HC37: Cancer, coagulation and thrombosis

The link between cancer and thrombosis

Cancer leads to coagulation, which leads to thrombosis. Coagulation itself also leads to cancer, and thrombosis may as well. The link between cancer and thrombosis was established almost 200 years ago by Jean-Baptiste Bouillaud and Armand Trousseau. Trousseau diagnosed himself with thrombosis and predicted he suffered of cancer. Months later, he indeed died of pancreatic cancer.

Dual problem:

Cancer and thrombosis are a dual clinical problem. Out of 7 million cancer-associated deaths, 1 million are attributed to thrombotic complications. Patients with cancer and thrombosis have an extremely bad prognosis. The second main cause of death in cancer patients is deep venous thrombosis/pulmonary embolism (VTE):

• 25% of all first VTE events are cancer-related
• The presence of malignancy results in a 7x increased risk of VTE
  • 0-3 months after cancer diagnosis: 54x increased risk
  • 3-12 months after cancer diagnosis: 14x increased risk
  • 12-36 months after cancer diagnosis: 4x increased risk
• 8% of all cancer patients experience VTE

The risk of developing VTE in case of cancer can be divided into 2 groups:

• Clinical risk factors
• Biological risk factors

Clinical risk factors

Stage:

Not all cancer types confer the same risk for VTE. Patients with pancreas, lung and/or brain cancer have the highest incidence rate of VTE. The more aggressive the tumor, the higher the risk. The risk of VTE reflects the stage of the disease → in remote cancers, the risk of VTE is highest.

Therapy:

Certain cancer treatments can increase the risk of VTE as well:

• Surgery: 2x increased risk of VTE in cancer patients
• Chemotherapy: the risk varies
• New antiangiogenic drugs
• Prolonged bed rest

Treatment of venous thrombosis in cancer patients usually consists of low molecular weight heparin (LMWH), which is more effective than vitamin K agonists. LMWH can be prescribed for 6 months. If VTE still is present after this, it is necessary to switch to vitamin K antagonists.

Biological risk factors

It is unknown which biological factors cause cancer-associated VTE. Possible causes are:

• Microparticles
• Neutrophil extracellular traps (NETs)
• Coagulation factors
• Localization of cancer cells to the blood (CTCs)
• Platelets/leukocytes
• Compression of blood vessels by the tumor

Microparticles:

Microparticles (MPs) are vesicles of 50 nM-1 μM shed from various cells. They are meant for intercellular communication and contain proteins and microRNA. TF+ microparticles (MP-TF) can be shed from:

• Platelets
• Leukocytes
• Endothelium
• Cancer cells

MP-TF predicts the chance of VTE → if more MP-TF+ is present, the cumulative incidence of VTE is higher:

• Healthy volunteers have low TF activity in plasma
• Cancer patients without VTE have low TF activity in plasma
• Cancer patients with VTE have high TF activity in plasma → microparticles play a role in VTE

A hypothesis is that once MP-TF is in the blood, it fuses with platelets, endothelial cells or the ECM. MPs can be measured with:

• Fluorescence-assisted cell sorting (FACS)
  • Cells flow through a machine with a laser → the size and number of cells are determined
  • Drawback: MPs <500 nM are often inaccurately measured
    • Physically impossible
• Functional assays/ELISA
  • Functional assay: MPs are put into a buffer → form complexes with FVIIa → FX is cleaved into FXa → the more activation, the more the substrate turns yellow
  • ELISA: antibodies specific for TF are spread on patient culture → a second antibody with an enzyme linked to it is added to recognize the other antibodies → the more TF is present, the more the substrate turns yellow
  • Drawback: inability to count MP numbers
• Nanoparticle tracking analysis
  • Tracks 10 nm-1000 nm particles based on light scattering/Brownian motion
  • Takes too long to use as a diagnostic or prognostic tool

Neutrophil extracellular traps:

Neutrophil extracellular traps (NETs) function to capture pathogens. DNA of neutrophils contains lots of enzymes which can degrade the pathogen. This usually is a defense mechanism. However, neutrophils can induce cancer-associated thrombosis → extracellular DNA forms a negative charged platform to bind platelets and activate coagulation factors. Because NETs contain DNA, they affect hemostasis:

• Histones activate platelets
• DNA facilitates inhibition of Tissue Factor Pathway Inhibitor
• DNA activates factor XII

Coagulation factors:

Studies of coagulation factors show that there are 4 groups:

• VT without cancer
• VT with cancer
  • Highest amount of vWF antigens → cancer must have something to do with vWF
  • Sometimes, tumors can also produce clotting factors
• No VT without cancer
• No VT with cancer

Knowledge gaps

The association between TF-MPs and cancer-associated thrombosis are not observed in every cancer type and not in all studies. Additionally, the involvement of NETs in cancer-associated thrombosis has only been demonstrated in vitro and in animal models. This results in 2 knowledge gaps:

• Are MPs and NETs really involved in cancer-associated thrombosis?
• Which other tumor characteristics are involved?

Mutated gene expression:

A hypothesis is that cancer-associated VTE is partially driven by tumor cell genomic events → mutated gene expression leads to VTE. KRAS mutations, for instance, increase the chance of VTE. Gene sets can be identified that are upregulated in colorectal tumors from patients with VTE. Despite seemingly different biological processes associated with cancer associated thrombosis before and during cancer diagnosis, a number of genes are upregulated in both groups. This opens up avenues for VTE prediction in cancer patients. Fibrin deposition in tumors reflect the VTE state in patients.

Proteins that are encoded by such genes need to be checked whether they are made by tumor genes or not. A CRC cohort compares 2 patients with the same type of cancer, of which 1 has VTE and 1 doesn’t:

1. Laser capture microdissection extracts tumor cells from a tumor
2. RNA is extracted from the tumor cells
3. RNA sequencing shows differentially expressed genes due to SNPs or mutations
   • Cancers with metabolic mutations have a high chance of developing VTE

Treatment:

Biological processes involved in VTE differ between patients with VTE before and with VTE after cancer diagnosis because of treatment.

Impact of hypercoagulation/VTE on cancer:

Hypercoagulation/VTE has an impact on the diagnosis and mechanisms of cancer:

• Diagnosis
  • Extensive screening of thrombosis patients indeed shows more tumors, but does not improve the survival rates
    • The tumors that cause thrombosis are often very aggressive
• Mechanisms: anticoagulant treatment potentially benefits the progression of cancer
  • Dalteparin (an LMWH) improves the 1-year survival of patients with a good prognosis (localized cancer)
    • Anticoagulation is only effective in unmetastasized patients
  • 6 months of anticoagulant treatment decreases the chance of developing cancer
  • Has an effect on cancer progression because:
    • Certain factors also have an effect on cells themselves → transmembrane receptors are activated
      • Thrombin and FXa → PAR1 is activated
      • TF-FVIIa and FXa → PAR2 is activated
      • Thrombin → PAR3 is activated
      • Thrombin → PAR4 is activated
    • Blood clotting itself can also modify cancer cells
      1. A metastasized cell expresses lots of TF → coagulation is activated
      2. Clotting capsulates the metastasized cell → the immune system cannot attack and induces migration