New thrombotic biomarkers in gastrointestinal tumors

Abstract

Venous thromboembolism is a relatively common comorbidity in cancer patients and the second leading cause of death in this population. Different anticoagulant treatments have been used to prevent this complication, but their low efficacy in unselected populations and the increased risk of bleeding associated with this tretaments have led to the abandonment of widespread thromboprophylaxis. This scenario stands out in gastrointestinal tumours, as although they are highly thrombogenic, they are also associated with a high risk of bleeding. Therefore, it is necessary to identify new thrombotic risk markers that can help select those patients who could benefit from primary thromboprophylaxis, despite the risk of bleeding. Thus, the objective of this thesis is to identify new thrombotic biomarkers in patients with colorectal cancer and advanced gastric cancer. In colorectal cancer, we focused on the study of hepsin, a serine protease involved in tumour invasion and activation of the extrinsic coagulation pathway. Retrospectively, we recruited nearly 300 patients with localized or metastatic colorectal cancer. Using immunohistochemistry, we measured hepsin staining intensity in the primary biopsy at diagnosis and correlated it with clinical variables such as cumulative incidence of thrombosis, metastatic relapse, overall survival, among others. Subsequently, we conducted in vitro and in vivo studies using colorectal cancer cells stably transfected with the hepsin gene, and determined the effect of its overexpression in migration, invasion, proliferation and thrombin generation assays. Finally, with a view toward therapeutic targeting, we used virtual screening and molecular docking techniques to identify FDA-approved drugs that could inhibit hepsin activity and suppress the effects of its overexpression in tumour cells. As main results, we described hepsin as a biomarker of thrombosis and metastatic relapse in patients with localized colorectal cancer. In line with this, hepsin overexpression in colorectal cancer cells significantly increased their invasive phenotype and thrombin generation capacity. Finally, we identified Venetoclax and Suramin as two new hepsin inhibitors that also suppressed its pro-tumoural and pro-thrombogenic effects. In conclusion, hepsin expressed in the primary tumour is a new potentially useful parameter for improving thrombotic risk prediction in patients with localized colorectal cancer, as well as a new target whose inhibition could prevent metastasis and thrombosis in these patients. In advanced gastric cancer, using a non-targeted expression array, we retrospectively compared gene expression in the primary tumour of 48 patients who developed venous thromboembolism during follow-up and 49 controls without this complication, matched by propensity score matching. Once genes significantly associated with the occurrence of venous thromboembolism were identified, we recruited a new cohort of 44 patients with this comorbidity and 39 controls. In the primary tumour, we measured the absolute expression of the previously identified genes by digital PCR and checked whether they followed the same trend concerning venous thromboembolism as in the first cohort, as well as their potential for stratifying patients at high risk. As main results, in the first cohort, we identified 15 genes whose differential expression was associated with the occurrence of venous thromboembolism, applicable to both intestinal and diffuse gastric cancer subtypes. In the validation cohort, 3 of these genes, EPS8, PRKD3 and SAA1, followed the same expression trend with respect to thrombosis, and their absolute expression distinguished two groups of patients with a significantly different risk of venous thromboembolism. In conclusion, we identified 3 genes whose expression in advanced gastric cancer could improve the prediction of venous thromboembolism.