Always supportive, you led by example and gave me every opportunity. ..... 4.2.2 Pre operative CAT results (without TM) . ...... cysts, PCOS) increase risks; .... absence or presence of metastasis to adjacent lymph Nodes (N), and the absence ..... keratinocytes in the skin; and a number of epithelial cell types, including those.
Hypercoagulability in Ovarian Cancer A thesis submitted to the University of Dublin, Trinity College for the degree of Doctor of Medicine in the Department of Obstetrics and Gynaecology, School of Medicine.
Feras Abu Saadeh M.B. Ch.B, M.R.C.O.G, M.R.C.P.I Supervisors Dr. Noreen Gleeson Dr. Lucy Norris
University of Dublin Trinity College
2013
DECLARATION
I declare that this thesis has not been submitted as an exercise for a degree at this or any other University. The work, upon which this thesis is based, was carried out in collaboration with a team of researchers and supervisors who are duly acknowledged in the text of the thesis. The Library may lend or copy this thesis upon request.
Signed:
Date:
________________________
_____________________
I
SUMMARY Ovarian cancer is the leading cause of death from all gynaecological cancers. Venous thromboembolism (VTE) is the second leading cause of death in cancer patients. The treatment and prevention of VTE is particularly challenging in gynaecological cancer particularly post surgery where the presence of a malignancy increases the risk of post operative VTE four fold. In addition, patients are at increased risk of VTE during chemotherapy. The link between cancer and VTE is known for decades, however the exact mechanism by which cancer increases the risk of VTE or how VTE increases mortality in cancer patients is not well established. In addition, there are no reliable markers of hypercoagulability that can be used to identify at risk patients. The aim of this project was (1) To investigate the role of TF and its inhibitor TFPI in VTE risk in ovarian cancer (2) To measure two new assays, calibrated automated thrombography (CAT) and microparticle TF (MP-TF) as markers of increased procoagulant activity in patients with ovarian malignancies before surgery and during the post operative period (3) To determine the effect of chemotherapy on procoagulant activity in ovarian cancer patients. In the first part of the study, TF and TFPI gene and protein expression in different histological types of malignant ovarian tumour was compared with benign ovarian tumours using TaqMan gene expression and ELISA. Immunohistochemistry was used to localize the source of TF. TF gene and protein expression was significantly higher in clear cell and endometroid ovarian carcinoma compared with benign ovarian tumours. TF expression was also increased in cases complicated II
by VTE regardless of histology compared with those who remained thrombosis free. A small non significant increase TFPI was found in clear cell cancers compared with benign cases. Immunohistochemistry showed that the increased TF was localized to the cancer cell. The CAT assay showed that Endogenous Thrombin Potential (ETP) was increased in patients with malignant disease prior to surgery compared with those with benign disease. Higher levels of ETP were found in patients with clear cell cancer compared with other subtypes. Five days post surgery (during LMWH prophylaxis), thrombin production was effectively reduced in all groups compared with pre-operative levels. Two weeks post surgery higher levels of peak thrombin production and ETP were observed in patients with malignant tumours compared with those with benign disease. There was no significant difference between the groups at 6 weeks post surgery. Low levels of MP-TF were observed in patients with malignant and benign ovarian cancer prior to surgery.
There were no
significant differences in MP-TF levels between the groups before surgery and in the post operative period. In the final part of the study, the effect of chemotherapy on procoagulant activity in ovarian cancer patients was evaluated. Chemotherapy, both adjuvant and neo adjuvant, did not increase procoagulant activity as determined by ETP.
To
determine sensitivity to the activated protein C (APC) pathway, ETP was measured following addition of thrombomodulin.
Patients who underwent
neoadjuvant chemotherapy were less sensitive to the anticoagulant effects of APC compared with those who were chemotherapy naive
Following 3 and 6 months
adjuvant chemotherapy, a reduced sensitivity to APC was also reported compared to pretreatment levels. III
In conclusion, this study showed that tumour TF has an important role in the pathogenesis of VTE in ovarian cancer patients and that the ETP assay is a promising biomarker for VTE risk in these patients. Hypercoagulability as determined by the ETP assay persists up to two weeks post surgery in patients with ovarian malignancies.
The recently recommended extension of LMWH
prophylaxis beyond the hospital stay is justified based on the increased and prolonged procoagulant activity in ovarian cancer patients following surgery. Chemotherapy does not directly affect thrombin production but does reduce the effectiveness
of
the
natural
anticoagulant
APC
hence
increasing
hypercoagulability. This may explain the high incidence of thrombosis during chemotherapy in cancer patients.
IV
ACKNOWLEDGEMENTS I would like to thank the following people who helped me during the preparation of this thesis: Dr. Noreen Gleeson, for the confidence she showed in appointing me specialist registrar, research registrar and then subspeciality registrar at gynaecology oncology. She provided the initial encouragement to begin this research and an optimistic outlook that helped me complete it. She was always generous with her time and knowledge. A big thank you
Dr. Lucy Norris, for excellent methodological advice, for all the labaratory expertise, for reading my thesis and abstracts over and over, for all her unwavering support and guidance. Really,this thesis would not have been made possible without her.
Dr. Sharon O’Toole, for providing access to biobank samples and for her help and guidance in the laboratory. For answering my silly questions. Dr. Ali Khashan, for his statistical expertise. Dr. Thomas Darcy, all registrars, SHOs, interns on the gynaecology oncology team. All theatre staff for their help in collecting patients specimens. Dr. Ream Langhe. Lynda Mc Evoy, Lynne Kelly and all other research fellows in TCD for collecting the samples and maintaining the tissue biobank. Dr. Dearbhaile O Donnell, and all medical oncology team for helping in collecting the samples for chemotherapy. Dr. John O’Leary, for allowing me to work in the central pathology lab (CPL).Every one in CPL especially Emma, Gary, Paul and Darragh for their advice on PCR. V
Prof. James ODonnell, for access to the CAT technology. Dr. Bashir Mohamed, for his help with immunohistochemistry. Ms. Christina Boccardo, for the administrative help. Gynaecology oncology fund at St James’s hospital and LEO Pharma Ireland for sponsoring this project. Finally, I want to express my deepest thanks to my parents Abu Adeeb and Om Adeeb. Always supportive, you led by example and gave me every opportunity.
VI
I dedicate this thesis to my incredible wife Nedaa and my wonderful little kids Omar,Yara and Noor. Nedaa, for your loving support, which never faltered through long days and late nights. You made this possible. Omar, Yara and Noor, I can not imagine my life without you
VII
PUBLICATIONS
F. Abu Saadeh, L. Norris, S. O'Toole, N. Gleeson. The role of tissue factor and tissue factor pathway inhibitor in ovarian cancer. Thrombosis Research, Volume 127, Supplement 3, February 2011, Page S149 F. Abu Saadeh, Norris. L, O' Ttool. S, Langhe. R, O' Leary. J, Gleeson. N. The Role of Tissue Factor and Tissue Factor Pathway Inhibitor and VEGF in Ovarian Cancer. International Journal Of Gynecological Cancer Volume 21, Supplement 3, October 2011 F. Abu Saadeh, L. Norris, S. O'Toole, R. Langhe, J.J. O'Leary, N. Gleeson. Does tissue factor and tissue factor pathway inhibitor over expression, play a role in the development of venous thromboembolism in ovarian cancer patients? Thrombosis Research, Volume 129, Supplement 1, April 2012, Page S170 F. Abu Saadeh, L. Norris, S. O'Toole, N. Gleeson. Procoagulant activity in patients with gynaecological malignancies and the effect of neoadjuvant chemotherapy. Thrombosis Research, Volume 129, Supplement 1, April 2012, Pages S189-S190 F. Abu Saadeh, L. Norris, S. O'Toole, R. Langhe, J.J. O'Leary, N. Gleeson. Why venous thromboembolism is a common complication of ovarian cancer? The International Journal of Gynecological Cancer – October 2012, vol 22, issue 8, supplement 3
VIII
F. Abu Saadeh, L. Norris, S. O'Toole, R. Langhe, N. Gleeson. Endogenous thrombin potential as a marker for venous thromboembolism in ovarian cancer. The International Journal of Gynecological Cancer – October 2012, vol 22, issue 8, supplement 3 F. Abu Saadeh, L. Norris, S. O'Toole, R. Langhe, N. Gleeson. ETP as a marker for venous thromboembolism in gynaecological cancer patients post surgery. The International Journal of Gynecological Cancer – October 2012, vol 22, issue 8, supplement 3
IX
TABLE OF CONTENTS DECLARATION ........................................................................................................ I SUMMARY .............................................................................................................. II ACKNOWLEDGEMENTS ....................................................................................... V PUBLICATIONS ................................................................................................... VIII TABLE OF CONTENTS .......................................................................................... X LIST OF TABLES .................................................................................................XVI LIST OF FIGURES .............................................................................................XVIII CHAPTER 1. INTRODUCTION ............................................................................ 1 1.1 Epithelial Ovarian Cancer............................................................................ 2 1.1.1 Introduction ........................................................................................... 2 1.1.2 Incidence ............................................................................................... 2 1.1.3 Etiology ................................................................................................. 6 1.1.4 Risk Factors .......................................................................................... 7 1.1.5 Protective Factor ................................................................................... 9 1.1.6 Histology ............................................................................................. 10 1.1.7 Staging ................................................................................................ 13
X
1.1.8 Grade .................................................................................................. 17 1.1.9 Current management of ovarian cancer.............................................. 19 1.2 Venous thromboembolism ......................................................................... 28 1.3 Venous thromboembolism and ovarian cancer ........................................ 31 1.4 Pathogenesis of venous thromboembolism .............................................. 33 1.4.1 Coagulation activation ......................................................................... 33 1.4.2 Regulation of coagulation.................................................................... 39 1.4.3 Fibrinolysis .......................................................................................... 43 1.4.4 Predictive markers of VTE .................................................................. 45 1.5 Coagulation activation in cancer................................................................ 51 1.6 Coagulation activation in ovarian cancer ................................................... 53 1.7 Effect of chemotherapy.............................................................................. 55 1.8 Low molecular weight heparin prophylaxis for VTE in cancer ................... 59 1.9 Summary of evidence leading to this research ......................................... 60 CHAPTER 2. PATIENTS AND METHODS ........................................................ 63 2.1 TCD Obstetrics and Gynaecology cancer bioresource ............................. 64 2.2 Ethical approval ......................................................................................... 65 2.3 Patients...................................................................................................... 66 XI
2.3.1 Patients for tissue factor study ............................................................ 66 2.3.2 Patients for CAT pre operative study .................................................. 72 2.3.3 Patients for CAT serial study .............................................................. 74 2.3.4 Patients for chemotherapy study ........................................................ 77 2.4 Blood and tissue processing ..................................................................... 79 2.4.1 Tissue Processing ............................................................................... 79 2.4.2 Blood sample processing .................................................................... 79 2.5 Methods..................................................................................................... 80 2.5.1 RNA extraction from fresh tissue ........................................................ 80 2.5.2 cDNA synthesis ................................................................................... 87 2.5.3 Taqman Real Time PCR ..................................................................... 90 2.5.4 Total protein extraction ....................................................................... 98 2.5.5 Protein Assay (BCA) ......................................................................... 100 2.5.6 Tissue Factor Pathway Inhibitor ELISA ............................................ 103 2.5.7 Tissue Factor ELISA ......................................................................... 110 2.5.8 Immunohistochemistry technique ..................................................... 116 2.5.9 Calibrated Automated Thrombogram ................................................ 117 2.5.10 Calibrated Automated Thrombogram with Thrombomodulin ............ 121 XII
2.5.11 Microparticles bioimmunoassay ....................................................... 124 2.6 Statistics .................................................................................................. 130 CHAPTER 3. TISSUE FACTOR, TISSUE FACTOR PATHWAY INHIBITOR AND VEGF EXPRESSION IN OVARIAN TUMOUR .................................................... 132 3.1 Introduction .............................................................................................. 133 3.2 Results..................................................................................................... 135 3.2.1 Clinicopathological details of patients ............................................... 135 3.2.2 TF mRNA expression in ovarian tumour tissue................................. 138 3.2.3 TF protein level in ovarian tumour tissue .......................................... 141 3.2.4 TFPI mRNA expression in ovarian tumour tissue ............................. 144 3.2.5 TFPI protein expression in ovarian tumour tissue ............................. 144 3.2.6 VEGF mRNA expression in ovarian tumour tissue ........................... 149 3.2.7 Immunohistochemistry ...................................................................... 152 3.2.8 Survival data ..................................................................................... 154 3.3 Discussion ............................................................................................... 155 CHAPTER 4.
PROCOAGULANT ACTIVITY PRE AND POST SURGERY IN
OVARIAN CANCER ............................................................................................ 159 4.1 Introduction .............................................................................................. 160 XIII
4.2 Results .................................................................................................... 162 4.2.1 Clinicopathological details of patients ............................................... 162 4.2.2 Pre operative CAT results (without TM) ............................................ 165 4.2.3 Pre operative CAT results (TM treated) ............................................ 169 4.2.4 Pre operative TF MP results ............................................................. 173 4.2.5 Serial CAT results (without TM) ........................................................ 174 4.2.6 Serial CAT results (TM treated) ........................................................ 180 4.3 Discussion ............................................................................................... 184 CHAPTER 5. EFFECT OF CHEMOTHERAPY ON PROCOAGULANT ACTIVITY IN OVARIAN CANCER PATIENTS ..................................................................... 190 5.1 Introduction.............................................................................................. 191 5.2 Results .................................................................................................... 193 5.2.1 Clinicopathological details of patients ............................................... 193 5.2.2 Preoperative CAT results (without TM) ............................................. 195 5.2.3 Preoperative CAT results (TM treated) ............................................. 198 5.2.4 Serial CAT results (without TM) ........................................................ 201 5.2.5 Serial CAT results (TM treated) ........................................................ 204 5.3 Discussion ............................................................................................... 207 XIV
CHAPTER 6. GENERAL DISCUSSION AND CONCLUSION ......................... 211 6.1 General discussion and conclusion ......................................................... 212 REFERENCES .................................................................................................... 216
XV
LIST OF TABLES Table 1-1 Summary information, incidence and prevalence in ovarian cancer in Ireland ... 3
Table 1-2 Hypotheses on physiologic susceptibilities to epithelial ovarian cancer .............. 7
Table 1-3 Histological classification of epithelial ovarian tumours according to FIGO....... 11
Table 1-4 Carcinoma of the ovary: FIGO nomenclature .................................................... 15
Table 1-5 Risk factors for Venous Thromboembolism ....................................................... 29
Table 1-6 Risk factors for thrombosis in cancer patients .................................................. 32
Table 1-7 The role of haemostatic constituents (coagulation and fibrinolytic proteins and platelets) in the growth and progression of cancer ............................................................ 53
Table 1-8 Khorana predictive model for chemotherapy-associated VTE .......................... 56
Table 2-1 Clinico pathological details of patients in TF study, group A ............................. 67
Table 2-2 Clinico pathological details of patients in TF study, group B ............................. 70
Table 2-3 Clinico pathological details of patients who participated in pre operative CAT study .................................................................................................................................. 73
Table 2-4 Clinico pathological details of patients participated in the serial CAT study ...... 76
Table 2-5 Clinico pathological details of patients participated in the chemotherapy study 78
Table 2-6 Example of RNA calculation for cDNA synthesis............................................... 88
Table 2-7 The volumes required of each component to prepare the 2X RT mastermix .... 88
XVI
Table 2-8 Volumes of each component required for a Taqman assay in one well of a 96well plate. ........................................................................................................................... 92
Table 2-9 List of gene targets selected for Taqman analysis of human cell systems ........ 93
Table 2-10 ABI Prism 7000 thermal cycler protocol........................................................... 95 Table 2-11 Example of Excel sheet to show how to calculate the fold changes by 2-ΔΔCT method ............................................................................................................................... 97
Table 2-12 Preparation and dilution scheme of BSA standards for the microplate BCA procedure ......................................................................................................................... 101
Table 2-13 Preparation of the standard samples for the TFPI ELISA.............................. 105
Table 2-14 Preparation of the standard samples for the TF ELIZA ................................. 112
Table 2-15 Preparation of the standard samples for the MP assay ................................. 127
Table 3-1 Clinico pathological details of group A. ............................................................ 136
Table 3-2 Clinico pathological details of group B. ............................................................ 137
Table 3-3 Summary of tissue factor immunohistochemistry in ovarian tumour ............... 153
Table 4-1 Clinicopathological details of pre operative group ........................................... 162
Table 4-2 Clinicopathological details of serial group........................................................ 163
Table 4-3 Details of patients complicated by VTE ........................................................... 164
Table 5-1 Clinicopathological details of the neoadjuvant group....................................... 193
Table 5-2 Clinicopathological details of serial chemotherapy group ................................ 194
XVII
LIST OF FIGURES Figure 1-1 Irish Cancer statistics (NCRI) 2007-2009. .......................................................... 3
Figure 1-2 Worldwide estimated age-standerised incidence rate of ovarian cancer ........... 4
Figure 1-3 Estimated age standarised mortality rate of ovarian cancer............................... 5
Figure 1-4 Representative examples of the major histological subtypes of EOC .............. 12
Figure 1-5 Carcinoma of the ovary. Staging ovarian cancer:............................................. 16
Figure 1-6 Same tumour showing different grades ............................................................ 18
Figure 1-7 Summary of literature demonstrating the survival ............................................ 21
Figure 1-8 Virchow’s Triad ................................................................................................. 30
Figure 1-9 Conventional cascade model of coagulation. ................................................... 34
Figure 1-10 Cell – based model of coagulation ................................................................. 36
Figure 1-11 TF/FVII/FX complex ....................................................................................... 37
Figure 1-12 Scheme of the coagulation-anticoagulation system ...................................... 39
Figure 1-13 Activated protein C pathway .......................................................................... 40
Figure 1-14 The antithrombin pathway ............................................................................. 41
Figure 1-15 TFPI pathway ................................................................................................ 42
Figure 1-16 The fibrinolytic system .................................................................................... 44
Figure 1-17 The dynamics of D-dimer formation . ............................................................. 46
XVIII
Figure 1-18 Calibrated Automated Thrombogram assay (CAT) curve ............................. 48 Figure 1-19 Role of TF+MPs in microvascular and venous thrombosis ............................. 51
Figure 1-20 Effect of cancer cells on coagulation system ................................................. 52
Figure 1-21 The prothrombotic effects of chemotherapy. .................................................. 58
Figure 2-1 Nandrop ND-1000 Spectrophotometer result of one sample ........................... 86 Figure 2-2. 5’to 3’nuclease activity of AmpliTaq Gold®. DNA Polymerase during PCR .... 91
Figure 2-3 Typical layout of a TaqMan PCR plate. ............................................................ 94
Figure 2-4 ABI Prism 7000 Sequence Detection System .................................................. 95
Figure 2-5 Typical amplification curve from the SDS software. ......................................... 96
Figure 2-6 An example of a standard curve using the BCA protein assay. ..................... 102
Figure 2-7 Preparation of the standard samples for the TFPI ELISA .............................. 106
Figure 2-8 An example of a standard curve for TFPI ....................................................... 108
Figure 2-9 An example of a standard curve for TF .......................................................... 115
Figure 2-10 CAT assay principle and parameters of the thrombin generation curve ...... 118
Figure 2-11 An example of a standard curve for micro-particles assay ........................... 129
Figure 3-1 Tissue factor mRNA expression in ovarian tumours. ..................................... 139
Figure 3-2 Tissue factor mRNA expression in patients with ovarian cancer the effect of VTE .................................................................................................................................. 140
Figure 3-3 Tissue factor protein concentration in ovarian tumours. ................................. 142
XIX
Figure 3-4 Tissue factor protein concentration in patients with ovarian cancer and (VTE) ......................................................................................................................................... 143
Figure 3-5 Tissue factor pathway inhibitor mRNA expression in ovarian tumours. ......... 145
Figure 3-6 Tissue factor pathway inhibitor mRNA expression in patients with ovarian cancer and (VTE). ............................................................................................................ 146
Figure 3-7 Tissue factor pathway inhibitor protein concentration in ovarian tumours. ..... 147
Figure 3-8 Tissue factor pathway inhibitor protein concentration in patients with ovarian cancer and (VTE) ............................................................................................................ 148
Figure 3-9 VEGF mRNA expression in ovarian tumour. .................................................. 150
Figure 3-10 Correlation between TF mRNA and VEGF mRNA expression in ovarian cancer cases not complicated with VTE .......................................................................... 150
Figure 3-11 VEGF mRNA expression in patients with ovarian cancer and venous thromboembolism (VTE) .................................................................................................. 151
Figure 3-12 Correlation between TF mRNA and VEG mRNA expression in ovarian cancer cases complicated with VTE ............................................................................................ 151
Figure 3-13 Representative IHC localization analyses of TF in epithelial ovarian tumour specimens.. ...................................................................................................................... 152
Figure 3-14 The effect of TF mRNA expression levels in ovarian cancer on overall survival. ............................................................................................................................ 154
Figure 4-1 Pre operative ETP level in patients with ovarian tumours .............................. 165
Figure 4-2 Pre operative peak thrombin levels in patients with ovarian tumours............. 166
XX
Figure 4-3 Pre operative time to peak thrombin production in patients with ovarian tumours ............................................................................................................................ 167
Figure 4-4 Pre operative Lag time to the start of thrombin production in patients with ovarian tumours, .............................................................................................................. 168
Figure 4-5 Pre operative ETP level after the addition of TM in patients with ovarian tumours, ........................................................................................................................... 169
Figure 4-6 Pre operative peak thrombin level after the addition of TM in patients with ovarian tumours, .............................................................................................................. 170
Figure 4-7 Pre operative time to peak thrombin after the addition of TM in patients with ovarian tumours, .............................................................................................................. 171
Figure 4-8 Pre operative lagtime after the addition of TM in patients with ovarian tumours ......................................................................................................................................... 172
Figure 4-9 Preoperative TF microparticle levels in patients with ovarian tumours........... 173
Figure 4-10 Serial ETP measurement in patients with ovarian tumours .......................... 175
Figure 4-11 Serial Peak thrombin measurement in patients with ovarian tumours.......... 177
Figure 4-12 Lagtime measurement (minutes) in patients with ovarian tumours .............. 178
Figure 4-13 Time to peak measurement (minutes) in patients with ovarian tumours ...... 179
Figure 4-14 Serial ETP (TM treated) measurement in patients with ovarian tumours ..... 180
Figure 4-15 Serial peak thrombin (TM treated) measurement in patients with ovarian tumours ............................................................................................................................ 181
Figure 4-16 Lagtime measurement (TM treated) in patients with ovarian tumours ......... 182
XXI
Figure 4-17 Time To peak measurement (TM treated) in patients with ovarian tumours. ......................................................................................................................................... 183
Figure 5-1 Pre operative ETP level (chemotherapy study). ............................................. 195
Figure 5-2 Preoperative peak thrombin production (chemotherapy study) ...................... 196
Figure 5-3 Lagtime for thrombin production (chemotherapy study) ……..……………….197
Figure 5-4 Time To peak for thrombin production (chemotherapy study) ........................ 197
Figure 5-5 ETP levels after adding (TM) (chemotherapy study) ...................................... 198
Figure 5-6 Peak thrombin levels after adding (TM) (chemotherapy study) ...................... 199
Figure 5-7 Lagtime for thrombin production after adding (TM) (chemotherapy study) .... 200
Figure 5-8 Time To peak thrombin production after adding (TM) (chemotherapy study) 200
Figure 5-9 Serial ETP (chemotherapy study)................................................................... 201
Figure 5-10 Serial Peak thrombin (chemotherapy study) ................................................ 202
Figure 5-11 Serial Lagtime and Time To peak (chemotherapy study) ............................. 203
Figure 5-12 Serial ETP following addition of (TM) (chemotherapy study) ....................... 204
Figure 5-13 Serial Peak thrombin following addition of (TM) (chemotherapy study) ....... 205
Figure 5-14 Serial Lagtime & Time To Peak following addition of TM (chemotherapy study) ............................................................................................................................... 206
XXII
ABBREVIATIONS aPTT
Activated Partial Thromboplastin Times
APC
Activated protein C
AB
Applied Biosystems
ACCP
American College of Chest Physicians
AJCC
American Joint Committee on Cancer
ANOVA
Analysis of Variance
ASCO
American Society of Clinical Oncology
AT
Antithrombin
AUC
Area Uncer Curve
BCA
Bicinchoninic Acid
BSO
Bilateral Salpingo Oophrectomy
CAT
Calibrated Automated Thrombogram
CATS
Cancer And Thrombosis Study
CCC
Clear Cell Carcinoma
cDNA
Complementary Deoxyribonucleic Acid
CI
Confidence Interval
CT
Computed Tomography
Ct
Threshold Cycle
CUS
Compression Ultrasonography Study
DD
D- Dimer
DEPC
Diethylene Pyrocarbonate
DIC
Disseminated Intravascular Coagulation
DNA
Deoxyribonucleic acid
DVT
Deep Vein Thrombosis XXIII
EDTA
Ethylenediaminetetra-acetic acid
ELISA
Enzyme-Linked ImmunoSorbent Assay
EMC
Endometroid Carcinoma
EOC
Epithelial Ovarian Cancer
EPCR
Endothelial Protein C Receptor
ETP
Endogenous Thrombin Potential
EU
European Union
FC
Fold Change
FF
Fresh Frozen
FFPE
Formalin Fixed Paraffin Embedded
FIGO
International Federation Of Gynecology And Obstetrics
FSS
Fertility-Sparing Surgery
G
Grade
G-CSF
Granulocyte Colony-Stimulating Factors
GCIG
Gynaecologic Cancer Intergroup
GOG
Gynaecologic Oncology Group
H&E
Haematoxylin and Eosin
HRP
Horseradish Peroxidase
HRT
Hormone Replacement Therapy
IDS
Interval Debulking Surgery
LETS
Leiden Thrombophilia Study
LITE
Longitudinal Investigation of Thromboembolism Etiology
LMP
Low malignant potential
LMWH
Low Molecular Weight Heparin
LN
Liquid Nitrogen
mg
Milligram XXIV
ml
Millilitre
MP
Microparticles
mRNA
Messenger Ribonucleic Acid
NCI
National Cancer Institute
NCRI
National Cancer Registry Ireland
ng
Nanogram
NICE
National Institute for health and Clinical Excellence
nM
Nanomolar
NPV
Negative Predictive Value
NTC
No Template Control
OC
Ovarian Carcinoma
OCP
Oral Contraceptive Pills
OS
Overall survival
OSE
Ovarian Surface Epithelium
PAI
Plasmingen Activator Inhibitor
PAI-1
Plasminogen Activator Inhibitors -1
PAI-2
Plasminogen Activator Inhibitors -2
PCOS
polyCystic Ovary Syndrome
PCR
Polymerase Chain Reaction
PE
Pulmonary Embolism
PFS
Progression free survival
PID
Pelvic Inflammatory Disease
PPP
Plasma Poor Platelets
PSC
Papillary Serous Adenocarcinomas
PT
Prothrombin Time
RNA
Ribonucleic acid XXV
RQ
Relative Quantitation
rRNA
Ribosomal RNA
RT
Radiotheraapy
SD
Standard Deviation
SEM
Standard Error of Mean
TAH
Total Abdominal Hysterectomy
TAT
Thrombin Antithrombin Complex
TCD
Trinity College Dublin
TF
Tissue Factor
TFPI
Tissue Factor Pathway Inhibitor
TGA
Thrombin Generation Assay
TM
Thrombomodulin
TMB
TetraMethylBenzidine
TNM
Tumour, Node, Metastasis
tPA
Tissue-type Plasminogen Activator
UICC
Union Internationale Centre de Cancer
uPA
urokinease Plasminogen Activator
VEGF
Vascular Endothelial Growth Factor
VTE
Venous Thrombo Embolism
WAI
Whole Abdominal Irradiation
WHO
World Health Organization
µl
Microlitre
µm
Micromole
XXVI
CHAPTER 1. INTRODUCTION
1
Introduction
Chapter 1
1.1 Epithelial Ovarian Cancer 1.1.1 Introduction Ovarian cancer is the most lethal gynaecological cancer. Worldwide, in 2008, approximately 225,000 women were diagnosed with ovarian cancer and 140,000 died from this disease 1. Ovarian cancer represents a major clinical challenge in gynaecological oncology. Dissemination of ovarian cancer differs from the classical metastatic patterns of tumours arising from other organ sites in that ovarian cancer seeds the peritoneal cavity with tumour cell nests that adhere to adjacent organs or float freely and lead to ascites formation. As a result, early ovarian cancer is asymptomatic. Most women present with advanced disease often spread as diffuse small-volume deposits or they present with complications like venous thromboembolism. Tumour metastasis to the peritoneal and/or pleural cavity is evident in two-thirds of cases at diagnosis and relapse is often detected in these anatomic sites 2. The majority of primary ovarian tumours (90 percent) are derived from surface epithelial cells; the remainder arises from other ovarian cell types (germ cell tumours, sex cord-stromal tumours). 1.1.2 Incidence There are approximately 315 new cases and 269 cancer-related deaths from ovarian cancer annually in Ireland, making it the second most common gynecologic malignancy and the most common cause of gynecologic cancer death (Figure 1.1).3 Over all it is the 4th most common cause of all cancer deaths in women. A summary of ovarian cancer statistics for Ireland is provided in Tables 1.1
2
Introduction
cervix, 2% melanom a, 3% lymphom a, 3%
Chapter 1
pancreas, 2%
Ovary , Colorecta 3% l, 8% Lung, 7%
leukemia, 3% brain, 3%
pancreas, 6%
lymphom a, 2% stomach, 3%
uterus, 3% nonmelanom a skin, 29%
Colorecta l, 10%
Lung, 17% other invasive, 30%
other invasive, 17% esophagu s, 3%
A
B
Ovary , 7%
Figure 1-1 Irish Cancer statistics (NCRI) 2007-2009. Pie chart displaying (A) Relative frequency of the main invasive cancers and (B) Relative frequency of the main causes of cancer death in women 3 Ireland has a relatively high incidence rate of up to 14.6 per 100,000 populations. Figure 1.2 shows the incidence rate of ovarian cancer across the world in 2008. The incidence rates are highest in Central America and Northern Europe and lowest in some parts of Africa and Asia. In comparison with other European countries, Ireland is among those with the highest incidence rate of ovarian cancers and Ireland has the highest mortality rates from ovarian cancer in Europe (Figure 1.3). Table 1-1 Summary information, incidence and prevalence in ovarian cancer in Ireland according to NCRI figures 2011 3 Rank among the common cancers in women % of all new cancer cases Average number of new cases per annum % of all cancer deaths Average number of deaths per anuum Average age at diagnosis of ovarian cancer Average age at death Chance of developing ovarian cancer • By age 65 • By age 75 5 year survival Rank among the common cancer in women 3
7th 3.8% 315 7% 269 63.5 69.5 1% 1.5% 38% 4th
Introduction
Chapter 1
Figure 1-2 Worldwide estimated age-standerised incidence rate of ovarian cancer per 100,000 population; all age (2008) 4 Thirty two per cent of ovarian cancers present in women aged 50-64 years, with just over 20% presenting in each of the 65-74 and 75 and older age groups. The median age at presentation is 63. The incidence increases with age. Between the ages of 70 and 74 years the age specific incidence is 57/100,000 women/year 5. Advanced age at diagnosis contributes to the mortality of the disease. One quarter of cases of ovarian cancer occur in women aged under 50. In terms of deaths, 42% are in women aged under 65, 27% in women aged 64-74 with the remaining 31% in women aged 75 and older.
4
Introduction
Chapter 1
Figure 1-3 Estimated age standarised mortality rate of ovarian cancer per 100,000 population. European union (2008) 4 Epithelial ovarian cancer (EOC) is predominantly a disease of peri- and postmenopausal women, with 80% to 90% of ovarian cancers occurring after the age of 40. One percent of EOC occurs before the age of 20. Ovarian cancers are mostly sporadic and only 5%-10% are familial. Hereditary ovarian cancers generally occur about 10 years earlier and are associated with a trend towards an earlier age at diagnosis with each successive generation 6, a phenomenon known as anticipation. While the overall lifetime risk of developing ovarian cancer in industrialized countries is ~2% 7, for women with a family history, the lifetime risk is estimated at 9.4% 8.
5
Introduction
Chapter 1
1.1.3 Etiology The etiology of epithelial ovarian cancer remains poorly understood. At the most basic level, the exact tissue of origin is not even clear, or at least not entirely consistent9,
10
. The ovary is surrounded by a single-cell layer of coelomic
epithelium, which has the potential to undergo malignant transformation and differentiate into a variety of cell types resembling those found in the fallopian tube, uterus, cervix and ovarian stroma. Until recently, it has been widely thought that most ovarian cancers develop from either this surface epithelium or postovulatory inclusion cysts
9, 11
. However, recently the distal fallopian tube also
has been thought to harbor the precursor lesions of serous ovarian cancer
12, 13
.
Prolonged exposure to hormones or other cytokines may induce inappropriate activation of pathways that could explain the müllerian-like features and morphologic heterogeneity of epithelial ovarian cancer. Several hypotheses have been proposed to explain the underlying physiological processes, that increase the risk of malignant transformation (Table 1-2) 14. Knowledge of the etiology of ovarian cancer helps us to understand the mechanisms underlying its risk and protective factors.
6
Introduction
Chapter 1
Table 1-2 Hypotheses on physiologic susceptibilities to epithelial ovarian cancer Hypothesis
Proposed mechanism
Best evidence
Incessant ovulation15, 16
OSE is damaged during ovulation and repair makes cells susceptible to mutations
Gonadotropin stimulation17, 18
Stimulatory effects of FSH and LH promote growth, increased cell divisions and mutations
Hormonal stimulation19, 20
High concentrations of androgens in the tumour microenvironment promote carcinogenesis; whereas progestins decrease risk
Inflammation21, 22
Damaged OSE with ovulation induces inflammation, which promotes reconstruction and mutation susceptibility
Risk of EOC decreases with decreased number of cycles, (pregnancy, lactation, and OC use) Increased EOC risk with infertility, PCOS; decreased risk with progesterone-only OCs;FSH upregulates many oncogenes Conditions of high circulating androgens (within inclusion cysts, PCOS) increase risks; progestin use decreases risk of EOC and induces OSE apoptosis Possible reduced risk with NSAID use; increased risk with talc or asbestos; abundance of inflammatory mediators in tumours
Abbreviations: OSE, ovarian surface epithelium; EOC, epithelial ovarian cancer; OC, oral contraceptives; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PCOS, polycystic ovarian syndrome
1.1.4 Risk Factors Numerous reproductive, environmental, and genetic risk factors have been identified. Early menarche, late menopause and nulliparity have been associated with an increased risk of ovarian cancer likely due to increased ovulation 23. After correcting for the effect of voluntary nulliparity, studies have found that infertility adds to EOC risk in nulliparous women. The association between fertility
7
Introduction
Chapter 1
drugs and EOC risk is not necessarily causal as women with refractory infertility are mostly exposed to prolonged treatment with fertility drugs 24, 25. A family history of ovarian cancer in a 1st-degree relative (i.e., mother, daughter, sister) triples a woman's lifetime risk of developing ovarian cancer. The risks further escalate with two or more afflicted 1st-degree relatives. Patients may be considered at increased risk due to their personal history (i.e., breast cancer prior to age 50, dual primary breast cancer) or due to family members having ovarian, breast, endometrial or colon cancer
26
. For women with family history the risk is
much higher and rates of 16-54% are given for those with defined BRCA germline mutations 27-29 Racial differences in ovarian cancer also exist with white women having the highest incidence of ovarian cancer among all racial and ethnic groups
30
.
Compared to black and hispanic women, the risk is elevated by 30 to 40% 31. In a recent pooled analysis of 12 cohort studies, height was associated with an increased ovarian cancer risk, especially in premenopausal women. Body mass index was not associated with ovarian cancer risk in postmenopausal women but was positively associated with risk in premenopausal women 32 Additional variables associated with ovarian cancer with various degrees of correlation include radiation exposure and psychotropic medication
33
.
No
statistically significant relations were found for consumption of specific dairy foods, galactose, caffeine or vitamin D and ovarian cancer risk dietary factors
34
.
Smoking, caffeine and alcohol intake are all potentially modifiable factors that have an unclear association with ovarian cancer risk. Alcohol is not associated with 8
Introduction
Chapter 1
ovarian cancer risk. Cigarette smoking may only increase the risk for mucinous ovarian tumours. High caffeine intake decreases ovarian cancer risk, particularly in women not using hormones 35. A prior history of pelvic inflammatory disease (PID) (PCOS)
37
36
, polycystic ovary syndrome
and endometriosis have been associated with an increased risk for
ovarian cancer. Endometriosis is associated with a higher risk of endometroid and clear cell histology in the ensuing cancer 38. 1.1.5 Protective Factor Pregnancy reduces the risk of ovarian carcinoma, the risks decrease with each live birth, eventually plateauing in women delivering five times
39
. One interesting
theory to explain this protective effect is that pregnancy may induce shedding of premalignant ovarian cells
40
. Alternatively, pregnancy may simply decrease the
number of lifetime ovulations and gonadotrophin secretion, thereby resulting in less capsular repair that when altered can result in a transforming event 41. Lactation suppresses the secretion of pituitary gonadotropins and leads to anovulation, especially in the initial months after delivery and in this respect, it protects against EOC 41, 42. Combined oral contraceptives (OCP) containing both oestrogens and progestins, suppress mid-cycle gonadotrophin surge and inhibit ovulation. In ovarian cancer epidemiology there is strong evidence that OCP use truly reduces ovarian cancer risk
43
. OCP use seems to protect against all histological types of EOC with the
possible exception of mucinous tumours 41. The overall protection for ever users is 30%. Reduced risks are present across all strata of parity and age at diagnosis, 9
Introduction
Chapter 1
with a stronger protection observed among nulliparous women increases with duration of OCP
44
44
. The protection
use with a 50% decline in risk after 5 years on
the pill 45. The reduced risk persists for at least 10 years after cessation of use 46. Tubal ligation and hysterectomy are associated with a 67% risk reduction for EOC, the mechanism being rather speculative, possibly through local disruption of ovarian blood supply. This protective effect appears to last for at least 20 to 25 years after surgery 47. 1.1.6 Histology The histological grouping of ovarian tumours encompasses variety of histological features, diverse morphology and a plethora of cell types. The task forces of the International Federation of Gynecology and Obstetrics (FIGO) endorse the histological typing of ovarian tumours as presented by the World Health Organisation (WHO)
48
, based on histogenetic principles which categorize ovarian
tumours with regard to their derivation from coelomic surface epithelial cells, germ cells and mesenchyme. According to FIGO, it is recommended that all ovarian epithelial tumours be classified
histologically
as
follows:
serous
tumours,
mucinous
tumours,
endometrioid tumours, clear cell tumours, Brenner tumours, undifferentiated tumours (too poorly differentiated to be placed in any other group), and mixed epithelial tumours (composed of 2 or more of the 5 major cell types of common epithelial tumours, which are usually specified), (Table 1.3). Malignant papillary serous adenocarcinomas(PSC) (resembling the fallopian tube) are the commonest, accounting for 40%-50% of malignant neoplasms 10
49
and
Introduction
Chapter 1
predominate in postmenopausal women
50
, almost always present as advanced
disease. They tend to be high grade and are the type most often seen in BRCA mutation carriers51. Table 1-3 Histological classification of epithelial ovarian tumours according to FIGO Serous Tumours • Serous Cystadenoma • Borderline Serous Cystadenoma • Serous Cystadenocarcinoma Mucinous Tumours • Mucinous Cystadenoma • Borderline Mucinous Cystadenoma • Mucinous Cystadenocarcinoma Endometroid Tumours • Endometroid cystadenoma • Borderline Endometroid cystadenoma • Endometroid cystadenocarcinoma Clear Cell Carcinoma • Benign clear cell tumour • Borderline clear cell tumour • Clear cell carcinoma Transitional Cell (Brenner) Tumour • Benign Brenner tumour • Borderline Brenner tumour • Malignant Brenner tumour Undifferentiated Carcinomas
Mixed Epithelial Tumours
Benign Tumour of low malignant potential Malignant Benign Low malignant potential Malignant Benign Low malignant potential Malignant Benign Low malignant potential Malignant Benign Low malignant potential Malignant A malignant tumour of epithelial structure that is too poorly differentiated to be placed in any other group These tumours are composed of two or more of the five major cell types of common epithelial tumours (types should be specified)
Clear cell carcinoma (CCC) of the ovary (resembling the endometrial glands in pregnancy) accounts for 5–25% of all EOC, depending on geographic location
52
.
In North America and Europe, CCC is the second most common histologic sub11
Introduction
Chapter 1
type of EOC, with an estimated prevalence of 1–12 % prevalence of CCC is 15–25%
55, 56
53, 54
. In Japan, the
, occurring most often in women in their 40s
56
.
Approximately 50% of women with clear cell tumours have associated endometriosis
57, 58
. Although 33% of clear cell tumours are diagnosed at stage I,
they tend to behave aggressively
52, 53, 56
. Endometrioid carcinomas (resembling
endometrium) comprise about 10-20% of EOC and are typically spread beyond the ovary at the time of diagnosis. These cancers are sometimes associated with endometriosis.
Mucinous tumours (resembling endocervix) are less frequent,
each accounting for less than 10% of all EOC, more likely to be found in younger women. 80% are benign and develop in one ovary (Fig. 1-4).
Figure 1-4 Representative examples of the major histological subtypes of EOC. (A) PSC high grade, (B) PSC low grade, (C) Endometroid adenocarcinoma, (D) CCC, (E) Mucinous adenocarcinoma 59 12
Introduction
Chapter 1
The histological similarity of ovarian epithelial tumours to epithelia in other portions of the female genital tract is not surprising, given that all of these epithelia, as well as the cells lining the peritoneal cavity, are thought to be derived from a common embryological precursor, the coelomic mesothelium. The heterogeneity of ovarian cancer can be roughly separated into two broad categories of carcinogenesis. Low-grade tumours (type 1) progress through a stepwise mutation process, grow more slowly, are less responsive to chemotherapy, and share molecular characteristics with low malignant potential (LMP) neoplasms. High-grade carcinomas (type 2) demonstrate greater genetic instability, are rapidly metastatic, relatively chemo sensitive and without a clear precursor lesion 14, 60. 1.1.7 Staging Cancer staging can be either clinical or pathological. Pathological staging is usually considered the "better" or "truer" stage because it allows direct examination of the tumour and its spread, contrasted with clinical staging, which is limited by the fact that the information is obtained by making indirect observations of a tumour, which is still in the body. However, clinical staging and pathological staging should complement each other. Accurate staging is recommended in order to optimize treatment. Ovarian cancer can be staged according to the AJCC/TNM or FIGO System (Table 1-4) 61. The first system describes the extent of the primary Tumour (T), the absence or presence of metastasis to adjacent lymph Nodes (N), and the absence or presence of distant Metastasis (M). 13
It was first introduced by the Union
Introduction
Chapter 1
Internationale Centre de Cancer (UICC) in 1958 and then accepted by the American Joint Committee on Cancer (AJCC). The FIGO system of classification, now the most commonly used staging system, was originally based on clinical examination, essentially of the anatomical extent of disease. Over the years, it has moved from a clinical basis to one of a surgical pathological nature and was last modified in 1988 (Figure 1-5)
14
Introduction
Chapter 1
Table 1-4 Carcinoma of the ovary: FIGO nomenclature (Rio de Janeiro 1988) and UICC/AJCC classification FIGO
UICC/AJCC
Stage I
Growth limited to the ovaries
Ia
T1aN0M0
Ib
T1bN0M0
Ic
T1cN0M0
Stage II IIa IIb IIc
Description
Growth limited to one ovary; no ascites present containing malignant cells. No tumour on the external surface; capsule intact Growth limited to both ovaries; no ascites present containing malignant cells. No tumour on the external surfaces; capsules intact Tumour either Stage Ia or Ib, but with tumour on surface of one or both ovaries, or with capsule ruptured, or with ascites present containing malignant cells, or with positive peritoneal washings Growth involving one or both ovaries with pelvic extension
T2aN0M0 T2bN0M0 T2cN0M0
Extension and/or metastases to the uterus and/or tubes Extension to other pelvic tissues Tumour either Stage IIa or IIb, but with tumour on surface of one or both ovaries, or with capsule(s) ruptured, or with ascites present containing malignant cells, or with positive peritoneal washings Tumour involving one or both ovaries with histologically confirmed peritoneal implants outside the pelvis and/or positive retroperitoneal or inguinal nodes. Superficial liver metastases equal Stage III. Tumour is limited to the true pelvis, but with histologically proven malignant extension to small bowel or omentum.
T3aN0M0
Tumour grossly limited to the true pelvis, with negative nodes, but with histologically confirmed microscopic seeding of abdominal peritoneal surfaces, or histologic proven extension to small bowel or mesentery Tumour of one or both ovaries with histologically confirmed implants, peritoneal metastasis of abdominal peritoneal surfaces, none exceeding 2 cm in diameter; nodes are negative Peritoneal metastasis beyond the pelvis >2 cm in diameter and/or positive retroperitoneal or inguinal nodes
Stage III
IIIa
IIIb T3bN0M0 IIIC T3cN0M0 AnyT,N1,M0 Stgae IV
AnyT,anyN,M 1
Growth involving one or both ovaries with distant metastases. If pleural effusion is present, there must be positive cytology to allot a case to Stage IV. Parenchymal solid organs (liver, spleen) metastasis
15
Introduction
Chapter 1
Figure 1-5 Carcinoma of the ovary. Staging ovarian cancer: primary tumour and metastases (FIGO and TNM) 2
16
Introduction
Chapter 1
1.1.8 Grade Tumour grade is a system used to classify cancer cells in terms of how abnormal they look under a microscope and how quickly the tumour is likely to grow and spread. This can refer to the appearance of the cells or to the percentage that appear to be dividing. The higher the grade, the more aggressive and fast growing is the cancer. Tumours are typically classified from least to most aggressive as grade 1 through 3. Historically, the most commonly used grading systems have been those proposed by FIGO, WHO, and the Gynecologic Oncology Group (GOG). The FIGO system uses three grades based on architectural criteria, i.e., the proportion of glandular or papillary structures relative to areas of solid tumour growth. Grades 1, 2, and 3 correspond to 50% solid growth, respectively (welldifferentiated, moderately differentiated, or poorly differentiated). The WHO system incorporates both architectural and cytological features, but these are not assigned based on quantitative criteria; as a consequence, this system can be considered rather subjective. In the GOG system, the grading method varies depending on the histological type of the tumour. Grade is related to prognosis in ovarian cancer, with patients with low-grade cancers doing better. Grade information is factored into treatment decisions for women with stage I disease.
•
Well-differentiated cancers have very clear boundaries and cells look relatively normal. They usually do not grow or spread rapidly.
17
Introduction
•
Chapter 1
Poorly differentiated cancers have less clearly defined boundaries and cells look very abnormal (Figure 1-6). They often grow and spread rapidly.
A unique feature of ovarian cancer is the subdivision of “low malignant potential” (LMP) or borderline tumours, which form one end of the pathologic grade (grade 0). As a result, the different histological subtypes are further classified as benign, borderline (low grade) and malignant (high-grade) to reflect their behaviour. Ovarian tumours are evaluated in the following grades:
•
GX: Grade cannot be assessed
•
GB: Borderline cancer (malignant)
•
G1: Well-differentiated cancer
•
G2: Moderately differentiated cancer
•
G3: Poorly differentiated or undifferentiated cancer
Figure 1-6 Same tumour showing different grades; (A) benign cystadenoma, (B)&(C) Borderline cystadenoma, (D) Grade 1 Cancer, (E) Grade 2 cancer, (F) Grade 3 cancer 62 18
Introduction
Chapter 1
1.1.9 Current management of ovarian cancer The first step in the management of patients with epithelial ovarian cancer is accurate diagnosis and thorough staging, with optimal surgical cytoreduction of metastatic disease, also referred to as debulking surgery. Postoperative platinumbased and taxane chemotherapy is administered to patients with a significant risk of recurrence. For bulky stage III/IV patients who are deemed not to be surgical candidates, neoadjuvant therapy is usually considered 63. Over the last three decades, 5 year survival for ovarian cancer patients has increased from 37 to 45%, related to more consistent use of cytoreductive surgery and combination chemotherapy with platinum compounds and taxanes 64. 1.1.9.1 Surgery Cytoreductive surgery is the cornerstone of the initial staging and treatment for most patients. Ovarian cancer is one of the few malignancies where surgeons will undertake cytoreductive operations, even if all macroscopic tumours cannot be removed. Reducing tumour burden to where no macroscopic tumour is left before chemotherapy is considered optimal cytoreduction. 1.1.9.1.1
Primary surgery
In patients with disease apparently confined to the pelvis, thorough staging is essential to define the correct extent of the disease at time of diagnosis. Considering the possible routes of spread, optimal surgical staging constitutes: midline laparotomy to allow thorough assessment of the abdomen and pelvis; a total abdominal hysterectomy, bilateral salpingo-oophorectomy, appendectomy 19
Introduction
Chapter 1
and infracolic omentectomy; biopsies of any peritoneal deposits; random biopsies of the pelvic and abdominal peritoneum; and retroperitoneal lymph node assessment
65
. Inadequate surgical staging can lead to understaging and
subsequently inadequate postoperative treatment, which can ultimately worsen patients’ prognosis. Thorough staging is the best treatment option in patients with presumed early-stage disease 66. Even in patients with advanced ovarian cancer, primary cytoreductive surgery is the cornerstone of the initial treatment
67-69
. The amount of residual disease after
primary surgery is generally considered the most important modifiable prognostic factor that influences survival of patients with advanced disease. Since Griffiths published the landmark study that first clearly delineated the inverse relationship between postoperative residual tumour size and patient survival
70
, nearly all
retrospective and prospective studies have confirmed that the extent of cytoreductive surgery and the amount of residual disease after primary surgery are the most important factors that influence the survival of patients with advanced ovarian cancer 71 (Figure 1-7). The tendency of ovarian cancer to remain clinically confined in the peritoneal cavity also reflects the ability to achieve optimal cytoreduction in 60% of women with advanced EOC. Optimal cytoreduction for advanced ovarian cancer is defined as removal of all disease 1 cm or larger in diameter
71, 72
, whereas complete
cytoreduction is defined as no visible residual disease at the end of surgery
62 63
.
Tumours amenable to cytoreduction are characterized by superficial implants without distant metastatic or parenchymal disease, are usually well differentiated
20
Introduction
Chapter 1
without deep-tissue invasion or dense adhesions and this contributes to an improvement in long-term outcomes.
Figure 1-7 Summary of literature demonstrating the survival (in months) differential according to the status of residual disease 71 The quoted sites of disease most frequently precluding optimal cytoreduction are the diaphragm, bowel and portal triad. Studies consistently show that gynaecologic oncologists are more likely than general surgeons to perform optimal surgery for ovarian cancer
73
. As a result, the National Institutes of Health, American College
of Obstetricians and Gynaecologists, royal College of Obstetricians and Gynaecologists and Society of Gynaecologic Oncologists all recommend that women with ovarian cancer be referred to a gynaecologic oncologist for their initial surgery 74, 75 The argument against aggressive surgical resection is based on the hypothesis that the initial extent of advanced disease correlates with the aggressiveness of 21
Introduction
Chapter 1
the underlying tumour biology and that this will ultimately dictate outcome, independent of the amount of residual disease at the end of the surgical procedure 76
. In maximal effort cytoreduction surgery, intestinal resection, splenectomy,
diaphragmatic resection and hepatic resection are undertaken as treatments of advanced ovarian cancer with acceptable morbidity, in the context of primary or secondary debulking 77. 1.1.9.1.2
Interval debulking
Interval debulking (cytoreductive) surgery (IDS) is the surgical procedure with debulking intent preceded and followed by chemotherapy during primary treatment of advanced epithelial ovarian cancer
78
. Many retrospective studies and
prospective non-randomized trials reported the beneficial effect of chemotherapy in patients with inoperable or bulky residual disease in advanced EOC. The rates of optimal resection in IDS after chemotherapy were reported ranging from 77% to 94%
79-82
. Other potential benefits of IDS after chemotherapy compared to
aggressive primary debulking surgery were, less blood loss, lower requirement of intensive care unit admission and shorter hospital stay
81, 82
. The quality of life
(QOL) of patients treated with IDS was also reported in one study to be better than those who had conventional treatment (primary debulking surgery followed by a complete and continual cycles of adjuvant chemotherapy) 83. Unlike the aforementioned advantages of IDS, there is still conflicting evidence regarding its survival benefit in comparison to conventional treatment. Many studies showed similar survival rates of patients who had IDS after few cycles of chemotherapy and those who had conventional treatment 22
82, 84, 85
. Only a few
Introduction
Chapter 1
studies reported significantly longer survival of patients having IDS
86, 87
and even
fewer studies showed an inferior result of IDS than optimal primary cytoreduction 88
.
1.1.9.1.3
Second look surgery and secondary debulking
Nowadays, second-look operation is not routinely used because of the lack of adequate data showing a clear survival benefit for patients undergoing this surgical procedure, and it should be reserved to patients enrolled in clinical research protocols or to selected cases 89. At least 60% of advanced stage (stage III–IV) ovarian cancer patients who are without clinical evidence of disease after completing primary therapy will ultimately develop recurrent disease
90
. Repeat or secondary cytoreductive surgery for
recurrent ovarian cancer is defined as an operative procedure performed at some time remote (disease- free interval of more than 6 to 12 months) from the completion of primary therapy with the intended purpose of tumour reduction
91
.
Although primary cytoreduction is universally considered the cornerstone of initial management for patients with advanced disease, the role of secondary cytoreductive surgery is still debated. In an attempt to identify ideal candidates for secondary surgery, Onda et al. suggested that patients with recurrent ovarian cancer should be considered ideal for secondary surgery when they have 3 or all of the following 4 factors at recurrence: (1) disease-free interval longer than 12 months, (2) no liver metastasis, (3) a solitary tumour, and (4) tumour size smaller than 6 cm. Exclusion criteria included (1) age of 75 years or older at recurrence, (2) bad performance status and (3) progressive disease during pre-surgical 23
Introduction
Chapter 1
chemotherapy. A complete secondary surgical cytoreduction was achievable in most of these selected patients and was associated with a significant survival benefit 92. 1.1.9.1.4
Fertility sparing surgery
EOC is largely a postmenopauseal disease. However 3-17% of all EOCs occur in woman under 40 years of age. In these patients, the preservation of reproductive and endocrine functions is considered. In general, fertility-sparing surgery (FSS) has been adopted for two groups of patients: (1) young women with borderline ovarian tumours -often diagnosed incidentally after oophorectomy or cystectomysince the rate of recurrence after a conservative treatment is very low (3%-6%) suggesting that eventual restaging after an incomplete primary staging is not required
93
. (2) young women with invasive ovarian cancer macroscopically
confined to one ovary, who desire further childbearing. These patients undergo a unilateral salpingo-oophorectomy with comprehensive staging. Again, the recurrence rate and the overall survival rate have been reported to be similar among patients with ovarian cancer who were treated conservatively and those who underwent a more aggressive surgical procedure
94
. Pregnancies after
fertility-sparing procedures have been reported 95. 1.1.9.2 Chemotherapy Ovarian cancer is a chemosensitive disease and combination chemotherapy results in complete remission in approximately 75% of patients. From 1960s to the present, primary chemotherapy for advanced ovarian cancer has evolved from single alkylating agents to cisplatin and cisplatin-based combinations, followed by 24
Introduction
Chapter 1
incorporation of paclitaxel and substitution of carboplatin for cisplatin
96, 97
.
Currently, the combination of paclitaxel and carboplatin (TC) is the standard firstline chemotherapy for ovarian cancer. In its most recent consensus statements on the management of ovarian cancer during the Fourth International Ovarian Cancer Consensus Conference, the Gynecologic Cancer InterGroup (GCIG) confirmed this. GCIG recommended the use of 175 mg/m2 paclitaxel, given intravenously (i.v.) over 3 h, followed by carboplatin as an i.v. infusion over 30–60 min at a dose adjusted to produce an area under the plasma concentration–time curve (AUC) of 5–6 mg·ml/min every 3 weeks for six cycles 98, 99. Almost all patients with ovarian cancer are treated with chemotherapy post surgery. The current recommendation for chemotherapy is as folllows: a. Unilateral, encapsulated, well-differentiated serous and endometrioid carcinoma (stage Ia grade 1, non-optimally staged) may be managed without adjuvant chemotherapy. b. Stage Ia, and Ib that has been comprehensively staged, well or moderately differentiated (grade 1/2) may be managed without adjuvant chemotherapy. c. Poorly or undifferentiated (grade 3) stage Ia/Ib disease should be offered adjuvant chemotherapy. d. Non serous histotypes, mucinous and clear cell, should be offered adjuvant therapy 65, 67. e. Stage Ic, II, III and IV should be offered chemotherapy Chemotherapy can be given before surgery (neoadjuvant). This is mainly indicated in a small (but constantly increasing) group of patients, mainly stage IIIc and IV 25
Introduction
Chapter 1
papillary serous carcinoma, where surgery cannot be performed either because complete cytoreduction is not achievable or because surgery carries a high morbidity rate. This approach produced similar overall and progression-free survival as primary debulking surgery followed by chemotherapy, but with less complications and a lower postoperative mortality 85, 100, 101. 1.1.9.3 Radiotherapy The use of whole abdominal irradiation (WAI) as consolidation therapy would appear to be a logical strategy given its ability to sterilize small tumour volumes, but the wide distribution of EOC in the abdomen cavity limits its application 102-104, 1.1.9.4 New agents Angiogenesis, the formation of new blood and lymphatic vessels from existing vasculature, is one of the critical processes for the growth, invasion, and metastasis of solid tumours
105
. This process is regulated by a number of growth
factor receptor pathways and cytokines, such as vascular endothelial growth factor (VEGF). In ovarian cancer, the expression of VEGF is higher than in benign or normal ovarian tissue and their levels correlate with the activation of signal transducers and activators of transcription (STATs) pathway, while preoperative serum VEGF expression levels were associated with advanced stage and worse survival 106, 107. Bevacizumab, whose approximate molecular weight is 149 kDa, is a recombinant humanized monoclonal IgG antibody that targets VEGF-A
105
. Bevacizumab binds
and neutralizes all biologically active forms of VEGF-A and then suppresses 26
Introduction
Chapter 1
tumour growth and inhibits metastatic disease progression by inhibiting neovascularization and regressing existing microvessels. In addition, bevacizumab is thought to improve the structure and function (normalization) of tumour vessels that are highly disorganized with numerous abnormalities. These morphological changes lead to functional changes (e.g. decreased interstitial fluid pressure, increased tumour oxygenation, and improved penetration of drugs in the tumours), which may improve the delivery of chemotherapeutic agents to the tumour. Thus, bevacizumab is thought to make tumours more sensitive to chemotherapy 108.
27
Introduction
Chapter 1
1.2 Venous thromboembolism Deep vein thrombosis (DVT) and pulmonary embolism (PE) are separate but related aspects of the same dynamic disease process known as venous thromboembolism (VTE). PE results from occlusion of pulmonary arteries by thromboemboli originating in the deep veins of the calf or pelvis in the majority of cases. This occlusion has variable, both mechanical and reflex effects of vascular occlusion with a consecutive perfusion defect as well as the release of vasoactive and other inflammatory mediators 109. VTE is a major health issue. An estimated 200,000 new cases occur in the United States, annually, consisting of 106,000 with DVT and 94,000 with PE which has an incidence of 23 per 100,000 patients per year-cases
110
, or one event per 1,000
person-years for the general population and reaching one event per 100 personyears for those over 85 years 111, 112. A comprehensive understanding of the pathogenesis of VTE is essential to enable the identification of patients at increased risk and who may therefore benefit from reinforced preventive measures and more aggressive therapies in terms of both type and duration. It is now widely accepted that the pathogenesis of VTE is in fact multifactorial, wherby any single cause (risk factor) predisposes to, but is not sufficient on its own, to trigger thrombosis. Consequently, all risk factors, whether congenital or acquired (Table 1-5), are relatively ‘‘innocent’’ when considered alone. However, when an individual with one or more hereditary abnormalities, encounters environmental hazards, that person may be propelled over the threshold that precipitates the development of thrombosis 110.
28
Introduction
Chapter 1
Table 1-5 Risk factors for Venous Thromboembolism Hereditary risk factors (primary)
Acquired risk factors (secondary)
Factor V Leiden mutation
Immobilization
Prothrombin gene mutation
Surgery within 3 months
Protein S deficiency
History of VTE
Protein C deficiency
Malignancy
Antithrombin (AT III) deficiency
Obesity
Heparin factor II deficiency
Cigarette smoking
Plasminogen deficiency
Hypertension
Factor XII deficiency
Antiphospholipid syndrome
Increased factor VIII coagulant activity
Congenital heart failure
Hyperhomocyseinemia
Nephrotic syndrome Sickle cell anaemia Essential thrombocythemia Hormonal therapy (OCP, HRT)
The pathogenesis of VTE is centered on three key factors known as Virchow’s triad, (a) the hypercoagulability of circulating blood, (b) change in the vessel wall and (c) stasis
113
(Figure 1-9). Stasis is thought to be a permissive factor and
alteration in blood constituents, including inflammatory mediators and changes in the vascular endothelium are the key events
114
. In arterial thrombosis, platelets
are the core component, but in venous thrombi the main constituent is fibrin, which facilitates the thrombus attaching to the vessel wall. The processes that initiate the formation of such thrombi are uncertain but inflammation
and
stasis
play a
major role. Inflammation activates the endothelium to release Weibel-Palade bodies containing Von Willebrand protein (VWF) and P-selectin, which facilitate the binding of leucocytes to the area. Inflammation triggers the release of inflammatory mediators, which can downregulate anticoagulant pathways thereby 29
Introduction
Chapter 1
promoting thrombus formation at a site of endothelial damage. Stasis can also activate the endothelium but in a different manner. Low flow rate of blood causing a buildup of prothrombotic components such as thrombin that would normally be inactivated by normally flowing blood
115
. As a result, the risk for DVT is increased
in bedridden patients and also those on long haul flights 112.
Figure 1-8 Virchow’s Triad
30
Introduction
Chapter 1
1.3 Venous thromboembolism and ovarian cancer Cancer is a substantial risk factor for the development of VTE, with an estimated 20% of cancer patients experiencing thrombosis at some time during the course of their disease
116
. Patients with cancer also contribute approximately 20% of the
new cases of VTE occurring in the community
117
. Even these numbers may
underestimate the degree to which cancer patients are afflicted by thrombosis, as autopsy studies have found venous thrombi in up to 60% of patients dying with cancer
118, 119
. Historically, the French physician, Armand Trousseau, is credited
with initially describing the relationship between VTE and cancer in a seminal book published in 1865
120
; But the first description of DVT in patients with cancer was
made by Bouillard in 1823, 42 years before Trousseau 121. Ovarian cancer, pancreatic cancers and glioblastoma are the three cancers with the highest risk of VTE
122
. Gynaecological malignancies and ovarian cancer in
particular have been associated with high rates of VTE even in the setting of appropriate prophylaxis
123, 124
. Ovarian cancer-related VTE is estimated at 12 per
1,000 patients, compared to the VTE risk in the general population at 117 per 100,000 capita124-126. The clear cell ovarian cancer appears to have an even higher propensity for VTE with rates from 11-27.3%
123, 127-130
. Patients with
advanced ovarian cancer often have many risk factors for thrombosis (Table1-6), including concomitant infection, prolonged bed rest, indwelling central venous catheters, direct vascular compression or invasion by the tumours and radical debulking cytoreductive surgery. Indeed, thrombosis is often the presenting symptom of patients with ovarian cancer
131
, This prothrombotic effect may be due
to direct release of procoagulant substances by the tumours 31
132
, or by signaling to
Introduction
Chapter 1
other cells such as monocytes, macrophages and endothelial cells to produce such substances 133. Table 1-6 Risk factors for thrombosis in cancer patients 134 Category
Risk factors
Patient related
Age
risk-factors
Race Medical co morbidities Obesity H/O thrombosis
Cancer related
Site (Lung, pancreas and ovary carry high risk)
risk-factors
Stage (high stage, especially with distal metastasis) Grade (high grade) Histology (adenocarcinoma higher than squamous carcinoma Time (highest in first 3–6 months)
Treatment related Chemotherapy risk-factors
Antiangiogenic agents Hormonal therapy Erythropoiesis-stimulating agents Transfusions Indwelling venous access devices Radiation Surgery
32
Introduction
Chapter 1
1.4 Pathogenesis of venous thromboembolism The haemostatic system maintains blood in a fluid state under normal conditions and responds to vessel injury by the rapid formation of a clot. Fibrin clot formation in response to tissue injury is the most clinically relevant event of haemostasis under normal physiological conditions. The explosive activation of the haemostatic system occurs as a result of the cascade system of coagulation in which inactive zymogens and cofactors are sequentially activated by proteolytic cleavage. The resulting fibrin production stimulates the fibrinolytic system, limiting fibrin deposition to the site of injury and a system of naturally occurring anticoagulants feeds back and prevents further activation of the coagulation pathway. The perfect haemostatic clot forms at a site of injury sealing the injured vessel but without propagation through the vascular tree. Thus, the “perfect clot” does not disrupt blood flow to other tissues. 1.4.1 Coagulation activation The first model of coagulation was proposed in 1960s, which consisted of a series of steps in which activation of each clotting factor led to sequential activation of another finally leading to a burst of thrombin generation
135, 136
. In this model, each
clotting factor existed as a proenzyme that could be converted to an active enzyme. In addition, the clotting sequences were divided into so- called extrinsic and intrinsic systems. The “extrinsic” system consisted of factor VIIa and tissue factor (TF), the latter being viewed as occurring only extrinsic to the circulating blood. By contrast, the factors in the “intrinsic” system were all thought to be intravascular. Both the “extrinsic” and the “intrinsic” pathways could activate factor X which, in complex with its cofactor Va, could convert prothrombin to thrombin. 33
Introduction
Chapter 1
The components of the extrinsic and common pathways are reflected clinically in the prothrombin time (PT); and components of the intrinsic and common pathways are reflected in the activated partial thromboplastin times (aPTT). Recent publications have changed this view to one that suggests there is no exclusion between the two and that they work synergistically 137, 138
Figure 1-9 Conventional cascade model of coagulation. The Waterfall/Cascade model consists of two separate initiations, intrinsic (contact) and extrinsic pathways, which ultimately merge at the level of Factor Xa (common pathway) 139
The more recently proposed model of hemostasis is a cell based model, in which it is proposed that hemostasis occurs in distinct, but overlapping, steps: initiation, amplification, and propagation
140
. The process requires the participation of 2 cell 34
Introduction
Chapter 1
types: TF-bearing cells and platelets. The initiation response is triggered by TF expressed on subendothelial pericytes and fibroblasts. Activated Factor VII (FVIIa), a serine protease that normally circulates in blood in low concentrations, binds to TF to activate Factor X to FXa. Subsequently, FXa (also a serine protease) generates trace amounts (0.1–1 nM) of thrombin. Any FXa that dissociates from the TF-bearing cell is rapidly inhibited in the fluid phase by TF pathway inhibitor (TFPI) or antithrombin (AT). Thus, the presence of inhibitors effectively localizes FXa activity to the surface on which it is formed. The procoagulant triggering reaction only proceeds when TF is exposed at a high enough level to overcome inhibition by TFPI and AT. In other words, FVIIa patrols the circulation in search of sites of vascular damage (i.e., where TF is exposed), and trace quantities of FXa and thrombin sound the “alarm” for any potential dangers. This activity is tightly monitored by naturally occurring inhibitors that prevent a “false alarm” or “too extensive of a response”
141
. In the amplification
phase, the small amounts of thrombin generated initially play a major role; activating platelets, and factor V and VIII and factor XI on platelet surface. By the end of this phase, the stage is set for large-scale thrombin generation in the propagation phase. The platelet is probably the only cell on which propagation of coagulation can occur effectively. The platelet surface is specialized to coordinate assembly of the tenase (FIXa/VIIIa) and prothrombinase (FXa/Va) complexes, leading to generation of large amount of thrombin, which leads the conversion of fibrinogen to fibrin. Fibrinogen is a plasma protein synthesized in the liver. Converted by thrombin to fibrin, it forms an insoluble polymer that seals the site of injury by forming a haemostatic plug 138, 141.
35
Introduction
Chapter 1
Figure 1-10 Cell – based model of coagulation which consists of three distinct, but overlapping phases; initiation, amplification and propagation 142
1.4.1.1 Tissue factor TF is also known as thromboplastin, CD142, and coagulation factor III. It is 47kDa integral membrane glycoprotein consisting of an extracellular domain, a single membrane-spanning domain, and a short cytoplasmic tail. First cloned independently by four different groups in 1987
143-146
, topographically, TF is a type
I integral membrane protein, which means that the amino-terminus of the protein is located outside the cell, whereas the carboxy - terminus is located inside the cell. The overall domain structure of TF is given diagrammatically in Figure 1-11 36
Introduction
Chapter 1
Figure 1-11 TF/FVII/FX complex 147 TF is mainly found on the surface of cells in which it is synthesized. TF is abundant in a variety of cell types distributed throughout the body, including adventitial cells surrounding all blood vessels larger than capillaries; differentiating keratinocytes in the skin; and a number of epithelial cell types, including those present in mucous membranes and many organ capsules
148
. The distribution of
TF can be rationalized by the requirement that it be present throughout the body, ready to trigger the clotting cascade at any time and place following vascular injury. This pattern of expression has been described as a protective hemostatic envelope surrounding the vasculature, organ structures, and the entire organism. In addition, TF is relatively abundant at anatomic sites in which the risk or the deleterious consequences of bleeding are high, such as the renal glomerulus, throughout the brain and placenta 149. In contrast to the high levels of TF in the vessel wall, only very low levels of TF are present in blood of healthy individuals. This so-called circulating TF consists 37
Introduction
Chapter 1
mainly of TF-positive micro particles (MP)
150
, which are submicron membrane
fragments derived from activated and/or apoptotic cells. Unstimulated monocytes express low levels of TF and may be the primary source of these MPs 150-152. TF expression can be induced by various pathological conditions; inflammation 153, 154
, sepsis
155
, atherosclerosis
156
, VTE157, and malignancy. The TF gene is
upregulated in many cancers; colorectal 158, pancreatic cancer 159, glioma 160, lung cancer
161, 162
and gastric carcinoma
163
. In addition to the role as a procoagulant
activator, TF participates in many tumour-related processes that contribute to malignant disease progression, including angiogenesis
164, 165
, metastasis
166-169
and malignant cell survival 170. Very limited research has been done on the role of TF in ovarian cancer. One study suggested that increased TF tumour expression in epithelial ovarian cancer increases the risk of VTE
171
. Another study showed a significant association
between tumour TF expression and clear cell carcinoma 172.
38
Introduction
Chapter 1
1.4.2 Regulation of coagulation Once a fibrin/platelet clot is formed over an area of injury, the clotting process must be limited to avoid thrombotic occlusion in adjacent normal areas of the vasculature. If the coagulation mechanism were not controlled, clotting could propagate throughout the entire vascular tree after even a modest procoagulant stimulus. Three important mechanisms regulate coagulation to ensure production of procoagulant activity confined to a site of injury.
Figure 1-12 Scheme of the coagulation-anticoagulation system 173 39
Introduction
Chapter 1
1.4.2.1 Activated protein C (APC) pathway This pathway consists of Protein C, Protein S, Thrombomodulin
(TM) and
endothelial protein C receptor (EPCR). Some of the thrombin formed during the coagulation process can diffuse away or be swept downstream from a site of vascular injury. When thrombin reaches an intact endothelial cell, it binds to TM on the endothelial surface. The endothelial thrombin/TM complex then activates protein C to activated protein C (APC), the cleavage of protein C to APC is accelerated by binding to EPCR on the surface of endothelial cells. APC becomes dissociated from EPCR and APC binds to its cofactor protein S and inactivates further thrombin generation by inactivating the cofactors FVa and FVIIIa, therefore shutting down the prothrombinase and tenase complexes.137
Disorders of the
APC pathway either genetic (Factor V Leiden) or acquired are associated with an increased risk of VTE 174
Figure 1-13 Activated Protein C pathway 175 40
Introduction
Chapter 1
1.4.2.2 Antithrombin Antithrombin, is a serine protease inhibitor expressed in the liver. FIXa, FXa, FXIa and thrombin are all inactivated by antithrombin. The ability of antithrombin to inhibit FIXa, FXa, FXIa, and thrombin is accelerated by heparin sulphate proteoglycans, which is the basis of the anticoagulant action of heparins. Antithrombin when bound to free thrombin and FXa removes these from the circulation thereby limiting their activity to the site of clot formation. When antithrombin binds thrombin it forms the thrombin-antithrombin (TAT) complex that can be measured in a clinical setting as a marker of thrombin generation
176
. A
variety of inherited mutations of the antithrombin gene exist and are associated with increased risk of thrombosis.
Figure 1-14 The antithrombin pathway 175
41
Introduction
Chapter 1
1.4.2.3 Tissue factor pathway inhibitor (TFPI) The initiation of coagulation through TF/FVIIa is shut down through the action of tissue factor pathway inhibitor (TFPI). TFPI is predominantly produced from the microvascular endothelium and is found in three distinct regions. The first is in the circulation and contains both free TFPI and TFPI bound to plasma lipoproteins. The second is found within the cytoplasm of platelets, and the largest (80%) is bound to the endothelium. Ten percent of the total TFPI is contained within platelets and is released in response to thrombin and other stimulants 177. TFPI inhibits the FXa/TF/FVIIa complex in a 2-stage process. First, TFPI binds and inactivates FXa. Secondly, TFPI/FXa/TF forms a quaternary complex with TF/FVIIa, thus inhibiting thrombin generation. TFPI also causes monocytes to degrade and internalize TF/FVIIa complexes on the cell surface 178.
Figure 1-15 TFPI pathway 175 42
Introduction
Chapter 1
1.4.3 Fibrinolysis Fibrinolysis is a mechanism that limits the formation of fibrin clots. The fibrinolytic system is a series of enzymes, which is initiated after the formation of fibrin, and functions by breaking down this protein into fibrin degradation products (Figure1.15). The
plasminogen
activator
system
consists
of
the
inactive
proenzyme
plasminogen, which,in the presence of fibrin is activated to the serine protease, plasmin, mainly by two other serine proteinases called urokinase and tissue-type plasminogen activators (uPA and tPA). Two endogenous inhibitors, plasminogen activator inhibitors -1 and -2 (PAI-1, PAI-2) control the activation of plasminogen. The final component of this system is a cell surface receptor for uPA, uPAR, which generates a highly efficient and focused proteolytic system at the cell membrane 179
.
Plasmin and plasminogen activators are present throughout the body in inactive forms (zymogens) that are activated under pathophysiological conditions. Plasmin has broad substrate specificity, and is involved in the process of fibrinolysis by lysing the fibrin clot. It also has an important role in degradation of the extracellular matrix during cell invasion and chemotaxis, and during tissue remodelling, which are essential steps of many physiological and pathological processes 180. The two plasminogen activators, uPA and tPA, are similar in structure but are coded for by two different genes. They also differ in distribution and biological function, as tPA primarily generates plasmin for thrombolysis and fibrinolysis
43
181
Introduction
Chapter 1
and uPA generates degradation of the matrix in both physiological and pathological processes The plasminogen activator system is involved at multiple stages of the metastatic cascade, and not just by catalysing degradation of the matrix as was originally thought. These stages include tumourigenesis, degradation, cell proliferation, cell migration, cell adhesion, angiogenesis, and intravasation 182. Plasminogen activation has been implicated in several human cancers including those of breast 183 prostate 184 colon, and rectum 185
Figure 1-16 The fibrinolytic system. Tissue plasminogen activator (tPA), urokinase type plasminogen activator (uPA), Plasminogen activator inhibitor 1 (PAI-1) 186
44
Introduction
Chapter 1
1.4.4 Predictive markers of VTE 1.4.4.1 D Dimer The D-dimer (DD) antigen is a unique marker of fibrin degradation that is formed by the sequential action of 3 enzymes: thrombin, factor XIIIa, and plasmin. First, thrombin cleaves fibrinogen producing fibrin monomers, which polymerize and serve as a template for factor XIIIa and plasmin formation. Second, thrombin activates plasma factor XIII bound to fibrin polymers to produce the active transglutaminase, factor XIIIa. Factor XIIIa catalyzes the formation of covalent bonds between D-domains in the polymerized fibrin. Finally, plasmin degrades the cross-linked fibrin to release fibrin degradation products and expose the D-dimer antigen, so DD reflects a global activation of blood coagulation and fibrinolysis 187. DD have been used for the exclusion of VTE,the diagnosis and monitoring of coagulation activation in disseminated intravascular coagulation (DIC), prediction of recurrent VTE and risk stratification of patients for VTE recurrence. When used in non-cancer patients, DD had a negative predictive value (NPV) of 99.1% -99.6%. In a large meta-analysis, Stein et al
188
demonstrated that a
negative D-dimer test by the rapid ELISA method is as diagnostically useful as a negative computed tomography (CT) or a negative compression ultrasonography study (CUS) in excluding PE and DVT, respectively. However, the NPV of DD testing in cancer patients is lower than in patients without cancer as a consequence of the higher prevalence of DVT in patients with cancer 189 DIC is a serious complication of sepsis, malignancy and other inflammatory condition. Early diagnosis is pivotal to initiation of appropriate management and 45
Introduction
Chapter 1
achieving better outcomes. DD is one of the diagnostic criteria used to diagnose DIC together with platelet count, prothrombin time and fibrinogen concentration 190 Elevated levels of DD have been used to predict risk of VTE in cancer patients 192
191,
. In one prospective cohort study, the highest risk of VTE was seen in patients
with elevated DD, who had a cumulative VTE incidence of 15% after 6 months of study inclusion compared to 5% in patients who had lower levels 192. DD has been also reported to predict risk of recurrent VTE in cancer patients after discontinuation of anticoagulation
193
and to assist in determining the proposed
duration of anticoagulation for VTE patients
194
. The Vienna Cancer and
thrombosis study group incorporated DD into the Khorana predictive model and found this has improved the sensitivity of the model 195, 196. There is growing evidence that DD antigen measurements can assist clinicians in numerous other clinical scenarios. Elevated levels are associated with poor prognosis and decreased survival in cancer patients 197-200.
Figure 1-17 The dynamics of D-dimer formation 187. 46
Introduction
Chapter 1
1.4.4.2 TAT Antithrombin binds to thrombin creating a stable thrombin-antithrombin complex (TAT), which is also used as a surrogate marker for coagulation activation. While antithrombin levels decrease, TAT complex has been observed to be increased in cancer providing further evidence of enhanced thrombin production in vivo.
201, 202
Many studies have investigated TAT levels in prothrombotic states to detect evidence of increased thrombin generation however its ability to predict VTE has had limited success. Patients with factor V Leiden mutation were shown to have high levels of TAT
203
. On the other hand, a population-based study failed to find
an association between TAT and factor V Leiden
204
. Another study has shown
that increased TAT level is present in
