauLectures in Clinical MedicineLectures in Clinical Medicine

“ “ I have always been struck with the I have always been struck with the frequency with which cancerous patients frequency with which cancerous patients are affected with painful oedema of the are affected with painful oedema of the superior or inferior extremities….”superior or inferior extremities….”

New Syndenham Society – 1865New Syndenham Society – 1865

VTE and Cancer

Professor Armand TrousseauProfessor Armand TrousseauMore Observations About Cancer and ThrombosisMore Observations About Cancer and Thrombosis

““In other cases, in which the absence of In other cases, in which the absence of appreciable tumour made me hesitate as to appreciable tumour made me hesitate as to the nature of the disease of the stomach, my the nature of the disease of the stomach, my doubts were removed, and I knew the doubts were removed, and I knew the disease to be cancerous when disease to be cancerous when phlegmasia phlegmasia alba dolens alba dolens appeared in one of the limbs.”appeared in one of the limbs.”

Lectures in Clinical Medicine, 1865Lectures in Clinical Medicine, 1865

VTE and Cancer

Trousseau’s SyndromeTrousseau’s Syndrome

Ironically, Trousseau died of gastric carcinoma six Ironically, Trousseau died of gastric carcinoma six months after writing to his student, Peter, on January months after writing to his student, Peter, on January 1st, 1867:1st, 1867:

““I am lost . . . the phlebitis that has just I am lost . . . the phlebitis that has just appeared tonight leaves me no doubt as to appeared tonight leaves me no doubt as to the nature of my illness”the nature of my illness”

VTE and Cancer

Trousseau’s SyndromeTrousseau’s Syndrome

► Occult cancerOccult cancer in patients with idiopathic in patients with idiopathic venous thromboembolismvenous thromboembolism

► ThrombophlebitisThrombophlebitis in patients with cancerin patients with cancer

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 1st 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 1st VTE vs. n=2131 control subjects

• CA patients = OR 7x VTE risk vs. non-CA patients

Effect of Malignancy on Risk of Effect of Malignancy on Risk of Venous Thromboembolism (VTE)Venous Thromboembolism (VTE)

00

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22.222.220.320.3

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2.62.6 1.11.13.63.6

VTE and Cancer

Cancer, Mortality, and VTECancer, Mortality, and VTEEpidemiology and RiskEpidemiology and Risk

► Patients with cancer have a 4- to 6-fold increased risk for Patients with cancer have a 4- to 6-fold increased risk for VTE vs. non-cancer patientsVTE vs. non-cancer patients

► Patients with cancer have a 3-fold increased risk for Patients with cancer have a 3-fold increased risk for recurrence of VTE vs. non-cancer patientsrecurrence of VTE vs. non-cancer patients

► Cancer patients undergoing surgery have a 2-fold Cancer patients undergoing surgery have a 2-fold increased risk for postoperative VTEincreased risk for postoperative VTE

► Death rate from cancer is four-fold higher if patient has Death rate from cancer is four-fold higher if patient has concurrent VTEconcurrent VTE

► VTE 2VTE 2ndnd most common cause of death in ambulatory most common cause of death in ambulatory cancer patients (tied with infection)cancer patients (tied with infection)

Heit et al Heit et al Arch Int Med Arch Int Med 2000;160:809-815 and 2002;162:1245-1248; Prandoni et al 2000;160:809-815 and 2002;162:1245-1248; Prandoni et al Blood Blood 2002;100:3484-3488; White et al 2002;100:3484-3488; White et al Thromb Haemost Thromb Haemost 2003;90:446-455; Sorensen et al 2003;90:446-455; Sorensen et al New Engl J New Engl J Med Med 2000;343:1846-1850); Levitan et al 2000;343:1846-1850); Levitan et al Medicine Medicine 1999;78:285-291; Khorana et al1999;78:285-291; Khorana et al J Thromb Haemost 2007;5:632-4

VTE and Cancer

Mechanisms of Cancer-Induced Thrombosis: Mechanisms of Cancer-Induced Thrombosis: The InterfaceThe Interface

1.1. Pathogenesis?Pathogenesis?

2.2. Biological significance?Biological significance?

3.3. Potential importance for cancer therapy?Potential importance for cancer therapy?

VTE and Cancer

““There appears in the cachexiae…a There appears in the cachexiae…a

particular condition of the blood that particular condition of the blood that

predisposes it to spontaneouspredisposes it to spontaneous

coagulation.”coagulation.”

Lectures in Clinical Medicine, 1865Lectures in Clinical Medicine, 1865

Trousseau’s Observations (continued)Trousseau’s Observations (continued)

VTE and Cancer

Copyright ©2007 American Society of Hematology. Copyright restrictions may apply.

Varki, A. Blood 2007;110:1723-1729

Multiple Mechanisms in Multiple Mechanisms in Trousseau's SyndromeTrousseau's Syndrome

Tissue Factor microparticlesTissue Factor microparticles

VTE and Cancer

Fibrinolytic activitiesFibrinolytic activities::t-PA, u-PA, u-PAR, t-PA, u-PA, u-PAR, PAI-1, PAI-2PAI-1, PAI-2

Procoagulant Activities Procoagulant Activities

FIBRINFIBRIN

Endothelial cellsEndothelial cells

IL-1, IL-1, TNF-TNF-VEGVEGFF

Tumor cells

MonocyteMonocyte

PMN leukocytePMN leukocyte

Activation of Activation of coagulationcoagulation

PlateletsPlatelets

Angiogenesis,Angiogenesis,Basement matrix Basement matrix degradationdegradation

Falanga and Rickles, Falanga and Rickles, New Oncology: ThrombosisNew Oncology: Thrombosis, 2005; , 2005; Hematology, Hematology, 20072007

Interface of Biology and CancerInterface of Biology and Cancer

VTE and Cancer

Pathogenesis of Thrombosis in Cancer – Pathogenesis of Thrombosis in Cancer – A Modification of Virchow’s TriadA Modification of Virchow’s Triad

1.1. StasisStasis Prolonged bed restProlonged bed rest Extrinsic compression of blood vessels by tumorExtrinsic compression of blood vessels by tumor

2.2. Vascular InjuryVascular Injury Direct invasion by tumorDirect invasion by tumor Prolonged use of central venous cathetersProlonged use of central venous catheters Endothelial damage by chemotherapy drugsEndothelial damage by chemotherapy drugs Effect of tumor cytokines on vascular endotheliumEffect of tumor cytokines on vascular endothelium

3.3. HypercoagulabilityHypercoagulability Tumor-associated procoagulants and cytokines (tissue factor, Tumor-associated procoagulants and cytokines (tissue factor,

CP, TNFCP, TNF, IL-1, IL-1, VEGF, etc.), VEGF, etc.) Impaired endothelial cell defense mechanisms (APC resistance; Impaired endothelial cell defense mechanisms (APC resistance;

deficiencies of AT, Protein C and S) deficiencies of AT, Protein C and S) Enhanced selectin/integrin-mediated, adhesive interactions Enhanced selectin/integrin-mediated, adhesive interactions

between tumor cells,vascular endothelial cells, platelets and host between tumor cells,vascular endothelial cells, platelets and host macrophagesmacrophages

VTE and Cancer

Mechanisms of Cancer-Induced Thrombosis: Mechanisms of Cancer-Induced Thrombosis: Clot and Cancer InterfaceClot and Cancer Interface



3.3. Potential importance for cancer therapy?Potential importance for cancer therapy?

VTE and Cancer

Activation of Blood Coagulation in CancerActivation of Blood Coagulation in CancerBiological Significance?Biological Significance?

► EpiphenomenonEpiphenomenon? ?

Is this a generic secondary event where Is this a generic secondary event where thrombosis is an incidental findingthrombosis is an incidental finding

oor, is clotting activation . . .r, is clotting activation . . .

► A Primary Event?A Primary Event?

Linked to malignant transformation Linked to malignant transformation

VTE and Cancer

TF

VEGF

Angiogenesis

Endothelial cellsEndothelial cells

IL-8IL-8

Blood CoagulationActivation

FIBRIN

PAR-2

Angiogenesis

FVII/FVIIaFVII/FVIIa

THROMBINTHROMBIN

Tumor Cell

TF

Falanga and Rickles, New Oncology:Thrombosis, 2005;1:9-16

Interface of Clotting Activation Interface of Clotting Activation and Tumor Biology and Tumor Biology

VTE and CancerCoagulation Cascade and Tumor BiologyCoagulation Cascade and Tumor Biology

TFTF ThrombinThrombin

Clotting-Clotting-dependentdependent


Clotting-Clotting-independentindependent


FibrinFibrin


PARsPARs

Fernandez, Patierno and Rickles. Sem Hem Thromb 2004;30:31; Ruf. J Thromb Haemost 2007;5:1584

VIIaVIIa XaXa

Angiogenesis, Tumor Angiogenesis, Tumor Growth and MetastasisGrowth and Metastasis

VTE and Cancer

Regulation of Vascular Endothelial Growth Factor Production and Regulation of Vascular Endothelial Growth Factor Production and Angiogenesis by the Cytoplasmic Tail of Tissue FactorAngiogenesis by the Cytoplasmic Tail of Tissue Factor

1.1. TF regulates VEGF expression in human cancer TF regulates VEGF expression in human cancer cell linescell lines

2.2. Human cancer cells with increased TF are more Human cancer cells with increased TF are more angiogenic (and, therefore, more “metastatic’) angiogenic (and, therefore, more “metastatic’) in in vivovivo due to high VEGF production due to high VEGF production

Abe et al Abe et al Proc Nat Acad SciProc Nat Acad Sci 1999;96:8663-8668; Ruf et al 1999;96:8663-8668; Ruf et al Nature MedNature Med 2004;10:502-509

VTE and Cancer

3.3. The cytoplasmic tail of TF, which contains three The cytoplasmic tail of TF, which contains three serine residues, appears to play a role in regulating serine residues, appears to play a role in regulating VEGF expression in human cancer cells, perhaps VEGF expression in human cancer cells, perhaps by mediating signal transductionby mediating signal transduction

4.4. Data consistent with new mechanism(s) by which Data consistent with new mechanism(s) by which TF signals VEGF synthesis in human cancer cells TF signals VEGF synthesis in human cancer cells may provide insight into the relationship between may provide insight into the relationship between clotting and cancerclotting and cancer

Abe et al Abe et al Proc Nat Acad SciProc Nat Acad Sci 1999;96:8663-8668; Ruf et.al. 1999;96:8663-8668; Ruf et.al. Nature MedNature Med 2004;10:502-509 2004;10:502-509

Regulation of Vascular Endothelial Growth Factor Production and Regulation of Vascular Endothelial Growth Factor Production and Angiogenesis by the Cytoplasmic Tail of Tissue FactorAngiogenesis by the Cytoplasmic Tail of Tissue Factor

VTE and Cancer

ActivationActivation of Blood Coagulation of Blood Coagulation in Cancer and Malignant Transformationin Cancer and Malignant Transformation

► Epiphenomenon vs. Epiphenomenon vs. Linked to Malignant Linked to Malignant Transformation?Transformation?

1.1. METMET oncogene induction produces DIC in human liver oncogene induction produces DIC in human liver carcinomacarcinoma (Boccaccio lab) (Boccaccio lab)

(Boccaccio et al (Boccaccio et al Nature 2005;434:396-400) 2005;434:396-400)

2.2. PtenPten loss and loss and EGFREGFR amplification produce TF activation amplification produce TF activation and pseudopalisading necrosis through JunD/Activator and pseudopalisading necrosis through JunD/Activator Protein-1 in human glioblastomaProtein-1 in human glioblastoma (Bratt lab)(Bratt lab)

(Rong et al (Rong et al Ca Res 2005;65:1406-1413; 2005;65:1406-1413; Ca Res 2009;69:2540-9)2009;69:2540-9)

3.3. K-K-rasras oncogene, p53 inactivation and TF induction in oncogene, p53 inactivation and TF induction in human colorectal carcinoma; TF and angiogenesis human colorectal carcinoma; TF and angiogenesis regulation in epithelial tumors by regulation in epithelial tumors by EGFR (EGFR (ErbB1ErbB1)) – – relationship to EMTsrelationship to EMTs (Rak lab) (Rak lab)

(Yu et al (Yu et al Blood 2005;105:1734-1741; Milson et al 2005;105:1734-1741; Milson et al Ca Res 2008;68:10068-76) 2008;68:10068-76)

VTE and Cancer

► METMET encodes a tyrosine kinase receptor for hepatocyte encodes a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF) growth factor/scatter factor (HGF/SF) Drives physiologicalDrives physiological cellular program of “invasive cellular program of “invasive

growth” (tissue morphogenesis, angiogenesis growth” (tissue morphogenesis, angiogenesis and repair)and repair)

Aberrant execution (e.g. hypoxia-induced Aberrant execution (e.g. hypoxia-induced transcription) is associated with neoplastic transcription) is associated with neoplastic transformation, invasion, and metastasistransformation, invasion, and metastasis

Boccaccio et al Boccaccio et al Nature 2005;434:396-4002005;434:396-400

““1. 1. METMET Oncogene Drives a Genetic Programme Oncogene Drives a Genetic Programme Linking Cancer to Haemostasis”Linking Cancer to Haemostasis”

Activation of Blood Coagulation Activation of Blood Coagulation in Cancer: Malignant Transformationin Cancer: Malignant Transformation

VTE and Cancer

► Mouse model of Trousseau’s SyndromeMouse model of Trousseau’s Syndrome

Targeted activated human Targeted activated human METMET to the mouse liver to the mouse liver with lentiviral vector and liver-specific promoter with lentiviral vector and liver-specific promoter slowly, progressive hepatocarcinogenesisslowly, progressive hepatocarcinogenesis

Preceded and accompanied by a thrombo-Preceded and accompanied by a thrombo-hemorrhagic syndrome hemorrhagic syndrome

Thrombosis in tail vein occurrs early and is followed Thrombosis in tail vein occurrs early and is followed by fatal internal hemorrhageby fatal internal hemorrhage

Syndrome characterized by Syndrome characterized by d-dimer and PT and d-dimer and PT and platelet count (DIC)platelet count (DIC)

““METMET Oncogene Drives a Genetic Programme Oncogene Drives a Genetic Programme Linking Cancer to Haemostasis”Linking Cancer to Haemostasis”


VTE and Cancer

Blood Coagulation Parameters in Mice Blood Coagulation Parameters in Mice Transduced with the Transduced with the MET MET OncogeneOncogene

TransgeneTransgene ParameterParameter

Time after Transduction (days)Time after Transduction (days)

0 30 900 30 90

GFPGFP

__________________

METMET

Platelets (x10Platelets (x1033))

D-dimer (µg/ml)D-dimer (µg/ml)

PT (s)PT (s)

________________________________

Platelets (x10Platelets (x1033))

D-dimer (µg/ml)D-dimer (µg/ml)

PT (s)PT (s)

968 656 800 968 656 800

<0.05 <0.05 <0.05<0.05 <0.05 <0.05

12.4 11.6 11.412.4 11.6 11.4

______________________________________________________________

974 350 150974 350 150

<0.05 0.11 0.22<0.05 0.11 0.22

12.9 11.8 25.112.9 11.8 25.1

VTE and Cancer

► Mouse model of Trousseau’s SyndromeMouse model of Trousseau’s Syndrome

Genome-wide expression profiling of hepatocytes Genome-wide expression profiling of hepatocytes expressing expressing METMET - - upregulation ofupregulation of PAI-1PAI-1 and and COX-COX-22 genes with 2-3x genes with 2-3x circulating protein levels circulating protein levels

Using either XR5118 (Using either XR5118 (PAI-1 inhibitorPAI-1 inhibitor) or Rofecoxib ) or Rofecoxib (Vioxx;(Vioxx; COX-2 inhibitorCOX-2 inhibitor) resulted in inhibition of ) resulted in inhibition of clinical and laboratory evidence for DIC in miceclinical and laboratory evidence for DIC in mice



VTE and Cancer


2. “2. “PtenPten and Hypoxia Regulate Tissue Factor and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation By Expression and Plasma Coagulation By

Glioblastoma”Glioblastoma”► PtenPten = tumor suppressor with lipid and protein = tumor suppressor with lipid and protein

phosphatase activityphosphatase activity

► Loss or inactivation of Loss or inactivation of Pten Pten (70-80% of (70-80% of glioblastomas) leads to Akt activation and glioblastomas) leads to Akt activation and upregulation of upregulation of RasRas/MEK/ERK/MEK/ERK signaling cascade signaling cascade

Rong et al Ca Res 2005;65:1406-1413

VTE and Cancer

► Glioblastomas characterized histologically by Glioblastomas characterized histologically by “pseudopalisading necrosis” “pseudopalisading necrosis”

► Thought to be wave of tumor cells migrating away Thought to be wave of tumor cells migrating away from a central hypoxic zone, perhaps created by from a central hypoxic zone, perhaps created by thrombosisthrombosis

► Pseudopalisading cells produce VEGF and IL-8 Pseudopalisading cells produce VEGF and IL-8 and drive angiogenesis and rapid tumor growth and drive angiogenesis and rapid tumor growth

► TF expressed by >90% of grade 3 and 4 malignant TF expressed by >90% of grade 3 and 4 malignant astrocytomas (but only 10% of grades 1 and 2)astrocytomas (but only 10% of grades 1 and 2)

““PtenPten and Hypoxia Regulate Tissue Factor Expression and and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation By Glioblastoma”Plasma Coagulation By Glioblastoma”

VTE and Cancer

Results:Results:

1.1. Hypoxia and Hypoxia and PTEN PTEN loss loss TF (mRNA, Ag and TF (mRNA, Ag and procoagulant activity); partially reversed with procoagulant activity); partially reversed with induction of induction of PTEN PTEN

2.2. Both Both AktAkt and and RasRas pathways modulated TF in pathways modulated TF in sequentially transformed astrocytes.sequentially transformed astrocytes.

3.3. Ex vivo Ex vivo data: data: TF (by IH-chemical staining) in TF (by IH-chemical staining) in pseudopalisades of # 7 human glioblastoma pseudopalisades of # 7 human glioblastoma specimensspecimens

““PtenPten and Hypoxia Regulate Tissue Factor Expression and and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation By Glioblastoma”Plasma Coagulation By Glioblastoma”

VTE and Cancer

Both Both AktAkt and and RasRas Pathways Modulate TF Pathways Modulate TF Expression By Transformed AstrocytesExpression By Transformed Astrocytes

N = NormoxiaN = Normoxia

H = HypoxiaH = Hypoxia

Similar data Similar data for EGFR – for EGFR – upregulationupregulationof TF via JunD/of TF via JunD/AP-1 transcriptionAP-1 transcription((CA Res CA Res 2009;69:2540-9)2009;69:2540-9)

VTE and Cancer

Pseudopalisading necrosis

Vascular Endothelium

H&EH&E

TF IHC

““PtenPten and Hypoxia Regulate Tissue Factor Expression and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation By Glioblastoma”and Plasma Coagulation By Glioblastoma”

VTE and Cancer


3. “Oncogenic Events Regulate Tissue Factor 3. “Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells: Implications For Expression In Colorectal Cancer Cells: Implications For Tumor Progression And Angiogenesis”Tumor Progression And Angiogenesis”

► Activation of K-Activation of K-ras ras oncogene and inactivation of oncogene and inactivation of p53 p53 tumor tumor suppressor suppressor TF expression in TF expression in human human colorectal cancer cellscolorectal cancer cells

► Transforming events dependent on MEK/MAPK and PI3KTransforming events dependent on MEK/MAPK and PI3K► Cell-associated and MP-associated TF activity linked to genetic Cell-associated and MP-associated TF activity linked to genetic

status of cancer cellsstatus of cancer cells► TF siRNA reduced cell surface TF expression, tumor growth and TF siRNA reduced cell surface TF expression, tumor growth and

angiogenesis angiogenesis ► TF may be required for K-TF may be required for K-ras-ras-driven phenotype driven phenotype

Yu et al Yu et al Blood 2005;105:1734-412005;105:1734-41

VTE and Cancer

““Oncogenic Events Regulate Tissue Factor Expression In Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells: Implications For Tumor Progression Colorectal Cancer Cells: Implications For Tumor Progression

And Angiogenesis”And Angiogenesis”TF expression in cancer cells parallels genetic tumor progression TF expression in cancer cells parallels genetic tumor progression

with an impact of K-with an impact of K-ras ras and and p53 p53 statusstatus


0

50

100

150

200

250

300

350

400

450

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40

60

80

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VTE and Cancer

““Oncogenic Events Regulate Tissue Factor Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells: Expression In Colorectal Cancer Cells:

Implications For Tumor Progression And Implications For Tumor Progression And Angiogenesis”Angiogenesis”

Effect of TF si mRNA on tumor growth in vitro and in vivo


VTE and Cancer

0

2

4

6

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10

12

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Effect of TF si mRNA on new vessel formation in colon cancerEffect of TF si mRNA on new vessel formation in colon cancer

““Oncogenic Events Regulate Tissue Factor Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells”Expression In Colorectal Cancer Cells”

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VTE and Cancer

““Oncogenic Events Regulate Tissue Factor Expression In Oncogenic Events Regulate Tissue Factor Expression In Colorectal Cancer Cells: Implications For Tumor Colorectal Cancer Cells: Implications For Tumor

Progression And Angiogenesis”Progression And Angiogenesis”

Matrigel Assay: (D) HCT 116; (E) SI-3 cells – vWF immunohistology


Similar amplification of TF with upregulated VEGF induced by mutated EGFR in glioblastoma and lung Similar amplification of TF with upregulated VEGF induced by mutated EGFR in glioblastoma and lung cancer cells, accompanied by epithelial-to-mesenchymal transition (EMT)cancer cells, accompanied by epithelial-to-mesenchymal transition (EMT) Milsom et al Milsom et al CA Res 2008;68:10068-762008;68:10068-76

VTE and Cancer

Kalluri and Kansaki Kalluri and Kansaki Nature Nature 2008;452:5432008;452:543

(21 nucleotides)*

* Kleinman et al * Kleinman et al NatureNature 2008;452:5912008;452:591

Class Effect of siRNA for Angiogenesis Inhibition Class Effect of siRNA for Angiogenesis Inhibition via Toll-Like Receptior 3 (TLR 3)via Toll-Like Receptior 3 (TLR 3)

VTE and Cancer

Mechanisms of Cancer-Induced Thrombosis: Mechanisms of Cancer-Induced Thrombosis: ImplicationsImplications



3.3. Potential importance for cancer Potential importance for cancer therapy?therapy?

VTE and Cancer


► QQ: What do all of these experiments in : What do all of these experiments in micemice have to do with real have to do with real patientspatients with cancer? with cancer?

► AA: They suggest two things:: They suggest two things:● Tumor cell-derived, Tumor cell-derived, TF-rich microparticlesTF-rich microparticles

(MPs) may be important as a predictive test (MPs) may be important as a predictive test for VTEfor VTE

● All patients with oncogene-driven cancer may All patients with oncogene-driven cancer may need prophylactic anticoagulation need prophylactic anticoagulation

VTE and Cancer

► Retrospective study Retrospective study

► Immunohistologic (IH) and microarray data on Immunohistologic (IH) and microarray data on expression of TF and VEGF, as well as microvascular expression of TF and VEGF, as well as microvascular density (MVD) in:density (MVD) in: Normal pancreas (10)Normal pancreas (10) Pre-malignant pancreatic lesions: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)

Resected or metastatic pancreatic adenoca (130)Resected or metastatic pancreatic adenoca (130)

► SurvivalSurvival

► VTE RateVTE Rate

Tissue Factor Expression, Angiogenesis, and Tissue Factor Expression, Angiogenesis, and Thrombosis in Human Pancreatic CancerThrombosis in Human Pancreatic Cancer

Khorana et al CKhorana et al Clin Cancer Reslin Cancer Res 2007;13:2870 2007;13:2870

VTE and Cancer

Immunohistologic Correlation of TF with the Expression of Immunohistologic Correlation of TF with the Expression of Other Angiogenesis Variables in Resected Pancreatic CancerOther Angiogenesis Variables in Resected Pancreatic Cancer

High TF High TF Low TFLow TF PP

expression expression expressionexpression________________________________________________________________________________________________________

VEGF expressionVEGF expression Negative Negative 1313 41 41 <0.0001<0.0001 PositivePositive 53 53 15 15

Microvessel densityMicrovessel density V6 per tissue core V6 per tissue core 2727 33 33 0.047 0.047 >6 per tissue core >6 per tissue core 39 39 23 23 Median Median 8 8 6 6 0.01 0.01

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Khorana et.al. Clin CA Res 2007:13:2870

VTE and Cancer

Symptomatic VTE in Pancreatic CancerSymptomatic VTE in Pancreatic Cancer

Khorana et al Clin CA Res 2007;13:2872

5/19; 26.3%5/19; 26.3%

1/22; 4.5%1/22; 4.5%

VTE and Cancer

Median Survival of #122 Median Survival of #122 Resected Pancreatic Cancer PatientsResected Pancreatic Cancer Patients

mon

ths

17.9

12.6

P = 0.16(HR 2.06; 0.74-5.7)

Khorana et al Clin CA Res 2007;13:2872

VTE and Cancer

1. 1. Does activation of blood coagulation affect Does activation of blood coagulation affect the biology of cancer positively or the biology of cancer positively or negatively?negatively?

2. 2. Can we treat tumors more effectively Can we treat tumors more effectively using using coagulation protein targets?coagulation protein targets?

3. 3. Can anticoagulation alter the biology of Can anticoagulation alter the biology of cancer?cancer?

Cancer and Thrombosis: Cancer and Thrombosis: Year 2009 State-of-the-Science UpdateYear 2009 State-of-the-Science Update


Key QuestionsKey Questions Key QuestionsKey Questions

VTE and Cancer

1. 1. Epidemiologic evidence is Epidemiologic evidence is suggestivesuggestive that VTE is a bad that VTE is a bad prognostic sign in cancerprognostic sign in cancer

2. 2. Experimental evidence is Experimental evidence is supportive supportive of the use of of the use of antithrombotic strategies for both prevention of antithrombotic strategies for both prevention of thrombosis and inhibition of tumor growth thrombosis and inhibition of tumor growth

3. 3. Results of recent, randomized clinical trials of LMWHs in Results of recent, randomized clinical trials of LMWHs in cancer patients indicate superiority to oral agents in cancer patients indicate superiority to oral agents in preventing recurrent VTE, as well as increasing survival preventing recurrent VTE, as well as increasing survival ((notnot due to prevention of VTE) due to prevention of VTE)


Tentative AnswersTentative Answers

VTE and Cancer

LMWH and Prolongation LMWH and Prolongation of Cancer Survivalof Cancer Survival

Mechanistic Explanations

VTECoagulation Proteases

Direct Heparin Other

VTE and Cancer

Heparins and Tumour BiologyHeparins and Tumour Biology

Multiple Potential Modes of Action

Angiogenesis Apoptosis Heparanase Adhesion

VTE and Cancer

Ex VivoEx Vivo Angiogenesis: Angiogenesis: Embryonic Chick Aortic RingsEmbryonic Chick Aortic Rings

Control Aortic Ring: Day 5 10U/ml Dalteparin-Treated Aortic Ring: Day 5

Fernandez, Patierno and Rickles. Proc AACR 2003;44:698 (Abstr. #3055)

VTE and Cancer

Effects of Low-Molecular Weight Heparin on Effects of Low-Molecular Weight Heparin on Lung Cancer Cell ApoptosisLung Cancer Cell Apoptosis

P<0.05P<0.05

Chen et al Chen et al Cancer Invest 2008;Cancer Invest 2008;26:718-2426:718-24

• G1 arrestG1 arrest• decrease in decrease in S phaseS phase• 3-fold 3-fold in p21 in p21WAF1 WAF1

and p27and p27KIP1 KIP1 ((p p <0.01)<0.01) • reversed apoptosisreversed apoptosis and G1 arrest with and G1 arrest with p21 or p27 siRNAp21 or p27 siRNA

VTE and Cancer

0

100

200

300

400

500

VEGFVEGF FGF-2FGF-2 TNF-TNF-

**

*

*

**

CytokineCytokine +enoxaparin+enoxaparin +dalteparin+dalteparin+UFH+UFH

§

§ §

ControlControl

** *

Marchetti et al. Thromb Res 2008;121:637-645

Heparins Inhibit Cytokine–Induced Heparins Inhibit Cytokine–Induced Capillary Tube FormationCapillary Tube Formation

§ = p<0.05 vs control, * = p<0.05 vs cytokine

Tub

e Le

ngth

(m

m/c

mT

ube

Leng

th (

mm

/cm 22

))

VTE and Cancer

LMWH and VEGF Antisense Oligonucleotides Inhibit LMWH and VEGF Antisense Oligonucleotides Inhibit Growth and Metastasis of 3LL Tumors in MiceGrowth and Metastasis of 3LL Tumors in Mice

► 40 mice with Lewis Lung Cancer (3LL)40 mice with Lewis Lung Cancer (3LL)► Rx qod x 15 with:Rx qod x 15 with:

● Control (saline)Control (saline)● VEGF antisense oligos (ASODN)VEGF antisense oligos (ASODN)● VEGF mismatch sense oligo (MSODN)VEGF mismatch sense oligo (MSODN)● LMWH (dalteparin)LMWH (dalteparin)● LMWH + ASODNLMWH + ASODN

► RESULTS: RESULTS: Growth InhibitGrowth Inhibit** Lung MetsLung Mets**

● ASODNASODN 47%47% 38%38%● LMWHLMWH 27%27% 38%38%● CombinedCombined 59%59% 25%25%

* * P < 0.05P < 0.05

Zhang YH et al Zhang YH et al Chinese Med JChinese Med J 2006;86:749-52 2006;86:749-52

VTE and Cancer

Inhibition of Binding of Selectins to Inhibition of Binding of Selectins to Human Colon Carcinoma by Heparins Human Colon Carcinoma by Heparins

Stevenson et al Clin Ca Res 2005;11:7003-11

VTE and Cancer

Heparin Inhibition of B16 Melanoma Heparin Inhibition of B16 Melanoma Lung Metastasis in MiceLung Metastasis in Mice

Stevenson et al Clin Ca Res 2005;11:7003-11

VTE and CancerCoagulation Cascade and Tumor BiologyCoagulation Cascade and Tumor Biology

TFTF ThrombinThrombin





FibrinFibrin


PARsPARs

Fernandez, Patierno and Rickles. Sem Hem Thromb 2004;30:31; Ruf. J Thromb Haemost 2007; 5:1584

VIIaVIIa XaXa

Angiogenesis, Tumor Angiogenesis, Tumor Growth and MetastasisGrowth and Metastasis

??

LMWHs LMWHs (e.g. (e.g. dalteparindalteparin); Non-anticoagulant heparins); Non-anticoagulant heparins

VTE and Cancer Cancer, Thrombosis, and the Biology of Malignancy Scientific Foundations for the Role...

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