Identificação de Proteínas Relacionadas à Resistência a Quimioterapia em Células Tumorais Circulantes de Pacientes com Câncer de Cólon Localmente Avançado

Abstract

The incidence of CRC in 2012 was approximately 1.36 million new cases causing 694,000 deaths worldwide. Recent works have been demonstrated that circulating tumor cells (CTCs) may be involved in metastatization process. Thus, its isolation is a strategy for clinical follow-up as a non-invasive method. This study's objectives are: 1) Using the ISETTM Technology (Isolation by Size of Epithelial Tumor Cells) to isolate CTCs from peripheral blood of patients with locally advanced colon cancer (stage III), to count CTCs and, to correlate their levels with imaging and progression-free survival; 2) To analyze in CTC markers involved in: drug resistance (TYMS, ERCC1, DPD, TIMP-1), epithelial-to-mesenchymal transition and invasion (MMP-2, MMP-9, TGF-beta), proliferation (Ki- 67) and dormancy (beta-galactosidase), and to correlate the presence of these markers with response to therapy and progression-free survival; 3) To verify mRNA expression of the same genes observed by immunocytochemistry plus epithelial-to-mesenchymal transition (EMT) transcription factors in CTCs, and to correlate with treatment response; 4) To verify if there is a relationship between the gene expression from platelets isolated simultaneously to CTCs, and to compare to original tumor (by medical records and literature data); 5) To identify the good responders and bad responders to treatment and to perform mRNA sequencing (from CTCs and platelets) aiming to identify new therapeutic targets; 6) To quantify, simultaneously to CTCs from locally advanced colon cancer patients, neutrophils and lymphocytes, and to compare these cells' ratio with progression free survival. Samples of 50 patients will be analyzed. Samples will be collected before surgery (baseline), after surgery and prior adjuvant chemotherapy (follow-up 1) after the end of the adjuvant chemotherapy (follow-up 2) and every 6 months until disease recurrence. CTCs are identified at all times by immunocytochemistry with specific antibodies for the markers cited above. All reactions will have a negative control (blood from healthy individuals) and positive control (the same blood spiked with colon tumor cells maintained in culture). It is expected by this work a better understanding of the mechanisms of recurrence, metastasis and treatment resistance in patients with locally advanced colon cancer by the analysis of CTCs and EMT transcription factors, and circulating tumor microemboli presence. It is expected, by platelets' molecular analysis and by performing quantification of neutrophils and lymphocytes, be able to identify new blood prognostic biomarkers that could direct clinicians to choose the best therapy. It is expected also to point new therapeutic targets, by platelets and CTCs gene expression, which could be used in the development of new drugs. It is expected also to get a better understanding of tumor biology, as well as the validation, in a different population, of the studies performed in our lab with the proteins TYMS and ERCC1, also related to resistance to chemotherapy.