Clotting, Cancer, and Controversies
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
Cancer and Venous Thromboembolism
VTE and Cancer
The Legacy of Armand Trousseau
(1801–1867)
Professor Armand Trousseau
VTE and Cancer
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
Professor Armand Trousseau
VTE and Cancer
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
Trousseau’s Syndrome
VTE and Cancer
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”
Trousseau’s Syndrome
VTE and Cancer
►
Occult cancer in patients with idiopathic
venous thromboembolism
►
Thrombophlebitis in patients with cancer
Effect of Malignancy on Risk of
Venous Thromboembolism (VTE)
VTE and Cancer
• 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. nonCA patients
40
30
28
22.2
20.3
19.8
20
14.3
10
3.6
2.6
5 to 10 years
4.9
1 to 3 years
Adjusted odds ratio
50
53.5
1.1
Type of cancer
Silver In: The Hematologist - modified from Blom et. al. JAMA 2005;293:715
> 15 years
3 to 12 months
0 to 3 months
Distant
metastases
Breast
Gastrointestinal
Lung
Hematological
0
Time since cancer diagnosis
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 2nd 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 J Thromb
Haemost 2007;5:632-4
VTE and Cancer
Mechanisms of Cancer-Induced Thrombosis:
The Interface
1. Pathogenesis?
2. Biological significance?
3. Potential importance for cancer therapy?
VTE and Cancer
Trousseau’s Observations (continued)
“There appears in the cachexiae…a
particular condition of the blood that
predisposes it to spontaneous
coagulation.”
Lectures in Clinical Medicine, 1865
VTE and Cancer
Multiple Mechanisms in
Trousseau's Syndrome
Tissue Factor microparticles
Varki, A. Blood 2007;110:1723-1729
Copyright ©2007 American Society of Hematology. Copyright restrictions may apply.
Interface of Biology and Cancer
VTE and Cancer
Tumor cells
Angiogenesis,
Basement matrix
degradation
Fibrinolytic activities:
t-PA, u-PA, u-PAR,
PAI-1, PAI-2
Procoagulant Activities
IL-1,
TNF-a,
VEGF
PMN leukocyte
Activation of
coagulation
FIBRIN
Platelets
Monocyte
Endothelial cells
Falanga and Rickles, New Oncology: Thrombosis, 2005; Hematology, 2007
Pathogenesis of Thrombosis in Cancer –
A Modification of Virchow’s Triad
VTE and Cancer
1. Stasis


Prolonged bed rest
Extrinsic compression of blood vessels by tumor
2. Vascular Injury




Direct invasion by tumor
Prolonged use of central venous catheters
Endothelial damage by chemotherapy drugs
Effect of tumor cytokines on vascular endothelium
3. Hypercoagulability



Tumor-associated procoagulants and cytokines (tissue factor, CP,
TNFa, IL-1, VEGF, etc.)
Impaired endothelial cell defense mechanisms (APC resistance;
deficiencies of AT, Protein C and S)
Enhanced selectin/integrin-mediated, adhesive interactions
between tumor cells,vascular endothelial cells, platelets and host
macrophages
VTE and Cancer
Mechanisms of Cancer-Induced Thrombosis:
Clot and Cancer Interface
1. Pathogenesis?
2. Biological significance?
3. Potential importance for cancer therapy?
Activation of Blood Coagulation in Cancer
VTE and Cancer
►
Biological Significance?
Epiphenomenon?
Is this a generic secondary event where
thrombosis is an incidental finding
or, is clotting activation . . .
►
A Primary Event?
Linked to malignant transformation
Interface of Clotting Activation
and Tumor Biology
VTE and Cancer
FVII/FVIIa
Tumor
Cell
TF
Blood Coagulation
Activation
VEGF
THROMBIN
FIBRIN
Angiogenesis
IL-8
PAR-2
Angiogenesis
TF
Endothelial cells
Falanga and Rickles, New Oncology:Thrombosis, 2005;1:9-16
Coagulation Cascade and Tumor Biology
VTE and Cancer
TF
Clottingdependent
VIIa
Clottingindependent
Thrombin
Xa
Clottingindependent
Clottingdependent
Fibrin
Clottingdependent
PARs
Angiogenesis, Tumor
Growth and Metastasis
Fernandez, Patierno and Rickles. Sem Hem Thromb 2004;30:31; Ruf. J Thromb Haemost 2007;5:1584
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 2004;10:502-509
VTE and Cancer
Regulation of Vascular Endothelial Growth Factor Production and
Angiogenesis by the Cytoplasmic Tail of Tissue Factor
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
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 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 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 Blood 2005;105:1734-1741; Milson et al Ca Res 2008;68:10068-76)
Activation of Blood Coagulation
in Cancer: Malignant Transformation
VTE and Cancer
“1. MET Oncogene Drives a Genetic Programme
Linking Cancer to Haemostasis”
►
MET encodes a tyrosine kinase receptor for hepatocyte
growth factor/scatter factor (HGF/SF) 

Drives physiological cellular program of “invasive

growth” (tissue morphogenesis, angiogenesis
and repair)
Aberrant execution (e.g. hypoxia-induced
transcription) is associated with neoplastic
transformation, invasion, and metastasis
Boccaccio et al Nature 2005;434:396-400
VTE and Cancer
►
“MET Oncogene Drives a Genetic Programme
Linking Cancer to Haemostasis”
Mouse model of Trousseau’s Syndrome

Targeted activated human MET to the mouse liver
with lentiviral vector and liver-specific promoter 
slowly, progressive hepatocarcinogenesis

Preceded and accompanied by a thrombohemorrhagic syndrome

Thrombosis in tail vein occurrs early and is followed
by fatal internal hemorrhage

Syndrome characterized by  d-dimer and PT and 
platelet count (DIC)
VTE and Cancer
Blood Coagulation Parameters in Mice
Transduced with the MET Oncogene
Time after Transduction (days)
Transgene
Parameter
0
30
90
Platelets (x103)
968
656
800
D-dimer (µg/ml)
<0.05
<0.05
<0.05
PT (s)
12.4
11.6
11.4
_________
________________
_______________________________
MET
Platelets (x103)
974
350
150
D-dimer (µg/ml)
<0.05
0.11
0.22
PT (s)
12.9
11.8
25.1
GFP
VTE and Cancer
►
“MET Oncogene Drives a Genetic Programme
Linking Cancer to Haemostasis”
Mouse model of Trousseau’s Syndrome

Genome-wide expression profiling of hepatocytes
expressing MET - upregulation of PAI-1 and COX2 genes with 2-3x  circulating protein levels

Using either XR5118 (PAI-1 inhibitor) or Rofecoxib
(Vioxx; COX-2 inhibitor) resulted in inhibition of
clinical and laboratory evidence for DIC in mice
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
VTE and Cancer
“Pten and Hypoxia Regulate Tissue Factor Expression
and Plasma Coagulation By Glioblastoma”
►
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)
VTE and Cancer
“Pten and Hypoxia Regulate Tissue Factor Expression
and Plasma Coagulation By Glioblastoma”
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
VTE and Cancer
Both Akt and Ras Pathways Modulate TF
Expression By Transformed Astrocytes
N = Normoxia
H = Hypoxia
Similar data
for EGFR –
upregulation
of TF via JunD/
AP-1 transcription
(CA Res 2009;69:2540-9)
VTE and Cancer
“Pten and Hypoxia Regulate Tissue Factor Expression
and Plasma Coagulation By Glioblastoma”
Pseudopalisading necrosis
H&E
TF IHC
Vascular
Endothelium
VTE and Cancer
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”
►
►
►
►
►
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 Blood 2005;105:1734-41
Activation of Blood Coagulation
in Cancer: Malignant Transformation
VTE and Cancer
TF expression in cancer cells parallels genetic tumor progression
with an impact of K-ras and p53 status
450
400
350
160
TF Activity (U/106 cells)
Mean Channel TF Flourescence
“Oncogenic Events Regulate Tissue Factor Expression In
Colorectal Cancer Cells: Implications For Tumor Progression
And Angiogenesis”
300
250
200
150
100
50
0
HKh-2
HCT116
del/+
+/+
mut/+
+/+
379.2
mut/+
del/del
140
120
100
80
60
40
20
0
HKh-2
HCT116
379.2
VTE and Cancer
Activation of Blood Coagulation
in Cancer: Malignant Transformation
“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
“Oncogenic Events Regulate Tissue Factor
Expression In Colorectal Cancer Cells”
VTE and Cancer
%VWF-Positive Area
Effect of TF si mRNA on new vessel formation in colon cancer
14
12
10
8
6
4
2
0
HCT116
SI-2
SI-3
MG only
VTE and Cancer
Activation of Blood Coagulation
in Cancer: Malignant Transformation
“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
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 CA Res 2008;68:10068-76
VTE and Cancer
Class Effect of siRNA for Angiogenesis Inhibition
via Toll-Like Receptior 3 (TLR 3)
(21 nucleotides)*
* Kleinman et al Nature
2008;452:591
Kalluri and Kansaki Nature 2008;452:543
VTE and Cancer
Mechanisms of Cancer-Induced Thrombosis:
Implications
1. Pathogenesis?
2. Biological significance?
3. Potential importance for cancer
therapy?
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
VTE and Cancer
Tissue Factor Expression, Angiogenesis, and
Thrombosis in Human Pancreatic Cancer
►
Retrospective study
►
Immunohistologic (IH) and microarray data on
expression of TF and VEGF, as well as microvascular
density (MVD) in:



Normal pancreas (10)
Pre-malignant pancreatic lesions:
• Intraductal papillary mucinous neoplasms (IPMN; 70)
• Pancreatic intrepithelial neoplasia (PanIN; 40)
Resected or metastatic pancreatic adenoca (130)
►
Survival
►
VTE Rate
Khorana et al Clin Cancer Res 2007;13:2870
VTE and Cancer
Immunohistologic Correlation of TF with the Expression of
Other Angiogenesis Variables in Resected Pancreatic Cancer
High TF
expression
Low TF
expression
P
____________________________________________________
VEGF expression
 Negative
13
41
<0.0001
 Positive
53
15
Microvessel density
 V6 per tissue core
27
33
0.047
 >6 per tissue core
39
23
 Median
8
6
0.01
---------------------------------------------------------------------------------------Khorana et.al. Clin CA Res 2007:13:2870
Symptomatic VTE in Pancreatic Cancer
VTE and Cancer
5/19; 26.3%
1/22; 4.5%
Khorana et al Clin CA Res 2007;13:2872
VTE and Cancer
Median Survival of #122
Resected Pancreatic Cancer Patients
17.9
months
12.6
Khorana et al Clin CA Res 2007;13:2872
P = 0.16
(HR 2.06;
0.74-5.7)
VTE and Cancer
Cancer and Thrombosis:
Year 2009 State-of-the-Science Update
Key Questions
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?
VTE and Cancer
Cancer and Thrombosis:
Year 2009 State-of-the-Science Update
Tentative Answers
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)
LMWH and Prolongation
of Cancer Survival
VTE and Cancer
Mechanistic Explanations
VTE
Coagulation
Proteases
Direct
Heparin
Other
Heparins and Tumour Biology
VTE and Cancer
Multiple Potential Modes of Action
Angiogenesis
Apoptosis
Heparanase
Adhesion
VTE and Cancer
Ex Vivo Angiogenesis:
Embryonic 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
Lung Cancer Cell Apoptosis
• G1 arrest
• decrease in
S phase
• 3-fold  in p21WAF1
and p27KIP1 (p <0.01)
• reversed apoptosis
and G1 arrest with
p21 or p27 siRNA
Chen et al Cancer Invest 2008;26:718-24
P<0.05
Heparins Inhibit Cytokine–Induced
Capillary Tube Formation
VTE and Cancer
Tube Length (mm/cm )
500
§
§
400
300
§
*
*
*
*
*
*
*
*
Control
*
200
100
2
0
VEGF
Cytokine
FGF-2
+UFH
TNF-a
+enoxaparin
+dalteparin
§ = p<0.05 vs control, * = p<0.05 vs cytokine
Marchetti et al. Thromb Res 2008;121:637-645
VTE and Cancer
►
►
40 mice with Lewis Lung Cancer (3LL)
Rx qod x 15 with:
●
●
●
●
●
►
LMWH and VEGF Antisense Oligonucleotides Inhibit
Growth and Metastasis of 3LL Tumors in Mice
Control (saline)
VEGF antisense oligos (ASODN)
VEGF mismatch sense oligo (MSODN)
LMWH (dalteparin)
LMWH + ASODN
RESULTS:
Growth Inhibit*
Lung Mets*
●
ASODN
LMWH
47%
27%
38%
38%
●
Combined
59%
25%
●
* P < 0.05
Zhang YH et al Chinese Med J 2006;86:749-52
VTE and Cancer
Inhibition of Binding of Selectins to
Human Colon Carcinoma by Heparins
Stevenson et al Clin Ca Res 2005;11:7003-11
VTE and Cancer
Heparin Inhibition of B16 Melanoma
Lung Metastasis in Mice
Stevenson et al Clin Ca Res 2005;11:7003-11
Coagulation Cascade and Tumor Biology
VTE and Cancer
TF
Clottingdependent
VIIa
Clottingindependent
Thrombin
Xa
Clottingdependent
Clottingindependent
?
PARs
Angiogenesis, Tumor
Growth and Metastasis
LMWHs (e.g. dalteparin); Non-anticoagulant heparins
Fernandez, Patierno and Rickles. Sem Hem Thromb 2004;30:31; Ruf. J Thromb Haemost 2007; 5:1584
Fibrin
Clottingdependent