Identificación de nuevos biomarcadores para cáncer colorrectal metastásicoAnálisis genómico y metabólomico

Abstract

Colorectal cancer (CRC) must be considered a real public health problem both because of its incidence, it is the third most frequent tumour in the world, representing 10% of all neoplasms, and because of its mortality, which places it as the second leading cause of death from cancer. In Spain and considering both sexes, CRC is the most frequent tumour, currently surpassing breast and lung tumours. Despite the important advances in the treatment of CRC in recent years and the improvement in its early diagnosis, a very important number of patients, between 20- 25%, present metastatic disease at the time of diagnosis and up to 50 % develop metastases throughout the course of the disease. Therefore, the determination of the metastatic stage of the disease at an early stage, as well as the development of strategies to determine its prognosis and the effectiveness of the applied therapy, is a priority field in cancer research. In this context, the increasingly in-depth knowledge of the etiopathogenesis of CRC, its heterogeneous nature, and the demonstration that multiple genetic and epigenetic alterations are capable of altering the control mechanisms of proliferation, differentiation and apoptosis cellular, are giving way, not only to the use of new molecular targets that allow a more precise and specific treatment, but to the determination of new biomolecules, which, used as biomarkers, can indicate the presence, evolution and / or response to treatment of the illness. Tumour biomarkers, understood as biological entities that can be detected in the different organic environments of cancer patients, are the molecules that are allowing the follow-up of the disease by monitoring its levels, its diagnosis (including population screening methods), its prognosis and even the follow-up of the therapeutic response. At present, there is a shortage of precise and sensitive markers for cancer in general and for CRC in particular. Thus, there are many studies that have investigated as a biomarker in CRC, DNA molecules released into the blood in process of cellular apoptosis or passage of living cells into the circulatory stream (circulating tumor cells or CTSs). Exosomes, small vesicles surrounded by a lipid bilayer that are involved in the transfer of miRNAs, mRNAs and proteins to a target cell, have also been proposed as biomarkers of this disease. Even the presence in the serum of patients with CRC of certain proteins has been extensively analyzed, despite which, only CEA and CA-19-9 are still used clinically, although they suffer from low sensitivity and specificity. Multiple studies have also been carried out to determine microRNA in patients with CRC that are specific for their diagnosis or prognosis but without definitive results. Among the molecules most widely analysed as CRC biomarkers are the presence of messenger RNA (mRNA) in peripheral blood. In this context, the mRNA of molecules related to the angiogenesis process have gained great interest in CRC, having tested the overexpression of molecules such as PTGS2 and GUCY2C as markers of the disease or of the presence of hidden metastases in patients with CRC. On the other hand, metabolomics, a discipline that evaluates the endogenous metabolites produced by the body, has revolutionised the field of biomarkers for different types of pathologies. Applied to cancer, metabolomics offers a molecular representation of the tumour phenotype that can be used for diagnosis, prognosis, and identification of clinically relevant subgroups. Tumour cells adapt their metabolism to uncontrolled proliferation that causes metabolic alterations that are reflected in the appearance or modulation of metabolite levels. In CRC, some specific metabolites (beta-hydroxybutyrate acid, cystamine, aspartic acid, among others) have been proposed for the diagnosis of CRC in both blood and urine, although the complexity to detect them and their high cost require more research efforts to take them into clinical practice. In this context, our work has had two fundamental objectives: i) On the one hand, to carry out a study that the utility of genes intensely related by the angiogenesis process, an essential phenomenon in the growth and metastatic expansion of a tumor, as new biomarkers easily detected in peripheral blood or serum of patients with metastatic CRC. For this we have selected five genes, GUCY2C, JAG1, PTGS2, PGF and MMP7, with demonstrated relevance in angiogenesis and we have determined the levels of circulating RNA both in serum and in peripheral blood mononuclear cells in a group of 59 patients with metastatic RCC faced with a group of 47 healthy controls. The presence of these markers in biological fluids was carried out by a highly sensitive technology such as digital PCR (dPCR) that would allow an easy development of its clinical application in a short period of time, whilst the analysis of the area under the curve (AUC) was used to estimate the predictive value of biomarkers both individually and in combinations (biological signature). ii) On the other hand, we have carried out a study to identify biomarkers based on the determination of metabolites in the serum of metastatic CRC patients that is useful in the diagnosis, prognosis and follow-up of these patients. In this case, and based on undirected metabolomics techniques, the sera of a group of 65 patients with metastatic CRC compared to 60 healthy controls were analysed. The samples were subjected to studies by reverse phase liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS), which allowed us to obtain a matrix to compare healthy controls with patients with CRC. The metabolites obtained were analyzed individually and also in the form of a "cluster" in order to obtain a possible biological trace.